Antinociceptive actions of central opioid delta receptors

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Antinociceptive actions of central opioid delta receptors

Heyman, Julius Scott, Ph.D. The University of Arizona, 1989

U·M·I 300 N. Zccb Rd. Ann Arbor. MI 48106

ANTINOCICEPTIVE ACTIONS OF

CENTRAL OPIOID DELTA RECEPTORS

by

Julius Scott Heyman

A Dissertation Submitted to the Faculty of the

COMMITIEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE)

In Partial Fulfillment of the Requirements

For the Degree of -

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1989 THE UNIVERStTY OF ARIZONA GRADUATE COLLEGE 2

As members of the Final Examination Committee, we certify that we have read

the dissertation prepared by ~Juu~l~i~u~s~S~C~Qatat~Hwe~ym~a~n~ ______entitled ANTINOCICEPTIVE ACTIONS OF CENTRAL OPIOID DELTA RECEPTORS

and recommend that it be accepted as fulfil:~ng the dissertation requirement for the Degree of Doctor of Philosophy

Date I I 1!/flJ'? emaSF. Burks Date

Date 811 )SS Date .fbh! Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my uirection and recommend that it be accepted as fulfilling the dissertation r~quirement.

Frank Porreca 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. 4

ACKNOWLEDGEMENTS

There are many people to whom I am grateful for the opportunity to have carried out the work described below. Firstly, I would like to thank Dr. Ronald J.

Tallarida for introducing me to the field of Pharmacology, and to Dr. Frank Porreca.

Through his wonderment, Dr. Porreca has shown me how the scientific process can be both fun and exciting. Also greatly appreciated, is the fact that Frank always treated me as a pee:!, and always had time to talk with me on both a professional and personal level. His guidance and friendship will always be appreciated. I would also like to thank the people associated with the Porreca laboratory, namely Margie

Malarchik, Sheila Mulvaney, Linda Nunan, Rita Wedell, Dr. Qi Jiang and Dr.

Russell J. Sheldon, whose friendship, technical and moral support, and ability to laugh at my jokes made my stay in Arizona one of the great times of my life.

None of the work included in this dissertation would ever have been possible, however, without the support of my family. I must first thank my wife Michelle and son Zachary for their love, t:nderstanding and limitless patience. It was from

Michelle and Zachary that I continually got my inspiration for the completion of this project. I must also thank my parents Irma and Louis, and my sister Lisa, whom I love very much, for having always supported me in everything I have done, and for having allowed me the luxury to follow my dreams. It is to my family, therefore, that

I dedicate this dissertation. 5

"Sometimes the light's all shining on me Other times I can barely see Lately it occurs to me What a long strange trip it's been"

Garcia, Lesh, Weir, Hunter 6

TABLE OF CONTENTS

LIST OF ILLUSTRATIONS...... 8

LIST OF TABLES...... 11

ABSTRACT...... 12

INTRODUCTION...... 14

The Anatomy of Pain Pathways ...... 17 Morphine and the Treatment of Pain ...... ; ...... 21 Discovery of Multiple Opioid Receptor Types ...... 23 Endogenous Opioid Ligands and Receptors ...... 30 Endogenous Pain Control Systems ...... 36 Statement of the Problem ...... 42 Approach ...... 46

MElHODS ...... 48

Animals...... 48 Chemicals ...... 48 Injection Techniques ...... 49 Intracerebroventricular injections ...... 49 Intrathecal injections ...... 49 Mouse Warm-Water Tail-Withdrawal Test ...... 50 Gastrointestinal Transit...... 51 Time-dependent Naloxone p~ Theory and Determination ...... 52 Theory...... 52 pA2 calculation ...... 55 Tolerance and Cross-tolerance Studies ...... 56 Antagonism Studies ...... 57 ICI 174,864 administration ...... 57 ,B-FNA pretreatment ...... 58 Naloxonazine pretreatment ...... 58 Modulation Studies ...... 58 Studies with morphine ...... 59 Studies with chemically diverse p. agonists ...... 60 Effect of p. selective antagonists on 0 modulation of p.-mediated antinociception ...... 60 7

RESULTS ...... 62

Estimation of the Naloxone p~ for Morphine, DAMGO and DPDPE ...... 62 Tolerance/Cross-tolerance Studies ...... 69 Antagonism Studies ...... 75 Effects of ICI 174,864 on morphine, DAMGO and DPDPE antinociception ...... 75 Effects of ,8-FNA pretreatment on morphine, DAMGO and DPDPE antinociception ...... 79 Effect of naloxonazine pretreatment on morphine, DAMGO and DPDPE antinociception ...... 85 Effect of naloxonazine pretreatment on the ability of morphine, DAMGO and DPDPE to inhibit gastrointestinal propulsion ...... 97 Modulation Studies ...... 98 Studies with morphine ...... , 98 Studies with chemically diverse p. agonists ...... 102 Effects of p. selective antagonists on 0 modulation of p. mediated antinociception ...... 102

DISCUSSION ...... 115

Supraspinal Studies ...... 116 Spinal Studies ...... 122 Estimation of the Naloxone p~ for morphine, DAMGO and DPDPE ...... 127 Summary of Direct 0 Involvement ...... 129 Modulation Studies ...... 130 Studies with morphine ...... 132 Studies with chemically diverse p. agonists ...... 135 Effect of p. selective antagonists on 0 modulation of p. mediated antinociception ...... , . . . , . . . .. 141 Summary ofIndirect 0 Involvement ...... 146 General Summary ...... 147

REFERENCES ...... 149 8

LIST OF ILLUSTRATIONS

Fig. 1. Spinal and supraspinal pain pathways ...... 19

Fig. 2. Proposed neurocircuitry of spinal dorsal horn pain pathway neurons . 40

Fig. 3. Time-response curves for i.c.v. morphine, DAMGO and DPDPE antinociception in the absence and presence of a single, i.c.v. dose of naloxone ...... 63

Fig. 4. Time-response curves for i.th. morphine, DAMGO and DPDPE antinociception in the absence and presence of a single, i.th. dose of naloxone ...... 64

Fig. 5. Dose-response lines at various times determined from time-response data for i.c.v. DPDPE in the absence and presence of naloxone ...... 65

Fig. 6. Plots of log (dose-ratio - 1) vs. time for i.c.v. and Lth. morphine, DAMGO and DPDPE ...... 66

Fig. 7. Dose-response lines for i.c.v. morphine in naive and morphine pretreated (100 mg/kg, s.c.) mice ...... 70

Fig. 8. Dose-response lines for i.c.v. DPDPE in naive and morphine pretreated (100 mg/kg, s.c.) mice ...... 71

Fig. 9. Dose-response lines for Lth. morphine in naive and morphine pretreated (100 mg/kg, s.c.) mice...... 72

Fig. 10. Dose-response lines for i.th. DPDPE in naive and morphine pretreated (100 mg/kg, s.c.) mice ...... 73

Fig. 11. Antinociceptive dose-response lines for i.c.v. administration of DAMGO, morphine and DPDPE in the absence or in the presence of i.c.v. ICI 174,864...... 76

Fig. 12. Antinociceptive dose-response lines for i.th. administration of morphine, DAMGO and DPDPE in the absence or in the presence of i.th. ICI 174,864...... 77 9

Fig. 13. Antinociceptive dose-response lines for i.c.v. morphine in naive, or in f3- FNA pretreated (18 nmol i.c.v. at - 4 hr) mice ...... 80

Fig. 14. Antinociceptive dose-response lines for i.c.v. DAMGO in naive or in f3-FNA pretreated (18 nmol i.c.v. at -4 hr) mice ...... " 81

Fig. 15. Antinociceptive dose-response lines for i.c.v. DPDPE in naive or in f3-FNA pretreated (18 nmol i.c.v. at -4 hr) mice ...... 82

Fig. 16. Effects of pretreatment with increasing doses of i.th. f3-FNA on equieffective antinociceptive doses of i.th. morphine or DPDPE ...... 84

Fig. 17. Effects of naloxonazine on antinociceptive responses to morphine 86

Fig. 18. Effects of naloxonazine on antinociceptive responses to DAMGO 87

Fig. 19. Effects of naloxonazine on antinociceptive responses to DPDPE ... 88

Fig. 20. Effects of naloxonazine on antinociceptive responses to i.th. morphine, DAMGO and DPDPE ...... 89

Fig. 21. Effects of naloxonazine on the anti transit effects of i.c.v. morphine and DAMGO ...... 91

Fig. 22. Effects of naloxonazine on the antitransit effects of i.th. morphine.. 92

Fig. 23. Effects of naloxonazine on the antitransit effects of i.th. DAMGO .. 93

Fig. 24. Effects of naloxonazine on the anti transit effects of i.th. DPDPE . .. 94

Fig. 25. DPDPE induced potentiation of morphine antinociception and reversal of potentiation ~Y IC:I !74!864 (4 nmol) following administration of compounds In the same I.C.V. InjectIOn ...... 99

Fig. 26. DPDPE induced potentiation of morphine antinociception in morphine pretreated mice ...... 100

Fig. 27. Effects of i.th. co-administration of a threshold antinociceptive dose of DPDPE on three morphine doses in naive mice ...... 101

Fig. 28. DPDPE-indueed potentiation of normorphine antinocieeption ..... 103

Fig. 29. DPDPE fails to potentiate i.e.v. DAMGO, PL017, etorphine, sufentanil, phenazocine, and f3-endorphin antinocieeption ...... 104 10

Fig. 30. Antinociceptive effects of i.c.v. etorphine (a), phenazocine (b) and·,8- endorphin (c) in the absence and presence of leI 174,864 ...... 105

Fig. 31. Time-response curve for i.c.v. DAMA (1..7 nmol) antinociception in the mouse. Data are mean and standard error ...... 107

Fig. 32. Dose-response relationship for i.c.v. DAMA antinociception in the mouse when tested at 20 min post injection ...... 108

Fig. 33. Effects of ,8-FNA pretreatment (18 nmol at -4 hr) on DPDPE-induced potentiation of morphine antinocke.ption ...... 109

Fig. 34. Effects of ,8-FNA pretreatment (18 nmol at -4 hr) on DAMA-induced attenuation of morphine antinociception ...... 110

Fig. 35. Effects of naloxonazine pretreatment (35 mg/kg, s.c. at -24 hr) on DPDPE­ induced potentiation of morphine antinociception ...... 112

Fig. 36. Effects of naloxonazine pretreatment (35 mg/kg, s.c. at -24 hr) on DAMA- induced attenuation of morphine antinociception ...... 113

Fig: 37. Profile of ~/6 selectivity based on the ratio of ~ values for displacing receptor selective ligands ...... 139 11

LIST OF TABLES

Table 1. A guide to the compounds used according to function and receptor selectivity ...... 47

Table 2. Time-dependent apparent p~ for naloxone against morphine, DAMGO and DPDPE after i.c.v. or i.th. administration ...... 68

Table 3. Effects of morphine pretreatment on the antinociceptive responses to morphine and DPDPE ...... 74

Table 4. Effects of leI 174,864 on the antinociception produced by morphine,

DAMGO and DPDPE ...... 0 ••••••••••••••••••••••••• 78

Table 5. Effects of ,B-FNA pretreatment on the antinociceptive properties of morphine, DAMGO and DPDPE ...... 83

Table 6. Effects of treatment with naloxonazine on the anti nociceptive responses to morphine, DAMGO and DPDPE ...... 90

Table 7. Effects of treatment with naloxonazine on the inhibition of gastrointestinal transit by morphine, DAMGO and DPDPE ..... 95

Table 8. Effects of s.c. naloxonazine pretreatment (at -24 hr) on opioid antinociception and antitransit effects in the mouse ...... 96

Table 9. Effects of i.c.v. ,B-FNA pretreatment (18 nmol at -4 hr) on the antinociceptive properties of DAMA ...... 106

Table 10. Effects of s.c. naloxonazine pretreatment (35 mg/kg at -24 hr) on the antinociceptive properties of DAMA ...... 111 12

ABSTRACT

Opioids produce many effects, perhaps the most clinically relevant of which is the relief of pain. The understanding of the functions mediated by opioid systems is complicated greatly by the presence of several opioid receptor types.

Understanding the functions associated with specific opioid receptors may lead to the development of receptor selective drugs which elicit only desirable effects. This dissertation addresses the possibility that supraspinal and spinal opioid s receptors mediate and/or modulate thermal antinociceptive processes in the mouse. A number of approaches were utilized in parallel in this investigation which included:

1) the determination of the naloxone pA2 in vivo against the opioid agonists

2 4 2 morphine (IL), [D-Ala , NMePhe , Gly-ol]enkephalin (DAMGO)(IL) and [D-Pen , D­ pen6]enkephalin (DPDPE)(s); 2) the investigation of possible cross-tolerance between morphine and DPDPE; and 3) antagonism studies using N,N-diallyl-Tyr­

Aib-Aib-Phe-Leu-Oh, (ICI 174,864)(8), ,B-funaltrexamine (,B-FNA)(IL) and naloxonazine (ILl). No differences were found in the apparent pA2 values for naloxone against morphine, DAMGO and DPDPE at either supraspinal or spinal sites. Cross-tolerance between morphine and DPDPE was not evident at supraspinal sites, but was demonstrated in the spinal cord. The antinociceptive effects of i.c.v. morphine and DAMGO were antagonized by ,B-FNA and naloxonazine, but not ICI

174,864. I.c.v. DPDPE antinociception was blocked by ICI 174,864, but not ,B-FNA or naloxonazine. Neither ICI 174,864 nor naloxonazine blocked the antinociceptive 13 effects of i.th. morphine or DAMGO. ICI 174,864, but not naloxonazine, antagonized i.th. DPDPE antinociception. I.th. morphine, but not Lth. DPDPE antinociception was blocked by ,B-FNA. Co-admini~iration of sub-effective doses of

DPDPE and DAMA was shown to potentiate and attenuate, respectively, i.c.v., morphine antinociception. This potentiation was evident in naive and morphine tolerant mice, and was blocked by ICI 174,864. The modulatory effects of DPDPE and DAMA were blocked by ,B-FNA, but not naloxonazine. In contrast to the supraspinal effects, i.th. DPDPE had no effect upon i.th. morphine antinociception.

Collectively, the data demonstrate that supraspinal and spinal opioid s receptors can directly mediate antinociception in the mouse. Additionally, supraspinal, but not spinal, s receptors are also capable of indirectly modulating antinociception. 14

INTRODUCTION

A symptom common to many medical maladies is pain. Pain is one life's most complex sensations. The complexity stems from the fact that pain has a sensory, as well as an emotional, or suffering component. The sensory aspect deals mainly with the identification of a stimulus as painful, whereas the suffering aspect of pain involves an emotional interpretation of the sensory information, along with an appropriate response, such as a desire to escape the pain. The pain sensation basically serves as a warning system in that it signals the brain that tissue damage is occurring, or is about to. The systems which convey pain information, therefore, serve man in a protective capacity. The unappealing nature associated with this sensation has been the impetus for millions to seek the aid of a physician for relief.

Although medical sophistication has increased greatly over the ages, methods in the treatment of pain have not advanced to the same degree. Thus, while many useful drugs have been developed for the relief of pain, such as the inhalation anesthetics and barbiturates, these compounds also profoundly affect other sensory systems such as touch, vision and hearing, as well as state of consciousness. Ideally, then, a pain relieving substance should inhibit pain sensation selectively without altering other types of information. Therefore, while our treatment of pain has advanced to some degree, these developments are far outweighed by our increased understanding of the underlying mechanisms of pain transmission and perception, and our desire for 15 an ideal pain relieving substance.

An important step in the understanding of pain, and therefore its treatment, has been the recognition of two basic types of pain: acute and chronic (Bonica,

1953). Such a classification is important as the etiology, physiopathology, anatomy and therapy for acute and chronic pain differ dramatically. Acute pain refers to pain which results from an acute injury or disease state and may last only as long as the tissue pathology itself (Bonica, 1953). Chronic pain, on the other hand, is believed to arise from a chronic tissue pathology which may develop from a chronic disease state or an untreated acute injury (Bonica, 1953). Indeed, these two types of pain may be transmitted and interpreted via entirely different mechanisms. For example, thalamic lesions have been shown to block chronic, but not acute pain in man (Richardson and Zomb, 1970). This dissertation has focussed on an investigation of acute pain mechanisms. The introduction will serve to review some relevant aspects of pain systems. As the studies within were carried out in animals, and because. man and animals are believed to experience noxious sensations differently, working definitions of pain and pain relief are in order.

As stated above, pain in man is believed to derive from both sensory

(physical) and emotional (psychic) components (Beecher, 1946; 1956). Working with wounded soldiers at World War II evacuation hospitals, Beecher (1946) asked the wounded to grade their pain so that the proper amount of pain-reliever could be given. Interestingly, even in the face of being severely injured, approximately one half of the wounded related feeling no pain. These findings were in direct contrast 16 with studies conducted in civilian hospitals where patients with tissue damage similar to that of the wounded soldiers normally reported feeling profound pain (Beecher,

1956). Based upon these differential pain assessments, Beecher distinguished the experience of pain in man into two components: the original sensation (sensory or physical component) and the reaction to the sensation (emotional or psychic component). In both man and animals, the sensory, or physical component of pain which includes the detection, transduction and transmission of noxious events, is termed nociception (Perl, 1980). Thus, blockade of the transmission of pain signals is called antinociception. Antinociception, therefore, is the inhibition of the afferent information associated with stimulation of sensory nerve endings. Man is believed to have the additional psychic component of pain which is thought to modify the sensory component of pain. For example, a simple headache can be a mildly painful event, but if one is told that the headache is due to a brain tumor, the anguish one experiences can greatly intensify the pain (Fields, 1984). In man, the narcotic analgesic drugs used to relieve pain are believed to inhibit both the physical and psychic components of pain (Jaffee and Martin, 1985) whereas animals are presumed not to experience the psychic component of pain. Accordingly, the relief of pain in man is referred to as analgesia while the inhibition of reactions to noxious stimuli in animals is termed antinociception, although the two terms are often used interchangeably in the literature. 17

The Anatomy of Pain Pathways

Mechanical, chemical and thermal stimuli are all capable of activating injury­

sensitive receptors which initiate nociceptive events. The injury-sensitive receptors,

termed nociceptors, are merely free nerve endings and have no special structure for

detecting tissue damage (Boivie and Perl, 1975). Nociceptors occur in many tissue

types, some of which include skin, blood vessels, subcutaneous tissue, muscle and joints. Upon activation, nociceptors initiate impulses which are transmitted along peripheral nerve fibers to the central nervous system.

Based upon their size, the 'afferent nerve fibers over which sensory signals

travel have been classified into three groups: A, Band C. Type A nerve fibers are

myelinated and have been subdivided further into subgroups Aa, A/3 and As. Type

A fibers are the largest of the three types of afferent fibers and accordingly, are the fastest conducting. Roughly 10%-25% of type A-s fibers transmit nociceptive information, and activation of these fibers is associated with sharp, burning pain: the remaining type A fibers, as well as all type B nerves do not carry nociceptive information. Type C nociceptive fibers are unmyelinated and thus slow conducting.

Approximately 50%-80% of C fibers respond to noxious stimuli and mediate long­ lasting burning pain (Kelly, 1985). Thus, the transmission of nociceptive information is confined to A-s and C fibers which have been termed primary afferent neurons.

The cell bodies of the primary afferent fibers are located in the spinal ganglia. 18 These fibers enter the dorsal horn of the spinal cord posteriorly and synapse upon cells which are located primarily in laminae I, II, III and V in the dorsal horn of the spinal cord (Melzack and Dennis, 1978; Kerr, 1980), with the majority of A-6 and

C fibers terminating in laminae I (marginal layer), II and III (substantia gelatinosa).

The primary afferent fibers terminate either directly on the cells of origin of the spinothalamic tract, or upon interneurons which, through a multisynaptic chain, terminate on spinothalamic neurons. The spinothalamic tract and its supraspinal counterpart, the trigeminothalamic tract, are made up of the neospinothalamic and paleospinothalamic tracts (Fig. 1). Axons in the neospinothalamic tract project monosynaptically and directly to the ventrolateral and posterior thalamus where they synapse upon neurons which project primarily to the somatosensory cortex (Price and Dubner, 1977). Information arriving via the faster neospinothalamic tract is believed to arrive rapidly and to permit the perception of the locale, intensity and duration of the noxious stimulus. Such information was termed "phasic" by Melzack and Dennis (1978). Clinically, this type of information relates to an acute, highly localized, sharp burning pain which is short in duration. Unlike the neospinothalamic tract, most neurons of the slower, polysynaptic paleospinothalamic tract terminate below the thalamus in the medulla, the reticular formation, and the midbrain with few fibers projecting directly to the thalamus. Information coming over the diffuse paleospinothalamic tract is eventually transmitted to the limbic forebrain structures which project profusely to numerous parts of the brain. Melzack and Dennis (1978) coined this "tonic" information: such information may be 19

Cerebral cortex: Superior frontal'

Posterior parietal cortex

Thalamus: Intralaminar nuclei ""'-----lH'-f': /I Ventrobasal complex ------'f'<1 To hypoth,llamlus\

Posterior nuclear W()U~)---+--J'---t\---'

Tectum ------,,...-

gray matter

Mesencephalon

Pons

Reticular formation

Medulla

<#_-Arller·ola,teroalsystem: Neospinothalamic Paleospinothalamic Spinal cord

Fig. 1. Spinal and supraspinal pain pathways. 20 responsible for the manifestation of the emotional and motivational aspects of pain. responsible for the manifestation of the emotional and motivational aspects of pain. Clinically, the type of pain transmitted over the paleospinothalamic tract is poorly localized, dull throbbing pain which follows the phasic pain response and lasts longer.

The areas of the central nervous system from the spinal cord up to and including the brains tern are basically involved in the relay of ascending pain transmission. The decoding of these signals, pain perception, is believed to occur in the thalamus, the limbic system and the cortex (Kelly, 1985). Fields (1984) defined pain perception as "... the awareness of a noxious sensation, appreciation of negative emotion, and attribution of meaning to the experience." Additionally,

Fields (1984) suggests that perception can be broken down into two parts: attention and cognition. It is the cognitive component of pain where the incoming information from the periphery regarding tissue damage is compiled with factors such as memory and discrimination to attribute meaning to the pain. Although it is a strongly held belief that thalamocortical ~nd corticothalamic circuits are involved intimately in the perception of pain, our understanding of the neuropsychological aspects of pain are, unfortunately, a measure of our ignorance of brain function at this time. 21

Morphine and the Treatment of Pain

Methods in the direct treatment of pain have not changed radically for centuries. Initially, opium, a crude extract of the poppy plant, Papaver Somnifemm, was used for pain relief. Later, the component of opium chiefly responsible for its antinociceptive effects was isolated by Serturner in 1803 and found to be the alkaloid morphine. The name morphine derives from Morpheus, the Greek god of dreams. Since the discovery of morphine, many compounds with morphine-like effects have been synthesized: these compounds generally fall into the category of drugs known as opioids, drugs whose actions are reversible by the antagonist naloxone, owing to similarities between their pharmacology and the pharmacology of the opium-derived morphine. Therefore, it is morphine which serves as the standard by which all opioids are measured.

Unlike the inhalation anesthetics and barbiturates which block the peripheral transmission of pain signals, morphine does nothing to either modify the responsiveness of free nerve endings to noxious stimuli or impede the conduction of pain transmission along peripheral nociceptive pathways. Instead, morphine works in the central nervous system at both supraspinal and spinal sites where it blocks the rostral transmission of nociceptive information and alters the conscious appreciation of the pain (described below in detail). Morphine, because it does not 22 profoundly affect other sensory systems such as touch, vision, hearing and state of

consciousness, is an ideal pain reliever. Unfortunately, the use of morphine has

many drawbacks which include a progressively decreasing effectiveness (tolerance),

production in man of a· consistent psychological craving and physical need for the

drug (psychological and physical dependence), constipation, drowsiness, nausea,

vomiting, mood changes including dysphoria and depression of respiration,

sometimes to the point of death. Morphine depresses respiratory function by

decreasing the sensitivity of the medullary respiratory center to carbon dioxide

(Chuang and Valdman 1963). Of these problems, the most ravaging has historically

been the development of dependence on morphine. Dependence has been defined

by the World Health Organization (1969) as: "the state, psychic and sometimes

physical, resulting from the interaction between a living organism and a drug,

characterized by behavioral and other responses that always include a compulsion

to take the drug on a continuous or periodic basis in order to experience its psychic

effects, and sometimes to avoid the discomfort of its absence. Tolerance mayor may not be present." The problems associated with morphine dependence have led to the search for strong (i.e., high efficacy) analgesic drugs that do not produce physical addiction in the way that morphine does; this search led to the development of the drug nalorphine in 1945. Nalorphine was unique in that it was found to antagonize the effects of morphine. Nalorphine was demonstrated to reverse morphine poisoning and to trigger acute abstinence in morphine addicts (Wilker et aI., 1953). Interestingly, notwithstanding its antagonistic properties, nalorphine could 23 also produce analgesia in man (Lasagna and Beecher, 1954). Interest in nalorphine

led to the further development of morphine and nalorphine congeners, perhaps the

most important of which was the pure opioid antagonist naloxone (Foldes et aI.,

1963; Lasagna, 1965, Blumberg et aI., 1966; Jasinski et aI., 1967; McClane and

Martin, 1967). The unique, mixed agonist/antagonist, profile of action of nalorphine

was eventually critical in the understanding that opioids produce their many effects

by actions at multiple types of opioid receptors.

Discovery of Multiple Opioid Receptor TXJ)es

Although the exact mechanism(s) by which opioids produce their numerous

effects is (are) poorly understood, a significant advance in the understanding of

opioid pharmacology has resulted from experiments demonstrating opioid actions

through specific receptors. Pharmacological indicators for drug action at specific

receptors include: 1) similar molecules producing similar effects, where changes in

the molecules can result in mild to drastic alterations in pharmacologic activity

(structure-activity relationships); 2) stereoisomers usually differ in pharmacological

activity or potency; and, 3) perhaps most important of all, competitive antagonists which can specifically block or reverse the actions of an agonist without affecting the

actions of other drugs. The opioids meet these criteria for action at receptors, and

are exemplified by morphine.

In the search for better analgesic drugs countless variations of the morphine molecule have been synthesized. While the structures of these drugs can vary 24

drastically, all possess a basic center and an aromatic moiety (Casey, 1971). It has

been shown that substitutions on the nitrogen moiety of morphine lead to products

of varying agonist and antagonist activity. Replacing the N-methyl with a 3 carbon

chain of any configuration generally produces an antagonist (Casey, 1971). N-butyl

and N-ethyl derivatives have little or no agonist or antagonist activity. Increasing

chain length beyond 3 carbons, however, restores agonist activity of the molecules

as compared to the analgesic activity of morphine (Beckett and Casey, 1965). Many

other substituted morphine analogs, too numerous to mention, have also been

synthesized which possess varying degrees of either agonist or antagonist activity.

Further evidence for opioid action at a specific receptor stems from the fact

that while morphine, which occurs naturally as the levo (-) enantiomer, has

historically been known to produce antinociception, the dextro (+) enantiomer of

morphine, available only by laboratory synthesis, is practically inactive in relieving pain (Goto et aI., 1957). The most convincing evidence for opioid action at a

specific receptor came from studies which demonstrated the selective antagonism of

opioid effects (Beckett and Casy, 1965). Thus, the opioids have been shown to

satisfy the requirements for action at a receptor.

In a 1967 review, Martin (1967) synthesized much of the available literature

concerning opioids and hypothesized that morphine and nalorphine initiated their

effects by action at two separate sites: this was the concept of receptor dualism.

Martin's theory of receptor dualism suggested that at least two receptors mediated the effects of opioids, and that compounds which were agonists at one receptor 25 could function as antagonists at the other. Martin et al. (1976) and Gilbert and

Martin (1976) later showed that while a number of effects could be produced by both morphine and nalorphine, nalorphine additionally induced dysphoria and hallucinations. The concept of receptor dualism thus led to the idea that each of the receptor types may be associated with a unique spectrum of effects. Thus, drugs may be designed which act at only one receptor type, perhaps producing only desirable effects.

In the mid 1970's, Martin and colleagues provided convincing evidence that multiple types of opioid receptors existed. Experiments using the chronic spinal dog, a preparation in which the spinal cord has been surgically severed at T-10, suggested the existence of not two, but three opioid receptor typ ;!s. Martin and coworkers

(1976) demonstrated that morphine, keto cyclazocine and SKF-10047 (N­ allylnormetazocine), produced distinct constellations of effects in the dog. Morphine produced miosis, bradycardia, hypothermia, antinociception and an indifference to environmental stimuli. Ketocyclazocine produced miosis, depressed the flexor reflex and led to sedation without altering the pulse rate or the skin twitch reflex. Martin and coworkers were careful to point out that keto cyclazocine did not produce effects opposite those of morphine, but which differed from morphine only in degree. In contrast, SKF-10047 elicited a number of effects which were quite different from those produced by morphine and ketocyclazocine; these included mydriasis, tachypnea, tachycardia and mania. The effects produced by morphine, ketocyclazocine and SKF-10047 were antagonized by the narcotic antagonist 26 naltrexone, thus indicating that these three compounds acted as agonists at an opioid

receptor. Additionally, the chronic administration of all three drugs resulted in a

tolerance to their agonist effects. Based on the observed differential

pharmacological profiles of morphine, ketocyclazocille and SKF-lO047, Martin

proposed the existence of three types of opioid receptor. These receptor types were

termed 1', /C and (J after the first letter of each receptor's prototype ligand, morphine,

ketocyclazocine and SKF-l0047, respectively.

The concept of three distinct opioid receptors was supported by the fact that

in addition to their different profiles of action, these compounds failed to substitute

for one another in the suppression of the withdrawal syndrome after induction of physical dependence in the chronic spinal dog (Martin et aI., 1976). Although

morphine suppressed withdrawal in the morphine-dependent dog, keto cyclazocine

did not. In fact, not only did keto cyclazocine fail to substitute for morphine in the

morphine-dependent dog, it only marginally precipitated withdrawal as well,

suggesting to Martin and his colleagues that ketocyclazocine had little affinity for the

morphine (I') receptor (Martin et aI., 1976). These findings were extended by

Gilbert and Martin (1976) with the use of two additional drugs, cyclazocine and

ethylketocyclazocine. Based on: a) the effects of cyclazocine and

ethylketocyclazocine on the morphine-dependent dog; b) the amount of naloxone

required to induce withdrawal in cyclazocine- and ethylketocyclazocine-dependent animals; and c) the similarity in pharmacological effects between cyclazocine and

SKF-10047, and ethylketocyclazocine and keto cyclazocine, Gilbert and Martin (1976) 27 postulated that neither cyclazocine or ethylketocyclazocine were capable of action

at the p. receptor in the capacity of either an agonist, antagonist or partial agonist.

Therefore, ethylketocyclazocine was thought to be a K. agonist while morphine acted

at both the p. and K. receptor. Cyclazocine was thus postulated to act at both the K.

and u receptor whereas SKF-10047 was believed to act solely at the u receptor.

Thus, Martin and colleagues provided evidence that not only did multiple types of

opioid receptors exist, but that ligands could be designed to interact with a specific

receptor type which would elicit the effects associated only with that receptor.

Whereas much of the early evidence for the existence of a specific opioid

receptor was inferential, 1973 saw the demonstration of a specific opioid receptor

in neural tissue. Using modified radioligand binding techniques originally suggested by Goldstein et aI. (1971) for the specific detection of opioid receptors, three groups indc.!pendently identified an opioid binding site which displayed saturability and a

high degree of stereospecificity in neural tissue (Pert and Snyder, 1973; Simon et aI.,

1973; Terenius, 1973). A number of groups had previously attempted to localize the opioid receptor, but were unsuccessful (Ingoglia and Dole, 1970; Berkowitz and

Way, 1971). Noting the methodological problems one could encounter in the study of opioid receptors, Goldstein and colleagues (Goldstein et aI, 1971) exploited the stereospecificity already observed for the opioids in an attempt to bind the opioid receptor. Goldstein and his group (Goldstein et aI, 1971) used the stereoisomers

(-) levorphanol and ( + ) dextrorphan and chose to measure the difference in binding of radioactive levorphanol in mouse brain homogenates under two conditions: 1) in 28 the presence of nonradioactive dextrorphan and; 2) in the presence of nonradioactive levorphanoI. They theorized that only the (-) enantiomer would displace levorphanol binding as the ( +) enantiomer was known to be pharmacologically inert. That is to say that dextrorphan was known to be without agonist or antagonist activity indicating no specific binding to the opioid binding site.

Thus, the difference between the two binding conditions would indicate stereospecific binding to an opioid receptor. Only 2% of the binding reported by

Goldstein et aI. (1971), however, was stereospecific. Using [3H]naloxone instead of

[3H]levorphanol, Pert and Snyder (1973) demonstrated high affinity saturable binding in rat brain homogenates. The criteria used by Pert and Snyder were similar to those of Goldstein et aI., (1971). Similar to the finding of Pert and Snyder (1973),

Simon et aI. (1973), using radioactive etorphine (a potent agonist), were also able to demonstrate high affinity binding in rat brain homogenates. Furthermore,

Terenius (1973), using a fraction of synaptosomal membranes from rat brain, demonstrated a greater reduction of radioactive dihydromorphine binding by nonradioactive (-) methadone than by ( + ) methadone. Thus, three separate groups had concurrently reported the detection of highly specific opioid binding sites. The affinity of the radiolabelled opioids for the identified binding site correlated well with the antinociceptive potency of opioids in vivo, suggesting that the binding site was indeed a pharmacologically relevant receptor. In support of the relevance of this binding site as a receptor, the same pattern of stereospecificity already noted for the pharmacological effects was found in binding affinity. 29

The identification of an endogenous receptor for opioids prompted the

question as to why nature should be so benevolent as to provide man with a

mechanism by which the alkaloids of the poppy could act to relieve pain and induce

euphoria. Perhaps there existed an endogenous ligand for this endogenous binding site. Therefore, the next logical step was to attempt to isolate and identify this putative endogenous ligand. The search for an endogenous opioid proved fruitful with the initial findings of Terenius and Wahlstrom (1974) and the subsequent discovery of Hughes et aI. (1975). A peptide that possessed opioid agonist activity and an apparent molecular weight of 1000 was found in pig brain, in beef brain, and in human cerebrospinal fluid (Terenius and Wahlstrom, 1974). Shortly thereafter,

Kosterlitz and coworkers (Hughes et aI., 1975) reported the isolation and purification of two pentapeptides from porcine brain, Tyr-Gly-Gly-Phe-Met, and Tyr­

Gly-Gly-Phe-Leu named [Met]- and [Leu] enkephc..1in respectively, both of which mimicked opioid effects in bioassays in vitro (Hughes et aI., 1975; Lord et aI., 1976,

1977): the effects of these peptides were antagonized by naloxone, the prototype opioid antagonist. These investigators named the pentapeptides "enkephalins" which translates from the Greek to mean "in the head".

With the discovery of the enkephalins (Hughes et aI., 1975) came the identification of an additional opioid receptor type, the s receptor (Lord et aI., 1976;

Lord et aI., 1977). The existence of the s receptor was based first on differences in the rank order of potency of several opioid agonists (Lord et aI., 1976, 1977) in their ability to: a) depress electrically-induced contractions of the guinea-pig ileum 30 myenteric plexus-longitudinal muscle preparation (GPI) (Gyang and Kosterlitz, 1966;

Kosterlitz and Watt, 1968) and the mouse vas deferens (MVD) bioassay (Henderson

et al., 1972; Hughes et al., 1975; Lord et al., 1976, 1977), two classic systems for

demonstrating opioid agonist and antagonist activity. [Leu]- and [Met]-enkephalin

were 50- and 9-fold more potent in the MVD than in the GPI, respectively, whereas

f3-endorphin (described below) was equipotent. Morphine, and the benzomorphan

MR2034, were more potent in the GPI (7- and 21-fold, respectively). Furthermore, while [Met]- and [Leu]-enkephalin were antagonized by the same concentration of

naloxone as morphine in the GPI, approximately 10 times as much naloxone was

needed to antagonize the effects of the pentapeptides as was required for morphine

in the MVD. As differences in rank order of potency have classically been

interpreted as demonstrating heterogeneous receptor populations in test systems

(Chang and Gaddum, 1933), it was believed that the receptor popUlations in the

GPI, MVD were not homogeneous. Therefore, the studies by Kosterlitz and coworkers (Lord et al., 1976; 1977) not only supported the concept of heterogeneous

opioid receptor types, but introduced a new type of opioid receptor, the {) receptor.

The designation {) came from vas ,deferens owing to the potency of [Met]- and [Leu]­ enkephalin in the MVD. Subsequently, the GPI is now thought of as essentially a

J.L assay whereas the MVD is used primarily as a test for {) activity.

Endogenous Opioid Ligands and Receptors

Following the discovery of the enkephalins, a number of endogenous opioid 31 ligands have now been identified. It is interesting to note that identification of endogenous opioid peptides and great advances made in molecular technology have gone hand in hand. Indeed, such an association prompted Costa to write, "The opioid peptides are the most extensively characterized of all neuropeptides and thus provide valuable model systems to investigate general features such as enzymatic mechanisms of biosynthesis and their regulation." (Costa et aI., 1987). As mentioned above, the first opioid peptides to be isolated and characterized were the pentapeptides [Met]- and [Leu]-enkephalin (Hughes et aI., 1975). At least sixteen additional opioid peptides have since been identified in brain, pituitary and adrenal tissues. The variety of endogenous opioid peptides stems from three distinct precursor opioid peptides which correspond to three distinct genes: Pro­ opiomelanocortin (POMC) (Mains et aI., 1977; Roberts and Herbert, 1977;

Nakanishi et aI., 1979), pro-enkephalin A (Kimura et aI., 1980) and pro-enkephalin

B (Kakidani et aI., 1982) which is also referred to as pro-dynorphin. The three precursors share a number of common features: (1) they are made up of approximately the same number of amino acids (260); (2) they contain repetitive amino acid sequences; (3) and all include a cysteine-containing amino terminal sequence preceded by a hydrophobic signal peptide which is needed for transport of the precursor across the endoplasmic reticulum membrane (Hollt, 1985).

Additionally, although not absolute, most of the identified cleavage products of the three precursors are flanked on both sides by paired basic amino acids, such as arginine and lysine, which serve as processing signals. It is interesting to note that 32

all of the opioid peptides derived from the three precursors have either an [Met]­

or [Leu]-enkephalin at their N-terrninus (Holt, 1985).

Interestingly, while all of the endogenous opioid ligands revealed to date have been pep tides, there is a growing body of evidence supporting the existence of an endogenous morphine. Early suggestions came from the discovery of substances in the mammalian brain which were recognized by antisera raised against morphine

(Gintzler et al., 1976; 1978). More recently, Spector and colleagues have reported the presence of immunoreactive morphine in toad skin (Oka et al., 1985): additionally, they located non-peptide opioids in the skin of the rabbit and rat, and in bovine adrenal gland and brain. Furthermore, Goldstein and coworkers have demonstrated the presence of immunoreactive morphine in bovine hypothalamus and adrenal gland (Goldstein et al., 1985; Weitz et al., 1986). These findings have been supported further in studies which demonstrated the transformation of thebaine, an intermediate in morphine synthesis in the poppy plant, to morphine in mammalian (Weitz et al., 1987) and rat (Kodaira and Spector, 1988) liver preparations as well as rat kidney and brain synaptosomes (Kodaira and Spector,

1988).

Although a rather large number of endogenous opioid peptides is known to exist, onlY.B-endorphin, [Met]- and [Leu]-enkephalin and dynorphin will be discussed further as these peptides are representative of the precursors POMC, proenkephalin

A and proenkephalin B, respectively. .B-endorphin is found in large amounts in the anterior pituitary gland where it is co-stored and co-released with the POMC 33 product ACTH (Guilleman et al., 1977; Hollt, 1983) . .8-endorphin is also found in

the intermediate lobe of the pituitary gland where it is co-localized and co-released

with a-MSH, which is also derived from PO Me. Much of this .8-endorphin, however,

appears to be biologically inactive (Zakarian and Smyth, 1979; 1982). Both the

anterior and intermediate lobes of the pituitary contribute to the immunoreactive

.8-endorphin found in the systemic circulation (Millan et al., 1982; Przewlocki et al.,

1982). The majority of brain .8-endorphin is basically found only in cell bodies in the

arcuate region of the hypothalamus (Bloom et al., 1978): from here, the .8-

endorphinergic axons project to many areas of the brain which are involved in

nociceptive processes including the septum, locus coeruleus, thalamus and the periaqueductal grey (Finley et al., 1981; Zakarian et al., 1982).

Similar to .8-endorphin, dynorphin can also be found in the central nervous system and in the pituitary gland. A high molecular weight form of dynorphin can be found in large amounts in the anterior lobe of the pituitary, although the function of such a large form is not clear at this time (Seizinger et al., 1984). Dynorphin containing nerve terminals, whose axons originate in the paraventricular and supraoptic nuclei are found in the neural lobe of the pituitary gland (Watson et al.,

1982; Millan et al., 1984). Dynorphin is also found in the periaqueductal grey, the limbic system and the thalamus (Watson et al., 1982; Khachaturian et al., 1983;

Millan et al., 1984; Millan et al., 1985). More significant is the dense population of dynorphin containing neurons in the dorsal horn of the spinal cord, especially in laminae I and V, which are critical locations of nociceptive processing. 34

Enkephalinergic neurons are more widely distributed in the central nervous system than ,B-endorphin containing neurons. Unlike ,B-endorphin, [Met]- and [Leu]­ enkephalin are not found in any significant concentrations in either the anterior or intermediate lobes of the pituitary. Nerve terminals containing [Met]-enkephalin which originate in the magnocellular paraventricular and supraoptic nuclei of the hypothalamus are found in the neural lobe of the pituitary gland (Martin et al.,

1983). Systemic [Met]-enkephalin, however, is believed to come from the adrenal medulla where [Met]-enkephalin is co-released with catecholamines (Yang et al.,

1980). [Met]-enkephalin is widely dis'ulbuted in the brain, and importantly is found in cell bodies and axons in the amygdala, thalamus, hypothalamus, dorsal raphe and periaqueductal grey, areas all believed to be involved in nociceptive processes

(Khachaturian et al., 1982). Additionally, [Met]-enkephalin is found in large amounts in laminae I, II and V of the dorsal horn of the spinal cord which receive primary afferent nociceptive information (Khachaturian et aI., 1982; Basbaum and

Fields, 1984; Cruz and Basbaum, 1985). It is believed that the enkephalinergic neurons receive descending serotonergic input from the periaqueductal grey and medullary reticular formation which stimulates (or disinhibits) the release of enkephalin which acts presynaptically on primary afferent sensory cells in the dorsal horn of the spinal cord to inhibit nociceptive processing.

Similar to the wide distribution of the endogenous opioid ligands, utilization of highly selective ligands in both autoradiographic and radioligand binding studies 35 performed in many species has provided evidence for the presence of 1-', s and It receptors in the central nervous system. As the distribution of opioid receptors in the rat is believed to be quite similar to that in man (Millan, 1986), the following discussion of opioid receptor distribution derives from studies in the rat except where indicated. These studies show I-' receptors to be present in striatal patches, laminae III and IV of the cortex, the thalamus and periaqueductal grey (Millan,

1986). The s receptor has been located in the pontine nucleus, parts of the amygdala, olfactory bulbs and deep cortex (Tempel and Zukin, 1987). It receptors have been located in high amounts in the hypothalamus, periaqueductal grey and claustrum (Millan, 1986). In the spinal cord, both I-' and It receptors are found in the substantia gelatinosa while s receptors can be found primarily in the marginal layer in both humans (Czlonkowski et al., 1983) and the rat (Tempel and Zukin,

1987). Clearly, the opioid receptors, as well as the opioid ligands, are in a position to play active roles in antinociceptive processes.

It is important to note that opioid action in man is not limited to the perception of pain via blockade of ascending nociceptive information (described above). In man, opioids also act to modify the conscious appreciation of pain, the acceptance of pain and the anxiety and stress associated with pain by actions at higher brain centers (Pawl, 1979). This is reflected by the distribution of opioid­ containing nerve fibers and receptors. Opioid systems found in the spinal cord, periaqueductal grey and medial parts of the thalamus act to decrease the direct transmission of nociceptive information, whereas opioid systems located in the amygdala, frontal cortex, hypothalamus and in the basal ganglia are associated not 36 with the transmission of pain signals, but with how pain is perceived (Pawl, 1979).

Endogenous Pain Control Systems

As the complexities of the endogenous opioid systems were beginning to unfold, the observation was made that electrical stimulation of the periaqueductal gray (PAG) in rats produced a profound level of antinociception (Reynolds, 1969;

Mayer et al., 1971; Mayer and Leibskind, 1974). The stimulation-produced antinociception was quite selective in that electrically stimulated animals remained alert and responsive to non-noxious stimuli while not responding to noxious stimuli.

These findings in the rat were then extended to humans with intractable pain

(Adams, 1976; Hosobuchi et al., 1977). The observation that stimulation of a discrete brain area was capable of eliciting an antinociceptive response suggested that some endogenous system for controlling pain existed. Interestingly, stimulation­ produced antinociception has a number of parallels to morphine-induced antinociception. Perhaps the strongest parallel is that stimulation-produced antinociception is naloxone reversible (Akil et al., 1972). Additionally, as seen with morphine-induced antinociception, stimulation-produced antinociception is prone to tolerance (Akil et al., 1972).

Basbaum and Fields (1978) proposed a three-tiered model for an endogenous pain control system where the major components include the PAG, a number of rostral ventral medullary nuclei (especially the midline nucleus raphe magnus 37 (NRM), and the dorsal horn of the spinal cord (Fig. 1). Based on the three-tiered model for pain control, it was proposed that stimulation of the P AG activates

excitatory connections between the P AG and the medullary raphe. Once activated, the raphe neurons, which project to the dorsal horn and nucleus caudalis of the trigeminal nerve via the dorsal lateral funiculus (DLF) (Basbaum and Fields, 1979), selectively inhibit dorsal horn and nucleus caudalis nociceptive neurons (Fields et al.,

1977).

The pain control model of Basbaum and Fields also (1978) links endogenous opioids with pain control at both supraspinal and spinal levels. In addition to, the presence of opioid peptides and receptors in the PAG (Atweh and Kuhar, 1977), functional evidence exists which links the opioid peptides with the endogenous pain control systems. Such evidence includes the findings that the antinociceptive effects of systemically administered opioids are reversed by microinjection naloxone into the

PAG (Tsou and Jang, 1964; Yeung and Rudy, 1980a), and that the antinociceptive effects of opioids microinjected into the PAG are reversed by the physical disruption of the spinal dorsolateral funiculus (Murfin et al., 1976). These findings led to the suggestion that a common neural mechanism underlies both opioid and stimulation­ produced antinociception (Mayer and Liebeskind, 1974; Mayer and Price, 1976).

The second opioidergic site in the descending pain control model of Basbaum and Fields (1978) is the spinal dorsal horn. As described above, this area of the spinal cord is rich in both opioid peptides and opioid receptors. It is believed that descending serotonergic raphe-spinal axons mediate their antinociceptive effects, at 38 least in part, through synaptic connections with opioidergic neurons in the cord (Fig.

2). Functional evidence for serotonergic-opioidergic connections was provided by

Zorman and coworkers (1982) who reported that spinally administered that naloxone antagonized the antinociception produced by stimulation of the raphe nuclei.

Additionally, serotonin-containing nerve terminals have been demonstrated to lie presynaptically to enkephalin-containing neurons in the dorsal horn (Basbaum et al., 1982; Glazer et al., 1981). A descending catecholaminergic system has also been implicated in pain control, but is less well understood (Basbaum and Fields, 1984).

The neurochemistry and circuitry of the endogenous pain control system will be discussed below.

The PAG would appear to sit in a key location for the control of nociception.

Anatomical studies have shown that the PAG receives a significant degree of input from the amygdala, insular cortex, the frontal cortex and the hypothalamus (Beitz,

1982a; Mantyh, 1983). Inasmuch as these areas of the brain are associated with the emotional component of pain, afferent input from these areas anatomically places the PAG in a position to be involved directly with the cognitive aspects of pain control. The PAG also receives input from the nucleus cuneiformis and pontine reticular formation of the brainstem. It is believed that these two areas of the brainstem provide a processing site for nociceptive input which stimulates PAG neurons (Gebhart, 1982): once activated, the PAG stimulates the rostral medulla.

A number of tracing methods have demonstrated the PAG connections to the rostral medulla (Gallagher and Pert, 1978; Abols and Basbaum, 1981; Mantyh, 1983). 39 The rostral medulla is the major source of neurons which project to the spinal

cord via the DLF (Leichnitz et aI., 1978; Martin et aI., 1978; Basbaum and Fields,

1979). A number of regions of the medulla contribute axons to the DLF which are

involved in antinociceptive processes. These include the nucleus raphe magnus

(NRM), the reticularis paragigantocellularis, the nucleus reticularis gigantocellularis

and the nucleus reticularis paragigantocellularis lateralis. Each of these medullary sites which send axons to the spinal cord via the DLF receive input from the PAG

(Beitz, 1982b; Mantyh, 1983). Additionally, when electrically stimulated, all are capable of eliciting antinociception (Zorman et at, 1981). Furthermore, to block completely the antinociceptive effect of midbrain stimulation, the NRM, the reticularis paragigantocellularis, the nucleus reticularis gigantocellularis and the nucleus reticularis paragigantocellularis lateralis must all be interrupted concurrently

(Sandkuhler et aI., 1982; Prieto et aI., 1983). Analogous to the PAG, local injection of opioids into the rostroventral medulla also produces antinociception. Using reasoning similar to that which they used for the PAG, Basbaum and Fields (1984) have suggested that medullary opioid-induced antinociception arises from the disinhibition of catecholaminergic interneurons. Such a disinhibition would, therefore, activate the medullary output neurons of the DRF.

Unlike the PAG and rostral medulla where opioids indirectly activate descending control systems, opioids produce their spinal antinociception by direct inhibition of ascending nociceptive signals. There is a good deal of evidence suggesting that spinal opioids act presynaptically to control primary afferent neurons. 40

Descending serotonergic 1 pathway

Enkephalinergic inhibitory interneuron

To spinothalamic tract

Fig. 2. Proposed neurocircuitry of spinal dorsal horn pain pathway neurons. 41 Primary afferents in the dorsal horn possess large populations of opioid receptors

(LaMotte et al., 1976; Atweh and Kuhar, 1977; Hiller et al., 1978; Fields et al.,

1980) and are inhibited by opioids both in VillO (Carstens et al., 1979) and in vitro

(Hentall and Fields, 1983). Additionally, opioids inhibit the release of substance P,

the putative neurotransmitter in small primary afferents, in both in vivo (Yaksh et

al., 1980; Kuraishi, 1983) and in vitro (Jessell and Iverson, 1977; Mudge et al., 1979)

systems, although an opioid-substance P synapse has yet to be conclusively

demonstrated.

Thus, opioids can act at numerous sites in the central nervous system to

inhibit the transmission of nociceptive signals. Although the circuitry is not

understood, there also appears to be some interplay between supraspinal and spinal

sites which mediate opioid-induced antinociception. Studies using isobolographic

techniques have demonstrated a synergism between the antinociceptive effects of

opioids administered in the brain and cord in both mice (Roerig et al., 1984) and

rats (Yeung and Rudy, 1980b). These findings suggested that the antinociception

produced by systemic administration of morphine may be the result of synergistic

action between supraspinal and spinal sites. Such a synergistic interaction between

the brain and cord has also been recently demonstrated for a second opioid

mediated effect, the inhibition of gastrointestinal transit in the mouse (Jiang et al.,

1987). Interestingly, where the antinociceptive effects of supraspinally administered opioids appear to be mediated primarily through descending serotonergic neurons,

the interactions between supraspinal and spinal opioids may be more dependent on 42 a descending noradrenergic system (Wigdor and Wilcox, 1987).

Statement of the Problem

Owing to the clinical importance of opioid use in the management of human pain, the study of opioid systems has great applicability. The many endogenous opioid ligands and receptors, however, greatly complicates the understanding of the functions mediated by opioid systems. It is desirable, therefore, to understand the functions mediated by activation of specific receptors. By correlating specific opioid ligands and receptors with distinct physiologic functions, we may achieve the development of receptor selective/specific drugs which are capable of eliciting only the desired effects of opioids. It is a long sought goal to have analgesic agents which do not produce physical dependence, respiratory depression, constipation or tolerance, always undesirable side effects associated with the clinical use of opioids for the relief of pain.

It is important to recall that while the existence of the p., It and (J receptors has been based on findings from studies carried out in vivo (Gilbert and Martin,

1976; Martin et aI., 1976), the existence of the s receptor has generally been accepted based on findings in vitro (Lord et aI., 1976, 1977). This discrepancy, therefore, makes it difficult to correlate in vivo functions with central 0 receptors despite the number of effects produced by administration of 0 ligands. The interpretation of receptor involvement in these effects is difficult due to the interaction of most opioids at one or more of the multiple receptor types which have 43 been described in detail above. This problem has been difficult to resolve due to the previous lack of compounds with both a high degree of selectivity for anyone opioid receptor type, and biological half-lives which were suitable for use in vivo.

Several compounds, however, have recently been synthesized which are stable in vivo and which show a high degree of selectivity for individual opioid receptor types.

The development of selective agonists and antagonists has finally begun to allow some insights as to the pharmacological, if not physiological, function of distinct opioid receptor types. Of vital importance to the determination of central 6 receptor

2 function is the highly selective 6 agonist [D-Pen , D-pen6]enkephalin (DPDPE), where Pen is penicillamine (Mosberg et aI., 1983), and the competitive 6 antagonist

N,N-dial(yl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), where Aib is a-aminoisobutyric acid (Cotton et aI., 1984).

Previous to the synthesis of DPDPE, there were a lack of compounds which possessed both a high degree of selectivity for the 6 receptor and which were resistant to enzymatic degradation allowing for their use in studies in vivo. Due to the extremely short half-lives of the enkephalins (Hambrook et aI., 1976), functions mediated by the 6 receptor have been difficult to investigate. To circumvent this problem, enkephalin analogs were synthesized with D-amino acids substituted along the enkephalin sequence making these compounds resistant to enzymatic degradation. Unfortunately, such substitutions resulted in a loss of 6 selectivity

(Mosberg et aI., 1983; Goldstein and James, 1984). In response to this dilemma, the cyclic enkephalin analog DPDPE was designed and synthesized at the University of

Arizona by Drs. Henry Mosberg and Victor Hruby (Mosberg et aI., 1983). DPDPE 44 is both highly selective for the s receptor and possesses stability suitable for use in vivo (Mosberg et aI., 1983). With DPDPE, the functions of central S receptors can more easily be addressed.

Although opioid antinociception has traditionally been associated with the p. receptor, especially supraspinally mediated antinociception (Audigier et aI., 1980;

Chang et aI., 1982; Chaillet et aI., 1984), evidence for the involvement of central S receptors has slowly been accumulating. More specifically, s receptors have been hypothesized to play a key role in both the mediation, and modulation of antinociceptive processes. The first evidence for S involvement, albeit indirect, came from studies with the enkephalin analog metkephamid (Frederickson et aI., 1981), which was 30-100-fold more selective for the s receptor than the p. receptor in vitro.

Metkephamid was a systemically active antinociceptive agent which was more potent than morphine. Of great clinical, as well as pharmacological, importance was the fact that metkephamid possessed a decreased tendency to produce respiratory depression, tolerance and physical dependence. Therefore, although the selectivity exhibited by metkephamid would suggest the likelihood of interaction at both p. and s sites in vivo, the compound was significant in suggesting that s receptors might be capable of mediating antinociception. Central S receptors were further implicated in the direct mediation of antinociceptive processes in the studies which initially characterized the pharmacology of DPDPE. In these studies, the p. selective agonists

2 4 [D-Ala , NMePhe , Gly-ol]enkephalin (DAMGO)(Handa et at, 1981) and the p. preferring morphine were shown to produce both antinociception and inhibit gastrointestinal propulsion after administration into the brain, whereas the s agonists 45

2 DPDPE and [D-Pen , L-pen6]enkephalin (DPLPE)(Mosberg et al., 1983) given by the same route produced only antinociception with no effect on gut propulsion

(Galligan et al., 1984; Porreca et al., 1984). Both the p. and 0 agonists, however, produced antinociceptive and antitransit effects following administration into the spinal cord (Porreca et al., 1984). It was reasoned therefore, that activation of the p. receptor produced both inhibition of gastrointestinal transit and antinociception.

If DPDPE and DPLPE acted at the p. receptor to produce antinociception, then these compounds should also inhibit gut propulsion when administered supraspinally.

As they did not, the antinociception must be the result of activity at 0 receptors.

In addition to directly mediating antinociception, central 0 receptors have been suggested to modulate p.-mediated antinociception. Sub-effective doses of the o selective [Leu]-enkephalin and [Leu]-enkephalin analogs were found to potentiate

(Vaught and Takemori, 1979; Vaught et al., 1982), while [Met]-enkephalin and

[Met]-enkephalin analogs attenuated morphine antinociception (Lee et al., 1980;

Vaught et al., 1982).

If central 0 receptors are indeed capable of affecting nociceptive processes, then perhaps drugs may then be developed which selectively activate 0 receptors to either directly produce antinociception, or to augment p.-mediated antinociception both in terms of potency and efficacy. Accordingly, the goal of the present study was to correlate central opioid 0 receptors, for which [Leu]- and [Met]-enkephalin are presumed to be the endogenous ligands (Lord et al., 1977), with: a) the direct production of antinociception: and b) the indirect modulation of antinociception in vivo. 46

Approach

Inasmuch as the compounds used in the present work are only selective, and

not specific for anyone type of opioid receptor, and because different receptors may

mediate similar functions, the results of a single approach would be unlikely to

reliably correlate function with a specific receptor type. By using a number of

approaches, however, one can attempt to establish distinct and independent lines of

evidence which converge at an unequivocal conclusion. Therefore, a number of

classic pharmacological methods were used in parallel to address the question of

central S receptor involvement in nociceptive processes. The following methods were used in parallel to address the question of central S receptors in the direct

mediation and indirect modulation of antinociception: 1) the determination of the naloxone pA2 against several selective agonists; 2) the determination of possible

cross-tolerance between the sand JL selective agonists; and 3) the differential antagonism of antinociception by highly selective antagonists. The compounds used in these studies along with their receptor selectivity are shown in Table 1. 47

TABLE 1

A guide to the compounds used according to function and receptor selectivity

Agonists:

Mu selective:

2 4 [D-Ala , NMePhe , Gly-ol]enkephalin (DAMGO) 3 4 [NMePhe , D-Pro ]morphiceptin (PL017) Morphine Normorphine Phenazocine Sufentanil

Delta selective:

2 [D-Pen , D-pen5]enkephalin where Pen is penicillamine (DPDPE) 2 5 [D-Ala , Met ]enkephalinamide (DAMA)

Non-selective:

.B-endorphin (human) Etorphine

Antagonists:

N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, where Aib is Q-aminoisobutyric acid (leI 174,864) (0 selective) .B-funaltrexamine (.B-FNA) (Jl selective) Naloxonazine (Jll selective) 48

METHODS

Animals

Male ICR mice (20-25 g) (Harlan, Indianapolis, IN) were used in all experiments. The animals were housed in groups of 5 in a temperature controlled room with a 12 h light cycle (lights on at 07:00 h). Food and water were available continuously, except in studies of gastrointestinal propUlsion where food was removed from the home cage 18 h prior to testing.

Chemicals

DPDPE (generously provided by Dr. Henry I. Mosberg, College of Pharmacy,

University of Michigan, Ann Arbor, MI), DADLE, DAMA, DAMGO, PL017, f3- endorphin (Penninsula Laboratories Inc.) and ICI 174,864 (Cambridge Research

Biochemicals) were dissolved in distilled water, frozen in aliquots and lyophilized; they were redissolved immediately before use. Morphine sulfate (Mallinckrodt Inc.), naloxone HCl (Sigma), f3-FNA (Research Biochemicals), phenazocine HCI, etorphine

HBr, sufentanil HCI (all generously provided by Dr. Alan Cowan, College of

Medicine, Temple University, Philadelphia, PA) and naloxonazine HCI (a generous gift from Dr. Diane DeHaven, NOVA Pharmaceutical Co.) were dissolved in distilled water just prior to administration. 49

Injection Techniques

Intracerebroventricular injections.

Intracerebroventricular (i.c.v.) injections were made into the lateral cerebral

ventricle according to the modified method of Haley and McCormick (1957) as

described previously by Porreca et al. (1984). The mice were lightly anesthetized with ether, an incision was made in the scalp and bregma was located. The injections were made 2 mm caudal and 2 mm lateral to bregma at a depth of 3 mm using a Hamilton microliter syringe with a 26-gauge needle.

Intrathecal injections.

The intrathecal (i.th.) injections were made into the spinal subarachnoid space using a modified version of the method of Hylden and Wilcox (1980) as previously reported (Porreca and Burks, 1983). Injections into the subarachnoid space were made in unanesthetized mice using a Hamilton microliter syringe fitted with a 30- gauge needle by direct lumbar puncture. All i.c.v. or i.th. injections were made in a volume of 5 ILL 50

Mouse Warm-Water Tail-Withdrawal Test

The test of antinociception used in the present investigation was the tail­ withdrawal test. Initially, antinociceptive testing was done over a time-course

described below. The reasons for initially carrying out a time-response study were

two-fold. First a time-response was needed for the determination of the time­ dependent apparent naloxone pA2 (described below). Second, the time-response studies allowed the determination of the time of peak effect for the agonists by both the i.c.v. and i.th. route of administration. Administering the compounds by these routes allowed for improved precision by delivering compounds close to their loci of action thereby permitting a dose unit more closely approximating tissue concentration as well as more predictable pharmacokinetics. The times of peak effect for all agonists by each route were then used for all other anti nociceptive studies. Warm (55°C) water was employed as the nociceptive stimulus according to the method of Jannsen et al. (1963). Prior to agonist administration, the tail of each animal was immersed in the water ann. the latency to a rapid flick recorded (control latency). Animals not flicking their tails within 5 sec were not used in the study. At test-dependent post injection times described below, testing was repeated. Animals not flicking their tails within 15 sec were assigned a maximal score of 100% antinociception to avoid tissue damage to the tail. Each response latency was then compared to the individual control latency for each mouse. Antinociception was 51 expressed as :

% antinociception = 100 x (test latenc.y - control latenc.y) (15 sec - control latency)

Gastrointestinal Transit

Gastrointestinal transit was evaluated as previously described by Porreca and

Burks (1983). Each mouse received a single injection of test compound or vehicle

followed by an immediate oral injection of 5Icr as sodium chromate-saline (0.5 J.lCi,

0.2 ml/mouse) (ICN Corporation, Irvine, CA). Thirty-five min after administration

of the marker, the animal was killed by cervical dislocation and the stomach and small bowel excised. The small intestine was placed on a ruled template and divided into 10 equal portions. Each of the 10 intestinal segments (and the stomach) was placed into individual and consecutive culture tubes and the radioactivity in each tube determined by gamma counting for 1 min. Movement of the marker along the intestine was calculated using the geometric center (G.c.) method (Miller et aI., 1981) in which values range from a low of 1.0 (all radioactivity present in the first intestinal segment) to a high of 10 (complete transit of the marker to the most distal intestinal segment). In order to compare the mean G.c. of the agonist groups, the G.c. for each animal was compared to the mean G.c. of the control (distilled water) group according to the formula:

% inhibition = 100 x (test G.C. - control G.c.) (1.0 - control G.c.) 52

Time-dependent Naloxone pA2 Theory and Determination

Theory.

The pA2 of a competitive antagonist was defined by Schild (1947) as the negative logarithm of the molar concentration of the antagonist which reduces the effect of a dose of agonist to that of half the agonist dose. The determination of pA2 was first introduced by Schild (1947) in an effort to impart an empirical nature to the strength of a competitive antagonist. pA2 is theoretically the same as the dissociation constant of an antagonist (KB) and can be used to help differentiate the receptor types through which agonists produce similar effects. If two agonists produce the same effect, it is possible that they do so by acting at the same receptor.

If, indeed, the two agonists in question are acting at the same receptor, then their actions should be blocked or reversed to the same extent by the same competitive antagonist; therefore, the dissociation constant, KB, for the competitive antagonist should be the same against both agonists. The determination of two different KB values for a competitive antagonist against two agonists producing the same effect is indicative of the two agonists acting at different receptors. Identical KB values, however, suggests that the two agonists are acting at the same receptor to produce their common effect.

Determination of in vivo pA2' however, presents some special problems as the 53 administered doses of agonist and antagonist are used in the calculation with the assumption that these doses are proportional to the tissue concentration. Further, the tissue concentration is assumed to be maximal at the time of peak effect and the time to peak effect can be different for each dose, thus it is often difficult, if not impossible, to make the measurement of effect at the appropriate time for each dose. These factors, therefore, make interpretation of in vivo pA2 values difficult

(see Tallarida et al., 1979 for review). A variation of the traditional pA2 method, therefore, has been employed. This method includes time as a variable and thus obviates many of the concerns of in vivo pA2 studies, such as making of measurements at the time of peak effect and the assumption of equilibrium conditions. This method has been previously derived (Tallarida et al., 1978) and validated (Porreca et al., 1981). Additionally, unlike studies in the past in which either the agonist or antagonist, or both, were delivered peripherally, all compounds in the present study were administered directly into the central nervous system.

The time-dependent form of the equation for competitive antagonism was employed as previously described by Tallarida and associates (Tallarida et al., 1978).

Briefly, the dissociation constant (KB) of a competitive antagonist of concentration

B is computed by the time-independent equation of Arunlakshana and Schild (1959):

log ([A']/[A] - 1) = log [B] - log KB (equation 1) where [A'] and [A] are equieffective agonist concentrations in the presence and in the absence of the antagonist [B]. If a concentration of antagonist [B] resulted in a dose ratio of 2, the left side term of the equation, log ([A']/[A]-1), would equal 54 o and [B] would simply reveal KB• As it is nearly impossible to choose a concentration of antagonist that produces a precise 2 fold shift in the dose-response line, in practice, several concentrations of antagonist are used which result in specific dose ratios. Plotting log (dose ratio - 1) against the corresponding -log [B] results in a straight line with a slope of -1 provided the antagonist acts in a competitive fashion. This plot is commonly called the Schild plot (Arunlakshana and Schild,

1959). The negative common logarithm of KB in molar units is called the pA2 where

pA2 = -log KB (equation 2) and can be determined graphically by determining the x-intercept of the Schild plot.

A modification of equation 1 which considers time assumes that both agonist and antagonist concentrations decrease exponentially after reaching maximum concentration. Thus, A = Aoe-a~ and B = Boe-,B~ where [Ao] and [Bol are the maximum concentrations, a and /3 are the rate constants for the disappearance of

[AJ and [BJ, respectively, and t is the time after peak effect. From these, the time dependent form of the Arunlakshana and Schild equation (equation 1) is:

log ([Ao]'/[Aol - 1) = log [Bol - /3log(e)t - log KB (equation 3)

If log ([Ao'J/[AoJ - 1) is plotted against time, a straight line results of slope =

-/3log( e) and intercept = (log Bo - log KB). As pA2 = -log KB, then pA2 = intercept - log Bo' Additionally, the rate constant, /3, for the disappearance of the antagonist, and resulting half-life, follows. It should be pointed out that by using the time­ dependent method one does not need to make assumptions regarding the time of peak effect of the agonist and the agonist plus antagonist. This consideration 55 increases the suitability of this approach for study in vivo.

pA2 calculation.

Warm (55°C) water was employed as the nociceptive stimulus according to the method of Jannsen et al. (1963) described above. After the determination of control responses, groups of mice then received i.c.v. or i.th. injection of distilled water

(vehicle) or naloxone, followed after 5 min by a dose of i.c.v. (in the same ventricle) or i.th. agonist. The dose of naloxone used varied, and was selected on its ability to produce an approximate 40% reduction in the maximum agonist effect (EmroJ for the high dose of each agonist. Testing took place after a further 5, 10, 20, 40, 50,

65, 80, 95, 110 and 125 min for both routes of administration, the antinociceptive response was calculated and time-response curves were plotted. From the time­ response curves, four time points which displayed good dose-response relationships were selected for each agonist by both routes; an example of this is seen in Figure

3. For each time selected, dose-response lines were constructed by standard regression techniques described by Tallarida and Murray (1986)(procedure 6). The dose-response lines were then tested for parallelism according to the program of

Tallarida and Murray (1986)(procedure 7). Dose ratios (A' / A) were determined by establishing the best parallel regression lines in the absence and in the presence of all naloxone doses again using the computer program of Tallarida and Murray

(1986)(procedure 8). The log (dose ratio - 1) for each time point selected was then 56 plotted against its respective time. pA2' which has been defined as the intercept on

the ordinate, - log Bo, was calculated by the computer program of Tallarida and

Murray (1986) (procedure 1O). Additionally, the 95% confidence limits (C.L.) for

the pA2 value of each agonist and route were estimated from the error of the intercept on the ordinate; thus the 95% c.L. was "t" times the standard error of the

intercept, where "t" represents the area under the t-distribution curve based on the

size of the sample. The 95% C.L. of the naloxone half-life was similarly determined from the confidence limits of the slope of the regression line obtained when plotting log (dose-ratio - 1) vs. time.

Tolerance and Cross-tolerance Studies

The test of antinociception was the tail withdrawal test described previously.

A single subcutaneous (s.c.) dose of morphine sulfate (100 mg/kg) was used to produce ~cute tolerance (Vaught and Takemori, 1979). Subcutaneous administration of distilled water (1 ml/kg) served as the control. Antinociceptive testing (tail withdrawal) was performed at a time when the drug pretreatment had no effect on control reaction times, generally 5 hr after morphine pretreatment. Treated mice whose control latencies were not within the control (1 ml/kg distilled water s.c.) reaction range were excluded from the study. Morphine and DPDPE were administered to the control and morphine pretreated mice and testing took place 20 min after i.e.v. and i.th. administration; i.th. DPDPE was tested at 10 min post 57 injection. These were the times of peak effect as determined from time-response

studies characterized above (Figs. 3 and 4).

Antagonism Studies

A number of receptor selective antagonists were used in these studies. These

antagonists included the competitive, s-selective ICI 174,864 (N,N-diallyl-Tyr-Aib­

Aib-Phe-Leu-OH, where Aib is a-aminoisobutyric acid)(Cotton et a1., 1984), the non­ surmountable JL-selective ,B-funaltrexamine (,B-FNA)(Portoghese et a1., 1980) and the long-acting JL antagonist, naloxonazine (Hahn et a1., 1982). Antinociception was measured using the tail withdrawal test described above. All agonists were tested

20 min after administration by both Lc.v. and Lth. routes with the exception of Lth.

DPDPE which was tested after 10 min. These were the times of peak agonist effect as determined from the time-response curves obtained previously (Figs. 3 and 4).

Gastrointestinal propulsion was determined as characterized above. All agonists were tested 35 min after their administration by both i.e.v. and i.th routes.

leI 174,864 administration.

ICI 174,864 (1.5 or 4 nmol) was administered concomitantly in the same i.c.v. or Lth. injection with the agonists and testing was carried out as described above.

Neither i.c.v. nor i.th. ICI 174,864 alone produced antinociception at the time of testing. S8

(3-FNA pretreatment.

The i.c.v (3-FNA pretreatment used was basically that described by Ward and

Takemori (1983). A single i.c.v. injection of 18 nmol was made 4 hr prior to testing.

For the i.th. experiments, graded doses of (3-FNA were given as a single i.th. pretreatment 4 hr prior to antinociception testing. Neither i.c.v. nor i.th. (3-FNA pretreatment produced antinociception alone at the time of testing. I.c.v. distilled water served as the control in these experiments.

Naloxonazine pretreatment.

All mice received a single s.c. injection of distilled water (vehicle control) or naloxonazine Hel (35 mg/kg) 24 hr prior to testing as described by Ling et al.

(1986). This pretreatment did not produce antinociception alone, nor did it affect gastrointestinal propulsion.

Modulation Studies

A series of modulation experiments was carried out in an effort to determine the effects of 0 agonists on J.L-mediated antinociception using the tail withdrawal test described previously. First, the effect of DPDPE co-administration on morphine antinociception at both supraspinal and spinal levels was studied. Secondly, the 59 effect of DPDPE and leI 174,864 on the antinociception produced by a series of chemically diverse IL agonists was studied. Finally, the effects of IL selective antagonists on S-IL interactions were addressed.

Studies with morphine.

Mter the determination of the i.c.v. DPDPE dose-response curve in naive mice, a dose of DPDPE (1.6 nmol) which produced barely detectable antinociception was chosen by extrapolation. To determine if this dose of DPDPE potentiated morphine antinociception, it was co-administered in the same i.c.v. injection as graded doses of morphine as described previously (Vaught et aI., 1982).

Testing took place 20 min after i.c.v. administration. The identical protocol was used for i.th. administration of DPDPE and morphine, the only difference being the dose of DPDPE (0.464 nmol) co-administered with morphine. To determine the mechanism through which DPDPE might modulate morphine antinociception, these experiments were repeated in morphine tolerant and in control mice in the presence of the S antagonist leI 174,864 (4 nmol i.c.v.) using the methods described above.

This dose of leI 174,864 was chosen as it completely antagonized the antinociceptive effects of DPDPE (Fig. 11). 60

Studies with chemically diverse J.L agonists.

Time-response and dose-response curves for the p. agonists morphine, normorphine, DAMGO, PL017, p-endorphin, etorphine, phenazocine and sufentanil were determined after i.c.v. administration using the tail withdrawal test. To determine the modulatory effects of DPDPE on the antinociception produced by the various p. agonists, DPDPE (1.6 nmol) was co-administered in the same i.c.v. injection with graded doses of the p. agonists as described above. Antinociceptive testing took place 20 min after i.c.v. injection. Additionally, the s selective antagonist leI 174,864 (4 nmol) was co-administered in the same i.c.v. injection as the agonists and antinociception was determined 20 min after injection.

Effect of p. selective antagonists on S modulation of p.-mediated antinociception.

Time- and dose-response lines for i.c.v. DPDPE (figs. 3 and 8, respectively), morphine (figs. 3 and 7, respectively) and DAMA (figs. 31 and 32, respectively) in control mice were obtained. Following the determination of control values, i.c.v.

DPDPE, morphine and DAMA dose-response curves were obtained in p-FNA (figs.

15, 13 and table 9, respectively) and naloxonazine (figs. 19, 17 and table 10, respectively) pretreated mice, doses of DPDPE and DAMA which produced barely detectable antinociception (0-5%) in the respective groups were chosen by extrapolation of the dose-response line. In order to determine if DPDPE and/or 61

DAMA were capable of modulating i.c.v. morphine antinociception in animals pretreated with ,B-FNA or naloxonazine, the 0 agonists were co-administered in the same i.c.v. injection with morphine as previously described. Testing took place 20 min after injection. 62

RESULTS

Estimation of the Naloxone pA2 for Morphine. DAMGO and DPDPE

Time-response curves for morphine, DAMGO and DPDPE in the absence

(top row) and in the presence (bottom row) of a single Lc.v. dose of naloxone (1.25,

1.25, and 2.5 nmol, respectively) are shown in Fig. 3 and for a single Lth. dose of naloxone (2.25, 1.25, and 0.25 nmol) are shown in Fig. 4. The agonists showed a duration of action ranging between 80 and 100 min after either Lc.v. or Lth. administration. From these paired curves, the response in the absence, and in the presence, of naloxone was plotted at each dose of agonist at various times. An example of such a plot for Lc.v. DPDPE is shown in Fig. 5. These plots for other agonists and routes were similar, showing a decrease in dose-ratio with time, indicative of the disappearance of naloxone. The log (dose-ratio - 1) obtained from plots such as those in Fig. 5 are shown for each agonist and route in Fig. 6. Each of the lines showed excellent correlation, strongly supporting the validity of the theoretical assumptions. Note that the slopes of these lines (Fig. 6) do not reflect whether antagonism is competitive (as shown by a slope of unity in the traditional

Schild plot) but are the result of the kinetics of naloxone disappearance from the biophase. From the plots in Fig. 6, the pA2 (and corresponding 95% CL.'s) were Uorphine OAGO DPOPE '00 '00 100 ~" w,j eo 80 .,; +1 60 U"'-l g60 r "'-1 ''.»~a .LJ - "-~ .~ 40 ~\r f:~j ""-1 ~ 20 (r~ / Q.3 ~ !..'l.x-""",,: .. , 90 '00110'20,30 Y 60 70 eo 90 ,00 110 '20'30 .. 10 20 3D"'" 40 50 60 70 80 90 '00110'20'30 o 10 20 30 40 50 60 70 eo 10 20 3D 40 ~me {min} Time (min) o Time (min)

'00 '00 '00

w,j 80 w,j 80 w,j 80 .,; .,; .,; +1 ~""-1 +, .. g 60 g 60 1 , I 60 ~ ~ ] -g 40 og 40 1 " rt--... ~ ~ ~ 20 .. .~~y it"' t.. .. 20 .. 0 0 Tim. (min) Time (min)

Fig. 3. Time-response curves for i.c.v. morphine. D~IGO and DPDPE antinociception in the absence

(top row) and presence (bottom row) of a single. i.c.v. dose of naloxone (1.25, 1.25 and 2.5 nmol respectively) given 5 min prior to agonist injection. Dose of agonist administered is denoted by the value next to each curve (nmol/mouse).

(;\ VI .....".,. ""'" O?DPE "Ml 100 100

eo 80

"-+ 0' r==:G":'>! 0 L' -~-~--~--==..::::Il--::' o 10 20 JO 40 50 60 70 60 90100"0'20130 0 TIfI'II:(min) Time (min) Time (m'fI)

100 100

.. 80 .,;

.~ '0 » ~:~/~/~ .! 6°f~' ~40 ~~J~ ~ ;§ {/~ ~ 'Of yi~k ... ! 20 t .. 20 i I ~

0 0 10 20 :lO 40 50 60 70 80 90 100 0 0 10 20 30 "0 50 60 70 Time (min) Time (min) Time (min)

Fig. 4. Time-response curves for i.th. morphine, DAMGO and DPDPE antinociception in the absence (top row) and presence (bottom row) of a single, i.th. dose of naloxone (2.25, 1.25 and 0.25 nmol respectively) given 5 min prior to agonist injection. Dose of ~gonist administered is denoted by the value next to each curve (nmol/mouse). ~ ~ 20 min 100 0 100· 25 min -"

W 80 W 80 vi vi +1 +1 I .2" 60 .~ 60 l ~ u 40 0 I0 " 1 " 40 r 1 /' /' 1 ~ 20 ~ ~ 20 Y ~ 0 0 4.6 15 30 46 150 4.6 15 30 46 150 Dose OPOPE (nmol. i.e.v.) Dose OPOPE (nmol. i.e.v.)

100. 35 min -<> 100· 40 min

W 80 W 80 vi vi +1 +1 0" 60 .2" 60 =a.. a. u ..u U 40 'u 40 0 0 ~ " ~ 20 ~ 20 ~ ~ 0 1 all 4.6 15 30 46 150 4.6 15 30 46 150 00•• OPOPE (nmol. i.e.v.) Dose CPOPE (nmol. i.c.v.)

Fig. 5. Dose-response lines at various times determined from time-response data for i.c.v. DPDPE in the absence (closed symbols) and presence (open symbols) of naloxone in the mouse tail with- drawal test. 0\ V1 66

1.0

1.4

0.4

0.2

10 20 30 40 10 20 30 40 50 nne (.00) nne (.00)

1.2 1.2 DAGO DAGO 1.0 (Le.v.) 1.0 (Lf.) _ O.B _ 0.8 R=O.BS R:::O.G9 ~ e 0.6 § 0.0 3 3 0.4 0.4

0.2 0.2

10 20 30 40 50 -0.1 10 20 30 40 50 nne (n'On) nne «mJ

0.6

1.0 OPOPE (i.e.v" 0.6 R=O.99

0.6

0.4

0.2

10 50 -0.2

-0.4

-0.0

-O,B

Fig. 6. Plots of log (dose-ratio - 1) vs. time for i.c.v. (left column) and i.th. (right column) morphine, DAMGO and DPDPE in the mouse tail-withdrawal test. See text for details. 67 obtained from each agonist and route (Table 2). Additionally, the naloxone half­ life (and 95% c.L.) against each agonist and route (given by the slope of the lines in Fig. 6) is shown in Table 2. Finally, the peak Dso (and 95% c.L.) (dose of agonist producing 50% antinociception) obtained from the time-response curves in the absence of naloxone (Figs. 3 and 4) are also shown in Table 2.

The apparent naloxone pA2 values (and 95% C.L) for i.c.v. morphine,

DAMGO and DPDPE were 10.39 (10.29-10.96), 9.99 (9.28-10.69) and 10.63 (10.14-

10.64), respectively. Similarly, after i.th. administration of morphine, DAMGO and

DPDPE, the pA2 values (and 95% c.L.) were 10.16 (9.91-10.43), 9.74 (9.5-9.98) and

10.15 (10.12-10.18), respectively. The overlapping 95% c.L. after i.c.v. administration indicate a lack of significant difference between these values when compared between agonists. The values seen after i.th. administration indicate no overlap between the naloxone pA2 against DAMGO and DPDPE, although the value against morphine was not different from either of the other agonists. The half-life of naloxone was found to range approximately between 9 and 17 min (see Table 2), values that compare well with those previously reported by the same, and other techniques (Porreca et aI., 1981; Tung and Yaksh, 1982). An exception was the briefer naloxone half-life found with i.c.v. morphine (6.6 min). Additionally, a comparison of Dso values of each compound after i.c.v. and i.th. administration shows that morphine and DAMGO are more potent i.c.v. (3.6 and 8.3 fold, respectively) while DPDPE is essentially equipotent by the two routes. 68

TABLE 2

Time-dependent apparent pA2 for naloxone against morphine, DAMGO and DPDPE after i.c.v. or i.th. administration. pA2 and tV2 were calculated using the time-dependent method described in the theory section while me agonist D50 values were calculated from the time of peak agonist effect. Peak effect for all agonists by either route was 20 min, except for i.th. DPDPE (= 10 min.).

Agonist Route pA2 (95% C.L.) tl/~ (95% C.L.) D50 (mm) (nmol)

Morphine i.c.v. 10.4(10.2-10.6) 6.6(5.6-.9) 0.6(.2-1.8)

DAMGO i.c.v. 9.9(9.3-10.7) 16.9(7.9-26.1 ) 0.01(0.006-0.2) DPDPE i.c. v. 10.63(10.3-10.9) 9.1(6.4-16.1) 9.4(5.7-16.28)

Morphine i.th. 10.16(9.61-10.43) 10.2(7.4-16.3 ) 2.27(.74-6.9)

DAMGO i.th. 9.74(9.5-9.98) 10.89(8.7-14.7) 0.08(.06-.12)

DPDPE i.th. 10.15(10.12-10.18) 14.22(13.4-15.0) 8.01(6.28-10.27) 69

Tolerance/Cross-tolerance Studies

Pretreatment with a single dose of subcutaneous morphine (100 mg/kg s.c.)

displaced the i.c.v. morphine dose-response line 7 fold to the right, indicating the

development of tolerance (Fig. 7), in agreement with previous reports (Vaught and

Takemori, 1979). However, morphine pretreatment failed to displace the i.c.v.

DPDPE dose-response line (Fig. 8) indicating a lack of Lc.v. cross-tolerance between

these agonists. The D50 values (and 95% c.L.) for Lc.v. morphine and DPDPE in

control and morphine pretreated mice are shown in table 3. The development of

tolerance to morphine at the spinal level is seen in Figure 9. A single pretreating dose of morphine (100 mg/kg, s.c.) produced a 25-fold rightward displacement of the

Lth. morphine dose-response line, indicating tolerance to this agonist. Similarly, the

Lth. DPDPE dose-response line was shifted 12-fold to the right, indicating cross­ tolerance at the spinal level between DPDPE and morphine (Fig. 10). Table 2 shows the D50 values (and 95% c.L.) for Lth. morphine and DPDPE in control and morphine pretreated. 70

100 0

W en 80 +1 I: 0 60 :tJ Q. Q) (,) '0 0 40 I: +i

0 0.3 0.9 1.8 3 9 15 18 Dose Morphine (nmol. i.e.v.)

Fig. 7. Dose-response lines for i.c.v. morphine in naive (closed symbols) and morphine pretreated (100 mg/kg, s.c.)(open symbols) mice. Testing occurred 5 hr after morphine pretreatment and 20 min after the i.e.v. administration of morphine. 71

100

t.J u; 80 +1 c 0 60 :tJa. Q) 0 '0 0 40 c :tJ «c ~ 20

0 4.6 9.3 15 30 46 Dose DPDPE(nmol, i.e.v.)

Fig. 8. Dose-response lines for i.c.v. DPDPE in naive (closed symbols) and morphine pretreated (100 mg/kg, s.c.)(open symbols) mice. Testing took place 5 hr after morphine pretreatment and 20 min after the i.c.v. administration of DPDPE. 72

100

W vi 80 +1 c 0 :p 60 a. Q) ·00 0 40 c :p c < ~ 20

0 0.3 0.9 1.8 3 9 30 90 Dose Morphine (nmol, i.th.)

Fig. 9. Dose-response lines for i.th. morphine in naive (closed symbols) and morphine pretreated (100 mg/kg, s.c.)(open symbols) mice. Testing took place 5 hr after morphine pretreatment and 20 min after the i.th. administration of morphine. 73

100

t.J vi 80 +1 c 0 :p 60 a. Q) 0 '0 0 40 c :p «c ~ 20

0 0.46 1.5 4.6 9.3 46.4 Dose DPDPE (nmol, Lth.)

Fig. 10. Dose-response lines for i.th. DPDPE in naive (closed symbols) and morphine pretreated (100 mg/kg, s.c.)(open symbols) mice. Testing took place 5 hr after morphine pretreatment and 10 min after the i.th. administration of DPDPE. 74

TABLE 3

Effects of morphine pretreatment on the antinociceptive responses to morphine and DPDPE

Agonist D50 (and 95% C.L's)(nmol/mouse) for antinociception are given for mice pretreated 5 hr before agonist administration with distilled water (I ml/kg, s.c.) or morphine (100 mg/kg, s.c.).

Agonist Route Water D50 Morphine D50 Ratio

Morphine i.c.v. 0.76(0.32-1.76) 4.93(2.36-10.37) 6.49 DPDPE i.c.v. 8.78(4.8-16.06) 12.07(6.97-20.88) 1.35

Morphine i.th. 1.41 (1.05-1.91) 34.74(9.8-123.0) 24.64

DPDPE i.th. 2.83( 1.89-4.26) 35.49(*) 12.54

*could not be calculated 75

Antagonism Studies

Effects of leI 174,864 on morphine, DAMGO and DPDPE antinociception.

The dose-response lines for i.c.v. DPDPE, DAMGO and morphine in the

absence and presence of i.c.v. lei 174,864 are shown in Figure 11. At the doses

tested, lei 174,864 had no antinociceptive effect alone. lei 174,864 (1.5 or 4 nmol)

produced a dose-related antagonism of i.c.v. DPDPE antinociception. In contrast,

lei 174,864 (4 nmol) had no effect on i.c.v morphine or DAMGO antinociception.

The D50 value (and 95% c.L.) of each i.c.v. agonist in the absence and presence of

lei 174,864 (4 nmol) is shown in Table 4. The effect of lei 174,864 on the agonists when given i.th. is seen in Figure 12. Similar to the i.c.v. results, i.th. lei 174,864

(4 nmol) completely abolished the antinociceptive effects of i.th. DPDPE.

Additionally, i.th. lei 174,864 failed to displace the i.th. dose-response lines for

DAMGO and morphine. The Dso values (and 95% c.L.) for the i.th. agonists in the

absence and presence of leI 174,864 are also seen in Table 4. 76

100

uJ 80 (J) +1 c: 0 60 +ia. Q) 0 '0 0 40 c: +i c: « 20 ~

o~----~--~--~------~--~~~~ 0.006 0.02 0.06 0.19 3 9 15 3046 Dose (nmol. i.e.v.)

Fig. 11. Antinociceptive dose-response lines for i.c.v. administration of DAMGO

(circles), morphine (squares) and DPDPE (triangles) in the absence (closed symbols) or in the presence (open symbols, closed diamonds) of i.c.v. leI 174,864. The antagonist was co-administered with the agonist at 4 nmol (open symbols, closed diamonds) or at 1.45 nmol (open triangles). 77

100

t.J vi 80 +1 c 0 :;; 60 a.. Q) 0 '0 0 40 c :;; «c ~ 20

0 0.06 0.2 0.6 1.8 3 9 15 30 46 Dose (nmol. i.th.)

Fig. 12. Antinociceptive dose-response lines for i.th. administration of morphine

(circles), DAMGO (triangles) and DPDPE (squares) in the absence (closed symbols) or in the presence (open symbols) of i.th. leI 174,864 (4 nmol). The antagonist was co-administered with the agonist at 4 nmol (open symbols). 78

TABLE 4

Effects of leI 174,864 on the antinociception produced by morphine, DAMGO and DPDPE

Antinociceptive Dso(and 95% C.L's)(nmoljmouse) for morphine, DAMGO and DPDPE given i.c.v. or i.th. in the absence or presence of ICI 174,864 (4 nmol) in the warm water tail withdrawal test.

Agonist Route Control ICI 174,864 Potency

Morphine i.c.v. 7.83 (4.9-12.4) 7.26 (5.26-10.07) 1.07

DAMGO Lc.v. 0.03 (0.02-0.04) 0.02 (0.01-0.261) 1.39 DPDPE i.c.v. 28.95 (23.9-35.2) * * Morphine Lth. 4.06 (2.51-6.34) 3.86 (2.63-5.71) 1.05 DAMGO Lth. 0.49 (0.31-0.74) 0.58 (0.25-1.29) 1.2 DPDPE Lth. 28.8 (21.1-39.17) * *

• Cannot be determined 79

Effects of f3-FNA pretreatment on morphine, DAMGO and DPDPE antinociception.

The effects of f3-FNA (18 muo! i.c.v. at -4 h) on the i.c.v. antinociceptive actions of morphine, DAMGO and DPDPE are shown in Figures 13, 14 and 15, respectively. f3-FNA alone had no antinociceptive effects in the tail withdrawal test.

Pretreatment with i.c.v. f3-FNA resulted in a 12-fold shift in the i.c.v. morphine dose­ response line (Fig. 13). Similarly, i.c.v. f3-FNA pretreatment also produced a rightward displacement of the i.c.v. DAMGO dose-response line; the rightward shift in this line, however, was 958-fold (Fig. 14). In contrast, Figure 15 shows that the same i.c.v. f3-FNA pretreatment had no effect on i.c.v. DPDPE antinociception. The

D50 values (and 95% C.L.) for i.c.v. morphine, DAMGO and DPDPE in control and f3-FNA pretreated mice are shown in Table 5. The dose-response line for the co­ administration of i.c.v. ICI 174,864 and morphine in f3-FNA pretreated mice is also seen in Figure 13. The presence of the 6 antagonist, leI 174,864, resulted in a further displacement to the right in these i.c.v. f3-FNA pretreated mice; the morphine dose-response line was shifted approximately 30-fold overall to the right of the control line. The effect of f3-FNA on antinociception at the spinal level was also studied. f3-FNA (0.19, 1.88 nmol) effectively antagonized both i.th. morphine and

DPDPE antinociception. Further decreasing the i.th. pretreatment dose of f3-FNA

(0.019, 0.009 nmol) resulted in selective antagonism of i.th. morphine, but not i.th.

DPDPE antinociception (Fig. 16). 80

100

LJen 80 +1 c 0 :;:; 60 a. Q) 0 '0 0 40 c :;:; «c ~ 20

0 3 9 18 30 90 180 Dose Morphine (nmol. i.e.v.)

Fig. 13. Antinociceptive dose-response lines for i.c.v. morphine in naive (closed circles), or in ,B-FNA pretreated (18 nmol i.c.v. at - 4 hr)(open circles) mice. The effects of co-administration of i.c.v. leI 174,864 (4 nmol) with morphine in ,B-FNA pretreated mice is shown by the triangles. 81

w en +1 c o :p a. Q) o '0 o c :p «c ~

0.006 0.02 0.06 0.19 5.8 19.5 48.6 116.8 Dose DAGO (nmol. i.e.v.)

Fig. 14. Antinociceptive dose-response lines for i.c.v. DAMGO in naive (closed circles) or in ,B-FNA pretreated (18 runol i.c.v. at -4 hr)( open circles) mice. 82

100

u.i en 80 +1 c: 0 60 :p a. Q) 0 '0 40 0 c: :p c: « 20 ~

0 4.6 15 46 Dose DPDPE (nmol. i.e.v.)

Fig. 15. Antinociceptive dose-response lines for i.c.v. DPDPE in naive (closed circles) or in ,B-FNA pretreated (18 nmol i.c.v. at -4 hr)(open circles) mice. 83

TABLE 5

Effects of P-FNA pretreatment on the antinociceptive properties of morphine, DAMGO and DPDPE

Antinociceptive D5,1l (and 95% C.L.'s)(nmol/mouse) for 3 agonists given i.c.v. to control or P-FNA pretreated tI8 nmol i.c.v. at -4 hr) mice in the warm water tail withdrawal test.

Agonist Control P-FNA Potency

Morphine 4.9 (3.61-6.64) 57.49 (23.65-139.69) 11.7

DAMGO 0.27 (0.017-0.036) 26.5 (14.54-48.25) 958.4

DPDPE 15.33 (11.48-20.48) 17.14 (10.22-28.7) 1.11 Control P-FNA P-FNA P-FNA (0.009 nm) (0.018 nm) (0.18 nm) 100 NS

W 80 ui + c 60 o +:ia. Q) o '0 40 o c +:i c < 20 ~

o I II 18 0 Sal 18 o Sal 18 o Sal 18 o Morphine o 46 0 46 0 46 0 46 DPDPE Dose (nmol, Lth.)

Fig. 16. Effects of pretreatment with increasing doses of i.th. S-FNA on equieffective anti-

nociceptive doses of i.th. morphine or DPDPE. Treatments are seen below each histogram with doses

shown in nmol/mouse. S-FNA pretreatment produced significant antagonism of morphine anti- nociception at all doses tested, but did not antagonize DPDPE antinociception at pretreatment doses of 0.018 and 0.009 nmol. 00 .j::o. 85

Effect of naloxonazine pretreatment on morphine, DAMGO and DPDPE antinociception.

The effects of naloxonazine pretreatmer,~ on i.c.v. morphine, DAMGO and

DPDPE antinociception are seen in Figures 17, 18 and 19, respectively. When tested 24 hr after the administration of naloxonazine, both the i.c.v. morphine and

DAMGO dose-response lines were displaced to the right (7- and 5-fold respectively) (Figs. 17 and 18). Whereas the displacement was not as great as seen at 24 hr post naloxonazine administration, both i.c.v. morphine and DAMGO antinociception were still markedly antagonized by naloxonazine when the pretreatment was carried out 48 hr prior to testing. This is evident from the approximate 3 fold displacement to the right of the dose-response lines for both morphine and DAMGO (Figs. 17 and 18). Unlike its effects on the J.£ agonists morphine and DAMGO, naloxonazine pretreatment had no effect on i.c.v. DPDPE antinociception at either 24 or 48 hr after administration (Fig. 19). In contrast to the supraspinal results, naloxonazine pretreatment failed to antagonize the antinociceptive properties of morphine and DAMGO when they were given i.th. as can be seen in Fig. 20). Additionally, and similar to the supraspinal results, the antinociceptive effects of Lth. DPDPE were not antagonized by pretreatment with naloxonazine (Fig. 20). The D50 values (and 95% c.L.) for i.c.v. and Lth. antinociception resulting from administration of these agonists in control and 86

100

t.J 80 en r +1 c 1 0 60 :;i Co Q) 0 '0 40 0 c :;i T C « 20 r J. ~

0 3 9 15 18 30 90 150 Dose Morphine (nmol, i.e.v.)

Fig. 17. Effects of naloxonazine on anti nociceptive responses to morphine.

Antinociceptive dose-response lines are given for i.c.v. morphine in mice pretreated with distilled water (1 ml/kg)(circles) and in naloxonazine pretreated (35 mg/kg, s.c.) mice at 24 hr (triangles) and 48 hr (squares) after treatment. 87

100

W 80 vi +1 c 0 60 :.p C- Q) 0 '0 40 0 c :.p c « 20 ~

0 0.006 0.02 0.06 0.19 0.58 Dose DAGO (nmol, i.e.v.)

Fig. 18. Effects of naloxonazine on antinociceptive responses to DAMGO.

Antinociceptive dose-response lines are given for i.c.v. DAMGO in mice pretreated with distilled water (1 mljkg)(circles) and in naloxonazine pretreated (35 mg/kg, s.c.) mice at 24 hr (triangles) and 48 hr (squares) after treatment. 88

100

LJ 80 vi +1 c: 0 60 +i C- O) 0 '0 40 0 c: +i c: « 20 ~

0 4.6 15 46 Dose DPDPE (nmol. i.c.v.)

Fig. 19. Effects of naloxonazine on antinociceptive responses to DPDPE.

Antinociceptive dose-response lines are given for i.c.v. DPDPE in mice pretreated with distilled water (1 ml/kg)(circles) and in naloxonazine pretreated (35 mg/kg, s.c.) mice at 24 hr (triangles) and 48 hr (squares) after treatment. 89

100 r

LJ 80 vi +1 C 0 60 :p a. Q) 0 '0 0 40 c :p c « 20 ~

o ~~----~------~----~--~----~~~--~ 0.06 0.19 0.58 1.8 3 9 15 30 46 Dose (nmol, i.th.)

Fig. 20. Effects of naloxonazine on anti nociceptive responses to Lth. morphine,

DAMGO and DPDPE. Antinociceptive dose-response lines are given for Lth. morphine (squares), DAMGO (circles) and DPDPE (triangles) in mice pretreated with distilled water (1 ml/kg)(closed symbols) or naloxonazine (35 mg/kg, s.c.)(open symbols) 24 hr before testing. 90

TABLE 6

Effects of treatment with naloxonazine on the antinociceptive responses to morphine, DAMGO and DPDPE

Agonist D60 values (and 95% c.L.'s) for antinociception are given for mice pretreated 24 hr before agonist administration with distilled water (I ml/kg s.c.) or naloxonazine (35 mg/kg s.c.). NXAZ, naloxonazine.

Water D60 NXAZ NXAZ/ and 95% C.L. and 95% C.L. water Agonist Route (nmol/mouse) (nmol/mouse) Ratio

Morphine i.c.v. 4.9 (3.6-6.6) 34.6 (23.5-53.76) 7.05

DAMGO i.c.v. 0.027 (0.02-0.04) 0.14 (0.07-0.29) 5.32

DPDPE i.c.v. 17.14 (10.2-18.7) 21.73 (14.46-32.66) 1.26

Morphine i.th. 4.63 (2.21- 9.77) 4.84 (0.44-51.8) 1.04

DAMGO i.th. 0.49 (0.31-0.76) 0.57 (0.14-2.58) 1.16

DPDPE i.th. 19.2 (12.1-30.61) 21.11 (14.81-30.11) 1.09 91

+' 'Ci) 100 c C L.. l- e 80 c :tJen Q) +'c 60 '0 L.. +'en c (!) 40 '+- 0 C 0 :tJ 20 :c:0 c ~ 0 0.006 0.02 0.06 0.19 0.9 3 9 30 Dose (nmol. i.e.v.)

Fig. 21. Effects of naloxonazine on the anti transit effects of i.c.v. morphine and

DAMGo. Dose-response lines are given for the inhibition of gastrointestinal transit by i.c.v. morphine (squares) and DAMGO (circles) in mice pretreated with distilled water ( 1 ml/kg)(closed symbols) or naloxonazine (35 mg/kg, s.c.)(open symbols) 24 hr before testing. 92

..... 100 'mc ~ I- 0 80 c +irn .....Q) c 60 '0 L...... rn 0 (!) 40 '+- 0 c 0 6 +i 20 1 :Ei :E c ~ ~ 0 0.9 3 9 30 Dose Morphine (nmol, i.th.)

Fig. 22. Effects of naloxonazine on the antitransit effects of i.th. morphine. Dose- response lines are given for the inhibition of gastrointestinal transit by i.th. morphine in mice pretreated with distilled water (1 ml/kg)(closed circles) or naloxonazine (35 mg/kg, s.c.)(open circles) 24 hr before testing. 93

....., 100 'enc

t-e o 80 c :;:; rn .....,Q) c 60 'e....., rn 0 (!) 40 0 -c 0 20 :c:;:; :c c 0 ~ 0.02 0.06 0.19 0.58 1.94 19.4 Dose DAGO (nmol. i.th.)

Fig. 23. Effects of naloxonazine on the antitransit effects of i.th. DAMGO. Dose- response lines are given for the inhibition of gastrointestinal transit by i.th. DAMGO in mice pretreated with distilled water (1 ml/kg)(closed circles) or naloxonazine (35 mg/kg, s.c.)(open circles) 24 hr before testing. 94

~ (f) 100 c 0 L. l- e 80 c +:i (f) .....Q) c 60 'e..... (f) 0 (!) 40 -0 c b- 0 +:i 20 1 :0:.c c ~ 0 3 9 30 90 Dose DPDPE (nmol. i.th.)

Fig. 24. Effects of naloxonazine on the anti transit effects of Lth. DPDPE. Dose- response lines are given for the inhibition of gastrointestinal transit by Lth. DPDPE in mice pretreated with distilled water (1 mljkg)(closed circles) or naloxonazine (35 mg/kg, s.c.)(open circles) 24 hr before testing. 9S

TABLE i

Effects of treatment with naloxonazine on the inhibition of gastrointestinal transit by morphine, DAMGO and DPDPE

Agonist D50 values (and 95% C.L.'s) for inhibition of gastrointestinal transit are given for mice pretreated 24 hr before agonist administration with distilled water (I ml/kg s.c.) or naloxonazine (35 mg/kg s.c.). NXAZ, naloxonazine.

Water D50 NXAZ D60 NXAZ/ and 95% C.L. and 95% C.L. water Agonist Route (nmol/mouse) (nmol/mouse) Ratio

Morphine i.c.v. 5.0 (3.34-7.53) 8.22 (6.2- 10.9) l.64

DAMGO i.c.v. 0.026 (0.003-0.28) 0.04 (0.005-2.9) 1.53

Morphine i.th 4.04 (2.66-6.09) 89.7 * 22.2 DAMGO i.th. 0.12 (0.08-0.18) 8.20 (3.42-19.66) 68.3

DPDPE i.th. 4.17 (0.2-8.3) 46.2 * 1l.08

* Cannot be determined 96

TABLE 8

Effects of s.c. naloxonazine pretreatment (at -24 hr) on opioid antinociceptioll and antitransit effects in the mouse

Agonists Given i.c.v. Agonists Given i.th. Antinociception Antitransit Antinociception Antitransit

Morphine + + DAMGO + + DPDPE * +

+ Effect blocked by naloxonazine pretreatment. - Effect not blocked by naloxonazine pretreatment. • No agonist effect by this route. 97 naloxonazine pretreated mice are shown in Table 6. As it was possible that the lack of effect of naloxonazine pretreatment on spinal antinociception stemmed from the inability of naloxonazine to enter the cord, the effect of naloxonazine on a second, opioid-mediated endpoint, the inhibition of gastrointestinal transit, was studied. It has previously been shown that opioids act in the spinal cord to inhibit the propulsion of a radiolabelled marker in the mouse (Porreca and Burks, 1983;

Porreca et at, 1984).

Effect of naloxonazine pretreatment on the ability of morphine, DAMGO and DPDPE to inhibit gastrointestinal propulsion.

While naloxonazine antagonized the antinociception produced by i.c.v. morphine and DAMGO, the ability of these J.L agonists to inhibit gastrointestinal transit when given i.c.v. was unchanged after naloxonazine pretreatment (Fig. 21).

As i.c.v. DPDPE does not affect gastrointestinal transit (Porreca et at, 1984) its interaction with naloxonazine was not studied. The inhibitory effects of i.th. morphine, DAMGO and DPDPE on gastrointestinal propulsion in distilled water and naloxonazine pretreated mice are presented in Figures 22, 23 and 24, respectively. Converse to the lack of naloxonazine effect on the antinociceptive properties of the agonists at the spinal level, the inhibitory effects of i.th. morphine,

DAMGO and DPDPE on gastrointestinal transit were greatly reduced in mice pretreated with naloxonazine 24 hr prior to testing. The D50 values (and 95% c.L.) 98 resulting from i.c.v. and i.th. administration of these agonists for inhibition of gastrointestinal transit in control and naloxonazine pretreated mice are shown in

Table 7. A summary of the effects of naloxonazine on antinociception and inhibition of gastrointestinal transit produced by either the i.c.v. or i.th. administration of morphine, DAMGO and DPDPE is seen in Table 8.

Modulation Studies

Studies with morphine.

When co-administered in the same i.c.v. injection as morphine, DPDPE (1.6 nmol) consistently and significantly potentiated the antinociception produced by morphine. An example illustrating the potentiation of morphine antinociception (3 nmol) produced by 1.6 nmol of DPDPE is shown in Figure 25. This dose of DPDPE

(1.6 nmol) produced minimal antinociception when given alone. Similarly, DPDPE was able to potentiate the i.c.v. anti nociceptive effects of morphine in mice pretreated with morphine (100 mg/kg s.c, at -5 h) (acute morphine tolerance)(Fig.

26). Potentiation of i.c.v. morphine antinociception by DPDPE was prevented by co­ administration of the 6 antagonist ICI 174,864 (4 nmol)(Fig. 25). ICI 174,864 failed to produce antinociception when given alone, and neither potentiated nor attenuated morphine antinociception at this dose (4 nmol) as described above. In contrast to the i.c.v. results, a sub-effective i.th. dose of DPDPE (0.46 nmol) which did not produce detectable antinociception when given alone failed to potentiate the Control ICI 174,864

100 r ~* W (J) 80 + c: 0 :z:; 60 Q. Q) 0 0 40 0 c: :z:; c:

Fig. 25. DPDPE induced potentiation of morphine antinociception and reversal of potentiation by leI 174,864 (4 nmol) following administration of compounds in the same i.c.v. injection.

The bars represent the mean and the S.E. of the agonist effects; asterisks indicate a significant difference (p<:O.05) when groups were compared to morphine antinociccption alone.

0 o 0.9 1.S 3 3 9 9 Morphine l.S O' 0.9 o l.S o 1.S DPDPE Dose (nmol, i.e.v.) Fig. 26. DPDPE induced potentiation of morphine antinociception in morphine pretreated mice.

Treatments are seen below each histogram with doses shown in nmol/mouse. The bars represent the mean and the S.E. of the agonist effects; asterisks indicate a significant difference (p

...... o o 101

100 NS w ~ vi 80 +1 c o :p 60 a. Q) o '0 o 40 c :p «c ~ 20

o o 0.9 0.9 3 3 9 9 Morphine 0.46 0 0.46 0 0.46 0 0.46 DPDPE Dose (nmol, i.th.)

Fig. 27. Effects of i.th. co-administration of a threshold anti nociceptive dose of

DPDPE on three morphine doses in naive mice. Treatments are seen below each histogram with doses shown in nmol/mouse. 102 antinociceptive effects of three doses of morphine (0.9, 3 and 9 nmol) (Fig.

27) compared to morphine antinociception alone.

Studies with chemically diverse I-' agonists.

Similar to its effects on morphine, DPDPE potentiated the antinociception produced by normorphine (Fig. 28). In contrast, DPDPE co-administration did not affect the antinociception produced by the peptide I-' agonists DAMGO (Fig. 29a) and PL017 (Fig. 29b), or the non-peptide I-' agonists etorphine (Fig. 29c), phenazocine (Fig. 29d), and sufentanil (Fig. 2ge). Furthermore, ,B-endorphin antinociception was unaltered by DPDPE (Fig. 29f). Additionally, co-administration of leI 174,864 at a dose (4 nmol) which was effective in antagonizing DPDPE antinociception had no significant effect on the antinociceptive effects of i.c.v. etorphine (Fig. 30a), phenazocine (Fig. 30b), or ,B-endorphin (Fig. 30c).

Effects of I-' selective antagonists on {j modulation of I-' mediated antinociception.

The effects of ,B-FNA pretreatment (18 nmol i.c.v., at -4 h) on the respective ability of DPDPE to potentiate, and DAMA to attenuate, i.c.v. morphine antinociception were investigated. As the ,B-FNA pretreatment used had no significant effect on the direct antinociceptive effects of i.c.v. DPDPE or DAMA

(Table 9), the modulatory doses for each {j agonist (1.6 nmol DPDPE and 0.17 nmol

DAMA) remained the same. As expected (Ward et aI., 1982; 1983; present study), however, morphine antinociception was significantly antagonized by ,B-FNA o~* __ 100 *T o * L.J 80 (J) +1 . 1 s::::: o 60 :;:; !~ ~t Q. (l) o ·u 40 o s::::: /1 :;:; s::::: <{ 20 ~ oT~.------r 1 49 o 0.11 0.36 1.10 3.68 11.1 Dose Normorphine (nmol, i.e.v.)

Fig. 28. DPDPE-induced potentiation of normorphine antinociception. Dose-pretreatment; thus, the doses of morphine used in 8-FNA pretreated mice were response curves for i.c.v. normorphine antinociception in the absence (closed circles) and presence (open circles) of non-agonist dose of DPDPE (1.6 nmo1); asterisks indicate a significant difference (pKO.OS)...... o CJ.l 104

@ 100 100 oJ ui I .. 110 j: Ji .. 20

0 0 O.OO~ 0.019 0.058 0.194 2.411 7.4~ 24.84 Do •• DACO (nmol, I.e,v.) Do .. PhenOlocln. (nmol, I,e.v.)

@ CD 100 100 ~ ::I.. 110 .. 60 § 60

1 40 I: ~ .. 20 .. 20

0 0.007 0.022 0.057 0,111 0.067 0.288 0.87 2.68 DoM Etorphlnl (nmof. I.e ..... ) 00 •• '-endorphln (nmol, I.e,y.)

Fig. 29. DPDPE fails to potentiate i.c.v. DAMGO, PL01?, etorphine, sufentanil, phenazocine, and ,8-endorphin antinociception. Dose-response curves for i.c.v. (a)

DAMGO, (b) PL01?, (c) etorphine, (d) phenazocine, (e) sufentanil and (f) ,8- endorphin in the absence (closed symbols) and presence (open symbols) of DPDPE

(1.6 nmol). lOS

@ 100 ,.....-....-,NS \J vi BO + c 0 60 '"Q. 40 ·ic '".:i 20 M

0 0 0.07 0.07 0.22 0.22 Etorphin. 4 0 4 0 IC1174.B64 00•• (nmol. i.e.v.)

NS r--A-o. 100

iJ vi 60 + g 60 ~ .~ 40

'".11 20 M

0 7.4 7.4 25 25 Phenazocine 0 ICI 174.664 Dose (nmol, i.e.v.)

NS © 100 r--A-o. iJ vi 60 + c 0 60 i .~ 40

~ 20 M

0 sol 0 0.66 0.66 2.66 2.66 p-endorphin 0 0 ICI 174.664 Dose (nmol. i.e.v.)

Fig. 30. Antinociceptive effects of i.c.v. etorphine (a), phenazocine (b) and /3- endorphin (c) in the absence and presence of leI 174,864 (4 nmol). The bars represent the mean and S.E. NS indicates no significant difference (p < 0.05). 106

TABLE 9

Effects of i.c.v. P-FNA pretreatment (18 nmol at -4 hr) on the anti nociceptive properties of DAMA

Dose DAMA %Antinociception ± S.E. (nmol) Control ,B-FNA pretreated

0.17 7.0 ± 4 6.0 ± 5 N.S.

0.51 21.0 ± 3 20.0 ± II N.S.

1.7 51.0 ± 13 38.9 ± 12 N.S.

N.S. indicates no significant difference when groups are compared to vehicle controls (5 JLl distilled water, i.c.v. at -4hr) where p < 0.05. 100

w 80 vi + § 60 :OJ I a. Q) o ·0 40 I o L--/-/j~l,____ I c :OJ ~ 20 j-j 1 '----i------l ~

o L'------~ o 10 20 30 40 50 Time (min)

Fig. 31. Time-response curve for i.c.v. D~~ (1.7 nmol) antinociception in the mouse. Data are mean and standard error. .... o " 100 W (f) 80 +1 c 0 :;:; 60 Q.. Q) U u 0 40 c :;:; «c 20 ~

0 0.17 0.51 1.7 5.1 Dose DAMA (nmol, i.e.v.)

Fig. 32. Dose-response relationship for i.c.v. DAMA antinociception in the mouse when tested at

20 min post injection. Data are mean and standard error. .... o 00 109

Control /1-FNA Pretreated

100 r-""-\* I&l 80 u; + NS c: 60 ~ :s.0 8 u 40 0 c :;:i ~ 20 lit

0 1.6 1.6 o Bal 1.6 o 1.6 DPDPE o 3 3 o 9 9 Morphine DoBe (nmol. i.e.v.)

Fig. 33. Effects of ,B-FNA pretreatment (18 nmol at -4 hr) on DPDPE-induced potentiation of morphine antinociception. ,B-FNA pretreatment abolishes the potentiation of morphine antinociception produced by DPDPE. Data shown are mean and standard error and asterisks indicate a significant difference from control

(p< 0.05, Student's t test). Control P-FHA Pretreated

100

..:J ~* u; sol NS + ~ flO

40 ti at 20

0 0.17 o 0.17 IGI 0.17 o 0.17 DAlIA o J J o 9 9 YotphIr.e Do. (nmol. Lc.v.)

Fig. 34. Effects of S-FNA pretreatment (18 nmol at -4 hr) on DAMA-induced attenuation of morphine antinociception. S-FNA pretreatment abolishes the attenuation of morphine antinociception by DAMA. Data shown are mean and standard error and asterisks indicate a significant difference from control (p

TABLE 10

Effects of s.c. naloxonazine pretreatment (35 mg/kg at -24 hr) on the antinociceptive properties of DAMA

Dose DAMA %Antinociception ± S.E. (nmol) Con trol Naloxonazine pretreated

0.17 6.0 ± 4 8.0 ± 4 N.S.

0.51 lI.3±3 15.0±9 N.S.

I.7 43.9 ± 11 36.4 ± 11 N.S.

N.S. indicates no significant difference when groups are compared to vehicle controls (I ml/kg distilled water, s.c. at -24 h) where p < 0.05. Control Naloxonazine Pretreated ~* 100 r * ~ w vi 80 + c 0 :p 60 Q. cu () 'u 0 40 c

~ 20 ~

0 1.6 0 1.6 sal 1.6 o 1.6 o 1.6 DPDPE 0 :5 :5 o 9 9 30 30 Morphine Dose (nmol. i.e.v.)

Fig. 35. Effects of naloxonazine pretreatment (35 mg/kg, s.c. at -24 hr) on DPDPE-induced potentiation of morphine antinociception. Naloxonazine pretreatment did not affect the potentiation of morphine antinociception produced by DPDPE. Data are mean and standard error and asterisks indicate a significant difference from controls (pKO.OS, Student's t test)...... N Control Naloxonazine Pretreated

100 ~*

W r--""--\* vi 80 l + c 0 :0:; 60 Q. CI) ·uu 0 40 c :0:; ~ 20 ~

0 o 3 3 sal o 30 30 90 90 Morphine 0.17 o 0.17 0.17 0 0.17 0 0.17 DAMA Dose (nmol, i.e.v.)

Fig. 36. Effects of naloxonazine pretreatment (35 mg/kg, s.c. at -24 hr) on DAMA-induced attenuation of morphine antinociception. Naloxonazine pretreatment did not affect the attenuation of morphine antinociception produced by DAMA. Data are mean and standard error and asterisks indicate a significant difference from controls (pKO.05, Student's t test)...... Vt 114 increased by 10 fold (3 nmol and 30 nmol, respectively, in naive and ,B-FNA groups shown in Figs. 33 and 34). Pretreatment with the I' antagonist ,B-FNA blocked the potentiating effect of DPDPE (Fig. 33) and the attenuating effect of DAMA (Fig.

34) when compared to control (5 1'1 distilled water i.c.v., at -4 h) groups. ,B-FNA had no antinociceptive effect alone in this test.

Naloxonazine pretreatment (35 mg/kg, s.c. at -24 h) had no effect on the direct antinociceptive effect produced by i.c.v. DPDPE (Fig. 19) or DAMA (Table

10); thus, no changes were required in the modulatory doses of these 0 agonists. As expected, based on findings in the present, and previous investigation (Ling et aI.,

1986) pretreatment with naloxonazine effectively antagonized i.c.v. morphine antinociception (Figs 35 and 36) and so the doses of morphine used were increased by 10 fold. Unlike the effects of pretreatment with ,B-FNA, naloxonazine pretreatment did not alter the ability of DPDPE to potentiate (Fig. 35) or of DAMA to attenuate (Fig. 36) morphine antinociception. 115

DISCUSSION

The goal of the current investigation was to correlate central opioid 0 receptors, for which Met- and Leu-enkephalin are presumed to be the endogenous ligands (Lord et a!., 1977), with antinociceptive properties in vivo. To do so, a number of pharmacological methods were used in parallel in hopes that the data would provide convergent evidence regarding a role for central 0 receptors in the direct mediation and indirect modulation of antinociception in the mouse. By demonstrating a role for the 0 receptor in antinociceptive processes, it may be possible to design pharmacological agents that can provide man with relief from pain without the undesirable, and sometimes toxic, side-effects associated with the traditional opioid analgesics such as morphine.

The first portion of the present study focussed on the ability of central 0 receptor activation to result in the direct production of antinociception. A role for supraspinal opioid 0 receptors in the direct mediation of antinociception has been demonstrated clearly in the present investigation. The lack of cross-tolerance between morphine and DPDPE coupled with the antagonism of DPDPE antinociception by the leI 174 864 (0 antagonist), but not ,a-FNA or naloxonazine

(IL and ILl antagonists, respectively), clearly shows that supraspinal 0 receptors can directly mediate antinociceptive processes.

Studies in the spinal cord also provided evidence that spinal 0 receptors are 116 capable of the direct mediation of antinociception. Unlike the findings of the supraspinal studies, cross-tolerance between morphine and DPDPE was present which would imply that these agonists were acting at a common receptor (0) to produce their effects. Studies with the 11 antagonist naloxonazine also supported the idea of a common site (0) of in the cord as this antagonist had no effect on the antinociception produced by the i.th. administration of either the 0 agonist DPDPE

or the J1. agonists DAMGO and morphine. Use of the 0 antagonist ICI 174,864 and the J1. antagonist ,B-FNA did, however, provided evidence that both spinal J1. and 0 opioid receptors are involved in the direct mediation of antinociceptive processes. ICI 174,864 antagonized i.th. DPDPE antinociception while having no effect on the

J1. agonists DAMGO and morphine. Additionally, the J1. antagonist ,B-FNA could be shown to antagonize Lth. morphine, but not DPDPE antinociception.

Supraspinal Studies

The first evidence that supraspinal 0 receptors can directly mediate antinociception came from studies in mice made acutely tolerant to morphine. The rationale for the use of morphine tolerance was that by comparing the antinociceptive potency of the 0 selective agonist DPDPE in control and morphine tolerant mice, inferences could then be made as to which opioid receptor DPDPE acted to produce its anti nociceptive effects at supraspinal sites.

The morphine pretreatment technique used has been demonstrated to reliably induce a state of acute tolerance (Vaught and Takemori, 1979) to morphine, and 117 has been used to demonstrate cross-tolerance between morphine and opioid peptides (Porreca et al., 1987a). This pretreatment did not produce any detectable antinociception at the time of testing. The presence of acute tolerance to morphine was demonstrated after the i.c.v. administration of morphine, in accordance with previous results (Vaught and Takemori, 1979). While i.c.v. tolerance could be demonstrated for morphine, no cross-tolerance could be shown between morphine and i.c.v. administration of the highly 6 selective DPDPE (this study). This result can be interpreted to indicate separate supraspinal antinociceptive sites of action for

DPDPE and morphine, presumably 6 and Jl. receptors, respectively.

To further characterize the direct antinociceptive properties of supraspinal 6 receptors, the next approach attempted to differentially antagonize the antinociceptive effects of morphine, DAMGO and DPDPE. Differential antagonism has historically been accepted as the strongest evidence for involvement of multiple receptors. In this portion of the study, a number of the most receptor selective opioid antagonists were used together with the selective agonists DAMGO (Jl.) and

DPDPE (6) already described above. Morphine was again used as a reference Jl. agonist. These antagonists included the 6 selective competitive peptide ICI 174,864

(Cotton et al., 1984), the non-surmountable Jl. selective non-peptide p-FNA

(Portoghese et al., 1980) and the long-acting Jl. selective naloxonazine (Hahn et al.,

1982).

The first antagonist used was the 6-selective ICI 174,864 (Cotton et al., 1984).

ICI 174,864 alone did not produce antinociception. The co-administration of ICI

174,864 along with DPDPE resulted in a dose-related antagonism of i.c.v. DPDPE 118

antinociception. The same antagonist treatment, however, failed to affect the

antinociceptive properties of either i.c.v. DAMGO or morphine. These findings

suggest that DPDPE is producing its antinociceptive effects at a supraspinal site, presumably the s receptor, which is different from that at which morphine and

DAMGO function (I' receptor). It should be noted that a previous study by Cowan

et al. (1985) found that i.c.v. ICI 174,864 produced postural abnormalities in rats, but

not in mice. That study also showed that ICI 174,864 produced analgesia in the

mouse writhing test at an i.c.v. dose of 14 nmol, but not at 7 nmol. Importantly,ICI

174,864 did not show any analgesic effects in tests employing heat as a noxious

stimulus. In any case, the dose employed in the present study is well below the

antinociceptive dose reported by Cowan et. al. (1985). Furthermore, the previous

study of Cowan et al. (1985) found that higher.doses of ICI 174,864 (i.e. 36 and 72

nmol) failed to antagonize the analgesic effects of DAMGO or DPDPE. Recent

. work has suggested that ICI 174,864 can act as an agonist both in vivo (Leander et

al., 1986) and in vitro (Cohen et al., 1986), possibly by actions at the s receptor.

Thus, it seems possible that high doses of this compound such as those studied by

Cowan et al. (1985) may in fact produce additive effects with DAMGO and DPDPE,

rather than the expected antagonism, which was found using lower doses in the

present work. Similar selective antagonism has been reported in another endpoint

by Dray and Nunan (1984), who showed that i.c.v. ICI 174,864 antagonized the

inhibitory effects of i.c.v. DPDPE on spontaneous rhythmic bladder contractions in

the anesthetized rat without blocking the effects of i.c.v. DAMGO. 119

The next antagonist used was the non-equilibrium Il selective ,B-FNA

(Portoghese et at, 1980). Pretreatment with ,B-FNA alone did not result in any antinociception. ,B-FNA markedly displaced the i.c.v. morphine dose-response line to the right, in agreement with previous findings (Ward and Takemori, 1983). A much greater shift in the i.c.v. DAMGO dose-response line resulted following i.c.v.

,B-FNA pretreatment (compared to morphine, 958 vs 12). This large difference in sensitivity may be due to the increased Il-selectivity of DAMGO (Handa et at,

1981). Conversely, ,B-FNA pretreatment failed to displace the i.c.v. DPDPE dose­ response line. Ward and Takemori (1983) have reported that ,B-FNA was able to

2 6 partially antagonize the antinociceptive actions of i.c.v. [D-Ala , D-Leu ]enkephalin (DADLE) (a proposed s agonist). This may be due to the fact that DADLE is much less s-selective than DPDPE (Corbett et at, 1983; Mosberg et at, 1983;

Galligan et at, 1984). While the precise site of action of ,B-FNA remains obscure, it is clear that this compound differentially antagonizes the antinociceptive effects of DAMGO and morphine, but not DPDPE. A previous study by Dray and colleagues (1985), involving centrally mediated inhibition of rat urinary bladder motility, showed that ,B-FNA lacked selectivity in antagonizing DAMGO and

DPDPE. That study, however, gave ,B-FNA acutely prior to the administration of the agonists, rather than at the 4 hr pretreatment employed here. Thus, these studies are not directly comparable.

Morphine is generally regarded as the prototype Il agonist and was Jsed for that purpose in the present study. It is widely accepted, however, that morphine is not highly Il selective as it been shown to have agonist properties at the s receptor 120 in vitro (Ward et al., 1982). The present findings described immediately below

reinforce this view. Pretreatment with the" selective ,8-FNA resulted in a 12-fold

shift to the right for i.c.v. morphine, but a 958-fold shift to the right for i.c.v. DAMao. One explanation for the large difference in the magnitude of

displacement may be due to the possible action of morphine at 0 receptors. The

action of morphine at 0 receptors is supported by the observed differences of the

slopes for the morphine dose-response lines in control and ,8-FNA pretreated mice.

In ,8-FNA pretreated mice, the slope of the morphine dose-response line is similar

to that for DPDPE. To examine this possibility, i.c.v. morphine was tested in ,8-

FNA pretreated mice (18 nmol i.c.v. at -4hr) in the absence and in the presence of

ICI 174,864 (4 nmol). Figure 11 shows that the 0 antagonist ICI 174,864 had no

effect, at this dose, on i.c.v. morphine antinociception in control mice. In ,8-FNA

pretreated mice, however, ICI 174,864 was able to displace the i.c.v. morphine dose­

response line to the right of the ,8-FNA displaced line (Fig. 13). A complete i.c.v.

ICI 174,864/morphine dose-response line in ,8-FNA pretreated mice could not be

achieved as the higher doses of morphine produced barrel rotation and seizures.

The antagonism of i.c.v. morphine antinociception by ICI 174,864 in ,8-FNA

pretreated mice is in good agreement with a study recently reported by Takemori

and Portoghese (1987). In their study, Takemori and Portoghese (1987) reported

that the naloxone apparent pA2 value against morphine in the mouse abdominal stretch (writhing) test changes significantly after ,8-FNA treatment suggesting that morphine interacts with non-" (0 and It) receptors to produce antinociception.

Additionally, Takemori and Portoghese (1987) have shown that the selective 0 121 antagonists ICI 174,864 (Cotton et al., 1984) and ICI 154,129 (Shaw et al., 1982), respectively, compounds which do not directly antagonize morphine tail withdrawal antinociception in control mice, significantly antagonize morphine antinociception in ,B-FNA pretreated mice. Furthermore, Gmerek and Woods (1985) have reported that ,B-FNA-precipitated withdrawal in the morphine-dependent rhesus monkey could not be suppressed by cumulative doses- of morphine while the same levels of deprivation-induced and naltrexone-precipitated withdrawal were reversible by cumulative doses of morphine. These findings indicate that the effects of ,B-FNA at the Jl. receptor are insurmountable in vivo. With this in mind, it would seem that any antinociception produced by morphine in the ,B-FNA pretreated mouse must be due to activation of receptors other than the Jl. receptor. As antinociception in the

,B-FNA pretreated mouse is antagonized by ICI 174,864, the receptor through which morphine produces its effects would appear to be s.

Following the studies with ICI 174,864 and ,B-FNA, the long-acting Jl. antagonist naloxonazine was used next. Based on the effects of naloxazone and its azine derivative, naloxonazine, on opioid binding as well as opioid-induced antinociception, respiratory depression, and a number of other parameters, Pasternak and co-investigators proposed the existence of opioid Jl. receptor subtypes in the central nervous system (Pasternak et al., 1980; Wolozin and Pasternak, 1981; Hahn et al., 1982; Pasternak and Wood, 1986); these have been termed Jl.l and Jl.2 where naloxonazine is thought to be selective for the high-affinity Jl.l receptor (see

Pasternak and Wood, 1986, for review). Although the existence of Jl. receptor subtypes remains controversial (Rourke and Shaw, 1983; Clark et al., 1986; Kenner 122 and Sarne, 1986), naloxonazine, nevertheless, represents a powerful tool for the possible differentiation of receptors and effects. Naloxonazine, therefore, was employed in an effort to compare the sensitivity of spinally and supraspinally administered agonists to this antagonist in opioid-induced antinociception.

Naloxonazine pretreatment 24 and 48 hr before testing significantly displaced the dose-response lines for i.c.v. morphine and DAMGO antinociception to the right.

The displacement at 24 hr was greater than that at 48 hr for both agonists. The sensitivity to naloxonazine of the J.L agonists is in agreement with the findings of Ling et al. (1985), who showed that pretreatment with this antagonist 24 hr before testing reduced the antinociception resulting from i.c.v. morphine in the rat. In addition, these findings correlate well with radioligand binding studies where naloxonazine pretreatment 24 hr before testing caused a marked decrease in binding of the J.L selective DAMGO in rat brain preparations (Ling et al., 1986). In contrast, naloxonazine pretreatment failed to antagonize i.c.v. DPDPE antinociception at both pretreatment times. This result suggests that i.c.v. DPDPE may produce its antinociceptive effects at a receptor other than the J.L receptor, a finding supported in this species by the findings with leI 174,864 and ,B-FNA stated above.

Spinal Studies

In order to determine the role of spinal opioid 0 receptors in the direct mediation of antinociception, the methods used for the supraspinal studies; i.e., tolerance/cross-tolerance and antagonism studies, were repeated in animals receiving 123 spinal agonist administration.

The tolerance/cross-tolerance studies revealed that, in contrast to the i.c.v. findings, cross-tolerance was seen between morphine and i.th. DPDPE. Interestingly, the displacement of the i.th. DPDPE dose-response line was of equal magnitude to that seen for morphine. The results of the i.th. experiments suggest, but do not establish, that morphine and DPDPE may be producing their spinal antinociception at the same receptor. The identity of this anti nociceptive receptor remains obscure, but has been suggested previously to be the 0 receptor (Ling and Pasternak, 1983;

Porreca et al., 1984).

It should be noted that other investigations of the spinal opioid receptors involved in antinociception which utilized the relatively nonselective 0 agonist

DADLE in cross-tolerance studies, have found results which suggest the presence of two receptor populations. In rats injected twice daily with s.c. morphine, Tung and

Yaksh (1982) reported the anti nociceptive effect of i.th. morphine was reduced by approximately 7-fold, indicating the development of tolerance. In contrast, rats receiving the same morphine pretreatment showed no change in the antinociceptive response to i.th. DADLE, suggesting a lack of cross-tolerance between the antinociceptive effects of these compounds. These results are not in agreement with those reported in the present study. Further complicating the interpretation of the data presented within are the results of Tseng (1982) who infused rats with DADLE and tested antinociceptive responses to i.th. DADLE or i.th. morphine. Both the

DADLE and morphine dose-response lines were shifted to the right in DADLE- 124

infused rats indicating tolerance to DADLE and corresponding cross-tolerance to

morphine. If, however, naloxone and DADLE were co-infused, tolerance to

DADLE was unaffected whereas cross-tolerance to morphine was blocked. These

results were interpreted as involvement of two receptor types with DADLE infusion being non-selective in activating both receptor populations.

Discrepancies between the present findings and the studies mentioned above could be due to several factors including: 1) species differences; 2) the relative non­ selectivity of the S agonist DADLE versus the use of the highly s selective DPDPE;

3) utilization of the i.th. cannula method of drug delivery (Yaksh and Rudy, 1976) whereas the present study utilized a technique for direct lumbar puncture (Hylden and Wilcox, 1980); 5) utilization of twice daily morphine injection or i.th. infusion of DADLE versus, the present method which relied on tolerance resulting from an acute injection of morphine; and 6) the use of morphine which may in and of itself be a complicating factor as this compound is not a highly selective opioid ligand.

A recent study using the highly I' selective morphiceptin analog PL017 revealed that while pretreatment with i.th. PL017 resulted in a marked rightward displacement of the i.th. PL017 dose-response line (61-fold), the same pretreatment resulted in only a 6 fold shift to the right of the i.th. DPDPE dose-response line suggesting little cross-tolerance between i.th. PL017 and i.th. DPDPE (Russell et al. 1987). Thus it would appear that both spinal I' and s receptors may be involved in the production of antinociception, and that the cross-tolerance between morphine and i.th. DPDPE may be due to the action of morphine at spinal s receptors. Lastly, the present 125

results in the mouse appear reliable in that cross-tolerance was seen after Lth.

DPDPE, but not after i.c.v. DPDPE, in morphine pretreated mice. These findings

could indicate that supraspinal opioid antinociception is mediated at both sand J.L

receptors whereas spinal opioid antinociception is mediated by a single receptor

subtype, presumably the S receptor. It should be noted, however, that the

observation of Lth. cross-tolerance between DPDPE and morphine, while suggesting

a common site of action, does not establish this point as the process may be the result of some post-receptor mechanism. If, indeed, both spinal J.L and s receptors are capable of mediating antinociception, the fact that there was no cross-tolerance between morphine and DPDPE in the brain, but cross-tolerance was demonstrated clearly at the spinal level is quite intriguing and may indicate differences between supraspinal and spinal s receptors; i.e., s receptor subtypes. The existence of s receptor subtypes has, in fact, been recently supported based on findings both in

2 vitro and in vivo with a novel enkephalin analog, [D-Ala , (2R,3S)-E-cyclopropyl­

Phe, Leu5]enkephalin (CP-OH)(Shimohigashi et al., 1982; 1984; 1987).

As was the case at the supraspinal level, the next approach to further characterize the antinociceptive actions of spinal s receptors was to use selective antagonists. The first antagonist used was the s selective ICI 174,864. Similar to the supraspinal results, the results at the spinal level demonstrate that ICI 174,864 completely blocked the antinociceptive effects of Lth. DPDPE without affecting Lth.

DAMGO or morphine. These results suggest that DPDPE produces its antinociceptive effects at a site different from that at which morphine and DAMGO work, presumably the sand J.L receptor respectively. 126 The effect of ,B-FNA on morphine and DPDPE antinociception was also examined at the spinal level. While high doses of ,B-FNA antagonized both morphine and DPDPE, lower doses (0.018 and 0.009 nmol) selectively blocked Lth. morphine but not Lth. DPDPE antinociception. This is in agreement with previous findings (Hylden and Wilcox, 1983) showing that Lth. ,B-FNA (0.94 nmol at -2 hr) pretreatment blocked the inhibition of substance P-induced behaviors (hind-limb scratching) by Lth. morphine, but not Lth. DADLE. It has been previously shown that DADLE has both p. and s activity (Galligan et al., 1984; James and Goldstein,

1984; Mosberg et al., 1983), a view supported by the fact that the dose of ,B-FNA

(0.94 nmol), used in previous work (Hylden and Wilcox, 1983), was selective against the p. effects of DADLE. The present finding, therefore, again suggests that activation of spinal 6, as well as p. receptors, can result in the direct production of antinociception.

The last antagonist used was naloxonazine. Unlike the results seen in the brain in the present study, naloxonazine pretreatment had no effect on the antinociception associated with Lth. morphine, DAMGO and DPDPE. Although the lack of effect on Lth. DPDPE was not surprising, the absence of naloxonazine antagonism of Lth morphine and DAMGO antinociception was unexpected. The lack of effect against spinal antinociception was shown not to be due to naloxonazine inactivity at the spinal level, however, as naloxonazine effectively blocked the gastrointestinal effects of the agonists following their i.th. administration. It is not likely, however, that all three of the agonists were acting at non-p' (6) spinal opioid receptors to produce their antinociception. As was stated above, the 6 selective 127 antagonist ICI 174,864 (Cotton et al., 1984) inhibited the antinociception produced by i.th. DPDPE but not i.th. morphine or DAMGO. It is important to note that spinal Ie receptors are not involved in antinociception when heat is used as the noxious stimulus (Porreca et al., 1984; 1987b). Thus, the receptor at which i.th. morphine and DAMGO act to produce antinociception would appear to be a naloxonazine-insensitive J.L receptor. The lack of binding data for the mouse spinal cord makes it difficult to correlate the naloxonazine-insensitive site with the putative

J.L2 receptor.

Estimation of the Naloxone pA2 for morphine. DAMGO and DPDPE

An additional approach, the determination of the apparent naloxone pA2' was taken in an effort to determine the direct antinociceptive actions of supraspinal opioid 0 receptors. The determination of the pA2 for an antagonist has historically been used in the differentiation of receptor types involved in a common effect.

Because the interpretation of the pA2 data is difficult, the results from these studies are discussed separately here.

The present approach involved the determination of this affinity constant at supraspinal and spinal sites using a time-dependent method (Tallarida et al., 1978).

Critically, both the agonist and the antagonist were given either directly into the brain or into the spinal subarachnoid space. Administration of the antagonist close to its site of action increases the precision of the determination of affinity. This is due to the fact that administering the compounds close to their loci of action permits 128 a dose unit more closely approximating tissue concentration as well as more . predictable pharmacokinetics. The values obtained, however, are not directly

comparable to studies using different routes of administration because of the

differences in the tissue drug concentration and pharmacokinetics (Tallarida et al.,

1979). The time-dependent method was originally derived by Tallarida and

colleagues (1978) as a check on the standard method of Schild (1947). The results were found to agree well with the traditional method in that study and in a

subsequent validation of the method when naloxone was given by a central (i.c.v.)

route (Porreca et al., 1981).

The results of the present study indicate a lack of significant difference

between the affinity of naloxone for the supraspinal receptors activated by DPDPE

and those activated by DAMGO and morphine. These findings are supported by

later work using the same agonists in the rat hot-plate test (Fang et al., 1986). A

difference in naloxone affinity against these receptor selective agonists would be

interpreted as suggesting that the agonists were acting at different receptor populations. Similar affinity suggests either that the agonists are activating the same

receptor population, or alternatively, that there is a fortuitous similarity in naloxone

affinity at two different receptor populations. Fortuitously similar naloxone affinity would appear to be the case here when the pA2 data are interpreted along with the

findings of the supraspinal studies discussed above which suggest that supraspinal 0,

as well as JL, receptors are capable of directly mediating antinociception.

An evaluation of the naloxone pA2 values after spinal administration shows 129 that the confidence limits of this antagonist against morphine overlap with those of

DAMGO and DPDPE. The affinity of naloxone appears to differ between i.th.

DPDPE and DAMGO. The magnitude of this difference is very small, however, and probably does not represent a meaningful difference. Thus, DPDPE and DAMGO, highly selective agonists for the sand 11 receptors, respectively, may be acting at different spinal receptors, 5 and 11 respectively, in the production of antinociception.

This interpretation is in agreement with the spinal findings discussed above.

Summary of Direct S Involvement

The goal of the first portion of this dissertation was to determine the antinociceptive properties of central opioid 5 receptors. The results of the present study demonstrate that activation supraspinal and spinal 5 receptors can result in the direct production of antinociception. The involvement of supraspinal S receptors in the direct production of antinociception is strongly supported based on: 1) the selective antagonism of DPDPE, but not morphine or DAMGO antinociception by leI 174,864; 2) the lack of effect of naloxonazine on DPDPE, but not morphine or

DAMGO antinociception; 3) the lack of effect of ,B-FNA on DPDPE, but not morphine or DAMGO antinociception; 4) the effectiveness of leI 174,864 against morphine antinociception in ,B-FNA, but not vehicle, pretreated mice; and 5) the lack of cross tolerance between morphine and DPDPE. The involvement of spinal s receptors in the direct production of antinociception is supported, but more complex based on the facts that: 1) leI 174,864 antagonizes DPDPE, but not morphine of 130 DAMGO antinociception; 2) naloxonazine pretreatment fails to affect DPDPE, morphine or DAMGO antinociception; 3) ,B-FNA antagonizes morphine, but not

DPDPE antinociception; and 4) morphine pretreatment results in acute tolerance to morphine and cross-tolerance between morphine and DPDPE.

Modulation Studies

Although the present study strongly supports the notion that supraspinal and spinal opioid 6 receptors playa role in the direct mediation of antinociception, data also exist which suggest that the enkephalins, putative endogenous ligands for the 6 receptor (Lord et aI., 1977) may also act indirectly to influence the anti nociceptive processes that are mediated by J.L receptors. Consequently, the interactions of 6 and

J.L agonists have been studied in an effort to characterize further the functional interactions of the J.L and 6 receptor as modulation may provide an additional means of providing pain relief in man.

Previous studies have demonstrated a modulation of the antinociception produced by the J.L preferring prototype agonist, morphine, by compounds with selectivity for the 6 receptor. I.c.v. administration of sub-antinociceptive doses of

Leu-enkephalin and Leu-enkephalin analogs have been shown to potentiate morphine antinociception as well as the development of acute morphine tolerance and dependence in the mouse (Vaught and Takemori, 1979; Barrett and Vaught,

1982; Vaught et aI., 1982). In contrast, sub-antinociceptive doses of Met-enkephalin and Met-enkephalin analogs attenuate morphine antinociception (Lee et aI., 1980; 131

Vaught et al., 1982). I.th. administration of sub-antinociceptive doses of Leu­ enkephalin have also been shown to potentiate i.th. morphine antinociception in the rat (Larson et al., 1980).

Based on observations of the modulation of antlnociception by 0 agonists, it has been hypothesized that some I' and 0 receptors may exist in an opioid receptor complex (Vaught et al., 1982). Further evidence for such a complex has been provided by mathematical analyses of radioligand binding data in brain membrane preparations from the rat (Rothman and Westfall, 1982a,b; Rothman and Westfall,

1983; Demoliou-Mason and Barnard, 1986; Rothman et al., 1987b) and mouse

(Barrett and Vaught, 1983). Furthermore, studies in vitro using the mouse vas deferens bioassay have also provided evidence for a 1'-0 receptor complex (Sanchez­

Blazquez et al., 1983). Finally, evidence in vivo for a 1'-0 receptor complex is not limited to systems mediating antinociception. A 1'-0 receptor complex has also been suggested to be involved in the reversal of endotoxic shock in the rat (Holaday and

D'Amato, 1983; D'Amato and Holaday, 1984).

The goal of the second portion of the current study, therefore, was to investigate further the functional interactions of I' and 0 receptors. To do so, studies were designed in which the effects of the 0 agonist DPDPE on wmediated antinociception could be observed. Additionally addressed, was the possibility that agonists with equal or near-equal affinity for the opioid I' and 0 receptor may be

"self-potentiating" (Vaught et al., 1982). Previous studies of 1'-0 interactions have generally used morphine as the I' agonist. Inasmuch as (a) morphine is I' preferring, but does not display a great deal of selectivity for the I' receptor (Mosberg et al., 132

1983), and (b) no specific endogenous ligand for the p. receptor has yet been identified, a series of agonists with varying selectivities for the p. receptor were studied: these included morphine, normorphine, DAMGO, PL017, etorphine, phenazocine, sufentanil and ,B-endorphin (human).

Studies with morphine.

The first step in studying p.-6 interactions was to investigate and characterize the effects of co-administration of the highly 6 selective agonist, DPDPE, with antinociception initiated by the prototype p. agonist morphine at both supraspinal and spinal sites. A dose of DPDPE which did not produce significant antinociception when given alone, potentiated i.c.v. morphine antinociception. In order to determine if the observed potentiation was due to an interaction of DPDPE with the 6 receptor, the modulatory dose of DPDPE was challenged with the selective 6 antagonist leI 174,864. The potentiation of i.c.v. morphine antinociception associated with DPDPE was prevented by 6 receptor blockade with leI 174,864. This finding suggests that the modulation of morphine antinociception results from the action of DPDPE at the 6 receptor. It is important to note that leI

174,864 had no antinociceptive effects alone, and did not directly antagonize or potentiate morphine antinociception as demonstrated in the antagonism studies described above. This result correlates well with the findings of Vaught et al. (1982) who showed that Met-enkephalin could counteract the potentiation of morphine produced by Leu-enkephalin in mice, and that Leu-enkephalin could prevent the 133 aUelluation of morphine antinociception produced by Met-enkephalin. These findings were interpreted as indicating that the modulatory effects may occur through a common opioid (6) receptor. Further support of this interpretation is provided by the fact that the same dose of DPDPE used in naive mice maintained its ability to potentiate i.c.v. morphine antinociception in acutely morphine-tolerant mice. The finding that DPDPE retains its potency as a potentiator of morphine antinociception in morphine-tolerant mice is in complete agreement with Lee et al. (1980) who demonstrated that the same dose of Met-enkephalin antagonizes morphine antinociception in morphine-tolerant mice. Lee and coworkers intimated that because a higher dose of Met-enkephalin was not needed to attenuate morphine antinociception in morphine-tolerant mice, it would appear that Met-enkephalin does not compete with morphine for the I' receptor to reduce morphine antinociception, but does so via a distinct (6) receptor. The current findings, therefore, support the previous findings which suggest that the enkephalins and enkephalin analogs produce their modulatory effects via an action at a receptor (6) distinct from that activated by morphine (I' receptor). Accordingly, as an increase in the dose of DPDPE is not required for modulation of morphine antinociception in morphine-tolerant mice, and because modulation is reversed by leI 174,864, it seems reasonable to conclude that the modulation of morphine antinociception occurs by action of DPDPE at the 6 receptor.

It could be argued that DPDPE potentiates morphine antinociception by simply displacing morphine from the 6 receptor, thereby increasing the relative concentration of morphine available at the I' receptor. If this hypothesis was correct, 134

then the 6 agonist DPDPE, and the 6 antagonist ICI 174,864 could (as a result of higher affinity for the 6 receptor) both be expected to displace morphine from binding to the 6 site and consequently raise the relative concentration of morphine

available at the I-' receptor, resulting in potentiation of antinociception. This was not the case, however, as the 6 antagonist ICI 174,864, at a dose which antagonized 6- mediated effects, had no antagonist effect on morphine antinociception and did not potentiate the effects of morphine in the above antagonist studies.

In contrast to potentiation seen at the supraspinal level, results at the spinal level demonstrate that DPDPE does not potentiate the antinociceptive effects of morphine. This result is consistent with the findings of the tolerance/cross-tolerance experiments discussed above which suggested that DPDPE and morphine may act at the same spinal opioid receptor to produce their pharmacological effects in that compounds acting at a common receptor would not functionally interact. The above studies with selective antagonists, however, indicate that morphine and DPDPE act at distinct spinal receptors, I-' and 6 respectively, thereby ruling out action at a common receptor as a reason for the lack of 1-'-6 interactions. In addition, the present finding that the 6 agonist DPDPE does not potentiate i.th. morphine antinociception in the mouse is in direct contrast to previous findings in the rat where Leu-enkephalin was able to potentiate i.th. morphine antinociception. The reasons for such a discrepancy are unclear, but may have to do with the choice of species, as well as other factors such as the precise method of intrathecal drug delivery (Yaksh and Rudy, 1976) (i.e., chronic i.th. cannula in the rat vs. direct lumbar puncture in the mouse). 135

Studies with chemically diverse JL agonists.

In addition to the interaction of DPDPE and morphine, the modulatory effect of DPDPE on the antinociception produced by several other structurally diverse f..L agonists was also studied in the present investigation. Similar to its effect on morphine, DPDPE also potentiated i.c.v. normorphine antinociception. In contrast,

DPDPE had no effect on the antinociception elicited by i.c.v. DAMGO, PL017, phenazocine, sufentanil or etorphine. Furthermore, DPDPE failed to potentiate (3- endorphin antinociception, which is in agreement with a previous study which showed that Leu-enkephalin did not potentiate (3-endorphin antinociception (Vaught et aI., 1982). While the ability of DPDPE to potentiate morphine and normorphine antinociception supports the concept of opioid JL-o receptor interactions via an opioid receptor complex, the present results suggest that of the JL compounds studied, only morphine and normorphine are selective for actions at this receptor complex.

One possible explanation for the differential effects of DPDPE on the JL agonists tested is self-potentiation. The concept of self-potentiation was introduced by Vaught et al. (1982) to explain the discrepancies between the affinity of certain agonists for the JL receptor in vitro and their antinociceptive potencies in vivo. As mentioned previously, there has traditionally been a strong correlation between affinity for the JL receptor in vitro and antinociceptive potency in tests where heat is used as the noxious stimulus. Discrepancies between affinity and potency began to arise with the advent of enkephalin analogs. Vaught et al. (1982) demonstrated 136 clearly that although the affinity of morphine for the J.I. receptor is greater than that

2 5 of the enkephalin analog [D-Ala , D-Leu ]enkephalin (DADLE), DADLE is a much more potent antinociceptive agent than morphine. The discrepancy between J.I. affinity and antinociceptive potency of DADLE was attributed to the affinity of

DADLE for the 0 receptor. Inasmuch as the affinity of DADLE for the 0 receptor is only 12-fold greater than its affinity for the J.I. receptor (Vaught et aI., 1982), it is conceivable that DADLE is capable of acting at both the J.I. and 0 receptor of the receptor complex. By activating both receptors of the J.I.-o receptor complex, the presence of DADLE at the 6 site of the receptor complex would thus, potentiate its own J.I.-mediated antinociception. The antinociceptive effects of phenazocine, etorphine and p-endorphin, therefore, could also be attributed to self-potentiation based upon the similar affinities of these compounds for the J.I. and 0 receptor

(Vaught et aI., 1982). If compounds are indeed self-potentiating, then no further potentiation by DPDPE would be expected.

While the concept of self-potentiation is alluring, it does not explain the lack of effect of DPDPE on DAMGO, PL017 and sufentanil antinociception in the current investigation. If the measured antinociceptive effect of the J.I. compounds were a self-potentiated effect, it is reasonable to expect that the possibility for self­ potentiation increases as the affinity for the 6 receptor increases. Such an increase would, thus, decrease the J.L/o affinity ratio which is an index of compound selectivity.

Conversely, as the J.I. affinity of an agonist increases (J.I./o increased), its ability for self-potentiation would decrease, and its susceptibility to potentiation by DPDPE 137 would increase. The rank order of s affinity (taken from literature sources), from

a highest to lowest, of the J.' agonists used in the present study is as follows: etorphine

> phenazocineB > ,a-endorphinb > morphineB.c > sufentanilB > DAMGOc > normorphineB > PL017c (data from BMagnan et aI., 1982; bMosberg et aI., 1983; cpersonal communication, Dr. Henry I. Yamamura). The ratio of J.'/s selectivity is presented graphically in figure 37.

To address the possibility of self-potentiation, an attempt was made to antagonize the antinociceptive effects of i.c.v. etorphine, phenazocine and ,a­ endorphin with the S antagonist leI 174,864 as these J.' agonists possess the greatest affinity for the s receptor amongst the compounds tested, and therefore, were most likely to be self-potentiating. A dose of 4 nmol leI 174,864 given i.c.v. has been shown above to completely abolish the antinociceptive effects of i.c.v. DPDPE while having no effect on i.c.v morphine antinociception. If, indeed, etorphine, phenazocine or ,a-endorphin were self-potentiating, this same dose of leI 174 864 would be expected to produce some reduction or attenuation in the antinociceptive effect. Such a reduction would be due to the elimination of the s (potentiating) component of these agonists. Figure 30 reveals, however, that leI 174,864 had no effect on the antinociceptive effects of i.c.v. etorphine, phenazocine or ,a-endorphin.

Thus, self-potentiation does not explain the lack of effect of DPDPE on etorphine, phenazocine or ,a-endorphin antinociception. Based on the thinking that self­ potentiation comes from the action of an agonist at both the J.' and s receptor of the receptor complex, and that decreasing s affinity of a J.' agonist increases the possibility that a compound will be potentiated by DPDPE, the antinociceptive 138

effects of DAMGO, PL017 and sufentanil in addition to morphine and normorphine,

should also have been potentiated by DPDPE. This was not the case, however, as

DPDPE affected neither DAMGO, PL017 or sufentanil antinociception.

An alternative explanation for the differential effects of DPDPE on ,,­ mediated antinociception is that not all " agonists are capable of activating the

"complexed" "receptor. If this is the case, the present data imply that differences

may exist between complexed and non-complexed " receptors, i.e. " receptor subtypes which have been discussed above. It is interesting to note that the specific

" agonists which are modulated by i.c.v. DPDPE in the present study (i.e. morphine and normorphine) are the same as those: (a) potentiated by DPDPE in the rat using the inhibition of the micturition reflex as the endpoint (Sheldon et aI., 1989) and

(b) antagonized by the /C agonists U50, 488H and dynorphin in the rat micturition reflex model (Sheldon et aI., 1987, 1989). The similarity in profile of modulated" agonists across species and effect not only supports the concept of a ,,-6 receptor complex, but also the existence of " receptor subtypes with characteristics which differ based on whether they reside in, or outside the receptor complex.

The criteria for activation of the complexed " receptor are not inherently obvious. Whilst there is some evidence for distinct" receptors in the guinea-pig ileum (Takemori and Portoghese, 1985) which may be selectively activated by peptide or non-peptide agonists (Ward et aI., 1986), such a distinction may not apply to the present data as neither the peptides DAMGO, PL017 or p-endorphin, nor the non-peptides phenazocine, etorphine or sufentanil were potentiated by DPDPE.

Regardless of physiochemical commonalities amongst the" agonists used, which 139

1000 • DPDPE 100

en <1> 10 DADLE :J • 0 > • Etorphine ~- 1 Phenazocine 'f- • p-endorphin 0 • 0 .1 :.;; • Sufentanil 0 a::: • Morphine\Normorphine .01 e DAGO

.001 .0006 • PL017

Fig. 37. Profile of ILls selectivity based on the ratio of ~ values for displacing receptor selective ligands. ~ values for PL017, DAMGO, morphine, DADLE and

DPDPE are based on displacement of the IL selective somatostatin analog eH]-[Cys2,

6 7 ~, Orn , Pen ]amide (CfOP)(Gulya et aI., 1986) and [3H]DPDPE (s) binding in rat brain membrane preparations. 140 could provide insight into the structure-activity relationship of complexed vs non­ complexed I" receptors, the differential potentiation of the various I" agonists is strongly suggestive of the existence of I" receptor subtypes.

Recently reported radioligand binding and autoradiography studies have provided evidence that not only supports the concept of a 1"-5 receptor complex, but which suggests that differences exist between the I" and 5 receptors in, and outside

2 of the receptor complex. It has been shown that the 5 agonist, eH)-[D-Ala , D­

Leu5]enkephalin eH-DADLE) labels two binding sites in vitro which are distinguished by the inhibitory mechanism of I" ligands (Rothman et aI., 1985a;

Rothman et aI., 1985c); while I" ligands are weak, competitive inhibitors at the higher affinity 3H-DADLE binding site, commonly identified as 5, they are potent, noncompetitive inhibitors at the lower affinity 3H-DADLE binding site. Based on the potent displacement of 3H-DADLE from the lower affinity site, Chang and

Cuatrecacas (1979) identified it as a I" binding site. However, a number of experimental manipulations demonstrate differences between I" binding sites and the lower affinity 3H-DADLE binding site. These include: (a) the ionic composition of the assay medium (Bowen et aI., 1981; Rothman et aI., 1984b); (b) the ability of the site directed acylating agent FIT (N-phenyl-N-[1-(2-(p-isothiocyanato )phenylethyl-4- piperadinyl]propanamide )-HCI to unmask lower affinity 3H-DADLE binding sites but not I" sites (Rothman et aI., 1985b); (c) the observation that the i.c.v. administration of p-funaltrexamine (p-FNA, Portoghese et aI., 1980) to rats 24 hr prior to preparation of brain membranes results in an approximately 60% decrease in the

Bmax of the lower affinity 3H-DADLE binding site without any alteration in 3H_[D_ 141

2 4 Ala , NMPhe , Gly-ol]enkephalin eH-DAMGO)(Handa et al., 1981) binding parameters (Rothman et al., 1984a; 1986; 1988) and (d) the finding that I' ligands are noncompetitive inhibitors of a I' binding site labelled by [3H]-naloxone (Rothman et al., 1985c) and [3H]-17 -cyclopropylmethyl-3, 14-dihydroxy-4,5-Q-epoxy-6-,B-fluoro­ morphinan eH-cycloFOXY)(Rothman et al., 1987b).

These reciprocal noncompetitive interactions between I' and 6 receptors led to the suggestion that (a) the lower affinity [3H]-DADLE binding site is the 6 binding site of an opioirl receptor complex (termed 6ex to indicate "in the complex") and that the noncompetitive interaction of I' ligands on the lower affinity 3H-DADLE binding site is mediated through an adjacent I' binding site (termed I'ex to indicate "in the complex"); (b) the higher affinity [3H]-DADLE binding site is a 6 binding site which is not part of the receptor complex (termed 6nex to indicate "not in the complex"), and (c) that I'-mediated antinociception occurs via binding of I' ligands to the I'ex binding site while the modulatory effects of 6 agonists on I'-mediated antinociception is mediated via binding to the 6 binding site of the opioid receptor complex (6ex) (Rothman et al., 1987a,b; Rothman et al., 1988).

Effect of I' selective antagonisis on 6 modulation of I' mediated antinociception.

Additional studies were carried out in an effort to further elucidate the possible functional interaction of I' and 6 receptors in the production of antinociception and to test the hypothesis that the modulatory effects of 6 agonists on morphine antinociception are mediated via actions at the 6ex receptor. To 142 address this hypothesis, it was desirable to try to disrupt the Jj-s receptor complex.

Recent studies in the rat have suggested that the irreversible Jj-antagonist, ,B-FNA may provide a means to disrupt the complex, as this compound has been shown to alkylate the receptor complex (Rothman et aI., 1986; 1988). Thus, the Jj modulatory

2 5 effects of DPDPE and [D-Ala , Met ]enkephalinamide (DAMA), s agonists previously shown to potentiate (present study) and antagonize (Vaught et aI., 1982) morphine antinociception, respectively, were studied in the absence and presence of the selective opioid antagonist, ,B-FNA (Portoghese et aI., 1980). Additionally, in an attempt to gain further insight into the nature of the Jjex and Jjnex sites, naloxonazine, an antagonist of the putative Jjl receptor was studied.

Doses of the s agonists DPDPE and DAMA which produced no significant antinociception when given alone were found to potentiate and attenuate i.c.v. morphine antinociception, respectively, in agreement with the present and previous studies (Lee et aI., 1980; Vaught et aI., 1982). These modulatory effects would appear to be due to an interaction of the 0 agonists with the 0 receptor as the 6 selective antagonist prevented both the potentiation and attenuation of morphine antinociception produced by DPDPE and DAMA respectively; it should be noted again that the dose of ICI 174,864 (4 nmol) used in the present study did not directly antagonize morphine antinociception.

Reasoning, therefore, that morphine antinociception may be modulated in part via a Jj-6 complex, attempts to disrupt this interaction were made using the non­ equilibrium Jj selective antagonist, ,B-FNA (Portoghese et aI., 1980; Ward et aI.,

1983). Previous work has shown that ,B-FNA pretreatment prevents the ability of ICI 143

154,129, a 0 selective antagonist (Shaw et aI., 1982) to reverse endotoxic shock

(Holaday and D'Amato, 1983; D'Amato and Holaday, 1984). Reasonable I-' selectivity has been demonstrated for ,B-FNA, however, as this antagonist does not inhibit 0nex binding in rat brain membrane preparations (Rothman et aI., 1984a;

Rothman et aI., 1987a,b), nor does it block the antinociceptive effects of 0 agonists given i.c.v. in mice (present study). Thus, the ability of ,B-FNA to prevent the effects of leI 154,129 was suggested to be the result of an alteration in the 1-'-0 receptor complex (Holaday and D'Amato, 1983; D'Amato and Holaday, 1984). Additionally, i.c.v. administration of ,B-FNA to rats 18-24 hr prior to preparation of brain membranes has been demonstrated to lead to a. selective alkylation of the opioid receptor complex which was reflected by a substantial decrease in the Bmax of the

0ex binding site, while the binding of [3H]-DADLE to the 0nex site was not significantly altered (Rothman et aI., 1986;1988). This apparent selective effect of

,B-FNA on the opioid receptor complex in vivo allowed the formulation of the following testable prediction: if the modulatory effects of sub-antinociceptive doses of 0 agonists on morphine antinociception are mediated via an opioid receptor complex, then pretreatment with ,B-FNA should prevent the ability of 0 agonists to modulate morphine antinociception.

The present study demonstrates that pretreatment with ,B-FNA abolishes both the potentiation and attenuation of morphine antinociception associated with

DPDPE and DAMA, respectively, a finding which might be interpreted as supporting the concept of functional uncoupling of the 1-'-0 complex given the observation of disruption of the opioid receptor complex by ,B-FNA in vivo (Rothman et aI., 1986; 144

Rothman et aI, 1988). An alternative explanation seems possible, however. Both the present study and work reported by Takemori and Portoghese (1987) have shown that after blockade of available I' receptors with ,B-FNA, morphine produces its antinociception at a non-I' site. Modulation by 0 agonists, therefore, would not be possible if both agonists were acting at the same receptor (oncx)' Furthermore, the competitive interactions of I' and 0 ligands at this binding site (oncx) in vitro are consistent with the observations in vivo which demonstrate that DPDPE and DAMA do not modulate morphine antinociception when morphine acts at the 0ncx site.

Further attempts to characterize the 1'-0 functional interaction were made using the long-lasting, proposed 1'1 antagonist naloxonazine. Naloxonazine pretreatment had no effect on i.c.v. DPDPE antinociception in agreement with the present and previous studies where this antagonist neither blocked i.c.v. DPDPE antinociception in the mouse (present study) or rat (Ling et al., 1986), nor altered binding of DPDPE in rat brain preparations (Clark et al., 1986). Similarly, naloxonazine pretreatment had no effect on i.c.v. DAMA antinociception (present study). In contrast to the lack of effect of naloxonazine on DPDPE and DAMA antinociception, this antagonist exhibited the expected long-lasting blockade of i.c.v. morphine antinociception in the mouse in agreement with the present study in this species as well as in the rat (Ling et al., 1986). Potentiation and attenuation of morphine antinociception by DPDPE and DAMA, respectively, were still evident in mice that received naloxonazine treatment prior to testing. Various interpretations might be made from these findings. For example, naloxonazine might alter the 145 conformation of the receptor complex in such a way that morphine binds with lower affinity, thus reducing the potency, but leaving the modulatory mechanisms intact.

As the mechanism of action for the long-lasting antagonism associated with naloxonazine is well not understood, continued action of morphine may be due to incomplete blockade of J.Lcx receptors. Alternatively, it is also possible that the J.L receptors in the receptor complex are naloxonazine insensitive (i.e., J.L2)'

As is often the case, the present findings in vivo are not in complete agreement with hypotheses drawn from findings in vitro. Based on evidence from binding studies with p-FNA, one would predict that all J.L agonists act at the J.Lcx receptor to produce their antinociception (Rothman et al., 1986; Rothman et al.,

1987a; Rothman et al., 1988). The present investigation shows that while morphine antinociception is potentiated by DPDPE, the antinociceptive effects of the J.L agonist

DAMGO are not, a finding which suggests that morphine and DAMGO may act at two distinct J.L receptors (J.Lcx and J.Lncx) respectively, to produce their antinociceptive effects. Although the antinociceptive effects of morphine and DAMGO were both found to be antagonized by pretreatment with p-FNA in the present study, a finding in agreement with binding studies and the above prediction, only morphine antinociception is potentiated by DPDPE. The lack of potentiation of DAMGO antinociception by DPDPE, therefore, is not directly in accord with the concept that the J.Lcx receptor is the sole site for the mediation of J.L antinociception. It is also important to note that while naloxonazine pretreatment has presently been shown to antagonize both i.c.v. morphine and DAMGO antinociception, this antagonist had 146 no effect on the ability of DPDPE or DAMA to modulate morphine antinociception.

The lack of effect of naloxonazine on the modulation of morphine antinociception coupled with the antagonism of both morphine and DAMGO antinociception by naloxonazine again suggests a discrepancy with the concept that antinociception is mediated solely at the J.'cx site. Although reasons which might account for these discrepancies can be formulated, it seems clear that further experiments are needed to resolve the inconsistencies noted above. Although inconsistencies do exist as to the identity of the specific site where J.' agonists act to produce their antinociceptive effects, it is important to note that the differential antagonism of the modulation observed with ,B-FNA and naloxonazine, nevertheless continues to support the concept of J.' receptor subtypes.

Summary of Indirect S Involvement

The goal of the second portion of the present study was to characterize further the functional interactions between J.' and s receptors. Functional interactions between sand J.' receptors, which result in the indirect modulation of antinociceptive processes, were shown to occur at supraspinal, but not spinal sites. The data clearly show that: 1) the antinociceptive effects of morphine were potentiated by DPDPE and attenuated by DAMA, and that these modulatory effects were prevented by leI

174,864 suggesting that modulation occurred via a S receptor; 2) of the J.' agonists tested, only morphine and normorphine were modulated by DPDPE; 3) pretreatment with ,B-FNA prevented the modulatory effects of DPDPE and DAMA on morphine antinociception while naloxonazine had no effect. These results, therefore, support 147

further the concept that supraspinal p. and 0 receptors interact functionally.

Furthermore, the findings of the present study suggest that the p. receptor of the

complex may be a specific p. receptor subtype which is activated only by certain

agonists represented by morphine and normorphine. This finding supports further

the concept of opioid receptor subtypes.

General Summaty

The goal of this investigation was to determine the role of supraspinal and

spinal opioid 0 receptors in the direct mediation, and indirect modulation of

antinociception in the mouse. Collectively, the data reported within this dissertation

suggest that: 1) supraspinal 0, as well as p, receptors are capable of directly

mediating antinociceptive processes; 2) supraspinal 0 receptors can indirectly

modulate p.-mediated antinociception; 3) spinal 0, as well as p.,receptors are capable

of directly mediating antinociceptive processes; and 4) subtypes of p. receptors may

exist.

These findings are not only of academic interest, but may also have direct

application to man. As central opioid 0 receptors appear to be intimately involved in the production of antinociception, the elucidation of the role(s) played by central o receptors could be of clinical importance. If so, it may be possible to exploit this fact by developing 0 selective drugs which can be used for antinociception, but which are free of the undesirable side-effects associated with morphine. Use of 0 selective

2 agonists shows great promise as the administration of the 0 agonist [D-Ala , D 148 -Leu5]enkephalin, DADLE, has been shown to produce significant pain relief in morphine-tolerant patients (Onofrio et at, 1983; Krames et at, 1986; Moulin et at,

1985). The development of 0 selective agonists may also lead to novel uses of pain relieving drugs in situations where their use has been prohibited previously. For example, narcotics are not normally used in childbirth as they cause a life threatening depression of respiration in the fetus. Ontogenic studies, however, show that opioid 0 receptors are not expressed in the newborns of a number of species

(Tavani et aI., 1985; McDowell and Kitchen, 1986), and are seen only one to two weeks after birth. If this is true in humans as well, 0 selective agonists could be used for pain relief in women in labor with no risk to the infant. Thus, the need for better understanding the functions mediated by the 0 receptor becomes apparent. 149

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