Effect of a novel long-acting GLP-1/GIP/glucagon triple agonist (HM15211) in a dyslipidemia animal models 1054-P Hyo Sang Jo1, Jae Hyuk choi1, Jung Kuk Kim1, Sang Don Lee1, Jong Soo Lee1, Sang Hyun Lee1, and In Young Choi1 1Hanmi Pharm. Co., Ltd, Seoul, South Korea BACKGROUND RESULTS Known targets of current dyslipidemia drugs, and suggested effects of HM15211 on lipid metabolism Lipid lowering efficacy in an animal model Enhanced LDL clearance by HM15211 Improved FFA metabolism by HM15211 Figure 1. Effect of HM15211 on blood cholesterol (CHO) in Figure 3. Effect of HM15211 on hepatic LDLR expression Figure 5. Effect of HM15211 on FFA metabolism related gene Diet HM15211 dyslipidemia hamsters (n=6) and LDL uptake expression in HepG2 cells (a) LDL-C level (b) HDL-C level (a) In HepG2 cells (b) In hamster liver (a) De novo lipogenesis Lipid 3 0 0 CTL HM15211 Evolocumab Vehicle HM15211 Evolocumab absorption Ezetimibe PCSK9 1 .5 2 0 0 1 .5

e 4 0 0 k

PCSK9 Ab LDLR LDLR*** n CTL

a

)

)

o

t

)

i

)

L L

LDLR p *** HM15211 10 μM

n

e L

3 0 0 s

d

u

d

o

/

/

T

s

)

s i

Fibrate Y

g

e

C

g

s a

e 1 .0 1 .0

P

.

s

r

s I

Intestine 2 0 0 β-actin 2 0 0 β-actin e

m

s

m

a e

1 8 0 r

p

D

(

(

v

r

e

LDL uptake ↑ *

c

x

r

O

p

C

C

n

e %

3 0 0 c

x

B

i

-

- (

DGAT PPARα↑ - 1 0 0

n

Niacin TG ↓ e

i

)

e

L

L L

* d

e * **

v

D L

l 0 .5

d

D

D

i

v l

L **

i

t

d o

L 0

t

H o

/ (c) LDL uptake in HepG2 cells

*

a F

a * **

F

l g

0 .l 5

(

d (

d ** e

1 6 0 e

Cholesterol ↓ o

VLDL ↓ LDL ↓ o m

4 0 0 R R

( 2 0 0 o

1 0 0 o

l *

l *** CTL 0 .0

e

C B

ABCA1 B -

*** k HM15211 0.01 μM a

L *** t

Statin HMG-CoA ) 3 0 0 HM15211 0.1 μM

HDL ↑ D

p

L u

L ***

T HM15211 Reductase HM15211 1 μM 0 .0

1 0 0 Y d

C Srebp1 Acc1 Acc2 Fas Scd1 P *** . o *** HM15211 10 μM

*** I 2 0 0

s

o

D v 0 l 5 0 Evolocumab 10 μg/mL

FGF-21 ↑ Apo A1 ↑ Apo A1 *** O

B

%

B

( -

L 1 0 0

(c) LDL/HDL ratio 0 D (b) β-oxidation Abbreviations L Normal hamster, Vehicle ***p<0.001 vs. CTL by One-way ANOVA Srebp1: Sterol regulatory element-binding protein 0 Acc1/2: Acetyl-CoA carboxylase 1/2 2 .0 Dyslipidemia hamster, Vehicle 1 5 Fas: Fatty acid synthase Evolocumab 219.2 nmol/kg, QW (1680 mg/month in human) HM15211 treatment increased LDLR expression in HepG2 cells and liver tissue, Scd1: Stearoyl-CoA desaturase1 Cpt1: Carnitine palmitoyltransferase1 As possessing single target, the efficacy of current dyslipidemia Rosuvastatin 3.0 mg/kg, QD (40 mg/day in human) which correlated with enhanced LDL uptake by HM15211 Vlcad: Very long-chain acyl-CoA dehydrogenase

n Hadha/b: Hydroxyacyl-CoA dehydrogenase

HM15211 1.6 nmol/kg, Q2D (4 mg/week in human) o

1 .5 )

drugs was limited. With GLP-1, GIP, and GCG triple-agonism, i Acox: Acyl-coA oxidase

e

s o

i 1 0

HM15211 3.1 nmol/kg ,Q2D (8 mg/week in human) s s

t Ehhadh: Enoyl-CoA Hydratase, 3-Hydroxyacyl CoA Dehydrogenase

a e HM15211 might provide more promising lipid lowering efficacy a §

r Acaa1: 3-ketoacyl-CoA thiolase

e

r

HMGCR inhibition by HM15211 r

p

L

c

x

n

D

e

i

1 .0

H

e

/

d

l v

L Figure 4. Effect of HM15211 on hepatic HMGCR expression,

i

o t

AIMS D 5

a

F

l

L ( . Previously, we showed that a long-acting GLP-1/GIP/Glucagon and enzymatic activity e ** R ** * ** 0 .5 ** triple agonist, HM15211, not only provided efficient weight loss, *** (a) In HepG2 cells (b) In hamster liver * *** but also improved lipid profiles in DIO mice. With its triple *** CTL HM15211 Rosuvastatin Vehicle HM15211 Rosuvastatin 0 ***p< 0.001 vs. vehicle by One-way ANOVA 5.7 Cpt1 Lcad Vlcad Hadha Hadhb Acox Ehhadh Acaa1 agonism, HM15211 could affect multiple pathways in lipid 0 .0 1 5 0 In mitochondria In peroxisome HMGCR HMGCR

metabolism, suggesting HM15211 as a novel therapeutic option In high fat- and high fructose-fed hamsters, HM15211 treatment showed superior

y

y

y t

t  HM15211 reduced and increased the expression of genes involved in de novo

t

i

i

i )

for dyslipidemia cholesterol lowering compared to lipid lowering agents )

)

v v

v β-actin

β-actin i

i

L

L i

L 1 0 0 t

t 1 0 0 lipogenesis and β-oxidation in HepG2 cells, respectively, suggesting favorable

t T

1) T

T

c c

. In the present study, we investigated the therapeutic effect of c

C

C

a

a

C

a

changes in lipid metabolism

.

.

.

R

R s

2) s

R

s v

HM15211 on dyslipidemia in disease animal model and its v

C

C v

Inhibition of lipid absorption by HM15211 § C

G

G

%

% G

(c) HMGCR activity % 5 0

(

(

(

M M

mode of action (MoA). M CONCLUSIONS

H H

Figure 2. Effect of HM15211 on lipid absorption during oil H )

L • In dyslipidemia hamsters, HM15211 provides greater CHO

tolerance test in normal mice (n=5) T METHODS 1 0 0 0 C lowering than commercial dyslipidemia drugs such as 0 2 4 4 8 1 2 0 0 . 0 2 4 4 8

1 2 0 0 s

) T im e (m in ) ) . To evaluate the therapeutic efficacy in dyslipidemia, high-fat and v T im e (m in )

) evolocumab and statin L

L 1 0 0 0

L d

d 7 5

/

/ %

d 1 0 0 0

g (

high-fructose diet hamsters were administered with HM15211, and g / 8 0 0 • In the series of mechanistic studies, responsible MoAs for

8 0 0 m 1 2 0 0 m C T L

1 2 0 0 g C T L (

( CTL

y t

blood lipids were monitored. Commercially available dyslipidemia )

)

)

m G

8 0 0 G i H M 1 5 2 1 1 1 0  M L 6 0 0 

( 6 0 0 HM15211 10 μM this potent CHO lowering by HM15211 are elucidated as

L

L T

1 0 0 0 T

1 0 0 0

v

5 0

d

d

d

i

/

/

/ a a R o s u v a ta tin 1  M t R o s u v a ta tin 1  M G μM

drugs such as evolocumab and rosuvastatin were used as g RosuvastatinR o s u v a ta tin 1  M 1

g g

m 4 0 0 6 0 0 m 4 0 0 8 0 0 c follows: (1) inhibition of lipid absorption, (2) enhanced LDL 8 0 0 T

m 8 0 0

m

s

s

m

(

(

a

(

a

a

l l

comparative control. At the end of study, liver tissue samples were d G

G 2 0 0

G 2 0 0 p

6 0 0 p clearance, (3) inhibition of CHO synthesis, and (4) improved o 6 0 0 R

T 2 5 T

T 4 0 0

***

o

a

C

a a prepared, and protein level of LDLR and HMGCR was evaluated l *** 0

m 4 0 0 FFA metabolism

m 4 0 0

m 4 0 0

G

B

s s

s 2 0 0 0 1 2 * * * 3 4

a

a

a M

. To evaluate the inhibitory effect of HM15211 on lipid absorption, oral l l l 2 0 0 p 2 0 0 0

p 2 0 0 *** *** p

T im e (h o u r s ) H • In conclusion, our results suggest that HM15211 might be a lipid tolerance test was performed. Briefly, overnight fasted normal 0 * * * 0 2 4 4 8 0 good therapeutic option for dyslipidemia patients mice were fed with olive oil, followed by blood TG monitoring 0 10 2 1 3 4 2 3 4 O liv e o il-fe d , V e h ic le T im e ( h r s ) T im e (h o u r s ) ***p<0.001 vs. CTL by One-way ANOVA T im e (h r s ) E v o lo c u m a b 5 1 .7 m g /k g , 4 2 0 m g /m o n th in h u m a n (1 X H E D ) . For in vitro MoA studies, cell lysates of HepG2 cells treated with §HMGCR: HMG-CoA reductase, rate limiting enzyme for CHO synthesis R o s u v a s ta tin 4 .9 m g /k g , 4 0 m g /d a y in h u m a n (1 X H E D ) REFERENCES HM15211 were subjected to western blot analysis (LDLR, and ***p<0.001 vs. Normal mice, vehicle by One-way ANOVA O liv e o il-fe d , V e h ic le E z e tim ib e 1 .2 m g /k g , 1 0 m g /d a y in h u m a n (1 X H E D ) Olive oil-fed, Vehicle  HM15211 treatment increased phosphorylation of HMGCR (data not shown), the • A Kharitonenkov et al, Mol Metab. 3, 221-9 (2013) HMGCR) and qPCR (lipid metabolism-related genes) EEvolocumabv o lo c u m a b 5 1 .7 m g51.7/k g , 4 2mg/kg0 m g /m o n(420th in h umg/monthm a n (1 X H E D ) in human) HM15211H M 1 5 2 1 1 0 .5 /0.5k g , 4 mg/kgm g /w e e k i(4n h mg/weeku m a n in human) . Additionally, LDL uptake and enzymatic activity of HMGCR in RRosuvastatino s u v a s ta tin 4 .9 m g /k4.9g , 4 0 mg/kg m g /d a y i n(40 h u m mg/daya n (1 X H E D ) in human) HM15211H M 1 5 2 1 1 1 .0 1.0m g /k mg/kgg , 8 m g /w e(8e k mg/weekin h u m a n in human) key enzyme for CHO synthesis, followed by its progressive degradation in HepG2 • E Osto et al, Circulation. 131, 871-81 (2015) E z e tim ib e 1 .2 m g /k g , 1 0 m g /d a y in h u m a n (1 X H E D ) cells and liver tissue, thereby substantially inhibiting the enzymatic activity of • JS Dron et al, Arterioscler Thromb Vas Biol. 38, 592-8 (2018) HepG2 cells were also determined after HM15211 treatment by BloodH M 1TG5 2 1 1 0 .was5 /k g , 4 msignificantlyg /w e e k in h u m a n reduced in HM15211 groups after olive oil challenge, HMGCR using commercially available kits demonstratingH M 1 5 2 1 1 1 .0 m g /k gthe, 8 m g /inhibitoryw e e k in h u m a n effect of HM15211 on lipid absorption

European Association for the Study of Diabetes (EASD) 55th Annual Meeting, Barcelona, Spain, 16 - 20 September 2019