Marlies Oorts

Drug-induced cholestasis detection in cryopreserved rat hepatocytes in sandwich culture.

INTRODUCTION In vitro identification of compounds that cause cholestasis in vivo still remains a problem in pharmaceutical R&D. Currently existing in vitro systems show poor predictivity towards the clinical situation. Recently, our research group developed a model, based on sandwich-cultured (rat) hepatocytes (SC(R)H), to detect compounds causing cholestasis via altered bile acid (BA) homeostasis (Chatterjee et al., 2014). In the present study, we assessed whether this model performs equally well with freshly-isolated and cryopreserved hepatocytes. METHODS We exposed sandwich cultures from rat hepatocytes before and after cryopreservation to the cholestatic compounds, cyclosporin A (CsA) and troglitazone (Tro), in the presence and in the absence of a BA mixture. At the end of the incubations, the capability of the hepatocytes to produce urea was measured to determine changes in the drug-induced cholestasis index (DICI). RESULTS The mean (± SEM) urea production was significantly higher in sandwich cultures from freshly-isolated hepatocytes (27.88 (± 0.96) nmol/cm(2)), compared to cultures from cryopreserved hepatocytes (22.86 (± 1.91) nmol urea/cm(2)). However, after normalization for confluence rate (based on light microscopic image analysis), it appeared that the urea production was similar for all the batches of SCRH. The mean (± SEM) DICI values for CsA 10 μM and Tro 75 μM were 0.89 (± 0.03) and 0.93 (± 0.03), respectively. Higher concentrations, CsA (≥ 15 μM) and Tro (≥ 100 μM), elicited a significant decrease in urea production when incubated in the presence of a BA mixture compared to the compound alone. This was the case for all the batches of SCRH, irrespective of cryopreservation history. DISCUSSION In conclusion, no significant differences were seen when the previously described in vitro cholestasis model was applied in SCRH before or after cryopreservation. This study demonstrates the robustness of the model, which implies that it can be used with SCRH obtained from both freshly-isolated and cryopreserved hepatocytes.

Primary Hepatocytes in Sandwich Culture

Hepatocytes in sandwich configuration constitute of primary hepatocytes cultured between two layers of extracellular matrix. Sandwich-cultured hepatocytes maintain expression of liver-specific proteins and gradually form intact bile canaliculi with functional biliary excretion of endogenous compounds and xenobiotics. Both freshly isolated and cryopreserved hepatocytes can be used to establish sandwich cultures. Therefore, this preclinical model has become an invaluable in vitro tool to evaluate hepatobiliary drug transport, metabolism, hepatotoxicity, and drug interactions. In this chapter, commonly used procedures to cultivate primary hepatocytes from human and rat in sandwich configuration are described.

MRP2 Inhibition by HIV Protease Inhibitors in Rat and Human Hepatocytes: A Quantitative Confocal Microscopy Study

Hepatic drug transporters play a pivotal role in the excretion of drugs from the body, in drug-drug interactions, as well as in drug-induced liver toxicity. Hepatocytes cultured in sandwich configuration are an advantageous model to investigate the interactions of drug candidates with apical efflux transporters in a biorelevant manner. However, the commonly used “offline” assays (i.e., that rely on measuring intracellular accumulated amounts after cell lysis) are time- and resource-consuming, and the data output is often highly variable. In the present study, we used confocal microscopy to investigate the inhibitory effect of all marketed HIV protease inhibitors (10 μM) on the apical efflux transporter multidrug resistance–associated protein 2 (MRP2; ABCC2) by visualizing the biliary accumulation of the fluorescent substrate 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF). This method was applied with sandwich-cultured human and rat hepatocytes. Alterations in the biliary excretion index of CDF were calculated on the basis of quantitative analysis of fluorescence intensities in the confocal images. In human hepatocytes, lopinavir followed by tipranavir, saquinavir, atazanavir, and darunavir were the most potent inhibitors of MRP2-mediated efflux of CDF. In rat hepatocytes, tipranavir inhibited Mrp2-mediated CDF efflux most potently, followed by lopinavir and nelfinavir. In conclusion, a comparison of these findings with previously published data generated in offline transporter inhibition assays indicates that this microscopy-based approach enables investigation of the inhibitory effect of drugs on efflux transporters in a very sensitive but nondestructive manner.

Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury.

Relationship between in vivo CYP3A4 activity, CYP3A5 genotype and systemic tacrolimus metabolite/parent drug ratio in renal transplant recipients and healthy volunteers.

CYP3A5 genotype is a major determinant of tacrolimus clearance, and has been shown to affect systemic tacrolimus metabolite/parent ratios in healthy volunteers, which may have implications for efficacy and toxicity. In a cohort of 50 renal transplant recipients who underwent quantification of CYP3A4 activity using the oral midazolam drug probe, we confirmed that CYP3A5 genotype is the single most important determinant of tacrolimus metabolite/parent ratio [CYP3A5 expressors displayed 2.7- and 2-fold higher relative exposure to 13-desmethyltacrolimus (DMT) and 31-DMT, respectively; P < 0.001]. There was, however, no relationship between CYP3A4 activity and tacrolimus metabolite/parent ratios. Additional analyses in 16 healthy volunteers showed that dual pharmacological inhibition of CYP3A4 and P-glycoprotein using itraconazole resulted in increased tacrolimus metabolite/parent ratios (+65%, +112%, and 25% for 13-, 15-, and 31-DMT, respectively; P < 0.01). This finding was confirmed in a cohort of nine renal transplant recipients who underwent tacrolimus pharmacokinetic assessments before and during CYP3A4 inhibition (58% increase in overall metabolite/tacrolimus ratio; P = 0.017).