AU2011200657B2 - Method of regulating glucose metabolism, and reagents related thereto - Google Patents

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, hyperlipoprotein-emia (such 5 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. )1

Description

- 1 AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicants: 1149336 Ontario, Inc. and New England Medical Center Hospitals, Inc. and Trustees of Tufts College Actual Inventors: William Bachovchin and Andrew G. Plaut and Daniel Drucker Address for Service is: SHELSTON IP 60 Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: METHOD OF REGULATING GLUCOSE METABOLISM, AND REAGENTS RELATED THERETO Details of Original Application No. 2007202745 dated 13 Jun 2007 The following statement is a full description of this invention, including the best method of performing it known to me/us: File: 28741AUP03 -2 METHOD OF REGULATING GLUCOSE METABOLISM, AND REAGENTS RELATED THERETO Related Application 5 The present application is a divisional application of Australian Patent Application No. 2007202745, which is incorporated in its entirety herein by reference. Field of the Invention 10 The present invention relates to a method for modifying glucose metabolism in an animal, comprising conjointly administering to the animal metformin and an inhibitor of dipeptidylpeptidase IV or a pharmaceutically acceptable salt thereof in an amount sufficient to modify glucose metabolism, wherein the amount or said inhibitor of dipeptidylpeptidase IV or pharmaceutically acceptable salt thereof is not 15 sufficient to suppress the immune system of the animal. Background of the Invention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of 20 common general knowledge in the field. 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 25 cells by an action on the cell membranes. The ingested glucose is normally converted in the liver to CO 2 and H 2 0 (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 30 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 -3 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 5 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 for 10 to 15% of all cases of diabetes mellitus. The 10 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 hyperglycemia that is not linked with DKA. Sporadic or persistent incidence of hyperglycemia can be controlled by administering insulin. 15 Uncontrolled hyperglycemia can damage the cells of the pancreas which produce insulin (-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 treatment of Diabetes mellitus. Both agents have the potential for producing hypoglycemia as a side effect, reducing the blood 20 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 25 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 entry of glucose into various "peripheral" tissues and (2) an increased liberation of glucose into the circulation from the liver. There is therefore 30 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.

-4 Endocrine secretions of pancreatic islets are regulated by complex control mechanisms driven not only by blood-borne metabolites such as glucose, amino acids, and catecholamines, but also by local paracrine influences. Indeed, pancreatic a-and p-cells are critically dependent on hormonal signals generating cyclic AMP 5 (cAMP) as a synergistic 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, somatostatin inhibits glucose-mediated insulin secretion. 10 The human hormone glucagon is a polypeptide honnone 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 secretory processing. However, the 15 principal recognized actions of pancreatic glucagon are to promote hepatic glycogenolysis 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). 20 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, 282: 260-266 (1979)) demonstrated that proglucagon is further processed into glucagon and a second peptide. Later experiments demonstrated that proglucagon is cleaved carboxyl to Lys-Arg or Arg-Arg residues (Lund et al., supra; 25 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 I (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 30 proglucagon 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).

- 4a GLP- I has been found to be a glucose-dependent insulinotropic agent (Gutniak et al. (1992) N. Engl. J. Bled. 326:1316-1322). GLP-l is now known to stimulate insulin secretion (insulinotropic action) causing glucose uptake by cells 5 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) Diabetologia 28:704; and Kreymann et al. (1987) Lancet 2:1300). Thus, 10 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- I analogs have been demonstrated which demonstrate insulinotropic action are known in the art. These variants and analogs include, for example, GLP-1(7-36), Gln 9 -GLP-1(7-37), D-Gln 9 -GLP-1(7-37), 15 acetyl-Lys 9 -GLP-1(7-37), Thri 6 -Lysis-GLP-1(7-37), and Lysis-GLP-1(7-37). Derivatives of GLP-1 include, for example, acid addition salts, carboxylate salts, lower alkyl esters, and amides (see, e.g., W091/11457). Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in 20 an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Although the invention has been described with reference to specific examples it will be appreciated by those skilled in the art that the invention may be embodied in many other forms. 25 It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. Summary of the Invention According to a first aspect of the present invention there is provided a method 30 for treating type I diabetes in an animal, comprising conjointly administering to the animal (i) metformin, and (ii) an inhibitor of dipeptidylpeptidase IV represented by the following formula: - 4b B-OR, R, O or a pharmaceutically acceptable salt thereof; wherein R, represents a C-terminally linked amino acid residue or amino acid 5 analog, a C-terminally linked peptide or peptide analog, or 0 s 0 R6 , R6 , or R f11/ ; R6, represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m-O-alkenyl, -(CH 2 )m-0 alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 ),-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S 10 alkenyl, -(CH 2 ),m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7 ,

R

8 O R R9 R9

NH

2 0

-(CH

2 )n-NH 2

-C-NH

2 , -(CH 2

)--C-O-R

7 0 0 _Il II -(CII,),-C-akyI, -(CH 2 ),-C-alkenyI, 0 0 -- (CI ),-C-akyny, or -(CH 2 )n-C-(CH 2 )m,,-R 7 ;

R

7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle;

R

8 and R 9 each independently represent hydrogen, alkyl, alkenyl, 15 (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O)

(CH

2 )m-R 7 ; or R 8 and R 9 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; RI, and R 12 each independently represent hydrogen, alkyl, or a 20 pharmaceutically acceptable salt; or RI and R 12 taken together with the 0 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; - 4c m is zero or an integer in the range of I to 8; n is an integer in the range of 1 to 8; the inhibitor inhibits dipeptidylpeptidase proteolysis of GLP-1 with a Ki of less than about 10 nM; and 5 the metformin and the inhibitor are administered in an amount sufficient to treat Type II diabetes but not sufficient to suppress the immune system of the animal. According to a second aspect of the present invention there is provided a 10 method for treating type II diabetes in an animal, comprising conjointly administering to the animal (i) a sulfonylurea, and (ii) an inhibitor of dipeptidylpeptidase IV represented by the following formula:

R

1 N /B-OR, 2

R

1 1O or a pharmaceutically acceptable salt thereof; 15 wherein Ri represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or 0 S , or

R

6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, 20 (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m-0-alkenyl, -(CH 2 )m-O alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S alkenyl, -(CH 2 )m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7 , R, O R8 -(CH12)m-N \ -(CH )--N , R9 R9

NH

2 O

-(CH

2

).--NH

2 -C- NH 2 , -(CH 2

).-C-O-R

7 0 0 II i - (CH 2 )-C--aikyi, -- (CH 2 )-C-aikenyi, - 4d 0 0 Il II

-(CH

2 ),-C-akynyI, or -(CH 2 )a-C-(CHI 2 ),-R7;

R

7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle;

R

8 and R 9 each independently represent hydrogen, alkyl, alkenyl, (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O) 5 (CH 2 )m-R 7 ; or Rs and RO 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;

R

1 and R 1 2 each independently represent hydrogen, alkyl, or a pharmaceutically acceptable salt; or R 1 and R 1 2 taken together with the 0 10 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; m is zero or an integer in the range of I to 8; n is an integer in the range of 1 to 8; the inhibitor inhibits dipeptidylpeptidase proteolysis of GLP-I with a 15 Ki of less than about 10 nM; and the sulfonylurea and the inhibitor are administered in an amount sufficient to treat Type II diabetes but not sufficient to suppress the immune system of the animal. 20 According to a third aspect of the present invention there is provided use of metformin and an inhibitor of dipeptidylpeptidase IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of Type II diabetes in a human without suppressing the immune system, wherein said inhibitor is represented by the following formula: B-OR,,

R

11 0 25 wherein R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or - 4e o S ) , or

R

6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m-O-alkenyl, -(CH 2 )m0 alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S 5 alkenyl, -(CH 2 )m,-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7 , R8 0 R 8

(CH

2 )m-N , - (H) - R9 R 9 -(CH2)n-NH2--C-NH2, -(CH[2),-C-0-R7 0II 0

(CH

2 ).-C-alkyl, -(C 2 ),-C-alkeny, 0 0

-(CH

2 ),-C-alkynyl, or -(CH 2 )n-C-(CH 2 )m-R 7 ;

R

7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle;

R

8 and R 9 each independently represent hydrogen, alkyl, alkenyl, 10 (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O)

(CH

2 )m-R 7 ; or R 8 and R 9 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; R, i and R 1 2 each independently represent hydrogen, alkyl, or a 15 pharmaceutically acceptable salt; or R, I and R 1 2 taken together with the 0 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; m is zero or an integer in the range of I to 8; n is an integer in the range of 1 to 8. 20 According to a fourth aspect of the present invention there is provided use of a sulfonylurea and an inhibitor of dipeptidylpeptidase IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of Type II diabetes in a human without suppressing the immune system, wherein said 25 inhibitor is represented by the following formula: - 4f R( B-OR, RuO0 wherein R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or 0 S 0

R

6 , R6, or R /; 5

R

6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH2)m-O-alkenyl, -(CH 2 )m-O alkynyl, -(CH 2 )m0-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S alkenyl, -(CH2)m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R7,

(C

2 )m-N -(CH,),-C-N R9 R9 NI, 0 11 - 11 -(CH2,)n-NH2-C-NH2, -(CH2)n-C-0--R7 0 0 10 -(CH12)n-C-aikyi, -- (CH2)n-Ckeyi, O O -(CHJ)-C--akynyi, or -(CH2)n-C-(CH2)m-R7;

R

7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle;

R

8 and R 9 each independently represent hydrogen, alkyl, alkenyl, (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O) 15 (CH 2 )m-R 7 ; or R 8 and R 9 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; Rii and R 1 2 each independently represent hydrogen, alkyl, or a pharmaceutically acceptable salt; or R, I and R 12 taken together with the 0 20 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; m is zero or an integer in the range of 1 to 8; - 4g n is an integer in the range of 1 to 8. According to a fifth aspect of the present invention there is provided a pharmaceutical formulation for the treatment of Type II diabetes without 5 suppressing the immune system, comprising (i) a pharmaceutically acceptable excipient, (ii) metformin, and (iii) an inhibitor of dipeptidylpeptidase IV represented by the following formula: R< /B-OR,, R,,O or a pharmaceutically acceptable salt thereof; 10 wherein R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or 0 s 0 R6-- , R-6- , or Re" ; ,or

R

6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, 15 (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 ),m-O-alkenyl, -(CH 2 )m-O alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S alkenyl, -(CH2)m-S-alkynyl,

-(CH

2 )m-S-(CH 2 )m-R 7 , R8 0 Rs

-(CH

2 )m-N \ - (CH2)--C-N , R9 R 9 NH,O -(CH1 2

)-NH,--C-NH

2 , -(CH 2

)-C-O-R

7 --- CH2.-C--lky, - (CH,)n--C- alkenyl, 0 0

-(CHI

2 ).-C-alkynyl, or -(C11 2 )n-C-(C-2),,-Rt 7 ; 20 R 7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle;

R

8 and R 9 each independently represent hydrogen, alkyl, alkenyl, (CH 2 )m,-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O)- - 4h (CH 2 )m-R 7 ; or R 8 and R 9 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; R, 1 and R 1 2 each independently represent hydrogen, alkyl, or a 5 pharmaceutically acceptable salt; or Ri 1 and R 1 2 taken together with the 0 B-O atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; m is zero or an integer in the range of I to 8; n is an integer in the range of I to 8; 10 the inhibitor inhibits dipeptidylpeptidase proteolysis of GLP-1 with a Ki of less than about 10 nM. According to a sixth aspect of the present invention there is provided a pharmaceutical formulation for the treatment of Type II diabetes without 15 suppressing the immune system, comprising (i) a pharmaceutically acceptable excipient, (ii) a sulfonylurea, and (iii) an inhibitor of dipeptidylpeptidase IV represented by the following formula: R N /B-OR,2

R,

1 O or a pharmaceutically acceptable salt thereof; 20 wherein R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or 0 S 0 o s o ,or ;

R

6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, 25 (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m,-O-alkenyl, -(CH 2 )m-O alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 )m,-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S alkenyl, -(CH 2 )m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7

,

- 4i R-8 O RS / II / -(CH2)m--N , - -(CH2)n-C-N , R9 R 9 NH2 0

-(CH

2

)

0

-NH

2

-C-NH

2 , -(CH 2 )-C-0- R 7 00 II _

(CH

2 )-,C--alky, -(CH 2 )n-C-akenyl, 0 0

-(CH

2 ).-C-alkynyl, or -(CH 2 )n--C-(CH 2 )m-R7;

R

7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle;

R

8 and R 9 each independently represent hydrogen, alkyl, alkenyl, 5 (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O)

(CH

2 )m-R 7 ; or R 8 and R 9 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;

R,

1 and R 1 2 each independently represent hydrogen, alkyl, or a 10 pharmaceutically acceptable salt; or R, 1 and R 12 taken together with the 0 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; m is zero or an integer in the range of 1 to 8; n is an integer in the range of 1 to 8; 15 the inhibitor inhibits dipeptidylpeptidase proteolysis of GLP-I with a Ki of less than about 10 nM. This invention relates to improved methods for reducing in animal subjects (including humans) in need of such treatment at least one of insulin resistance, 20 hyperinsulinemia, and hyperglycemia and abating Type II diabetes. The invention also relates to improved methods for reducing at least one of body fat stores, hyperlipidemia, hyperlipoproteinemia, and for abating atherosclerosis. The invention further relates to methods for interfering with glucose and/or lipid metabolism in a manner beneficial to the host. 25 The invention also relates to 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.

- 4j The present invention further relates to a method for regulating, and altering on a long term basis, the glucose and lipogenic responses of vertebrate animals including humans. The invention relates to methods for producing long lasting beneficial 5 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, hyperinsulinemia, blood glucose levels, the amount of body fat stores, blood lipoprotein levels, effective treatment for diabetes, obesity and/or atherosclerosis. 10 Brief Description of the Drawings A preferred embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: Figure 1 is a diagrammatic representation of the synthesis of a boro proline compound. 15 Figure 2 is a glucose tolerance curve which shows that a single injection of PBP-I 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-l. 20 Figure 3 shows that a single injection of PBP-2 improves glucose levels in blood. Figure 4 shows that treatment with PBP-3 under "chronic" conditions also results in lowering of the blood sugar levels. Figures 5A and 5B compare the ability of Pro-boro-pro to lower plasma 25 glucose levels in GLP-I 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 30 1) and gastric inhibitory peptide (GIP) are insulinotropic agents, e.g., being agents which can stimulate, or cause the stimulation 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-l is a glucoincretin both in man and other mammals (Dupre et al. 5 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-l(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 10 have identical effects on insulin secretion in beta cells (Mojsov et al. (1987) ._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-l has been studied in nondiabetic and type II diabetic subjects. Subcutaneous and intravenous GLP-1 are both rapidly degraded in a time 15 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. Overvicw of the Invention The present invention provides methods and compositions for modification and 20 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 25 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-I and accordingly increase the plasma half-life of that hormone. 30 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 35 compounds have EC50's for immunosuppression in the tM or greater range. Thus, a -6 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 5 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-l may have other receptors. However, in light of the correlation with DPIV inhibition. in preferred 10 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 convience, as "DPIV inhibitors", it will be understood that such nomenclature is not intending to limit the subject 15 invention to a particular mechanisim of action, 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 20 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 tum, produces the sensation of being full. 25 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 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 30 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 -7 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 hal-f-life of other. proglucagon-derived peptides, such as glicentin (corresponding to PG 1-69), 5 oxyntomodulin (PG 33-69), glicentin-related pancreatic polypeptide (GRPP, PG 1-30), intervening peptide-2 (IP-2, PG 11 1-122amide), 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 epithelium. See, for example, Drucker et al. (1996) PNAS 10 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 homone-releasing factor (GHRF). GHRF is a member of the family of homologous 15 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 20 certain growth honnone 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 yield, leaner livestock. Likewise, the DPIV inhibitors of the subject invention can be used to alter the 25 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 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 30 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 35 methods are useful as insulinotropic agents, or to potentiate the insulinotropic effects of -8 such molecules as GLP-l. 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. 5 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 10 peptidomimetics) which are optimized, e.g., generally by selection of the Cct 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 ct-diketone or a peptidyl ct-keto ester, a peptide 15 haloalkylketone, a peptide sulfonyl fluoride, a peptidyl boronate, a peptide epoxide, a peptidyl diazomethanes, a peptidyl phosphonate, isocourarins, benzoxazin-4-ones, carbamates.-isotyantes, 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; 20 Bey et al.. EPO 364,344; Grubb et al., WO 88/10266; -iguchi et al., EPO 393,457; Ewoldt et al., Molecular Immunologv 29(6):713-721 (1992); Hernandez et al., Journal of Medicinal Chemistry 35(6): 1121-1129 (1992); Vlasak et al., J ViroloN\y 63(5):2056-2062 (1989); Hudig et al.. J Immunol 147(4):1360-1368 (1991); Odake et al., Biochemistry 30(8):2217 2227 (1991): Vijavalakshmi et al., Biochemistry 30(8):2175-2183 (1991); Kam et al. 25 Thrombosis and Haemostasis 64(1):133-137 (1990); Powers et al., J Cell 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 Immunoloav 26(8):793-798 (1989); Orlowski et al., Archives of Biochemistry and Biophysics 30 269(l):125-136 (1989); Zunino et al., Biochimica et Biophysica Acta. 967(3):331-340 (1988); Kam et al., Biochemistry 27(7):2547-2557 (1988); Parkes et aL., Biochen 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., Arch. Biochem. Biophys. 27:346-358 (1989); Hanada et al., Proteinase Inhibitors: Medical and Biological Aspects, Katunuma et al., Eds., 35 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. Bioohvs. Res. Commun.

-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 5 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 R1 N-z R3 10 wherein A represents a 4-8 membered heterocycle including the N and the Ccl. 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=NR 5 , o 0 y R 5 o S-X, -P-X - - R2 or R5 0 R5 R, 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, or 0 S 0 li || . II R-C-, R,-C- , R6-S 0 20 R-, 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 - 10 (such as a carboxyl, an ester, a formate, or a ketone), a thiocafbonyl (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, -(CH?)m-R 7 , -(CH,)m-OH, -(CH2)m-O-loer alkyl,

(CH

2 )m,-O-lower alkenyl, -(CH 2 )n-O-(CH 2 )m-R 7 , -(CHF)m-SHI,

-(CH

2 ) -S -lower alkyl, 5 (CH 2 )m-S-Iower alkenyl, -(CH2)n-S-(CH 2 )m-R 7 . if X is N, R 3 represents hydrogen, if X is C, R 3 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 thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a 10 sulfonamido. -(CH-1 2 )m,-R 7 , -(CH-)mn-OH, -(CH2)m-O-lower alkyl, -(CH,)m-0-lower alkenyi,

-(CH

2 )n-O-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH-)mn-S-lower alkyl, -(CH2)m-S-!ower alkenyl, -(CHl)n-S-(CH-)m-R 7 ;

R

5 represents H, an alkyl, an alkenyl, an alkynyl,

-C(X

1

)(X

2

)X

3 , -(CH,)m-R 7 , (CH2)n-OH, -(CHi)n-O-alkyl, -(CH2)n-O-alkenyl, -(C-2)n-O-alkynvl,

-(CH

2 )n-O 15 (CH-)m-R 7 , -(CH 2 )n-SH. -(CH-)n-S-alkyl, -(CH2)n-S-alkenyl, -(CH-,)n-S-alkvnvl. (CH 2 )n-S-(C

H

2 )m-R 7 , -C(O)C(O)NH 2 , -C(O)C(O)OR' 7 ;

R

6 represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, an arvl, -(CFI)M

R

7 , -(CH 2 )m-OH, -(CH2)m-0-alkyl, -(CH2)m-0-alkenyl,

-(CH

2 )m-O-alkynyl,

-(CH

2 )m -O

(CH

2 )m-R 7 , -(CH')m-SF, -(CH1)m-S-alkyl -(CH-)m -S-alkenyl -(CHi)m-S-alkynvl 20 (CH2)m-S-(CH 2 )m-R 7 ,

R

7 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 25 Yi and Y-) can independently or together be OH, or a group capable of being hydrolyzed to a hydroxyl group, including cyclic derivatives where Yj and Y- are connected via a ring having from 5 to 8 atoms in the ring structure (such as pinacol or the like),

R

5 0 represents 0 or S; 30

R

5 1 represents

N

3 , SH2, NH 2 , NO 2 or OR' 7 ;

R

5 2 represents hydrogen, a lower alkyl, an amine, OR' 7 , or a pharmaceutically acceptable salt, or R 5 1 and R 5 2 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 XI represents a halogen; X- and X 3 each represent a hydrogen or a halogen m is zero-or an-inte-ger in the range of - to 8; and n is an integeriri therange.of 1 to 8. 5 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. In preferred embodiments, W represents B or RS In preferred embodiments, RI is

R

3 6 R 4 N 15 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 Cc 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 20 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. 25 In preferred embodiments, XI is a fluorine, and X2 and X3, if halogens, are fluorine.

- 12 Also deemed as equivalents are any compounds wh ich can be hydrolytically converted into any of the aforementioned compounds including boronic acid esters and halides, and carbonyl equivalents including acetals, hemiacetal.5, ketals, and hemiketals, and.. cyclic dipeptide analogs. 5 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: R1~'_ - B O OR wherein R, represents a C-terminally linked amino acid residue or amino acid analog, or a 0 S 0 terminally linked peptide or peptide analog, or R 6 -C-, -C-, 15 C 5 C R 6 represents hydrogen, a halogen, a alkyl, a alkenyl, a alkvnyl, an aryl, -(CH2)m

R

7 , -(CH)m-OH, -(CH), 1 -O-alkyl, -(CH 2 )m,-0-alkenyl, -(CH 2 )m-0-alkynyl., -(CH-)mI-0

(CH

2 )m-R 7 , -(CH 2 )m-SH, -(C1-1)m-S-alkyl, -(CH)m-S-alkenyl, -(CH-,)m-S-alkynyl, (CH 2 )m-S-(CH 9 )m-R 7 , 0RS RS

NH

2 0 -(CH2)m-N -- (CH2)n- -N -- (CH2)n-NH2-C-NH2 -(CHI)n--0-R7

R

9 ' R 9 ' 0 0 0 0 -- (CH2)n-"-alkyl, -(CH2)n---alkenyl, -(CH2)n- alkynyl , or -(CH)n- -(CH2)rI-R 7

R

7 represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle; - 13 RS and R 9 each independently represent hydrogen, alkyl. alkenyl, -(CH2)m-R 7 , C(=D)-alkyl, -C(=0)-alkenyl, -C(=0)-alkynyl, -C(=0)-(CH 9 )m-R 7 , or R 8 and R 9 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; 5 R I I and R 12 each independently represent hydrogen, a alkyl, or a pharmaceutically acceptable salt, or R 1 I and R 12 taken together with the 0-B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; m is zero or an integer in the range of 1 to 8; and n is an integer in the range of 1 to 10 8. In other embodiments, the subject DPIV inhibitors include an aldehyde analogs of proline or prolyl derivatives. Exemplary aldehyde-derived inhibitors of the present invention are represented by the general formula: 15 0 R1'-N wherein R, represents a C-terminally linked amino acid residue or amino acid analog, or a 0 S 0 )Il il I| terminally linked peptide or peptide analog. or R-C , R-C-

C

20 R 6 represents hydrogen, a halogen, a alkyl, a alkenyl, a alkvnyl, an aryl, -(CH2)m,

R

7 , -(CHi)m-OH. -(CH 2 )m-0-alkyl, -(CH 2 )m-0-alkenyl, -(CH,)m-0-alkynyl, -(CH 2 )m-0

(CH

2 )m-R 7 , -(CH 9 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S-alkenyl, -(CH 2 )m-S-alkynyl, (CH 2 )m-S-(CH 2 )m-R 7 , R0 O RS NH 2 0 -(CH')m-N -(CH2)n- - -(CH2)n-NH2-C-NH2, -(CH2)n-C-0-R7 R9 'R9' 0 0 0 0 25 ,n- -KY -(C H)n--alkenyi, -(CH2)n-akyTh'l , or -(CH2-)n(CH2)V1-R7 - 14

R

7 represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;

R

8 and R 9 each independently represent hydrogen, alkyl, alkenyl, -(CH2)m-R 7 , 5 C(=0)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl,

-C(=O)-(CH

2 )m-R 7 , or RS and R 9 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; and m is zero or an integer in the range of I to 8; and n is an integer in the range of 1 to ) 8. 10 In yet further embodiments, the subject DPIV inhibitors are halo-methyl ketone analogs of an amino acid. Exemplary inhibitors of this class include compounds represented by the general formula: 0 R1-'--N X2 X 2 15 wherein R, represents a C-terminally linked arnino acid residue or amino acid analog, or a 0 S 0 terminally linked peptide or peptide analog, or R--C--, R-C-, R--S- Il C- O

R

6 represents hydrogen, a halogen, a alkyl. a alkenyl, a alkynyl, an aryl, -(CH2)m R7, -(CHI)ml-OH, -(C1-I)mn-O-alkyl, -(CH-))m-0-alkenyl, -(CH-))m-0-alkynyl, -(CH-))m-0 20 (CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl. -(CH,)m-S-alkenyl,

-(CH

9 )m-S-alkynyl, (CH2)m-S-(CH 2 )mn.-R 7

,

- 15

R

8 . 0 R 8 H -(CH2)m-N -(CH2)n-C-N R9 -(CH 2 )n-NH-C-NH 2 -(CH2)n-C-0-R7 0 0 0 0 -(CH2)n -alkyl , -(CH2)n 4 -alkenyl, -- (CH)n- 4 -akynyl , or -(CH)n--(CH 2 )m--R 7

R

7 represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;

R

8 and R 9 each independently represent hydrogen, alkyl. alkenyl, -(CH 2 ')m-R 7 , 5 C(=0)-alkyl, -C(=0)-alkenyl, -C(=0)-alkynyl, -C(=0)-(CH 2 )m-P 7 , or R 8 and R 9 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; XI, X 2 and X 3 each represent a hydrogen or a halogen; and m is zero or an integer in the range of I to 8; and n is an integer in the range of 1 to 10 8. In preferred embodiments, the DPIV inhibitor is a peptide or peptidomimetic including a prolyl group or analog thereof in the PI specificity position. and a nonpolar amino acid in the P2 specificity position, e.g.. a nonpolar amino acid such as alanine, 15 leucine, isoleucine, valine, praline, phenylalanine. tryptophan or methionine. or an analog thereof. For example. the DPIV inhibitor may include an Ala-Pro or Pro-Pro dipeptide sequence or equivalent thereof, and be represented in the general formulas: R2 R2 R2 A A R32 A N W R30-N N w R30 R3 R3 0 or 20 In preferred embodiments, the ring.A is a 5, 6 or 7 membered ring. e.g., represented by the formula ) -- qN -16 In preferred embodiments, R32 is a small hydrophobic group, e.g., a lower alkyl or a halogen. In preferred embodiments, R30 represents a C-terminally linked amino acid residue or amino acid analog, or a C-terminally linked peptide or peptide analog, or an amino 5 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. 10 Another representative class of compounds for use in the subject method include peptide and peptidomimetics of (D)-Ala-(L)-Ala, e.g., preserving the diasteromeric orientation. Such inhibitors include compounds represented by the general formula: R N L W R1 D 0 R wherein 15 W represents a functional group which reacts with an active site residue of the targeted protease. as for example, -CN, -CI=NR 5 0 0 y seC o1 o1 / I5 o -- X -P-X B, - -- Rs 2 or R5 O Rsi R, 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, or 0 S 0

R

6 -C-, R 6 -C- R 6

-S

0 20

R

3 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 thioformate), an amino. an acylamino. an amido, a cyano, a nitro. an azido, a sulfate, a sulfonate, a sulfonamido. -(CH 2 )mn-R 7 , -(CH2)m-OH, 25 (CH 2 )m-0-lower alkyl, -(CH,)m,-0-lower alkenyl, -(CH 2 ),-0-(CH 2 )m 1

-R

7 , -(CH 2 )m-SH, (CH-))m-S-lower alkyi, -(CH-)m,-S-lowver alkenyl, -(Cl-H2),-S-(C H-)m,-Ry; - 17

R

5 represents H, an alkyl., an alkenyl, an alkynyl, -C(X 1

)(X

2

)X

3 , -(CH 2 )m-R 7 , (CH-2)n-OH, -(CH 2 )n-O-alkyl, -(CH 2 )n-O-alkenyl, -(CH 2 )n-0-alkynyl, -(CH2)n-O (CH-,)m-R 7 , -(CH 2 )n-SH, -(CH,)n-S-alkyl, -(CH-,)n-S-alkenyl, -(CH 2 )n-S-alkynyl, (CH2)n-S-(CH 2 )m-R 7 , -C(O)C(O)NH 2 , -C(O)C(O)OR' 7 ; 5 R 6 represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl. an aryl, -(CH2)m

R

7 , -(CH 2 )m-OH, -(CI 2 )m-0-alkyl, -(CH 2 )m-O-alkenyl, -(CH 2 )m-O-alkynyl; -(CH 2 )m-0

(CH

2 )m-R 7 , -(CH2)m-SH, -(CH 2 )m-S-alkyl. -(CH 2 )m-S-alkenyl, -(CH 2 )m-S-alkynyl, (CH)m-S-(CH 2 )m-R 7 ,

R

7 represents, for each occurrence, a substituted or unsubstituted aryl, aralkyl., 10 cycloalkyl, cycloalkenyl, or heterocycle; R'7 represents, for each occurrence, hydrogen, or a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R

6 1 and R 6 2, indepedently, represent small hydrophobic groups: Yi and Y, can independently or together be 011, or a group capable of being 15 hydrolyzed to a hydroxyl group, including cyclic derivatives where Y 1 and Y2 are connected via a ring having from 5 to 8 atoms in the ring structure (such as pinacol or the like),

R

50 represents 0 or S;

R

51 represents

N

3 . SH2, NH2-, NO-2 or OR' 7 ; 20 R 5 2 represents hydrogen, a lower alkyl, an amine, OR' 7 , or a pharmaceutically acceptable salt, or R 5 1 and R 5 2 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 XI represents a halogen;

X

2 and X 3 each represent a hydrogen or a halogen 25 m is zero or an integer in the range of I to 8; and n is an integer in the range of 1 to 8. In preferred embodiments, Rl is

R

36 R 38 0 wherein R36 is a small hydrophobic group, e.g., a lower alkyl or a halogen and R38 is 30 hydrogen, or, R36 and R37 together form a 4-7 membered heterocycle including the N and the Cct 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, R3 is a hydrogen, or a small hydrophobic group such as a 5 lower alkyl or a halogen. In preferred embodiments, RS is a hydrogen, or a halogentated lower alkyl. In preferred embodiments, X1 is a fluorine, and X2 and X3, if halogens, are fluorine. In preferred embodiments, R 6 1 and R 62 , independently. represent low alkyls, such 10 as methyl, ethyl, propyl, isopropyl, tert-butyl or the lik.; Also included are such peptidomimetics as olefins, phosphonates., aza-amino acid analogs and the like. Also deemed as equivalents are any compounds which can be hydrolytically converted into any of the aforementioned compounds including boronic acid esters and 15 halides, and carbonyl equivalents including acetals,hemiacetals, ketals, and hemiketals. and cyclic dipeptide analogs. As used herein, the definition of each expression, e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure. 20 The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g.. from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as 25 acetic, propionic, succinic, glycolic, stearic, lactic, malic. tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2 acetoxybenzoic. fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. The pharmaceutically acceptable salts of the present invention can be synthesized 30 from the subject compound which contain a basic or acid moiety by conventional chemical methods. Generally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid or base in a suitable solvent. The pharmaceutically acceptable salts of the acids of the -19 subject compounds are also readily prepared by conventional procedures such as treating an acid of Formula I with an appropriate amount of a base such as an alkali or alkaline earth methyl hydroxide (e.g. sodium, potassium, lithium, calcium or magnesium) or an organic base such as an amine, piperidine, pyrrolidine, benzylamine and the like, or a quatemary 5 ammonium hydroxide such as tetramethylammonium hydroxide and the like. Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g. the ability to inhibit proteolysis of GLP-1 or other peptide hormone or precursor thereof), wherein one or more simple variations of substituents are made which do not 10 adversely affect the efficacy of the compound in use in the contemplated method. In )eneral, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using, readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in I S themselves known, but are not mentioned here. ii. D einiliois '~ ~ For convenience, before further description of the present invention, certain terms employed in the specification, examples, and appended claims are collected here. 20 The term "alkvl" refers to the radical of saturated aliphatic groups. including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Cl-C 3 0 for straight chain, C 3

-C

3 0 for branched chain), and more 25 preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. Moreover, the term "alkyl" (or "lower alkyl") as used throughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or 30 more carbons of the hydrocarbon backbone. Such substituents can include, for example, a halogen, a hvdroxyl, a. carbonyl (such as a carboxyl, an ester, a formyl. or a ketone). a thiocarbonyl (such as a thioester, a thioacetate., or a thioformate), an alkoxyl, a phosphoryl, a phosphonate, a phosphinate, an amino, an amido, an amidine. an imine, a cyano. a nitro. an azido., a sulfiydryl, an alkylthio. a sulfate, a sulfonate, a sulfamoyl, a sulfonamido. a 35 sulfonyl, a heterocyclyl, an aralkyl. or an aromatic or heteroaromatic moiety. It will be - 20 understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, 5 sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes. carboxylates, and esters), -CF 3 , -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls. carbonyl-substituted alkyls, -CF 3 , CN, and the like. 10 The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group). The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. 15 Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group,. as defied above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl. 20 The term "aryl" as used herein includes 5-, 6- and 7-membercd single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, ) furan., thiophene, imidazole, oxazole, thiazole., triazole, pyrazole, pyridine, pyrazine. pyridazine and pyrimidine. and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic 25 ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl. hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl., silyl, ether, alkylthio, sulfonyl, sul fonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF 3 , -CN, or the like. The term "aryl" also includes polycyclic 30 ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are' "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls. aryls and/or heterocyclyls. The terms "heterocvclvl" or "heterocyclic group" refer to 3- to 10-membered ring 35 structures, more preferably 3- to 7-membered rings. whose ring structures include one to -21 four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups includefor example, thiophene, thianthrene, furan, pyran. isobenzofuran. chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, 5 isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine; phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine. morpholine, lactones, lactams such as azetidinones and pyrrolidinones. sultans, sultones, and the like. The heterocyclic ring can be substituted 10 at one or more positions with such substituents as described above. as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl. amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF 3 . -CN, or the like. 15 The terms "polycyclyl" or "polycyclic group" refer to two or more rings (e.g. cycloalkyls, cycloalkenyls, cycloalkynyls. aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings ate "fused rings" Riigs that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, 20 halogen, alkyl, aralkyl, alkenyl, alkynyl. cycloalkyl. hydroxyl, amino, nitro, sulthydryl, imino, amido, phosphonate, phosphinate, carbonyl; carboxyl. silyl. ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, CF 3 -CN. or the like. The term "carbocycle", as used herein, refers to an aromatic or non-aromatic ring in 25 which each atom of the ring is carbon. The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorous. As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH: the tem "hydroxyl" means -OH; and the term 30 "sulfonyl" means -SO2)-. The terms "amine" and "amino" are art recognized and refer to both unsubstituted and substituted amines., e.g., a moiety that can be represented by the general formula: Rio - 1 + _N -- N--Ro or

R

9 wherein R 9 , R 10 and R' 10 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)m-R8, or R 9 and R 1 0 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R8 represents an aryl, 5 a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of ] to 8. In preferred embodiments, only one of R 9 or R10 can be a carbonyl, e.g.,

R

9 , R 10 and the nitrogen together do not form an imide. In even more preferred embodiments, R 9 and R 10 (and optionally R' 1 0 ) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)m-RS. Thus. the term "alkylamine" as used herein means an 10 amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R 9 and R 10 is an alkyl group. The term "acylamino" is art-recognized and refers to a moiety that can be represented by the general formula: -- N RI R.. 15 wherein R9 is as defined above, and R'i1 represents a hydrogen, an alkyl, an alkenyl or -(CH2)nRg, where m and Rg are as defined above. The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula: 0 N __R9 Ri 20 wherein R 9 , R 10 are as defined above. Preferred embodiments of the amide will not include imides which may be unstable. The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2))m-R8, wherein m and RS are defined 25 above. Representative alkylthio groups include methyIthio, ethyl thio, and the like.

-23 The term "carbonyl" is art recognized and includes such moieties as can be represented by the general formula: X-Ru , or_ _ , wherein X is a bond or represents an oxygen or a. sulfur; and RI 1 -represents a hydrogen, an 5 alkyl, an alkenyl, -(CH2))m-RS or a pharmaceutically acceptable salt, R'l I represents a hydrogen, an alkyl, an alkenyl or -(CH2)m-R8, where m and R 8 are as defined above. Where X is an oxygen and R 1 I or R'l I is not hydrogen, the formula represents an "ester". Where X is an oxygen. and R 1 I is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when RI I is a hydrogen. the formula represents a 10 "carboxylic acid". Where X is an oxygen, and R'l I is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where X is a sulfur and R 1 I or R'l I is not hydrogen, the formula represents a "thiolester." Where X is a sulfur and R 1 I is hydrogen, the formula represents a "thiolcarboxylic acid." Where X is a sulfur and RI I' is hydrogen, the formula represents a "thiolformate." On the other hand, where X is a bond., and Ri 1 is not hydrogen, the above formula represents a "ketone" group. Where X is a bond, and RI I is hydrogen, the above formula represents an "aldehyde" group. The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group. as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include 20 methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -0-alkyl, -0 alkenyl, -O-alkynyl, -O-(CH2)m-RS, where m and R8 are described above. The term "sulfonate" is art recognized and includes a moiety that can be represented 25 by the general formula: 0

-S-OR

4 1 0 in which R 4 1 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl. The term "sulfate" is art recognized and includes a moiety that can be represented by the general formula: - 24 0

---

S--OR41 II 0 in which R41 is as defined above. The term "sulfonamido" is art recognized and includes a moiety that can be represented by the general formula: 0 || 11 -- N-S-R' 5 0 in which R 9 and R'll are as defined above. The term "sulfamoyl" is art-recognized and includes a moiety that can be represented by the general formula: 0 R -S-N

..............

II.- \ o 9 10 in which R 9 and R 10 are as defined above. The terms "sulfoxido" or -sulfinyl". as used herein. refers to a moiety that can be represented by the general formula: 0 in which R 4 4 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl. 15 cycloalkyl. heterocyclyl, aralkyl, or aryl. A "phosphoryl" can in general be represented by the formula: Q, I wherein Ql represented S or 0, and R 4 6 represents hydrogen. a lower alkyl or an aryl. When used to substitute, e.g., an alkyl, the phosphoryl group of the phosphorylalkyl can be 20 represented by the general formula: -25 Qi Qi -Q-P-O- -Qp--OR4 I , or 2 1|

OR

4 OR 4 wherein QI represented S or 0, and each R 4 6 independently represents hydrogen, a lower alkyl or an aryl, Q2 represents 0, S or N. When QI is an S. the phosphoryl moiety is a "phosphorothioate". 5 A "phosphoramidite" can be represented in the general formula: 0 0 -- Q--P-O- -- Q-----ORg , or 2 | N(R)RIO N(R,)RIO wherein R 9 and R 10 are as defined above, and Q2 represents 0. S or N. A "phosphonamidite" can be represented in the general formula:

R

4 S

R

4 8 -Q;- -- o-- p--ORs 2~W - orQT.2 O 4 N(Rg) RIO N(R,) RIO 10 wherein R 9 and R 10 are as defined above, Q- represents 0, S or N. and R4S represents a lower alkyl or an aryl. Q2 represents 0, S or N. A "selenoalkyl" refers to an alkyl group having a substituted seleno group attached thereto. Exemplary "selenoethers" which may be substituted on the alkyl are selected from one of -Se-alkyl. -Se-alkenyl. -Se-alkynyl, and -Se-(CH)nrR 7 , m and R 7 being defined 15 above. Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls. It will be understood that "substitution" or "substituted with" includes the implicit 20 proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible 25 substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched. carbocyclic and heterocyclic, aromatic and -26 nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described hereinabove. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible 5 substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds. A "small" substituent is one of 10 atoms or less. By the terms "amino acid residue" and "peptide residue" is meant an amino acid or 10 peptide molecule without the -OH of its carboxyl group. In general the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-LUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For instance Met, Ile, Leu, Ala and Gly represent "residues"of methionine, isoleucine. leucine. alanine and glycine, respectively. By the 15 residue is meant a radical derived from the corresponding a-amino acid by eliminating the OH portion of the carboxyl group and the H portion of the a-amino group. The term "amino acid side chain" is that paftof'ah'a'mino acid exclusive of the - CI(NH-,)COOH portion, as defined by K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33; examples of such side chains of the 20 common amino acids are -CHiCH-2SCH; (the side chain of methionine). -CH 2

(CH

3

)

CH-,CH

3 (the side chain of isoleucine), -C-I 2

CH(CH

3

)

2 (the side chain of leucine) or H (the side chain of glycine). For the most part, the amino acids used in the application of this invention are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or 25 catabolic products of such amino acids which contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine. valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine. and tryptophan, and those amino acids and 30 amino acid analogs which have been identified as constituents of peptidylglycan bacterial cell walls. The term amino acid residue further includes analogs, derivatives and congeners of any specific amino acid referred to herein. as well as C-terminal or N-terminal protected amino acid derivatives (e.g. modified with an N-terminal or C-terminal protecting group). 35 For example, the present invention contemplates the use of amino acid analogs wherein a -27 side chain is lengthened or shortened while still providing a' carboxyl, amino or other reactive precursor functional group for cyclization, as well as amino acid analogs having variant side chains with appropriate functional groups). For instance, the subject compound can include an amino acid analog such as, for example. cyanoalanine, canavanine, djenkolic 5 acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5 hydroxytryptophan, 1-methylhistidine, 3-rne-thylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having side chains which are suitable herein will be recognized by those skilled in the art and are included in the scope of the present invention. 10 Also included are the (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomeric forms. The configuration of the amino acids and amino acid residues herein are designated by the appropriate symbols (D), (L) or (DL), furthermore when the configuration is not designated the amino acid or residue can have the configuration (D), (L) or (DL). It will be noted that the structure of some of the 15 compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of this invention. Such isomers can be obtained in substantially pure form -by, classical separation techniques and by sterically controlled synthesis. For the purposes of this application, unless expressly noted to the contrary, a named amino acid shall be construed 20 to include both the (D) or (L) stereoisomers. The phrase "protecting group" as used herein means substituents which protect the reactive functional group from undesirable chemical reactions. Examples of such protecting groups include esters of carboxylic acids and boronic acids, ethers of alcohols and acetals and ketals of aldehydes and ketones. For instance, the phrase "N-terminal protecting 25 group" or "amino-protecting group" as used herein refers to various amino-protecting groups which can be employed to protect the N-terminus of an amino acid or peptide against undesirable reactions during synthetic procedures. Examples of suitable groups include acyl protecting groups such as, to illustrate, formyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl; aromatic urethane protecting groups as, for 30 example, benzyloxycarbonyl (Cbz); and aliphatic urethane protecting groups such as t butoxycarbonyl (Boc) or 9 -Fluorenylmethoxycarbonyl (FMOC). As noted above- certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D) 35 isomers. (L)-isomers, the racenic mixtures thereof, and other mixtures thereof. as falling within the scope of the invention. Additional asymmetric carbon'atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended. to be included in this invention. If, for instance, a particular enantiomer of a compound of the present invention is 5 desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an.acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed ) 10 by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements. CAS version, Handbook of Chemistry and 15 Physics, 67th Ed.. 1986-87, inside cover. Also for purposes of this invention, the term "hydrocarbon" is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds which can be substituted or unsubstituted. 20 A compound is said to have an "insulinotropic activity" if it is able to stimulate, or cause the stimulation of, the synthesis or expression of the hormone insulin. ) iii. Exemplary' Formilations 25 A. .4gonisni of GLP-I effects The inhibitors useful in the subject methods possess, in certain embodiments, the ability to lower blood glucose levels, to relieve obesity, to alleviate impaired glucose tolerance, to inhibit hepatic glucose neogenesis, and to lower blood lipid levels and to 30 inhibit aldose reductase. They are thus useful for the prevention and/or therapy of hyperglycemia, obesity, hyperlipidemia. diabetic complications (including retinopathy, nephropathy, neuropathy, cataracts, coronary artery disease and arteriosclerosis) and furthermore for obesity-related hypertension and osteoporosis.

- 29 Diabetes mellitus is a disease characterized by hypergfycemia occurring from a relative or absolute decrease in insulin secretion, decreased insulin sensitivity or insulin resistance. The morbidity and mortality of this disease result from vascular, renal, and neurological complications. An oral glucose tolerance test is a clinical test used to diagnose 5 diabetes. In an oral glucose tolerance test, a patient's physiological response to a glucose load or challenge is evaluated. After ingestin' the glucose, the patient's physiological response to the glucose challenge is evaluated. Generally, this is accomplished by determining the patient's blood glucose levels (the concentration of glucose in the patient's plasma, serum or whole blood) for several predetennined points in time. 110 As described in the appended examples, we demonstrate that, in vivo, high affinity inhibitors of DPIV are biologically active with respect to regulation of glucose metabolism. For example, a single injection of the inhibitor Pro-boro-Pro (see examples for structure) was alone sufficient to improve glucose control. A single injection of Pro-boro-Pro was also observed to potentiate the response to a subtherapeutic dose of GLP-1. We have also 15 observed that chronic (>5 days) treatment with Pro-boro-Pro alone lowers both fasting blood sugars, and the glycemic excursion to oral glucose challenge. As indicated above, the inhibitors useful in the subject method can be peptide- or peptidomimetic-derived inhibitors of the target proteolytic activity, or can be a non-peptide compound identified. e.g., by drug screening assays described herein. With respect to 20 DPIV inhibitors, a salient feature of the subject method is the unexpected finding that certain DPIV inhibitors have antidiabetic activity at concentrations significantly lower than the EC50 of the compound as an immunosuppressant. Thus, an animal can be dosed under a regimen designed to provide a blood serum concentration of inhibitor at or about the EC50 for antidiabetic effects, and still be sufficiently below the EC50 for 25 immunosuppressive activity so as to avoid complications resulting from that activity. Indeed, for certain of the subject inhibitors, it is anticipated that dosing can be at least an order of magnitude or more greater than the antidiabetic EC50, yet still remain sufficiently below a dose producing any significant immunosuppression. As discussed further below, a variety of assays are available in the art for identifying 30 potential inhibitors of DPIV and the like, as well as assessing.thevarious..biological... activities (including side-effects and toxicity) of such an inhibitor. B. Agonism of other peplide hormones -30 In another embodiment, the subject agents can be used:to agonize (e.g., mimic or potentiate) the activity of other polypeptide hormones. To illustrate, the present invention provides a method-for agonizing the action of GLP-2. It has been determined that GLP-2 acts as a trophic agent., to promote growth of 5 gastrointestinal tissue. The effect of GLP-2 is marked particularly by increased growth of the small bowel, and is therefore herein referred to as an "intestinotrophic" effect. In still other embidiments, the subject method can be used to increase the half-life of other proglucagon-derived peptides, such as glicentin, oxyntomodulin, glicentin-related pancreatic polypeptide (GRPP), and/or intervening peptide-2 (IP-2). For example, glicentin 10 has been demonstrated to cause proliferation of intestinal mucosa and also inhibits a peristalsis of the stomach, and has thus been elucidated as useful as a therapeutic agent for digestive tract diseases, thus leading to the present invention. Thus, in one aspect, the present invention relates to therapeutic and related uses of DPIV inhibitors for promoting the growth and proliferation of gastrointestinal tissue, most 15 particularly small bowel tissue. For instance, the subject method can be used as part of a regimen for treating injury,, inflammation or rejection of intestinal tissue, e.g., where enhanced growth and repair of the intestinal mucosal epithelial is desired. With respect to small bowel tissue, such grovh is measured conveniently as a increase in small bowel mass and length, relative to an untreated control. The effect of 20 subject inhibitors on small bowel also manifests as an increase in the height of the crypt plus villus axis. Such activity is referred to herein as an "intestinotrophic" activity. The efficacy of the subject method may also be detectable as an increase in crypt cell proliferation and/or a decrease in small bowel epithelium apoptosis. These cellular effects may be noted most significantly in relation to the jejunum, including the distal jejunum and 25 particularly the proximal jejunum, and also in the distal ileum. A compound is considered to have "intestinotrophic effect" if a test animal exhibits significantly increased small bowel weight, increased height of the crypt plus villus axis, or increased crypt cell proliferation or decreased small bowel epithelium apoptosis when treated with the compound (or genetically engineered to express it themselves). A model suitable for determining such 30 gastrointestinal growth is described by US Patent 5,834,428. In general, patients who would benefit from either increased small intestinal mass and consequent increased small bowel mucosal function are candidates for treatment by the subject method. Particular conditions that may be treated include the various forms of sprue including celiac sprue which results from a toxic reaction to ct-gliadin from wheat, and is 35 marked by a tremendous loss of villae of the bowel: tropical sprue which results from - 31 infection and is marked by partial flattening of the village; hypocgammaglobulinemic sprue which is observed commonly in patients with common variable immunodeficiency or hypogammaglobulinemia and is marked by significant decrease in villus height. The therapeutic efficacy of the treatment may be monitored by enteric biopsy to examine the 5 villus morphology, by biochemical assessment of nutrient absorption, by patient weight gain, or by amelioration of the symptoms associated with these conditions. Other conditions that may be treated by the subject method; or for which the subject method may be useful prophylactically, include radiation enteritis, infectious or post-infectious enteritis, regional enteritis (Crohn's disease), small intestinal damage due to toxic or other chemotherapeutic 10 agents, and-patients with short bowel syndrome. More generally, the present invention provides a therapeutic method for treating digestive tract diseases. The term "digestive tract" as used herein means a tube through which food passes, including stomach and intestine. The term "digestive tract diseases" as used herein means diseases accompanied by a qualitative or quantitative abnormality in the 15 digestive tract mucosa, which include, e. g., ulceric or inflammatory disease; congenital or acquired digestion and absorption disorder including malabsorption syndrome; disease caused by loss of a mucosal barrier -function of the. -gut;. and protein-losing gastroenteropathy. The ulceric disease includes, e.g., gastric ulcer, duodenal ulcer, small intestinal ulcer, colonic ulcer and rectal ulcer. The inflammatory disease include, e.g., 20 esophagitis, gastritis, duodenitis, enteritis, colitis, Crohn's disease, proctitis, gastrointestinal Behcet, radiation enteritis, radiation colitis, radiation proctitis, enteritis and medicamentosa. The malabsorption syndrome includes the essential malabsorption syndrome such as disaccharide-decomposing enzyme deficiency, glucose-galactose malabsorption, fractose malabsorption; secondary malabsorption syndrome. e.g., the disorder caused by a mucosal 25 atrophy in the digestive tract through the intravenous or parenteral nutrition or elemental diet, the disease caused by the resection and shunt of the small intestine such as short gut syndrome, cul-de-sac syndrome; and indigestible malabsorption syndrome such as the disease caused by resection of the stomach, e.g., dumping syndrome. The term "therapeutic agent for digestive tract diseases" as used herein means the 30 agents for the prevention and treatment of the digestive tract diseases, which include, e.g., the therapeutic agent for digestive tract ulcer, the therapeutic agent for inflammatory digestive tract disease, the therapeutic agent for mucosal atrophy in the digestive tract and the therapeutic agent for digestive tract wound, the amelioration agent for the function of the digestive tract including the agent for recovery of the mucosal barrier function and the 35 amelioration agent for digestive and absorptive function. The ulcers include digestive ulcers and erosions, acute ulcers, namely, acute mucosal lesions.

The subject method, because of promoting proliferation of intestinal mucosa, can be used in the treatment and prevention of pathologic conditions of insufficiency in digestion and absorption, that is, treatment and prevention of mucosal atrophy, or treatment of hypoplasia of the digestive tract tissues and decrease in these tissues by surgical removal as 5 well as improvement of digestion and absorption. Further, the subject method can be used in the treatment of pathologic mucosal conditions due to inflammatory diseases. such as enteritis, Crohn's disease and ulceric colitis and also in the treatment of reduction in function of the digestive tract after operation, for example, in damping syndrome as well as in the treatment of duodenal ulcer in conjunction with the inhibition of peristalsis of the 10 stomach and rapid migration of food from the stomach to the jejunum. Furthermore, glicentin can effectively be used in promoting cure of surgical invasion as well as in improving functions of the digestive tract. Thus, the present invention also provides a therapeutic agent for atrophy of the digestive tract mucosa, a therapeutic agent for wounds in the digestive tract and a drug for improving functions of the digestive tract which 15 comprise glicentin as active ingredients. 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 polypeptide family, as DPIV has been implicated in the 20 processing of those peptides in a manner which alters receptor selectivity. Neuropeptide Y (NPY) is believed to act in the regulation vascular smooth muscle tone, as well as regulation of blood pressure. NPY also decreases cardiac contractility. NPY is also the most powerful appetite stimulant known (Wilding et al., (1992) J_ Endocrinology 132:299-302). The centrally evoked food intake (appetite stimulation) effect 25 is predominantly mediated by NPY Yl receptors and causes increase in body fat stores and obesity (Stanley et al., (1989) Physioloav and Behavior 46:173-177). According to the present invention, a method for treatment of anorexia comprises administering to a host subject an effective amount of a DPIV inhibitor to stimulate the appetite and increase body fat stores which thereby substantially relieves the symptoms of 30 anorexia. A method for treatment of hypotension comprises administering to a host subject an effective amount of a*DPIV inhibitor of the present invention to mediate vasoconstriction and increase blood pressure which thereby substantially relieves the symptoms of hypotension.

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 5 inhibitory peptide (GIP) and helodermin. Kubiak et al. (1994) Peptide Res 7:153. GHRF is secreted by the hypothalanius, 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 10 used in veterinary practice, for example, to develop higher yield milk production and higher yield. leaner livestock. C. Examples ofpeptidyl DPIV inhibitors In the case of DPIV inhibitors, a preferred class of inhibitors are peptidyl 15 compounds based on the dipeptides Pro-Pro or Ala-Pro. Another preferred class of peptidyl inhibitors are compounds based on-the dipeptide.(D)-Ala-(L)-Ala. In many embodiments, it will be desirable to provide the peptidyl moiety as a peptidomimetic, e.g., to increase bioavailability and/or increase the serum half-life relative to the equivalent peptide. For instance, a variety of peptide backbone analogs are available in the art and be readily 20 adpated for use in the subject methods. In an exemplary embodiment, the peptidomimetic can be derived as a retro-inverso analog of the peptide. To illustrate, certain of the subject peptides can be generated as the retro-inverso analog (shown in its unprotected state): R 0 C 'H HN N OH HN L NB H I R' 0 0 OH 25 Such retro-inverso analogs can be made according to the methods known in the art, such as that described by the Sisto et al. U.S. Patent 4,522,752. For example, the illustrated retro-inverso analog can be generated as follows. The geminal diamine corresponding to the N-terminal amino acid analogs is synthesized by treating an N-Boc-protected amino acid (having the sidechain R) with ammonia under HOBT-DCC coupling conditions to 30 yield amide, and then effecting a Hofmann-type rearrangement with 1,1-bis (trifluoroacetoxv)iodobenzene (TIB), as described in Radhakrishna et al. (1979) J. Org - 34 Chem. 44:1746. The product amine salt is then coupled to a side-chain protected (e.g., as the benzyl ester) N-Fmoc D-enatiomer of the second amino acid residue (e.g., having a sidechain R') under standard conditions to yield the pseudodipeptide. The Fmoc (fluorenylmethoxycarbonyl) group is removed with piperidine in dimethylformamide, and 5 the resulting amine is trimethylsilylated with bistrimethylsilylacetamide (BSA) before condensation with suitably alkylated, side-chain protected derivative of Meldrum's acid, as described in U.S. Patent 5,061,811 to Pinori et al., to yield the retro-inverso tripeptide analog. The pseudotripeptide is then coupled with (protected) boro-proline under standard conditions to give the protected tetrapeptide analog. The protecting groups are removed to 10 release the final product, which is purified by HPLC. In another illustrative embodiment, the peptidomimetic can be derived as a retro enantio analog of the peptide. HO O C0 0 R H B\ D -H N D COOR H IH 0 R Retro-enantio analogs such as this can be synthesized using D-enatiomers of commercially 15 available D-amino acids or other amino acid analogs and standard solid- or solution-phase peptide-synthesis techniques. In still another illustrative embodiment, trans-olefin derivatives can be made with the subject boronophenylalanine analogs. For example, an exemplary olefin analog is: ) R CH3 H H2NN -'-OH

H

2 N--Y O R ' 0 OH 20 The trans olefin analog can be synthesized according to the method of Y.K. Shue et al. (1 987) Tetrahedron Letters 28:3225. Still another class of peptidomimetic boronophenylalanine derivatives include the phosphonate derivatives, such as: R H 0 CH

H

2 N NNQ OH O--I 'OOOH O R1 0 OH -35 The synthesis of such phosphonate derivatives can be adapted from known synthesis schemes. See, for example, Loots et al. in Peptides. Chemistry and Biolog, (Escom Science Publishers, Leiden, 1988, p. 118); Petrillo et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co. 5 Rockland, IL, 1985). D. Non-peptidyl DPIV inhibitors The pharmaceutical industry has developed a variety of different strategies for assessing millions of compounds a year as potential lead compounds based on inhibitory .10 activity against an enzyme. DPIV and other proteolytic enzymes targeted by the subject method are amenable to the types of high throughput screening required to sample large arrays of compounds and natural extracts for suitable inhibitors. As an illustrative embodiment, the ability of a test agent to inhibit DPIV can be assessed using a colorimetric or fluorometric substrate, such as Ala-Pro-paranitroanilide. 15 See US Patent 5.,462,928. Moreover, DPIV can be purified, and is accordingly readily amenable for use in such high throughput formats as multi-well plates. Briefly, DPIV is purified from pig kidney cortex (Barth et al. (1974) Acta Biol Med Germ 32:157; Wolf et al. (1972) Acta Bio Mes Germ 37:409) or human placenta (Puschel et al. (1982) Eur J Biochein 126:359). An illustrative reaction mixture includes 50pM 20 sodium H-pes (pH7.8), 10aM Ala-Pro-paranitroanilide., 6 milliunits of DPIV, and 2% (v/v) dimethylformamide in a total volume of 1.0 mL. The reaction is initiated by addition of enzyme, and formation of reaction product (paranitroanilide) in the presence and absence of a test compound can be detected photometrically, e.g., at 410nnm. Exemplary compounds which can be screened for activity against DPIV (or other 25 relevant enzymes) include peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries, such as isolated from animals, plants, fungus and/or microbes. E. Assays of"Insulinotropic Activity 30 In selecting a compound suitable for use in the subject method, it is noted that the insulinotropic property of a compound may be determined by providing that compound to animal cells, or injecting that compound into animals and monitoring the release of immunoreactive insulin (IRI) into the media or circulatory system of the animal, - 36 respectively. The presence of IRI can be detected through the use of a radioimmunoassay which can specifically detect insulin. The db/db mouse is a genetically obese and diabetic strain of mouse. The db/db mouse develops hyperglycemia and hyperinsulinemia concomitant with its development of 5 obesity and thus serves as a model of obese type 2 diabetes (NIDDM). The db/db mice can purchased from, for example, The Jackson Laboratories (Bar -Iarbor, Me.). In an exemplary embodiment, for treatment of the mice with a regimen including a DPIV inhibitor or control, sub-orbital sinus blood samples are taken before and at some time (e.g., 60 minutes) after dosing of each animal. Blood glucose measurements can be made by any 10 of several conventional techniques. such as using a glucose meter. The blood glucose levels - of the control and DPIV inhibitor dosed animals are compared The metabolic fate of exogenous GLP- I can also be followed in either nondiabetic and type II diabetic subjects, and the effect of a candidate DPIV inhibitor determined. For instance, a combination of high-pressure liquid chromatography (HPLC), specific 15 radioimmunoassays (RIAs), and a enzyme-linked immunosorbent assay (ELISA), can be used, whereby intact biologically active GLP-1 and its metabolites can be detected. See, for example, Deacon et al. (1995) Diabetes 44:1126-1131,. To illusItrIate, after GLP-f administration, the intact peptide can be measured using an NH2-terminally directed RIA or ELISA, while the difference in concentration between these assays and a COOH-terrninal 20 specific RIA allowed determination of NH2-tenninally truncated metabolites. Without inhibitor, subcutaneous GLP-1 is rapidly degraded in a time-dependent manner, forming a metabolite which co-elutes on HPLC with GLP-1(9-36) amide and has the same immunoreactive profile. For instance, thirty' minutes after subcutaneous GLP-1 administration to diabetic patients (n = 8), the metabolite accounted for 88.5 + 1.9% of the 25 increase in plasma immunoreactivity determined by the COOH-terminal RIA, which was higher than the levels measured in healthy subjects (78.4 + 3.2%; n = 8; P < 0.05). See Deacon et al., supra. Intravenously infused GLP-l was also extensively degraded. F. Pharmaceutical Formulations 30 The inhibitors can be administered in various forms, depending on the disorder to be treated and the age, condition and body weight of the patient, as is well known in the art. For example, where the compounds are to be administered orally, they may be formulated as tablets, capsules, granules, powders or syrups; or for parenteral administration, they may be formulated as injections (intravenous. intramuscular or subcutaneous), drop infusion 35 preparations or suppositories. For application by the ophthalmic mucous membrane route.

- 37 they may be formulated as eyedrops or eye ointments. These formulations can be prepared by conventional means, and, if desired, the active ingredient may be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent or a coating agent. 5 Although the dosage will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the drug, in general, a daily dosage of from 0.01 to 2 0 00 mg of the compound is recommended for an adult human patient, and this may be administered in a single dose or in divided doses. 10 Glucose metabolism can be altered, and symptoms associated with type II diabetes can be decreased or eliminated, in accordance with a "timed" administration of DPIV inhibitors wherein one or more appropriate indices for glucose metabolism and/or type I diabetes can be used to assess effectiveness of the treatment (dosage and/or timing): e.g. glucose tolerance, glucose level, insulin level, insulin sensitivity, glycosylated hemoglobin. 15 An effective time for administering DPIV inhibitors needs to be identified. This can be accomplished by routine experiment as described below, using one or more groups of animals (preferably af leasi 5 aiinials per group). In animals, insulinotropic activity by DPIV inhibitor treatment can be assessed by administering the inhibitor at a particular time of day and measuring the effect of the 20 administration (if any) by measuring one or more indices associated with glucose metabolism, and comparing the post-treatment values of these indices to the values of the same indices prior to treatment. The precise time of administration and/or amount of DPIV inhibitor that will yield the most effective results in terms of efficacy of treatment in a given patient will depend 25 upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, disease type and stage, general physical condition. responsiveness to a given dosage and type of medication), route of administration, etc. However, the above guidelines can be used as the basis for fine-tuning the treatment. e.g., determining the optimum time and/or amount of administration, which 30 will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing. While the subject is being treated, glucose metabolism is monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour period. Treatment (amounts, times of administration and type of medication) may be adjusted 35 (optimized) according to the results of such monitoring. The patient is periodically - 38 reevaluated to determine extent of improvement by measuring the same parameters, the first such reevaluation typically occurring at the end of four weeks from the onset of therapy, and subsequent reevaluations occurring every 4 to 8 weeks during therapy and then every 3 months thereafter. Therapy may continue for several months or even years with six months 5 being a typical length of therapy for humans. Adjustments to the amount(s) of drug(s) administered and possibly to the time of administration may be made based on these reevaluations. For example, if after 4 weeks of treatment one of the metabolic indices has not improved but at least one other one has, the dose could be increased by 1/3 without changing the time of administration. 10 Treatment can be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. The phrase "therapeutically-effective amount" as used herein means that amount of, 15 e.g., a DPIV inhibitor(s), which is effective for producing some desired therapeutic effect by inhibiting, for example, the proteolysis of a peptide hormone at a reasonable benefitl/risk ratio applicable to any medical treatment. The phrase "pharmaceutically acceptable" is employed herein to refer to those DPIV inhibitors, materials, compositions, and/or dosage forms which are, within the scope of 20 sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically-acceptable carrier" -as used herein means a pharnaceutically-acceptable material, composition or vehicle, such as a liquid or solid 25 filler, diluent, excipient., solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) 30 sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, 35 such as propylene glycol; (11) polvols, such as glycerin, sorbitol, mannitol and . -39 polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydro\ide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in 5 pharmaceutical formulations. The term "pharmaceutically-acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of DPIV inhibitors. These salts can be prepared in situ during the final isolation and purification of the DPIV Inhibitors, or by separately reacting a purified DPIV inhibitor in its free base form with a suitable organic or inorganic 10 acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate., oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66:1 15 19) In other cases, the DPIV inhibitor useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically- acceptable salts" in these instances refers to the relatively non-toxic, 20 inorganic and organic base addition salts of a DPIV inhibitor(s). These salts can likewise be prepared in situ during the final isolation and purification of the DPIV inhibitor(s), or by separately reacting the purified DPIV inhibitor(s) in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or 25 tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra). 30 Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents. release agents, coating agents, sweetening, flavoring and perfunin.; agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble 35 antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium -40 metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and 5 the like. Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may .onveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount 10 of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount 15 will range from about I per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent. Methods of preparing these formulations or compositions include the step of bringing into association a DPIV inhibitor(s) with the carrier and, optionally, one or more 20 accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a DPIV inhibitor with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets. lozenges (using a flavored basis, usually sucrose and acacia or 25 tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a DPIV inhibitor(s) as an active ingredient. A compound may also be administered as a bolus, 30 electuary or paste. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, 35 mannitol, and/or silicic acid: (2) binders, such as, for example. carboxymethylcellulose, - 41 alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such .as glycerol; (4) disintegrating agents. such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as'quaternary ammonium compounds; (7) 5 wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a 10 similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, 15 gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent. 20 Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxvpropylmethyl cellulose in varying proportions to provide the desired 25 release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they 30 release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract. optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. 35 Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions. syrups and elixirs. In addition to the - 42 active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as; for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, prop-ylene glycol, I,3-butylene glycol, oils (in particular, cottonseed, groundnut, 5 corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. 10 Suspensions, in addition to the active DPIV inhibitor(s) may contain suspending agents as. for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Formulations for rectal or vaginal administration may be presented as a suppository, 15 which may be prepared by rnixing one or more DPIV inhibitor(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent. 20 Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forn-s for the topical or transdermal administration of a DPIV inhibitor(s) include powders. sprays. ointments, pastes, creams, lotions, gels, solutions, patches and 25 inhalants. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to DP1V inhibitor(s), excipients., such as animal and vegetable fats, oils, waxes, paraffins, starch, 30 tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to a DPIV inhibitor(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide. calcium silicates and polyamide powder. or mixtures of these substances. Sprays can additionally contain customary propellants.

-43 such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. The DPIV inhibitor(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles 5 containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and 10 stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. 15 Transdermal patches have the added advantage of providing controlled delivery of a DPIV inhibitor(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer 20 matrix or gel. Ophthalmic formulations, eye ointments, powders. solutions and the like, are also contemplated as being within the scope of this invention. Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more DPIV inhibitor(s) in combination with one or more 25 pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. 30 Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol. and the like), and suitable mixtures thereof, vegetable oils, such as olive.oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by - 44 the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of 5 microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum 10 monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of 15 dissolution which, in turn. may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of DPIy inhibitor(s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the 20 ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping .the drug in liposomes or microemulsions which are compatible with body tissue. When the DPIV inhibitor(s) of the present invention are administered as 25 pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. The preparations of agents may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they 30 are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually 35 by injection, and includes, without limitation, intravenous, intramuscular, intraarterial.

-45 intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral 5 administration" and "administered peripherally" as used herein mean the administration of a DPIV inhibitor, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. These DPIV inhibitor(s) may be administered to humans and other animals for 10 therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. Regardless of the route of administration selected, the DPIV inhibitor(s), which may be used in a suitable hydrated form. and/or the pharmaceutical compositions of the present 15 invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, 20 and mode of administration, without being toxic to the patient. G. Conjoint administration Another aspect of the invention provides a conjoint therapy wherein one or more other therapeutic agents are administered with the protease inhibitor. Such conjoint 25 treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. In one embodiment, a DPIV inhibitor is conjointly administered with insulin or other insulinotropic agents, such as GLP-1 or a gene therapy vector which causes the ectopic expression of GLP-l. 30 In another illustrative embodiment, the subject inhibitors can be conjointly administered with a an MI receptor antagonist. Cholinergic agents are potent modulators of insulin release that act via muscarinic receptors. Moreover, the use of such agents can have the added benefit of decreasing cholesterol levels, while increasing HDL levels. Suitable muscarinic receptor antagonists include substances that directly or indirectly block -46 activation of muscarinic cholinergic receptors. Preferably, such substances are selective. (or are used in amounts that promote such selectivity) for the MI receptor. Nonlimiting examples include quaternary amines (such as methantheline, ipratropium, and propantheline). tertiary amines (e.g. dicyclomine, scopolamine) and tricyclic amines (e.g. 5 telenzepine). Pirenzepine and methyl scopolamine are preferred. Other suitable muscarinic receptor antagonists include benztropine (commercially available as COGENTINfrom Merck), hexahydro-sila-difenidol hydrochloride (HHSID hydrochloride disclosed in Lambrecht et al. (19S9) Trends in Pharmacol _Sci. 10(Suppl):60; ( +/- )-3-quinuclidinyl xanthene-9-carboxylate hemioxalate (QNX-herioxalate; Birdsall et al., Trends in 10 Pharmacol. Sci. 4:459, 1983; telenzepine dihydrochloride (Coruzzi et al. (1989) Arch. Int. Pharrnacodvn. Ther. 302:232; and Kawashima et al. (1990) Gen. Pharmacol. 21:17) and atropine. The dosages of such muscarinic receptor antagonists will be generally subject to optimization as outlined above. In the case of lipid metabolism disorders, dosage optimization may be necessary independently of whether administration is timed by 15 reference to the lipid metabolism responsiveness window or not. In terms of regulating insulin and lipid metabolism and reducing the foregoing disorders, the subject DPIV inhibitors may also act synergistically with prolactin inhibitors such as d2 dopamine agonists (e.g. bromocriptine).. Accordingly, the subject method can include the conjoint administration of such prolactin inhibitors as prolactin-inhibiting ergo 20 alkaloids and prolactin-inhibiting dopamine agonists. Examples of suitable compounds include 2-bromo-alpha-ergocriptine, 6-methyl-8 beta-carbobenzyloxyaminoethyl-10-alpha ergoline. 8-acvlaminoergolines, 6-methyl-8-alpha-(N-acvl)amino-9-ergoline, 6-methyl-8 alpha-(N-phenylacetyl)amino-9-ergoline, ergocormine, 9,10-dihydroergocornine, D-2-halo 6-alkyl-8-substituted ergolines, D-2-bromo-6-methyl-8-cyanomethylergoline, carbidopa, 25 benserazide and other dopadecarboxylase inhibitors, L-dopa, dopamine and non toxic salts thereof. The DPIV inhibitors used according to the invention can also be used conjointly with agents acting on the ATP-dependent potassium channel of the p-cells, such as glibenclamide, glipizide, gliclazide and AG-EE 623 ZW. The DPIV inhibitors may also 30 advantageously be applied in combination with other oral agents such as metformin and related compounds or glucosidase inhibitors as, for example, acarbose. Exemplification 35 The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration - 47 of certain aspects and embodiments of the present invention, and are not intended to limit the invention. Example 1: Synthesis of BoroProline 5 Referring to Figure 1, the starting compound I is prepared essentially by the procedure of Matteson et al. (Organometallics 3:1284, 1984), except that a pinacol ester is substituted for the pinanediol ester. Similar compounds such as boropipecolic acid and 2 azetodine boronic acid can be prepared by making the appropriate selection of starting material to yield the pentyl and propyl analogs of compound I. Further. Cl can be 10 substituted for Br in the formula. and other diol protecting groups can be substituted for pinacol in the formula, e.g., 2. 3-butanediol and alphapinanediol. Compound II is prepared by reacting compound I with [(CH 3

)O

3 Si] 2 N-Li* . In this reaction hexamethyldisilazane is dissolved in tetrahydrofuran and an equivalent of n butyllithium added at -78'C After warming to room temperature (20'C) and cooling to 15 78'C, an equivalent of compound I is added in tetrahydrofuran. The mixture is allowed to slowly come to room temperature and to stir overnight. The alpha-bis[trimethylsilane] protected amine is isolated by evaporating solvent and adding hexane under anhydrous conditions. Insoluble residue is removed by filtration under a nitrogen blanket. yielding a hexane solution of compound II. 20 Conpound III., the N-trimethysilyl protected form of boroProline is obtained by the thermal cyclization of compound It during the distillation process in which compound II is heated to 100-150'C and distillate is collected which boils 66-62'C at 0.06-0.10 mm pressure. Compound IV, boroProline-pinacol hydrogen chloride, is obtained by treatment of 25 compound III with HCI:dioxane. Excess HC and by-products are removed by trituration with ether. The final product is obtained in a high degree of purity by recrystallization from ethyl acetate. The boroProline esters can also be obtained by treatment of the reaction mixture obtained in the preparation of compound II with anhydrous acid to yield 1-amino-4 30 bromobutyl boronate pinacol as a salt. Cyclization occurs after neutralizing the salt with base and heating the reaction. Example 2: Preparation of boroProline-pinacol -48 The intermediate, 4-Bromo-1-chlorobutyl boronate pinacol, was prepared by the method in Matteson et al. (Organometallics 3:1284, 1984) except that conditions were modified for large scale preparations and pinacol was substituted for the pinanediol protecting group. 5 3-bromopropyl boronate pinacol was prepared by hydrogenboronation of allyl bromide (173 ml, 2.00 moles) with catechol borane (240 ml, 2.00 moles).. Catechol borane was added to allyl bromide and the reaction heated for 4 hours at 100'C under a nitrogen atmosphere. The product, 3-bromopropyl boronate catechol (bp 95-102'C, 0.25 mm), was isolated in a yield of 49% by distillation. The catechol ester (124 g, 0.52 moles) was 10 transesterified with pinacol (61.5 g, 0.52 moles) by mixing the component in 50 ml of THF and allowing them to stir for 0.5 hours at 0 0 C and 0.5 hours at room temperature. Solvent was removed by evaporation and 250 ml of hexane added. Catechol was removed as a crystalline solid. Quantitative removal was achieved by successive dilution to 500 ml and to 1000 ml with hexane and removing crystals at each dilution. Hexane was evaporated and 15 the product distilled to yield 177g (bp 60-64'C, 0.35 mm). 4-Bromo-l-chlorobutyl boronate pinacol was prepared by homologation of the corresponding propel boronate. Methylene chloride (50.54 ml, 0.7 13 moles) was dissolved in 500 ml of THF, 1.54N n-butyllithium in hexane (480 ml, 0.780 moles) was slowly added at -100'C. 3-Bromopropyl boronate pinacol (178 g. 0.713 moles) was dissolved in 500 ml 20 of THG, cooled to the freezing point of the solution, and added to the reaction mixture. Zinc chloride (54.4 , 0.392 moles) was dissolved in 250 ml of THG, cooled to 0 0 C, and added to the reaction mixture in several portions. The reaction was allowed to slowly warm to room temperature and to stir overnight. Solvent was evaporated and the residue dissolved in hexane (1 liter) and washed with water (1 liter). Insoluble material was discarded. After 25 drying over anhydrous magnesium sulfate and filtering, solvent was evaporated. The product was distilled to yield 147 g (bp 110-1I12 0 C, 0.200 mm). N-Trimethylsilyl-boroProline pinacol was prepared first by dissolving hexamethyldisilizane (20.0 g, 80.0 mmoles) in 30 ml of THF, cooling the solution to 78'C, and adding 1.62N n-butyllithium in hexane (49.4 ml, 80.0 mmoles). The solution was 30 allowed to slowly warm to room temperature. It was recooled to -78*C. and 4-bromo-l chlorobutyl boronate pinacol (23.9 g, 80.0 mmoles) added in 20 ml of THF. The mixture was allowed to slowly warm to room temperature and to stir ovemight. Solvent was removed by evaporation and dry hexane (400 ml) added to yield a precipitant which was removed by filbration under a nitrogen atmosphere. The filtrate was evaporated and the 35 residue distilled, yielding 19.4 g of the desired product (bp 60-62'C, 0.1-0.06 mm).

-49 H-boroProline-pinacol.HCI (boroProline-pinacol.HCI) was' prepared by cooling N trimethylsilyl-boroProline pinacol (16.0 g, 61.7 mmoles) to -78C and adding 4N HCL:dioxane 46 ml, 185 mmoles). The mixture was stirred 30 minutes at -78*C and 1 hour at room temperature. Solvent was evaporated and the residue triturated with ether to yield a 5 solid. The crude product was dissolved in chloroform and insoluble material removed by filtration. The solution was evaporated and the product crystallized from ethyl acetate to yield 11.1 g of the desired product (mp 156.5-157 0 C). Example 3: Synthesis of boroProline Peptides 10 General methods of coupling of N-protected peptides and amino acids with suitable side-chain protecting groups to H-boroProline-pinacol are applicable. When needed, side chain protecting and N-terminal protecting groups can be removed by treatment with anhydrous HCl, HBr, trifluoroacetic acid, or by catalytic hydrogenation. These procedures are known to those skilled in the art of peptide synthesis. 15 The mixed anhydride procedure of Anderson et al. (J. Am. Chem. Soc. 89:5012, 1984) is preferred for peptide coupling. Referring again to Figure 1, the mixed anhydride of an N-protected amino acid or a peptide is prepared by dissolving the peptide in tetrahydrofuran and adding one equivalent of N-nethylmorpholine. The solution is cooled to -20'C and an equivalent of isobutyl chloroformate is added. After 5 minutes, this mixture 20 and one equivalent of triethylamine (or other sterically hindered base) are added to a solution of H-boroPro-pinacol dissolved in either cold chloroform of tetrahydrofuran. The reaction mixture is routinely stirred for one hour at -20*C and I to 2 hours at room temperature (20'C). Solvent is removed by evaporation, and the residue is dissolved in ethyl acetate. The organic solution is washed with 0.20N hydrochloric acid, 5% aqueous 25 sodium bicarbonate, and saturated aqueous sodium chloride. The organic phase is dried over anhydrous sodium sulfate. filtered. and evaporated. Products are purified by either silica gel chromatography or gel permeation chromatography using Sephadex TM LH-20 and methanol as a solvent. Previous studies have shown that the pinacol protecting group can be removed in 30 situ by preincubation in phosphate buffer prior to running biological experiments (Kettner et al., J. Biol. Chem. 259:15106, 1984). Several other methods are also applicable for removing pinacol groups from peptides, including boroProline, and characterizing the final product. First. the peptide can be treated with diethanolamine to yield the corresponding diethanolamine boronic acid ester. which can be readily hydrolyzed by treatment with - 50 aqueous acid or- a sulfonic acid substituted polystyrene resin as described in Kettner et al. (supra). Both pinacol and pinanediol protecting groups can be removed by treating with BC13 in methylene chloride as described by Kinder et al. (J. Med. Chem. 28:1917). Finally, the free boronic acid can be converted to the difluoroboron derivative (-BF2) by treatment 5 with aqueous HF as described by Kinder et al. (supra). Similarly, different ester groups can be introduced by reacting the free boronic acid with various di-hydroxy compounds (for example, those containing heteroatoms such as S or N) in an inert solvent. 10 Example 4: Preparation of H-Ala-boroPro Boc-Ala-boroPro was prepared by mixed anhydride coupling of the N-Boc protected alanine and H-boroPro prepared as described above. H-Ala-boroPro (Ala boroPro) was prepared by removal of the Boc protecting group at 0 0 C in 3.5 molar excess of 4N HCI-dioxane. The coupling and deblocking reactions were performed by standard 15 chemical reaction. Ala-boroPro has a Ki for DP-lV of in the nanomolar range. Boc-blocked Ala-boroPro has no affinity for DP-IV. The two diastereomers of Ala-boroPro-pinacol, L-Ala-D-boroPro-pinacol and L Ala-L-boroPro-pinacol, can be partially separated by silica gel chromatography with 20% methanol in ethyl acetate as eluant. The early fraction appears by NMR analysis to be 95% 20 enriched in one isomer. Because this fraction has more inhibits DP-IV to a greater extent than later fractions (at equal concentrations) it is probably enriched in the L-boroPro (L Ala-L-boroPro-pinacol) isomer. Example 5: Glucose Tolerance Test 25 Experiments show that Pro-boro-pro clearly lowers blood sugar based upon results from an oral glucose challenge in mice. The first two experiments are "acute" experiments wherein the mice were injected with a single dose of Pro-boro-pro. In the first set of experiments mice were injected with 150 pLg of Pro-boro-pro (PBP-1) and then subjected to an oral glucose tolerance test within an hour. 8 tg of GLP- I was also administered to these 30 mice five minutes prior to administration of glucose. See Figure 2. In a second set of experiments mice were injected with Pro-boro-pro (PBP-2) one hour prior to an oral glucose challenge test. Figure 3 presents the results of these experiments. Each set of experiments was also performed using saline as a control.

- 51 The third set of experiments were "chronic" experiments wherein the mice were injected twice daily with Pro-boro-pro for four days, followed by an oral glucose challenge. These results are presented in Figure 4. 5 Example 6: Glucose Tolerance Test, comparison of normal and GLP-1 receptor -/- mice GLP-1 receptor gene "knock-out" causes glucose intolerance in transgenic mice. Gallwitz B; Schmidt WE Z Gastroenterol (1997) 35: 655-8. Figure 5 compares the ability of Pro-boro-pro to lower plasma glucose levels in normal and GLP-1 receptor -/- transgenic mice. 10 All of the above-cited references and publications are hereby incorporated by reference. Equivalents 15 Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation. many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (36)

1. A method for treating type II diabetes in an animal, comprising conjointly administering to the animal (i) metformin, and (ii) an inhibitor of 5 dipeptidylpeptidase IV represented by the following formula: R, N R 0B-OR 1 or a pharmaceutically acceptable salt thereof; wherein R, represents a C-terminally linked amino acid residue or amino acid 10 analog, a C-terminally linked peptide or peptide analog, or 0 s 0 ,or R 6/; R 6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, (CH 2 )m,-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m-O-alkenyl, -(CH 2 )m-O alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S 15 alkenyl, -(CH 2 )m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7 , R0 O R 8 -(CH2)m-N , -(CH 2 )a-C-N , R 9 R9 NH 2 0 II || -(CH 2 )n-NH2-C-NH2, -(CH 2 )--C-o--Ry 0 0 -(C14z),-C--alky1, -(CH,),-C-alkenyl, O O -(CH)n-C--alkynyl, or -(C 2 )n-C--(CH 2 )m-R 7 ; R 7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle; R 8 and R 9 each independently represent hydrogen, alkyl, alkenyl, 20 (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=0)-alkenyl, -C(=0)-alkynyl, or -C(=0) (CH 2 )m-R 7 ; or R 8 and R 9 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; - 53 RII and R 1 2 each independently represent hydrogen, alkyl, or a pharmaceutically acceptable salt; or R, and R 12 taken together with the 0 B-O atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; 5 m is zero or an integer in the range of I to 8; n is an integer in the range of 1 to 8; the inhibitor inhibits dipeptidylpeptidase proteolysis of GLP-1 with a Ki of less than about 10 nM; and the metformin and the inhibitor are administered in an amount 10 sufficient to treat Type II diabetes but not sufficient to suppress the immune system of the animal.

2. A method according to claim 1, wherein said conjointly administering is achieved by separate dosing of the metformin and the inhibitor. 15

3. A method according to claim 1, wherein said conjointly administering is achieved by administering the metformin and the inhibitor in the same composition. 20

4. A method for treating type II diabetes in an animal, comprising conjointly administering to the animal (i) a sulfonylurea, and (ii) an inhibitor of dipeptidylpeptidase IV represented by the following formula: B-OR 12 R 11 O or a pharmaceutically acceptable salt thereof; 25 wherein R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or 0 S 0 ,osr R6--' , R6 ', or R6 -"r - 54 R 6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m-0-alkenyl, -(CH 2 )m-0 alkynyl, -(CH 2 )m0-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S alkenyl, -(CH 2 )m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7 , 0 -(CII 2 )m-N , -- (CH 2 ).-C-N , R 9 R9 NH, O -CH2)h-NH2-C--NH2, -(CH2).--C-o-R7 0 0

5 (CH 2 ).-C-aikyi, -(CH 2 ).-C-akenyi, 0 0 || |1 -(CI 2 ).-C-akynyI, or -(CH 2 )n--C-(CH 2 )m-R 7 ; R 7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle; Rs and R 9 each independently represent hydrogen, alkyl, alkenyl, (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O) 10 (CH 2 )m-R 7 ; or R 8 and R 9 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; R, and R 12 each independently represent hydrogen, alkyl, or a pharmaceutically acceptable salt; or R, and R 1 2 taken together with the 0 15 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; m is zero or an integer in the range of 1 to 8; n is an integer in the range of I to 8; the inhibitor inhibits dipeptidylpeptidase proteolysis of GLP-1 with a 20 Ki of less than about 10 nM; and the sulfonylurea and the inhibitor are administered in an amount sufficient to treat Type II diabetes but not sufficient to suppress the immune system of the animal. 25 5. A method according to claim 4, wherein said sulfonylurea is selected from the group consisting ofglibenclamide, glipizide and gliclazide. - 55

6. A method according to claim 4, wherein said conjointly administering is achieved by separate dosing of the sulfonylurea and the inhibitor.

7. A method according to claim 4, wherein said conjointly administering is 5 achieved by administering the sulfonylurea and the inhibitor in the same composition.

8. A method according to claim 4, further comprising conjointly administering (iii) metformin. 10

9. A method according to claim 8, wherein said conjointly administering is achieved by separate dosing of the sulfonylurea, metformin and the inhibitor. 15

10. A method according to claim 8, wherein said conjointly administering is achieved by administering the sulfonylurea, metformin and the inhibitor in the same composition.

11. A method according to any one of the preceding claims, wherein the 20 inhibitor has a Ki for inhibition of dipeptidylpeptidase proteolysis of GLP-1 of 1.0 nm or less.

12. A method according to any one of claims 1 to 10, wherein the inhibitor has a molecular weight less than 7500 amu. 25

13. A method according to any one of claims I to 10, wherein R 1 is a C terminally linked amino acid residue or amino acid analog.

14. A method according to claim 13, wherein R, is: H 0 30 HNC \ -56

15. A method according to any one of the preceding claims, , wherein R, I is hydrogen and R 12 is hydrogen.

16. A method according to claim 1 or claim 8, wherein the metformin and the 5 inhibitor are administered orally.

17. A method according to claim 4 or claim 8, wherein the sulfonylurea and the inhibitor are administered orally. 10

18. A method according to claim 8, wherein the metformin, the sulfonylurea and the inhibitor are administered orally.

19. A method according to any one of the preceding claims, wherein said animal is a human. 15

20. Use of metformin and an inhibitor of dipeptidylpeptidase IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of Type II diabetes in a human without suppressing the immune system, wherein said inhibitor is represented by the following 20 formula: B-OR, 2 R 1 1 0 wherein R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or 0 s 0 O S R6--/, -j , or Re-", ,or 25 R. 6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, (CH 2 )m.-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m-O-alkenyl, -(CH 2 )m-0- - 57 alkynyl, -(CH 2 )m0-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S alkenyl, -(CH 2 )m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7 , /R, O Rs (CH 2 )m-N -C-N , R19 R 9 NH 2 0 -CH2)n-NHt2-C--NH2, -(CH2)n-C-0-R7 0 0 -(C11,),-C-alkyl1, -(CH,)n--C-alkenyl, 0 0 -(CH 2 ),-C-alkyny, or -(CH2),,-C-(CH2),,,-R7; 5 R 7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle; R 8 and R 9 each independently represent hydrogen, alkyl, alkenyl, (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O) (CH 2 )m-R 7 ; or R 8 and R 9 taken together with the N atom to which they are attached complete a heterocyclic ring having from 4 to 8 atoms in the ring 10 structure; Ra and R 1 2 each independently represent hydrogen, alkyl, or a pharmaceutically acceptable salt; or R, and R 12 taken together with the 0 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; 15 m is zero or an integer in the range of I to 8; n is an integer in the range of I to 8.

21. Use of a sulfonylurea and an inhibitor of dipeptidylpeptidase IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament 20 for the treatment of Type II diabetes in a human without suppressing the immune system, wherein said inhibitor is represented by the following formula: R 1 R B-OR,, R,wee wherein - 58 R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or 0 S O 4/; R 6 , or; R 6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, 5 (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m-0-alkenyl, -(CH 2 )m-0 alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S alkenyl, -(CH 2 )m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7 , R80 R 8 (CH 2 )m-N -(CH 2 )a-C-N , Ro R9 NH, 0 IIII -0 -R -CHI 2 )--NH 2 -C-NH 2 , -(CH2),-C-0-R 7 0 0 (CHI)--C--akyl, -- (CH 2 ).-C-alkenyl, 0 0 -(CH 2 )a-C-akynyI, or -(CH 2 )-C-(CH 2 )m-RI; 10 R 7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle; R 8 and R 9 each independently represent hydrogen, alkyl, alkenyl, (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=0) (CH 2 )m-R 7 ; or R 8 and R 9 taken together with the N atom to which they are attached complete a heterocyclic ring having from 4 to 8 atoms in the ring 15 structure; R 1 and R 12 each independently represent hydrogen, alkyl, or a pharmaceutically acceptable salt; or R, and R 1 2 taken together with the 0 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; 20 m is zero or an integer in the range of I to 8; n is an integer in the range of I to 8.

22. Use according to claim 21, wherein said sulfonylurea is selected from the group consisting of glibenclamide, glipizide and gliclazide. 25 - 59

23. Use according to claim 21 or claim 22, further comprising metformin.

24. Use according to any one of claims 20 to 23, wherein R 1 is a C-terminally linked amino acid residue or amino acid analog. 5

25. Use according to claim 24, wherein R, is: H 0 10

26. Use according to any one of claims 20 to 25, wherein R, 1 is hydrogen and R 12 is hydrogen.

27. A pharmaceutical formulation for the treatment of Type II diabetes without suppressing the immune system, comprising (i) a pharmaceutically 15 acceptable excipient, (ii) metformin, and (iii) an inhibitor of dipeptidylpeptidase IV represented by the following formula: B-OR,, R 1 1 O or a pharmaceutically acceptable salt thereof; wherein 20 R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or 0 S0 , or R 6S1 R6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 ),m-O-alkenyl, -(CH 2 )m-O 25 alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m,-S-alkyl, -(CH 2 )m-S alkenyl, -(CH 2 )m-S-alkynyl, -(CH 2 ),-S-(CH 2 )m-R 7 , -60 R8 0 -(CI2)m-N , -(CH 2 )n-C-N , R9 R9 NH, 0 II ~ | (CH2)--NH2-C-NH2, -(CH 2 )a-C-0-R 7 0 0 -(CH)-C-alkyl, -(CH 2 )a-C-alkenyl, 0 0 -(CH-T,),-C-alkyny, or -(CH 2 )n-C-(CH 2 ),-R 7 ; R 7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle; Rs and R 9 each independently represent hydrogen, alkyl, alkenyl, 5 (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=0) (CH 2 )m-R 7 ; or R 8 and R 9 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; R, and R 12 each independently represent hydrogen, alkyl, or a 10 pharmaceutically acceptable salt; or R, and R 12 taken together with the 0 B-O atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; m is zero or an integer in the range of I to 8; n is an integer in the range of I to 8; 15 the inhibitor inhibits dipeptidylpeptidase proteolysis of GLP-l with a Ki of less than about 10 nM.

28. A pharmaceutical formulation for the treatment of Type II diabetes without suppressing the immune system, comprising (i) a pharmaceutically 20 acceptable excipient, (ii) a sulfonylurea, and (iii) an inhibitor of dipeptidylpeptidase IV represented by the following formula: , N /-OR, R 1 10 or a pharmaceutically acceptable salt thereof; wherein - 61 R, represents a C-terminally linked amino acid residue or amino acid analog, a C-terminally linked peptide or peptide analog, or o s 0 r 11,or R 6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, 5 (CH 2 )m-R 7 , -(CH 2 )m-OH, -(CH 2 )m-O-alkyl, -(CH 2 )m-O-alkenyl, -(CH 2 )m0 alkynyl, -(CH 2 )m-O-(CH 2 )m-R 7 , -(CH 2 )m-SH, -(CH 2 )m-S-alkyl, -(CH 2 )m-S alkenyl, -(CH 2 )m-S-alkynyl, -(CH 2 )m-S-(CH 2 )m-R 7 , R8 0 -(CH 2 )m-N , -(CH2).-C-N , R9 R 9 NH 2 0 -(CH42).-NH2-C-NH2i. -- (CH2).-C-0-R7 0 0 Il II -(CH,),-C-alkyi, -(CH 2 ).-C-alkenyl, 0 0 -- (CH 2 )--C-alkynyl, or -(CI-)--C-(CH 2 )m,-R7; 10 R 7 represents aryl, cycloalkyl, cycloalkenyl, or a heterocycle; R 8 and R 9 each independently represent hydrogen, alkyl, alkenyl, (CH 2 )m-R 7 , -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, or -C(=O) (CH 2 )m-R 7 ; or R 8 and R 9 taken together with the N atom to which they are attached complete a heterocyclic ring having from 4 to 8 atoms in the ring 15 structure; R, and R 1 2 each independently represent hydrogen, alkyl, or a pharmaceutically acceptable salt; or R, 1 and R 1 2 taken together with the 0 B-0 atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; 20 m is zero or an integer in the range of I to 8; n is an integer in the range of I to 8; the inhibitor inhibits dipeptidylpeptidase proteolysis of GLP-I with a Ki of less than about 10 nM. - 62

29. A pharmaceutical formulation according to claim 28, further comprising metformin.

30. A pharmaceutical formulation according to any one of claims 27 to 29, 5 wherein R, is a C-terminally linked amino acid residue or amino acid analog.

31. A pharmaceutical formulation according to claim 30, wherein Ri is: H 0 10 HN

32. A pharmaceutical formulation according to any one of claims 27 to 31, wherein R 1 is hydrogen and R 12 is hydrogen. 15

33. A method for treating type I diabetes in an animal, said method substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples. 20

34. Use of metformin and an inhibitor of dipeptidylpeptidase IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of Type II diabetes in a human without suppressing the immune system, said use substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying 25 drawings and/or examples.

35. Use of a sulfonylurea and an inhibitor of dipeptidylpeptidase IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of Type II diabetes in a human without suppressing the 30 immune system, said use substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples. - 63

36. A pharmaceutical formulation substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples. 5 Dated this 18th day of August 2011 Shelston IP Attorneys for: 10 1149336 Ontario, Inc. and New England Medical Center Hospitals, Inc. and Trustees of Tufts College