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: kikim@glaceum.com

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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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HSG4112 reduces body weight and liver fat

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HSG4112 improves histological features of NASH

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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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.

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HSG4112 improves NAFLD activity score

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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HSG4112 improves liver transcriptome signatures of fibrogenesis and inflammation

COL6A1

TIMP1

COL1A1

TGFB COL5A3

TIMP2

MMP9TIMP3

COL6A2

GFAP

PDGF

COL5A2COL3A1

COL5A1

EGF

COL1A2

TIMP4

MMP2

CD146

A-SMA

COL6A3

MMP13

Down-regulated Up-regulated

Adjusted p = 0.05

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TGFB

F4/80

CD163IL-10

CD68

CD86TNFA

CCR1

IL-1B

IL-1A

RANTESMCP-1

CCR2

MAC-2

CD14

Down-regulated Up-regulated

Adjusted p = 0.05

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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

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frequency

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volume

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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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Lobular inflammation

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012345678

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NAFLD Activity Score (NAS) Fibrosis score