Inhibition of human CYP2B6 by N,N′,N″-triethylenethiophosphoramide is irreversible and mechanism-based
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.
Section snippets
Chemicals
ThioTEPA was obtained from Wyeth Pharma GmbH. NADP+, NADPH, diethyldithiocarbamate (DDC), sulfaphenazole, superoxide dismutase (SOD), N-acetylcysteine (N-Ac), DMSO, gluthathione (GSH), 7-ethoxycoumarin, coumarin, umbelliferone, quinidine and sodium hydrosulfite were purchased from Sigma. Furafylline was a kind gift from U. Fuhr (University of Cologne, Germany). Glycerine was purchased from Roth and Emulgen 911 was provided by Kao-Atlas. Glucose-6-phosphate (GP) was obtained from Roche
Effect of thioTEPA on P450 monooxygenase activities
To determine the selectivity of inhibition by thioTEPA towards catalytic activities representing the most relevant drug metabolizing CYPs, recombinant enzymes coexpressed with OR were analysed with appropriate assays using CYP isoform-specific inhibitors as positive controls (Table 1). Bupropion-hydroxylation, as a specific marker reaction for CYP2B6 [21], [22] was inhibited by almost 90% at 10 μM concentration. Incubations of human liver microsomes with 10 μM thioTEPA and NADPH-regenerating
Discussion
The cytostatic agent, thioTEPA, has previously been shown to be a specific inhibitor of CYP2B6 [14]. In their work, the authors concluded that the inhibition type was non-competitive, but they did not assess the actual mechanism of inhibition. The present in vitro study provides additional data on the cytochrome P450 interaction specificity of thioTEPA and demonstrates that it acts as a potent mechanism-based inhibitor of human CYP2B6. These conclusions are based on experiments with human liver
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