FASN inhibition targets multiple drivers of NASH by reducing steatosis, inflammation and fibrosis in preclinical models

Figures

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  Figure 1. In human in vitro models, FASN inhibition directly reduces cellular triglyceride content, fibrogenic marker expression, pro-inflammatory cytokine production and Th17 cell development. (a) Effect of FASNi on TG content in Liver Micro Tissues (LMTs) were treated for 10 days with TVB-2640 in the presence of glucose, insulin and fatty acids, and assayed for TG levels. Mean ± SD of 12 replicates for vehicle and 6 for each TVB-2640 condition. *p < 0.05 by unpaired T test versus vehicle. (b) Effect of FASNi on cellular proliferation in LX-2 cells. *p < 0.05 by unpaired T test versus vehicle. (c) Effect of FASNi on expression of fibrogenic genes in LX-2 cells. *p < 0.01, **p < 0.05, ****p < 0.0001 by unpaired T test versus vehicle. Quantitative PCR analysis was performed after 48 h of treatment (solid bars) with TVB-3664 followed by washout to remove drug and a further 48 h incubation (striped bars). (d) Effect of FASNi on protein expression of Col1α1 and αSMA in LX-2 cells. Immunocytochemistry in cells exposed to 50 or 150 nM TVB-3664 for 48 h. Three independent experiments are indicated by n = 1, 2 or 3. (e) Effect of FASNi on protein expression of Col1α1 and αSMA in primary human…

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  Figure 2. FASN inhibition prevents development of liver steatosis and fibrosis in a preventative NASH diet induced model. (a) Effect of FASNi on fibrosis and lipid accumulation. Liver sections at the end of study stained with Mason’s Trichrome or adipophilin, from vehicle or TVB-3664 (3 mg/kg) treated animals on a form of WD, or vehicle treated animals on a normal diet. Representative examples are shown. The scoring per animal is provided in Supplementary Table S1. (b) Histological scoring results of H&Estained liver sections. The mean of 5 animals is indicated by horizontal line. Scoring system: 0 (normal), 1 (minimal), 2 (mild), 3 (moderate), 4 (marked). The scoring per animal is provided in Supplementary Table S2. (c) IL-1β levels measured from serum collected at the end of the treatment period from vehicle or TVB-3664 treated animals.

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  Figure 3. FASN inhibition reduces liver fat, metabolic parameters and NASH histology severity scores in a NASH diet induced therapeutic mouse model. (a) Effect of FASNi on liver histology. Left: Representative images of H&E stained liver sections at the end of the treatment period with vehicle or TVB-3664 (10 mg/kg). Magnification 10x, scale bar = 200 µm. Right: Liver fat (steatosis) determined by histological scoring. Mean ± SEM (n = 11/12 per group). ***p < 0.001 vs vehicle, One-Way ANOVA with Dunnett’s multiple comparison test. (b) Effect of FASNi on liver NAS score. Histological scoring of liver at the end of the treatment period. The number of animals with a higher (worse, pink), no change (grey) or lower (improved, blue) histology score in liver samples at end of study compared to prestudy biopsies for each animal is indicated by the height of the bar. ***p < 0.001 vs vehicle, Fisher’s exact test followed by adjustment for multiple correction using the Bonferroni method. (c) Effect of FASNi on plasma ALT, AST, cholesterol and triglyerides at the end of the treatment period. Mean ± SEM. **p < 0.01, ***p < 0.001 vs vehicle, One-Way ANOVA with Dunnet’s multiple comparison test.

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  Figure 4. FASN inhibition reduces established fibrosis and hepatocellular carcinoma tumor formation in the FAT-NASH (CCl4) Model. (a) Effect of FASNi on liver fibrosis by conventional pathology. Left: Photomicrograph of representative sirius red/fast green stained liver sections from vehicle or TVB-3664 treatment (5 mg/kg). Right: Quantification of fibrillar collagen using BioQuant software. *p < 0.0 vs vehicle, unpaired two tailed Mann–Whitney test. (b) Effect of FASNi on liver fibrosis by AI based digital pathology. Quantification of total collagen area using Second Harmonic Generation. Left: Representative images from vehicle and TVB-3664 treated. Collagen is represented in green. Right: Graph shows mean/SD of all animals. An expanded view of representative images is shown in Supplementary Fig. S2. (c) Effect of FASNi on the development of hepatocellular carcinoma. Number of liver tumors per animal at the end of treatment with vehicle or TVB-3664. (d) Effect of FASNi on appearance of livers at the end of treatment with vehicle or TVB-3664. Representative images are shown.

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  Figure 5. Gene and protein expression, and NAS score in the FAT-NASH (CCl4) Model. (a) Effect of FASNi on mRNA expression of fibrogenic genes. liver tissues measured by RT-qPCR treated with vehicle or TVB-3664 (5 mg/kg). (b) Effect of FASNi on protein expression of fibrogenic genes. Western blot analysis of protein expression of Col1A1 and αSMA in liver tissue from animals treated with vehicle or TVB-3664. (c) Effect of FASNi on liver histology by H&E. Representative photomicrographs of H&E stained liver sections in vehicle (PEG400) and TVB-3664. Steatosis (yellow arrow), hepatocyte ballooning (red arrow) and lobular inflammation (black arrow) are indicated. Statistical analysis was by unpaired two tailed Mann–Whitney test. *p ≤ 0.05 vs vehicle group. (d) Effect of FASNi on liver histology by NAS score Analysis performed blinded at the end of treatment with vehicle or TVB-3664. Lines represent Mean/SD. (e) Effect of FASNi on plasma AST, ALT, total cholesterol and triglycerides. Lines represent Mean/SD. Statistical analysis was by unpaired two tailed Mann–Whitney test. *p ≤ 0.05 vs vehicle group, **p ≤ 0.01 vs vehicle group, ***p ≤ 0.001 vs vehicle group.