Sang-Ku Yoo l v 1Glaceum Inc., Suwon, South Korea; 2Gubra,...
1
Anti-obesity drug HSG4112 ameliorates liver histopathology and improves NAFLD activity score in a diet-induced obese and biopsy-confirmed mouse model of NASH Kyungil Kim 1* , Mette V. Ø stergaard 2 , Sanne S. Veidal 2 , Keun-Wan Lim 1 , Yunsun Park 1 , Henrik H. Hansen 2 , Michael Feigh 2 , Sang-Ku Yoo 1 1 Glaceum Inc., Suwon, South Korea; 2 Gubra, Hørsholm, Denmark. *Corresponding author: [email protected] 0 5 10 15 20 25 30 35 40 45 25 30 35 40 45 Treatment day Body weight (g S.E.M.) *** DIO-NASH vehicle DIO-NASH HSG4112 Figure 1 | (A) Body weight; (B) Cumulative food intake. DIO-NASH: Two-way ANOVA with Dunnett’s multiple comparison test. (C) Liver mass and lipids. DIO-NASH: One-way ANOVA with Dunnett’s multiple comparison test. **p<0.01, ***p<0.001. 0 1 2 3 4 Liver weight (g SEM) DIO-NASH Vehicle DIO-NASH HSG4112 *** 0 50 100 150 Liver total triglycerides (mg/g liver SEM) 0 5 10 15 Liver total cholesterol (mg/g liver SEM) ** 21% ↓ 0 5 10 15 20 25 30 35 40 45 0 1 2 3 4 Treatment day Daily food intake (g S.E.M.) C A HSG4112 reduces body weight and liver fat B HSG4112 improves histological features of NASH Figure 3 | Quantitative liver histology. Left panels: Levels of liver histopathology markers are expressed as fractional area (% of total section area, panels A-D) and total liver content (mg per liver, % area multiplied with total liver weight, panels E-H). *p<0.05, ***p<0.001 vs. DIO-NASH vehicle, one-way ANOVA with Dunnett’s multiple comparison test. Right panels: Representative photomicrographs of stained liver sections from DIO-NASH mice treated with vehicle and HSG4112. 0 10 20 30 Liver lipids (% area SEM) DIO-NASH Vehicle DIO-NASH HSG4112 *** 0 200 400 600 800 1000 Liver lipid content (total mg/liver SEM) *** 0 1 2 3 4 5 Liver galectin-3 (% area SEM) *** 0 50 100 150 200 Liver galectin-3 content (total mg/liver SEM) *** 0 1 2 3 4 5 Liver -SMA (% area SEM) *** 0 50 100 150 200 Liver -SMA content (total mg/liver SEM) *** 0 2 4 6 8 10 Liver collagen 1a1 (% area SEM) 0 100 200 300 400 Liver collagen 1a1 content (total mg/liver SEM) * A E B F C G D H DIO-NASH vehicle DIO-NASH HSG4112 Lipids (HE staining) α-SMA Galectin-3 Collagen 1a1 HSG4112 improves plasma biochemistry Figure 4 | Plasma biochemistry. (A-C) Liver injury markers (ALT, AST, alkaline phosphatase); (D) Leptin. **p<0.01, ***p<0.001 vs. DIO-NASH vehicle, one-way ANOVA with Dunnett’s multiple comparison test. 0 100 200 300 Plasma ALT (U/L SEM) DIO-NASH vehicle DIO-NASH HSG4112 *** 0 100 200 300 400 Plasma AST (U/L SEM) *** 0 50 100 150 200 Plasma alkaline phosphatase (U/L SEM) *** 0 2000 4000 6000 8000 10000 Plasma leptin (pg/mL SEM) *** A B C D HSG4112 improves NAFLD activity score Figure 5 | Differential expression of hepatic genes associated with fibrogenesis and inflammation following HSG4112 treatment compared to vehicle dosing. Volcano plot of differentially expressed genes (DEGs) associated with fibrogenesis (left panel) and monocyte recruitment (right panel). X-axis indicates average fold change (log2) in gene expression compared to DIO-NASH vehicle. A p-value less than 0.05 (y-axis, data points above the dotted horizontal line) was considered statistically significant regulation compared to vehicle controls. Genes in grey color are not significantly regulated (p>0.05). Monocyte Recruitment (inflammation) Stellate Cell Activation (fibrogenesis) Figure 2 | Liver histopathology scores. The NAFLD activity score (NAS) and fibrosis staging system were applied to liver pre-biopsies and terminal samples for scoring of steatosis, lobular inflammation, hepatocyte ballooning and fibrosis as outlined by Kleiner et al for DIO-NASH mice. *p<0.05, **p<0.01 vs. DIO-NASH vehicle, Fisher’s Exact Test with Bonferroni’s multiple comparisons test. Fibrosis score Vehicle HSG4112 0 2 4 6 8 10 12 Lower Same Higher Number of animals NAS Vehicle HSG4112 0 2 4 6 8 10 12 Number of animals ** ** Steatosis score Vehicle HSG4112 0 2 4 6 8 10 12 Number of animals Inflammation score Vehicle HSG4112 0 2 4 6 8 10 12 Number of animals * * Ballooning score Vehicle HSG4112 0 2 4 6 8 10 12 Number of animals HSG4112 improves liver transcriptome signatures of fibrogenesis and inflammation COL6A1 TIMP1 COL1A1 TGFB COL5A3 TIMP2 MMP9 TIMP3 COL6A2 GFAP PDGF COL5A2 COL3A1 COL5A1 EGF COL1A2 TIMP4 MMP2 CD146 A-SMA COL6A3 MMP13 Down-regulated Up-regulated Adjusted p = 0.05 -1 0 1 0 2 4 6 8 Log2 (Fold-change) -log10 (p-value) TGFB F4/80 CD163 IL-10 CD68 CD86 TNFA CCR1 IL-1B IL-1A RANTES MCP-1 CCR2 MAC-2 CD14 Down-regulated Up-regulated Adjusted p = 0.05 -1.0 -0.5 0.0 0.5 1.0 0.0 2.5 5.0 7.5 10.0 log2 (Fold-change) -log10 (p-value) OVERVIEW HSG4112, a synthetic new chemical entity, is a first-in-class oral small molecule in clinical development for the treatment of obesity. As a leptin sensitizer, HSG4112 markedly reduces adiposity and liver fat accumulation in a diet-induced obese (DIO) mouse model through increased energy expenditure. As these therapeutic effects are highly relevant to the treatment of obesity-associated liver complications, notably non-alcoholic steatohepatitis (NASH), we characterized the effects of HSG4112 in biopsy-confirmed DIO-NASH mouse model. 6-week treatment of HSG4112 robustly improved histopathological, biochemical, and transcriptional endpoints of NASH. Longer treatment of HSG4112 is expected to mitigate fibrosis in NASH. METHODS Male C57BL/6JRj mice were fed AMLN diet high in trans-fat, fructose, and cholesterol for 33 weeks prior to liver biopsy sampling. Only animals with biopsy-confirmed steatosis (score ≥2) and fibrosis (stage ≥F1) were included and stratified into treatment groups. DIO-NASH mice received vehicle (PO, QD) or HSG4112 (100 mg/kg, PO, QD) for 6 weeks (44 days). Pre-post liver biopsy histology was performed for within-subject evaluation of changes in NAFLD Activity Score (NAS) and fibrosis stage. Terminal liver histomorphometry was evaluated by quantitative imaging analysis. Terminal blood and liver biochemistry analyses were performed. RNA-sequencing was performed on liver samples. STUDY DESIGN AMLN diet- induction Day 0 First Dose Week 6 Termination In vivo study period Assay/Histology Week -4 Liver Pre-biopsy + Histology Week -1 Stratification Week -37 • Plasma & liver biochemistry • Histopathological scoring • Quantitative histological image analysis • RNA-sequencing + bioinformatics Group # Animal Gender Strain Number of anima ls Treatment Administration r oute Dosing frequency Dosing volume Dosing concentration 1 DIO-NASH Male C57BL/6JRj 12 Vehicle PO QD 5 ml/kg - 2 DIO-NASH Male C57BL/6JRj 11 HSG4112 PO QD 5 ml/kg 100 mg/kg CONCLUSIONS • HSG4112 improves metabolic, biochemical parameters and quantitative histopathology in a biopsy-confirmed DIO-NASH mouse model • HSG4112 improves NAFLD activity score, primarily driven by reduced inflammation • HSG4112 improves liver transcriptome signatures of fibrogenesis and inflammation; mitigation of fibrosis is expected in prolonged treatment • HSG4112 is a potent molecule for targeting obesity and NASH RESULTS Vehicle Pre Post 0 1 2 3 Lobular inflammation HSG4112 Pre Post 0 1 2 3 Lobular inflammation Vehicle Pre Post 0 1 2 3 4 5 6 7 8 NAS HSG4112 Pre Post 0 1 2 3 4 5 6 7 8 Vehicle Pre Post 0 1 2 3 4 Fibrosis HSG4112 Pre Post 0 1 2 3 4 NAFLD Activity Score (NAS) Fibrosis score
Transcript of Sang-Ku Yoo l v 1Glaceum Inc., Suwon, South Korea; 2Gubra,...
Anti-obesity drug HSG4112 ameliorates liver histopathology and improves NAFLD activity score in a diet-induced obese and biopsy-confirmed mouse model of NASH
Kyungil Kim1*, Mette V. Ø stergaard2, Sanne S. Veidal2, Keun-Wan Lim1 , Yunsun Park1, Henrik H. Hansen2, Michael Feigh2, Sang-Ku Yoo1
1Glaceum Inc., Suwon, South Korea; 2Gubra, Hørsholm, Denmark. *Corresponding author: [email protected]
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Figure 1 | (A) Body weight; (B) Cumulative food intake. DIO-NASH: Two-way ANOVA with Dunnett’s multiplecomparison test. (C) Liver mass and lipids. DIO-NASH: One-way ANOVA with Dunnett’s multiple comparison test.**p<0.01, ***p<0.001.
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Figure 3 | Quantitative liver histology. Left panels: Levels of liver histopathology markers are expressed as fractionalarea (% of total section area, panels A-D) and total liver content (mg per liver, % area multiplied with total liver weight,panels E-H). *p<0.05, ***p<0.001 vs. DIO-NASH vehicle, one-way ANOVA with Dunnett’s multiple comparison test.Right panels: Representative photomicrographs of stained liver sections from DIO-NASH mice treated with vehicle andHSG4112.
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Figure 4 | Plasma biochemistry. (A-C) Liver injury markers (ALT, AST, alkaline phosphatase); (D) Leptin. **p<0.01,***p<0.001 vs. DIO-NASH vehicle, one-way ANOVA with Dunnett’s multiple comparison test.
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Figure 5 | Differential expression of hepatic genes associated with fibrogenesis and inflammation followingHSG4112 treatment compared to vehicle dosing. Volcano plot of differentially expressed genes (DEGs) associated withfibrogenesis (left panel) and monocyte recruitment (right panel). X-axis indicates average fold change (log2) in geneexpression compared to DIO-NASH vehicle. A p-value less than 0.05 (y-axis, data points above the dotted horizontalline) was considered statistically significant regulation compared to vehicle controls. Genes in grey color are notsignificantly regulated (p>0.05).
Monocyte Recruitment (inflammation)Stellate Cell Activation (fibrogenesis)
Figure 2 | Liver histopathology scores. The NAFLD activity score (NAS) and fibrosis staging system were applied to liverpre-biopsies and terminal samples for scoring of steatosis, lobular inflammation, hepatocyte ballooning and fibrosis asoutlined by Kleiner et al for DIO-NASH mice. *p<0.05, **p<0.01 vs. DIO-NASH vehicle, Fisher’s Exact Test withBonferroni’s multiple comparisons test.
Fibrosis score
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COL6A1
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OVERVIEWHSG4112, a synthetic new chemical entity, is a first-in-class oral small molecule in clinical
development for the treatment of obesity. As a leptin sensitizer, HSG4112 markedly reduces
adiposity and liver fat accumulation in a diet-induced obese (DIO) mouse model through
increased energy expenditure. As these therapeutic effects are highly relevant to the
treatment of obesity-associated liver complications, notably non-alcoholic steatohepatitis
(NASH), we characterized the effects of HSG4112 in biopsy-confirmed DIO-NASH mouse
model. 6-week treatment of HSG4112 robustly improved histopathological, biochemical, and
transcriptional endpoints of NASH. Longer treatment of HSG4112 is expected to mitigate
fibrosis in NASH.
METHODSMale C57BL/6JRj mice were fed AMLN diet high in trans-fat, fructose, and cholesterol for 33
weeks prior to liver biopsy sampling. Only animals with biopsy-confirmed steatosis (score ≥2)
and fibrosis (stage ≥F1) were included and stratified into treatment groups. DIO-NASH mice
received vehicle (PO, QD) or HSG4112 (100 mg/kg, PO, QD) for 6 weeks (44 days). Pre-post
liver biopsy histology was performed for within-subject evaluation of changes in NAFLD
Activity Score (NAS) and fibrosis stage. Terminal liver histomorphometry was evaluated by
quantitative imaging analysis. Terminal blood and liver biochemistry analyses were
performed. RNA-sequencing was performed on liver samples.
STUDY DESIGNAMLN diet-induction
Day 0First Dose
Week 6 Termination
In vivo study period Assay/Histology
Week -4Liver
Pre-biopsy +Histology
Week -1Stratification
Week -37
• Plasma & liver biochemistry• Histopathological scoring• Quantitative histological image analysis• RNA-sequencing + bioinformatics
Group
#Animal Gender Strain
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of anima
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TreatmentAdministration r
oute
Dosing
frequency
Dosing
volume
Dosing
concentration
1 DIO-NASH Male C57BL/6JRj 12 Vehicle PO QD 5 ml/kg -
2 DIO-NASH Male C57BL/6JRj 11 HSG4112 PO QD 5 ml/kg 100 mg/kg
CONCLUSIONS
• HSG4112 improves metabolic, biochemical parameters and quantitative histopathology in a biopsy-confirmed DIO-NASH mouse model
• HSG4112 improves NAFLD activity score, primarily driven by reduced inflammation
• HSG4112 improves liver transcriptome signatures of fibrogenesis and inflammation; mitigation of fibrosis is expected in prolonged treatment
• HSG4112 is a potent molecule for targeting obesity and NASH
RESULTS
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