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The application of a dihydropyridine-pyridinium salt redox system to drug delivery to the brain

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Title:
The application of a dihydropyridine-pyridinium salt redox system to drug delivery to the brain
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Brewster, Marcus Eli, 1957-
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English
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xiii, 160 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Blood brain barrier ( jstor )
Capillaries ( jstor )
Dihydropyridines ( jstor )
Dosage ( jstor )
Drug carriers ( jstor )
Hydrochlorides ( jstor )
Molecules ( jstor )
Neutral amino acids ( jstor )
Oxidation ( jstor )
Plasmas ( jstor )
Berberine Alkaloids -- pharmacology ( mesh )
Brain -- drug effects ( mesh )
Dissertations, Academic -- Medicinal Chemistry -- UF ( mesh )
Medicinal Chemistry thesis Ph.D ( mesh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida.
Bibliography:
Includes bibliographical references (leaves 150-159).
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Also available online.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Marcus Eli Brewster III.

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University of Florida
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THE APPLICATION OF A DIHYDROPYRIDINE-PYRIDINIUM SALT
REDOX SYSTEM TO DRUG DELIVERY TO THE BRAIN






BY


MARCUS ELI BREWSTER III






















A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA 1982
































Copyright 1982

by

Marcus Eli Brewster III
























TO MOTHER













ACKNOWLEDGEMENTS

I would like to express my gratitude to Professor Nicholas S. Bodor. His genius and kindness will always be an inspiration to me. I would also like to thank the other members of my committee, Dr. Merle Battiste, Dr. Kenneth Sloan, Dr. Margaret James, and Dr. James Simpkins, for their advice and help.

This work would truly not have been possible but for the guidance of a number of post-doctoral fellows and technicians, especially Dr. Hassan Farag, Dr. Cynthia Luiggi, Dr. Thorsteinn Loftsson, Mrs. Jirina Vlasak, Mrs. Nancy Gildersleeve, and Mr. Edward Phillips. I would also like to thank Jane and C.J. Rogers, Dr. Yasuo Oshiro and Dr. Tadao Sato for their assistance.

Although space is limited, I want to recognize a few friends who made graduate school bearable: Linda and John Hirschy, Mark V. Davis, R.E. Golightly, Mrs. James E. Ray, Newton Galloway, Mike Morris, Richard Panarese, Wayne and Anita Riggins, Mike and Kay Dempsey, Chuck Hartsfield, Mike Gibbons, Raun and Cissy Kilgo, Jeff and Jane Dean, Ernie Lee, and the late Chip Connally. Chip was a dear friend. I would like to thank Scott and Margie Makar for their cordiality and hospitality.

I would also to like to acknowledge the P.C.'s, Dr. Frank Davis, Mr. Jim Templeton, and Dr. Gary Visor, the last of a dying breed. Two special people deserve mention here because of their guidance early in my scientific career, Professor G.L. Ware, and Mrs. Frances Kenning. Special thanks are accorded to my Amazonian, sybaritic amanuensis, Cynthia Jordan. And, last but certainly not least, I would like to thank my family without
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whom I would not have been able to pursue an academic career. The anticancer testing was graciously performed by Otsuka Pharmaceutical Co., Ltd. This work was supported by Grant GM 27167 from the National Institute of General Medical Sciences.












































v













TABLE OF CONTENTS

CHAPTER PAGE

ACKNOWLEDGEMENTS .........................................i v

LIST OF TABLES.......................................... vii

LIST OF FIGURES........................................i x

ABSTRACT.............................................. xi i

1 INTRODUCTION........................................... 1

Blood-Brain Barrier .................................. 2
Prodrugs and Drug Delivery Systems ..................... 31
Statement of the Problem ............................. 41

2 MATERIALS AND METHODS .................................. 47

Synthesis ........................................... 49
Characterization of Dihydroberberine.................... 59
Animal Studies....................................... 63

3 RESULTS AND DISCUSSION ................................. 68

Synthesis and Characterization of Dihydroberberine .......68
Theoretical Studies on the Dihydropyridine~pyridinium
Redox System........................................ 81
Further Studies on the Chemical and Biological Properties
of Dihydroberberine................................. 104
In Vivo Studies ..................................... 116
Con-clus ions ......................................... 147

BIBLIOGRAPHY .......................................... 150

BIOGRAPHICAL SKETCH.................................... 160












vi












LIST OF TABLES

TABLE PAGE

1-1 Blood Brain Barrier Transport Systems......................... 14

3-1 Proton Assignments of the 1H NMR of Dihydroberberine (2)....... 74 3-2 Carbon Assignments of the 13C NMR of Dihydroberberine (2)...... 76 3-3 Distribution Coefficients for Berberine (1) and Dihydroberberine Hydrochloride (3) in Chloroform/pH 7.4 Buffer and in
l-Octanol/pH 7.4 Buffer....................................... 78

3-4 The Heats of Formation, Vertical Ionization Potentials, and
Dipole Moments of the Isoquinoline Model (30) and the Dihydroisoquinoline Model (31)....................................... 86

3-5 Bond Lengths in Angstroms between Various Atoms of the Isoquinoline Model (30) and the Dihydroisoquinoline Model (31).... 87 3-6 Bond Angles in Degrees between Various Atoms of the Isoquinoline Model (30) and the Dihydroisoquinoline Model (31)......... 88 3-7 Charge Density at Various Atoms of the Isoquinoline Model (30)
and the Dihydroisoquinoline Model (31)........................ 89

3-8 Dihedral Angles between Various Atoms of the Isoquinoline Model
(30) and the Dihydroisoquinoline Model (31) ................... 90

3-9 Differences in the Heats of Formations (AAHf) of (30) (31),
2-PAM 1,4-Dihydro-2-PAM and 2-PAM : 1,6-Dihydro-2-PAM........ 91

3-10 A Comparison of Bond Lengths in Angstroms of the Pyridine (32) 2
1,2-Dihydropyridine (33) System and the Isoquinoline (30) :
Dihydroisoquinoline (31) Model System......................... 94

3-11 A Comparison of the Bond Angles in Degrees of the Pyridine (32)i1,2-Dihydropyridine (33) System and the Isoquinoline (30) 2
Dihydroisoquinoline (31) Model System.......................... 96

3-12 A Comparison of the Atomic Charge Densities of the Pyridine (32)+
1,2-Dihydropyridine (33) System and the Isoquinoline (30)
Dihydroisoquinoline (31) Model System.......................... 97
3-13 The Rate of Oxidation of Dihydroberberine in Various Media...... 105



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TABLE PAGE

3-14 The Relative Rates of Oxidation of Dihydroberberine (2),
1-Methyl-1 ,4-dihydronicotinamide (21), and l-Benzyl-1 ,4dihydronicotinamide (22) in Dilute Hydrogen Peroxide......... 108 3-15 Proton Assignments of the 1H NMR of the l-Methyl-l,4-dihydronicotinic Acid Ester (27)................................... 112

3-16 The Rates of Oxidation and Corresponding Correlation Coefficients of Various 1-Methyl-1,4-dihydronicotinic Acid Esters
and l-Benzyl-l1,4-dihydronicotinamide (22)................... 114
3-17 The Effect of Glucose on the Movement of Berberine into Red
Blood Cells................................................. 118

3-18 Slow Infusion of Dihydroberberine (3)....................... 131

3-19 Efflux of 3H-Inulin from the Brain after Intracerebral Ventricular Administration........................................ 139
3-20 In Vivo Metabolism of Berberine and Dihydroberberine in the
at (RHPLC).................................................. 140
3-21 In Vivo Metabolism of Berberine and Dihydroberberine in the
E-t T C)................................................... 142

3-22 Probit Analysis of the LD50 Study.......................... 145

3-23 Effect of Berberine (1) and Dihydroberberine Hydrochloride (3)
Against P388 Lymphocytic Leukemia........................... 146





















viii













LIST OF FIGURES

FIGURE PAGE

1-1 This Schematic Illustration Represents an Endothelial Cell
Derived from either a Muscle (ECm) or Brain (ECb) Capillary.
In this Figure, (ma) is the Macula Adherens or Loose Junction, (zo) is the Zona Occludens or Tight Junction, (mv) are Microvesicles, and (bl) is the Basal Lamina. This Figure was Modified from Reference 1, Page 162 by Permission .................. 6

1-2 A Proposed Carrier-mediated Chemical Delivery System with
Specificity for the Brain. The Drug Molecule to be Transported is Represented by the (0 ) .............................. 40

1-3 The Proposed Drug Delivery Scheme .............................. 43

3-1 Synthesis of Dihydroberberine (2) and its Hydrochloride
Salt (3) ................................................... 69

3-2 Ultraviolet Spectrum of Dihydroberberine (2) in 95% Ethanol .... 70

3-3 Infrared Spectrum of Dihydroberberine (2) (KBr) ................ 71

3-4 Mass Spectrum (70 eV, EI) of Dihydroberberine (2) ............. 72

3-5 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of Dihydroberberine in CDCI3. The Insert Represents the Region between
2.86 and 3.46 at 100 MHz........................ .............. 73

3-6 The 13C Nuclear Magnetic Resonance Spectrum (100 MHz, CDCI3)
of Dihydroberberine (2) ........................................ 75

3-7 Demethylation of Berberine (1) and Methylation of Berberrubin (4) ...................................................... 80

3-8 Structures and Salient Numbering Protocols for Berberine (1),
Dihydroberberine (2), the Isoquinoline Model (30), the Dihydroisoquinoline Model (31), Pyridine (32), and 1,2-Dihydropyridine (33) .................................................. 83

3-9 The Highest Occupied Molecular Orbital of the Dihydroisoquinoline Model (31) ................................................ 92

3-10 A Computer-assisted Drawing of the Most Stable Conformation
of the Isoquinoline Model (30) at 25"C ......................... 99



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Figure Page
3-11 A Computer-assisted Drawing of the Most Stable Conformation
of the Isoquinoline Model (30) at 250C. This View is Oriented so that the Interatomic Axis between Atoms 26 and 2 is Perpendicular to the Plane of the Page.......................... 100

3-12 A Computer-assisted Drawing of the Most Stable Conformation
of the 1,2-Dihydroisoquinoline Model (31) at 250C.............. 102

3-13 A Computer-assisted Drawing of the Most Stable Conformation
of the 1,2-Dihydroisoquinoline Model (31) at 250C. This View
is Oriented so that an Imaginary Axis between Atoms 2 and 5
is Perpendicular to the Plane of the Page ..................* 103

3-14 Spectral Changes of Dihydroberberine (2) upon Oxidation to
Berberine (1) in pH 5.8 Phosphate Buffer at 260C. Traces
were made every 10 Min ....................................... 106

3-15 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of (27)
in CDC13.............................................. 111ll

3-16 The Rates of Oxidation of Various l-Methyl-l1,4-dihydronicotinic
Acid Esters (23), (24), (25), (26), (27), (28) and l-Benzyl1,4-Dihydronicotinamide (22) at 370C in 40% Human Plasma (l),
6% Brain Homogenate (A) and 3.5% Liver Homogenate (0) ........ 113
3-17 Partitioning of 26.5 mg of Berberine (1) from Plasma (A) into
Red Blood Cells (A) and of 26.5 mg of Dihydroberberine Hydrochloride (3) from Plasma (0) into Red Blood Cells (0). The
Volume of Blood Used in each Experiment was 75 ml .............. 117
3-18 Distribution of Berberine in the Brain after iv Administration
of Berberine (1) (*) at a Dose of 55 mg/Kg or of Dihydroberberine Free Base (2) (0) at a Dose of 55 mg/Kg ............... 120

3-19 Efflux of Berberine from the Brain after iv Administration of
either 55 mg/Kg of Dihydroberberine Hydrochloride (3) (0)
or 55 mg/Kg of Berberine (1) (*). Analysis was for (1) only
and not Unoxidized (2)........................................ 121
3-20 Efflux of Berberine (1) and Unoxidized Dihydroberberine (2)
(A) after iv Administration of 55 mg/Kg of Dihydroberberine
Hydrochloride (3)............................................. 122
3-21 A Comparison of the Efflux of Berberine (1) (0) and Berberine
(1) and Unoxidized Dihydroberberine (2) (A) after a Dose of
55 mg/Kg of Dihydroberberine Hydrochloride (3) Administered
iv........ .................................................... 123
3-22 Distribution of Berberine after iv Administration of 35 mg/Kg
of Berberine (1) into the Kidney ((), Liver (l), Lung (0),
and Brain (A ) ............................................... 125


x







Figure Page
3-23 Distribution of Berberine after iv Administration of 55
mg/Kg of Dihydroberberine Hydrochloride (3) into the Kidney
(), Liver (0), Lung (0), and Brain (A) .................. 127
3-24 A Comparison of the Efflux of Berberine from Lungs when Administered iv as 55 mg/Kg of Dihydroberberine Hydrochloride
(3) (0) or 35 mg/Kg of Berberine (1) (0) .............. 128
3-25 A Comparison of the Efflux of Berberine from the Kidneys when
Berberine (1) is Administered iv at a Dose of 35 mg/Kg (*) and Dihydroberberine Hydrochloride (3) when Administered iv
at a Dose of 55 mg/Kg (0) ......................** 129
3-26 A Comparison of the Efflux of Berberine from the Liver when
Berberine (1) is Administered iv at a Dose of 35 mg/Kg (U) and Dihydroberberine Hydrochloride (3) when Administered iv
a Dose of 55 mg/Kg ( ]) ......................................130
3-27 A Comparison of the Efflux of the Total Berberine, i.e. (1)
and (2) from the Brain when either 55 mg/Kg of Dihydroberberine Hydrochloride (3) is Administered iv (A) or 55 mg/Kg
of Dihydroberberine (2) and 200 mg/Kg of l-Methyl-l1,4dihydronicotinamide (21) is Administered iv (0) .............134
3-28 Efflux of l-Benzylnicotinamide Bromide (7) from the Brain after iv Administration of 60 mg/Kg of l-Benzyl-1,4-dihydronicotinamide (22) (A ) ...................................... 135
3-29 Efflux of Berberine from the Brain after icv Injection of
either 50 ug of Berberine (1) (0) or 50 pg of Berberine (1)
and 1000 ug of 1-Methylnicotinamide Iodide (6) (A).......... 137
3-30 The LDs5o Dose-response Curve of Berberine (1) (A) and Dihydroberberine Hydrochloride (3) ()). Doses of (1) or (2)
were Administered ip in CD-1 Mice...........................144















xi












Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


THE APPLICATION OF A DIHYDROPYRIDINE-PYRIDINIUM SALT
REDOX SYSTEM TO DRUG DELIVERY TO THE BRAIN


By

MARCUS ELI BREWSTER III

AUGUST 1982

Chairman: Nicholas S. Bodor
Major Department: Medicinal Chemistry

This work has been concerned with the design and testing of a broadly applicable drug delivery system which is specific for the brain. The method developed for this purpose is based on a dihydropyridine-pyridinium salt redox system and on the blood-brain barrier. In the proposed delivery system, a pharmacologically active agent which contains a pyridinium nucleus would be reduced to its corresponding dihydropyridine. After systemic administration, the highly lipoidal dihydropyridine would partition into the brain as well as into the periphery. In both locations oxidation

would occur. In the systemic circulation, the charge species would be rapidly eliminated by renal or biliary mechanisms while in the brain the compound would be retained.

The prototype compound which was chosen for inclusion in this scheme was berberine. This alkaloid has a high in vitro activity against various cancer systems but its in vivo activity is low. The first step in the application of the described delivery system to berberine is the synthesis



xii








of dihydroberberine which was accomplished using sodium borohydride in pyridine. The dihydroberberine was analyzed by various spectroscopic means and a number of physical properties were measured. Theoretical calculations using a MINDO/3 approach were also undertaken.

In order to verify the proposed scheme, the delivery of berberine to the brain and the retention of berberine in the brain had to be shown. When dihydroberberine or its hydrochloride salt were injected systemically, high levels of berberine were found in the brain and its efflux from the brain was slow. If, however, berberine is injected systemically, no detectable levels are found in the brain. When dihydroberberine is slowly infused the concentration of berberine rises in the brain and at

forty-five minutes, this concentration is specifically higher in the brain than in any other organ analyzed. The mechanism of efflux of berberine from the brain was investigated and appears to be mediated by a passive

process, perhaps the bulk flow of cerebral spinal fluid. Dihydroberberine was found to be less toxic than berberine. Preliminary anticancer data indicate that dihydroberberine is more effective in increasing the life span of animals injected intercerebrally with P388 lymphocytic leukemia

than is berberine.
















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CHAPTER I

INTRODUCTION

A method for delivering drugs specifically to a particular organ would be valuable. Properly designed, a drug delivery system should concentrate an agent at its site of action and reduce its concentration in other locations. The results of these manipulations would not only be an increase in the efficacy of an agent, but also a decrease in its toxicity.

A site specific system designed for the central nervous system (CNS) would be especially useful. The reason for this, aside from the inherent importance of the brain, is that the entry of many pharmacologically active agents into the CNS is impeded by a set of specialized barriers present at the blood-brain interface. This barrier system, termed the blood-brain barrier (BBB), is composed of numerous enzymatic and anatomical components.

This dissertation will present a general method for the specific delivery of drugs to the brain and give an example which substantiates the method. In order to provide an adequate background for a discussion of drug delivery to the brain, a review of the BBB is necessary. The introductory material is then continued with a cursory historical account of drug delivery systems and prodrugs. Because of the importance and great interest of anticancer agents, emphasis is placed on this topic in this section. The closing section of the first chapter will state specific aspirations as they apply to the present research.





2


Blood-Brain Barrier

Structural and Enzymatic Considerations of the Blood-Brain Barrier

The existence of a barrier which separates central nervous tissue from the general circulation was first postulated by Ehrlich at the end of the 19th century.'',2',3'4'5 In a series of pioneering experiments, he injected a number of dyes into laboratory animals and found that, while the visceral organs were highly stained, the brain was conspicuously uncolored. It was later discovered that these dyes bind extensively to plasma proteins so that the actual barrier then described was one to these complexes. Many small hydrophilic compounds cannot, however, pass into the brain so that this barrier is presented to a wide variety of compounds. Early in the study of the BBB, the obstruction was considered absolute but this idea was soon dispensed with since the nutritional requirements of the brain necessitate the equilibration of a number of compounds between the general circulation and the CNS.3

The morphological basis of the BBB had been a very controversial subject until relatively recently. Historically, three hypotheses have been put forward to explain this impermeability to blood-borne substances.' All are based on structural differences between the cerebral vascular system and the systemic circulation. It was proposed that the small extracellular space characteristic of mammalian brains prohibited the accumulation of compounds and, as such, constituted a barrier. It was shown, however, that some animals with large extracellular spaces have a well-defined barrier to a number of substances.6 The suggestion was also made that the general impermeability of the brain to blood-borne substances was due to astrocytic end feet which surround the capillaries, forming an envelope, or due to the endothelial cell lining of the cerebral capillaries.' In order to study this question, electron microscopic evaluation is necessary.





3

Progress was somewhat slowed because of the lack of appropriate electron microscopic tracers.7 The first tracers used were saccharated iron oxides8 or ferritin (molecular weight 560,000) whose limits of resolution were close to the thickness of the endothelium itself. It was not until the introduction of horseradish peroxidase (HRP) and microperoxidase (MP) that the exact structure of the BBB could be deduced. Horseradish peroxidase is a relatively small enzyme (molecular weight 43,000) which, unlike ferritin, does not contain an electron-dense core but rather produces a material which has a high affinity for osmium tetraoxide and other radiopaque substances.7

By using HRP, Reese and Karnovsky demonstrated the inability of the marker to pass from the lumen of the cerebral capillary.9,10 In fact, HRP was never found in the extracellular space surrounding the capillary. Additionally, when HRP was injected directly into the brain it readily passed the astrocytic end processes and was stopped at the endothelial membrane.

The anatomical basis of the BBB was, therefore, isolated to the endothelial lining of the cerebral capillaries and not a perivascular site. There are several ultrastructural differences between systemic capillaries and cerebral capillaries which account for their general impermeability.1,4

The manner in which endothelial cells of the cerebral capillaries are joined is distinct from systemic capillaries. Cerebral junctions are characterized as tight or closed junctions meaning the cells closely approximate each other. These junctions gird the cell circumferentially, forming a zona occludens and providing an absolute barrier to HRP. Structurally, the junctions consist of aligned intramembranous ridges and grooves which are in close apposition.1" These tight junctions have been examined by thin section electron microscopy and attempts are now underway to examine them by a freeze-fracture technique.4,2 This method, which allows a longitudinal





4


view of the capillaries, will add greatly to the structural knowledge of these tight junctions but the method is technically difficult. Recently, a freeze-fracture technique was applied to the cerebral vasculature of a chameleon, which possesses a BBB similar to that of mammals.13 In these animals a series of ridges and grooves is seen. The ridges are connected to neighboring ridges by an anastomosing network. In general, the more complex this system is, i.e. the number of ridges it has, the tighter the junction is. The tightness of the junction can be assayed not only structurally but also by measuring ionic conductance and resistance through the junction. The ionic conductance is low for most ions.12,14

Systemic capillaries lack this closed junction. Morphologically, this can be traced to a lack of continuity in the intercellular appositions. In cardiac muscle, for example, the ratio of junctional width to the width of the cell membrane is 2.4, while in cerebral capillaries this is reduced to 1.7.10 These open junctions allow a high degree of nonspecific transport of nutrients and other compounds into systemic capillaries. Materials pass easily between these leaky cells, while in the brain the sealing of the intercellular fissures severely restricts this nonspecific transport. Since intercellular transport is removed, only intracellular transport remains. Lipophilic compounds can readily pass through these phospholipoidal membranes,

but hydrophilic compounds and compounds with high molecular weights are excluded. Systems are available to transport small hydrophilic nutrients, and this will be discussed later.

A second difference between cerebral and systemic capillaries is the

paucity of vesicles and vesicular transport in the CNS.4,7 Vesicular transport is a process for transcellular transport and, as such, vesicles are transported from the luminal to the abluminal membrane. Pinocytotic activity, on the other hand, is concerned with the nutritional requirements of





5


the cell and, therefore, involves vesicular movement from the luminal membrane to a cell organelle, presumably a lysosome.

Cerebral endothelial vesicles are usually uncoated and few in number compared to other systems. Using electron microscopic morphometry, five vesicles per micrometer luminally and thirty-forty vesicles per micrometer abluminally were found.4 In the diaphragm and in myocardial vessels the values are much higher, being seventy-eight and eighty-nine vesicles per micrometer, respectively. This lower content of vesicles is another mechanism by which the CNS can limit nonspecific influx. A third difference is the lack of fenestra in the cerebral capillaries. These differences are demonstrated schematically in Figure 1-1.

Cerebral vessels have a number of perivascular accessory structures which appear to be involved in BBB function.4 While it is known that the astrocytic end feet are not involved as a barrier per se, their role in attenuating BBB action is interesting. These glial end feet may be involved

with regulation of amino acid flux. They also appear to engulf protein which breeches the BBB and, therefore, may act as a second line of defense.15 Phagocytic pericytes which are present abluminally may play a similar role. It is possible that the basement member of endothelial capillaries acts as a mass filter preventing large molecules from penetrating it.

In addition to these structural features the BBB maintains a number of enzymes which appear to augment barrier function.2,16,17 Since optimal neuronal control requires a careful balancing between neurotransmitter release, metabolism, and uptake, it is of vital importance to restrict the entry of blood-borne neurotransmitters into the CNS. It is not surprising, therefore, to find high concentrations of such enzymes as catechol-O-methyl transferase (COMT), monoamine oxidase (MAO), y-aminobutyric acid transaminase (GABA-T) and aromatic amino acid decarboxylase (DOPA decarboxylase)





6









;bi





zo
MUSCLE CAPILLARY AIN























Figure 1-I. This Schematic Illustration Represents an Endothelial Cell
Derived from either a Muscle (ECm) or Brain (ECb) Capillary.
In this Figure, (ma) is the Macula Adherens or Loose Junction, (zo) is the Zona Occludens or Tight Junction, (my) are Microvesicles, and (bl) is the Basal Lamina. This Figure was Modified from Reference 1, Page 162 by Permission.





7


in the BBB. Recently, a distributional study of COMT in the brain indicated that this enzyme is present in many sites including several which lack a structural BBB.18 This distribution may aid in the exclusion of

neurotransmitters from structurally unprotected areas. The presence of DOPA decarboxylase explains partially the need for giving such large doses of L-dihydroxyphenylalanine (DOPA) in the treatment of CNS dopamine deficiencies to achieve appropriate therapeutic cerebral levels. The enzymatic BBB may also play a role in the exclusion of lipophilic compounds which might otherwise passively diffuse through the BBB. This is suggested by the presence of pseudo (butyryl) cholinesterase in the cerebral capillaries.4 This enzyme is not found in noncerebral capillaries. The occurrence of y-glutamyltranspeptidase has also been described and may account for some protection from peptide infiltration.2,19 An early proposal that y-glutamyltranspeptidase is involved with carrier systems is questionable. Acid phosphatase activity, which is a marker for lysosomes and

pre-lysosomes or phagosomes, is present in the endothelial cells.'5 These organelles appear to be involved in the degradation of endocytosed material and, as such, can be considered a component of the BBB. The endothelial cells of the cerebral microcirculation contain a large number of enzymes but care must be taken in ascribing a certain enzyme to a barrier role.17 There are numerous enzymes which, while present in the endothelial cells, do not serve in any capacity other than general cellular functioning. Molecular Carriers Involved in BBB Transport

The aspects of the BBB which have been discussed thus far give an indication of its relative impermeability toa number of blood-borne substances, but do not explain the movement of essential nutrients into the CNS. This

transport is brought about by a number of carriers which are situated in the endothelial cells. These carriers are generally assumed to be proteinaceous.





8


They are equilibrative, i.e. nonenergy dependent and bidirectional in nature and can be saturated.1'20'21 The net movement of compounds is

always along a concentration gradient and since nutrients are readily utilized as soon as they pass into the brain, this gradient is in the direction of the brain.

A number of specific carriers for compounds have been described. The first to be characterized was one for hexoses.1,12'22 This carrier displays saturable kinetics and can be competitively inhibited. The hexose carrier is stereospecific and has a high affinity for a-D-glucose with a Km between

6.0 and 9.0 mM. Other sugars with affinity for this carrier include, in order of decreasing affinity, 2-deoxy-D-glucose, 3-O-methylglucose, s-Dglucose, D-mannose, D-galactose and D-xylose.22 The Km of D-fructose and

L-glucose is very high.

The carrier is Na+ independent and is inhibited by phloretin, a noncompetitive inhibitor, more than phlorizin, a competitive inhibitor.20 The Vmax is similar for all sugars tested which indicates the rate limiting step for transport is not the association of the sugar with the carrier but, rather, the movement of the carrier across the membrane complex. This carrier demonstrates exchange diffusion, i.e. the carrier moves more rapidly when loaded than when empty.22,23

At a Km of 7 mM, the concentration of glucose required to produce saturation is about 126 mg% so that under physiological conditions, the system is about half saturated. In normal situations the rate determining step in glucose utilization is the hexokinase step. This can be shifted to BBB transport of glucose in conditions of hypoglycemia.21 While substrate flux is usually thought of as being related only to plasma levels of glucose,

recent studies have indicated that intracerebral glucose concentrations can alter carrier kinetics.





9


The effects of insulin on the transport of glucose are controversial *224 Several reports have indicated no effect on either unidirectional or net flux after insulin infusion. This is curious in that insulin would not be expected to pass the BBB. The presence of insulin receptors in cerebral capillaries may explain this enigma in that insulin may bind to a receptor on the luminal surface and its effects may be mediated to the abluminal surface by a second messenger.24

Also controversial is the possibility that a low and high affinity

system is operating in glucose transport.20'22'23 There is a nonspecific flux associated with glucose of 7%. Some authors attribute this to diffusion but an alternate hypothesis has been proposed. This involves the presence of two systems on the carrier: a high affinity, low capacity system and a low affinity, high capacity system. Most data have been collected in isolated vessel preparation but there is some support of this proposal from in vivo experiments. Gjedde showed the putative low affinity system to be stereoselective and to have a Kmn of 1.0 M,23 compared with a Km of 1.1 mM for the high affinity system. He contends that a single set of kinetic parameters does not adequately describe the system and that this high-low affinity system better correlates with the data. A suggestion that a protein tetrameter with both low and high affinity sites was the carrier has also been made. At the choriod plexus there appears to be ouiban sensitive, Na+ dependent glucose flux and this is apparently important in cerebral spinal fluid (CSF) homeostasis.22

Three carriers have been described for amino acid transport.'"12'20 These carriers have affinity for neutral, basic, and acidic amino acids. In general, essential amino acids, which are large and bulky, are transported in preference to nonessential amino acids.20 In all of these




10

systems, net flux is small compared to unidirectional flux since amino acids derived from proteins are constantly being lost and the magnitude of this loss is similar to uptake.

The transport of neutral amino acids has been described by Christensen and these generalizations apply to the BBB.25,26 Four neutral amino acid transport systems have been shown to occur in Ehrlich ascites, a model cell system, and in several other systems. An L- or leucine-preferring system is characterized by high affinity for phenylalanine, leucine, tyrosine, tryptophan and several other large essential amino acids. It is Na+ independent, bidirectional and equilibrative in nature. The definition of this system can be made by observing the flux of 2-aminonorbornane-2-carboxylic acid which is exclusively transported by this carrier. The A- or alaninepreferring system is characterized by a Na+ dependence, an energy dependence, and the ability to concentrate substrates. The system has affinity for glycine, proline, alanine, serine, threonine and several other small amino acids. The defining compound for this system is a-(methylamino)isobutyric acid. Two other amino acid transport carriers have also been described but they are not well characterized. There is also an ASC (alanine-serine-cysteine-preferring) system and a Gly (glycine-preferring) system.

Since generally only large essential amino acids (L-system substrates)

are transported through the BBB, the L-system is assumed to be the dominant mechanism in amino acid uptake.,20 The absence, however, of the A-system has been questioned. It was recently argued that the A system is present

but has a different distribution than the L-system. Betz and Goldstein discovered a system whose characteristics are similar to the A system but which is located abluminally.27 This system may act as an active mechanism for





11


efflux of these amino acids from the brain parenchyma or, more probably, for concentrating them in the endothelial cytosol. The necessity for this concentration is related to a proposal that the L and A systems may

act together in the transport of amino acids. This hypothesis is based on a possible equilibration of amino acids between the two carriers in the cytoplasm of the endothelial cell.26

In many cases the Km value for an amino acid is similar to its plasma concentrations. This being the case, slight changes in the blood levels of an amino acid may alter their disposition. The utilization of tryptophan, for example, which is a precursor of serotonin, is partially determined by its availability and its movement across the BBB.21'28 A similar situation may exist in certain circumstances with tyrosine, which is the precursor for dopamine, norepinephrine and epinephrine.

Recently, a hypothesis was forwarded to explain the regulation and induction of these carriers.29'30,31 It has been suggested that an amino acid is produced abluminally in large amounts and transported on the neutral amino acid carrier. The amino acids in such a system should have a high Km and, therefore, be easily displaced from the carrier at the luminal side or in transit. These carriers, like the hexose carriers, exhibit exchange diffusion. The proposed amino acid in this regulatory role is

glutamine. Glutamine is synthesized from glutamic acid and ammonia by glutamine synthetase in astrocytes, a perivascular locus. The concentration of glutamine is ten times higher than the concentration of any other neutral amino acid and its local concentration in the vicinity of the BBB is predicted to be still higher.30

This hypothesis was formulated on the basis of several interesting observations. In cases where ammonia and, presumably, glutamine levels





12

increase cerebrally, as in porto-systemic shunts, the uptake of phenylalanine and other neutral amino acids increases. Also, if glutamine synthetase is inhibited by methionine sulfoximine, the uptake of essential amino acids is reduced.29 While this is an attractive proposal, several authors have questioned it on the grounds that many amino acids compete for this neutral amino acid carrier and the role of glutamine, therefore, may be relatively unimportant.32

Relatively little work has been done with basic and acidic amino

acids. The acidic system has affinity for glutamate and is fully saturated at physiological concentrations. Basic amino acids are transported on a distinct carrier. This carrier is equilibrative, saturable and bidirectional and has been described as a Ly+ or lysine-preferring system.

A monocarboxylic acid carrier has been described which demonstrates

affinity for lactate, pyruvate, acetate, propionate, butyrate, 6-aminolevulinic acid and ketone bodies.1,20 Ketone bodies, such as -hydroxybutyrate and acetoacetate, are produced in a number of stressful circumstances, including starvation.33 The carrier possesses similar characteristics to those which have already been described. It is stereospecific and pH sensitive.20 The transport of the substrates on the carrier increases with a decrease in pH. This has been interpreted to mean either that a proton is cotransported with the acid or that hydroxide acts as a high affinity competitive inhibitor. Only the ionized acid is transported by this system and this has led to the proposal that an R-NH3+ moiety is present in the active site. The unionized acid can pass through the membrane by simple diffusion.

The Km of lactate is similar to the plasma level of lactate. This being the case, rapid rises in systemic lactate levels, such as the ones which accompany exercise, are not transferred to the brain immediately.20





13

A carrier for choline was described in 1978.34 Choline cannot be

synthesized de novo in the brain but this precursor is required for such important cellular components as acetylcholine and phosphatidylcholine. The carrier conforms generally to those characteristics specified for other systems. It has affinity for choline, hemicholinium, dimethylaminoethanol (deanol), tetraethylammonium, tetramethylammonium, cartitine and spermine but not for NH4. The distribution of this carrier is not uniform. Choline

uptake decreases with age and this correlates with an age-dependent diminution of the carrier. The rate determining step in choline utilization is

its movement across the BBB.

A carrier with affinity for nucleosides such as adenosine, guanosine and inosine has been described.1,20 Additionally, a system for transporting purine bases has been isolated. This system transports adenine, guanine and hypoxanthine but not pyrimidines. This is interesting in light of the fact that pyrimidines can be synthesized in the brain from NH4+ and aspartate, while purines cannot. This carrier is very active in neonates, but its activity diminishes with age.35

Recently, a carrier was described for thyroid hormones.36'37 The

carrier has affinity for triiodotyrosine (T3) and thyroxine (T4) but not for tyrosine, leucine or potassium iodide. The transport of T3 is saturable and inhibited by T4. The carrier is weakly stereospecific and of high affinity. The presence of a carrier for T3 and T4 is interesting because these compounds are fairly lipophilic. The bulkiness of the molecule, however, tends to decrease its ability to pass membranes. A carriermediated transporthas also been proposed for thiamine.38 These systems are summarized in Table 1-1.





14



Table 1-1 Blood-Brain Barrier Transport Systems


Representative Km Vmax
Transport system Substrate (mM) (nmol min-Ig-1)


Hexose Glucose 9 1600

Neutral Amino Acid Phenylalanine 0.12 30

Acidic Amino Acid Glutamate

Basic Amino Acid Lysine 0.10 6

Monocarboxylic Acid Lactate 1.9 120

Amine Choline 0.22 6

Nucleoside Adenosine 0.018 0.7

Purine Adenine 0.027 1

Thyroid Hormone T3 0.001 0.17

Thiamine Thiamine





15

In addition to these carrier systems, there are a number of active

efflux mechanisms. Two of these systems are located in the choriod plexus and have affinity for organic ions. A system for the disposition of anions has been described and divided into two subsystems.20,39,4'41,42 An L or liver system with affinity for prostaglandins, 5-hydroxyindole acetic acid and probenicid, as well as a K or kidney system with affinity for p-aminohippuric acid and phenol red, has been proposed. Additionally, a carrier for the efflux of iodide has been described.43,4 A cationic system is also present in the choriod plexus and this species has affinity for N-methylnicotinamide, decamethonium and hexamethonium ions.4s46 These systems appear to be important in the removal of metabolic acids and bases. This, as well as the anionic system, is energy-dependent and can be competitively inhibited. The presence of several energy (ATP)-dependent systems in the capillaries indicates that this site may also be important for active efflux.19 The high density of mitochondria in cerebral capillaries is further support for this location.47 Movement of Compounds Across the BBB

The BBB, therefore, consists of a relatively impermeable membrane

superimposed on which are mechanisms for allowing the entrance of essential nutrients and the exit of metabolic wastes. If a compound is to gain access to brain parenchyma, it may do so via several routes. If the agent has affinity for one of the carriers previously described, it may diffuse across the BBB by association with this carrier. A compound which has a high intrinsic lipophilicity can diffuse passively through the phospholipoidal cell membrane matrix.1'4,12 The pK of a compound with ionizable groups is also important, since only the unionized species diffuses across the BBB rapidly.48'49 The ability of a substance to enter into the cell membrane





16

is often correlated with its in vitro octanol:water partition coefficient.5051 This is a measure of lipophilicity, and can be correlated with biological effects.52 These correlations can be extended to the permeability of compounds through the BBB. Several examples of this correlation have appeared in the literature, including the opiates morphine, codeine, and heroin.52 Good correlation is obtained between lipophilicity, the ability to pass

the BBB, and narcotic efficacy.

The increase in the ability of a compound to pass membranes can, all too often, be correlated with an increase in undesirable side effects. An example of this was shown in a series of B-blockers in which lipophilic members of this pharmacologic group, i.e. propranolol penetrated the BBB rapidly but demonstrated a number of deleterious psychiatric manifestations.53 Conversely, s-blockers of lower lipophilicity exhibit an attenuation of these side effects.

These two avenues, namely passive diffusion and carrier mediation, represent the major components of influx. Other minor mechanisms may also allow the entry of substrates into the CNS. The cell bodies of many neurons are located centrally while their axons may penetrate into the

periphery. These axons can take up material and transport it in a retrograde fashion to the CNS.7,54 This retrograde axoplasmic transport has been observed in such areas as the nucleus ambiguus and the abducen nucleus.

In some cases, however, the endocytosed material is reacted with lysosomes and, therefore, this transport route may have a protective function.

There are several areas of the brain which lack a BBB.1,8,55 These include such locations near the ventricles as the area postrema, the subfornical organ, the median eminence of the neurohyphosis, the organum vasculosum of the lamina terminalis, and the choriod plexus. Collectively,





17

these areas are termed the circumventricular organ. In addition, the pineal gland lacks a BBB. These areas constitute a small fraction of the total surface area of the BBB and may allow a limited nonspecific flux.

These loci do have important pharmacological ramifications as demonstrated by the action of a series of atypical neuroleptics.56,57 These compounds are so named because they provoke certain symptoms of dopaminergic blockade but not others. Specifically, metoclopramide exerts an antiemetic action but not an antischizophrenic effect. This dichotomy was explained by the fact that metoclopramide does not penetrate the BBB. The site of action of antiemetic agents is at the chemoreceptive trigger zone, which is located in the area postrema and is outside the BBB. The apparent inability of metoclopramide to produce the full spectrum of changes which occurs as a consequence of dopaminergic blockade may be related to its pharmacokinetics and, specifically, its inability to pass the BBB. This relegates the compound to only those sites of action not protected by the BBB. Other pharmacologically important sites outside the BBB include the median eminence, which controls prolactin secretion. In some areas, while the BBB is not absent it is diminished. These areas include certain arteriolar segments whose diameters are between 15-30 pm. '5'9 In these segments limited protein extravasation, or leakage of compounds from the lumen of the capillary to the extracellular spaces, has been observed.

The ability of small peptides to penetrate the BBB is an extremely controversial point.21,60,61,62 Systemic administration of certain centrally active peptides elicits a central response. Differences concerning

the interpretation of these data are significant. One view is that the flux of these small peptides across the BBB is low indicating, perhaps, some nonspecific route accounts for their entry.21,62 The presence of peptidyl





18

receptors luminally, whose stimulation results in the generation of a second messenger, has been proposed in this respect. A different conclusion is that small peptides have a significant flux across the BBB. The peptides which have been investigated thus far include stabilized enkephalins and endorphins, a nonapeptide which induces delta-sleep, melanin stimulating hormone (a-MSH) and melanin inhibiting factor one (MIF-l).60,61,63

The BBB plays a major role in CSF homeostasis.12'64 Cerebral spinal

fluid is produced at the choriod plexus and drains from the ventricals through the foramina of Magendie and Luschka into the ventral aspects of

the brain.65 This fluid serves many important mechanical and nutritive functions. Cerebral spinal fluid flow is constant with a t, of renewal of about two hundred and seventy minutes. The ventricular volume of CSF is about 23 ml while the subarachnoid volume is 117 ml. Cerebral spinal fluid, along with any dissolved materials, leaves the subarachnoid space via the arachnoid villi, which protrude into a venous sinous. The arachnoid villi act as a one-way valve and prevent backflow.66 This loss of CSF provides a slow mechanism for nonspecific efflux of compounds from the CNS. This mechanism rids the brain of polar compounds such as metabolic wastes at a fairly constant rate regardless of molecular weight. If a compound is fairly polar and does not have affinity for any passive or active efflux mechanism, it will leave the CNS by CSF bulk flow. Therefore, while lipophilicity is very important for influx to the brain, the efflux of a compound is only partially dependent on this parameter.46,67,68,69,70

Since the ionic environment in which neurons function is so important, the composition of the CSF is strictly maintained within narrow limits. The CSF is not simply an ultrafiltrate of plasma, and several concentration gradients are produced. The maintenance of these gradients can, in part, be attributed to the low ionic conductance of the BBB. This is especially





19

important for K+, since a low K +csf/K+ plasma ratio apparently acts to stabilize neurons.12,71'72

One of the major factors in influencing diffusion into the CNS is the degree to which a substance is bound to plasma proteins. It had been assumed for a long time that only the free, dialyzable fraction of the total plasma concentration of a compound was available for diffusion.7 This is important for a great number of compounds. The major species involved in this binding are serum albumins, which tend to bind molecules loosely but to a large extent, and globulins, which bind with high affinity and low capacity. The premise that only the unbound species is capable of diffusion has, however, been challenged.

Steroids have profound central effects and gain entry into the CNS by simple diffusion across the BBB, even though they are highly bound to plasma proteins.5 This diffusion correlates well with the octanol:water partition coefficient of the steroids and inversely with the tendency of the molecules to form hydrogen bonds.73 Different steroids are taken up differently, however, and this is in large part related to protein binding. There are several globulins which bind specific steroids. These include sex hormone binding globulin (SHBG), which is found in man but not rats, cortical binding globulin (CBG), estradiol binding globulin (EBG) which, in fetal and neonatal rats, may be synonymous with ca-fetoprotein, progesterone binding globulin (PBG), which is found in pregnant guinea pigs and also, thyroid hormone binding globulin (TBG), which is found in man.

Those steroids which are bound to globulins such as cortisone to CBG in rats have a small flux into the brain while those steroids which are bound more highly to albumins such as progesterone, estrogen or testosterone in the rat, easily diffuse through the BBB.73 The reason for this is that





20

binding to albumin is sufficiently weak that the capillary transit time in the brain is long enough to allow dissociation of the compound from the macromolecule. In the case of globulins, however, the binding is tighter and the turnover is only of the order of 3-10%//second. The sojourn through the cerebral capillaries is not, therefore, sufficient to release

the bound material. It is not, therefore, the plasma-protein-bound fraction which is unavailable for transport but, rather, the globulin-bound fraction.

These principles also apply to free fatty acids such as palmitate. In this case, however, there appear to be high and low affinity sites on the albumin molecule so that the rate of dissociation of the palmitate bound to the site with the lower Km may be slow compared with capillary time, while the low affinity site may not.74 Melatonin, which is also highly protein bound, shows a significant diffusion through the BB8.75

Thyroid hormones which are transported into the CNS by carriers are also bound by plasma proteins.36'37 The carrier, whose Km is lower than that of the albumin site, is able to compete successfully with the albumin for binding. This is effectively a stripping of the compound from its albumin site. The effect is also seen with tryptophan and other amino acids.28

The BBB in Pathological and Experimentally Altered States

The integrity of the BBB is known to be impaired in a number of pathological or experimentally-induced conditions.4'76'77 The effect produced can be the result of changes of the structural components of cerebral capillaries such as the junction or vesicular activity and, as such, results in generalized increases in permeability. Alternatively, the carrier systems may be compromised and this may lead to specific changes in permeabi1i ty.





21

Generalized increases in permeability result in a number of deleterious events. Since the BBB is relatively permeable to water, but not to most other substances, osmotic gradients can be rapidly changed. If plasma proteins and other compounds are allowed to freely enter the CNS, they will bring with them large amounts of water, with the result being cerebral edema.1,20

The morphological basis of these changes in any particular situation can be highly controversial. This is especially the case with hypertonic treatment of cerebral capillaries. It has been known for some time that hypertonic solutions of such solutes as glucose, sucrose, urea, arabinose, lactamide and several others can increase the permeability of the cerebral capillaries to protein and other small polar compounds. Rapoport explains this phenomenon as the result of osmotic shrinkage of the endothelial cells,

resulting in a pulling apart of the tight junctions.5,71 This has been challenged. If HRP is injected after a hypertonic solution, the HRP reaction product does not form a continuous line from the luminal to the abluminal surfaces at the junction or at any other location.4'20 It was suggested, therefore, that increased vesicular transport accounted for the increased permeability, perhaps as a result of increases in local blood pressure. 77

In any case, if the concentration of the hypertonic solution is close to the critical opening concentration, the opening of the BBB is transient and does not produce acute edema.5 This procedure may have therapeutic applications. In many cases it is desirable to introduce highly polar compounds into the brain and this osmotic opening of the BBB may provide an avenue for that purpose. Methotrexate is a folate inhibitor used in the treatment of cancer. The pKa of the carboxylic acid functions of this





22

molecule is 4.7 and at physiological pH methotrexate is 99.8% ionized and, as such, passes the BBB very slowly, if at all. After pretreatment with hypertonic arabinose, the concentration of methotrexate showed a fifty-fold increase in the CNS.78

Hypertension is a major health problem and can produce a number of debilitating complications.79 Hypertension increases the extravasation of albumin, sucrose, and other polar compounds.80,81'82 This has been studied in a number of animal models of hypertension including those in which the increased system blood pressure is induced with either amphetamine, ephedrine, AramineR or bicuculline.

The basis for this increased permeability has been debated frequently and appears to be related to increased vesicular transport since ultrastructural investigations do not indicate capillary lesions or junctional openings.77 Increased vesicular transport has, in fact, been implicated in a number of situations in which capillary permeability increases. The factors that affect vesicular formation which are described here also apply in those cases.

The mechanism by which hypertension elicits an increase in vesicular

activity is not clear. The increased hydrostatic pressure may act to induce an invagination. Also, there are a number of substances associated with hypertension that induce vesicular formation.4'77'83 These include the catecholamines, serotonin, and histamine. Joo explained the increased vesicular transport in terms of a cyclic 5'-adenine monophosphate (cAMP) stimulation mediated by a catalyzed adenyl cyclase, since cAMP can directly induce vesicular formation.84,85

Both serotonin and histamine have been shown to increase protein extravasation and both act to catalyze specific adenyl cyclase. A perivascular





23

source of both of these compounds is available, since histamine is stored in perivascular mast cells and serotonin in platelets.' The action of histamine is partially reversible by H2 antagonists.86 Recently, cyclic guanine monophosphate (cGMP) has been isolated in cerebral capillaries and this may play a role in vesicular transport.87 These effects are seen peripherally as well as centrally.

The major controversy surrounding this area is whether hypertension

itself is responsible for extravasation, or if some humoral agent produced as a result of hypertension is culpable. It is difficult to look at this in vivo since many agents can increase blood pressure and increase vesicular transport.

Additional evidence that vesicles are important in extravasation is indicated by the decreased protein flux in capillaries treated with compounds which decrease vesicular formation. These compounds include imidazole, which alters cAMP function by inhibiting the inactivation of phosphodiesterase, thioridazine, a phenothiazine which decreases vesicular fusion with the cell membrane,and desipramine.80 The anionic transport blocking agent 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid disodium (SITS) inhibits exocytosis.88 This compound, which also inhibits protein extravasation in hypertension, inhibits ATP-evoked adrenalin release from chromaffin granules as well as serotonin secretion from platelets. Increased vesicular transport has also been related to the formation of transendothelial channels.5s4'89 These channels may be the result of the simultaneous opening of a chain of vesicles and may provide an avenue for nonspecific flux in hypertension. The presence of this channel is highly debated, however.





24

The nature of the hypertension itself can be a factor in increased

capillary permeability.82 While acute hypertension is very likely to produce a protein influx, chronic hypertension may protect the system in the likelihood of an acute rise in blood pressure. The reason for this is probably the hypertrophy of vascular muscles. This hypertrophy leads to an increased vascular resistance and thickening of the vessel wall providing a mechanism for handling greater pressures. This is consistent with the observation that vasoconstriction decreases capillary permeability, while vasodilation increases this parameter.

In some cases hypertension may lead to the rare malady, hypertensive encephalopathy.8l This disease is related to the inability of the cerebral vasculature to acclimate to acute severe hypertension. In this instance cerebral edema occurs and this may cause swelling of the nervous tissue.

This yields an increased cerebral pressure and may prevent vasodilation caused by a variety of stimuli.71

Hypervolemia may also result in an increase in protein extravasation.90 If mice, whose blood is about 1.3 ml, are treated systemically with high volumes (1 ml) of saline, extravasation of HRP can be documented. At lower volumes, however, no changes in permeability are observed.

Since, in these experiments, the blood pressure increases only by 20 mm Hg, the increased capillary permeability may not be due to hydrostatic pressure. In order to open the BBB, pressure increases on the

order of 100 mm Hg are required.

The vasculature of primary brain tumors and of metastatic secondary

brain cancers exhibits a generalized degradation.7'91'92 This breakdown can be characterized by the presence of gap junctions, fenestra and open endothelial junctions, indicating the typical electron microscopic





25


structure of the tight junctions is destroyed. Although it has been suggested that these deficiencies should render cerebral tumors susceptible to treatment,93 the clinical evidence does not support this. The therapy which has, thus far, been directed to brain neoplasms has been disappointing at best. Neurosurgery may have reached its practical limit and most

agree that major new advances must come from the field of chemotherapy.91 It is interesting to note that many agents are effective against certainperipheral tumors, but not their secondary brain metastases.

The unresponsiveness of cerebral tumors has been explained by two

phenomena. First, it is known that the greatest display of disintegration of the capill1ary structure occurs in the central sl ow growi ng porti on of the tumor, and as one moves to the periphery, the abnormalities decrease.92 This area of the tumor would be expected to show the highest resistance to chemotherapeutic agents. Secondly, since only a relatively small area of the brain vasculature is disturbed, any drug which reaches the neoplasm would rapidly diffuse into the outlying areas, thereby diminishing its concentration and effectiveness at the tumor site.78 Few suggestions have been forwarded to circumvent this problem. One involves osmotic opening of the BBB followed by methotrexate treatment, but this has a very limited usefulness because of problems with the accompanying edema. 78

Many experimental models of brain injury are associated with an increase in capillary permeability.4'57,94 These models include injury induced by dropped weights, pendulums, hammers, spring-mounted weights, blasting caps, rotary strikers, compressed air guns, humane stunners and accelerating devices. Generally, these procedures have been applied to the cat.





26


The leakage of proteins from the capillaries is usually proportional to the amount of injury, but even in severe injury the endothelial cell is intact and shows no sign of lesion.94 The explanation most often proposed for this extravasation is increased vesicular activity. In most injuries there is an increase in serotonin and norepinephrine levels, as well as the production of a number of humoral agents. The increased vesicular content, which appears first in the arterioles and subsequently in the capillaries, may be mediated by stimulation of cAMP. There are also, however, increases in blood pressure and that may act to increase vesicular transport.

Cerebral infarcts also disrupt BBB function, as evidenced by an increased albumin concentration in the CSF of infarct victims.95 Correlation, however, between infarct size and location and the quantity of albumin leaked has not revealed any significance.

Both electroconvulsive shock and pentylenetetrazole-induced seizures result in an increased capillary permeability to a number of plasma markers.1,,54 The degree of permeability increase is proportional to the

number of shocks or compounds given. The morphological basis for this extravasation is not known and both junctional opening and increased vesicular transport have been suggested.77 It is interesting to note that, in both of these procedures, the blood pressure rises and this may be an important factor.

Induced ischemia also produces a marked increase in the permeability of the cerebral vasculature.54,77 The experimental model which is most often used is that of the Mongolian gerbil. In this species, about half of the individuals lack arterial connections between cerebral and vertebral

systems. After carotid occlusion and development of ischemia, HRP leaks





27

from the capillary lumen. In these investigations, there is no indication of endothelial cellular damage and, therefore, the extravasation of HRP is attributed to increased vesicular transport. The stimuli for this may be

release of serotonin from platelets inducing vesicular formation secondary to vasoconstriction and increased blood pressure. Focal edema induced by a number of means, such as ultraviolet exposure, also increases vesicular

transport and HRP uptake.

Nonionizing radiation has long been implicated in BBB disruption.

Microwaves and x-rays have been studied extensively in this regard. The effects of microwaves are highly controversial. One report states that exposure of rats to 2450 MHz at 10 mW/cm2 for two hours produces a marked extravasation.96 This level is considered safe for human exposure in the United States, but not in other countries. The biological effects of microwaves can be subclassified as changes resulting from gross thermal effects or nonthermal effects. In this study, the body temperatures of the animals were constant and the altered permeability of the BBB was

attributed to microwave-stimulated serotonin release from platelets, although other mechanisms are possible. Conversely, a recent publication indicates that much higher levels of radiation are required to cause protein leakage, specifically 3000 mW/cm27. At these levels, cerebral temperature increases significantly and changes can be attributed to gross

thermal effects.

Porto-caval anastomosis, which causes severe liver dysfunction, has also been implicated in BBB breakdown.54'77 This disintegration has been termed hepatic encephalopathy. As in a myriad of other circumstances, increased vesicular transport has been implicated as the mechanism of extravasation.





28


Several situations involving autoimmune afflictions and induced autoimmunity, such as experimental allergic encephalomyelitis (EAE) which is used as a model of multiple sclerosis, demonstrate an increased capillary permeability.98 In EAE, the cerebral vessels are said to cuff or deform and this abnormality correlates with BBB breakdown. This may be important

in the general progression of multiple sclerosis, as vascular changes are among the first changes which precede demylelination.

Agents which solubilize and fluidize membranes may also act to increase BBB permeability.99,"000,' Dimethylsulfoxide (DMSO), in very high concentrations, has been thought to increase the flux of such plasma markers as inulin and mannitol. Its effects are said to be derived from its lytic action on membrane although interaction by micellular formation cannot be ruled out. Nortriptyline has a similar effect. Ethanol, in large doses, can also cause opening of the BBB to sucrose. It was postulated that this generalized increase in permeability may increase the susceptibility of the CNS to bacterial and viral infection. It has been shown, however, in acute and chronic doses of ethanol that were compatible with continued life, that there was no alteration in the BBB.102,103

Many heavy metals have been associated in both general and specific changes in the BBB.1,56,14 Mercury (II) Hg+, in high concentrations, i.e. >80 Pm, causes a generalized increase in the permeability of cerebral capillaries to sugars and protein markers but in low concentrations affects only the hexose carrier. Ionic lead has a similar effect.

Anumber of conditions can alter BBB function in more subtle, yet no less damaging ways. In these instances, changes occur at the level of a specific carrier or carriers and, as such, only a particular type of compound is involved. These changes can result from physical alteration of





29


the carrier by denaturation. Additionally, diminution or increase of the blood level of a transportable compound can affect the utilization of the compound. In some cases, the rate limiting step in metabolism of a nutrient can be transferred from an enzyme to transit of the agent across the 888.21

Glucose transport is very important to CNS function. Under normal circumstances, the rate-determining step in glucose metabolism involves the enzyme hexokinase. If, however, the plasma concentration of glucose falls, as in hypoglycemia, or cerebral metabolism increases, as in relative hypoglycemia, the limiting step in utilization is shifted to transport of glucose across the BBB.20

In severe hypoxia (P02 <10 mmn), a number of progressive changes associated with glucose flux occur.' In the dog, after one minute of oxygen deprivation, the influx of glucose into the brain is unchanged but efflux decreases. Ten minutes after initiation of hypoxia, influx and efflux of glucose decrease. This has been explained by a postulated modulator which, in the absence of hypoxia, is bound to the carrier and alters transport. In other animal models, different results are obtained.

The transport of amino acids and, especially, neutral amino acids in certain disease states, has been the subject of much research. The neutral amino acid carrier is responsible for the transport of such neurotransmitter precursors as tryptophan, tyrosine and histidine and, as such, any interruption of this supply can have tremendous neurological consequences. Nowhere is this more apparent than in phenylketonuria.1,20 This syndrome, which is associated with high blood levels of phenylalanine, has been linked to mental retardation. This condition of high phenylalanine levels can act to competitively inhibit other substrates of this carrier, such as tyrosine, tryptophan, and histidine. This is made possible because of





30

the similar Km values for these amino acids in addition to the similarity between the Km's and plasma levels of these amino acids. Hyperphenylalaninemia also can reduce protein synthesis by a similar mechanism. Treatment of this disease involves a phenylalanine restricted diet and 5-hydroxytryptophan supplements.

In hepatic encephalopathy, the neutral amino acid carrier is induced and, as previously discussed, this induction may be related to increase in ammonia and glutamine levels. Dihydroxyphenylalanine (L-DOPA), which is used as a dopamine source in the treatment of parkinsonism, is transported by the neutral amino acid carrier. It has been shown that ethanol increases DOPA transport as well as the transport of tyrosine, tryptophan and cmethyldopa into the CNS.03

The monocarboxylic acid carrier appears to be a major organ for eliminating metabolic acid wastes. As was previously discussed, the Km of lactate is close to its plasma concentration so that rises in lactate, such as those that accompany exercise, are only slowly translated to the CNS. In the case of anoxia, however, where cerebral levels of lactate rise, the systemic dissipation of this byproduct is slowed by the same phenomenon.20 In the brain the loss of lactate is carrier and not diffusionlimited, unlike other organs. In hypoglycemia, lactate may act as an energy source.105 This is true in neonates, where lactate uptake is much higher than in adults. The Km of the monocarboxylic acid carrier is also correspondingly higher in neonates.

Ketone bodies, which have an affinity for the monocarboxylic acid carrier, are produced during fasting and can act as a metabolic energy source. These bodies include a-hydroxybutyric acid and acetoacetic acid. The enzyme responsible for their production, -hydroxybutyrate dehydrogenase, is





31


induced by starvation. The utilization of the surrogate food sources is

limited by BBB transit.

In starvation, the monocarboxylic acid carrier is said to be induced, although this is controversial. A recent paper demonstrated a lower Km and Vmax for carriers in starved animals compared with control animals.33 The increased flux of a-hydroxybutyrate and other substrates was attributed in this article to an increase in the diffusional component.

A number of therapeutic agents have affinity for these carriers. These include such anionic compounds as probenicid, penicillin, and aspirin.20 This affinity could potentially lead to competition and a decreased ability to eliminate metabolic acids from the CNS. It has been shown, however, that the concentrations required to cause inhibition are far above therapeutic levels.

The BBB is a complex system of enzymes, protein carriers, and vessels which impart to the brain a selective interface. This interface prevents potentially damaging substances from entering the brain without impeding the entry of nutrients or the exit or metabolites and excretory products. In adverse circumstances, the permeability of the barrier can be increased, resulting in a number of deleterious effects.

Prodrugs and Drug Delivery Systems

The BBB excludes a number of pharmacologically active agents and, as such, treatment of many cerebral diseases is severely limited. In order to increase the effectiveness of drugs which are active against central maladies, the pharmacokinetic profile of the agent must be augmented and, specifically, the transit time of the drug in the brain must be increased. If a method were available to implement these alterations, an agent could

be delivered specifically to the brain. This specificity should increase





32


the therapeutic index of an agent since not only is the concentration of

the agent increased in the vicinity of the bioreceptor but, of equal importance, the peripheral concentration of the drug is reduced decreasing any associated toxicity.

Unfortunately, there are very few methods for circumventing the BBB and these are of limited usefulness. The direct administration of drugs into the CNS;i.e., an intrathecal injection has been used to deliver the folate antagonist, methotrexate, to the brain. This method is not very satisfactory since the distribution of methotrexate in the brain is uneven and slow.46 Additionally, since the ventricular volume of the CSF is small, increases in intracerebral pressure can occur with repeated injections. This is particularly dangerous when the intracerebral pressure is already high as it is in CNS cancers. Repeated lumbar puncturing also carries a risk.

A general method which can be applied to delivery of drugs to the brain is the prodrug approach.106-"10 The term prodrug was coined by Albert and refers to the result of a transient chemical modification of a pharmacologically active agent. This change imparts to the compound an improvement in some deficient physiochemical property such as water solubility or membrane permeability. Ideally, a prodrug is biologically inactive but reverts to the parent compound in vivo. This transformation can be mediated by an enzyme or may occur chemically due to some designed

instability in the agent. The aim of these manipulations is to increase the concentration of the active agent at its site of action and thereby, increase its efficacy. While potentially there are many different types of proderivatives, most thus far synthesized are simple esters and amides. These compounds are transformed to the parent acid, alcohol or amine by the ubiquitous hydrolases which are present in vivo. Many anticancer





33


agents have lent themselves to this type of manipulation. Several amides, for example, of the highly water soluble anticancer agent guanazole have been made. This series of lipoidal compounds hydrolyzed in vivo to yield the parent drug.111 A more sophisticated prodrug is cyclophosphamide, which is inactive in vitro. The agent is activated by P450 mixed function oxidases in the liver to the potent alkylating agent,N,N-bis(chloroethyl)phosphordiamidic acid.109 This drug is extensively used in cancer chemotherapy.

One of the most important applications of prodrugs is in the sustained release of therapeutically active agents. Cytosine arabinoside is used as an S-phase specific antimetabolite, but suffers from rapid metabolism by cytidine deaminase requiring a continual administration of the drug. This problem produced the prodrug cyclocytidine which is not a substrate for cytidine deaminase and which slowly releases the cytosine arabinoside by ring cleavage.112

The influx of a compound to any organ can be related by the equation Kp = QE

where Kp is the clearance of a drug by an organ, Q is the blood flow through that organ and E is the extraction coefficient.113 The extraction coefficient is related to the lipophilicity or octanol-water partition coefficient of a compound which is in turn related to the ability of a compound to partition into phospholipoidal membranes and consequently into organs. By increasing the lipophilicity of a compound with the prodrug approach, one can increase the entry of a compound into its site of action but this is not specific and, in general, all organs are exposed to a greater tissue burden.113 Nonspecificity is, therefore, one of the major drawbacks of the prodrug approach. This is especially important





34


with cytotoxic agents. While increased membrane permeability makes a compound more effective locally as a cytotoxic agent, there is almost always a disproportionate rise in systemic toxicity. This has severely

restricted anticancer prodrugs of this type.

Several types of toxicities are also associated with prodrugs.114

Theoretically, a prodrug should be metabolized only to the parent compound but the formation of toxic metabolites by the prodrug is possible. This occurs with such compounds as phenacetin, a prodrug of acetaminophen. Another possible toxic reaction may be brought about by enzymatic or glutathione depletion. The compound thiamine tetrahydrofurfuryl disulfide,

a prodrug of thiamine, requires glutathione-mediated disulfide bond cleavage for activation and, therefore, toxicity may arise from the associated depletion of glutathione.

The idea of using prodrugs to increase the specificity of delivery has been considered. This has proved, however, to be difficult and not very fruitful. Two basic approaches have been taken in this regard: sitedirected or site-activated delivery and delivery by the association of a drug with a macromolecular carrier.

The first method does not attempt to concentrate a compound at a particular location, but is based on site-specific activation of the prodrug. For this to be possible, the enzyme responsible for the activation must be located specifically, or at least in high relative concentrations in a particular organ. The finding, for example, that y-glutamyl peptidase is present in high concentrations in the kidney led to a number of compounds substituted with the y-glutamyl group."3 Sulfamethiazide and L-DOPA were derivatized in this manner in order to achieve renal delivery.





35


This approach has been extensively applied to cancer chemotherapy.114 The philosophy behind this application is related to the many differences that occur between cancer cells and normal cells. These variations are basically the result of the altered metabolism of neoplastic cells. Many alkylating agents have been synthesized in an attempt to capitalize on these differences. The lower pH of tumor cells has been exploited in a
series of aziridines which are more active at the pH of tumors than at physiological pH. The greater reducing power of cancerous sites has led to the development of a number of biologically inactive azo compounds which upon reduction yield potent cytotoxic agents.115 Examples of these aretetrazolium mustard and azomustard, both of which are reduced to aniline

mustards. The inactivity of the parent compound is due to the delocalization of the nucleophilic nitrogen lone pair by the conjugated ring system.
A number of 0-phosphate esters have been synthesized in order to take advantage of the high levels of acid phosphatase which are characteristic of human neoplasms.115s The 0-phosphate esters of p-hydroxy mustard and

estradiol mustard were prepared as specific agents to be used in prostate cancers. The enzyme, y-glutamyl transpeptidase, is also found in high concentrations in tumor cells so that y-glutamyl derivatives of cytosine arabinoside and phenylene-diamine mustard have been proposed. The presence of hydrolytic esters has also been established in neoplastic formations. These include esterases and a-glucuronidases.112,'11s The cytotoxic agent, aniline mustard, is converted in the liver as a result of a first pass effect to its 0-glucuronide. Tumors which contain high a-glucuronidase

activity convert the 0-glucuronide to the potent alkylating agent p-hydroxyaniline mustard.





36


While these compounds have demonstrated some promise as anticancer

drugs, for the most part they have not lived up to their potential. There are a number of reasons for this failing. The chemical manipulation of these agents may act to decrease their accessibility to a site of action. Additionally, if the agent is fairly lipophilic, it may "leak" from its site of action before exerting a pharmacological effect.113 In cancers there may also be diffusion limitations because of the restrictions in blood flow.

A second approach for increasing the specificity of an agent for a

particular organ involves the coupling of a pharmacologically active agent with a macromolecule.116 The specificity derived from such a system is related to the interaction between cells and endogenous and exogenous biopolymers and macromolecules. Albumin, for example, is actively endocytosed by various macrophages.117 An anticancer compound could be coupled to albumin and the complex taken up by a macrophage tumor. This would then be

directed to a lysosome where the drug would be hydrolyzed from its albumin carrier and exert its pharmacological action specifically. This has also been suggested as a means of treating DNA viruses which implant in macrophages. Anthracyclines, such as daunorubicin and adriamycin, intercalate into DNA. A drug carrier has been devised in which fragments of DNA containing intercalated anthracyclines are administered systemically.14,1"18 This intercalated complex is inactive but can be endocytosed and broken down in lysosomes by DNAases. Again, the aim is to release the cytotoxic

agent in the vicinity of the malignancy and thereby increase its efficacy. Antibodies have also been studied as specific delivery carriers, but research has been hampered by the inhomogeneity of tumor-specific antibodies.





37


The idea of including a drug into liposomes formed in vitro has received a great deal of attention.117,119 In these systems, the drug is inactive since it is enclosed in the phospholipoidal matrix of the liposome and, as in the case of the albumin conjugates, these packets are taken up by endocytosis. Again, the system should specifically exert its action at the site of influx. While these approaches are promising theoretically, they have not met with much success. These carrier complexes are the subject of several recent books.117,119
A general method was recently proposed for the specific delivery of drugs to the brain. This system was based on the results of some work with N-methylpyridinium-2-carbaldoxime chloride (2-PAM).120,121,122,123 This pyridinium quaternary compound is the agent of choice for the treatment of organophosphate poisonings, and exerts its action by reactivating deactivated cholinesterases. The problem with this agent is its highly polar nature. Organophosphates such as diisopropyl fluorophosphate (DFP) and paraoxon are very lipophilic and easily penetrate the BBB. The brain is, therefore, very susceptible to acetylcholinesterase inactivation by these agents. The highly polar 2-PAM has a very low activity in the brain since it is almost totally excluded by the BBB. To deal with this problem the dihydro adduct of 2-PAM, pro-2-PAM,was synthesized as a prodrug.








N CH:NOH N CH:NOH
14@ I
CH3 CI CH3
2-PAM pro-2-PAM





38

The rather involved synthesis of pro-2-PAM yielded the enamine salt which corresponds to the 3-protonated dihydro compound. The pKa of the tertiary nitrogen in this enamine salt was determined potentiometrically and spectrophotometrically to be 6.32 + 0.6 and the oxime was estimated to have a pKa of -11. With a pKa of 6.32, it would be predicted that about 90% of the enamine would exist as the free base at physiological pH (7.4). Since this dihydro free base is far more lipophilic than the

parent quaternary, its ability to penetrate membranes is greatly enhanced. This pro-2-PAM is rapidly converted to the parent 2-PAM at physiological pH and the t has been determined to be 1.04 min by pharmacokinetic modeling. This rapid conversion of the pro-2-PAM to 2-PAM is desirable since ideally, the only metabolism of the prodrug is to the parent compound.

A number of experiments were carried out using 2-PAM and pro-2-PAM. From the linear descending portion of a semilog plot of blood concentration versus time, the biological t, of 2-PAM and pro-2-PAM was calculated. The t, for comparable doses of 2-PAM and pro-2-PAM differed by more than
2
60 min, and since the conversion of pro-2-PAM 2-PAM is rapid, this difference was assumed to be the altered distribution of pro-2-PAM. This indicates that even though the rate of conversion of pro-2-PAM is rapid, it is long enough for the enhanced distributional characteristics of the pro-2-PAM to be expressed.

Blood levels of 2-PAM were significantly higher after an oral dose of pro-2-PAM than with a dose of 2-PAM. If pro-2-PAM is administered intravenously, there are no new metabolites formed. If brain concentrations of 2-PAM are examined after 2-PAM and pro-2-PAM dosing, the concentration of 2-PAM in the brain is 13-fold higher in the case of pro-2-PAM administration. If brain acetylcholinesterase is inactivated using DFP, the





39

extent of reactivation observed in mice injected with pro-2-PAM shows a dramatic increase over 2-PAM treated animals.

A further experiment with 2-PAM showed that this small quaternary

salt was rapidly lost from the CNS and this loss was attributed to an active efflux process.124 These results differ from those obtained by Ross and Froden who attempted to deliver a quaternary compound to the brain as its uncyclized w-haloalkyl amine.12,126 They found that the loss of the quaternary compound was slow (t, = 38 hours) and concluded that the
2
efflux of the quaternary was comparable to its influx. This difference in the rate efflux may be related to differences in molecular size, shape or charge.

This preliminary work led to a proposed drug delivery system (Figure 1-2) which is specific for the brain and which is generally applicable.124 In this proposal, a pharmacologically active agent, whose ability to pass the BBB is low, is chemically linked to a pyridinium carrier. This carrier could be envisioned as a nicotinamide or nicotinic acid ester. This complex would be reduced under conditions which would yield the dihydropyridine. This complex is then injected systemically and,because of the increased lipophilicity of the dihydropyridine, partitions into the brain as well as into the periphery. In both locations, oxidation (kox) should occur. The rate of this oxidation is somewhat controllable by the judicious placement of ring substituents on the pyridinium nucleus. Systemically, the charged polar oxidized species should be eliminated rapidly by the kidney and/or liver (kout2), while in the brain the compound, because of its charge and size, would be retained i.e., kout2 > koutl*

Also, in both locations, cleavage of the drug from its carrier should occur (kcleavage). In the brain, the small nontoxic pyridinium carrier is





40
















R REDUCTION

x

N
R
BRAIN PERIPHERY


N N




RxkO R kg



eavage kc leavag N
R R


k out 1Q O O O'I
N N kot2
R R
maI kout4



Figure 1-2. A Proposed Carrier-mediated Chemical Delivery System with
Specificity for the Brain. The Drug Molecule to be Transported is Represented by the (0).





41


rapidly eliminated, kout3* If the cleavage of the drug from its carrier occurs at an appropriate rate i.e., kcleavage > koutl a sustained release of the agent to the brain could also be obtained. Again, the cleaved polar drug would be rapidly eliminated systemically. By these manipulations a compound can be delivered specifically to the brain while its systemic concentration is kept low. This reduces any associated systemic toxicity of the agent and increases its therapeutic index dramatically.

In this system the positively charged carrier complex cleaves to

yield the active compound. The dihydropyridine is not, therefore, a prodrug but rather a pro-prodrug or, better stated, a chemical delivery system. This drug delivery system is based on the naturally occurring reduced nicotinamide adenine dinucleotide (NADH) -_ oxidized nicotinamide (NAD+) system. These endogenous coenzymes are important in electron transferring chains and their suitability to this purpose is related to the chemistry of dihydropyridines and enamines. In the described delivery system the relative unstability, greater lipophilicity and predictability

of chemistry of the dihydromoieties are exploited. Additionally, since this method relies on enzymatic activation by an endogenous system (NADH dehydrogenase) whose substrates closely approximate the delivery compounds, any toxicity associated with the oxidation should be minimal. In this system the BBB has not been simply circumvented but rather used as an integral part of the delivery scheme.

Statement of the Problem

The chemical delivery system proposed by Bodor et al. should demonstrate a broad applicability since the agent to be delivered is simply attached to a particular carrier.124 In certain instances, however, this

system can be simplified. If a pyridinium nucleus is an integral structural





42

component of a molecule, as it is in many pharmacologically active agents, the molecule is provided with an internal delivery moiety. The drug to be delivered and the carrier are therefore merged into one molecule. The pharmacokinetics of this scheme is also simpler than those involved with the carrier system since no cleavage is necessary of the delivered molecule from the carrier. In this approach, which appears in Figure 1-3, an appropriately chosen pharmacologically active compound i.e., one which contains a pyridinium moiety, would be reduced to its corresponding dihydropyridine. This lipophilic species would penetrate the BBB as well

as into the systemic circulation. After a period of time determined by the stability of the compound, it would be oxidized to the parent quaternary salt (kox). Systemically, this agent would rapidly be eliminated by filtration or by tubular secretory mechanisms (kout2). In the CNS, however, since the ability of the compound to freely diffuse would be lost, it would be delivered fairly specifically (kout2 > koutl). The transit time of the drug in the brain would depend upon a number of factors including the participation of the compound in any active efflux processes. It would be hoped that in most cases the rate at which the compound entered the brain would be faster and ideally, much faster than the rate at which the compound left the brain (koutl).

This system, unlike the carrier mediated chemical delivery system, can be considered a prodrug. This prodrug should, however, demonstrate a specificity for the brain because of its design, the characteristics

of the BBB, and the chemistry of dihydropyridines. Again, this specificity should increase the therapeutic index of the drug delivered.

Several criteria were used in choosing a molecule for these delivery approaches. Obviously, the compound should contain a pyridinium moiety





43
















N
SREDUCT I ON





N

BRAIN PERIPHERY











N INe

Hkout1 kout 2

V





Figure 1-3. The Proposed Drug Delivery System.





44

which is reducible to some dihydropyridine species which is stable enough to be isolated. The compound should be active in vitro. Since quaternary compounds are to be considered, the lack of any in vivo activity might be ascribed to transport or distributional problems. A review of the literature showed two groups of compounds which looked particularly suitable. These include the substituted benzophenanthridinium salts and the protoberbine alkaloids which have the basic skeleton:


CD D

eAB A B NC
N
"R
benzophenanthridinium ion protoberbine

Members of these groups contain an N-substituted isoquinoline moiety which can be reduced by a number of agents. A number of these dihydroisoquinoline compounds are stable.127 These agents show a wide range of effects including antineoplastic and antibiotic activity.128-136 The specific compound chosen was berberine. C"
O







OCH3 Cr
berberine

Berberine, which has the chemical name 5,6-dihydro-9,10-dimethoxybenz[g]-[1,3]benzodioxolo[5,6-a]quinolizinium chloride, has a rather high in vitro activity against several cancer types including Ehrlich and lymphoma ascites.131,133 Its in vivo action is, however, very low.132,133





45


Berberine is widely distributed in the plant kingdom and is found

in such families as menispermaceae and berberidaceae, to name only two.137 The compound was isolated by 1826 by Pelletan and Chevallier.

Biosynthetically, berberine is interesting because of the presence of the so-called berberine bridge. This term refers to the single carbon atom between the nitrogen and the methoxylated aromatic nucleus. Two postulates for the formation of this have been suggested. The first involves formaldehyde in a Mannich-type ring closure while the second involves an oxidative cyclization of an N-methyl group.138,139 Labelling experiments favor the second mechanism.139

There are several total syntheses for berberine in the literature and the chemistry of the protoberbine alkaloids is well reviewed.140-142 Berberine has a variety of pharmacological actions and several therapeutic uses. Pharmacologically, it exhibits a depressive action on excitable

tissues,143 induces hypotension and tachycardia,144,145s and inhibits a number of enzymes including histaminease (human pregnancy plasma diamine oxidase),146 cholinesterase,"4 dopamine-adenyl cyclase,'47 and cationdependent ATP phosphorylases.148 Berberine also possesses an antiheparin149 and local anesthetic activity.144 Because of the affinity of berberine for dopaminergic receptors5is0 and alcohol dehydrogenases,1' it has been

used to characterize geometric and stereospecific requirements for substrate binding to these enzymes. Berberine has long been known as an antibiotic. The alkaloid causes mutations in certain bacterium by affecting nonchromosomal genetic material.134 Berberine has been used mostly in India to treat cholera,'s2 diarrhea,15 leshmaniasis and other parasitic

infections.





46

Biochemically, berberine acts to inhibit DNA, RNA and protein synthesis.143 It has been suggested that many compounds which are structurally similar to berberine exert their cytotoxic activity via alkylation at the iminium site.128 A more thoroughly studied, and perhaps more accurate, hypothesis is that berberine acts by intercalating between the

base pairs of DNA. This intercalation can be explained by a modified neighbor-exclusion model.154 A model which has been used to explain the intercalation of berberine is one in which the greater portion of rings A, B and D are intercalated.155 As berberine complexes, it assumes a more rigid planar species as may be indicated by the increase in fluorescence quantum yield. This intercalation does, however, slightly bend the double helix. The unwinding angle of DNA due to intercalation of berberine is

less than for the more planar molecules such as corylene. The effect of the positive charge on intercalation has also been noted.'56

Berberine, therefore, is appropriately suited as a candidate for inclusion in the drug delivery system described. In order to verify the original hypothesis of site-specific delivery, a number of experiments are required. Dihydroberberine must be synthesized and its stability assayed. The ability of dihydroberberine to penetrate the BBB and concentrate in the brain must also be demonstrated. Additionally, the toxicity and anticancer activity of the agent should be investigated.












CHAPTER 2

MATERIALS AND METHODS

Elemental analyses of compounds synthesized were performed by Galbraith Laboratories, Inc., Knoxville, Tennesse, or Atlantic Microlab, Inc., Atlanta, Georgia. Uncorrected melting points were determined using a Thomas-Hoover melting point apparatus. Ultraviolet spectra (UV) were recorded on a Beckman 25, Cary 210 or Cary 219 spectrophotometer. An

Apple II Plus microprocessor was dedicated to the Cary 210 instrument. Infrared spectra (IR) were taken on a Beckman 4210 high resolution and a Beckman Acculab 1 infrared spectrophotometer. Samples were analyzed as a thin film between sodium chloride windows or as a potassium bromide pellet. Nuclear magnetic resonance spectra (NMR) were obtained from either a Varian T60 or Joel-JNM-FX 100 Fourier transform spectrometer. The samples were dissolved in deuterated chloroform (CDCI3), deuterated dimethylsulfoxide ((CD3)2SO), deuterated pyridine (C5D5N), deuterated methanol (CD30D), deuterated acetonitrile (CD3CN), deuterium oxide (D20) or trifluoroacetic acid (TFA). Chemical shifts in parts per million were reported relative to the internal standard tetramethylsilane except in aqueous systems where sodium 3-trimethylsilylpropanesulfonate is used. Mass spectra were obtained using a DuPont 21-491B double focusing magnetic sector mass spectrometer to which was dedicated a Hewlett Packard 2100A computer. In all determinations, the ionizing voltage was 70 eV.

High pressure liquid chromatography (HPLC) was performed on either a Waters Associates system consisting of a Model U6K injector, a Model 6000A solvent delivery system and a Model 440 absorbance detector or a

47





48

ternary Beckman system consisting of three Model 112 solvent delivery systems, a Model 160 absorbance detector and a Model 421 controller. In some studies, a Varian FluorichromR detector was used with one of the Beckman pumps. Thin-layer chromatography (TLC) was performed on EM Reagents Cat. 5751 Aluminum Oxide 60 F-254 precoated plates (layer thickness

0.25 mm) or Analtech, Inc. UniplateR, which are precoated with silica gel G at a thickness of 0.25 mmn. Tissues were homogenized by a VirTis 45 homogenizer or by a teflon pestle and ground glass tube. In potentiometric titrations, a Radiometer-Copenhagen PHM 84 Research pH meter was used. A Sage Instrument Co. Model 341A syringe pump was used for infusions. For radionuclide studies, a Packard Tri-Carb 460 CD Liquid Scintillation system was employed.

In the theoretical studies, the MINDO/3 program was modified to suit the University of Florida's IBM System/370 computer. The molecule drawing program, X3DMOL, was developed by E.W. Phillips.

All chemicals used were of reagent grade. Berberine was obtained from Sigma Co. while nicotinic acid, nicotinamide, methyl iodide, benzyl bromide, butanol, hexanol, octanol, decanol, sodium borohydride and sodium

dithionite were obtained from Aldrich Co. Phenethyl alcohol was purchased from Matheson, Coleman and Bell. Tritiated inulin, SolueneR and scintillation cocktails were obtained from New England Nuclear. Solvents were of

chromatographic purity and were obtained from Fisher Co. Pyridine (Aldrich Co.) was refluxed over CaH2 and distilled before using. In cases where oxygen was to be excluded, solutions were made from water which, after boiling for fifteen minutes, was cooled with a stream of pyrogallolscrubbed nitrogen passing through it. A phosphate buffer (pH 7) was made by dissolving 1.183 g of potassium dihydrogen phosphate and 4.320 g of disodium hydrogen phosphate in water and diluting to 1.0 z.





49

Synthesis

Dihydroberberine (2)

Seven grams of berberine chloride (1) were dried over phosphorous pentoxide at 80C in a vacuum oven for eight hours. Five grams of the dried salt (0.013 moles) were added to a suspension of dry pyridine and 0.6 g of sodium borohydride (NaBH4). The solution was stirred under N2 for twenty minutes at room temperature, at which time 0.5 g more of NaBH4 were added. The liquid was then poured into 800 ml of ice water. The ensuing precipitate was dried overnight at 50*C over phosphorous pentoxide. The crude material was recrystallized from benzene-petroleum ether (low boiling). The yield was 35%: Melting point 156-1580C, Literature 157-159C; 1H NMR (CDCI3) 5 7.1 (1 H, s), 6.7 (2 H, s), 6.4 (1 H, s),

5.9 (3 H, s), 4.3 (2 H, s), 3.4 (6 H, s), 3.1 (2 H, t), 2.9 (2 H, t); MS m/e 338 (M+ + 1) 22%, 337 (M+) 100%, 322 (M' 15) 27%, 278 (M+ 59) 13%; UV (95% ethanol) 370 Xmax; IR (KBr) v (C-H) 3005 and 2970, (C-C, C-N) 1600 and 1482, (C-O-C asym) 1227 and 1270, (C-O-C sym) 1028 and 1083; 13C NMR (see figure 3-6); Elemental analysis calculated %: C, 71.21; H, 5.60; N, 4.15. Found %: C, 71.19; H, 5.70; N, 4.14. Dihydroberberine Hydrochloride (3)

One gram of (2) was dissolved in a minimal amount of CH2Cl2. Anhydrous hydrogen chloride, produced by dropping concentrated sulfuric acid (H2SO4) on sodium chloride (NaCl), was bubbled through the solution yielding a yellow precipitate. The material was recrystallized in aqueous ethanol.

9-Demethylberberine (Berberrubin) (4)

Seven grams of (1) were dried for two hours at 100C in a vacuum oven and then were heated to 200C for an additional thirty minutes. The deep purple material produced was dissolved in hot water and extracted with





50


chloroform (CHCI3). The organic layer was reduced to dryness and the residue dissolved in hot water. The solution was filtered and then made acidic with an excess of hydrochloric acid (HCl). The ensuing precipitate (yellow-brown solid) was collected by filtration and redissolved in hot water. The solution was filtered and made basic with potassium hydroxide. The solution turned deep purple and crystallization was induced by scratching: IH NMR (CD30D) 6 9.0 (s, 1 H), 7.8 (s, 1 H), 7.2 (m, 2 H),

6.8 (m, 2 H), 5.8 (s, 2 H), 3.7 (s, 3 H), 4.4 (m, 2 H), 3.0 (m, 2 H); UV (95% ethanol) 276 nm and 238 nm Xmax, others 512 nm; IR (KBr) v (C-H) 3000 and 2900, (C-C, C-N) 1635, 1571, 1509 and 1472, (C-0-C asym) 1221 and 1289, (C-0-C sym) 1035 and 1144; Elemental analysis calculated %: C, 71.03; H, 4.67; N, 4.36. Found %: C, 71.20; H, 4.81; N, 4.42.

Berberine Iodide (5)

A solution of 1.0 g of (4) in acetone was prepared. A 1.0 M excess of methyl iodide was added and the solution was allowed to reflux for several hours. The characteristic yellow color of berberine appeared and TLC, NMR, IR, and UV confirmed the methylation.
3-(Aminocarbonyl )-l-methylpyridinium Iodide/l-Methylnicotinamide Iodide (6*

Five grams of nicotinamide (0.041 moles) were dissolved in 50 ml of dry methanol. A molar excess of methyl iodide (11.6 g) was added to the stirring mixture and after one hour of refluxing, a precipitate formed. This was filtered and washed. The material was recrystallized from aqueous methanol: Melting point 101-1030C, Literature 102-105C; UV (H20) 224 mm and 265 mm xmax; NMR and IR were identical with the literature.


*The nicotinic acid derivatives are given a systematic name followed by a common name





51

3-(Aminocarbonyl )-l-(phenylmethyl )pyridinium Bromide/1-Benzylnicotinamide
Bromide (7

Ten grams of nicotinamide (0.083 moles) were dissolved in 150 ml of methanol. A molar excess of benzyl bromide (28.0 g) was added and the mixture was allowed to reflux for several hours. Upon cooling, a white solid appeared. This was filtered, washed and recrystallized from methanol: Melting point 206-2080C, Literature 2050C; IH NMR (D20 or TFA)

6 9.5 (1 H, s), 9.0 (2 H, m), 8.2 (1 H, m), 7.5 (5 H, s), 5.9 (2 H, s); IR (NaCl) v (N-H asym) 3300, (N-H sym) 3148, (-C(=0)NH2 Amide I) 1690, (Amide II) 1643, (C-N-C) 1388.
3-Pyridinecarboxylic Acid Ethyl Ester/Ethyl Nicotinate (8)

Forty-two grams (0.34 moles) of nicotinic acid were mixed with 55 ml of absolute ethanol and 25 ml of concentrated H2SO 4. The mixture was refluxed in an oil bath for four hours at which time the solution was poured over ice and made slightly basic with ammonia. The aqueous solution was extracted with ethyl ether. The organic layer was dried with sodium sulfate (Na2SO4) and the solvent evaporated under reduced pressure. The product was a clear liquid and the yield was 55%: 1H NMR (CDC13) 6 9.2 (I H, s), 8.8 (1 H, m), 8.2 (1 H, m), 7.4 (1 H, m), 4.4 (2 H, q), 1.4 (3 H, t); IR (NaCl) v (C-H) 2990, (C=O) 1728, (-C-C(=0)0) 1286, (0-C-C) 1112, (y CH) 742, (a ring) 703.

3-Pyridinecarboxylic Acid Butyl Ester/Butyl Nicotinate (9)
Forty-two grams (0.34 moles) of nicotinic acid were dissolved in
55 ml of 1-butanol and 25 ml of concentrated H2S0I4 was slowly added. The solution was heated to reflux in an oil bath for three hours, at which time the solution was poured over ice and made slightly basic with ammonia. The mixture was extracted with ethyl ether. The separated ether layer
was dried over Na2SO4 and the solvent removed under reduced pressure.





52

The product was a clear liquid and the yield was 62%: 1H NMR (CDC13)
9.2 (1 H, s), 8.8 (1 H, m), 8.2 (1 H, m), 7.4 (1 H, m), 4.3 (2 H, t), 1.6 (4 H, m), 0.97 (3 H, t); IR (NaCl) v (C-H) 2962, (C=0) 1730, (C-C, C-N) 1594, (-C-C(=0)0) 1288, (0-C-C) 1117, (y CH) 742, (6 ring) 702. 3-Pyridinecarbonyl chloride Hydrochloride/Nicotinoyl Chloride Hydrochloride
(10)
Forty-one grams (0.33 moles) of nicotinic acid were stirred in an ice bath with 110 ml of thionyl chloride (SOCl2) slowly added. After the addition was complete, the mixture was refluxed for three hours. The SOC12 was removed under reduced pressure and traces of SOC12 were azeotroped off with benzene. The white crystalline product was obtained in 94% yield; NMR and IR were identical with the literature. 3-Pyridinecarboxylic Acid Hexyl Ester/Hexyl Nicotinate (11)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry distilled pyridine and 5.73 ml (0.062 moles) of 1-hexanol. The solution was refluxed in an oil bath for six hours. The solution was then poured over ice which had been made basic with ammonia. The aqueous solution was extracted with ethyl ether, the organic layer dried over Na2SO, and the solvent removed under reduced pressure. The yield was 60%: 1H NMR (CDC13)
6 9.1 (1 H, s), 8.7 (1 H, m), 8.2 (1 H, m), 7.3 (1 H, m), 4.3 (2 H, t),
1.2 (8 H, m), 0.87 (3 H, t); IR (NaCl) v (C-H) 2938 and 2961, (C=0) 1726, (C-C, C-N) 1590, (-C-C(=0)0) 1282, (0-C-C) 1111, (y CH) 741, (8 ring) 702. 3-Pyridinecarboxylic Acid Octyl Ester/Octyl Nicotinate (12)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry pyridine and 9.75 ml (0.062 moles) of 1-octanol. The solution was heated to reflux in an oil bath and the progress of the reaction monitored with TLC. After eight hours, the liquid was poured over ice which had been made basic with ammonia. The aqueous solution was extracted with ethyl ether. The





53

ether layer was dried over sodium sulfate (Na2SO4) and the solvent removed under reduced pressure. The yield was 58%: 1H NMR (CDC13) 6 9.1 (1 H, s),
8.7 (1 H, m), 8.2 (1 H, m), 7.3 (1 H, m), 4.3 (2 H, t), 1.3 (12 H, m), 0.88 (3 H, t); IR (NaCl) v (C-H) 2920 and 2950, (C=0) 1723, (C-C, C-N) 1589, (-C-C(=0)0) 1277, (0-C-C) 1109, (y CH) 732, (s ring) 693. 3-Pyridinecarboxylic Acid Decyl Ester/Decyl Nicotinate (13)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry pyridine and 11.82 ml (0.062 moles) of 1-decanol. The solution was refluxed in an oil bath for eight hours. The liquid was then poured over ice which had been made basic with ammonia. The aqueous solution was extracted with ethyl ether, the organic layer dried over Na2SO, and the solvent removed under reduced pressure. The yield was 64%: 1H NMR (CDC13) 6 9.2 (1 H, s),

8.7 (1 H, m), 8.2 (1 H, m), 7.2 (1 H, m), 4.3 (2 H, t), 1.2 (16 H, m),
0.83 (3 H, t); IR (NaCl) v (C-H) 2915 and 2946, (C=0) 1720, (C-C, C-N) 1584, (-C-C(=0)0) 1275, (0-C-C) 1105, (y CH) 732, ( ring) 692. 3-Pyridinecarboxylic Acid -Phenylethyl Ester/6-Phenethyl Nicotinate (14)
Twenty-six grams (0.15 moles) of (10) were dissolved in 200 ml of
pyridine. To this stirring solution was added dropwise 18.3 g (0.15 moles) of B-phenethyl alcohol. The solution was refluxed in an oil bath for several hours. The solution was then poured over ice and made slightly basic with ammonia. This solution was then extracted with ether. The organic layer was dried over Na2SO4 and evaporated under reduced pressure. The yield was 62%: 1H NMR (CDC13) 6 9.1 (1 H, s), 8.6 (1 H, m), 8.0 (1 H,m),
7.2 (6 H, s), 4.4 (2 H, t), 2.9 (2 H, t); IR (NaCl) v (C-H) 3030 and 2959, (C=0) 1722, (C-C, C-N) 1585, (-C-C(=0)0) 1276, (0-C-C) d 1120, 1105, (y CH) 737, (a ring) 695.




54

3-(Ethoxycarbonyl)-l-methylpyridinium lodide/Ethyl N Methyl nicotinate
Iodide 15
Ten grams of (8) (0.066 moles) were mixed with methanol and with a molar excess of methyl iodide (18.7 g). The solution was refluxed for several hours. The solvent was removed, yielding a red-orange oil which solidified on cooling to a yellow solid. The solid was recrystallized from acetone-ether: 1H NMR (CDC13, (CD3)2SO), 6 9.6 (2 H, m), 9.0 (1 H, m),
8.4 (1 H, m), 4.8 (3 H, s), 4.5 (2 H, q), 1.4 (3 H, t); IR (NaCl) v (C-H) 3008, (C=0) 1723, (-C-C(=O)0) 1303, (0-C-C) d 1102, 1117, (y CH ) 742, (0 ring) 654.
3-(Butoxycarbonyl)-1-methylpyridinium lodide/Butyl N-Methylnicotinate
Iodide (16)
Ten grams of (9) (0.056 moles) were dissolved in 60 ml of acetone
and a molar excess of methyl iodide (15.9 g) was added. The solution was refluxed for two hours. The solvent was removed, leaving a yellow solid which was recrystallized from acetone-ether: 1H NMR (CDC13, (CD3) SO)
6 9.9 (2 H, m), 9.0 (1 H, m), 8.6 (1 H, m), 4.5 (3 H, s), 4.4 (2 H, t),
0.93 (3 H, t); IR (NaCl) v (C-H) 2950, (C=0) 1720, (-C-C(=0)0) 1291, (0-C-C) 1100, (y CH) 735, (a ring) 651. 3-(Hexoxycarbony1)-l-methylpyridinium I odide/Hexyl N Methylnicotinate
Iodide (17)
Ten grams of (11) (0.048 moles) were dissolved in 60 ml of acetone, and a molar excess of methyl iodide (13.7 g) was added. The solution was refluxed for two hours. The solvent was removed, producing an orange oil: 1H NMR (CDC13, (CD3)2S0) 6 9.3 (2 H, m), 8.9 (1 H, m), 8.3 (1 H, m),
4.7 (3 H, s), 4.3 (2 H, t), 0.90 (3 H, t); IR (NaCl) v (C-H) 2944, (C=0) 1723, (-C-C(=0)O) 1295, (0-C-C) 1111, (y CH) 738, (a ring) 654.





55

3-(Octoxycarbonyl)-l-methylpyridinium lodide/Octyl N Methylnicotinate
Iodide (18)
Ten grams of (12) (0.043 moles) were mixed with 60 ml of acetone and with a molar excess of methyl iodide (12.1 g). The solution was allowed to reflux for two hours at which time the solvent was removed, yielding an oil which was resistant to crystallization: 'H NMR (CDC13, (CD3)2SO) 6 9.9 (2 H, m), 9.0 (1 H, m), 8.6 (1 H, m), 4.6 (3 H, s), 4.4 (2 H, t), 0.92 (3 H, t); IR (NaCl) v (C-H) 2960, (C=0) 1731, (-C-C(=0)0) 1302, (0-C-C) 1120, (y CH) 749, (8 ring) 668.
3-(Decoxycarbonyl)-1-methylpyridinium lodide/Decyl N Methylnicotinate
Iodide (19)
Ten grams of (13) (0.038 moles) were dissolved in 60 ml of acetone and a molar excess of methyl iodide (10.8 g) was added. The solution was refluxed for two hours. The solvent was removed, leaving an orange oil: 1H NMR (CDC13, (CD3)2SO) 6 9.7 (2 H, m), 9.0 (1 H, m), 8.5 (1 H, m), 4.8 (3 H, s), 4.4 (2 H, t), 0.95 (3 H, t); IR (NaCl) v (C-H) 2944, (C=0) 1725, (-C-C(=0)0) 1297, (0-C-C) 1113, (y CH) 741, (8 ring) 658. 3-( B-Phenylethoxycarbonyl )-l-methylpyridini um lodide/p-Phenethyi N-Methylnicotinate Iodide (20)
Ten grams of (14) (0.044 moles) were dissolved in 60 ml of acetone. A 1.0 M excess of methyl iodide (12.5 g) was added to the liquid and the system was allowed to reflux for two hours. The solvent was removed, yielding a solid which was recrystallized from acetone: 1H NMR (CDC13, (CD3)2S0) 6 9.4 (2 H, m), 8.7 (1 H, m), 8.2 (1 H, m), 7.2 (5 H, s), 4.7 (3 H, s),4.6(2 H, t), 3.1 (2 H, t); IR (NaCl) v (C-H) 2995 and 3023, (C=0) 1724, (-C-C(=0)0) 1290, (0-C-C) d 1135 and 1124, (y CH) 731, (a ring) 657.





56

1,4-Dihydro-l1-methyl-3-pyridinecarboxamide/l1-Methyl -1 ,4-Dihydronicotinamide (21)

Four and six-tenths grams of sodium hydrogen carbonate (NaHCO3) and 2.64 g of (6) (0.019 moles) were dissolved in 100 ml of water and cooled in an ice bath. To this stirring solution was added 6.96 grams of sodium dithionite (Na2S204). A stream of nitrogen (N) covered the reaction mixture. After one hour, the reaction was stopped and the solution was extracted with several aliquots of CHC13. The CHC13 layer was removed under reduced pressure, yielding an orange oil. The oil was dissolved in a minimal amount of CHC13 and tritrated with petroleum ether. From this, an oil appeared and this was removed and dried in vacuo: 1H NMR (D 0) 6 6.9 (1 H, s), 5.7 (1 H, d), 4.8 (1 H, m), 3.2 (2 H, s), 3.0 (3 H, s); UV 355 nm Xmax; Elemental analysis calculated %: C, 60.87; H, 7.25; N, 20.20. Found %: C, 60.92; H, 7.29; N, 20.36 (C7HION20).
1 ,4-Dihydro--(phenylmethyl)-3-pyridinecarboxamide/l1-Benzyl-l ,4-dihydronicotinamide (22)

To 100 ml of water were added 4.6 g of NaHC03 and 2.93 g of (7) (0.013 moles). The solution was cooled and 6.96 g of Na2S204 were added. After two hours of stirring under N2, a precipitate formed. The solution was filtered. The solid was recrystallized from aqueous methanol, giving lemon-yellow needles: IH NMR (D20) 6 7.2 (6 H, s), 7.1 (1 H, s), 5.7 (1 H, m), 4.7 (1 H, m), 4.2 (2 H, s), 3.1 (2 H, m), in CDC13 two protons at 5.86 appear; UV 357 nm Xmax; Elemental analysis calculated %: C, 72.29; H, 6.58; N, 12.97. Found %: C, 72.09; H, 6.60; N, 12.84 (C13H 1N20). l1,4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Ethyl Ester/Ethyl 1 ,4-Dihydro-N-methylnicotinate (23)

Four and six-tenths grams of NaHCO3 and 2.75 g of (15) (0.016 moles)

were dissolved in 100 ml of water and cooled in an ice bath. To this stirring solution was slowly added 6.96 g of Na2S204. Two hundred milliliters





57

of ethyl ether were then added so that the dihydro would be extracted upon formation. This two-phase system avoided tetrahydropyridine production. The reaction proceeded for one hour under nitrogen. The ether layer was removed and the aqueous layer extracted. The combined ether fractions were dried over Na2SO4 and the solvent removed under reduced pressure. The resulting orange-red oil was dried in vacuo: 'H NMR (CDC13) 6 6.9 (1 H, d),

5.6 (1 H, m), 4.8 (1 H, m), 4.1 (2 H, g), 3.1 (2 H, m), 2.9 (3 H, s), 1.2 (3 H, t); UV 358 nm xmax; Elemental analysis calculated %: C, 64.67; H, 8.17; N, 8.43. Found %: C, 64.65; H, 7.88; N, 8.34 (C9Hz3N02). 1,4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Butyl Ester/Butyl 1,4-Dihydro-N-methylnicotinate (24)

A solution of 4.6 g of NaHCO3 and 3.12 g of (16) (0.016 moles) was

prepared in 100 ml of water. The solution was cooled and 6.96 g of Na2S204 were added. Two hundred milliliters of ethyl ether were added and this mixture stirred under nitrogen for one hour. The ether layer was removed, dried with Na2SO4 and reduced in volume. The resulting oil was dried in vacuo: IH NMR (CDC13) 6 6.9 (1 H, s), 5.6 (1 H, m), 4.7 (1 H, m), 4.1 (2 H, t), 3.1 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 358 nm Xmax; Elemental analysis calculated %: C, 67.69; H, 9.07; N, 7.23. Found %: C, 67.58; H, 8.82; N, 7.09 (CjjH17N02).

1,4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Hexyl Ester/Hexyl 1,4Dihydro-N-methylnicotinate (25)

A solution of 4.6 g of NaHCO3 and 3.57 g of (17) (0.016 moles) was prepared in 100 ml of water. The solution was cooled and 6.96 g of Na2S204 were added. Two hundred milliliters of ethyl ether were added

and the mixture stirred under nitrogen for one hour. The ether layer was separated, dried and reduced in volume. The dried oil was orange in color: 1H NMR (CDC13) 6 6.8 (1 H, s), 5.5 (1 H, m), 4.6 (1 H, m), 4.0 (2 H, t),





58

3.1 (2 H, m), 3.0 (3 H, s), 0.9 (3 H, t); UV 359 nm xmax; Elemental analysis calculated %: C, 69.96; H, 9.73; N, 6.32. Found %: C, 69.82; H, 9.46; N, 6.28 (C13 H 21NO 2).

1,4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Octyl Ester/Octyl 1,4-Dihydro-N-methylnicotinate (26)

A solution of 4.6 g of NaHCO3 and 4.02 g of (18) (0.016 moles) was prepared in 5% aqueous methanol. The solution was cooled and 6.96 g of Na2S204 were slowly added. Two hundred milliliters of ethyl ether were added and the mixture stirred under nitrogen for one hour. The ether layer was separated, dried and reduced in volume. The oil was dried in vacuo: 1H NMR (CDCl3) 6 6.9 (1 H, m), 5.6 (1 H, m), 4.7 (1 H, m), 4.0 (2 H, t),

3.0 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 358 nm Amax; Elemental analysis calculated %: C, 70.69; H, 9.97; N, 5.50. Found %: C, 70.76; H, 9.68; N, 5.88 (CisH2sNO2.*H20).

1,4-Dihydro-l-methy1-3-pyridinecarboxylic Acid Decyl Ester/Decyl 1,4-Dihydro-N-methylnicotinate (27)

A solution of 4.6 g of NaHCO3 and 4.46 g of (19) (0.016 moles) was prepared in 5% aqueous methanol. The solution was cooled and 6.96 g of Na 2S204 were slowly added. To this solution was added 200 ml of ethyl ether and the mixture stirred under N2 for one hour. The ether layer was separated, dried and reduced in volume. The dried oil was orange in color: 1H NMR (CDCl3) 6 6.9 (1 H, m), 5.6 (1 H, m), 4.7 (1 H, m), 4.0 (2 H, t),

3.0 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 359 nm Xmax; Elemental analysis calculated %: C, 73.12; H, 10.39; N, 5.02. Found %: C, 73.16; H, 10.48; N, 5.03 (C H29N02).

1,4-Dihydro-l-methyl-3-pyridinecarboxylic Acid 6-Phenylethyl Ester/a-Phenethyl 1,4-Dihydro-N-methylnicotinate (28)

Four and six-tenths grams of NaHCO3 and 3.89 g of (20) (0.016 moles) was dissolved in 100 ml of water. The solution was cooled and 6.96 g of





59

Na 2S204 were added. Two hundred milliliters of ethyl ether were added and the mixture stirred over N2 for one hour. The ether layer was separated, dried and reduced in volume. The dried oil was orange in color: 1H NMR (CDCI3) 6 7.2 (5 H, s), 6.9 (1 H, m), 5.7 (1 H, m), 4.7 (1 H, m), 4.2 (2 H, t), 3.0 (2 H, m), 2.9 (2 H, t), 2.8 (3 H, s); UV 355 nm Xmax; Elemental analysis calculated %: C, 70.18; H, 6.63; N, 5.46. Found %: C, 70.27; H,

7.00; N, 5.10 (C15H17N02 H20).
Characteri zati on of Di hydroberberi ne Distribution Coefficients

Fifty milliliters of a cold 1 x 10-4 M solution of berberine (1) in

pH 7.4 buffer were partitioned against 50 ml of CHCl3 or 50 ml of 1-octanol. The concentration of (1) was determined spectrophotometrically in the organic and aqueous layer. A stock solution of 2.7 x 10-3 M dihydroberberine hydrochloride (3) was made in methanol. An aliquot of this, sufficient to produce a 1 x 10-4 M solution, was pipetted into 50 ml of cold pH 7.4 buffer and extracted immediately with either CHC13 or 1-octanol. After allowing for oxidation, the concentration of (1) in the organic and aqueous layer was determined spectrophotometrical ly. Potentiometric pKa Determination of Dihydroberberine (2)

Due to the extreme water insolubility of (2) (< 3 pg/ml), all determinations were done in 25% methanolic solutions. A titration curve was generated by adding 10 wl aliquots of NaOH to a 1.0 mM solution of (3). The pKa was determined by inspection of the titration curve. During the

experiments, all solutions were covered with a stream of nitrogen. Spectrophotometric pKa Determination of Dihydroberberine (2)

The pKa of (2) was determined by measuring the absorbance difference at 355 nm in basic, acidic, and buffered media. The relationship that allows this determination appears below:





60


pKa = pH log %obs aHA
aA- aobs

where a obs is the absorbance in buffer, aA- is the absorbance in base and XHA is the absorbance in acidic media. Oxidation of Dihydroberberine (2) by Silver Nitrate

Two hundred milligrams of (2) were dissolved in 95% aqueous ethanol. Upon addition of a 10% solution of silver nitrate, a black precipitate formed. Centrifugation and analysis of the supernatant shows stoichiometric oxidation of (2).

Oxidation of Dihydroberberine (2) by Diphenylpicrylhydrazyl Free Radical (DPP')

Two hundred milligrams of (2) were dissolved in acetonitrile. To this was added a solution of DPP" in acetonitrile which caused an immediate disappearance of the purple color due to DPP'. Ultraviolet analysis confirmed this oxidation.

Oxidation of Dihydroberberine (2) by Concentrated Hydrogen Peroxide

Two hundred milligrams of (2) were dissolved in 95% aqueous ethanol. A 30% solution of hydrogen peroxide was added and the system monitored by UV. The analysis demonstrated a rapid and complete oxidation of (2). Oxidation of Dihydroberberine (2) in Buffers

The oxidation of (2) was determined by UV and an HPLC method. In the UV method, a solution of (3) was prepared and pipetted into buffers of various pH and at various temperatures. The changes of absorption at 460 nm were measured with time. Data acquisition was facilitated by an Apple II microprocessor and an enzyme kinetic software package. In the HPLC method, two buffers, pH 5.8 and pH 7.4, were used. The samples were maintained at 37*C in a water bath. At certain times, 5 pl of the solulution were injected onto a pBondapak C18 reverse-phase column, and the





61


peak heights analyzed. The mobile phase was 60:40 acetonitrile: pH 6.2

phosphate buffer and the flow rate was 2 ml/min. Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydropyridines (21) and (22 in Hydrogen Peroxide

Solutions of (3), (21) and (22) were prepared. An aliquot of these solutions was added to a standardized solution of hydrogen peroxide (H202) (0.18 M). The appearance of the 460 nm peak of (1) or the disappearance of

the 359 nm peak of the dihydronicotinamides was measured. This determination was made using the enzyme kinetics software package. Quantitation of the Oxidation of Dihydroberberine (21) and Various Dihydropyridines (22)-(28) in Plasma

Freshly drawn 80% human plasma was obtained from Civitan Regional Blood Center. The oxidation of (3) was determined by HPLC and the oxidation of

(3), (22), (23), (24), (25), (26), (27), and (28) by UV. In the HPLC analysis, a solution of (3) was added to 80% plasma and maintained at 370C. At certain times, 1.0 ml of plasma was removed and treated with 3 ml of acetonitrile. The solution was centrifuged and 5 vl of the supernatant was analyzed by a pBondapak C18 reverse-phase column with a mobile phase of 60:40 acetonitrile: pH 6.2 phosphate buffer. The peak heights were analyzed and concentrations obtained from a standard curve. In the UV method, 40% plasma was maintained at 370C in a kinetic cell. A solution of either

(3) or one of the various dihydronicotinates was added to this and the appearance of the 460 nm absorbance of (1) or disappearance of the 359 nm absorbance of dihydronicotinates was observed. Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydropyridines (22)-(28) in Liver Homogenate

The determination of the rate of oxidation of (3) in a liver homogenate by an HPLC method and (3), (22), (23), (24), (25), (26), (27), and (28) by a UV method was performed. In the HPLC method, 14 g of fresh rat liver





62

were homogenized in 45 ml of cold phosphate-buffered saline. To this was added a solution of (3), and the system was maintained at 37.0C. At various times, 1.0 ml of the solution was removed and the protein precipitated with 3 ml of acetonitrile. The sample was centrifuged and 5 jil of the supernatant analyzed using a pBondapak C1. reverse-phase column with a mobile phase of 60:40 acetonitrile: pH 6.2 phosphate buffer. The peak heights were analyzed and concentrations obtained from a standard curve. The UV method involved homogenizing 7 g of rat liver in 15 ml of pH 7.4 phosphate buffer and diluting the homogenate to 200 ml (3.5% w/v). The homogenate was centrifuged and the supernatant was used. To this was added a solution of (3) or one of the dihydropyridines. The appearance of the 460 nm peak of berberine or the disappearance of the 359 nm peak of the

dihydronicotinate was then measured. Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydropyridines (22)-(28) in Brain Homogenate

Again, both an HPLC and UV method were employed in the determination of the rate of oxidation of (3), (22), (23), (24), (25), (26), (27) and

(28). In the HPLC method, a solution of (3) was added to a 20% brain homogenate in pH 7.4 phosphate buffer. The homogenate was maintained at 370C in a water bath. At various times, 1.0 ml of the homogenate was mixed with 3 ml of acetonitrile. The sample was centrifuged and the supernatant analyzed by the same method used in the liver homogenates. In the UV method, 2 g of freshly obtained rat brain were homogenized in 33 ml of pH 7.4 phosphate buffer, yielding a 6.0% w/v homogenate. The homogenate was centrifuged and the supernatant was used in the determinations. To

this was added a solution of (3) or one of the dihydronicotinates. The appearance of 460 nm absorption of (1) or disappearance of the 359 nm peak

of the dihydronicotinate was measured.





63

In VitM Distribution of Berberine (1) and Dihydroberberine (2) in Whole
Blood

Solutions of (1) or (3) were added to 60 or 75 ml of freshly drawn

heparinized sheep's blood maintained at 37C. At various times, 4 ml of blood were withdrawn. The blood was centrifuged and the plasma removed. One milliliter of the plasma was treated with 9 ml of acetonitrile and the supernatant was analyzed spectrophotometrically. The entire volume of the packed red blood cells was treated with 8 ml of acetonitrile and centrifuged. The supernatant was again analyzed spectrophotometrically. A standard curve was obtained by preparing solutions of known concentration in plasma or packed red blood cells. Recovery from the red blood cells was 71.4%.

Effect of Glucose on the Distribution of Berberine (1) in Whole Blood

Glucose was added to a volume of blood so that a concentration of 200 mg% was obtained. The above procedure was then repeated.

Animal Studies

In Vivo Characterization of Berberine (1) and Dihydroberberine (2)
White Sprague-Dawley rats, who weighed between 200-250 g, were anesthetized intramuscularly with InovarR (0.13 ml/Kg). Injections were made intravenously into the external jugular vein. The doses used include 55 mg/Kg of (2) in dimethylsulfoxide (DMSO), 55 mg/Kg of (3) in 20-25% aqueous ethanol, 55 mg/Kg of (1) in DMSO or 35 mg/Kg of (1) in DMSO. At certain times after the injection, the chest cavities of the rats were opened, the vena cava severed and the heart perfused with normal saline. Afterwards, the animals were decapitated and the brains removed. In certain experiments, the lungs, liver, and kidneys were also excised. The organs were then homogenized in a minimal amount of water, usually 2 ml, and extracted with 8 ml of acetonitrile. A standard curve of (1) in the





64

organ homogenate was constructed. Analysis was performed by injecting

5 jIl of the supernatant onto a liBondapak C18 reverse-phase column with a mobile phase of 60:40 acetonitrile: phosphate buffer. The flow was 2 ml/ min. Under these conditions, the retention time of (1) was 3.8 min and

(2), 9.3 min.

Slow Infusion of Dihydroberberine Hydrochloride (3)

Rats were anesthetized and prepared as above. A dose of 55 mg/Kg of

(3) was prepared in a volume of 1.0 ml. The vehicle was 20% aqueous ethanol. This dose was infused into the external jugular vein over a period of either thirty or forty-five minutes. At the end of the perfusion, the animals were decapitated, their organs collected and analyzed. Effect of l-Methyl-l,4-dihydronicotinamide (21) on the Efflux of Berberine
from the Brain

Rats were anesthetized and prepared as above. Animals were injected with 200 mg/Kg of (21) in aqueous ethanol intravenously. After fifteen minutes, the standard dose of 55 mg/Kg of (3) was given. The animals were sacrificed at various times after the injection, selected organs were removed and homogenized, and the samples analyzed by HPLC. In. iv Characterization of l-Benzyl-l,4-dihydronicotinamide (22)

Rats were anesthetized and cut down as above. Doses between 60 mg/Kg and 400 mg/Kg of (22) were administered. At various times after the injection, the animals were perfused, decapitated, and the brains removed.

The brains were then frozen in liquid nitrogen and stored at OC until they were analyzed, at which time the brains were thawed, homogenized in 2 ml of water, and extracted with 8 ml of acetonitrile.

Analysis was made by HPLC using a pBondapak C1. reverse-phase column and a mobile phase of 40:60 acetonitrile: 1 x 10-3 M sodium heptanesulfonate. The dihydronicotinamide had a retention time of 3.4 min and the





65

quaternary compound had one of 10.6 min at a flow rate of 2 ml/min. A standard curve was constructed in brain homogenates. Intracerebral Ventricular (icv) Administration

Sprague-Dawley rats were anesthetized with 70 -l/l00 g of pentobarbital sodium, and atropine sulfate (0.05 mg/Kg) was given, if necessary. Injections of (1), (6), and 3H-inulin (29) were made into the lateral ventricles of rat brains. The injections were made with the aid of a stereotaxic instrument and the site of the injection was -0.4 mm anteriorposterior, 1.5 mm medial-lateral and -3.0 mm dorsal-ventral relative to the bregma. The dose of (1) infused was 50 pg and the infusion volume was between 3 and 5 pl. The vehicle was DMSO and the infusion rate was 5 pIl/5 min. In several experiments, (1) was coinjected with 1000 jig of

(6). In another set of experiments, a dose of 2.3 pCi of (29) was injected icv.

At specific times after the infusions, the animals were decapitated. The brain was homogenized in 1.0 ml of water and (1) extracted with 4 ml of acetonitrile. Analysis of (1) was by HPLC with a mobile phase consisting of 50:50 acetonitrile: pH 6.2 phosphate buffer. A pBondapak C18 reverse-phase column was used and a standard curve constructed using brain homogenates. For radionuclide analysis, the brains were homogenized in

8 ml of water. Twenty-five hundredths of a milliliter of this homogenate were added to 0.75 ml of SolueneR. After the sample dissolved, 12 ml of the scintillation cocktail were added. The samples were counted for five minutes and disintegrations per minute (dpm) were obtained by using a standard quench curve.
Limited Metabolic Studies

Rats treated intravenously (iv) with 55 mg/Kg of (1) or 55 mg/Kg of

(3) were housed in metabolic cages. Urine was collected and extracted with





66

CHCI3. The aqueous layer was then extracted with 3-methyl-l-butanol. The organic layers were reduced to dryness and then reconstituted with a small volume (50 vii) of methanol. This residue was used for HPLC and TLC analysis. Five microliters of each sample were analyzed by HPLC. A pBondapak C18 reverse-phase column and a mobile phase of 50:50 acetonitrile: pH 6.2 phosphate buffer were used. The TLC analysis consisted of spotting 5 pl of each sample on an alumina plate and eluting the system with cyclohexane: chloroform: acetic acid 45:45:10 or methanol. The plates were developed with iodine vapor or iodoplatinate spray reagent. Toxicity

White CD-l mice were employed in this study, the average mass of which was 22.6 2 g. The mice were segregated into groups of 10, and 8-10 groups were used in each study. The doses given were determined by preliminary studies in which the LDo and the LD10o were obtained using small groups of animals. The doses were then prepared in equal increments between the two extremes, but there was additional emphasis placed at the lower end of the curve. Since this study was concerned with acute toxicity, the animals were injected intraperitoneally, and the number dead recorded after twenty-four hours. The groups were, however, observed an additional forty-eight hours to ensure an accurate appraisal of acute toxicity. Food and water were given ad libitum. The injection volume was 75-100 pl. The data were analyzed by fitting them to a sigmoid curve and
by the method of Probits.

Anti cancer Acti vi ty
Male BDF mice (20.6 0.3 g) were used in this study. A suspension of P388 lymphocytic leukemia cells was injected intraperitoneally (ip) (1 x 106 cells) or intracerebrally (2.5 x I05 cells). The survival time of animals treated with various doses of (1) or (3) compared to the controls





67


was recorded. Berberine or dihydroberberine hydrochloride were given ip

3 times a day on day 2, 6, and 10 in 0.5% carboxymethylcellulose.













CHAPTER 3

RESULTS AND DISCUSSION

Synthesis and Characterization of Dihydroberberine

The initial step in the application of the proposed drug delivery system to berberine (1) is the preparation of its dihydro adduct. The first synthesis of dihydroberberine (2) involved disproportionation of

(1) in strong base and was performed by Gadamer in 1905.157,158 The mechanism of this reaction involves nucleophilic attack of hydroxide to the carbon adjacent to the nitrogen resulting in the formation of a transient amino alcohol. This intermediate collapses to oxyberberine and dihydroberberine, presumably via a hydride transfer. Several other syntheses for (2) have appeared in the literature and these involve the direct reduction of (1) by zinc amalgam,'59,160 complex metal hydrides162 or sodium borohydride.163,164 Historically, (2) has been of interest because of its spectroscopic properties,165-167 and as an intermediate in certain synthetic schemes.164 The use of (2) as a drug or in a drug delivery system is novel to this thesis.

In the present work (1) was reduced, as shown in Figure 3-1, by sodium borohydride in dry pyridine. Spectroscopic analysis showed that the yellow crystalline material produced was (2). The UV, IR, and MS are shown in Figures 3-2, 3-3, and 3-4 respectively, and are consistent with the assigned structure.167-169 The 1H NMR is presented in Figure 3-5, and the proton assignments in Table 3-1. The 13C NMR is shown in Figure 3-6, and the corresponding carbon assignments in Table 3-2. These assignments were made by comparing (2) to a number of model systems.170 The synthesized

68







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74


Table 3-1 Proton Assignments of the 1H NMR of Dihydroberberine (2)


Hd
0 Hd

bH 0

Ha Hd
HC Hb

f H Hh
H CO N
HCH

H



Proton PPM (6)

a 7.1

b 6.7

c 6.4

d 5.9

e 4.3

f 3.4

g 3.1
h 2.9







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76


Table 3-2. Carbon Assignments of the 13C NMR of Dihydroberberine (2)



0 14



13C







16




Carbon Carbon
Number PPM (6) Number PPM (6)
1 150.301 2 01l.457

2 147.182 12 107.753

3 146.597 13 103.659

4 144.452 14 100.881

5 141 .528 15 96.202
6 128.661 16 60.576

7 128.466 17 55.848

8 124.470 18 49.270

9 122.033 19 48.929

10 118.670 20 29.725


(CDC13: 78.267, 77.040, 75.732)





77


material gave an elemental analysis in good agreement with that predicted for (2). Also, the reduced material reacted rapidly with such oxidizing agents as hydrogen peroxide, silver nitrate, and l,l-diphenyl-2-picrylhydrazyl free radical (DPP') to yield (1). These data support the successful synthesis of (2).

The hydrochloride of dihydroberberine (3) was synthesized, as illustrated in Figure 3-1, by treating a concentrated solution of (2), in methylene chloride with dry hydrogen chloride (HCl) gas. The hydrochloride reverts to (2) at pH above 7.0. Analysis of the regenerated material demonstrated no addition of HCl or any other nucleophile to the molecule.

This addition is known to occur with several dihydropyridines. The hydrochloride is many times more soluble in aqueous solutions than the free base.

If (2) is to be successful in a drug delivery system described in Figure 1-3, it should demonstrate a greater lipophilicity than (1). Dihydroberberine is expected to be less polar than (1) because of the loss of the positive charge. To investigate the relative lipid solubility of

(2) compared to (1), the two compounds were extracted with organic solvents and their distribution (partition) coefficients compared.171

The two solvent systems which were used included an octanol-pH 7.4 phosphate buffer and a chloroform-pH 7.4 phosphate buffer. The alcoholbuffer system was chosen as one of the extracting systems because of its ability to,in some ways, mimic the partitioning of compounds in vivo. The data from Table 3-2 show that in both systems (2) has a high affinity for the organic phase while (1) exhibits a high affinity for the aqueous phase.
The chemistry of dihydroberberine is largely a result of its enamine character.172 Dihydroberberine, like all enamines, is in equilibrium with





78


Table 3-3. Distribution Coefficients for Berberine (1) and Dihydroberberine Hydrochloride (3) in Chloroform/pH 7.4 Buffer and in
l-Octanol/pH 7.4 Buffer

Distribution Coefficient

Compound Chloroform/pH 7.4 Buffer l-Octanol/pH 7.4 Buffer


Berberine (1) < 0.001 0.062

Dihydroberberine 5.33 2.59
Hydrochloride (3)





79

its corresponding imine and this unusual situation allows enamines, dihydroberberine included, to be substrates in both nucleophilic and electrophilic reactions. This equilibrium tends to concentrate a negative charge on the carbon a to the nitrogen. Protonation usually occurs, for this reason, at the a-carbon rather than the nitrogen. At physiological pH,

a portion of the dihydroberberine molecules will exist in a C-protonated state. The pKa which is an indication of the degree of this ionization is important since only the unprotonated free base is available for diffusion across membranes. The pKa of (2) was determined to be 6.80 + 0.05
by both a spectrophotometric and potentiometric method. At a physiological pH of 7.4, a substantial portion of (2) will thus exist in the unionized, freely diffusable form. Because of the water insolubility of the dihydroberberine free base, all pKa determinations were carried out in 25% methanolic solutions.

Demethylation of Berberine

A number of other synthetic schemes were explored in an attempt to

obtain a method for preparing radiolabeled (1), should spectroscopic method prove too insensitive. A radiolabelled compound would also greatly expedite whole body distribution studies. In radiolabeling a compound, one of the important factors governing the selection of a synthetic route is

yield. The scheme which was chosen involves pyrolyzing (1) at 2000C.173,174 Berberine loses methyl chloride,as shown in Figure 3-7,to form the deep purple zwitterionic berberrubin. This method would allow the placement of either a 14C or 3H label at the nine position of (1) by reacting berberrubin with the appropriately tagged methyl iodide or methyl sulfate.

Methylation of berberine with cold methyl iodide was performed to demonstrate the viability of the scheme.






80





















Cl)



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S
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T 4-0 r 0
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81

Theoretical Studies on the
Dihydropyridine : Pyridinium Redox System

If the drug delivery system described in Figure 1-3 is to be used to its potential, then, a knowledge of its basic chemistry is required. The reason for this is not only to understand the subtle chemistry inherent in this particular system, but also to allow prediction, extrapolation and generalization of the system to other examples. The ability to discern, for instance, those factors that add to or detract from the stability of a molecule would allow attenuation of a particular property by molecular manipulation. A thorough chemical knowledge of a particular compound would also allow an intelligent prediction as to its suitability to the scheme.

While a few ionization potentials have been measured, the general instability of dihydropyridines often precludes their investigation by experimental means. Because of this limitation, these compounds have lent themselves well to theoretical study. While the dihydronicotinamides, because of their biological relevance, have been the subject of copious reports, larger dihydro systems have received little attention.175'176 In order to gain a greater chemical insight into the proposed drug delivery system in general, and the berberine (1) dihydroberberine (2) system in particular, a theoretical investigation was undertaken.

Berberine is a rather large molecule and, as such, would be expected to present problems in terms of computational time. It must be remembered that 3N-6 (where N is the number of atoms) independent variables are required for a molecule and large molecules can easily cost $8000-$10000 in computer time. Because of this, and also in an attempt to generalize the calculations so that they would be applicable to a number of compounds, model was developed for the (1) -* (2) system. The compounds chosen as





82

the model, 3H,4H-dihydro-7,8-dihydroxybenzo[b]quinolizinium ion (30) and 3H,4H,6H-trihydro-7,8-dihydroxybenzo[b]quinolizine (31) along with their numbering protocol, are shown in Figure 3-8.
This quaternary and its dihydro adduct, while simpler than the

(1) 2 (2) system, do not differ greatly in structure or in general chemistry. Compounds (30) and (31) have not been investigated and no mention of them has appeared in the literature. Some theoretical work on the unsubstituted benzo[b]quinolizinium ion and much on the isoquinoline molecule has been published. The benzo[b]quinolizinium ion was investigated by Galasso in 1968 and charge densities and T-bond orders were reported.177 All of these reported studies have employed simplistic theoretical treatments such as PPP,178 IOC-w-technique,179 HMO,180 SSP,181 and CNDO/2.182 The corresponding dihydrocompounds have not been studied.
In order to investigate the isoquinoline model (30) 1 dihydroisoquinoline model (31) system, a MINDO/3 method was selected.183 This approach is a semi-empirical self-consistent field molecular orbital method in which all valence electrons are treated. The MINDO/3 program is the culmination of the series MINDO,184 MINDO/2,1s'85,1s86 and MINDO/2'.187 This program was developed to solve chemical problems quickly and efficiently using a quantum mechanical framework. Unlike the methods of Pople, whose major aim

was to reproduce nonempirical calculations, M.J.S. Dewar parametrized MINDO so that the system would produce useful and chemically accurate data. This program is extremely versatile and has been applied to a number of chemical problems including reactions. Only the most cursory details of MINDO/3 are appropriate here, as the subject is well reviewed elsewhere.183,188

In quantum mechanical approaches like MINDO, a molecular orbital is the result of the interaction of a wavefunction of an electron with nuclei and other electrons present in the molecule.189 Mathematically, the







83











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84


Hamiltonian for such a system consists of the kinetic energy term for the

movement of the electrons and the potential energy terms for electronnuclei attraction and electron-electron repulsi on.

In systems with only one electron,e.g. H, H2 + or He+, the differential equation which constitutes the Hamiltonian can be separated and exactly solved. If, however, more than one electron is present, the electrons interact and the differential equation is no longer separable or exactly solvable. Because of this problem, approximations and simplifications are incorporated into the Schriidinger equation. These include considering the molecular orbital ('T) as a linear combination of atomic orbitals (p):189

i
Additional simplifications are obtained by neglecting certain electron repulsion terms since these values are close to zero. These approximations allow the application of quantum mechanical approaches like MINDO to rather complex chemical problems without extremely large expenditures of capital or computer time.

The MINDO/3 program provides the following data: optimized geometries

of the most stable ground state conformation at 250C, the heat of formation (AHf) in kcal/mole at 250C of the optimized structures, the atomic charge distribution, the dipole moments, the total electronic energy, the vertical ionization potentials, the bond order matrix, and the eigen vectors and eigen values.

The eigen values and eigen vectors allow a thorough examination of the contributions of the individual atomic orbitals to the molecular orbital and thus of the electronic structure of the molecule. This can be indicative of many of the chemical proclivities of a molecule. The MINDO





85


approach allows one to examine compounds and reactions which are not approachable by conventional means. For example, the stability of very reactive species can be assayed, their structures determined, and qualitative aspects of their chemistry elucidated.

The input for the program includes, for each atom, the atomic number, the approximate bond distances between adjoining atoms, the approximate bond angle, and the approximate twist angle out of the plane. As previously mentioned, the number of parameters needed is 3N-6 where N is the number of atoms. Convergence is assumed when the difference between two successive calculations is less than 0.1 kcal/mole.

The two models (30) and (31) were analyzed and the results are presented in Table 3-4 through 3-8. The heat of formation calculated for (30) was 95.1 kcal/mole and that of (31) was -39.3 kcal/mole. While the use of AHf for comparisons is restricted to structural isomers, the relative stability of a system can be calculated by comparing the differences in AHf (AAHf) from one system to another. The AAHf of the isoquinoline model

(30) -1 dihydroisoquinoline model (31) pair is 134.4 kcal/mole. As shown in Table 3-9, this value is smaller than that obtained from simple dihydropyridine -( pyridinium systems indicating a greater stability of (31) relative to simple dihydropyridines.190 The stabilization of (31) indicates that it should be less reactive than simple dihydropyridines. The basis of this stabilization is derived from the extended aromatic conjugation of (31). Another contribution to the relative stabilization can be seen by an examination of the highest occupied molecular orbital (HOMO) which is represented in Figure 3-9 as a linear combination of atomic orbitals. In looking at the HOMO, only the magnitude of the coefficients is important since the sign simply represents the phase of the orbital. A







86









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Full Text
41
rapidly eliminated, If the cleavage of the drug from its carrier
occurs at an appropriate rate i.e., kc-¡eavage > kouti a sustained release
of the agent to the brain could also be obtained. Again, the cleaved
polar drug would be rapidly eliminated systemically. By these manipula
tions a compound can be delivered specifically to the brain while its
systemic concentration is kept low. This reduces any associated systemic
toxicity of the agent and increases its therapeutic index dramatically.
In this system the positively charged carrier complex cleaves to
yield the active compound. The dihydropyridine is not, therefore, a pro
drug but rather a pro-prodrug or, better stated, a chemical delivery sys
tem. This drug delivery system is based on the naturally occurring re
duced nicotinamide adenine dinucleotide (NADH) t oxidized nicotinamide
(NAD+) system. These endogenous coenzymes are important in electron trans
ferring chains and their suitability to this purpose is related to the
chemistry of dihydropyridines and enamines. In the described delivery
system the relative unstability, greater lipophilicity and predictability
of chemistry of the dihydromoieties are exploited. Additionally, since
this method relies on enzymatic activation by an endogenous system (NADH
dehydrogenase) whose substrates closely approximate the delivery compounds,
any toxicity associated with the oxidation should be minimal. In this
system the BBB has not been simply circumvented but rather used as an in
tegral part of the delivery scheme.
Statement of the Problem
The chemical delivery system proposed by Bodor et al. should demon
strate a broad applicability since the agent to be delivered is simply
attached to a particular carrier.124 In certain instances, however, this
system can be simplified. If a pyridinium nucleus isanintegral structural


9
The effects of insulin on the transport of glucose are controversial.2224
Several reports have indicated no effect on either unidirectional or net
flux after insulin infusion. This is curious in that insulin would not be
expected to pass the BBB. The presence of insulin receptors in cerebral
capillaries may explain this enigma in that insulin may bind to a receptor
on the luminal surface and its effects may be mediated to the abluminal
surface by a second messenger.24
Also controversial is the possibility that a low and high affinity
system is operating in glucose transport.202223 There is a nonspecific
flux associated with glucose of 7%. Some authors attribute this to diffu
sion but an alternate hypothesis has been proposed. This involves the pres
ence of two systems on the carrier: a high affinity, low capacity system
and a low affinity, high capacity system. Most data have been collected in
isolated vessel preparation but there is some support of this proposal from
in vivo experiments. Gjedde showed the putative low affinity system to be
stereoselective and to have a Km of 1.0 M,23 compared with a Km of 1.1 mM
for the high affinity system. He contends that a single set of kinetic
parameters does not adequately describe the system and that this high-low
affinity system better correlates with the data. A suggestion that a pro
tein tetrameter with both low and high affinity sites was the carrier has
also been made. At the choriod plexus there appears to be ouiban sensi
tive, Na+ dependent glucose flux and this is apparently important in cere
bral spinal fluid (CSF) homeostasis.22
Three carriers have been described for amino acid transport.11220
These carriers have affinity for neutral, basic, and acidic amino acids.
In general, essential amino acids, which are large and bulky, are trans
ported in preference to nonessential amino acids.20 In all of these


145
Table 3-22. Probit Analysis of the LD50 Study
Dose
mg/Kg
In [Dose]
% Dead
Probit
Berberine
10
2.303
0
17
2.833
0
24
3.178
10
3.718
31
3.434
20
4.150
38
3.637
50
5.000
45
3.807
70
5.524
59
4.078
90
6.282
80
4.382
100
8.719
Dihydroberberine
25
3.219
0
Hydrochloride
33.3
3.506
0
41.6
3.728
10
3.718
50
3.912
30
4.476
58.3
4.066
40
4.747
66.6
4.199
60
5.253
75
4.317
80
5.842
83.3
4.422
90
6.282
100
4.605
100
8.719


70
Figure 3-2. Ultraviolet Spectrum of Dihydroberberine ^2) in 95%
Ethanol


12
increase cerebrally, as in porto-systemic shunts, the uptake of phenyl
alanine and other neutral amino acids increases. Also, if glutamine syn
thetase is inhibited by methionine sulfoximine, the uptake of essential
amino acids is reduced.29 While this is an attractive proposal, several
authors have questioned it on the grounds that many amino acids compete
for this neutral amino acid carrier and the role of glutamine, therefore,
may be relatively unimportant.32
Relatively little work has been done with basic and acidic amino
acids. The acidic system has affinity for glutamate and is fully saturated
at physiological concentrations. Basic amino acids are transported on a
distinct carrier. This carrier is equilibrative, saturable and bidirec
tional and has been described as a Ly+ or lysine-preferring system.
A monocarboxylic acid carrier has been described which demonstrates
affinity for lactate, pyruvate, acetate, propionate, butyrate, 5-aminolevu-
linic acid and ketone bodies.120 Ketone bodies, such as e-hydroxybutyrate
and acetoacetate, are produced in a number of stressful circumstances,
including starvation.33 The carrier possesses similar characteristics to
those which have already been described. It is stereospecific and pH sen
sitive.20 The transport of the substrates on the carrier increases with a
decrease in pH. This has been interpreted to mean either that a proton is
cotransported with the acid or that hydroxide acts as a high affinity com
petitive inhibitor. Only the ionized acid is transported by this system
and this has led to the proposal that an R-NH3+ moiety is present in the
active site. The unionized acid can pass through the membrane by simple
diffusion.
The Km of lactate is similar to the plasma level of lactate. This
being the case, rapid rises in systemic lactate levels, such as the ones
which accompany exercise, are not transferred to the brain immediately.20


Table
3-5. Bond Lengths in Angstroms
Dihydroisoquinoline Model
between Various Atoms
(31)
of the
Isoquinoline Model
(30) and the
Isoquinoline Model (30)
Dihydroisoquinoline Model (31)
Atom
Number
Bond
Length
Atom
Number
Bond
Length
Atom
Number
Bond
Length
Atom
Number
Bond
Length
1-2
1.342
13-14
1.389
1-2
1.451
13-14
1.397
2-3
1.433
14-4
1.433
2-3
1.517
11-18
1.321
3-4
1.467
11-18
1.321
3-4
1 .445
12-17
1.337
4-5
1.444
12-17
1.337
4-5
1.468
2-27
1.132
5-6
1.388
2-26
1 .115
5-6
1.370
2-26
1.130
6-1
1.415
5-19
1.110
6-1
1 .401
5-19
1.110
6-7
1.480
7-20
1.103
6-7
1.485
7-20
1 .103
7-8
1.345
8-21
1 .106
7-8
1.343
8-21
1 .106
8-9
1.487
9-22,23
1.118
8-9
1.489
9-23,22
1 .118
9-10
1.518
10-24,25
1.123
9-10
1 .521
10-24,25
1 .123
10-1
1 .480
13-16
1.106
10-1
1 .452
13-16
1 .106
3-11
1.467
14-15
1.104
3-11
1.444
14-15
1 .104
11-12
1.418
18-27
0.952
11-12
1 .432
18-28
0.951
12-13
1.433
17-28
0.950
12-13
1.417
17-29
0.951
00
--4


14
I l I I
30 60 90 120
Time (min)
Figure 3-29. Efflux of Berberine from the Brain after icv Injection of either 50 yg
of Berberine (1) () or 50 pg of Berberine (1) and 1000 yg of 1-Methyl -
nicotinamide Iodide (6) (A)


142
Table 3-21. In Vivo Metabolism of Berberine and Dihydroberberine in
the Rat (TLC)
TLCa
Compound
Rf of Spots Observed
Berberine
Chloroform Extract
0.15*
0.125x
0.21x
0.34
Isoamyl Alcohol
0.14*
Extract
0.34
Dihydroberberine
Chloroform Extract
0.21*
0.35
Isoamyl Alcohol
0.21*
Extract
0.26x
0.29
0.60
Corresponds to berberine standard
xThese spots were found in blank animals injected with the anesthetic
aFive microliters of the sample were spotted on alumina plates and eluted
with cyclohexane:chloroform:acetic acid 45:45:10


25
structure of the tight junctions is destroyed. Although it has been sug
gested that these deficiencies should render cerebral tumors susceptible
to treatment,93 the clinical evidence does not support this. The therapy
which has, thus far, been directed to brain neoplasms has been disappoint
ing at best. Neurosurgery may have reached its practical limit and most
agree that major new advances must come from the field of chemotherapy.91
It is interesting to note that many agents are effective against certain-
peripheral tumors, but not their secondary brain metastases.
The unresponsiveness of cerebral tumors has been explained by two
phenomena. First, it is known that the greatest display of disintegration
of the capillary structure occurs in the central, slow growing portion of
the tumor, and as one moves to the periphery, the abnormalities decrease.92
This area of the tumor would be expected to show the highest resistance to
chemotherapeutic agents. Secondly, since only a relatively small area of
the brain vasculature is disturbed, any drug which reaches the neoplasm
would rapidly diffuse into the outlying areas, thereby diminishing its
concentration and effectiveness at the tumor site.78 Few suggestions have
been forwarded to circumvent this problem. One involves osmotic opening
of the BBB followed by methotrexate treatment, but this has a very limited
usefulness because of problems with the accompanying edema.78
Many experimental models of brain injury are associated with an in
crease in capillary permeability.45794 These models include injury
induced by dropped weights, pendulums, hammers, spring-mounted weights,
blasting caps, rotary strikers, compressed air guns, humane stunners and
accelerating devices. Generally, these procedures have been applied to
the cat.
*


Figure 3-15. Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of (27) in CDC13


11
efflux of these amino acids from the brain parenchyma or, more probably,
for concentrating them in the endothelial cytosol. The necessity for
this concentration is related to a proposal that the L and A systems may
act together in the transport of amino acids. This hypothesis is based on
a possible equilibration of amino acids between the two carriers in the
cytoplasm of the endothelial cell.26
In many cases the Km value for an amino acid is similar to its plasma
concentrations. This being the case, slight changes in the blood levels
of an amino acid may alter their disposition. The utilization of trypto
phan, for example, which is a precursor of serotonin, is partially deter
mined by its availability and its movement across the BBB.21,28 A similar
situation may exist in certain circumstances with tyrosine, which is the
precursor for dopamine, norepinephrine and epinephrine.
Recently, a hypothesis was forwarded to explain the regulation and
induction of these carriers.29,3,31 It has been suggested that an amino
acid is produced abluminally in large amounts and transported on the neu
tral amino acid carrier. The amino acids in such a system should have a
high Km and, therefore, be easily displaced from the carrier at the luminal
side or in transit. These carriers, like the hexose carriers, exhibit ex
change diffusion. The proposed amino acid in this regulatory role is
glutamine. Glutamine is synthesized from glutamic acid and ammonia by
glutamine synthetase in astrocytes, a perivascular locus. The concentra
tion of glutamine is ten times higher than the concentration of any other
neutral amino acid and its local concentration in the vicinity of the BBB
is predicted to be still higher.30
This hypothesis was formulated on the basis of several interesting
observations. In cases where ammonia and, presumably, glutamine levels


118
Table 3-17. The Effect of Glucose on the Movement of Berberine into
Red Blood Cells
Berberine
Concentration (yg/ml) Plasma3
Time (min)
Control
+ 200 mg% Glucose
5
360.94
352.29
30
300.39
309.04
60
274.95
276.48
90
267.29
255.62
120
259.17
272.92
aThis is a representative experiment selected from a group of three


107
/mole sec. A number of oxidants were investigated to study the oxida
tion of (2). Unfortunately, these produced rates of oxidation which
were far too rapid to analyze by simple means.
The rate of oxidation of (2) in various biological media was also
determined and these results are presented in Table 3-13. Second-order
kinetics were again observed. In brain homogenates the correlation co
efficients were much higher than those obtained in buffer. In the major
ity of the determinations, pseudo first-order rate constants at specific
concentrations were calculated. The values obtained allow a comparison
from system to system. The t^, for example, of oxidation of a 5 x 10-5 M
solution of (2) in plasma, liver homogenate, and brain homogenate was
calculated to be forty-eight minutes, thirty-one minutes, and approximately
twenty-five minutes, respectively. The oxidation of (2) by a second-order
process is very characteristic of many dihydropyridines.
The relative stabilities of dihydropyridines were investigated by com
paring their rates of oxidation in dilute hydrogen peroxide. This system was
reproducible and, as shown in Table 3-14, gave data of good quality. The
t calculated from the pseudo first-order rate constants obtained at 1 xlO4M
2
for (2), 1-methyl-1,4-dihydronicotinamide (21), and 1-benzyl-l,4-dihydro
nicotinamide (22) were 25.6 min, 1.2 min, and 11.5 min, respectively.
These results are consistent with the greater stabilization of (2) compared
to simpler systems, which was predicted by the theoretical calculations.
The rate constants are relatively small because of the slightly acidic nature
of the peroxide. At the end of the experiment, (1) was analyzed to make
sure that nucleophilic addition of the peroxide to (2) did not occur.
The Mechanism of Oxidation of Dihydroberberine
A knowledge of the mechanism of oxidation of dihydropyridine could
be helpful in applying the drug delivery system. The mechanism of oxidation


34
with cytotoxic agents. While increased membrane permeability makes a
compound more effective locally as a cytotoxic agent, there is almost
always a disproportionate rise in systemic toxicity. This has severely
restricted anti cancer prodrugs of this type.
Several types of toxicities are also associated with prodrugs.114
Theoretically, a prodrug should be metabolized only to the parent compound
but the formation of toxic metabolites by the prodrug is possible. This
occurs with such compounds as phenacetin, a prodrug of acetaminophen.
Another possible toxic reaction may be brought about by enzymatic or glu
tathione depletion. The compound thiamine tetrahydrofurfuryl disulfide,
a prodrug of thiamine, requires glutathione-mediated disulfide bond cleav
age for activation and, therefore, toxicity may arise from the associated
depletion of glutathione.
The idea of using prodrugs to increase the specificity of delivery
has been considered. This has proved, however, to be difficult and not
very fruitful. Two basic approaches have been taken in this regard: site-
directed or site-activated delivery and delivery by the association of a
drug with a macromolecular carrier.
The first method does not attempt to concentrate a compound at a par
ticular location, but is based on site-specific activation of the prodrug.
For this to be possible, the enzyme responsible for the activation must
be located specifically, or at least in high relative concentrations in a
particular organ. The finding, for example, that y-glutamyl peptidase is
present in high concentrations in the kidney led to a number of compounds
substituted with the y-glutamyl group.113 Sulfamethiazide and L-DOPA were
derivatized in this manner in order to achieve renal delivery.


2
Blood-Brain Barrier
Structural and Enzymatic Considerations of the Blood-Brain Barrier
The existence of a barrier which separates central nervous tissue
from the general circulation was first postulated by Ehrlich at the end
of the 19th century.12345 In a series of pioneering experiments, he
injected a number of dyes into laboratory animals and found that, while the
visceral organs were highly stained, the brain was conspicuously uncolored.
It was later discovered that these dyes bind extensively to plasma proteins
so that the actual barrier then described was one to these complexes. Many
small hydrophilic compounds cannot, however, pass into the brain so that this
barrier is presented to a wide variety of compounds. Early in the study of
the BBB, the obstruction was considered absolute but this idea was soon dis
pensed with since the nutritional requirements of the brain necessitate
the equilibration of a number of compounds between the general circulation
and the CNS.3
The morphological basis of the BBB had been a very controversial sub
ject until relatively recently. Historically, three hypotheses have been
put forward to explain this impermeability to blood-borne substances.1
All are based on structural differences between the cerebral vascular sys
tem and the systemic circulation. It was proposed that the small extra
cellular space characteristic of mammalian brains prohibited the accumula
tion of compounds and, as such, constituted a barrier. It was shown, how
ever, that some animals with large extracellular spaces have a well-defined
barrier to a number of substances.6 The suggestion was also made that the
general impermeability of the brain to blood-borne substances was due to
astrocytic end feet which surround the capillaries, forming an envelope,
or due to the endothelial cell lining of the cerebral capillaries.1 In
order to study this question, electron microscopic evaluation is necessary.


ABSORBANCE
Figure 3-14. Spectral Changes of Dihydroberberine (2) upon Oxidation to Berberine (1)
in pH 5.8 Phosphate Buffer at 26C. Traces were made every 10 min.


119
the animals were opened, the heart perfused with saline, and the brain
removed. High pressure liquid chromatography was used in analysis.
The initial experiments showed that after administration of 55 mg/Kg
of (1) in dimethyl sulfoxide (DMSO), no (1) could be detected in the brain
at any time. When, however, 55 mg/Kg of (2) in DMSO were administered,
a high concentration of (1) was observed in the brain as shown in Figure
3-18. The free base was not exceptionally stable in solution, however,
and was also very water insoluble. For these reasons the hydrochloride
(3) was prepared and used in subsequent experiments. If 55 mg/Kg of (3)
in 20% aqueous ethanol are injected systemically, the concentration of (1)
in the brain is again found to be relatively high. This is presented in
Figure 3-19. The concentration of (1) achieved in the brain after admin
istration of (2) or (3) was similar (approximately 50 yg/g tissue). The
loss of (1) from the brain after administration of (3) is slow and the
t, of this loss, calculated from the terminal portion of the log concen-
2
tration versus time relation, is approximately eleven hours.
It should be emphasized that in these experiments only the concen
tration of (1) was measured even though unoxidized (2) was present. To
obtain a more complete picture of the behavior of (1) in the brain, the
total berberine concentration, i.e. (1) and (2) was measured and this
appears in Figure 3-20. The concentration of (2) is high at early time
points but diminishes rapidly. The initial rate of disappearance of (2),
obtained by subtracting the concentration of (1) in the brain from the
total concentration (Figure 3-21), yields a tx of thirty-four minutes
2
which is of the same magnitude as the value obtained from brain homogenates.
The slope of the terminal portion of the curve in Figure 3-20 yields a
ti of 5.7 hours.
2


''!*> v^yv hv t ^ ^ ^ 4w *WV>VfjAi^4nvjy^^
cn
Figure 3-6. The 13C Nuclear Magnetic Resonance Spectrum (100 MHz, CDC13) of
Dihydroberberine (2)


Figure 3-5 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of Dihydroberberine in CDC13.
The Insert Represents the Region between 2.8s and 3.46 at 100 MHz


TABLE OF CONTENTS
CHAPTER PAGE
ACKNOWLEDGEMENTS iv
LIST OF TABLES vii
LIST OF FIGURES ix
ABSTRACT xii
1 INTRODUCTION 1
Blood-Brain Barrier 2
Prodrugs and Drug Delivery Systems 31
Statement of the Problem 41
2 MATERIALS AND METHODS 47
Synthesis 49
Characterization of Dihydroberberine 59
Animal Studies 63
3 RESULTS AND DISCUSSION 68
Synthesis and Characterization of Dihydroberberine 68
Theoretical Studies on the Dihydropyridine£Pyridinium
Redox System 81
Further Studies on the Chemical and Biological Properties
of Dihydroberberine 104
In Vivo Studies 116
Conclusions 147
BIBLIOGRAPHY 150
BIOGRAPHICAL SKETCH 160
vi


102
Figure 3-12. A Computer-assisted Drawing of the Most Stable
Conformation of the 1,2-Dihydroisoquinoline
Model (31) at 25C


44
which is reducible to some dihydropyridine species which is stable enough
to be isolated. The compound should be active in vi tro. Since quaternary
compounds are to be considered, the lack of any in vivo activity might be
ascribed to transport or distributional problems. A review of the litera
ture showed two groups of compounds which looked particularly suitable.
These include the substituted benzophenanthridinium salts and the proto-
berbine alkaloids which have the basic skeleton:
benzophenanthridinium ion protoberbine
Members of these groups contain an N-substituted isoquinoline moiety
which can be reduced by a number of agents. A number of these dihydroiso
quinoline compounds are stable.127 These agents show a wide range of
effects including anti neoplastic and antibiotic activity. 128-136 The
Berberine, which has the chemical name 5,6-dihydro-9,10-dimethoxybenz-
[g]-[l,3]benzodioxolo[5,6-a]quinolizinium chloride, has a rather high
in vitro activity against several cancer types including Ehrlich and lym
phoma ascites.131133 Its in vivo action is, however, very low.132133


77
material gave an elemental analysis in good agreement with that predicted
for (2). Also, the reduced material reacted rapidly with such oxidizing
agents as hydrogen peroxide, silver nitrate, and 1,l-diphenyl-2-picryl-
hydrazyl free radical (DPP*) to yield (1). These data support the suc
cessful synthesis of (2).
The hydrochloride of dihydroberberine (3) was synthesized, as illus
trated in Figure 3-1, by treating a concentrated solution of (2), in methy
lene chloride with dry hydrogen chloride (HC1) gas. The hydrochloride
reverts to (2) at pH above 7.0. Analysis of the regenerated material
demonstrated no addition of HC1 or any other nucleophile to the molecule.
This addition is known to occur with several dihydropyridines. The hydro
chloride is many times more soluble in aqueous solutions than the free
base.
If (2) is to be successful in a drug delivery system described in
Figure 1-3, it should demonstrate a greater lipophilicity than (1). Di
hydroberberine is expected to be less polar than (1) because of the loss
of the positive charge. To investigate the relative lipid solubility of
(2) compared to (1), the two compounds were extracted with organic sol
vents and their distribution (partition) coefficients compared.171
The two solvent systems which were used included an octanol-pH 7.4
phosphate buffer and a chloroform-pH 7.4 phosphate buffer. The alcohol-
buffer system was chosen as one of the extracting systems because of its
ability to,in some ways, mimic the partitioning of compounds in vivo.
The data from Table 3-2 show that in both systems (2) has a high affinity
for the organic phase while (1) exhibits a high affinity for the aqueous
phase.
The chemistry of dihydroberberine is largely a result of its enamine
character.172 Dihydroberberine, like all enamines, is in equilibrium with


Table 3-11. A Comparison of the Bond Angles in Degrees of the Pyridine (32) t 1,2-Dihydropyridine (33)
System and the Isoquinoline (30) t Dihydroisoquinoline (31) Model System
Pyridine (32)
Dihydropyridine (33)
Isoquinoline Model (30)
Dihydroisoquinoline Model (31)
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
2-1-6
119.8
2-1-6
124.0
2-1-6
121.6
2-1-6
121.8
3-4-5
119.8
3-4-5
117.9
3-4-5
115.7
3-4-5
116.3
2-3-4
118.2
4-5-6
120.1
4-5-6
125.5
4-5-6
126.4
1-2-3
122.0
1-6-5
121.4
1-6-5
116.2
1-6-5
117.9
8-3-2
120.6
6-1-13
119.0
6-1-10
121 .9
6-1-10
123.4
7-2-3
120.6
1-2-0
123.2
3-2-26
120.4
1-2-26
108.3
1-2-27
108.8
3-4-9
120.1
6-5-11
119.9
4-5-19
117.8
4-5-19
116.3
12-6-5
122.9
1-6-7
117.1
1-6-7
116.5
10-4-5
119.6
6-7-8
123.7
6-7-8
123.6
9-3-4
121.8
11-3-4
118.6
11-3-4
118.7
9-10-24
109.7
9-10-24
108.0
1
9-10-25
109.8
9-10-25
108.6
UD
cn


39
extent of reactivation observed in mice injected with pro-2-PAM shows a
dramatic increase over 2-PAM treated animals.
A further experiment with 2-PAM showed that this small quaternary
salt was rapidly lost from the CNS and this loss was attributed to an ac
tive efflux process.124 These results differ from those obtained by Ross
and Froden who attempted to deliver a quaternary compound to the brain
as its uncyclized aj-haloalkyl amine.125126 They found that the loss of
the quaternary compound was slow (t = 38 hours) and concluded that the
2
efflux of the quaternary was comparable to its influx. This difference
in the rate efflux may be related to differences in molecular size, shape
or charge.
This preliminary work led to a proposed drug delivery system (Figure
1-2) which is specific for the brain and which is generally applicable.124
In this proposal, a pharmacologically active agent, whose ability to pass
the BBB is low, is chemically linked to a pyridinium carrier. This car
rier could be envisioned as a nicotinamide or nicotinic acid ester. This
complex would be reduced under conditions which would yield the dihydro
pyridine. This complex is then injected systemically and,because of the
increased lipophilicity of the dihydropyridine, partitions into the brain
as well as into the periphery. In both locations, oxidation (kox) should
occur. The rate of this oxidation is somewhat controllable by the judi
cious placement of ring substituents on the pyridinium nucleus. Systemi
cally, the charged polar oxidized species should be eliminated rapidly
by the kidney and/or liver (kout2)> while in the brain the compound, be
cause of its charge and size, would be retained i.e., kQUt2 > ^0utT
Also, in both locations, cleavage of the drug from its carrier should
occur (kcleavage)* In the brain, the small nontoxic pyridinium carrier is


Table 3-8. Dihedral Angles between Various Atoms of the Isoquinoline Model (30) and the Dihydroisoquinoline
Model (31)
Isoquinoline Model (30) Dihydroisoquinoline Model (31)
Atom
Number
Dihedral
Angle,0
Atom
Number
Dihedral
Angle,0
Atom
Number
Dihedral
Angle,6
Atom
Number
Dihedral
Angle,6
14-13-12-11
-1.1
11-12-13-16
180.0
14-13-12-11
0.0
14-13-12-17
180.0
13-12-11-3
0.0
14-13-12-17
180.0
13-12-11-3
0.0
13-12-11-18
180.0
12-11-3-4
0.0
13-12-11-18
180.0
12-11-3-4
0.0
8-6-7-20
180.0
11-3-4-5
179.7
1-6-7-20
176.9
11-3-4-5
180.0
9-7-8-21
180.0
3-4-5-6
0.5
6-7-8-21
181.0
3-4-5-6
3.1
10-8-9-22
123.8
4-5-6-1
1.6
10-8-9-22
123.8
4-5-6-1
1.3
10-8-9-23
236.1
5-6-1-2
-2.7
10-8-9-23
236.1
5-6-1-2
-9.1
1-9-10-24
122.6
2-1-6-7
177.4
1-9-10-24
122.6
5-1-6-7
180.0
1-9-10-25
234.9
1-6-7-8
-3.6
1-9-10-25
234.9
1-6-7-8
1.6
6-4-5-19
180.0
6-7-8-9
0.3
3-4-5-19
180.0
6-7-8-9
0.1
13-12-17-29
92.0
2-6-1-10
179.1
13-12-17-28
87.9
2-6-1-10
180.2
3-11-18-28
180.0
12-13-14-15
180.0
3-11-18-27
174.7
12-13-14-15
180.0
3-1-2-26
125.4
4-3-2-26
179.4
11-12-13-16
180.0
3-1-2-27
235.0


55
3-(0ctoxycarbonyl)-l-methylpyridinium Iodide/Octyl N Methylnicotinate
Iodide (18)
Ten grams of (12) (0.043 moles) were mixed with 60 ml of acetone and
with a molar excess of methyl iodide (12.1 g). The solution was allowed
to reflux for two hours at which time the solvent was removed, yielding an
oil which was resistant to crystallization: XH NMR (CDC13, (CD3)oS0) 6 9.9
(2 H, m), 9.0 (1 H, m), 8.6 (1 H, m), 4.6 (3 H, s), 4.4 (2 H, t), 0.92 (3 H,
t); IR (NaCl) v (C-H) 2960, (C=0) 1731, (-C-C(=0)0) 1302, (0-C-C) 1120,
(y CH) 749, (6 ring) 668.
3-(Decoxycarbonyl)-l-methylpyridinium Iodide/Decyl N Methylnicotinate
Iodide (19)
Ten grams of (13) (0.038 moles) were dissolved in 60 ml of acetone
and a molar excess of methyl iodide (10.8 g) was added. The solution was
refluxed for two hours. The solvent was removed, leaving an orange oil:
!H NMR (CDC13, (CD3)2S0) 6 9.7 (2 H, m), 9.0 (1 H, m), 8.5 (1 H, m), 4.8
(3 H, s), 4.4 (2 H, t), 0.95 (3 H, t); IR (NaCl) v (C-H) 2944, (C=0) 1725,
(-C-C(=0)0) 1297, (0-C-C) 1113, (y CH) 741, (g ring) 658.
3-(g-Phenylethoxycarbonyl)-l-methy1pyridinium Iodide/g-Phenethyl N-Methyl-
nicotinate Iodide (20)
Ten grams of (14) (0.044 moles) were dissolved in 60 ml of acetone.
A 1.0 M excess of methyl iodide (12.5 g) was added to the liquid and the
system was allowed to reflux for two hours. The solvent was removed,
yielding a solid which was recrystallized from acetone: XH NMR (CDC13,
(CDo) SO) 6 9.4 (2 H, m), 8.7 (1 H, m), 8.2 (1 H, m), 7.2 (5 H, s), 4.7
(3 H, s), 4.6 (2 H, t), 3.1 (2 H, t); IR (NaCl) v (C-H) 2995 and 3023,
(C=0) 1724, (-C-C(=0)0) 1290, (0-C-C) d 1135 and 1124, (y CH) 731, (g ring)
657.


92
HOMO = 0-449 PzCNi} + -042 pz(C^ '221 pz(C3) *247 Pz(Clt)
+ 0.479 pz(C5) + 0.291 pz(C6) 0.074 pz(C7) 0.174 pz(C8) + 0.013 pz(C9)
+ 0.018 P2(C10) + 0.130 pz(C11) + 0.343 p (C12) + 0.106 PZ(C13) 0.284 pz(C14)
+ 0.005 s(H15) + 0.006 s(H16) 0.085 p (017) 0.079 Pz(018) 0.008 s(H19)
+ 0.008 s(H20) 0.010 s(H21) + 0.041 s(H22) 0.034 s(H23) + 0.113 s(H24)
- 0.082 s(H25) + 0.138 s(H26) 0.179 s(H27) 0.005 s(H28) 0.092 sCH29)
Figure 3-9. The Highest Occupied Molecular Orbital of the Dihydroisoquino
line Model (31)


46
Biochemically, berberine acts to inhibit DNA, RNA and protein syn
thesis.143 It has been suggested that many compounds which are structur
ally similar to berberine exert their cytotoxic activity via alkylation
at the iminium site.128 A more thoroughly studied, and perhaps more
accurate, hypothesis is that berberine acts by intercalating between the
base pairs of DNA. This intercalation can be explained by a modified
neighbor-exclusion model.154 A model which has been used to explain the
intercalation of berberine is one in which the greater portion of rings A,
B and D are intercalated.155 As berberine complexes, it assumes a more
rigid planar species as may be indicated by the increase in fluorescence
quantum yield. This intercalation does, however, slightly bend the double
helix. The unwinding angle of DNA due to intercalation of berberine is
less than for the more planar molecules such as corylene. The effect of
the positive charge on intercalation has also been noted.156
Berberine, therefore, is appropriately suited as a candidate for in
clusion in the drug delivery system described. In order to verify the
original hypothesis of site-specific delivery, a number of experiments
are required. Dihydroberberine must be synthesized and its stability
assayed. The ability of dihydroberberine to penetrate the BBB and concen
trate in the brain must also be demonstrated. Additionally, the toxicity
and anti cancer activity of the agent should be investigated.


147
in increasing the life span (ILS) of the animals compared to a control
group. However, when the leukemia is inoculated intracerebrally, (3) is
significantly more effective in increasing life span than is (1). The
1o ILS actually falls when (1) is administered. This is indicative of
the toxic peripheral effects of (1). These preliminary anticancer studies
again support the original hypothesis. The dihydropyridine is capable of
passing the BBB and oxidizing to (1) where it may exert its cytotoxic
acti vity.
Cone!usions
This dissertation has presented a broadly applicable drug delivery
scheme which is specific for the brain. This delivery method is based on
a dihydropyridine-pyridinium salt redox system and on the multifaceted
nature of the BBB. There are two major aspects of this delivery scheme.
In the first, which is a chemical delivery system, a pyridinium carrier
is attached to a drug molecule. The second, and the one with which this
dissertation has dealt, is a prodrug system in which the carrier moiety
is an integral component of the molecule. This system is simpler than the
first but since the molecule to be delivered must contain a pyridinium par
tial structure, it is less general. In both cases the basis of the brain
specific delivery is related to the greater lipophilicity of dihydropyri-
dines, the ease of their oxidation and subsequent elimination peripherally
and the difficulty with which large pyridinium compounds leave the CNS.
In order to substantiate the proposed method, it was applied to a
salient example, berberine (1). This anti cancer alkaloid contains a pyri
dinium moiety which is reducible and whose product of reduction is stable.
The physical and chemical properties of dihydroberberine (2) were examined.
Its rate of oxidation was found to be rapid in a number of media but not
as rapid as the rate of oxidation of simple dihydropyridines. Dihydroberberine


21
Generalized increases in permeability result in a number of deleteri
ous events. Since the BBB is relatively permeable to water, but not to
most other substances, osmotic gradients can be rapidly changed. If plas
ma proteins and other compounds are allowed to freely enter the CNS, they
will bring with them large amounts of water, with the result being cere
bral edema.120
The morphological basis of these changes in any particular situation
can be highly controversial. This is especially the case with hypertonic
treatment of cerebral capillaries. It has been known for some time that
hypertonic solutions of such solutes as glucose, sucrose, urea, arabinose,
lactamide and several others can increase the permeability of the cerebral
capillaries to protein and other small polar compounds. Rapoport explains
this phenomenon as the result of osmotic shrinkage of the endothelial cells,
resulting in a pulling apart of the tight junctions.571 This has been
challenged. If HRP is injected after a hypertonic solution, the HRP reac
tion product does not form a continuous line from the luminal to the ablu-
minal surfaces at the junction or at any other location.420 It was sug
gested, therefore, that increased vesicular transport accounted for the
increased permeability, perhaps as a result of increases in local blood
pressure.77
In any case, if the concentration of the hypertonic solution is close
to the critical opening concentration, the opening of the BBB is transient
and does not produce acute edema.5 This procedure may have therapeutic
applications. In many cases it is desirable to introduce highly polar
compounds into the brain and this osmotic opening of the BBB may provide
an avenue for that purpose. Methotrexate is a folate inhibitor used in the
treatment of cancer. The pKa of the carboxylic acid functions of this


DOSE/GRAM TISSUE
3.5
TIME (MIN)
Figure 3-23. Distribution of Berberine after i v Administration of 55 mg/Kg of Dihydrober-
berine Hydrochloride (3) into the Kidney (<>) > Liver (), Lung (O), and
Brain (A)
ro
si
fj.g BERBERINE/GRAM TISSUE


TO MOTHER


82
the model, 3H,4H-dihydro-7,8-dihydroxybenzo[b]quinolizinium ion (30) and
3H,4H,6H-trihydro-7,8-dihydroxybenzo[b]quinolizine (31) along with their
numbering protocol, are shown in Figure 3-8.
This quaternary and its dihydro adduct, while simpler than the
(1) t (2) system, do not differ greatly in structure or in general chem
istry. Compounds (30) and (31) have not been investigated and no mention
of them has appeared in the literature. Some theoretical work on the
unsubstituted benzo[b]quinolizinium ion and much on the isoquinoline mole
cule has been published. The benzo[b]quinolizinium ion was investigated
by Galasso in 1968 and charge densities and Tr-bond orders were reported.177
All of these reported studies have employed simplistic theoretical treat
ments such as PPP,178 IOC-w-technique,179 HMO,180 SSP,181 and CNDO/2.182
The corresponding dihydrocompounds have not been studied.
In order to investigate the isoquinoline model (30) t dihydroisoquino
line model (31) system, a MIND0/3 method was selected.183 This approach
is a semi-empirical self-consistent field molecular orbital method in which
all valence electrons are treated. The MIND0/3 program is the culmination
of the series MINDO,184 MIND0/2,185,186 and MIND0/2'.187 This program was
developed to solve chemical problems quickly and efficiently using a quan
tum mechanical framework. Unlike the methods of Pople, whose major aim
was to reproduce nonempirical calculations, M.J.S. Dewar parametrized
MINDO so that the system would produce useful and chemically accurate data.
This program is extremely versatile and has been applied to a number of
chemical problems including reactions. Only the most cursory details of
MINDO/3 are appropriate here, as the subject is well reviewed elsewhere.183188
In quantum mechanical approaches like MINDO, a molecular orbital is
the result of the interaction of a wavefunction of an electron with nuclei
and other electrons present in the molecule.189 Mathematically, the


76
Table 3-2. Carbon Assignments of the 13C NMR of Dihydroberberine (2)
Carbon
Number
PPM (5)
Carbon
Number
PPM (6)
1
150.301
11
111 .457
2
147.182
12
107.753
3
146.597
13
103.659
4
144.452
14
100.881
5
141.528
15
96.202
6
128.661
16
60.576
7
128.466
17
55.848
8
124.470
18
49.270
9
122.033
19
48.929
10
118.670
20
29.725
(CDC13: 78.267, 77.040, 75.732)


31
induced by starvation. The utilization of the surrogate food sources is
limited by BBB transit.
In starvation, the monocarboxylic acid carrier is said to be induced,
although this is controversial. A recent paper demonstrated a lower Km
and Vmax for carriers in starved animals compared with control animals.33
The increased flux of B-hydroxybutyrate and other substrates was attributed
in this article to an increase in the diffusional component.
A number of therapeutic agents have affinity for these carriers. These
include such anionic compounds as probenicid, penicillin, and aspirin.20
This affinity could potentially lead to competition and a decreased ability
to eliminate metabolic acids from the CNS. It has been shown, however,
that the concentrations required to cause inhibition are far above thera
peutic levels.
The BBB is a complex system of enzymes, protein carriers, and vessels
which impart to the brain a selective interface. This interface prevents
potentially damaging substances from entering the brain without impeding
the entry of nutrients or the exit or metabolites and excretory products.
In adverse circumstances, the permeability of the barrier can be increased,
resulting in a number of deleterious effects.
Prodrugs and Drug Delivery Systems
The BBB excludes a number of pharmacologically active agents and, as
such, treatment of many cerebral diseases is severely limited. In order
to increase the effectiveness of drugs which are active against central
maladies, the pharmacokinetic profile of the agent must be augmented and,
specifically, the transit time of the drug in the brain must be increased.
If a method were available to implement these alterations, an agent could
be delivered specifically to the brain. This specificity should increase


81
Theoretical Studies on the
Pi hydropyridine £ Pyridinium Redox System
If the drug delivery system described in Figure 1-3 is to be used to
its potential, then, a knowledge of its basic chemistry is required. The
reason for this is not only to understand the subtle chemistry inherent
in this particular system, but also to allow prediction, extrapolation
and generalization of the system to other examples. The ability to dis
cern, for instance, those factors that add to or detract from the stability
of a molecule would allow attenuation of a particular property by mole
cular manipulation. A thorough chemical knowledge of a particular com
pound would also allow an intelligent prediction as to its suitability
to the scheme.
While a few ionization potentials have been measured, the general in
stability of dihydropyridines often precludes their investigation by ex
perimental means. Because of this limitation, these compounds have lent
themselves well to theoretical study. While the dihydronicotinamides,
because of their biological relevance, have been the subject of copious
reports, larger dihydro systems have received little attention.175176
In order to gain a greater chemical insight into the proposed drug deliv
ery system in general, and the berberine (1) Z dihydroberberine (2) sys
tem in particular, a theoretical investigation was undertaken.
Berberine is a rather large molecule and, as such, would be expected
to present problems in terms of computational time. It must be remembered
that 3N-6 (where N is the number of atoms) independent variables are re
quired for a molecule and large molecules can easily cost $8000-$10000
in computer time. Because of this, and also in an attempt to generalize
the calculations so that they would be applicable to a number of compounds,
a model was developed for the (1) Z (2) system. The compounds chosen as


17
these areas are termed the circumventricular organ. In addition, the
pineal gland lacks a BBB. These areas constitute a small fraction of the
total surface area of the BBB and may allow a limited nonspecific flux.
These loci do have important pharmacological ramifications as demon
strated by the action of a series of atypical neuroleptics.5657 These
compounds are so named because they provoke certain symptoms of dopaminer
gic blockade but not others. Specifically, metoclopramide exerts an anti
emetic action but not an anti schizophrenic effect. This dichotomy was
explained by the fact that metoclopramide does not penetrate the BBB. The
site of action of antiemetic agents is at the chemoreceptive trigger zone,
which is located in the area postrema and is outside the BBB. The apparent
inability of metoclopramide to produce the full spectrum of changes which
occurs as a consequence of dopaminergic blockade may be related to its
pharmacokinetics and, specifically, its inability to pass the BBB. This
relegates the compound to only those sites of action not protected by the
BBB. Other pharmacologically important sites outside the BBB include the
median eminence, which controls prolactin secretion. In some areas, while
the BBB is not absent it is diminished. These areas include certain ar-
teriolar segments whose diameters are between 15-30 ym. In these
segments limited protein extravasation, or leakage of compounds from the
lumen of the capillary to the extracellular spaces, has been observed.
The ability of small peptides to penetrate the BBB is an extremely
controversial point.21606162 Systemic administration of certain cen
trally active peptides elicits a central response. Differences concerning
the interpretation of these data are significant. One view is that the flux
of these small peptides across the BBB is low indicating, perhaps, some
nonspecific route accounts for their entry.2162 The presence of peptidyl


66
CHC13- The aqueous layer was then extracted with 3-methyl-1-butanol.
The organic layers were reduced to dryness and then reconstituted with
a small volume (50 yl) of methanol. This residue was used for HPLC and
TLC analysis. Five microliters of each sample were analyzed by HPLC.
A yBondapak Cis reverse-phase column and a mobile phase of 50:50 aceto
nitrile: pH 6.2 phosphate buffer were used. The TLC analysis consisted
of spotting 5 yl of each sample on an alumina plate and eluting the sys
tem with cyclohexane: chloroform: acetic acid 45:45:10 or methanol. The
plates were developed with iodine vapor or iodoplatinate spray reagent.
Toxicity
White CD-I mice were employed in this study, the average mass of
which was 22.6 2 g. The mice were segregated into groups of 10, and
8-10 groups were used in each study. The doses given were determined by
preliminary studies in which the LDo and the LDioo were obtained using
small groups of animals. The doses were then prepared in equal incre
ments between the two extremes, but there was additional emphasis placed
at the lower end of the curve. Since this study was concerned with acute
toxicity, the animals were injected intraperitoneally, and the number dead
recorded after twenty-four hours. The groups were, however, observed an
additional forty-eight hours to ensure an accurate appraisal of acute
toxicity. Food and water were given ad libitum. The injection volume was
75-100 yl. The data were analyzed by fitting them to a sigmoid curve and
by the method of Probits.
Anti cancer Activity
Male BDF mice (20.6 0.3 g) were used in this study. A suspension
of P388 lymphocytic leukemia cells was injected intraperitoneally (ip)
(1 x 106 cells) or intracerebrally (2.5 x 105 cells). The survival time
of animals treated with various doses of (1) or (3) compared to the controls


91
134.40
kcal/mole
Anti
Syn
134.42
13S.20
2-PAM
1,6-dihydro-2-PAM


Table 3-23. Effect of Berberine (1) and Dihydroberberine Hydrochloride (3) Against P388 Lymphocytic
Leukemia
P388 i.p.
P388 intracerebrally
Drug
Dose
mg/Kg
Number
of mice
Surv. Time
(days)
%ILS
Surv. Time
(days)
%ILS
Berberine (1)
5
6
11.17
.40
10.8
9.83
.17
1.7
10
6
11.33
.42
12.4
9.50
.22
-1.8
20
6
10.50
.50
4.2
8.67
.56
-10.3
Dihydroberberine
5
6
10.83
.17
7.4
10.50
1 .31
8.6
Hydrochloride (3)
10
6
11.00
.30
9.1
10.00
.37
3.4
20
6
11.17
.17
10.8
11.33
1.63
17.2
Vehicle
5 (ml/Kg)
12
10.08 .15
9.67 .14


CHAPTER 3
RESULTS AND DISCUSSION
Synthesis and Characterization of Dihydroberberine
The initial step in the application of the proposed drug delivery
system to berberine (1) is the preparation of its dihydro adduct. The
first synthesis of dihydroberberine (2) involved disproportionation of
(1) in strong base and was performed by Gadamer in 1905.157,158 The
mechanism of this reaction involves nucleophilic attack of hydroxide to
the carbon adjacent to the nitrogen resulting in the formation of a tran
sient amino alcohol. This intermediate collapses to oxyberberine and di
hydroberberine, presumably via a hydride transfer. Several other syntheses
for (2) have appeared in the literature and these involve the direct re
duction of (1) by zinc amalgam,159160 complex metal hydrides161162 or
sodium borohydride.163164 Historically, (2) has been of interest because
of its spectroscopic properties,165-167 and as an intermediate in certain
synthetic schemes.164 The use of (2) as a drug or in a drug delivery
system is novel to this thesis.
In the present work (1) was reduced, as shown in Figure 3-1, by sodi
um borohydride in dry pyridine. Spectroscopic analysis showed that the
yellow crystalline material produced was (2). The UV, IR, and MS are shown
in Figures 3-2, 3-3, and 3-4 respectively, and are consistent with the
assigned structure.167-169 The XH NMR is presented in Figure 3-5, and the
proton assignments in Table 3-1. The 13C NMR is shown in Figure 3-6, and
the corresponding carbon assignments in Table 3-2. These assignments were
made by comparing (2) to a number of model systems.170 The synthesized
68


6
Figure 1-1. This Schematic Illustration Represents an Endothelial Cell
Derived from either a Muscle (ECm) or Brain (EC^) Capillary.
In this Figure, (ma) is the Macula Adherens or Loose Junction,
(zo) is the Zona Occludens or Tight Junction, (mv) are Micro
vesicles, and (bl) is the Basal Lamina. This Figure was Modi
fied from Reference 1, Page 162 by Permission.


109
of simple dihydropyridines, particularly dihydronicotinamides, has been
extensively studied since these partial structures occur in the NADHJNAD+
system. Many models of this system have been used and most are simple,
substituted 1,4-dihydronicotinamides.
In the classic work of Abeles and Westheimer, the oxidation of sub
stituted 1,4-dihydronicotinamides by thiobenzophenones was studied.194
Like most dihydropyridines, these exhibit second-order kinetics: first-
order with respect to the dihydropyridine and first-order with respect
to the thiobenzophenone. The mechanism of oxidation proposed by this
group was that of a concerted hydride transfer from the dihydronicotin
amide to the thiocarbonyl carbon. This mechanism has been modified over
the years.195"197 Most recently, Ohno has described a system in which the
oxidation proceeds through a charge transfer complex.198199 The initial
step in this process is an electron transfer, followed by a proton trans
fer followed, in turn, by a subsequent electron transfer. In free radi
cal oxidations Eisner, using substituted 1,4-dihydronicotinamides and the
oxidant, diphenyl pi cryIhydrazyl free radical (DPP*), again found a second-
order oxidative process with the rate-determining step being the initial
abstraction of a hydrogen.200 This is followed mechanistically by the
formation of the quaternary compound. The oxidation of other dihydropy-
ridines, such as the free radical oxidation of dihydroanthracene or dihy-
drophenanthrene, has also been reported.201>202 The kinetics of enzymatic
oxidation of dihydronicotinamides have also been studied. In 1980 Porter
and Bright published an article on the oxidation of substituted 1,4-dihy-
dronicotinamides by lumiflavins and old yellow enzyme.203 Kinetically,
a second-order oxidation was found to take place, mediated by a charge
transfer or biradical complex.


49
Synthesis
Dihydroberberine (2)
Seven grams of berberine chloride (1) were dried over phosphorous
pentoxide at 80C in a vacuum oven for eight hours. Five grams of the
dried salt (0.013 moles) were added to a suspension of dry pyridine and
0.6 g of sodium borohydride (NaBh^). The solution was stirred under
for twenty minutes at room temperature, at which time 0.5 g more of
NaBH4 were added. The liquid was then poured into 800 ml of ice water.
The ensuing precipitate was dried overnight at 50C over phosphorous pen
toxide. The crude material was recrystallized from benzene-petroleum
ether (low boiling). The yield was 35%: Melting point 156-158C, Litera
ture 157-159C; XH NMR (CDC13) 7.1 (1 H, s), 6.7 (2 H, s), 6.4 (1 H, s),
5.9 (3 H, s), 4.3 (2 H, s), 3.4 (6 H, s), 3.1 (2 H, t), 2.9 (2 H, t);
MS m/e 338 (M+ + 1) 22%, 337 (M+) 100%, 322 (M+ 15) 27%, 278 (M+ 59)
13%; UV (95% ethanol) 370 xmax; IR (KBr) v (C-H) 3005 and 2970, (C-C, C-N)
1600 and 1482, (C-0-C asym) 1227 and 1270, (C-0-C sym) 1028 and 1083;
13C NMR (see figure 3-6); Elemental analysis calculated %: C, 71.21;
H, 5.60; N, 4.15. Found %: C, 71.19; H, 5.70; N, 4.14.
Pi hydroberberine Hydrochloride (3)
One gram of (2) was dissolved in a minimal amount of CH2C12. Anhy
drous hydrogen chloride, produced by dropping concentrated sulfuric acid
(H2S04) on sodium chloride (NaCl), was bubbled through the solution yield
ing a yellow precipitate. The material was recrystallized in aqueous
ethanol.
9-Demethylberberine (Berberrubin) (4)
Seven grams of (1) were dried for two hours at 100C in a vacuum oven
and then were heated to 200C for an additional thirty minutes. The deep
purple material produced was dissolved in hot water and extracted with


64
organ homogenate was constructed. Analysis was performed by injecting
5 yl of the supernatant onto a yBondapak Cis reverse-phase column with a
mobile phase of 60:40 acetonitrile: phosphate buffer. The flow was 2 ml/
min. Under these conditions, the retention time of (1) was 3.8 min and
(2), 9.3 min.
Slow Infusion of Dihydroberberine Hydrochloride (3)
Rats were anesthetized and prepared as above. A dose of 55 mg/Kg of
(3) was prepared in a volume of 1.0 ml. The vehicle was 20% aqueous etha
nol. This dose was infused into the external jugular vein over a period
of either thirty or forty-five minutes. At the end of the perfusion, the
animals were decapitated, their organs collected and analyzed.
Effect of 1-Methyl-1,4-dihydronicotinamide (21) on the Efflux of Berberine
from the Brain
Rats were anesthetized and prepared as above. Animals were injected
with 200 mg/Kg of (21) in aqueous ethanol intravenously. After fifteen
minutes, the standard dose of 55 mg/Kg of (3) was given. The animals were
sacrificed at various times after the injection, selected organs were re
moved and homogenized, and the samples analyzed by HPLC.
In Vivo Characterization of l-Benzyl-1,4-dihydronicotinamide (22)
Rats were anesthetized and cut down as above. Doses between 60 mg/Kg
and 400 mg/Kg of (22) were administered. At various times after the in
jection, the animals were perfused, decapitated, and the brains removed.
The brains were then frozen in liquid nitrogen and stored at 0C until
they were analyzed, at which time the brains were thawed, homogenized
in 2 ml of water, and extracted with 8 ml of acetonitrile.
Analysis was made by HPLC using a yBondapak C18 reverse-phase column
and a mobile phase of 40:60 acetonitrile: 1 x 10"3 M sodium heptanesul-
fonate. The dihydronicotinamide had a retention time of 3.4 min and the


of dihydroberberine which was accomplished using sodium borohydride in
pyridine. The dihydroberberine was analyzed by various spectroscopic means
and a number of physical properties were measured. Theoretical calcula
tions using a MINDO/3 approach were also undertaken.
In order to verify the proposed scheme, the delivery of berberine to
the brain and the retention of berberine in the brain had to be shown.
When dihydroberberine or its hydrochloride salt were injected systemi-
cally, high levels of berberine were found in the brain and its efflux
from the brain was slow. If, however, berberine is injected systemically,
no detectable levels are found in the brain. When dihydroberberine is
slowly infused the concentration of berberine rises in the brain and at
forty-five minutes, this concentration is specifically higher in the brain
than in any other organ analyzed. The mechanism of efflux of berberine
from the brain was investigated and appears to be mediated by a passive
process, perhaps the bulk flow of cerebral spinal fluid. Dihydroberberine
was found to be less toxic than berberine. Preliminary anticancer data
indicate that dihydroberberine is more effective in increasing the life
span of animals injected intercerebrally with P388 lymphocytic leukemia
than is berberine.
xi i i


20
binding to albumin is sufficiently weak that the capillary transit time
in the brain is long enough to allow dissociation of the compound from
the macromolecule. In the case of globulins, however, the binding is
tighter and the turnover is only of the order of 3-10%/second. The sojourn
through the cerebral capillaries is not, therefore, sufficient to release
the bound material. It is not, therefore, the plasma-protein-bound frac
tion which is unavailable for transport but, rather, the globulin-bound
fraction.
These principles also apply to free fatty acids such as palmitate.
In this case, however, there appear to be high and low affinity sites on
the albumin molecule so that the rate of dissociation of the palmitate
bound to the site with the lower Km may be slow compared with capillary
time, while the low affinity site may not.74 Melatonin, which is also
highly protein bound, shows a significant diffusion through the BBB.75
Thyroid hormones which are transported into the CNS by carriers are
also bound by plasma proteins.3637 The carrier, whose Km is lower than
that of the albumin site, is able to compete successfully with the albumin
for binding. This is effectively a stripping of the compound from its
albumin site. The effect is also seen with tryptophan and other amino
acids.28
The BBB in Pathological and Experimentally Altered States
The integrity of the BBB is known to be impaired in a number of patho
logical or experimentally-induced conditions.47677 The effect produced
can be the result of changes of the structural components of cerebral capil
laries such as the junction or vesicular activity and, as such, results
in generalized increases in permeability. Alternatively, the carrier sys
tems may be compromised and this may lead to specific changes in perme-
ability.


36
While these compounds have demonstrated some promise as anti cancer
drugs, for the most part they have not lived up to their potential. There
are a number of reasons for this failing. The chemical manipulation of
these agents may act to decrease their accessibility to a site of action.
Additionally, if the agent is fairly lipophilic, it may "leak" from its
site of action before exerting a pharmacological effect.113 In cancers
there may also be diffusion limitations because of the restrictions in
blood flow.
A second approach for increasing the specificity of an agent for a
particular organ involves the coupling of a pharmacologically active agent
with a macromolecule.116 The specificity derived from such a system is
related to the interaction between cells and endogenous and exogenous bio
polymers and macromolecules. Albumin, for example, is actively endocytosed
by various macrophages.117 An anti cancer compound could be coupled to al
bumin and the complex taken up by a macrophage tumor. This would then be
directed to a lysosome where the drug would be hydrolyzed from its albumin
carrier and exert its pharmacological action specifically. This has also
been suggested as a means of treating DNA viruses which implant in macro
phages. Anthracyclines, such as daunorubicin and adriamycin, intercalate
into DNA. A drug carrier has been devised in which fragments of DNA con
taining intercalated anthracyclines are administered systemically.114118
This intercalated complex is inactive but can be endocytosed and broken
down in lysosomes by DNAases. Again, the aim is to release the cytotoxic
agent in the vicinity of the malignancy and thereby increase its efficacy.
Antibodies have also been studied as specific delivery carriers, but re
search has been hampered by the inhomogeneity of tumor-specific antibodies.


Figure 3-11. A Computer-assisted Drawing of the Most Stable Conformation of the
Isoquinoline Model (30) at 25C. This View is Oriented so that the
Interatomic Axis between Atoms 26 and 2 is Perpendicular to the Plane
of the Page
o
o


13
A carrier for choline was described in 1978.34 Choline cannot be
synthesized de novo in the brain but this precursor is required for such
important cellular components as acetylcholine and phosphatidylcholine.
The carrier conforms generally to those characteristics specified for other
systems. It has affinity for choline, hemicholinium, dimethyl ami noethanol
(deanol), tetraethyl ammonium, tetramethylammonium, cartitine and spermine
but not for NH4+. The distribution of this carrier is not uniform. Choline
uptake decreases with age and this correlates with an age-dependent diminu
tion of the carrier. The rate determining step in choline utilization is
its movement across the BBB.
A carrier with affinity for nucleosides such as adenosine, guanosine
and inosine has been described.120 Additionally, a system for transport
ing purine bases has been isolated. This system transports adenine, guanine
and hypoxanthine but not pyrimidines. This is interesting in light of the
fact that pyrimidines can be synthesized in the brain from NH4+ and aspar
tate, while purines cannot. This carrier is very active in neonates, but
its activity diminishes with age.35
Recently, a carrier was described for thyroid hormones.3637 The
carrier has affinity for triiodotyrosine (T3) and thyroxine (T4) but not
for tyrosine, leucine or potassium iodide. The transport of T3 is satur
able and inhibited by T4. The carrier is weakly stereospecific and of high
affinity. The presence of a carrier for T3 and T4 is interesting because
these compounds are fairly lipophilic. The bulkiness of the molecule,
however, tends to decrease its ability to pass membranes. A carrier-
mediated transport has also been proposed for thiamine.38 These systems
are summarized in Table 1-1.


140
Table 3-20. In Vivo Metabolism of Berberine and Dihydroberberine in the Rat
(HPLC)
HPLCa
Retention time of Peak Height
Compound Peaks Observed (min) Relative to Berberine
Berberine
Chloroform Extract
1.8X
2.0X
2.5X
2.9
4.2*
5.6
0.73
0.44
0.39
0.05
1.0
0.01
Isoamyl Alcohol 1.7X
Extract
2.5X
3.6

4.0
4.8
Pi hydro berberine
Chloroform Extract 1.8X
2.1X
2.6X
3.0

4.2
4.7
1.4
0.28
1.0
0.09
0.52
0.37
0.35
0.04
1.0
5.6
0.01


LIST OF TABLES
TABLE PAGE
1-1 Blood Brain Barrier Transport Systems 14
3-1 Proton Assignments of the 1H NMR of Dihydroberberine (2) 74
3-2 Carbon Assignments of the 13C NMR of Dihydroberberine (2) 76
3-3 Distribution Coefficients for Berberine (1) and Dihydrober
berine Hydrochloride (3) in Chloroform/pH 7.4 Buffer and in
1-Octanol/pH 7.4 Buffer 78
3-4 The Heats of Formation, Vertical Ionization Potentials, and
Dipole Moments of the Isoquinoline Model (30) and the Dihydro-
isoquinoline Model (31) 86
3-5 Bond Lengths in Angstroms between Various Atoms of the Iso
quinoline Model (30) and the Dihydroisoquinoline Model (31).... 87
3-6 Bond Angles in Degrees between Various Atoms of the Isoquino
line Model (30) and the Dihydroisoquinoline Model (31) 88
3-7 Charge Density at Various Atoms of the Isoquinoline Model (30)
and the Di hydroisoquinoline Model (31) 89
3-8 Dihedral Angles between Various Atoms of the Isoquinoline Model
(30) and the Dihydroisoquinol ine Model (31) 90
3-9 Differences in the Heats of Formations (AAHf) of (30) X (31),
2-PAM t 1,4-Dihydro-2-PAM and 2-PAM X 1,6-Dihydro-2-PAM 91
3-10 A Comparison of Bond Lengths in Angstroms of the Pyridine (32)X
1,2-Dihydropyridine (33) System and the Isoquinoline (30) %
Dihydroisoquinol ine (31) Model System 94
3-11 A Comparison of the Bond Angles in Degrees of the Pyridine (32)i
1,2-Dihydropyridine (33) System and the Isoquinoline (30) X
Dihydroisoquinol ine (31) Model System 96
3-12 A Comparison of the Atomic Charge Densities of the Pyridine (32)t
1,2-Dihydropyridine (33) System and the Isoquinoline (30) X
Dihydroisoquinol ine (31) Model System 97
3-13 The Rate of Oxidation of Dihydroberberine in Various Media 105


o
en
Figure 3-1. Synthesis of Dihydroberberine (2) and its Hydrochloride Salt (3).


no
The mechanism of oxidation of dihydropyridines is, therefore, depen
dent on the oxidant and the conditions under which oxidation takes place.
A series of experiments was performed to investigate the mechanism of oxi
dation of simple dihydronicotinates and (2), and to determine if the oxi
dation of the dihydropyridines is mediated by an enzyme or by some other
species such as dissolved oxygen.
In these experiments a homologous series of 1-methyl-1,4-dihydronico-
tinic acid esters was synthesized. The NMR of a representative compound
is presented in Figure 3-15, and the corresponding proton assignments in
Table 3-15. The rate of oxidation of (22), (23), (24), (25), (26), (27),
and (28) in 40% human plasma, 6% brain homogenate, or 3.5% liver homogenate
was measured. This was done by determining the rate of disappearance of
the 359 nm absorption of the dihydronicotinamide with time. Since the
rate of ester hydrolysis is slower for nicotinic acid esters and much
slower for 1-methylnicotinic acid esters than the values obtained, the
results clearly represent the oxidation process of the dihydropyridine
and not hydrolysis. In all determinations the concentration of the dihy-
dropyridines was 5 x 105 M. The results of this experiment are shown
in Figure 3-16 and Table 3-16. Both in plasma and in buffer, the rate
of oxidation as measured by the pseudo first-order rate constant, is rela
tively slow and the correlation coefficients are relatively small. There
is also little effect on the rate constants by the molecular structure.
This indicates a nonspecific oxidative route. In organ homogenates, there
is a marked acceleration in the rate of oxidation as well as an increase
in the correlation coefficients. There is also a large dependence upon
the rate by the structure of the molecule and, in general, as the chain
length increases, the rate decreases. These three changes acceleration
of the rate, linearization of the data, and the greater reliance of the


Figure 3-13. A Computer-assisted Drawing of the Most Stable Conformation of the 1,2-Dihydro-
isoquinoline Model (31) at 25C. This View is Oriented so that an Imaginary
Axis between Atoms 2 and 5 is Perpendicular to the Plane of the Page
o
CJ


152
44. H. Davson and M. Pollay, J_. Physiol., 167, 239 (1963).
45. L. S. Schanker, L. D. Prockop, J. Schov and P. Sisodia, Life Sci.,
515 (1962).
46. L. S. Schanker, Antimicrob. Ag. Chemother., 1044 (1965).
47. W. H. Oldendorf and W. J. Brown, Proc. Soc. Ex£. Biol. Med., 149,
736 (1975).
48. W. Oldendorf, L. Braun and E. Cornford, Stroke, 10, 577 (1979).
49. B. B. Brodie, H. Kurz and L. S. Schanker, i. Pharmacol. Exp. Ther.,
130, 20 (1960).
50.V. A. Levin, J. Med. Chem., 23, 682 (1980).
51. J. E. Hardebo and B. Nilsson, Acta Physiol. Scand., 107, 153 (1979).
52. W. H. Oldendorf, L. Braun, S. Hyman and S. Z. Oldendorf, Science,
178, 984 (1972).
53.J. M. Cruickshank, G. Neil-Dwyer, M. M. Cameron and J. McAinsh, Clin.
Sci., 59, 453s (1980).
54. E. Westergaard, Adv. Neurol., 28, 55 (1980).
55. C. W. Wilson and B. B. Brodie, J. Pharmacol. Exp. Ther., 133, 332
(1961).
56. J. M. Jacobs, Environ. Health Per., 26, 107 (1978).
57. L. J. Herberg and T. B. Wishart, Pharmacol. Biochem. Behav., 12,
871 (1980).
58. E. Westergaard and M. W. Brightman, Comp. Neur., 152, 17 (1974).
59. C. K. Petito and D. E. Levy, Lab Invest., 43, 262 (1980).
60.S. I. Rapoport, W. A. Klee, K. D. Pattiqrew and K. Ohno, Science,
207, 84 (1980).
61. A. J. Kastin, C. Nissen, A. V. Schally and D. H. Coy, Pharmacol.
Biochem. Behav., 11 717 (1979).
62. A. Kastin, C. Nissen, D. H. Coy, Pharmacol. Biochem. Behav., 15,
955 (1981).
63. R. Sankar, F. R. Domer and A. J. Kastin, Peptides, 2, 345 (1981).
64. M. L. Friis, 0. B. Paulson and M. M. Hertz, Microvas. Res., 20,
71 (1980).


Page
Figure
3-11 A Computer-assisted Drawing of the Most Stable Conformation
of the Isoquinoline Model (30) at 25C. This View is Oriented
so that the Interatomic Axis between Atoms 26 and 2 is Perpen
dicular to the Plane of the Page 100
3-12 A Computer-assisted Drawing of the Most Stable Conformation
of the 1,2-Dihydroisoquinoline Model (31) at 25C 102
3-13 A Computer-assisted Drawing of the Most Stable Conformation
of the 1,2-Dihydroisoquinoline Model (31) at 25C. This View
is Oriented so that an Imaginary Axis between Atoms 2 and 5
is Perpendicular to the Plane of the Page 103
3-14 Spectral Changes of Dihydroberberine (2) upon Oxidation to
Berberine (1) in pH 5.8 Phosphate Buffer at 26C. Traces
were made every 10 Min 106
3-15 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of (27)
in CDC1 Ill
3-16 The Rates of Oxidation of Various 1-Methyl-1,4-dihydronicotinic
Acid Esters (23), (24), (25), (26), (27), (28) and 1-Benzyl-
1,4-Dihydronicotinamide (22) at 37C in 40% Human Plasma (),
6% Brain Homogenate (A) and 3.5% Liver Homogenate () 113
3-17 Partitioning of 26.5 mg of Berberine (1) from Plasma (A) into
Red Blood Cells (A) and of 26.5 mg of Dihydroberberine Hydro
chloride (3) from Plasma (O) into Red Blood Cells (). The
Volume of Blood Used in each Experiment was 75 ml 117
3-18 Distribution of Berberine in the Brain after iv Administration
of Berberine (1) (A) at a Dose of 55 mg/Kg or of Dihydrober
berine Free Base (2) () at a Dose of 55 mg/Kg 120
3-19 Efflux of Berberine from the Brain after iv Administration of
either 55 mg/Kg of Dihydroberberine Hydrochloride (3) ()
or 55 mg/Kg of Berberine (1) (A). Analysis was for (1) only
and not Unoxidized (2) 121
3-20 Efflux of Berberine (1) and Unoxidized Dihydroberberine (2)
(A) after iv Administration of 55 mg/Kg of Dihydroberberine
Hydrochloride (3) 122
3-21 A Comparison of the Efflux of Berberine (1) () and Berberine
(1) and Unoxidized Dihydroberberine (2) (A) after a Dose of
55 mg/Kg of Dihydroberberine Hydrochloride (3) Administered
iv 123
3-22 Distribution of Berberine after iv Administration of 35 mg/Kg
of Berberine (1) into the Kidney (Ah Liver (), Lung (#),
and Brain (A) 125
x


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE APPLICATION OF A DIHYDROPYRIDINE-PYRIDINIUM SALT
REDOX SYSTEM TO DRUG DELIVERY TO THE BRAIN
By
MARCUS ELI BREWSTER III
AUGUST 1982
Chairman: Nicholas S. Bodor
Major Department: Medicinal Chemistry
This work has been concerned with the design and testing of a broadly
applicable drug delivery system which is specific for the brain. The
method developed for this purpose is based on a dihydropyridine^pyridinium
salt redox system and on the blood-brain barrier. In the proposed delivery
system, a pharmacologically active agent which contains a pyridinium nu
cleus would be reduced to its corresponding dihydropyridine. After sys
temic administration, the highly lipoidal dihydropyridine would partition
into the brain as well as into the periphery. In both locations oxidation
would occur. In the systemic circulation, the charge species would be
rapidly eliminated by renal or biliary mechanisms while in the brain the
compound would be retained.
The prototype compound which was chosen for inclusion in this scheme
was berberine. This alkaloid has a high in vitro activity against various
cancer systems but its in vivo activity is low. The first step in the ap
plication of the described delivery system to berberine is the synthesis
XT 1


37
The idea of including a drug into liposomes formed in vitro has re
ceived a great deal of attention.117119 In these systems, the drug is
inactive since it is enclosed in the phospholipoidal matrix of the lipo
some and, as in the case of the albumin conjugates, these packets are
taken up by endocytosis. Again, the system should specifically exert its
action at the site of influx. While these approaches are promising the
oretically, they have not met with much success. These carrier complexes
are the subject of several recent books.117119
A general method was recently proposed for the specific delivery of
drugs to the brain. This system was based on the results of some work
with N-methylpyridinium-2-carbaldoxime chloride (2-PAM).120121>122123
This pyridinium quaternary compound is the agent of choice for the treat
ment of organophosphate poisonings, and exerts its action by reactivating
deactivated cholinesterases. The problem with this agent is its highly
polar nature. Organophosphates such as diisopropyl fluorophosphate (DFP)
and paraoxon are very lipophilic and easily penetrate the BBB. The brain
is, therefore, very susceptible to acetylcholinesterase inactivation by
these agents. The highly polar 2-PAM has a very low activity in the brain
since it is almost totally excluded by the BBB. To deal with this prob
lem the dihydro adduct of 2-PAM, pro-2-PAM,was synthesized as a prodrug.

ch3
2-PAM
pro-2-PAM


149
compound is greatly enhanced. This was demonstrated when the delivery
scheme was applied to the berberine (1)idihydroberberine (2) example.
This method is potentially extendable to any drug which contains a
pyridinium moiety. A number of anti cancer agents such as nitidine, cora
lyne, and fagaronine fit these criteria. Phenothiazines and 8-blockers,
among others, could also be modified in this way to attain specific deli
ery to the brain.


115
rate on structure indicate the involvement of an enzyme in the oxida
tion. One of the enzymes which is said to be responsible for the oxida
tion of dihydropyridines is NADH dehydrogenase.204 Since this family of
enzymes is membrane bound, it would be present in the organ homogenate
but not in plasma. The results are consistent with this distribution.
Enzymes involved in the oxidation of dihydropyridines have as their endo
genous substrate NADH. This molecule is not substituted at the amide
nitrogen and one would expect compounds which more closely resemble NADH
to be better substrates for the enzyme than molecules which greatly devi
ate from this structure. One would predict that the shorter chain ana
logs (23) and (24), and the N-benzyl compound (22) would be oxidized
more rapidly than the longer chain analogs and this is, in fact, the case
An additional observation which supports this hypothesis is that as the
homogenates age, the rate constants as well as the correlation coeffi
cient decreases. This is consistent with the time-dependent denaturation
characteristic of this type of enzymatic system.
These trends also occur in the oxidation of (2). In plasma and buf
fer, the data indicates a relatively slow oxidation with poor correlation
In brain homogenate, there is an acceleration and a linearization in the
rate constants. These results indicate that although oxidation of (2)
can be mediated by oxygen, in tissues like the brain and liver, an enzy
matic oxidation can occur. The effect of protein binding on the rate of
oxidation of (2) was investigated by changing the concentrations of the
homogenates. One would expect (2) to bind to proteins but the effects
of this complexation on oxidation were not large.
Membrane Permeability of Dihydroberberine and Berberine
In order to investigate the relative ability of dihydroberberine (2)
to penetrate membranes, the behavior of (2) and (1) in a model system was


26
The leakage of proteins from the capillaries is usually proportional
to the amount of injury, but even in severe injury the endothelial cell
is intact and shows no sign of lesion.94 The explanation most often pro
posed for this extravasation is increased vesicular activity. In most in
juries there is an increase in serotonin and norepinephrine levels, as well
as the production of a number of humoral agents. The increased vesicular
content, which appears first in the arterioles and subsequently in the
capillaries, may be mediated by stimulation of cAMP. There are also, how
ever, increases in blood pressure and that may act to increase vesicular
transport.
Cerebral infarcts also disrupt BBB function, as evidenced by an in
creased albumin concentration in the CSF of infarct victims.95 Correla
tion, however, between infarct size and location and the quantity of al
bumin leaked has not revealed any significance.
Both electroconvulsive shock and pentylenetetrazole-induced seizures
result in an increased capillary permeability to a number of plasma
markers.1454 The degree of permeability increase is proportional to the
number of shocks or compounds given. The morphological basis for this ex
travasation is not known and both junctional opening and increased vesi
cular transport have been suggested.77 It is interesting to note that,
in both of these procedures, the blood pressure rises and this may be an
important factor.
Induced ischemia also produces a marked increase in the permeability
of the cerebral vasculature.5477 The experimental model which is most
often used is that of the Mongolian gerbil. In this species, about half
of the individuals lack arterial connections between cerebral and vertebral
systems. After carotid occlusion and development of ischemia, HRP leaks


54
3-(Ethoxycarbonyl )-1-methy1pyridinium Iodide/Ethyl N Methylnicotinate
Iodide (15)
Ten grams of (8) (0.066 moles) were mixed with methanol and with a
molar excess of methyl iodide (18.7 g). The solution was refluxed for
several hours. The solvent was removed, yielding a red-orange oil which
solidified on cooling to a yellow solid. The solid was recrystallized
from acetone-ether: *H NMR (CDC13, (CD3)oS0), 5 9.6 (2 H, m), 9.0 (1 H, m),
8.4 (1 H, m), 4.8 (3 H, s), 4.5 (2 H, q), 1.4 (3 H, t); IR (NaCl) v (C-H)
3008, (C=0) 1723, (-C-C(=0)0) 1303, (0-C-C) d 1102, 1117, (y CH ) 742,
(6 ring) 654.
3-(Butox.ycarbon\
/~\ )-l-methylpyridinium Iodide/Butyl N Methyl nicotinate
Iodide 1
)
Ten grams of (9) (0.056 moles) were dissolved in 60 ml of acetone
and a molar excess of methyl iodide (15.9 g) was added. The solution was
refluxed for two hours. The solvent was removed, leaving a yellow solid
which was recrystallized from acetone-ether: XH NMR (CDC13, (CD3)oS0)
6 9.9 (2 H, m), 9.0 (1 H, m), 8.6 (1 H, m), 4.5 (3 H, s), 4.4 (2 H, t),
0.93 (3 H, t); IR (NaCl) v (C-H) 2950, (C=0) 1720, (-C-C(=0)0) 1291,
(0-C-C) 1100, (y CH) 735, (b ring) 651.
3-(Hexoxycarbonyl)-1-methylpyridiniurn Iodide/Hexyl N Methylnicotinate
Iodide (17)
Ten grams of (11) (0.048 moles) were dissolved in 60 ml of acetone,
and a molar excess of methyl iodide (13.7 g) was added. The solution was
refluxed for two hours. The solvent was removed, producing an orange oil:
lH NMR (CDC13, (CD3)2S0) <5 9.3 (2 H, m), 8.9 (1 H, m), 8.3 (1 H, m),
4.7 (3 H, s), 4.3 (2 H, t), 0.90 (3 H, t); IR (NaCl) v (C-H) 2944, (C=0)
1723, (-C-C(=0)0) 1295, (0-C-C) 1111, (y CH) 738, (8 ring) 654.


DOSE/GRAM TISSUE
2.0
_i 1 1 1 1 i i i i i l i l i i l l l
20 60 100 140 180 220 260 300 340
TIME (MIN)
Figure 3-24. A Comparison of the Efflux of Berberine from Lungs when Administered i v
as 55 mg/Kg of Dihydroberberine Hydrochloride (3) (O) or 35 mg/Kg of
Berberine (1) ()
ro
00


whom I would not have been able to pursue an academic career. The anti can
cer testing was graciously performed by Otsuka Pharmaceutical Co., Ltd.
This work was supported by Grant GM 27167 from the National Institute of
General Medical Sciences.
v


85
approach allows one to examine compounds and reactions which are not
approachable by conventional means. For example, the stability of very
reactive species can be assayed, their structures determined, and quali
tative aspects of their chemistry elucidated.
The input for the program includes, for each atom, the atomic number,
the approximate bond distances between adjoining atoms, the approximate
bond angle, and the approximate twist angle out of the plane. As previously
mentioned, the number of parameters needed is 3N-6 where N is the number
of atoms. Convergence is assumed when the difference between two succes
sive calculations is less than 0.1 kcal/mole.
The two models (30) and (31) were analyzed and the results are pres
ented in Table 3-4 through 3-8. The heat of formation calculated for (30)
was 95.1 kcal/mole and that of (31) was -39.3 kcal/mole. While the use of
AHf for comparisons is restricted to structural isomers, the relative sta
bility of a system can be calculated by comparing the differences in AHf
(AAHf) from one system to another. The AAHf of the isoquinoline model
(30) Z dihydroisoquinoline model (31) pair is 134.4 kcal/mole. As shown
in Table 3-9, this value is smaller than that obtained from simple dihy
dropyridine Z pyridinium systems indicating a greater stability of (31)
relative to simple dihydropyridines.190 The stabilization of (31) indi
cates that it should be less reactive than simple dihydropyridines. The
basis of this stabilization is derived from the extended aromatic conjuga
tion of (31). Another contribution to the relative stabilization can be
seen by an examination of the highest occupied molecular orbital (HOMO)
which is represented in Figure 3-9 as a linear combination of atomic orbi
tals. In looking at the HOMO, only the magnitude of the coefficients is
important since the sign simply represents the phase of the orbital. A


50
chloroform (CHC13). The organic layer was reduced to dryness and the
residue dissolved in hot water. The solution was filtered and then made
acidic with an excess of hydrochloric acid (HC1). The ensuing precipi
tate (yellow-brown solid) was collected by filtration and redissolved in
hot water. The solution was filtered and made basic with potassium hy
droxide. The solution turned deep purple and crystallization was induced
by scratching: *H NMR (CD30D) 6 9.0 (s, 1 H), 7.8 (s, 1 H), 7.2 (m, 2 H),
6.8 (m, 2 H), 5.8 (s, 2 H), 3.7 (s, 3 H), 4.4 (m, 2 H), 3.0 (m, 2 H);
UV (95% ethanol) 276 nm and 238 nm A others 512 nm; IR (KBr) v (C-H)
MiaX
3000 and 2900, (C-C, C-N) 1635, 1571, 1509 and 1472, (C-0-C asym) 1221
and 1289, (C-0-C sym) 1035 and 1144; Elemental analysis calculated %:
C, 71.03; H, 4.67; N, 4.36. Found %: C, 71.20; H, 4.81; N, 4.42.
Berberine Iodide (5)
A solution of 1.0 g of (4) in acetone was prepared. A 1.0 M excess
of methyl iodide was added and the solution was allowed to reflux for
several hours. The characteristic yellow color of berberine appeared and
TLC, NMR, IR, and UV confirmed the methylation.
3-(Aminocarbonyl )-l-methylpyridinium Iodide/1-Methylnicotinamide Iodide (6)*
Five grams of nicotinamide (0.041 moles) were dissolved in 50 ml of
dry methanol. A molar excess of methyl iodide (11.6 g) was added to the
stirring mixture and after one hour of refluxing, a precipitate formed.
This was filtered and washed. The material was recrystallized from aque
ous methanol: Melting point 101-103C, Literature 102-105C; UV (H20)
224 mm and 265 mm Amax; NMR and IR were identical with the literature.
*The nicotinic acid derivatives are given a systematic name followed by
a common name


58
3.1 (2 H, m), 3.0 (3 H, s), 0.9 (3 H, t); UV 359 nm Amax; Elemental analy
sis calculated %: C, 69.96; H, 9.73; N, 6.32. Found %: C, 69.82; H, 9.46;
N, 6.28 (C13H2iN02).
1.4-Dihydro-1 -methyl-3-pyridinecarboxylic Acid Octyl Ester/Octyl 1,4-Dihy-
dro-N-methylnicotinate (26)
A solution of 4.6 g of NaHC03 and 4.02 g of (18) (0.016 moles) was
prepared in 5% aqueous methanol. The solution was cooled and 6.96 g of
Na2S204 were slowly added. Two hundred milliliters of ethyl ether were
added and the mixture stirred under nitrogen for one hour. The ether layer
was separated, dried and reduced in volume. The oil was dried in vacuo:
XH NMR (CDC13) 6 6.9 (1 H, m), 5.6 (1 H, m), 4.7 (1 H, m), 4.0 (2 H, t),
3.0 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 358 nm xmax; Elemental analy
sis calculated %: C, 70.69; H, 9.97; N, 5.50. Found %: C, 70.76; H, 9.68;
N, 5.88 (C15H25N02-i-H20).
1.4-Dihydro-1-methyl-3-pyridinecarboxylic Acid Decyl Ester/Decyl 1,4-Dihy-
dro-N-methylnicotinate (27)
A solution of 4.6 g of NaHC03 and 4.46 g of (19) (0.016 moles) was
prepared in 5% aqueous methanol. The solution was cooled and 6.96 g of
Na2S204 were slowly added. To this solution was added 200 ml of ethyl
ether and the mixture stirred under N2 for one hour. The ether layer was
separated, dried and reduced in volume. The dried oil was orange in color:
XH NMR (CDC13) 6.9 (1 H, m), 5.6 (1 H, m), 4.7 (1 H, m), 4.0 (2 H, t),
3.0 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 359 nm xmax; Elemental analy
sis calculated %: C, 73.12; H, 10.39; N, 5.02. Found %: C, 73.16; H, 10.48;
N, 5.03 (C17H2gN02).
1.4-Dihydro-1-methyl-3-p.yridi necarboxyl ic Acid B-Phenylethyl Ester/B-Phen-
ethyl 1,4-Dihydro-N-methylnicotinate (28)
Four and six-tenths grams of NaHC03 and 3.89 g of (20) (0.016 moles)
was dissolved in 100 ml of water. The solution was cooled and 6.96 g of


Figure 3-1G. The Rates of Oxidation of Various 1-Methyl-1,4-dihydronicotinic Acid Esters
(23), (24), (25), (26), (27), (23) and 1-Benzyl-l,4-Dihydronicotinamide
(22) at 37C in 40% Human Plasma (), 6% Brain Homogenate (A) and
3.5% Liver Homogenate ()


151
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(1975).
203. D. J. Porter and H. J. Bright, J_. Biol. Chem., 255, 7362 (1980).
204. J. Rydstrom, J. B. Hoek and L. Ernster in "The Enzymes," Vol. XIII
Oxidation-Reduction, P. D. Boyer (ed.), Academic Press, New York (1976).
205. F. T. Schein and C. Hanna, Arch. Int. Pharmacodyn., 104, 317 (1960).
206. V. B. Schatz, B. C. O'Brien, W. M. Chadduck, A. M. Kanter, A. Burger
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2128 (1969).
208. J. Yamahara, K. Goto and T. Sawada, Jap, vh Pharmacog., 26, 53 (1972)
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209. S. Sakurai, M. Tezuka and 0. Tamemasa, Applied Pharmacol., 11, 351
(1976) (Oyo Yakuri).
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57 (1974).
211. T. Furuya, Bul 1. Osaka Med. School, 2, 18 (1956).
212. V. V. Berezhinskaya, E. E. Aleshinskaya and E. A. Trutneva, Russian
Pharmacol. Toxicol., 31, 129 (1968).


155
108. N. Bodor in "Design of Biopharmaceutical Properties Through Prodrugs
and Analogs," E. B. Roche ( ed. ), APnA Academy of Pharmaceutical
Sciences, Washington, D.C. (1976) p. 98.
109. N. Bodor, Drugs of the Future, 6_, 165 (1981 ).
110. N. Bodor in "Optimization of Drug Delivery," H. Bundgaard, A. B. Han
sen and H. Kofod (eds.) Alfred Benzon Symposium, Vol. 17, Munksgaard,
Copenhagen (1982) p. 156.
111. C. D. Selassi, E. J. Lein and T. A. Khwaja, J. Pharm. Sci., 70, 1281
(1981).
112. T. A. Connors, Chem. and Ind., 11, 447 (1980).
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114. J. W. Gorrod, Chem. and Ind., 11, 458 (1980).
115. P. Workman and J. A. Double, Biomed., 28, 255 (1978).
116. G. Gregoriadis, Nature, 265, 407 (1977).
117. G. Gregoriadis (ed.) "Drug Carriers in Biology and Medicine,"
Academic Press, New York (1978).
118. W. B. Pratt and R. W. Ruddon, "The Anticancer Drugs," Oxford Univer
sity Press, New York (1979).
119. R. L. Juliano (ed.) "Drug Delivery Systems," Oxford University Press,
New York (1980).
120. N. Bodor, E. Shek and T. Higuchi, Science, 190, 155 (1975).
121. N. Bodor, E. Shek and T. Higuchi, J_. Med. Chem., 19, 102 (1976).
122. E. Shek, N. Bodor and T. Higuchi, vh Med. Chem., 19, 108 (1976).
123. E. Shek, N. Bodor and T. Higuchi, Med. Chem., 19, 113 (1976).
124. N. Bodor, R. G. Roller and S. J. Selk, J_. Pharm. Sci., 67, 685 (1978).
125. S. B. Ross and 6. Froden, Eur. Pharmacol., 13, 46 (1970).
126. S. B. Ross, J_. Pharm. Pharmacol., 27, 322 (1975).
127. S. F. Dyke, Adv. Heterocyclic Chem., 14, 279 (1972).
128. F. R. Stermitz, K. A. Larson and D. K. Kim, J^. Med. Chem., 16, 939
(1973).
129. R. K. Zee-Cheng and C. C. Cheng, J_. Med. Chem., 18, 66 (1975).
F. R. Stermitz, J. P. Gillespie, L. G. Amoros, R. Romero, T. A. Stermitz,
K. A. Larson, S. Earl and J. E. Ogg, J_. Med. Chem., 18, 708 (1975).
130.


18
receptors lumirally, whose stimulation results in the generation of a
second messenger, has been proposed in this respect. A different conclu
sion is that small peptides have a significant flux across the BBB. The
peptides which have been investigated thus far include stabilized enkepha
lins and endorphins, a nonapeptide which induces delta-sleep, melanin stim
ulating hormone (a-MSH) and melanin inhibiting factor one (MIF-1).606163
The BBB plays a major role in CSF homeostasis.1264 Cerebral spinal
fluid is produced at the choriod plexus and drains from the ventricals
through the foramina of Magendie and Luschka into the ventral aspects of
the brain.65 This fluid serves many important mechanical and nutritive
functions. Cerebral spinal fluid flow is constant with a ti of renewal
of about two hundred and seventy minutes. The ventricular volume of CSF
is about 23 ml while the subarachnoid volume is 117 ml. Cerebral spinal
fluid, along with any dissolved materials, leaves the subarachnoid space
via the arachnoid villi, which protrude into a venous sinous. The arach
noid villi act as a one-way valve and prevent backflow.66 This loss of
CSF provides a slow mechanism for nonspecific efflux of compounds from
the CNS. This mechanism rids the brain of polar compounds such as metabolic
wastes at a fairly constant rate regardless of molecular weight. If a
compound is fairly polar and does not have affinity for any passive or
active efflux mechanism, it will leave the CNS by CSF bulk flow. Therefore,
while lipophilicity is very important for influx to the brain, the efflux
of a compound is only partially dependent on this parameter.4667686970
Since the ionic environment in which neurons function is so important,
the composition of the CSF is strictly maintained within narrow limits.
The CSF is not simply an ultrafiltrate of plasma, and several concentration
gradients are produced. The maintenance of these gradients can, in part,
be attributed to the low ionic conductance of the BBB. This is especially


Table 3-16. The Rates of Oxidation and Corresponding Correlation Coefficients of Various 1-Methyl-l ,4-
dihydronicotinic Acid Esters and 1-Benzyl-l,4-dihydronicotinamide (22)
Pseudo First Order Rate Constants x 10"4sec_1
Compound Brain Liver
Number Plasma Correlation Homogenate Correlation Homogenate Correlation
(23)
1.19

0.14
.970
4.87
+
0.25
.9998
6.49

0.51
.998
(24)
0.90

0.11
.921
4.80
+
0.27
.9998
4.97

0.19
.998
(25)
0.97

0.10
.962
3.75

0.31
.9998
3.40

0.55
.981
(26)
0.81

0.03
.971
3.08

0.33
.998
3.29

0.16
.974
(27)
0.71

0.05
.940
3.02

0.44
.9991
3.25

0.39
.993
(28)
0.82

0.06
.976
3.88

0.09
.9994
4.25

0.91
.998
(22)
0.90

0.05
.990
3.53

0.17
.9992
4.14

0.16
.9990


Table 3-6. Bond Angles in Degrees between Various Atoms of the Isoquinoline Model (30) and the
Dihydroisoquinoline Model (31)
Isoquinoline Model (30) Dihydroisoquinoline Model (31)
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
2-1-6
121.6
3-2-26
120.4
2-1-6
121 .8
1-2-26
108.3
3-4-5
115.7
13-14-15
199.9
3-4-5
116.3
1-2-27
108.8
4-5-6
125.5
12-13-16
119.7
4-5-6
126.4
13-14-15
119.9
1-6-5
116.2
13-12-17
117.2
1-6-5
117.9
12-13-16
119.7
1-6-7
117.1
12-11-18
128.1
1-6-7
116.5
13-12-17
117.2
6-7-8
123.7
6-7-20
114.5
6-7-8
123.6
12-11-18
128.1
7-8-9
121.9
7-8-21
121.3
7-8-9
121.8
6-7-20
114.5
6-1-10
121.9
8-9-22
110.7
6-1-10
123.4
7-8-21
121.3
1-3-4
118.6
8-9-23
108.9
11-3-4
118.7
8-9-22
109.8
2-11-3
120.5
9-10-24
109.7
12-11-3
120.4
8-9-23
109.0
1-12-13
119.1
9-10-25
109.8
11-12-13
119.3
9-10-24
108.0
2-13-14
121.8
12-17-28
114.3
12-13-14
121.1
9-10-25
108.6
4-5-19
117.8
11-18-27
114.3
4-5-19
116.3
12-17-29
113.9
11-18-28
113.9


500
-J 1 I I l I I I I
40 80 120 160 200 240 280 320 360
TIME (MIN)
Figure 3-17. Partitioning of 2G.5 mg of Berberine (1) from Plasma (A) into Red Blood
Cells (A) and of 26.5 mg of Dihydroberberine Hydrochloride (3) from Plasma
(O) into Red Blood Cells (). The Volume of Blood Used in each Experiment
was 75 ml


CHAPTER 1
INTRODUCTION
A method for delivering drugs specifically to a particular organ would
be valuable. Properly designed, a drug delivery system should concentrate
an agent at its site of action and reduce its concentration in other loca
tions. The results of these manipulations would not only be an increase in
the efficacy of an agent, but also a decrease in its toxicity.
A site specific system designed for the central nervous system (CNS)
would be especially useful. The reason for this, aside from the inherent
importance of the brain, is that the entry of many pharmacologically active
agents into the CNS is impeded by a set of specialized barriers present at
the blood-brain interface. This barrier system,, termed the blood-brain
barrier (BBB), is composed of numerous enzymatic and anatomical components.
This dissertation will present a general method for the specific deliv
ery of drugs to the brain and give an example which substantiates the method.
In order to provide an adequate background for a discussion of drug deliv
ery to the brain, a review of the BBB is necessary. The introductory mate
rial is then continued with a cursory historical account of drug delivery
systems and prodrugs. Because of the importance and great interest of an
ticancer agents, emphasis is placed on this topic in this section. The
closing section of the first chapter will state specific aspirations as they
apply to the present research.
1


108
Table 3-14. The Relative Rates of Oxidation of Dihydroberberine (2),
1-Methyl-1,4-dihydronicotinamide (21), and l-Benzyl-1,4-
di hydronicotinamide (22) in Dilute Hydrogen Peroxide
Compound
Pseudo First
Order Rate Constant
Correlation
Relative Rate
of Oxidation
(2)
4.51 x 10'4
0.998
1.0
(21)
9.83 x 10"3
0.99999
21.7
(22)
1.00 x 10'3
0.999
2.2


156
131. M. Cushman, F. W. Dekow, and L. B. Jacobsen, J. Med. Chem., 22,
331 (1979).
132. A. Hoshi, T. Ikekawa, Y. Ikeda, S. Shirakawa, M. liga, K. Kuretani and
F. Fukuoka, Gann, 67, 321 (1976).
133. I. F. Shvarev and A. L. Tsetlin, Farmakol. Toksikol., 35, 73 (1972).
134. F. E. Hahn and J. Ciak in "Antibiotics III Mechanism of Action of
Antimicrobial and Antitumor Agents," J. W. Corcoran and F. E. Hahn
(eds.), Springer-Verlag, New York, (1975) p. 577.
135. M. Vincrov, B. Brzdovicov and D. Kostlov, Farm. Obzor, 46,
543 (1977).
136. M. Pitea and C. Margineanu, Cluj. Med., 45, 465 (1972).
137. W. H. Perkin, J. Chem. Soc., 55, 63 (1899).
138. T. A. Greissman and D. H. Grout, "Organic Chemistry of Secondary
Plant Metabolism," Freeman, Cooper and Co., San Francisco, California
(1969) p. 411.
139. D. H. Barton, R. H. Hesse and G. W. Kirby, J_. Chem. Soc., 6379 (1965).
140. G. D. Pandey and K. P. Tiwari, Heterocycles, 14, 59 (1980).
141. V. Deulofeu, D. Giacopello and A. L. Margni, Anal. Fis. Quim., 62B,
536 (1966).
142. G. D. Pandey and K. P. Tiwari, Ind. J_. Chem., 18B, 545 (1979).
143. W. A. Creasey, Biochem. Pharmacol., 28, 1081 (1979).
144. M. Sabir and N. K. Bhide, Ind. J_. Physiol. Pharmacol., 111 (1971).
145. M. Sabir, M. H. Akhter and N. K. Bhide, Ind. J. Physiol Pharmacol.,
9 (1978).
146. A. B. Vaidya, T. G. Rajagopalan, A. G. Kale and R. J. Levine,
J_. Postgrad. Med., 26, 28 (1980).
147. H. Sheppard and C. R. Brughardt, Biochem. Pharmacol, 27, 1113 (1978).
148. L. R. Meyerson, K. D. McMurtrey and V. E. Davis, Neurochem. Res.,
239 (1978).
149. M. Sabir and N. K. Bhide, Ind. J_. Physiol. Pharmacol., 97 (1971 ).
150. Y. C. Clement-Cormier, L. R. Meyerson, H. Phillips, and V. E. Davis,
Biochem. Pharmacol., 28, 3123 (1979).
151. J. Kovr, E. Drrova, and L. Skursky", Eur. J. Biochem., 101, 507
(1979).


TI ME ( min)
Figure 3-21. A Comparison of the Efflux of Berberine (1) () and Berberine (1) and co
Unoxidized Dihydroberberine (2) (A) after a Dose of 55 mg/Kg of
Dihydroberberine Hydrochloride (3) Administered i v


78
Table 3-3. Distribution Coefficients for Berberine (1) and Dihydrober-
berine Hydrochloride (3) in Chloroform/pH 7.4 Buffer and in
1-Octanol/pH 7.4 Buffer
Distribution
Coefficient
Compound
Chloroform/pH 7.4 Buffer
1-Octanol/pH 7.4 Buffer
Berberine (1)
< 0.001
0.062
Dihydroberberine
Hydrochloride (3)
5.33
2.59


101
out of the plane. In applying the same considerations to this molecule
as to (30), one would expect that this system would interact less with
DNA than would (30). This is due to the further destruction of the aro
matic nucleus resulting not only in a more nonplanar structure, but also
in a structure with a lower propensity to interact electronically with
DNA. The loss of the positive charge should also reduce macromolecular
intercalation because of the lowered coulombic interaction between (31)
and the anionic phosphate backbone of DNA.156 In extending these results
to dihydroberberine (2), one would predict a lower cytotoxicity and, there
fore, toxicity of (2) relative to (1). The optimized structure of (31)
at 25C is presented in Figures 3-12 and 3-13.
To summarize, the MINDO/3 calculation predicts the dihydro model (31)
and, presumably, (2) to be more stable than simple dihydropyridines because
of extended conjugation and hyperconjugation. The model (30) is predicted
to undergo nucleophilic attack at the carbon adjacent to the nitrogen, and
protonation and electrophilic reaction at the carbon 6 to the nitrogen of
the enamine. The calculations show localization of double and single
bonds on the reduction of (30) to (31). These data are in good agreement
with what is known about chemistry of this genre of compounds. The calcu
lation suggests that the reason berberine does not intercalate as well as
totally aromatic compounds is not because of steric problems associated
with the carbon skeleton but, rather, electronic differences and the steric
effects of added hydrogens and, finally, MIND0/3 predicts that (31) and
presumably, (2) are less toxic than (30) and (1). These results should
be applicable not only to the (1) X (2) system, but other conjugated di
hydropyridine systems as well.


95
of (30), the bond connecting carbons 5-6 shortens, indicating a greater
double bond character at this location, while the bonds between carbons 2-3
and between the carbon and nitrogen at position 1-2 lengthen, indicating
an increased single bond character at these locations. This correlates
well with the structural formalism and also mirrors those changes that
occur in the reduction of (32) to (33). A similar study of bond angles is
presented in Table 3-11.
The charge densities at specific atoms can be indicative of the type
of chemistry that a compound undergoes. A study of the charge densities
of atoms in (30) t (31) is presented in Table 3-7 and a comparison of this
system to the (32) t (33) system appears in Table 3-12. The charge densi
ties of the pyridinium nucleus of (30) reveal the most highly charged de
ficient center is at carbon C2. One would expect nucleophilic attack at
this electropositive position and, in fact, this is what is observed.
These observations can be extended to berberine (1) since it is known that
hydroxide, hydride, and acetonide attack (1) at this position. In general,
nucleophiles attack pyridines at the carbon adjacent to the nitrogen.172
The charge densities of the atoms in (31) indicate a highly electronegative
center at C5. One would therefore expect protonation and electrophilic
attack at this location. This, again, is borne out experimentally. In
the case of dihydroberberine (2), protonation as well as alkylation occurs
here and, in general, this type of reaction is well known in the chemistry
of enamines.191-193 These properties and trends are also seen in the
(32) t (33) pair.
The planarity of a pharmacologically active aromatic molecule, espe
cially anti neoplastic agents, is an extremely important parameter and many
correlations between activity and toxicity and planarity have been made.


148
was shown to be more lipophilic than berberine (1) and also better able to
penetrate biological membranes. In an attempt to delve into the basic
chemistry of these relatively unstable compounds, a model system was de
veloped for them and this was examined by a MINDO/3 approach. The results
obtained from this study were consistent with experimental data and were
extendable to the berberine (1) ^ dihydroberberine (2) pair. In addition,
several predictions were made concerning the biological activity of (2)
relative to (1) and these were found to be valid.
Dihydroberberine (2) or its hydrochloride salt (3) was injected iv
into rats and when the brains were analyzed, high levels of (1) were found.
No berberine (1) was found in the brain after systemic administration of
(1). The rate at which (1) left the brain after its delivery by (3) was
slow and the t of the efflux was eleven hours. If dihydroberberine hy-
2
drochloride (3) is slowly infused iv, the concentration of (1) rises in
the brain with time but falls in all other organs tested. At forty-five
minutes the concentration of (1) is highest in the brain. The efflux of
berberine (1) from the CNS appears to be mediated by a passive process,
perhaps the bulk flow of CSF.
Dihydroberberine hydrochloride (3) was shown to be less toxic than
(1) in vivo in accordance with predictions made by the theoretical studies.
Additionally, (3) was shown to be less effective than (1) in inhibiting
the growth of KB cells in vitro. While the two compounds are equipotent
in increasing the life span of mice injected ip with a suspension of P388
lymphocytic leukemia cell, (3) is more potent in increasing the life span
of mice who were inoculated intracerebrally with the P388 cell line.
These data verify the proposed drug delivery scheme. By concentrat
ing a pharmacologically active agent at its site of action and by reducing
its concentration in other locations, the therapeutic index of the delivered


o
o
Figure 3-7. Demethylation of Berberine (1) and Hethylation of Berberrubin (4).
03
O


138
the limitations of diffusion do not apply and (1) may simply be lost by
the bulk flow of CSF.
To determine the rate of CSF flow in the animal system used in these
experiments, the rate of efflux of 3H-inulin (29) from the brain was mea
sured. This radionuclide was injected icv in DMSO at a dose of 2.3 yCi.
As shown in Table 3-19, the t of efflux of (29) is two hours, in good
agreement with previously reported values.45
The loss of (1) from the brain appears not to be mediated by an ac
tive process. Simple bulk flow of CSF seems to be sufficient to remove
(1) from the brain. The rate-limiting step in the cerebral elimination
of (1) does not appear to be the actual efflux process but rather, the
redistribution of (1) out of brain cells and membranes by this poorly
mobile species to those areas where elimination can take place.
Limited Metabolic Studies
The metabolism of (1) after the administration of (1) or (3) was
studied in the rat. Urine collected for three days after a dosing of (1)
or (3) was extracted with chloroform or 3-methyl-1-butanol. The alcohol
was used because it is reported to extract berberine and similar alkaloids
efficiently from aqueous solutions.210 The results of the HPLC and TLC
analyses are shown in Tables 3-20 and 3-21, respectively. As one can see,
there is very little metabolism of (1) and the major component of the
urine from animals dosed with either (1) or (3) was (1). There were sev
eral peaks which could not be attributed to the anesthetic and these were
found in the urine of all tested animals. The size and retention time
of these peaks was again similar.
These data are consistent with the few articles which have been pub
lished concerning the metabolism of (1). While this metabolism is reported


84
Hamiltonian for such a system consists of the kinetic energy term for the
movement of the electrons and the potential energy terms for electron-
nuclei attraction and electron-electron repulsion.
In systems with only one electron, e.g. H, H2+ or He+, the differen
tial equation which constitutes the Hamiltonian can be separated and
exactly solved. If, however, more than one electron is present, the elec
trons interact and the differential equation is no longer separable or
exactly solvable. Because of this problem, approximations and simplifi
cations are incorporated into the Schrodinger equation. These include
considering the molecular orbital (v) as a linear combination of atomic
orbitals ():189
'f = E Ci i
Additional simplifications are obtained by neglecting certain electron
repulsion terms since these values are close to zero. These approximations
allow the application of quantum mechanical approaches like MINDO to rather
complex chemical problems without extremely large expenditures of capital
or computer time.
The MINDO/3 program provides the following data: optimized geometries
of the most stable ground state conformation at 25C, the heat of forma
tion (AHf) in kcal/mole at 25C of the optimized structures, the atomic
charge distribution, the dipole moments, the total electronic energy, the
vertical ionization potentials, the bond order matrix, and the eigen vec
tors and eigen values.
The eigen values and eigen vectors allow a thorough examination of
the contributions of the individual atomic orbitals to the molecular or
bital and thus of the electronic structure of the molecule. This can be
indicative of many of the chemical proclivities of a molecule. The MINDO


LIST OF FIGURES
FIGURE PAGE
1-1 This Schematic Illustration Represents an Endothelial Cell
Derived from either a Muscle (ECm) or Brain (ECb) Capillary.
In this Figure, (ma) is the Macula Adherens or Loose Junction,
(zo) is the Zona Occludens or Tight Junction, (mv) are Micro
vesicles, and (bl) is the Basal Lamina. This Figure was Modi
fied from Reference 1, Page 162 by Permission 6
1-2 A Proposed Carrier-mediated Chemical Delivery System with
Specificity for the Brain. The Drug Molecule to be Trans
ported is Represented by the (O) 40
1-3 The Proposed Drug Delivery Scheme 43
3-1 Synthesis of Dihydroberberine (2) and its Hydrochloride
Salt (3) 69
3-2 Ultraviolet Spectrum of Dihydroberberine (2) in 95% Ethanol.... 70
3-3 Infrared Spectrum of Dihydroberberine (2) (KBr) 71
3-4 Mass Spectrum (70 eV, El) of Dihydroberberine (2) 72
3-5 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of Dihydro
berberine in CDCI3. The Insert Represents the Region between
2.86 and 3.46 at 100 MHz 73
3-6 The 13C Nuclear Magnetic Resonance Spectrum (100 MHz, CDC13)
of Dihydroberberine (2) 75
3-7 Demethylation of Berberine (1) and Methylation of Berber-
rubin (4) 80
3-8 Structures and Salient Numbering Protocols for Berberine (1),
Dihydroberberine (2), the Isoquinoline Model (30), the Dihydro-
isoquinoline Model (31), Pyridine (32), and 1,2-Dihydro-
pyridine (33) 83
3-9 The Highest Occupied Molecular Orbital of the Dihydroisoquino
line Model (31) 92
3-10 A Computer-assisted Drawing of the Most Stable Conformation
of the Isoquinoline Model (30) at 25C 99
ix


Figure 3-27. A Comparison of the Efflux of the Total Berberine, i.e. (1) and (2) from the
Brain when either 55 mg/Kg of Dihydroberberine Hydrochloride (3) is Administered
i v (A) or 55 mg/Kg of Di hydroberberi ne (2) and 200 mg/Kg of l-Methyl-1,4-
dmydronicotinamide (21) is Administered iv (#)


Table 3-13. The Rate of Oxidation of Dihydroberberine in Various Media
Pseudo First
Corre-
Second Order
Medium (C)
Order Rate Constant
lation
Concentration (M)
Rate Constant
pH 5.8 Buffer (37C)
7.17
x 10"4sec_1
0.900
2 x 10-4
3.99
x 104sec_1
0.880
1 x 104
4.44 0.53 ,mole_1sec-1
2.87
x 104sec_1
0.920
5 x 10'5
6% Brain Homogenate
2.51
x 10_3sec_1
0.995
1.22 x 104
(37C)
1.99
x 10_3sec-1
0.997
9.79 x 10"5
20.17 0.24 mole-1sec-1
9.59
x 104sec-1
0.995
4.89 x 105
40 or 80% Plasma
2.37
x 10_4sec_1
0.993
5 x 10"5
(37C)
30% Liver Homogenate
3.74
X
o
1
-r
to
rd
o
i
0.995
5 x 10"5
(37C)


32
the therapeutic index of an agent since not only is the concentration of
the agent increased in the vicinity of the bioreceptor but, of equal im
portance, the peripheral concentration of the drug is reduced decreasing
any associated toxicity.
Unfortunately, there are very few methods for circumventing the BBB
and these are of limited usefulness. The direct administration of drugs
into the CNSji.e., an intrathecal injection has been used to deliver the
folate antagonist, methotrexate, to the brain. This method is not very
satisfactory since the distribution of methotrexate in the brain is uneven
and slow.46 Additionally, since the ventricular volume of the CSF is
small, increases in intracerebral pressure can occur with repeated injec
tions. This is particularly dangerous when the intracerebral pressure is
already high as it is in CNS cancers. Repeated lumbar puncturing also
carries a risk.
A general method which can be applied to delivery of drugs to the
brain is the prodrug approach.106-110 The term prodrug was coined by
Albert and refers to the result of a transient chemical modification of a
pharmacologically active agent. This change imparts to the compound an
improvement in some deficient physiochemical property such as water solu
bility or membrane permeability. Ideally, a prodrug is biologically in
active but reverts to the parent compound in vivo. This transformation
can be mediated by an enzyme or may occur chemically due to some designed
instability in the agent. The aim of these manipulations is to increase
the concentration of the active agent at its site of action and thereby,
increase its efficacy. While potentially there are many different types
of proderivatives, most thus far synthesized are simple esters and amides.
These compounds are transformed to the parent acid, alcohol or amine by
the ubiquitous hydrolases which are present in vivo. Many anticancer


24
The nature of the hypertension itself can be a factor in increased
capillary permeability.82 While acute hypertension is very likely to pro
duce a protein influx, chronic hypertension may protect the system in the
likelihood of an acute rise in blood pressure. The reason for this is
probably the hypertrophy of vascular muscles. This hypertrophy leads to
an increased vascular resistance and thickening of the vessel wall pro
viding a mechanism for handling greater pressures. This is consistent with
the observation that vasoconstriction decreases capillary permeability,
while vasodilation increases this parameter.
In some cases hypertension may lead to the rare malady, hypertensive
encephalopathy.81 This disease is related to the inability of the cerebral
vasculature to acclimate to acute severe hypertension. In this instance
cerebral edema occurs and this may cause swelling of the nervous tissue.
This yields an increased cerebral pressure and may prevent vasodilation
caused by a variety of stimuli.71
Hypervolemia may also result in an increase in protein extravasation.90
If mice, whose blood is about 1.3 ml, are treated systemically with high
volumes (1 ml) of saline, extravasation of HRP can be documented. At
lower volumes, however, no changes in permeability are observed.
Since, in these experiments, the blood pressure increases only by
20 mm Hg, the increased capillary permeability may not be due to hydro
static pressure. In order to open the BBB, pressure increases on the
order of 100 mm Hg are required.
The vasculature of primary brain tumors and of metastatic secondary
brain cancers exhibits a generalized degradation.79192 This breakdown
can be characterized by the presence of gap junctions, fenestra and open
endothelial junctions, indicating the typical electron microscopic


70i
60-
50-
L
3
CO 40
(O
U 30-
$
CP
N
CP
20
{

i
i
I 0-
10 20 30 40 50 60 70 80 90
TIME (min)
Figure 3-18. Distribution of Berberine in the Brain after iv Administration of Berberine (1)
() at a Dose of 55 mg/Kg or of Dihydroberberine Free Base (2) () at a Dose
of 55 mg/Kg
ro
o


51
3-(Ami nocarbonyl)-!-(phenyl methyl)pyridinium Bromide/1-Benzyl nicotinamide
Bromide (7)~
Ten grams of nicotinamide (0.083 moles) were dissolved in 150 ml of
methanol. A molar excess of benzyl bromide (28.0 g) was added and the
mixture was allowed to reflux for several hours. Upon cooling, a white
solid appeared. This was filtered, washed and recrystallized from metha
nol: Melting point 206-208C, Literature 205C; LH NMR (D20 or TFA)
6 9.5 (1 H, s), 9.0 (2 H, m), 8.2 (1 H, m), 7.5 (5 H, s), 5.9 (2 H, s);
IR (NaCl) v (N-H asym) 3300, (N-H sym) 3148, (-C(=0)NH2 Amide I) 1690,
(Amide II) 1643, (C-N-C) 1388.
3-Pyridinecarboxylic Acid Ethyl Ester/Ethyl Nicotinate (8)
Forty-two grams (0.34 moles) of nicotinic acid were mixed with 55 ml
of absolute ethanol and 25 ml of concentrated H2S04. The mixture was
refluxed in an oil bath for four hours at which time the solution was
poured over ice and made slightly basic with ammonia. The aqueous solu
tion was extracted with ethyl ether. The organic layer was dried with
sodium sulfate (Na2S04) and the solvent evaporated under reduced pressure.
The product was a clear liquid and the yield was 55%: *H NMR (CDCI3) 6 9.2
(1 H, s), 8.8 (1 H, m), 8.2 (1 H, m), 7.4 (1 H, m), 4.4 (2 H, q), 1.4
(3 H, t); IR (NaCl) v (C-H) 2990, (C=0) 1728, (-C-C(=0)0) 1286, (0-C-C)
1112, (Y CH) 742, (b ring) 703.
3-Pyridinecarboxylic Acid Butyl Ester/Butyl Nicotinate (9)
Forty-two grams (0.34 moles) of nicotinic acid were dissolved in
55 ml of 1-butanol and 25 ml of concentrated H2S04 was slowly added. The
solution was heated to reflux in an oil bath for three hours, at which
time the solution was poured over ice and made slightly basic with ammonia.
The mixture was extracted with ethyl ether. The separated ether layer
was dried over Na2S04 and the solvent removed under reduced pressure.


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56
1.4-Pi hydro-1-methyl-3-pyridinecarboxamide/1-Methyl-1,4-Dihydronicotin
amide (21)
Four and six-tenths grams of sodium hydrogen carbonate (NaHC03) and
2.64 g of (6) (0.019 moles) were dissolved in 100 ml of water and cooled
in an ice bath. To this stirring solution was added 6.96 grams of sodium
dithionite (Na2S204). A stream of nitrogen (N2) covered the reaction mix
ture. After one hour, the reaction was stopped and the solution was ex
tracted with several aliquots of CHC13. The CHC13 layer was removed under
reduced pressure, yielding an orange oil. The oil was dissolved in a min
imal amount of CHC13 and tritrated with petroleum ether. From this, an
oil appeared and this was removed and dried in vacuo: XH NMR (D20) 6 6.9
(1 H, s), 5.7 (1 H, d), 4.8 (1 H, m), 3.2 (2 H, s), 3.0 (3 H, s); UV 355 nm
Amav; Elemental analysis calculated %: C, 60.87; H, 7.25; N, 20.20. Found
%: C, 60.92; H, 7.29; N, 20.36 (C7H1QN20).
1.4-Dihydro-l-(phenylmethyl )-3-p.yridinecarboxamide/1-Benzyl-1 ,4-dihydro-
nicotinamide (22)' *
To 100 ml of water were added 4.6 g of NaHC03 and 2.93 g of (7) (0.013
moles). The solution was cooled and 6.96 g of Na2S204 were added. After
two hours of stirring under N2, a precipitate formed. The solution was
filtered. The solid was recrystallized from aqueous methanol, giving
lemon-yellow needles: XH NMR (D20) 6 7.2 (6 H, s), 7.1 (1 H, s), 5.7 (1 H,
m), 4.7 (1 H, m), 4.2 (2 H, s), 3.1 (2 H, m), in CDC13 two protons at 5.86
appear; UV 357 nm X ; Elemental analysis calculated %: C, 72.29; H, 6.58;
max
N, 12.97. Found %: C, 72.09; H, 6.60; N, 12.84 (C13H14N20).
1.4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Ethyl Ester/Ethyl 1,4-Di-
hydro-N-methylnicotinate (23~)~
Four and six-tenths grams of NaHC03 and 2.75 g of (15) (0.016 moles)
were dissolved in 100 ml of water and cooled in an ice bath. To this stir
ring solution was slowly added 6.96 g of NaS204. Two hundred milliliters


40
R
Figure 1-2. A Proposed Carrier-mediated Chemical Delivery System with
Specificity for the Brain. The Drug Molecule to be Trans
ported is Represented by the (O)-


131
Table 3-18. Slow Infusion of Dihydroberberine (3)
30 min
Organ
Concentration (yg/g)
after infusion
Concentration (yg/g)
30 min after iv bolus
A(yg/g)
Brain
135.95
13
91.8 20
+ 44
Kidney
185.5
26
351.8 54
-166
Lung
71.4
10
210.2 14
-139
Liver
101.2
23
67.8 11
+ 33
45 min
Organ
Concentration (yg/g)
after infusion
Concentration (yg/g)
45 min after iv bolus
A
Brain
162.2
8
88
+ 74
Kidney
121.4
19
315
-194
Lung
62.8
6
165
-102
Liver
79.4
10
52
+ 27


DRUG CONCENTRATION/xg/g WET TISSUE
Figure 3-19.
TIME (min)
Efflux of Berberine from the Brain after iv Administration ¡>f eitlier b5 mq/Kg
of Dih.ydroberberine Hydrochloride (3) () r jj mg/Kg of Berberin ( ) ()
Analysis vias for (1) only and not Unoxidized (2)
ro


22
molecule is 4.7 and at physiological pH methotrexate is 99.8% ionized and,
as such, passes the BBB very slowly, if at all. After pretreatment with
hypertonic arabinose, the concentration of methotrexate showed a fifty-fold
increase in the CNS.78
Hypertension is a major health problem and can produce a number of
debilitating complications.79 Hypertension increases the extravasation
of albumin, sucrose, and other polar compounds.808182 This has been
studied in a number of animal models of hypertension including those in
which the increased system blood pressure is induced with either ampheta-
p
mine, ephedrine, Aramine or bicuculine.
The basis for this increased permeability has been debated frequently
and appears to be related to increased vesicular transport since ultra-
structural investigations do not indicate capillary lesions or junctional
openings.77 Increased vesicular transport has, in fact, been implicated
in a number of situations in which capillary permeability increases. The
factors that affect vesicular formation which are described here also ap
ply in those cases.
The mechanism by which hypertension elicits an increase in vesicular
activity is not clear. The increased hydrostatic pressure may act to
induce an invagination. Also, there are a number of substances associated
with hypertension that induce vesicular formation.47783 These include
the catecholamines, serotonin, and histamine. Joo explained the increased
vesicular transport in terms of a cyclic 5'-adenine monophosphate (cAMP)
stimulation mediated by a catalyzed adenyl cyclase, since cAMP can directly
induce vesicular formation.8485
Both serotonin and histamine have been shown to increase protein ex
travasation and both act to catalyze specific adenyl cyclase. A perivascular


157
152. R. B. Sack and J. L. Froehlich, Infect. Immun., 35, 471 (1982).
153. M. H. Akhter, M. Sabir, and N. K. Bhide, Ind. J_. Med. Res., 70,
233 (1979).
154. J. D. McGhee and D. H. von Hippel, J_. Mol. Biol., 86, 469 (1974).
155. M. W. Davidson, I. Lopp, S. Alexander, and W. D. Wilson, Nucleic Acid
Res., 4, 2697 (1977).
156. L. Kapicak and E. J. Gabbay, Am. Chem. Soc., 97, 404 (1975).
157. J. Gadamer, Arch. Pharm., 248, 670 (1910).
158. J. Gadamer, Arch. Pharm., 243, 31 (1905).
159. W. Awe and H. Unger, Chem. Ber., 70B, 472 (1937).
160. W. Awe, Chem. Ber., 67B, 836 (1934).
161. H. Scmid and P. Karrer, Helv. Chim. Acta, 32, 960 (1949).
162. W. Awe, H. Wichmann and R. Beurhop, Chem. Ber., 90, 1997 (1957).
163. S. Bose, Ind. Chem. Soc., 32, 450 (1955).
164. I. W. Elliot, J_. Heterocyclic Chem., 4, 639 (1967).
165. S. Pavelka and J. Kovr, Col 1. Czech. Chem. Commun., 41 3654 (1976).
166. S. Pavelka and J. Kovr, Col 1. Czech. Chem. Commun., 41 3157 (1976).
167. L. Sebe, A. Seishi, N. Murase and Y. Shibata, J. Chinese Chem. Soc.,
14, 135 (1967).
168. B. Witkop, J_. Amer. Chem. Soc., 78, 2873 (1956).
169. G. Habermehh, J. Schunck and G. Schaden, Liebigs Ann. Chem., 742,
138 (1970).
170. D. W. Huqhes, H. L. Holland and D. B. MacLean, Can. J. Chem., 54,
2252 (1976).
171. A. Leo, C. Hansch and D. Elkins, Chem. Rev., 71 525 (1971).
172. U. Eisner and J. Kuthan, Chem. Rev., 72, 1 (1972).
173. G. Frerichs, Arch. Pharm., 248, 278 (1910).
174. G. Frerichs and. P. Stoepel, Arch. Pharm., 251, 321 (1913).
175. B. Pullman and A. Pullman, Proc. Natl. Acad. Sci. USA, 45, 136 (1959).
176. J. Kuthan and L. Musil, Coll. Czech. Chem. Commun., 42, 857 (1977).


THE APPLICATION OF A DIHYDROPYRIDINE-PYRIDINIUM SALT
REDOX SYSTEM TO DRUG DELIVERY TO THE BRAIN
BY
MARCUS ELI BREWSTER III
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1982


35
This approach has been extensively applied to cancer chemotherapy.114
The philosophy behind this application is related to the many differences
that occur between cancer cells and normal cells. These variations are
basically the result of the altered metabolism of neoplastic cells. Many
alkylating agents have been synthesized in an attempt to capitalize on
these differences. The lower pH of tumor cells has been exploited in a
series of aziridines which are more active at the pH of tumors than at
physiological pH. The greater reducing power of cancerous sites has led
to the development of a number of biologically inactive azo compounds
which upon reduction yield potent cytotoxic agents.115 Examples of these
are tetrazolium mustard and azomustard, both of which are reduced to aniline
mustards. The inactivity of the parent compound is due to the delocaliza
tion of the nucleophilic nitrogen lone pair by the conjugated ring system.
A number of O-phosphate esters have been synthesized in order to take
advantage of the high levels of acid phosphatase which are characteristic
of human neoplasms.115 The O-phosphate esters of p-hydroxy mustard and
estradiol mustard were prepared as specific agents to be used in prostate
cancers. The enzyme, y-glutamyl transpeptidase, is also found in high con
centrations in tumor cells so that y-glutamyl derivatives of cytosine ara-
binoside and phenylene-diamine mustard have been proposed. The presence
of hydrolytic esters has also been established in neoplastic formations.
These include esterases and g-glucuronidases.112,115 The cytotoxic agent,
aniline mustard, is converted in the liver as a result of a first pass
effect to its O-glucuronide. Tumors which contain high B-glucuronidase
activity convert the O-glucuronide to the potent alkylating agent p-hydroxy-
aniline mustard.


112
Table 3-15. Proton Assignments of the XH NMR of the 1-Methyl-1,4-dihydro-
nicotinic Acid Ester (27)
OCH2(CH2)8CH
g
3
Proton
a
b
c
d
e
f
g
PPM (6)
6.9
5.6
4.7
4.0
3.0
2.9
0.9


ACKNOWLEDGEMENTS
I would like to express my gratitude to Professor Nicholas S. Bodor.
His genius and kindness will always be an inspiration to me. I would also
like to thank the other members of my committee, Dr. Merle Battiste,
Dr. Kenneth Sloan, Dr. Margaret James, and Dr. James Simpkins, for their
advice and help.
This work would truly not have been possible but for the guidance of
a number of post-doctoral fellows and technicians, especially Dr. Hassan
Farag, Dr. Cynthia Luiggi, Dr. Thorsteinn Loftsson, Mrs. Jirina Vlasak,
Mrs. Nancy Gildersleeve, and Mr. Edward Phillips. I would also like to
thank Jane and C.J. Rogers, Dr. Yasuo Oshiro and Dr. Tadao Sato for their
assistance.
Although space is limited, I want to recognize a few friends who
made graduate school bearable: Linda and John Hirschy, Mark V. Davis,
R.E. Golightly, Mrs. James E. Ray, Newton Galloway, Mike Morris, Richard
Panarese, Wayne and Anita Riggins, Mike and Kay Dempsey, Chuck Hartsfield,
Mike Gibbons, Raun and Cissy Kilgo, Jeff and Jane Dean, Ernie Lee, and
the late Chip Connally. Chip was a dear friend. I would like to thank
Scott and Margie Makar for their cordiality and hospitality.
I would also to like to acknowledge the P.C.'s, Dr. Frank Davis,
Mr. Jim Templeton, and Dr. Gary Visor, the last of a dying breed. Two
special people deserve mention here because of their guidance early in my
scientific career, Professor G.L. Ware, and Mrs. Frances Kenning. Special
thanks are accorded to my Amazonian, sybaritic amanuensis, Cynthia Jordan.
And, last but certainly not least, I would like to thank my family without
iv


Table 3-7.
Charge Density at Various
Model (31)
Atoms of the
Isoquinoline Model
(30) and the
Dihydroisoquino1ine
Isoquinoline
Model (30)
Dihydroisoquinoline Model
(31)
Atom
Number
Charge
Density
Atom
Number
Charge
Density
Atom
Number
Charge
Density
Atom
Number
Charge
Density
1
+0.0779
15
+0.0432
1
-0.1534
15
+0.0044
2
+0.1827
16
+0.0533
2
+0.2443
16
+0.0104
3
-0.1151
17
-0.4266
3
-0.1434
17
-0.4508
4
+0.1029
18
-0.4276
4
+0.0979
18
-0.4283
5
-0.0429
19
+0.0512
5
-0.1597
19
+0.0055
6
+0.1080
20
+0.0514
6
+0.1737
20
+0.0081
7
-0.0446
21
+0.0491
7
-0.0311
21
+0.0001
8
+0.0359
22
+0.0221
8
+0.0024
22
-0.0257
9
+0.0546
23
+0.0128
9
+0.0724
23
-0.0280
10
+0.1104
24
-0.0011
10
+0.1777
24
-0.0610
11
+0.3261
25
+0.0010
11
+0.2993
25
-0.0472
12
+0.2019
26
+0.0429
12
+0.1775
26
-0.0663
13
+0.0340
27
+0.2847
13
-0.0206
27
-0.0788
14
-0.0582
28
+0.2699
14
-0.0652
28
+0.2463
29
+0.2394


15
In addition to these carrier systems, there are a number of active
efflux mechanisms. Two of these systems are located in the choriod plexus
and have affinity for organic ions. A system for the disposition of anions
has been described and divided into two subsystems.2039404142 An L
or liver system with affinity for prostaglandins, 5-hydroxyindole acetic
acid and probenicid, as well as a K or kidney system with affinity for
p-aminohippuric acid and phenol red, has been proposed. Additionally, a
carrier for the efflux of iodide has been described.4344 A cationic sys
tem is also present in the choriod plexus and this species has affinity
for N-methylnicotinamide, decamethonium and hexamethonium ions.4546
These systems appear to be important in the removal of metabolic acids and
bases. This, as well as the anionic system, is energy-dependent and can
be competitively inhibited. The presence of several energy (ATP)-dependent
systems in the capillaries indicates that this site may also be important
for active efflux.19 The high density of mitochondria in cerebral capil
laries is further support for this location.47
Movement of Compounds Across the BBB
The BBB, therefore, consists of a relatively impermeable membrane
superimposed on which are mechanisms for allowing the entrance of essential
nutrients and the exit of metabolic wastes. If a compound is to gain access
to brain parenchyma, it may do so via several routes. If the agent has
affinity for one of the carriers previously described, it may diffuse across
the BBB by association with this carrier. A compound which has a high in
trinsic lipophilicity can diffuse passively through the phospholipoidal
cell membrane matrix.1412 The pK of a compound with ionizable groups is
also important, since only the unionized species diffuses across the BBB
rapidly.4849 The ability of a substance to enter into the cell membrane


158
177.V. Galasso, Gazz. Chim. Italia., 100, 421 (1970).
178. R. W. Wagner, P. Hochmann and M. A. El-Bayoumi, J_. Mol. Spec., 54,
167 (1975).
179. G. P. Bahuguna and B. Krishna, Ind. J_. Chem., 16A, 933 (1978).
180. M. Hi rota, H. Masuda, Y. Hamada and Isao Takeuchi, Bull. Chem. Soc.
Japan, 52, 1498 (1979).
181. A. Sheinkman, M. Mestetschkin, A. Kutchenenko, N. Kliviov, U. Poltavets,
G. Malltseva, L. Palagutchkina and I. Visotskii, Khim. Geterotsik.
Soedinenii, 1096 (1974).
182. P. van de Weijer and D. van der Meer, Theor. Chim. Acta, 38, 223 (1975).
183. R. C. Bingham, M. J. S. Dewar and D. H. Lo, J_. Amer. Chem. Soc., 97,
1285 (1975).
184. N. C. Baird and M. J. S. Dewar, J. Amer. Chem. Soc., 91 352 (1969).
185.M. J. S. Dewar and E. Haselbach, J. Amer. Chem. Soc., 92, 590 (1970).
186.N. Bodor, M. J. S. Dewar, A. Harget and E. Haselbach, J_. Amer. Chem.
Soc., 92, 3854 (1970).
187. N. Bodor, M. J. S. Dewar and D. H. Lo, J_. Amer. Chem. Soc., 94, 5304
(1972).
188. J. N. Murrell and A. J. Harget, "Semi-empirical Self-consistent-field
Molecular-orbital Theory of Molecules," Wiley-Interscience, New York
(1972).
189. I. N. Levine, "Quantum Chemistry, 2nd Edition," Allyn and Bacon, Inc.,
Boston (1974).
190. N. Bodor and R. Pearl man, J.. Amer. Chem. Soc., 100, 4946 (1978).
191. N. Viswanathan and V. Balakrishnan, Ind. J_. Chem., 16B, 1100 (1978).
192. T. Takemoto and Y. Kondo, J_. Pharm. Soc. Japan, 82, 1408 (1962).
193. H. W. Bersch, Arch. Pharm., 283, 192 (1950).
194. R. H. Abeles, R. F. Hutton and F. H. Westheimer, J. Amer. Chem. Soc.,
79, 712 (1957).
195. J. J. Steffens and D. M. Chipman, J_. Amer. Chem. Soc., 93, 6694 (1971 ).
196. D. J. Creighton, J. Hajdu, G. Mooser and D. S. Sigman, J. Amer. Chem.
Soc., 95, 6855 (1973).
197.J. Hajdu and D. S. Sigman, J_. Amer. Chem. Soc., 98, 6060 (1976).


104
Further Studies on the Biological and
Chemical Properties of Dihydroberberine
The rate and nature of the oxidation of the dihydropyridines included
in the delivery scheme is important to the proper functioning of the system.
A dihydropyridine must be stable enough to be formulated and stored. The
in vivo rate of oxidation must, however, be rapid enough to efficiently
transform the delivering species and thereby avoid competing metabolisms.
The rate of oxidation of dihydroberberine (2) was therefore studied
in a variety of media, and in a number of different situations. One prob
lem which hampered these determinations was the extreme water insolubility
of the free base. The rate of oxidation of (2) was determined by both
HPLC and UV methods. The UV procedure involved measuring the appearance
of the 460 nm absorbance of berberine (1) with time, while the HPLC deter
minations were made by calculating the appearance of the absorbance due to
(1) or disappearance of the absorbance due to (2). In most cases agreement
between the two methods was good. In all determinations the spectrum of (1)
showed no change within the timeframe of the experiment. Initial oxida
tion studies were performed in areated buffer. At a pH of 7.4, (2) oxi
dized very rapidly and erratically. Buffers of lower pH were then used
to partially stabilize (2) by shifting the equilibrium in favor of the
hydrochloride in an effort to yield a more reproducible system. This
shift reduces the electronic density at the nitrogen, and precludes the
participation of the nitrogen lone pair in oxidative reactions.172 The
lower pH also greatly facilitates solubilization. The rate of oxidation
of (2) at a pH of 5.8 is shown in Table 3-13, and the spectral changes
that are characteristic of this oxidation are shown in Figure 3-14.
Although the correlation coefficients are not good, second order kinetics
are indicated with a calculated second-order rate constant of 44.4 0.53


I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Nicholas S. Bodor, Chairman
Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Kenneth B.
Assistant Professor of
Medicinal Chemistry
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Margaret 0.
Assistant Professor of
Medicinal Chemistry
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
k/.
jmes W. Simpkins
jsistant Professor of
Pharmacy
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Merle A. Battiste
Professor of Chemistry


65
quaternary compound had one of 10.6 min at a flow rate of 2 ml/min. A
standard curve was constructed in brain homogenates.
Intracerebral Ventricular (icv) Administration
Sprague-Dawley rats were anesthetized with 70 yl/100 g of pentobar
bital sodium, and atropine sulfate (0.05 mg/Kg) was given, if necessary.
Injections of (1), (6), and 3H-inulin (29) were made into the lateral
ventricles of rat brains. The injections were made with the aid of a
stereotaxic instrument and the site of the injection was -0.4 mm anterior-
posterior, 1.5 mm medial-lateral and -3.0 mm dorsal-ventral relative to
the bregma. The dose of (1) infused was 50 yg and the infusion volume
was between 3 and 5 yl. The vehicle was DMS0 and the infusion rate was
5 yl/5 min. In several experiments, (1) was coinjected with 1000 yg of
(6). In another set of experiments, a dose of 2.3 yCi of (29) was injected
icv.
At specific times after the infusions, the animals were decapitated.
The brain was homogenized in 1.0 ml of water and (1) extracted with 4 ml
of acetonitrile. Analysis of (1) was by HPLC with a mobile phase consist
ing of 50:50 acetonitrile: pH 6.2 phosphate buffer. A yBondapak Ci8 re
verse-phase column was used and a standard curve constructed using brain
homogenates. For radionuclide analysis, the brains were homogenized in
8 ml of water. Twenty-five hundredths of a milliliter of this homogenate
were added to 0.75 ml of SolueneR. After the sample dissolved, 12 ml of
the scintillation cocktail were added. The samples were counted for five
minutes and disintegrations per minute (dpm) were obtained by using a
standard quench curve.
Limited Metabolic Studies
Rats treated intravenously (iv) with 55 mg/Kg of (1) or 55 mg/Kg of
(3) were housed in metabolic cages. Urine was collected and extracted with


' 10
systems, net flux is small compared to unidirectional flux since amino
acids derived from proteins are constantly being lost and the magnitude
of this loss is similar to uptake.
The transport of neutral amino acids has been described by Christensen
and these generalizations apply to the BBB.2526 Four neutral amino acid
transport systems have been shown to occur in Ehrlich ascites, a model cell
system, and in several other systems. An L- or leucine-preferring system
is characterized by high affinity for phenylalanine, leucine, tyrosine,
tryptophan and several other large essential amino acids. It is Na+ inde
pendent, bidirectional and equilibrative in nature. The definition of this
system can be made by observing the flux of 2-aminonorbornane-2-carboxylic
acid which is exclusively transported by this carrier. The A- or alanine-
preferring system is characterized by a Na+ dependence, an energy depen
dence, and the ability to concentrate substrates. The system has affinity
for glycine, proline, alanine, serine, threonine and several other small
amino acids. The defining compound for this system is a-(methyl ami no)-
isobutyric acid. Two other amino acid transport carriers have also been
described but they are not well characterized. There is also an ASC
(alanine-serine-cysteine-preferring) system and a Gly (glycine-preferring)
system.
Since generally only large essential amino acids (L-system substrates)
are transported through the BBB, the L-system is assumed to be the dominant
mechanism in amino acid uptake.120 The absence, however, of the A-system
has been questioned. It was recently argued that the A system is present
but has a different distribution than the L-system. Betz and Goldstein dis
covered a system whose characteristics are similar to the Asystembut which is
located abluminally.27 This system may act as an active mechanism for


30
the similar Km values for these amino acids in addition to the similarity
between the Km's and plasma levels of these amino acids. Hyperphenylala-
ninemia also can reduce protein synthesis by a similar mechanism. Treat
ment of this disease involves a phenylalanine restricted diet and 5-hydroxy-
tryptophan supplements.
In hepatic encephalopathy, the neutral amino acid carrier is induced
and, as previously discussed, this induction may be related to increase in
ammonia and glutamine levels. Dihydroxyphenylalanine (L-DOPA), which is
used as a dopamine source in the treatment of parkinsonism, is transported
by the neutral amino acid carrier. It has been shown that ethanol increases
DOPA transport as well as the transport of tyrosine, tryptophan and a-
methyldopa into the CNS.103
The monocarboxylic acid carrier appears to be a major organ for elim
inating metabolic acid wastes. As was previously discussed, the Km of
lactate is close to its plasma concentration so that rises in lactate,
such as those that accompany exercise, are only slowly translated to the
CNS. In the case of anoxia, however, where cerebral levels of lactate
rise, the systemic dissipation of this byproduct is slowed by the same
phenomenon.20 In the brain the loss of lactate is carrier and not diffusion-
limited, unlike other organs. In hypoglycemia, lactate may act as an energy
source.105 This is true in neonates, where lactate uptake is much higher
than in adults. The Km of the monocarboxylic acid carrier is also corre
spondingly higher in neonates.
Ketone bodies, which have an affinity for the monocarboxylic acid car
rier, are produced during fasting and can act as a metabolic energy source.
These bodies include B-hydroxybutyric acid and acetoacetic acid. The en
zyme responsible for their production, e-hydroxybutyrate dehydrogenase, is


52
The product was a clear liquid and the yield was 62%: XH NMR (CDC13)
9.2 (1 H, s), 8.8 (1 H, m), 8.2 (1 H, m), 7.4 (1 H, m), 4.3 (2 H, t),
1.6 (4 H, m), 0.97 (3 H, t); IR (NaCl) v (C-H) 2962, (C=0) 1730, (C-C,
C-N) 1594, (-C-C(=0)0) 1288, (0-C-C) 1117, (y CH) 742, (g ring) 702.
3-P.yridinecarbonylchloride Hydrochloride/Nicotinoyl Chloride Hydrochloride
(10)
Forty-one grams (0.33 moles) of nicotinic acid were stirred in an ice
bath with 110 ml of thionyl chloride (S0C12) slowly added. After the
addition was complete, the mixture was refluxed for three hours. The
S0C12 was removed under reduced pressure and traces of S0C12 were azeo-
troped off with benzene. The white crystalline product was obtained in
94% yield; NMR and IR were identical with the literature.
3-Pyridinecarboxylic Acid Hexyl Ester/Hexyl Nicotinate (11)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry dis
tilled pyridine and 5.73 ml (0.062 moles) of 1-hexanol. The solution was
refluxed in an oil bath for six hours. The solution was then poured over
ice which had been made basic with ammonia. The aqueous solution was ex
tracted with ethyl ether, the organic layer dried over Na2S0M and the
solvent removed under reduced pressure. The yield was 60%: XH NMR (CDC13)
9.1 (1 H, s), 8.7 (1 H, m), 8.2 (1 H, m), 7.3 (1 H, m), 4.3 (2 H, t),
1.2 (8 H, m), 0.87 (3 H, t); IR (NaCl) v (C-H) 2938 and 2961, (C=0) 1726,
(C-C, C-N) 1590, (-C-C(=0)0) 1282, (0-C-C) lili, (y CH) 741, (g ring) 702.
3-Pyridinecarboxylic Acid Octyl Ester/Octyl Nicotinate (12)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry pyri
dine and 9.75 ml (0.062 moles) of 1-octanol. The solution was heated to
reflux in an oil bath and the progress of the reaction monitored with TLC.
After eight hours, the liquid was poured over ice which had been made basic
with ammonia. The aqueous solution was extracted with ethyl ether. The


Figure 3-30. The LD50 Dose-response Curve of Berberine (1) (A) and Dihydroberberine
Hydrochloride (3) (). Doses of (1) or (2) were Administered i p in CD-I Mice
-pi
-p*


60
pKa = pH log
aobs ~ aHA
a._ a ,
A obs
where aQbs is the absorbance in buffer, a^- is the absorbance in base and
a,, is the absorbance in acidic media.
HA
Oxidation of Dihydroberberine (2) by Silver Nitrate
Two hundred milligrams of (2) were dissolved in 95% aqueous ethanol.
Upon addition of a 10% solution of silver nitrate, a black precipitate
formed. Centrifugation and analysis of the supernatant shows stoichio
metric oxidation of (2).
Oxidation of Dihydroberberine (2) by Pi phenyl pi cry!hydrazyl Free Radical (DPP*)
Two hundred milligrams of (2) were dissolved in acetonitrile. To this
was added a solution of DPP in acetonitrile which caused an immediate dis
appearance of the purple color due to DPP-. Ultraviolet analysis confirmed
this oxidation.
Oxidation of Dihydroberberine (2) by Concentrated Hydrogen Peroxide
Two hundred milligrams of (2) were dissolved in 95% aqueous ethanol.
A 30% solution of hydrogen peroxide was added and the system monitored by
UV. The analysis demonstrated a rapid and complete oxidation of (2).
Oxidation of Dihydroberberine (2) in Buffers
The oxidation of (2) was determined by UV and an HPLC method. In the
UV method, a solution of (3) was prepared and pipetted into buffers of
various pH and at various temperatures. The changes of absorption at
460 nm were measured with time. Data acquisition was facilitated by an
Apple II microprocessor and an enzyme kinetic software package. In the
HPLC method, two buffers, pH 5.8 and pH 7.4, were used. The samples were
maintained at 37C in a water bath. At certain times, 5 pi of the solu-
lution were injected onto a yBondapak C18 reverse-phase column, and the


57
of ethyl ether were then added so that the dihydro would be extracted
upon formation. This two-phase system avoided tetrahydropyridine produc
tion. The reaction proceeded for one hour under nitrogen. The ether layer
was removed and the aqueous layer extracted. The combined ether fractions
were dried over Na2S04 and the solvent removed under reduced pressure. The
resulting orange-red oil was dried in vacuo: NMR (CDC1J 6 6.9 (1 H, d),
5.6 (1 H, m), 4.8 (1 H, m), 4.1 (2 H, g), 3.1 (2 H, m), 2.9 (3 H, s), 1.2
(3 H, t); UV 358 nm Xm3 ; Elemental analysis calculated %: C, 64.67; H, 8.17;
max
N, 8.43. Found %: C, 64.65; H, 7.88; N, 8.34 (C9H13N02).
1.4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Butyl Ester/Butyl 1,4-Dihy-
dro-N-methylnicotinate (24)
A solution of 4.6 g of NaHC03 and 3.12 g of (16) (0.016 moles) was
prepared in 100 ml of water. The solution was cooled and 6.96 g of Na2S20i+
were added. Two hundred milliliters of ethyl ether were added and this
mixture stirred under nitrogen for one hour. The ether layer was removed,
dried with Na2S04 and reduced in volume. The resulting oil was dried in
vacuo: NMR (CDC13) 6.9 (1 H, s), 5.6 (1 H, m), 4.7 (1 H, m), 4.1
(2 H, t), 3.1 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 358 nm Xmax; Ele
mental analysis calculated %: C, 67.69; H, 9.07; N, 7.23. Found %: C,
67.58; H, 8.82; N, 7.09 (CuH17N02).
1.4-Di hydro-1-methyl-3-pyridinecarbox,ylic Acid Hexyl Ester/Hexyl 1,4-
Dihydro-N-methylnicotinate (25)
A solution of 4.6 g of NaHC03 and 3.57 g of (17) (0.016 moles) was
prepared in 100 ml of water. The solution was cooled and 6.96 g of
Na2S204 were added. Two hundred milliliters of ethyl ether were added
and the mixture stirred under nitrogen for one hour. The ether layer was
separated, dried and reduced in volume. The dried oil was orange in color:
XH NMR (CDC13) 6 6.8 (1 H, s), 5.5 (1 H, m), 4.6 (1 H, m), 4.0 (2 H, t),


Copyright 1982
by
Marcus Eli Brewster III


48
ternary Beckman system consisting of three Model 112 solvent delivery
systems, a Model 160 absorbance detector and a Model 421 controller. In
some studies, a Varan FluorichromR detector was used with one of the
Beckman pumps. Thin-layer chromatography (TLC) was performed on EM Re
agents Cat. 5751 Aluminum Oxide 60 F-254 precoated plates (layer thickness
0.25 mm) or Analtech, Inc. UniplateR, which are precoated with silica gel
G at a thickness of 0.25 mm. Tissues were homogenized by a VirTis 45
homogenizer or by a teflon pestle and ground glass tube. In potentiomet-
ric titrations, a Radiometer-Copenhagen PHM 84 Research pH meter was used.
A Sage Instrument Co. Model 341A syringe pump was used for infusions.
For radionuclide studies, a Packard Tri-Carb 460 CD Liquid Scintillation
system was employed.
In the theoretical studies, the MINDO/3 program was modified to suit
the University of Florida's IBM System/370 computer. The molecule draw
ing program, X3DM0L, was developed by E.W. Phillips.
All chemicals used were of reagent grade. Berberine was obtained
from Sigma Co. while nicotinic acid, nicotinamide, methyl iodide, benzyl
bromide, butanol, hexanol, octanol, decanol, sodium borohydride and sodium
dithionite were obtained from Aldrich Co. Phenethyl alcohol was purchased
from Matheson, Coleman and Bell. Tritiated inulin, SolueneR and scintilla
tion cocktails were obtained from New England Nuclear. Solvents were of
chromatographic purity and were obtained from Fisher Co. Pyridine (Aldrich
Co.) was refluxed over CaH2 and distilled before using. In cases where
oxygen was to be excluded, solutions were made from water which, after
boiling for fifteen minutes, was cooled with a stream of pyrogallol-
scrubbed nitrogen passing through it. A phosphate buffer (pH 7) was made
by dissolving 1.183 g of potassium dihydrogen phosphate and 4.320 g of
disodium hydrogen phosphate in water and diluting to 1.0 a.


16
is often correlated with its in vitro octanol :water partition coefficient.5051
This is a measure of lipophilicity, and can be correlated with biological
effects.52 These correlations can be extended to the permeability of com
pounds through the BBB. Several examples of this correlation have appeared
in the literature, including the opiates morphine, codeine, and heroin.52
Good correlation is obtained between lipophilicity, the ability to pass
the BBB, and narcotic efficacy.
The increase in the ability of a compound to pass membranes can, all
too often, be correlated with an increase in undesirable side effects. An
example of this was shown in a series of B-blockers in which lipophilic
members of this pharmacologic group, i.e. propranolol penetrated the BBB
rapidly but demonstrated a number of deleterious psychiatric manifestations.53
Conversely, B-blockers of lower lipophilicity exhibit an attenuation of
these side effects.
These two avenues, namely passive diffusion and carrier mediation,
represent the major components of influx. Other minor mechanisms may
also allow the entry of substrates into the CNS. The cell bodies of many
neurons are located centrally while their axons may penetrate into the
periphery. These axons can take up material and transport it in a retro
grade fashion to the CNS.754 This retrograde axoplasmic transport has
been observed in such areas as the nucleus ambiguus and the abducen nucleus.
In some cases, however, the endocytosed material is reacted with lysosomes
and, therefore, this transport route may have a protective function.
There are several areas of the brain which lack a BBB.1855 These
include such locations near the ventricles as the area postrema, the sub
fornical organ, the median eminence of the neurohyphosis, the organum vas-
culosum of the lamina terminal is, and the choriod plexus. Collectively,


153
65. T. H. Maren in "Medical Physiology," 14th ed., V. B. Mountcastle
(ed.j, C.V. Mosby Co., St. Louis (1980) p. 1218.
66. K. Welch and V. Friedman, Brain, 83, 454 (1960).
67. L. D. Prockop, L. S. Schanker and B. B. Brodie, Science, 134,
1424 (1961).
68. H. Davson, C. R. Kleeman and E. Levin, J_. Physiol., 161 126 (1962).
69. S. E. Mayer, R. P. Maickel and B. B. Brodie, J_. Pharmacol. Exp. Ther.,
128, 41 (1960).
70. A. R. Rothman, E. J. Freireich, J. R. Gaskins, C. S. Patlak and
D. P. Rail, Am. vh Physiol., 201 1145 (1961 ).
71. S. I. Rapoport, M. Ohata and E. D. London, Fed. Proc., 40, 2322
(1981).
72. N. Ogata, N. Hori and N. Katsuda, Brain Res., 110, 371 (1976).
73. W. M. Pardridge and L. J. Meitus, Clin. Invest., 64, 145 (1979).
74. W. M. Pardridge and L. J. Meitus, J_. Neurochem., 34, 463 (1980).
75. W. M. Pardridge and L. J. Meitus, J_. Neurochem., 34, 1761 (1980).
76. M. W. Brightman, I. Klatzo, Y. Olsson and T. S. Reese, Neurol.
Sci., 10, 215 (1970).
77. E. Westergaard, Acta Neuropath., 39, 181 (1977).
78. K. Ohno, W. R. Fredericks and S. I. Rapoport, Surg. Neurol., 12,
323 (1979).
79. H. R. Kaplan, Fed. Proc., 40, 2250 (1981).
80. B. B. Johansson, Acta Pharmacol. et Toxicol., 48, 242 (1981).
81. H. B. Dinsdale, Adv. Neurol., 20, 341 (1978).
82. S. M. Mueller and D. D. Heistad, Hypertension, 2, 809 (1980).
83. E. Westergaard, J_. Neural Trans. Suppl., 14, 9 (1978).
84. F. Joo, Experientia, 28, 1470 (1972).
85. F. Joo, Z. Rakonczay and M. Wollemann, Experientia, 31, 582 (1975).
86. P. M. Gross, G. M. Teasdale, W. J. Angerson and A. M. Harper,
Brain Res., 210, 396 (1980).


61
peak heights analyzed. The mobile phase was 60:40 acetonitrile: pH 6.2
phosphate buffer and the flow rate was 2 ml/min.
Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro-
pyridines (21) and (22) in Hydrogen Peroxide
Solutions of (3), (21) and (22) were prepared. An aliquot of these
solutions was added to a standardized solution of hydrogen peroxide (H202)
(0.18 M). The appearance of the 460 nm peak of (1) or the disappearance of
the 359 nm peak of the dihydronicotinamides was measured. This determina
tion was made using the enzyme kinetics software package.
Quantitation of the Oxidation of Dihydroberberine (21) and Various Dihydro-
pyridines (22)-(28) in Plasma
Freshly drawn 80% human plasma was obtained from Civitan Regional Blood
Center. The oxidation of (3) was determined by HPLC and the oxidation of
(3), (22), (23), (24), (25), (26), (27), and (28) by UV. In the HPLC analy
sis, a solution of (3) was added to 80% plasma and maintained at 37C. At
certain times, 1.0 ml of plasma was removed and treated with 3 ml of aceto
nitrile. The solution was centrifuged and 5 pi of the supernatant was
analyzed by a pBondapak Cis reverse-phase column with a mobile phase of
60:40 acetonitrile: pH 6.2 phosphate buffer. The peak heights were ana
lyzed and concentrations obtained from a standard curve. In the UV method,
40% plasma was maintained at 37C in a kinetic cell. A solution of either
(3) or one of the various dihydronicotinates was added to this and the
appearance of the 460 nm absorbance of (1) or disappearance of the 359 nm
absorbance of dihydronicotinates was observed.
Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro-
pyridines (22)-(28) in Liver Homogenate
The determination of the rate of oxidation of (3) in a liver homogenate
by an HPLC method and (3), (22), (23), (24), (25), (26), (27), and (28)
by a UV method was performed. In the HPLC method, 14 g of fresh rat liver


99
Figure 3-10. A Computer-assisted Drawing of the Most Stable
Conformation of the Isoquinoline Model (30) at
25C


% DOSE/GRAM TISSUE
TIME (MIN)
Figure 3-25. A Comparison of the Efflux of Berberine from the Kidneys when Berberine (1) is
Administered i v at a Dose of 35 mg/Kg () and Dihydroberberine Hydrochloride
(3) when Administered i v at a Dose of 55 mg/Kg (O)


42
component of a molecule, as it is in many pharmacologically active agents,
the molecule is provided with an internal delivery moiety. The drug to
be delivered and the carrier are therefore merged into one molecule. The
pharmacokinetics of this scheme is also simpler than those involved with
the carrier system since no cleavage is necessary of the delivered mole
cule from the carrier. In this approach, which appears in Figure 1-3,
an appropriately chosen pharmacologically active compound i.e., one which
contains a pyridinium moiety, would be reduced to its corresponding di
hydropyridine. This lipophilic species would penetrate the BBB as well
as into the systemic circulation. After a period of time determined by
the stability of the compound, it would be oxidized to the parent quater
nary salt (kox). Systemically, this agent would rapidly be eliminated
by filtration or by tubular secretory mechanisms (kg^)- In the CNS,
however, since the ability of the compound to freely diffuse would be lost,
it would be delivered fairly specifically (kout2 > kouti) The transit
time of the drug in the brain would depend upon a number of factors in
cluding the participation of the compound in any active efflux processes.
It would be hoped that in most cases the rate at which the compound entered
the brain would be faster and ideally, much faster than the rate at which
the compound left the brain (kouf|)*
This system, unlike the carrier mediated chemical delivery system,
can be considered a prodrug. This prodrug should, however, demonstrate
a specificity for the brain because of its design, the characteristics
of the BBB, and the chemistry of dihydropyridines. Again, this specificity
should increase the therapeutic index of the drug delivered.
Several criteria were used in choosing a molecule for these delivery
approaches. Obviously, the compound should contain a pyridinium moiety


Figure Page
3-23 Distribution of Berberine after iv Administration of 55
mg/Kg of Dihydroberberine Hydrochloride (3) into the Kidney
(Oh Liver (), Lung (O), and Brain (A) 127
3-24 A Comparison of the Efflux of Berberine from Lungs when Ad
ministered iv as 55 mg/Kg of Dihydroberberine Hydrochloride
(3) (O) or 35 mg/Kg of Berberine (1) () 128
3-25 A Comparison of the Efflux of Berberine from the Kidneys when
Berberine (1) is Administered iv at a Dose of 35 mg/Kg (A)
and Dihydroberberine Hydrochloride (3) when Administered iv
at a Dose of 55 mg/Kg (O) 129
3-26 A Comparison of the Efflux of Berberine from the Liver when
Berberine (1) is Administered iv at a Dose of 35 mg/Kg (B)
and Dihydroberberine Hydrochloride (3) when Administered iv
a Dose of 55 mg/Kg () 130
3-27 A Comparison of the Efflux of the Total Berberine, i.e. (1)
and (2) from the Brain when either 55 mg/Kg of Dihydrober
berine Hydrochloride (3) is Administered iv (A) or 55 mg/Kg
of Dihydroberberine (2) and 200 mg/Kg of 1-Methyl-l,4-
dihydronicotinamide (21) is Administered iv () 134
3-28 Efflux of 1-Benzylnicotinamide Bromide (7) from the Brain af
ter iv Administration of 60 mg/Kg of 1-Benzyl-1,4-dihydro
nicotinamide (22) (A) 135
3-29 Efflux of Berberine from the Brain after icv Injection of
either 50 yg of Berberine (1) () or 50 yg of Berberine (1)
and 1000 yg of 1-Methylnicotinamide Iodide (6) (A) 137
3-30 The LD50 Dose-response Curve of Berberine (1) (A) and Dihy
droberberine Hydrochloride (3) (). Doses of (1) or (2)
were Administered ip in CD-I Mice 144
xi


136
from the N-benzyl quaternary compound (7) from the brain is 5.8 hours,
which is twice as fast as the efflux of (1).
These studies indicate that there was no effect of the quaternary
compound N-methylnicotinamide (6) on the efflux of (1) from the brain.
The concentration difference, however, between (6) and (2) was only a
factor of four at the injection, and this difference is even less at the
level of the brain. To overcome this quantitative objection, a series of
intracerebral ventricular (icv) injections was performed. The purpose of
injecting the compound icv was to allow direct introduction of high con
centrations of (1) and (1) with the putative competitive inhibitor, (6),
into the brain. The injections were made into the lateral ventricles of
rats with the aid of a stereotaxic instrument and an infusion pump. A
dose of 50 yg of (1) or 50 yg of (1) and 1000 yg of (6) was administered
and the compounds were dissolved in DMS0. The volume of the dose was 3-5
yl. The results of these studies are shown in Figure 3-29. It can be
seen that (6) has little effect on the efflux of (1) from the brain. The
tx obtained from the terminal portion of the curves was 3.8 hours for the
2
efflux of (1) and 3.1 hours for the efflux of (1) when coinjected with (6)
The fact that the t obtained for (1) in this experiment is much slower
2
than that obtained when (1) is administered systemically as (3) is not
surprising. These icv injections are similar to intrathecal injections
in that even or complete distribution does not occur. Therefore, (1) is
basically restricted to the CSF. In the case of systemic administration
of (3), however, the distribution of (2) is fairly complete and even in
the brain. The efflux of (1) after administration of (3) is limited by
the diffusion of (1) from the brain tissue, and from the discussion of the
BBB, this is a slow and inefficient process. When (1) is administered icv


4
view of the capillaries, will add greatly to the structural knowledge of
these tight junctions but the method is technically difficult. Recently,
a freeze-fracture technique was applied to the cerebral vasculature of a
chameleon, which possesses a BBB similar to that of mammals.13 In these
animals a series of ridges and grooves is seen. The ridges are connected
to neighboring ridges by an anastomosing network. In general, the more com
plex this system is, i.e. the number of ridges it has, the tighter the junc
tion is. The tightness of the junction can be assayed not only structurally
but also by measuring ionic conductance and resistance through the junction.
The ionic conductance is low for most ions.1214
Systemic capillaries lack this closed junction. Morphologically, this
can be traced to a lack of continuity in the intercellular appositions.
In cardiac muscle, for example, the ratio of junctional width to the width
of the cell membrane is 2.4, while in cerebral capillaries this is reduced
to 1.7.10 These open junctions allow a high degree of nonspecific transport
of nutrients and other compounds into systemic capillaries. Materials pass
easily between these leaky cells, while in the brain the sealing of the
intercellular fissures severely restricts this nonspecific transport. Since
intercellular transport is removed, only intracellular transport remains.
Lipophilic compounds can readily pass through these phospholipoidal membranes,
but hydrophilic compounds and compounds with high molecular weights are ex
cluded. Systems are available to transport small hydrophilic nutrients,
and this will be discussed later.
A second difference between cerebral and systemic capillaries is the
paucity of vesicles and vesicular transport in the CNS.47 Vesicular trans
port is a process for transcellular transport and, as such, vesicles are
transported from the luminal to the abluminal membrane. Pinocytotic acti
vity, on the other hand, is concerned with the nutritional requirements of


45
Berber'ne is widely distributed in the plant kingdom and is found
in such families as menispermaceae and berber'daceae, to name only two.137
The compound was isolated by 1826 by Pelletan and Chevallier.
Biosynthetically, berberine is interesting because of the presence
of the so-called berberine bridge. This term refers to the single carbon
atom between the nitrogen and the methoxylated aromatic nucleus. Two post
ulates for the formation of this have been suggested. The first involves
formaldehyde in a Mannich-type ring closure while the second involves an
TOO IOQ #
oxidative cyclization of an N-methyl group. Labelling experiments
favor the second mechanism.139
There are several total syntheses for berberine in the literature
and the chemistry of the protoberbine alkaloids is well reviewed.140-142
Berberine has a variety of pharmacological actions and several therapeutic
uses. Pharmacologically, it exhibits a depressive action on excitable
tissues,143 induces hypotension and tachycardia,144145 and inhibits a
number of enzymes including histaminease (human pregnancy plasma diamine
oxidase),146 cholinesterase,144 dopamine-adenyl cyclase,147 and cation-
dependent ATP phosphorylases.148 Berberine also possesses an antiheparin149
and local anesthetic activity.144 Because of the affinity of berberine
for dopaminergic receptors150 and alcohol dehydrogenases,151 it has been
used to characterize geometric and stereospecific requirements for sub
strate binding to these enzymes. Berberine has long been known as an anti
biotic. The alkaloid causes mutations in certain bacterium by affecting
nonchromosomal genetic material.134 Berberine has been used mostly in
India to treat cholera,152 diarrhea,153 leshmaniasis and other parasitic
infections.


TABLE PAGE
3-14 The Relative Rates of Oxidation of Dihydroberberine (2),
1-Methyl-1,4-dihydronicotinamide (21), and 1-Benzyl-1,4-
dihydronicotinamide (22) in Dilute Hydrogen Peroxide 108
3-15 Proton Assignments of the 3H NMR of the 1-Methyl-1,4-dihydro-
nicotinic Acid Ester (27) 112
3-16 The Rates of Oxidation and Corresponding Correlation Coef
ficients of Various 1-Methyl-l,4-dihydronicotinic Acid Esters
and 1-Benzyl-l ,4-di hydronicotinamide (22) 114
3-17 The Effect of Glucose on the Movement of Berberine into Red
Blood Cells 118
3-18 Slow Infusion of Dihydroberberine (3) 131
3-19 Efflux of 3H-Inulin from the Brain after Intracerebral Ventri
cular Administration 139
3-20 In Vivo Metabolism of Berberine and Dihydroberberine in the
RatTHPLC) 140
3-21 In Vivo Metabolism of Berberine and Dihydroberberine in the
Rat-(TLC) 142
3-22 Probit Analysis of the LD50 Study 145
3-23 Effect of Berberine (1) and Dihydroberberine Hydrochloride (3)
Against P388 Lymphocytic Leukemia 146
vi i i


DIHYDROBERBERINE
ro
Figure 3-4- Mass Spectrum (70 eV, El) of Dihydroberberine (2)


62
were homogenized in 45 ml of cold phosphate-buffered saline. To this was
added a solution of (3), and the system was maintained at 37.0C. At vari
ous times, 1.0 ml of the solution was removed and the protein precipitated
with 3 ml of acetonitrile. The sample was centrifuged and 5 yl of the
supernatant analyzed using a yBondapak C18 reverse-phase column with a
mobile phase of 60:40 acetonitrile: pH 6.2 phosphate buffer. The peak
heights were analyzed and concentrations obtained from a standard curve.
The UV method involved homogenizing 7 g of rat liver in 15 ml of pH 7.4
phosphate buffer and diluting the homogenate to 200 ml (3.5% w/v). The
homogenate was centrifuged and the supernatant was used. To this was added
a solution of (3) or one of the dihydropyridines. The appearance of the
460 nm peak of berberine or the disappearance of the 359 nm peak of the
dihydronicotinate was then measured.
Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro
pyridines (22)-(28) in Brain Homogenate
Again, both an HPLC and UV method were employed in the determination
of the rate of oxidation of (3), (22), (23), (24), (25), (26), (27) and
(28). In the HPLC method, a solution of (3) was added to a 20% brain homo
genate in pH 7.4 phosphate buffer. The homogenate was maintained at 37C
in a water bath. At various times, 1.0 ml of the homogenate was mixed
with 3 ml of acetonitrile. The sample was centrifuged and the supernatant
analyzed by the same method used in the liver homogenates. In the UV
method, 2 g of freshly obtained rat brain were homogenized in 33 ml of
pH 7.4 phosphate buffer, yielding a 6.0% w/v homogenate. The homogenate
was centrifuged and the supernatant was used in the determinations. To
this was added a solution of (3) or one of the dihydronicotinates. The
appearance of 460 nm absorption of (1) or disappearance of the 359 nm peak
of the dihydronicotinate was measured.


33
agents have lent themselves to this type of manipulation. Several amides,
for example, of the highly water soluble anti cancer agent guanazole have
been made. This series of lipoidal compounds hydrolyzed in vivo to yield
the parent drug.111 A more sophisticated prodrug is cyclophosphamide,
which is inactive in vitro. The agent is activated by P450 mixed function
oxidases in the liver to the potent alkylating agent, N,N-bis(chloroethyl)-
phosphordiamidic acid.109 This drug is extensively used in cancer chemo
therapy.
One of the most important applications of prodrugs is in the sustained
release of therapeutically active agents. Cytosine arabinoside is used
as an S-phase specific antimetabolite, but suffers from rapid metabolism
by cytidine deaminase requiring a continual administration of the drug.
This problem produced the prodrug cyclocytidine which is not a substrate
for cytidine deaminase and which slowly releases the cytosine arabinoside
by ring cleavage.112
The influx of a compound to any organ can be related by the equation
Kp = QE
where Kp is the clearance of a drug by an organ, Q is the blood flow
through that organ and E is the extraction coefficient.113 The extraction
coefficient is related to the lipophilicity or octanol-water partition
coefficient of a compound which is in turn related to the ability of a
compound to partition into phospholipoidal membranes and consequently in
to organs. By increasing the lipophilicity of a compound with the pro
drug approach, one can increase the entry of a compound into its site of
action but this is not specific and, in general, all organs are exposed
to a greater tissue burden.113 Nonspecificity is, therefore, one of the
major drawbacks of the prodrug approach. This is especially important


5
the cell and, therefore, involves vesicular movement from the luminal mem
brane to a cell organelle, presumably a lysosome.
Cerebral endothelial vesicles are usually uncoated and few in number
compared to other systems. Using electron microscopic morphometry, five
vesicles per micrometer luminally and thirty-forty vesicles per micrometer
abluminally were found.4 In the diaphragm and in myocardial vessels the
values are much higher, being seventy-eight and eighty-nine vesicles per
micrometer, respectively. This lower content of vesicles is another mecha
nism by which the CNS can limit nonspecific influx. A third difference
is the lack of fenestra in the cerebral capillaries. These differences
are demonstrated schematically in Figure 1-1.
Cerebral vessels have a number of perivascular accessory structures
which appear to be involved in BBB function.4 While it is known that the
astrocytic end feet are not involved as a barrier per se, their role in
attenuating BBB action is interesting. These glial end feet may be involved
with regulation of amino acid flux. They also appear to engulf protein
which breeches the BBB and, therefore, may act as a second line of defense.15
Phagocytic pericytes which are present abluminally may play a similar role.
It is possible that the basement member of endothelial capillaries acts as
a mass filter preventing large molecules from penetrating it.
In addition to these structural features the BBB maintains a number of
enzymes which appear to augment barrier function.21617 Since optimal
neuronal control requires a careful balancing between neurotransmitter
release, metabolism, and uptake, it is of vital importance to restrict the
entry of blood-borne neurotransmitters into the CNS. It is not surprising,
therefore, to find high concentrations of such enzymes as catechol-0-methyl
transferase (COMT), monoamine oxidase (MAO), y-aminobutyric acid trans
aminase (GABA-T) and aromatic amino acid decarboxylase (DOPA decarboxylase)


Figure 3-S. Structures and Salient Numbering Protocols for Berberine (1), Dihydroberberine (2),
the Isoquinoline Model (30), the Dihydroisoquinoline Model (31), Pyridine (32), and
1,2-Dihydropyridine (33).


3
Progress was somewhat slowed because of the lack of appropriate electron
microscopic tracers.7 The first tracers used were saccharated iron oxides8
or ferritin (molecular weight 560,000) whose limits of resolution were close
to the thickness of the endothelium itself. It was not until the intro
duction of horseradish peroxidase (HRP) and microperoxidase (MP) that the
exact structure of the BBB could be deduced. Horseradish peroxidase is a
relatively small enzyme (molecular weight 43,000) which, unlike ferritin,
does not contain an electron-dense core but rather produces a material
which has a high affinity for osmium tetraoxide and other radiopaque sub
stances .7
By using HRP, Reese and Karnovsky demonstrated the inability of the
marker to pass from the lumen of the cerebral capillary.910 In fact, HRP
was never found in the extracellular space surrounding the capillary. Addi
tionally, when HRP was injected directly into the brain it readily passed
the astrocytic end processes and was stopped at the endothelial membrane.
The anatomical basis of the BBB was, therefore, isolated to the endothelial
lining of the cerebral capillaries and not a perivascular site. There are
several ultrastructural differences between systemic capillaries and cere
bral capillaries which account for their general impermeability.14
The manner in which endothelial cells of the cerebral capillaries are
joined is distinct from systemic capillaries. Cerebral junctions are char
acterized as tight or closed junctions meaning the cells closely approximate
each other. These junctions gird the cell circumferentially, forming a
zona occludens and providing an absolute barrier to HRP. Structurally,
the junctions consist of aligned intramembranous ridges and grooves which
are in close apposition.11 These tight junctions have been examined by thin
section electron microscopy and attempts are now underway to examine them
by a freeze-fracture technique.412 This method, which allows a longitudinal


Table 3-4. The Heats of Formation, Vertical Ionization Potentials, and Dipole Moments of the Isoquinoline
Model (30) and the Dihydroisoquinoline Model (31).
ISOQUINOLINE MODEL (30)
DIHYDROISOQUINOLINE MODEL (31)
95.1
HEAT OF FORMATION
-39.3
(kcal/mol)
11.56(h)
VERTICAL I.P.
7.1 9(tt-Pn)
12.94(h-Pn)
(eV)
8.31(h)
9.07(Pn)
9.44(a)
9.58(a)
15.43
DIPOLE MOMENT
1.73
(Debye)


143
be to minimal in vivo, two minor metabolites have been identified. Furuya
described a urinary metabolite which apparently contained a carboxylic acid
moiety.211 Another study reported that very small amounts of tetrahydro-
berberine were present in the urine.208 Identification of the metabolites
found in the present study was not attempted.
The importance of these studies to the proposed drug delivery scheme
is related to the requirement of the scheme that the principal and, ideally,
only metabolism of (2) is to (1). This was shown to be the case. In ad
dition, no metabolite was present in the urine extracts of animals dosed
with (3) which was not present in the urine extracts of animals dosed
with (1).
Toxicity and Anti cancer Activity of Dihydroberberine
The toxicity of (1) and (3) was determined in mice and the results
are shown in Figure 3-30 and Table 3-22. The data were analyzed by the
method of Probits as well as by fitting the data to a sigmoid dose-response
curve. The lethal dose for 50% mortality (LD50) for (1) was found to be
37.0 mg/Kg, and that of (3), 58.2 mg/Kg. The injections were made in-
traperitoneally. The value obtained for (1) was in good agreement with
other values reported in the literature.144212 The toxicity of (1) is
60% higher than that of (3), substantiating a prediction made by the
theoretical calculations. The anti cancer activity of (1) and (3) is
presented in Table 3-23. First, the ability of (1) or (3) to inhibit
the growth of KB cells in vitro was investigated. The ID50 calculated
for (3) was 2.2 yg/ml while that calculated for (1) was 0.95 yg/ml.
This is again constant with the higher toxicity of (1) relative to (3).
The next series of experiments involved inoculating mice with P388
lymphocytic leukemia cells. If this is done ip, (1) and (3) are equipotent


93
relatively large contribution to the HOMO is made by the methylene hydro
gens 27, 28 and 24, 25 and the nonaromatic carbon, C2. This phenomenon
is termed hyperconjugation. Hyperconjugation also occurs in simple 1,2-
and 1,4-dihydropyridines but, because of the additional methylene inter
actions, the effect is slightly larger in the case of (31).190 The large
contribution to the HOMO by the nitrogen lone pair is also noted.
The vertical ionization potentials (IP) of (30) and (31) were calcu
lated using Koopman's Theorem which simply states that the ionization po
tential is the negative of orbital energy. The calculated values are
presented in Table 3-4 and are 11.56 eV for (30) and 7.19 eV for (31).
In addition, the character of the orbital which loses the electron can be
obtained by examination of the HOMO and, for (30), a ir-type system is in
volved while for (31) a mixed u-PN type orbital occurs. These values are
similar to values obtained from other systems and are consistentwith the
molecular structures.190 The dipole moments calculated for (30) and (31)
also appear in Table 3-4 and, again, are consistent with the molecular
structure.
It is instructive to compare changes that occur upon reduction of
simple dihydropyridines to those that occur upon reduction of (31). This
cmparison demonstrates the similarity in chemistry between simple and
more highly conjugated dihydropyridines and also demonstrates the extreme
usefulness of the computational method. Tables 3-10 to 3-12 are comparisons
of the pyridine (32) J dihydropyridine (33) system and the pyridinium
nucleus of (30) £ (31) system. The values for the (32) £ (33) system
were calculated by a MIND0 procedure by Bodor and Pearl man in 1976.190 The
numbering of these various compounds appears in Figure 3-8.
Table 3-10 shows the bond lengths in the isoquinoline model system,
(30) + (31), and the simple pyridine system, (32) £ (33). Upon reduction


59
Na2So04 were added. Two hundred milliliters of ethyl ether were added and
the mixture stirred over N2 for one hour. The ether layer was separated,
dried and reduced in volume. The dried oil was orange in color: 1H NMR
(CDC13) 7.2 (5 H, s), 6.9 (1 H, m), 5.7 (1 H, m), 4.7 (1 H, m), 4.2 (2 H,
t), 3.0 (2 H, m), 2.9 (2 H, t), 2.8 (3 H, s); UV 355 nm Amax; Elemental
analysis calculated %: C, 70.18; H, 6.63; N, 5.46. Found %: C, 70.27; H,
7.00; N, 5.10 (C15H17N02-^-H20).
Characterization of Dihydroberberine
Distribution Coefficients
Fifty milliliters of a cold 1 x 10"4 M solution of berberine (1) in
pH 7.4 buffer were partitioned against 50 ml of CHC13 or 50 ml of 1-octanol.
The concentration of (1) was determined spectrophotometrically in the organ
ic and aqueous layer. A stock solution of 2.7 x 103 M dihydroberberine
hydrochloride (3) was made in methanol. An aliquot of this, sufficient to
produce a 1 x 104 M solution, was pipetted into 50 ml of cold pH 7.4 buf
fer and extracted immediately with either CHC13 or 1-octanol. After allow
ing for oxidation, the concentration of (1) in the organic and aqueous layer
was determined spectrophotometrically.
Potentiometric pKfl Determination of Dihydroberberine (2)
Due to the extreme water insolubility of (2) (< 3 ug/ml), all deter
minations were done in 25% methanolic solutions. A titration curve was
generated by adding 10 ul aliquots of NaOH to a 1.0 mM solution of (3).
The pKa was determined by inspection of the titration curve. During the
experiments, all solutions were covered with a stream of nitrogen.
Spectrophotometric pKa Determination of Dihydroberberine (2)
The pKa of (2) was determined by measuring the absorbance difference
at 355 nm in basic, acidic, and buffered media. The relationship that
allows this determination appears below:


126
The tissue distribution of dihydroberberine hydrochloride (3) was
investigated in an analogous manner. The compound (3) is taken up by tis
sues to a greater extent than (1) and is handled well by the kidney. The
concentration of (1) observed in the brain is high. A comparison of the
time courses of (1) after administration of either (1) or (3), in various
organs appears in Figures 3-24 to 3-26. These results shown are consistent
with the greater ability of (3) to penetrate tissues. The liver (Figure
3-26) is noteworthy because (1) is known to be significantly excreted by
a biliary mechanism.
In order to better illustrate the specificity of the (1) Z (2) sys
tem, (3) was infused intravenously. The results of these infusions are
presented in Table 3-18. In these administrations the standard dose of
55 mg/Kg of (3) was given over a period of either thirty or forty-five
minutes. If a comparison is made between the concentration of (1) in
various organs at the end of the infusion and the concentration of (1)
obtained at thirty or forty-five minutes after a bolus iv injection, the
specificity inherent in the system can be demonstrated. If the system
is not specific, one would expect to see an overall decrease in the con
centration of (1) after an iv infusion of (3) compared to the bolus.
The results show that at thirty minutes, there is a higher concentration
of (1) in the brain compared to the iv bolus and a decrease in the concen
tration of (1) in the lungs and kidneys. The liver shows a moderate in
crease. At forty-five minutes these trends continue. A comparison of
the data at thirty and forty-five minutes shows an increase of the con
centration of (1) in the brain and a reduction in all other organs. The
concentration of (1) in the brain is higher than in any other tissue ana
lyzed at forty-five minutes.


116
observed. The model system which was chosen in this study was that of
the red blood cell. In this experiment (1) or (2) were placed in a known
volume of whole blood, and at various times the concentration was deter-

mined in the plasma or packed red blood cells. The results are shown in
Figure 3-17. As one can see, the initial rate of penetration of (2) into
red blood cells is rapid and greater than that of (1). The initial con
centration is also higher. This affinity for the red blood cells is mir
rored by a disappearance of (2) from the plasma. With time, equilibra
tion occurs in the system. Berberine penetrates the red blood cell slow
ly and reaches a maximum concentration much later than does (2). This is
another indication of the increased membrane mobility of (2) relative to
(1). The two curves finally converge to the same value, as the oxidation
of (2) to berberine takes place. Creasey indicated that there was a re
lationship between glucose transport and the transport of berberine into
red blood cells.143 To investigate this possibility, the behavior of ber
berine in a red blood cell system which contained 200 mg% glucose was ob
served. As shown in Table 3-17, there is very little effect of glucose
on the entry of berberine into the red blood cells from the plasma.
In Vivo Studies
The preliminary studies indicate that dihydroberberine possesses all
of those characteristics required of a compound which is to be applied to
the drug delivery system proposed in Figure 1-3. The substantiation of
this drug delivery scheme requires not only the demonstration of the deliv
ery of (1) after administration of (2) but also the specific retention of
(1) in the brain. The first priority of the in vivo system was to show
delivery into the brain of (1). The protocol used in these studies in
volved injecting rats with either berberine (1), dihydroberberine (2),
or its hydrochloride (3). After a period of time the chest cavities of


150
_J I I I I I I I I I I
20 40 60 80 100 120 140 160 180 200 220
Time (min)
Figure 3-28. Efflux of 1-Benzylnicotinamide Bromide (7) from the Brain after i v
of 60 mg/Kg of 1-Benzyl-l ,4-dihydronicotinamide (22) (A)
Administration
U>
cn


DOSE/GRAM TISSUE
Figure 3-22. Distribution of Berberine after iv Administration of 35 mg/Kg of Berberine (1)
into the Kidney (), Liver (), Lung (), and Brain (A)
fj.q BERBERINE/GRAM TISSUE


19
important for K+, since a low K+csf/K+p]asma ratio apparently acts to
stabilize neurons.12,7172
One of the major factors in influencing diffusion into the CNS is the
degree to which a substance is bound to plasma proteins. It had been
assumed for a long time that only the free, dialyzable fraction of the
total plasma concentration of a compound was available for diffusion.7
This is important for a great number of compounds. The major species in
volved in this binding are serum albumins, which tend to bind molecules
loosely but to a large extent, and globulins, which bind with high affinity
and low capacity. The premise that only the unbound species is capable
of diffusion has, however, been challenged.
Steroids have profound central effects and gain entry into the CNS
by simple diffusion across the BBB, even though they are highly bound to
plasma proteins.5 This diffusion correlates well with the octanol:water
partition coefficient of the steroids and inversely with the tendency of
the molecules to form hydrogen bonds.73 Different steroids are taken up
differently, however, and this is in large part related to protein binding.
There are several globulins which bind specific steroids. These include
sex hormone binding globulin (SHB6), which is found in man but not rats,
cortical binding globulin (CBG), estradiol binding globulin (EBG) which,
in fetal and neonatal rats, may be synonymous with a-fetoprotein, proges
terone binding globulin (PBG), which is found in pregnant guinea pigs and
also, thyroid hormone binding globulin (TBG), which is found in man.
Those steroids which are bound to globulins such as cortisone to CBG
in rats have a small flux into the brain while those steroids which are
bound more highly to albumins such as progesterone, estrogen or testosterone
in the rat, easily diffuse through the BBB.73 The reason for this is that


23
source of both of these compounds is available, since histamine is stored
in perivascular mast cells and serotonin in platelets.4 The action of
histamine is partially reversible by H2 antagonists.86 Recently, cyclic
guanine monophosphate (cGMP) has been isolated in cerebral capillaries and
this may play a role in vesicular transport.87 These effects are seen
peripherally as well as centrally.
The major controversy surrounding this area is whether hypertension
itself is responsible for extravasation, or if some humoral agent produced
as a result of hypertension is culpable. It is difficult to look at this
in vivo since many agents can increase blood pressure and increase vesi
cular transport.
Additional evidence that vesicles are important in extravasation is
indicated by the decreased protein flux in capillaries treated with com
pounds which decrease vesicular formation. These compounds include imida
zole, which alters cAMP function by inhibiting the inactivation of phos
phodiesterase, thioridazine, a phenothiazine which decreases vesicular
fusion with the cell membrane, and desipramine.80 The anionic transport
blocking agent 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid
disodium (SITS) inhibits exocytosis.88 This compound, which also inhibits
protein extravasation in hypertension, inhibits ATP-evoked adrenalin
release from chromaffin granules as well as serotonin secretion from plate
lets. Increased vesicular transport has also been related to the formation
of transendothelial channels.5489 These channels may be the result of the
simultaneous opening of a chain of vesicles and may provide an avenue for
nonspecific flux in hypertension. The presence of this channel is highly
debated, however.


74
Table 3-1 Proton Assignments of the NMR of Dihydroberberine (2)
H
Proton
a
b
c
d
e
f
g
h
PPM (5)
7.1
6.7
6.4
5.9
4.3
3.4
3.1
2.9


98
This characteristic is accessible by MINDO/3. The program calculates a
dihedral angle which is a measure of the deviation from planarity of a
molecule. Pyridine (32) is planar and 1,2-dihydropyridine (33) is planar
within one degree. The dihedral angles of (30) t (31) are shown in
Table 3-8. In the case of (30), the overall deviation from planarity is
less than 3.6. In berberine (1), which has an additional benzene ring
annulated at the C7-C3 position, this difference should be less because
of the planarizing effects of the added aromatic system. This would tend
to cast doubt on the proposal that the reason berberine does not inter
calate into deoxyribonucleic acid (DNA), as well as totally aromatic mole
cules such as coralyne, is due to the buckling of the C ring.135155
This nonplanarity is attributed to the partial saturation of that ring.
The present study, however, indicates that this deviation is slight and
probably plays a minor role in attenuation of the action of (1).
A more plausible reason for the lower intercalative ability of (1)
is because of lower electronic interactions. When a molecule intercalates
into DNA, there exists an electronic interaction between the base pairs
of DNA and the ir-cloud of the intercalating aromatic compound. The greater
the stabilization of this complex, the greater is the DNA-molecular inter
action. In berberine, there is a partial destruction of the aromatic
system which lowers any electronic interaction. The hydrogens added to
the C-ring act to increase the effective thickness of the molecule and
this may play a role in decreasing macromolecular complexation. These
structural concerns are apparent in Figures 3-10 and 3-11, which are the
fully optimized structure for (30).
The dihydro model (31) contains one more sp3 center than does (30),
and this has a slight deplanarizing effect with the molecule twisting 9.1


PERCENT TRANSMISSION
WAVELENGTH IN MICRONS
3 3 5 I 4.5 5 5 5 6 6.5 7 7.5 8 9 0 II 12 14 16
V/A' EMUMbEf* CM1
Figure 3-3 Infrared Spectrum of Dihydroberberine (2) (KBr)
PERCENT TRANSMISSION


133
The first experiments involved injecting rats with 200 mg/Kg of
1-methyl-1,4-dihydronicotinamide (21) in aqueous ethanol followed fifteen
minutes later by an injection of the standard dose of 55 mg/Kg of (3).
The results of this study are shown in Figure 3-27. The dihydronicotin
amide (21) was used as a proform of quaternary compound (6) in an attempt
to generate high levels of (6) in the brain. The results do not show, how
ever, any significant difference in the efflux of berberine (1) between
pretreated and unpretreated animals.
In an analogous study 1-benzylnicotinamide (7) and its corresponding
dihydro adduct (22) was employed. Unlike the 1-methyl derivatives, this
pair of compounds possesses a UV chromophore which greatly simplifies quan
titation. A preliminary study was performed to determine the time at which
the maximum concentration (tmax) of (7) after the administration of 1-
benzyl-1,4-dihydronicotinamide (22) occurred. The results of this experi
ment are shown in Figure 3-20. After injection of (7) no detectable levels
of (7) in the brain could be observed. The reason the tmax is important
is that, ideally, the lag time between the injection of the antagonist,
i.e. (7) and (2) is the time required for (7) to reach a maximum concen
tration. Unfortunately, however, the toxicity of dihydrobenzyl derivative
(22) proved to be much higher than that of dihydromethyl derivative (21)
and the maximum dose which could be given was only 60 mg/Kg and, because
*
of this, further studies with this compound were abandoned.
A number of observations concerning this figure are germane to the
topic of the efflux of (1) from the brain. In the case of administration
of this N-benzyldihydropyridine (22), no dihydronicotinamide is present at
early time points at the dose used in the experiment. This is not the
case with (2). This is consistent with the greater stability of (31) and
hence, (2), predicted by the theoretical calculations. The t, of efflux
2


14
Table 1-1 Blood-Brain Barrier Transport Systems
Transport system
Representative
Substrate
Km
(mM)
vmax
(nmol min^g"1)
Hexose
G1ucose
9
1600
Neutral Amino Acid
Phenylalanine
0.12
30
Acidic Amino Acid
G1utamate
Basic Amino Acid
Lysine
0.10
6
Monocarboxylic Acid
Lactate
1.9
120
Amine
Choline
0.22
6
Nucleoside
Adenosine
0.018
0.7
Purine
Adenine
0.027
1
Thyroid Hormone
t3
0.001
0.17
Thiamine
Thiamine


139
Table 3-19. Efflux of 3H
Ventricular
-Inulin from the Brain after
Administration
Intracerebral
Time (min)
% Dose/g
In % Dose/g
Wet Tissue
Wet Tissue
15
9.28%
2.23
30
7.94%
2.07
45
6.52%
1.87
60
5.54%
1.71
120
4.71%
1.55
Corr. = 0.930
k = 6.23 x 103min_1
t. = 1.9 hrs
2


63
In Vitro Distribution of Berberine (1) and Dihydroberberine (2) in Whole
Blood
Solutions of (1) or (3) were added to 60 or 75 ml of freshly drawn
heparinized sheep's blood maintained at 37C. At various times, 4 ml of
blood were withdrawn. The blood was centrifuged and the plasma removed.
One milliliter of the plasma was treated with 9 ml of acetonitrile and the
supernatant was analyzed spectrophotometrically. The entire volume of the
packed red blood cells was treated with 8 ml of acetonitrile and centri
fuged. The supernatant was again analyzed spectrophotometrically. A stan
dard curve was obtained by preparing solutions of known concentration in
plasma or packed red blood cells. Recovery from the red blood cells was
71.4%.
Effect of Glucose on the Distribution of Berberine (1) in Whole Blood
Glucose was added to a volume of blood so that a concentration of
200 mg% was obtained. The above procedure was then repeated.
Animal Studies
In Vivo Characterization of Berberine (1) and Dihydroberberine (2)
White Sprague-Dawley rats, who weighed between 200-250 g, were anes
thetized intramuscularly with Inovar^ (0.13 ml/Kg). Injections were made
intravenously into the external jugular vein. The doses used include
55 mg/Kg of (2) in dimethylsulfoxide (DMS0), 55 mg/Kg of (3) in 20-25%
aqueous ethanol, 55 mg/Kg of (1) in DMS0 or 35 mg/Kg of (1) in DMS0.
At certain times after the injection, the chest cavities of the rats were
opened, the vena cava severed and the heart perfused with normal saline.
Afterwards, the animals were decapitated and the brains removed. In cer
tain experiments, the lungs, liver, and kidneys were also excised. The
organs were then homogenized in a minimal amount of water, usually 2 ml,
and extracted with 8 ml of acetonitrile. A standard curve of (1) in the


Table 3-10. A Comparison of Bond Lengths in Angstroms of the Pyridine (32) Z 1,2-Dihydropyridine (33)
System and the Isoquinoline (30) Z Dihydroisoquinoline (31) Model System
Pyridine (32)
Atom Bond
Number Length
Dihydropyridine (33)
Atom Bond
Number Length
Isoquinoline Model (30) Dihydroisoquinoline Model (31)
Atom Bond Atom Bond
Number Length Number Length
1-2
1.336
1-2
1.432
1-2
1.342
1-2
1.451
2-3
1.402
2-3
1.492
2-3
1.433
2-5
1.517
3-4
1.406
3-4
1.357
3-4
1.467
3-4
1.445
4-5
1.406
4-5
1.453
4-5
1.444
4-5
1 .468
5-6
1 .402
5-6
1 .351
5-6
1.388
5-6
1.370
6-1
1.336
6-1
1.363
6-1
1.415
6-1
1.401
7-2
1.107
7-2
1.134
2-26
1.115
2-26
1 .130
8-2
1.134
2-27
1.132
8-3
1.105
9-3
1.105
3-11
1 .467
3-11
1.444
9-4
1.114
10-4
1.105
4-14
1.433
5-11
1.101
5-19
1 .110
5-19
1.110
6-12
1 .113
6-8
1.480
6-7
1 .485
UD


132
These data verify the drug delivery scheme devised. After adminis
tration of (3) high concentrations of (1) are obtained specifically in
the brain, while the concentrations in other organs are reduced. The
reason that the slow infusion of (3) enhances the specificity is related
to a number of factors. Dihydroberberine is a lipophilic compound. The
iv bolus injection presents to the tissues a large concentration of (2)
and the result of this large burden is an inordinately long transit time
of (2) in peripheral sites. This obfuscates the kinetics. By slowly
administering (3), the tissue burden is greatly reduced, and the designed
improvements in the bidirectional characteristics of the delivery molecule
can be fully demonstrated. This system in general, and (2) in particular,
allows specific delivery of quaternary compounds to the brain. This sys
tem is designed to reduce systemic levels of an agent and thereby reduce
any accompanying toxicity.
Efflux of Berberine from the Brain
The mechanism by which berberine leaves the brain is not known but
it is important to the quaternary scheme. According to the original postu
lation, large quaternary compounds like (2) should leave the brain slowly,
presumably by passive processes such as movement in the CSF. Small qua
ternary compounds, on the other hand, are substrates for active carriers
which rapidly remove these compounds from the brain extracellular fluid.1,5
The next series of experiments was designed to determine the mechanism of
efflux of (1) from the brain. If the efflux of (2) is mediated by a spe
cific carrier, it should be possible to demonstrate the competitive inhi
bition of the efflux of (2) by introducing into the system a large con
centration of another agent which has affinity for the cationic pump, such
as 1-methylnicotinamide (6) and 1-benzylnicotinamide (7).


124
This indicates a faster efflux for the total berberine concentration
than for the efflux of (1) alone. The reason for this is that, aside
from efflux of (1) from the brain, a component of the oxidation of (2)
and for the efflux of (2) also occurs in this rate. Since the oxidation
of (2) is not immediate, a significant quantity of it is present at later
times and this population of molecules will redistribute out of the brain
as a function of the blood concentration of (2) as would any lipophilic
compound. This added equilibrium complicates the kinetics of the original
scheme.
The distribution of berberine (1) in various tissues after an intra
venous dose of 35 mg/Kg is shown in Figure 3-22. The lower dose was used
because of the higher toxicity of (1). These results show a high concen
tration of (1) in the kidney and, to reiterate, no quantitatable amount
in the brain. Berberine is rapidly lost from the tissue and this contri
butes to its relatively short biological t^. The distribution of (1) in
the tissues has been previously studied.205-209 The results obtained from
a number of these studies are consistent with those reported here. Ber
berine is rapidly lost from the tissues and effectively excreted by the
kidney and by a biliary route. In literature reports the concentration of
(1) in the brain was always undetectable or the lowest of all other tissues
examined. In those cases where (1) was detected, the amounts found were
usually on the order of 50-200 ng/g tissue which is below the limit of
detection in this study. These values are low in both relative and abso
lute terms. Since the effective concentration of (1) in in vitro systems
is on the order of 1.0 yg/g, these levels would be ineffective therapeuti
cally. These studies also indicate berberine is not absorbed to a great
extent from the intestine, again reflecting its highly polar nature.


29
the carrier by denaturation. Additionally, diminution or increase of the
blood level of a transportable compound can affect the utilization of the
compound. In some cases, the rate limiting step in metabolism of a nutrient
can be transferred from an enzyme to transit of the agent across the BBB.21
Glucose transport is very important to CNS function. Under normal cir
cumstances, the rate-determining step in glucose metabolism involves the
enzyme hexokinase. If, however, the plasma concentration of glucose falls,
as in hypoglycemia, or cerebral metabolism increases, as in relative hypo
glycemia, the limiting step in utilization is shifted to transport of glu
cose across the BBB.20
In severe hypoxia (p02 <10 mm), a number of progressive changes as
sociated with glucose flux occur.1 In the dog, after one minute of oxygen
deprivation, the influx of glucose into the brain is unchanged but efflux
decreases. Ten minutes after initiation of hypoxia, influx and efflux of
glucose decrease. This has been explained by a postulated modulator which,
in the absence of hypoxia, is bound to the carrier and alters transport.
In other animal models, different results are obtained.
The transport of amino acids and, especially, neutral amino acids in
certain disease states, has been the subject of much research. The neutral
amino acid carrier is responsible for the transport of such neurotrans
mitter precursors as tryptophan, tyrosine and histidine and, as such, any
interruption of this supply can have tremendous neurological consequences.
Nowhere is this more apparent than in phenylketonuria.1*20 This syndrome,
which is associated with high blood levels of phenylalanine, has been linked
to mental retardation. This condition of high phenylalanine levels can
act to competitively inhibit other substrates of this carrier, such as
tyrosine, tryptophan, and histidine. This is made possible because of


154
87. I. Karnushina, I. Toth, E. Dux and F. Joo, Brain Res., 189, 588
(1980).
88. J. E. Hardebo and B. B. Johansson, Acta Neuropathol., 51, 33
(1980).
89. A. S. Lossinsky, A. W. Vorbrodt, H. M. Wisniewski and L. Iwanowski,
Acta Neuropathol., 53, 197 (1981).
90.J. C. Horton and T. Hedley-Whyth, Brain Res., 169, 610 (1979).
91.E. A. Neuwelt and E. P. Frenkel, Ann. Int. Med., 93, 137 (1980).
92. D. M. Long, J_. Neurosurq., 51 53 (1979).
93. N. A. Vick, J. D. Khandekar and D. D. Bigner, Arch. Neurol., 34,
523 (1977).
94. J. T. Poulishock, D. P. Becker, H. G. Sullivan and J. D. Miller,
Brain Res., 153, 223 (1978).
95. S. Al-Kassab, T. S. Olsen and E. B. Skriver, Acta Neurol. Scand.,
64, 438 (1981).
96. E. N. Albert and J. M. Kerns, Brain Res., 230, 153 (1981).
97.J. C. Lin and M. F. Lin, Rad. Res., 89, 77 (1982).
98. P. M. Daniel, D. K. C. Lamard, 0. E. Pratt, J. Neurol. Sci., 52,
211 (1981).
99. D. M. Keane, I. Gray and J. A. Panuska, Cryobiology, 14, 592 (1977).
100.J. J. Brink and D. G. Stein, Science, 158, 1479 (1967).
101. J. J. Kocsis, S. Hankaway and W. H. Vogel, Science, 160, 1472
(1968).
102. S. C. Phillips, J. Neurol. Sci., 50, 81 (1981).
103. T. Eriksson, S. Liljequist, A. Carlsson, J. Pharm. Pharmacol.,
31_, 636 (1979).
104. M. M. Hertz, T. G. Bolwig, P. Grandjean and E. Westergaard,
Acta Neurol. Scand., 63, 286 (1981).
105. J. Hellmann, R. C. Vannucci and E. E. Nardis, Pediatr. Res., 16,
40 (1982).
106. A. A. Sinkula and S. H. Yalkowsky, J_. Pharm. Sci., 64, 181 (1975).
107.V. Stella in "Pro-drugs as Novel Drug Delivery Systems," T. Higuchi
and V. Stella (eds.), ACS Symposium Series Vol. 14, American Chemical
Society, Washington, D.C. (1975) p. 1.


79
its corresponding imine and this unusual situation allows enamines, dihy-
droberberine included, to be substrates in both nucleophilic and electro
philic reactions. This equilibrium tends to concentrate a negative charge
on the carbon 8 to the nitrogen. Protonation usually occurs, for this
reason, at the 8-carbon rather than the nitrogen. At physiological pH,
a portion of the dihydroberberine molecules will exist in a C-protonated
state. The pKa which is an indication of the degree of this ionization
is important since only the unprotonated free base is available for dif
fusion across membranes. The pKa of (2) was determined to be 6.80 0.05
by both a spectrophotometric and potentiometric method. At a physiological
pH of 7.4, a substantial portion of (2) will thus exist in the unionized,
freely diffusable form. Because of the water insolubility of the dihydro
berberine free base, all pKa determinations were carried out in 25% metha
nol ic solutions.
Demethylation of Berber'ne
A number of other synthetic schemes were explored in an attempt to
obtain a method for preparing radiolabeled (1), should spectroscopic method
prove too insensitive. A radiolabel led compound would also greatly expe
dite whole body distribution studies. In radiolabeling a compound, one
of the important factors governing the selection of a synthetic route is
yield. The scheme which was chosen involves pyrolyzing (1) at 200C.173,174
Berberine loses methyl chloride, as shown in Figure 3-7, to form the deep
purple zwitterionic berberrubin. This method would allow the placement
of either a 14C or 3H label at the nine position of (1) by reacting ber
berrubin with the appropriately tagged methyl iodide or methyl sulfate.
Methylation of berberine with cold methyl iodide was performed to demon
strate the viability of the scheme.


28
Several situations involving autoimmune afflictions and induced auto
immunity, such as experimental allergic encephalomyelitis (EAE) which is
used as a model of multiple sclerosis, demonstrate an increased capillary
permeability.98 In EAE, the cerebral vessels are said to cuff or deform
and this abnormality correlates with BBB breakdown. This may be important
in the general progression of multiple sclerosis, as vascular changes are
among the first changes which precede demylelination.
Agents which solubilize and fluidize membranes may also act to in
crease BBB permeability.99100101 Dimethyl sulfoxide (DMSO), in very high
concentrations, has been thought to increase the flux of such plasma mark
ers as inulin and mannitol. Its effects are said to be derived from its
lytic action on membrane although interaction by micellular formation can
not be ruled out. Nortriptyline has a similar effect. Ethanol, in large
doses, can also cause opening of the BBB to sucrose. It was postulated
that this generalized increase in permeability may increase the suscepti
bility of the CNS to bacterial and viral infection. It has been shown,
however, in acute and chronic doses of ethanol that were compatible with
continued life, that there was no alteration in the BBB.102103
Many heavy metals have been associated in both general and specific
changes in the BBB.156104 Mercury (II) Hg+, in high concentrations,
i.e. >80 pm, causes a generalized increase in the permeability of cerebral
capillaries to sugars and protein markers but in low concentrations affects
only the hexose carrier. Ionic lead has a similar effect.
A number of conditions can alter BBB function in more subtle, yet no
less damaging ways. In these instances, changes occur at the level of a
specific carrier or carriers and, as such, only a particular type of com
pound is involved. These changes can result from physical alteration of


67
was recorded. Berberine or dihydroberberine hydrochloride were given ip
3 times a day on day 2, 6, and 10 in 0.5% carboxymethy1 cellulose.


DOSE/GRAM TISSUE
Figure 3-26. A Comparison of the Efflux of Berberine from the Liver when Berberine (1)
is Administered i v at a Dose of 35 mg/Kg () and Dihydroberberine Hydro
chloride (3) when Administered i v at a Dose of 55 mg/Kg ()
GJ
O


TI ME (min)
Figure 3-20. Efflux of Berberine (1) and Unoxidized Dihydroberberine (2) (A) after i v
Administration of 55 mg/Kg of Dihydroberberine Hydrochloride (3)
IX)
ro


53
ether layer was dried over sodium sulfate (Na2S04) and the solvent removed
under reduced pressure. The yield was 58%: XH NMR (CDC13) 6 9.1 (1 H, s),
8.7 (1 H, m), 8.2 (1 H, m), 7.3 (1 H, m), 4.3 (2 H, t), 1.3 (12 H, m),
0.88 (3 H, t); IR (NaCl) v (C-H) 2920 and 2950, (C=0) 1723, (C-C, C-N)
1589, (-C-C(=0)0) 1277, (0-C-C) 1109, (y CH) 732, (g ring) 693.
3-Pyridinecarboxylic Acid Decyl Ester/Decyl Nicotinate (13)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry pyri
dine and 11.82 ml (0.062 moles) of 1-decanol. The solution was refluxed in
an oil bath for eight hours. The liquid was then poured over ice which
had been made basic with ammonia. The aqueous solution was extracted with
ethyl ether, the organic layer dried over Na2S04, and the solvent removed
under reduced pressure. The yield was 64%: XH NMR (CDC13) 8.7 (1 H, m), 8.2 (1 H, m), 7.2 (1 H, m), 4.3 (2 H, t), 1.2 (16 H, m),
0.83 (3 H, t); IR (NaCl) v (C-H) 2915 and 2946, (C=0) 1720, (C-C, C-N) 1584,
(-C-C(=0)0) 1275, (0-C-C) 1105, (y CH) 732, (g ring) 692.
3-Pyridinecarboxylic Acid g-Phenylethyl Ester/g-Phenethyl Nicotinate (14)
Twenty-six grams (0.15 moles) of (10) were dissolved in 200 ml of
pyridine. To this stirring solution was added dropwise 18.3 g (0.15 moles)
of g-phenethyl alcohol. The solution was refluxed in an oil bath for
several hours. The solution was then poured over ice and made slightly
basic with ammonia. This solution was then extracted with ether. The
organic layer was dried over Na2S04 and evaporated under reduced pressure.
The yield was 62%: *H NMR (CDC13) 9.1 (1 H, s), 8.6 (1 H, m), 8.0 (1 H, m),
7.2 (6 H, s), 4.4 (2 H, t), 2.9 (2 H, t); IR (NaCl) v (C-H) 3030 and 2959,
(C=0) 1722, (C-C, C-N) 1585, (-C-C(=0)0) 1276, (0-C-C) d 1120, 1105, (y CH)
737, (g ring) 695.


THE APPLICATION OF A DIHYDROPYRIDINE-PYRIDINIUM SALT
REDOX SYSTEM TO DRUG DELIVERY TO THE BRAIN
BY
MARCUS ELI BREWSTER III
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1982

Copyright 1982
by
Marcus Eli Brewster III

TO MOTHER

ACKNOWLEDGEMENTS
I would like to express my gratitude to Professor Nicholas S. Bodor.
His genius and kindness will always be an inspiration to me. I would also
like to thank the other members of my committee, Dr. Merle Battiste,
Dr. Kenneth Sloan, Dr. Margaret James, and Dr. James Simpkins, for their
advice and help.
This work would truly not have been possible but for the guidance of
a number of post-doctoral fellows and technicians, especially Dr. Hassan
Farag, Dr. Cynthia Luiggi, Dr. Thorsteinn Loftsson, Mrs. Jirina Vlasak,
Mrs. Nancy Gildersleeve, and Mr. Edward Phillips. I would also like to
thank Jane and C.J. Rogers, Dr. Yasuo Oshiro and Dr. Tadao Sato for their
assistance.
Although space is limited, I want to recognize a few friends who
made graduate school bearable: Linda and John Hirschy, Mark V. Davis,
R.E. Golightly, Mrs. James E. Ray, Newton Galloway, Mike Morris, Richard
Panarese, Wayne and Anita Riggins, Mike and Kay Dempsey, Chuck Hartsfield,
Mike Gibbons, Raun and Cissy Kilgo, Jeff and Jane Dean, Ernie Lee, and
the late Chip Connally. Chip was a dear friend. I would like to thank
Scott and Margie Makar for their cordiality and hospitality.
I would also to like to acknowledge the P.C.'s, Dr. Frank Davis,
Mr. Jim Templeton, and Dr. Gary Visor, the last of a dying breed. Two
special people deserve mention here because of their guidance early in my
scientific career, Professor G.L. Ware, and Mrs. Frances Kenning. Special
thanks are accorded to my Amazonian, sybaritic amanuensis, Cynthia Jordan.
And, last but certainly not least, I would like to thank my family without
iv

whom I would not have been able to pursue an academic career. The anti can
cer testing was graciously performed by Otsuka Pharmaceutical Co., Ltd.
This work was supported by Grant GM 27167 from the National Institute of
General Medical Sciences.
v

TABLE OF CONTENTS
CHAPTER PAGE
ACKNOWLEDGEMENTS iv
LIST OF TABLES vii
LIST OF FIGURES ix
ABSTRACT xii
1 INTRODUCTION 1
Blood-Brain Barrier 2
Prodrugs and Drug Delivery Systems 31
Statement of the Problem 41
2 MATERIALS AND METHODS 47
Synthesis 49
Characterization of Dihydroberberine 59
Animal Studies 63
3 RESULTS AND DISCUSSION 68
Synthesis and Characterization of Dihydroberberine 68
Theoretical Studies on the Dihydropyridine£Pyridinium
Redox System 81
Further Studies on the Chemical and Biological Properties
of Dihydroberberine 104
In Vivo Studies 116
Conclusions 147
BIBLIOGRAPHY 150
BIOGRAPHICAL SKETCH 160
vi

LIST OF TABLES
TABLE PAGE
1-1 Blood Brain Barrier Transport Systems 14
3-1 Proton Assignments of the 1H NMR of Dihydroberberine (2) 74
3-2 Carbon Assignments of the 13C NMR of Dihydroberberine (2) 76
3-3 Distribution Coefficients for Berberine (1) and Dihydrober
berine Hydrochloride (3) in Chloroform/pH 7.4 Buffer and in
1-Octanol/pH 7.4 Buffer 78
3-4 The Heats of Formation, Vertical Ionization Potentials, and
Dipole Moments of the Isoquinoline Model (30) and the Dihydro-
isoquinoline Model (31) 86
3-5 Bond Lengths in Angstroms between Various Atoms of the Iso
quinoline Model (30) and the Dihydroisoquinoline Model (31).... 87
3-6 Bond Angles in Degrees between Various Atoms of the Isoquino
line Model (30) and the Dihydroisoquinoline Model (31) 88
3-7 Charge Density at Various Atoms of the Isoquinoline Model (30)
and the Di hydroisoquinoline Model (31) 89
3-8 Dihedral Angles between Various Atoms of the Isoquinoline Model
(30) and the Dihydroisoquinol ine Model (31) 90
3-9 Differences in the Heats of Formations (AAHf) of (30) X (31),
2-PAM t 1,4-Dihydro-2-PAM and 2-PAM X 1,6-Dihydro-2-PAM 91
3-10 A Comparison of Bond Lengths in Angstroms of the Pyridine (32)X
1,2-Dihydropyridine (33) System and the Isoquinoline (30) %
Dihydroisoquinol ine (31) Model System 94
3-11 A Comparison of the Bond Angles in Degrees of the Pyridine (32)i
1,2-Dihydropyridine (33) System and the Isoquinoline (30) X
Dihydroisoquinol ine (31) Model System 96
3-12 A Comparison of the Atomic Charge Densities of the Pyridine (32)t
1,2-Dihydropyridine (33) System and the Isoquinoline (30) X
Dihydroisoquinol ine (31) Model System 97
3-13 The Rate of Oxidation of Dihydroberberine in Various Media 105

TABLE PAGE
3-14 The Relative Rates of Oxidation of Dihydroberberine (2),
1-Methyl-1,4-dihydronicotinamide (21), and 1-Benzyl-1,4-
dihydronicotinamide (22) in Dilute Hydrogen Peroxide 108
3-15 Proton Assignments of the 3H NMR of the 1-Methyl-1,4-dihydro-
nicotinic Acid Ester (27) 112
3-16 The Rates of Oxidation and Corresponding Correlation Coef
ficients of Various 1-Methyl-l,4-dihydronicotinic Acid Esters
and 1-Benzyl-l ,4-di hydronicotinamide (22) 114
3-17 The Effect of Glucose on the Movement of Berberine into Red
Blood Cells 118
3-18 Slow Infusion of Dihydroberberine (3) 131
3-19 Efflux of 3H-Inulin from the Brain after Intracerebral Ventri
cular Administration 139
3-20 In Vivo Metabolism of Berberine and Dihydroberberine in the
RatTHPLC) 140
3-21 In Vivo Metabolism of Berberine and Dihydroberberine in the
Rat-(TLC) 142
3-22 Probit Analysis of the LD50 Study 145
3-23 Effect of Berberine (1) and Dihydroberberine Hydrochloride (3)
Against P388 Lymphocytic Leukemia 146
vi i i

LIST OF FIGURES
FIGURE PAGE
1-1 This Schematic Illustration Represents an Endothelial Cell
Derived from either a Muscle (ECm) or Brain (ECb) Capillary.
In this Figure, (ma) is the Macula Adherens or Loose Junction,
(zo) is the Zona Occludens or Tight Junction, (mv) are Micro
vesicles, and (bl) is the Basal Lamina. This Figure was Modi
fied from Reference 1, Page 162 by Permission 6
1-2 A Proposed Carrier-mediated Chemical Delivery System with
Specificity for the Brain. The Drug Molecule to be Trans
ported is Represented by the (O) 40
1-3 The Proposed Drug Delivery Scheme 43
3-1 Synthesis of Dihydroberberine (2) and its Hydrochloride
Salt (3) 69
3-2 Ultraviolet Spectrum of Dihydroberberine (2) in 95% Ethanol.... 70
3-3 Infrared Spectrum of Dihydroberberine (2) (KBr) 71
3-4 Mass Spectrum (70 eV, El) of Dihydroberberine (2) 72
3-5 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of Dihydro
berberine in CDCI3. The Insert Represents the Region between
2.86 and 3.46 at 100 MHz 73
3-6 The 13C Nuclear Magnetic Resonance Spectrum (100 MHz, CDC13)
of Dihydroberberine (2) 75
3-7 Demethylation of Berberine (1) and Methylation of Berber-
rubin (4) 80
3-8 Structures and Salient Numbering Protocols for Berberine (1),
Dihydroberberine (2), the Isoquinoline Model (30), the Dihydro-
isoquinoline Model (31), Pyridine (32), and 1,2-Dihydro-
pyridine (33) 83
3-9 The Highest Occupied Molecular Orbital of the Dihydroisoquino
line Model (31) 92
3-10 A Computer-assisted Drawing of the Most Stable Conformation
of the Isoquinoline Model (30) at 25C 99
ix

Page
Figure
3-11 A Computer-assisted Drawing of the Most Stable Conformation
of the Isoquinoline Model (30) at 25C. This View is Oriented
so that the Interatomic Axis between Atoms 26 and 2 is Perpen
dicular to the Plane of the Page 100
3-12 A Computer-assisted Drawing of the Most Stable Conformation
of the 1,2-Dihydroisoquinoline Model (31) at 25C 102
3-13 A Computer-assisted Drawing of the Most Stable Conformation
of the 1,2-Dihydroisoquinoline Model (31) at 25C. This View
is Oriented so that an Imaginary Axis between Atoms 2 and 5
is Perpendicular to the Plane of the Page 103
3-14 Spectral Changes of Dihydroberberine (2) upon Oxidation to
Berberine (1) in pH 5.8 Phosphate Buffer at 26C. Traces
were made every 10 Min 106
3-15 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of (27)
in CDC1 Ill
3-16 The Rates of Oxidation of Various 1-Methyl-1,4-dihydronicotinic
Acid Esters (23), (24), (25), (26), (27), (28) and 1-Benzyl-
1,4-Dihydronicotinamide (22) at 37C in 40% Human Plasma (),
6% Brain Homogenate (A) and 3.5% Liver Homogenate () 113
3-17 Partitioning of 26.5 mg of Berberine (1) from Plasma (A) into
Red Blood Cells (A) and of 26.5 mg of Dihydroberberine Hydro
chloride (3) from Plasma (O) into Red Blood Cells (). The
Volume of Blood Used in each Experiment was 75 ml 117
3-18 Distribution of Berberine in the Brain after iv Administration
of Berberine (1) (A) at a Dose of 55 mg/Kg or of Dihydrober
berine Free Base (2) () at a Dose of 55 mg/Kg 120
3-19 Efflux of Berberine from the Brain after iv Administration of
either 55 mg/Kg of Dihydroberberine Hydrochloride (3) ()
or 55 mg/Kg of Berberine (1) (A). Analysis was for (1) only
and not Unoxidized (2) 121
3-20 Efflux of Berberine (1) and Unoxidized Dihydroberberine (2)
(A) after iv Administration of 55 mg/Kg of Dihydroberberine
Hydrochloride (3) 122
3-21 A Comparison of the Efflux of Berberine (1) () and Berberine
(1) and Unoxidized Dihydroberberine (2) (A) after a Dose of
55 mg/Kg of Dihydroberberine Hydrochloride (3) Administered
iv 123
3-22 Distribution of Berberine after iv Administration of 35 mg/Kg
of Berberine (1) into the Kidney (Ah Liver (), Lung (#),
and Brain (A) 125
x

Figure Page
3-23 Distribution of Berberine after iv Administration of 55
mg/Kg of Dihydroberberine Hydrochloride (3) into the Kidney
(Oh Liver (), Lung (O), and Brain (A) 127
3-24 A Comparison of the Efflux of Berberine from Lungs when Ad
ministered iv as 55 mg/Kg of Dihydroberberine Hydrochloride
(3) (O) or 35 mg/Kg of Berberine (1) () 128
3-25 A Comparison of the Efflux of Berberine from the Kidneys when
Berberine (1) is Administered iv at a Dose of 35 mg/Kg (A)
and Dihydroberberine Hydrochloride (3) when Administered iv
at a Dose of 55 mg/Kg (O) 129
3-26 A Comparison of the Efflux of Berberine from the Liver when
Berberine (1) is Administered iv at a Dose of 35 mg/Kg (B)
and Dihydroberberine Hydrochloride (3) when Administered iv
a Dose of 55 mg/Kg () 130
3-27 A Comparison of the Efflux of the Total Berberine, i.e. (1)
and (2) from the Brain when either 55 mg/Kg of Dihydrober
berine Hydrochloride (3) is Administered iv (A) or 55 mg/Kg
of Dihydroberberine (2) and 200 mg/Kg of 1-Methyl-l,4-
dihydronicotinamide (21) is Administered iv () 134
3-28 Efflux of 1-Benzylnicotinamide Bromide (7) from the Brain af
ter iv Administration of 60 mg/Kg of 1-Benzyl-1,4-dihydro
nicotinamide (22) (A) 135
3-29 Efflux of Berberine from the Brain after icv Injection of
either 50 yg of Berberine (1) () or 50 yg of Berberine (1)
and 1000 yg of 1-Methylnicotinamide Iodide (6) (A) 137
3-30 The LD50 Dose-response Curve of Berberine (1) (A) and Dihy
droberberine Hydrochloride (3) (). Doses of (1) or (2)
were Administered ip in CD-I Mice 144
xi

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE APPLICATION OF A DIHYDROPYRIDINE-PYRIDINIUM SALT
REDOX SYSTEM TO DRUG DELIVERY TO THE BRAIN
By
MARCUS ELI BREWSTER III
AUGUST 1982
Chairman: Nicholas S. Bodor
Major Department: Medicinal Chemistry
This work has been concerned with the design and testing of a broadly
applicable drug delivery system which is specific for the brain. The
method developed for this purpose is based on a dihydropyridine^pyridinium
salt redox system and on the blood-brain barrier. In the proposed delivery
system, a pharmacologically active agent which contains a pyridinium nu
cleus would be reduced to its corresponding dihydropyridine. After sys
temic administration, the highly lipoidal dihydropyridine would partition
into the brain as well as into the periphery. In both locations oxidation
would occur. In the systemic circulation, the charge species would be
rapidly eliminated by renal or biliary mechanisms while in the brain the
compound would be retained.
The prototype compound which was chosen for inclusion in this scheme
was berberine. This alkaloid has a high in vitro activity against various
cancer systems but its in vivo activity is low. The first step in the ap
plication of the described delivery system to berberine is the synthesis
XT 1

of dihydroberberine which was accomplished using sodium borohydride in
pyridine. The dihydroberberine was analyzed by various spectroscopic means
and a number of physical properties were measured. Theoretical calcula
tions using a MINDO/3 approach were also undertaken.
In order to verify the proposed scheme, the delivery of berberine to
the brain and the retention of berberine in the brain had to be shown.
When dihydroberberine or its hydrochloride salt were injected systemi-
cally, high levels of berberine were found in the brain and its efflux
from the brain was slow. If, however, berberine is injected systemically,
no detectable levels are found in the brain. When dihydroberberine is
slowly infused the concentration of berberine rises in the brain and at
forty-five minutes, this concentration is specifically higher in the brain
than in any other organ analyzed. The mechanism of efflux of berberine
from the brain was investigated and appears to be mediated by a passive
process, perhaps the bulk flow of cerebral spinal fluid. Dihydroberberine
was found to be less toxic than berberine. Preliminary anticancer data
indicate that dihydroberberine is more effective in increasing the life
span of animals injected intercerebrally with P388 lymphocytic leukemia
than is berberine.
xi i i

CHAPTER 1
INTRODUCTION
A method for delivering drugs specifically to a particular organ would
be valuable. Properly designed, a drug delivery system should concentrate
an agent at its site of action and reduce its concentration in other loca
tions. The results of these manipulations would not only be an increase in
the efficacy of an agent, but also a decrease in its toxicity.
A site specific system designed for the central nervous system (CNS)
would be especially useful. The reason for this, aside from the inherent
importance of the brain, is that the entry of many pharmacologically active
agents into the CNS is impeded by a set of specialized barriers present at
the blood-brain interface. This barrier system,, termed the blood-brain
barrier (BBB), is composed of numerous enzymatic and anatomical components.
This dissertation will present a general method for the specific deliv
ery of drugs to the brain and give an example which substantiates the method.
In order to provide an adequate background for a discussion of drug deliv
ery to the brain, a review of the BBB is necessary. The introductory mate
rial is then continued with a cursory historical account of drug delivery
systems and prodrugs. Because of the importance and great interest of an
ticancer agents, emphasis is placed on this topic in this section. The
closing section of the first chapter will state specific aspirations as they
apply to the present research.
1

2
Blood-Brain Barrier
Structural and Enzymatic Considerations of the Blood-Brain Barrier
The existence of a barrier which separates central nervous tissue
from the general circulation was first postulated by Ehrlich at the end
of the 19th century.12345 In a series of pioneering experiments, he
injected a number of dyes into laboratory animals and found that, while the
visceral organs were highly stained, the brain was conspicuously uncolored.
It was later discovered that these dyes bind extensively to plasma proteins
so that the actual barrier then described was one to these complexes. Many
small hydrophilic compounds cannot, however, pass into the brain so that this
barrier is presented to a wide variety of compounds. Early in the study of
the BBB, the obstruction was considered absolute but this idea was soon dis
pensed with since the nutritional requirements of the brain necessitate
the equilibration of a number of compounds between the general circulation
and the CNS.3
The morphological basis of the BBB had been a very controversial sub
ject until relatively recently. Historically, three hypotheses have been
put forward to explain this impermeability to blood-borne substances.1
All are based on structural differences between the cerebral vascular sys
tem and the systemic circulation. It was proposed that the small extra
cellular space characteristic of mammalian brains prohibited the accumula
tion of compounds and, as such, constituted a barrier. It was shown, how
ever, that some animals with large extracellular spaces have a well-defined
barrier to a number of substances.6 The suggestion was also made that the
general impermeability of the brain to blood-borne substances was due to
astrocytic end feet which surround the capillaries, forming an envelope,
or due to the endothelial cell lining of the cerebral capillaries.1 In
order to study this question, electron microscopic evaluation is necessary.

3
Progress was somewhat slowed because of the lack of appropriate electron
microscopic tracers.7 The first tracers used were saccharated iron oxides8
or ferritin (molecular weight 560,000) whose limits of resolution were close
to the thickness of the endothelium itself. It was not until the intro
duction of horseradish peroxidase (HRP) and microperoxidase (MP) that the
exact structure of the BBB could be deduced. Horseradish peroxidase is a
relatively small enzyme (molecular weight 43,000) which, unlike ferritin,
does not contain an electron-dense core but rather produces a material
which has a high affinity for osmium tetraoxide and other radiopaque sub
stances .7
By using HRP, Reese and Karnovsky demonstrated the inability of the
marker to pass from the lumen of the cerebral capillary.910 In fact, HRP
was never found in the extracellular space surrounding the capillary. Addi
tionally, when HRP was injected directly into the brain it readily passed
the astrocytic end processes and was stopped at the endothelial membrane.
The anatomical basis of the BBB was, therefore, isolated to the endothelial
lining of the cerebral capillaries and not a perivascular site. There are
several ultrastructural differences between systemic capillaries and cere
bral capillaries which account for their general impermeability.14
The manner in which endothelial cells of the cerebral capillaries are
joined is distinct from systemic capillaries. Cerebral junctions are char
acterized as tight or closed junctions meaning the cells closely approximate
each other. These junctions gird the cell circumferentially, forming a
zona occludens and providing an absolute barrier to HRP. Structurally,
the junctions consist of aligned intramembranous ridges and grooves which
are in close apposition.11 These tight junctions have been examined by thin
section electron microscopy and attempts are now underway to examine them
by a freeze-fracture technique.412 This method, which allows a longitudinal

4
view of the capillaries, will add greatly to the structural knowledge of
these tight junctions but the method is technically difficult. Recently,
a freeze-fracture technique was applied to the cerebral vasculature of a
chameleon, which possesses a BBB similar to that of mammals.13 In these
animals a series of ridges and grooves is seen. The ridges are connected
to neighboring ridges by an anastomosing network. In general, the more com
plex this system is, i.e. the number of ridges it has, the tighter the junc
tion is. The tightness of the junction can be assayed not only structurally
but also by measuring ionic conductance and resistance through the junction.
The ionic conductance is low for most ions.1214
Systemic capillaries lack this closed junction. Morphologically, this
can be traced to a lack of continuity in the intercellular appositions.
In cardiac muscle, for example, the ratio of junctional width to the width
of the cell membrane is 2.4, while in cerebral capillaries this is reduced
to 1.7.10 These open junctions allow a high degree of nonspecific transport
of nutrients and other compounds into systemic capillaries. Materials pass
easily between these leaky cells, while in the brain the sealing of the
intercellular fissures severely restricts this nonspecific transport. Since
intercellular transport is removed, only intracellular transport remains.
Lipophilic compounds can readily pass through these phospholipoidal membranes,
but hydrophilic compounds and compounds with high molecular weights are ex
cluded. Systems are available to transport small hydrophilic nutrients,
and this will be discussed later.
A second difference between cerebral and systemic capillaries is the
paucity of vesicles and vesicular transport in the CNS.47 Vesicular trans
port is a process for transcellular transport and, as such, vesicles are
transported from the luminal to the abluminal membrane. Pinocytotic acti
vity, on the other hand, is concerned with the nutritional requirements of

5
the cell and, therefore, involves vesicular movement from the luminal mem
brane to a cell organelle, presumably a lysosome.
Cerebral endothelial vesicles are usually uncoated and few in number
compared to other systems. Using electron microscopic morphometry, five
vesicles per micrometer luminally and thirty-forty vesicles per micrometer
abluminally were found.4 In the diaphragm and in myocardial vessels the
values are much higher, being seventy-eight and eighty-nine vesicles per
micrometer, respectively. This lower content of vesicles is another mecha
nism by which the CNS can limit nonspecific influx. A third difference
is the lack of fenestra in the cerebral capillaries. These differences
are demonstrated schematically in Figure 1-1.
Cerebral vessels have a number of perivascular accessory structures
which appear to be involved in BBB function.4 While it is known that the
astrocytic end feet are not involved as a barrier per se, their role in
attenuating BBB action is interesting. These glial end feet may be involved
with regulation of amino acid flux. They also appear to engulf protein
which breeches the BBB and, therefore, may act as a second line of defense.15
Phagocytic pericytes which are present abluminally may play a similar role.
It is possible that the basement member of endothelial capillaries acts as
a mass filter preventing large molecules from penetrating it.
In addition to these structural features the BBB maintains a number of
enzymes which appear to augment barrier function.21617 Since optimal
neuronal control requires a careful balancing between neurotransmitter
release, metabolism, and uptake, it is of vital importance to restrict the
entry of blood-borne neurotransmitters into the CNS. It is not surprising,
therefore, to find high concentrations of such enzymes as catechol-0-methyl
transferase (COMT), monoamine oxidase (MAO), y-aminobutyric acid trans
aminase (GABA-T) and aromatic amino acid decarboxylase (DOPA decarboxylase)

6
Figure 1-1. This Schematic Illustration Represents an Endothelial Cell
Derived from either a Muscle (ECm) or Brain (EC^) Capillary.
In this Figure, (ma) is the Macula Adherens or Loose Junction,
(zo) is the Zona Occludens or Tight Junction, (mv) are Micro
vesicles, and (bl) is the Basal Lamina. This Figure was Modi
fied from Reference 1, Page 162 by Permission.

7
in the BBB. Recently, a distributional study of COMT in the brain indi
cated that this enzyme is present in many sites including several which
lack a structural BBB.18 This distribution may aid in the exclusion of
neurotransmitters from structurally unprotected areas. The presence of
DOPA decarboxylase explains partially the need for giving such large doses
of L-dihydroxyphenylalanine (DOPA) in the treatment of CNS dopamine defi
ciencies to achieve appropriate therapeutic cerebral levels. The enzymatic
BBB may also play a role in the exclusion of lipophilic compounds which
might otherwise passively diffuse through the BBB. This is suggested by
the presence of pseudo (butyryl) cholinesterase in the cerebral capil
laries.4 This enzyme is not found in noncerebral capillaries. The occur
rence of y-glutamyltranspeptidase has also been described and may account
for some protection from peptide infiltration.219 An early proposal
that Y-glutamyltranspeptidase is involved with carrier systems is ques
tionable. Acid phosphatase activity, which is a marker for lysosomes and
pre-lysosomes or phagosomes, is present in the endothelial cells.15 These
organelles appear to be involved in the degradation of endocytosed material
and, as such, can be considered a component of the BBB. The endothelial
cells of the cerebral microcirculation contain a large number of enzymes
but care must be taken in ascribing a certain enzyme to a barrier role.17
There are numerous enzymes which, while present in the endothelial cells,
do not serve in any capacity other than general cellular functioning.
Molecular Carriers Involved in BBB Transport
The aspects of the BBB which have been discussed thus far give an indi
cation of its relative impermeability to a number of blood-borne substances,
but do not explain the movement of essential nutrients into the CNS. This
transport is brought about by a number of carriers which are situated in
the endothelial cells. These carriers are generally assumed to be proteinaceous.

8
They are equilibrative, i.e. nonenergy dependent and bidirectional in
nature and can be saturated.120,21 The net movement of compounds is
always along a concentration gradient and since nutrients are readily
utilized as soon as they pass into the brain, this gradient is in the di
rection of the brain.
A number of specific carriers for compounds have been described. The
first to be characterized was one for hexoses.11222 This carrier displays
saturable kinetics and can be competitively inhibited. The hexose carrier
is stereospecific and has a high affinity for a-D-glucose with a Km between
6.0 and 9.0 mM. Other sugars with affinity for this carrier include, in
order of decreasing affinity, 2-deoxy-D-glucose, 3-0-methylglucose, B-D-
glucose, D-mannose, D-galactose and D-xylose.22 The Km of D-fructose and
L-glucose is very high.
The carrier is Na+ independent and is inhibited by phloretin, a non
competitive inhibitor, more than phlorizin, a competitive inhibitor.20
The Vmax is similar for all sugars tested which indicates the rate limiting
step for transport is not the association of the sugar with the carrier but,
rather, the movement of the carrier across the membrane complex. This car
rier demonstrates exchange diffusion, i.e. the carrier moves more rapidly
22 2 3
when loaded than when empty.
At a Km of 7 mM, the concentration of glucose required to produce sat
uration is about 126 mg% so that under physiological conditions, the system
is about half saturated. In normal situations the rate determining step in
glucose utilization is the hexokinase step. This can be shifted to BBB
transport of glucose in conditions of hypoglycemia.21 While substrate flux
is usually thought of as being related only to plasma levels of glucose,
recent studies have indicated that intracerebral glucose concentrations can
alter carrier kinetics.

9
The effects of insulin on the transport of glucose are controversial.2224
Several reports have indicated no effect on either unidirectional or net
flux after insulin infusion. This is curious in that insulin would not be
expected to pass the BBB. The presence of insulin receptors in cerebral
capillaries may explain this enigma in that insulin may bind to a receptor
on the luminal surface and its effects may be mediated to the abluminal
surface by a second messenger.24
Also controversial is the possibility that a low and high affinity
system is operating in glucose transport.202223 There is a nonspecific
flux associated with glucose of 7%. Some authors attribute this to diffu
sion but an alternate hypothesis has been proposed. This involves the pres
ence of two systems on the carrier: a high affinity, low capacity system
and a low affinity, high capacity system. Most data have been collected in
isolated vessel preparation but there is some support of this proposal from
in vivo experiments. Gjedde showed the putative low affinity system to be
stereoselective and to have a Km of 1.0 M,23 compared with a Km of 1.1 mM
for the high affinity system. He contends that a single set of kinetic
parameters does not adequately describe the system and that this high-low
affinity system better correlates with the data. A suggestion that a pro
tein tetrameter with both low and high affinity sites was the carrier has
also been made. At the choriod plexus there appears to be ouiban sensi
tive, Na+ dependent glucose flux and this is apparently important in cere
bral spinal fluid (CSF) homeostasis.22
Three carriers have been described for amino acid transport.11220
These carriers have affinity for neutral, basic, and acidic amino acids.
In general, essential amino acids, which are large and bulky, are trans
ported in preference to nonessential amino acids.20 In all of these

' 10
systems, net flux is small compared to unidirectional flux since amino
acids derived from proteins are constantly being lost and the magnitude
of this loss is similar to uptake.
The transport of neutral amino acids has been described by Christensen
and these generalizations apply to the BBB.2526 Four neutral amino acid
transport systems have been shown to occur in Ehrlich ascites, a model cell
system, and in several other systems. An L- or leucine-preferring system
is characterized by high affinity for phenylalanine, leucine, tyrosine,
tryptophan and several other large essential amino acids. It is Na+ inde
pendent, bidirectional and equilibrative in nature. The definition of this
system can be made by observing the flux of 2-aminonorbornane-2-carboxylic
acid which is exclusively transported by this carrier. The A- or alanine-
preferring system is characterized by a Na+ dependence, an energy depen
dence, and the ability to concentrate substrates. The system has affinity
for glycine, proline, alanine, serine, threonine and several other small
amino acids. The defining compound for this system is a-(methyl ami no)-
isobutyric acid. Two other amino acid transport carriers have also been
described but they are not well characterized. There is also an ASC
(alanine-serine-cysteine-preferring) system and a Gly (glycine-preferring)
system.
Since generally only large essential amino acids (L-system substrates)
are transported through the BBB, the L-system is assumed to be the dominant
mechanism in amino acid uptake.120 The absence, however, of the A-system
has been questioned. It was recently argued that the A system is present
but has a different distribution than the L-system. Betz and Goldstein dis
covered a system whose characteristics are similar to the Asystembut which is
located abluminally.27 This system may act as an active mechanism for

11
efflux of these amino acids from the brain parenchyma or, more probably,
for concentrating them in the endothelial cytosol. The necessity for
this concentration is related to a proposal that the L and A systems may
act together in the transport of amino acids. This hypothesis is based on
a possible equilibration of amino acids between the two carriers in the
cytoplasm of the endothelial cell.26
In many cases the Km value for an amino acid is similar to its plasma
concentrations. This being the case, slight changes in the blood levels
of an amino acid may alter their disposition. The utilization of trypto
phan, for example, which is a precursor of serotonin, is partially deter
mined by its availability and its movement across the BBB.21,28 A similar
situation may exist in certain circumstances with tyrosine, which is the
precursor for dopamine, norepinephrine and epinephrine.
Recently, a hypothesis was forwarded to explain the regulation and
induction of these carriers.29,3,31 It has been suggested that an amino
acid is produced abluminally in large amounts and transported on the neu
tral amino acid carrier. The amino acids in such a system should have a
high Km and, therefore, be easily displaced from the carrier at the luminal
side or in transit. These carriers, like the hexose carriers, exhibit ex
change diffusion. The proposed amino acid in this regulatory role is
glutamine. Glutamine is synthesized from glutamic acid and ammonia by
glutamine synthetase in astrocytes, a perivascular locus. The concentra
tion of glutamine is ten times higher than the concentration of any other
neutral amino acid and its local concentration in the vicinity of the BBB
is predicted to be still higher.30
This hypothesis was formulated on the basis of several interesting
observations. In cases where ammonia and, presumably, glutamine levels

12
increase cerebrally, as in porto-systemic shunts, the uptake of phenyl
alanine and other neutral amino acids increases. Also, if glutamine syn
thetase is inhibited by methionine sulfoximine, the uptake of essential
amino acids is reduced.29 While this is an attractive proposal, several
authors have questioned it on the grounds that many amino acids compete
for this neutral amino acid carrier and the role of glutamine, therefore,
may be relatively unimportant.32
Relatively little work has been done with basic and acidic amino
acids. The acidic system has affinity for glutamate and is fully saturated
at physiological concentrations. Basic amino acids are transported on a
distinct carrier. This carrier is equilibrative, saturable and bidirec
tional and has been described as a Ly+ or lysine-preferring system.
A monocarboxylic acid carrier has been described which demonstrates
affinity for lactate, pyruvate, acetate, propionate, butyrate, 5-aminolevu-
linic acid and ketone bodies.120 Ketone bodies, such as e-hydroxybutyrate
and acetoacetate, are produced in a number of stressful circumstances,
including starvation.33 The carrier possesses similar characteristics to
those which have already been described. It is stereospecific and pH sen
sitive.20 The transport of the substrates on the carrier increases with a
decrease in pH. This has been interpreted to mean either that a proton is
cotransported with the acid or that hydroxide acts as a high affinity com
petitive inhibitor. Only the ionized acid is transported by this system
and this has led to the proposal that an R-NH3+ moiety is present in the
active site. The unionized acid can pass through the membrane by simple
diffusion.
The Km of lactate is similar to the plasma level of lactate. This
being the case, rapid rises in systemic lactate levels, such as the ones
which accompany exercise, are not transferred to the brain immediately.20

13
A carrier for choline was described in 1978.34 Choline cannot be
synthesized de novo in the brain but this precursor is required for such
important cellular components as acetylcholine and phosphatidylcholine.
The carrier conforms generally to those characteristics specified for other
systems. It has affinity for choline, hemicholinium, dimethyl ami noethanol
(deanol), tetraethyl ammonium, tetramethylammonium, cartitine and spermine
but not for NH4+. The distribution of this carrier is not uniform. Choline
uptake decreases with age and this correlates with an age-dependent diminu
tion of the carrier. The rate determining step in choline utilization is
its movement across the BBB.
A carrier with affinity for nucleosides such as adenosine, guanosine
and inosine has been described.120 Additionally, a system for transport
ing purine bases has been isolated. This system transports adenine, guanine
and hypoxanthine but not pyrimidines. This is interesting in light of the
fact that pyrimidines can be synthesized in the brain from NH4+ and aspar
tate, while purines cannot. This carrier is very active in neonates, but
its activity diminishes with age.35
Recently, a carrier was described for thyroid hormones.3637 The
carrier has affinity for triiodotyrosine (T3) and thyroxine (T4) but not
for tyrosine, leucine or potassium iodide. The transport of T3 is satur
able and inhibited by T4. The carrier is weakly stereospecific and of high
affinity. The presence of a carrier for T3 and T4 is interesting because
these compounds are fairly lipophilic. The bulkiness of the molecule,
however, tends to decrease its ability to pass membranes. A carrier-
mediated transport has also been proposed for thiamine.38 These systems
are summarized in Table 1-1.

14
Table 1-1 Blood-Brain Barrier Transport Systems
Transport system
Representative
Substrate
Km
(mM)
vmax
(nmol min^g"1)
Hexose
G1ucose
9
1600
Neutral Amino Acid
Phenylalanine
0.12
30
Acidic Amino Acid
G1utamate
Basic Amino Acid
Lysine
0.10
6
Monocarboxylic Acid
Lactate
1.9
120
Amine
Choline
0.22
6
Nucleoside
Adenosine
0.018
0.7
Purine
Adenine
0.027
1
Thyroid Hormone
t3
0.001
0.17
Thiamine
Thiamine

15
In addition to these carrier systems, there are a number of active
efflux mechanisms. Two of these systems are located in the choriod plexus
and have affinity for organic ions. A system for the disposition of anions
has been described and divided into two subsystems.2039404142 An L
or liver system with affinity for prostaglandins, 5-hydroxyindole acetic
acid and probenicid, as well as a K or kidney system with affinity for
p-aminohippuric acid and phenol red, has been proposed. Additionally, a
carrier for the efflux of iodide has been described.4344 A cationic sys
tem is also present in the choriod plexus and this species has affinity
for N-methylnicotinamide, decamethonium and hexamethonium ions.4546
These systems appear to be important in the removal of metabolic acids and
bases. This, as well as the anionic system, is energy-dependent and can
be competitively inhibited. The presence of several energy (ATP)-dependent
systems in the capillaries indicates that this site may also be important
for active efflux.19 The high density of mitochondria in cerebral capil
laries is further support for this location.47
Movement of Compounds Across the BBB
The BBB, therefore, consists of a relatively impermeable membrane
superimposed on which are mechanisms for allowing the entrance of essential
nutrients and the exit of metabolic wastes. If a compound is to gain access
to brain parenchyma, it may do so via several routes. If the agent has
affinity for one of the carriers previously described, it may diffuse across
the BBB by association with this carrier. A compound which has a high in
trinsic lipophilicity can diffuse passively through the phospholipoidal
cell membrane matrix.1412 The pK of a compound with ionizable groups is
also important, since only the unionized species diffuses across the BBB
rapidly.4849 The ability of a substance to enter into the cell membrane

16
is often correlated with its in vitro octanol :water partition coefficient.5051
This is a measure of lipophilicity, and can be correlated with biological
effects.52 These correlations can be extended to the permeability of com
pounds through the BBB. Several examples of this correlation have appeared
in the literature, including the opiates morphine, codeine, and heroin.52
Good correlation is obtained between lipophilicity, the ability to pass
the BBB, and narcotic efficacy.
The increase in the ability of a compound to pass membranes can, all
too often, be correlated with an increase in undesirable side effects. An
example of this was shown in a series of B-blockers in which lipophilic
members of this pharmacologic group, i.e. propranolol penetrated the BBB
rapidly but demonstrated a number of deleterious psychiatric manifestations.53
Conversely, B-blockers of lower lipophilicity exhibit an attenuation of
these side effects.
These two avenues, namely passive diffusion and carrier mediation,
represent the major components of influx. Other minor mechanisms may
also allow the entry of substrates into the CNS. The cell bodies of many
neurons are located centrally while their axons may penetrate into the
periphery. These axons can take up material and transport it in a retro
grade fashion to the CNS.754 This retrograde axoplasmic transport has
been observed in such areas as the nucleus ambiguus and the abducen nucleus.
In some cases, however, the endocytosed material is reacted with lysosomes
and, therefore, this transport route may have a protective function.
There are several areas of the brain which lack a BBB.1855 These
include such locations near the ventricles as the area postrema, the sub
fornical organ, the median eminence of the neurohyphosis, the organum vas-
culosum of the lamina terminal is, and the choriod plexus. Collectively,

17
these areas are termed the circumventricular organ. In addition, the
pineal gland lacks a BBB. These areas constitute a small fraction of the
total surface area of the BBB and may allow a limited nonspecific flux.
These loci do have important pharmacological ramifications as demon
strated by the action of a series of atypical neuroleptics.5657 These
compounds are so named because they provoke certain symptoms of dopaminer
gic blockade but not others. Specifically, metoclopramide exerts an anti
emetic action but not an anti schizophrenic effect. This dichotomy was
explained by the fact that metoclopramide does not penetrate the BBB. The
site of action of antiemetic agents is at the chemoreceptive trigger zone,
which is located in the area postrema and is outside the BBB. The apparent
inability of metoclopramide to produce the full spectrum of changes which
occurs as a consequence of dopaminergic blockade may be related to its
pharmacokinetics and, specifically, its inability to pass the BBB. This
relegates the compound to only those sites of action not protected by the
BBB. Other pharmacologically important sites outside the BBB include the
median eminence, which controls prolactin secretion. In some areas, while
the BBB is not absent it is diminished. These areas include certain ar-
teriolar segments whose diameters are between 15-30 ym. In these
segments limited protein extravasation, or leakage of compounds from the
lumen of the capillary to the extracellular spaces, has been observed.
The ability of small peptides to penetrate the BBB is an extremely
controversial point.21606162 Systemic administration of certain cen
trally active peptides elicits a central response. Differences concerning
the interpretation of these data are significant. One view is that the flux
of these small peptides across the BBB is low indicating, perhaps, some
nonspecific route accounts for their entry.2162 The presence of peptidyl

18
receptors lumirally, whose stimulation results in the generation of a
second messenger, has been proposed in this respect. A different conclu
sion is that small peptides have a significant flux across the BBB. The
peptides which have been investigated thus far include stabilized enkepha
lins and endorphins, a nonapeptide which induces delta-sleep, melanin stim
ulating hormone (a-MSH) and melanin inhibiting factor one (MIF-1).606163
The BBB plays a major role in CSF homeostasis.1264 Cerebral spinal
fluid is produced at the choriod plexus and drains from the ventricals
through the foramina of Magendie and Luschka into the ventral aspects of
the brain.65 This fluid serves many important mechanical and nutritive
functions. Cerebral spinal fluid flow is constant with a ti of renewal
of about two hundred and seventy minutes. The ventricular volume of CSF
is about 23 ml while the subarachnoid volume is 117 ml. Cerebral spinal
fluid, along with any dissolved materials, leaves the subarachnoid space
via the arachnoid villi, which protrude into a venous sinous. The arach
noid villi act as a one-way valve and prevent backflow.66 This loss of
CSF provides a slow mechanism for nonspecific efflux of compounds from
the CNS. This mechanism rids the brain of polar compounds such as metabolic
wastes at a fairly constant rate regardless of molecular weight. If a
compound is fairly polar and does not have affinity for any passive or
active efflux mechanism, it will leave the CNS by CSF bulk flow. Therefore,
while lipophilicity is very important for influx to the brain, the efflux
of a compound is only partially dependent on this parameter.4667686970
Since the ionic environment in which neurons function is so important,
the composition of the CSF is strictly maintained within narrow limits.
The CSF is not simply an ultrafiltrate of plasma, and several concentration
gradients are produced. The maintenance of these gradients can, in part,
be attributed to the low ionic conductance of the BBB. This is especially

19
important for K+, since a low K+csf/K+p]asma ratio apparently acts to
stabilize neurons.12,7172
One of the major factors in influencing diffusion into the CNS is the
degree to which a substance is bound to plasma proteins. It had been
assumed for a long time that only the free, dialyzable fraction of the
total plasma concentration of a compound was available for diffusion.7
This is important for a great number of compounds. The major species in
volved in this binding are serum albumins, which tend to bind molecules
loosely but to a large extent, and globulins, which bind with high affinity
and low capacity. The premise that only the unbound species is capable
of diffusion has, however, been challenged.
Steroids have profound central effects and gain entry into the CNS
by simple diffusion across the BBB, even though they are highly bound to
plasma proteins.5 This diffusion correlates well with the octanol:water
partition coefficient of the steroids and inversely with the tendency of
the molecules to form hydrogen bonds.73 Different steroids are taken up
differently, however, and this is in large part related to protein binding.
There are several globulins which bind specific steroids. These include
sex hormone binding globulin (SHB6), which is found in man but not rats,
cortical binding globulin (CBG), estradiol binding globulin (EBG) which,
in fetal and neonatal rats, may be synonymous with a-fetoprotein, proges
terone binding globulin (PBG), which is found in pregnant guinea pigs and
also, thyroid hormone binding globulin (TBG), which is found in man.
Those steroids which are bound to globulins such as cortisone to CBG
in rats have a small flux into the brain while those steroids which are
bound more highly to albumins such as progesterone, estrogen or testosterone
in the rat, easily diffuse through the BBB.73 The reason for this is that

20
binding to albumin is sufficiently weak that the capillary transit time
in the brain is long enough to allow dissociation of the compound from
the macromolecule. In the case of globulins, however, the binding is
tighter and the turnover is only of the order of 3-10%/second. The sojourn
through the cerebral capillaries is not, therefore, sufficient to release
the bound material. It is not, therefore, the plasma-protein-bound frac
tion which is unavailable for transport but, rather, the globulin-bound
fraction.
These principles also apply to free fatty acids such as palmitate.
In this case, however, there appear to be high and low affinity sites on
the albumin molecule so that the rate of dissociation of the palmitate
bound to the site with the lower Km may be slow compared with capillary
time, while the low affinity site may not.74 Melatonin, which is also
highly protein bound, shows a significant diffusion through the BBB.75
Thyroid hormones which are transported into the CNS by carriers are
also bound by plasma proteins.3637 The carrier, whose Km is lower than
that of the albumin site, is able to compete successfully with the albumin
for binding. This is effectively a stripping of the compound from its
albumin site. The effect is also seen with tryptophan and other amino
acids.28
The BBB in Pathological and Experimentally Altered States
The integrity of the BBB is known to be impaired in a number of patho
logical or experimentally-induced conditions.47677 The effect produced
can be the result of changes of the structural components of cerebral capil
laries such as the junction or vesicular activity and, as such, results
in generalized increases in permeability. Alternatively, the carrier sys
tems may be compromised and this may lead to specific changes in perme-
ability.

21
Generalized increases in permeability result in a number of deleteri
ous events. Since the BBB is relatively permeable to water, but not to
most other substances, osmotic gradients can be rapidly changed. If plas
ma proteins and other compounds are allowed to freely enter the CNS, they
will bring with them large amounts of water, with the result being cere
bral edema.120
The morphological basis of these changes in any particular situation
can be highly controversial. This is especially the case with hypertonic
treatment of cerebral capillaries. It has been known for some time that
hypertonic solutions of such solutes as glucose, sucrose, urea, arabinose,
lactamide and several others can increase the permeability of the cerebral
capillaries to protein and other small polar compounds. Rapoport explains
this phenomenon as the result of osmotic shrinkage of the endothelial cells,
resulting in a pulling apart of the tight junctions.571 This has been
challenged. If HRP is injected after a hypertonic solution, the HRP reac
tion product does not form a continuous line from the luminal to the ablu-
minal surfaces at the junction or at any other location.420 It was sug
gested, therefore, that increased vesicular transport accounted for the
increased permeability, perhaps as a result of increases in local blood
pressure.77
In any case, if the concentration of the hypertonic solution is close
to the critical opening concentration, the opening of the BBB is transient
and does not produce acute edema.5 This procedure may have therapeutic
applications. In many cases it is desirable to introduce highly polar
compounds into the brain and this osmotic opening of the BBB may provide
an avenue for that purpose. Methotrexate is a folate inhibitor used in the
treatment of cancer. The pKa of the carboxylic acid functions of this

22
molecule is 4.7 and at physiological pH methotrexate is 99.8% ionized and,
as such, passes the BBB very slowly, if at all. After pretreatment with
hypertonic arabinose, the concentration of methotrexate showed a fifty-fold
increase in the CNS.78
Hypertension is a major health problem and can produce a number of
debilitating complications.79 Hypertension increases the extravasation
of albumin, sucrose, and other polar compounds.808182 This has been
studied in a number of animal models of hypertension including those in
which the increased system blood pressure is induced with either ampheta-
p
mine, ephedrine, Aramine or bicuculine.
The basis for this increased permeability has been debated frequently
and appears to be related to increased vesicular transport since ultra-
structural investigations do not indicate capillary lesions or junctional
openings.77 Increased vesicular transport has, in fact, been implicated
in a number of situations in which capillary permeability increases. The
factors that affect vesicular formation which are described here also ap
ply in those cases.
The mechanism by which hypertension elicits an increase in vesicular
activity is not clear. The increased hydrostatic pressure may act to
induce an invagination. Also, there are a number of substances associated
with hypertension that induce vesicular formation.47783 These include
the catecholamines, serotonin, and histamine. Joo explained the increased
vesicular transport in terms of a cyclic 5'-adenine monophosphate (cAMP)
stimulation mediated by a catalyzed adenyl cyclase, since cAMP can directly
induce vesicular formation.8485
Both serotonin and histamine have been shown to increase protein ex
travasation and both act to catalyze specific adenyl cyclase. A perivascular

23
source of both of these compounds is available, since histamine is stored
in perivascular mast cells and serotonin in platelets.4 The action of
histamine is partially reversible by H2 antagonists.86 Recently, cyclic
guanine monophosphate (cGMP) has been isolated in cerebral capillaries and
this may play a role in vesicular transport.87 These effects are seen
peripherally as well as centrally.
The major controversy surrounding this area is whether hypertension
itself is responsible for extravasation, or if some humoral agent produced
as a result of hypertension is culpable. It is difficult to look at this
in vivo since many agents can increase blood pressure and increase vesi
cular transport.
Additional evidence that vesicles are important in extravasation is
indicated by the decreased protein flux in capillaries treated with com
pounds which decrease vesicular formation. These compounds include imida
zole, which alters cAMP function by inhibiting the inactivation of phos
phodiesterase, thioridazine, a phenothiazine which decreases vesicular
fusion with the cell membrane, and desipramine.80 The anionic transport
blocking agent 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid
disodium (SITS) inhibits exocytosis.88 This compound, which also inhibits
protein extravasation in hypertension, inhibits ATP-evoked adrenalin
release from chromaffin granules as well as serotonin secretion from plate
lets. Increased vesicular transport has also been related to the formation
of transendothelial channels.5489 These channels may be the result of the
simultaneous opening of a chain of vesicles and may provide an avenue for
nonspecific flux in hypertension. The presence of this channel is highly
debated, however.

24
The nature of the hypertension itself can be a factor in increased
capillary permeability.82 While acute hypertension is very likely to pro
duce a protein influx, chronic hypertension may protect the system in the
likelihood of an acute rise in blood pressure. The reason for this is
probably the hypertrophy of vascular muscles. This hypertrophy leads to
an increased vascular resistance and thickening of the vessel wall pro
viding a mechanism for handling greater pressures. This is consistent with
the observation that vasoconstriction decreases capillary permeability,
while vasodilation increases this parameter.
In some cases hypertension may lead to the rare malady, hypertensive
encephalopathy.81 This disease is related to the inability of the cerebral
vasculature to acclimate to acute severe hypertension. In this instance
cerebral edema occurs and this may cause swelling of the nervous tissue.
This yields an increased cerebral pressure and may prevent vasodilation
caused by a variety of stimuli.71
Hypervolemia may also result in an increase in protein extravasation.90
If mice, whose blood is about 1.3 ml, are treated systemically with high
volumes (1 ml) of saline, extravasation of HRP can be documented. At
lower volumes, however, no changes in permeability are observed.
Since, in these experiments, the blood pressure increases only by
20 mm Hg, the increased capillary permeability may not be due to hydro
static pressure. In order to open the BBB, pressure increases on the
order of 100 mm Hg are required.
The vasculature of primary brain tumors and of metastatic secondary
brain cancers exhibits a generalized degradation.79192 This breakdown
can be characterized by the presence of gap junctions, fenestra and open
endothelial junctions, indicating the typical electron microscopic

25
structure of the tight junctions is destroyed. Although it has been sug
gested that these deficiencies should render cerebral tumors susceptible
to treatment,93 the clinical evidence does not support this. The therapy
which has, thus far, been directed to brain neoplasms has been disappoint
ing at best. Neurosurgery may have reached its practical limit and most
agree that major new advances must come from the field of chemotherapy.91
It is interesting to note that many agents are effective against certain-
peripheral tumors, but not their secondary brain metastases.
The unresponsiveness of cerebral tumors has been explained by two
phenomena. First, it is known that the greatest display of disintegration
of the capillary structure occurs in the central, slow growing portion of
the tumor, and as one moves to the periphery, the abnormalities decrease.92
This area of the tumor would be expected to show the highest resistance to
chemotherapeutic agents. Secondly, since only a relatively small area of
the brain vasculature is disturbed, any drug which reaches the neoplasm
would rapidly diffuse into the outlying areas, thereby diminishing its
concentration and effectiveness at the tumor site.78 Few suggestions have
been forwarded to circumvent this problem. One involves osmotic opening
of the BBB followed by methotrexate treatment, but this has a very limited
usefulness because of problems with the accompanying edema.78
Many experimental models of brain injury are associated with an in
crease in capillary permeability.45794 These models include injury
induced by dropped weights, pendulums, hammers, spring-mounted weights,
blasting caps, rotary strikers, compressed air guns, humane stunners and
accelerating devices. Generally, these procedures have been applied to
the cat.
*

26
The leakage of proteins from the capillaries is usually proportional
to the amount of injury, but even in severe injury the endothelial cell
is intact and shows no sign of lesion.94 The explanation most often pro
posed for this extravasation is increased vesicular activity. In most in
juries there is an increase in serotonin and norepinephrine levels, as well
as the production of a number of humoral agents. The increased vesicular
content, which appears first in the arterioles and subsequently in the
capillaries, may be mediated by stimulation of cAMP. There are also, how
ever, increases in blood pressure and that may act to increase vesicular
transport.
Cerebral infarcts also disrupt BBB function, as evidenced by an in
creased albumin concentration in the CSF of infarct victims.95 Correla
tion, however, between infarct size and location and the quantity of al
bumin leaked has not revealed any significance.
Both electroconvulsive shock and pentylenetetrazole-induced seizures
result in an increased capillary permeability to a number of plasma
markers.1454 The degree of permeability increase is proportional to the
number of shocks or compounds given. The morphological basis for this ex
travasation is not known and both junctional opening and increased vesi
cular transport have been suggested.77 It is interesting to note that,
in both of these procedures, the blood pressure rises and this may be an
important factor.
Induced ischemia also produces a marked increase in the permeability
of the cerebral vasculature.5477 The experimental model which is most
often used is that of the Mongolian gerbil. In this species, about half
of the individuals lack arterial connections between cerebral and vertebral
systems. After carotid occlusion and development of ischemia, HRP leaks

27
from the capillary lumen. In these investigations, there is no indication
of endothelial cellular damage and, therefore, the extravasation of HRP is
attributed to increased vesicular transport. The stimuli for this may be
release of serotonin from platelets inducing vesicular formation secondary
to vasoconstriction and increased blood pressure. Focal edema induced by
a number of means, such as ultraviolet exposure, also increases vesicular
transport and HRP uptake.
Nonionizing radiation has long been implicated in BBB disruption.
Microwaves and x-rays have been studied extensively in this regard. The
effects of microwaves are highly controversial. One report states that
exposure of rats to 2450 MHz at 10 mW/cm2 for two hours produces a marked
extravasation.96 This level is considered safe for human exposure in the
United States, but not in other countries. The biological effects of
microwaves can be subclassified as changes resulting from gross thermal
effects or nonthermal effects. In this study, the body temperatures of
the animals were constant and the altered permeability of the BBB was
attributed to microwave-stimulated serotonin release from platelets,
although other mechanisms are possible. Conversely, a recent publication
indicates that much higher levels of radiation are required to cause pro-
9 7
tein leakage, specifically 3000 mW/cm2. At these levels, cerebral tem
perature increases significantly and changes can be attributed to gross
thermal effects.
Porto-caval anastomosis, which causes severe liver dysfunction, has
also been implicated in BBB breakdown.5477 This disintegration has been
termed hepatic encephalopathy. As in a myriad of other circumstances,
increased vesicular transport has been implicated as the mechanism of
extravasation.

28
Several situations involving autoimmune afflictions and induced auto
immunity, such as experimental allergic encephalomyelitis (EAE) which is
used as a model of multiple sclerosis, demonstrate an increased capillary
permeability.98 In EAE, the cerebral vessels are said to cuff or deform
and this abnormality correlates with BBB breakdown. This may be important
in the general progression of multiple sclerosis, as vascular changes are
among the first changes which precede demylelination.
Agents which solubilize and fluidize membranes may also act to in
crease BBB permeability.99100101 Dimethyl sulfoxide (DMSO), in very high
concentrations, has been thought to increase the flux of such plasma mark
ers as inulin and mannitol. Its effects are said to be derived from its
lytic action on membrane although interaction by micellular formation can
not be ruled out. Nortriptyline has a similar effect. Ethanol, in large
doses, can also cause opening of the BBB to sucrose. It was postulated
that this generalized increase in permeability may increase the suscepti
bility of the CNS to bacterial and viral infection. It has been shown,
however, in acute and chronic doses of ethanol that were compatible with
continued life, that there was no alteration in the BBB.102103
Many heavy metals have been associated in both general and specific
changes in the BBB.156104 Mercury (II) Hg+, in high concentrations,
i.e. >80 pm, causes a generalized increase in the permeability of cerebral
capillaries to sugars and protein markers but in low concentrations affects
only the hexose carrier. Ionic lead has a similar effect.
A number of conditions can alter BBB function in more subtle, yet no
less damaging ways. In these instances, changes occur at the level of a
specific carrier or carriers and, as such, only a particular type of com
pound is involved. These changes can result from physical alteration of

29
the carrier by denaturation. Additionally, diminution or increase of the
blood level of a transportable compound can affect the utilization of the
compound. In some cases, the rate limiting step in metabolism of a nutrient
can be transferred from an enzyme to transit of the agent across the BBB.21
Glucose transport is very important to CNS function. Under normal cir
cumstances, the rate-determining step in glucose metabolism involves the
enzyme hexokinase. If, however, the plasma concentration of glucose falls,
as in hypoglycemia, or cerebral metabolism increases, as in relative hypo
glycemia, the limiting step in utilization is shifted to transport of glu
cose across the BBB.20
In severe hypoxia (p02 <10 mm), a number of progressive changes as
sociated with glucose flux occur.1 In the dog, after one minute of oxygen
deprivation, the influx of glucose into the brain is unchanged but efflux
decreases. Ten minutes after initiation of hypoxia, influx and efflux of
glucose decrease. This has been explained by a postulated modulator which,
in the absence of hypoxia, is bound to the carrier and alters transport.
In other animal models, different results are obtained.
The transport of amino acids and, especially, neutral amino acids in
certain disease states, has been the subject of much research. The neutral
amino acid carrier is responsible for the transport of such neurotrans
mitter precursors as tryptophan, tyrosine and histidine and, as such, any
interruption of this supply can have tremendous neurological consequences.
Nowhere is this more apparent than in phenylketonuria.1*20 This syndrome,
which is associated with high blood levels of phenylalanine, has been linked
to mental retardation. This condition of high phenylalanine levels can
act to competitively inhibit other substrates of this carrier, such as
tyrosine, tryptophan, and histidine. This is made possible because of

30
the similar Km values for these amino acids in addition to the similarity
between the Km's and plasma levels of these amino acids. Hyperphenylala-
ninemia also can reduce protein synthesis by a similar mechanism. Treat
ment of this disease involves a phenylalanine restricted diet and 5-hydroxy-
tryptophan supplements.
In hepatic encephalopathy, the neutral amino acid carrier is induced
and, as previously discussed, this induction may be related to increase in
ammonia and glutamine levels. Dihydroxyphenylalanine (L-DOPA), which is
used as a dopamine source in the treatment of parkinsonism, is transported
by the neutral amino acid carrier. It has been shown that ethanol increases
DOPA transport as well as the transport of tyrosine, tryptophan and a-
methyldopa into the CNS.103
The monocarboxylic acid carrier appears to be a major organ for elim
inating metabolic acid wastes. As was previously discussed, the Km of
lactate is close to its plasma concentration so that rises in lactate,
such as those that accompany exercise, are only slowly translated to the
CNS. In the case of anoxia, however, where cerebral levels of lactate
rise, the systemic dissipation of this byproduct is slowed by the same
phenomenon.20 In the brain the loss of lactate is carrier and not diffusion-
limited, unlike other organs. In hypoglycemia, lactate may act as an energy
source.105 This is true in neonates, where lactate uptake is much higher
than in adults. The Km of the monocarboxylic acid carrier is also corre
spondingly higher in neonates.
Ketone bodies, which have an affinity for the monocarboxylic acid car
rier, are produced during fasting and can act as a metabolic energy source.
These bodies include B-hydroxybutyric acid and acetoacetic acid. The en
zyme responsible for their production, e-hydroxybutyrate dehydrogenase, is

31
induced by starvation. The utilization of the surrogate food sources is
limited by BBB transit.
In starvation, the monocarboxylic acid carrier is said to be induced,
although this is controversial. A recent paper demonstrated a lower Km
and Vmax for carriers in starved animals compared with control animals.33
The increased flux of B-hydroxybutyrate and other substrates was attributed
in this article to an increase in the diffusional component.
A number of therapeutic agents have affinity for these carriers. These
include such anionic compounds as probenicid, penicillin, and aspirin.20
This affinity could potentially lead to competition and a decreased ability
to eliminate metabolic acids from the CNS. It has been shown, however,
that the concentrations required to cause inhibition are far above thera
peutic levels.
The BBB is a complex system of enzymes, protein carriers, and vessels
which impart to the brain a selective interface. This interface prevents
potentially damaging substances from entering the brain without impeding
the entry of nutrients or the exit or metabolites and excretory products.
In adverse circumstances, the permeability of the barrier can be increased,
resulting in a number of deleterious effects.
Prodrugs and Drug Delivery Systems
The BBB excludes a number of pharmacologically active agents and, as
such, treatment of many cerebral diseases is severely limited. In order
to increase the effectiveness of drugs which are active against central
maladies, the pharmacokinetic profile of the agent must be augmented and,
specifically, the transit time of the drug in the brain must be increased.
If a method were available to implement these alterations, an agent could
be delivered specifically to the brain. This specificity should increase

32
the therapeutic index of an agent since not only is the concentration of
the agent increased in the vicinity of the bioreceptor but, of equal im
portance, the peripheral concentration of the drug is reduced decreasing
any associated toxicity.
Unfortunately, there are very few methods for circumventing the BBB
and these are of limited usefulness. The direct administration of drugs
into the CNSji.e., an intrathecal injection has been used to deliver the
folate antagonist, methotrexate, to the brain. This method is not very
satisfactory since the distribution of methotrexate in the brain is uneven
and slow.46 Additionally, since the ventricular volume of the CSF is
small, increases in intracerebral pressure can occur with repeated injec
tions. This is particularly dangerous when the intracerebral pressure is
already high as it is in CNS cancers. Repeated lumbar puncturing also
carries a risk.
A general method which can be applied to delivery of drugs to the
brain is the prodrug approach.106-110 The term prodrug was coined by
Albert and refers to the result of a transient chemical modification of a
pharmacologically active agent. This change imparts to the compound an
improvement in some deficient physiochemical property such as water solu
bility or membrane permeability. Ideally, a prodrug is biologically in
active but reverts to the parent compound in vivo. This transformation
can be mediated by an enzyme or may occur chemically due to some designed
instability in the agent. The aim of these manipulations is to increase
the concentration of the active agent at its site of action and thereby,
increase its efficacy. While potentially there are many different types
of proderivatives, most thus far synthesized are simple esters and amides.
These compounds are transformed to the parent acid, alcohol or amine by
the ubiquitous hydrolases which are present in vivo. Many anticancer

33
agents have lent themselves to this type of manipulation. Several amides,
for example, of the highly water soluble anti cancer agent guanazole have
been made. This series of lipoidal compounds hydrolyzed in vivo to yield
the parent drug.111 A more sophisticated prodrug is cyclophosphamide,
which is inactive in vitro. The agent is activated by P450 mixed function
oxidases in the liver to the potent alkylating agent, N,N-bis(chloroethyl)-
phosphordiamidic acid.109 This drug is extensively used in cancer chemo
therapy.
One of the most important applications of prodrugs is in the sustained
release of therapeutically active agents. Cytosine arabinoside is used
as an S-phase specific antimetabolite, but suffers from rapid metabolism
by cytidine deaminase requiring a continual administration of the drug.
This problem produced the prodrug cyclocytidine which is not a substrate
for cytidine deaminase and which slowly releases the cytosine arabinoside
by ring cleavage.112
The influx of a compound to any organ can be related by the equation
Kp = QE
where Kp is the clearance of a drug by an organ, Q is the blood flow
through that organ and E is the extraction coefficient.113 The extraction
coefficient is related to the lipophilicity or octanol-water partition
coefficient of a compound which is in turn related to the ability of a
compound to partition into phospholipoidal membranes and consequently in
to organs. By increasing the lipophilicity of a compound with the pro
drug approach, one can increase the entry of a compound into its site of
action but this is not specific and, in general, all organs are exposed
to a greater tissue burden.113 Nonspecificity is, therefore, one of the
major drawbacks of the prodrug approach. This is especially important

34
with cytotoxic agents. While increased membrane permeability makes a
compound more effective locally as a cytotoxic agent, there is almost
always a disproportionate rise in systemic toxicity. This has severely
restricted anti cancer prodrugs of this type.
Several types of toxicities are also associated with prodrugs.114
Theoretically, a prodrug should be metabolized only to the parent compound
but the formation of toxic metabolites by the prodrug is possible. This
occurs with such compounds as phenacetin, a prodrug of acetaminophen.
Another possible toxic reaction may be brought about by enzymatic or glu
tathione depletion. The compound thiamine tetrahydrofurfuryl disulfide,
a prodrug of thiamine, requires glutathione-mediated disulfide bond cleav
age for activation and, therefore, toxicity may arise from the associated
depletion of glutathione.
The idea of using prodrugs to increase the specificity of delivery
has been considered. This has proved, however, to be difficult and not
very fruitful. Two basic approaches have been taken in this regard: site-
directed or site-activated delivery and delivery by the association of a
drug with a macromolecular carrier.
The first method does not attempt to concentrate a compound at a par
ticular location, but is based on site-specific activation of the prodrug.
For this to be possible, the enzyme responsible for the activation must
be located specifically, or at least in high relative concentrations in a
particular organ. The finding, for example, that y-glutamyl peptidase is
present in high concentrations in the kidney led to a number of compounds
substituted with the y-glutamyl group.113 Sulfamethiazide and L-DOPA were
derivatized in this manner in order to achieve renal delivery.

35
This approach has been extensively applied to cancer chemotherapy.114
The philosophy behind this application is related to the many differences
that occur between cancer cells and normal cells. These variations are
basically the result of the altered metabolism of neoplastic cells. Many
alkylating agents have been synthesized in an attempt to capitalize on
these differences. The lower pH of tumor cells has been exploited in a
series of aziridines which are more active at the pH of tumors than at
physiological pH. The greater reducing power of cancerous sites has led
to the development of a number of biologically inactive azo compounds
which upon reduction yield potent cytotoxic agents.115 Examples of these
are tetrazolium mustard and azomustard, both of which are reduced to aniline
mustards. The inactivity of the parent compound is due to the delocaliza
tion of the nucleophilic nitrogen lone pair by the conjugated ring system.
A number of O-phosphate esters have been synthesized in order to take
advantage of the high levels of acid phosphatase which are characteristic
of human neoplasms.115 The O-phosphate esters of p-hydroxy mustard and
estradiol mustard were prepared as specific agents to be used in prostate
cancers. The enzyme, y-glutamyl transpeptidase, is also found in high con
centrations in tumor cells so that y-glutamyl derivatives of cytosine ara-
binoside and phenylene-diamine mustard have been proposed. The presence
of hydrolytic esters has also been established in neoplastic formations.
These include esterases and g-glucuronidases.112,115 The cytotoxic agent,
aniline mustard, is converted in the liver as a result of a first pass
effect to its O-glucuronide. Tumors which contain high B-glucuronidase
activity convert the O-glucuronide to the potent alkylating agent p-hydroxy-
aniline mustard.

36
While these compounds have demonstrated some promise as anti cancer
drugs, for the most part they have not lived up to their potential. There
are a number of reasons for this failing. The chemical manipulation of
these agents may act to decrease their accessibility to a site of action.
Additionally, if the agent is fairly lipophilic, it may "leak" from its
site of action before exerting a pharmacological effect.113 In cancers
there may also be diffusion limitations because of the restrictions in
blood flow.
A second approach for increasing the specificity of an agent for a
particular organ involves the coupling of a pharmacologically active agent
with a macromolecule.116 The specificity derived from such a system is
related to the interaction between cells and endogenous and exogenous bio
polymers and macromolecules. Albumin, for example, is actively endocytosed
by various macrophages.117 An anti cancer compound could be coupled to al
bumin and the complex taken up by a macrophage tumor. This would then be
directed to a lysosome where the drug would be hydrolyzed from its albumin
carrier and exert its pharmacological action specifically. This has also
been suggested as a means of treating DNA viruses which implant in macro
phages. Anthracyclines, such as daunorubicin and adriamycin, intercalate
into DNA. A drug carrier has been devised in which fragments of DNA con
taining intercalated anthracyclines are administered systemically.114118
This intercalated complex is inactive but can be endocytosed and broken
down in lysosomes by DNAases. Again, the aim is to release the cytotoxic
agent in the vicinity of the malignancy and thereby increase its efficacy.
Antibodies have also been studied as specific delivery carriers, but re
search has been hampered by the inhomogeneity of tumor-specific antibodies.

37
The idea of including a drug into liposomes formed in vitro has re
ceived a great deal of attention.117119 In these systems, the drug is
inactive since it is enclosed in the phospholipoidal matrix of the lipo
some and, as in the case of the albumin conjugates, these packets are
taken up by endocytosis. Again, the system should specifically exert its
action at the site of influx. While these approaches are promising the
oretically, they have not met with much success. These carrier complexes
are the subject of several recent books.117119
A general method was recently proposed for the specific delivery of
drugs to the brain. This system was based on the results of some work
with N-methylpyridinium-2-carbaldoxime chloride (2-PAM).120121>122123
This pyridinium quaternary compound is the agent of choice for the treat
ment of organophosphate poisonings, and exerts its action by reactivating
deactivated cholinesterases. The problem with this agent is its highly
polar nature. Organophosphates such as diisopropyl fluorophosphate (DFP)
and paraoxon are very lipophilic and easily penetrate the BBB. The brain
is, therefore, very susceptible to acetylcholinesterase inactivation by
these agents. The highly polar 2-PAM has a very low activity in the brain
since it is almost totally excluded by the BBB. To deal with this prob
lem the dihydro adduct of 2-PAM, pro-2-PAM,was synthesized as a prodrug.

ch3
2-PAM
pro-2-PAM

38
The rather involved synthesis of pro-2-PAM yielded the enamine salt
which corresponds to the 3-protonated dihydro compound. The pKa of the
tertiary nitrogen in this enamine salt was determined potentiometrically
and spectrophotometrically to be 6.32 0.6 and the oxime was estimated
to have a pKa of -11. With a pKa of 6.32, it would be predicted that
about 90% of the enamine would exist as the free base at physiological
pH (7.4). Since this dihydro free base is far more lipophilic than the
parent quaternary, its ability to penetrate membranes is greatly enhanced.
This pro-2-PAM is rapidly converted to the parent 2-PAM at physiological
pH and the tx has been determined to be 1.04 min by pharmacokinetic model-
2
ing. This rapid conversion of the pro-2-PAM to 2-PAM is desirable since
ideally, the only metabolism of the prodrug is to the parent compound.
A number of experiments were carried out using 2-PAM and pro-2-PAM.
From the linear descending portion of a semilog plot of blood concentra
tion versus time, the biological tx of 2-PAM and pro-2-PAM was calculated.
2
The tx for comparable doses of 2-PAM and pro-2-PAM differed by more than
2
60 min, and since the conversion of pro-2-PAM -* 2-PAM is rapid, this dif
ference was assumed to be the altered distribution of pro-2-PAM. This in
dicates that even though the rate of conversion of pro-2-PAM is rapid, it
is long enough for the enhanced distributional characteristics of the
pro-2-PAM to be expressed.
Blood levels of 2-PAM were significantly higher after an oral dose
of pro-2-PAM than with a dose of 2-PAM. If pro-2-PAM is administered in
travenously, there are no new metabolites formed. If brain concentrations
of 2-PAM are examined after 2-PAM and pro-2-PAM dosing, the concentration
of 2-PAM in the brain is 13-fold higher in the case of pro-2-PAM admin
istration. If brain acetylcholinesterase is inactivated using DFP, the

39
extent of reactivation observed in mice injected with pro-2-PAM shows a
dramatic increase over 2-PAM treated animals.
A further experiment with 2-PAM showed that this small quaternary
salt was rapidly lost from the CNS and this loss was attributed to an ac
tive efflux process.124 These results differ from those obtained by Ross
and Froden who attempted to deliver a quaternary compound to the brain
as its uncyclized aj-haloalkyl amine.125126 They found that the loss of
the quaternary compound was slow (t = 38 hours) and concluded that the
2
efflux of the quaternary was comparable to its influx. This difference
in the rate efflux may be related to differences in molecular size, shape
or charge.
This preliminary work led to a proposed drug delivery system (Figure
1-2) which is specific for the brain and which is generally applicable.124
In this proposal, a pharmacologically active agent, whose ability to pass
the BBB is low, is chemically linked to a pyridinium carrier. This car
rier could be envisioned as a nicotinamide or nicotinic acid ester. This
complex would be reduced under conditions which would yield the dihydro
pyridine. This complex is then injected systemically and,because of the
increased lipophilicity of the dihydropyridine, partitions into the brain
as well as into the periphery. In both locations, oxidation (kox) should
occur. The rate of this oxidation is somewhat controllable by the judi
cious placement of ring substituents on the pyridinium nucleus. Systemi
cally, the charged polar oxidized species should be eliminated rapidly
by the kidney and/or liver (kout2)> while in the brain the compound, be
cause of its charge and size, would be retained i.e., kQUt2 > ^0utT
Also, in both locations, cleavage of the drug from its carrier should
occur (kcleavage)* In the brain, the small nontoxic pyridinium carrier is

40
R
Figure 1-2. A Proposed Carrier-mediated Chemical Delivery System with
Specificity for the Brain. The Drug Molecule to be Trans
ported is Represented by the (O)-

41
rapidly eliminated, If the cleavage of the drug from its carrier
occurs at an appropriate rate i.e., kc-¡eavage > kouti a sustained release
of the agent to the brain could also be obtained. Again, the cleaved
polar drug would be rapidly eliminated systemically. By these manipula
tions a compound can be delivered specifically to the brain while its
systemic concentration is kept low. This reduces any associated systemic
toxicity of the agent and increases its therapeutic index dramatically.
In this system the positively charged carrier complex cleaves to
yield the active compound. The dihydropyridine is not, therefore, a pro
drug but rather a pro-prodrug or, better stated, a chemical delivery sys
tem. This drug delivery system is based on the naturally occurring re
duced nicotinamide adenine dinucleotide (NADH) t oxidized nicotinamide
(NAD+) system. These endogenous coenzymes are important in electron trans
ferring chains and their suitability to this purpose is related to the
chemistry of dihydropyridines and enamines. In the described delivery
system the relative unstability, greater lipophilicity and predictability
of chemistry of the dihydromoieties are exploited. Additionally, since
this method relies on enzymatic activation by an endogenous system (NADH
dehydrogenase) whose substrates closely approximate the delivery compounds,
any toxicity associated with the oxidation should be minimal. In this
system the BBB has not been simply circumvented but rather used as an in
tegral part of the delivery scheme.
Statement of the Problem
The chemical delivery system proposed by Bodor et al. should demon
strate a broad applicability since the agent to be delivered is simply
attached to a particular carrier.124 In certain instances, however, this
system can be simplified. If a pyridinium nucleus isanintegral structural

42
component of a molecule, as it is in many pharmacologically active agents,
the molecule is provided with an internal delivery moiety. The drug to
be delivered and the carrier are therefore merged into one molecule. The
pharmacokinetics of this scheme is also simpler than those involved with
the carrier system since no cleavage is necessary of the delivered mole
cule from the carrier. In this approach, which appears in Figure 1-3,
an appropriately chosen pharmacologically active compound i.e., one which
contains a pyridinium moiety, would be reduced to its corresponding di
hydropyridine. This lipophilic species would penetrate the BBB as well
as into the systemic circulation. After a period of time determined by
the stability of the compound, it would be oxidized to the parent quater
nary salt (kox). Systemically, this agent would rapidly be eliminated
by filtration or by tubular secretory mechanisms (kg^)- In the CNS,
however, since the ability of the compound to freely diffuse would be lost,
it would be delivered fairly specifically (kout2 > kouti) The transit
time of the drug in the brain would depend upon a number of factors in
cluding the participation of the compound in any active efflux processes.
It would be hoped that in most cases the rate at which the compound entered
the brain would be faster and ideally, much faster than the rate at which
the compound left the brain (kouf|)*
This system, unlike the carrier mediated chemical delivery system,
can be considered a prodrug. This prodrug should, however, demonstrate
a specificity for the brain because of its design, the characteristics
of the BBB, and the chemistry of dihydropyridines. Again, this specificity
should increase the therapeutic index of the drug delivered.
Several criteria were used in choosing a molecule for these delivery
approaches. Obviously, the compound should contain a pyridinium moiety

43
Figure 1-3. The Proposed Drug Delivery System.

44
which is reducible to some dihydropyridine species which is stable enough
to be isolated. The compound should be active in vi tro. Since quaternary
compounds are to be considered, the lack of any in vivo activity might be
ascribed to transport or distributional problems. A review of the litera
ture showed two groups of compounds which looked particularly suitable.
These include the substituted benzophenanthridinium salts and the proto-
berbine alkaloids which have the basic skeleton:
benzophenanthridinium ion protoberbine
Members of these groups contain an N-substituted isoquinoline moiety
which can be reduced by a number of agents. A number of these dihydroiso
quinoline compounds are stable.127 These agents show a wide range of
effects including anti neoplastic and antibiotic activity. 128-136 The
Berberine, which has the chemical name 5,6-dihydro-9,10-dimethoxybenz-
[g]-[l,3]benzodioxolo[5,6-a]quinolizinium chloride, has a rather high
in vitro activity against several cancer types including Ehrlich and lym
phoma ascites.131133 Its in vivo action is, however, very low.132133

45
Berber'ne is widely distributed in the plant kingdom and is found
in such families as menispermaceae and berber'daceae, to name only two.137
The compound was isolated by 1826 by Pelletan and Chevallier.
Biosynthetically, berberine is interesting because of the presence
of the so-called berberine bridge. This term refers to the single carbon
atom between the nitrogen and the methoxylated aromatic nucleus. Two post
ulates for the formation of this have been suggested. The first involves
formaldehyde in a Mannich-type ring closure while the second involves an
TOO IOQ #
oxidative cyclization of an N-methyl group. Labelling experiments
favor the second mechanism.139
There are several total syntheses for berberine in the literature
and the chemistry of the protoberbine alkaloids is well reviewed.140-142
Berberine has a variety of pharmacological actions and several therapeutic
uses. Pharmacologically, it exhibits a depressive action on excitable
tissues,143 induces hypotension and tachycardia,144145 and inhibits a
number of enzymes including histaminease (human pregnancy plasma diamine
oxidase),146 cholinesterase,144 dopamine-adenyl cyclase,147 and cation-
dependent ATP phosphorylases.148 Berberine also possesses an antiheparin149
and local anesthetic activity.144 Because of the affinity of berberine
for dopaminergic receptors150 and alcohol dehydrogenases,151 it has been
used to characterize geometric and stereospecific requirements for sub
strate binding to these enzymes. Berberine has long been known as an anti
biotic. The alkaloid causes mutations in certain bacterium by affecting
nonchromosomal genetic material.134 Berberine has been used mostly in
India to treat cholera,152 diarrhea,153 leshmaniasis and other parasitic
infections.

46
Biochemically, berberine acts to inhibit DNA, RNA and protein syn
thesis.143 It has been suggested that many compounds which are structur
ally similar to berberine exert their cytotoxic activity via alkylation
at the iminium site.128 A more thoroughly studied, and perhaps more
accurate, hypothesis is that berberine acts by intercalating between the
base pairs of DNA. This intercalation can be explained by a modified
neighbor-exclusion model.154 A model which has been used to explain the
intercalation of berberine is one in which the greater portion of rings A,
B and D are intercalated.155 As berberine complexes, it assumes a more
rigid planar species as may be indicated by the increase in fluorescence
quantum yield. This intercalation does, however, slightly bend the double
helix. The unwinding angle of DNA due to intercalation of berberine is
less than for the more planar molecules such as corylene. The effect of
the positive charge on intercalation has also been noted.156
Berberine, therefore, is appropriately suited as a candidate for in
clusion in the drug delivery system described. In order to verify the
original hypothesis of site-specific delivery, a number of experiments
are required. Dihydroberberine must be synthesized and its stability
assayed. The ability of dihydroberberine to penetrate the BBB and concen
trate in the brain must also be demonstrated. Additionally, the toxicity
and anti cancer activity of the agent should be investigated.

CHAPTER 2
MATERIALS AND METHODS
Elemental analyses of compounds synthesized were performed by Gal
braith Laboratories, Inc., Knoxville, Tennesse, or Atlantic Microlab,
Inc., Atlanta, Georgia. Uncorrected melting points were determined using
a Thomas-Hoover melting point apparatus. Ultraviolet spectra (UV) were
recorded on a Beckman 25, Cary 210 or Cary 219 spectrophotometer. An
Apple II Plus microprocessor was dedicated to the Cary 210 instrument.
Infrared spectra (IR) were taken on a Beckman 4210 high resolution and a
Beckman Acculab 1 infrared spectrophotometer. Samples were analyzed as
a thin film between sodium chloride windows or as a potassium bromide pel
let. Nuclear magnetic resonance spectra (NMR) were obtained from either
a Vari an T60 or Joel-JNM-FX 100 Fourier transform spectrometer. The sam
ples were dissolved in deuterated chloroform (CDC13), deuterated dimethyl
sulfoxide ((CD3)2S0), deuterated pyridine (C5D5N), deuterated methanol
(CD30D), deuterated acetonitrile (CD3CN), deuterium oxide (D20) or tri-
fluoroacetic acid (TFA). Chemical shifts in parts per million were re
ported relative to the internal standard tetramethylsilane except in aque
ous systems where sodium 3-trimethylsilylpropanesulfonate is used. Mass
spectra were obtained using a DuPont 21-491B double focusing magnetic sec
tor mass spectrometer to which was dedicated a Hewlett Packard 2100A com
puter. In all determinations, the ionizing voltage was 70 eV.
High pressure liquid chromatography (HPLC) was performed on either
a Waters Associates system consisting of a Model U6K injector, a Model
6000A solvent delivery system and a Model 440 absorbance detector or a
47

48
ternary Beckman system consisting of three Model 112 solvent delivery
systems, a Model 160 absorbance detector and a Model 421 controller. In
some studies, a Varan FluorichromR detector was used with one of the
Beckman pumps. Thin-layer chromatography (TLC) was performed on EM Re
agents Cat. 5751 Aluminum Oxide 60 F-254 precoated plates (layer thickness
0.25 mm) or Analtech, Inc. UniplateR, which are precoated with silica gel
G at a thickness of 0.25 mm. Tissues were homogenized by a VirTis 45
homogenizer or by a teflon pestle and ground glass tube. In potentiomet-
ric titrations, a Radiometer-Copenhagen PHM 84 Research pH meter was used.
A Sage Instrument Co. Model 341A syringe pump was used for infusions.
For radionuclide studies, a Packard Tri-Carb 460 CD Liquid Scintillation
system was employed.
In the theoretical studies, the MINDO/3 program was modified to suit
the University of Florida's IBM System/370 computer. The molecule draw
ing program, X3DM0L, was developed by E.W. Phillips.
All chemicals used were of reagent grade. Berberine was obtained
from Sigma Co. while nicotinic acid, nicotinamide, methyl iodide, benzyl
bromide, butanol, hexanol, octanol, decanol, sodium borohydride and sodium
dithionite were obtained from Aldrich Co. Phenethyl alcohol was purchased
from Matheson, Coleman and Bell. Tritiated inulin, SolueneR and scintilla
tion cocktails were obtained from New England Nuclear. Solvents were of
chromatographic purity and were obtained from Fisher Co. Pyridine (Aldrich
Co.) was refluxed over CaH2 and distilled before using. In cases where
oxygen was to be excluded, solutions were made from water which, after
boiling for fifteen minutes, was cooled with a stream of pyrogallol-
scrubbed nitrogen passing through it. A phosphate buffer (pH 7) was made
by dissolving 1.183 g of potassium dihydrogen phosphate and 4.320 g of
disodium hydrogen phosphate in water and diluting to 1.0 a.

49
Synthesis
Dihydroberberine (2)
Seven grams of berberine chloride (1) were dried over phosphorous
pentoxide at 80C in a vacuum oven for eight hours. Five grams of the
dried salt (0.013 moles) were added to a suspension of dry pyridine and
0.6 g of sodium borohydride (NaBh^). The solution was stirred under
for twenty minutes at room temperature, at which time 0.5 g more of
NaBH4 were added. The liquid was then poured into 800 ml of ice water.
The ensuing precipitate was dried overnight at 50C over phosphorous pen
toxide. The crude material was recrystallized from benzene-petroleum
ether (low boiling). The yield was 35%: Melting point 156-158C, Litera
ture 157-159C; XH NMR (CDC13) 7.1 (1 H, s), 6.7 (2 H, s), 6.4 (1 H, s),
5.9 (3 H, s), 4.3 (2 H, s), 3.4 (6 H, s), 3.1 (2 H, t), 2.9 (2 H, t);
MS m/e 338 (M+ + 1) 22%, 337 (M+) 100%, 322 (M+ 15) 27%, 278 (M+ 59)
13%; UV (95% ethanol) 370 xmax; IR (KBr) v (C-H) 3005 and 2970, (C-C, C-N)
1600 and 1482, (C-0-C asym) 1227 and 1270, (C-0-C sym) 1028 and 1083;
13C NMR (see figure 3-6); Elemental analysis calculated %: C, 71.21;
H, 5.60; N, 4.15. Found %: C, 71.19; H, 5.70; N, 4.14.
Pi hydroberberine Hydrochloride (3)
One gram of (2) was dissolved in a minimal amount of CH2C12. Anhy
drous hydrogen chloride, produced by dropping concentrated sulfuric acid
(H2S04) on sodium chloride (NaCl), was bubbled through the solution yield
ing a yellow precipitate. The material was recrystallized in aqueous
ethanol.
9-Demethylberberine (Berberrubin) (4)
Seven grams of (1) were dried for two hours at 100C in a vacuum oven
and then were heated to 200C for an additional thirty minutes. The deep
purple material produced was dissolved in hot water and extracted with

50
chloroform (CHC13). The organic layer was reduced to dryness and the
residue dissolved in hot water. The solution was filtered and then made
acidic with an excess of hydrochloric acid (HC1). The ensuing precipi
tate (yellow-brown solid) was collected by filtration and redissolved in
hot water. The solution was filtered and made basic with potassium hy
droxide. The solution turned deep purple and crystallization was induced
by scratching: *H NMR (CD30D) 6 9.0 (s, 1 H), 7.8 (s, 1 H), 7.2 (m, 2 H),
6.8 (m, 2 H), 5.8 (s, 2 H), 3.7 (s, 3 H), 4.4 (m, 2 H), 3.0 (m, 2 H);
UV (95% ethanol) 276 nm and 238 nm A others 512 nm; IR (KBr) v (C-H)
MiaX
3000 and 2900, (C-C, C-N) 1635, 1571, 1509 and 1472, (C-0-C asym) 1221
and 1289, (C-0-C sym) 1035 and 1144; Elemental analysis calculated %:
C, 71.03; H, 4.67; N, 4.36. Found %: C, 71.20; H, 4.81; N, 4.42.
Berberine Iodide (5)
A solution of 1.0 g of (4) in acetone was prepared. A 1.0 M excess
of methyl iodide was added and the solution was allowed to reflux for
several hours. The characteristic yellow color of berberine appeared and
TLC, NMR, IR, and UV confirmed the methylation.
3-(Aminocarbonyl )-l-methylpyridinium Iodide/1-Methylnicotinamide Iodide (6)*
Five grams of nicotinamide (0.041 moles) were dissolved in 50 ml of
dry methanol. A molar excess of methyl iodide (11.6 g) was added to the
stirring mixture and after one hour of refluxing, a precipitate formed.
This was filtered and washed. The material was recrystallized from aque
ous methanol: Melting point 101-103C, Literature 102-105C; UV (H20)
224 mm and 265 mm Amax; NMR and IR were identical with the literature.
*The nicotinic acid derivatives are given a systematic name followed by
a common name

51
3-(Ami nocarbonyl)-!-(phenyl methyl)pyridinium Bromide/1-Benzyl nicotinamide
Bromide (7)~
Ten grams of nicotinamide (0.083 moles) were dissolved in 150 ml of
methanol. A molar excess of benzyl bromide (28.0 g) was added and the
mixture was allowed to reflux for several hours. Upon cooling, a white
solid appeared. This was filtered, washed and recrystallized from metha
nol: Melting point 206-208C, Literature 205C; LH NMR (D20 or TFA)
6 9.5 (1 H, s), 9.0 (2 H, m), 8.2 (1 H, m), 7.5 (5 H, s), 5.9 (2 H, s);
IR (NaCl) v (N-H asym) 3300, (N-H sym) 3148, (-C(=0)NH2 Amide I) 1690,
(Amide II) 1643, (C-N-C) 1388.
3-Pyridinecarboxylic Acid Ethyl Ester/Ethyl Nicotinate (8)
Forty-two grams (0.34 moles) of nicotinic acid were mixed with 55 ml
of absolute ethanol and 25 ml of concentrated H2S04. The mixture was
refluxed in an oil bath for four hours at which time the solution was
poured over ice and made slightly basic with ammonia. The aqueous solu
tion was extracted with ethyl ether. The organic layer was dried with
sodium sulfate (Na2S04) and the solvent evaporated under reduced pressure.
The product was a clear liquid and the yield was 55%: *H NMR (CDCI3) 6 9.2
(1 H, s), 8.8 (1 H, m), 8.2 (1 H, m), 7.4 (1 H, m), 4.4 (2 H, q), 1.4
(3 H, t); IR (NaCl) v (C-H) 2990, (C=0) 1728, (-C-C(=0)0) 1286, (0-C-C)
1112, (Y CH) 742, (b ring) 703.
3-Pyridinecarboxylic Acid Butyl Ester/Butyl Nicotinate (9)
Forty-two grams (0.34 moles) of nicotinic acid were dissolved in
55 ml of 1-butanol and 25 ml of concentrated H2S04 was slowly added. The
solution was heated to reflux in an oil bath for three hours, at which
time the solution was poured over ice and made slightly basic with ammonia.
The mixture was extracted with ethyl ether. The separated ether layer
was dried over Na2S04 and the solvent removed under reduced pressure.

52
The product was a clear liquid and the yield was 62%: XH NMR (CDC13)
9.2 (1 H, s), 8.8 (1 H, m), 8.2 (1 H, m), 7.4 (1 H, m), 4.3 (2 H, t),
1.6 (4 H, m), 0.97 (3 H, t); IR (NaCl) v (C-H) 2962, (C=0) 1730, (C-C,
C-N) 1594, (-C-C(=0)0) 1288, (0-C-C) 1117, (y CH) 742, (g ring) 702.
3-P.yridinecarbonylchloride Hydrochloride/Nicotinoyl Chloride Hydrochloride
(10)
Forty-one grams (0.33 moles) of nicotinic acid were stirred in an ice
bath with 110 ml of thionyl chloride (S0C12) slowly added. After the
addition was complete, the mixture was refluxed for three hours. The
S0C12 was removed under reduced pressure and traces of S0C12 were azeo-
troped off with benzene. The white crystalline product was obtained in
94% yield; NMR and IR were identical with the literature.
3-Pyridinecarboxylic Acid Hexyl Ester/Hexyl Nicotinate (11)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry dis
tilled pyridine and 5.73 ml (0.062 moles) of 1-hexanol. The solution was
refluxed in an oil bath for six hours. The solution was then poured over
ice which had been made basic with ammonia. The aqueous solution was ex
tracted with ethyl ether, the organic layer dried over Na2S0M and the
solvent removed under reduced pressure. The yield was 60%: XH NMR (CDC13)
9.1 (1 H, s), 8.7 (1 H, m), 8.2 (1 H, m), 7.3 (1 H, m), 4.3 (2 H, t),
1.2 (8 H, m), 0.87 (3 H, t); IR (NaCl) v (C-H) 2938 and 2961, (C=0) 1726,
(C-C, C-N) 1590, (-C-C(=0)0) 1282, (0-C-C) lili, (y CH) 741, (g ring) 702.
3-Pyridinecarboxylic Acid Octyl Ester/Octyl Nicotinate (12)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry pyri
dine and 9.75 ml (0.062 moles) of 1-octanol. The solution was heated to
reflux in an oil bath and the progress of the reaction monitored with TLC.
After eight hours, the liquid was poured over ice which had been made basic
with ammonia. The aqueous solution was extracted with ethyl ether. The

53
ether layer was dried over sodium sulfate (Na2S04) and the solvent removed
under reduced pressure. The yield was 58%: XH NMR (CDC13) 6 9.1 (1 H, s),
8.7 (1 H, m), 8.2 (1 H, m), 7.3 (1 H, m), 4.3 (2 H, t), 1.3 (12 H, m),
0.88 (3 H, t); IR (NaCl) v (C-H) 2920 and 2950, (C=0) 1723, (C-C, C-N)
1589, (-C-C(=0)0) 1277, (0-C-C) 1109, (y CH) 732, (g ring) 693.
3-Pyridinecarboxylic Acid Decyl Ester/Decyl Nicotinate (13)
Ten grams of (10) (0.062 moles) were dissolved in 100 ml of dry pyri
dine and 11.82 ml (0.062 moles) of 1-decanol. The solution was refluxed in
an oil bath for eight hours. The liquid was then poured over ice which
had been made basic with ammonia. The aqueous solution was extracted with
ethyl ether, the organic layer dried over Na2S04, and the solvent removed
under reduced pressure. The yield was 64%: XH NMR (CDC13) 8.7 (1 H, m), 8.2 (1 H, m), 7.2 (1 H, m), 4.3 (2 H, t), 1.2 (16 H, m),
0.83 (3 H, t); IR (NaCl) v (C-H) 2915 and 2946, (C=0) 1720, (C-C, C-N) 1584,
(-C-C(=0)0) 1275, (0-C-C) 1105, (y CH) 732, (g ring) 692.
3-Pyridinecarboxylic Acid g-Phenylethyl Ester/g-Phenethyl Nicotinate (14)
Twenty-six grams (0.15 moles) of (10) were dissolved in 200 ml of
pyridine. To this stirring solution was added dropwise 18.3 g (0.15 moles)
of g-phenethyl alcohol. The solution was refluxed in an oil bath for
several hours. The solution was then poured over ice and made slightly
basic with ammonia. This solution was then extracted with ether. The
organic layer was dried over Na2S04 and evaporated under reduced pressure.
The yield was 62%: *H NMR (CDC13) 9.1 (1 H, s), 8.6 (1 H, m), 8.0 (1 H, m),
7.2 (6 H, s), 4.4 (2 H, t), 2.9 (2 H, t); IR (NaCl) v (C-H) 3030 and 2959,
(C=0) 1722, (C-C, C-N) 1585, (-C-C(=0)0) 1276, (0-C-C) d 1120, 1105, (y CH)
737, (g ring) 695.

54
3-(Ethoxycarbonyl )-1-methy1pyridinium Iodide/Ethyl N Methylnicotinate
Iodide (15)
Ten grams of (8) (0.066 moles) were mixed with methanol and with a
molar excess of methyl iodide (18.7 g). The solution was refluxed for
several hours. The solvent was removed, yielding a red-orange oil which
solidified on cooling to a yellow solid. The solid was recrystallized
from acetone-ether: *H NMR (CDC13, (CD3)oS0), 5 9.6 (2 H, m), 9.0 (1 H, m),
8.4 (1 H, m), 4.8 (3 H, s), 4.5 (2 H, q), 1.4 (3 H, t); IR (NaCl) v (C-H)
3008, (C=0) 1723, (-C-C(=0)0) 1303, (0-C-C) d 1102, 1117, (y CH ) 742,
(6 ring) 654.
3-(Butox.ycarbon\
/~\ )-l-methylpyridinium Iodide/Butyl N Methyl nicotinate
Iodide 1
)
Ten grams of (9) (0.056 moles) were dissolved in 60 ml of acetone
and a molar excess of methyl iodide (15.9 g) was added. The solution was
refluxed for two hours. The solvent was removed, leaving a yellow solid
which was recrystallized from acetone-ether: XH NMR (CDC13, (CD3)oS0)
6 9.9 (2 H, m), 9.0 (1 H, m), 8.6 (1 H, m), 4.5 (3 H, s), 4.4 (2 H, t),
0.93 (3 H, t); IR (NaCl) v (C-H) 2950, (C=0) 1720, (-C-C(=0)0) 1291,
(0-C-C) 1100, (y CH) 735, (b ring) 651.
3-(Hexoxycarbonyl)-1-methylpyridiniurn Iodide/Hexyl N Methylnicotinate
Iodide (17)
Ten grams of (11) (0.048 moles) were dissolved in 60 ml of acetone,
and a molar excess of methyl iodide (13.7 g) was added. The solution was
refluxed for two hours. The solvent was removed, producing an orange oil:
lH NMR (CDC13, (CD3)2S0) <5 9.3 (2 H, m), 8.9 (1 H, m), 8.3 (1 H, m),
4.7 (3 H, s), 4.3 (2 H, t), 0.90 (3 H, t); IR (NaCl) v (C-H) 2944, (C=0)
1723, (-C-C(=0)0) 1295, (0-C-C) 1111, (y CH) 738, (8 ring) 654.

55
3-(0ctoxycarbonyl)-l-methylpyridinium Iodide/Octyl N Methylnicotinate
Iodide (18)
Ten grams of (12) (0.043 moles) were mixed with 60 ml of acetone and
with a molar excess of methyl iodide (12.1 g). The solution was allowed
to reflux for two hours at which time the solvent was removed, yielding an
oil which was resistant to crystallization: XH NMR (CDC13, (CD3)oS0) 6 9.9
(2 H, m), 9.0 (1 H, m), 8.6 (1 H, m), 4.6 (3 H, s), 4.4 (2 H, t), 0.92 (3 H,
t); IR (NaCl) v (C-H) 2960, (C=0) 1731, (-C-C(=0)0) 1302, (0-C-C) 1120,
(y CH) 749, (6 ring) 668.
3-(Decoxycarbonyl)-l-methylpyridinium Iodide/Decyl N Methylnicotinate
Iodide (19)
Ten grams of (13) (0.038 moles) were dissolved in 60 ml of acetone
and a molar excess of methyl iodide (10.8 g) was added. The solution was
refluxed for two hours. The solvent was removed, leaving an orange oil:
!H NMR (CDC13, (CD3)2S0) 6 9.7 (2 H, m), 9.0 (1 H, m), 8.5 (1 H, m), 4.8
(3 H, s), 4.4 (2 H, t), 0.95 (3 H, t); IR (NaCl) v (C-H) 2944, (C=0) 1725,
(-C-C(=0)0) 1297, (0-C-C) 1113, (y CH) 741, (g ring) 658.
3-(g-Phenylethoxycarbonyl)-l-methy1pyridinium Iodide/g-Phenethyl N-Methyl-
nicotinate Iodide (20)
Ten grams of (14) (0.044 moles) were dissolved in 60 ml of acetone.
A 1.0 M excess of methyl iodide (12.5 g) was added to the liquid and the
system was allowed to reflux for two hours. The solvent was removed,
yielding a solid which was recrystallized from acetone: XH NMR (CDC13,
(CDo) SO) 6 9.4 (2 H, m), 8.7 (1 H, m), 8.2 (1 H, m), 7.2 (5 H, s), 4.7
(3 H, s), 4.6 (2 H, t), 3.1 (2 H, t); IR (NaCl) v (C-H) 2995 and 3023,
(C=0) 1724, (-C-C(=0)0) 1290, (0-C-C) d 1135 and 1124, (y CH) 731, (g ring)
657.

56
1.4-Pi hydro-1-methyl-3-pyridinecarboxamide/1-Methyl-1,4-Dihydronicotin
amide (21)
Four and six-tenths grams of sodium hydrogen carbonate (NaHC03) and
2.64 g of (6) (0.019 moles) were dissolved in 100 ml of water and cooled
in an ice bath. To this stirring solution was added 6.96 grams of sodium
dithionite (Na2S204). A stream of nitrogen (N2) covered the reaction mix
ture. After one hour, the reaction was stopped and the solution was ex
tracted with several aliquots of CHC13. The CHC13 layer was removed under
reduced pressure, yielding an orange oil. The oil was dissolved in a min
imal amount of CHC13 and tritrated with petroleum ether. From this, an
oil appeared and this was removed and dried in vacuo: XH NMR (D20) 6 6.9
(1 H, s), 5.7 (1 H, d), 4.8 (1 H, m), 3.2 (2 H, s), 3.0 (3 H, s); UV 355 nm
Amav; Elemental analysis calculated %: C, 60.87; H, 7.25; N, 20.20. Found
%: C, 60.92; H, 7.29; N, 20.36 (C7H1QN20).
1.4-Dihydro-l-(phenylmethyl )-3-p.yridinecarboxamide/1-Benzyl-1 ,4-dihydro-
nicotinamide (22)' *
To 100 ml of water were added 4.6 g of NaHC03 and 2.93 g of (7) (0.013
moles). The solution was cooled and 6.96 g of Na2S204 were added. After
two hours of stirring under N2, a precipitate formed. The solution was
filtered. The solid was recrystallized from aqueous methanol, giving
lemon-yellow needles: XH NMR (D20) 6 7.2 (6 H, s), 7.1 (1 H, s), 5.7 (1 H,
m), 4.7 (1 H, m), 4.2 (2 H, s), 3.1 (2 H, m), in CDC13 two protons at 5.86
appear; UV 357 nm X ; Elemental analysis calculated %: C, 72.29; H, 6.58;
max
N, 12.97. Found %: C, 72.09; H, 6.60; N, 12.84 (C13H14N20).
1.4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Ethyl Ester/Ethyl 1,4-Di-
hydro-N-methylnicotinate (23~)~
Four and six-tenths grams of NaHC03 and 2.75 g of (15) (0.016 moles)
were dissolved in 100 ml of water and cooled in an ice bath. To this stir
ring solution was slowly added 6.96 g of NaS204. Two hundred milliliters

57
of ethyl ether were then added so that the dihydro would be extracted
upon formation. This two-phase system avoided tetrahydropyridine produc
tion. The reaction proceeded for one hour under nitrogen. The ether layer
was removed and the aqueous layer extracted. The combined ether fractions
were dried over Na2S04 and the solvent removed under reduced pressure. The
resulting orange-red oil was dried in vacuo: NMR (CDC1J 6 6.9 (1 H, d),
5.6 (1 H, m), 4.8 (1 H, m), 4.1 (2 H, g), 3.1 (2 H, m), 2.9 (3 H, s), 1.2
(3 H, t); UV 358 nm Xm3 ; Elemental analysis calculated %: C, 64.67; H, 8.17;
max
N, 8.43. Found %: C, 64.65; H, 7.88; N, 8.34 (C9H13N02).
1.4-Dihydro-l-methyl-3-pyridinecarboxylic Acid Butyl Ester/Butyl 1,4-Dihy-
dro-N-methylnicotinate (24)
A solution of 4.6 g of NaHC03 and 3.12 g of (16) (0.016 moles) was
prepared in 100 ml of water. The solution was cooled and 6.96 g of Na2S20i+
were added. Two hundred milliliters of ethyl ether were added and this
mixture stirred under nitrogen for one hour. The ether layer was removed,
dried with Na2S04 and reduced in volume. The resulting oil was dried in
vacuo: NMR (CDC13) 6.9 (1 H, s), 5.6 (1 H, m), 4.7 (1 H, m), 4.1
(2 H, t), 3.1 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 358 nm Xmax; Ele
mental analysis calculated %: C, 67.69; H, 9.07; N, 7.23. Found %: C,
67.58; H, 8.82; N, 7.09 (CuH17N02).
1.4-Di hydro-1-methyl-3-pyridinecarbox,ylic Acid Hexyl Ester/Hexyl 1,4-
Dihydro-N-methylnicotinate (25)
A solution of 4.6 g of NaHC03 and 3.57 g of (17) (0.016 moles) was
prepared in 100 ml of water. The solution was cooled and 6.96 g of
Na2S204 were added. Two hundred milliliters of ethyl ether were added
and the mixture stirred under nitrogen for one hour. The ether layer was
separated, dried and reduced in volume. The dried oil was orange in color:
XH NMR (CDC13) 6 6.8 (1 H, s), 5.5 (1 H, m), 4.6 (1 H, m), 4.0 (2 H, t),

58
3.1 (2 H, m), 3.0 (3 H, s), 0.9 (3 H, t); UV 359 nm Amax; Elemental analy
sis calculated %: C, 69.96; H, 9.73; N, 6.32. Found %: C, 69.82; H, 9.46;
N, 6.28 (C13H2iN02).
1.4-Dihydro-1 -methyl-3-pyridinecarboxylic Acid Octyl Ester/Octyl 1,4-Dihy-
dro-N-methylnicotinate (26)
A solution of 4.6 g of NaHC03 and 4.02 g of (18) (0.016 moles) was
prepared in 5% aqueous methanol. The solution was cooled and 6.96 g of
Na2S204 were slowly added. Two hundred milliliters of ethyl ether were
added and the mixture stirred under nitrogen for one hour. The ether layer
was separated, dried and reduced in volume. The oil was dried in vacuo:
XH NMR (CDC13) 6 6.9 (1 H, m), 5.6 (1 H, m), 4.7 (1 H, m), 4.0 (2 H, t),
3.0 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 358 nm xmax; Elemental analy
sis calculated %: C, 70.69; H, 9.97; N, 5.50. Found %: C, 70.76; H, 9.68;
N, 5.88 (C15H25N02-i-H20).
1.4-Dihydro-1-methyl-3-pyridinecarboxylic Acid Decyl Ester/Decyl 1,4-Dihy-
dro-N-methylnicotinate (27)
A solution of 4.6 g of NaHC03 and 4.46 g of (19) (0.016 moles) was
prepared in 5% aqueous methanol. The solution was cooled and 6.96 g of
Na2S204 were slowly added. To this solution was added 200 ml of ethyl
ether and the mixture stirred under N2 for one hour. The ether layer was
separated, dried and reduced in volume. The dried oil was orange in color:
XH NMR (CDC13) 6.9 (1 H, m), 5.6 (1 H, m), 4.7 (1 H, m), 4.0 (2 H, t),
3.0 (2 H, m), 2.9 (3 H, s), 0.9 (3 H, t); UV 359 nm xmax; Elemental analy
sis calculated %: C, 73.12; H, 10.39; N, 5.02. Found %: C, 73.16; H, 10.48;
N, 5.03 (C17H2gN02).
1.4-Dihydro-1-methyl-3-p.yridi necarboxyl ic Acid B-Phenylethyl Ester/B-Phen-
ethyl 1,4-Dihydro-N-methylnicotinate (28)
Four and six-tenths grams of NaHC03 and 3.89 g of (20) (0.016 moles)
was dissolved in 100 ml of water. The solution was cooled and 6.96 g of

59
Na2So04 were added. Two hundred milliliters of ethyl ether were added and
the mixture stirred over N2 for one hour. The ether layer was separated,
dried and reduced in volume. The dried oil was orange in color: 1H NMR
(CDC13) 7.2 (5 H, s), 6.9 (1 H, m), 5.7 (1 H, m), 4.7 (1 H, m), 4.2 (2 H,
t), 3.0 (2 H, m), 2.9 (2 H, t), 2.8 (3 H, s); UV 355 nm Amax; Elemental
analysis calculated %: C, 70.18; H, 6.63; N, 5.46. Found %: C, 70.27; H,
7.00; N, 5.10 (C15H17N02-^-H20).
Characterization of Dihydroberberine
Distribution Coefficients
Fifty milliliters of a cold 1 x 10"4 M solution of berberine (1) in
pH 7.4 buffer were partitioned against 50 ml of CHC13 or 50 ml of 1-octanol.
The concentration of (1) was determined spectrophotometrically in the organ
ic and aqueous layer. A stock solution of 2.7 x 103 M dihydroberberine
hydrochloride (3) was made in methanol. An aliquot of this, sufficient to
produce a 1 x 104 M solution, was pipetted into 50 ml of cold pH 7.4 buf
fer and extracted immediately with either CHC13 or 1-octanol. After allow
ing for oxidation, the concentration of (1) in the organic and aqueous layer
was determined spectrophotometrically.
Potentiometric pKfl Determination of Dihydroberberine (2)
Due to the extreme water insolubility of (2) (< 3 ug/ml), all deter
minations were done in 25% methanolic solutions. A titration curve was
generated by adding 10 ul aliquots of NaOH to a 1.0 mM solution of (3).
The pKa was determined by inspection of the titration curve. During the
experiments, all solutions were covered with a stream of nitrogen.
Spectrophotometric pKa Determination of Dihydroberberine (2)
The pKa of (2) was determined by measuring the absorbance difference
at 355 nm in basic, acidic, and buffered media. The relationship that
allows this determination appears below:

60
pKa = pH log
aobs ~ aHA
a._ a ,
A obs
where aQbs is the absorbance in buffer, a^- is the absorbance in base and
a,, is the absorbance in acidic media.
HA
Oxidation of Dihydroberberine (2) by Silver Nitrate
Two hundred milligrams of (2) were dissolved in 95% aqueous ethanol.
Upon addition of a 10% solution of silver nitrate, a black precipitate
formed. Centrifugation and analysis of the supernatant shows stoichio
metric oxidation of (2).
Oxidation of Dihydroberberine (2) by Pi phenyl pi cry!hydrazyl Free Radical (DPP*)
Two hundred milligrams of (2) were dissolved in acetonitrile. To this
was added a solution of DPP in acetonitrile which caused an immediate dis
appearance of the purple color due to DPP-. Ultraviolet analysis confirmed
this oxidation.
Oxidation of Dihydroberberine (2) by Concentrated Hydrogen Peroxide
Two hundred milligrams of (2) were dissolved in 95% aqueous ethanol.
A 30% solution of hydrogen peroxide was added and the system monitored by
UV. The analysis demonstrated a rapid and complete oxidation of (2).
Oxidation of Dihydroberberine (2) in Buffers
The oxidation of (2) was determined by UV and an HPLC method. In the
UV method, a solution of (3) was prepared and pipetted into buffers of
various pH and at various temperatures. The changes of absorption at
460 nm were measured with time. Data acquisition was facilitated by an
Apple II microprocessor and an enzyme kinetic software package. In the
HPLC method, two buffers, pH 5.8 and pH 7.4, were used. The samples were
maintained at 37C in a water bath. At certain times, 5 pi of the solu-
lution were injected onto a yBondapak C18 reverse-phase column, and the

61
peak heights analyzed. The mobile phase was 60:40 acetonitrile: pH 6.2
phosphate buffer and the flow rate was 2 ml/min.
Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro-
pyridines (21) and (22) in Hydrogen Peroxide
Solutions of (3), (21) and (22) were prepared. An aliquot of these
solutions was added to a standardized solution of hydrogen peroxide (H202)
(0.18 M). The appearance of the 460 nm peak of (1) or the disappearance of
the 359 nm peak of the dihydronicotinamides was measured. This determina
tion was made using the enzyme kinetics software package.
Quantitation of the Oxidation of Dihydroberberine (21) and Various Dihydro-
pyridines (22)-(28) in Plasma
Freshly drawn 80% human plasma was obtained from Civitan Regional Blood
Center. The oxidation of (3) was determined by HPLC and the oxidation of
(3), (22), (23), (24), (25), (26), (27), and (28) by UV. In the HPLC analy
sis, a solution of (3) was added to 80% plasma and maintained at 37C. At
certain times, 1.0 ml of plasma was removed and treated with 3 ml of aceto
nitrile. The solution was centrifuged and 5 pi of the supernatant was
analyzed by a pBondapak Cis reverse-phase column with a mobile phase of
60:40 acetonitrile: pH 6.2 phosphate buffer. The peak heights were ana
lyzed and concentrations obtained from a standard curve. In the UV method,
40% plasma was maintained at 37C in a kinetic cell. A solution of either
(3) or one of the various dihydronicotinates was added to this and the
appearance of the 460 nm absorbance of (1) or disappearance of the 359 nm
absorbance of dihydronicotinates was observed.
Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro-
pyridines (22)-(28) in Liver Homogenate
The determination of the rate of oxidation of (3) in a liver homogenate
by an HPLC method and (3), (22), (23), (24), (25), (26), (27), and (28)
by a UV method was performed. In the HPLC method, 14 g of fresh rat liver

62
were homogenized in 45 ml of cold phosphate-buffered saline. To this was
added a solution of (3), and the system was maintained at 37.0C. At vari
ous times, 1.0 ml of the solution was removed and the protein precipitated
with 3 ml of acetonitrile. The sample was centrifuged and 5 yl of the
supernatant analyzed using a yBondapak C18 reverse-phase column with a
mobile phase of 60:40 acetonitrile: pH 6.2 phosphate buffer. The peak
heights were analyzed and concentrations obtained from a standard curve.
The UV method involved homogenizing 7 g of rat liver in 15 ml of pH 7.4
phosphate buffer and diluting the homogenate to 200 ml (3.5% w/v). The
homogenate was centrifuged and the supernatant was used. To this was added
a solution of (3) or one of the dihydropyridines. The appearance of the
460 nm peak of berberine or the disappearance of the 359 nm peak of the
dihydronicotinate was then measured.
Quantitation of the Oxidation of Dihydroberberine (2) and Various Dihydro
pyridines (22)-(28) in Brain Homogenate
Again, both an HPLC and UV method were employed in the determination
of the rate of oxidation of (3), (22), (23), (24), (25), (26), (27) and
(28). In the HPLC method, a solution of (3) was added to a 20% brain homo
genate in pH 7.4 phosphate buffer. The homogenate was maintained at 37C
in a water bath. At various times, 1.0 ml of the homogenate was mixed
with 3 ml of acetonitrile. The sample was centrifuged and the supernatant
analyzed by the same method used in the liver homogenates. In the UV
method, 2 g of freshly obtained rat brain were homogenized in 33 ml of
pH 7.4 phosphate buffer, yielding a 6.0% w/v homogenate. The homogenate
was centrifuged and the supernatant was used in the determinations. To
this was added a solution of (3) or one of the dihydronicotinates. The
appearance of 460 nm absorption of (1) or disappearance of the 359 nm peak
of the dihydronicotinate was measured.

63
In Vitro Distribution of Berberine (1) and Dihydroberberine (2) in Whole
Blood
Solutions of (1) or (3) were added to 60 or 75 ml of freshly drawn
heparinized sheep's blood maintained at 37C. At various times, 4 ml of
blood were withdrawn. The blood was centrifuged and the plasma removed.
One milliliter of the plasma was treated with 9 ml of acetonitrile and the
supernatant was analyzed spectrophotometrically. The entire volume of the
packed red blood cells was treated with 8 ml of acetonitrile and centri
fuged. The supernatant was again analyzed spectrophotometrically. A stan
dard curve was obtained by preparing solutions of known concentration in
plasma or packed red blood cells. Recovery from the red blood cells was
71.4%.
Effect of Glucose on the Distribution of Berberine (1) in Whole Blood
Glucose was added to a volume of blood so that a concentration of
200 mg% was obtained. The above procedure was then repeated.
Animal Studies
In Vivo Characterization of Berberine (1) and Dihydroberberine (2)
White Sprague-Dawley rats, who weighed between 200-250 g, were anes
thetized intramuscularly with Inovar^ (0.13 ml/Kg). Injections were made
intravenously into the external jugular vein. The doses used include
55 mg/Kg of (2) in dimethylsulfoxide (DMS0), 55 mg/Kg of (3) in 20-25%
aqueous ethanol, 55 mg/Kg of (1) in DMS0 or 35 mg/Kg of (1) in DMS0.
At certain times after the injection, the chest cavities of the rats were
opened, the vena cava severed and the heart perfused with normal saline.
Afterwards, the animals were decapitated and the brains removed. In cer
tain experiments, the lungs, liver, and kidneys were also excised. The
organs were then homogenized in a minimal amount of water, usually 2 ml,
and extracted with 8 ml of acetonitrile. A standard curve of (1) in the

64
organ homogenate was constructed. Analysis was performed by injecting
5 yl of the supernatant onto a yBondapak Cis reverse-phase column with a
mobile phase of 60:40 acetonitrile: phosphate buffer. The flow was 2 ml/
min. Under these conditions, the retention time of (1) was 3.8 min and
(2), 9.3 min.
Slow Infusion of Dihydroberberine Hydrochloride (3)
Rats were anesthetized and prepared as above. A dose of 55 mg/Kg of
(3) was prepared in a volume of 1.0 ml. The vehicle was 20% aqueous etha
nol. This dose was infused into the external jugular vein over a period
of either thirty or forty-five minutes. At the end of the perfusion, the
animals were decapitated, their organs collected and analyzed.
Effect of 1-Methyl-1,4-dihydronicotinamide (21) on the Efflux of Berberine
from the Brain
Rats were anesthetized and prepared as above. Animals were injected
with 200 mg/Kg of (21) in aqueous ethanol intravenously. After fifteen
minutes, the standard dose of 55 mg/Kg of (3) was given. The animals were
sacrificed at various times after the injection, selected organs were re
moved and homogenized, and the samples analyzed by HPLC.
In Vivo Characterization of l-Benzyl-1,4-dihydronicotinamide (22)
Rats were anesthetized and cut down as above. Doses between 60 mg/Kg
and 400 mg/Kg of (22) were administered. At various times after the in
jection, the animals were perfused, decapitated, and the brains removed.
The brains were then frozen in liquid nitrogen and stored at 0C until
they were analyzed, at which time the brains were thawed, homogenized
in 2 ml of water, and extracted with 8 ml of acetonitrile.
Analysis was made by HPLC using a yBondapak C18 reverse-phase column
and a mobile phase of 40:60 acetonitrile: 1 x 10"3 M sodium heptanesul-
fonate. The dihydronicotinamide had a retention time of 3.4 min and the

65
quaternary compound had one of 10.6 min at a flow rate of 2 ml/min. A
standard curve was constructed in brain homogenates.
Intracerebral Ventricular (icv) Administration
Sprague-Dawley rats were anesthetized with 70 yl/100 g of pentobar
bital sodium, and atropine sulfate (0.05 mg/Kg) was given, if necessary.
Injections of (1), (6), and 3H-inulin (29) were made into the lateral
ventricles of rat brains. The injections were made with the aid of a
stereotaxic instrument and the site of the injection was -0.4 mm anterior-
posterior, 1.5 mm medial-lateral and -3.0 mm dorsal-ventral relative to
the bregma. The dose of (1) infused was 50 yg and the infusion volume
was between 3 and 5 yl. The vehicle was DMS0 and the infusion rate was
5 yl/5 min. In several experiments, (1) was coinjected with 1000 yg of
(6). In another set of experiments, a dose of 2.3 yCi of (29) was injected
icv.
At specific times after the infusions, the animals were decapitated.
The brain was homogenized in 1.0 ml of water and (1) extracted with 4 ml
of acetonitrile. Analysis of (1) was by HPLC with a mobile phase consist
ing of 50:50 acetonitrile: pH 6.2 phosphate buffer. A yBondapak Ci8 re
verse-phase column was used and a standard curve constructed using brain
homogenates. For radionuclide analysis, the brains were homogenized in
8 ml of water. Twenty-five hundredths of a milliliter of this homogenate
were added to 0.75 ml of SolueneR. After the sample dissolved, 12 ml of
the scintillation cocktail were added. The samples were counted for five
minutes and disintegrations per minute (dpm) were obtained by using a
standard quench curve.
Limited Metabolic Studies
Rats treated intravenously (iv) with 55 mg/Kg of (1) or 55 mg/Kg of
(3) were housed in metabolic cages. Urine was collected and extracted with

66
CHC13- The aqueous layer was then extracted with 3-methyl-1-butanol.
The organic layers were reduced to dryness and then reconstituted with
a small volume (50 yl) of methanol. This residue was used for HPLC and
TLC analysis. Five microliters of each sample were analyzed by HPLC.
A yBondapak Cis reverse-phase column and a mobile phase of 50:50 aceto
nitrile: pH 6.2 phosphate buffer were used. The TLC analysis consisted
of spotting 5 yl of each sample on an alumina plate and eluting the sys
tem with cyclohexane: chloroform: acetic acid 45:45:10 or methanol. The
plates were developed with iodine vapor or iodoplatinate spray reagent.
Toxicity
White CD-I mice were employed in this study, the average mass of
which was 22.6 2 g. The mice were segregated into groups of 10, and
8-10 groups were used in each study. The doses given were determined by
preliminary studies in which the LDo and the LDioo were obtained using
small groups of animals. The doses were then prepared in equal incre
ments between the two extremes, but there was additional emphasis placed
at the lower end of the curve. Since this study was concerned with acute
toxicity, the animals were injected intraperitoneally, and the number dead
recorded after twenty-four hours. The groups were, however, observed an
additional forty-eight hours to ensure an accurate appraisal of acute
toxicity. Food and water were given ad libitum. The injection volume was
75-100 yl. The data were analyzed by fitting them to a sigmoid curve and
by the method of Probits.
Anti cancer Activity
Male BDF mice (20.6 0.3 g) were used in this study. A suspension
of P388 lymphocytic leukemia cells was injected intraperitoneally (ip)
(1 x 106 cells) or intracerebrally (2.5 x 105 cells). The survival time
of animals treated with various doses of (1) or (3) compared to the controls

67
was recorded. Berberine or dihydroberberine hydrochloride were given ip
3 times a day on day 2, 6, and 10 in 0.5% carboxymethy1 cellulose.

CHAPTER 3
RESULTS AND DISCUSSION
Synthesis and Characterization of Dihydroberberine
The initial step in the application of the proposed drug delivery
system to berberine (1) is the preparation of its dihydro adduct. The
first synthesis of dihydroberberine (2) involved disproportionation of
(1) in strong base and was performed by Gadamer in 1905.157,158 The
mechanism of this reaction involves nucleophilic attack of hydroxide to
the carbon adjacent to the nitrogen resulting in the formation of a tran
sient amino alcohol. This intermediate collapses to oxyberberine and di
hydroberberine, presumably via a hydride transfer. Several other syntheses
for (2) have appeared in the literature and these involve the direct re
duction of (1) by zinc amalgam,159160 complex metal hydrides161162 or
sodium borohydride.163164 Historically, (2) has been of interest because
of its spectroscopic properties,165-167 and as an intermediate in certain
synthetic schemes.164 The use of (2) as a drug or in a drug delivery
system is novel to this thesis.
In the present work (1) was reduced, as shown in Figure 3-1, by sodi
um borohydride in dry pyridine. Spectroscopic analysis showed that the
yellow crystalline material produced was (2). The UV, IR, and MS are shown
in Figures 3-2, 3-3, and 3-4 respectively, and are consistent with the
assigned structure.167-169 The XH NMR is presented in Figure 3-5, and the
proton assignments in Table 3-1. The 13C NMR is shown in Figure 3-6, and
the corresponding carbon assignments in Table 3-2. These assignments were
made by comparing (2) to a number of model systems.170 The synthesized
68

o
en
Figure 3-1. Synthesis of Dihydroberberine (2) and its Hydrochloride Salt (3).

70
Figure 3-2. Ultraviolet Spectrum of Dihydroberberine ^2) in 95%
Ethanol

PERCENT TRANSMISSION
WAVELENGTH IN MICRONS
3 3 5 I 4.5 5 5 5 6 6.5 7 7.5 8 9 0 II 12 14 16
V/A' EMUMbEf* CM1
Figure 3-3 Infrared Spectrum of Dihydroberberine (2) (KBr)
PERCENT TRANSMISSION

DIHYDROBERBERINE
ro
Figure 3-4- Mass Spectrum (70 eV, El) of Dihydroberberine (2)

Figure 3-5 Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of Dihydroberberine in CDC13.
The Insert Represents the Region between 2.8s and 3.46 at 100 MHz

74
Table 3-1 Proton Assignments of the NMR of Dihydroberberine (2)
H
Proton
a
b
c
d
e
f
g
h
PPM (5)
7.1
6.7
6.4
5.9
4.3
3.4
3.1
2.9

''!*> v^yv hv t ^ ^ ^ 4w *WV>VfjAi^4nvjy^^
cn
Figure 3-6. The 13C Nuclear Magnetic Resonance Spectrum (100 MHz, CDC13) of
Dihydroberberine (2)

76
Table 3-2. Carbon Assignments of the 13C NMR of Dihydroberberine (2)
Carbon
Number
PPM (5)
Carbon
Number
PPM (6)
1
150.301
11
111 .457
2
147.182
12
107.753
3
146.597
13
103.659
4
144.452
14
100.881
5
141.528
15
96.202
6
128.661
16
60.576
7
128.466
17
55.848
8
124.470
18
49.270
9
122.033
19
48.929
10
118.670
20
29.725
(CDC13: 78.267, 77.040, 75.732)

77
material gave an elemental analysis in good agreement with that predicted
for (2). Also, the reduced material reacted rapidly with such oxidizing
agents as hydrogen peroxide, silver nitrate, and 1,l-diphenyl-2-picryl-
hydrazyl free radical (DPP*) to yield (1). These data support the suc
cessful synthesis of (2).
The hydrochloride of dihydroberberine (3) was synthesized, as illus
trated in Figure 3-1, by treating a concentrated solution of (2), in methy
lene chloride with dry hydrogen chloride (HC1) gas. The hydrochloride
reverts to (2) at pH above 7.0. Analysis of the regenerated material
demonstrated no addition of HC1 or any other nucleophile to the molecule.
This addition is known to occur with several dihydropyridines. The hydro
chloride is many times more soluble in aqueous solutions than the free
base.
If (2) is to be successful in a drug delivery system described in
Figure 1-3, it should demonstrate a greater lipophilicity than (1). Di
hydroberberine is expected to be less polar than (1) because of the loss
of the positive charge. To investigate the relative lipid solubility of
(2) compared to (1), the two compounds were extracted with organic sol
vents and their distribution (partition) coefficients compared.171
The two solvent systems which were used included an octanol-pH 7.4
phosphate buffer and a chloroform-pH 7.4 phosphate buffer. The alcohol-
buffer system was chosen as one of the extracting systems because of its
ability to,in some ways, mimic the partitioning of compounds in vivo.
The data from Table 3-2 show that in both systems (2) has a high affinity
for the organic phase while (1) exhibits a high affinity for the aqueous
phase.
The chemistry of dihydroberberine is largely a result of its enamine
character.172 Dihydroberberine, like all enamines, is in equilibrium with

78
Table 3-3. Distribution Coefficients for Berberine (1) and Dihydrober-
berine Hydrochloride (3) in Chloroform/pH 7.4 Buffer and in
1-Octanol/pH 7.4 Buffer
Distribution
Coefficient
Compound
Chloroform/pH 7.4 Buffer
1-Octanol/pH 7.4 Buffer
Berberine (1)
< 0.001
0.062
Dihydroberberine
Hydrochloride (3)
5.33
2.59

79
its corresponding imine and this unusual situation allows enamines, dihy-
droberberine included, to be substrates in both nucleophilic and electro
philic reactions. This equilibrium tends to concentrate a negative charge
on the carbon 8 to the nitrogen. Protonation usually occurs, for this
reason, at the 8-carbon rather than the nitrogen. At physiological pH,
a portion of the dihydroberberine molecules will exist in a C-protonated
state. The pKa which is an indication of the degree of this ionization
is important since only the unprotonated free base is available for dif
fusion across membranes. The pKa of (2) was determined to be 6.80 0.05
by both a spectrophotometric and potentiometric method. At a physiological
pH of 7.4, a substantial portion of (2) will thus exist in the unionized,
freely diffusable form. Because of the water insolubility of the dihydro
berberine free base, all pKa determinations were carried out in 25% metha
nol ic solutions.
Demethylation of Berber'ne
A number of other synthetic schemes were explored in an attempt to
obtain a method for preparing radiolabeled (1), should spectroscopic method
prove too insensitive. A radiolabel led compound would also greatly expe
dite whole body distribution studies. In radiolabeling a compound, one
of the important factors governing the selection of a synthetic route is
yield. The scheme which was chosen involves pyrolyzing (1) at 200C.173,174
Berberine loses methyl chloride, as shown in Figure 3-7, to form the deep
purple zwitterionic berberrubin. This method would allow the placement
of either a 14C or 3H label at the nine position of (1) by reacting ber
berrubin with the appropriately tagged methyl iodide or methyl sulfate.
Methylation of berberine with cold methyl iodide was performed to demon
strate the viability of the scheme.

o
o
Figure 3-7. Demethylation of Berberine (1) and Hethylation of Berberrubin (4).
03
O

81
Theoretical Studies on the
Pi hydropyridine £ Pyridinium Redox System
If the drug delivery system described in Figure 1-3 is to be used to
its potential, then, a knowledge of its basic chemistry is required. The
reason for this is not only to understand the subtle chemistry inherent
in this particular system, but also to allow prediction, extrapolation
and generalization of the system to other examples. The ability to dis
cern, for instance, those factors that add to or detract from the stability
of a molecule would allow attenuation of a particular property by mole
cular manipulation. A thorough chemical knowledge of a particular com
pound would also allow an intelligent prediction as to its suitability
to the scheme.
While a few ionization potentials have been measured, the general in
stability of dihydropyridines often precludes their investigation by ex
perimental means. Because of this limitation, these compounds have lent
themselves well to theoretical study. While the dihydronicotinamides,
because of their biological relevance, have been the subject of copious
reports, larger dihydro systems have received little attention.175176
In order to gain a greater chemical insight into the proposed drug deliv
ery system in general, and the berberine (1) Z dihydroberberine (2) sys
tem in particular, a theoretical investigation was undertaken.
Berberine is a rather large molecule and, as such, would be expected
to present problems in terms of computational time. It must be remembered
that 3N-6 (where N is the number of atoms) independent variables are re
quired for a molecule and large molecules can easily cost $8000-$10000
in computer time. Because of this, and also in an attempt to generalize
the calculations so that they would be applicable to a number of compounds,
a model was developed for the (1) Z (2) system. The compounds chosen as

82
the model, 3H,4H-dihydro-7,8-dihydroxybenzo[b]quinolizinium ion (30) and
3H,4H,6H-trihydro-7,8-dihydroxybenzo[b]quinolizine (31) along with their
numbering protocol, are shown in Figure 3-8.
This quaternary and its dihydro adduct, while simpler than the
(1) t (2) system, do not differ greatly in structure or in general chem
istry. Compounds (30) and (31) have not been investigated and no mention
of them has appeared in the literature. Some theoretical work on the
unsubstituted benzo[b]quinolizinium ion and much on the isoquinoline mole
cule has been published. The benzo[b]quinolizinium ion was investigated
by Galasso in 1968 and charge densities and Tr-bond orders were reported.177
All of these reported studies have employed simplistic theoretical treat
ments such as PPP,178 IOC-w-technique,179 HMO,180 SSP,181 and CNDO/2.182
The corresponding dihydrocompounds have not been studied.
In order to investigate the isoquinoline model (30) t dihydroisoquino
line model (31) system, a MIND0/3 method was selected.183 This approach
is a semi-empirical self-consistent field molecular orbital method in which
all valence electrons are treated. The MIND0/3 program is the culmination
of the series MINDO,184 MIND0/2,185,186 and MIND0/2'.187 This program was
developed to solve chemical problems quickly and efficiently using a quan
tum mechanical framework. Unlike the methods of Pople, whose major aim
was to reproduce nonempirical calculations, M.J.S. Dewar parametrized
MINDO so that the system would produce useful and chemically accurate data.
This program is extremely versatile and has been applied to a number of
chemical problems including reactions. Only the most cursory details of
MINDO/3 are appropriate here, as the subject is well reviewed elsewhere.183188
In quantum mechanical approaches like MINDO, a molecular orbital is
the result of the interaction of a wavefunction of an electron with nuclei
and other electrons present in the molecule.189 Mathematically, the

Figure 3-S. Structures and Salient Numbering Protocols for Berberine (1), Dihydroberberine (2),
the Isoquinoline Model (30), the Dihydroisoquinoline Model (31), Pyridine (32), and
1,2-Dihydropyridine (33).

84
Hamiltonian for such a system consists of the kinetic energy term for the
movement of the electrons and the potential energy terms for electron-
nuclei attraction and electron-electron repulsion.
In systems with only one electron, e.g. H, H2+ or He+, the differen
tial equation which constitutes the Hamiltonian can be separated and
exactly solved. If, however, more than one electron is present, the elec
trons interact and the differential equation is no longer separable or
exactly solvable. Because of this problem, approximations and simplifi
cations are incorporated into the Schrodinger equation. These include
considering the molecular orbital (v) as a linear combination of atomic
orbitals ():189
'f = E Ci i
Additional simplifications are obtained by neglecting certain electron
repulsion terms since these values are close to zero. These approximations
allow the application of quantum mechanical approaches like MINDO to rather
complex chemical problems without extremely large expenditures of capital
or computer time.
The MINDO/3 program provides the following data: optimized geometries
of the most stable ground state conformation at 25C, the heat of forma
tion (AHf) in kcal/mole at 25C of the optimized structures, the atomic
charge distribution, the dipole moments, the total electronic energy, the
vertical ionization potentials, the bond order matrix, and the eigen vec
tors and eigen values.
The eigen values and eigen vectors allow a thorough examination of
the contributions of the individual atomic orbitals to the molecular or
bital and thus of the electronic structure of the molecule. This can be
indicative of many of the chemical proclivities of a molecule. The MINDO

85
approach allows one to examine compounds and reactions which are not
approachable by conventional means. For example, the stability of very
reactive species can be assayed, their structures determined, and quali
tative aspects of their chemistry elucidated.
The input for the program includes, for each atom, the atomic number,
the approximate bond distances between adjoining atoms, the approximate
bond angle, and the approximate twist angle out of the plane. As previously
mentioned, the number of parameters needed is 3N-6 where N is the number
of atoms. Convergence is assumed when the difference between two succes
sive calculations is less than 0.1 kcal/mole.
The two models (30) and (31) were analyzed and the results are pres
ented in Table 3-4 through 3-8. The heat of formation calculated for (30)
was 95.1 kcal/mole and that of (31) was -39.3 kcal/mole. While the use of
AHf for comparisons is restricted to structural isomers, the relative sta
bility of a system can be calculated by comparing the differences in AHf
(AAHf) from one system to another. The AAHf of the isoquinoline model
(30) Z dihydroisoquinoline model (31) pair is 134.4 kcal/mole. As shown
in Table 3-9, this value is smaller than that obtained from simple dihy
dropyridine Z pyridinium systems indicating a greater stability of (31)
relative to simple dihydropyridines.190 The stabilization of (31) indi
cates that it should be less reactive than simple dihydropyridines. The
basis of this stabilization is derived from the extended aromatic conjuga
tion of (31). Another contribution to the relative stabilization can be
seen by an examination of the highest occupied molecular orbital (HOMO)
which is represented in Figure 3-9 as a linear combination of atomic orbi
tals. In looking at the HOMO, only the magnitude of the coefficients is
important since the sign simply represents the phase of the orbital. A

Table 3-4. The Heats of Formation, Vertical Ionization Potentials, and Dipole Moments of the Isoquinoline
Model (30) and the Dihydroisoquinoline Model (31).
ISOQUINOLINE MODEL (30)
DIHYDROISOQUINOLINE MODEL (31)
95.1
HEAT OF FORMATION
-39.3
(kcal/mol)
11.56(h)
VERTICAL I.P.
7.1 9(tt-Pn)
12.94(h-Pn)
(eV)
8.31(h)
9.07(Pn)
9.44(a)
9.58(a)
15.43
DIPOLE MOMENT
1.73
(Debye)

Table
3-5. Bond Lengths in Angstroms
Dihydroisoquinoline Model
between Various Atoms
(31)
of the
Isoquinoline Model
(30) and the
Isoquinoline Model (30)
Dihydroisoquinoline Model (31)
Atom
Number
Bond
Length
Atom
Number
Bond
Length
Atom
Number
Bond
Length
Atom
Number
Bond
Length
1-2
1.342
13-14
1.389
1-2
1.451
13-14
1.397
2-3
1.433
14-4
1.433
2-3
1.517
11-18
1.321
3-4
1.467
11-18
1.321
3-4
1 .445
12-17
1.337
4-5
1.444
12-17
1.337
4-5
1.468
2-27
1.132
5-6
1.388
2-26
1 .115
5-6
1.370
2-26
1.130
6-1
1.415
5-19
1.110
6-1
1 .401
5-19
1.110
6-7
1.480
7-20
1.103
6-7
1.485
7-20
1 .103
7-8
1.345
8-21
1 .106
7-8
1.343
8-21
1 .106
8-9
1.487
9-22,23
1.118
8-9
1.489
9-23,22
1 .118
9-10
1.518
10-24,25
1.123
9-10
1 .521
10-24,25
1 .123
10-1
1 .480
13-16
1.106
10-1
1 .452
13-16
1 .106
3-11
1.467
14-15
1.104
3-11
1.444
14-15
1 .104
11-12
1.418
18-27
0.952
11-12
1 .432
18-28
0.951
12-13
1.433
17-28
0.950
12-13
1.417
17-29
0.951
00
--4

Table 3-6. Bond Angles in Degrees between Various Atoms of the Isoquinoline Model (30) and the
Dihydroisoquinoline Model (31)
Isoquinoline Model (30) Dihydroisoquinoline Model (31)
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
2-1-6
121.6
3-2-26
120.4
2-1-6
121 .8
1-2-26
108.3
3-4-5
115.7
13-14-15
199.9
3-4-5
116.3
1-2-27
108.8
4-5-6
125.5
12-13-16
119.7
4-5-6
126.4
13-14-15
119.9
1-6-5
116.2
13-12-17
117.2
1-6-5
117.9
12-13-16
119.7
1-6-7
117.1
12-11-18
128.1
1-6-7
116.5
13-12-17
117.2
6-7-8
123.7
6-7-20
114.5
6-7-8
123.6
12-11-18
128.1
7-8-9
121.9
7-8-21
121.3
7-8-9
121.8
6-7-20
114.5
6-1-10
121.9
8-9-22
110.7
6-1-10
123.4
7-8-21
121.3
1-3-4
118.6
8-9-23
108.9
11-3-4
118.7
8-9-22
109.8
2-11-3
120.5
9-10-24
109.7
12-11-3
120.4
8-9-23
109.0
1-12-13
119.1
9-10-25
109.8
11-12-13
119.3
9-10-24
108.0
2-13-14
121.8
12-17-28
114.3
12-13-14
121.1
9-10-25
108.6
4-5-19
117.8
11-18-27
114.3
4-5-19
116.3
12-17-29
113.9
11-18-28
113.9

Table 3-7.
Charge Density at Various
Model (31)
Atoms of the
Isoquinoline Model
(30) and the
Dihydroisoquino1ine
Isoquinoline
Model (30)
Dihydroisoquinoline Model
(31)
Atom
Number
Charge
Density
Atom
Number
Charge
Density
Atom
Number
Charge
Density
Atom
Number
Charge
Density
1
+0.0779
15
+0.0432
1
-0.1534
15
+0.0044
2
+0.1827
16
+0.0533
2
+0.2443
16
+0.0104
3
-0.1151
17
-0.4266
3
-0.1434
17
-0.4508
4
+0.1029
18
-0.4276
4
+0.0979
18
-0.4283
5
-0.0429
19
+0.0512
5
-0.1597
19
+0.0055
6
+0.1080
20
+0.0514
6
+0.1737
20
+0.0081
7
-0.0446
21
+0.0491
7
-0.0311
21
+0.0001
8
+0.0359
22
+0.0221
8
+0.0024
22
-0.0257
9
+0.0546
23
+0.0128
9
+0.0724
23
-0.0280
10
+0.1104
24
-0.0011
10
+0.1777
24
-0.0610
11
+0.3261
25
+0.0010
11
+0.2993
25
-0.0472
12
+0.2019
26
+0.0429
12
+0.1775
26
-0.0663
13
+0.0340
27
+0.2847
13
-0.0206
27
-0.0788
14
-0.0582
28
+0.2699
14
-0.0652
28
+0.2463
29
+0.2394

Table 3-8. Dihedral Angles between Various Atoms of the Isoquinoline Model (30) and the Dihydroisoquinoline
Model (31)
Isoquinoline Model (30) Dihydroisoquinoline Model (31)
Atom
Number
Dihedral
Angle,0
Atom
Number
Dihedral
Angle,0
Atom
Number
Dihedral
Angle,6
Atom
Number
Dihedral
Angle,6
14-13-12-11
-1.1
11-12-13-16
180.0
14-13-12-11
0.0
14-13-12-17
180.0
13-12-11-3
0.0
14-13-12-17
180.0
13-12-11-3
0.0
13-12-11-18
180.0
12-11-3-4
0.0
13-12-11-18
180.0
12-11-3-4
0.0
8-6-7-20
180.0
11-3-4-5
179.7
1-6-7-20
176.9
11-3-4-5
180.0
9-7-8-21
180.0
3-4-5-6
0.5
6-7-8-21
181.0
3-4-5-6
3.1
10-8-9-22
123.8
4-5-6-1
1.6
10-8-9-22
123.8
4-5-6-1
1.3
10-8-9-23
236.1
5-6-1-2
-2.7
10-8-9-23
236.1
5-6-1-2
-9.1
1-9-10-24
122.6
2-1-6-7
177.4
1-9-10-24
122.6
5-1-6-7
180.0
1-9-10-25
234.9
1-6-7-8
-3.6
1-9-10-25
234.9
1-6-7-8
1.6
6-4-5-19
180.0
6-7-8-9
0.3
3-4-5-19
180.0
6-7-8-9
0.1
13-12-17-29
92.0
2-6-1-10
179.1
13-12-17-28
87.9
2-6-1-10
180.2
3-11-18-28
180.0
12-13-14-15
180.0
3-11-18-27
174.7
12-13-14-15
180.0
3-1-2-26
125.4
4-3-2-26
179.4
11-12-13-16
180.0
3-1-2-27
235.0

91
134.40
kcal/mole
Anti
Syn
134.42
13S.20
2-PAM
1,6-dihydro-2-PAM

92
HOMO = 0-449 PzCNi} + -042 pz(C^ '221 pz(C3) *247 Pz(Clt)
+ 0.479 pz(C5) + 0.291 pz(C6) 0.074 pz(C7) 0.174 pz(C8) + 0.013 pz(C9)
+ 0.018 P2(C10) + 0.130 pz(C11) + 0.343 p (C12) + 0.106 PZ(C13) 0.284 pz(C14)
+ 0.005 s(H15) + 0.006 s(H16) 0.085 p (017) 0.079 Pz(018) 0.008 s(H19)
+ 0.008 s(H20) 0.010 s(H21) + 0.041 s(H22) 0.034 s(H23) + 0.113 s(H24)
- 0.082 s(H25) + 0.138 s(H26) 0.179 s(H27) 0.005 s(H28) 0.092 sCH29)
Figure 3-9. The Highest Occupied Molecular Orbital of the Dihydroisoquino
line Model (31)

93
relatively large contribution to the HOMO is made by the methylene hydro
gens 27, 28 and 24, 25 and the nonaromatic carbon, C2. This phenomenon
is termed hyperconjugation. Hyperconjugation also occurs in simple 1,2-
and 1,4-dihydropyridines but, because of the additional methylene inter
actions, the effect is slightly larger in the case of (31).190 The large
contribution to the HOMO by the nitrogen lone pair is also noted.
The vertical ionization potentials (IP) of (30) and (31) were calcu
lated using Koopman's Theorem which simply states that the ionization po
tential is the negative of orbital energy. The calculated values are
presented in Table 3-4 and are 11.56 eV for (30) and 7.19 eV for (31).
In addition, the character of the orbital which loses the electron can be
obtained by examination of the HOMO and, for (30), a ir-type system is in
volved while for (31) a mixed u-PN type orbital occurs. These values are
similar to values obtained from other systems and are consistentwith the
molecular structures.190 The dipole moments calculated for (30) and (31)
also appear in Table 3-4 and, again, are consistent with the molecular
structure.
It is instructive to compare changes that occur upon reduction of
simple dihydropyridines to those that occur upon reduction of (31). This
cmparison demonstrates the similarity in chemistry between simple and
more highly conjugated dihydropyridines and also demonstrates the extreme
usefulness of the computational method. Tables 3-10 to 3-12 are comparisons
of the pyridine (32) J dihydropyridine (33) system and the pyridinium
nucleus of (30) £ (31) system. The values for the (32) £ (33) system
were calculated by a MIND0 procedure by Bodor and Pearl man in 1976.190 The
numbering of these various compounds appears in Figure 3-8.
Table 3-10 shows the bond lengths in the isoquinoline model system,
(30) + (31), and the simple pyridine system, (32) £ (33). Upon reduction

Table 3-10. A Comparison of Bond Lengths in Angstroms of the Pyridine (32) Z 1,2-Dihydropyridine (33)
System and the Isoquinoline (30) Z Dihydroisoquinoline (31) Model System
Pyridine (32)
Atom Bond
Number Length
Dihydropyridine (33)
Atom Bond
Number Length
Isoquinoline Model (30) Dihydroisoquinoline Model (31)
Atom Bond Atom Bond
Number Length Number Length
1-2
1.336
1-2
1.432
1-2
1.342
1-2
1.451
2-3
1.402
2-3
1.492
2-3
1.433
2-5
1.517
3-4
1.406
3-4
1.357
3-4
1.467
3-4
1.445
4-5
1.406
4-5
1.453
4-5
1.444
4-5
1 .468
5-6
1 .402
5-6
1 .351
5-6
1.388
5-6
1.370
6-1
1.336
6-1
1.363
6-1
1.415
6-1
1.401
7-2
1.107
7-2
1.134
2-26
1.115
2-26
1 .130
8-2
1.134
2-27
1.132
8-3
1.105
9-3
1.105
3-11
1 .467
3-11
1.444
9-4
1.114
10-4
1.105
4-14
1.433
5-11
1.101
5-19
1 .110
5-19
1.110
6-12
1 .113
6-8
1.480
6-7
1 .485
UD

95
of (30), the bond connecting carbons 5-6 shortens, indicating a greater
double bond character at this location, while the bonds between carbons 2-3
and between the carbon and nitrogen at position 1-2 lengthen, indicating
an increased single bond character at these locations. This correlates
well with the structural formalism and also mirrors those changes that
occur in the reduction of (32) to (33). A similar study of bond angles is
presented in Table 3-11.
The charge densities at specific atoms can be indicative of the type
of chemistry that a compound undergoes. A study of the charge densities
of atoms in (30) t (31) is presented in Table 3-7 and a comparison of this
system to the (32) t (33) system appears in Table 3-12. The charge densi
ties of the pyridinium nucleus of (30) reveal the most highly charged de
ficient center is at carbon C2. One would expect nucleophilic attack at
this electropositive position and, in fact, this is what is observed.
These observations can be extended to berberine (1) since it is known that
hydroxide, hydride, and acetonide attack (1) at this position. In general,
nucleophiles attack pyridines at the carbon adjacent to the nitrogen.172
The charge densities of the atoms in (31) indicate a highly electronegative
center at C5. One would therefore expect protonation and electrophilic
attack at this location. This, again, is borne out experimentally. In
the case of dihydroberberine (2), protonation as well as alkylation occurs
here and, in general, this type of reaction is well known in the chemistry
of enamines.191-193 These properties and trends are also seen in the
(32) t (33) pair.
The planarity of a pharmacologically active aromatic molecule, espe
cially anti neoplastic agents, is an extremely important parameter and many
correlations between activity and toxicity and planarity have been made.

Table 3-11. A Comparison of the Bond Angles in Degrees of the Pyridine (32) t 1,2-Dihydropyridine (33)
System and the Isoquinoline (30) t Dihydroisoquinoline (31) Model System
Pyridine (32)
Dihydropyridine (33)
Isoquinoline Model (30)
Dihydroisoquinoline Model (31)
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
Atom
Number
Bond
Angle0
2-1-6
119.8
2-1-6
124.0
2-1-6
121.6
2-1-6
121.8
3-4-5
119.8
3-4-5
117.9
3-4-5
115.7
3-4-5
116.3
2-3-4
118.2
4-5-6
120.1
4-5-6
125.5
4-5-6
126.4
1-2-3
122.0
1-6-5
121.4
1-6-5
116.2
1-6-5
117.9
8-3-2
120.6
6-1-13
119.0
6-1-10
121 .9
6-1-10
123.4
7-2-3
120.6
1-2-0
123.2
3-2-26
120.4
1-2-26
108.3
1-2-27
108.8
3-4-9
120.1
6-5-11
119.9
4-5-19
117.8
4-5-19
116.3
12-6-5
122.9
1-6-7
117.1
1-6-7
116.5
10-4-5
119.6
6-7-8
123.7
6-7-8
123.6
9-3-4
121.8
11-3-4
118.6
11-3-4
118.7
9-10-24
109.7
9-10-24
108.0
1
9-10-25
109.8
9-10-25
108.6
UD
cn

Table 3-12. A Comparison of the Atomic Charge Densities of the Pyridine (32) t 1,2-Dihydropyridine
(33) System and the Isoquinoline (30) t Dihydroisoquinoline (31) Model System
Pyridine (32)
Dihydropyridine (33)
Isoquinoline Model (30)
Dihydroisoquinol ine Model(31
Atom
Number
Charge
Density
Atom
Number
Charge
Density
Atom
Number
Charge
Density
Atom
Number
Charge
Density
1
-0.1641
1
-0.1174
1
+0.0779
1
-0.1534
2
+0.1351
2
+0.2577
2
+0.1827
2
+0.2443
3
-0.0659
3
-0.1162
3
-0.1151
3
-0.1434
4
+0.0732
4
+0.0911
4
+0.1029
4
+0.0979
5
-0.0657
5
-0.1678
5
-0.0429
5
-0.1597
6
+0.1348
6
+0.1670
6
+0.1080
6
+0.1737
7
-0.0151
7
-0.0903
26
+0.0429
26
-0.6663
8
-0.0899
27
-0.0788
8
+0.0005
9
+0.0083
11
+0.3261
11
+0.2993
9
-0.0182
10
+0.0101
14
-0.0582
14
-0.0562
10
+0.0001
11
+0.0184
19
+0.0512
19
+0.0055
11
-0.0147
12
-0.0173
7
-.0446
7
-0.0311
13
+0.0663
10
+0.1104
10
+0.1777

98
This characteristic is accessible by MINDO/3. The program calculates a
dihedral angle which is a measure of the deviation from planarity of a
molecule. Pyridine (32) is planar and 1,2-dihydropyridine (33) is planar
within one degree. The dihedral angles of (30) t (31) are shown in
Table 3-8. In the case of (30), the overall deviation from planarity is
less than 3.6. In berberine (1), which has an additional benzene ring
annulated at the C7-C3 position, this difference should be less because
of the planarizing effects of the added aromatic system. This would tend
to cast doubt on the proposal that the reason berberine does not inter
calate into deoxyribonucleic acid (DNA), as well as totally aromatic mole
cules such as coralyne, is due to the buckling of the C ring.135155
This nonplanarity is attributed to the partial saturation of that ring.
The present study, however, indicates that this deviation is slight and
probably plays a minor role in attenuation of the action of (1).
A more plausible reason for the lower intercalative ability of (1)
is because of lower electronic interactions. When a molecule intercalates
into DNA, there exists an electronic interaction between the base pairs
of DNA and the ir-cloud of the intercalating aromatic compound. The greater
the stabilization of this complex, the greater is the DNA-molecular inter
action. In berberine, there is a partial destruction of the aromatic
system which lowers any electronic interaction. The hydrogens added to
the C-ring act to increase the effective thickness of the molecule and
this may play a role in decreasing macromolecular complexation. These
structural concerns are apparent in Figures 3-10 and 3-11, which are the
fully optimized structure for (30).
The dihydro model (31) contains one more sp3 center than does (30),
and this has a slight deplanarizing effect with the molecule twisting 9.1

99
Figure 3-10. A Computer-assisted Drawing of the Most Stable
Conformation of the Isoquinoline Model (30) at
25C

Figure 3-11. A Computer-assisted Drawing of the Most Stable Conformation of the
Isoquinoline Model (30) at 25C. This View is Oriented so that the
Interatomic Axis between Atoms 26 and 2 is Perpendicular to the Plane
of the Page
o
o

101
out of the plane. In applying the same considerations to this molecule
as to (30), one would expect that this system would interact less with
DNA than would (30). This is due to the further destruction of the aro
matic nucleus resulting not only in a more nonplanar structure, but also
in a structure with a lower propensity to interact electronically with
DNA. The loss of the positive charge should also reduce macromolecular
intercalation because of the lowered coulombic interaction between (31)
and the anionic phosphate backbone of DNA.156 In extending these results
to dihydroberberine (2), one would predict a lower cytotoxicity and, there
fore, toxicity of (2) relative to (1). The optimized structure of (31)
at 25C is presented in Figures 3-12 and 3-13.
To summarize, the MINDO/3 calculation predicts the dihydro model (31)
and, presumably, (2) to be more stable than simple dihydropyridines because
of extended conjugation and hyperconjugation. The model (30) is predicted
to undergo nucleophilic attack at the carbon adjacent to the nitrogen, and
protonation and electrophilic reaction at the carbon 6 to the nitrogen of
the enamine. The calculations show localization of double and single
bonds on the reduction of (30) to (31). These data are in good agreement
with what is known about chemistry of this genre of compounds. The calcu
lation suggests that the reason berberine does not intercalate as well as
totally aromatic compounds is not because of steric problems associated
with the carbon skeleton but, rather, electronic differences and the steric
effects of added hydrogens and, finally, MIND0/3 predicts that (31) and
presumably, (2) are less toxic than (30) and (1). These results should
be applicable not only to the (1) X (2) system, but other conjugated di
hydropyridine systems as well.

102
Figure 3-12. A Computer-assisted Drawing of the Most Stable
Conformation of the 1,2-Dihydroisoquinoline
Model (31) at 25C

Figure 3-13. A Computer-assisted Drawing of the Most Stable Conformation of the 1,2-Dihydro-
isoquinoline Model (31) at 25C. This View is Oriented so that an Imaginary
Axis between Atoms 2 and 5 is Perpendicular to the Plane of the Page
o
CJ

104
Further Studies on the Biological and
Chemical Properties of Dihydroberberine
The rate and nature of the oxidation of the dihydropyridines included
in the delivery scheme is important to the proper functioning of the system.
A dihydropyridine must be stable enough to be formulated and stored. The
in vivo rate of oxidation must, however, be rapid enough to efficiently
transform the delivering species and thereby avoid competing metabolisms.
The rate of oxidation of dihydroberberine (2) was therefore studied
in a variety of media, and in a number of different situations. One prob
lem which hampered these determinations was the extreme water insolubility
of the free base. The rate of oxidation of (2) was determined by both
HPLC and UV methods. The UV procedure involved measuring the appearance
of the 460 nm absorbance of berberine (1) with time, while the HPLC deter
minations were made by calculating the appearance of the absorbance due to
(1) or disappearance of the absorbance due to (2). In most cases agreement
between the two methods was good. In all determinations the spectrum of (1)
showed no change within the timeframe of the experiment. Initial oxida
tion studies were performed in areated buffer. At a pH of 7.4, (2) oxi
dized very rapidly and erratically. Buffers of lower pH were then used
to partially stabilize (2) by shifting the equilibrium in favor of the
hydrochloride in an effort to yield a more reproducible system. This
shift reduces the electronic density at the nitrogen, and precludes the
participation of the nitrogen lone pair in oxidative reactions.172 The
lower pH also greatly facilitates solubilization. The rate of oxidation
of (2) at a pH of 5.8 is shown in Table 3-13, and the spectral changes
that are characteristic of this oxidation are shown in Figure 3-14.
Although the correlation coefficients are not good, second order kinetics
are indicated with a calculated second-order rate constant of 44.4 0.53

Table 3-13. The Rate of Oxidation of Dihydroberberine in Various Media
Pseudo First
Corre-
Second Order
Medium (C)
Order Rate Constant
lation
Concentration (M)
Rate Constant
pH 5.8 Buffer (37C)
7.17
x 10"4sec_1
0.900
2 x 10-4
3.99
x 104sec_1
0.880
1 x 104
4.44 0.53 ,mole_1sec-1
2.87
x 104sec_1
0.920
5 x 10'5
6% Brain Homogenate
2.51
x 10_3sec_1
0.995
1.22 x 104
(37C)
1.99
x 10_3sec-1
0.997
9.79 x 10"5
20.17 0.24 mole-1sec-1
9.59
x 104sec-1
0.995
4.89 x 105
40 or 80% Plasma
2.37
x 10_4sec_1
0.993
5 x 10"5
(37C)
30% Liver Homogenate
3.74
X
o
1
-r
to
rd
o
i
0.995
5 x 10"5
(37C)

ABSORBANCE
Figure 3-14. Spectral Changes of Dihydroberberine (2) upon Oxidation to Berberine (1)
in pH 5.8 Phosphate Buffer at 26C. Traces were made every 10 min.

107
/mole sec. A number of oxidants were investigated to study the oxida
tion of (2). Unfortunately, these produced rates of oxidation which
were far too rapid to analyze by simple means.
The rate of oxidation of (2) in various biological media was also
determined and these results are presented in Table 3-13. Second-order
kinetics were again observed. In brain homogenates the correlation co
efficients were much higher than those obtained in buffer. In the major
ity of the determinations, pseudo first-order rate constants at specific
concentrations were calculated. The values obtained allow a comparison
from system to system. The t^, for example, of oxidation of a 5 x 10-5 M
solution of (2) in plasma, liver homogenate, and brain homogenate was
calculated to be forty-eight minutes, thirty-one minutes, and approximately
twenty-five minutes, respectively. The oxidation of (2) by a second-order
process is very characteristic of many dihydropyridines.
The relative stabilities of dihydropyridines were investigated by com
paring their rates of oxidation in dilute hydrogen peroxide. This system was
reproducible and, as shown in Table 3-14, gave data of good quality. The
t calculated from the pseudo first-order rate constants obtained at 1 xlO4M
2
for (2), 1-methyl-1,4-dihydronicotinamide (21), and 1-benzyl-l,4-dihydro
nicotinamide (22) were 25.6 min, 1.2 min, and 11.5 min, respectively.
These results are consistent with the greater stabilization of (2) compared
to simpler systems, which was predicted by the theoretical calculations.
The rate constants are relatively small because of the slightly acidic nature
of the peroxide. At the end of the experiment, (1) was analyzed to make
sure that nucleophilic addition of the peroxide to (2) did not occur.
The Mechanism of Oxidation of Dihydroberberine
A knowledge of the mechanism of oxidation of dihydropyridine could
be helpful in applying the drug delivery system. The mechanism of oxidation

108
Table 3-14. The Relative Rates of Oxidation of Dihydroberberine (2),
1-Methyl-1,4-dihydronicotinamide (21), and l-Benzyl-1,4-
di hydronicotinamide (22) in Dilute Hydrogen Peroxide
Compound
Pseudo First
Order Rate Constant
Correlation
Relative Rate
of Oxidation
(2)
4.51 x 10'4
0.998
1.0
(21)
9.83 x 10"3
0.99999
21.7
(22)
1.00 x 10'3
0.999
2.2

109
of simple dihydropyridines, particularly dihydronicotinamides, has been
extensively studied since these partial structures occur in the NADHJNAD+
system. Many models of this system have been used and most are simple,
substituted 1,4-dihydronicotinamides.
In the classic work of Abeles and Westheimer, the oxidation of sub
stituted 1,4-dihydronicotinamides by thiobenzophenones was studied.194
Like most dihydropyridines, these exhibit second-order kinetics: first-
order with respect to the dihydropyridine and first-order with respect
to the thiobenzophenone. The mechanism of oxidation proposed by this
group was that of a concerted hydride transfer from the dihydronicotin
amide to the thiocarbonyl carbon. This mechanism has been modified over
the years.195"197 Most recently, Ohno has described a system in which the
oxidation proceeds through a charge transfer complex.198199 The initial
step in this process is an electron transfer, followed by a proton trans
fer followed, in turn, by a subsequent electron transfer. In free radi
cal oxidations Eisner, using substituted 1,4-dihydronicotinamides and the
oxidant, diphenyl pi cryIhydrazyl free radical (DPP*), again found a second-
order oxidative process with the rate-determining step being the initial
abstraction of a hydrogen.200 This is followed mechanistically by the
formation of the quaternary compound. The oxidation of other dihydropy-
ridines, such as the free radical oxidation of dihydroanthracene or dihy-
drophenanthrene, has also been reported.201>202 The kinetics of enzymatic
oxidation of dihydronicotinamides have also been studied. In 1980 Porter
and Bright published an article on the oxidation of substituted 1,4-dihy-
dronicotinamides by lumiflavins and old yellow enzyme.203 Kinetically,
a second-order oxidation was found to take place, mediated by a charge
transfer or biradical complex.

no
The mechanism of oxidation of dihydropyridines is, therefore, depen
dent on the oxidant and the conditions under which oxidation takes place.
A series of experiments was performed to investigate the mechanism of oxi
dation of simple dihydronicotinates and (2), and to determine if the oxi
dation of the dihydropyridines is mediated by an enzyme or by some other
species such as dissolved oxygen.
In these experiments a homologous series of 1-methyl-1,4-dihydronico-
tinic acid esters was synthesized. The NMR of a representative compound
is presented in Figure 3-15, and the corresponding proton assignments in
Table 3-15. The rate of oxidation of (22), (23), (24), (25), (26), (27),
and (28) in 40% human plasma, 6% brain homogenate, or 3.5% liver homogenate
was measured. This was done by determining the rate of disappearance of
the 359 nm absorption of the dihydronicotinamide with time. Since the
rate of ester hydrolysis is slower for nicotinic acid esters and much
slower for 1-methylnicotinic acid esters than the values obtained, the
results clearly represent the oxidation process of the dihydropyridine
and not hydrolysis. In all determinations the concentration of the dihy-
dropyridines was 5 x 105 M. The results of this experiment are shown
in Figure 3-16 and Table 3-16. Both in plasma and in buffer, the rate
of oxidation as measured by the pseudo first-order rate constant, is rela
tively slow and the correlation coefficients are relatively small. There
is also little effect on the rate constants by the molecular structure.
This indicates a nonspecific oxidative route. In organ homogenates, there
is a marked acceleration in the rate of oxidation as well as an increase
in the correlation coefficients. There is also a large dependence upon
the rate by the structure of the molecule and, in general, as the chain
length increases, the rate decreases. These three changes acceleration
of the rate, linearization of the data, and the greater reliance of the

Figure 3-15. Proton Nuclear Magnetic Resonance Spectrum (60 MHz) of (27) in CDC13

112
Table 3-15. Proton Assignments of the XH NMR of the 1-Methyl-1,4-dihydro-
nicotinic Acid Ester (27)
OCH2(CH2)8CH
g
3
Proton
a
b
c
d
e
f
g
PPM (6)
6.9
5.6
4.7
4.0
3.0
2.9
0.9

Figure 3-1G. The Rates of Oxidation of Various 1-Methyl-1,4-dihydronicotinic Acid Esters
(23), (24), (25), (26), (27), (23) and 1-Benzyl-l,4-Dihydronicotinamide
(22) at 37C in 40% Human Plasma (), 6% Brain Homogenate (A) and
3.5% Liver Homogenate ()

Table 3-16. The Rates of Oxidation and Corresponding Correlation Coefficients of Various 1-Methyl-l ,4-
dihydronicotinic Acid Esters and 1-Benzyl-l,4-dihydronicotinamide (22)
Pseudo First Order Rate Constants x 10"4sec_1
Compound Brain Liver
Number Plasma Correlation Homogenate Correlation Homogenate Correlation
(23)
1.19

0.14
.970
4.87
+
0.25
.9998
6.49

0.51
.998
(24)
0.90

0.11
.921
4.80
+
0.27
.9998
4.97

0.19
.998
(25)
0.97

0.10
.962
3.75

0.31
.9998
3.40

0.55
.981
(26)
0.81

0.03
.971
3.08

0.33
.998
3.29

0.16
.974
(27)
0.71

0.05
.940
3.02

0.44
.9991
3.25

0.39
.993
(28)
0.82

0.06
.976
3.88

0.09
.9994
4.25

0.91
.998
(22)
0.90

0.05
.990
3.53

0.17
.9992
4.14

0.16
.9990

115
rate on structure indicate the involvement of an enzyme in the oxida
tion. One of the enzymes which is said to be responsible for the oxida
tion of dihydropyridines is NADH dehydrogenase.204 Since this family of
enzymes is membrane bound, it would be present in the organ homogenate
but not in plasma. The results are consistent with this distribution.
Enzymes involved in the oxidation of dihydropyridines have as their endo
genous substrate NADH. This molecule is not substituted at the amide
nitrogen and one would expect compounds which more closely resemble NADH
to be better substrates for the enzyme than molecules which greatly devi
ate from this structure. One would predict that the shorter chain ana
logs (23) and (24), and the N-benzyl compound (22) would be oxidized
more rapidly than the longer chain analogs and this is, in fact, the case
An additional observation which supports this hypothesis is that as the
homogenates age, the rate constants as well as the correlation coeffi
cient decreases. This is consistent with the time-dependent denaturation
characteristic of this type of enzymatic system.
These trends also occur in the oxidation of (2). In plasma and buf
fer, the data indicates a relatively slow oxidation with poor correlation
In brain homogenate, there is an acceleration and a linearization in the
rate constants. These results indicate that although oxidation of (2)
can be mediated by oxygen, in tissues like the brain and liver, an enzy
matic oxidation can occur. The effect of protein binding on the rate of
oxidation of (2) was investigated by changing the concentrations of the
homogenates. One would expect (2) to bind to proteins but the effects
of this complexation on oxidation were not large.
Membrane Permeability of Dihydroberberine and Berberine
In order to investigate the relative ability of dihydroberberine (2)
to penetrate membranes, the behavior of (2) and (1) in a model system was

116
observed. The model system which was chosen in this study was that of
the red blood cell. In this experiment (1) or (2) were placed in a known
volume of whole blood, and at various times the concentration was deter-

mined in the plasma or packed red blood cells. The results are shown in
Figure 3-17. As one can see, the initial rate of penetration of (2) into
red blood cells is rapid and greater than that of (1). The initial con
centration is also higher. This affinity for the red blood cells is mir
rored by a disappearance of (2) from the plasma. With time, equilibra
tion occurs in the system. Berberine penetrates the red blood cell slow
ly and reaches a maximum concentration much later than does (2). This is
another indication of the increased membrane mobility of (2) relative to
(1). The two curves finally converge to the same value, as the oxidation
of (2) to berberine takes place. Creasey indicated that there was a re
lationship between glucose transport and the transport of berberine into
red blood cells.143 To investigate this possibility, the behavior of ber
berine in a red blood cell system which contained 200 mg% glucose was ob
served. As shown in Table 3-17, there is very little effect of glucose
on the entry of berberine into the red blood cells from the plasma.
In Vivo Studies
The preliminary studies indicate that dihydroberberine possesses all
of those characteristics required of a compound which is to be applied to
the drug delivery system proposed in Figure 1-3. The substantiation of
this drug delivery scheme requires not only the demonstration of the deliv
ery of (1) after administration of (2) but also the specific retention of
(1) in the brain. The first priority of the in vivo system was to show
delivery into the brain of (1). The protocol used in these studies in
volved injecting rats with either berberine (1), dihydroberberine (2),
or its hydrochloride (3). After a period of time the chest cavities of

500
-J 1 I I l I I I I
40 80 120 160 200 240 280 320 360
TIME (MIN)
Figure 3-17. Partitioning of 2G.5 mg of Berberine (1) from Plasma (A) into Red Blood
Cells (A) and of 26.5 mg of Dihydroberberine Hydrochloride (3) from Plasma
(O) into Red Blood Cells (). The Volume of Blood Used in each Experiment
was 75 ml

118
Table 3-17. The Effect of Glucose on the Movement of Berberine into
Red Blood Cells
Berberine
Concentration (yg/ml) Plasma3
Time (min)
Control
+ 200 mg% Glucose
5
360.94
352.29
30
300.39
309.04
60
274.95
276.48
90
267.29
255.62
120
259.17
272.92
aThis is a representative experiment selected from a group of three

119
the animals were opened, the heart perfused with saline, and the brain
removed. High pressure liquid chromatography was used in analysis.
The initial experiments showed that after administration of 55 mg/Kg
of (1) in dimethyl sulfoxide (DMSO), no (1) could be detected in the brain
at any time. When, however, 55 mg/Kg of (2) in DMSO were administered,
a high concentration of (1) was observed in the brain as shown in Figure
3-18. The free base was not exceptionally stable in solution, however,
and was also very water insoluble. For these reasons the hydrochloride
(3) was prepared and used in subsequent experiments. If 55 mg/Kg of (3)
in 20% aqueous ethanol are injected systemically, the concentration of (1)
in the brain is again found to be relatively high. This is presented in
Figure 3-19. The concentration of (1) achieved in the brain after admin
istration of (2) or (3) was similar (approximately 50 yg/g tissue). The
loss of (1) from the brain after administration of (3) is slow and the
t, of this loss, calculated from the terminal portion of the log concen-
2
tration versus time relation, is approximately eleven hours.
It should be emphasized that in these experiments only the concen
tration of (1) was measured even though unoxidized (2) was present. To
obtain a more complete picture of the behavior of (1) in the brain, the
total berberine concentration, i.e. (1) and (2) was measured and this
appears in Figure 3-20. The concentration of (2) is high at early time
points but diminishes rapidly. The initial rate of disappearance of (2),
obtained by subtracting the concentration of (1) in the brain from the
total concentration (Figure 3-21), yields a tx of thirty-four minutes
2
which is of the same magnitude as the value obtained from brain homogenates.
The slope of the terminal portion of the curve in Figure 3-20 yields a
ti of 5.7 hours.
2

70i
60-
50-
L
3
CO 40
(O
U 30-
$
CP
N
CP
20
{

i
i
I 0-
10 20 30 40 50 60 70 80 90
TIME (min)
Figure 3-18. Distribution of Berberine in the Brain after iv Administration of Berberine (1)
() at a Dose of 55 mg/Kg or of Dihydroberberine Free Base (2) () at a Dose
of 55 mg/Kg
ro
o

DRUG CONCENTRATION/xg/g WET TISSUE
Figure 3-19.
TIME (min)
Efflux of Berberine from the Brain after iv Administration ¡>f eitlier b5 mq/Kg
of Dih.ydroberberine Hydrochloride (3) () r jj mg/Kg of Berberin ( ) ()
Analysis vias for (1) only and not Unoxidized (2)
ro

TI ME (min)
Figure 3-20. Efflux of Berberine (1) and Unoxidized Dihydroberberine (2) (A) after i v
Administration of 55 mg/Kg of Dihydroberberine Hydrochloride (3)
IX)
ro

TI ME ( min)
Figure 3-21. A Comparison of the Efflux of Berberine (1) () and Berberine (1) and co
Unoxidized Dihydroberberine (2) (A) after a Dose of 55 mg/Kg of
Dihydroberberine Hydrochloride (3) Administered i v

124
This indicates a faster efflux for the total berberine concentration
than for the efflux of (1) alone. The reason for this is that, aside
from efflux of (1) from the brain, a component of the oxidation of (2)
and for the efflux of (2) also occurs in this rate. Since the oxidation
of (2) is not immediate, a significant quantity of it is present at later
times and this population of molecules will redistribute out of the brain
as a function of the blood concentration of (2) as would any lipophilic
compound. This added equilibrium complicates the kinetics of the original
scheme.
The distribution of berberine (1) in various tissues after an intra
venous dose of 35 mg/Kg is shown in Figure 3-22. The lower dose was used
because of the higher toxicity of (1). These results show a high concen
tration of (1) in the kidney and, to reiterate, no quantitatable amount
in the brain. Berberine is rapidly lost from the tissue and this contri
butes to its relatively short biological t^. The distribution of (1) in
the tissues has been previously studied.205-209 The results obtained from
a number of these studies are consistent with those reported here. Ber
berine is rapidly lost from the tissues and effectively excreted by the
kidney and by a biliary route. In literature reports the concentration of
(1) in the brain was always undetectable or the lowest of all other tissues
examined. In those cases where (1) was detected, the amounts found were
usually on the order of 50-200 ng/g tissue which is below the limit of
detection in this study. These values are low in both relative and abso
lute terms. Since the effective concentration of (1) in in vitro systems
is on the order of 1.0 yg/g, these levels would be ineffective therapeuti
cally. These studies also indicate berberine is not absorbed to a great
extent from the intestine, again reflecting its highly polar nature.

DOSE/GRAM TISSUE
Figure 3-22. Distribution of Berberine after iv Administration of 35 mg/Kg of Berberine (1)
into the Kidney (), Liver (), Lung (), and Brain (A)
fj.q BERBERINE/GRAM TISSUE

126
The tissue distribution of dihydroberberine hydrochloride (3) was
investigated in an analogous manner. The compound (3) is taken up by tis
sues to a greater extent than (1) and is handled well by the kidney. The
concentration of (1) observed in the brain is high. A comparison of the
time courses of (1) after administration of either (1) or (3), in various
organs appears in Figures 3-24 to 3-26. These results shown are consistent
with the greater ability of (3) to penetrate tissues. The liver (Figure
3-26) is noteworthy because (1) is known to be significantly excreted by
a biliary mechanism.
In order to better illustrate the specificity of the (1) Z (2) sys
tem, (3) was infused intravenously. The results of these infusions are
presented in Table 3-18. In these administrations the standard dose of
55 mg/Kg of (3) was given over a period of either thirty or forty-five
minutes. If a comparison is made between the concentration of (1) in
various organs at the end of the infusion and the concentration of (1)
obtained at thirty or forty-five minutes after a bolus iv injection, the
specificity inherent in the system can be demonstrated. If the system
is not specific, one would expect to see an overall decrease in the con
centration of (1) after an iv infusion of (3) compared to the bolus.
The results show that at thirty minutes, there is a higher concentration
of (1) in the brain compared to the iv bolus and a decrease in the concen
tration of (1) in the lungs and kidneys. The liver shows a moderate in
crease. At forty-five minutes these trends continue. A comparison of
the data at thirty and forty-five minutes shows an increase of the con
centration of (1) in the brain and a reduction in all other organs. The
concentration of (1) in the brain is higher than in any other tissue ana
lyzed at forty-five minutes.

DOSE/GRAM TISSUE
3.5
TIME (MIN)
Figure 3-23. Distribution of Berberine after i v Administration of 55 mg/Kg of Dihydrober-
berine Hydrochloride (3) into the Kidney (<>) > Liver (), Lung (O), and
Brain (A)
ro
si
fj.g BERBERINE/GRAM TISSUE

DOSE/GRAM TISSUE
2.0
_i 1 1 1 1 i i i i i l i l i i l l l
20 60 100 140 180 220 260 300 340
TIME (MIN)
Figure 3-24. A Comparison of the Efflux of Berberine from Lungs when Administered i v
as 55 mg/Kg of Dihydroberberine Hydrochloride (3) (O) or 35 mg/Kg of
Berberine (1) ()
ro
00

% DOSE/GRAM TISSUE
TIME (MIN)
Figure 3-25. A Comparison of the Efflux of Berberine from the Kidneys when Berberine (1) is
Administered i v at a Dose of 35 mg/Kg () and Dihydroberberine Hydrochloride
(3) when Administered i v at a Dose of 55 mg/Kg (O)

DOSE/GRAM TISSUE
Figure 3-26. A Comparison of the Efflux of Berberine from the Liver when Berberine (1)
is Administered i v at a Dose of 35 mg/Kg () and Dihydroberberine Hydro
chloride (3) when Administered i v at a Dose of 55 mg/Kg ()
GJ
O

131
Table 3-18. Slow Infusion of Dihydroberberine (3)
30 min
Organ
Concentration (yg/g)
after infusion
Concentration (yg/g)
30 min after iv bolus
A(yg/g)
Brain
135.95
13
91.8 20
+ 44
Kidney
185.5
26
351.8 54
-166
Lung
71.4
10
210.2 14
-139
Liver
101.2
23
67.8 11
+ 33
45 min
Organ
Concentration (yg/g)
after infusion
Concentration (yg/g)
45 min after iv bolus
A
Brain
162.2
8
88
+ 74
Kidney
121.4
19
315
-194
Lung
62.8
6
165
-102
Liver
79.4
10
52
+ 27

132
These data verify the drug delivery scheme devised. After adminis
tration of (3) high concentrations of (1) are obtained specifically in
the brain, while the concentrations in other organs are reduced. The
reason that the slow infusion of (3) enhances the specificity is related
to a number of factors. Dihydroberberine is a lipophilic compound. The
iv bolus injection presents to the tissues a large concentration of (2)
and the result of this large burden is an inordinately long transit time
of (2) in peripheral sites. This obfuscates the kinetics. By slowly
administering (3), the tissue burden is greatly reduced, and the designed
improvements in the bidirectional characteristics of the delivery molecule
can be fully demonstrated. This system in general, and (2) in particular,
allows specific delivery of quaternary compounds to the brain. This sys
tem is designed to reduce systemic levels of an agent and thereby reduce
any accompanying toxicity.
Efflux of Berberine from the Brain
The mechanism by which berberine leaves the brain is not known but
it is important to the quaternary scheme. According to the original postu
lation, large quaternary compounds like (2) should leave the brain slowly,
presumably by passive processes such as movement in the CSF. Small qua
ternary compounds, on the other hand, are substrates for active carriers
which rapidly remove these compounds from the brain extracellular fluid.1,5
The next series of experiments was designed to determine the mechanism of
efflux of (1) from the brain. If the efflux of (2) is mediated by a spe
cific carrier, it should be possible to demonstrate the competitive inhi
bition of the efflux of (2) by introducing into the system a large con
centration of another agent which has affinity for the cationic pump, such
as 1-methylnicotinamide (6) and 1-benzylnicotinamide (7).

133
The first experiments involved injecting rats with 200 mg/Kg of
1-methyl-1,4-dihydronicotinamide (21) in aqueous ethanol followed fifteen
minutes later by an injection of the standard dose of 55 mg/Kg of (3).
The results of this study are shown in Figure 3-27. The dihydronicotin
amide (21) was used as a proform of quaternary compound (6) in an attempt
to generate high levels of (6) in the brain. The results do not show, how
ever, any significant difference in the efflux of berberine (1) between
pretreated and unpretreated animals.
In an analogous study 1-benzylnicotinamide (7) and its corresponding
dihydro adduct (22) was employed. Unlike the 1-methyl derivatives, this
pair of compounds possesses a UV chromophore which greatly simplifies quan
titation. A preliminary study was performed to determine the time at which
the maximum concentration (tmax) of (7) after the administration of 1-
benzyl-1,4-dihydronicotinamide (22) occurred. The results of this experi
ment are shown in Figure 3-20. After injection of (7) no detectable levels
of (7) in the brain could be observed. The reason the tmax is important
is that, ideally, the lag time between the injection of the antagonist,
i.e. (7) and (2) is the time required for (7) to reach a maximum concen
tration. Unfortunately, however, the toxicity of dihydrobenzyl derivative
(22) proved to be much higher than that of dihydromethyl derivative (21)
and the maximum dose which could be given was only 60 mg/Kg and, because
*
of this, further studies with this compound were abandoned.
A number of observations concerning this figure are germane to the
topic of the efflux of (1) from the brain. In the case of administration
of this N-benzyldihydropyridine (22), no dihydronicotinamide is present at
early time points at the dose used in the experiment. This is not the
case with (2). This is consistent with the greater stability of (31) and
hence, (2), predicted by the theoretical calculations. The t, of efflux
2

Figure 3-27. A Comparison of the Efflux of the Total Berberine, i.e. (1) and (2) from the
Brain when either 55 mg/Kg of Dihydroberberine Hydrochloride (3) is Administered
i v (A) or 55 mg/Kg of Di hydroberberi ne (2) and 200 mg/Kg of l-Methyl-1,4-
dmydronicotinamide (21) is Administered iv (#)

150
_J I I I I I I I I I I
20 40 60 80 100 120 140 160 180 200 220
Time (min)
Figure 3-28. Efflux of 1-Benzylnicotinamide Bromide (7) from the Brain after i v
of 60 mg/Kg of 1-Benzyl-l ,4-dihydronicotinamide (22) (A)
Administration
U>
cn

136
from the N-benzyl quaternary compound (7) from the brain is 5.8 hours,
which is twice as fast as the efflux of (1).
These studies indicate that there was no effect of the quaternary
compound N-methylnicotinamide (6) on the efflux of (1) from the brain.
The concentration difference, however, between (6) and (2) was only a
factor of four at the injection, and this difference is even less at the
level of the brain. To overcome this quantitative objection, a series of
intracerebral ventricular (icv) injections was performed. The purpose of
injecting the compound icv was to allow direct introduction of high con
centrations of (1) and (1) with the putative competitive inhibitor, (6),
into the brain. The injections were made into the lateral ventricles of
rats with the aid of a stereotaxic instrument and an infusion pump. A
dose of 50 yg of (1) or 50 yg of (1) and 1000 yg of (6) was administered
and the compounds were dissolved in DMS0. The volume of the dose was 3-5
yl. The results of these studies are shown in Figure 3-29. It can be
seen that (6) has little effect on the efflux of (1) from the brain. The
tx obtained from the terminal portion of the curves was 3.8 hours for the
2
efflux of (1) and 3.1 hours for the efflux of (1) when coinjected with (6)
The fact that the t obtained for (1) in this experiment is much slower
2
than that obtained when (1) is administered systemically as (3) is not
surprising. These icv injections are similar to intrathecal injections
in that even or complete distribution does not occur. Therefore, (1) is
basically restricted to the CSF. In the case of systemic administration
of (3), however, the distribution of (2) is fairly complete and even in
the brain. The efflux of (1) after administration of (3) is limited by
the diffusion of (1) from the brain tissue, and from the discussion of the
BBB, this is a slow and inefficient process. When (1) is administered icv

14
I l I I
30 60 90 120
Time (min)
Figure 3-29. Efflux of Berberine from the Brain after icv Injection of either 50 yg
of Berberine (1) () or 50 pg of Berberine (1) and 1000 yg of 1-Methyl -
nicotinamide Iodide (6) (A)

138
the limitations of diffusion do not apply and (1) may simply be lost by
the bulk flow of CSF.
To determine the rate of CSF flow in the animal system used in these
experiments, the rate of efflux of 3H-inulin (29) from the brain was mea
sured. This radionuclide was injected icv in DMSO at a dose of 2.3 yCi.
As shown in Table 3-19, the t of efflux of (29) is two hours, in good
agreement with previously reported values.45
The loss of (1) from the brain appears not to be mediated by an ac
tive process. Simple bulk flow of CSF seems to be sufficient to remove
(1) from the brain. The rate-limiting step in the cerebral elimination
of (1) does not appear to be the actual efflux process but rather, the
redistribution of (1) out of brain cells and membranes by this poorly
mobile species to those areas where elimination can take place.
Limited Metabolic Studies
The metabolism of (1) after the administration of (1) or (3) was
studied in the rat. Urine collected for three days after a dosing of (1)
or (3) was extracted with chloroform or 3-methyl-1-butanol. The alcohol
was used because it is reported to extract berberine and similar alkaloids
efficiently from aqueous solutions.210 The results of the HPLC and TLC
analyses are shown in Tables 3-20 and 3-21, respectively. As one can see,
there is very little metabolism of (1) and the major component of the
urine from animals dosed with either (1) or (3) was (1). There were sev
eral peaks which could not be attributed to the anesthetic and these were
found in the urine of all tested animals. The size and retention time
of these peaks was again similar.
These data are consistent with the few articles which have been pub
lished concerning the metabolism of (1). While this metabolism is reported

139
Table 3-19. Efflux of 3H
Ventricular
-Inulin from the Brain after
Administration
Intracerebral
Time (min)
% Dose/g
In % Dose/g
Wet Tissue
Wet Tissue
15
9.28%
2.23
30
7.94%
2.07
45
6.52%
1.87
60
5.54%
1.71
120
4.71%
1.55
Corr. = 0.930
k = 6.23 x 103min_1
t. = 1.9 hrs
2

140
Table 3-20. In Vivo Metabolism of Berberine and Dihydroberberine in the Rat
(HPLC)
HPLCa
Retention time of Peak Height
Compound Peaks Observed (min) Relative to Berberine
Berberine
Chloroform Extract
1.8X
2.0X
2.5X
2.9
4.2*
5.6
0.73
0.44
0.39
0.05
1.0
0.01
Isoamyl Alcohol 1.7X
Extract
2.5X
3.6

4.0
4.8
Pi hydro berberine
Chloroform Extract 1.8X
2.1X
2.6X
3.0

4.2
4.7
1.4
0.28
1.0
0.09
0.52
0.37
0.35
0.04
1.0
5.6
0.01

141
Table 3-20-continued.
HPLC
Compound
Retention Time of Peak Height
Peaks Observed (min) Relative to Berberine
Dihydroberberine
Isoamyl Alcohol
Extract
1.9X
5.1
2.5X
1.3
3.4
0.18
4.1*
1.0
4.8
0.09
Corresponds to berberine standard
xThese peaks were found in blank animals injected with the anesthetic
a
Five microliters of the sample were injected on a yBondapak C18 reverse
phase column. The mobile phase was acetonitrile:pH 6.2 phosphate buffer
60:40 and the flow rate was 2.0 ml/min

142
Table 3-21. In Vivo Metabolism of Berberine and Dihydroberberine in
the Rat (TLC)
TLCa
Compound
Rf of Spots Observed
Berberine
Chloroform Extract
0.15*
0.125x
0.21x
0.34
Isoamyl Alcohol
0.14*
Extract
0.34
Dihydroberberine
Chloroform Extract
0.21*
0.35
Isoamyl Alcohol
0.21*
Extract
0.26x
0.29
0.60
Corresponds to berberine standard
xThese spots were found in blank animals injected with the anesthetic
aFive microliters of the sample were spotted on alumina plates and eluted
with cyclohexane:chloroform:acetic acid 45:45:10

143
be to minimal in vivo, two minor metabolites have been identified. Furuya
described a urinary metabolite which apparently contained a carboxylic acid
moiety.211 Another study reported that very small amounts of tetrahydro-
berberine were present in the urine.208 Identification of the metabolites
found in the present study was not attempted.
The importance of these studies to the proposed drug delivery scheme
is related to the requirement of the scheme that the principal and, ideally,
only metabolism of (2) is to (1). This was shown to be the case. In ad
dition, no metabolite was present in the urine extracts of animals dosed
with (3) which was not present in the urine extracts of animals dosed
with (1).
Toxicity and Anti cancer Activity of Dihydroberberine
The toxicity of (1) and (3) was determined in mice and the results
are shown in Figure 3-30 and Table 3-22. The data were analyzed by the
method of Probits as well as by fitting the data to a sigmoid dose-response
curve. The lethal dose for 50% mortality (LD50) for (1) was found to be
37.0 mg/Kg, and that of (3), 58.2 mg/Kg. The injections were made in-
traperitoneally. The value obtained for (1) was in good agreement with
other values reported in the literature.144212 The toxicity of (1) is
60% higher than that of (3), substantiating a prediction made by the
theoretical calculations. The anti cancer activity of (1) and (3) is
presented in Table 3-23. First, the ability of (1) or (3) to inhibit
the growth of KB cells in vitro was investigated. The ID50 calculated
for (3) was 2.2 yg/ml while that calculated for (1) was 0.95 yg/ml.
This is again constant with the higher toxicity of (1) relative to (3).
The next series of experiments involved inoculating mice with P388
lymphocytic leukemia cells. If this is done ip, (1) and (3) are equipotent

Figure 3-30. The LD50 Dose-response Curve of Berberine (1) (A) and Dihydroberberine
Hydrochloride (3) (). Doses of (1) or (2) were Administered i p in CD-I Mice
-pi
-p*

145
Table 3-22. Probit Analysis of the LD50 Study
Dose
mg/Kg
In [Dose]
% Dead
Probit
Berberine
10
2.303
0
17
2.833
0
24
3.178
10
3.718
31
3.434
20
4.150
38
3.637
50
5.000
45
3.807
70
5.524
59
4.078
90
6.282
80
4.382
100
8.719
Dihydroberberine
25
3.219
0
Hydrochloride
33.3
3.506
0
41.6
3.728
10
3.718
50
3.912
30
4.476
58.3
4.066
40
4.747
66.6
4.199
60
5.253
75
4.317
80
5.842
83.3
4.422
90
6.282
100
4.605
100
8.719

Table 3-23. Effect of Berberine (1) and Dihydroberberine Hydrochloride (3) Against P388 Lymphocytic
Leukemia
P388 i.p.
P388 intracerebrally
Drug
Dose
mg/Kg
Number
of mice
Surv. Time
(days)
%ILS
Surv. Time
(days)
%ILS
Berberine (1)
5
6
11.17
.40
10.8
9.83
.17
1.7
10
6
11.33
.42
12.4
9.50
.22
-1.8
20
6
10.50
.50
4.2
8.67
.56
-10.3
Dihydroberberine
5
6
10.83
.17
7.4
10.50
1 .31
8.6
Hydrochloride (3)
10
6
11.00
.30
9.1
10.00
.37
3.4
20
6
11.17
.17
10.8
11.33
1.63
17.2
Vehicle
5 (ml/Kg)
12
10.08 .15
9.67 .14

147
in increasing the life span (ILS) of the animals compared to a control
group. However, when the leukemia is inoculated intracerebrally, (3) is
significantly more effective in increasing life span than is (1). The
1o ILS actually falls when (1) is administered. This is indicative of
the toxic peripheral effects of (1). These preliminary anticancer studies
again support the original hypothesis. The dihydropyridine is capable of
passing the BBB and oxidizing to (1) where it may exert its cytotoxic
acti vity.
Cone!usions
This dissertation has presented a broadly applicable drug delivery
scheme which is specific for the brain. This delivery method is based on
a dihydropyridine-pyridinium salt redox system and on the multifaceted
nature of the BBB. There are two major aspects of this delivery scheme.
In the first, which is a chemical delivery system, a pyridinium carrier
is attached to a drug molecule. The second, and the one with which this
dissertation has dealt, is a prodrug system in which the carrier moiety
is an integral component of the molecule. This system is simpler than the
first but since the molecule to be delivered must contain a pyridinium par
tial structure, it is less general. In both cases the basis of the brain
specific delivery is related to the greater lipophilicity of dihydropyri-
dines, the ease of their oxidation and subsequent elimination peripherally
and the difficulty with which large pyridinium compounds leave the CNS.
In order to substantiate the proposed method, it was applied to a
salient example, berberine (1). This anti cancer alkaloid contains a pyri
dinium moiety which is reducible and whose product of reduction is stable.
The physical and chemical properties of dihydroberberine (2) were examined.
Its rate of oxidation was found to be rapid in a number of media but not
as rapid as the rate of oxidation of simple dihydropyridines. Dihydroberberine

148
was shown to be more lipophilic than berberine (1) and also better able to
penetrate biological membranes. In an attempt to delve into the basic
chemistry of these relatively unstable compounds, a model system was de
veloped for them and this was examined by a MINDO/3 approach. The results
obtained from this study were consistent with experimental data and were
extendable to the berberine (1) ^ dihydroberberine (2) pair. In addition,
several predictions were made concerning the biological activity of (2)
relative to (1) and these were found to be valid.
Dihydroberberine (2) or its hydrochloride salt (3) was injected iv
into rats and when the brains were analyzed, high levels of (1) were found.
No berberine (1) was found in the brain after systemic administration of
(1). The rate at which (1) left the brain after its delivery by (3) was
slow and the t of the efflux was eleven hours. If dihydroberberine hy-
2
drochloride (3) is slowly infused iv, the concentration of (1) rises in
the brain with time but falls in all other organs tested. At forty-five
minutes the concentration of (1) is highest in the brain. The efflux of
berberine (1) from the CNS appears to be mediated by a passive process,
perhaps the bulk flow of CSF.
Dihydroberberine hydrochloride (3) was shown to be less toxic than
(1) in vivo in accordance with predictions made by the theoretical studies.
Additionally, (3) was shown to be less effective than (1) in inhibiting
the growth of KB cells in vitro. While the two compounds are equipotent
in increasing the life span of mice injected ip with a suspension of P388
lymphocytic leukemia cell, (3) is more potent in increasing the life span
of mice who were inoculated intracerebrally with the P388 cell line.
These data verify the proposed drug delivery scheme. By concentrat
ing a pharmacologically active agent at its site of action and by reducing
its concentration in other locations, the therapeutic index of the delivered

149
compound is greatly enhanced. This was demonstrated when the delivery
scheme was applied to the berberine (1)idihydroberberine (2) example.
This method is potentially extendable to any drug which contains a
pyridinium moiety. A number of anti cancer agents such as nitidine, cora
lyne, and fagaronine fit these criteria. Phenothiazines and 8-blockers,
among others, could also be modified in this way to attain specific deli
ery to the brain.

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BIOGRAPHICAL SKETCH
Marcus Eli Brewster III was bom in Jacksonville, Florida, on October
14, 1957, the 891st anniversary of the Battle of Hastings. He graduated
from S. Wolfson High School in 1975. He then enrolled at Mercer Univer
sity in Macon, Georgia, where he earned his B.S. cum laude in 1978 with a
major in biology and a minor in chemistry. At Mercer he was a member of
Lambda Chi Alpha social fraternity, Beta Beta Beta biological honor so
ciety and Gamma Sigma Epsilon chemical fraternity. After a summer posi
tion as a historian of the War between the States, he entered graduate
school at the University of Florida, College of Pharmacy. Four years
later he was granted a Ph.D. He is a member of American Chemical Society,
American Pharmaceutical Association, American Association for Advancement
of Science, and Society for Applied Spectroscopy.
160

I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Nicholas S. Bodor, Chairman
Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Kenneth B.
Assistant Professor of
Medicinal Chemistry
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Margaret 0.
Assistant Professor of
Medicinal Chemistry
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
k/.
jmes W. Simpkins
jsistant Professor of
Pharmacy
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Merle A. Battiste
Professor of Chemistry

This dissertation was submitted to the Graduate Faculty of the College
of Pharmacy and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of doctor of Philosophy.
August 1982
7%
Dean,
fge of Pharmacy
Dean for Graduate Studies
and Research



BIOGRAPHICAL SKETCH
Marcus Eli Brewster III was bom in Jacksonville, Florida, on October
14, 1957, the 891st anniversary of the Battle of Hastings. He graduated
from S. Wolfson High School in 1975. He then enrolled at Mercer Univer
sity in Macon, Georgia, where he earned his B.S. cum laude in 1978 with a
major in biology and a minor in chemistry. At Mercer he was a member of
Lambda Chi Alpha social fraternity, Beta Beta Beta biological honor so
ciety and Gamma Sigma Epsilon chemical fraternity. After a summer posi
tion as a historian of the War between the States, he entered graduate
school at the University of Florida, College of Pharmacy. Four years
later he was granted a Ph.D. He is a member of American Chemical Society,
American Pharmaceutical Association, American Association for Advancement
of Science, and Society for Applied Spectroscopy.
160


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This dissertation was submitted to the Graduate Faculty of the College
of Pharmacy and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of doctor of Philosophy.
August 1982
7%
Dean,
fge of Pharmacy
Dean for Graduate Studies
and Research


38
The rather involved synthesis of pro-2-PAM yielded the enamine salt
which corresponds to the 3-protonated dihydro compound. The pKa of the
tertiary nitrogen in this enamine salt was determined potentiometrically
and spectrophotometrically to be 6.32 0.6 and the oxime was estimated
to have a pKa of -11. With a pKa of 6.32, it would be predicted that
about 90% of the enamine would exist as the free base at physiological
pH (7.4). Since this dihydro free base is far more lipophilic than the
parent quaternary, its ability to penetrate membranes is greatly enhanced.
This pro-2-PAM is rapidly converted to the parent 2-PAM at physiological
pH and the tx has been determined to be 1.04 min by pharmacokinetic model-
2
ing. This rapid conversion of the pro-2-PAM to 2-PAM is desirable since
ideally, the only metabolism of the prodrug is to the parent compound.
A number of experiments were carried out using 2-PAM and pro-2-PAM.
From the linear descending portion of a semilog plot of blood concentra
tion versus time, the biological tx of 2-PAM and pro-2-PAM was calculated.
2
The tx for comparable doses of 2-PAM and pro-2-PAM differed by more than
2
60 min, and since the conversion of pro-2-PAM -* 2-PAM is rapid, this dif
ference was assumed to be the altered distribution of pro-2-PAM. This in
dicates that even though the rate of conversion of pro-2-PAM is rapid, it
is long enough for the enhanced distributional characteristics of the
pro-2-PAM to be expressed.
Blood levels of 2-PAM were significantly higher after an oral dose
of pro-2-PAM than with a dose of 2-PAM. If pro-2-PAM is administered in
travenously, there are no new metabolites formed. If brain concentrations
of 2-PAM are examined after 2-PAM and pro-2-PAM dosing, the concentration
of 2-PAM in the brain is 13-fold higher in the case of pro-2-PAM admin
istration. If brain acetylcholinesterase is inactivated using DFP, the


7
in the BBB. Recently, a distributional study of COMT in the brain indi
cated that this enzyme is present in many sites including several which
lack a structural BBB.18 This distribution may aid in the exclusion of
neurotransmitters from structurally unprotected areas. The presence of
DOPA decarboxylase explains partially the need for giving such large doses
of L-dihydroxyphenylalanine (DOPA) in the treatment of CNS dopamine defi
ciencies to achieve appropriate therapeutic cerebral levels. The enzymatic
BBB may also play a role in the exclusion of lipophilic compounds which
might otherwise passively diffuse through the BBB. This is suggested by
the presence of pseudo (butyryl) cholinesterase in the cerebral capil
laries.4 This enzyme is not found in noncerebral capillaries. The occur
rence of y-glutamyltranspeptidase has also been described and may account
for some protection from peptide infiltration.219 An early proposal
that Y-glutamyltranspeptidase is involved with carrier systems is ques
tionable. Acid phosphatase activity, which is a marker for lysosomes and
pre-lysosomes or phagosomes, is present in the endothelial cells.15 These
organelles appear to be involved in the degradation of endocytosed material
and, as such, can be considered a component of the BBB. The endothelial
cells of the cerebral microcirculation contain a large number of enzymes
but care must be taken in ascribing a certain enzyme to a barrier role.17
There are numerous enzymes which, while present in the endothelial cells,
do not serve in any capacity other than general cellular functioning.
Molecular Carriers Involved in BBB Transport
The aspects of the BBB which have been discussed thus far give an indi
cation of its relative impermeability to a number of blood-borne substances,
but do not explain the movement of essential nutrients into the CNS. This
transport is brought about by a number of carriers which are situated in
the endothelial cells. These carriers are generally assumed to be proteinaceous.


27
from the capillary lumen. In these investigations, there is no indication
of endothelial cellular damage and, therefore, the extravasation of HRP is
attributed to increased vesicular transport. The stimuli for this may be
release of serotonin from platelets inducing vesicular formation secondary
to vasoconstriction and increased blood pressure. Focal edema induced by
a number of means, such as ultraviolet exposure, also increases vesicular
transport and HRP uptake.
Nonionizing radiation has long been implicated in BBB disruption.
Microwaves and x-rays have been studied extensively in this regard. The
effects of microwaves are highly controversial. One report states that
exposure of rats to 2450 MHz at 10 mW/cm2 for two hours produces a marked
extravasation.96 This level is considered safe for human exposure in the
United States, but not in other countries. The biological effects of
microwaves can be subclassified as changes resulting from gross thermal
effects or nonthermal effects. In this study, the body temperatures of
the animals were constant and the altered permeability of the BBB was
attributed to microwave-stimulated serotonin release from platelets,
although other mechanisms are possible. Conversely, a recent publication
indicates that much higher levels of radiation are required to cause pro-
9 7
tein leakage, specifically 3000 mW/cm2. At these levels, cerebral tem
perature increases significantly and changes can be attributed to gross
thermal effects.
Porto-caval anastomosis, which causes severe liver dysfunction, has
also been implicated in BBB breakdown.5477 This disintegration has been
termed hepatic encephalopathy. As in a myriad of other circumstances,
increased vesicular transport has been implicated as the mechanism of
extravasation.


Table 3-12. A Comparison of the Atomic Charge Densities of the Pyridine (32) t 1,2-Dihydropyridine
(33) System and the Isoquinoline (30) t Dihydroisoquinoline (31) Model System
Pyridine (32)
Dihydropyridine (33)
Isoquinoline Model (30)
Dihydroisoquinol ine Model(31
Atom
Number
Charge
Density
Atom
Number
Charge
Density
Atom
Number
Charge
Density
Atom
Number
Charge
Density
1
-0.1641
1
-0.1174
1
+0.0779
1
-0.1534
2
+0.1351
2
+0.2577
2
+0.1827
2
+0.2443
3
-0.0659
3
-0.1162
3
-0.1151
3
-0.1434
4
+0.0732
4
+0.0911
4
+0.1029
4
+0.0979
5
-0.0657
5
-0.1678
5
-0.0429
5
-0.1597
6
+0.1348
6
+0.1670
6
+0.1080
6
+0.1737
7
-0.0151
7
-0.0903
26
+0.0429
26
-0.6663
8
-0.0899
27
-0.0788
8
+0.0005
9
+0.0083
11
+0.3261
11
+0.2993
9
-0.0182
10
+0.0101
14
-0.0582
14
-0.0562
10
+0.0001
11
+0.0184
19
+0.0512
19
+0.0055
11
-0.0147
12
-0.0173
7
-.0446
7
-0.0311
13
+0.0663
10
+0.1104
10
+0.1777


8
They are equilibrative, i.e. nonenergy dependent and bidirectional in
nature and can be saturated.120,21 The net movement of compounds is
always along a concentration gradient and since nutrients are readily
utilized as soon as they pass into the brain, this gradient is in the di
rection of the brain.
A number of specific carriers for compounds have been described. The
first to be characterized was one for hexoses.11222 This carrier displays
saturable kinetics and can be competitively inhibited. The hexose carrier
is stereospecific and has a high affinity for a-D-glucose with a Km between
6.0 and 9.0 mM. Other sugars with affinity for this carrier include, in
order of decreasing affinity, 2-deoxy-D-glucose, 3-0-methylglucose, B-D-
glucose, D-mannose, D-galactose and D-xylose.22 The Km of D-fructose and
L-glucose is very high.
The carrier is Na+ independent and is inhibited by phloretin, a non
competitive inhibitor, more than phlorizin, a competitive inhibitor.20
The Vmax is similar for all sugars tested which indicates the rate limiting
step for transport is not the association of the sugar with the carrier but,
rather, the movement of the carrier across the membrane complex. This car
rier demonstrates exchange diffusion, i.e. the carrier moves more rapidly
22 2 3
when loaded than when empty.
At a Km of 7 mM, the concentration of glucose required to produce sat
uration is about 126 mg% so that under physiological conditions, the system
is about half saturated. In normal situations the rate determining step in
glucose utilization is the hexokinase step. This can be shifted to BBB
transport of glucose in conditions of hypoglycemia.21 While substrate flux
is usually thought of as being related only to plasma levels of glucose,
recent studies have indicated that intracerebral glucose concentrations can
alter carrier kinetics.


CHAPTER 2
MATERIALS AND METHODS
Elemental analyses of compounds synthesized were performed by Gal
braith Laboratories, Inc., Knoxville, Tennesse, or Atlantic Microlab,
Inc., Atlanta, Georgia. Uncorrected melting points were determined using
a Thomas-Hoover melting point apparatus. Ultraviolet spectra (UV) were
recorded on a Beckman 25, Cary 210 or Cary 219 spectrophotometer. An
Apple II Plus microprocessor was dedicated to the Cary 210 instrument.
Infrared spectra (IR) were taken on a Beckman 4210 high resolution and a
Beckman Acculab 1 infrared spectrophotometer. Samples were analyzed as
a thin film between sodium chloride windows or as a potassium bromide pel
let. Nuclear magnetic resonance spectra (NMR) were obtained from either
a Vari an T60 or Joel-JNM-FX 100 Fourier transform spectrometer. The sam
ples were dissolved in deuterated chloroform (CDC13), deuterated dimethyl
sulfoxide ((CD3)2S0), deuterated pyridine (C5D5N), deuterated methanol
(CD30D), deuterated acetonitrile (CD3CN), deuterium oxide (D20) or tri-
fluoroacetic acid (TFA). Chemical shifts in parts per million were re
ported relative to the internal standard tetramethylsilane except in aque
ous systems where sodium 3-trimethylsilylpropanesulfonate is used. Mass
spectra were obtained using a DuPont 21-491B double focusing magnetic sec
tor mass spectrometer to which was dedicated a Hewlett Packard 2100A com
puter. In all determinations, the ionizing voltage was 70 eV.
High pressure liquid chromatography (HPLC) was performed on either
a Waters Associates system consisting of a Model U6K injector, a Model
6000A solvent delivery system and a Model 440 absorbance detector or a
47


43
Figure 1-3. The Proposed Drug Delivery System.


141
Table 3-20-continued.
HPLC
Compound
Retention Time of Peak Height
Peaks Observed (min) Relative to Berberine
Dihydroberberine
Isoamyl Alcohol
Extract
1.9X
5.1
2.5X
1.3
3.4
0.18
4.1*
1.0
4.8
0.09
Corresponds to berberine standard
xThese peaks were found in blank animals injected with the anesthetic
a
Five microliters of the sample were injected on a yBondapak C18 reverse
phase column. The mobile phase was acetonitrile:pH 6.2 phosphate buffer
60:40 and the flow rate was 2.0 ml/min