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

doi:10.1016/j.jpba.2012.10.016

Journal of Pharmaceutical and Biomedical Analysis

Volume 74, 23 February 2013, Pages 126-132

https://doi.org/10.1016/j.jpba.2012.10.016 Get rights and content

Abstract

A sensitive and specific CYP cocktail assay for simultaneous measurement of the activities of major human cytochrome P450 enzymes (CYP1A2 (phenacetin), CYP3A4/5 (midazolam), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin) and CYP2D6 (dextromethorphan)) in primary cultures of human hepatocytes, was developed and validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Hepatocyte incubation medium was processed by a solid phase extraction (SPE) using Oasis SPE extraction cartridges prior to chromatography. The metabolites derived from each of the substrates were simultaneously quantitated using the corresponding stable isotope-labeled internal standards by a positive electrospray ionization mode using multiple reactions monitoring with a single eight minute run. The mean accuracy was in the range of 98–114%. The interday and intraday precision over the concentration ranges evaluated for all the analytes were lower than 15%, and 14%, respectively. All the generated metabolites were stable under the conditions used for sample analysis. Additionally, the interaction of a cocktail substrate on other CYP substrates was also analyzed. Due to substantial inter-substrate interaction, chlorzoxazone (CYP2E1) and bupropion (CYP2B6) were removed from the initial seven probes CYP cocktail assay. Therefore, the final CYP cocktail assay consisting of five probes provides a robust method to simultaneously measure activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5 in primary cultures of human hepatocytes.

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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Published by Elsevier B.V.

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

Abstract

Introduction

Section snippets

Chemicals and materials

Results and discussion

Conclusions

Acknowledgement

J. Pharmacol. Toxicol. Methods

J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci.

J. Pharmacol. Toxicol. Methods

Toxicol. Appl. Pharmacol.

Talanta

Clin. Ther.

The effect of cytochrome P450 metabolism on drug response, interactions, and adverse effects

Am. Fam. Physician

The conduct of in vitro and in vivo drug–drug interaction studies: a pharmaceutical research and manufacturers of America (PhRMA) perspective

Drug Metab. Dispos.

In vitro inhibition and induction of human cytochrome P450 enzymes by mirabegron, a potent and selective beta3-adrenoceptor agonist

Xenobiotica

Human hepatocytes as a tool for studying toxicity and drug metabolism

Curr. Drug Metab.

Reliable high-throughput method for inhibition assay of 8 cytochrome P450 isoforms using cocktail of probe substrates and stable isotope-labeled internal standards

Drug Metab. Pharmacokinet.

In vitro LC–MS cocktail assays to simultaneously determine human cytochrome P450 activities

Biopharm. Drug Dispos.