title: In vitro evaluation of the disposition of A novel cysteine protease inhibitor.
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 parasite Trypanosoma 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 (apparent K(m) = 14.0 +/- 4.5 microM and apparent V(max) = 3460 +/- 3190 pmol. mg(-1). min(-1), n = 4), beta-hydroxy-homoPhe K11777 (K(m) = 16.8 +/- 3.5 microM and V(max) = 1260 +/- 1090 pmol. mg(-1). min(-1), n = 4), and N-desmethyl K11777 (K(m) = 18.3 +/- 7.0 microM and V(max) = 2070 +/- 1830 pmol. mg(-1). 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 microM, 54-62% inhibition) and antibodies against human CYP3A4/5 (100 microg of antibodies/100 microg microsomal protein, 55-68% inhibition). CYP2D6 metabolized K11777 to its N-desmethyl metabolite with an apparent K(m) (9.2 +/- 1.4 microM) lower than for CYP3A4 (25.0 +/- 4.0 microM) and human liver microsomes. The apparent K(m) for N-oxide K11777 formation by cDNA-expressed FMO3 was 109 +/- 11 microM. Based on the intrinsic formation clearances and the results of inhibition experiments (CYP2D6, 50 microM 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 (IC(50) = 0.06 microM) and efficacious (maximum inhibition 85%) NADPH-dependent inhibitor of human CYP3A4 mediated 6'beta-hydroxy lovastatin formation, suggesting that K11777 is not only a substrate but also a mechanism-based inhibitor of CYP3A4.
authors.affiliation: Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco, California 94143-0446, USA.
authors.forename: W
authors.initials: W
authors.lastname: Jacobsen
authors.forename: U
authors.initials: U
authors.lastname: Christians
authors.forename: L Z
authors.initials: LZ
authors.lastname: Benet
mesh_terms: D015853:Cysteine Proteinase Inhibitors / Q000493:pharmacokinetics*; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D015853:Cysteine Proteinase Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human