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 ± 4.5 μM and apparentVmax = 3460 ± 3190 pmol · mg−1 · min−1, n = 4), β-hydroxy-homoPhe K11777 (Km = 16.8 ± 3.5 μM and Vmax = 1260 ± 1090 pmol · mg−1 · min−1, n = 4), andN-desmethyl K11777 (Km = 18.3 ± 7.0 μM and Vmax = 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 μM, 54–62% inhibition) and antibodies against human CYP3A4/5 (100 μg of antibodies/100 μg microsomal protein, 55–68% inhibition). CYP2D6 metabolized K11777 to its N-desmethyl metabolite with an apparent Km (9.2 ± 1.4 μM) lower than for CYP3A4 (25.0 ± 4.0 μM) and human liver microsomes. The apparent Km forN-oxide K11777 formation by cDNA-expressed FMO3 was 109 ± 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.
Footnotes
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Send reprint requests to: Leslie Z. Benet, Ph.D., Professor, Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco, CA 94143-0446. E-mail: benet{at}itsa.ucsf.edu
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This study was supported in part by National Institutes of Health Grant CA72006 (to L.Z.B), the Deutsche Forschungsgemeinschaft Grant Ch 95/6-2 (to U.C.), and National Institutes of Health NCRR Grant 01614.
- Abbreviations used are::
- K11777
- N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl
- CYP
- cytochrome P450
- FMO3
- flavin-containing monooxygenase 3
- MS
- mass spectrometry
- HLF
- human liver (female)
- HLM
- human liver (male)
- LOD
- below limit of detection
- Received November 11, 1999.
- Accepted July 26, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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