AU2014374945A1 - Novel polypeptides - Google Patents

Abstract

The present invention relates to an isolated polypeptide comprising (a) the amino acid sequence set forth in SEQ ID NO: 1; or (b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, nucleic acids, vectors and host cells encoding the same, as well as therapeutic treatments based thereon.

Description

NOVEL POLYPEPTIDES

Field of the Invention

The present invention relates to novel polypeptides, to nucleic acids encoding the same and to compositions comprising the same, as well as to their uses in therapeutic methods.

Background of the Invention

Disorders of carbohydrate metabolism occur in many forms. The most common disorders are acquired. Acquired or secondary derangements in carbohydrate metabolism, such as diabetic ketoacidosis, hyperosmolar coma, and hypoglycemia, all affect the central nervous system. Many forms and variants of peripheral nerve disease also are seen in diabetes. The remaining disorders of carbohydrate metabolism are the rare inborn errors of metabolism (i.e. genetic defects).

The acquired disorders of carbohydrate metabolism are fairly common, both in the United States and internationally. Hypoglycemia is a common cause of neurological disease, especially acute mental deterioration, memory loss, disorientation, obtundation, and coma, among both alcoholics and patients with diabetes who are treated with insulin. Hyperinsulinemia from other causes is rare, but pancreatic tumors could be the cause. Diabetes, with its various neurological complications, is among the most common disorders treated in adult patients.

Diabetes (Diabetes mellitus) is the most common endocrine disease, and is characterized by abnormalities of glucose metabolism. The abnormal glucose metabolism associated with this disease results in hyperglycemia (high blood glucose levels) and eventually causes complications of multiple organ systems, including eyes, kidneys, nerves, and blood vessels. Patients with persistent hyperglycemia or abnormal glucose tolerance are generally diagnosed with the disease, although most commonly patients initially present with excessive urination (polyuria) and frequent drinking due to extreme thirst (polydipsia). These typical initial symptoms result from the osmotic effects of hyperglycemia. The pathogenesis of diabetes mellitus is typically associated with pancreatic dysfunction, particularly of the beta cells of the pancreatic islets of Langerhans. This dysfunction may lead to destruction of the islet beta cells, which produce insulin, a glucose regulatory peptide hormone. Diabetes mellitus has been generally categorized as insulin dependent or type I, versus non-insulin dependent, or type II.

The principal three forms or diabetes are:

Type I: Results from the body's failure to produce insulin. Treatment usually involves insulin administration.

Type II: Results from a condition in which the body fails to use insulin properly, combined with relative insulin deficiency. Many people destined to develop type II diabetes spend many years in a state of Pre-diabetes, a condition that occurs when a person's blood glucose levels are higher than normal but not high enough for a diagnosis of type II diabetes.

Gestational diabetes: Pregnant women who have never had diabetes before but who have high blood sugar (glucose) levels during pregnancy are said to have gestational diabetes. Gestational diabetes affects about 4% of all pregnant women. It may precede development of type II (or rarely type I).

Many other forms of diabetes are categorized separately from these. Examples include congenital diabetes due to genetic defects of insulin secretion, cystic fibrosis-related diabetes, steroid diabetes induced by high doses of glucocorticoids, and several forms of monogenic diabetes.

However, this terminology has evolved as the disease has become better understood. For example, it has been found that in some patients suffering from non-insulin dependent diabetes, the disease progresses into an insulin dependent form, while in other patients insulin dependence does not develop.

Patients are thus often categorized in terms of the mechanisms of pathogenesis of islet destruction, and the designation type I is now used to refer to autoimmune islet pathogenesis, i.e., to diabetes caused by islet- specific autoimmune attack, and is so used herein. The term insulin dependent diabetes mellitus (IDDM) refers to Type I diabetes that has progressed to a stage where enough autoimmune destruction of the pancreatic beta cells has occurred to produce overt symptoms. The term pre-IDDM refers to an autoimmune condition that can be detected by biopsy or by analysis of autoimmune responses, in which pancreatic islet beta cells are being subject to a specific autoimmune attack to an extent where some cells may be subject to destruction. In pre-IDDM, however, the destruction (if any) has not progressed to an extent sufficient to require the administration of insulin. Since there can be a point in the early stages of Type I diabetes in which overt symptoms are observed but some islet function remains (known as the "honeymoon period", not all Type I diabetes is classified as IDDM, and not all pre-IDDM presents without overt symptoms.

The metabolic complications associated with the abnormal metabolism caused by insulin insufficiency can affect numerous organ systems. The most common acute metabolic complication is that of diabetic ketoacidosis, characterized by severe hyperglycemia (and resulting hypovolemia caused by osmotic diuresis) as well as metabolic acidosis induced by excess free fatty acid release and the production of ketone bodies.

In addition to the acute metabolic complication of ketoacidosis, the diabetic patient is susceptible to a series of late complications that cause considerable morbidity and premature mortality. Atherosclerosis occurs more extensively and earlier in diabetics than in the general population as a result of abnormalities in both glucose and lipid metabolism. This vascular pathology can lead to, inter alia, coronary artery disease, stroke, and peripheral vascular disease with gangrene. Retinopathy is another vascular complication of diabetes. Diabetic retinopathy is a leading cause of blindness, and is initiated by increased permeability of retinal capillaries which can progress to occlusion, hemorrhage, aneurysm formation, and neovascularization known as proliferative retinopathy.

As mentioned above, meticulous control of blood glucose has been associated with amelioration of the late complications of Type I diabetes, suggesting that preservation or restoration of beta cell function could reduce or eliminate the majority of the pathologic complications of the disease.

The inherited disorders of carbohydrate metabolism are rare. Severe defects of the pyruvate dehydrogenase (PDH) complex and the benign chemical anomaly called pentosuria have been reported in very few (2-6) patients.

Hypoglycemia, diabetic ketoacidosis, and hyperosmolar coma are all potentially fatal but potentially curable conditions. In WO 2011/016026, the present inventors have discovered novel secreted proteins, namely PRT5, PRT6, PRT7 and PRT8. In particular, it has been shown that PRT8 is able to significantly lower the glucose level in blood.

However, it has been now surprisingly found by the inventor that a shortened PRT8 peptide, namely sbPRT8, has an improved biological activity over the full length PRT8 peptide, and is able to lower blood glucose level, even more significantly, while other PRT8 fragments have not shown any substantial activity. Therefore, an object of the present invention is to provide polypeptides having an improved activity in comparison to the one of PRT8, as well as nucleic acid, vectors, and cells encoding the same. Another object of the present invention is to provide methods and uses of sbPRT8 for therapeutic treatment of diseases or disorders associated with an excess of glucose in blood (i.e. hyperglycemia). These and other uses and objects of the invention will become apparent as the description proceeds.

Summary of the Invention

It is an object of the present invention to provide an isolated polypeptide comprising (a) the amino acid sequence set forth in SEQ ID NO: 1; or (b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, wherein said polypeptide is able to lower glucose level in blood. In a specific embodiment, the isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. In another specific embodiment, the polypeptide is modified at the N-terminal end with an acetyl moiety and at the C-terminal end with an amide moiety.

The invention further provides a pharmaceutical composition comprising an isolated polypeptide as defined above and at least one pharmaceutically acceptable carrier, excipient, or diluent.

The invention further provides a therapeutically effective amount of the polypeptide described above for use for in lowering blood glucose level in a subject. In some specific embodiments, said subject suffers from a disease or disorder associated with hyperglycemia. Such disease or disorder may be selected, but is not limited to, diabetes mellitus type 1, diabetes mellitus type 2, metabolic syndrome, obesity, nephropathy, retinopathy, cardiovascular diseases, glands dysfunction, pancreas diseases, sepsis, intracranial diseases, and post- surgery stress. The invention still further provides a therapeutically effective amount of the polypeptide described above for use in lowering plasma triglycerides (TG) blood level in a subject. The invention further provides a therapeutically effective amount of the polypeptide as described above for use in lowering the level of glycated hemoglobin (HbAlc) in blood of a subject.

The invention further provides isolated nucleic acid molecules encoding a polypeptide comprising (a) the amino acid sequence set forth in SEQ ID NO: 1; or (b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, expression vectors comprising the same, and host cells comprising said expression vector. Brief Description of the Figures

Figures 1A-1B: Effects of sbPRT8 on C57B1/6 mice treated with streptozotocyn (STZ)

Figure 1A: Graph showing glucose levels in C57B1/6 mice injected with STZ (IP injection of 50 mg/kg for 4 days) followed by a 5 weeks treatment with either PRT8 or sbPRT8, and IP injected with 1 mg/kg of glucose (IPGTT);

Legend: -♦- 100 μg/mice of PRT8; - A - 20 μg/mice of sbPRT8; -■- 100 μg/mice sbPRT8; Gluc.= glucose; T.= Time after glucose injection

Figure IB: Graph showing glucose levels in C57B1/6 mice injected with STZ (IP injection of 50mg/kg for 4 days) followed by a 4 weeks treatment with either PRT8 or sbPRT8, and IP injected with 1 mg/kg of glucose (IPGTT);

Legend: -A - 100 μg/mice PRT8; -■- 100 μg/mice sbPRT8; -♦- 300 μg/mice of SbPRT8.

Detailed Description of the Invention

WO 2011/016026 disclosed novel proteins named PRT5, PRT6, PRT7 and PRT8. PRT8 was found particularly in the pancreas and testis, and also in the liver. In particular, it has been shown that PRT8 is able to significantly lower the glucose level in blood.

In the present invention, it has been surprisingly found by the inventors that a shortened PRT8 peptide, namely sbPRT8, has an improved biological activity over the full length PRT8 peptide, and is able to lower blood glucose level, even more significantly.

Thus, in a first aspect, the present invention provides an isolated polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1, as well as homologs and derivatives thereof, wherein said polypeptide lower glucose level in blood (i.e. decrease the glucose concentration in blood).

The term "polypeptide" is used herein to denote a polypeptide or a protein. The polypeptide may be obtained synthetically, through genetic engineering methods, expression in a host cell, or through any other suitable means. Unless indicated otherwise, a polypeptide is generally composed of naturally- occurring L-amino acids. In some embodiments, the polypeptide of the invention is modified at the C- terminal end and/or at the N-terminal end. In one specific embodiment, the peptide is modified as follows: acetyl-(Peptide Sequence)-amide. In some other specific embodiments, the polypeptide of the invention is modified with one or more groups selected from a biotin group, a fluorescent group or a cysteine residue.

With respect to a modified polypeptide, fragments, homologues and derivatives thereof, in connection with the present invention, it is understood that it retains the biological activity of the polypeptide of SEQ ID NO: 1. In order to determine whether a polypeptide retains a biological activity qualitatively similar to that of the unmodified molecule, one or more assays can be carried out, such as for example an in vitro, in vivo or a clinical experiment in which a modified polypeptide is compared to the corresponding unmodified one that is assayed in parallel or in a separately conducted experiment. A modified polypeptide or variant polypeptide may be a polypeptide that includes at least two, three or more copies of sbPRT8 optionally separated by an amino acid spacer. A modified polypeptide may be also a polypeptide having a degree of identity being at least 70%, preferably at least 80%, more preferably at least 90% and particularly at least 95% with SEQ ID NO:l.

Also provided by the invention are polypeptides derived from sbPRT8, e.g., modified polypeptides in which one or more amino acids are replaced by another amino acid by conservative substitution. As used herein, "conservative substitution" refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (He, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gin, or Glu is a conservative substitution.

In one embodiment, only one substitution is made in the amino acid sequence. In another embodiment, two substitutions are made. In a further embodiment, three substitutions are made. The maximum number of substitutions should not exceed the number of amino acids which leaves at least 70%, desirably at least 80%, preferably at least 90%, most preferably at least 95% of the amino acids in the unsubstituted sequence. In one particular embodiment, the substitutions which include up to 3, at times up to 6 amino acid residues substituted by others, are conservative substitutions. In a further embodiment, one or more amino acids may be replaced by D-amino acids, preferably the corresponding D-amino acids. In a particular embodiment, all of the amino acids are D-amino acids.

Preferred substitutions are changes that would not be expected to alter the secondary structure of the polypeptide, i.e., conservative changes. The following list shows amino acids (right side) that may be exchanged for the original amino acids (left side).

Original Residue Exemplary Substitution

Ala Gly; Ser

Arg Lys

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Ala; Pro

His Asn; Gin

lie Leu; Val

Leu lie; Val

Lys Arg; Gin; Glu

Met Leu; Tyr; He

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val He; Leu Amino acids can also be grouped according to their essential features, such as charge, size of the side chain, and the like. The following list shows groups of similar amino acids. Preferred substitutions would exchange an amino acid present in one group with an amino acid from the same group, as follows:

1. Small aliphatic, nonpolar: Ala, Ser, Thr, Pro, Gly;

2. Polar negatively charged residues and their amides: Asp, Asn, Glu, Gin; 3. Polar positively charged residues: His, Arg, Lys;

4. Large aliphatic nonpolar residues: Met, Leu, He, Val, Cys;

5. Large aromatic residues: Phe, Tyr, Trp.

The preferred conservative amino acid substitutions as detailed above are expected to substantially maintain or increase the function or activity of the protein of the invention, as detailed herein below. Of course, any amino acid substitutions, additions, or deletions are considered to be within the scope of the invention where the resulting polypeptide retains its original functions. For example, a conservative substitution is one in which the polypeptide of the invention is still recognized by the antibodies which recognize the wild- type polypeptide.

The polypeptide of the invention can be produced by conventional chemical methods, such as solid phase synthesis (using e.g. FMOC and BOC techniques), and solution phase synthesis. These proteins or polypeptides may also be produced in bacterial or insect cells or other eukaryotic transcriptional in vivo system. Following production, the polypeptide are purified from the cells in which they have been produced. Polypeptide purification and isolation methods are known to the person of skill in the art. Advantageously, the polypeptide may be produced as a fusion with a second protein, such as Glutathione-S-transferase (GST) or the like, or a sequence tag, such as the Histidine tag (His-tag) sequence. The use of fusion or tagged proteins simplifies the purification procedure. The polypeptide of the invention can also be synthesized in cell-free systems, using, for example, cell extracts or ribosomes. The polypeptide of the invention may be further modified to improve their function, affinity, or stability. For instance, cyclization may be used to impart greater stability and/or overall improved performance upon a polypeptide. A number of different cyclization methods have been developed, including side chain cyclization and backbone cyclization. These methods are well documented in the prior art. A particular method of cyclization involves stabilization of an amphipathic alpha-helix by using para-substituted amino acid derivatives of a benzene ring. Another particular method of cyclization is backbone cyclization. Another method of cyclization which involves backbone- to side chain connections may also be used.

Nonetheless, according to the invention, the polypeptide of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different organic moieties which are not naturally occurring or synthetic amino acids. As an example for such extension, the polypeptide may be extended at the N-terminus and/or C-terminus thereof with an N-acetyl group.

In order to improve polypeptide structure, the polypeptide of the invention can be coupled through their N-terminus to a lauryl-cysteine (LC) residue and/or through their C-terminus to a cysteine (C) residue, or to other residue/s suitable for linking the polypeptide to adjuvant/s for immunization.

In another aspect, the present invention provides an isolated nucleic acid encoding a polypeptide as set forth in SEQ ID NO: 1 and any derivatives and analogs thereof.

As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double- stranded, but preferably it is double- stranded DNA. The term "isolated nucleic acid molecule" is intended to include nucleic acid molecules which are separated from other nucleic acid molecules and which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

Derivatives within the scope of the invention also include polynucleotide derivatives. Polynucleotide or nucleic acid derivatives differ from the sequences described or known in nucleotide sequence. For example, a polynucleotide derivative may be characterized by one or more nucleotide substitutions, insertions, or deletions.

A nucleic acid molecule of the present invention, i.e. a nucleic acid molecule having a nucleotide sequence encoding sbPRT8, can be generated using standard molecular biology techniques and the sequence information provided herein.

In yet another aspect the present invention provides a vector comprising an isolated nucleic acid molecule encoding the polypeptide as set forth in SEQ ID NO: 1 or any functional derivative or analog thereof. In one embodiment of said vector, said nucleic acid molecule is operably linked to a promoter. In another embodiment, said vector is an expression vector. As used herein, the term "vector" is intended to include a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector may be characterized by one or a small number of restriction endonuclease sites at which such DNA sequences may be cut in a determinable fashion without the loss of an essential biological function of the vector, and into which a DNA fragment may be spliced in order to bring about its replication and cloning. A vector may further contain a marker suitable for use in the identification of cells transformed with the vector. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "operatively linked" or "operably linked" is intended to mean that molecules are functionally coupled to each other in that the change of activity or state of one molecule is affected by the activity or state of the other molecule. Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the DNA sequence encoding the polypeptide or protein of interest. For example, a promoter nucleotide sequence is operably linked to a DNA sequence encoding the polypeptide of interest if the promoter nucleotide sequence controls the transcription of the DNA sequence encoding the protein of interest. Typically, two polypeptides that are operatively linked are covalently attached through peptide bonds. In another further aspect the present invention provides a cell comprising the vector as described above, said vector comprising an isolated nucleic acid molecule encoding the polypeptide as set forth in SEQ ID NO: 1 and any derivatives or analog thereof. In one embodiment, said cell is a host cell selected from the group consisting of: a plant cell, an insect cell, a fungal cell, a bacterial cell or a mammalian cell. The terms "host cell" and "recombinant host cell" are used interchangeably herein. A "host cell" includes any cultivatable cell that can be modified by the introduction of heterologous DNA. Preferably, a host cell is one in which a transcriptional regulatory protein can be stably expressed, post- translationally modified, localized to the appropriate sub-cellular compartment, and made to engage the appropriate transcription machinery. The choice of an appropriate host cell will also be influenced by the choice of detection signal. For example, reporter constructs, as described above, can provide a selectable or screenable trait upon activation or inhibition of gene transcription in response to a transcriptional regulatory protein; in order to achieve optimal selection or screening, the host cell phenotype will be considered. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell of the present invention includes prokaryotic cells and eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. Eukaryotic cells include, but are not limited to, yeast cells, plant cells, fungal cells, insect cells (e.g., baculovirus), mammalian cells, and the cells of parasitic organisms, e.g., trypanosomes. As used herein, the term "yeast" includes not only yeasts in a strict taxonomic sense, i.e., unicellular organisms, but also yeast-like multicellular fungi of filamentous fungi. Exemplary species include Kluyverei lactis, Schizosaccharomyces pombe, and Ustilaqo maydis, with Saccharomyces cerevisiae being preferred. Other yeast which can be used in practicing the present invention are Neurospora crassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris, Candida tropicalis, and Hansenula polymorpha.

Mammalian host cell culture systems include established cell lines such as HeLa cells, COS cells, L cells, 3T3 cells, Chinese hamster ovary (CHO) cells, embryonic stem cells, etc.

In another further aspect, the present invention provides a composition comprising an isolated polypeptide sbPRT8 wherein said polypeptide comprises the sequence as set forth in SEQ ID NO: 1, or any analogs of derivatives thereof, as defined above.

In one embodiment, any composition provided in the present invention may further comprise a pharmaceutically acceptable adjuvant, carrier, excipient, or diluent.

By the term "pharmaceutically acceptable carrier" it is meant any one of inert, non-toxic materials, which do not react with the active ingredient. The carrier is selected at times based on the desired form of the formulation. The carrier may also at times have the effect of the improving the delivery or penetration of the active ingredient to the target tissue, for improving the stability of the drug, for slowing clearance rates, for imparting slow release properties, for reducing undesired side effects etc. The carrier may also be a substance that stabilizes the formulation (e.g. a preservative), for providing the formulation with an edible flavor, etc. The carriers may be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the antibodies of the invention, and by the route of administration. The carrier may include additives, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. In addition, the carrier may be an adjuvant, which, by definition are substances affecting the action of the active ingredient in a predictable way. Typical examples of carriers include (a) liquid solutions, where an effective amount of the active substance is dissolved in diluents, such as water, saline, natural juices, alcohols, syrups, etc.; (b) capsules (e.g. the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers), tablets, lozenges (wherein the active substance is in a flavor, such as sucrose and acacia or tragacanth or the active substance is in an inert base, such as gelatin and glycerin), and troches, each containing a predetermined amount of active agent as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; (e) suitable emulsions; (f) liposome formulation; and others.

In another embodiment, compositions of the invention may also optionally further comprise additional active agents, such as, but not limited to antibiotics, cytokines, lymphokines, growth factors, hormones, anti-oxidants, vitamins, etc.

In another further aspect the present invention provides the use of a polypeptide sbPRT8 for the preparation of a medicament for the treatment of a disease or disorder associated with hyperglycemia, in particular a disease or disorder selected from the group consisting of diabetes mellitus type 1, diabetes mellitus type 2, metabolic syndrome, obesity, nephropathy, retinopathy, cardiovascular diseases, glands dysfunction, pancreas diseases, sepsis, intracranial diseases, and post-surgery stress. As referred to herein, the term "effective amount" means an amount necessary to achieve a selected result, which at present, involves the amount of sbPRT8, or biologically active derivatives thereof, necessary for treating a disorder.

Said therapeutic effective amount, or dosing, is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting one hour to several hours, one day to several days, or until a cure is effected or a diminution of the disease state is achieved. Persons of ordinary skill can readily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of each polypeptide or protein of the invention, or compositions comprising thereof, and can generally be estimated based on EC50, found to be effective in in vitro as well as in in vivo animal models. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times, concentrations, and adjustment to the employed polypeptide or protein.

The terms "treat, treating or treatment" as used herein mean ameliorating one or more clinical indicia of disease activity in a patient having a disease or disorder. "Treatment" refers to therapeutic treatment.

By "patient" or "subject in need" is meant any mammal for which treatment of a disorder or disease is desired in order to overcome said disorder or disease, particularly a human subject.

Usually, a "therapeutically effective amount" is also determined by the severity of the disease in conjunction with the preventive or therapeutic objectives, the route of administration and the patient's general condition (age, sex, weight and other considerations known to the attending physician). Various methods of administration may be used for delivering sbPRT8 described in the invention to a subject in need. Said polypeptide, or compositions comprising the same may be delivered via intravenous (i.v.), intramuscular (i.m.), intraperitoneal (i.p.), or topical injections, or through any other route found suitable by the man skilled in the art. In order to be effective therapeutically, the polypeptides or proteins of the invention should be prepared in a way that would enable their stability in the system following administration. As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, congenital malformations, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, chronic or permanent health defects resulting from disease.

The terms "disease", "disorder", "condition" and "illness" are interchangeably used herein.

When referring herein to a subject, said subject may be a mammal, human or non-human. Non-human mammals include, but are not limited to cows, horses, dogs, cats, mice, rats, guinea-pigs, etc. Usually the subject is a human, particularly a patient, or a healthy individual.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, a "disease or disorder associated with hyperglycemia" refers to diseases or disorders in which an excessive amount of glucose circulates in the blood plasma. This is generally a glucose level higher than 11.1 mmol/1 (200 mg/dl), but symptoms may not start to become noticeable until even higher values such as 15-20 mmol/1 (~250-300 mg/dl). According to the American Diabetes Association guidelines, a subject with a consistent range between 100 and 126 is considered hyperglycemic, while above 126 mg/dl or 7 mmol/1 is generally held to have diabetes. Chronic levels exceeding 7 mmol/1 (125 mg/dl) can produce organ damage. Diseases or disorders associated with hyperglycemia include diabetes mellitus (type 1 and type 2), nephropathy, retinopathy, cardiovascular diseases (e.g. stroke, myocardial infarction), glands dysfunction (e.g. thyroid, adrenal, or pituitary), pancreas diseases, sepsis, intracranial diseases (e.g encephalitis, brain tumors, brain bleeds, meningitis). Prolonged, major surgeries can temporarily increase glucose levels. Recent results also suggest that in a non-fasted state, acute hyperglycemia increases plasma triglycerides (TG) by stimulating hepatic TG secretion, in a manner which is independent of either plasma insulin or free fatty acids levels. As used herein, "glycated hemoglobin" (also referred to as hemoglobin Ale, HbAlc, AlC, Hblc, or HbAlc) is a form of hemoglobin that is measured primarily to identify the average plasma glucose concentration over prolonged periods of time. It is formed in a non-enzymatic glycation pathway by hemoglobin's exposure to plasma glucose. Normal levels of glucose produce a normal amount of glycated hemoglobin. As the average amount of plasma glucose increases (hyperglycemia), the fraction of glycated hemoglobin increases in a predictable way. In diabetes mellitus, higher amounts of glycated hemoglobin have been associated with cardiovascular disease, nephropathy, and retinopathy.

The following Examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the intended scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. Comparative Example 1

Effect of sbPRT8 administration on glucose levels of C57B1/6 mice treated with streptozotocyn (STZ) as compared to PRT8

administration In the following experiments, a sbPRT8 peptide blocked with an acetyl moiety at the N-terminal end and an amide moiety (N¾) at the C-terminal end has been employed. The effect of sbPRT8 administration on glucose levels was examined and compared to the effect obtained with PRT8 in mice treated with streptozocin (STZ). As known in the art, STZ is a chemical compound that is particularly toxic to the insulin-producing beta cells of the pancreas in mammals. It is used in medicine for treating certain cancers of the Islets of Langerhans and used in medical research to produce an animal model for Type 1 diabetes as well as Type 2 diabetes.

Experience 1:

C57B1/6 males mice were purchased from Harlan Laboratories Ltd., Jerusalem, Israel, After 7 days of rest, mice were IP injected with 50 mg/kg STZ for 4 days. Five days after STZ injection, mice were treated as follows:

- Group A (control) - 100 μg/mice of PRT8 (control)

- Group B (treatment) - 20 μg/mice of sbPRT8 (treatment 1)

- Group C (control) - 100 μg/mice of sbPRT8 (treatment 2)

After 5 weeks of treatment mice were starved for food only for 12h and then were IP injected with lmg/kg of glucose solution (IPGTT). The level of glucose in mice blood was measured with Accucheck glucometer (Roche) at t = 0, 15, 30, 60, 90 and 120 minutes following glucose injection.

Experience 2:

C57B1/6 males mice were purchased from Harlan Laboratories Ltd., Jerusalem, Israel. After 7 days of rest, mice were IP injected with 50 mg/kg STZ for 4 days. Five days after STZ injection, mice were treated as follows:

- Group A (control) - 100 μg/mice of PRT8 (control)

- Group B (treatment) - 100 μg/mice of sbPRT8 (treatment 1)

- Group C (control) - 300 μg/mice of sbPRT8 (treatment 2) After 4 weeks of treatment mice were starved for food only for 12h and then were IP injected with 1 mg/kg of glucose solution (IPGTT). The level of glucose in mice blood was measured with Accucheck glucometer (Roche) at t = 0, 15, 30, 60, 90 and 120 minutes following glucose injection. The results are shown in Figures 1A-1B. In all the treatments, glucose levels peaked in the first 30 minutes following injection. While injection of PRT8 enables a significant reduction of blood glucose level in mice (see also Example 9), the injection of sbPRT8 shows a surprising and very significant improvement in said reduction of blood glucose level. These results clearly demonstrate that the peptide of SEQ ID NO: 1 (sbPRT8), which is a C- terminal fragment of PRT8, has an improved glucose reduction activity over the full length PRT8 peptide. Similar experiments were conducted with two other PRT8 fragments of SEQ ID NO: 2 (PRT8 N-ter) and SEQ ID NO: 3 (PRT8 C-ter), and contrary to the results obtained with sbPRT8, no significant blood glucose reduction was observed. From the aforesaid, it can be concluded that the short peptide sbPRT8 shows a significantly improved potential in glucose metabolism regulation and blood glucose level reduction over the full length PRT8 peptide. sbPRT8 may be thus of great interest for treating diseases or disorders associated with hyperglycemia. Furthermore, sbPRT8 may be used to decrease the blood level of glycated hemoglobin (HbAlc level) and lower the plasma triglycerides (TG) level, since both levels are closely related to blood glucose concentration.

All the above description and examples have been provided for the purpose of illustration and are not intended to limit the invention in any way.

Claims (9)

1. An isolated polypeptide comprising (a) the amino acid sequence set forth in SEQ ID NO: 1; or (b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, wherein said polypeptide reduces glucose concentration in blood.

2. An isolated polypeptide according to claim 1, consisting of the amino acid sequence set forth in SEQ ID NO: 1.

3. An isolated polypeptide according to claim 1 or 2, wherein said polypeptide is modified at the N-terminal end with an acetyl moiety and at the C-terminal end with an amide moiety.

4. A pharmaceutical composition comprising an isolated polypeptide according to any one of claims 1 to 3 and at least one pharmaceutically acceptable carrier, excipient, or diluent.

5. An isolated polypeptide of any one of claims 1 to 3 or a pharmaceutical composition of claim 4 for use in the treatment of a disease or disorder associated with hyperglycemia, wherein said disease or disorder is selected from the group consisting of diabetes mellitus type 1, diabetes mellitus type 2, metabolic syndrome, obesity, nephropathy, retinopathy, cardiovascular diseases, glands dysfunction, pancreas diseases, sepsis, intracranial diseases, and post-surgery stress.

6. An isolated polypeptide of any one of claims 1 to 3 or a pharmaceutical composition of claim 4 for use in lowering the blood glucose level in a subject.

7. An isolated polypeptide of any one of claims 1 to 3 or a pharmaceutical composition of claim 4 for use in lowering plasma triglycerides (TG) blood level in a subject.

8. An isolated polypeptide of any one of claims 1 to 3 or a pharmaceutical composition of claim 4 for use in lowering the level of glycated hemoglobin (HbAlc) in blood.

9. An isolated nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 3.

An expression vector comprising the nucleic acid molecule of claim 9.

A host cell comprising the expression vector of claim 10.