Engineered hepatocellular models for drug development

“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. 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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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