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) in human liver microsomes. For HCY, the estimated values (mean +/- SD; n = 3) of Km1 and Km2 were 0.095 +/- 0.072 and 5.09 +/- 4.30 mM, and the estimated values of Vmax1 and Vmax2 were 0.138 +/- 0.070 and 1.55 +/- 0.50 nmol/min/mg protein. For DCCY, Km1 and Km2 were 0.046 +/- 0.017 and 8.58 +/- 5.84 mM, and Vmax1 and Vmax2 were 0.006 +/- 0.003 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-expressed human P450 isozyme studies, CYP2C9 and CYP3A4/5 seemed to be the major P450 isoforms responsible for HCY formation at low (0.1 mM) and high (0.7 and 5 mM) concentrations of CY, respectively. Although orphenadrine inhibition was observed in human liver microsomes (which has been taken to indicate CYP2B6 catalysis), orphenadrine inhibited cDNA-expressed CYP3A4 formation of HCY to the same extent observed in human liver microsomes, and the addition of orphenadrine to incubations containing sulfaphenazole (a specific inhibitor of CYP2C9) or troleandomycin (a specific CYP3A inhibitor) did not increase inhibition beyond that observed with sulfaphenazole or troleandomycin alone. Similar studies indicated that CYP3A4/5 was the major P450 isoform responsible for 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 identified due to its very low formation rate.
Ren
S
S
Department of Pharmaceutics, University of Washington, Seattle 98195, USA.
Yang
J S
JS
Kalhorn
T F
TF
Slattery
J T
JT
D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000493:pharmacokinetics; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009966:Orphenadrine / Q000494:pharmacology; D010084:Oxidation-Reduction; D011355:Prodrugs / Q000378:metabolism* / Q000493:pharmacokinetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014217:Troleandomycin / Q000494:pharmacology
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; C012358:4-hydroxycyclophosphamide; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; D009966:Orphenadrine; D014217:Troleandomycin; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; C510163:CYP3A4 protein, human