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Therapeutics


relevant endpoint readouts that capture pathologi- cal aspects of NASH.


A new era of game-changing NASH models Over the past few years, enormous strides have been made in cell-based NASH model develop- ment. Several research groups have engineered advanced models for studying NASH (see Figure 2). For example, in 2016 scientists at Hemoshear established an in vitroNASH model that combined flow technology with primary cell culture in a tran- swell format. In their model, primary hepatocytes cultured on a collagen-matrix are separated from stellate cells and Kupffer cells by a polycarbonate membrane. Upon exposure to lipotoxic stimuli, key hallmarks of NASH, including lipid loading, changes in hepatocyte metabolism and stellate cell activation, can be recapitulated8. Researchers at AstraZeneca and the University


of Gothenburg have furthered the field by collabo- rating on 3D spheroid co-cultures of HepG2 with LX-2 cell lines. FFA treatment in this co-culture model leads to significant lipid loading and stellate cell activation9. Compared to the Hemoshear model, this approach achieves much greater throughput, as it utilises a fraction of the number of cells, in a 96-well format. The team also used a monoculture of individual donor primary hepato- cytes in a similar spheroid format to demonstrate important differences in lipid metabolism depen- dent on the genetic background of individual donors10. Aiming to combine the advantages of primary


liver cells within a scalable 3D culture system, InSphero developed a 3D human liver NASH model that includes all liver cell types thought to be involved in the development of NASH: hepato- cytes, hepatic stellate cells, liver endothelial cells and Kupffer cells. This model is disease tunable in that treatment with FFA, sugars and inflammatory stimuli induce lipid accumulation in hepatocytes (steatosis), release pro-inflammatory cytokines and chemokines from Kupffer cells (inflammation) and deposit fibril collagens (fibrosis) by stellate cells11 (see Figure 3). As in humans, this model progresses over multiple stages upon induction with NASH stimuli, but the process is condensed down to two weeks. With this compact induction protocol, this model offers a significant advantage over mouse models, which typically require sever- al months to achieve experimental maturity. Furthermore, since combination therapies may be needed for effectively fighting NASH, this approach provides a promising tool for high


Drug Discovery World Fall 2019


throughput screening with various mechanisms of action. Each of these advanced human cell-based


approaches can be used to complement findings from mouse models and optimise use of animal testing in discovery programmes. Aside from issues such as relevance, scalability and predictivity, rodent models are most useful for single ‘snapshot’ endpoints, typically collected at the completion of the study (eg sacrifice of the animals and histopathological assessment). For progressive complex diseases like NASH, it is especially crucial to gain more insight in the dynamics and kinetics of the underlying mechanisms. The latest genera- tion of advanced in vitro models retain longevity, and when combined with newer sophisticated methods such as live cell imaging, allow deconvo- lution of the sequential steps in pathophysiology of NASH. From another perspective, they can also be used to generate a large amount of relevant data (eg, combination therapies, multiple timepoints, numerous endpoints) in a much shorter timeframe than possible with an animal study. Theoretically, this should allow for a much more rapid prioritisa- tion of promising drug combinations.


A drug developer’s wish list for in vitro human NASH models Of course, there are still major hurdles to be over- come with cell-based models. Even the most advanced in vitro human NASH models currently lack extra-hepatic contributions to the progression of NASH, such as circulating blood macrophages (important for inflammation and further activation of stellate cells), intestinal cells (important for FXR-stimulated release of FGF-19), or gut micro- bial imbalances. Furthermore, the increasing com- plexity of cellular models makes robust experimen- tal readouts more challenging to implement. The amount of data increases remarkably in complex cellular assays, strengthening the need for appro- priate tools for data management, analysis and interpretation. The rapid progress in cutting-edge technologies, such as high-content imaging (see Figure 3) or single cell sequencing, as well as the increasing availability of sophisticated bioinfor- matic tools is encouraging, however, fitting these technologies to the needs of complex in vitro mod- els remains a major challenge. The interpretation of results – putting them in


context with existing data from in vivo and in vitro studies – is also a challenge with 3D models. Ironically, the difficulty here is due to the complex- ity of model. As with animal studies, it is not easy to determine pathways leading to an unexpected


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4 Grant, C (2019). A Big Fatty Opportunity for Big Pharma. The Wall Street Journal, Jan 18, 2019, U.S. Edition. https://www.wsj.com/articles/a- big-fatty-opportunity-for-big- pharma-11547807401. 5 National Institutes of Health (NIH) U.S. National Library of Medicine ClinicalTrials.gov. https://clinicaltrials.gov/. 6 Farell, G, Schattenberg, JM, Leclercq, I, Yeh, MM, Goldin, R, Teoh, N, Schuppan, D (2018). Mouse Models of Nonalcoholic Steatohepatitis: Toward Optimization of Their Relevance to Human Nonalcoholic Steatohepatitis. Hepatology, 69: 2241-2257. doi:10.1002/hep.30333. https://aasldpubs.onlinelibrary. wiley.com/doi/abs/10.1002/hep. 30333. 7 Asgharpour, A, Cazanave, SC, Pacana, T, Seneshaw, M, Vincent, R, Banini, BA, Kumar, DP, Daita, K, Min, H, Mirshahi, F, Bedossa, P, Sun, X, Hoshida, Y, Koduru, SV, Contaifer, D, Warncke, UO, Wijesinghe ,DS, Sanyal, AJ (2016). A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer. Journal of Hepatology, 65(3):579-588. doi:10.1016/j.jhep.2016.05.005. https://www.journal-of- hepatology.eu/article/S0168- 8278(16)30190-8/fulltext. 8 Feaver, RE, Banamathi, KC, Lawson, MJ, Hoang, SA, Marukian, S, Blackman, BR, Figler, RA, Sanyal, AJ, Wamhoff, BR, Dash, A (2016). Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis. JCI Insight, 1(20)e90954. doi:10.1172/ jci.insight.90954. https://insight. jci.org/articles/view/90954. 9 Pingitore, P, Sasidharan, K, Ekstrand, M, Prill, S, Lindén, D, Romeo, S (2019). Human Multilineage 3D Spheroids as a Model of Liver Steatosis and Fibrosis. Int. J. Mol. Sci, 20(7):1629. https://www.mdpi. com/1422-0067/20/7/1629.


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