Life Sciences, Vol. 52, pp.

PL 199-203 Printed in the USA

Pergamon Press

PHARMACOLOGY LETTERS Accelerated Communication

ETONITAZENE:

AN OPIOID SELECTIVE FOR THE MU R E C E P T O R TYPES

M a r j o r i e S. Moolten, J o r d a n B. Fishman, J i n - C h u n g Chen and Kristin R. Carlson* D e p a r t m e n t of Pharmacology, U n i v e r s i t y of M a s s a c h u s e t t s Medical Center, Worcester, MA 01655 (Submitted February 2, 1993; accepted February 5, 1993; received in final form February 22, 1993)

Abstract. S p e c i f i c r a d i o l i g a n d binding p r o t o c o l s were u t i l i z e d to compare the affinity of m o r p h i n e and the h i g h - p o t e n c y opioid e t o n i t a z e n e at mul, mu2, delta, kappa I and sigma receptors. Both e t o n i t a z e n e and m o r p h i n e d i s p l a y e d a mul-selective b i n d i n g profile; however, e t o n i t a z e n e had a 2500-fold higher a f f i n i t y at this r e c e p t o r type. The latter result is c o n s i s t e n t with the relative p o t e n c i e s of m o r p h i n e and e t o n i t a z e n e in various b e h a v i o r a l tests.

Introduction M a n y drugs, including opioids, serve as r e i n f o r c e r s when ingested by rodents and other species. Thus, oral drug selfa d m i n i s t r a t i o n is a practical and accepted model of drug abuse in humans (for review see i). One factor w h i c h has limited the u s e f u l n e s s of the oral route is the b i t t e r n e s s of opioids in solution. This p r o b l e m can be overcome by e m p l o y i n g very dilute solutions of a h i g h - p o t e n c y drug. E t o n i t a z e n e (ETZ) is a synthetic opioid w h o s e effects are q u a l i t a t i v e l y similar to morphine. It is, however, a p p r o x i m a t e l y 1000-2000 times more potent than m o r p h i n e in b e h a v i o r a l tests such as analgesia (2,3), catalepsy (3), s u p p r e s s i o n of w i t h d r a w a l symptoms (4), ability to serve as a p o s i t i v e reinforcer (3), and identification as an opioid in a drug d i s c r i m i n a t i o n task (4,5). At b e h a v i o r a l l y active c o n c e n t r a t i o n s the taste of ETZ does not appear aversive to rats (6), and the drug is used f r e q u e n t l y in oral s e l f - a d m i n i s t r a t i o n studies (e.g., 6-8). For such a useful drug, s u r p r i s i n g l y little is known about the i n t e r a c t i o n of ETZ at the various types of opioid receptor. The only study of w h i c h we are aware was done a decade ago, and, of necessity, used ligands that are relatively u n s e l e c t i v e (9). The results of that study indicated that ETZ binds to mu sites with high a f f i n i t y and to delta sites with low affinity. The p r e s e n t study c a p i t a l i z e d on the recent introduction of p r o t o c o l s that define opioid r e c e p t o r types with high specificity, enabling us to compare the b i n d i n g a f f i n i t y of ETZ and m o r p h i n e at mul, mu2, delta, and kappa I opioid sites, as well as at the n o n - o p i o i d sigma site. * C o r r e s p o n d i n g author: P h a r m a c o l o g y Dept, U. M a s s a c h u s e t t s Medical Center, Worcester, MA 01655 0024-3205/93 $6.00 + .00 Copyright 1993 Pergamon Press Ltd All rights reserved.

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Methods Frozen rat brains and guinea pig c e r e b e l l u m were p u r c h a s e d from P e l - F r e e z e (Rogers, AK). R a d i o l a b e l l e d ligands 3H-DADLE ([3H]-[DAla2-D-LeuS]-enkephalin), 3H-DAGO ([3H]-[Tyr-D-Ala-Gly-NMe-Phe-Glyol]-enkephalin), ~ - D P D P E ([3H]-[D-Pen2,D-PenS]-enkephalin),3H-U69,593, and 3H-SKF-10,047 were obtained from New England Nuclear/Dupont. M o r p h i n e sulfate was p u r c h a s e d from Sigma and ETZ was o b t a i n e d from the R e s e a r c h T e c h n o l o g y Branch, NIDA. U n l a b e l e d DPDPE and DSLET ([D-Ser2-LeuS-Thr6]-enkephalin) were purchased from Peninsula L a b o r a t o r i e s (Belmont, CA). B i n d i n g to mul, m u 2 , delta, kappa I and sigma receptors was c h a r a c t e r i z e d a c c o r d i n g to p r e v i o u s l y p u b l i s h e d p r o t o c o l s (10-12); details are p r o v i d e d 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 4C in i0 volumes of the relevant buffer (pH 7.5 at 22C). The h o m o g e n a t e was c e n t r i f u g e d at 35,000xg for 30 min at 4C. The m e m b r a n e fractions were r e s u s p e n d e d in i0 volumes of buffer, incubated for 20 min at room temperature to dissociate endogenous ligands, r e c e n t r i f u g e d and, finally, r e s u s p e n d e d in buffer at a p r o t e i n c o n c e n t r a t i o n of 5 mg/ml. M e m b r a n e s were incubated at 22C for the times listed in Table 1 with a K n c o n c e n t r a t i o n of the r a d i o l i g a n d d e t e r m i n e d in pilot e q u i l i b r i u m binding studies (data not shown). C o n c e n t r a t i o n s of c o m p e t i n g drugs ranged from 0.0001 nM to i00 ~M (0.0001 pM to 1 ~M for ETZ c o m p e t i t i o n at the mu I site). N o n s p e c i f i c b i n d i n g was d e t e r m i n e d in the p r e s e n c e of a 500-fold excess of ETZ or morphine. All plasticware employed in the assays was silicone-coated (Sigmacote) to prevent peptides from sticking. I n c u b a t i o n s were t e r m i n a t e d by f i l t r a t i o n onto p r e s o a k e d (0.5% p o l y e t h y l a m i n e for 20 min) GF/B filters, followed by six ice-cold buffer washes, using a Brandel Model 48R v a c u u m manifold. R a d i o a c t i v i t y was d e t e r m i n e d by liquid s c i n t i l l a t i o n spectrometry. Assays were p e r f o r m e d in triplicate, and at least three separate e x p e r i m e n t s were c o n d u c t e d for each c o m p e t i n g drug at each receptor. ICs0 values were derived from the c o m p e t i t i o n curves using the LIGAND p r o g r a m (14). K i values were c a l c u l a t e d from the ICs0 values a c c o r d i n g to the m e t h o d of Cheng and Prusoff (15). Results The results are shown in Table 2. Compared to morphine, ETZ had an a p p r o x i m a t e l y 2500-fold greater affinity at the mu I site and a 10-fold greater affinity at the mu 2 site. In contrast, the a f f i n i t y of ETZ at the delta site was c o m p a r a b l e to that of morphine, and it had a lower affinity at the kappa I receptor. Both ETZ and m o r p h i n e competed only weakly at the sigma receptor.

Discussion The o b s e r v e d K i values for m o r p h i n e at the v a r i o u s receptor types are consistent with those previously reported in the l i t e r a t u r e (11,13,16). M o r p h i n e e x h i b i t e d the h i g h e s t a f f i n i t y for

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TABLE Characterization Receptor mu I mu 2 delta kappa I sigma Radioligand 0.7 n M 3H-DADLE + i0 n M D P D P E a 2.0 nM 3H-DAGO + 5 nM DSLET c 2.0 n M 3H-DPDPE

of O p i o i d

Receptor

Types Incubation T i m e (min) 150 60 60 60 30

Buffer 30 m M T r i s - H ~ l + 30 m M M g C I 2 30 m M T r i s - H C l 30 m M T r i s - H C l 30 m M T r i s - H C l i0 m M H E P E S - K O H + 0.5 m M E D T A r e c e p t o r s ; UAdded non-mu 2 binding.

2.1 n M 3HU69,593 20 n M 3H-SKF10,047 to d e l t a to b l o c k

aAdded to b l o c k b i n d i n g b i n d i n g (11,12); CAdded

to e n h a n c e

TABLE Competitive

B i n d i n g of M o r p h i n e a n d E t o n i t a z e n e to O p i o i d R e c e p t o r T y p e s

K i (riM) Receptor mu I mu 2 delta kappa I sigma Values are Mean SEM. Morphine I.i 4.2 310.0 15.8 0.i0 0.42 Etonitazene 0.00042 0.41 423.0 900.0 0.00014 0.01 56.0 219.0

44.0 9.0

> 1 ~M

> 1 ~M

m u I r e c e p t o r s (i.I nM), a f o u r - f o l d l o w e r a f f i n i t y for m u 2 r e c e p t o r s , a n d a 1 5 - f o l d l o w e r a f f i n i t y for k a p p, a I sites. L i k e m o r p h i n e , ETZ , w a s m o s t p o t e n t at c o m p e t i n g for b l n d l n g to m u I r e c e p t o r s (0.42 pM). H o w e v e r , t h e mu2/mu I a f f i n i t y r a t i o for ETZ w a s 976, c o m p a r e d to 4 for m o r p h i n e , a n d it w a s m o r e t h a n 5 0 - f o l d less p o t e n t at t h e k a p p a I receptor. The two compounds had comparable affinities at d e l t a receptors and sigma receptors. T h e p i c o m o l a r a f f i n i t y for ETZ at m u I r e c e p t o r s o b s e r v e d in t h e p r e s e n t s t u d y is s u b s t a n t i a l l y h i g h e r t h a n t h ~ a f f i n i t y r e p o r t e d b y R i c e et al. (9). These authors utilized H-morphine or H-[DAlaLM~5]~nkephalinamide, in t h e a b s e n c e of m a g n e s i u m , to label

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opioid receptors. Under these conditions, w h i c h s e l e c t for m u 2 binding, l e s s t h a n 25% of t h e t o t a l s p e c i f i c b i n d i n g r e p r e s e n t s b i n d i n g to h i g h a f f i n i t y , l o w d e n s i t y m u I s i t e s (ii) In c o n t r a s t , w e u t i l i z e d m u I- a n d m u 2 - s e l e c t i v e b i n d i n g p r o t o c o l s (ii-13). Thus, t h e d i s c r e p a n c y b e t w e e n o u r r e s u l t s a n d t h o s e of R i c e et al. m a y w e l l r e f l e c t t h e s e d i f f e r e n c e s in t h e s e l e c t i v i t y of t h e t w o b i n d i n g assays. B i n d i n g a f f i n i t y at o p i o i d r e c e p t o r t y p e s is t h o u g h t to be c o r r e l a t e d w i t h t h e i r p h y s i o l o g i c a l a c t i o n s in vivo. Thus, b i n d i n g to t h e m u r e c e p t o r is c o r r e l a t e d w i t h a n a l g e s i c p o t e n c y (11,12,17), while blndlng to m u 2 r e c e p t o r s has b e e n l i n k e d to r e s p i r a t o r y depression, physical dependence, and the inhibition of g a s t r o i n t e s t i n a l m o t i l i t y (17-19). T h e o b s e r v e d b i n d i n g p r o f i l e s of ETZ and morphine are consistent with this hypothesis. In particular, t h e e x t r e m e l y h i g h a f f i n i t y of ETZ for m u I r e c e p t o r s c o m p a r e d to m o r p h i n e is c o n s i s t e n t w i t h t h e o b s e r v a t i o n t h a t ETZ is 1000-2000 times more potent at inducing analgesia and other b e h a v i o r a l e f f e c t s (2-5). In t h i s respect, it r e s e m b l e s fentanyl, another potent opioid analgesic displaying a mul-selective binding p r o f i l e a n d a K i at t h e m u I s i t e of 7 p M (13). T h e s e l e c t i v i t y and p o t e n c y of c o m p o u n d s s u c h as ETZ a n d f e n t a n y l underscore their superiority to morphine as reinforcers in oral drug selfadministration paradigms.
I ,

Acknowledqements T h i s w o r k w a s s u p p o r t e d b y N I D A D A 0 6 5 3 9 (KRC) a n d t h e O f f i c e of Naval Research (JBF). W e t h a n k Dr. D e b r a N i e h o f f Deutsch for m a n u s c r i p t r e v i e w a n d comment. References i. R.A. MEISCH and M.E. CARROLL, Methods of Assessing the of Abused Drugs. M.A. B o z a r t h (ed),

Reinforcing
2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14.

Properties

143-160, S p r i n g e r - V e r l a g , N e w Y o r k (1987). A. WIKLER, W.R. MARTIN, F.T. PESCOR and C.G. EADES, Psychopharmacologia 5 55-76 (1963). M. SALA, D. BRAIDA, P. C A L C A T E R R A , M.P. L E O N E a n d E. GORI, Eur. J. Pharm. 217 37-41 (1992). C.P. F R A N C E a n d J.H. WOODS, J. Pharm. exp. Ther. 254 6 2 6 - 6 3 2
(1990) .

H.E. S H A N N O N a n d S.G. H O L T Z M A N , J. Pharm. exp. Ther. 201 55-66 (1974). K.R. C A R L S O N , Pharm. Biochem. Behav. 3_44 4 1 9 - 4 2 3 (1989). M.E. C A R R O L L a n d R.A. MEISCH, P s y c h o p h a r m a c o l o g y 6_441-7 (1979). T. SUZUKI, F.R. G E O R G E and R.A. MEISCH, Pharm. Biochem. Behav. 42 5 7 9 - 5 8 6 (1992). K.C. RICE, A.E. J A C O B S O N , T.R. BURKE, JR., B.S. BAJWA, R.A. S T R E A T Y a n d W.A. KLEE, S c i e n c e 220 3 1 4 - 3 1 6 (1983). R.A. LAHTI, M.M. MICKELSON, J.M. MCCALL and P.F. YON VOIGTLANDER, Eur. J. Pharm. 109 2 8 1 - 2 8 4 (1985). J.A. CLARK, R. H O U G H T E N a n d G.W. P A S T E R N A K , Mol. Pharm. 3_44 308317 (1988).

K. K I N O U C H I ,
40 3 8 2 - 3 8 4

K.M. S T A N D I F E R
(1990).

a n d G.W. P A S T E R N A K ,

Biochem.

Pharm.

J.-C. CHEN, E.R. SMITH, M. CAHILL, R. C O H E N and J.B. FISHMAN, L i f e Sci. 5-2 3 8 9 - 3 9 6 (1993). P.J. M U N S O N a n d D. R O D B A R D , Anal. B i o c h e m . 107 2 2 0 - 2 3 9 (1980).

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15. 16. 17. 18. 19.

Y. C H E N G a n d W.H. P R U S O F F , B i o c h e m . P h a r m a c o l . 222 3 0 9 9 - 3 1 0 6 (1973). P.J. H O R A N a n d I.K. HO, Pharm. Biochem. Behav. 3__44 8 4 7 - 8 5 4 (1989). G.W. P A S T E R N A K a n d P.J. WOOD, L i f e Sci. 38 1 8 8 9 - 1 8 9 8 (1986). J.S. H E Y M A N , C.L. W I L L I A M S , T.F. BURKS, H.I. M O S B E R G a n d F. P O R R E C A , J. Pharm. exp. Ther. 245 2 3 8 - 2 4 3 (1988). R.A. LUTZ, R.A. CRUCIANI, T. COSTA, P.J. MUNSON a n d D. R O D B A R D , B i o c h e m . B i o p h y s . Res. Comm. 122 2 6 5 - 2 6 9 (1984).