Naloxonazine, a potent, long-lasting inhibitor of opiate binding sites

Naloxazone, the hydrazone derivative of naloxone, has proven useful in studies of opiate binding site heterogeneity both in vivo and in vitro based on its long-acting inhibition of high affinity, or mu1, binding sites. However, the need for high doses of naloxazone to inactivated the mu1 sites raised the possibility that its actions might result from lower concentrations of a more active compound. We now present evidence suggesting that this more active compound is the azine derivative of naloxone. In acidic solutions, approximately 35% of naloxazone, spontaneously rearranges to the azine, naloxonazine. Unlike naloxazone, naloxonazine is relatively stable in solution. It does not appreciably dissociate into naloxone and naloxazone and no additional compounds can be detected. Under assay conditions under which no azine formation can be detected, no inhibition of binding of either 3H-dihydromorphine or 3H-DADL is found after incubating tissue with naloxazone at concentrations up to 2000 nM followed by extensive washing. Naloxonazine, on the other hand, produces a potent, dose-dependent inhibition of binding which is resistant to washing. Despite the washes, naloxonazine at 50 nm abolishes high affinity binding with some inhibition seen at concentrations down to 10 nM.