Abstract
The principal enzyme involved in the oxidation of mifepristone is cytochrome P450 3A4 (CYP3A4), which undergoes mechanism-based inactivation by the drug. However, no information is available on the interaction with CYP3A5, the second most abundant CYP3A enzyme in adult human liver. Oxidation of mifepristone by recombinant CYP3A4 produced mono- and didemethylated products and one C-hydroxylated metabolite, as reported previously. However, CYP3A5 produced only the demethylated metabolites. The apparent Vmax andKM values for formation of the monodemethylated product by CYP3A4 and CYP3A5 were 46 and 30 nmol/min/nmol P450, and 36 and 16 μM, respectively. Unlike CYP3A4, CYP3A5 was not inactivated by mifepristone. The basis of this differential susceptibility was explored using site-directed mutants in which a CYP3A4 residue was converted to its 3A5 counterpart. Surprisingly, none of these replacements caused a significant decrease in CYP3A4 inactivation by mifepristone. Examination of selected CYP3A4 mutants at 20 other positions indicated that the relative formation rate of the C-hydroxylated product could not account for the differential susceptibility of CYP3A4 and 3A5. Together these results indicate that mifepristone fails to orient itself in the CYP3A5 active site in such a way that its propylenic group is accessible for oxidation, thus rendering CYP3A5 unable to produce the C-hydroxylated product or putative ketene that leads to enzyme inactivation. Identification of mifepristone as a selective mechanism-based inactivation of CYP3A4 may be very useful in distinguishing between the two major CYP3A enzymes collectively responsible for the oxidative metabolism of over half of the drugs currently in use.
Footnotes
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This work was supported by Grants GM54995 (to J.R.H.), DK26506 (to M.A.C.), and Core Center Grant ES06676 from the National Institutes of Health.
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↵2 Nonenzymatic hydrolysis of ketene intermediates is known to produce the corresponding acid derivatives. Our attempts to isolate and/or identify the mifepristone acid derivative by HPLC and LC-MS were unsuccessful. These experiments were performed with wild-type CYP3A4 as well as two mutants, S119A and A370F, which form the maximal and almost negligible amount of C-hydroxylated mifepristone, respectively.
- Abbreviations used are::
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammino]-1-propanesulfonic acid
- DOPC
- dioleoylphosphatidylcholine
- PCR
- polymerase chain reaction
- HPLC
- high-performance chromatography
- LC-MS
- liquid chromatography-mass spectometry
- APCI
- atmospheric pressure chemical ionization
- SRS
- substrate recognition site
- Received April 12, 2002.
- Accepted June 3, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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