Delivery of a kyotorphin analog into the central nervous system by molecular packing and sequential metabolism

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Title:
Delivery of a kyotorphin analog into the central nervous system by molecular packing and sequential metabolism design, synthesis and pharmacology
Alternate title:
Design, synthesis and pharmacology
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xiv, 150 leaves : ill. ; 29 cm.
Language:
English
Creator:
Chen, Pei, 1962-
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Subjects

Subjects / Keywords:
Drug Delivery Systems   ( mesh )
Endorphins -- analogs & derivatives   ( mesh )
Endorphins -- chemical synthesis   ( mesh )
Endorphins -- pharmacology   ( mesh )
Brain -- drug effects   ( mesh )
Protein Engineering   ( mesh )
Drug Design   ( mesh )
Tyrosine   ( mesh )
Arginine -- pharmacology   ( mesh )
Lysine -- pharmacology   ( mesh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
Bibliography:
Includes bibliographical references (leaves 137-149).
Statement of Responsibility:
by Pei Chen.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 49817526
ocm49817526
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AA00011190:00001

Full Text











DELIVERY OF A KYOTORPHIN ANALOG INTO THE CENTRAL
NERVOUS SYSTEM BY MOLECULAR PACKING AND SEQUENTIAL
METABOLISM: DESIGN, SYNTHESIS AND PHARMACOLOGY










By

PEI CHEN


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

UNIVERSITY OF FLORIDA


1996


































DEDICATED TO MY MOTHER















ACKNOWLEDGMENTS

I would like to express my gratitude to my academic advisor, Dr. Nicholas

S. Bodor, for his intellectual guidance throughout my work here at the Center for

Drug Discovery, which has made me realize the importance of choosing the right

academic supervisor. I understand, though, that it is not enough to just say thank

you to someone who has given me timely and critical support, both spiritually and

materially, which has enabled me to pursue the freedom of life and freedom of

academia.

I would like to thank the members of my supervisory committee, Dr. Laszlo

Prokai, Dr. William J. Millard, Dr. James W. Simpkins and Dr. Alan Katritzky,

for their advice and help.

I would like to acknowledge the help I received form Dr. Whei-Mei Wu and

Dr. Hassan Farag.

I would also like to thank Mrs. Laurie Johnston, Mrs. Julie Berger, Mrs.

Kathy Eberst, and Mr. Robert Wong for their cordial assistance.

Finally, I would like to thank my wife and my family for their caring,

understanding, love, and support, without that I would not have been able to make

progress personally and academically.















TABLE OF CONTENTS


pagge

ACKNOWLEDGMENTS ..................................................................iii

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

LIST OF FIGURES......................................................................ix

ABBREVIATIONS....................................................................... xi

ABSTRACT............................................................................... xiii

CHAPTERS

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

Neuropeptides and Neuromodulation............................................. 1

Neuropeptides as Potential Neuropharmaceticals................................ 4

The Opioids......................................................................... 7

The History of Opioid Peptides and Their Receptors........................... 9

Opioid Actions Related to the Central Nervous System (CNS) .............13

Opioid Actions Related to the Peripheral Tissues ............................14

The Enkephalins...................................................................20

Kyotorphin..........................................................................21

Peptide and Protein Drug Delivery.............................................25










page

Neuropeptide-Degrading Enzymes..............................................27

The Enzymatic Barrier For Peptide and Protein Drug Delivery..............32

The Blood-Brain Barrier (BBB) and Its Selective Permeability Properties..34

The Blood-Brain Barrier (BBB) For Peptide and Protein Drug Delivery.... 39

The Chemical Delivery System (CDS) ........................................41

The Principles of Redox-Based Chemical Delivery System ................43

Delivery of Peptides into the CNS via CDS ..................................45

Chemical Delivery Systems for Kyotorphin and a Kyotorphin Analog ....48

2. MATERIALS AND METHODS.................................................58

M materials ........................................................................... 58

Synthetic Protocol for the Chemical Delivery Systems of Kyotorphin......59

Synthetic Protocol for the Chemical Delivery Systems and
the Brain Targeted Redox Analog of Tyr-Lys................................ 61

Typical Experimental Procedures for Chemical Synthesis...................66

Pharmacology Studies.............................................................70

3 CHEMICAL SYNTHESIS.......................................................75

Synthesis of Kyotorphin-CDS's (the Boc- Method) .........................75

Synthesis of Kyotorphin-CDS's (the Fmoc Method)..........................80

Synthesis of KAYK-CDS's ......................................................87

Synthesis of BTRA.................................................................96











page

4 RESULTS AND DISCUSSION.................................................100

Peptide Synthesis -- The DCC/HOBt Method......................................100

Synthesis of Kyotorphin-CDS's ...............................................102

Synthesis of KAYK-CDS's and BTRA........................................109

Pharmacology Studies of KAYK-CDS's and BTRA......................... 112

5 CONCLUSIONS...................................................................134

LIST OF REFERENCES...................................................................137

BIOGRAPHICAL SKETCH..............................................................150















LIST OF TABLES


Table page


1. Correlation of Opioid Action with Receptor Localization in the CNS....... 15

2. Some Kyotorphin Analogs........................................................24

3. Some Peptidases Present in the CNS.................................................29

4. Blood-Brain Barrier Transport Systems and Their Substrates..............38

5. The Intermediates and Kyotorphin-CDS Synthesized
by the Boc M ethod............................................................... 102

6. The Intermediates and Kyotorphin-CDS Synthesized
by the Fmoc M ethod............................................................. 103

7. The Intermediates and KAYK-CDS's and BTRA Synthesized............. 110

8. The Tail Flick Latency Period Prior to and After i.v.
Administration of Different Doses of CDS-P .................................113

9. Tail Flick Latency Period Prior To and After i.v.
Administration of Different KAYK-CDS's and BTRA...................... 116

10. Two-Factor Anova with Replication for Comparison
of the Rat Tail Flick Latency Prior to and After
i.v. Administration of KAYK-CDS's and BTRA
over the Six Hour Testing Period.................................................118

11. The Rat Tail-Flick Latency Period Prior to and After i.v.
Administration of CDS-PP and Its Major Intermediates.................... 122

12. The Rat Tail-Flick Latency Period Prior to and After i.v.
Administration of BTRA and Its Major Intermediates...................... 125











Table


page


13. The Effect of Naloxone on Rat Tail Flick Latency Periods
After i.v. Administration of the CDS-PP and BTRA........................ 130















LIST OF FIGURES


Figure page

1. The Structure of Morphine.......................................................... 7

2. The Mechanism of the Redox-Based CDS for Enhanced and
Sustained Delivery of Drugs to the Brain........................................44

3. The Chemical Delivery System for Peptides...................................46

4. The Sequential Metabolism of the CDS of peptide.............................47

5. The Structures of KTP, KAYK, and Their Basic CDS's ......................49

6. The Structure of CDS-P -- a CDS for KAYK
1,4-Dihydrotrigonellyl-Pro-Tyr-Lys(Boc)-Cholesteryl Ester................50

7. The Structure of Boc-Tyr-Nys-Cholesteryl Ester (BTRA)................... 51

8. The Isoelectronic/isosteric Effects of Tyr-Lys and Tyr-Nys in vivo........52

9. The Sequential Metabolism of CDS-P in vivo................................. 54

10. The Sequential Metabolism of CDS-PA in vivo............................... 55

11. The Sequential Metabolism of CDS-PP in vivo............................... 56

12. The Sequential Metabolism of BTRA in vivo.................................. 57

13. CDS-KTP-A -- A Kyotorphin-CDS.............................................59

14. The Synthetic Scheme for Kyotorphin-CDS
by Using the Boc Method........................................................60

15. The Synthetic Scheme for Kyotorphin-CDS
by Using the Fmoc Method.......................................................62










Figure


16. The Structures of KAYK-CDS's & BTRA.................................... 63

17. The Synthetic Scheme for CDS-P................................................64

18. The Synthetic Scheme for BTRA.................................................67

19. The Mechanism of Zincke Reaction.............................................69

20. The Mechanism of the DCC/HOBt Method........................................101

21. Removal of the s-NO2 group of Kyotorphin-CDS by Hydrogenesis........ 105

22. The Proposed Synthetic Scheme for the Addition of a Labile
Protection Group to s-NH2 Group of Arginine................................. 108

23. The Dose Response of CDS-P after i.v. Administration in Rat............ 114

24. The Rat Tail-Flick Latency Periods Different KAYK-CDSs
and BTRA after i.v Administration of......................................... 117

25. The Time Response of Rat Tail-Flick Latency of Different KAYK
Brain-Targeted Delivery Systems after i.v. Administration................ 119

26. The Structures of CDS-PP and Its Major Intermediates..................... 121

27. The Rat Tail-Flick Latency Periods after i.v. Administration
of CDS-PP and Its Major Intermediates........................................123

28. The Structures of BTRA and Its Major Intermediates....................... 124

29. The Rat Tail-Flick Latency Periods After i.v.
Administration of BTRA and Its Major Intermediates....................... 126

30. The Time Responses of BTRA and YNC after i.v administration......... 127

31. The Biocoversion of YNC in vivo............................................. 128

32. The Effects of Naloxone on Rat Tail Flick Latency Periods
After i.v. Administration of CDS-PP and BTRA..............................131


Dage















ABBREVIATIONS


Ala (A):
Arg (R):
BBB:
BTRA:
Boc:
CDS:
CDS-P:

CDS-PA:
CDS-PP:

CNS:
DADLE:
DCC:
DCU:
DIEA:
DMAP:
DMF:
DMSO:
DNP:
Et3N:
FAB:
g:
Fmoc:
HOBt:
i.v.:
KAYK:
KAYK-CDS:
KTP:
KTP-CDS:
HPLC:
LH:
Lys (K):
ml:
mmol:
MS


alanine
arginine
blood-brain barrier
the brain-targeted redox analog for Tyr-Lys (a kyotorphin analog)
tert-Butyloxycarbonyl
chemical delivery system
the chemical delivery system for Tyr-Lys with a single proline as
spacer
the chemical delivery system for Tyr-Lys with Pro-Ala as the spac
the chemical delivery system for Tyr-Lys with double prolines as
spacer
central nervous system
D-Ala -D-Leus-enkephalin
dicyclohexylcarbodiimide
dicyclohexylurea
diisopropylethylamine
4-dimethylaminopyridine
dimethylformamide
dimethylsulfoxide
dinitrophenyl
triethylamine
fast atom bombardment ionization
gram
9-florenylmethoxycarbonyl
N-hydroxybenzotriazole
intravenously
Tyr-Lys (a kyotorphin analog)
the chemical delivery system for Tyr-Lys (a kyotorphin analog)
kyotorphin
the chemical delivery system for kyotorphin
high performance liquid chromatography
luteinizing hormone
lysine
milliliter
millimole
mass spectrometer


the

;er
the










Nys: the redox analog of the natural diamino acid lysine
Nys: the quarternized form of the redox analog of the natural diamino acid
PAG: the periaqueductal gray
PG: propylene glycol
POMC: proopiomelanocortin
Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl
Pro (P): proline
s.c.: subcutaneously
TFA: trifluoroacetic acid
TLC: thin layer chromatography
TRH: thyroid-releasing hormone
Tyr (Y): tyrosine
VTA: ventral tegmental area















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



DELIVERY OF A KYOTORPHIN ANALOG INTO THE CENTRAL
NERVOUS SYSTEM BY MOLECULAR PACKING AND SEQUENTIAL
METABOLISM: DESIGN, SYNTHESIS AND PHARMACOLOGY


By

PEI CHEN

May, 1996


Chairman: Nicholas S. Bodor
Major Department: Pharmaceutics


Kyotorphin (KTP -- L-Tyrosyl-L-Arginine) is an endogenous neuropeptide which

exhibits analgesic action through induction of the release of enkephalin in the brain. Tyr-

Lys (KAYK) is a kyotorphin analog which has almost the same analgesic effect as

kyotorphin. The peptide sequence, Tyr-Arg or Tyr-Lys, was incorporated into brain-

targeted delivery systems to achieve their brain delivery for potential pain-relief

treatment. These brain-targeted delivery systems disguise the peptide nature of Tyr-Arg

or Tyr-Lys and provide necessary lipophilic functions to penetrate the blood-brain barrier

(BBB) by passive transport. Through sequential enzymatic bioactivation, the target










peptide, Tyr-Arg or Tyr-Lys, is retained in the brain and released in pharmacologically

significant amounts in situ.

A brain-targeted chemical delivery system for Tyr-Arg (KTP-CDS) 'was

synthesized. It was found that the Boc method for the synthesis of KTP-CDS is not

practical. The Fmoc method is suitable to synthesize KTP-CDS. However, the KTP-

CDS proposed has to be further modified to become a complete molecular package for

brain delivery. The modification was done by replacing arginine with lysine. The

replacement avoided the difficulties of dealing with the &-NH2 group of arginine. A

series of brain-targeted chemical delivery systems for Tyr-Lys (KAYK-CDS) and a the

brain targeted redox analog for Tyr-Lys (BTRA) was synthesized by a stepwise

procedure in solution starting with esterification of lysine derivatives with cholesterol and

followed by coupling of the respective peptide cholesterol ester with Boc- and Fmoc-

amino acids using the DCC/HOBt method.

Analgesic actions of the CDS's and BTRA of Tyr-Lys were observed by

increased tail-flick time of rats, which indicated the successful delivery of Tyr-Lys into

the brain. All CDS's and BTRA showed analgesic activity upon systemic administration,

the extent of which depends on the number and the nature of the spacer.















CHAPTER 1
INTRODUCTION


The Neuropeptides and Neuromodulation

Over the past 30 years there have been two developments in neuroscience

which have fundamentally influenced research on neural substrates of behavior. The

first was anatomical demonstration of the brain's monoaminergic pathways,

followed by extensive behavioral and pharmacological investigation of the nature

and function of these neurons. These studies fostered notions about the role of

neurotransmitters in the regulation of behavior, as well as forming the basis of many

of the current biological theories of mental illness. The second important

development was the discovery and characterization of peptide-containing neurons in

the brain.

Extracellular chemical messenger molecules may be broadly classified into

three categories: ions (Na, K, Ca2+, Cl-, etc.), amines and amino acids

catecholaminee, serotonin, histamine, glutamic acid), and peptides. Peptides are

defined as anywhere in size from two amino acids to proteins of a molecular weight

of 100,00 or more. While the major neuroactive ions and amino acids/amines were

isolated and chemically defined by the 1960s, the identification and structural

elucidation of the neuropeptides date to as recently as 1969 with the elucidation of











the chemical structure of TRH (throtropin-releasing hormone). Since then, numerous

neuropeptides have been identified.

Decades ago it was thought that the brain peptidergic substances were

restricted to the hypothalamic-pituitary axis, where hypothalamic releasing factors

specifically controlled release of pituitary hormones. Throughout the 1970s, the

development of radioimmunological assays and immunohistochemistry led to the

demonstration of numerous different peptides in neuronal networks throughout the

brain. About this time, it was suggested that peptides represented a whole new

generation of neurotransmitters, and that study of those substances could provide

important clues to the mechanisms of neuronal communication (Bloom, 1973;

Iversen, 1974). Since then, the number of peptides demonstrated in nervous tissue

has grown rapidly, a process which still continues. In parallel, considerable efforts

have been aimed at understanding the behavioral function of neuropeptides, within

domains of study as diverse as learning and memory, response to stress and pain,

ingestive behavior, and motivation.

Researchers were originally quite unwilling to accept such large, complex

molecules as peptides, which were presumably synthesized at ribosomes within the

neuronal cell body, as synaptic transmitters. We now know that many of these

substances have a specific synaptic function; moreover, recent developments in

molecular genetic studies suggest that specific mechanisms at the transcriptional

level may play an important role in information encoding and ultimately in global












behavioral functions (Bloom, 1979; Schmitt, 1984). For example, opioid peptides

are derived from three distinct prohormones, proopiomelanocortin, proenkephalin,

and prodynorphin. In some cases proenkephalin- and prodynorphin-derived peptides

may be co-localized, since they appear to have the same distribution. The substantial

nigra and VTA (ventral tegmental area) both contain dynorphin and enkephalin. In

the study by Bannon, amounts of these two peptides were measured in the VTA

after footshock stress (Bannon et al., 1986). Footshock markedly depleted dynorphin

but left enkephalin levels unchanged. In support of differential actions of the opioid

peptides, the work of Kanamatsu showed that dynorphin and met-enkephalin are

altered differentially by repeated electroconvulsive shock treatment (Kanamatsu et

al., 1986). Evidence such as these had important consequences for concepts about

the neural coding of behavior. The information coding potential inherent in these

molecules and their associated genetic operation is great. The existence of multiple

forms of the same peptide or similar peptides suggests that peptides may play a

fundamental role in the elaboration of complex, adaptive behavior.

The discovery of a wide range of biologically active peptides

(adrenocorticortropin, a-endorphin, 13-endorphin, bradykinin, dynorphin, gastrin,

growth hormone, insulin, kyotorphin, leu-enkephalin, met-enkephalin, neurotensin,

secretin, substance P, TRH, vasopressin, etc.) in the nervous system and growing

knowledge of their characteristics has contributed to new theories of information

transfer and chemical integrative process in the brain (Bloom, 1979; Schmitt, 1984).











Considering the number of different amino acid combinations, different chain

lengths, and multiple forms of related peptides, the actual number of peptides in the

brain could be very large indeed. It is a truly formidable and baffling task for the

behavioral neuroscientist to incorporate this geometrically expanding body of

knowledge into working hypotheses about behavior. Thus the study and

understanding of mammalian behavior, which are never simple, become even more

complex. One can hope nevertheless that in the future research an integration of

refined behavioral techniques and paradigms with knowledge of the basic

neurobiology of peptides may contribute at least partially to this understanding.



Neuropeptides as Potential Neuropharmaceuticals

Disorders of the brain are surprisingly common. These disorders include

headache, alcohol abuse, anxiety/phobia, sleep disorders, depression/mania, drug

abuse, obsessive-compulsive disorder, Alzheimer's disease, stroke, epilepsy, and

Parkinson's disease. The drugs currently used for disorders of the brain such as

codeine (headache), diazepam (anxiety), amitryptiline (depression), and phenytoin

(epilepsy) are all lipid-soluble pharmaceuticals that readily cross BBB following oral

administration. These drugs were discovered with the traditional "trial-and-error"

method of drug discovery wherein up to 10,000 different compounds were screened

with in vivo bioassays (Waldrop, 1990). This approach excluded drugs that may

have interacted with particular receptors in the brain but were drugs that had poor











BBB transport properties owing to the low lipid solubility of the compound, such as

neuropeptides.

The multiplicity of biological actions of peptides in the brain suggests that

these agents may be utilized as neuropharmaceuticals in the treatment of a variety of

disorders of the brain and spinal cord. However, as with any potential

neuropharmaceutical, peptides must be able to undergo transport into the brain from

blood. Neuropeptide investigation in the mid and late 1970s underscored the

importance of a through understanding of the mechanisms by which peptides are

transported between blood and the brain.

Shortly after the discovery of peptides such as TRH or 3-endorphin, these

agents were infused intravenously into humans for the treatment of psychiatric

disorders such as schizophrenia or depression (Barchas & Elliott, 1986). No

consistent effects on the brain were found with these agents in humans. In the case

of 3-endorphins, the peptide had profound effects on the central nervous system in

rats following the injection of the peptide directly into the ventricular compartment.

However, no effects were found after the systemic administration of very large

doses of 3-endorphin into rats or humans. The failure of any central actions of 3-

endorphin following peripheral administration is due to the presence of a barrier

between blood and the brain, the blood-brain barrier (BBB).

The lack of transport of most neuroactive peptides through the BBB parallels

the absence of transport of the neuroactive amino acids or monoamines such as












dopamine. In the 1960s, the neutral amino acid L-dihydroxyphenylalanine (L-dopa)

was developed for the treatment of Parkinson's disease, a degenerative condition of

the brain wherein the caudate putamen region of the brain forms inadequate amounts

of dopamine. L-dopa can be transported into the brain via the BBB neutral amino

acid transport system and, subsequent to this transport, is converted to dopamine by

brain aromatic amino acid decarboxylase (Wade & Katzman, 1975). Unfortunately,

peripheral tissues are also endowed with aromatic amino acid decarboxylase, and

this results in the peripheral conversion of L-dopa to dopamine.

The use of L-dopa in Parkinson's disease is the paradigm of single

neurotransmitter repletion therapy of neurologic disease and illustrates at least two

important principles applicable to the use of neuropeptides as neuropharmaceuticals.

The first principle is that neurotransmitter or neuromodulator requires a drug

delivery system to cross the BBB. The second principle is the need to slow the rapid

enzymatic inactivation of the neuropharmaceuticals so that adequate amounts can

reach appropriate sites within the brain.

In this dissertation, I will apply these principles to the use of kyotorphin, an

endogenous opioid neuropeptide, as a potential neuropharmaceutical in the treatment

of pain.











The Opioids

The term opiate was once used to designate drugs derived from opium --
morphine, codeine, and the many semisynthetic congeners of morphine. Soon after

the development of totally synthetic entities with morphine like actions, the word
opioid was introduced to refer in a generic sense to all drugs, natural and synthetic,
with morphine-like actions.
There are now many compounds that produce analgesia and other effects
similar to those produced by morphine (Fig. 1). Some of these may have some

special properties, but none has proven to be clinically superior in relieving pain.



HO



N-CH3
^Y^^^N- CH3


Figure 1. The Structure of Morphine











Morphine remains the standard against which new analgesics are measured.

Because the laboratory synthesis of morphine is difficult, the drug is still obtained

from opium or extracted from poppy straw.

In man, morphine-like drugs produce analgesia, drowsiness, changes in

mood, and mental clouding, etc. A significant feature of the analgesia is that it

occurs without loss of consciousness. When therapeutic doses of morphine are given

to patients with pain, they report that the pain is less intense, less discomforting, or

entirely gone. The relief of pain by morphine-like opioids is relatively selective, in

that other sensory modalities (touch, vibration, vision, hearing, etc.) are not

affected. Continuous dull pain is relieved more effectively than sharp intermittent

pain, but with sufficient amounts of morphine, it is possible to relieve the severe

pain such as those associated with renal or biliary colic.

Opioid-induced analgesia is due to actions at several sites within the CNS and

involves several systems of neurotransmitters. Although opioids do not alter the

threshold or responsitivity of afferent nerve endings to noxious stimulation or impair

the conduction of the nerve impulse along peripheral nerves, they may decrease

conduction of impulses of primary afferent fibers when they enter the spinal cord

and decrease activity in other sensory endings. There are opioid binding sites (ji

receptors) on the terminal axons of primary afferents within laminae I and II

substantiala gelatinosa) of the spinal cord and in the spinal nucleus of the terminal

nerve. Morphine-like drugs acting at this site are thought to decrease the release of












neurotransmitters, such as substance P, that mediate transmission of pain impulses.

Enkephalinergic nerve fibers in the dorsal horn of the spinal cord, which appear to

come from interneurons, are usually inhibitory to dendrites and some of nerves

whose cell bodies may be in deeper laminae (IV and V). It can be inferred that in

the spinal cord, separate p and 8 receptors participate in inhibiting transmission of

pain impulses.

Other than analgesia, morphine can produce nausea, vomiting, feeling of

drowsiness, inability to concentrate, difficulty in mentation, apathy, lessened

physical activity, reduced visual acuity, body warmth, relief of stress, and euphoria

(Duggan & North, 1983; Martin, 1983)



The History of Opioid Peptides and Their Receptors

Morphine was the first natural opioid to be identified and characterized. This

alkaloid was isolated as one of the analgesic components of opium. The question of

why a plant alkaloid should bind to stereospecific receptors on neuronal membranes

and display such dramatic effects on the mammalian nervous system was answered

by an extensive search for an endogenous opioid ligand to the mammalian nervous

system. Initial studies were aimed at the isolation of alkaloids, but it soon became

apparent from many experiments that peptides may be endogenous ligands for the

opioid receptors.











The Opioid Peptides

The early evolution of concepts of endogenous opioids and multiple opioid

receptors had its inception in a concerted program to develop safe, non-addicting

substitutes for opioids. Although the search for safer and less-abusable analgesics

has not been entirely successful, ideas concerning multiple opioid receptors and

endogenous opioid transmitters were evolved during the process.

In the early 1970s, two pentapeptides with opioid-like activity were extracted

from porcine brain and characterized (Hughes et al., 1975). These two peptides

differed only in the C-terminal amino acid and were named methionine enkephalin

(Tyr-Gly-Gly-Phe-Met) and leucine enkephalin (Tyr-Gly-Gly-Phe-Leu). The

isolation of the other two families of opioid peptides, the 13-endorphins and the

dynorphins, was also accomplished in 1975 (Bradbury et al., 1975; Cox et al.,

1975).

Each family of opioid peptides is derived from a genetically distinct

precursor polypeptide and has a characteristic anatomical distribution. These

precursors are now commonly designated as proenkephalin (also proenkephalin A),

pro-opiomelanocortin (POMC), and prodynorphin (also proenkephalin B). Each of

these precursors contains a number of biologically active peptides, both opioid and

nonopioid, that have been detected in blood and various tissues. Proteolytic

processing of the precursors generates the active peptides which are expressed via

peptide receptor systems at the target cell -- the opioid peptide receptors.











In addition to products belonging to these major families, other opioid

peptides have been identified in the brain (kyotorphin) and frog skin (dermorphin).



The Opioid Peptide Receptors

When considering the most basic requirements for a neurotransmitter system,

one must include certain specific receptors as well as ligands. In fact, in the case of

the endogenous opioids the existence of opioid receptors preceded the discovery of

the opioid peptide ligands. It has been possible to study opioid receptors by using

receptor binding assays. In this technique the interaction of a radio-labeled opioid

ligand with the receptor may be monitored in vitro. Extensive characterization of the

opioid receptor has taken place using these biochemical techniques and some

important conclusions may be summarized as follows:

1) The receptors appear to be proteins (Miller & Dawson, 1980; Pasternak &

Snyder, 1976; Zukin & Zukin, 1984). High affinity, saturable, stereo-specific

binding sites may be labeled by using a variety of ligands, including opioid agonists

opioid antagonists, and opioid peptides.

2) Multiple opioid receptors exist. Martin originally suggested that three

different categories of receptor existed based on the analysis of the effects of a wide

range of narcotics in the chronic spinal dog preparation. These categories were

designated p (mu), K (kappa), and a (sigma) (Gilbert & Martin, 1976; Martin et al.,

1974, 1976). In addition to these categories, Kosterlitz and colleagues suggested a












further category designated 8 (delta) on the basis of differential effects of opioid

peptides and narcotic drugs on the guinea pig ileum and mouse vas deferens

bioassays (Kosterlitz et al., 1979). The psychotropic actions of opioids and opioid

peptides are mediated by interaction with g, 6, and K opioid receptors and the

related a receptor. The pt receptor is operationally defined as the high-affinity site at

which morphine-like opioids produce analgesia and other classical opioid effects.

The 6 receptor is operationally defined as the receptor that is found in peripheral

tissues such as the mouse vas deferens as well as in the CNS (Chang et al., 1979),

and that exhibits a higher affinity for naturally occurring enkephalins than for

morphine. The ic receptor is the receptor at which ketocyclazocine-like opioids

produce analgesia as well as their unique ataxic and sedative effects (Gilbert &

Martin, 1976; Martin et al., 1976). It has been defined as a receptor highly selective

for dynorphin -- a 17 amino acid opioid peptide (Chavkin et al., 1982). Actions at

all three of these sites are reversible by a specific opioid antagonist -- naloxone, with

decreasing sensitivity going from p to 6 to K receptors. The a receptor was proposed

to be the site at which the psychotomimetic and stimulatory effects of N-

allylnormetazocine, cyclazocine, and related opioid are mediated (Martin et al.,

1976; Zukin & Zukin, 1981).












In summary, analgesia is associated with g and Ki receptors, while dysphoria

or psychotomimetic effects is ascribed to a receptors. 6 receptor is involved in

altercation of affective behavior.



Opioid Actions Related to the Central Nervous System (CNS)

The distribution of opioid receptors in the CNS is revealed in detail by the

autoradiographic method which generally agrees well with the biochemical binding

studies. Using [3H]diprenorphine, an antagonist with high affinity with opioid

binding sites, Kuhar's group developed the first autoradiographic technique used to

localize opioid receptors at the light microscopic level. In 1977, Atweh and Kuhar

published a detailed description of the distribution of [3H]diprenorphine binding

throughout the central nervous system (Atweh & Kuhar, 1977 a, b, c), providing an

excellent correlation between regions capable of mediating the various opioid actions

(analgesia, autonomic reflexes, endocrine effects, behavioral and mood effects, and

motor rigidity) and those containing receptors.



Analgesia

Opioids and the opioid peptides have the unique ability to selectively relieve
the subjective component of pain without affecting primary sensory modalities, such
as touch, vibration, vision, and hearing. Opioids can modulate pain sensation at
several levels of the CNS. Most experimental work has focused on the spinal cord,











specifically, laminae I and II and the Periaqueductal gray (PAG). The physiology of
this system has been extensively studied (Basbaum, 1984).


Respiratory Depression

Classical p. and 8 opioids and the opioid peptides consistently depress
respiration in all species that have been tested. These actions appear to involve both
decreased responsiveness of the CNS centers to CO2 and a decrease in the CNS
respiratory frequency controller (Eckenhoff & Oech, 1960; McQueen, 1983).


Other Opioid Actions Mediated by the CNS.

The other opioid actions mediated by the CNS, such as autonomic reflexes,
endocrine effects, behavioral and mood effects, and motor rigidity, are summarized
in Table 1.



Opioid Actions Related to the Peripheral Tissues

Gastrointestinal Tract

Bioassays of intestinal extracts confirmed that the presence of enkephalin-like

peptides in intestinal extracts (Hughes et al., 1977). In all mammalian studied,

neurons in the myenteric plexus staining with anti-enkephalin antisera have been

found. The relative number of these enkephalin-containing cells appears to vary both

with regard to location along the gastrointestinal tract from esophagus to colon and

among species. In general, fibers from the enkephalin-containing cells run from and

between myenteric plexus ganglia and distribute extensively through the circular





















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and longitudinal muscle layers. Some fibers also innervate the submucus plexus and

the mucosa, but enkephalin cell bodies are rare or absent in the submucus plexus.

Opioid receptors in the gastrointestinal tract were found in intestine, esophageal

sphincter, stomach circular muscle, duodenum, ileum, and rectum among species

(Elde et al., 1976; Linnoila et al., 1978; Jessen et al., 1980; Schultzberg et al.,

1980; Uddman et al., 1980).

Opioids can exert several different actions on gastrointestinal motility, such

as reduced gastrointestinal propulsion and secretion. The overall result of these

actions is a decline in propulsive activity, resulting in a delay in the passage of

gastrointestinal contents. The reduction in propulsion of gastrointestinal contents

allows longer time for fluid reabsorption. A reduction in fluid secretion also

contributes to the production of dry, compacted feces (Powell, 1981). It is possible

that opioids can act at both central and peripheral sites to alter gastrointestinal

function after systemic administration (Schulz et al., 1979; Manara & Bianchetti,

1985). Those actions contributed to the antidiarrheal action of opioids.



Adrenal Gland

The presence of enkephalins in adrenal medulla has been revealed by

Schutlzberg (Schutlzberg et al., 1978). Subsequently, adrenal medullary tissue has

been used as a major source of enkephalin precursors (Lewis et al., 1980) and of












proenkephalin mRNA (Noda et al., 1982; Comb et al., 1982). Enkephalins and the

larger molecular weight peptides are released from adrenal medulla together with

catecholamine in response to potassium perfusion of the gland or stimulation of the

splanchnic nerve (Viveros et al., 1979; Chaminade et al., 1984).



The Sympathetic Nervous System

The presence of enkephalin-like material in sympathetic ganglia has been

demonstrated and, as in adrenal gland, has been shown to consist of both the

pentapeptide enkephalins and higher molecular weight enkephalin containing

peptides (Di Giulio et al., 1978). The immunocytochemical localization of

enkephalin peptides in sympathetic ganglia of guinea pig, rat, and human has also

been described (Schutlzberg et al., 1979; Hervonen et al., 1981).



Blood Vessels

The ability of enkephalins to depress vasoconstrictor response to sympathetic

nerve stimulation in the rabbit isolated ear artery was first noted by Knoll (Knoll,

1976) and was confirmed by other groups soon after (Ronal et al., 1982; Illes et al.,

1983). The locus of action has been shown to be at the presynaptic nerve terminal.

In this tissue, morphine is less effective than the opioid peptides.

Enkephalins can also exert non-opioid effects on blood vessels. Pulmonary

vasoconstriction induced by leu-enkephalin is not reversed by naloxone or











diprenorphine. Other enkephalins, including met-enkephalin and DADLE (D-Ala2-

D-Leu5-enkephalin), do not induce the same vasoconstrictor effect (Crooks et al.,

1984). Thus, a non-opioid mechanism is assumed to be responsible for this effect.



The Heart

Endogenous opioids have been reported to be present in guinea pig and rat

heart (Hughes et al., 1977; Spampinato & Goldstein, 1983; Lang et al., 1983).

Proenkephalin mRNA has also been found in rat heart (Howells et al., 1985). They

found that the heart contained larger amounts of enkephalin mRNA than any other

tissue and 95 % of the mRNA was found in the ventricles which contain very low

concentrations of enkephalin, relative to both atria and to other tissue like brain.

Pharmacological studies suggested that the opioid binding site might be located on

the autonomic innervation of the heart (Saunders & Thornhill, 1985).

Intravenous administration of morphine and some opioid peptides in rat has

been shown to produce a transient bradycardia that is blocked by atropine or

vagotomy (Fennessy & Rattray, 1971; Wei et al., 1980). The effect is peripherally

mediated, since it is antagonized by the quaternary antagonist N-methylnaloxonium

(Kiang et al., 1983).











Sensory Nerves

Endogenous enkephalins have been found in tooth pulp (Kudo et al., 1983)

and in the carotid body (Wharton et al., 1980). In the spinal cord, there are high

concentrations of enkephalins and dynorphins in the substantial gelatinosa (Hunt et

al., 1980; Vincent et al., 1982), where collateral's of primary sensory neuron's

synapse with regulatory interneurons. Thus, endogenous opioids appear to be

strategically located for the regulation of transmission from the primary afferent

neuron to the central nervous system. The opioids might reduce the output of

transmitter from primary afferent neurons, reducing their ability to activate

secondary sensory neurons (Werz & Macdonald, 1982, 1984).



Reproductive Tract Tissues

All three classes of endogenous opioid peptides have been found in the

tissues of the reproductive tract (Sharp et al., 1980; Margioris et al., 1983; Lim et

al., 1983; Pintar et al., 1984). The functions of reproductive tract endogenous

opioids are still unclear.



In summary, the localization of opioid receptors and enkephalin-containing

neurons in the thalamus, Periaqueductal gray matter, and substantial gelatinosa of the

spinal cord suggests a role for endogenous opioid peptides in pain modulation at

these three levels (Hokfelt et al., 1977). A major end point of all studies of opioid











and opioid peptide action is the development of new compounds with clinical utility.

To date, opioids have been used primarily as analgesics and antidirrheals. The

importance of opioid mechanisms in a wide range of neuropharmacological actions,

however, suggests that these agents may have a much wider use. The development

of an agent which is capable of producing desired actions with fewer side-effects is

desired.



The Enkephalins

The enkephalins were first isolated and sequenced by Hughes (Hughes et al.,

1975) from a mixture of two pentapeptides present in the extracts of porcine brain

which differed only by possessing either a Met or Leu residue at their C-terminal.

The two peptides were named met-enkephalin (NH2-Tyr-Gly-Gly-Phe-Met-OH) and

leu-enkephalin (NH2-Tyr-Gly-Gly-Phe-Phe-OH). Later the same two compounds

were obtained from bovine brain (Simantov & Snyder, 1976).

Under suitable assay conditions, the enkephalins have many of the biological

characteristics of the alkaloid opioids; however, their susceptibility to enzymatic

break down and short duration of action made their study difficult. Met- and leu-

enkephalin both bind to opioid receptors on brain cell membrane preparations with

comparable affinities to morphine when experiments are performed at 0C or in the

presence of proteolytic enzyme inhibitors (Chang et al., 1976). In systems where

enzymatic activity is higher, such as vas deferens and ileum assays, the peptides are












considerably less active than morphine. For induction of analgesia, very high doses

of met-enkephalin must be directly introduced into the brain and leu-enkephalin is

even less active (Beluzzi et al., 1976; Buescher et al., 1976).

Early interest in the enkephalins centered on whether these endogenous

substances would induce tolerance and withdrawal symptoms in a similar way to the

morphinoids. This was unfortunately the case in a number of paradigms. For

instances, several investigators have detected cross-tolerance between morphine and

enkephalins and some of their analogs (Waterfield et al., 1976; Bhargava, 1978).

Apart from analgesic activity, both met- and leu-enkephalin have effects on

the secretion of several pituitary hormones. Principal among these are stimulatory

actions on growth hormone and prolactin release which appear to be mediated by

hypothalamic growth hormone-releasing hormone and possibly catecholamines,

respectively (Dupont et al., 1979; Meites et al., 1979; Cusan et al., 1977). Both

enkephalins were also found to significantly decrease serum LH luteinizingg

hormone; Meites et al., 1979). Because of the multi-type opioid receptors, it is not

surprising that the enkephalins and their analogs exhibit behavioral properties which

cannot be duplicated by using the morphinoids.



Kyotorphin (KTP)

Kyotorphin (KTP, L-Tyrosyl-L-Arginine) is an endogenous neuropeptide that

exhibits analgesic action by mediation of the release of endogenous enkephalins from












nerve terminals inside the brain. The name of kyotorphin means an endorphin-like

substance which was discovered in Kyoto, Japan. The analgesic potency of

kyotorphin is 4.2 times that of met-enkephalin (Takagi et al., 1980).

Kyotorphin administered intracisternally elicits a naloxone-reversible

antinociception. This peptide does not bind to g-, 8-, and K-sub-types of opioid

receptors, but does induce an enhancement of met-enkephalin release in brain and

spinal cord slices (Takagi et al., 1979). Thus, kyotorphin-induced analgesia may be

attributed to the enhanced release of met-enkephalin in the brain stem and spinal

dorsal horn involved in pain transmission.

Its analgesic effects may also result from its inhibition of the enzymatic

hydrolysis of other endogenous opioid peptides (Takagi et al., 1979). The selective

inhibition by kyotorphin and neo-kyotorphin (Thr-Ser-Lys-Tyr-Arg) on enkephalin-

degrading enzymes suggests that kyotorphin might protect the released met-

enkephalin. Thus, kyotorphin may not only induce the release of met-enkephalin,

but also stabilize the released neuropeptide (Hazato et al., 1986). Satoh has

suggested that the analgesic actions of kyotorphin and D-kyotorphin result from two

different mechanisms: 1) enkephalin releasing mechanism in the PAG and spinal

dorsal horn, and 2) a mechanism without involvement of enkephalin-releasing

actions in the NRPG (nucleus reticularis paragigantocellularis) (Satoh et al., 1985).

Kyotorphin is formed through two discrete pathways; one is formation from

L-arginine and L-tyrosine by a specific synthetase (Ueda et al., 1987); the other











pathway is formation through the processing of precursor proteins by a neutral

protease (Yoshihara et al., 1988).

It was reported that kyotorphin is lower in concentration in patients with

persistent pain, which suggests that kyotorphin acts as a putative neuromediator

and/or an endogenous pain modulator in the human brain (Nishimura, 1991). As

with stimulation-produced analgesia, several studies suggest that Chinese traditional

acupuncture analgesia may be mediated, at least in part, by endogenous opioid

peptides (Mayer et al., 1977).

Analgesia, opioid receptor binding, and neurochemical effects of kyotorphin

were studied in rat. It was found that while kyotorphin, in vivo, causes naloxone

reversible analgesia, and affects dopamine metabolism and acetylcholine turnover in

the same manner as does morphine and other opioid agents, the dipeptide does not

bind to pL-, 6-, and ic-opioid receptors in vitro (Rackham et al., 1982). The above

data support the concept that there is an indirect action of kyotorphin on opioid

receptors. Stone also concluded that the naloxone-reversible analgesic effects of

kyotorphin and D-phenylalanine may be mediated indirectly, rather than through an

activation of opioid receptors (Stone, 1983).

Kyotorphin may also play a neurotransmitter/neuromodulator role in the

brain or may increase transmitter release from preganglionic nerve terminals inside

CNS (Ueda et al., 1982; Hirai et al., 1985). It was reported that specific high and

low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin











receptor is functionally coupled to stimulation of phospholipase C through Gi (Ueda

et al., 1989).

Kyotorphin is rapidly hydrolyzed in the brain (Ueda et al., 1985). Specific

kyotorphin-hydrolyzing enzymes -- KTPase I and II have been identified recently

(Akasaki et al., 1991). Kyotorphin is also easily degraded by other unspecific

hydrolyzing enzymes, such as enkephalin-degrading aminopeptidase as well as other

peptidases (Akasaki & Tsuji, 1991).

Many kyotorphin analogs has been synthesized and tested. Table 2 shows

some of the kyotorphin analogs that have shown analgesic activities (Rolka et al.,

1983; Carcia-Lopez et al., 1987; Carcia-Lopez et al., 1988).


Table 2. Some Kyotorphin Analogs

Names of the Compounds

D-Tyr-Arg Arg-Tyr

Tyr-D-Arg cyclo(Tyr-Arg)

Tyr-Lys cyclo(Trp-Arg)

D-Tyr-Lys Lys-Trp(NPS)

Tyr-Orn Lys-Trp(NPS)-OMe

D-Tyr-Orn Arg-Trp(NPS)-OMe












The structure-activity relationship of a dipeptide with met-enkephalin-

releasing action includes an aromatic acid with a basic nitrogen on the ring (Tyr or

Trp ) and a basic amino acid (Arg or Lys or Orn). The basic side-chain amine group

of the basic amino acid, which is positively charged in vivo, is critical to the

activities, although the side-chain length is not a critical factor.

Tyrosyl-lysine (KAYK) is a kyotorphin analog which exhibits the same

analgesic effect as kyotorphin when administered i.c., 1 mg/kg, in rats (Rolka et al.,

1983).



Peptide and Protein Drug Delivery

The search for effective and safe drugs continues to be a major effort,

involving the pharmaceutical industry, universities, and government. The

complexities of discovering and testing new drugs have become enormous as a result

of the many aspects of safety, efficacy, and economics that determine acceptability

of a drug. The concept of drug carriers or drug delivery systems has been embraced

with great enthusiasm by many as the solution. For example, toxicity would be

reduced by delivering a drug to its target in higher concentrations, reducing harmful

systemic effects by decreasing the quantity of administered drug needed to produce a

desired effect. Toxicity could also be reduced by administering the drug in a non-

toxic form that is activated only at the site of action. More broadly, drug delivery

systems could modify various parameters, such as pharmacokinetics, so as to permit












the effective use of drugs that by themselves are not useful and efficacious. Finally,

the rational and scientific design of drugs could be greatly facilitated by their

development in conjunction with an appropriate carrier system. In reality, drug

carriers became entrapped in many of the same problems that they were intended to

solve, such as potential toxicity, efficacy, and so forth. Nevertheless, their potential

remains bright. The difficulty of finding new pharmacological therapies for diseases

not already amenable to existing drugs is so great that if drug carriers were to lead

to the successful treatment of only a single important disease, that success alone

would easily justify the research time and funds that have been expended on the

development of drug carriers. It seems likely that drug carriers will be an increasing

important aspect of pharmacology and therapeutics in the future.

Hormones, serum proteins, and enzymes have been used as drugs ever since

the commercial introduction of insulin, thyroid hormone, and factor VIII from 1920

through 1940. Molecular biology has now given us the tools to expand the range of

peptide- and protein-based drugs to combat poorly controlled diseases. While there

has been rapid progress in molecular biology, this has not been matched by the

progress in the formulation and development of peptide and protein delivery

systems. This is due, in part, to the lack of appreciation for the unique demands

imposed by the physicochemical and biological properties of peptides and protein

drugs on route of delivery as well as on delivery system design and formulation.

These properties include molecular size, short plasma half-life, requirement for











specialized mechanisms for transport across biological membranes, susceptibility to

breakdown in both physical and biological environments, tendency to under go self-

association, complex feedback control mechanisms, and particular dose-response

relationships.



Neuropeptide-Degrading Enzymes

Termination of the biological actions of neuropeptides is very important to

organisms and appears to occur predominantly through metabolism by cell-surface

membrane peptidases (Lynch & Snyder, 1986). One of the enzymes that has been most

investigated is a plasma membrane metalloendopeptidase that participates in the

metabolism of the enkephalins. When studied in the brain, the enzyme has hence been

termed "enkephalinase", although the enzyme is widely distributed and probably

participates in the metabolism of other neuropeptides (Matsas et al., 1983). The

nomenclature "endopeptidase-24.11" is now regarded as a more appropriate name

(Hersh, 1986). A number of other membrane exopeptidases and endopeptidases

hydrolyzing neuropeptides have been isolated and characterized, including peptidyl

dipeptidase A (angiotensin-converting enzyme; ACE) and aminopeptidase N (AP). Some

of the peptidases presented in the CNS are listed in Table 3. There are many other

peptidases which might present in the CNS, such as carboxypeptidase P (EC3.4.17.-),

carboxypeptidase N (EC 3.4.17.3), aminopeptidase A (EC 3.4.11.7), aminopeptidase P

(EC 3.4.11.9), and aminopeptidase W (Turner et al., 1989).











Table 3. Some Peptidases Present in the CNS


Enzyme Active site Specificity Specific inhibitors

Endopeptidase-24.11 -O-O-X-O Phosphoramidon
EC 3.4.24.11 Zn2+ Thiorphan
hydrophobic
Angiotensin-Converting -O-O-X-X- Captopril
Enzymes Zn2 Enalaprilat
non-specific
EC 3.4.15.1 Lisnopril
Endopeptidase-24.15 -O-O-X-O-O- N-(1(R,S)-carboxy-2-
EC 3.4.24.15 Zn'? phenylethyl)-Ala-Ala-

Phe-pAB
Aminopeptidase N X-O-O-O- Amastatin
EC 3.4.11.2 Zn2+ T Bestatin
NH2- terminal
Actinonin
Dipeptidyl peptidase IV -O-X-O-O- Diisopropyl-
EC 3.4.14.5 serine fluorophosphate
Pro or Ala
(Dip-F)
Post-proline cleaving -O-Pro-O-O- Dip-F
enzyme serine Z-Pro-prolinal
EC 3.4.21.26
Pyroglutamyl peptidase metallo- Glp-His-Pro-NH2 ?
II peptidase
EC 3.4.19.-
Microsomal dipeptidase -0-0- Cilastatin
EC 3.4.13.11 Zn2+ T
___(non-specific)











Endopeptidase-24.11

Endopeptidase-24.11 (enkephalinase; neutral metallo-endopeptidase; EC

3.4.24.11) is a widely distributed enzyme. In the nervous system, the enzyme has been

implicated in the metabolism of the enkephalins which are hydrolyzed at the Gly3-Phe4

bond with the release of Tyr-Gly-Gly. The broad substrate specificity of endopeptidase-

24.11, however, suggests that it may also participate in the degradation of tachykinins

(e.g. substance P), neurotensin, cholecystokinin, kyotorphin, and other neuropeptides.

Endopeptidase-24.11 is an integral membrane glycoprotein of Mr 85,000 -

95,000, depending on the tissue, and in most species, exists as a dimer in the plasma

membrane. One Zn2 is bound at the active site of the enzyme which is essential for

its catalytic activity. Endopeptidase-24.11 hydrolyses peptide bond involving the

amino groups of hydrophobic residues, (X-Y- where Y is Phe, Leu, Ile, Ala, Val,

Tyr, or Trp). Several specific inhibitors of the enzyme exist, of which the most

widely used is Phosphoramidon Thiorphan (Turner et al., 1985).



Peptidyl Dipeptidase A

Peptidyl Dipeptidase A (angiotensin-converting enzyme; ACE; kininase II;

EC 3.4.15.1) is widely distributed throughout the mammalian body, with the lung

and kidney having the highest concentration of enzyme per gram of tissue. At the

vascular surface of the lung, the enzyme converts angiotensin I to angiotensin II and











also inactivates bradykinin (Soffer, 1976). In the brain, the striatum, substantial nigra

and choroid plexus are enriched in ACE. The endogenous substrates for ACE in the

brain are unclear, although it has been implicated in the metabolism of C-terminally

extended enkephalin peptides, neurotensin, and substance P.

The enzyme is a glycoprotein of Mr about 13,000. It has one Zn2+ bound at

its active site which is essential for its catalytic activity. Originally, ACE was

classified as a peptidyl dipeptidase. Recently, certain endopeptidase and tripeptidase

activity have been attributed to the enzyme.



Aminopeptidase N

Aminopeptidase N (AP-N, aminopeptidase M; alanyl aminopeptidase; EC

3.4.11.2) is an integral membrane glycoprotein of Mr 160,000. The enzyme has

a very broad distribution. It is highly concentrated in the vasculature in the CNS.

The active site of AN-P contains one Zn2+

Aminopeptidase N has a broad specificity, releasing N-terminal amino acids

from unblocked di, tri, and oligopeptides. When the N-terminal amino acid is Ala,

hydrolysis is most rapid. Aminopeptidase N is the major aminopeptidase releasing

Tyr from met- and leu-enkephalin (Matsas et al., 1985), and may function to

degrade other neuropeptides with free N-terminus. Hydrolysis is more rapid with

extended peptides than with dipeptides when chain length is considered.











Dipeptidyl Peptidase IV

Dipeptidyl Peptidase IV (DPP-IV; EC 3.4.14.5) is a plasma membrane

peptidase widely distributed in mammalian tissues. It has been reported to present in

the brain where it appears to have a diffuse distribution. In kidney, the enzyme

exists as a dimer with subunit Mr of 130,000. DPP-IV is a serine peptidase and is

therefore inhibited by diisopropylflurophosphate (Kenny et al., 1976).

The specificity of the enzyme is to remove dipeptides from the N-terminus of

unblocked oligopeptides as follows: W-X-Y- -+ W-X + Y-, when X= Pro or Ala.

The rates with Ala are about one-fifth those with Pro.



Post-Proline Cleaving Enzyme

Post-Proline Cleaving Enzyme (post-proline endopeptidase; PPCE; Prolyl

endopeptidase; EC 3.4.21.26) is a dimeric enzyme of subunit Mr 62,000 77,000.

The enzyme is widely distributed in mammalian tissues, with brain being one of the

richest sources. The enzyme cleaves peptide bonds on the C-terminal side of proline

as follows: -V-Pro-X-Y- -> -V-Pro + X-Y-. PPCE is a serine peptidase sensitive to

inhibition by diisopropylflurophosphate (Wilk, 1983).



Microsomal Dipeptidase

Microsomal Dipeptidase (renal dipeptidase; dehydropeptidase I; EC

3.4.13.11) is an integral membrane glycol protein of Mr 47,000 (Hooper et al.,












1987). The enzyme has been identified in kidney, pancreas, liver, spleen, lung, and

brain. The active site of microsomal dipeptidase contains one Zn2+

Microsomal dipeptidase has a broad specificity towards dipeptides, including

dipeptides in which the C-terminal residue has the D-configuration. Longer peptides

are not substrates for the enzyme. In the nervous system the enzyme may serve to

hydrolyze dipeptide neurotransmitters (e.g., kyotorphin or glycyl-glutamine).

Dipeptides can also arise as secondary metabolites from the hydrolysis of other

neuropeptides by membrane peptidases.



The Enzymatic Barrier For Peptide and Protein Drug Delivery

Potential peptide and protein drugs are subject to degradation by numerous

enzymes or enzyme systems throughout the body. This degradation can come in two

forms: 1) hydrolytic cleavage of peptide bonds by proteases, such as enkephalinases,

and 2) chemical modification of the peptides or proteins, such as oxidation and

phosphorylation. Hydrolysis is by far the more common. Therefore, a major

challenge in peptide and protein drug delivery is to overcome the enzymatic barrier

that limits the amount of peptide and protein drugs from reaching their targets.

Degradation usually begins at the site of administration and can be extensive. Even

when the subcutaneous or intramuscular route is used, less than complete

bioavailability is often observed. For instance, the subcutaneous or intramuscular

bioavailability of TRH, a tripeptide, in mice is only 67.5 % and 31.1%, respectively











(Redding & Schally, 1972). The enzymatic barrier has three essential features, as

follows.

First, since proteases and other proteolytic enzymes are ubiquitous, peptides

and proteins are usually susceptible to degradation in multiple sites, including the

site of administration, blood, liver, kidney and vascular endothelia, etc.

Consequently, peptides and proteins must be protected against degradation in more

than one anatomical site for them to reach their target sites intact.

Second, almost all the peptidases and proteases capable of degrading a given

peptide or protein are likely to be present in a given anatomical site where the

peptide or protein is located (Palmieri & Ward, 1983; Ward, 1984; Palmieri et al.,

1985). The implication is that protecting a peptide or protein from degradation by

one protease/peptidase may not necessarily lead to marked increase in its stability or

in the amount of peptide/protein reaching its site of action (Dodda Kashi & Lee,

1986).

Third, a given peptide or protein is usually susceptible to degradation at more

than one linkage within the molecular backbone; each locus of hydrolysis is

mediated by a certain peptidase/protease. Often, even when one linkage is modified

to circumvent one peptidase/protease, the rest of the peptide molecule is still

vulnerable to other peptidases/proteases. This usually manifests itself as a shift in the

relative proportion of the various degradation products of a given peptide.











Proteases and peptidases are essentially hydrolyses; hence they have the

ability to cleave peptide bonds with the addition of water. Proteases/peptidases

rarely show absolute specificity in their action, hence any protease has the potential

to hydrolyze more than one substrate (Given et al., 1985). The problem is especially

true in the case of kyotorphin. Since kyotorphin is only a dipeptide, it lacks any

stereo hindrance which may slow down an unspecific peptidase/protease.

Clearly, in order to promote the delivery of peptides and proteins from any

route of administration, the many components of the enzymatic barrier must be

controlled. This can be achieved to some extent by modifying the peptide or protein

structure, through co-administration of protease inhibitors, or by using the

formulation approach.



The Blood-Brain Barrier (BBB) and Its Selective Permeability Properties

The Blood-Brain Barrier (BBB)

The BBB is a membranous barrier that is highly resistant to solute free

diffusion and which segregates brain interstitial fluid from the circulating blood. The

BBB is comprised of two plasma membranes in series, which are the lumenal and

antilumenal membranes of the brain capillary endothelium that are separated by

about 0.3 pm of endothelial cytosol.

There are several ultrastructural differences between systemic capillaries and

cerebral capillaries which explain the difference in their permeabilities. The main











difference is in the manner how endothelial cells in cerebral capillaries are joined.

Cerebral junctions are characterized as tight or closed junctions, which grid the cell

circumferentially, forming zona occuludens and acting like a zipper which closes the

interendothelial pores that normally exist in microvascular endothelial barrier in

peripheral tissue (Rapoport, 1976). This unique architecture prevents the bulk

movement of materials between cells and forces compounds to diffuse directly

through the phospholipid cell membrane if they are to gain access to the brain

parenchyma. A second main difference between cerebral and systemic capillaries is

the paucity of vesicles and vesicular transport in the CNS (Brightman, 1977).

Vesicular transport is a process for transcellular transport. Vesicles are transported

from the luminal to the abluminal membrane. Pinocytotic activity, on the other

hand, is concerned with the nutritional requirements of the cell and, as such,

involves vesicular movement from the luminal membranes to a cell organelle,

presumably by a lysosome. Cerebral endothelial vesicles are usually uncoated and

few in number compared to other systems. This lower vesicle content is another

mechanism by which the CNS can limit nonspecific influx and have clearly evolved

to protect the delicate environment necessary for optimal neural functioning. A

third difference is the lack of fenestrae in the cerebral capillaries.

In addition to these structural features, the BBB maintains a number of

enzymes which appear to augment its barrier function (Levin, 1977). 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), gamma-aminobutyric acid transaminase (GABA-T),

and aromatic amino acid decarboxylase (DOPA decarboxylase) in the BBB. The

presence of DOPA decarboxylase explains partially the need for giving such large

doses of L-dihydroxyphenylalanine (L-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 some lipophilic compounds which otherwise

might passively diffuse through the barrier.



The Selective Permeability Properties of the BBB

Lipid soluble drugs with a molecular weight of less than 600 readily diffuse

through the BBB via lipid mediation based on the high lipid solubility of the drug

(Oldendorf, 1974). The prototype example of increasing lipid solubility of a drug

compound by masking polar functional groups is the morphine-heroin model. The

morphine structure was shown in Figure 1. The relatively low lipophilicity of

morphine is attributed to the two hydroxyl groups on the ring nucleus. Methylation

of one of these hydroxyl groups results in the conversion of morphine to codeine,

and codeine is transported across the BBB approximately tenfold faster than is

morphine. Acetylation of both of the morphine hydroxy groups results in the











conversion of morphine to heroin, and heroin is transported through the BBB

approximately 100-fold faster than is morphine (Oldendorf et al., 1972).

Some drugs and peptides are bound by plasma proteins. For example, some

acidic drugs and peptides are tightly bound to albumin, some lipophilic amine drugs

are bound by both albumin and az-acid glycoprotein, and some lipid-soluble

peptides such as cyclosporin are bound by lipoproteins. These plasma protein-bound

drugs may be transported across the BBB by plasma protein-mediated transport. This

process arises from enhanced dissociation reactions that occur within the lumen of

the capillary and which are catalyzed by transient interactions between the plasma

protein and the glycocalyx surface of the brain capillary endothelium (Pardridge,

1987).

Most nutrients in the circulation are water-soluble compounds that would not

traverse the BBB in the absence of special carrier-mediated transport systems that are

embedded within both the lumenal and antilumenal membranes of the BBB. These

carriers and some representative nutrients they transport are listed in Table 4. The

substrates listed are not the only substrates those carriers transport.

Insulin can cross the BBB via receptor-mediated peptide transcytosis which

involves three sequential steps: 1) receptor-mediated endocytosis at the lumenal or

blood side of the BBB, 2) movement of the ligand-receptor complex through the

endothelial cytoplasm, and 3) receptor-mediated exocytosis of the ligand into the

brain interstitial fluid at the antilumenal or brain side of the BBB.














Table 4. Blood-Brain Barrier Transport Systems and Their Substrates

Carrier Representative Substrates

Hexose Carrier Glucose, Mannose, 2-deoxyglucose

Monocarboxylic Acid Carrier Lactic acid

Amine Carrier Choline

Neutral Amino Acid Carrier Neutral amino acids and their analogs
e.g. phenylalanine, L-dopa, a-methyl-dopa
Acidic Amino Acid Carrier Glutamate

Basic Amino Acid Carrier Arginine, Lysine

Nucleoside Carrier Adenosine, guanosine, uridine

Purine Base Carrier Adenine

Thyroid Hormone Carrier T3, T4

Thiamin Carrier Thiamin


In contrast to carrier-mediated transport of small nutrients, which takes place

over a time period of milliseconds to seconds, receptor-mediated transcytosis takes

place over a time period of minutes to hours.

In summary, the BBB consists of a relatively impermeable membrane super-

imposed on which are mechanisms for allowing the entrance of essential nutrients

and the exit of metabolic waste. The permeability of the BBB to molecules in the











general circulation is very selective, which acts to protect the CNS from periodic

peripheral changes that might disturb neurofunction.



The Blood-Brain Barrier (BBB) For Peptide and Protein Drug Delivery

The BBB is the main obstacle for the development of centrally active

peptides. Since only those agents with sufficient affinity with the lipid membrane

will penetrate the BBB, hydrophilic molecules (including peptides) are excluded

(Levin, 1980). The BBB is also distinct from the peripheral capillary system in that

high concentrations of various lytic enzymes are present, including some highly

active neuropeptide degrading enzymes, such as enkephalinase, aminopeptidase,

endopeptidase. This enzymatic barrier also prevents the uptake of blood-borne

neurotransmitters and neuromodulators (Levin, 1977).

While the BBB acts to protect the CNS from periodic peripheral changes that

might disturb neurofunction, it also restricts the movement of many potentially

important drugs or hormones, which limits the treatment of cerebral diseases.

Varieties of amino acids and peptides have been considered for therapeutic use, such

as GABA (aminobutyric acid), DOPA (dihydroxyphenylalnine), enkephalin, KTP,

TRH, etc. Unfortunately, the BBB prevents significant uptake of these amino

acids/peptides. Therefore, a general method for improving the delivery of amino

acids and peptides into the CNS would be desirable.











The overall membrane transport properties are very important for a drug, as

these are governing its absorption, distribution, and elimination of its intact form, as

well as affecting its binding, affinity, and other important characteristics. One

approach for improving brain uptake is via prodrugs.

A prodrug is a pharmacologically inactive compound that results from

transient chemical modification of a biologically active specie. When the BBB is

considered, increased drug penetration is usually well correlated with the

lipophilicity (Levin, 1980). In order to improve the entry of a hydroxy-, amino-, or

carboxylic acid-containing drug, esterification or amidation may be performed. This

greatly enhances the lipophilicity of the drug. As a result, the drug can better enter

the brain parenchyma. Once inside the CNS, hydrolysis of the lipophilic modifying

group will release the active compound.

Unfortunately, simple prodrugs suffer from several important limitations.

While increasing the lipophilicity of a molecule may increase its chance of crossing

the BBB, the uptake of the compound into the other tissue is likewise augmented.

This nonselectivity of delivery is detrimental when kyotorphin is considered.

Kyotorphin is an enkephalin releasing hormone. Enkephalin receptors are

widespread in the human gut, including distal small intestine, colon, cystic duct, gall

bladder, pancreas, and esophagus; and are particularly high in pyloric antral mucosa

and duodenal G-cells (Uddman et al., 1980). Therefore, the nontarget site toxicity

might be exacerbated. In addition, while drug uptake into the CNS is increased, its












efflux is also enhanced. This results in poor tissue retention of the drugs and short

biological action. Finally, while the only metabolism associated with prodrugs

should be by conversion to the parent drug, other routes can occur that may

contribute to the toxicity. These effects, poor selectivity, poor retention, and the

possibility of inactive catabolism, often conspire to decrease, not to increase, the

therapeutic index of a drug when masked as a prodrug. Therefore, it is very

important to find methods which enable delivery of drugs specifically to a particular

organ, or site. This requires more than simply optimizing overall membrane

transport characteristics. Among the various possible ways to achieve site specific or

organ specific delivery, the "chemical delivery system" (CDS) designed by Dr.

Bodor is the most flexible and offers possibilities for specific delivery not only to the

brain, skin, eye, but to other organs and sites. Properly designed, a CDS should

concentrate the desired active agent at its site of action and reduce its concentration

in other locations. The main result of this manipulation is not only an increase in the

efficacy of the drug entity, but also a decrease in its toxicity.



The Chemical Delivery System (CDS)

As mentioned above, although the acquired lipophilicity in a prodrug

approach may be enough to assure the drug of penetrating the BBB into the CNS by

passive transport, it does not ensure the drug to stay inside the CNS. Moreover, a

simple prodrug approach is prone to the enzymatic barrier. In a perfect approach,











the target drug should acquire enough lipophilicity to penetrate the BBB; and once it

is inside the CNS, it should stay inside the CNS. It should also disguise the target

peptide/protein enough to confuse the various enzymes during the passage of the

substances from the general circulation to the brain.

The CDS is defined as a biologically inert molecule requiring several steps

for conversion to the active drug, thereby enhancing drug delivery to the target site

(Bodor, 1987).

There are three criteria for a brain-targeting CDS. First, it should be

lipophilic enough to allow for brain uptake. Second, after the brain penetration,

retention of the lipophilic molecule is required to prevent its efflux from the CNS.

Lastly, the conversion intermediate should be degraded enzymatically according to

the designed route to release the active molecule over a long period. Since both the

BBB and enzymatic degradation prevent the passage of the peptides from the general

circulation to the brain tissue, the CDS, an enzyme-based strategy, is an excellent

candidate for peptide brain-delivery.

Since it was first proposed by Dr. Bodor in 1978, the brain-targeting CDS

has been extensively applied to various neurotransmitters and other pharmaceuticals.

Among those, peptides are included as a major consideration.














The Principles of the Redox-Based Chemical Delivery System.

The principle of the CDS that permits enhanced and sustained delivery of

drugs to the brain is schematically shown in Figure 2.

The CDS employs interconversion of a lipophilic dihydropyridine moiety to a

hydrophilic pyridium salt moiety that is an analog to the NADH < = > NAD co-

enzyme system. This lipophilic dihydropyridine carrier is covalently linked to an

active drug to form the Drug-CDS that is able to penetrate the BBB. Upon systemic

administration, the CDS can partition into several body compartments due to its

enhanced lipophilicity; some of those (i.e., the CNS) are inaccessible to the

unmanipulated compound. At this point, the CDS is simply working as a lipidal

prodrug. The carrier molecule is specially designed, however, to undergo an

enzymatically mediated oxidation that converts the membrane-permeable

dihydrotrigonellinate (the lipophilic dihydropyridine moiety) to a hydrophilic,

membrane-impermeable, trigonellinate salt (the hydrophilic pyridium salt moiety) in

vivo. The polar carrier-drug conjugate is then trapped behind the BBB. Any of this

oxidized salt that is present in the periphery will be rapidly lost as it is now an

excellent candidate for elimination by kidney and liver. The conjugate that is trapped

behind the BBB then slowly hydrolyzes to release the active drug in a sustained

manner.






































0 + K1 Rapid
SCH3 Elimination
S0O-Dumg

S Trapped /\
N






|CH3
.7N- I7
I + Receptor
--L K3





Figure 2. The Mechanism of the Redox-Based CDS for Enhanced
and Sustained Delivery of Drugs to the Brain











By the system design, concentration of the active drug is low in the

periphery, which minimizes dose-related side effects and toxicity. In addition, the

active drug in the CNS is present mostly as an inactive conjugate, which offers two

advantages -- lower central toxicity and increased dose interval.



Delivery of Peptides into the CNS via the CDS

For many smaller drugs, a simple redox targetor has proved to be applicable

(Bodor & Brewster, 1983; Bodor & Simpkins, 1983). For peptides, however, the

attachment of 1,4-dihydrotrigonelline to the NH2-terminus alone will not furnish

sufficient increase in lipophilicity and will only protect peptides against

aminopeptidases. The unmodified COOH-terminal of the molecule will decrease the

lipophilicity as well as be susceptible to cleavage by numerous exo- and endo-

peptidases. In the strategy called "molecular packaging," the peptide unit of the

CDS appears only as a perturbation on the bulky molecule dominated by lipophilic

modifying groups that assure the BBB penetration and irrecognition by peptidases.

A chemical delivery system for peptide brain-delivery has been proposed

based on all the considerations, as shown in Figure 3. A centrally active peptide

sequence (P) is placed in a molecular package that disguises its peptide nature and

provides biolabile, lipophilic functions to penetrate the BBB by passive transport.

The design incorporates an 1,4-dihydrotrigonellinate targetor (T) at the NH2-

terminal of the peptide via a spacer (S) and a cholesterol ester at the C-terminal.











(L)






(T) 0 (p)

S)- Target Peptide-O

N
I


Figure 3. The Chemical Delivery System for Peptides



Because of the low amidase activity of the brain tissue, a spacer (S) is used to

separate the peptide sequence (P) from the targetor part of the CDS. The spacer is

selected based on the peptidolytic activity prevalent at the site of action, so that the

release of the desired peptide is favored over the degradation induced by other

peptidases. The brief description of its metabolism was shown in Figure 4.

After the molecular package penetrates the BBB and enters the CNS, the 1,4

dihydrotrigonellinate targetor (T) undergoes an enzymatically-mediated oxidation to

become a hydrophilic, membrane impermeable trigonellinate salt (T+) which traps

the whole molecule (II) behind the BBB and inside the CNS. Hydrolysis of this

molecule provides a polar targetor-peptide conjugate (III) which is a substrate for

peptide degrading enzymes, such as dipeptidyl peptidases and post proline cleaving

enzymes.





















































'U
rj2
'U
0
rt


-

'U

0



-U


I I .


U

Cd




























0


iiz


0




'4-








+











Through the sequential enzymatic degrading, the final biologically active peptide is

released in a pharmacologically significant amount inside the brain. The spacer is

used to ensure a precise cleavage between the peptide and the targetor, not among

the amino acid residues of the parent peptide.



The Chemical Delivery Systems for Kyotorphin and a Kyotorphin Analog

Basic amino acids, such as arginine and lysine, have a basic side-chain amine

group which is positively charged at pH 7.4 -- the pH in vivo. The side-chain group

make basic amino acids very hydrophilic and therefore it is more difficult for them

to penetrate the BBB than other amino acids. Since kyotorphin also has a very short

half-life in plasma (Ueda et al., 1985; Akasaki & Tsuji, 1991), it is very

challenging to try to deliver kyotorphin or its analog into the brain.

A possible approach is the derivatization of the peptide to produce a transport

form that is markedly more lipophilic than the parent peptide as well as resistant

towards the various peptidases. Yet, it must remain cleavable by enzyme-catalyzed

hydrolysis at a prescribed joint to have the sustained brain-specific release of the

parent peptide in situ.

The efficacy of this kind of peptide delivery system depends not only on the

effective lipophilicity of the molecular package, but also on minimal exposure of the

target peptide to vascular peptidases. Therefore, the CDS approach seems to be an

excellent candidate for brain-targeted delivery of kyotorphin.















The structures of KTP (kyotorphin) and KAYK (Kyotorphin Analog -


Tyrosyl-Lysine) and their basic CDS's are shown in Figure 5.


0
NOH NH

H( HNH NH2


H2

NH NOH
0 ,NH2


OH


Kyotorphin (L-Tyr-L-Aig)




OH




CH3 -N



1,4-Dihydrotrigone lyl-Prc




OH




CN H
CH3 -N
< 0 H


L-Tyr-L-Lys (a Kyotophin analog)


-Tyr-Arg-Cholesteryl Ester


0 NH

N0 H
*^--C**sNH2


1,4-Dihydrotrigonellyl-Pro-Tyr-Lys-Cholesteryl Ester


Figure 5. The Structure of KTP, KAYK, and Their Basic CDS's











The proline is used as the spacer that may be replaced by alanine. Double

amino acids may also be used as the spacer.

Since the side-chain NH2 groups of arginine and lysine are very basic (pKa

> 10), they would exist mostly positively charged in vivo (pH 7.4), which

would decrease the lipophilicity of the whole molecular package. Therefore,

additional protection should be considered to ensure the desired high-lipophilic

nature of the CDS. Such a CDS for KAYK (Tyr-Lys) with a proline as the spacer is

shown in Figure 6.







0
0







Figure 6. The Structure of CDS-P--a CDS for KAYK
1,4-Dihydrotrigonellyl-Pro-Tyr-Lys(Boc)-Cholesteryl Ester



The side chain NH2 group of lysine offers another way to deliver KAYK into

the CNS which is unsuitable for Tyr-Arg, as shown in Figure 7.




















0 NQ

CONH2

Figure 7. The Structure of Boc-Tyr-Nys-Cholesteryl Ester (BTRA)



In this molecular package, the lysine moiety of KAYK is replaced by Nys --

the redox analog of the natural diamino acid lysine. The replacement is applicable

theoretically due to the isoelectronic/isosteric effects of Tyr-Lys and Tyr-Nys in

vivo, as shown in Figure 8. The whole molecular package is called KAYK brain-

targeted redox analog (BTRA).

Compared to arginine, lysine is less basic and is easier to convert to potential

bioreversible lipophilic derivatives, such as CDS and BTRA.

When KTP-CDS/KAYK-CDS/BTRA is administered systemically, as

discussed in the scheme for the general CDS (Fig. 2), KTP/KAYK-CDS/BTRA can

partition into several body compartments; some of those are inaccessible to the

unaltered KTP/KAYK. The design of CDS/BTRA allows for an enzymatically














TyrNH yN NH 0 NH
HO) sNH2 HO N
O 0

Tyr-Lys Tyr-Nys








TyryrNHO 0
NHN Tyr
HONl3 HO
0 +

Tyr-Lys+ Tyr-Nys+



Figure 8. The Isoelectronic/isosteric Effects of Tyr-Lys and Tyr-Nys in vivo



mediated oxidation that converts the membrane-permeable dihydrotrigonellinate to a

membrane-impermeable hydrophilic trigonellinate salt, a reaction that occurs

throughout the organism. The polar salt is then trapped behind the BBB and is held

within the CNS. The peripherally distributed polar salt is rapidly eliminated via

kidney and liver. For example, in the case of CDS-P, the polar CNS trapped drug --

Trigonellyl-L-Pro-L-Tyr-L-Lys(Boc)-Cholesteryl Ester will be slowly hydrolyzed to








53


release Tyrosyl-lysine inside the CNS over a sustained time. Since the peripheral

concentration of Trigonellyl-L-Pro-L-Tyr-L-Lys(Boc)-Cholesteryl Ester is very low,

the systemic toxicity is minimized. The sequential metabolism of CDS-P is shown

in Figure 9, and those of CDS-PA, CDS-PP, and BTRA are shown in Figure 10-12.

















c00
0
0
o o* z a 6

o o rd
00


H W
00
00





+ o<
+ 0
0 0
o6
0 0
0=







0 a
zS





0 o











00 o = Z







/\O
5= UD( 5-0

00

00
00



0 =0

0 H



















\ I' E






oI




0 -
E 00








"a I













o 8 o
a- 6
cF-







0
0/
+cu

















el ,



0





0
A 0 0-,



0


U, CU


H Y3 00
0 0 I c
ci ^ ^t o^J^0
^ .M o=<


J 1 /^"^CU
G) -/~**0


























0 0



+0







00
o
0 00


CA

Cu-~
-+U











-0I



S=0 ClI-HC















CHAPTER 2
MATERIALS AND METHODS


Materials

All chemicals used were reagent grade or peptide synthesis grade. All

solvents used were A.C.S. Reagent Grade. Amino acid derivatives were L-

configured and purchased from BaChem Inc., Torrance, CA. All solvents were

purchased from Fisher Scientific.

Melting points were taken on a Fisher-Jones melting point apparatus and are

uncorrected. All synthesized products were characterized by FAB (fast atom

bombardment) mass spectroscopy by using a Kratos MS80RFA Mass Spectrometer,

Manchester, U. K. TLC (thin layer chromatography) determinations were carried

out on silica-gel 60 coated foil -- Merck DC-Alufolien Kiesegel (silica gel) 60 F254.

Elemental analyses of compounds synthesized were performed by Atlantic

Microlab, Inc., Atlanta, GA.

Regular column chromatography was performed using silica gel (100-200

mesh) and appropriate mobile phase; flash column chromatography was performed

using silica gel (200-400 mesh) and appropriate mobile phase under the pressure of

10 psi.











Synthetic Protocol for the CDS's (Chemical Delivery Systems) of Kyotorphin

Both Boc and Fmoc methods were used to synthesize the CDS's for the

brain-targeted delivery of the kyotorphin. There were six CDS's projected: 1) CDS

with one alanine as the spacer (CDS-KTP-A, Fig. 13), 2) CDS with double alanine

(Ala-Ala) as the spacer (CDS-KTP-AA); 3) CDS with Pro-Ala as the spacer (CDS-

KTP-PA); 4) CDS with one proline as the spacer (CDS-KTP-P); 5) CDS with

double prolines (Pro-Pro) as the spacer (CDS-KTP-PP); and 6) CDS with Ala-Pro

as the spacer (CDS-KTP-AP).


Figure 13.


CDS-KTP-A


The synthetic scheme of KTP-CDS-A by using the Boc method is shown in

Figure 14.

















S0NOH N-NO2
of NH-

Boc-Arg(NO2)-OH

1. Cholestrol I 2. TFA
DCC, DMAP






NH
Arg(NO2)-Cholesteryl Ester

1. Boc-Tyr-OH 2. TFA
HOBt, DCC


O

CN'

Nicotinic Acid Ala-OtBu Ester

1. HOBt, DCC 2. TFA



: M -O O H

Nicotinyl-Ala-OH






CHH
0 0

1, o NHlyNH


1,4-Dihydrotrigonellyl-Ala-Tyr-Arg-Cholesteryl Ester


Tyr-Arg(NO2)-Cholesteryl Ester


1. Side-chain
Deprotection


2. Na2S204


Me2SO4


NH-


Nicotinyl-Ala-Tyr-Arg(NO2)-Cholesteryl Ester


Trigonellyl-Ala-Tyr-Arg(N2)-Cholesteryl Ester


Figure 14. The Synthetic Scheme for KTP-CDS-A
by Using the Boc Method


+ H2tO
0











Ala-OtBu ester was replaced by Pro-OtBu ester for the synthesis of

Nicotinyl-Pro-OH in the synthesis of 1,4-Dihydrotrigonellyl-Pro-Tyr-Arg-

Cholesteryl Ester (KTP-CDS-P). An extra amino-acid residue (alanine or proline)

was added at the N-terminal of NH2-Tyr-Arg(N02)-Cholesteryl Ester and then

coupled with Nicotinyl-Ala-OH or Nicotinyl-Pro-OH during the synthesis to

generate the other four KTP-CDS's.

The synthetic scheme of the KTP-CDS-A by using the Fmoc method is

shown in Figure 15. Ala-OtBu ester was replaced by Pro-OtBu ester for the

synthesis of Nicotinyl-Pro-OH in the synthesis of 1,4-Dihydrotrigonellyl-Pro-Tyr-

Arg-Cholesteryl Ester (KTP-CDS-P). An extra amino-acid residue (alanine or

proline) was added after the finish of NH2-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester

during the synthesis to generate the other four KTP-CDS's.



Synthetic Protocol for CDS's (Chemical Delivery Systems) and
BTRA (Brain Targeted Redox Analog) of Tyr-Lys

The Fmoc method was used to synthesize the brain-targeted delivery systems

of Tyr-Lys. There are four brain-targeted delivery systems to be synthesized: 1)

CDS with one proline as the spacer (CDS-P), 2) CDS with double prolines as the

spacer (CDS-PP), 3) CDS with Pro-Ala as the spacer (CDS-PA), and 4) BTRA.

Their structures are shown in Figure 16. The synthetic scheme of CDS-P was shown

in Figure 17.

























o-. )-o N S 0-
NH NH0 O 0
Fmoc-Arg(Pmc)-OH

1. Cholesterol 2. Piperidine
DCC, DMAP





O N-Pbc

NH2
Arg(Pmc)-Cholesteryl Ester

2. Piperidine 1. Fmoc-Tyr(OtBu)-OH
HOBt, DCC


Q)OH + H2ON-.O+


Nicotinic Acid Ala

1. HOBt, DCC 2. TFA


OtBuEster


0

Nitiyl-la-OH


Nicotinyl-Ala-OH


1,4-Dihydrotrigonellyl-A-TyArg-Cholesteryl Ester


Tyr(OtBu)-Arg(Pme)-Cholestcryl Ester


Me2SO4


Nicotinyl-Ala-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester


Trigonellyl-Ala-Tyr(OtBu)-Arg(Pm)-Cholesteryl Ester


Figure 15. The Synthetic Scheme for KTP-CDS-A
by Using the Fmoc Method






















1,4-Dihydrotrigonellyl-Pro-Tyr-Lys(Boc)-Cholesteryl Ester -- CDS-P


1,4-Dihydrotrigonellyl-Pro-Pro-Tyr-Lys(Boc)-Cholesteryl Ester -- CDS-PP


HO




H3C y N O -- NH



1,4-Dihydrotrigonellyl-Pro-Ala-Tyr-Lys(Boc)-Cholesteryl Ester -- CDS-PA


Boc-Tyr-Nys-Cholesteryl Ester -- BTRA

Figure 16. The Structures of KAYK-CDS's & BTRA


or
0











































1 <
t s
B N

A a
SQ d
0 ^S
En

U2)


\


0
0 n
0 o


v I-



+



U


0
a=


r
U-


40 Q

t&

F -
0



..




H
E5
WU


















04



w&-A
8 jo '

zz-



0
oo




oo
t ^ t



0

1-.
s> a y= / \
I~4 5b )O

44





/ 4)
6
8S




o Uo
4)w
\) c
4) 0
'5" ^ 3 5
c ^0
_ _\_ 0
^ o^0F-
0 6 U

F- F I ~


A'











An extra amino-acid residue (alanine or proline) was added at the N-

terminal of Tyr- Lys(Boc)-Cholesteryl Ester and then coupled with Nicotinyl-Pro-

OH during the synthesis to generate CDS-PA and CDS-PP -- the other two CDS's

of the Lys-Tyr. The synthetic scheme of BTRA is shown in Figure 18.



Typical Experimental Procedures for Chemical Synthesis

To prepare cholesterol esters of Arg(N02/Pmc) and Lys(Boc/Fmoc), DCC

(dicyclohexylcarbodiimide) was used as the dehydrating agent and DMAP

(dimethylaminopyridine) was used as the catalyst. Throughout the synthesis, all

amide bonds were coupled by using the DCC/HOBt method. The Fmoc and Boc

groups were removed by Piperidine/CH2Cl2 (1:3) and TFA/CH2Cl2 (1:1),

respectively. The Pmc and O-tBu groups were removed by TFA/H20 (19:1). The

N-alkylation in preparation of the trigonellyl compounds was performed by using

dimethyl sulfate (Me2SO4) in ethyl ether or methalene chloride. Nys+ was formed

by Zincke reaction (Fig. 19). The reduction of the quaternary salts to the

corresponding 1,4-dihydrotrigonellinate derivative was carried out by using sodium

dithionite (Na2S204) as the reducing agent in a mixture of methanol and deaerated

aqueous sodium bicarbonate (NaHCO3).

In all cases, the intermediates synthesized were purified by silica gel

chromatography, except for those otherwise noted. In order to avoid side-reactions,

all the coupling reactions were performed at an initial temperature of 0C and then






















4 6



U +


0
r, .gO^;00
+ Qg

o=^

0=p
0 W
0
2 U
Ss O

a
S I &
< 1-U


0


0
8
-- o i

V-f ^8
I -- O


\


I
c'~I


I-.
0
(12
w
0
112
0
U

2
IL.



















S
'a
0
I;-)
'U
"a

0
U


a
z
9
Cl


-


66
Nu
2En
z z
















+
0=I


C











at room temperature. The N-methylation was performed at room temperature with

an excess of the alkylating agent. The reduction of the quaternary salts with sodium

dithionite was accomplished at 0C in an oxygen-free environment.

A liquid-phase peptide synthesis method has been applied for the syntheses of

all KAYK-CDS's and BTRA. The liquid-phase method has been selected because it

is most suitable for the synthesis of peptides of less than 10 amino-acid residues or

of peptides with the carboxyl terminal which is not amenable to solid-phase

techniques (such as a labile cholesterol ester). The other advantages of the liquid-

phase technique are that it is very easy to scale-up to multi-gram scale and much less

expensive compared to solid-phase technique.



Pharmacology Studies

The Animals

Male Sprague-Dawley rats weighing 250-300 g were purchased from Harlan

Sprague Dawley Inc. (Indianapolis, Indiana, USA) and used in all the experiments.

Animals were housed one per cage in room temperature (- 25C) on a 14 hour

light cycle. Purina lab Chow and water were provided ad libitum.

All the animal studies were conducted in accordance with the guidelines set

forth in the Declaration of Helsinki and the Guiding Principles in the Care and Use

of Animals (DHEW Publication, NIH-80-23).











The Testing Method

Drugs were dissolved in vehicle (PG/DMSO, 2:1) and were injected into the

animals through the tail vein. When administered icv., the minimum dose of

kyotorphin which can produce the analgesic effect in rat is about 1 mg/kg (Rolka et

al., 1983). Accordingly, CDS-P at doses of 0.0030, 0.0075, 0.0148, 0.0223

mmol/kg (equimolar to 1.0, 2.5, 5.0, and 7.5 mg/kg of kyotorphin) were

administered to monitor the dose-response. Vehicle and 0.0223 mmol/kg of drugs

(equimolar to 7.5 mg/kg of kyotorphin) were also administered to study the

pharmacological activities of the other KAYK brain-targeted delivery systems and

their important intermediates.

Tail-flick latency, an index of spinal cord mediated analgesia (D' Amour &

Smith, 1941), was measured to evaluate the analgesic effects of the brain-targeted

delivery systems for KAYK. Time between presentation of a focused beam of light

and the reflexive removal of the tail from the stimulus was defined as the tail-flick

latency period (7), and the tail-flick latency difference between each time point and

control was defined as the change in tail flick latency. In the absence of response, a

cut-off period of 1 min was used. Each drug at each dose was tested on six animals.

Control latency (To) was obtained 10 minutes prior to the drug

administration; the test latencies (T15, T30, T60, T120, T180, T240, T300, T360 ) were

measured at 15 min, 30 min, 1 hr, 2, hr, 3 hr, 4 hr, 5 hr, and 6 hr after the

administration of drugs. The instrument used was a Model 33 Tail Flick Analgesia












Meter (Litc, Inc., Landing, N. J.). The beam dial was set at 90 and the sensitivity

was at eight.



The Effect of Naloxone on CDS-PP and BTRA Induced Analgesia

To test the effect of naloxone on the KAYK brain-targeted delivery systems,

rats were treated with the CDS-PP and BTRA (0.0223 mmol/kg) as described

previously; and the tail-flick latencies were recorded at 15 and 30 minutes after the

administration of the drugs. Subsequently, naloxone hydrochloride, 2 mg/kg,

dissolved in 0.9% saline, was injected subcutaneously, and the tail-flick latency

periods were recorded at 45, 60, 90, and 120 minutes (15, 30, 60, and 90 minutes

after the administration of naloxone).



Statistical Analysis

Anova was used to process all the data generated from the pharmacological

experiments.

Generally, analysis of variance, or anova, is a statistical procedure used to

determine whether means from two or more samples are drawn from populations

with the same mean. Anova is one of the most powerful and useful statistical

procedure utilized in the analysis of research data. The analysis of variance allows

us to make what is known as a test of significance between or among means, the

results being given on the basis of the probability. The test of significance is cast in












the form of accepting or rejecting what is termed a null hypothesis, the hypothesis of

no difference between or among means. If the null hypothesis were rejected, we are

guided by the fact that the probability of finding a difference as large as or larger

than that obtained in the experiment is quite small (p<0.05 or p<0.01), and we

concluded that there is a significant difference between the treatment means. If the

null hypothesis is rejected at the 5 percent level (p<0.05), we state that there is a

"significant" difference between two means. If the null hypothesis is rejected at the

1 percent level (p<0.01), we state that there is a "highly significant" difference

between two means (Damon & Harvey, 1987).

Single-Factor Anova performs simple analysis of variance, which tests the

hypothesis that means from several samples are equal. Two-Factor with Replication

Anova performs an extension of the single-factor anova that includes more than one

sample for each group of data. In all the anova performed in this study, the a value

was set at 0.05.

Single-Factor Anova was used to compare the maximum analgesic effects

(the mean of maximum response) induced by KAYK brain-targeted delivery systems

with those of vehicle, kyotorphin, intermediates of CDS-PP and BTRA and among

KAYK brain-targeted delivery systems themselves.

Two-Factor with Replication Anova was used to compare the analgesic

effects (the mean of maximum response) induced by KAYK brain-targeted delivery








74


systems across the testing period with those of vehicle, kyotorphin, intermediates of

CDS-PP and BTRA and among KAYK brain-targeted delivery systems themselves.

The variances of the data were measured by standard deviation.














CHAPTER 3
CHEMICAL SYNTHESIS


Synthesis of Kyotorphin-CDS's (the Boc- Method)



Synthesis of Arg(NO)-Cholesteryl Ester

N-a-Boc-Arginine(N02)-OH (3.72 g, 10.0 mmol) in 75 ml CH2C12/DMF

(9:1) was stirred at 0C and cholesterol (3.88 g, 10.0 mmol) dissolved in 75 ml

CH2C12 was added, which was followed immediately by DCC (2.44 g, 11.0 mmol)

dissolved in 15 ml CH2C12 and DMAP (1.68 g, 14.0 mmol) dissolved in 25 ml

CH2C12. The mixture was stirred for 48 hours at room temperature. The DCU

formed was filtered off and the solvent was removed in vacuo. The crude material

was dissolved in CHC13 and washed successively with 10% citric acid solution

(3x100 ml), 5% NaHCO3 solution (3x100 ml), and saturated NaCl solution (100

ml). The organic phase was dried over anhydrous Na2SO4 and evaporated to give a

solid -- Boc-Arg(N02)-Cholesteryl Ester. The product was dissolved in 60 ml

CH2Cl2/TFA (1:1) and stirred at room temperature for half an hour. Then the

solvent was removed in vacuo to minimum and precipitated from ethyl ether to give

a white solid -- Arg(N02)-Cholesteryl Ester (3.62 g, 51.81%). TLC Rf=0.35,

CH30H/CH2CI2 (1:9). Mass spectrum: m/z=611 (M+Na) .











Synthesis of Tyr-Arg(NO,)-Cholesteryl Ester

Arg(N02)-Cholesteryl ester (3.50 g, 5.0 mmol) in 35 ml CH2Cl2/DMF (9:1)

was stirred with Et3N (0.53 g, 5.0 mmol) at 0C. Boc-Tyr-OH (1.7 g, 5.0 mmol)

dissolved in 35 ml CH2Cl2/DMF (1:1) was added, which was followed immediately

by DCC (1.22 g, 5.5 mmol) dissolved in 15 ml CH2Cl2 and HOBt (0.91 g, 6.75

mmol) dissolved in 15 ml DMF. The mixture was stirred for 24 hours at room

temperature. The DCU formed was filtered off and the solvent was removed in

vacuo. The crude material was dissolved in CHC13 and washed successively with

10% citric acid solution (3x100 ml), 5% NaHCO3 solution (3x100 ml), and

saturated NaCI solution (100 ml). The organic phase was dried over anhydrous

Na2SO4 and evaporated to give a solid -- N-a-Boc-Tyr-Lys(N02)-Cholesteryl Ester.

The peptide ester was deprotected with TFA/CH2Cl2 (1:1) in an identical manner

illustrated in the preceding step to give a white solid -- Tyr-Arg(N02)-Cholesterol

Ester (3.53 g, 81.1%). TLC Rf=0.56, CH3OH/CH2CI2 (1:8); Mass spectrum:

m/z=774, (M+Na)+.



Synthesis of Nicotinvl-Ala-OH

Ala-OtBu ester*HCI (1.82 g, 10.0 mmol) and Et3N (1.01 g, 10.0 mmol) in

25 ml CH2Cl2 were stirred at 0C. Nicotinic acid (1.85 g, 15.0 mmol) dissolved in

25 ml DMF was added, which was followed immediately by DCC (2.42 g, 11.0

mmol) dissolved in 15 ml CH2C12 and HOBt (1.82 g, 13.0 mmol) dissolved in 15











ml DMF. The mixture was stirred for 24 hours at room temperature. The DCU

formed was filtered off and the solvent was removed in vacuo. The crude material

was dissolved in CH2Cl2 and washed successively with 10% citric acid solution

(3x100 ml), 5% NaHCO3 solution (3x100 ml), and saturated NaCI solution (100

ml). The organic phase was dried over anhydrous Na2SO4 and evaporated to give a

solid -- Nicotinyl-Ala-OtBu. The product was dissolved in 50 ml 95 % TFA in H20

and stirred for one hour at room temperature. Then the majority of the solvent was

removed and the material was recrystallized from ethyl ether to give a white solid --

Nicotinyl-Ala-OH (1.56 g, 80.41%). TLC Rf=0.16, CH3OH/CH2Cl2 (1:9); Mass

spectrum: m/z=195 (M+H).



Synthesis of Nicotinyl-Ala-Tyr-Arg(NOI)-Cholesteryl Ester

Tyr-Arg(N02)-Cholesteryl Ester (1.5 g, 2.0 mmol) and Et3N (0.2 g 2.0

mmol) dissolved in 25 ml CH2Cl2/DMF (9:1) were stirred at 0C. Nicotinyl-Ala-

OH (0.39 g, 2.0 mmol) in 25 ml DMF/CH2Cl2 (1:1) was added, which was

followed immediately by DCC (0.44 g, 2.2 mmol) dissolved in 15 ml CH2C12 and

HOBt (0.38 g, 2.7 mmol) dissolved in 10 ml DMF. The mixture was stirred for 96

hours at room temperature. The DCU yielded was filtered off and the solvent was

removed in vacuo. The material obtained was dissolved in CH2Cl2 and washed

successively with 10% citric acid solution (3x100 ml), 5% NaHCO3 solution (3x100

ml), and saturated NaCl solution (100 ml). The organic phase was dried over











anhydrous Na2SO4 and the solvent was removed in vacuo. Silica-gel column

chromatography (5% CH3OH and 10% Et20 in CH2C2 ) afforded a white solid --

Nicotinyl-Ala-Tyr-Arg(N02)-Cholesteryl Ester (1.32 g, 67.28%). TLC Rf=0.37,

CH30H/CH2Cl2 (1:9); Mass spectrum: m/z=928 (M+H)+.



Synthesis of Trigonellyl-Ala-Tyr-Arg(NO,)-Cholesteryl Ester

Nicotinyl-Ala-Tyr-Arg(N02)-Cholesteryl Ester (0.49g, 5.0 mmol) in 50 ml

CH2Cl2 was stirred at 0C, dimethylsulfate (0.32 g, 25 mmol) was added. The

mixture was stirred for overnight at room temperature. The solvent was evaporated

to minimum and the product was precipitated from ethyl ether to afford a white solid

-- Trigonellyl-Ala-Tyr-Arg(N02)-Cholesteryl Ester (0.45 g, 91.84%). TLC Rf=0,

CH3OH/CH2Cl2 (1:9). Mass spectrum: m/z=942, M+.



Synthesis of Ala-Tyr-Arg(NO,)-Cholesteryl Ester

Tyr-Arg(N02)-Cholesteryl ester (4.33 g, 5.0 mmol) and Et3N (0.53 g, 5.0

mmol) in 35 ml CH2Cl2/DMF (9:1) were stirred at 0C. Boc-Ala-OH (0.67 g, 5.0

mmol) dissolved in 25 ml CH2C12 was added, which was followed immediately by

DCC (1.22 g, 5.5 mmol) dissolved in 15 ml CH2Cl2 and HOBt (0.91 g, 6.75

mmol) dissolved in 10 ml DMF. The mixture was stirred for 24 hours at room

temperature. The DCU yielded was filtered off and the solvent was removed in

vacuo. The crude material was dissolved in CHC13 and washed successively with











10% citric acid solution (3x100 ml), 5% NaHCO3 solution (3x100 ml), and

saturated NaCi solution (100 ml). The organic phase was dried over anhydrous

Na2SO4 and evaporated to give a solid -- N-a-Boc-Ala-Tyr-Arg(N02)-Cholesteryl

Ester. The peptide ester was deprotected with TFA in CH2CI2 (1:1) in an identical

manner illustrated in the previous step to give a white solid -- Ala-Tyr-Arg(NO2)-

Cholesterol Ester (3.69 g, 78.2%). TLC Rf=0.41, CH3OH/CH2CI2 (1:9). Mass

spectrum: m/z=845, (M+Na).



Synthesis of Nicotinyl-Ala-Ala-Tyr-Arg(NO)-Cholesteryl Ester

Ala-Tyr-Arg(N02)-Cholesteryl Ester (1.65 g, 2.0 mmol) and Et3N (0.2 g 2.0

mmol) dissolved in 25 ml CH2CI2/DMF (9:1) were stirred at 0C. Nicotinyl-Ala-

OH (0.39 g, 2.0 mmol) in 25 ml DMF/CH2CI2 (1:1) was added, which was

followed immediately by DCC (0.44 g, 2.2 mmol) dissolved in 15 ml CH2Cl2 and

HOBt (0.38 g, 2.7 mmol) dissolved in 10 ml DMF. The mixture was stirred for 96

hours at room temperature. The DCU yielded was filtered off and the solvent was

removed in vacuo. The material obtained was dissolved in CHC13 and washed

successively with 10% citric acid solution (3x100 ml), 5% NaHCO3 solution (3x100

ml), and saturated NaCl solution (100 ml). The organic phase was dried over

anhydrous Na2SO4 and the solvent was removed in vacuo. Silica-gel column

chromatography (5% CH30OH and 10% Et2O in CH2CI2) afforded a white solid --











Nicotinyl-Ala-Ala-Tyr-Arg(NO2)-Cholesteryl Ester (1.32 g, 67.28%). TLC

Rf=0.45, CH3OH/CH2Cl2 (1:9). Mass spectrum: m/z=999 (M+H) .



Synthesis of Kyotorphin-CDS's (the Fmoc Method)



Synthesis of Arg(Pmc)-Cholesteryl Ester

N-a-Fmoc-Arg(Pmc)-OH (6.60 g, 10.0 mmol) in 100 ml CH2C12 was stirred

at 0C and cholesterol (3.88 g, 10.0 mmol) dissolved in 75 ml CH2Cl2 was added,

which was followed immediately by DCC (2.44 g, 11.0 mmol) dissolved in 15 ml

CH2Cl2 and DMAP (1.68 g, 14.0 mmol) dissolved in 25 ml CH2CI2. The mixture

was stirred for 48 hours at room temperature. The DCU yielded was filtered off and

the solvent was removed in vacuo to give a solid -- N-a-Fmoc-Arg(Pmc)-Cholesteryl

Ester. The product was dissolved in 60 ml Piperidine/CH2Cl2 (1:3) and was stirred

at room temperature for half an hour. Then the solvent was removed in vacuo. The

crude material was dissolved in CHC13 and washed with distilled water (100 ml)

three times, dried over anhydrous Na2SO4, and evaporated to give a solid. Silica-

gel column chromatography (7% CH3OH and 10% Et20O in CH2Cl2 ) afforded a

white solid -- Arg(Pmc)-Cholesteryl Ester (5.68 g, 70.4%). TLC Rf=0.50,

CH30H/CH2Cl2 (1:9). Mass spectrum: m/z=809 (M+H) .











Synthesis of Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester

Arg(Pmc)-Cholesteryl Ester (4.04 g, 5.0 mmol) in 35 ml CH2CI2 was stirred

at 0C and Fmoc-Tyr(OtBu)-OH (2.32 g, 5.0 mmol) dissolved in 35 ml CH2C12

was added, which was followed immediately by DCC (1.22 g, 5.5 mmol) dissolved

in 15 ml CH2C12 and HOBt (0.91 g, 6.75 mmol) dissolved in 15 ml DMF. The

mixture was stirred for 24 hours at room temperature. The DCU yielded was filtered

off and the solvent was removed in vacuo to give a solid -- N-a-Fmoc-Tyr(OtBu)-

Arg(Pmc)-Cholesteryl Ester. The peptide ester was deprotected in 35 ml

Piperidine/CH2Cl2 (1:3) in an identical manner as illustrated in the previous step.

Silica-gel column chromatography (5% CH3OH and 10% Et20O in CH2CI2) afforded

a white solid -- Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester(3.88 g, 75.4%). TLC

Rf=0.45, CH3OH/CH2Cl2 (1:9). Mass spectrum: m/z=1029 (M+H).



Synthesis of Nicotinyl-Ala-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester

Nicotinyl-Ala-OH (0.39 g, 2.0 mmol) in 25 ml DMF/CH2CI2 (1:1) was

stirred at 0C and Tyr(O-tBu)-Arg(Pmc)-Cholesteryl-Ester (2.05 g, 2.0 mmol)

dissolved in 25 ml CH2Cl2 was added, which was followed immediately by DCC

(0.44 g, 2.2 mmol) dissolved in 15 ml CH2Cl2 and HOBt (0.38 g, 2.7 mmol)

dissolved in 10 ml DMF. The mixture was stirred for 96 hours at room temperature.

The DCU yielded was filtered off and the solvent was removed in vacuo. The

material obtained was dissolved in CH2CL2 and washed with IN HCI (3x100 mi),











5% NaHCO3 (3x100 ml), and saturated NaCI solution (100 ml), respectively. Then

the organic phase was dried over anhydrous Na2SO4 and removed in vacuo. Silica-

gel column chromatography (5% CH3OH and 10% Et20 in CH2C12 ) afforded a

white solid -- Nicotinyl-Ala-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester (1.43 g,

60.91%). TLC Rf=0.57, CH3OH/CH2CI2 (1:9); Mass spectrum: m/z=1205

(M+H).



Synthesis of Trigonellyl-Ala-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester

Nicotinyl-Ala-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester (0.50 g, 0.41 mmol)

in 25 ml CH2C!2 was stirred at 0C and dimethylsulfate (0.26 g, 20 mmol) was

added. The mixture was stirred for overnight at room temperature. The solvent then

was removed at reduced pressure. The product was dissolved in CH3OH and

recrystallized from ethyl ether to afford a white solid -- Trigonellyl-Ala-Tyr(O-tBu)-

Arg(Pmc)-Cholesteryl Ester (0.475 g, 95.12%). TLC Rf=0, CH3OH/CH2CI2 (1:9;

Mass spectrum: m/z= 1219, M .



Synthesis of Trigonellyl-Ala-Tyr-Arg-Cholesteryl Ester

Trigonellyl-Pro-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (0.25 g, 0.21 mmol)

was dissolved in 25 ml TFA/H20 (19:1) and stirred at 0C for an hour. Then the

solvent was removed at reduced pressure and the product was triturated with Et20

three times and was recrystallized from Et2O to afford a white solid -- Trigonellyl-











Ala-Tyr-L-Arg-Cholesteryl Ester (0.15 g, 79.72%). TLC Rf=0, CH3OH/CH2Cl2

(1:9); Mass spectrum: m/z=896, M.



Synthesis of 1.4-Dihydrotrigonellyl-Ala-Tyr-Arg-Cholesteryl Ester

Trigonellyl-Ala-Tyr-Arg-Cholesteryl Ester (0.1 g, 0.11 mmol) in 15 ml H20

was stirred at 0C under argon and NaHCO3 (0.092 g, 1.1 mmol) and Na2SO4

(0.184 g, 1.1 mmol) was added little by little. The mixture was stirred at 0C under

argon for an hour. Then saturated NaCl solution (50 ml) was added to dilute the

solution which was then extracted with 25 ml CH2CI2. The organic phase was

separated, washed with deaerated water several times, dried over anhydrous

Na2SO4, and evaporated to afford a yellow solid -- 1,4-Dihydrotrigonellyl-Ala-Tyr-

Arg-Cholesteryl Ester (0.056 g, 56.2%). TLC Rf=0.33, CH3OH/CH2Cl2 (1:9). UV

spectrum showed the typical absorption peak for dihydro-compounds at 350 nm.



Synthesis of Nicotinyl-Pro-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester

Nicotinyl-Ala-OH (0.22 g, 1.0 mmol) in 15 ml DMF/CH2Cl2 (1:1) was

stirred at 0C and Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (0.99 g, 1.0 mmol)

dissolved in 25 ml CH2Cl2 was added, which was followed immediately by DCC

(0.22 g, 1.1 mmol) dissolved in 15 ml CH2Cl2 and HOBt (0.19 g, 1.35 mmol)

dissolved in 10 ml DMF. The mixture was stirred for 96 hours at room temperature.

The DCU yielded was filtered off and the solvent was removed in vacuo. The











material obtained was dissolved in CH2Cl2 and washed with IN HCI (3x100 ml),

5% NaHCO3(3x100 ml), and saturated NaCI (100 ml) solution, respectively. Then

the organic phase was dried over anhydrous Na2SO4 and removed in vacuo. Silica-

gel column chromatography (5% CH3OH and 10% Et2O in CH2Cl2 ) afforded a

white solid -- Nicotinyl-Pro-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester (0.71 g,

57.61%). TLC Rf=0.51, CH3OH/CH2Cl2 (1:9). Mass spectrum: m/z=1231,

(M+H)+.



Synthesis of Ala-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester

Tyr(OtBu)-Arg(Pmc)-Cholesteryl ester (2.5 g, 2.5 mmol) in 35 ml CH2C12

was stirred at 0C and Fmoc-Ala-OH (0.78 g, 2.5 mmol) dissolved in 25 ml CH2C12

was added, which was followed immediately by DCC (0.61 g, 2.75 mmol) dissolved

in 15 ml CH2CI2 and HOBt (0.46 g, 3.38 mmol) dissolved in 15 ml DMF. The

mixture was stirred for 24 hours at room temperature. The DCU yielded was filtered

off and the solvent was removed in vacuo to give a solid -- N-a-Fmoc-Ala-

Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester. The peptide ester was deprotected in 35 ml

Piperidine/CH2Cl2 (1:3) in an identical manner illustrated in the previous step.

Silica-gel column chromatography (5% CH3OH and 10% Et2O in CH2C2 ) afforded

a white solid -- Ala-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (2.54 g, 77.75%). TLC

Rf=0.47, CH3OH/CH2Cl2 (1:9). Mass spectrum: m/z= 1101, (M+H)+.











Synthesis of Nicotinyl-Ala-Ala-Tvr(O-tBu)-Arg(Pmc)-Cholesteryl Ester

Nicotinyl-Ala-OH (0.39 g, 2.0 mmol) in 25 ml DMF/CH2Cl2 (1:1) was

stirred at 0C and Ala-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (2.02 g, 2.0 mmol)

dissolved in 25 ml CH2Cl2 was added, which was followed immediately by DCC

(0.44 g, 2.2 mmol) dissolved in 15 ml CH2Cl2 and HOBt (0.38 g, 2.7 mmol)

dissolved in 10 ml DMF. The mixture was stirred for 96 hours at room temperature.

The DCU yielded was filtered off and the solvent was removed in vacuo. The

material was dissolved in CH2Cl2, washed with IN HCI (3x100 ml), 5% NaHCO3

(3x100 ml), and saturated NaCl solution (100 ml), respectively. Then the organic

phase was dried over anhydrous Na2SO4 and removed in vacuo. Silica-gel column

chromatography (5% CH3OH and 10% Et20O in CH2CI2 ) afforded a white solid --

Nicotinyl-Ala-Ala-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (1.82 g, 71.37%). TLC

Rf=0.55, CH3OH/CH2Cl2 (1:9). Mass spectrum: m/z=1276, (M+H) .



Synthesis of Trigonellyl-Ala-Ala-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester

Nicotinyl-Ala-Ala-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (0.64g, 5.0 mmol)

in 25 ml CH2CI2 was stirred at 0C, dimethylsulfate (0.32 g, 25 mmol) was

added. The mixture was stirred for overnight at room temperature. The solvent then

was removed at reduced pressure. The product was dissolved in CH3OH and

precipitated from ethyl ether to afford a white solid -- Trigonellyl-Ala-Ala-











Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (0.58 g, 89.92%). TLC Rf=0,

CH30H/CH2Cl2 (1:9). Mass spectrum: m/z= 1290, M+.



Synthesis of Trigonellyl-Ala-Ala-Tyr-Arg-Cholesteryl Ester

Trigonellyl-Ala-Ala-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (0.26 g, 0.20

mmol) was dissolved in 25 ml TFA/H20 (19:1) and was stirred at 0C for an hour.

Then the solvent was removed at reduced pressure and the product was triturated

with Et20O three times and was recrystallized from Et2O to afford a white solid --

Trigonellyl-Ala-Ala-Tyr-L-Arg-Cholesteryl Ester (0.17 g, 87.90%). TLC Rf=0,

CH30H/CH2C12 (1:9). Mass spectrum: m/z=967, M+.



Synthesis of Nicotinyl-Pro-Ala-Tyr(O-tBu)-Arg(Pmc)-Cholesteryl Ester

Nicotinyl-Pro-OH (0.44g, 2.0 mmol) in 25 ml DMF/CH2Cl2 (1:1) was

stirred at 0C and Ala-Tyr(OtBu)-Arg(Pmc)-Cholesteryl Ester (2.20 g, 2.0 mmol)

dissolved in 25 ml CH2Cl2 was added, which was followed immediately by DCC

(0.44 g, 2.2 mmol) dissolved in 15 ml CH2C12 and HOBt (0.38 g, 2.7 mmol)

dissolved in 10 ml DMF. The mixture was stirred for 96 hours at room temperature.

The DCU yielded was filtered off and the solvent was removed in vacuo. The

material was dissolved in CH2C12, washed with IN HCl (3x100 ml), 5% NaHCO3

(3x100 ml), and saturated NaCI solution (100 ml), respectively. Then the organic

phase was dried over anhydrous Na2SO4 and removed in vacuo. Silica-gel column