Enhanced Cyclophosphamide and Ifosfamide Activation in Primary Human Hepatocyte Cultures: Response to Cytochrome P-450 Inducers and Autoinduction by Oxazaphosphorines

Abstract

The anticancer oxazaphosphorine prodrugs cyclophosphamide and ifosfamide are activated in human liver by a 4-hydroxylation reaction catalyzed by multiple cytochrome P450 (CYP) enzymes. In the present study, we used a cultured human hepatocyte model to identify possible inducers of the CYP-catalyzed activation of these two anticancer prodrugs. Treatment of primary cultures of human hepatocytes with phenobarbital, dexamethasone, or rifampin elevated hepatocyte microsomal oxazaphosphorine 4-hydroxylation by up to 200–400% of control for both drug substrates. These inductions were associated with corresponding increases in immunoreactive CYP2B6, CYP2C8, CYP2C9, and CYP3A4, all previously shown to catalyze oxazaphosphorine activation. Rifampin (1 µm, 96-h exposure) was a particularly potent inducer of ifosfamide and cyclophosphamide 4-hydroxylation, as well as of CYP3A protein levels and CYP3A-dependent testosterone 6β-hydroxylation. CYP3A4, CYP2C8, and CYP2C9 protein levels were also increased by exposure of the hepatocytes to cyclophosphamide or ifosfamide (50 µm), which thereby enhanced their own rates of 4-hydroxylation in the cultured hepatocytes. In one human hepatocyte culture that contained the polymorphically expressed CYP3A5 in addition to the more widely expressed CYP3A4, only CYP3A4 was induced by cyclophosphamide, ifosfamide, and rifampin. These studies: (a) demonstrate an underlying metabolic basis for the clinically important oxazaphosphorine autoinduction pharmacokinetics seen with these drugs in cancer patients; and (b) identify rifampin and other CYP inducers as potentially useful for increasing the rates of cyclophosphamide 4-hydroxylation and ifosfamide 4-hydroxylation in human liver in a manner that could favorably impact the clinical pharmacokinetics of these anticancer prodrugs.