PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 5 :
`(11) International Publication Number:
`
`,
`
`= A61K 31/00
`
`W0 99f38501 5 August 1999 (05.08.99)
`
`(43) International Publication Date:
`
`(21) International Application Number:
`
`PCT/US99/02294
`
`(22) International Filing Date:
`
`2 February 1999 (0202.99)
`
`(30) Priority Data:
`60/073,409
`
`2 February 1998 (0202.98)
`
`US
`
`(71) Applicant (for all designated States except US): TRUSTEES
`OF TUFTS UNIVERSITY [US/US]; Tufts University, Med-
`ford, MA 02155 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): BACI-IOVCHIN, William,
`W.
`[US/US]; 7 Warwick Street, Melrose, MA 02176
`(US). PLAUT, Andrew, G.
`[US/US]; 22 Peacock Farm
`Road, Lexington, MA 02421 (US). DRUCKER, Daniel, J.
`[CA/CA]; 19 Fernwood Road, Toronto, Ontario M6B 3G3
`(CA).
`
`(74) Agents: VINCENT, Matthew, P. et al.; Foley, Hoag & Eliot,
`LLP, One Post Office Square, Boston, MA 02109 (US).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD,
`GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP,
`KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
`MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TR, Tl‘, UA, UG, US, UZ, VN, YU,
`ZW, ARIPO patent (GH, GM, KE, LS, MW, SD, SZ, UG,
`ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI,
`FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
`SN, TD, TG).
`
`Published
`Without international search report and to be republished
`upon receipt of that report.
`
`(54) Title: METHOD OF REGULATING GLUCOSE METABOLISM, AND REAGENTS RELATED THERETO
`
`(57) Abstract
`
`The present invention provides methods and compositions for modification and regulation of glucose and lipid metabolism, generally
`to reduce insulin resistance, hyperglycemia, hyperinsulinemia,obesity, hyperlipidemia, hyper1ipoprotein—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 disorders,
`especially those associated with diabetes, obesityand/or atherosclerosis.
`
`AURO - EXHIBIT 1011
`
`

`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`CU
`CZ
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cote d’Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
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`

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`wo 99/33501
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`PCT/US99/02294
`
`Method ofRegulating 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
`
`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 converted in the liver to CO2 and H20
`
`(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 metabolized 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 insulin-
`
`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-dependent (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 arising from long-standing diabetes are blindness,
`
`kidney failure, and limb amputations. Insulin—dependent diabetes mellitus (IDDM) accounts
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`for 10 to 15% of all cases of diabetes mellitus. The action of IDDM is to cause
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`hyperglycemia (elevated blood glucose concentration) and a tendency towards diabetic
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`ketoacidosis (DKA). Currently treatment requires chronic administration of insulin. Non-
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`insulin dependent diabetes mellitus (NIDDM) is marked by hyperglycemia that is not
`
`linked with DKA. Sporadic or persistent incidence of hyperglycemia can be controlled by
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`administering insulin. Uncontrolled hyperglycemia can damage the cells of the pancreas
`
`which produce insulin (the B-islet cells) and in the long term create greater insulin
`
`deficiencies. Currently, oral sulfonylureas and insulin are the only two therapeutic agents
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`available in the United States. for treatment of Diabetes mellitus. Both agents have the
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`potential
`
`for producing hypoglycemia as a side effect,
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`reducing the blood glucose
`
`concentration to dangerous levels. There is no generally applicable and consistently
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`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 abnormalities. In most NIDDM
`
`subjects, the fundamental defects to which the abnormalities can be traced are (1) a reduced
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`entry of glucose into various "peripheral" tissues and (2) an increased liberation of glucose
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`into the circulation from the liver. There is therefore an extracellular glucose excess and an
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`intracellular glucose deficiency. There is also a decrease in the entry of amino acids into
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`muscle and an increase in lipolysis. Hyperlipoproteinemia is also a complication of
`
`diabetes. The cumulative effect of these diabetes-associated abnormalities is severe blood
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`vessel and nerve damage.
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`Endocrine secretions of pancreatic islets are regulated by complex control
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`mechanisms driven not only by blood-bome metabolites such as glucose, amino acids, and
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`catecholamines, but also by local paracrine influences.
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`Indeed, pancreatic OL- and fi—cells
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`are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a
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`synergistic messenger for nutrient-induced hormone release. The major pancreatic islet
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`hormones, glucagon, insulin and somatostatin, interact with specific pancreatic cell types to
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`modulate the secretory response. Although insulin secretion is predominantly controlled by
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`blood glucose levels, somatostatin 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 intestinal peptide and glicentin.
`
`These peptides variously regulate carbohydrate metabolism, gastrointestinal motility and
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`secretory processing. However, the principal recognized actions of pancreatic glucagon are
`
`to promote hepatic glycogenolysis and glyconeogenesis, resulting in an elevation of blood
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`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 translated from a 360 base pair
`
`gene and is processed to form proglucagon (Lund, et al., supra). Patzelt, et al. (Nature,
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`282:260-266 (1979)) demonstrated that proglucagon is further processed into glucagon and
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`a second peptide. Later experiments demonstrated that proglucagon is cleaved carboxyl to
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`Lys-Arg or Arg-Arg residues (Lund et al., _s1_1;ga; and Bell et al. (1983) me 3022716-
`
`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 proglucagon by cells of the intestinal mucosa, is released into the circulation after
`
`nutrient intake (Holst et al. (1987) 1~"_l§§_S_Litt 211:169; Orskov et al. (1987) Diabetologia
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`301874; Conlon J (1988) Diabetologia 312563).
`
`GLP-1 has been found to be a glucose-dependent insulinotropic agent (Gutniak et
`
`al. (1992) N. Engl. J. Bled. 32621316-1322).
`
`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
`
`79:6l6; Schmidt et al. (1985) Diabetologia 28:704; and Kreymann et al. (1987) Lancet
`
`2:l300). 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 known in the art. These variants and analogs include,
`for example, GLP-l(7-36), Gln9-GLP-l(7—37), D-Gln9-GLP-1(7-37), acetyl-Lys9—GLP—l(7—
`
`37), Thr16-Lyslg-GLP-l(7-37), and Lyslg-GLP-l(7-37). Derivatives of GLP-1 include, for
`
`example, acid addition salts, carboxylate salts, lower alkyl esters, and amides (see, e.g.,
`
`W091/11457).
`
`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.
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`It is yet another object of this invention to provide improved methods for the long-
`
`term reduction and abatement 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 resistance), blood insulin levels,
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`hyperinsulinemia, blood glucose levels, the amount of body fat stores, blood lipoprotein
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`levels, and thus to provide effective treatments for diabetes, obesity and/or atherosclerosis.
`
`Brief Description of the Drawings
`
`Figure 1
`
`is a diagrammatic representation of the synthesis of a boro proline
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`15
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`compound.
`
`Figure 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-l potentiates the response to a sub-therapeutic dose of GLP-1.
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`Figure 3 shows that a single injection of PBP-2 improves glucose levels in blood.
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`Figure 4 shows that treatment with PBP-3 under “chronic” conditions also results in
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`lowering of the blood sugar levels.
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`Figures 5Aand 5B compare the ability of Pro-boro-pro to lower plasma glucose
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`levels in GLP-1 receptor -/- transgenic mice.
`
`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-l (GLP-l) and
`
`gastric inhibitory peptide (GIP) are insulinotropic agents, e.g., being agents which can
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`stimulate, or cause the stimulation of, the synthesis or expression of the hormone insulin,
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`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—1ike peptide-1 is a glucoincretin both in man and other mammals (Dupre et al.
`
`fly, and Kreymann et al. (1987) Imnjcet 2:300). It is part of the preproglucagon molecule
`
`(Bell et al. (1983) Nature 3042368) which is proteolytically processed in intestinal L cells to
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`GLP-l(l-37) and GLP-1(7-36)amide or GLP-1(7-37) (Mojsov et al. (1986) J. Biol. Chem.
`
`261 :1 1880; 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
`
`792616; and Weir et al. (1989) Diabetes 382338). They are the most potent gluco-incretins
`
`so far described and are active at concentrations as low as one to ten picomolar.
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`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 4421126-1131.
`
`i. Overview of the Invention
`
`The present invention provides methods and compositions for modification and
`
`regulation of glucose and lipid metabolism, generally to reduce insulin resistance,
`
`hyperglycemia, hyperinsulinemia, obesity, hyperlipidemia, hyperlipoprotein-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 disorders, 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 EC50’s for immunosuppression in the uM or greater range. Thus, a
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`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, correlatively, able to improve glucose tolerance,
`
`though not
`
`necessarily through mechanisms involving DPIV inhibition per se.
`
`Indeed, the results
`
`described in Example 6 (and Figure 5) demonstrating an effect in mice lacking a GLP-1
`
`receptor suggest that the subject method may not include a mechanism 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,
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`inhibitors with Ki Values in the picomolar and even femtamolar range are
`
`contemplated. Thus, while the active agents are described herein, for convience, as “DPIV
`
`inhibitors”, it will be understood that such nomenclature is not intending to limit the subject
`
`invention to a particular mechanisim of action.
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`For instance, in certain embodiments the method involves administration of a DPIV
`
`inhibitor, preferably at a predetermined 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 intolerance,
`
`insulin resistance, hyperglycemia, hyperinsulinemia
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`and Type II diabetes).
`
`In other embodiments, the method involves administration 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.
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`The leading theory is that the fat cells of most obese people probably produce enough
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`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 accordingly a great deal of research
`
`towards utilizing preparations of GLP-1 as an apepitite 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.
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`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
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`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
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`regulatory peptides, and the other enzymes involved in these processes may be suitable
`
`targets for pharmacological intervention 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 l-30),
`
`intervening peptide-2 (IP-2, PG lll-122amide), and glucagon—like peptide-2 (GLP-2, PG
`
`126-158).
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`GLP-2, for example, has been identified as a factor responsible for inducing
`
`proliferation of intestinal epithelium.
`
`See, for example, Drucker et al. (1996) PNAS
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`93:791l.
`
`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 peptide (VIP), peptide
`
`histidine isoleucine (PHI), pituitary adenylate cyclase activating peptide (PACAP), gastric
`
`inhibitory 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 deficient 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
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`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
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`subject method can be used to alter the pharmacokinetics of Peptide YY and neuropeptide
`
`Y, both members of the pancreatic 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 pharmaceutical 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 embodiment, the inhibitors have hypoglycemic and antidiabetic
`
`activities, and can be used in the treatment of disorders marked by abberrant glucose
`
`metabolism (including storage). In particular embodiments, the compositions of the subject
`
`methods are useful as insulinotropic agents, or to potentiate the insulinotropic effects of
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`-3-
`
`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
`
`preferably 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 optimized, e.g., generally by selection of the COL 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
`
`facilitates inhibition of a serine—, cysteine- or aspartate-type protease, as appropriate. For
`
`example, the inhibitor can be a peptidyl on-diketone or a peptidyl on-keto ester, a peptide
`
`haloalkylketone, a peptide sulfonyl fluoride, a peptidyl boronate, a peptide epoxide, a
`
`peptidyl diazomethanes,
`
`a peptidyl phosphonate,
`
`isocoumarins, benzoxazin-4-ones,
`
`carbamates, 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
`
`Chemistg 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); Odakc et al., Biochemistfl 30(8):2217-
`
`2227 (1991); Vijayalakshmi et al., Biochemistry 30(8):2175-2183 (1991); Kam et al.
`
`,
`
`Thrombosis and Haemostasis 64(1):133-137 (1990); Powers et al.,
`
`J_(_3_eL Biochem
`
`39(1):33-46 (1989); Powers et al., Proteinase Inhibitors, 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 Biochemistg and Biophysics
`
`269(1):125-136 (1989); Zunino et al., Biochimica et Biophysica Acta. 967(3):331-340
`
`(1988); Kam et al., Biochemistfl 27(7):2547-2557 (1988); Parkes et al., Biochem J.
`
`230:509-516 (1985); Green et al., J. Biol. Chem. 256:1923—1928 (1981); Angliker et al.,
`Biochem. J. 241:871—875 (1987); Puri et al., Ar_c£_Biochem. Biophys. 27:346-358 (1989);
`
`Hanada et al., Proteinase Inhibitors: Medical and Biological Aspects, Katunuma et al., Eds.,
`
`Springer-Verlag pp. 25-36 (1983); Kajiwara et al., Biochem. Int. 15:935-944 (1987); Rao et
`
`al., Thromb. Res. 47:635—637 (1987); Tsujinaka et al., Biochem. Biophys. Res. Commun.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
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`

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`W0 99/38501
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`PCT/US99/02294
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`-9-
`
`153:1201—1208 (1988)). See also U.S. Patents Bachovchin et al. 4,935,493; Bachovchin et
`
`al. 5,462,928; Powers et al. 5,543,396; Hanko et al. 5,296,604; and the PCT publication of
`
`Ferring PCT/GB94/02615.
`
`In other embodiments, the inhibitor is a non-peptidyl compound, e.g., which can be
`
`identified by such drug screening assays as described herein. These inhibitors can be,
`
`merely to illustrate, synthetic organis, natural products, nucleic acids or carbohydrates.
`
`A representative class of compounds for use in the method of the present invention
`
`are represented by the general formula;
`
`R2
`
`A
`
`/
`R3
`
`10
`
`wherein
`
`A represents a 4-8 membered heterocycle including the N and the COL carbon;
`
`Z represents C or N;
`
`W represents a functional group which reacts with an active site residue of the
`
`15
`
`targeted protease, as for example, -CN, -CH=NR5,
`
`R1 represents a C-terrninally linked amino acid residue or amino acid analog, or a
`
`C-
`
`terminally linked peptide or peptide analog, or
`
`an amino—protecting group, or
`
`if
`
`E
`
`‘H
`
`0
`
`20
`
`R2 is absent or represents one or more substitutions to the ring A, each of which can
`
`independently be a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl
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`

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`W0 99/38501
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`PCT/US99/02294
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`-10-
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`(such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a
`
`thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido,
`
`a sulfate, a sulfonate, a sulfonamido, -(CH2)m-R7, -(CH2)m-OH, -(CH2)m-O-lower alkyl, -
`
`(CH2)m-O-lower alkenyl,
`
`-(CH2)n—O—(CH2)m—R7, —(CH2)m—SH, —(CH2)m-S—lower alkyl,
`
`-
`
`(CH2)m-S-lower alkenyl, -(CH2)n—S—(CH2)m-R7;
`
`if X is N, R3 represents hydrogen, if X is C, R3 represents hydrogen or a halogen, a
`
`lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a
`
`formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioforrnate), an
`
`amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a
`
`sulfonamido,
`
`-(CH2)m-R7,
`
`-(CH2)m-OH, —(CH2)m-O-lower
`
`alkyl,
`
`-(CH2)m-O-lower
`
`alkenyl,
`
`-(CH2)n-O-(CH2)m-R7, -(CH2)m-SH,
`
`-(CH2)m-S-lower alkyl, -(CH2)m-S-lower
`
`alkenyl, -(CH2)n—S-(CH2)m-R7;
`
`R5 represents H, an alkyl, an alkenyl, an alkynyl, -C(X1)(X2)X3, -(CH2)m-R7, —
`
`(CH2)n-OH, —(CH2)n-O-alkyl,
`
`-(CH2)n-O-alkenyl,
`
`-(CH2)n-O-alkynyl, —(CH2)n-O-
`
`(CH2)m-R7,
`
`-(CH2)n-SH,
`
`-(CH2)n-S-alkyl,
`
`-(CH2)n-S-alkenyl,
`
`-(CH2)n-S-alkynyl,
`
`-
`
`(CH2)n-S-(CH2)m-R7, -C(O)C(O)NH_7_, -C(O)C(O)OR’7;
`
`R6 represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, an aryl, -(CH2)m-
`
`R7, -(CH2)m-OH, -(CH2)m-O-alkyl, -(CH2)m-O-alkenyl, -(CH2)m-O-alkynyl, -(CH2)m-O-
`
`(CH2)m-R7,
`
`-(CH2)m-SH,
`
`-(CH2)m-S-alkyl,
`
`-(CH2)m-S-alkenyl,
`
`-(CH2)m-S-alkynyl,
`
`-
`
`10
`
`15
`
`20
`
`(CH2)m-S-(CH2)m-R7=
`
`R7 represents, for each occurrence, a substituted or unsubstituted aryl, aralkyl,
`
`cycloalkyl, cycloalkenyl, or heterocycle;
`
`R’7 represents, for each occurrence, hydrogen, or a substituted or unsubstituted
`
`alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and
`
`Y1 and Y2 can independently or together be OH, or a group capable of being
`
`hydrolyzed to a hydroxyl group,
`
`including cyclic derivatives where Y1 and Y2 are
`
`connected via a ring having from 5 to 8 atoms in the ring structure (such as pinacol or the
`
`like),
`
`R50 represents 0 or S;
`
`R51 represents N3, SH2, NH2, N02 or OR’7;
`
`25
`
`30
`
`R52 represents hydrogen, a lower alkyl, an amine, OR’-,, or a pharmaceutically
`
`acceptable salt, or R51 and R52 taken together with the phosphorous atom to which they
`
`are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure
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`

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`wo 99/33501
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`PCT/US99/02294
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`-11-
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`X] represents a halogen;
`
`X2 and X3 each represent a hydrogen or a halogen
`
`m is zero or an integer in the range of 1 to 8; and n is an integer in the range of 1 to
`
`In preferred embodiments, the ring A is a 5, 6 or 7 membered ring, e.g., represented
`
`by the formula
`
`)n
`
`‘N
`
`and more preferably a 5 or 6 membered ring. The ring may, optionally, be further
`
`10
`
`substituted.
`
`Y
`In preferred embodiments, W represents ——}3/\ 1
`Y2
`
`O
`or AL
`
`R5
`
`In preferred embodiments, R1 is
`
`15
`
`20
`
`25
`
`wherein R36 is a small hydrophobic group, e.g., a lower alkyl or a halogen and R38 is
`
`hydrogen, or, R36 and R37 together form a 4-7 membered heterocycle including the N and
`
`the Con carbon, as defined for A above; and R40 represents a C-terminally linked amino
`
`acid residue or amino acid analog, or a C-terminally linked peptide or peptide analog, or an
`
`amino-protecting group
`
`In preferred embodiments, R2 is absent, or represents a small hydrophobic group
`
`such as a lower alkyl or a halogen.
`
`In preferred embodiments, R3 is a hydrogen, or a small hydrophobic group such as a
`
`lower alkyl or a halogen.
`
`In preferred embodiments, R5 is a hydrogen, or a halogentated lower alkyl.
`
`In preferred embodiments, X1 is a fluorine, and X2 and X3, if halogens, are
`fluorine.
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`

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`wo 99/33501
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`PCT/US99/02294
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`_12_
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`Also deemed as equivalents are any compounds which can be hydrolytically
`
`converted into any of the aforementioned compounds including boronic acid esters and
`
`halides, and carbonyl equivalents including acetals, hemiacetals, ketals, and hemiketals, and
`
`cyclic dipeptide analogs.
`
`Longer peptide sequences are needed for the inhibition of certain proteases and
`
`improve the specificity of the inhibition in some cases.
`
`In preferred embodiments,
`
`the subject method utilizes, as a DPIV inhibitor, a
`
`boronic acid analogs of an amino acid. For example, the present invention contemplates the
`
`use of boro-prolyl derivatives in the subject method. Exemplary boronic acid derived
`
`10
`
`inhibitors of the present invention are represented by the general formula:
`
`/N
`
`Rl
`
`PR”
`B \
`
`OR11
`
`wherein
`
`R1 represents a C-terminally linked amino acid residue or amino acid analog, or a
`
`(ll
`fin
`‘I?
`terminally linked peptide or peptide analog, or R6““C"“ , R6-'C_‘ 2 R6—fi—‘ ;
`0
`
`15
`
`C_
`
`R6 represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, an aryl, -(CH2)m-
`
`R7, -(CH2)m-OH, -(CH2)m-O-alkyl, -(CH2)m—O-alkenyl, -(CH2)m-O-alkynyl, -(CH2)m—O-
`
`(CH2)m-R7,
`
`-(CH2)m-SH,
`
`-(CH7_)m-S-alkyl,
`
`-(CH2)m-S—alkenyl,
`
`-(CH2)m-S—alkynyl,
`
`-
`
`(CH2)m'S'(CH2)m"R7a
`
`20
`
`—<cH2>n—3—N’\
`
`4?
`
`—(CH2)n
`
`-alkyl, —(CH2)n
`
`3
`
`‘alkenyl,
`
`4?
`
`--(CH2)n
`
`-alkynyl ,or —(CH2)n— ‘(CH2)nTR7
`
`(3
`
`R7 represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;
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`

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`WO 99/38501
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`PCT/US99/02294
`
`-13-
`
`R8 and R9 each independently represent hydrogen, alkyl, alkenyl, -(CH2)m-R7, -
`
`C(=O)-alkyl, —C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)—(CH2)m-R7,
`
`or R3 and R9 taken together with the N atom to which they are attached complete a
`
`heterocyclic ring having from 4 to 8 atoms in the ring structure;
`
`R11 and R12 each independ