MECHANISM OF CLOCINNAMOX BLOCKADE OF OPIOID RECEPTORS - EVIDENCE FROMIN-VITRO AND EX-VIVO BINDING AND BEHAVIORAL ASSAYS

In behavioral experiments, the cinnamoylaminomorphinone clocinnamox (C CAM) has been shown to act as an insurmountable antagonist of mu, but not delta or kappa opioid agonists. In contrast, CCAM displayed only m oderate mu selectivity (29:6:1 for mu:delta:kappa) in radioligand disp lacement experiments using mouse brain membranes. In the present study , the apparent discrepancy between the high mu selectivity of the insu rmountable functional antagonism of CCAM and its only moderate mu sele ctivity in in vitro binding experiments was resolved by in vitro washo ut experiments and ex vivo binding experiments involving all three opi oid receptor types. In the ex vitro washout experiments, CCAM-mediated mu receptor binding inhibition could not be reversed even after allow ing 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-Ala(2),N-Me-Phe(4), Gly(5)-ol]-enkephalin ([H -3]DAMGO) binding (tested at greater than or equal to 5 K-d) by 90%, paralleled by an 88% decrease in mu receptor density in equilibrium s aturation binding assays. Ex vivo [H-3]DAMGO binding returned to contr ol levels with a T-1/2 of 2.7 to 4.2 days (independent of the CCAM dos e), the effect being predominantly due to a recovery of mu receptor de nsity. The CCAM inhibition of ex vivo [H-3]DAMGO binding was dose-depe ndent and could be prevented in part by simultaneous administration of the protein synthesis inhibitor cylcoheximide. In contrast to the ex vivo binding of [H-3]DAMGO, ex vivo binding of p-[H-3]Cl-[D-Pen(2),D-P en(5)]enkephalin or (-)-[H-3]bremazocine (in the presence of 1 mu M DA MGO and 1 mu M [D-Pen(5),D-Pen(5)]enkephalin) was not affected by CCAM pretreatment. Finally, ex vivo [H-3]DAMGO binding inhibition data cor related well with mu receptor population changes estimated by Black an d Leff anal ysis of behavioral (antinociception) experiments (T-1/2 of receptor reappearance, 3.2 days). Thus, although CCAM reversibly inte racted with mu, delta and kappa opioid receptors, only binding to mu r eceptors was wash-resistant. Binding of methoclocinnamox, a codeinone CCAM precursor with mu agonistic activity, seemed to be at least parti ally reversible, even at mu receptors.