A sensitive and specific CYP cocktail assay for the simultaneous assessment of human cytochrome P450 activities in primary cultures of human hepatocytes using LC–MS/MS
Introduction
Cytochrome P450 enzymes (CYP450) are commonly involved in clinically important drug–drug interactions (DDI). Among the various human CYP450 enzymes, CYP3A4/5, CYP2C9, CYP2C19, CYP2D6 and CYP1A2 isoforms account for the metabolism of approximately 90% of drugs [1]. In order to avoid unwanted DDI and associated toxicities in human, several in vitro DDI studies are routinely performed in human liver microsomes (HLM) and primary hepatocytes for the prediction of in vivo DDI [2]. While HLM can only be used for short term CYP inhibitory studies, the primary cultures of human hepatocytes have an added advantage that they can be useful for long term CYP induction and short term/long term CYP inhibition studies.
In a conventional DDI study, the CYP activities are measured individually for the assessment of CYP isoform susceptible for inhibition/induction by drugs [3]. Because of the limited availability of human livers [4] and the extensive time [5] required to perform individual CYP enzyme activity, there is a need to minimize the amount of time and human liver microsomes or hepatocytes needed to perform in vitro DDI studies. Hence, the CYP cocktail approach was adopted by many researchers to simultaneously assess various CYP activities [6], [7], [8]. In a CYP cocktail assay, the preferred and acceptable probe substrate for individual CYP isoform will be mixed together as a cocktail and then incubated with suitable in vitro system (HLM or hepatocytes) for the simultaneous measurement of different CYP enzymes activities.
Although several CYP cocktail assays were developed for HLM [9] or microsomes obtained from primary cultures of human hepatocytes [10], very limited number of CYP cocktail assays have been reported for the simultaneous assessment of CYP activities directly in the primary cultures of human hepatocytes [11], [12], [13], [14]. In addition, the use of stable isotope labeled internal standards for each of CYP probe substrate was very limited in a CYP cocktail assay that was performed in primary cultures of human hepatocytes. Therefore the objective of this study was to develop and validate a sensitive CYP cocktail assay for the simultaneous measurement of major human cytochrome P450 enzymes (CYP3A4/5, CYP2C9, CYP2C19, CYP2D6 and CYP1A2) activities in primary cultures of human hepatocytes using stable isotope labeled internal standards using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Additionally, the CYP cocktail assay was cross validated by comparing the individual and simultaneous incubation of CYP substrates in primary cultures of human hepatocytes to identify any potential interactions among the CYP substrates used in the CYP cocktail assay.
Section snippets
Chemicals and materials
Phenacetin, dextromethorphan hydrobromide monohydrate, bupropion, chlorzoxazone and acetaminophen were obtained from Sigma–Aldrich (St. Louis, MO, USA). Diclofenac sodium salt and dextrorphan-d-tartrate were purchased from MP Biomedical Inc. (Solon, OH, USA). S-mephenytoin, midazolam, hydroxy bupropion, 4′-hydroxy diclofenac, (S)-4-hydroxy mephenytoin, 1′-hydroxy midazolam and deuterated internal standards such as acetaminophen-D4, (±)-4-hydroxy mephenytoin-d3, dextrorphan-d3, tartrate salt,
Results and discussion
Because of the characteristic resemblance to human liver, the primary cultures of human hepatocytes are routinely used for in vivo prediction of DDI [16]. The objective of this study was to develop a sensitive and specific CYP cocktail assay for assessing the activities of major CYP450 enzymes involved for the metabolism based DDI.
The selection of CYP cocktail substrates was based on the recent FDA guidance over the preferred and acceptable CYP substrates (//www.fda.gov/drugs/developmentapproval-process/developmentresources/druginteractionslabeling/ucm093664.htm
Conclusions
This CYP cocktail assay consisting of five probe drugs provides a sensitive, specific and robust method to measure drug–drug interactions involving CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 in primary cultures of human hepatocytes. This assay can be used to assess the CYP activities in human fetal hepatocytes or human liver microsomes.
Acknowledgement
This study was partially supported by NICHD, OPRU network grant 5U10 HD047905.
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