Gene polymorphisms and contents of cytochrome P450s have only limited effects on metabolic activities in human liver microsomes

Abstract

Extensive inter-individual variations in pharmacokinetics are considered as a major reason for unpredictable drug responses. As the most important drug metabolic enzymes, inter-individual variations of cytochrome P450 (CYP) activities are not clear in human liver. In this paper, metabolic activities, gene polymorphisms and protein contents of 10 CYPs were determined in 105 human normal liver microsomes. The results indicated substantial inter-individual variations in CYP activities, with the greatest being CYP2C19 activity (> 600-fold). Only half of 10 CYP isoforms and 26 gene polymorphism sites had limited effects on metabolic activities, such as CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP3A4/5, others had almost no effects. Compared with their respective wild type, K_m, V_max, and CL_int decreased by 51.6%, 88.7% and 70.7% in CYP2A6*1/*4 genotype, V_max and CL_int decreased by 32.8% and 60.2% in CYP2C9*1/*3 genotype, K_m increased by 118.4% and CL_int decreased by 65.2% in CYP2D6 100TT genotype, respectively. Moreover, there were only 4 CYP isoforms, CYP1A2, CYP2A6, CYP2E1 and CYP3A5, which had moderate or weak correlations between V_max values and corresponding contents. In conclusions, the genotypes and contents of some CYPs have only limited effects on metabolic activities, which imply that there are other more important factors to influence inter-individual variations.

Introduction

The cytochrome P450 (CYP) superfamily plays a considerable role in the metabolism of endogenous and exogenous compounds. The first three families (CYP1-3) are responsible for the oxidation of about 90% of drugs (Martiny and Miteva, 2013). The 10 most significant drug metabolizing CYPs in the human liver include CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP2D6 and CYP3A4/5 (Wrighton and Stevens, 1992).

It was well known that there were obvious inter-individual variations in pharmacokinetics of CYP substrates. Ujjin et al. (2002) reported that an approximately 150-fold variation in the amount of 7-hydroyxcoumarin excreted in urine in 202 subjects. The results of Fux et al. (2005) showed that the variation in concentration of metoprolol concentration was up to 150-fold. Metabolism plays an important role in pharmacokinetics and CYPs are mainly responsible for metabolism, so extensive inter-individual variations in function of CYPs are considered as a major reason for unpredictable drug responses and drug toxicity (Wolbold et al., 2003). Personalized medicine is an emerging practice of medicine that uses an individual biological information to optimize the management of their disease. So knowledge of the inter-individual variation and its influencing factors are helpful for personalized medicine.

Generally, gene polymorphism of CYP is thought to mainly affect pharmacokinetics by altering the CYP activity, resulting different phenotypes (Martiny and Miteva, 2013). Many studies had attributed these differences in pharmacokinetics to gene polymorphism, so dosage regimen was usually adjusted according to genotype. However administering drug adjustment based on genotype was not suited for some patients. For example, CYP2C9*2 and CYP2C9*3 genotypes only explained 5–20% of the overall variability in warfarin dose used in previous studies (Bazan et al., 2014, Perera et al., 2013). Moreover, the results about effects of gene polymorphisms in vitro were not completely consistent with in vivo effects because the pharmacokinetics of drug might not directly reflect the metabolic activity of CYP. For example, Marchand et al. (1999) reported that oral clearance of chlorzoxazone decreased with the number of variant C2 alleles, but the results of Inoue et al. (2000) reported that C1/C2 polymorphisms may not cause significant alterations in the catalytic activity of CYP2E1 in human liver microsomes (HLMs). So we could not simply attribute changes in phenotype to gene polymorphism.

To evaluate the effect of gene polymorphisms on drug metabolism directly, human-derived systems, such as HLMs, should be used. HLMs have become more important in the process of drug discovery and development (Yang et al., 2012). Now, some studies have been carried out in HLMs (Hesse et al., 2004, Picard et al., 2011b, Yoshida et al., 2003). Hesse et al. (2004) reported the effect of gene polymorphisms on inter-individual variability in CYP2B6 activity, while others had focused on the effect of gene polymorphisms on CYP2A6 and CYP3A activities in this system (Picard et al., 2011a, Yoshida et al., 2003). Although these studies were developed in HLMs, many previous in vitro studies were confounded by inadequate polymorphism sites, smaller sizes and not clear background of HLMs (Hesse et al., 2004, Yamazaki et al., 1998), indicating a need for further studies using normal HLM with larger sample size.

In addition, the obvious inter-individual variations were found in subjects with same genotype. The difference among the subjects with same genotype was referred as intra-genotype individual variation. For instance, a six-fold inter-individual variation in omeprazole clearance was reported in people carrying the CYP2C19*1/*1 genotype (Hu et al., 2007). It suggested that there are other factors involved in drugs metabolism except gene polymorphisms, such as content of CYP isoform.

It was reported that the inter-individual variations in expression of CYP isoforms were extensive. The inter-individual variations of CYP2B6 (Xie et al., 2003) and CYP3A4 (Wolbold et al., 2003) determined by Western Blot were from 50-fold to 700-fold. Moreover, many studies showed that the contents of CYP isoforms had significantly correlation with their activities, such as CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6, CYP2E1 and CYP3A4 (Bahadur et al., 2002b, Lee et al., 2009, Liu et al., 2014, Lucas et al., 1993, Xie et al., 2003). But the data of CYP isoforms content were usually obtained from human liver with not clear background by Western Blot, which made them less objective when evaluating the influence of content on CYP activity in HLMs. Now the contents of CYPs could be quantified by LC-MS/MS with high accuracy and precision.

Up to now, many reports have demonstrated that both the gene polymorphisms and contents of CYPs had significant effects on CYP activities (Bahadur et al., 2002b, Lee et al., 2009, Xie et al., 2003). Based on these reports, it seems that the variations in CYP activities almost cause by gene polymorphisms and contents of CYPs and no other factors contribute to the variations. In fact, there are many factors affecting enzyme activities, such as protein post translational modification, protein-protein interaction and so on (Hughes et al., 2007). As one kind of protein, CYPs must be without exception. It is necessary to further evaluate the limits and extends of effects of gene polymorphisms and contents on the activities 10 CYP isoforms.

In the present study, the gene polymorphisms and metabolic activities of 10 CYP isoforms were assessed in 105 isolated human normal liver microsomes. The protein contents of 10 CYPs detected by LC-MS/MS were used to analyze their effects on activities of CYPs.