In vitro and in vivo effects of kelatorphan on enkephalin metabolism in rodent brain

Biologically relevant assays were used to compare the potency of kelatorphan (N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine) as inhibitor of the peptidase-induced metabolism of enkephalins to that of bestatin, a non-specific inhibitor of aminopeptidase and thiorphan, a highly potent blocker of the neutral endopeptidase (EC 3.4.24.11) designated as enkephalinase. Kelatorphan almost completely inhibited the formation of the three metabolites [ 3H]Tyr, [ 3H]Tyr-Gly and [ 3H]Tyr-Gly-Gly produced by incubation of [ 3H][Tyr 1,Met 5]enkephalin with rat striatal slices. Co-administered with [Met 5]enkephalin in mouse brain, kelatorphan was able to prevent by 80% the degradation of the exogenous peptide. Moreover, a mixture of thiorphan (1 μM) and bestatin (20 μM) or kelatorphan alone (20 μM) induced a 2.2 to 2.5-fold increase in endogenous [Met 5]enkephalin overflow after evoked depolarization of superfused rat striatal slices. In this assay, kelatorphan was the only compound to increase by 63% the basal level of released [Met 5]enkephalin. Kelatorphan was about 100 times less potent than bestatin to inhibit the total rat striatal aminopeptidases, but as efficient (IC 50 = 4 × 10 −7 M) as bestatin to inhibit a minor aminopeptidase activity resembling aminopeptidase M. Therefore the reported enhanced analgesic potency of kelatorphan with regard to the association of bestatin and thiorphan is very likely related to its ability to almost completely inhibit enkephalin-degrading enzymes (including the Tyr-Gly releasing peptidase) and to its better selectivity for the biologically relevant aminopeptidase M. Kelatorphan would be a valuable probe, preferable to the association of bestatin and thiorphan, to investigate the physiological functions regulated by a phasic enkephalinergic activity.