DIFFERENTIAL ACTIVATION OF CYCLOPHOSPHAMIDE AND IFOSPHAMIDE BY CYTOCHROME-P-450-2B AND CYTOCHROME-P450-3A IN HUMAN LIVER-MICROSOMES

The present study identifies the specific human cytochrome P-450 (CYP) enzymes involved in hydroxylation leading to activation of the antica ncer drug cyclophosphamide and its isomeric analogue, ifosphamide. Sub stantial interindividual variation (4-9-fold) was observed in the hydr oxylation of these oxazaphosphorines by a panel of 12 human liver micr osomes, and a significant correlation was obtained between these 2 act ivities (r = 0.85, P < 0.001). Enzyme kinetic analyses revealed that h uman liver microsomal cyclophosphamide 4-hydroxylation and ifosphamide 4-hydroxylation are best described by a 2-component Michaelis-Menten model composed of both low K(m) and high K(m) P-450 4-hydroxylases. To ascertain whether one or more human P-450 enzymes are catalytically c ompetent in activating these oxazaphosphorines, microsomal fractions p repared from a panel of human B-lymphoblastoid cell lines stably trans formed with individual P-450 complementary DNAs were assayed in vitro for oxazaphosphorine activation. Expressed CYP2A6, -2B6, -2C8, -2C9, a nd -3A4 were catalytically competent in hydroxylating cyclophosphamide and ifosphamide. Whereas CYP2C8 and CYP2C9 have the characteristics o f low K(m) oxazaphosphorine 4-hydroxylases, CYP2A6, -2B6, and -3A4 are high K(m) forms. In contrast, CYP1A1, -1A2, -2D6, and -2E1 did not pr oduce detectable activities. Furthermore, growth of cultured CYP2A6- a nd CYP2B6-expressing B-lymphoblastoid cells, but not of CYP-negative c ontrol cells, was inhibited by cyclophosphamide and ifosphamide as a c onsequence of prodrug activation to cytotoxic metabolites. Experiments with P-450 form-selective chemical inhibitors and inhibitory anti-P-4 50 antibodies were then performed to determine the contributions of in dividual P-450s to the activation of these drugs in human liver micros omes. Orphenadrine (a CYP2B6 inhibitor) and anti-CYP2B IgG inhibited m icrosomal cyclophosphamide hydroxylation to a greater extent than ifos phamide hydroxylation, consistent with the 8-fold higher activity of c omplementary DNA-expressed CYP2B6 with cyclophosphamide. In contrast, troleandomycin, a selective inhibitor of CYP3A3 and -3A4, and anti-CYP 3A IgG substantially inhibited microsomal ifosphamide hydroxylation bu t had little or no effect on microsomal cyclophosphamide hydroxylation . By contrast, the CYP2D6-selective inhibitor quinidine did not affect either microsomal activity, while anti-CYP2A antibodies had only a mo dest inhibitory effect. Overall, the present study establishes that li ver microsomal CYP2B and CYP3A preferentially catalyze cyclophosphamid e and ifosphamide 4-hydroxylation, respectively, suggesting that liver P-450-inducing agents targeted at these enzymes might be used in canc er patients to enhance drug activation and therapeutic efficacy.