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

Gerald Zernig, Timothy Burke, John W. Lewis, James H. Woods

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37 Scopus citations


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 ≤ 5 * K(d)) 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 T( 1/4 ) 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]Cl[D-Pen2,D- Pen5]enkephalin or (-)-[3H]bremazocine (in the presence of 1 μM DAMGO and 1 μM [D-Pen5,D-Pen5]enkephalin) was not affected by CCAM protreatment. Finally, ex vivo [3H]DAMGO binding inhibition data correlated well with mu receptor population changes estimated by Black and Left analysis of behavioral (antinociception) experiments (T( 1/4 ) 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.

Original languageEnglish (US)
Pages (from-to)23-31
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number1
StatePublished - Oct 1996
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology


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