Lipolytic, Energy Expenditure, and Insulinotropic Effects of HM12525A: A Novel Long-Acting...

Lipolytic, Energy Expenditure, and Insulinotropic Effects of HM12525A: A Novel Long-Acting GLP-1/Glucagon Dual Agonist

SY Jung1,, YJ Park1 , JK Kim1, JS Lee1, YM Lee1, YH Kim1, JH Kang1, M Trautmann2, M Hompesch2, SC Kwon1 1Hanmi Pharm. Co., Ltd., Seoul, South Korea, 2Profil Institute, Chula Vista, CA, USA

Test materialsRelative activity (cAMP assay)

GLP : Glucagon% of GLP-1 % of Glucagon

GLP-1 100 ---

Glucagon --- 100

HM12525A 30 37 1 : 1

BACKGROUND

Potential beneficial effects of GLP-1/Glucagon dual agonist Energy expenditure & Lipolysis Insulin secretion & Food intake

GLP-1/Glucagon dual agonist

Liver TG ↓ Ketogenesis ↑ FGF21 ↑

BrainFood intake ↓

Fat Cells Lipolysis ↑ Lipogenesis ↓ Energy Expenditure ↑

PancreasInsulin secretion ↑

StomachGastric emptying ↓

HM12525A is a long acting GLP-1/Glucagon dual agonist withbalanced dual agonism at GLP-1 and Glucagon receptors

METHODS

P866

a) Energy expenditure

0 24 48 72 96 120 144 168

8

12

16

Time (hr)

En

erg

y E

xp

en

dit

ure

(Kca

l/kg

/hr)

0 24 48 72 96 120 144 1680

100

200

300

400

Time (hr)

Lo

co

mo

tor

Acti

vit

y (

co

un

t)

0 24 48 72 96 120 144 168

1500

2000

2500

3000VehicleHM12525A, 3 nmol/kg/week

Time (hr)

VO 2

(m

L/k

g/h

r)

0 24 48 72 96 120 144 168

1500

2000

2500

3000VehicleHM12525A, 3 nmol/kg/week

Time (hr)

VO 2 (m

L/kg

/h

r)

b) Locomotor activity

HM12525A increased energy expenditure without locomotor activity change.

Figure 4. Energy expenditure in DIO mice (n=10, 4 weeks)

Figure 3. Body weight loss in DIO mice (n=6, 2 weeks)

Improved BWL by Increase of Energy Expenditure

0 2 4 6 8 10 12 1460

70

80

90

100

110

VehicleVictoza 100nmol/kg/day

HM12525A 3 nmol/kg/weekHM12525A 5 nmol/kg/week

Time (Days)

No

rm

ali

zed

BW

%

0 2 4 6 8 10 12 140

10

20

30

40

Vehicle

LAPS OXM25 3 nmol/kg/weekLAPS OXM25 5 nmol/kg/week

Victoza 100nmol/kg/day

Time (Days)

Accu

mu

late

d F

oo

d i

nta

ke (

g)

0 2 4 6 8 10 12 1460

70

80

90

100

110

VehicleLiraglutide 100 nmol/kg/dayHM12525A 3 nmol/kg/weekHM12525A 5 nmol/kg/week

Time(days)

No

rm

alized

B

W %

-1%

-10%

-17%

-31%

a) Body weight loss b) Cumulative food intake

HM12525A showed more potent weight loss even with less food intake inhibition compared with liraglutide.

0 2 4 6 8 10 12 1460

70

80

90

100

110

Vehicle

Liraglutide 100 nmol/kg/day

HM12525A 3 nmol/kg/week

HM12525A 5 nmol/kg/week

Time(days)

No

rm

alized

B

W %

-51%-36%

RESULTS

Figure 1. HbA1c and body weight reduction in db/db mice (n=7, 4 weeks)

a) HbA1c b) Body weight gain

HM12525A decreased HbA1c as well as body weight gain in db/db mice.

0

2

4

6

8

10

Vehicle

HM12525A 6 nmol/kg/week

Liraglutdie 60 nmol/kg/day (1X HED, Diabetes)

Liraglutide 100 nmol/kg/day (1X HED, Obesity)

*** *** ***

6.7 6.5 6.37.7Hb

A1

C (%

)

** p<0.01, *** p<0.001 vs vehicle

Improved Glycemic Control by GLP-1R Activation

AIMS1. To assess glycemic control of HM12525A 2. To assess body weight and fat mass changes by HM12525A 3. To assess liver function improvement by HM12525A

Db/db mice (n=7) were treated (s.c) with HM12525A once a week, or liraglutide once daily, for 4 weeks respectively. Blood glucose levels were measured using a glucometer.

For ipGTT, overnight fasted C57BL/6J mice were administrated with either HM12525A or liraglutide (i.v.), and the 2 g/kg of glucose was subsequently administrated (i.p.). Blood glucose and the serum insulin level was determined at 0, 30, 60, 120 min using commer-cially available kit.

26 weeks HFD induced C57BL/6J mice (n=6) were treated (s.c) with HM12525A once a week, or with liraglutide once daily for 2 weeks respectively. The body weight and food in-take was monitored daily.

Energy Expenditure as well as home-cage activity were assessed by using a combined indirect calorimetry system for 1 week after the first administration in DIO mice. O2 con-sumption and CO2 production were measured every 10 min for a total of 7 days to de-termine the respiratory quotient and energy expenditure. Home-cage locomotor activity was determined using a multidimensional infrared light beam system. .

To differentiate into adipocytes, confluent 3T3-L1 cells (day 1) were incubated with 0.5 mM IBMX, 0.5 μM dexamathasone, and 1 μg/ml insulin added to DMEM/10% FBS for 2 days. On day 3, the medium was changed to DMEM/FBS 10% with 1 μg/ml insulin, which was repeated every 2 days. The cells were fully differentiated into adipocytes after 10 to 14 days incubation of differentiation medium. HM12525A was co-incubated in the pres-ence or absence of GCGR antagonist from day 3 to the end of experiment. On day 14, to evaluate the effects of HM12525A on lipid droplet formation, Oil-Red O staining was con-ducted. Briefly, the cells were fixed with 4% paraformaldehyde for 30 min, followed by in-cubation of Oil-Red O solution for 1 hr. After washing, Oil-Red O stained lipid droplet was determined using optical microscope

0

5

10

15

20

25

Vehicle

Liraglutide 60 nmol/kg/day (1X HED, Diabetes)

Lirglutide 100 nmol/kg/day (1X HED, Obesity)

HM12525A 6 nmol/kg/week

** **

***

Body w

eig

ht change (%

)

European Association for the Study of Diabetes 50th Annual Meeting; Vienna, Austria; September 15-19, 2014 For any questions, please contact Hanmi Pharm. Co., Ltd., Phone: +82-31-371-5141; cjchoi@hanmi.co.kr Hanmi Hanmi Pharm. Co., Ltd.

CONCLUSIONS

REFERENCES

HM12525A showed glucose lowering efficacy in normal and db/db mice by balanced activity on both GLP-1 and glucagon receptors.

Potent body weight loss was driven by fat mass reduction and the lean/muscle mass did not changed, because of increased energy expenditure.

HM12525A showed therapeutic potential in NASH animal model

1. Diabetes (2009) 58 : 2258 ~ 2266. 2. Nat. Rev Endocrinol. (2014) 10 : 24 ~ 363. Diabetes (2013) 62 : 1131 ~1138

Figure 10. Clinical development milestone

HM12525A 2015 2016 2017 2018 2019 2020 2021 NDA submission

First In Human (SAD + MAD)

T2DM 2019

Obesity2020P3P2

P3

P1

P2

0

2

4

6

8

10

12

LAPSGLP/Glucagon

GLP/Glucagon 25

ex v

ivo

T c

ell

activ

atio

n

fre

qu

en

cy o

f R

esp

on

se

(%

)

2%4%

0

2

4

6

8

10

HM12525A

Non conjugated active moiety

ex vivo

T

cell activatio

n

fre

qu

en

cy o

f R

esp

on

se

(%

)

Immunogenic threshold

Figure 9. Ex vivo T cell activation of HM12525A

Both non-conjugated active moiety and HM12525A showed negligible T cell activation, which is below the immunogenic threshold among 50 healthy human donors.

Figure 8. Liver function improvement in MCD-diet db/db mice (12 days)

2014

Immunogenic Potential

Improved Liver Function in NASH Animal Model

Body weight(MCD + db/db mice, n=7)

0 2 4 6 8 10 12 14-40

-30

-20

-10

0

10

Time (days)

Body

wei

ght c

hang

e(%

)vs

pre

dose

1.0

1.5

2.0

2.5

MCD + vehicle

HM12525A, 0.7 nmol/kg/Q2D

HM12525A, 1.4 nmol/kg/Q2D

Live

r w

eigh

t (g

)

Liver weight(MCD + db/db mice, n=7)

******

SREBP-1c

0.0

0.5

1.0

1.5Vehicle

HM12525A, 0.7HM12525A, 1.4

SREB

P-1c

mRN

A fol

dvs

vehic

le

**p<0.01, ***p<0.001 vs vehicle by Anova test

**

SREPB-1c mRNA level(MCD + db/db mice, n=3-6)

1.0

1.5

2.0

2.5

MCD + vehicle

HM12525A, 0.7 nmol/kg/Q2D

HM12525A, 1.4 nmol/kg/Q2D

HM11260C, 0.7 nmol/kg/Q2D

HM11260C, 1.4 nmol/kg/Q2D

HM11260C, 2.9 nmol/kg/Q2D

Liraglutide, 30 nmol/kg/BID

Liraglutide, 50 nmol/kg, BID

Liv

er w

eig

ht (

g)

HM12525A showed reduction of liver weight and SREPB-1c mRNA level in MCD- diet db/db mice which indicates NASH could be improved by HM12525A

Figure 2. ipGTT in normal mice (n=7)

a) ipGTT b) AUCipGTT

**p<0.01, ***p<0.001 vs vehicle by Anova test

HM12525A improved glucose tolerance dose-dependently in normal mice.

Figure 6. Body composition change in normal and DIO mice (n=10, 4 weeks)

Lipid and Fat Mass Reduction by Lipolytic Activity

0

20

40

60

Body

weigh

t (g)

Body weight

-23 %***

Body fat mass

0

5

10

15

20

25

Fat m

ass (g

)

-48.8 %***

0

50

100

150

200Normal VehicleVehicle (DIO)HM12525A (DIO), 3 nmol/kg/week

***

AS

T(I

U/L

)

0

20

40

60

Vehicle (HFD)

LAPS OXM25 3 nmol/kg

Vehicle (RC)

Body lean

(g)

*** ; P<0.001, vs DIO vehicle

0

100

200

300

400

500

Vehicle (HFD)

LAPS OXM25 3 nmol/kg

Vehicle (RC)

Muscle (m

g)

Body muscle massLean body mass

Not significant

Not significant

No lean mass and body muscle reduction was observed by HM12525A treatment

Figure 7. Reduction of lipid droplet formation in 3T3-L1 adipocytes

*Glucagon antagonist : (des His1, Glu9) GCG (1-29)

+ Glucagon antagonist *VehicleHM12525A, 100 nM

VehicleEpinephrine

1000 nMHM12525A

100 nM

HM12525A reduced lipid droplet formation in 3T3-L1 adipocytes via glucagon action.

Figure 5. Changes in energy expenditure mediator expression by HM12525A in white adipocytes (3T3-L1 cells)

3T3-L1 (White adipocyte)

6h~9 day

UCP-1 mRNA 3T3-L1 differentiation

HM12525A increased the expression of UCP-1 in white adipocytes, suggesting browning of WAT by HM12525A.

-24 0 0.25 0.5 1 20

200

400

600

vehicleHM12525A 3nmol/kg

HM12525A 5nmol/kgHM12525A 7nmol/kgVictoza 100nmol/kg

Time (hr)

Bloo

d Gluc

ose (

mg/dL

)

0

200

400

600

800

vehicle

HM12525A 3 nmol/kgHM12525A 5 nmol/kg

***

Dunnett's Multiple Comparison Test* : P<0.05** : P<0.01*** : P<0.001

HM12525A 7 nmol/kg

Liraglutide 100 nmol/kg

***

******

AU

C0-2h

r(m

g/d

L*d

ay)

0

200

400

600

800

vehicleVictoza 100nmol/kgHM12525A 3nmol/kg

***

HM12525A 5nmol/kgHM12525A 7nmol/kg

*** ******

AU

C 0-2

hr(m

g/d

L*d

ay)

-24 0 0.25 0.5 1 20

200

400

600

Vehicle

HM12525A 3 nmol/kgHM12525A 5 nmol/kgHM12525A 7 nmol/kg

Liraglutide 100 nmol/kg

Time (hr)

Blo

od G

luco

se (m

g/dL

)

UCP1 mRNA in 3T3-L1 cells (Mouse adipocyte, Q-PCR)

100

200

300

400

500Control

Epinephrine 1 M

HM12525A 10 M

HM12525A 1 M

Rel

ativ

e m

RN

A le

vel

(% o

f co

ntro

l)

***

***

0

2

4

6

8

10

Vehicle

HM12525A 6 nmol/kg/week

Liraglutdie 60 nmol/kg/day (1X HED, Diabetes)

Liraglutide 100 nmol/kg/day (1X HED, Obesity)

*** *** ***

6.7 6.56.3

7.7

Hb

A1

C (

%)

SAD : 2014. 4Q MAD : 2015. 2Q

22.4%(+9.3 g)

13.9%(+5.7 g)

1.0%(+0.4 g)

14.5%(+5.7 g)