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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; [email protected] 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)
