An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure
Graphical abstract
Introduction
Cytochromes P450 (P450) are the major phase I metabolic enzymes involved in the oxidative biotransformation of xenobiotics as well endogenous compounds. High inter-individual differences in P450 activities have been described in many publications, and major sources of this variability are associated with genetic features (e.g., gene expression regulation, polymorphism, gender, and age), environmental influences (e.g., stress, diet, and life style) and/or xenobiotic exposure (e.g., drug therapy, dietary supplements, environmental pollutants, and toxic substances). As a consequence, the perturbations of their activities (inhibition/reduction or increase/induction) may lead to a significant variation in the concentration of a xenobiotic and its metabolites at the target site, i.e., enhanced clearance, production of toxic metabolites or toxic accumulation of the parent compound. The inhibition of P450 has been shown to produce unexpected severe effects in drug pharmacokinetics and clinical responses, particularly due to drug-drug interactions (DDIs) (Zanger and Schwab, 2013).
The evaluation of the P450 interaction potential of a xenobiotic must thus be conducted to predict the risks in the case of co-exposure. DDI investigations in the early discovery process have already been achieved to reduce the frequency of costly late failures of the drug candidates and to promote safer medical treatment. In contrast to the clinical environment, where DDIs could also be investigated in vivo thanks to dedicated clinical studies, drug-toxicant interactions cannot be easily performed for evident ethical reasons. However, because toxicants are ubiquitous in the environment and humans are chronically and/or acutely exposed throughout their entire lives, their impact on P450 activities should be more thoroughly investigated, even if official recommendations in this context are not yet enforced (Pelkonen et al., 2008).
According to the 3Rs principle (Replace, Reduce and Refine) for promoting a more reasonable and ethical animal testing, in vitro approaches have been further recommended to evaluate and/or anticipate the impact of toxicants on P450 activities. Hepatocytes are currently the gold standard in vitro tool for performing P450 induction studies, whereas for studying the inhibition phenomenon at the enzyme level, human liver microsomes (HLM) can be judiciously employed as an in vitro native phase I enzyme source thanks to their commercial availability, ease of handling and reliable in vivo extrapolation (Parkinson et al., 2010).
Nowadays, except for numerous academic settings, in vitro P450 inhibition assays are generally conducted using higher-end robotic systems or high-throughput-MS methods (e.g., RapidFire® and Phytronix®), which drastically increase the efficiency of the assays (Haarhoff et al., 2016, Wu et al., 2007, Wu et al., 2012). Alternatively, the cocktail approach has been developed to efficiently and rapidly monitor the activities of several microsomal P450 isoforms within a single test, reducing the time and assay costs when expensive robotic handling instruments are not available (Lahoz et al., 2008). However, when an extended substrate mixture is used for an overall P450 inhibition screening, the following numerous challenges have been identified: (i) an enhanced risk of probe-probe interactions, (ii) a more difficult implementation according to the different optimal incubation conditions for both rapid-and slow-turnover substrates and (iii) the need for efficient separation-based analytical techniques such as LC/MS methods for the reliable analysis of the high number of analytes. Above all, the combination of high-turnover substrates together with low-turnover substrates has often been recognized as the major issue for implementing unique optimal incubation conditions. Indeed, to allow the detection of low amounts of a metabolite, a relatively high concentration of a slow-turnover substrate along with a high protein concentration and incubation time have to be employed. This practice indirectly promoted the occurrence of probe-probe interactions and simultaneously created disadvantaged conditions for high-turnover substrates. Due to the presence of probe interactions not fully characterized as well as sub-optimal assay conditions for each substrate, the reliability of inhibition investigations could be reduced (Spaggiari et al., 2014a). Recently, the separation of critical substrates into two distinct cocktails has been successfully proposed to overcome probe interactions and facilitate cocktail design. Authors also highlighted the importance of chromatographically separating metabolites and substrates still present in the incubation mixture prior to analysis to avoid potential and relevant analytical interferences due to co-elution, which could not be adjusted using a single analytical standard. Besides pooled samples could be analyzed to save analysis time, this methodology duplicated reagent consumption and manipulations (Dinger et al., 2014a). Alternatively, with the powerful separation capabilities of ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), including its high sensitivity, selectivity and resolution, it was possible to overcome some of the difficulties of cocktail approach. Analytical interferences were corrected using stable isotope-labeled metabolites to correct potential co-elutions (Kozakai et al., 2012).
In this study, an alternative analytical strategy was developed for the original design of an extensive cocktail assay for a microsomal P450 direct reversible inhibition screening (IC50 assay) for the most important isoforms involved in the biotransformations of clinically used drugs, namely, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. An optimized UHPLC separation of the eleven substrates and their P450-specific metabolites was combined with highly sensitive MS/MS detection to achieve favorable analytical and metabolic conditions for optimal cocktail incubation. As a novel aspect, P450 microsomal activities were expressed by combining metabolite formation with substrate depletion (i.e., metabolic ratio) rather than metabolite formation only in the analysis. Afterwards, the inhibition curves and subsequent IC50 values were compared to those obtained with the single probe approach. Finally, this assay demonstrated to be a promising safety assessment tool for the evaluation of environment-related direct reversible inhibition of P450 (e.g., pesticides, cosmetics, food additives, phytochemicals, diet, etc.), which is currently not systematically addressed in the panel of toxicological screening tests.
Section snippets
Chemicals, reagents, test compounds and other materials
Acetonitrile (MeCN), methanol (MeOH) and water of ULC/MS grade were purchased from Biosolve (Valkenswaard, Netherlands). Formic acid was obtained from Merck (Darmstadt, Germany). (S)-(+)-N-3-benzylnirvanol (98%), chlorzoxazone (98%), O-desmethylastemizole (98%) hydroxybupropion (95%), 6-hydroxychlorzoxazone (97%), 5-hydroxyomeprazole (98%) and omeprazole (98%) were purchased from Toronto Research Chemicals (Ontario, Canada). Acetaminophen (99%), ammonium hydroxide, amodiaquine dihydrochloride
Cocktail composition and analytical method development
For the first time in the context of the cocktail approach, eleven P450 probe substrates, namely, phenacetin (1A2), coumarin (2A6), bupropion (2B6), amodiaquine (2C8), diclofenac (2C9), omeprazole (2C19), dextromethorphan (2D6), chlorzoxazone (2E1), astemizole (2J2), midazolam and testosterone (3A subfamily), have been selected for simultaneously monitoring the most clinically and toxicologically relevant P450 isoforms in HLM. According to our recent survey of the published in vitro cocktails,
Conclusion
The presented cocktail approach included, for the first time, eleven probe substrates for direct reversible inhibition screening of xenobiotics affecting the ten most relevant P450 isoforms in human metabolism. Thanks to the high analytical sensitivity, it was possible to minimize probe-probe interactions in the cocktail and drastically reduce both the incubation time and reagent consumption. The IC50 values obtained were in agreement with reference values obtained by the single probe methods.
Abbreviations
DDI drug-drug interactions DT dwell time ESI electrospray ionization HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid pHLM pooled human liver microsomes LLOQ lower limit of quantification MeCN acetonitrile MeOH methanol Me2SO dimethyl sulfoxide NADPH β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt NOEC no observed effect concentration OP organophosphorothionate P450 cytochrome P450 PBBK physiologically-based biokinetics QqQ/MS triple-quadrupole mass spectrometer S/N signal-to-noise
Funding sources
No funding sources.
Transparency document
Acknowledgments
The authors wish to thank Dr. Szabolcs Fekete for his valuable help regarding the Drylab®2010 Plus modeling software.
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