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 )

***

***

***