Life Sciences, Vol. 52, pp. PL 199-203 Pergamon Press Printed in the USA

PHARMACOLOGY LETTERS

Accelerated Communication

ETONITAZENE: AN OPIOID SELECTIVE FOR THE MU RECEPTOR TYPES

Marjorie S. Moolten, Jordan B. Fishman, Jin-Chung Chen and Kristin R. Carlson*

Department of Pharmacology, University of Massachusetts Medical Center, Worcester, MA 01655

(Submitted February 2, 1993; accepted February 5, 1993; received in final form February 22, 1993)

Abstract. Specific radioligand binding protocols were utilized to compare the affinity of morphine and the high-potency opioid etonitazene at mul, mu2, delta, kappa I and sigma receptors. Both etonitazene and morphine displayed a mul-selective binding profile; however, etonitazene had a 2500-fold higher affinity at this receptor type. The latter result is consistent with the relative potencies of morphine and etonitazene in various behavioral tests.

Introduction

Many drugs, including opioids, serve as reinforcers when ingested by rodents and other species. Thus, oral drug self- administration is a practical and accepted model of drug abuse in humans (for review see i). One factor which has limited the usefulness of the oral route is the bitterness of opioids in solution. This problem can be overcome by employing very dilute solutions of a high-potency drug. Etonitazene (ETZ) is a synthetic opioid whose effects are qualitatively similar to morphine. It is, however, approximately 1000-2000 times more potent than morphine in behavioral tests such as analgesia (2,3), catalepsy (3), suppression of withdrawal symptoms (4), ability to serve as a positive reinforcer (3), and identification as an opioid in a drug discrimination task (4,5). At behaviorally active concentrations the taste of ETZ does not appear aversive to rats (6), and the drug is used frequently in oral self-administration studies (e.g., 6-8).

For such a useful drug, surprisingly little is known about the interaction of ETZ at the various types of opioid receptor. The only study of which we are aware was done a decade ago, and, of necessity, used ligands that are relatively unselective (9). The results of that study indicated that ETZ binds to mu sites with high affinity and to delta sites with low affinity. The present study capitalized on the recent introduction of protocols that define opioid receptor types with high specificity, enabling us to compare the binding affinity of ETZ and morphine at mul, mu2, delta, and kappa I opioid sites, as well as at the non-opioid sigma site.

*Corresponding author: Pharmacology Dept, U. Massachusetts Medical Center, Worcester, MA 01655

0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.

PL-200 Etonitazene on Opioid Receptor Binding Vol. 52, No. 18, 1993

Methods

Frozen rat brains and guinea pig cerebellum were purchased from Pel-Freeze (Rogers, AK). Radiolabelled ligands 3H-DADLE ([3H]-[D- Ala2-D-LeuS]-enkephalin), 3H-DAGO ([3H]-[Tyr-D-Ala-Gly-NMe-Phe-Gly- ol]-enkephalin), ~-DPDPE ([3H]-[D-Pen2,D-PenS]-enkephalin),3H-U69,593, and 3H-SKF-10,047 were obtained from New England Nuclear/Dupont. Morphine sulfate was purchased from Sigma and ETZ was obtained from the Research Technology Branch, NIDA. Unlabeled DPDPE and DSLET ([D-Ser2-LeuS-Thr6]-enkephalin) were purchased from Peninsula Laboratories (Belmont, CA).

Binding to mul, mu2, delta, kappa I and sigma receptors was characterized according to previously published protocols (10-12); details are provided in (13) and summarized in Table i. Briefly, membranes were prepared by homogenizing rat brains (minus cerebellum) or, for kappa I assays, guinea pig cerebellum, at 4°C in i0 volumes of the relevant buffer (pH 7.5 at 22°C). The homogenate was centrifuged at 35,000xg for 30 min at 4°C. The membrane fractions were resuspended in i0 volumes of buffer, incubated for 20 min at room temperature to dissociate endogenous ligands, recentrifuged and, finally, resuspended in buffer at a protein concentration of 5 mg/ml.

Membranes were incubated at 22°C for the times listed in Table 1 with a K n concentration of the radioligand determined in pilot equilibrium binding studies (data not shown). Concentrations of competing drugs ranged from 0.0001 nM to i00 ~M (0.0001 pM to 1 ~M for ETZ competition at the mu I site). Nonspecific binding was determined in the presence of a 500-fold excess of ETZ or morphine. All plasticware employed in the assays was silicone-coated (Sigmacote) to prevent peptides from sticking. Incubations were terminated by filtration onto presoaked (0.5% polyethylamine for 20 min) GF/B filters, followed by six ice-cold buffer washes, using a Brandel Model 48R vacuum manifold. Radioactivity was determined by liquid scintillation spectrometry. Assays were performed in triplicate, and at least three separate experiments were conducted for each competing drug at each receptor. ICs0 values were derived from the competition curves using the LIGAND program (14). K i values were calculated from the ICs0 values according to the method of Cheng and Prusoff (15).

Results

The results are shown in Table 2. Compared to morphine, ETZ had an approximately 2500-fold greater affinity at the mu I site and a 10-fold greater affinity at the mu 2 site. In contrast, the affinity of ETZ at the delta site was comparable to that of morphine, and it had a lower affinity at the kappa I receptor. Both ETZ and morphine competed only weakly at the sigma receptor.

Discussion

The observed K i values for morphine at the various receptor types are consistent with those previously reported in the literature (11,13,16). Morphine exhibited the highest affinity for

Vol. 52, No. 18, 1993 Etonitazene on Opioid Receptor Binding PL-201

TABLE 1

Characterization of Opioid Receptor Types

Receptor Radioligand Buffer Incubation Time (min)

mu I 0.7 nM 3H-DADLE 30 mM Tris-H~l 150 + i0 nM DPDPE a + 30 mM MgCI 2

mu 2 2.0 nM 3H-DAGO 30 mM Tris-HCl 60 + 5 nM DSLET c

delta 2.0 nM 3H-DPDPE 30 mM Tris-HCl 60

kappa I 2.1 nM 3H- 30 mM Tris-HCl 60 U69,593

sigma 20 nM 3H-SKF- i0 mM HEPES-KOH 30 10,047 + 0.5 mM EDTA

aAdded to block binding to delta receptors; UAdded to enhance binding (11,12); CAdded to block non-mu 2 binding.

TABLE 2

Competitive Binding of Morphine and Etonitazene to Opioid Receptor Types

K i (riM)

Receptor Morphine Etonitazene

mu I I.i ± 0.i0 0.00042 ± 0.00014

mu 2 4.2 ± 0.42 0.41 ± 0.01

delta 310.0 ± 44.0 423.0 ± 56.0

kappa I 15.8 ± 9.0 900.0 ± 219.0

sigma > 1 ~M > 1 ~M

Values are Mean ± SEM.

mu I receptors (i.I nM), a four-fold lower affinity for mu 2 receptors, and a 15-fold lower affinity for kappa sites. Like morphine, ETZ

, , I

was most potent at competing for blndlng to mu I receptors (0.42 pM). However, the mu2/mu I affinity ratio for ETZ was 976, compared to 4 for morphine, and it was more than 50-fold less potent at the kappa I receptor. The two compounds had comparable affinities at delta receptors and sigma receptors.

The picomolar affinity for ETZ at mu I receptors observed in the present study is substantially higher than th~ affinity reported by Rice et al. (9). These authors utilized H-morphine or H-[D- AlaLM~5]~nkephalinamide, in the absence of magnesium, to label

PL-202 Etonitazene on Opioid Receptor Binding Vol. 52, No. 18, 1993

opioid receptors. Under these conditions, which select for mu 2 binding, less than 25% of the total specific binding represents binding to high affinity, low density mu I sites (ii) In contrast, we utilized mu I- and mu2-selective binding protocols (ii-13). Thus, the discrepancy between our results and those of Rice et al. may well reflect these differences in the selectivity of the two binding assays.

Binding affinity at opioid receptor types is thought to be correlated with their physiological actions in vivo. Thus, binding to the mu receptor is correlated with analgesic potency (11,12,17),

° ° I ,

while blndlng to mu 2 receptors has been linked to respiratory depression, physical dependence, and the inhibition of gastrointestinal motility (17-19). The observed binding profiles of ETZ and morphine are consistent with this hypothesis. In particular, the extremely high affinity of ETZ for mu I receptors compared to morphine is consistent with the observation that ETZ is 1000-2000 times more potent at inducing analgesia and other behavioral effects (2-5). In this respect, it resembles fentanyl, another potent opioid analgesic displaying a mul-selective binding profile and a K i at the mu I site of 7 pM (13). The selectivity and potency of compounds such as ETZ and fentanyl underscore their superiority to morphine as reinforcers in oral drug self- administration paradigms.

Acknowledqements

This work was supported by NIDA DA06539 (KRC) and the Office of Naval Research (JBF). We thank Dr. Debra Niehoff Deutsch for manuscript review and comment.

References

i. R.A. MEISCH and M.E. CARROLL, Methods of Assessing the Reinforcing Properties of Abused Drugs. M.A. Bozarth (ed), 143-160, Springer-Verlag, New York (1987).

2. A. WIKLER, W.R. MARTIN, F.T. PESCOR and C.G. EADES, Psychopharmacologia 5 55-76 (1963).

3. M. SALA, D. BRAIDA, P. CALCATERRA, M.P. LEONE and E. GORI, Eur. J. Pharm. 217 37-41 (1992).

4. C.P. FRANCE and J.H. WOODS, J. Pharm. exp. Ther. 254 626-632 (1990) .

5. H.E. SHANNON and S.G. HOLTZMAN, J. Pharm. exp. Ther. 201 55-66 (1974).

6. K.R. CARLSON, Pharm. Biochem. Behav. 3_44 419-423 (1989). 7. M.E. CARROLL and R.A. MEISCH, Psychopharmacology 6_441-7 (1979). 8. T. SUZUKI, F.R. GEORGE and R.A. MEISCH, Pharm. Biochem. Behav.

42 579-586 (1992). 9. K.C. RICE, A.E. JACOBSON, T.R. BURKE, JR., B.S. BAJWA, R.A.

STREATY and W.A. KLEE, Science 220 314-316 (1983). i0. R.A. LAHTI, M.M. MICKELSON, J.M. MCCALL and P.F. YON

VOIGTLANDER, Eur. J. Pharm. 109 281-284 (1985). ii. J.A. CLARK, R. HOUGHTEN and G.W. PASTERNAK, Mol. Pharm. 3_44 308-

317 ( 1 9 8 8 ) . 12. K. KINOUCHI, K.M. STANDIFER and G.W. PASTERNAK, Biochem. Pharm.

40 382-384 ( 1 9 9 0 ) . 13. J.-C. CHEN, E.R. SMITH, M. CAHILL, R. COHEN and J.B. FISHMAN,

Life Sci. 5-2 389-396 (1993). 14. P.J. MUNSON and D. RODBARD, Anal. Biochem. 107 220-239 (1980).

Vol. 52, No. 18, 1993 Etonitazene on Opioid Receptor Binding PL-203

15. Y. CHENG and W.H. PRUSOFF, Biochem. Pharmacol. 222 3099-3106 (1973).

16. P.J. HORAN and I.K. HO, Pharm. Biochem. Behav. 3__44 847-854 (1989).

17. G.W. PASTERNAK and P.J. WOOD, Life Sci. 38 1889-1898 (1986). 18. J.S. HEYMAN, C.L. WILLIAMS, T.F. BURKS, H.I. MOSBERG and F.

PORRECA, J. Pharm. exp. Ther. 245 238-243 (1988). 19. R.A. LUTZ, R.A. CRUCIANI, T. COSTA, P.J. MUNSON and D.

RODBARD, Biochem. Biophys. Res. Comm. 122 265-269 (1984).