Oral Glucose Tolerance Test Glucose mgd L Insulin

Φαινόμενο Ινκρετίνης Oral Glucose Tolerance Test Glucose (mg/d. L) Insulin (pmol/L) 50 g glucose Nauck MA, et al. J Clin Endocrinol Metab. 1986; 63: 492 -8. 4

Φαινόμενο Ινκρετίνης OGTT and Matched IV Infusion Glucose (mg/d. L) Nauck MA, et al. J Clin Endocrinol Metab. 1986; 63: 492 -8. Insulin (pmol/L) 5

Οι ΙΝΚΡΕΤΙΝΕΣ GLP-1: Glucagon-Like Peptide– 1 H A E G T F T S D V S S Y L E G Q A A K F L R I V E W G G GIP: Glucose-Dependent Insulinotropic Peptide A E G T F I S D Y S I A M D K I H Q K K G K A L L W N V F D Q D N W Q K T Q I N H Y *Amino acids shown in gold are homologous with the structure of glucagon. 9

Synthesis and Secretion of GLP-1 and GIP K-Cell (jejunum) L-Cell (ileum) Pro. GIP Proglucagon GLP-1 [7 -37] GIP [1 -42] GLP-1 [7 -36 NH 2] 10

Mεταβολισμός GLP-1 και GIP Active Hormones Capillary GLP-1 [7 -36 NH 2] GIP [1 -42] DP P-4 Inactive Metabolites Dipeptidyl peptidase-4 (DPP-4) GLP-1 [9 -36 NH 2] GIP [3 -42] ØUbiquitous, specific protease ØCleaves N-terminal dipeptide ØInactivates > 50% of GLP-1 ~ 1 min > 50% of GIP in ~ 7 min 11

Μεταβολισμός GLP-1 και GIP L-Cell K-Cell Proglucagon Pro. GIP GLP-1 [7 -37] Capillary GIP [1 -42] GLP-1 [7 -36 NH 2] GLP-1[7 -36 NH 2] ACTIVE GIP [1 -42] ACTIVE DPP-4 GLP-1 [9 -36 NH 2] INACTIVE Dipeptidyl peptidase-4 (DPP-4) • Ubiquitous, specific protease • Cleaves N-terminal dipeptide • Inactivates > 50% of GLP-1 in ~ 1 min > 50% of GIP in ~ 7 min GIP [3 -42] INACTIVE 12

30 -Minute Εκκριση Total and Intact GLP-1 and GIP in σε Υγιη Ατομα GLP-1 Vilsbøll T, et al. J Clin Endocrinol Metab. 2003; 88: 2706 -13. GIP 13

Τόπος Δράσης -Cell ATP GLP-1 -cell Adipocyte Brain Stomach Small intestine ATP Ac Ac Gs c. AMP PKA Insulin Gs GIP -cell Adipocyte Brain Adrenal Pituitary 14

Glucose-Dependent Insulinotropic Effect of GLP-1 and GIP in Healthy Subjects Test Infusion GL Kreymann B, et al. Lancet. 1987; 2: 1300 -4. Test Infusion GL 15

Impaired Glucose Tolerance in GLP-1 Receptor Knockout Mice * * * r -/- ** * ** +/+ -/- **P <. 01, *P <. 05 Scrocchi LA, et al. Nat Med. 1996; 2: 1254 -8. 16

Impaired Glucose Tolerance in GIP Receptor Knockout Mice * ** r -/- r *** -/- * ***P <. 001, **P <. 01, *P <. 05. Miyawaki K, et al. Proc Natl Acad Sci U S A. 1999; 96: 14843 -7. 17

GLP-1 Decreases Glucagon and Glucose Levels in Patients with Type 1 Diabetes GLP-1 or Placebo P <. 001 * * * * * GLP-1 Creutzfeldt WO, et al. Diabetes Care. 1996; 19: 580 -6. * * GLP-1 * * P <. 001 18

GLP-1 Decreases Gastric Emptying of a Solid Meal in Healthy Subjects GLP-1 Saline Näslund E, et al. Am J Physiol. 1999; 277: R 910 -R 916. 19

Effect of GLP-1 on -Cell Mass in Diabetic Rats -Cell Proliferation -Cell Apoptosis -Cell Mass P <. 001 P <. 05 Farilla L, et al. Endocrinology. 2002; 143: 4397 -408. 20

Comparison of Physiology of GLP-1 and GIP GLP-1 GIP L-cells in ileum and colon K-cells in duodenum and jejunum Indirect/neuronal Direct Inhibits glucagon Yes No Slows gastric emptying Yes No Stimulation of -cell growth/mass Yes -cell, stomach, nervous system -cell, adipose tissue Antagonist Exendin [9 -39] GIP [7 -30] Receptor KO mice Yes, with IGT Site of production Response to stimuli Major target tissues 21

Το φαινόμενο Ινκρετίνης στον ΣΔ τύπου 2 Plasma Glucose (mg/d. L) 240 180 90 0 30 Type 2 DM *P <. 05 20 10 * * * * 0 C-Peptide (nmol/L) Plasma Glucose (mg/d. L) Control 240 180 90 0 IV Oral 30 20 10 0 Time (min) Nauck M, et al. Diabetologia. 1986; 29: 46 -52. Time (min) 23

Εκκριση Ινκρετινών μετα απο γευμα σε ασθενεις με ΣΔ τυπου 2 GIP GLP-1 P = 0. 095 P < 0. 001 NGT IGT Toft-Nielsen MB, et al. J Clin Endocrinol Metab. 2001; 86: 3717 -23. T 2 DM 24

Διαφορετική ινσουλινοτρόπος δραση των ινκρετινών στον ΣΔ τυπου 2 Hyperglycemic Clamp Saline or GIP or GLP-1 GIP Saline -15 -10 0 5 Vilsböll T, et al. Diabetologia. 2002; 45: 1111 -9. 10 15 20 30 45 60 75 90 105 120 150 25

IV χορηγηση GLP-1 σε ασθενεις με ΣΔ τυπου 2 Glucose (mg/d. L) C-Peptide (nmol/L) GLP-1 Infusion * * * GLP-1 Saline * *P <. 05 Nauck MA, et al. Diabetologia. 1993; 36: 741 -4. Glucagon (pmol/L) GLP-1 Infusion * ** ** 26

6 εβδομαδες συνεχους χορηγησης GLP-1 σε ασθενεις με ΣΔ τυπου 2 Patients assigned GLP-1 Patients assigned saline Meal Week 0 Zander M, et al. Lancet. 2002; 359: 824 -30. Week 1 Week 6 27

Effects of 4 Weeks’ Treatment with a DPP-4 Inhibitor in Patients with Type 2 Diabetes Glucose GLP-1 meal Insulin Glucagon Time (min) Ahren B, et al. JCEM. 2004; (in press). Time (min) 28

4 εβδομαδες θεραπεια με ινκρετινομιμητικα φαρμακα ( GLP-1 Receptor Agonist ) σε αθενεις με ΣΔ τυπου 2 Placebo Meal | P <. 001 Drug (all arms) BD = before breakfast and dinner Fineman MS, et al. Diabetes Care. 2003; 26: 2370 -7. 29

At 30 Weeks, PPG Remained Low among Patients Treated with GLP-1 Analogue Postprandial Plasma Glucose (mmol/L) Week 30 Meal Tolerance Test* 16 15 14 13 12 Injection Meal Placebo (n=23) 5 µg exenatide (n=27) 10 µg exenatide (n=27) 11 10 9 8 7. 5 0 0 25 50 75 100 125 150 175 Time (Min) GLP-1=glucagon-like peptide-1; PPG=postprandial glucose *Exendin-4 or placebo were administered at time zero; subjects in all treatment arms were maintained on metformin-sulfonylurea therapy. Adapted from Kendall DM, et al. Diabetes Care. 2005; 28: 1083– 1091. 30

Effect of Bedtime Administration of a Long-acting GLP-1 Derivative in Patients with T 2 DM Breakfast Placebo Drug Juhl CB, et al. Diabetes. 2002; 51: 424 -9. Breakfast Placebo Drug 32

Glucose Tolerance, Insulin, and GLP-1 Levels in DPP-4 Deficient Mice +/+ DPP-4 -/- *** * * ** -30 0 30 60 120 180 ***P <. 001, *P <. 05 Marguet D, et al. Proc Natl Acad Sci U S A. 2000; 97: 6874 -9. 33

GLP-1 Analogues Enhance Glucagon Secretion in Hypoglycemic Conditions 300 Glucose Clamp Steps (mmol/L): 5. 0 * 4. 0 * 3. 2 2. 7 * Recovery Plasma Glucagon (ng/L) 250 Exenatide† (n=11) Placebo (n=11) 200 150 100 50 0 – 30 0 30 60 90 120 150 180 210 240 270 300 330 360 Time (Min) GLP-1=glucagon-like peptide-1 *P <0. 05 †Exenatide infused at. 066 pmol/kg/min for 270 minutes. Adapted from Degn KB, et al. Diabetes. 2004; 54: 2397– 2403. 34

Inhibition of DPP-4 Increases Active GLP-1 Meal Intestinal GLP-1 release GLP-1 t½=1– 2 min Active GLP-1 DPP-4 GLP-1 inactive (>80% of pool) DPP-4=dipeptidyl peptidase-4; GLP-1=glucagon-like peptide-1 Adapted from Rothenberg P, et al. Diabetes. 2000; 49(suppl 1): A 39. Abstract 160 -OR. Adapted from Deacon CF, et al. Diabetes. 1995; 44: 1126 -1131. 38

Blocking DPP-4 Can Improve Incretin Activity and Correct the Insulin: Glucagon Ratio in T 2 DM Incretin response diminished Insulin Further impaired islet function Hyperglycemia Glucagon DPP-4 inhibitor Incretin activity prolonged Insulin Improved islet function Improved glycemic control Glucagon DPP-4=dipeptidyl peptidase-4; T 2 DM=type 2 diabetes mellitus Adapted from Unger RH. Metabolism. 1974; 23: 581– 593. Ahrén B. Curr Enzyme Inhib. 2005; 1: 65– 73. 39

Vildagliptin – a potent and selective DPP-4 inhibitor H HO O N N N · Highly selective DPP-4 inhibitor · Has a high affinity for the human enzyme · Reversible inhibition X-ray crystallographic structure of vildagliptin (green) bound to the active site (yellow) of human DPP-4=dipeptidyl peptidase-4 40

Vildagliptin Provides Potent and Dose-dependent Inhibition of DPP-4 in Patients with T 2 DM Placebo (n=16) Vilda 10 mg (n=16) Vilda 25 mg (n=16) Vilda 50 mg (n=16) Vilda 100 mg (n=15) Vilda 200 mg (n=16) Vilda 400 mg (n=16) DPP-4=dipeptidyl peptidase-4; T 2 DM=type 2 diabetes mellitus; vilda=vildagliptin He YL, et al. In press. 41

Vildagliptin Enhances Islet Cell Function by Increasing Insulin and Decreasing Glucagon Secretion OGTT 30 Min after Single Oral Dose of Vildagliptin (100 mg) 75 g Glucose 120 Insulin (pmol/L) Placebo (n=16) Vildagliptin 100 mg (n=15) 100 Dose 80 60 40 20 0 − 90 − 60 − 30 0 30 60 90 120 150 180 210 240 270 300 22. 5 Glucose (mmol/L) 17. 5 12. 5 7. 5 140 Glucagon (ng/L) 120 100 80 60 OGTT=oral glucose tolerance test *P <0. 01. He YL, et al. In press. Time 42

Vildagliptin Enhances GLP-1 Levels in Patients with T 2 DM Active GLP-1 (pmol/L) 16. 0 Meal Placebo (n=16) * Vildagliptin 100 mg (n=16) * * 12. 0 * * ** 8. 0 * * * 4. 0 0. 0 17: 00 20: 00 GLP-1=glucagon-like peptide-1 *P <0. 05 Balas B, et al. J Clin Endocrinol Metab. 2007; Epub ahead of print. 23: 00 02: 00 05: 00 08: 00 Time 43

Vildagliptin Suppresses Glucagon Secretion 20 Meal Placebo (n=16) Delta Glucagon (ng/L) 10 Vildagliptin 100 mg (n=16) 0 − 10 − 20 * − 30 * − 40 − 50 − 60 17: 00 * * * 20: 00 23: 00 *P <0. 05 vs placebo Balas B, et al. J Clin Endocrinol Metab. 2007; Epub ahead of print. * * * 02: 00 * 05: 00 08: 00 Time 44

Vildagliptin Suppresses Endogenous Glucose Production Time Meal Delta EGP (mg/kg/min) 17: 00 0 20: 00 23: 00 02: 00 05: 00 08: 00 − 0. 3 − 0. 6 * − 0. 9 * − 1. 2 − 1. 5 ** * * *P <0. 05 vs placebo Balas B, et al. J Clin Endocrinol Metab. 2007; Epub ahead of print. * ** * * *** Placebo (n=16) Vildagliptin 100 mg (n=16) 45

Vildagliptin Increases Levels of Active Incretins in Patients with T 2 DM N=9 Intact GIP (pmol/L) Intact GLP-1 (pmol/L) 35 250 30 200 25 150 20 100 15 50 0 – 2 0 2 4 // 10 // 5 6 8 10 12 14 Time (h) Vildagliptin Day 1 16 0 – 2 0 2 // // 4 6 8 10 12 14 Time (h) Vildagliptin Day 28 GIP=glucose-dependent insulinotropic peptide; GLP-1=glucagon-like peptide-1; T 2 DM=type 2 diabetes mellitus Adapted from Mari A, et al. J Clin Endocrinol Metab. 2005; 90: 4888– 4894. 46

Vildagliptin Improves First-Phase Insulin Response in Drug-Naïve T 2 DM Patients 180 * * 160 * Vildagliptin 100 mg daily Week 12 Insulin (pmol/L) 140 Vildagliptin 100 mg daily Week 0 120 100 80 >3 -fold increase in AIRg (P <0. 001 vs Week 0, P <0. 001 vs placebo) 60 40 − 6 − 4 − 2 0 2 4 6 8 10 12 Time (Min) AIRg=acute insulin response to glucose; T 2 DM=type 2 diabetes mellitus *P <0. 05 D’Alessio DA, et al. Poster 454 -P presented at ADA 2006. 47

Vildagliptin: Enhances Insulin Sensitivity Duration: 6 weeks Vildagliptin vs placebo Hyperinsulinemic Euglycemic Clamp Glucose Rd (mg/kg/min) 7. 0 6. 0 * 6. 1 Vildagliptin 100 mg daily (n=8) 5. 4 Placebo (n=8) 5. 0 4. 0 3. 0 2. 0 1. 0 0. 0 Rd=rate of disappearance *P <0. 05 Azuma K, et al. Diabetes. 2007 (submitted). 48

Vildagliptin Reduces Postprandial Total Serum Triglycerides after 4 Weeks of Treatment Vildagliptin 100 mg daily* Week 0 Week 4 4. 0 Total Serum TG (mmol/L) Placebo** 3. 5 3. 0 2. 5 2. 0 1. 5 1. 0 Week 4 3. 5 3. 0 2. 5 2. 0 1. 5 1. 0 − 1 0 1 2 3 4 5 Time (h) 6 7 8 TG=triglycerides ITT population (intention-to-treat). *Adapted from Matikainen N, et al. Diabetologia. 2006; 49: 2049– 2057. **Study LAF 2217. Data on file, Novartis Pharmaceuticals, 2 December 2005, p. 36. − 1 0 1 2 3 4 5 6 7 8 Time (h) 49

Vildagliptin Reduces Chylomicron Tryglycerides after 4 Weeks of Treatment Vildagliptin 100 mg daily 0. 8 Week 0 Chylomicron TG (mmol/L) Week 4 0. 6 0. 4 0. 2 0. 0 -1 0 1 2 3 4 5 6 7 8 Time (h) TG=triglycerides Matikainen N, et al. Diabetologia. 2006; 49: 2049– 2057. 50

T 1/2 (Min) for Gastric Emptying Vildagliptin: No Effect on Gastric Emptying 150 143 145 Placebo Vildagliptin 100 mg daily 125 100 75 50 25 0 T 1/2=time to empty 50% of stomach contents. Difference not statistically significant Vella A, et al. Diabetes. 2007: Epub ahead of print. 51

Vildagliptin Enhances -cell Function and Insulin Sensitivity over 52 Weeks Insulin Sensitivity 300 0. 050 0. 045 * * * 0. 040 0. 035 0. 030 0. 025 m. L · min-1 · m -2 pmol/L 30 min/(mmol/L) Insulin Secretion 275 * * 250 225 200 0 12 24 52 Time (Week) nmol. C-peptide · mmolglucose-1 · m. L-1 · m-2 Patients on Stable Metformin Therapy Adaptation Index 14 12 * * † 10 8 6 0 12 24 52 Time (Week) Vildagliptin 100 mg daily / metformin Placebo / metformin *P <0. 05 vs placebo; †P <0. 01 vs placebo. Adapted from Ahrén B, et al. Diabetes Care. 2005; 28: 1936– 1940. 52

Vildagliptin: Improves β-cell Mass (Neonatal Rat Pancreatic Growth Model) Insulin Vildagliptin 60 mg/kg 21 days Vehicle 100 80 60 40 20 0 Vehicle Vilda 2. 5 2. 0 1. 5 P <0. 05 1. 0 0. 5 0. 0 Day 7 Vehicle -cell Mass (mg) P <0. 001 Apop. Tag-Positive Cells (%) Brd. U-Positive Cells (%) 120 -cell mass Apoptosis Replication Vilda=vildagliptin Duttaroy A, et al. Diabetes. 2005; 54 (suppl 1): A 141. Abstract 572 -P and poster presented at ADA. P <0. 05 0. 14 0. 12 0. 10 0. 08 0. 06 0. 04 0. 02 0. 00 Vehicle Vilda Day 21 53

ΕΥΧΑΡΙΣΤΩ Meal Intestinal GLP-1 release GLP-1 t½=1– 2 min Active GLP-1 DPP-4 GLP-1 inactive (>80% of pool) 55