HepaRG™ can be used in various applications including in vitro ADME testing, inhibition assays, drug metabolism and clearance, cholestasis, virology (HBV, HCV) and viral infection. Below, a few examples of applications with references are listed.
Drug-induced liver injury (DILI) and drug-drug interactions (DDIs) are concerns when developing safe and efficacious compounds.
HepaRG cells have a full complement of drug metabolising enzymes (DME, Phase I and II), drug transporters and nuclear receptors (CAR, PXR, and AhR). So that, HepaRG cell model, recognized by the FDA (Guidance for inductry 2006), can be used as a single batch or donor of human hepatocytes for determining if the Investigational drug is an Inducer of metabolizing enzymes in vitro. In addition, the variation in induction responses was very high in primary human hepatocytes; whereas, values for HepaRG cells were reproducible. The CYP induction responses in HepaRG cells are well within the range exhibited by primary human hepatocytes and can therefore be considered to be representative of a typical primary human hepatocyte (PHH) donor that can be used in multiple induction experiments conducted over years to generate reproducible control data.
In Vitro Drug Interaction Studies-Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions. FDA Guidance for industry
Cholestasis is defined as the decrease or suppression of bile flow due to impaired secretion by hepatocytes or to obstruction of bile at any level of the excretory pathway. A key functional parameter for good bile flow from hepatocytes is contraction of the bile canaliculi. Evidence shows that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis. Models and methods for detecting and characterizing drug-associated cholestatic disorders have been attempted. One problem relates to finding suitable hepatic models for in vitro studies. Hepatocyte polarization with bile canalicular formation is a complex mechanism which includes cytoskeletal, tight junctional and intracellular trafficking components. HepaRG cells which express phases 1 and 2 drug metabolizing enzymes and transporters, and form bile canaliculi structures, can be used to mimic features of intrahepatic cholestasis induced by cholestatic drug treatment and to characterize the mechanisms involved in the initiation of the lesions. Since it has been demonstrated that 12 tested cholestatic drugs could cause bile canaliculi constriction and dilatation in HepaRG cell cultures associated with different disturbances of the ROCK/MLCK. All fluorescent marker compounds driven to the biliary pole by transporters can be used as well for High Content Screening with imaging analysis. Exemplary assay is performed using CDFDA as marker compound for detecting the bile canaliculi deformation resulting in constriction or dilatation of the canalicular lumen. All those data suggest that HepaRG cells represent a suitable, easy-to-use liver model for identification of drug-induced cholestasis. In addition, the early alterations of bile canaliculi dynamics associated with Rho kinase/myosin light chain kinase pathway are the new potential predictive biomarkers of drug-induced cholestasis using HepaRG.
Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs.. Sci Rep. 2016 May 12;6:24709.
Drug-induced liver injury (DILI) and drug-drug interactions (DDIs) are concerns when developing safe and efficacious compounds. HepaRG cells have a full complement of drug metabolising enzymes (DME, Phase I and II), drug transporters and nuclear receptors (CAR, PXR, and AhR). So that, HepaRG cell model, recognized by the FDA (Guidance for inductry 2006), can be used as a single batch or donor of human hepatocytes for determining if the Investigational drug is an Inducer of metabolizing enzymes in vitro. In addition, the variation in induction responses was very high in primary human hepatocytes; whereas, values for HepaRG cells were reproducible. The CYP induction responses in HepaRG cells are well within the range exhibited by primary human hepatocytes and can therefore be considered to be representative of a typical primary human hepatocyte (PHH) donor that can be used in multiple induction experiments conducted over years to generate reproducible control data.
In vitro assessment of mitochondrial toxicity to predict drug-induced liver injury. Drug-Induced Liver Toxicity pp 283-300
Drug-induced liver injury (DILI) is a major cause of attrition during both early and late stages of drug development and a leading reason for drug withdrawal in postmarketing. Hepatocytes constitute approximately 80% of total liver mass making drug-induced hepatotoxicity a serious concern for the pharmaceutical industry.
Current models to predict hepatotoxicity in preclinical stages have shown many limitations. Interspecies differences in expression and activities of enzymes and transporters involved in drug metabolism and pharmacokinetics are a major limitation of animal models. The use of primary human hepatocytes, the gold standard for studying hepatotoxicity, is limited by the lack of availability, the interdonor variability and the rapid loss of hepatic functions that makes them unsuitable for long term hepatotoxicity prediction. Although 3D cultures retain their molecular phenotypes and hepatic functions for multiple weeks in culture, their predictive capacity of hepatotoxicants has not been fully assessed and their use for high throughput screening is not yet optimized.
Differentiated HepaRG cells express the main hepatic functions, including phase I and II enzymes, transporters, and nuclear receptors at levels comparable to those found in primary human hepatocytes and maintain this expression almost stable over 4 weeks. The ability of HepaRG cells to remain differentiated in culture over extended period give this model a definite advantage of studying the delayed onset of hepatotoxicity. HepaRG cells represent a unique metabolically competent cell model for in vitro chronic toxicity studies. This model has proven high sensitivity, specificity, and accuracy of flagging human hepatotoxic compounds. The utility and performance of HepaRG are particularly highlighted in the investigation of delayed hepatotoxicity.
The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine. Toxicol Appl Pharmacol. 2020 Sep 15, 403:115163
BSEP (bile salt export pump), located on the canalicular membrane of hepatocytes, is the major efflux transporter for the secretion of bile acids (BA) from hepatocytes into bile in humans. Because that bile secretory failure via BSEP inhibition can lead to cholestatic DILI in humans, the European Medicines Agency Guideline on the Investigation of Drug Interactions (2012) recommends in vitro screening of BSEP inhibition!. Differentiated HepaRG hepatocytes are highly polarized cells exhibiting specialized apical (canalicular) and sinusoidal (basolateral) domains. As well HepaRG cells express canalicular transporters, including BSEP, MRP2, MDR1, and MDR3. Therefore, HepaRG cells could be useful to mechanistically better understand drug-induced alterations of BA influx and efflux. In addition, two novel bile acid fluorescent probed are synthesized by Starlight which are potential specific for BESP transporter. The inhibition of BSEP in HepaRG cells by incubation of candidate drugs and BA fluorescent probes result in a decreased accumulation of BA fluorescent probes inside the bile canaliculi networks in HepaRG cells. Therefore, measurement of BSEP inhibition on the efflux of fluoresence probes by candidate drugs allow to predict BSEP-drug candidate interactions. HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.
Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes. Toxicol Sci. 2015, 145(1): 157–168.
A new proof of principle for the use of HeapRG-HSCs to generate complex in vitro spheroid cultures that better mimic the complexity of the liver as well as liver function. In this 3D co-culture, HepaRG-HSCs promote maintenance of hepatocyte metabolic functionality while being able to respond to hepatocyte-mediated toxicity, activating and promoting intra-spheroid fibrogenesis, one of the main drug-associated adverse liver outcomes. Since the potential use of HepaRG/HSC spheroids for the assessment of fibrogenesis is evaluated by exposure of the spheroids to TGF-b and thioacetamide (TAA) and measure of the expression of fibrogenic markers and secretion of pro-collagen type I and increased phalloidin and collagen staining. This relevant human hepatic organoid model is suitable for testing drug-induced liver fibrosis and liver fibrosis modeling in vitro.
Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro. Biomaterials, 2016, 78, 1-10
The human liver cell line HepaRG, after differentiation, resembles PHHs with respect to many hepatocyte-specific markers including the expression of cytochrome P450 enzymes, liver-specific transcription factors, and transporter proteins like the HBV-specific receptor, sodium taurocholate co-transporting polypeptide (NTCP). So far, the HepaRG cell line is the only one allowing both studies on HBV/HDV infection and liver-specific drug toxicity and metabolism. So that HepaRG cell line is used as a suitable in vitro cell culture systems for investigations of virus-host interactions becouse of its efficiently support virus infection.
Hepatitis B Virus Infection of HepaRG Cells, HepaRG-hNTCP Cells, and Primary Human Hepatocytes. Hepatitis B Virus pp 15-25
FADU assay is designed for automated genotox risk assessment via DNA strand break analysis in one 96-well plate of adherent HepaRG cell culture. The DNA damage detection id based on progressive DNA unwinding under highly controlled conditions of alkaline pH, time and temperature. A fluorescent dye id used as marker for double stranded DNA and decrease in the flurescence intensity indicates an increase in DNA unwinding and consequently a greater number of DNA straind breaks. Controls are performed in parallel with the experimentally treated cells.
The automated FADU-assay, a potential high-throughput in vitro method for early screening of DNA breakage.. ALTEX. 2011;28(4):295-303