EXPERIMENTAL MODELS FOR NASH NAFLD LIVER DISEASES.pptx

THIS PPT GIVES AN IDEA ABOUT PHARMACOLOGICAL SCREENING METHODS.PHARMACOLOGICAL SCREENING MODELS
PHARMACY MODELS

1. PHARMACOLOGICAL SCREENING MODELS FOR NAFLD Presented by Payaam Vohra 23PCM3895 2 nd Semester

2. NAFLD in the general population Metabolic risk factors (T2DM, obesity, unhealthy diet, sedentary lifestyle) Genetic polymorphisms PNPLA3 or TM6SF2 1 1.5–6.5% 4 ~0.5% each 4–5 person NASH responsible for > 50% cases of cryptogenic cirrhosis 6 20–25% 2 ?? 10–20% 3 NASH NAFLD CIRRHOSIS Chronic inflammation/NASH Fibrosis HCC, hepatocellular carcinoma; NAFLD, non- alcoholic fatty liver disease; NASH, non- alcoholic steatohepatitis; PNPLA3, patatin- like phospholipase domain-containing protein 3; TM6SF2, transmembrane 6 superfamily member 2; T2DM, type 2 diabetes mellitus. 1. Meroni M, et al. Int J Mol Sci 2020;21:2986; 2. Abeysekera K, et al. Lancet Gastroenterol Hepatol 2020;5:295–305; 3. Hashimoto E, et al. JGH 2013;28(Suppl 4):64–70; 4. Younossi ZM, et al. Clin Liver Dis (Hoboken) 2018;11:92–4; 5. Dam- Larsen S, et al. Gut 2004;53:750–5; 6. Golabi P, et al. Medicine 2018;97:e11518

3. NAFLD/NASH pathogenesis Fructose + Glucose Acetyl CoA Circulating FFA  Adipose tissue  Chylomicrons Triglycerides Liver FFA Triglycerides Liver injury Apoptosis /Inflammation NASH Fibrosis HCC VLDL Hypertriglyceridemia IR Lipogenesis de novo 60% 25% 15% “Multi- hit pathogenesis” ROS ROS Mitochondrial β-oxidation Modifying factors Genes Intestinal microbiome Adipokines Lipotoxic RES Stress lipids PNPLA3 Cytokines Adipokines Acetyl CoA, acetyl coenzyme A; FFA, free fatty acid; HCC, hepatocellular carcinoma; IR, insulin resistance; NAFLD, non-alcoholic fatty liver disease; NASH, non- alcoholic steatohepatitis; PNPLA3, patatin- like phospholipase domain-containing protein 3; ROS, reactive oxygen species; VLDL, very-low- density lipoprotein Softic S, et al. Dig Dis Sci 2016;61:1282–93; Bril F, et al. Diabetes Care 2019;42:1

4. Atherosclerosis Cardiovascular diseases Type 2 diabetes Prediabetic state Obesity Metabolic syndrome NAFLD/NASH IRS FFA GLUCOSE Triglycerydes PAI FFA, free fatty acid; IRS, insulin receptor substrate; NAFLD, non- alcoholic fatty liver disease; NASH, non- alcoholic steatohepatitis; PAI, plasminogen activator inhibitor type 1 Meroni M, et al. Int J Mol Sci 2020;21:2986

5. PATHOGENESIS FOR NASH

6. Lau JK, Zhang X, Yu J. Animal models of non-alcoholic fatty liver disease: current perspectives and recent advances. J Pathol . 2017 Jan;241(1):36-44. doi : 10.1002/path.4829. Model Summary of diet composition Obese Steatosis NASH Fibrosis High‐fat diet The classic reported HFD model comprised 71% fat, 11% carbohydrates, and 18% protein Yes Yes Yes (mild) Yes ob / ob  mice NA Yes Yes No (does not develop spontaneously) No (resistant to fibrosis) db / db  mice NA Yes Yes No (does not develop spontaneously) No (does not develop spontaneously) Methionine and choline‐deficient diet Diet usually consists of sucrose (40% of energy) and fat (10%); however, it is deficient in methionine and choline No Yes Yes Yes High‐cholesterol diet Often fed in conjunction with high fat (15%) or high cholate (0.5%) Yes Yes Yes Yes foz / foz  mice NA Yes Yes Yes Yes Choline‐deficient high‐fat diet 20% protein, 35% carbohydrate, and 45% fat, without choline added Yes Yes Yes Yes Choline‐deficient  l ‐amino acid‐defined diet 28.9 kcal/g  l ‐glutamic acid, 15.8 kcal/g  l ‐aspartic acid, 12.7 kcal/g  l ‐arginine, and 10.5 kcal/g  l ‐leucine, without choline bitartrate Yes Yes Yes Yes Choline‐deficient  l ‐amino acid‐defined diet + carbon tetrachloride 28.9 kcal/g  l ‐glutamic acid, 15.8 kcal/g  l ‐aspartic acid, 12.7 kcal/g  l ‐arginine, and 10.5 kcal/g  l ‐leucine, without choline bitartrate, with CCl 4  injection No Yes Yes Yes High‐fat diet + streptozotocin 24.8% protein, 46.7% nitrogen‐free extract, and 14.4% fat, with 200‐µg streptozotocin injection Yes Yes Yes Yes Hepatocyte‐specific PTEN‐deficient mice NA Yes Yes Yes Db / db  mice + DEN NA Yes Yes Yes ? Animal models of non‐alcoholic fatty liver diseases L ist of Dietary Models for NASH

7. MODELS FOR NASH/NAFLD In vitro models Cell lines Pros Cons Primary cell cultures Hepatocytes from NAFLD patients/rodents Kupffer cells/stellate cells/ iNKT cells from human patients/rodents Mimic in vivo settings Isolation problems Ethical issues Varying reproducibility in experiments Limited culture time Immortalized cell lines RAW 264.7, AML-12, J774A, HepG2, HuH7, H4IIE, H4IIEC3, PAV-1, LX2 Continuous growth Easy to culture Stable phenotype Expression of several enzymes and nuclear factors alter according to the immortalization method Co-culture models RAW 264.7 and AML-12 Human hepatocytes and adipocytes Mimic in vivo liver architecture Important tools in cellular cross talk studies Difficult to cultivate 3D cultures H35 rat hepatoma cell line Mimic in vivo liver architecture Liver specific differentiation and function Tools for transcriptional regulation studies Difficult to cultivate

8. Myeloid cell-derived osteopontin (OPN) protects from diet-induced non-alcoholic fatty liver disease in mice Han H, et al. NAFLD Summit 2019; P06-20 BACKGROUND & AIMS ​ OPN is an extracellular cytokine upregulated in patients with NASH and in myeloid cells in mouse models of NASH Anti-inflammatory effects but an unknown MoA Aim: to investigate the possible NASH protective effects of OPN in myeloid cells METHODS ​ Mice overexpressing OPN in myeloid cells ( Opn Mye Tg ) were generated using  Opn - Stop fl / fl  and recombination with  Lyz2.Cre  mice Opn Mye Tg and Cre + littermates fed Western (high fat) diet or isocaloric diet (up to 6 months) Histological and biochemical assessments and RNA sequencing were performed RESULTS ​ Control mice (particularly males) developed a typical NASH phenotype Steatosis, hepatocyte ballooning degeneration, inflammation and chicken-wire fibrosis Opn Mye Tg  mice (particularly males) were protected from NASH Significant reduction in hepatic triglycerides, cholesterol, immune cell infiltration, and pro- fibrogenic signals

9. Myeloid cell-derived osteopontin (OPN) protects from diet-induced non-alcoholic fatty liver disease in mice Han H, et al. NAFLD Summit 2019; P06-20 RESULTS (Cont.) ​ Female Opn Mye Tg  mice were protected from insulin resistance Opn Mye Tg had a significantly reduced hepatic expression of genes involved in fatty acid transport, beta-oxidation, and cholesterol biosynthesis Opn Mye Tg mice presented with upregulation of the urea cycle Myeloid cells from Opn Mye Tg mice revealed significantly increased Arg2 protein expression, correlated with Opn and associated with downregulation of Nos2 or iNos , Tnf , and Il1b mRNAs CONCLUSIONS OPN in myeloid cells regulates hepatic fatty acid transport, cholesterol biosynthesis, and the urea cycle. Moreover, OPN regulates expression of Arg2 in myeloid cells and reduces pro-inflammatory signalling . Overall, these events contribute to protection from NASH

10. Inhibition of alpha 2A adrenergic receptors reduces liver inflammation and fibrosis in experimental NASH Thomsen KL, et al. NAFLD Summit 2019; P04-11 BACKGROUND & AIMS ​ NAFLD is increasing worldwide; patients with NASH have increased risk of cirrhosis and HCC, whilst fibrogenesis correlates with long-term morbidity and mortality Norepinephrine (NE), through Adra2aR, may trigger development and progression of NAFLD NE stimulates pro-inflammatory cytokines from Kupffer cells; blocking Adra2aR reduces stellate cell activation Aim: To study Adra2aR function in a rat NASH model and Adra2aR antagonism as potential therapy METHODS ‌ Following treatment, formalin-fixed liver sections were stained with H&E and picrosirius red, and CPA was measured Fibrogenic and pro-inflammatory changes were investigated in liver tissue using qPCR, ELISA, and CD68 immunohistochemistry Sprague Dawley rats Diet only 8 weeks CPA, fibrogenic and pro-inflammatory changes assessed HFHC Chow Control Adra2a antagonist 0.4g/kg/day - ve control Diet + treatment 8 weeks R

11. Inhibition of alpha 2A adrenergic receptors reduces liver inflammation and fibrosis in experimental NASH CONCLUSIONS Adra2aR antagonism reduces fibrosis and inflammatory progression in NAFLD, associated with reduced immune activation. Adra2aR antagonism may slow NAFLD progression. The therapeutic potential of Adra2aR warrants further research RESULTS ‌ HFHC increased liver/body weight ratio, NAS score, and CPA, all reduced by YoHCl (Table) TIMP1 expression was increased in HFHC animals and significantly reduced by YoHCl (8.4 [6.1–9.2] vs 13.5 [9.2–25.8], p=0.03) HFHC diet increased CD68 staining (p<0.0001), expression of chemokines (Ccl3, Cx3Cl1, CxCl1, CxCl5; all p<0.001) and protein expression of CxCl5 and Cx3Cl1 All these factors were reduced by YoHCl , indicating lower hepatic inflammation Thomsen KL, et al. NAFLD Summit 2019; P04-11

12. Propionic acid intervention in obese Ldlr -/-.Leiden mice attenuates NASH development, but negatively affects cognition Gart E, et al. NAFLD Summit 2019; P02-16 BACKGROUND & AIMS ​ In the context of the obesity epidemic, which is associated with NASH development and cognitive impairment, there is an increasing interest in elucidating the health effects of short-chain fatty acids (SCFAs) Aim: to explore the effects of the SCFA propionic acid (PA) on obesity-associated NASH development, as well as cognitive dysfunction and structural brain tissue integrity METHODS ‌ Ldlr -/-.Leiden mice Run-in for 17 weeks Analysis of liver and brain tissue HFD Chow PA + HFD Healthy reference Treatment diet for 12 weeks HFD Assessment of cognition and metabolic and inflammatory risk factors

13. Propionic acid intervention in obese Ldlr -/-.Leiden mice attenuates NASH development, but negatively affects cognition Gart E, et al. NAFLD Summit 2019; P02-16 CONCLUSIONS PA treatment during obesity had favourable metabolic effects, reducing body weight gain, improving metabolic risk factors and reducing the development of NASH and associated fibrosis. Simultaneously, PA had detrimental effects on the brain, reducing synaptogenesis and affecting spatial memory. Altogether, the results from this study indicate that while the beneficial metabolic effects of PA treatment seem promising, it can also have negative effects on brain functioning and cognition, and should therefore be treated with caution RESULTS ‌ PA reduced body weight (independent of food intake), fasting insulin levels and systolic blood pressure PA reduced hepatic lipid accumulation, especially cholesterol ester storage, and hepatic inflammation with a corresponding trend towards a reduction in serum amyloid A and hepatic collagen content PA-fed mice showed an increased latency in finding the platform in the Morris water maze, suggesting decreased spatial memory. In addition, we observed alterations in tissue integrity and gene expression in the hippocampus, a brain region important in memory consolidation

14. Establishment of a 3D human liver model to recapitulate NASH progression in vitro Ströbel S, et al. NAFLD Summit 2019; P06-05 BACKGROUND & AIMS ​ Progression to NASH eventually leads to liver cirrhosis/failure​ Aim:  Creation of a 96-well platform based human  in vitro  3D NASH model containing relevant primary liver cell types and following disease specific stimuli to recapitulate hallmarks of NASH such as, steatosis, inflammation and fibrosis METHODS ​ A microtissue model was developed to incorporate relevant primary liver cells like hepatocytes, endothelial cells, Kupffer cells and hepatic stellate cells​ A protocol for induction of NASH with free fatty acids and LPS in medium containing high levels of sugars was developed using liver microtissues to recapitulate NASH pathogenesis and for drug efficacy testing ​ Characteristic end points were measured: ​ Tissue lipid content ( high-content imaging / triglyceride assay ( biochemical assay ))​ Secretion of proinflammatory markers (Luminex)​ Fibrosis (pro-collagen type I secretion [ELISA] and deposition of fibril collagens [histology])​ NGS ( TempO -seq,  BioSpyder )

15. Establishment of a 3D human liver model to recapitulate NASH progression in vitro RESULTS ​ NASH-treated microtissues have shown key physiological manifestations of the disease compared with the control such as:​ Increased tissue lipid accumulation  ​ Secretion of pro-inflammatory markers: TNF- α , IL-6, IL-8, MCP-1, MIP- 1α , and IP-10  ​ Elevated secretion of pro-collagen type I and deposition of fibril collagens ​ NGS analysis of NASH-treated microtissues exhibited differential gene expression signatures for inflammation and TGF-β signalling compared with controls CONCLUSIONS A  human 3D NASH model that recapitulates the key biological aspects of the disease (inflammation, steatosis, and fibrosis) is presented in a novel platform. Traditional biochemical and histological assays were implemented for increased throughput compatibility. ​ Furthermore, with the integration of high-content imaging and sequencing technologies more in-depth information of disease and drug efficacy can be gained. ​ In conclusion, the microtissue based model is a powerful tool for assessing efficacy of anti-NASH drugs.

16. Drugs investigated in phase 3 trials Drug Mechanism Trial Endpoint Obeticholic acid 1 Agonist FXR REGENERATE Improvement of fibrosis (≥ 1 stage) w/out worsening of NASH OR NASH resolution w/out worsening of fibrosis Cenicriviroc 2 Antagonist CCR2/CCR5 AURORA Improvement of fibrosis (≥ 1 stage) w/out worsening of NASH Elafibranor 3 Agonist PPAR α/δ RESOLVE- IT NASH resolution w/out worsening of fibrosis Resmetirom 4 Selective agonist THR- β MAESTRO- NASH NASH resolution . Impact on mortality, cirrhosis development and all hepatic complications Aramchol 5 Modulator of stearylo- CoA desaturase ARMOR NASH resolution w/out worsening of fibrosis OR improvement of fibrosis (≥ 1 stage) w/out worsening of NASH CCR2, C- C chemokine receptor type 2; CCR5, C- C chemokine receptor type 5; FXR, farnesoid X receptor; NASH, non- alcoholic steatoheaptitis; PPAR, peroxisome proliferator- activated receptors; THR- β, thyroid hormone receptor beta 1. clinicaltrials.gov/ct2/show/NCT02548351; 2. clinicaltrials.gov/ct2/show/NCT03028740; 3. clinicaltrials.gov/ct2/show/NCT02704403; 4. clinicaltrials.gov/ct2/show/NCT03900429; 5. clinicaltrials.gov/ct2/show/NCT04104321

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