Inhibition of Human CYP2B6 by N,N ,N -Triethylenethiophosphoramide Is
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Biochemical Pharmacology 69 (2005) 517–524 www.elsevier.com/locate/biochempharm Inhibition of human CYP2B6 by N,N0,N00-triethylenethiophosphoramide is irreversible and mechanism-based Tanja Richter, Matthias Schwab, Michel Eichelbaum, Ulrich M. Zanger* Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376 Stuttgart, Germany Received 25 June 2004; accepted 11 October 2004 Abstract The chemotherapeutic agent N,N0,N00-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 À1 À1 restored by extensive dialysis. The maximal rates of inactivation (Kinact) were 0.16 min in human liver microsomes and 0.17 min in membrane preparations expressing recombinant CYP2B6. The half-maximal inactivator concentrations (KI) were 3.8 mM in human liver microsomes and 2.2 mM 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. # 2004 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450; CYP2B6; thioTEPA; Mechanism-based inhibition; Suicide-inhibition; Cyclophoshamide 1. Introduction CYP enzymes responsible for metabolism of thioTEPA to TEPA were only recently identified as CYP3A4 N,N0,N00-Triethylenethiophosphoramide (thioTEPA) is a and CYP2B6 [4]. TEPA itself and, to a lesser extent, polyfunctional alkylating agent that has been used in the the degradation product N,N0-diethylene N00-2-chloroethyl- treatment of breast, ovarian and bladder cancer for almost phosphoramide (monochloro-TEPA), contribute to the five decades as well as in high-dose chemotherapy regi- antineoplastic potential [5]. Alternatively, thioTEPA mens with subsequent bone marrow transplantation [1]. undergoes extensive phase II metabolism leading to con- ThioTEPA is mainly metabolized to N,N0,N00-triethylene- jugation with glutathione catalyzed by glutathione S-trans- phosphoramide (TEPA), a pharmacologically active com- ferases A1-1 and P1-1, and this step is believed to pound, by oxidative desulfuration. In rat, 50–80% of the contribute to drug-resistance to alkylating agents [6].In active metabolite TEPA is generated by CYP2B1 and addition, drug–drug interactions have also been suspected CYP2C11 [2,3]. It is interesting to note that the human to be involved in drug non-response or increased toxicity of chemotherapy [7]. ThioTEPA is commonly applied in Abbreviations: CP, cyclophosphamide; CYP, cytochrome P450; ESI, combination with cyclophosphamide (CP) and carboplatin electrospray ionisation; HPLC, high-performance liquid chromatography; using standard chemotherapy protocols [8]. The CTCb OR, NADPH:cytochrome-P450-oxidoreductase; thioTEPA, N,N0,N00- regime includes all three drugs which are administered triethylenethiophosphoramide simultaneously as 96-h continuous infusions [9] whereas a * Corresponding author. Tel.: +49 711 81 01 37 04; fax: +49 711 85 92 95. consecutive administration of thioTEPA, CP, and carbo- E-mail address: [email protected] (U.M. Zanger). platin over a period of 4 days using short-time infusions 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2004.10.008 518 T. Richter et al. / Biochemical Pharmacology 69 (2005) 517–524 was used in the CTC chemotherapeutic protocol [10].CPis by Kao-Atlas. Glucose-6-phosphate (GP) was obtained a cancer chemotherapeutic prodrug which unfolds its from Roche Diagnostics GmbH. Glucose-6-phosphate- alkylating activity only after metabolism to 4-hydroxy- dehydrogenase (GPD) was purchased from Calbiochem. CP. Huitema et al. [11] found significantly reduced plasma- Ketoconazole was obtained from Ultrafine Chemicals. S- levels of 4-hydroxy-CP in patients using a high-dose Mephenytoin was a kind gift from U.A. Meyer (Biozen- chemotherapy regime with thioTEPA when thioTEPA trum, Basel, Switzerland). Propafenone, verapamil and was administered prior to CP. Moreover, investigations metabolites were obtained from Knoll. Bupropion hydro- 2 in human liver microsomes provided evidence for a chloride, hydroxybupropion hydrochloride and [ H3]- reduced conversion of CP to 4-hydroxy-CP after co-incu- hydroxybupropion hydrochloride, 40-hydroxymepheny- 2 0 2 bation with thioTEPA [10]. Multiple CYP enzymes can toin, [ H3]-4 -hydroxymephenytoin and [ H5]-nirvanol catalyze CP 4-hydroxylation in vitro, including CYP2B6, were synthesized as described by Richter et al. [23]. CYP2C9 and CYP3A4 [12], with CYP2B6 playing the major role [13]. Indeed, thioTEPA has been identified as a 2.2. Cytochromes P450 and human liver microsomes specific inhibitor of human CYP2B6 activity characterised by S-mephenytoin-N-demethylation [14], but the mechan- Recombinant CYPs co-expressed with NADPH-P450 ism of inhibition has not been investigated in detail. oxidoreductase (OR) in insect cells (supersomesTM) were Expression and function of CYP2B6 is highly variable purchased from BD Biosciences. Human liver microsomes [15] and thus the contribution of CYP2B6 for CP-hydro- were prepared from surgically removed liver tissue as xylation depends on individual expression levels and gen- described previously [15]. The study was approved by otypes [16]. Several mutations have been identified which the Ethics Committees of the Medical Faculties of the affect both expression and catalytic activity [15,16]. Charite´, Humboldt-University Berlin and of the University Although there are conflicting results with regard to of Tu¨bingen and written informed consent was obtained dose-dependent clearance of thioTEPA [1], several studies from each patient. using high dose chemotherapy protocols demonstrated an inverse relationship between clearance and dose [17,18], 2.3. Determination of catalytic CYP activities indicating either a saturable step in elimination or enzyme inhibition. In contrast to reversible enzyme inactivation, Details for each assay are described below. All assays the loss of enzyme activity caused by irreversible inactiva- were performed with recombinant CYPs (2.5–5 pmol) or tion persists even after elimination of the inhibitor, and de human liver microsomes (50–100 mg protein) in a final novo biosynthesis of new enzyme is the only means by volume of 250 ml with inhibitors as indicated. After equi- which activity can be restored. Clinical and pharmacoki- librating the reaction mixture at 37 8C for 3 min, preincu- netic consequences of irreversible drug inhibition are thus bation with inhibitors was started by adding 25 ml of 10- quite complicated, depending on the duration, dose and fold concentrated NADPH-regenerating system (final con- frequency of administration [19]. In addition, many drugs centrations, 5 mM MgCl2, 4 mM glucose 6-phosphate, which inhibit their own metabolism are mechanism-based 0.5 mM NADP+ and 4 U/ml glucose 6-phosphate dehy- inhibitors of CYPs [20]. In this study we investigated the drogenase) and performed for the indicated times at 37 8C. mechanism of inhibition of the human CYP2B6 by thio- Subsequently, enzyme reactions were started by the addi- TEPA using the specific probe drug, bupropion [21,22]. tion of substrate. Reactions were terminated and processed The results demonstrate that inhibition is mechanism- as described below. based, contrary to present knowledge [14].Thefindings Bupropion-hydroxylation [21,22] was performed with have implications for the clinical use and for application CYP2B6 or human liver microsomes at concentrations protocols in chemotherapy. indicated in 0.1 M sodium phosphate buffer pH 7.4, and with clopidogrel (1 mM) as a CYP2B6 control inhibitor [23]. Enzyme reactions were carried out using 50 mM 2. Materials and methods bupropion with different incubation times for different assays to allow a sensitive detection of inhibition. The 2.1. Chemicals reactions were stopped by adding 50 ml of 1N HCl. After addition of the internal standard d3-OH-bupropion ThioTEPA was obtained from Wyeth Pharma GmbH. (100 pmol), the samples were centrifuged at 16,000 Â g NADP+, NADPH, diethyldithiocarbamate (DDC), sulfa- for 5 min. The supernatant was directly injected into phenazole, superoxide dismutase (SOD), N-acetylcysteine the HPLC system. The metabolite hydroxy-bupropion (N-Ac), DMSO, gluthathione (GSH), 7-ethoxycoumarin, was separated and detected by HPLC-ESI-mass spectro- coumarin, umbelliferone, quinidine and sodium hydrosul-