Abstract
K02 (morpholine-urea-Phe-Hphe-vinylsulfone), a newly developed peptidomimetic, acts as a potent cysteine protease inhibitor, especially of cathepsins B and L (which are associated with cancer progression) and cruzain (a cysteine protease of Trypanosoma cruzi, which is responsible for Chagas’ disease). Here we investigated features of the disposition of K02 using in vitro systems, characterizing the interaction of the drug with human cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp), a mediator of multidrug resistance (MDR) to cancer chemotherapy and a countertransporter in the intestine that limits oral drug bioavailability. P-gp functions as an ATP-dependent drug efflux pump to reduce intracellular cytotoxic concentrations. An HPLC assay was developed to analyze K02 and its metabolites formed in human liver microsomes. Three major primary metabolites were determined by LC/MS/MS to be hydroxylated products of the parent compound. A rabbit anti-CYP3A polyclonal antibody (200 μl antibody/mg microsomal protein) produced 75–94% inhibition of the formation of these three hydroxylated metabolites. Ketoconazole (5 μM), a selective CYP3A inhibitor, produced up to 75% inhibition, whereas other CYP-specific inhibitors,i.e. quinidine (CYP2D6), 7,8-benzoflavone (CYP1A2), and sulfaphenazole (CYP2C9), showed no significant effects. An identical metabolite formation profile for K02 was observed with cDNA-expressed human CYP3A4 (Gentest). These data demonstrate that K02 is a substrate for CYP3A. Formation of 1′-hydroxymidazolam, the primary human midazolam metabolite, was markedly inhibited by K02 viacompetitive processes, which suggests the potential for drug-drug interactions of K02 with other CYP3A substrates. K02 significantly inhibited the photoaffinity labeling of P-gp with azidopine and LU-49888, a photoaffinity analogue of verapamil. Transport studies with [14C]K02, using MDR1-transfected Madin-Darby canine kidney cell monolayers in the Transwell system, demonstrated that the basolateral-to-apical flux of K02 acrossMDR1-transfected Madin-Darby canine kidney cells was markedly greater than the apical-to-basolateral flux (ratio of 63 with 10 μM [14C]K02). This suggests that K02 is also a P-gp substrate. These studies are important for formulating strategies to increase the absorption and/or decrease the elimination of K02 and to optimize its delivery to malignant cells and parasite-infected host cells.
Footnotes
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Send reprint requests to: Dr. Leslie Z. Benet, Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, 513 Parnassus Avenue, San Francisco, CA 94143-0446.
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This study was supported in part by National Institutes of Health Grants GM26691 and CA72006. This work was presented in part at the 10th Annual Meeting of the American Association of Pharmaceutical Scientists (Seattle, WA, October 27–31, 1996).
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↵1 McKerrow JH (Department of Pathology, Veterans Affairs Medical Center, San Francisco, CA), personal communication.
- Abbreviations used are::
- CYP
- cytochrome P450
- P-gp
- P-glycoprotein
- MDR
- multidrug resistance
- MDCK
- Madin-Darby canine kidney
- Received May 12, 1997.
- Accepted December 18, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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