Compositions and methods for treating diabetes, hypertension and hypercholesterolemia

Abstract

To provide a polypeptide corresponding in sequence homology to an active region of a 40s ribosomal protein S2 ("RPS2") that is beneficial as a therapeutic for oral and iv administration.SOLUTION: Provided is an isolated polypeptide that comprises or consists of a 40s ribosomal protein S2 (RPS2) or a fragment thereof, having a specific amino acid sequence.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] Diabetes mellitus, or simply diabetes, when left uncontrolled, leads to hyperglycemia, or high levels of sugar in the blood. Over time, hyperglycemia can cause serious damage to many body systems, especially nerves and blood vessels. There are many types of diabetes. In type 1 diabetes, the pancreas produces little or no insulin. Treatment of type 1 diabetes requires insulin injections. In type 2 diabetes, the most common form of diabetes, the pancreas does not produce enough insulin, or the insulin produced is largely ineffective due to cellular resistance, or both. The World Health Organization states that type 2 diabetes accounts for 90% of diabetes cases worldwide. Treatment of type 2 diabetes includes not only drug and insulin therapy, but also a healthy diet and exercise.

[0002] Complications of chronic hyperglycemia in diabetes include endothelial damage, proliferative retinopathy, neuropathy, nephropathy, hypertension, and ischemic heart disease. Diabetes is one of the leading causes of heart disease, stroke, kidney failure, blindness, and limb amputation, and therefore is a cost to the economies of all developed countries.

[0003] Pharmacological intervention is necessary for the treatment of many cases of type 2 diabetes. Many medications have been approved for type 2 diabetes, including sulfonylureas, dipeptidyl peptidase IV (DPP-IV) inhibitors, meglitinides, biguanides, thiazolidinediones, and α-glucosidase inhibitors. However, these medications cause unwanted side effects, such as stomach upset, hypoglycemia, weight gain, liver damage, rash, headache, and respiratory infections. Furthermore, these medications are often used together as combination therapy to achieve enhanced efficacy. However, the use of multiple medications increases the likelihood of unwanted side effects. Nearly 50% of patients with type 2 diabetes eventually require insulin administration.

[0004] Insulin administration remains the only treatment option for type 1 diabetes. Furthermore, treatment of type 1 diabetes with insulin inevitably leads to long-term complications caused by daily cycles of hyperglycemia and hypoglycemia due to the difficulty in determining the exact insulin dose required for changing physiological conditions.

[0005] Hypertension, hypercholesterolemia, and hyperglycemia are frequently present in patients with both types of diabetes, especially type 2 diabetes. The combination of hypertension, high cholesterol, and diabetes significantly increases the risk of heart attack and stroke. Currently, the drugs used to treat these three conditions (hypertension, high cholesterol, and diabetes) have varying degrees of side effects. Therefore, it would be useful for a therapeutic composition that lowers blood glucose while also regulating blood pressure and cholesterol without the side effects seen with currently used drugs. Such a composition would have utility in the treatment of hypertension, cholesterol, as well as diabetes. Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to novel pharmaceutical compositions and methods for treating clinical diseases, including those associated with high blood pressure and elevated blood sugar levels (hyperglycemia), such as diabetes, stroke, peripheral vascular disease, pulmonary hypertension, metabolic syndrome, hypercholesterolemia, and atherosclerosis.

[0007] Surprisingly, polypeptides corresponding by sequence homology to the 40s ribosomal protein S2 active domain ("RPS2") have been found to be effective as oral and intravenous therapeutic agents and have been shown to promote lowering of blood glucose levels, lowering of insulin resistance, lowering of hepatic glucose production, lowering of glucagon levels, lowering of blood pressure (systolic and diastolic levels), and lowering of blood cholesterol levels.

[0008] Provided herein are methods and compositions for pharmaceutical compositions comprising RPS2 polypeptides, RPS2 peptide analogs, and / or mixtures thereof. In one embodiment, the RPS2 polypeptide has the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the RPS2 polypeptide has an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In yet another embodiment, the RPS2 polypeptide has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0009] In one embodiment, the RPS2 polypeptide has the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the RPS2 polypeptide has an amino acid sequence that has at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In yet another embodiment, the RPS2 polypeptide has an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0010] In one embodiment, the RPS2 polypeptide has an amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the RPS2 polypeptide has an amino acid sequence that has at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In yet another embodiment, the RPS2 polypeptide has an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0011] In a first aspect, the present invention provides an isolated polypeptide comprising 40s ribosomal protein S2 (RPS2), or a fragment or analog thereof, having an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4. In one embodiment, the present invention provides an isolated polypeptide consisting of 40s ribosomal protein S2 (RPS2), or a fragment thereof, having an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4.

[0012] In one embodiment, the present invention provides an isolated polypeptide comprising, or consisting of, a 40s ribosomal protein S2 (RPS2) having an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the present invention provides an isolated polypeptide comprising, or consisting of, a 40s ribosomal protein S2 (RPS2) having an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the present invention provides an isolated polypeptide comprising, or consisting of, a 40s ribosomal protein S2 (RPS2) having an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the present invention provides an isolated polypeptide comprising, or consisting of, a 40s ribosomal protein S2 (RPS2) having an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0013] In one embodiment, RPS2 or a fragment thereof has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4. In one embodiment, RPS2 has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, a fragment of RPS2 has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the fragment of RPS2 has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the fragment of RPS2 has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0014] In one embodiment, RPS2 or a fragment thereof has the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4. In one embodiment, RPS2 has the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, a fragment of RPS2 has the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, a fragment of RPS2 has the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, a fragment of RPS2 has the amino acid sequence set forth in SEQ ID NO: 4.

[0015] In one embodiment, RPS2 or a fragment thereof consists of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4. In one embodiment, RPS2 consists of the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, a fragment of RPS2 consists of the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, a fragment of RPS2 consists of the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, a fragment of RPS2 consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0016] In a second aspect, the present invention provides a formulation comprising at least one polypeptide according to the present invention, which may be at least one polypeptide according to the first aspect.

[0017] In one embodiment, the formulation is an oral pharmaceutical formulation. In one embodiment, the formulation is a parenteral pharmaceutical formulation. In one embodiment, the formulation is a topical pharmaceutical formulation.

[0018] In one embodiment, the formulation comprises one or more pharmaceutically acceptable carriers and / or one or more pharmaceutically acceptable diluents and / or one or more pharmaceutically acceptable excipients. The formulation may comprise one or more pharmaceutically acceptable carriers. The formulation may comprise one or more pharmaceutically acceptable diluents. The formulation may comprise one or more pharmaceutically acceptable excipients.

[0019] In one embodiment, the formulation is an aqueous pharmaceutical formulation. The peptide may be present in the aqueous pharmaceutical formulation at a concentration of 0.05 to 5 μg / L. For example, the peptide may be present at a concentration of 0.08 to 3 μg / L, or at a concentration of 0.1 to 1 μg / L. The peptide may be present at a concentration of at least 0.05 μg / L, e.g., at least 0.7, 0.1, or 0.5 μg / L. The peptide may be present at a concentration of 5 μg / L or less, e.g., 4, 3, or 2 μg / L or less (e.g., 1 μg / L or less). The aqueous pharmaceutical formulation may contain a buffer. The buffer may have a pH of about 7 to 8, e.g., about 7.2 to about 7.6. The buffer may have a physiological pH (a pH of about 7.4, e.g., a pH of 7.3 to 7.4). The buffer may be phosphate-buffered saline.

[0020] In a third aspect, the present invention provides a pharmaceutical agent comprising a polypeptide of the present invention or a formulation of the present invention. The polypeptide of the present invention may be at least one polypeptide as described in the first aspect. The formulation of the present invention may be a formulation as described in the second aspect.

[0021] In a fourth aspect, the present invention provides a polypeptide of the present invention or a formulation of the present invention for use in a method for treating a disease. The polypeptide of the present invention may be at least one polypeptide as described in the first aspect. The formulation of the present invention may be a formulation as described in the second aspect. The disease may be at least one of type 1 and / or type 2 diabetes, hyperglycemia, hypercholesterolemia, hypertension, and metabolic syndrome. The disease may be type 1 and / or type 2 diabetes. The disease may be hyperglycemia. The disease may be hypercholesterolemia. The disease may be hypertension. The disease may be metabolic syndrome.

[0022] In a fifth aspect, the present invention provides a polypeptide of the present invention or a formulation of the present invention for use in a method for treating type 1 and / or type 2 diabetes. The polypeptide of the present invention may be at least one polypeptide according to the first aspect. The formulation of the present invention may be a formulation according to the second aspect. The use may result in reduced hepatic glucose production, and / or reduced cholesterol levels, and / or reduced glucagon levels, and / or reduced blood pressure. The use may result in reduced hepatic glucose production. The use may result in reduced cholesterol levels. The use may result in reduced glucagon levels. The use may result in reduced blood pressure.

[0023] In a sixth aspect, the present invention provides the use of at least one polypeptide according to the invention for the manufacture of a medicament for the treatment of type 1 and / or type 2 diabetes. The polypeptide of the invention may be at least one polypeptide according to the first aspect.

[0024] In a seventh aspect, the present invention provides use of at least one polypeptide according to the invention for the manufacture of a medicament for treating at least one of hyperglycemia, and / or hypercholesterolemia, and / or hypertension, and / or metabolic syndrome. The polypeptide of the invention may be at least one polypeptide according to the first aspect. The medicament is capable of treating hyperglycemia. The medicament is capable of treating hypercholesterolemia. The medicament is capable of treating hypertension. The medicament is capable of treating metabolic syndrome.

[0025] In an eighth aspect, the present invention provides a method for treating at least one of diabetes, and / or hyperglycemia, and / or hypercholesterolemia, and / or hypertension in a subject, the method comprising administering to the subject a polypeptide of the present invention or a formulation of the present invention. The polypeptide of the present invention may be at least one polypeptide described in the first aspect. The formulation of the present invention may be a formulation described in the second aspect. The method may comprise administering an effective amount of the polypeptide or the formulation. The method may treat diabetes (e.g., type 1 and / or type 2 diabetes). The method may treat hyperglycemia. The method may treat hypercholesterolemia. The method may treat hypertension.

[0026] The present invention provides pharmaceutical compositions comprising a therapeutically effective amount of an RPS2 polypeptide or peptide analog corresponding to one or more of SEQ ID NOS: 1, 2, 3, or 4, and one or more pharmaceutically acceptable carriers, and / or one or more pharmaceutically acceptable diluents, and / or one or more pharmaceutically acceptable excipients. The polypeptide therapeutics of the present invention can be formulated for administration to a subject in need of treatment as oral, parenteral, topical, aqueous, solid, lyophilized, or transdermal formulations. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 is a graph showing the relationship between body weight in diabetic animals (Zucker diabetic rat animal model) administered metformin and / or IMG-1 intravenously (10 μg) and orally (200 μg) and the consistency of weight gain between animals. [Figure 2] FIG. 2 is a graph showing that treatment of diabetic animals with IMG-1 formulations (oral and IV) resulted in normalized blood glucose levels compared to controls and metformin. [Figure 3]FIG. 3 is a graph showing that treatment of diabetic animals with IMG-1 formulations (oral and IV) resulted in normalized blood pressure (systolic and diastolic) levels compared to controls and metformin. [Figure 4] FIG. 4 is a bar graph showing that treatment of diabetic animals with IMG-1 formulations (oral and IV) resulted in a reduction in HbA1c levels compared to controls and metformin. [Figure 5] FIG. 5 is a bar graph showing that treatment of diabetic animals with IMG-1 formulations (oral and IV) does not affect insulin concentrations (mcU / mL). [Figure 6] FIG. 6 is a bar graph showing that treatment of diabetic animals with IMG-1 formulations (oral and IV) resulted in a decrease in glucagon levels compared to controls and metformin. [Figure 7] Figure 7 is a bar graph showing that mean cholesterol levels (mg / dL) measured from diabetic animals administered IMG-1 (oral and intravenous) were lower compared to untreated controls: treated animals averaged <170 mg / dL, while controls averaged 224 mg / dL. [Figure 8] FIG. 8 is a bar graph showing that IMG-1 formulation is effective in lowering blood glucose levels in an animal model of type 1 diabetes, and that the reduction is significant compared to control (untreated) diabetic-induced animals. [Figure 9] 9 is a graph showing the clearance of IMG-1 (measured as pg / mL) from serum over 24 hours in a Sprague-Dawley animal model. Results shown are designated BES17-02-1, 2, 2 (representing three animals). [Figure 10] FIG. 10 is a graph showing blood glucose levels in Sprague-Dawley animals treated with IMG-1 formulation. [Figure 11] FIG. 11 is a graph showing that IMG-1 formulations administered at doses of 1.0 μg to 1000.0 μg have no effect on creatinine levels in Sprague-Dawley rats. [Figure 12] FIG. 12 is a graph showing that IMG-1 formulations administered at doses of 1.0 μg to 1000.0 μg have no effect on alanine aminotransferase levels in Sprague-Dawley rats. [Figure 13] FIG. 13 is a bar graph showing that IMG-1 formulation administered at a dose of 2 μg (intravenous) reduces insulin resistance in diabetes-induced obese (DIO) mice compared to controls, based on glucose infusion rate (mg / kg / m). [Figure 14] FIG. 14 is a bar graph showing that IMG-1 formulation administered at a dose of 2 μg (intravenous) inhibits hepatic glucose production (HGP) in DIO mice compared to controls. [Figure 15] FIG. 15 is a bar graph showing that IMG-1 formulation at a dose of 2 μg (intravenous) had no effect on whole-body glucose turnover, glycolysis, or glycogen synthesis in DIO mice, essentially equivalent to controls. [Figure 16] FIG. 16 is a bar graph showing that IMG-1 formulation at a dose of 2 μg (intravenous) has no effect on skeletal muscle or white fat glucose uptake. [Figure 17] 17 is a bar graph showing the activity of truncated RPS2 peptides designated (IMG-1L and IMG-1S) compared to full-length RPS2 (IMG-1) compared to untreated. Preparations of IMG-1L, corresponding to a C-terminal fragment of the full-length protein, showed similar activity in vitro compared to IMG-1 (full-length), while preparations of IMG-1S (corresponding to an N-terminal fragment of the full-length protein) appeared to have no activity, as measured by optical density from an MTT assay using endothelial cells. DETAILED DESCRIPTION OF THE INVENTION

[0028] The following detailed description is provided to aid those skilled in the art in practicing the present invention, but should not be construed as limiting the present invention, as modifications and variations in the embodiments disclosed herein can be made by those skilled in the art without departing from the scope and spirit of the present invention. All publications and other references cited in this application are incorporated herein by reference in their entirety.

[0029] definition The following terms are used herein to describe different aspects of the present invention. These terms are used for descriptive purposes only and are not intended to limit the scope of any aspect of the present invention.

[0030] As used herein, "active ingredient," "active compound," "active ingredient," and / or "active agent" can be used interchangeably and refer to a polypeptide, peptide fragment, or analog thereof having an amino acid sequence having the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 4, or a combination thereof. Formulations of the present invention having such active ingredients / compounds are collectively referred to as "IMG-1" formulations, without implying a specific dose or concentration of the active compound.

[0031] As used herein, "effective amount" refers to the amount of active ingredient / compound that, when administered to a subject, is effective in lowering blood glucose levels to less than 200 mg / dl, lowering cholesterol to less than 200 mg / dl, and / or lowering blood pressure to less than 140 / 90 mmHg.

[0032] As used herein, the terms "pharmaceutical formulation," "pharmaceutical composition," "formulation," or "composition" refer (interchangeably) to a liquid (aqueous, gel, or ointment) or solid form containing an amount of an active compound, prepared to be suitable for administration directly or after reconstitution to a mammal, such as a human or other animal. If necessary, the formulation may contain pharmaceutically acceptable carriers and / or additives, such as detergents / surfactants (e.g., PEG, Tween (20, 80, etc.), Pluronic), excipients, antioxidants (e.g., ascorbic acid, methionine), colorants, flavoring agents, preservatives, stabilizers, buffers, chelating agents (e.g., EDTA), suspending agents, tonicity agents, binders, disintegrants, lubricants, glidants, and taste-masking agents. The pharmaceutical compositions of the present invention may contain other active ingredients in combination with the RPS2 polypeptides and / or polypeptide analogs described herein.

[0033] As used herein, the terms "treat," "treating," or "treatment," and other grammatical equivalents as used herein, include alleviating, reducing, or ameliorating a disease or symptom, preventing additional symptoms, improving or preventing the underlying metabolic cause of a symptom, inhibiting a disease or condition, e.g., preventing the onset of a disease or condition, alleviating a disease or condition, causing the alleviation of a disease or condition, alleviating a condition caused by a disease or condition, or halting the symptoms of a disease or condition, and preventing it. This term further includes achieving therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to the eradication or amelioration of the underlying disease being treated. Therapeutic benefit is also achieved by the eradication or amelioration of one or more physiological symptoms associated with an underlying disease, such that an improvement is observed in the patient, even though the patient may still be afflicted by the underlying disease. For prophylactic benefit, the composition can be administered to a patient at risk of developing a particular disease or who reports one or more physiological symptoms of a disease, even if the patient has not been diagnosed with the disease.

[0034] As used herein, a "therapeutically effective amount" for treating a condition such as diabetes or hypertension is an amount of active compound capable of achieving a clinically relevant endpoint in a patient or patient population, such as lowering blood glucose levels in diabetes, or lowering blood pressure in hypertension, or lowering cholesterol in hypertriglyceridemia or hypercholesterolemia. As a non-limiting example, administration of an effective amount of an IMG-1 composition has been shown in animal studies to lower blood glucose levels to less than 200 mg / dL, blood pressure to less than 140 / 90 mmHg, and total cholesterol to less than 200 mg / dL (in diabetic animal models).

[0035] The present invention provides pharmaceutical compositions for treating one or more diseases in a subject having one or more of diabetes (type 1 and / or type 2), hypertension, hypercholesterolemia, vascular disease, or metabolic syndrome. The pharmaceutical compositions comprise a purified or synthetic RPS2 polypeptide or peptide analog corresponding to one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or active regions thereof, in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients. The pharmaceutical compositions can be formulated for oral, parenteral, or transdermal / topical administration to a subject in need of treatment.

[0036] In another embodiment, the present invention provides an aqueous pharmaceutical formulation comprising a purified or synthetic RPS2 peptide, analog thereof, or active domain thereof, and a buffer such as phosphate buffered saline (PBS), wherein the formulation has a pH within the normal physiological range (about 7.4), and the RPS2 polypeptide or peptide analog has an amino acid sequence set forth in one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0037] Methods for preventing, delaying the onset of, or reducing the severity of one or more conditions having hyperglycemia, hypertension, and / or hypercholesterolemia are also provided. The present invention also provides methods and compositions for improving protein expression in cell culture, particularly yeast, bacterial, and mammalian cell cultures, including media for growing cells for protein expression and cell culture production media optimized for protein expression.

[0038] The present invention is also described through examples and experimental results which are intended as examples and are not intended to be exhaustive and should be understood as being non-limiting.

[0039] 40s RPS2 peptide and amino acid sequences Surprisingly, it has been discovered that purified and isolated 40s ribosomal protein S2 (RPS2), including truncated fragments, when administered to a subject, exhibit therapeutic benefits, including the ability to lower blood glucose levels, normalize blood glucose levels, lower blood cholesterol levels, lower and normalize blood pressure levels, lower hemoglobin A1c levels, lower glucagon levels, and reduce insulin resistance. For reference, 40s RPS2 is a protein belonging to the S5P family of ribosomal proteins. In humans, the RPS2 gene encodes a ribosomal protein that is a component of the 40S subunit and is located in the cytoplasm.

[0040] The RPS2 amino acid sequence does not contain a typical nuclear localization signal. Using deletion mutant analysis and rpS2-β-galactosidase chimeric proteins, we determined the putative identity of a nuclear targeting domain in RPS2. A central domain spanning 72–75 amino acids is necessary and sufficient for targeting the chimeric β-galactosidase to the nucleus. This nuclear targeting domain does not share significant similarity with previously characterized nuclear localization signals in ribosomal proteins or other nuclear proteins.

[0041] The full-length RPS2 amino acid sequence is a 293 amino acid sequence designated SEQ ID NO: 1. A truncated C-terminal fragment of SEQ ID NO: 1 is a 159 amino acid fragment designated SEQ ID NO: 2. The non-conventional nuclear localization signal is a 75 amino acid fragment located at amino acids 161-235 of SEQ ID NO: 1 and designated SEQ ID NO: 3. An 87 amino acid fragment located at amino acids 135-221 of SEQ ID NO: 1 is designated SEQ ID NO: 4. A truncated N-terminal fragment of SEQ ID NO: 1 is a 134 amino acid fragment designated SEQ ID NO: 5.

[0042] The RPS2 polypeptide of SEQ ID NO: 1 is 94%-100% identical to all animals when compared by sequence analysis using the BLAST database (https: / / blast.ncbi.nlm.nih.gov). The 159 amino acid C-terminal region of SEQ ID NO: 2 is 99-100% identical to phyla within the Animalia kingdom. The 75 amino acid nuclear localization sequence corresponding to SEQ ID NO: 3 shares 99-100% identity with all animals and bacteria. While animal / bacterial protein sequences average 98-100% identity, identity to plants can be as low as 77%.

[0043] The amino acid sequence of RPS2 of the present invention (293 amino acids) is shown below and designated SEQ ID NO: 1. MADDAGAAGGPGGPGGPGMGNRGGFRGGFGSGIRGRGRGRGRGRGRGARGGKAEDKEWMPVTKLGRLVKDMKIKSLEEIYLFSLPIKESEIIDFFLGASLKDEVLKIMPVQKQTRAGQRTRFKAFVAIGDYNGHVGLGVKCSKEVATAIRGAIILAKLSIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDDCYTSARGCTATLGNFAKATFDAISKTYSYLTPDLWKETVFTKSPYQEFTDHLVKTHTRVSVQRTQAPAVATT

[0044] The RPS2 C-terminal fragment of the present invention is a 159 amino acid fragment of approximately 18 kDa. This C-terminal fragment is shown below and designated SEQ ID NO: 2: GHVGLGVKCSKEVATAIRGAIILAKLIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDDCYTSARGCTATLGNFAKATFDAISKTYSYLTPDLWKETVFTKSPYQEFTDHLVKTHTRVSVQRTQAPAVATT

[0045] The 75 amino acid nuclear localization signal of the present invention (corresponding to amino acids 161-235 of SEQ ID NO: 1) is shown below and designated SEQ ID NO: 3: SIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDDCYTSARGCTATLGN

[0046] The 87 amino acid sequence corresponding to a portion of the nuclear localization signal near the N-terminus of SEQ ID NO: 1, from amino acids 135 to 221, is shown below and designated SEQ ID NO: 4: GHVGLGVKCSKEVATAIRGAIILAKLSIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDD

[0047] The RPS2 N-terminal fragment of the present invention is an approximately 13 kDa fragment and is designated SEQ ID NO: 5. MADDAGAAGGPGGPGGPGMGNRGGFRGGFGSGIRGRGRGRGRGRGRGRGARGGKAEDKEWMPVTKLGRLVKDMKIKSLEEIYLFSLPIKESEIIDFFLGASLKDEVLKIMPVQK QTRAGQRTRFKAFVAIGDYN

[0048] The amino acids contained in the amino acid sequences of the present invention can be post-translationally modified according to methods well known in the art. For example, modification of N-terminal glutamine (Gln) residues to pyroglutamic acid (pGlu) residues by pyroglutamylation is well known to those skilled in the art. Of course, such post-translationally modified amino acids are included in the amino acid sequences and fall within the scope of the present invention.

[0049] Purification of RPS2 and RPS2 expression in vitro The RPS2 gene, encoding the human 40S ribosomal protein S2, and various peptide fragments were subcloned into the pMAL-5 vector (New England Biolabs). The RPS2 gene was amplified using a forward primer (sequence: atggcggatgacgccggtgc) and a reverse primer (sequence: ctatgttgtagccacagctgg). The resulting PCR fragment was phosphorylated and purified from low-melting-point agarose. The resulting sequence was ligated into the pMAL-5 vector. After ligation and incubation, the transformants were plated on LB plates containing 100 μg / ml ampicillin and incubated overnight at 37°C to generate transformants for in vivo protein production.

[0050] Generation of RPS2 protein in vivo The RPS2 transformant was inoculated into 5 ml of broth and grown to 2 x 10 cells / ml. This culture was used to inoculate 200 ml of LB amp 0.2% glucose to an A600 of approximately 0.5. The culture was then induced by adding ITPG (isopropylthio-β-galactoside) to a final concentration of 0.3 mM and grown for an additional 4 hours at 30°C. The cells were then centrifuged and resuspended in 25 ml of column buffer per liter of culture. The cells were lysed by freeze-thawing and subsequently passaged through a 20-gauge needle. The lysed cells were centrifuged, and the supernatant was diluted by adding 125 ml of cold CB for every 25 ml of crude extract. The diluted crude extract was applied to a 15 ml amylose column and washed with 12 column volumes of CB. Protein was eluted with CB and maltose (10 mM). The resulting eluate was incubated with 1 μl of Factor Xa diluted to 200 μg / ml for 4 hours at room temperature. The fusion protein cleavage mixture was dialyzed at pH 8.0, the amylase column was washed with buffer, and the fusion protein cleavage mixture was loaded onto the column. The flow-through was collected, and the amount of isolated protein was determined by bicinchoninic acid assay (BSA) and assessed by ELISA.

[0051] For in vivo animal studies, the purified RPS2 protein corresponding to SEQ ID NO: 1 in solution after column purification was diluted with PBS to the desired concentration (for oral or intravenous administration), and the resulting solution was filtered through a 0.22 μm filter to filter-sterilize the formulation for administration to animal subjects, as described in the Examples.

[0052] Similar studies were performed using the bacterial RPS2 gene and yielded similar results in vivo compared to human RPS2. Various studies were conducted to test the safety and efficacy of isolated RPS2 polypeptides and peptide fragments as therapeutic agents, including testing IMG formulations in diabetic animal models and toxicity studies in controlled animal models. The studies conducted and results obtained are presented herein as examples, along with the accompanying figures. [Example]

[0053] Type 2 diabetes animal model The Zucker Diabetic Fatty (ZDF) strain of rats is widely known and commonly used to study obesity and type 2 diabetes associated with hypertension and high cholesterol. The ZDF strain is an inbred rat model of early-onset diabetes, and all fa / fa male rats develop diabetes at 10–12 weeks of age when fed a special diet of Purina 5008 (Charles River Laboratories International, Inc., MA, USA). The phenotype is homogeneous, primarily due to the fact that the strain is genetically inbred and the special dietary treatment is provided.

[0054] Zucker Diabetic Fatty (ZDF) rats were fed a special diet of Purina 5008 (Charles River Laboratories International, Inc.) to promote weight gain. Prior to the study, the animals' blood glucose levels were assessed, and only rats with blood glucose levels of 200 mg / dL or higher were used in the study. The animals were randomly divided into four groups: untreated (n = 7), metformin (200 mg / kg, n = 7) once daily, 10 μg of IMG-1 administered intravenously (IV, n = 8) once daily, and 200 μg of IMG-1 administered orally (PO, n = 9) once daily. The animals were maintained on this diet for 35 days, and their body weights were measured twice weekly. After 35 days, the animals were sacrificed. Figure 1 shows the distribution of body weights (grams) per group measured over a 30-day period.

[0055] Fasting blood glucose (FBG) levels were assessed in ZDF rats throughout the experiment using the Accutrend Plus system. IMG-1-treated ZDF rats, regardless of the mode of administration, showed a significant decrease in blood glucose levels as early as 3 days after treatment (mean FBG levels of 179 mg / dl with IV administration of IMG-1 and 135 mg / dl with PO administration of IMG-1, compared with FBG levels of 281 mg / dl and 258 mg / dl in the untreated and metformin-treated groups, respectively), and by day 7, all IMG-1-treated animals had normal FBG levels (levels below 200 mg / dl). Untreated controls and metformin-treated animals had significantly elevated FBG levels throughout the study, with mean levels exceeding 400 mg / dl (481 mg / dl and 468 mg / dl, respectively) at the end of the study. FIG. 2 shows that IMG-1 normalizes blood glucose levels in a diabetic animal model, with blood glucose levels being lower in animals treated with IMG-1 versus controls and metformin.

[0056] The blood pressure of ZDF rats was monitored twice weekly using a tail-cuff blood pressure monitor (CODA, Kent Scientific). Within 3 days of treatment, rats treated with IMG-1 began to exhibit lower blood pressure; ZDF rats administered orally with IMG-1 exhibited normal BP by day 7, whereas rats administered intravenously with IMG-1 exhibited normal BP by day 10 (120 / 82 mmHg and 115 / 80 mmHg for IV and PO administration of IMG-1, respectively). Untreated and metformin-treated ZDF rats had hypertension (>140 / 90 mmHg, compared with 141 / 95 mmHg for untreated and 142 / 99 mmHg for metformin treatment). Figure 3 shows systolic and diastolic blood pressure levels in IMG-1-treated animals compared with control and metformin-treated animals, demonstrating that treatment with IMG-1 normalizes blood pressure in a diabetic animal model.

[0057] To assess HbA1c levels, blood samples were collected from all animals at sacrifice on days 0, 15, and 35. Bioassays were performed on the collected blood and serum. Hemoglobin A1c (HbA1c) levels were measured in all animals at all three time points. As shown in Figure 4, mean Hb1Ac levels were not significantly different on day 0 (9.7-10.8), and on day 15, Hb1Ac levels were significantly lower in the IMG-1-treated group compared with both the untreated and metformin groups (10.2 IV, 9.5 PO vs. 12.1 untreated, 11.0 metformin-treated). By day 35, IMG-1-treated animals had significantly lower levels (7.4 IV, 7.5 PO) than both untreated and metformin-treated animals (12.3 untreated, 11.8 metformin-treated), as well as their initial starting levels (10.4 IV, 10.5 PO).

[0058] Insulin levels were measured in all animals at three time points, along with Hb1Ac levels. As shown in Figure 5, unlike the Hb1Ac levels in Figure 4, there were no significant differences in insulin levels between the untreated and IMG-1-treated groups at any of the three time points. Furthermore, the results observed in the control group were consistent with the literature.

[0059] Although there was no change in insulin levels in IMG-1, metformin, and control animals, there was a significant decrease in glucagon levels in both IV and PO IMG-1-treated animals on Day 15 (from 115 pg / ml and 119 pg / ml to 90 pg / ml and 92 pg / ml, respectively) and Day 35 (89 pg / ml and 92 pg / ml). Control and metformin-treated animals did not have significantly different glucagon levels throughout the study. Figure 6 shows that IMG-1 reduces glucagon levels in a diabetic animal model. The decrease in glucagon levels compared to controls was consistent between oral and IV administration of IMG-1.

[0060] Cholesterol levels in ZDF rats were assessed 48 hours after a single injection of 20 μg of IMG-1, after continuous access to IMG-1 in drinking water, or untreated (n=4). IMG-1-treated animals had significantly reduced cholesterol levels compared to untreated cohorts. Figure 7 shows that IMG-1 reduces cholesterol in a diabetic animal model. Untreated animals had cholesterol levels of 224 mg / dL, while IMG-1-treated animals had cholesterol levels of 171 mg / dL (IV) and 156 mg / dL (oral), respectively. [Example]

[0061] Type 1 diabetes animal model Since IMG-1 has been shown to normalize blood glucose levels and reduce HA1C in a type 2 diabetes animal model, we investigated whether IMG-1 could affect blood glucose levels in a type 1 diabetes animal model. Diabetes can be induced in mice by using streptozotocin (STZ), a compound that has selective toxicity to pancreatic β cells and is a widely used chemical for the induction of experimental diabetes in rodents. STZ is an antibiotic produced by the bacterium Streptomyces achromogens. It contains a glucose molecule (deoxygenated form) bound to a highly reactive methylnitrosourea moiety, which exerts cytotoxic effects on pancreatic β cells.

[0062] To investigate the efficacy of IMG-1 in the STZ-induced diabetes model, 20 male C57BL / 6J mice, aged 3–4 months, were administered STZ by IP injection for 5 days to promote the development of hyperglycemia. After STZ injection, baseline blood glucose levels were measured after a 4-hour fast and used to select mice into two study groups: the control group (placebo, n = 7) and the IMG-1 group (n = 8 / group). The animals were treated with a once-daily oral gavage of either control or 33 μg of IMG-1 (PO) for 3 weeks. During the treatment period, fasting blood glucose levels were measured every 2–3 days, as were insulin and glucagon levels. On day 18 after STZ treatment, fasting blood glucose levels in the control group increased from 216 mg / dL to 319 mg / dL, and one animal in the control group (<14%) had to be euthanized due to failure to thrive. However, the blood glucose levels of the IMG-1 group only increased slightly from 209 mg / dl to 237 mg / dl, and by day 18, the IMG-1 group was shown to have significantly lower blood glucose than the control animals, as shown in Figure 8. [Example]

[0063] Toxicity and PK studies The clearance and toxicity of IMG-1 formulations were assessed using a Sprague-Dawley model. Three male Sprague-Dawley rats were treated intravenously with 20 μg of the active compound (corresponding to SEQ ID NO: 1), followed by blood sampling at intervals: 0, 5, 15, 30, 60, 90 minutes, 2 hours, 4 hours, 6 hours, 24 hours, and 48 hours after IMG-1 administration. As shown in Figure 9, serum levels (pg / mL) of the IMG-1 active compound measured over a 6-hour period showed that the active compound was detectable in the blood of all three animals by 2 hours after administration, with the third animal showing detectable levels up to 4 hours after treatment. Blood glucose levels were also assessed in the Sprague-Dawley model along with the active compound levels. Although IMG-1 administration dramatically reduced blood glucose levels in ZDF rats, a single dose of IMG-1 formulation (20 μg) administered to Sprague-Dawley rats did not promote hypoglycemia within 48 hours, as seen by blood glucose levels over 48 hours in Sprague-Dawley animals (see Figure 9 ).

[0064] To evaluate the toxicity of the IMG-1 formulation, a pilot dose-finding toxicity assay was conducted using Sprague-Dawley rats. The study consisted of five groups, with three female and three male animals per group. Treatment groups received a single dose of one of the following IMG-1 concentrations: 1.0 μg, 10 μg, 100 μg, or 1000 μg. The control group received no treatment. Clinical observations were performed hourly for four hours after dosing and daily for a total of 14 days for all five groups. Blood samples were collected from each of the five groups (treated and untreated) on days 7 and 14 after dosing. No adverse clinical observations were observed in any of the animals. Creatinine levels were measured to assess renal function, and alanine aminotransferase levels were measured to assess liver function in each of the five groups. As shown in Figures 10 and 11, respectively, no discernible differences were observed between animals treated with the IMG-1 formulation and untreated animals in both creatinine and alanine aminotransferase levels. [Example]

[0065] Insulin clamp studies The most widely accepted test method for quantifying insulin secretion and resistance is the euglycemic insulin clamp, which measures how well an animal metabolizes glucose, or how sensitive an animal is to insulin. In this procedure, glucose is clamped at basal levels (100–150 mg / dL) by infusing glucose at various rates while infusing exogenous insulin to maintain constant plasma insulin levels above fasting levels.

[0066] To evaluate insulin action and glucose metabolism in IMG-1-treated animals (n = 8) versus controls (n = 8), diet-induced obese (DIO) C57BL / 6J mice were subjected to a 2-hour hyperinsulinemic-euglycemic clamp. Prior to the clamp, 2 μg of IMG-1 formulation was administered intravenously via the tail vein (per animal) 48 and 24 hours before the clamp compared with PBS-treated animals. IMG-1-treated animals had significantly higher steady-state glucose infusion rates (38.8 mg / kg / mL) during the clamp than PBS-treated animals (30 mg / kg / mL). As shown in Figure 12, treatment with IMG-1 reduced insulin resistance in DIO mice. As shown in Figure 13, hepatic glucose production (HGP) was also dramatically suppressed in all IMG-1-treated animals. Furthermore, hepatic insulin action (the percentage of HGP suppression) was increased in all IMG-1-treated animals during the hyperinsulinemic-euglycemic clamp. However, IMG-1 did not affect whole-body glucose turnover, glycolysis, or glycogen synthesis in any of the treated animals, as evidenced by Figure 14, which shows that the levels of glucose turnover, glycolysis, and glycogen synthesis were approximately equal between control and treated animals. The results of the clamping procedure also indicated that glucose metabolism in skeletal muscle and adipose tissue was not significantly different in either IMG-1- or PBS-treated animals during the clamp assay, as evidenced by Figure 15, which shows that skeletal muscle glucose uptake and white adipose tissue glucose uptake were approximately identical between untreated and treated animals. [Example]

[0067] Identification of RPS2 active regions The full-length purified RPS2 protein was digested with hydroxylamine (NHOH), which cleaves RPS2 at two positions (Asn at position P1 and Gly at position P1, amino acid 134) to yield two subunits, designated RPS2-short (IMG-1S) at approximately 13 kDa and RPS2-long (IMG-1L) at approximately 18 kDa. The resulting fragments were separated on a non-denaturing polyacrylamide gel, visualized, and then electroeluted from the gel. This fragment was used to prepare a formulation for testing in cell culture.

[0068] Human dermal microvascular endothelial cells (CADMEC / HMVEC) provide an excellent model system for studying many aspects of endothelial function and disease, particularly those related to the microvasculature. The MTT assay is a colorimetric assay for assessing cellular metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes can, under defined conditions, reflect the number of viable cells present. These enzymes can reduce the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to its insoluble formazan, which has a purple color. To evaluate the effect of IMG formulations on cellular activity, CADMEC cultures, such as those available from Cell Applications, Inc., were treated with one of various formulations: 0 μg / mL full-length protein (termed IMG-1), 0.5 μg / mL C-terminal fragment (termed IMG-1L), or 0.5 μg / mL N-terminal fragment (termed IMG-1S). Treated cultures were grown for 72 h. After 72 hours of cell growth, 100 μL of cell culture was treated with 10 μL of 12 mM MTT and incubated at 37°C for 4 hours. After 4 hours of incubation with MTT, 100 μL of SDS-HCl solution was added to the MTT-treated cell culture to solubilize the crystals and incubated for an additional 4 hours. Absorbance was then read at 570 nm using a microplate spectrophotometer (similar to BioRad's xMark™). As shown in Figure 17, at 72 hours, there was a significant increase in relative optical density (OD) in cells treated with IMG-1 and IMG-1L formulations (164% and 157%, respectively), whereas IMG-1S did not appear to affect cell growth or untreated levels. IMG-1L corresponds to a C-terminal fragment of the full-length protein, while the IMG-1S formulation corresponds to an N-terminal fragment of the full-length protein. Therefore, the C-terminus of RPS2, as well as its fragments and / or analogs, may have therapeutic value along with the full-length RPS2 protein.

[0069] Exemplary IMG-1 Formulations The present invention provides pharmaceutical compositions having one or more polypeptides, peptides, and / or analogs corresponding to one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and / or SEQ ID NO: 4.

[0070] Formulations of the present invention containing purified or synthetic peptides or peptide analogs corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4 can be formulated according to methods available to those skilled in the art. In one embodiment, a pharmaceutical formulation contains an RPS2 polypeptide or analog corresponding to one of SEQ ID NOs:1-4, or an active peptide region thereof, in a range of less than 20 μg to 150 μg, in a solid dosage form or solution. In one embodiment, the formulation contains the peptide at a concentration of 0.05-5 μg / L. In another embodiment, the formulation contains the peptide at a concentration of 0.1-1 μg / L. In yet another embodiment, the formulation contains the peptide at a concentration of 50-150 μg / kg for oral formulations and 5-15 μg / kg for intravenous formulations. The concentration of the active ingredient and corresponding dose will depend, in part, on the subject's body weight, the route of administration, the condition / disorder being treated, and the severity of the condition.

[0071] In one embodiment, the pharmaceutical formulation of the present invention further comprises one or more absorption enhancers including one or more detergents, surfactants, bile salts, Ca chelators, fatty acids, medium chain glycerides, acylcarnitines, alkanoylcholines, N-acetylated α-amino acids, N-acetylated non-α-amino acids, chitosan, mucoadhesive polymers, and phospholipids.

[0072] In one embodiment, exemplary excipients useful in the present invention include buffers, salts, surfactants, polyols / disaccharides / polysaccharides, amino acids, and antioxidants. Exemplary buffers that maintain a pH level of 4.7 to 7.4 include acetate, citrate, histidine, succinate, phosphate, and hydroxymethylaminomethane (Tris). Exemplary surfactants include polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188. Lyophilized formulations can use one or a mixture of polyols / disaccharides / polysaccharides (e.g., mannitol, sorbitol, sucrose, trehalose, and dextran 40). Sugars comprise the majority of lyophilized formulations and are known to serve as stabilizers for therapeutic proteins. Sodium chloride (NaCl) is commonly used with protein formulations. Examples of antioxidants include ascorbic acid, methionine, and ethylenediaminetetraacetic acid (EDTA).

[0073] In other embodiments, the pharmaceutical formulations of the present invention further comprise a surface modification with one or more lipophilic moieties. In yet another embodiment, the pharmaceutical formulations optionally comprise an active agent co-administered with a concentrated solution of one or more carrier molecules.

[0074] In yet another embodiment, the pharmaceutical formulation further comprises one or more synthetic bioadhesive polymers comprising polyacrylic acid or cellulose derivatives. Examples of polyacrylic acid-based polymers include, but are not limited to, carbopol, polycarbophil, polyacrylic acid (PAAc), polyacrylate, poly(methyl vinyl ether-co-methacrylic acid), poly(2-hydroxyethyl methacrylate), poly(methacrylate), poly(alkyl cyanoacrylate), poly(isohexyl cyanoacrylate), and poly(isobutyl cyanoacrylate). Cellulose derivatives include, but are not limited to, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, and methylhydroxyethyl cellulose. Additionally, semi-natural bioadhesive polymers include chitosan and various gums such as guar, xanthan, poly(vinylpyrrolidone), and polyvinyl alcohol.

[0075] In yet another embodiment, the pharmaceutical formulation of the present invention further comprises a gastrointestinal mucoadhesive patch system (GI-MAPS) containing an active agent with a layered film contained in an enteric capsule. The GI-MAPS comprises a backing layer containing the water-insoluble polymer ethyl cellulose (EC), a surface layer containing an enteric pH-sensitive polymer such as hydroxypropyl methylcellulose phthalate, Eudragit L100, or S100, a coating layer containing an adhesive layer, and a peptide-containing intermediate layer consisting of a cellulose membrane attached to the backing layer. After oral administration, the surface layer dissolves at the target intestinal site and adheres to the small intestinal wall, where it adheres to the mucosa, forming a closed space at the target site in the gastrointestinal mucosa. As a result, both the active substance and the absorption enhancer coexist within the closed space, forming a high concentration gradient between the system and enterocytes, which contributes to enhanced peptide absorption.

[0076] It will be apparent to those skilled in the art that features described with respect to any of the above-described embodiments may be applicable interchangeably between different embodiments. The above-described embodiments are examples for illustrating various features of the present invention.

[0077] Throughout the description and claims of this specification, the terms "having" and "comprising," and variations thereof, mean "including but not limited to," and they are not intended to exclude, and do not exclude, other moieties, additives, ingredients, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating the plural as well as the singular, unless the context requires otherwise.

[0078] Any feature, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, except where incompatible. All features disclosed in this specification (including any accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the embodiments described above. The invention extends to any novel, or any novel combination of, features disclosed in this specification (including any accompanying claims, abstract, and drawings), or any novel, or any novel combination of steps of any method or process so disclosed.

[0079] The reader's attention is drawn to all articles and documents in connection with this application that are filed contemporaneously or earlier herewith and that are made available for public inspection hereby, the contents of all such articles and documents being incorporated herein by reference.

Claims

1. An isolated polypeptide consisting of an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in sequence ID number 2.

2. A polypeptide as described in claim 1, wherein the polypeptide consists of the amino acid sequence set forth in sequence ID number 2.

3. A composition comprising a polypeptide consisting of an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in sequence ID number 2, said composition being used to lower at least one value selected from the group consisting of blood glucose level, cholesterol level, glucagon level, and blood pressure in a patient in need thereof.

4. A composition according to claim 3, wherein the polypeptide consists of an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in sequence ID number 2, or wherein the polypeptide consists of the amino acid sequence set forth in sequence ID number 2.

5. A composition according to claim 3 or 4, wherein the composition is an oral pharmaceutical composition.

6. A composition according to claim 3 or 4, wherein the composition is a parenteral pharmaceutical composition.

7. A composition according to any one of claims 3 to 6, wherein the composition comprises one or more pharmaceutically acceptable carriers, and / or one or more pharmaceutically acceptable diluents, and / or one or more pharmaceutically acceptable excipients.

8. A composition according to any one of claims 3 to 7, wherein the composition is an aqueous pharmaceutical composition.

9. A composition described in any one of claims 3 to 8, wherein the polypeptide is present at a concentration of 0.05 to 5 μg / L.

10. A composition according to any one of claims 3 to 8, wherein the polypeptide is present at a concentration of 0.1 to 1 μg / L.

11. The composition of claim 8, wherein the aqueous pharmaceutical composition comprises a buffer, and the buffer has a physiological pH.

12. The composition of claim 11, wherein the buffer is phosphate buffered saline.

13. A drug comprising a polypeptide according to claim 1 or 2, or a composition according to any one of claims 3 to 12.

14. Use of a polypeptide comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in sequence ID number 2 for the manufacture of a medicament for the treatment of type 2 diabetes.

15. Use of a polypeptide comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in sequence ID number 2 for the manufacture of a medicament for treating at least one of hyperglycemia, and / or hypercholesterolemia, and / or hypertension, wherein the treatment of hyperglycemia is by lowering blood glucose levels to a level below 200 mg / dL.

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