Comparison of selective and complete inhibitors of enkephalin-degrading enzymes on morphine withdrawal syndrome

European Journal of Pharmacology,

165 (1989) 199-207

199

Elsevier EJP 50832

Comparison of selective and complete inhibitors of enkephalindegrading enzymes on morphine withdrawal syndrome R. Maldonado 1,4, V. Daug6 1, j. Callebert 3, J.M. Villette J. Feger 1 and B.P. Roques 2,,

3, M.-C.

Fourni6-Zaluski 2,

Laboratoire de Pharmacologie et 2 Ddpartement de Chimie Organique, U 266 INSERM, UA 498 CNRS, Facultd des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75006 Paris and 3 Laboratoire de Biochimie et Neuroendocrinologie, Hopital Saint Louis, Paris, France

Received 8 December1988, revised MS received2 March 1989, accepted 28 March 1989

We investigated the effects of thiorphan, a selective inhibitor of endopeptidase 24.11 'enkephalinase', kelatorphan ((R)-3-(N-hydroxy-carboxamido-2-benzylpropanoyl)-L-alarfine),and RB 38 A ((R)-3-(N-hydroxycarboxamido-2-benzylpropanoyl)-L-phenylalanine) two almost complete inhibitors of enkephalin metabolism, on the naloxone-precipitated morphine withdrawal syndrome in rats. Inhibitors administered intracerebroventricularly reduced several symptoms of the withdrawal syndrome. Jumping, chewing and tooth chattering were decreased by all drugs. The rise in plasma corticosterone and the hypothermia were reduced by kelatorphan and RB 38 A whereas rhinorrhea was blocked by thiorphan, tremor by kelatorphan and diarrhoea by RB 38 A. Other signs remained unchanged. These data suggest that an increase in opioid receptor occupancy by endogenous opioid peptides, protected from biotransformation specially by mixed inhibitors reduced the severity of the morphine abstinence symptoms in rats. Morphine withdrawal; Naloxone; Enkephalinase inhibitors; Kelatorphan; Thiorphan

1. Introduction

The administration of fl-endorphin (Tseng et al., 1976), dynorphin-(1-13) (Wen and Ho, 1982; Wen et al., 1984) and morphine-like pituitary peptides (Blasig and Herz, 1976) has been reported to be effective to reduce withdrawal syndromes in animals chronically treated with morphine and in heroin addicts. The enkephalins (Tseng et al., 1976) and dynorphin however (Tulunay et al., 1981) were relatively inefficient,

* To whom all correspondence should be addressed at D6partement de Chimie Organique, U 266 INSERM, UA 498 CNRS. 4 R. Maldonado is on leave from Unidad de Neuropsicofarmacologia, Departamento de Neurociencias, Facultad de Medicina, Cadiz, Spain.

very likely due to rapid degradation of these opioid peptides in vivo. In agreement with this, blockade of the degradation of endogenous opioid peptides by the unspecific peptidase inhibitors, apotropin and bacitracin (Pinsky et al., 1982), or by the specific endopeptidase 24.11 'enkephalinase' inhibitors, phosphoramidon (Dzoljic et al., 1986) and thiorphan (Haffmans and Dzoljic, 1987), were shown to minimize the severity of the morphine withdrawal syndrome in rats. In addition the peptidase-resistant enkephalin analogs, F K 33-824 and metkephamid, completely suppressed the opiate withdrawal syndrome in monkeys (Gmerek et al., 1983). Together, these data suggest that the strength of the narcotic withdrawal syndrome attenuation is related to the level of opioid receptor occupancy by exogenous or endogenous agonists.

0014-2999/89/$03.50 © 1989 Elsevier SciencePublishers B.V. (BiomedicalDivision)

200 Therefore it was of interest to investigate the effects of selective or mixed enkephalin-degrading enzyme inhibitors, such as thiorphan, or kelatorphan, (R)-3-(N-hydroxycarboxamido-2-benzylpropanoyl)-L-alanine, and RB 38 A, (R)-3-N-hydroxycarboxamido-2-benzylpropanoyl)-L-phenylalanine, on the naloxone-precipitated withdrawal syndrome in morphine-dependent rats. In contrast to thiorphan (Roques et al., 1980), kelatorphan (Fournir-Zaluski et al., 1984) and RB 38 A (Xie et al., in press) completely inhibited in vivo enkephalin catabolism (Waksman et al., 1985; Bourgoin et al., 1986) and consequently had much higher analgesic potencies than thiorphan (Fournir-Zaluski et al., 1984; Dickenson et al., 1987). RB 38 A has IC50 values of 2.5 ___0.7 and 2.5 ___0.6 nM for the endopeptidase 24.11 enkephalinase and the dipeptidylaminopeptidase, respectively. Moreover the inhibitory potency of RB 38 A for aminopeptidase N is about 3 times higher than that of kelatorphan (130 + 10 versus 380 ± 50 nM) (Xie et al., in press). In this study the effects of intracerebroventricularly (i.c.v.) injected thiorphan, kelatorphan and RB 38 A on the naloxoneprecipitated withdrawal syndrome in rats chronically treated with morphine were compared. Corticosterone levels in plasma, weight loss, colonic temperature and behavioral changes were measured to assess the effects.

2. Materials and methods

2.1. Animals and surgery One hundred and twenty male Sprague-Dawley rats (Depr6), ranging in weight from 200 to 220 g, were used in this study. The animals were anaesthetized with ketamine (100 m g / k g i.p.) and unilateral stainless-steel guide cannulas (25 gauge) were stereotaxically implanted 1 mm above the final injection site. The cannulas were secured to the skull with stainless-steel screws and dental cement. The guide cannulas were kept open with wire stylets. The cannulas were implanted with the bregma taken as the origin f o r coordinates: A -0.8, L + 1.6, V - 3 . 7 from the skull (Paxinos and Watson, 1982). After surgery, the animals

were housed in cages with free access to water and food.

2.2. Induction of physical dependence One week after surgery the rats were divided into eight groups (n = 15) corresponding to morphine treatment groups (four) and saline control groups (four). Saline and morphine chlorhydrate were injected s.c. dally at 9 a.m. and 6 p.m., in a volume of 2.5 ml/kg. The morphine dose was increased progressively from 8 to 45 m g / k g over a period of 5 days. The first and second number inside the parentheses represent the dose of morphine (mg/kg) injected at 9 a.m. and 6 p.m., respectively on consecutive days: 1st day (8, 15), 2nd day (20, 25), 3rd day (30, 35), 4th day (40, 45), 5th day (45 at 9 a.m. only).

2.3. Morphine withdrawal The withdrawal syndrome was precipitated 2 h after the final morphine injection by injecting naloxone chlorhydrate (5 m g / k g s.c.) in a volume of 2.5 m l / k g (s.c.). Immediately after naloxone injection, the animals were placed individually into test chambers consisting of round boxes (30 cm diameter x 35 cm height) and withdrawal signs were evaluated during a 30 min period. Two classes of signs were distinguished: counted and checked signs. Jumping, wet dog shake, tooth chattering and chewing were counted. Ptosis, diarrhoea, tremor, eye twitch, rhinorrhea, lacrimation and piloerection were evaluated over 10 min periods with one point being given for the presence of each sign during each period (maximum score: 3). Colonic temperature was determined by inserting a lubricated temperature probe 6 cm into the rectum of the rats. The temperature was recorded 2 min later using a thermometer (Model TE 3, Ellab instruments, Carrieri). Body weight and colonic temperature were determined 2 min before, and 30 and 60 min after naloxone injection. The rats were decapitated 65 rain after naloxone injection and blood was collected into test tubes for corticosterone assay.

201

2.4. Corticosterone assay Corticosterone was measured by radioimmunoassay using an antiserum raised in rabbits against corticosterone-3 cmo coupled to bovine serum albumin by the mixed anhydride method of Erlanger et al. (1957) and using [1,2,6,7-3H]corticosterone (Amersham) as radiolabelled ligand. Briefly, 100/~1 of rat serum were added to 100 /zl of radiolabelled corticosterone (5000 cpm) and 800/zl of assay buffer (0.05 M sodium phosphate pH 7.4 containing 0.1% gelatine). After extraction with 10 ml of cyclohexane/ethylacetate (50:50, v/v), the dried extracts were dissolved in 6 ml of assay buffer. Aliquots (500 and 200 /tl) of the extracts were assayed directly in duplicate by adding 100/zl of diluted antiserum (1/1800), 100/~1 of tritiated corticosterone (10000 cpm) and assay buffer to a final volume of 700/~1. The standard curve ranged from 15 to 2000 pg/tube of corticosterone. After 16 h incubation at 4°C, bound and free fractions were separated by adding 500/~1 of a dextran-coated charchaol suspension followed by centrifugation. The bound fractions were added to 10 ml of scintillation liquid and counted in a LKB liquid scintillation counter. Corticosterone concentrations were calculated by interpolation from the standard curve and were corrected for experimental losses during extraction. The method had a sensitivity of 7 pg/tube and intra- and inter-assay variation of 4.7 and 6.8%, respectively. The antiserum used had a low cross-reactivity with most steroids, as determined according to Abraham (1974): cortisol 0.005%; ll-deoxycortisol 0.005%; 21-deoxycortisol 0.18%; ll-deoxycorticosterone 5%; 21-deoxycorticosterone 0.16%; progesterone 0.002%; 17-hydroxyprogesterone, pregnenolone and 17-hydroxypregnenolone < 0.001%. 2.5. Lc.v. injection and drugs Lateral ventricle administration (i.c.v.) was performed with an injection apparatus consisting of 30.5 gauge stainless-steel needles attached to a 10 /~l microsyringe (Hamilton) by polyethylene tubing. The localization in the ventricle was checked in each animal by the ability of saline in the

catheter to flow rapidly when the extremity of catheter was placed higher, as a consequence of negative pressure in the ventricle. Thiorphan (100 /~g), kelatorphan (32 /~g), RB 38 A (12 /zg) and control solutions were administered i.c.v, by means of an infusion pump (Precinorm) in a constant volume of 5/~1 at a rate of 0.0416/~l/s 30 rain before the naloxone injection. The needle was left in situ for 30 s to allow diffusion of the drug away from the guide cannula. All drugs were dissolved in saline (0.9% NaC1). As the hot plate test was not appropriate for evaluating the analgesic potencies of the various inhibitors in rats, the doses used in this study were identical to the analgesic EDs0s in the hot-plate test with mice: thiorphan (100 /~g), kelatorphan (32 /~g) and RB 38 A (12 #g) (Schmidt et al., in preparation). It should be noted that the mixed inhibitor RB 38 A is effective in the tail flick test in rats while thiorphan is inactive in this assay (Schmidt et al., in preparation). The inhibitors of enkephalin-degrading enzymes: thiorphan, kelatorphan and RB 38, were synthesized in the laboratory of B.P. Roques. Naloxone HC1 was obtained from Sigma laboratolies.

2.6. Stat&tical analys& Changes in colonic temperature and weight loss were analyzed using repeated measures analysis of variance (ANOVA). The factors were group and TABLE 1 Number of wet dog shakes after naloxone in rats treated chronically with saline (5 days): effect of pretreatment with saline, thiorphan 100 jag, kelatorphan 32 /xg or RB 38 12 /~g, 30 rain before naloxone (5 mg/kg). Values are means + S.E.M. Number of animals per group = 15. Treatment Saline Saline Saline Saline

chronic + chronic + chronic + chronic +

Number of wet dog shakes saline thiorphan kelatorphan RB 38 A

0.30 + 0.21 3.60 5:1.26 b 1.40 _ 0.67 a 6.40 + 2.47 b

a p < 0.05, b p < 0.01 vs. saline + saline group (Mann-Whitney U-test.

202 time. If a significant effect of treatment or interaction with treatment was observed, one-way analysis of variance was used to determine the significance at each time point. Paired comparisons were made using the Newman-Keuls test. Plasma corticosterone levels were analyzed using single-factor ANOVA. Individual results were evaluated statistically using the Kruskal-Wallis H-test. Comparisons of treatment groups were made using the Mann-Whitney U-test.

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3.1.1. Non-dependent animals Table 1 shows that chronic saline injection failed to elicit any withdrawal signs after naloxone administration except for wet dog shakes which appeared in rats treated with thiorphan (100/~g/5 /~1 i.c.v.), kelatorphan (32/~g/5 ~tl i.c.v.) or RB 38 A ( 1 2 / ~ g / 5 / t l i.c.v.). 3.1.2. Morphine-dependent animals Naloxone (5 m g / k g s.c.) given to morphine-dependent rats 30 min saline (5/xl i.c.v.) precipitated a withdrawal syndrome characterized by numerous behavioral changes (figs. 1 and 2). Pretreatment (30 rain prior to naloxone) with thiorphan (100 /1g/5 /~l i.c.v.), kelatorphan (32 /xg/5 #l i.c.v.) or RB 38 A (12 /~g/5 /xl i.c.v.) reduced significantly the severity of naloxone-induced abstinence symptoms. Thus jumping, tooth chattering and chewing were strongly reduced by the three drugs (fig. 1). Furthermore fig. 2 shows that rhinorrhea was reduced by thiorphan, tremor by kelatorphan and diarrhoea by RB 38 A. However, wet dog shake, ptosis, eye twitch, piloerection and lacrimation were not significantly modified by the three drugs, although RB 38 A, the most potent of the inhibitors tested, showed a tendency to reduce the latter effect. 3.2. Weight loss 3.2.1. Non-dependent animals Small weight losses were observed in non-dependent rats after naloxone administration (less

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Fig. 1. Counted withdrawal signs after naloxone in rats treated chronically with morphine: effect of pretreatment with saline, thiorphan 100 #g, kelatorphan 32 #g or RB 38 12 /~g, 30 rain before naloxone (5 mg/kg). Values are means+S.E.M. Number of animals per group =15. *P < 0.05, **P < 0.01 vs. morphine+ saline group (Mann-WhitneyU-test).

than 1% body weight within 60 min). No significant differences were observed between the different groups (data not shown).

3.2.2. Morphine-dependent animals After naloxone administration, the morphinedependent rats lost 5% of their body weight within 60 min. This weight loss was not changed by pretreatment with thiorphan, kelatorphan or RB 38 A (data not shown). 3.3. Colonic temperature 3.3.1. Non-dependent animals No significant change was observed in colonic temperature in non-dependent rats (36.9°C before and 37 and 36.9 ° C at 30 and 60 min, respectively, after naloxone administration). Thiorphan, kelatorphan or RB 38 A administered acutely did not modify this parameter (data not shown).

203 38"

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3.3.2. Morphine-dependent animals After naloxone, the rats treated chronically with morphine showed hypothermia ( - 1 . 7 and - 2 ° C at 30 and 60 min, respectively, after naloxone administration) (fig. 3). Thiorphan did not modify the hypothermia significantly. However, kelatorphan and RB 38 A reversed the hypothermia 60 min after naloxone administration (fig. 3).

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Fig. 3. Colonic temperature before and 30 and 60 m i n after naloxone in morphine chronically treated rats after various pretreatments (saline, thiorphan 100/~g, kelatorphan 32/xg or RB 38 12 /~g) 30 rain before naloxone (5 m g / k g ) . N u m b e r of animals per group = 15. The values are means. * P < 0.05 vs. morphine + saline group at the same time interval. ~ " P < 0.01 vs. saline + saline group (Newman-Keuls test).

was significantly decreased by kelatorphan and RB 38 A while thiorphan only slightly modified this parameter. A

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204

4. Discussion

The selective 'enkephalinase' inhibitor, thiorphan, and the mixed inhibitors, kelatorphan and RB 38 A, administered i.c.v, at concentrations corresponding to their EDs0 values in the hot-plate test in mice were now shown to attenuate the severity of the morphine withdrawal syndrome in rats. Several well-characterized abstinence symptoms such as jumping, tooth chattering and chewing were decreased by all inhibitors. In addition, hypothermia was attenuated by both kelatorphan and RB 38 A, while rhinorrhea was modified by thiorphan only, tremor by kelatorphan only and diarrhoea by RB 38 A only. Some of the withdrawal symptoms remained unchanged (weight loss, ptosis, eye twitch and piloerection). The main purpose was to compare a selective (thiorphan) and two complete (kelatorphan and RB 38 A) inhibitors of enkephalin-degrading enzymes. Since we have found a correlation between the inhibitory potency of these drugs towards the different enkephalin-degrading enzymes and their antiwithdrawal effects, only one dose of each inhibitor was used. The inhibitors were injected 30 rain before naloxone and it could be assumed that the resulting increase in endogenous opioid peptides inhibited the binding of the competitive antagonist to opioid receptors and, consequently, reduced the withdrawal syndrome. This hypothesis is supported by the fact that inhibitors of enkephalindegrading enzymes increase the [MetS]enkephalin content of mouse striatum (Zhang et al., 1982) and the release of endogenous [MetS]enkephalin by the rat spinal cord in vitro and in vivo (Bourgoin et al., 1986). Both jumping and tooth chattering, which have been associated with severe opiate withdrawal (Blasig et al., 1973; Williams and Thorn, 1984), are widely accepted as reliable indices for quantifying the degree of physical dependence in rats. The wet dog shake has also been used by many investigators to evaluate morphine abstinence effects, although this behaviour reflects mild opiate withdrawal (Blasig et al., 1973; Williams and Thorn, 1984; Buccafusco et al., 1984). The signs classified as peripheral manifestations of the withdrawal syndrome (diarrhoea, rhinorrhea,

lacrimation and piloerection) seem to be modified preferentially by peripherally acting drugs, although some centrally acting drugs may also modulate some of these effects (Frederickson and Pinsky, 1975; Galligan and Burks, 1982). In the present study i.c.v, injected inhibitors of enkephalindegrading enzymes modified these signs inconsistently. The rise in plasma corticosterone may be correlated with, but is not repsonsible for, stress-induced states, dysphoria and major depressive illness (Gold et al., 1981). Moreover this rise has been shown to be a sensitive indicator of the severity of opiate withdrawal (Zimmerman et al., 1975; Eisenberg and Sparber, 1979). In this study the rise in plasma corticosterone was slightly decreased by thiorphan but was significantly decreased by the mixed inhibitors, kelatorphan and RB 38 A. Treatment with thiorphan, kelatorphan or RB 38 A induced wet dog shakes in non-dependent rats (table 1). It is consistent with this that there are reports that wet dog shakes occur after treatment with enkephalins (Drust et al., 1981; Cowan and Tortella, 1982) or phosphoramidon (Rupreht et al., 1983). This behavior was attenuated but not suppressed by naloxone (Drust et al., 1981; Ukponmwan et al., 1985). Several neurotransmitter systems are involved in wet dog shake behaviour: the serotoninergic (Yap and Taylor, 1983), noradrenergic (Handley and Singh, 1986), dopaminergic (Araki and Aihara, 1985), GABAergic (Handley and Singh, 1985) and opioid systems (Asthon and Wauquier, 1982; Araki and Aihara, 1985). Nevertheless, despite their own wet dog shake-inducing effects, the three inhibitors tested tended to reduce this particular behavior in physically dependent rats (fig. 2). Our results are in agreement with the report by Haffmans and Dzoljic (1987) that the i.c.v, administration of thiorphan (40/tg) reduced escape behavior and mastication, while wet dog shakes and tooth chattering remained unchanged. RB 38 A and kelatorphan were more effective than thiorphan to reduce withdrawal symptoms. This greater efficacity can be related to results of studies showing that RB 38 A and kelatorphan are much more efficient than thiorphan to protect in

205

vivo and in vitro enkephalins from degrading enzymes (Waksman et al., 1985; Bourgoin et al., 1986) and consequently have greater analgesic potencies (Fournir-Zaluski et al., 1984; Dickenson et al., 1987; Schmidt et al., in preparation). Cowan et al. (in press) reported that rats, infused centrally with selective agonists for /~ (DAGO; 8.2 nmol/168 h), 8 (DPDPE; 2062 nmol/168 h) and x (U-50,488H; 70015 nmol/168 h) opioid receptors, showed high, low and negligible levels of naloxone-precipitated withdrawal syndrome, respectively. Accordingly, /~ ag0nists suppressed the morphine withdrawal syndrome (Blasig et al., 1973) while the selective x opioid agonist, U-50,488, attenuated this syndrome only at doses which also produce severe stupor (Katz e t al., 1982). [D-Ala2,D-LeuS]enkephalin, an enkephalin analogue exhibiting a 3-fold higher affinity for 6 than for /~ opioid receptors was also reported to attenuate morphine abstinence (Wen, 1980; Wen et al., 1984). Furthermore, no withdrawal signs were obtained after administration of the 6 antagonist ICI 154,124 in rats implanted with morphine pellets (Priestley et al., 1985) while the potent and highly selective cyclic/~ opioid antagonist, D-Phe-Cys-Tyr-D-Trp-OrnThr-Pen-Thr-NH2, induced a precipiated morphine withdrawal syndrome in rats at a concentration lower than naloxone (Gulya et al., 1988). Likewise the high sensitivity of adrenergic neurons of the locus coeruleus, a brain region which contains a much higher (although not exclusive) concentration of /~ opioid binding sites, induced by chronic morphine is usually considered as one of the causes of the withdrawal syndrome (Aghajanian et al., 1978; Nestler and Tallman, 1988). It has been reported that the caudal part of the periaqueductal gray matter is a brain region in which morphine induces analgesia (Jacquet et al., 1977) and could be an important site of action for the development of physical morphine dependence (Laschka et al., 1976). In line with this, wet dog shakes and tooth chattering were found to decrease after direct administration of thiorphan into the periaqueductal gray matter (Haffmans and Dzoljic, 1987). Autoradiographic studies have shown much higher densities of the /~ opioid receptor subtype than of the 8 subtype in the peri-

aqueductal gray matter (Quirion et al., 1983; Waksman et al., 1986). Therefore it should be interesting to test this hypothesis through evaluation of the antiwithdrawal effects produced by selective # and 8 agonists and by the various enkephalin-degrading enzyme inhibitors injected directly into the periaqueductal gray matter. As the enkephalins have a better affinity for 8 than for/~ opioid receptors (Chang et al., 1979; Zajac et al., 1983; Paterson et al., 1984) the greater efficiency of RB 38 A and kelatorphan as compared to that of thiorphan could be explained by a higher local concentration of the opioid peptides induced by the mixed inhibitors at the ~t receptors. In agreement with this, the /~ receptor has been proposed as the main receptor type implicated in morphine dependence and its withdrawal syndrome (Delanger et al., 1984). Nevertheless, it cannot be excluded that endogenous opioid peptides may also modulate the withdrawal syndrome by activating 8 receptors. In addition to opiates, various compounds have been used to try to attenuate the morphine-induced abstinence syndrome, suggesting that various neuronal pathways involving different neuromediators are implicated in physical dependence to opioids. Thus benzodiazepines decrease jumps, tooth chattering and tremor, but the wet dog shake remains unmodified or markedly increased by diazepam and flunitrazepam respectively (Gibert-Rahola et al., in press). Similarly, clonidine, a drug used to attenuate the withdrawal syndrome in man, blocks wet dog shakes but significantly increases the mean number of jumps in rats (Tseng et al., 1975; Romandini et al., 1984). Furthermore clonidine has no effect on the rise in plasma corticosterone in morphine withdrawal (Eisenberg, 1983). In conclusion, our results show clearly that an increase in opioid receptor occupation by endogenous opioid peptides, protected from peptidaseinduced inactivation, effectively reduced the severity of the opiate withdrawal syndrome in rats. These findings suggest that inhibition of enkephalin catabolism, especially by mixed inhibitors, may be of some value in the treatment of opiate addicts.

206

Acknowledgements This research was supported by a grant from INSERM (CRE 876010). R. Maldonado is a fellowship from Ministerio de Educacion y Ciencia (Spain). We would like to thank N. Beau, Laboratoire de Math6matiques, for helpful assistance with statistical analyses. We acknowledge S. Turcaud for the synthesis of inhibitors.

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