As our fledgling DMPK group looks to expand our portfolio of assays one of the main driving factors when evaluating a new method is the cost effectiveness of bringing a method in house as compared to employing a CRO. We run a microsome stability screen as one of a panel of assays in our initial triage of compounds which upon progression are then assessed in a broader range of (generally more expensive) assays including CYP isoform inhibition, MDCK-MDR1 and Hepatocyte stability among others. The average cost among four of the leading CROs offering the hepatocyte stability assay is ≈£600 per compound per species. To purchase cryo-preserved hepatocytes and run the assay in the same format as the CROs would be ≈£100 per compound per species, a significant saving but still an expensive assay. In addition, primary human hepatocytes (PHHs) are subject to inter-individual phenotypic variability; whilst suspensions of freshly isolated hepatocytes demonstrate near in-vivo levels of metabolic competence this is significantly reduced within 2-3 hours limiting the utility of assessing low metabolised compounds; rapid loss of canalicular and basolateral efflux transport in freshly isolated PHHs may also affect metabolic outcomes. The metabolic competence of PHHS is therefore not an intrinsic property but dependent upon the culture environment. Despite these drawbacks clearance intrinsic clearance in PHH suspensions is by the far the most widely used format and still represents the significantly more accurate indicator of intrinsic hepatic in vivo clearance when compared to data derived from rat/dog hepatocytes/allometric scaling or hepatocyte subcellular fractions.
The development of a more practical, stable, sustainable and less costly model of hepatocyte metabolism is therefore highly desired. The ideal alternative cell line for intrinsic clearance in suspension would therefore (apart from being cheaper than PHH) stably express the entire array of CYPs, Phase II enzymes and (as much at least as freshly isolated suspension PHH) transporter proteins at physiologically relevant levels. Immortalised and hepatoma derived cell lines including HepG2, Huh7 and Fa2N-4 have been investigated as such alternatives but all lack significant expression for large swathes of Liver-specific function. HepaRG cells demonstrate significant advantage over these cell lines and recent papers (1,2) suggest interest in using these cells in the pharmaceutical setting may be growing especially with the recent availability of pre-differentiated cryo-preserved HepaRG cells.
Originally described by Gripon et al (3) HepaRG cells are a hepatoma cell line that can be differentiated into a phenotype that closely resembles mature hepatocyte which exhibit cellular interactions, drug metabolism/transport, and drug induction responsiveness comparable to PHH cultures. After 6 weeks under differentiating conditions in culture (2 % DMSO, 50 µM hydrocortisone hemisuccinate) the expression of the main phase I & II enzymes and transport proteins averages between 20-100% of levels exhibited in freshly isolated PHH (with the notable exceptions of CYP7A1, GSTA1, MDR1, MPR1 which demonstrate a >5x increase (4)) but which closely mirror the levels expressed in sandwich/3D cultured PHH. CYPs 2C9, 2D6 and 3A5 also all contain poor metaboliser alleles but this reflects the typical Caucasian allotype of the original donor. Chr 22 (which encodes 2D6) also only has one copy in HepaRG cells contributing further to low expression levels. Additionally HepaRG cells demonstrate cytosolic sequestering of the transcription activator CAR and translocation to the nucleus upon activation by phenobarbital, a crucial PHH hallmark lacking in most hepatic cell lines.
Whilst differentiated HepaRG cells demonstrate a reasonable level of hepatic function and morphology after ≈10 days in culture (compared to cultured PHH) such that their utility as a model for hepatotoxicity has been clearly demonstrated (5) they still fall short of being a suitable substitute for performing intrinsic clearance studies in suspension. One problem is that the cells need to be maintained in 2% DMSO to enable adequate differentiation to a hepatocyte phenotype via activation by, for instance, AP-1 (induces cell cycle arrest and differentiation) which also exerts transcription regulation across a broad array of metabolic genes. Efforts are currently being made to develop methods to differentiate HepaRG cells without the use of DMSO (6) and with the emergence of CRISPR as a means to rapidly and easily modify genes (e.g. replacing low metabolic alleles) perhaps it won’t be too long before we have cost effective, stable, widely available and consistent source of cells with which to perform reliable Intrinsic clearance assays.
- Ferguson, S et al (2016) ‘Contextualising Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures’ Drug Metabolism and Disposition 44: 1463-1479
- Plevris, J et al (2016) ‘Human Hepatic HepaRG Cells Maintain an Organotypic Phenotype with High Intrinsic CYP450 Activity/Metabolism and Significantly Outperform Standard HepG2/C3A Cells for Pharmaceutical and Therapeutic Applications’ Basic & Clinical Pharmacology & Toxicology 15 JUL 2016, DOI: 10.1111/bcpt.12631
- Gripon, P et al (2002) ‘Infection of a Human Hepatoma Cell Line by Hepatitis B Virus’ Proceedings of the National Academy of Sciences of the United States of America, 99 (24): 15655–15660
- Andersson, T et al (2008) ‘Evaluation of HepaRG Cells as an in Vitro Model for Human Drug Metabolism Studies’ Drug Metabolism and Disposition 36 (7): 1444–1452
- Sakharov, D (2016) ‘Maintenance of High Cytochrome P450 Expression in HepaRG Cell Spheroids in DMSO-Free Medium’ Bulletin of Experimental Biology and Medicine 161 (1): 138-142
Blog written by Marcus Hanley