OXIDATION OF CYCLOPHOSPHAMIDE TO 4-HYDROXYCYCLOPHOSPHAMIDE AND DESCHLOROETHYLCYCLOPHOSPHAMIDE IN HUMAN LIVER-MICROSOMES

We have investigated the formation of 4-hydroxycyclophosphamide (HCY) and deschloroethylcyclophosphamide (DCCY) from cyclophosphamide (CY) i n human liver microsomes, For HCY, the estimated values (mean +/- SD; n = 3) of K-m1 and K-m2 were 0.095 +/- 0.072 and 5.09 +/- 4.30 mM, and the estimated values of V-max1 and V-max2 were 0.138 +/- 0.070 and 1. 55 +/- 0.50 nmol/min/mg protein, For DCCY, K-m1 and K-m2 were 0.046 +/ - 0.017 and 8.58 +/- 5.84 mM, and V-max1 and V-max2 were 0.006 +/- 0.0 03 and 0.274 +/- 0.214 nmol/min/mg protein, At CY concentrations of 0. 1, 0.7, and 5 mM, HCY respectively accounted for 95.7 +/- 1.3, 95.1 +/ - 2.4, and 90.7 +/- 2.7% of the total products of CY (HCY + DCCY; n = 6), In a separate experiment, 98.7 +/- 11.9% (n = 3) of CY loss could be accounted for by the formation of HCY at 0.1 mM CY, On the basis of cytochrome P450 (CYP) isoform-specific chemical inhibitor and cDNA-ex pressed human P450 isozyme studies, CYP2C9 and CYP3A4/5 seemed to be t he major P450 isoforms responsible for HCY formation at low (0.1 mM) a nd high (0.7 and 5 mM) concentrations of CY, respectively, Although or phenadrine inhibition was observed in human liver microsomes (which ha s been taken to indicate CYP2B6 catalysis), orphenadrine inhibited cDN A-expressed CYP3A4 formation of HCY to the same extent observed in hum an liver microsomes, and the addition of orphenadrine to incubations c ontaining sulfaphenazole (a specific inhibitor of CYP2C9) or troleando mycin (a specific CYP3A inhibitor) did not increase inhibition beyond that observed with sulfaphenazole or troleandomycin alone, Similar stu dies indicated that CYP3A4/5 was the major P450 isoform responsible fo r DCCY formation at high (0.7 and 5 mM) concentrations of CY, The P450 isoform responsible for DCCY formation at 0.1 mM CY could not be iden tified due to its very low formation rate.