Cyclo (Leu-Gly) + haloperidol: Effects on dopamine receptors and conditioned avoidance responding

Peptides, Vol. 8, pp. 39-44. Copyright©PergamonJournals Ltd., 1987. Printedin the U.S.A.

0196-9781/87$3.00 + .00

Cyclo (Leu-Gly) + Haloperidol: Effects on Dopamine Receptors and Conditioned Avoidance Responding A N D R E W J. B E A N , 1 R O B E R T J. E L G I N , JR., D I R E E S E M. C O O P E R A N D G R E G O R Y E. M A R T I N

D e p a r t m e n t o f Biological Research, M c N e i l Pharmaceutical, Spring House, P A 19477-0776 R e c e i v e d 18 April 1986 BEAN, A. J., R. J. ELGIN, JR., D. M. COOPER AND G. E. MARTIN. Cyclo (Leu-Gly) + haloperidol: Effects on doparnine receptors and conditioned avoidance responding. PEPTIDES 8(1) 39-44, 1987.--Behavioral effects of cyclo (Leu-Gly) (cLG), administered either acutely or chronically, were assessed in combination with haloperidol in the rat. cLG administered chronically, produced a significant reduction in the increase in apomorphine-induced stereotypy produced by chronic haloperidol infusion. On the other hand, the same dose of cLG which reduced this induction of dopamine receptor supersensitivity due to chronic haloperidol treatment, failed to produce a change in the potency of haloperidol in blocking conditioned avoidance responding in the rat. Furthermore, degeneration-induced supersensitivity of dopamine neurons, produced by unilateral destruction of the nigrostriatal pathway, was not reduced by acute or chronic treatment with cLG as measured by apomorphine-induced rotation. These data suggest that cLG may decrease motor system side effects thought to be caused by chronic antipsychotic administration without affecting the therapeutic efficacy of the antipsychotic agent. Cyclo (Leu-Gly) Haloperidol Conditioned avoidance 6-Hydroxydopamine Tardive dyskinesia

Dopamine

Stereotypy

Rotation

morphine [5]. Since cLG has a longer half-life than MIF, has been shown to penetrate into the brain [18], and seemingly exerts no effects on postsynaptic DA receptors either in vivo [31] or in vitro [6], it may be useful as an adjunctive therapy for patients taking antipsychotic medication. It is clear that the peptide can somehow modulate the neuronal mechanism whereby receptor numbers increase. The purpose of the present experiments was twofold. First, cLG was chronically administered to rats concomitantly administered haloperidol (HAL), to determine whether it reduced the behavioral supersensitivity to a dopamine agonist that normally develops under such conditions. Secondly, the effects of chronic treatment with cLG on the efficacy of H A L in an animal test of antipsychotic activity, the conditioned avoidance paradigm [20], were ascertained. Although several reports have examined the effects of cLG on the supersensitivity produced by HAL, no reports have addressed the question of whether or not this treatment reduces the efficacy of HAL. In an additional experiment, the effect of chronic treatment with cLG on the contraversive turning response evoked in the rat with a unilateral 6-hydroxydopamine lesion in the substantia nigra was ascertained. This contraversive turning response is thought to be mediated by supersensitive DA receptors in the striatum [301

DURING chronic treatment with antipsychotic agents, whose primary action is blockade of dopamine (DA) receptors [13], DA receptors proliferate both in animal and human tissue [2, 8, 27]. The increase has been observed in laboratory animals after chronic antipsychotic treatment using receptor binding techniques [22, 23, 27], and in examining behavioral responses following the administration of a DA agonist [2, 17, 27-29]. The consequences of this increase in receptor number are twofold. First, there is an apparent tolerance to the effect of the drug [1,24]. Secondly, the increment in DA receptors may underlie the appearance of some tardive dyskinesias [2, 19, 32]. The prevention of the increase in receptor numbers during chronic treatment with antipsychotic agents might therefore prevent the appearance of tardive dyskinesia which is among the most serious sideeffects of chronic antipsychotic treatment. In this regard, it is most interesting to note that several reports have appeared demonstrating that a tripeptide, melanocyte stimulating hormone release-inhibiting factor (MIF, prolyl-leucylglycinamide) or a stable analog of its metabolite leucineglycine, cyclo (leucyl-glycinamide) (cLG), can apparently block the augmentation of DA receptors that is normally produced by long-term antipsychotic administration [7, 10, 11, 21, 26, 31]. This same peptide is reported to block the development of tolerance to repeated injections of

~Requests for reprints should be addressed to Andrew J. Bean at his present address: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.

39

40

BEAN, ELGIN, COOPER AND MARTIN TABLE 1 RATINGSCALE USED FOR STEREOTYPYSCORING Score 0 1 2 3 4 5

Behavior asleep or inactive grooming or alert discontinuous sniffing continuous sniffing continuous sniffing and licking cage grids continuous sniffing and biting cage lids

METHOD

Conditioned Avoidance Responding (CAR) Male Fisher 344 rats (Charles River Farms, Kingston, NY), weighing 200-225 g housed singly with food and water available ad lib, were trained to perform the CAR task which consisted of a lever press in order to avoid an impending 0.7 mA foot shock. The test session consisted of 60 discrete trials spaced once a minute. The conditioned stimuli (paired light and tone) were presented for 15 sec followed by 5 sec of shock in the absence of a lever press. The animal could also escape the shock by depressing the lever during the 5 sec shock interval. Each study was performed using drug naive rats. For single dose studies (study 1), cLG dissolved in distilled water (2 mg/kg SC) was administered 1 hr prior to various doses of HAL (0.08--0.16 mg/kg IP, n = 10 rats/dose) which was given 1 hr prior to CAR testing. In one chronic study (study 2), cLG was infused continuously at a rate of 2.0-2.3 mg/kg/day for 21 days via a subcutaneously implanted Alza 2ML2 osmotic minipump in ten animals. After two weeks of infusion, the 2ML2 minipump was removed and a second pump implanted to insure a continuous three-week infusion. HAL, 0.12 mg/kg IP, was administered 14 and 21 days after the pumps were implanted, I hr prior to testing the animals in the CAR task. All pumps were removed prior to CAR testing. To examine the effects of simultaneous chronic treatment with cLG and HAL (study 3), four groups of six rats each were used, and two minipumps were implanted in each animal. In group 1, both pumps were loaded with sterile water; in group 2, one pump was loaded with sterile water and the other with cLG (2-2.3 mg/kg/day, 14 days); in group 3, one pump was loaded with sterile water and the other with HAL (1-1.2 mg/kg/day, 14 days); whereas group 4 was given cLG in one pump and H A L in the other. After a two-week infusion period, all pumps were removed from the animals. In study 1, dose response curves for blockade of CAR were determined and EDs0 values (dose calculated to cause a 50% reduction in the number of avoidance responses) computed using linear regression with an analysis of variance. Data from experiments 2 and 3 were analyzed using analysis of variance in conjunction with Dunnett's multiple comparison test.

Stereotyped Behavior Male Sprague-Dawley rats (Charles River Farms, Kingston, NY), weighing 200-225 g with food and water available ad lib were used. The stereotypy rating procedure was modified

from that reported by Creese and Iversen [14]. Briefly, after weighing, each rat is placed in a Plexiglas box with a grid floor and ceiling both fashioned from stainless steel. No testing was begun until each rat was observed to have a stereotypy score equal to zero in the test box (Table 1). Rats were then given drug or control injections and, based on the scoring system shown in Table 1, were given individual stereotypy scores at 10-rain intervals for the 2 hr period following drug treatment. All scoring was done by a rater unaware of the treatment given each rat. In studies in which the induction of stereotypy was measured, the peak scoring from six consecutive rating periods was chosen for each rat and the percent of the maximum stereotypy score was calculated in the following manner. The rat's score + the maximum possible score (=30) x 100 = percent maximum stereotypy. ED,~0 values were determined using linear regression analysis and relative potencies were compared using an analysis of variance. In studies in which blockade of stereotypy was measured, the peak scores for six consecutive time points were determined and percent inhibition of stereotypy was calculated as follows: maximum possible score (=30) - observed score + maximum possible score (=30) x 100 = percent inhibition of stereotypy. Once again, linear regression analysis was used to determine EDs0 values and analysis of variance was used to determine significant differences. The first experiment in this section was designed to ascertain whether chronic treatment with HAL did increase the amount of stereotypy evoked by apomorphine. Furthermore, would such an increase be blunted by concomitant treatment with cLG? In three groups of six animals, two 2ML2 osmotic minipumps were implanted SC. In one group 0.~Yb saline was infused from both pumps. In a second group, one pump dispensed 0.9% saline while the other infused HAL (1.0-1.2 mg/kg/day). In the final group, one pump dispensed cLG (2.0-2.3 mg/kg/day) and the other HAL (0.1-1.2 mg/kg/day). Chronic infusions were maintained for two weeks, after which all pumps were removed. The stereotypy evoked by 0.6 mg/kg SC of apomorphine was compared among the three groups using the Student's t-test. The effect of acute administration of cLG on apomorphine-induced stereotypy was determined by treating groups of animals with cLG (2 mg/kg SC) 0.5 hr prior to apomorphine in doses ranging from 0.3-3.0 mg/kg SC (n=5 or 6/group), while other groups given the same doses of apomorphine were pretreated with vehicle (sterile water). ED.~0 values between groups were compared using the parallel line bioassay method (Finney) [16]. Finally, a test was run to determine whether acutely administered cLG (2 mg/kg SC) alters the action of HAL in blocking apomorphine-induced stereotyped behavior. HAL alone in doses of 0.05, 0.1, 0.2 and 0.4 mg/kg IP, or HAL in these doses preceded immediately by cLG (2 mg/kg SC) was administered IP 1 hr prior to the administration of apomorphine (0.8 mg/kg SC) to groups of six rats each. Separate groups of animals were given 0.8 mg/kg SC of apomorphine alone. The ED.~0 value for HAL in blocking apomorphineinduced stereotypy was compared between cLG-HAL and HAL alone-treated groups using probit analysis and a parallel line bioassay.

Turning in Rats With a Unilateral 6-Hydroxydopamine (6OHDA) Lesion in the Substantia Nigra In male Sprague-Dawley rats (Charles River Farms, Kingston, NY) weighing 250-300 g at the time of surgery, a

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FIG. I. Effects of 60 min pretreatment with cLG on haloperidol blockade of conditioned avoidance responding.

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rotometers for 90 min to establish a baseline turning rate. Subsequently, in two groups of rats, either sterile water or c L G (2 mg/kg SC) were injected once per day for 14 consecutive days. On the 14th day, 6 hr after the final injection, the amount of turning produced by 0.15 mg/kg SC of apomorphine was again measured and compared with the control values using Student's t-test.

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unilateral 6-OHDA lesion of the substantia nigra was performed following the method of Ungerstedt [30]. Rats were anesthetized with Ketamine (100 mg/kg IP) and 6-OHDA was unilaterally injected via a 33 gauge needle (8 t~g/4/~1) at a rate of 1/xl/min into the substantia nigra. Lesioned rats exhibiting >300 turns per 90 min, in response to amphetamine challenge (2 mg/kg IP, ipsilaterally directed to the lesioned side) and apomorphine challenge (0.25 mg/kg SC, contralaterally directed) when tested two-four weeks post-operatively were used. To determine the acute effects of cLG, it was administered to lesioned rats (2 or 8 mg/kg SC, n=6/group) and rotation was measured in automated rotometers for 90 min. Whether c L G alters apomorphine-induced (0.15 mg/kg SC) rotation was studied by pretreating lesioned rats with c L G (2 mg/kg SC, - 1 or - 2 4 hr) before administering apomorphine. To determine the effects of chronic cLG, rats were first tested with apomorphine (0.15 mg/kg SC) and placed in the

RESULTS

Conditioned Avoidance Responding The acute administration of cLG (2.0 mg/kg SC, 1 hr before HAL), failed to alter the potency of H A L in blocking CAR activity in the rats in study 1 (Fig. 1). As shown in Fig. 1, the ED.~0 values for HAL-induced block of CAR did not differ between the group which had been given cLG acutely and the group which had been given vehicle. In addition, the chronic SC infusion of c L G over a three-week period in study 2 failed to diminish the CAR blocking efficacy of H A L (0.12 mg/kg IP), given either two or three weeks after the initiation of the chronic c L G infusions (Fig. 2). In study 3, H A L administered via osmotic minipumps exerted an anti-CAR effect for the duration of the two-week infusion period as shown in Fig. 3. More importantly, the simultaneous infusion of cLG did not reduce H A L ' s efficacy in CAR during the two-week time period as also shown in Fig. 3. In a follow-up test two weeks after cessation of the chronic infusions, no significant differences were detected between the different treatment groups in their performance in CAR (Fig. 3).

Effect of cLG on Apomorphine-lnduced Stereotypy Following a two-week infusion of H A L (1.0-1.2 mg/kg/d SC), there was a significant increase in the stereotypy score evoked by apomorphine given in a dose of 0.6 mg/kg SC (Fig. 4). The mean stereotypy score in HAL-treated rats rose significantly from the 57.3% value of the vehicle-infused animals to a value of 78.2%. In animals in which c L G was chronically infused concomitant with H A L , however, apomorphine (0.6 mg/kg SC) produced the same amount of stereotypy (59.3%) as in the vehicle-treated control animals. In groups of drug-naive rats, the pretreatment with a single dose of c L G (2 mg/kg SC) failed to alter the potency of

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apomorphine in producing stereotyped behavior as shown in Fig. 5. When administered acutely in a dose of 2 mg/kg SC, c L G failed to alter the potency of H A L in blocking the induction of stereotypy induced by apomorphine (0.8 mg/kg SC) as shown in Fig. 6.

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cLG, given in doses of 2 or 8 mg/kg SC, failed to elicit turning in either direction in rats with a unilateral 6-OHDA lesion in the substantia nigra. Furthermore, as shown in Fig. 7, pretreatment with a single dose of 2 mg/kg SC of c L G either 1 or 24 hr earlier exerted no effect on rotation induced by apomorphine. Fourteen daily SC injections of c L G (2 mg/kg SC) also failed to alter the amount of turning evoked by apomorphine in lesioned rats (Fig. 8). Following the two-week dosing period, the number of turns elicited by apomorphine (0.15 mg/kg SC) was 162±32 in the group given daily cLG, whereas 159_ + 18 contralateral turns were completed by animals dosed chronically with the vehicle.

TREATMENTGROUPS

FIG. 8. Effects of 14-day treatment with cLG or vehicle on apomorphine-induced (0.15 mg/kg SC) contralateral rotation.

DISCUSSION

The present data demonstrate that neither chronic nor acute injections of cLG alter the potency of H A L in blocking CAR in the rat. Moreover, the same 2 mg/kg dose of cLG does block the development of behavioral supersensitivity to chronic antipsychotic treatment. These findings suggest that cLG may prevent the development of supersensitivity of DA receptors occurring during chronic antipsychotic treatment

CYCLO (LEU-GLY) + H A L O P E R I D O L

43

but should not reduce the therapeutic potency of the antipsychotic agent. Although others have shown that c L G can prevent the behavioral supersensitivity to apomorphine induced by chronic H A L [7, 26, 31] and the increase in receptor number that this treatment produces [7, 10, 11, 21, 26, 31] the present experiments demonstrate for the first time that the efficacy of H A L in a test thought to reflect clinical efficacy is not compromised by this treatment. It is not yet clear how cLG acts to prevent the alterations in receptor number or the increase in receptor sensitivity that is produced by chronic treatment with H A L or morphine [4,31]. The increased sensitivity of animals to the behavioral actions of apomorphine following chronic treatment with antipsychotic agents has been used as an animal model of tardive dyskinesia [2, 27, 29]. If this model is indeed a useful model for such dyskinesias (which has been recently questioned [32]) then the importance of the present results are clear, cLG, which by itself fails to alter the action of dopamine agonists or antagonists at the dopamine receptor, may block the development of tardive dyskinesia via prevention of postsynaptic receptor proliferation. This is not an original suggestion since it has been proposed by other authors based on chronic or acute treatment with c L G using behavioral or receptor binding indices of drug action [7,11]. The present study extends the previous finding, however, to include the fact that H A L does not lose its potency in a test of antipsychotic efficacy. Apomorphine is thought to produce contralateral turning in rats with a unilateral 6-hydroxydopamine-lesion in the substantia nigra via an action exerted at supersensitive DA receptor sites in the denervated striatum [30]. Treatment with cLG did not reverse the effects of neuronal degeneration-induced supersensitivity of postsynaptic dopamine receptors in the striatum of these rats as reflected

by the fact that c L G failed to produce a decrease in apomorphine-induced turning. Although it has been shown that c L G can inhibit the dopamine receptor supersensitivity that arises following intracerebroventricular injection of 6-OHDA in mice [25], recent studies suggest that c L G inhibits drug-induced supersensitivity via a mechanism that does not include the nigrostriatal system [31]. Although Chiu et al. [12] have demonstrated that there is a binding site for the structurally-related M I F in the striatum, the precise site of action of c L G is not yet clear. Pharmacokinetic differences between constant infusion and once per day injections may have played a role in c L G ' s lack of ability to reverse lesion-induced supersensitivity. However, the supersensitivity that develops after dopamine receptor blockade in the striatum may be different than that which occurs following neuronal degeneration. Alternately, the fact that the supersensitivity of DA receptors in 6-OHDA lesioned rats is a chronic effect may be another crucial difference in the two supersensitivities. M I F has been studied as a dopamine agonist in treating Parkinson's disease, but with no substantial therapeutic benefit [3,9]. In the clinic, the therapeutic effects of MIF in alleviating tardive dyskinesias has also been studied. Transient improvements in dyskinesia scores were seen, but the improvements were judged to be little better than the placebo effect [15]. It would be of interest to examine the effect of c L G in patients with tardive dyskinesia since c L G has a longer half-life than M I F and, as reported here, fails to diminish the potency of H A L while blocking the development of supersensitivity of DA receptors that usually develops with chronic antipsychotic treatment. ACKNOWLEDGEMENT The authors wish to thank Cathy Braun for her careful preparation of this manuscript.

REFERENCES 1. Asper, H., M. Battiolini, H. R. Burki, H. Lavener, W. Ruch and G. Stille. Tolerance phenomena with neuroleptics: catalepsy, apomorphine stereotypies and striatal dopamine metabolism in the rat after single and repeated administration of loxapine and haloperidol. Eur J Pharmacol 22: 287-294, 1973. 2. Baldessarini, R. J. and D. Tarsy. The pathophysiologicai basis of tardive dyskinesia. In: Tardive Dyskinesia: Research and Treatment, edited by W. E. Fann, R. C. Smith, J. M. Davis and E. F. Domino. New York: Spectrum Publications, Inc., 1980, pp. 181-199. 3. Barbeau, A., M. Roy and A. J. Kastin. Double-blind evaluation of oral L-prolyl-L-leucyl-glycineamide in Parkinson's disease. Can Med Assoc J 114: 120--122, 1976. 4. Bhargava, H. N. Binding of [3H]spiroperidol to striatal membranes of rats treated chronically with morphine: Influence of Pro-Leu-Gly-NH2 and cyclo (Leu-Gly). Neuropharmacology 22: 1357-1361, 1983. 5. Bhargava, H. N. Cyclo (Leucyl-glycine) inhibits the development of morphine-induced analgesic tolerance and dopamine receptor supersensitivity in rats. Life Sci 27:117-123, 1980. 6. Bhargava, H. N. The effect of melanotropin release inhibiting factor, its metabolites and analogs on [3H]spiroperidol and [aH]apomorphine binding sites. Gen Pharmacol 14: 60%614, 1983. 7. Bhargava, H. N. and R. F. Ritzmann. Inhibition of neurolepticinduced dopamine receptor supersensitivity by cyclo(Leu-Gly). Pharmacol Biochem Behav 13: 633-636, 1980.

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44 17. Gianutsos, G., R. B. Drawbaugh, M. D. Hynes and H. Lal. Behavioral evidence for dopaminergic supersensitivity after chronic haloperidol. Life Sei 14: 887-898, 1984. 18. Hoffman, P. L., R. Walter and M. Bulat. An enzymatically stable peptide with activity in the central nervous system: its penetration through the blood-CSF barrier. Brain Res 122: 87-94, 1977. 19. Kobayashi, R. M., J. Z. Fields, P. E. Hruska, K. Beaumont and H. I. Yamamura. Brain neurotransmitter receptors and chronic antipsychotic drug treatment: A model for tardive dyskinesia. In: Anirnal Models in Psychiatry and Neurology, edited by I. Hanin and E. Usdin. New York: Pergamon, 1977, pp. 405-409. 20. Kuribara, H. and S. Tadokora. Correlation between antiavoidance activities of antipsychotic drugs in rats and daily clinical doses. Pharmaeol Biochem Behav 14: 181-192, 1981. 21. LeDouarin, C., D. Fage and B. Scatton. Effects of cyclo (Leu-Gly) on neurochemical indices of striatal dopaminergic supersensitivity induced by prolonged haloperidol treatment. Lift, Sci 34: 393-399, 1983. 22. Muller, P. and P. Seeman. Brain neurotransmitter receptors after long-term haloperidol: dopamine, acetylcholine, serotonin, c~-noradrenergic, and naloxone receptors. Life Sei 21: 17511758, 1977. 23. Muller, P. and P. Seeman. Dopaminergic supersensitivity after neuroleptics: Time course and specificity. Psyehopharmacology (Berlin) 60: 1-11, 1978.

BEAN, ELGIN, COOPER AND MARTIN 24. Puff, S. K. and H. Lal. Tolerance to the behavioral and neurochemical effects of haloperidol and morphine in rats chronically treated with morphine or haloperidol. Naunyn Schmiedebergs Arch Pharmaeol 282: 155-170, 1974. 25. Ritzmann, R. F, and H. N. Bhargava. The effects of cyclo (Leu-Gly) on chemical denervation supersensitivity of dopamine receptors by intracerebroventricular injection of 6-hydroxydopamine in mice. Life Sei 27: 2075-2080, 1980. 26. Ritzmann, R. F., J. M. Lee and J. Z. Fields. Peptide inhibition of morphine-induced dopaminergic supersensitivity. Lift, Sei 31: 2287-2290, 1982. 27. Seeman, P. Brain dopamine receptors. Pharmacol Rev 32: 22%313, 1981. 28. Smith, R. C. and J. M. Davis. Behavioral evidence for supersensitivity after chronic administration of haloperidol, clozapine and thioridazine. Life Sci 19: 725-732, 1976. 29. Tarsy, D. and R. J. Baldessarini. Behavioral supersensitivity to apomorphine following chronic treatment with drugs which interfere with the synaptic function of catecholamines. Neuropharmacoh)gy 13: 927-940, 1974. 30. Ungerstedt, U. Postsynaptic supersensitivity after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine system. Acta Physiol Seand [Suppl] 367: 6%93, 1971. 31. Versteeg, D. H. G. Neurohypophyseal hormones and brain neurochemistry. Pharmacol Ther 19: 297-325, 1983. 32. Waddington, J. L. Further anomalies in the dopamine receptor supersensitivity hypothesis of tardive dyskinesia. Trends Neurosei 8: 200, 1985.