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. Herein we provide detailed methods for the hepatocyte imaging assay technology (HIAT) and the bile flux imaging assay technology (BIAT) to evaluate drug-induced liver injury (DILI) potentials for drug candidates. Optimized culturing methods for primary human hepatocytes, both freshly isolated and prequalified cryopreserved cells, are also presented. The applications of these high-content cellular imaging technologies in the evaluation of p38 and Her2 kinase inhibitors are highlighted to illustrate the usefulness of the research methodology in a compound screening as well as mechanistic investigative setting.
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References
Duan, W., and Wong, W. S. (2006) Targeting mitogen-activated protein kinases for asthma. Curr. Drug Targets 7, 691–698.
Wong, K. K. (2009) Recent Developments in Anti-Cancer Agents Targeting the Ras/Raf/ MEK/ERK Pathway. Recent Patents Anticancer Drug Discov. 4, 28–35.
Schindler, J. F., Monahan, J. B., and Smith, W. G. (2007) p38 pathway kinases as anti-inflammatory drug targets. J. Dent. Res. 86, 800–811.
Hendriks, B. S., Hua, F., and Chabot, J. R. (2008) Analysis of mechanistic pathway models in drug discovery: p38 pathway. Biotechnol. Prog. 24, 96–109.
Konecny, G. E., Pegram, M. D., Venkatesan, N., Finn, R., Yang, G., Rahmeh, M., Untch, M., Rusnak, D. W., Spehar, G., Mullin, R. J., Keith, B. R., Gilmer, T. M., Berger, M., Podratz, K. C., and Slamon, D. J. (2006) Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res. 66, 1630–1639.
Moulder, S. L., Yakes, F. M., Muthuswamy, S. K., Bianco, R., Simpson, J. F., and Arteaga, C. L. (2001) Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res. 61, 8887–8895.
Dominguez, C., Powers, D. A., and Tamayo, N. (2005) p38 MAP kinase inhibitors: many are made, but few are chosen. Curr. Opin. Drug Discov. Devel. 8, 421–430.
Guo, F., Letrent, S. P., Munster, P. N., Britten, C. D., Gelmon, K., Tolcher, A. W., and Sharma, A. (2008) Pharmacokinetics of a HER2 tyrosine kinase inhibitor CP-724,714 in patients with advanced malignant HER2 positive solid tumors: correlations with clinical characteristics and safety. Cancer Chemother. Pharmacol. 62, 97–109.
Xu, J. J., Hendriks, B. S., Zhao, J., and de Graaf, D. (2008) Multiple effects of acetaminophen and p38 inhibitors: towards pathway toxicology. FEBS Lett. 582, 1276–1282.
Antoine, D. J., Williams, D. P., and Park, B. K. (2008) Understanding the role of reactive metabolites in drug-induced hepatotoxicity: state of the science. Expert Opin. Drug Metab. Toxicol. 4, 1415–1427.
Obach, R. S., Kalgutkar, A. S., Soglia, J. R., and Zhao, S. X. (2008) Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18 drugs with consideration of intrinsic clearance and daily dose. Chem. Res. Toxicol. 21, 1814–1822.
Zhao, S. X., Dalvie, D. K., Kelly, J. M., Soglia, J. R., Frederick, K. S., Smith, E. B., Obach, R. S., and Kalgutkar, A. S. (2007) NADPH-dependent covalent binding of [3H]paroxetine to human liver microsomes and S-9 fractions: identification of an electrophilic quinone metabolite of paroxetine. Chem. Res. Toxicol. 20, 1649–1657.
Xu, J. J., Henstock, P. V., Dunn, M. C., Smith, A. R., Chabot, J. R., and de Graaf, D. (2008) Cellular imaging predictions of clinical drug-induced liver injury. Toxicol. Sci. 105, 97–105.
Olson, H., Betton, G., Robinson, D., Thomas, K., Monro, A., Kolaja, G., Lilly, P., Sanders, J., Sipes, G., Bracken, W., Dorato, M., Van Deun, K., Smith, P., Berger, B., and Heller, A. (2000) Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul. Toxicol. Pharmacol. 32, 56–67.
LeCluyse, E. L., Audus, K. L., and Hochman, J. H. (1994) Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. Am. J. Physiol. 266, C1764–1774.
Liu, X., LeCluyse, E. L., Brouwer, K. R., Gan, L. S., Lemasters, J. J., Stieger, B., Meier, P. J., and Brouwer, K. L. (1999) Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration. Am. J. Physiol. 277, G12–21.
Hariparsad, N., Carr, B. A., Evers, R., and Chu, X. (2008) Comparison of immortalized Fa2N-4 cells and human hepatocytes as in vitro models for cytochrome P450 induction. Drug Metab. Dispos. 36, 1046–1055.
Le Vee, M., Jigorel, E., Glaise, D., Gripon, P., Guguen-Guillouzo, C., and Fardel, O. (2006) Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line. Eur. J. Pharm. Sci. 28, 109–117.
Munster, P. N., Britten, C. D., Mita, M., Gelmon, K., Minton, S. E., Moulder, S., Slamon, D. J., Guo, F., Letrent, S. P., Denis, L., and Tolcher, A. W. (2007) First study of the safety, tolerability, and pharmacokinetics of CP-724,714 in patients with advanced malignant solid HER2-expressing tumors. Clin. Cancer Res. 13, 1238–1245.
Feng, B., Xu, J. J., Bi, Y. A., Mireles, R., Davidson, R., Duignan, D. B., Campbell, S., Kostrubsky, V. E., Dunn, M. C., Smith, A. R., and Wang, H. F. (2009) Role of hepatic transporters in the disposition and hepatotoxicity of a HER2 tyrosine kinase inhibitor CP-724,714. Toxicol. Sci. 108, 492–500.
Bi, Y. A., Kazolias, D., and Duignan, D. B. (2006) Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab. Dispos. 34, 1658–1665.
Kreamer, B. L., Staecker, J. L., Sawada, N., Sattler, G. L., Hsia, M. T., and Pitot, H. C. (1986) Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell Dev. Biol. 22, 201–211.
Vinken, M., Henkens, T., Vanhaecke, T., Papeleu, P., Geerts, A., Van Rossen, E., Chipman, J. K., Meda, P., and Rogiers, V. (2006) Trichostatin a enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes. Toxicol. Sci. 91, 484–492.
Papeleu, P., Vanhaecke, T., and Rogiers, V. (2006) Histone deacetylase inhibition: A differentiation therapy for cultured primary hepatocytes? Curr. Enz. Inhib. 2, 91–104.
Acknowledgments
The authors would like to thank Drs. David de Graaf, Jeffery Chabot, Peter Henstock, Michael Aleo, Denise Robinson-Gravatt, and other research program team members for their support in the development and validation of HIAT and BIAT protocols.
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Xu, J.J., Dunn, M.C., Smith, A.R., Tien, E.S. (2012). Assessment of Hepatotoxicity Potential of Drug Candidate Molecules Including Kinase Inhibitors by Hepatocyte Imaging Assay Technology and Bile Flux Imaging Assay Technology. In: Kuster, B. (eds) Kinase Inhibitors. Methods in Molecular Biology, vol 795. Humana Press. https://doi.org/10.1007/978-1-61779-337-0_6
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DOI: https://doi.org/10.1007/978-1-61779-337-0_6
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