Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse
Graphical abstract
Introduction
Cytochrome P450 (CYP) enzymes are responsible for most biotransformation steps of xenobiotics and endogenous molecules (Ortiz-de-Montellano, 2005). Variations of their activity by inhibition or induction can influence the pharmacokinetics and thereby the effect of drugs (of abuse). Enzyme inhibition by co-administered drugs (of abuse) and/or genetic variations of their expression can increase the risk of adverse reactions (Gasche et al., 2004) or reduce the desired effect (Stamer et al., 2007). Such drug–drug interactions were described as a major reason for hospitalization or even death (Lazarou et al., 1998). Therefore, new drug candidates have to be screened before approval and corresponding data are available on the Internet (http://medicine.iupui.edu/clinpharm/ddis/main-table/). In contrast, inhibition data of most drugs of abuse (DOA), particularly the so-called novel psychoactive substances (NPS), are not available because such drugs are marketed and consumed without any safety pharmacology testing. But these data could be of clinical and forensic relevance if the patient, e.g., polydrug users, takes more than one drug of abuse or is under treatment with therapeutic drugs. Only few studies about the CYP inhibition potential of DOA were published showing that DOA such as 2,5-dimethoxy-4-iodo-amfetamine (DOI), methylone, or some amphetamine analogs could inhibit CYP2D6 in the same range as clinically relevant inhibitors such as fluoxetine (Ewald and Maurer, 2008, Pedersen et al., 2013, Pritzker et al., 2002, Wu et al., 1997). Therefore, the classic drugs of abuse and the NPS should be screened for their inhibition potential toward all relevant CYPs. IC50 values, representing the inhibitor concentration, at which the enzyme activity is reduced by 50%, should be determined. For that purpose, the concentration of the metabolites of model substrates formed by the corresponding CYPs in absence or presence of different concentrations of the potential inhibitors had to be determined. Various inhibition assays were described using different substrates and/or model inhibitors for five to nine CYPs (Dierks et al., 2001, Kim et al., 2005, Lee and Kim, 2013, Qiao et al., 2014, Qin et al., 2014, Turpeinen et al., 2005). Some of these assays showed limitations concerning the analytical method (Dierks et al., 2001, Kim et al., 2005, Lee and Kim, 2013, Turpeinen et al., 2005), the number of tested CYPs (Dierks et al., 2001, Lee and Kim, 2013, Qiao et al., 2014, Qin et al., 2014), or the IC50 values comparability between single and cocktail incubation (Turpeinen et al., 2005). Unfortunately, all of these cocktail assays did not test for reproducibility of the IC50 values over several days, an important task for data evaluation (Dierks et al., 2001, Kim et al., 2005, Lee and Kim, 2013, Qiao et al., 2014, Qin et al., 2014, Turpeinen et al., 2005). The limitations concerning the analytical methods have been discussed recently in the article describing a validated, sensitive, and selective liquid chromatography-high resolution–tandem mass spectrometry (LC-HR–MS/MS) method for quantification of the specific CYP substrate metabolites used for the present study (Dinger et al., 2014).
Therefore, in the following a new inhibition cocktail assay for nine CYP is presented based on this method. The assay should be proven comparability of the IC50 values between single and cocktail assay, reproducibility over several days, and the applicability using DOA as test inhibitors.
Section snippets
Chemicals and reagents
Alpha-naphthoflavone, amodiaquine 2HCl, coumarin, deethyl amodiaquine, dextrorphan HBr, diclofenac, DOI, 5-hydroxy coumarin, isocitrate, isocitrate dehydrogenase (IDH), R,S-3,4-methylendioxy-N-methylamphetamine (MDMA), omeprazole, sulfaphenazole, superoxide dismutase (SOD), trimethoprim, and verapamil HCl were obtained from Sigma–Aldrich (Steinheim, Germany), bupropion HCl and 4-hydroxy bupropion HCl from GlaxoSmithKline (Munich, Germany), 4-hydroxy diclofenac from Pombiotech (Saarbrücken,
Determination of cross-inhibitions
Interferences for both reactions resulting in reduced Km and Vmax values (Fig. 2) could be observed. As O-deethyl phenacetin, a phenacetin metabolite formed by CYP1A2, is also metabolized by CYP2E1, the interaction potential of these two reactions in the cocktail assay had to be checked. Therefore, the enzyme kinetic constants (Km and Vmax) of chlorzoxazone, a CYP2E1 substrate, were determined with and without O-deethyl phenacetin. To use real incubation conditions, the O-deethyl phenacetin
Conflict of interest
The authors declare that there are no conflicts of interest.
Transparency document
Acknowledgements
The authors like to thank their colleagues Achim Caspar, Andreas G. Helfer, Golo M.J. Meyer, Julian A. Michely, Armin A. Weber, Jessica Welter, and Carina S.D. Wink for their support.
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