Miia Turpeinen

Inhibition and induction of human cytochrome P450 enzymes: current status

Variability of drug metabolism, especially that of the most important phase I enzymes or cytochrome P450 (CYP) enzymes, is an important complicating factor in many areas of pharmacology and toxicology, in drug development, preclinical toxicity studies, clinical trials, drug therapy, environmental exposures and risk assessment. These frequently enormous consequences in mind, predictive and pre-emptying measures have been a top priority in both pharmacology and toxicology. This means the development of predictive in vitro approaches. The sound prediction is always based on the firm background of basic research on the phenomena of inhibition and induction and their underlying mechanisms; consequently the description of these aspects is the purpose of this review. We cover both inhibition and induction of CYP enzymes, always keeping in mind the basic mechanisms on which to build predictive and preventive in vitro approaches. Just because validation is an essential part of any in vitro–in vivo extrapolation scenario, we cover also necessary in vivo research and findings in order to provide a proper view to justify in vitro approaches and observations.

Functional pharmacogenetics/genomics of human cytochromes P450 involved in drug biotransformation

We investigated the elimination routes for the 200 drugs that are sold most often by prescription count in the United States. The majority (78%) of the hepatically cleared drugs were found to be subject to oxidative metabolism via cytochromes P450 of the families 1, 2 and 3, with major contributions from CYP3A4/5 (37% of drugs) followed by CYP2C9 (17%), CYP2D6 (15%), CYP2C19 (10%), CYP1A2 (9%), CYP2C8 (6%), and CYP2B6 (4%). Clinically well-established polymorphic CYPs (i.e., CYP2C9, CYP2C19, and CYP2D6) were involved in the metabolism of approximately half of those drugs, including (in particular) NSAIDs metabolized mainly by CYP2C9, proton-pump inhibitors metabolized by CYP2C19, and beta blockers and several antipsychotics and antidepressants metabolized by CYP2D6. In this review, we provide an up-to-date summary of the functional polymorphisms and aspects of the functional genomics of the major human drug-metabolizing cytochrome P450s, as well as their clinical significance.

STABLE EXPRESSION, ACTIVITY AND INDUCIBILITY OF CYTOCHROMES P450 IN DIFFERENTIATED HepaRG CELLS

HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.

The Functional Role of CYP2B6 in Human Drug Metabolism: Substrates and Inhibitors In Vitro, In Vivo and In Silico

CYP2B6 metabolizes a number of drug substrates, that are usually non-planar, neutral or weakly basic, fairly lipophilic with one or two hydrogen bond acceptors, on which it catalyses various oxidative reactions. For bupropion, cyclophosphamide, ifosfamide, pethidine, ketamine and propofol, these reactions represent major metabolic or activation pathways and for their kinetics CYP2B6 function is of considerable importance. For the rest of the substrates found, CYP2B6 contributes to overall metabolism or to a single pathway, but probably not to a materially significant extent. Among inhibitors, thiotepa, ticlopidine and clopidogrel have been characterised extensively in terms of selectivity and potency. Thiotepa is the most selective of the inhibitors, but is not useful as an in vivo inhibitor, whereas ticlopidine and clopidogrel can be used as CYP2B6-selective probes in human clinical studies. Bupropion hydroxylation is a selective, and consequently useful, in vivo probe for CYP2B6. Computational approaches are being developed to the extent that predictions on affinity of chemicals to CYP2B6 are becoming reliable enough as a first screen of new drug molecules and other chemicals. With validated in vitro and in vivo substrates (e.g. bupropion) and inhibitors (e.g. ticlopidine), it is expected that pharmacological (including pharmacogenetic) and clinical significance of CYP2B6 will be delineated more fully in the near future.

Liquid chromatography-mass spectrometry in in vitro drug metabolite screening

A combination of high performance liquid chromatography (HPLC) and mass spectrometry (LC/MS) has proven its status as the most powerful analytical tool for screening and identifying drug metabolites in modern drug discovery. These techniques have become irreplaceable for drug metabolism laboratories, providing high amounts of information from a wide variety of samples. This review focuses on the most common and useful applications of these techniques when working on in vitro metabolism, more specifically with screening and identification of chemically stable or reactive metabolites formed via biotransformation reactions. Matching specific tasks and suitable instruments is a recurring consideration; for many reasons, the time-of-flight or orbitrap mass spectrometry provides clearly increased efficiency in metabolite profiling compared to other types of mass spectrometry.

Pharmacogenomics of human liver cytochrome P450 oxidoreductase: multifactorial analysis and impact on microsomal drug oxidation

AIMS NADPH:CYP oxidoreductase (POR) is an essential component of several enzyme systems, including the microsomal CYP monooxygenases. We investigated genetic and nongenetic POR variability and its impact on drug-oxidation activities in human liver microsomes. MATERIAL AND METHODS POR mRNA, protein and activity, as well as ten major drug-oxidation activities, were measured in the microsomes of 150 Caucasian surgical liver samples. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometric assays were established to determine the frequency of 46 selected POR SNPs. Multivariate log-linear regression models, including main effects and two-way interaction terms, and analyses of variance were used to identify statistically significant relationships. RESULTS POR phenotypes were less variable within the study population as compared with CYP phenotypes. Intronic SNPs g.18557G>A (intron 2), g.25676C>T (intron 3) and g.30986 G>A (intron 10) were associated with various drug-oxidation activities. The common allele POR*28 (A503V) was not associated with any activity or expression changes. Haplotype analysis identified two novel composite alleles POR*36 (P228L plus A503V) and POR*37 (A503V plus V631I). CONCLUSION Models that integrate POR and microsomal CYP function are complex and depend on the CYP isozyme, the substrate and numerous genetic and nongenetic factors. Intronic POR variants may influence microsomal CYP activities. These data provide a basis for further studies towards inclusion of POR polymorphisms in pharmacogenomic strategies.

Functional expression, inhibition and induction of CYP enzymes in HepaRG cells

Practically all human hepatocyte cell lines are deficient in major cytochrome P450 (CYP)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the recently developed HepaRG cell line to determine the spectrum of most important CYP enzyme activities involved in xenobiotic metabolism (CYP1A1/2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) and the effect of the prototypical CYP-inducer phenobarbital and a panel of known CYP-selective inhibitors on these activities. Comparison of these activities was carried out with two human primary hepatocyte populations. We show that excluding CYP2A6 and CYP2E1, HepaRG cells express high functional levels of most of the major xenobiotic metabolising CYPs. These activities were found to be selectively inhibited and induced by prototypical CYP-selective inhibitors and inducer at comparable levels to primary hepatocytes. In conclusion, HepaRG cells may be a promising cell line for various applications, which currently employ hepatic subcellular preparations or cultured primary hepatocytes.

Characterization of Diuron N-Demethylation by Mammalian Hepatic Microsomes and cDNA-Expressed Human Cytochrome P450 Enzymes

Diuron, a widely used herbicide and antifouling biocide, has been shown to persist in the environment and contaminate drinking water. It has been characterized as a “known/likely” human carcinogen. Whereas its environmental transformation and toxicity have been extensively examined, its metabolic characteristics in mammalian livers have not been reported. This study was designed to investigate diuron biotransformation and disposition because metabolic routes, metabolizing enzymes, interactions, interspecies differences, and interindividual variability are important for risk assessment purposes. The only metabolic pathway detected by liquid chromatography/mass spectometry in human liver homogenates and seven types of mammalian liver microsomes including human was demethylation at the terminal nitrogen atom. No other phase I or phase II metabolites were observed. The rank order of N-demethyldiuron formation in liver microsomes based on intrinsic clearance (Vmax/Km) was dog > monkey > rabbit > mouse > human > minipig > rat. All tested recombinant human cytochrome P450s (P450s) catalyzed diuron N-demethylation and the highest activities were possessed by CYP1A1, CYP1A2, CYP2C19, and CYP2D6. Relative contributions of human CYP1A2, CYP2C19, and CYP3A4 to hepatic diuron N-demethylation, based on average abundances of P450 enzymes in human liver microsomes, were approximately 60, 14, and 13%, respectively. Diuron inhibited relatively potently only CYP1A1/2 (IC50 4 μM). With human-derived and quantitative chemical-specific data, the uncertainty factors for animal to human differences and for human variability in toxicokinetics were within the range of the toxicokinetics default uncertainty/safety factors for chemical risk assessment.

Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts

Hormone replacement therapy is effectively used to prevent postmenopausal bone loss. Variation in response to the therapy is, however, frequently seen. In addition, the direct effects of sex steroids on isolated human bone marrow stromal cells have been reported to vary depending on the donor, but the biological mechanisms are not understood. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and testosterone in human-bone-marrow-derived mesenchymal stem cell (MSC) cultures from both female and male donors of various ages. The osteoblast differentiation capacity and activity of the MSCs were quantified in vitro by measuring alkaline phosphatase activity and calcium deposition. We show here that also the osteoblast responses of MSCs to sex hormones vary widely depending on the donor. When the results from all donors were analyzed together, treatment with E2 increased calcium deposition significantly by MSCs of both sexes but ALP activity only in the male MSCs. Testosterone had no effect on ALP activity nor calcium deposition in either sex. To further characterize the individual variation, we investigated estrogen receptor alpha PvuII restriction site polymorphism with PCR. Restriction fragment-length polymorphism was assigned as P or non-P, P signifying the absence of the restriction site. Our results indicate that higher basal osteoblast differentiation capacity of MSCs is associated with the presence of the P allele in females, whereas higher response to sex steroids treatment is associated with the non-P allele. These results could help explain the contradictory effects of E2 on osteoblasts in vitro and might also provide new insights to understanding the differences in responses to hormone replacement therapy.

Cytochrome P450 2B6: function, genetics, and clinical relevance

Abstract Cytochrome P450 (CYP) 2B6 belongs to the set of important hepatic drug-metabolizing CYPs. It makes up roughly 3%–6% of total hepatic CYP content and metabolizes several pharmaceuticals including bupropion, efavirenz, cyclophosphamide, pethidine, ketamine and propofol. The enzyme is susceptible to drug-drug interactions by enzyme induction and inhibition. In addition to drugs, CYP2B6 is able to both detoxify and bioactivate a number of procarcinogens and environmental agents including pesticides and herbicides. There is an extensive interindividual variability in the expression of CYP2B6, which is in part explained by extensive genetic polymorphism. CYP2B6 is one of the most polymorphic CYP genes in humans with over 100 described SNPs, numerous complex haplotypes and distinct ethnic and racial frequencies. This review summarizes the basic properties of CYP2B6 and the main characteristics of clinical relevance.