Lipolytic , Energy Expenditure, and Insulinotropic Effects of HM12525A:

1. Lipolytic, Energy Expenditure, and Insulinotropic Effects of HM12525A: A Novel Long-Acting GLP-1/Glucagon Dual Agonist P866 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 Improved Liver Function in NASH Animal Model RESULTS BACKGROUND Figure 5. Changes in energy expenditure mediator expression by HM12525A in white adipocytes (3T3-L1 cells) Figure 8. Liver function improvement in MCD-diet db/dbmice (12 days) Improved Glycemic Control by GLP-1R Activation Potential beneficial effects of GLP-1/Glucagon dual agonist Liver weight (MCD + db/db mice, n=7) SREPB-1c mRNA level (MCD + db/db mice, n=3-6) Body weight (MCD + db/db mice, n=7) ~9 day • Energy expenditure  & Lipolysis  • Insulin secretion  & Food intake  6h Figure 1. HbA1c and body weight reduction in db/dbmice (n=7, 4 weeks) UCP-1 mRNA 3T3-L1 differentiation b) Body weight gain a) HbA1c 3T3-L1 (White adipocyte) 22.4% (+9.3 g) Brain Food intake ↓ Liver TG ↓ Ketogenesis ↑ FGF21 ↑ 14.5% (+5.7 g) 13.9% (+5.7 g) *** *** UCP1 mRNA in 3T3-L1 cells (Mouse adipocyte, Q-PCR) ** *** 1.0% (+0.4 g) **p<0.01, ***p<0.001 vs vehicle by Anova test ** p<0.01, *** p<0.001 vs vehicle GLP-1/Glucagon dual agonist *** • HM12525A decreased HbA1c as well as body weight gain in db/db mice. • HM12525A showed reduction of liver weightandSREPB-1c mRNA level in MCD- • diet db/dbmice which indicates NASH could be improved by HM12525A Pancreas Insulin secretion ↑ Fat Cells Lipolysis ↑ Lipogenesis ↓ Energy Expenditure ↑ Figure 2. ipGTT in normal mice (n=7) Immunogenic Potential • HM12525A increased the expression of UCP-1 in white adipocytes, suggesting • browning of WAT by HM12525A. b) AUCipGTT a) ipGTT Figure 9. Ex vivo T cell activation of HM12525A Stomach Gastric emptying ↓ Lipid and Fat Mass Reduction by LipolyticActivity Immunogenic threshold HM12525A is a long acting GLP-1/Glucagon dual agonist with balanced dual agonism at GLP-1 and Glucagon receptors Figure 6. Body composition change in normal and DIO mice (n=10, 4 weeks) Body muscle mass Body fat mass Lean body mass Body weight -48.8 % -23 % Not significant 4% *** *** 2% **p<0.01, ***p<0.001 vs vehicle by Anova test • Both non-conjugated active moiety and HM12525A showed negligible T cell • activation, which is below the immunogenic threshold among 50 healthy human • donors. • HM12525A improved glucose tolerance dose-dependently in normal mice. Not significant AIMS Figure 10. Clinical development milestone • To assess glycemic control of HM12525A • To assess body weight and fat mass changes by HM12525A • To assess liver function improvement by HM12525A 2014 HM12525A 2015 2016 2017 2018 2019 2020 2021 NDA submission SAD : 2014. 4Q MAD : 2015. 2Q First In Human (SAD + MAD) *** ; P<0.001, vs DIO vehicle P1 Improved BWL by Increase of Energy Expenditure METHODS 2019 T2DM • No lean mass and body muscle reduction was observed by HM12525A treatment P2 P3 -36% Figure 3. Body weight lossin DIO mice (n=6, 2 weeks) • 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 commercially available kit. • 26 weeks HFD induced C57BL/6J mice (n=6) were treated (s.c) with HM12525A once a week, or with liraglutide oncedaily for 2 weeks respectively. The body weight and food intake 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 consumption and CO2 production were measured every 10 min for a total of 7 days to determine 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 presence 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 conducted. Briefly, the cells were fixed with 4% paraformaldehyde for 30 min, followed by incubation of Oil-Red O solution for 1 hr. After washing, Oil-Red O stained lipid droplet was determined using optical microscope -51% Figure 7. Reduction of lipid droplet formation in 3T3-L1 adipocytes 2020 a) Body weight loss b) Cumulative food intake Obesity P2 P3 Epinephrine 1000 nM • HM12525A • 100 nM Vehicle -1% CONCLUSIONS -10% • 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 -17% Figure 4. Energy expenditure in DIO mice (n=10, 4 weeks) b) Locomotor activity a) Energy expenditure • HM12525A, 100 nM -31% Vehicle • + Glucagon antagonist * REFERENCES • HM12525A showed more potent weight loss even with less food intake • inhibition compared with liraglutide. • *Glucagon antagonist : (des His1, Glu9) GCG (1-29) 1. Diabetes (2009) 58 : 2258 ~ 2266. 2. Nat. Rev Endocrinol. (2014) 10 : 24 ~ 36 3. Diabetes (2013) 62 : 1131 ~1138 • HM12525A reduced lipid droplet formation in 3T3-L1 adipocytes via glucagon action. • HM12525A increased energy expenditure without locomotor activity change. For any questions, please contact Hanmi Pharm. Co., Ltd., Phone: +82-31-371-5141; cjchoi@hanmi.co.kr Hanmi Pharm. Co., Ltd. European Association for the Study of Diabetes 50th Annual Meeting; Vienna, Austria; September 15-19, 2014 Hanmi