HepaRG™ cells express multiple functional Phase 1 and 2 drug metabolising enzyme (DME) activities at comparable levels to those in cultures of primary human hepatocytes (Table 1).
The high batch-to-batch reproducibility of DME levels allows for routine high throughput analysis of compound clearance. Moreover, the sustained presence of DMEs over weeks (Guillouzo et al., Chem Biol Interact., 2007) makes HepaRG™ ideal for measuring the clearance of low clearance compounds.
Table 1. Phase 1 and 2 drug metabolising enzyme activities in HepaRG™ and primary human hepatocytes.
Activities are nmole metabolite/h/mg protein; Adapted from Gripon et al., PNAS 2002
Using HepaRG™ cells and standard high-throughput methodology, the prediction of in-vivo intrinsic clearance of the majority of a set of 26 drugs was mostly within 2-fold of the observed values (Lübberstedt et al., J Pharmacol Toxicol Methods, 2010; Zanelli et al., DMD, 2012). The greater under-prediction of more highly cleared compounds by HepaRG™ cells is also characteristic for primary human hepatocytes and is thought to be an inherent feature of cell-based clearance models. Indeed, the correlation between the intrinsic clearance in HepaRG™ cells and primary human hepatocytes was close to unity, confirming that overall metabolism of the substrates was comparable in both cell types. HepaRG™ cells are polymorphic for CYP2C9, which is reflected in the clearance of its corresponding substrates (e.g. tolbutamide for CYP2C9 (shown in the figure 1). Despite being polymorphic for CYP2D6, the in-vivo clearance of drugs metabolised mainly by this CYP was only under-predicted by 3-fold.
These findings support the use of HepaRG™ cells in high-throughput clearance screening assays.
HepaRG™ cells have a full complement of DMEs, cofactors and transporter proteins and metabolites produced in human hepatocytes can also be generated in HepaRG™ cells (e.g. aflatoxin B1, as shown in the figure 2). As such, HepaRG™ cells can be considered as a single batch or donor of human hepatocytes.
Together with the high stability of the enzymes, this makes these cells ideal for producing relevant drug metabolites in sufficient quantities to enable metabolite identification.
Transcription factors and nuclear receptors responsible for regulating DME and transporters (e.g. AhR, CAR, PXR, and PPARa) are highly expressed in HepaRG™ cells, enabling them to respond to prototypical CYP inducers. Maximal induction of CYP mRNAs peak at 24 h of treatment and include CYP1A1/2, CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 (Kanebratt and Andersson, DMD, 2008).
As with primary hepatocytes, CYP2D6 is not induced in HepaRG™ cells. Detection of induction of CYP activities after 48 h is also possible and shows that the entire process of DME production - from transcription factor activation, to mRNA and protein synthesis and post-translational modification – takes place in these cells. The CYP induction responses are stable over one month and the inter-batch reproducibility is very good. Notably, the nuclear receptor, CAR, has been shown to be present and functional, since incubation of HepaRG™ cells with the CAR specific inducer, CITCO, induces CYP2B6 mRNA and activities (See Figure 3). This is particularly important because some cell lines lack this nuclear receptor and therefore, unlike HepaRG™ cells, are not suitable for the type of CYP induction assays recommended by the FDA and EMA.
A study by Yajima et al., (DMD, 2014) reported the variability of CYP1A2, CYP2B6 and CYP3A4 induction responses across 15 different batches of primary human hepatocytes and compared them with those in 3 different batches of HepaRG™ cells. Unsurprisingly, the variation in induction responses was very high in primary human hepatocytes; whereas, values for HepaRG™ cells were reproducible (Table 2). The CYP induction responses in HepaRG™ cells were well within the range exhibited by primary human hepatocytes and can therefore be considered to be representative of a typical primary hepatocyte donor that can be used in multiple induction experiments conducted over years to generate reproducible control data.
Table 2. CYP induction responses in HepaRG cells and primary human hepatocytes.
Values are fold induction of CYP activities, mean ± SD, ranges are in parentheses. Adapted from Yajima et al., (DMD, 2014)
Prediction models such as the “Relative Induction Score” (“RIS”, employing induction EC50 and Maximum fold induction values) and F2 values (the F2 is the concentration resulting in a 2-fold induction (see figure 4)) have been employed successfully with HepaRG™ cells to predict in-vivo CYP3A4 induction. EC50 values for CYP3A4 induction in HepaRG™ cells treated with rifampicin (0.42 µM for mRNA) correlated well with those reported in primary human hepatocytes and PXR reporter gene assays (Kaneko et al., Xenobiotica, 2009, Kanebratt and Andersson, DMD, 2008). A calibration curve for HepaRG™ cells using known potent, mild and non-inducers of CYP3A4 can be used to compare the in-vitro potency of test compounds and therefore the potential to cause in-vivo CYP3A4 induction. Using these models, excellent correlations between the in-vitro and in-vivo induction of CYP3A4 by selected compounds have been demonstrated (Kanebratt KP, Andersson, DMD 2008; Grime et al., Curr Drug Metab 2010; Templeton et al., DMD, 2011).
The validation study for CYP induction using HepaRG™ cells is complete and the report is currently undergoing ESAC review. We are hopeful that the ESAC opinion will be delivered by September. We intend to issue a draft EURL ECVAM Recommendation on the methods for consultation with our regulatory advisory network (PARERE), our stakeholder forum (ESTAF) and partners of the International Cooperation on Alternative Testing Methods (ICATM).
In addition, we are preparing a draft OECD Test Guideline, which we hope will be submitted to the OECD in time for it to be considered at the next WNT meeting in April 2015.
The high levels of DMEs in HepaRG™ cells makes them ideal for inhibition screening studies.
The IC50 values of a panel of CYP inhibitors using HepaRG™ cells correlated very well with those determined in primary human hepatocytes (Table 3, Turpeinen et al., TIV, 2009), suggesting that HepaRG™ cells represent a promising model for this important endpoint. Time-dependent CYP inhibition can also be investigated using these cells. This time-dependent effect has been demonstrated in HepaRG™ cells for ritonavir, which both induces CYP3A mRNA and inhibits CYP3A4 activity (Grime et al., Curr Drug Metab, 2010).
Table 3. Comparison of the CYP selective inhibition in HepaRG™ cells and primary human hepatocytes (PHH)
Adapted from Turpeinen et al., TIV, 2009
It is important to assess the drug-drug interaction (DDI) potential of drugs acting on uptake and efflux transporters since they can affect the distribution and excretion of co-administered drugs.
HepaRG™ cells express substantial levels of uptake and efflux drug transporters (see Table 4) and form tight junctions and bile canaliculi, making them ideal for uptake and biliary secretion studies. Like CYPs, transporters are regulated by transcription factors, as shown in HepaRG™ cells by the induction of MDR1, MRP2 and BSEP expression by rifampicin, phenobarbital and chenodeoxycholic acid, respectively. The presence of efflux transporters is best demonstrated by staining the bile canaliculi for p-glycoproteins (Figure 5). After only a few hours in culture, both HepaRG™ cells and human hepatocytes form round pockets which are intensely stained for P-glycoprotein. The active efflux of basolateral transports can be visualised using the probe, CDFA. This ester is cleaved in cells to become fluorescent and the fluorescent metabolite is subsequently removed from the cell into the bile canaliculi via the MRP2 transporter.
Transporter studies “highlight the potential interest of using HepaRG™ cells for in vitro pharmacological and toxicological studies.” (Le Vee et al., Eur J Pharmaceut Sci 2006)
Figure 5. HepaRG™ cells form polarised structures with bile canaliculi identical to primary human hepatocytes with functional efflux properties (A). The function of the MRP2 transporter is demonstrated using the probe, CDFA, which is metabolised to a fluorescent green product and pumped from the cell via MRP2 into bile canaliculi (B).
Figure 1. Comparison of CLint in HepaRG™ cells and human hepatocytes
The solid line is the line of identity; the dashed line is the line of regression. Adapted from Zanelli et al., DMD, 2012.
Figure 2: Aflatoxin B1 metabolic profile in (A) HepaRG™ cells and (B) human hepatocytes
Adapted from Aninat et al., DMD 2006.
(A) HepaRG™ cells
(B) Primary Human Hepatocytes
Figure 3. Concentration-dependent induction of CYP mRNA in HepaRG™ cells
Figure 4. Correlation of AUC/F2 of CYP3A4 mRNA induction in HepaRG™ cells with in-vivo induction (% decrease in AUC of CYP3A4 probe drugs).
Adapted from Kanebratt and Andersson, DMD, 2008
Table 4. A comparison of uptake and efflux transporter expression in different cell types
Adapted from Le Vee et al., Eur J Pharmaceut Sci 2006