ENANTIOSELECTIVE METABOLISM AND CYTOTOXICITY OFR-IFOSFAMIDE ANDS-IFOSFAMIDE BY TUMOR CELL-EXPRESSED CYTOCHROMES P450

Abstract

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 K m value of 200 M, ϳ5-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.Ifosfamide (IFA) and its structural isomer cyclophosphamide (CPA) are clinically effective anticancer alkylating agent prodrugs used to treat a broad range of neoplasms. Both prodrugs are activated in the liver by a cytochrome P450-catalyzed 4-hydroxylation reaction. The primary 4-hydroxy metabolite exists in equilibrium with the ring-opened aldo(iso)phosphamide, which undergoes ␤-elimination to yield the therapeutically active, DNA cross-linking metabolite (iso)phosphoramide mustard and acrolein, a reactive aldehyde (Sladek, 1988;Zalupski and Baker, 1988). An alternative, P450-catalyzed N-dechloroethylation reaction inactivates the parent drug and generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA). Although CPA and IFA are very similar in chemical structure and mechanism of action, they differ strikingly with respect to their pharmacokinetic and pharmacodynamic properties. CPA is metabolized predominantly by 4-hydroxylation, catalyzed by CYP2B and CYP2C enzymes, whereas IFA undergoes extensive N-dechloroethylation, catalyzed by CYP2B and CYP3A enzymes (Huang and Waxman, 1999;Roy et al., 1999b;Chen et al., 2004). The formation of CAA as a major byproduct of IFA N-dechloroethylation is associated with the neurotoxicity and urotoxicity seen in patients treated with IFA b...

MicroRNAs Regulate CYP3A4 Expression via Direct and Indirect Targeting

“…Lastly, we assessed the impact of miRNA pathways on the sensitivity of cells to cyclophosphamide, which may be activated by CYP3A4 to produce cytotoxic alkylating mustards (Chen et al, 2005;Chen et al, 2006). As expected, the induction of CYP3A4 by VD3 led to a significantly higher cyclophosphamide-induced cytotoxicity ( (Takagi et al, 2008), RXR␣/NR2B1 (Ji et al, 2009), and VDR/NR1I1 (this study), and control transcriptional regulation of CYP3A4.…”

A Mouse Model with Liver-Specific Deletion and Global Suppression of the NADPH-Cytochrome P450 Reductase Gene: Characterization and Utility for in Vivo Studies of Cyclophosphamide Disposition

“…Plasma levels of 4-OH-CPA were determined using an HPLC method described previously (Chen et al, 2005). Blood samples for determination of 4-OH-CPA were processed as described previously (Yu et al, 1999).…”

Colorectal Cancer-Specific Cytochrome P450 2W1: Intracellular Localization, Glycosylation, and Catalytic Activity

“…Thus, CYP1B1 has been evaluated as drug target for aryl oxime prodrugs in tumor xenografts, and a number of compounds, including isoflavonoids, stilbenes, pyrrolo-indole, and pyrrolo-quinoline derivatives have been patented and are under investigation as CYP1B1-targeted prodrugs (McFadyen and Murray, 2008). The other example of gene-directed enzyme prodrug therapy approach is the introduction of exogenous CYP2B6 gene into the tumor cells followed by the treatment with phosphamides (Chen et al, 2005). However, using CYP1B1 and CYP2B6 as drug targets is problematic because of their expression in many tissues, which is prone toward unwanted toxic side effects.…”