(12)
`
`United States Patent
`Bachovchin et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,157.429 B1
`*Jan. 2, 2007
`
`US007 157429B1
`
`(54) METHOD OF REGULATING GLUCOSE
`METABOLISM, AND REAGENTS RELATED
`THERETO
`
`(*) Notice:
`
`(75) Inventors: William A. Bachovchin, Melrose, MA
`(US); Andrew G. Plaut, Lexington,
`MA (US); Daniel Drucker, Toronto
`(CA)
`(73) Assignee: Trustees of Tufts College, Medford,
`MA (US)
`0
`-
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is Subject to a terminal dis-
`claimer.
`(21) Appl. No.: 09/628,225
`9
`Jul. 28, 2000
`
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`WO 89.03.223
`93,08259
`95/15307
`WO 96.14857
`WO 97/40832
`WO 98/19998
`98.25644
`
`4f1989
`* 4f1993
`* 6/1995
`5, 1996
`11, 1997
`5, 1998
`* 6/1998
`
`OTHER PUBLICATIONS
`Deacon et al. Dipeptidyl Peptidase IV Inhibition . . . Diabetes. vol.
`47, pp. 764-769, May 1998.*
`Bell et al., 1983. “Exon duplication and divergence in the human
`preproglucagon gene”. Nature 304(5924):368-71.
`Bell et al., 1983, "Hamster preproglucagon contains the sequence of
`glucagon and two related peptides', Nature 302(5910):716-8.
`Conlon, 1988, “Proglucagon-derived peptides: nomenclature,
`biosynthetic relationships and physiological roles'. Diabetologia
`31(8):563-6.
`
`(Continued)
`Primary Examiner Jeffrey Edwin Russel
`E. togey Agent, or Firm—Dana M. Gordon; Foley
`Oag
`(7)
`
`ABSTRACT
`
`(22) Filed:
`(51) Int. Cl.
`(2006.01)
`A6 IK 38/04
`(52) U.S. Cl. .............................. 514/18: 514/2: 514/19;
`5145,514/423,514,626
`(58) Field of Classification Search .................... 514/2,
`514/18, 19, 119, 423, 626; 530/330,331,
`530/300
`See application file for complete search history.
`References Cited
`U.S. PATENT DOCUMENTS
`
`(56)
`
`The present invention provides methods and compositions
`for modification and regulation of glucose and lipid metabo
`lism, generally to reduce insulin resistance, hyperglycemia,
`hyperinsulinemia, obesity, hyperlipidemia, hyperlipopro
`teinemia (Such as chylomicrons, VLDL and LDL), and to
`regulate body fat and more generally lipid stores, and, more
`generally, for the improvement of metabolism disorders,
`especially those associated with diabetes, obesity and/or
`6, 1985 Sisto et al. ................. 530/314
`4,522,752 A
`atherosclerosis. The compositions of the present invention
`5,061,811 A ck 10, 1991 Pinori et al................. 549,274
`include dipeptidylpeptidase inhibitors, which are able to
`...
`i-S Eise al. .
`- - - -
`inhibit the proteolysis of GLP-1 and accordingly increase
`5,953.30. A
`9, 1999 Drucker.
`. 514/12
`6,011,155 A
`1/2000 villhauer. 53, the plasma half-life of that hormone. The subject inhibitors
`6,803,357 B1 * 10/2004 Bachovchin et al. .......... 5142
`may be peptidyl, peptidomimetic (e.g. boronyl peptidomi
`metics), or non-peptidyl nitrogen containing heterocycles.
`FOREIGN PATENT DOCUMENTS
`
`DE
`
`196 16486
`
`* 10/1997
`
`21 Claims, 5 Drawing Sheets
`
`14
`
`12-
`
`-- +/-- NPBP
`-o- +/-- N SALINE
`
`s
`8
`
`10
`
`C
`D g 8
`
`f
`
`6
`
`4-
`O
`
`
`
`i - - - - -
`.
`.
`.
`.
`.
`.
`.
`.
`O 20 30 40 50 60 70 8O 90 100 liO 20 30 40 50
`TIME (MINUTES)
`
`Merck Exhibit 2182, Page 1
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`US 7,157,429 B1
`Page 2
`
`OTHER PUBLICATIONS
`Coruzzi et al., 1989, “Gastric antisecretory activity of telenzepine,
`a new M1-selective muscarinic antagonist: comparison with
`pirenzepine”, Arch Int Pharmacodyn Ther 302:232-41.
`Deacon et al., 1995, “Both subcutaneously and intravenously
`administered glucagon-like peptide 1 are rapidly degraded from the
`NII2-terminus in type II diabetic patients and in healthy subjects',
`Diabetes 44(9): 1126-31.
`Dupre, 1991, “Influences of the gut on the endocrine pacreas' The
`Endocrine Pancreas (Raven Press, new York) pp. 253-281.
`Ebert et al., 1987, “Gastrointestinal peptides and insulin secretion'.
`Diabetes Met. Rev. 3:1-26.
`Gutniak et al., 1992, “Antidiabetogenic effect of glucagon-like
`peptide-1 (7-36)amide in normal subjects and patients with diabetes
`mellitus”, N Engl J Med 326(20): 1316-22.
`Habener et al., 1991, “BioSyntesies of glucagon” The Endocrine
`Pancreas (Raven Press. New York) pp. 53-71.
`Holst et al., 1987. “Truncated glucagon-like peptide 1, an insulin
`releasing hormone from the distal gut', FEBS Lett. 211(2): 169-74.
`Kawashima et al., 1990, “Pharmacological differentiation of
`presynaptic M1 muscarinic receptors modulating acetylcholine
`release from postsynaptic muscarinic recptors in guinea-pig ileum'.
`Gen Pharmacol 21(1):17-21.
`Kinder et al., 1985. “Acylamino boronic acids and difluoroborane
`analogues of amino acids: potent inhibitors of chymotrypsin and
`elastase”, J Med Chem 28(12): 1917-25.
`Kreymann et al., 1987, “Glucagon-like peptide-1 7-36: a physi
`ological incretin in man'. Lancet 2(8571): 1300-4.
`Kubiak et al., 1994, "Metabolism of mouse growth hormone
`releasing factor, mGRF(1-42)OH, and selected analogs from the
`bovine GRF series in mouse and bovine plasma in vitro', Pept Res
`7(3): 153-61.
`Lambrecht et al., 1989, "Pharmacology of hexahydro-difenidol,
`hexahydro-sila-difenidol and related selective muscarinic antago
`nists'. Trends Pharmacol Sci 10(Suppl):60.
`Lund et al., 1982, "Pancreatic preproglucagon cDNA contains two
`glucagon-related coding sequences arranged in tandem'. Proc Natl
`Acad Sci U S A79(2):345-9.
`Matteson et al., 1984, “Synthesis and properties of pinanediol
`O-amino boronic acids'. Organometallics 3:1284.
`Mojsov et al., 1986, "Preproglucagon gene expression in pancreas
`and intestine diversities at the level of post-translational process
`ing”, J Biol Chem 261 (25): 11880-9.
`Mojsov et al., 1987. “Insulinotropin: glucagon-like peptide 1 (7-37)
`co-encoded in the glucagon gene is a potent stimulator of insulin
`release in the prefused rat pancreas'. J. Clin Invest 79(2):616-9.
`Mojsov, 1992, "Structural requirements for biological activity of
`glucagon-like peptide-1”, Int J Pept Protein Res 40(3–4):333-43.
`Orskov et al., 1987. “Pancreatic and intestinal processing of
`proglucagon in man', Diabetologia 30(11):874-81.
`
`Patzelt et al., 1979. “Identification and processing of proglucagon in
`pancreatic islets'. Nature 282(5736):260-6.
`Pospisilik, John A. et al. Metabolism of Glacagon by Dipeptidyl
`Peptidase IV (CD26). Regulatory Peptides 96, 133-141 (2001).
`Radhakrishna et al., 1979, "New method for direct conversion of
`amides to amines', J Org Chem 44:1746.
`Schmidt et al., 1985, "Glucagon-like peptide-1 but not glucagon
`like peptide-2 stimulates insulin release from isolated rat pancreatic
`islets”. Diabetologia 28(9):704-7.
`Shue et al., 1987. “Amide bond surrogates: a general synthetic route
`to trans carbon-carbon double bond isosteres'. Tetrahedron Letters
`28:3225.
`Stanley et al., 1989, “Repeated hypothalamic stimulation with
`neuropeptide Yincreases daily carbohydrate and fat intake and body
`weight gain in female rats'. Physiol Behav 46(2):173-7.
`Weir et al., 1989, “Glucagonlike peptide I (7-37) actions on endo
`crine pancreas'. Diabetes 38(3):338-42.
`Wilding et al., 1992, “Increased neuropeptide Y content in indi
`vidual hypothalamic nuclei, but not neuropeptide Y mRNA, in
`diet-induced obesity in rats'. J. Endocrinol 132(2):299-304.
`Balkan et al. Improved insulin Secretion and oral glucose tolerance
`after in vivo inhibition of DPP-IV in obese Zucker rats. Diabetologia
`Suppl. 40, A131 Abstract (1997).
`Coutts et al. Structure-Activity Relationships of Boronic Acid
`Inhibitors of Dipeptidyl Peptidase IV. 1. Variation of the P2 Position
`of Xaa-boroPro Dipeptides. J. Med. Chem. 39, 2087-2094 (1996).
`Deacon et al. Degradation of Glucagon-Like Peptide-1 by Human
`Plasma in Vitro Yields an N-Terminally Truncated Peptide that is a
`Major Endogenous Metabolite in Vivo. J. Clin. Endocrin. 83,
`952-957 (1995).
`Holst, J. J. & Deacon, C. F. Inhibition of the Activity of Dipeptidyl
`Peptidase IV as a Treatment for Type 2 Diabetes. Diabetes 47.
`1663-1670 (1998).
`Kieffer et al. Degradation of Glucose-Dependent Insulinotropic
`Polypeptide and Truncated Glucagon-Like Peptide 1 in Vitro and in
`Vivo by Dipeptidyl Peptidase IV. Endocrin. 136,3585-3596 (1995).
`Mentlein et al. Dipeptidyl-peptidase IV hydrolyses gastric inhibi
`tory polypeptide, glucagons-like peptide-1 (7-36)amide, peptide
`histidine methionine and is responsible for their degradation in
`human serum. Eur: J. Biochem. 214,829-835 (1993).
`Mentlein et al. Proteolytic processing of neuropeptide Y and peptide
`YY by dipeptidyl peptidase IV. Regulatory Peptides 49, 133-144
`(Dec. 10, 1993).
`Pederson et al. Improved Glucose Tolerance in Zucker Fatty Rats by
`Oral Administration of the Dipeptidyl Peptidase IV Inhibitor
`Isoleucine Thiazolidide. Diabetes 47, 1253-1258 (Aug. 1998).
`* cited by examiner
`
`Merck Exhibit 2182, Page 2
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`U.S. Patent
`
`Jan. 2, 2007
`
`Sheet 1 of 5
`
`US 7,157,429 B1
`
`D
`Br-(CH-CH-B
`M
`N O-k
`
`C
`
`O-K
`7
`
`B.Ch., H-B,
`
`(2)
`
`N
`/ N.
`Si
`Si
`AM / N
`
`C
`
`O-k
`
`M/
`Si
`N
`
`(3)
`
`(4)
`
`O-K
`O-K
`
`B,
`
`C - * *
`
`H
`
`N
`
`O-K
`O-k
`
`(4)
`
`H
`N
`
`0
`Q
`H
`O-K
`B
`HC Boc-Ala-Boc--&-N B
`Nn.
`Y -
`O \
`CH
`U O
`
`Fig. 1
`
`Merck Exhibit 2182, Page 3
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`U.S. Patent
`
`Jan. 2, 2007
`
`Sheet 2 of 5
`
`US 7,157.429 B1
`
`4
`
`2-
`
`s
`E
`
`-- +/- W PBP
`-o- +/-- M. SALINE
`
`O
`O
`P
`CD
`
`& s
`
`Ol
`
`4- - - - - - - - - - - - - - -
`O
`O 20 30 40 50 60 70 80 90 OO O 20 30 40 50
`TIME (MINUTES)
`
`Fig. 2
`
`4.
`
`
`
`
`
`
`
`s 12
`8
`9 10
`2
`(D
`O S 8
`C
`
`6
`
`4-
`
`-- PBP
`-o- SALNE
`
`I.
`
`- 20 30 40 so so 7o so go too lio 20 30
`TIME (MINUTES)
`Fig. 3
`
`Merck Exhibit 2182, Page 4
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`U.S. Patent
`
`Jan. 2, 2007
`
`Sheet 3 of 5
`
`US 7,157.429 B1
`
`-- PBP
`-O- SALNE
`
`2
`
`8O ---
`
`6
`
`O
`
`50
`
`- I
`OO
`
`--
`150
`
`TIME (MINUTES)
`
`Fig. 4
`
`Merck Exhibit 2182, Page 5
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`U.S. Patent
`
`Jan. 2, 2007
`
`Sheet 4 of 5
`
`US 7,157.429 B1
`
`4
`s t
`s, 10.
`< 8
`y u) CCO 8.
`a Q 6
`
`CD
`
`4
`O
`
`
`
`is 12
`<3
`10
`58
`
`CD
`
`6
`O
`
`
`
`s S. 9
`S 7
`5 5
`3 -
`O
`
`-- +/- MPBP
`-o- +/-- M. SALNE
`
`-T- -
`-
`OO
`50
`50
`TIME (MINUTES)
`
`Fig. 5A-1
`
`-o- +/- MPBP
`-o- +/-- WSALINE
`
`OO
`50
`TIME MINUTES)
`
`Fig. 5A-2
`
`50
`
`-o- +/- MPBP
`-o- +/-- M. SALNE
`
`- - - - - - ------
`50
`OO
`50
`TIME (MINUTES)
`
`Fig. 5A-3
`
`Merck Exhibit 2182, Page 6
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`U.S. Patent
`
`Jan. 2, 2007
`
`Sheet 5 of 5
`
`US 7,157.429 B1
`
`
`
`Ol2345
`O234.5
`
`
`
`N.
`
`SALNE PBP
`n=5
`n=5
`
`Fig. 5B-1
`
`
`
`SAINE PBP
`n=4
`n=4
`Fig. 5B-2
`
`SALNE
`n=5
`
`n=5
`
`Fig. 5B-3
`
`Merck Exhibit 2182, Page 7
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`US 7,157,429 B1
`
`1.
`METHOD OF REGULATING GLUCOSE
`METABOLISM, AND REAGENTS RELATED
`THERETO
`
`FUNDING
`
`Work described herein was supported by funding from the
`National Institutes of Health. The United States Government
`has certain rights in the invention.
`BACKGROUND OF THE INVENTION
`
`2
`entry of glucose into various "peripheral" tissues and (2) an
`increased liberation of glucose into the circulation from the
`liver. There is therefore an extracellular glucose excess and
`an intracellular glucose deficiency. There is also a decrease
`in the entry of amino acids into muscle and an increase in
`lipolysis. Hyperlipoproteinemia is also a complication of
`diabetes. The cumulative effect of these diabetes-associated
`abnormalities is severe blood vessel and nerve damage.
`Endocrine secretions of pancreatic islets are regulated by
`complex control mechanisms driven not only by blood
`borne metabolites such as glucose, amino acids, and cat
`echolamines, but also by local paracrine influences. Indeed,
`pancreatic C- and f-cells are critically dependent on hor
`monal signals generating cyclic AMP (cAMP) as a syner
`gistic messenger for nutrient-induced hormone release. The
`major pancreatic islet hormones, glucagon, insulin and
`somatostatin, interact with specific pancreatic cell types to
`modulate the secretory response. Although insulin Secretion
`is predominantly controlled by blood glucose levels. Soma
`tostatin inhibits glucose-mediated insulin secretion.
`The human hormone glucagon is a polypeptide hormone
`produced in pancreatic A-cells. The hormone belongs to a
`multi-gene family of structurally related peptides that
`include secretin, gastric inhibitory peptide, vasoactive intes
`tinal peptide and glicentin. These peptides variously regulate
`carbohydrate metabolism, gastrointestinal motility and
`secretory processing. However, the principal recognized
`actions of pancreatic glucagon are to promote hepatic gly
`cogenolysis and glyconeogenesis, resulting in an elevation
`of blood sugar levels. In this regard, the actions of glucagon
`are counter regulatory to those of insulin and may contribute
`to the hyperglycemia that accompanies Diabetes mellitus
`(Lund et al. (1982) PNAS, 79:345–349).
`Preproglucagon, the Zymogen form of glucagon, is trans
`lated from a 360 base pair gene and is processed to form
`proglucagon (Lund, et al. Supra). Patzelt, et al. (Nature,
`282:260–266 (1979)) demonstrated that proglucagon is fur
`ther processed into glucagon and a second peptide. Later
`experiments demonstrated that proglucagon is cleaved car
`boxyl to Lys-Arg or Arg-Arg residues (Lund et al., Supra;
`and Bell et al. (1983) Nature 302:716–718). Bell et al. also
`discovered that proglucagon contained three discrete and
`highly homologous peptide regions which were designated
`glucagon, glucagon-like peptide 1 (GLP-1), and glucagon
`like peptide 2 (GLP-2). GLP-1 has attracted increasing
`attention as a humoral stimulus of insulin secretion. In
`humans, this 29-amino acid peptide, cleaved from proglu
`cagon by cells of the intestinal mucosa, is released into the
`circulation after nutrient intake (Holst et al. (1987) FEBS
`Lett 211:169: Orskov et al. (1987) Diabetologia 30:874;
`Conlon J (1988) Diabetologia 31:563).
`GLP-1 has been found to be a glucose-dependent insuli
`notropic agent (Gutniak et al. (1992) N. Engl. J. Bled.
`326:1316–1322). GLP-1 is now known to stimulate insulin
`secretion (insulinotropic action) causing glucose uptake by
`cells which decreases serum glucose levels (see, e.g.,
`Mojsov, S., Int. J. Peptide Protein Research, 40:333–343
`(1992)). For instance, it has been shown to be a potent
`insulin secretagogue in experimental models and when
`infused into humans (Gutniak et al., Supra; Mojsov et al.
`(1988) J Clin Invest 79:616; Schmidt et al. (1985) Diabe
`tologia 28:704; and Kreymann et al. (1987) Lancet 2:1300).
`Thus, GLP-1 is a candidate for the role of an “incretin”.
`having augmentary effects on glucose-mediated insulin
`release.
`It is also noted that numerous GLP-1 analogs have been
`demonstrated which demonstrate insulinotropic action are
`
`Diabetes adversely affects the way the body uses Sugars
`and starches which, during digestion, are converted into
`glucose. Insulin, a hormone produced by the pancreas,
`makes the glucose available to the body’s cells for energy.
`In muscle, adipose (fat) and connective tissues, insulin
`facilitates the entry of glucose into the cells by an action on
`the cell membranes. The ingested glucose is normally con
`verted in the liver to CO and H2O (50%); to glycogen (5%);
`and to fat (30–40%), the latter being stored in fat depots.
`Fatty acids from the adipose tissues are circulated, returned
`to the liver for re-synthesis of triacylglycerol and metabo
`lized to ketone bodies for utilization by the tissues. The fatty
`acids are also metabolized by other organs. Fat formation is
`a major pathway for carbohydrate utilization.
`The net effect of insulin is to promote the storage and use
`of carbohydrates, protein and fat. Insulin deficiency is a
`common and serious pathologic condition in man. In insu
`lin-dependent (IDDM or Type I) diabetes the pancreas
`produces little or no insulin, and insulin must be injected
`daily for the survival of the diabetic. In noninsulin-depen
`dent (NIDDM or Type II) diabetes the pancreas retains the
`ability to produce insulin and in fact may produce higher
`than normal amounts of insulin, but the amount of insulin is
`relatively insufficient, or less than fully effective, due to
`cellular resistance to insulin.
`Diabetes mellitus (DM) is a major chronic illness found in
`humans with many consequences. Some complications aris
`ing from long-standing diabetes are blindness, kidney fail
`ure, and limb amputations. Insulin-dependent diabetes mel
`litus (IDDM) accounts for 10 to 15% of all cases of diabetes
`mellitus. The action of IDDM is to cause hyperglycemia
`(elevated blood glucose concentration) and a tendency
`towards diabetic ketoacidosis (DKA). Currently treatment
`requires chronic administration of insulin. Non-insulin
`dependent diabetes mellitus (NIDDM) is marked by hyper
`glycemia that is not linked with DKA. Sporadic or persistent
`incidence of hyperglycemia can be controlled by adminis
`tering insulin. Uncontrolled hyperglycemia can damage the
`cells of the pancreas which produce insulin (the f-islet cells)
`and in the long term create greater insulin deficiencies.
`Currently, oral sulfonylureas and insulin are the only two
`therapeutic agents available in the United States, for treat
`ment of Diabetes mellitus. Both agents have the potential for
`producing hypoglycemia as a side effect, reducing the blood
`glucose concentration to dangerous levels. There is no
`generally applicable and consistently effective means of
`maintaining an essentially normal fluctuation in glucose
`levels in DM. The resultant treatment attempts to minimize
`the risks of hypoglycemia while keeping the glucose levels
`below a target value. The drug regimen is combined with
`control of dietary intake of carbohydrates to keep glucose
`levels in control.
`In either form of diabetes there are widespread abnor
`malities. In most NIDDM subjects, the fundamental defects
`to which the abnormalities can be traced are (1) a reduced
`
`10
`
`15
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Merck Exhibit 2182, Page 8
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`3
`known in the art. These variants and analogs include, for
`example, GLP-1 (7-36), Gln-GLP-1 (7-37), D-Gln-GLP-1
`(7-37), acetyl-Lys-GLP-1 (7-37), Thr-Lyss-GLP-1 (7-
`37), and Lyss-GLP-1 (7-37). Derivatives of GLP-1 include,
`for example, acid addition salts, carboxylate salts, lower
`alkyl esters, and amides (see, e.g., WO91/11457).
`
`OBJECTS OF THE INVENTION
`
`It is one object of this invention to provide improved
`methods for reducing in animal Subjects (including humans)
`in need of Such treatment at least one of insulin resistance,
`hyperinsulinemia, and hyperglycemia and abating Type II
`diabetes. Another object is to provide improved methods for
`reducing at least one of body fat stores, hyperlipidemia,
`hyperlipoproteinemia, and for abating atherosclerosis. It is
`another object of this invention to provide methods for
`interfering with glucose and/or lipid metabolism in a manner
`beneficial to the host.
`It is yet another object of this invention to provide
`improved methods for the long-term reduction and abate
`ment of at least one of the foregoing disorders based on a
`therapeutic regimen administered over the short-term.
`It is still another object of the present invention to provide
`a method for regulating, and altering on a long term basis,
`the glucose and lipogenic responses of Vertebrate animals,
`including humans.
`In particular, it is an object of the invention to provide
`methods for producing long lasting beneficial changes in one
`or more of the following: the sensitivity of the cellular
`response of a species to insulin (reduction of insulin resis
`tance), blood insulin levels, hyperinsulinemia, blood glucose
`levels, the amount of body fat stores, blood lipoprotein
`levels, and thus to provide effective treatments for diabetes,
`obesity and/or atherosclerosis.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a diagrammatic representation of the synthesis
`of a boro proline compound.
`FIG. 2 is a glucose tolerance curve which shows that a
`single injection of PBP-1 improves glucose levels in blood.
`The glucose concentration is measured before and at
`30-minute intervals after the test dose of glucose. This figure
`demonstrates that a single injection of PBP-1 potentiates the
`response to a sub-therapeutic dose of GLP-1.
`FIG. 3 shows that a single injection of PBP-2 improves
`glucose levels in blood.
`FIG. 4 shows that treatment with PBP-3 under “chronic’
`conditions also results in lowering of the blood Sugar levels.
`FIGS. 5A and 5B compare the ability of Pro-boro-pro to
`lower plasma glucose levels in GLP-1 receptor-f- transgenic
`1CC.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`Glucose-induced insulin secretion is modulated by a
`number of hormones and neurotransmitters. In particular,
`two gut hormones, glucagon-like peptide-1 (GLP-1) and
`gastric inhibitory peptide (GIP) are insulinotropic agents,
`e.g., being agents which can stimulate, or cause the stimu
`lation of the synthesis or expression of the hormone insulin,
`are thus called gluco-incretins (Dupre, in The Endocrine
`Pancreas, E. Samois Ed. (Raven Press, New York, (1991),
`253–281); and Ebert et al. (1987) Diabetes Metab. Rev. p3).
`Glucagon-like peptide-1 is a glucoincretin both in man and
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`other mammals (Dupre et al. Supra, and Kreymann et al.
`(1987) Lancet 2:300). It is part of the preproglucagon
`molecule (Bell et al. (1983) Nature 304:368) which is
`proteolytically processed in intestinal L cells to GLP-1 (1-
`37) and GLP-1 (7-36) amide or GLP-1 (7-37) (Mojsov et al.
`(1986) J. Biol. Chem. 261:11880; and Habener et al.: The
`Endocrine Pancreas, E. Samois Ed. (Raven Press, New York
`(1991), 53-71). Only the truncated forms of GLP-1 are
`biologically active and both have identical effects on insulin
`secretion in beta cells (Mojsov et al. (1987) J. Clin. Invest
`79:616; and Weir et al. (1989) Diabetes 38:338). They are
`the most potent gluco-incretins so far described and are
`active at concentrations as low as one to ten picomolar.
`The metabolic fate of exogenous GLP-1 has been studied
`in nondiabetic and type II diabetic subjects. Subcutaneous
`and intravenous GLP-1 are both rapidly degraded in a
`time-dependent manner, for instance, having a half-life in
`diabetic patients of substantially less than 30 minutes. See,
`for example, Deacon et al. (1995) Diabetes 44:1126–1131.
`i. Overview of the Invention
`The present invention provides methods and composi
`tions for modification and regulation of glucose and lipid
`metabolism, generally to reduce insulin resistance, hyperg
`lycemia, hyperinsulinemia, obesity, hyperlipidemia, hyper
`lipoprotein-emia (Such as chylomicrons, VLDL and LDL),
`and to regulate body fat and more generally lipid stores, and,
`more generally, for the improvement of metabolism disor
`ders, especially those associated with diabetes, obesity and/
`or atherosclerosis. As described in greater detail below, the
`Subject method includes the administration, to an animal, of
`a composition including one or more dipeptidylpeptidase
`inhibitors, especially inhibitors of the dipeptidylpeptidase
`IV (DPIV) enzyme or other enzyme of similar specificity,
`which are able to inhibit the proteolysis of GLP-1 and
`accordingly increase the plasma half-life of that hormone.
`Preferably, the compounds utilized in the subject method
`will produce an EC50 for the desired biological effect of at
`least one, two, three and even four orders of magnitude less
`than the EC50 for that compound as an immunosuppressant.
`Indeed, a salient feature of Such compounds as the peptidyl
`boronates is that the inhibitors can produce, for example, an
`EC50 for inhibition of glucose tolerance in the nanomolar or
`less range, whereas the compounds have EC50s for immu
`nosuppression in the LM or greater range. Thus, a favorable
`therapeutic index can be realized with respect to the
`unwanted sideeffect of immunosuppression.
`While not wishing to bound by any particular theory, it is
`observed that compounds which inhibit DPIV are, correla
`tively, able to improve glucose tolerance, though not nec
`essarily through mechanisms involving DPIV inhibition per
`se. Indeed, the results described in Example 6 (and FIG. 5)
`demonstrated an effect in mice lacking a GLP-1 receptor
`Suggest that the Subject method may not include a mecha
`nism of action directly implicating GLP-1 itself, though it
`has not been ruled out that GLP-1 may have other receptors.
`However, in light of the correlation with DPIV inhibition, in
`preferred embodiments, the Subject method utilizes an agent
`with a Ki for DPIV inhibition of 1.0 nm or less, more
`preferably of 0.1 nm or less, and even more preferably of
`0.01 nM or less. Indeed, inhibitors with Ki values in the
`picomolar and even femtamolar range are contemplated.
`Thus, while the active agents are described herein, for
`convenience, as “DPIV inhibitors', it will be understood that
`Such nomenclature is not intending to limit the Subject
`invention to a particular mechanism of action.
`
`Merck Exhibit 2182, Page 9
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

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`US 7,157,429 B1
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`For instance, in certain embodiments the method involves
`administration of a DPIV inhibitor, preferably at a prede
`termined time(s) during a 24-hour period, in an amount
`effective to improve one or more aberrant indices associated
`with glucose metabolism disorders (e.g., glucose intoler
`ance, insulin resistance, hyperglycemia, hyperinsulinemia
`and Type II diabetes).
`In other embodiments, the method involves administra
`tion of a DPIV inhibitor in an amount effective to improve
`aberrant indices associated with obesity. Fat cells release the
`hormone leptin, which travels in the bloodstream to the brain
`and, through leptin receptors there, Stimulates production of
`GLP-1. GLP-1, in turn, produces the sensation of being full.
`The leading theory is that the fat cells of most obese people
`probably produce enough leptin, but leptin may not be able
`to properly engage the leptin receptors in the brain, and so
`does not stimulate production of GLP-1. There is accord
`ingly a greater deal of research towards utilizing prepara
`tions of GLP-1 as an appetite Suppressant. The Subject
`method provides a means for increasing the half-life of both
`endogenous and ectopically added GLP-1 in the treatment of
`disorders associated with obesity.
`In a more general sense, the present invention provides
`methods and compositions for altering the pharmokinetics of
`a variety of different polypeptide hormones by inhibiting the
`proteolysis of one or more peptide hormones by DPIV or
`some other proteolytic activity. Post-secretory metabolism is
`an important element in the overall homeostasis of regula
`tory peptides, and the other enzymes involves in these
`processes may be Suitable targets for pharmacological inter
`vention by the subject method.
`For example, the Subject method can be used to increase
`the half-life of other proglucagon-derived peptides, such as
`glicentin (corresponding to PG 1-69), oxyntomodulin (PG
`33-69), glicentin-related pancreatic polypeptide (GRPP, PG
`1-30), intervening peptide-2 (IP-2, PG 111-122amide), and
`glucagon-like peptide-2 (GLP-2, PG 126-158).
`GLP-2, for example, has been identified as a factor
`responsible for inducing proliferation of intestinal epithe
`lium. See, for example, Drucker et al. (1996) PNAS 93:7911.
`The Subject method can be used as part of a regimen for
`treating injury, inflammation or resection of intestinal tissue,
`e.g., where enhanced growth and repair of the intestinal
`mucosal epithelial is desired.
`DPIV has also been implicated in the metabolism and
`inactivation of growth hormone-releasing factor (GHRF).
`GHRF is a member of the family of homologous peptides
`that includes glucagon, secretin, vasoactive intestinal pep
`tide (VIP), peptide histidine isoleucine (PHI), pituitary ade
`nylate cyclase activating peptide (PACAP), gastric inhibi
`tory peptide (GIP) and helodermin. Kubiak et al. (1994)
`Peptide Res 7:153. GHRF is secreted by the hypothalamus,
`and stimulates the release of growth hormone (GH) from the
`anterior pituitary. Thus, the subject method can be used to
`improve clinical therapy for certain growth hormone defi
`cient children, and in clinical therapy of adults to improve
`nutrition and to alter body composition (muscle VS. fat). The
`Subject method can also be used in Veterinary practice, for
`example, to develop higher yield milk production and higher
`yield, leaner livestock.
`Likewise, the DPIV inhibitors of the subject invention can
`be used to alter the plasma half-life of secretin, VIP, PHI,
`PACAP, GIP and/or helodermin. Additionally, the subject
`method can be used to alter the pharmacokinetics of Peptide
`YY and neuropeptide Y, both members of the pancreatic
`
`6
`polypeptide family, as DPIV has been implicated in the
`processing of those peptides in a manner which alters
`receptor selectivity.
`Another aspect of the present invention relates to phar
`maceutical compositions of dipeptidylpeptidase inhibitors,
`particularly DPIV inhibitors, and their uses in treating and/or
`preventing disorders which can be improved by altering the
`homeostasis of peptide hormones. In a preferred embodi
`ment, the inhibitors have hypoglycemic and antidiabetic
`activities, and can be used in the treatment of disorders
`marked by abberrant glucose metabolism (including Stor
`age). In particular embodiments, the compositions of the
`Subject methods are useful as insulinotropic agents, or to
`potentiate the insulinotropic effects of Such molecules as
`GLP-1. In this regard, the present method can be useful for
`the treatment and/or prophylaxis of a variety of disorders,
`including one or more of hyperlipemia, hyperglycemia,
`obesity, glucose tolerance insufficiency, insulin resistance
`and diabetic complications.
`In general, the inhibitors of the subject method will be
`small molecules, e.g., with molecular weights less than 7500
`amu, preferably less than 5000 amu, and even more prefer
`ably less than 2000 amu and even 1000 amu. In preferred
`embodiments, the inhibitors will be orally active.
`In certain embodiments, the subject inhibitors are peptidyl
`compounds (including peptidomimetics) which are opti
`mized, e.g., generally by selection of the CO. Substituents, for
`the substrate specificity of the targeted proteolytic activity.
`These peptidyl compounds will include a functional group,
`Such as in place of the Scissile peptide bond, which facili
`tates inhibition of a serine-, cysteine- or aspartate-type
`protease, as appropriate. For example, the inhibitor can be a
`peptidyl C-diketone or a peptidyl C.-keto ester, a peptide
`haloalkylketone, a peptide sulfonyl fluoride, a peptidyl bor
`onate, a peptide epoxide, a peptidyl diazomethanes, a pep
`tidyl phosphonate, isocoumarins, benzoxazin-4-ones, car
`bamates, isocyantes, isatoic anhydrides or the like. Such
`functional groups have bee provided in other protease
`inhibitors, and general routes for their synthesis are known.
`See, for example, Angelastro et al., J. Med Chem. 33:11-13
`(1990); Bey et al., EPO 363,284: Bey et al., EPO 364,344;
`Grubb et al., WO 88/10266; Higuchi et al., EPO 393.457;
`Ewoldt et al., Molecular Immunology 29(6):713–721
`(1992); Hernandez et al., Journal of Medicinal Chemistry
`35(6): 1121–1129 (1992); Vlasak et al., J Virology 63(5):
`2056–2062 (1989);
`Hudig et
`al.,
`J Immunol
`147(4): 1360–1368 (1991); Odake et al., Biochemistry 30(8):
`2217-2227 (1991); Vijayalakshmi et al., Biochemistry
`30(8):2175–2183 (1991); Kam et al., Thrombosis and Hae
`mostasis 64(1):133-137 (1990); Powers et al., J Cell Bio
`chem 39(1):33-46 (1989); Powers et al., Proteinase Inhibi
`tors, Barrett et al., Eds. Elsevier, pp.55-152 (1986); Powers
`et al., Biochemistry 29(12):3108–3118 (1990); Oweida et
`al., Thrombosis Research 58(2):391-397 (1990); Hudig et
`al., Molecular Immunology 26(8):793–798 (1989); Orlowski
`et al., Archives of Biochemistry and Biophysics 269(1):
`125–136 (1989); Zunino et al., Biochimica et Biophysica
`Acta. 967(3):331-340 (1988); Kam et al., Biochemistry
`27(7):2547.