In vitro evaluation of the disposition of a novel cysteine protease inhibitor

K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl) is a potent, i rreversible cysteine protease inhibitor. Its therapeutic targets are cruzai n, a cysteine protease of the protozoan parasite Trypanosoma cruzi, and cat hepsins B and L, which are associated with cancer progression. We evaluated the metabolism of K11777 by human liver microsomes, isolated cytochrome P4 50 (CYP) enzymes, and flavin-containing monooxygenase 3 (FMO3) in vitro. K1 1777 was metabolized by human liver microsomes to three major metabolites: N-oxide K11777 (apparent K-m = 14.0 +/- 4.5 mu M and apparent V-max = 3460 +/- 3190 pmol . mg(-1) . min(-1), n = 4), beta-hydroxy-homoPhe K11777 (K-m = 16.8 +/- 3.5 mu M and V-max = 1260 +/- 1090 pmol . mg(-1) . min(-1), n = 4), and N-desmethyl K11777 (K-m = 18.3 +/- 7.0 mu M and V-max = 2070 +/- 18 30 pmol . mg(-1) . min(-1), n = 4). All three K11777 metabolites were forme d by isolated CYP3A and their formation by human liver microsomes was inhib ited by the CYP3A inhibitor cyclosporine (50 mu M, 54-62% inhibition) and a ntibodies against human CYP3A4/5 (100 mu g of antibodies/100 mg microsomal protein, 55-68% inhibition). CYP2D6 metabolized K11777 to its N-desmethyl m etabolite with an apparent K-m (9.2 +/- 1.4 mu M) lower than for CYP3A4 (25 .0 +/- 4.0 mu M) and human liver microsomes. The apparent K-m for N-oxide K 11777 formation by cDNA-expressed FMO3 was 109 +/- 11 mu M. Based on the in trinsic formation clearances and the results of inhibition experiments (CYP 2D6, 50 mu M bufuralol; FMO3 mediated, 100 mM methionine) using human liver microsomes, it was estimated that CYP3A contributes to >80% of K11777 meta bolite formation. K11777 was a potent (IC50 = 0.06 mu M) and efficacious (m aximum inhibition 85%) NADPH-dependent inhibitor of human CYP3A4 mediated 6 'beta-hydroxy lovastatin formation, suggesting that K11777 is not only a su bstrate but also a mechanism-based inhibitor of CYP3A4.