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“ENGINEERED HEPATOCELLULAR MODELS FOR DRUG DEVELOPMENT" ABHISHEK ANANTHANARAYANAN B.TECH, SRMIST A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and has been written by me in entirety. I have duly acknowledged all sources of information which have been used in the thesis This thesis has not been submitted for any degree in any university previously Abhishek Ananthanarayanan 11th July 2013 Table of contents Page No Summary Acknowledgement List of figures List of tables List of symbols Chapter Introduction to drug development 11 1.1 Introduction to drug development process 1.2 Need for in vitro models 1.3 Structure function relationship of the liver and hepatocyte microenvironment 1.4 Cell types in the liver 1.4.1 Hepatocytes 1.4.2 Endothelial cells 1.4.3 Kupffer cells 1.4.4 Stellate cells 1.4.5 Oval cells 1.4.6 Pit cells 1.5 In vitro cellular models for drug development 1.6 Tissue engineering approaches and paradigms 1.7 Toolbox development for precision tissue engineering 1.7.1 Biomaterials for cellular assembly 1.7.2 Micro and nano- scale construct technologies 1.8 Purpose driven liver tissue engineering for applications 1.8.1 Cell models for pathogen testing 1.8.2 Hepatotoxicity testing Chapter Outline and specific aim of the thesis 2.1 Specific Aim 2.1.1 Hypothesis 2.1.2 Rationale 2.1.3 Experimental design 2.2 Specific Aim 2.2.1 Hypothesis 36 2.2.2 Rationale 2.2.3 Experimental design Chapter Scalable spheroid model of human hepatocytes to study HCV infection and replication 3.1 Introduction 3.2 Background 3.3 HCV and host interactions 3.4 Viral proteins mediating entry 3.4.1 CD-81 3.4.2 SCARB-1 3.4.3 Claudin-1 3.4.4 Occludin-1 3.5 Mechanism of entry 3.6 Hepatitis C replication 3.7 HCV proteases 3.7.1 NS2-3 3.7.2 NS3-4A 3.7.3 NS 4B 3.7.4 NS 5A 3.7.5 NS 5B 3.8 Viral replication complex 3.9 Packaging and assembly 3.10Evasion of host responses by the virus 3.11Methods 3.11.1 Huh 7.5 cell culture 3.11.2 Human hepatocyte culture 3.11.3 Cell seeding 3.11.4 Synthesis of cellulosic scaffold 3.11.5 Scanning electron microscope 3.11.6 Live/dead staining 3.11.7 Immunostaining 3.11.8 Real time PCR 3.11.9 HCVpp synthesis 3.11.10 HCVpp entry and inhibition assay 3.11.11 Quantifying viral replication 3.12 Results 3.12.1 Characterization of spheroids in the scaffold 3.12.2 Characterization of spheroids using SEM 3.12.3 Characterization of presence of apical and basolateral domain in spheroid cultured cells 41 3.12.4 Assessment of viability 3.12.5 Characterization of cellular phenotype 3.12.6 Expression of viral entry marker and pseudoparticle entry 3.12.7 HCV live viral replication 3.13 Discussion 3.14 Conclusion Chapter 81 Co-culture of rat hepatocytes and NIH 3T3 fibroblasts suppresses drug induced CYP 450 responses via TGF β1 mediated transcription factor inhibition 4.1 Introduction 4.2 Background 4.2.1 Factors eliciting toxicity 4.2.2 Liver enriched nuclear factors 4.2.3 CYP 450 enzymes 4.2.4 Phase II enzymes 4.2.5 Transporters 4.3 General mechanism of toxicity and liver injury 4.3.1 Aryl hydrocarbon receptor 4.3.2 Pregnane X receptor 4.3.3 Constitutive androstane receptor 4.3.4 Farsenoid X receptor 4.3.5 Peroxisome proliferator-activated receptor 4.4 Biotransformation, CYP induction and how it leads to toxicity 4.4.1 Phase I 4.4.2 Phase II 4.4.3 Phase III 4.5 APAP metabolism as an example for toxic metabolite mediated hepatotoxicity 4.6 Cytokines and their roles in liver disease 4.7 Materials and methods 4.7.1 NIH-3T3 culture 4.7.2 Rat hepatocyte isolation and culture 4.7.3 Hepatocyte synthetic function 4.7.4 CYP induction assay 4.7.5 RT-PCR 4.7.6 LC/MS measurement of CYP specific metabolites 4.7.7 Hepatocyte excretory function 4.7.8 TGF β1 pulldown 4.7.9 EROD assay 4.7.10Measurement of drug sensitivity 4.8 Results 4.8.1 Characterization of synthetic and metabolic function of sandwich and co-culture 4.8.2 Comparing drug sensitivity and drug induction between co-culture and sandwich culture 4.8.3 TGF β1 is an important regulator of hepatocyte function 4.8.4 TGF β1 is an important factor regulating CYP induction in co-culture 4.8.5 TGF β1 and co-culture enhance hepatocyte excretion 4.9 Discussion 4.10 Conclusion Chapter 119 Recommendations for future research 5.1 Liver tissue engineering 5.2 Hepatotoxicity testing and improving predictivity 5.3 Hepatitis C infections and drug development References 125 Summary: Primary hepatocytes of adult human and rodent origin are essential components for developing drugs against infectious pathogens and for studying drug mediated liver toxicity. One of the key drawbacks limiting the use of these primary hepatocytes in vitro is their rapid loss of differentiated function, polarity, inability to recapitulate drug responses accurately and failure to capture the life cycle of pathogens. Although multiple platforms have been developed to improve functional maintenance of hepatocytes in culture, there is little understanding on the utility of these models for applications like toxicology and infections by various liver specific pathogens. In this thesis we have studied the utility of spheroid cultures of human hepatocytes to support hepatitis C infection and replication and sandwich culture of rat hepatocytes and coculture of rat hepatocytes with fibroblasts for drug testing applications. Spheroid culture models of human hepatocytes and human hepatoma cells maintain and enhance liver specific functions, while localizing various liver specific proteins at domains similar to that found in vivo. These spheroid models maintain polarity over prolonged cultures and support glycoprotein mediated HCV entry. Huh 7.5 also support higher levels of replication of HCV virus in vitro. This makes it a suitable model to screen for drugs inhibiting HCV entry and replication. Rat hepatocyte culture with fibroblasts (co-culture) enhances hepatocyte specific synthetic and metabolic functions. However co-culture of hepatocytes with fibroblasts inhibits drug-induced CYP 450 responses. We found that TGFβ1 is an important cytokine in co-culture responsible for repression of drug-induced responses. Soluble factor mediated repression of drug-induced CYP 450 responses makes co-culture an unsuitable model to study drug induction/inhibition and drug-drug interactions. We have analyzed the strengths of different hepatocyte culture models and demonstrated the strengths of different models for applications pertaining to drug development. Acknowledgements: I am indebted to my supervisor Prof Hanry Yu for giving me independence and freedom to define my thesis and also execute it with tremendous amounts of support. The idea of doing this thesis came about after talks with different pharmaceutical industries like Johnson and Johnson and Hoffman La Roche pharmaceuticals who were interested like rest of the pharmaceutical world in the major points of focus in this thesis namely Hepatotoxicity and HCV replication in vitro. Prof Yu was instrumental in my visit and collaborative efforts with both these pharmaceutical giants to understand the needs of the industry. To my thesis advisor Dr Michael McMillian, I owe my deepest gratitude for many insightful discussions and making me understand the nuances of mechanistic toxicity and understanding the biology behind various toxic responses. To Dr Miriam Triyatni, Dr Surya Sankuratri and Dr Stefan Hart for discussions on characterization of the model for Hepatitis C infection and useful discussions on HCV biology and the problems the pharmaceutical industry is facing to combat this virus. I would like to also thank my colleagues Bramasta, Narmada, Justin, Inn Chuan, George Annene, Yee Han, Yi Chin and Derek Phan for all the wonderful discussions and support over the last years and making it a wonderful sojourn I would also like to thank all LCTE members and members at Institute at Bioengineering and Nanotechnology past and present for letting me to work with them over these years in a conducive lab environment. Finally I would like to thank my parents for being my advisors, friends, philosophers and guides all my life without which this dissertation would not be possible. List of publications: 1. Ananthanarayanan.A. et al. 2011 “ Systems Biology in Biomaterials and Tissue Engineering”. Comprehensive Biomaterials. Elsevier 2. Ananthanarayanan.A., et al. 2011 “ Purpose driven biomaterials research in liver tissue engineering” 29 (8):110-8. Trends in Biotechnology (Cover March 2011) 3. Shufang Zhang, Wenhao Tong, Baixue Zheng, Thomas Adi Kurnia Susanto, Lei Xia, Chi Zhang, Abhishek Ananthanarayanan, Xiaoye Tuo, Sakbhan Rashidah Binte, Rui Rui Jia, Ciprian Iliescu, Kah Hin Chai, Michael McMillian, Shali Shen, Hwa Liang Leo, Hanry Yu. 2011. A robust high throughput sandwich cell based drug screening platform. 32 (4):1229-41. Biomaterials 4. Lei Xia, Yinghua Qu, Sakban Rashidah Binte, Xin Hong, Wenxia Zhang, Bramasta Nugraha, Wenhao Tong, Abhishek Ananthanarayanan, BaiXue Zheng, Ian Yin-Yan, Rui Rui Jia, Michael McMillian, Jose Silvia, Shanon Dallas, Hanry Yu. 2012“Tethered spheroids as a hepatocyte in vitro model for drug hepatotoxicity screening” 33 (7):2165-76 5. Hanry Yu and Abhishek Ananthanarayanan. “Introduction to Cellular and Tissue engineering”. 2013 Imaging in cellular and Tissue engineering. Taylor and Francis 6. Baixue Zheng and Abhishek Ananthanarayanan.2013”Confocal Microscopy for Cellular Imaging: High-Content Screening”. Imaging in cellular and Tissue engineering. Taylor and Francis 7. Ananthanarayanan. A et al. 2013. “Scalable spheroid model of human hepatocytes for HCV infection and replication” (Manuscript to be submitted) 8. Ananthanarayanan et al. 2013. “ Co-culture of rat hepatocytes with NIH 3T3 suppresses drug induced responses via TGF β1mediated transcription factor inhibition” (manuscript to be submitted) levels of infection and persistent replication also allow for the study of the accurate drug responses that are activated by various strains of the virus. However the difficulty in obtaining high quality human hepatocytes from donors makes it very difficult to use in vitro systems with primary human hepatocytes for routine drug screening. We envision a future where we will obtain human hepatocytes from sources such as humanized mice where fresh human hepatocytes can be obtained from mice, which have been implanted with liver cells of human donors supporting HCV infection. Other alternatives can be iPS-derived hepatocytes, which can support HCV replication. This will allow us to screen for differences in metabolic capacities of different donors and also identify the differences in drug responses among different patient lines. This will help us estimate the efficacy of the drug among different ethnic groups/different patients well before the clinical trials. The 3D spheroid system, which allows for hepatitis C replication holds great promise for the future and the salient features of this system allow it to be scalable amenable to screening in 96 well plate format allowing for high throughput and routine screening of anti-viral drugs and also a system for basic biological research to understand hostvirus responses. The system can also be extrapolated for the study of HBV and characterized for infections by other pathogens. 124 References [1] DiMasi JA, Hansen RW, Grabowski HG. The price of innovation: new estimates of drug development costs. J Health Econ. 2003;22:151-‐85. [2] Burbaum JJ, Ohlmeyer MH, Reader JC, Henderson I, Dillard LW, Li G, et al. A paradigm for drug discovery employing encoded combinatorial libraries. Proc Natl Acad Sci U S A. 1995;92:6027-‐31. [3] Venkatesh S, Lipper RA. Role of the development scientist in compound lead selection and optimization. J Pharm Sci. 2000;89:145-‐54. [4] Jemnitz K, Veres Z, Monostory K, Kobori L, Vereczkey L. Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes. Toxicol In Vitro. 2008;22:961-‐7. [5] Kubinyi H. Drug research: myths, hype and reality. Nat Rev Drug Discov. 2003;2:665-‐8. [6] Flint M, von Hahn T, Zhang J, Farquhar M, Jones CT, Balfe P, et al. Diverse CD81 proteins support hepatitis C virus infection. J Virol. 2006;80:11331-‐42. [7] Brass V, Moradpour D, Blum HE. Hepatitis C virus infection: in vivo and in vitro models. J Viral Hepat. 2007;14 Suppl 1:64-‐7. [8] Lampertico P, Malter JS, Gerber MA. Development and application of an in vitro model for screening anti-‐hepatitis B virus therapeutics. Hepatology. 1991;13:422-‐6. [9] Nelson DR, Soldevila-‐Pico C, Reed A, Abdelmalek MF, Hemming AW, Van der Werf WJ, et al. Anti-‐interleukin-‐2 receptor therapy in combination with mycophenolate mofetil is associated with more severe hepatitis C recurrence after liver transplantation. Liver Transpl. 2001;7:1064-‐70. [10] Hendriksen CF. Replacement, reduction and refinement alternatives to animal use in vaccine potency measurement. Expert Rev Vaccines. 2009;8:313-‐ 22. [11] Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci. 2002;65:166-‐76. [12] Rappaport AM, Borowy ZJ, Lougheed WM, Lotto WN. Subdivision of hexagonal liver lobules into a structural and functional unit; role in hepatic physiology and pathology. Anat Rec. 1954;119:11-‐33. [13] Cunningham CC, Van Horn CG. Energy availability and alcohol-‐related liver pathology. Alcohol Res Health. 2003;27:291-‐9. [14] Katz NR. Metabolic heterogeneity of hepatocytes across the liver acinus. J Nutr. 1992;122:843-‐9. [15] LeCluyse EL, Bullock PL, Parkinson A, Hochman JH. Cultured rat hepatocytes. Pharm Biotechnol. 1996;8:121-‐59. [16] Wisse E, Braet F, Luo D, De Zanger R, Jans D, Crabbe E, et al. Structure and function of sinusoidal lining cells in the liver. Toxicol Pathol. 1996;24:100-‐11. [17] Roberts RA, Ganey PE, Ju C, Kamendulis LM, Rusyn I, Klaunig JE. Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis. Toxicol Sci. 2007;96:2-‐15. [18] Sleyster EC, Knook DL. Relation between localization and function of rat liver Kupffer cells. Lab Invest. 1982;47:484-‐90. [19] Sato M, Suzuki S, Senoo H. Hepatic stellate cells: unique characteristics in cell biology and phenotype. Cell Struct Funct. 2003;28:105-‐12. 125 [20] Wang X, Tang X, Gong X, Albanis E, Friedman SL, Mao Z. Regulation of hepatic stellate cell activation and growth by transcription factor myocyte enhancer factor 2. Gastroenterology. 2004;127:1174-‐88. [21] Moreira RK. Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med. 2007;131:1728-‐34. [22] Lowes KN, Croager EJ, Olynyk JK, Abraham LJ, Yeoh GC. Oval cell-‐mediated liver regeneration: Role of cytokines and growth factors. Journal of gastroenterology and hepatology. 2003;18:4-‐12. [23] Newsome PN, Hussain MA, Theise ND. Hepatic oval cells: helping redefine a paradigm in stem cell biology. Current topics in developmental biology. 2004;61:1-‐28. [24] Nakatani K, Kaneda K, Seki S, Nakajima Y. Pit cells as liver-‐associated natural killer cells: morphology and function. Med Electron Microsc. 2004;37:29-‐36. [25] Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209-‐18. [26] Ananthanarayanan A, Narmada BC, Mo X, McMillian M, Yu H. Purpose-‐driven biomaterials research in liver-‐tissue engineering. Trends Biotechnol.29:110-‐8. [27] Bayad J, Bagrel D, Sabolovic N, Magdalou J, Siest G. Expression and regulation of drug metabolizing enzymes in an immortalized rat hepatocyte cell line. Biochem Pharmacol. 1991;42:1345-‐51. [28] Carlile DJ, Zomorodi K, Houston JB. Scaling factors to relate drug metabolic clearance in hepatic microsomes, isolated hepatocytes, and the intact liver: studies with induced livers involving diazepam. Drug Metab Dispos. 1997;25:903-‐11. [29] Brandon EF, Raap CD, Meijerman I, Beijnen JH, Schellens JH. An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons. Toxicol Appl Pharmacol. 2003;189:233-‐46. [30] Farkas D, Tannenbaum SR. In vitro methods to study chemically-‐induced hepatotoxicity: a literature review. Curr Drug Metab. 2005;6:111-‐25. [31] Sivaraman A, Leach JK, Townsend S, Iida T, Hogan BJ, Stolz DB, et al. A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction. Curr Drug Metab. 2005;6:569-‐91. [32] LeCluyse EL, Alexandre E, Hamilton GA, Viollon-‐Abadie C, Coon DJ, Jolley S, et al. Isolation and culture of primary human hepatocytes. Methods Mol Biol. 2005;290:207-‐29. [33] Foy E, Li K, Sumpter R, Jr., Loo YM, Johnson CL, Wang C, et al. Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-‐inducible gene-‐I signaling. Proc Natl Acad Sci U S A. 2005;102:2986-‐91. [34] Gebhardt R, Hengstler JG, Muller D, Glockner R, Buenning P, Laube B, et al. New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures. Drug Metab Rev. 2003;35:145-‐213. [35] Dunn JC, Yarmush ML, Koebe HG, Tompkins RG. Hepatocyte function and extracellular matrix geometry: long-‐term culture in a sandwich configuration. FASEB J. 1989;3:174-‐7. [36] Griffith LG, Naughton G. Tissue engineering-‐-‐current challenges and expanding opportunities. Science. 2002;295:1009-‐14. [37] MacNeil S. Progress and opportunities for tissue-‐engineered skin. Nature. 2007;445:874-‐80. 126 [38] Sauer IM, Obermeyer N, Kardassis D, Theruvath T, Gerlach JC. Development of a hybrid liver support system. Ann N Y Acad Sci. 2001;944:308-‐19. [39] Bruns H, Kneser U, Holzhuter S, Roth B, Kluth J, Kaufmann PM, et al. Injectable liver: a novel approach using fibrin gel as a matrix for culture and intrahepatic transplantation of hepatocytes. Tissue Eng. 2005;11:1718-‐26. [40] Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater. 2005;4:518-‐24. [41] Roelandt P, Pauwelyn KA, Sancho-‐Bru P, Subramanian K, Ordovas L, Vanuytsel K, et al. Human embryonic and rat adult stem cells with primitive endoderm-‐like phenotype can be fated to definitive endoderm, and finally hepatocyte-‐like cells. PLoS One. 2010;5:e12101. [42] Williams DF. On the nature of biomaterials. Biomaterials. 2009;30:5897-‐ 909. [43] Khademhosseini A, Langer R. Microengineered hydrogels for tissue engineering. Biomaterials. 2007;28:5087-‐92. [44] Khademhosseini A, Langer R, Borenstein J, Vacanti JP. Microscale technologies for tissue engineering and biology. Proc Natl Acad Sci U S A. 2006;103:2480-‐7. [45] Du Y, Lo E, Ali S, Khademhosseini A. Directed assembly of cell-‐laden microgels for fabrication of 3D tissue constructs. Proc Natl Acad Sci U S A. 2008;105:9522-‐7. [46] Tsuda Y, Morimoto Y, Takeuchi S. Monodisperse cell-‐encapsulating peptide microgel beads for 3D cell culture. Langmuir. 2010;26:2645-‐9. [47] Liu Tsang V, Chen AA, Cho LM, Jadin KD, Sah RL, DeLong S, et al. Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels. FASEB J. 2007;21:790-‐801. [48] Zhao D, Ong SM, Yue Z, Jiang Z, Toh YC, Khan M, et al. Dendrimer hydrazides as multivalent transient inter-‐cellular linkers. Biomaterials. 2008;29:3693-‐702. [49] Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials. 2005;26:6415-‐22. [50] Ong SM, He L, Thuy Linh NT, Tee YH, Arooz T, Tang G, et al. Transient inter-‐ cellular polymeric linker. Biomaterials. 2007;28:3656-‐67. [51] Mo X, Li Q, Yi Lui LW, Zheng B, Kang CH, Nugraha B, et al. Rapid construction of mechanically-‐ confined multi-‐ cellular structures using dendrimeric intercellular linker. Biomaterials. 2010;31:7455-‐67. [52] Ohashi K, Yokoyama T, Yamato M, Kuge H, Kanehiro H, Tsutsumi M, et al. Engineering functional two-‐ and three-‐dimensional liver systems in vivo using hepatic tissue sheets. Nat Med. 2007;13:880-‐5. [53] Tsuda Y, Kikuchi A, Yamato M, Nakao A, Sakurai Y, Umezu M, et al. The use of patterned dual thermoresponsive surfaces for the collective recovery as co-‐ cultured cell sheets. Biomaterials. 2005;26:1885-‐93. [54] Toh YC, Zhang C, Zhang J, Khong YM, Chang S, Samper VD, et al. A novel 3D mammalian cell perfusion-‐culture system in microfluidic channels. Lab Chip. 2007;7:302-‐9. [55] Zhang C, Zhao Z, Abdul Rahim NA, van Noort D, Yu H. Towards a human-‐on-‐ chip: culturing multiple cell types on a chip with compartmentalized microenvironments. Lab Chip. 2009;9:3185-‐92. 127 [56] V.N Goral YCH, O.N. Petzold, J.S. Clark, P.k. Yuen, R.A. Faris. Perfusion based microfluidic device for 3D dynamic primary human hepatocyte culture in absence of biological or synthetic matrices or coagulants. MicroTas. Groningen, Netherlands2010. [57] Hui EE, Bhatia SN. Micromechanical control of cell-‐cell interactions. Proc Natl Acad Sci U S A. 2007;104:5722-‐6. [58] Khetani SR, Bhatia SN. Microscale culture of human liver cells for drug development. Nat Biotechnol. 2008;26:120-‐6. [59] Zhang S, Xia L, Kang CH, Xiao G, Ong SM, Toh YC, et al. Microfabricated silicon nitride membranes for hepatocyte sandwich culture. Biomaterials. 2008;29:3993-‐4002. [60] Feng ZQ, Chu XH, Huang NP, Leach MK, Wang G, Wang YC, et al. Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-‐I collagen-‐coated poly(L-‐lactic acid). Biomaterials. 2010;31:3604-‐12. [61] Ghibaudo M, Trichet L, Le Digabel J, Richert A, Hersen P, Ladoux B. Substrate topography induces a crossover from 2D to 3D behavior in fibroblast migration. Biophys J. 2009;97:357-‐68. [62] Ng S, Han R, Chang S, Ni J, Hunziker W, Goryachev AB, et al. Improved hepatocyte excretory function by immediate presentation of polarity cues. Tissue Eng. 2006;12:2181-‐91. [63] Mee CJ, Grove J, Harris HJ, Hu K, Balfe P, McKeating JA. Effect of cell polarization on hepatitis C virus entry. J Virol. 2008;82:461-‐70. [64] Harris HJ, Farquhar MJ, Mee CJ, Davis C, Reynolds GM, Jennings A, et al. CD81 and claudin coreceptor association: role in hepatitis C virus entry. J Virol. 2008;82:5007-‐20. [65] Chong TW, Smith RL, Hughes MG, Camden J, Rudy CK, Evans HL, et al. Primary human hepatocytes in spheroid formation to study hepatitis C infection. J Surg Res. 2006;130:52-‐7. [66] Ploss A, Khetani SR, Jones CT, Syder AJ, Trehan K, Gaysinskaya VA, et al. Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures. Proc Natl Acad Sci U S A. 2010;107:3141-‐5. [67] Bissig KD, Wieland SF, Tran P, Isogawa M, Le TT, Chisari FV, et al. Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J Clin Invest. 2010;120:924-‐30. [68] Yang PL, Althage A, Chung J, Chisari FV. Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci U S A. 2002;99:13825-‐30. [69] Brophy CM, Luebke-‐Wheeler JL, Amiot BP, Khan H, Remmel RP, Rinaldo P, et al. Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function. Hepatology. 2009;49:578-‐86. [70] Kienhuis AS, Wortelboer HM, Maas WJ, van Herwijnen M, Kleinjans JC, van Delft JH, et al. A sandwich-‐cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling. Toxicol In Vitro. 2007;21:892-‐901. [71] Keum YS, Han YH, Liew C, Kim JH, Xu C, Yuan X, et al. Induction of heme oxygenase-‐1 (HO-‐1) and NAD[P]H: quinone oxidoreductase (NQO1) by a phenolic antioxidant, butylated hydroxyanisole (BHA) and its metabolite, tert-‐ butylhydroquinone (tBHQ) in primary-‐cultured human and rat hepatocytes. Pharm Res. 2006;23:2586-‐94. 128 [72] Williams DP. Toxicophores: investigations in drug safety. Toxicology. 2006;226:1-‐11. [73] Obach RS, Kalgutkar AS, Soglia JR, Zhao SX. 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. 2008;21:1814-‐22. [74] Dykens JA, Will Y. The significance of mitochondrial toxicity testing in drug development. Drug Discovery Today. 2007;12:777-‐85. [75] Seidle T, Robinson S, Holmes T, Creton S, Prieto P, Scheel J, et al. Cross-‐ Sector Review of Drivers and Available 3Rs Approaches for Acute Systemic Toxicity Testing. Toxicological Sciences. 2010;116:382-‐96. [76] Allen JW, Khetani SR, Bhatia SN. In vitro zonation and toxicity in a hepatocyte bioreactor. Toxicol Sci. 2005;84:110-‐9. [77] Chao P, Maguire T, Novik E, Cheng KC, Yarmush ML. Evaluation of a microfluidic based cell culture platform with primary human hepatocytes for the prediction of hepatic clearance in human. Biochem Pharmacol. 2009;78:625-‐32. [78] Du Y, Han R, Ng S, Ni J, Sun W, Wohland T, et al. Identification and characterization of a novel prespheroid 3-‐dimensional hepatocyte monolayer on galactosylated substratum. Tissue Eng. 2007;13:1455-‐68. [79] Cosgrove BD, King BM, Hasan MA, Alexopoulos LG, Farazi PA, Hendriks BS, et al. Synergistic drug-‐cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-‐associated idiosyncratic drug hepatotoxicity. Toxicol Appl Pharmacol. 2009;237:317-‐30. [80] Dash A IW, Hoffmaster K, Sevidal S, Kelly J, Obach RS, Griffith LG, Tannebaum SR. Liver tissue engineering in the evaluation of drug safety. Expert Opinion on Drug Metabolism & Toxicology. 2009;5:1159-‐74. [81] Ploss A, Khetani SR, Jones CT, Syder AJ, Trehan K, Gaysinskaya VA, et al. Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures. Proc Natl Acad Sci U S A.107:3141-‐5. [82] Nugraha B, Hong X, Mo X, Tan L, Zhang W, Chan PM, et al. Galactosylated cellulosic sponge for multi-‐well drug safety testing. Biomaterials.32:6982-‐94. [83] Farkas D, Tannenbaum SR. Characterization of chemically induced hepatotoxicity in collagen sandwiches of rat hepatocytes. Toxicol Sci. 2005;85:927-‐34. [84] Chia SM, Lin PC, Yu H. TGF-‐beta1 regulation in hepatocyte-‐NIH3T3 co-‐ culture is important for the enhanced hepatocyte function in 3D microenvironment. Biotechnol Bioeng. 2005;89:565-‐73. [85] Zeisel MB, Fofana I, Fafi-‐Kremer S, Baumert TF. Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies. J Hepatol.54:566-‐76. [86] Blight KJ, McKeating JA, Rice CM. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol. 2002;76:13001-‐14. [87] Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med. 2005;11:791-‐6. [88] Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A. 2005;102:9294-‐9. [89] Vinken M, Vanhaecke T, Rogiers V. Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis? Toxicol In Vitro.26:541-‐4. 129 [90] Xia L, Ng S, Han R, Tuo X, Xiao G, Leo HL, et al. Laminar-‐flow immediate-‐ overlay hepatocyte sandwich perfusion system for drug hepatotoxicity testing. Biomaterials. 2009;30:5927-‐36. [91] Du Y, Han R, Wen F, Ng San San S, Xia L, Wohland T, et al. Synthetic sandwich culture of 3D hepatocyte monolayer. Biomaterials. 2008;29:290-‐301. [92] Chisari FV. Unscrambling hepatitis C virus-‐host interactions. Nature. 2005;436:930-‐2. [93] Oze T, Hiramatsu N, Song C, Yakushijin T, Iio S, Doi Y, et al. Reducing Peg-‐IFN doses causes later virologic response or no response in HCV genotype patients treated with Peg-‐IFN alfa-‐2b plus ribavirin. J Gastroenterol.47:334-‐42. [94] Oze T, Hiramatsu N, Yakushijin T, Mochizuki K, Imanaka K, Yamada A, et al. The efficacy of extended treatment with pegylated interferon plus ribavirin in patients with HCV genotype and slow virologic response in Japan. J Gastroenterol.46:944-‐52. [95] Brown RS. Hepatitis C and liver transplantation. Nature. 2005;436:973-‐8. [96] Hoofnagle JH. Course and outcome of hepatitis C. Hepatology. 2002;36:S21-‐ 9. [97] Alter MJ. Prevention of spread of hepatitis C. Hepatology. 2002;36:S93-‐8. [98] Sulkowski MS, Mast EE, Seeff LB, Thomas DL. Hepatitis C virus infection as an opportunistic disease in persons infected with human immunodeficiency virus. Clin Infect Dis. 2000;30 Suppl 1:S77-‐84. [99] Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-‐borne non-‐A, non-‐B viral hepatitis genome. Science. 1989;244:359-‐62. [100] Lohmann V, Koch JO, Bartenschlager R. Processing pathways of the hepatitis C virus proteins. J Hepatol. 1996;24:11-‐9. [101] Penin F, Dubuisson J, Rey FA, Moradpour D, Pawlotsky JM. Structural biology of hepatitis C virus. Hepatology. 2004;39:5-‐19. [102] Nielsen SU, Bassendine MF, Burt AD, Martin C, Pumeechockchai W, Toms GL. Association between hepatitis C virus and very-‐low-‐density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients. J Virol. 2006;80:2418-‐28. [103] Seigneuret M. Complete predicted three-‐dimensional structure of the facilitator transmembrane protein and hepatitis C virus receptor CD81: conserved and variable structural domains in the tetraspanin superfamily. Biophys J. 2006;90:212-‐27. [104] Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, et al. Binding of hepatitis C virus to CD81. Science. 1998;282:938-‐41. [105] Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudo-‐ particles containing functional E1-‐E2 envelope protein complexes. J Exp Med. 2003;197:633-‐42. [106] Bartosch B, Vitelli A, Granier C, Goujon C, Dubuisson J, Pascale S, et al. Cell entry of hepatitis C virus requires a set of co-‐receptors that include the CD81 tetraspanin and the SR-‐B1 scavenger receptor. J Biol Chem. 2003;278:41624-‐30. [107] Zhang J, Randall G, Higginbottom A, Monk P, Rice CM, McKeating JA. CD81 is required for hepatitis C virus glycoprotein-‐mediated viral infection. J Virol. 2004;78:1448-‐55. [108] Boucheix C, Rubinstein E. Tetraspanins. Cell Mol Life Sci. 2001;58:1189-‐ 205. 130 [109] Rocha-‐Perugini V, Montpellier C, Delgrange D, Wychowski C, Helle F, Pillez A, et al. The CD81 partner EWI-‐2wint inhibits hepatitis C virus entry. PLoS One. 2008;3:e1866. [110] Brazzoli M, Bianchi A, Filippini S, Weiner A, Zhu Q, Pizza M, et al. CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes. J Virol. 2008;82:8316-‐29. [111] Tseng CT, Klimpel GR. Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions. J Exp Med. 2002;195:43-‐9. [112] Rhainds D, Brissette L. The role of scavenger receptor class B type I (SR-‐BI) in lipid trafficking. defining the rules for lipid traders. Int J Biochem Cell Biol. 2004;36:39-‐77. [113] Rigotti A, Trigatti B, Babitt J, Penman M, Xu S, Krieger M. Scavenger receptor BI-‐-‐a cell surface receptor for high density lipoprotein. Curr Opin Lipidol. 1997;8:181-‐8. [114] Trigatti BL, Rigotti A, Braun A. Cellular and physiological roles of SR-‐BI, a lipoprotein receptor which mediates selective lipid uptake. Biochim Biophys Acta. 2000;1529:276-‐86. [115] Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, et al. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J. 2002;21:5017-‐25. [116] Bartosch B, Verney G, Dreux M, Donot P, Morice Y, Penin F, et al. An interplay between hypervariable region of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-‐density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies. J Virol. 2005;79:8217-‐29. [117] Voisset C, Callens N, Blanchard E, Op De Beeck A, Dubuisson J, Vu-‐Dac N. High density lipoproteins facilitate hepatitis C virus entry through the scavenger receptor class B type I. J Biol Chem. 2005;280:7793-‐9. [118] Burlone ME, Budkowska A. Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J Gen Virol. 2009;90:1055-‐70. [119] Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk B, et al. Claudin-‐1 is a hepatitis C virus co-‐receptor required for a late step in entry. Nature. 2007;446:801-‐5. [120] Furuse M, Shinichi W, Suyama Y, Takahashi K, Kajikawa H. Ischaemia-‐ induced vascular vulnerability resulting in intracerebral haemorrhage with ipsilateral internal carotid artery occlusion. Neurol Sci. 2008;29:367-‐9. [121] Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J, Blasig IE. Structure and function of claudins. Biochim Biophys Acta. 2008;1778:631-‐45. [122] Meertens L, Bertaux C, Cukierman L, Cormier E, Lavillette D, Cosset FL, et al. The tight junction proteins claudin-‐1, -‐6, and -‐9 are entry cofactors for hepatitis C virus. J Virol. 2008;82:3555-‐60. [123] Ploss A, Evans MJ, Gaysinskaya VA, Panis M, You H, de Jong YP, et al. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature. 2009;457:882-‐6. [124] Cocquerel L, Voisset C, Dubuisson J. Hepatitis C virus entry: potential receptors and their biological functions. J Gen Virol. 2006;87:1075-‐84. [125] Feldman GJ, Mullin JM, Ryan MP. Occludin: structure, function and regulation. Adv Drug Deliv Rev. 2005;57:883-‐917. 131 [126] Benedicto I, Molina-‐Jimenez F, Bartosch B, Cosset FL, Lavillette D, Prieto J, et al. The tight junction-‐associated protein occludin is required for a postbinding step in hepatitis C virus entry and infection. J Virol. 2009;83:8012-‐20. [127] Benedicto I, Molina-‐Jimenez F, Barreiro O, Maldonado-‐Rodriguez A, Prieto J, Moreno-‐Otero R, et al. Hepatitis C virus envelope components alter localization of hepatocyte tight junction-‐associated proteins and promote occludin retention in the endoplasmic reticulum. Hepatology. 2008;48:1044-‐53. [128] Lavillette D, Bartosch B, Nourrisson D, Verney G, Cosset FL, Penin F, et al. Hepatitis C virus glycoproteins mediate low pH-‐dependent membrane fusion with liposomes. J Biol Chem. 2006;281:3909-‐17. [129] Roohvand F, Maillard P, Lavergne JP, Boulant S, Walic M, Andreo U, et al. Initiation of hepatitis C virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein. J Biol Chem. 2009;284:13778-‐91. [130] Spahn CM, Kieft JS, Grassucci RA, Penczek PA, Zhou K, Doudna JA, et al. Hepatitis C virus IRES RNA-‐induced changes in the conformation of the 40s ribosomal subunit. Science. 2001;291:1959-‐62. [131] Andrade RJ, Lucena MI, Fernandez MC, Pelaez G, Pachkoria K, Garcia-‐Ruiz E, et al. Drug-‐induced liver injury: an analysis of 461 incidences submitted to the Spanish registry over a 10-‐year period. Gastroenterology. 2005;129:512-‐21. [132] Pavlovic D, Neville DC, Argaud O, Blumberg B, Dwek RA, Fischer WB, et al. The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-‐ alkyl-‐chain iminosugar derivatives. Proc Natl Acad Sci U S A. 2003;100:6104-‐8. [133] Branch AD, Stump DD, Gutierrez JA, Eng F, Walewski JL. The hepatitis C virus alternate reading frame (ARF) and its family of novel products: the alternate reading frame protein/F-‐protein, the double-‐frameshift protein, and others. Semin Liver Dis. 2005;25:105-‐17. [134] Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol. 2007;5:453-‐63. [135] Pietschmann T, Kaul A, Koutsoudakis G, Shavinskaya A, Kallis S, Steinmann E, et al. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci U S A. 2006;103:7408-‐13. [136] Grakoui A, McCourt DW, Wychowski C, Feinstone SM, Rice CM. A second hepatitis C virus-‐encoded proteinase. Proc Natl Acad Sci U S A. 1993;90:10583-‐7. [137] Hijikata M, Mizushima H, Akagi T, Mori S, Kakiuchi N, Kato N, et al. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993;67:4665-‐75. [138] Wolk B, Sansonno D, Krausslich HG, Dammacco F, Rice CM, Blum HE, et al. Subcellular localization, stability, and trans-‐cleavage competence of the hepatitis C virus NS3-‐NS4A complex expressed in tetracycline-‐regulated cell lines. J Virol. 2000;74:2293-‐304. [139] Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, et al. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature. 2003;426:186-‐9. [140] Li K, Foy E, Ferreon JC, Nakamura M, Ferreon AC, Ikeda M, et al. Immune evasion by hepatitis C virus NS3/4A protease-‐mediated cleavage of the Toll-‐like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci U S A. 2005;102:2992-‐7. 132 [141] Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R, et al. Cardif is an adaptor protein in the RIG-‐I antiviral pathway and is targeted by hepatitis C virus. Nature. 2005;437:1167-‐72. [142] Johnson CL, Gale M, Jr. CARD games between virus and host get a new player. Trends Immunol. 2006;27:1-‐4. [143] Levin MK, Gurjar M, Patel SS. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase. Nat Struct Mol Biol. 2005;12:429-‐35. [144] Frick DN, Rypma RS, Lam AM, Gu B. The nonstructural protein protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J Biol Chem. 2004;279:1269-‐80. [145] Egger D, Wolk B, Gosert R, Bianchi L, Blum HE, Moradpour D, et al. Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol. 2002;76:5974-‐84. [146] Ago H, Adachi T, Yoshida A, Yamamoto M, Habuka N, Yatsunami K, et al. Crystal structure of the RNA-‐dependent RNA polymerase of hepatitis C virus. Structure. 1999;7:1417-‐26. [147] Lyle JM, Bullitt E, Bienz K, Kirkegaard K. Visualization and functional analysis of RNA-‐dependent RNA polymerase lattices. Science. 2002;296:2218-‐22. [148] Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE, Bienz K, et al. Identification of the hepatitis C virus RNA replication complex in Huh-‐7 cells harboring subgenomic replicons. J Virol. 2003;77:5487-‐92. [149] Ye J, Wang C, Sumpter R, Jr., Brown MS, Goldstein JL, Gale M, Jr. Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci U S A. 2003;100:15865-‐70. [150] Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell. 2005;19:111-‐22. [151] Andre P, Perlemuter G, Budkowska A, Brechot C, Lotteau V. Hepatitis C virus particles and lipoprotein metabolism. Semin Liver Dis. 2005;25:93-‐104. [152] Molina-‐Jimenez F, Benedicto I, Dao Thi VL, Gondar V, Lavillette D, Marin JJ, et al. Matrigel-‐embedded 3D culture of Huh-‐7 cells as a hepatocyte-‐like polarized system to study hepatitis C virus cycle. Virology.425:31-‐9. [153] Hamilton G. Multicellular spheroids as an in vitro tumor model. Cancer Lett. 1998;131:29-‐34. [154] Mitry RR, Hughes RD, Dhawan A. Progress in human hepatocytes: isolation, culture & cryopreservation. Semin Cell Dev Biol. 2002;13:463-‐7. [155] Liu S, Yang W, Shen L, Turner JR, Coyne CB, Wang T. Tight junction proteins claudin-‐1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J Virol. 2009;83:2011-‐4. [156] Molina-‐Jimenez F, Benedicto I, Dao Thi VL, Gondar V, Lavillette D, Marin JJ, et al. Matrigel-‐embedded 3D culture of Huh-‐7 cells as a hepatocyte-‐like polarized system to study hepatitis C virus cycle. Virology. 2012;425:31-‐9. [157] Sainz B, Jr., TenCate V, Uprichard SL. Three-‐dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol J. 2009;6:103. [158] Streetz K, Fregien B, Plumpe J, Korber K, Kubicka S, Sass G, et al. Dissection of the intracellular pathways in hepatocytes suggests a role for Jun kinase and IFN regulatory factor-‐1 in Con A-‐induced liver failure. J Immunol. 2001;167:514-‐ 23. 133 [159] Jones CT, Catanese MT, Law LM, Khetani SR, Syder AJ, Ploss A, et al. Real-‐ time imaging of hepatitis C virus infection using a fluorescent cell-‐based reporter system. Nat Biotechnol. 2010;28:167-‐71. [160] Hantz O, Parent R, Durantel D, Gripon P, Guguen-‐Guillouzo C, Zoulim F. Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-‐like cells. J Gen Virol. 2009;90:127-‐35. [161] Schwartz RE, Trehan K, Andrus L, Sheahan TP, Ploss A, Duncan SA, et al. Modeling hepatitis C virus infection using human induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2012;109:2544-‐8. [162] Xia L, Sakban RB, Qu Y, Hong X, Zhang W, Nugraha B, et al. Tethered spheroids as an in vitro hepatocyte model for drug safety screening. Biomaterials.33:2165-‐76. [163] Zhang S, Tong W, Zheng B, Susanto TA, Xia L, Zhang C, et al. A robust high-‐ throughput sandwich cell-‐based drug screening platform. Biomaterials.32:1229-‐ 41. [164] Bhatia SN, Balis UJ, Yarmush ML, Toner M. Microfabrication of hepatocyte/fibroblast co-‐cultures: role of homotypic cell interactions. Biotechnol Prog. 1998;14:378-‐87. [165] Riccalton-‐Banks L, Liew C, Bhandari R, Fry J, Shakesheff K. Long-‐term culture of functional liver tissue: three-‐dimensional coculture of primary hepatocytes and stellate cells. Tissue Eng. 2003;9:401-‐10. [166] Zinchenko YS, Schrum LW, Clemens M, Coger RN. Hepatocyte and kupffer cells co-‐cultured on micropatterned surfaces to optimize hepatocyte function. Tissue Eng. 2006;12:751-‐61. [167] Tuschl G, Hrach J, Walter Y, Hewitt PG, Mueller SO. Serum-‐free collagen sandwich cultures of adult rat hepatocytes maintain liver-‐like properties long term: a valuable model for in vitro toxicity and drug-‐drug interaction studies. Chem Biol Interact. 2009;181:124-‐37. [168] Zhang P, Tian X, Chandra P, Brouwer KL. Role of glycosylation in trafficking of Mrp2 in sandwich-‐cultured rat hepatocytes. Mol Pharmacol. 2005;67:1334-‐41. [169] Annaert PP, Turncliff RZ, Booth CL, Thakker DR, Brouwer KL. P-‐ glycoprotein-‐mediated in vitro biliary excretion in sandwich-‐cultured rat hepatocytes. Drug Metab Dispos. 2001;29:1277-‐83. [170] Chandra P, Lecluyse EL, Brouwer KL. Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-‐cultured rat hepatocytes. In Vitro Cell Dev Biol Anim. 2001;37:380-‐5. [171] Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, et al. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul Toxicol Pharmacol. 2000;32:56-‐67. [172] Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Hynan LS, et al. Acetaminophen-‐induced acute liver failure: results of a United States multicenter, prospective study. Hepatology. 2005;42:1364-‐72. [173] Mehendale HM. Tissue repair: an important determinant of final outcome of toxicant-‐induced injury. Toxicol Pathol. 2005;33:41-‐51. [174] Lee WM. Drug-‐induced hepatotoxicity. N Engl J Med. 2003;349:474-‐85. [175] Boelsterli UA. Disease-‐related determinants of susceptibility to drug-‐ induced idiosyncratic hepatotoxicity. Curr Opin Drug Discov Devel. 2003;6:81-‐ 91. 134 [176] Ostapowicz G, Fontana RJ, Schiodt FV, Larson A, Davern TJ, Han SH, et al. Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann Intern Med. 2002;137:947-‐54. [177] Tam YK. Individual variation in first-‐pass metabolism. Clin Pharmacokinet. 1993;25:300-‐28. [178] Schwabe RF, Brenner DA. Mechanisms of Liver Injury. I. TNF-‐alpha-‐ induced liver injury: role of IKK, JNK, and ROS pathways. Am J Physiol Gastrointest Liver Physiol. 2006;290:G583-‐9. [179] Jaeschke H, Bajt ML. Intracellular signaling mechanisms of acetaminophen-‐ induced liver cell death. Toxicol Sci. 2006;89:31-‐41. [180] Schrem H, Klempnauer J, Borlak J. Liver-‐enriched transcription factors in liver function and development. Part I: the hepatocyte nuclear factor network and liver-‐specific gene expression. Pharmacol Rev. 2002;54:129-‐58. [181] Xu L, Hui L, Wang S, Gong J, Jin Y, Wang Y, et al. Expression profiling suggested a regulatory role of liver-‐enriched transcription factors in human hepatocellular carcinoma. Cancer Res. 2001;61:3176-‐81. [182] Donato MT, Castell JV. Strategies and molecular probes to investigate the role of cytochrome P450 in drug metabolism: focus on in vitro studies. Clin Pharmacokinet. 2003;42:153-‐78. [183] Wrighton SA, Stevens JC. The human hepatic cytochromes P450 involved in drug metabolism. Crit Rev Toxicol. 1992;22:1-‐21. [184] Li P, Wang GJ, Robertson TA, Roberts MS. Liver transporters in hepatic drug disposition: an update. Curr Drug Metab. 2009;10:482-‐98. [185] Faber KN, Muller M, Jansen PL. Drug transport proteins in the liver. Adv Drug Deliv Rev. 2003;55:107-‐24. [186] Pang KS. Safety testing of metabolites: Expectations and outcomes. Chem Biol Interact. 2009;179:45-‐59. [187] Hewitt NJ, Lechon MJ, Houston JB, Hallifax D, Brown HS, Maurel P, et al. Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev. 2007;39:159-‐234. [188] Willson TM, Kliewer SA. PXR, CAR and drug metabolism. Nat Rev Drug Discov. 2002;1:259-‐66. [189] Quattrochi LC, Vu T, Tukey RH. The human CYP1A2 gene and induction by 3-‐methylcholanthrene. A region of DNA that supports AH-‐receptor binding and promoter-‐specific induction. J Biol Chem. 1994;269:6949-‐54. [190] Whitlock JP, Jr. Induction of cytochrome P4501A1. Annu Rev Pharmacol Toxicol. 1999;39:103-‐25. [191] Mimura J, Fujii-‐Kuriyama Y. Functional role of AhR in the expression of toxic effects by TCDD. Biochim Biophys Acta. 2003;1619:263-‐8. [192] Kazlauskas A, Poellinger L, Pongratz I. Evidence that the co-‐chaperone p23 regulates ligand responsiveness of the dioxin (Aryl hydrocarbon) receptor. J Biol Chem. 1999;274:13519-‐24. [193] Hankinson O, Brooks BA, Weir-‐Brown KI, Hoffman EC, Johnson BS, Nanthur J, et al. Genetic and molecular analysis of the Ah receptor and of Cyp1a1 gene expression. Biochimie. 1991;73:61-‐6. 135 [194] Brunnberg S, Andersson P, Lindstam M, Paulson I, Poellinger L, Hanberg A. The constitutively active Ah receptor (CA-‐Ahr) mouse as a potential model for dioxin exposure-‐-‐effects in vital organs. Toxicology. 2006;224:191-‐201. [195] Swanson HI, Bradfield CA. The AH-‐receptor: genetics, structure and function. Pharmacogenetics. 1993;3:213-‐30. [196] Gonzalez-‐Fraguela ME, Cespedes EM, Arencibia R, Broche F, Gomez AA, Castellano O, et al. [Indicators of oxidative stress and the effect of antioxidant treatment in patients with primary Parkinson disease]. Rev Neurol. 1998;26:28-‐ 33. [197] Zaher H, Fernandez-‐Salguero PM, Letterio J, Sheikh MS, Fornace AJ, Jr., Roberts AB, et al. The involvement of aryl hydrocarbon receptor in the activation of transforming growth factor-‐beta and apoptosis. Mol Pharmacol. 1998;54:313-‐ 21. [198] Fallone F, Villard PH, Seree E, Rimet O, Nguyen QB, Bourgarel-‐Rey V, et al. Retinoids repress Ah receptor CYP1A1 induction pathway through the SMRT corepressor. Biochem Biophys Res Commun. 2004;322:551-‐6. [199] Androutsopoulos VP, Tsatsakis AM, Spandidos DA. Cytochrome P450 CYP1A1: wider roles in cancer progression and prevention. BMC Cancer. 2009;9:187. [200] Kliewer SA, Goodwin B, Willson TM. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr Rev. 2002;23:687-‐702. [201] Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA. The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest. 1998;102:1016-‐23. [202] Pascussi JM, Jounaidi Y, Drocourt L, Domergue J, Balabaud C, Maurel P, et al. Evidence for the presence of a functional pregnane X receptor response element in the CYP3A7 promoter gene. Biochem Biophys Res Commun. 1999;260:377-‐81. [203] Pascussi JM, Gerbal-‐Chaloin S, Fabre JM, Maurel P, Vilarem MJ. Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation. Mol Pharmacol. 2000;58:1441-‐50. [204] Staudinger J, Liu Y, Madan A, Habeebu S, Klaassen CD. Coordinate regulation of xenobiotic and bile acid homeostasis by pregnane X receptor. Drug Metab Dispos. 2001;29:1467-‐72. [205] Dussault I, Lin M, Hollister K, Wang EH, Synold TW, Forman BM. Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR. J Biol Chem. 2001;276:33309-‐12. [206] Drocourt L, Pascussi JM, Assenat E, Fabre JM, Maurel P, Vilarem MJ. Calcium channel modulators of the dihydropyridine family are human pregnane X receptor activators and inducers of CYP3A, CYP2B, and CYP2C in human hepatocytes. Drug Metab Dispos. 2001;29:1325-‐31. [207] Moore LB, Goodwin B, Jones SA, Wisely GB, Serabjit-‐Singh CJ, Willson TM, et al. St. John's wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc Natl Acad Sci U S A. 2000;97:7500-‐2. [208] Sueyoshi T, Negishi M. Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annu Rev Pharmacol Toxicol. 2001;41:123-‐ 43. 136 [209] Watt AJ, Garrison WD, Duncan SA. HNF4: a central regulator of hepatocyte differentiation and function. Hepatology. 2003;37:1249-‐53. [210] Handschin C, Meyer UA. Induction of drug metabolism: the role of nuclear receptors. Pharmacol Rev. 2003;55:649-‐73. [211] Tzameli I, Pissios P, Schuetz EG, Moore DD. The xenobiotic compound 1,4-‐ bis[2-‐(3,5-‐dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR. Mol Cell Biol. 2000;20:2951-‐8. [212] Zhang J, Huang W, Chua SS, Wei P, Moore DD. Modulation of acetaminophen-‐induced hepatotoxicity by the xenobiotic receptor CAR. Science. 2002;298:422-‐4. [213] Forman BM, Goode E, Chen J, Oro AE, Bradley DJ, Perlmann T, et al. Identification of a nuclear receptor that is activated by farnesol metabolites. Cell. 1995;81:687-‐93. [214] Kostadinova R, Wahli W, Michalik L. PPARs in diseases: control mechanisms of inflammation. Curr Med Chem. 2005;12:2995-‐3009. [215] Mehendale HM. PPAR-‐alpha: a key to the mechanism of hepatoprotection by clofibrate. Toxicol Sci. 2000;57:187-‐90. [216] Kane CD, Francone OL, Stevens KA. Differential regulation of the cynomolgus, human, and rat acyl-‐CoA oxidase promoters by PPARalpha. Gene. 2006;380:84-‐94. [217] Keller BJ, Yamanaka H, Thurman RG. Inhibition of mitochondrial respiration and oxygen-‐dependent hepatotoxicity by six structurally dissimilar peroxisomal proliferating agents. Toxicology. 1992;71:49-‐61. [218] Corton JC, Lapinskas PJ, Gonzalez FJ. Central role of PPARalpha in the mechanism of action of hepatocarcinogenic peroxisome proliferators. Mutat Res. 2000;448:139-‐51. [219] Wakabayashi K, Tamura A, Saito H, Onishi Y, Ishikawa T. Human ABC transporter ABCG2 in xenobiotic protection and redox biology. Drug Metab Rev. 2006;38:371-‐91. [220] Guastadisegni C, Balduzzi M, Mancuso MT, Di Consiglio E. Liver mitochondria alterations in chloroform-‐treated Sprague-‐Dawley rats. J Toxicol Environ Health A. 1999;57:415-‐29. [221] Kukielka E, Cederbaum AI. NADPH-‐ and NADH-‐dependent oxygen radical generation by rat liver nuclei in the presence of redox cycling agents and iron. Arch Biochem Biophys. 1990;283:326-‐33. [222] James LP, Mayeux PR, Hinson JA. Acetaminophen-‐induced hepatotoxicity. Drug Metab Dispos. 2003;31:1499-‐506. [223] Thummel KE, Lee CA, Kunze KL, Nelson SD, Slattery JT. Oxidation of acetaminophen to N-‐acetyl-‐p-‐aminobenzoquinone imine by human CYP3A4. Biochem Pharmacol. 1993;45:1563-‐9. [224] Kitteringham NR, Powell H, Clement YN, Dodd CC, Tettey JN, Pirmohamed M, et al. Hepatocellular response to chemical stress in CD-‐1 mice: induction of early genes and gamma-‐glutamylcysteine synthetase. Hepatology. 2000;32:321-‐ 33. [225] Johansson I, Ekstrom G, Scholte B, Puzycki D, Jornvall H, Ingelman-‐ Sundberg M. Ethanol-‐, fasting-‐, and acetone-‐inducible cytochromes P-‐450 in rat liver: regulation and characteristics of enzymes belonging to the IIB and IIE gene subfamilies. Biochemistry. 1988;27:1925-‐34. 137 [226] Goeptar AR, Scheerens H, Vermeulen NP. Oxygen and xenobiotic reductase activities of cytochrome P450. Crit Rev Toxicol. 1995;25:25-‐65. [227] Gibson JD, Pumford NR, Samokyszyn VM, Hinson JA. Mechanism of acetaminophen-‐induced hepatotoxicity: covalent binding versus oxidative stress. Chem Res Toxicol. 1996;9:580-‐5. [228] Park BK, Kitteringham NR, Maggs JL, Pirmohamed M, Williams DP. The role of metabolic activation in drug-‐induced hepatotoxicity. Annu Rev Pharmacol Toxicol. 2005;45:177-‐202. [229] Gao B. Cytokines, STATs and liver disease. Cell Mol Immunol. 2005;2:92-‐ 100. [230] Poynard T, Leroy V, Cohard M, Thevenot T, Mathurin P, Opolon P, et al. Meta-‐analysis of interferon randomized trials in the treatment of viral hepatitis C: effects of dose and duration. Hepatology. 1996;24:778-‐89. [231] Weng HL, Ciuclan L, Liu Y, Hamzavi J, Godoy P, Gaitantzi H, et al. Profibrogenic transforming growth factor-‐beta/activin receptor-‐like kinase signaling via connective tissue growth factor expression in hepatocytes. Hepatology. 2007;46:1257-‐70. [232] Rodriguez-‐Antona C, Donato MT, Pareja E, Gomez-‐Lechon MJ, Castell JV. Cytochrome P-‐450 mRNA expression in human liver and its relationship with enzyme activity. Arch Biochem Biophys. 2001;393:308-‐15. [233] Jasmund I, Bader A. Bioreactor developments for tissue engineering applications by the example of the bioartificial liver. Adv Biochem Eng Biotechnol. 2002;74:99-‐109. [234] Wang WW, Khetani SR, Krzyzewski S, Duignan DB, Obach RS. Assessment of a micropatterned hepatocyte coculture system to generate major human excretory and circulating drug metabolites. Drug Metab Dispos.38:1900-‐5. [235] Kang IK, Kim GJ, Kwon OH, Ito Y. Co-‐culture of hepatocytes and fibroblasts by micropatterned immobilization of beta-‐galactose derivatives. Biomaterials. 2004;25:4225-‐32. [236] Qian L, Krause DS, Saltzman WM. Enhanced growth and hepatic differentiation of fetal liver epithelial cells through combinational and temporal adjustment of soluble factors. Biotechnol J.7:440-‐8. [237] Donato MT, Castell JV, Gomez-‐Lechon MJ. Co-‐cultures of hepatocytes with epithelial-‐like cell lines: expression of drug-‐biotransformation activities by hepatocytes. Cell Biol Toxicol. 1991;7:1-‐14. [238] Kidambi S, Yarmush RS, Novik E, Chao P, Yarmush ML, Nahmias Y. Oxygen-‐ mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance. Proc Natl Acad Sci U S A. 2009;106:15714-‐9. [239] Gramatzki D, Pantazis G, Schittenhelm J, Tabatabai G, Kohle C, Wick W, et al. Aryl hydrocarbon receptor inhibition downregulates the TGF-‐beta/Smad pathway in human glioblastoma cells. Oncogene. 2009;28:2593-‐605. [240] Muller GF, Dohr O, El-‐Bahay C, Kahl R, Abel J. Effect of transforming growth factor-‐beta1 on cytochrome P450 expression: inhibition of CYP1 mRNA and protein expression in primary rat hepatocytes. Arch Toxicol. 2000;74:145-‐52. [241] Schuster N, Krieglstein K. Mechanisms of TGF-‐beta-‐mediated apoptosis. Cell Tissue Res. 2002;307:1-‐14. 138 [242] Zhang C, Chia SM, Ong SM, Zhang S, Toh YC, van Noort D, et al. The controlled presentation of TGF-‐beta1 to hepatocytes in a 3D-‐microfluidic cell culture system. Biomaterials. 2009;30:3847-‐53. [243] Pocar P, Fischer B, Klonisch T, Hombach-‐Klonisch S. Molecular interactions of the aryl hydrocarbon receptor and its biological and toxicological relevance for reproduction. Reproduction. 2005;129:379-‐89. [244] Southam AD, Easton JM, Stentiford GD, Ludwig C, Arvanitis TN, Viant MR. Metabolic changes in flatfish hepatic tumours revealed by NMR-‐based metabolomics and metabolic correlation networks. J Proteome Res. 2008;7:5277-‐85. [245] Janakiraman V, Sastry S, Kadambi JR, Baskaran H. Experimental investigation and computational modeling of hydrodynamics in bifurcating microchannels. Biomed Microdevices. 2008;10:355-‐65. [246] Di Ventura B, Lemerle C, Michalodimitrakis K, Serrano L. From in vivo to in silico biology and back. Nature. 2006;443:527-‐33. [247] Howe K, Gibson GG, Coleman T, Plant N. In silico and in vitro modeling of hepatocyte drug transport processes: importance of ABCC2 expression levels in the disposition of carboxydichlorofluroscein. Drug Metab Dispos. 2009;37:391-‐9. [248] Cosgrove BD, Alexopoulos LG, Hang TC, Hendriks BS, Sorger PK, Griffith LG, et al. Cytokine-‐associated drug toxicity in human hepatocytes is associated with signaling network dysregulation. Mol Biosyst.6:1195-‐206. [249] Billiar TR, Curran RD, Ferrari FK, Williams DL, Simmons RL. Kupffer cell:hepatocyte cocultures release nitric oxide in response to bacterial endotoxin. J Surg Res. 1990;48:349-‐53. [250] Groneberg DA, Grosse-‐Siestrup C, Fischer A. In vitro models to study hepatotoxicity. Toxicol Pathol. 2002;30:394-‐9. [251] Pennie WD, Tugwood JD, Oliver GJ, Kimber I. The principles and practice of toxigenomics: applications and opportunities. Toxicol Sci. 2000;54:277-‐83. [252] Ellinger-‐Ziegelbauer H, Stuart B, Wahle B, Bomann W, Ahr HJ. Characteristic expression profiles induced by genotoxic carcinogens in rat liver. Toxicol Sci. 2004;77:19-‐34. [253] McMillian M, Nie A, Parker JB, Leone A, Kemmerer M, Bryant S, et al. Drug-‐ induced oxidative stress in rat liver from a toxicogenomics perspective. Toxicol Appl Pharmacol. 2005;207:171-‐8. [254] Kong AN, Owuor E, Yu R, Hebbar V, Chen C, Hu R, et al. Induction of xenobiotic enzymes by the MAP kinase pathway and the antioxidant or electrophile response element (ARE/EpRE). Drug Metab Rev. 2001;33:255-‐71. [255] Pawlotsky JM, Germanidis G, Neumann AU, Pellerin M, Frainais PO, Dhumeaux D. Interferon resistance of hepatitis C virus genotype 1b: relationship to nonstructural 5A gene quasispecies mutations. J Virol. 1998;72:2795-‐805. [256] Saito T, Hirai R, Loo YM, Owen D, Johnson CL, Sinha SC, et al. Regulation of innate antiviral defenses through a shared repressor domain in RIG-‐I and LGP2. Proc Natl Acad Sci U S A. 2007;104:582-‐7. 139 [...]... Brenner [25]) 1.5 In vitro cellular models for drug development The choice of the cell type to be used for drug development application depends mainly on factors such as application and cost In vitro cellular models are mainly used for early screening of toxic events and mechanistic evaluations of drug toxicity and propagation of liver specific pathogens [26] However in vitro models have significant disadvantages... xenobiotics (e.g drugs and pathogens) Here is discussed the trends in these two areas, for rational tissue engineering approach for better maintenance of liver phenotype for the above mentioned applications 1.7 Toolbox development for precision liver tissue engineering in vitro Engineering microtissue constructs with bottom-up approaches for in vivo and in vitro applications requires the development of... cure chronic hepatitis C of genotype 1 and 2 1.2 Need for in vitro models From these pitfalls observed with the current strategy, there is a huge unmet need for in vitro models for developing new drug entities and predictive toxicology The concept of fail early, fail cheap will have considerable economic value to pharmaceutical companies These models could also have tremendous implications by contributing... level and also aid in identification of novel targets and leads [8] For various toxicology applications these models could aid in understanding mechanisms of toxicity and can aid in understanding the toxicity detected during preclinical studies This information can be then obtained and rational drug design can be performed [9] These models can also 14 be used to ascertain human risk during preclinical... and absence of TGF β1 normalized to uninduced controls Figure 39: Hepatocyte excretory function 6 List of Tables: Table 1: Various cellular models for drug development and their advantages and disadvantages Table 2: Integration of various tissue engineered models into applications Table 3: Viral evasion strategies Table 4: Human hepatocyte specific primers Table 5: Characterization of spheroid number... physiologically relevant as possible Over the years many models have 21 been developed or utilized for studying liver toxicity and to study the life cycle of the hepatitis C virus in vitro Below is a schematic of the different in vitro models and the characterization of these based on application and practicality Figure 8 :In vitro and in vivo models used in drug development (Adapted with permission from Brandon... even today for covering the wound and preventing infection, without concerns for finer skin-features, such as wrinkles or hair follicles, that are important for aesthetics and perspiration, as reviewed by McNeil [37] Liver tissue engineering research began with the development of hybrid liver-support systems [38] and cell-seeded scaffolds for stimulating liver regeneration [39] These initial efforts employed...List of figures: Figure 1 Schematic of drug discovery process Figure 2: Reasons for drug failure Figure 3 Global in vitro toxicology market estimates Figure 4: Various liver functions Figure 5: Lobular model of the liver Figure 6: Acinar model of the liver Figure 7: Various liver cells and their arrangement Figure 8: in vitro and in vivo models used in drug development Figure 9: Various technologies... makes it a very time consuming and expensive process [1] Figure 1: Schematic of drug discovery process (Adapted from www.gsdpharmaconsulting.com) The discovery phase starts with identification of the best targets specific to a disease Identification of drug targets allows for chemists and biologists to perform targeted drug discovery by high throughput screening of existing chemical or biological libraries... number of compounds from early phases of drug discovery to compounds entering clinical trials It is less than 0.1% of the compounds developed initially are suitable for testing on human subjects [3] Due to such stringent control and safety and efficacy assessment it takes between 6-9 years for one 12 compound to enter the market as a marketable drug [1] It can therefore be observed that there is a huge . ! ! ENGINEERED HEPATOCELLULAR MODELS FOR DRUG DEVELOPMENT& quot; ABHISHEK ANANTHANARAYANAN B.TECH, SRMIST A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. In vitro cellular models for drug development 1.6 Tissue engineering approaches and paradigms 1.7 Toolbox development for precision tissue engineering 1.7.1 Biomaterials for cellular assembly. List of symbols 8 Chapter 1 Introduction to drug development 11 1.1 Introduction to drug development process 1.2 Need for in vitro models 1.3 Structure function relationship of the
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