Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse

Highlights

• Development and validation of an all-in-two CYP inhibition cocktail assay.

• Testing for chemical reactivity between inhibitors and the test substrate metabolites.

• IC₅₀ values were comparable between single and cocktail incubations.

• IC₅₀ values of the model inhibitors were reproducible over several days.

• The CYP2D6 inhibitor DOI inhibited also CYP1A2, CYP2B6, CYP2C19, and CYP3A.

Abstract

Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution–MS/MS. The IC₅₀ values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC₅₀ values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI).

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.