Bone protective effect of a novel long-acting GLP-1/GIP/Glucagon triple agonist (HM15211) in the obese-osteoporosis rodent model
1105-P
Sang Don Lee1, Jong Suk Lee1, Eun Jin Park1, Sang-Hyun Lee1, Jong Soo Lee1, In Young Choi1, Young Hoon Kim1, and Sun Jin Kim1
1Hanmi Pharm. Co., Ltd, Seoul, Korea
American Diabetes Association’s (ADA) 78th Scientific Sessions, Orlando, Florida, USA; June 22‐26, 2018
ABSTRACT METHODS
BACKGROUND
• Bone homeostasis effects of GCG2, GLP-13 and GIP4
• Increased fracture risk associated to weight loss1
Bone mass loss
↓ Mechanical effect
↓ Insulin and IGF-1
↓ Estrogen, Adiponectin and Ghrelin
↓ Calcium absorption
Increased Fall Risk
Disorder of bone quality
Mineralization disorder
Bone mass loss
Weight
Loss
SD ♀5wks old
D-56
OVX
60% kcal fat diet Serum Serum Serum
Necropsy
D0 D14 D28
Progress of in vivo study
HM15211 Q3D
Liraglutide BID
Reduction of body weight and food intake
Figure 1. Body weight change and food intake
➢ HM15211 administration significantly decreased body weight and
food intake, respectively.
0 3 6 9 12 15 18 21 24-40
-20
0
20
+ 7.5%+ 8.8%
- 3.0% **
- 15.2% ***
- 35.7% ***
(a) Body weight change
△B
od
y w
eig
ht
(%)
RESULTS
(b) Accumulative food intake0-24day
0
50
100
150
102 g 103g
80g
80g
50g**
Ac
cu
mu
lati
ve
Fo
od
In
tak
e0-2
4d
ay
Improvement of bone biochemical markers
REFERENCES
1. Francisco J. A. de Paula and Clifford J. Rosen, Arq Bras Endocrinol
Metabol. 2010 Mar;54(2):150-7.
2. Francesc Villarroya et al., Nat Rev Endocrinol. 2017 Jan;13(1):26-35.
3. Guojing Luo et al., Br J Clin Pharmacol. 2016 Jan;81(1):78-88.
4. Katsushi Tsukiyama et al., Mol Endocrinol. 2006 Jul;20(7):1644-51.
Figure 2. Serum levels of Glu-OC, OPG and P1NP
➢ Bone bio chemical markers (Glu-OC, OPG and P1NP) were
dose dependently improved on HM15211 dosing group,
respectively.
OVX vehicle, Q2DShame vehicle, Q2D
OVX Liraglutide 25 nmol/kg, BID (3 mg/day in human)
OVX HM15211 2.2 nmol/kg, Q2D (4 mg/week in human)OVX HM15211 4.4 nmol/kg, Q2D (8 mg/week in human)
(a) Glu-OC (bone resorption marker)
Glu
-OC
(n
g/m
l)
-4 14 280
50
100
150
200
***
***
******
*
***
Day
Prevention of BMD loss following weight loss
(a) Femurs BMD and weight loss
△B
od
y w
eig
ht (%
)
Fe
mu
rs B
MD
(g
/cm
2)
0.0
0.1
0.2
0.3
0.4
0.5
-40
-20
0
20*
**
***
***
28
Day
Figure 3. BMD and µ-CT image of femurs
OVX
Vehicle
Shame
Vehicle
(b) µ-CT image of FemursOVX
Liraglutide
25 nmol/kg
OVX
HM15211
2.2 nmol/kg
OVX
HM15211
4.4 nmol/kgSevere weight loss is often associated with reduction in bone mineral density
(BMD) and an imbalance between bone formation and resorption in obese
people. As a consequence, there can be an increased risk of bone fractures
with body weight loss. Several studies have proposed that the gut hormones,
gastric inhibitory polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and
glucagon (GCG), might be modulators of bone growth and remodeling.
HM15211 is a novel long-acting GLP-1/GIP/Glucagon agonist that is being
developed for the treatment of obesity. In this study, we investigated whether
treatment with HM15211 prevents bone loss under a severe weight loss
condition, and the underlying mechanism of action.
To investigate bone protection efficacy of HM15211 and liraglutide in obese-
osteoporosis rats model for chronic treatment. After 4 weeks subcutaneous
treatment of HM15211 showed lower serum level of decarboxylated
osteocalcin (Glu-OC) and higher serum levels of osteoprotegerin (OPG) and
procollagen type I pro-peptide (P1NP) compared with those of vehicle and
liraglutide treated groups. Consequently, HM15211 showed comparable BMD
of femurs with vehicle group while it showed greater weight loss compared to
liraglutide.
For elucidating the underlying molecular mechanism, related marker gene
expression was investigated using the MC3T3-E1 cell (mouse osteoblast cell).
HM15211 led to significant increase in type1 collagen-α1 and carboxylated
osteocalcin (Gla-OC) expression, which were blunted by inhibition of GIPR-
mediated signaling.
These results suggest that HM15211 might provide potent weight loss without
the otherwise inevitable bone loss.
➢ Even severe weight loss condition, HM15211 prevented
BMD loss of femurs
MoA studies for bone protection
Figure 4. Bone protection mechanism in MC3T3-E1 cell
CONCLUSIONS
➢ HM15211 improved osteoblast differentiation and showed
anti-apoptotic effect. Additionally, GIP antagonist reversed the
beneficial effect of HM15211 on bone protection.
• Lower serum level of Glu-OC and higher serum levels of OPG
and P1NP were observed compared with those of vehicle and
liraglutide treated groups in obese-osteoporosis rats model.
• HM15211 showed comparable BMD of femurs with vehicle
while it showed greater weight loss compared to liraglutide in
obese-osteoporosis rats model.
• HM15211 led to significant increase in collagen and Gla-OC
expression, which were blunted by inhibition of GIPR-
mediated signaling in osteoblast cell.
• These results suggest that HM15211 might provide potent
weight loss without bone loss
Figure 5. Schematic summary for bone protective effect
GIPR
Pre-osteoblast
Apoptosis
2) Induction of
osteoblast differentiation
HM15211
3) Inhibition of mature osteoblast apoptosis
1) Reduced bone
resorption
• To investigate MoA for bone protection of HM15211, MC3T3-E1 cells were treated
with HM15211. Osteoblast differentiation related markers (RUNX2, OCN, ALP
and Col1α) were analyzed using real-time PCR. Additionally, collagen protein
expression change and anti-apoptotic effect were evaluated using commercial kit.
• Diet induced obesity (DIO) osteoporosis rat model was induced by surgical
oophorectomy (OVX) and fed 60% kcal fat diet to immatured 5 weeks old female
sprague dawley (SD) rats for 8 weeks. Serum levels of bone biochemical markers
(Glu-OC, OPG and P1NP) were measured by commercial ELISA kits. BMD of
femurs were monitored using a high resolution in vivo µ-CT system (n = 7 /group).
(b) OPG (osteoclastogenesis inhibition marker)
& P1NP (bone formation marker)
OP
G (
pg
/ml)
280
50
100
150
200
250
***
Day
P1
NP
(n
g/m
l)
2820
40
60
80
***
Day
28
Day28
Day
GLP-1
GIP
GCG
Mesenchymal
progenitor
Osteoblast
Hematopoietic
progenitor
OsteoclastOsteoblast apoptosis
↑ IGFBP2 & WNT10b
↑ Osteoblastogenesis
↑ OPG gene expression
↓ Osteoclastogenesis
↑ Intracellular cAMP
↓ Osteoblast apoptosis
Br J Clin Pharmacol. 2016 Jan;81(1):78-88.
(a) Osteoblast differentiation marker genes
R U N X 2 O C N AL P C o l1 A
1
2
3
Ge
ne
ex
pre
ss
ion
(fo
ld i
nc
re
as
e)
**
***
**
C o n tro l
H M 1 5 2 1 1 u M
H M 1 5 2 1 1 1 0 u M
Ge
ne
ex
pre
ssio
n
(fo
ld i
nc
rea
se)
RUNX2 OCN ALP Col1α
Control
HM15211 1 µM
HM15211 10 µM
(b) Collagen expression in conditioned media
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
Co
lla
ge
n C
on
ce
ntr
ati
on
(μg/m
l)
* *
C o n tro l
L A P SG IP 1 u M
H M 1 5 2 1 1 1 u M
H M 1 5 2 1 1 1 0 u M
H M 1 5 2 1 1 1 u M + G IP in h ib ito r 1 0 u M
H M 1 5 2 1 1 1 0 u M + G IP in h ib ito r 1 0 0 u M
Co
lla
ge
n c
on
ce
ntr
ati
on
(µg
/ml)
ControlLAPSGIP 1 µM
HM15211 1 µM
HM15211 10 µM
HM15211 1 µM + GIP inhibitor 10 µM
HM15211 10 µM + GIP inhibitor 100 µM
9 0 0 0
1 2 0 0 0
1 5 0 0 0
2 8 0 0 0
Lu
min
os
ce
nc
e
(RL
U)
***
***
***
F B S (% ) 1 0 0 0 0 0 0 0
H M 1 5 2 1 1 (u M ) 0 0 2 2 2 2 2
G IP in h ib ito r (u M ) 0 0 0 0 .3 1 1 .2 5 5 2 0
***
(c) Inhibition of osteoblast apoptosis
GIP inhibitor
Lu
min
os
ce
nce
(RL
U)
FBS (%)
HM15211 (µM)
GIP inhibitor (µM)
10
0
0
0
0
0
0
2
0
0
2
0.31
0
2
1.25
0
2
5
0
2
20
*~***p<0.05~0.001 vs. vehicle by One-way ANOVA
*~***p<0.05~0.001 vs. vehicle by One-way ANOVA
*~***p<0.05~0.001 vs. vehicle by One-way ANOVA
Nature. 481, 314-20 (2012), Nat Rev Cancer. 5, 21-8 (2005)
*~***p<0.05~0.001 vs. vehicle by One-way ANVAO
0
1
2
3
4
NA
FL
D a
cti
vit
y s
co
re
*
**
0
2 0 0
4 0 0
6 0 0
8 0 0
Hy
dro
xy
pro
lin
e (
nm
ol/
g l
ive
r)
***
**
0
2
4
6
8
Re
lati
ve
ex
pre
ss
ion
(Fo
ld i
nc
re
as
e)
** ** ** **
††
††
Effect of a novel long-acting GLP-1/GIP/Glucagon triple agonist (HM15211) in a NASH and fibrosis animal model
In Young Choi1, Jung Kuk Kim1, Jong Suk Lee1, Eun Jin Park1, Dae Jin Kim1, Young Hoon Kim1, and Sun Jin Kim1
1Hanmi Pharm. Co., Ltd, Seoul, South Korea
American Diabetes Association’s (ADA) 78th Scientific Sessions, Orlando, FL, USA; June 22-26, 2018
ABSTRACTNonalcoholic steatohepatitis (NASH), a potential consequence of NAFLD,may lead to end stage liver disease including cirrhosis and hepatocellularcarcinoma. Despite its severity and prevalence, NASH currently lackseffective treatment. In this respect, we developed a novel long-acting, GLP-1/GIP/Glucagon triple agonist, HM15211. Previously, we showed thatHM15211 exerts potent reductions in body weight and hepatic TG(triglycerides) in DIO mice, and showed a liver preferential distribution,suggesting HM15211 as a potential treatment option for NASH. Here, weevaluated the therapeutic effect of HM15211 on NASH and fibrosis by usingDIO mice and MCD-diet mice.
In DIO mice, chronic HM15211 treatment favorably reprogrammed hepaticlipid metabolism as indicated by decrease in lipogenesis related geneexpression (SREBP-1C, ACC1, ACC2, FAS and SCD1) and increase in β-oxidation gene expression (PGC-1α, and CPT-1). In addition, HM15211significantly improved NAFLD activity score and hepatic TG in AMLN dietmice, clearly demonstrating beneficial effect of HM15211 on hepatic lipidmetabolism.
In MCD-diet mice, chronic HM15211 treatment led to significant decrease inhepatic TG (-82.6% vs. vehicle) and TBARS (oxidative stress marker, -60.7%vs. vehicle), which coincided with significant reduction in ALT and bilirubin.Time course MRI analysis also confirmed the progressive steatosisresolution by HM15211, but not by liraglutide. Moreover, qPCR analysisindicated that HM15211 not only reduced the expression of genes involved inhepatic inflammation and HSC activation, but also inhibited fibrosis relatedgene expression. Consistently, HM15211, but not liraglutide, significantlyreduced NAFLD activity score (1.3 for HM15211, 3.4 for liraglutide, and 2.7for vehicle). As to fibrosis improvement, only HM15211 significantly reducedhepatic hydroxyproline contents (-47.8% vs. vehicle) in MCD-diet mice.
Based on these observations, HM15211 may offer a therapeutic potential forNASH and fibrosis as well as obesity.
BACKGROUND
METHODS• To investigate the effects of HM15211 on hepatic lipid metabolism related gene
expression, liver tissue samples were prepared after 4 weeks treatment of HM15211
in DIO mice. Then, the cDNA was synthesized from prepared liver tissues, and
indicated gene expression (de novo lipogenesis: SREBP-1C, ACC1, ACC2, FAS and
SCD1; β-oxidation: PGC-1α, CPT-1, LCAD, ACADVL) was determined via real time
quantitative PCR (qPCR) using cognate primers.
• NAS (NAFLD activity score) and hepatic TG level were determined at the end of
study after 4 weeks treatment of HM15211 in AMLN-diet mice.
• Therapeutic potential of HM15211 in NASH and fibrosis was evaluated in MCD-diet
mice (6 weeks induction). After 4 weeks treatment of HM15211, liver tissue samples
were prepared to measure hepatic TG, TBARS (oxidative stress marker),
Inflammation & HSC activation related marker gene expression (TNF-α, F4/80, TGF-
β and α-SMA) and fibrosis related marker gene expression (Collagen-1α, and TIMP-
1). To non-invasively monitor the changes in hepatic lipid contents, each mouse was
subjected to MRI analysis every 2 weeks.
• To determine NAS (NAFLD activity score), the same region of each liver tissue was
subjected to H&E staining. For fibrosis analysis, Sirius red staining and hepatic
hydroxyproline analysis were performed
Liver preferential distribution of HM15211
RESULTS
Figure 1. Time-dependent tissue distribution of HM15211 in SD
rats (n=3)
CONCLUSIONS
REFERENCES
• HM15211, a novel long-acting triple agonist, improved hepatic lipid
metabolism related gene expression in DIO mice and reduced NASH
prognosis markers in AMLN diet-mice
• HM15211 reduced NASH prognosis related markers including hepatic lipid
contents, oxidative stress, blood ALT, and bilirubin in MCD-diet mice
• HM15211 reduced the expression of genes responsible for hepatic
inflammation, HSC activation, and fibrosis in MCD-diet mice
• The therapeutic effects of HM15211 on NASH is further demonstrated as
reduction of NAS and hepatic hydroyproline contents
• Finan B et al., Sci Transl Med. 5, 209ra(151) (2013)
• Neuschwander-Tetri BA et al., Lancet. 385, 956-65 (2015)
• Finan B et al., Nat Med. 21, 27-36 (2015)
• Harriman G et al., Proc Natl Acd Sci USA. 113, E1796-805 (2016)
1106-P
HM15211 was preferentially distributed to the liver, which is a main target
organ for glucagon action. High glucagon activity in HM15211 might make the
liver preferential distribution.
Figure 5. Effect of HM15211 on hepatic NASH/fibrosis marker
gene expression in MCD-diet mice (n=7)
(a) Inflammation & HSC activation marker gene expression
(b) Fibrosis marker gene expression
HM15211 not only reduced hepatic inflammation and HSC activation related
marker gene expression, but also inhibited fibrosis related gene expression.
Figure 6. Therapeutic effect of HM15211 on NASH and fibrosis
in MCD-diet mice (n=7)
(a) NAFLD activity score †††
(b) Hepatic hydroxyproline
Consistently, HM15211 significantly reduced NAS and hepatic hydroxyproline
(hepatic fibrosis marker).HM15211, long-acting GLP-1/GIP/Glucagon tri-agonist, might
have therapeutic potential in NASH by various MoA in liver.
Kupffer cell
activation
Macrophage
infiltration
ROS
Normal
NAFLD NASH Cirrhosis
Dyslipidemia
Excess
dietary lipid
Oxidative stress
Fatty acid pool ↑
HSC activation,
followed by
increased fibrogenic
properties
Expected benefit by HM15211 treatment on NASH progression
Lipid metabolism reprograming
Hepatic lipid profile improvement
Oxidative stress reduction
Anti-inflammation
Liver function protection
Fibrosis improvement
Inflammation
Fibrosis
0
1
2
3
4
5
Re
lati
ve
ex
pre
ss
ion
(Fo
ld i
nc
re
as
e)
**** ***
T N F - F 4 /8 0 T G F - -S M A
0
5
1 0
1 5
2 0
2 5
3 0
He
pa
tic
TB
AR
S1
) (n
mo
l/m
g)
***
*
N o r m a l m ic e , V e h ic le
M C D m ic e , V e h ic le
L ir a g lu t id e 5 0 n m o l/k g , B ID ( 3 m g /d a y in h u m a n )
H M 1 5 2 1 1 0 .3 6 n m o l/k g , Q 2 D (0 .5 m g /w k in h u m a n )
H M 1 5 2 1 1 0 .7 2 n m o l/k g , Q 2 D (1 .0 m g /w k in h u m a n )
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
Normal vehicle Liraglutide 50 nmol/kg, BID (3 mg/day in human)
HM15211 0.72 nmol/kg, Q2D (1 mg/wk in human)MCD, vehicle
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
*~***p<0.05~0.001 vs. vehicle by One-way ANOVA, †††p<0.001 vs. Liraglutide by One-way ANOVA
Liver preferential
distributionHM15211
*~***p<0.05~0.001 vs. MCD mice, vehicle by One-way ANOVA,††p<0.01 vs. Liraglutide by One-way ANOVA
0
1 0 0 0
2 0 0 0
3 0 0 0
HM
15
21
1 C
on
c.
(ng
/mL
fo
r S
eru
m,
ng
/g f
or T
iss
ue
) 4 hr 48 hr 168 hr
Improved hepatic lipid metabolism in DIO mice
Figure 2. Effect of HM15211 on in hepatic lipid metabolism
related gene and blood lipid profiles in DIO mice (n=7)
(a) De novo lipogenesis related gene
0 .0
0 .5
1 .0
1 .5
Fo
ld i
nc
re
as
e
*
** **
S R E B P -1 C A C C 1 A C C 2 F A S S C D 1
0
2
4
6
Fo
ld i
nc
re
as
e ***
*
***
*
***
NASH and fibrosis improvement in animal models
Figure 3. Effect of HM15211 on NASH prognosis markers in
AMLN-diet mice (n=7)
0
1
2
3
4
5
NA
FL
D a
cti
vit
y s
co
re
*
**
**
0
1 0 0
2 0 0
3 0 0
He
pa
tic
TG
(m
g/d
L)
***
*** ***
(b) NAFLD activity score (a) Hepatic TG
0
5
1 0
1 5
2 0
2 5
3 0
He
pa
tic
TB
AR
S1
) (n
mo
l/m
g)
***
*
N o r m a l m ic e , V e h ic le
M C D m ic e , V e h ic le
L ir a g lu t id e 5 0 n m o l/k g , B ID ( 3 m g /d a y in h u m a n )
H M 1 5 2 1 1 0 .3 6 n m o l/k g , Q 2 D (0 .5 m g /w k in h u m a n )
H M 1 5 2 1 1 0 .7 2 n m o l/k g , Q 2 D (1 .0 m g /w k in h u m a n )
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
Normal vehicle HM15211 1.44 nmol/kg, Q2D (2 mg/wk in human)
HM15211 2.88 nmol/kg, Q2D (4 mg/wk in human)AMLN, vehicle
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
In AMLN-diet induced NASH mice, HM15211 treatment significantly reduced
NAS and hepatic TG.
*~***p<0.05~0.001 vs. AMLN mice, vehicle by One-way ANOVA
(b) β-oxidation related gene
0
1 0 0 0
2 0 0 0
3 0 0 0
HM
15
21
1 C
on
c.
(ng
/mL
fo
r S
eru
m,
ng
/g f
or
Tis
su
e)
S e ru m
L iv e r
H e a rt
L u n g
L a rg e I .
S p le e n
P a n c re a s
A d ip o s e t is s u e
S m a ll I.
S to m a c h
M u s c le
HM15211 treatment not only reduced de novo lipogenesis related gene
expression but also improved β-oxidation related gene expression.
*~***p<0.05~0.001 vs. vehicle by One-way ANOVA
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
Vehicle
Liraglutide 50 nmol/kg, BID (3 mg/day in human)
HM15211 1.44 nmol/kg, Q2D (2 mg/wk in human)
Figure 4. Effect of HM15211 on NASH prognosis markers in
MCD-diet mice (n=7)
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
He
pa
tic
TG
(m
g/g
liv
er)
**
*
0
5
1 0
1 5
2 0
2 5
3 0
He
pa
tic
TB
AR
S (
nm
ol/
mg
liv
er)
***
*
(b) Hepatic TBARS
††
(a) Hepatic TG
†
0
5
1 0
1 5
2 0
2 5
3 0
He
pa
tic
TB
AR
S1
) (n
mo
l/m
g)
***
*
N o r m a l m ic e , V e h ic le
M C D m ic e , V e h ic le
L ir a g lu t id e 5 0 n m o l/k g , B ID ( 3 m g /d a y in h u m a n )
H M 1 5 2 1 1 0 .3 6 n m o l/k g , Q 2 D (0 .5 m g /w k in h u m a n )
H M 1 5 2 1 1 0 .7 2 n m o l/k g , Q 2 D (1 .0 m g /w k in h u m a n )
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
Normal vehicle Liraglutide 50 nmol/kg, BID (3 mg/day in human)
HM15211 0.72 nmol/kg, Q2D (1 mg/wk in human)MCD, vehicle
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
*~***p<0.05~0.001 vs. MCD mice, vehicle by One-way ANOVA††p<0.01 vs. Liraglutide by One-way ANOVA
MCD, Veh, week 2 211 1 mg HED, week 2Lira 3 mg HED week 2Normal, baseline
MCD, Veh, week 4 211 1 mg HED, week 4Lira 3 mg HED week 4MCD, Veh, baseline
HM15211 reduced blood lipid contents, followed by reduction of TBARS, a
well-known lipid peroxidation marker. In addition, HM15211 could improved
liver function as indicated by reduced blood ALT and bilirubin.
(c) Representative MRI
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
Blo
od
AL
T
(IU
/L)
*** ***
0 .0
0 .5
1 .0
1 .5
Blo
od
to
tal
bil
iru
bin
(mg
/dL
)
***
***
(d) Blood ALT (e) Blood total bilirubin
0
5
1 0
1 5
2 0
2 5
3 0
He
pa
tic
TB
AR
S1
) (n
mo
l/m
g)
***
*
N o r m a l m ic e , V e h ic le
M C D m ic e , V e h ic le
L ir a g lu t id e 5 0 n m o l/k g , B ID ( 3 m g /d a y in h u m a n )
H M 1 5 2 1 1 0 .3 6 n m o l/k g , Q 2 D (0 .5 m g /w k in h u m a n )
H M 1 5 2 1 1 0 .7 2 n m o l/k g , Q 2 D (1 .0 m g /w k in h u m a n )
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
Normal vehicle
MCD, vehicle
Liraglutide 50 nmol/kg, BID (3 mg/day in human)
HM15211 0.72 nmol/kg, Q2D (1 mg/wk in human)
0
5 0
1 0 0
1 5 0
He
pa
tic
TG
(m
g/g
)
* * *
V e h ic l e
L ir a g lu t id e 5 0 n m o l/k g , B ID (3 m g /d a y in h u m a n )
H M 1 5 2 1 1 1 .4 4 n m o l/k g , Q 2 D (2 m g /w k in h u m a n )
(c) H&E (above) and sirius red (below) staining
100μm
Normal, vehicle MCD, vehicle MCD, Liraglutide MCD, HM15211
100μm
PGC-1α CPT-1 LCAD ACADVL
TNF-α F4/80 TGF-β α-SMA
Collagen-1α TIMP-1
0
5 0
1 0 0
1 5 0
2 0 0
IFN
- (
pg
/ml)
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
*
0
3 0
6 0
9 0
1 2 0
IL-1
(
pg
/ml)
V e h ic le
H M 1 5 2 1 1 1 .0 8 n m o l/k g
d b /m v e h ic le
d b /d b D 0 (5 w )
*
**
ABSTRACTHM15211 is a novel long-acting GLP-1/GIP/Glucagon triple agonist that isbeing developed for the treatment of obesity and non-alcoholic fatty liverdisease (NASH). Accumulated evidences have shown that obesity, type 2diabetes, and NASH increase the risk of developing progressiveneurodegenerative disease such as Parkinson’s disease (PD) andAlzheimer’s disease (AD). In addition to peripheral contributions, each ofincretins consisting HM15211 have neuroprotective effects in several braindiseases like AD, PD, and ischemia.
Previously, we demonstrated that HM15211 exerted neuroprotective effects inMPTP induced subacute Parkinson’s disease mice model. Here, weevaluated 1) the neuroprotective effects of HM15211 in chronicMPTP/probenecid Parkinson’s disease model, and 2) the protection ofAlzheimer’s disease progression in db/db mice.
Chronic Parkinson’s disease mice model was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in combination with probenecid injection,twice a week for 5 weeks. HM15211 was administered once a week for 6weeks. Dopaminergic neuronal death by MPTP/probenecid was protected byHM15211, which was derived from anti-inflammatory effect by HM15211. Also,HM15211 decreased alpha synuclein in striatum of chronic mice PD model.Together with these efficacies, HM15211 significantly improved theMPTP/probenecid-induced motor impairments in behavior tests (rotarod, poletest, and traction test).
The db/db mice are well-established diabetic model and it was reported thatdb/db mice increase amyloid beta 1-42. Thus, we chose db/db mice toelucidate the prophylactic effect of HM15211 on Alzheimer’s disease. Afteronce every 2 days subcutaneous administration for 12 weeks, HM15211reversed inflammatory cytokines, which was increased in db/db mice. Also,increased amyloid beta 1-42 in db/db mice was decreased by HM15211.
Based on these observations, HM15211 might be a potential therapeuticoption for the neurodegenerative disease.
BACKGROUND
• Neuroprotective effects of GLP-12, glucagon3 and GIP4
CONCLUSIONS
• In MPTP/Probenecid induced chronic Parkinson’s disease model,
HM15211 inhibited the increase of alpha synuclein, which is the most
prominent pathological biomarker of Parkinson’s disease.
• In aged db/db mice, pathological characters of Alzheimer’s disease
such as Aβ1-42 and AGE accumulations were shown. These were
reversed by HM15211 treatment.
• These neuroprotective effects of HM15211 are derived from anti
inflammatory effect through the altered cytokine expression and
reduced lipid peroxidation (data not shown).
• Based on these results, the novel long-acting GLP-1 / GIP / Glucagon
tri-agonist, HM15211 might have therapeutic potential for
neurodegenerative diseases.
Neuroprotective effects of HM15211, a novel long-acting GLP-1/GIP/Glucagon triple agonistin the neurodegenerative disease models
Jeong A Kim1, Sang Don Lee1, Sang-Hyun Lee1, Sung Min Bae1, Young Hoon Kim1, In Young Choi1, and Sun Jin Kim1
1Hanmi Pharm. Co., Ltd, Seoul, Korea
American Diabetes Association’s (ADA) 78th Scientific Sessions, Orlando, FL, USA; June 22‐26, 2018
• Chronic Parkinson’s disease mice model was induced by 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) in combination with probenecid
intraperitoneal injection, twice a week for 5 weeks and HM15211 was
subcutaneously administered once a week for 6 weeks.
• Db/db mice are well-established diabetic model. It has been reported that
db/db mice increase amyloid beta 1-425. Thus we chose db/db mice to
elucidate the prophylactic effect of HM15211 on the development of
Alzheimer’s disease. Six weeks old db/db mice were subcutaneously
treated with HM15211, once every two days for 12 weeks.
1107-P
RESULTS
HM15211 administration restored MPTP/P induced motor function
impairment in (a) traction test, (b) pole test and (c) rotarod test.
Figure 2. Dopaminergic neuroprotection by HM15211
(b) Pole test (T-Total)
HM15211 administration protected MPTP/P induced dopaminergic
neuronal cell damage in the striatum and the substantia nigra (a, b) and
also effectively inhibited the α-synuclein toxicity, which was induced by
MPTP/P (c).
Figure 3. Anti-inflammatory effects of HM15211
In striatum of MPTP/P chronic PD mouse model, HM15211 reduced the
area covered by microglia (a, b) and reversed the induction of IFN-γ (c)
and the reduction of IL-10 (d) levels.
The Aβ1-42 levels in cortex was increased in db/db mice, but HM15211
prevented the accumulation of Aβ1-42(a). Also, HM15211 effectively
decreased the AGE (Advanced glycation end product), which is a factor in
worsening of neurodegenerative disease.
Neuroprotection in chronic PD mice
40xSu
bs
tan
tia
nig
ra
100x
Str
iatu
m
40x
Vehicle MPTP/PMPTP/P + HM15211
5.03 nmol/kg REFERENCES
1. Claudio Procaccini et al., Metabolism. 65(9):1376-90 (2016)
2. Yazhou Li et al., Proc Natl Acad Sci U S A. Jan 27;106(4):1285-90 (2009)
3. Rami Abu Fanne et al., Am J Physiol Regul Integr Comp Physiol 301:
R668–R673 (2011)
4. Yanwei Li et al., Neuropharmacology. 101, 255e263 (2016)
5. Son SM et al., Diabetes. 61(12):3126-38 (2012)
(c) IFN-γ
Figure 1. Motor function restoring effects of HM15211
(a) Traction test (c) Rotarod test
Functional evaluation in chronic PD mice
(d) IL-10
METHODS
GLP-1
Glucagon
GIP
Neuroprotection
Peripheral
contributions
Peripheral-CNS
Crosstalk
↑ Neurite outgrowth
↑ Progenitor proliferation
↓ inflammation
↓ Glutamate neurotoxicity
↑ Progenitor proliferation
↓ inflammation
Mechanisms of neuroprotection in chronic PD mice
AD pathological resolution in db/db mice
Figure 5. Reduced inflammation by HM15211
Mechanisms of neuroprotection in db/db mice
HM15211 decreased of IL-1β (a) and IFN-γ (b) levels of db/db mice cortex.
Also, HM15211 reduced activated microglia in cortex and hippocampus of
db/db mice brain (c).
(a) IL-1β (b) IFN-γ
(c) Reduction of activated microglia in cortex and hippocampus
Figure 4. Inhibited accumulation of Aβ1-42 and AGE by
HM15211
(a) Microglia staining (Iba1)
(a) Aβ1-42 (b) AGE
Diabetes / ObesityIncreased insulin resistance
Accumulation of AGE : Vasculature
Impaired glucose metabolism (Peripheral & brain)
Hyperactivation of RAGE
Release of proinflammatory factors
Reactive oxygen species
Cytokines
Worsening of diabetes
Increased risk of Alzheimer’s disease
Accumulation of Aβ, AGE : Brain
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
Am
ylo
id b
eta
1-4
2 (
% v
s.
ve
hic
le)
V e h ic le
H M 1 5 2 1 1 1 .0 8 n m o l/k g
d b /m v e h ic le
d b /d b D 0 (5 w )
***
***
***
0 .0
0 .3
0 .6
0 .9
1 .2
1 .5
1 .8
AG
E (
ug
/ml)
V e h ic le
H M 1 5 2 1 1 1 .0 8 n m o l/k g
d b /m v e h ic le
d b /d b D 0 (5 w )
***
***
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
IFN
- (
pg
/ml)
V e h ic le
H M 1 5 2 1 1 1 .0 8 n m o l/k g
d b /m v e h ic le
d b /d b D 0 (5 w )
***
**
db/m_vehicledb/db _D0 (6w) db/db_Vehicledb/db_HM15211
1.08 nmol/kg
Co
rte
xH
ipp
o_
CA
1H
ipp
o_
DG
400x
400x
400x
0
1
2
3
4
Tra
ctio
n t
es
t (
sc
or
e 0
~3
)
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
*****
Vehicle
MPTP 25 mg/kg (sc, twice weekly)
+ Probenecid 250 mg/kg (ip, twice weekly)
MPTP/P +
HM15211 5.03 nmol/kg (sc, QW)
0
1
2
3
4
Tra
ctio
n t
es
t (
sc
or
e 0
~3
)
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
*****
0
1
2
3
4
Tra
ctio
n t
es
t (
sc
or
e 0
~3
)
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
*****
0
2 0
4 0
6 0
Po
le t
es
t (T
-to
tal,
s) V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
***
***
0
5 0
1 0 0
1 5 0
2 0 0
Ro
ta
ro
d (
fa
llin
g l
ate
nc
y,
s)
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
*** ***
0
5 0
1 0 0
1 5 0
2 0 0
TH
+ c
ell
s i
n S
ub
sta
nti
a n
igra
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
***
*
0
2
4
6
8
-s
yn
uc
lein
(n
g/m
l) V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
*****
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
Iba
1 +
are
a i
n S
tria
tu
m
(% v
s.
ve
hic
le)
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
**
*
0
1 0 0 0
2 0 0 0
3 0 0 0
IL-1
0 (
pg
/ml)
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
******
(b) Iba1 + area
0
1
2
3
4
Tra
ctio
n t
es
t (
sc
or
e 0
~3
)
V e h ic le
M P T P 2 5 m g /k g (s c , tw ic e w e e k ly ) +
P ro b e n e c id 2 5 0 m g /k g ( ip , tw ic e w e e k ly )
M P T P /P +
L A P S -H M T 2 1 1 5 .0 3 n m o l/k g (s c , Q W )
*****
(a) Dopaminergic neuron staining (TH; tyrosine hydroxylase)
(b) TH+ cell number (c) α-synuclein
• Obesity is one of the risk factors of neurological disorder1
Alzheimer’s disease Parkinson’s disease
Insulin resistance, T2DM ↑ PD
↑ Insulin levels ↑ α-synuclein aggregation
Leptin ↑ survival of DA cells
↑ BMI, T2DM ↑ AD risk
Leptin/insulin resistance ↑ AD
Leptin ↓ Aβ, p-tau
Tra
ctio
n te
st (s
co
re 0
~3
)
Po
le te
st (T
-to
tal, s
)
*~***p<0.05~0.001 vs. MPTP/P by One-way ANOVA
Ro
taro
d(f
alli
ng
la
ten
cy,
s)
TH
+ c
ells
in
su
bsta
ntia n
igra
α-s
yn
ucle
in(n
g/m
l)
Iba
1 +
are
a in
str
iatu
m
(% v
s. ve
hic
le)
IFN
-γ(p
g/m
l)
IL-1
0 (
pg/m
l)
IL-1
β(p
g/m
l)
IFN
-γ(p
g/m
l)
Aβ
1-4
2(%
vs.
ve
hic
le)
AG
E (
μg
/ml)
Vehicle MPTP/P
Str
iatu
m
200x
MPTP/P + HM15211
5.03 nmol/kg
400x
*~***p<0.05~0.001 vs. db/db (18w) vehicle by One-way ANOVA
db/db (18w) vehicle
db/m (18w) vehicle
db/db D0 (6w)
db/db (18w) HM15211 1.08 nmol/kg, Q2D
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
Am
ylo
id b
eta
1-4
2 (
% v
s.
ve
hic
le)
V e h ic le
H M 1 5 2 1 1 1 .0 8 n m o l/k g
d b /m v e h ic le
d b /d b D 0 (5 w )
***
***
***
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
Am
ylo
id b
eta
1-4
2 (
% v
s.
ve
hic
le)
V e h ic le
H M 1 5 2 1 1 1 .0 8 n m o l/k g
d b /m v e h ic le
d b /d b D 0 (5 w )
***
***
***0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
Am
ylo
id b
eta
1-4
2 (
% v
s.
ve
hic
le)
V e h ic le
H M 1 5 2 1 1 1 .0 8 n m o l/k g
d b /m v e h ic le
d b /d b D 0 (5 w )
***
***
***
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
Am
ylo
id b
eta
1-4
2 (
% v
s.
ve
hic
le)
V e h ic le
H M 1 5 2 1 1 1 .0 8 n m o l/k g
d b /m v e h ic le
d b /d b D 0 (5 w )
***
***
***