Abstract
The anticancer prodrug ifosfamide (IFA) contains a chiral phosphorous atom and is administered in the clinic as a racemic mixture of R-IFA and S-IFA. Hepatic cytochrome P450 (P450) enzymes exhibit enantioselective preferences in the metabolism of R-IFA and S-IFA; however, the impact of this selectivity on P450-dependent anticancer activity is not known. Presently, the metabolism and cytotoxicity of R-IFA and S-IFA were determined in 9L gliosarcoma and Chinese hamster ovary tumor cells expressing an IFA-activating P450 enzyme and by in vitro steady-state kinetic analysis using cDNA-expressed P450 enzymes. Tumor cells expressing P450 enzyme CYP3A4 were the most sensitive to R-IFA cytotoxicity, whereas tumor cells expressing CYP2B1 or CYP2B6 were most sensitive to cyclophosphamide (CPA), an isomer of IFA. Correspondingly, CYP3A4-expressing cells and cDNA-expressed CYP3A4 metabolized R-IFA to yield the active, 4-hydroxylated metabolite at a 2- to 3-fold higher rate than they metabolized S-IFA or CPA. CYP2B cells and cDNA-expressed CYP2B enzymes metabolized CPA almost exclusively by 4-hydroxylation, whereas R-IFA and S-IFA were substantially converted to inactive, N-dechloroethylated metabolites. Further investigation revealed that CYP3A1, a rat enzyme, exhibited superior kinetic properties compared with the human enzyme CYP3A4, with R-IFA and S-IFA both metabolized with high catalytic efficiency by 4-hydroxylation and with a Km value of 200 μM, ∼5-fold lower than CYP3A4. Based on these kinetic parameters and metabolic profiles, R-IFA is expected to exert greater anticancer activity than S-IFA or CPA against tumors that express CYP3A enzymes, whereas tumors expressing CYP2B enzymes may be more sensitive to CPA treatment.
Footnotes
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Supported in part by National Institutes of Health Grant CA49248 (to D.J.W.).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.004788.
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ABBREVIATIONS: IFA, ifosfamide; CPA, cyclophosphamide; P450, cytochrome P450; CAA, chloroacetaldehyde; deCl, N-dechloroethylation; P450R, P450 reductase; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; IRES, internal ribosome entry site; CHO, Chinese hamster ovary; MEM, minimal essential medium; HPLC, high-performance liquid chromatography.
- Received March 22, 2005.
- Accepted May 20, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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