Inhibition of human CYP2B6 by N,N′,N″-triethylenethiophosphoramide is irreversible and mechanism-based

Abstract

The chemotherapeutic agent N,N′,N″-triethylenethiophosphoramide (thioTEPA) is frequently used in high-dose chemotherapy regimens including cyclophosphamide. Previous studies demonstrated partial inhibition by thioTEPA of the cytochrome P4502B6 (CYP2B6)-catalyzed 4-hydroxylation of cyclophosphamide, which is required for its bioactivation. The aim of our study was to investigate the detailed mechanism of CYP2B6 inhibition by thioTEPA. Using human liver microsomes and recombinant P450 enzymes we confirmed potent inhibition of CYP2B6 enzyme activity determined with bupropion as substrate. ThioTEPA was found to inhibit CYP2B6 activity in a time- and concentration-dependent manner. The loss of CYP2B6 activity was NADPH-dependent and could not be restored by extensive dialysis. The maximal rates of inactivation (K_inact) were 0.16 min⁻¹ in human liver microsomes and 0.17 min⁻¹ in membrane preparations expressing recombinant CYP2B6. The half-maximal inactivator concentrations (K_I) were 3.8 μM in human liver microsomes and 2.2 μM in recombinant CYP2B6. Inhibition was attenuated by the presence of alternative active site ligands but not by nucleophilic trapping agents or reactive oxygen scavengers, further supporting mechanism-based action. Inactivated CYP2B6 did not lose its ability to form a CO-reduced complex suggesting a modification of the apoprotein, which is common for sulfur-containing compounds. Pharmacokinetic consequences of irreversible inactivation are more complicated than those of reversible inactivation, because the drug's own metabolism can be affected and drug interactions will not only depend on dose but also on duration and frequency of application. These findings contribute to better understanding of drug interactions with thioTEPA.

Introduction

N,N′,N″-Triethylenethiophosphoramide (thioTEPA) is a polyfunctional alkylating agent that has been used in the treatment of breast, ovarian and bladder cancer for almost five decades as well as in high-dose chemotherapy regimens with subsequent bone marrow transplantation [1]. ThioTEPA is mainly metabolized to N,N′,N″-triethylenephosphoramide (TEPA), a pharmacologically active compound, by oxidative desulfuration. In rat, 50–80% of the active metabolite TEPA is generated by CYP2B1 and CYP2C11 [2], [3]. It is interesting to note that the human CYP enzymes responsible for metabolism of thioTEPA to TEPA were only recently identified as CYP3A4 and CYP2B6 [4]. TEPA itself and, to a lesser extent, the degradation product N,N′-diethylene N″-2-chloroethylphosphoramide (monochloro-TEPA), contribute to the antineoplastic potential [5]. Alternatively, thioTEPA undergoes extensive phase II metabolism leading to conjugation with glutathione catalyzed by glutathione S-transferases A1-1 and P1-1, and this step is believed to contribute to drug-resistance to alkylating agents [6]. In addition, drug–drug interactions have also been suspected to be involved in drug non-response or increased toxicity of chemotherapy [7]. ThioTEPA is commonly applied in combination with cyclophosphamide (CP) and carboplatin using standard chemotherapy protocols [8]. The CTCb regime includes all three drugs which are administered simultaneously as 96-h continuous infusions [9] whereas a consecutive administration of thioTEPA, CP, and carboplatin over a period of 4 days using short-time infusions was used in the CTC chemotherapeutic protocol [10]. CP is a cancer chemotherapeutic prodrug which unfolds its alkylating activity only after metabolism to 4-hydroxy-CP. Huitema et al. [11] found significantly reduced plasma-levels of 4-hydroxy-CP in patients using a high-dose chemotherapy regime with thioTEPA when thioTEPA was administered prior to CP. Moreover, investigations in human liver microsomes provided evidence for a reduced conversion of CP to 4-hydroxy-CP after co-incubation with thioTEPA [10]. Multiple CYP enzymes can catalyze CP 4-hydroxylation in vitro, including CYP2B6, CYP2C9 and CYP3A4 [12], with CYP2B6 playing the major role [13]. Indeed, thioTEPA has been identified as a specific inhibitor of human CYP2B6 activity characterised by S-mephenytoin-N-demethylation [14], but the mechanism of inhibition has not been investigated in detail. Expression and function of CYP2B6 is highly variable [15] and thus the contribution of CYP2B6 for CP-hydroxylation depends on individual expression levels and genotypes [16]. Several mutations have been identified which affect both expression and catalytic activity [15], [16]. Although there are conflicting results with regard to dose-dependent clearance of thioTEPA [1], several studies using high dose chemotherapy protocols demonstrated an inverse relationship between clearance and dose [17], [18], indicating either a saturable step in elimination or enzyme inhibition. In contrast to reversible enzyme inactivation, the loss of enzyme activity caused by irreversible inactivation persists even after elimination of the inhibitor, and de novo biosynthesis of new enzyme is the only means by which activity can be restored. Clinical and pharmacokinetic consequences of irreversible drug inhibition are thus quite complicated, depending on the duration, dose and frequency of administration [19]. In addition, many drugs which inhibit their own metabolism are mechanism-based inhibitors of CYPs [20]. In this study we investigated the mechanism of inhibition of the human CYP2B6 by thioTEPA using the specific probe drug, bupropion [21], [22]. The results demonstrate that inhibition is mechanism-based, contrary to present knowledge [14]. The findings have implications for the clinical use and for application protocols in chemotherapy.