@article {Jacobsen1343, author = {Wolfgang Jacobsen and Uwe Christians and Leslie Z. Benet}, title = {In Vitro Evaluation of the Disposition of A Novel Cysteine Protease Inhibitor}, volume = {28}, number = {11}, pages = {1343--1351}, year = {2000}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl) is a potent, irreversible cysteine protease inhibitor. Its therapeutic targets are cruzain, a cysteine protease of the protozoan parasiteTrypanosoma cruzi, and cathepsins B and L, which are associated with cancer progression. We evaluated the metabolism of K11777 by human liver microsomes, isolated cytochrome P450 (CYP) enzymes, and flavin-containing monooxygenase 3 (FMO3) in vitro. K11777 was metabolized by human liver microsomes to three major metabolites:N-oxide K11777 (apparentKm = 14.0 {\textpm} 4.5 μM and apparentVmax = 3460 {\textpm} 3190 pmol {\textperiodcentered} mg-1 {\textperiodcentered} min-1, n = 4), β-hydroxy-homoPhe K11777 (Km = 16.8 {\textpm} 3.5 μM and Vmax = 1260 {\textpm} 1090 pmol {\textperiodcentered} mg-1 {\textperiodcentered} min-1, n = 4), andN-desmethyl K11777 (Km = 18.3 {\textpm} 7.0 μM and Vmax = 2070 {\textpm} 1830 pmol {\textperiodcentered} mg-1 {\textperiodcentered} min-1, n = 4). All three K11777 metabolites were formed by isolated CYP3A and their formation by human liver microsomes was inhibited by the CYP3A inhibitor cyclosporine (50 μM, 54{\textendash}62\% inhibition) and antibodies against human CYP3A4/5 (100 μg of antibodies/100 μg microsomal protein, 55{\textendash}68\% inhibition). CYP2D6 metabolized K11777 to its N-desmethyl metabolite with an apparent Km (9.2 {\textpm} 1.4 μM) lower than for CYP3A4 (25.0 {\textpm} 4.0 μM) and human liver microsomes. The apparent Km forN-oxide K11777 formation by cDNA-expressed FMO3 was 109 {\textpm} 11 μM. Based on the intrinsic formation clearances and the results of inhibition experiments (CYP2D6, 50 μM bufuralol; FMO3 mediated, 100 mM methionine) using human liver microsomes, it was estimated that CYP3A contributes to \>80\% of K11777 metabolite formation. K11777 was a potent (IC50 = 0.06 μM) and efficacious (maximum inhibition 85\%) NADPH-dependent inhibitor of human CYP3A4 mediated 6'β-hydroxy lovastatin formation, suggesting that K11777 is not only a substrate but also a mechanism-based inhibitor of CYP3A4. The American Society for Pharmacology and Experimental Therapeutics}, issn = {0090-9556}, URL = {https://dmd.aspetjournals.org/content/28/11/1343}, eprint = {https://dmd.aspetjournals.org/content/28/11/1343.full.pdf}, journal = {Drug Metabolism and Disposition} }