Predictive Utility of In Vitro Rifampin Induction Data Generated in Fresh and Cryopreserved Human Hepatocytes, Fa2N-4, and HepaRG Cells

Templeton, Ian E.; Houston, J. Brian; Galetin, Aleksandra

doi:10.1124/dmd.111.040824

Cited by 18 publications

(14 citation statements)

References 41 publications

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“…8B). Induction of CYP2B6 in response to indirect activators (i.e., PB and phenytoin) has dmd.aspetjournals.org not been observed with any immortalized hepatic cell lines (i.e., HepG2, Huh-7, Fa2N-4) to date, which has been attributed to insufficient expression and cell-signaling functionality to sequester CAR within the cytosol or translocate it to the nucleus (Sueyoshi et al, 2008;Templeton et al, 2011). CYP2B6 induction in cryo-HepaRG cultures (three independent lots) was further contextualized against the range of responses observed in SC-PHH and showed comparable (and substantially less variable) CYP2B6 induction in response to PB (Fig.…”

Section: Active Uptake Transport Characterizationmentioning

confidence: 99%

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

Jackson

Chamberlain

et al. 2016

Drug Metabolism and Disposition

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Over the last decade HepaRG cells have emerged as a promising alternative to primary human hepatocytes (PHH) and have been featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG cells that require timeconsuming culture (∼28 days) for full differentiation. Recently, a cryopreserved, predifferentiated format of HepaRG cells (termed here "cryo-HepaRG") has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether HepaRG cells in this format fully retain their hepatic characteristics. Therefore, we systematically investigated the hepatocyte functionality of cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich-culture and suspension formats. These evaluations uncovered a novel adaptation period for the cryo-HepaRG format and demonstrated the impact of extracellular matrix on cryo-HepaRG functionality. Pharmacologically important drug-metabolizing alleles were genotyped in HepaRG cells and poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor activators comparable to that of sandwich-cultured PHH. Finally, we show for the first time that cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations for the use of this cell model and demonstrate that cryo-HepaRG are suitable for metabolism and toxicology screening.

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Section: Active Uptake Transport Characterizationmentioning

confidence: 99%

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

Jackson

Chamberlain

et al. 2016

Drug Metabolism and Disposition

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“…Among these alternatives, the human HepaRG cell line is one of the most suitable human hepatic cell lines due to the retention of key liver functionality (Kanebratt and Andersson, 2008;Turpeinen et al, 2009;Templeton et al 2011). This model is considered useful for the evaluation of DDIs as most of the common CYP isoform activities have been measured in this cell line and shown to be both selectively inhibited and induced by prototypical CYP-selective inhibitors and inducers at comparable levels to those of primary cultures of human hepatocytes (Turpeinen et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

“…Predictive mathematical models incorporating either induction alone or induction in combination with inhibition mechanisms have been applied by a number of authors (Fahmi et al, 2008b;Shou et al, 2008;Fahmi and Ripp, 2010;Kirby et al, 2011;Templeton et al 2011). Dynamic models based on an inducer concentration-time profile to account for the change in enzyme expression have also been proposed (Almond et al, 2009;Fahmi et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Normalization Methods To Predict CYP3A4 Induction in Six Fully Characterized Cryopreserved Human Hepatocyte Preparations and HepaRG Cells

Vermet

Raoust

Ngo

et al. 2015

Drug Metabolism and Disposition

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Prediction of drug-drug interactions due to cytochrome P450 isoform 3A4 (CYP3A4) overexpression is important because this CYP isoform is involved in the metabolism of about 30% of clinically used drugs from almost all therapeutic categories. Therefore, it is mandatory to attempt to predict the potential of a new compound to induce CYP3A4. Among several in vitro-in vivo extrapolation methods recently proposed in the literature, an approach using a scaling factor, called a d factor, for a given hepatocyte batch to provide extrapolation between in vitro induction data and clinical outcome has been adopted by leading health authorities. We challenged the relevance of the calibration factor determined using a set of 15 well-known clinical CYP3A4 inducers or the potent CYP3A4 inducer rifampicin only. These investigations were conducted using six batches of human hepatocytes and an established HepaRG cell line. Our findings show that use of a calibration factor is preferable for clinical predictions, as shown previously by other investigators. Moreover, the present results also suggest that the accuracy of prediction through calculation of this factor is sufficient when rifampicin is considered alone, and the use of a larger set of fully characterized CYP3A4 clinical inducers is not required. For the established HepaRG cell line, the findings obtained in three experiments using a single batch of cells show a good prediction accuracy with or without the d factor. Additional investigations with different batches of HepaRG cell lines are needed to confirm these results.

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“…In the 48-h induction studies (Table 3), DMSO-treated HuH-7 cells showed minimal induction by both rifampicin and dexamethasone compared to HepaRG cells. These results suggested that HuH-7 cells may not be an ideal model for CYP3A4 induction compared with other in vitro models such as primary human hepatocytes and HepaRG cells (Gerets et al 2012; Templeton et al 2011). …”

Section: Discussionmentioning

confidence: 96%

Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

et al. 2015

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A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1% dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC50 within 2.5 fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7 cells, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity.

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Predictive Utility of In Vitro Rifampin Induction Data Generated in Fresh and Cryopreserved Human Hepatocytes, Fa2N-4, and HepaRG Cells

Cited by 18 publications

References 41 publications

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures

Evaluation of Normalization Methods To Predict CYP3A4 Induction in Six Fully Characterized Cryopreserved Human Hepatocyte Preparations and HepaRG Cells

Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

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