sang-ku yoo l v 1glaceum inc., suwon, south korea; 2gubra,...
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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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Treatment day
Body w
eig
ht
(g S
.E.M
.)
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DIO-NASH vehicle
DIO-NASH HSG4112
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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21% ↓
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Treatment day
Da
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od inta
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.E.M
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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 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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A E
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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.
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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
Veh
icle
HSG41
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LowerSameHigher
Num
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NAS
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HSG41
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Num
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Steatosis score
Veh
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HSG41
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Inflammation score
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HSG41
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Ballooning 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
-1 0 1
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Log2 (Fold-change)
-log1
0 (
p-v
alu
e)
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
-1.0 -0.5 0.0 0.5 1.0
0.0
2.5
5.0
7.5
10.0
log2 (Fold-change)
-log1
0 (
p-v
alu
e)
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
Number
of anima
ls
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
Vehicle
Pre Post
0
1
2
3
Lobula
r in
flam
mation
HSG4112
Pre Post
0
1
2
3
Lobular inflammation
Vehicle
Pre Post
012345678
NA
S
HSG4112
Pre Post
012345678
Vehicle
Pre Post
0
1
2
3
4
Fib
rosis
HSG4112
Pre Post
0
1
2
3
4
NAFLD Activity Score (NAS) Fibrosis score
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