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Design, synthesis and pharmacological evaluation of a chemical delivery system for drug targeting to lung tissue

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Title:
Design, synthesis and pharmacological evaluation of a chemical delivery system for drug targeting to lung tissue
Creator:
Saah, Maurice Philip, 1960-
Publication Date:
Language:
English
Physical Description:
xiv, 139 leaves : illustrations; 29 cm.

Subjects

Subjects / Keywords:
Research ( mesh )
Drug Evaluation ( mesh )
Drug Design ( mesh )
Chlorambucil -- chemical synthesis ( mesh )
Chlorambucil -- analogs and derivatives ( mesh )
Chlorambucil -- pharmacology ( mesh )
Cromolyn Sodium -- chemical synthesis ( mesh )
Cromolyn Sodium -- analogs and derivatives ( mesh )
Cromolyn Sodium -- pharmacology ( mesh )
Thioctic Acid ( mesh )
Drug Delivery Systems ( mesh )
Lung -- drug effects ( mesh )
Department of Medicinal Chemistry thesis Ph. D ( mesh )
Medicinal Chemistry thesis, Ph. D
Dissertations, Academic -- College of Pharmacy -- Department of Medicinal Chemistry -- UF ( mesh )
Dissertations, Academic -- Medicinal Chemistry -- UF
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )
Academic theses ( lcgft )
Academic theses ( fast )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 131-138).
General Note:
Typescript.
General Note:
Vita.
General Note:
Local note: Major Adviser : Nicholas S. Bodor.
Statement of Responsibility:
by Maurice Saah.

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Full Text
DESIGN, SYNTHESIS AND PHARMACOLOGICAL
EVALUATION OF A CHEMICAL DELIVERY SYSTEM
FOR DRUG TARGETING TO LUNG TISSUE
By
MAURICE SAAH
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
1994


Dedicated
to
my
Mother, Sarah;
my Sisters, Theresa, Lillian and Sally;
my Brothers, Patrick, William and Edmund;
my Wife, Betty;
and our Daughter Sheryl
with love and deep appreciation.


ACKNOWLEDGMENTS
I thank Dr. Nicholas Bodor, my advisor and chair of the supervisory
committee for my dissertation. The excellent guidance, patience and kind support
received from Dr. Bodor have been the most important factors in bringing this
project to fruition. To him I am deeply indebted for the enrichment of my
evolution as a pharmaceutical scientist.
Special thanks go to Drs. Luis Muga, James Simpkins and Hassan Farag for
their advice and assistance at various stages of my program and to all the members
of my supervisory committee, Drs. Nicholas Bodor, Margaret James, Richard
Hammer, James Simpkins, and Luis Muga, for the attention and interest in the
project.
The timely help and advice I received from Dr. Emy Wu throughout the
project are deeply appreciated and warrant special mention. Also of special
mention is Dr. Ede Marvenyos whose initial efforts spurred my interest in this
project. I also thank Drs. Laszlo Prokai, and Gabor Somogyi and other members
at the Center for Drug Discovery: Joan Martignago, Laurie Johnston, Julie Berger,
Kathy Eberst, Gizella Somogyi, Dr. Kerry Estes and Robert Wong for their
willingness to help. I would also like to thank all friends and colleagues at the
Center: Angela, Martha, Kumar, Ouyang and others who helped make this tenure
a pleasant experience. I cherish the support received from my wife, Betty; my best
friend, Jeanne-Marie; my mother Sarah, cousin, Clara; aunts, Adelaide and Letitia;
and my brothers and sisters.
iii


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vii
LIST OF FIGURES ix
KEY TO SYMBOLS AND ABBREVIATIONS xii
ABSTRACT xiii
CHAPTERS
1. INTRODUCTION
Drug Design and Need for Drug -Targeting 1
Drug Targeting Systems 8
Biological Drug Targeting Systems 8
Physical and Biophysical Targeting Systems 9
Chemical Molecular Systems 9
2. FEASIBILITY OF DRUG TARGETING TO LUNG
Physiology and Pharmacology of the lung 13
Feasibility and Possible Mechanisms
of Selective Delivery to Lung Tissue 15
Lipoic Acid 19
Chlorambucil 22
Cromolyn (DSCG) 23
Mechanism of Action of DSCG 24
Need for Improved Delivery of DSCG 25
Predicted in-vivo Metabolism of CDS 27
3. RESEARCH DESIGN AND SPECIFIC OBJECTIVES
Objective 31
Synthesis 31
IV


Chlorambucil CDS 32
Cromolyn CDS 33
Evaluation of Mixed Disulfide Bond Formation as a
Probable Mechanism for CDS Selectivity for Lung 35
Enzyme Inhibition Studies 36
Kinetics of Mixed-Disulfide Bond Formation 36
Tissue Sulfhydryl 37
Research Protocol 37
4. EXPERIMENTAL METHODOLOGY
Materials 39
Methods
Synthesis 41
Chlorambucil CDS (MS-2) 41
Cromolyn CDS-1 (MS-4) 42
Cromolyn CDS-2 (MS-17) 44
HPLC Assays 48
Drug recovery efficacy of HPLC assay 49
OctanolAVater Partition Coefficients (Log P) 50
In-Vitro Stability 50
Hydrolysis Products of CDS 51
In-Vivo Distribution Studies 52
Chlorambucil and CDS in rats and rabbits 52
Cromolyn and CDS: Distribution in rats and rabbits 52
Determination of Tissue Sulfhydryl 54
In-Vitro Binding Studies 56
Inhibition of CDS hydrolysis by BPNP 56
Effect of BPNP on hydrolysis of MS-4 57
Determination of bound and unbound drug 57
Mixed disulfide binding in the presence of NEM 58
Effect of [BPNP] on Binding of MS-4 59
Time dependence of mixed disulfide binding of MS-4
to bovine serum albumin 60
to a model tissue (rabbit lung) 60
to rabbit lung tissue compared with liver 60
Dependence of binding on initial drug concentration 61
Comparative in-vitro tissue binding of MS-4 via
mixed disulfides 61
Dependence of binding on homogenate concentration .... 61
Data Treatment and Statistical Evaluation 61
v


5 RESULTS AND DISCUSSION
Syntheses 63
Chlorambucil CDS 66
Cromolyn-CDSs 66
Cromolyn CDS-2, MS-17 75
HPLC Assays 76
Chlorambucil and CDS 76
Cromolyn and CDSs: Improved Detection
of Cromoglycate Anion 76
OctanolAVater Partition Coefficients 79
In-Vitro Stability 80
Hydrolysis Products of CDS 82
In-Vivo Distribution Studies 83
Chlorambucil and CDS: Distribution in Rats and Rabbits .... 84
Cromolyn and CDS: Distribution in Rats and Rabbits 87
Kinetics of Mixed Disulfide-Bond Formation 103
Effect of BPNP on the Hydrolysis of MS-4 104
Effect of Inhibitors on Binding of MS-4 via
Mixed Disulfides 105
Time Dependence of Mixed Disulfide Binding of CDS 107
Dependence of MS-4 Binding on the Initial
Drug Concentration Ill
Comparative Binding of MS-4 In-Vitro 113
Dependence of Binding on Homogenate Concentration 114
Tissue Sulfhydryl 114
6 CONCLUSIONS 120
APPENDIX
SAMPLE CALCULATIONS 126
REFERENCES 131
BIOGRAPHICAL SKETCH 139
vi


LIST OF TABLES
TABLE Page
1 Recovery of Cromolyn as the Cromoglycate Anion
from Whole Rat Blood 78
2 Recovery Efficiencies of Cromolyn from Rat Tissues
Using Method B 78
3 Octanol-Water Partition coefficients 79
4 In-vitro Stabilities of CDSs Compared to Parent Drugs 80
5. In-vitro Stability of a Model CDS, MS-4, in
Various Rat Tissue Media: (25% Homogenate) 80
6 Percent Composition of Organs/Tissues Relative to
Body Weight in the Rat and Rabbit 83
7 In-Vivo Distribution of Drug 5 Minutes After
Administration of Chlorambucil and CDS; 85
8 In-Vivo Distribution of Drug 30 Minutes
After Administration of Cromolyn and CDS 86
9 In-Vivo Distribution of Drug in Rabbit Tissues 30 Minutes
After Administration of CDS and Cromolyn 87
10 Distribution of Drug in Rat Tissues with Time After Administration
of 6.2 gmol/kg of CDS-1 (MS-4) and Parent (Cromolyn) 89
11 Drug Distrbution in Rat Lung with Time 90
12 Drug Distribution in Rat Blood with Time 90
vii


13 Drug Distribution in Rat Liver with Time 91
14 Drug Distribution in Rat Kidney with Time 92
15 Recovery of Drug with Time After Administration to Rats 99
16 Dependence of Hydrolysis Rate Constants and Half-lives of MS-4
on BPNP Concentration in Rat Liver Homogenate 104
17 Effect of [BPNP] on Binding of MS-4 to BSA 105
18 Binding of MS-4 in Presence of Inhibitors (BPNP and NEM) 107
19 Binding of MS-4 to BSA (50 mg Protein/mL) with Time 108
20 Dependence of Binding on Time of Incubation 109
21 Binding of MS-4 With Time to Rabbit Lung Tissue
Compared with the Liver and BSA 110
22 Dependence of MS-4 Binding in Liver Tissue on the
Initial Drug Concentration In-Vitro Ill
23 Comparative Binding of MS-4 to Various Rabbit Tissues In-Vitro ... 113
24 Dependence of MS-4 Binding in Rabbit or
Liver Tissue on Homogenate Concentration 114
25 Tissue Sulfhydryl Content in the Rabbit as Total Thiol, T-SH;
Non-Protein Thiol, NP-SH; and Protein-Bound Thiol, P-SH .... 115
26 Estimated Magnitudes of Binding of MS-4
in Respective Rabbit Tissues 116
viii


LIST OF FIGURES
FIGURE Page
1 The Metabolic Fate of a Conventional Drug After Administration 4
2 The Soft Drug Concept 5
3 The Prodrug Approach 6
4 The CDS Approach 10
5 General Structure of the Human Lung 14
6 Mechanism for Enhanced Delivery of Spirothiazolidine
Derivatives 16
7 Proposed Mechanism of Delivery by the Lipoic Acid CDS 18
8 Coenzymatic Function of Lipoic Acid 21
9 Predicted Metabolism of Chlorambucil CDS 28
10 Metabolism of Cromolyn CDS 29
11 Mechanism of Esterification with DCC 32
12 Synthesis of Chlorambucil CDS (MS-2) 42
13 Synthesis of Cromolyn CDS-1 (MS-4) 43
14 Scheme for the Synthesis of Cromolyn CDS-2 47
15 !H-NMR Spectrum of Lipolol 64
16 UV Spectrum of Lipolol 65
IX


17 H-NMR Spectrum of Chlorambucil CDS 67
18 Formation of Lipoyl Bromide Dimer 69
19 IR Spectrum of Lipoly Bromide 71
20 1 H-NMR Spectrum of Cromolyn CDS-1 72
21 UV Spectrum of Cromolyn 73
22 UV Spectrum of Cromolyn CDS-1 (MS-4) 74
23 In-Vitro Hydrolysis Products of CDSs in Rabbit Blood 82
24 In-Vivo Distribution of Total Drug as Chlorambucil with Time
in Rats After i.v. Administration of CDS and Parent 84
25 In-Vivo Distribution of Chlorambucil in Rabbit 30 Minutes
After Administration of Chlorambucil and CDS 86
26 In-Vivo Distribution in Rabbit Following Cromolyn
and CDS Administration 88
27 Drug Distribution in Lung After CDS/Cromolyn Administration 93
28 Distribution of Cromoglycate in Rat Lung with Time
After Administration of CDS or Parent Drug 93
29 In-Vivo Concentrations of Drug in Rat Tissues with Time After
Administration of CDS vrs. Parent Drug (Cromolyn) 94
30 Percent of Total Drug in Respective Rat Tissues with Time
After Administration of CDS and Parent Drug (Cromolyn) 95
31 Percent of Recovered Drug in Respective Rat Tissues with Time
After Administration of CDS and Parent Drug (Crm) 96
32 In-vivo Distribution of Drug (CDS + Metabolites) in Rat
Tissues with Time After CDS Administration 97
33 Percent of Administered Retained in All Tissues with Time 99
x


34 Mean Percent Distribution of Total Drug With Time
in Whole Rat Tissues 101
35 Mean Percent Distribution of Recovered Drug with
Time in Whole Rat Tissues 102
36 Effect of BPNP on Binding of MS-4 to BSA 106
37 In-Vitro Binding of MS-4 in Rat Lung Tissue with Time 110
38 Dependence of Binding on Initial Drug Concentration 112
39 Percent Binding and Initial Drug Concentration 113
40 Tissue Sulfhydryl Content of the Rabbit 116
41 Tissue Sufhydryl, Mixed Disulfide Binding
and Magnitude of Binding 117
XI


KEY TO SYMBOLS AND ABBREVIATIONS
v
vas
^as
Ss
vs
gg
gL
pM
!H-NMR
AUC
CDS
cone.
DMSO
g
HPLC
kg
m
mL
mM
mmol
n
nm
nmol
q
r
t
T */2
v/v
X
i.d.
vibrational frequency (IR spectroscopy)
asymmetrical stretching
asymmetrical bending vibration
scissoring vibration
symmetrical stretching
microgram
microliter
micromolar
Proton magnetic resonance
Area Under Curve
Chemical Delivery System
concentration
dimethyl sulphoxide
gram
high-performance liquid chromatography
kilogram
multiplet
milliliter
millimolar
millimole
number of determinations or sample size
nanometer
nanomole
quintet
linear regression correlation coefficeint
triplet (nmr spectroscopy)
half-life
volume to volume ratio
wavelenght
internal diameter
Xll


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
DESIGN, SYNTHESIS AND PHARMACOLOGICAL
EVALUATION OF A CHEMICAL DELIVERY SYSTEM
FOR DRUG TARGETING TO LUNG TISSUE
By
Maurice Saah
August 1994
Chairman: Nicholas Bodor, Ph.D.
Major Department: PharmacyMedicinal Chemistry
In conventional therapy, the achievement of therapeutically effective levels
of drug at its pathophysiologically relevant site is very often adversely mitigated
by systemic toxicity resulting from a lack of selectivity in drug pharmacokinetics.
It is therefore obvious that the development of a mechanism to selectively deliver
drugs to, in this instance, lung tissue, could raise the therapeutic index and thereby
optimize the clinical usefulness of drugs used to treat lung diseases, especially
lung cancer. This dissertation intends to expostulate the application of a novel
chemical delivery system (CDS) approach to a delivery mechanism for drug
targeting to lung tissue using the l,2-dithiolane-3-pentyl moiety of lipoic acid as
the 'targetor moiety'. It describes the synthesis, and the physicochemical and
pharmacological evaluation of a CDS modeling the lipolyl and other ester


derivatives of chlorambucil (an antineoplastic agent), and cromolyn (a
bischromone used in anti-asthma prophylaxis) as compared with their respective
parent drugs. The chlorambucil CDS was synthesized by esterifying the alkanol
derivative of lipoic acid with chlorambucil using dicyclohexyl carbodiimide as the
coupling agent. The cromolyn esters were prepared by respective multistep
synthetic procedures each culminating in the reaction of the alkyl bromide
derivative of lipoic acid with the disodium salt of the bischromone compound. All
the esters were highly lipophilic, unlike the parent compounds. The in-vitro and
in-vivo kinetic and pharmacokinetic studies showed the respective CDSs were
sufficiently stable in buffer and biological media, hydrolyzed rapidly into the
respective active parent drugs, and significantly enhanced delivery of the active
compound to lung tissue in comparison with the underivatized parent compounds
used in conventional therapy. A proposed mechanism for the selective delivery
suggested the involvement of drug binding through mixed disulfide linkages to
tissue proteins. In-vitro binding studies showed the CDS did indeed bind to tissue
proteins, and that the magnitude of binding was greatest in the lung. This factor,
coupled with the fact that the total venous return after intravenous administration
goes first to the lung probably helped the lung to maximally sequester the drug.
XIV


CHAPTER 1
INTRODUCTION
Drug Design and the Need For Drug Targeting:
A singular and arguably, the most important parameter of a drug that needs
to be optimized in the drug development process is the therapeutic index (TI),
which represents the ratio between the median toxic dose, TD50, and the median
effective dose, ED50:
TI = TD50/ED50
In conventional therapy, the parenteral or enteral administration of a drug
has usually involved, a more or less, passive distribution of the drug entity
throughout the body. This has very often lead to the achievement of
therapeutically effective levels of drug in a target organ/tissue woefully mitigated
by systemic toxicity. It is now therefore widely accepted that clinical usefulness
(expressed by the therapeutic index) of drugs in therapy would be enhanced if
drugs were to concentrate and exert their therapeutic effect selectively in/on the
target tissue. Drug targeting as one approach or as a part of an integrated approach
will therefore improve TI by raising the median toxic dose and/or decreasing the
median effective dose.
Lung disease (including lung cancers and other respiratory ailments)
constitute one of the leading causes of morbidity and mortality in the United
States, hence any attempt to improve therapy in the management of lung disease
1


2
should certainly present an area of immense interest to pharmaceutical scientists
especially in the area of lung cancer treatment where systemic toxicity constitutes
a major problem.
Drug design in the last two decades has taken on a more rational approach.
No longer do medicinal chemists rely on chance discovery of drugs as had been
the case in the past for many presently important types of drugs (Jacobsen. 1976;
and Austel, 1981), but use is made of information about the various bio-molecular
features (receptors, enzyme systems or metabolic pathways) as potential targets for
pharmacological intervention, permitting the rational design of agonists and
inhibitors to mitigate the pathophysiological process. Since the bio-molecular
processes underlying many disease states are not usually well understood, such
approach has not always been feasible. For example, since the development of the
receptor theory, some success has been achieved with some CNS active agents.
However since most receptors are generally distributed throughout the body while
the disease may be localized, a receptor based intervention could sometimes
produce undesired effects elsewhere. For instance the neurotransmitter dopamine
is released at specific parts of the brain and localized by the blood brain barrier
(BBB) therein to produce the desired anticholinergic action locally. In Parkinson's
disease, there is dopamine deficiency in the striatum. The peripheral
administration of dopamine (even in the form that could penetrate the BBB) could
result in side effects such as dryness of the mouth, cycloplegia, mydriasis and
tachycardia (Korolkovas, 1988) due to its central anti-cholinergic activity.
Besides, there are still many situations where selectivity at receptor sites is not
enough or where no such receptors are yet known to exist or where the target
disease stems from little understood biological response. So additionally,
medicinal chemists have used a variety of empirical and semi-empirical structure
activity relationships to enhance pharmacological activity through improved


3
delivery across membranes (improved bioavailability) notably, the improved
delivery across the BBB (Bodor et al., 1975), and the establishment of a
correlation between physicochemical parameters and biological activity by Hansch
(1981), and its subsequent myriads of applications.
In spite of these advances, relatively few compounds having even maximal
activity actually proceed to become clinically useful drugs (Bodor 1984), and even
when they do sometimes have to be used under trying circumstances with extremes
of caution. The reason being the accompanying and unexpected toxicities and
other pharmacological effects of the drug. From a schematic depiction of the
metabolic fate of a conventional drug in-vivo (.Figure 7), Bodor (1984),
summarizes the toxic effects of a drug as resulting from a combination of factors
which include all the other pharmacological effects of the drug (D) itself, the
effects of the direct and indirect metabolites (Dj... Dn, Mj... Mj and Mj... Mq
respectively), reactive intermediates (I*] ... I*m), and the various compounds (IC¡
... ICn) resulting from the interactions of these intermediates with cellular
components. The overall toxicity of a drug could then be described as the
summation of the toxicity due to the drug itself, which essentially is its lack of
selectivity, and the toxicities due to its various metabolic products.
Thus for the medicinal chemist, a rational drug design aims at improving
the therapeutic index of a drug by taking into consideration selectivity in drug
delivery and intervention of toxic pathways in drug metabolism through structural
manipulations. Hence, the primary focus of a rational drug development as
suggested by Bodor (1984) is to optimize activity rather than merely maximizing
it. These considerations have ultimately resulted in the prodrug and soft drug
concepts (Bodor, 1984), the hard drug concept (Ariens and Simonis, 1977) and the
novel chemical delivery system approach (Bodor 1984 and 1992).


4
Drug (D)
ko
Delivery
Process
Direct Elimination ^kl0r k2
of Unchanged
or Conjugated D
D
Jiv k
Reactive
Intermediates
k k
1 *CJ v
ICj... ICn
Toxic Metabolites
or Products
Figure 1: The Metabolic Fate of a Conventional Drug After Administration


5
Structural modifications on a drug molecule result in the alteration of some
pharmacodynamic and physicochemical aspects of the drug resulting in an
optimization of its metabolism, activity, delivery and elimination processes. Thus
depending on the specific design goals, it may be possible to design a drug having
predictable metabolism and/or other properties on a rational basis by the
introduction of certain labile or 'vulnerable groups' into the drug molecule. The
identification of certain such 'vulnerable moieties' by Ariens (1977) as parts of
drug molecules responsible for bio-inactivation, metabolism or elimination makes
the feasibility of this approach especially probable, and could also form the basis
for Bodor's (1984) novel concept of "structure-metabolism relationships"
analogous to Hansch's (1981) quantitative structure-activity relationships and its
myriads of applications.
Direct Elimination
Proo
Del
M ,
M 2 Mk
inactive
metabolite
inactive
metabolites
Elimination
Figure 2: The Soft Drug Concept (Bodor, 1984):
The therapeutically active soft drug (SD) is inactivated in one metabolic
step. This eliminates the pathways to the formation of toxic metabolites.


6
The soft drug {Figure 2) above, defined as "a biologically active
therapeutically useful chemical compound (drug) characterized by a predictable
and controllable in vivo destruction (metabolism) into non-toxic moieties after
achieving their therapeutic role" (Bodor 1984; 261), enables the separation of
therapeutic properties from toxic affects.
In the prodrug approach {Figure 3), the drug as an inactive moiety (PD) is
designed to undergo mainly the metabolism required to activate it in-vivo, without
substantial direct elimination and thereafter follow the scheme illustrated in Figure
1. The hard drug on the other hand, as exemplified by cromoglycic acid, is a non-
metabolizable active compound that can elicit its pharmacological activity and
undergo elimination unchanged.
elimination
/
DRUG
chemical in viy
transformations ^ ^ k 2
G)+ 0
in vivo
metabolism
and
disposition
Figure 3: The Prodrug Approach
The inactive prodrug is activated in-vivo where it enhances
therapeutic index through optimized delivery and elimination.


7
These approaches to a rational drug design have not been without
limitations. The prodrug approach can at best improve bioavailability through
alteration of pharmacodynamic parameters, and protect against some unwanted
degradations such as those occurring in the gastrointestinal tract or during the
hepatic first pass. It cannot substantially influence the formation of reactive or
toxic intermediates although it may indirectly reduce toxicity through optimized
delivery and elimination.
The hard drug by design necessitates 'blocking' of the metabolically
sensitive parts of the drug molecule. This will have to be achieved at the expense
of favorable pharmacokinetics. In other words, the drug would have to be very
lipophilic or very hydrophilic. In the highly lipophilic form, it can deposit in
adipose tissue and organelles resulting in extremely prolonged half-lives and
possibly long term physical and biochemical damage to tissues. With the highly
water soluble form, the in-vivo half-life would be too short to afford the drug any
significant degree of usefulness. Besides, such metabolic stability is only
idealistic as the various xenobiotic metabolizing enzymes particularly the
cytochrome P-450, have been known to attack and alter even the most highly
stable compounds, and according to Bodor (1984), it appears to be the general rule
that the more difficult the metabolism of a chemical, the more likely it is to form
highly reactive intermediates.
Thus, in spite of some successes, especially with the soft drug and prodrug
concepts, these approaches seem idealistic at best. Furthermore, since the disease
is usually localized, whereas the drug passively distributes throughout the body, it
behoves medicinal chemists to adopt an integrated approach with emphasis also on
improving selectivity or achieving drug targeting.


8
Drug Targeting Systems
Drug targeting involves directing the therapeutic agent to a desired site of
action with minimum or no pharmacological interaction with other tissues. In
general, drug targeting may be achieved by;
i.exploiting the selectivity of biological receptors and finding agents
which will selectively act at these sites (Gardner, 1986);
ii. having the drug in an inactive or chemically modified form (prodrug
and related approaches) to be activated at, or 'homed in' on, the
required site of action; or
iii. encapsulating the drug moiety in liposomes or other particulate
carriers to be released at an intended site.
The various approaches that have been employed to achieve drug targeting
may be in the form of biological, physical or chemical molecular systems.
Biological Drug Targeting Systems.
These include the use of biomolecules such as monoclonal antibodies, small
peptides, hormones, glycoconjugates or lectins that show recognition for specific
components in/on tissue or cell surfaces. An example is provided by the potential
use in drug-targeting of the copolymer of N-(2-hydroxypropyl)-methacrylamide
(HPMA) variously derivatized with certain amino sugars that show recognition for
specific sugar receptors on certain mammalian cell types (Seymour et al., 1987).
A number of sugar-receptor interaction involving several mammalian cell types
have been widely investigated and documented. These include the;
i. galactose-receptors of mammalian hepatocytes, Kupffer cells,
liver endothelial cells and bone marrow:


9
ii. N-acetylglucosamine receptors of avian hepatocytes;
iii. mannose-6-phosphate receptor on a variety of mammalian
tissues;
iv. receptor with high affinity for fucose on mouse L1210 Leukemia
cells; and
v. mannose/N-acetylglucosamine receptors on alveolar
macrophages and on a variety of reticuloendothelial cells.
Physical and Biophysical Drug Targeting Systems.
The use of localized polymeric reservoir devices, liposomes, microcapsules,
nanospheres and other particulate devices such as resealed erythrocytes and other
cellular reservoir carriers constitute the physical systems. Here, the drug is
essentially chemically unmodified, and targeting is achieved by physical
sequestration of the carrier by virtue of size, surface (charge, magnetic properties
or antibody coating) characteristics, and a combination of anatomical and
(patho)physiological events of both the passive and active types (Tomlinson,
1986). Such physical targeting systems are useful for persistent and sustained
presentation of drug at such discrete compartments as the eye, joints, respiratory
tract and the GI tract, and also for the treatment of diseases affecting the cells of
the reticuloendothelial system (RES) such as Leischmaniasis, Guacher's disease
and Leprosy. Polymer-drug conjugate targeting systems whose mode of selective
sequestration is size fall under this category on account of which cellular uptake
is by phagocytosis and pinocytosis by certain cells of the RES.
Chemical Molecular Systems.
Although many opportunities exist for drug targeting, each approach has its
own limitations, and medicinal chemists are more interested in this means of drug


10
targeting. The chemical molecular systems for drug targeting are based on the soft
drug, prodrug and the related Chemical Delivery System (CDS) approaches and
involve chemically modifying the parent drug to incorporate the desired targeting
capability.
T + D
[TD] + P
[TDP] + F
[CDS] n
successive enzymatic
reactions
F, P, or F
inactive
/
Active Drug
at Target Site
[TD] + F
in vivo metabolism
and disposition
Figure 4: The CDS Approach:
The parent drug (D) is chemically transformed into the CDS by the
incorporation of targetor (T) and/or other modifier moieties (P and/or F)
For improved delivery of drugs, the prodrug and related approaches seem
most viable and have been credited with many successes including improved
delivery across biological barriers notably improved delivery across the blood-
brain-barrier based on the dihydropyridine/pyridinium salt bioreversible systems
(Bodor et al., 1975). The prodrug approach has also been extensively used for


11
improving the pharmacokinetics and even some aesthetic attributes (taste etc.) of
many drugs. For targeting purposes however, Bodor (1984) has proposed that the
simple prodrug form cannot effectively achieve site specificity unless a given
enzyme is concentrated in certain areas or organs that will cause activation
specifically at these. Rather the CDS approach will present a more viable system
that is capable of achieving site specificity. In the CDS approach, the drug is
transformed by several synthetic steps into the inactive derivative. The resulting
CDS, in vivo, is then expected to undergo successive and predictable enzymatic
transformation ultimately resulting in the selective release of drug at the desired
site. The CDS approach is illustrated diagrammatically in Figure 4. The
modification involves the attachment of monomolecular units generally
comparable in size or smaller than the parent molecule (to contrast from targeting
systems involving polymer-drug conjugates).
The modification in the CDS by design provide a site-specific or site
enhancing delivery of the drug through the incorporation of, most importantly, the
'targeting moiety' (T) in addition to none or several other moieties or
functionalities (P or F) introduced to enhance such parameters as water solubility,
partition coefficient, or to protect against premature metabolism (Bodor, 1984 &
1992). Although the approach is somewhat related, the prodrug or soft-drug in
general does not contain a targeting moiety, but may contain some or all of the
other characteristics of the CDS.
The CDS gets transported to the desired site by non-specific transport
wherefore the various physicochemical or enzymatic reactions occur to release or
sequester the drug at that site. Targeting by the CDS may result from enzymatic-
physical-chemical means, site specific enzyme activation, or receptor based. An
enzymatic -physical-chemical based targeting system may be exemplified by the
dihydropyridinium (DHP) pyridinium (P) salt redox based system for drug


12
targeting to the brain. The CDS (Drag-DHP conjugate) penetrates the blood brain
barrier on account of its high lipophilicity. In the brain (as in other parts of the
body), the CDS undergoes oxidation (chemical) into the hydrophilic quaternary
form (D-QP+) which on account of its hydrophilicity and hence inability to
traverse the BBB remains 'locked' in the brain (physical) whereas that produced at
other parts of the body gets rapidly eliminated. Enzymatic reactions then release
the drug from the pyridinium salt form trapped in the brain (Bodor et al., 1975 and
Bodor, 1984).
The system for site selective delivery described in this dissertation, as may
be more obvious in the subsequent discussion, is also based on the physical-
chemical-enzymatic meansphysical in the reference to the transportation
processes and the appurtenant factors that take the drug to its target site, and
chemico-enzymatic for the processes that selectively sequestrate the lung at the
target site.


CHAPTER 2
FEASIBILITY OF DRUG TARGETING TO LUNG
Physiology and Pharmacology of the Lung
The general structure of the human lung is as shown in Figure 5.
Morphologically, the lung may be divided into parenchymal (alveoli, alveolar
ducts and capillaries), and non-parenchymal tissues (conductive airways, blood
vessels, connective tissue and pleura), comprising altogether a total of about 40
different cell types. The alveoli tissues consist of various cell types which include
macrophages. These macrophages are mobile and metabolically active species
possessing phagocytic, microbicidal and cytocidal activity.
Humans can tolerate oxygen deprivation for only about five minutes. The
major function of the lungs, as part of the respiratory system, is to act as the gas
exchanger (oxygenation of blood and removal of carbon dioxide). For this
purpose, the lungs present a very large surface area, about 72 m2about 35 times
the total skin surface area (Hollinger, 1985a), to communicate between the body
and the environment. However, by this very nature, the respiratory system also
represents a significant portal for the introduction of either noxious or therapeutic
agents. Therefore, in addition to the fact that the lung is also capable of metabolic
functions (xenobiotic metabolism and detoxification), it is susceptible to a wide
variety of disorders namely, bacterial and fungal infections, interstitial lung
disease (ILD) which includes drug/xenobiotic induced diseases, collagen
13


14
vascular disease and fibrosis (cystic fibrosis), connective tissue disorders like
Systemic Lupus Erythematosus (SLE) and lung cancers. Lung cancer along with
the other respiratory tract disorders is the leading cause of morbidity and mortality
in the United States (Miller and Johnson, 1989).
Carina
Right primary
bronchus
Thyroid cartilage
Cricoid cartilage
Trachea
Tertiary
bronchiole
Secondary
bronchiole
Superior
lobe
Middle
lobe
Inferior
lobe
Left primary
bronchus
Superior
lobe
Pulmonary
artery
Left
pulmonary
vein
Inferior
lobe
Pleura
Right lung
showing airways
Left lung showing
pulmonary blood
vessels
Eiguge 5: General Structure of the Human Lung (Washington et al., 1989)


15
Feasibility and Possible Mechanisms
of Selective Delivery to Luna Tissue
The lungs possess the next highest levels of nearly all the metabolic
enzymes found in the liver (Hollinger, 1985b; & Damani, 1987) and in some cases
even higher levels (although in only a few specific cell types). For example, N-
acetyl transferase, an enzyme that acts on para-aminobenzoic acid has 75% higher
activity in the rabbit lung, and Gluthathione -S-Transferases, a group of enzymes
which catalyze the conjugation of glutathione with some xenobiotics, have about
three times higher specific activity in rat lung than in the liver (Damani, 1987).
Additionally, in contrast to all other tissues, the lungs receive the total venous
return first, so it is in an ideal position to regulate (through metabolism and/or
sequestration) the concentration of substrates in the blood before they reach the
arterial circulation (Alabaster, 1977). This implies that after intravenous
administration, a drug reaches the lungs before the liver hence avoiding problems
that may be associated with a hepatic first pass and thereby permitting a more
efficacious sequestration of the drug entity in the lung.
Nevertheless a mechanism for successful CDS for lung delivery, need make
use of enzyme systems, carrier systems, binding reactions or metabolic pathways
occurring (more extensively) in the lungs: For instance, in certain species (e.g.
rabbit, rat and hamster), the specific activities of some oxygenases are higher in
the lungs than in the liver (Bodor, 1984; and Philpot et al., 1977). This factor may
be of importance for mechanisms involving oxygenase catalyzed redox-based
systems such as may probably be the case with the system described in this
dissertation. Also, certain chemicals such as 5-hydroxy-tryptamine, noradrenaline
and amphetamines that accumulate in the lung have been found to do so through
carrier mediated sodium dependent transport systems. Others accumulate through
surfactant binding (e.g. methadone), and protein binding (Philpot et al., 1977).


16
During transdermal delivery studies and evaluation of endogenous
substances as natural soft drugs (Bodor, 1984), it was observed that with the
corticosteroid derivatives of 3-spirothiazolidine (an a-, B- unsaturated ketone),
there was enhanced deposition of progesterone to rat lung tissue after
intravenous administration of the mono- or bis- spirothiazolidine derivatives,
thereby making spirothiazolidine attractive for use in, and generating an interest in
the development of a CDS for drug targeting to lung tissue.
Figure 6: Mechanism for the Enhanced Delivery of
Spirothiazolidine Derivatives (Bodor, 1984)
It was suggested that the mechanism of delivery (Figure 6) was via an
oxidative binding mechanism involving disulfide linkage formation of the


17
intermediate imminium salt (2) of the spirothiazolidine nucleus and cysteinyl
residues of lung tissue proteins. The bound form (3) will then hydrolyze easily to
release the drug (4).
Lipoic acid (l,2-dithiolane-3-pentanoic acid or thioctic acid), on account of
its potential to also form disulfide linkages was investigated for use as the
targeting moiety in a CDS for selective drug delivery to lung tissue. A proposed
mechanism of drug delivery by the lipoic acid CDS is depicted in Figure 7, below.
The probable involvement of disulfide bond formation in the suggested mechanism
was made more conceivable by the report of Livesey et al. (1990) that both the
thiol and disulfide metabolites of aminoalkylphosphorous acid (a class of
compounds that provide cellular protection against radiation and chemo
therapeutic drug toxicity) are capable of binding to rat tissue through mixed
disulfide bond formation.
It may be noted that the oxidative binding may be enzyme enhanced. There
is evidence suggesting that disulfide/sulfhydryl drug and protein thiol bond
formation in vivo is non-specific (Ziegler, 1985), may be catalyzed by a 'disulfide
interchange protein' (Goldberger et al., 1963) since the in vitro rates of disulfide
formation under non-physiological conditions were slower than the in-vivo rates.
The enzymes catalyzing the thiol- disulfide exchange reactions are present in the
cytosol and membrane fractions (cited in, Ziegler, 1985). One such enzyme, the
protein disulfide isomerase, PDI, has been shown to catalyze, the scission as well
as disulfide bond formation between disulfide/sulfhydryl drugs and tissue
sulfhydryl groups (Edman et al., 1985 & Darrow et al., 1988).
These findings suggest the probable involvement of disulfide-bond
formation in the mechanism of accumulation of the lipoic acid based chemical
delivery system for drug targeting to lung tissue as shown in Figure 7.


18
RCOCr(CH
2)5N0
s~s
CDS
' RCOO- (CH2)5 ^
SH SH
v /
^ Enzymatic
'
in vivo redox
reactions
Figure 7: Proposed Mechanism of Delivery by the Lipoic Acid CDS:
In the scheme, the inactive lipophilic CDS after administration enters lung tissue. In
the pharmacokinetic phase the lipolvl moiety undergoes disulfide exchange with the
sulfhydrvl groups of cvsteme residues of lung protem. This serves to anchor the CDS
while esterases release the active parent drug.


19
In addition, a review of available literature revealed reports that some sulfur
containing compounds, sulfhydryl and disulfide containing drugs covalently bind
with sulfhydryl groups of tissue proteins in rats (Livesey et al., 1990; & Miwa et
al., 1988), and in rabbit and human tissue (Tabachnick, 1982). Naturally
therefore, one may expect a possible relationship may exist between
disulfide/sulfhydryl drug binding and PD1 distribution and tissue sulfhydryl. The
relative tissue distribution/activity of PDI in the rat has been estimated to be of the
order (Edman et al., 1985):
liver > pancreas and kidney > lung > testes and spleen > heart > brain.
It has also been observed that, for the rabbit, tissue sulfhydryl content
(extracted with hot 80% ethanol), in mmoles/gram wet weight (or mL wet volume
for blood), was of the order (Ellman, 1959):
kidney (10.1) > Lung (2.2) > Liver (1.9) Blood 5.36 x 10'3
Biologically, sulfhydryl groups occur in tissue mainly as the amino acid
cysteine (Perret and Rudge, 1985). Cysteine, along with its disulfide cystine
occurs in body fluids and cells as well as in most animal proteins. Homocysteine
is found as the free thiol, its homodisulfide and mixed disulfides in man. Reduced
glutathione (GSH), the cysteine-containing tripeptide, is found in high
concentrations in most mammalian tissues where it fulfills many roles including
maintenance of the oxidation status of the cell.
Lipoic Acid:
The proposed use of lipoic acid in the CDS is made even more attractive by
the fact that lipoic acid is non-toxic and it is required as a co-enzyme for acyl
transfer and redox reactions in living systems (Kijima et al., 1984).


20
Alpha-Lipoic acid (i.e. 1.2-dithiolane-3-pentanoic acid, or thioctic acid) is
the internal disulfide of 6,8-dithioctanoic acid. It is the coupler of electron and
group transfers catalyzed by a-keto acid dehydrogenase multienzyme complexes.
As coenzyme, lipoic acid forms part of these enzyme complexes which are
centrally involved in carbohydrate metabolism. The role of lipoic acid within the
pyruvate dehydrogenase complex in catalyzing the reaction of pyruvate with
NAD+ and Coenzyme A (CoA) is illustrated in Figure 8 (Zubay, 1986). The
enzyme complex consists of three proteins: a pyruvate dehydrogenase which is
thiamine pyrophosphate (TPP) dependent and designated Ej-TPP; dihydrolipoyl
transacetylase (E2-lipoyl-S2) which contains the oxidized form of lipoic acid
covalently bonded through amide linkages to the e-amino group of lysine; and a
dihydrolipoyl dehydrogenase, a flavoprotein designated E3-FAD.
In the scheme illustrated below, pyruvate is decarboxylated by EpTPP.
The resulting activated acyl group is accepted by lipoic acid (1) which eventually
then transfers it to CoA (to be utilized in intermediary metabolism). The Lipoic
acid gets reduced to dihydrolipoic acid, DLAc, (3) which is then oxidized by FAD
(4) to give back lipoic acid, the reaction being coupled with the formation of
NADH (a reducing equivalent in in-vivo redox reactions). The chemical aspect of
the conenzymatic activity of lipoic acid is thus to mediate the transfer of electrons
and activated acyl groups resulting from the decarboxylation and oxidation of a-
keto acids within the complexes. In this process, lipoic acid is itself transiently
reduced, and this reduced form is the acceptor of the activated acyl group. This
dual role of electron and acyl group acceptor enables lipoic acid to couple the two
processes (Zubay, 1986).


21
CH.COCOj
CO,
1
Figure 8: Coenzymatic Function of Lipoic Acid (Zubay, 1986)


22
Chlorambucil:
HOOC(CH2)3-^)-N
^ch2ch2ci
ch2ch2ci
Chlorambucil
Chlorambucil, (4-p-[bis(2-chloroethyl)amino]phenylbutyric acid), is a
bifunctional nitrogen mustard alkylating agent that has shown wide clinical
activity as an antineoplastic agent particularly against chronic lymphocytic
leukemia and malignant lymphomas (Oppitz et al., 1989; & Greig et al., 1990)
primarily due to its alkylation reaction with DNA. It also binds to nuclear proteins
causing disturbances leading to cell death (Riches and Harrap, 1975). The
alkylation proceeds through a cyclic ethyleneimmonium ion or carbonium ion
intermediate which is susceptible to attack by nucleophiles.
Chlorambucil
i
t
+
CH2CH2CI
the ethyleneimmonium ion


23
This carbonium ion intermediate which is essential for pharmacological
activity is also readily formed in aqueous solution where it is attacked by
nucleophiles such as water. Thus chlorambucil and other nitrogen mustards have
been reportred to have very short shelf-lives in aqueous solutions Chlorambucil
12 minutes, and Mephalan about 25 minutes (Ehrsson et al., 1980). Active
metabolites of chlorambucil include 3,4-dehydrochlorambucil and phenylacetic
acid formed in vivo via (1-oxidation. Although the drug is rapidly absorbed from
the gastrointestinal tract, ionization at physiological pH limits its ability to
penetrate cells. Besides, there are problems associated with its use which include
a wide variety of major but usually dose limiting toxicity involving hematologic
(e.g. myelosuppression and anemia), gastrointestinal (nausea, anorexia),
neurologic (CNS effects seizures, ataxia etc.), dermatologic and even mutagenic
and carcinogenic effects, introduction of the lipoyl group to mask the ionizable
carboxylic acid group will increase its lipophilicity and hence enhance its cellular
uptake in addition to the desired targeting capability and ultimately reduce its
toxicity.
Cromolyn (DSCG):
Cromolyn, in the form of its sodium salt, disodium cromoglycate, DSCG, is
a bischromone, the disodium salt of l,3-bis-(2-carboxychromon-5-yloxy)-2-
hydroxypropane.
Disodium Cromoglycate


24
DSCG is used primarily in the prophylactic treatment of asthma, and
allergic rhinitis. Since its first introduction in 1968, it has increasingly and
successfully been used in many medical conditions including allergic disorders of
the upper respiratoiy tract, eye and ear problems, food sensitive enteropathies, a
variety of skin conditions such as eczema (Kuzemko, 1989), and more recently it
has been reported to inhibit benzo(a)pyrene induced tumors in rats (Vlckova et al.,
1989).
Mechanism of action of DSCG
in the asthmatic response, the antibody IgE is produced by lymphoid tissue
in response to the extrinsic allergen. The IgE becomes fixed to mast cells in the
bronchial walls in the sensitized individual. Exposure to further allergen results in
antigen-antibody reaction occurring on the surface of the mast cell which results
in the release of the mediators of anaphylaxis which include histamine, slow-
reacting substance of anaphylaxis (SRS-A) which produces prolonged
bronchoconstriction, eosinophil chemotactic factor (ECF-A), bradykinin and
others. This constitutes the early asthmatic response. The mediators also cause
alteration in capillary permeability, resulting in the entry of IgG and leukocytes
into the bronchial connective tissue resulting in a Type III delayed complement-
fixing reaction (the late asthmatic reaction) leading to leukocyte damage, release of
lysosomes, local tissue damage and release of prostaglandins and other mediators
which result in further bronchoconstriction and other symptoms (Brewis, 1975).
The DSCG exerts its anti-allergic activity by inhibiting the release of mediators
(such as histamine, and some leukotrienes) from mast cells and thereby preventing
the onset of asthmatic symptoms due to the bronchoconstrictor and inflammatory
effects of these mediators. There are two main phases of the asthmatic response,
the early asthmatic response, EAR, and two stages of the late asthmatic responses,


25
LARs (Holgate, 1989). Following allergen challenge, the EAR and the first LAR
but not the second LAR are inhibited by the prior administration of DSCG
(Holgate, 1989). The lack of effect of DSCG on the second LAR may relate to the
limited bioavailability of DSCG such that by the onset of the second LAR (72
hours), the drug has been completely cleared from the body. This is supported by
Matolli et al. (1987), who demonstrated that the administration of DSCG between
the EAR and the first LAR inhibited the second LAR. DSCG acts by inhibiting
mast cell degranulation and thereby preventing the release of the preformed
mediators of anaphylaxis. It also interferes with the migration and function of
eosinophils. DSCG has been found to inhibit the cellular uptake of Ca2+
(Hemmerich et al., 1991). The mechanism by which DSCG acts is not precisely
known but it is believed to work by inhibiting the flux of calcium, Ca2+, ions
(Vlckova et al., 1986, & Holgate, 1989). The uptake of Ca2+ being an essential
trigger to activate microfilament contraction responsible for exocytotic secretion.
The antimutagenic activity of DSCG reported by Vlckova et al., 1986, is also
believed to operate via this mechanism. The known pharmacological activity of
DSCG therefore lies in its ability to block pathological exocytotic secretion.
Exocytotic response requires contact with cytoplasm by signaling mechanisms, in
addition to a rise in cellular Ca2+. The rise in intracellular Ca2+ is known to be a
factor which triggers exocytosis in response to external stimuli at the plasma
membrane. With DSCG blocking the influx of Ca2+, the preserved Ca2+ balance
is thought to be a factor for the preservation of cell functions within the
physiological range (Vlckova et al., 1986)
Need for Improved Delivery of DSCG:
Although DSCG is the safest anti-asthma drug currently in use (Kuzemko,
1989), it has very poor bioavailability as it is too polar and very water soluble. It


26
is thus poorly absorbed, and rapidly cleared from the body. After oral
administration, only 1% of the dose is absorbed in man, and after inhalation, less
than 8% of the dose reaches the lung and is absorbed systemically (Cox et al.,
1970, & AHFS Drug Information, 1990). After intravenous administration, the
drug is rapidly cleared from the plasma and tissues, and accumulated in the liver or
kidney prior to biliary or urinary excretion. In rats, 85% of the injected dose is
cleared from the plasma at a rate with half-life of about 8 minutes. In man, the
corresponding plasma or lung Ty2 is about 30 minutes (Cox et al., 1970).
For the treatment of asthma, DSCG is clinically effective only after
inhalation administration. Pulmonary drug delivery is primarily used to treat
conditions of the upper respiratory tract. The drug delivered must be deposited
onto the bronchial epithelium in therapeutic quantities. But generally, only about
10% of the dose delivered remains in the lungs (Newman et al., 1982). The
process of drug delivery via the inhalation route is inefficient due to a combination
of factors which include the mode of delivery, the point of inspiration relative to
the triggering device and respiratory variable such as the breathing pattern. The
anatomy and pathophysiological state of the respiratory tract can also markedly
affect the drug delivery process. Additionally, structures in the upper respiratory
tract function to minimize the inhalation of particulate materials (Washington et
al., 1989; and Ganderton and Jones, 1987). The problems inherent with inhalation
delivery especially in pediatric cases, the limited bioavailability, and the other
possible uses of DSCG make the need for other modes of administration as well as
improved or targeted delivery to lung welcome. The proposed lipoyl derivative is
expected to improve the lipid solubility of DSCG and therefore enhance its
bioavailability irrespective of route of administration. This relatively more lipid
soluble derivative will be more able to penetrate the cells, be retained in tissues
longer (i.e. longer biological half-life), enhance selective delivery to lung tissue


27
and also, possibly, enable the administration of the drug by additional routes other
than inhalation.
Predicted In-Vivo Metabolism of CDS
In the work presented in this dissertation, the l,2-dithiolane-3-pentyl
moiety of D,L -lipoic acid was investigated for use as a targeting moiety, based on
a concept related to the CDS approach, for drug targeting to lung tissue, using
Chlorambucil and Cromolyn Sodium as the respective model parent drugs. Three
final derivatives were prepared and studied namely, the Chlorambucil CDS or
MS-2; Cromolyn CDS-1 or MS-4; and Cromolyn CDS-2 or MS-17. Some aspects
of the in-vivo metabolism of the CDS, characteristic of a rationally designed drug,
may be fairly accurately predicted. Both model CDS's were expected to undergo
ester hydrolysis to release the respective parent drugs. Chlorambucil (Figure 9),
as previously known (Greig et al., 1990), undergoes further metabolism via (3-
oxidation to produce the metabolites 3,4-dehydrochlorambucil and phenylacetic
mustard both of which like the parent drug are reactive as alkylating agents (these
were probably the two metabolites detected during the HPLC assay of the
hydrolysis products of the CDS graphically presented in Figure 24, page 84). The
predicted metabolism of the cromolyn CDS is also shown in Figure 10. In both
pathways, the liberated lypoyl moiety would be expected to participate in its
known in-vivo coenzymatic function or undergo degradation and elimination
characteristic of thiol metabolism.
Thiol/disulfide compounds in general undergo metabolic transformations by
desulfuration or transfuration into hydrogen sulfide, sulfates or other sulfur
compounds. In-vivo, thiols and disulfides readily interconvert.
-SH + V202 <=> -S-S- + H20.


28
Keilin (1930) has shown that this reaction is catalyzed by cytochrome C and
cytochrome oxidase acting together.
(CH2>5'OOC'(CH2>3
/CH2CH2CI
N
^ch2ch2ci
Chlorambucil CDS
I
H00C(CH2)3~O"Nv
^ch2ch2ci
'ch2ch2ci
Chlorambucil
rr
S- s
(CH2)5-OH
LIPOLOL
I
<-
J
1
1
^CH^CHoCI
1
in-vivo redox
ch2ch2ci
reactions
3,4 Dehydrochlorambucil
HOOC-CH 2
H^-N
ch2ch2ci
ch2ch2ci
Thiol
Metabolism
Phenylacetic Mustard
Figure 9: Predicted Metabolism of Chlorambucil CDS


29
o2nov^
O COO-lipoyl
Cromolyn CDS-2 (MS-17)
carboxylesterase
T
COO'
Cromolycate Nitrate (MS-16)
nitrate
reductase
COO"
/
cromoglycate
(active drug)
Direct Elimination
Urinary & Biliary routes
GSSH
HO
O COO-lipoyl
Cromolyn CDS-1 (MS-4)
2
X
thiol
metabolism
Lipoamide
1
In-vivo Redox &
acyl transfer reactions
Figure 10: Metabolism of Cromolyn CDSs


30
The pathways for thiol metabolism are mostly oxidative and while not
precisely known for each individual thiol/disulfide, the metabolic pathway for the
lipoyl moiety may be expected to follow a general scheme and resemble that of
aspargusic acid, a dithiolane compound, (Waring et al., 1987). The dithiolane
compound is reduced to the dithiol which then undergoes S-methylation. In
humans, the enzymes thiol methyltransferase TMT, and thiopurine
methyltransferase, TPMT, catalyze the S-methylation of thiols (Bremer &
Greenberg, 1961; Drummer et al., 1982; and Glauser et al., 1993). TMT is
membrane bound and catalyzes preferentially the S-methylation of aliphatic -SH
compounds whereas TPMT is cytoplasmic and catalyzes the S-methylation of
aromatic and heterocyclic -SH groups such as lipoic acid. The S-methyl
intermediate may then undergo [3-oxidation of the ring carbons (Waring et al.,
1987), liberating methanethiol. Subsequent oxidation, methylation and
dimerization would result in a variety of products such as dimethyl sulfide,
dimethyl sulfoxide, dimethyl sulphone and methanethiol. Additionally, the thiol
may be oxidized to sulfate or sulfite through a variety of oxidizing systems. No
distinctive pathway for the oxidation is known, but it is known to involve
xanthine oxidase or the cytochrome oxidase systems (Kun, 1967).


CHAPTER 3
RESEARCH DESIGN AND SPECIFIC OBJECTIVES
Objective
The main objective of the work described in this dissertation was the
development of a novel chemical delivery system to achieve the selective delivery
of drugs to lung tissue and thereby lessen unwanted side effects, especially in the
treatment of lung cancers. The system is also expected to enhance the
bioavailability of highly polar hydrophilic drugs like cromoglycic acid, and
increase the duration of action of drug in the lung by prolonging its retention in
lung tissue through probable formation of disulfide linkages and thereby ensuring,
to some extent, a 'slow release' of the anchored drug entity. By these newly
incorporated capabilities, the system will also work to optimize the therapeutic
index of drugs used in the management of lung diseases.
Synthesis
In the design process, employing a variation of the CDS approach
(illustrated in Figure 5), each of the two model drugs used in this work were
converted into the corresponding CDSs according to the following schemes
respectively:
31


32
Chlorambucil CDS
The synthesis of the chlorambucil CDS, analogous to the species TD' in
Figure 5, was achieved by the direct esterification of the corresponding alcohol of
lipoic acid, the targetor moiety, T, with the carboxylic acid group of chlorambucil,
(the parent drug, D), using Dicyclocarbodiimide (DCC) as a coupling agent. The
DCC converts the acid chlorambucil, (I) into a compound with a better leaving
group (5) and thereby helping drive the reaction via the following mechanism:
R C.
+ DCC
O
2
3
ESTER PRODUCT
Figure 11: Mechanism of Esterification with DCC


33
(CH 2)5 -OOC-(CH 2)3-^)-N^
ch2ch2ci
CH2CH 2CI
s- S
Chlorambucil CDS, (MS-2)
Cromolyn CDS
In a similar approach, the lipolyl group (T) was coupled to the cromolyn
parent drug (D) to give MS-4, the 'Cromolyn CDS-1' or TD'. Also. In an attempt
to enhance the aqueous solubility of the cromolyn CDS-1, a modifier group 'F in
the form of the nitrate (N02) group was introduced to replace the OH- group on
the carbon-2 of the propanyl link between the two chromone heterocycles to give
the 'Cromolyn CDS-2'. This site was chosen primarily because it was assumed to
have no role in the pharmacological activity of the drug (Cairns et al, 1972), and
also because it seemed to be the easiest to nitrate.
HO
CH"CH?-0 O
SS -I
2
Cromolyn CDS-1, (MS-4)


34
(CH2)5^y^
SS
2
Cromolyn CDS-2, (MS-17)
Application of the DCC esterification method to the cromolyn system
resulted in a very poor yield less than 5%. This might have been due to the
following two reasons:
1. The anion (2) generated from the reaction of the DCC with the acid form of
cromolyn (step 1 in the mechanism described in Figure 11 above) was much
less nucleophilic due to the electron withdrawing effect of the aromatic
chromone nucleus of the cromoglycic acid. Hence the formation of the species
(4) leading to the formation of the intermediate compound (5), having the
better leaving group (step 2) was not very favored.
2. Under the conditions used for the esterification, i.e., refluxing 60 80C in
solution for prolonged period -- more than 24 hours, lipolol (or the dithiolane
ring) is known to be unstable and undergoes polymerization (Walton et al.,
1955; & Brown and Edwards, 1969). Indeed the bulk of product extracted
from the reaction was a sticky plastic-like substance.
With these considerations taken into account, the aim then was to find a
method which required little or no heating and involving solubilization of the
lipolyl species for the least time possible. These conditions were realized through
the an adaptation of a method developed by Pfeffer et al., (1972) in which the


35
corresponding alkyl halide of lipoic acid was reacted with the disodium salt
(cromolyn) in hexamethyltriphosphoramide (HMPA or HMPT). This method
resulted in quantitative yield (over 85% pure product), occurred at moderate
temperatures (35 40C) and was complete in about 30 minutes.
Evaluation of Mixed-Disulfide-Bond Formation as a
Probable Mechanism for CDS Selectivity for Lung Tissue
In-vivo experiments described later on in this dissertation showed that there
was enhanced delivery of chlorambucil and cromoglycic acid (cromolyn) to rat
and rabbit lung tissues through the lipoic acid CDS, and of progesterone to rat lung
tissue via the 3-spirothiazolidine based CDS (Bodor, 1984). It was suggested that
the mechanism of this enhanced delivery involves the probable formation, in-vivo,
of mixed disulfide linkages between free sulfhydryl groups generated on the CDS
and tissue sulfhydryl mainly in the form of cysteinyl residues. In order to
experimentally establish or support the proposed mechanism, a series of in-vitro
binding experiments were designed which involved incubating the drug/CDS with
respective tissue homogenates and determining:
1. The amounts of free unbound drug and drug bound through disulfide
linkages by treatment of the bound drug fraction (in the form of
precipitated protein) with 1,4-dithiotreitol, DTT (a disulfide
reducing agent). Any amounts of drug detected following this
treatment indicated the drug originally bound by disulfide linkages;
2. The extent of CDS/drug-tissue binding in the presence of N-
ethylmaleimide (NEM), a sulfhydryl blocking agent; and also,


36
3. The relationship, if any, between tissue sulfhydryl content and drug
binding.
4. The absence of mixed disulfide binding in the absence of NEM and
its occurrence in the absence of NEM is evidence of mixed disulfide
binding.
Enzyme Inhibition Studies
Since a model CDS consisted of the active (parent) drug attached to the
targeting moiety by an ester linkage, the metabolic conversion of the CDS to the
active drug was presumed to occur via ester hydrolysis catalyzed by a
carboxylesterase (B-esterase) enzyme. It was therefore necessary to inhibit these
enzymes in the tissue homogenate so that a reasonable amount of the intact CDS
was maintained throughout the binding studies. The inhibitor used for this
purpose was the B-esterase inhibitor bis-(p-nitrophenyl)phosphate (BPNP) sodium
salt (Sterri & Fonnum, 1987; and Yamada et al., 1992) and the method for the
esterase inhibition was adapted from Yamada et al. (1992). Studies were included
to establish the optimum concentration of inhibitor needed to reasonably inhibit
the esterases.
Kinetics of Mixed Disulfide-Bond Formation
With the presence of BPNP in the homogenate during the binding studies it
was necessary to determine also whether its presence had any effect on the extent
of CDS binding. In addition, further kinetic studies were conducted to shed some
light on such aspects of the mixed-disulfide binding with respect to time,
concentration and tissue type.


37
Tissue Sulfhvdrvl
Since binding to tissue sulfhydryl was suspected to be a probable
mechanism of site selective delivery by the CDS, the tissue sulfhydryl content for
the various tissues were determined in an attempt to relate the extent of mixed-
disulfide binding to tissue sulfhydryl in order to expatiate on differences, if any, in
the binding of the CDS to the various tissues and help provide an estimate of the
'magnitude of binding' per tissue type.
Research Protocol
The experimental protocol described in this dissertation comprised
1. Synthesis of the various chemical delivery systems/derivatives for
chlorambucil and cromolyn respectively.
2. HPLC assays for detection of respective parent drugs and CDS/
derivatives in biological fluids.
3. Determination of physicochemical parameters such as octanol/water
partition coefficient (or Log P), and in-vitro stability in buffer and
various biological media.
4. Comparative in-vivo studies in rats and Rabbits to determine the ability
of CDS to selectively deliver drug to lung tissue relative to parent drugs.
5. Determination of tissue sulfhydryl (Total-Thiol, Protein bound Thiol,
and Non Protein Bound Thiol)
6. In-vitro binding and related studies to experimentally support the
proposed mechanism of delivery by the CDS comprising:
i. Enzyme inhibition Studies.


38
ii. In vitro binding experiments, namely
Determination of free unbound drug/CDS, and drug or
CDS bound to tissue via mixed disulfides linkages.
Determination of bound drug/CDS in presence of the
tissue sulfhydryl-blocker, N-ethylmaleimide,
Effect of BPNP on disulfide binding.
iii. Evaluation of kinetics of binding with respect to
Concentration dependence and
Dependence of binding on time.


CHAPTER 4
EXPERIMENTAL METHODOLOGY
Materials
All chemicals and reagents used were reagent grade. Solvents and reagents
used for the High Performance Liquid Chromatography (HPLC) and Ultra-Violet
spectrophotometric analyses were HPLC or spectroscopic grade. Chlorambucil
and cromolyn were obtained from Sigma Chemical Company, St. Louis, Missouri.
Other chemicals and solvents were obtained from Sigma Chemical Company,
Aldrich Chemical Company, Milwaukee, Wisconsin, and also from Fisher
Scientific Company.
For the analytical and compound characterization methods, thin-layer
chromatography (TLC) was performed using EM Science DC-plastic foil plates
coated to a thickness of 0.2 mm with silica gel 60 containing Fluorescent (254)
indicator. Spots were detected by exposure to short-wavelength UV light. All
melting points were determined using the Fisher Johns Melting Point Apparatus,
and were uncorrected. iH-NMR data were measured in CDCI3 or in dimethyl-/6
sulfoxide, (ig-DMSO, (for the polar compounds that would not dissolve in the
CDCI3) on a Varan EM-390 NMR Spectrophotometer and chemical shifts
reported in parts per million relative to tetramethylsilane. Infrared Spectra were
recorded with a Perkin Elmer 1420 Ratio Recording Infrared Spectrophotometer
39


40
and were made in KBr pellets (~ 1 mg of compound in 100-mg pellet), paste in
mineral oil, or neat liquid. Ultraviolet (UV) spectra as well the UV spectro-
photometric analyses were performed using the Varan CARY 210 UV/Visible
Spectrophotometer with quartz cuvettes up to A, < 200 nm and an optical path
width of 10 mm. Mass spectrometry was performed by Fast Atom Bombardment
(FAB) on the Kratos MFC 500 Mass Spectrometer. Elemental analyses of the
purified specimens were performed by the Atlantic MicroLab Inc., Atlanta,
Georgia. Instrumentation for the HPLC assays included a Spectra Physics SP
8810 Precision Isocratic Pump, the SP 8450 UV/VIS Detector (operated at
wavelengths of 240 nm for cromolyn and its derivatives, and at 254 nm for
chlorambucil and its derivative) and the SP 4290 Integrator. The HPLC sample
loop size was 20 pL. The HPLC method used was reverse-phase (with ion-pairing
or ion suppression in the case of cromoglycate estimation). The stationary phase
columns used were Hypersil ODS-2 Qg and Cg Columns, both 100 mm x 4.0 mm
internal diameter with guard columns of similar packing. The ion-pairing reagent
was benzyltributylammonium chloride (BTBA-C1).
All in-vivo studies were conducted in laboratory animals 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 83).
The animals used in the studies were male New Zealand Albino rabbits weighing
about 3 Kg each (obtained from Kel Farm, Alachua, Florida), and white male
Sprague Dawley rats each weighing between 300 and 350 grams (obtained from
Harlan Sprague Dawley Inc., Indianapolis, Indiana). Human blood used in the in
vitro stability studies, was obtained from this investigator himself, and from a staff
member at the Center for Drug Discovery (University of Florida College of
Pharmacy) where this research was conducted. Dog blood was obtained from the
Animal Resources unit of the University of Florida Veterinary School.


41
Methods
Synthesis
Chlorambucil CDS. (MS-2)
a. 1,2-dithiolane-3-pentanol, (dl-lipolol), MS-1; Lipoic acid, (2.06g, 10
mmol) was placed in a flame dried 250 mL flask fitted with a stirrer, dropping
funnel, and connected to an oil bubbler to maintain a Nitrogen atmosphere. 70 mL
of anhydrous chloroform was added followed by 1.0 M catecholborane, in
tetrahydrofuran (50 mL, 50 mmol) dropwise. The mixture was refluxed (70 80
C) for about 6 hours. 20 mL of cold water was then added dropwise and the
organic solvent evaporated in vacuo. 50 mL of dichloromethane was added, and
the mixture extracted with one 25 mL aliquot of water followed by six 25 mL
aliquots of 1.0M NaOH to remove the catechol. The organic portion was dried
with sodium sulfate, filtered and solvent evaporated in vacuo. The crude product
was purified by column chromatography (silica gel, methylene chloride/ethyl
acetate, 4:1).
b. Lipolyl ester of Chlorambucil (i.e. the Chlorambucil-CDS or MS-2):
DL-lipolol, MS-1, (1.92g, 10 mmol) in 50 mL of dichloromethane (CH2CI2) was
added to chlorambucil (3.04g, 10 mmol) in 50 mL of CL^C^. An excess of DCC,
1,3-dicyclocarbodiimide, (8.25g, 40.0 mmol) in 20 mL of CH2CI2 was then added.
A catalytic amount of 4-dimethylaminopyridine (DMAP)approximately 1 mg,
was added to the reaction mixture and stirred at room temperature until the
reaction was complete (about 24 hoursmonitored by TLC). At the end of the
reaction, the mixture was washed with two 20 mL portions of water. The organic
layer was then evaporated in vacuo. The crude product, a yellowish brown
viscous oil, was purified by column chromatography on silica gel using toluene as


42
the eluant. The overall scheme for the synthesis of the chlorambucil CDS is
summarized in the following scheme
(CH2)4COOH
s-s
1. Catecholborane

2. H,0
(CH2)5-OH
Lipoic Acid
Chlorambucil
(CH
2^5
-OOC-(CH2 )3
-H
CH2CH2 Cl
CH2CH 2C1
s-s
MS-2
Figure 12: Synthesis of Chlorambucil-CDS (MS-2)
Cromolyn CDS-L (MS-4)
a. 5-bromo-3-penty¡-1,2-dithiolane, (Lipolyl bromide)-, Lipolol, (0.58g, 3
mmol) and triphenylphosphine (2.37g, 9 mmol) were dissolved in 15 mL of
anhydrous tetrahydrofuran, THF. Zinc bromide (0.68g, 3 mmol) in 10 mL THF
was added followed by diethyl azidodicarboxylate, DEADC, (1.57g, 9 mmol) in 5
mL THF. The mixture was stirred at room temperature under nitrogen atmosphere
for about 2 hours (i.e. until lipolol had disappeared). Methanol (2.0 mL) was then


43
added to destroy the excess reagent. After 5 minutes, the mixture was extracted
with ether (20 mL), and the ether layer washed with 12 mL aliquots of water,
saturated Na2CC>3, and saturated NaCl successively, and the organic solvent
evaporated in vacuo. The product was then extracted twice into hexane, leaving a
residue of triphenylphosphine oxide. After evaporation of the hexane fraction, the
solid residue was again extracted by trituration with hexane, and the organic
extract evaporated in vacuo to give the crude product. The crude product was
purified by column chromatography on silica (50g, eluant was Ethyl Acetate/
Hexane, 1:9).
S~S
DEADC, Anhyd. THF S S
Lipolol
Lipoyl Bromide
O COONa 2
Cromolyn
HO
I
CH~CH2_o o
S-S J 2
MS-4
Figure 13: Synthesis of Cromolyn-CDS-1 (MS-4)


44
b. 1,3-bis(2-Upolyloxycarbonyl-chromon-5-yloxy)propane i.e. the Lipolyl
ester of cromolyn or the Cromolyn-CDS, (MS-4): Hexamethylphosphoramide,
HMPA, 10.0 mL, was added to disodium cromoglycate, DSCG, (0.77 g, 1.50
mmol) in a 50 mL flask with stirring at room temperature and nitrogen atmosphere.
Lipolyl bromide (1.42 g, 6.0 mmol) was added and the mixture warmed to 45 -
50C for about 40 minutes to aid in the dissolution of the DSCG. Stirring was
continued for about two hours at room temperature (or until all of the cromolyn
has disappeared) and the reaction mixture poured into 35 mL of water. The
mixture was then extracted with two 20 mL portions of CH2Cl2, and the combined
organic layers washed with several 20 mL portions of water and dried (Na2SC>4).
The crude oily product was purified by column chromatography (silica gel,
Chloroform/Ethyl acetate 10:1, v/v). Fractions containing the diester were pooled
together and evaporated in vacuo, the resulting oily product was recrystallized
from ether by dissolving it in 2 mL of chloroform and adding the resulting solution
dropwise to 50 mL of ether with stirring. The yellowish white crystals obtained
were filtered, washed with cold ether and vacuum dried in the presence of P205.
The scheme for the synthesis is summarized in Figure 13.
Cromolyn CDS-2. (MS-17)
a. \,3-bis(2-ethoxycarbonylchromon-5-yloxy)-2-hydroxypropane,
(Cromolyn diethyl ester, MS-11): DSCG (2.05g, 4 mmol) was placed in a 50 mL
two-necked flask. 15 mL HMPA was added with stirring and N2 atmosphere.
Bromoethane (3.5g or 4.5 mL, 32 mmol, 400 % mol equivalents) was added. The
mixture was warmed to about 45 50C for about 30 minutes until a clear solution
formed. The reaction mixture was poured into 50 mL water and extracted with
two 25 mL portions CH2C12. The organic layer was then washed with two 25 mL


45
portions water dried (Na2S04) and evaporated in vacuo. The resulting product
was redissolved in chloroform and added dropwise to 100 mL ether with stirring.
The white crystals obtained were filtered, washed with cold ether, and then dried.
b. 7,3-bis(2-ethoxycarbonylchromon-5-yloxy)-propan-2-nitrate, (diethyl
cromolyn nitrate ester, MS-15): The nitrating agent, Acetylnitrate, CH3C(0)-
ONO2 was prepared by the method of Manstch and Bodor (cited in Bodor et al.,
1980). Acetic anhydride (20 mL) was placed in a 50 mL conical flask in an ice-
water bath (approx. 4C) and 2 mL of concentrated HNO3 added. The mixture
was stirred in the cold for two hours and the resulting solution containing the
acetylnitrate was used directly in the subsequent reaction.
Cromolyn diethyl ester, MS-11, (1.05g, 2 mmol) was dissolved in 25 mL of
chloroform, and 10 mL of the nitrating reagent solution added. A clear yellow
solution formed which soon turned cloudy. The mixture was stirred at room
temperature for about 30 minutes and then poured into 50 mL water. The
chloroform layer was separated, washed with water, dried (MgSC>4) and
evaporated in vacuo. The resulting solid crude product was dissolved in methanol
and chromatographed over silica (mobile phase chloroform/methanol, 10:1). The
fractions containing the nitrate ester were pooled together concentrated and
recrystalyzed by adding dropwise to ether as before.
c. l,3-bis(2-carboxychromon-5-yloxy)-propan-2-nitrate disodium salt,
cromolyn nitrate disodium salt, MS-16: Diethyl cromolyn nitrate ester, MS-15,
(0.57g, 1.0 mmol) was suspended with stirring in 5 mL absolute ethanol and
treated dropwise with 2 mL of IN NaOH (2.0 mmol). The resulting mixture was
refluxed for 1 hour and after tap cooling, about 10 mL ethanol was added. The
mixture was chilled in the freezer compartment to precipitate the sodium salt. The
solid was filtered off, dissolved in a minimum amount of hot water, filtered,
reheated to boiling. The hot solution was then added slowly to boiling ethanol (15


46
mL). The solution was cooled under the tap and refrigerated for about one hour.
A small amount of ether (10 mL) was added to aid precipitation. The precipitated
sodium salt was filtered and dried (vacuum, P2O5).
d. \,3-bis(2-lipolyloxycarbonylchromon-5-yloxy)pwpan-2-nitrate, i. e.
Cromolyn lipolyl nitrate ester, Cromolyn CDS-2, or MS-17: Hexamethyl-
phosphoramide, HMPA, 10.0 mL, was added to cromolyn nitrate disodium salt,
MS-16, (0.38 g, 0.72 mmol) in a 50 mL flask with stirring at room temperature and
nitrogen atmosphere. Lipolyl bromide (0.44 g, 1.80 mmol) was added and the
mixture warmed to 45 50C. The thick slurry turned into a light clear liquid.
Stirring was continued at this temperature for about two hours, or until the
disodium salt, MS-16, had disappeared). The reaction mixture was then poured
into 25 mL of water and extracted with two 15 mL portions of CH2CI2, and the
combined organic layers washed with two 10 mL portions of water and dried
(Na2S04) and evaporated in vacuo. The crude oily product was purified by
column chromatography (silica gel, Chloroform/Ethyl acetate 10:1, v/v). Fractions
containing the diester were pooled together and evaporated in vacuo, the resulting
oily product was recrystallized from ether by dissolving it in 2 mL of chloroform
and adding the resulting solution dropwise to 25 mL of ether with stirring. The
yellowish white crystals obtained were filtered, washed with cold ether and dried
(vacuum, P2O5).
The multi-step synthesis of the cromolyn CDS-2 is summarized in the
following scheme (Figure 14).


47
HO
I
chtch2-o o
O COONa
Disodium
Cromoglycate
1 J
o 'COOC2H5
Cromolyn Diethyl Ester, MS-11
O
II
ch3c -ono,
Acetyl Nitrate
Reagent
o2no
o2no
-CHo-O O 1
NaOH, Ethanol
1
CH
ch2-0
0
1
ax
^
Reflux
(Y
COONaJ
2
o COOC2H5
Cromolyn Nitrate Disodium Salt
MS-16
o2no
Figure 14: Scheme for the Synthesis of Cromolyn CDS-2


48
HPLC Assays:
Reversed phase HPLC methods were used to resolve and quantitatively
estimate the respective drug/CDS's and their metabolites in the various biological
and other media. Reversed phase HPLC with ion-pairing was used for the
estimation of cromolyn. The respective efficiencies of the drug recovery methods
from biological media were well over 95% in all cases. Concentrations were
estimated from appropriate calibration curves based on concentration versus area
under curve (AUC) plots, all of which showed linearity (r = 0.999) for the range of
0.1 to 10.0 pM.
For the estimation of chlorambucil and the chlorambucil CDS (MS-2), the
HPLC system included a Cjg stationary phase column (100 mm x 4.0 mm i.d.) and
73.5% Acetonitrile/0.5% Acetic Acid/27% Water as the mobile phase. The UV-
detector was set at wavelength of 254 nm and a sensitivity of 0.01. The solvent
used for drug/CDS recovery from biological media was 5%DMSO/95%
Acetonitrile. For a 'one run' simultaneous resolution of chlorambucil and CDS the
flow rate was adjusted manually according to the following routine: 0.6 mL/min
from 0 3.5 minutes; and 1.5 mL/minute from 3.5 minute to the end of run. The
sensitivity of the HPLC system was 0.09 pg/mL for both drugs.
For the cromoglycic acid CDSs, MS-4 and MS-17, the stationary phase
column used was the same as above, the mobile phase was 75% Acetonitrile/0.1%
Acetic Acid/water run at a flow rate of 1.0 mL/min, and the retention times were;
MS-4, 6.5 minutes, and MS-17, 10.0 minutes. The solvent for drug recovery was
5%DMSO/35% Methanol/60% Acetonitrile and the lower limit of detection was
just less than 0.03 pg/mol for both MS-4, and MS-17.
For the recovery and estimation of cromoglycate anion from biological
media, a novel and simple HPLC procedure (Method B) described below, was


49
developed, and used. In addition this method was compared to a recently
published HPLC procedure (Method A), (Gardiner, 1984), according to the
procedures described below.
In one study, 200 pL of Cromolyn CDS-1 (i.e. MS-4) was added to 2800
pL of whole fresh rat blood, vortexed for 20 seconds and incubated at 37C for 30
minutes. Then, 250 pL samples of the incubation mixture containing the
hydrolysis product of the CDS were removed and analyzed for cromoglycate anion
separately by the two different methods mentioned above, as described below:
1. For the 'Method A', the procedure used included as follows:-
Fifty microliters each of 2N HC1 and saturated NaCl solutions were added to
0.25 mL sample of the incubation mixture.
Two milliliters of ethyl acetate were added, and the mixture vortexed for about
30 seconds. The organic layer was saved and pooled with further two more
extractions with 2.0 mL ethyl acetate.
The pooled organic mixture was evaporated to dryness on a water bath, the
residue reconstituted with 1 mL of mobile phase, centrifuged and the
supernatant analyzed on the HPLC.
2. In 'Method B', the following procedure was used for the drug recovery:
A quarter of a milliliter aliquot of the incubation mixture was added to 0.75 mL
of the protein-precipitating/drug-extracting solvent (composed of
DMSO/Methanol/Acetonitrile, 5:35:60 v/v) in a microcentrifuge tube, vortexed
for 20 seconds, centrifuged, and the supernatant diluted two times with de
ionized water before injecting on the HPLC.
Drug recovery efficacy of HPLC assay
Using 'Method B', samples of respective rat and rabbit tissue homogenates
(25%, 1.8 mL) were spiked with 200 pL of standard 1.0 mM Cromolyn, to an


50
initial concentration of 100 pM cromoglycate anion. After vortexing for 15
seconds, aliquots were taken and extracted via Method B and analyzed on the
HPLC.
OctanolAVater Partition Coefficients (Log P)
For the determination of the OctanolAVater partition coefficients, the
following procedure was used:
Approximately 10 mg of the compound was placed in 2 mL of anhydrous n-
Octanol in a 15 mL tube and shaken vigorously for about 2 minutes and
filtered.
An equal volume of phosphate buffer pH 7.4 was added and the tube sealed
with a plastic cap.
The mixture was equilibrated by repeated inversion of up to 200 times for five
minutes (a method adapted from Craig et al., 1947).
The mixture was allowed to stand for 30 minutes for the phases to fully
separate and the respective phases analyzed separately for the drug.
For the water soluble compounds, (cromolyn or MS-16), the solute was first dis
solved in the pH 7.4 buffer, and an equal volume of n-Octanol added instead.
In Vitro Stability
The in-vitro stabilities of the parent compounds or CDS in isotonic
physiological buffer and various biological media obtained from a number of
species (man, dog, rat, and rabbit) were evaluated by determining the pseudo-first-
order hydrolysis rate constants and hence the respective half-lives from the
disappearance of the drug by linear regression of the natural logarithm of the


51
HPLC peak areas versus time plots for the respective drugs/CDS in the respective
tissue homogenate or buffer media.
The procedure described below for the cromolyn CDS was essentially the
same as that used to estimate the stabilities of the other drugs/CDS in biological
media from the other species or buffer.
Adult white male Sprague Dawley rat weighing about 300g was sacrificed by
decapitation. Blood and tissues were immediately harvested. The tissues
homogenized in cold isotonic phosphate buffer pH 7.4, diluted to 25% and
stored in ice (0 4C) until ready for use. (The blood or tissue homogenates
were used within two hours of harvesting)
Two hundred microliters of 1.0 mM Cromolyn-CDS-1 (MS-17), dissolved in
DMSO/Propylene Glycol/Ethanol (1:2:4 v/v) and kept at 37C, were added to
2800 pL of blood or tissue homogenate pre-incubated at 37C with vortex
mixing. The initial drug concentration was 66.7 pM.
Immediately, and at subsequent time intervals, 200 pL aliquots of the
incubation mixture were pipetted into a micro centrifuge tubes containing 800
pL of the cold drug extracting solvent, vortexed for 20 seconds, and
centrifuged at 13 G for 7 minutes.
The supernatants were diluted two times with deionized water, and 20 pL
portion analyzed on the HPLC.
Hydrolysis Products of CDS
One half of a milliliter aliquots of 1.0 mmole stock solutions of each CDS
were respectively added to 4.5 mL portions of fresh rabbit blood (giving a final
concentration of 100 pM) and incubated at 37C. At various time intervals,
aliquots of the mixture were withdrawn and analyzed for each CDS and its
respective metabolite(s).


52
In- Vivo Distribution Studies
Chlorambucil and CDS in rats and rabbits
Comparison of the in-vivo distribution of the chlorambucil CDS (MS-2)
and parent drug was performed according to the following procedure :-
Fifteen millimolar stock solutions of Chlorambucil and the CDS (MS-2) were
prepared by dissolving in DMSO/Propylene Glycol/Ethanol (1:3:6 v/v) solvent
mixture.
The drugs were administered, intravenously through the ear vein, to white male
New Zealand rabbits weighing between 2.8 and 3.0 kg, at a rate of 0.4 mL / kg
of body weight resulting in a dose of 6 pmoles/kg of each drug (i.e. 1.825
mg/kg of chlorambucil or 2.871 mg/kg of MS-2).
Blood (about 0.25 mL) was drawn at 5, 15 and 30 minute intervals where
possible and mixed with two volumes of cold 5% DMSO in Acetonitrile and
stored in dry ice until ready for analysis by HPLC.
At predetermined time points, each respective rabbit was sacrificed by injection
with an over-dose of pentobarbital. The respective tissue samples were
immediately excised, homogenized with two volumes of cold Acetonitrile and
stored in dry ice until ready for analysis.
The homogenate was then centrifuged, diluted 1:2 with the mobile phase and
then analyzed for chlorambucil and/or CDS where applicable.
For the rats, the dosage rate used was the same as it was for the rabbits and drug
administration was via the tail vein.
Cromolyn and CDS: Distribution in rats and rabbits
A stock solution of the CDS for intravenous administration was prepared by
dissolving about 200 mg of the drug in a little amount of Dimethylsulfoxide


53
(DMSO) and adding propylene glycol (PG) followed by ethanol to give solvent
proportions of 1 DMSO/2 PG/4 Ethanol. The resulting mixture was then the
filtered through a micro-filter and thereafter diluted up to 2000 times on a portion
of the stock solution, and several 20 mL aliquots injected on the HPLC and
concentrations estimated by extrapolation from a calibration curve. The first
dilution was made with DMSO/Acetonitrile (1:1 v/v) and subsequent ones with the
respective mobile phase.
A corresponding stock solution of cromolyn equimolar with its CDS was
prepared similarly. Because of the low solubilization rate of cromolyn in the
above solvent system, the cromolyn solution was made by dissolving the cromolyn
first in a minimum of water and then adding the other solvents to approximate the
proportions mentioned above.
Adult male New Zealand Rabbits weighing between 2.75 and 3.25 kg, and
white male Sprague-Dawley rats (300 350 g) were used. The respective drugs
were administered to the rabbits via the ear vein at the rate of 0.42 mL/kg (i.e.
2.62 pmol/kg or 2.14 mg/kg CDS and 1.34 mg/kg Cromolyn), and to the rats
through the tail vein at 1.0 mL/kg (i.e. 6.22 pmol/kg or 5.08 mg CDS/kg and 3.19
mg Cromolyn/kg).
At pre-determined time intervals, the animals were sacrificed (the rabbits by
the injection of an overdose of pentobarbital, and the rats by decapitation).
Following each sacrifice, tissues were immediately harvested stored frozen in dry
ice, and when needed, subsequently thawed and homogenized (1:3) in the
extracting solvent system. A portion of each homogenate was centrifuged, diluted
with deionized water (cromolyn estimation) or mobile phase (CDS), and analyzed
on the HPLC.


54
Determination of Tissue Sulfhvdrvl
The procedure was based on the methods by Ellman (1959), and Sedlak &
Lindsay (1968). The basis of the assay was that DTNB, 5,5'-dithiobis-(2-
nitrobenzoic acid), a disulfide compound is quantitatively reduced by -SH groups
to form 1 mole of 2-nitro-5-mercaptobenzoic acid per mole of -SH. The
nitromercaptobenzoic anion has an intense yellow color which is used to
spectrophotometrically measure -SH.
The materials used in the assay included:-
- L-cysteine Hydrochloride H2O
- Reduced Glutathione (GSH)
- 5,5'-dithiobis-(2-nitrobenzoic acid), DTNB
- Tris-EDTA Buffer 0.2M, pH 8.2, and 0.4M, pH 8.9
- Ethylenediaminetetraacetic acid-disodium (EDTA-Na2),
- Trichloroacetic acid. (TCA)
The solutions for the assay were prepared as follows:-
- 0.01M DTNB (99.1 mg in 25 mL Methanol)
- Tris EDTA buffer, 0.2M, pH 8.2 was prepared by dissolving
6.05 g Tris, Tris[hydroxymethyl]aminomethane, in deionized
water, adding 25 mL of 0.2M EDTA, and water to make up
the volume to 250 mL, and adjusting pH to 8.2 with IN HC1.
- Tris-EDTA buffer, 0.4M, pH 8.9 was similarly prepared.
Standard solutions of GSH, and Cysteine (for the calibration) were made in
0.02M EDTA to prevent oxidation. The tissue homogenates were prepared from
freshly sacrifice animals. The organs were excised and immediately placed in ice
bath after accurate weighing, and homogenized in 0.02M EDTA and diluted to
give 5% homogenate. All solutions were degassed with a vigorous stream of


55
Nitrogen gas for 2 3 minutes prior to use. The respective types of tissue thiols
were then determined as follows
Total thiols were estimated as follows:-
One and a half milliliters of 0.2M Tris buffer pH 8.2, and 0.1 mL of 0.01M
DTNB were mixed with 0.5 mL 5% tissue homogenate in 15 mL test tube, and
the mixture diluted up to 10 mL with 7.9 mL Methanol.
The tubes were capped and allowed to stand with occasional shaking for 15
minutes, after which the mixture was filtered and allowed to continue standing
at room temperature for an additional 15 minutes.
The absorbance of the clear filtrates were read at 412 nm against a reagent
blank (i.e. with no homogenate).
Non-protein thiols were determined according to the following procedure
Five milliliter aliquots of homogenate were mixed with 4.0 mL Di-water and
1.0 mL of 50% TCA.
The mixture was allowed to stand 15 minutes with intermittent shaking and
subsequent filtering and continued standing for further 15 minutes.
A two milliliter aliquot of the clear filtrate was mixed with 4 mL of 0.4 M Tris
buffer, pH 8.9 and 0.1 mL 0.01M DTNB added and mixed.
The absorbance was read at 412 nm, within 5 minutes of addition of the
DTNB, against a reagent blank.
The corresponding thiol concentrations were estimated by extrapolation from
calibration curves prepared by the procedure for total thiols using standard
solutions of cysteine in the range 0 10"5 M concentrations.
Protein-bound thiols (P-SH) were simply obtained by subtracting non
protein thiols (NP-SH) from the total thiols (T-SH).


56
In-Vitro Binding Studies
Inhibition of CDS hydrolysis by BPNP
The kinetics of hydrolysis of the respective CDS, namely; Chlorambucil
CDS (MS-2); Cromolyn CDS-1 (MS-4); and Cromolyn CDS-2 (MS-17); in the
presence and absence of a carboxylesterase inhibitor were studied. The inhibitor
selected for use was BPNP, bis-(p-nitrophenyl)phosphate sodium salt (Sterri and
Fonnum, 1987; and Yamada et al., 1992), and the method for the esterase
inhibition was adapted from Yamada et al. (1992).
The materials used included:-
- BPNP, bis-(p-nitrophenyl)phosphate sodium salt
- Isotonic Phosphate Buffer pH 7.4
- 25% tissue homogenate and/or whole blood
- MS-2, MS-4, and MS-17 (1.0 mM Stock Solutions).
The procedure used was as follows:
The BPNP (0.2 mL, 10 mM in phosphate buffer pH 7.4) was added to 1.8 mL
whole serum or 25% tissue homogenate giving a final inhibitor concentration
of 1 mM in the homogenate, vortexed for 30 seconds and pre-incubated at
37C for 15 minutes.
To the incubation mixture, 0.1 mL of 1.0 mM CDS was added, vortexed 15
seconds, and returned to incubation at 37C.
At time intervals, 0.1 mL aliquots were removed and 0.9 mL 5% DMSO in
Acetonitrile added, vortex mixed, and centrifuged at 13 G for 5 minutes.
Aliquots of the supernatants (20 pL) were injected unto HPLC to analyze for
CDS.
For the controls (i.e. hydrolysis in absence of inhibitor), 0.2 mL aliquots of the
phosphate buffer were substituted for BPNP.


57
Effect of [BPNP1 on hydrolysis of MS-4
Cromolyn CDS-1, MS-4 was selected as a model CDS, and the effect of
various concentrations of the selected inhibitor on its hydrolysis was investigated.
The materials used were the same as for the above, except only one drug, MS-4,
was investigated here, and procedure used is described as follows:
Stock BPNP solutions (2.5 mM and 25 mM respectively) were prepared in
phosphate buffer (0.05M, and pH 7.4).
Portions of 25% rat liver homogenate (1.8 mL) were placed in test-tubes with
the appropriate amounts of stock BPNP and buffer solutions to give the
respective BPNP concentrations in the range 0 4.55 mM indicated.
The mixtures were vortexed and pre-incubated at 37C for 20 minutes after
which 0.1 mL aliquots of the CDS (MS-4 1.0 mM solution in 5%
DMSO/Acetonitrile) were added to each tube giving a final drug concentration
of about 50 pM drug in each.
At time intervals, 0.1 mL aliquots of the reaction mixture was taken into 0.9
mL of 5% DMSO/Acetonitrile, vortexed for 15 seconds, centrifuged 5 minutes
and the supernatant analyzed by HPLC for CDS.
ii. Determination of bound and unbound drug
A measured quantity of drug or CDS was incubated with the appropriate
tissue homogenate or Bovine Serum Albumin (BSA) for a predetermined length of
time and analyzed as follows for free unbound drug, and drug bound via mixed
disulfide bond formation.
The unbound drug was determined as follows:-
One half milliliter of Acetonitrile was added to 0.5 mL aliquot of the
incubation mixture (i.e. x2 dilution), and centrifuged for 30 seconds;
An aliquot, 0.5 mL, of the supernatant was added to 0.5 mL Acetonitrile and


58
centrifuged again, 5 minutes (i.e. further x2 dilution);
A portion of the supernatant was then diluted 3x with the mobile phase and
analyzed by HPLC (total of 12 times dilution).
Drug bound via mixed -disulfide linkages was also determined by the
follwoing procedure:-
The protein pellet from above was suspended in, and washed about three times
with ethanol to remove all traces of drug not covalently bonded to the protein;
The washed pellet was re-suspended in 0.5 mL of 50 mM MOPS containing
25mM DTT and incubated at 37C for 1 hour.
At the end of this reduction step, the protein was reprecipitated by adding 0.75
mL of Acetonitrile (i.e. 2.5 times dilution), centrifuged, and the supernatant
analyzed for the CDS by the HPLC system.
Mixed disulfide binding in the presence of NEM
Samples of the respective CDSs MS-2, MS-4 and MS-17, respectively,
were incubated with various tissue homogenates or whole blood, in the presence
and absence of BPNP and/or NEM (N-ethylmaleimide), a sulfhydryl blocker. The
respective amounts of bound and unbound drug were determined.
The materials and procedure used were as follows respectively:
- Freshly excised tissues (whole blood, liver, lung and Kidney)
- Bis-(p-nitrophenyl)phosphate (BPNP)
- 4-Morpholinopropane sulfonic acid, MOPS
- 1,4-Dithiotreitol (DTT)
- Phosphate Buffer, 0.1M, pH 7.4
Twenty five percent tissue homogenate, 1.8 mL, was mixed with 0.2 mL of 10
mM BPNP to give a final concentration of 1.0 mM BPNP, (or 0.2 mL


59
phosphate buffer in place of BPNP for the control) and pre-incubated at 37C
for 15 minutes.
For binding in the presence of the sulfhydryl blocker, 0.1 mL of 10 mM NEM
(or water for the control) was added, at this point, after the pre-incubation, and
vortexed for 1 minute (final concentration of NEM = 0.48 mM).
A hundred microliters of the CDS (1.0 mM in DMSO) were added with
vigorous mixing (resulting in an initial drug concentration in homogenate of
0.45 fiM) and incubated at 37C for 1 hour and subsequently analyzed for
mixed disulfide bound drug and unbound drug by the methods described
above.
Effect of TBPNP1 on binding of MS-4
Bovine Serum Albumin. BSA, is a model protein with a single -SH group.
An amount of BSA of protein concentration (50 mg/mL) approximating to that of
33% tissue homogenate was used instead of an actual tissue homogenate owing to
the desire to eliminate the differential hydrolysis of the CDS with the different
BPNP concentrations in the tissue homogenates as a mitigating factor in the
binding.
The effect of the B-esterase inhibitor, BPNP, on the binding was
determined as follows:-
- BSA in phosphate buffer (68.75 mg protein / mL)
- 50 mM Morpholinopropane sulfonic acid, pH 8.0 in 25 mM
DTT in methanol.
- BPNP
A portion, 1.6 mL, of the stock BSA solution is placed in a test tube with the
appropriate concentrations of BPNP and buffer to make volume up to 2.0 mL.
The mixture was pre-incubated for 15 minutes at 37C.


60
The CDS, 0.15 mL 1.0 mM MS-4 is added with vortexing to give a final drug
concentration in mixture of 69.8 gM.
Incubation is continued for 1 hour after which 0.5 mL aliquots were withdrawn
and analyzed for free unbound drug, and drug bound by mixed disulfide, using
the procedures already described above.
Time dependence of mixed disulfide binding of MS-4
i. to bovine serum albumin:
The procedure used was similar to that described above except that no
BPNP was added and the incubation times of the CDS in the tissue homogenate
were varied to correspond to the desired time intervals before being analyzed for
the bound and unbound drug components.
ii. to a model tissue (rabbit limp:
Five and a half milliliters of 33.3% Rabbit Liver homogenate in isotonic buffer
pH 7.4 were placed in a 15 mL test tube, 0.3 mL of 25 mM BPNP and 0.3 mL
of 1.2 mM MS-4 were added with vortex mixing (giving final concentrations
of; homogenate: 29.8%; BPNP:- 1.23 mM; and Drug:- 60 pM), and then
incubated at 37C.
After intervals within the range of 0 to 26 hours, samples were withdrawn and
analyzed, as before, for bound and unbound drug concentrations.
iii. to rabbit lung tissue compared to liver:
The procedure used was similar to the above except that the incubation
times used were set at 0, 30, and 75 minutes.
Dependence of binding on initial drug concentration
To 1.86 mL of 33.3% rabbit liver homogenate in a test tube, 120 pL of 25 mM
BPNP in phosphate buffer was added and incubated at 37C for 15 minutes.


61
Various amounts of stock MS-4 solution and solvent for the stock solution
were added to make the total volume up to 2200 pi to correspond to the various
initial drug concentrations in the range of 6 to 240 pM.
The mixtures were incubated for 1 hour at 37C and then analyzed by HPLC
for free unbound drug and drug bound by mixed disulfides.
Comparative in-vitro tissue binding of MS-4 via mixed disulfides
To 1.8 mL of 33% respective rabbit tissue homogenates or blood, 120 pL of 25
mM BPNP was added, vortex mixed and pre-incubated at 37C for 15 minutes.
Fifty microliters of 1.2 mM MS-4 were then added resulting in an initial drug
concentration of 30 pM, and the incubation continued for one hour, after which
sample were taken and analyzed for the bound and unbound drug
concentrations.
Dependence of drug binding on homogenate concentration
Rabbit liver homogenates 15, 25, 35 and 50 % respectively were prepared.
A portion of each homogenate (1.85 mL) was pre-incubated as before with 100
pL of the esterase inhibitor BPNP, after which 50 pL of the drug, MS-4, was
added (the initial drug concentration in each homogenate being 30 pM).
The mixture was incubated for an hour and then analyzed for bound and
unbound drugs.
Data Treatment and Statistical Evaluation
All data presented in the dissertation are reported as the mean value plus or
minus the standard error of the mean (SEM), unless otherwise stated. A standard


62
t-test for unpaired data were used when comparisons were made within a group
whereas for paired data, the t-test was used. Assuming a normal distribution with
the population variance equal and unknown, the difference between two sets of
data was considered significant when the P value was more than 0.05. For
comparison within a group comprising more than two sets of unpaired data, a one
way analysis of variance (ANOVA) was performed and the difference was
considered significant when the computed F value was more than a critical value.


CHAPTER 5
RESULTS AND DISCUSSION
Syntheses
The respective CDSs, MS-2, MS-4, and MS-17 were prepared as esters.
The chlorambucil CDS (MS-2) was a yellow oil whereas the cromolygcic acid
CDS's were both light yellow crystalline solids. The compounds along with the
intermediate compounds were characterized by spectral methods (NMR, IR, UV
and MS), purity was assessed by TLC, HPLC and elemental analyses. Melting
points were also determined, and for the liquid compounds boiling points could
not be determined due to decomposition at high temperatures. The spectral and
other physical characteristics including percentage composition from elemental
analysis are presented below. Introduction of the l,2-dithiolane-3-pentyl moiety
was achieved through esterification. A commercially available starting material
lipoic acid, first had to be converted into the alcohol (lipolol).
l,2-dithiolane-3-pentanol, (dl-lipolol), MS-1: Yield:- Yellow oil 88.5 %;
TLC:- Rp0.69 (mobile phase methylene-chloride/ethyl acetate, 4:1); UV (in
CH2CI2):- 330 nm (intact dithiolane ring, Furr et al., (1979); IR (neat
liquid, NaCl discs):- v 3360 (s, broad, hydrogen bonded -OH str.). !H-NMR (in
CDCI3): Spectrum is presented in Figure 15 below, and the following observed
chemical shift (8) values were in close agreement with spectra obtained by
Kabalka et al., 1977, and with most of the theoretically estimated/calculated values
using data obtained from Silverstein et al., 1981:- 1.5 (broad, 8H, alkyl),
63


varan instrument division
I 'I""
I (X I I'
I () K l i\Vi It
FCOUII I IOS
I H roUPl INt. IOWI It
Mir.! AMI'I 5000
0.1
o SWI I I1 Wllllll
nil. IU IOWI M 0.05 mi*. I Nf) OI SWf I I
5 "1 NUCI I II.
10 iii-iii /i no ni i TMS
0 >>< SAMI'I I II Ml' RT
t HI MAI t *1 M.S.
5/8/92
DAN
( MHVINI
MS-1
CDC13 SII I 11;11M N
Figure 15: *H-NMR Spectrum for Lipolol:
Characteristic chemical shifts showed a quintet at 2.4 correspon-ding to the ring -CH2-, and a
triplet at 3.15 for the methylene protons a to the S (i.e. -CH2S-)
EM360/390 NMR SPECTROMETER


65
(broad, 8H, alkyl), 2.4 (quintet, 2H, ring CH2), 3.15 (t, 2H, -CH2S-), 3.6 (m, 3H, -
CHRS- and -CH2O-), and 4.0 (t, 1H, -OH). The triplet at 5 = 0.9 which
disappears upon formation of the R-Br derivative, may be due to the hydroxylic
proton. In deuterochloroform, the hydroxylic peak is usually found between 8 2
and 4, however, at very low solvent concentrations, the peak shifts to higher fields
close to 8 0.5 (Silverstein et al., 1981).
UPOLOL
Figure 16: UV Spectrum of Lipolol:
Absorption maxima at X 328 nm, characteristic of the
intact dithiolane ring is shown.


66
Chlorambucil CDS
l,2-dithiolane-3-pentyl-5-{4-[p-bis(2-chloroethyl)amino] phenyl butyrate},
MS-2. Yield:- Yellow oil, 30%; Elemental Analysis: Calculated for
C22H33NS2CI2O2: C, 55.21155.28; H, 6.95; N, 2.93; S, 13.40; and Cl, 14.82
Found: C, 55.28; H, 7.01; N, 2.94; S, 13.33; Cl, 14.73. 1HINMR (in CDC13):-
Spectrum is presented in Figure 17 the observed chemical shift (5) values were
largely in close agreement with calculated values: 1.2 1.8 (broad, 8H, -CH2-,
lipoyl alkyl), 2.3 -2.7 (multiplet 6H, -0C(0)-(CH2)6-), 3.2 (t, 6H, mustard -N-
CH2- and lipoyl -CH2S-), 4.18 (t, 2H, -OCH2-), and 6.7 & 7.1 (both doublets,
4H, aromatic, para-substitution).
Cromolyn-CDSs
Taking into account the considerations discussed in Chapter 4, the involved
syntheses were achieved via the reaction of the alkyl bromide derivative of lipoic
acid with the disodium salts of the appropriate bischromone compound in HMPA.
Yields were quantitative, and the esterification reactions were over in about 30
minutes. The reactions worked just fine using 1:1 mole equivalents of the
reactants. One of the earliest reports of the use of this reaction (Shaw et al., 1973)
had suggested a 1:4 (disodium salt: alkyl bromide) mole equivalents ratio.
a. Lipolyl bromide, MS-9, (5-bromo-3-pentyl-l,2-dithiolane). Yield:- brownish
yellow oil, 65.4 %. TLC:- Aluminum sheet silica gel 60 F254, 0.2 mm layer
thickness, RE = 0.73 (mobile phase = 10% ethyl Acetate/Hexane).


varan instrument division paio sito, ca;
1 20 1.63 4.18
3 8 2.25 2.04 2.64 /TT\ *
^y/(CH2)3CH2CH2-OOC-CH2CH2CH2-\Q>-N
3 06 3 63
'CH2CH2CI
S-S
^ch2ch2ci
Calculated chemical shift values for MS-2
ppm
10
LOCK POS.
LOCK POWER
DECOUPLE POS.
DECOUPLING POWER _
8000
ppm SPECTRUM AMPI SWEEP TIME_
_mG 0.05
FILTER
-Ppm
_ sec SWEEP WIDTH_
. min NUCLEUS
10 7FRn ___ TMS
ppm ZERO REF
.SAMPLE: MS2 OPERATOR. _
DATE.
mG RF POWER _
0.1
mG END OF SWEEP ^ ppm SAMPLE TEMP SOLVENT: CDCI3 SPECTRUM NO.
Figure 17: ^H-NMR Spectrum for Chlorambucil CDS:
Spectrum showed 5-values of 6.6 and 7.1 corresponding to the 4 para-substituted aromatic
hydrogens at the alkyl and mustard sides of ring respectively, 4.18(t) for the CH2-O, 3.5(t)
for -CHRS (a) and 3.15(t) for -CH2S (b) of the dithiolane ring.
On
EM360/390 NMR SPECTROMETER


68
1H-NMR:- 8-values (CDCI3): 1.5 (broad, 8H, alkyl), 1.8 (q, 2H, -CH2-, P to Br),
2.2 (q, 2H, ring -CH2-), 3.15 (t, 2H, -CH2S-), 3.3 3.6 (m, 3H, -CHRS- and -
CH2Br). IR (neat liquid on NaCl discs):- v 1440 (8S CH2, -CH2-S or -CH2-Br),
650 (medium, 8S CH2Br), 2050 & 1675), UV: Absorption maxima at 330 nm
(characteristic of intact dithiolane ring). Elemental Analysis: Calculated for
C8H15S2Br; C, 37.65; H, 5.92; S, 25.22; Br, 31.30: Found; C, 37.83; H, 5.89;
S, 25.22; Br, 31.05.
A comment on the synthesis of lipolyl bromide needs special mention. For
the synthesis, the commercially available starting material, lipoic acid, had to be
converted to the alcohol and then to the alkyl bromide. One frequently used
method of converting alcohols to alkyl halide is by treatment with N-
bromosuccinimide (NBS) and triphenylphosphine (TPP). However this did not
work for this case as NBS has been known to attack disulfide linkages (Buchel and
Conte, 1967) thereby destroying the dithiolane ring. Loss of the dithiolane ring
was evidenced by the fact that the UV spectrum of the resulting product initially
thought to be lipolyl bromide did not show the characteristic maxima of absorption
at 330 nm, a characteristic of the intact dithiolane ring (Furr et al., 1979). Also
results of the elemental analysis of the product had indicated an empirical formula
of C8Hj5SBr2 instead of the desired C8Hi5S2Br. A thorough review of the
synthetic methods and available literature revealed that this product must have
been a dimeric form of lipolyl bromide possibly formed through the mechanism
below (Figure 18) deduced from the work of Buchel and Conte, 1967; and the
observed deposition of yellow elemental sulfur on the glassware during work up.
What lead to its detection were the results of elemental analysis that corresponded
to the dimeric structure, and the absence of the absorption maxima at 330 nm or
free sulfhydryl groups (IR v 2600 2555).


69
Br
Lipolol
Br
o C
+ PPh
then;
Lipoyl Bromide
Br
S
/S
Br
R
sulfido free
radical
ry^ rrR
S Ss s
^ rearrangement
R^n rrR
Br SS Br
Y"!
Dimer
Emp. Formula: C 8 H15SBr2
R = -(CH 2 )5 -Br
+
S
elemental sulfur
(yellow deposit)
Figure 18: Formation of Lipolyl Bromide Dimer:


70
In the mechanism, the desired product, lipolyl bromide is formed alright,
but it undergoes further reaction resulting from the attack by a bromine atom
generated from the NBS leading to the formation of sulfido- free radical bromide
intermediate which then undergoes further reactions including rearrangement
(Buchel and Conte, 1967) to eliminate sulfur atoms (yellow deposit).
It is interesting to note that this dimeric product shared very similar spectral
characteristics with the desired monomeric product. Furthermore, the
ferricyanide/cyanide test, Furr et al., 1979 for disulfide bond in the inteimediate
alkyl bromide and subsequent CDS molecules had been positive, and the CDS
upon in-vitro hydrolysis had yielded the cromoglycate.
b. 1,3-bis[2-(1,2-dithiolane-3-pentyloxycarbonychromone-5-yloxy) ]-2-
hydroxy-propane, i.e Lypoyl ester of cromolyn or Cromolyn CDS-1: Yield:- 85.6
%: Melting point: 78 81 C: Molecular Weight = 817. 1HINMR:- (CDCI3) 8-
values:- 1.5 (broad, 16H, alkyl), 2.4 (q, 4H ring -CH2-); 3.15 (t, 4H, -CF^S-); 3.4
(multiplet, 2H, ring -CHRS-), 4.4 (10H, propane bridge and -OH), 6.95 (doublet,
4H, aromatic, positions #3 & #8), 7.2 (t, 2H, aromatic #6), 7.5 (t, 2H, aromatic,
#7). IRj. (Paste in mineral oil)vibrational frequency, v, values:- 3525 cm-1
(O-H stretching due to free OH); 3450 (overtone of C=0 stretch); 1745 (C=0
stretch); 1650 (aromatic ester); 1465 (5S CH2 scissoring -CH2OCK)). The *H-
NMR, UV and IR spectral data (Figures 20 23) were in complete agreement with
those obtained by Cox et al. (1977) for corresponding structural features of the bis-
chromone compound. Elemental Analysis:- Calculated for C39H440jiS4-
(0.5H2O): C, 56.71; H, 5.49; O, 22.27; and S, 15.52. Found: C, 56.44; H,
5.49; and S, 15.56.


d,CH2
-CHjS- or -CHrdr
3000
2500
1800
1600
1200 1000
WAVENUMBER (CM ')
800
CHrBr
600
SOLVFNT Sample in Nujol
REMARKS Nfl C=S (due to absence of absorption at
SCAN MODE
CONCENTRATION
220 2050; & 1675)
SLIT TIME CONSTANT
CR1 PATH NaCl Discs
.. .
REFERENCE
No S-H present (absence of 2600 2555 weak)
No PR 5100 4367
Figure 19: IR Spectrum of Lipoyl Bromide:
Characteristic frequencies of absorption (v) shown were at 1440 and 1470 (strong, 8S CH2) corresponding
to -CH2-S- and -CH2-Br respectively; and 1240 (strong, coCF^) for -CH2-Br.


varan instrument division
1
I NI OI SWfc l I
ppm
LOCK POS.
LOCK POWER
DECOUPLE POS
DECOUPLING POWER
ppm SPECTRUM AMPL.
0.2
8000
mG
mG RF POWER
0.15
SWEEP TIME
sec SWEEP WIDTH
mG END OF SWEEP
10
o
mm NUCLEUS
ppm ZERO REF
ppm SAMPLE TEMP
1H
TMS
RT
SAMPLE: MS-4
CDS
C SOI VENT
CDC13
OPERATOR
6/2/92
DATE
<*>-'P TUI IM NO
Figure 20: ^-NMR Spectrum for Cromolyn CDS-1:
Spectrum showed the characteristic cromolyn absorptions of the aromatic hydrogens
(6.9 7.5) and the ten hydrogens of the propanyl bridge & -OH at 8= 4.4, in addition
to the characteristic lipoyl absorptions.
M
EM360/390 NMR SPECTROMETER


% ABSORBANCE
73
CROMOLYN
(in methanol)
WAVELENGTH (nm)
Figure 21: UV Spectrum of Cromolyn
Absorption bands at 238 nm (strong) and a weak band at
319 nm charatenstic of the oione structure (insert) of
cromolyn are shown.


% Absorbance
74
Figure 22: UV Spectrum of Cromolyn CDS (MS-4):
The spectrum for cromolyn (Figure 21) showed the characteristic absorption
maxima at 238 (strong) and at 319 nm, both corresponding to the enone structure
of the bischromone nuclei. These features were retamed m the CDSs (MS-4. &
MS-17) both of which showed a shift in X,max from 319 to 325 nm due to the
presence of the intact dithiolane ring.


75
Cromolyn CDS-2, MS-17
a. 1,3-bis(2-ethoxycarbonylchromon-5-yloxy)-2-hydroxypropane,
(Cromolyn diethyl ester, MS-11): Yield:- 77%; Melting point:- 184 185C,
(In close agreement with reported literature value of 182 183C, (Cairns et al.,
1972); TLC:- Rf = 0.79 (chloroform/methanol, 10:1); iH-NMR:- 8-values
(CDCI3) 1.35 (t, 6H, ethyl -CH3), 4.25-4.6 (broad multiplet, 10H, 2-propanyl
bridge hydrogens and -OH, in agreement with Cox et al., 1970), and aromatic
hydrogens 6.95 (d, 4H); 7.2 (t, 2H); & 7.6 (t, 2H). Mass Spectroscopy
(electrospray ionization):- molecular-ion peak [M + Na]+ = 547.
b. 1,3-bis(2-ethoxycarbonylchromon-5-yloxy)-propan-2-numtc\ (diethyl
cromolyn nitrate ester, MS-15): Yield:- white powder, 75%; Melting point: 172 -
173C; TLC:- Rf=0.81 (chloroform/methanol, 10:1); 1H-NMR> results similar
to spectrum for MS-11 with one less H at 8 value 4.4. Elemental Analysis:-
Calculated for C27H23N013 as C, 56.94; H, 4.07; N, 2.46; and O, 36.53.
Found:- C, 56.71; H, 4.21; and N, 2.52.
c. l,3-bis(2-carboxychromon-5-yloxy)-propan-2-nitrate disodium sail
(cromolyn nitrate disodium salt, MS-16): Yield:- white powder, 51%; Melting
point:- approximately 230C (with decomposition). Elemental analysis:-
Calculated for C23Hi3N0i3Na2-(2H20) as C, 46.56; H, 2.89; N, 2.36; O, 40.44;
and Na, 7.75. Found:- C, 46.62; H, 2.91; N, 2.37; and Na, 7.67.
d. l,3-bis(2-lipolyloxycarbonylchromon-5-yloxy)propan-2-nitrate,
cromolyn lipolyl nitrate ester (i.e. Cromolyn CDS-2, or MS-17): Yield:-
Yellowish crystalline solid, 80.5%; Melting point: 102 104 C; 1H-NMR>
(CDCI3); chemical shift (8) values:- 1.5 (broad, 16H, alkyl), 2.4 (q, 4H ring -
CH2-); 3.15 (t, 4H, CH2S-); 3.4 (multiplet, 2H, ring -CHRS-), 4.4 (9H,
propane bridge, Cox et al, 1970), 6.95 (doublet, 4H, aromatic, positions #3 & #8),
7.2 (t, 2H, aromatic #6), 7.6 (t, 2H, aromatic, #7). Elemental analysis:- Calculated


76
for C39H43013NS4 as C, 54.32; H, 5.03; O, 24.14; N, 1.62 and S, 14.88.
Found:- C, 54.24; H, 5.01; N, 1.68 and S, 14.80.
HPLC Assays
Chlorambucil and CDS:
Chlorambucil is unstable in aqueous solutions (Erhsson et al., 1979). This
results from the fact that the ethyleneimmonium ion or carbonium ion intermediate
which is responsible for its therapeutic alkylating activity is also readily formed in
aqueous solutions where it is readily attacked by nucleophiles such as water
(Loftsson et al., 1989). The mechanism of nitrogen mustard hydrolysis is known
to involve the attack of the unprotonated nitrogen to expel chloride, forming the
cyclic intermediate. This is followed by the attack of water or other nucleophiles
such as N-guanine (as occurs in the mechanism of its therapeutic alkylating
activity). The availability of a free electron pair on the nitrogen is essential for
this reactivity, and protonation of this nitrogen eliminates this reactivity (Chatterji
et al., 1981). Therefore the mobile phase and extraction solvent for the recovery
of Chlorambucil and its CDS were acidified up to 0.5% with acetic acid to prevent
the hydrolytic degradation of drug during the quantitative estimation.
Cromolyn and its CDSs:
Improved Detection of Cromoglycate Anion
Methods for the estimation of the cromoglycate anion in biological media
have mostly involved some rather cumbersome procedures involving
radioimmunoassay (Brown et al., 1983); colorimetric (Moss et al., 1971); or
polarographic methods (Fogg and Fayad, 1978); or radiotracer techniques


77
(Hemmerich et al., 1991). Very few HPLC procedures have been published in
recent times but these along with most of the other methods, have involved the use
of specialized/customized columns, analyte limitation and/or complicated
multistep extraction/drug recovery or sample concentration procedures ultimately
resulting in lowered extraction efficiencies (Gardner, 1984; Ishikura et al., 1987;
and Yoshimi et al., 1992). In recent years HPLC assays have increasingly
supplanted other methods for the quantitative estimation of bioactive compounds
in biological systems on account of its simplicity, speed and general reliability.
The paucity of HPLC assays for the estimation of cromolyn, a frequently
studied compound, underscores the difficulties that other investigators have
probably encountered in the development of a suitable HPLC assay. For this
study, a new, fairly sensitive and simple HPLC assay (Method B) was developed
the results of which, as presented in Table 1, proved more accurate and sensitive
than one of the simplest HPLC procedures so far, recently published by Gardner,
1984 (Method A). In comparing the two methods for the estimation of the
cromoglycate anion, the published (Method A) was adapted to the HPLC
detection system for method B. Effectively, it was the methods for drug recovery
from biological media that were compared. The results (Table 1) showed that
Method A had a drug recovery efficiency of about 75% from rat blood (in close
agreement with the author's reported 70% from human plasma) whereas Method B
produced an efficiency of 92%. Considering the actual concentration of cromolyn
in the lysate based on the assumption that complete hydrolysis had occurred,
Method B was more accurate besides having a slightly greater sensitivity. (The
minimum detectable were 0.1 and 0.05 pM for A and B, respectively).
Employing Method B, the recovery efficiencies of cromoglycate from the
respective tissue types in the rat were compared, and the results presented in Table
2. The results showed that recovery from the blood was a little lower (about 93%)


78
than for the other tissues (approximately 97% and over). This suggested that some
binding of cromolyn had probably occurred in the blood. Albumin in the blood is
known to bind many drugs. In addition, a 'cromoglycate binding protein' has been
identified (Hemmerich et al., 1992), and although its distribution in the body has
not been studied and there is a possibility that it might play a part in the binding.
For the other tissue types, there was no significant difference at P < 0.05, in the
recovery rates indicating that the procedure for drug recovery from biological
media could not be a significant source of error in the differences in the various
tissue drug concentrations assayed.
Table 1: Recovery of Cromolyn as the Cromoglycate Anion from Whole
Rat Blood*; Comparison of Methods A and B
Method
[Cromolyn]
Recovered
Recovery
Efficiency (%)
A
5.00 0.12
75.0 2.7
B
6.17 0.19
92.4 3.0
* Based on the following: 1. Initial fCDS-1] in media = 6.67 pM
2. Assumption that all CDS-1 hydrolyzed into
cromoglycic acid within 30 minutes.
Table 2: Recovery Efficiencies of Cromolyn from Rat Tissues (by Method B)
Tissue
Recovery
Efficiency (%)
Blood
92.6 2.1
Liver
99.1 1.3
Lung
97.4 2.0
Kidney
96.8 1.8


79
Octanol/Water Partition Coefficients
Table 3, below, lists the experimentally determined octanol/water partition
coefficients of the CDSs and the respective parent drugs. Unlike the parent
compounds the respective CDSs were highly lipophilic. The octanol/water
partition coefficients, Log P, for cromolyn and chlorambucil were in close
agreement with values obtained by other investigators (Oppitz et al., 1989; and
Yoshimi et al., 1992 respectively). That of the cromolyn CDS-2 (MS-17) was
only slightly lower than for the cromolyn CDS-1 (MS-4). The purpose for the
synthesis of MS-17 was to increase the aqueous solubility and thereby lower the
Log P. The presence of the nitrate group on the #2 carbon of the propanyl bridge
of the bis-chromone compound did not seem to have influenced the aqueous
solubility that much perhaps owing to the highly hindered nature of that site. MS-
16, the disodium salt of MS-17 was of comparable solubility to cromolyn. The
high lipophilicity of the CDSs compared to the parent drugs, translated into
increased bioavailability and long in-vivo half-lives for the CDSs.
Table 3: Octanol Water Partition Coefficients
Compound
Octanol/
Water
Log P
Cromolyn
0.02 0.01
-1.70 0.15
Cromolyn CDS-1 (MS-4)
743.4 62.1
2.87 0.04
Cromolyn CDS-2 (MS-17)
705.7 41.7
2.85 0.02
MS-16
0.03 0.01
- 1.56 0.14
Chlorambucil (ionized)
3.24 0.51
-0.51 0.21
Chlorambucil CDS, (MS-2)
551.1 48.9
2.74 0.17


80
In-Vitro Stability
The in-vitro stabilities of the CDSs in comparison with the parent drugs are
presented in Tables 4 and 5 below.
Table 4: In-vitro Stabilities of CDSs Compared to Parent Drugs
Medium
Half-life, T j/2, (in minutes)
Chlorambucil
* Cromolyn
CDS
(MS-2)
Parent
CDS-1
(MS-4)
CDS-2
(MS-17)
Human Blood
140.911.5
153.310.2
85.5 9.8
105.1 8.7
Rabbit Blood
26.7 7.1
360.437.8
23.7 5.4
48.3 6.3
Rat Blood
0.4 0.1
1.7 0.5
3.1 0.2
18.6 3.4
Buffer pH 7.4
503.725.9
79.8 11.1
223.U18.5
250.621.7
*Cromolyn was stable in all the media tested.
Table 5: In-Vitro Stability of Model CDS in Various Rat Tissues:
(MS-4; 25% Tissue Homogenate or Blood)
Tissue
k (x 10-2)
T i/2 (min)
r
Blood
13.5 3.4
5.49 1.43
0.9542
Liver
6.4 0.5
10.86 0.79
0.9879
Lung
7.6 0.8
9.19 0.59
0.9946
Kidney
5.5 0.3
12.61 0.59
0.9914
k = Hydrolysis Rate Constants
T1/2= Half-life
r = correlation coefficient


81
For chlorambucil, there was no significant difference (P > 0.05) in stability
between the CDS and the parent drug in human blood, but stability of the CDS in
buffer pH 7.4 was substantially greater than for the parent drug. The higher
stability of CDS in physiological buffer relative to biological media was suggestive
of the probable involvement of hydrolytic enzymes in the biological matrix. It was
reported by Erhsson et al., 1980, that the half life of chlorambucil in aqueous
media was 12 minutes, however, this study showed a half-life of nearly 80 minutes
in aqueous media (buffer pH 7.4). For cromolyn the two CDSs showed
comparable stability in human blood and in buffer but significant differences in
rodents' blood, with the CDS-2 being the more stable of the two. The parent drug
cromolyn was completely stable in all the media tested. This was in agreement
with the knowledge that cromolyn is apparently non-metabolizable in all of the
mammalian species studied, including humans (Cox, 1967).
In general, the kinetic studies data showed that the CDSs of both parent
drugs were unstable in rat blood (Tj/2 < 3 minutes) but fairly stable in rabbit (T j/2
~ 25 minutes) and in human (Ti/2 ~ 85 minutes) bloods. In fact, the fairly good
stability in human blood could ensure several 'circulatory passes' in the blood
stream without much degradation and thereby allow sufficient sequestration of the
CDS to occur in the lung. While the stability of the CDSs showed no specific
trends, the interspecies variation were probably a mere reflection of the different
distribution of B-esterase enzymes among the species.
The in-vitro stability of a model CDS, MS-4, shown in Table 5 indicated no
significant differences (P < 0.05) in respective rat tissues except in blood
suggesting a nearly equal activity of the esterase enzymes in the tissues but higher
activity in blood. This was in line with the fact that P-esterases are non-specific
enzymes of ubiquitous nature in biological tissue.


82
Hydrolysis Products of CDS
Further in-vitro hydrolysis studies, Figure 23 showed that the respective
CDSs hydrolyzed completely and fairly rapidly in freshly obtained biological
tissue media to yield the respective parent compounds.
Drug Cone. (pM)
Chlorambucil CDS (MS-2)
Cromolyn CDS (MS-4)
Drug Cone. (/M)
Figure 23: In-Vitro Hydrolysis Products of CDSs in Rabbit Blood.


83
In-Vivo Distribution Studies
The drugs were administered to rats and rabbits according to the procedure
described in Chapter 4. The mean percentage composition of the respective organs
and tissues of several animals relative to the body weight were estimated (Table 6)
in order to enable the calculation of the total amounts of drug in a given tissue.
From these estimates, the amount of drug present in a given tissue, expressed as a
percentage of the total drug administered (designated '% of Total') and of the sum
total of drug remaining in each respective tissues investigated (i.e. '% of
Recovered') at a given time point. Within limits of the procedure used therefore,
drug stored in adipose tissue, muscle, and in other body parts not investigated
including drug excreted in urine prior to sacrifice or stored in the bladder or bile
were not included in the later estimation and were collectively treated as excreted
drug.
Table 6: Percent Composition of Organs/Tissues Relative to Body
Weight in the Rat and Rabbit
Tissue
Rat
Rabbit
Blood
7.00
7.00
Lung
0.50 0.02
0.58 0.02
Liver
3.25 0.09
2.92 0.05
Brain
0.47 0.02
0.25 0.01
Heart
0.34 0.01
N.D.


84
Chlorambucil and CDS: Distribution in Rats and Rabbits
In the study with rats (Figure 24), there was no significant difference in
the lung delivery capability of the CDS in comparison to the parent drug. This
may relate to the instability of the chlorambucil-CDS in rat blood (in-vitro half-life
less than 45 seconds), such that upon administration, the CDS almost
instantaneously hydrolyzed to the parent drug. In rabbits where the CDS was
5 Minutes After Administration
b
30 Minutes After Administration
Liver Lung Blood
I CDS
I Chloramb.
Figure 24: In-Vivo Distribution of Total Drug as Chlorambucil in
Rats After i.v. Administration of CDS and Parent.


85
sufficiently stable, the in-vivo distribution (Tables 7 & 8 and Figure 24, above)
showed that in all cases, substantially higher concentrations of chlorambucil were
delivered to the lungs when the CDS was administered as compared to the parent
drug. For example 30 minutes after administration of the CDS, about 22 x 10-2
pmol/mL of chlorambucil (more than 20 fold) was delivered to the lung as
compared to only about 1.0 x 10~2 gmol/mL after administration of the parent
chlorambucil.
In the blood (Table 8), the CDS concentration was shown to have decreased
with time within 30 minutes as that of the parent drug, chlorambucil, increased
following the CDS administration. This was expected because the CDS
hydrolyzed in-vivo to give chlorambucil.
Table 7: In-vivo Distribution of Drug 5 Minutes After Intravenous
Administration of Chlorambucil, and CDS: (6 pmol/kg) in Rabbits.
Tissue
Drug Concentration in Tissue(pmol x 10-2 /g or mL)
CDS (MS-2) Administration
Chlorambucil
Admin.
[Chloramb]
[MS-2]
Total [Drug]
[Chloramb.]
Blood
0.60
0.77
1.37
5.76
Brain
0.65
0.10
0.75
N.D.
Kidney
1.42
2.49
3.91
10.33
Liver
4.92
1.98
6.90
2.22
Lung
8.02
38.10
46.12
9.36


86
Table 8: In-vivo Distribution of Drug 30 Minutes After intravenous
Administration of Chlorambucil, and CDS to Rabbits.
Tissue
Drug Concentration in Tissue (gmol x 10-2 /g or mL)
CDS (MS-2) Administration
Chlorambucil
Admin.
[Chloramb.]
[MS-2]
Total [Drug]
[Chloramb.]
Blood (5')
0.92 + 0.38
0.35 + 0.01
1.28 + 0.37
2.27 + 0.50
Blood (15')
1.02 + 0.10
0.22 + 0.02
1.24 + 0.09
1.79 + 0.35
Blood (30')
1.08 + 0.09
0.15 + 0.01
1.23 + 0.10
0.04 + 0.01
Brain*
0.52
Trace
0.52
0.14
Kidney
0.95 + 0.18
1.54 + 0.22
2.49 + 0.27
1.57 + 0.35
Liver
3.40+ 1.25
1.06 + 0.24
4.45 + 1.00
1.69 + 0.57
Lung
13.08+1.01
9.24+1.37
22.33 + 1.62
0.77 + 0.19
* Brain: Only one rabbit was investigated.
Figure 25: In-Vivo Distribution of Chlorambucil in Rabbit 30 minutes
After Administration of Chlorambucil and CDS.


Full Text
DESIGN, SYNTHESIS AND PHARMACOLOGICAL
EVALUATION OF A CHEMICAL DELIVERY SYSTEM
FOR DRUG TARGETING TO LUNG TISSUE
By
MAURICE SAAH
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
1994

Dedicated
to
my
Mother, Sarah;
my Sisters, Theresa, Lillian and Sally;
my Brothers, Patrick, William and Edmund;
my Wife, Betty;
and our Daughter Sheryl
with love and deep appreciation.

ACKNOWLEDGMENTS
I thank Dr. Nicholas Bodor, my advisor and chair of the supervisory
committee for my dissertation. The excellent guidance, patience and kind support
received from Dr. Bodor have been the most important factors in bringing this
project to fruition. To him I am deeply indebted for the enrichment of my
evolution as a pharmaceutical scientist.
Special thanks go to Drs. Luis Muga, James Simpkins and Hassan Farag for
their advice and assistance at various stages of my program and to all the members
of my supervisory committee, Drs. Nicholas Bodor, Margaret James, Richard
Hammer, James Simpkins, and Luis Muga, for the attention and interest in the
project.
The timely help and advice I received from Dr. Emy Wu throughout the
project are deeply appreciated and warrant special mention. Also of special
mention is Dr. Ede Marvenyos whose initial efforts spurred my interest in this
project. I also thank Drs. Laszlo Prokai, and Gabor Somogyi and other members
at the Center for Drug Discovery: Joan Martignago, Laurie Johnston, Julie Berger,
Kathy Eberst, Gizella Somogyi, Dr. Kerry Estes and Robert Wong for their
willingness to help. I would also like to thank all friends and colleagues at the
Center: Angela, Martha, Kumar, Ouyang and others who helped make this tenure
a pleasant experience. I cherish the support received from my wife, Betty; my best
friend, Jeanne-Marie; my mother Sarah, cousin, Clara; aunts, Adelaide and Letitia;
and my brothers and sisters.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vii
LIST OF FIGURES ix
KEY TO SYMBOLS AND ABBREVIATIONS xii
ABSTRACT xiii
CHAPTERS
1. INTRODUCTION
Drug Design and Need for Drug -Targeting 1
Drug Targeting Systems 8
Biological Drug Targeting Systems 8
Physical and Biophysical Targeting Systems 9
Chemical Molecular Systems 9
2. FEASIBILITY OF DRUG TARGETING TO LUNG
Physiology and Pharmacology of the lung 13
Feasibility and Possible Mechanisms
of Selective Delivery to Lung Tissue 15
Lipoic Acid 19
Chlorambucil 22
Cromolyn (DSCG) 23
Mechanism of Action of DSCG 24
Need for Improved Delivery of DSCG 25
Predicted in-vivo Metabolism of CDS 27
3. RESEARCH DESIGN AND SPECIFIC OBJECTIVES
Objective 31
Synthesis 31
IV

Chlorambucil CDS 32
Cromolyn CDS 33
Evaluation of Mixed Disulfide Bond Formation as a
Probable Mechanism for CDS Selectivity for Lung 35
Enzyme Inhibition Studies 36
Kinetics of Mixed-Disulfide Bond Formation 36
Tissue Sulfhydryl 37
Research Protocol 37
4. EXPERIMENTAL METHODOLOGY
Materials 39
Methods
Synthesis 41
Chlorambucil CDS (MS-2) 41
Cromolyn CDS-1 (MS-4) 42
Cromolyn CDS-2 (MS-17) 44
HPLC Assays 48
Drug recovery efficacy of HPLC assay 49
OctanolAVater Partition Coefficients (Log P) 50
In-Vitro Stability 50
Hydrolysis Products of CDS 51
In-Vivo Distribution Studies 52
Chlorambucil and CDS in rats and rabbits 52
Cromolyn and CDS: Distribution in rats and rabbits 52
Determination of Tissue Sulfhydryl 54
In-Vitro Binding Studies 56
Inhibition of CDS hydrolysis by BPNP 56
Effect of BPNP on hydrolysis of MS-4 57
Determination of bound and unbound drug 57
Mixed disulfide binding in the presence of NEM 58
Effect of [BPNP] on Binding of MS-4 59
Time dependence of mixed disulfide binding of MS-4
to bovine serum albumin 60
to a model tissue (rabbit lung) 60
to rabbit lung tissue compared with liver 60
Dependence of binding on initial drug concentration 61
Comparative in-vitro tissue binding of MS-4 via
mixed disulfides 61
Dependence of binding on homogenate concentration .... 61
Data Treatment and Statistical Evaluation 61
v

5 RESULTS AND DISCUSSION
Syntheses 63
Chlorambucil CDS 66
Cromolyn-CDSs 66
Cromolyn CDS-2, MS-17 75
HPLC Assays 76
Chlorambucil and CDS 76
Cromolyn and CDSs: Improved Detection
of Cromoglycate Anion 76
OctanolAVater Partition Coefficients 79
In-Vitro Stability 80
Hydrolysis Products of CDS 82
In-Vivo Distribution Studies 83
Chlorambucil and CDS: Distribution in Rats and Rabbits .... 84
Cromolyn and CDS: Distribution in Rats and Rabbits 87
Kinetics of Mixed Disulfide-Bond Formation 103
Effect of BPNP on the Hydrolysis of MS-4 104
Effect of Inhibitors on Binding of MS-4 via
Mixed Disulfides 105
Time Dependence of Mixed Disulfide Binding of CDS 107
Dependence of MS-4 Binding on the Initial
Drug Concentration Ill
Comparative Binding of MS-4 In-Vitro 113
Dependence of Binding on Homogenate Concentration 114
Tissue Sulfhydryl 114
6 CONCLUSIONS 120
APPENDIX
SAMPLE CALCULATIONS 126
REFERENCES 131
BIOGRAPHICAL SKETCH 139
vi

LIST OF TABLES
TABLE Page
1 Recovery of Cromolyn as the Cromoglycate Anion
from Whole Rat Blood 78
2 Recovery Efficiencies of Cromolyn from Rat Tissues
Using Method B 78
3 Octanol-Water Partition coefficients 79
4 In-vitro Stabilities of CDSs Compared to Parent Drugs 80
5. In-vitro Stability of a Model CDS, MS-4, in
Various Rat Tissue Media: (25% Homogenate) 80
6 Percent Composition of Organs/Tissues Relative to
Body Weight in the Rat and Rabbit 83
7 In-Vivo Distribution of Drug 5 Minutes After
Administration of Chlorambucil and CDS; 85
8 In-Vivo Distribution of Drug 30 Minutes
After Administration of Cromolyn and CDS 86
9 In-Vivo Distribution of Drug in Rabbit Tissues 30 Minutes
After Administration of CDS and Cromolyn 87
10 Distribution of Drug in Rat Tissues with Time After Administration
of 6.2 gmol/kg of CDS-1 (MS-4) and Parent (Cromolyn) 89
11 Drug Distrbution in Rat Lung with Time 90
12 Drug Distribution in Rat Blood with Time 90
vii

13 Drug Distribution in Rat Liver with Time 91
14 Drug Distribution in Rat Kidney with Time 92
15 Recovery of Drug with Time After Administration to Rats 99
16 Dependence of Hydrolysis Rate Constants and Half-lives of MS-4
on BPNP Concentration in Rat Liver Homogenate 104
17 Effect of [BPNP] on Binding of MS-4 to BSA 105
18 Binding of MS-4 in Presence of Inhibitors (BPNP and NEM) 107
19 Binding of MS-4 to BSA (50 mg Protein/mL) with Time 108
20 Dependence of Binding on Time of Incubation 109
21 Binding of MS-4 With Time to Rabbit Lung Tissue
Compared with the Liver and BSA 110
22 Dependence of MS-4 Binding in Liver Tissue on the
Initial Drug Concentration In-Vitro Ill
23 Comparative Binding of MS-4 to Various Rabbit Tissues In-Vitro ... 113
24 Dependence of MS-4 Binding in Rabbit or
Liver Tissue on Homogenate Concentration 114
25 Tissue Sulfhydryl Content in the Rabbit as Total Thiol, T-SH;
Non-Protein Thiol, NP-SH; and Protein-Bound Thiol, P-SH .... 115
26 Estimated Magnitudes of Binding of MS-4
in Respective Rabbit Tissues 116
viii

LIST OF FIGURES
FIGURE Page
1 The Metabolic Fate of a Conventional Drug After Administration 4
2 The Soft Drug Concept 5
3 The Prodrug Approach 6
4 The CDS Approach 10
5 General Structure of the Human Lung 14
6 Mechanism for Enhanced Delivery of Spirothiazolidine
Derivatives 16
7 Proposed Mechanism of Delivery by the Lipoic Acid CDS 18
8 Coenzymatic Function of Lipoic Acid 21
9 Predicted Metabolism of Chlorambucil CDS 28
10 Metabolism of Cromolyn CDS 29
11 Mechanism of Esterification with DCC 32
12 Synthesis of Chlorambucil CDS (MS-2) 42
13 Synthesis of Cromolyn CDS-1 (MS-4) 43
14 Scheme for the Synthesis of Cromolyn CDS-2 47
15 !H-NMR Spectrum of Lipolol 64
16 UV Spectrum of Lipolol 65
IX

17 H-NMR Spectrum of Chlorambucil CDS 67
18 Formation of Lipoyl Bromide Dimer 69
19 IR Spectrum of Lipoly Bromide 71
20 1 H-NMR Spectrum of Cromolyn CDS-1 72
21 UV Spectrum of Cromolyn 73
22 UV Spectrum of Cromolyn CDS-1 (MS-4) 74
23 In-Vitro Hydrolysis Products of CDSs in Rabbit Blood 82
24 In-Vivo Distribution of Total Drug as Chlorambucil with Time
in Rats After i.v. Administration of CDS and Parent 84
25 In-Vivo Distribution of Chlorambucil in Rabbit 30 Minutes
After Administration of Chlorambucil and CDS 86
26 In-Vivo Distribution in Rabbit Following Cromolyn
and CDS Administration 88
27 Drug Distribution in Lung After CDS/Cromolyn Administration 93
28 Distribution of Cromoglycate in Rat Lung with Time
After Administration of CDS or Parent Drug 93
29 In-Vivo Concentrations of Drug in Rat Tissues with Time After
Administration of CDS vrs. Parent Drug (Cromolyn) 94
30 Percent of Total Drug in Respective Rat Tissues with Time
After Administration of CDS and Parent Drug (Cromolyn) 95
31 Percent of Recovered Drug in Respective Rat Tissues with Time
After Administration of CDS and Parent Drug (Crm) 96
32 In-vivo Distribution of Drug (CDS + Metabolites) in Rat
Tissues with Time After CDS Administration 97
33 Percent of Administered Retained in All Tissues with Time 99
x

34 Mean Percent Distribution of Total Drug With Time
in Whole Rat Tissues 101
35 Mean Percent Distribution of Recovered Drug with
Time in Whole Rat Tissues 102
36 Effect of BPNP on Binding of MS-4 to BSA 106
37 In-Vitro Binding of MS-4 in Rat Lung Tissue with Time 110
38 Dependence of Binding on Initial Drug Concentration 112
39 Percent Binding and Initial Drug Concentration 113
40 Tissue Sulfhydryl Content of the Rabbit 116
41 Tissue Sufhydryl, Mixed Disulfide Binding
and Magnitude of Binding 117
XI

KEY TO SYMBOLS AND ABBREVIATIONS
v
vas
^as
Ss
vs
gg
gL
pM
!H-NMR
AUC
CDS
cone.
DMSO
g
HPLC
kg
m
mL
mM
mmol
n
nm
nmol
q
r
t
T */2
v/v
X
i.d.
vibrational frequency (IR spectroscopy)
asymmetrical stretching
asymmetrical bending vibration
scissoring vibration
symmetrical stretching
microgram
microliter
micromolar
Proton magnetic resonance
Area Under Curve
Chemical Delivery System
concentration
dimethyl sulphoxide
gram
high-performance liquid chromatography
kilogram
multiplet
milliliter
millimolar
millimole
number of determinations or sample size
nanometer
nanomole
quintet
linear regression correlation coefficeint
triplet (nmr spectroscopy)
half-life
volume to volume ratio
wavelenght
internal diameter
Xll

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
DESIGN, SYNTHESIS AND PHARMACOLOGICAL
EVALUATION OF A CHEMICAL DELIVERY SYSTEM
FOR DRUG TARGETING TO LUNG TISSUE
By
Maurice Saah
August 1994
Chairman: Nicholas Bodor, Ph.D.
Major Department: PharmacyMedicinal Chemistry
In conventional therapy, the achievement of therapeutically effective levels
of drug at its pathophysiologically relevant site is very often adversely mitigated
by systemic toxicity resulting from a lack of selectivity in drug pharmacokinetics.
It is therefore obvious that the development of a mechanism to selectively deliver
drugs to, in this instance, lung tissue, could raise the therapeutic index and thereby
optimize the clinical usefulness of drugs used to treat lung diseases, especially
lung cancer. This dissertation intends to expostulate the application of a novel
chemical delivery system (CDS) approach to a delivery mechanism for drug
targeting to lung tissue using the l,2-dithiolane-3-pentyl moiety of lipoic acid as
the 'targetor moiety'. It describes the synthesis, and the physicochemical and
pharmacological evaluation of a CDS modeling the lipolyl and other ester

derivatives of chlorambucil (an antineoplastic agent), and cromolyn (a
bischromone used in anti-asthma prophylaxis) as compared with their respective
parent drugs. The chlorambucil CDS was synthesized by esterifying the alkanol
derivative of lipoic acid with chlorambucil using dicyclohexyl carbodiimide as the
coupling agent. The cromolyn esters were prepared by respective multistep
synthetic procedures each culminating in the reaction of the alkyl bromide
derivative of lipoic acid with the disodium salt of the bischromone compound. All
the esters were highly lipophilic, unlike the parent compounds. The in-vitro and
in-vivo kinetic and pharmacokinetic studies showed the respective CDSs were
sufficiently stable in buffer and biological media, hydrolyzed rapidly into the
respective active parent drugs, and significantly enhanced delivery of the active
compound to lung tissue in comparison with the underivatized parent compounds
used in conventional therapy. A proposed mechanism for the selective delivery
suggested the involvement of drug binding through mixed disulfide linkages to
tissue proteins. In-vitro binding studies showed the CDS did indeed bind to tissue
proteins, and that the magnitude of binding was greatest in the lung. This factor,
coupled with the fact that the total venous return after intravenous administration
goes first to the lung probably helped the lung to maximally sequester the drug.
XIV

CHAPTER 1
INTRODUCTION
Drug Design and the Need For Drug Targeting:
A singular and arguably, the most important parameter of a drug that needs
to be optimized in the drug development process is the therapeutic index (TI),
which represents the ratio between the median toxic dose, TD50, and the median
effective dose, ED50:
TI = TD50/ED50
In conventional therapy, the parenteral or enteral administration of a drug
has usually involved, a more or less, passive distribution of the drug entity
throughout the body. This has very often lead to the achievement of
therapeutically effective levels of drug in a target organ/tissue woefully mitigated
by systemic toxicity. It is now therefore widely accepted that clinical usefulness
(expressed by the therapeutic index) of drugs in therapy would be enhanced if
drugs were to concentrate and exert their therapeutic effect selectively in/on the
target tissue. Drug targeting as one approach or as a part of an integrated approach
will therefore improve TI by raising the median toxic dose and/or decreasing the
median effective dose.
Lung disease (including lung cancers and other respiratory ailments)
constitute one of the leading causes of morbidity and mortality in the United
States, hence any attempt to improve therapy in the management of lung disease
1

2
should certainly present an area of immense interest to pharmaceutical scientists
especially in the area of lung cancer treatment where systemic toxicity constitutes
a major problem.
Drug design in the last two decades has taken on a more rational approach.
No longer do medicinal chemists rely on chance discovery of drugs as had been
the case in the past for many presently important types of drugs (Jacobsen. 1976;
and Austel, 1981), but use is made of information about the various bio-molecular
features (receptors, enzyme systems or metabolic pathways) as potential targets for
pharmacological intervention, permitting the rational design of agonists and
inhibitors to mitigate the pathophysiological process. Since the bio-molecular
processes underlying many disease states are not usually well understood, such
approach has not always been feasible. For example, since the development of the
receptor theory, some success has been achieved with some CNS active agents.
However since most receptors are generally distributed throughout the body while
the disease may be localized, a receptor based intervention could sometimes
produce undesired effects elsewhere. For instance the neurotransmitter dopamine
is released at specific parts of the brain and localized by the blood brain barrier
(BBB) therein to produce the desired anticholinergic action locally. In Parkinson's
disease, there is dopamine deficiency in the striatum. The peripheral
administration of dopamine (even in the form that could penetrate the BBB) could
result in side effects such as dryness of the mouth, cycloplegia, mydriasis and
tachycardia (Korolkovas, 1988) due to its central anti-cholinergic activity.
Besides, there are still many situations where selectivity at receptor sites is not
enough or where no such receptors are yet known to exist or where the target
disease stems from little understood biological response. So additionally,
medicinal chemists have used a variety of empirical and semi-empirical structure
activity relationships to enhance pharmacological activity through improved

3
delivery across membranes (improved bioavailability) notably, the improved
delivery across the BBB (Bodor et al., 1975), and the establishment of a
correlation between physicochemical parameters and biological activity by Hansch
(1981), and its subsequent myriads of applications.
In spite of these advances, relatively few compounds having even maximal
activity actually proceed to become clinically useful drugs (Bodor 1984), and even
when they do sometimes have to be used under trying circumstances with extremes
of caution. The reason being the accompanying and unexpected toxicities and
other pharmacological effects of the drug. From a schematic depiction of the
metabolic fate of a conventional drug in-vivo (.Figure 7), Bodor (1984),
summarizes the toxic effects of a drug as resulting from a combination of factors
which include all the other pharmacological effects of the drug (D) itself, the
effects of the direct and indirect metabolites (Dj... Dn, Mj... Mj and Mj... Mq
respectively), reactive intermediates (I*] ... I*m), and the various compounds (IC¡
... ICn) resulting from the interactions of these intermediates with cellular
components. The overall toxicity of a drug could then be described as the
summation of the toxicity due to the drug itself, which essentially is its lack of
selectivity, and the toxicities due to its various metabolic products.
Thus for the medicinal chemist, a rational drug design aims at improving
the therapeutic index of a drug by taking into consideration selectivity in drug
delivery and intervention of toxic pathways in drug metabolism through structural
manipulations. Hence, the primary focus of a rational drug development as
suggested by Bodor (1984) is to optimize activity rather than merely maximizing
it. These considerations have ultimately resulted in the prodrug and soft drug
concepts (Bodor, 1984), the hard drug concept (Ariens and Simonis, 1977) and the
novel chemical delivery system approach (Bodor 1984 and 1992).

4
Drug (D)
ko
Delivery
Process
Direct Elimination ^kl0r k2
of Unchanged
or Conjugated D
D
Jiv k
Reactive
Intermediates
k k
1 *CJ v
ICj... ICn
Toxic Metabolites
or Products
Figure 1: The Metabolic Fate of a Conventional Drug After Administration

5
Structural modifications on a drug molecule result in the alteration of some
pharmacodynamic and physicochemical aspects of the drug resulting in an
optimization of its metabolism, activity, delivery and elimination processes. Thus
depending on the specific design goals, it may be possible to design a drug having
predictable metabolism and/or other properties on a rational basis by the
introduction of certain labile or 'vulnerable groups' into the drug molecule. The
identification of certain such 'vulnerable moieties' by Ariens (1977) as parts of
drug molecules responsible for bio-inactivation, metabolism or elimination makes
the feasibility of this approach especially probable, and could also form the basis
for Bodor's (1984) novel concept of "structure-metabolism relationships"
analogous to Hansch's (1981) quantitative structure-activity relationships and its
myriads of applications.
Direct Elimination
Proo
Del
M ,
M 2 Mk
inactive
metabolite
inactive
metabolites
Elimination
Figure 2: The Soft Drug Concept (Bodor, 1984):
The therapeutically active soft drug (SD) is inactivated in one metabolic
step. This eliminates the pathways to the formation of toxic metabolites.

6
The soft drug {Figure 2) above, defined as "a biologically active
therapeutically useful chemical compound (drug) characterized by a predictable
and controllable in vivo destruction (metabolism) into non-toxic moieties after
achieving their therapeutic role" (Bodor 1984; 261), enables the separation of
therapeutic properties from toxic affects.
In the prodrug approach {Figure 3), the drug as an inactive moiety (PD) is
designed to undergo mainly the metabolism required to activate it in-vivo, without
substantial direct elimination and thereafter follow the scheme illustrated in Figure
1. The hard drug on the other hand, as exemplified by cromoglycic acid, is a non-
metabolizable active compound that can elicit its pharmacological activity and
undergo elimination unchanged.
elimination
/
DRUG
chemical in viy
transformations ^ ^ k 2
G)+ 0
in vivo
metabolism
and
disposition
Figure 3: The Prodrug Approach
The inactive prodrug is activated in-vivo where it enhances
therapeutic index through optimized delivery and elimination.

7
These approaches to a rational drug design have not been without
limitations. The prodrug approach can at best improve bioavailability through
alteration of pharmacodynamic parameters, and protect against some unwanted
degradations such as those occurring in the gastrointestinal tract or during the
hepatic first pass. It cannot substantially influence the formation of reactive or
toxic intermediates although it may indirectly reduce toxicity through optimized
delivery and elimination.
The hard drug by design necessitates 'blocking' of the metabolically
sensitive parts of the drug molecule. This will have to be achieved at the expense
of favorable pharmacokinetics. In other words, the drug would have to be very
lipophilic or very hydrophilic. In the highly lipophilic form, it can deposit in
adipose tissue and organelles resulting in extremely prolonged half-lives and
possibly long term physical and biochemical damage to tissues. With the highly
water soluble form, the in-vivo half-life would be too short to afford the drug any
significant degree of usefulness. Besides, such metabolic stability is only
idealistic as the various xenobiotic metabolizing enzymes particularly the
cytochrome P-450, have been known to attack and alter even the most highly
stable compounds, and according to Bodor (1984), it appears to be the general rule
that the more difficult the metabolism of a chemical, the more likely it is to form
highly reactive intermediates.
Thus, in spite of some successes, especially with the soft drug and prodrug
concepts, these approaches seem idealistic at best. Furthermore, since the disease
is usually localized, whereas the drug passively distributes throughout the body, it
behoves medicinal chemists to adopt an integrated approach with emphasis also on
improving selectivity or achieving drug targeting.

8
Drug Targeting Systems
Drug targeting involves directing the therapeutic agent to a desired site of
action with minimum or no pharmacological interaction with other tissues. In
general, drug targeting may be achieved by;
i.exploiting the selectivity of biological receptors and finding agents
which will selectively act at these sites (Gardner, 1986);
ii. having the drug in an inactive or chemically modified form (prodrug
and related approaches) to be activated at, or 'homed in' on, the
required site of action; or
iii. encapsulating the drug moiety in liposomes or other particulate
carriers to be released at an intended site.
The various approaches that have been employed to achieve drug targeting
may be in the form of biological, physical or chemical molecular systems.
Biological Drug Targeting Systems.
These include the use of biomolecules such as monoclonal antibodies, small
peptides, hormones, glycoconjugates or lectins that show recognition for specific
components in/on tissue or cell surfaces. An example is provided by the potential
use in drug-targeting of the copolymer of N-(2-hydroxypropyl)-methacrylamide
(HPMA) variously derivatized with certain amino sugars that show recognition for
specific sugar receptors on certain mammalian cell types (Seymour et al., 1987).
A number of sugar-receptor interaction involving several mammalian cell types
have been widely investigated and documented. These include the;
i. galactose-receptors of mammalian hepatocytes, Kupffer cells,
liver endothelial cells and bone marrow:

9
ii. N-acetylglucosamine receptors of avian hepatocytes;
iii. mannose-6-phosphate receptor on a variety of mammalian
tissues;
iv. receptor with high affinity for fucose on mouse L1210 Leukemia
cells; and
v. mannose/N-acetylglucosamine receptors on alveolar
macrophages and on a variety of reticuloendothelial cells.
Physical and Biophysical Drug Targeting Systems.
The use of localized polymeric reservoir devices, liposomes, microcapsules,
nanospheres and other particulate devices such as resealed erythrocytes and other
cellular reservoir carriers constitute the physical systems. Here, the drug is
essentially chemically unmodified, and targeting is achieved by physical
sequestration of the carrier by virtue of size, surface (charge, magnetic properties
or antibody coating) characteristics, and a combination of anatomical and
(patho)physiological events of both the passive and active types (Tomlinson,
1986). Such physical targeting systems are useful for persistent and sustained
presentation of drug at such discrete compartments as the eye, joints, respiratory
tract and the GI tract, and also for the treatment of diseases affecting the cells of
the reticuloendothelial system (RES) such as Leischmaniasis, Guacher's disease
and Leprosy. Polymer-drug conjugate targeting systems whose mode of selective
sequestration is size fall under this category on account of which cellular uptake
is by phagocytosis and pinocytosis by certain cells of the RES.
Chemical Molecular Systems.
Although many opportunities exist for drug targeting, each approach has its
own limitations, and medicinal chemists are more interested in this means of drug

10
targeting. The chemical molecular systems for drug targeting are based on the soft
drug, prodrug and the related Chemical Delivery System (CDS) approaches and
involve chemically modifying the parent drug to incorporate the desired targeting
capability.
T + D
[TD] + P
[TDP] + F
[CDS] n
successive enzymatic
reactions
F, P, or F
inactive
/
Active Drug
at Target Site
[TD] + F
in vivo metabolism
and disposition
Figure 4: The CDS Approach:
The parent drug (D) is chemically transformed into the CDS by the
incorporation of targetor (T) and/or other modifier moieties (P and/or F)
For improved delivery of drugs, the prodrug and related approaches seem
most viable and have been credited with many successes including improved
delivery across biological barriers notably improved delivery across the blood-
brain-barrier based on the dihydropyridine/pyridinium salt bioreversible systems
(Bodor et al., 1975). The prodrug approach has also been extensively used for

11
improving the pharmacokinetics and even some aesthetic attributes (taste etc.) of
many drugs. For targeting purposes however, Bodor (1984) has proposed that the
simple prodrug form cannot effectively achieve site specificity unless a given
enzyme is concentrated in certain areas or organs that will cause activation
specifically at these. Rather the CDS approach will present a more viable system
that is capable of achieving site specificity. In the CDS approach, the drug is
transformed by several synthetic steps into the inactive derivative. The resulting
CDS, in vivo, is then expected to undergo successive and predictable enzymatic
transformation ultimately resulting in the selective release of drug at the desired
site. The CDS approach is illustrated diagrammatically in Figure 4. The
modification involves the attachment of monomolecular units generally
comparable in size or smaller than the parent molecule (to contrast from targeting
systems involving polymer-drug conjugates).
The modification in the CDS by design provide a site-specific or site
enhancing delivery of the drug through the incorporation of, most importantly, the
'targeting moiety' (T) in addition to none or several other moieties or
functionalities (P or F) introduced to enhance such parameters as water solubility,
partition coefficient, or to protect against premature metabolism (Bodor, 1984 &
1992). Although the approach is somewhat related, the prodrug or soft-drug in
general does not contain a targeting moiety, but may contain some or all of the
other characteristics of the CDS.
The CDS gets transported to the desired site by non-specific transport
wherefore the various physicochemical or enzymatic reactions occur to release or
sequester the drug at that site. Targeting by the CDS may result from enzymatic-
physical-chemical means, site specific enzyme activation, or receptor based. An
enzymatic -physical-chemical based targeting system may be exemplified by the
dihydropyridinium (DHP) pyridinium (P) salt redox based system for drug

12
targeting to the brain. The CDS (Drag-DHP conjugate) penetrates the blood brain
barrier on account of its high lipophilicity. In the brain (as in other parts of the
body), the CDS undergoes oxidation (chemical) into the hydrophilic quaternary
form (D-QP+) which on account of its hydrophilicity and hence inability to
traverse the BBB remains 'locked' in the brain (physical) whereas that produced at
other parts of the body gets rapidly eliminated. Enzymatic reactions then release
the drug from the pyridinium salt form trapped in the brain (Bodor et al., 1975 and
Bodor, 1984).
The system for site selective delivery described in this dissertation, as may
be more obvious in the subsequent discussion, is also based on the physical-
chemical-enzymatic meansphysical in the reference to the transportation
processes and the appurtenant factors that take the drug to its target site, and
chemico-enzymatic for the processes that selectively sequestrate the lung at the
target site.

CHAPTER 2
FEASIBILITY OF DRUG TARGETING TO LUNG
Physiology and Pharmacology of the Lung
The general structure of the human lung is as shown in Figure 5.
Morphologically, the lung may be divided into parenchymal (alveoli, alveolar
ducts and capillaries), and non-parenchymal tissues (conductive airways, blood
vessels, connective tissue and pleura), comprising altogether a total of about 40
different cell types. The alveoli tissues consist of various cell types which include
macrophages. These macrophages are mobile and metabolically active species
possessing phagocytic, microbicidal and cytocidal activity.
Humans can tolerate oxygen deprivation for only about five minutes. The
major function of the lungs, as part of the respiratory system, is to act as the gas
exchanger (oxygenation of blood and removal of carbon dioxide). For this
purpose, the lungs present a very large surface area, about 72 m2about 35 times
the total skin surface area (Hollinger, 1985a), to communicate between the body
and the environment. However, by this very nature, the respiratory system also
represents a significant portal for the introduction of either noxious or therapeutic
agents. Therefore, in addition to the fact that the lung is also capable of metabolic
functions (xenobiotic metabolism and detoxification), it is susceptible to a wide
variety of disorders namely, bacterial and fungal infections, interstitial lung
disease (ILD) which includes drug/xenobiotic induced diseases, collagen
13

14
vascular disease and fibrosis (cystic fibrosis), connective tissue disorders like
Systemic Lupus Erythematosus (SLE) and lung cancers. Lung cancer along with
the other respiratory tract disorders is the leading cause of morbidity and mortality
in the United States (Miller and Johnson, 1989).
Carina
Right primary
bronchus
Thyroid cartilage
Cricoid cartilage
Trachea
Tertiary
bronchiole
Secondary
bronchiole
Superior
lobe
Middle
lobe
Inferior
lobe
Left primary
bronchus
Superior
lobe
Pulmonary
artery
Left
pulmonary
vein
Inferior
lobe
Pleura
Right lung
showing airways
Left lung showing
pulmonary blood
vessels
Eiguge 5: General Structure of the Human Lung (Washington et al., 1989)

15
Feasibility and Possible Mechanisms
of Selective Delivery to Luna Tissue
The lungs possess the next highest levels of nearly all the metabolic
enzymes found in the liver (Hollinger, 1985b; & Damani, 1987) and in some cases
even higher levels (although in only a few specific cell types). For example, N-
acetyl transferase, an enzyme that acts on para-aminobenzoic acid has 75% higher
activity in the rabbit lung, and Gluthathione -S-Transferases, a group of enzymes
which catalyze the conjugation of glutathione with some xenobiotics, have about
three times higher specific activity in rat lung than in the liver (Damani, 1987).
Additionally, in contrast to all other tissues, the lungs receive the total venous
return first, so it is in an ideal position to regulate (through metabolism and/or
sequestration) the concentration of substrates in the blood before they reach the
arterial circulation (Alabaster, 1977). This implies that after intravenous
administration, a drug reaches the lungs before the liver hence avoiding problems
that may be associated with a hepatic first pass and thereby permitting a more
efficacious sequestration of the drug entity in the lung.
Nevertheless a mechanism for successful CDS for lung delivery, need make
use of enzyme systems, carrier systems, binding reactions or metabolic pathways
occurring (more extensively) in the lungs: For instance, in certain species (e.g.
rabbit, rat and hamster), the specific activities of some oxygenases are higher in
the lungs than in the liver (Bodor, 1984; and Philpot et al., 1977). This factor may
be of importance for mechanisms involving oxygenase catalyzed redox-based
systems such as may probably be the case with the system described in this
dissertation. Also, certain chemicals such as 5-hydroxy-tryptamine, noradrenaline
and amphetamines that accumulate in the lung have been found to do so through
carrier mediated sodium dependent transport systems. Others accumulate through
surfactant binding (e.g. methadone), and protein binding (Philpot et al., 1977).

16
During transdermal delivery studies and evaluation of endogenous
substances as natural soft drugs (Bodor, 1984), it was observed that with the
corticosteroid derivatives of 3-spirothiazolidine (an a-, B- unsaturated ketone),
there was enhanced deposition of progesterone to rat lung tissue after
intravenous administration of the mono- or bis- spirothiazolidine derivatives,
thereby making spirothiazolidine attractive for use in, and generating an interest in
the development of a CDS for drug targeting to lung tissue.
Figure 6: Mechanism for the Enhanced Delivery of
Spirothiazolidine Derivatives (Bodor, 1984)
It was suggested that the mechanism of delivery (Figure 6) was via an
oxidative binding mechanism involving disulfide linkage formation of the

17
intermediate imminium salt (2) of the spirothiazolidine nucleus and cysteinyl
residues of lung tissue proteins. The bound form (3) will then hydrolyze easily to
release the drug (4).
Lipoic acid (l,2-dithiolane-3-pentanoic acid or thioctic acid), on account of
its potential to also form disulfide linkages was investigated for use as the
targeting moiety in a CDS for selective drug delivery to lung tissue. A proposed
mechanism of drug delivery by the lipoic acid CDS is depicted in Figure 7, below.
The probable involvement of disulfide bond formation in the suggested mechanism
was made more conceivable by the report of Livesey et al. (1990) that both the
thiol and disulfide metabolites of aminoalkylphosphorous acid (a class of
compounds that provide cellular protection against radiation and chemo
therapeutic drug toxicity) are capable of binding to rat tissue through mixed
disulfide bond formation.
It may be noted that the oxidative binding may be enzyme enhanced. There
is evidence suggesting that disulfide/sulfhydryl drug and protein thiol bond
formation in vivo is non-specific (Ziegler, 1985), may be catalyzed by a 'disulfide
interchange protein' (Goldberger et al., 1963) since the in vitro rates of disulfide
formation under non-physiological conditions were slower than the in-vivo rates.
The enzymes catalyzing the thiol- disulfide exchange reactions are present in the
cytosol and membrane fractions (cited in, Ziegler, 1985). One such enzyme, the
protein disulfide isomerase, PDI, has been shown to catalyze, the scission as well
as disulfide bond formation between disulfide/sulfhydryl drugs and tissue
sulfhydryl groups (Edman et al., 1985 & Darrow et al., 1988).
These findings suggest the probable involvement of disulfide-bond
formation in the mechanism of accumulation of the lipoic acid based chemical
delivery system for drug targeting to lung tissue as shown in Figure 7.

18
RCOCr(CH
2)5N0
s~s
CDS
' RCOO- (CH2)5 ^
SH SH
v /
^ Enzymatic
'
in vivo redox
reactions
Figure 7: Proposed Mechanism of Delivery by the Lipoic Acid CDS:
In the scheme, the inactive lipophilic CDS after administration enters lung tissue. In
the pharmacokinetic phase the lipolvl moiety undergoes disulfide exchange with the
sulfhydrvl groups of cvsteme residues of lung protem. This serves to anchor the CDS
while esterases release the active parent drug.

19
In addition, a review of available literature revealed reports that some sulfur
containing compounds, sulfhydryl and disulfide containing drugs covalently bind
with sulfhydryl groups of tissue proteins in rats (Livesey et al., 1990; & Miwa et
al., 1988), and in rabbit and human tissue (Tabachnick, 1982). Naturally
therefore, one may expect a possible relationship may exist between
disulfide/sulfhydryl drug binding and PD1 distribution and tissue sulfhydryl. The
relative tissue distribution/activity of PDI in the rat has been estimated to be of the
order (Edman et al., 1985):
liver > pancreas and kidney > lung > testes and spleen > heart > brain.
It has also been observed that, for the rabbit, tissue sulfhydryl content
(extracted with hot 80% ethanol), in mmoles/gram wet weight (or mL wet volume
for blood), was of the order (Ellman, 1959):
kidney (10.1) > Lung (2.2) > Liver (1.9) Blood 5.36 x 10'3
Biologically, sulfhydryl groups occur in tissue mainly as the amino acid
cysteine (Perret and Rudge, 1985). Cysteine, along with its disulfide cystine
occurs in body fluids and cells as well as in most animal proteins. Homocysteine
is found as the free thiol, its homodisulfide and mixed disulfides in man. Reduced
glutathione (GSH), the cysteine-containing tripeptide, is found in high
concentrations in most mammalian tissues where it fulfills many roles including
maintenance of the oxidation status of the cell.
Lipoic Acid:
The proposed use of lipoic acid in the CDS is made even more attractive by
the fact that lipoic acid is non-toxic and it is required as a co-enzyme for acyl
transfer and redox reactions in living systems (Kijima et al., 1984).

20
Alpha-Lipoic acid (i.e. 1.2-dithiolane-3-pentanoic acid, or thioctic acid) is
the internal disulfide of 6,8-dithioctanoic acid. It is the coupler of electron and
group transfers catalyzed by a-keto acid dehydrogenase multienzyme complexes.
As coenzyme, lipoic acid forms part of these enzyme complexes which are
centrally involved in carbohydrate metabolism. The role of lipoic acid within the
pyruvate dehydrogenase complex in catalyzing the reaction of pyruvate with
NAD+ and Coenzyme A (CoA) is illustrated in Figure 8 (Zubay, 1986). The
enzyme complex consists of three proteins: a pyruvate dehydrogenase which is
thiamine pyrophosphate (TPP) dependent and designated Ej-TPP; dihydrolipoyl
transacetylase (E2-lipoyl-S2) which contains the oxidized form of lipoic acid
covalently bonded through amide linkages to the e-amino group of lysine; and a
dihydrolipoyl dehydrogenase, a flavoprotein designated E3-FAD.
In the scheme illustrated below, pyruvate is decarboxylated by EpTPP.
The resulting activated acyl group is accepted by lipoic acid (1) which eventually
then transfers it to CoA (to be utilized in intermediary metabolism). The Lipoic
acid gets reduced to dihydrolipoic acid, DLAc, (3) which is then oxidized by FAD
(4) to give back lipoic acid, the reaction being coupled with the formation of
NADH (a reducing equivalent in in-vivo redox reactions). The chemical aspect of
the conenzymatic activity of lipoic acid is thus to mediate the transfer of electrons
and activated acyl groups resulting from the decarboxylation and oxidation of a-
keto acids within the complexes. In this process, lipoic acid is itself transiently
reduced, and this reduced form is the acceptor of the activated acyl group. This
dual role of electron and acyl group acceptor enables lipoic acid to couple the two
processes (Zubay, 1986).

21
CH.COCOj
CO,
1
Figure 8: Coenzymatic Function of Lipoic Acid (Zubay, 1986)

22
Chlorambucil:
HOOC(CH2)3-^)-N
^ch2ch2ci
ch2ch2ci
Chlorambucil
Chlorambucil, (4-p-[bis(2-chloroethyl)amino]phenylbutyric acid), is a
bifunctional nitrogen mustard alkylating agent that has shown wide clinical
activity as an antineoplastic agent particularly against chronic lymphocytic
leukemia and malignant lymphomas (Oppitz et al., 1989; & Greig et al., 1990)
primarily due to its alkylation reaction with DNA. It also binds to nuclear proteins
causing disturbances leading to cell death (Riches and Harrap, 1975). The
alkylation proceeds through a cyclic ethyleneimmonium ion or carbonium ion
intermediate which is susceptible to attack by nucleophiles.
Chlorambucil
i
t
+
CH2CH2CI
the ethyleneimmonium ion

23
This carbonium ion intermediate which is essential for pharmacological
activity is also readily formed in aqueous solution where it is attacked by
nucleophiles such as water. Thus chlorambucil and other nitrogen mustards have
been reportred to have very short shelf-lives in aqueous solutions Chlorambucil
12 minutes, and Mephalan about 25 minutes (Ehrsson et al., 1980). Active
metabolites of chlorambucil include 3,4-dehydrochlorambucil and phenylacetic
acid formed in vivo via (1-oxidation. Although the drug is rapidly absorbed from
the gastrointestinal tract, ionization at physiological pH limits its ability to
penetrate cells. Besides, there are problems associated with its use which include
a wide variety of major but usually dose limiting toxicity involving hematologic
(e.g. myelosuppression and anemia), gastrointestinal (nausea, anorexia),
neurologic (CNS effects seizures, ataxia etc.), dermatologic and even mutagenic
and carcinogenic effects, introduction of the lipoyl group to mask the ionizable
carboxylic acid group will increase its lipophilicity and hence enhance its cellular
uptake in addition to the desired targeting capability and ultimately reduce its
toxicity.
Cromolyn (DSCG):
Cromolyn, in the form of its sodium salt, disodium cromoglycate, DSCG, is
a bischromone, the disodium salt of l,3-bis-(2-carboxychromon-5-yloxy)-2-
hydroxypropane.
Disodium Cromoglycate

24
DSCG is used primarily in the prophylactic treatment of asthma, and
allergic rhinitis. Since its first introduction in 1968, it has increasingly and
successfully been used in many medical conditions including allergic disorders of
the upper respiratoiy tract, eye and ear problems, food sensitive enteropathies, a
variety of skin conditions such as eczema (Kuzemko, 1989), and more recently it
has been reported to inhibit benzo(a)pyrene induced tumors in rats (Vlckova et al.,
1989).
Mechanism of action of DSCG
in the asthmatic response, the antibody IgE is produced by lymphoid tissue
in response to the extrinsic allergen. The IgE becomes fixed to mast cells in the
bronchial walls in the sensitized individual. Exposure to further allergen results in
antigen-antibody reaction occurring on the surface of the mast cell which results
in the release of the mediators of anaphylaxis which include histamine, slow-
reacting substance of anaphylaxis (SRS-A) which produces prolonged
bronchoconstriction, eosinophil chemotactic factor (ECF-A), bradykinin and
others. This constitutes the early asthmatic response. The mediators also cause
alteration in capillary permeability, resulting in the entry of IgG and leukocytes
into the bronchial connective tissue resulting in a Type III delayed complement-
fixing reaction (the late asthmatic reaction) leading to leukocyte damage, release of
lysosomes, local tissue damage and release of prostaglandins and other mediators
which result in further bronchoconstriction and other symptoms (Brewis, 1975).
The DSCG exerts its anti-allergic activity by inhibiting the release of mediators
(such as histamine, and some leukotrienes) from mast cells and thereby preventing
the onset of asthmatic symptoms due to the bronchoconstrictor and inflammatory
effects of these mediators. There are two main phases of the asthmatic response,
the early asthmatic response, EAR, and two stages of the late asthmatic responses,

25
LARs (Holgate, 1989). Following allergen challenge, the EAR and the first LAR
but not the second LAR are inhibited by the prior administration of DSCG
(Holgate, 1989). The lack of effect of DSCG on the second LAR may relate to the
limited bioavailability of DSCG such that by the onset of the second LAR (72
hours), the drug has been completely cleared from the body. This is supported by
Matolli et al. (1987), who demonstrated that the administration of DSCG between
the EAR and the first LAR inhibited the second LAR. DSCG acts by inhibiting
mast cell degranulation and thereby preventing the release of the preformed
mediators of anaphylaxis. It also interferes with the migration and function of
eosinophils. DSCG has been found to inhibit the cellular uptake of Ca2+
(Hemmerich et al., 1991). The mechanism by which DSCG acts is not precisely
known but it is believed to work by inhibiting the flux of calcium, Ca2+, ions
(Vlckova et al., 1986, & Holgate, 1989). The uptake of Ca2+ being an essential
trigger to activate microfilament contraction responsible for exocytotic secretion.
The antimutagenic activity of DSCG reported by Vlckova et al., 1986, is also
believed to operate via this mechanism. The known pharmacological activity of
DSCG therefore lies in its ability to block pathological exocytotic secretion.
Exocytotic response requires contact with cytoplasm by signaling mechanisms, in
addition to a rise in cellular Ca2+. The rise in intracellular Ca2+ is known to be a
factor which triggers exocytosis in response to external stimuli at the plasma
membrane. With DSCG blocking the influx of Ca2+, the preserved Ca2+ balance
is thought to be a factor for the preservation of cell functions within the
physiological range (Vlckova et al., 1986)
Need for Improved Delivery of DSCG:
Although DSCG is the safest anti-asthma drug currently in use (Kuzemko,
1989), it has very poor bioavailability as it is too polar and very water soluble. It

26
is thus poorly absorbed, and rapidly cleared from the body. After oral
administration, only 1% of the dose is absorbed in man, and after inhalation, less
than 8% of the dose reaches the lung and is absorbed systemically (Cox et al.,
1970, & AHFS Drug Information, 1990). After intravenous administration, the
drug is rapidly cleared from the plasma and tissues, and accumulated in the liver or
kidney prior to biliary or urinary excretion. In rats, 85% of the injected dose is
cleared from the plasma at a rate with half-life of about 8 minutes. In man, the
corresponding plasma or lung Ty2 is about 30 minutes (Cox et al., 1970).
For the treatment of asthma, DSCG is clinically effective only after
inhalation administration. Pulmonary drug delivery is primarily used to treat
conditions of the upper respiratory tract. The drug delivered must be deposited
onto the bronchial epithelium in therapeutic quantities. But generally, only about
10% of the dose delivered remains in the lungs (Newman et al., 1982). The
process of drug delivery via the inhalation route is inefficient due to a combination
of factors which include the mode of delivery, the point of inspiration relative to
the triggering device and respiratory variable such as the breathing pattern. The
anatomy and pathophysiological state of the respiratory tract can also markedly
affect the drug delivery process. Additionally, structures in the upper respiratory
tract function to minimize the inhalation of particulate materials (Washington et
al., 1989; and Ganderton and Jones, 1987). The problems inherent with inhalation
delivery especially in pediatric cases, the limited bioavailability, and the other
possible uses of DSCG make the need for other modes of administration as well as
improved or targeted delivery to lung welcome. The proposed lipoyl derivative is
expected to improve the lipid solubility of DSCG and therefore enhance its
bioavailability irrespective of route of administration. This relatively more lipid
soluble derivative will be more able to penetrate the cells, be retained in tissues
longer (i.e. longer biological half-life), enhance selective delivery to lung tissue

27
and also, possibly, enable the administration of the drug by additional routes other
than inhalation.
Predicted In-Vivo Metabolism of CDS
In the work presented in this dissertation, the l,2-dithiolane-3-pentyl
moiety of D,L -lipoic acid was investigated for use as a targeting moiety, based on
a concept related to the CDS approach, for drug targeting to lung tissue, using
Chlorambucil and Cromolyn Sodium as the respective model parent drugs. Three
final derivatives were prepared and studied namely, the Chlorambucil CDS or
MS-2; Cromolyn CDS-1 or MS-4; and Cromolyn CDS-2 or MS-17. Some aspects
of the in-vivo metabolism of the CDS, characteristic of a rationally designed drug,
may be fairly accurately predicted. Both model CDS's were expected to undergo
ester hydrolysis to release the respective parent drugs. Chlorambucil (Figure 9),
as previously known (Greig et al., 1990), undergoes further metabolism via (3-
oxidation to produce the metabolites 3,4-dehydrochlorambucil and phenylacetic
mustard both of which like the parent drug are reactive as alkylating agents (these
were probably the two metabolites detected during the HPLC assay of the
hydrolysis products of the CDS graphically presented in Figure 24, page 84). The
predicted metabolism of the cromolyn CDS is also shown in Figure 10. In both
pathways, the liberated lypoyl moiety would be expected to participate in its
known in-vivo coenzymatic function or undergo degradation and elimination
characteristic of thiol metabolism.
Thiol/disulfide compounds in general undergo metabolic transformations by
desulfuration or transfuration into hydrogen sulfide, sulfates or other sulfur
compounds. In-vivo, thiols and disulfides readily interconvert.
-SH + V202 <=> -S-S- + H20.

28
Keilin (1930) has shown that this reaction is catalyzed by cytochrome C and
cytochrome oxidase acting together.
(CH2>5'OOC'(CH2>3
/CH2CH2CI
N
^ch2ch2ci
Chlorambucil CDS
I
H00C(CH2)3~O"Nv
^ch2ch2ci
'ch2ch2ci
Chlorambucil
rr
S- s
(CH2)5-OH
LIPOLOL
I
<-
J
1
1
^CH^CHoCI
1
in-vivo redox
ch2ch2ci
reactions
3,4 Dehydrochlorambucil
HOOC-CH 2
H^-N
ch2ch2ci
ch2ch2ci
Thiol
Metabolism
Phenylacetic Mustard
Figure 9: Predicted Metabolism of Chlorambucil CDS

29
o2nov^
O COO-lipoyl
Cromolyn CDS-2 (MS-17)
carboxylesterase
T
COO'
Cromolycate Nitrate (MS-16)
nitrate
reductase
COO"
/
cromoglycate
(active drug)
Direct Elimination
Urinary & Biliary routes
GSSH
HO
O COO-lipoyl
Cromolyn CDS-1 (MS-4)
2
X
thiol
metabolism
Lipoamide
1
In-vivo Redox &
acyl transfer reactions
Figure 10: Metabolism of Cromolyn CDSs

30
The pathways for thiol metabolism are mostly oxidative and while not
precisely known for each individual thiol/disulfide, the metabolic pathway for the
lipoyl moiety may be expected to follow a general scheme and resemble that of
aspargusic acid, a dithiolane compound, (Waring et al., 1987). The dithiolane
compound is reduced to the dithiol which then undergoes S-methylation. In
humans, the enzymes thiol methyltransferase TMT, and thiopurine
methyltransferase, TPMT, catalyze the S-methylation of thiols (Bremer &
Greenberg, 1961; Drummer et al., 1982; and Glauser et al., 1993). TMT is
membrane bound and catalyzes preferentially the S-methylation of aliphatic -SH
compounds whereas TPMT is cytoplasmic and catalyzes the S-methylation of
aromatic and heterocyclic -SH groups such as lipoic acid. The S-methyl
intermediate may then undergo [3-oxidation of the ring carbons (Waring et al.,
1987), liberating methanethiol. Subsequent oxidation, methylation and
dimerization would result in a variety of products such as dimethyl sulfide,
dimethyl sulfoxide, dimethyl sulphone and methanethiol. Additionally, the thiol
may be oxidized to sulfate or sulfite through a variety of oxidizing systems. No
distinctive pathway for the oxidation is known, but it is known to involve
xanthine oxidase or the cytochrome oxidase systems (Kun, 1967).

CHAPTER 3
RESEARCH DESIGN AND SPECIFIC OBJECTIVES
Objective
The main objective of the work described in this dissertation was the
development of a novel chemical delivery system to achieve the selective delivery
of drugs to lung tissue and thereby lessen unwanted side effects, especially in the
treatment of lung cancers. The system is also expected to enhance the
bioavailability of highly polar hydrophilic drugs like cromoglycic acid, and
increase the duration of action of drug in the lung by prolonging its retention in
lung tissue through probable formation of disulfide linkages and thereby ensuring,
to some extent, a 'slow release' of the anchored drug entity. By these newly
incorporated capabilities, the system will also work to optimize the therapeutic
index of drugs used in the management of lung diseases.
Synthesis
In the design process, employing a variation of the CDS approach
(illustrated in Figure 5), each of the two model drugs used in this work were
converted into the corresponding CDSs according to the following schemes
respectively:
31

32
Chlorambucil CDS
The synthesis of the chlorambucil CDS, analogous to the species TD' in
Figure 5, was achieved by the direct esterification of the corresponding alcohol of
lipoic acid, the targetor moiety, T, with the carboxylic acid group of chlorambucil,
(the parent drug, D), using Dicyclocarbodiimide (DCC) as a coupling agent. The
DCC converts the acid chlorambucil, (I) into a compound with a better leaving
group (5) and thereby helping drive the reaction via the following mechanism:
R C.
+ DCC
O
2
3
ESTER PRODUCT
Figure 11: Mechanism of Esterification with DCC

33
(CH 2)5 -OOC-(CH 2)3-^)-N^
ch2ch2ci
CH2CH 2CI
s- S
Chlorambucil CDS, (MS-2)
Cromolyn CDS
In a similar approach, the lipolyl group (T) was coupled to the cromolyn
parent drug (D) to give MS-4, the 'Cromolyn CDS-1' or TD'. Also. In an attempt
to enhance the aqueous solubility of the cromolyn CDS-1, a modifier group 'F in
the form of the nitrate (N02) group was introduced to replace the OH- group on
the carbon-2 of the propanyl link between the two chromone heterocycles to give
the 'Cromolyn CDS-2'. This site was chosen primarily because it was assumed to
have no role in the pharmacological activity of the drug (Cairns et al, 1972), and
also because it seemed to be the easiest to nitrate.
HO
CH"CH?-0 O
SS -I
2
Cromolyn CDS-1, (MS-4)

34
(CH2)5^y^
SS
2
Cromolyn CDS-2, (MS-17)
Application of the DCC esterification method to the cromolyn system
resulted in a very poor yield less than 5%. This might have been due to the
following two reasons:
1. The anion (2) generated from the reaction of the DCC with the acid form of
cromolyn (step 1 in the mechanism described in Figure 11 above) was much
less nucleophilic due to the electron withdrawing effect of the aromatic
chromone nucleus of the cromoglycic acid. Hence the formation of the species
(4) leading to the formation of the intermediate compound (5), having the
better leaving group (step 2) was not very favored.
2. Under the conditions used for the esterification, i.e., refluxing 60 80C in
solution for prolonged period -- more than 24 hours, lipolol (or the dithiolane
ring) is known to be unstable and undergoes polymerization (Walton et al.,
1955; & Brown and Edwards, 1969). Indeed the bulk of product extracted
from the reaction was a sticky plastic-like substance.
With these considerations taken into account, the aim then was to find a
method which required little or no heating and involving solubilization of the
lipolyl species for the least time possible. These conditions were realized through
the an adaptation of a method developed by Pfeffer et al., (1972) in which the

35
corresponding alkyl halide of lipoic acid was reacted with the disodium salt
(cromolyn) in hexamethyltriphosphoramide (HMPA or HMPT). This method
resulted in quantitative yield (over 85% pure product), occurred at moderate
temperatures (35 40C) and was complete in about 30 minutes.
Evaluation of Mixed-Disulfide-Bond Formation as a
Probable Mechanism for CDS Selectivity for Lung Tissue
In-vivo experiments described later on in this dissertation showed that there
was enhanced delivery of chlorambucil and cromoglycic acid (cromolyn) to rat
and rabbit lung tissues through the lipoic acid CDS, and of progesterone to rat lung
tissue via the 3-spirothiazolidine based CDS (Bodor, 1984). It was suggested that
the mechanism of this enhanced delivery involves the probable formation, in-vivo,
of mixed disulfide linkages between free sulfhydryl groups generated on the CDS
and tissue sulfhydryl mainly in the form of cysteinyl residues. In order to
experimentally establish or support the proposed mechanism, a series of in-vitro
binding experiments were designed which involved incubating the drug/CDS with
respective tissue homogenates and determining:
1. The amounts of free unbound drug and drug bound through disulfide
linkages by treatment of the bound drug fraction (in the form of
precipitated protein) with 1,4-dithiotreitol, DTT (a disulfide
reducing agent). Any amounts of drug detected following this
treatment indicated the drug originally bound by disulfide linkages;
2. The extent of CDS/drug-tissue binding in the presence of N-
ethylmaleimide (NEM), a sulfhydryl blocking agent; and also,

36
3. The relationship, if any, between tissue sulfhydryl content and drug
binding.
4. The absence of mixed disulfide binding in the absence of NEM and
its occurrence in the absence of NEM is evidence of mixed disulfide
binding.
Enzyme Inhibition Studies
Since a model CDS consisted of the active (parent) drug attached to the
targeting moiety by an ester linkage, the metabolic conversion of the CDS to the
active drug was presumed to occur via ester hydrolysis catalyzed by a
carboxylesterase (B-esterase) enzyme. It was therefore necessary to inhibit these
enzymes in the tissue homogenate so that a reasonable amount of the intact CDS
was maintained throughout the binding studies. The inhibitor used for this
purpose was the B-esterase inhibitor bis-(p-nitrophenyl)phosphate (BPNP) sodium
salt (Sterri & Fonnum, 1987; and Yamada et al., 1992) and the method for the
esterase inhibition was adapted from Yamada et al. (1992). Studies were included
to establish the optimum concentration of inhibitor needed to reasonably inhibit
the esterases.
Kinetics of Mixed Disulfide-Bond Formation
With the presence of BPNP in the homogenate during the binding studies it
was necessary to determine also whether its presence had any effect on the extent
of CDS binding. In addition, further kinetic studies were conducted to shed some
light on such aspects of the mixed-disulfide binding with respect to time,
concentration and tissue type.

37
Tissue Sulfhvdrvl
Since binding to tissue sulfhydryl was suspected to be a probable
mechanism of site selective delivery by the CDS, the tissue sulfhydryl content for
the various tissues were determined in an attempt to relate the extent of mixed-
disulfide binding to tissue sulfhydryl in order to expatiate on differences, if any, in
the binding of the CDS to the various tissues and help provide an estimate of the
'magnitude of binding' per tissue type.
Research Protocol
The experimental protocol described in this dissertation comprised
1. Synthesis of the various chemical delivery systems/derivatives for
chlorambucil and cromolyn respectively.
2. HPLC assays for detection of respective parent drugs and CDS/
derivatives in biological fluids.
3. Determination of physicochemical parameters such as octanol/water
partition coefficient (or Log P), and in-vitro stability in buffer and
various biological media.
4. Comparative in-vivo studies in rats and Rabbits to determine the ability
of CDS to selectively deliver drug to lung tissue relative to parent drugs.
5. Determination of tissue sulfhydryl (Total-Thiol, Protein bound Thiol,
and Non Protein Bound Thiol)
6. In-vitro binding and related studies to experimentally support the
proposed mechanism of delivery by the CDS comprising:
i. Enzyme inhibition Studies.

38
ii. In vitro binding experiments, namely
Determination of free unbound drug/CDS, and drug or
CDS bound to tissue via mixed disulfides linkages.
Determination of bound drug/CDS in presence of the
tissue sulfhydryl-blocker, N-ethylmaleimide,
Effect of BPNP on disulfide binding.
iii. Evaluation of kinetics of binding with respect to
Concentration dependence and
Dependence of binding on time.

CHAPTER 4
EXPERIMENTAL METHODOLOGY
Materials
All chemicals and reagents used were reagent grade. Solvents and reagents
used for the High Performance Liquid Chromatography (HPLC) and Ultra-Violet
spectrophotometric analyses were HPLC or spectroscopic grade. Chlorambucil
and cromolyn were obtained from Sigma Chemical Company, St. Louis, Missouri.
Other chemicals and solvents were obtained from Sigma Chemical Company,
Aldrich Chemical Company, Milwaukee, Wisconsin, and also from Fisher
Scientific Company.
For the analytical and compound characterization methods, thin-layer
chromatography (TLC) was performed using EM Science DC-plastic foil plates
coated to a thickness of 0.2 mm with silica gel 60 containing Fluorescent (254)
indicator. Spots were detected by exposure to short-wavelength UV light. All
melting points were determined using the Fisher Johns Melting Point Apparatus,
and were uncorrected. iH-NMR data were measured in CDCI3 or in dimethyl-/6
sulfoxide, (ig-DMSO, (for the polar compounds that would not dissolve in the
CDCI3) on a Varan EM-390 NMR Spectrophotometer and chemical shifts
reported in parts per million relative to tetramethylsilane. Infrared Spectra were
recorded with a Perkin Elmer 1420 Ratio Recording Infrared Spectrophotometer
39

40
and were made in KBr pellets (~ 1 mg of compound in 100-mg pellet), paste in
mineral oil, or neat liquid. Ultraviolet (UV) spectra as well the UV spectro-
photometric analyses were performed using the Varan CARY 210 UV/Visible
Spectrophotometer with quartz cuvettes up to A, < 200 nm and an optical path
width of 10 mm. Mass spectrometry was performed by Fast Atom Bombardment
(FAB) on the Kratos MFC 500 Mass Spectrometer. Elemental analyses of the
purified specimens were performed by the Atlantic MicroLab Inc., Atlanta,
Georgia. Instrumentation for the HPLC assays included a Spectra Physics SP
8810 Precision Isocratic Pump, the SP 8450 UV/VIS Detector (operated at
wavelengths of 240 nm for cromolyn and its derivatives, and at 254 nm for
chlorambucil and its derivative) and the SP 4290 Integrator. The HPLC sample
loop size was 20 pL. The HPLC method used was reverse-phase (with ion-pairing
or ion suppression in the case of cromoglycate estimation). The stationary phase
columns used were Hypersil ODS-2 Qg and Cg Columns, both 100 mm x 4.0 mm
internal diameter with guard columns of similar packing. The ion-pairing reagent
was benzyltributylammonium chloride (BTBA-C1).
All in-vivo studies were conducted in laboratory animals 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 83).
The animals used in the studies were male New Zealand Albino rabbits weighing
about 3 Kg each (obtained from Kel Farm, Alachua, Florida), and white male
Sprague Dawley rats each weighing between 300 and 350 grams (obtained from
Harlan Sprague Dawley Inc., Indianapolis, Indiana). Human blood used in the in
vitro stability studies, was obtained from this investigator himself, and from a staff
member at the Center for Drug Discovery (University of Florida College of
Pharmacy) where this research was conducted. Dog blood was obtained from the
Animal Resources unit of the University of Florida Veterinary School.

41
Methods
Synthesis
Chlorambucil CDS. (MS-2)
a. 1,2-dithiolane-3-pentanol, (dl-lipolol), MS-1; Lipoic acid, (2.06g, 10
mmol) was placed in a flame dried 250 mL flask fitted with a stirrer, dropping
funnel, and connected to an oil bubbler to maintain a Nitrogen atmosphere. 70 mL
of anhydrous chloroform was added followed by 1.0 M catecholborane, in
tetrahydrofuran (50 mL, 50 mmol) dropwise. The mixture was refluxed (70 80
C) for about 6 hours. 20 mL of cold water was then added dropwise and the
organic solvent evaporated in vacuo. 50 mL of dichloromethane was added, and
the mixture extracted with one 25 mL aliquot of water followed by six 25 mL
aliquots of 1.0M NaOH to remove the catechol. The organic portion was dried
with sodium sulfate, filtered and solvent evaporated in vacuo. The crude product
was purified by column chromatography (silica gel, methylene chloride/ethyl
acetate, 4:1).
b. Lipolyl ester of Chlorambucil (i.e. the Chlorambucil-CDS or MS-2):
DL-lipolol, MS-1, (1.92g, 10 mmol) in 50 mL of dichloromethane (CH2CI2) was
added to chlorambucil (3.04g, 10 mmol) in 50 mL of CL^C^. An excess of DCC,
1,3-dicyclocarbodiimide, (8.25g, 40.0 mmol) in 20 mL of CH2CI2 was then added.
A catalytic amount of 4-dimethylaminopyridine (DMAP)approximately 1 mg,
was added to the reaction mixture and stirred at room temperature until the
reaction was complete (about 24 hoursmonitored by TLC). At the end of the
reaction, the mixture was washed with two 20 mL portions of water. The organic
layer was then evaporated in vacuo. The crude product, a yellowish brown
viscous oil, was purified by column chromatography on silica gel using toluene as

42
the eluant. The overall scheme for the synthesis of the chlorambucil CDS is
summarized in the following scheme
(CH2)4COOH
s-s
1. Catecholborane

2. H,0
(CH2)5-OH
Lipoic Acid
Chlorambucil
(CH
2^5
-OOC-(CH2 )3
-H
CH2CH2 Cl
CH2CH 2C1
s-s
MS-2
Figure 12: Synthesis of Chlorambucil-CDS (MS-2)
Cromolyn CDS-L (MS-4)
a. 5-bromo-3-penty¡-1,2-dithiolane, (Lipolyl bromide)-, Lipolol, (0.58g, 3
mmol) and triphenylphosphine (2.37g, 9 mmol) were dissolved in 15 mL of
anhydrous tetrahydrofuran, THF. Zinc bromide (0.68g, 3 mmol) in 10 mL THF
was added followed by diethyl azidodicarboxylate, DEADC, (1.57g, 9 mmol) in 5
mL THF. The mixture was stirred at room temperature under nitrogen atmosphere
for about 2 hours (i.e. until lipolol had disappeared). Methanol (2.0 mL) was then

43
added to destroy the excess reagent. After 5 minutes, the mixture was extracted
with ether (20 mL), and the ether layer washed with 12 mL aliquots of water,
saturated Na2CC>3, and saturated NaCl successively, and the organic solvent
evaporated in vacuo. The product was then extracted twice into hexane, leaving a
residue of triphenylphosphine oxide. After evaporation of the hexane fraction, the
solid residue was again extracted by trituration with hexane, and the organic
extract evaporated in vacuo to give the crude product. The crude product was
purified by column chromatography on silica (50g, eluant was Ethyl Acetate/
Hexane, 1:9).
S~S
DEADC, Anhyd. THF S S
Lipolol
Lipoyl Bromide
O COONa 2
Cromolyn
HO
I
CH~CH2_o o
S-S J 2
MS-4
Figure 13: Synthesis of Cromolyn-CDS-1 (MS-4)

44
b. 1,3-bis(2-Upolyloxycarbonyl-chromon-5-yloxy)propane i.e. the Lipolyl
ester of cromolyn or the Cromolyn-CDS, (MS-4): Hexamethylphosphoramide,
HMPA, 10.0 mL, was added to disodium cromoglycate, DSCG, (0.77 g, 1.50
mmol) in a 50 mL flask with stirring at room temperature and nitrogen atmosphere.
Lipolyl bromide (1.42 g, 6.0 mmol) was added and the mixture warmed to 45 -
50C for about 40 minutes to aid in the dissolution of the DSCG. Stirring was
continued for about two hours at room temperature (or until all of the cromolyn
has disappeared) and the reaction mixture poured into 35 mL of water. The
mixture was then extracted with two 20 mL portions of CH2Cl2, and the combined
organic layers washed with several 20 mL portions of water and dried (Na2SC>4).
The crude oily product was purified by column chromatography (silica gel,
Chloroform/Ethyl acetate 10:1, v/v). Fractions containing the diester were pooled
together and evaporated in vacuo, the resulting oily product was recrystallized
from ether by dissolving it in 2 mL of chloroform and adding the resulting solution
dropwise to 50 mL of ether with stirring. The yellowish white crystals obtained
were filtered, washed with cold ether and vacuum dried in the presence of P205.
The scheme for the synthesis is summarized in Figure 13.
Cromolyn CDS-2. (MS-17)
a. \,3-bis(2-ethoxycarbonylchromon-5-yloxy)-2-hydroxypropane,
(Cromolyn diethyl ester, MS-11): DSCG (2.05g, 4 mmol) was placed in a 50 mL
two-necked flask. 15 mL HMPA was added with stirring and N2 atmosphere.
Bromoethane (3.5g or 4.5 mL, 32 mmol, 400 % mol equivalents) was added. The
mixture was warmed to about 45 50C for about 30 minutes until a clear solution
formed. The reaction mixture was poured into 50 mL water and extracted with
two 25 mL portions CH2C12. The organic layer was then washed with two 25 mL

45
portions water dried (Na2S04) and evaporated in vacuo. The resulting product
was redissolved in chloroform and added dropwise to 100 mL ether with stirring.
The white crystals obtained were filtered, washed with cold ether, and then dried.
b. 7,3-bis(2-ethoxycarbonylchromon-5-yloxy)-propan-2-nitrate, (diethyl
cromolyn nitrate ester, MS-15): The nitrating agent, Acetylnitrate, CH3C(0)-
ONO2 was prepared by the method of Manstch and Bodor (cited in Bodor et al.,
1980). Acetic anhydride (20 mL) was placed in a 50 mL conical flask in an ice-
water bath (approx. 4C) and 2 mL of concentrated HNO3 added. The mixture
was stirred in the cold for two hours and the resulting solution containing the
acetylnitrate was used directly in the subsequent reaction.
Cromolyn diethyl ester, MS-11, (1.05g, 2 mmol) was dissolved in 25 mL of
chloroform, and 10 mL of the nitrating reagent solution added. A clear yellow
solution formed which soon turned cloudy. The mixture was stirred at room
temperature for about 30 minutes and then poured into 50 mL water. The
chloroform layer was separated, washed with water, dried (MgSC>4) and
evaporated in vacuo. The resulting solid crude product was dissolved in methanol
and chromatographed over silica (mobile phase chloroform/methanol, 10:1). The
fractions containing the nitrate ester were pooled together concentrated and
recrystalyzed by adding dropwise to ether as before.
c. l,3-bis(2-carboxychromon-5-yloxy)-propan-2-nitrate disodium salt,
cromolyn nitrate disodium salt, MS-16: Diethyl cromolyn nitrate ester, MS-15,
(0.57g, 1.0 mmol) was suspended with stirring in 5 mL absolute ethanol and
treated dropwise with 2 mL of IN NaOH (2.0 mmol). The resulting mixture was
refluxed for 1 hour and after tap cooling, about 10 mL ethanol was added. The
mixture was chilled in the freezer compartment to precipitate the sodium salt. The
solid was filtered off, dissolved in a minimum amount of hot water, filtered,
reheated to boiling. The hot solution was then added slowly to boiling ethanol (15

46
mL). The solution was cooled under the tap and refrigerated for about one hour.
A small amount of ether (10 mL) was added to aid precipitation. The precipitated
sodium salt was filtered and dried (vacuum, P2O5).
d. \,3-bis(2-lipolyloxycarbonylchromon-5-yloxy)pwpan-2-nitrate, i. e.
Cromolyn lipolyl nitrate ester, Cromolyn CDS-2, or MS-17: Hexamethyl-
phosphoramide, HMPA, 10.0 mL, was added to cromolyn nitrate disodium salt,
MS-16, (0.38 g, 0.72 mmol) in a 50 mL flask with stirring at room temperature and
nitrogen atmosphere. Lipolyl bromide (0.44 g, 1.80 mmol) was added and the
mixture warmed to 45 50C. The thick slurry turned into a light clear liquid.
Stirring was continued at this temperature for about two hours, or until the
disodium salt, MS-16, had disappeared). The reaction mixture was then poured
into 25 mL of water and extracted with two 15 mL portions of CH2CI2, and the
combined organic layers washed with two 10 mL portions of water and dried
(Na2S04) and evaporated in vacuo. The crude oily product was purified by
column chromatography (silica gel, Chloroform/Ethyl acetate 10:1, v/v). Fractions
containing the diester were pooled together and evaporated in vacuo, the resulting
oily product was recrystallized from ether by dissolving it in 2 mL of chloroform
and adding the resulting solution dropwise to 25 mL of ether with stirring. The
yellowish white crystals obtained were filtered, washed with cold ether and dried
(vacuum, P2O5).
The multi-step synthesis of the cromolyn CDS-2 is summarized in the
following scheme (Figure 14).

47
HO
I
chtch2-o o
O COONa
Disodium
Cromoglycate
1 J
o 'COOC2H5
Cromolyn Diethyl Ester, MS-11
O
II
ch3c -ono,
Acetyl Nitrate
Reagent
o2no
o2no
-CHo-O O 1
NaOH, Ethanol
1
CH
ch2-0
0
1
ax
^
Reflux
(Y
COONaJ
2
o COOC2H5
Cromolyn Nitrate Disodium Salt
MS-16
o2no
Figure 14: Scheme for the Synthesis of Cromolyn CDS-2

48
HPLC Assays:
Reversed phase HPLC methods were used to resolve and quantitatively
estimate the respective drug/CDS's and their metabolites in the various biological
and other media. Reversed phase HPLC with ion-pairing was used for the
estimation of cromolyn. The respective efficiencies of the drug recovery methods
from biological media were well over 95% in all cases. Concentrations were
estimated from appropriate calibration curves based on concentration versus area
under curve (AUC) plots, all of which showed linearity (r = 0.999) for the range of
0.1 to 10.0 pM.
For the estimation of chlorambucil and the chlorambucil CDS (MS-2), the
HPLC system included a Cjg stationary phase column (100 mm x 4.0 mm i.d.) and
73.5% Acetonitrile/0.5% Acetic Acid/27% Water as the mobile phase. The UV-
detector was set at wavelength of 254 nm and a sensitivity of 0.01. The solvent
used for drug/CDS recovery from biological media was 5%DMSO/95%
Acetonitrile. For a 'one run' simultaneous resolution of chlorambucil and CDS the
flow rate was adjusted manually according to the following routine: 0.6 mL/min
from 0 3.5 minutes; and 1.5 mL/minute from 3.5 minute to the end of run. The
sensitivity of the HPLC system was 0.09 pg/mL for both drugs.
For the cromoglycic acid CDSs, MS-4 and MS-17, the stationary phase
column used was the same as above, the mobile phase was 75% Acetonitrile/0.1%
Acetic Acid/water run at a flow rate of 1.0 mL/min, and the retention times were;
MS-4, 6.5 minutes, and MS-17, 10.0 minutes. The solvent for drug recovery was
5%DMSO/35% Methanol/60% Acetonitrile and the lower limit of detection was
just less than 0.03 pg/mol for both MS-4, and MS-17.
For the recovery and estimation of cromoglycate anion from biological
media, a novel and simple HPLC procedure (Method B) described below, was

49
developed, and used. In addition this method was compared to a recently
published HPLC procedure (Method A), (Gardiner, 1984), according to the
procedures described below.
In one study, 200 pL of Cromolyn CDS-1 (i.e. MS-4) was added to 2800
pL of whole fresh rat blood, vortexed for 20 seconds and incubated at 37C for 30
minutes. Then, 250 pL samples of the incubation mixture containing the
hydrolysis product of the CDS were removed and analyzed for cromoglycate anion
separately by the two different methods mentioned above, as described below:
1. For the 'Method A', the procedure used included as follows:-
Fifty microliters each of 2N HC1 and saturated NaCl solutions were added to
0.25 mL sample of the incubation mixture.
Two milliliters of ethyl acetate were added, and the mixture vortexed for about
30 seconds. The organic layer was saved and pooled with further two more
extractions with 2.0 mL ethyl acetate.
The pooled organic mixture was evaporated to dryness on a water bath, the
residue reconstituted with 1 mL of mobile phase, centrifuged and the
supernatant analyzed on the HPLC.
2. In 'Method B', the following procedure was used for the drug recovery:
A quarter of a milliliter aliquot of the incubation mixture was added to 0.75 mL
of the protein-precipitating/drug-extracting solvent (composed of
DMSO/Methanol/Acetonitrile, 5:35:60 v/v) in a microcentrifuge tube, vortexed
for 20 seconds, centrifuged, and the supernatant diluted two times with de
ionized water before injecting on the HPLC.
Drug recovery efficacy of HPLC assay
Using 'Method B', samples of respective rat and rabbit tissue homogenates
(25%, 1.8 mL) were spiked with 200 pL of standard 1.0 mM Cromolyn, to an

50
initial concentration of 100 pM cromoglycate anion. After vortexing for 15
seconds, aliquots were taken and extracted via Method B and analyzed on the
HPLC.
OctanolAVater Partition Coefficients (Log P)
For the determination of the OctanolAVater partition coefficients, the
following procedure was used:
Approximately 10 mg of the compound was placed in 2 mL of anhydrous n-
Octanol in a 15 mL tube and shaken vigorously for about 2 minutes and
filtered.
An equal volume of phosphate buffer pH 7.4 was added and the tube sealed
with a plastic cap.
The mixture was equilibrated by repeated inversion of up to 200 times for five
minutes (a method adapted from Craig et al., 1947).
The mixture was allowed to stand for 30 minutes for the phases to fully
separate and the respective phases analyzed separately for the drug.
For the water soluble compounds, (cromolyn or MS-16), the solute was first dis
solved in the pH 7.4 buffer, and an equal volume of n-Octanol added instead.
In Vitro Stability
The in-vitro stabilities of the parent compounds or CDS in isotonic
physiological buffer and various biological media obtained from a number of
species (man, dog, rat, and rabbit) were evaluated by determining the pseudo-first-
order hydrolysis rate constants and hence the respective half-lives from the
disappearance of the drug by linear regression of the natural logarithm of the

51
HPLC peak areas versus time plots for the respective drugs/CDS in the respective
tissue homogenate or buffer media.
The procedure described below for the cromolyn CDS was essentially the
same as that used to estimate the stabilities of the other drugs/CDS in biological
media from the other species or buffer.
Adult white male Sprague Dawley rat weighing about 300g was sacrificed by
decapitation. Blood and tissues were immediately harvested. The tissues
homogenized in cold isotonic phosphate buffer pH 7.4, diluted to 25% and
stored in ice (0 4C) until ready for use. (The blood or tissue homogenates
were used within two hours of harvesting)
Two hundred microliters of 1.0 mM Cromolyn-CDS-1 (MS-17), dissolved in
DMSO/Propylene Glycol/Ethanol (1:2:4 v/v) and kept at 37C, were added to
2800 pL of blood or tissue homogenate pre-incubated at 37C with vortex
mixing. The initial drug concentration was 66.7 pM.
Immediately, and at subsequent time intervals, 200 pL aliquots of the
incubation mixture were pipetted into a micro centrifuge tubes containing 800
pL of the cold drug extracting solvent, vortexed for 20 seconds, and
centrifuged at 13 G for 7 minutes.
The supernatants were diluted two times with deionized water, and 20 pL
portion analyzed on the HPLC.
Hydrolysis Products of CDS
One half of a milliliter aliquots of 1.0 mmole stock solutions of each CDS
were respectively added to 4.5 mL portions of fresh rabbit blood (giving a final
concentration of 100 pM) and incubated at 37C. At various time intervals,
aliquots of the mixture were withdrawn and analyzed for each CDS and its
respective metabolite(s).

52
In- Vivo Distribution Studies
Chlorambucil and CDS in rats and rabbits
Comparison of the in-vivo distribution of the chlorambucil CDS (MS-2)
and parent drug was performed according to the following procedure :-
Fifteen millimolar stock solutions of Chlorambucil and the CDS (MS-2) were
prepared by dissolving in DMSO/Propylene Glycol/Ethanol (1:3:6 v/v) solvent
mixture.
The drugs were administered, intravenously through the ear vein, to white male
New Zealand rabbits weighing between 2.8 and 3.0 kg, at a rate of 0.4 mL / kg
of body weight resulting in a dose of 6 pmoles/kg of each drug (i.e. 1.825
mg/kg of chlorambucil or 2.871 mg/kg of MS-2).
Blood (about 0.25 mL) was drawn at 5, 15 and 30 minute intervals where
possible and mixed with two volumes of cold 5% DMSO in Acetonitrile and
stored in dry ice until ready for analysis by HPLC.
At predetermined time points, each respective rabbit was sacrificed by injection
with an over-dose of pentobarbital. The respective tissue samples were
immediately excised, homogenized with two volumes of cold Acetonitrile and
stored in dry ice until ready for analysis.
The homogenate was then centrifuged, diluted 1:2 with the mobile phase and
then analyzed for chlorambucil and/or CDS where applicable.
For the rats, the dosage rate used was the same as it was for the rabbits and drug
administration was via the tail vein.
Cromolyn and CDS: Distribution in rats and rabbits
A stock solution of the CDS for intravenous administration was prepared by
dissolving about 200 mg of the drug in a little amount of Dimethylsulfoxide

53
(DMSO) and adding propylene glycol (PG) followed by ethanol to give solvent
proportions of 1 DMSO/2 PG/4 Ethanol. The resulting mixture was then the
filtered through a micro-filter and thereafter diluted up to 2000 times on a portion
of the stock solution, and several 20 mL aliquots injected on the HPLC and
concentrations estimated by extrapolation from a calibration curve. The first
dilution was made with DMSO/Acetonitrile (1:1 v/v) and subsequent ones with the
respective mobile phase.
A corresponding stock solution of cromolyn equimolar with its CDS was
prepared similarly. Because of the low solubilization rate of cromolyn in the
above solvent system, the cromolyn solution was made by dissolving the cromolyn
first in a minimum of water and then adding the other solvents to approximate the
proportions mentioned above.
Adult male New Zealand Rabbits weighing between 2.75 and 3.25 kg, and
white male Sprague-Dawley rats (300 350 g) were used. The respective drugs
were administered to the rabbits via the ear vein at the rate of 0.42 mL/kg (i.e.
2.62 pmol/kg or 2.14 mg/kg CDS and 1.34 mg/kg Cromolyn), and to the rats
through the tail vein at 1.0 mL/kg (i.e. 6.22 pmol/kg or 5.08 mg CDS/kg and 3.19
mg Cromolyn/kg).
At pre-determined time intervals, the animals were sacrificed (the rabbits by
the injection of an overdose of pentobarbital, and the rats by decapitation).
Following each sacrifice, tissues were immediately harvested stored frozen in dry
ice, and when needed, subsequently thawed and homogenized (1:3) in the
extracting solvent system. A portion of each homogenate was centrifuged, diluted
with deionized water (cromolyn estimation) or mobile phase (CDS), and analyzed
on the HPLC.

54
Determination of Tissue Sulfhvdrvl
The procedure was based on the methods by Ellman (1959), and Sedlak &
Lindsay (1968). The basis of the assay was that DTNB, 5,5'-dithiobis-(2-
nitrobenzoic acid), a disulfide compound is quantitatively reduced by -SH groups
to form 1 mole of 2-nitro-5-mercaptobenzoic acid per mole of -SH. The
nitromercaptobenzoic anion has an intense yellow color which is used to
spectrophotometrically measure -SH.
The materials used in the assay included:-
- L-cysteine Hydrochloride H2O
- Reduced Glutathione (GSH)
- 5,5'-dithiobis-(2-nitrobenzoic acid), DTNB
- Tris-EDTA Buffer 0.2M, pH 8.2, and 0.4M, pH 8.9
- Ethylenediaminetetraacetic acid-disodium (EDTA-Na2),
- Trichloroacetic acid. (TCA)
The solutions for the assay were prepared as follows:-
- 0.01M DTNB (99.1 mg in 25 mL Methanol)
- Tris EDTA buffer, 0.2M, pH 8.2 was prepared by dissolving
6.05 g Tris, Tris[hydroxymethyl]aminomethane, in deionized
water, adding 25 mL of 0.2M EDTA, and water to make up
the volume to 250 mL, and adjusting pH to 8.2 with IN HC1.
- Tris-EDTA buffer, 0.4M, pH 8.9 was similarly prepared.
Standard solutions of GSH, and Cysteine (for the calibration) were made in
0.02M EDTA to prevent oxidation. The tissue homogenates were prepared from
freshly sacrifice animals. The organs were excised and immediately placed in ice
bath after accurate weighing, and homogenized in 0.02M EDTA and diluted to
give 5% homogenate. All solutions were degassed with a vigorous stream of

55
Nitrogen gas for 2 3 minutes prior to use. The respective types of tissue thiols
were then determined as follows
Total thiols were estimated as follows:-
One and a half milliliters of 0.2M Tris buffer pH 8.2, and 0.1 mL of 0.01M
DTNB were mixed with 0.5 mL 5% tissue homogenate in 15 mL test tube, and
the mixture diluted up to 10 mL with 7.9 mL Methanol.
The tubes were capped and allowed to stand with occasional shaking for 15
minutes, after which the mixture was filtered and allowed to continue standing
at room temperature for an additional 15 minutes.
The absorbance of the clear filtrates were read at 412 nm against a reagent
blank (i.e. with no homogenate).
Non-protein thiols were determined according to the following procedure
Five milliliter aliquots of homogenate were mixed with 4.0 mL Di-water and
1.0 mL of 50% TCA.
The mixture was allowed to stand 15 minutes with intermittent shaking and
subsequent filtering and continued standing for further 15 minutes.
A two milliliter aliquot of the clear filtrate was mixed with 4 mL of 0.4 M Tris
buffer, pH 8.9 and 0.1 mL 0.01M DTNB added and mixed.
The absorbance was read at 412 nm, within 5 minutes of addition of the
DTNB, against a reagent blank.
The corresponding thiol concentrations were estimated by extrapolation from
calibration curves prepared by the procedure for total thiols using standard
solutions of cysteine in the range 0 10"5 M concentrations.
Protein-bound thiols (P-SH) were simply obtained by subtracting non
protein thiols (NP-SH) from the total thiols (T-SH).

56
In-Vitro Binding Studies
Inhibition of CDS hydrolysis by BPNP
The kinetics of hydrolysis of the respective CDS, namely; Chlorambucil
CDS (MS-2); Cromolyn CDS-1 (MS-4); and Cromolyn CDS-2 (MS-17); in the
presence and absence of a carboxylesterase inhibitor were studied. The inhibitor
selected for use was BPNP, bis-(p-nitrophenyl)phosphate sodium salt (Sterri and
Fonnum, 1987; and Yamada et al., 1992), and the method for the esterase
inhibition was adapted from Yamada et al. (1992).
The materials used included:-
- BPNP, bis-(p-nitrophenyl)phosphate sodium salt
- Isotonic Phosphate Buffer pH 7.4
- 25% tissue homogenate and/or whole blood
- MS-2, MS-4, and MS-17 (1.0 mM Stock Solutions).
The procedure used was as follows:
The BPNP (0.2 mL, 10 mM in phosphate buffer pH 7.4) was added to 1.8 mL
whole serum or 25% tissue homogenate giving a final inhibitor concentration
of 1 mM in the homogenate, vortexed for 30 seconds and pre-incubated at
37C for 15 minutes.
To the incubation mixture, 0.1 mL of 1.0 mM CDS was added, vortexed 15
seconds, and returned to incubation at 37C.
At time intervals, 0.1 mL aliquots were removed and 0.9 mL 5% DMSO in
Acetonitrile added, vortex mixed, and centrifuged at 13 G for 5 minutes.
Aliquots of the supernatants (20 pL) were injected unto HPLC to analyze for
CDS.
For the controls (i.e. hydrolysis in absence of inhibitor), 0.2 mL aliquots of the
phosphate buffer were substituted for BPNP.

57
Effect of [BPNP1 on hydrolysis of MS-4
Cromolyn CDS-1, MS-4 was selected as a model CDS, and the effect of
various concentrations of the selected inhibitor on its hydrolysis was investigated.
The materials used were the same as for the above, except only one drug, MS-4,
was investigated here, and procedure used is described as follows:
Stock BPNP solutions (2.5 mM and 25 mM respectively) were prepared in
phosphate buffer (0.05M, and pH 7.4).
Portions of 25% rat liver homogenate (1.8 mL) were placed in test-tubes with
the appropriate amounts of stock BPNP and buffer solutions to give the
respective BPNP concentrations in the range 0 4.55 mM indicated.
The mixtures were vortexed and pre-incubated at 37C for 20 minutes after
which 0.1 mL aliquots of the CDS (MS-4 1.0 mM solution in 5%
DMSO/Acetonitrile) were added to each tube giving a final drug concentration
of about 50 pM drug in each.
At time intervals, 0.1 mL aliquots of the reaction mixture was taken into 0.9
mL of 5% DMSO/Acetonitrile, vortexed for 15 seconds, centrifuged 5 minutes
and the supernatant analyzed by HPLC for CDS.
ii. Determination of bound and unbound drug
A measured quantity of drug or CDS was incubated with the appropriate
tissue homogenate or Bovine Serum Albumin (BSA) for a predetermined length of
time and analyzed as follows for free unbound drug, and drug bound via mixed
disulfide bond formation.
The unbound drug was determined as follows:-
One half milliliter of Acetonitrile was added to 0.5 mL aliquot of the
incubation mixture (i.e. x2 dilution), and centrifuged for 30 seconds;
An aliquot, 0.5 mL, of the supernatant was added to 0.5 mL Acetonitrile and

58
centrifuged again, 5 minutes (i.e. further x2 dilution);
A portion of the supernatant was then diluted 3x with the mobile phase and
analyzed by HPLC (total of 12 times dilution).
Drug bound via mixed -disulfide linkages was also determined by the
follwoing procedure:-
The protein pellet from above was suspended in, and washed about three times
with ethanol to remove all traces of drug not covalently bonded to the protein;
The washed pellet was re-suspended in 0.5 mL of 50 mM MOPS containing
25mM DTT and incubated at 37C for 1 hour.
At the end of this reduction step, the protein was reprecipitated by adding 0.75
mL of Acetonitrile (i.e. 2.5 times dilution), centrifuged, and the supernatant
analyzed for the CDS by the HPLC system.
Mixed disulfide binding in the presence of NEM
Samples of the respective CDSs MS-2, MS-4 and MS-17, respectively,
were incubated with various tissue homogenates or whole blood, in the presence
and absence of BPNP and/or NEM (N-ethylmaleimide), a sulfhydryl blocker. The
respective amounts of bound and unbound drug were determined.
The materials and procedure used were as follows respectively:
- Freshly excised tissues (whole blood, liver, lung and Kidney)
- Bis-(p-nitrophenyl)phosphate (BPNP)
- 4-Morpholinopropane sulfonic acid, MOPS
- 1,4-Dithiotreitol (DTT)
- Phosphate Buffer, 0.1M, pH 7.4
Twenty five percent tissue homogenate, 1.8 mL, was mixed with 0.2 mL of 10
mM BPNP to give a final concentration of 1.0 mM BPNP, (or 0.2 mL

59
phosphate buffer in place of BPNP for the control) and pre-incubated at 37C
for 15 minutes.
For binding in the presence of the sulfhydryl blocker, 0.1 mL of 10 mM NEM
(or water for the control) was added, at this point, after the pre-incubation, and
vortexed for 1 minute (final concentration of NEM = 0.48 mM).
A hundred microliters of the CDS (1.0 mM in DMSO) were added with
vigorous mixing (resulting in an initial drug concentration in homogenate of
0.45 fiM) and incubated at 37C for 1 hour and subsequently analyzed for
mixed disulfide bound drug and unbound drug by the methods described
above.
Effect of TBPNP1 on binding of MS-4
Bovine Serum Albumin. BSA, is a model protein with a single -SH group.
An amount of BSA of protein concentration (50 mg/mL) approximating to that of
33% tissue homogenate was used instead of an actual tissue homogenate owing to
the desire to eliminate the differential hydrolysis of the CDS with the different
BPNP concentrations in the tissue homogenates as a mitigating factor in the
binding.
The effect of the B-esterase inhibitor, BPNP, on the binding was
determined as follows:-
- BSA in phosphate buffer (68.75 mg protein / mL)
- 50 mM Morpholinopropane sulfonic acid, pH 8.0 in 25 mM
DTT in methanol.
- BPNP
A portion, 1.6 mL, of the stock BSA solution is placed in a test tube with the
appropriate concentrations of BPNP and buffer to make volume up to 2.0 mL.
The mixture was pre-incubated for 15 minutes at 37C.

60
The CDS, 0.15 mL 1.0 mM MS-4 is added with vortexing to give a final drug
concentration in mixture of 69.8 gM.
Incubation is continued for 1 hour after which 0.5 mL aliquots were withdrawn
and analyzed for free unbound drug, and drug bound by mixed disulfide, using
the procedures already described above.
Time dependence of mixed disulfide binding of MS-4
i. to bovine serum albumin:
The procedure used was similar to that described above except that no
BPNP was added and the incubation times of the CDS in the tissue homogenate
were varied to correspond to the desired time intervals before being analyzed for
the bound and unbound drug components.
ii. to a model tissue (rabbit limp:
Five and a half milliliters of 33.3% Rabbit Liver homogenate in isotonic buffer
pH 7.4 were placed in a 15 mL test tube, 0.3 mL of 25 mM BPNP and 0.3 mL
of 1.2 mM MS-4 were added with vortex mixing (giving final concentrations
of; homogenate: 29.8%; BPNP:- 1.23 mM; and Drug:- 60 pM), and then
incubated at 37C.
After intervals within the range of 0 to 26 hours, samples were withdrawn and
analyzed, as before, for bound and unbound drug concentrations.
iii. to rabbit lung tissue compared to liver:
The procedure used was similar to the above except that the incubation
times used were set at 0, 30, and 75 minutes.
Dependence of binding on initial drug concentration
To 1.86 mL of 33.3% rabbit liver homogenate in a test tube, 120 pL of 25 mM
BPNP in phosphate buffer was added and incubated at 37C for 15 minutes.

61
Various amounts of stock MS-4 solution and solvent for the stock solution
were added to make the total volume up to 2200 pi to correspond to the various
initial drug concentrations in the range of 6 to 240 pM.
The mixtures were incubated for 1 hour at 37C and then analyzed by HPLC
for free unbound drug and drug bound by mixed disulfides.
Comparative in-vitro tissue binding of MS-4 via mixed disulfides
To 1.8 mL of 33% respective rabbit tissue homogenates or blood, 120 pL of 25
mM BPNP was added, vortex mixed and pre-incubated at 37C for 15 minutes.
Fifty microliters of 1.2 mM MS-4 were then added resulting in an initial drug
concentration of 30 pM, and the incubation continued for one hour, after which
sample were taken and analyzed for the bound and unbound drug
concentrations.
Dependence of drug binding on homogenate concentration
Rabbit liver homogenates 15, 25, 35 and 50 % respectively were prepared.
A portion of each homogenate (1.85 mL) was pre-incubated as before with 100
pL of the esterase inhibitor BPNP, after which 50 pL of the drug, MS-4, was
added (the initial drug concentration in each homogenate being 30 pM).
The mixture was incubated for an hour and then analyzed for bound and
unbound drugs.
Data Treatment and Statistical Evaluation
All data presented in the dissertation are reported as the mean value plus or
minus the standard error of the mean (SEM), unless otherwise stated. A standard

62
t-test for unpaired data were used when comparisons were made within a group
whereas for paired data, the t-test was used. Assuming a normal distribution with
the population variance equal and unknown, the difference between two sets of
data was considered significant when the P value was more than 0.05. For
comparison within a group comprising more than two sets of unpaired data, a one
way analysis of variance (ANOVA) was performed and the difference was
considered significant when the computed F value was more than a critical value.

CHAPTER 5
RESULTS AND DISCUSSION
Syntheses
The respective CDSs, MS-2, MS-4, and MS-17 were prepared as esters.
The chlorambucil CDS (MS-2) was a yellow oil whereas the cromolygcic acid
CDS's were both light yellow crystalline solids. The compounds along with the
intermediate compounds were characterized by spectral methods (NMR, IR, UV
and MS), purity was assessed by TLC, HPLC and elemental analyses. Melting
points were also determined, and for the liquid compounds boiling points could
not be determined due to decomposition at high temperatures. The spectral and
other physical characteristics including percentage composition from elemental
analysis are presented below. Introduction of the l,2-dithiolane-3-pentyl moiety
was achieved through esterification. A commercially available starting material
lipoic acid, first had to be converted into the alcohol (lipolol).
l,2-dithiolane-3-pentanol, (dl-lipolol), MS-1: Yield:- Yellow oil 88.5 %;
TLC:- Rp0.69 (mobile phase methylene-chloride/ethyl acetate, 4:1); UV (in
CH2CI2):- 330 nm (intact dithiolane ring, Furr et al., (1979); IR (neat
liquid, NaCl discs):- v 3360 (s, broad, hydrogen bonded -OH str.). !H-NMR (in
CDCI3): Spectrum is presented in Figure 15 below, and the following observed
chemical shift (8) values were in close agreement with spectra obtained by
Kabalka et al., 1977, and with most of the theoretically estimated/calculated values
using data obtained from Silverstein et al., 1981:- 1.5 (broad, 8H, alkyl),
63

varan instrument division
I 'I""
I (X I I'
I () K l i\Vi It
FCOUII I IOS
I H roUPl INt. IOWI It
Mir.! AMI'I 5000
0.1
o SWI I I1 Wllllll
nil. IU IOWI M 0.05 mi*. I Nf) OI SWf I I
5 "1 NUCI I II.
10 iii-iii /i no ni i TMS
0 >>< SAMI'I I II Ml' RT
t HI MAI t *1 M.S.
5/8/92
DAN
( MHVINI
MS-1
CDC13 SII I 11;11M N
Figure 15: *H-NMR Spectrum for Lipolol:
Characteristic chemical shifts showed a quintet at 2.4 correspon-ding to the ring -CH2-, and a
triplet at 3.15 for the methylene protons a to the S (i.e. -CH2S-)
EM360/390 NMR SPECTROMETER

65
(broad, 8H, alkyl), 2.4 (quintet, 2H, ring CH2), 3.15 (t, 2H, -CH2S-), 3.6 (m, 3H, -
CHRS- and -CH2O-), and 4.0 (t, 1H, -OH). The triplet at 5 = 0.9 which
disappears upon formation of the R-Br derivative, may be due to the hydroxylic
proton. In deuterochloroform, the hydroxylic peak is usually found between 8 2
and 4, however, at very low solvent concentrations, the peak shifts to higher fields
close to 8 0.5 (Silverstein et al., 1981).
UPOLOL
Figure 16: UV Spectrum of Lipolol:
Absorption maxima at X 328 nm, characteristic of the
intact dithiolane ring is shown.

66
Chlorambucil CDS
l,2-dithiolane-3-pentyl-5-{4-[p-bis(2-chloroethyl)amino] phenyl butyrate},
MS-2. Yield:- Yellow oil, 30%; Elemental Analysis: Calculated for
C22H33NS2CI2O2: C, 55.21155.28; H, 6.95; N, 2.93; S, 13.40; and Cl, 14.82
Found: C, 55.28; H, 7.01; N, 2.94; S, 13.33; Cl, 14.73. 1HINMR (in CDC13):-
Spectrum is presented in Figure 17 the observed chemical shift (5) values were
largely in close agreement with calculated values: 1.2 1.8 (broad, 8H, -CH2-,
lipoyl alkyl), 2.3 -2.7 (multiplet 6H, -0C(0)-(CH2)6-), 3.2 (t, 6H, mustard -N-
CH2- and lipoyl -CH2S-), 4.18 (t, 2H, -OCH2-), and 6.7 & 7.1 (both doublets,
4H, aromatic, para-substitution).
Cromolyn-CDSs
Taking into account the considerations discussed in Chapter 4, the involved
syntheses were achieved via the reaction of the alkyl bromide derivative of lipoic
acid with the disodium salts of the appropriate bischromone compound in HMPA.
Yields were quantitative, and the esterification reactions were over in about 30
minutes. The reactions worked just fine using 1:1 mole equivalents of the
reactants. One of the earliest reports of the use of this reaction (Shaw et al., 1973)
had suggested a 1:4 (disodium salt: alkyl bromide) mole equivalents ratio.
a. Lipolyl bromide, MS-9, (5-bromo-3-pentyl-l,2-dithiolane). Yield:- brownish
yellow oil, 65.4 %. TLC:- Aluminum sheet silica gel 60 F254, 0.2 mm layer
thickness, RE = 0.73 (mobile phase = 10% ethyl Acetate/Hexane).

varan instrument division paio sito, ca;
1 20 1.63 4.18
3 8 2.25 2.04 2.64 /TT\ *
^y/(CH2)3CH2CH2-OOC-CH2CH2CH2-\Q>-N
3 06 3 63
'CH2CH2CI
S-S
^ch2ch2ci
Calculated chemical shift values for MS-2
ppm
10
LOCK POS.
LOCK POWER
DECOUPLE POS.
DECOUPLING POWER _
8000
ppm SPECTRUM AMPI SWEEP TIME_
_mG 0.05
FILTER
-Ppm
_ sec SWEEP WIDTH_
. min NUCLEUS
10 7FRn ___ TMS
ppm ZERO REF
.SAMPLE: MS2 OPERATOR. _
DATE.
mG RF POWER _
0.1
mG END OF SWEEP ^ ppm SAMPLE TEMP SOLVENT: CDCI3 SPECTRUM NO.
Figure 17: ^H-NMR Spectrum for Chlorambucil CDS:
Spectrum showed 5-values of 6.6 and 7.1 corresponding to the 4 para-substituted aromatic
hydrogens at the alkyl and mustard sides of ring respectively, 4.18(t) for the CH2-O, 3.5(t)
for -CHRS (a) and 3.15(t) for -CH2S (b) of the dithiolane ring.
On
EM360/390 NMR SPECTROMETER

68
1H-NMR:- 8-values (CDCI3): 1.5 (broad, 8H, alkyl), 1.8 (q, 2H, -CH2-, P to Br),
2.2 (q, 2H, ring -CH2-), 3.15 (t, 2H, -CH2S-), 3.3 3.6 (m, 3H, -CHRS- and -
CH2Br). IR (neat liquid on NaCl discs):- v 1440 (8S CH2, -CH2-S or -CH2-Br),
650 (medium, 8S CH2Br), 2050 & 1675), UV: Absorption maxima at 330 nm
(characteristic of intact dithiolane ring). Elemental Analysis: Calculated for
C8H15S2Br; C, 37.65; H, 5.92; S, 25.22; Br, 31.30: Found; C, 37.83; H, 5.89;
S, 25.22; Br, 31.05.
A comment on the synthesis of lipolyl bromide needs special mention. For
the synthesis, the commercially available starting material, lipoic acid, had to be
converted to the alcohol and then to the alkyl bromide. One frequently used
method of converting alcohols to alkyl halide is by treatment with N-
bromosuccinimide (NBS) and triphenylphosphine (TPP). However this did not
work for this case as NBS has been known to attack disulfide linkages (Buchel and
Conte, 1967) thereby destroying the dithiolane ring. Loss of the dithiolane ring
was evidenced by the fact that the UV spectrum of the resulting product initially
thought to be lipolyl bromide did not show the characteristic maxima of absorption
at 330 nm, a characteristic of the intact dithiolane ring (Furr et al., 1979). Also
results of the elemental analysis of the product had indicated an empirical formula
of C8Hj5SBr2 instead of the desired C8Hi5S2Br. A thorough review of the
synthetic methods and available literature revealed that this product must have
been a dimeric form of lipolyl bromide possibly formed through the mechanism
below (Figure 18) deduced from the work of Buchel and Conte, 1967; and the
observed deposition of yellow elemental sulfur on the glassware during work up.
What lead to its detection were the results of elemental analysis that corresponded
to the dimeric structure, and the absence of the absorption maxima at 330 nm or
free sulfhydryl groups (IR v 2600 2555).

69
Br
Lipolol
Br
o C
+ PPh
then;
Lipoyl Bromide
Br
S
/S
Br
R
sulfido free
radical
ry^ rrR
S Ss s
^ rearrangement
R^n rrR
Br SS Br
Y"!
Dimer
Emp. Formula: C 8 H15SBr2
R = -(CH 2 )5 -Br
+
S
elemental sulfur
(yellow deposit)
Figure 18: Formation of Lipolyl Bromide Dimer:

70
In the mechanism, the desired product, lipolyl bromide is formed alright,
but it undergoes further reaction resulting from the attack by a bromine atom
generated from the NBS leading to the formation of sulfido- free radical bromide
intermediate which then undergoes further reactions including rearrangement
(Buchel and Conte, 1967) to eliminate sulfur atoms (yellow deposit).
It is interesting to note that this dimeric product shared very similar spectral
characteristics with the desired monomeric product. Furthermore, the
ferricyanide/cyanide test, Furr et al., 1979 for disulfide bond in the inteimediate
alkyl bromide and subsequent CDS molecules had been positive, and the CDS
upon in-vitro hydrolysis had yielded the cromoglycate.
b. 1,3-bis[2-(1,2-dithiolane-3-pentyloxycarbonychromone-5-yloxy) ]-2-
hydroxy-propane, i.e Lypoyl ester of cromolyn or Cromolyn CDS-1: Yield:- 85.6
%: Melting point: 78 81 C: Molecular Weight = 817. 1HINMR:- (CDCI3) 8-
values:- 1.5 (broad, 16H, alkyl), 2.4 (q, 4H ring -CH2-); 3.15 (t, 4H, -CF^S-); 3.4
(multiplet, 2H, ring -CHRS-), 4.4 (10H, propane bridge and -OH), 6.95 (doublet,
4H, aromatic, positions #3 & #8), 7.2 (t, 2H, aromatic #6), 7.5 (t, 2H, aromatic,
#7). IRj. (Paste in mineral oil)vibrational frequency, v, values:- 3525 cm-1
(O-H stretching due to free OH); 3450 (overtone of C=0 stretch); 1745 (C=0
stretch); 1650 (aromatic ester); 1465 (5S CH2 scissoring -CH2OCK)). The *H-
NMR, UV and IR spectral data (Figures 20 23) were in complete agreement with
those obtained by Cox et al. (1977) for corresponding structural features of the bis-
chromone compound. Elemental Analysis:- Calculated for C39H440jiS4-
(0.5H2O): C, 56.71; H, 5.49; O, 22.27; and S, 15.52. Found: C, 56.44; H,
5.49; and S, 15.56.

d,CH2
-CHjS- or -CHrdr
3000
2500
1800
1600
1200 1000
WAVENUMBER (CM ')
800
CHrBr
600
SOLVFNT Sample in Nujol
REMARKS Nfl C=S (due to absence of absorption at
SCAN MODE
CONCENTRATION
220 2050; & 1675)
SLIT TIME CONSTANT
CR1 PATH NaCl Discs
.. .
REFERENCE
No S-H present (absence of 2600 2555 weak)
No PR 5100 4367
Figure 19: IR Spectrum of Lipoyl Bromide:
Characteristic frequencies of absorption (v) shown were at 1440 and 1470 (strong, 8S CH2) corresponding
to -CH2-S- and -CH2-Br respectively; and 1240 (strong, coCF^) for -CH2-Br.

varan instrument division
1
I NI OI SWfc l I
ppm
LOCK POS.
LOCK POWER
DECOUPLE POS
DECOUPLING POWER
ppm SPECTRUM AMPL.
0.2
8000
mG
mG RF POWER
0.15
SWEEP TIME
sec SWEEP WIDTH
mG END OF SWEEP
10
o
mm NUCLEUS
ppm ZERO REF
ppm SAMPLE TEMP
1H
TMS
RT
SAMPLE: MS-4
CDS
C SOI VENT
CDC13
OPERATOR
6/2/92
DATE
<*>-'P TUI IM NO
Figure 20: ^-NMR Spectrum for Cromolyn CDS-1:
Spectrum showed the characteristic cromolyn absorptions of the aromatic hydrogens
(6.9 7.5) and the ten hydrogens of the propanyl bridge & -OH at 8= 4.4, in addition
to the characteristic lipoyl absorptions.
M
EM360/390 NMR SPECTROMETER

% ABSORBANCE
73
CROMOLYN
(in methanol)
WAVELENGTH (nm)
Figure 21: UV Spectrum of Cromolyn
Absorption bands at 238 nm (strong) and a weak band at
319 nm charatenstic of the oione structure (insert) of
cromolyn are shown.

% Absorbance
74
Figure 22: UV Spectrum of Cromolyn CDS (MS-4):
The spectrum for cromolyn (Figure 21) showed the characteristic absorption
maxima at 238 (strong) and at 319 nm, both corresponding to the enone structure
of the bischromone nuclei. These features were retamed m the CDSs (MS-4. &
MS-17) both of which showed a shift in X,max from 319 to 325 nm due to the
presence of the intact dithiolane ring.

75
Cromolyn CDS-2, MS-17
a. 1,3-bis(2-ethoxycarbonylchromon-5-yloxy)-2-hydroxypropane,
(Cromolyn diethyl ester, MS-11): Yield:- 77%; Melting point:- 184 185C,
(In close agreement with reported literature value of 182 183C, (Cairns et al.,
1972); TLC:- Rf = 0.79 (chloroform/methanol, 10:1); iH-NMR:- 8-values
(CDCI3) 1.35 (t, 6H, ethyl -CH3), 4.25-4.6 (broad multiplet, 10H, 2-propanyl
bridge hydrogens and -OH, in agreement with Cox et al., 1970), and aromatic
hydrogens 6.95 (d, 4H); 7.2 (t, 2H); & 7.6 (t, 2H). Mass Spectroscopy
(electrospray ionization):- molecular-ion peak [M + Na]+ = 547.
b. 1,3-bis(2-ethoxycarbonylchromon-5-yloxy)-propan-2-numtc\ (diethyl
cromolyn nitrate ester, MS-15): Yield:- white powder, 75%; Melting point: 172 -
173C; TLC:- Rf=0.81 (chloroform/methanol, 10:1); 1H-NMR> results similar
to spectrum for MS-11 with one less H at 8 value 4.4. Elemental Analysis:-
Calculated for C27H23N013 as C, 56.94; H, 4.07; N, 2.46; and O, 36.53.
Found:- C, 56.71; H, 4.21; and N, 2.52.
c. l,3-bis(2-carboxychromon-5-yloxy)-propan-2-nitrate disodium sail
(cromolyn nitrate disodium salt, MS-16): Yield:- white powder, 51%; Melting
point:- approximately 230C (with decomposition). Elemental analysis:-
Calculated for C23Hi3N0i3Na2-(2H20) as C, 46.56; H, 2.89; N, 2.36; O, 40.44;
and Na, 7.75. Found:- C, 46.62; H, 2.91; N, 2.37; and Na, 7.67.
d. l,3-bis(2-lipolyloxycarbonylchromon-5-yloxy)propan-2-nitrate,
cromolyn lipolyl nitrate ester (i.e. Cromolyn CDS-2, or MS-17): Yield:-
Yellowish crystalline solid, 80.5%; Melting point: 102 104 C; 1H-NMR>
(CDCI3); chemical shift (8) values:- 1.5 (broad, 16H, alkyl), 2.4 (q, 4H ring -
CH2-); 3.15 (t, 4H, CH2S-); 3.4 (multiplet, 2H, ring -CHRS-), 4.4 (9H,
propane bridge, Cox et al, 1970), 6.95 (doublet, 4H, aromatic, positions #3 & #8),
7.2 (t, 2H, aromatic #6), 7.6 (t, 2H, aromatic, #7). Elemental analysis:- Calculated

76
for C39H43013NS4 as C, 54.32; H, 5.03; O, 24.14; N, 1.62 and S, 14.88.
Found:- C, 54.24; H, 5.01; N, 1.68 and S, 14.80.
HPLC Assays
Chlorambucil and CDS:
Chlorambucil is unstable in aqueous solutions (Erhsson et al., 1979). This
results from the fact that the ethyleneimmonium ion or carbonium ion intermediate
which is responsible for its therapeutic alkylating activity is also readily formed in
aqueous solutions where it is readily attacked by nucleophiles such as water
(Loftsson et al., 1989). The mechanism of nitrogen mustard hydrolysis is known
to involve the attack of the unprotonated nitrogen to expel chloride, forming the
cyclic intermediate. This is followed by the attack of water or other nucleophiles
such as N-guanine (as occurs in the mechanism of its therapeutic alkylating
activity). The availability of a free electron pair on the nitrogen is essential for
this reactivity, and protonation of this nitrogen eliminates this reactivity (Chatterji
et al., 1981). Therefore the mobile phase and extraction solvent for the recovery
of Chlorambucil and its CDS were acidified up to 0.5% with acetic acid to prevent
the hydrolytic degradation of drug during the quantitative estimation.
Cromolyn and its CDSs:
Improved Detection of Cromoglycate Anion
Methods for the estimation of the cromoglycate anion in biological media
have mostly involved some rather cumbersome procedures involving
radioimmunoassay (Brown et al., 1983); colorimetric (Moss et al., 1971); or
polarographic methods (Fogg and Fayad, 1978); or radiotracer techniques

77
(Hemmerich et al., 1991). Very few HPLC procedures have been published in
recent times but these along with most of the other methods, have involved the use
of specialized/customized columns, analyte limitation and/or complicated
multistep extraction/drug recovery or sample concentration procedures ultimately
resulting in lowered extraction efficiencies (Gardner, 1984; Ishikura et al., 1987;
and Yoshimi et al., 1992). In recent years HPLC assays have increasingly
supplanted other methods for the quantitative estimation of bioactive compounds
in biological systems on account of its simplicity, speed and general reliability.
The paucity of HPLC assays for the estimation of cromolyn, a frequently
studied compound, underscores the difficulties that other investigators have
probably encountered in the development of a suitable HPLC assay. For this
study, a new, fairly sensitive and simple HPLC assay (Method B) was developed
the results of which, as presented in Table 1, proved more accurate and sensitive
than one of the simplest HPLC procedures so far, recently published by Gardner,
1984 (Method A). In comparing the two methods for the estimation of the
cromoglycate anion, the published (Method A) was adapted to the HPLC
detection system for method B. Effectively, it was the methods for drug recovery
from biological media that were compared. The results (Table 1) showed that
Method A had a drug recovery efficiency of about 75% from rat blood (in close
agreement with the author's reported 70% from human plasma) whereas Method B
produced an efficiency of 92%. Considering the actual concentration of cromolyn
in the lysate based on the assumption that complete hydrolysis had occurred,
Method B was more accurate besides having a slightly greater sensitivity. (The
minimum detectable were 0.1 and 0.05 pM for A and B, respectively).
Employing Method B, the recovery efficiencies of cromoglycate from the
respective tissue types in the rat were compared, and the results presented in Table
2. The results showed that recovery from the blood was a little lower (about 93%)

78
than for the other tissues (approximately 97% and over). This suggested that some
binding of cromolyn had probably occurred in the blood. Albumin in the blood is
known to bind many drugs. In addition, a 'cromoglycate binding protein' has been
identified (Hemmerich et al., 1992), and although its distribution in the body has
not been studied and there is a possibility that it might play a part in the binding.
For the other tissue types, there was no significant difference at P < 0.05, in the
recovery rates indicating that the procedure for drug recovery from biological
media could not be a significant source of error in the differences in the various
tissue drug concentrations assayed.
Table 1: Recovery of Cromolyn as the Cromoglycate Anion from Whole
Rat Blood*; Comparison of Methods A and B
Method
[Cromolyn]
Recovered
Recovery
Efficiency (%)
A
5.00 0.12
75.0 2.7
B
6.17 0.19
92.4 3.0
* Based on the following: 1. Initial fCDS-1] in media = 6.67 pM
2. Assumption that all CDS-1 hydrolyzed into
cromoglycic acid within 30 minutes.
Table 2: Recovery Efficiencies of Cromolyn from Rat Tissues (by Method B)
Tissue
Recovery
Efficiency (%)
Blood
92.6 2.1
Liver
99.1 1.3
Lung
97.4 2.0
Kidney
96.8 1.8

79
Octanol/Water Partition Coefficients
Table 3, below, lists the experimentally determined octanol/water partition
coefficients of the CDSs and the respective parent drugs. Unlike the parent
compounds the respective CDSs were highly lipophilic. The octanol/water
partition coefficients, Log P, for cromolyn and chlorambucil were in close
agreement with values obtained by other investigators (Oppitz et al., 1989; and
Yoshimi et al., 1992 respectively). That of the cromolyn CDS-2 (MS-17) was
only slightly lower than for the cromolyn CDS-1 (MS-4). The purpose for the
synthesis of MS-17 was to increase the aqueous solubility and thereby lower the
Log P. The presence of the nitrate group on the #2 carbon of the propanyl bridge
of the bis-chromone compound did not seem to have influenced the aqueous
solubility that much perhaps owing to the highly hindered nature of that site. MS-
16, the disodium salt of MS-17 was of comparable solubility to cromolyn. The
high lipophilicity of the CDSs compared to the parent drugs, translated into
increased bioavailability and long in-vivo half-lives for the CDSs.
Table 3: Octanol Water Partition Coefficients
Compound
Octanol/
Water
Log P
Cromolyn
0.02 0.01
-1.70 0.15
Cromolyn CDS-1 (MS-4)
743.4 62.1
2.87 0.04
Cromolyn CDS-2 (MS-17)
705.7 41.7
2.85 0.02
MS-16
0.03 0.01
- 1.56 0.14
Chlorambucil (ionized)
3.24 0.51
-0.51 0.21
Chlorambucil CDS, (MS-2)
551.1 48.9
2.74 0.17

80
In-Vitro Stability
The in-vitro stabilities of the CDSs in comparison with the parent drugs are
presented in Tables 4 and 5 below.
Table 4: In-vitro Stabilities of CDSs Compared to Parent Drugs
Medium
Half-life, T j/2, (in minutes)
Chlorambucil
* Cromolyn
CDS
(MS-2)
Parent
CDS-1
(MS-4)
CDS-2
(MS-17)
Human Blood
140.911.5
153.310.2
85.5 9.8
105.1 8.7
Rabbit Blood
26.7 7.1
360.437.8
23.7 5.4
48.3 6.3
Rat Blood
0.4 0.1
1.7 0.5
3.1 0.2
18.6 3.4
Buffer pH 7.4
503.725.9
79.8 11.1
223.U18.5
250.621.7
*Cromolyn was stable in all the media tested.
Table 5: In-Vitro Stability of Model CDS in Various Rat Tissues:
(MS-4; 25% Tissue Homogenate or Blood)
Tissue
k (x 10-2)
T i/2 (min)
r
Blood
13.5 3.4
5.49 1.43
0.9542
Liver
6.4 0.5
10.86 0.79
0.9879
Lung
7.6 0.8
9.19 0.59
0.9946
Kidney
5.5 0.3
12.61 0.59
0.9914
k = Hydrolysis Rate Constants
T1/2= Half-life
r = correlation coefficient

81
For chlorambucil, there was no significant difference (P > 0.05) in stability
between the CDS and the parent drug in human blood, but stability of the CDS in
buffer pH 7.4 was substantially greater than for the parent drug. The higher
stability of CDS in physiological buffer relative to biological media was suggestive
of the probable involvement of hydrolytic enzymes in the biological matrix. It was
reported by Erhsson et al., 1980, that the half life of chlorambucil in aqueous
media was 12 minutes, however, this study showed a half-life of nearly 80 minutes
in aqueous media (buffer pH 7.4). For cromolyn the two CDSs showed
comparable stability in human blood and in buffer but significant differences in
rodents' blood, with the CDS-2 being the more stable of the two. The parent drug
cromolyn was completely stable in all the media tested. This was in agreement
with the knowledge that cromolyn is apparently non-metabolizable in all of the
mammalian species studied, including humans (Cox, 1967).
In general, the kinetic studies data showed that the CDSs of both parent
drugs were unstable in rat blood (Tj/2 < 3 minutes) but fairly stable in rabbit (T j/2
~ 25 minutes) and in human (Ti/2 ~ 85 minutes) bloods. In fact, the fairly good
stability in human blood could ensure several 'circulatory passes' in the blood
stream without much degradation and thereby allow sufficient sequestration of the
CDS to occur in the lung. While the stability of the CDSs showed no specific
trends, the interspecies variation were probably a mere reflection of the different
distribution of B-esterase enzymes among the species.
The in-vitro stability of a model CDS, MS-4, shown in Table 5 indicated no
significant differences (P < 0.05) in respective rat tissues except in blood
suggesting a nearly equal activity of the esterase enzymes in the tissues but higher
activity in blood. This was in line with the fact that P-esterases are non-specific
enzymes of ubiquitous nature in biological tissue.

82
Hydrolysis Products of CDS
Further in-vitro hydrolysis studies, Figure 23 showed that the respective
CDSs hydrolyzed completely and fairly rapidly in freshly obtained biological
tissue media to yield the respective parent compounds.
Drug Cone. (pM)
Chlorambucil CDS (MS-2)
Cromolyn CDS (MS-4)
Drug Cone. (/M)
Figure 23: In-Vitro Hydrolysis Products of CDSs in Rabbit Blood.

83
In-Vivo Distribution Studies
The drugs were administered to rats and rabbits according to the procedure
described in Chapter 4. The mean percentage composition of the respective organs
and tissues of several animals relative to the body weight were estimated (Table 6)
in order to enable the calculation of the total amounts of drug in a given tissue.
From these estimates, the amount of drug present in a given tissue, expressed as a
percentage of the total drug administered (designated '% of Total') and of the sum
total of drug remaining in each respective tissues investigated (i.e. '% of
Recovered') at a given time point. Within limits of the procedure used therefore,
drug stored in adipose tissue, muscle, and in other body parts not investigated
including drug excreted in urine prior to sacrifice or stored in the bladder or bile
were not included in the later estimation and were collectively treated as excreted
drug.
Table 6: Percent Composition of Organs/Tissues Relative to Body
Weight in the Rat and Rabbit
Tissue
Rat
Rabbit
Blood
7.00
7.00
Lung
0.50 0.02
0.58 0.02
Liver
3.25 0.09
2.92 0.05
Brain
0.47 0.02
0.25 0.01
Heart
0.34 0.01
N.D.

84
Chlorambucil and CDS: Distribution in Rats and Rabbits
In the study with rats (Figure 24), there was no significant difference in
the lung delivery capability of the CDS in comparison to the parent drug. This
may relate to the instability of the chlorambucil-CDS in rat blood (in-vitro half-life
less than 45 seconds), such that upon administration, the CDS almost
instantaneously hydrolyzed to the parent drug. In rabbits where the CDS was
5 Minutes After Administration
b
30 Minutes After Administration
Liver Lung Blood
I CDS
I Chloramb.
Figure 24: In-Vivo Distribution of Total Drug as Chlorambucil in
Rats After i.v. Administration of CDS and Parent.

85
sufficiently stable, the in-vivo distribution (Tables 7 & 8 and Figure 24, above)
showed that in all cases, substantially higher concentrations of chlorambucil were
delivered to the lungs when the CDS was administered as compared to the parent
drug. For example 30 minutes after administration of the CDS, about 22 x 10-2
pmol/mL of chlorambucil (more than 20 fold) was delivered to the lung as
compared to only about 1.0 x 10~2 gmol/mL after administration of the parent
chlorambucil.
In the blood (Table 8), the CDS concentration was shown to have decreased
with time within 30 minutes as that of the parent drug, chlorambucil, increased
following the CDS administration. This was expected because the CDS
hydrolyzed in-vivo to give chlorambucil.
Table 7: In-vivo Distribution of Drug 5 Minutes After Intravenous
Administration of Chlorambucil, and CDS: (6 pmol/kg) in Rabbits.
Tissue
Drug Concentration in Tissue(pmol x 10-2 /g or mL)
CDS (MS-2) Administration
Chlorambucil
Admin.
[Chloramb]
[MS-2]
Total [Drug]
[Chloramb.]
Blood
0.60
0.77
1.37
5.76
Brain
0.65
0.10
0.75
N.D.
Kidney
1.42
2.49
3.91
10.33
Liver
4.92
1.98
6.90
2.22
Lung
8.02
38.10
46.12
9.36

86
Table 8: In-vivo Distribution of Drug 30 Minutes After intravenous
Administration of Chlorambucil, and CDS to Rabbits.
Tissue
Drug Concentration in Tissue (gmol x 10-2 /g or mL)
CDS (MS-2) Administration
Chlorambucil
Admin.
[Chloramb.]
[MS-2]
Total [Drug]
[Chloramb.]
Blood (5')
0.92 + 0.38
0.35 + 0.01
1.28 + 0.37
2.27 + 0.50
Blood (15')
1.02 + 0.10
0.22 + 0.02
1.24 + 0.09
1.79 + 0.35
Blood (30')
1.08 + 0.09
0.15 + 0.01
1.23 + 0.10
0.04 + 0.01
Brain*
0.52
Trace
0.52
0.14
Kidney
0.95 + 0.18
1.54 + 0.22
2.49 + 0.27
1.57 + 0.35
Liver
3.40+ 1.25
1.06 + 0.24
4.45 + 1.00
1.69 + 0.57
Lung
13.08+1.01
9.24+1.37
22.33 + 1.62
0.77 + 0.19
* Brain: Only one rabbit was investigated.
Figure 25: In-Vivo Distribution of Chlorambucil in Rabbit 30 minutes
After Administration of Chlorambucil and CDS.

87
Cromolyn and CDS: Distribution in Rats and Rabbits
The distribution of cromolyn and its two CDSs were studied in rats and
rabbits. Both CDSs were found to have appreciable in vitro half-lives in rat (about
3 minutes for CDS-1 and 18 minutes for CDS-2), thereby permitting a worthwhile
study in rats unlike with the chlorambucil CDS. After the initial study in both rats
and rabbits, it was determined that the CDS-1 and CDS-2 were stable enough in
the rat to warrant continued used of this animal for further investigation. The
initial studies showed a rather similar trend of drug distribution in both animals
and also for both CDSs, so for convenience (animal size and cost), the study in
rabbits were not replicated and instead rats were used. For a similar reason the
study with the cromolyn CDS-2 was discontinued.
Table 9: In-Vivo Distribution of Drug in Rabbit Tissues 30 Minutes After
Administration of CDS-1 and Parent (Cromolyn): (MS-4), 2.62 pmol/kg
Tissue*
Concentration of Drug in Tissue (nmol/g)
CDS Administration
Cromolyn
Admin.
CDS-1
Cromolyn
Total Drug
Blood: 5'
2.33
7.60
9.93
3.2
Blood: 15'
1.80
4.01
5.81
2.5
Blood: 30'
0.87
2.9
3.77
0.2
Liver
42.66
6.85
49.51
4.7
Brain
0.27
~o
0.27
~o
Kidney
27.91
1.94
29.91
9.79
Lung
88.59
5.28
93.87
Trace
* Blood was sampled from the ear vein at 5, 15 and 30 minutes respectively.

88
The preliminary data from the rabbit study was presented here (Table 9, & Figure
26) just to illustrate that similarity and also to demonstrate the possible
applicability of the CDS to different animal species.
H CDS-(MS-4) CDS-Crm 1] Total (CDS) [EBP-Crm
Figure 26: In-Vivo Distribution in Rabbit Following Cromolyn and CDS Administration:
30 minutes after adminstration of 2.62 pmol/kg of drug, most of the drug remained
in the lung with the CDS. whereas with the parent drug only traces were detected.
A summary of the in-vivo drug distribution with time in rats following the
administration of cromolyn-CDS-1 and the parent drug, cromolyn, respectively is
shown in Table 10, below. From the data in Table 10, Tables 11 to 14, below,
were prepared for each respective tissues. Each table showed for each time point
the drug concentration (pM) from which the total amount of drug present in that
whole tissue/organ was estimated and expressed as a percentage of the
administered dose (i.e. % Total) and also as a percentage of the amount retained in
all the tissues combined after the given time (i.e. % Recovered).

89
Table 10: Distribution of Drug in Rat Tissues With Time After Administration
of 6.2 pmol/kg of CDS-1 (MS-4), and Parent (Cromolyn)
Concentration of Drug in Tissue (nmol/g)
Time (min)
CDS Administration
Cromolyn
/Tissue
CDS-1
Cromolyn
Total Drug
Admin.
5: Blood
5.1 1.8
2.7 0.6
7.8 1.6
3.2 0.9
Lung
285.3 25.6
3.5 0.8
288.8 26.1
2.5 0.5
Liver
59.0 10.0
2.9 0.1
62.0 10.1
17.0 4.4
Kidney
1.8 0.1
7.7 0.2
9.5 0.2
46.7 3.2
Brain
1.8 0.2
0
1.8 0.2
0
Heart
2.0
0
2.0
0
15: Blood
1.1 0.3
2.2 0.4
3.3 0.3
1.4 0.3
Lung
208.6 19.2
1.8 0.2
210.4 19.1
1.9 0.7
Liver
75.0 12.2
3.4 0.8
78.4 5.6
17.0 4.4
Kidney
1.7 0.5
3.5 1.2
5.2 0.8
19.4 0.5
Brain
0.9 0.1
0.1 0.0
1.0 0.1
0
Heart
0.9
0.1
1.0
-
30: Blood
1.0 0.2
0
1.0 0.2
0
Lung
190.1 16.5
2.7 1.4
192.8 16.8
2.4 0.4
Liver
37.3 6.6
2.9 1.1
40.6 5.9
0.8 0.1
Kidney
3.3 1.2
2.6 1.1
5.9 1.7
5.5 1.4
Brain
0.7 0.1
0
0.7 0.1
N.D.
Heart
1.1
N.D.
N.D.
N.D.
60: Blood
0
0
0
0
Lung
229.8 34.6
4.4 0.1
234.2 34.5
0.2 0.1
Liver
39.5 10.4
1.1 0.1
40.6 10.5
0.2 0.1
Kidney
4.5 0.9
1.3 0.1
5.8 0.8
3.0 0.4
Brain
0
N.D.
N.D.
N.D.
120: Lung
192.4 22.7
3.2 0.2
190.5 22.8
0
Liver
51.7 5.8
0.9 0.2
52.5 5.6
0
Kidney
1.8 0.3
0.9 0.2
2.6 0.2
0
240: Lung
105.8 15.2
2.3 0.5
108.1 15.3
N.D.
Liver
47.2 11.8
0.8 0.2
53.1 12.0
N.D.
Kidney
6.7 1.0
0.5 0.1
7.2 1.2
N.D.
500: Lung
44.9 7.3
0.5 0.1
45.4 7.3
N.D.
Liver
49.2 4.4
0.5 0.2
49.8 4.3
N.D.
Kidney
1.6 0.2
0.1 0.1
1.8 0.2
N.D.
* N.D. = Not performed

90
Table 11: Drug Distribution to Rat Lung Tissue with Time
' Time (min)/
Amount of Drug in Whole Tissue
Drug Admin.
(pM)
(gmol x 10 -2)
% of Total
% of Recov.
5: CDS
288.8 26.1
50.5 4.6
23.2 2.1
35.8 3.1
Parent
2.5 0.5
0.4 0.1
0.2 0.1
1.2 0.3
15: CDS
210.4 19.1
36.8 3.3
16.9 1.5
27.7 3.4
Parent
1.9 0.7
0.4 0.1
0.2 0.1
2.2 0.1
30: CDS
192.8 16.8
33.7 2.9
15.5 1.4
40.9 1.5
Parent
2.4 0.4
0.4 0.1
0.2 0.1
15.9 1.9
60: CDS
234.2 34.5
39.5 8.1
18.2 3.7
39.7 5.7
Parent
0
0
0
0
120: CDS
190.5 22.7
32.1 5.3
14.8 1.3
32.2 3.7
Parent
0
0
0
0
240: CDS
108.1 15.3
18.9 2.7
8.7 1.2
24.3 5.8
Parent
-
-
-
-
500: CDS
45.4 7.3
8.0 1.3
3.7 0.6
12.6 2.6
I Parent
-
-
-
-
Table 12: Drug Distribution in Rat Blood with Time
Time (min)/
Drug Concentration in Whole Tissue
Drug Admin
(pM)
(gmol x 10 *2)
% of Total
% of Recov
5: CDS
7.8 1.6
19.1 0.4
8.8 1.8
13.4 1.5
Parent
3.2 0.9
7.7 0.2
3.6 1.0
20.0 6.4
15: CDS
3.3 0.3
8.1 0.8
3.7 0.4
6.1 0.8
Parent
1.4 0.3
3.5 0.7
1.6 0.3
18.9 2.8
30: CDS
1.0 0.2
2.5 0.4
1.2 0.2
3.1 0.2
Parent
0
0
0
0
60: CDS
0
0
0
0
Parent
0
0
0
0

91
Table 13: Drug Distribution in Rat Liver with Time
Time (min)/
Drug Concentration in Whole Tissue
Drug Admin.
(gM)
(pmol x 10 "2)
% of Total
% of Recov.
5: CDS
62.0 10.1
69.4 11.3
32.4 5.2
48.6 1.9
Parent
17.0 4.4
19.7 0.4
9.1 1.9
49.8 7.6
15: CDS
78.0 5.6
87.8 14.2
40.3 6.5
65.1 3.8
Parent
8.5 0.8
9.5 0.1
4.4 2.2
52.6 2.4
30: CDS
40.2 6.0
45.0 6.6
20.7 3.0
54.2 2.2
Parent
0.8 0.
0.9 0.2
0.4 0.1
33.5 9.4
60: CDS
40.6 10.5
45.5 1.2
20.9 5.4
51.2 3.1
Parent
0.2 0.1
0.2 0.1
0.1 0.0
21.6 6.0
120: CDS
52.5 5.6
58.8 6.3
27.0 2.9
59.7 5.7
Parent
0
0
0
0
240: CDS
53.1 12.0
59.5 13.4
27.3 2.9
73.4 6.2
Parent
0
0
0
0
500: CDS
49.5 4.3
55.7 4.8
25.6 2.2
86.8 2.6
Parent
0
0
0
0
An additional summary of the in-vivo distribution of drug with time in the rat
following the intravenous administration of Cromolyn CDS-1 and the parent drug
respectively is also presented in Figures 27 31. These results showed that after the
CDS administration, high levels of drug mainly in the form of the intact CDS remained in
the lung throughout an 8 hour period and that the CDS was being hydrolyzed at a steady
rate to give the desired metabolite, the cromoglycate, throughout the entire 8 hour period
in the lung at levels comparable to the cromoglycate levels achieved only during the first
30 minutes following administration of the parent drug, cromolyn, as illustrated in Figure
28, below.

92
Table 14: In-Vivo Distribution of Drug in Rat Kidney with Time
Time (min)/
Drug Concentration in Whole Tissue
Drug Admin.
(pM)
(mol x 1 O'2)
% of Total
% of Recov.
5: CDS
9.5 0.2
2.3 0.1
1.1 0.2
1.7 0.2
Parent
46.7 3.2
1.3 0.2
5.6 0.4
29.1 1.7
15: CDS
5.2 0.8
1.3 0.2
0.6 0.1
1.0 0.3
i Parent
19.4 0.5
4.7 0.1
2.2 0.1
26.4 1.7
30: CDS
5.9 1.7
1.4 0.4
0.7 0.2
1.8 0.7
Parent
5.5 1.4
1.4 0.3
0.6 0.2
50.6 8.3
60: CDS
5.8 0.8
1.4 0.2
0.7 0.1
1.6 0.1
Parent
3.0 0.4
0.7 0.1
0.4 0.1
75.5 7.2
120: CDS
2.6 0.2
0.6 0.1
0.3 0.1
0.7 0.1
Parent
0
0
0
0
240: CDS
27.2 1.1
0.2 0.1
0.8 0.1
2.3 0.6
Parent
-
-
-
-
500: CDS
1.8 0.2
0.4 0.1
0.2 0.0
0.7 0.1
Parent
-
-
-
-
Overall, considering the estimated rate of disappearance of the CDS from the lung
(as well as other tissues), the amounts of cromolyn detected after either the CDS or
parent drug administration were very low. This could be explained by the fact that
owing to the extremely high hydrophilicity of cromolyn, it was rapidly cleared
from the lungs and other tissues as soon as it was absorbed or generated from the
CDS.

93
Total (MS-4 + CDS-Crm ) -*CDS-Crm # P-Crm
Figure 27: Drug Distribution in Lung After CDS or Cromolyn Administration:
(6.2 pmol/kg dose to Rats); MS-4 was the intact CDS, CDS-CRM
represented cromoglvcate arising from the hydrolysis of CDS, and
P-CRM was the cromoglvcate from the parent drug, cromolyn.
Time in Minutes (not to scale)
Figure 28: Distribution of Cromoglycate in Rat Lung with Time After CDS or
Parent Drug Administration: (this chart highlights the lower
portion of the previous one).

Tissue Cone of Drug ipM)
300
Time After Drug Admin (min)
Type of Tissue and Drug Administered
Muver-CDS juver-Crm HHl-ung-CDS Hl-ung-Crm HI Kidney-CDS fH Kidney-Crm BIood-CDS EU Blood-Crm
SO
-fc.
Figure 29: Drug Concentrations in Rat Tissues With Time After Administration of CDS vrs. Parent Drug (Cromolyn)
(There was more than a 100 fold increase in total drug delivered to the lung by CDS compared to parent drug)

50
40 -
5 15 30 60 120 240 500
Time After Drug Admin (min)
Type of Tissue and Drug Administered
Hl-iver-CDS 1 Liver-Crm CLng-CDS 0 Lung Crm l Kidney-CDS 0Kidney-Crm 0Blood-CDS DHDl Blood-Crm
Figure 30. Percent of Total Drug Administered in Respective Rat Tissues with Time
After Administration of CDS and Parent Drug (Crm).

% of Recovered Drug
100
Time After Drug Admin (min)
Type of Tissue and Drug Administered
Liver-CDS fH Liver-Crm CLung-CDS H Lung-Crm Hi Kidney-CDS K¡dney-Crm I^BIood-CDS fflD Blood-Crm
Figure 31: Percent of Recovered Drug in Respective Rat Tissues with Time
After Administration of CDS and Parent Drug
VO
On

97
At the dose administered, the in-vivo half-life of the CDS, MS-4, in the
lung was about 200 minutes. The in-vitro half-life in the lung was only about 8
minutes (Table 4). This large difference could be attributed to a number of factors
including :-
1. The in-vitro system was a one compartment fluid-like system hence presented
a much larger surface area for the enzyme catalyzed reaction compared to the
in-vivo system which consisted largely of more compact multi-compartment
cell matrixes with transport problems and enzyme accessibility to overcome.
2. The CDS, due to its high lipophilicity could have been stored in adipose
tissue, or in the lipophilic membranes of cells and thereby establishing some
sort of equilibrium with the CDS in the lung cell cytosol, thereby replenishing
it as hydrolysis occurred.
3. Intracellular factors such as drug-binding may influence hydrolysis of CDS.
For distribution in the other tissues. Figure 32 showed that following
the CDS administration, the concentration of drug (as CDS + Metabolite) in liver
and kidney, remained approximately constant over the 8 hours period, showing a
steady state clearance rate. The levels of drug in the liver and kidney were fairly
high, particularly in the liver. This may be explained by the fact that these two
organs were the principal conduits for the excretion of the drug.
Relative to the kidney, much higher levels of the drug were found in the
liver after CDS administration in contrast to the higher levels found in the kidney
relative to the liver after administration of the parent drug cromolyn (Figures 34 &
35). An explanation for this paradox (a paradox because, both molecules, CDS
and the parent, were large therefore expected to be excreted principally by the
biliary route) may be due in the main to the differences in lipophilicity and tissue
binding capability. Cromolyn is very hydrophilic and does not covalently bind to
tissue, thus renal deposition was very much favored as the drug gets into the

98
kidney but owing to its large size does not get easily past the kidney's glomerular
filtration system so gets deposited until it is taken back into circulation to the liver.
On the other hand, the high lipophilicity of CDS prevented its facile clearance
from the tissues. Hence, in the first place, not very much of it could reach the
kidney anyway, and moreover, its ability to covalently bind to tissues through
disulfide linkages, effectively 'trapped' most of the CDS in the liver.
Time (minutes)
BLOCO LUNG UVER KCffY
Figure 32: In-Vivo Distribution of Drug (CDS + Metabolite) in Rat Tissues with Time
After CDS Administration. Drug concentration in lung dropped gradually over
tune, whereas it remained almost constant in the Liver and Kidney.

99
Table 15: Recovery of Drug with Time After Administration
To Rats: CDS-1 versus. Parent
Time
(minutes)
Percent of Dru
g Recovered*
CDS-1 (MS-4)
Administration
Cromolyn
Administration
5
65.8 8.2
18.1 1.1
15
61.7 6.6
8.3 0.7
30
38.0 4.3
1.2 0.1
60
40.4 8.2
0.5 0.1
120
45.5 4.9
Trace
240
36.8 5.4
0
500
29.5 1.7
N.D.
Jjc
i.e. Total amount of drug at each time point in the six
tissue types as percent of total drug administered.
Fraction (%) of
Administered Drug
80
5 15 30 60 120 240 500
Time After Drug Administration
MS-4 B Cromolyn
Figure 33: Percent of Administered Drug Retained in All Tissues with Time

100
Table 15, and Figure 33 above, compared the drug recovery rates for
Cromolyn and the CDS from rat tissues with time after administration. The data,
along with those shown in Tables 10 14, and Figures 34 and 35, showed that
after CDS administration significantly large amounts of drug were retained in the
tissues as opposed to rapid elimination from nearly all the tissues within 60
minutes following the parent drug administration. In the lung (Table 10 and
Figure 30), about 20% of the total drug administered remained present in that
tissue after 120 minutes compared to less than 1% for the parent drug
administration. Even after 500 minutes about 30% of the administered dose still
remained in the tissue after CDS administration compared to the parent drug which
was no longer detectable in any tissue after only 60 minutes (Tables 10 14, and
Figures 29 & 31).
Figures 30 & 34 showed a comparison of the mean distribution of drug in
the respective tissues with time expressed as a percentage of the administered dose
following CDS or parent drug administration, and in Figures 31 & 35, as the
percentage of drug retained in the tissues (i.e. % of Recovered). Figure 35a
showed that upon administration of the CDS, most of the administered drug was
retained in the lung and liver at all times. The corresponding amounts in the liver
were higher than in the lung. This resulted from the mere fact that the liver was
over six times the size of the lung (Table 6). In terms of concentrations however,
the lungs retained much more drug per gram of tissue than did the liver.
Compared with the parent drug administration as shown in. Figure 29 or 34,
whereas about 20% of the administered dose was found in the lung for the period
up to 30 minutes following CDS administration, only about 0.2% was found in the
lung for the same period following the parent drug administration. Detection of
small amounts of CDS in the brain within the first few minutes of administration
(Table 10) may be a reflection of the high lipophilicity of the CDS.

% of Total Drug % 0f Total Drug
101
CDS Administration to Rats
Lung -X- Kidney Liver --Blood
Parent Drug (Cromolyn) Administration
0 40 80 120
Time (minutes)
Lung Kidney Liver -- Blood
Figure 34: Mean Percent Distribution of Total Drug With Time in Whole
Rat Tissues: This is a representation of the amount of drug present in each tissue
at a given time expressed as a percentage of the amount of CDS or parent
originally administered.

102
a.
CDS Administration to Rats
100
* Lung x Kidney Liver Blood
b. Parent Drug (Cromolyn) Administration
Time (minutes)
~ Lung -i- Kidney Liver Blood
Figure 35: Mean Percent Distribution of Recovered Drug in Whole Rat
Tissues With Time: This represents the percentage of drug in a given tissue at
the given time expressed as a percentage of the sum total of drug retained in the
tissues (i.e. unexcreted drug).

103
Higher percentages of the administered drug were found in the liver and
kidneys relative to other tissues following parent drug administration, and the
percentages fell rapidly with time until after 60 minutes no drug was detectable in
any of the tissues suggesting a very rapid clearance rate.
Of the recovered drug (i.e. drug retained in the tissues), Figures 31 or 35a
& b, again it was observed that most of the drug remaining in the tissues was in the
liver followed by the lung, after the CDS administration. With time, as the
proportion in the lung fell from about 40% to about 15% over the eight hour
period, that in the liver rose from about 50 to 85%. This seemed to suggest that as
the drug (in the form of intact CDS and cromoglycate) slowly cleared from the
lungs it was accumulating in the liver. The amount in the kidney was constant
throughout suggesting a steady state renal clearance rate. Accumulation in the
liver was consistent with the biliary route being the major means of excretion of
cromoglycate and its analogs. The drug is stored in the bile prior to its excretion.
Kinetics of Mixed Disulfide-Bond Formation
The mechanism of delivery of the lipoic acid based chemical delivery
system has been suggested to involve the formation of mixed-disulfide linkages
between CDS and tissue sulfhydryl proteins. Several sulfur containing
compounds, sulfhydryl and disulfide containing drugs have been reported to
covalently bind with sulfhydryl groups of tissue proteins in rats (Livesey et al.,
1990; & Miwa et al., 1988) and in rabbit and human tissue (Tabachnick et al.,
1982). In order to experimentally investigate the suggested mechanism, a senes of
in-vitro binding experiments were designed to demonstarte that binding due mixed
disulfide linkages did indeed occur, that the binding was enzyme catalysed, and

104
that there was a preponderance of mixed disulfide drug binding in the lung relative
to the other tissues. The results are presented here as follows:
Effect of BPNP on the Hydrolysis of MS-4
Bis-(p-nitrophenyl) phosphate, BPNP, was used to inhibit the ester
hydrolysis of the CDS in the in-vitro binding experiments. At the inhibitor
concentration of 1.0 mM in 25% rat Liver homogenate, the half-life of a model
CDS, MS-4, at an initial concentration of 50pM was about 260 minutes compared
to 12 minutes in the control, i.e. it produced over 20 fold inhibition, (Table 15). In
another experiment, at this concentration, BPNP inhibited the hydrolysis of the
Table 16: Dependence of Hydrolysis Rate Constants and Half-Lives of
MS-4 on BPNP Concentration in Rat Liver Homogenate.*
[BPNP]
(mM)
k (x 10-3)
Half-life
(minutes)
Correlation
Coefficient
0
58.2 4.7
12.0 1.0
0.9842
0.01
45.7 + 3.0
15.0 1.0
0.9890
0.05
33.7 + 4.6
21.0 2.8
0.9951
0.10
18.3+0.6
38.0 1.4
0.9926
0.50
4.85 0.44
144.3 13.4
0.9827
1.00
2.70 0.11
257.6 11.1
0.9715
2.00
1.99 0.24
353.8 43.0
0.9317
3.80
1.04 0.06
668.7 37.9
0.8938
4.55
0.89 0.02
793.3 16.7
0.9512
* Average of tvvo determinations deviation from mean .

105
model CDS, MS-4, in 25% Rabbit Blood by over 60 fold (it produced a Ty2 =
1433 min compared to 24 minutes for the control). Thus the B-esterase inhibitor,
BPNP, was used at concentrations between 1.0 and 1.25 mM in the in-vitro
binding experiments to inhibit the hydrolysis of the CDS. Rabbit tissues were
used as the model for the in-vitro binding studies because of the greater stability of
the CDS in rabbit tissues as opposed to rat tisssue.
Effect of Inhibitors on Binding of MS-4 via mixed disulfides
Further experiments using bovine serum albumin (BSA) demonstrated that
the presence of BPNP had no effect on the mixed disulfide binding of the CDS
(Table 17, & Figure 36 below).
Table 17: Effect of [BPNP] on Binding of MS-4 to BSA:
[BPNP]
(mM)
Bound Drug
(pM)
Unbound
Drug (pM)
Percent
Binding
0.00
0.26 0.02
19.40 0.13
1.33 0.09
0.01
0.23 0.06
19.17 0.05
1.17 0.28
0.05
0.21 0.07
18.91 0.68
1.12 0.11
0.10
0.30 0.13
19.31 0.36
1.55 0.67
0.50
0.26 0.04
19.63 0.32
1.33 0.20
1.00
0.23 0.07
19.20 0.12
1.16 0.33
2.50
0.23 0.12
19.36 0.42
1.17 0.63
4.55
0.38 0.17
21.14 0.67
1.75 0.79

106
The importance of this study was to demonstrate that the presence of
BPNP, the carboxyl esterase inhibitor introduced into the incubation mixture, was
not going to be a source of error in the binding experiments. From the data above,
it was determined that no significant differences (at P > 0.05) existed in the in-
vitro binding of the model CDS to rabbit liver tissue sulfhydryl at the
concentrations of BPNP used.
Figure 36: Effect of BPNP on Binding of MS-4 to BSA
The data in Table 18, below, showed that in the absence of no inhibitor (the
control study), no drug was detectable after the one hour incubation period as
neither bound nor as unbound drug. This was because within the one hour period
used for evaluation of the binding in 33% rabbit liver homogenate, all the CDS had
been completely hydrolyzed (the in-vitro half-life in 25% rat lung homogenate

107
being only about 10 minutes). In the presence of NEM only, again no drug was
detected because NEM did not inhibit the hydrolysis of the CDS. However, in the
presence of BPNP only, there was some binding of drug because BPNP inhibited
the activity of the hydrolytic enzymes and thereby enabling the CDS to remain
intact for long enough to bind and be detected. In the presence of both NEM and
BPNP some unbound drug was detected, but no binding. This was because, BPNP
did inhibit the hydrolysis of the CDS permitting it to remain intact long enough to
be detected, but NEM being a sulfhydryl blocker, inhibited any binding by mixed
disulfides.
Table 18: Binding of MS-4 in Presence of Inhibitors (BPNP or NEM)
Type of Inhibitor(s) Added, and Drug Concentrations in pM
BPNP and NEM
BPNP only
NEM only
None (Control)
UD
BD
UD
BD
UD
BD
UD
BD
2.57
0
2.75
0.21
0
0
0
0
2.81
0
2.51
0.27
0
0
0
0
Duplicate determinations both presented above
UD = Free Unbound Drug
BD = Amount of Drug bound through Mixed Disulfides.
25% Rat Lung Homogenate
Initial Drug concentration in homogenate = 45 pM
[BPNP] = 1.0 mM
[NEM] = 0.46 mM
Time Dependence of Mixed Disulfide Binding of CDS
The mixed disulfide binding of CDS (MS-4), in vitro, was found to be time
dependent, and showed saturation kinetics with time in both BSA (Table 19,

108
below) and in a model rabbit tissue, the lung (Figure 37). From the results shown
in Table 19, the maximum binding in BSA, expressed as a percentage of total drug
or as the amount of bound drug at a given time point was achieved within 6
hours. In the lung, the amount of bound drug showed saturation after about 12
hours, liable 20 and Figure 37, and within the time range (0 26 hours) used in
the study the reaction rate profile closely resembled that of a typical of enzyme
catalyzed reaction as suggested by Zeffren and Hall, 1973.
Table 19: Binding of MS-4 to BSA (50 mg Protein/mL) with Time:
Time
(hours)
Bound Drug
(AUC)
Unbound
Drug (AUC)
Percent
Binding
0
2304156
8918
0.39
6
3065016
56153
1.80
12
3051144
50615
1.66
24
1178808
20270
1.72
The observed increased binding with time was suggestive of enzyme
involvement in the mixed disulfide binding of the CDS, and the reaction profile
may be suggestive of enzyme induction or activation in the first three hours,
marked by the exponential increase in the amount of bound drug. The subsequent
portions of the curve indicated a drop in rate resulting from a number of possible
factors including saturation of the enzyme systems involved in the binding. On
the other hand, the increase in binding with time could also have resulted from the
fact that cellular glutathione (GSH) which is believed to inhibit mixed disulfide

109
bond formation between drug and protein sulfhydryl (Ziegler, 1985; and Miwa et
al., 1988) was itself undergoing 'destruction'oxidation in the presence of
atmospheric oxygen in the in-vitro system, with time, and thereby decreasing its
inhibitory effects on the binding.
Table 20: Binding of MS-4 to Rabbit Lung with Time
| Time
(hours)
Drug Concentration in Tissue
(nmol/g)
% Binding
Bound Drug
Unbound Drug
0.00
0.71 0.04
127.47 2.65
0.55 0.03
1 0.08
0.76 0.18
82.39 20.05
0.92 0.22
0.33
1.38 0.58
150.75 4.93
0.91 0.38
0.50
1.97 0.45
133.28 10.51
1.45 0.33
i 1.08
2.06 0.18
92.03 20.04
2.19 0.19
3.00
3.23 0.56
105.14 17.67
2.98 0.52
6.17
2.96 0.27
65.86 31.47
4.30 0.39
12.75
4.74 0.76
65.33 32.50
6.77 1.09
26.00
5.06 0.38
32.34 9.75
13.52 1.02
( Mean of two determinations Deviation from the Mean)
The data in Table 21, below, showed a preliminary study comparing the
dependence of binding with time in the liver, lung, and BSA of protein
concentration approximating to that of the tissue homogenates. The general trend
was increased binding with time, however in comparing the three media, it was
observed that the rate of increase in binding was of the order :
Lung Liver > BSA

110
Figure 37: In-vitro Binding of MS-4 in Rabbit Lung Tissue with Time:
The reaction profile showed saturation charateristic in enzyme catalysis.
Table 21: Binding of MS-4 with Time to Rabbit Lung Tissue
Compared with the Liver and BSA.
Time
Bound drug & (Unbound drug) (pM)*
% Binding in Medium
(min)
Liver
Lung
BSA
Liver
Lung
BSA
0
Trace
(7.45)
0.154
(7.39)
0.046
(7.336)
Trace
2.31
0.63
30
0.143
(7.29)
0.293)
(7.31)
0.126
(7.286)
2.42
3.21
1.73
75
0.232
(7.30)
0.743
(7.24)
0.135
(7.319)
3.22
9.80
1.83
* Single determinations only
A possible explanation for the observed order was that the binding was
enzyme catalyzed. In BSA which obviously lacked the enzymes present in the
tissue homogenates, the rate was very low. On the other hand, the rate was much

Ill
higher in the lung than in the liver presumable because the enzyme system
catalyzing the binding was more active in the lung a contributing basis for the
selective delivery of the CDS to lung tissue.
Dependence of MS-4 Binding on the Initial Drug Concentration
The extent of drug binding as a function of initial drug concentration was
evaluated to determine whether the binding was saturable and thereby provide
further evidence for the possible involvement of enzymes in the binding. The
binding was evaluated in vitro using rabbit lung as a model tissue.
Table 22: Dependence of MS-4 Binding in Liver Tissue on the
Initial Drug Concentration In-Vitro
Initial
[MS-4]
(pM)
Amount of Drug in Tissue
(gmol x 10 3)/g
% Binding
Bound Drug
Unbound Drug
6
0.08 0.01
2.24 0.35
3.59 0.42
12
0.11 0.03
5.23 0.56
2.01 0.66
24
0.19 0.02
7.42 0.86
2.52 0.09
36
0.30 0.04
9.49 3.96
3.06 1.01
60
1.29 0.27
21.42 1.47
5.69 1.53
90
4.31 0.54
22.16 3.15
16.28 1.49
120
10.47 0.57
35.17 3.77
22.93 2.42
180
17.62 1.20
42.18 3.52
29.75 2.39
240
21.1 1.85
50.5 4.10
29.17 2.39

112
O 50 100 150 200 250
Initial CDS Cone. (pM)
Figure 38: Dependence of Binding on Initial Drug Concentration
The binding was also found to be dependent on the initial drug
concentration when the drug was evaluated in rabbit lung as a model tissue. It was
also observed that the mixed disulfide binding of the CDS in vitro, increased
exponentially with the initial drug concentration, (Table 23 & Figure 38) for the
range 36 120 pM and began leveling off with further increase in drug
concentration resulting in the sigmoidal curve (Figures 3 7 and 38) characteristic
of enzyme catalyzed reactions (Zubay, 1986).
The pattern of time and concentration dependency of the binding was
very' highly suggestive of enzyme involvement in the binding. The logarithmic
part of the curve, i.e. the exponential increase in binding with increasing initial
drug concentration may result from the formation of an intermediate enzyme
substrate complex (Zubay, 1986), enzyme activation or induction. At higher
initial drug (substrate) concentrations, the percentage binding actually began to
decrease as may be seen in Figure 39 indicating that the binding was saturable.

113
Figure 39: Percent Binding and Initial Drug Concentration.
The percentage of bound drug decreases at high initial drug
concentration indicating possible saturation of binding sites.
ComparativeTissue Binding of MS-4 In-Vitro
The results of the in-vitro binding (Table 23) showed the binding was
greatest in lung and kidney than in the liver and blood.
Table 23: Comparative Binding of MS-4 to Various
Rabbit Tissues In-Vitro.
Tissue
Amount of drug in Tissue
(gmol x 10 3)/g or mL
% Binding
Bound Drug
Unbound Drug
Blood
0.36 0.01
27.61 3.48
1.30 0.16
Lung
0.98 0.08
31.39 4.35
3.29 0.53
Kidney
1.23 0.28
28.21 5.87
4.34 2.05
Liver
0.44 0.07
33.32 5.83
1.31 0.40

114
Dependence of Binding on Homogenate Concentration
Table 24, below, showed that the extent of binding in vitro (in a model
tissue-rabbit liver) was not significantly dependent on the concentration of the
tissue homogenate. It could be inferred that in dilute homogenate, i.e. homogenate
of concentration less than 25%, the binding was lower, whereas at homogenate
concentrations between 25 and 50, there was no significant difference.
Table 24: Dependence of MS-4 Binding on Homogenate Concentration
[Homogenate]
(%)
Drug Concentration in Tissue (pM)
% Binding
Bound (x 10'1)
Unbound
15
1.21
13.31
0.88
25
1.43
12.80
1.07
35
1.40
12.06
1.10
50
1.60
12.60
1.26
Tissue Sulfhvdryl
Biologically, sulfhydryl groups occur in tissue mainly as the amino acid
cysteine (Perret and Rudge, 1985). Cysteine along with its disulfide cystine
occurs in body fluids and cells as well as in most animal proteins. Homocysteine
is found as the free thiol, its homodisulfide and mixed disulfides in man. Reduced
Glutathione (GSH), the cysteine containing tripeptide, is found in high
concentrations in most mammalian tissues where it fulfills many roles including

115
the maintenance of the oxidation status of the cell. Tissue thiols are of two types -
protein bound (T-SH) and non-protein bound (NP-SH).
Table 25: Tissue Sulfhydryl Content in The Rabbit as Total Thiol, T-SH;
Non-Protein Thiol, NP-SH and Protein-bound Thiol (P-SH)
Tissue
Tissue Thiol Coi
Total Thiol
ncentration (mmol
Non-Protein
x 102/g or mL)
Protein-Bound
Blood
39.70 7.88
2.37 0.28
33.02 3.18
Kidney
30.86 0.75
5.20 0.15
26.00 1.63
Liver
50.42 0.98
11.36 0.44
39.46 3.97
Lung
24.57 0.99
4.71 0.18
20.10 1.00
The Protein Thiol, P-SH, (as well as Total Thiol, T-SH) content in the
rabbit were of the order :-
Liver > Blood > Kidney > Lung
The comparative study showed that the mixed-disulfide binding in the respective
tissues, Table 23, was of the order:
Kidney > Lung Liver > Blood
From the estimated 'magnitude of binding' values for MS-4 (Table 24), the extent
of in-vitro binding in the tissues, after 1 hour incubation, were of the order of less
than 5 pmol drug per mole of tissue P-SH content. This suggested an
overwhelming abundance of 'free' -SH groups available for binding under the right
concentration or equilibrium conditions.

116
Blood Kidney Liver Lung
t-sh Unp-sh 11 P-SH
Figure 40: Tissue Sulfhydryl Content of Rabbit.
Table 26: Estimated Magnitudes of In-Vitro Binding
of MS-4 in Respective Rabbit Tissues
Tissue
Bound Drug
gmol xl0'3/g
P-SH
mmol xl0_1/g
Magnitude of
Binding (in
pmol Drug/mol
P-SH)
Blood
0.36 0.01
3.30 0.29
1.09 0.01
Kidney
1.23 0.08
2.60 0.06
4.73 0.21
Liver
0.44 0.28
3.95 0.12
1.12 0.02
Lung
0.98 0.07
2.01 0.10
4.88 0.04

117
Figure 41: Tissue Sulfhydryl, Mixed Disulfide Binding and Magnitude of Binding
It may be noted that, in this study, since the bound drug fraction was
estimated from the precipitated protein, it was well assumed that only the tissue
P-SH was of importance in drawing a meaningful correlation between the mixed
disulfide drug binding in-vitro and tissue sulfhydryl.
The observed order of drug-tissue disulfide binding did not correlate with
the observed tissue sulfhydryl content. It might have been reasonable to expect the
binding to increase with increasing tissue sulfhydryl (or tissue P-SH) content, but
rather the reversed correlation was observed. Tissues with the highest -SH content
such as the liver and blood exhibited the least extent of binding, whereas the lung
and kidney with the lowest tissue P-SH content of the tissues evaluated, had the
highest amounts of mixed disulfide bound drug (i.e. the magnitude of in-vitro
binding in pmol drug bound per mole of tissue P-SH was found to be highest in
the lungs and kidneys, Table 26 and Figure 41).

118
Some explanations for this apparent paradox were as follows:
1. The CDS binding to tissue by mixed disulfide in-vitro may be enzyme
catalyzed as has been known to occur with disulfide and sulfhydryl containing
drugs (Pihlajaniemi, 1991). One such enzyme being the protein disulfide
isomerase, PDI. Evidence for the possible involvement of enzyme catalysis in
this study was also implicit from the fact that the percentage binding in BSA,
liver and lung tissues, with time was of the order;
Lung Liver BSA, (Tables 3, 6, & 7).
In other words, binding was much higher in the tissue homogenates than in
BSA where enzymes that may be found in the homogenates were obviously
absent. Also the fact that binding in a model tissue was logarithmically
dependent upon time, and the initial drug concentration was suggestive of the
involvement of enzymes enzyme induction and/or activation might have been
occurring.
2. Also mixed disulfide linkages between the drug and tissue sulfhydryl my occur
with tissue sulfhydryl as well as with tissue disulfide.
3. Also, -SH groups readily interconvert with disulfides in an equilibrium
reaction. In a tissue with high concentration of -SH groups, the extent of inter-
and intra- protein sulfhydryl to disulfide interchange will be higher, and
especially so if PDI activity is also high in that tissue. PDI catalyzes the
isomerization of both intramolecular and intermolecular disulfide bonds and
has also been shown to catalyze, via sulfhydryl-disulfide interchange, the
scission as well as disulfide bond formation between disulfide/sulfhydryl drugs
and tissue sulfhydryl groups (Edman et al., 1985 & Darrow et al., 1988). For
such a tissue, for example the liver, it may be argued that for the foregoing
reasons, there was less free -SH available for drug binding due to competition
from the mentioned inter- and intra- protein reactions. In fact, the relative

119
activity of PDI in the rat tissues has been estimated by various investigators to
be essentially of the order, (Edman et al., 1985; Chandler & Varandani, 1972):
Liver Kidney ~ Lung > testes and spleen > heart > brain.
The low binding in the blood in spite of its high T-SH might have been due to
its assumed deficiency in PDI activity (PDI activity in blood was not
mentioned in the reference).
4. Also mixed disulfide bond formation is greatly modified by the presence of
molecular oxygen (enhance: Keilin, 1967; and Freedman, 1984); endogenous
peroxides and other oxygen reactive species (enhance: Zeigler, 1985);
oxidized glutathione, GSSG, (enhance: Darby and Creighton, 1993); and
reduced glutathione, GSH (inhibit: Watabe et al., 1986; and Miwa et al., 1988).
Oxygenated tissues may contain endogenous peroxides as well as other
reactive oxygen species (Zeigler, 1985). The lung tissues are much more
oxygenated than any other tissues, besides there is a higher tissue concentration
of GSSG in the lung than in the liver (Philpot, 1977) which in turn has much
higher levels of GSH (Chasseaud, 1976; and Miwa et al., 1988 ).
5. Other enzyme systems, particularly those with oxygenase activities, may be
involved in the binding. In rats, rabbits, and hamsters, it has been reported that
some oxygenase activities are highest in the lungs (Philpot et al., 1977).

CHAPTER 6
CONCLUSIONS
One of the most important properties of a drug, with regard to drug
delivery, is an optimum octanol -water partition coefficient (Log P). Transport
within the body primarily occurs via the hydrophilic phases of plasma and extra
cellular fluid to enable the pharmacogen to reach its target cell, and transport
across biological membrane barriers occur via lipid phases. From the log P values
estimated in the work reported in this dissertation, the respective CDS were much
more lipophilic than the parent drugs. The low lipophilicity of the parent
compounds (Log P < 0), as was especially the case for cromolyn translated into its
low cell permeation and high tissue clearance rates as implied from the data in
Table 15. The highly lipophilic CDS (Log P > 0), on the other hand showed
greater tissue retention.
The studies also showed that the CDS needed to be sufficiently stable in-
vivo in order for it to have its own characteristic distribution profile necessary for
comparing it with the parent drug. An index of in-vivo stability was approximated
from in-vitro stability data using freshly prepared tissue homogenates. For
instance the in vivo distribution of chlorambucil in the rat after CDS
administration was no significantly different from that of the parent drug because
the chlorambucil CDS was so unstable in the rat (less than 40 seconds) that it
almost instantaneously hydrolyzed into the parent drug upon administration. On
the other hand, the cromolyn CDS was sufficiently stable in the rat (up to 10
120

121
minutes) to allow some evaluation of its pharmacokinetics. The stability of the
CDS (MS-4) in the respective rat tissues (except blood), in terms of the half-lives
were approximately the same, about 9-11 minutes (Table 4). The half-life in
blood was about half as much (5.5 minutes). This reflects a possible difference in
the distribution of esterase enzymes in the blood as opposed to other tissues.
The in-vivo distribution showed that following intravenous administration
at all the time points investigated, there was substantially enhanced delivery of the
respective parent drugs to lung tissue by the various CDSs compared to the
underivatized parent drugs. For example, the chlorambucil CDS MS-2, after 30
minutes of administration produced over 20 fold increase in delivering
chlorambucil to rabbit lung tissue than when the parent drug was administered,
Figure 26. The results of the study with the cromolyn CDS were even more
impressive. Significantly large quantities of drug as cromoglycate were delivered
to lungs, and the presence of large quantities of the unhydrolyzed CDS retained in
that tissue served as a reservoir for the sustained release of cromolyn for over eight
hours.
In the studies with cromolyn as a model, administration of the CDS,
resulted in much less drug being excreted over a longer period of time and hence
in the chlorambucil study, there was much less chlorambucil in the kidney with the
CDS. For example, 5 minutes after administration, chlorambucil concentration in
the kidney was 3.91 x 10*2 pmol/g after CDS administration compared with
10.33 x 10'2 pmol/g after parent drug administration (Table 7). Much less drug
was being excreted since with the CDS more of the drug was being retained in the
lung (especially) and the liver.
The B-esterase inhibitor, bis-(p-nitrophenyl) phosphate, BPNP, at a
concentration of about 1 mM was effective in inhibiting the ester hydrolysis of the
model CDS, MS-4, in the rat or rabbit tissue homogenate reasonably well enough

122
to maintain the CDS intact for duration of the in-vitro binding study. The BPNP
did not seem to have had any effect on the binding of the drug via mixed disulfide
bond formation.
The binding of MS-4 with rabbit liver, and lung homogenates and BSA
showed that the binding was time dependent, and also dependent on the initial
drug concentration. The extent of binding increased with time and with increasing
initial drug concentration. The pattern of time dependence kinetics suggests the
involvement of enzymes. The increase with time suggested enzyme induction.
Increase of binding with drug concentration may also suggest enzyme induction,
but this might also have merely resulted from the fact that mixed disulfide bond
formation between -SH and -S-S- is an equilibrium reaction, the higher the
concentration of one 'reactant' (e.g. the initial drug concentration), the greater the
equilibrium shifts to more binding. Within the time and initial drug concentration
ranges used in the study, it was concluded that the binding of the CDS was not
convincingly saturable.
Comparison of the drug binding in BSA and the tissue homogenates Lung
and Liver (Table 22) suggested the possibility of enzyme involvement. The
binding was much higher in the liver and lung than in BSA of equivalent protein
concentration. In-vitro, the amount of drug bound to tissue was of the order;
Kidney > Lung > Liver > Blood.
But magnitude of binding (rate of binding with respect to tissue sulfhydryl)
indicated that the extent of binding was greatest in the lung, and generally was of
the order:
Lung > Kidney > Liver > Blood
This order did not correlate with the observed tissue sulfhydryl (P-SH or T-SH)
content as should have been expected (rather it was the exact opposite). From this
it was suggested that the mixed disulfide binding was enzyme catalyzed, and in a

123
tissue with high -SH content and high PDI activity, inter- and intra-molecular
mixed disulfide reactions between the endogenous proteins probably competed
with the binding of the CDS by mixed disulfides. Another explanation suggested
was the possible involvement of cofactors such as molecular oxygen, or other
enzyme systems having greater distribution in the lung than in other tissues as has
been found to be the case for glutathione S-transferases (a group of enzymes
which catalyze the glutathione conjugation with some xenobiotics) which have
three times higher activity in the lung than in the liver (Damani, 1987). These
factors along with the fact that after intravenous administration, the total cardiac
output first goes to the lungs, probably and rather fortuitously, contributed to the
enhanced delivery of drug to the lung by the CDS.
The results of the in-vitro binding studies strongly suggested the
involvement of mixed disulfide linkage formation in the mechanism of selective
delivery by the CDS. Evidence for the formation of mixed disulfide-linkages came
from the fact that upon treatment of bound fraction (protein precipitate) with
dithiotreitol, DTT, drug originally bound via mixed disulfide linkages was
released, and that in the presence of N-ethylmaleimide, NEM, a sulfhydiyl
blocker, no binding took place. DTT breaks disulfide bonds and NEM blocks its
formation.
The CDS resulted in a much greater retention of the administered drug with
(and hence much less excretion) with the greatest percentage of the total drug
remaining in the lung in liver, with the lung having a much higher drug
concentration per gram of tissue than the Liver.
In the estimation of the cromoglycate dianion in biological media, a
comparison of a method published by Gardner, 1984, with a novel method
developed in this study showed that the HPLC assay developed and used in this
project, afforded a much simpler, convenient and accurate, one step recovery of

124
cromoglycate from biological media. The solvent used for protein
precipitation/drug recovery comprised a 5:35:60 v/v mixture of DMSO/Methanol/
Acetonitrile. This solvent system proved effective with drug recovery efficiencies
approaching 100% and thus along with the HPLC system proved to be more
accurate and more sensitive than the published method of Gardner, (1984) and
more convenient to use than the methods that previous investigators have
traditionally employed.
Compared to the parent drug the CDS was effective in delivering cromolyn
and chlorambucil to the rat and rabbit lung tissues at a nearly steady rate for longer
than 8 hours in the rat following one administration of 6.2 pmol/kg of a model
CDS, the Cromolyn CDS-1, whereas the parent drug administration was only
effective in producing a comparable level of cromolyn in the lung for only about
30 minutes following administration (Figure 29).
Concern about increased systemic toxicity resulting from the CDS on
account of its higher lipophilicity and observed higher retention/deposition in
tissues other than lung need not be a cause for alarm, because for instance,
whereas these increases were of the order of 2 to 3 fold for the chlorambucil CDS,
the increase for lung tissue was over 20 fold. The resulting 20 fold decrease in
effective (and hence the vastly lowered dose that may thus be required for clinical
therapy) could more than offset the increased tissue retention of the drug
associated with the CDS administration.
Finally, in conclusion, the CDS, as designed, appeared to have served to
anchor the drug (cromolyn or chlorambucil) in the lung tissue and provided a
significant and sustained release of the active drug over a protracted period of
time. The reason for the selectivity of the CDS in delivery to lung tissue was due
in part, at least, to the higher magnitude of mixed disulfide binding in the lung.
The binding was enzyme catalyzed, and probably involved cofactors such as

125
molecular oxygen, and/or other enzyme systems or inhibitors whose distribution
favored the preponderance of binding in the lung. The similarity of the overall
results with respect to the two model drugs, chlorambucil and cromolyn, suggests
the possible universal applicability of the discussed delivery system for drug
targeting to lung tissue.

APPENDIX
SAMPLE CALCULATIONS
A.Extraction Efficiency
Initial drug concentration in Blood before hydrolysis =
Dilution Factor in samples for HPLC analysis =
Final drug concentration in analyte =
65.0 pM
10
6.5 pM
Estimated [Cromolyn] by Extraction
Extraction Efficiency = x 100 %
Actual [Cromolyn] in Lysate
B.Partition Coefficients
The Partition Coefficient, P, was calculated from the relationship,
[Drug] in Octanol Phase
P =
[Drug] in Aqueous Phase
C.Stability Studies
For each drug, stability in terms of its hydrolysis rate constant, k, and the
half-life (T1/2, in minutes) in each respective media was determined by plotting
the log of Drug concentration at time t, values against the time t, and determining
the slope by linear regression, whereby:
Slope = -2.303 k, and
0.693
Half-life, (Ti/2) =
k
126

127
D. In-vivo Distribution
Calculation of drug concentrations in tissues:
At the time 5 minutes after administration of CDS-1, the average MS-4 concentra
tion in Rat blood was given by the AUC value 64578;
From the Calibration equation for MS-4,
y = 104952.72x 1095.39 (r = 0.9985)
[MS-4] = 0.63 gM x Dilution Factor of 8
= 5,07 uM
= 5,07 nmol/mL or g (Table 7)
The corresponding concentration, x, of cromolyn arising from the hydrolysis of the
CDS, (i.e. [P-Cromolyn]) in the blood 5 minutes after CDS administration was
calculated from the calibration equation:
y = 165582.64x 4775.81 (r = 0.9978)
Thus for AUC = 64827
[Cromolyn] =2.7 pM and,
Total Drug = [MS-4] + [Cromolyn]
= 5.1 +2.7
= 7.8 gM
= 7.8 nmol/mL or g
Amount of drug (as 'total drug') in whole tissue was obtained for the case of the
cromolyn CDS-1 administration by the following relationship:
Amount = Concentration (nmol/mL or g) x Total Mass or Volume of Tissue
Thus for blood (composition 7% of Body Weight of 350g Rat), 5 minutes After
Administration of CDS :
Amount = (7.8 nmol/mL or g)(0.07)(350g)
= 191 nmol
= 1.91 x 10 pinol

128
The amount of drug in a given whole tissue expressed as a percentage of the total
amount administered (i.e.) %TD) was obtained as follows:
Concentration of Stock Drug solution Administered = 6.2 mM
Dosage Rate = 1.0mL/kg
Thus total Amount Administered = 2.17 pmole
Hence for blood, 5 min after CDS-1 administration,
% TD = (0.019/2.17) (100)
= 8,8 %
and %Recov.
(8.8/65.8) (100)
13,4%
The amount of drug present in a given tissue as a percentage of the total amount
remaining in the animal at the time of sacrificing is given by '% Recov.'
E. Drug Bound By Mixed Disulfides:
Amount of drug bound to tissue by mixed disulfide, [BD], was given by:
BD] = x (Df-1 x Df-2)
where:
X = Concentration corresponding to the AUC as given by the
calibration equation: {x = (AUC + 1905.4)/104952.7}
Df-1 = Dilution factor resulting from sample preparation for HPLC analysis
Df-2 = Dilution resulting from tissue homogenate preparation
Thus, for the AUC value of 6176, i.e. the average value corresponding to the
mixed disulfide bound drug concentration in liver tissue (Dependence of Binding
on Initial Drugl Run 1, [MS-4]jjppai = 6 pM), the amount of bound drug was:
= 0.0770 pM x 2 x 100/28.2
= 0.546 pM
= 0.546 pmol/ lOOOg
= 0,054 x 10~2 umol/u.

129
For samples corresponding to the initial MS-4 concentrations of 6.0, 12.0, 24.0,
and 36.0 pM respectively,
Df-1 = 2 (Bound drug), or 3 (Unbound Drug)
For [MS-4]¡mtmi = 60.0 and 90.0 pM;
Df-1 = 2 (bound), and 6 (unbound)
For [MS-4]mitiai = 120 pM;
Df-1 = 2 (bound), and 9 (unbound).
For all the samples the factor Df-2 = 100/28.2
(This factor resulted from the dilution due the final homogenate
concentration which was 28.2%).
F. Comparative Tissue Binding:
For [BD] ; Df-1 =2 (all)
Df-2 = 100/45.7 (for Liver & Kidney only)
Df-2 = 1 (for Blood)
- 100/30.5 (Lung)
For [Unbound Drug]; Df-1 = 3, and Df-2 = 1
G. Tissue Sulfhydryl:
The linear regression equation from calibration curve based on the method of total-
thiols, (T-SH) was:
Y = (6.942 x 10-4)Y- (4.917 x 10'3)
where
n = 15,
r = 0.9998
Y = absorbance
-SH concentration (in pM)

130
Thus the T-SH for Blood, Run 1, where average absorbance was 0.6907, the -SH
content was given by;
X= 0,6907 + (4,917 x 10-3) x JO. x J00
(6.942 x 10-4) 0.5 5
= 4000817 fiM
= 0.40 moles/1000 mL
= 0.40 mmoles /mL
<=> 40,0 x 10~2 mmol /mL.
The factor 10/0.5 resulted from the dilution of 0.5 mL of the sample to 10 mL in
the assay; whereas the factor 100/5 resulted from the dilution of the homogenate to
5% before commencing the analyses.
Similarly, for the Non-Protein Thiols, NP-SH, the average absorbance was 0.1305
for blood, Run 1. The NP-SH content is then given by:
0,1305 + (4,917 x 10-3) x 10 x 6.1
6.942 x 10-4 5 2
x 100
5
H. Magnitude of In-Vitro Binding:
For the estimation of the 'Magnitude of In-Vitro Binding', the following expression
does provide some relative values when binding in various tissues are compared
based on the amount of drug binding per mole of tissue Protein-Thiol. Thus for
blood or a given tissue;
amount of drug bound/ mL or gm blood or tissue
Magnitude of Binding =
P-SH content/ mL or gm blood or tissue
= 0.36 x 10-3 pmol/ mL
= 3.3 x 10'1 x mmol/ mL
= 1.09 u mol MS-4/ mole P-SH (for blood)

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BIOGRAPHICAL SKETCH
Maurice Philip Saah was bom on February 2, 1960 in Cape Coast, Ghana.
He had his primary education at the Catholic Jubilee School in Cape Coast,
Ghana, high school at the St. Augustine's College also in Cape Coast, and received
his B.Sc. (honors) double major degree in chemistry and biochemistry from the
University of Ghana, Legn Accra, Ghana in 1980. After joining the work force
for some time in industry and academia, he briefly attended graduate school at the
University of South Florida at Tampa, Florida, and then transferred to the
University of Florida, Gainesville, Florida, where he enrolled in the College of
Pharmacy in 1989. There he joined Dr. Bodor's select group of graduate students
and a cream of internationally diverse crop of researchers at the Center for Drag
Discovery, and graduated with a Ph.D. degree in the Summer of 1994.
139

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Nicholas Bodor, Chair
Graduate Research Professor of
Medicinal Chemistry and
Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
H.O. Margaret O. James
Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Richard Hammer
Professor of Pharmaceutics
and Medicinal Chemistry

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
ames Simpkins
Professor of Pharmacodynamics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Luis M. Muga
Professor of Chemistry
This dissertation was submitted to the Graduate Faculty of the College of
Pharmacy and to the Graduate School of University of Florida and was accepted as
partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Dean, Graduate School