Evaluation of enkephalinase inhibition in the living mouse, using [3H]acetorphan as a probe.

A novel in vivo binding test was developed in order to evaluate the degree of occupancy of enkephalinase (EC 3.4.24.11), a membrane-bound metallopeptidase, in cerebral and peripheral tissues of mice treated with enkephalinase inhibitors. The probe selected for this purpose was the prodrug [3H]acetorphan, a lipophilic diesterified derivative of the potent enkephalinase inhibitor thiorphan readily releasing the latter by tissue hydrolysis. In order to validate the in vivo binding assay, [3H]thiorphan binding to membranes was first studied in vitro. [3H]Thiorphan binding to cerebral and peripheral tissues (lung and kidney) was saturable over a low nonspecific binding, occurring with a KD of 0.6 nM consistent with the Ki of the compound as enkephalinase inhibitor. [3H]Thiorphan binding varied largely among various tissues and was highly correlated with the catalytic activity of enkephalinase, thus indicating a selective labeling of the peptidase. After the i.v. administration of [3H]acetorphan a large fraction of the radioactivity remained bound to membranes isolated by a rapid filtration assay. Bound radioactivity mainly corresponded to [3H] thiorphan as identified by high-performance liquid chromatography analysis of kidney membranes, whereas unchanged [3H]acetorphan was not detectable. In vivo binding generated by [3H]acetorphan was saturable, with maximum binding sites values which were in rather good agreement with corresponding maximum binding sites values of [3H]thiorphan binding in vitro, particularly in brain. Specific in vivo binding was calculated as the difference between total and a generally low, nonspecific binding evaluated in mice receiving a large dose of nonlabeled acetorphan. Specific in vivo binding varied largely among tissues and generally reflected the abundance of enkephalinase molecules in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)