Studies on opioid delta receptor mediated antinociception,opioid antinociceptive tolerance and physical dependence

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STUDIES ON OPIOID DELTA RECEPTOR MEDIATED ANTINOCICEPTION, OPIOID ANTINOCICEPTIVE TOLERANCE

AND PHYSICAL DEPENDENCE

by

Edward James Bilsky

A Dissertation Submitted to the Faculty of the

COMMITTEE 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

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THE DNIVERSITY OF ARIZONA ® GRADUATE COLLEGE

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

read the dissertation prepared by Edward James Bilsky

entitled Studies on Opioid Delta Receptor Mediated Antinociception,

Opioid Antinociceptive Tolerance and Physical Dependence

and recommend that it be accepted as fulfilling the dissertation

requirement for the Degree of Doctor of Philosophy

Date

^/-u-rC Date

Date

- 'iG Date

l( j j q c , Dat^ 7

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 direction and recomm^d that it be accepted as fulfilling the dissertation requirement.

Dissietrtation Director QAAJL^

Dat«

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 acknowledgment 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 judgment 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

ACKNOWLEDGMENTS

" Without love in the dream it will never come true " -Robert Hunter-

The culmination of my graduate studies has taken considerable time and effort. There are many people who have directly or indirectly helped me along the way. First and foremost is my Ph.D. advisor Frank Porreca. Frank and his wife Gabriella have always treated me as a friend of the family. Their generosity and hospitality have extended from the workplace to a more personal level. I have always been taken care of. More importantly, Frank has guided my intellectual growth with expertise and passion. He has a genuine concern for my development as a research scientist, spending many extra hours working with me. He has also provided many opportunities for me to teach at the University. For all of this and anything else I have forgot­ten to mention, I sincerely thank Frank. I look forward to working with him in our future endeavors.

The other faculty members on my dissertation committee also deserve special recognition. I thank them for all their advice and comments on my research at the University of Arizona. Josephine Lai has been a close collaborator on the antisense projects and I thank her for all of her help. I have also shared some good times with Jay Gandoifi. I especially enjoyed the late Softball games followed by the even later recollections by Jay of the San Francisco scene in the late 1960s. Ed French and Paul Consroe have also been good friends and have given me additional opportunities to teach at the University.

There have been many post-docs, graduate students and staff in the Porreca laboratory that have helped me along the way. Mike Nichols deserves special recognition as we have survived over 11 years of sharing the same dorm, laboratories, office spaces, as well as football and Softball fields. Through it all we have remained fnends. As Mike gets ready to head to Minnesota, I wish him and Wanda all the best.

Some additional members of the Porreca lab to mention: Todd Vanderah is a unly unique person. He is one of the most positive and unselfish people I know. We had some great times while we were students here. I thank Robert "Dr. Num Num" Horvath for all his help with the intrathecal injections and the admirable job he did as our laboratory manager. Mike "Flash" Ossipov has been a great resource of pharmacological knowl­edge and comic relief. His leather jacket, carton of cigarettes and half-a-six-pack of beer will forever be etched in my memories of the Department camping trips. Di Bian, C.J. Kovelowski and Sandy Wegert have also been an integral part of the Porreca lab experience. I also thank the rest of the Porreca lab (former and current members) especially Peg Davis, Kent Menkens and Ken Wild.

There are a number of faculty and staff that have also helped me along. These include our Depart­ment Chair Glenn Sipes, Sandi Sledge and Anita Finnell in the graduate office and Jane Ageton and Karia Hayes in the front office. A special thanks also to our Department Business manager, Terri Vorholzer. She and her husband Steve have become close family friends. At work, Terri has always looked out for me and when­ever I needed something, she has taken care of it right away. I truly appreciate all that she has done for me.

Finally, I would like to thank Steve Stratton. When I was planning my move out here. Ken Wild located a roommate for me. I drove from beautiful Troy, New York to Tucson in three days, pulling into the stately Rio Vista apartment complex at 1 a.m. There at the door was my new roommate Steve. He not only had a can of ice-cold coca cola waiting on the table, but had also made my bed and had clean towels and a fuzzy rug set up in the bathroom. Over the next five years he cooked, cleaned and provided hours of entertainment (Nintendo and his previous girlfriends) and somehow never complained. I can only think of one time we argued in those five years-I had just gotten my ass kicked in an epic battle of Streetfighter and had thrown the Nintendo controller onto the floor (You don't mess with his Nintendo controllers). Unfortunately, we were unable to celebrate our 7 year anniversary (common law marriage) as he moved in with his girlfriend Kaihy this past summer. I had some great times with Steve while we were roommates and hope to continue our friendship wherever we end up.

5

DEDICATION

This dissertation is dedicated to my parents Erwin and Betty Bilsky and to my wife

Jill. My parents have always supported my educational and personal goals even when it

meant significant sacrifice on their part. They have provided sound guidance and counsel

even when I was too foolish or smbbom to heed it, and always managed to remain patient

and loving. As I prepare to start my own family, I will do my best to emulate them. My wife

Jill has had the dubious honor of witnessing the final two years of my graduate studies. Her

patience and support have not gone unnoticed. I thank her for everything that she has added

to my life. I love you dearly Jill.

6

TABLE OF CONTENTS

LIST OF FIGURES 11

LIST OF TABLES 14

ABSTRACT 15

L INTRODUCTION 17

A Brief History of Opiates, Opioids and Opioid Receptors 20

Agonists, partial agonists and antagonists 23

Endogenous Opioid Receptors 25

Endogenous Opioids 26

Cloning of opioid receptors 29

Cloning of the "orphan" receptor 30

Transection studies 33

Site-directed mutagenesis of the opioid receptors 37

Opioid receptor chimeras 40

Localization of opioid receptors in pain pathways 42

Gene knock-out studies of opioid receptors 47

Pharmacological Classification of Delta Opioid Receptors 47

Ligands for Delta Opioid Receptors 49

Delta Opioid Receptors Mediating Antinociception 53

Subtypes of Opioid Delta Receptors 55

Evidence in vivo for the existence of opioid delta receptor subtypes 55

Evidence in vitro for the existence of opioid delta receptor subtypes 60

Delta Opioid Agonists and Antagonists as Potential Therapeutic Agents 67

Dependence liability of delta opioid agonists 68

Opioid delta receptor mediated suppression of morphine abstinence 72

Delta opioid receptors and addictive processes 74

Summary of Research Goals 77

7

TABLE OF CONTENTS - Continued

II. IN VITRO PHARMACOLOGICAL PROFILE OF SNC 80 79

Speciflc Aims: 79

Introduction 80

Materials and Methods 82

Animals 82

GPI and MVD bioassays 82

Radioligand binding experiments 83

Chemicals 85

Radioligands 85

Statistics 85

Results 86

Bioassays 86

Radioligand binding studies 88

Discussion 89

III. IN VIVO PHARMACOLOGICAL PROFILE OF SNC 80 91

Speciflc Aims: 91

Introduction 92

Methods 92

Animals 92

Injections 92

Antinociceptive tests 93

Antagonist studies 94

Chemicals 95

Statistics 95

Results 96

Behavioral and antinociceptive effects 96

Antagonist studies 98

Discussion 102

8

TABLE OF CONTENTS - Continued

IV. IN vrvo CHARACTERIZATION OF ANTISENSE EFFECTS 105

Specific Aims: 105

Introduction 106

Materials and Methods 108

Animals 108

Injections 108

Synthesis of oligodeoxynucleotides 109

Antinociceptive testing 110

Statistical analysis 110

Chemicals HI

Results 112

In vivo assessment of DOR antisense and mismatch administration 112

Antisense ODN targeting a conserved region of all three cloned opioid receptors 118

Discussion 122

v. IN VITRO CHARACTERIZATION OF ANTISENSE EFFECTS 132

Specific Aims: 132

Introduction 133

Methods 133

Antisense ODNs 133

In vivo administration of ODNs 134

Radioligand binding studies 135

NG 108-15 cell procedures 136

Immunostaining and imaging ofNG 108-15 cells and mouse spinal cord 137

Results 139

Radioligand binding studies in DOR antisense treated mice 139

NG 108-15 cell studies 142

Spinal cord studies 143

Discussion 147

9

TABLE OF CONTENTS - Continued

VI. NMDA RECEPTOR ANTAGONISTS AND OPIOID TOLERANCE 150

Specific Aims: 150

Introduction 151

Methods 153

Animal subjects 153

Drugs and injections 153

Administration of NMDA antagonists 154

Tolerance regimen 155

Cold-water swim-stress paradigm 156

Antinociceptive testing and statistical analysis 156

Results 158

Discussion 166

VII. ADDITIONAL STUDIES ON OPIOID TOLERANCE AND PHYSICAL

DEPENDENCE 173

Speciflc Aims: 173

Introduction 174

Materials and Methods 179

Animal subjects 179

Injection techniques / 79

Tests for antinociception 179

Effects of protein kinase inhibitors on morphine antinociception 180

Acute morphine dependence 180

Acute morphine tolerance / 82

Results 183

Discussion 190

VIIL RECENT STUDIES 196

Speciflc Aims: 197 Further studies with DOR antisense 197

Studies with DARPP-32 knockout mice 203

10

TABLE OF CONTENTS - Continued

IX. SYNTHESIS AND CONCLUSIONS 212

1. Development of selective, nonpeptidic, 5 opioid agonists 212

Summary 2/2

Future studies 213

2. Development of antisense techniques for the study of 5 receptors 214

Summary 214

Future studies 214

3. Mechanisms of opioid tolerance and physical dependence 216

Summary 2/6

APPENDIX A. ABBREVLVTIONS AND DEFINITIONS 220

APPENDIX B. LIST OF PUBLICATIONS 223

Refereed Journal Articles 223

Book Chapters 225

APPENDIX C. HUMAN/ANIMAL SUBJECTS APPROVAL 226

REFERENCES 227

11

LISTOFnGURES

l-l. Chemical structures of morphine, heroin and naloxone 24

1-2. Classification of opioid receptors 59

2-1. Chemical structures ofBW 373U86 and SNC 80 80

2-2. Effects of SNC 80 in the mouse vas deferens bioassay 86

2-3. Radioligand competition curves with SNC 80 88

3-1. Dose- and time-response curves for i.c.v. and i.th. SNC 80 97

3-2. Dose- and time-response curves for i.p. SNC 80 98

3-3. Naltrindole antagonism of SNC 80 antinociception 99

3-4. Selectivity of naltrindole for 5 receptors 99

3-5. ICl 174,864 antagonism of SNC 80 antinociception 101

3-6. 8 subtype selectivity of SNC 80 antinociception / 01

4-1. Sites at which antisense ODNs may disrupt protein synthesis 107

4-2. Dose-response effects of DOR antisense/mismatch ODN 112

4-3. Time-course study of DOR antisense/mismatch effects 113

4-4. Time-course of [D-Ala^, Glu*]deltorphin antinociception 114

4-5. DOR antisense/mismatch and ji and K opioid antinociception 115

4-6. Dose-response curves with [D-Ala^, Glu*]deltorphin or DPDPE in DOR

antisense/mismatch treated mice 117

4-7. Dose-response curves with DAMGO or etorphine in COR antisense/

mismatch treated mice 119

4-8. Dose-response curves with U69,593 or bremazocine in COR antisense/

mismatch treated mice 120

4-9. Dose-response curves with [D-Ala-, GlU*]deltorphin or DPDPE in COR

antisense/mismatch treated mice 121

12

LIST OF FIGURES - Continued

5-1. pH]Naltrindole whole-brain binding in DOR antisense

or mismatch ODN treated mice 139

5-2. Time-course of DOR antisense/mismatch ODN treatment

on [^HJNaltrindole binding 141

5-3. Effects of DOR antisense ODN on DOR immunofluorescence in NG108-15 cells. 144

5-4. Effects of DOR mismatch ODN on DOR immunofluorescence in NG108-15 cells 144

5-5. Covisualization of DOR immunofluorescence and Texas-red

ODN in NG 108-15 cells treated with DOR antisense 145

5-6. DOR immunoreactivity in the dorsal horn of mice treated with

vehicle, DOR antisense or mismatch ODN 145

5-7. Control experiments for i.th. DOR antisense treatment 146

6-1. Antinociceptive effects ofMKSOl and LY235959 158

6-2. Effects ofMK801 on the development of antinociceptive

tolerance to s.c. or i.c.v. morphine 160

6-3. Effects ofLY235959 on the development of antinociceptive

tolerance to s.c. or i.c.v. morphine 161

6-4. Effects ofMK801 on the development of antinociceptive

tolerance to s.c. or i.c.v. fentanyl 162

6-5. Effects ofMK801 or LY235959 on the development of

antinociceptive tolerance to i.c.v. DAMGO 163

6-6. Effects ofMK801 or LY235959 on the development of

antinociceptive tolerance to i.c.v. [D-Ala^, GlU*Jdeltorphin 164

6-7. MK801 and antinociceptive tolerance to swim-stress 165

7-1. Time course of acute morphine abstinence 183

13

LIST OF FIGURES - Continued

7-2. Effects of kinase inhibitors on morphine antinociception 184

7-3. Effects of kinase inhibitors on acute morphine dependence 185

7-4. Precipitation of morphine abstinence by opioid antagonists 187

7-5. Attenuation of naloxone-precipitated withdrawal with CTAP 187

7-6. Time-course for the development of acute morphine tolerance 188

7-7. Effects of kinase inhibitors on acute morphine tolerance 189

8-1. DOR antisense/mismatch and i.e.v. SNC 80 antinociception 198

8-2. DOR antisense/mismatch and s.c. morphine antinociception 198

8-3. DOR antisense/mismatch and acute morphine dependence 200

8-4. DOR antisense/mismatch and i.c.v. morphine antinociception 201

8-5. Antinociceptive actions of several i.c.v. opioid agonists in mice

pretreated with DOR antisense or mismatch ODN 201

8-6. Antinociceptive responses to i.c.v. morphine in three different strains of mice 205

8-7. Baseline tail-flick latencies in three different strains of mice 205

8-8. Effects of naloxone on the baseline tail-flick and. hot-plate

latencies in the 129 SvEv strain of mice 206

8-9. Precipitation of acute morphine abstinence by naloxone in three

different strains of mice 207

8-10.Precipitation of chronic morphine abstinence by naloxone in

three different strains of mice 207

9-1. Proposed scheme for the regulation of[i receptor activity 2 / 7

14

LIST OF TABLES

1-1. [^HJNaltrindole binding in Citellus lateralis 65

2-1. Summary of 5 agonists in the MVD and GPI bioassays 87

4-1. Summary table of COR antisense/mismatch ODN treatment

on opioid mediated antinociception 118

5-1. Summary of pH]Naltrindole binding in DOR antisense

or mismatch ODN treated mice 140

5-2. Summary of [^HJDAMGO and [^H]U69,593 binding in

DOR antisense or mismatch ODN treated mice 140

6-1. Summary of the effects ofMKSOl on the development

of opioid tolerance 159

6-2. Summary of the effects ofLY235959 on the development

of opioid tolerance 159

7-1. Injection schedules for acute morphine tolerance experiments 181

8-1. Summary of morphine antinociception and acute morphine

abstinence in ICR, wild-type and DARPP-32 knockout mice 208

8-2. Summary of acute morphine abstinence in ICR, wild-type

and DARPP-32 knockout mice 209

15

ABSTRACT

The central hypothesis of this dissertation is that agonists and antagonists acting at the

5 opioid receptor will have therapeutic applications in treating acute and chronic pain states

and in the treatment of drug addiction. It is further hypothesized that 5 compounds will have

better therapeutic profiles than currently available opioids that act predominantly at the |i

receptor. In advancing the central hypothesis, selective nonpeptidic 5 compounds, that readily

cross the blood brain barrier after systemic administration, were tested. BW373U86, a non­

peptidic ligand with moderate selectivity and activity at 5 opioid receptors represented a

lead compound. A structurally related molecule, SNC80, displayed an improved selectivity

and activity profile compared to BW373U86. Importantly, SNC80 produced antinocicep-

tion following systemic administration which was blocked by 5, but not p., selective antago­

nists. The pharmacology of 6 opioid receptors was further studied using antisense oligode-

oxynucleotides that disrupted the synthesis of 6 receptors in vivo and in vitro. The experi­

ments provided further evidence for distinct 5 receptor subtypes and demonstrated the util­

ity of the antisense approache in studying neurochemical processes in vivo. Several studies

addressed the phenomenon of opioid tolerance and physical dependence, two processes

which compromise the clinical application of currently available opioid analgesics. The

observation that NMDA receptor antagonists block the development of antinociceptive tol­

16

erance to repeated administrations of morphine was confirmed. The results were extended

by demonstrating that NMDA antagonists did not block antinociceptive tolerance to more

selective 5 or agonists. These studies caution against the generalization that an effect seen

with morphine is applicable to all opioid agonists. Further hypotheses regarding the mecha­

nisms of opioid tolerance and physical dependence were tested using inhibitors of protein

kinases and putative neutral and inverse opioid antagonists. These studies advanced the

hypothesis that opioid receptor phosphorylation may play a critical role in the development

of opioid antinociceptive tolerance and physical dependence. In summary, this dissertation

has provided strong evidence that nonpeptidic 5 selective opioid agonists and antagonists

can be developed and that these compounds will have therapeutic applications in the treat­

ment of pain and addictive disorders.

17

I. INTRODUCTION

...thou hast the keys of Paradise, O just, subtle, and mighty opium! Thomas De Quincey, 1822

The history of opium and opioids is arguably one of the richest and most interest­

ing in the annals of ancient and modem medicine, sociology and the arts. References to the

medical and recreational use of opium dates back many thousands of years and to this date,

extracts of opium continue to be the basis of effective relief from pain. The use and misuse

of opium has also influenced many poets and writers, has had enormous socioeconomic

impact on cultures throughout the world and even brought nations to war. The scientific

advances associated with the chemistry, pharmacology and physiology of opioids has been

equally as impressive.

From a medical and social perspective, there is a need for medications that produce

safe, effective and nonaddicting analgesia, and for medications that aid in recovery from

drug addiction. Despite significant efforts since the 1940's to synthesize and develop novel

nonaddictive drugs to treat pain and manage drug addiction, and in spite of the many poten­

tial types of opioid receptors as targets, progress has been disappointing. Clinical applica­

tion of narcotics continues to be limited to substances that have significant abuse potential

and that share the same basic mechanism of action (Jaffe and Martin, 1990; Jaffe, 1990). In

18

addition to standard morphine-like drugs (that is, opioid p. receptor agonists), the most

important development has been the identification of mixed partial agonists such as penta­

zocine, and compounds of lower efficacy at opioid |i, receptors such as buprenorphine (Jaffe

and Martin, 1990). To date, no opioid compounds of clinical significance are available that

have a mechanism of action other than through the n receptor. The K opioid receptor ago­

nist enadoline (CI-977)(Leighton et al., 1987; Hunter et al., 1990), is currently in clinical

trials. Enadoline may provide effective analgesia without some of the negative side-effects

of ^ receptor agonists such as physical dependence, respiratory depression, nausea and

vomiting, and constipation. However, it is not yet clear whether the well-established prob­

lems of dysphoria and diuresis (Pfeiffer et al., 1986; Peters et al., 1987; Peters and Gay lor,

1989) associated with compounds acting at K receptors can be surmounted. Preliminary

clinical trials with enadoline have also been disappointing (Pande et al., 1996a,b).

The only other notable recent development in the opioid analgesic field is the cur­

rent effort to introduce tramadol to the U.S. market (Raffa et al., 1992, 1993). Tramadol,

which also acts in part through |i receptors, was synthesized more than 20 years ago; the

results of preclinical and clinical findings were published as early as 1978 (see Raffa et al.,

1992). Initial reports of the use of tramadol in the United States have been positive. Trama­

dol provides analgesia comparable to codeine with fewer reported side-effects.

19

Even less successful than the development of novel opioid analgesics has been the

effort to introduce new medications for the treatment of drug addiction. Pharmacological

treatment for dmg addictions still relies heavily on methadone therapy (Dole and Nyswander,

1965; 1966). The "next generation" of compounds (after methadone and naltrexone) has all

but failed to materialize; LAAM (1-a-acetylmethadol) is only now being introduced to clinical

practice (Best et al., 1996; Jaffe, 1990; Prendergast et al., 1995), and although recent data

suggest some applicability for buprenorphine in opioid (Best et al., 1996; Mello and

Mendelson, 1980) and cocaine (Mello et al., 1989) abuse, these pharmacotherapies have

yet to be proven successful in humans.

The development of new medications has thus been disappointing. A brighter note,

however, stems from the substantial progress which has been made in understanding mecha­

nisms by which opioids produce their effects. The recent elucidation of the molecular

structure of |i, 5 and k opioid receptors extends investigations of opioid mechanisms to the

most fundamental level. As the exploration of the pharmacology of opioid receptors has

proceeded with the development of appropriate tools, it is now clear that the results of such

studies suggest that opioid 5 and K receptors, and possibly subtypes of these receptors, may

be appropriate targets for the development of clinically useful compounds. These potential

therapeutic applications include partial 5 and K agonists and antagonists as well.

20

This dissertation focuses on the 5 opioid receptor. The primary goal of the research

is to help develop 6 opioid agonists and antagonists that have therapeutic efficacy in treat­

ing acute and chronic pain and addiction to various classes of drugs. As mentioned, there is

reasonable expectations that 5 agonists and antagonist will have advantages over currently

available |i. opioids. To help accomplish the research goals, novel compounds and tech­

niques were developed, and then applied, to studying aspects of 5 opioid pharmacology.

The research focused on the mechanisms of 6-mediated spinal and supraspinal antinocice-

ption, and the more general area of opioid tolerance and physical dependence.

A Brief History of Opiates, Opioids and Opioid Receptors

The opium poppy (Papaver somniferum /.), is an annual herb that is widely culti­

vated in several temperate and subtropical regions of the world (Merlin, 1984). It has been

used for its nutritive, medicinal and mood-enhancing properties for thousands of years.

Early use of the plant probably included boiling, steeping or soaking the capsules to obtain

a dilute mixture of its psychoactive ingredients (Merlin, 1984). At some point, it was found

that cutting the unripe capsule of the plant produced a thickened juice or sap that could be

easily collected. This sap, crude opium, contains over twenty alkaloids termed opiates. Two

of these opiates, morphine and codeine, produce the prominent pharmacological actions of

21

opium (e.g., analgesia, constipation and euphoria).

Scientific advances in the 1800's revolutionized the use and misuse of many sub­

stances including morphine. At the start of the 19th century, it was an almost universal

belief that plants could synthesize compounds of only an acid or neutral nature and that

alkalies were substances of a very different character, related more to metals (Macht, 1915).

In 1806, William Sertiimer published experiments detailing the isolation of the first alka­

loid morphine from opium. Not only did this discovery open a new field of chemistry, it

also resulted in the preparation of a pure compound which was 10-times as potent as raw

opium. The therapeutic effects of morphine were now more predictable than with the crude

extract of opium.

The isolation of morphine was actually preceded by the isolation of another opium

alkaloid, narcotine, in 1803 by the French pharmacist Derosne (Macht, 1915). It was not

until 1817, however, that Robiquet established the alkaloid character of the substance.

Robiquet followed up his studies on opium extracts by isolating codeine in 1832. Another

French chemist, Joseph Pelletier, was involved in the isolation of a number of other opium

alkaloids including thebaine and narceine in the early 1830s.

The development of the hypodermic syringe in 1853 by Pravaz and Wood made the

delivery of drugs into the body even more accurate. The hypodermic syringe also allowed

22

morphine to be directly injected into a vein, resulting in an almost instantaneous delivery of

a large dose of the drug to the brain. The widespread use of i.v. morphine as an analgesic in

the Civil War highlighted the physical dependence and abuse liability of opiates. In fact,

there were so many soldiers physically dependent on morphine, the illness became known

as Soldier's disease.

Further developments in the late 1800s included the synthesis of the opioid heroin

(diacetylmorphine) in 1874. Heroin has greater lipid solubility than morphine. This increased

lipid solubility allows heroin to enter the brain more rapidly than morphine. The quick

"rush" associated with an i.v. injection of heroin has been described by many heroin users

as a whole body orgasm, and further increases the abuse liability of the compound. Incred­

ibly, heroin was marketed by Bayer laboratories in 1898 as a nonaddicting substitute for

morphine and codeine.

The 20th century has seen further rapid advances in our understanding of opioid

pharmacology. The actual structure of morphine was worked out by Gulland and Robinson

in 1925. The isolation and synthesis of novel opioid compounds including opioid antago­

nists also continued. The discoveries of receptors in the CNS that bind morphine, the isola­

tion of endogenous morphine-like compounds and the cloning of at least three opioid recep­

tors have all significantly advanced our understanding of opioids and opioid receptors. These

23

advances, detailed below, have also led to additional pharmacological treatments for pain,

narcotic overdose and substance abuse.

Agonists, partial agonists and antagonists

World War n served as a impetus for the development of novel opioid analgesics.

Germany and its allies were isolated from opium producing parts of the world. Purely syn­

thetic opioids such as meperidine and methadone were developed in response to the short­

age of morphine (Terrenius, 1993). By altering the structure of available opioids such as

morphine and thebaine, or by synthesizing other novel structures, many new compounds

were further tested for morphine-like activity. The isolated guinea-pig ileum preparation

was one of several in vitro bioassays that proved useful in characterizing the actions of

opioids (Paton, 1957; Gyang and Kosterlitz, 1966). Electrical stimulation of the muscle in a

tissue bath produces contractions which are inhibited by morphine and other opioid ago­

nists. Some of the novel compounds were extremely potent, being 10,000 times more po­

tent than morphine. Other compounds did not produce a full morphine like effect and were

termed partial agonists. Many modifications produced compounds that lacked any biologi­

cal activity. Interestingly, some of these compounds could block or reverse the actions of

morphine. These were termed antagonists. Naloxone is one example of how a small modi­

fication of the morphine molecule can turn the molecule into an antagonist (naloxone) or

increase its potency (heroin) (see Figure I-l). Structure-activity requirements for agonist,

partial agonist and antagonist activity were soon being mapped out. From these and other

observations it was suggested that these compounds were acting at some type of receptor or

receptors in the CNS to produce their effects.

H,COCO OCOCH

Morphine Heroin

N CH2-CH=CH2

HO o o

Naloxone

Figure I-L Chemical structures of morphine, heroin and naloxone. Small changes in the structure of morphine can produce dramatic changes in the potency and efficacy of the compound.

25

Endogenous Opioid Receptors

In 1972, the pharmacodynamics of morphine and other opioid agonists was not well

understood. The following is taken from the first edition of Drugs, Society and Human

Behavior by Oakley Ray (Ray, 1972).

Most researchers, though, have accepted the idea that there are specific receptors on neurons that are sensi­

tive to narcotic agents. These agents have their ejfects as a result of combination with these receptors. These

receptors are not those affected by the neurotransmitters and, in the absence of narcotics, presumably are

unused throughout life.

The rest of the decade saw monumental discoveries in the field of opioid research

that elucidated the biochemical actions of morphine and the physiological role of endog­

enous opioids. Definitive evidence for opioid receptors was obtained in the early 1970s

using a novel technique termed radioligand binding. Initial attempts to detect opioid recep­

tors were worked out by Goldstein and colleagues (Goldstein et al., 1971) but suffered from

poor signal to noise ratios. Opioids could be tagged with a radioactive marker and their

specific binding to a receptor quantitated. Subsequent smdies with radioactive opioids hav­

ing higher specific activity, and with optimal conditions which reduced nonspecific bind­

26

ing, demonstrated conclusively that these compounds were, in fact, binding at specific re­

ceptors (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). This was based on

several criteria, including (a) the binding showed high stereospecificity similar to what was

seen pharmacologically; (b) the binding was saturable suggesting a finite number of recep­

tors and (c) the binding affinity of a number of opioids correlated well with their potency in

pharmacological assays. In addition, the distribution of opioid receptors was higher in re­

gions thought to regulate the affective component of the pain response including the amygdala

and hypothalamus (Kuhar et al., 1973).

Endogenous Opioids

With the demonstration of opioid receptors in the mammalian CNS came the ques­

tion of why they were there. It seemed unlikely that animals would develop receptors to

bind the extract of a geographically isolated plant. Furthermore, none of the known neu­

rotransmitters at that time had an anatomical distribution similar to the opioid receptors or

affected opioid binding. The search began for endogenous compounds which could activate

opioid receptors. In 1974, several laboratories isolated brain extracts with suspected opioid

activity. These extracts could inhibit opioid binding (Pasternak et al., 1975; Terenius and

Wahlstrom, 1975) or electrically induced contractions of the GPI (Hughes, 1975; Kraulis et

27

al., 1975). The pentapeptides Leu-enkephalin and Met-enkephalin were subsequently iso­

lated and sequenced from pig brains in 1975 (Hughes et al., 1975b).

The actions of these peptides, and their analogs, were subsequently studied in great

detail. It was demonstrated that they could produce many of the same effects as morphine

including analgesia (Belluzzi et al., 1976; Buscher et al., 1976; Loh et al., 1976; Pert et al.,

1977), physical dependence (Wei and Loh, 1976) and pleasure as indexed by conditioned

place preference testing (Stapleton et al., 1979). Importantly, the effects of the enkephalins

were naloxone reversible. An additional study further supported the role of endogenous

opioids in the physiological modulation of pain. Electrical stimulation of the periaqueduc­

tal grey (PAG) could produce strong analgesia similar to morphine (Mayer and Liebeskind,

1974). This analgesic effect showed tolerance to repeated stimulation and cross-tolerance

to morphine (Mayer and Hayes, 1975). Furthermore, the effect was also blocked by nalox­

one suggesting that endogenous opioids were released following nerve excitation in the

PAG (Akil et al., 1976).

The discovery and characterization of the enkephalins was soon followed by the

isolation of two additional classes of endogenous opioid peptides. The proopiomelanocortin

(POMC) family includes the large precursor peptide POMC which is expressed in the pitu­

itary. A number of peptide products of POMC, including P-endorphin, are released into the

28

blood stream in response to stress (Li and Chung, 1976). The third class of endogenous

opioids discovered were the dynorphins (Goldstein et al., 1979,1981). Along with the three

classes of opioid peptides there were now three opioid genes (POMC, proenkephalin and

prodynorphin), each giving rise to their respective precursors.

At approximately the same time as the endogenous opioid peptide families were

being characterized, it also became apparent that there were multiple opioid receptors. The

opioid antagonist naloxone proved to be a valuable pharmacological tool for classifying

opioid receptors. Any compound which produced an effect that could be blocked or re­

versed by naloxone was termed an opioid. Based on extensive pharmacological testing, it

was shown that some of the tested opioids (a) produced effects that were slightly different

from morphine and (b) differed in their sensitivity to naloxone antagonism. The putative

receptor for morphine was termed the |i receptor. In addition, opioid 6 and k receptors were

postulated based on enkephalin activity in another tissue bioassay (the mouse vas deferens)

and the actions of ketocyclazocine in the spinally transected dog, respectively (Lord et al.,

1977; Martin et al., 1976). The synthesis of selective antagonists for N, 6 and K receptors,

further supported the existence of multiple opioid receptors while the recent cloning of

three opioid receptors has confirmed their existence and elucidated their structure and func­

tion.

29

Cloning of opioid receptors

Using very similar expression cloning strategies, Evans at al. (1992) and Kieffer et

al. (1992) independently cloned the cDNA encoding the mouse 6 opioid receptor (DOR)

from the hybrid NG 108-15 cell line. Using this sequence and the presumed homology

between opioid receptors, other groups identified cDNA clones encoding the (MOR) and

k (KOR) opioid receptors,, as well as the DOR receptor, in mouse, rat and human (Chen et

al., 1993; Fukuda et al., 1993; Knapp et al., 1994; Li et al., 1993; Mansson et al., 1994;

Meng et al„ 1993; Minami et al., 1993; Nishi et al., 1993;1994; Thompson et al., 1993;

Wang et al., 1993; Yasuda et al., 1993). The cloned opioid receptors were between 372 and

400 amino acids in length. Using the deduced amino acid sequences, hydrophobicity analy­

sis suggested that these receptors contained seven putative transmembrane domains, a fea­

ture common to the G-protein coupled receptor (GPR) family. The overall sequence homol­

ogy between the MOR, DOR and KOR was high (approximately 70%) and there was a high

degree of conservation between species. The transmembrane and intracellular regions of

the receptors shared the highest degree of homology (-75% and -65%, respectively) while

the extracellular regions were more divergent (-35-40%).

In addition to verifying the primary amino acid structures of the receptors, the clon­

ing of the MOR, DOR and KOR allowed the transfection and functional expression studies,

30

site-directed mutagenesis, deletions and the construction of receptor chimeras to investi­

gate the mechanisms of ligand affinity, selectivity and signal transduction. The anatomical

distribution of the opioid receptor mRNAs or the receptor protein were also conducted

using in situ hybridization or antibody techniques, respectively. Studies using gene "knock­

out" mice that lack the mu (p.) receptor have been used to study the actions of this opioid

receptor on whole-animal health and general behavior as well as on specific systems of

interest (i.e., pain and analgesia). It is anticipated the development of delta (5) and kappa

(K) opioid receptor "knock-out" mice will soon be reported. Related experiments using

antisense oligodeoxynucleotides (ODNs) designed to selectively "knock-down" opioid re­

ceptors, presumably by interfering with protein synthesis, have also been applied in the

study of 5 and K opioid receptor pharmacology. Some of the progress using each of these

approaches is detailed below.

Cloning of the "orphan " receptor

In addition to the three classical opioid receptors, a novel seven transmembrane

protein with substantial sequence similarity to the opioid receptors has been identified

(Bunzow et a!., 1994; Fukuda et al., 1994; Mollereau et al., 1994; Vanderah, 1995; Wick et

al.. 1994) and termed the "orphan" receptor due to a lack of high affinity binding to known

31

opioid and non-opioid ligands (Bunzow et al., 1994; Fukuda et al., 1994; Mollereau et al.,

1994). The orphan receptor is widely and unevenly distributed in the adult rat with hybrid­

ization most prominent in extracts of lung, liver and adrenal. Detection of orphan receptor

mRNAhas also been reported in extracts of kidney, aorta, heart, spinal cord, gut and testes

(Harrison, et al., 1993) and low levels were detected in brain tissue including the midbrain

and the pons (Mansour et al., 1996). Etorphine has been reported to inhibit forskolin-

induced accumulation of cAMP in Chinese hamster ovary (CHO) cells stably transfected

with the orphan receptor (Mollereau et al., 1994). However, the etorphine concentrations

required for this effect were approximately three times higher than necessary to produce

similar effects with the cloned classical opioid receptors.

A heptadecapeptide has been independently identified by two laboratories (Meunier

et al., 1995; Reinscheid, et al., 1995) and proposed to be the endogenous ligand for the

orphan receptor. The peptide has been termed orphanin FQ or nociceptin (Meunier et al.,

1995; Reinscheid, et al., 1995) and has been shown to interact with G-proteins. The orphanin

FQ/nociceptin (OFQ/N) peptide stimulated [•^•''SIGTPTS binding in various brain regions

including cortex, amygdala, hypothalamus, thalamus and brain stem (Sim et al., 1996). In

contrast, there was no significant OFQ/N peptide-stimulated P^SJOTPyS binding in the

caudate-putamen and cerebellum, areas that normally contain relatively high levels of opioid

32

receptors in the rat (Sim et al., 1996). Further, in situ hybridization and immunohistochemical

studies for the OFQ/N peptide by Mansour et al. (1996) have identified dense labelling in

the medial and central nuclei of the amygdala, bed nucleus of the stria terminalis, lateral

and laterodorsal septum, medial and lateral preoptic areas, arcuate nucleus and median

eminence. These high levels in the limbic, hypothalamic nuclei and median eminence sug­

gest that the peptide may play a role in emotional responsiveness and hormonal release

(Mansour et al., 1996). Immunohistochemistry and in situ hybridization have identified the

OFQ/N protein and mRNA in the superficial layers of the dorsal horn in the L4-L5 region of

rats, however dorsal root ganglion (DRG) neurons were not labelled by OFQ/N antibodies

nor by its mRNA probes (Zhang et al., 1996).

Direct central administration of OFQ/N to mice was reported as having a hyperalge-

sic action in the mouse tail-flick assay (using radiant heat as the noxious stimulus) but not in

the 58°C hot-plate test (Reinscheid, et al., 1995). On the other hand, Meunier et al., 1995,

reported that intracerebroventricular (i.c.v.) administration of OFQ/N to mice produced a

hyperalgesic action in the hot-plate test (unspecified temperature). The doses used by these

two groups differed by 180-fold, with the latter group using much lower doses (i.e., 5 to 55

pmol) while the former group used doses ranging between I nmol and 10 nmol. Meunier

and colleagues also reported that the treatment of mice with an antisense ODN to the or­

33

phan receptor once daily for 4 days resulted in a significant increase in hot-plate latency

(i.e., antinociception). These studies have led to the suggestion that orphanin FQ is an

endogenous pronociceptive or hyperalgesic agent and that the peptide exerts tonic activity

at the orphan receptor to lower the threshold to noxious stimuli (i.e., to enhance the percep­

tion of pain). The hypothesis that OFQ/N is pronociceptive or hyperalgesic iz controversial

at present, and remains to be established.

Transection studies

The cloned opioid receptors can be expressed in cultured mammalian cells by trans-

fection of the cDNA clone. Importantly, the cDNA clones can be transfected into cell lines

that do not normally express opioid receptors and thus represent a simplified functional

system in which to smdy opioid pharmacology. Opioid |i agonists can inhibit the formation

of cAMP via pertussis toxin-sensitive G-proteins in cells transiently or stably transfected

with the MOR (Arden et al., 1995; Chen et al., 1993, 1996; Wang et al., 1996). Similar

transduction mechanisms are seen in DOR and KOR transfected cells (Kong et al., 1993.

1994; Yasuda et al., 1993). More detailed analysis of the coupling of the 5 receptor with G-

proteins has been performed by Law and Reisine (1994). Activation of the 5 receptor alters

the association of the receptor with G^, and G.^ and may represent one of the first steps in

34

its signal transduction pathway.

The development of tolerance and physical dependence to repeated administration

of opioids is poorly understood. Cells transfected with a particular opioid receptor may

offer a near ideal system in which to study the mechanisms of opioid tolerance and physical

dependence. It has been suggested that opioid tolerance involves both desensitization and

down regulation of the receptor. The rapid desensitization of the receptor may further in­

volve the uncoupling of the receptor with the G-protein or the internalization of the receptor

into the intracellular compartment (Trapaidze et al., 1996) while down-regulation of the

receptor may be one of the changes responsible for longer-term changes associated with

chronic tolerance.

Compensatory changes in the adenylyl cyclase system have been measured in CHO

cells stably transfected with the MOR (Yu, 1996). The selective agonist DAMGO re­

duced forskolin-stimulated cAMP levels by approximately 30%. Following chronic mor­

phine exposure, the inhibitory effects of DAMGO in these cells was potentiated without

affecting the ECj^ value of the agonist (Chen et al., 1996). There was also no change in the

affinity of DAMGO for opioid receptors or in the number of fi receptors as measured by

radioligand binding. Chronic morphine exposure in these same cells, followed by a period

with no morphine (withdrawal) produced an increase in basal and forskolin-stimulated cAMP

35

levels (Chen et al., 1996). Compensatory increases in adenylyl cyclase, but not in the num­

ber of cell surface opioid receptors, may therefore, be critical to the development of |i

opioid tolerance and physical dependence.

An intriguing difference between morphine and other p, agonists has recently been

discovered using MOR transfected cells. Assuming that agonists with different chemical

structures can induce different active receptor conformations, one might expect different

downstream events to be associated with each agonist class. Indeed, while both morphine

and DAMGO lowered cAMP levels in HEK 293 cells transfected with the p. receptor, only

DAMGO was capable of internalizing the receptor (Arden et al., 1995; Keith et al., 1996).

Similar effects were seen with jj. receptors in the guinea pig ileum (Stemini et al., 1996).

Treatment in vivo with etorphine produced a rapid internalization of |i. receptors. In con­

trast, treatment in vivo with morphine did not trigger p. receptor endocytosis. These results

suggest that the mechanisms of tolerance differ for etorphine and morphine (Stemini et al..

1996). Interestingly, whereas morphine enhanced the rate of p. receptor phosphorylation

(Arden et al., 1995), DAMGO decreased |i receptor phosphorylation (Wang and Sadee.

unpublished observations). These results may help explain the differential action of NMDA

antagonists on the development of antinociceptive tolerance to morphine and DAMGO

(Bilsky et al., 1997a).

36

Similar findings were seen in HEK 293 cells transfected with the DOR. Cells pre-

treated with the 5 agonist DPDPE for 10 min showed rapid densensitization and phosphory­

lation (Pei et al., 1995). Protein kinase A (PKA) and protein kinase C (PKC) appear to be

unimportant for the agonist-promoted receptor phosphorylation. In contrast, the P-adrener-

gic receptor kinase-1 (PARKl), and possibly other G-protein coupled receptor kinases

(GRKs) may play a critical role in regulating the phosphorylation and desensitization of 6

receptors expressed in HEK 293 cells. (Pei et al., 1995). A second 5 agonist, DADLE, also

produced a rapid internalization of the DOR in HEK 293 or CHO cells (Keith et al., 1996;

Trapaidze et al., 1996). Morphine, however, failed to induce a detectable redistribution of 6

receptors from the plasma membrane (Keith et al., 1996). Using deletions and point muta­

tions, the authors demonstrated the importance of the middle region of the C-terminal tail in

regulating 6 receptor internalization in CHO cells.

Investigation of the mechanisms of K opioid tolerance has lagged behind studies

with p. or 5 opioid tolerance. Historically, there has been an absence of cell lines containing

high levels of K receptors. In addition, morphine has been used as a prototypical opioid

agonist. The affinity of morphine for K receptors is significantly lower than for ^ or 5

receptors. Development of selective K opioid agonists and the cloning and transfection of

the KOR into cell lines that do not normally express opioid receptors should be useful in

37

studying the mechanisms of K tolerance. CHO cells transfected with KOR, for example,

have demonstrated that the K agonists U50,488H and U69,593 inhibit forskolin-stimulated

cAMP accumulation (Avidor-Reiss et al., 1996; Raynor et al., 1994). There are, however,

conflicting reports of whether the K receptor desensitizes. Raynor et al. (1994) detected K-

agonist induced desensitization of mouse KORs transiently transfected in COS-7 cells which

was sensitive to manipulations of PARK. Avidor-Reiss et al. (1996), however, detected no

significant desensitization in CHO cells. Future studies should resolve these findings and

address whether internalization of the K receptor takes place.

Site-directed mutagenesis of the opioid receptors

Structure/activity studies using opioid ligands have indicated that a basic amine in

opioid alkaloids or the protonated amino terminus in opioid peptides is critical for ligand

recognition. A positively charged nitrogen appears to be necessary for ligand binding to p.,

5 or K opioid receptors (Barnard, 1993: Rees and Hunter, 1990). It was postulated that a

negatively charged amino acid side chain of the cloned opioid receptors may form an elec­

trostatic bond with the positively charged nitrogen (Befortet al., 1996a). One potential site

of interaction was the Asp'^® residue found in the 3rd transmembrane domain (TMIII) of the

cloned mouse DOR as this site is conserved in GPRs activated by cationic neurotransmit-

38

ters. This hypothesis was tested by using point mutations of the mouse DOR (Befort et al.,

1996a). Surprisingly, substitution of Asp with Ala (carboxylate elimination) did not signifi­

cantly affect opioid ligand binding to the 6 receptor, suggesting that the negative charge of

Asp'^ was not critical for high affinity binding. In contrast, replacing the carboxylate group

with an amide group (Asp'^ to Asn'^) resulted in a strong decrease in ligand affinity. The

binding of peptidic ligands was affected to a greater extent than the binding of alkaloid

ligands, further suggesting the existence of multiple binding sites on the 5 receptor. Inter­

estingly, similar mutations on the corresponding Asp residue of the cloned mouse MOR

(Asp'"*' to Ala'"*') strongly modified the receptor pharmacology in a manner that was distinct

from the above mentioned mutations of the mouse DOR (Befort et al., 1996a).

Kong et al. (1993) targeted the only other negatively charged residue located in the

putative a helices of the DOR (transmembrane region II Asp'^). They found that a Asp'^ to

Asn'' substitution decreased the binding of 6 selective opioid agonists without affecting

alkaloid ligand binding. The results of Kong et al. (1993) and Befort et al. (1996a) suggest

that there is no universal negatively charged attachment point for opioid peptides in the 6

receptor binding pocket (Befort et al., 1996a). Other anionic sites on the 5 receptor may,

however, be critical to the formation of a stable ligand/receptor complex or, conversely,

nonionic interactions may predominate.

39

More recent studies from the laboratory of Brigitte Kieffer (Befort et al., 1996b)

have looked at the importance of aromatic residues in the transmembrane domains of opioid

receptors. These aromatic residues have been suggested to be important in maintaining

receptor structure and may provide ligand binding sites for opioid ligands. Three-dimen­

sional computer modeling of the mouse DOR, for example, suggest that these aromatic

residues may face the inner side of the helices bundle to form an aromatic pocket. The

hydrophobic portion common to opioid ligands may fit into this pocket and result in a stable

configuration. This hypothesis was investigated by substituting the transmembrane amino

acids that have aromatic side chains (Trp and Phe) with Ala. Inhibition studies using a

variety of opioid ligands were then conducted against pH]naltrindole, a selective 5 receptor

antagonist, in wild-type and mutant receptors. The role of hydroxyl groups on transmem­

brane Tyr residues was also investigated by substituting Tyr with Phe or Ala.

The general conclusion of the study was that the aromatic residues are part of a

general opioid binding domain and are not involved in agonist/antagonist differentiation or

6 selectivity (Befort et al., 1996b). Specifically, the mutations tested in the study affected

the binding of nonselective opioids (e.g., naloxone) to a greater extent than the binding of

selective 5 ligands (e.g., naltrindole). This suggests that there are additional specific inter­

action sites on the 5 receptor that contribute to 6 selective binding and confer 5 selectivity.

40

This view is consistent with studies from Yamamura and colleagues which suggest that the

third extracellular loop is important for determining the selectivity of antagonists at the

human DOR (Li et al., 1996) as well as for agonists at this receptor (Varga et al., 1996).

Further, peptidic and nonpeptidic ligands may bind differentially on the DOR as shown by

point mutations aimed at Trp^ of the human DOR (Li et al., 1995). These mutants showed

that this residue was crucial for the binding of SNC 121, a selective and nonpeptidic 6

agonist, but did not affect the binding of either [4'-ClPhe'']DPDPE or [D-Ala^, Glu"*]deltorphin,

peptidic 8 agonists, or of naltrindole, a nonpeptidic 5 antagonist (Li et al., 1995).

By deleting segments of the cloned opioid receptors it is possible to ask whether a

particular segment is critical to the binding affinity, selectivity or actions of various ligands.

A deletion of the first 64 N-terminal amino acids of the cloned MOR, for example, had no

effect on the binding of the |i selective opioid agonist DAMGO (Surratt et al., 1994). The

affinity of naloxone was also not affected by this deletion. This would indicate that the N-

terminal of the |i receptor is not involved in the binding of these ligands.

Opioid receptor chimeras

The construction of opioid receptor chimeras has been useful in determining the

role of receptor domains in ligand binding, selectivity and agonist activity. Meng et al.

41

(1996) constructed chimeras of the cloned DOR and either the cloned KOR or MOR. Their

experiments demonstrate that TM6 and the third extracellular loop (EL3) of the DOR is

critical to 5 opioid receptor selectivity. The high affinity binding of 5 selective opioid pep­

tides may also require the EL2 region of the DOR. The EL2 displays a great deal of hetero­

geneity across the three cloned opioid receptors and may play an important role in discrimi­

nating between the endogenous enkephalins and dynorphins (Meng et al., 1996).

Receptor chimeras have also been used to study receptor coupling and opioid toler­

ance. The third intracellular loop of many G-protein coupled receptors is critical for the

coupling of the receptor to the G-proteins. By constructing chimeric DOR and somatostatin

receptors, Reisine and colleagues demonstrated that the third intracellular loop of the DOR

is needed for agonist-induced inhibition of adenylyl cyclase (Reisine et al., 1994). This

intracellular loop, and its corresponding Ser^"*^, Ser^'*' and Ser^"*' residues, also appears to be

critical to desensitization of the receptor (Reisine et al., 1994). Specifically, the Ser^"*' resi­

due may serve as an acceptor of pARK. It has been proposed that PARK phosphorylates the

Ser residue on the DOR and KOR, leading to a desensitization of the receptors (Raynor et

al., 1994; Reisine, 1995).

42

Localization of opioid receptors in pain pathways

The expression of opioid receptor mRNA can be measured using in situ hybridiza­

tion histochemistry. The anatomical distribution of each receptor type might be inferred

from these data. As mentioned, this technique may offer greater selectivity than techniques

using conventional radioligand binding and autoradiography. The distribution of (Delfs et

al., 1994; Mansouretal., 1994b; Minami et al., 1994), 6 (Bzedega et al., 1993; Mansour et

al., 1993) and K (DePaoli et al., 1994; Mansour et al., 1994a; Minami et al., 1993) opioid

receptor mRNA has been reviewed (Satoh and Minami, 1995).

As predicted from the autoradiography studies, there are significant levels of opioid

receptor mRNA in many cortical, diencephalic, brainstem and spinal cord regions. There

are some specific regions of interest that will be mentioned here. The locus coeruleus, which

contains the majority of the brain's noradrenergic neurons, expresses high levels of MOR

mRNA. The noradrenergic neurons have terminals that innervate many other brain regions.

These terminals may, in tum, have n opioid receptors on their surface, a fact which may

relate to the lack of correlation between the distribution of receptors and their mRNAs.

The immunolabelling of opioid receptors has been used to further investigate recep­

tor distribution in the DRG, spinal cord, brainstem and cerebral cortex. Advantages of

using the specific receptor antibodies include ultrastructural localization of the receptors

and the covisualization of two or more receptor populations on the same neurons. All three

opioid receptors are expressed primarily in nociceptive C- and A5 fibers of the DRG cells

as measured by immunohistochemistry (Arvidsson et al., 1995a,b; Dado et al., 1993; Ji et

al., 1996). The distribution of DOR mRNA in the rodent spinal cord is of interest. Autora­

diography and immunohistochemical localization indicates highest levels of 5 opioid re­

ceptors in the outer laminae (laminae I and II) of the dorsal horn (Arvidsson et al., 1995a;

Dado et al., 1993; Gouarderes et al., 1993; Lai et al., 1996). This distribution fits with the

hypothesis that 5 opioid agonists suppress the transmission of pain signals from the primary

sensory afferents that terminate in laminae I and II onto projections neurons that form the

spinothalamic tract. In contrast, levels of DOR mRNA are low to moderate throughout

laminae I-VI. Moderate levels of DOR mRNAare, however, seen in the ventral horn (lami­

nae Vn and VO) with highest levels expressed in the motor neurons of laminae IX (Satoh

and Minami, 1995). One possible explanation for the discrepancy between the high levels

of 5 opioid receptors in laminae I and II and the relative low levels of DOR mRNA may be

the presence of DOR mRNA in the DRG. Presumably, 5 receptors synthesized in the DRG

are transported to the central terminal of the primary afferent. The presynaptic 5 receptors

are contained on the nociceptive A8 and C-fibers which terminate in laminae I and II.

Intraplantar injection of carrageenan, a model of peripheral pain and inflammation.

44

differentially regulates the expression of fi, 5 and K opioid receptors (Ji et al., 1996). That is,

carrageenan injection significantly increased MOR-immunoreactivity while decreasing DOR-

and KOR-immunoreactivity 1 and 3 days after inflammation. In contrast to the carrag-

eenan-induced changes in the DRG, spinal cord MOR-, DOR and KOR immunoreactivity

was affected to a much lesser extent, albeit the pattern was the same as seen in the DRG (Ji

et al., 1996). The slight increase in spinal MOR-immunoreactivity following inflammation

is in agreement with behavioral, binding and electrophysiological studies (Besse et al., 1992;

Hyldenetal., 1991; Stanfaet al., 1992). Satoh and colleagues (Maekawaetal., 1995) have

examine the expression of mRNAs for opioid receptors in lumbar spinal cord of rats with

unilateral hindpaw inflammation produced by Mycobacterium butyricum. Their studies

showed that 11 days after the adjuvant, |I and K, but not 6, receptor mRNA was increased in

lamina I and H. These studies suggest a prominent role for lumbar opioid |i receptors in

inflammatory pain states.

Nociceptive signals entering at the level of the spinal cord are regulated not only by

intrinsic intemeurons but by descending pathways that originate in the brainstem. Activa­

tion of a population of cells in the PAG produces excitation of neurons in the rostral ventral

medulla (RVM)(Basbaum and Fields, 1984). The RVM cells project to, and inhibit nocice­

ptive cells in the spinal cord. Kalyuzhny et al. (1996) have recently used antibodies for the

45

MOR and DOR, along with retrograde tract-tracing from the RVM and spinal cord, to fur­

ther investigate the role of opioids in this descending antinociceptive system. They provide

evidence that both p.- and 6-receptors are involved in the brainstem antinociceptive circuits.

Furthermore, they propose that opioids exert mainly indirect effects on PAG-RVM projec­

tion neurons but have both direct and indirect effects on bulbospinal neurons. Finally, they

demonstrate that some of the serotonergic neurons that project to the dorsal horn of the

spinal cord also express p. opioid receptors.

The ultrastructural localization of opioid receptors has also been reported in the

locus coeruleus (LC) (Van Bockstaele et al., 1996), nucleus accumbens (Svingos et al.,

1996) and human frontal cortex (Schmidt et al., 1994). In the LC, MOR immunolabelling

was localized to parasynaptic and extrasynaptic portions of the plasma membranes of

perikarya and dendrites (Van Bockstaele et al., 1996). Many of the cells that were positive

for MOR-immunolabelling were also positive for tyrosine hydroxylase. Furthermore, the

MOR positive cells were usually postsynaptic to unlabeled axon terminals that had charac­

teristics of excitatory-type synapses. These results would suggest an important postsynaptic

role for |i opioid receptor regulation of excitatory responses to catecholamine-containing

neurons in the LC.

46

The nucleus accumbens is another CNS site that has a large population of opioid

receptors. Ultrastnictural studies in the rat nucleus accumbens indicated MOR-immunore-

activity was localized predominantly to extrasynaptic sites along neuronal plasma mem­

branes (Svingos et al., 1996). The majority of these neuronal profiles were dendrites and

dendritic spines. Interestingly, these MOR-labeled sites were closely associated with

[Leu^]enkephalin-labeled terminals and other unlabeled terminals forming either inhibitory

or excitatory type synapses (Svingos et al., 1996). Similar types of studies have shown an

ultrastructural extrasynaptic localization of MOR-immunolabeling in the superficial layers

of the rat cervical spinal cord that is in close proximity with [Leu^]enkephalin-labeled

terminals (Cheng et al., 1996).

The K opioid receptor has been localized to specific layers of the rat cortex using

autoradiography. Schmidt et al. (1994) used a monoclonal antibody against the KOR to

study the cellular and subcellular distribution of the K opioid receptor in human frontal

cortex. Immunoprecipitate was seen on free ribosomes and cell membranes of neuronal

perikarya. Immunolabel was localized in apical dendrites where it was in part associated

with microtubules. Neither nuclei nor synaptic specializations were immunolabeled (Schmidt

et al., 1994). Interestingly, there was no evidence for postsynaptic membrane labeling of K

opioid receptors in human frontal cortex.

47

Gene knock-out studies of opioid receptors

The genes that encode the cloned opioid receptors can be selectively inactivated.

This approach has only recently been reported for opioid receptors. Kieffer and colleagues

(Matthes et al. 1996) disrupted the MOR gene in mice by homologous recombination. The

homozygous mice showed no obvious morphological abnormalities and did not differ from

their littermates in terms of health or growth. Radioligand saturation studies showed a com­

plete loss of [ H]DAMGO (|j. receptor selective ligand) binding, while 6 [•''H]NTI and K

[^H]CI-977 (K receptor selective ligand) binding were unaffected. In contrast to studies

with wild-type mice, morphine did not produce any signs of antinociception, reward or

physical dependence in the mutant mice. Future studies should continue to advance our

understanding of the endogenous opioid systems and the mechanisms through which exog­

enous opioids produce their effects.

Pharmacological Classiflcation of Delta Opioid Receptors

As mentioned, the molecular classification of opioid receptors has had a tremen­

dous impact on opioid pharmacology. The pharmacological identification of types and sub­

types of opioid receptors has, however, made rapid progress due, in part, to the increasing

availability of highly selective opioid agonists and antagonists. Such compounds have

48

been critical in allowing investigators to develop reasonable confidence that particular opioid

receptor types are involved in the transduction of specific effects, such as antinociception.

As with many other systems, it is now clear that the opioid receptors can all be linked to the

mediation of particular effects, and that multiple receptor involvement in a particular effect

is the rule, rather than the exception.

The recent cloning of MOR, DOR and KOR provides a long-awaited structural

basis for the classification of opioid receptor types. Whether multiple subtypes of these

three opioid receptors will be identified or whether other explanations exist for the many

pharmacological studies which suggest that the opioid receptor types can be further classi­

fied into subtypes remains to be determined. Though future work will be necessary to

elucidate this issue, the pharmacological classification of opioid receptors according to type,

and to subtype, nevertheless allows the continuation of efforts aimed at the development of

highly selective molecules which may prove to be of clinical importance in specific appli­

cations. Of particular interest is the pharmacological identification of subtypes of opioid 5

receptors, and the possible importance of such receptor types in the development of appro­

priate molecules for the relief of pain, particularly in the potential treatment of chronic or

persistent pain states or perhaps in selected conditions such as those involving visceral

pain.

49

Ligands for Delta Opioid Receptors

From a historical point of view, the events leading to the discovery of opioid 5

receptors (Lord et al., 1977), and the approaches towards the uncovering of their pharma­

cology, was distinctly different from the process involved in the identification of p. and k

receptors (Martin et al., 1976). While opioid and K receptors were postulated on the basis

of activity of compounds in vivo, the 6 receptor was proposed on the basis of activity of

compounds in isolated organ bioassays in vitro. Thus, the functional consequences of acti­

vation of the opioid 6 receptor by the enkephalins (Hughes 1975; Hughes et al., 1975b)

were not obvious. Further, due to the rapid enzymatic breakdown (Hambrook et al., 1976)

of [Met^jenkephalin and [Leu^Jenkephalin, the proposed endogenous ligands of the 5 re­

ceptor, evaluation of 6 receptor mediated effects in vivo was virtually impossible. Attempts

were made to stabilize the endogenous peptides by incorporation of unnatural D-amino

acids into the peptide sequence resulting in compounds such as [D-Ala*.

Met^]enkephalinamide (DAME)(Pert et al., 1976) and subsequently, other groups devel­

oped compounds such as [D-Ala-, D-Leu']enkephalin (DADLE). Although these approaches

provided stable molecules, these compounds demonstrated only a very limited selectivity

for the 5 receptor (i.e., approximately 90-fold for DADLE)(Mosberg et al., 1983), a factor

which complicated the assessment of the actions of these substances in vivo. Additionally,

50

due to the peptidic nature of these substances, assessment of their pharmacology in vivo had

to be approached using site-directed injections via the i.c.v. or intrathecal {i.th.) routes rather

than by systemic administration. For these reasons, advances in the understanding of 5

pharmacology has been hampered.

The issue of 5 receptor selectivity has been largely overcome by careful and cre­

ative synthetic efforts in which peptides have been modified in order to retain biological

stability and to achieve selectivity ratios for the 6 receptor of up to 20,000-fold (Kramer et

al., 1993). These types of selectivity ratios begin to approach receptor specificity and ef­

fects observed with central administration of these peptide analogues can be ascribed to 5

receptors with considerable confidence. Efforts from the group at the University of Arizona

resulted in a series of compounds including DPDPE (Mosberg et al., 1983). DPDPE was

the first compound with sufficient selectivity for the 6 receptor (approximately 3,000 fold

in comparing the ICJQ values in the GPI and MVD bioassays)(Mosberg et al., 1983) and

suitable stability for studies in vivo (Porreca et al., 1984; Galligan et al., 1984) to provide

information about the possible functions of opioid 5 receptors.

Erspamer and colleagues have isolated a family of peptides from frog skin, which

are generally termed the deltorphins (Erspamer et al., 1989; Kreil et al., 1989) and which

have become highly important in the elucidation of opioid 6 receptor pharmacology. This

51

family of compounds has high 5 selectivity in receptor binding, in bioassays and in studies

in vivo. Evaluation of the antinociceptive pharmacology of the deltorphins has shown that

[D-Ala^, GIu'']deItorphin produces antinociception following i.c.v. administration to mice

and that this antinociceptive effect is selectively antagonized by ICI 174,864 but not by p-

E^A (Jiang et al., 1990b). This observation, made with appropriate controls for the recep­

tor selectivity of the antagonists, provides strong support for the concept that iP-Ala^,

Glu'*]deltorphin, like DPDPE, produces supraspinal antinociception via opioid 5 receptors.

Another important development was the synthesis of ICI 174,864, a highly selective

antagonist for the 5 receptor (Cotton et al., 1984). These tools have led to a rapid increase

in the amount of information available with regard to the pharmacology of opioid 5 recep­

tors in vivo and in vitro. In spite of the selectivity of compounds like DPDPE and other

peptidic agonists, these compounds remain generally unsuitable for systemic studies. Ulti­

mately, the potential of 6 opioid agonists will remain unknown until systemically active and

highly subtype-selective compounds can be identified and tested in a variety of species and

paradigms. This has begun to be addressed in this dissertation.

Most of the currently available nonpeptidic molecules are antagonists (e.g., naltrin-

dole, naltriben, BNTX) developed within the laboratories of Drs. Portoghese and Takemori

(see Takemori and Portoghese, 1992, for review). Although these have been highly useful

52

and important compounds, they suffer from rather poor (i.e., < 100-fold) selectivity for the

5 receptor; by the selectivity standards of peptidic opioid 5 agonists (i.e., > 1000-fold),

these antagonists would have been rejected, a fact which underscores the need for the de­

velopment of highly selective nonpeptidic 5 ligands.

Recently, a nonpeptidic 5 agonist, BW 373U86, was developed by the Burroughs-

Wellcome group (Chang et al., 1993). BW 373U86 provides an important structural lead

that will be critical in the synthesis of novel nonpeptidic 5 agonists; unfortunately, it also

shows relatively poor 6 selectivity (i.e., approximately 15-fold for 5 vs. [i receptors in ra­

dioligand binding and about 700-fold in bioassay)(Wild et al., 1993a). BW 373U86, how­

ever, is an important molecule in that it (a) appears to act as an agonist via an atypical 5

mechanism (i.e., does not demonstrate the typical G-shift associated with agonists)(Wild et

al., 1993a; Childers et al., 1993) and (b) provides a lead structure which can serve as the

basis for development of more selective nonpeptidic opioid 5 ligands. Although difficult to

classify fully by the standards of reference peptidic 5 agonists, BW 373U86 appears to be a

partial opioid 5 agonist that suppresses the development and expression of dependence to

morphine through a 5 receptor mechanism (Lee et al., 1993). This finding, together with

other observations which demonstrate that 6 subtype selective antagonists (see below)

(Abdelhamid et al., 1991; Miyamoto et al., 1993) suppress the development and expression

53

of dependence to morphine serve as the basis for further investigation of the potential of

such compounds for battling the problems associated with addiction. The development of

more selective nonpeptidic 5 agonists is addressed in Qiapters 2 and 3.

Delta Opioid Receptors Mediating Antinociception

Opioids have been shown to produce antinociception at both supraspinal and spinal

sites (Audigier et al., 1980; Challet et al., 1984; Porreca et al., 1984; Jensen and Yaksh,

1986). On the basis of the relationship between binding affinity, bioassay potency and

antinociceptive potency, Audigier et al. (1980) and Challet et al. (1984) have suggested the

exclusive involvement of supraspinal opioid |x receptors in antinociception. In contrast, the

studies of Galligan et al. (1984) and Porreca et al. (1984) suggested a role for supraspinal 5

receptors also, in antinociception. This question was examined in detail through the use of

selective antagonists for the 5 (ICI 174,864) and |i ( P-FNA) receptors at both supraspinal

and spinal sites in the mouse tail-flick test (Heyman et al., 1987). At both supraspinal and

spinal sites, ICI 174,864 was found to produce a dose-related antagonism of the antinoci­

ceptive effects of DPDPE, but not those of the |i agonists, DAMGO (Handa et al., 1981)

and morphine. In contrast, pretreatment with P-FNA antagonized the antinociception of

DAMGO and morphine, but not that of DPDPE. Vaught and colleagues studied |j, deficient

54

mice (CXBK mice) and showed that the potency of i.c.v. morphine and DAMGO were

greatly reduced (i.e., no Aj^ was achievable) in this strain while the potency of DPLPE (also

a highly selective 5 agonist, Mosberg et al., 1983) was virtually unaffected (Vaught et al.,

1988), offering direct evidence of involvement of supraspinal 8 receptors in antinocicep-

tion. Further, Jensen and Yaksh (1986) used a microinjection study to reveal brain sites in

the rat, such as the broadly defined medullary reticular formation, in which opioid 5 ago­

nists produce antinociceptive effects while opioid p. agonists are only partially active or

inactive. These studies provided a method of separation of the involvement of |i and 5

receptors in supraspinal antinociception and strongly suggested that both receptors may be

important in mediating analgesic actions in humans. The issue of involvement of 5 recep­

tors in supraspinal antinociception has been reviewed (Heyman et al., 1988).

Similarly, the involvement of spinal 5 opioid receptors in antinociception has been

supported by extensive research (e.g., Drower et al., 1991; Heyman et al., 1987; Ling and

Pasternak, 1983; Malmberg and Yaksh, 1992; Porreca et al., 1984; Stewart and Hammond.

1993,1994; Tung and Yaksh, 1982; Yaksh et al., 1980) and is not controversial. From these

studies, it would appear likely that systemically active 5 agonists which reach both su­

praspinal and spinal sites would be effective as analgesics in man.

55

Subtypes of Opioid Delta Receptors

Evidence in vivo for the existence of opioid delta receptor subtypes

The strongest pharmacological evidence for the existence of subtypes of opioid 5

receptors have come from experiments in vivo. Several different lines of evidence have

provided a strong basis for such conclusions. In particular, such studies have been based on

(a) the observation of two-way differential antagonism of 5 receptor-mediated antinocicep-

tion produced by selective 5 receptor antagonists, and (b) the demonstration of a two-way

lack of antinociceptive cross-tolerance between the highly selective opioid 5 selective ago­

nists.

I.c.v. administration of either DPDPE or [D-Ala^ Glu'*]deltorphin, two highly 5-

selective opioid peptides, produced antinociception in the mouse warm-water tail-flick test

which was blocked by ICI 174,864 but not by P-FNA (Heyman et al., 1987; Jiang et al.,

1990b). Taken together with the finding that the doses of p-FNA and ICI 174,864 em­

ployed in these studies were respectively able to antagonize, and fail to antagonize, the

known opioid |j. agonists, morphine and DAMGO, such observations provided a reasonable

basis for the conclusion that DPDPE and [D-Ala^, Glu'*]deltorphin were producing their

antinociceptive actions via supraspinal opioid 5 receptors in the mouse.

56

DALCE was characterized in this same antinociceptive assay, as well as other as­

says, to be initially a reversible opioid 5 agonist (Bowen et al., 1987; Calcagnetti et al..

1989) and to subsequently bind to the 5 receptor in an "irreversible" fashion, presumably as

a result of covalent linkage between sulfhydryl groups in the receptor and the cysteine

residue in position 6 (Bowen et al., 1987). Pretreatment of animals with DALCE produced

a dose- and time-related antagonism of the antinociceptive actions of DPDPE, but not of

morphine (Jiang et al., 1991). Interestingly, however, DALCE pretreatment at the same

doses and times failed to antagonize the antinociceptive actions of [D-Ala^, Glu'*]deltorphin.

This finding could suggest that (a) [D-Ala^ Glu'^Jdeltorphin is more efficacious than DPDPE

or (b) that [D-Ala^, Glu^Jdeltorphin and DPDPE, though both 6 agonists (i.e., producing ICI

174,864-sensitive and P-FNA-insensitive antinociception) were in fact acting at subtypes

of opioid 5 receptors.

These possibilities were distinguished with the aid of 5'-NTll, a novel irreversible

and selective opioid 6 receptor antagonist (Portoghese et al., 1990). Thus, S'-NTII was

demonstrated to antagonize the antinociceptive actions of i.c.v. [D-Ala-, Glu'']deltorphin in

a dose- and time-related fashion, but to have no effect on antinociceptive actions of DPDPE

or of morphine (Jiang et al., 1991). In the absence of the data with DALCE, such findings

with an irreversible antagonist would again raise the possibility of differences in efficacy

57

between the two 5 agonists. However, taken together, the data with these two noncompeti­

tive antagonists demonstrating two-way differential antagonism of DPDPE and [D-Ala-,

Glu'*]deitorphin, strongly suggest a valid distinction of the 5 receptors mediating the antino­

ciceptive actions of these two agonists. On this basis, compounds producing ICI 174,864-

sensitive and P-FNA insensitive antinociception which were antagonized by DALCE, but

not by 5'-NTn, were hypothesized to act via opioid 5, receptors while compounds antago­

nized by 5'-NTII, but not by DALCE, were hypothesized to act via the opioid 5, receptor

(Jiang et al., 1991; Mattia et al., 1991a,b).

If such a hypothesis were accurate, then classical pharmacological theory predicts

that induction of tolerance to an agonist acting at one receptor subtype should have no

effect on the actions of an agonist acting at a second receptor subtype. This hypothesis was

tested by repeated administration of DPDPE or [D-Ala-, Glu'*]deltorphin to mice by i.c.v.

injection. While tolerance to repeated DPDPE was developed as demonstrated by a 4.8-

fold rightward displacement of the DPDPE dose-effect curve, the antinociceptive dose-

response curve for [D-Ala-, Glu"']deltorphin was unaffected (i.e., no cross-tolerance to [D-

Ala-, Glu^Jdeltorphin was demonstrated in mice tolerant to DPDPE)(Mattia et al., 1991b).

Similarly, while tolerance was developed to repeated [D-Ala^, Glu'']deltorphin as demon­

strated by a 37-fold rightward displacement of the [D-Ala^ Glu'*]deltorphin dose-effect

58

curve, the antinociceptive dose-response curve for DPDPE was unaffected (i.e., no cross-

tolerance to DPDPE was demonstrated in mice tolerant to [D-Ala^, Glu'*]deltorphin)(Mattia

et al., 199 lb). Additionally, in mice rendered tolerant to the antinociceptive actions of the p.

agonist, DAMGO, no cross-tolerance was observed to either DPDPE or [D-Ala^,

Glu'']deltorphin (Mattia et al., 1991b). These findings of two-way lack of antinociceptive

cross-tolerance between two highly selective opioid 5 agonists, as well as to a highly selec­

tive |j. receptor agonist, support the prediction of data based on two-way differential antago­

nism which implicates the presence of subtypes of the opioid 5 receptor.

Takemori and colleagues have used NTI and the benzofuran analogue NTB to reach

similar conclusions of the involvement of subtypes of opioid 5 receptors in antinociception

(Sofuoglu et al., 1991). Recently, BNTX has been identified as a selective, nonpeptidic 5,

antagonist (Portoghese et al., 1992), with limited selectivity (i.e., 3-10 fold) for 5 vs. |i

receptors (Porreca et al., unpublished observations).

On the basis of such information, we have proposed a classification of subtypes of

opioid 5 receptors (see Figure 1-2) as the opioid 5, receptor (i.e., mainly activated by DPDPE

and sensitive to antagonism by DALCE and BNTX) and the opioid 6., receptor (i.e., acti­

vated by [D-Ala-, Glu'*]deltorphin and sensitive to antagonism by S'-NTII and NTB; both

the 5, and 5^ receptors are insensitive to the |i antagonist, P-FNA and naloxonazine, and

59

5 K

Agonist

Antagonists

DPDPE

DALCE BNTX

[D-Ala^ Glu^Jdeltorphin

5'NTII NTB

Figure 1-2. Classification of opioid receptors with 5 receptor subtypes indicated. Representative S subtype selective agonists and antagonists are also noted. See abbreviations for a more complete description.

both subtypes are blocked by the 8 selective antagonist ICI 174,864 (Mattia et al., 1992).

DPDPE may not be completely selective for the opioid 5 receptor, though its direct antino­

ciceptive effects appear to be mediated almost entirely at this site (Vanderah et al., 1994);

the |i-modulatory actions of DPDPE (see below) are associated with the opioid 5^ receptor

(Porreca et al., 1992; Vanderah et al., 1993). We have no evidence in vivo for actions of [D-

Ala^, Glu'']deltorphin at receptors other than the 5^ receptor (Vanderah et al., 1994).

Substantial evidence has also indicated the existence of subtypes of opioid 6 recep­

tors at the spinal level in the rat. For example, Stewart and Hammond (1993) have em­

ployed i.th. injections of naltriben to distinguish between the antinociceptive actions of

DPDPE and [D-Ala^ Glu'']deltorphin. In these studies, NTB antagonized the actions of [D-

Ala-, Glu'']deltorphin, but not DPDPE, or the p. receptor agonists. The data are significant

because they indicate that both DPDPE and [D-Ala^, Glu'^Jdeltorphin act via 5 receptors in

60

the rat spinal cord, and that both compounds can produce antinociceptive effects via appar­

ently different 5 receptors. These findings were consistent with conclusions from other

studies using site directed administration of selective agonists and antagonists in the rat

(Malmberg and Yaksh, 1992; Tiseo and Yaksh, 1993).

An interesting finding of potential clinical significance related to the i.th. potency of

DPDPE and [D-Ala^, Glu'^ldeltorphin in rats rendered hyperalgesic by administration of

carrageenan in the paw (Stewart and Hammond, 1994). The activity of these selective 5

ligands were increased in this hyperalgesic state, suggesting that under conditions of in­

flammation, 5 receptors may be a particularly important target for promoting efficient pain

relief (Stewart and Hammond, 1994).

Evidence in vitro for the existence of opioid delta receptor subtypes

A variety of approaches in vitro have provided further evidence for the existence of

subtypes of opioid 6 receptors. The behavioral observations, for example were also con­

firmed using electrophysiological methods (Glaum et al., 1994). In the latter smdies, exci­

tatory postsynaptic currents were evoked electrically and were fully reduced by either DPDPE

or DAMGO and only partially reduced by [D-Ala^, Glu'*]deltorphin; only [D-Ala%

Glu'']deltorphin effects were sensitive to competitive antagonism by NTB. In addition to

61

confirming the existence of subtypes of 5 receptors, this study also showed that these opio­

ids did not decrease the amplitude of a postsynaptic current, suggesting a presynaptic site of

action in which activation of either 5, or 5^ receptors can produce an inhibition of excitatory

glutaminergic afferent transmission in the spinal cord (Glaum et al., 1994).

Studies in which function is evaluated continue to indicate that the effects of [D-

Ala^ Glu'*]deltorphin and DPDPE can be readily distinguished by selective antagonists.

For example, a recent study by Cox and colleagues has shown that the inhibition of adenylyl

cyclase produced by DPDPE was more readily reversed by BNTX (5,) than by NTB (5,) in

both the caudate-putamen and the nucleus accumbens (Buzas et al., 1994). In addition, the

effects of [D-AIa-, Glu'*]deltorphin were more readily antagonized by NTB than by BNTX

in these same brain regions (Buzas et al., 1994). Such observations continue to support the

view that subtypes of the 5 receptor exist and can be functionally demonstrated.

Vaughn et al. (1990) have studied a cyclopropyl-phenylalanine substimted enkephalin

[D-AIa^, (2R, 3S)-VEPhe"', Leu^jenkephalin methyl ester (CP-OMe)(Shimohigashi et al.,

1987; 1988) in competition studies against [-^HIDPDPE in rat brain and in mouse vas defer­

ens. Such smdies were based on suggestions by Shimohigashi and colleagues (Shimohigashi

et al., 1987; 1988) that 6 receptors in these two tissues might be different. The studies of

Vaughn et al. (1990) showed that while CP-OMe had high affinity in rat brain, it exhibited

62

33-fold lower affinity in mouse vas deferens, a finding which correlated with the weak

activity of this compound in the mouse vas deferens. More recently. Fang et al. (1994) have

studied the characteristics of pHJNTI (Yamamura et al., 1992) binding in mouse brain and

in mouse vas deferens. Their findings showed that multiple binding sites were recognized

through competition assays with selective agonists in mouse brain, but that only a single

site was identified in mouse vas deferens. These studies have provided evidence for hetero­

geneity of 5 receptors in mouse brain and raise the possibility that opioid 5 receptors in

MVD differ from those in mouse brain.

Investigations of opioid 5 receptor binding characteristics in mouse or rat brain with

a variety of ligands proved somewhat disappointing, however, in that clear conditions of

binding to the proposed subtypes of this receptor in a single tissue have not been estab­

lished. Indications of 6 subtypes have been reported using ['H]deltorphin I (i.e., pH][D-

Ala-, Asp'*]deltorphin) and pH]DPDPE (Negri et al. 1991). In that study, deltorphin I inhib­

ited binding of pH]DPDPE monophasically, while DPDPE inhibited the binding of

pHJdeltorphin I biphasically. These investigators interpreted the data to suggest the pres­

ence of subtypes of opioid 5 receptors, although it is clearly possible that these data may

reflect the presence of two affinity states of a single receptor type.

63

Support in vitro for subtypes of opioid 5 receptors has also been reported by Roth-

man and colleagues (Xu et al., 1991,1992,1993). In these studies, the site-directed acylating

agent BIT was used to deplete rat brain membranes of opioid p, receptors and competition

studies using pH] DADLE with a series of 5 selective ligands were conducted. In some

cases, two binding sites were identified, a finding consistent with the presence of subtypes

of opioid 5 receptors. Again, however, the possibility of multiple affinity states could not

be eliminated. Recently, Takemori and colleagues have used radioligand approaches to

suggest the existence of subtypes of opioid 5 receptor subtypes in mouse brain using

['H]DPDPE and pH]DSLET (Sofiioglu et al., 1992). However, these data have been ques­

tioned, in part, due to the possibility that the binding experiments were not conducted at

equilibrium.

Recent investigations of opioid receptor systems in the ground squirrel hibemator,

Citellus lateralis, have been undertaken with the aim of providing in vitro correlation for

studies which demonstrate a failure of this species to develop physical dependence to mor­

phine following exposure to this opiate during the hibernating state (Beckman et al., 1981).

Our studies in this species with ['H]Nn, a selective opioid 5 receptor antagonist (Yamamura

et al., 1992) which does not identify multiple affmity states in rat or mouse brain (Yamamura

et al., 1992), have resulted in the observation of two distinct binding sites, indicative of

64

subtypes of opioid 6 receptors in this tissue (Wild, Bilsky and Porreca., unpublished obser­

vations). These data appear to represent the first clear demonstration of opioid 5 receptor

subtypes using radioligand binding techniques in a single tissue from the same species. In

these studies, saturation analysis of pHJNTI binding to whole brain membranes prepared

from Citellus lateralis indicated that the data fit a two-site model. This biphasic model

discerned two sites of high affinity with the highest affinity site comprising 18% of the total

number of sites labeled by pH]NTI. The dissociation constants (K, values) and binding

capacities (B^ values) for the two sites are given in Table 1-1.

In competition studies, the potency of NTI was determined for the inhibition of

[^H]DAMGO and pH]U69,593 from the and K-receptors, respectively, in this species.

The K. values for NTI at |j. and K receptors were 19 and 9.4 nM, respectively, suggesting

that the two sites identified in saturation studies represented two 6 sites.

We have also characterized the binding of [^H]NTI in rat and mouse brain (Yamamura

et al., 1992; Wild et al., 1993a). In these studies, pH]NTI was shown to be a highly selec­

tive 5 receptor ligand with high affinity for 6 sites (K. of 56.2 pM)(Yamamura et al, 1992);

selective ligands for opioid N and K receptors competed for pH]NTI binding sites with low

affinity (i.e., micromolar range) while 6 ligands competed for these sites with high affinity

(i.e., nanomolar range)(Wild et al., 1993a). Critically, NTI has previously been character-

65

Table 1-1. Binding of pH]NTI in whole brain homogenate of Citellus lateralis.

Ko(nM) Bj^ (fmol/mg protein)

Site 1 Site 2 Site 1 Site 2

0.(H±0.01 0.78 ±0.17 13.0 ± 1.6 59.1 ±4.6

ized to be a selective antagonist at opioid 5 receptors both in vivo and in vitro (Portoghese et

al., 1988; Takemori and Portoghese, 1992). Characterization of pH]NTI binding in mouse

brain also revealed a lack of regulation by guanine nucleotides and sodium, consistent with

its profile as a 6 antagonist (Wild et al., 1993a). On this basis, it is clear that pHjNTI does

not distinguish between different affinity states of the opioid 5 receptor.

The data from tissues prepared from nonhibemating Citellus lateralis show a sig­

nificant difference from those of mice or rats. In contrast to studies using brain homogenates

of these latter species (Yamamura et al., 1992; Fang et al., 1994), pH]NTI was shown to

label two sites in Citellus lateralis (Wild, Bilsky and Porreca, unpublished observations).

Due to the antagonist nature of [-^HINTI, it would appear unlikely that the two sites distin­

guished by this ligand represent different affinity states of the opioid 5 receptor. It is un­

66

clear why pH]Nn does not recognize multiple sites in the brain of mouse and rat, even

though one of these species has been used to pharmacologically demonstrate subtypes of

opioid 5 receptors (Jiang et al., 1991; Sofiioglu et al., 1991). It seems possible that, al­

though subtypes of opioid 8 receptors may be present in rats and mice, the relative amounts

of these subtypes may not be conducive to detection using radioligand binding techniques.

In contrast, Citellus lateralis appears to possess enough of both subtypes of the opioid 6

receptor to allow detection by these methods in vitro. Thus, Citellus lateralis is likely to be

a useful rodent species for the smdy of opioid 5 receptors and their subtypes both in vitro

and in vivo.

Additional studies that have recently been published, use different approaches to

investigate populations of 6 receptors in vitro. Hiller and colleagues (Hiller et al., 1996)

used autoradiographic comparison of pH]DPDPE and [-^IflDSLET binding in rat brain slices.

Densitometric image analysis showed a general similarity in the distribution of 5, and 6,

receptors. There were, however, CNS regions that displayed significant differences in the

binding levels of these ligands. The dorsomedial hypothalamus and nucleus accumbens, for

example, displayed higher levels of binding for ['H]DSLET (6, receptors). Another study

(Bausch et al.. 1995) used an antibody generated against the carboxy terminal of the cloned

DOR to map out the distribution of 5 receptors. They found that the immunolabeling corre­

67

lated better with pH]Nn (S/Sj) binding than with pH]DPDPE (5,) binding. The results

further support the existence of distinct populations of 8 receptors and suggests that the

recently cloned DOR corresponds to the 5^ subtype.

Delta Opioid Agonists and Antagonists as Potential Therapeutic Agents

The discovery of opioid analgesics which act via non-p. receptor mediated mecha­

nisms has been a goal of opioid research for many years. Based on a significant body of

work with highly selective peptidic ligands, it would appear that opioids which selectively

act via 6 receptors should have advantages over currently available opioid analgesics. In

particular, potential advantages include the production of analgesia with (a) decreased (or

no) development of physical dependence (Cowan et al., 1988) (b) lack of depression (and

possible stimulation) of respiratory function (Cheng et al., 1993; Dr. Thomas H. Kramer,

unpublished observations), (c) little (or no) adverse gastrointestinal effects (Galligan et al.,

1984; Porreca et al., 1983; Sheldon et al., 1990) and (d) potentially beneficial modulatory

actions (i.e., enhancement of potency and efficacy) on the analgesic actions of standard

opioids such as morphine (Vaught and Takemori, 1979; Jiang et al., 1990c).

The potential advantages of compounds acting via 5 receptors have been derived

from smdies with opioid peptides which have high selectivity for the 5 opioid receptor.

68

including DPDPE and [D-Ala^, Glu'^]deItorphin (Mosberg et al., 1983; Erspamer et al., 1989).

However, the exploration of 6 receptor pharmacology has been hampered by the lack of

systemic availability of peptidic ligands, though some progress has been made in this area

(Polt et al., 1994). Though useful following site-directed administration, the inability of

these compounds to cross the blood-brain barrier has not allowed an evaluation of the con­

sequences of systemic activation of 5 receptors. The availability of a selective, systemi-

cally-active (presumably nonpeptidic) 5 opioid agonist is thus a desirable research objec­

tive and is addressed in Chapters 2 and 3.

Dependence liability of delta opioid agonists

One of the main goals of opioid research has always been to develop strong analge­

sics which are devoid of dependence liability associated with compounds such as mor­

phine. Following the development of DPDPE, the question of dependence liability at the 5

receptor was obvious and was investigated by Porreca and Cowan (Cowan et al., 1988).

Experiments tested whether opioid antagonist-induced physical dependence signs would

result following prolonged exposure to DPDPE. These experiments were the first, and to

the best of our knowledge, the only experiments investigating the dependence liability of

selective compounds acting at the 6 receptor. The mode! used was well established previ­

69

ously by Wei and colleagues (Wei and Loh, 1976) and the study was designed to evaluate

the development of physical dependence relative to agonists selective at |I, 5 and K recep­

tors. For this purpose, we evaluated the development of physical dependence following

infusion of equiantinociceptive doses of DPDPE, DAMGO, morphine and U50,488H into

the Aqueduct of Sylvius of the rat. The animals received infusions of the compounds for a

period of 70 hr. At the time of testing, each rat received s.c. naloxone (3 mg/kg) or ICI

174,864 (3 mg/kg) and signs of withdrawal were evaluated for 30 min. The results demon­

strated three levels of abstinence following administration of antagonists. First, rats receiv­

ing infusions of distilled water or U50,488H showed negligible abstinence scores; second,

rats receiving DPDPE showed low to moderate abstinence scores; and third, rats receiving

DAMGO or morphine showed a high abstinence score indicating a severe degree of physi­

cal dependence.

Thus, while the data were clear in indicating that agonists at 6 receptors provide

clear advantage relative to agonists at |j. receptors in that a much lower degree of physical

dependence is produced, it was unclear whether the abstinence syndrome observed may

have been related to spill-over activity of DPDPE at |i receptors. It is possible that in spite

of the relatively high degree of selectivity for DPDPE at 5 receptors, this compound may

nevertheless have some actions at jj. sites following prolonged infusion. The question of

70

whether agonists with selectivity for the 5 receptor of 10-100 times greater than that of

DPDPE produce even lower dependence liability was recently investigated in the mouse.

The possibility of subtypes of opioid receptors raised the possibility that agonists at one of

these sites would produce negligible dependence liability. Our initial results indicate that

[D-Ala^, Glu'*]deltorphin, a compound which acts at the opioid 5^ receptor, has virtually no

physical dependence liability when infused by the i.c.v. route in mice (Horan and Porreca,

unpublished observations). In this regard, it should be noted that while K agonists were

found to be "disliked" by rats in the conditioned place pairing paradigm, 6 agonists such as

DPDPE show relatively neutral or positive profiles (Shippenberg et al., 1987) while some 6

agonists such as [D-Ala^ Glu'*]deltorphin show no positive or aversive effects in condi­

tioned place pairing experiments in the mouse (Menkens and Porreca, unpublished obser­

vations).

While the data from these experiments utilizing infusion into the lateral cerebral

ventricle or into the Aqueduct of Sylvius are suggestive of a lower dependence liability of

opioid 5 agonists, it could be argued that the compounds are not reaching an appropriate site

of action for the development of a state of physical dependence, which may require activa­

tion of sites distal to the site of infusion, perhaps including the spinal cord. These studies

are unlike experiments which traditionally evaluate the dependence state resulting from

71

repeated administration of systemically-active substances such as morphine and are thus

not directly comparable to such states. Additionally, the dependence state and the type of

abstinence syndrome expressed by repeated activation of the 5 receptor, or subtypes of the

5 receptor, may not be comparable to those produced by |i agonists and will require the

evaluation of a variety of species, states and endpoints in addition to jumping, diarrhea,

body temperature and weight loss. For these reasons, the possible existence of a state of 5

dependence will require evaluation in mice, rats and monkeys following repeated systemic

exposure to subtype selective opioid 5 agonists. It will be important to standardize the

nature of the 5 abstinence syndrome, if one exists, in a fashion which will allow direct

comparison of severity to that induced by opioid |j, agonists. The existence of subtypes of

opioid 5 receptors also allows the possibility of differences in the nature of dependence and

abstinence elicited by activation of each receptor subtype.

Preliminary studies utilizing this approach with racemic BW 373U86 have been

carried out (Lee et al., 1993); BW 373U86 was infused by osmotic minipump and rats

challenged with either naloxone or NTI. Virtually no signs of abstinence were observed,

suggesting that compounds of this type may have low physical dependence liabilities. Such

observations support the preliminary assessment that opioid 5 agonists may be effective

analgesics in humans with no, or reduced, physical dependence and abuse liability.

72

Opioid delta receptor mediated suppression of morphine abstinence

Recent studies from Takemori and colleagues have suggested that occupation of

opioid 5, receptors by either NTI or 5'-N l Ll prevents the development of tolerance to mor­

phine antinociception as well as the development of physical dependence (or the expression

of abstinence) to morphine in mice (Abdelhamid et al., 1991). This study utilized doses of

the 5^ antagonists which did not antagonize the acute antinociceptive actions of morphine

and showed that withdrawal jumping in mice was blocked in animals pretreated with these

compounds. It is critical to note that the doses of antagonists used in this study did not

antagonize the acute effects of morphine, and thus, could not be due to a blockade of p.

receptors. The findings were repeated using either an acute tolerance/dependence para­

digm or in a chronic model involving the implantation of morphine pellets. Our laboratory

has confirmed these observations in the mouse showing that pretreatment 24 hr before test­

ing with either S'-NTII or [D-Ala-, Cys'*]deltorphin , both highly selective and long-acting

antagonists at opioid 5, receptors (Horan et al., 1993; Wild et a!., 1993b) blocked the ex­

pression of withdrawal jumping in mice challenged with naloxone. It should be empha­

sized that these pretreatments had no effect on the acute antinociceptive actions of mor­

phine, or other p. agonists such as DAMGO.

73

These findings are provocative and exciting. Further work from the Takemori labo­

ratory shows that NTB, but not BNTX, blocks the development of tolerance and depen­

dence to morphine, suggesting the involvement of opioid receptors in this effect (Miyamoto

et al., 1993). This finding is also supported in our laboratory by the observation of a lack of

effect of DALCE (an opioid 5, antagonist) on the expression of the morphine abstinence

syndrome following naloxone challenge in the mouse. These observations with NTB, NTI,

5'-NTn and [D-Ala^, Cys'']deltorphin, all compounds which block 5, receptors (all except

NTI are selective for this subtype), together with the lack of activity of DALCE, suggests

that occupation of the 6^ receptor can alter the actions of morphine in some fashion (Bilsky

and Porreca, unpublished observations). Interestingly, we have recently reported that 8^, but

not 6,, antagonists block the modulatory effects of 6 agonists on morphine antinociception

(Porreca et al., 1992), suggesting that 6^ receptors may reside in the hypothesized func­

tional or physical opioid p.-5 receptor complex (see Rothman et al., 1993, for review). The

modulation of morphine antinociception via an opioid 6, receptor appears to be consistent

with the view that 5^ receptors are also involved in the modulation of morphine antinoci­

ceptive tolerance and physical dependence (Porreca et al., 1992). The modulatory action of

these |i ligands are clearly mediated through 5 receptors as the acute actions of morphine, or

other |i agonists, are not antagonized (Porreca et al., 1992; Heyman et al., 1989a,b).

74

The findings of Takemori and colleagues (Abdelhamid et al., 1991; Miyamoto et al.,

1993) and of our laboratory in mice have also been confirmed in the rat using BW 373U86

(Lee et al., 1993). In that study, BW 373U86 was infused by minipump in rats receiving

repeated injections of morphine. It was shown that BW 373U86 prevented the develop­

ment of the motor signs of morphine abstinence (jumping, wet-dog shakes, head shakes,

forelimb tremors, digging and teeth chattering) in a dose-related fashion (Lee et al., 1993).

These findings suggested that occupation of the opioid 5 receptor can prevent the develop­

ment of tolerance and dependence to morphine. Again, while BW 373U86 is difficult to

classify in terms of 5 receptor subtype selectivity, it would most closely appear to be a

partial opioid 5, agonist. The suggestion from these studies is that occupation of the opioid

6, receptor by either an antagonist, or a partial agonist, can effectively modulate the devel­

opment of dependence to morphine, and perhaps other |i opiates.

Delta opioid receptors and addictive processes

Vertebrate animals have developed unique anatomical and neurochemical systems

that reinforce goal-directed behaviors such as feeding, drinking, social coexistence and sex.

These behaviors ensure the survival of the individual and the continued existence of the

species. The successful completion of each goal leads to a rewarding stimulus (good feel­

75

ings, pleasure, etc.) which further reinforces the behavior, making it more likely to be re­

peated.

There is considerable evidence to suggest that drugs of abuse activate the same

biochemical pathways as natural rewards such as a palatable meal or stimulating social

interaction. In fact, drugs such as cocaine may activate these systems so strongly that the

individual spends increasing amounts of time acquiring and administering the drug. Natural

rewards become insignificant or unimportant compared to the euphoria of the cocaine rush.

It is these supraphysiological drug rewards that contribute to addiction.

Neuroanatomical substrates that are critical to reward include dopamine cell bodies

that originate in the ventral tegmental area (VTA) of the midbrain. These neurons project to

more rostral areas of the brain via the medial forebrain bundle (MFB). They terminate in a

variety of limbic structures including the nucleus accumbens, hypothalamus, amygdala,

hippocampus and limbic cortical areas (e.g., cingulate cortex). These and other connections

give rise to a complex but well coordinated response to a variety of external and internal

stimuli that helps insure the survival of the individual and the species.

There is considerable evidence that suggests that endogenous and exogenous opio­

ids can regulate neuronal activity in the above described mesolimbic and mesocortical dopam­

ine systems. Opioid 5 receptors, for example, are located in relatively high levels in the

76

nucleus accumbens (Hiller et al., 1996; Mansour and Watson, 1993). In addition, the VTA

contains enkephalinergic neurons that may originate in the nucleus accumbens (Groenewegen

and Russchen, 1984; Khachaturian et al., 1985). Opioid 5 receptor agonists in the VTA also

increase the release of dopamine in the nucleus accumbens (Devine et al., 1993; Di Chiara

and Imperato, 1988; Spanagel et al., 1992). At the more behavioral level, 5 opioid agonists

increase forward locomotion (Calenco-Choukroun et al., 1991), are self-administered (Devine

and Wise, 1990) and produce conditioned place preferences (Menkens et al., unpublished

observations).

The regulation of dopamine release by endogenous 5 opioids and their receptors has

provided a potential target for novel pharmacological treatments for drug addiction. There

is evidence to suggest that 6 opioid antagonists such as naltrindole (NTI) may block some

of the reinforcing effects of cocaine and other psychostimulants (Heidbreder et al., 1993;

Jones and Holtzman, 1992; Jones et al., 1993; Menkens et al., 1992; Reid et al., 1993;

Shippenberg and Heidbreder, 1995). By blocking the reinforcing effects of drugs of abuse,

or by attenuating dopamine mediated responses to conditioned stimuli associated with these

drugs, 8 antagonists may turn out to have some efficacy in helping treat drug addiction.

These issues will be further explored using novel 5 opioid compounds and techniques.

77

Summary of Research Goals

This introductory chapter should convince the reader that there is a need for further

developing opioid agonists and antagonist which have better therapeutic profiles than cur­

rently available opioids. In addressing this need, it is obvious that we need to have a better

understanding of chemistry, pharmacology and physiology of endogenous and exogenous

opioids. With this additional information, we will be better able to develop these future

medications.

As mentioned, opioid agonists and antagonist that act through 6 opioid receptors

may offer significant advantages over currently available n opioid agonists and antagonists.

Unfortunately, the most selective 5 opioid agonists are peptide molecules which cross the

blood-brain barrier poorly. Chapters 2-4 report on the pharmacology of a novel nonpeptidic

opioid agonist that has a selectivity ratio for 5 receptors over p. and K receptors that ap­

proaches the selectivity ratios of the peptide agonists DPDPE and [D-Ala^, Glu'*]deltorphin.

Chapters 4 and 5 characterize a novel pharmacological technique. We then use the tech­

nique to further investigate the possibility of subtypes of 5 opioid receptors. Specifically,

antisense oligodeoxynucleotides complementary to the cloned 5 opioid receptor, or to all

three cloned opioid receptors (|i, 5 and K) were used to selectively interfere with the syn­

thesis of target opioid receptors. The resulting knock-down of the receptors may be superior

78

(in terms of selectivity) to what can be accomplished with conventional antagonists. Chap­

ters 6 and 7 assess another important aspect of opioid pharmacology which is not well

understood. Repeated administration of opioid agonists leads to the development of toler­

ance. The observation that morphine tolerance can be attenuated by coadministration of an

NMDA antagonist has potential clinical significance. The generality of this finding to addi­

tional opioids, including 5-selective agonists, is addressed in Chapter 6. Chapter 7 looks at

the role of post-receptor mechanisms (e.g., protein kinases that are activated subsequent to

opioid receptor activation) in mediating morphine tolerance. Chapter 8 concludes the ex­

perimental section of the dissertation by presenting some additional work that has emerged

from the dissertation research.

79

II. IN VITRO PHARMACOLOGICAL PROFILE OF SNC 80

Specific Aims:

There is a significant impetus to synthesize selective nonpeptidic 5 opioid agonists. In

vitro characterization techniques provide useful information on the selectivity and agonist

activity of these novel compounds. SNC 80, an enantiomer of the promising 8 opioid ligand

BW 373U86, was evaluated to:

a. Determine if SNC 80 binds to opioid receptors (inhibition studies with selective ji,

5 and K radioligands).

b. Calculate the selectivity ratios for SNC 80 binding to |I, 5 and K receptors (compare

the Kj values of SNC 80 at each receptor)

c. Determine if SNC 80 has opioid agonist activity in two standard in vitro opioid

bioassays (isolated mouse vas deferens and guinea pig ileum bioassays).

d. Determine which opioid receptor(s) SNC 80 is acting at to produce the measured

effects in these bioassays (pretreat tissues with selective |i and 6 antagonists).

e. Calculate the approximate |i/5 selectivity ratios for SNC 80 by comparing the deter­

mined ICjQ values in each bioassay.

80

Introduction

A number of laboratories have recently reported on the pharmacology of BW 373U86

(Figure 2-1), the first reported nonpeptidic opioid agonist with some selectivity for 6 opioid

receptors (Chang et al., 1993; Comer et al., 1993a,b; Dykstra et al., 1993; Lee et al., 1993;

Wild et al., 1993a). Though the pharmacology of BW 373U86 was somewhat disappoint­

ing in terms of a relative lack of systemic activity and significant degree of toxicity, this

compound represents a lead structure for the development of nonpeptidic 6 opioid agonists.

BW 373U86 displayed significant selectivity for 5 receptors in bioassays (approximately

700-fold) with nanomolar potency in the MVD (5 bioassay)(Chang et al. 1993). Inhibition

(±)-BW373U86 (+)-SNC80 (relative configuration) (absolute configuration)

Figure 2-1. Relative configuration of (±) BW 373U86 and absolute configuration of(+) SNC 80.

81

studies with BW 373U86 against selective radioligands for p., 5 and K receptors revealed a

very limited (approximately 20-fold) selectivity for 5 receptors in rodent brain and spinal

cord tissue (Chang et al., 1993; Wild et al., 1993a). In spite of the somewhat favorable

profile of BW 373U86 in vitro, the actions of this compound in vivo suggested a mixed

profile of actions at |X and 5 receptors. BW 373U86 was not effective in the mouse tail-flick

or tail-pinch tests following i.e.v., i.p. or p.o. administration, though i.th. BW 373U86 pro­

duced naloxone-sensitive antinociception in these assays. Critically, however, BW 373U86

produced significant toxicity, characterized behaviorally as convulsions and barrel rolling,

following i.c.v. or i.p. administration (Comer et al., 1993b). This finding was unexpected as

peptides with 5 receptor selectivity such as [D-AIa^, Glu'']deUorphin and DPDPE had not

been associated with toxicity of this nature.

One factor which complicated the interpretation of the pharmacology of BW 373U86

was the use of this compound as a racemic mixture. Numerous examples have been re­

ported where significant pharmacological differences exist between enantiomers of a given

compound (e.g., Raffaet al., 1992, 1993; VonVoigtlander and Lewis, 1988). Furthermore,

slight changes in the structure of a molecule can produce striking changes in the activity of

the compound. For this reason, we have begun a systematic investigation of the pharmacol­

ogy of the resolved, and chemically modified enantiomers of BW 373U86. SNC 80 (Figure

82

2-1), the methyl ether of one enantiomer of BW 373U86, has recently been synthesized

(Calderon et al., 1994) and shown to display significant selectivity for 6 opioid receptors.

Sections 11 and 11 of this dissertation present a more extensive characterization of the phar­

macological profile of SNC 80 in vitro and in vivo. The data indicate that SNC 80 should be

useful as a systemically-active and highly selective opioid 5 receptor agonist.

Materials and Methods

Animals

Male, ICR mice (20-30 g) and male Hartley guinea pigs (250-500 g)(Harlan Indus­

tries, Cleveland, OH) were housed in groups of 5 and 4, respectively in Plexiglas chambers

with food and water available ad libitum prior to any procedures. Animals were maintained

on a 12 hr light/dark cycle in a temperature controlled animal colony. Studies were carried

out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted

and promulgated by the National Institutes of Health.

GPI and MVD bioassays

Animals were sacrificed by cervical dislocation and the vasa deferentia or ileum

were removed. Tissues were tied to gold chain with suture silk, suspended in 20 ml baths

83

containing 37°C oxygenated (95% 0^ 5% COj) Krebs-bicarbonate solution (magnesium

free for the MVD) and allowed to equilibrate for 15 min. The tissues were then stretched to

optimal length previously determined to be 1 g tension (0.5 g for the MVD) and allowed to

equilibrate for 15 min. The tissues were stimulated transmurally between platinum wire

electrodes at 0.1 Hz, 0.4-ms pulses (2.0 ms pulses for MVD) and supra-maximal voltage.

Drugs were added to the baths in 14-60 p,l volumes. The agonists remained in contact with

the tissue for 3 min before addition of the next cumulative dose, until maximum inhibition

was reached.

Percent inhibition was calculated by using the average contraction height for 1 min

preceding the addition of the agonist divided by the maximal height after exposure to the

dose of the agonist. ICj^ values represent the mean of four tissues and these values and their

associated 95% confidence limits were determined by fitting the mean data to the Hill equa­

tion by using the computerized nonlinear least-squares method.

Radioligand binding experiments

Mice were killed by cervical dislocation and the brains (including cerebella) were

rapidly removed and placed in 20 volumes of ice cold 50 mM:5 mM Tris:MgCl, buffer (pH

7.4). The tissues were then homogenized using a polytron set at the lowest setting and

84

centrifuged at 48,000 x g for 20 min. The pellet was resuspended in 20 volumes of fresh

buffer and incubated at 25°C for 30 min to dissociate and remove endogenous opioid pep­

tides. Following this incubation period, membranes were centrifuged and resuspended in

fresh buffer two more times. Membranes were resuspended in fresh 50 niM:5 mM Tris:MgCl,

buffer (pH 7.4). All other components of the incubation were prepared in an assay buffer of

50mM:5 mM TrisrMgCl,containing 1.0 mg/ml bovine serum albumin (final concentration)

and 100 fiM phenylmethylsulfonylfluoride (final concentration) at pH 7.4.

The affinity of SNC 80 for |I, 5 and K receptors was determined by competing unla­

beled SNC 80 against [^H]DAMGO (1 nM, selective ^.-receptor ligand), pH]NTI (0.3 nM,

selective 5-receptor ligand) or [^H]U69,593 (1 nM, selective K-receptor ligand). All experi­

ments were performed in duplicate with total binding, nonspecific binding (10 fiM nalox­

one) and 10 inhibitor concentrations determined. Incubation times for all three radioligands

was 5 hr. Incubations were terminated by rapid filtration through Whatman GF/B filter

strips previously soaked in 0.1% polyethylenimine for at least 1 hr. The filtered membranes

were washed three times with 4 ml of ice-cold 0.9% saline and radioactivity measured

using a liquid scintillation counter with >42% efficiency.

85

Chemicals

SNC 80 (M.W. 545) was synthesized as previously described (Calderon et al., 1994).

CTAP was synthesized by solid phase methods at The University of Arizona Chemistry

Department (Victor Hruby and colleagues). ICI 174,864 was purchased from Cambridge

Research Biochemicals. All drugs were dissolved in either distilled water or 20% DMSO

immediately prior to use.

Radioligands

PHJDAMGO (55.0 Ci/mmol), pH]Nn (30.0 Ci/mmol) and pH]U69,593 (47.0 CM

mmol) were purchased from New England Nuclear (Cambridge, MA).

Statistics

Radioligand binding data were analyzed using a microcomputer and InPlot4 graph­

ing software, a curve-fitting package that utilizes nonlinear, least-squares regression analy­

sis based on models for 1 or 2 independent binding sites. In all experiments, inhibition

curves were best Fit by a 1 site curve as determined by the F-ratio test. Summary data are

presented as K values (± S.E.M.) and represent at least three separate experimental deter­

minations.

86

Results

Bioassays

The mean responses of the vasa deferentia (expressed as percent inhibition of twitch

height) to each concentration of SNC 80 are shown in Figure 2-2. SNC 80 produced con­

centration-dependent inhibition of electrically-stimulated twitch contractions in both the

MVD (Figure 2-2) and GPI (data not shown) bioassays.

100 r

£ O

C

C ec 0)

0 SNC80 • w/iai74^(lfiM)

V W/CTAP(1MM)

0.1 1 10 100 1000

Concentration of SNC 80 (nM)

10000

Figure 2-2. Concentration-response curves for SNC 80 inhibition of contractions in the mouse isolated vas deferens in controKopen circles) tissues or tissues pretreated with ICI 174,864 (Santagonist. closed circles) or CTAP (IX antagonist, triangles).

87

The calculated ICj^ values for SNC 80, DPDPE and [D-Ala^, Glu^Jdeltorphin in these

bioassays are shown in Table 2-1.

Concentration-effect curves were constructed in the presence of the 5-seIective an­

tagonist ICI 174,864 or the |i selective antagonist CTAP (Figure 2-2). Pretreatment with

ICI 174,864 resulted in a 236-fold rightward shift in the SNC 80 curve. In contrast, pretreat­

ment with CTAP produced only a 1.9-fold rightward shift.

Table 2-1. Agonist ICso values (nM) for SNC 80, DPDPE, md[D-Ala, Glu* jdeltorphin in the GPI and MVD bioassays.

Compound GPI

(Mean ± S.E.M.)

MVD

(Mean ± S.E.M.)

GPI/MVD

(fi/5) Ratio

SNC 80 5457 ± 2052 2.73 ± 0.5 1996

DPDPE 7300 ± 1700 5.1 ± 0.5 1800

[D-Ala-, Glu^ldeltorphin 15000 ± 1000 0.85 ± 0.07 17000

88

Radioligand binding studies.

SNC 80 produced concentration-dependent inhibition of pH]DAMGO, pH]Nn and

['H]U69,593 binding in mouse whole brain membranes (Figure 2-3). The calculated K.

values for SNC 80 at 6, and K sites were 1.78, 881.5 and 441.8 nM, respectively. A

comparison of the calculated K. values against each radioligand, revealed that SNC 80 was

approximately 495- and 248-fold more potent in inhibiting [-^HINTI binding than

[^H]DAMGO or pH]U69,593 binding.

100

c o

p

c CC

10 0.1 1 100 1000 10000 100000

Concentration of SNC 80 (nM)

Figure 2-3. Representative competition curves for SNC 80 against ['H]DAMGO (open circles), ['HINT! (closed circles) or ['H]U69,593 (triangles) in mouse whole brain assays.

89

Discussion

The development of systemically-available, 5 selective opioid agonists may offer

therapeutic advantages over currently available opioid analgesics. Previous studies with

the racemic mixture of BW 373U86 have demonstrated that this non-peptidic compound

has activity at 5 opioid receptors (Chang et al., 1993; Wild et al., 1993), but demonstrates

poor systemic activity, antinociceptive profile and 5 receptor selectivity in vivo. Here, the

profile of SNC 80, the methyl ether of one enantiomer of BW 373U86 (Calderon et al.,

1994) has been investigated both in vivo (section HI) and in vitro (this section). The profile

of this compound was found to be markedly different from that of BW 373U86.

Studies with SNC 80 in the MVD and GPI preparations, bioassays generally consid­

ered to reveal 5 and |X receptor activity, respectively, demonstrated that SNC 80 shows

approximately 2,000-fold selectivity for 5 (rather than |i) receptors. This degree of selec­

tivity is similar to that seen with the best 5-selective opioid peptides, such as [D-Ala*,

Glu'*]deltorphin and greater than that observed with DPDPE. The selective antagonism of

SNC 80 inhibition in the MVD bioassay by ICI 174,864, but not CTAP, confirms that the

agonist actions of SNC 80 in this tissue are 6-receptor mediated.

Inhibition studies using radioligands selective for opioid 5 and K receptors

further confirm the selectivity of SNC 80 for 5 opioid receptors. SNC 80 potently inhibited

90

the binding of pH]Nn to mouse whole brain membranes while being significantly less

potent in competing for sites labeled by [^H]DAMGO or pH]U69,593. The binding selec­

tivity profile of SNC 80 for 6 receptors was an order of magnitude greater than that reported

with BW 373U86 (Chang et al., 1993) and comparable to that seen with DPDPE (Mosberg

et al., 1983; Calderon et al., 1994). It should be noted that the degree of selectivity seen

with SNC 80 in the present radioligand binding experiments is less than that previously

reported (Calderon et al., 1994). This discrepancy appears to be due to methodological

differences in the radioligand binding assay previously employed to assess the affinity of

SNC 80 at |j. receptors, as the affinity SNC 80 for 5 receptors was similar in both studies.

In summary, the in vitro data from the present study indicate that SNC 80, an ana­

logue of one enantiomer of BW 373U86, is a selective 6 opioid receptor agonist.

91

in. IN VIVO PHARMACOLOGICAL PROFILE OF SNC 80

Specific Aims:

The in vitro experiments with SNC 80 were promising. SNC 80 displayed significant

selectivity for 5 over fi and K opioid receptors in two in vitro models. SNC 80 also appeared

to be acting as an agonist at 5 receptors. Further studies were needed to assess the in vivo

activity of SNC 80. The experiments in this chapter addressed the following issues.

a. Determine if SNC 80 produces antinociception in standard nociceptive bioassays

following central (i.e.v. or i.th.) or systemic (i.p. orp.o.) administration.

b. Calculate the antinociceptive potency of SNC 80 in these tests following the differ­

ent routes of administration.

c. Determine whether the antinociceptive effects are: (1) opioid mediated; (2) 5 recep­

tor mediated and (3) 6 receptor subtype mediated using selective opioid antagonists.

d. Make gross behavioral observations concerning the toxicity and behavioral effects

of SNC 80 following its administration by the various routes.

92

Introduction

Based on the favorable in vitro profile of SNC 80, further evaluation of the com­

pound in vivo was performed.

Methods

Animals

Male, ICR mice (20-30 g) (Harlan Industries, Cleveland, OH) were housed in groups

of 5 in Plexiglas chambers with food and water available ad libitum prior to any procedures.

Animals were maintained on a 12 hr light/dark cycle in a temperature controlled animal

colony. Studies were carried out in accordance with the Guide for the Care and Use of

Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Injections.

Administration of compounds by the i.c.v. route was accomplished by direct injec­

tion into the right lateral ventricle as previously described (Porreca et al., 1984). Briefly,

mice were lightly anesthetized with ether and a slit was made along the midline of the scalp.

An injection was made with a 10 |il Hamilton syringe at a point 2 mm caudal and 2 mm

lateral from Bregma. The injection was made at a depth of 3 mm in a volume of 5 |il. The

93

i.th. administration of compounds was made with a 10 Hamilton syringe (5 p,l volume)

and a 30 gauge needle using the method of Hylden and Wilcox (1980) as modified by

Porreca and Burks (1983). Mice were lightly anesthetized with ether and the needle in­

serted between the L5-L6 vertebrae, l.p. injections were given using a I ml syringe with a

30 gauge needle in a 10 ml/kg volume. Oral injections were administered using a 1 ml

syringe and a 22 gauge animal feeding needle.

Antinociceptive tests

Antinociception was assessed using the 52°C warm-water tail-flick assay and the

52°C hot-plate test. In the tail-flick assay, the latency to the first sign of a rapid tail-flick

was taken as the behavioral endpoint (Janssen et al., 1963). Each mouse was first tested for

baseline latency by immersing its tail in the water and recording the time to response. Mice

not responding within 5 sec were excluded from further tesdng. SNC 80 was then adminis­

tered and tested at various subsequent times. A maximum score was assigned to animals

not responding within 15 sec to avoid tissue damage. Antinociception was calculated as %

Antinociception = 100 x (test latency-control latency)/(15-control latency). In the hot-plate

test, mice were placed on the heated surface, and the time to lick the back paws or to elicit

an escape jump was recorded. Percent antinociception was calculated as 100 x (test latency-

94

control latency)/(60-control latency). A maximum score was assigned to animals not re­

sponding within 60 sec to avoid tissue damage. Mice not responding within 20 sec in the

predrug control were excluded from further study. All testing was performed in mice that

had completely recovered from anesthetic.

Antagonist studies

In order to determine if the antinociceptive effects of SNC 80 were mediated by

opioid receptors, mice were pretreated with various opioid selective, and subtype selective,

antagonists. The nonselective opioid antagonist naloxone (3 nmol), and the 6 antagonist

ICI 174,864 (Cotton et al., I984)(4.4 nmol) were given i.c.v. while NTI, a nonpeptidic 5

antagonist (Portoghese et al., 1988), was given by the i.p. route (20 mg/kg); these antago­

nists were given 10 min prior to SNC 80 and antinociceptive testing took place either 10 or

30 min after the i.c.v. or i.p. administration of agonist. The non-equilibrium antagonists (3-

FNA (|i antagonist, Jiang et al., 1990b)(18.8 nmol), DALCE (5, antagonist, Bowen et al.,

1987; Jiang etal., 1990a, 1991)(4.5 nmol) and Cys-DELT(5T antagonist, Horanetal., 1993)(3

nmol) were all administered i.c.v. 24 hr prior to SNC 80. These doses and times have previ­

ously been shown to offer selectivity for the intended opioid receptor type and to be maxi­

mally effective in this assay (Heyman et al., 1987; Horan et al., 1993; Jiang et al., 1990a,b).

95

Chemicals

SNC 80 was synthesized as previously described (Calderon et al., 1994). DPDPE

and P-FNA were obtained through the National Institute of Drug Abuse Drug Supply Pro­

gram. NTI and naloxone were purchased from Research Biochemicals Inc. DALCE, [D-

Ala-, Glu'^Jdeltorphin and Cys-DELT were synthesized by solid phase methods in the labo­

ratory of Dr. Victor Hruby. ICI 174,864 was purchased from Cambridge Research Bio­

chemicals (Atlantic Beach, NY). All drugs were dissolved in either distilled water, saline or

20% DMSO immediately prior to use. In all cases, animals received an appropriate vehicle

treatment as a control.

Statistics

For antinociceptive tests, dose-response lines were constructed at times of agonist

peak effect and analyzed using linear regression by using a modification of the method of

Tallarida and Murray (1986). A minimum of 10 mice were used at each dose level. All A,^

values (95% confidence limits) shown are calculated from the linear portion of the dose-

response curve. Single dose studies used analysis of variance (ANOVA) followed where

appropriate by Student's t-test for between groups comparisons (significance was set at /? <

0.05).

96

Results

Behavioral and antinociceptive effects

Administration of SNC 80 by the /.c.u, Lth. or i.p. routes evaluated in these studies

did not reveal evidence of significant behavioral effects. To this end, non-quantitative as­

sessment of animals did not show obvious changes in righting reflex, locomotor activity,

Straub tail, piloerection or stereotypy. Some mild evidence of sedation was observed at the

highest doses tested by the i.c.v. and i.p. route (i.e., 3(X) nmol and 100 mg/kg, respectively).

In this case the animals appeared to be sitting quietly in their cages but responded readily to

any stimulation with no obvious signs of ataxia or other obvious behavioral abnormality.

SNC 80 produced dose- and time-related antinociception following i.c.v. or i.th. ad­

ministration in the tail-flick (Figure 3-1 A,B) and i.c.v. administration in the hot-plate (Fig­

ure 3-lC) tests. I.p. SNC 80 also produced dose- and time-related antinociception in the

tail-flick test (Figure 3-2). Peak antinociceptive effects were reached 10 min after i.c.v. and

i.th. administration (Figure 3-1 A,B), and 30 min after i.p. (Figure 3-2) administration, with

antinociception persisting for approximately 40-60 min. The calculated A^^ values (and

95% C.I.) for i.c.v., i.th. and i.p. administration of SNC 80 in the tail-flick test were 104.9

(63.7 - 172.7) nmol, 69 (51.8 - 92.1) nmol and 57 (44.5-73.1) mg/kg, respectively. The

calculated for i.c.v. SNC 80 in the hot-plate test was 91.9 (60.3 -140) nmol.

97

100 SNC 80 dose; O SOOnznol • lOOnznol V 60nmol • 30nmoi

c o a 0) u 'u c c c <

Time (minutes)

100 SNC 80 dose; O 300nmol • 100 nmot V 60nmol • 30nmol

c o CL Qi U 'C O c

20 30 10 40

Time (minutes)

100 SNC 80 dose: O 300nmol • 100 nmol V 60 nmol c o

c

Time (minutes)

Figure 3-1. Dose- and time-response curves for i.c.v. (A) and i.th. (B) SNC 80 in the warm-water tail-flick test and i.c.v. (C) SNC 80 in the hot-plate test

98

100 SNC 80 dose:

O 100 mg/kg • 60 mg/kg V 30 mg/kg

20 60 40 80

Time (minutes)

Figure 3-2. Dose- and time-response curves for i.p. SNC 80 in the warm-water tail-flick test.

Antagonist studies

The antinociceptive effects of i.c.v. SNC 80 (300 nmol) in the hot-plate test were

antagonized by pretreatment with i.p. NTI, reducing the antinociceptive effect from 77.6 ±

9 to 18.7 ± 6.3 in control and NTI-treated mice, respectively (Figure 3-3). Similarly, the i.p.

administration of an approximate dose of SNC 80 (1(K) mg/kg) produced significant

antinociception in the hot-plate and tail-flick tests which was blocked by i.p. pretreatment

with the same dose of NTI (Figures 3-3 and 3-4). This dose of NTI blocked the antinocicep­

tive effects of DPDPE and [D-Ala-, Glu^jdeltorphin without affecting the antinociceptive

actions of morphine in the tail-flick test (Figure 3-4). The doses of DPDPE and [D-Ala^

99

100 r

vd c/i

c o a. 0) u

'u o G

c <

c a O)

SNC 80 300 nmol, i.c.v.

w/naltrindole 20mg/kg,i.p.

SNC 80 100mg/kg,i.p.

w/naltrindole 20 mg/kg, i.p.

Figure 3-3. Antinociceptive effects of a single dose of i.c.v. (A) or i.p. (B) SNC 80 in vehicle-pretreated or NTI-pretreated (20 mg/kg, i.p.) mice in the hot-plate test.

tu C/3 M

C 0 O, 01 u 'u o C

'•4-* c <

c (8 01 2

100

75

50

25

SNC 80 100 mg/kg, i.p.

Morphine DPDPE [D-Ala , Glu Ideltorphin 10 mg/kg, i.p 30 runol, i.c.v. 19 runol, i.c.v.

Figure 3-4. Antinociceptive effects of a fixed i.p. dose of SNC 80 or morphine, or an i.c.v. dose of DPDPE or [D-Ala-, Glw'Jdeltorphin in control (closed bars) or NTI-pretreated (20 mg/kg, i.p.) mice.

100

Glu'^]deltorphin have previously been characterized to act via opioid 5, and 5, receptors,

respectively (Jiang et al., 1991; Mattia et al., 1991b). The Lc.v. administration of naloxone

significantly, but not completely, antagonized the antinociceptive effect of i.p. SNC 80 (100

mg/kg)(data not shown). The Lc.v. or Lth. administration of ICI 174,864, a 5 antagonist,

produced a significant, but partial, block of the antinociceptive effects of Lp. SNC 80 in the

tail-flick test (Figure 3-5). The coadministration of Lc.v. and Lth. ICI 174,864 (4.4 nmol at

each site) produced a virtually complete block of the antinociceptive effects of Lp. SNC 80

(Figure 3-5). Pretreatment of animals with the selective antagonist 3-FNA (18.8 nmol,

Lc.v, Lth. or Lc.v./Lth.) had no effect on Lp. SNC 80 antinociception (Figure 3-5); this pre­

treatment dose and time have previously been shown to selectively antagonize the actions

of Lc.v. p. agonists (Heyman et al., 1987). The antinociceptive actions of i.e.v. SNC 80 were

blocked by Lc.v. pretreatment with either DALCE (S^ selective antagonist) or Cys-DELT

(5j selective antagonist)(Figure 3-6). Pretreatment with both DALCE and Cys-DELT pro­

duced a greater inhibition of SNC 80 antinociception than was observed with either antago­

nist alone. Pretreatment with Lc.v. |3-FNA had no effect on the Lc.v. SNC 80 dose-response

curve (Figure 3-6).

101

100

a c/i +L 75 c o a, 0) u 50 u O .5 c < 25

C CQ (U

i.c.v./i.th. antagonists i.th. antagonists I.C.V. antagonists

SNC80 w/P-FNA w/IQ 174,864 w/P-FNA w/IQ 174,864 w/P-FNA w/IQ 174,864 100 mg/kg, i.p. 18.8 nmol 4.4 nmol 18.8 nmol 4.4 nmol 18.8 nmol 4.4 nmol

Figure 3-5. Antinociceptive effects ofi.p. SNC 80 in control mice, or in mice pretreated with i.c.v., i.th. or

i.c.vA.th. FNA orlCI 174,864.

100 r

C o o, a. 60

u O .5 c < 5^

O SNC 80 control

• SNC80/DALCE

V SNC 80/[Cys''ldeltorphin

• SNC80/[Cys''ldeItorphin/DALCE

• SNCSO/P-FNA

Dose SNC 80 (i.c.v., nmol)

Figure 3-6. Antinociceptive effects of i.c.v. SNC 80 in control mice (open circles) or in mice pretreated with i.c.v. DALCE (closed circles, 4.4 nmol at -24 hr), [Cys* jdeltorphin (open triangles, 3 nmol at -24 hr). P-FNA (open squares, 18.8 nmol at -24 hr) or both DALCE and [Cys^Jdeltorphin.

102

Discussion

The profile of SNC 80 in vivo was significantly improved when compared to that

seen with BW 373U86. In this regard, SNC 80 produced dose- and time-related

antinociception in the tail-flick test following i.c.v. and i.th. administration, and critically,

following systemic {i.p.) administration. SNC 80 was also active in producing antinociception

in the hot-plate test. The antinociceptive effects of the compound were selectively antago­

nized by NTI, suggesting activity at opioid 6 receptors. Also, recent studies have shown

that SNC 80 produces 5 opioid mediated antinociception in the formalin test in rats after

i.p., i.th. or i.paw administration (Bilsky et al., 1997b; Nichols and Porreca, unpublished

observations). These studies suggest that SNC 80 is active in many models of acute nocic­

eption in rats and mice and that it may also be active in models of tonic nociception.

Preliminary experiments also demonstrated that p.o. SNC 80 produced a moderate

degree of antinociception, suggesting that oral availability is possible, though not promi­

nent with this molecule. Despite the relatively low potency of SNC 80 following /.c.v., i.th.

or i.p. administration, a full agonist effect was seen following all routes of administration.

Importantly, at the highest doses which could be tested due to solubility limits, no signs of

toxicity (i.e., convulsions) were observed. Further, no evidence of Straub-tail or hyperex-

citability was observed, though these very high doses did produce some signs of sedation.

103

Studies characterizing SNC 80 antinociception with antagonists confirm the actions

of this compound to be mediated via opioid 5 receptors. The systemically active 6 selective

antagonist NTI (Portoghese et al., 1988) blocked the antinociceptive actions of i.p. SNC 80

in the tail-flick and hot-plate tests; in the tail-flick test this dose of NTI did not antagonize

i.p. morphine, but effectively blocked the antinociceptive actions of i.e.v. DPDPE or IP-

Ala^, Glu'^Jdeltorphin, confirming the selectivity of the antagonist for the 5 receptor. The

ic.v. administration of ICI 174,864, a peptidic 5 antagonist, partially blocked the actions of

i.p. SNC 80. This partial blockade of SNC 80 antinociception was also seen following i.c.v.

treatment with naloxone. The partial blockade seen with these i.c.v. antagonists against

systemically administered SNC 80 may reflect the action of this agonist at both supraspinal

and spinal sites as revealed by the direct i.th. injections. Coinjection of i.c.v. and i.th. ICI

174,864 produced a virtually complete block of the effects of SNC 80 in the tail-flick test.

The 6 receptor selectivity of i.p. SNC 80 was further confirmed by the lack of antagonism

seen with i.c.v. |3-FNA, a |i antagonist, which has been demonstrated previously to selec­

tively block fi-mediated antinociception (Heyman et al., 1987; Jiang et al., 1990b).

The proposed 5 subtype selective antagonists DALCE (6,) and Cys-DELT (62) (Jiang

et al., 1991; Mattia et al., 1991a) were used to assess the potential role of supraspinal 5

opioid receptor subtypes in SNC 80 antinociception. Administration of either DALCE or

104

Cys-DELT produced significant antagonism of SNC 80 effects, and the combination of

these two antagonists produced an even greater antagonism. These data suggest that SNC

80 does not display selectivity for proposed subtypes of 5 receptors, in contrast to the ac­

tions of DPDPE (predominately 5,) and [D-Ala^, Glu'*]deltorphin selective) in this spe­

cies. In agreement with the data following Lp. administration of SNC 80, the lack of an­

tagonism of i.c.v. SNC 80 by i.e.v. pretreatment with P-FNA suggests that opioid recep­

tors do not mediate the antinociceptive actions of SNC 80.

In summary, the agonist profile of this compound in vivo is consistent with its char­

acteristics in vitro and demonstrates the importance of possible differences in pharmacol­

ogy of resolved enantiomers of a given compound. The profile of SNC 80 appears to be

particularly encouraging in that no signs of toxicity were observed. Continued evaluation

of SNC 80, and many related analogues that may have improved potency, is underway with

the goal of the development of therapeutically useful 5 analgesics.

105

IV. IN VrVO CHARACTERIZATION OF ANTISENSE EFFECTS

Specific Aims:

The next two chapters of the dissertation investigate 5 opioid receptor pharmacology

using a novel technique, antisense oligodeoxynucleotide (ODN) mediated "knock-down"

of receptors. The experiments also further characterize the mechanisms of the antisense

effect. The specific aims of the project are to:

a. Determine if in vivo treatment with antisense ODN directed at the cloned DOR can

reduce the antinociceptive actions of 5 selective opioid agonists.

b. Optimize the dosing and time-effect parameters of antisense ODN administration in

the bioassays used.

c. Address the specificity of the antisense ODN treatment for the 6 opioid receptor

using mismatch ODN controls and selective |I and K opioid agonists (see methods).

d. Determine if the 5 antisense ODN can differentiate 5| and 5^ receptors using 5 sub­

type selective agonists.

e. Assess the effects of an antisense ODN common to all three cloned opioid receptors

on the antinociception of |X, 5 and K selective agonists (this might provide further

evidence for 5 opioid receptor subtypes (see discussion)

106

Introduction

The recent cloning of three types of opioid receptors (the |i, 5 and K opioid

receptors)(Evans et al., 1992; Kieffer et al., 1992; Chen et al., 1993; Minami et al., 1993;

Yasuda et al., 1993) from tissues derived from mouse or rat provides a long-awaited struc­

tural basis for the classification of opioid receptor types. However, despite the strong phar­

macological evidence for subtypes of opioid 5 receptors, no subtypes have been identified

at the molecular level to date. Recently, the administration of antisense ODNs to cloned

receptors in vivo has been used to identify the functional importance of specific receptor

subtypes (Wahlestedt et al., 1993b; Zhou et al., 1994; Raffa and Porreca, 1996). We, along

with others, have used this antisense strategy to further investigate the pharmacology of

opioid receptors (Adams etal., 1994; Bilsky et al., 1994, 1996a; Chien et al., 1994; Lai et

al., 1994; Rossi et al., 1994; Standifer et al., 1994; Tseng et al. 1994). The results of these

studies support recent evidence which shows that treatment with antisense ODNs comple­

mentary to the cDNA of a specific receptor can be successfully employed to apparently

disrupt the biosynthesis of the receptor in vivo (see Figure 4-1).

Although the preliminary findings in vivo with antisense ODNs to the DOR were

supportive of the hypothesis of subtypes of opioid 5 receptors, potential questions regard­

ing the time-course and specificity of the effect still remained. To that end, the present

107

Transcription

Capping/ Polyadenylation

Splicing

Transport

Protein

Translation

I AAAAA

I AAAAA

Nucleus AAAAA

AAAAA

AAAAA

Cytoplasm

Figure 4-1. Possible sites at which antisense ODNs may disrupt protein synthesis. The antisense ODN may (I) form a triple helix with the DNA in the cell nucleus; (2) the antisense ODN may bind the nascent RNA and prevent transcription; (3) the antisense may bind to the exon and prevent transcription; (4) the antisense ODN may bind to the exon-intron junction and prevent splicing or bind to the mRNA and prevent transport from the nucleus to the cell cytoplasm (5). In the cell cytoplasm, antisense ODN may bind to the 5'-CAP (6), bind to the AUG initiation codon or to the 3'-untranslated region of the mRNA (8), all of which prevent translation. Recent studies measuring the cellular uptake of labelled ODNs indicate that they are preferentially taken up and kept in the cell nucleus. This may indicate a more direct action on gene expression (e.g., inhibition ofpre-RNA splicing or inhibition of transport of mRNA out of the cell nucleus (Iverson et at., 1992). Figure modified from Pilowsky et ai, 1994; Crooke, 1992 and Phillips and Gyurko, 1995).

108

investigation extends our initial findings and provides a more detailed characterization of

the effects of treatment with DOR antisense ODNs on the antinociceptive response to selec­

tive opioid 5 and K agonists. Further, this investigation has also employed an antisense

ODN directed to a conserved region of the cloned opioid receptors as a control to evaluate

the specificity of the antisense strategy. The data continue to support the hypothesis of

subtypes of opioid 6 receptors.

Materials and Methods

Animals

Male, ICR mice (20-30 g) (Harlan Industries, Cleveland, OH) were housed in groups

of 4 in Plexiglas chambers with food and water available ad libitum prior to any experimen­

tal procedures. The mice were maintained on a 12 hr light/dark cycle in a temperature

controlled animal colony. Studies were carried out in accordance with the Guide for the

Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes

of Health.

Injections

Compounds were injected by the i.c.v. route as described in Chapter 3.

109

Synthesis of oligodeoxynucleotides

Antisense and mismatch ODNs derived from the 5' end of the coding sequence of

the cloned DOR (nucleotides 7 to 26) were synthesized by Midland Certified Reagent Co.

(Midland, TX). Two 20 base oligos, for the DOR, of the following sequence were used in

these experiments: 5'-GCA CGG OCA GAG GGC ACC AG-3' (antisense) and 5'-GCA

GCG GCA AGG GGC CAC AG-3' (mismatch). The specificity of these sequences to the

DOR were verified by a search for sequence homology (Genepro, Riverside, WA) to the

gene sequences of the cloned DOR, MOR and KOR to ensure minimal cross-hybridization.

A second set of ODNs, derived from sequences common to all three cloned opioid recep­

tors, were also designed (common antisense/mismatch ODN). These 20 base ODNs of the

following sequence were used in these experiments in vivo: 5'-ATG TAG ATG TTG GTG

GCG GT-3' (antisense) and 5'-AGT TAG AGT TTG GTG CGG GT-3' (mismatch). They

corresponded to the following nucleotide positions: 244-263 (5), 274-293 (K) and 301 -320

(ji). ODNs were reconstituted in sterile deionized water immediately prior to the first injec­

tion. Separate aliquots of the oligos were stored at 2°C for up to 6 days of the procedures.

All ODNs (12.5 fig/injection) were administered i.c.v., twice daily at 12 hr intervals for up

to six days.

1 1 0

Antinociceptive testing

Antinociception was assessed at least 12 hr after the last ODN administration using

the 55°C warm-water tail-flick test. The latency to the first sign of a rapid tail-flick was

taken as the behavioral endpoint (Jannsen et al., 1963). Each mouse was first tested for

baseline latency by immersing its tail in the water and recording the time to response. Mice

not responding within 5 sec were excluded from further testing. Mice were then adminis­

tered selective opioid agonists and tested at time of agonist peak effect. A maximum score

was assigned (100%) to animals not responding within 15 sec to avoid tissue damage.

Antinociception was calculated by the following formula: % antinociception = 100 x (test

latency-control latency)/(15-control latency). All evaluation of antinociception was done

by a single individual who was blinded to the ODN treatments.

Statistical analysis

For antinociceptive tests, dose-response lines were constructed at times of agonist

peak effect and analyzed using linear regression by using a modification of the method of

Tallarida and Murray (1986). All A^^ values (95% confidence limits) shown are calculated

from the linear portion of the dose-response curve. Single dose data were analyzed using

one-way analysis of variance (ANOVA) followed by Student's t-test for between groups

I l l

comparisons (significance set at p<0.05). A minimum of 10 mice were used at each dose

level.

Chemicals

DPDPE and [D-Ala^, Glu^Jdeitorphin were synthesized by solid phase methods as

previously described (Erspamer et al., 1989; Misicka et al., 1991; Mosberg et al., 1983).

DAMGO was purchased from Bachem (Torrance, CA). Bremazocine was purchased from

Research Biochemicals Inc. (Natick, MA). Sufentanil, etorphine, U69,593 and PLC 17 were

obtained through the NIDA drag supply program. All drugs were dissolved in either dis­

tilled water or 20% DMSO immediately prior to use.

1 1 2

Results

In vivo assessment of DOR antisense and mismatch administration

Previous work (Bilsky et al., 1994; Lai et al., 1994; Wahlestedtet al., 1993a,b) has

demonstrated that repeated i.c.v. injections of 12.5 p.g antisense oligos can selectively in­

hibit receptor mediated agonist effects in mice. To further investigate the actions of ODNs,

doses of DOR antisense and mismatch (1, 3 or 10 jig/injection) were administered twice

daily for three days. DOR antisense, but not mismatch, dose-dependently reduced the

antinociceptive response to an A„ dose of [D-Ala^ Glu'']deltorphin (18 nmol) (Figure 4-2).

5 mismatch 8 antisense

1 3 10 Dose of Oligodeoxynucleotide (pig/injection)

Figure 4-2. Antinociceptive effect of an A^ dose of [D-Ala-, Glu* Ideltorphin in mice pretreated with varying doses of DOR antisense or mismatch ODN (see text for details). An asterisk indicates a significant (p<0.05) difference from mismatch control. Each bar represents the mean of 9 to 10 mice.

113

The effects of [D-Ala^, Glu'*]deltorphin (18 nmol, 12 hrs after the last ODN injection)

were also tested in animals receiving from 1 to 6 days (twice-daily for a total of 2-12 injec­

tions) of DOR antisense ODN treatment (Figure 4-3). There was a reduction of antinocice-

ption across the first three days of treatment (D1-D3) which was nearly complete by the

third day. The reduction of antinociceptive effect was maintained across a subsequent three

days of treatment (D4-D6). In animals that had received 6 days of antisense treatment, the

antinociceptive response to [D-Ala^, Glu^ldeltorphin began to recover by the second day

after ODN treatment ended and reached control levels by recovery day 4 (R4).

I 5 Mismatch B S Antisense

Days of ODN treatmerxt (D) and recovery (R)

Figure 4-3. The antinociceptive response of a fixed, submaximal dose ofi.c.v. [D-Alar, Glu'Jdeltorphin (10 nmol) in separate groups of vehicle treated mice or in mice treated for up to 6 days (D1-D6) with DOR antisense or mismatch ODN. Recovery of the antinociceptive response was evaluated in separate groups of mice that had received 6 days of DOR antisense or mismatch ODN treatment followed by 1 to 4 days (RI-R4) of no treatment. Each group of animals was tested only once.

1 1 4

A time course to the antinociceptive action of a fixed i.e.v. dose of [D-Ala-,

GIu'*]deltorphin (18 nmol) was constructed in control, DOR antisense or mismatch ODN

treated mice (Figure 4-4). Control and mismatch ODN treated mice showed an approximate

80% antinociceptive response 10 min after the [D-Ala^, Glu'*]deltorphin injection. These

animals maintained measurable antinociception up to 60 min post-injection. In contrast,

DOR antisense ODN treated mice showed no measurable antinociception at any time tested

from 10 to 60 min.

100 Q Control # S antisaise

V 5 mismatch

cu

0 20 40 60

Time (minutes)

Figure 4-4. The antinociceptive time-course of a fixed, submaximal i.c.v. dose of [D-Ala\ Glu'jdeltorphin (10 nmol) in vehicle (control) mice or mice treated for 3 days with DOR antisense or mismatch ODN.

115

Approximate doses of |I or K selective opioid agonists were administered i.e.v. to

mice treated with DOR antisense or mismatch ODN, or vehicle. The antinociceptive re­

sponses to the ^ selective agonists sufentanil (0.9 nmol), DAMGO (0.2 nmol) or PL017

(0.2 nmol) (Figure 4-5 A) or the K agonists U69,593 (60 nmol) and bremazocine (30 nmol)

(Figure 4-5B) were unaffected by DOR antisense or mismatch ODN treatment.

CD Control

• S mismatch

Sufentanil DAMGO PL017

U69^93

Figure 4-5. The antinociceptive responses to a fixed sumbmaximal dose of selective opioid fi (A) or K (B) agonists in mice pretreated for 3 days with vehicle, DOR antisense, or DOR mismatch ODN.

1 1 6

The antinociceptive effect of an dose of the 6 selective agonist [D-Ala%

Glu'^]deltorphin was inhibited by 3 day pretreatment with DOR antisense, but not mismatch,

ODN (data not shown). Dose-response curves for [D-Ala^, Glu^Jdeltorphin were constructed

in vehicle treated and in DOR antisense or mismatch ODN-treated mice (Figure 4-6A); the

calculated Ajo values (nmol/mouse, 95% C.I.) were 5.6 (3.5-9.0), 38.8 (27.1-55.8) and 6.3

(4.3-9.1), respectively, indicating an approximate 6.9- and 1.1-fold rightward shift in the

[D-Ala^, Glu'']deltorphin dose-effect curve following DOR antisense or mismatch ODN

treatment, respectively.

In contrast to the rightward shift of the [D-Ala^, GIu'*]deltorphin dose-response curve,

DOR antisense ODN had no effect on the antinociceptive actions of DPDPE (Figure 4-6B).

The calculated A^^ values (nmol/mouse, 95% C.I.) for control, DOR antisense or mismatch

ODN treated animals were 9.3 (7.6-12.9), 11.3 (6.9-15.2) and 9.4 (7.5-12.4), respectively.

1 1 7

100

80

C o

g- 60 u

*u O

•S .n •S 40

20

O Control • 5 antisense V 5 mismatch

,1 ^ U J. • » ' « ' »

1 10 100

Dose [D-Ala^, Glu^Jdeltorphin (nmol, i.c.v)

100

80

c .2

60 u u o c C 40 <

20

B

' • » .

10 100

Dose DPDPE (nmol, i.c.v)

Figure 4-6. Antinociceptive dose-effect curves of i.c.v. [D-Alcf, Glu'Jdeltorphin (A) or DPDPE (B) after 3-day treatment with vehicle (control), DOR antisense or mismatch ODN. Each point represents the mean of 9 to 10 mice.

1 1 8

Antisense ODN targeting a conserved region of all three cloned opioid receptors

The administration of COR antisense, but not mismatch, ODN inhibited the antino­

ciceptive actions of the )i selective agonist DAMGO and etorphine (Figure 4-7A,B), the K

selective agonists U69,593 and bremazocine (Figure 4-8A,B) and the 5 selective agonists

[D-Ala^, Glu'^Jdeltorphin, and DPDPE (Figure 4-9A,B). Three day pretreatment with COR

antisense, but not mismatch, ODN produced an approximately 10.5, 12.1,2.8, 6.3, 11, and

>30-fold rightward shift in the dose-response curves for i.e. v. administration of DAMGO,

etorphine, U69,593, bremazocine, [D-Ala^, Giu^Jdeltorphin and DPDPE, respectively. Cal­

culated Aj^ values (95% C.I.) for each agonist/treatment group are depicted in Table 4-1.

Table 4-1. Calculated A^^ values (nmol) and 95% C.L. for selective agonists in control, and 3-day-treated COR antisense and mismatch ODN-treated mice.

Agonist Control COR antisense COR mismatch

DAMGO 0.05 (0.03 - 0.07) 0.48 (0.17 - 1.38) 0.04 (0.03 - 0.06)

DPDPE 9.8 (7.2 - 13.2) >300 10.2 (6.9 - 15.0)

[D-Ala^ Glu-*] deltorphin

7.8 (5.6 - 10.7) 85.6 (39.5 - 185.4) 7.7 (6.9 - 15.0)

U69,593 24.2 (16.7 - 35.0) 66.9 (52.1 - 85.9) 24.5 (18.1 - 33.2)

Bremazocine 10.5 (8.1 - 13.6) 66.5 (47.1 - 93.8) 7.1 (5.7 - 10.5)

Etorphine 0.02 (0.01 -0.03) 0.2 (0.16-0.31) 0.03 (0.02 - 0.04)

119

100 O Control # Common antisense V Common mismatch

c o a, <u y 'S O .5

0.3 3 0.03 0.01 1 0.1

Dose DAMGO (nmol, i.e. v.)

100

c o

IX O) u 'u o •S c <

0.003 0.01 0.1 0.3 1 0.03 3

Dose Etorphine (nmol, i.e. v.)

Figure 4-7. The antinociceptive dose-response curve for Lc.v. DAMGO (jx agonist) (A) or etorphine (B) in mice treated for 3 days with vehicle, COR antisense or mismatch ODN. Each point represents the mean of 9 to W mice.

120

100

80

c o cu tu CJ

• •M u o 40

C <

60

20

C o a 0) u

• 4

O c

'•w c <

100

80

60

40

20

O Control # Coinmon antisense V Common mismatch

I I 10 30 60

Dose of U69^93 (nmol, i.c.v.)

O Control # Corrunon antisense V Common mismatch

100

B

I » I I I I I I I I I. I

1 10 100

Dose of Bremazodne (nmol, i.c.v.)

Figure 4-8. Antinociceptive dose-effect curves of the K agonists U69,593 (A) or bremazodne (B) given i.c.v. after 3 days of treatment with vehicle, COR antisense or mismatch ODN. Each point represents the mean of 9 to 10 mica.

121

c o cu 0) u u o

C o a cu u u O .S

100

80

60

•E 40

20

O Control # Common antisense + V Common mismatch

1 3 10 30 100 300

.2 r-uA^ Dose [D-Ala , Glu ]deltorphin (nmol, i.c.v)

100

80

60

40

20

O Control 9 Common antisense V Common mismatch

B

3 10 30 100

Dose DPDPE (nmol, i.c.v.)

300

Figure 4-9. Antinociceptive dose-effect curves of the S agonists [D-Alar, Glu" Jdeltarphin (A) or DPDPE (B) given i. c. v. after 3 days of treatment with vehicle, COR antisense or mismatch ODN. Each point represents the mean of 9 to 10 mice.

122

Discussion

Historically, the discovery and classification of receptor types, and subtypes, has

relied on selective agonists and antagonists. The use of molecular cloning techniques and

the expression of cDNAs encoding novel receptor proteins has allowed for additional ap­

proaches to the study of drug-receptor interactions. For example, several studies have dem­

onstrated that synthetic antisense oligodeoxynucleotides may effectively interfere with the

biosynthesis of a particular receptor, presumably by interacting specifically with the tran­

scripts encoding that polypeptide, thereby reducing the transcript pool for translation of the

protein (Wahlestedt et al., 1993a,b;Zhouetal., 1994). Antisense strategy offers high affin­

ity and specificity for its target by sequence complementarity; thus the resulting "knock­

down" effect on the density and the function of the target protein can be directly correlated

with its structural identity (Wahlestedt, 1994). The present experiments describe the results

of such an antisense strategy by using a sequence that targets the cloned DOR specifically

(Bilsky et al., 1994; Lai et al., 1994), as well as a region that target all three cloned opioid

receptor types. The results of these experiments support the concept that antisense ODNs

to the opioid receptors produce highly specific alterations in the pharmacological responses

to opioid agonists in mice.

123

Previous experiments with proposed receptor selective antagonists have demon­

strated a difference in pharmacological profile for DPDPE and [D-Ala^, Glu'']deltorphin,

two agonists mediating their antinociceptive actions at opioid 6 receptors (Jiang et al., 1991;

Mattia et al., 1991; Sofuoglu et al., 1991). The data with DOR antisense ODN treatments

similarly demonstrate a difference in the pharmacological profile of these two 6 agonists.

Construction of full i.e. v. dose-response curves with DPDPE and [D-Ala^, Glu'*]deltorphin

after three day treatment with DOR antisense or mismatch showed a significant rightward

displacement only of the [D-Ala^, Glu'*]deltorphin curve. The lack of effect of the DOR

antisense ODN treatment on the DPDPE dose-response curve suggests that the antisense

ODN may interfere selectively with a subtype of the cloned DOR, in agreement with data

from pharmacological characterization using antagonists. Additionally, our previous ex­

periments have shown that administration of DPDPE was fully effective in the same DOR

antisense ODN treated mice that showed no response to [D-Ala^, Glu^jdeltorphin while [D-

Ala-, Glu'*]deltorphin was ineffective in the same DOR antisense ODN treated mice which

had shown a full response to DPDPE (Lai et al., 1994).

Treatment with DOR antisense ODN produced a parallel rightward displacement in

the [D-Ala% Glu"']deltorphin dose-response curve. This apparent competitive profile of the

data may be due to the presence of spare DOR receptors, thus a "knock-down" of the recep­

124

tor pool by the antisense ODN conceivably may allow enough residual 5 receptors to pro­

duce a full effect. Alternatively, there may be a difference in distribution of the DOR antisense

ODN compared to that of [D-Ala^ Glu^Jdeltorphin, such that the agonist produces

antinociception at sites distal to antisense-affected areas. In this regard, however, previous

in vivo binding studies have shown that concentrations of ['"I]deltorphin I remained higher

in the circumventricular, rather than in deep structures, of the mouse brain (Mokhtari et al.,

1993) arguing against this possibility. Moreover, recent autoradiographic experiments in

rats has shown that i.c.v. administration of DOR antisense ODN, as described here for mice,

uniformly decreases 5 receptors labeled by ['"I][D-Ala^, Glu"']deltorphin across all ana­

tomical regions of the rat brain (Cha and Rothman, personal communication). These data

lead to the suggestion that the ODN distributes throughout the brain following i.c.v. admin­

istration. Thus, the parallel rightward shift of the [D-Ala-, Glu^jdeltorphin dose-response

curve most likely reflects the presence of spare receptors. Future experiments with radiola­

beled ODN will address the issue of ODN distribution.

The time-course studies with antisense oligos provides further information regard­

ing the specificity of the DOR antisense ODN effect. DOR antisense, but not mismatch,

ODN treatment produced a nearly complete antinociceptive blockade of a fixed i.c.v. dose

of [D-Ala% Glu'*]deltorphin across the full 60 min time course of the antinociceptive re­

125

sponse. The blockade of antinociception was time-related and reversible in that separate

groups of animals treated for 6 days with DOR antisense displayed antinociceptive responses

to [D-Ala^, Glu'^ldeltorphin that were not significantly different from control groups within

3 days of cessation of antisense treatment. The time-related nature of the DOR antisense

ODN treatment would suggest that the perturbation which is achieved is not the result of

generalized neurotoxicity or effects on non-opioid receptor systems. Further, the suggested

turnover rate of DOR synthesis appears to be consistent with the recovery of the antinoci­

ceptive response within 3 days. In this regard, previous studies using the irreversible 5,

antagonist 5'-NTn similarly showed an approximate 60 hr delay in recovery of antinocicep­

tive function of [D-Ala^ Glu'*]deltorphin (Jiang et al., 1991).

It is interesting to note that the data from the present experiments with a DOR anti-

sense ODN confirm previous preliminary reports (Lai et al., 1994; Bilsky et al., 1994) and

continue to differ from paradigms in which the 6 agonists are administered by the Uh. route

(Bilsky et al., 1994; Standifer et al., 1994). That is, route-dependent differences are ob­

served in the antinociceptive sensitivity of DPDPE to DOR antisense ODN. Thus, i.c.v.

DPDPE is not affected by the DOR antisense ODN used in the present studies, or a second

DOR antisense ODN used previously (Bilsky et al., 1994). However, administration of a

DOR antisense ODN by the i.th. routes showed no effects against the antinociceptive re­

126

sponses to a selective P. or K agonist but failed to discriminate between the antinociceptive

responses to both DPDPE and to [D-Ala^ Glu'*]deltorphin (Bilsky et al., 1994; Standifer et

al., 1994). While these findings appear paradoxical, it should be emphasized that these data

with antisense ODNs are completely consistent with finding previously reported using pro­

posed 5 subtype selective antagonists. Spinal administration of S'-NTU has been shown to

antagonize the antinociceptive response of both DPDPE and [D-Ala^, Glu'']deltorphin sug­

gesting the overriding importance of the S'-NTII- sensitive (i.e., ) receptor in mediation

of 6 antinociception at the spinal level in mice (Mattia et al., 1992). Additionally, these

findings with antagonists also suggested that the 5 agonists could not be considered specific

for the proposed subtypes of 8 receptors, a finding which has been confirmed using specific

conditions of receptor blockade in vivo (Vanderah et al., 1994).

An important control experiment which would further substantiate the findings with

DOR antisense ODN was the use of an antisense ODN targeted to a conserved region of the

three cloned opioid receptors. Such an antisense ODN should be effective in blocking the

antinociceptive response to opioid agonists, including that to DPDPE if this compound

were producing its antinociceptive response via an opioid receptor containing the same

conserved sequence. The results of these studies show that treatment with a COR antisense,

but not mismatch, ODN for 3 days produces a significant rightward shift in the antinocicep-

127

tive response of opioid agonists that lack opioid receptor subtype selectivity (e.g., etor-

phine), as well as of agonists with selectivity for P. (DAMGO), K (U69,593, bremazocine)

and 5 ([D-Ala^, Glu'*]deItorphin, DPDPE) receptors. Supraspinal blockade of both CD-

Ala^, Glu^jdeltorphin and DPDPE suggests that the putative 5, and 5^ receptors share a

conserved nucleotide sequence that encodes residues 82-88 of DOR, but differ at the start

of the coding region. The discriminating power of the ODNs is large because as few as 20-

30% base pair mismatches (4-6 mismatches out of a 20 base sequence) would abolish the

specificity of the ODN. This would imply that the differential effect of the DOR antisense

ODN on the antinociceptive response of [D-Ala^ Glu'*]deltorphin and DPDPE is based on

the ligands' differential interaction with two structurally related, but distinct receptors.

These data fit the characteristics of receptor subtypes within the G-protein coupled

receptor family. However, homology screening has yet to identify a cDNA that encodes a

5 receptor that is structurally distinct from that isolated previously. This lack of definitive

structural evidence for 5 receptor subtypes does not disqualify the body of pharmacological

evidence which supports the heterogeneity of the 5 receptors, and the data presented here

implicate further that this heterogeneity may have a structural basis. Receptor heterogene­

ity has been shown to arise primarily from discrete genes (e.g. muscarinic, cholinergic,

serotonergic and adrenergic receptors) and alternative splicing (e.g. dopamine and prostag-

128

landin receptors) within the G-protein coupled receptor family. Other possible mechanisms

include posttranslational processing of the protein, the protein's interaction with other mo­

lecular components, as well as RNA editing, although to date none of these has been

demonstrated to be responsible for the synthesis of receptor subtypes within this gene fam­

ily. As our data suggest a structural distinction at the primary sequence between the puta­

tive subtypes, they argue against the role of posttranslational processing or the possibility of

the protein's interaction with other cellular components. Only definitive evidence against

the existence of multiple gene products that exhibit the properties of the 5 receptor may

disprove the hypothesis of receptor subtypes.

An interesting finding of the experiments with COR antisense ODNs is the degree

of rightward displacement of the dose-effect curves for agonists with differing profile. In

mice treated for 3 days with COR antisense, but not mismatch ODN, the dose-response

curves for selective agonists at ^i, 5,, 5, and K receptors showed 10.5-, >30-, 11- and 2.8-

fold rightward displacements. One possible explanation for the differential shifts in the

dose-effect curves of these agonists with selectivity for these putative receptors would be

the differences in the relative numbers of these sites in mouse brain. It might be hypoth­

esized that the receptor type with the fewest relative number of receptors should be the most

sensitive to antisense ODN "knock-down". In this regard, saturation studies with

129

pH]DAMGO, pH]NTI and pH]U69,593 show that fi, 5 and k receptors constimte 43.9,

46.5 and 9.6%, respectively, of the total opioid receptor population (see Tables 5-1 and 5-2).

From these data, it would appear that K mediated antinociception should be the most sensi­

tive to treatment with COR antisense ODN, a suggestion which was not supported by the

data. Additionally, the data correlating the effect of COR antisense ODN treatment on the

antinociceptive effects of etorphine and bremazocine, two relatively nonselective agonists

showed a similar reduction in effect.

The degree of the antisense ODN-induced rightward shift could potentially be a

function of several factors including the relative numbers of opioid receptor subtypes, the

number of spare receptors of each receptor type, the anatomical distribution of the

"knocked-down" receptors, the efficacy of the test agonist and the degree to which a recep­

tor type is "knocked-down". Treatment with antisense ODNs are unlikely to alter the brain

distribution of a drug administered via the i.c.v. route, suggesting that the anatomical distri­

bution of the selective agonist following i.c.v. administration is probably not a significant

factor. Similarly, administration of DOR antisense ODN to rats uniformly decreased 5 re­

ceptors labeled by ['^I]deltorphin across all anatomical regions of the brain (Cha and Roth-

man, personal communication) suggesting that the anatomical distribution of the

"knocked-down" receptors is also probably not a significant factor. The degree to which

130

each receptor type is "knocked down" will clearly affect the degree of the rightward shift.

The degree of the "knock down" will also depend on several factors including uptake of the

ODN by different populations of nerves and the turnover rate of the receptor. A receptor

with a longer half-life will likely be less affected by the COR antisense ODN than a recep­

tor with a shorter half-life. Thus, given the multiple factors which can contribute to the

antisense ODN-induced rightward shift, it is not surprising that the observed shifts differ as

a function of the receptor subtype activated by the selective agonist. Indeed, it is interesting

to note that the antisense ODN-induced rightward shift should be the same for two ligands

acting through the same receptor. Thus, the fact that the COR antisense ODN shifts the

DPDPE dose-response curve to the right much more than the [D-Ala-, Glu'*]deltorphin

dose-response curve is additional evidence that the 6, receptor is distinct from the cloned

5, receptor.

The observation that some 5-mediated effects are not attenuated by DOR antisense

ODN treatment predicts that there should exist a 5 binding site which is not affected by

administration of DOR antisense ODN. Administration of DOR antisense ODN decreases 5

receptors assayed under standard assay conditions with a variety of ligands including [^H]NTI

(see Chapter 5), [-^HJCD-Ala-, D-Leu']enkephalin (Cha et al., 1995), as well as

pH][pCl]DPDPE, ['"I][D-Ala% Glu'^Jdeltorphin and pH]SNC-12I (Chaand Rothman, per­

131

sonal communication). This indicates that these ligands all label a receptor synonymous

with the cloned 5 receptor. The recent report of a 5-Iike binding site in rat brain which is

insensitive to DOR antisense ODN (Cha et al., 1995) is consistent with the above predic­

tion. It will be of interest to determine the effect of COR antisense ODN on this novel 5

binding site.

In summary, the current study further characterizes the actions of antisense ODNs

targeting the cloned 5, p. and K opioid receptors. These data continue to support the hypoth­

esis which suggest the existence of subtypes of opioid 5 receptors. The selective actions of

the DOR antisense ODN to inhibit the antinociceptive actions of [D-Ala^, Glu'']deltorphin,

but not DPDPE, following Lc.v. treatment, is consistent with the classification of multiple

supraspinal 8 receptor subtypes mediating the antinociceptive actions of the 5 ligands, and

of the classification of the cloned DOR as a 6, receptor. The continued characterization of

antisense ODN effects on DOR-mediated behaviors is ongoing with the goals of under­

standing the mechanism(s) of antisense action and the neurophysiological role of opioid 5

receptors.

132

V. IN VITRO CHARACTERIZATION OF ANTISENSE EFFECTS

Specific Aims:

The administration of 5 antisense ODNs in vivo appears to be an effective method to

selectively decrease an agonist/receptor-mediated event There are, however, additional ques­

tions regarding the specificity and mechanisms of the observed effect. Specifically, is there

a correlation between the behavioral data and a disruption of synthesis of 6 opioid recep­

tors? Chapter 5 addresses some of these issues using:

a. Radioligand binding studies that measure the number of |j., 5 and K opioid receptors

in mouse brain following vehicle, DOR antisense or mismatch ODN treatments in

vivo.

b. Immunofluorescence visualization and quantification of 5 opioid receptors in the

mouse spinal cord following i.th. vehicle, DOR antisense or mismatch ODN admin­

istration.

c. Measuring the accumulation of a labelled Texas-red ODN into cultured NG 108-15

cells and correlating this accumulation with changes in 5 opioid receptor levels

following DOR antisense or mismatch ODN treatment.

133

Introduction

The previous chapter hypothesizes that the Ic.v. administration of antisense ODNs

can selectively reduce the number of targeted receptor proteins that are expressed in vivo.

This "knock-down" of receptors leads to a rightward shift in the corresponding agonist

dose-response curve. The present studies further evaluated the specificity and selectivity of

the antisense "knock-down" of DOR in cultured NG 108-15 cells and in spinal cords of

DOR antisense treated mice. Parts of these studies were a collaborative effort with Dr.

Robert Elde and his laboratory at the University of Minnesota.

Methods

Antisense ODNs

Three ODNs were used in these studies, (i) A Texas red-conjugated ODN (5'-CTG

TOG CCC CTT GCC GCT GC-3'), complementary to the mismatch control sequence for

the cloned DOR from NG 108-15 cells (see below). The ODN was synthesized by solid

phase procedures, conjugated with Texas-red at the 5' end of the ODN and purified by

reverse phase HPLC (Midland Certified Reagent Co., Midland, TX). This Texas-red ODN

was reconstituted in nuclease free water and stored in the dark at 4°C. (ii) DOR antisense

ODN (5'-GCA CGG GCA GAG GGC ACC AG-3'); complementary to nucleotides 7-26 of

134

the DOR coding region, (iii) Mismatch control ODN (5'-GCA GCG OCA AGO GGC CAC

AG-3'). All three ODN sequences were screened through the Gen-Bank database to ensure

that these sequences were not likely to cross-react with other gene sequences found in NG

108-15 cells.

In vivo administration of ODNs

I.c.v. injections were performed as detailed in Chapter 4. Additional mice that had

received vehicle, 5 antisense or mismatch ODN for up to 6 days (and others allowed to

recover for up to an additional 3 days) were used for the radioligand binding studies. These

opioid naive mice were sacrificed 12 hr after the last ODN injection (or at 24,48 and 72 hr

in the case of the recovery groups). I.th. injections of ODN were made by direct lumbar

puncture into the subarachnoid space between L5 and L6 in unanesthetized male ICR mice

(20-30 g) using a Hamilton microliter syringe fitted with a 30 gauge needle. ODNs (12.5

|ig/injection) were injected in a volume of 5 |il ( see Bilsky et al., 1994). Injections were

made twice daily for three day. All studies involving animals were carried out in accordance

with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by

the National Institutes of Health.

135

Radioligand binding studies

In binding studies, mice were sacrificed by cervical dislocation and the brains (in­

cluding cerebella) were rapidly removed and placed in 20 volumes of ice cold 50mM:5 mM

Tris:MgC12 buffer (pH 7.4). The tissues were homogenized using a polytron set at the low­

est setting and then centrifuged at 48,000 x g for 20 min. The pellet was resuspended in 20

volumes of fresh buffer and incubated at 25°C for 30 min to dissociate and remove endog­

enous opioid peptides. Following this incubation period, membranes were centrifuged and

resuspended in fresh buffer twice more as above. All assays were carried out in the Tris/

MgClj buffer supplemented with 0.5 mg/ml bovine serum albumin and 100 |iM

phenylmethylsulfonylfluoride. In saturation smdies, twelve concentrations of pH]DAMGO

(55.0 Ci/mmol), pH]NTI (30.0 Ci/mmol) or pH]U69,593 (47.0 Ci/mmol) (New England

Nuclear, Cambridge, MA) were used to label |i, 5 or k receptors, respectively. All experi­

ments were performed in duplicate. Nonspecific binding of the radioligands was deter­

mined in the presence of 10 |iM naloxone. Incubation times for all three radioligands was 5

hr. This time has previously been determined to be sufficient to allow the binding reactions

to reach equilibrium. Incubations were terminated by rapid filtration through Whatman GF/

B filter strips previously soaked in 0.1% polyethylenimine for at least 1 hr. The filtered

membranes were washed three times with 4 ml of ice-cold 0.9% saline and radioactivity

136

measured using a liquid scintillation counter with >42% efficiency. Radioligand binding

data were analyzed using a microcomputer and InPIot4 graphing software, a curve-fitting

package that utilizes nonlinear least-squares regression analysis based on models for 1 or 2

independent binding sites. In all experiments, saturation curves were best fit by a 1 site

curve as determined by the F-ratio test. Summary data are presented as mean and

values (± S.E.M.) and represent at least three separate experimental determinations.

NG 108-15 cell procedures

NG 108-15 cells were cultured in 5% fetal calf seruni/5% newborn calf serum/45%

Ham's F-12/45% Dulbecco's modified Eagle's medium/lOO U ml"' penicillin/100 jig ml"'

streptomycin. For experiments, the cells were initially seeded onto cover slips at 100,000

cells/50mm^ Petri dish in normal media 24 hr prior to treatment. On the day of the experi­

ments, the cells were washed twice with serum free medium and treated with antisense or

mismatch ODN (in serum-free medium) at a final concentration of 5 |iM for a total of 4

days in an attempt to mimic the time course in vivo (Bilsky et al., 1994), during which the

ODN-containing medium was refreshed daily. Initial experiments showed that NG 108-15

cells remained viable in the absence of serum over a period of several days if they were

initially established in relatively high density (50% confluency) in serum-containing me­

137

dium. These culturing conditions were designed to circumvent the lability of ODN in serum

incubation (Akhtar et al., 1991). Cells cultured under these conditions did not actively di­

vide; overnight loading of the cells with eosin-dextran showed that the cells were metaboli-

cally viable and expressed a substantial amount of DOR-ir (Lai et al., 1996). On Day 5, the

cells were rinsed briefly and incubated for 24 hr with 5 of Texas-red ODN (in a total

volume of 40 fil with the cover slip inverted). At the end of this incubation, the cells were

processed for immunohistochemical analysis.

Immunostaining and imaging ofNG 108-15 cells and mouse spinal cord

For immunostaining of NG 108-15 cells, the media were aspirated and the cells washed

twice with serum-free medium. Immunolabeling was carried out as described previously

(Lynch et al., 1991) using an antiserum raised against a fusion protein containing the C-

terminal 35 amino acid peptide from the DOR (Lai et al., 1996). For spinal cord studies,

mice were perfusion fixed 12 hr after their last injection as previously described (Arvidsson

et al., 1995a). The spinal cords were dissected from animals, cut at a 14 jim thickness using

a cryostat, and mounted onto gel coated slides for immunohistochemical analysis.

Immunostaining of mouse spinal cord sections was carried out as previously described

using an antiserum raised against peptide corresponding to amino acids 3-n of DOR (see

138

Arvidsson et al., 1995a; 1:1000) and secondary antisera (donkey anti-rabbit) conjugated to

cyanine 3.18 (Jackson ImmunoResearch).

Confocal laser microscopy was performed using aBioRad 1000 confocal microscope

equipped with a krypton/argon laser as described previously (Arvidsson et al., 1995b). For

NG 108-15 cells, confocal images were captured with a Leica TCS-4D laser scanning con­

focal microscope and SCANWARE software. Laser optics for fluorescein included excita­

tion centered at 488nm and a 30 nm band pass emission filter centered at 530 nm. For

Texas-red, the excitation was centered at 568nm and emission was captured with a 590 nm

long pass filter. Image analysis was performed using customized software on a Silicon

Graphics IRIS 10/900. Fluorescence intensity of a cell was corrected for background fluo­

rescence. Specific labelling was defined as a level of in-cell fluorescence which was sig-

nificantiy greater than background. For Texas-red ODN loading, because the NG 108-15

cells do not exhibit significant autofiuorescence at the specified wavelength, specific label­

ing was expressed as arbitrary units of fluorescence (AUF) per unit area (}Xm^) above that of

out-of-cell background. For immunofluorescence, nonspecific labeling was expressed as

AUF/|im^ measured from cells stained with a secondary fluorophore-labelled antibody alone.

139

Results

Radioligand binding studies in DOR antisense treated mice

Representative saturation curves with pH]Nn in brain membranes from mice treated

i.c.v. for 3 days (twice daily injections) with DOR-antisense, mismatch ODN or vehicle are

depicted in Figure 5-1 A. Antisense, but not mismatch, ODN treatment produced a signifi­

cant reduction in calculated values without a change in the value (Figure 5-IB).

o Control • 5 mismatch • 5 antisense

0 200 400 600 800 1000 1200

Free radioligand concentration (pM)

0.8

0.2

0.0 0.0 10.0 20.0 30.0 40.0 500

Bound (pM)

Figure 5-1. Saturation curves (A) and Rosenthal plots (B) of whole brain binding with ['HJrmltrindole after J days of vehicle. DOR antisense or mismatch ODN treatment.

140

Table 5-1 presents the calculated mean and values for pH]NTI in mice treated

for 3 days with vehicle, DOR antisense or mismatch ODN. Treatment with DOR antisense

ODN produced no significant changes in calculated or values of P, and K binding

compared to brain tissues from control or mismatch DOR ODN treated mice (Table 5-2).

Table 5-1. Calculated [^H]NTI B^ (fmol/mg protien) and (pM) values in mice treated for 3 days with DOR antisense or mismatch ODN.

Treatment B max

Control 41.3 ± 1.5 63.0 ± 13.0

DOR antisense 33.2" ± 1.3 68.4 ± 11.4

DOR mismatch 40.9 ± 1.2 68.2 ± 11.2

' Significant differance from control, Student's t-test, p <: 0.05.

Table 5-2. Calculated pH]DAMGO and pH]U69,593 B^ (fmol/mg protein) and Kj (nM) values in mice treated for 3 days with DOR antisense or mismatch ODN

Treatment [^H]DAMGO

B max

['H]DAMGO K,

['H]U69,593 B max

[•'H]U69,593

Control 39.0 ± 2.0 1.5710.36 8.6 ±0.1 1.63 ±0.26

DOR antisense 40.3 ± 1.2 1.46 + 0.38 7.8 ± 0.5 1.57 ±0.25

DOR mismatch 41.7 ±4.7 1.26 ±0.27 8.7 ± 0.3 1.58 ±0.21

141

Saturation studies with pH]naltrindoIe in brain tissue obtained from mice treated for

up to 6 days (and also animals allowed up to 3 days of recovery) with DOR antisense, or

DOR mismatch, ODNs were also conducted. Calculated B values for aniisense-treated max

groups (expressed as % of respective mismatch controls) are shown in Figure 5-2. The

values for the mismatch ODN treated groups were not significantly different than vehicle

controls, while that for antisense ODN treated groups was significantly reduced by day 3 of

treatment and sustained across days 4-6. Return of 6 opioid receptor density to mismatch

levels was nearly complete by the second day of recovery (Figure 5-2).

Q 6 mismatch B 5 antisense

x

D1 D2 D3 D4 D5 D6 R1 R2 R3

Days of 6-antisense/6-inismatch treatment (D) and recovery (R)

Figure 5-2. Calculated values in mice treated with DOR antisense ODN for up to 6 days (DI-D6). The data are expressed as the respective mean % of DOR mismatch control values. Recovery of DOR number was followed in mice that had received 6 days of DOR antisense ODN treatment for 1-3 days (RI-R3). Each bar represents the mean of at lest three separate experiments.

142

NG 108-15 cell studies

NG 108-15 cells incubated with the Texas-red ODN exhibited variable degrees of

accumulation of the tagged ODN within a population. The uptake characteristics of the

Texas-red ODN were similar in cells which had been incubated with the tagged ODN only

(data not shown), or which had been preincubated with antisense or mismatch ODN (non-

tagged) for up to 4 days prior to loading with the tagged ODN (Figure 5-3), indicating that

the cells were not adversely affected by prolonged exposure to these ODNs. Fluorescence

of the ODN was found both in the cytoplasm and the nucleus. Figure 5-3A points out a cell

which exhibited a robust accumulation of Texas-red (red immunofluorescence). This cell

had a lower level of DOR antibody staining (green immunofluorescence) when compared

with surrounding cells which accumulated much less ODN (Figure 5-3B). In contrast, in

cells that had been pretreated with the mismatch ODN control, significant amounts of DOR

immunostaining could be seen despite high levels of accumulated Texas-red ODN (Figure

5-4). Because we could not determine directly the stoichiometric ratio of FTTC-conjugated

second antibody to DOR molecules, we were not able to correlate directly the density of

DOR to that of tagged ODN uptake. Thus, semi-quantitative comparison was made be­

tween ODN accumulation and DOR density by correlating the level of Texas-red and FITC

fluorescence in the same cell. There was a significant inverse correlation between the im-

143

munoreactivity of DOR and the amount of Texas-red ODN accumulated in these cells

(p<0.01; linear regression; correlation coefficient = -0.71. Furthermore, cells which accu­

mulated over 25,000 AUF/nm^ of Texas-red all showed markedly reduced DOR immunore-

activity. In contrast, no correlation was observed in cells which had been pretreated with the

mismatch control ODN prior to labeling with Texas-red ODN (P=0.06; two-tailed t test).

Figure 5-5 is a confocal superimposition of Texas-red ODN-ir and DOR-ir in additional

cells. The inverse correlation between cells that have high levels of Texas-red ODN-ir and

low levels of DOR-ir can be seen. The red fluorescence in the nuclear region is due in part

to autofluorescence in the Texas-red range.

Spinal cord studies

DOR-ir was located primarily in the superficial layers of the dorsal horn of the spinal

cord (Figure 5-6). Following i.th. treatment with DOR antisense ODN, DOR-ir was greatly

reduced in the lumbar spinal cord (Figure 5-6, antisense). No reduction in DOR-ir was seen

after i.th. injections of vehicle or mismatch control ODN. No obvious abnormalities were

present in the superficial laminae as a result of these treatments. Levels of [Leu^]enkephalin-

ir, |i opioid receptor-ir, serotonin (5-HT) receptor-ir and calcitonin gene related peptide

(CGRP)-ir were also similar in DOR antisense or mismatch ODN treated mice (Figure 5-7).

144

Figure 5-3. Texas-red ODN and FITC immunofluorescence of DOR in NG108-15 cells pretreated with antisense ODNfor the DOR. A, Texas-red fluorescence (inAUF/fini?) is 6,250 (upper l^) and 24,500 (lower right); B, FITC immunofluorescence (inAUF/jMi^) is 13,900 (upper l^) and 3,620 (lower right). Magnification: 400X.

Figure 5-4. Texas-red ODN and FFTC immunofluorescence of DOR in NGI08-15 cells pretreated with mismatch control ODN for DOR. A, Texas-red fluorescence (inAUF/fM^) is (clockwise from top right): 17,000: 5,800 and63,000. B, FITC immunofluorescence (in AUF/fM^) is (clockwise from top right): 21,900; 25,300:19.300 and 24,700. Magnification: 400X.

145

Figure 5-5. ConfocaL superimposition offluorescence imaging ofNG 108-15 cells pretreated with DOR antisense ODN showing accumulation of Texas-red ODN (red) arul immunofluorescence of DOR (green). In general, cells that display high levels of Texas-red ODNfluorescence show low levels of DOR fluorescence. In contrast, celb having low levels of Texas-red ODN show higher levels of DOR fluorescence. Note that the redfluorescence in the nuclear region is due in part to autofluorescence in the Texas-red range. Magnification: 100 x

Naive Vehlde

Mismatch Antisense

Figure 5-6. Immunofluorescent confocal imaging ofDOR-ir in the dorsal horn of the mouse lumbar spinal cord from untreated mice (Naive) or mice that had recived i.th. injections of nuclease-free water (Vehicle), mismatch control ODN (Mismatch), or DOR antisense ODN (Antisense). Note the marked reduction in DOR-ir in the superficial dorsal horn after antisense treatment that was not evident after i.th. injections of nuclease free water or mismatch control ODN. Scale = 200 fJm.

146

Mismatch

Figure 5-7. Immunofluorescent confocal imaging of (A) leucine enkephalin invnuno reactivity (L-Enk). (B) opioid receptor immunoreactivity (mu-ir), (D) serotonin immunoreactivity (5-HT), and (D) CGRP immunoreactivity in the dorsal horn of the mouse lumbar spinal cordfrom mice that had recived i.th. injections of mismatch control ODN (Mismatch), or DOR antisense ODN (Antisense). There were no significant differences between the two treatment groups in any of the measured parameters. Scale = 200 jj/n.

147

Discussion

The putative mechanism of action of antisense ODNs is an inhibition of the synthesis

of the targeted protein, thereby blocking the function of that protein by reducing its abun­

dance in the tissue. Thus, the most direct means of evaluating antisense-mediated "knock­

down" of the target protein is by measuring the changes in the level of the target protein

after antisense treatment. Our data support this mode of action of DOR antisense ODN by

demonstrating a significant reduction in (a) supraspinal mouse 5 receptors labelled with

['H]NTI; (b) immunoreactivity of the DOR in mouse spinal cord tissue and (c) immunore-

activity of the DOR in NG 108-15 cells after DOR-antisense, but not mismatch, ODN treat­

ment.

The specificity of the DOR antisense ODN treatment in vivo is further supported by

determination of the density of 5 receptors in mouse whole brain. Saturation studies using

pH]NTI indicated a significant decrease in 5 receptor density, without change in NTI affin­

ity, following 3 days of DOR antisense, but not mismatch, ODN. Treatment for an addi­

tional 1-3 days with DOR antisense, but not mismatch, ODN produced a further reduction

in receptor number showing an approximately 35% change compared to controls. Similar

results have also been reported in the rat where 3 days of treatment with DOR antisense

decreased 6 receptors labeled by [^H][D-Ala-, D-Leu^]enkephalin by about 50% (Chaet al..

148

1995). Studies targeting different receptor populations, including dopamine and GAB A

receptors, have also provided support for the specificity of the antisense technique and its

effects on receptor populations (Karle and Nielsen, 1995; Zhou et al., 1994).

The changes in antinociceptive response appear to correlate well with the reduction in

6 receptor density and, further, this reduction in 5 receptor density was reversible with a

similar time course to return of antinociceptive fiinction. While it could be speculated that

the decrease in antinociceptive response was most likely due to a decrease in receptor num­

ber without change in agonist affinity, it should be emphasized that these binding studies

were done with [^H]NTI, an antagonist, and that changes in agonist affinity may also be

possible. Nevertheless, these findings provide a molecular basis for the observed DOR

antisense ODN mediated inhibition of [D-Ala-, Glu'*]deltorphin antinociception.

Interestingly, there was a differential uptake of Texas-red ODN into NG 108-15 cells.

The density of DOR was reduced only in cells which had accumulated a significant amount

of tagged ODN, suggesting further that the effect of the antisense ODN depends critically

on the intracellular localization of the ODN. The DOR immunostaining observed in a popu­

lation of DOR antisense ODN-treated cells was 26% that of mismatch control ODN-treated

cells. The reduction in the level of the DOR in the NG 108-15 cells is consistent with

previous findings that DOR antisense ODN treatment resulted in a reduction in the binding

149

sites for the 5 receptor-selective ligand pH][D-Pen^, D-Pen^]enkephalin in NG 108-15 cells

(Standifer et al., 1994) and [^H]naltrindole in mouse brain (Chapter 4 and Bilsky et al.,

1996a).

Mismatch ODN treatment had no effect on the DOR-ir in either NG 108-15 cells or the

mouse spinal cord. This argues against the possibility of nonspecific effects of the ODN

treatment. Furthermore, the dosage of ODN used did not compromise cell viability in vitro

or precipitate behavioral toxicity in vivo. Furthermore, i.th. treatment with DOR antisense

ODN did not produce any changes in the levels of a number of endogenous peptides or in

the level of |i opioid or serotonin 5-HT receptors in the mouse spinal cord. Together, these

data indicate that the DOR antisense ODN targets selectively the DOR in the NG 108-15

cells and at least one subtype of 5 receptors in the mouse brain and spinal cord. Importantly,

the selectivity of this effect is based on the sequence of the ODN.

150

VI. NMDA RECEPTOR ANTAGONISTS AND OPIOID TOLERANCE

Specific Aims:

Chapters 6 and 7 address mechanisms of opioid tolerance and physical dependence.

These poorly understood phenomenon frequently occur following repeated opioid agonist

administration and may complicate the therapeutic use of these compounds. A number of

interrelated mechanisms have been proposed that may regulate the development and ex­

pression of opioid tolerance. Chapter 6 addresses the involvement of NMDA receptors in

the development of antinociceptive tolerance to morphine and more selective p, and 5 opioid

agonists. Specifically, the aims of the studies were to:

a. Determine if systemic administration of NMDA receptor antagonists can inhibit the

development of antinociceptive tolerance to morphine administered (1) systemi-

cally and (2) centrally (e.g., Lc.v.).

b. Determine if systemic administration of NMDA receptor antagonists can inhibit the

development of antinociceptive tolerance to centrally administered selective |i and

8 opioid agonists

c. Using a paradigm that produces 6-mediated antinociception through activation of

endogenous opioid peptides, assess the activity of an NMDA antagonist on the de­

velopment of antinociceptive tolerance.

151

Introduction

Repeated administration of opioids can lead to the development of tolerance in both

laboratory animals and humans (Wei et al., 1969; Foley et al., 1991). Tolerance limits the

analgesic actions of these compounds and can complicate the management of patients with

persistent pain (Inturrisi, 1990; Foley, 1991). There is evidence, however, that the analgesic

actions of opioids, and the development of tolerance to this effect, are mediated through

separable physiological mechanisms. For example, pretreatment with the noncompetitive

NMDA antagonist MK801, or the competitive NMDA antagonist LY235959, can block

development of antinociceptive tolerance to morphine without affecting its acute

antinociceptive actions (Trujillo and Akil, 1991; Marek et al., 1991; Tiseo and Inturrisi,

1993). It is, therefore, a research goal to further understand the mechanisms responsible for

opioid tolerance and to develop therapeutic agents which minimize this side-effect.

Morphine is commonly used in the laboratory as the prototypic opioid ^ agonist

based on its prominence in clinical pharmacotherapy and its relatively good affinity and

reasonable selectivity for opioid |i receptors in laboratory animals (Chang et al., 1983;

Emmerson et al., 1994; Horan and Ho, 1989; Mignat etal., 1995). Morphine, however, also

has moderate affinity for 5 and K receptors (Chang et al., 1983; Emmerson et al., 1994;

Horan and Ho, 1989; Mignat et al., 1995). It is, therefore, unclear if the actions of NMDA

152

antagonists to block the development of antinociceptive tolerance to morphine is compli­

cated by potential activity of this agonist at non-n opioid receptors. Additionally, it is

unclear whether such anti-tolerance activity of NMDA antagonists applies to agonists at

other opioid receptor types (e.g., 6 and K) although the latter issue has begun to be ad­

dressed in recent investigations of NMDA antagonists and opioid K agonists (Elliott et al.,

1994; Bhargava andThorat, 1994). For these reasons, the present study has investigated the

effects of such NMDA antagonists on the development of antinociceptive tolerance to: (a)

selective opioid p. receptor agonists; (b) a selective 5 receptor agonist; and (c) 5-receptor

mediated antinociception elicited by activation of endogenous opioids.

The experimental design included an assessment of the effects of MK801 and

LY235959, noncompetitive and competitive NMDA antagonists, respectively, on the de­

velopment of antinociceptive tolerance to more selective and 5 opioid agonists with chemi­

cal structures which differ from that of morphine; the (i selective agonists DAMGO (Handa

etal., 1981; Mattiaetal., 1991b), PLO17 (Chang etal., 1983) and fentanyl (Maguire et al.,

1992), and the 5 selective agonist [D-Ala^ Glu'*]deltorphin (Jiang et al., 1990b; Mattia et

al., 1991b) were used in these studies together with a paradigm known to produce antinoci­

ception through endogenous opioids acting at 6 receptors (i.e., swim-stress).

153

Methods

Animal subjects

Male, ICR mice (20-30 g, Harlan Industries) were housed in groups of 5 in Plexiglas

chambers with food and water available ad libitum. Mice were maintained on a 12 hr light/

dark cycle in a temperature controlled animal colony throughout the experimental regimen.

Studies were carried out in accordance with the Guide for the Care and Use of Laboratory

Animals as adopted and promulgated by the National Institutes of Health.

Drugs and injections

Morphine sulfate, DAMGO and PL017 (all obtained from the NIDA) and fentany!

(Sigma Chemical Co.) were dissolved in distilled water (i.c.v. injections) or physiological

saline (systemic injections). The 6-selective agonist [D-Ala^ Glu'*]deltorphin was synthe­

sized by standard methods in Dr. "Victor Hmby's laboratory. The compound was dissolved

in 20% DMSO and brought to volume in distilled water. The NMDA antagonists MK801

(Research Biochemicals Inc.) and LY235959 (Lilly Research Laboratories) were dissolved

in physiological saline. Administration of agonists by the i.c.v. route was accomplished by

direct injection into the right lateral ventricle using a 10 |il Hamilton syringe (5 ^il volume)

as previously described (Porreca et al., 1984). Additionally, the use of peptidic agonists

154

requiring direct administration at supraspinal sites allowed a determination of the result of

NMDA blockade on antinociception resulting firom activation of opioid receptors at su­

praspinal sites. Lp. and s.c. injections were given using a 1 ml syringe with a 30 gauge

needle in a 10 ml/kg volume.

Administration of NMDA antagonists

The NMDA antagonists MK801 or LY235959 were first tested for acute antinoci­

ceptive actions in the 55°C tail-flick test. In order to insure that mild antinociceptive re­

sponses of the NMDA antagonists alone did not occur, these compounds were also evalu­

ated using 50°C warm-water as the nociceptive stimulus. To assess whether NMDA antago­

nists would block antinociceptive tolerance to selective |J. or 5 agonists, separate groups of

mice were pretreated 30 min prior to the agonist administration with either i.p. saline, MK801

(0.1 mg/kg) or LY235959 (3 mg/kg); these doses were chosen on the basis previous studies

(Trujillo and Akil, 1991; Marek et al., 1991; Tiseo and Inturrisi, 1993). The antagonists

were also tested for their ability to modify the acute antinociceptive actions of selective n or

5 agonists by pretreating (-30 min) naive mice with Lp. saline, MK801 (0.1 mg/kg) or

LY235959 (3 mg/kg). The respective agonists were then administered and the mice tested

for antinociception. Additionally, in order to determine if repeated administration of the

155

compounds might produce changes in behavior, thermal response threshold or activity of

opioid agonists, separate groups of mice received repeated injections of these NMDA an­

tagonists at the same doses and injection schedule for 3 days and the mice were observed

periodically throughout the treatment regimen and tested in the tail-flick assay on day 4.

Tolerance regimen

Tolerance to systemic morphine was induced by twice daily (8 a.m. and 5 p.m.) s.c.

injections of increasing doses of morphine for 3 days (10, 30,30, 100, 100 and 100 mg/kg,

respectively). A modified schedule of 5 s.c. injections of fentanyl per day (7 and 10 a.m. and

1,4 and 7 p.m.) for 3 days was used to induce fentanyl tolerance. Doses administered were

0.02 mg/kg (injections 1 and 2), 0.04 mg/kg (injections 3 and 4) and 0.06 mg/kg (injections

5-15). For supraspinal studies, mice were treated with approximate A^ doses of morphine

(10 nmol), fentanyl (1 nmol), DAMGO (0.2 nmol), PL017 (0.2 nmol) or [D-Ala-,

Glu'']deltorphin (20 nmol), twice daily (8 a.m. and 5 p.m.) for 3 days. Separate groups of

mice received the vehicle used for [D-Ala^, Glu"*]deltorphin and were evaluated for possible

antinociceptive or behavioral effects both after the first injection and after the same number

of injections used in the tolerance paradigm.

156

Cold-water swim-stress paradigm

Antinociceptive actions of endogenous opioids were elicited using a swim-stress

paradigm as previously described (Vanderah et al., 1992). Mice were immersed in 5°C

water in a Plexiglas cylinder (17 cm in diameter and 23 cm deep) for 3 min. Upon extraction

from the cylinder they were patted dry with a paper towel and placed in a holding cage lined

with paper towels. For determination of antinociceptive response to a single swim-stress

exposure (i.e., control antinociceptive response), the mice were tested in the tail-flick test

10 min after the swim session; this time has previously been shown to represent the peak

antinociceptive effect (Vanderah et al., 1992). To assess the development of tolerance to

this effect (previously described, Vanderah et al., 1992), and the action of MK801, this

paradigm was repeated twice daily (8 a.m. and 5 p.m.) for three days and antinociception

was assessed 10 min following the final swim exposure (i.e., on the morning of the fourth

day). Separate groups of animals were pretreated 30 min before the swim-stress exposures

with MK801 as described above.

Antinociceptive testing and statistical analysis

On the morning of the fourth day following the agonist pretreatment regimen, sepa­

rate groups of mice received graded doses of the same pretreatment agonist (i.e , "toleragen").

157

Antinociception was assessed using the 55°C warm-water tail-flick assay at time of agonist

peak effect (10 min for Lc.v. agonists, 30 min for j.c. agonists). Antinociception was calcu­

lated as described in Chapter 3. Dose-response lines were analyzed by linear regression. In

all cases, groups of 8-10 mice were used at each dose level and mice were tested for

antinociceptive response twice - i.e., once for baseline determination and once after admin­

istration of drugs (on day 4). All Aj^ values and 95% confidence limits shown are calcu­

lated from regression analysis of the linear portion of the dose-response curve as described

by Tallarida and Murray (1986). Shift ratios were calculated by dividing the antinociceptive

Ajp value of the test group by the antinociceptive A^^ value of the control (naive) animals.

For groups of animals subjected to cold-water swim-stress, antinociceptive responses were

compared using a paired Smdent's t-test.

158

Results

Neither NMDA antagonist produced a significant antinociceptive effect when given

alone (Figure 6-1) nor did they produce overt signs of behavioral toxicity at these doses

after either acute or repeated administration. LY235959 produced mild sedation in some

mice; this effect was observed throughout the treatment period.

c _o o. 01 u

"u o c 'S c <

100

80

60

40

20

MK801 (0.1 mg/kg, Ip.);

O 50"C - • 55"C

10 15 20 25 30 35 40 45

u o c c <

100

80

LY235959 (3 mg/kg, i.p.);

O 50"C • 55"C

C a ••= 60 o u

40

20

0<

-20 -j_

10 15 20 25 30

Time (min)

J 35 40 45

Figure 6-1. Time course of antinociception produced by the i.p. administration of either MK801 (0.1 mglkg, top panel) or LY235959 (3 mglkg, bottom panel) in the 50°C or 55°C warm-water tail-flick test. Antinociception is expressed as mean % antinociception (±S.E.M.) of 8-10 mice per group.

159

Pretreatment with either NMDA antagonists did not affect the acute antinociceptive

effects of any of the appropriate tested agonists (Tables 6-1 and 6-2). These compounds also

failed to elicit any antinociceptive actions when tested after repeated administration on the

fourth day.

Tablet. Effect of i-p. MK801 pretreatment on the development of xnhnodceptive tolerance to morphine and |i and 5 selective ai^onists.

Shifts represent chanses in A« vahies from dose-response curves.

Agonist

(Route)

Control Am

(95% CD

ControI/MKBOl A«

(95%CL)

Tolerance A«

(95% CD

Tolerance/MKSOt Am

(9S%CU

Cantrol/MK801

Shift

Tolentf^ce

Shrft

MK801 /Tolenmce

Shift

Morphine

(sc.)

8.9mg/kK

(6.2-119 mit/lcR)

93 mg/kg

(6.1-13.6 me/ke) (I2.4-36.«nie/k*)

103 mg/kg

(6.9-U4mR/kK)

1.1 3.1 1.2

Morphine

(i.c.v.)

4.1 runol

(2.7-6J (unol)

27 nmol

(1.4'5.2 runol)

21.2 runol

(17.1.26.4 nmol)

4.8 nmol

(16-8.8 nmol)

0.7 51 12

Fentiinyl

(5.C.)

0.2 mg/kR

(0 14^.25 mi5/kE)

0.2 mg/kg

(0.12-0.22 mK/ke)

0J7 mg/kg

(0.29^39 mK/kK)

038 mg/kg

(0.2-0.4 mg/kg)

1.0 18 1 9

Fentanyl

(i.cv 1

0.5 runol

(0.3-0.7 tunoH

0.23 nmol

<0.12-0.44 nmol)

19 runol

(t.M.9nmcU

3.7 nmol

a7-5.l nmol)

05 6.4 80

DAMGO

(lev)

0.03 nmol

(0.02-0.04 nmol)

0.05 nnwl

(0.04-0.09 nmol)

0.6 runol

(03-1.1 nnwiO

0.9 runol

(05-1.6 lunol)

1.7 200 307

PL017

(tc.v)

O.I runol

(0 07-0.2 runol)

0.11 nmol

(0.07-0.17 runol)

O.S5ftnu>l

(0.4-1.6 runol)

0.9 runol

(03-15 runol)

1.1 71 76

Dettorphm 11

(i c V )

65 nmol

(4.2-10.1 lunol)

6.9 nmol

(4.ft^.8 runol)

31.4 nmol

(177-56 nmol)

375 runol

(225-617 nmol)

1.1 4.8 5.8

TrfbleZ Effect of ip LY23S959 (LY) pretreatment on the development of aruirwciceptJve tolerartce to morphine aiul |i and 5 selective tiKcnists.

Shifts represent chances in A«. values from dose-response curves.

Agonist

iRoute)

Control A <m

(^SXCL)

Control/LY A«

(95%CL)

TotenuKeA*

(95% CU)

Tolenmce/LY A«

(95%C.U)

Control/LY

Shift

Tolerance

Shift

LY/TolerarKe

Shift

Morphine

(sc.)

7 8 mg/kg

(5 5-11.2 me/kc)

9 6 runol

(65-10.2 nmol)

273 mg/kg

(214-36.8 mc/kit)

54 mg/kg

(3 64.lmic/ke)

1.2 35 07

Morphtne

(i.c.v)

3 J nmol

(2.4-4.7 runol)

3.1 nmol

(2^.2 nmol)

21.2 nmol

(17.1-26.4 runol)

3.1 nmol

(1.6-5.9 runol)

0.9 6.4 09

DAMGO

(i-c.v.)

0.03 runol

(0.02-0.04 nmol)

0.05 runol

(0.03^.11 nmol)

0.6 ruiwl

(03-1.1 luiral)

03 runol

(0.08-13 nmol)

1.7 200 1 1 0

Femwyl

(i-c.v.)

0 5 nmot

(0J-0-7nmol)

0.4nmc(

(03-0.6 nmol)

19Turu>l

(1.8-4.9 nmol)

11 ruiwl

(16-5.3 nmol)

0.8 5.8 62

Deltorphm II

(i c.v J

14 8 nmol

(10.6-207 nmoO

13.9 lunol

no 9-17.7 nmol)

31.4 nmol

(17.7-56 rvnol)

375 runol

(215^7 nmol)

0.9 11 15

160

The administration of MK801, 30 min prior to each morphine injection (s.c. or :.c.v.)

throughout the 3 day tolerance regimen reliably blocked the development of antinocicep­

tive tolerance to this agonist (Figure 6-2 and Table 6-1).

100 r O Control Chronicmorphine

V Chronic morphine/MK801

C _o

Q, OJ

'G o c

1 10 100

Dose Morphine (mg/kg, s.c.)

100

80

60

O Control

# Chronic morphine

V Chronic morphine/ MK801

S 40 c <

20

10 100

Dose Morphine (ninol, i.c.v.)

Figure 6-2. Dose-response curves for s.c. (top panel) or i.c.v. morphine in control (open circles), morphine-tolerant (closed circles) and MKSQUmorphine-treated (open triangles) mice. Repeated injections of morphine produced antinociceptive tolerance as shown by the significant rightward shift in the dose-response curves and in the calculated value (see Table 6-1). Such rightward shifts in the dose-response curves were prevented by pretreatment with MK801 throughout the toleratice regimen (Table 6-1).

161

Similarly, LY235959 pretreatment blocked the development of antinociceptive toler­

ance to both S.C.. and i.c.u morphine (Figure 6-3 and Table 6-2).

100 O Control

9 Chronic morphine

^ Chronic morphine/ LY235959 C

o

u o c

10 1 100

Dose Morphine (mg/kg, s.c.)

100 O Control

# Chronic morphine

V Chronic morphine/ LY235959 C

o D, 0) U o O c

1 100

Dose Morphine (nmol, i.e.v.)

Figure 6-3. Dose-response curves for s.c. (top panel) or i.c.v. morphine in control (open circles), morphine-tolerant (closed circles) and LY235959/morphine-treated (open triangles) mice. Repeated injections of morphine produced antinociceptive tolerance as shown by the significant rightzvard shift in the dose-response curves and in the calculated value (see Table 6-2). Such rightzvard shifts in the dose-response curves were prevented by pretreatment with LYZ35959 throughout the tolerance regimen (Table 6-2).

162

Repeated Lc.v. administration of fentanyl produced significant antinociceptive toler­

ance (Figure 6-4 and Table 6-1). In contrast to the results seen with Lc.v. morphine, pre-

treatment with MK801 or LY235959 did not prevent the development of antinociceptive

tolerance to Lc.v. fentanyl (Figure 6-4, Tables 6-1 and 6-2). A moderate, but significant,

degree of antinociceptive tolerance was produced by repeated s.c. injections with fentanyl

(Figure 6-4, Table 6-1). Similar to the Lc.v. results, pretreatment with MK801 did not block

the development of tolerance to s.c. fentanyl (Figure 6-4 and Table 6-1).

too

_2 a. «J 60

•§ C 1 « <

20

O Cuntmi

0 Chronic imtanyl V Chronic fentanyl/MKSOI

0.01 0.1 I

Dose Fentanyl (mg/kg, s.c.)

c o

too

80

O Omtrul

9 Chnmic fentanyi

V Chronic fmtanvl/ T MKBOl h

= 40

<

20

O.I

Dose Fentanyl (nmol, i.cv.)

Figure 6-4. Dose-response curves for s.c. (top panel) or i.c.v. (bottom panel) fentanyl in control (open circles), fentanyl-tolerant (closed circles) and MKSOHfentanyl-treated (open triangles) mice. Repeated injections of fentanyl produced tolerance as indexed by the significant rightward shifts in the calculated values (see Table 6-1) which was not prevented by pretreatment ivith MK801.

163

Repeated i.c.v. injections of DAMGO produced significant antinociceptive tolerance

which was not altered by either MK801 or LY235959 pretreatment (Figure 6-5, Tables 6-1

and 6-2). MK801 pretreatment also did not block the development of antinociceptive toler­

ance to PL017 (results summarized in Table 6-1).

r O Control 0 Chronic DAMGO V Chronic DAMGO/

80 - MK801 C 0 •x: cx 01 'G o c c <

0.001 0.01 0.1 1 10

100 O Control 0 Chronic DAMGO V Chronic DAMGO/

LY235959 C o o-OJ y u o c c <

0.001 0.01 0.1 1 10

Dose DAMGO (runol, i.c.v.)

Figure 6-5. Dose-response curves fori.c.v. DAMGO in control (open circles), DAMGO-tolerant (closed circles) and DAMGO/NMDA antagonist-pretreated (open triangles) mice. Repeated i.c.v. injections of DAMGO produced tolerance as indexed by the significant rightward shifts in the calculated A^^ values (see Tables 6-1 and 6-2) which was not prevented by pretreatment with either MK801 (top panel) or LY235959 (bottom panel) throughout the tolerance regimen.

164

The selective 5 opioid agonist [D-Ala^, Glu'*]deltorphin produced a fiill antinocicep­

tive effect following ic.v. adniinistration (Figure 6-6). Repeated i.e.v. injections produced

significant antinociceptive tolerance which was not blocked by pretreatment with MK801

or LY235959 (Figure 6-6, Tables 6-1 and 6-2) 100

c _o a. (U u 'u o c •c c <

80

60

40

20

O Control 9 Chronic Delt II ^ Chronic Dell II/MK801

10 100

100

C o D-<u

'D o c c <

O Control # Chronic Delt n ^ Chronic Delt U/

LY235959

Dose [D-AIa^ GIu'']deItorphin (nmol, i.e.v.)

Figure 6-6. Dose-response curves for i.c.v. [D-Ala\ Glu*]deltorphin (Delt II) in control (open circles), [D-Ala^, Glu*]deltorphin-treated (closed circles) and [D-Ala\ Glu*]deltorphinl NMDA-antagonist pretreated (open triangles) mice. Repeated i.c.v. injections of [D-Ala\ Glti*]deltorphin produced tolerance as indexed by the significant rightward shifts in the calculated [D-Ala^, Glu*Jdeltorphin values (see Tables 6-1 and 6-2) which was not prevented by pretreatment with either MK801 (top panel) or LY235959 (bottom panel).

165

The cold-water swim stress paradigm was used to further assess the effects of the

NMDA antagonists on tolerance to 5 opioid mediated antinociception. Tolerance to the

antinociceptive effects of the swim stress developed across the 3 day procedure as seen in

Figure 6-7. MK801 pretreatment did not block the development of antinociceptive toler­

ance (Figure 6-7).

100 r

Acute Swim Stress

Acute Swim Stress/

MK801

Chronic Swim Stress

Chronic Svnm Stress/

MK801

Treatment Group

Figure 6-7. The antinociceptive effect of acute cold-zuater swim-stress (CWSS) is depicted in naive and MK801-pretreated mice. Repeated CWSS (twice daily for 3 days) produced antinociceptive tolerance which was not blocked by pretreatment with NK801 throughout the tolerance regimen. Antinociception is expressed as mean % antinociception (±S.EM.) of 8-10 mice per group.

166

Discussion

These data confirm and extend previous studies indicating that the development of

antinociceptive tolerance to morphine can be prevented by pretreatment with NMDA an­

tagonists (Trujillo and Akil, 1991; Marek et al., 1991; Tiseo and Inturrisi, 1993). Specifi­

cally, as reported by others, the development of antinociceptive tolerance to systemic mor­

phine is prevented by pretreatment with NMDA antagonists. Additionally, it is clear that

the development of tolerance elicited by repeated i.c.v. injections of morphine is also sensi­

tive to systemic administration of either of these noncompetitive or competitive NMDA

antagonists. The latter finding suggests that the development of antinociceptive tolerance

following repeated supraspinal administration is an appropriate paradigm for investigation

of the effects of NMDA antagonists on more selective opioid agonists which are not ame­

nable to systemic delivery. In this regard, the use of the central route of administration was

necessitated by the peptidic nature of some of these highly selective agonists (i.e., DAMGO,

PL017 and [D-Ala^ Glu'*]deltorphin).

In contrast to the findings with morphine, neither NMDA antagonist blocked the

development of antinociceptive tolerance to either i.c.v. DAMGO or fentanyl, and MK801

did not block the development of tolerance to t.c.v. PL017. Additionally, MK801 did not

block the development of antinociceptive tolerance to s.c. fentanyl. These results indicate

167

that there may be significant mechanistic differences between the development of mor­

phine tolerance and tolerance to more selective opioid |i agonists. The reasons for such

differences are not clear. An obvious difference between morphine and the most selective ji

agonists (DAMGO, PL017) is the alkaloid and peptidic nature of these molecules, respec­

tively, which could have accounted for these results. This issue was addressed by attempt­

ing to block the development of antinociceptive tolerance to fentanyl, a nonpeptidic |j,-

selective agonist. The failure of MK801 to block the development of antinociceptive toler­

ance to either i.c.v. or s.c. fentanyl suggests that the peptidic nature of the agonist was not of

overriding importance in whether the effect was observed.

The mechanisms by which morphine tolerance occurs and the blockade of this phe­

nomenon by NMDA antagonists are not well understood. Mechanistically, |i. opioid recep­

tor activation can increase NMDA-gated currents in trigeminal neurons (Chen and

Huang, 1991) resulting in an elevation of intracellular calcium. These increases in intracel­

lular calcium have been observed in brain synaptosomes of morphine tolerant mice (Welch

and Olson, 1991). By blocking the NMDA receptor-mediated calcium influx and the asso­

ciated rise in intracellular calcium, NMDA antagonists may reduce Ca^*-mediated intracel­

lular changes which are critical to morphine tolerance. Two such changes, cellular redistri­

bution and activation of Ca-*-sensitive PKC and nitric oxide (NO) by Ca-'^-calmodulin de­

168

pendent NO synthase, have been suggested to be important because of their known role in

neuronal plasticity (Mao et al., 1995a). Morphine tolerance, for example, is associated with

an increase in membrane bound PKC levels (Mayer et al., 1995). In addition, activation of

|X receptor kinases, possibly sensitive to Ca^*, may be involved in antinociceptive tolerance

to morphine (Sadee et al., 1994).

These hypotheses have begun to be tested experimentally by pharmacologically

interfering with kinase activity and NO production. GMl ganglioside, an intracellular in­

hibitor of PKC translocation and activation (Vaccarino et al., 1987; Favaron et al., 1988;

Magal et al., 1990), attenuates the development of antinociceptive tolerance to morphine

(Mayer et al., 1995). Furthermore, MK801 can block the increases in PKC immunoreactiv-

ity in the spinal cord associated with morphine tolerance (Mao et al., 1995b). Other kinase

inhibitors can also prevent tolerance to morphine in vitro (Wang et al., 1994) and in vivo

(Bilsky et al., 1996b). Inhibitors of NO synthase also block the development of morphine

tolerance, but not tolerance to kappa opioid agonists, in vivo (Elliott et al., 1994; Kolesnikov

et al., 1993). The connection between lowered NO levels and morphine tolerance remains

to be clarified. However, a major NO signaling cascade involves activation of guanylyl

cyclase, increased cGMP levels, and subsequent phosphorylation and activation of DARPP-

32, a major neuronal regulatory protein (Tsou et al., 1993). The phosphorylated DARPP-32

169

inhibits protein phosphatase I which is known to dephosphorylate neurotransmitter recep­

tors. Whether this signaling cascade regulates p. receptor phosphorylation, and thereby tol­

erance, remains to be tested.

It is possible that the biochemical changes associated with the acute and chronic

actions of morphine may be different from those produced by injections of the p. selective

agonists DAMGO, PL017 or fentanyl and the 5 selective agonist [D-Ala^, Glu'*]deltorphin.

The differences in opioid receptor selectivity of these compounds may also be of impor­

tance in the failure of the NMDA antagonists to block the development of tolerance. Mor­

phine produces the majority of its actions at the opioid |i receptor, but is probably less than

100-fold selective for this receptor (Chang et al., 1983; Emmerson et al., 1994; Horan and

Ho, 1989; Maguire et al., 1992). Repeated administration of morphine at the high doses

associated with tolerance paradigms is likely to elicit interactions between morphine and 5

and K, as well as |I, opioid receptors. It seems possible that the state of tolerance resulting

from the interaction of morphine with multiple opioid receptors may be different from that

seen with agonists which may predominately interact with only the p. or 5 receptor. This

possibility is supported by studies from Takemori and colleagues which have shown that

selective blockade of 5 receptors prevents the development of morphine tolerance but not

acute antinociception (Abdelhamid et al., 1991). Furthermore, Kest et al. (1997) have shown

170

that treatment with an antisense ODN to the delta opioid receptor (DOR-1) inhibits the

development of morphine tolerance and dependence. However, failure of NMDA antago­

nists to block tolerance to both and 6 selective agonists suggest that opioid receptor sub­

type selectivity cannot account for the selective suppression of morphine tolerance.

Additional differences between morphine and the other more selective |j. and 5 ago­

nists used may explain the presented results. Morphine, for example, is likely to be a partial

agonist at the opioid p. receptor, while some of the tested compounds (e.g., DAMGO and

fentanyl) may have higher efficacy (Adams et al., 1990; Kennedy and Henderson, 1991;

Kramer etal., 1993; Miller et al., 1986; Mjanger and Yaksh, 1991). Differences in efficacy

may also account for the ability of NMDA antagonists to block the development of

antinociceptive tolerance. Finally, it should be noted that some of the agonists tested here

may exhibit pharmacokinetic differences compared to morphine and that such factors may

be important in accounting for the observed results. In spite of this concern, it should be

noted that fentanyl was given on a sustained dosing paradigm in order to elicit a significant

development of tolerance, and that this effect was not altered by the NMDA antagonists.

Recently, an alternative hypothesis has emerged capable of resolving the discrepant

effects of NMDA antagonists on tolerance to various opioid agonists. Assuming that ago­

nists with different chemical structures can induce different active receptor conformations.

171

one might expect different downstream events to be associated with each agonist class.

Indeed, while both morphine and DAMGO lowered cAMP levels in HEK 293 cells trans-

fected with the fi, receptor, only DAMGO was capable of internalizing the [L receptor (Arden

et al., 1995). Moreover, whereas morphine enhanced the rate of p, receptor phosphorylation

(Arden et al., 1995), DAMGO decreased receptor phosphorylation (Wang and Sadee,

unpublished observations). The selective effect of NMDA inhibitors on the actions of mor­

phine, but not other p. agonists, may therefore be related to specific and different actions of

these compounds at the receptor. One would therefore predict that NO synthase inhibitors,

if also affecting fi receptor phosphorylation, are similarly selective in reversing tolerance to

morphine but not to the other agonists tested; this hypothesis remains to be tested. The

ability of different agonists to induce different downstream events represents a new para­

digm of receptor control which may serve in the development of opioid analgesics with

modified long-term sequelae.

While the reasons for the observed differences in sensitivity to the NMDA antago­

nists remains to be clarified, these results demonstrate that blockade of opioid tolerance by

NMDA antagonists is not a general phenomenon, and at least with the compounds evalu­

ated to this point, appear selective for tolerance induced by morphine. Potential clinical

applications of NMDA antagonists in reversing tolerance therefore may be limited to mor­

172

phine and its congeners. Fortunately, morphine is the compound with the most clinical

significance and so, this approach may ultimately be of therapeutic significance.

173

VII. ADDITIONAL STUDIES ON OPIOID TOLERANCE AND

PHYSICAL DEPENDENCE

Specific Aims:

Chapter 7 continues investigating the mechanisms of opioid tolerance and also ad­

dresses issues of physical dependence associated with morphine administration. These smdies

represent a collaboration between the laboratories of Dr. Frank Porreca and Dr. Wolgang

Sadee. Dr. Sadee has proposed a novel mechanism that may contribute to opioid tolerance

and physical dependence (see the chapter introduction that follows). The in vivo work pre­

sented here is a test of some of these hypotheses. The specific aims of the studies are out­

lined below.

a. Further characterize an acute model of morphine tolerance and physical dependence

in mice.

b. Using parameters established in specific aim a, test the hypothesis that protein ki­

nase inhibitors may attenuate the expression of acute morphine tolerance and physi­

cal dependence.

c. Using parameters established in specific aim a, test the hypothesis that opioid an­

tagonists with neutral or negative intrinsic activity (see below) may have differen­

tial effects on the expression of acute morphine tolerance and physical dependence.

174

Introduction

The mechanisms underlying opioid tolerance and dependence are poorly under­

stood. Biochemical and molecular changes to opioid receptors, and the second messengers

with which they interact, may play critical roles in mediating these phenomena. Changes in

receptor number and coupling efficiency to their effector systems could lead to tolerance

(Puttfarcken et al., 1988). Opioid dependence, on the other hand, has been associated with

changes in the cyclic AMP (cAMP) system. This system, including protein kinase A (PKA),

is upregulated as a compensatory response to chronic opioid receptor mediated inhibition

of adenylyl cyclase (AC)(see Nestler, 1992; Wang et al., 1994a). Furthermore, this upregu-

lation occurs in areas that are involved in opioid withdrawal, including the locus ceruleus

(Alreja and Aghajanian, 1991).

On the basis of studies in the human neuroblastoma cell line SH-SY5Y, Sadee and

colleagues proposed that opioid agonist stimulation results in a gradual conversion of the fX

opioid receptor into a sensitized or constituti vely active state termed fi* (Wang et al., 1994a).

In this model, the opioid dependent state is characterized by an upregulated cAMP second

messenger system, counterbalanced by the presence of the sensitized or activated |J.* state.

Increasing dependence is seen to parallel increasing conversion to the |i* receptor state.

Paradoxically, increasing constitutive |i receptor activation can also contribute to tolerance.

175

If all p. receptors are converted to n*, leading to a new intracellular equilibrium of the

cAMP signaling system, p. agonists canno longer exert any effect (assuming that the

state is already fully active and thus unresponsive to agonists). Alternatively, in response to

a sensitized/activated p.* state, changes in the downstream signaling cascade may also con­

tribute to tolerance.

The postulated n* receptor state stipulates the existence of antagonists with nega­

tive intrinsic activity (inverse agonists) and neutral antagonists, as shown for other recep­

tors with elevated basal activity, such as the S-HT^^ receptor (Barker et al., 1994). Whereas

negative antagonists should block |i* activity, neutral antagonists do not, but they may

prevent the inverse effect of a negative antagonist. The increasing potency of naloxone to

elicit withdrawal in increasingly morphine dependent animals suggests this antagonist to

have negative intrinsic activity at ^*. Indeed, in SH-SY5Y cells pretreated with morphine,

followed by a thorough washing of the cells to remove any residual agonist, the addition of

naloxone caused a significant increase in cAMP levels, suggesting the presence of fX* and

that naloxone is a negative antagonist (Wang et al., 1994a). Moreover, the p. selective an­

tagonist CTAP failed to affect cAMP levels in morphine pretreated cells but suppressed the

effects of naloxone, therefore, CTAP was classified as a neutral antagonist at fi*. However,

one cannot exclude the possibility that naloxone acts as an agonist at a sensitized |i* state.

176

activation of which results in elevated cAMP levels in morphine dependent tissue. Further­

more, such a naloxone effect could be blocked by the antagonist CTAP. Both hypotheses,

involving distinct intrinsic properties of naloxone and CTAP, are based on a gradual con­

version of |i to a sensitized/activated ji* state during agonist pretreatment. It is unlikely that

the unusual effects of naloxone in dependent tissues are mediated by receptors other than

the |j, opioid receptor since identical observations were made in a cell line transfected with

the |j. opioid receptor gene (Wang et al., 1994b).

The mechanism of conversion of (j. to (i* may involve protein kinases and receptor

phosphorylation. The general kinase inhibitor H7, for example, prevents and reverses the

naloxone induced cAMP increase in morphine pretreated SH-SY5Y cells, whereas the con­

gener H8 did not (Wang et al., 1994a). At the concentrations used (100 ^.M), both H7 and

H8 are expected to inhibit PKAand PKC (Hidaka et al, 1984); therefore, the selective effect

of H7 on formation cannot be accounted for by inhibition of PKA or PKC. Rather, a

class of protein kinases with selectivity for G-protein coupled receptors (GRKs)(Lefkowitz.

1993) may play a role in |i* formation. To demonstrate directly the effect of H7 and H8 on

receptor phosphorylation, Arden et al. (1995) transfected an epitope tagged |j. receptor

construct into HEK 293 cells and measured-^^P-phosphate incorporation into the |i receptor.

Whereas treatment of cells with 100 |iM H8 had no effect, 100 |iM H7 strongly suppressed

177

receptor phosphorylation (Wang et al., 1994b, and unpublished results), indicating that

H7 does act as a n receptor icinase inhibitor.

These observations led to several predictions on the in vivo effects of naloxone,

CTAP, H7 and H8, assuming that p.* represents a key element in narcotic tolerance and

dependence. First, the proposed neutral antagonist CTAP should elicit less withdrawal and

inhibit naloxone induced withdrawal in morphine dependent animals. Second, H7, but not

H8, should rapidly reverse morphine tolerance and dependence in morphine treated mice,

by blocking the receptor kinase(s) and allowing phosphatases to revert p.* to the ji ground

state.

In a preliminary series of experiments, these predicted results were observed in

mice pretreated with a single large dose of morphine (Wang et al., 1994a), consistent with

the hypothesis that fi,* plays a role in acute narcotic tolerance and dependence. However,

multiple alternative in vivo effects of the drugs used could confound this interpretation

involving a |i* state. The present studies extend previous findings on the in vivo effects of

naloxone, CTAP, H7 and H8, by further characterizing the effects of these agents on the

expression of morphine tolerance and dependence in mice. This study focuses on acute

tolerance and dependence following a single dose of morphine, because chronic dosing

with the protein kinase inhibitors was limited by toxicity, particularly when given system!-

178

cally. However, a recent study independently demonstrated the ability of H7 to prevent

tolerance to morphine in mice during a 3-day i.c.v. infusion (Narita et al., 1994). Further­

more, it was demonstrated that an interaction between both spinal and supraspinal sites is

required to elicit full naloxone induced withdrawal jumping in mice.

179

Materials and Methods

Animal subjects.

Male ICR mice (20-30 g, Harlan Industries, Cleveland, OH) were maintained on a

12/12 hr light/dark cycle and provided food and water ad libitum prior to the experimental

procedures. All animal experiments were performed under an approved protocol in accor­

dance with institutional guidelines and in accordance with the Guide for the Care and Use

of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Injection techniques.

I.c.v. injections were made into the left lateral ventricle as described by Porreca et

al. (1984). Mice were injected under light ether anesthesia and recovered within several

minutes. I.th. injections were made into the subarachnoid space (L5-L7) in unanesthetized

mice by the methods of Hylden and Wilcox (1980) as modified by Porreca and Burks (1983).

Tests for antinociception.

Antinociception was assessed using the 55°C warm-water tail-flick test. Mice were

firmly held over the water-bath and lowered so that their tails were quickly immersed 3-4

cm into the water. A baseline measurement for latency of a rapid tail withdrawal was taken

180

for each mouse prior to any experimental procedure. Any mouse not responding within 5

sec was excluded from any further testing. To prevent tissue damage, a testing cutoff of 15

sec was used. Percent antinociception was calculated according to the following formula:

% Antinociception = 100 x (postdrug latency-predrug latency)/(15-predrug latency).

Effects of protein kinase inhibitors on morphine antinociception.

The effects of the protein kinase inhibitors were assessed on morphine antinocicep­

tion. Mice were pretreated 30 min prior to morphine injection with either distilled water, H7

(50 nmol, i.c.v.) or H8 (50 nmol, i.c.v.). Morphine (1,3,6 or 10 nmol) was injected i.c.v. and

antinociception assessed 10 min later.

Acute morphine dependence.

Mice were made acutely dependent on morphine by s.c. injection of a 100 mg/kg

dose of morphine sulfate (Yano and Takemori, 1979). Separate groups of mice received an

i.c.v. injection of distilled water, H7 (50 nmol) or H8 (50 nmol) 3.5 hr later and were ob­

served for 15 min for signs of opioid withdrawal (jumping) or general toxicity. Withdrawal

was precipitated at various times after morphine administration by injection of either nalox­

one (0.1 - 10 mg/kg, i.p; 10,30 or 100 nmol i.c.v.-, 30 nmol i.c.v. and i.th. concurrently) or D-

181

Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NHj (CTAP) (0.3 or l.O ntnol either i.e.v. or Lth., or

both as indicated) an opioid p. antagonist (Kramer et al., 1989). Due to the expected poor

penetration of peptide to the central nervous system and based on pilot experiments, CTAP

was coadministered by the i.c.v. and i.th. routes. Controls assessed the effects of coinjected

i.c.v. and Ith. naloxone (10 or 30 nmol) or naloxone methiodide (10 or 30 nmol, an opioid

antagonist which crosses the blood-brain barrier poorly)(Iorio and Frigeni, 1984; Jones and

Holtzman, 1992). Mice were then placed in Plexiglas cylinders and observed for a 15 min

period with the number of vertical jumps recorded. To test the ability of kinase inhibitors to

reverse the dependent state, lightly anesthetized mice were injected 3.5 hrs after morphine

administration with either i.c.v. saline, H7(I0, 30 or 50 nmol) (inhibitor of several kinases

including PKA and PKC) or H8 (50 nmol) (inhibitor of several kinases including PKA and

Table 7-1. Injection schedules for acute morphine tolerance experiments

Pretreatment

(s.c., -5hr)

Kinase Inhibitor

(i.e.v., -60 min)

Test Dose

(s.c., -30 min)

Saline (10 mg/kg) Distilled water (5 |j.l) Morphine (3-25) mg/kg)

Morphine (100 mg/kg) Distilled water (5 pJ) Morphine (10-40) mg/kg)

Morphine (100 mg/kg) H7 (50 nmol) Morphine (3-25) mg/kg)

Morphine (100 mg/kg) H8 (50 nmol) Morphine (10-40) mg/kg)

182

PKC, with moderate preference for PKA)(Hidaka et al., 1984; Nixon et al., 1991; Raya et

al., 1992). Withdrawal was then precipitated with naloxone as described above.

Acute morphine tolerance.

Mice were injected s.c. with a 100 mg/kg dose of morphine sulfate as a pretreat-

ment. Antinociception was assessed every hour for 8 hours in separate groups of animals

using the 55°C warm-water tail-flick test. A second dose of morphine (25 mg/kg , s.c., an

Agg dose in naive animals) was administered to additional groups of pretreated animals 30

min prior to each hourly test, starting with the 2 hr time point. The relative response to the

second morphine dose compared to control mice was used as an index of tolerance. The

effects of protein kinase inhibition on morphine tolerance was assessed by using the injec­

tion schedule presented in Table 7-1. Dose-response curves for morphine were then gener­

ated by an injection of a second s.c. dose of morphine (3-40 mg/kg, -30 min) followed 30

min later by a test for antinociception.

183

Results

Mice were made acutely dependent to morphine (100 mg/kg, j.c.) and tested for

naloxone-precipitated (3 mg/kg, i.p.) withdrawal jumping at various times after morphine

injection. The data indicated that maximal dependence, as measured by jumping, occurred

4 hr after morphine injection (Figure 7-1).

Hours after morphine injection (100 mg/kg, s.c.)

Figure 7-1. Mice were made acutely dependent to morphine (100 mg/kg, s.c.) and tested for naloxone-precipitated (3 mg/kg. i.p.) withdrawal (jumping) at various times after morphine injection. The data indicated that maximal dependence, as measured by jumping, could be elicited 4 hr after morphine injection.

184

The kinase inhibitors H7 and H8 were tested for possible nonspecific actions on gen­

eral behavior as well as for effects on morphine antinociception. Both kinase inhibitors

produced some brief behavioral effects (i.e., tremors) immediately after injection. Neither

kinase inhibitor precipitated jumping in naive or morphine treated mice (data not shown).

Pretreatment (50 nmol at -30 min) with either H7 or H8 had no effect on the antinociceptive

morphine dose-response curve (Figure 7-2).

O Morphine control

0 Morphine/H7 (50 runol, i.c.v.)

80 - V Morphine/H8 (50 nmol, i.c.v.)

100 r

C <

0 1 3

Dose Morphine (nmol, i.c.v.) 10

Figure 7-2. The effects of the kinase inhibitors H7 and H8 were assessed on morphine antinociception in the 55°C tail-flick test. Pretreatment (50 nmol at -30 min) with either H7 or H8 had no effect on the morphine dose-response curve.

185

The effects of the kinase inhibitors H7 and H8 were also assessed on naloxone-pre-

cipitated jumping behavior in mice made acutely dependent to a 100 mg/kg s.c. dose of

morphine (-4 hr). In morphine treated animals, naloxone (0.1-10.0 mg/kg, Lp.) produced

dose-dependent jumping (Figure 7-3, as previously reported, Wang et al., 1994a). H7 (i.e. v.)

pretreatment dose-dependently inhibited jumping without producing behavioral effects at

the time of testing. In contrast, pretreatment with H8 (50 nmol, -30 min, i.c.v.) did not

inhibit jumping behavior (see Wang et al., 1994a).

120 O Naloxone Control ^ w/H7 (50 nmol, i.c.v.)

V w/H7 (30 nmol, i.c.v.) • w/H7 (10 nmol, i.c.v.)

OH 100

80

60

40

20

0 0.1

Dose Naloxone (mg/kg, i.p.)

Figure 7-3. The effects of the kinase inhibitor H7 were assessed in mice made acutely dependent to morphine (100 mg/kg, S.C.. -4 hr). In morphine treated animals, naloxone (0.1-IO.O mg/kg, i.p.) produced dose-dependent jumping. Pretreatment with H7 dose-dependently inhibited jumping without ejecting gross motor behavior

186

The putative neutral p. antagonist CTAP was tested for its ability to elicit jumping in

acutely morphine dependent mice. Because of the presumed inability of this peptide to

cross the blood-brain barrier following systemic administration, site directed injections were

used to precipitate withdrawal. For comparison, single i.c.v. or Uh. injections of graded

doses of naloxone or naloxone methiodide (up to 100 nmol) were administered. Surpris­

ingly, neither antagonist produced robust jumping in morphine-treated (i.e., dependent) mice

(data not shown). In contrast to the lack of effect of the opioid antagonists at either supraspi­

nal or spinal sites alone, the effects of concurrent i.c.v. and Uh. administration of naloxone

or naloxone methiodide produced significant jumping (Figure 7-4). The Uh.H.c.v. coinjection

of CTAP (0.3 or 1.0 nmol) produced significantly less jumping in morphine-dependent

mice (Figure 7-4). These doses of CTAP produced equieffective antagonism of morphine

antinociception compared to 30 nmol of naloxone (data not shown). CTAP was also tested

for its ability to block naloxone-induced jumping in acutely morphine dependent mice.

Naloxone (3 mg/kg, i.p.) elicited jumping that was significantly reduced by either i.c.v. or

i.th. cotreatment with CTAP (0.3 nmol, -10 min) (Figure 7-5).

187

60 r

Naloxone (30 runol/30 nmol)

Naloxone methiodide (10 nmol/10 nmol)

CTAP (03 nmol/0.3 nmol)

CTAP (1 nmol/I nmol)

Figure 7-4. Concurrent i.c.v. and i.th. administration of either naloxone (30 nmol) or naloxone methiodide (ID nmol)precipitatedjumping in morphine (100 mg/kg, s.c., -4hr)pretreated mice. In contrast, the n antagonist CTAP (0.3 or 1.0 nmol, i.c.vA.th.) produced only limited jumping in morphine treated mice.

100

Naloxone (3 mg/kg, i.p.)

Naloxone/CTAP (03 nmol, i.c.v.)

Naloxone/CTAP (03 nmol, i.th.)

Figure 7-5. The jU antagonist CTAP was tested for its ability to block ruiloxone-induced jumping in acutely morphine dependent mice. Naloxone (3 mg/kg, i.p.) elicited jumping that was attenuated by either i.c.v. or i.th. pretreatment with CTAP (0.3 nmol. -10 min).

188

Acute tolerance to a s.c. 100 mg/kg dose of morphine was assessed by testing the

antinociceptive effect of a second dose morphine in the 55°C tail-flick test. Mice re­

ceived the second dose of morphine at various times after the pretreatment dosing. The

time-course suggests that maximal tolerance occurs 4-6 hr after the first dose of morphine

(Figure 7-6).

O Morphine (100 mg/kg, s.c.)/Saline (s.c.)

9 Morphine (100 mg/kg, s.c.)/Morphine (25 mg/kg, s.c.)

0 1 2 3 4 5 6 7 8

Time after first morphine injection (hours)

Figure 7-6. Acute tolerance to a s.c. 100 mg/kg dose of morphine waj assessed by testing the antinociceptive effect of a subsequent A^,dose morphine (25 mg/kg, s.c.) in the 55°C tail-flick test. Mice received the second dose of morphine at various times after the first administration. The time-course suggests that maximal tolerance occurs 4-6 hrs after the first dose of morphine.

189

The effects of kinase inhibitors H7 and H8 were also assessed on acute morphine

tolerance (Figure 7-7 and also Table 7-1 for injection schedules). The calculated Aj^ value

(and 95% C.L.) for s.c. morphine antinociception in naive mice was 8.5 mg/kg (6.3-II.4

mg/kg). Pretreatment with morphine produced a 2.5-fold rightward shift in the morphine

dose-response curve with a calculated value (and 95% C.L.) of 22.9 mg/kg (16.3-32.1

mg/kg). Administration of H7, but not H8, reversed tolerance with calculated values

(and 95% C.L.) of 9.0 mg/kg (5.8-14.1 mg/kg) and 19.3 mg/kg (15.1-27.4 mg/kg), respec­

tively.

100 Q Morphine control

^ Acute tolerance

^ Acute tolerance/

H7 (50 tunol, i.c.v.) C o o, O) u u o c c

<

10 100 1

Dose of Morphine (mg/kg, s.c.)

Figure 7-7. The effects of kinase inhibitors H7 and H8 were assessed on acute morphine tolerance (see Methods section and Table 7-1 for procedures). Pretreatment with morphine (100 mg/kg, s.c., -5 hr) produced a 2.5-fold rightward shift in the control morphine dose-response curve indicating the development of tolerance. The i.c.v. administration ofH7 (50 nmol), but not H8 (50 nmol), blocked the development of tolerance.

190

Discussion

Acute treatment with a large dose of morphine produced tolerance and dependence

within 4-5 hours after the injection. The degree of dependence can be measured by with­

drawal jumping elicited by naloxone (Yano and Takemori, 1977) which was maximal 4 hrs

after the morphine injection. We first tested whether the kinase inhibitors H7 and H8 would

affect naloxone induced withdrawal jumping. If a phosphorylated state played a role in

dependence, and naloxone acts as a negative antagonist at or exerts selective effects on

|j.*, we would expect that H7, but not H8 would suppress withdrawal jumping if injected

shortly before the naloxone dose. Reversal of the p.* state in SH-SY5Y cells was rapid (<20

min), possibly because net receptor dephosphorylation is rapid once phosphorylation is

suppressed by addition of a receptor kinase inhibitor (Wang et al., 1994a). Therefore, the

kinase inhibitors were injected 30 min before the naloxone dose, at a time when much of the

administered morphine was already excreted from the body. Indeed, H7, but not H8, blocked

naloxone induced withdrawal jumping in a dose-dependent fashion. In contrast, treatment

with the same dose of H7 and H8 did not affect i.e. v. morphine antinociception (Figure 7-2)

nor precipitated overt withdrawal behavior in morphine pretreated mice. Suppression of

naloxone induced withdrawal jumping did not appear to be related to behavioral toxicity as

both kinase inhibitors produced only brief effects (<5 min) which were no longer present at

191

the time of withdrawal testing. Furthermore, this mouse model of dependence is resistant to

nonspecific CNS depression (Wei et al., 1969).

Direct measurements of n receptor phosphorylation revealed that pretreatment with

H7, but not H8 (100 ^iM each), suppressed |j. receptor phosphorylation in HEK 293 cells

transfected with the jx opioid receptor gene (Sadee et al. 1994; Wang et al., 1994b; Wang et

al., unpublished results). Since both H7 and H8 are known PKA and PKC inhibitors, these

two protein kinases may not contribute to |i* formation. Rather, H7, but not H8, appears to

inhibit a receptor kinase distinct from PKA and PKC (Wang et al., 1994a). Moreover, the

present data support the hypothesis that H7 suppresses naloxone induced withdrawal jump­

ing by preventing p. receptor phosphorylation in vivo, thereby reverting p.* to p. However,

we cannot exclude the possibility that H7 acted by another, yet unknown mechanism that

was unaffected by H8.

The conversion of p receptors to the p.* state may also account for some aspects of

morphine tolerance. As an agonist would no longer exert any effect on a constitutively

active receptor (although it may further enhance partial constitutive activity), complete tol­

erance to the drug is expected if all p. receptors were converted to p*. Since |i and n* are

expected to exist in an equilibrium, even if only a very slow one, full conversion to |i* is

unlikely to occur. Therefore, tolerance occurring by such a mechanism would be expected

192

to be partial, as seen in SH-S Y5Y neuroblastoma cells (Yu et al., 1988). However, |J.* may

also represent a sensitized receptor state responsive to further agonist stimulation, and tol­

erance may have, at least in part, resulted from a compensatory change in the second mes­

senger cascade. In vivo, maximal antinociceptive tolerance was found to occur 5 hours after

a single large dose of morphine (100 mg/kg). Strikingly, i.c.v. injection of H7 30 min before

the challenge dose of morphine (60 min before testing), completely reversed the tolerance

to morphine, whereas H8 had no effect. These data agree with in vitro results demonstrating

reversal of morphine tolerance in SH-SY5Y cells (Wang et al., 1994a). Tolerance may have

resulted from fewer JJ. receptors remaining sensitive to the agonist because of |i* formation,

or from the upregulated cAMP system, or both. However, we again cannot exclude other

H7 effects not shared by H8.

Site-directed administration of antagonists revealed that both supraspinal and spinal

sites were necessary to elicit withdrawal jumping in the acute morphine-dependence model

in mice. Both naloxone and naloxone methiodide (antagonists with proposed negative in­

trinsic activity) were able to induce a significant degree of jumping following concurrent

i.c.v./i.th. administration. The lower effective dose of naloxone methiodide, than of nalox­

one, may result from its higher polarity restricting diffusion out of the CNS. In contrast,

CTAP (a proposed neutral antagonist) precipitated significantly less jumping in morphine-

193

pretreated mice. These data in vivo are consistent with predictions from the model gener­

ated in vitro that the sensitized or active {i* receptor state plays a role in the dependent state.

CTAP, as a proposed neutral antagonist, would not be expected to block activity, and

therefore, it should elicit less withdrawal than the negative antagonist naloxone. Cotreatment

with CTAP also attenuated the withdrawal jumping elicited by administration of naloxone

to morphine-pretreated mice. These data are again consistent with the view that CTAP may

be acting as a neutral antagonist which binds to |X* but does not affect its activity, while

naloxone has negative intrinsic activity but is prevented from interacting with |x* by CTAP.

Alternatively, naloxone may act as a partial excitatory agonist at a sensitized ji* state, and

CTAP blocks this excitatory action. More direct analysis of receptor activity by measuring

OTP exchange at the relevant G proteins is currently in progress using HEK 293 cells trans-

fected with the jj, receptor gene in order to distinguish between these hypotheses.

Administration of the antagonist CTAP, or the closely related analog CTOP, has

previously been shown to precipitate some signs of withdrawal In morphine-dependent

mice and rats (Gulyaetal., 1988; Maldonadoetal., 1992; Shook etal., 1987). For example,

CTOP was assessed for its ability to precipitate withdrawal in both acute and chronic mor-

phine-dependence models In mice (Gulya et al., 1988). The authors were able to measure

some signs of CTOP precipitated withdrawal Including decreases In body temperature and

194

body weight. However, the extent of maximal effect on loss of body weight was greater

with naloxone than with CTOP. These investigators also did not observe stereotyped jump­

ing following CTOP administration. Similar to CTOP, i.c.v. CTAP produces weight loss in

morphine dependent mice (Shook et al., 1987). There is, however, a greatly reduced occur­

rence of diarrhea and jumping compared to ix.v. or s.c. naloxone. Assessment of i.c.v. CTAP-

precipitated withdrawal in rats implanted with morphine pellets for 72 hrs (Maldonado et

al., 1992) showed that a reduction occurred in the CTAP global withdrawal score compared

to the scores following i.c.v. methylnaloxonium or systemic naloxone. The differences were

not likely due to the interaction of the latter two antagonists with delta or kappa opioid

receptors (see Maldonado et al., 1992). Furthermore, systemic sites of action do not medi­

ate the withdrawal-jumping in mice as naloxone methiodide (up to 100 mg/kg, 5.c.) did not

precipitate this behavior (unpublished observations).

These combined data show that CTAP produces significantly less withdrawal than

naloxone at equipotent doses (as judged by antagonism of morphine antinociception). In

the tolerant-dependent state, part of (i, receptor activity may occur via p.*, and part by re­

sidual morphine activity at |i. This latter component of \i opioid activity is expected to be

antagonized by a neutral antagonist which could account for the observed effects with CTAP.

However, we cannot rule out that the distinct effects of CTAP were caused by other mecha­

195

nisms. For example, CTAP may block excitatory activation of by nziloxone. Moreover,

the different structures of the peptide CTAP and the alkaloid naloxone could result in differ­

ences in tissue distribution and distinct secondary effects at other receptor targets. Never­

theless, negative antagonism of naloxone and neutral antagonism of CTAP can account for

the in vivo data presented here and elsewhere.

The results of the present study are consistent with the hypothesis that sensitization

or constitutive activation of a p. receptor may occur via phosphorylation. Furthermore, this

process may be mediated by an H7-sensitive protein kinase other than PBCA and PKC. Con­

version of ji receptors to the sensitized/constitutively active \i* state can account for some

aspects of acute antinociceptive tolerance and dependence. Differentiation of CTAP and

naloxone as antagonists with distinct intrinsic efficacies is supported by their distinct ef­

fects on withdrawal jumping. However, additional independent evidence will be required to

classify the effects of CTAP and naloxone at the and |i* receptor states. Experiments are

in progress to determine whether this model may also be valid for repeated administration

of morphine and other [J. agonists. Regardless of the precise mechanisms, the unexpected

differences between naloxone and CTAP, and the dramatic reversal of tolerance and depen­

dence by the kinase inhibitor H7, promises new approaches to narcotic addiction.

196

Vra. RECENT STUDIES

Specific Aims:

This section presents preliminary data related to the specific aims of the previous chap­

ters. These experiments use compounds and techniques developed over the course of the

dissertation. The format of this chapter is informal, with brief descriptions of each experi­

mental protocol followed by the preliminary results. The specific aims are provided below.

a. Use the antisense approach to further investigate the pharmacological actions of

morphine and SNC 80.

b. Further investigate the role of intracellular proteins in regulating morphine toler­

ance and physical dependence by using a mutant strain of mice that have the DARPP-

32 protein "knocked out".

197

Further Studies with DOR Antisense

One of the specific aims of Chapters 4 and 5 was to develop the antisense technique as

a pharmacological tool in the study of protein mediated processes. One area of research has

focused on the actions of opioid agonists including SNC 80 and morphine. Based on the

results of Chapters 2 and 3, one might predict that the antinociceptive actions of SNC 80

would be at least partially blocked by DOR antisense treatment. We tested this hypothesis

by administering DOR antisense or mismatch i.c.v. for 3 days using the procedures outlined

in Chapter 4. Treatment with DOR antisense, but not mismatch, ODN produced a partial,

but significant, reduction in the antinociceptive actions of SNC 80 (300 nmol, i.c.v, -10

min)(Figure 8-1). The incomplete blockade of SNC 80 antinociception may be due to the

compounds agonist effects on 5,, as well as opioid receptors (see Chapter 3).

In contrast to SNC 80, the actions of morphine are thought to be mediated primarily

through p. opioid receptors. Pretreatment with a |i opioid antagonist, for example, blocks

many of the agonist effects of morphine, including antinociception and self-administration

behavior. The administration of fJ. antagonists also blocks the development of antinocicep­

tive tolerance and physical dependence to morphine. It was, therefore, not surprising to find

that i.c.v. DOR antisense treatment had no significant effect on the antinociceptive dose-

response curve for s.c. morphine (Figure 8-2).

198

100 r-

c O

Dh a; u u o c:

«4-'

C <

SNC 80 SNC 80/ 5 antisense

SNC 80/ 6 mismatch

Figure 8-1. The i.c.v. administration of DOR antisense, but not mismatch, ODN attenuates the antinociceptive response of an approximate i.c.v. dose of SNC 80.

100

80 C o 4-i

a a; CJ u o

•S 40 c <

60

20

O Control

0 S Antisense ^ 5 Mismatch

10 30

Dose Morphine (mg/kg, s.c.)

Figure 8-2. The i.c.v. administration of DOR antisense ODN had no effect an the antinociceptive actions of s.c. morphine.

199

There is, however, evidence suggesting that morphine also interacts with 5 and K opioid

receptors both in vivo and in vitro (Abdelhamid et al., 1991; Miyamoto et al., 1993; Suzuki

et al., 1994; Takemori and Portoghese, 1987). Takemori and colleagues, for example, have

proposed that 5 opioid receptors are involved in the development of morphine tolerance and

physical dependence in mice (Abdelhamid et al., 1991). Furthermore, they have demon­

strated that the 5^ subtype is critical for this effect (Miyamota et al., 1993). We further tested

this hypothesis by treating mice for 3 days with DOR antisense or mismatch ODN. On the

morning of the fourth day, we assessed the effects of the ODN treatments using the acute

morphine dependence model described in Chapter 7.

The antagonist dose-response curve for naloxone precipitated jumping is depicted in

Figure 8-3. DOR antisense, but not mismatch, ODN treatment significantly decreased nalox­

one precipitated jumping confirming that 5 opioid receptors are critical to the development

of morphine dependence in mice. The results also further support the hypothesis that the

cloned DOR corresponds to the 5 receptor pharmacologically characterized as the 8^ sub-

type.

Our experimental plan was to further confirm our work with 5 opioid receptors and

morphine using the antisense approach. Administration of subantinociceptive effective doses

of 8 agonists can modulate the potency and efficacy of some |i agonists such as morphine.

200

120 Morphine control

Morphine/5 antisense Morphine/5 mismatdi 100

o 0) Si s p c c fC O)

0.1 1 10 Dose of naloxone (mg/kg, i.p.)

Figure 8-3. DOR antisense, but not mismatch, ODN treatment attenuated the development and/or expression of acute morphine dependence in mice.

Thus, in cases of severe nociceptive stimuli, or in situations of reduced maximal effect due

to chronic exposure to an agonist (tolerance), the efficacy of morphine could be enhanced

(see Roihman et al., 1993 for a review). The hypothesis was that DOR antisense treatment

would block the modulatory actions of 5 agonists on morphine antinociception. We were

testing this hypothesis using the central administration of both morphine and 5 agonists in

an effort to separate spinal/supraspinal synergy of morphine antinociception from the mor­

phine/5 receptor synergy. It was, therefore, necessary to assess the effects of i.c.v. DOR

antisense on i.c.v. morphine antinociception.

201

100

80

60

X 3 4 0

20 -

O C o n t r o l 9 5 A n t i s e n s e ^ 5 M i s m a t c h

0 . 3 _i I I I I I I I

1 3 1 0 3 0

Dose Morphine (nmol, i.c.v.)

Figure 8-4. I.e. v. treatment with DOR antisense, but not mismatch, ODN attenuated the antinociceptive actions of i.c.v. morphine.

120 r • Control

B Antisense

H Missense

s 80

Morphine Morphine-6-p- Biphalin Nonnorphine Sufentanil (10 nmol) glucoronide 0.1 nmol (30nincl) (0.9 nmol)

(10 nmol) Treatment

DAMGO (0.2 nmol)

PL017 (0.2 nmol)

Figure 8-5. Antinociceptive responses to approximate A^ doses of several opioid agonists in mice pretreated with vehicle, DOR antisense or mismatch ODN.

202

Surprisingly, Lc.v. administration of DOR antisense ODN shifted the i.c.v. morphine

dose-response curve to the right, indicative of antagonism (Figure 8-4). This effect is not,

however, seen in mice pretreated with i.e.v. 5 selective antagonists (Porreca et al., 1992;

Heyman et al., 1989a,b). It is difficult to explain these discrepancies, though the observa­

tion has been confirmed several more times in our laboratory.

To further investigate this effect, additional mice were treated with i.e.v. DOR anti-

sense or mismatch ODN for 3 days. They were then administered a number of additional

opioid agonists including |X selective compounds (DAMGO, PL 017, sufentanil), compounds

structurally related to morphine (morphine-6-P-glucoronide and normorphine) and a unique

compound (biphalin) thought to interact with 6 and |Li receptors to produce an extremely

potent antinociceptive effect (see Horan et al., 1993). Figure 8-5 depicts the results from

these studies. DOR antisense or mismatch treatment had no effect on the antinociceptive

actions of an i.c.v. doses of normorphine, sufentanil, DAMGO or PL 017. In contrast,

DOR antisense, but not mismatch, significantly reduced the antinociceptive actions of mor­

phine, morphine-6-P-glucoronide and biphalin.

It should be emphasized that the antinociceptive effects of |i agonists are not all modu­

lated by 5 agonists. Morphine is one of only a few compounds that shows this effect. The

antinociceptive effect of DAMGO, for example, is not modulated by 6 agonists. It may be

203

that compounds sensitive to 6 agonist modulation, through some unique supraspinal mecha­

nism (receptor complex?), are sensitive to DOR antisense treatment. Biphalin, for example,

may represent a new class of peptide compounds that activates this complex to produce its

extremely potent antinociceptive effects. Further work, possibly using antisense techniques,

will hopefully clarify these interesting findings.

Studies with DARPP-32 Knockout Mice

Chapter 7 describes ongoing experiments that are investigating the role of intracellular

processes in the development of morphine tolerance and physical dependence. Sadee and

colleagues have proposed that phosphorylation of the ^ opioid receptor produces a consti-

tutively activated state that contributes to the aforementioned processes. A novel dopamine

and cAMP-reguIated phosphoprotein (DARPP) may play an important role in regulating

the intracellular activity of protein phosphatases, and thus receptor phosphorylation. Selec­

tive inhibitors of these intracellular proteins are, however, generally unavailable. The anti-

sense approach is, therefore, an attractive alternative to assessing the role of these proteins

in the actions of opioid agonists such as morphine.

Related to antisense "knock-down" of a protein is the "knock-out" of a particular gene.

This technique, in which a target gene is ablated by homologous recombination, has been

204

used to study a number of systems including the dopamine D, receptor and its involvement

in cocaine-reward (Miner et al., 1995) and opioid m receptors (Matthes et al., 1996). In

collaboration with Wolfgang Sadee and Alan Feinberg, I have conducted some preliminary

studies using mice that have the DARPP-32 protein knocked-out. The hypothesis is that the

DARPP-32 knock-out mice will display less tolerance and physical dependence to mor­

phine compared to control mice.

The knock-out mice were derived from two separate progenitor strains (129 SvEv and

C57 black mice). It was first necessary to compare these new strains with ICR control mice

in models of morphine antinociception and physical dependence. Control 129 SvEv and

C57 black mice showed antinociceptive responses to morphine (10 nmol, i.c.v.) that were

similar those seen in ICR mice (Figure 8-6). There were, however, significant differences in

the baseline latencies for the 129 SvEv strain. These mice had an elevated baseline latency

in the 55°C warm-water tail-flick test (Figure 8-7) and the 55°C hot-plate test (data not

shown). These elevated baseline latencies were partially blocked by naloxone (3 mg/kg,

s.c.) pretreatment (Figure 8-8). To further complicate the experiments, acute (Figure 8-9)

or chronic (Figure 8-10) exposure to morphine produced significantly fewer naloxone pre­

cipitated jumps in the 129 SvEv mice. Despite these differences, we proceeded with a pilot

study using mutant DARPP-32 knockout mice.

205

100

c o

•4-1 80 OH 0) u

'C 60 o 60

.S r;

40

d OS 20 01 20

s

I C R C 5 7 b l a c k 1 2 9 S v E v T a c

Strain of Mice Figure 8-6. Antinociceptive response to an approximate A^ i.c.v. dose of morphine in three strains of mice.

U q; CO

u C a» 4-> CC HJ c as QJ

S

8

7

6

5

4

3

2

1

0 ICR C57 black 129 SvEv Tac

Strain of Mice

Figure 8-7. Baseline tail-flick latencies in the 55°C warm-water tail-flick test in three strains of mice.

206

50 r

U 40 QJ CO

30

c 0)

KS hJ 20

10

• Baseline

• Test

0 Tail-Flick Hot-Plate

Nociceptive Test

Figure 8-8. Tail-flick and hot-plate latencies in 129 SvEv mice before and after an injection of a 3 mg/kg, s.c. dose of naloxone. Naloxone partially blocked the elevated baseline latencies seen in this strain of mice.

Due to the limited numbers of DARPP-32 knockout mice that were available, we were

able to test only 10 mutant and 10 wild-type controls in our assays. Unfortunately, we were

not able to identify which progenitor strain (C57 black versus 129 SvEv) a particular mouse

came from. Our observations with these mice in the acute and chronic morphine depen­

dence assays are summarized in Tables 8-1 and 8-2, respectively.

207 100 r

80 -

60 -

40 -

20 -

O ICR

# C57 Black

V 129/SvEv

Dose Naloxone (mg/kg, i.p.)

Figure 8-9. Naloxone precipitated jumps in three different strains of mice treated with a single 100 mg/kg s.c. dose of morphine. Naloxone was given 4 hr after morphine injection (see Chapter 7 for a detailed description of the procedure).

120 r

ICR 129 SvEv TAC

Strain of Mouse

Figure 8-10. Naloxone precipitated jumps in ICR and 129 SvEv strains of mice treated with a single s. c. 75 mg pellet of morphine for three days. Naloxone (3 mg/kg, i.p.) was given on the morning of the fourth day.

208

Table 8-1. Acute morphine antinociception and dependence (as assayed by naloxone precipitated jumping) in ICR control. Wild-type (129 SvEv and C57 black) and DARPP-32 knockout mice.

ICR controte

Mouse • Bodywelght Base Test*30 min %A-30 Test-3 hr hr # Jumps

36 2.1 15 100 15 100 31

2 37 1.8 15 100 IS 100 9

3 38 2 15 100 15 100 12

4 36 1.5 15 100 15 100 0

5 33 1.6 15 100 15 100 1

6 36 2.3 15 100 15 100 27

7 31 2.6 15 100 15 100 16

B 29 2 15 100 15 100 27

9 35 1.9 15 100 15 100 29

11 36 2 15 100 15 100 0

12 35 2.6 15 100 15 100 12

13 38 2.1 15 100 15 100 29 14 34 1.9 15 100 15 too 42

15 36 1.8 15 100 15 100 36

10 38 2.3 15 100 15 100 23

Mean: 35.2 2.0 15 100 15 100 19.6

SBA 0.86 0.11 0.00 0.00 0.00 0.00 4.50

Wild type

Mouse • Bodywelght Bess Test'30 min %A-30 Test-3 hr %A-3 hr f Jumps

3656 38 4.2 15 100 15 100 8

6009 39 4.3 15 100 15 100 1

5887 52 4.5 15 100 15 100 1

6011 37 2.9 15 100 15 100 6

5892 50 7.2 15 100 15 100 0

6022 38 4.7 15 100 15 100 19

6041 40 5.2 15 too 15 100 0

6020 37 2-7 15 100 15 100 23

6031 33 4.2 15 100 15 100 0

3655 35 2.7 15 100 15 100 17

Mean: 39.9 4.26 15 100 IS 100 7.5

S3A 2.06 0.45 0.00 0.00 0.00 0.00 2.98

0ARP-a2 knoclcoutt

Mouse • Bodvweiqht Base Test*30 min %A-3a Test-3 hr %A-3 hr • Jumps

6012 37 2.1 15 100 IS 100 0 3653 27 2.7 15 100 15 100 0 6010 33 4.9 15 100 IS 100 10

6016 34 4.7 15 too 15 100 6

5866 40 2.8 15 100 15 100 5 6039 37 3.4 15 100 15 100 14

6043 46 3 15 100 15 100 1 1

6028 35 2.8 15 100 15 100 0

36S4 31 3.2 15 100 15 100 33

Mean: 35.6 3.3 15.0 100.0 15.0 100.0 8.8

SBA 1 92 0.33 0.00 0.00 0.00 0.00 3.69

Morphine. 100 mg/kg. s.c., -4 hr Naloxone. 3 mg/kg. i.p-. 0 mtn Jumping oDserved for 15 mtn following naloxone injection Antinoaception tested 30 min and 3 hr alter morphine injection

209

Table 8-2. Chronic morphine dependence (as assayed by naloxone precipitated jumping) in ICR control. Wild-type (129 SvEv and C57 black) and DARPP-32 knockout mice.

ICR controls Mouse # Bodyweiqht # Jumps

1 35 84 2 39 28 3 36 125 4 37 111 5 39 54 6 25 226 7 29 16 8 38 58 9 33 129 10 37 27

Mean: 34.8 85.8 SB4: 1.53 21.42

Wild type Mouse # Bodyweiqht » Jumps

6009 34 96 5887 45 14 6011 30 100 5892 42 85 6022 33 45 6041 35 120 6031 31 42 3655 28 55

Mean; 34.75 69.6 SBA 1.97 12.00

DARP-32 knockouts Mouse # Bodyweiqht # Jumps

3653 24 0 5866 35 35 6039 35 79 6043 37 52 6028 33 20 3654 30 95

Mean: 32.3 46.8 SQA 1.67 12.68

Morphine, 75 mg pellet, s.c., -3 days Naloxone, 10 mg/kg, i.p., 0 min Jumping observed for 15 min following naloxone injection

210

At this time, we can not conclude if the DARPP-32 protein is involved in morphine

tolerance and physical dependence. Baseline latencies were elevated in the Wild-type and

DARPP-32 knock-out mice. This may reflect differences in the C57 black and 129 SvEv

progenitor strains of mice. The antinociceptive response to a large dose of morphine ap­

peared to be similar in all three strains, as one would predict from the species studies de­

tailed earlier. Mice pretreated with an acute dose of morphine (100 mg/kg, s.c., -4hr) dis­

played typical jumping behavior when challenged with a dose of naloxone (3 mg/kg, i.p.).

Both the wild-type and knock-out mice displayed fewer jumps than the ICR controls, again

possibly due to the progenitor strains used. Chronic morphine treatment (75 mg s.c. pellet

for 3 days) produced more robust naloxone-induced jumping. The DARPP-32 knock-out

mice had the fewest number of jumps of the three groups tested. However, it is impossible

to conclude if this was due to the knockout of the DARPP-32 gene or if it was due to strain

differences.

There are, however, a number of important conclusions that can be drawn from the

current studies:

(1) The 129 SvEv strain of mouse looks particularly interesting in terms of its nocice­

ptive and opioid responses. This strain appears to have an elevated nociceptive threshold in

two different tests. This elevated threshold is also opioid-sensitive, as an injection of nalox-

211

one attenuates it. It should be noted that naloxone generally has no effect on baseline laten­

cies in ICR mice. We are pursuing the observations made with this strain of mouse in an

effort to understand this unique opioid profile. Further assessments of the opioid physiol­

ogy in this strain will include gastrointestinal motility studies and additional nociceptive

studies using different models of pain.

(2) These studies demonstrate the importance of appropriate controls in assessing the

effects of any treatment. Proper controls are especially critical in knock-out type experi­

ments. It is important to match the strain of mice with the particular hypothesis that is being

tested. We are currently awaiting additional mice that have been bred from a single progeni­

tor strain (the C57 black strain). It should also be kept in mind that any change in an organ­

isms genome may produce unanticipated effects. These include effects not only on the tar­

get system, but on other systems as well.

212

IX. SYNTHESIS AND CONCLUSIONS

The central hypothesis of this dissertation is that agonists and antagonists acting at 5

opioid receptors will have therapeutic applications in treating acute and chronic pain states

and in the treatment of drug addiction. To advance this hypothesis, several research themes

were investigated, each with a number of specific aims. The results of each theme are sum­

marized below along with future research opportunities.

1. Development of selective, nonpeptidic, 5 opioid agonists (Chapters 2 and 3)

Summary

Previous work with the opioid peptide agonists DPDPE and [D-Ala^ Glu'']deltorphin

have suggested that 5 selective agonists may have therapeutic advantages over currently

available analgesics such as morphine. Peptides generally cross the blood-brain barrier poorly,

thus requiring the development of nonpeptidic, 5 selective, opioid agonists that are active

following systemic administration.

SNC 80 represents a second generation of compounds related to BW 373U86. In vitro

testing (radioligand binding and isolated tissue assays) of SNC 80 indicated that this com­

pound has a selectivity profile for 5 opioid receptors that is comparable to the reference

213

peptide compounds DPDPE and [D-Ala^, Glu^]deltorphin, and is significantly greater than

those of the parent compound BW 373U86. More importantly, SNC 80 has an improved in

vivo profile compared to BW 373U86. SNC 80 produces a full agonist effect in two differ­

ent nociceptive assays following spinal, supraspinal or systemic administration. Further,

the antinociceptive effect is blocked by administration of 5, but not |i, selective opioid

antagonists. Based on these and other studies, SNC 80 represents the first nonpeptidic 5

selective opioid agonist.

Future Studies

Though the pharmacology of SNC 80 looks promising, further development of

nonpeptidic 5 opioid agonists is needed. SNC 80, for example, is not particularly potent and

solubility problems did not allow us to test higher doses of the compound in the tolerance

and p.o. administration studies. Furthermore, though SNC 80 did not appear to produce BW

373U86-like convulsions in our hands, more detailed analysis suggests that there may still

be some seizure-like activity associated with the compound (Dr. Jim Woods, personal com­

munication). The synthesis and pharmacological evaluation of compounds based on the

SNC 80 structure, and other novel structures, is on going. The design of these compounds

will be guided by structure-activity smdies similar to those presented in Chapters 2 and 3.

214

2. Development of antisense techniques for the study of 5 receptors (Chapters 4 and 5)

Summary

The use of antisense ODNs to selectively knock-down targeted proteins is an emerg­

ing tool for the neurosciences. We used the antisense technique to further study the physi­

ological role of 6 receptors in nociception. In vivo treatment with DOR antisense selec­

tively attenuates 5^ mediated antinociception without affecting fi, 8, or K mediated

antinociception. Administration of a mismatch ODN has no effect on any of the opioid

agonists tested, indicating that the selectivity of the 18-ODN antisense sequence is very

high. The in vitro experiments correlated the behavioral effects with a reduction in the

number of 5 receptors present at spinal or supraspinal sites. Collectively, these experiments

reaffirm the utility of the antisense approach and support the hypothesis of 5 receptor sub­

types as distinct gene products.

Future Studies

The antisense techniques developed in this dissertation can be (a) used to further study

functional opioid systems and (b) be applied to other protein systems (see theme 3 below).

In terms of the opioid receptors, DOR antisense could be administered in vivo followed by

autoradiographic localization of 5, and 5, opioid receptors in vitro (Hiller et al., 1996). This

215

study may provide more accurate distribution maps of the 5, receptor than those currently

available with the radioligand pH]DPDPE. This type of approach may also lead to a better

understanding of the function of 6, receptors and to the cloning of a second DOR gene, if in

fact one does exist. The administration of an antisense ODN complementary to a common

region of all three cloned opioid receptors (Chapter 4) may be an important tool in this

regard.

As mentioned in the introduction, 5 opioid receptors are involved in many other pro­

cesses besides antinociception. These include (a) modulation of morphine potency and effi­

cacy (Porreca et al., 1992), (b) the regulation of dopamine release in the nucleus accumbens

(Di Chiara and Imperato, 1988, Spanagel et al., 1990, 1992), (c) the reinforcing effects of

cocaine (Menkens et al., 1992; Reid et al.. 1993), (d) development and expression of behav­

ioral sensitization to cocaine (Heidbreder et al., 1996) and (e) aspects of feeding, drinking

and possibly even olfaction. The antisense approach, with its potentially increased selectiv­

ity over conventional antagonists, can be used to study each of these processes using tech­

niques from cell cultures to whole animals. Some of these processes have begun to be

studied in our laboratory including the modulatory role of 5 receptors in |i mediated

antinociception (Chapter 8) and in the reinforcing effects of cocaine (Menkens et al., un­

published experiments).

216

3. Mechanisms of opioid tolerance and physical dependence (Chapters 6 and 7)

Summary

As mentioned, the mechanisms of opioid tolerance and physical dependence are poorly

understood. Cellular (e.g., NMDA receptors) and intracellular (NO synthase, protein ki­

nases) processes may regulate these states. The administration of NMDA receptor antago­

nists, for example, can block the development and expression of morphine antinociceptive

tolerance. A major problem with these studies has been the use of morphine as a prototypic

|i selective agonist. Morphine, especially at the doses used in these studies, has effects on 6

and K opioid receptors.

The research reported here suggests that the effects of NMDA antagonists on opioid

antinociceptive tolerance may be limited to morphine-like drugs. NMDA antagonists failed

to alter the antinociceptive tolerance that occurs to repeated administration of more selec­

tive |i (DAMGO, PL 017) and 5 ([D-Ala^ Glu^]deltorphin) agonists.

The activation of opioid receptors by morphine can also perturb intracellular proteins

and ion levels (see Chapter 7). Based on this and previous work, Sadee and colleagues have

proposed a working model of how some aspects of opioid tolerance and physical depen­

dence occurs (see Figure 9-1 and Sadee et al., 1994).

217

Kinases

PPase 1

T PKA/PKG

PPase 2B Calcineurin

cGMP

DARP-32

DARP-32

Guanylyl cyclase

L-Arginine

Ca^"^ channels (N, L, NMDA)

Ca -calmodulin

Figure 9-1. A proposed scheme for the regulation of fi receptor activity through phosphorylation and dephosphorylation of the receptor. It is hypothesized that the phosphorylated receptor accounts for some of the observed tolerance and physical dependence that occurs following chronic morphine exposure (see text). Each component of the proposed scheme can, in theory, be tested using receptor antagonists, kinase inhibitors and antisense ODNs.

218

The proposed scheme can be summarized as follows (see Chapter 7 and Sadee et al.,

1994 for additional details). Chronic morphine exposure leads to an increased number of

phosphorylated \l receptors (p.*) which are constitutively active. These |i* receptors, which

inhibit the formation of cAMP, are counterbalanced by upregulated levels of adenylyl cy­

clase (which increases cAMP levels). Any mechanism which inhibits the phosphorylation

of the receptor or enhances the action of protein phosphatases (PPase I) should prevent or

reverse the morphine tolerant and dependent states. The H7 compound, for example, is

hypothesized to inhibit a kinase that converts n to p.*.

Additional observations have suggested possible regulation sites which modulate the

activity of PPase 1. Chronic morphine exposure leads to elevated levels of intracellular

calcium. Blockade of calcium channels by NMDA antagonists prevents the association of

the ion with calmodulin. Nitric oxide (NO) synthase activity is enhanced by the calcium/

calmodulin complex, so there should be a decrease in NO levels. It is predicted that an NO

synthase inhibitor should have the same effect on morphine tolerance as an NMDA antago­

nist (in fact, both treatments inhibit morphine tolerance and dependence). Decreased NO

levels results in a lower activity level for guanylyl cyclase and therefore decreased cGMP

formation.

219

The cGMP molecules regulate the activity of protein kinase A (PKA) or protein kinase

G (PKG). These kinases activate DARP-32 by phosphorylating the protein. It is proposed

that DARP-32 inhibits PPase I. If this inhibition is reduced (by knockout of the DARP-32

gene or by inhibiting the phosphorylation of DARP-32) then PPase 1 activity is increased.

This in turn returns the (X* state back to the ^ state.

Tolerance and dependence studies are ongoing, with the goal being to continue testing

and modifying the above hypotheses. The difficulties with the DARP-32 knockout experi­

ments presented in Chapter 8 will hopefully be resolved. We have also begun to test the

effects of NO synthase inhibitors on opioid tolerance. It is predicted that these inhibitors,

which block morphine tolerance, will not block the antinociceptive tolerance to selective |J.

and 6 agonists.

In summary, this dissertation has advanced our understanding of 5 opioid pharmacol­

ogy and supports the hypothesis that 8 opioid agonists and antagonists will be clinically

effective as analgesics and as treatments for drug addiction. The major findings of this

dissertation include (1) the development of a 5 selective, nonpeptidic opioid agonist; (2) the

characterization and use of antisense ODNs as selective pharmacological tools in the study

of 5 opioid receptors and (3) advances in our understanding of the mechanisms of opioid

tolerance and physical dependence.

220

APPENDK A. ABBREVIATIONS AND DEFINITIONS

An extensive list of compounds and terms are used throughout the text. In

an effort to provide clarity and save space, many of these compounds and terms

are abbreviated. The following is a list of abbreviations used throughout the text

and a brief description of each.

|3-CNA, P-chlomaltrexamine (irreversible |i. antagonist)

P-FNA, P-funaltrexamine (irreversible (i antagonist)

BNTX, 7-benzylidenenaltrexone (5j antagonist)

CP-OMe, D-Ala^ (2R, 3S)-A^he'', Leu®]enkephalin methyl ester

BW373U86, (±)-4-[(a-R*)-a-[(2S*, 5R*)-4 allyl-2,5-dimethyl-l-piperazinyl]-3-

hydroxyben2yl]-N,N-diethylbenzamide (nonpeptidic 5 partial agonist)

DADLE, [D-Ala^ Leu®]enkephalin

[D-Ala^, Asp'^l-deltorphin, H^N-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH,

[D-Ala^ Cys'']-deltorphin, HjN-Tyr-D-Ala-Phe-Cys-Val-Val-Gly-NHj (irreversible

Sj antagonist)

[D-Ala^ Glu'*]- deltorphin, HjN-Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH^ (selective 8^

agonist)

DALCE, [D-Ala^ Leu^' Cys®]enkephalin (irreversible 5j antagonist)

DAME, D-Ala^, Met®] enkephalin

DAMGO, [D-Ala^, NMPhe''' Gly-ol]enkephalin (selective and potent fi agonist)

221

DOR, delta opioid receptor (refers to the cloned delta opioid receptor)

DPDPE, cyclic [D-Pen^ D-Pen®]enkephalin where Pen is penicillamine (selective

5j agonist)

DPLPE, cyclic [D-Pen^ L-Pen^erikephalin where Pen is penicillamine

DSLET, [D-Ser^, Leu®' Thr®]enkephalin

GPI, guinea pig-isolated ileum longitudinal muscle/myenteric plexus (bioassay

for p. opioid receptor activity)

ICI 174,864, N,N-diallyl-Tyr-(Aib)2-Phe-Leu-OH (Aib=a-aminoisobut3nic acid) (se­

lective 5 antagonist)

i.e.v., rntracerebroventricular (into the cerebral ventricles)

i.th., intrathecal (into the subarachnoid space of the spinal cord)

LAAM, levo-alpha-acetylmethadol (long acting |i opioid agonist)

MVD, moiase-isolated vas deferens (bioassay for 5 opioid receptor activity)

NalBzoh, naloxone benzolhydrate (fi^ antagonist)

nor-BNI, nor-binaltorphimine (selective K antagonist)

NTB, naltriben (5^ antagonist)

NTI, naltrindole (selective 5 antagonist)

5'-NTil, naltrindole-5'-isothiocyanate (selective 5^ antagonist)

PD 117,302, (±)-trans-N-methyl-N-[2-(l-pyrrolidinyl)-cyclohexyl]benzo[b]-

thiophene-4-acetamide

222

U50,488, trans-(±)-3,4-dichloro-N-methyl-N-[2-(l-pyrrolidinyl)-cyclohexyl]-

benzeneacetamide methanesulfonate (K agonist)

U69,593, (5a, 7a, 8P)-(+)-N-methyl-N-(7-(l-pyrrolidinyl)-l-oxaspiro(4,5)dec-8-

yl)benzeneacetainide (selective K agonist)

223

APPENDIX B. LIST OF PUBLICATIONS

Titles appearing in bold represent published work from this dissertation

Refereed Journal Articles

1. Bilsky, E.J., Hui, Y., Hubbell, CL. and Reid, L.D. Methylenedioxymethamphetamine's capacity to estab­lish place preferences and modify intake of an alcoholic beverage. Pharmacology Biochemistn/ and Be­havior 37, ^3-638,1990.

2. Bilsky, E.J., Marglin, S.H., and Reid, L.D. Using antagonists to assess neurochemical coding of a drugs ability to establish a CPP. Pharmacology Biochemistry and Behavior 37,425-431,1990.

3. Bilsky, E.J., Hubbell, CL, Delconte, J.D. jmd Reid, LD. MDMA produces a conditioned place preference and elicits ejaculation in male rats: A modulatory role for the endogenous opioids. Pharmacology Bio­chemistry and Behavior 40,443-447,1991.

4. Bilsky, E.J. and Reid, L.D. MDL72222, a serotonin S-HT, receptor antagonist, blocks MDMA's ability to establish a conditioned place preference. Pharmacology Biochemistry and Behavior 39,509-512,1991.

5. Hubbell, C.L., Chrisbacher, GA., Bilsky, E.J. and Reid, L.D. Manipulations of the renin-angiotensin sys­tem and intake of a sweetened alcoholic beverage among rats. Alcohol 9,53-61,1991.

6. Reid, LD., Delconte, J.D., Nichols, M.L., Bilsky, E.J., and Hubbell, CL Tests of opioid deficiency hypoth­eses of alcoholism. Alcohol 8,147-"257,1991.

7. Bilsky, E.J., Montegut, M.L., Delong, C.L and Reid, LD. Opioidergic modulation of cocaine conditioned place preferences. Life Sciences 50, PL 85-90,1992.

8. Menkens, K., Bilsky, E.J., Wild, K.D., Portoghese, P.S., Reid, L.D. and Porreca, F. Cocaine place prefer­ence is blocked by the opioid delta antagonist, naltrindole. European Journal of Pharmacology 219,345-346,1992.

9. Reid, L.D., Hubbell, CL., Glaccum, M.B., Bilsky, E.J., Portoghese, PS. and Porreca, F. Naltrindole, an opioid delta receptor antagonist, blocks cocaine-induced facilitation of responding for rewarding brain stimulation. Life Sciences, 52, PL 67-71,1993.

10. Horan, P.J., Mattia, A., Bilsky, E.J., Webber, S.J., Davis, T.P., Yamamura, H.L, Malatynska, E., Appleyard, S.M., Slaninova, J., Misicka, A., Lipkowski, A.W., Hruby, V.J. and Porreca, F. Antinociceptive profile of biphalin, a dimeric enkephalin analog. The Journal of Pharmacology and Experimental Therapeutics 265, 1446-1454,1993.

11. Wild, K.D., McCormick, J., Bilsky, E.J., Vanderah, T.W., McNutt, R.W., Qiang, K.J. and Porreca, F. Anti­nociceptive actions of BW 373U86: A novel, nonpeptidic delta opioid agonist, in the mouse. The Journal of Pharmacology and Experimental Therapeutics, 2&7,858-865,1993.

IZ Bilsky, E.J., Bernstein, R.N., Pasternak, G.W., Hruby, V.J., Patel, D., Porreca, F. and Lai, J. Selective inhibition of [D-Ala^ GIu'']Deltorphin antinodception by supraspinal, but not spinal, administra­tion of an antisense oligodeoxynucleotide to an opioid delta receptor. Life Sciences 55, PL37-PL43, 1994.

224

13. Calderon, S.N., Rothman, R.B., Porreca, F., Flippen-Anderson, J., McNutt, R.W., Xu, H., Smith, L.E., Bilsky, E.J., Menkens, K.L. and Rice, K.C Probes for narcotic mediated phenomena. SNC80: A highly selective, nonpeptide 5 opioid receptor agonist.. Journal of Medicinal Chemistry 37,2125-2128,1994.

14. Haaseth, R.C., Horan, P.J., Bilsky, E.J., Davis, P., Zalewska, T., Slaninova, J., Yamamura, H.I., Weber, S.J., Davis, T.P., Porreca, F., and Hruby, V.J. [L-AIa^]DPDPE: A new enkephalin analog with a unique opioid receptor activity profile. Further evidence of delta opioid receptor multiplicity, foumal of Me­dicinal Oiemistry 37,1572-1577,1994.

15. Lai, J., Bilsky, E.J., Rothman, R.B., and Porreca, F. Treatment with antisense oligodeoxynucleotide to the opioid 5 receptor selectively inhibits 52~agonist antinodception. NeuroReport 5,1049-1052,1994.

16. Polt, R, Porreca, F., Szabo, LZ., Bilsky, E.J., Davis, P., Abbruscato, T.J., Davis, T.P., Horvath, R., Yamamura, H.I. and Hruby, V.J. Glycopeptide enkephalin analogues produce analgesia in mice: Evidence for pen­etration of the blood-brain barrier. Proceedings of the National Academy of Sciences 91,7114-7118,1994.

17. Qian, X., Kover, K.E., Shenderovich, M.D., Lou, B-S., Misicka, A., Zalewska, T., Horvath, R., Davis, P., Bilsky, E.J., Porreca, F., Yamamura, H.I., Hruby, V.J. Newly discovered stereochemical requirements in the side chain conformation of 5 opioid agonists for recognizing opioid 5 receptors. The Journal of Medicinal Chemistry 37,1746-1757,1994.

18. Wang, Z., Bilsky, E.J., Porreca, F. and Sadee, W. Constitutive n opioid receptor activation as a regtila-tory mechanism tmderlying narcotic tolerance and dependence. Life Sciences 54, PL 339-350,1994.

19. Beutler, AS., Banck, M5., Bach, F.S., Gage, F.H., Porreca, F., Bilsky, E.J. and Yaksh, T.Y. Retrovirus medi­ated expression of an artificial P-endorphin precursor in primary fibroblasts. Journal ofNeurochemistry 64, 475-481,1995.

20. Bilsky, E.J., Calderon, S.N., Wang, T., Bernstein, R.N., Davis, P., Hruby, V.J., McNutt, R.W., Rothman, R.B., Rice, K.C. and Porreca, F. SNC 80, a selective, non-peptidic and systemically-active opioid delta agonist. The Journal of Pharmacology and Experimental Therapeutics 273,359-366,1995.

21. Lai, J., Bilsky, E.J. and Porreca, F. Treatment with antisense oligodeoxynucleotide to a conserved se­quence of opioid receptors inhibits antinociceptive effects of delta subtjrpe selective ligands. Jour­nal of Receptor and Signal Transduction Research 15,643-650,1995.

22. Reid, L.D., Glick, S.D., Menkens, K.A., French, E.D., Bilsky, E.J. and Porreca, F. Cocaine self-administra­tion and naltrindole, a delta-selective opioid antagonist. Neuroreport, 6,1409-1412,1995.

23. Bilsky, E.J., Bernstein, R.N., Hruby, V.J., Rothman, R.B., Lai, J. and Porreca, F. Characterization of antinociception to opioid receptor selective agonists following antisense oligodeoxjmucleotide-mediated "knock-down" of opioid receptors in vivo. The Journal of Pharmacology and Experimen­tal Therapeutics, 277:491-501,1996.

24. Bilsky, E.J., Bernstein, R.N., Wang, Z.J., Sadee, W. and Porreca, F. Effects of neutral and negative antagonists and protein kinase inhibitors on acute morphine dependence and antinociceptive tol­erance in mice. Journal of Pharmacology and Experimental Therapeutics, 277:484-490,1996.

25. Lai, J., Riedl, M., Stone, L, Arvidsson, U., Bilsky, E.J., Wilcox, G., Elde, R. and Porreca, F. Immunofluo­rescence analysis of antisense oligodeoxynucleotide-mediated "knock-down" of the 5 opioid re­ceptor in vitro and in vivo. Neuroscience letters, 213:205-208,1996.

26. Bilsky, E.J., Wang, T., Lai, J. and Porreca, F. Selective blockade of peripheral delta opioid agonist induced antinociception by intrathecal administration of delta receptor antisense oligodeoxynucle-otides. Neuroscience letters, In press, 1997.

225

27. Bilsky, EJ., Intutiisi, CE., Sadee, W., Hraby, V.J. and Poneca, F. Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective mu or delta opioid agonists in mice. Pain, In press, 1997.

28. Calderon, S.N., Rice, K.C, Rothman, R.B., Porreca, P., Flippen-Anderson, J.L, Kayakiri, H., Xu, H., Becketts, K., Smith, L.E., Bilsky, E.J., Davis, P. and Horvath, R. Probes for narcotic receptor mediated phenom­ena. 23. Synthesis, opioid receptor binding and bioassay of the highly selective delta agonist SNC 80 cmd related novel nonpeptide delta opioid receptor ligands. Journal of Medicinal Qiemistry, In press, 1997.

Book Chapters

1. Porreca, F., Bilsky, E.J. and Lai, J. Pharmacological characterization of opioid delta and kappa recep­tors. In: L. F. Tseng (Ed.), The Pharmacology of Opioid Peptides. Reading; United Kingdom: Harwood Academic Publishers, pp. 219-248,1995.

2. Zaki, P., Bilsky, E.J., Vanderah, T.W., Lai, J., Evans, CJ. and Porreca, F. Opioid receptor types and subtypes: The delta receptor as a model. Annual Review of Pharmacology and Toxicology, 36:379-401,1996.

3. Porreca, F. and BUsky, EJ. Opioids in acute and chronic pain: Lessons from the cloned receptors. Proceedings of the VTH World Congress on Pain, lASP press, in press, 1997.

226

APPENDIX C. HUMAN/ANIMAL SUBJECTS APPROVAL

TtaUMvatsmror

tnalUuliorat Afuowi Cm klvl * 7UcMR,Afima IIS721 uidUacCiinunint* UlCSON AMZDNA

VcriCcMioa of Review Uy Tile InMitutioail Ammil Cue nd Use Cammiuee 0ACUC)

Final Approval Granted

PHS Aiiunuwe No. A0248-0i - USUANo.

TnUai PROTOCOJ. CONTROL fSiJlZS

"Antinociceptive EfTecu orOpioids"

PRINCU'AL tNVESnOATOIWliPARTMHNT:

Fmnk PorrcCR, PbD St, Kdward Bilsky - Phannacology

SUHMISSION DATK; M«y 12,1994 APPROVAL DATR June 23,19»4

QRANTINOAORNCY:

NIDA

The Univcnity oTAnzDiui InoiUiliunii Animal Can: «ad UM CoouniUa; reviews all iicctiuai of prapoole rcUiing to aniinal cafe aid UM; HW afauw: naned propuol bit been granliai Floal Appnirai aooordtnit lo the review poiieiai oT ihe lACUC.

NOTES:

*«* pull ippruval of litis conliul number is valid Uirou^*: June 22, 1997

• vita pniactt or VM pniai ciaal pM Iks ataovc uutai txpinun date, Uir rrindpal limalgator win aulMii) > new protocol pnpoHirorlblltmw. FMIo<ito|IACUCMvi<iir,ancwPnitiKulCiiniralNmbcriiiilEiq«aUiinllMawU)baMiipi^

*•* Conlinued approval fijrUiisprojcel was vonfiraicd: ••• Rcvi»uiis(ifaay). arc listed below;

MichacI A. Cusanovich, Ph.D. Vice Prcsidem fir Reaearch

DATE.- Jmuavl^. 19^7

227

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