Antinociception after intracerebroventricular administration of naltrindole in the mouse

European Journal of Pharmacology, 214 (1992) 273-276

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© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 21044 Short communication

Antinociception after intracerebroventricular administration of naltrindole in the mouse A w i l d a S t a p c l f c l d a, D o n n a L. H a m m o n d b a n d M i c h a e l F. R a f f e r t y

a

'~Neurological Diseases Research, G.D. Searle & Co., Chicago, IL 60077, U.S.A. and h Department of Anesthesia and Critical Care, University of Chicago, Chicago, IL 60637, U.S.A.

Received 12 December 1991,revised MS received21 February 1992,accepted 25 February 1992

Intracerebroventricular (i.c.v.) injection of the 6-opioid receptor antagonist naltrindole hydrochloride (2.2-22.2 nmol) in mice produced a dose-dependent increase in tail flick and hot plate latencies with respective EDs0 and 95% confidence limits of 10.6 (8.3-13.9) and 16.4 (9.2-62.3) nmol. This increase in response latencies was antagonized by 1 mg/kg s.c. naloxone or by i.c.v. coadministration of 1.4 nmol ICI-174,864, a selective peptidergic 6-receptor antagonist. Pretreatment 24 h earlier with the irreversible ~-receptor antagonist /3-funaltrexamine (6 nmol i.c.v.) or 1 h earlier with the selective K-receptor antagonist nor-binaltorphimine (100 nmol i.c.v.) did not attenuate the antinociceptive effects of naltrindole. These data indicate that high doses of naltrindole may have agonist activity at supraspinal 6-opioid receptors in the mouse. Naltrindole; Antinociception; 6-Opioid receptors

1. Introduction

antagonist in vitro. This compound, which has been named naltrindole hydrochloride, has been reported to selectively antagonize the antinociceptive effects of centrally administered 6-receptor agonists such as cyclic[D-Pen 2, D-PenS]enkephalin (DPDPE) or [DSer 2, Leu 5, Thr6]enkephalin (DSLET) (Calcagnetti and Holtzman, 1990; Drower et al., 1991; Portoghese et al., 1990; Sofuoglu et al., 1991). In this paper, we describe the results of a series of studies designed to further characterize the properties of naltrindole in the mouse. Our results indicate that, following i.c.v, administration of doses ranging from 2.2 to 22.2 nmol, naltrindole produced antinociception as measured by the tail flick and hot plate tests. This effect was persistent, dose-dependent, and appeared to be mediated via activation of supraspinal 6-opioid receptors. The apparent agonist activity of naltrindole occurred at doses that were much higher than those previously reported to antagonize the effects of prototypic 6-receptor agonists in the mouse (Portoghese et al., 1990; Sofuoglu et al., 1991).

Ever since t h e recognition of the g-opioid receptor in the CNS and periphery, the pharmacology and function of these sites has been the subject of investigation. Progress over the last several years toward the resolution of questions concerning the behavioral relevance of 6 opioid receptors in the CNS has been slowed, however, by the lack of appropriate tools. Several laboratories have succeeded in developing highly selective peptide g-receptor agonists (see Hruby and Gehrig, 1989 for review), which have been used extensively in local administration studies in intact animals. However, in general, these compounds have very poor bioavailability and present synthetic challenges to the chemists, factors which limit their usefulness in the characterization of 6-receptor-mediated pharmacology after systemic administration. In addition, until very recently the only selective antagonists for g-receptors were peptides having the same limitations as the agonists (see Hruby and Gehig, 1989 for review). Recently, Portoghese and coworkers (1990) described a novel derivative of naltrexone which was found to be a potent and highly selective 6-receptor

2. Materials and methods

Correspondence to: M.F. Rafferty, Neurological Diseases Research, G.D. Searle & Co., 4901 Searle Parkway, Skokie, IL 60077, U.S.A. Tel. 1.708.9824716, fax 1.708.9824714.

Male albino mice (Charles River Laboratory, Portage, MI; C D - 1 / H A M / I C R 20-30 g) were used. Animals were housed with food and water freely available. They were used only once in this study. Thermal

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nociceptive threshold was determined using the tail flick and 55°C hot plate tests as previously described (Drower et al., 1991). Cut-off latencies of 12 s for the tail flick and 40 s for the hot plate were chosen to prevent tissue damage. The first experiment evaluated the dose-response relationship of naltrindole. After measurement of baseline response latencies, either vehicle or naltrindole (2.2-22.2 nmol) was injected i.c.v, in a volume of 5/xl and tail flick latency and hot plate latency were redetermined at fixed intervals for as long as 4 h posttreatment. Two-way analyses of variance for repeated measures were used to compare the effects of naltrindole to those of vehicle. Newman-Keuls test was used to make multiple post-hoc comparisons between individual mean values. The EDs0 values (defined as the dose that produced the half-maximal increase in response latency) were determined from linear regression analysis of the individual data; 95% confidence limits were determined using Fieller's theorem. The second experiment characterized the opioid nature of the increase in tail flick and hot plate latencies produced by naltrindole. After determination of baseline response latencies, mice were injected with 22.2 nmol i.c.v, naltrindole. Ten minutes later, after the measurement of tail flick and hot plate latencies, 1 m g / k g s.c. naloxone hydrochloride was injected. Tail flick latency and hot plate latency were then reassessed 10, 30, 45 and 60 min later. To determine whether the increase in tail flick latency or hot plate latency was mediated by/z-opioid receptors, mice were pretreated with vehicle or 6 nmol i.c.v./3-funaltrexamine (/3-FNA). Twenty-four hours later, animals in both treatment groups were injected with 22.2 nmol i.c.v, naltrindole and tail flick and hot plate latencies were redetermined 10, 20, 35 and 50 min later. To determine whether the increase in response latencies was mediated by 8-opioid receptors, 1.4 nmol ICI-174,864 was coadministered i.c.v, with 22.2 nmol naltrindole in a total volume of 5 /zl; tail flick latency and hot plate latency were redetermined 10, 20, 35, 50 and 70 min later. Finally, to determine whether the increase in tail flick and hot plate latencies was mediated by K-opioid receptors, mice were pretreated with 100 nmol i.c.v, nor-binaltorphimine (nor-BNI). One hour later, the mice were injected i.c.v, with 22.2 nmol naltrindole. Tail flick and hot plate latencies were determined 10, 20, 35 and 50 min later. Two-way analyses of variance for repeated measures were used to compare the effects of drug treatment to that of vehicle. Newman-Keuls test was used to make multiple post-hoc comparisons among individual mean values. Naltrindole hydrochloride was prepared in our laboratories according to reported procedures (Portoghese et al., 1990). ICI-174,864, nor-BNI and naloxone hydrochloride were purchased from commercial suppliers.

Water was used as the vehicle for i.c.v, administration, while saline was used as the vehicle for s.c. administration.

3. Results

I.c.v. administration of 2.2-22.2 nmol naltrindole dose dependently increased tail flick latency and hot plate latency in the mouse (fig. 1). Response latencies were maximally increased within 10 min of injection of 6.7 or 22.2 nmol and remained elevated for at least 40 min (P < 0.01 both tests). The EDs0 and 95% confidence limits for naltrindole 20 min after injection were 10.6 (8.3-13.9) and 16.4 (9.2-62.3) nmol i.c.v, in the tail flick and hot plate tests, respectively. In a subsequent experiment, it was determined that tail flick and hot plate latencies remained significantly elevated for at least 4 h after i.c.v, administration of 22.2 nmol naltrindole (data not shown). The increase in tail flick and hot plate latencies produced by 22.2 nmol i.c.v, naltrindole was significantly antagonized by administration of 1 m g / k g s.c. naloxone (P < 0.05 both tests; fig. 2). The antagonism was apparent within 10 min of the injection of naloxone and persisted for at least 55 min. The increase in tail flick latency and hot plate latency produced by 22.2 nmol i.c.v, naltrindole was also completely antagonized by coadministration of 1.4 nmol ICI-174,864 (P < 0.05

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Time (rain) Fig. 1. Effects of intracerebroventricularly administered naltrindole on response latencies in the (A) tail flick and (B) hot plate tests. Each point represents the m e a n ± S.E.M. of determinations made in 8 - 1 0 mice. Asterisks and daggers denote values that differ significantly from those in vehicle-treated mice at the corresponding time point (* P < 0.05; * P <0.01). (e) Vehicle; ( I ) 2.2 nmol; ( 0 ) 6.7 nmol; ( • ) 22.2 nmol.

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Fig. 2. Effects of 1 mg/kg s.c. naloxone, 1.4 nmol i.c.v. ICI-174,864, 6 nmol i.c.v. /3-FNA or 100 nmol i.c.v, nor-BNI on the increase in response latency produced by 22.2 nmol i.c.v, naltrindole in the (A) tail flick and (B) hot plate tests. Bars depict the mean+S.E, of determinations made in 8-12 mice. For ease of presentation, only the data obtained 10 min after injection of naltrindole are depicted. See text for order of antagonist administration. Response latencies determined in mice receivingi.c.v, injection of vehicle are provided for comparative purposes. Daggers denote values that differ significantly from those in mice that received naltrindole alone (P < 0.01).

both tests; fig. 2); this antagonism persisted for the duration of the experiment. In contrast, pretreatment with 6 nmol i.c.v. /3-FNA did not antagonize the increase in either tail flick latency or hot plate latency produced by 22.2 nmol i.c.v, naltrindole at any time point (fig. 2; P > 0.4 both tests). This dose of/3-FNA, however, completely antagonized the increase in tail flick latency and hot plate latency produced by a supramaximal dose of morphine sulfate (15 nmol i.c.v.; data not shown). Pretreatment 1 h earlier with 100 nmol i.c.v, nor-BNI also failed to antagonize the increase in either tail flick latency or hot plate latency produced by 22.2 nmol i.c.v, naltrindole at any time point (fig. 2; P > 0.4 both tests).

4. Discussion

The principal finding of this study was that i.c.v. administration of naltrindole in the mouse produced antinociception in the tail flick and hot plate tests. This effect appeared to be mediated by the 6-opioid receptor as it was antagonized by either naloxone or the peptidergic g-receptor antagonist ICI-174,864, but not by the irreversible/z-receptor antagonist/3-FNA or by

the selective x-receptor antagonist nor-BNI. The doses o f / 3 - F N A and nor-BNI used in this study were previously shown to antagonize the antinociceptive effects of selective/z- (Ward et al., 1982; unpublished observations) or K-receptor agonists (Takemori et al., 1988), respectively. The present results, which indicate that the antinociceptive effects of high doses of naltrindole are mediated by a g-opioid receptor, are not at variance with previous reports that naltrindole selectively antagonized the antinociceptive effects of i.c.v, or intrathecally administered g-receptor agonists such as D P D P E or DSLET. It is important to note that in these previous studies the effective antagonist doses of naltrindole were in the picomolar range, 1000 times less than those used in the present study (Portoghese et al., 1990; Sofuoglu et al., 1991). Indeed, Takemori et al. (1990) previously noted that higher doses of naltrindole and other related compounds produced antinociception in the writhing test in the mouse, although the mechanism responsible for this effect was not specified. Finally, the peptidergic g-opioid receptor antagonist ICI-174,864 has similarly been reported to have agonist activity at the g-opioid receptor at high doses (Cohen et al., 1986). These data and the results of the present study therefore indicate that investigations of the role of supraspinal 6-opioid receptors must pay particular attention to the dose range and take into consideration the agonist properties of naltrindole at doses in excess of 2.2 nmol i.c.v, in the mouse. Finally, several investigators have recently postulated the existence of subtypes of the g-opioid receptor. Porreca and coworkers have proposed two subtypes of the g-opioid receptor that can be distinguished on the basis of their sensitivity to antagonism by the irreversible g-receptor antagonists [D-AlaZ,LeuS,Cys6] enkephalin or naltrindole 5'-isothiocyanate (Jiang et al., 1991). Thus, [D-Ala2,LeuS,Cys6]enkephalin antagonized the increase in tail flick latency produced by DPDPE, but not [D-AlaZ]deltorphin II, whereas the irreversible g-selective receptor antagonist naltrindole 5'-isothiocyanate antagonized the effect of [D-Ala 2] deltorphin II, but not that of DPDPE. Takemori has similarly proposed two subtypes of the g-receptor based on the differential antagonism of D S L E T and D P D P E by the benzofuran analog of naltrindole and, to a lesser extent, by naltrindole itself (Sofuoglu et al., 1991). At present, it is not known whether the agonist actions of high doses of naltrindole may be mediated a different subtype of the 6-opioid receptor than that at which its antagonist activity is expressed.

Acknowledgements

We wish to thank Mr. Peter Yonan, Neurological Diseases Research, G.D. Searle & Co. for the synthe-

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sis of naltrindole. This work was supported in part by PHS Grant DA06736 to D.L.H. References Calcagnetti, D.J. and S.G. Holtzman, 1990, Delta opioid antagonist, naltrindole, selectively blocks analgesia induced by DPDPE, but not DAGO or morphine, Pharmacol. Biochem. Behav. 38, 185. Cohen, M.L., R.T. Shuman, J.J. Osborne and P.D. Gesellchen, 1986, Opioid agonist activity of ICI 174,864 and its carboxypeptidase degradation product, LY281217, J. Pharmacol. Exp. Ther. 238, 769. Drower, E.J., A. Stapelfeld, M.F. Rafferty, B.R. DeCosta, K.C. Rice and D.L. Hammond, 1991, Selective antagonism by naltrindole of the antinociceptive effects of the delta opioid agonist cyclic[Dpenicillamine2-D-penicillamineS]enkephatin in the rat, J. Pharmacol. Exp. Ther. 259, 725. Hruby, V.J. and C.A. Gehig, 1989, Recent developments in the design of receptor specific opioid peptides, Med. Res. Rev. 9, 343. Jiang, Q., A.E. Takemori, M. Sultana, P.S. Portoghese, W.D. Bowen, H.I. Mosberg and F. Porreca, 1991, Differential antagonism of

opioid delta antinociception by [D-AIa 2, Leu 5, Cys6]enkephalin (DALCE) and naltrindole 5'-isothiocyanate (naltrindoleI): Evidence for 6 receptor subtypes, J. Pharmacol. Exp. Ther. 257, 1069. Portoghese, P.S., M. Sultana and A.E. Takemori, 1990, Design of peptidomimetic g-opioid receptor antagonists using the messageaddress concept, J. Med. Chem. 33, 1714. Sofuoglu, M., P.S. Portoghese and A.E. Takemori, 1991, Differential antagonism of delta opioid agonists by naltrindole and its benzofuran analog (NTB) in mice: evidence for delta opioid receptor subtypes, J. Pharmacol. Exp. Ther. 257, 676. Takemori, A.E., B.G. Ho, J.S. Naeseth and P.S. Portoghese, 1988, Nor-binaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays, J. Pharmacol. Exp. Ther. 246, 255. Takemori, A.E., M. Sofuoglu, M. Sultana and P.S. Portoghese, 1990, Pharmacology of highly selective, non-peptide delta opioid receptor antagonists, in: New Leads in Opioid Research, eds. J.M. Van Ree, A.H. Mulder, V.M. Wiegant and T.B. Van Wimersa Greidamus (Excerpta Medica, Amsterdam) p. 277. Ward, S.J., P.S. Portoghese and A.E. Takemori, 1982, Pharmacological characterization in vivo of the novel opiate,/3-funaltrexamine, J. Pharmacol. Exp. Ther. 220, 494.