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N,N',N''-Triethylenethiophosphoramide (Thio-TEPA) Oxygenation by Constitutive Hepatic P450 Enzymes and Modulation of Drug Metabolism and Clearance in Vivo by P450-inducing Agents¹

Department of Biological Chemistry and Molecular Pharmacology and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

The cancer chemotherapeutic drug N,N',N''-triethylenethiophosphoramide (thio-TEPA) is oxidatively desulfurated to yield the active metabolite N,N',N''-triethylenephosphoramide (TEPA) in a reaction catalyzed by the phenobarbital-inducible rat liver P450 enzyme IIB1. In the current study, the role of constitutively expressed P450 enzymes in thio-TEPA metabolism was studied using purified P450s, isolated liver microsomes, and intact rats. Metabolism of thio-TEPA (100 µM) to TEPA by uninduced adult female and male rat liver microsomes proceeded at initial rates of 0.10 and 0.28 nmol TEPA formed/min/mg microsomal protein, respectively. Although these rates are low compared to those catalyzed by phenobarbital-induced liver microsomes (3.5 nmol TEPA/min/mg), they are sufficient to contribute to the systemic metabolism of this drug. Thio-TEPA metabolism catalyzed by uninduced female liver microsomes was 70% inhibitable by antibodies selectively reactive with P450 IIC6. For the uninduced male liver microsomes, which exhibit a severalfold higher rate of thio-TEPA metabolism, enzyme activity was only 15–20% inhibitable by these antibodies but was 80–85% inhibited by an anti-P450 IIC6 monoclonal antibody cross-reactive with P450 IIC11, which is expressed only in the males. Consistent with these observations, purified P450s IIC11 and IIC6 both oxidized thio-TEPA in reconstituted systems (turnover, 1.1 and 0.3 min^-1 P450^-1, respectively, at 100 µM substrate), while several other constitutive hepatic P450s exhibited significantly lower or undetectable activities (turnover, 0.15 min^-1 P450^-1). Metabolism of thio-TEPA by purified P450 IIC11 was associated with a time-dependent inactivation of the cytochrome analogous to that previously shown to accompany thio-TEPA metabolism catalyzed by P450 IIB1. Depletion of hepatic P450 IIC11 by cisplatin treatment of adult male rats led to a 70% reduction of TEPA formation catalyzed by the isolated liver microsomes, suggesting that cisplatin may influence thio-TEPA pharmacokinetics when these two drugs are given in combination. The extent to which hepatic P450s contribute to thio-TEPA metabolism and clearance in vivo was assessed by monitoring thio-TEPA and TEPA pharmacokinetics in rats that exhibit widely differing rates of microsomal thio-TEPA metabolism, i.e., uninduced female and male rats, and male rats treated with the P450 IIB1 inducers clofibrate and phenobarbital. In accord with the microsomal activities, conversion of thio-TEPA to TEPA was less extensive and thio-TEPA elimination slower in female than in male rats. Clofibrate and phenobarbital both accelerated thio-TEPA clearance by increasing metabolism to TEPA; this was evidenced by a 4-fold increase in the peak plasma level of the oxo-metabolite in the inducer-pretreated animals. These findings demonstrate that liver P450 enzymes have a major impact on thio-TEPA metabolism and clearance and further suggest ways in which thio-TEPA pharmacokinetics might be modulated in vivo to achieve improved therapeutic effects.

¹ Supported in part by Grant CN-14 from the American Cancer Society [D. J. W.].

² To whom correspondence should be addressed, at Dana-Farber Cancer Institute, JF-525, 44 Binney Street, Boston, MA 02115.

Received 11/26/90. Accepted 2/22/91.