Assessment of Hepatotoxicity Potential of Drug Candidate Molecules Including Kinase Inhibitors by Hepatocyte Imaging Assay Technology and Bile Flux Imaging Assay Technology
Abstract:Kinases are members of a major protein family targeted for drug discovery and development. Given the ubiquitous nature of many kinases as well as the broad range of pathways controlled by these enzymes, early safety assessments of small molecule inhibitors of kinases are crucial in identifying new molecules with sufficient therapeutic window for clinical development. Failure or attrition of drug candidates in late-stage pipelines due to hepatotoxicity is a significant challenge in the drug development field. H… Show more
“…The bile canaliculi were stained by cholyl lysyl fluorescein (CLF), a substrate for BSEP. The clinical development of CP-724,714, a small molecule Her2 inhibitor for oncology indications, was stopped in phase 2 due to jaundice and cholestatic liver damage (for further experimental details, refer to Xu et al 2012)Fig. 48
Left : Hepatocytes on collagen (I) spot after 5 days in culture (from Jones et al 2009).…”
Section: Use Of In Vitro Systems For Predicting Liver Toxicitymentioning
confidence: 99%
“…ROS generation and GSH depletion as occurs during oxidative stress, mitochondrial membrane potential change as occurs during mitochondrial injury, inhibition of BSEP as occurs during drug-induced intrahepatic cholestasis (Boelsterli 2003). At a practical level, these phenotypic screens further employed a panel of optimized fluorescent probes to measure these modes of action specifically (Xu et al 2012). The concept of phenotypic screens can be extended to include other toxicologically relevant end points by using additional fluorescent or other markers to probe other pathophysiological processes including steatosis, phospholipidosis, apoptosis, endoplasmic reticulum (ER) stress, protein trafficking and transport dysfunctions.…”
Section: Use Of In Vitro Systems For Predicting Liver Toxicitymentioning
confidence: 99%
“…Another realization is that when clinical C max is not yet available for a new drug candidate, it can be replaced with predicted efficacious concentrations ( C eff ). Furthermore, when comparing drugs modulating the same biological target, such a comparison based on predicted C eff can be very effective in differentiating toxic from safer drug candidates (Xu et al 2012). …”
Section: Use Of In Vitro Systems For Predicting Liver Toxicitymentioning
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.
“…The bile canaliculi were stained by cholyl lysyl fluorescein (CLF), a substrate for BSEP. The clinical development of CP-724,714, a small molecule Her2 inhibitor for oncology indications, was stopped in phase 2 due to jaundice and cholestatic liver damage (for further experimental details, refer to Xu et al 2012)Fig. 48
Left : Hepatocytes on collagen (I) spot after 5 days in culture (from Jones et al 2009).…”
Section: Use Of In Vitro Systems For Predicting Liver Toxicitymentioning
confidence: 99%
“…ROS generation and GSH depletion as occurs during oxidative stress, mitochondrial membrane potential change as occurs during mitochondrial injury, inhibition of BSEP as occurs during drug-induced intrahepatic cholestasis (Boelsterli 2003). At a practical level, these phenotypic screens further employed a panel of optimized fluorescent probes to measure these modes of action specifically (Xu et al 2012). The concept of phenotypic screens can be extended to include other toxicologically relevant end points by using additional fluorescent or other markers to probe other pathophysiological processes including steatosis, phospholipidosis, apoptosis, endoplasmic reticulum (ER) stress, protein trafficking and transport dysfunctions.…”
Section: Use Of In Vitro Systems For Predicting Liver Toxicitymentioning
confidence: 99%
“…Another realization is that when clinical C max is not yet available for a new drug candidate, it can be replaced with predicted efficacious concentrations ( C eff ). Furthermore, when comparing drugs modulating the same biological target, such a comparison based on predicted C eff can be very effective in differentiating toxic from safer drug candidates (Xu et al 2012). …”
Section: Use Of In Vitro Systems For Predicting Liver Toxicitymentioning
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.
“…Table 3 to the 100 · the Human C max Values Reported in Table 1 Compounds The DILI drugs tested in our study, although widely studied using vesicle membrane assays, have limited reports on their effects in SCH, especially when CMFDA was used as a fluorescent probe. Of the 12 DILI drugs we tested, only 2 compounds, namely, cyclosporin A, 12 and erythromycin estolate, 30 have previously been demonstrated to inhibit the transportation of CLF to the canalicular space in sandwich-cultured human hepatocytes. Rifampicin, benzbromarone, and cyclosporin A have previously been shown to eliminate 5,(6)-carboxy-2¢,7¢-dichlorofluorescein (CDF) accumulation in the canaliculi of sandwich-cultured rat hepatocyte.…”
Section: Discussionmentioning
confidence: 99%
“…30 Each compound was tested at 9 concentrations with the top concentration above its 100· human C max (maximum drug plasma concentration) or 1000 lM if there was no C max information. The various concentrations of compounds were incubated with hepatocytes first for 1 hour at 37°C.…”
Inhibition of hepatic efflux transporters (BSEP, MRP2) is one of the important mechanisms in drug-induced liver injury and in particular cholestasis. The inhibitory effect of compounds on these two hepatic transporters is commonly delineated using inverted membrane vesicles from Sf9 insect cells overexpressing the BSEP or MRP2 protein. However, due to lack of bioactivation and metabolism of the compound, and the lack of the expression and functionality of other sinusoidal efflux and uptake transporters in the cell-free assay system, it is difficult to fully understand the role of a given hepatic transporter in cholestasis. Therefore, our aim was to characterize and develop a competent cell-based bile canalicular imaging assay for measuring the overall inhibitory effects of compounds on biliary transport. Since species differences have been reported for some compounds, we developed the assay using both rat and human hepatocytes. Two fluorescent dyes, cholyl-lysyl-fluorescein (CLF) and carboxymethyl flourescein diacetate (CMFDA), have been reported in the literature to measure BSEP and MRP2 inhibition. We show in our study that neither CLF nor CMFDA is a specific substrate for either BSEP/Bsep or MRP2/ Mrp2 as demonstrated by generating K m values for each transporter. Most of the drugs tested inhibited CLF accumulation and exhibited less potency toward CMFDA accumulation in hepatocytes. We found the highest concordance between IC 50 values for inhibition of CLF accumulation in human hepatoyctes and IC 50 values generated in the human BSEP vesicle assay. This suggests that the human vesicle-based assays could suffice as a primary screen to avoid future potential human liver injury.
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