In vitro Phase I- and Phase II-Drug Metabolism in The Liver of Juvenile and Adult Göttingen Minipigs.

PURPOSE: In view of pediatric drug development, juvenile animal studies are gaining importance. However, data on drug metabolizing capacities of juvenile animals are scarce, especially in non-rodent species. Therefore, we aimed to characterize the in vitro biotransformation of four human CYP450 substrates and one UGT substrate in the livers of developing Göttingen minipigs. METHODS: Liver microsomes from late fetal, Day 1, Day 3, Day 7, Day 28, and adult male and female Göttingen minipigs were incubated with a cocktail of CYP450 substrates, including phenacetin, tolbutamide, dextromethorphan, and midazolam. The latter are probe substrates for human CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. In addition, the UGT multienzyme substrate (from the UGT-GloTM assay), which is glucuronidated by several human UGT1A and UGT2B enzymes, was also incubated with the porcine liver microsomes. RESULTS: For all tested substrates, drug metabolism significantly rose postnatally. At one month of age, 60.5 and 75.4% of adult activities were observed for acetaminophen and dextrorphan formations, respectively, while 35.4 and 43.2% of adult activities were present for 4-OH-tolbutamide and 1'-OH-midazolam formations. Biotransformation of phenacetin was significantly higher in 28-day-old and adult females compared with males. CONCLUSIONS: Maturation of metabolizing capacities occurred postnatally, as described in man.

In vitro studies on the metabolism of trabectedin (YONDELIS) in monkey and man, including human CYP reaction phenotyping.

Trabectedin (YONDELIS) is a potent anticancer agent which was recently approved in Europe for the treatment of soft tissue sarcoma. The drug is currently also in clinical development for the treatment of ovarian carcinoma. In vitro experiments were conducted to investigate the hepatic metabolism of [(14)C]trabectedin in Cynomolgus monkey and human liver subcellular fractions. The biotransformation of trabectedin was qualitatively similar in 12,000 x g supernatants of both species, and all human metabolites were also produced by the monkey. The trabectedin metabolites were identified by QTOF mass spectrometry, and HPLC co-chromatography with reference compounds. Trabectedin was metabolized via different biotransformation pathways. Most of the metabolic conversions occurred at the trabectedin A domain including mono-oxidation and di-oxidation, carboxylic acid formation with and without additional oxidation, and demethylation either without (N-demethylation to ET-729) or with additional mono-, di- or tri-oxidation. Another metabolite resulted from O-demethylation at the trabectedin C subunit, and in addition, aliphatic ring opening of the methylene dioxybridge at the B domain was detected. Overall, demethylation and oxidation played a major role in phase I metabolism of the drug. Human cDNA expressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4 and 3A5 in E. coli membranes, but not CYP1B1, 2C19, and 4A11 were able to metabolize [(14)C]trabectedin. Experiments with chemical inhibitors and CYP inhibitory antibodies indicated that, at therapeutic levels, CYP3A4 is the main human CYP isoform involved in trabectedin's hepatic metabolism. In monkey and human liver microsomes, trabectedin was not substantially metabolized by glucuronidation.

The ontogeny of drug metabolizing enzymes and transporters in the rat.

Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is important for adequate interpretation of the findings during safety studies in juvenile animals. The present study was designed to collect information on plasma concentrations of total protein and albumin, enzyme activity and mRNA expression of cytochrome P450 isoenzymes (CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1), carboxylesterase and thyroxin glucuronidation (T4-GT) activity in liver microsomes, and mRNA expression of transporters (Mdr1a/b, Mrp1-3 and 6, Bsep and Bcrp, Oct1-2, Oat1-3 and Oatp1a4) in liver, kidney and brain tissue during development in Sprague-Dawley rats. Enzyme activities were determined by measuring the metabolism of marker substrates; expression of mRNAs was assessed using RTq-PCR. There were considerable differences in the ontogeny of the individual cytochrome P450 isoenzymes. In addition, ontogeny patterns of enzyme activity did not always parallel ontogeny patterns of mRNA expression. Ontogeny of the transporters depended on the transporter and the organ studied. Changes in mRNA expression of the various transporters during development are likely to result in altered elimination and/or tissue distribution of substrates, with concomitant changes in hepatic metabolism, renal excretion and passage through the blood-brain barrier. Consideration of the ontogeny of metabolizing enzymes and transporters may improve the design and interpretation of results of toxicity studies in juvenile animals.

Expression and induction potential of cytochromes P450 in human cryopreserved hepatocytes.

Fresh human hepatocytes are still considered as the "gold standard" to screen in vitro for cytochrome P450 (P450) induction. However, sparse availability of good quality human liver tissue for research purposes and the demand for standardized cell populations, together with the need for proper storage of the cells not immediately required, have resulted in the development of cryopreservation techniques that provide adequate viability and plateability of hepatocytes after thawing. This study aimed at validating cryopreserved human hepatocytes as a model to investigate P450 induction. Cryopreserved cells from four different donors were plated and cultured for 48 h, followed by incubation in the presence of typical P450 inducers. During the experiments, quality of the cultured cells was monitored both physiologically and morphologically. Concomitantly, the activity of CYP1A2, 2B6, 2C9, 2E1, and 3A4 was measured together with their mRNA and protein expression. Determination of CYP1A2, 2B6, 2C9, 2E1, and 3A4 activity in control versus prototypical inducer-treated hepatocytes revealed a maximal significant mean 11.6-, 2.8-, 1.9-, 1.5-, and 9.0-fold induction over their basal expression, respectively. Protein expression analysis of these P450s confirmed these results. Moreover, a mean 44.9-, 3.5-, 3.2-, and 13.8-fold induction of CYP1A2, 2B6, 2C9, and 3A4 mRNA was observed. Our data demonstrate that cryopreserved human hepatocytes are a valuable tool to study the induction of CYP1A2, 2B6, 2C9, 2E1, and 3A4.