enkephalin--ala(2)-mephe(4)-gly(5)- and clocinnamox

In search of a truly high-efficacy (i.e., tau > 100) mu opioid analgesic, we determined the efficacy (tau) and apparent in vivo affinity (KA) of the high-potency alkoxymorphinan 14-methoxymetopon. However, in the present study, 14-methoxymetopon's efficacy proved to be only 1.5-fold higher than that of morphine (tau, 19 vs. 12). KA values were 2,900 nmol/kg for 14-methoxymetopon and 46,000 nmol/kg for morphine (Ki for [3H]DAMGO binding, 0.33 vs 3.4 nmol/l). Thus, the 24-fold higher potency of methoxymetopon could be fully accounted for by its 16-fold higher apparent in vivo affinity and its only 1.5-fold higher efficacy. Furthermore, the 10-fold higher affinity of 14-methoxymetopon for the mu opioid receptor - as previously determined in radioligand binding assays - was confirmed in the present behavioral tests of thermal antinociception.

Topics: Analgesics, Opioid; Animals; Binding, Competitive; Cinnamates; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hot Temperature; Male; Mice; Mice, Inbred C57BL; Morphine; Morphine Derivatives; Narcotic Antagonists; Narcotics; Pain Measurement; Receptors, Opioid, mu; Signal Transduction

Naltrexone (NTX) exhibited approximately 3-fold higher affinity for sites labeled by [3H]U69,593 (putative kappa 1-selective ligand) than [3H]bremazocine (non-selective ligand) in the presence of mu and delta receptor blockade in monkey brain membranes. This led us to test an hypothesis that NTX could display in vivo antagonist selectivity for kappa 1-versus non-kappa 1-mediated effects. Six opioid agonists were characterized by NTX apparent pA2 analysis in a 50 degrees C water tail-withdrawal assay in rhesus monkeys. Constrained NTX pA2 values (95% confidence limits) were: alfentanil, 8.66 (8.47-8.85); ethylketocyclazocine, 7.97 (7.93-8.01); U69,593, 7.64 (7.49-7.79); U50,488, 7.55 (7.42-7.67); bremazocine, 6.92 (6.73-7.12); enadoline, 6.87 (6.69-7.05). Pretreatment with clocinnamox, an irreversible mu antagonist, confirmed that mu receptors were not involved in the antinociception produced by the kappa agonists, U69,593, U50,488, bremazocine and enadoline; however, both mu and kappa receptors mediated the antinociceptive effects of ethyl-ketocyclazocine. The apparent NTX pA2 profile of opioid agonists correlated highly with the radioligand binding studies, which indicates that U69,593 and U50,488 produced antinociception by acting on kappa-1 receptors, whereas bremazocine and enadoline probably acted via non-kappa-1 receptors. This study provides further functional evidence of kappa opioid receptor multiplicity in primates and suggests that NTX may be a useful tool to study this phenomenon in vivo.

Topics: Analgesics, Opioid; Animals; Cinnamates; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Macaca mulatta; Male; Morphine Derivatives; Naltrexone; Narcotic Antagonists; Receptors, Opioid, kappa

The effect of a mu-opioid receptor irreversible antagonist on the development of tolerance to fentanyl was determined in mice. Mice were injected with saline or clocinnamox (3.2 mg/kg, i.p.) and 4 h later mice implanted s.c. with a placebo pellet or an osmotic minipump that infused fentanyl (0.165 mg/kg per day) for 3 days. Fentanyl pumps and placebo pellets were removed on the third day following implantation and 4 h later mu-opioid receptor saturation binding studies in whole brain ([3H][D-Ala2,MePhe4,Gly-ol5]enkephalin: DAMGO) or fentanyl analgesic dose-response studies (tailflick assay) were conducted. Fentanyl infusions and clocinnamox both significantly reduced the potency of fentanyl by 2.8- and 2.4-fold, respectively. When fentanyl and clocinnamox were administered together, a significant 5.0-fold reduction in fentanyl potency relative to the saline-placebo group was observed, which represents an additive effect of clocinnamox and fentanyl. The ED50 of fentanyl in clocinnamox-treated mice was shifted 2.1-fold by fentanyl infusion relative to the clocinnamox-placebo group. This is comparable to the 2.8-fold shift in the ED50 produced by fentanyl infusion in saline-treated mice. In binding studies, fentanyl produced a small (-9%) reduction in Bmax, while clocinnamox significantly reduced (-41%) mu-opioid receptor density without altering affinity (Kd). In the clocinnamox-fentanyl group, there was a 50% reduction in Bmax, which is similar to the additive effect observed in analgesia studies. These data indicate that changes in mu-opioid receptor density prior to the development of tolerance to fentanyl do not impact on the magnitude of tolerance.

Topics: Analgesics; Animals; Brain Chemistry; Cinnamates; Dose-Response Relationship, Drug; Down-Regulation; Drug Implants; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Fentanyl; Kinetics; Male; Mice; Morphine Derivatives; Narcotic Antagonists; Narcotics; Pain Measurement; Reaction Time; Receptors, Opioid, mu

Clocinnamox is a long-lasting, nonequilibrium, mu-opioid receptor antagonist in mice and monkeys. The present studies examined the in vivo and ex vivo effects of clocinnamox in rats. Under control conditions, morphine dose-dependently increased tail-withdrawal latencies from 50 degrees C water, with a mean ED50 of 7.3 +/- 1.1 mg/kg. Clocinnamox antagonized the antinociceptive effects of morphine. 1.0 mg/kg clocinnamox displaced the morphine dose-response curve 4-fold to the right of the control curve and 10 mg/kg clocinnamox eliminated morphine's antinociceptive effects at doses up to 1000 mg/kg for at least seven days. There was a gradual recovery of the antinociceptive response to morphine; however, the morphine dose-response curve did not return to its original position by five weeks after 10 mg/kg clocinnamox. Whole brain membranes were prepared from separate groups of rats for determination of binding parameters of [3H][D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO). Clocinnamox dose-dependently decreased [3H]DAMGO binding ex vivo and the decreased binding was a result of changes in Bmax. The control Bmax for [3H]DAMGO was 234 +/- 8 fmol/mg protein; in membranes prepared from rats pretreated with 10 mg/kg clocinnamox, the Bmax value for [3H]DAMGO was 54 +/- 2 fmol/mg protein. The Bmax values for [3H]DAMGO binding after an injection of 10 mg/kg clocinnamox returned towards control values gradually, four weeks after clocinnamox the Bmax was 178 +/- 10 fmol/mg protein. These results suggest that clocinnamox is a long-lasting, nonequilibrium mu-opioid receptor antagonist in rats.

Topics: Analgesics, Opioid; Animals; Cinnamates; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Kinetics; Membranes; Morphine; Morphine Derivatives; Narcotic Antagonists; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Time Factors

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.

Topics: Animals; Behavior, Animal; Benzomorphans; Cinnamates; Cycloheximide; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Mice; Morphine Derivatives; Narcotic Antagonists; Receptors, Opioid, mu

The effects of the systemically active irreversible opioid receptor antagonist clocinnamox (C-CAM; 14 beta-(p-chlorocinnamoylamino)-7,8-dihydro-N- cyclopropylmethyl normorphinone mesylate) on mu receptor binding to cerebral membranes and on mu opioid analgesia were assessed using mice. After systemic administration, C-CAM produced a dose-dependent decrease in the Bmax values of both [3H]DAMGO ([D-Ala2, N-MePhe4, Gly5-ol][tyrosyl-3,5-3H]enkephalin) and [3H]naltrexone without affecting the Kd value of either ligand. After administration of 3.2 mg/kg of C-CAM, [3H]DAMGO binding recovered gradually, returning to control levels by 8 days. This time course of recovery was similar to that observed with 3.2 mg/kg of C-CAM against morphine analgesia in the warm-water tail-withdrawal assay. The analgesic effect of the mu agonist etonitazene also was assessed in the assay. C-CAM produced dose-dependent rightward and slight downward shifts of the etonitazene dose-effect curve. The analgesic activity of etonitazene had still not returned to base-line levels 12 days after administration of 32 mg/kg of C-CAM, a time at which [3H]DAMGO binding had returned to control levels. In addition, the apparent pA2 values of etonitazene with naltrexone in the tail-withdrawal assay were assessed at 4, 8 and 12 days after the administration of 32 mg/kg of C-CAM, and none were found to be different from the control pA2 value. These results support the notion that C-CAM is an irreversible mu receptor antagonist and suggest that post-treatment, perhaps newly synthesized, mu receptors are similar to mu receptors in control membranes.

Topics: Analgesia; Animals; Benzimidazoles; Cinnamates; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Mice; Morphine Derivatives; Naltrexone; Narcotic Antagonists; Receptors, Opioid, mu