C F DIO - gubra.dk
1
Metabolic, biochemical, histopathological, and transcriptomic effects of resmetirom (MGL-3196) in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH Authors: Michael Feigh 1 , Jacob Nøhr-Meldgaard 1 , Sanne S. Veidal 1 , Martin Rønn Madsen 1 , Henrik H. Hansen 1 1 Gubra, Hørsholm Kongevej 11B, Hørsholm, Denmark Corresponding author: Michael Feigh - [email protected] Background & Aim Resmetirom (MGL-3196), a selective THR-β agonist, has been recently been reported to improve liver histological outcomes in a clinical trial for non- alcoholic steatohepatitis (NASH). The present study aimed to evaluate the metabolic, biochemical, histopathological and transcriptomic effects of resmetirom treatment in the Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mouse model of fibrosing NASH. CONCLUSION + Resmetirom (MGL-3196) reduces hepatomegaly, plasma ALT and liver total cholesterol. + Resmetirom promotes ≥2-point significant improvement in NAFLD Activity Score. + Fibrosis stage was unaffected by Resmetirom. + Resmetirom reduces quantitative histological markers of steatosis and stellate cell activation. + Resmetirom largely unaffected transcriptomic suppression of fibrosis-associated gene expression + These findings agree with clinical findings, further highlighting clinical translatability of the GAN DIO-NASH mouse model Hepatic transcriptomic profile for fibrosis Study outline Improvement in metabolic and biochemical parameters Improvement in NAFLD Activity Score Improvement in quantitative histology of steatosis and stellate cell activation Figure 2. Resmetirom (MGL-3196) improves liver histopathological scores in GAN DIO-NASH mice. Histopathological scores were determined by Gubra Histopathological Objective Scoring Technique (GHOST) deep learning-based image analysis. (A) NAFLD Activity Score (NAS). (B) Fibrosis stage. (C) Comparison of individual pre-post NAS and individual pre-post Fibrosis stage. **p<0.01 to corresponding DIO-NASH vehicle group (One-sided Fisher's exact test with Bonferroni correction). Bottom panels: Representative HE and PSR photomicrographs used for GHOST evaluation. Figure 3. Resmetirom (MGL-3196) improves quantitative liver histological markers in GAN DIO-NASH mice. Histomorphometric assessments were performed by GHOST deep learning-based image analysis on scoring-associated variables (panels A-B) and conventional IHC image analysis (panels C-F). (A) % hepatocytes with lipid droplets. (B) Number of inflammatory foci. (C) % area of galectin- 3. (D) % area of PSR. (E) % area of collagen-1a1. (F) % area of alpha-smooth muscle actin (α-SMA) as marker for stellate cell activation. Mean ± SEM. *p<0.05, ***p<0.001 to corresponding DIO-NASH vehicle group (Dunnett’s test one-factor linear model). Bottom panels: Representative galectin-3, collagen 1a1 and α-SMA photomicrographs (scale bar, 100 μm). Figure 4. Resmetirom (MGL-3196) unaffected fibrosis-associated genes in GAN DIO-NASH mice. (A) Principal component analysis (PCA) of samples based on top 500 most variable gene expression levels. (B) Venn diagram depicting shared and separate differentially expressed genes in treatment groups. (C) Regulation of hepatic extracellular matrix (ECM) candidate genes (log2-fold change compared to DIO-NASH vehicle mice). Blue colour gradients indicate significantly (p<0.05) down-regulated gene expression. White boxes indicate genes not significantly regulated (p>0.05) compared to DIO-NASH vehicle mice. Figure 1. Resmetirom (MGL-3196) improves hepatomegaly and biochemical parameters in GAN DIO-NASH mice. (A) Terminal body weight (g). (B) Terminal liver weight. (C) Terminal plasma alanine aminotransferase (ALT). (D) Terminal plasma aspartate aminotransferase (AST). (E) Terminal plasma triglycerides. (F) Terminal plasma total cholesterol. (G) Terminal liver total cholesterol. (H) Terminal liver triglycerides. **p<0.01, ***p<0.001 compared to corresponding DIO-NASH vehicle control (Dunnett’s test one-factor linear model). Galectin-3 α-SMA Collagen-1a1 FIBROSIS AI NAS AI A B C A B C D E F A B C D E F A B C Week -37 GAN diet-induction Week -1 Stratification Randomization Week -4 Liver Pre-biopsy + Histology Day 0 First Dose Stratification/ Randomization + Baseline GAN diet-maintenance Week 12 ✚ NAFLD Activity Score (HE) ≥5 ✚ Fibrosis Stage (PSR) ≥1 ✚ Plasma - ALT ✚ Termination - Liver weight In vivo study period GAN diet-maintenance Assay/Histology Terminal Biochemistry: ✚ Liver lipids (TG/TC) Liver biopsy histology: ✚ NAFLD Activity Score (HE) (pre-post) ✚ Fibrosis Stage (PSR) (pre-post) ✚ Terminal morphometric image analysis: - Hepatocytes with lipids (HE) - Inflammatory foci (HE) - Inflammation (Gal-3) (IHC) - Fibrosis (PSR) - Collagen (Col1a1) (IHC) - Stellate cell activation (α-SMA) (IHC) Liver RNAseq and bioinformatic analysis Group # Animal Gender Strain Number of animals Treatment Administration route Dosing Frequency Dosing volume Dosing concentration 1 LEAN-CHOW Male C57BL/6JRj 10 Vehicle PO QD 5 ml/kg - 2 DIO-NASH Male C57BL/6JRj 16 Vehicle PO QD 5 ml/kg - 3 DIO-NASH Male C57BL/6JRj 16 MGL-3196 PO QD 5 ml/kg 1 mg/kg Chow Vehicle MGL-3196 0 10 20 30 40 50 Body weight (g) *** Chow Vehicle MGL-3196 0 1 2 3 4 5 Liver weight (g) *** *** Chow Vehicle MGL-3196 0 100 200 300 400 Plasma ALT (U/L) *** *** Chow Vehicle MGL-3196 0 20 40 60 80 Liver triglycerides (mg/g liver) *** Chow Vehicle MGL-3196 0 5 10 15 20 25 Liver total cholesterol (mg/g liver) *** ** 0 1 2 3 4 5 6 7 8 9 101112 60 80 100 120 Treatment week Body weight (% of day 0) NAFLD Activity Score Chow Vehicle MGL-3196 0 20 40 60 80 100 % of mice ** Fibrosis stage Chow Vehicle MGL-3196 0 20 40 60 80 100 % of mice Chow Vehicle Pre Post 0 1 2 3 4 5 6 7 8 NAFLD Activity Score DIO-NASH Vehicle Pre Post 0 1 2 3 4 5 6 7 8 NAFLD Activity Score DIO-NASH MGL-3196 Pre Post 0 1 2 3 4 5 6 7 8 NAFLD Activity Score Chow Vehicle Pre Post 0 1 2 3 4 Fibrosis stage DIO-NASH Vehicle Pre Post 0 1 2 3 4 Fibrosis stage DIO-NASH MGL-3196 Pre Post 0 1 2 3 4 Fibrosis stage Worsening No change 1 point improvement ≥2 point improvement Chow Vehicle MGL-3196 0 20 40 60 80 % hepatocytes with lipid droplets *** * Chow Vehicle MGL-3196 0 5 10 15 20 25 Number of inflammatory foci *** Chow Vehicle MGL-3196 0 2 4 6 Galectin-3 (% area) *** Chow Vehicle MGL-3196 0 1 2 3 4 α-SMA (% area) *** ** Chow Vehicle MGL-3196 0 1 2 3 PSR (% area) *** Chow Vehicle MGL-3196 0 2 4 6 8 Col1a1 (% area) *** Chow Vehicle DIO-NASH Vehicle DIO-NASH MGL-3196 -15 -10 -5 0 5 10 -30 -20 -10 0 10 20 PC1 (65%) PC2 (11%) 7373 472 1076 Chow Vehicle vs DIO-NASH Vehicle DIO-NASH Resmetirom (MGL-3196) vs DIO-NASH Vehicle MGL-3196 Chow Vehicle -3 -2 -1 0 Log2 fold change ✚ Fibrosis %FA (PSR)
Transcript of C F DIO - gubra.dk
Metabolic, biochemical, histopathological, and transcriptomic effects of resmetirom (MGL-3196) in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH
Authors: Michael Feigh1, Jacob Nøhr-Meldgaard1, Sanne S. Veidal1, Martin Rønn Madsen1, Henrik H. Hansen1
1Gubra, Hørsholm Kongevej 11B, Hørsholm, Denmark
Corresponding author: Michael Feigh - [email protected]
Background & Aim
Resmetirom (MGL-3196), a selective THR-β agonist,has been recently been reported to improve liver
histological outcomes in a clinical trial for non-
alcoholic steatohepatitis (NASH). The present studyaimed to evaluate the metabolic, biochemical,
histopathological and transcriptomic effects of
resmetirom treatment in the Gubra-Amylin NASH(GAN) diet-induced obese (DIO) mouse model of
fibrosing NASH.
CONCLUSION
+ Resmetirom (MGL-3196) reduces hepatomegaly, plasma ALT and liver total cholesterol.
+ Resmetirom promotes ≥2-point significant improvement in NAFLD Activity Score.
+ Fibrosis stage was unaffected by Resmetirom.
+ Resmetirom reduces quantitative histological markers of steatosis and stellate cell activation.
+ Resmetirom largely unaffected transcriptomic suppression of fibrosis-associated gene expression
+ These findings agree with clinical findings, further highlighting clinical translatability of the GAN DIO-NASH mouse model
Hepatic transcriptomic profile for fibrosis
Study outline Improvement in metabolic and biochemical parameters
Improvement in NAFLD Activity Score Improvement in quantitative histology of steatosis and stellate cell activation
Figure 2. Resmetirom (MGL-3196) improves liver histopathological scores in GAN DIO-NASH mice.Histopathological scores were determined by Gubra Histopathological Objective Scoring Technique (GHOST)deep learning-based image analysis. (A) NAFLD Activity Score (NAS). (B) Fibrosis stage. (C) Comparison ofindividual pre-post NAS and individual pre-post Fibrosis stage. **p<0.01 to corresponding DIO-NASH vehiclegroup (One-sided Fisher's exact test with Bonferroni correction). Bottom panels: Representative HE and PSRphotomicrographs used for GHOST evaluation.
Figure 3. Resmetirom (MGL-3196) improves quantitative liver histological markers in GAN DIO-NASH mice.Histomorphometric assessments were performed by GHOST deep learning-based image analysis on scoring-associated variables (panels A-B)and conventional IHC image analysis (panels C-F). (A) % hepatocytes with lipid droplets. (B) Number of inflammatory foci. (C) % area of galectin-3. (D) % area of PSR. (E) % area of collagen-1a1. (F) % area of alpha-smooth muscle actin (α-SMA) as marker for stellate cell activation. Mean ±SEM. *p<0.05, ***p<0.001 to corresponding DIO-NASH vehicle group (Dunnett’s test one-factor linear model). Bottom panels: Representativegalectin-3, collagen 1a1 and α-SMA photomicrographs (scale bar, 100 µm).
Figure 4. Resmetirom (MGL-3196) unaffected fibrosis-associated genes in GAN DIO-NASH mice.(A) Principal component analysis (PCA) of samples based on top 500 most variable gene expression levels.(B) Venn diagram depicting shared and separate differentially expressed genes in treatment groups. (C)Regulation of hepatic extracellular matrix (ECM) candidate genes (log2-fold change compared to DIO-NASHvehicle mice). Blue colour gradients indicate significantly (p<0.05) down-regulated gene expression. Whiteboxes indicate genes not significantly regulated (p>0.05) compared to DIO-NASH vehicle mice.
Figure 1. Resmetirom (MGL-3196) improves hepatomegaly and biochemical parameters in GAN DIO-NASH mice. (A) Terminal body weight (g). (B) Terminal liver weight. (C) Terminalplasma alanine aminotransferase (ALT). (D) Terminal plasma aspartate aminotransferase (AST). (E) Terminal plasma triglycerides. (F) Terminal plasma total cholesterol. (G) Terminalliver total cholesterol. (H) Terminal liver triglycerides. **p<0.01, ***p<0.001 compared to corresponding DIO-NASH vehicle control (Dunnett’s test one-factor linear model).
Galectin-3 α-SMACollagen-1a1FIBROSIS AINAS AI
A B
C
A B C
D E F
A B C
D E F
A B
C
Week -37
GAN diet-induction
Week -1Stratification
Randomization
Week -4Liver
Pre-biopsy +
Histology
Day 0First Dose
Stratification/Randomization
+ BaselineGAN diet-maintenance
Week 12
✚ NAFLD Activity Score (HE) ≥5✚ Fibrosis Stage (PSR) ≥1
✚ Plasma- ALT✚ Termination- Liver weight
In vivo study periodGAN diet-maintenance Assay/Histology
Terminal Biochemistry:✚ Liver lipids (TG/TC)
Liver biopsy histology:✚ NAFLD Activity Score (HE) (pre-post)✚ Fibrosis Stage (PSR) (pre-post)✚ Terminal morphometric image analysis:- Hepatocytes with lipids (HE)- Inflammatory foci (HE)- Inflammation (Gal-3) (IHC)- Fibrosis (PSR) - Collagen (Col1a1) (IHC) - Stellate cell activation (α-SMA) (IHC)
Liver RNAseq and bioinformatic analysis
Group
#
Animal Gender Strain Number of
animals
Treatment Administration
route
Dosing
Frequency
Dosing
volume
Dosing
concentration
1 LEAN-CHOW Male C57BL/6JRj 10 Vehicle PO QD 5 ml/kg -
2 DIO-NASH Male C57BL/6JRj 16 Vehicle PO QD 5 ml/kg-
3 DIO-NASH Male C57BL/6JRj 16 MGL-3196 PO QD 5 ml/kg 1 mg/kg
Chow Vehicle MGL-31960
10
20
30
40
50
Body w
eig
ht (g
)
***
Chow Vehicle MGL-31960
1
2
3
4
5
Liv
er
weig
ht (g
)
******
Chow Vehicle MGL-31960
100
200
300
400
Pla
sm
a A
LT
(U
/L)
***
***
Chow Vehicle MGL-31960
20
40
60
80
Liv
er
trig
lycerides
(mg/g
liv
er)
***
Chow Vehicle MGL-31960
5
10
15
20
25
Liv
er
tota
l chole
ste
rol
(mg/g
liv
er)
***
**
0 1 2 3 4 5 6 7 8 9 10 11 1260
80
100
120
Treatment week
Body w
eig
ht
(% o
f d
ay 0
)
NAFLD Activity Score
Chow Vehicle MGL-31960
20
40
60
80
100
% o
f m
ice **
Fibrosis stage
Chow Vehicle MGL-31960
20
40
60
80
100
% o
f m
ice
ChowVehicle
Pre Post
0
1
2
3
4
5
6
7
8
NA
FLD
Activity S
core
DIO-NASHVehicle
Pre Post
0
1
2
3
4
5
6
7
8
NA
FLD
Activity S
core
DIO-NASHMGL-3196
Pre Post
0
1
2
3
4
5
6
7
8
NA
FLD
Activity S
core
ChowVehicle
Pre Post
0
1
2
3
4
Fib
rosis
sta
ge
DIO-NASHVehicle
Pre Post
0
1
2
3
4
Fib
rosis
sta
ge
DIO-NASHMGL-3196
Pre Post
0
1
2
3
4
Fib
rosis
sta
ge
Fibrosis stage
C+V V S V L0
20
40
60
80
100
% o
f m
ice
WorseningNo change1 point improvement≥2 point improvement
*
B
Chow Vehicle MGL-31960
20
40
60
80
% h
epato
cyte
s w
ith
lipid
dro
ple
ts
***
*
Chow Vehicle MGL-31960
5
10
15
20
25
Num
ber
of
inflam
mato
ry foci
***Chow Vehicle MGL-3196
0
2
4
6
Gale
ctin-3
(%
are
a)
***
Chow Vehicle MGL-31960
1
2
3
4
α-S
MA
(%
are
a)
***
**
Chow Vehicle MGL-31960
1
2
3
PS
R (
% a
rea)
***
Chow Vehicle MGL-31960
2
4
6
8
Col1
a1 (
% a
rea)
***
ChowVehicle
DIO-NASHVehicle
DIO-NASHMGL-3196
-15
-10
-5
0
5
10
-30 -20 -10 0 10 20
PC1 (65%)
PC
2 (
11%
)
7373 4721076
Chow Vehicle vsDIO-NASH Vehicle
DIO-NASH Resmetirom (MGL-3196) vsDIO-NASH Vehicle
MGL-3196
Chow Vehicle
-3
-2
-1
0
Lo
g2
fold
ch
an
ge
✚ Fibrosis %FA (PSR)
