Oxazaphosphorine Metabolism by the Human Cytochrome P450s and Some Commonly Expressed P450 Polymorphic Variants.

Abstract

The oxazaphosphorines, cyclophosphamide (CPA) and ifosfamide (IFO), are commonly used cancer therapeutics. Hallmarks of oxazaphosphorine-treatment include variable patient response and severe adverse drug reactions. These deleterious effects are related to the metabolism of the oxazaphosphorines. Improved understanding of CPA and IFO metabolism is important for the optimization of treatment regimens utilizing these drugs. This dissertation has focused on advancing our knowledge of the two primary metabolic pathways for oxazaphosphorine metabolism, hydroxylation and N-dechloroethylation, by the human cytochrome P450s (P450s) (CYP2B6, CYP2C9, CYP2C19 and CYP3A4). Hydroxylation of the oxazaphosphorines activates the drugs, whereas N-dechloroethylation results in inactivation and the formation of toxic metabolites. We demonstrated that CYP2B6 is 38-fold more efficient than the other P450s for the activation of CPA and that only CYP3A4 catalyzed the inactivation of CPA. For IFO, CYP3A4 was 2.8-fold more efficient at activation of IFO than the other P450s and multiple P450s catalyzed the inactivation of IFO. As the P450s are highly polymorphic enzymes, and polymorphic variants may have altered catalytic activities, we assessed commonly expressed variants for alterations in the rate of oxazaphosphorine metabolism. We observed statistically significant differences in the activation of both of the drugs by some of the polymorphic variants when compared to the wild type enzymes. We also evaluated the ability to favor the activation pathway of oxazaphosphorine metabolism in two ways; specific P450 inhibition in human liver microsomes (HLM) and deuterium labeling of the oxazaphosphorines. Inhibition of P450s in HLM was effective for favoring the activation of CPA, but not IFO. Deuterium labeling of CPA and IFO, resulted in inhibition of inactivation for both drugs but it was more evident with IFO, likely because IFO undergoes inactivation more readily than CPA. Thus, different methods are better for increasing the ratio of activation to inactivation for of each the drugs. Taken together, the work compiled in this thesis has enhanced our mechanistic understanding of the metabolism of the oxazaphosphorines. Our hope is that this information will encourage future investigations focused on the improvement of current cancer chemotherapies and the use of pharmacogenomics to design enhanced chemotherapeutic regimens.