(12) United States Patent
`Greig et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,576,050 B2
`Aug. 18, 2009
`
`US00757605OB2
`
`(54) GLP-1 EXENDIN-4 PEPTIDE ANALOGS AND
`USES THEREOF
`
`(75) Inventors: Nigel Greig, Phoenix, MD (US);
`Josephine Egan, Baltimore, MD (US);
`Maire Doyle, Baltimore, MD (US);
`Harold Holloway, Middle River, MD
`(US); Tracy Ann Perry, Baltimore, MD
`(US)
`(73) Assignee: The United States of America as
`represented by the Department of
`Health and Human Services,
`Washington, DC (US)
`
`(*) Notice:
`
`(21) Appl. No.:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 634 days.
`10/485,140
`
`(22) PCT Filed:
`(86). PCT No.:
`
`Jul. 30, 2002
`PCT/USO2A24141
`
`S371 (c)(1),
`Apr. 1, 2004
`(2), (4) Date:
`(87) PCT Pub. No.: WO03/011892
`PCT Pub. Date: Feb. 13, 2003
`
`(65)
`
`Prior Publication Data
`US 2004/O242853 A1
`Dec. 2, 2004
`
`Related U.S. Application Data
`(60) Provisional application No. 60/309,076, filed on Jul.
`31, 2001.
`
`(51) Int. Cl.
`(2006.01)
`A6 IK 38/26
`(2006.01)
`C07K I4/605
`(52) U.S. Cl. ............................. 514/2; 514/12:530/308:
`530/324
`(58) Field of Classification Search ....................... None
`See application file for complete search history.
`References Cited
`
`(56)
`
`U.S. PATENT DOCUMENTS
`3,710,795 A
`1/1973 Higuchi et al.
`6,268.479 B1* 7/2001 Stern et al. .................. 530/350
`6,429,197 B1
`8/2002 Collidge et al.
`7,157,429 B1
`1/2007 Bachovchin ................ 514,200
`2002/01 15605 A1* 8/2002 During et al. ................. 514/12
`2003,0004162 A1
`1/2003 Treadway ....
`514,228.2
`2003/005O227 A1
`3/2003 Kondo ........................... 514/2
`2004, OO18981 A1
`1/2004 Dong .......................... 514/12
`2005/0239854 Al 10/2005 Sugiyama ................... 424/449
`
`
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`WO
`
`O658568 A1
`1304121
`WO95/31214
`
`6, 1995
`4/2003
`11, 1995
`
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`12/1997
`WO97/.46584
`7, 1998
`WO 98.30231 A2
`8, 1999
`WO99/O385O1
`8, 1999
`WO99/4O788 A1
`9, 1999
`WO99.43705
`9, 1999
`WO99/46283
`12/1999
`WO99, 67278
`3, 2000
`WOOO16797 A2
`3, 2000
`WO 200016797 A2
`6, 2000
`WOOO,34332
`T 2000
`WOOO 41546 A
`8, 2000
`WOOOf 47219
`WOOOf 66142 A 11, 2000
`WOOOf73331 A 12/2000
`WO 01/04156 A1
`1, 2001
`WOO1, 51078 A
`T 2001
`WOO2,46227 A
`6, 2002
`
`OTHER PUBLICATIONS
`Wang et al. ‘Glucagon-like Peptide-1 Can Reverse the Age-related
`Decline in Glucose Tolerance in Rats J. Clin. Invest. 199799: 2883
`2889.*
`Oka et al. Endogenous GLP-1 is involved in beta-amyloid protein
`induced memory impairment and hippocampal neuronal death in
`rats.
`Siram et al. Experimental Allergic Encephalomyelitis a Misleading
`Model of Multiple Sclerosis. Ann Neurol. vol. 58, pp. 939-945.
`2005.*
`Citron, Martin. Alzheimer's Disease: Treatments in Discovery and
`Development. Nature Neurroscience Supplment. vol. 5, pp. 1055
`1057. Nov. 2002.
`File Medline on STN. An No. 2005478947. Simmons, Zachary.
`“Management Strageies for Patients with Amyotrpholic Lateral Scle
`rosis from diagnosis Through Death.” The Neurologist (Sep. 2005),
`vol. 11, No. 5, pp. 257-270. Abstract only.*
`AZZouz, Mimoun "Gene Therapy for ALS: Progress and Prospects'
`Biochemical et Biophysica Acta (2006), vol. 1762 pp. 112-1127.*
`Margolis et al. Diagonsis of Huntington Disease” Clinical Chemis
`try, vol. 49, No. 10 pp. 1726-1732 (2003).*
`Korczyn et al. Emerging Therapies in the Pharmacological Treat
`ment of Parkinson's Disease' Drugs vol. 62, No. 5 pp. 775-786.
`20O2.*
`Steinman et al. How to Successfully Apply Animal Studies in
`Experimental Allergic Encephalomyelitis to Research on Multiple
`Sclerosis' Ann Neurol. vol. 60, pp. 12-21, 2006.*
`(Continued)
`Primary Examiner Anish Gupta
`(74) Attorney, Agent, or Firm Ballard Spahr, LLP
`
`(57)
`
`ABSTRACT
`
`The invention relates to novel polypeptide analogues of
`GLP-1 and exendin-4. The polypeptide, in a preferred
`embodiment, is insulinotropic and long-acting. Preferably,
`the polypeptides insulinotropic effect is comparable to or
`exceeds the effect of an equimolar amount of GLP-1 or exen
`din-4. The invention also relates to a method of treating a
`Subject with diabetes, comprising administering to the Sub
`ject the polypeptide of the invention in an amount that has an
`insulinotropic effect. The invention also relates to methods of
`using GLP-1, exendin-4, and polypeptide analogues thereof
`for neuroprotective and neurotrophic effects.
`
`15 Claims, 28 Drawing Sheets
`
`MPI EXHIBIT 1028 PAGE 1
`
`MPI EXHIBIT 1028 PAGE 1
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`

`

`US 7,576.050 B2
`Page 2
`
`OTHER PUBLICATIONS
`Pateletal. Pharmacotherapy of Cognitive Impariment in Alzheimer's
`Disease: A Review J. Geriatr. Psychiatry Neruol. vol.8 pp. 81-95.
`1995.*
`Pinderhuges et al. Evidence-Based Apporach to Management of
`Feverin Patients With End-Stage Dementia.J. of Palliative Med. vol.
`6, No. 3, 2003.*
`Okamoto et al. “Treatment of Vascular Dementia.” Ann. New York
`Academy of Science. pp. 507-512. 2002.*
`Poewe, W. “The Need for Neuroprotective Therapies in Parkinson's
`Disease.” Neurology, vol. 66 Supp. 4, pp. s2-s9.*
`Bressler et al., “Pharmacological regulation of blood glucose levels
`in non-insulin-dependent diabetes mellitus.” Arch. Int. Med.
`157:836-848 (1997).
`Calvo et al., “Structural characterization by affinity cross-linking of
`glucagon-like peptide-1 (7-36) amide receptor in rat brain.” J.
`Neurochem, 64(1):299-306 (1995).
`Campos et al., “Divergent tissue-specific and developmental expres
`sion of receptors for glucagons and glucagon-like peptide-1 in the
`mouse.” Endocrinology 134: 2156-64 (1994).
`Chen et al., “Tissue-specific expression of unique mRNAS that
`encode proglucagon-derived peptides or exendin-4 in the lizard. J.
`Biol. Chem. 272: 4108-41 15(1997).
`De Ore et al., “The effect of GLP-1 on insulin release in young and
`old rats in the fasting state and during an intravenous glucose toler
`ance test.” J. Gernotol. 52: B245-249 (1997).
`Drucker et al., “Glucagon-like peptide I stimulates insulin gene
`expression and increases cyclic AMP levels in a rat islet cell line.”
`Proc. Natl. Acad. Sci. 84: 3434-3438 (1987).
`Egan etal. “Glucagon-like peptide-1 (7-36) amide (GLP-1) enhances
`insulin-stimulated glucose metabolism in 3T3-I1 adipocytes: one of
`several potential exrtapancreatic sites of GLP-1 action.” Endocrinol
`ogy 135: 2070–2075, 1994.
`Elahi et al., “The insulinotropic actions of glucose-dependent
`insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (7-37)
`in normal and diabetic subjects.” Regul. Pep. 51: 63-74 (1994).
`Elahi, et al. “The effect of age and glucose concentration on insulin
`Secretion by the isolated perfused pancreas.' Endocrinology 1 16;
`11-16, 1985.
`Federal Register 63, Friday, Feb. 20, 1998, p. 8652.
`Fehmann et al. "Cell and Molecular Biology of the Incretin Hor
`mones Glucagon-Like Peptide-I and Glucose-dependent Insulin
`Releasing polypeptide.” Endocrine Rev. 16:390-410, 1995.
`Fehmann et al. "Cell and Molecular Biology of the Incretin Hor
`mones Glucagon-Like Peptide-I and Glucose-Dependent Insulin
`Releasing Polypeptide.” Endocrine Rev. 16:390-410 (1995).
`Fehmann et al., “Insulinotropic hormone glucagon-like peptide-I(7-
`37) stimulation of proinsulin gene expression and proinsulin
`biosynthesis in insulinoma BTC-1 cells,” Endocrinology 130: 159
`166 (1992).
`Gefel et al. “Glucagon-Like Peptide I Analogs: Effects on Insulin
`Secretin and Adenosine 3',5'-Monophosphate Formation.” Endocri
`nology 126:2164-68, 1990.
`Geula and MeSulam, “Cortical cholinergic fibers in aging and
`Alzheimer's disease: a morphometric study.” Neuroscience 33: 469
`81 (1989).
`Ghazzi et al., “Cardiac and glycemic benefits of troglitaZone treat
`ment in NIDDM.” Diabetes 46: 433-439. (1997).
`Göke et al., “Distribution of GLP-1 binding sites in the rat brain:
`evidence that exendin-4 is aligand of brain GLP-1 binding sites.” Eur:
`J. Neurosci: 2294-2300 (1995).
`Göke et al., “Exendin-4 is a high potency agonist and truncated
`exendin-(9-39)-amide in an antagonist at the Glucagon-like Peprtide
`1-(7-36)-amide receptor of insulin-secreting-B-cells,” J. Biol. Chem.
`268: 19650-19655 (1993).
`Greig et al., “Once daily injection of exendin-4 to diabetic mice
`achieves long-term beneficial effects on blood glucose concentra
`tions.” Diabetologia 42:45-50 (1999).
`Gromada et al. "Glucagon-Like Peptide 1 (7-36) Amide Stimulates
`Exocytosis in Human Pancreatic B-Cells by both Proximal and Distal
`Regulatory Steps in Stimulus-secretion Coupling.” Diabetes 47:57
`65, 1998.
`
`Dev. Neuroscience 19:27
`
`Gutniak et al., “Antidiabetogenic effect of glucagon-like peptide-1
`(7-36 amide) in normal subjects and patients with diabetes mellitus.”
`N. Eng. J. Med. 326: 1316- 1322 (1992).
`Guz et al. “Expression of murine STF-1, aputative insulin gene
`transcription factor, in 3 cells of pancreas, duodenal epithelium and
`pancreatic excrine and endocrine progenitors during ontogengy.”
`Development 121: 11-18, 1995.
`Hawes et al. “Distinct pathways of G-and G-mediated mitogen
`activated protein kinase activation.” J. Biol. Chem. 270: 17148
`17153, 1995.
`Holz et al. “Activation of a cAMP-regulated Ca"-signaling pathway
`in pancreatic beta-cells by the insulinotropic hormone glucagon-like
`peptide-1.” J. Boil. Chem. 270: 17749-17757, 1995.
`Hosokawa et al. “Mechanism of impaired glucose-potentiated insulin
`Secretion in diabetic 90% pancreatectomy rats. Study using
`glucagonlike peptide-1 (7-37).” J. Clin. Invest. 97: 180-1860, 1996.
`Jin et al., “Distribution of glucagonlike peptide I (GLP-I), glucagon,
`and glicentin in the rat brain: An Immunocytochemical Study,” J.
`Comp. Neurol. 271: 519-32. (1988).
`Kimura et al. “High concentrations of cholecystokinin octapeptide
`Suppress protein kinase c activity in guinea pig pancreatic acini.”
`Peptides 17: 917-925, 1996.
`Lahiri et al., "Cholinesterase inhibitors, 3-amyloid precursor protein
`and amyloid 3-peptides in Alzheimer's disease.” Acta. Neurol. Scand
`Suppl 176: 60-67.(2000).
`Lee, “NeuroD and Neurogenesis,
`32(1997).
`Malhotraetal. “Exendin-4, a new peptide from heloderma suspectum
`vemon, potentiates cholecystokinin-induced amylase from rat pan
`creatic acini.” Regul. Pept. 41: 149-156, 1992.
`Mark et al., “Amyloid -peptide impairs glucose transport in hip
`pocampal and cortical neurons: involvement of membrane lipid
`peroxidation.” J. Neurosci. 17: 1046-1054(1997).
`Mashima et al. "Betacellulin and activin A. coordinately convert
`amylase-secreting AR42J cells into insulin-secreting cells.” J. Clin.
`Invest. 97: 1647-1654, 1996.
`Mashima et al. “Formation of Insulin-Production Cells from pancre
`atic Acinar AR42J Cells by Hepatocyte Growth Factor.” Endocrinol
`ogy 137: 3969-3976, 1996.
`Mattson et al., “Neurotrophic factors attenuate glutamate-induced
`accumulation of peroxides, elevation of intracellular Ca" concentra
`tion, and neurotoxicity and increase antioxidant enzyme activities in
`hippocampal neurons.” J. Neurochem 65 (4): 1740-1751. (1995).
`Moceri et al., “Early-life risk factors and the development of
`Alzheimer's disease.” Neurology. 54: 415-420 (2000).
`Montrose-Rafizadeh et al. “Incretin hormones regulate glucose-de
`pendent insulin secretion in RIN 1046-38 cells: mechanism of
`action.” Endocrinology 135: 589-594, 1994.
`Montrose-Rafizadeh et al., “High potency antagonists of the pancre
`atic glucagon-like peptide-1 receptor.” J. Biol. Chem. 272: 21201
`21206 (1997).
`Montrose-Rafizadeh et al., “Incretin hormones regulate glucose-de
`pendent insulin secretion in RIN 1046-38 cells: mechanisms of
`action.” Endocrinology 135: 589-594 (1994).
`Montrose-Rafizadeh et al., “Overexpression of glucagon-like
`peptide-1 receptor in an insulin-secreting cell line enhances glucose
`responsiveness.” Mol, Cell Endocrinol. 130 (1-2): 109-117. (1997).
`Montrose-Rafizdeh et al. “Novel signal transduction and peptide
`specificity of gluccagon-like peptide receptor in 3T3-L1 adipocytes.”
`J. Cell. Physiol. 172: 275-280, 1997.
`Nathan et al. “Insulinotropic action of glucagonlike peptide-I- (7-37)
`in diabetic and nondiabetic subjects.” Diabetes Care 15: 270-276
`(1992).
`Naucket al. "Normalization of fasting hyperglycaemia by exogenous
`glucagon-like peptide 1 (7-36 amide) in type 2 (non-insulin depen
`dent) diabetic patients.” Diabetologia 36: 741-744 (1993).
`Naucket al. "Preserved incretin activity of Glucagon-like peptide 1
`(7-36 amide) but not of synthetic human gastric inhibitory
`polypeptide inpatients with Type-2 diabetes mellitus.”.J. Clin. Invest.
`91: 301-307 (1993).
`Naya et al. “Diabetes, defective pancreatic morphogenesis, and
`abnormal enteroendocrine differentiation in Beta2/NeuroD-deficient
`mice.” Genes Dev. 11: 2323-2334 (1997).
`
`MPI EXHIBIT 1028 PAGE 2
`
`MPI EXHIBIT 1028 PAGE 2
`
`

`

`US 7,576.050 B2
`Page 3
`
`Noma, "Overexpression of NeuroD in PC12 cells alters morphology
`and enhances epXression of the adenylate kinase isozyme 1 gene.”
`Molecular Brain Research 67:53-63 (1999).
`Orskov, "Glucagon-like peptide-1, a new hormone of the entero
`insular axis.” Diabetologia 35: 701-711 (1992).
`Ott et al. "Diabetes mellitus and the risk of dementia: The Rotterdam
`Study.” Neurology 53: 1937-42 (1999).
`Perfetti et al. "Age-dependent reduction on insulin Secretion and
`insullin mRNA in isolated islet from rats.” Am. J. Physiol. 269:
`E983-990, 1995.
`Perry et al., “Behavioural, histological and immunocytochemical
`consequences following 192 IgG-Saporin immunolesions of the basal
`forebrain cholinergic System.” Brain Res. Bull. 54: 29-48 (2001).
`Ritzel et al., “Pharmacokinetic insulinotropic, and glucagonostatic
`properties of GLP-1 (7-36 amide after subcutaneous injection in
`healthy volunteers. Dose-Response-Relationships.” Diabetologia
`38: 720-725 (1995).
`Satoh et al., "Characterization of human and rat glucagon-like
`peptide-1 receptors in the neurointermediate lobe: lack of coupling to
`either stimulation of inhibition of adenylyl cyclase.” Endocrinology
`141: 1301-9 (2000).
`Shughrue et al., “Glucagon-like peptide-1 receptor (GLP1-R) mRNA
`in the rat hypothalamus.” Endocrinol. 137 (11): 5159-62 (1996).
`Suzuki et al., “An increased percentage of long amyloid f protein
`Secreted by familial amyloid 3 protein precursor (3 APP-717) mutants.”
`Science 264: 1336-1340. (1994).
`Teitelman “Induction of beta-cell neogenesis by islet injury.” Diabe
`tes Metabolism Rev. 12:91-102, 1996.
`Thorens et al. “Expression cloning of the pancreatic beta cell receptor
`for the gluco-incretin hormone glucagon-like peptide 1.” Proc. Natl.
`Acad. Sci. USA 89:8641-8645, 1992.
`Thorens et al. “Glucagon-like peptide-I and the control of insulin
`secretion in the normal state and in NIDDM, Diabetes 42: 1219
`1225 (1993).
`Thorens et al., “Cloning and functional expression of the human islet
`GLP-1 receptor Demonstration that exendin-4 is an agonist and
`exendin-(9-39) an antagonist of the receptor.” Diabetes 42: 1678
`1682 (1993).
`Valverde and Villanueva-Penacarrillo et al. "Invitro insulinomimetic
`effects of GLP-1 in liver, muscle and fat.” Acta Physiologica
`Scandinavica. 157:359-360, 1996.
`Wang et al. "GIP regulates glucose transporters, hexokinases, and
`glucose-induced insulin secretion in RIN 1046-38 cells.” Mol. Cell.
`Endo. 116: 81-87 (1996).
`Wang et al. "Glucagon-like peptide-1 is a phsyiological incretin in
`rat.” J. Clin. Invest. 95:417-421, 1995.
`Wang et al. Glucagon-like peptides-1 can reverse the age related
`decline in glucose tolerance in rats. J. Clin. Invest. 99: 2883-2889,
`1997.
`Wang et al., “Glucagon-like peptide-1 affects gene transcription and
`messenger ribonucleic acid stability of components of the insulin
`secretory system in RIN 1046-38 cells,” Endocrinology 136: 4910
`4917 (1995).
`Wei et al., “Tissue-specific expression of the human receptor for
`glucagon-like peptide-I: brain, heart and pancreatic forms have the
`same deduced amino acid sequences.” FEBS Letters 358(3): 219-224
`(Jan. 30, 1995).
`Widmann et al. “Desensitization and phosporylation of the glucagon
`like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol. 12
`Myristate 13-acetate.” Mol. Endocrinol. 10: 62-75, 1996.
`Willms et al., “Gastric emptying, glucose responses, and insulin
`Secretion after a liquid test meal: effects of exogenous glucagon-like
`
`peptide-1 (GLP-1)-(7-36) amide in Type 2 (noninsulin-dependent)
`diabetic patients.” Clin. Endocrinol. Metab. 81: 327-332 (1996).
`Yada et al. "Glucagon-like peptide-1-(7-36) amide and a rise in cyclic
`adenosine 3',5'-monophosphate increase cyOSolic free Ca' in rat
`pancreatic f-cells by enhancing Ca"channel activity.” Endocrinol
`ogy 133: 1685-1692.
`Drucker, "Minireview: The glucagon-like peptides,” Endocrinology
`142:521-527 (2001).
`Iwai, et al., “Effects of glucagon-like peptide-1 on LTP in B-amyloid
`protein (1-42)-treated hippocampal slices.” Soc. Neurosci. Abstr.
`26(12): 1116 Abstract No. 420.7 (2000).
`Kondo, et al., “Effects of endogenous GLP-1 on neurite elongation in
`rat primary cultured hippocampal neurons. Jpn. J. Pharmacol.
`85(1):276, P-866 (2001).
`Lahiri, et al., “Exendin-4 (Ex-4) revives PC12 cells from nerve
`growth factor (NGF)-mediated cell death and apoptosis.” Soc.
`Neurosci. Abstr. 27(2):2620, Abstract No. 983.16 (2001).
`Lovshin, et al. "Glucagon-like peptide (GLP)-2 action in the murine
`central nervous system is enhanced by elimination of GLP-1 receptor
`signaling.” J. Biol. Chem. 276(24):21489-21499 (Jun. 2001).
`Oka, et al., “Endogenous GLP-1 is involved in B-amyloid protein
`induced memory impairment and hippocampal neuronal death in
`rats.” Brain Research 878:194-198 (2000).
`Oka, et al., “Behavioral studies indicate a role for glucagon-like
`peptide-1 in memory and learning in rat.' Society for Neuroscience
`25:1862, 742.13 (1999).
`Oka, et al., Partial translation of “Effect of a GLP-1, on B-amyloid
`protein-induced memory impairment and hippocampal neuronal
`death.” The 22". Annual Meeting of the Japanese Neuroscience Soci
`ety, Program Abstract, p. 146, I-P-207 (1999).
`Perry, et al., “Evidence of GLP-1 mediated neuroprotection in an
`animal model of pyridoxine-induced peripheral sensory neuropathy.”
`Experimental Neurology 203:293-301 (2007).
`Perry, et al., “A Novel Neurotrophic Property of Glucagon-Like
`Peptide 1: A Promoter of Nerve Growth Factor-Mediated Differen
`tiation in PC12 Cells.” The Journal of Pharmacology and Experi
`mental Therapeutics 300(3):958-966 (Mar. 2002).
`Suzuki, et al., “A role of endogenous GLP-1 in amnesia and neuronal
`death induced by continuous I.C.V. infusion of B-amyloid protein in
`rat.” Jpn. J. Pharmacol. 82(1):236P. P-468 (2000).
`Doyle et al., Insertion of an N-terminal 6-Aminohexanoic Acid after
`the 7 Amino Acid Position of Glucagon-Like Peptide-1 Produces a
`Long-Acting Hypolycemic Agent, Endocrinology 2001, Vol. 142,
`No. 10, pp. 4462-4468.
`Ahren et al., No Correlation Between Insulin and Islet Amyloid
`Polypeptide After Stimulation with Glucagon-like Peptide-1 in Type
`2 Diabetes, EP Journal of Endocrinology, 1997, vol. 137, pp. 643
`649.
`Koide et al., “Biosynthesis of a Protein Containing A Nonprotein
`Amino Acid By Escherichia coli: L-2-Aminohexanoic Acid At Posi
`tion 21 in Human Epidermal Growth Factor.” Proc. Natl. Acad. Sci.
`USA., vol. 85, pp. 6237-6241, (1988).
`Doyle et al., Insertion of an N-terminal 6-Aminohexanoic Acid after
`the 7 Amino Acid Position of Glucagon-Like Peptide-1 Produces a
`Long-Acting Hypolycemic Agent, Endocrinology 2001, Vol. 142,
`No. 10, pp. 4462-4468.
`Ahren et al., No Correlation Between Insulin and Islet Amyloid
`Polypeptide After Stimulation with Glucagon-like Peptide-1 in Type
`2 Diabetes, EP Journal of Endocrinology, 1997, vol. 137, pp. 643
`649.
`* cited by examiner
`
`MPI EXHIBIT 1028 PAGE 3
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`Sheet 1 of 28
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`MPI EXHIBIT 1028 PAGE 5
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`Aug. 18, 2009
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`(9P:ON
`
`ai03s)
`
`GlDAS)6LP:ON
`
`(8h:ON
`
`MPI EXHIBIT 1028 PAGE 6
`
`MPI EXHIBIT 1028 PAGE 6
`
`MPI EXHIBIT 1028 PAGE 6
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 4 of 28
`
`US 7.576,050 B2
`
`FERENOFBSA (s
`
`1
`0
`
`
`
`5
`0
`
`2
`0
`0
`
`2
`
`50
`
`30
`
`
`
`INSULIN SECRETION in RIN 1048-36 cells
`
`
`
`
`
`MPI EXHIBIT 1028 PAGE 7
`
`MPI EXHIBIT 1028 PAGE 7
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 5 of 28
`
`US 7,576,050 B2
`
`
`
`O
`
`S
`
`25
`20
`15
`Insulin released (egg protein)
`
`30
`
`3S
`
`Figure 3
`
`MPI EXHIBIT 1028 PAGE 8
`
`MPI EXHIBIT 1028 PAGE 8
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 6 of 28
`
`US 7.576,050 B2
`
`ck:
`
`Basal
`
`
`
`GLP.
`GLP.1Gly
`GLP. Ahid
`GLP.l. (Aha'),
`GLP-l (Ahn)
`GLPO3
`GLP.O.S
`
`G.P.D.
`G.L.P.D2
`
`GLP.D2
`G.L.P.A)
`
`0
`
`2S
`2
`S
`0.5
`Entracellular cAMP (pmol/g protein)
`
`3
`
`Figure 4
`
`MPI EXHIBIT 1028 PAGE 9
`
`MPI EXHIBIT 1028 PAGE 9
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 7 of 28
`
`US 7.576,050 B2
`
`
`
`
`
`s
`
`O
`N
`
`O
`
`O
`
`O
`V
`
`C. 3
`(XBLIJ0%) WIBOElu
`
`Figure 5
`
`MPI EXHIBIT 1028 PAGE 10
`
`MPI EXHIBIT 1028 PAGE 10
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 8 of 28
`
`US 7.576,050 B2
`
`3L-1 (43).
`-> (tal (As8S)
`- K.P.
`
`
`
`O-3
`
`lor-7
`
`lo-6
`
`lor-5
`
`Reptide-Concentration (two
`Figure 6
`
`MPI EXHIBIT 1028 PAGE 11
`
`MPI EXHIBIT 1028 PAGE 11
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 9 of 28
`
`US 7,576,050 B2
`
`Acuteeffectiveness of GP1Analogs in Zve feRat
`
`
`
`sEx4WO
`GR-1 (GY8.
`Ex4
`GR1
`
`O
`
`100
`
`2.0D
`3DO
`4OOD
`SO
`6O
`Concentrationofinsulinin Plasma(ught)
`
`700
`
`800
`
`Figure 7
`
`MPI EXHIBIT 1028 PAGE 12
`
`MPI EXHIBIT 1028 PAGE 12
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 10 of 28
`
`US 7,576,050 B2
`
`18000
`
`
`
`16000
`
`14000
`
`12000
`
`1OOOO
`
`8000
`
`4000
`
`2000
`
`15
`
`20
`
`25
`
`O
`
`5
`
`10
`time (hours)
`
`Figure 8
`
`MPI EXHIBIT 1028 PAGE 13
`
`MPI EXHIBIT 1028 PAGE 13
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 11 of 28
`
`US 7.576,050 B2
`
`2SO
`
`
`
`g
`g
`
`4.
`
`D
`
`40
`
`30
`
`s
`g
`
`20
`
`O
`
`O
`
`--Zucker Rats GLP-l Gly
`--Zucker Rats GLP-Ahab
`-e-Wistar Rats GLP. Ahab
`
`A
`
`O
`
`OO
`
`200
`
`3OO
`
`400
`
`SOO
`
`Time (mins)
`
`Figure 9
`
`MPI EXHIBIT 1028 PAGE 14
`
`MPI EXHIBIT 1028 PAGE 14
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 12 of 28
`
`US 7.576,050 B2
`
`GLP-1
`GG
`GLP-1 ET
`Ex- 4
`Ex (1-3O)
`GG1
`
`20
`
`
`
`
`
`OO
`
`
`
`80
`
`60
`
`40
`
`20
`
`O
`
`S.
`
`.
`
`-
`
`s
`s
`
`-20
`0.
`
`10
`
`O
`0.
`Peptide Concentration (M)
`
`0.
`
`Figure 10A
`
`MPI EXHIBIT 1028 PAGE 15
`
`MPI EXHIBIT 1028 PAGE 15
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 13 of 28
`
`US 7,576,050 B2
`
`
`
`0.
`
`IO
`0.
`0.
`Peptide Concentration (M)
`
`0.
`
`Figure 10B
`
`MPI EXHIBIT 1028 PAGE 16
`
`MPI EXHIBIT 1028 PAGE 16
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 14 of 28
`
`US 7.576,050 B2
`
`
`
`20
`
`OO
`
`80
`
`60
`
`40
`
`20
`
`O
`
`&
`.
`
`r
`Pl
`-
`
`S.
`
`-20
`0.
`
`0.
`0.
`0.
`Peptide Concentration (M)
`
`O
`
`Figure 10C
`
`MPI EXHIBIT 1028 PAGE 17
`
`MPI EXHIBIT 1028 PAGE 17
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 15 of 28
`
`US 7,576,050 B2
`
`SO
`
`O
`
`SO
`
`-
`-00
`150
`100
`
`r
`& so
`
`o
`
`-50.
`
`-100
`
`
`
`5
`
`9
`
`p
`
`is
`S.
`
`s
`
`10
`
`0.
`
`10
`
`Sngfind NGF
`
`25
`
`50
`
`to
`
`33
`
`33
`
`330
`
`NGF
`(ngfml)
`
`Exerdin-
`(gfm)
`
`luginal Exendin-4
`
`NGF
`(ng)
`
`End 4-WOT
`(ig/ml)
`
`GLP
`(ug/ml)
`
`B
`
`D
`
`Figure 11
`
`MPI EXHIBIT 1028 PAGE 18
`
`MPI EXHIBIT 1028 PAGE 18
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 16 of 28
`
`US 7,576,050 B2
`
`--
`
`+ :
`
`& 3 & S $ 3 &
`3
`(pabanun on 2Apeal) saunau auto
`Sao Joaquinu u oscalou! usaid
`
`Figure l2
`
`MPI EXHIBIT 1028 PAGE 19
`
`MPI EXHIBIT 1028 PAGE 19
`
`

`

`U.S. Patent
`
`Aug.18, 2009
`
`Sheet 17 of 28
`
`US 7,576,050 B2
`
`~
`
`=
`
`tx
`+&
`Ooz
`aeww
`Blo tv Is
`Zl> os
`e
`A
`= _|4
`ma +t
`O+ x
`6
`Z| ZS
`
`E
`
`G+ o
`Z mz
`ELe
`iwy
`Tele
`m=
`O+ cao
`2 Maia
`_— “1a
`+ &
`a2
`
`eo
`
`OZ
`
`z 3£ao O
`
`o
`
`~*~
`
`9
`
`2
`38
`8
`@
`7
`Stjs> JIQeLA JO uoniodolg
`
`*®
`
`Figure 13
`
`MPI EXHIBIT 1028 PAGE 20
`
`MPI EXHIBIT 1028 PAGE 20
`
`MPI EXHIBIT 1028 PAGE 20
`
`

`

`U.S. Patent
`
`US 7.576,050 B2
`
`C
`
`Figure 14
`
`MPI EXHIBIT 1028 PAGE 21
`
`MPI EXHIBIT 1028 PAGE 21
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 19 Of 28
`
`US 7,576,050 B2
`
`
`
`l-ATO t-aiT0
`
`1- ATO
`
`HON
`
`qußuç
`BION
`
`8
`azueco plot
`
`asue pod
`
`Figure 15
`
`MPI EXHIBIT 1028 PAGE 22
`
`MPI EXHIBIT 1028 PAGE 22
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 20 of 28
`
`US 7,576,050 B2
`
`A
`
`s 100
`E 80
`2
`is
`60
`c.
`40
`CD
`2O
`
`N
`
`O -
`
`12
`10
`
`C
`
`90
`8O
`7O
`8 60
`.9
`5 50
`8 40
`30
`2O
`1O
`O
`
`Cl
`
`SS
`
`B
`
`
`
`5 10
`8
`6
`4
`2
`
`E
`
`>
`
`o
`
`-8
`s
`10
`10
`10
`10
`GLP-1 concentration (M)
`
`s
`10
`
`s
`
`O
`
`O
`
`5
`
`15
`10
`Time (min)
`
`2O
`
`25
`
`3O
`
`Control
`
`Glutamate
`
`Glutamate +
`GLP-1
`
`GLP-1
`
`Glutamate --
`Exerdin-4
`
`Exendin-4
`
`Figure l6
`
`MPI EXHIBIT 1028 PAGE 23
`
`MPI EXHIBIT 1028 PAGE 23
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 21 of 28
`
`US 7,576,050 B2
`
`
`
`Figure l7
`
`MPI EXHIBIT 1028 PAGE 24
`
`MPI EXHIBIT 1028 PAGE 24
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 22 of 28
`
`US 7,576,050 B2
`
`
`
`Ibotenic acid
`
`Vehicle Exendin-4
`
`GLP-1
`
`Vehicle Exendin-4
`
`GLP-1
`
`Figure 18
`
`MPI EXHIBIT 1028 PAGE 25
`
`MPI EXHIBIT 1028 PAGE 25
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 23 of 28
`
`US 7,576,050 B2
`
`50
`
`- d.
`100
`t
`to 8 50
`d
`2
`0
`a 9
`2.
`
`50
`
`5 CD -100
`SS
`
`-150
`
`
`
`5
`
`NGF
`(ng/ml)
`
`O. 1
`
`1.O
`
`Ex4
`(ug/ml)
`
`10 5ng/ml O.1
`NGF
`--
`0.1 g/ml
`Ex4
`
`1 0
`
`3.3
`
`33
`
`330
`
`Ex4-WO
`(ug/ml)
`
`GLP-1
`(pg/ml)
`
`Figure 19
`
`MPI EXHIBIT 1028 PAGE 26
`
`MPI EXHIBIT 1028 PAGE 26
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 24 of 28
`
`US 7,576,050 B2
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`600
`
`
`
`500
`
`400
`
`300
`
`200
`
`100
`
`Control
`(n = 9)
`
`3.3 ug
`GLP-1
`(n = 3)
`
`0.2 ug
`6.6 pug
`GLP-1 exendin4
`(n = 4)
`(n = 3)
`
`2.0 ug
`NGF
`(n = 5)
`
`Figure 20
`
`MPI EXHIBIT 1028 PAGE 27
`
`MPI EXHIBIT 1028 PAGE 27
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 25 of 28
`
`US 7,576,050 B2
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`
`
`-- GLP-1 Gly8
`- GLP-1
`. . .
`. . . Ex (1-3O)
`-0--GLP-1 Gly8 Ex (31-39)
`
`
`
`
`
`20
`
`OO
`
`80
`
`60
`
`40
`
`0.
`
`0.
`
`0.
`
`0.
`
`0.
`
`Peptide Concentration (M)
`
`Figure 21
`
`MPI EXHIBIT 1028 PAGE 28
`
`MPI EXHIBIT 1028 PAGE 28
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 26 of 28
`
`US 7,576,050 B2
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`
`
`2O)
`
`ISO
`
`O
`
`Figure 22A
`
`MPI EXHIBIT 1028 PAGE 29
`
`MPI EXHIBIT 1028 PAGE 29
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 27 of 28
`
`US 7,576,050 B2
`
`-e- GLP-1 Gly8 Ex (31–39)
`-- GLP-1 Gly8 Ex(31-36)
`... O. GLP-1 Gly8 Ex(1-33)
`-0- - GLP-1 Ex (1-39)
`
`
`
`
`
`200
`
`ISO
`
`OO
`
`SO
`
`Figure 22B
`
`MPI EXHIBIT 1028 PAGE 30
`
`MPI EXHIBIT 1028 PAGE 30
`
`

`

`U.S. Patent
`
`Aug. 18, 2009
`
`Sheet 28 of 28
`
`US 7,576,050 B2
`
`O
`
`
`
`da %
`
`“, &
`
`D &
`2. *
`o
`28
`O
`GD
`
`2
`D
`
`t
`I
`A)
`
`S
`
`1.2
`S
`
`a 6 *
`% & 6. 2s O %
`
`Abdominal Fat Lost
`(% initial total body fat)
`
`sh
`O
`
`Nd
`O
`
`Cud
`O
`
`a
`O
`
`Figure 23
`
`MPI EXHIBIT 1028 PAGE 31
`
`MPI EXHIBIT 1028 PAGE 31
`
`

`

`1.
`GLP-1 EXENDIN-4 PEPTIDE ANALOGS AND
`USES THEREOF
`
`CROSS REFERENCE TO RELATED
`APPLICATION
`
`This application claims priority to U.S. Application Ser.
`No. 60/309,076, filed Jul. 31, 2001.
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`This invention relates generally to glucagon-like peptide-1
`(GLP-1), exendin-4 and their peptide analogs. The invention
`also relates to their uses in the treatment of diabetes and
`neurodegenerative conditions.
`2. Background Art
`Pancreatic beta cell dysfunction and the concomitant
`decrease in insulin production can result in diabetes mellitus.
`In type 1 diabetes, the beta cells are completely destroyed by
`the immune system, resulting in an absence of insulin pro
`ducing cells (Physician's Guide to Insulin DependentType II
`Diabetes Mellitus: Diagnosis and Treatment, American Dia
`betes Association, 1988). In type 2 diabetes, the beta cells
`become progressively less efficient as the target tissues
`become resistant to the effects of insulin on glucose uptake.
`Thus, beta cells are absent in people with type 1 diabetes and
`are functionally impaired in people with type 2 diabetes.
`Beta cell dysfunction currently is treated in several differ
`ent ways. In the treatment of type 1 diabetes or the late stages
`of type 2 diabetes, insulin replacement therapy is necessary.
`Insulin therapy, although life-saving, does not restore nor
`moglycemia, even when continuous infusions or multiple
`injections are used in complex regimes. For example, post
`prandial levels of glucose continue to be excessively high in
`individuals on insulin replacement therapy. Thus, insulin
`therapy must be delivered by multiple daily injections or
`continuous infusion and the effects must be carefully moni
`tored to avoid hyperglycemia, hypoglycemia, metabolic aci
`dosis, and ketosis.
`People with type 2 diabetes are generally treated with
`drugs that stimulate insulin production and secretion from the
`beta cells and/or improve insulin sensitivity. A major disad
`Vantage of these drugs, however, is that insulin production
`and secretion is promoted regardless of the level of blood
`glucose. Thus, food intake must be balanced against the pro
`motion of insulin production and secretion to avoid hypogly
`cemia or hyperglycemia. In recent years several new agents
`have become available to treat type 2 diabetes. These include
`metformin, rosiglitaZone, pioglitaZone, and acarbose (see
`Bressler and Johnson, 1997). However, the drop in hemoglo
`bin Alc obtained by these newer agents is less than adequate
`(Ghazzi et al., 1997), suggesting that they will not improve
`the long-term control of diabetes mellitus.
`Glucagon-like peptide-1 (GLP-1), a hormone normally
`secreted by neuroendocrine cells of the gut in response to
`food, has been suggested as a new treatment for type 2 dia
`betes (Gutniak et al., 1992: Nauck et al., J. Clin. Invest.
`1993). It increases insulin release by the beta cells even in
`Subjects with long-standing type 2 diabetes (Nauck et al.,
`Diabetologia, 1993). GLP-1 treatment has an advantage over
`insulin therapy because GLP-1 stimulates endogenous insu
`lin secretion, which turns off when blood glucose levels drop
`(Naucket al., Diabetologia, 1993: Elahi et al., 1994). GLP-1
`promotes euglycemia by increasing insulin release and Syn
`thesis, inhibiting glucagon release, and decreasing gastric
`emptying (Nauck et al., Diabetologia, 1993: Elahi et al.,
`
`10
`
`15
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`US 7,576,050 B2
`
`2
`1994; Wills et al., 1996; Nathan et al., 1992; De Ore et al.,
`1997). GLP-1 also induces an increase in hexokinase mes
`senger RNA levels (Wang et al., Endocrinology 1995; Wang
`et al., 1996). GLP-1 is known to have a potent insulin-secret
`ing effect on beta cells (Thorens and Waeber, 1993: Orskov,
`1992) and to increase insulin biosynthesis and proinsulin
`gene expression when added to insulin-secreting cell lines for
`24 hours (Drucker et al., 1987: Fehmann and Habener, 1992).
`In studies using RIN 1046-38 cells, twenty-four hour treat
`ment with GLP-1 increased glucose responsiveness even
`after the GLP-1 had been removed for an hour and after
`several washings of the cells (Montrose-Rafizadeh et al.,
`1994). Thus, GLP-1 is an insulinotropic agent known to have
`biological effects on Bcells even after it has been metabolized
`from the system. GLP-1 is a product of posttranslational
`modification of proglucagon. The sequences of GLP-1 and its
`active fragments GLP-1 (7-37) and GLP-1 (7-36) amide are
`known in the art (Fehmann et al., 1995). Although GLP-1 has
`been proposed as a therapeutic agent in the treatment of
`diabetes, it has a short biological half-life (De Ore et al.,
`1997), even when given by a bolus subcutaneously (Ritzel et
`al., 1995). GLP-1 degradation (and GLP-1 (7-36) amide), in
`part, is due to the enzyme dipeptidyl peptidase (DPP1V),
`which cleaves the polypeptide between amino acids 8 and 9
`(alanine and glutamic acid).
`Exendin-4 is a polypeptide produced in the salivary glands
`of the Gila Monster lizard (Goke et al., 1993). The amino acid
`sequence for exendin-4 is known in the art (Fehmann et al.
`1995). Although it is the product of a uniquely non-mamma
`lian gene and appears to b