Validated method for rapid inhibition screening of six cytochrome P450 enzymes by liquid chromatography–tandem mass spectrometry

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Abstract

In vitro drug interaction data can be used in guiding clinical interaction studies, or, the design of new candidates. To make such a claim, it must be assured that the in vitro data obtained is confident. To meet this need, a rapid liquid chromatography–tandem mass spectrometry (LC/MS/MS) method has been validated and employed for routine screening of new chemical entities for inhibition of six major human cytochrome P450 (CYP) isoforms using cDNA-expressed CYPs. Probe substrates were used near the Michaelis–Menten constant (Km) concentration values for CYP1A2 (phenacetin), CYP2C9 (tolbutamide), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam and dextromethorphan). The major metabolites of CYP-specific probe substrates were quantified. The LC/MS/MS method was found to be accurate and precise within the linear range of 1.0–2000 ng/ml for each analyte in enzyme incubation mixture. The lower limit of quantification (LLOQ) was 1.0 ng/ml. The limit of detection (LOD) for the tested analytes was 0.48 ng/ml or better based on signal-to-noise ratio >3. The inhibition potential of the six CYP isoforms has been evaluated using their known selective inhibitors. The 50% inhibitory concentrations (IC50 values) measured by this method demonstrated high precision and are consistent with the literature values.

Introduction

Drug–drug interactions (DDIs) are of great interest to pharmaceutical scientists, clinicians, and patients. The inhibition of cytochrome P450 (CYP) activities has been recognized as the pivotal cause of the DDIs in clinic, which may alter the metabolism, potential toxicity, and efficacy of drugs. Among numerous CYP enzymes identified to date, six human hepatic CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) have been found to play dominant roles in drug metabolism, which are involved in many clinical important drug interactions [1]. The withdrawal of terfenadine and mibefradil from the market due to inhibition-based interactions involving CYP3A4 and CYP2D6 exemplify the medical relevance of CYP inhibition [2], [3]. For the inhibition assessment, a common strategy is to monitor the effect of test compounds on the metabolism of CYP probe substrates using human liver microsomes or recombinant CYPs. The data obtained from in vitro inhibition study can help guide clinical development programs by identifying drug–drug interactions that ought to be investigated, or guide the design of new candidates [4], [5], [6], [7]. However, these data often vary considerably from one laboratory to the next, even when the experimenters claim to use the same assays and methods [5]. Therefore, a systematic and reliable approach is needed to verify the conclusions [4], [5], [6], [7].

At present, numerous high-throughput approaches using fluorogenic substrates to measure CYP activities have been described [8], [9], as well as substrate “cocktail” experiments that simultaneously measure more than one CYP activity [10], [11], [12], [13], [14], [15], [16], [17], [18]. Recently Kim et al. [19] described a high-throughput method that allowed the simultaneous evaluation of the metabolic activities of nine major human CYP isoforms (i.e., CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) on their well-known specific probe substrates using human hepatic microsomes. The total run time was 6.5 min for each incubation sample. However, only one substrate (midazolam) was selected for CYP3A4 inhibition screening. CYP3A4 is the most abundant isoform in human liver. This isoform accounts for approximately 30% of the total human liver microsomal CYP protein and is involved in the metabolism of approximately 50% of all market drugs. Inhibition of this enzyme by coadministered drugs has been shown to result in adverse clinical DDIs, including fatalities [2]. CYP3A4 exhibits atypical enzyme kinetics depending on the substrate used. For some inhibitors, qualitative differences and quantitative differences of up to 300-fold in 50% inhibitory concentrations (IC50 values) can be obtained depending on the substrate being used in the assay [20], [21]. Therefore, as a minimum requirement for in vitro inhibition studies, it was recommended that at least two CYP3A4 substrates should be used [22], [23], [24], [6]. Additionally, human hepatic microsomes used in these “cocktail” experiments is the greater contributor to the effect of nonspecific binding compared with cDNA-expressed CYPs, which may result in false negative errors in prediction of the risk of drug interaction [25], [26].

In this research, we developed a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the evaluation of inhibition potential of drugs on six major human CYP isoforms using cDNA-expressed CYPs. For CYP3A4 inhibition studies, two substrates, midazolam and dextromethorphan were employed. To our best knowledge, the analytical method for simultaneous determination of 3-methoxymorphinan (3MM), dextrorphan (DX), acetaminophen (AP), 4′-hydroxymephenytoin (OH-MP) and 1′-hydroxymidazolam (OH-MDZ) in incubation has not been reported. Furthermore, the LC/MS/MS method for the simultaneous determination of multi-components in a very short run-time has been fully validated.

Section snippets

Materials

cDNA-expressed CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were purchased from BD Gentest (Woburn, MA, USA). All the CYPs were expressed in microsomes of insect cells (BTI-5B1-4) infected with a baculovirus containing human CYP and NADPH-CYP reductase cDNA inserts, and the cytochrome b5 was co-expressed. The selective CYP substrates used in this method are listed in Table 1.

S-Mephenytoin, chlorzoxazone, and 3-methoxymorphinan (3MM) were purchased from BD Gentest Co. (Woburn, MA, USA).

Mass spectrometry

During the early stage of method development, attempt was made to simultaneously determine seven probe metabolites (AP, OH-MP, DX, OH-MDZ, 3MM, OH-TB, and OH-CZ). However, under (+) ESI conditions, OH-CZ and OH-MP produced no MS signal. When (+) APCI interface was used, intensive [M + H]+ peak of OH-MP was observed, while the MS response of OH-CZ was still very low. The other analytes, including AP, DX, OH-MDZ, 3MM, and OH-TB, provided high MS response. Further research showed that OH-CZ, OH-MP,

Conclusions

This paper describes the development and validation of a new LC/MS/MS method for a rapid quantitative analysis of seven CYP probe metabolites. It offers some advantages over previously published methods for evaluation of CYP activity and inhibition. All of the substrates and metabolites are readily available commercially (e.g. phenacetin, tolbutamide, dextromethorphan, AP, OH-TB, and DX could be purchased from Sigma); high sensitivity permits decreasing the amount of cDNA expressed CYPs

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