Objectives P450 oxidoreductase (POR) donates electrons to all microsomal cytochrome P450s, including drug-metabolizing and steroidogenic enzymes. Severe POR mutations cause skeletal malformations and disordered steroidogenesis. The POR polymorphism A503V is found on ~28% of human alleles and decreases activities of CYP3A4 and steroidogenic CYP17, but not the activities of steroidogenic CYP21 or drug-metabolizing CYP1A2 and CYP2C19. CYP2D6 metabolizes about 25% of clinically used drugs; we assessed the capacity of POR variants to support the activities of human CYP2D6. Methods N-27 forms of wild type (WT), Q153R, A287P, R457H and A503V POR, and WT CYP2D6 were expressed in E.coli. POR proteins in bacterial membranes were reconstituted with purified CYP2D6. Support of CYP2D6 was measured by metabolism of EOMCC (2H-1-benzopyran-3-carbonitrile,7-(ethoxy-methoxy)-2-oxo-(9Cl)), dextromethorphan and bufuralol. Km and Vmax were determined in three triplicate experiments for each reaction; catalytic efficiency is expressed as Vmax/Km. Results Compared to WT POR, disease-causing POR mutants A287P and R457H supported no detectable CYP2D6 activity with EOMCC, but A287P supported ~ 25% activity with dextromethorphan and bufuralol. Q153R had increased function with CYP2D6 (128% with EOMCC; 198% with dextromethorphan; 153% with bufuralol). A503V supported decreased CYP2D6 activity: 85% with EOMCC, 62% with dextromethorphan and 53% with bufuralol. Conclusions POR variants have different effects depending on the substrate metabolized. Disease-causing POR mutations R457H and A287P had poor activities, suggesting that diminished drug metabolism should be considered in affected patients. The common A503V polymorphism impaired CYP2D6 activities with two commonly-used drugs by 40–50%; potentially explaining some genetic variation in drug metabolism.

There are a considerable number of reports identifying and characterizing genetic variants within the CYP2C9 coding region. Much less is known about polymorphic promoter sequences that also might contribute to interindividual differences in CYP2C9 expression. To address this problem, approximately 10,000 base pairs of CYP2C9 upstream information were resequenced using 24 DNA samples from the Coriell Polymorphism Discovery Resource. Thirty-one single-nucleotide polymorphisms (SNPs) were identified; nine SNPs were novel, whereas 22 were reported previously. Using both sequencing and multiplex single-base extension, individual SNP frequencies were determined in 193 DNA samples obtained from unrelated, selfreported Hispanic Americans of Mexican descent, and they were compared with similar data obtained from a non-Latino white cohort. Significant interethnic differences were observed in several SNP frequencies, some of which seemed unique to the Hispanic population. Analysis using PHASE 2.1 inferred nine common (Ͼ1%) variant haplotypes, two of which included the g.3608CϾT (R144C) CYP2C9*2 and two the g.42614AϾC (I359L) CYP2C9*3 SNPs. Haplotype variants were introduced into a CYP2C9/luciferase reporter plasmid using site-directed mutagenesis, and the impact of the variants on promoter activity assessed by transient expression in HepG2 cells. Both constitutive and pregnane X receptor-mediated inducible activities were measured. Haplotypes 1B, 3A, and 3B each exhibited a 65% decrease in constitutive promoter activity relative to the reference haplotype. Haplotypes 1D and 3B exhibited a 50% decrease and a 40% increase in induced promoter activity, respectively. These data suggest that genetic variation within CYP2C9 regulatory sequences is likely to contribute to differences in CYP2C9 phenotype both within and among different populations.

Background : Determination of cytochrome P450 enzyme-mediated kinetics in vitro can be useful for predicting drug dosing and clearance in humans. Expressed P450s, human liver microsomes, human hepatocytes (both fresh and cryopreserved), and human liver slices are used to estimate K m and V max values for determination of intrinsic clearance of the drug for scale-up to predict in vivo clearance. Objective : To describe the advantages and disadvantages of the various in vitro systems used to estimate kinetic parameters for disposition of drugs and the various kinetic profiles that can be observed. Methods : A review of the literature was conducted to evaluate the utility of the various in vitro preparations, the methods for determining kinetic parameters and the types of kinetic profiles that may be observed. Results/conclusions : The choice of in vitro system for determining kinetic parameters will depend on the objective of the studies, as each system has advantages and disadvantages. Kinetic parameter determinations must be carefully assessed to assure that the correct kinetic model is applied and the most accurate kinetic parameters are determined.

1. Members of the cytochrome P450 3A (CYP3A) subfamily metabolize numerous compounds and serve as the loci of drug-drug interactions (DDIs). Because of high amino acid sequence identity with human CYP3A, the cynomolgus monkey has been proposed as a model species to support DDI risk assessment. 2. Therefore, the objective of this study was to evaluate 35 known inhibitors of human CYP3A using human (HLM) and cynomolgus monkey (CLM) liver microsomes. Midazolam was employed as substrate to generate IC values (concentration of inhibitor rendering 50% inhibition) in the absence and presence of a preincubation (30 mins) with NADPH. 3. In the absence of preincubation, the IC values generated with CLM were similar to those obtained with HLM (86% within 2-fold; 100% within 3-fold difference). However, significant differences (up to 48-fold) in preincubation IC were observed with 17% of the compounds (raloxifene, bergamottin, nicardipine, mibefradil, ritonavir, and diltiazem). 4. Our results indicate that in most cases the cynomolgus monkey can be a viable DDI model. However, significant species differences in time-dependent CYP3A inhibition can be observed for some compounds. In the case of raloxifene, such a difference can be ascribed to a specific CYP3A4 amino acid residue.

In vitro studies play an important role in characterizing biotransformation reactions. Kinetic parameters are determined during the early phases of drug discovery and development and provide invaluable information needed to predict in vivo metabolism and assess potential interactions with enzyme effectors. In order to obtain reliable and accurate in vitro data that reflect the in vivo situation, one must employ well‐defined enzyme kinetic practices and reaction conditions. There are multiple factors to consider when conducting in vitro kinetic experiments such as enzyme concentration, addition of co‐substrates, buffering systems, and the effects that each of these may have on the experimental results. Of equal importance is to be aware of the potential caveats that may lead to erroneous results. An overview of the appropriate experimental conditions for kinetic studies is described, as well an explanation of which kinetic model is most appropriate when describing the kinetic results. Kinetic characterizations of most enzymatic reactions are governed by hyperbolic kinetics that can be described by the Michaelis–Menten equation. In some instances, atypical kinetic profiles have been observed. Atypical kinetics may result from multiple substrate molecules occupying the enzyme active site simultaneously. An overview of the fundamental concepts underlying both typical and atypical kinetic analysis is provided. In addition, the graphical representation and interpretation of the data is reviewed. In summary, the focus of this chapter is on general enzyme kinetic principles. These principles are presented in the context of defining drug metabolic clearance and drug inhibition potential. It is our hope to provide valuable insight into the complexities of in vitro kinetics and the many challenges that surround quantitative prediction in drug metabolism.