In Vitro and in Silico Identification and Characterization of Thiabendazole as a Mechanism-Based Inhibitor of CYP1A2 and Simulation of Possible Pharmacokinetic Drug-Drug Interactions Roslyn S. Thelingwani, Simbarashe P. Zvada, Hugues Dolgos, Anna-Lena B. Ungell, and Collen M. Masimirembwa Department of Drug Metabolism and Pharmacokinetics/PD and Toxicology, African Institute of Biomedical Science and Technology, Harare, Zimbabwe (R.S.T., S.P.Z., C.M.M.); African Institute of Biomedical Science and Technology-University of Capetown C-Lab, Department of Chemistry, University of Capetown, South Africa (R.S.T.); and Department of Discovery DMPK and Bioanalytical Chemistry, AstraZeneca, Mo ¨ lndal, Sweden (H.D., A.-L.B.U.) Received September 15, 2008; accepted March 18, 2009 ABSTRACT: Thiabendazole (TBZ) and its major metabolite 5-hydroxythiabenda- zole (5OH-TBZ) were screened for potential time-dependent inhi- bition (TDI) against CYP1A2. Screen assays were carried out in the absence and presence of NADPH. TDI was observed with both compounds, with k inact and K I values of 0.08 and 0.02 min 1 and 1.4 and 63.3 M for TBZ and 5OH-TBZ, respectively. Enzyme inactiva- tion was time-, concentration-, and NADPH-dependent. Inactiva- tion by TBZ was irreversible by dialysis and oxidation by potassium ferricyanide, and there was no protection by glutathione. 5OH-TBZ was a weak TDI of CYP1A2, and enzyme activity was recovered by dialysis. IC 50 determination of TBZ and 5OH-TBZ showed both compounds to be potent inhibitors, with IC 50 values of 0.83 and 13.05 M, respectively. IC 50 shift studies also demonstrated that TBZ was a TDI of CYP1A2. In silico methods identified the thiazole group as a TDI fragment and predicted it as the site of metabolism. The observation pointed to epoxidation of the thiazole and the benzyl rings of TBZ as possible routes of metabolism and mech- anisms of TDI. Drug-drug interaction (DDI) simulation studies using SimCyp showed good predictions for competitive inhibition. How- ever, predictions for mechanism-based inhibition (MBI)-based DDI were not in agreement with clinical observations. There was no TBZ accumulation upon chronic administration of the drug. The in vitro MBI findings might therefore not be capturing the in vivo situation in which the proposed bioactivation route is minor. This might be the case for TBZ in which, in vivo, UDP glucuronosyl- transferases and sulfanotransferase metabolize and eliminate the 5OH-TBZ. Thiabendazole (TBZ) is a broad-spectrum antihelminthic for many animal species and is used to treat parasitic infections in humans (Brown et al., 1961; Hennekeuser et al., 1969; Walton et al., 1999). It has also been used as an agricultural fungicide for pre- and posthar- vest treatment of fruit and vegetables and as a preservative in many consumer food products (Szeto et al., 1993; Arenas and Johnson, 1994; Walton et al., 1999; Groten et al., 2000). Studies have shown that thiabendazole is extensively metabolized in humans and in ani- mals (Tocco et al., 1966). Several routes of biotransformation of thiabendazole have been proposed (Fig. 1). The major route is the CYP1A2-catalyzed hydroxylation to 5-hydroxythiabendazole, which is further metabolized to glucuronide and sulfate conjugates (Coulet et al., 1998). Other metabolites identified include 4-hydroxythiabenda- zole, 2-acetylbenzimidazole, N-methylthiabendazole, and benzimid- azole (Fujitani et al., 1991). TBZ has also been shown to induce members of the CYP1A and CYP2B family in rats in vivo (Price et al., 2004) and rabbit CYP1A2 in vitro (Aix et al., 1994), which implies the possibility of the compound inducing its own metabolism. However, TBZ has not been shown to induce CYP1A2 in humans in vitro (Bapiro et al., 2002). No studies have been done on its potential to induce CYP1A2 in vivo in humans. Although TBZ is considered a safe drug in humans, studies in mice have shown some toxicity. It has been associated with nephrotoxicity, resulting in severe kidney damage (Mizutani et al., 1990; Tada et al., 1992; Fujitani et al., 1999); and teratogenicity, resulting in impairment of limb development and toxicity to the embryo (Ogata et al., 1984). Isolated cases of hepatotoxicity have also been reported in humans after TBZ administration (Manivel et al., 1987; Bion et al., 1995). Toxicity to the embryo has been associated with covalent binding of This work was supported in part by the African Institute of Biomedical Science and Technology (Zimbabwe) [Grant DMPK-004]; International Programme in the Chemical Sciences (Sweden); European Union (AntiMal Project); and AstraZen- eca (Sweden). R.T. is a recipient of the European Union AntiMal project Ph.D. scholarship for a joint project between African Institute of Biomedical Science and Technology and the University of Capetown. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.024604. ABBREVIATIONS: TBZ, thiabendazole; P450, cytochrome P450; CHC, 3-cyano-7-hydroxycoumarin; TDI, time-dependent inhibition/inhibitor; MBI, mechanism-based inhibition/inhibitor; CEC, 3-cyano-7-ethoxycoumarin; fm, fraction metabolized; 5OH-TBZ, 5-hydroxythiabendazole; AUC, area under the curve; ACN, acetonitrile. 0090-9556/09/3706-1286–1294$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 6 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 24604/3475881 DMD 37:1286–1294, 2009 Printed in U.S.A. 1286 at ASPET Journals on January 21, 2016 dmd.aspetjournals.org Downloaded from