Metabolism of medroxyprogesterone acetate (MPA) via CYP enzymes in vitro and effect of MPA on bleeding time in female rats in dependence on CYP activity in vivo

Abstract

Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced breast cancer, although it is known to cause thrombosis as a serious side effect. Recently, we found that cytochrome P450 3A4 (CYP3A4) mainly catalyzed the metabolism of MPA via CYP in human liver microsomes. However, the metabolic products of MPA in humans and rats have not been elucidated. In addition, it is not clear whether thrombosis could be induced by MPA itself or by its metabolites. In this study, we determined the overall metabolism of MPA as the disappearance of the parent drug from an incubation mixture, and identified the enzymes catalyzing the metabolism of MPA via CYP in rats. Moreover, the effects of CYP-modulators on MPA-induced hypercoagulation in vivo were examined. Intrinsic clearance of MPA in rat liver microsomes was increased by treatment with CYP3A-inducers. The intrinsic clearance of MPA in liver microsomes of rats treated with various CYP-inducers showed a significant correlation with CYP3A activity, but not CYP1A activity, CYP2B activity or CYP2C contents. Among the eight recombinant rat CYPs studied, CYP3A1, CYP3A2 and CYP2A2 catalyzed the metabolism of MPA. However, since CYP3A2 and CYP2A2 are male-specific isoforms, CYP3A1 appears to be mainly involved in the metabolism of MPA in liver microsomes of female rats. In an in vivo study, pretreatment of female rats with SKF525A, an inhibitor of CYPs including CYP3A1, significantly (p < 0.05) enhanced MPA-induced hypercoagulation, whereas pretreatment with phenobarbital, an inducer of CYPs including CYP3A1, reduced it. These findings suggest that CYP-catalyzed metabolism of MPA is mainly catalyzed by CYP3A1 and that MPA-induced hypercoagulation is predominantly caused by MPA itself in female rats.

Introduction

Medroxyprogesterone acetate (MPA) is one of the drugs most commonly used in endocrine therapy for advanced or recurrent breast cancer and endometrial cancer. However, it is known that MPA can cause serious adverse effects such as thrombosis as well as other side effects such as weight gain, hypertension, nausea and cushingoid effects (Etienne et al., 1992). Although a higher frequency of toxicity has been seen at higher doses (Etienne et al., 1992), it is not clear whether MPA actually contributes to the occurrence of thrombosis, because thrombosis is caused by many factors, including tumors, surgery and genetic mutations of some coagulation factors (Rosendaal, 1999). Recently, it has been reported that MPA shortened bleeding time in rats in a dose-dependent manner (Nobukata et al., 1999). These results suggest that MPA itself and/or its metabolites might stimulate blood coagulation.

MPA exhibits low oral bioavailability (<10%), which may be due to numerous factors, including metabolism in the intestinal mucosa and liver (Stockdale and Rostom, 1989). In fact, MPA has been shown to undergo extensive and rapid metabolism in humans (Stockdale and Rostom, 1989) and in experimental animals (Rautio et al., 1985). Recently, we reported that MPA was metabolized by cytochrome P450 3A4 (CYP3A4) in human liver microsomes (Kobayashi et al., 2000). This finding agrees with the observation reported by Ohtsu et al. (1998) that the plasma concentration of MPA was lower than that of MPA alone when dexamethasone (DEX), a CYP3A inducer Pichard et al., 1990, Morris and Davila, 1996 or phenobarbital (PB), an inducer of CYP2B, CYP2C and CYP3A (Waxman and Azaroff, 1992) was coadministered with MPA in patients with breast cancer. Similarly, PB administered with MPA to rats greater decrease in the plasma concentration of MPA, while SKF525A (a CYP inhibitor) administered with MPA to rats caused a smaller decrease in the plasma concentration of MPA compared to that in the case of administration of MPA alone (Saarni et al., 1983). These results suggest that plasma decay of MPA depends mainly on the metabolism of MPA by CYP, although it is not clear which CYP isoform(s) is responsible for the metabolism of MPA in rats.

Since the metabolic products of MPA in rats have not been elucidated, we determined the metabolism of MPA as the disappearance of the parent drug from an incubation mixture, and identified the rat CYP isoforms involved in the CYP-catalyzed metabolism of MPA by using liver microsomes of DEX-, PB- or β-naphthoflavone (BNF, a CYP1A inducer; Daujat et al., 1992, Morris and Davila, 1996)-treated female rats, and microsomes from baculovirus-infected insect cells expressing individual rat CYP isoforms in the present study. Since CYP1A, CYP2B, CYP2C and CYP3A are major CYP isoforms in female rat liver, their probe activities or contents in liver microsomes were determined. Moreover, we examined the effects of SKF525A and PB on change in bleeding time by a single po administration of MPA in female rats to elucidate whether MPA itself or its metabolites stimulates blood coagulation.