An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics

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.

Introduction

Adverse reactions associated with exposure of individuals to drugs (ADRs) or xenobiotics are a common and a significant cause of morbidity and mortality (Lazarou et al., 1998, Meyer, 2000). From a clinical perspective, ADRs may be classified as augmented reactions (type A), which are predictable from the known pharmacology and often represent an exaggeration of the pharmacological effects of the drug that may be reversed by dose reduction, or as idiosyncratic reaction (type B), which are unpredictable from the knowledge of the basic pharmacology of the drug and show marked individual susceptibility and no simple dose dependency. From a chemical point of view, this classification can be expanded to reactions (type C), which are predictable from the chemistry of the drug or xenobiotics, and to reactions (type D), which are delayed reactions that occur many years after treatment (Park et al., 1998). Pharmacogenetic, toxicogenetic and other host-dependent factors have been identified to be important in predisposition to type A reactions (Meyer and Gut, 2002). In contrast, most type B, type C and type D ADRs are mediated by toxic metabolites (Gut et al., 1995, Park et al., 1998, Park et al., 2004).

There are many examples of metabolites or reactive intermediates of essentially non-toxic drugs or chemicals that have been shown to exert adverse drug reactions. Some of these ADRs are so serious that drug treatment had to be limited and in some cases the drug had to be withdrawn from the market with enormous consequences for both patients and pharmaceutical industries (e.g. troglitazone, zomepirac and benoxaprofen) (Nassar and Lopez-Anaya, 2004, Wolfgang and Johnson, 2002). Because of this, pharmaceutical companies are making significant efforts to predict ADRs and, currently, several tools and strategies are considered to address the formation of reactive metabolites and the potential consequence for the overall safety profile on candidate drugs.

Reactive metabolites are a common product of phase I oxidation reactions mediated by cytochrome P450 (CYP)-dependent mixed function oxygenases, although also examples of other phase I (e.g. flavin-mono oxygenases; FMOs) and phase II drug metabolizing reactions have been described (Zhou et al., 2005). The generation of such reactive metabolites may produce adverse reactions via different inter-related process such as formation of free radicals, oxidation of thiols and covalent binding with endogenous macromolecules, resulting in the oxidation of cellular components or inhibition of normal cellular functions (Riley et al., 1988). Sometimes, and for unknown reasons, covalently modified proteins may be immunogenic and elicit an immune response (Knowles et al., 2000, Park et al., 2000, Uetrecht, 1999).

In contrast with adverse reactions that are usually dose dependent and due to the pharmacology of the drug (e.g. type A), ADRs caused by toxic metabolites are difficult to predict accurately because metabolic activation of the drug is required to observe the toxic effect. In addition, significant species differences in drug metabolism, chemical instability of the reactive metabolites, and the presence of detoxification pathways are just some of the several factors, which make the screening for toxic metabolites a real challenge.

Over the past years, several approaches have been used to detect metabolism-mediated toxicity in vitro. Because covalent binding of drugs to proteins has been associated with drug toxicity (Zhou et al., 2005), it is rather common practice within large pharmaceutical companies to determine the extent of irreversible binding to protein using radiolabeled drug and human liver microsomes (Day et al., 2005, Kitteringham et al., 1988).

Human hepatocytes represent another in vitro system for the evaluation of metabolism mediated toxicity (Gomez-Lechon et al., 2003, Li et al., 1999), but the poor availability of human liver, the high cost and the significant variability among human hepatocytes preparations make this tool not applicable to an high-throughput screening in drug discovery. More recently, cell lines that have been genetically modified to express a single or multiple drug-metabolising enzyme(s) have been developed (Bull et al., 2001, Dai and Cederbaum, 1995, Lin et al., 1999, Nakagawa et al., 1996, Philip et al., 1999, Wu and Cederbaum, 1996). However, most of these stably transfected cell lines have very low metabolic activity, compared to hepatocytes or liver microsomes. Finally, the immortalized human hepatocyte cell line Fa2N-4 has been recently described as an in vitro tool to study drug metabolism issues (Mills et al., 2004), but like for stably transfected cell lines, also the use of Fa2N-4 cells to address reactive metabolites is limited due to low enzymatic activities.

In the present study, we describe the development of two in vitro models to evaluate the activation of xenobiotics to toxic metabolites. In the first method, we used human CYP3A4 cDNA expressed microsomes (as activating system), in combination with HepG2 cells (as target system).

In the second model, a HepG2 cell line transiently transfected with CYP3A4 was developed. Transient transfection was preferred above stable transfection, because of the higher metabolic activity observed in these cells.

HepG2 cells were selected as target cells for evaluating toxicity because they are derived from human liver and have been extensively used as the test system for the prediction of toxicity, carcinogenicity and cell mutagenicity in humans. Moreover, HepG2 cells contain the co-enzymes NADPH-cytochrome P450 reductase and cytochrome b5, required for CYP mediated drug metabolism (Rodriguez-Antona et al., 2002, Yoshitomi et al., 2001).

Because of the general importance of CYP3A4 in drug metabolism (50% of the drugs in use today are metabolised by CYP3A4) (Guengerich, 2001, Nelson, 1999, Rendic and Di Carlo, 1997) and the several examples of the involvement of CYP3A4 in the formation of reactive metabolites, it was decided to focus on CYP3A4 as an activating system.

In both systems, the metabolic activity of CYP3A4 was determined by incubation with testosterone and midazolam, two well known substrate markers of CYP3A4 metabolism.

For the detection of any effect due to toxic metabolites, reproducible and sensitive endpoints are fundamental. After evaluation of the available assays it was decided that the MTT and ATP were the most suitable endpoints.

The CYP3A4 inhibitor ketoconazole, control microsomes and a control vector plasmid were included to make sure that toxic effects were due to CYP3A4 activity.

To minimize the effect of detoxification, in particular the role of reduced glutathione (GSH), HepG2 cells were treated with l-buthionine S,R-sulphoximine (BSO) in order to deplete GSH levels.

To verify the applicability of both models ten compounds that require metabolic activation to manifest their toxic effects were evaluated. In addition, two compounds known to be metabolised by CYP3A4 into non toxic metabolites were included as well. Compounds tested and their reactive metabolites are reported in Table 1 and compound chemical structures are shown in Fig. 1.