Compositions and Methods for Treating Diabetes, Hypertension, and Hypercholesterolemia

Abstract

Compositions for treating diabetes, hyperglycemia, hypercholesterolemia, hypertension, etc. in a subject are provided. [Solution] The present invention provides compositions comprising a therapeutically effective amount of a polypeptide, peptide, or analog having a specific sequence and one or more of a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, and / or a pharmaceutically acceptable excipient. 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. Also disclosed are methods for treating at least one of diabetes, hyperglycemia, hypercholesterolemia, hypertension, and / or metabolic syndrome in a subject, comprising administering to the subject a polypeptide or formulation of the present invention.

Description

Background Art

[0001] True diabetes or simply diabetes, if left uncontrolled, results in hyperglycemia or elevated blood sugar levels. Over time, hyperglycemia causes serious damage to many body systems, especially the nerves and blood vessels. There are many types of diabetes. Type 1 diabetes occurs when the pancreas produces little or no insulin. Treatment for type 1 diabetes requires insulin injections. The most common type of diabetes, type 2 diabetes, occurs when the pancreas cannot produce enough insulin, or the insulin produced is mostly ineffective due to cellular resistance, or both. The World Health Organization has defined that 90% of diabetes cases worldwide are type 2 diabetes. Treatment for type 2 diabetes includes not only drug treatment and insulin therapy, but also a healthy diet and exercise.

[0002] Complications of chronic hyperglycemia in diabetes include endothelial dysfunction, 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 amputations, and thus is damaging the economies of all developed countries.

[0003] In many cases of type 2 diabetes, pharmacological intervention is necessary for treatment. Many drugs have been approved for type 2 diabetes, such as sulfonylureas, dipeptidyl peptidase-IV (DPP-IV) inhibitors, meglitinides, biguanides, thiazolidinediones, and α-glucosidase inhibitors. However, these drugs cause undesirable side effects such as nausea, hypoglycemia, weight gain, liver damage, rash, headache, and respiratory infections. Furthermore, these drugs are often used together as combination therapy to obtain a stronger drug effect. However, using multiple drugs in combination increases the likelihood of undesirable side effects. Almost 50% of type 2 diabetes patients will ultimately require insulin administration.

[0004] Insulin administration remains the only treatment option for type 1 diabetes. Furthermore, treating type 1 diabetes with insulin is difficult to determine the exact insulin dosage required for changes in physiological conditions, so long-term complications caused by daily cycles of hyperglycemia and hypoglycemia are inevitable.

[0005] Hypertension, hypercholesterolemia, and hyperglycemia are often present in both types of diabetic patients, especially type 2 diabetic patients. The combination of hypertension, hypercholesterolemia, and diabetes significantly increases the risk of heart attack and stroke. Currently, the drugs used to treat these three conditions (hypertension, hypercholesterolemia, and diabetes) have various degrees of side effects. Therefore, it would be useful to have a therapeutic composition that lowers blood glucose without the side effects seen in currently used drugs and also regulates blood pressure and cholesterol. Such a composition would be useful in the treatment of hypertension, cholesterol along with diabetes.

Summary of the Invention

Means for Solving the Problems

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

[0007] Surprisingly, a polypeptide corresponding to the sequence homology in the 40s ribosomal protein S2 active region ("RPS2") has been found to be effective as an orally administered and intravenous therapeutic agent, and has been shown to promote a decrease in blood glucose level, a decrease in insulin resistance, a decrease in hepatic glucose production, a decrease in glucagon level, a decrease in blood pressure (systolic and diastolic levels), and a decrease in blood cholesterol level.

[0008] This specification provides methods and compositions of pharmaceutical compositions having an RPS2 polypeptide, an RPS2 peptide analog, and / or a mixture 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 having 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 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: 2.

[0010] In one embodiment, the RPS2 polypeptide has the amino acid sequence set forth in SEQ ID NO: 3. 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: 3. 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: 3.

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

[0012] In one embodiment, the present invention provides an isolated polypeptide having or consisting of a 40S ribosomal protein S2 (RPS2) having an amino acid sequence with 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 having or consisting of a 40S ribosomal protein S2 (RPS2) having an amino acid sequence with 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 having or consisting of a 40S ribosomal protein S2 (RPS2) having an amino acid sequence with 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 having or consisting of a 40S ribosomal protein S2 (RPS2) having an amino acid sequence with 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, 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: 3. 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: 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, the fragment of RPS2 consists of the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the fragment of RPS2 consists of the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the 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 having at least one polypeptide according to the present invention. At least one polypeptide according to the present invention 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 has 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 have one or more pharmaceutically acceptable carriers. The formulation may have one or more pharmaceutically acceptable diluents. The formulation may have 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 may be present 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, for example, 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, for example 4, 3, or 2 μg / L or less (for example 1 μg / L or less). The aqueous pharmaceutical formulation may contain a buffer. The buffer may have a pH of about 7 to 8, for example, the buffer may have a pH of about 7.2 to about 7.6. The buffer may have a physiological pH (a pH of about 7.4, for example 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 medicament having the polypeptide of the present invention or the 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 the formulation described in the second aspect.

[0021] In a fourth aspect, the present invention provides the polypeptide of the present invention or the formulation of the present invention for use in a method of treating a disease. 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 the formulation 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 the polypeptide of the present invention or the formulation of the present invention for use in a method of treating type 1 and / or type 2 diabetes. 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 the formulation described in the second aspect. The use can result in a decrease in hepatic glucose production, and / or a decrease in cholesterol levels, and / or a decrease in glucagon levels, and / or a decrease in blood pressure. The use can result in a decrease in hepatic glucose production. The use can result in a decrease in cholesterol levels. The use can result in a decrease in glucagon levels. The use can result in a decrease in blood pressure.

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

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

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

[0026] The present invention provides a pharmaceutical composition 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 therapeutic agent of the present invention can be formulated for administration to a subject in need of treatment as an oral formulation, a parenteral formulation, a topical formulation, an aqueous formulation, a solid formulation, a lyophilized formulation, or a transdermal formulation.

Brief Description of the Drawings

[0027]

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Mode for Carrying Out the Invention

[0028] The following detailed description is provided to assist those skilled in the art in practicing the present invention. However, 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, and thus the present invention should not be construed as being limited thereto. All publications and other references cited in this application are hereby incorporated by reference in their entirety.

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

[0030] As used herein, the terms "active ingredient", "active compound", "active component", 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. The formulation of the present invention having the active ingredient / compound is generically referred to as the "IMG-1" formulation without meaning a specific dose or concentration of the active compound.

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

[0032] As used herein, the terms "pharmaceutical preparation", "pharmaceutical composition", "preparation", or "composition" (interchangeably) refer to a liquid (aqueous, gel, or ointment) or a solid form containing an amount of active compound, and are prepared to be suitable for direct or reconstituted administration to mammals such as humans or other animals. Optionally, the preparation may contain pharmaceutically acceptable carriers and / or additives. For example, 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, isotonic agents, binders, disintegrants, lubricants, flow promoters, and flavor correctives. 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 grammatically equivalent terms as used herein include the alleviation, reduction, or improvement of a disease or symptom, the prevention of additional symptoms, the improvement or prevention of the underlying metabolic cause of the symptom, the suppression of a disease or condition, e.g., the prevention of the onset of a disease or condition, the alleviation of a disease or condition, the cause of the alleviation of a disease or condition, the alleviation of a condition caused by a disease or condition, or the cessation of symptoms of a disease or condition, and prevention. This term further includes achieving a therapeutic and / or prophylactic benefit. A therapeutic benefit means the eradication or improvement of the underlying disease being treated. Also, a therapeutic benefit is achieved by the eradication or improvement of one or more of the physiological symptoms associated with the underlying disease such that improvement is observed in the patient, even though the patient may still be afflicted with the underlying disease. For a prophylactic benefit, the composition can be administered to a patient at risk of developing a particular disease or a patient reporting one or more physiological symptoms of a disease even if the disease has not been diagnosed.

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

[0035] The present invention provides a pharmaceutical composition 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 composition comprises 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 their active regions, in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients. The pharmaceutical composition 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 having a purified or synthetic RPS2 peptide, an analog thereof, or an active region, and a buffer such as phosphate buffered saline (PBS), the formulation having a pH within the normal physiological range (about 7.4), and the RPS2 polypeptide or peptide analog having an amino acid sequence as 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 are also provided for preventing, delaying the onset of, or reducing the severity of one or more conditions having hyperglycemia, hypertension, and / or hypercholesterolemia. The invention also provides methods and compositions for improving protein expression in cell culture, particularly yeast, bacteria, and mammalian cell culture, including media for growing cells for protein expression and cell culture production media optimized for protein expression.

[0038] The invention is also described through examples and experimental results that are intended to be illustrative, not intended to be comprehensive, and are to be understood as not being limited thereto.

[0039] 40s RPS2 peptide and amino acid sequence Surprisingly, purified and isolated 40s ribosomal protein S2 (RPS2) containing a truncated fragment, when administered to a subject, exhibits therapeutic advantages 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 is a component of the 40S subunit and encodes a ribosomal protein located in the cytoplasm.

[0040] The RPS2 amino acid sequence does not contain a typical nuclear localization signal. Deletion mutant analysis and rpS2-β-galactosidase chimeric proteins were used to determine the putative identification of the nuclear targeting domain in RPS2. The central domain having 72 - 75 amino acids is necessary and sufficient to target 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 named SEQ ID NO: 1. The truncated C-terminal fragment of SEQ ID NO: 1 is a 159-amino acid fragment named 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 is named SEQ ID NO: 3. The 87-amino acid fragment located at amino acids 135-221 of SEQ ID NO: 1 is named SEQ ID NO: 4. The truncated N-terminal fragment of SEQ ID NO: 1 is a 134-amino acid fragment named SEQ ID NO: 5.

[0042] When the RPS2 polypeptide of SEQ ID NO: 1 is compared by sequence analysis performed using the BLAST database (https: / / blast.ncbi.nlm.nih.gov), it is 94%-100% homologous among all animals. The C-terminal region consisting of 159 amino acids of SEQ ID NO: 2 is 99-100% homologous to the phyla of the animal kingdom. The 75-amino acid nuclear localization sequence corresponding to SEQ ID NO: 3 shares 99-100% homology between all animals and bacteria. While the animal / bacterial protein sequences are on average 98-100% homologous, the homology to plants can be as low as about 77%.

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

[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 named SEQ ID NO: 2. GHVGLGVKCSKEVATAIRGAIILAKLIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDDCYTSARGCTATLGNFAKATFDAISKTYSYLTPDLWKETVFTKSPYQEFTDHLVKTHTRVSVQRTQAPAVATT

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

[0046] An 87-amino acid sequence corresponding to a part of the nuclear localization signal near the N-terminus of amino acids 135 to 221 of SEQ ID NO: 1 is shown below and named SEQ ID NO: 4. GHVGLGVKCSKEVATAIRGAIILAKLSIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDD

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

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

[0049] Purification of RPS2 and RPS2 expression in vitro The RPS2 gene encoding human 40S ribosomal protein S2 and various peptide portions 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 obtained sequence was ligated into the pMAL-5 vector. After ligation and incubation, it was spread on an LB plate containing 100 μg / ml ampicillin and incubated overnight at 37°C to produce transformants for in vivo protein production.

[0050] Production of RPS2 protein in vivo The RPS2 transformant was inoculated into 5 ml of broth and grown to 2×108 cells / ml. Using this culture, 200 mL of LB amp 0.2% glucose was inoculated to an A600 of approximately 0.5. The culture was then induced by adding IPTG (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-thaw and subsequently passed through a 20-gauge needle. The lysed cells were centrifuged and the supernatant was diluted by adding 125 ml of cold CB per 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. The 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, the amount of isolated protein was determined by a bicinchoninic acid assay (BSA) assay, and the amount of protein was evaluated by ELISA.

[0051] For in vivo animal experiments, the purified RPS2 protein corresponding to SEQ ID NO: 1 in the solution after column purification was diluted with PBS to the desired concentration (depending on oral or intravenous administration). 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 conducted using the gene for bacterial RPS2, resulting in similar results in vivo compared to human RPS2. Various tests were conducted to test the safety and efficacy of the isolated RPS2 polypeptide and peptide fragments as therapeutic agents, including tests of the IMG formulation in a diabetic animal model and toxicity tests in a control animal model. The studies conducted and the results obtained are presented herein as examples, together with the accompanying drawings.

Example

[0053] Type 2 diabetes animal model The Zucker diabetic fatty (ZDF) strain of rats is widely known and is commonly used to study type 2 diabetes associated with obesity, as well as hypertension and hypercholesterolemia. 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 and is mainly due to the fact that the strain is genetically inbred and that a special dietary regimen is provided.

[0054] Zucker diabetic fatty (ZDF) rats were fed a special diet of Purina 5008 (Charles River Laboratories International, Inc.) to increase body weight. The blood glucose levels of the animals were evaluated before the test, and only rats with blood glucose levels of 200 mg / dl or higher were used in the test. The animals were randomly divided into four groups: untreated (n = 7), once-daily metformin (200 mg / kg, n = 7), once-daily intravenous administration (IV) of 10 μg of IMG-1 (n = 8), and once-daily oral administration (PO) of 200 μg of IMG-1 (n = 9). The animals were fed this diet for 35 days, and their body weights were measured twice a week. After 35 days, the animals were sacrificed. Figure 1 shows the distribution of body weight (grams) per group measured over 30 days.

[0055] Fasting blood glucose (FBG) levels were evaluated in ZDF through experiments using the Accutrend Plus system. ZDF rats treated with IMG-1 showed a significant decrease in blood glucose levels as early as 3 days after treatment regardless of the administration mode (for the mean FBG levels of 179 mg / dl in the IV administration of IMG-1 and 135 mg / dl in the PO administration of IMG-1, the FBG levels in the untreated group and metformin-treated group were 281 mg / dl and 258 mg / dl respectively). By day 7, all IMG-1-treated animals had normal FBG levels (levels less than 200 mg / dl). Untreated controls and metformin-treated animals had significantly elevated FBG levels throughout the study, and the mean levels exceeded 400 mg / dl (481 mg / dl and 468 mg / dl respectively) at the end of the study. Figure 2 shows that IMG-1 normalizes blood glucose levels in a diabetic animal model, and blood glucose levels decreased in animals treated with IMG-1 compared to controls and metformin.

[0056] The blood pressure of ZDF rats was monitored twice a week using a tail-cuff blood pressure monitor (CODA, a monitor from Kent Scientific). Rats treated with IMG-1 started to show lower blood pressure within 3 days of treatment. ZDF rats orally administered IMG-1 showed normal BP by day 7, while rats intravenously administered IMG-1 showed normal BP by day 10 (120 / 82 mmHg and 115 / 80 mmHg for the IV and PO administrations of IMG-1 respectively). Untreated ZDF rats and metformin-treated ZDF rats had hypertension (>140 / 90 mmHg, 141 / 95 mmHg untreated and 142 / 99 mmHg metformin-treated). Figure 3 shows the systolic and diastolic blood pressure levels in animals treated with IMG-1 compared to controls and metformin, indicating that treatment with IMG-1 normalizes blood pressure in a diabetic animal model.

[0057] To evaluate HbA1c levels, blood was collected from all animals at sacrifice on day 0, day 15, and day 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, there was no significant difference in the mean Hb1Ac level on day 0 (9.7 - 10.8). On day 15, the Hb1Ac level was significantly lower in the IMG-1 treated group compared to both the untreated group and the metformin group (10.2 IV and 9.5 PO vs. 12.1 untreated and 11.0 metformin treated). By day 35, the IMG-1 treated animals were significantly lower than both the untreated and metformin treated animals (12.3 untreated and 11.8 metformin treated), as well as their initial starting levels (10.4 IV and 10.5 PO) (7.4 IV and 7.5 PO).

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

[0059] There was no change in insulin levels in IMG-1, metformin, and control animals, but there were significant decreases in glucagon levels in both IMG-1 treated animals by IV and IMG-1 treated animals by PO 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). There was no significant difference in glucagon levels in control and metformin treated animals throughout the study. Figure 6 shows that IMG-1 decreases glucagon levels in a diabetic animal model. The decrease in glucagon levels when compared to the control was consistent between oral administration and IV administration of IMG-1.

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

Example

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

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

Example

[0063] Toxicity and PK Studies Using the Sprague-Dawley model, access to the clearance and toxicity of the IMG-1 formulation was achieved. Three male Sprague-Dawley rats were treated with 20 μg of the active compound (corresponding to SEQ ID NO: 1) by intravenous administration, followed by blood sampling at intervals. Specifically, at 0, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 2 hours, 4 hours, 6 hours, 24 hours, and 48 hours after IMG-1 administration. As shown in Figure 9, by the serum levels (pg / mL) of the IMG-1 active compound measured over 6 hours, the active compound was detected in the blood of all three animals by 2 hours after administration, and the third animal showed detectable levels up to 4 hours after treatment. Along with the levels of the active compound, blood glucose levels were evaluated in the Sprague-Dawley model. IMG-1 administration dramatically decreased the blood glucose levels in ZDF rats, but a single-dose IMG-1 formulation (20 μg) administered to Sprague-Dawley rats did not promote hypoglycemia within 48 hours, as seen by the 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 performed using Sprague-Dawley rats. The study consisted of 5 groups, using 3 female and 3 male animals per group. The treatment groups were given a single dose of one of the following IMG-1 concentrations: 1.0 μg, 10 μg, 100 μg, or 1000 μg. The control group was not treated. Clinical observations were made hourly for 4 hours after administration and daily for a total of 14 days for all 5 groups. Blood samples were taken from each of the 5 groups (treated and untreated) on days 7 and 14 after administration. No adverse clinical observations were seen in any of the animals. In each of the 5 groups, creatinine levels were measured to evaluate renal function and alanine aminotransferase levels were measured to evaluate liver function. As shown in Figures 10 and 11 respectively, no distinguishable differences were seen between the animals treated with the IMG-1 formulation and the untreated animals in terms of the levels of both creatinine and alanine aminotransferase.

Example

[0065] Insulin clamp study The most widely accepted test method for quantifying insulin secretion and resistance is the euglycemic insulin clamp method, which measures how well an animal metabolizes glucose or how sensitive the animal is to insulin. In this procedure, glucose is fixed at a basal level (100 - 150 mg / dl) by infusing glucose at various rates while infusing exogenous insulin to maintain a constant plasma insulin level higher than during fasting.

[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 the IMG-1 formulation was administered intravenously (per animal) via the tail vein 48 and 24 hours before the clamp test, compared to PBS-treated animals. IMG-1 treated animals had a significantly higher steady-state glucose infusion rate (38.8 mg / kg / mL) during clamping compared to PBS-treated animals (30 mg / kg / mL). As shown in Figure 12, treatment with IMG-1 decreased insulin resistance in DIO mice. As shown in Figure 13, hepatic glucose production (HGP) was also dramatically suppressed in all animals treated with IMG-1. Furthermore, hepatic insulin action (suppression rate of HGP) increased in all IMG-1 treated animals during the hyperinsulinemic euglycemic clamp. However, as is clear from Figure 14, which shows that the levels of glucose metabolic turnover, glycolysis, and glycogen synthesis were nearly equal between control and treated animals, IMG-1 did not affect whole-body glucose metabolic turnover, glycolysis, or glycogen synthesis in any of the treated animals. As is also clear from Figure 15, which shows that skeletal muscle glucose uptake and white adipose tissue glucose uptake were nearly identical between untreated and treated animals, the results of the clamping procedure also showed that glucose metabolism in skeletal muscle and adipose tissue did not differ significantly between IMG-1 or PBS-treated animals during the clamp assay.

Example

[0067] Identification of the RPS2 Active Region The full-length purified RPS2 protein was digested with hydroxylamine (NH2OH) that cleaved RPS2 at two positions (Asn at position P1 and amino acid 134 of Gly at position P1) to obtain two subunits, which were named RPS2-short (IMG-1S) of approximately 13 kDa and RPS2-long (IMG-1L) of approximately 18 kDa. The obtained fragments were separated by non-denaturing polyacrylamide gel, visualized, and then electroeluted from the gel. Formulations for testing in cell culture were prepared using these fragments.

[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 analysis for evaluating cell 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 cell viability, CADMEC cultures, such as those available from Cell Applications, Inc., were treated with one of various formulations (0 μg / mL full-length protein (referred to as IMG-1), 0.5 μg / mL C-terminal fragment (referred to as IMG-1L), or 0.5 μg / mL N-terminal fragment (referred to as IMG-1S)). The treated cultures were grown for 72 hours. After 72 hours of cell growth, 100 μL of the cell culture was treated with 10 μL of 12 mM MTT and incubated at 37 °C for 4 hours. After incubation with MTT for 4 hours, 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 absorbance spectrophotometer (similar to BioRad's xMark™). As shown in Figure 17, at 72 hours, there was a significant increase in relative optical OD in cells treated with the IMG-1 and IMG-1L formulations (164% and 157%, respectively), while IMG-1S did not appear to affect cell growth and was at untreated levels. IMG-1L corresponds to the C-terminal fragment of the full-length protein, while the IMG-1S formulation corresponds to the N-terminal fragment of the full-length protein. Thus, the C-terminal of RPS2, as well as its fragments and / or analogs, have therapeutic value together with the full-length RPS2 protein.

[0069] Exemplary IMG-1 formulation The present invention provides a pharmaceutical composition 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] The formulations of the present invention having a purified or synthetic peptide or peptide analog 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, the pharmaceutical formulation has an RPS2 polypeptide or analog corresponding to one of SEQ ID NOs:1-4, or an active peptide region thereof, in the range of less than 20 μg to 150 μg, as a solid dosage form or a solution. In one embodiment, the formulation has a peptide present at a concentration of 0.05 to 5 μg / L. In another embodiment, the formulation has a peptide present at a concentration of 0.1 to 1 μg / L. In yet another embodiment, the formulation has a peptide present at a concentration of 50 to 150 μg / kg for an oral formulation and 5 to 15 μg / kg for an intravenous formulation. The concentration of the active ingredient and the corresponding dosage will depend, in part, on the body weight of the subject, the route of administration, the symptom / disorder to be treated, and the severity of the symptom.

[0071] In one embodiment, the pharmaceutical formulation of the present invention further has one or more absorption promoters including one or more of detergents, surfactants, bile salts, Ca2+ chelating agents, 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 acetates, citrates, histidine, succinates, phosphates, and hydroxymethylaminomethane (Tris). Exemplary surfactants include polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188. For lyophilized formulations, one or a mixture of polyols / disaccharides / polysaccharides (e.g., mannitol, sorbitol, sucrose, trehalose, and dextran 40) can be used. Sugars make up the majority of the lyophilized formulation 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 have surface modification by one or more lipophilic moieties. In yet another embodiment, the pharmaceutical formulation has an active agent that is selectively 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 having polyacrylic acid or a cellulose derivative. 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(isobutyl cyanoacrylate), and poly(isobutyl cyanoacrylate). Examples of cellulose derivatives include, but are not limited to, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, and methyl hydroxyethyl cellulose. Further, examples of semi-natural bioadhesive polymers include chitosan and various gums such as guar, xanthan, poly(vinyl pyrrolidone), and polyvinyl alcohol.

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

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

[0077] Throughout the description and claims of this specification, the terms "have" and "include" and their variations mean "include but are not limited to", and they are not intended to exclude, nor do they exclude, other parts, additives, components, or steps. Throughout the description and claims of this specification, unless the context requires otherwise, the singular form encompasses the plural form. In particular, when an indefinite article is used, unless the context otherwise requires, this specification should be understood as contemplating not only the singular form but also the plural form.

[0078] Features, characteristics, compounds, chemical moieties, or groups described in connection with a particular aspect, embodiment, or example of the present invention are applicable to other aspects, embodiments, or examples described herein, unless incompatible. All features disclosed in this specification (including the appended claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of the above embodiments. The present invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any appended claims, abstract, and drawings), or any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0079] The reader's attention is directed to all papers and documents filed in connection with this application and simultaneously or previously with this specification and made available to the public for inspection using this specification, and the entire contents of such papers and documents are hereby incorporated by reference into this specification.

Claims

1. A pharmaceutical composition for use in the treatment of hyperglycemia, comprising a peptide of array ID number 1, wherein said treatment is to reduce the blood glucose level to a level below 200 mg / dL.

2. The pharmaceutical composition according to claim 1, wherein the composition is an oral pharmaceutical composition.

3. The pharmaceutical composition according to claim 1, wherein the peptide is present at a concentration of 0.1 to 1 μg / L.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition comprises one or more pharmaceutically acceptable carriers, one or more pharmaceutically acceptable diluents, or one or more pharmaceutically acceptable excipients.

5. Use of a peptide of array ID number 1 in the manufacture of a pharmaceutical composition for the treatment of hyperglycemia, wherein said treatment is to reduce the blood glucose level to a level below 200 mg / dL.

6. The use according to claim 5, wherein the peptide is present in the pharmaceutical composition at a concentration of 0.05 to 5 μg / L.

7. The use according to claim 5, wherein the peptide is present in the pharmaceutical composition at a concentration of 0.1 to 1 μg / L.

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