Inhibition of enkephalinase activity attenuates naloxone-precipitated withdrawal symptoms

Gen. Pharmac. Vol. 18, No. 1, pp. 103-105, 1987 Printed in Great Britain

0306-3623/87 $3.00+0.00 Pergamon Journals Ltd

INHIBITION OF ENKEPHALINASE ACTIVITY ATTENUATES NALOXONE-PRECIPITATED WITHDRAWAL SYMPTOMS JUDITH HAFFMANSand MIHAILO R. DZOLJIC Department of Pharmacology, Medical Faculty, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands [Tel: (010)-463-9111] (Received 24 March 1986) A b s t r a c t - - l . In this study we examined the effects of the enkephalinase inhibitor, thiorphan, on the

naloxone-precipitated withdrawal syndrome in chronic morphine dependent rats. 2. Intracerebroventricular administration of thiorphan (40/~g/2 #1) in morphine dependent rats, inhibited the severity of the naloxone-precipitated abstinential syndrome. 3. Administration of thiorphan (20 #g/0.5/zl) in the periaqueductal grey matter of morphine dependent rats, in addition to explosive motor behaviour and ipsilateral rotation, also significantly suppressed most of the naloxone-precipitated withdrawal symptoms. 4. It is suggested that a decreased biotransformation of endogeneous opioid peptides might replace the relative shortage of morphine during withdrawal in opiate addicted subjects and attenuate the abstinence symptoms. INTRODUCTION Physical dependence on opiates, reflecting adaptive processes to chronic administration of these drugs, is unmasked when drug supply is interrupted or when an opiate antagonist is administered. Some recent data indicate that exogenous administration of opioid peptides suppress the opiate withdrawal symptoms in animals (Bl/isig et al., 1973) and m a n (Wen and Ho, 1982). Furthermore, it was found that a mixture of the unspecific peptidase inhibitors aprotonin and bacitracin, which are supposed to block the biodegradation of enkephalins, tend to suppress an expression of the narcotic withdrawal syndrome in the rat (Pinsky et al., 1982). Recently, we reported that more specific enkephalinase inhibitor phosphoramidon decreases the handling-induced convulsions after withdrawal of nitrous oxide (Dzoljic et al., 1984). This rrlight be of relevance to opiate addiction, since nitrous oxide can be considered as an opioid receptor agonist (Gillman and Lichtigfeld, 1981) or a releaser of endogenous opioid-like substances (Ngai and Fink, 1982; Quock et al., 1985). These data justify further study on the relationship between the opiate withdrawal syndrome and an increased activity of the endorphinergic system. This can be achieved by using more specific enkephalinase inhibitors resulting in an inhibition of the biotransformation of endogenous opioid peptides. For this purpose thiorphan was selected because of its good selectivity towards enkephalinase A or dipeptidylcarboxypeptidase (Roques et al., 1980). The naloxone-precipitated withdrawal syndrome in thiorphan-pretreated rats was examined and compared with similarly induced withdrawal without pretreatment with the enkephalinase inhibitor. In addition to the intracerebroventricular (i.c.v.) administration, thiorphan was also injected into the periaqueductal grey matter (PAG), since it was suggested that this area is one of the anatomical regions

important for the development of physical dependence to morphine (Laschka et al., 1976). MATERIALS AND METHODS

Animals Male Wistar rats (200-225 g) were anaesthetized by Hypnorm (fluanison/fentanyl base, Duphar, 0.4 ml/100 g, s.c.). A steel cannula for injections of artificial liquor (CSF) or thiorphan into the PAG (coordinates: AP:+0.6mm, L : - 0 . 5 mm, H : - 6 mm from dura with lambda 0 and the head in horizontal position) or into the left lateral ventricle (coordinates: AP: -0.1 mm, L: + 1.5 rnm, H: - 2 mm from dura with bregma 0, De Groot, 1972) was implanted stereotaxically. Placement of the cannula was checked after each experiment histologically. The animals were housed single with food and water available ad libitum and were allowed 3 days recovery before the experiments started. Each rat was only tested once. Chronic morphine treatment To induce morphine dependence, three morphine containing pellets (75 rag) were implanted subcutaneously on the back of the animals under light ether anaesthesia (Bl/isig et al., 1973). For control, placebo pellets containing the excipient but no morphine were implanted under identical conditions. Abstinence syndrome The abstinence syndrome was precipitated 70-74 hr after the pellet implantation by injecting naloxone (4 mg/kg, i.p.) dissolved in saline. The pellets were not removed before naloxone injection. Withdrawal was precipitated only once in the same rat. Procedure For the experimental procedure the rat was placed into a box (base area: 40 x 40cm; height: 45cm). Thiorphan (20~g/0.5~ul), dissolved in artificial cerebrospinal fluid (CSF) with slight warming, or CSF (0.5 pl) was administered into the PAG I0 min prior to naloxone. In a second group of animals a dose of 40 #g of thiorphan in 2/~1 CSF was administered intracerebroventricularly (i.c.v.) 10min prior to naloxone (4 mg/kg, i.p.). The observation period

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JUDITH HAFFMANS a n d M1HAILO R. DZOLJIC

Table 1. Naloxonc (4mg/kg, i.p.)-precipitated withdrawal symptoms (mean +_ SEM) in chronic morphine dependent rats, pretreated with CSF (2 ~1, i.c.v, or 0.5 jzl into PAG) or thiorphan (40/zg/2/zl, i.c.v, or 20 ttg/0.5 #l into PAG), injected intracerebroventricularly (i.c.v.) or into the periaqueductal grey (PAG), 10min prior to naloxone. (*P < 0.05; +, presence of sign; - , absence of sign) i.c.v. Withdrawal signs Teeth chattering Wet dog shakes (WDS) Grooming

PAG

CSF (n = 8)

Thiorphan (n = 9)

CSF (n = 9)

5.9 _+0.8 14.5_+0.9 4.8 _+ 0.8

4.2 _+0.8 16.4+2.3 3.6 _+ 0.7

11.2 -+ 4.8 19.4_+4.3 9.2 _+ 1.9

0.1 +_0.06 11.8-+2.8" -* 8.2 -+ 1.2 4.7 _+0.9* 1.7_+ 1" 44.1_+7" 3.5 +_0.8 2.8 _+1.6 + +

0.2 -+0.1 3.2_+ 1.5 8.3 + 1.5 26.2 + 8.2 18.5_+5.5 67.5_+11 7.7 +__2.1 2.8 _+1.2 + + +

Scratching 0.3 +_0.1 Penile licking 5.2-+2 Writhing 2.3 _+0.5 Rearing 10.2 _+2.3 Digging 29.8 _+7.7 Head hiding 14-+3.5 Chewing 85.7_+11 Paw tremor 2.3 +_0.5 Head shakes 2.5 +_0.6 Diarrhoea + Scream on touch + Hostility towards handling +

Thiorphan (n = 11) 4.8 + 3.5* 5.6+2.5* 1.4 _+ 0.4*

0.5 +_0.3 -* 1.3 -+0.4* 8.4 -+3.4* 6.4_+3* 62.7_+9.9 0.1 _+0.06* 2.1 _+1.4 -

started 15 min before the injection of thiorphan/CSF into the PAG or left lateral ventricle and lasted up to 30 min after naloxone administration. Two classes of signs were distinguished: counted and checked signs. The incidence of the following signs were counted: teeth chattering, "wet dog shakes" (WDS), grooming, scratching, penile licking, writhing, rearing, digging, head hiding (escape behaviour), chewing, paw tremor and head shakes. The presence of the following signs was checked: diarrhea, scream on touch and hostility on handling. The explosive motor behaviour (EMB) was characterized as a fearful hyper-reactivity with high leaps in repeated attempts to escape from the box (Jacquet and Marks, 1976).

morphine dependent rats resulted in an abstinential syndrome (Table 1). Similar administration of naloxone in morphine dependent rats pretreated with thiorphan (40/~ g/2/zl, i.c.v., 10min prior to naloxone) resulted in a significantly less severe abstinential syndrome. For example, the frequency of writing, digging, head hiding (escape behaviour) and chewing were decreased and diarrhoea was completely absent (Table 1). However, other signs of the withdrawal syndrome remained unchanged except penile licking which was significantly increased.

Statistical analysis

Morphine dependent rats, injected into the P A G with CSF (0.5/~1) were aggressive on handling. No other behavioural symptoms could be observed. Naloxone (4mg/kg, i.p.), 10min after CSF (0.5#1, into the PAG), precipitated withdrawal symptoms (Table 1). Thiorphan (20 /~g/0.5 #1) administered into the PAG, resulted in an immediate explosive motor behaviour (EMB, high undirected leaps) lasting for about 30 sec and followed by hyper-reactivity and ipsilateral rotation in 8 out of 11 rats. However, thiorphan (20/~g/0.5/zl, 10 min prior) antagonized naloxone-precipitated withdrawal symptoms (Table 1), such as teeth chattering, WDS, grooming, penile licking, rearing, digging, head hiding and paw tremor, while other signs were unaffected. In addition, the checked signs like diarrhoea, scream on touch and hostility towards handling were abolished by thiorphan in this group of animals (Table 1).

A non-parametric test (Mann-Whitney U-test) was used for calculation of the significance of differences of mean values (P < 0.5). RESULTS

Non-dependent rats Control rats, which had been implanted with placebo pellets and injected with CSF into the P A G or lateral ventricle, showed normal exploring behaviour (walking episodes, standing up on the hindlegs, sniffing and occasional digging). Thiorphan (40/~g/2/~1) administered into the left lateral ventricle induced an increase in locomotion (not evaluated) for about 4 min, but no other behavioural changes could be observed. Naloxone (4mg/kg, i.p., 10 rain after thiorphan or CSF) or saline had no effect on the animal's behaviour. Thiorphan (20/~g/0.5 ~tl) injected into the P A G of sham operated animals resulted in hyper-reactivity (increased locomotion, rearing on hind legs, sniffing, digging) for about 2.Smin. Administration of naloxone (4 mg/kg, i.p.), 10 rain after thiorphan, did not affect the behaviour of the rat with exception of some grooming after 20 min.

Chronic morphine dependent rats Intracerebroventricular (i.c.v.) administration of CSF/thiorphan. Administration of naloxone (4mg/kg, i.p.), 10min after CSF (2#1, i.c.v.) in

Administration of CSF/thiorphan into the PAG.

DISCUSSION

In this study the enkephalinase inhibitor thiorphan, given either i,c.v, or into the PAG, alleviated several symptoms of the morphine withdrawal syndrome, such as digging, head hiding and diarrhoea. Several symptoms were decreased only by i.c.v, administration of thiorphan (writhing and chewing) or after injection of this drug into the P A G (teeth chattering, WDS, grooming, rearing and paw tremor). However, some of the withdrawal symptoms were unaltered by both types of drug administration

Inhibition of enkephalinase (scratching and head shakes). This means that thiorphan, injected into the P A G or lateral ventricle of morphine dependent rats, does not attenuate equally all abstinential symptoms. It might indicate that enkephalinase inhibitors selectively interfere with the nervous mechanism(s) underlying one or several individual withdrawal signs without affecting the mechanisms responsible for other withdrawal symptoms. In our experiments this might be due to the local administration of thiorphan (i.c.v. or into the PAG) and the corresponding unequal distribution of this drug in various brain regions. A phenomenon of unequal suppression of withdrawal symptoms has been observed with other agents as well. For example, agents, which facilitate 5-HT transmission can block the jumping of morphine dependent rats but have no effect on ptosis, diarrhea and WDS (Romandini et al., 1984). Similarly, clonidine blocked WDS, while the mean number of jumps was raised significantly (Tseng et al., 1975). Differences between the control values of some naloxone-precipitated withdrawal symptoms after administration of CSF, i.c.v, or into the PAG, might be due to differences in the routes of administration. It is important to note that after administration of the enkephalinase inhibitor into the P A G more withdrawal symptoms were attentuated compared to the i.c.v, pretreatment. Penile licking, which showed increased frequency after thiorphan (i.c.v.), was even completely abolished after administration of this drug into the PAG. These phenomena are probably a reflection of the role of the P A G in corresponding withdrawal symptoms and the concentration of the enkephalinase inhibitors, which reach this region via two different ways of administration. Namely, it has been suggested that the caudal parts of the P A G are important sites of action for the development of physical dependence on morphine (Laschka et al., 1976). Furthermore, it was reported that the midbrain P A G is a unique site of several morphine actions such as analgesia (Jacquet et al., 1977) and catatonia (Jacquet and Marks, 1976) and fearful hyper-reactivity characterized by EMB (Jacquet et al., 1977). Although fl-endorphin injected into the PAG never resulted in EMB, (Jacquet, 1978) thiorphan, in our experiments, affecting probably the level of endogenous enkephalins, induced EMB. Several studies indicate that enkephalins are potent ligands for the 6 opioid receptors (Lord et al., 1977). This might suggest that the raised levels of enkephalins, and consequently activation of the excitatory 6 opioid receptors (Haffmans and Dzoljic, 1983), induces EMB. This aspect and the possibility that the different opioid receptors mediate the different withdrawal signs, need further experiments with more specific #- and 6 opiate agonists and antagonists. In conclusion, this study indicates that a decrease of the biotransformation of endogenous enkephalins and the corresponding increase of the opioid receptor saturation by endogenous opioid peptides, inhibits some symptoms of the abstinential syndrome. Evidently, an enkephalinase inhibitor which could penetrate the blood-brain barrier and inhibits in addition to enkephalinase A, aminopeptidases as well, could

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be a more promising tool in the treatment of opiate withdrawal. Acknowledgement--This research was supported by the

Praeventiefonds, Den Haag, The Netherlands. REFERENCES

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