Effects of Naloxonazine on Opioid Analgesia in The

Home , Naloxonazine

(

EFFECTS OF NALOXONAZINE ON OPIOID ANALGESIA IN THE

FORMALIN AND THE TAIL-IMMERSION TESTS

Lei Chen

A chesis submitted to the Faculty of

Gradua te s tudies and Re s earch in par t ial

fulfillment of the requirements for the

degree of Master of Science

Department of Pharmacology & Therapeutics

McGill University

Montreal, Quebec April 1990

The in terae t ion of naloxonazine, a putative long

lasting or "irreversible" mUI reeeptor antagonist, with

morphine, morphine-6-glucuronide (M6G) and sufentanil was

studied in two nociceptive tests using rats, the formalin

t~st and the tail-immersion test. Also, the displacement of

[3H]naloxone binding by selective opioid agonists in the rat

brain membrane was performed after naloxonazine pretreatment

in vivo.

The specifie i ty of naloxonazine was dependant on the

nociceptive tes t used. In the tail-immersion test,

intracranial naloxonazine (1 ug 4 hours before testing)

ptoduced a nonpara11el right shift of the dose effect

relations of aIl three agonists studied, consistent with long

lasting "irreversib1e" antagonist properties of naloxonazine.

In the formalin test, the same naloxonazine pretreatment

regimen produeed parallel right shift of the morphine dose

effect relation but failed to alter the effects of M6G and

sufentanil, suggesting e i the r "reversible" antagonist

properties or a more complex meehanism. Displacement binding

assays suggest that naloxonazine interacts with mu and delta

opio id receptor sites.

The data imply thp.t naloxonazine interacts in a long

lasting manner with more th an one opioid receptor subtypes.

An allosteric interaction between opioid receptor subtypes is

proposed to explain the effects in the formalin test. ( i RESUME

.' Les interactions entre la naloxonazine (un

antagoniste Il irréversible" des récepteurs mu, à action prolongée) et la morphine, la morphine glucoroconjuguée en position 6 (MGG) , ainsi que le sufentanil, ont été étudiées

chez le rat à l'aide de deux épreuves noclceptives: la formaline et l'immersion caudale. Le déplacement de la naloxone tritiée de ses liaisons opioides par différents

agonistes sélectifs a également été évalué suite à un

traitement préalable à la naloxonazine in vivo. La spécificité de la naloxonazine s'avéra dépendre de l'épreuve utilisée. Dans l'épreuve d'immersion caudale,

l'injection intraventriculaire de naloxonazine (1 ug 4 heures

auparavant) produisit un déplacement non-parallèle vers la droite de la courbe dose-réponse des trois agonistes étudiés; ceci s'accorde avec les propriétés antagonistes "irréversibles" et de longue durée de cette substance. Par

contre, lors du test à la formaline, le même pré-traitement

à la naloxonazine produisit ur 1éplacement parallèle vers ln droite de la courbe dose-réponse de la morphine, alors qu'il

laissa le MGr. et le sufentanil inchangés; ceci suggère soit une action 3.ntaSJoniste "réversible" de la naloxonazine, ou encore un mécanisme plus complexe. L'étude du déplacement de i la naloxone tritiée de ses sites de liaison indiquent que la \ 1 naloxonazine agit au niveau des récepteurs opio ides mu et t delta. .., , f ii r ,

f \ Les données impliquent que la naloxonazine réagit réciproquement de manière prolongée avec plusieurs sous- groupes de récepteurs opioides. Un mode d'interaction allostérique entre les divers sous-groupes de récepteurs opioides est proposé afin d'expliquer les résultats au test

à la formaline .

.. 1

;' , l " iii r1,. TABLE OF CONTENTS

Chapter

INTRODUCTION. : 1 " 1 t· 1. SELECTED REVIEW OF THE LITERATURE .... J

1.1 Mu Recl~ptor Subtypes and Na loxonaz ine. J

1.2 Sufentllnil, a Mu Agonist ......

1.3 Morphine-6-Glucuronide, an Active Metabolite of Morphine ...... 8

1.4 Two Animal Models of Pain: The Tail-immer&ion and the Formalin Tests ..... 1 1

l.4l The taU-immersion test .... 12 1.42 The formalin test .... 1 3

II. METHODS ...... 1 1

2.1 Purpose and Design ... 11

2.2 Animal Preparation: .. 1 Il

2.21 Subjecl:s. 18 2.2? Surgery .. 18

2.3 Procedure ..... 1 C)

2.31 Habituation ... 1 f) 2.32 Nociception testing. 19

2.4 Drugs and Their Adminstration ... 22

2.5 Binding Assay.

2.6 Data Analysis.

III. RESULTS ...... ï6

3.1 Effects of Naloxonazine in the Tail-immersion Test ......

3.2 Effects of Naloxonazine in the Formalin Test ...... 28

3.3 Effects of Naloxonazine Pretreatment in Vivo on the Displacement Binding of Opioids ...... 30

iv IV. DISCUSSION ...... 47

4.1 Effects of Naloxonazine on Opioid Antinociception in the Tail-immersion Test ...... 47

4.2 Effects of Naloxonazine on Opioid Antinociception in the Fcrmalin Test ... 50

4.3 Effects of Naloxonazine Pretreatment on Opioids Displacement Binding to the Rat Brain Membrane ...... 54

4.4 General Discussions .... 56

V. CONCLUSION ...... 63

REFERENCES ...... 65

.. v LIST OF TABLES

Table

1. SLOPES AND ED50'S FOR SUFENTANIL WITH NALOXONAZINE OR NALOXONAZINE VEHICLE PRETREATMENT IN THE TAI L- IMMERS ION TEST ...

2. SLOPES AND ED50'S FOR MORPHINE, MORPHINE-6-GLUCURONIDE AND SUFENTANIL WITH NALOXONAZINE OR NALOXONAZINE VEHICLE PRETREATMENT IN THE FORMALIN TEST......

3. SUMMARY OF EFFECTS OF NALOXONAZINE (ICV) ON DOSE EFFECT RELATIONS OF THREE AGONISTS IN TWO TESTS ......

vi { LIST OF FIGU!?ES

Figures

1. EFFECTS OF NALOXONAZI~E IN THE TAIL- IMMERSION TEST ...... 33

2. EFFECTS OF NALOXONAZIN~ PRETREATMENT ON MORPHINE ANTINOCICEPTION IN THE TAIL-IMMERSION TEST ...... 35

1. EFFECTS OF NALOXONAZINE PRETREATMENT ON THE ANTINOCICEPTIVE EFFECTS OF M6G IN THE TAIL- IMMERSION TEST ...... 35

4. DOSE EFFECT RELATIONS FOR MORPHINE WITH NALOXONAZINE 1 ug OR NALOXONAZINE VEHICLE 4 HOURS BEFORE IN THE FORMALIN TEST ...... 37

5. DOSE EFFECT ~ELATION~ FOR MORPHINE-6-GLUCURONIDE WITH NALOXONAZINE 1 ug OR NALOXONAZINE VEHICLE 4 HOUR BEFORE IN THE FORMALIN TEST ...... , 39

6. DOSE EFFECT RELATIONS OF SUFENTANIL WITH NALOXONAZINE 1 ug OR NALOXONAZINE VEHICLE 4 HOUR BEFORE IN THE FORMALIN TEST ...... , 39

7. DISPLACEMENT CURVES OF NALOXONE TO [3 H]NALOXONE WITH NALOXONAZINE 1 ug OR NALOXONAZINE VEHICLE 4 HOURS BEFORE SACRIFICE ...... '" ...... 41

8. DISPLACEMENT CURVES OF 13H1NALOXONE BY [D-A1a, N-methy1-Phe~, Gly5-o11ENKEPHALIN (DAMPGO) AFTER NALOXONAZINE 1 ug OR NALOXONAZINE VEHICLE 4 HOURS BEFORE SACRIFICE ...... 43

9. DISPLACEMENT CURVES OF [3H)NALOXONE BY [D-A1a2 -D-Leu5 jENKEPHALIN (DADLE) AFTER NALOXONAZINE 1 ug OR VEHICLE 4 HOURS BEFORE SACRIFICE ...... 43

{ vii 10. THE PROPOSED "MU1" RECEPTOR SUBSTRATE MEDIATING ANTINOCICEPTION IN THE TAIL-IMMERSION TEST ...... 60

11. THE PROPOSED MU-DELTA RECEPTOR SUBSTRATE MEDIATING ANTINOr.ICEPTION IN THE FORMALIN TEST...... 62

{tt

,- viii ACKNOWLEDGEMENTS ( 1 should l ike to thank my research direc tor, Dr.

Frances v. Abbott for patience, guida'lce and

encouragement in this research proj ect and for being a

fr iend in need.

1 wi sh to thank the D~partment 0 f Pha rmaco 10 gy

and Therapeutics for the privilege of pursuing my studies

in the Department. Thanks are also extended to Shanghai

Medical University where l did my medical studies for five

years.

Dr. Roberta Palmour introduced me to the in vitro

binding assay employed in part of the project. 1 gratefully

acknowledge her help. l thank Dr, George Kunos for

introducing me to the Department and for his encouragement.

1 acknowledge the technical guidance of Mr. Larry

Yelen and Colette Oblin; Dr Simon Young, Miss Sandra

Loscome aud Miss Anna-marie Babey who aIl helped to provide

a friendly and stimulating research environment.

1 am grateful to the Medical Research Gouncil of

Canada for financial support given throughout my studies.

To Babara l thank for her proof reading the whole

thesis and for her continues encouragement.

Very special thanks go to my parents for their

whole-hearted support which made this thesis possible; To

my daughter Mingna, and last, but not least to my husband

for their patience and support throughout my thesis work,

ix INTRODUCTION

Naloxonazine, a derivative of na1oxone, ls [l

symmetric compound with two na1oxone substituents bridged

by hydrazine. Like naloxone, naloxonazine has op i 0 id

antagonist properties but with high affinity for che "muI"

receptor subtype, where it produces long lasting blockade

(Simone et al. 1986; Hahn & Pasternak, 1982). In fact, the

mUl receptor subtype, is defined primarily on the basis of

long lasting or "wash resistant" block"lde by naloxazonc

(Pasternak et al. 1980b; Wolozin,~ Pasternak, 1981), [l

metabolic precursor of na1oxonazine (Hahn & Pasternak,

1982) .

However. a1though there is evidence showing long " lasting or "irreversible" antagonist properties of

naloxonazine in vitro, in antinociceptive tests in vivo

naloxonazine produces parallel shift of the op i 0 id

antlnociceptive dose effect relation to the right (Ling et

al. 1985; Heyman et al. 1988). From a pharmacologicLll

standpoint, this charac te ris t le suggests reversible

antagonism (Goldstein et al. 1974; Ta11arida and Jacobs

1979). In the na1oxonazine studies, rats were pretreated

r·, with systemie naloxonazine and tested 24 hours later, when

t the animal is presumed to have eliminated any frec 1 t ) 1 , l r nal oxonaz i ne and only the drug i rrever s ib ly bound to the

reeeptors remains. However, very large doses of

naloxonazine were used (35 mg/kg, s.e.; 10 mg/kg, Lv.) in

these experiments and little is known about the kineties

metabolism of naloxonazine. The assumption that the drug is

eliminated is based on e1imination of 3H-label on an

unspeeified position in the molecule (Ling et al. 1986).

The purpose 0 f the preSf'n t inve S t iga t ions was ta

examine the effects of naloxonazine given

intraventrieularly on opioid antinoeiception and receptor

binding. In this method, the agent is administered in a

small dose, directly into the central nervous system, and

it is presumed that it is th en di1uted by distribution

throughout the body of the rat so that on1y specifie

lasting CNS effect are present. This technique e1iminates

the prob1em of residual unknown metabolites at the time of

testing and has been used successful1y with other

irreversible opioid antagonists (Ward et a1. 1981) .

Antinoeiception was assessed using a heat pain threshold

test and the forma1in test whieh assesses a rat's response

to a minor tissue injury. These two tests differ in both the neural and pharmacologica1 mechanisms whereby opioids produce ana1gesia (Franklin & Abbott, 1989).

2 Chapter One

SELECTED REVIEW OF THE LITERATURE

1.1 Mu Receptor Subtypes and Na1oxonazine

Shortly after the initial demonstration of

stereospecific receptor binding of opiates (Goldstein pt al. 1971) pharmaeclogical studies suggested the presencp of subpopulations of opioid reeeptors whieh were named after proto typ ie drugs: mu (morphine), kappa (ke to c yc1 az 0 e i ne) and sigma (SKF10,047) (Martin et al. 1976). The discovery of the enkephalins (Hughes et al. 1975) led to the description of a fourth reeeptor subtype, delta o t- enkephalin reeeptors (Lord et al. 1977). Of these receptor subtypes, mu reeeptors are elosely assoeiated with two of the cardinal properties of morphine ana1gesia and respiratory depression (Wood et al. 1982; Mcgilliard &

Takemori, 1978; Goode et al. 1979). A1though mu agonists a1most invariab1y produce both analgesia and respira tory depression, Megi11iard and Takemori (1978) found that the in vivo apparent pA2 values for na1oxone were significantly higher for antinociception than for respira tory depression.

This suggests that there are two distinct binding sites underlying ana1gesia and respiratory depression.

3 In 1980, Pasternak and co11eagues reportec. that l they had identified an long-lasting or "irreversible"

antagonist, naloxaz one, later found to be activated

f 0 llowi ng convers ion to naloxonaz ine (Hahn & Pasternak,

1982), which dissociated the analgesic and respiratory

depressant effects of opioids. Zhang and Pasternak (1981),

treated rats with na1oxazone (300 mg/kg, s.e.) 24 h before,

sacrificing rats to perform opioid saturation and

competition binding assays. In paralle1, they tested

naloxazone pretreated rats in the tai1-flick test. In the

binding assays, aIl the opioids tested yie1ded curvilinear

Scatchard plots which could be broken into high and low

affini ty binding components. Naloxazone selectively

inhibited the high affinity binding of aIl the opioids. The

analgesic effects of opioids in the tail-flick test were

a1so inhibited. In later studies, they showed that the

respiratory depressant effects were unaltered (Ling et al.

1985) .

The high affini ty si te was named "mul" and has

been held to be responsible for opioid analgesia (Zhang &

Pasternak, 1981). The low affinity sites remaining after

naloxazone treatment included two receptor subtypes. One of

them preferentially binds morphine, i. e. a "mu" site.

Unlike the mUl site, which binds opiates and enkephalins

equally weIl and with greater affinity, the second mu site

4 has a low affini ty for enkepha1 ins. This site was named 111\.12

and proposed to be responsible for respiratory depression

(Pasternak et al. 1980a). The third site bound morphine

wi th abouta 10 - fo1d lower affini ty than the mU2 site and

was believed to correspond with the delta receptor.

Naloxazone, and later naloxonazine, have been

used to define other mUl and mU2 receptor mediated effects

in vivo. On the basis of these in vivo studies, it has bcen

proposed that opioid receptors are involved in

prolactin secretion (Spiegel et al. 1982), cata1epsy (Ling

& Pasternak, 1982) free and deprivation-induced feeding

(Simone et a1. 1985) and acety1choline turnover (Zsilla et

a1. 1977). The lower affinity sites (mu2) has been proposcd

mediate other pharmacological actions of opioids, such as

respiratory depression (Ling et al. 1985), regulation of

growth hormone release (Wood & Pasternak, 1983) and

dopamine turnover (Wood et al. 1982).

There are, however, data that are discrepant with

the concept of na1oxonazine as an irreversible antagonist

of the mUl receptor which is defined on the bas is 0 f

naloxonazine: 1) Na1oxonazine (10 mg/kg i.v.), produced a

paralle1 right shift of morphine antinociception d05e

affect relation (Ling et al. 1985 ) which is a

characteristic of reversible antagonist (Goldstein et al.

5 1974; Tal1arida and Jacobs 1979). 2) There are also sorne

interesting data on the effects of na1oxonazine on opioid

modulation of reflex contraction of the urinary b1adder. In

this mode 1, Dray and coworkers (1987) showed na1oxonaz ine

antagonized the specifie de1 ta agonist [D-pen2 ,D-

pen5 ] enkepha1in (DPDPE) more than 24 h in the dose of 1.0

to 6.5 ug per rat administered intracerebroventricu1arly.

Wh il e , the effects of [D-A1a2 ,N-methy1-Phe4 ,D-

1eu5 ] Enkepha1in (DAMPGO) a mu agoni st were b locke d les s

than 3 hour s . 3) Using computer fitting program "Ligand" which a110ws ana1ysis of saturation and competition data at

the same time instead of separate1y, Cruciani et al (1987)

showed that naloxonazine binding fits a three sites receptor model that correspond to mUl, mu and delta receptor respectively. The selectivity was lO-fo1d greater for binding to the mU1 receptor. However, they fai1ed to demonstrate the "irreversible properties" of naloxonazine.

4) ln the wash-resistant disp1acement binding assay (i.e. the b rain membrane s we re washed ex tens ive ly to el imina te reversib1y bound antagonist), naloxonazine displaced opioid agonists in a biphasic manner (Hahn et al. 1982). This is consistent with the in vivo data presented by Ling et al.,

(1986) who found that pretreatment with naloxonazine produced a biphasic antagonism to morphine antinociceptive dose effect relation in tai1-flick test. The biphasic nature of the curve imp1ied at 1east two sites with

6 , r 1 f ' fi(. ~ ~ ," differing sensitivities toward naloxonazine induced both ~ 1 , l .- wash-resistant inhibition and antagonism. They conciuded ," , l,.' ~. 1 that the selectivity of wash-resistant inhibition for high } " f;~ , affinity binding site (muI) is concentration dependent and " ~ ~ the non-mul sites will be inhibited in a wash-resistant

manner in the presence of higher concentration of

naloxonazine (Hahn et al. 1982).

1.2 Sufentanil. a Mu Agonist

Sufentanil is a potent derivative of fentanyi

(Niemegeers et al. 1976). lt is '+521 times more potent than

, , 1 morphine in the tail-flick test. lts primary clinical use , 1 1 1 is as an intraoperative anaesthetic adj unct in patients

undergoing major surgery, particularly heart surgery (De

Lange et al. 1983). Using opioid sensitive periphernl

tissue preparations, combined with selectively induced

tolerance of opioid receptor subtypes, sufentanil was

demonstrated to be very specifie for mu-opioid receptou,

(Wuster et al. 1980).

In binding s tudies , [3 H ]sufentanil was

demonstrated to be a superior ligand for mu-opiold

receptors (Leysen et al. 1983). lt has high stereospecific

(90% of total), opioid analgesic agonis t and antagonis t

compatible binding ability with Kd value of 0.13 nM. lt was

7 demonstrated that the inhibition constants of opioids

measured in vitro for stereospecifie [3H]sufentanil binding

in rat forebrain membranes correlated highly with the

analgesie poteney of the compounds in the tail-fliek test.

'T'his suggests that [3H]sufentanil labels mu reeeptor sites

that medlate analgesia in thermal pain tests.

1.3 Morphine-6-Glucuronide. an Active Metabolite of

Morphine

Morphine is a potent, clinical1y important ana1ges ie. However, studies examining the pharmacodynamies of morphine have frequently failed to demonstrate the relationship between plasma morphine levels and the behaviora1 effeets of morphine (Dahlstrom & Paa1zow, 1975;

Dahlstrom et al. 1978) . One explanation is that the ana1gesic effect of morphine is due, in part, to a slow1y eliminated me tabol i te with different pharmacodynamie properties. Morphine ean be glueuronidated at both the phenolic group ("3" earbon) and the a1eohol group "6" earbon to produee morphine-3-glueuronide and morphine-6- glueuronlde (Yoshimura et al. 1969) . The phenolie glueuronide, morphine-3-g1ueuronide, is the primary metabo1ite. However, in humans 8 to 10 % of the total glueuronide is M6G (Sawe, 1986).

8 "; 1- - There is evidence to suggest that morphine-6- 1 , >! glucuronide (M6G) is involved in the analgesic effects ai r"r- t· \ l4 l morphine. Despite the hydrophilic nature of M6G, C _ ,fI" ~ ~ labeled M6G penetrates the brain (Yoshimura et al. 1973). î In 1971, Shimomura and coworkers used the hot plate method ~" ï. ( .( to show tha t M6G i s more po ten t than the p aren t c ompo u ne!, .~ < morphine, fol1owing subcutaneous administration in micl'. ~

~ These results were confirmed by experiments in rats d

decade later. Pasternak (1987) showed that the M6G is 20-

fo1d more potent than morphine following microinjectioll

into the periaqueductal gray in the tail-flick test. Abbott

and Palmour (1988) demonstrated tha t MGG

(intracerebroventricular adminstration) was 60-fold mort'

potent than morphine itself in formalin test and 14)-200

fold more potent in the tail-immersion test. Systemica11y,

M6 G was approx ima te ly equipo ten t wi th morph i ne.

M6G has been examined in a displacement binding

assay using brain membranes and has been shown to disp1ac0

other opioid ligands from binding sites. In genera1, thC'

affinity of M6G for opioid receptors in these studies is 3

to 10 fold lower than that of morphine (Oguri et al. 1987;

Abbott, 1989) or naloxone (Christensen & Jurgensen, 1987).

Oguri and co-workers (1987) showed that M6G has 1.8-[01d

higher potency competing with leucine-enkephalin (a e!Edta

agonist) binding than against morphine. Abbott and Pa1mour

9 (1988) showed that at very low concentrations « 10- 9 M),

M6G enhanced the binding of etorphine, dihydromorphine and

naloxone. Such an effect did not occur at low

concentrations of morphine.

Cl inical reports al sa sugge s t tha t M6G may b e

responsib1e ;or a significant component of analgesia and

respiratory depression following morphine administration.

Pharmacokinetic studies in humans indicate tha t wi th

chronic dosing, the b100d 1eveis of M6G are higher than

those of morphine itse1f (Sawe, 1986) . Osborne and

associates (1986) reported three patients with impaired

rena1 function who experienced pro1onged respiratory

depression foilowing morphine adminstration. At the time of

initiation of the study (40-153 hours after cessation of

dosing), these patients had c1assical, naloxone reversible

signs of opiate intoxication with no measurable morphine in

their plasma. However, substantiai leveis of M6G were

present. This suggests that M6G may be responsible for the

respiratory depression in these patients.

Subsequently, Osborne et al. (1988) demonstrated

that 1.0 mg per 70 kg body weight M6G administered by slow

intravenous injection produced a profound analgesic effect

in five patients. These data strongly suggest that the

analgesic and respiratory depressant effects of morphine

" 10 may be due, in part, to M6G.

1.4 Two Animal Models of Pain, the Tail-immersion and thp

Formalin Tests

The primary purpose in the development of animal

pain models was to screen for potentia1 analgesic drugs

(Janssen et a1. 1963). In this context, the most important

characteristies of a test are that it eorrect1y identifies

compounds that are analgesie in pathological pain in humdns

and correctly eliminates eompounds without this activity.

The tail-fliek test which measures the lateney of a motor

response to heat: stimuli was developed mainly for this

purpose. Sinee the discovery of the endogenous opioids,

there has been increasing use of animal pain models for tilt>

exploration of the neuroanatomieal, neurophysiologieal and

neuropharmacological mechanisms of analgesia (Franklin and

Abbott, 1989). In this context, the pain test becomes 11

mode1 of pathological pain in humans and the relationship

between the processes involved in the effect of a drug on

the tested response, and those involved in its effeet in

humans assumes critieal importance. The formalin test in

whieh a tissue injury is produced, was developed as a model

of pain involving tissue pathology.

1i ! ( 1.41 The tai1-immersion test

The tail-immersion test (Janssen et al., 1963),

is a common variant of the radiant heat tail-flick test

(D'Amour & Smith, 1941) and measures the withdrawal latency

from noxious heat in rat. The tai1- flick test was adapted

from a test measuring the heat pain tbreshold in hum ans

(Hardy et al. 1940). As observed in humans (Schumacher et

al. 1941), the individual variation, under a variety of

conditions for determining the pain threshold, was found to

be very smal1 in the tail-flick test (D'Amour & Smith,

1941).

The tail-f1ick response is a simple spinal reflex

that can be elicited after spinal cord transaction

(Bonnycast1e et al. 1953; Sinclair et al. 1988). However,

i t i s modul a ted by neural sys tems tha t de s cend from the

brain stem and a major component of the antinociceptive

effects of morphine are believed to be mediated by these

bulbospinal systems ( Bas b a um & Fie 1 d s, 19 84). Les ion S 0 f

the caudal periaqueductal gray (Abbott et al. 1982b;

Thorn-Gray et al. 1981), the nucleus raphe magnus (Llewelyn

et al. 1986 ; Proudfit and Anderson, 1975) and the

dorsolateral funiculus (Basbaum et al., 1977; Ryan et al.,

1985) all attenuate morphine analgesia in the tail-flick

12 test. Microinjection 5-HT agonists and antagonists into

local brain stem regions combined with systemic morphinC'

indicated that 5-HT systems play a major role in the opioie!

antinociception in the tail-flick test (Roberts, 1984).

The pharmacological research indicates that the

tai1-f1ick test can accurately and sensitively detect

morphine-like drugs (Franklin &. Abbott, 1989; D'Amour &.

Smith, 1941; Tyers, 1980) i.e. mu receptor agonists

1.42 The formalin test

The forma1in test developed by Dubuisson ane!

Dennis (1977) is an animal model of acute, tissue injury-

induced pain in which the behavioral response to a minor

.1 [ tissue inj ury is assessed. In contrast to the

immersion test, the formalin test asses ses pain levcl

rather than thresho1d. Severa1 1ines of experimental

evidence suggest that the formalin test is different from

the heat pain threshold tests ln the neural mechanisms

underlying both pain and analgesia.

First, the neural substrates underlying morphine'

analgesia involved in the formalin test are different frolll

those involved in the tail-flick test. Unlike in the tail-

f1ick test, les ions 0 f the nucleus raphe magnus and r ' .... 1, 13 1 dorso1atera1 funiculus fail to alter rno:-phine's { antinoeieeptive effects in the forrnalin test (Ryan et al.

1985; Abbott & Melzaek, 1982a). Microinjeetion of

na I t (exone in to PAG does not antagoni ze sy s ternie morph i ne

antinociceptive effect in the forrnalin test (Helrnstetter &

Landeira-Fernandez, 1989).

Secondly, 5 -HT mechanisrns appear to an tagonize

ra~her than potentiate morphine in the formalin test.

Lesions of the median raphe nucleus (Abbott & Melzack,

1982a) and pharmacological manipulation of 5-HT (Abbott et

al. 1987) in rats indieate that 5-HT antagonizes rather

than faeilitating morphine. In experiments using tryptophan

uptake competitor L-valine, Abbott and coworkers (1986)

showed that reducing serotonin synthesis redueed pain in

habituated rats but inereased pain in non-habituated rats

in forma1in test. This su~gests that different serotonin

systems may be invo1ved in the forma1in test and ean be

aetivated under sorne conditions, speeifiea11y when animaIs

are stressed.

Third1y, the naloxone dose ratio for morphine is

2.9-fold lower in the formalin test than that the tai1-

immersion test (Abbott et al., 1986) which suggests that

different opioid reeeptor meehanisms are involved in the

test. On the other hand, beta-funaltrexamine an alkylating

14 agent with se1ectivity for the mu receptor subtype

(Takemori et al. 1980; Tam et al. 1985) antagonized the

antinociceptive effect of morphine comp1ete1y (Chen & 1 Abbott, 1988) which exc1udes the invo1vement of kappa receptor and favours the invo1vement of mu, possibly, delta

receptors (c. f. Fanse10w et al. 1989, Abbott et al.,

submitted)

In summary, the two pain tests are dlfferent in

several aspects:

1) In the tail-immersion test, pain occurs prior to

injury and reflex withdrawa1 from noxious heat i5 t, assessed, whi1e in the formalin test pain arises from

1 injured tissue and the continuing behavioral response 1 to the injury is assessed.

2) The neural substrates for morphine antinociception arc

different in two tests. In the tail-immersion test, a

major component of opioid antinociception is mediated

by descending bulbospinal inhibitory systems and th('

integrity of dorsal raphe and raphe magnus 5 -liT

neurons is important, while in the formalin test these

systems are not involved. Instead, 5~HT systems,

possibly ascending to the forebrain, antagonize rather

than potentiate morphine antinociception.

15 { 3) Naloxone dose ratios indicate that the two tests also

differ in the characteristics of the opioid reeeptor

me d ia ting anal ges ia. The mu receptor i s invo 1 ved in

the antinocieeptive effeet of the tail-immersion test,

while mu and delta reeeptors are suggested to be

involved in the formalin test.

The charac teris t ies of two tests are very

di fferen t. In the present experiments, both these

experimental models of pain were used to explore the opioid

receptor meehanisms involved in analgesia.

,,'

16 1 J, 1

Chapter Two

METHODS

2.1 Purpose and Design:

The purpose of this study was to investigate the

effects of na1oxonazine on opioid analgesia in two animal

models of pain; the tail-immersion test, in which reflex

withdrawal from noxious heat is assessed, and the formu1in

test, in which the behavioral response to a minor tissue

injury is assessed. In these experiments, three opioid

agonists were used: morphine, an active metabolite of

morphine, morphine-6-glucuronide (Pasternak et al. 1987;

Abbott & Palmour, 1988) and a potent synthetic mu-agonist,

sufentanil (Leysen et al. 1983; Wuster et al. 1980). AlI

of these agents are mu agonists with varying potencies in

the two pain tests. The relative potencies of morphine:

M6G: sufentanil are 1: 129: 2187 in the tail-immersion test

and 1: 78: 301 in the formalin test (Abbott et al. 1986a;

Abbott & Palmour, 1988). AlI three agonists pro duce an

unambiguous maximal effect in the tail-immersion test:.

However, in the formalin tes t, sufentanil doe 5 no t

completely block pain behaviour at subtoxic doses (Abbott

et al. 1986a).

17 As indlcated in the in t roduc t i on, na1oxonaz ine ( has usually been given systemica11y in a re1ative1y large

dose 24 hours before testing. In the present experiments

intro c e rebroventr icu1ar adminstration was used. Two

strategies were used to ensure specificity. First, three

doses of naloxonazine were givpn. The two highest doses

produced equiva1ent effects and subsequent testing was done

with the lower of these in order to minimize nonspecific

effects. Second, the disp1acement of other opioid ligands

was examined in brain membranes after in vivo treatment of

rats with this dose to determine if an argument could be

made that naloxonazine eliminates a single binding site.

2.2 An ima1 Prepara t ion

2.21 Subjects

Adul t ma 1 e Long - Evans rats were purchased from

Charles River Ltd. Canada (S t . Cons tant, Quebec). They

weighed 280-320 g at time of surgery. During the

experiments, the rats were maintained on ad libitum food

and water in group cages of 2 -4 in the co1ony room on a

12:12 1ight:dark cycle (lights on 7:00, off 19:00). AH

testing was performed between 9: 00 and 18: 00.

2.22 Surgery ( 18 Five to seven days prior to testing, the rdt~

were anaesthetized with sodium pentobarbital 65 mg/kg

(i.p.). A stainless 23 gauge steel guide cannula was

implanted with the tip 1 mm above the lateral ventricle

(coordinates: l. 3 AP, 1,.8 L, 3.0 below bregma) using

standard stereotactic techniques (Paxinos & Watson, 1986).

The implant was anchored to the skull with three screws

embedded in dental acry1ic. Rats were 1eft to recover for 5

days after surgery before pain testing began.

2.3 Procedure

2.31 Habituation

Prior to experimenta1 sessions, rats were brought

into the laboratory, hand1ed briefly and placed in the

forma1in test boxes dai1y for 10 minutes over a 3-day

period. This fami1iarization with the testing environmenl

e1iminates stress-induced 5-HT activity (Kelly and Franklin

1984)

2.32 Nociception testing

1) The tail-immersion test .

.- 19 The test was performed by the experimenter

ho lding the rat gently and dipping the distal 5 em of the

tail in a beaker of water whieh was maintained at 55--±.

o. SoC by a tissue bath regulator. The lateney for the rat

to curl i ts tai l out of the water was determined by means

of a foot-operated timer. The latency is considered as a

me asure 0 f sens i ti vi ty to hea t pain. In order to avoid

burning of the tail, the tai1 was dried with a towe1

immediate1y and a cei1i.ng of 15 sec was imposed on the

wi thdrawa 1 la te ne ies. The rats we re re turned to the i r home

cages between tests.

The tail-immersion test was carried ou~ on groups

of rats (N 4-8) 4 hours after administration of

na1oxonazine or its vehicle. The test was repeated every 10

minutes after administration of morphine or its vehicle,

every 15 minutes after M6G or its vehicle and every 5

minutes after sufentani1 or its vehiele. Data for 30-40

minutes after s.e. morphine, 45-60 minutes after i.e.v. M6G

and 10-20 minutes after s.e. sufentani1 were used to

ca1eu1ate peak effects. These sehedu1e were ehosen on the

basis of the time effeet relations of the three agonists,

M6G having the longest duration of action and the slowest

rise time and sufentani1 having the shortest time effect

curve (Abbott et al. 1986a; Abbott & Palmour, 1988).

( 20 2) The formalin test

The formalin test, adapted from Dubuisson and r Dennis (1977), was carried out in a 30 x 30 x 30 cm p1exig1ass chamber with a mirror mounted under the Eloor ut

1 45 0 to a110w an unobstructed view of the paws. Formalin (50

u1 of a 2.5 % solution) was inj ected subcutaneous ly in ta

the plantar surface of one rear paw and the rat was placed

in the chamber. Pain scores peak about 5 min after farmalin

injection and then drop before rising to a stable level

sorne 20-25 min after injection. Stable levels of behavior,d

pain persist for at least 30 min. Drug injections were>

timed so that peak effects occurred 30 ta 50 min after

formalin injection. Pain scoring was do ne during this

period. A hand-held computer was used onto which thE'

observer entered the momentary pain scores continuously

according to the fullowing criteria:

o Weight is barn evenly on both rear paws.

1 - The injected paw i5 favored durillg

locomotion, 1ying or sitting.

2 - The injected paw 1s elevated with at JlI0~t

the nai1s touching the floor.

3 - The injected paw is groomed or chewed. t, A pain score was calculated u5ing the following

21 ------

. formula: \

Pain score - --.------.. ------.---- •. --

Where to, tl. t2 and t3 are the number of seconds

rats spent in each behavioral rating category.

2.4 Drugs and Their Adminstration

Na loxonaz ine hydrochloride ( Gift from Dr.

G.Pasternak Department of Neuro logy, Memorial Sloan

Kettering Cancer Center, New York) was dissolved in 0.2 %

=,cetic acid. Morphine sulfate (kindly donated by Sabex

Canada Ltd, Quebec) , morphine-6-glucuronide (Salford

Ultrafine Ltd, Manchester UK) anc1 sufentanil citrate (gift

of Janssen Pharmaceutical, Beerse, Belgium), were dissolved

in distilled water for systemic or intraventricular

injection. Intraventricular adminstration volume was 5 ul

injected over 60 sec through a 30 gauge cannula connected

to a Hamilton gas tight syringe. Subcutaneous injection

volumes were 1 ml/kg body weight and administered in the

back of the rat.

[3H ] naloxone was purchased from New England

Nuclear (Boston MA). Naloxone HGl ( gift of Endo

Laboratories, Rahway, New Jersey) , [D-Ala 2 ,N-methyl-

22 Phe 4 ,Gly5- o 1]enkephalin (Sigma Chemicals Ltd) and [D-

Ala 2 ,D-Leu5 ]enkephalin acetate salt (Sigma Chemicals Ltd) were dissolved at millimolar concentrations in distilll'd water and appropriate log dilutions were made for bindlllg assays.

2.5 Binding Assay

Rats with naloxonazine or vehicle pretreatmelll were decapitated and their brains were rapidly removed The cerebellum, which contains negligible 3H-opiate binding wa., excised, and the remainder of the brain was immediately placed in 50 ml of 50 mM Tris-HeL buffer at pH 7.7. The' brain was homogenized with a Polytron (setting 4.5 for 10

sec) followed by 3 cycles of manual homogenization in [l

Glenco dounce homogenizer. The homo gena te was the 11 centrifuged at 20,000 g for 20 min. The pellet WH!:. resuspended and centrifuged again. The displacement bindin~ assays of [3H]naloxone by nal.oxone, DAMPGO and DADLE werc performed with 10 mg brain (wet weight) suspended in l IlIl

Tris buffer, pH 7.7 in the presence of EDTA (0.1 mM) at L,OC for 4 h.

AlI determinations were performed in triplicatc

Nonspecific binding was defined by 1 uM na l oxone>

23 [ 3 H ]naloxone Wé:.S used as the labelled ligand at

concentration of app roxima tely l nM. Unlabe lIed naloxone 1

DAMPGO and DADLE were present in concentrations ranging from 10 -10 M to 10 - 7 M. The incubation was terminated by filtration under vacuum over Millipore AP filters. The filters were th en washed with three 4 ml aliquots of cold

50 mM Tri s - Hel , pH 7. 7. Dr i e d fil ter s we r e c 0 un t e d b Y l 2 Il

Rackbeta Liquid Scintillation Counter, using Liquifluor

(N ew Eng land Nuc lear, Bos ton MA) .

2.6 Data Analysis

Pain scores in both tests for the peak effect pe r i od 0 f the op ioid agoni s ts we re converted to % maximum possible effect (MPE) by the formula:

(E-Emin)

MPE - x 100

The Emin represents the mean score of 6 8 identically treated rats receiving vehicle injections and

Emax was arbitrarily defined as 0 for the formalin test and as 15 sec for the tail-immersion test.

For the dose effect relation data, the mean %

24 ------

MPE's were plotted against log dose and a straight line W3b

fitted by computer using Sigma-Plot (Version 3.10, 1987).

Statistical estimates of the slope and MPE50 and their

respective standard errors were calcu1ated from the data

for individual animals by jackknifing the regression lines

and interpolating MPESO's (Mosteller & Tukey, 1968).

Jackknifing is a method of directly assessing variability

of statistics which offers ways to set sensible confidence

limits in complex situations. Differences in slopes and

MPESO's were tested using Student's t-test. The

displacement binding data were analyzed using the EBDA

computer program (McPherson, 1985) and were expressed as a

percentage of control against log concentration of drug.

The total specifie binding of membrane with naloxonazine

vehicle treated was set as 100 %. The displacement binding

curves were fitted using non-linear regression program in

Sigma-Plot.

25 ( Chapter Three

RESULTS

3.1 Effects of Na1oxonazine in the Tail-immersion Test

Fig.1A shows tail-immersion latencies after

pretreatment wiLh naloxonazine in the absence of any of the

agonists. The rats received a vehicle injection at the time

morphine or sufentanil or M6G would have been administered.

Naloxonazine pretreatment by itself did not alter the

baseline of tail-immersion Iateneies (ANOVA, F(3,19)=1.33,

p ~ 0.05). These data were pooled to prol/ide a baseline

measure for ealculation of % MPE.

Fig. lB represents the dose effeet relations for

sufentanil (s.e.) following pretreatment with naloxonazine

[0.2 ug, 1 ug or 6.5 ug (i.e.v.)] in the tail-immersion

test. All three doses of naloxonazine produeed nonparallel

shifts of the dose effect relation for sufentanil. Slopes

of regression lines for the sufen tani l dose e ffee t

relations (Table 1) are: with naloxonazine vehicle, 199 ±

27; naloxonazine 0.2 ug, 90 ± 15; naloxonazine 1 ug, 106 +

19; naloxonazine 6.5 ug, 90 ± 28. AlI three doses of

naloxonazine pretreatment significantly redueed the slopes

of the sufentanil dose effect relation (tO.2 = 3.63; tl =

26 2.82; tb.5 - 2.77: for all the three, P < 0.01). Increasing

the naloxonazine pretreatment dose to 1 ug not only altered

the slope of the sufentanil dose effect relation, but a1so

altered the ED50 value (M ±. SE: 9.7 ±. 0.5). Increasing the

dose of naloxonazine to 6.5 ug did not increase the ED50 of

sufentanil further (Table 1).

These data constitute the only behavioural

evidence for "noncompetitive" antagonism of opioid effect

by na loxonaz ine. As such, the da ta are incomp le te b e caus e

naloxonazine did not block the respiratory depressant

effects of sufentanil. This made it impossible to determine

if the max ima1 e ffec t was re duced becaus e hi ghe r do ses 0 f

sufentanil produced marked cyanosis. On the basis of the

similarity of the effects of 1.0 ug and 6.5 ug naloxonazine

in this test, subsequent experiments were done using 1.0 ug

naloxonazine. The principle under1ying this decision is

that: the lowest dose of an antagonist required to produce

ail e1"tect will have the fewest nonspecific effects (cf

S~wyn0K et al. 1979).

The antinociceptive effects of morphine (Fig. 2)

and morphine-6-glucuronide (M6G) (Fig 3) after pretreatment

1 cl with 1 ug naloxonazine or na1oxonazine vehicle were tested.

Naloxonazine antagonized the effects of higher doses of

morphine (t6 - 4.20, P < 0.01) and M6G (tsoo - 2.27, P <

27 0.05), but not those of lower doses of morphine (t3 .. 1.55,

p ~ 0.05) and M6G (tlOO - 1.01; t250 -1.76, P ~ 0.05).

These results are similar to those produced by sufentanil

and imply a reductlon in slopes of the dose effect

relations. As with sufentanil, the respiratory depressant

e ffec t s of morphine and M6G preel uded te s ting the hi gher

doses.

These data suggest that in the tail-immersion

test, opioid antinocieeption is produeed by opioid receptor

subtype(s) that are sensitive to antagonism by naloxonazine

and may be a single opioid receptor.

3.2 Effects of Naloxonazine in the Formalin Test

Fig.4 shows the dose effect rela tions for

morphine in the formalin test after naloxonazine 1 ug or

naloxonazine vehicle (1. c.v.) 4 h before testing. Unlike

the tail-immersion test, naloxonazine pretreatment did not

al ter the slope of morphine dose effect relation but

produced a parallel shift of morphine dose effect relation

to the right. As shown in Fig. 5 and Fig. 6, pretreatment 1 1. 1 1 with 1 ug naloxonazine did not alter the antinocieeptive ·1, effects of M6G or sufentanil. Fig. 6 aiso shows that the anti'1ocieeptive potency of sufentanil in the formalin test

is much Iower than in the tail-immersion test and maximal effects are not observed at subtoxic doses. This 15

consistent with the previous observations (Abbott et al.

1986a) .

>, > r \ <, Table 2. presents statistical data for morphine.

M6G and sufentanil dose effect relations in the formalin

test. The EDSO of morphin(~ is altered by 1 ug naloxonazillp

pretreatment (t=- 6.2, p ~ 0.01), while the slope is

unchanged (t-1.64, P > 0.05). There is no significant

difference between naloxonazine pretreatment ùncl

naloxonazi ne vehi c le pre trea tmen t in the dose effect

relations of M6G and sufentanil.

Table 3 summarizes the effects of naloxonazine

pretreatment on the antinociception produced by morphin(l,

M6G and sufentanil in the two tests. These data sugge<::t

that, in the tail-immersion test, the antinociceptive

effect is mediated by opioid receptor subtypes which are:

b locke d by naloxonaz ine in a manne r cons i s tent w i th known

non-competitive systems (i.e. a decrease in slope). In

contrast in the formalin test the antinociceptive effect of

morphine appears to be "competitively" blocked by

naloxonazine in the sense tbat a parallel shift of the:

dose-effect relation was observed. The antinociceptive

effects of M6G and sufentani 1 were unaltered by

naloxonazine pretreatment in the formalin ~est.

29 , 3.3 Effects of Naloxonazine Pretreatment in Vivo on the " Disp1acement Binding of Opioids Using iden t ica1 conditions for naloxonazine

pretreatment in vivo, three opioid agonists were used to

disp1aee [3H]Naloxone binding in ra t brain membrane

receptors to see whether a specifie opioid receptor subtype

b10cked by naloxonazine cou1d be identified.

As shown in Fig.7, [3H]na1oxone displacement by

naloxone was reduced by naloxo~azine pretreatment on1y at a

single eompeting dose of naloxone (Sx10- 1 0M) and that by

only 17.2 %. A1though this is a very sma1l proportion of

total naloxone binding, it dose suggest that naloxonazine

preferentia11y oecupies high affinity naloxone binding

site s .

Na1oxonazine pretreatment decreased [D-A1a, N-

methyl-Phe 4 ,GlyS-ol]enkepha1in (DAMPGO) displacement of

[3 H]naloxone binding by 23.4 % (Fig. 8). DAMPGO is a

selective mu agonist (Fang et al. 1986; Gillan &

Kosterlitz, 1982). The effect of na1oxonazine on DAMPGO-

specifie [3 H]naloxone binding oceurs only at DAMPGO

c once n tra tians 10wer than 2 nM wh i ch sugges ts tha t this

nal ox onaz ine sensitive site has sorne eharacteristies

similar to those of mu sites. ( 30 However. as shown in Fig.9 na lox onaz i ne pre t rea tmen t is even more poten t ly di rec te d aga i ns t a

(3H ]naloxone binding site displaced by [D-Ala 2 -D

LeuS ] enkephalin (DADLE). a delta agonist. As shown in the graph, the first phase of the (3 H ]naloxone-DADLE disp lacement curve was obliterated by naloxonazine

pre t reatmen t . After na loxonaz ine trea tmen t 1 (3 H ]naloxone binding in the presence of 2 nM DADLE was only 54 6 % of that in vehicle-treated animals. The second pha&e of

[3H]naloxone binding, displaced by DADLE concentrations greater th an 80 nM, did not differ between vehicle- and naloxonazine- treated rats.

All these binding experiments were repeated ut least once and similar results were obtained.

31 Fig.l Effects of naloxonazine in the tail-immersion test.

A) The tail-immersion test was performed after pretreatment

wi th three doses of naloxonazine (icv) 4 h before testing.

B) Dose effect relation for sufentanil after pretreatment with three doses of naloxonazine 4 hours before tail- immersion testing.

32 14

~ 12 .,u ~10 ~ ~ 8 ~ ~ 6 o ~ 4 ...J ~ 2

o----~~~~~~~~~~~~~ __ __ o 0.2 1 6.5 NALOXONAZINE (ug i.e.v.)

. ,

100 Ovehicle en~ • nxozine 0.2 pg LtJ â nxozine 1.0 pg i 80 ... nxozine 6.5 pg ~ 0 60 it ..J ~ 40

:s0 ~ 20 • 0 , ~ \ 1 2 5 10 SUfENTANIL (ug/kg s.c.)

33 Fig.2 Effects of naloxonazine pretreatment on morphine

antinociception in the tail-immersion test.

Fig.3 Effects of naloxonazine pretreatment on the

antinociceptive effects of M6G in the tail-irnrnersion test.

- 34 - 100 c:J vehicle ~ nxozine 1~g en~ ; 80 o~ 60 2 ...J 40 ~ t Z

0:::~ 20 Wa..

MORPHINE (mg/kg s.c.)

100 c:::J vehicle ~ ~ nxozine 1~ i 80 l:S 60 it -J 40 ~ 120

O~--~~~L---~ __~ ____~~~~_ 100 2~ 500 WORPHINE-6-GlUCURONIDE (ng I.e.v.)

35 Fig.4 Dose effect relations for morphine with naloxonazine

1 ug or naloxonazine vehicle 4 hours before in the formalin

tes t. Efich point represents the me an ± SE. Naloxonazine produced a parallel shift the morphine dose effect relation ta the right.

36 -,

~ 100 Ovehicle (1) .nxozine lM T ~ 80 t; 60 ~ z 40 ~ ~ Cl:: . \ ~ 20 t- u~ oL------=~---__;;f------a:: W l ~-20L------~--~------~ 1 2 5 10 MORPHINE (mg/kg s.c.)

37 Fig.5 Dose effeet relations of morphine-6-g1ueuronide

(M6G) wi th naloxonaz ine 1 ug or naloxonaz ine vehiele (iev)

4 hours before in the formalin test. Eaeh point represents the rnean ± SE. Data show that the antinociceptive effect of

M6G was not altered by naloxonazine in the formalin test

Fig.6 Dose effect relations of sufentanil with naloxonazine 1 ug or na1oxonazine vehicle (i.e.v.) 4 h before in the formalin tes t. Data show that the antinocieeptive effeet of sufentanil was not bloeked by naloxonazine in the formalin test. Eaeh point represents the mean ± SE. Note that the potency of sufentanil is mueh lower than in the taU- immersion test and on1y about 50 % analgesia Is obtained at subtoxic doses.

38 ------

~ 120 Ovehiele en • nxozÎne 1Jj9 ~ 100 T ~ 80 ....en ~ z 60 40 a::~ ~ 20

(.)~ 15 0 ~ -20 ~ 50 100 1000 MORPHINE-6-GLUCURONIDE (ng i.e.v.) -- \

Ovehicle • nxozine 1JS9

z 40 ~ a: 20 ~ ~ 0 u« LaJ 0.. -20+------+- 1 1 2 5 10 20 SUFENTANIL (ug/kg s.c.)

39 Fig.? Displacement curves of naloxone to [3H1naloxone with

naloxonazine 1 ug Ci.c.v.) or r,aloxonazine vehicle 4 h

before sacrifice. Each point represents the mean ± SE of

triplicate determinations of binding. The data show naloxonazine pretreatment reduced [3H)naloxone binding only at the lowest concentration of naloxone.

40 Ovehicle 100r'~ • nxozine 1JI.9 ""' BO T ~ ....~ c 0 u 60 ....,~ ...., \ <.!)z 40 ë5z éD 20 ~ 0 1 1E-9 1E-8 1E-7 NALOXONE ( M)

41 Fig.8 Displacement curves of (3H ]naloxone by [D-Ala, N

methyl-Phe 4 , Gly5- o1 ]enkephalin (DAMPGO) after naloxonazine

1 ug or naloxonazine vehicle 4 h before sacrifice. Each

point represents the mean ± SE of triplicate determinations

of binding. The data show naloxonazine pretreatment altered

the first portion of DAMPGO displacement curve.

Fig.9 Displacement curve of [3H ]Naloxone by [D-Ala2 -D-

Leu 5 ]enkephalin (DADLE) after naloxonazine 1 ug or vehicle

4 h before sacrifice. Each point represents the mean ± SE of triplicate determinations of binding. The Data show naloxonazine pretreatment altered the displacement curve in

é1 greater extent.

42 Ovehlcle • nxazine 1J"9 ...... 80 e +Jc 8 60 ~

(!) 40 z ë5 z ID 20

o 1E-10 1E-9 1E-8 1E-7

-' \ DAMPGO ( M ) ;j

Ovehicle • nxozine 1JI9 e 4J C 0 () ~ • • (!) -z ë5 • z • âi 20

o 2E-9 1E-8 1E-7 DADLE (M ) TABLE 1. SLOPES AND E050'S FOR ·SUFENTANIL WITH NALOXONAZINE OR NALOXONAZINE VEHICLE PRETREATMENT IN THE TAIL-IMMERSION TEST

Naloxonaz/ne C 0.2 1 6.5 Cug)

N 20 15 15 15

r 0.9 0.81 0.84 0.71

( , Siope. 199 ! 27 90! 15 106 ! 19 90 ~ 28 ~.. t (elope) 3.63 2.82 2.77

P (slope) ~ 0.01 ~ 0.01 ~ 0.01

ED50 (ug) 7.7! 0.2 7.4 ! 0.5 9.7 ! 0.5 10.0 :!. 1.0

t CEDSO) 0.55 3.70 2.30

P (ED50) ~ 0.05 ! 0.01 ! 0.05

i> .. '

44 • _,__ ~o, __ ,_. ____ " __'-. ____F __ ~~"·_ ,-.---",? -.-~,.~~ ~-~'-,.-.,..",..'T"'l"Z"' ... .,. .. - ...... -~~...... ,....--, ...... -, ~..,..,~... --,.,....

-'t .-

TABLE 2. SLOPES AND EOSO'S FOR MORPHINE, MORPHINE-6-GLUCURONIDE AND SUFENTANIL WITH NALOXONAZINE OR NALOXONAZINE VEHICLE PRETREATMENT IN THE FORMALIN TEST

Agonist Morphine M6G Sufentanil Naloxonazine 0 1 Cug) 0 1 0 1

N 17 24 18 18 18 18 t- IJ'I r 0.83 0.70 0.76 0.67 0.51 0.63

SI opes 176 + 30 292 .! 64 141 + 30 108 !. 32 66 !. 29 81 ~ 25 t (slope) 1.64 0.76 0.41

P (slope) ~ 0.05 ~ 0.05 > 0.05 ED50 Cug) 4.94 !. 0.41 7.92 !. 0.25 233.8 + 57.1 246.7 !. 88.8 10.44 + 1.69 9.81 + 1.11 t CED50) 6.19 0.122 1.53 P CED50) < 0.01 > 0.05 ) 0.05 TABLE 3 SUMMARY OF EFFECTS OF NALOXONAZINE (ICV) ON DOSE EFFECT RELATIONS FOR THREE AGONISTS IN TWO PAIN TESTS

Agonist. Formalin test T ail immersion test

Sufentanil no antagonism non-parallel shi ft

Morphine parallel shift non-parallel shi ft

M-6-G no antagonism non-parallel shift

l \ .. 46 Chapter Four

.' DISCUSSION

Naloxonazine was developed primarily as a long 1 f; lasting "irreversible" mUl antagonist (Hahn and Pasternak,

1982) . However, there is sorne contradictory evidence

regarding both the receptor specificity and irreversible

antagonist properties of naloxonazine. The purpose of th('

present experiments was to use two different pain tests to

investigate the properties of naloxonazine and the

substrates of opioid antinociception. lt was found that

naloxonazine' s antagonist properties differ according lü

the nociceptive test and opioid agonist used.

4.1 Effects of Naloxonazine on Opioid Antinociception in

the Tail-immersion Test

In the tail-immersion test, naloxonazine by

itself did not alter baseline withdrawal latencies. l t

produced nonparallel right shifts of the dose (·[[pet

relations for sufentanil. Quantitative estimates of dOSl'

effect relation parameters for sufentanil indieate thal

when the naloxonazine dose was l ug (i.c.v.) or higher, th!·

slope was redueed and the EDSO was inereased. Pretreatm('nt

with 0.2 ug of naloxonazine (i.e.v.) reduced the slope of

the dose effeet relation but did not inerease th2 ED')()

47 significantly. Similar results were observed when morphine

or M6G were used as agonists. These results constitute the

only behavioural data suggesting that naloxonazine has

"irreversible" antagonistic properties. As such, they are

incomplete since respiratory depression precluded testing

higher doses of the agonist to determine if the maximal

effect was reduced. These results conflict with those

reported by Ling et al. , (1985). They showed that naloxonazine given systemically 24 h (10 mg/kg i.v.) before

testing produced paraI leI shift of morphine and DAMPGO dose effect relations to the right in the tai1-flick test.

The parallel shifts of op io id dose effect relations that have been described previously (Ling et al. ,1985) could be explained by slow elimination or dissociation of a competitive antagonist. The relative1y high dose of naloxonazine used systemically in Ling et al. ' s experiments suggest the possibility that a significant blood concentration of naloxonazine or an active metabolite was present when the pain test was performed. By its chemical structure, naloxonazine is a symme tr i c compound wi th two naloxone subs ti tuents br idge d by hydrazin. lt 1s proposed to be a "bifunctional" rnolecule of naloxone (Hahn and Pasternak, 1982) in that both "ends" may b ind to site s s imul taneous ly, grea tly enhanc i ng the affinity and increasing potency relative to naloxone. The

48 blood concentration of naloxonazine would be estimated to

be similar to those produced by a dose of 0.075 mg/kg, if

10 mg/kg were administered 24 h before sinee the half life

of naloxonazine is estimated to be 3 hours (Ling et al.,

1986). This eou1d be significant since na1oxone in the dose

of 0.1 mg/kg (s.e.) produces a 16-fold right shift of

opioid dose effeet relations in the tail-immersion test

(Abbott et al., 1986). In addition, naloxonazine has been

demonstrated to possess both Ireversib1e" and

"irreversible" (i.e.wash-resistant) antagonist properties

The specifie lIirreversible" antagonist properties of

naloxonazine are exhibited in vitro only at the lowcr

concentration of naloxonazine or after extensive washing of

the naloxonazlne treated membrane (Hahn et al. 1982; Hahn

et al. 1985) . Thus it is possible that sufficient

naloxonazine remains in circulation as much as 24 h ùfter

systemic pretreatment to act as a competitive antagonist a~

found by Ling et al. Also, the formation of an activ(·

metabolite after systemic adminstration of a high dose of

naloxonazine ean not be ruled out.

The results presented here for the tail-immersion

test provide evidence that naloxonazine can produce a non-

parellel shift dose effect relation which is consisle'lll

with lIirreversible" antagonist properties suggested ln

vitro binding studies (Hahn & Pas te rnak, 1982; Johnson & ~. 49 , Pasternak, 1984). These non-para11el antagonist properties { are demonstrated by using a 10w dose administered centra11y

and relying on redistribution ta reduce the concentration

avai1ab1e for competitive binding.

4.2 Effects of Naloxonazine on Opioid antinociception in

the Formalin Test

ln the formalin test, naloxonazine produced a

"parallel" shift of morphine dose effect relation to the

right. This is characteristic of competitive antagonist

(Goldstein et al. 1974; Tallarida and Jacobs 1979).

The trivial explanation for the parallel

antagonism is that slow leaching of naloxonazine from

nonspecific binding sites leads to a significant

concentration of free drug which interacts reversibly and

non-selectively with sorne opioid receptors (Hahn et al.

1982; Hahn et al. 1985) . The total concentration of

naloxonazine wou1d be expected to be similar to those

produced by a dose of 0.003 mg/kg at most, if 1 ug/rat dose

were administered. This concentration of naloxonazine is

unlikely to produce significant antagonism after 4 hours of

re-distribution. Thus, slow leaching of naloxonazine from

nonspecific binding sites is insufficient to explain the

parallel shift of morphine dose e ffec t relations by f .; 50 naloxonazine pretreatment in the present experiment

Clearly, sorne other mechanisms must be involved in the parallel antagonism produced by this " irreversible" antagonist.

The opioid receptor sub type s involved in modulation of pain are complex. Activation of either mu or delta receptors results in analgesia (Porreca et al. 1987,

Mathiasen et al. 1987). Furthermore, subanalgesic doses of a delta agonist or antagonist can modulate the a!1algesic effect of a mu agonist (Vaught et al. 1982). Recent evidence suggests that mu and delta receptors may eXIst both independently and as a mu-delta receptor complex

(Heyman et al. 1989). The subtype of mu receptor in the mu- de 1 ta comp lex is suggested to be a mU2' Within the receptor complex, it is proposed that allosteric modulation of either the affinity or the coupling of one component of the receptor complex, could be caused by binding of an agonisl or antagonist at the other component (Bowen et al 1981,

Vaught & Takemori, 1977; Holaday et al. 1986).

In the formalin test, evidence suggests that the opioid receptor types involved are different from thosc which mediate opioid antinociception in the tail-immersion test and that mu receptor activation is particularly important in the latter. For example sufentanil, a potent

51 mu agonist, is far less potent in the formalin test th an in the tai1-immersion test (present study; Abbott et al.,

1986) . In the forma 1 in test, beta-funaltrexamine an alkylating agent with se1ectivity for the mu receptor subtype (Takemori et al. 1980; Tarn et al. 19.85) was found to b lock the analge sic e ffec t of mo rphine comp 1 e te 1y , suggesting the involvement of mu receptors (Chen and

Abbott, 1988; Abbott submitted). lt has been reported that

DPDPE, a delta-selective pept ide has ant inoc icepti ve effects in the formalin test (Fanselow et al. 1989) .

Furthermore ICI-174864, a partial delta agonist (Cotton et al. 1981) poten t iate s the antinociceptive effects of c10nidine while naloxone antagonizes these effects

(Mastrianni et al. 1989).

The data presented here, taken in conjunction with previous work discussed above cou1d be explained if several assumptions were made. First, analgesia in the tai1- immersion test is proposed to be mediated by interaction cf an opioid agonist with the "mu1" receptor for which naloxonazine is a "noncompetitive" antagonist

(Fig.10). Second, the receptor substrate of the antinociceptive effects of opioids in the forma1in test is a mU2-delta comp1ex as illustrated in Fig. 11. AIl three agoni s ts i nterac t with the receptor which is modulated by an associated "delta" receptor. Sufentanil and

52 M6G are shown with tip truncated to imp1y that the bindin!j ... or coupling is una1tered under circumstances that reduc('

the potency of morphine. The binding of na1oxonazine ta th"

"delta" receptor, might then alter the conformation of th"

"mu2" receptor and reduce the affinity or coup1ing of

t morphine to the receptor comp1ex (Fig.11 B). The affini ly

or coupling of the M6G and sufentanil would remJin

una l tere d becaus e of di ffe rence s in the s truc ture 0 f the

compounds. Note that M6G is drawn as a mo1ecule that could

potential1y bind to the "delta" receptor and increase "mu2"

binding. This is do ne to exp1ain the increased bindinfj of

opioid ligands observed by Abbott and Pa1mour (1988) nt low

concentrations of M6G.

There are sorne data consistent with

a110steric models proposed in the forma1in test. In the

displacement binding of etorphine, dihydromorphine and

naloxone, M6G at the dose lower than 10- 9 M enhnnced the

binding of three opioids. M6G displaced three opioids in d

dose dependent manner when the dose of M6G higher than

lO-9 M (Abbott and Palmour, 1988). This suggested that M6G

bound to two opioid receptor types. One of them ean compete

three opioids used, the other can modulate the receptor

which bound three opioids used. This modulation of opioid

binding by M6G was observed in vivo, too. The subanalge~,ic

doses of M6G increased the slopes of the dose effccl , ' ",

53 relati'lns for morphine in the formalin test in a

preliminary studied in our laboratory (unpublished).

4.3 Effects of Naloxonazine Pretreatment on Opioids

Displacement Binding to the Rat Brain Membrane

(3H]naloxone is a general opioid ligand. lts

binding to opioid receptors can be displaced to a greater

or lesser extent by all opioids (Pert & Snyder, 1973).

[3H]Naloxone has higher affinity in vitro for the mu

receptor and lower affinity (around lÜ-fold) for delta and

kappa receptors (Pfeiffer & Herz, 1982).

With the large body of evidence for the existence

of multiple subtypes of opioid receptors (Martin et al.

1976; Lord et al. 1977), many investigators have attempted

to assign specifie pharmaco10gica1 properties to one or

more of these various receptors. For this purpose, many

opioid subtype specifie agonists and antagonists have been

developed, including DAMPGO for mu, OAOLE and DPOPE for

delta and EKC and U50,488 for kappa receptors. Nonetheless,

the binding selectivity profiles indicate that no matter

how specifie a given analogue is for one subtype of opioid

receptor, there is a1so residual binding to other opioid

receptor subtypes as the concentration of compound i5

increased (Goldstein, 1987). However, as discussed by ( 54 Goldstein (1987), a competing ligand with very high selectivity for the secondary sites of radioligand binding wi Il first (at low concentration) displace whatever radioligand is bound to those sites, then will yield

As i11ustrated in Fig.7, Fig.8 and Fig.9,

Na1oxone has high affinity for both mu and delta r e cep t 0 r s. DAM P G0 i sas e 1 e c t ive 1 i g and for mur e cep t 0 r ) whi1e DADLE is a delta selective ligand. The results presented here suggest that naloxonaz i ne pretreatmcnl reduced binding to both the mu and delta receptors) but with higher potency towards the delta receptor in present conditions. This is consistent with the findings of Dray et al., (1987). Naloxonazine antagonized both DAMPGO(mu) - and

DPDPE(delta) - induced inhibition of reflex urinary bladder contraction and the effect of naloxonazine on DPDPE was demons t r a ted to b e more prolonged than tha t 0 f DAM PCO )

55 suggesting that naloxonazine binds "irreversibly" to delta

receptors. The results are also consis tent wi th the

findings of Cruciani (1987). They showed that while t naloxonazine has a high affinity for mUl site, it may a1so act -'.: mU2 and delta sites 1

4.4 General Discussions

Naloxonazine antagonizes three opioid agonists

differently in the two pain tests. This is consistent with

the bi-phasic antagonistic properties of na1oxonazine in

vivo (Ling et al., 1986) and bi-phasic disp1acement curve

in vitro (Hahn et al. 1982) as discussed in the literature

review.

These resul ts suggest that naloxonazine has

different properties in the tail-immersion and the formalin

tests. In the taU-immersion test, na1oxonazine displayed

irreversible properties which confirms that it i5 a long

lasting "irreversible" antaganist. In the formalin test,

naloxonazine effects were c1early "reversible".

In the displacement binding experiment,

naloxonazine pretreatment decreased both the binding of

DAMPGO (mu) and DADLE (de l ta) up to 23.4% and 46.4%

respectively which confirmed the blockade of bath mu and ( 56 delta receptors by naloxonazine under the present naloxonazine pretreatment regimen.

Combining the results observed both in vivo and in vi tro, it is proposed here that one of the antinociceptive systems activated by opioid agonists in the formalin test is mediated by mu-delta receptor complex.

Although there is evidence to suggesting allosteric inte rac ti on be tween op io id recep tor sub type s, the us e 0 [ this model to explain the antinociceptive effect of opioid in the formalin test is first presented here. To further examine the hypothesis, both in vivo and in vitro experiments will be performed. The dose effect relations for morphine with subanalgesic dose of M6G in the formalit\ test as well as the displacement binding experiments of e torphine , dihydromorphine and naloxone by M6G will be perfûrmed again but with the pretreatment of naloxonazine.

According to the proposed model, the potentiation of morphine .snalgesic effect by subanalgesia dose of M6G and enhancement of three opioid binding by low concentration of

M6G will be obliterated by the pretreatment of naloxonazine. Further experiments using specifie mu and delta agonist or antagonist will be proposed. Such as, dose effect relations for morphine will be tested with the pretreatment of ICl-174,864, a delta receptor antagonist

(Costa and Herz, 1986) or its vehicle or DPDPE, a Séllective

57 delta agonist in the formalin test. It is expected that ( parallel right shift of dose effect relation for morphine

will be observed in the pretreatment of delta antagonist

and the opposite alteration may be observed in the

pretreatment of delta agonist. AIso, saturation binding

assays of morphine and sufentanil in the rat brain membrane

will be performed after pretreatment with naloxonazine in

vivo. Both Bmax of high affinity sites and kd of low

affinity sites of morphine and Bmax of sufentanil are

expected to be decreased following pretreatment wi th

naloxonazine.

58 Fig.ID The proposed opioid receptor substrates mediating

antinociception in the tail-immersion test.

A) AlI three agonists can bind to and activate the "muI"

receptor.

B) Naloxonazine pretreatment blocks the "mul" receptor and

reduces the antinociceptive effects of the agonists in il manner that is consistent with irreversible antagonism.

59 . \ 1..

MORPHINE [:>

ANTINOCICEPTION SUFENTANIL D IN THE TAIL·IMMERSION TEST

M·S-G D

. \

MORPHINE c:> , SUFENTANIL D , ANTINOCICEPTION BLOCKED

M·e·G D

NAlOXONAZlNE

, 1

.. ) Fig. Il The proposed opioid receptor substrates mediating

antinociception in the formalin test. 1, 1 ,) •

A) All three agonists interact with the "mu2" receptor.

Sufentani1 and M6G are shown with the tip truncated ta

imp1y that the binding or coupling is una1tered under

circumstances that reduce the potency of morphine. Note

also that M6G is drawn as a mo1ecule that could potentially

bind to the "delta" receptor and increase "mu2" binding.

B) Na1oxonazine binds to the delta receptor and alters the

affinity or coup1ing of the "mu2" receptor 50 that the

effects of morphine are decreased but not those of

sufentanil or M6G.

61 MORPHINE [>

SUFENTANIL () , ..... ANnNOCICEPTION IN THE FORMAlIN TEST M·8·G D

MORPHINE [>

DECREASED POTENCY SUFENTANIL (:) -==--- OF MORPHINE

M·6-G D

NALOXONAZINE

62 Chapter Five

CONCLUSION , .i

The results presented here support the

proposition that naloxonazine is a long lasting antagolli&t

It clearly has sorne selectivity in that resplt"atory

de pre s san t e f f e c t s 0 f 0 P i 0 id s are no tan t a go n i z e d li 0 W e v l' r ,

the data presented here suggest that it interacts witb 1lI01l'

than one opioid receptor subtype. In the tail-imlllel"&1011

test, naloxonazine produced a nonparallel shift the do'..('

effect relations of sufentanil, morphine and M6G to lh('

right. This is consistent with long lasting "irreversiblp"

antagonist properties. Whe ther the receptor subtypl'

involved is a subset of mu receptors, named mUl hy

Pasternak and his colleagues is not clear. In the forllla] jll

test, naloxonazine produced a parallel shift of lIlorphillt'

dose effect relation but failed to alter the dose ef[ect

relation of sufentanil or M6G. On the bas is of evidence i Il

the literature, it has been argued that the effect'.. in lll(

formalin test may result from long la5ting blockade of

delta receptors. , .- These data add to other evidence that the opiold l , receptor subtypes involved in the formalin te & t dr(; i. : ~ different from those involved in the tail-immersion test ,f

63 An allosteric interaction between mu-delta receptor l subtypes is proposed in the antinociceptive effect of

opioid in the formalin test. To the ex tent tha t the

formalin test is a valid model of pain associated with

damaged tissue in human, an understanding of interactions

of opioid receptor subtypes in this pain model may le ad to

novel strategies for the development of analgesic agents.

64 .. REFERENCES:

Abbott, F.V. & Melzack, R. "Brainstem lesions dissocLllv

neural mechanisms of morphine ana1gesia in different ktndc,

of pain" Brain Res., 251: 149-155 (1982a).

Abbott, F.V.,Me1zaek, R. & Samuel, C. "Morphine ané.llge~i

in the tai1-fliek and formalin pain tests is mediate·ct by

different neural systems" Exp.Neurol., 75: 644-651 (1982b)

Abbott, F.V.,Franklin, K.B.J. & Libman, R.B. "A dose-ratio

study of mu and kappa agonists in formalin and therllldl

pain" Life Sei., 39: 2017-2024 (1986a).

Abbott, F.V.,Franklin, K.B.J. & Conne11, B. "The strps<, oi

a nove1 environment reduees forma1i~ pain: possible rol~ 01

serotonin" Eur.J.Pharmaeol., 126: 141-144 (1986b).

Abbott, F.V. ,Rubin, M.B. & Franklin, K. B J "5-IIT

precursors antagonize morphine analgesia in the [ormallll

test" Soc.Neurosci.Abs., 13: 1590 (1987).

Abbott, F.V. & Pa1mour, R.M. "Morphine-6-g1ueurollidp

a n a 1 g e sic e f f e c t san d r e cep t 0 r b in di n g pro fil e i n LI t <, "

Life Sei., 43: 1685-1695 (1988).

65 ~. tr'''' - " r-1r.:.1~""'I"Plr~~""''''''''9''''i!IoI''''''''''''''''''''''''''''______------

Abbott, F.V. "Morphine-6-glucuronide produces place

preference conditioning in the rat"

Pharmacol.Biochem.Behav., submitted:(1989).

Basbaum, A. 1. ,Marley, N. , O'Keef, J. & Clanton, C.H. ,

"ReversaI of morphine and stimulus produced analgesia by

subtotal spinal cord lesions" Pain, 3: 43-56 (1977).

Basbaum, A.1. & Fields, H.L "Endogenous pain control

mechanisms: bra i ns tem pa thways and endorphi n ci r c ui try"

Ann. Rev.Neurosci., 7: 309-338 (1984).

Bonnycastle, D.D. ,Cook, L. & Ipsen, J.Jr. "Action of sorne

ana1gesic drugs in intact and chronic spinal rats" Acta

Pharmaco1.Toxicol., 9: 322-336 (1953).

Bowen, W. D. ,Gentleman, S. , Herkenham, M. & Pert, C.B

"Interconverting mu and beta forms of the opiate receptor

in rat striatal patches" Proc.Natl.Acad.Sci., 78: 4818-4822

(1981) .

Chen, L. & Abbott, F. V. "0p ioid receptor subtypes mediating

antinociception in the formalin and tail-immersion test"

Soc.Neurosci.Abs., 14: 713 (1988).

Il Christensen, C.B. & Jurgensen, L.N. "Morphine-6-glucuronide

has high affini ty for the opioid receptol'''

Pharmacol.Toxicol., 60: 75-76 (1987).

Costa, T. & Herz, A. "Antagonists with negative intrinsic

activity at delta opioid receptors coupled to GPT-binding

proteins", Pro.Natl.Acad.Sci.USA, 86: 7321-7325 (1986).

Co t ton, R., G i 1 es, M. G. ,M i Il e r , L., S ha w , J. S . & T i 1II lU C" D

"ICI-l74864 a highly selective antagonist for the opioid

; delta-receptor" Eur.J.Pharmacol., 97: 331-332 (1981). ,1 f

Cruc i .:ini , R.A. ,Lutz, R.A. ,Munson, P.J. & Rodbard, D.

"Na1oxonazine effects on the interaction of enkephalin

ana10gs with Mu-l, Mu and Delta opioid binding sites in l'dl

brain" J.Pharmacol.Exp.Ther., 242: 15-20 (1987).

D'Amour, F.E. & Smith, D.L. liA method for determining los!>

of pain sensation" J.Pharmacol., 72: 74-79 (1941).

Dah1s trom, B.E. & Paa1zow, L.K. "PharmacokinetlC& of

Morphine in plasma and discrete areas of the rdt braitl"

J.Pharmacokinet.Biopharmacol., 3: 293-302 (1975)

Dahlstrom, B.E.,Paalzo~." L.K.,Segre, G .. & Agren, /\ J

Relation between morphine pharmacokinetics and ana1gesia"

67 • l J .Pharmacokinet.Biopharmacol., 6: 41-53 (1978).

De Lange, S. ,Boscoe, M.J. ,Stanley, T.H. & Pace, N.

"Comp ar ison of sufe Itani 1-02 and fentany1- 02 for cor onary

artery surgery" Eur.J.Pharmacol., 87: 209-225 (1983).

Dray, A. ,Nunan, L. & Wire, w. "Naloxonazine and

1 opioid-induced inhibition of reflex urinary b1adder

contractions" Neuropharmacol., 26: 67-74 (1987). t Dubuisson, D. & Dennis, S.G. "The formalin test: A

quantitative study of ,the ana1gesic effects of morphine,

meperidine and brain stem stimulation in rats and cats"

Pain, 4: 161-174 (1977).

Fang, F.G.,Fie1ds, H.L. & Lee, N.M. "Action at mu receptor

is sufficient to exp1ain the supraspinal analgesic effects

of opiates" J.Pharmacol.Exp.Ther., 238: 1039-1043 (1986).

Fanselow, I1.S.,Calcagnetti, D.J. Oc Helmstetter, F.J. "Delta

opioid antagonis t, 16 -me cyprenorphine, selectively

attenuates conditiona1 fear- and DPDPE-induced ana1gesia on

the formaI in test" Pharmacol.Biochem.Behav., 32: 469-473

(1989).

Franklin, K.B.J. & Abbott, F.V. Neuromethods: Vol. 13,

68 ~sychopharmaco1ogy (Bou1ton, A.A. , Baker, c, B. &

Greenshaw, A. J ., eds.), Humana Press, C1ifton, NJ. pp.

145-216, (1989).

Gi11an, M.G.C. & Koster1itz, H.W. "Spectrurn of the mu-, de1ta- and kappa-binding sites in homogenates of rat brain"

Br.J.Pharmacol., 77: 461-469 (1982).

Go1dstein, A. ,Lowney, L.I. & Pal, B.K. "Stereospecific and nonspecific interactions of the morphine congener

1evorphano1 in subce11u1ar fractions of mouse brain"

Proc.Natl.Acad.Sci., 68: 1742-1747 (1971).

Go1dstein, A., Aronow,L., Kalman, S.M. "Princip1es of drug action: the basis of pharmac01ogy", A Wi1ey biomedica1- hea1th publication. John Wi1ey and Sons, Inc. (1974).

Go1dstein, A. "Binding se1cctivity profiles for ligands of multiple receptor types focus on opioid receptors" TIPS, 8.

456-459 (1987).

Gaode, P.G.,Rhodes, K.F. & Waterfa11, J.F. "The analgesic and respiratory effects af meptazina1, morphine and pentazocine in the rat" J.Pharmacol., 31: 793-795 (1979).

Hahn, E. F. & Pasternak, G.W. "Na1oxonazine, a patent,

69 long-lasting inhibitor of opiate binding sites" L1fe Sci.,

31: 1385-1388 (1982).

Hahn, E.F.,Carroll-buatti, M. Pasternak, G.W.

"Irreversible opiate agonists and antagonists: the

14 - hydroxydihydromorph inone az ines" J. Ne urosc i ., 2: 572 - 576

(1982) .

Hahn, E.F. ,Nishimura, S.,Goodman, R.R. & Pasternak, G.W.

"Irreversible opiate agonists and antagonists.II. Evidence against a bivalent mechanism of action for opiate azines and diacylhydrazones" J .Pharmacol. Exp.Ther., 235: 839-845

(1985) .

Hardy, J.O. ,Wolff, H.G. & Goodell, H. "Studies on pain.

Measurement of the effect of morphine, codeine and other opiates on the pain thresho1d and an analysis of their rel a t ion t 0 the pa in exp e rie n ce Il J. Cl in. 1 n "Te st., 1 9: 649

(1940).

Helmstetter, F.J. & Landeira-Fernandez, J. "Conditional hypoa1gesia on the forma1in test i5 blocked by naltrexone applied to the PAG" Soc.Neurosci.Abs., 15: 155 (1989).

Heyman, J.S. ,Williams, C.L. ,Burkes, T.F. ,Mosberg, H.I. &

Porreca, F. "Dissociation of opioid antinociception and

70 central gastrointestinal propulsion in the mouse: studies

with naloxonazine" J.Pharmacol.Exp.Ther. , 245 : 238-243

(1988) .

Heyman, J.S.,Jiang, Q. ,Rothman, R.B. ,Mosberg, H. l &

Porreca, F. "Modulation of mu-mediated antinociception by

delta agonists: characterization with antagonists"

Eur.J.Pharmacol., 169: 43-52 (1989).

Holaday, J.W., Torte11a, F.C., Maneckjee, R. & Long, J.B.

NIDA Monograph Series. "Opiate Receptor Subtypes and Brai 11

Function" Vol 71 (eds.), US Govt., Washjngton pp. 173-188,

(1986) .

Hughes, J. ,Smith, T.W. ,Kosterlitz, H.W ,Fothergill,

L.A. ,Morgan, B.A. & Morris, H.R. "Identification of two

related pentapeptides from the brain with potent opiate

agonist activity" Nature, 258: 577-579 (1975).

Janssen, P.S.J. ,Neimegeers, C.J.E. & Dony, J.G.H. "The

. t' inhibitory effect of fentanyl and other morphine-1ik2

ana1gesics on the warm water induced tdil withdrawal reflex

in rats" Arzneim. -Forsch., 13: 502-507 (1963).

Johnson, N. & Pasternak, G.W. "Binding of [3 H ]Naloxonaziné

t 0 rat br a in me mb r an es" Mo 1. Ph a r mac 0 1., 2 6: 4 7 7 - 4 8 3 (1 9 8 Il ) .

71 Kelly, S.J. & Franklin, K.B.J. "Evidence that stress

augments morphine ana1gesia by increasing brain tryptophan"

Neurosci.Lett., 44: 305-310 (1984).

Leysen, J.E.,Gommeren, W. & Niemegeers, J.E.

,,[3 H ]sufentani1, a superior ligand for mu-opiate receptors:

bindi.g properties and regiona1 distribution in rat brain

and spinal cord" Eur.J.Pharmacol., 87: 209-225 (1983)

Ling, G.S.F. & Pasternak, G.W. "Morphine cata1epsy in the

rat: invo1vement of mu-1 (high affinity) opioid binding

sites" Neurosci.Lett, 32: 193-196 (1982).

Ling, G.S.F .• Spiege1. K. ,Lockhart, S.H. & Pasternak, G.H.

"Separation of opioid ana1ges ia from respi ra tory

depression: evidence for different receptor mechanisms"

J.Pharmacol.Exp.Ther., 232: 149-155 (1985).

Ling, G.S.F.,Simantov, R.,C1~rk, J.A. & Pasternak, G.\L

"Na1oxonazine actions in vivo" Eur.J .Pharmacol., 129: 33-38

(1986) .

Llewelyn, M.B. ,Azami, J. & Roberts, M.H.T. "Brainstem

mechanisms of antinoc iception: effects of e1ectrical

stimulation and injection of morphine into the nucleus

raphe magnus" Neuropharmacol., 25(7): 727-735 (1986).

- Lord, J.A.H. ,Waterfield, A.A. ,Hughes, J. & Kosterlitz, H.W

" Endogenous opioid peptides: multiple agonists d Ild

receptors" Nature, 267: 495-499 (1977).

Martin, W.R. ,Eades, C.G.,Thompson, J.A.,Huppler, RE.

Gilbert, P.E. "The effects of morphine- and nalorphine-likp

drugs in the nondependent and morphine-dependent chrollll

spinal dog" J.Pharmacol.Exp.Ther., 197: 517-532 (1976)

Mastrianni, J.A.,Abbott, F.V. & Kunos, G. "Actlvdlioll of

central mu-opioid receptors is invo1ved in cIo Il i d i Ill'

analgesia in rats" Brain Res., 479: 283-289 (1989).

Mathiasen, J.R. ,Raffa, R.B. & Vaught, J.L. "C57BL/6S-bg

( Bei g e ) mie e : d i f fer e n t i a 1 sen s i t i vit yin the t ail - f 1 ici:

test to centrally administered mu- and delt.1-opioid

receptor agonists" Life Sei., 40: 1989 (1987)

Mcgilliard, K.L. & Takemori, A.E. "Antagonism by lIaio;,:oll{

of na r c 0 tic - i n duc e d r e spi rat 0 r y de pre s s ion and and il'. (' S Iii"

J.Pharmacol.Exp.Ther., 207: 494-503 (1978).

McPherson, G.A. Kinetic, ERDA. Ligand. Lowry A co110cti OH of radioligand binding analysis programs, Els8vier Science:

Publishers BV, (1985 )

73 Moste11er, F. & Tukey, J.W. Handbook of Social psycho1ogy

(Lindsey, G. & Aronson, E., eds.), Addison-Wesley, Reading

Mass. pp. 80-203, (1968).

Niemegeers, C.J.E.,Schel1ekens, K.H.L.,Van Bever, W.F.M. &

Janssen, P.A.J. "Sufentanil a very potent and extremely safe intravenous morphine-1ike compound in mice, rats and dogs" Arzneim. -Fot'sch., 26: 1551-1556 (1976).

Oguri, K.,Isomi, Y-M.,Junko, S.,Takaaki, H. & Hidetoshi, Y.

"Enhanced binding of morphine and nalorphine to opioid delta receptor by glucuronate and sulfate eonjugations at the 6-position" Life Sei., 41: 1457-1464 (1987).

Osborne, R.,Joe1, S.,Trew, D. & Slevin, M. "Analgesie aetivity of morphine-6-glucuronide" Lancet, 1: 828 (1988).

Osborne, R.J.,Joe1, S. P. & Slevin, M. L. "Morphine intoxication in rena1 failure: the role of morphine-6-g1ucuronide" British Medical Journal, 292:

1548-1549 (1986).

Pasternak, G.W.,Chi1ders, S.R. & Snyder, S.H. "Na1oxazoe, a long lasting opiate antagonist: Effeets on ana1gesia in intact animaIs and on opiate receptor binding in vitro"

74 J.Pharm.Exp.Ther., 214: 455-462 (1980a).

Pasternak, G.W. ,Zhang, A. & Teeott, L. "Deve1opmental differences between high and 10w affinity opiate binding sites: Their relationship to ana1gesia and respiLltorv depression" Life Sei., 27: 1185-1190 (1980b).

Pasternak, G.W.,Bodnar, R.J.,C1ark, J.A. & lnturrisi, CE

"Morphine-6-g1ucuronide, A potent mu agonist" Lifp Sei

41: 2845-2849 (1987).

Paxinos, G. & Watson, C. The rat brain in stel'eot<1'll' eoordinates, Academic press, Sydney, Orlando, S<111 DiC'go,

New York, Austin, London (1986)

P e r t , C . B . & Sn y der, S. H . " Pro p e r t i e s 0 f 0 pia te - r ccc' Il lo r binding in rat brain" Proc. Natl Aead. Sc i . 7 O. 2 2 4 3 - 2 ') l, /

(1973) .

P f e if fer, A . & He r z , A. "0 i s cri min a t ion 0 f th r e e 0 p i.1 l c' receptor binding sites with the use of a computerized curV0 fitting technique" Mol.Pharmaeol., 21: 266-271 (1982)

Porreea, F. , Heyman, J.S.,Mosberg, H.I.,Omnaas, J . R. f:.

Vaught, J.L. "Role of mu and delta reeeptors in L he supraspinal and sp i nal analgesic effects of

75 [D-Pen2,D-Pen5]enkephalin in the mouse"

J.Pharmacol.Exp.Ther., 241: 393 (1987).

Proudfit, H.K. & Anderson, E.G. "Morphine analgesia

bloekade by raphe magnus lesions", Brain Res., 98: 612-618

(1975) .

Roberts, M.H.T. "5-Hydroxytryptamine and antinoeieeption"

Neuropharmaeol., 23: 1529-1536 (1984).

Ryan, S.M.,Watkins, L.R.,Mayer, D.J. & Maier, S.F. "Spinal

pain suppression mechanisms may differ for phasie and tonie

pain" Brain Res., 334: 173-175 (1985).

Sawe, J. Advanees in Pain Researeh and Therapy, Vol 8

(Fo1ey, K.M. & Inturrisi, C.E., eds.), Raven Press, New

York pp. 45-55, (1986).

Sawynok, J.,Pinsky, C. & La Bella, P.S. "On the specifieity of naloxone as an opiate antagonist" Life Sci., 25:

1621-1632 (1979).

Schumacher, A,G.,Goodell, H.,Hardy, J.D. & Wolff, H.G.

"Uniformity of the pain threshold in man" Science, 92:

110-112 (1941).

76 Shimomura, K. ,Kamata, O.,Ueki, S. , Ida, S. ,OtUl'l,

K. ,Yoshimura, H. & Tsukamoto, H. "Analgesie effectb 01 morphine glucuronide" Tohoku J.Exp.Med., 105: 45-52 (1911)

Simone, D.A. ,Bodnar, R.J. & Pasternak, G.W. "Invo1vement 01 opioid reeeptors subtypes in rat feeding behavlor" ~

Sei., 36: 829-833 (1985).

Sim 0 ne, D. A. ,B 0 d na r , R . J . , P 0 r t z lin e, T. & Pas ter n il k, G \~

"Antagonism of morphine ana1gesia I>y i n t r ace r e b r 0 ven tri cul a r n a 1 ° x 0 n il Z 1 Il C· "

Pharmaeol.Biochem.Behav., 24: 1721-1727 (1986).

Sinclair, J.G.,Main, C.D. & Lo, G.F. "Splfldl v'> supraspina1 actions of morphine on the rat t d i 1 - f 1 1 (. l' reflex" Pain, 33: 357-362 (1988).

Spiegel, K.,Kourides, I.A. & Pasternak, G.W. "Prolactll1 dllcl growth hormone release by morphine in the rat. dlffet'l'lIl reeeptor meehanisms" Science, 217: 745-747 (1982)

Takemori, A. E. ; Larson, D. L. & Portoghese, P S " Tht· irreversible narcotic antagonist and reversib1p aeonist ie properties of the fumarate methyl ester derivativp of na1trexone" Eur.] .Pharmaeol., 70: /145-451 (1980).

77 Ta11arida, R.J. & Jacobs, L.S. Dose-response Relation in

Pharmacology, Springer-Ver1ag, New York, (1979).

Tarn, S.W.; Nickolson, V.J. & Liu-Chen, L.

funa1trexamine binds covalently to brain opioid receptors"

Eur.J.Pharmacol, 119: 259-260 (1985).

Thorn-Gray, B.E.,Levitt, R.A.,Hi11, J. & Ward, K. "A

neuroanatomical study of analgesia and catatonia induced by

etorphine" Neuropharmaco1., 20: 763-767 (1981).

Tyers, M. B. "A classification of opiate receptors that

mediate antinociception in animaIs" Br.J.Pharmacol., 69:

503-512 (1980).

Vaught, J.L. & Takemori, A.E. "Interactions of leucine and

methionine enkephalin with morphine in vitro and in vivo"

Pharmacolo.,&.1n, 19: 189 (1977).

Vaught, J.L.,Rothman, R.B. & Westfall, T.C. "Mu and delta

receptors: their role in analgesia and in the differential

effects of opioid peptides on analgesia" Life Sei., 30:

1443-1455 (1982).

Ward, S. J. ,Portoghese, P.S. & Takemori, A.E.

"Pharmacological characterization in vivo of the novel

78 opiate, beta-funa1trexamine" J,Pharmaco1,Exp,Ther, 220 .

494-498 (1981).

Wo1ozin, B.L. & Pasternak, G,W. "Classification of lI1u1tip1p

morphine and enkepha1in binding sites in the central

nervous system" Proc.Natl.Acad,Sci., 78: 6181-6185 (1981)

Wood, P.L"Richard, J.W. & Thakur, M. "Mu opiate receptot'~

differentiation with kappa agonists" Li fe Sel , 31

2313-2317 (1982).

Wood, P.L. & Pasternak, G.W. "Specifie opioie!

isoreceptor regu1ation of nigrostriatal neurons: in vivo

evidence with na1oxonazine" Neurosci.Lett 37: L 9 1 - L C)J

(1983).

Wuster, M ,Schulz, R. & Herz, A. "The direction of OpiOld

agonists towards mu-,delta- and epsilon-receptor!:. 111 tllC' vas de ferens of the mous e and the ra t" Li fe S (' i 27

163-170 (1980),

Yoshimura, H. ,Oguri, K. & Tsukamoto, H "Isolation Lllle!

ide n tif i c a t ion 0 f m 0 r phi n e glu c u r 0 nid es in uri n e and h li (.

of rabbits" Biochemical Pharmacol., 18: 279-286 (1969)

Yoshj mura 1 H, 1 Ida, S, ,Oguri, K. & Tsukamoto, F

79 , " Bioehemica1 bas is for analgesic activity of .\ • morphine-6-g1ucuronide-I" Bi ochemi cal Pharmaco1. , 22:

1423-1430 (1973).

2hang, Z. & Pasternak, G.W. "Opiates and enkepha1ins: a

cornmon binding site mediates their ana1gesic actions in

rats" Life Sei., 29: 843-851 (1981).

2si11a, G.,Racagni, G.,Cheney, D.L. & Costa, E. "Constant

rate infusion of deuterated phosphorycho1ine to measure the

effeets of morphine on acety1cho1ine turnover rate in

specifie nue1ei of rat brain" Neuropharmacol., 16: 25-31

(1977) .