NZ719672B2 - Tripeptide compositions and methods for treatment of diabetes - Google Patents

Abstract

Disclosed is an oral dosage form comprising as the sole active product ingredient, at least one peptide consisting of the amino acid sequence GGL, GLL, GGdL, GdLL, GLdL or GdLdL, or a pharmaceutically acceptable salt of the peptide, and a pharmaceutically acceptable excipient.

Description

PATENTS FORM NO. 5 Our ref: JX 236695NZPR DIVISIONAL APPLICATION FILED OUT OF NZ 614080 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Tripeptide compositions and methods for treatment of diabetes We, The Regents of the University of Michigan of Office Of The Technology Transfer, 600 Huron Parkway, 2nd Floor, Ann Arbor, 48109-2590, an, United States of America hereby declare the ion, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following ent: (Followed by page 1a) 103840182_1.docx:JX:ewa PEPTIDE COMPOSITIONS AND METHODS FOR TREATING PATIENTS This application claims the benefit of U.S. Provisional Patent Application No. 61/441,748 filed February 11, 2011. The ional application is hereby incorporated by reference in its entirety. ent of Government Interest This invention was made with government support under HL68878 and HL89544 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

Field of the Invention The present invention is directed to peptide compositions and methods of using the peptide compositions to treat prediabetes, diabetes, obesity, high blood pressure and metabolic syndrome.

Background Our bodies turn the food we eat into the sugar glucose. Blood transports glucose to cells which convert it into energy. Normally, a protein hormone called n controls the level of glucose in the blood. When there are defects in n production, insulin action, or both, high levels of glucose in the blood result. Diabetes is the group of diseases characterized by these defects.

The three most common forms of diabetes are type 1 es, type 2 diabetes and gestational diabetes. Type 1 diabetes (previously known as n—dependent diabetes mellitus or juvenile—onset diabetes) usually develops in childhood or adolescence. It occurs when the body’s immune system destroys the cells of the pancreas that e n.

People with type 1 diabetes must monitor the level of sugar in their blood multiple times a day and take insulin (via injections or a pump) to maintain an appropriate level. Gestational diabetes occurs when pregnant women become intolerant to e. Gestational diabetes also requires treatment to maintain riate glucose blood levels and avoid complications in the infant. Woman who have gestational diabetes are at sed risk for developing type 2 diabetes.

(Followed by page 2) W0 2012/109561 PCT/U82012/024684 Type 2 diabetes ously known as sulin—dependent diabetes mellitus or adult—onset diabetes) usually develops in adulthood. It develops as cells first do not use insulin properly and then the pancreas loses its ability to produce insulin. Many people with type 2 es control their blood glucose with a meal plan, exercise program, losing weight and taking oral medication. Some people with type 2 es need take insulin as well.

Diabetes is serious because too much sugar in the blood can damage the eyes, kidneys, nerves and heart. Complications of diabetes e heart disease, stroke, hypertension, blindness, other eye problems (such as ic retinopathy), kidney disease, nervous system disease (such as impaired sensation or pain in the feet or hands, slowed digestion of food, carpal tunnel syndrome and erectile dysfunction), amputations, periodontal disease, susceptibility to other illnesses (such as pneumonia and influenza), impaired mobility and depression. Uncontrolled diabetes can result in acute life—threatening events such as diabetic ketoacidosis and hyperosmolar coma.

Diabetes is the leading cause of kidney failure, aumatic lower limb amputations and new cases of blindness among adults in the United States. Diabetes is a major cause of heart disease and stroke. Diabetes was the seventh leading cause of death in the United States in 2007. Overall, the risk for death among people with diabetes is about twice that of people of similar age t diabetes. According to the Centers for Disease Control and Prevention, as of January 2011, diabetes affects 25.8 n people, 8.3% of the United States population. Another 79 million American adults are estimated to have prediabetes, a condition in which blood sugar levels are higher than normal but not high enough to be diagnosed as diabetes. Prediabetes is sometimes called impaired fasting glucose or impaired e tolerance. Prediabetes itself raises ’s risk of type 2 diabetes, heart disease and stroke. Many prediabetics develop type 2 diabetes within ten years.

In addition to lifestyle interventions, prediabetic and type 2 diabetic patients are often treated with medications to address cations of diabetes. Doctors ibe medications to control blood pressure and blood lipids to reduce cardiovascular complications. Often, in younger and heavier patients with normal kidney on, s prescribe the oral drug metformin to more directly address the defects causing diabetes.

Metformin suppresses hepatic glucose production, increases insulin sensitivity, es peripheral glucose uptake, increases fatty acid oxidation and decreases absorption of glucose from the gastrointestinal tract. Metformin, though, is contraindicated in people with any PCT/U52012/024684 condition that could increase the risk of lactic acidosis, including kidney disorders, lung disease and liver e.

Other more recently approved drugs do not appear to be more effective than metformin and each has its own set of contraindications. For example, rosiglitazone was one of the first insulin—sensitizers used as an anti-diabetic drug. It renders fat cells more sensitive to insulin. Annual sales of rosiglitazone peaked at approximately $2.5 billion in 2006. e rosiglitazone can be associated with an increased risk of cardiovascular events, the European Medicines Agency recommended the drug be suspended from the European market. The US. Food and Drug Agency has allowed it to remain on the market but it became subject to significant restrictions as of September 23, 2010.

A precursor to n called human proinsulin C—peptide, and fragments of C- peptide, have also been investigated for the treatment of diabetes. See, International Publication Nos. W0 98/13384, , , WC 29095 and . See also, Ohtomo et al., Diabetologia, 41: 287-291 (1998); Sato er al., Cell. M01. Life Sal, 6]: 727-732 (2004); Hach et al., Exp. Diabetes Res; 1-6 (2008) and Ido er al., Science, 277: 563-566 (1997).

Food proteins are composed of twenty different amino acids and scientists have studied the effect of individual amino acids when ingested with e. See, Gannon and Nuttall, IUBMB Life, 62: 660-668 (2010); Gannon et al., Metabolism, 37: 1081-1088 (1988); Gannon et al., Am. J. Clin. NuII‘., 76: 1302—1307 (2002) and Kalogeropoulou et al., lism, 57: 752 (2008). The amino acids leucine and glycine have been reported to attenuate the serum glucose response and stimulate onal insulin secretion. This effect requires the ingestion of significant amounts of the amino acids , with accompanying bad taste, unbalanced amino acid intake and concerns of ing renal function.

Glucagon-like peptide—1 (GLP-1) is an incretin hormone. Incretin hormones are secreted by inal cells in response to nutrient ingestion. The primary physiological function of GLP-1 appears to be related to glycemic control. GLP—l stimulates insulin release, inhibits glucagon secretion, reduces gastric emptying and augments satiety. In ts with type 2 es the incretin effect is reduced, buting to impaired glycemic control. Administration of GLP—1 to patients has been reported to restore blood glucose regulation via endogenous insulin secretion. GLP—1 administration has also been reported to reduce energy intake through its actions of delaying gastric emptying and sing satiety, W0 20121‘109561 PCT/U52012/024684 therefore it may induce weight loss. Two GLP—receptor agonists/analogues are currently approved for treatment of type 2 diabetes mellitus, exenatide (Byetta®), and utide (Victoza®) and others are in clinical development. A once-weekly formulation of exenatide (Bydureon®) has also been approved. See, Barnett et al., Diabetes, Obesity and Metabolism. accepted article published online (2011).

In addition, studies have demonstrated that agonists of the GLP-1 receptor also effect cardiovascular related functions such as heart rate and blood pressure. See, Giievc er 411., British J. Pharm, 157a: 1340-1351 (2009). In a particular study, Dahl ensitive (DSS) rats were fed a high salt diet: and treated with an exenatide c (AC3174) alone or in combination with an ACE inhibitor (captopril). AC3174 had ypertensive, insulin- sensitizing, and renoprotective effects comparable to that of captopril. See, Liu et al., vascular Diabetology, 9(32): 1-10 .

There thus exists a need in the art for new treatments for prediabetes, diabetes and their complications. There also exists in the an: a need for new treatments for obesity, high blood re and metabolic syndrome.

The present invention provides ts and methods for treating prediabetes, diabetes, obesity, high blood pressure, metabolic syndrome, poor glycemic control, and reduced n secretion.

The invention provides a method for treating a condition comprising administering to a patient an effective amount of a composition comprising at least one peptide consisting of the amino acid sequence GGL, GLG, LGL, LLG, LGG or GLL, or a phannaceutically acceptable salt of the peptide, wherein the condition is prediabetes, diabetes, obesity, high blood pressure, metabolic syndrome, poor glycemic control, or reduced insulin secretion. In addition, the invention provides a method for treating a condition comprising administering to a patient an effective amount of a composition compiising at least one peptide consisting of the amino acid sequence GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL, or dLGdL, or a pharmaceutically able salt of the peptide, wherein the condition is prediabetes, diabetes, obesity, high blood pressure, metabolic syndrome, poor glycemic control, or reduced insulin secretion.

Also provided is a method of preventing, ng, or ameliorating a diabetesassociated complication in a diabetic t compiising administering to the patient an effective amount of a composition comprising at least one peptide consisting of the amino acid sequence GGL, GLG, LGL, LLG, LGG or GLL, or a pharmaceutically acceptable salt of the peptide, wherein the diabetes-associated complication is a cardiovascular disease, chronic kidney disease, kidney failure, bladder problems, erectile dysfunction, gastroporesis, an eye disease, a diabetic athy, foot or skin , or lower extremity amputation. In addition, the invention provides a method of preventing, reducing, or ameliorating a diabetes— associated complication in a diabetic patient comprising administering to the patient an effective amount of a composition comprising at least one peptide consisting of the amino acid sequence GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceutically acceptable salt of the peptide, wherein the diabetes— associated cation is a cardiovascular disease, chronic kidney e, kidney failure, bladder problems, erectile dysfunction, gastroporesis, an eye disease, a diabetic athy, foot or skin ulcers, or lower extremity amputation.

In all of the foregoing methods, the peptides can be acetylated at the N-terminus, amidated at the C—terminus, or both. The composition can be administered by an oral, eritoneal, ocular, ermal, intranasal, subcutaneous, intramuscular or intravenous route.

The pharmaceutical compositions provided by the invention include a composition comprising at least one peptide consisting of the amino acid ce GGL, GLG, GLL, GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceutically able salt of the peptide, and a pharmaceutically acceptable excipient. They also include a composition wherein the pharmaceutical composition comprises at least one e consisting of the amino acid sequence GGL, GLG, or GLL, or a pharmaceutically acceptable salt of the peptide, and a pharmaceutically able excipient. In the compositions, the peptides can be acetylated at the N—terminus, amidated at the C-terminus, or both.

The ion es a kit for administering a pharmaceutical composition compn'sing at least one peptide consisting of the amino acid sequence GGL, GLG, GLL, GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceutically acceptable salt of the peptide, and a pharmaceutically acceptable excipient, wherein the kit comprises the composition, instructions for administration of the composition and a device for stering the composition to the patient. Additionally, the invention provides a kit wherein the pharmaceutical composition WO 09561 comprises at least one peptide consisting of the amino acid sequence GGL, GLG, or GLL. In the kits, the peptides can be acetylated at the N—terminus, amidated at the C-terminus, or both.

Cardiovascular diseases (CVD) are the primary cause of mortality among diabetic patients, accounting for almost two out of three deaths. Thus, minimization of risk of CVD is a critical clinical goal in the management of prediabetic and diabetic patients. The present invention provides ts and methods that improve glycemic control and rently decrease the risk of cardiovascular events and other diabetes—related complications.

Brief Summary of the Figures Figure 1 shows the effect of glycine on blood glucose after oral load e and e in C57BL/6J mice.

Figure 2 shows the effect of leucine on blood e after oral load leucine and glucose in C57BL/6J mice.

Figure 3 shows Diapin inhibits the increase of blood glucose after oral load of glucose in KKay diabetic mice.

Figure 4 Show Diapin inhibits the increase of blood glucose after oral load of starch in KKay diabetic mice.

Figure 5 shows Diapin reduces random blood e in KKay ic mice.

Figure 6 shows Diapin stimulates insulin secretion in KKay diabetic mice 30 min after oral load of glucose and Diapin.

Figure 7 shows Diapin stimulates GLP—l secretion in KKay diabetic mice 30 min after oral load of glucose and Diapin.

Figure 8 shows Diapin decreases the blood e level of KKay diabetes mouse in a time— and dose-dependent manner.

Figure 9 shows Diapin has no effect on fasting blood glucose levels in C57BL/6J mice.

Figure 10 shows Diapin inhibits the increase in blood glucose after the ip injection of glucose into C57BL/6J mice.

Figure 11 shows another peptide GGH has no effect on blood glucose after the ip injection of glucose into C57BL/6J mice.

WO 09561 2012/024684 Figure 12 shows the effect of two other peptides, LGG and LGL, on blood glucose after the ip injection of glucose into 6J mice.

Figure 13 shows the effect of the e LLG on blood glucose after oral load of glucose in C57BL/6J mice.

Figure 14 shows the effect of the peptides GLG and GLL on blood glucose after oral load of glucose in C57BL/6J mice.

Figure 15 shows the effect of Diapin and Diapin with an amidated C-terminus on blood glucose after oral administration of glucose in C57BL/6J mice.

Figure 16 shows the effect of Diapin and Diapin with an acetylated N-terminus on blood glucose after oral administration of glucose in C57BU6J mice.

Figure 17 shows the effect of Diapin with both an amidated C— and acetylated N- terminus on blood glucose after oral administration of glucose in C57BL/6J mice.

Figure 18 shows the effect of Diapin given at 30min prior to oral glucose administration on blood glucose in C57BL/6J mice.

Figure 19 shows the effect of Diapin given at 1 hour prior to oral glucose administration on blood glucose in C57BL/6J mice.

Figure 20 shows the effect of Diapin and dipeptides on blood e level in C57BL/6J mice after oral glucose administration.

Figure 21 shows the effect of Diapin and dipeptides on blood glucose level in C57BL/6] mice after oral glucose administration.

Figure 22 shows the effect of Diapin in ob/ob mice after oral glucose stration.

Figure 23 shows the effect of Diapin on blood glucose level in db/db mice after oral glucose administration.

Figure 24 shows the effect of Diapin on blood glucose level in high fat diet-induced diabetic mice after oral glucose administration.

Figure 25 shows the effect of D—Diapin (composed of D—isomer amino acids) on lowering blood glucose level in C57BL/6J mice after oral glucose administration.

Figure 26 shows the effect of Diapin on blood e level in C57BL/6J mice after oral glucose administration and ip Diapin administration.

Figure 27 shows the effect of d dipeptides on blood glucose level in C57BL/6J mice after oral glucose administration.

Detailed Description In one aspect, the invention provides peptides to be administered to prediabetic or ic patients. Examples of peptides of the invention are GGL (termed “Diapin” herein), GLG, LGL, LLG, LGG and GLL. Other examples of peptides of the invention are GL and LG. The invention also provides for peptides GGL, GLG, LGL, LLG, LGG and GLL in which each leucine is ndently in the form of the L—isomer or the D—isomer. Other examples of the peptides of the ion are LG and GL in which e is in the D— isomeric form. es of the invention may be chemically synthesized or derived by digestion of proteins by methods known in the art.

As used herein, the singular forms “ 7’ “ , an”, and “the” include plural references unless the context y dictates ise.

It is known in the art that it is possible to substitute a chemically similar amino acid for an amino acid in a peptide or protein without negatively affecting the ty of the peptide or protein. Therefore, it is specifically contemplated that a glycine or leucine residue in a e of the invention may be substituted with a chemically similar amino acid residue such as a different aliphatic amino acid residue or an amino acid isomer. Other aliphatic amino acids are alanine, valine and isoleucine. It is also specifically contemplated that chemically similar amino acids may be added to one or both ends of a peptide of the invention without negatively affecting the activity of the peptide.

With the exception of glycine, the common amino acids all contain at least one chiral carbon atom. These amino acids therefore exist as pairs of stereoisorners designated as the L—isomer and the D-isomer. Most naturally occurring proteins and peptides are composed exclusively of the L—isomeric form. D—isomeric amino acids can affect the mation of a peptide or protein and may lead to increased stability or a change in activity.

In some embodiments of the peptide, Leucine is replaced with ine. For example in some embodiments the peptide is Glycine-Glycine—Leucine (GGL), or is Glycine— Glycine-D-Leucine (GGdL), or is Glycine—Leucine—Glycine (GLG), or is Glycine—D—Leucine— PCT/U82012/024684 Glycine (GdLG), or is Leucine—Leucine—Glycine (LLG), or is D-Leucine-Leucine— Glycine(dLLG), or is Leucine—D-Leucine—Glycine (LdLG), or is D—LeucineD—Leucine— Glycine (deLG), or is Leucine-Leucine-Glycine (LLG), or is D-Leucine-Leucine-Glycine (dLLG), or is Leucine—D—Leucine—Glycine (LdLG), or is D-Leucine-D-Leucine—Glycine (deLG), or is Leucine—Glycine—Glycine (LGG), or is D—Leucine-Glycine-Glycine (dLGG), or is e-Leucine—Leucine (GLL), or is Glycine—D~Leucine-Leucine (GdLL), or is Glycine-Leucine—D—Leucine (GLdL), or is Glycine—D—Leucine-D-Leucine (GdeL), or is Leucine—Glycine (LG), or is D~Leucine~Glycine (dLG), or is Glycine—Leucine (GL), or is Glycine—D—Leucine (GdL). The peptides of the invention may be used individually or used as a mixture of two or more peptides. With respect to a mixture, each possible subcombination of peptides is specifically plated by the invention.

In some ments, peptides of the invention are chemically modified. In some embodiments peptides of the invention are acetylated at the N—terminus. In some embodiments, peptides of the invention are amidated at the C—terminus. In some embodiments, peptides of the invention are acetylated at the N—tenninus and amidated at the C-tenninus. Peptides are ated or amidated by methods known in the art. In some embodiments of the present disclosure, the peptide is glycosylated, carboxylated, phosphorylated, esterified, or converted into an acid addition salt and/or optionally dimerized, polymerized, pegylated, or otherwise conjugated.

In some embodiments, the peptides comprise one or more non—peptide bonds in place of peptide bond(s). For example, the peptides comprise in place of a peptide bond, an ester bond, an ether bond, a thioether bond or an amide bond.

In another aspect, compositions of at least one of the peptides of the invention are provided. Examples of compositions of the invention are compositions comprising one or more of the peptides GGL, GLG, LGL, LLG, LGG and GLL, or a pharmaceutically acceptable salt thereof. Other examples of compositions of the invention are itions comprising one or more of the peptides GL and LG, or ceutically acceptable salts f. Other examples of compositions of the invention are compositions comprising GGL, GLG, GLL, LLG, LGG, LGL, GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL, dLGdL, or a pharmaceutically acceptable salt thereof. The compositions of the invention may include other ents, ing other amino acids.

With respect to the itions, each possible subcombination of es is ically contemplated by the invention.

W0 20121‘109561 2012/024684 As used , the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, tion, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A “pharmaceutically acceptable salt” means any non-toxic salt or salt of an ester of a compound of this ion that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention. Pharmaceutically acceptable salts are well known in the art. For example, Berge er al. describe pharmaceutically acceptable salts in detail in J.

Pharmaceutical Sciences, 66: 1-19 (1977).

It is contemplated the peptides of the invention, or mixtures thereof, can be used as the sole active product ingredient in the composition. Accordingly in an aspect of the invention, compositions of one or more of the peptides of the invention are provided wherein the peptide or peptides of the invention are the sole active ingredient. Thus, an embodiment of the present disclosure is a composition ting ially of at least one peptide consisting of the amino acid sequence GGL, GLG, LGL, LLG, LGG or GLL, or ceutically acceptable salts thereof. Another embodiment is a composition consisting essentially of at least one peptide consisting of the amino acid sequence GL or LG, or pharmaceutically acceptable salts thereof. Yet another ment is a composition consisting essentially of at least one peptide ting of the amino acid sequence GGL, GLG, GLL, LLG, LGG, LGL, GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL, dLGdL, or a pharmaceutically acceptable salt thereof. With respect to the compositions, each possible bination of peptides is specifically contemplated by the invention.

In yet a further aspect, the invention provides a composition sing at least one peptide of the invention and a pharmaceutically acceptable excipient.

In some embodiments, a pharmaceutical composition comprises at least one peptide consisting of the amino acid sequence GGL, GLG, GLL, GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceutically acceptable salt of the peptide, and a ceutically acceptable excipient. In some embodiments, the pharmaceutical composition ses at least one peptide consisting of the amino acid sequence GGL, GLG, or GLL, or a ceutically acceptable salt of the peptide, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises the peptide consisting of the amino acid sequence PCT/U82012/024684 GGL, or a pharmaceutically acceptable salt of the peptide, and a pharmaceutically acceptable ent. With respect to the pharmaceutical compositions, each possible subcombination of peptides is specifically contemplated by the invention.

Pharmaceutical compositions of the invention are formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., ing upon the particular mode of administration and dosage form. The compositions are generally formulated to e a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the ation and route of administration. In alternative embodiments, the pH is adjusted to a range from about pH 5.0 to about pH 8. In various aspects, the compositions comprise a therapeutically effective amount of at least one peptide as described herein, together with one or more pharmaceutically acceptable excipients. The compositions may include a second active ingredient useful in the treatment or prevention of ial growth (for example and without limitation, anti-bacterial or icrobial agents).

Suitable excipients include, for example, carrier les that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, ycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients include antioxidants (for example and without limitation, ic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol) wetting or emulsifying agents, pH ing substances, and the like.

Pharmaceutical compositions suitable for the delivery of peptides of the present invention and methods for their preparation will be readily apparent to those d in the art.

Such compositions and methods for their preparation may be found, for example, in Remington’s Pharmaceutical Sciences, The Science and Practice of Pharmacy, 20th Edition, Lippincott ms & White, Baltimore, Md. (2000). The es of the present invention may be formulated to be immediate and/or modified release.

In yet another aspect, the invention es a method for treating a condition comprising administering to a patient an ive amount of a composition comprising at least one peptide consisting of the amino acid sequence GGL, GLG, LGL, LLG, LGG or W0 2012/109561 GLL, or a pharmaceutically able salt of the peptide, wherein the condition is betes, diabetes, obesity, high blood pressure, metabolic syndrome, poor glycemic control, or reduced insulin secretion. The invention also provides a method for treating a condition comprising administering to a patient an effective amount of a composition comprising at least one peptide consisting of the amino acid sequence GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL, or dLGdL, or a pharmaceutically acceptable salt of the peptide, wherein the condition is prediabetes, diabetes, obesity, high blood pressure, metabolic me, poor glycemic control, or reduced insulin secretion. With respect to the s, each possible subcombination of peptides is specifically contemplated by the invention.

In some embodiments, the invention provides a method for treating a prediabetic or diabetic patient ses stering to the patient a composition comprising at least one of the peptides GGL, GLG, LGL, LLG and GLL. In some ments, the invention also provides a method for treating a prediabetic or diabetic patient comprising administering to the patient a composition comprising at least one of peptide GL or LG. In some embodiments, the es is type 1 diabetes. In some embodiments, the diabetes is type 2 diabetes. The amount of the composition administered is therapeutically effective to achieve at least one of the following: reducing blood glucose levels, stimulating insulin secretion, stimulating GLP—l secretion, reducing n resistance, and improving glycemic control.

The term “treating” (or other forms of the word such as “treatment” or “treat”) is used herein to mean that administration of a composition of the t invention mitigates a condition in a patient and/or reduces, ts, or eliminates a particular teristic or event associated with a condition. Thus, the term "treatment" includes, preventing a condition from occum'ng in a patient, particularly when the patient is predisposed to acquiring the condition; reducing or ting the condition; and/or ameliorating or reversing the ion. Insofar as the s of the present invention are directed to preventing conditions, it is tood that the term "prevent" does not require that the condition be completely thwarted. Rather, as used herein, the teim preventing refers to the ability of the skilled artisan to identify a population that is susceptible to condition, such that administration of the compositions of the present invention may occur prior to onset of the condition. The term does not imply that the condition must be tely avoided.

An " effective amount" as used herein refers to an amount of a peptide of the ion sufficient to exhibit a detectable therapeutic effect. The effect is detected by, for 2012/024684 example, an improvement in al condition, or a prevention, reduction or amelioration of complications. The precise effective amount for a patient will depend upon the patient's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts fora given situation are determined by routine experimentation that is Within the skill and judgment of the clinician.

In some embodiments, the invention provides methods for treating obesity, high blood re or metabolic syndrome. Accordingly, one embodiment of the invention is a method for treating obesity comprising administering to a patient an effective amount of composition comprising at least one peptide consisting of the amino acid sequence GGL, GLG, LGL, LLG, LGG, GLL, LG, or GL, or a ceutically acceptable salt f. Still another embodiment is a method for treating high blood pressure comprising administering to a patient an effective amount of a composition comprising at least one peptide consisting of the amino acid sequence GGL, GLG, LGL, LLG, LGG, GLL, LG, or GL, or a pharmaceutically acceptable salt thereof. Another embodiment is a method for treating metabolic me comprising administering to a patient an effective amount of a ition comprising at least one peptide consisting of the amino acid ce GGL, GLG, LGL, LLG, LGG, GLL, LG, or GL, or a pharmaceutically acceptable salt thereof. In any of the ing embodiments, one or more leucine in the peptide is independently ed with the D—isomer of leucine. With respect to the methods, each possible subcombination of peptides is specifically contemplated by the invention.

In still another aspect, the invention provides methods for preventing, reducing and/or ameliorating diabetes—associated complications in a prediabetic or diabetic t sing administering to the patient a composition comprising at least one peptide consisting of the amino acid sequence GGL, GLG, IrGL, LLG, LGG or GLL. The invention also provides methods for preventing, reducing and/or ameliorating diabetes—associated cations in a prediabetic or diabetic patient comprising administering to the t a composition comprising at least one of the peptides consisting of the amino acid sequence GL or LG. It also provides a method of preventing, ng, or ameliorating a diabetes— associated complication in a diabetic patient comprising stering to the patient an effective amount of a composition comprising at least one peptide consisting of the amino acid sequence GGL, GLG, LGL, LLG, LGG or GLL, or a pharmaceutically acceptable salt of the peptide. It also provides a method of preventing, reducing, or ameliorating a diabetes- PCT/U82012/024684 associated complication in a diabetic patient comprising administering to the patient an ive amount of a composition comprising at least one peptide consisting of the amino acid sequence GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL or dLGdL, or a ceutically acceptable salt of the peptide. With respect to the methods, each possible subcombination of peptides is specifically contemplated by the invention. In some embodiments, the diabetes is type 1 diabetes. In some embodiments, the diabetes is type 2 diabetes. The administration is of an amount of the ition that is therapeutically effective to prevent, reduce or ameliorate at least one diabetes-associated complication including, but not limited to, the following: a cardiovascular disease [e.g., coronary artery disease (sometimes called ischemic heart disease), al vascular es (such as stroke or transient ischemic attacks), heart failure, atherosclerosis, or peripheral arterial disease], chronic kidney disease, kidney failure, r problems, erectile dysfunction, poresis, an eye disease (such as diabetic retinopathy, cataract or glaucoma), a diabetic neuropathy (peripheral, mic, proximal or focal), foot or skin ulcers, or lower extremity amputation.

The compounds of the t invention may be administered by any suitable route.

For example, compositions of the invention can be administered by the oral, ocular, ermal, intraperitoneal (“ip”), asal, subcutaneous, intramuscular or intravenous route.

Formulations suitable for oral stration include, for e, solid, semi-solid and liquid systems such as, tablets; soft or hard capsules containing multi- or nano— particulates, liquids, or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays. In some embodiments the peptides of the present invention are formulated for oral stration using delivery vehicles known in the art, including but not limited to, microspheres, mes, enteric coated dry emulsions or nanoparticles.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable ons, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active peptides, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, lizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3—butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, PCT/U82012/024684 tetrahydrofuifuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include nts such as g agents, emulsifying and suspending agents, ning, flavoring, and perfuming agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active peptide is mixed with at least one inert, pharmaceutically acceptable excipient or cairier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar~~agar, calcium carbonate, potato or tapioca starch, alginic acid, certain tes, and sodium ate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid hylene glycols, sodium lauryl sulfate, and mixtures f. In the case of capsules, tablets and pills, the dosage form may also comprise buffering . The active compounds can also be in microencapsulated form with one or more excipients as noted above. Solid compositions of a similar type may also be employed as fillers in soft and hard—filled gelatin capsules using such ents as lactose or milk sugar as well as high lar weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as c coatings and other coatings well known in the pharmaceutical formulating art. Injectable ations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

Among the able vehicles and solvents that may be ed are water, Ringer's solution, U.S.P. and isotonic sodium de solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed ing synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the ation of ables. The injectable formulations can be sterilized, for example, by filtration through a bacterial—retaining filter, or by incorporating W0 2012I109561 steiilizing agents in the form of e solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Treatment of pre-diabetic or diabetic patients with itions of the invention in combination with other diabetes drugs known in the art is specifically contemplated. In some embodiments, treatment with compositions of the invention allows a reduction in the dose of the other diabetes drug or drugs and therefore reduces the side effects associated with the other chug or dings. In some embodiments, the other diabetes drug is insulin. In some embodiments, the other diabetes drug is a biguanide (such as metformin). In some embodiments, the other diabetes drug is a thiazolidinedione (such as pioglitazone). In some embodiments, the other diabetes drug is a DPP—4 inhibitor (such as iptin). In other words, compositions of the t invention can be used in combination with other dings such as those used as rd of care for the ion being treated. In some embodiments the drug is a statin (including but not limited to, atorvastatin, lovastatin, simvastatin, pravastatin rosuvastatin, fluvastatin, and pitastatin). In some embodiments, the drug is a blood pressure lowering drug [including but not limited to, Angiotensin-converting enzyme (ACE) inhibitors such as captopril, lisinopril, and ramipril; Angiotensin II receptor rs such as losaitan, olmesartan and valsartan; beta blockers such as metoprolol, l and penbutolol; and calcium channel blockers such as pine, diltiazem and nifedipine].

In still another aspect, the invention provides a kit for administering a composition of invention to a patient in need thereof, where the kit ses a composition of invention, instructions for use of the composition and a device for administering the composition to the patient. In some embodiments, a kit for administering a pharmaceutical composition comprises at least one peptide consisting of the amino acid sequence GGL, GLG, GLL, LLG, LGL, LGG, GGdL, GdLG, GdLL, GLdL, GdeL, dLLG, LdLG, deLG, dLGG, dLGL, LGdL or dLGdL, or a pharmaceutically acceptable salt of the peptide, and a ceutically acceptable excipient, wherein the kit comprises the composition, instructions for administration of the composition and a device for administering the composition to the patient. In some embodiments, the kit comprises a pharmaceutical composition sing at least one peptide consisting of the amino acid sequence GGL, GLG, or GLL. With respect to the kits, each possible bination of peptides is ically contemplated by the invention.

W0 2012.1109561 Examples The invention will be more fully understood by reference to the following examples which detail exemplary embodiments of the invention.

Example 1 The s of the amino acids glycine and leucine on blood glucose were ined.

Effect of e on blood glucose after oral load glycine and glucose An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab (Bar Harbor, Maine). Fasted mice were given glucose (2mg/g body weight, diamond line in Figure 1, n=10) or glycine (0.35mg/g bw, square line in Figure 1, n=10) and glucose by gavaging. Blood glucose was measured at 0, 30, 60, 90, and 120 min after giving Blood glucose levels at any time point in the glycine group were not significantly changed ed to the control group.

Effect of leucine on blood glucose after oral load leucine and glucose ] An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. Fasted mice were given glucose (2mg/g body weight, diamond line in Figure 2, n=10) or Leucine (0.35mg/g bw, square line in Figure 2, n=10) and glucose by gavaging.

Blood glucose was measured at 0, 30, 60, 90, 120 min after giving glucose.

Example 2 Diapin (peptide GGL of the ion) potently attenuates blood glucose levels when orally ed with either glucose or starch in a diabetic mouse. Moreover, Diapin also reduces blood glucose levels under sting condition in KKay diabetic mice [Yamauchi er al., Nat. Med, 7(8): 971—946 (2001)]. See Figure 3.

Diapin inhibits the increase of blood glucose after oral load of glucose in diabetic mice Blood glucose levels at 30, 60, 90 and 120 min in Diapin group was significantly lower than those in the control.

W0 2012f109561 Diapin inhibits the se in blood glucose after oral load of starch in diabetic mice An experiment was performed in adult male KKay diabetic mice purchased from the Jackson Lab. In the l group (diamond line in Figure 4, n=lO), starch was orally administered at dose of 2 mg/g bw. In the Diapin group (square line in Figure 4, n=9), starch and Diapin were orally administered at dose of 2 mg/g bw and lmg/g bw, respectively. Blood glucose was measured at 0, 30, 60, 90 and 120 min after gavaging starch and Diapin.

Blood glucose levels at 30, 60, 90 and 120 min in the Diapin group was significantly lower than those in the control.

Diapin reduces random blood glucose in diabetic mice An experiment was performed in adult male KKay diabetic mice purchased from the Jackson Lab. Under non—fasting condition, in the control group (diamond line in Figure 5, n=9), distilled water was orally given and in the Diapin group (square line in Figure 5, n=9), Diapin was orally administered at lmg/g bw. Blood glucose was measured at O, 30, 60, 90, 120, 150 and 180 min after gavaging of Diapin.

Blood glucose levels at 60, 90, 120, 150 and 180 min in the Diapin group were significantly lower than those in the control.

Example 3 Diapin per se stimulates insulin secretion in KKay diabetic mice. Furthermore, Diapin also ses GLP—l ion in diabetic mice.

Diapin stimulates insulin secretion in diabetic mice after oral load of glucose and Diapin An experiment was performed in adult male KKay diabetic mice. Under fasting conditions in the control group (white bar in Figure 6, n21 1), glucose was orally administered at dose of 1i5mg/g bw. In the Diapin group (black bar in Figure 6, n=l l), Diapin and glucose were orally stered at 1mg/g bw and 1.5mg/g bw, respectively. Blood s were collected at 30 min after oral administration of e and Diapin. Blood glucose was monitored with FreeStyle e meter and insulin was measured by ELISA , Cat# 80-INSMS—E01).

Diapin stimulated insulin secretion in the KKay diabetic mice.

WO 09561 PCT/U82012/024684 Diapin stimulates GLP—l secretion in diabetic mice after oral load of glucose and Diapin An experiment was performed in adult male KKay diabetic mice. Under fasting conditions, in the l group (white bar in Figure 7, n=11), glucose was orally administered at dose of 1.5mg/g bw and in the Diapin group (black bar in Figure 7, n=1 1), Diapin and glucose were orally administered at lmg/g bw and 1.5mg/g bw, respectively.

Blood samples were collected at 30 min after oral administration of glucose and Diapin.

GLP—l was measured by ELISA (Alpco, Cat# 43—GP1HU—E01).

Diapin also increases GLP—l secretion in ic mice.

Example 4 Diapin ingested with diet decreases random blood glucose levels in KKay diabetic mice in a time— and ependent manner.

An experiment was med in adult male KKay diabetic mice. The mice were divided into three groups of 10 animals each and fed, ad libitum, regular chow (control), chow, chow mixed with 6g Diapin/kg, or chow mixed with 12g Diapin/kg for the duration of the experiment. Blood glucose levels were measured weekly in the early morning at initiation (week 0), and weekly fter for 4 weeks. Results are shown in Figure 8.

Blood glucose levels in both groups fed with chow mixed with Diapin were significantly lower than those in the control.

Example 5 Diapin does not reduce blood e levels in non-diabetic C57BL/6J mice when blood glucose levels are at normal levels.

Diapin has no effect on fasting blood glucose levels in C57BL/6J mice An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. The mice were given water (diamond line in Figure 9, n=6) or Diapin (2mg/g bw, square line in Figure 9, n=6). Blood glucose was measured at O, 30, 60, 90, 120, 150 and 180 min after oral administration of Diapin.

There was no significant difference in blood glucose levels between the groups.

PCT/U82012/024684 Example 6 Diapin, LGL, LGG reduce blood glucose levels in non-diabetic C57BL/6J mice after e is loaded intraperitoneally. In comparison, the peptide GGH does not.

Diapin inhibits the increase of blood glucose after the ip injection of glucose An experiment was performed in adult male 6J mice purchased from Jackson Lab. Fasted mice were given water (diamond line in Figure 10, n=lO) or Diapin orally g bw, square line in Figure 10, n=lO). Glucose was given by ip ion at 10 minutes after the oral stration of Diapin. Blood glucose was measured at 0, 30, 60, 90, 120 min after giving glucose.

Blood glucose levels at 30, 60, 90 and 120 min in Diapin group were significantly lower than those in the control.

The peptide GGH has no significant effect on blood glucose after the ip ion of glucose An experiment was peiformed in adult male C57BL/6J mice purchased from Jackson Lab. Fasted mice were given water (diamond line in Figure 11, n=lO) or GGH (lmg/g bw, square line in Figure 11, n=lO). Glucose was given by ip injection at 10 minutes after oral administration of GGH. Blood glucose was measured at O, 30, 60, 90, 120 min after giving glucose.

Blood glucose levels at any time point in the GGH group were not significantly changed compared to the control mice.

The peptides LGG and LGL inhibit the increase in blood glucose after the ip injection of An ment was performed in adult male C57BL/6J mice purchased from Jackson Lab. Fasted mice were given water (diamond line in Figure 12, n=lO) or LGL (lmg/g bw, square line in Figure 12, n=10) or LGG (lmg/g bw, triange line in Figure 12, n=lO). Glucose was given by ip injection 10 minutes after the oral administration of LGG or LGL. Blood glucose was measured at O, 30, 60, 90, 120 min after giving glucose.

Blood glucose levels at 30, 60, 90 and 120 min in LGL group are significantly lower than those in the control. The peptide LGG reduces the blood glucose levels at 30 and 60 min.

W0 2012/109561 Male].

The peptides LGL, GLG, LLG, and GLL significantly reduce blood glucose levels in non-diabetic C57BL/6J mice loaded with glucose.

Effect of LLG on blood glucose after oral load of glucose An experiment was performed in adult male 6J mice purchased from Jackson Lab. In the control group (diamond line in Figure 13, n=lO), glucose was orally administered at close of 21ng/g bw. In the Diapin and LLG group (square line or triangle line, n=9), glucose and Diapin or glucose and LLG were orally administered at dose of 2mg/g bw and lmg/g bw, respectively. Blood glucose was measured at 0, 30, 60, 90 and 120 min after gavaging glucose and Diapin.

LLG showed similar effects to Diapin.

Effect of es GLG and GLL on blood glucose after oral load of glucose An experiment was performed in adult male C57BL/6J mice sed from Jackson Lab. In the control group (diamond line in Figure 14, n=10), glucose was orally administered at dose of 2mg/g bw. In the GLG and GLL group (triangle line or circle line in Figure 14, n=9), glucose and GLG or GLL were orally administered at dose of 2mg/g bw and lmg/g bw, respectively. Blood glucose was measured at O, 30, 60, 90 and 120 min after gavaging glucose and GLG or GLL.

Peptides GLG and GLL each showed similar effects to Diapin.

Example 8 Amidation and acetylation do not decrease Diapin glucose reduction. Diapin was amidated by the method described in Bergstrom et (11., J. Biol Chem, 280: 23114-23121 (2005) and/or acetylated by the method described in John et (11., Eur. J. Med. Res, 13: 73—78 Effect of ion on Diapin An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. In the control group nd line in Figure 15, n=10), glucose was orally administered at dose of 2mg/g bw. In the Diapin group gle line in Figure 15, n=9), glucose and Diapin were orally administered at dose of 2mg/g bw and lmg/g bw, respectively. In the amidated Diapin group (square line in figure 15, n=9), e and amidated Diapin were orally administered at dose of 2mg/g bw and 1mg/g bw, respectively.

Blood glucose was measured at 0, 0.5, 1, 1.5 and 2 hours after gavaging e, Diapin and amidated Diapin.

Blood glucose levels at O, 0.5, l, 1.5 and 2 hours in the Diapin group and amidated Diapin group were significantly lower than those in the control.

Effect of acetylation of Diapin An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. In the Diapin group (diamond line in Figure 16, n=10), glucose and Diapin were orally administered at dose of 2g/kg bw and Ig/kg bw, respectively. In the acetylated Diapin group (square line in Figure 16, n=lO), glucose and acetylated Diapin were orally administered at dose of 2g/kg bw and lg/kg bw, respectively. Blood glucose was measured at 0, 0.5, l, 1.5 and 2 hours after gavaging e, Diapin, and acetylated Diapin.

Blood glucose levels at O, 0.5, 1, 1.5 and 2 hours in the Diapin group were not significantly different from those in the acetylated Diapin group.

Effect of dual modification on Diapin An experiment was med in adult male C57BL/6J mice sed from Jackson Lab. In the Diapin group (triangle line in Figure 17, n=9), glucose and Diapin were orally administered at dose of 2g/kg bw and Iglkg bw, respectively. In the amidated/acetylated Diapin group (square line, n=9), glucose and amidated/acetylated Diapin were orally administered at dose of 2g/kg bw and Ig/kg bw, tively. Blood glucose was measured at 0, 0.5, 1, 1.5 and 2 hours after gavaging glucose, Diapin and amidated/acetylated Diapin.

Blood glucose levels at 0, 0.5, 1, 1.5 and 2 hours in the Diapin group were not significantly different from those in the amidated/acetylated Diapin group.

Example 9 Diapin reduces blood glucose levels when orally stered prior to glucose administration.

PCT/U52012/024684 Effect of Diapin given at 30min prior to oral glucose administration An experiment was performed in adult male 6J mice purchased from Jackson Lab. Fasted mice were given water (diamond line in Figure 18, n=10) or Diapin (1mg/g bw, square line in Figure 18, n=10), then oral gavage glucose 2g/kg bw after 30min.

Blood glucose was ed at 0, 30, 60 and 120 min after giving glucose.

Blood glucose levels at 30, 60 and 120 min in the Diapin group were significantly lower than those in control group.

Effect of Diapin given at 1 hour prior to oral glucose administration An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. Fasted mice were given water (diamond line in Figure 19, n=10) or Diapin (lmg/g bw, square line in Figure 19, n=10), then oral gavage glucose 2g/kg bw after 1 hour.

Blood glucose was measured at O, 30, 60 and 120 min after giving glucose.

Blood glucose levels at 30, 60 min in the Diapin group were significantly lower than those in control group.

Example 10 ide GG does not cantly reduce blood glucose levels in C57BL/6J mice after oral glucose administration Effect of Diapin and dipeptides GG and GL on blood glucose level after oral e administration An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. Fasted mice were given glucose 200mg/kg bw (diamond line in Figure 20, n=10) or glucose 200mg/kg bw plus Diapin (square line in Figure 20, ling/g bw, n=10), GG e line in Figure 20, 0.67mg/g bw, n=10) or GL (triangle line in Figure 20, 0.67mg/g bw, n=10). Blood glucose was measured at 30, 60, 90 and 120 min after giving glucose. Diapin served as a ve control in the experiment.

Diapin significantly reduced blood glucose levels at 30, 60, 90 and 120 min.

Peptide GL reduced the blood glucose level at 30 min while peptide GG did not significantly reduce blood glucose levels in comparison to Diapin. 2012/024684 Effect of Diapin and dipeptide LG on blood glucose level after oral e administration An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. Fasted mice were given glucose kg bw (triangle line in Figure 21, n=10), glucose 200mg/kg bw plus Diapin (square line in Figure 21, lmg/g bw, n=10). or LG (triangle line in Figure 21, 0.67mg/g bw, n=10). Blood glucose was measured at 30, 60, 90 and 120 min after giving glucose. Diapin served as a positive control in the experiments.

The dipeptide LG showed a more transient affect than did Diapin in reducing blood glucose levels. Diapin reduced blood glucose levels at all ed time points, whereas the dipeptide LG reduced blood glucose levels only at 30 and 60 min.

Example 11 Diapin lowers blood glucose level in ob/ob mice [Liu et al., Diabetes, l409— 16 (2003)] after oral e administration.

An experiment was performed in adult male B6.V—Lepob/J mice purchased from Jackson Lab. The fasted mice were given glucose 2 mg/g (n=10) or glucose 2 mg/g bw plus Diapin (lmg/g bw, n=10) by gavaginga Blood e levels were measured at 30, 60, 90 and 120 min after giving glucose and Diapin.

As shown in Figure 22, Diapin inhibits the increase of blood glucose after oral load of glucose in ob/ob mice.

Example 12 Diapin also lowers blood glucose level in db/db mice [Chen et al., Cell, (3):49l-495 (1996) and Hummel et al., Science, 153 (740):1127—1128 (1966)] after oral glucose stration.

The experiment was performed in adult male BKS.Cg~m +/+ Leprdb/J mice purchased from Jackson Lab. The fasted mice were given glucose 2 mg/g bw (n=10) or glucose 2 mg/g bw plus Diapin (lmg/g bw, n=10) by gavaging. Blood glucose levels were measured at 30, 60, 90 and 120 min after giving glucose and Diapin.

As shown in Figure 23, Diapin inhibits the increase of blood glucose after oral load of glucose in db/db mice.

PCT/U82012/024684 Example 13 Diapin lowers blood glucose level in high fat nduced diabetic mice [Tomas et al., Diabetes Obes. Metab, 26—33 (2011) and Dezaki et al., Diabetes, 55 (12):3486-93 (2006)] after oral glucose administration.

Wild type male C57BL/6J mice purchased from Jackson Lab were fed with high fat diet [rodent diet with 60% of calories from fat (Research Diets Inc. Cat#: D12492)] for eight weeks to induce obesity with insulin resistance mouse model. Then, the fasted mice were given glucose 2mg/g bw (nle) or glucose 2mg/g bw plus Diapin (1mg/g bw, n=10) by gavaging. Blood glucose levels were measured at 30, 60, 90 and 120 min after giving glucose and Diapin.

As shown in Figure 24, Diapin inhibits the increase of blood glucose after oral load of glucose in high fat diet—induced diabetic mice.

Example 14 e-G1ycine—D-Leucine (D—Diapin) has an extended effect on lowering blood glucose level in C57BL/6J mice after oral glucose stration.

An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. The fasted mice were given glucose 2 mg/g bw (n=10) or glucose 2 mg/g bw plus D—Diapin (lmg/g bw, n=10) or Diapin (lmg/g bw, n=10) by gavaging. Blood glucose levels were measured at 30, 60, 90, 120, and 180 min after giving e.

As shown in Figure 25, D—Diapin is more effective than Diapin in lowering blood glucose levels after oral load of glucose in C57BL/6J mice.

Example 15 Diapin lowers blood glucose level in C57BL/6J mice after oral glucose administration and ip Diapin stration.

The experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. The fasted mice were given glucose 2mg/g bw (n=l4) or glucose 2mg/g bw plus Diapin (lmg/g bw, ip, n=l4) or Diapin (lmg/g bw, n=14) by gavaging. Blood glucose was ed at 30, 60, 90 and 120 min after giving glucose and Diapin. Results are shown in Figure 26.

W0 2012l109561 Example 16 Modified dipeptides had ent effects on blood e level in C57BL/6J mice after oral glucose administration. The modified dipeptides tested were an amidated GL dipeptide and the er of dipeptide LG.

An experiment was performed in adult male C57BL/6J mice purchased from Jackson Lab. The fasted mice were given glucose 2mg/g bw (n=12) or glucose 2mg/g bw plus Diapin (lmg/g bw, ip, n=12) or dipeptide (0.67mg/g bw, n212) by gavaging. Blood glucose was measured at 30, 60, 90 and 120 min after giving glucose and peptide. Results are shown in Figure 27.

While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the an.

Accordingly, only such tions as appear in the claims should be placed on the invention.

All nts cited in this application are hereby incorporated by reference in their entirety for their disclosure described.

Claims

Claims (18)

We claim:

1. An oral dosage form comprising as the sole active product ingredient, at least one peptide consisting of the amino acid sequence GGL, GLL, GGdL, GdLL, GLdL or GdLdL, or a pharmaceutically acceptable salt of the peptide, and a pharmaceutically able excipient.

2. The oral dosage form of claim 2, comprising as the sole active product ingredient at least one peptide consisting of the amino acid sequence GGL or GLL.

3. The oral dosage form of claim 2, comprising as the sole active product ient the peptide consisting of the amino acid sequence GGL.

4. The oral dosage form of claim 1, wherein the peptide is acetylated at the N-terminus, amidated at the C-terminus, or both.

5. The oral dosage form of claim 4, comprising as the sole active product ingredient at least one peptide consisting of the amino acid ce GGL or GLL.

6. The oral dosage form of claim 4, comprising as the sole active product ient the peptide consisting of the amino acid sequence GGL.

7. The oral dosage form according to any one of claims 4-6, wherein the peptide is acetylated at the N-terminus.

8. The oral dosage form according to any one of claims 4-6, wherein the peptide is amidated at the C-terminus.

9. The oral dosage form according to any one of claims 4-6, wherein the peptide is acetylated at the N-terminus and amidated at the C-terminus.

10. A kit comprising an oral dosage form wherein the oral dosage form comprises as the sole active product ingredient, at least one peptide ting of the amino acid sequence ed from GGL, GLL, GGdL, GdLL, GLdL or GdLdL, or a pharmaceutically acceptable salt of the peptide, wherein the peptide may be acetylated at the N—terminus, amidated at the C—terminus, or both, and a pharmaceutically acceptable excipient, n the kit is adapted to administer the oral dosage form, and comprises the oral dosage form, instructions for administration of the oral dosage form and a device for stering the oral dosage form to the patient.

11. The kit according to claim 10, wherein the peptide is acetylated at the N—tenninus.

12. The kit according to claim 10, wherein the e is amidated at the C—terminus.

13. The kit according to claim 10, wherein the peptide is ated at the N—terminus and amidated at the C—terminus.

14. The kit ing to claim 10, wherein the peptide is not acetylated at the N—terminus, amidated at the C-terminus, or both.

15. The kit ing to any one of claims 10-13, wherein the oral dosage form comprises as the sole active product ingredient at least one peptide consisting of the amino acid sequence GGL or GLL.

16. The kit according to any one of claims 10—15, wherein the oral dosage form is a solid dosage form.

17. The oral dosage form according to claim 1, substantially as herein described with reference to any one of the accompanying examples and/or figures.

18. The kit according to claim 10, substantially as herein described with reference to any one of the accompanying examples and/or figures. WO 09561 PCT/U