53-LB Super-Agonistic Activation of GLP-1 by CA Exendin-4 (exendin-4 analog) with Fast Constant In Young Choi, Sung Hee Park, Sang Youn Hwang, Gyu Hwang Lee, Young Hoon Kim, Se Chang Kwon* Hanmi Pharm. Co., Ltd, Seoul, South Korea *Se Chang Kwon, Tel: +82-31-371-5001; e-mail: [email protected] Abstract Results Conclusions Exendin-4 is a glucagon-like peptide-1 (GLP-1) receptor used for the Receptor Kinetics A Model for Strong . Receptor kinetics based on SPR analysis revealed that CA Exendin-4 dissociates treatment of type 2 diabetes. We have developed a novel, N-terminal modified 5 fold faster than native Exendin-4 from human GLP-1 receptor (GLP-1R). Exendin-4 analog, CA Exendin-4, which showed potent insulinotropic and glucose (a) Exendin-4 (b) CA Exendin-4 Exendin-4 CA Exendin-4 How lowering activity. Surface Plasmon resonance (SPR) analysis revealed CA Exendin-4 Table 1. Binding affinity of CA . CA Exendin-4 showed greater in vitro activity compared with Exendin-4 in cyclic GLP-1R dissociated 5.5-fold faster than native Exendin-4 from the human GLP-1 receptor Exendin-4 and Exendin-4 Fast Dissociation GLP-1R AMP accumulation and insulin release. 5 folds Signaling Signaling (GLP-1R). CA Exendin-4 showed greater in vitro cyclic AMP accumulation and insulin (nM) rapid to human GLP-1R Contribute to Receptor . 200 dissociation Significantly less internalized GLP-1R was confirmed in the CA Exendin-4 treated release in a rat insulinoma cell line. We hypothesized that the fast dissociation of CA 100 desensitization Less k k K Strong ? by slower internalization 50 a d D cells. The fast dissociation of CA Exendin-4 might elicit less internalization of Exendin-4 might elicit less internalization of GLP-1R so that it enhanced GLP-1 -1 -1 -1 dissociation By fast 25 (M s ) (s ) (nmol/L) dissociation signaling. In the receptor internalization assay, significantly less internalization of 12.5 GLP-1R so that it can enhance GLP-1 signaling. 6.25 Exendin-4 4.9 x 105 1.1 x 10-3 2.2 GLP-1R was confirmed in CA Exendin-4 treated cells. We conjugated human Fc . The binding properties of human Fc conjugated CA Exendin-4 (HM11260C) and fragment to CA Exendin-4 (HM1160C) and Exendin-4 (HM11260A), to provide for CA Exendin-4 2.0 x 105 6.0 x 10-3 30 Exendin-4 (HM11260A) were conserved similarly with CA Exendin-4 and Exendin- prolonged half-life. The binding properties to GLP-1R were conserved similarly with 4 even after conjugation. CA Exendin-4 and Exendin-4 even after conjugation. The glucose lowering efficacy of Keep signaling HM11260C was compared with that of HM11260A in db/db mice, and the result showed Figure 1. SPR Sensorgram on immobilized GLP-1R of CA Exendin-4 and Exendin-4 Weaker signaling . The results of the glucose lowering study in db/db mice showed that much more much more potent were achieved in HM11260C treated group. These hGLP-1 receptor was immobilized onto a CM5 chip and probed with Exendin-4 and CA Exendin-4 respectively. Figure 4. A receptor desensitizing model for strong efficacy of CA Exendin-4 potent efficacies were achieved in the HM11260C treated group. results suggest that the rapid dissociation kinetics on the GLP-1 receptor can trigger The sensorgram of CA Exendin-4 showed a 5-fold more rapid dissociation rate compared with Exendin-4. Fast Dissociation of CA Exendin-4 induces less internalizing of the receptors and leads to continuous strong signaling without desensitization. . These results suggest that the rapid dissociation kinetics at the GLP-1 receptor

super-agonistic activation resulting in more potent efficacy outcomes. can trigger super-agonistic activation and result in more potent efficacy Correlation of Strong Potency Application of LAPSCOVERY (Fc conjugation) outcomes. (a) cAMP stimulation (b) Insulin release (a) LAPSCOVERY (b) Receptor Kinetics of LAPS-Exendin-4

(RINm5F, murine cell) (RINm5F, murine cell) HM11260A HM11260C Introduction (LAPS-Exendin-4) (LAPS-CA Exendin-4) 50 . Exendin-4 is a 39 amino acid peptide derived from the saliva of Gila monster and is a glucagon-like Exendin-4 Exendin-4 4 folds (nM) rapid peptide-1 (GLP-1) receptor agonist through high sequence homology to mammalian GLP-1[1]. It has 200 40 CA Exendin-4 CA Exendin-4 200 dissociation prolonged action compared to GLP-1 with resistance to dipeptidyl peptidase-IV (DPP-IV) and shows anti- 100 Aglycosylated Aglycosylated Aglycosylated 30 Aglycosylated 50 References diabetic actions such as glucose-dependent insulin secretion, appetite suppression, expansion of β-cell Site specific 25 Conjugation & Purification mass and a delay of gastric emptying [2-4] 12.5 1. Alan J. Garber: Long-acting glucagon-like peptide 1 receptor : a review of their efficacy and 150 6.25 20 association dissociation , Diabetes Care 2011, 34(S2): S279-284 . We developed CA Exendin-4 which showed advantages in insulinotropic and glucose lowering activity + association dissociation 2. Briones M, Bajaj M: Exenatide: a GLP-1 receptor agonist as novel therapy for Type 2 diabetes mellitus. Fc Fc Fc through its N-terminal modifications. In the current stud y, the potentials of CA Exendin-4 in diabetic 10 Fc

Conc. of cAMP (nM) ka kd KD Expert Opin Pharmacother 2006, 7(8): 1055-1064. treatment had been assessed. In vitro studies were conducted to investigate the superiority of CA 100 (M-1s-1) (s-1) (nmol/L)

Exendin-4 compared with Exendin-4; binding affinity to the human GLP-1 receptor, insulinotropic potency, Insulin conc. (% of control) 0 3. Monika Malm-Erjefält, Inga Bjørnsdottir, Jan Vanggaard, Hans Helleberg, Uffer Larsen, Berend From Bacterial LAPSLanglenatide-Exendin-4 4 -3 0.001 0.01 0.1 1 10 100 1000 Fermentation HM11260A 2.4 x 10 1.0 x 10 42.8 and receptor mediated were compared. In vivo studies were performed to compare the glucose 0.0001 0.01 1 100 10000 (LAPS-(HM11260C)Exendin-4; HM11260C) Oosterhuis, Jan Jaap van Lier, Milan Zdravkovic, Anette K. Olsen: Metabolism and of the once- lowering efficacy in normal Sprague Dawley rats. Conc. of exendin-4 derivatives (nM) Conc. of ex-4 derivatives (nM) HM11260C 1.3 x 104 4.2 x 10-3 317 daily human glucagon-like peptide-1 anlaog liraglutide in healthy male subjects and its in vitro . degaradation by dipeptidyl peptidase IV and neutral endopeptidase, Metabolism and Disposition One of major drawbacks with the use of Exendin-4 as Figure 2. Correlation of cAMP stimulating and insulinotropic activities of CA Exendin-4 and Exendin-4 Figure 5. Receptor kinetics of LAPS-Exendin-4 (a) Structure of LAPS-Exednin-4 (HM11260C) 2010, 38(11): 1944-1953. diabetes treatment is frequent administration (BID) N’- H G E G T F CA Exendin-4 was 2-fold more potent with EC of 0.89 nM than Exendin-4 with EC of 2.06 nM (P<0.05) as (b) SPR sensorgrams of HM11260A and HM11260C on immobilized GLP-1R because of its short half-life (3.3-4 h) in vivo [5, 6]. To 50 50 4. Scott E. Kanoski, Samantha M. Fortin, Myrtha Arnold, Harvey J. Grill, Matthew R. Hayes: Peripheral and overcome this, LAPSCOVERY was applied to CA well as maximal magnitude of cAMP synthesis (a). The magnitude of maximal insulinotropic activity was 2-fold LAPSCOVERY was applied to Exendin-4 (HM11260A) and CA Exendin-4 (HM11260C). central GLP-1 Effects of peripherally administered GLP-1 receptor agonists, Liraglutide and Exendin-4. Exendin-4 higher on CA Exendin-4 (P<0.05) (b). cAMP stimulation and insulinotropic activity of CA Exendin-4 were Exendin-4 (LAPS-Exendin-4, HM11260C) which is Dissociation rate (kd) of HM11260C was 4-folds faster than HM11260A. The rapid dissociation kinetics of CA Endocrinol 2011, 152(8): 3103-3112. composed of CA Exendin-4 chemically conjugated correlated well. Exendin-4 was well maintained by the LAPSCOVERY application. 5. Young Min Cho, Catherine E. Merchant, Timothy J. Kieffer: Targeting the glucagon receptor family for

with a recombinant human immunoglobulin (IgG4) Fc diabetes and obesity therapy. Pharmacology and Therapeutics 2012, 135: 247-278. fragment through a non-peptidyl linker at its N- H G E G T F Receptor Mediated Internalization Glucose Lowering Efficacy of HM11260C 6. Dennis Kim, Michael trautmann, Leigh MacConell, Mark Fineman, Kristin Taylor, Prajakti A. Kothare, terminus. The receptor kinetics of HM11260C and in Effects of once-weekly dosing of a long-acting release formulation of exenatide on glucose control and vivo efficacy were compared with LAPSCOVERY- CA Exendin-4 Receptor Internalization (hGLP-1R transformed U2OS cell) (a) Blood Glucose in db/db mice (n=5, 4 weeks) (b) ) in rats (n=3, single) body weight in subjects with type 2 diabetes, Diabetes Care 2007, 30: 1487-1493. 10000 HM11260A applied Exendin-4 (HM11260A). Vehicle Langlenatide 800 800 HM11260A 11 nmol/kg Exendin-4 EC50: 3.23 nM HM11260C 11 nmol/kg 1000 CA Exendin-4 600 600 Further Information Methods Concentration (ng/mL as protein) 100 0 100 200 300 400 . The receptor kinetics were measured by a SPR (Surface Plasmon Resonance) assay using immobilized 400 Less Desensitization 400 Time (hr) [Acknowledgments] extracellular domain of the human GLP-1 receptor which is fused to the GST (glutathione S transferase). (c) HbA1c in db/db mice (n=5, 4 wks) . This study was supported by a grant of the Korea Drug Development Fund R&D Project.(KDDF-201204-03) 8 Vehicle The hGLP-1R/GST was expressed in transformed CHO cells and purified by GST affinity chromatography. % of control 200 HM11260A 11 nmol/kg 200 Langlenatide 11 nmol/kg . The authors wish to thank following individuals and study staff for their contributions; . 7 Intracellular cyclic AMP and insulin released to the assay medium were measured, following treatment EC50: 5.88 nM ** 6.7% * Profil Institute for Clinical Research, for scientific advice on this poster

with CA Exendin-4 and Exendin-4 in a rat insulinoma cell, RINm5F. In vivo glucose lowering efficacy was Blood Glucose (mg/dL) 6 0 5.9% investigated by intraperitonial glucose tolerance test following a single dose of study drug in normal rats. 0 *** Dr. Michael Trautmann, for assistance with the preparation of this poster. 0.01 1 100 10000 5 0 5 10 15 20 25 5.0% . Internalization of GLP-1 receptor was assessed using the PathHunter™ eXpress Kit (DiscoveRx HbA1c at 4 weeks (%) Conc. of GLP-1 agonists (nM) 4 Corporation. Ltd., UK). Receptor mediated clearance was measured on human GLP-1 receptor Time (day) * P<0.05, ** P<0.01, *** P<0.001 [Developmental Plans] transformed CHO cells. 100 nM of Exendin-4 and CA Exendin-4 were spiked into assay medium, and Figure 3. Internalization of human GLP-1 receptors by CA Exendin-4 and Exendin-4 ® Figure 6. Glucose lowering efficacies of HM11260C and HM11260A in db/db mice (a) Blood . Single ascending dose studies in healthy volunteers (Korea) and T2DM patients (Europe) completed remaining Exendin-4 or CA exendin-4 was measured by ELISA. Human GLP-1 receptor internalization was measured using of PathHunter eXpress GLP1R Activated GPCR glucose profiles monitored for 4 weeks (b) Comparison of PK profiles in normal rats (c) . For comparison of HM11260C and HM11260A, in vivo glucose lowering efficacy was investigated in db/db Internalization Assay (DiscoveRX, CA). The maximal receptor internalization by CA Exendin-4 was 2.3 fold . Multiple ascending dose study is ongoing (USA), and the interim results are available on ADA 2013 poster mice. Following weekly administration blood glucose changes were monitored and compared over 4 lower than Exendin-4 (Emax of 268.0% vs. 620.5% of control), while their EC50 were 5.88 nM and 3.23 nM Comparison of HbA1c after 4 weeks treatment. session 59-LB weeks, and HbA1c levels were compared at the end of the treatment period. respectively. Weekly administration of HM11260C for 4 weeks revealed prolonged and potent glycemic control compared with that of HM11260A. 59-LB HM11260C, a New Generation Long Acting GLP-1R Agonist with a unique Pharmacokinetic Profile Improves Glucose Control and GI tolerability; a Phase IIa Clinical Trial in Type 2 Diabetes Mellitus

JaHoon Kang *1) , SooMin Choi 1) , In Young Choi 1), Se Chang Kwon 1), Michael Trautmann 2), Marcus Hompesch 2), JeeWoong Son 1) *JaHoon Kang, Tel: +82-2-410-9041; e-mail: [email protected] 1) Hanmi Pharm. Co., Ltd, Seoul, South Korea, 2) Profil Institute for Clinical Research, Inc., CA, USA. Hanmi Abstract Results

A very long T1/2(~ 180 hrs) and no burst absorption (Tmax: ~144 hrs) of HM11260C was Baseline and Demographic characteristics Effect of HM11260C on glycaemic control Effect of HM11260C on body weight confirmed in a previous single ascending dose study in T2DM. The aim of this double-blind, . No significant differences were seen between the treatment groups and placebo with regard to randomized, placebo controlled phase Ⅱa study in patients with T2DM is to investigate Figure 1. The reduction in HbA1c from baseline Change from baseline body weight baseline characteristics. safety, tolerability, PK and PD of HM11260C when treated repeatedly with weekly or monthly Placebo (n=6-8) Placebo (n=8) W1 W2 W3 M1 M2 M3 regimens. HM11260C was administered subcutaneously over an 8-week period for weekly Weekly regimen Monthly regimen HM11260C 1 mg/week (n=8-9) HM11260C 8 mg/month (n=6-7) Weekly Monthly Population HM11260C 2 mg/week (n=9) HM11260C 12 mg/month (n=8-9) HM11260C HM11260C HM11260C HM11260C HM11260C HM11260C HM11260C Placebo HM11260C Placebo (kg) Placebo Placebo regimens or a 9-week period for monthly regimens. Mean ± SD HM11260C 4 mg/week (n=9) HM11260C 16 mg/month (n=5-6) 1 mg/week 2 mg/week 4 mg/week 8 mg/mo 12 mg/mo 16 mg/mo # 0.5 0.5 (N=6-8) (N=8) (N=27) (N=9) (N=24) (N=8) (N=9) (N=9) (N=9) (N=6-7) (N=9) (N=5-6) 53.19 ± 6.83 53.67±7.63 49.58±9.91 52±8.64 Data through Day 57 (Weekly) or Day 78 (Monthly) from 68 patients in W1 (1 mg/wk), W2 (2 Age (yrs) Gender 0.0 0.0 Day 57 Day 78 -0.09 mg/wk), W3 (4 mg/wk), M1 (8 mg/mo), M2 (12 mg/mo) and M3 (16 mg/mo) are reported. Key Female, n (%) 16 (59.3) 3 (66.7) 7 (29.2) 5 (62.5) -0.14 (%) demographics were (active vs. placebo; mean [SD]): age, 51.5 [8.5] vs. 52.9 [8.0] years; HbA1c, Male, n (%) 11 (40.7) 6 (33.3) 17 (70.8) 3 (37.5) (%) ** * Baseline 86.9±9.4 107.2±17.6 104.3±18.5 81.1±16.2 101.6±14.5 90.5±8.1 109.9±21.0 97.4±24.5 -0.5 ** -0.5 Mean ± SD 1c 8.3 [1.0] vs. 8.0 [0.9] %. At Day 57/78, patients treated with weekly regimens or monthly Race 1c *** 21 (77.8) 8 (88.9) 19 (79.2) 5 (62.5) *** regimens experienced clinically significant improvements from baseline HbA1c, fasting White, n (%) * -0.92 *** Change from -1.0 -1.0 *** -0.99 0.26 -1.09 -1.28 -2.44 0.12 0.50 0.22 -2.76 African-American, n (%) 6 (22.2) 1 (11.1) 5 (20.8) 3 (37.5) ** baseline plasma glucose and body weight compared with placebo. Most common AEs were nausea, -1.02 ** [0.89] [0.76] [0.74] [0.80] [0.69] [0.83] [0.67] [0.87] HbA Weight (kg) 98.2±20.7 88.5±10.5 101.5±20.0 102.6±14.5 HbA ** -1.09 LS mean [SE] vomiting and diarrhea. The weekly regimen showed fewer GI AEs with most events reported **-1.24 ∆ BMI (kg/m2) 34.4±4.8 31.3±3.5 34.1±5.4 36.9±4.9 ∆ after the first injection. No treatment effect was observed on vital signs, laboratory and ECG. -1.5 -1.5 * -1.51 HbA1c (%) 8.3±1.0 7.9±0.9 8.2±0.9 8.0±0.9 p-value 0.2687 0.201 0.0261* 0.7302 0.9211 0.0159* Duration of Diabetes 57 56 60 39 -2.0 -2.0 (months) * HM11260C demonstrated meaningful improvements in blood glucose control and good 1 29 57 1 29 57 78 * p<0.05, ** p <0.01, *** p<0.001 vs. placebo. History of Metformin tolerability after repeated treatment in all weekly and monthly cohorts. Further development 27 44 53 39 Day Day of HM11260C is warranted to explore its full potential as mono and combination therapy in (months) * ±SE). patients with T2DM. * Data are presented as median. Data are presented as LS mean change from baseline ( * p<0.05, ** p <0.01, *** p<0.001 vs. placebo. # M3 cohort (16 mg/month) included only 8 patients, since it met the dose- escalation stopping criteria. Conclusions Figure 2. The percent of patients attaining A1c goal < 7% at Day 57/78 . Safety and GI tolerability Placebo Placebo Weekly and monthly administrations of HM11260C were generally well HM11260C 1mg/week HM11260C 8mg/month tolerated. The most frequent treatment related adverse events were GI Background . Of 68 patients, 166 AEs in HM11260C and 55 AEs in placebo were reported, and primarily mild-to- HM11260C 2mg/week HM11260C 12mg/month moderate in intensity. HM11260C 4mg/week HM11260C 16mg/month events such as nausea, vomiting, and diarrhea. . GLP-1 analogs are increasingly used as treatment for patients with T2DM who no longer . GI related AEs were the most frequently reported in HM11260C treated patients. 80 80 achieve good glucose control on oral anti-diabetes medications. The main drawbacks are 63% . None of the subjects had an abnormal and clinically significant electrocardiogram finding at any time point. 60% . Repeated treatments of HM11260C led to clinically meaningful reductions frequent GI adverse events, the need for frequent injections or cumbersome preparations for 60 60 . There was no significant shift in laboratory assessment or vital signs for any treatment group. 50% 50% injection. in HbA1c and fasting blood glucose in patient with T2DM.

Time course of GI events 40 38% 40

goal (%) . We have developed a GLP-1 receptor agonist with special properties with an extended half-life 25% goal (%) . Body weight loss was observed across the HM11260C treated groups. 1C

Placebo HM11260C 1mg/week HM11260C 2mg/week HM11260C 4mg/week 1C using the LAPS-technology. 20 20 14% A A . Percent attaining 7 7 0% Percent attaining The observed glucose-lowering effects and its safety profile support . A single dose study demonstrated pharmacokinetics which may allow either once-weekly or 0 0 6 6 further clinical development of HM11260C for patients of T2DM. even once-monthly injections of a ready-to-use preparation. A < 7% (at Day 57) 5 5 1c A1c < 7% (at Day 78) 4 4 3 3 Nausea 2 Vomiting 2 Table 1. Change from baseline fasting plasma glucose 1 1

Study design 0 episodes) of (numer 0 References (number of episodes) of (number W1 W2 W3 M1 M2 M3 1 - 28 29 - 56 57 1 - 28 29 - 56 57 Weekly Monthly . This was a double-blind, randomized, placebo-controlled, multiple ascending-dose Phase Ⅱ HM11260C HM11260C HM11260C HM11260C HM11260C HM11260C (mg/dL) Placebo Placebo . Days Days 1 mg/week 2 mg/week 4 mg/week 8 mg/mo 12 mg/mo 16 mg/mo Briones M, Bajaj M et al. Exenatide: a GLP-1 receptor agonist as novel therapy for Type 2 study in 6 groups of 12 patients with T2DM each receiving s.c. injections of HM11260C or (N=6-8) (N=8) (N=8-9) (N=9) (N=9) (N=6-7) (N=8-9) (N=5-6) diabetes mellitus. Expert Opin Pharmacother 2006, 7(8):1055-1064. placebo in a 3:1 ratio. Placebo HM11260C 8mg/month HM11260C 12mg/month HM11260C 16mg/month . Pharmacokinetic and pharmacodynamic effects of a single dose of the long-acting GLP-1R Day 57 Day 78 nd . 7 7 agonist HM11260C in subjects with type 2 diabetes mellitus, 72 ADA 2012, PA, USA. Dose escalation was stopped if there are more than 3 people with mild vomiting, more than 6 6 Baseline 182.9±51.2 182.3±25.7 185.8±51.1 176.6±59.7 153.1±32.8 167.1±23.8 177.2±62.2 175±35.1 2 people with moderate vomiting, or more than 1 person with severe vomiting in any one ± 5 5 Mean SD cohort (CTCAE : 2009, version 4.0). 4 4 [Acknowledgement] 3 3 Change from 1.63 -36.16 -26.43 -65.73 -2.78 -31.4 -20.5 -35.1

Nausea baseline . 2 Vomiting 2 [12.9] [11.2] [10.5] [10.5] [9.9] [11.2] [9.2] [12.4] This study is supported by a grant of the Korean Health Technology R&D Project, Ministry . LS mean [SE] Eight times of weekly regimen 1, 2, and 4mg, and three times of monthly regimen 8, 12 and 16 1 1 of Health & Welfare, Republic of Korea (KDDF-201204-03)

mg of HM11260C were administered. No dose titration was applied on all cohorts. 0 episodes) of (numer 0 (number of episodes) of (number 1 - 28 29 - 56 57 - 78 1 - 28 29 - 56 57 - 78 p-value 0.0357* 0.1033 0.0004*** 0.0684 0.2091 0.0558

Days . Eligible participants were T2DM with a stable dose of metformin for at least 3 months, whose Days All nausea or vomiting episodes were included regardless of relationship with study . * p<0.05, ** p <0.01, *** p<0.001 vs. placebo. A1C was between 7.0 and 10%, inclusive.