Potent and selective inactivation of human liver microsomal cytochrome P-450 isoforms by L-754,394, an investigational human immune deficiency virus protease inhibitor.

L-754,394, N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-(1,1-dimethylethylaminocarbonyl )-4- [(furo[2,3-b]pyridin-5-yl)methyl]piperazin-1-yl]-4(S)-hydroxy-2(R) - phenylmethylpentanamide, is a potent and specific inhibitor of the human immune deficiency virus (HIV) protease. The drug selectively inhibited human liver microsomal CYP 3A4-dependent testosterone 6 beta-hydroxylase and CYP 2D6-dependent bufuralol 1'-hydroxylase activities in a time- and concentration-dependent manner in the presence of an NADPH-generating system. L-754,394 was found to be a very potent inactivator of CYP 3A4. Thus, for testosterone 6 beta-hydroxylase, the inactivation kinetic constants, Kl and kinact, were 7.5 microM and 1.62 min-1, respectively, and the partition ratio (moles product formed per moles enzyme inactivated) was approximately 1.35. To a lesser extent, L-754,394 also was an inactivator of CYP 2D6, for which the corresponding values for Kl, kinact and partition ratio were 32 microM, 0.18 min-1 and 40, respectively. CYP 3A4 inactivation was reduced markedly by ketoconazole, a selective CYP 3A4 inhibitor. Similarly, CYP 2D6 inactivation also was prevented by quinidine, a specific competitive inhibitor of this isoform. However, exogenously added nucleophiles (GSH, semicarbazide and N-acetylcysteine) failed to protect against P-450 inactivation. These results suggest that the inactivation process likely is mediated by a reactive metabolite of L-754,394 that alkylates, and thereby destroys, the enzyme. Furthermore, this electrophilic intermediate may not be released into the medium before the inactivation event.