Therapeutics
Figure 3: Inducible in vitro NASH model. In humans, the progression of NASH starts with lipid accumulation in the hepatocytes (steatosis). High content imaging of this 3D multicellular human liver model shows the changes in model phenotype under healthy control conditions (A) and after NASH induction with a specialised media that contains higher sugar levels and free fatty acids (lipids). Treatment with low (C) and high (D) concentrations of an anti- steatotic clinical drug candidate leads to a decrease in intracellular lipids. Nile Red staining (magenta) captures a normal amount of lipids (green) in the control and after treatment with high drug concentrations of the drug, whereas steatotic hepatocytes are abnormally enlarged and filled with lipid vacuoles. Hoechst staining for nuclei (blue) further highlights macrovesicular steatosis (engorgement of hepatocytes by lipids that displace nuclei), mimicking the fatty liver disease state in humans. Photo courtesy of InSphero AG, imaged on a Yokogawa high-content screening system
References 1Younossi, ZM, Koenig, AB, Abdelatif, D, Fazel, Y, Henry, L, Wymer, M (2016). Global epidemiology of nonalcoholic fatty liver disease – Meta analytic assessment of prevalence, incidence, and outcomes. Hepatology, 64:73- 84. doi:10.1002/hep.28431.
https://aasldpubs.onlinelibrary.
wiley.com/doi/full/10.1002/hep. 28431. 2 Diehl, AM, Day, C (2017). Cause, pathogenesis, and treatment of nonalcoholic steatohepatitis. The New England Journal of Medicine, 377:2064-2072. doi: 10.1056/NEJMra1503519.
https://www.nejm.org/doi/full/1 0.1056/NEJMra1503519. 3 Charlton, MR, Burns, JM, Pedersen, RA, Watt, KD, Heimbach, JK, Dierkhising, RA (2011). Frequency and outcomes of liver transplantation for nonalcoholic steatohepatitis in the United States. Gastroenterology. Oct;141(4):1249-53. doi: 10.1053/
j.gastro.2011.06.061.
https://www.gastrojournal.org/a rticle/S0016-5085(11)00911- 5/fulltext?referrer=https%3A%2
F%2Fwww.ncbi.nlm.nih.gov%2F.
and the extracellular environment results in signif- icant changes in cell morphology, polarity and function. For example, primary hepatocytes are known to become dedifferentiated and quickly lose their metabolic functionality (within 2-3 days) in 2D culture, whereas HepG2 simply lack these metabolic capabilities. As hepatocyte metabolism plays a crucial in NASH pathophysiology, the time window for disease induction and drug efficacy testing is therefore quite limited when working in 2D. While 2D cultured HepG2 cells and primary hepatocytes cultured are still quite useful for inves- tigating lipid loading and de novo lipogenesis (DNL) aspects of NASH, the primary limitation of these models is the lack of the downstream non- parenchymal cell contributions to the progression of NASH. Similarly, stellate cell lines (eg LX-1 and LX-2)
Continued on page 65 64
and primary hepatic stellate cells have been valu- able for studying the fibrotic aspect of NASH, because stellate cells play a pivotal role in the ini- tiation, progression and regression of liver fibro- sis. However, as with 2D hepatocyte monocul- ture, 2D stellate cell monoculture models ignore the contribution and interplay of other liver cell types essential for NASH initiation and progres- sion. In addition, 2D stellate cell models harbour another serious liability: stellate cells become spontaneously activated upon attaching to plastic surfaces in culture. These artificially-activated stellate cells should not be used to evaluate the efficacy of drug candidates that target slowly pro- gressing activation processes, as observed in NASH. The shortcomings of 2D models are particularly problematic for investigators working on new ther-
apies for complex diseases, such as NASH. Recent advances in cell culture techniques are now enabling more complex and robust disease models. For example, it is now possible to incorporate mul- tiple cell types, along with biochemical and biome- chanical micro-environments, to better mimic in vivo pathophysiology. These ‘next gen’ models are driving greater understanding of the underlying mechanisms and progression of disease and should, in turn, help identify the most promising drug can- didates to advance to clinical trials. It is also impor- tant to consider that NASH develops in humans over the course of years, if not decades. Researchers focusing on NASH model development must tackle the monumental task of condensing what occurs in humans over 10 or more years… in a dish, within an extremely accelerated timeframe. In summary, to meet the needs of today’s pharma
drug discovery efforts, in vitro NASH models must:
l Recreate the pathophysiology of the progression of NAFLD and NASH in humans, from lipid accu- mulation in hepatocytes (steatosis), inflammatory response (hepatitis) and fibrotic scarring (fibrosis). l Mimic chronic drug exposure of clinical treat- ment programmes by maintaining longevity in cul- ture for longitudinal, repeat-dose treatments. l Reflect the human response to disease inhibitors/inducers, enable monitoring of path- ways perturbed in the disease state and predict patient response to treatment dosing in the clinic. l Deliver robust functionality to ensure repro- ducible and comparable results over multiple experimental assays. l Enable scalable, screening-compatible, clinically-
Drug Discovery World Fall 2019
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68
