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An in Vitro Approach to Detect Metabolite Toxicity Due to CYP3A4

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An in Vitro Approach to Detect Metabolite Toxicity Due to CYP3A4 Toxicology 216 (2005) 154–167 An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics Luisella Vignati ∗, Elisa Turlizzi 1, Sonia Monaci, Pietro Grossi, Ruben de Kanter, Mario Monshouwer 2 Department of Pre-Clinical Development, Nerviano Medical Sciences S.r.l., V.le Pasteur, 10, 20014, Nerviano, MI, Italy Received 22 June 2005; received in revised form 3 August 2005; accepted 3 August 2005 Available online 19 September 2005 Abstract Many adverse drug reactions are caused by the cytochrome P450 (CYP) dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-mediated toxicity and to improve safety of drug candidates, we developed two in vitro cell-based assays by combining an activating system (human CYP3A4) with target cells (HepG2 cells): in the first method we incubated microsomes containing cDNA-expressed CYP3A4 together with HepG2 cells; in the second approach HepG2 cells were transiently transfected with CYP3A4. In both assay systems, CYP3A4 catalyzed metabolism was found to be comparable to the high levels reported in hepatocytes. Both assay systems were used to study ten CYP3A4 substrates known for their potential to form metabolites that exhibit higher toxicity than the parent compounds. Several endpoints of toxicity were evaluated, and the measurement of MTT reduction and intracellular ATP levels were selected to assess cell viability. Results demonstrated that both assay systems are capable to metabolize the test compounds leading to increased toxicity, compared to their respective control systems. The co-incubation with the CYP3A4 inhibitor ketoconazole confirmed that the formation of reactive metabolites was CYP3A4 dependent. To further validate the functionality of the two assay systems, they were also used as a “detoxification system” using selected compounds that can be metabolized by CYP3A4 to metabolites less toxic than their parent compounds. These results show that both assay systems can be used to screen for metabolic activation, or de-activation, which may be useful as a rapid and relatively inexpensive in vitro assay for the prediction of CYP3A4 metabolism-mediated toxicity. © 2005 Published by Elsevier Ireland Ltd. Keywords: Metabolism-mediated toxicity; Adverse drug reactions (ADRs); Reactive metabolites; In vitro screening; CYP3A4; Cytotoxicity 1. Introduction Abbreviations: BSO, l-buthionine S,R-sulphoximine; CYP, Adverse reactions associated with exposure of indi- cytochrome P450; DMEM, Dulbecco’s Modified Eagle’s Medium; viduals to drugs (ADRs) or xenobiotics are a com- DMSO, dimethyl-sulphoxide; GSH: reduced glutathione; MTT, 3- (4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, mon and a significant cause of morbidity and mortality phosphate buffered saline (Lazarou et al., 1998; Meyer, 2000). From a clinical ∗ Corresponding author. Tel.: +39 0331 581335; perspective, ADRs may be classified as augmented reac- fax: +39 0331 581105. tions (type A), which are predictable from the known E-mail address: [email protected] (L. Vignati). pharmacology and often represent an exaggeration of 1 Present address: Drug profiling, Sienabiotech S.p.A., via Fiorentina, 1, 53100 Siena, Italy. the pharmacological effects of the drug that may be 2 Present address: Pharmacokinetics and Drug Metabolism, Amgen reversed by dose reduction, or as idiosyncratic reaction Inc., 1120 Veterans Blvd, South San Francisco, CA 94080, USA. (type B), which are unpredictable from the knowledge 0300-483X/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.tox.2005.08.003 L. Vignati et al. / Toxicology 216 (2005) 154–167 155 of the basic pharmacology of the drug and show marked Over the past years, several approaches have been individual susceptibility and no simple dose dependency. used to detect metabolism-mediated toxicity in vitro. From a chemical point of view, this classification can be Because covalent binding of drugs to proteins has been expanded to reactions (type C), which are predictable associated with drug toxicity (Zhou et al., 2005), it from the chemistry of the drug or xenobiotics, and to is rather common practice within large pharmaceuti- reactions (type D), which are delayed reactions that occur cal companies to determine the extent of irreversible many years after treatment (Park et al., 1998). Pharmaco- binding to protein using radiolabeled drug and human genetic, toxicogenetic and other host-dependent factors liver microsomes (Day et al., 2005; Kitteringham et al., have been identified to be important in predisposition 1988). to type A reactions (Meyer and Gut, 2002). In contrast, Human hepatocytes represent another in vitro sys- most type B, type C and type D ADRs are mediated by tem for the evaluation of metabolism mediated toxicity toxic metabolites (Gut et al., 1995; Park et al., 1998, (Gomez-Lechon et al., 2003; Li et al., 1999), but the poor 2004). availability of human liver, the high cost and the sig- There are many examples of metabolites or reactive nificant variability among human hepatocytes prepara- intermediates of essentially non-toxic drugs or chemicals tions make this tool not applicable to an high-throughput that have been shown to exert adverse drug reactions. screening in drug discovery. More recently, cell lines that Some of these ADRs are so serious that drug treatment have been genetically modified to express a single or had to be limited and in some cases the drug had to multiple drug-metabolising enzyme(s) have been devel- be withdrawn from the market with enormous conse- oped (Bull et al., 2001; Dai and Cederbaum, 1995; Lin et quences for both patients and pharmaceutical industries al., 1999; Nakagawa et al., 1996; Philip et al., 1999; Wu (e.g. troglitazone, zomepirac and benoxaprofen) (Nassar and Cederbaum, 1996). However, most of these stably and Lopez-Anaya, 2004; Wolfgang and Johnson, 2002). transfected cell lines have very low metabolic activity, Because of this, pharmaceutical companies are making compared to hepatocytes or liver microsomes. Finally, significant efforts to predict ADRs and, currently, several the immortalized human hepatocyte cell line Fa2N-4 has tools and strategies are considered to address the for- been recently described as an in vitro tool to study drug mation of reactive metabolites and the potential conse- metabolism issues (Mills et al., 2004), but like for sta- quence for the overall safety profile on candidate drugs. bly transfected cell lines, also the use of Fa2N-4 cells to Reactive metabolites are a common product of phase address reactive metabolites is limited due to low enzy- I oxidation reactions mediated by cytochrome P450 matic activities. (CYP)-dependent mixed function oxygenases, although In the present study, we describe the development of also examples of other phase I (e.g. flavin-mono oxyge- two in vitro models to evaluate the activation of xenobi- nases; FMOs) and phase II drug metabolizing reactions otics to toxic metabolites. In the first method, we used have been described (Zhou et al., 2005). The genera- human CYP3A4 cDNA expressed microsomes (as acti- tion of such reactive metabolites may produce adverse vating system), in combination with HepG2 cells (as reactions via different inter-related process such as for- target system). mation of free radicals, oxidation of thiols and covalent In the second model, a HepG2 cell line transiently binding with endogenous macromolecules, resulting in transfected with CYP3A4 was developed. Transient the oxidation of cellular components or inhibition of nor- transfection was preferred above stable transfection, mal cellular functions (Riley et al., 1988). Sometimes, because of the higher metabolic activity observed in and for unknown reasons, covalently modified proteins these cells. may be immunogenic and elicit an immune response HepG2 cells were selected as target cells for eval- (Knowles et al., 2000; Park et al., 2000; Uetrecht, 1999). uating toxicity because they are derived from human In contrast with adverse reactions that are usually dose liver and have been extensively used as the test system dependent and due to the pharmacology of the drug (e.g. for the prediction of toxicity, carcinogenicity and cell type A), ADRs caused by toxic metabolites are diffi- mutagenicity in humans. Moreover, HepG2 cells con- cult to predict accurately because metabolic activation of tain the co-enzymes NADPH-cytochrome P450 reduc- the drug is required to observe the toxic effect. In addi- tase and cytochrome b5, required for CYP mediated drug tion, significant species differences in drug metabolism, metabolism (Rodriguez-Antona et al., 2002; Yoshitomi chemical instability of the reactive metabolites, and the et al., 2001). presence of detoxification pathways are just some of Because of the general importance of CYP3A4 in the several factors, which make the screening for toxic drug metabolism (50% of the drugs in use today are metabolites a real challenge. metabolised by CYP3A4) (Guengerich, 2001; Nelson, 156 L. Vignati et al. / Toxicology 216 (2005) 154–167 Table 1 Test compounds and their metabolites Compound tested (Main) toxic metabolite ADR Therapeutic use Reference Albendazole Sulfoxide Hepatotoxicity Anthielminthic Rawden et al. (2000) Carbamazepine o-Quinone, iminoquinone Skin rash, hepatic disorders Anticonvulsant Pirmohamed et al. (1992) Dapsone Nitroso-dapsone Hemolysis Antileprosy Coleman (1995) Flutamide Nitroradicals Hepatotoxicity Anticancer Berson et al. (1993)
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