1. European Association for the Study of Diabetes 50 th 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 The Long-acting Basal Insulin ( LAPS Insulin 115) Offers Once-weekly Dosing Potential With Favorable PK, PD And Mitogenic Profiles IY Choi 1, SY Hwang 1,, JY Kim 1, SY Jung 1, DJ Kim 1, YM Lee 1, YH Kim 1, M Trautmann 2, M Hompesch 2, JW Son 1, SC Kwon 1 1 Hanmi Pharm. Co., Ltd., Seoul, South Korea, 2 Profil Institute, Chula Vista, CA, USA P933 Hanmi Hanmi Pharm. Co., Ltd. METHODS RESULTS  Pharmacokinetic analysis of LAPS Insulin 115 in animal models Test materials were s.c. injected to normal or renal failure SD rats or normal pigs. Renal failure SD rat model was induced by 4 mpk of cisplatin. Serum concentration of test articles were determined using ELISA and PK parameters were calculated by a non- compartmental method. Table 1. Receptor binding affinity, metabolic and mitogenic potencies relative to human insulin BACKGROUND LAPS Insulin 115 as a once weekly basal insulin  Lower peak-to-trough ratio from long duration of action  Improved patient adherence by once-weekly administration  Ideal combination partner with weekly GLP-1 agonist [EASD 2014 Poster #972, Combination of LAPS Insulin 115 and LAPS CA-Exendin-4] Insulin 115 analog conjugated with a constant region of a human immunoglobulin fragment via non-peptidyl linker. AIMS  Investigation of in vitro receptor pharmacology, metabolic, and mitogenic activity of LAPS Insulin 115  Evaluation of pharmacokinetics and pharmacodynamics of LAPS Insulin 115 in normal and diabetic animal models  Prediction of human pharmacokinetic profile of LAPS Insulin 115 based on animals’ PK data Test materials Receptor binding (%)Mitogenic potency (%) Mitogenic/Metabolic potency § ratio InsulinIGF-1SaOS-2MCF-7SaOS-2MCF-7 Human insulin 100 11 IGF-1 0.6 17,984 6,255 1,527 2,234545 Insulin AspB10 248 449 756 912 2.53.0 Insulin 115 86 84 119 88 <1 LAPS Insulin 115 1.7 Too low to be determined 5.3 5.4 <1  Both LAPS Insulin 115 and Insulin 115 showed reduced affinities on IR and IGF-1R compared with human insulin. In addition, LAPS Insulin 115 and Insulin 115 showed mitogenic/metabolic potency which was comparable to human insulin. Figure 2. Ex vivo T cell activation of Insulin 115 and LAPS Insulin 115  Insulin 115 showed negligible activation of T cells from 50 human donors, which is below the immunogenic threshold. In addition, LAPS Insulin 115 completely attenuated the residual T cell activation. 0% 8% Immunogenic threshold Key requirements for once weekly insulin Additional requirements  Excellent Device  No CV safety concerns  PK matched with weekly GLP-1 for combination therapy  Longer half-life  Flat profile  Low Variability (intra-patient)  Low Risk of hypos  Flexible timing/dosing  Little or no weight gain  Pharmacokinetic prediction in human Human serum concentration-time was predicted by C ss -MRT method from PK parameters of mice, rats and dogs. Human CL was derived from rule of exponent methods, and human V d was from allometry applied correction factor.  Phamacodynamic analysis of LAPS Insulin 115 in db/db mice In an acute study, 4-hr fasting blood glucose level was measured every day after s.c administration of test articles in db/db mice. In a chronic study, HbA1c level was measured after 4-wk administration in db/db mice with Q2D dosing interval to mimic human QW dosing.  Ex vivo T cell activation (Antitope limited, UK) Test articles were incubated for 8 days with PBMC from 50 donors. Cell proliferation was meausred by [ 3 H]-Thymidine incorporation and T-cell activation was identified with IL-2 secretion by Elispot assay. Figure 1. Mitogenic signaling in MCF-7 cells IGF-1R p-ERK1/2 ERK1/2 p-IGF-1R p-AKT AKT Actin h-Insulin Control IGF-1 Asp B10 Insulin 115 IGlargine IGF-1R phosphorylation AKT phosphorylation ERK1/2 phosphorylation Insulin IGF-1R IRS-1/2 PI3K Apoptosis SHC RAS/RAF Cell proliferation IGF-1 receptor signaling IGF-1 AKTERK1/2  Insulin 115 triggered mitogenic signals comparable with human insulin, whereas positive controls (IGF-1 and AspB10) showed significantly higher mitogenic signals.  In vitro receptor binding affinity and mitogenic signaling in MCF-7 Binding affinity of test materials was measured in competition between unlabeled and 125 I-labeled materials on the IR- or IGF-1R/CHO membrane. Mitogenic signaling was assayed in MCF-7 cells and the phosphorylation level was analyzed by Western blot. In vitro Binding, Proliferation, and ex vivo T Cell Activation § Metabolic potencies were obtained from lipogenesis assay from rat primary adipocytes LAPS Insulin 115 t 1/2 = 44.1 hrs LAPS Insulin t 1/2 = 18.5 hrs 20K PEG Insulin t 1/2 = 6.7 hrs IDeg t 1/2 = 2.9 hrs Figure 3. PK in normal and renal failure model rats  LAPS Insulin 115 showed PK/PD correlation in normal pigs. Figure 4. PK/PD in pigs (n=3, s.c.) LAPS Insulin 115 t 1/2 = 76.6 hrs LAPS Insulin t 1/2 = 23 hrs (a) PK in normal rats (n=5, s.c.) Figure 6. Human PK simulation of LAPS Insulin115 by weekly injection  Pharmacokinetics in humans was predicted based on PK of three different animals. LAPS Insulin 115 demonstrated extended half-life and lower peak-to-trough ratio than LAPS Insulin. In addition, inter-day variability of LAPS Insulin 115 was ranged from 1.0-1.1. REFERENCES CONCLUSIONS  In vitro biological properties showed that Insulin 115 and LAPS Insulin 115 did not increase the mitogenic potency when compared with insulin.  An extended PK profiles and prolonged glucose lowering efficacies suggest that LAPS Insulin 115 may achieve a basal insulin profile suitable for once weekly use.  The extended human PK modeling suggests that LAPS Insulin 115 is an ideal combination partner with LAPS CA-Exendin-4 [EASD 2014 Poster #972]. 1. Diabetic Medicine (2013) 30: 1293–1297., 2. Diabetes (2011) 60 (Suppl. 1):LB11 (37-LB) 3. PLoS ONE (2010) 5: e9540., 4. PLoS ONE (2012) 7: e34274. Figure 5. Dose-sparing and glucose lowering in db/db mice (n=7, s.c.)  LAPS Insulin 115 showed prolonged glucose lowering effect compared to LAPS Insulin in db/db mice at the same dose and even with a 6 fold lower dose.  LAPS Insulin 115 achieved a similar HbA1c reduction with a 4 fold lower dose. *p<0.05, **p<0.01 vs vehicle by Anova test ** *** * -2.0 -3.4 -1.8 -2.6 -3.3 4 fold reduced dose (b) 4-week repeated dose (a) Single dose 6 fold reduced dose Weekly injected steady state profile Single week Profile LAPS Insulin 115, t 1/2 = 132 hrs Weekly Peak-to-Trough Ratio = 1.6 LAPS Insulin, t 1/2 = 55 hrs Weekly Peak-to-Trough Ratio = 4.0 LAPS Insulin 115, Daily Peak-to-Trough Ratio = ~1.1 Figure 7. Development plan of LAPS Insulin 115 and QUANTUM project  QUANTUM is a proprietary name of Hanmi’s diabetes & obesity pipeline PK/PD Correlation and Dose Sparing Effect Human PK and Developmental Plan LAPS Insulin, Daily Peak-to-Trough Ratio = ~1.3 1.11.01.1  LAPS Insulin 115 showed extended half-life compared with LAPS Insulin and daily insulins.  LAPS Insulin 115 showed a comparable PK profile in a renal failure rat model (4 mg/kg of cisplatin injected, ip, rats) compared with normal rats. (b) PK comparison in normal and renal failure model rats (n=5, s.c.) Insulin 115 (172 nmol/kg, sc) LAPS Insulin 115 (22.3 nmol/kg, sc) Insulin (172 nmol/kg, sc)