Mechanism of clocinnamox blockade of opioid receptors: evidence from in vitro and ex vivo binding and behavioral assays

G Zernig, T Burke, JW Lewis and JH Woods

Department of Pharmacology, University of Michigan, Ann Arbor, USA.

In behavioral experiments, the cinnamoylaminomorphinone clocinnamox (CCAM) has been shown to act as an insurmountable antagonist of mu, but not delta or kappa opioid agonists. In contrast, CCAM displayed only moderate mu selectivity (29:6:1 for mu:delta:kappa) in radioligand displacement experiments using mouse brain membranes. In the present study, the apparent discrepancy between the high mu selectivity of the insurmountable functional antagonism of CCAM and its only moderate mu selectivity in in vitro binding experiments was resolved by in vitro washout experiments and ex vivo binding experiments involving all three opioid receptor types. In the ex vitro washout experiments, CCAM- mediated mu receptor binding inhibition could not be reversed even after allowing for 8 hr hr dissociation, whereas the CCAM inhibition of delta and kappa receptor binding was time-dependently reversed. In the ex vivo binding experiments, 1 hr pretreatment of mice with 10 mg/kg of CCAM i.p. decreased ex vivo [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin ([3H]DAMGO) binding (tested at > or = 5 * Kd) by 90%, paralleled by an 88% decrease in mu receptor density in equilibrium saturation binding assays. Ex vivo [3H]DAMGO binding returned to control levels with a T1/2 of 2.7 to 4.2 days (independent of the CCAM dose), the effect being predominantly due to a recovery of mu receptor density. The CCAM inhibition of ex vivo [3H]DAMGO binding was dose-dependent and could be prevented in part by simultaneous administration of the protein synthesis inhibitor cylcoheximide. In contrast to the ex vivo binding of [3H]DAMGO, ex vivo binding of p-[3H]CI-[D-Pen2, D-Pen5]enkephalin or (-)-[3H]bremazocine (in the presence of 1 microM DAMGO and 1 microM [D- Pen6, D-Pen5]enkephalin) was not affected by CCAM pretreatment. Finally, ex vivo [3H]DAMGO binding inhibition data correlated well with mu receptor population changes estimated by Black and Leff analysis of behavioral (antinociception) experiments (T1/2 of receptor reappearance, 3.2 days). Thus, although CCAM reversibly interacted with mu, delta and kappa opioid receptors, only binding to mu receptors was wash-resistant. Binding of methoclocinnamox, a codeinone CCAM precursor with mu agonistic activity, seemed to be at least partially reversible, even at mu receptors.

Volume 279, Issue 1, pp. 23-31, 10/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				A. C. Barrett, E. S. Smith, and M. J. Picker Use of Irreversible Antagonists to Determine the Relative Efficacy of {micro}-Opioids in a Pigeon Drug Discrimination Procedure: Comparison of {beta}-Funaltrexamine and Clocinnamox J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 1061 - 1070. [Abstract] [Full Text] [PDF]

				E. A. Walker and A. M. Young Clocinnamox Distinguishes Opioid Agonists According to Relative Efficacy in Normal and Morphine-Treated Rats Trained to Discriminate Morphine J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 101 - 110. [Abstract] [Full Text] [PDF]

				J. H. Broadbear, T. L. Sumpter, T. F. Burke, S. M Husbands, J. W. Lewis, J. H. Woods, and J. R. Traynor Methocinnamox Is a Potent, Long-Lasting, and Selective Antagonist of Morphine-Mediated Antinociception in the Mouse: Comparison with Clocinnamox, beta -Funaltrexamine, and beta -Chlornaltrexamine J. Pharmacol. Exp. Ther., September 1, 2000; 294(3): 933 - 940. [Abstract] [Full Text]

				J. A. Vivian, M. B. DeYoung, T. L. Sumpter, J. R. Traynor, J. W. Lewis, and J. H. Woods kappa -Opioid Receptor Effects of Butorphanol in Rhesus Monkeys J. Pharmacol. Exp. Ther., July 1, 1999; 290(1): 259 - 265. [Abstract] [Full Text]

				J. P. McLaughlin, K. P. Hill, Q. Jiang, A. Sebastian, S. Archer, and J. M. Bidlack Nitrocinnamoyl and Chlorocinnamoyl Derivatives of Dihydrocodeinone: In Vivo and In Vitro Characterization of µ-Selective Agonist and Antagonist Activity J. Pharmacol. Exp. Ther., April 1, 1999; 289(1): 304 - 311. [Abstract] [Full Text]

				R. C. Pitts, R. M. Allen, E. A. Walker, and L. A. Dykstra Clocinnamox Antagonism of the Antinociceptive Effects of Mu Opioids in Squirrel Monkeys J. Pharmacol. Exp. Ther., June 1, 1998; 285(3): 1197 - 1206. [Abstract] [Full Text]