A 96-well plate assay for CYP4503A induction using cryopreserved human hepatocytes.
ABSTRACT A reliable and practical CYP3A induction assay with cryopreserved human hepatocytes in a 96-well format was developed. Various 96-well plates with different basement membrane were evaluated using prototypical inducers, rifampicin, phenytoin, and carbamazepine. Thin-layer (TL) Matrigel was found to yield the highest basal and induced levels of CYP3A activity as determined by testosterone 6β-hydroxylation. Concentration-dependent CYP3A induction of rifampicin was reproducible with the EC(50) values of 0.36 ± 0.28 μM from four batches of human hepatocytes using the 96-well plate with TL Matrigel. The rank order of induction potency for nine inducers or noninducers at a concentration of 10 μM were well comparable among the multiple donors, by expressing the results as percentage of change compared with the positive control, 10 μM rifampicin. Cotreatment of avasimibe or efavirenz with 10 μM rifampicin was found to reduce CYP3A activities induced by rifampicin at a lower rate than treatment with rifampicin alone, whereas treatment with phenobarbital and carbamazepine had no effect. From a comparison of induced CYP3A activities and gene expression levels, there were compounds that would cause induction of CYP3A4 mRNA but not activity, presumably due to their inhibitory effect on CYP3A activity. The cotreatment assay of test compound with rifampicin allows us to exclude the false-negative results caused by the cytotoxicity and/or the mechanism-based inactivation, when the drug candidate's ability for CYP3A induction is evaluating the enzyme activity. This 96-well plate assay, which is robust, reproducible, and convenient, has demonstrated the paramount applicability to the early drug discovery stage.
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ABSTRACT: Rilpivirine and etravirine are second generation non-nucleoside reverse transcriptase inhibitors approved recently by the United States Food and Drug Administration for the treatment of human immunodeficiency virus-1 infection. Pregnane X receptor (PXR) is a member of the superfamily of nuclear receptors that regulate the expression of various genes controlling diverse biological functions. The present study investigated the effects of rilpivirine and etravirine on the activity of human PXR (hPXR), including the mode of activation, and compared them to those of efavirenz, nevirapine, and delavirdine, which are first generation non-nucleoside reverse transcriptase inhibitors. In transiently transfected HepG2 cells, rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, activated human, mouse, and rat PXR. Results from mechanistic studies indicated that rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, bound to the ligand-binding domain of hPXR, as assessed by a transactivation assay and by a competitive ligand-binding assay using time-resolved fluorescence resonance energy transfer; triggered nuclear translocation of a green fluorescence protein-tagged hPXR, as visualized by confocal imaging; and recruited steroid receptor coactivator-1 (SRC-1), SRC-2, and SRC-3 to hPXR, as demonstrated by mammalian two-hybrid assays. Rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, increased hPXR target gene (CYP3A4) expression in primary cultures of human hepatocytes. In summary, select non-nucleoside reverse transcriptase inhibitors activated human and rodent PXR. Rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, were identified as agonists of hPXR, as assessed in mechanistic experiments, and inducers of CYP3A4, as determined in primary cultures of human hepatocytes.Biochemical pharmacology 04/2013; · 4.25 Impact Factor
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ABSTRACT: Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of the current study was to find out mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100μM) was cytotoxic only after CYP-induction by rifampicin. Clopidogrel (10 and 100μM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100μM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10µM and 100µM) in HepG2/CYP3A4, but not in HepG2 cells/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10µM and 100µM) in HepG2/CYP3A4 supersome. Co-incubation with 1μM ketoconazole or 10mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 or HepG2/CYP3A4 supersome, respectively. HepG2/CYP3A4 incubated with 100μM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites which are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.Free Radical Biology & Medicine 06/2013; · 5.27 Impact Factor
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ABSTRACT: Abstract 1. Human hepatocytes that had been cold-preserved in SureTran(TM) matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012). 2. After 5 d of cold preservation, the viability was still more than 70%, and after 8 d it was around 60%. In hepatocytes that had been cold-preserved for 3 d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8 µM rifampicin for 72 h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3 d and thereafter treated with 1 mM phenobarbital for 72 h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3 d, while the activity increased in cells treated with 0.3-25 µM β-naphthoflavone for 72 h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers. 3. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2 d. 4. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.Xenobiotica 04/2013; · 1.98 Impact Factor