CI 988, an antagonist of the cholecystokinin-B receptor, potentiates endogenous opioid-mediated antinociception at spinal level

Neuropeptides(1997) 31 (3), 287-291 © PearsonProfessionalLtd 1997

CI 988, an antagonist of the cholecystokinin.B receptor, potentiates endogenous opioid.mediated antinociception at spinal level X.-J. Xu,* A. Elfvin,* J.-X. Hao,* M. C. Fournie-Zaluski,* B. P. RoquesJ Z. Wiesenfeld-Hallin* *Department of Medical Laboratory Sciences and Technology, Section of Clinical Neurophysiology, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden tINSERM U-266, Paris, France

Summary The effects of RB 101 {N-[(R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-l-oxo-propyl]-Lphenylalanine benzyl ester}, a complete inhibitor of enkephalin-degrading enzymes and CI 988, a selective antagonist of the cholecystokinin (CCK)-B receptors, on the flexor reflex in decerebrate, spinalized, unanaesthetized rats were assessed. Intravenous RB 101 induced a dose-dependent depression of the flexor reflex with a threshold dose of 20 mg/kg and an EDso of 25.3 mg/kg. Subcutaneous CI 988 at 1 mg/kg, which by itself did not influence the flexor reflex, strongly enhanced the reflex depressive effect of RB 101. The dose-response c u r v e for RB 101 was shifted to the left and the duration of reflex depression was significantly prolonged. The results confirmed and extended previous behavioural data indicating that blockade of CCK-B receptors potentiated antinociception elicited by endogenous opioids protected from enzymatic degradation. Furthermore, the spinal cord is an important site of interaction between the endogenous opioid and CCK systems.

INTRODUCTION

The endogenous opioidergic system plays an important role in numerous functions, including nociceptive modulation. Similarly to other neuropeptides, endogenous opioids are inactivated by enzymatic degradation. ~ A large number of membrane-bound and soluble enzymes are known to act on enkephalins and dynorphins, among which neutral endopeptidase (NEP; E.C. 3.4.24.11) and aminopeptidase N (APN; E.C. 3.4.11.2) appear to be the most important under physiological conditions.2-5 It has been suggested that protection of endogenous opioids from degradation may provide analgesia with reduced Received 15 January 1997 Accepted 7April 1997 Correspondence to: Dr Xiao-Jun Xu, Section of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Tel: +46 8 7461775; Fax: +46 8 7748856; E-mail: Xiao-Jun.Xu @ neurophys.hs.slLse

side-effects compared to exogenous opiates, such as morphine. For this reason, inhibitors of enzymes such as NEP and APN have been developed (see ref. 6 for review). The most interesting and best characterized compound to date is RB 101, a dual inhibitor of enkephalin-degrading enzymes which readily penetrates the blood brain barrier. 7 Systemic RB 101 has been shown to induce antinociception in rodents in a number of behavioural nociceptive tests. 8 More interestingly, repeated injection of RB 101 did not induce physical dependence, tolerance or cross-tolerance to morphine. 9,1° It is well established that the neuropeptide cholecystokinin (CCK) is an endogenous opioid antagonist. Thus, CCK has been shown to reduce antinociception induced by exogenous or endogenous opioids in behavioural and electrophysiological studies (see refs 11 and 12 for reviews). The antagonism by CCK of opioid-induced analgesia is present in normal conditions, as blockade of the actions of endogenous CCK with potent and specific 287

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receptor antagonists or receptor antisense oligonucleotides resulted in an enhancement of opioid-induced antinociception. 13-17 The antiopioid effect of CCK is mediated in rats by CCK-B receptors, which predominate in the rodent central nervous system. 18,19 It has been reported recently that antinociception induced by systemic RB 101 is also strongly potentiated by selective CCK-B receptor antagonists, 2°,2~ indicating a functional antagonism between the endogenous opioidergic and CCK systems in modulating nociception, s The antinociceptive effect of RB 101 and its potentiation by CCK-B receptor antagonists have only been studied so far in behavioural experiments and the site of interaction is not clear, as the drugs have been administered systemically in intact animals. Thus, the present studies were carried out to examine the effects of systemic RB 101 in a physiological preparation, the flexor reflex in decerebrate, spinalized, unanaesthetized rats. Furthermore, the interaction between RB 101 and C1988, a high affinity CCK-B receptor antagonist, 22 was examined. MATERIALS AND METHODS

All experiments were carried out with the approval of the local research ethics committee. Twenty female SpragueDawley rats weighing 200-250 g (B and K Universal, Stockholm, Sweden) were used. The magnitude of the polysynaptic hamstring flexor reflex in response to activation of high threshold afferents was examined after the animals had been decerebrated and spinalized. The rats were briefly anaesthetized with methohexital (Brietal, Lilly, Indianapolis, USA; 70 mg/kg, i.p.), ventilated and decerebrated by aspiration of the forebrain and midbrain. One jugular vein was cannulated for intravenous (i.v.) drug administration. The spinal cord was exposed by a laminectomy at mid-thoracic level and sectioned at Th8-9. The flexor reflex was elicited by supramaximal electric shocks applied to the sural nerve innervation area in the left foot (0.5 ms, 10 mA, l/rain) that activated A- and C-afferents. The flexor reflex was recorded as electromyographic activity via stainless steel needle electrodes inserted into the ipsilateral posterior biceps femoris/semitendinosus muscles. The number of action potentials exceeding the level of spontaneous activity was integrated over 2 s and recorded on a chart recorder (Gould 2400 S). During the experiments the heart rate and rectal temperature of the rat were monitored. RB 101 {N-[(R,S)-2-benzyl-3[(S)(2-amino-4-methyhhio)butyl dithio]-l-oxo-propyll-L-phenylalanine benzyl ester} was synthesized as described previously7 and dissolved in a vehicle containing ethanol (10%), cremophor EL (10%, Sigma) and saline (80%). RB 101 and its vehicle Neuropeptides (1997) 31(3), 287-291

were injected i.v. in a volume of 0.2 ml, except at 40 mg/ kg where 0.4 ml of the solution was administered because of the limitation of its solubility. CI 988, kindly provided by Dr J. Hughes (Parke Davis Neuroscience Research Centre, Cambridge, UK) (see ref. 22 for structure), was dissolved in 0.9% saline and injected subcutaneously (s.c.). A stable reflex baseline (defined as <15% variability) was established for at least 20-30rain before drug administration. The dose-response curve for RB 101 in depressing the flexor reflex was constructed with or without administration of a fixed dose of CI 988 (1 mg/kg). Data were expressed as mean + SEM and analysed by regression analysis and ANOVA. RESULTS

The vehicle of RB 101 injected i.v. did not significantly influence flexor reflex excitability, although in some experiments a brief facilitation of the reflex was observed after vehicle injection. I.v. RB 101 at 5 and 10 mg/kg also elicited reflex facilitation in some experiments with no subsequent inhibition (Fig. 1). At 20mg/kg, RB 101 induced a moderate, short-lasting depression of the flexor reflex in the majority of experiments (Figs 1 and 2). Stronger and more prolonged depression of the reflex was observed after 40 mg/kg RB 101 (Fig. 1). Regression analysis indicated an ED50 of 25.3 mg/kg for RB 101 for depressing the flexor reflex (Fig. 3). S.c. CI 988 at 1 mg/kg 1-5 rain before i.v. administration of vehicle did not influence the reflex (data not shown). Five mg/kg RB 101 administered after pretreatment with CI 988 still did not depress the flexor reflex (Figs 1 and 3), whereas 10 mg/kg RB 101 in combination with CI 988 caused reflex depression in all experiments (Figs 1 and 3). C1988 also enhanced the depressive effect of 20 mg/kg RB 101 and markedly prolonged its duration of action (Figs 1-3). The dose-response curve for RB 101 in the presence of CI 988 was shifted to the left with an EDso of 12.1 mg/kg for reflex depression (Fig. 3). DISCUSSION

The present study showed that systemic RB 101, a mixed inhibitor of enkephalin-degrading enzymes, dosedependently depressed the spinal nociceptive flexor reflex. Furthermore, the depressive effect of RB 101 was enhanced and prolonged by CI 988, a selective CCK-B receptor antagonist. These electrophysiological results therefore confirmed previous behavioural observations.8,20,21 As the flexor reflex model employed a spinalized preparation and the reflex was elicited by electrical stimulation, it is unlikely that the antinociceptive effect of RB 101 observed in the present study was derived © Pearson Professional Ltd 1997

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from supraspinal or peripheral action. Thus, the results indicate that blockade of degradation of enkephalins in the spinal cord results in antinociception. This is not surprising, as exogenous opioids exert powerful antinociception at spinal level and both interneurons and descending fibres containing enkephalin are present in the spinal cord. 2~ Moreover, previous studies have established a spinal site of action for other enkephalin-degrading enzyme inhibitors such as kelatorphma. 4,24 The reflex depressive effect of RB 101 was strongly potentiated by CI 988, a selective CCK-B receptor © Pearson Professional Ltd 1997

Fig. 3 Summary of the dose-related depression of the flexor reflex induced by RB 101 alone (0) or in the presence of CI 988 (11). Regression lines are drawn according to the formula Y=-116X+113 (R~=0.67) and Y=-111X+70 (R2=0.74), respectively. ANOVA indicated that both regressions are highly significant (F~,~s=28.6and F1,14=36.3 respectively, P<0.0001 ).

antagonist. This result is again in agreement with previous behavioural studies with both CI 988 and other CCK-B receptor antagonists. 2°, 21 CI 988 did not induce reflex depression in the present experiments, whereas a previous study showed that this dose of CI 988 injected i.v. induced moderate reflex depression.14 The reasons for Neuropeptides (1997) 31(3), 287-291

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t h e difference b e t w e e n t h e p r e s e n t a n d p r e v i o u s r e s u k s are u n k n o w n . Nevertheless, as CI 988 b y itself did n o t depress t h e flexor reflex, t h e leftward-shift of t h e doseresponse curve for RB 101 in t h e presence of CI 988 supports a potentiating, r a t h e r t h a n additive, effect of t h e CCK-B antagonist. The m e c h a n i s m s b y w h i c h CCK-B r e c e p t o r antagonists p o t e n t i a t e a n t i n o c i c e p t i o n i n d u c e d b y e x o g e n o u s or e n d o g e n o u s opioids h a v e b e e n addressed. It h a s b e e n h y p o t h e s i z e d t h a t i n c r e a s e d s t i m u l a t i o n of opioid receptors b y either e x o g e n o u s l y a d m i n i s t e r e d opioids or b y i n c r e a s e d e n d o g e n o u s content, s u c h as after RB 101 administration, m a y stimulate t h e release of CCK, w h i c h in t u r n c o u l d r e d u c e a n d curtail t h e action of opioids t h r o u g h a n u m b e r of m e c h a n i s m s . 8, 25-27 Several g r o u p s h a v e r e p o r t e d i n c r e a s e d release of CCK-LI from t h e spinal cord after m o r p h i n e t r e a t m e n t in vivo a n d in vitro27-29 A c u t e m o r p h i n e t r e a t m e n t m a y also increase g e n e expression a n d tissue c o n t e n t for CCK in several b r a i n regions a n d t h e spinal cord. 3° As a novel analgesic, RB 101 has several advantages over traditional opioids. No signs of withdrawal were o b s e r v e d after administration of opioid antagonists in animals chronically treated with RB 101.1° Chronic administration of RB 101 did not induce tolerance or cross-tolerance with morphine, a n d unlike morphine, RB 101 did n o t induce physical dependence. 9 One disadvantage of this comp o u n d is t h a t t h e antinociception i n d u c e d b y RB 101 is relatively brief. Thus, t h e finding t h a t t h e c o m b i n a t i o n of selective CCK-B receptor antagonists with RB 101 e n h a n c e d a n d p r o l o n g e d t h e effects of RB 101 m a y have significant practical implications. Moreover, it was reported recently t h a t RB 101 exerted an antidepressantlike action w h i c h is reinforced b y CCK-B antagonists. 31,32 This finding, t o g e t h e r with t h e fact t h a t CCK-B receptor antagonists are anxiolytic, = indicate a possible application for a c o m b i n a t i o n of RB 101 with a CCK-B antagonist in reducing t h e e m o t i o n a l c o m p o n e n t of pain.

ACKNOWLEDGEMENTS The p r e s e n t s t u d y was s u p p o r t e d b y t h e Biomed II p r o g r a m m e of t h e E u r o p e a n C o m m i s s i o n (project BMH4CT95-0172), t h e Swedish Medical Research Council (07913, 11011, 11012), Astra Pain Control AB, t h e Bank of Sweden Tercentenary Foundation, t h e Swedish Society of Medicine a n d research funds of t h e Karolinska Institute.

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CCK-B antagonist and opioid analgesia

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Neuropeptides (1997) 31(3), 287-291