ABSTRACT: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 sulfoxidedependent. 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.

Both clopidogrel and ticlopidine significantly inhibited the CYP2B6-catalyzed bupropion hydroxylation. Patients receiving either clopidogrel or ticlopidine are likely to require dose adjustments when treated with drugs primarily metabolized by CYP2B6.

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Drug metabolism profoundly affects drug action, because almost all drugs are metabolised in the body and thus their concentrations and elimination rates are dependent on metabolic activity. Drug metabolism contributes substantially to interindividual differences in drug response and is also often involved in drug interactions, resulting in either therapeutic failure or adverse effects. Knowledge about the metabolism of a new chemical entity and its affinity to drugmetabolising enzymes helps in the drug development process by providing important information for the selection of a lead compound from among a number of substances pharmacologically equally effective in their therapeutic response. In drug development protocols, metabolism characteristics should be assessed very early during the development process. This has been made possible by the advances made especially in analytical capabilities and in in vitro technologies that are employed to predict in vivo metabolite profile, pharmacokinetic parameters and drug-drug interaction potential.The principal route of elimination of drugs from the body is enzymatic biotransformation. The oxidative reactions are mainly catalysed by cytochrome P450 (CYP) enzymes (phase I metabolism) (Rendic & DiCarlo 1997;Guengerich & Rendic 2002) and, after that, by conjugating enzymes (phase II metabolism). Especially glucuronidation, catalysed by the several UDP-glucuronosyltransferase isoforms is an important route of phase II drug metabolism in humans. Some prodrugs need to be metabolically activated before they are pharmacologically active. This activation usually occurs via hydrolytic enzymes, or in some cases, by CYP enzymes.Many drug-metabolizing enzymes constitute extensive and complex families, even superfamilies, with many individual members exhibiting distinct, but often overlapping selectivities towards substrates and inhibitors. Furthermore, expression of most drug-metabolizing enzymes varies between individuals due to genetic, host and environmental factors and some diseases (Pacifici & Pelkonen 2001). These factors produce huge inter-individual variation in the rate and metabolic pathways of drugs. One example of genetic factors influencing the inter-individual and inter-population variation is the polymorphic expression of many P450 and glucuronosyl transferase enzymes in the population (Ingelman-Sundberg et al. 1999). The frequency of poor and ultrarapid metabolisers varies markedly between ethnic Author for correspondence: Olavi Pelkonen, Department of Pharmacology & Toxicology, University of Oulu, POB 5000, FIN-90014 Oulu, Finland (fax π358 8537 5247, e-mail olavi.pelkonen/oulu.fi).groups. Some external factors, such as dietary compounds, cigarette smoking, alcohol and drugs may cause induction or repression of the expression of certain P450s. However, in vitro systems for predicting induction are not covered here to any extent and a reader is referred to recent reviews (Pelkonen et al. 2002b;Honkakoski 2003). The need to assess metabolism in early drug d...

Hormone replacement therapy markedly inhibited the CYP2B6-catalyzed hydroxylation of bupropion, whereas a combination oral contraceptive had only a modest effect on CYP2B6 activity. Patients receiving hormone replacement therapy or oral contraceptives may need dose adjustment when treated with drugs metabolized by CYP2B6.

A sensitive and rugged LC/MSMS method was developed for a comprehensive in vitro metabolic interaction screening assay with N-in-1 approach reported earlier. A cocktail consisting of ten cytochrome P450 (CYP)-selective probe substrates with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8) tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were simultaneously analysed with a single LC/MSMS run. Altogether, 13 metabolites and internal standard phenacetin were analysed in multiple reaction mode. Polarity switching mode was utilized to acquire negative ion mode electrospray data for hydroxychlorzoxazone and positive ionization data for the rest of the analytes. Fast gradient elution was applied, giving total injection cycle of 8 min. The method was modified for two different LC/MSMS systems, and was validated for linear range, detection limit, accuracy and precision for each metabolite. In addition, cocktail inhibition system was further tested using monoclonal anti-CYP antibodies as inhibitors for each probe reaction.

Reproducibility of product ion spectra acquired using a liquid chromatography/triple-quadrupole mass spectrometry (LC/MS/MS) instrument over a 4-year period, and with three other LC/MS/MS instruments, one from the same manufacturer and two from a different manufacturer, was examined. The MS/MS spectra of 30 drug substances were generated in positive electrospray ionization mode at low, medium, and high collision energies (20, 35, and 50 eV). Purity and Fit score percentages against a 400-compound LC/MS/MS spectral library were calculated using an algorithm in which fragment intensity ratios and weighting factors were included. The long-term reproducibility study was conducted using a brand A instrument; after 4 years the reproducibility of the product ion spectra was still 94%, expressed as average Purity score. The inter-laboratory study involved two parts. Firstly, two LC/MS/MS spectral libraries, created independently in separate laboratories using brand A instruments, were compared with each other. The average Fit and Purity scores of spectra from one library against the other were better than 93 and 91%, respectively, when the same collision energies were used. Secondly, for the comparison of product ion spectra between brand A and brand B instruments, fragmentation conditions were first standardized for amitriptyline as the standard analyte. The average Fit scores of brand B spectra against the brand A spectral library varied between 79 and 85% at all three collision energies. These results indicate that, after standardizing the instrumental conditions, LC/MS/MS spectral libraries of drug substances are suitable for inter-laboratory use.

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