Analgesic and immunomodulatory effects of codeine and codeine 6-glucuronide

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Analgesic and immunomodulatory effects of codeine and codeine 6-glucuronide
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
        Page v
    Abstract
        Page vi
        Page vii
    Chapter 1. Background and significance
        Page 1
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    Chapter 2. Methods
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    Chapter 3. Results
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    Chapter 4. Discussion
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    References
        Page 132
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    Biographical sketch
        Page 149
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Full Text







ANALGESIC AND IMMUNOMODULATORY EFFECTS OF
CODEINE AND CODEINE 6-GLUCURONIDE














By


VINAYAK JAYA SRINIVASAN


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



UNIVERSITY OF FLORIDA


1996














ACKNOWLEDGMENTS


I would like to take this opportunity to offer my heartfelt appreciation to Dr.
Ian Tebbett. His continous guidance and encouragement was the driving force
for this research project. I would like to express my sincere thanks to the other
members of my committee--Dr. Donna Wielbo, Dr. Kenneth Sloan, Dr. Hartmut
Derendorf and Dr. Roger Bertholf--for all their advice and suggestions throughout
the project.
I want to thank Dr. James Simpkins, Dr. Janet Karlix and Dr. Guenther
Hochhaus for giving me an opportunity to work in their labs in order to generate

some of my data. I also would like to thank Jim Ketcham for his invaluable help
in putting all my posters, papers and this dissertation together.
I would also like to convey my thanks to Shawn Toffolo and Becky
Frieburger for their help in my work. I also deeply appreciate the support of all
the graduate students and the other faculty members and secretaries in the
department.
I want to express my deepest gratitute to my brother, my relatives and all
my friends who have stayed by me through the thick and thin. Finally, I want to
dedicate this work to my parents because I did it for them.














TABLE OF CONTENTS



ACKNOW LEDGMENTS ................................................................................... ii


ABSTRACT ...................................................................................................... vi


CHAPTERS

1. BACKGROUND AND SIGNIFICANCE .......................................... I

1.1 Opioids and Pain ....................................................................... 2
1.1.1 Pain Transmission ........................................................ 3
1.1.2. Pain Perception ........................................................... 4
1.2 Codeine ..................................................................................... 4
1.2.1 Administration and Dosage ........................................... 5
1.2.2 Pharmacological Actions ............................................... 6
1.2.3 Toxicity ......................................................................... 7
1.2.4 Therapeutic Uses ........................................................... 7
1.2.5 Drug Dependence and Tolerance ................................ 8
1.2.6 Analytical Techniques ................................................... 9
1.2.7 Pharmacokinetics in Man .............................................. 10
1.2.7.1 Absorption ........................................................ 11
1.2.7.2 Distribution ........................................................ 11
1.2.7.3 Metabolism ......................................................... 12
1.2.7.4 Elimination ........................................................ 14
1.2.8 Pharm acokinetics in Rats ............................................... 16
1.2.8.1 Absorption ........................................................ 16
1.2.8.2 Distribution ........................................................ 17
1.2.8.3 Metabolism ......................................................... 18
1.2.8.4 Elim ination ........................................................ 19
1.3 Drug Glucuronidation .................................................................. 20
1.3.1 Overview ........................................................................ 20
1.3.2 Direct Pharmacological Activity .................................... 21
1.4 Evaluation of Analgesia in Small Animals ........................ 26
1.4.1 Introduction ...................................................................... 26











1.4.2 Time Course of Analgesic Effect ..................................... 27
1.5 Genetic Polym orphism ................................................................ 28
1.6 Im m unomodulation ..................................................................... 29
1.6.1 Opioid Receptors ........................................................... 30
1.6.2 Effects on Lym phocytes ................................................ 31
1.6.3 Effects on Myleoid Cells ............................................... 32
1.6.4 Effects on Natural Killer Cells ........................................ 34
1.6.5 Mechanism of Action ..................................................... 34
1.7 Receptor Binding ......................................................................... 37
1.8 Hypotheses ................................................................................ 39
1.9 Specific Objectives ....................................................................... 39

2. M ETHODS ..................................................................................... 41

2.1 Specific Objective #1: Analytical Method .................................... 41
2.1.1 Materials ....................................................................... 41
2.1.2 Extraction Procedure .................................................... 41
2.1.2.1 Human urine ...................................................... 41
2.1.2.2 Rat plasma ......................................................... 42
2.1.2.3 Rat brain .......................................................... 42
2.1.3 Chromatographic Conditions ......................................... 43
2.1.3.1 HPLC system 1 ................................................. 43
2.1.3.2 HPLC system 2 ................................................. 44
2.2 Specific Objective #2: Synthesis of Codeine 6-glucuronide ...... 45
2.2.1 Reaction Step I ............................................................. 45
2.2.1.1 Dry benzene ...................................................... 47
2.2.1.2 Fresh silver carbonate ...................................... 47
2.2.2 Reaction Step II ............................................................. 48
2.2.3 Reaction Step III ........................................................... 48
2.3 Specific Objective #3: Analgesic Activities of Codeine and
Codeine 6-glucuronide ....................................................................... 48
2.3.1 Intracerebroventricular Route Studies ........................... 49
2.3.1.1 Surgery ............................................................ 50
2.3.1.2 Tail flick method ................................................ 50
2.3.2 Subcutaneous Route Studies ........................................ 51
2.3.3 Intravenous Route Studies ............................................ 51
2.3.4 Statistics ......................................................................... 52
2.4 Specific Objective #4: Immune Studies with Human
T Lymphocytes ................................................................................... 52
2.4.1 Method ........................................................................... 53
2.4.2 Statistics ....................................................................... 55
2.5 Specific Objective #5: Receptor Binding Studies ........................ 55











2.5.1 M aterials ....................................................................... 55
2.5.2 M ethod ......................................................................... 56
2.6 Specific Objective #6: Plasma and Brain Concentrations ........ 57

3. RESULTS ...................................................................................... 59

3.1 HPLC Developm ent .................................................................... 59
3.1.1 Extraction Recoveries ................................................... 61
3.1.2 Range / Linearity of Standard Curve ............................. 61
3.1.3 Specificity ....................................................................... 62
3.1.4 Sensitivity / Limit of Detection and Quantitation ........ 63
3.1.5 Precision and Accuracy .................................................. 63
3.1.6 Stability ......................................................................... 65
3.2 Synthesis ..................................................................................... 65
3.3 Analgesia Studies ...................................................................... 66
3.3.1 Intracerebroventricular Route ........................................ 66
3.3.2 Subcutaneous Route .................................................... 70
3.3.3 Intravenous Route ........................................................ 83
3.4 Im m une Studies ............................................................................... 105
3.5 Receptor Binding Studies ................................................................. 111
3.6 Plasm a and Brain Concentrations .................................................... 116

4. DISCUSSIO N ..................................................................................... 121


REFERENCES ................................................................................................. 132


BIO G RA PHICAL SKETCH ............................................................................... 149















v














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

ANALGESIC AND IMMUNOMODULATORY EFFECTS OF

CODEINE AND CODEINE 6-GLUCURONIDE

By

VINAYAK JAYA SRINIVASAN

May 1996


Chairman: Dr. Ian Ronald Tebbett
Major Department: Pharmaceutics


The interactions between opioid analgesics and the human immune

system can have important clinical consequences. A better understanding of
these interactions is needed due to the widespread use and abuse of opiates. In
recent years, an increased knowledge and awareness in this area has generated

a considerable surge in research. Narcotics are predominantly used to alleviate
pain and discomfort in patients with trauma or undergoing major surgery.
However, they are also known to cause impairment of the immune system.
Subsequently, this could lead to patients becoming predisposed to infectious
diseases as a result of the immunosuppressive effects of narcotics.
An HPLC system was successfully developed for the analysis of codeine
and its metabolites in various biological samples, that is, plasma, urine and brain
tissue. Codeine 6-glucuronide and an intermediate compound were synthesized
using a modification of the Koenigs-Knorr reaction. The synthetic procedure was












efficient and reproducible. Analgesia studies with the tail flick method showed
that codeine 6-glucuronide and the intermediate exhibited a higher analgesic

activity compared to codeine when administered intracerebroventricularly.
However, both compounds were not as active as codeine when administered by
subcutaneous and intravenous routes. Immunomodulatory studies showed that
the glucuronide metabolites of codeine and morphine were less
immunosuppressive compared to their parent compounds, especially at

physiologically relevant concentrations. Receptor binding profiles of codeine 6-
glucuronide and the intermediate were similar to codeine, indicating that they
possessed activity towards the p-opioid receptors.

The overall goal of the project was to correlate the analgesic and
immunomodulatory effects of codeine and codeine 6-glucuronide. This would

result in a better understanding of the significance of high levels of codeine 6-
glucuronide present in the plasma and urine in man after codeine administration.
Further, this may lead to the development of glucuronide analogs for the
management and treatment of pain in immunocompromised patients.














CHAPTER 1
BACKGROUND AND SIGNIFICANCE


Pain is an unpleasant sensation that can disturb the comfort, thought,
sleep, and normal daily activity of a person. Pain signals are considered to be

part of a protective mechanism designed to indicate the presence of a potentially
dangerous condition. Thus, it is considered to be symptomatic of an underlying
condition that requires attention and treatment. Pain is the net effect of complex
interactions of ascending and descending neurosystems which include

biochemical, physiological, psychological, and neurocortical processes. Also,
since pain is a very subjective experience, only the patient can describe its
intensity. This subjectivity makes it difficult to assess the activity of analgesics in
humans.

Analgesics are defined as drugs that can relieve pain without causing loss
of consciousness. The most potent analgesics are referred to as narcotics and
act directly on the central nervous system. Narcotics as a group include the
opioids, which are considered to be the most effective analgesics available. The
opioid family, whose name derives from opium, includes agents such as
morphine, codeine, meperidine and methadone. While opioid is a general term

for any drug, natural or synthetic, that has actions similar to morphine, the term
opiate is more specific and applies only to compounds present in opium such as
morphine and codeine. Apart from acting as analgesics, opioids produce a
variety of pharmacological actions on various tissues in the body.











1.1 Opioids and Pain

Opioids represent the main class of drugs in the clinical management of
mild to moderate pain in various cases of medical illness, and relieve pain
primarily through direct actions on receptors in the central nervous system.

Opioid analgesics include natural alkaloids from opium (morphine, codeine),
synthetic surrogates (methadone, meperidine) and endogenous peptides
(enkephalins, 13-endorphins).

Opioids act at receptor sites both within and outside the central nervous

system. Binding studies with various drugs and ligands in the brain and other
tissues suggest the presence of a multitude of opioid receptors. The three
important receptor types are designated as mu (j), kappa (K:) and delta (8). The
effects mediated by the p. receptors include supraspinal analgesia, respiratory

depression and euphoria. The K receptors mediate analgesia at the level of the
spinal cord, along with sedation and miosis. The 8 receptors are also thought to

be involved in analgesia, both at the spinal and supraspinal sites. However, their
role in this regard remains controversial (Jaffe and Martin, 1985).
The body produces three families of peptides that are capable of
interacting with opioid receptors--enkephalins, 3-endorphins and dynorphins.
These endogenous opioids have a high affinity for the p., K and 8 receptors,

respectively. They are present throughout the body and serve as hormones and

neurotransmitters. It is thought that morphine and other opioid analgesics mimic
the actions of these endogenous ligands by binding to the opioid receptors.
These interactions are presumed to give rise to the observed pharmacological

effects.















1. 1. 1 Pain Transmission


Pain generally begins with a noxious stimulus that injures or destroys

tissues. Endogenous chemical substances such as histamine, bradykinins,

prostaglandins, and others are then released from the damaged tissues and

nerve terminals. The released chemicals bind to "pain receptors" or nociceptors

present along the afferent nerve fibers, depolarizing the nerve membranes and

initiating an action potential. This causes the generation of a pain impulse which

is then transmitted via the afferent fibers to the spinal cord as shown in the figure

below (Figure 1-1). When the pain signals arrive at the spinal cord, they are in

turn relayed to the higher centers of the brain--thalamus and cortex.


Cortex


System


Pe*r-AqueduCtai Gray



R~ph* MOMLatral Reiculr
Formation


Nt~lceu = r 4edial Reticulalr



MEDULLA


Spno-Tharmni -
Tract

SPINAL
CORD From
Primary Afferent
Pain Fibres


Figure 1-1 : The pain modulating system (adapted from Puntillo, 1988).











1.1.2 Pain Perception

Although some responses are reflexive in nature (e.g., knee jerk), the
perception and appraisal of pain usually occurs in the higher centers of the brain.
These systems are known to be responsible for attention, mood, motivation and
arousal. Thus, pain is perceived in the thalamic and forebrain levels and
evaluated in the cortex (Puntillo, 1988).
The perception and reaction to pain varies with each individual. It is now

evident that a host of biochemical substances, including neurotransmitters and
endogenous opioids, can modulate pain by either facilitating or inhibiting the
transmission of pain impulses at various levels of the nervous system. There is
evidence that the pain suppression system is mediated in part by endogenous

opioids along the descending pathway, which relays processed information in
response to the pain stimulus (Figure 1-1). The administration of exogenous
opioids, like morphine and codeine, is thought to enhance this pain suppression
system. However, the exact relationship between the analgesic effect of opioids
and the role of pain modulators is yet to be clearly established.


1.2 Codeine

Codeine (Figure 1-2) is a naturally occurring alkaloid in which the phenolic
hydroxyl group of morphine is replaced by a methoxy group. It was isolated in

1832 by Robiquet from the opium plant, papaver somniferum. Barbier in 1834
was the first to report its analgesic activity in humans (Baselt and Cravey, 1989).











H43CO
3



t3 9





Figure 1-2 The structure of codeine (adapted from Muhtadi and Hassan,
1981).


Codeine is a white crystalline powder which, when made anhydrous,
melts at 154-156 C. The phosphate salt is more soluble than the sulfate salt and
hence it is used more commonly. The free base is sparingly soluble in water, but
freely soluble in alcohol. Codeine is a monoacidic, weak base with a pKa value

of 8.2 (Baselt and Cravey, 1989). It exhibits a characteristic UV absorbance peak
in water at 284.8 nm (Grasselli and Ritchey, 1975). The anhydrous base has a

nominal molecular weight of 300.


1 .2.1 Administration and Dosage

Codeine is usually given orally as a phosphate or sulfate salt for the relief
of cough and mild to moderate pain. The phosphate salt may also be given
parentally for the relief of pain by intramuscular or subcutaneous injection.
As an analgesic, the usual oral dose is 30 to 60 mg every four or six
hours, as needed for the relief of pain. For treatment of cough, the usual adult
dose is 10 to 20 mg every four to six hours, not to exceed 120 mg. As with other












opiate agonists, the smallest effective dose must be given in order to minimize

the development of tolerance and physical dependence.

1.2.2 Pharmacological Actions

Codeine, like morphine, acts by blocking excitatory synaptic transmission
in the central nervous system and relieves pain and anxiety primarily by raising

the pain threshold. It exerts a combination of depressing and stimulating effects

on the central nervous system and various peripheral organs. Important CNS
effects include analgesia, euphoria, sedation and respiratory depression.
Supression of the cough reflex is a well-recognized action of opioids, particularly

codeine. Miosis (constriction of pupils) is another pharmacological action seen
with virtually all opioid agonists. Codeine can also cause activation of the brain
stem chemoreceptor trigger zone to produce nausea and vomiting.

Peripheral effects of codeine include increasing the tone and decreasing

the rhythmic contractions of different types of smooth muscles. In the
gastrointestinal tract this produces constipation, which may be troublesome in
ordinary analgesic therapy, but useful in the treatment of diarrhea (Jaffe and
Martin, 1985). Urethral and biliary tract spasms are usually increased by

codeine, but the analgesia produced may outweigh these undesirable effects. It
can, however, be life threatening in cases of asthma when combined with
repiratory depression.
Other effects of codeine include central vasomotor capacity depression
and dilatation of some vessels, including the coronary arteries. On the whole,

these circulatory effects are small and probably result from a combination of












central actions and peripheral histamine release. Some anticholinergic activity
may be present, but is probably not critical (Way and Adler, 1962).


1.2.3 Toxicity

Codeine shares the toxic potentials of the opiate agonists. The most

common side effects observed after the administration of therapeutic doses of
codeine include dizziness, sedation, nausea, vomiting, sweating and a feeling of

light-headedness. Other adverse effects that can be seen include euphoria,
dysphoria, weakness, headache, insomnia, anorexia, gastrointestinal distress,

bradycardia and even urinary retention.
Toxic effects of opioid overdose produce a classic triad of signs : coma,
respiratory depression and constricted pupils. Breathing becomes shallow and

irregular and may slow to as low as 2-4 breaths per minute. A severe overdose
of codeine can cause respiratory depression, cyanosis, extreme somnolence
which can progress to a coma, and severe hypotension with bradycardia. This
could lead to apnea, circulatory collapse, cardiac failure and finally death.

Codeine toxicity can be treated successfully with an intravenous administration
of the narcotic antagonist naloxone (Cutting, 1972 ; Jaffe and Martin, 1985
McEvoy, 1990).


1.2.4 Therapeutic Uses

Codeine is a mild analgesic indicated for symptomatic relief of moderate
pain. It is considered to be 1/10 to 1/6 as potent as morphine as an analgesic. It
is also used as an antitussive, alone or in combination with other antitussives or












expectorants, in the symptomatic relief of non-productive cough (Cutting, 1972;
Jaffe and Martin, 1985 ; McEvoy, 1990).

1.2.5 Drug Dependence and Tolerance

The major limitation of opioids is that they characteristically produce "drug
habituation" or "addiction". Drug dependence is marked by tolerance-the gradual

deveopment of resistance to the effects of the drug after repeated administration.
Tolerance is manifested by a decline in the effectiveness of a drug, requiring a
gradual increase in the dosage in order to maintain the initial effect.

Psychic dependence is a clinical term to indicate habituation. It is defined

as compulsive use of a drug by an individual who feels euphoric and a sense of
well-being from its chronic use. This kind of dependence is seen to a lesser or

greater extent with numerous agents like caffeine, nicotine, salicylates and
bromides as well as narcotic analgesics.
Physical dependence, on the other hand, deals with the biochemical and
physiological adaptation of tissues to a new chemical environment after repeated
use of a drug. The drug becomes necessary for normal tissue function and its

withdrawal causes an abnormal cellular response referred to as "abstinence
syndrome". This situation is usually characterized by effects opposite to those of

the pharmacological effects of the drug.
The phenomenon of physical dependence can actually be visualized as
being due to the prolonged occupation of the receptor sites within the cells of the
central nervous system by opioid analgesics. This receptor-drug interaction leads
to adaptive changes in the latent cellular excitability. These changes then
manifest themselves during drug abstinence as symptoms of withdrawal. The












intensity of the withdrawal syndrome is proportional to the amount and duration
of drug administration.


1.2.6 Analytical Techniques

Until recently there was little pharmacokinetic data described in the
literature regarding low doses of codeine. This was mainly due to the lack of
analytical techniques of sufficient sensitivity and specificity. Earlier studies relied

on colorimetric assays which were not very sensitive (Woods, Muehlenbeck and
Mellett, 1956). Johannesson and Woods (1964), Yeh and Woods (1969, 1970)
used high doses of radiolabeled codeine and were able to measure codeine and

biotransformed morphine in rat plasma.
As codeine undergoes extensive metabolism, forming active metabolites,
there is a need to develop an assay to precisely determine the extent of
formation of each metabolite and quantify its potential contribution to the overall
analgesia and/or toxicity associated with codeine. The older analytical methods

to determine the levels of codeine and some of its metabolites included
radioimmunoassay (Findlay et al., 1977 ; Gintzler et al., 1976), gas

chromatography (Jain et al., 1977 ; Kogan and Chedchel, 1976) and gas
chromatography-mass spectroscopy (Ebbighausen et al., 1973 ; Cone et al.
1983) techniques.
Although radioimmunoassays offer the sensitivity required for the
detection of these compounds, differentiation between very similar species like
morphine 6-glucuronide and morphine 3-glucuronide cannot be achieved. The
ability to identify and quantify the above metabolites is important, since morphine
6-glucuronide is known to be pharmacologically active. Gas chromatography and












mass spectroscopy can offer both sensitivity and specificity required for
examination of codeine and morphine, but the techniques involve time-
consuming derivatization steps and are not suitable for the glucuronides. Many
researchers have turned their attention to the development of rapid, sensitive

and specific HPLC methods for the detection of opiates.
Numerous HPLC-based methods have been reported with ultraviolet

(Persson et al., 1989), fluorescence (Chen et al., 1989 ; Tsina et al., 1982) and
electrochemical (Harris et al., 1988 ; Svensson 1986 ; Verway-van Wissen et al.,

1991 ; Besner et al., 1989 and Bedford and White, 1985) detection systems.

HPLC with fluorescence detection requires the conversion of some compounds
to fluorescent products before analysis. Electrochemical detection does not allow

simultaneous detection of all the compounds, due to differing redox potentials.
HPLC with ultraviolet detection would therefore seem to be the method of choice
for developing an assay for all of the compounds of interest.

A potential problem with using a reversed phase HPLC system is that the
polar glucuronides elute very close to the solvent front and are prone to being
hidden by co-eluting endogenous substances (Chari et al., 1991). An alternate
method has been described for morphine and its metabolites using a normal
phase system (Wielbo et al., 1993).


1.2.7 Pharmacokinetics in Man

There is an abundance of literature describing the pharmacokinetics of
codeine in man (Quiding et al., 1986 ; Guay et al., 1987 ;Chen et al., 1991 ; Yue
et al., 1989 a, 1990 a, b ; Shah and Mason, 1990 a ; Way and Adler, 1962 ;
Findlay et al., 1977, 1978, 1986 ; Hull et al., 1982 ; Rogers et al., 1982 ; Persson












et al., 1992 ; Vree and Verway-van Wissen, 1992). Despite this extensive
documentation, the relevance of some of the active metabolites of codeine is not
clear. The extent of formation of individual metabolites and their potential
contributions to the analgesic efficacy seen after codeine administration need to

be assessed in detail.

1.2.7.1 Absorption

Codeine is well absorbed following oral and intramuscular administration
in man. Its bioavailability after oral administration was found to be 50-60%
(Rogers et al., 1982). At a dose of 60 mg, a peak plasma concentration (Cmax)

of around 100-200 ng/ml was seen within one to two hours after administration
(Mohammed et al., 1993). Chen et al. (1991) observed that the mean tmax (time

at which the peak plasma concentration is observed) for codeine occurred about
1 hour after oral administration in the case of both single and chronic dosing.
However, the mean peak plasma concentration after chronic dosing was

significantly higher than after single dosing. This was also observed by Quiding
et al. (1986) and can in part explain why some subjects experience a greater
analgesic effect after chronic dosing compared to single dosing.

1.2.7.2 Distribution
After the drug is absorbed into the blood, it is distributed to tissues in the
body. However, only the free drug concentration can equilibrate with these
tissues. The main interaction in blood is between the plasma proteins and the
drug molecules and this is usually a reversible physical process. Thus, the
binding of drugs to plasma proteins is a dynamic process. Codeine has been
shown to be bound to plasma proteins to an extent of 25-30% (Findlay et al.,
1977). Baselt and Cravey (1989) reported a volume of distribution of about












3.5 I/kg for codeine, indicating an extensive distribution in the various tissues of
the body.

1.2.7.3 Metabolism
Codeine is primarily metabolized in the liver, the major site being the
microsomes in the endoplasmic reticulum. Lesser, but significant sites include

the central nervous system, kidney, lung and placenta. Metabolism is
predominantly via conjugation with glucuronic acid at the 6-position (Yue et al.,
1989 a, b, 1990 a, b ; Chen et al., 1991 ; Vree and Verway-van Wissen, 1992).
Other metabolic pathways include 0-demethylation to morphine and N-
demethylation to norcodeine (Sindrup et al., 1990). These primary metabolites of
codeine are further metabolized to their glucuronides as outlined in Figure 1-3.

The hepatic biotransformation of codeine to morphine has led many to
believe that that codeine may exert its analgesic effect through partial conversion

to morphine (Adler and Latham, 1950 ; Findlay et al., 1978 ; Yue et al., 1990 a).
This assumption is supported by the low affinity of codeine to the p. opiate

receptor and by the marked in vivo analgesic efficacy of morphine, morphine-6-
glucuronide and normorphine (Lasagna and Kornfeld, 1958 ; Osborne et al.,
1988). However, studies by Quiding et al. (1986) and Shah and Mason (1990 a)

have questioned the possible role of morphine in codeine analgesia because of
the very low concentrations of morphine seen after both single and repeated

doses of codeine.
Chen et al. (1991) described codeine 6-glucuronide pharmacokinetics in
detail after single and chronic oral codeine administration. There was no
difference in the plasma tmax (1.28 versus 1.13 h) and Cmax (1.43 versus 1.38
pgg/ml) for single and chronic dosing, respectively. The mean AUCs for codeine












and codeine 6-glucuronide at steady state were not significantly different for

either single or chronic dosing. The average ratio of the AUCC6G "AUCCod was

Codeine-6-glucuronide


IGlucuronidation
NCH3




OH
CH3o 0' N
0-demethylationV Codeine


Glucuronidation


M3G, M6G


CH3(


Morphine
N-demethylation


-demethylation
K NH



o


Norcodeine

0-demethylation


Normorphine


Figure 1-3: The various metabolic pathways of codeine.


about 15 for both single and chronic dosing regimens. Vree and Verway-van

Wissen (1992) reported similar values for the various pharmacokinetic
parameters of codeine 6-glucuronide except in the case of tl/2 and AUC ratio.












While they reported t1/2 values for codeine and codeine 6-glucuronide as 1.5
and 2.8 hours respectively, Chen's group found the t1/2 values for both
compounds to be similar, about 3 hours. Vree's group reported a AUCC6G
AUCCod ratio of 10 compared with 15 by Chen's group.
Quiding et al. (1986) reported plasma concentrations of morphine both
after single and multiple doses of 60 mg of codeine to be about 2-3% of that of
codeine. Shah and Mason (1990 a) found that Morphine AUC values ranged
from 2-5% after a 60 mg oral dose of codeine. However, Vree and Verway-van
Wissen (1992) reported that no free morphine could be detected in the plasma of
human volunteers who took 30 mg of codeine orally. Any morphine formed was

immediately glucuronidated at the 3- and 6- positions to form the corrosponding
glucuronides. Both glucuronides were detected in the plasma with the morphine
3-glucuronide concentrations being higher than those of morphine 6-glucuronide.

Very small amounts of normorphine, norcodeine and its glucuronide conjugate,
norcodeine 6-glucuronide, were also detected in the plasma.

1.2.7.4 Elimination
Codeine and its metabolites are excreted almost exclusively by the
kidneys. A very small fraction is eliminated as free codeine (about 5%). The
major portion of the administered dose appearing in the urine consists of
biotransformed products. Urinary recoveries of codeine and its metabolites
indicate that codeine 6-glucuronide is the major metabolite formed from codeine.
There are also trace amounts of morphine and its glucuronides along with
normorphine and its glucuronide conjugate (Chen et al., 1991). The excretion of
codeine and its metabolites in 24 hour urine as a% of the dose of administered
codeine is summarized in Table 1-1. Adler et al. (1955) observed that, after a












single dose of codeine, urinary excretion was almost complete in 24 hours,
although trace amounts of codeine and morphine stayed in the body for several
days before being completely eliminated. A small percentage of the dose (0.02-

0.17%), consisting mostly of free codeine and some metabolites, was also
detected in the feces.



Codeine I Metabolites % Excreted in 24 Hour Urine

Codeine 8-16
Morphine 0.5-1
Codeine 6-glucuronide 48 69
Morphine 6-glucuronide 0.5 2
Morphine 3-glucuronide 5- 8
Norcodeine 2 10


Table 1-1 : The urinary excretion data in humans after a 60 mg oral dose
of codeine. (from Yue et al., 1990 a).

Codeine has an elimination half-life of about 2-3 hours (Findlay et al.,
1977, 1978 ; Quiding et al., 1986 ; Yue et al., 1990 a, b). Chen et al. (1991)
reported that elimination half-lives for both codeine (3.2 versus 2.9 h) and

codeine 6-glucuronide (3.2 vs 3.3 h) after both single and chronic dosing,
respectively, were not significantly different. The renal clearance of codeine is
between 67 and 265 ml/min. The creatinine clearance values of codeine in
healthy volunteers has been reported to be in the range of 90 to 132 ml/min.,
indicating that in addition to glomerular filtration, codeine can undergo active
secretion into the lumen of the proximal tubules (Chen et al., 1991).











1.2.8 Pharmacokinetics in Rats

The physiological disposition of codeine in various experimental animals
after relatively high doses has been extensively studied. Studies done in male
rats have shown that about half the dose (55%) of codeine undergoes 0-
demethylation (Yeh and Woods, 1969). Morphine formed by the metabolic

conversion of codeine has been found in the plasma, urine, bile and feces
(Johannesson and Shou, 1963 ; Johannesson and Woods, 1964 ; Yoshimura et
al., 1970). Morphine has also been determined to be present in the brain of rats

following large doses of codeine (Dahlstrom and Paalzow, 1976).
Traditionally, the major routes of administration in rats have been

subcutaneous (s.c.) and intraperitoneal (i.p.) injections. Numerous
pharmacological experiments with rats to determine analgesic activity of codeine,

morphine (Johannesson and Shou, 1963 ; Yeh and Woods, 1969, 1970 ; Oguri

et al., 1990) and the 3- and 6-glucuronide metabolites (Yoshimura et al., 1973 ;
Shimomura et al., 1971 ; Abbott and Palmour, 1988 ; Sullivan et al., 1989; Gong
et al., 1991 ; Paul et al., 1989 ; Pasternak et al., 1987 ; Smith et al., 1990), have
utilized both s.c. and i.p. routes of administration. There are a few reports in the
literature regarding intravenous administration of codeine and morphine in rats

(Dahlstrom and Paalzow, 1976 ; Shah and Mason, 1991 ; Bhargava and Villar,
1992 ; Thurston et al., 1993). Shah and Mason (1990 b) also administered

codeine orally in rats as well as by i.v. injection and compared the two routes of
administration.

1.2.8.1 Absorption
Shah and Mason (1990 b) described codeine pharmacokinetics after an
oral dose of 5 mg/kg in rats. A solution of codeine phosphate was made by












dissolving it in 2-4 ml of physiological saline. The drug was then carefully
delivered via gastric intubation to the fasting animals. Codeine was rapidly

absorbed after the 5 mg/kg oral dose. The mean peak plasma concentrations

were 101.3 + 42.4 ng/ml around 6.4 + 4.5 min after dosing. After 4 hours, no
codeine could be detected in the whole blood with the HPLC method used in the
study. A large intersubject variation was observed in the absorption of codeine.
This variation may be partly explained by factors such as gastric motility and
intestinal transit time.
The mean bioavailability was calculated from oral AUC, Di.v. / i.v. AUC

* Doral as 0.08 + 0.03, that is., only 8% of the ingested dose of codeine reaches

the systemic circulation. Absorption of opiates in general is thought to occur by
passive diffusion rather than by processes invoving energy expenditure
(Christensen et al., 1984). Incomplete absorption was not considered to be a
major factor as the amounts of free codeine found in the feces were negligible

following oral administration.

1.2.8.2 Distribution

Miller and Elliot (1955) conducted several distribution experiments in the
rat. They showed that codeine was well distributed, and capable of leaving the
the blood and concentrating in parenchymatous tissues such as liver, kidney,
lung, adrenal glands and brain. This is reflected in the large volume of

distribution of the terminal portion (Vd area = 5.1 I/kg), obtained from the
concentration-time profile after intravenous codeine administration.

Codeine given i.v. appears to fit a two compartment body model with a
rapid distribution and a short terminal elimination phase. This indicates that

codeine is rapidly metabolized and /or excreted. Elimination rate from the central












compartment (0.0569 min-1) is more than the terminal rate constant (P), due to
distribution of codeine into the peripheral compartment. The mean ratio of 0.92

for K12 / K21 (ratio of the inetrcompartmental rate constants) shows
approximately an equivalent distribution of codeine between the central and

peripheral compartments. Linear pharmacokinetics was exhibited by codeine at

i.v. doses of 1-4 mg/kg (Shah and Mason, 1990 b).

1.2.8.3 Metabolism
There are a few studies in the literature that examine the metabolism of

codeine in rats. These were done mostly in the 1960s and were conducted with

radioactive codeine. Plasma concentrations after a 2 mg/kg s.c. injection of
codeine showed the presence of free codeine, free morphine and conjugated
morphine (in the ratio of 10 : 3 : 1). No conjugated codeine was seen either in

the plasma or brain (Yeh and Woods, 1969). The conjugated morphine was later
characterized as morphine 3-glucuronide. It was reaffirmed that morphine 6-

glucuronide and codeine 6-glucuronide were not formed in rats (Yeh and Woods,

1970). However, using thin layer chromatography, a Japanese group
(Yoshimura, 1970) detected traces of codeine 6-glucuronide (0.2%) in the urine

of rats, showing that a small amount of this metabolite can be formed in rats.
This has been supported by Oguri et al. (1990) who used a specific HPLC
method and found the urinary recovery of this compound to be about 1% (Table
1-2). Recent findings (Oguri et al., 1990 ; Lawrence et al., 1992) have shown that
morphine 6-glucuronide could not be detected in plasma or urine of rats after
codeine administration.











1.2.8.4 Elimination

In vivo codeine disposition studies using radiolabeled carbon-14 have
shown that about 74% of the injected radioactivity is excreted as free codeine,
free morphine and morphine conjugate via the pulmonary, biliary, intestinal and

urinary routes in male rats (Yeh and Woods, 1969). Of this 74%, 20-40% is

eliminated in the expired air as C02 via the pulmonary pathway.



Codeine I Metabolites % Excreted in 24 Hour Urine

Codeine 1-2
Morphine 4-5
Codeine 6-glucuronide 0.1 0.3
Morphine 6-glucuronide Not detected
Morphine 3-glucuronide 23 24
Norcodeine Not detected


Table 1-2: Urinary excretion data as a% of a dose of codeine (from
Oguri et al., 1990).


A significant part of an i.v. dose of codeine has been shown to undergo
enterohepatic recirculation to the extent of 10-30% (Walsh and Levine, 1975). It
is known that codeine can decrease gastrointestinal tract motility, thereby
increasing transit time of morphine glucuronide in the intestinal tract. This would
then allow longer exposure to bacterial glucuronide hydrolysis which, in turn,
would favor enterohepatic recycling. Yeh and Woods (1969), using radioactively-
labeled tracers, reported the following amounts recovered from intact bile : free
codeine (1.3%), free morphine (0.9%) and conjugated morphine (43.1%).












Oguri et al. (1990) saw a substantial interspecies difference in the

metabolism of codeine (Table 1-3). This can influence considerably the nature
and duration of the pharmacological and toxicological activities of codeine. The
development of molecular aspects of gene evolution has been applied
extensively to explain species differences seen in drug metabolism (Nebert and
Gonzalez, 1987).


Codeine/ Metabolites Mouse Rat Guinea Pig Rabbit
(n=25) (n=4) (n=4) (n=3)
Codeine 6.8 + 0.7 1.6 + 0.2 1.6 + 0.2 2.2 + 0.5
Morphine 0.8 + 0.2 4.3 + 0.4 0.2 + 0.1 1.3 + 0.3
Codeine 6-glucuronide 1.6 + 0.2 0.2 + 0.1 39.8 + 3.9 24.5 + 3.7
Morphine 6-glucuronide nd nd 0.7 + 0.1 1.9 + 0.3
Morphine 3-glucuronide 7.6 + 1.0 23.9 + 2.8 1.6 + 0.2 17.9 + 1.4
Norcodeine 9.0 + 0.8 nd nd nd

nd = not detected

Table 1-3: Urinary excretion data (% of dose) 24 hours after codeine
administration in various species (from Oguri et al., 1990).

1.3 Drug Glucuronidation


A major pathway for drug metabolism and excretion is the generation of
water soluble glucuronide metabolites. Since many drugs exhibit structural

features that allow conjugation without previous phase I reactions,
glucuronidations are viewed as first line detoxification mechanisms. Most












glucuronides of drugs are considered to be inactive and rapidly eliminated.
Therefore, glucuronide metabolites of drugs are often neglected in
pharmacodynamic and pharmacokinetic studies and are not taken into account
when evaluating drug effects.
The pharmacological and toxicological relevance of glucuronidation was
pioneered by Dutton and is summarized in a comprehensive review by Kroemer
and Klotz (1992). The general reaction scheme which describes the conjugation
of glucuronic acid to various drugs in the presense of glucuronosyl transferase

enzymes is shown in Figure 1-4. This reaction mediates the formation of ether,
ester, thiolic, N- and C- glucuronides.

Uridine diphosphate glucuronosyltransferases (UGTs) are enzymes
located in the endoplasmic reticulum and therefore form a part of the microsomal
fraction. UGTs are generally 50 to 60 kD in size and span the entire membrane

of the endoplasmic reticulum. There is a small C-terminal domain located in the
cytoplasm with the active site directed toward the lumen of the endoplasmic
reticulum.


1.3.2 Direct Pharmacological Activity

The best documented example of glucuronide activity is that of morphine.
Morphine is conjugated to give morphine 3-glucuronide and morphine 6-
glucuronide in the liver (Wahlstrom et al., 1988 ; Coughtrie et al., 1989).

Shimomura et al. (1971) observed that morphine 6-glucuronide had a direct
analgesic effect in the hot plate test. Subsequently, the same group
demonstrated that both morphine 6-glucuronide and morphine 3-glucuronide can
penetrate the blood brain barrier (Yoshimura et al., 1973). Using the tail flick














H COON
S H UOP glucuronosy S
transferase
OO/H
OH./ +UOP
HH
No OOH UO ---
ON
GA
OH OH O



Figure 1-4: Glucuronidation of a substrate (H-S) by reaction with
uridine diphosphate-glucuronic acid (UDP-GA) in the presence of UDP-
glucuronosyl transferase enzymes (from Kroemer and Klotz, 1992).


latency tests in rats, they found that morphine 6-glucuronide was 20-fold more

potent than morphine after direct microinjection into the periaquaductal gray area

of the brain. Morphine 3-glucuronide in this experimental design did not produce

any effect.
The observations of Pasternak have been confirmed by a number of

investigators (Paul et al., 1989), who performed detailed characterization of
morphine 6-glucuronide. After peripheral administration to rats the analgesic
effect was twice that of morphine itself. After intrathecal administration, morphine

6-glucuronide was reported to be 650 times more potent than the parent

compound. Smith et al. (1990) showed that morphine 3-glucuronide had no
analgesic activity but could act as a potent antagonist of morphine and morphine
6-glucuronide induced analgesia in rats. In this context Woolf (1981) reported

that morphine 3-glucuronide was capable of inducing hyperalgesia in rats.












Recent investigations of force field and quatum mechanical characterization of
morphine 3-glucuronide and morphine 6-glucuronide reveal an unexpectedly
high degree of lipophilicity (Carrupt et al., 1991).
The question which then arises is whether this detailed pharmacological

evidence for the contribution of glucuronides to the net drug effects of morphine
is matched by clinical observations. Joel et al. (1985) speculated that morphine
6-glucuronide may contribute to the clinical efficacy of morphine. Hanks et al.

(1987) tried to explain the potency of repeated oral doses of morphine as due to
accumulation of morphine 6-glucuronide. Direct clinical evidence for the

analgesic action of morphine 6-glucuronide was obtained by Osborne et al.
(1988), who injected morphine 6-glucuronide, 1.0 mg/kg to 5 patients and 0.5
mg/kg to 1 patient. Five patients reported total pain relief within 30 minutes and

the analgesia lasted for 1-7 hours. Pain and pain relief were monitored by visual
analog scales (Figure 1-5).


Patient A Patient B
Pharmacokinetic Parameters (Normal Renal (Impaired Renal
Function) Function)
Clearance (I/kg) ** 89 26
Volume of Distribution (I) 14.7 16.4
Elimination Half-life (h) 1.9 7.4
Area Under the Curve (nmol I'h) ** 370 1319


** indicates that the parameters were significantly different.


Table 1-4: Pharmacokinetic parameters of two patients after a 1 mg/kg
intravenous dose of morphine 6-glucuronide (from Osborne et al., 1988).










































2 3 A
TIME (hours)


5 6 7 8


Figure 1-5 : A visual analog scale used for monitoring the extent of pain

relief (from Osborne et al., 1988).


patient B


TIME (hours)


Figure 1-6: The pharmacokinetic profile of two patients after 1 mg/kg i.v.
administration of morphine 6-glucuronide (from Osborne et al., 1988).


100 T


80


460



40



20


250



200


0 5



100
LU

o
0


1














Pharmacokinetic indices for two patients are shown in Table 1-4. Patient
A had normal renal function while patient B had chronic renal impairment. The

elimination of morphine 6-glucuronide was closely related to renal function. No

morphine or morphine 3-glucuronide levels were detected in the plasma at any

time. Plasma morphine 6-glucuronide levels for the two patients are shown in

Figure 1-6.
Hannah et al. (1990) investigated the analgesic efficacy of intrathecal

morphine 6-glucuronide in comparison with morphine in 3 patients with chronic

cancer pain. The doses required for controlled analgesia were 393 + 227 mg/24

h and and 227 + 114 mg/24 h for morphine and morphine 6-glucuronide

administration, respectively.

Conjugation of drugs by glucuronosyl transferases plays an important role
in the overall picture of drug disposition. The resulting glucuronides represent

metabolites that are not always inactive and may in fact contribute to drug action

either directly (by producing analgesia as in the case of morphine 6-glucuronide)

or indirectly (by release of the parent compound via hydrolysis as in

enterohepatic recycling). Moreover, some glucuronic acid conjugates are not
rapidly excreted. Their disposition can be modulated at different levels of

distribution, metabolism and excretion, thereby modifying net drug action.

Therefore, glucuronic acid conjugates should be taken into account when
pharmacokinetic and pharmacodynamic characterization of drugs are

determined.











1.4 Evaluation of Analgesia in Small Animals


1.4.1 Introduction

Eddy (1928, 1932) is credited as being the first to describe methods for
determining analgesia in animal experiments by exerting a variable pressure on
the distal part of the cat tail. Friend and Harris (1948) used a pair of forceps,

whereby pressure could be exerted on the tail of the rat. Green et al. (1951)
produced pain by exerting pressure on the tip of the tail using a syringe piston

system.
Macht and Macht (1940) were the first to describe a method in which
electrical stimulation was used to produce pain in rats. They implanted two

electrodes in the skin of the scrotum. The rats reacted with a squeak response
when the voltage was increased over a certain threshold. Luckner and Magun
(1951) implanted two electrodes in the upper part of the tail. Collins et al. (1964)
implanted an electrode in the rectum and another in the upper part of the tail in

rats.
Eddy et al. (1950) placed mice in a cylindrical glass container, the base of
which was a copper plate. This plate was maintained at a temperature of 50-55
oC by a hot water bath placed below the copper plate. The mice responded to

the heat by licking their forepaws and trying to jump out of the cylinder. This

technique is called Eddy's hot plate method and is a good pain model for the rat
and mouse. D'Amour and Smith (1941) irradiated the tail tip with heat from a light
source of defined strength. This tail flick method is also widely used for
determinating analgesia in small animals.












A modification of the tail flick method was described by Berglund and
Simpkins (1988). It involves measurement of the withdrawal time of the tail when
a beam of light was focused on it. This was done both before drug

administration, and at regular intervals thereafter. The former measurement was
called baseline latency and the latter test latency. The instrument used was a
model 33 tail flick analgesia meter (litc Inc., Landing, NJ, USA). and consisted of

an incandescent light bulb with the beam intensity and sensitivity dial set at 75
and 8, respectively. The time between presentation of a focused beam of light
and removal of the tail was recorded as the latency period. However, in the

absence of any response, a cut-off period of 40 seconds was used to prevent
tissue damage. This was considered as the maximal suppression of pain.


1.4.2 Time Course of Analgesic Effect

D'Amour and Smith (1941) were the first to determine the analgesic effect

of morphine and codeine administered by i.p. injection. They found the effect to
be maximal at 30 minutes post-injection for both drugs. Ercoli and Lewis (1945)
injected equianalgesic doses of morphine and codeine, both intraperitoneally
and subcutaneously. They found that both drugs had their greatest effect 30-60

minutes after administration, with the effect persisting for 60-120 minutes.
Following higher doses, the degree of analgesia was found to be more complete.
Miller and Elliott (1955) were the first researchers to make a serious
attempt to look into changes in the amounts of codeine and morphine in the
brain with time. These investigators administered 25 mg/kg codeine-N-14CH3 by
subcutaneous injection to rats. These rats were killed after 15, 30, 60 and 150

minutes respectively. The spinal cord and the brain were removed, and the












concentrations of these drugs were determined in the spinal cord, hypothalamus,
cerebellum, medulla oblongata, mid-brain and parts of the hemispheres.
Miller and Elliott (1955) reported that concentrations of codeine rose
sharply from the 15th to the 30th minute, while a slower rise was seen from the

30th to the 60th minute. The highest concentration of codeine was measured 60
minutes after drug administration. Another important fact to be considered is that
relatively small amounts of both codeine and morphine enter the brain, and it is

these concentrations which are responsible for producing analgesia. These
results indicate a relationship between drug concentrations of codeine and

morphine in the brain and the degree of analgesia produced. The exact
relationship is yet to be firmly established.


1.5 Genetic Polymorphism

Genetic variation is an important cause of the large differences seen in
drug metabolism between individuals. A number of isoenzymes in the

cytochrome P450 family are involved in the oxidative metabolism of several
essential drugs (Nebert et al., 1987). The entire population can be divided into
extensive and poor hydroxylators based on the extent to which they metabolize
certain drugs like mephenytoin, debrisoquine, sparteine and procainamide. It is

well known that O-demethylation of codeine, leading to the formation of the
analgesically active metabolites morphine and morphine-6-glucuronide, is
catalyzed by cytochrome P450 1ID6 isoenzyme (Nebert et al., 1989) and
cosegregates with the debrisoquine/sparteine oxidative polymorphism.
Five to ten percent of caucasians are classified as "poor metabolizers"
(PMs), since they lack the specific isoenzyme for O-demethylation (Yue et al.,












1990 a, b), while the remainder, capable of 0-demethylating codeine, are termed
"extensive metabolizers" (EMs). PMs are rare in the native Chinese population

compared to Caucasians (Yue et al., 1989 a) indicating inter-ethnic differences in
drug metabolism. This pharmacogenetic bias translates clinically into the fact

that the PMs may not derive as much analgesic effect as the EMs. There are no
data in the literature regarding genetic factors influencing the N-demethylation of

codeine.
Glucuronidation reactions are catalyzed by a group of isoenzymes with
overlapping specificities. Glucuronidation of codeine has been shown to be
induced by smoking and oral contraceptives (Yue et al., 1990 c). It is also

affected by the co-administration of other drugs like diazepam and

chloramphenicol.


1.6 Immunomodulation

The immune process consists of a number of concerted events which
include recognition and processing of foreign antigens, proliferation and

differentiation of responder cells, and the production of proteins and peptides for
the amplification and mediation of the immune response. In this regard, the
immune system is not isolated and autonomous in nature. In fact it is a part of an
interactive and communicative triad, that also includes the nervous and

endocrine systems.
The immune system can affect brain functions as evidenced by the
release of neurotransmitters and enhanced brain activity in certain localized
regions after activation of the immune system following bacterial infection
(Saphier, 1987). A number of centrally acting pharmacological agents have been












shown to affect immune function directly or indirectly via the hypothalamo-

pituitary-adrenal (HPA) axis. Within this axis, feedback loops have been
identified which, when activated by certain immune products such as cytokines,

are responsible for the production of glucucorticoids, leading to marked
immunomodulatory effects. These effects are enhanced in stressful situations
(Herz, 1993). This is further indicative of the relationship between the brain and
the immune system, either by neuronal pathways or by modulation of endocrine

system
Drugs of abuse, particularly opioids, can produce deleterious effects on

the immune system. From an immunological standpoint, it is necessary to
determine whether the effects of drug abuse were due to the drugs, or the

consequence of needle sharing and poor nutrition--both of which are frequently
observed factors associated with drug addiction. Earlier epidemiological findings

suggested that increased infections were caused by sharing of unsterilized and
contaminated needles. However, subsequent clinical studies have focused on

the increasing evidence that the opioids themselves are capable of affecting the
host defense mechanisms by directly acting on the immune system.


1.6.1 Opioids Receptors

The concept of opioids as immunomodulators is based on studies
showing the presence of classical opioid receptors on the surface of cells of the
immune system. Ovadia et al. (1989) demonstrated that rat lymphocytic
membranes possess a certain GTP binding protein that couples opioid receptors

to adenylate cyclase in response to the action of an opioid on the lymphocytes.
Wybran et al. (1979) reported an opioid involvement in immune function based












on their observations with human T lymphocytes. They indicated that the opioid-
induced suppression of lymphocytes was reversible with naloxone.
Carr et al. (1994) found that naltrexone antagonized both the analgesic
and immunosuppressive effects of mice, suggesting the involvement of the same
receptor for both actions, that is, the p. receptor. This inference is supported by

the findings of Ward et al. (1984) which showed that a ji-selective antagonist P-
funaltrexamine, and not the 8-selective antagonist naltrindole (Portoghese et al.,
1988) or the K-selective antagonist nor-binaltorphimine (Portoghese et al., 1987),

attenuated morphine-induced immune suppression.


1.6.2 Effects on Lymphocytes

Lymphocytes are the primary immunocytes responsible for cell-mediated
(T lymphocytes) as well as humoral (B lymphocytes) immunity as seen in Figure
1-7. Measurement of the effect of various drugs on the proliferative capacity of
lymphocytes is the most common in vitro assessment of functional cell-mediated
responses. A large number of lymphocyte markers are suppressed following

acute and/or chronic administration of opioids (Herz, 1993).
Naloxone-reversible reduction in the number of circulating lymphocytes in
morphine-treated rabbits has been reported along with effects on the various
subpopulations of lymphocytes (Herz, 1993). Arora et al. (1990) found an
increase in the T helper to T suppressor ratio following morphine treatment. T
lymphocyte rosette formation was one of the first functional measures of T cell
function to be studied for comparing the immunosuppressive potential of various
drugs. Wybran et al. (1979) showed that the suppressive effects of opioid
agonists, in particular morphine, on the T cell rosette formation was












stereospecific for levorotatory forms, naloxone reversible, and produced
tolerance.
Humoral immunity, on the other hand, involves antibody responses to
drugs which are recognized as antigens by the immune system. Primary
antibody responses were seen to be suppressed by the action of opioids in
sheep erythrocytes (Lefkowitz and Chiang, 1975). Plaque-forming responses,
which are indicative of antibody production, were suppressed in splenocytes
obtained from mice implanted with morphine pellets (Bryant et al., 1990).


1.6.3 Effects on Myeloid Cells

Cells of myeloid origin include monocytes, macrophages, neutrophils,
mast cells, basophils and eosinophils. In addition to serving as mediators of
inflammatory responses, they are involved in numerous functions critical to early
immune response. These functions include antigen presentation, antibody
production, lysis of tumor cells, phagocytosis of foreign particles and the release
of immune response mediators such as interferons, cytokines, transforming

growth factor (TGF) and tumor necrosis factor (TNF).
In humans, morphine exposure leads to a depression of the phagocytic
properties associated with myeloid cells (Tubaro et al., 1985). Macrophages,
which are activated by the stimulatory agent y-interferon, were inhibited in

morphine-pelleted mice and the tumoricidal activity of the macrophages was
seen to be completely abolished (Bryant et al., 1988 a). Chronic morphine
administration in mice also inhibited macrophage colony formation as the result
of a decreased expression of the macrophage stimulating factor (Herz, 1993).














H .mat.polet st1 ee



Myelotd progenitor Lymphold progenitor


Neutrophll
* Phagocylosis
* Inflammation


Eoainophll
* Antiparasitic
SInflammatlion
" Phagocytosis


Baophil Mast Cell
* Inflammation
* Allergic response


Natural Killer Cell
* Tumor surveillance
t Antigen Independent
cytotoxicity


T Lymphocytes
* Cell mediated
cytlotoxlcity
* Lymphokline production


a Lymphocytes
'Antibody Productlog, O
CO


:Antigen presentation
Inflammation
Phagocytosis
Monokine production


Figure 1-7: The Classification and function of lymphoid cells (adapted from Herz, 1993).


Monocyle











1.6.4 Effects on Natural Killer Cells

Natural killer cells represent another type of immune cell and constitute
only about 5% of the total leukocyte population. They are large cells possessing
an intrinsic activity for non-specific killing and lysis of a variety of tumor cells. In
addition to serving as scavengers of malignant cells, natural killer cells also play

a regulatory role in antibody production.
Shavit et al. (1984) demonstrated that daily doses of subcutaneous
morphine (50 mg/kg) suppressed natural killer cell tumoricidal activity (NK
activity), and that naltrexone administration abolished this effect. This is strong

evidence that the natural killer cytotoxic effect is centrally-mediated. Shavit et al.
(1986 a) injected small quantities of morphine directly into the lateral ventricles
and found that the NK activity was markedly suppressed. Novick et al. (1989)
reported a marked decrease in NK activity in heroin addicts on methadone

maintenance therapy.
NK activity is usually below normal basal levels in HIV patients and
declines as the disease progresses. Endogenous opiate peptides, leucine-
enkephalin and methionine-enkephalin have been reported to increase the

cytolytic capacity of the natural killer cells (Wybran et al., 1987 ; Oleson and

Johnson, 1989). This indicates that such candidates can potentiate the NK cell
responses and restore immunocompetence in the case of pre-AIDS and AIDS

patients.

1.6.5 Mechanism of Action

Opioids have been shown to down-regulate immune responses. The
exact mechanism by which this immunosuppression occurs has yet to be












established. A direct mechanism of action is thought to be through lymphocyte
opioid receptors (Figure 1-8). The second hypothesis is that the immune effects
are indirectly mediated, either by the activation of the hypothalamic-adrenal-

pituitary (HPA) axis with subsequent increase in the production of adrenal

corticosteriods or by the release of catecholamines as a result of sympathetic

innervation.
It has been observed that systemic administration of morphine
suppresses the activity of NK cells in the rat (Shavit et al., 1984, 1986 a, b). The

same group also reported that central administration of morphine produced the
similar results, but required doses one third of those administered systemically.
The NK suppression was blocked by naltrexone. N-Methyl morphine, a morphine

analog which cannot cross the blood brain barrier, had no effect on the NK
cytotoxicity when administered systemically. This is further evidence that the
immunosuppressive effects of morphine are mediated by opioid receptors in the

brain.
Morphine and other opioids are known activators of the HPA axis, and
induce glucocorticoid output. Corticosterone has been implicated as possessing

potential immunosuppressive effects as it was able to dose-dependently
suppress NK activity in vitro. This effect was also observed in vivo in mice
implanted with morphine pellets (Freier and Fuchs, 1994). A glucocorticoid
receptor antagonist, RU 38486, blocked morphine-induced suppression of NK
activity in a dose-dependent fashion. Naltrexone administration antagonized the
morphine-induced elevation in serum corticosterone. This suggests that
suppression of NK activity is linked to glucocorticoid elevation, which is the result


















MORPHINE

0 IMMUNE SYSTEM: bone
marrow, lymph nodes, thymus,
spleen, somatic tissues, vascular
space


Figure 1-8: The mechanisms of opioid-induced immunosuppression (from
Peterson et al., 1990).

studies have shown that exposure of immune cells to opiates, especially
morphine, results in a variety of functional disturbances (Chao et al., 1992, 1993;
Peterson et al., 1991,1993).
There are many reports in the literature implicating opiates as
immunomodulatory agents. Most of these studies have focused on morphine, the

narcotic of choice in case of severe pain associated with trauma and cancer, and
have shown that morphine possesses potent immunosuppressant activity. There
are, however, no studies that investigate the immunosuppressive effects of
codeine or any of the glucuronide metabolites of codeine and morphine.

A technique that simulates cellular immune response in vitro is the mixed
lymphocyte reaction (MLR). It is based on the observation that lymphocytes from
a mixture of genetically different individuals with different HLA (Human
Leukocyte Antigen) types react with each other and proliferate. This test is
performed by preventing the response of one set of lymphocytes (donor) through












radiation which then allows only the other set of lymphocytes (recipient) to
proliferate as shown in Figure 1-9.


One way Q
Irradion
mitonycin C


~123456
Days




Figure 1-9: A mixed lymphocyte reaction with cell proliferation.


Thus, opioids possess receptors which are capable of modifying immune
functions. The concentrations achieved with analgesic doses of opioids are
similar to those reported in the in vitro immunomodulatory experiments. It is also

apparent that stress-induced activation of the endogenous opioid networks can
contribute to various immunological changes. The focus for future research must
include an understanding of the role of opioids in regulating immunity and their
interaction with other immune function mediators.


1.7 Receptor Binding

In the case of morphinans, the aromatic ring and the basic nitrogen atom
are necessary for analgesic activity. Substitutions at the phenolic hydroxyl group
(position 3) and the alcoholic hydroxyl group (position 6) have been shown to
cause pharmacological profiles which are opposite in nature. While additions at












the 6-position were seen to enhance opioid receptor binding affinities, changes
at the 3-position significantly decreased receptor binding (Labella et al., 1979).
The structure activity relationships of morphine and its 3- and 6-glucuronide
metabolites have been evaluated in several studies (Yaksh et al., 1986 ; Gong

et al., 1991 ; Shimomura et al., 1971 ; Pasternak et al., 1987).
On the basis of these results, investigators speculated that the inactivity of
morphine 3-glucuronide compared with morphine 6-glucuronide could be due to
the differences in the level of receptor binding. Christensen and Jorgensen

(1987) showed that morphine 6-glucuronide, but not morphine 3-glucuronide,
had a high affinity for the opiate receptors isolated from bovine brains in

competition with 3H-naloxone. Subsequent investigation by Pasternak et al.
(1987) identified that morphine 6-glucuronide interacts with P- but not K,-

receptors.
The greater potency of morphine 6-glucuronide compared to morphine in
antinociception studies has been reported by various investigators (Abbott and
Palmour, 1988 ; Sullivan et al., 1989 ; Paul et al., 1989). In contrast to morphine

and morphine 6-glucuronide analgesia, morphine 3-glucuronide produces
hyperalgesia, respiratory stimulation and behavioral excitation by non-opioid

mechanisms (Yaksh et al., 1986 ; Pelligrino et al., 1989). These studies suggest
that morphine 3-glucuronide can actually antagonize the effects of both
morphine and morphine 6-glucuronide. This is clinically important not only for the
analgesic effect, but also for the respiratory depression associated with morphine
administration.











1.8 Hypotheses

The hypotheses of this project are based on the overall aim of the project,

that is, to examine and compare the analgesic and immunomodulatory effects of

codeine and codeine 6-glucuronide.
1. Codeine 6-glucuronide, like morphine 6-glucuronide, possesses

analgesic activity.
2. The glucuronide metabolites of codeine and morphine are less

immunosuppressive than their parent compounds.


1.9 Specific Objectives

1. Develop and validate a reliable and sensitive HPLC-UV based assay

for the quantitation of codeine, morphine, codeine 6-glucuronide,
morphine 6-glucuronide and morphine 3-glucuronide in biological

samples.
2. Chemically synthesize codeine 6-glucuronide utilizing a modification

of the Koenigs-Knorr reaction.
3. Assess the immunomodulatory effects of codeine, morphine and their
6-glucuronide metabolites in human T lymphocytes (in vitro).
4. Compare the analgesic potencies of codeine and codeine 6-glucuronide
in the rat using the tail flick method after intracerebroventricular (i.c.v.),
subcutaneous (s.q.) and intravenous (i.v.) routes of administration.
5. Determine the p opioid receptor binding affinities of codeine and

codeine 6-glucuronide.
6. Analyze plasma and brain concentrations of codeine and their






40





metabolites at the peak analgesic response time after administration of

codeine and codeine 6-glucuronide by various routes as described in

specific objective #3.











CHAPTER 2
METHODS

2.1 Specific Objective #1:
Analytical Method

An isocratic HPLC method was developed using a ultraviolet absorbance
detector along with an efficient solid phase extraction method to analyze
physiological concentrations of codeine, codeine 6-glucuronide, morphine,
morphine 6-glucuronide and morphine 3-glucuronide in various biological

samples, that is, human urine, rat plasma and rat brain. The method was first
developed using blank human urine. It was then validated using rat plasma and

brain samples.


2.1.1 Materials

HPLC grade methanol and acetonitrile were used (Fischer Scientific,
Fairlawn, NJ, USA). Analytical grade potassium dihydrogen phosphate and 85%
v/v o-phosphoric acid were supplied by Sigma (St. Louis, MO, USA) as were

codeine, morphine, morphine 6-glucuronide and morphine 3-glucuronide. A
sample of codeine 6-glucuronide was donated by the National Institute on Drug

Abuse (NIDA, Rockville, MD, USA).

2.1.2 Extraction Procedure


2.1.2.1 Human urine
Solid phase extraction was performed with 3 ml Clean Screen@ columns
(Worldwide Monitoring, Horsham, CA, USA) containing 40 micron bonded silica












particles. The cartridges were placed on a 12 station Vac-Elut (Varian, Harbor
City, CA, USA). The columns were conditioned with methanol (3 ml), distilled
water (3 ml) and 0.025 M phosphate buffer pH 3 (1 ml). A 1 ml sample of urine

mixed with 2 ml of phosphate buffer pH 3 was then loaded onto the column. The
cartridges were air-dried for 30 seconds and then washed with 0.025 M
phosphate buffer pH 3 (1 ml), followed by methanol (1 ml). The columns were
air-dried again for 30 seconds before eluting the compounds with 3 ml of 5%
freshly prepared ammoniacal methanol solution. The eluent was evaporated to

dryness under a stream of nitrogen gas and the residue was reconstituted in 150
j1 of the mobile phase and 50 jil was injected into the HPLC system.

2.1.2.2 Rat plasma
Extractions were performed with 10 ml Clean Screen columns. The
columns were conditioned with methanol (10 ml), distilled water (10 ml) and
0.025 M phosphate buffer pH 3 (2 ml). A 200 pL sample of rat plasma was mixed
with 400 [tL of 0.025 M phosphate buffer pH 3 and loaded onto the column. The

cartridges were air-dried for 30 seconds and then washed with 0.05 M acetate
buffer pH 4.5 (2 ml), followed by methanol (2 ml). The columns were air-dried for
30 seconds before eluting the compounds with 3 ml of 10% freshly prepared
ammoniacal methanol solution. The eluent was evaporated to dryness under a
stream of nitrogen gas. The residue was reconstituted in 150 gl of the mobile
phase and 50 g1 was injected into the HPLC system.

2.1.2.3 Rat brain
Brain samples were accurately weighed and an aliquot of 0.025 M
phosphate buffer pH 3 (1 ml) was added to each sample. The samples were then
homogenized and transferred to borosilicate tubes. After addition of a further 2












ml 0.025 M phosphate buffer pH 3, the samples were placed in a shaker for 10
minutes. The samples were then centrifuged at 4000 rpm for 20 minutes. The

supernatant was removed and loaded onto 10 ml Clean Screen columns. The
columns were conditioned with methanol (10 ml), distilled water (10 ml) and
0.025 M phosphate buffer pH 3 (2 ml). After the samples were loaded, the

cartridges were air-dried for 30 seconds and washed with 0.01 M acetate buffer
pH 4.5 (2 ml) followed by methanol (2 ml). The columns were air-dried for 30
seconds before eluting the compounds with 3 ml of 10% freshly prepared
ammoniacal methanol solution. The eluent was evaporated to dryness under a
stream of nitrogen gas. The residue was reconstituted in 150 P.1 of the mobile
phase and 50 p.1 was injected into the HPLC system.


2.1.3 Chromatographic Conditions


2.1.3.1 HPLC system 1
This system was used to examine human urine samples for the presence
of opiates. It consisted of a Waters 501 multi-solvent pump set at a flow rate of
0.9 ml/min. The mobile phase consisted of 82% acetonitrile and 18% phosphate
buffer (0.05 M potassium dihydrogen phosphate adjusted to a final pH of 3 with

85% v/v o-phosphoric acid). Separation of the compounds was achieved on a 20
cm x 4.5 mm I.D. Accubond diol column with a 5 micron particle size (J & W
Scientific Inc., Folsam, CA, USA). A Spectra-Physics Focus multiwavelength
forward optical scanning detector (San Jose, CA) set at 220, 230 and 280 nm
was used to detect eluting compounds. Data acquisition and analysis was

performed with Autolab software loaded on a 386 IBM computer.












Apart from chromatographic analysis, the Autolab software provided the
option to examine the UV spectra of the compounds in the chromatograms. All of
the compounds of interest exhibited a maxima in their UV spectra around 285

nm. A specific opiate of interest could be further characterized using derivative
spectroscopy. In derivative spectroscopy, the first or higher derivative of

absorbance with respect to wavelength is recorded versus wavelength. In this

way the ability to detect and measure minor spectral features is considerably

enhanced (Willard, 1986).

2.1.3.2 HPLC system 2
This system was used to determine concentrations of codeine, morphine
and their glucuronides in rat plasma and brain samples. It consisted of a Waters
501 multi-solvent pump set at a flow rate of 0.7 ml/min. The mobile phase
consisted of 88% acetonitrile and 12% phosphate buffer (0.05 M potassium
dihydrogen phosphate adjusted to a final pH of 3 with 85% v/v o-phosphoric
acid). Separation of the compounds was achieved on a 20 cm x 4.5 mm I.D.
Accubond diol column with a 5 micron particle size (J & W Scientific Inc.,

Folsam, CA, USA). Sample injection was automated by the use of a Waters'M
717 Plus autosampler. A Waters 486 tunable absorbance detector set at 220 nm
was used to detect eluting compounds. A Millenium 2010 Chromatography
Manager software was used to acquire data. All the instruments were controlled

by a 386 NEC Powermate computer, which also stored and processed the
acquired chromatographic data.











2.2 Specific Objective #2:
Synthesis of Codeine 6-glucuronide

Codeine 6-glucuronide is not available commercially. In order to evaluate
its pharmacological activity in the rat, relatively large amounts of this compound

(100-200 mg) were required. The National Institute of Drug Abuse (NIDA) is the

only agency which provides samples of this compound, basically for analytical

purposes (5 mg). For this reason, it was decided to synthesize codeine 6-
glucuronide using a reproducible method described in the literature (Yoshimura

et al., 1968). The initial attempt to synthesize codeine 6-glucuronide with some
modifications of the Koenigs-Knorr reaction provided a good yield of the product,
comparable to that reported in literature. A scheme of the synthetic route (Figure

2-1) is shown below.


2.2.1 Reaction Step I

In the first step, codeine monohydrate (1 g) was dissolved in 200 ml of dry
benzene in a three-necked flask attached to a condenser with a drying tube

containing drierite (CaSO4) and a Dean-Starke trap. The trap was used to
periodically distill benzene and to maintain anhydrous conditions in the flask. The
solution was heated at 150 0C with an oil bath. Small amounts (250 mg) of
freshly prepared silver carbonate along with equal portions of a solution

containing 5 g of the acetobromo derivative of glucuronic acid, that is, methyl
2,3,4-tri-0-acetyl-l-bromo-l-deoxy-D-glucupyranuronate (obtained from NBS
Biologicals Inc., Herts, UK), in 100 ml of dry benzene were added every hour

over two seven hour periods.











Me( O Br
a A
AAJO XAc Me
MeN (Protected Glucuronic Acid)

Silver Carbonate
/ \ in Benzene M
0 H Meoc0
Codeine A A
"Intermediate"
Sodium Methoxidel
Me in Methanol
"'N Me
"N
1. Barium Hydroxide

0 2. Oxalic Acid MO
0OC
OH M H H

Codeine 6-glucuronide


Figure 2-1 : Synthetic route of codeine-6-glucuronide using the Koenigs-Knorr
reaction (adapted from Yoshimura et al., 1968).


Thin layer chromatography (TLC) was performed at regular intervals
during the heating period to monitor the extent of the reaction. The solvent used

for the TLC was a mixture of methanol and methylene chloride (1:4). Small
portions (1 Ll) were removed from the boiling mixture and spotted on TLC plates
(Brinkman Polygram Sil G/UV 254). With increasing time, the spot representing
the starting material codeine, disappeared and another spot with a higher Rf

value appeared. This indicated the formation of a product assumed to be methyl
[codein-6-yl 2,3,4-tri-O-acetyl-l-bromo-l-deoxy-D-glucupyranosiduronate, that is,
the 6-glucuronide of codeine with intact acetyl and methyl groups on the
glucuronic acid moiety, and subsequently referred to as the intermediate. The












structure and identity of the compound in CDCl3 was confirmed by IH NMR

(Varian EM 390 Spectrometer).
After the heating was stopped, the contents of the flask were filtered and

the clear filtrate was evaporated to dryness with a rotory evaporator. The residue
was redissolved in absolute ethanol and evaporated to dryness. The solid
residue was then transferred to a column and chromatographed using 500 ml
each of ethyl acetate, ethyl acetate : ethanol (60 :40), ethanol: methanol (50 :

50) and methanol. Various fractions were collected and concentrated to dryness.
The dried fractions which indicated the presence of the compound of interest
were recrystallized using methanol. The melting point of the compound was 113-

1160C, in agreement with the value previously reported (Yoshimura et al., 1968)

2.2.1.1 Dry benzene
A key point to guarantee the success of the first step was to ensure that
the benzene used was absolutely dry. This was achieved by boiling benzene in

the same apparatus as the reaction was done and removing the water with the
Dean-Starke trap. Boiling for about 4 hours ensured removal of all the water

present in the benzene.

2.2.1.2 Fresh silver carbonate
Freshly prepared silver carbonate is another essential prerequisite in the
first step of the reaction, as the silver carbonate used as a catalyst is susceptible
to oxidation in the presence of moisture and light. The procedure for preparing
"active" silver carbonate (Wolfrom and Lineback, 1963) involves adding an

aqueous solution of anhydrous sodium carbonate (1.6 g in 7.5 ml of distilled
water) dropwise into a mechanically stirred solution of 8 g of silver nitrate
dissolved in 20 ml of water. A solution of 1 g of anhydrous sodium hydrogen












carbonate in 12.5 ml of water was then added to the above in 2-3 portions. The
mixture foams and a yellowish precipitate is formed. The solid recovered by
filtration was silver carbonate.


2.2.2 Reaction Step II

The intermediate compound from step I (0.6 g) was suspended in a test

tube with 3.5 ml of absolute methanol. A 1 % solution of sodium methoxide in
methanol (2 ml) was added and the mixture was stirred with a magnetic stirrer.
The solution was evaporated to dryness in vacuo.


2.2.3 Reaction Step III

The dried residue from step II was dissolved in 2.2 ml of a 0.43N Ba(OH)2

solution and stirred for about 4 hours before leaving it overnight in a refrigerator

at 40 C. The barium salt which precipitated on cooling was dissolved in 4 ml of
distilled water and adjusted to pH 6 with 2N oxalic acid solution. The solution

was refrigerated overnight and the barium oxalate formed was removed by
filtration. The filtrate was evaporated to dryness and the residue was
recrystallized from methanol. The compound decomposed at 225-230 C, which
was slightly lower than that reported in the reference paper.


2.3 Specific Obiective #3:
Analgesic Activities of Codeine and Codeine 6-glucuronide


The polar glucuronide metabolites of codeine and morphine do not cross
the blood brain barrier (BBB) as efficiently as their parent compounds. With this
in mind, initial studies were designed to bypass the BBB and determine if












codeine 6-glucuronide produced analgesia in pain-induced rats by using
standard antinociceptive tests, that is, tail flick and hot plate methods. This was
done by delivering the compounds directly into the brain of rats via the
intracerebroventricular route (i.c.v.). The next step was to compare the activities

of codeine and codeine 6-glucuronide after subcutaneous and intravenous
administrations using the same methods as in the i.c.v. studies for measuring the

effects.
During the synthetic procedure, the intermediate compound formed after

the first reaction step was isolated and characterized. This intermediate is the
glucuronic acid moiety attached to the 6-position of codeine with intact

acetyl/methyl groups. The antinociceptive activity of this intermediate was also

investigated.


2.3.1. Intracerebroventricular Route Studies

These studies involved investigation of the antinociceptive effects of

codeine, codeine 6-glucuronide and the intermediate compared to the standard

analgesic drug, morphine. All drugs were dissolved in physiological saline (pH
4.5 5.5) and administered at doses of 100 pg/5 pl for codeine, 10 pg/5 pl for
codeine 6-glucuronide and the intermediate, and a dose of 5 pg/5 pl for
morphine. Each compound was administered to groups of 6 rats. One group of

rats was injected with saline only and served as a control group. Another group
was not injected with anything to control for any possible effects of the surgical
procedure on the pharmacodynamic measurements. Measurements were made
at the following time points after administration of the compounds : 0, 10, 20, 30,
40, 60, 90, 120, 150 and 180 minutes.











2.3.1.1 Surgery
After the animals were acquired, they were housed in cages in a room

with a 12 hour light-dark cycle. The animals were fed on standard laboratory rat
chow and tap water ad libitum. Each rat was anesthetized with 30-50 mg/kg
sodium pentobarbital intraperitoneally and stereotaxically fitted with a 23 gauge
intracerebroventricular stainless steel guide cannula. The co-ordinates used for
the stereotaxic apparatus were : 1.0 mm lateral; 1.0 mm caudal to the bregma

and 5.0 mm below the skull surface (Paxinos and Watson, 1986). The rats were
allowed to recover for 3-5 days prior to starting the antinociceptive experiments.


2.3.1.2 Tail flick method

Analgesia was determined using the tail flick method of Berglund and
Simpkins (1988) and previously described in section 1.4. Reduction in pain was
expressed as the% of the maximum possible effect (% MPE) calculated as:



% MPE = (test latency baseline latency) 100
(cut-off period baseline latency)



The area under the effect curve (AUEC) was determined from individual%
MPE versus time graphs using trapezoidal calculation. The AUEC is considered
a good indicator for comparing the intensity of effect during a certain time period.
It also provides a good estimate of the duration of effect produced by each
compound. Comparison of total AUEC values are, therefore, considered to
reflect the relative effectiveness of the compounds.











2.3.2 Subcutaneous Route Studies

After the intracerebroventricular route, studies were performed by injecting

the test compounds subcutaneously, that is, under the nape of the neck.
Codeine, codeine 6-glucuronide and the intermediate were dissolved in
physiological saline (pH 4.5 5.5) and injected at a dose of 10 mg/kg. A higher

dose of 20 mg/kg of codeine was also administered to a group of 6 rats. The
volume of injection was 1 mi/kg body weight of the rat. Each rat was also

administered saline in a separate study and therefore, acted as its own control.
Assessment of the reduction in pain was determined by the tail flick
method as described for the intracerebroventricular route. The intervals between

time points for measuring the response were, however, greater than in the
intracerebroventricular studies so as to take the absorption factor into

consideration. The measurement time points were 0, 15, 30, 45, 60, 90, 120,

180, 240, 300 and 360 minutes.


2.3.3 Intravenous Route Studies

In these studies, the jugular vein of rats was catheterized so that

compounds could be directly injected into the general blood circulation. Animals
were first anesthetized with ketamine/xylazine (45:9 mg/100 g body weight of rat)
intraperitoneally. A heparinized (100 U/ml) catheter (PE50 tubing, 0.58 mm X

0.965 mm) was then placed in the right jugular vein. The catheters were
stoppered and exteriorized between the scapulae to avoid chewing. The rats
were allowed 1-2 days to recover from the anesthesia and surgery. Drugs were
dissolved in physiological saline (pH 4.5 5.5) and injected through the catheter.
Codeine, codeine 6-glucuronide and the intermediate were administered at a












dose of 10 mg/kg. The injection volume was 1 mg/kg body weight of the rat.
Physiological saline (500 ltl) was also injected to ensure that the drug reached

the systemic circulation and did not remain in the dead volume of the catheter.
As in the intracerebroventricular and subcutaneous studies, the tail flick
method was used to determine the analgesic effect. As there was no absorption

phenomenon to consider, periods between measurement times were less than in

the subcutaneous studies but greater than the intracerebroventricular studies,
that is, 0, 10, 20, 30, 40, 60, 90, 120, 180, 240 and 300 minutes.

2.3.4 Statistics


The area under the effect curve data for each compound was compared

to its saline treatment using a paired Student's t-test. The area under the effect

curve (AUEC) of compounds were also compared with each other using an
unpaired Student's t-test.


2.4 Specific Obiective #4:
Immune Studies with Human T Lymphocytes


In vitro immune studies on the drugs were performed with human T
lymphocytes found in peripheral blood mononuclear cells (PBMCs) which were
isolated from the blood of healthy volunteers using the Ficoll-Hypaque density

gradient centrifugation procedure. The isolated T lymphocytes were then
stimulated by mitogens phytohemagglutin (PHA) and phorbol 12-myristate-13-
acetate (PMA), activating the resting T cells and enabling them to become
transformed into lymphoblast cells. These transformed cells are then capable of
synthesizing DNA, dividing rapidly and proliferating.












When the lymphocytes reached their peak proliferation, they were labeled

with 1 pCi 3H thymidine after a period of 48 hours (for the PHNPMA assay) and

120 hours (for the mixed lymphocyte reaction assay). The cells incorporated the
radiolabeled thymidine into their DNA and counts were done using a scintillation

counter. The differences in cell count determined with and without drugs was
used as a measure of drug-induced changes in lymphocyte proliferation.

Immunosuppression produced by drugs was expressed in terms of% inhibition of
proliferation and calculated as:



% Inhibition of Proliferation = (CPM without drug CPM with drug), 100
(CPM without drug)




2.4.1 Method

Peripheral blood (20 ml) was obtained from a healthy volunteer and
transferred to a conical tube. To this conical tube 20 ml of RPMI tissue culture
media containing 5% albumin was added (this culture media was developed at

Rosewell Park Memorial Institute and hence the name, RPMI). A 10 ml Ficoll-
Hypaque solution was drawn up in a pipette and carefully delivered to the bottom
of the tube. The tube was then placed in a balanced centrifuge for 30 minutes at
1200 rpm. A white layer containing lymphocytes formed between the culture
media and red cells. The lymphocyte cells were transferred into another conical
tube and diluted with 50 ml of the RPMI cell culture media. The tube was
centrifuged again for 10 minutes at 1200 rpm. A "pellet" of cells was seen to be
formed at the bottom of the tube. The supernatant was decanted until 1 ml of the












media remained in the tube with the pellet. The tube was then slightly agitated to

disperse the pellet homogeneously in the media and a cell-count was done to
determine the total number of cells/ml using tryphan blue stain under a light
microscope (Fischer Scientific, Fairlawn, NJ, USA) at a magnification of 40 X.
The cell suspension was diluted with RPMI media to obtain a cell-count of lx 106

cells/ml for PMA and PHA assays and 2x1 06 cells/ml for the MLR assay.
Stock solutions and dilutions of the drug to be tested were made in RPMI

cell culture media. A 96-well U-bottom cell plate was used and 100 PI drug
solutions of each concentration of each drug was pipetted into the plate in
triplicate. To each drug-containing well was added, 100 pl of either PHA (5
pg/ml) or PMA (50 ng/ml) containing T lymphocytes. The controls in the assay

consisted of T cells without the drug, T cells containing the mitogens (PHA or

PMA) but without the drug, and a control with only the cell culture media. The cell
plate was covered on the top and placed in an incubator until it was radiolabeled.

Standard drug dilutions were done in the same way as in the case of
PHAIPMA assays. Lymphocytes from two separate individuals were used for this
assay. The cells of one individual was selected to be irradiated and labeled as
the donor. This was done in a cell irradiator with a 137Cs source at 2500 rads for

4 minutes. To 100 pl of drug solution of varying concentrations, 50 pl of the
irradiated cells and 50 pl of the plain cells from the other individual were added.
The cell plate was covered and incubated until it was radiolabeled.
The cell plates in the PHAIPMA assay (on day 2) and MLR assay (day 5)
were taken out of the incubator and placed on the work surface. 3H-Thymidine (1
pCi) diluted with the RPMI culture media was taken out of the refrigerator. Using












a Hamilton microsyringe stored in ethanol, each cell well was given 10 pl of the
3H-thymidine solution.

Cells were harvested onto nitromethylcellulose paper via a cell harvester
attached to a vacuum pump. The cell plate was discarded in a radioactive waste
box. Scintillation vials were set up and labeled appropriately. After the paper had
dried, forceps were used to punch out individual circles (each representing a cell

well) which were placed into the corresponding scintillation vials. Scintillation

fluid (3 ml) was then added to each vial and the vials were capped tightly.
Radioactivity counts were determined using a scintillation counter.


2.4.2 Statistics

The% inhibition of proliferation data of the compounds were compared to
each other using an unpaired Student's t-test.


2.5 Specific Obiective #5:
Receptor Binding Studies


2.5.1 Materials

Tris-HCI, bovine serum albumin (BSA), morphine sulfate and naloxone
HCI were obtained from Sigma Chemicals (St. Louis, MO, USA). Sodium chloride

was purchased from Fischer Scientific Inc. (Fairlawn, NJ, USA). Codeine
phosphate was obtained from Westlab Pharmacy, Gainesville, FL. Codeine 6-
glucuronide and the intermediate were synthesized using the Keonigs-Knorr

reaction. 3H-DAGO ([ D-ala 2, N-methyl-phe4, glyol5 1[ tyrosyl-3,5-3H ] enkephalin)
was used as a competitive ligand for the binding studies and was purchased
from Amersham (Arlington Heights, IL).














Receptor binding studies were carried out using brain tissues from
Sprague-Dawley rats (200-300 g) using a modification of the procedure reported
by Hochhaus et al. (1988). Brain tissue was homogenized in 60 volumes of 50
mM Tris-HCI buffer (pH 7.4) containing 100 mM sodium chloride. The

homogenate was incubated in a water bath for 1 hour at 20 C. After incubation,
the homogenate was centrifuged at 20,000 rpm for 20 minutes at 4 C. The pellet
formed at the bottom was washed twice with 50 mM Tris-HCI buffer. The
membrane suspension was vortexed for 2 minutes to ensure homogeneity.
Competitive binding studies were performed after adding 1% w/v of
bovine serum albumin (BSA) to the membrane suspensions. The suspension
(880 .I) was placed in polyethylene tubes and incubated with 20 FI of the tracer
(1 nM 3H-DAGO, a j.-receptor selective agonist) and 100 RI of the competing

ligand (various concentrations of the drugs dissolved in 50 mM Tris-HCI buffer).
This mixture was incubated in a water bath for 1 hour at 20 C. After incubation,

the bound and unbound fractions were separated by filtration using Whatman
GF/C filters. The filter paper containing the retained radioactivity was transferred

to scintillation vials and 3 ml of scintillation fluid was added to each vial. The vials
were capped tightly and shaken to enable the radioactivity to be distributed in the

scintillation cocktail. The vials were left overnight and tritium counts were
performed using a liquid scintillation counter.
Specific binding of morphine, codeine, codeine 6-glucuronide and the
intermediate to the ji-receptor was determined by competitive displacement of

the radiolabeled tracer by various concentrations of the test compounds. The
non-specific binding (NS) was determined with a relatively high concentration of












morphine (1 IM). The total binding (T) was determined from vials in which no
drug was added. The counts per minute (CPMs) obtained from a scintillation
counter were then plotted versus increasing drug concentrations. The data was
fitted using an Emax model from the Scientist program (Micromath Scientific Inc.,
Salt Lake City, UT) as described in Equation 1. The raw data (CPMs) for each
compound were standardized by transforming them into% of total binding and
plotting them as a function of increasing drug concentration (Equation 2). The
equations used for fitting the data are as follows:


Equation 1




CPM=T- T*CN
CPMT C -NN
IC5ON + CN + NS









CPM
% Total Binding 100
T


2.6 Specific Objective #6:
Plasma and Brain Concentrations


The objective of this study was to determine plasma and brain
concentrations at the peak analgesic response time in the rat. This was achieved












by the administration of codeine or codeine 6-glucuronide to groups of rats (n=6)
by intracerebroventricular, subcutaneous and intravenous routes (described in
section 2.3). The peak response time from the tail flick experiments after each
route of administration was used as the time at which plasma and brain samples
were collected.
Rats were first anesthetized with metofane 5 minutes prior to the peak
response time. At the appropriate time, the rats were decapitated using a

guillotine. Trunk blood samples were collected in vacutainers containing EDTA
as an anticoagulant. Plasma was obtained by centrifuging the blood at 2500 rpm

for 20 minutes. Brain samples were obtained after removing the skull and other
membranes attached to the brain. Both the plasma and brain samples were
stored in a freezer at -80 C. The samples were analyzed using the HPLC
method described in section 2.1.














CHAPTER 3
RESULTS


3.1 HPLC Development


An HPLC-UV based method was successfully developed for the analysis
of codeine, morphine, codeine 6-glucuronide, morphine 6-glucuronide and
morphine 3- glucuronide in biological samples, that is, human urine, rat plasma

and rat brain. A typical chromatogram obtained after injection of a standard

solution containing 100 ng/ml of each compound is represented in Figure 3-1
(system 1). This system uses a multiwavelength scanning UV detector and

allows the simultaneous examination of the same chromatogram at different

wavelengths in the'scanning range (Figure 3-2).


0.0100

0.0030

0.0060

0.0039

0.0019

-0.0001


M


MW WO


11.20


14.40 17.60
T=m(min)


20.80


Figure 3-1 : A standard solution containing 100 ng/ml of each of all the
compounds using system 1.








60







C M






0.0030 220
0.0024 240
0.0018 260
0.0012 280
0.0006 300
0.0000 320
8.00 11.20 14.40 17.60 20.80 24.00

Time (mn)



Figure 3-2 The same chromatogram as in Figure 3-1 showing the
multiwavelength capabilities of the detector.



The software also allows examination of the UV spectra at any time point

in the acquired chromatogram (Figure 3-3). This can be used to identify the

presence of an opiate, since all opiates exhibit a UV maxima of 285 nm. The

differential UV spectra further helps to confirm the identity an opiate of interest

(Figure 3-4). This is done by following minute changes in the spectra of an opiate

and matching it with the spectra obtained from a standard solution of the same

opiate.


0.1000 15. 527

0.0800

0.0600

0.0400

0.0200

0.0000 i I I I I


Wavelength (k)
Figure 3-3: A typical UV spectra which is exhibited by all opiates.






61




15.527 15.527"









220 240 260 280 300 320
Wavelength ( X)


Figure 3-4: A normal (-) and second derivative (--) spectra of morphine.

3.1.1 Extraction Recoveries

Extraction recoveries for each compound were determined by comparing

the peak area of an extracted standard to an unextracted standard. The elution
solvent of methylene chloride-isopropanol-ammonium hydroxide recommended
by Worldwide Monitoring for opiates gave good recoveries for codeine and
morphine only, but not for the glucuronide metabolites. However, the use of a

5% ammonium hydroxide solution in methanol enabled the efficiency of

extraction of polar glucuronides from human urine samples to be increased. This
elution solvent gave clean extracts and excellent recoveries in excess of 80% for
all the compounds of interest. The% extraction recoveries for the various
compounds of interest in human urine are represented in Table 3-1.

3.1.2 Range / Linearity of Standard Curve

After the initial development of the chromatographic system and extraction

procedure, calibration curves were prepared in drug-free human plasma and












urine. Standards were set up for each compound of interest in the biological
matrix (plasma/urine) at concentrations of 0, 10, 50, 100, 200, 400 and 500
ng/ml. Each point on the calibration curve was taken as the average of two
determinations. The standard curve was determined from calibrators by the

linear least squares fit to the equation y = mx + b, where x = concentration, y =
drug peak area, m = slope of the line and b = y-intercept of the line. Linear
correlations between the area under the peak and concentration of each
compound was performed by regression analysis using the Microsoft Excel
program. It was seen that each compound of interest had a good correlation with

an R2 of 0.99 or better over the range 0-500 ng/ml (Figures 3-5 and 3-6).



Compound % Extraction recovery sd (n=4)

Codeine 92 +6

Morphine 90 + 8

Codeine 6-glucuronide 86 + 4

Morphine 6-glucuronide 85 + 8

Morphine 3-glucuronide 83 + 6


Table 3-1 :% extraction recoveries for compounds of interest in human
urine.

3.1.3 Specificity


Drug-free plasma and urine samples were analyzed with and without the

drugs. This was performed to show that the drug peaks were well separated from












the solvent front and did not have any interference from endogenous materials
also eluting from the column.

3.1.4 Sensitivity/Limit of Detection and Quantitation

The detection limit was determined as the concentration of the sample

which corresponds to the signal that is twice that of the baseline noise. When
extracted from human urine using Clean Screen columns, the minimum
quantifiable concentrations of the various compounds using system 1 were : 5
ng/ml for codeine and morphine; 10 ng/ml for codeine 6-glucuronide, morphine
6-glucuronide and morphine 3-glucuronide.


3.1.5 Precision and Accuracy

Precision is defined as a measure of the closeness between replicate

concentrations of a sample and its mean value. It is expressed as the% relative
standard deviation about the mean (standard deviation/mean *100). Replicate (n
= 7) analysis of the samples were done on the same day (intra-day) and over a
period of two weeks (inter-day). Both intra-day and inter-day precision were
determined to be less than 10% for all the compounds. The intra-day and inter-
day precision values for codeine are shown in Table 3-2. Accuracy is expressed

as the difference between the actual value and the mean measured value for
each concentration. Accuracy determined from calibration graphs was less than
10%.























Morphine
Codeine


0 50 100 150 200 250 300 350 400 450 500
Concentration (ng/ml)


Figure 3-5 : A typical calibration curve for codeine and morphine
in spiked urine.


-.*- M6G

-M3G

AC6G


0 50 100 150 200 250 300 350 400 450 500
Concentration (nglml)


Figure 3-6 : A typical calibration curve for codeine 6-glucuronide,
morphine 6-glucuronide and morphine 3-glucuronide in spiked urine.











3.1.6Stability

Stability of extracted plasma, urine and brain samples were determined

over a 24 hour period by replicate analyses during the assay development and
validation. The variation in peak areas of samples determined from replicate

analysis was less than 10%.


Concentration Intra-day variability Inter-day variability

(nglml) (%) (%)

10 6.4 9.7

50 3.3 5.8

100 4.1 5.3

200 4.3 6.7

400 4.5 7.2

500 6.2 8.7


Table 3-2 : Intra-day and inter-day variability from replicate analysis of
standards containing various concentrations of codeine.




In the first step of the synthetic procedure, the key to the success of the
reaction was maintaining anhydrous conditions in the flask. This was achieved
by using a Dean-Starke trap, which allowed distillation of benzene and water at
regular intervals. The completion of the reaction was determined by the
disappearance of the starting material by thin layer chromatography. The
structure and identity of the compound formed after the first step was confirmed












by 1H NMR. It indicated the presence of acetyl and methyl groups and the

attachment of a glucuronic acid group to codeine. The melting point of the
compound was 113-1160C in agreement with the value reported by Yoshimura et

al. (1968). The yield of this reaction was 70%.

Codeine 6-glucuronide was recrystallized from methanol and decomposed
at 225-230 C, slightly lower than the value reported by Yoshimura et al. (1968).

This may be due to the fact that the product was apparently anhydrous
compared to the half-hydrate product reported in the reference paper. Absolute

methanol was used to recrystallize the compound instead of a water-methanol
mixture used in the reference. The identity of the product was confirmed by

comparing the retention time of the chromatographic peak to an analytical
standard by using the HPLC method previously described (section 2.1). The
overall yield of the reaction was about 16%.


3.3 Analgesia Studies


3.3.1 Intracerebroventricular Route

Intracerebroventricular administration of morphine, codeine, codeine 6-
glucuronide and the intermediate produced significant antinociceptive responses
in the rats. All the compounds tested produced a peak response about 20
minutes after administration. Each rat was also administered saline which
produced minimal changes to baseline responses. The data set for the effect of
surgery on the response time and the analgesic responses are summarized in
Tables 3-3 and 3-4, respectively. It was observed that the surgical procedure












itself had no effect on the response time (Figure 3-7) and the analgesic response
(Figure 3-8) and over a 3 hour period.



Time(min) 1 2 3 4 5 Mean SD SEM


0 11.8 8.7 8.1 7.8 8.4 9.0 1.6 0.7


10 11.5 6.8 9.5 11.2 8.1 9.4 2.0 0.9


20 9.2 7.0 10.3 9.6 10.3 9.3 1.4 0.6


30 7.2 8.5 8.2 11.1 11.8 9.4 2.0 0.9


40 7.4 9.4 9.6 8.8 11.3 9.3 1.4 0.6


60 12.2 12.2 11.4 9.6 10.8 11.2 1.1 0.5


90 9.7 9.1 10.3 9.3 11.1 9.9 0.8 0.4


120 9.0 11.2 10.8 8.4 9.4 9.8 1.2 0.5


150 10.8 9.6 11.8 8.7 9.7 10.1 1.2 0.5


180 11.3 9.2 11.6 9.2 10.1 10.3 1.1 0.5


Table 3-3 : The data for the effect of intracerebroventricular
surgery on the response time (in seconds).


I-
















Time(min) 1 2 3 4 5 Mean SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


10 -1.1 -6.1 4.4 10.6 -0.9 1.4 6.3 2.8


20 -9.2 -5.4 6.9 5.6 6.0 0.8 7.5 3.4


30 -16.3 -0.6 0.3 10.2 10.8 0.9 11.0 4.9


40 -15.6 2.2 4.7 3.1 9.2 0.7 9.5 4.3


60 1.4 11.2 10.3 5.6 7.6 7.2 3.9 1.8


90 -7.4 1.3 6.9 4.7 8.5 2.8 6.3 2.8


120 -9.9 8.0 8.5 1.9 3.2 2.3 7.4 3.3


150 -3.5 2.9 11.6 2.8 4.1 3.6 5.4 2.4


180 -1.8 1.6 11.0 4.3 5.4 4.1 4.7 2.1



Table 3-4 The data for the effect of intracerebroventricular
surgery on the analgesic responses (in% MPE).







69







40



030
U
0



!,-
,.

10



0.
0 30 60 90 120 150 180

Time (min)

Figure 3-7 : The effect of the intracerebroventricular surgical procedure
on the response time.



100
9o
80
0 70.



E
S40.
S30.
~20
10

0
0 30 60 90 120 150 180

Time (min)


Figure 3-8 : The effect of the intracerebroventricular surgical procedure
on the analgesic response.












The response time (Tables 3-5 to 3-9) for all the treatments was

transformed into% of maximum effect data (Tables 3-10 to 3-14). The average
values from the data sets were then plotted as a function of time (Figures 3-9
and 3-10). Morphine was the most effective of the compounds, producing a
maximal suppression of pain, that is, 100% of the maximum possible effect

(MPE) in the rats. Codeine 6-glucuronide and the intermediate also exhibited
marked increases in antinociceptive responses, that is, up to 89 + 6 and 81 + 9%
of MPE ( standard error of the mean or SEM), respectively. Codeine also
produced analgesia, with a peak effect of 63 + 2% of MPE. The% MPE at peak
response time for all the compounds is summarized in Table 3-15. The area

under the effect curve (AUEC) was determined from individual% MPE versus

time graphs for each treatment using trapezoidal calculation (Figure 3-11) and is
summarized in (Table 3-16).


3.3.2 Subcutaneous Route


Significant responses were seen with codeine, but not with codeine 6-

glucuronide or the intermediate after subcutaneous administration. There was
also a difference in the peak response time. While codeine produced a peak
response 30 minutes after administration, codeine 6-glucuronide and the
intermediate showed a peak response 45 minutes after they were administered.
The response time (Tables 3-17 to 3-20) for all the treatments was transformed

into% of maximum effect data (Tables 3-21 to 3-24). The average values from
the data sets were then plotted as a function of time (Figures 3-12 and 3-13).
Codeine exhibited a significant response, that is, 61 + 5% of the MPE (Figure 3-












13). On the other hand, codeine 6-glucuronide and the intermediate produced
very poor responses, that is, 17 + 3 and 11 + 2% of MPE, respectively. The%



Time(min) 1 2 3 4 5 6 MEAN SD SEM


0 10.7 10.4 10.4 13.3 11.9 13.1 11.6 1.3 0.5


10 28.1 18.9 18.8 30.6 26.3 18.6 23.6 5.4 2.2


20 33.3 40.0 38.1 40.0 38.9 31.6 37.0 3.6 1.5


30 29.2 40.0 33.3 40.0 37.2 29.7 34.9 4.9 2.0


40 24.8 40.0 28.3 36.6 31.8 23.6 30.9 6.5 2.7


60 22.6 33.7 24.2 32.7 27.7 21.7 27.1 5.2 2.1


90 15.2 27.6 22.7 30.5 26.9 18.6 23.6 5.8 2.4


120 13.3 24.4 20.9 26.9 24.8 18.0 21.4 5.1 2.1


150 13.4 20.1 19.7 17.7 19.9 17.2 18.0 2.6 1.0


180 12.1 11.3 16.8 15.1 12.6 16.6 14.1 2.4 1.0



Table 3-5 : The data for the effect of intracerebroventricular
administration of 10 gg of codeine 6-glucuronide on the response time
(in seconds).
















Time(min) 1 2 3 4 5 6 MEAN SD SEM


0 9.8 9.4 11.3 8.3 12.1 9.4 10.1 1.4 0.6


10 11.1 16.3 16.4 15.8 18.4 16.6 15.8 2.5 1.0


20 28.8 27.6 28.4 29.7 29.3 29.7 28.9 0.8 0.3


30 24.3 25.9 26.7 27.8 27.1 26.8 26.4 1.2 0.5


40 21.5 22.7 23.6 24.4 25.2 24.5 23.7 1.4 0.6


60 18.8 21.1 19.5 21.1 22.1 23.7 21.1 1.8 0.7


90 16.6 19.8 16.9 17.9 18.6 20.9 18.5 1.7 0.7


120 15.1 16.5 15.5 14.2 16.8 19.6 16.3 1.9 0.8


150 13.6 11.4 12.1 11.7 15.9 16.2 13.5 2.1 0.9


180 10.1 9.2 10.7 8.4 13.7 11.3 10.6 1.9 0.8



Table 3-6 : The data for the effect of intracerebroventricular
administration of 100 jig of codeine on the response time (in seconds).
















Time (min) 1 2 3 4 5 6 MEAN SD SEM


0 14.4 11.4 14.2 10.0 12.9 12.7 12.6 1.7 0.7


10 40.0 14.6 22.3 33.1 19.7 22.6 25.4 9.4 3.8


20 40.0 28.1 40.0 40.0 28.1 32.8 34.8 5.9 2.4


30 40.0 24.4 36.4 40.0 27.3 30.7 33.1 6.7 2.7


40 28.3 19.3 28.8 40.0 21.0 24.6 27.0 7.4 3.0


60 18.1 19.8 24.9 40.0 20.3 20.4 23.9 8.2 3.3


90 18.6 15.3 21.7 29.3 17.7 17.8 20.1 5.0 2.0


120 14.7 11.1 19.2 27.8 16.9 16.3 17.7 5.6 2.3


150 13.6 10.7 18.6 22.7 15.8 14.7 16.0 4.2 1.7


180 12.8 9.8 16.4 14.6 13.2 12.3 13.2 2.2 0.9



Table 3-7 : The data for the effect of intracerebroventricular
administration of 10 jig of the intermediate on the response time
(in seconds).
















Time(min) 1 2 3 4 5 6 MEAN SD SEM


0 11.2 12.6 12.3 12.6 8.7 9.3 11.1 1.7 0.7


10 30.2 33.6 24.2 27.3 26.9 21.7 27.3 4.2 1.7


20 40.0 40.0 40.0 40.0 40.0 40.0 40.0 0.0 0.0


30 40.0 40.0 40.0 40.0 40.0 40.0 40.0 0.0 0.0


40 40.0 40.0 40.0 40.0 40.0 38.7 39.8 0.5 0.2


60 33.6 40.0 40.0 40.0 40.0 40.0 38.9 2.6 1.1


90 24.7 40.0 40.0 36.6 34.2 40.0 35.9 6.0 2.4


120 18.9 28.9 26.7 29.1 27.7 27.3 26.4 3.8 1.6


150 18.6 19.7 20.1 20.6 19.9 22.1 20.2 1.2 0.5


180 13.8 16.6 14.3 14.7 12.3 13.4 14.2 1.4 0.6



Table 3-8 : The data for the effect of intracerebroventricular
administration of 5 gg of morphine on the response time (in seconds).
















Time(min) 1 2 3 4 5 6 MEAN SD SEM


0 11.2 10.4 10.3 11.0 11.4 8.7 10.5 1.0 0.4


10 10.6 11.7 11.2 12.8 13.1 9.9 11.6 1.2 0.5


20 12.1 12.3 12.3 13.2 13.6 10.2 12.3 1.2 0.5


30 14.4 11.6 14.3 12.6 12.9 10.9 12.8 1.4 0.6


40 15.2 10.9 14.8 13.9 13.9 11.3 13.3 1.8 0.7


60 14.1 11.1 12.5 14.1 12.6 10.6 12.5 1.5 0.6


90 12.9 10.8 13.8 12.7 13.1 9.7 12.2 1.6 0.6


120 11.4 11.2 14.1 13.6 13.6 11.3 12.5 1.4 0.6


150 11.0 11.6 12.2 12.1 13.4 11.5 12.0 0.8 0.3


180 10.5 10.5 9.8 11.2 13.3 10.8 11.0 1.2 0.5


Table 3-9 : The data for the effect of intracerebroventricular
administration of saline on the response time (in seconds).















Time(min) 1 2 3 4 5 6 MEAN SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


10 4.3 22.5 17.8 23.7 22.6 23.5 19.1 7.6 3.1


20 62.9 59.5 59.6 67.5 61.6 66.3 62.9 3.4 1.4


30 48.0 53.9 53.7 61.5 53.8 56.9 54.6 4.4 1.8


40 38.7 43.5 42.9 50.8 47.0 49.3 45.4 4.5 1.8


60 29.8 38.2 28.6 40.4 35.8 46.7 36.6 6.8 2.8


90 22.5 34.0 19.5 30.3 23.3 37.6 27.9 7.2 2.9


120 17.5 23.2 14.6 18.6 16.8 33.3 20.7 6.8 2.8


150 12.6 6.5 2.8 10.7 13.6 22.2 11.4 6.7 2.7


180 1.0 -0.7 -2.1 0.3 5.7 6.2 1.8 3.4 1.4



Table 3-10 : The data for the effect of intracerebroventricular
administration of 100 pg of codeine on the analgesic responses
(in% MPE).















Time (min) 1 2 3 4 5 6 Mean SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


10 59.4 28.7 28.4 64.8 51.2 20.4 42.2 18.6 7.6


20 77.1 100.0 93.6 100.0 96.1 68.8 89.3 13.1 5.4


30 63.1 100.0 77.4 100.0 90.0 61.7 82.0 17.3 7.1


40 48.1 100.0 60.5 87.3 70.8 39.0 67.6 23.2 9.5


60 40.6 78.7 46.6 72.7 56.2 32.0 54.5 18.3 7.5


90 15.4 58.1 41.6 64.4 53.4 20.4 42.2 20.3 8.3


120 8.9 47.3 35.5 50.9 45.9 18.2 34.5 17.2 7.0


150 9.2 32.8 31.4 16.5 28.5 15.2 22.3 9.9 4.0


180 4.8 3.0 21.6 6.7 2.5 13.0 8.6 7.4 3.0



Table 3-11 : The data for the effect of intracerebroventricular
administration of 10 pg of codeine 6-glucuronide on the analgesic
responses (in% MPE).















Time(min) 1 2 3 4 5 6 Mean SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


10 66.0 76.6 43.0 53.6 58.1 40.4 56.3 13.8 5.6


20 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0.0 0.0


30 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0.0 0.0


40 100.0 100.0 100.0 100.0 100.0 95.8 99.3 1.7 0.7


60 77.8 100.0 100.0 100.0 100.0 100.0 96.3 9.1 3.7


90 46.9 100.0 100.0 87.6 81.5 100.0 86.0 20.7 8.4


120 26.7 59.5 52.0 60.2 60.7 58.6 53.0 13.2 5.4


150 25.7 25.9 28.2 29.2 35.8 41.7 31.1 6.4 2.6


180 9.0 14.6 7.2 7.7 11.5 13.4 10.6 3.1 1.3



Table 3-12 : The data for the effect of intracerebroventricular
administration of 5 pg of morphine on the analgesic responses
(in% MPE).















Time(min) 1 2 3 4 5 6 Mean SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


10 100.0 11.2 31.4 77.0 25.1 36.3 46.8 34.1 13.9


20 100.0 58.4 100.0 100.0 56.1 73.6 81.4 21.3 8.7


30 100.0 45.5 86.0 100.0 53.1 65.9 75.1 23.7 9.7


40 54.3 27.6 56.6 100.0 29.9 43.6 52.0 26.4 10.8


60 14.5 29.4 41.5 100.0 27.3 28.2 40.1 30.6 12.5


90 16.4 13.6 29.1 64.3 17.7 18.7 26.6 19.2 7.8


120 1.2 -1.0 19.4 59.3 14.8 13.2 17.8 21.9 8.9


150 -3.1 -2.4 17.1 42.3 10.7 7.3 12.0 16.8 6.8


180 -6.3 -5.6 8.5 15.3 1.1 -1.5 1.9 8.5 3.5



Table 3-13 : The data for the effect of intracerebroventricular
administration of the 10 g.g of the intermediate on the analgesic
responses (in% MPE).
















Time(min) 1 2 3 4 5 6 Mean SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


10 -2.1 4.4 3.0 6.2 5.9 3.8 3.6 3.0 1.2


20 3.1 6.4 6.7 7.6 7.7 4.8 6.1 1.8 0.7


30 11.1 4.1 13.5 5.5 5.2 7.0 7.7 3.7 1.5


40 13.9 1.7 15.2 10.0 8.7 8.3 9.6 4.8 2.0


60 10.1 2.4 7.4 10.7 4.2 6.1 6.8 3.3 1.3


90 5.9 1.4 11.8 5.9 5.9 3.2 5.7 3.5 1.4


120 0.7 2.7 12.8 9.0 7.7 8.3 6.9 4.4 1.8


150 -0.7 4.1 6.4 3.8 7.0 8.9 4.9 3.4 1.4


180 -2.4 0.3 -1.7 0.7 6.6 6.7 1.7 4.0 1.6



Table 3-14 : The data for the effect of intracerebroventricular
administration of saline on the analgesic responses (in% MPE).












_<> Cod
(100 mcg)
-- C6G
(10 mcg)
--n-- Mor
(5 mcg)
A_. Int
(10 mcg)
._Saline


0 30 60 90 120 150 180

Time (min)


* (mcg = micrograms).

Figure 3-9: Effect of Response Time to ICV Administration
of Saline, Codeine, Intermediate, Codeine 6-Glucuronide and Morphine.


Compound % MPE Peak Time
Morphine (5 pg) 100 + 20 min
Codeine (100 p g) 63 + 2 20 min
Codeine 6-glucuronide (10 pg) 89 + 6 20 min
Intermediate (10 jig) 81 + 9 20 min


Table 31-5: % of maximum possible effect for various compounds at
the peak response time after i.c.v. administration.













100.- see C6G
90 ;(10 mcg)
Int
80 (10 mcg)
S& 70 ;-Cod
7(100 mcg)
60-- Saline
UE 50.- -- Mor
40 (5 mcg)
S 40
E 30
0

10
0

0 30 60 90 120 150 180
Time (min)

(mcg = micrograms).

Figure 3-10: Analgesic responses of rats to i.c.v. administration
of saline, codeine, intermediate, codeine 6-glucuronide and morphine.


Compound Total AUECQ4 h
Morphine (5 l.g) 11545 + 744
Codeine (100 pig) 4462 + 546
Codeine 6-glucuronide (10 jig) 8156 + 1744
Intermediate (10 jig) 6416 + 2602


Table 3-16: Total AUEC0.3 h values after i.c.v. administration.






83





15000


12000


(9000.


6000
0
I-

3000


0.
C6G Sal Cod Sal Int Sal Mor Sal

Figure 3-11 -: The total area under the effect curve (AU ECo.3 h) after
i.c.v. administration.


The area under the effect curve (AUEC) was determined from individual% MPE
versus time graphs for each treatment using trapezoidal calculation (Figure 3-14)

and is summarized in Table 3-26.


3.3.3 Intravenous Route


Intravenous administration resulted in significant increases in the
responses associated with both codeine 6-glucuronide and the intermediate.

Codeine also produced a greater response by this route compared to the
subcutaneous route. As in the case of subcutaneous administration, there were
differences in the peak response times.














Time(min) 1 2 3 4 5 6 Mean SD SEM


0 10.9 9.9 11.6 8.7 10.8 12.2 10.7 1.2 0.5


15 18.2 16.3 17.4 18.3 20.5 22.3 18.8 2.2 0.9


30 26.9 27.7 28.9 27.3 29.9 30.2 28.5 1.4 0.6


45 21.3 25.1 26.3 26.6 28.7 28.6 26.1 2.7 1.1


60 20.6 24.4 24.7 25.9 28.3 27.8 25.3 2.8 1.1


90 18.7 22.2 23.6 25.4 27.9 27.7 24.3 3.5 1.4


120 17.6 20.1 21.1 23.5 25.8 25.4 22.3 3.2 1.3


180 16.8 18.7 20.3 21.7 22.6 23.3 20.6 2.5 1.0


240 14.9 16.6 17.5 20.2 20.6 21.1 18.5 2.5 1.0


300 13.8 14.8 15.2 18.3 17.7 18.4 16.4 2.0 0.8


360 12.3 12.2 13.7 15.1 13.2 13.8 13.4 1.1 0.4



Table 3-17: The data for the effect of subcutaneous administration of
10 mg/kg of codeine on the response time (in seconds).














Time (min) 1 2 3 4 5 6 Mean SD SEM


0 8.8 10.6 10.3 9.4 13.1 13.2 10.6 1.8 0.6


15 12.3 12.1 12.5 12.2 11.3 10.9 11.9 1.6 0.5


30 14.7 14.8 12.8 13.6 12.1 12.2 13.5 2.3 0.8


45 18.2 17.3 14.7 13.9 12.7 13.7 15.3 3.6 1.2


60 16.8 16.5 12.9 13.7 14.6 13.9 14.9 2.9 1.0


90 16.7 14.9 12.6 12.9 13.3 12.6 13.9 2.9 1.0


120 13.0 14.5 13.5 12.7 13.1 12.2 13.2 1.8 0.6


180 11.4 13.7 11.7 11.2 12.7 12.3 11.8 1.6 0.5


240 9.8 12.1 11.2 11.9 11.9 11.9 11.3 1.2 0.4


300 10.4 11.3 11.8 10.8 11.3 11.8 11.3 0.7 0.2


360 8.9 11.0 11.9 10.3 10.8 11.2 10.6 1.1 0.4



Table 3-18 : The data for the effect of subcutaneous administration of
10 mg/kg of codeine 6-glucuronide on the response time (in seconds).













Time(min) 1 2 3 4 5 6 Mean SD SEM


0 9.5 9.9 10.1 10.2 12.1 10.1 10.0 1.0 0.3


15 9.8 11.5 10.6 10.4 11.3 9.5 10.3 0.8 0.3


30 10.2 13.3 10.6 12.3 10.9 11.2 11.3 1.1 0.4


45 12.5 13.8 11.3 13.8 12.6 11.6 13.0 1.3 0.5


60 13.2 13.7 12.5 13.9 13.1 12.8 13.2 0.7 0.2


90 13.4 12.6 12.9 12.9 13.3 12.7 13.0 1.2 0.4


120 12.7 12.7 11.7 12.6 12.8 11.9 12.6 1.1 0.4


180 11.9 13.2 12.3 11.8 12.2 11.3 12.1 0.6 0.2


240 12.6 12.5 12.6 11.9 11.7 10.8 11.9 1.1 0.4


300 12.7 11.9 10.7 11.1 11.6 10.7 11.4 0.8 0.3


360 12.0 10.7 9.6 11.4 10.3 10.2 10.8 1.0 0.3



Table 3-19 : The data for the effect of subcutaneous administration
of 10 mg/kg of the intermediate on the response time (in seconds).












Time(min) 1 2 3 4 5 6 MEAN SD SEM


0 8.1 8.5 8.6 9.8 9.6 10.8 9.8 1.4 0.5


15 9.8 10.0 8.1 10.7 9.3 9.2 9.8 0.9 0.3


30 10.1 10.8 10.2 10.2 10.0 9.9 10.3 0.5 0.2


45 11.0 10.2 11.3 10.1 10.1 11.2 10.9 0.7 0.3


60 9.8 11.1 9.8 10.0 9.9 11.7 10.6 0.8 0.3


90 8.9 10.8 9.8 10.2 10.2 12.2 10.6 1.0 0.4


120 8.6 9.3 10.7 10.3 10.3 11.9 10.3 1.0 0.4


180 10.3 8.9 11.3 9.5 10.7 11.7 10.5 0.9 0.3


240 10.6 9.4 10.7 9.7 9.8 10.6 10.1 0.5 0.2


300 11.2 8.6 11.3 9.9 9.9 10.4 9.9 1.0 0.4


360 11.1 9.7 10.6 10.1 10.4 10.2 10.2 0.6 0.2



Table 3-20 : The data for the effect of subcutaneous administration
of saline on the response time (in seconds).












Time(min) 1 2 3 4 5 6 Mean SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


15 25.1 21.3 20.4 30.7 33.2 36.3 27.8 6.6 2.7


30 55.0 59.1 60.9 59.4 65.4 64.7 60.8 3.9 1.6


45 35.7 50.5 51.8 57.2 61.3 59.0 52.6 9.2 3.8


60 33.3 48.2 46.1 55.0 59.9 56.1 49.8 9.6 3.9


90 26.8 40.9 42.3 53.4 58.6 55.8 46.3 12.0 4.9


120 23.0 33.9 33.5 47.3 51.4 47.5 39.4 11.0 4.5


180 20.3 29.2 30.6 41.5 40.4 39.9 33.7 8.4 3.4


240 13.7 22.3 20.8 36.7 33.6 32.0 26.5 8.9 3.6


300 10.0 16.3 12.7 30.7 23.6 22.3 19.3 7.7 3.1


360 4.8 7.6 7.4 20.4 8.2 5.8 9.0 5.7 2.3



Table 3-21 : The data for the effect of subcutaneous administration of
10 mg/kg of codeine on the analgesic responses (in% MPE).












Time(min) 1 2 3 4 5 6 Mean SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


15 4.5 5.1 10.2 7.4 2.8 9.2 6.9 3.0 1.0


30 11.6 14.3 21.6 8.4 5.9 13.7 12.8 6.1 2.0


45 28.4 22.8 28.3 14.8 2.5 14.7 16.6 9.8 3.3


60 27.1 20.1 21.9 8.8 4.7 14.1 13.9 7.3 2.4


90 18.8 14.6 21.2 7.7 0.6 11.4 11.1 7.7 2.6


120 9.6 13.3 16.3 10.8 5.6 10.8 11.3 3.9 1.3


180 4.1 10.5 5.7 4.7 1.6 5.9 5.7 3.2 1.1


240 0.0 5.1 3.5 3.0 3.7 8.2 4.7 2.1 0.7


300 1.0 2.4 2.8 5.1 8.1 4.6 4.6 2.3 0.8


360 2.7 1.4 -3.5 5.4 3.1 2.9 1.9 3.3 1.1



Table 3-22 : The data for the effect of subcutaneous administration of
10 mg/kg of the Intermediate on the analgesic responses (in% MPE).













Time(min) 1 2 3 4 5 6 Mean SD SEM

0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


15 0.3 1.0 5.3 1.7 3.8 0.7 1.6 2.8 1.0


30 3.3 2.3 11.3 1.7 8.9 7.0 4.5 5.7 2.0


45 19.0 9.8 13.0 4.0 14.7 12.1 11.1 4.2 1.5


60 15.7 12.1 12.6 8.0 11.2 12.4 10.0 3.6 1.3


90 18.6 12.8 9.0 9.4 7.0 9.1 8.6 2.8 1.0


120 18.0 10.5 9.3 5.4 7.7 8.1 7.2 2.9 1.0


180 10.5 7.9 11.0 7.4 9.3 5.4 6.9 3.7 1.3


240 11.4 10.2 8.6 8.4 3.5 5.7 5.8 4.3 1.5


300 8.8 10.5 6.6 2.0 5.1 3.0 4.2 4.2 1.5


360 9.5 8.2 2.7 -1.7 4.8 4.0 1.9 5.2 1.8



Table 3-23: The data for the effect of subcutaneous administration
of 10 mg/kg of codeine 6-glucuronide on the analgesic responses
(in% MPE).












Timer(min) 1 2 3 4 5 6 Mean SD SEM


0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


15 11.3 11.6 13.1 13.9 8.3 0.7 8.0 6.5 2.5


30 15.6 17.5 16.9 20.8 16.2 -1.1 11.8 9.7 3.7


45 -2.8 13.4 2.1 20.2 17.8 -7.1 6.4 10.7 4.0


60 -1.1 14.4 7.6 21.5 15.0 -4.9 7.7 9.7 3.7


90 -4.3 9.1 3.8 18.6 12.7 -0.4 5.5 8.3 3.1


120 -1.1 7.2 10.3 17.0 13.1 -2.8 6.2 7.7 2.9


180 -5.0 11.6 -3.1 17.7 12.4 -1.8 4.7 9.0 3.4


240 -1.4 10.0 5.5 15.8 10.5 -3.2 5.1 7.4 2.8


300 -6.7 5.9 2.1 18.3 10.5 -7.4 2.8 9.5 3.6


360 -5.0 3.8 -7.9 12.6 9.9 -1.8 1.0 7.9 3.0



Table 3-24 : The data for the effect of subcutaneous administration
of saline on the analgesic responses (in% MPE).































0 60 120 180 240 300 360

Time (min)



Figure 3-12 Effect of response time to s.q. administration
of saline, codeine, intermediate and codeine 6-glucuronide.


Compound % MPE Peak Time
Codeine (10 mg/kg) 61 + 5 30 min
Codeine 6-glucuronide ( 10 mg/kg) 11 + 2 45 min
Intermediate (10 mg/kg) 17 + 3 45 min


Ta"e325: % of maximum possible effect for various compounds at
the peak response time after s.q. administration.












100

90

80

70

60

50
40

30

20

10

0


_oCod
10 mglkg
_ Int
10 mglkg
- C6G
10 mglkg
--*-Saline


0 60 120 180 240 300 360

Time (min)


Figure 3-13: Analgesic responses to s.q. administration
of saline, codeine, intermediate and codeine 6-glucuronide.


Compound Total AUECo4 h

Codeine (10 mg/kg) 10886 + 646
Codeine 6-glucuronide (10 mg/kg) 1765 + 159
Intermediate (10 mg/kg) 2321 + 269



Iab -26: Total AUEC0- h values after s.q. administration.