Discovery of DPP-4 Inhibitors as Antidiabetic Agents Tesfaye Biftu Merck & Co., Inc. Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved History of Diabetes • c. 1552 B.C. – 1st reference to Rx for polyuria by Hesy- Ra, 3rd Dynasty Ebers Papyrus • c. 100 B.C. – The father of medicine in India, Susruta of the Hindus, diagnosed diabetes • c. 30 B.C. ~ 50 A.D. – 1st clinical description of diabetes, Aulus Cornelius Celsus • c. 150 A.D. – The name “diabetes” was first introduced from the Greek word for “siphon” by Aretaeus of Cappadocia Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved ……….continued • Discovery of Insulin (1921) – An Instant Hit – First Human Clinical Trial 1922 – Noble Price 1923 Benting & MacLoed – Commercial Production 1923 Eli Lilly – First Total Synthesis 1965 Chinese Team • Discovery of Glucagon (1923) – Largely Unnoticed – Sequenced in 1957 – Rediscovered in 1970’s for its role in diabetes • Discovery of Incretins – Gastrointestinal Hormones – 1932 The term “incretin” was first used – 1970 GIP was isolated – 1982 GLP-1 was sequenced Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Diabetes is a Growing Worldwide Epidemic Europe 2000: 33.3 million 2030: 48 million Middle East 2000: 15.2 million 2030: 42.6 million Africa The Americas 2000: 7 million Asia and Australia 2000: 33 million 2030: 18.2 million 2000: 82.7 million 2030: 66.8 million 2030: 190.5 million Prevalence (%) in persons 35-64 yrs <3 3-5 6-8 >8 • 2000: 171 million cases worldwide; 2030: 366 million cases predicted • >90% is Type 2 diabetes: obesity is a major risk factor Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved WHO Trends among U.S. Adults: 1994 to 2004 1994 2004 <4% 4-4.9% 5-5.9% 6+% missing data Ø 20,800,000 People in US (7%) have diabetes (2005) Ø 6th Leading cause of death in US (2002), by death certificate Ø >90% Type 2, associated with metabolic syndrome Taken from CDC diabetes website: http://www.cdc.gov/diabetes Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Current Medications for Treatment of Diabetes Drug Class Side Effects Sulfonylureas Hypoglycemia, weight gain Meglitinides Hypoglycemia, weight gain Metformin GI intolerance Thiazolidinediones Edema, weight gain α-Glucosidase inhibitors GI intolerance Insulin Hypoglycemia, weight gain Safer and more effective medications are needed Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Role of Active GLP-1 and GIP in Glucose Homeostasis • Glucagon-Like-Peptide (GLP-1) and Glucose-Dependent- Insulotropic-Polypeptide (GIP) are secreted in response to food intake (glucose-dependent mechanism) • GLP-1 and GIP - (↑)stimulate insulin biosynthesis & secretion • GLP-1 inhibits - (↓) glucagon secretion • GLP-1 and GIP - (↓)lower blood sugar level Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved …. but Active GLP-1 (or GIP) is Cleaved Rapidly by DPP-4 Stabilize GLP-1 (Active) HA EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2 Inhibit DPP-4 t½ ~ 1 min GLP-1 (Inactive) EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2 Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Dipeptidyl Peptidase IV (DPP-4) • First Isolated from rat liver in 1966 • Identical to CD26, a marker for activated T cells • Its role in energy homeostasis was discovered which led to the first patent application in 1996 • Cell surface serine dipeptidase belonging to the prolyl oligopeptidase family • Widely expressed, and has Specificity for P1 Pro >> Ala Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Selective Inhibitors F O O Me O S N NH2 O Me O N N N N N NH2 S NH F N 2 I CF3 DPP-4 Selective QPP Selective DPP-8/9 Selective Enzyme IC50, nM DPP-9 > 100,000 11,000 55 DPP-8 69,000 22,000 38 FAP > 100,000 > 100,000 > 100,000 DPP-4 27 1900 30,000 PEP > 100,000 > 100,000 > 100,000 QPP/DPP-2 > 100,000 19 14,000 APP > 100,000 > 100,000 > 100,000 prolidase > 100,000 > 100,000 > 100,000 Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Comparative Toxicity Study QPP DPP-8/9 DPP-4 2 wk. Rat Toxicity Selective Selective Selective Alopecia ü Thrombocytopenia ü Reticulocytopenia ü ü Enlarged spleen ü Mortality ü Acute dog toxicity Bloody diarrhea ü G. Lankas, et al., Diabetes 2005, 54, 2988 Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Potential Importance of Selective Inhibition for the Treatment of Type 2 Diabetes • Conclusion – “Off-target” peptidase inhibition (i.e. inhibition of other DPP family peptidases such as DPP8/9) can produce severe toxicity in preclinical species • Variables that may determine degree of toxicity – Cell penetration • Unlike DPP-4, DPP8/9 are intracellular DPP-4 proteins – Extent of inhibition of DPP8 and/or DPP9 • Not known if inhibition of both enzymes (or how much) is required to produce toxicities – Intraspecies differences in DPP8/9 inhibition from Demuth et al. Biochim. Biophys. Acta 2005, 1751, 33 Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved DPP-4 Inhibitor Program - Objective Identify a potent and selective DPP-4 inhibitor for the treatment of type 2 diabetes mellitus with the following characteristics: • >1000-fold selectivity over other proline peptidases, especially DPP-8 and DPP-9 • Half-life suitable for BID or preferably QD dosing • Structure lacking reactive electrophile as a serine trap, e.g., O O B(OH)2 H CN H N N 2 N R N R R' X Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Common Lead Discovery Methods • Biological screening of compounds • Substrate or active-structure based design • Enzyme – inhibitor crystal structure Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Screening Leads Cl • β-Amino acid proline amides H O N IC = 1.9 µM NH2 O O 50 O N N H • β-Amino piperazines H2N IC50 = 11 µM N O N N NHSO2Me H Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Recovery of Radioactivity after IV (1 mg/kg) or Oral (2 mg/kg) Administration to Rats T NH2 O N F NH Radioactivity Recovered in 24 hrs (% Dose) Collection Period Intravenous Oral Bile 28.3 ± 2.6% 38.2 ± 4.7% Urine 18.1 ± 2.0% 14.0 ± 4.2% Feces 1.0 ± 0.2% 1.4 ± 0.6% Total 47.4 ± 3.9% 54.6 ± 4.9% PK Rat: • high clearance (>150 mL/min/kg) and • short half-life. Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Y. Teffera Metabolites identified in microsomes, hepatocites, and bile of PO/IV treated rats Microsomes: piperazine ring oxidation. Hepatocytes: Conjugation of the primary amino group (glucuronic acid and glucose). Oxidation of the piperazine, the phenyl and the fluoro-phenyl rings. Glutathione conjugation on the fluoro-phenyl ring; d) amide bond hydrolysis. Bile of PO treated rats: carbamoyl glucuronide conjugate on the piperazine ring. Glutathione conjugation of the fluoro-phenyl ring, piperazine ring oxidation, and N- glucuronidation of the primary amine were also observed. Bile of IV treated rats: Glutathione conjugation on the fluoro-phenyl ring, piperazine and phenyl ring oxidation, and N-glucuronidation of the primary amine. Urine of PO treated rats: Acid formed by the de-ethylation and oxidative de- amination of the piperazine ring. Oxidation of the piperazine ring, glucuronidation of the primary amine, and amide bond hydrolysis were also observed. Urine of IV treated rats: excreted mainly parent compounds Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Efforts made to stabilize metabolism NH2 O NH2 O N F X = NH, O N F N Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Bicyclic Piperazine Replacements N N N N N N N N N N N N R N R N N N N R R R O N R S N N N N N N R O N R R O N S R' R N R R R N N N N O N N N R' N R' R R N N N N O O R" R" R R N N N N N N N N R" N R R R N N N N R' R' N N N N N N N N N R R R R R N N N N Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Left Hand Side SAR NH2 O R N N N N CF3 Rat PK R DPP-4 IC50 Clp t½ Frat (nM) (mL/min/kg) (h) (%) 3,4-diF 130 51 1.8 44 2,5-diF 27 43 1.6 50 2,4,5-triF 18 60 1.7 76 MK-0431 = 100 JANUVIA™ (sitagliptin phosphate) D. Kim, et al., J. Med. Chem. 2005, 48, 141 Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved F F Sitagliptin: In Vitro Potency NH2 O N N and Selectivity N F N CF3 IC50, (nM) Selectivity Ratio DPP-9 > 100000 > 5000 DPP-8 48000 2700 DPP-4 FAP > 100000 > 5000 Gene DPP-4 18 1 Family DPP-6 not active not active PEP > 100000 > 5000 Other Proline QPP/DPP-2 > 100000 > 5000 Specific Enzymes APP > 100000 > 5000 prolidase > 100000 > 5000 Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved F F PK Properties of Sitagliptin NH2 O N N 1 mg/kg IV, 2 mg/kg PO N F N CF3 Clp Vd t½ PO AUCnorm PO Cmax F species (mL/min/kg) (mL/kg) (h) (µM·h/mpk) (µ M ) (% ) Mouse 64 3.8 0.8 0.41 0.51 61 Rat 60 6.4 1.7 0.52 0.33 76 Dog 6.0 9.0 4.9 8.3 2.2 100 Monkey 28 2.3 3.7 1.0 0.33 68 Copyright © 2010 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved Pharmacodynamics of Sitagliptin: OGTT in Lean Mice 20 min