WO2001072320A1

WO2001072320A1 - A method of treatment of metabolic disorders and agents useful for same

Info

Publication number: WO2001072320A1
Application number: PCT/AU2001/000348
Authority: WO
Inventors: Gregory Royce Collier; Kenneth Russell Walder; Paul Zev Zimmet; Lakshmi Kantham
Original assignee: Autogen Research Pty Ltd
Current assignee: Autogen Research Pty Ltd
Priority date: 2000-03-28
Filing date: 2001-03-28
Publication date: 2001-10-04
Anticipated expiration: 2002-09-28
Also published as: AUPQ656500A0

Abstract

A method is disclosed for regulating energy expenditure, insulin resistance and/or muscle proteolysis in a muscle cell by modulating the level and/or functional activity of a target protein selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein. Also disclosed is a method for treating one or more metabolic disorders such as insulin resistance, obesity and Type II diabetes mellitus, by contacting a muscle cell with an agent which modulates the level and/or functional activity of a said protein.

Description

A method of treatment of metabolic disorders and agents useful for same

FIELD OF THE INVENTION

The present invention relates generally to a method for modulating a cellular function such as energy expenditure and/or insulin resistance in a cell. More particularly, the present invention is directed to a method for regulating the level and/or functional activity of a myofibrillar protein and/or proteolysis in a muscle cell, which thereby affects energy expenditure and/or insulin resistance. The present invention extends to a wide range of therapeutic and/or prophylactic applications which includes, but is not limited to, the treatment and/or prophylaxis of metabolic disorders such as insulin resistance, obesity and Type II diabetes mellitus. The present invention is also useful, inter alia, for the development of methods for diagnosing metabolic disorders and for screening of agents useful in modulating energy expenditure and/or insulin resistance.

BACKGROUND OF THE INVENTION

Metabolic disorders such as obesity and diabetes are serious health problems particularly in industrialised societies where lack of exercise by individuals is thought to exacerbate the problem. Obesity results from an imbalance between calorific intake and energy expenditure. Various forms of diabetes exist including insulin dependent diabetes mellitus (type I diabetes) where the insulin producing β cells in the islets of the pancreas are destroyed in an autoimmune response. Many of the pathological consequences of obesity, which include hypertension, hyperlipidaemia and non-insulin dependent (Type II) diabetes mellitus, are thought to involve insulin resistance. However, the molecular basis for these metabolic disorders is poorly understood.

Insulin elicits a wide range of biological responses by binding to its cell surface receptor and has a major function in regulating the level of plasma glucose by facilitating uptake of glucose into major storage sites such as muscle or adipocytes. Insulin resistance occurs when the body's biological response to insulin is smaller than expected. There are many proposed causes of insulin resistance, including down regulation of insulin receptors or insulin sensitive glucose transporters and elevations of free fatty acids and lipoproteins. Current preventative measures and therapies for obesity and diabetes are inadequate and accordingly there is a need to develop new therapeutic strategies to selectively combat and/or prevent metabolic disorders such as insulin resistance, diabetes and obesity.

In work leading up to the present invention, the inventors unexpectedly discovered elevated levels of calpain expression in muscle and lower myofibril titin levels in obese, diabetic subjects relative to controls. Calpains, are a group of ubiquitous Ca²⁺- activated cytosolic cysteine proteases, which are hypothesised to participate in cytoskeletal remodelling events, cellular adhesion, shape change, and motility by the site-specific regulatory proteolysis of membrane- and actin-associated cytoskeletal proteins (Beckerle et ah, 1987, Cell 51: 569-577; Yao et ah, 1993, Am. J. Physioh 265(pt. 1): C36-46; and Shuster et ah, 1995, J. Cell Biol. 128: 837-848). Calpains may also act as regulators of transcription factors, thereby controlling expression of key genes in various cellular processes. (Chen F., et ah, 1997, Arch Biochem. Biophys. 342(2): 383-8). Calpains occur in various different forms including m-, μ, p94 and NCL2 forms, which appear to be activated at different Ca²⁺ concentrations and which differ in terms of tissue specificity and stability. Calpains have also been implicated in the pathophysiology of cerebral and myocardial ischaemia, platelet activation, NF-kB activation, Alzheimer's disease, muscular dystrophy, cataract progression and rheumatoid arthritis.

Calpastatins are endogenous physiological inhibitors of calpains. Calpastatins have four internally repeated domains, each of which independently binds a calpain molecule in its active, Ca²⁺-bound conformation with high affinity (Mellgren et ah, The Regulation of Calpains by Interaction with Calpastatins, and Malci et ah, Structure- Function Relationship of Calpastatins, both in Intracellular Calcium-Dependent Proteolysis, Mellgren and Murachi, Eds, CRC Press, Boca Raton, Fla., 1990; and Yang et ah, 1994, J. Biol. Chem. 269: 18977-18984). There is considerable interest in inhibitors of calpain (Wang et ah, 1994, Trends in Pharm. Sci. 15: 412-419; Mehdi, 1991, Trends in Biochem. Sci. 16: 150-153).

Titin is a myofibrillar protein of muscle cells. Myofibrils are the contractile apparatus in the cytoplasm of striated or skeletal muscle cells. Myofibrils comprise thick (myosin) and thin (actin filaments) which occur in parallel bundles in the axis of contractions. Myofibrillar bundles form part of the sarcomere, which is the contractile unit of a muscle cell. Titin is a large (3 MDa) abundant protein in the sarcomere and is thought to span the entire half-sarcomere, between the Z disc (the Z disc defines the outer boundary of the sarcomere) and the M-line. Titin is thought to be important in the temporal and spatial control of sarcomere assembly and in providing tension in the myofibrils by binding to both thick and thin filaments. Titin contains two binding sites for p94 calpain (also referred to as calpain 3), one in the I-band and one in the C-terminal region of the protein.

The precise physiological role of the calpain-calpastatin system is poorly understood despite the identification of a large number of calpain substrates. However, it is known that imbalance of the calpain-calpastatin system is closely related to certain disease states. For example, in patients with muscular dystrophy, the level of calpain is high, and the degeneration of myofibrils and neurofilaments by calpain is accelerated, which seems to be related to the onset of this disease. Also, in cells infected with adult T-cell leukemia viruses, the activity levels of calpain and of receptors for interleukin 2 are abnormally high. These abnormalities suggest that change of the receptor activity for calpain affects proteins of the cell skeleton, and then the cells come to have an abnormal reaction to growth factors and the like, possibly causing the appearance of the disease (Adachi et ah, 1985, Seikagaku, 57: 1202). Further, calpain cleaves the protein of crystalline lens; when this reaction is excessive in its extent, it may cause cataracts. Also, calpastatin is identical to the stabilising factor of steroid-receptor complexes that is required for the signal transduction (Bodwell et ah, 1985, J. Biol. Chem., 260: 2601-2604). Also, in hypertensive rats, the calpastatin level is abnormally low (Pontremoli et ah, 1987, Biochem. Biophys. Res. Commun., 145: 1287-12948).

However, it has been hitherto unknown that an imbalance between calpains, calpastatin and myofibrillar proteins may underlie the pathophysiology of obesity, insulin- resistance and type II diabetes.

SUMMARY OF THE INVENTION

The inventors have reduced their discovery to practice in a novel method for regulating energy expenditure, insulin resistance and/or muscle proteolysis in a muscle cell, and in a method for treating one or more metabolic disorders such as insulin resistance, obesity and Type II diabetes mellitus, by contacting a muscle cell with an agent which modulates the level and/or functional activity of a target protein selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein. Furthermore, the identification of these targets permits the selection or rational design of agents that modulate the activity of one or more of these proteins for use inter alia in the treatment of metabolic disorders such as insulin resistance and/or obesity.

Accordingly, in one aspect of the present invention, there is provided a method for modulating a function of a muscle cell, said function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

In another aspect, the present invention contemplates a method for modulating the energy expenditure of a muscle cell, comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

hi yet another aspect, the present invention encompasses a method for modulating insulin resistance of a muscle cell, comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

hi still yet another aspect, the present invention envisions a method for modulating proteolysis in a muscle cell, comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

i another aspect, the invention provides a method for modulating a function of a muscle cell, said function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of a calpain.

In yet another aspect, the invention provides a method for modulating a function of a muscle cell, said function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of a calpastatin.

In another aspect, the invention contemplates a method for modulating a function of a muscle cell, said function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of a myofibrillar protein.

Preferably, the calpain is calpain 3, which is also referred to as P94 calpain.

Suitably, the muscle cell is a striated muscle cell.

The myofibrillar protein is preferably titin.

In one embodiment, the agent may modulate the level and/or functional activity of a single said member. In another embodiment, the agent may modulate the level and/or functional activity of two or more of said members.

In one embodiment, the agent increases the level and/or functional activity of the myofibrillar protein.

In another embodiment, the agent increases the level and/or functional activity of the calpastatin.

In yet embodiment, the agent decreases the level and/or functional activity of the calpain.

In still yet another embodiment, the agent increases the level and/or functional activity of the calpain.

In a further embodiment, the agent modulates the level and/or functional activity of the calpain to a normal reference level and/or functional activity. Accordingly, in one embodiment of this type, the agent may decrease the level and/or functional activity of the calpain to the normal reference level and/or functional activity. In an alternate embodiment of this type, the agent increases the level and/or functional activity of the calpain to the normal reference level and/or functional activity. Suitably, said normal reference level and/or functional activity is other than a level and/or functional activity associated with a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes.

In another aspect, the invention resides in a composition for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said composition comprising an agent which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

In a further aspect, the invention extends to a composition for treatment and/or prophylaxis of obesity, said composition comprising an agent which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

hi another aspect, the invention contemplates a composition for treatment and/or prophylaxis of insulin resistance, said composition comprising an agent which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

In yet another aspect, the invention encompasses a composition for treatment and/or prophylaxis of diabetes, said composition comprising an agent which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

In yet another aspect, the invention encompasses a composition for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said composition comprising an agent which modulates the level and/or functional activity of a calpain, and optionally together with a pharmaceutically acceptable carrier. Ih a further aspect, the invention extends to a composition for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said composition comprising an agent which modulates the level and/or functional activity of a calpastatin, and optionally together with a pharmaceutically acceptable carrier.

hi another aspect, the invention contemplates a composition for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said composition comprising an agent which modulates the level and/or functional activity of a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

According to another aspect of the invention, there is provided a method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent as broadly described above for a time and under conditions sufficient to modulate the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

hi yet another aspect, the invention provides a method for treatment and/or prophylaxis of obesity, comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent as broadly described above for a time and under conditions sufficient to modulate the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

In another aspect, the invention extends to a method for treatment and/or prophylaxis of insulin resistance, comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent as broadly described above for a time and under conditions sufficient to modulate the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

In another aspect, the invention encompasses a method for treatment and/or prophylaxis of diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent as broadly described above for a time and under conditions sufficient to modulate the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

In another aspect, the invention contemplates a method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent as broadly described above for a time and under conditions sufficient to modulate the level and/or functional activity of a calpain.

In yet another aspect, the invention encompasses a method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent as broadly described above for a time and under conditions sufficient to modulate the level and/or functional activity of calpastatin.

In a further aspect, the invention envisions a method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent as broadly described above for a time and under conditions sufficient to modulate the level and/or functional activity of a myofibrillar protein.

In yet another aspect, the invention extends to a method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising: - contacting a preparation comprising a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein or a genetic sequence encoding said member with a test agent; and

- detecting a change in the level and/or functional activity of said member or an expression product of said genetic sequence. In another aspect, the invention features a method of screening for an agent which modulates energy expenditure, comprising:

- contacting a preparation comprising a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein or a genetic sequence encoding said member with a test agent; and

- detecting a change in the level and/or functional activity of said member or an expression product of said genetic sequence.

In still yet another aspect, the invention provides a method of screening for an agent which modulates insulin resistance, comprising: - contacting a preparation comprising a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein or a genetic sequence encoding said member with a test agent; and

In yet another aspect, the invention extends to a method of screening for an agent which modulates muscle proteolysis, said method comprising:

- contacting a preparation comprising a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein or a genetic sequence encoding said member with a test agent; and - detecting a change in the level and/or functional activity of said member or an expression product of said genetic sequence.

In yet another aspect, the invention contemplates a method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising: - contacting a preparation comprising a calpain or a genetic sequence encoding said calpain with a test agent; and

- detecting a change in the level and/or functional activity of said calpain or an expression product of said genetic sequence. According to another aspect of the invention, there is provided a method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising: - contacting a preparation comprising a calpastatin or a genetic sequence encoding said calpastatin with a test agent; and

- detecting a change in the level and/or functional activity of said calpastatin or an expression product of said genetic sequence.

In another aspect, the invention resides in a method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising:

- contacting a preparation comprising a myofibrillar protein or a genetic sequence encoding said myofibrillar protein with a test agent; and

- detecting a change in the level and/or functional activity of said myofibrillar protein or an expression product of said genetic sequence.

In a further aspect, the invention envisions a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition to develop or progress said disorder, wherein said disorder is associated with an aberrant concentration of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, said method comprising:

- contacting a biological sample with an antigen-binding molecule which binds said at least one member; and

- measuring the concentration of a complex comprising said at least one member and the antigen binding molecule in said contacted sample; and - relating said measured complex concentration to the concentration of said at least one member in said sample.

In yet another aspect, the invention contemplates a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition to develop or progress said disorder, wherein said disorder is associated with an aberrant concentration of a calpain, said method comprising: - contacting a biological sample with an antigen-binding molecule which binds said calpain; and

- measuring the concentration of a complex comprising said calpain and the antigen binding molecule in said contacted sample; and - relating said measured complex concentration to the concentration of said calpain in said sample.

In another aspect, the invention provides a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition to develop or progress said disorder, wherein said disorder is associated with an aberrant concentration of a calpastatin, said method comprising:

- contacting a biological sample with an antigen-binding molecule which binds said calpastatin; and

- measuring the concentration of a complex comprising said calpastatin and the antigen binding molecule in said contacted sample; and - relating said measured complex concentration to the concentration of said calpastatin in said sample.

In still yet another aspect, the invention resides in a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition to develop or progress said disorder, wherein said disorder is associated with an aberrant concentration of a myofibrillar protein, said method comprising:

- contacting a biological sample with an antigen-binding molecule which binds said myofibrillar protein; and

- measuring the concentration of a complex comprising said myofibrillar protein and the antigen binding molecule in said contacted sample; and

- relating said measured complex concentration to the concentration of said myofibrillar protein in said sample. In yet another aspect of the invention, there is provided a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising:

- measuring aberrant expression in a muscle cell of a polynucleotide encoding a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

In a further aspect of the invention, there is provided a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising: - measuring aberrant expression in a muscle cell of a polynucleotide encoding a calpain.

In another aspect, the invention extends to a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising: - measuring aberrant expression in a muscle cell of a polynucleotide encoding a calpastatin.

In a further aspect, the invention features a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising: - measuring aberrant expression in a muscle cell of a polynucleotide encoding a myofibrillar protein.

In another aspect, the invention extends to a method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising: - detecting an aberrant genetic sequence encoding a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, or an aberrant genetic sequence encoding a modulatory protein that modulates the level and/or functional activity of said member or an expression product of a said genetic sequence. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graphical representation showing titin gene expression in skeletal muscle of Psammomys obesus in lean, insulin sensitive Psammomys obesus (group A), overweight and insulin resistant (group B) and obese, diabetic Psammomys obesus (group C). Titin gene expression is elevated in group A relative to groups B and C. (*p<0.05 compared with group A animals).

Figure 2A is a graphical representation showing the relationship between percent body fat in Psammomys obesus and titin gene expression in skeletal muscle (1^=0.24, p=0.001).

Figure 2B is a graphical representation showing the relationship between plasma insulin levels and titin gene expression in skeletal muscle of Psammomys obesus (r²=0.15, p=0.003).

Figure 3 is a graphical representation of the level of muscle specific calpain (calpain 3) expressed in skeletal muscle of Psammomys obesus in lean, insulin-sensitive animals (Group A), overweight and insulin resistant (IGT, Group B) and obese, diabetic animals (Group C).

Figure 4A shows a graphical representation of the relationship between calpain 3 (p94) gene expression in skeletal muscle of Psammomys obesus and blood glucose (r=- 0.43, ρ=0.013), plasma insulin (r=-0.37, p=0.034), carbohydrate oxidation (r=0.57, p=009) and energy expenditure (r=0.54, p=0.013).

Figure 4B shows the relationship between calpain 3 (p94) gene expression in skeletal muscle of Psammomys obesus and fat mass (r=-0.35, p=O.049).

Figure 4C shows the relationship between calpain 3 (p94) gene expression in skeletal muscle of human subjects and fat mass (r=-0.44, p=0.022) and central abdominal fat mass (r=-0.49, p=0.034).

Figure 5 is a graphical representation of the level of calpastatin gene expression in skeletal muscle of Psammomys obesus in lean, insulin sensitive animals (group A), overweight and insulin resistant (group B) and obese, diabetic animals (group C) (*p<0.05 compared with group A animals). Figure 6 is a schematic representation showing the proposed consequence of altered p94 calpain on titin levels and the consequence of decreased titin levels such as obesity and type 2 diabetes.

DETAILED DESCRIPTION

1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

The articles "a " and "an " are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "aberrant polynucleotide" "aberrant genetic sequence" and the like refers to a polynucleotide resulting from a substitution, deletion and/or addition of one or more nucleotides in a "normal" reference polynucleotide.

The term "aberrant polypeptide" refers to a polypeptide resulting from a substitution, deletion and/or addition of one or more amino acid residues in a "normal" reference polypeptide.

By "agent" is meant a naturally occurring or synthetically produced molecule which interacts either directly or indirectly with a target member, the level and/or functional activity of which is to be modulated.

By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. As used herein, the term "binds specifically" and the like refers to antigen- binding molecules that bind a target antigen of interest but do not significantly bind other antigens.

The term "biological sample" as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from an animal. The biological sample may be selected from the group consisting of whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, skin biopsy, and the like. Preferably, the biological sample is a tissue biopsy, more preferably a muscle tissue biopsy.

Throughout this specification, unless the context requires otherwise, the words

"comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

As used herein, the term "function " refers to a biological, enzymatic, or therapeutic function.

By "modulating" is meant increasing or decreasing, either directly or indirectly, the level and/or functional activity of a target molecule. For example, an agent may indirectly modulate the said level/activity by interacting with a molecule other than the target molecule. In this regard, indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a first nucleic acid molecule, wherein an expression product of the first nucleic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide.

As used herein, the term "myofibrillar protein BDP-specific antigen-binding molecule" refers to an antigen-binding molecule that recognises calpain-generated myofibrillar protein BDPs, but not intact myofibrillar protein.

The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotide residues, but the term can refer to molecules of any length, although the term "polynucleotide" or "nucleic acid" is typically used for large oligonucleotides.

By "operably linked" is meant that transcriptional and translational regulatory polynucleotides are positioned relative to a polypeptide-encoding polynucleotide in such a manner that the polynucleotide is transcribed and the polypeptide is translated.

The term "patient" refers to patients of human or animal origin and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that "patient" does not imply that symptoms are present. Suitable animals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (e.g. sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g. rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g. cats, dogs) and captive wild animals (e.g. foxes, deer, dingoes).

By "pharmaceutically-acceptable carrier" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in topical or systemic administration.

The term "polynucleotide" or "nucleic acid^'' as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotide residues in length.

"Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. By "primer" is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerising agent. The primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerisation agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotide residues, although it can contain fewer nucleotide residues. Primers can be large polynucleotides, such as from about 200 nucleotide residues to several kilobases or more. Primers can be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridise and serve as a site for the initiation of synthesis. By "substantially complementary", it is meant that the primer is sufficiently complementary to hybridise with a target polynucleotide. Preferably, the primer contains no mismatches with the template to which it is designed to hybridise but this is not essential. For example, non- complementary nucleotide residues can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotide residues or a stretch of non-complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridise therewith and thereby form a template for synthesis of the extension product of the primer.

"Probe " refers to a molecule that binds to a specific sequence or sub-sequence or other moiety of another molecule. Unless otherwise indicated, the term "probe" typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridisation conditions. Probes can be labelled directly or indirectly.

By "reporter molecule" as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that allows the detection of a complex comprising an antigen-binding molecule and its target antigen. The term "reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

By "therapeutically effective amount", or "effective amount" in the context of the treatment of a metabolic disorder, is meant the administration of that amount of active to an individual in need of such treatment, either in a single dose or as part of a series, that is effective for treatment of that disorder. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

2. Method of modulating cellular metabolic function

The present invention is predicated in part on the discovery that obese, diabetic subjects have aberrant levels of calpain expression in muscle as well as aberrant myofibril titin levels relative to normal subjects. The inventors have found unexpectedly that different activity levels of calpain could alter or modulate processing of titin. Not wishing to be bound by any one particular theory or mode of operation, the inventors consider that alterations in muscle proteolysis, particularly the calpain-calpastatin-titin system, may be implicated in the pathophysiology of obesity, insulin-resistance and type II diabetes. Accordingly, it is believed that modulating the levels of calpains could result in altered levels of myofibrillar titin, which could alter inter alia sarcomere assembly, passive tension of muscle, and glucose uptake and/or utilisation leading inter alia to obesity, insulin resistance and type II diabetes.

The invention therefore provides a method for modulating a function of a muscle cell wherein said function is selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis. The method comprises contacting the cell, which is preferably a striated muscle cell, with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

In one embodiment, the agent is capable of decreasing the level and/or functional activity of a calpain, preferably calpain 3. Any suitable calpain inhibitor may be used in this regard. For example,

to US Patent Serial Nos 6,015,787, 5,834,508, 5,770,693, 5,691,368, 5,679,680, 5,554,767, 5,506,243 and 5,336,783, which describe a plethora of calpain inhibitors.

Alternatively, a calpastatin may be used as the calpain inhibitor or a polynucleotide sequence encoding a calpastatin. In this respect, reference may be made to the calpastatin or calpastatin-like polynucleotide and polypeptide sequences disclosed, for example, in GenBank under Accession No U26724 and in US Patent Serial Nos 5,629,165 and 5,189,144.

Also within the scope of the invention are oligoribonucleotide sequences, that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of calpain-encoding mRNA. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions of a gene encoding a calpain, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridisation of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyse endonucleolytic cleavage of calpain RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridisation with complementary oligonucleotides, using ribonuclease protection assays.

Both anti-sense RNA and DNA molecules and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesising oligodeo^ r bϋnuclefttides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesise antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or

3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

The present invention also contemplates use in the above method of calpain inhibitors identified by a method described for example in Section 3, infra.

hi another embodiment, the agent is capable of enhancing the level and/or functional activity of a calpain, preferably calpain 3. An agent of this type may be prepared, for example, by methods described in Section 3, infra. Alternatively, such an agent may comprise a calpain-encoding polynucleotide or a calpain polypeptide as set forth, for example in GenBank under Accession No AF127764 and AAD28253, respectively.

In another embodiment, the agent is capable of increasing the level and/or functional activity of a calpastatin. Such agent may be identified by a method described for example in Section 3, infra.

hi yet a preferred embodiment, the agent is capable of increasing the level and/or functional activity of a myofibrillar protein, which is preferably titin. Such agent may be identified by a method described for example in Section 3, infra.

It will be understood that the agent need not necessarily modulate the level and/or functional activity of only a single target member, but may modulate the activity of two or more of the calpain, the calpastatin and the myofibrillar protein. The modulatory agent of the invention will suitably affect or modulate one or more cellular functions selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis.

For example, energy expenditure can be measured in vivo using several different methods including doubly-labelled water (as for example described by Schoeller DA, et ah, 1982. J Appl Physiol. 53(4): 955-9) and indirect calorimetry (as for example described by Ravussin E, et ah, 1988. N Engl J Med. 318(8): 467-72). In both methods, the rate of production of carbon dioxide is measured, while oxygen consumption is also determined in calorimetry studies. The production of carbon dioxide and consumption of oxygen enable accurate calculation of energy expenditure using standard equations.

Insulin resistance can be measured in vivo in a number of ways, all of which involve determining how rapidly glucose is removed from the blood. Such methods include the hyperinsulinemic, euglycemic clamp (as for example described by Ravussin E, et ah, 1983, J Clin Invest. 72(3): 893-902), oral glucose tolerance test (as for example described by Steele R, et al., 1968, Diabetes 17(7): 415-21) and intravenous glucose tolerance test (as for example described by Bergman RN, et ah, 1981 J Clin Invest. 68(6): 1456-67). Insulin resistance may also be measured in vitro using techniques such as glucose uptake (as for example described by Sarabia V, et ah, 1990, Biochem Cell Biol.6S(2): 536-542) and oxidation assays (as for example described by Hepp D, et ah, 1967, Metabolism 16(5): 393-401.

3. Identification of calpain and myofibrillar protein modulators

The invention also features a method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis. The method comprises contacting a preparation comprising a member selected from the group consisting of a calpain, a myofibrillar protein and a calpastatin or a genetic sequence encoding said member with a test agent and detecting a change in the level and/or functional activity of said member or an expression product of said genetic sequence.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2j§ K) iQθltQn Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited^" to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.

Small (non-peptide) molecule modulators of a member described above are particularly preferred. In this regard, small molecules are particularly preferred because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, and/or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene (e.g., by interacting with the regulatory region or transcription factors involved in gene expression); or affect the activity of a gene by inhibiting or enhancing the binding of accessory molecules.

Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogues. Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.

Screening for modulatory agents according to the invention can be achieved by any suitable method. For example, the method may include contacting a cell, preferably a muscle cell, comprising a genetic sequence expressing a target protein selected from the group consisting of a calpain, a myofibrillar protein and a calpastatin, with an agent suspected of having said modulatory activity and screening for the modulation of that protein, or the modulation of expression of the genetic sequence encoding that protein, or the modulation of the activity or expression of a downstream cellular target of said protein. Detecting such modulation can be achieved utilising techniques including, but not restricted to, Western blotting, ELIS A, and RT-PCR. It will be understood that a genetic sequence from which the target protein of interest is regulated or expressed may be naturally occurring in the cell which is the subject of testing or it may have been introduced into the host cell for the purpose of testing. Further, the naturally-occurring or introduced sequence may be constitutively expressed - thereby providing a model useful in screening for agents which down-regulate expression of an encoded product of the sequence wherein said down regulation can be at the nucleic acid or expression product level - or may require activation - thereby providing a model useful in screening for agents that up-regulate expression of an encoded product of the sequence. Further, to the extent that a polynucleotide is introduced into a cell, that polynucleotide may comprise the entire coding sequence which codes for the target protein or it may comprise a portion of that coding sequence (e.g., a domain such as a protein binding domain) or a portion that regulates expression of a product encoded by the polynucleotide (e.g., a promoter). For example, the promoter that is naturally associated with the genetic sequence may be introduced into the cell which is the subject of testing. In this regard, where only the promoter is utilised, detecting modulation of the promoter activity can be achieved, for example, by operably linking the promoter to a suitable reporter polynucleotide including, but not restricted to, luciferase, β-galactosidase and CAT. Modulation of expression may be determined by measuring the activity associated with the reporter polynucleotide.

In another example, the subject of detection could be a downstream regulatory target of the target protein, rather than target protein itself or the reporter molecule operably linked to a promoter of a gene encoding a protein the expression of which is regulated by the target protein.

These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the genetic sequence encoding the target protein or expression product itself or which modulate the expression of an upstream molecule, which subsequently modulates the expression of the genetic sequence encoding the target protein or expression product activity. Accordingly, these methods provide a mechanism of detecting agents which either directly or indirectly modulate the expression and/or activity of a target protein according to the invention. In a preferred embodiment, a method of identifying agents that inhibit calpain activity is provided in which a purified myofibrillar protein, preferably titin, is incubated with a calpain in the presence and absence of a candidate agent under conditions in which the calpain is activated, and the level of calpain-generated myofibrillar protein breakdown products (BDP) formed is measured by a suitable assay, which is preferably an immunoassay using a myofibrillar protein BDP-specific antigen-binding molecule; such an antigen-binding molecule does not bind intact myofibrillar protein. Such antigen-binding molecule may be produced using routine methods known to those skilled in the art. An agent tests positive if the amount of myofibrillar protein BDPs present in the sample containing the agent is less than that present in the sample lacking the agent. A detailed protocol for the above method is described, for example, in U.S. Patent Serial No. 5,871,712 (to Siman).

In an alternate embodiment, a method of identifying agents that enhance calpain activity is provided in which a purified myofibrillar protein, preferably titin, is incubated with a calpain in the presence and absence of a candidate agent under conditions in which the calpain is, or would be, activated, and the level of calpain-generated myofibrillar protein breakdown products (BDP) formed is measured by a suitable assay, which is preferably the immunoassay referred to above. An agent tests positive if the amount of myofibrillar protein BDPs present in the sample containing the agent is more than that present in the sample lacking the agent.

The amount of bound antigen-binding molecule can be determined by analysis of enzymatic, chromogenic, radioactive, fluorescent, or luminescent reporter molecules that are attached to either the antigen-binding molecule which binds to the myofibrillar protein BDPs or to a secondary antigen-binding molecule which recognises the antigen-binding molecule which binds to the myofibrillar protein BDPs. Immunoassays which may be used in the invention include, but are not limited to, ELISA, cell-based ELISA, filter-binding ELISA, inhibition ELISA, Western blots, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, scintillation proximity assays, fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis, and immunoassays employing an avidin-biotin or a streptavidin-biotin detection system. In an alternate method, a calpain inhibitor can be identified by measuring the ability of a candidate agent to decrease calpain activity in a cell, preferably a muscle cell and more preferably a striated muscle cell, hi this method, a cell that expresses calpain and the myofibrillar protein is exposed to, or cultured in the presence and absence of, the candidate agent under conditions in which calpain is activated in the cell, and the level of calpain-generated myofibrillar protein BDPs formed is measured in an immunoassay using a myofibrillar protein BDP-specific antigen-binding molecule. An agent tests positive if the amount of calpain-generated myofibrillar protein BDPs present in the sample prepared from the cells that were exposed to or cultured in the presence of the agent is less than that present in the sample from the control cells. Alternatively, an agent that enhances the level and/or functional activity of calpain can be identified by measuring the ability of a candidate agent to increase calpain activity in a cell, preferably a muscle cell and more preferably a striated muscle cell, hi this method, a cell that expresses calpain and the myofibrillar protein is exposed to, or cultured in the presence and absence of, a candidate agent under conditions in which calpain is activated in the cell, and the level of calpain- generated myofibrillar protein BDPs formed is measured in an immunoassay using a myofibrillar protein BDP-specific antigen-binding molecule. An agent tests positive if the amount of calpain-generated myofibrillar protein BDPs present in the sample prepared from the cells that were exposed to or cultured in the presence of the agent is more than that present in the sample from the control cells.

In an alternate embodiment, a calpain inhibitor can be identified by measuring the ability of a candidate agent to decrease the level and/or functional activity of calpain in an animal, which is preferably a mammal such as a gerbil, mouse, or rat. In this method, a candidate agent is administered to the animal, and the level of calpain activity is determined using the immunoassays described above. A compound tests positive if the amount of myofibrillar protein BDPs present in a sample taken from the animal to which the agent had been administered is less than that present in an equivalent sample from an untreated animal. Alternatively, an agent that enhances the level and/or functional activity of calpain can be identified by measuring the ability of a candidate agent to increase the level and/or functional activity of calpain in an animal by administering the agent to the animal, and the level of calpain activity is determined using the immunoassays described above. A compound tests positive if the amount of myofibrillar protein BDPs present in a sample taken from the animal to which the agent had been administered is more than that present in an equivalent sample from an untreated animal.

In yet another embodiment, random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to a calpain or to a functional domain thereof. Identification of molecules that are able to bind to a calpain may be accomplished by screening a peptide library with a recombinant soluble calpain. The calpain may be purified, recombinantly expressed or synthesised by any suitable technique. A suitable purification procedure is disclosed for example by Seubert et α/.(1987, Synapse, 1: 20-24).

To identify and isolate the peptide/solid phase support that interacts and forms a complex with the calpain, it is necessary to label or "tag" the calpain. The calpain may be conjugated to any suitable reporter molecule, including enzymes such as alkaline phosphatase and horseradish peroxidase and fluorescent reporter molecules such as fluorescein isothyiocynate (FITC), phycoerythrin (PE) and rhodamine. Conjugation of any given reporter molecule, with calpain, may be performed using techniques that are routine in the art. Alternatively, calpain expression vectors may be engineered to express a chimeric calpain containing an epitope for which a commercially available antigen-binding molecule exist. The epitope specific antigen-binding molecule may be tagged using methods well known in the art including labelling with enzymes, fluorescent dyes or colored or magnetic beads.

For example, the "tagged" calpain conjugate is incubated with the random peptide library for 30 minutes to one hour at 22°C to allow complex formation between calpain and peptide species within the library. The library is then washed to remove any unbound calpain protein. If calpain has been conjugated to alkaline phosphatase or horseradish peroxidase the whole library is poured into a petri dish containing a substrate for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCLP) or 3,3',4,4"-diamnobenzidine (DAB), respectively. After incubating for several minutes, the peptide/solid phase-calpain complex changes colour, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescent tagged calpain molecule has been used, complexes may be isolated by fluorescent activated sorting. If a chimeric calpain protein expressing a heterologous epitope has been used, detection of the peptide/calpain complex may be accomplished by using a labelled epitope specific antigen-binding molecule. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing.

4. Diagnosis of a metabolic disorder

The invention also extends to a method for diagnosis of a metabolic disorder including obesity, insulin resistance and/or diabetes or a predisposition for the disorder.

4.1. Detection of aberrant antigen concentrations

In one embodiment, a biological sample is contacted with an antigen-binding molecule which binds an antigen selected from the group consisting of a calpain, a myofibrillar protein and a calpastatin. The concentration of a complex comprising the antigen and the antigen-binding molecule is measured in the contacted sample and the measured complex concentration is then related to the concentration of the antigen in the sample. The presence of the metabolic disorder is diagnosed if the concentration of the antigen corresponds to an aberrant concentration.

Any suitable technique for determining formation of the complex may be used. For example, an antigen-binding molecule according to the invention, having a reporter molecule associated therewith may be utilised in immunoassays. Such immunoassays include, but are not limited to, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic techniques (ICTs), Western blotting which are well known those of skill in the art. For example, reference may be made to "CURRENT PROTOCOLS IN IMMUNOLOGY" (1994, supra) which discloses a variety of immunoassays that may be used in accordance with the present invention. Immunoassays may include competitive assays as understood in the art or as for example described infra. It will be understood that the present invention encompasses qualitative and quantitative immunoassays.

Suitable immunoassay techniques are described for example in U.S. Patent Serial

Nos 4,016,043, 4, 424,279 and 4,018,653. These include both single-site and two-site assays of the non-competitive types, as well as the traditional competitive binding assays. These assays also include direct binding of a labelled antigen-binding molecule to a target antigen. Two site assays are particularly favoured for use in the present invention. A number of variations of these assays exist all of which are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antigen-binding molecule such as an unlabelled antibody is immobilised on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, another antigen-binding molecule, suitably a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may be either qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including minor variations as will be readily apparent. In accordance with the present invention, the sample is one that might contain an antigen according to the invention and therefore preferably comprises a cell, preferably a tissue biopsy and more preferably a muscle tissue biopsy.

h the typical forward assay, a first antibody having specificity for the antigen or antigenic parts thereof is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient and under suitable conditions to allow binding of any antigen present to the antibody. Following the incubation period, the antigen-antibody complex is washed and dried and incubated with a second antibody specific for a portion of the antigen. The second antibody has generally a reporter molecule associated therewith that is used to indicate the binding of the second antibody to the antigen. The amount of labelled antibody that binds, as determined by the associated reporter molecule, is proportional to the amount of antigen bound to the immobilised first antibody.

An alternative method involves immobilising the antigen in the biological sample and then exposing the immobilised antigen to specific antibody that may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound antigen may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

From the foregoing, it will be appreciated that the reporter molecule associated with the antigen-binding molecule may include the following:

(a) direct attachment of the reporter molecule to the antigen-binding molecule;

(b) indirect attachment of the reporter molecule to the antigen-binding molecule; i.e., attachment of the reporter molecule to another assay reagent which subsequently binds to the antigen-binding molecule; and

(c) attachment to a subsequent reaction product of the antigen-binding molecule.

The reporter molecule may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), a radioisotope and a direct visual label.

In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

A large number of enzymes suitable for use as reporter molecules is disclosed in US Patent Serial Nos 4,366,241, U.S. 4,843,000, and U.S. 4,849,338. Suitable enzymes useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzymes may be used alone or in combination with a second enzyme that is in solution. Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethyhhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower et al. (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in US Patent Serial No 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in US Patent Serial Nos 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to the skilled artisan. The substrates to be used with the specific enzymes are generally chosen for the production of, upon hydrolysis by the corresponding enzyme, a detectable colour change. Examples of suitable enzymes include those described supra. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.

Alternately, fluorescent compounds, such as fluorescein, rhodamine and the lanthanide, europium (EU), may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. The fluorescent-labelled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest, hnmunofluorometric assays (IFMA) are well established in the art and are particularly useful for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.

4.2. Detection of aberrant polynucleotide expression

In another embodiment, the method for diagnosis comprises measuring aberrant expression in a muscle cell of a polynucleotide encoding a member selected from the group consisting of a calpain, a myofibrillar protein and a calpastatin. Aberrant expression of the said polynucleotide may be determined using any suitable technique. For example, a labelled polynucleotide encoding a said member may be utilised as a probe in a Northern blot of a RNA extract obtained from the muscle cell. Preferably, a nucleic acid extract from the animal is utilised in concert with oligonucleotide primers corresponding to sense and antisense sequences of a polynucleotide encoding a said member, or flanking sequences thereof, in a nucleic acid amplification reaction such as RT PCR. A variety of automated solid-phase detection techniques are also appropriate. For example, very large scale immobilised primer arrays (VLSIPS™) are used for the detection of nucleic acids as for example described by Fodor et ah, (1991, Science 251:767-777) and Kazal et a , (1996, Nature Medicine 2:753-759). The above generic techniques are well known to persons skilled in the art.

4.3. Detection of aberrant polynucleotides

It is believed that aberrant polynucleotides including mutant polynucleotides and alternately spliced variant polynucleotides, are linked to one or more of the metabolic disorders discussed above, wherein the aberrant polynucleotides encode at least one protein selected from the group consisting of a calpain, a myofibrillar protein and a calpastatin, or encode a modulatory protein or an expression product that modulates the level and/or expression of said at least one protein. Accordingly, the invention extends to a method for diagnosing a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition therefor, wherein the method comprises detecting a said aberrant polynucleotide.

4.3.1. Screening for aberrant polypeptides

Screening or diagnosis of the disorders mentioned above, or a predisposition to develop such disorders, in a patient may be possible by detecting an aberrant polypeptide linked to this disorder, wherein the aberrant polypeptide is an aberrant calpain, calpastatin or myofibrillar protein or an aberrant modulatory protein that modulates the level and or functional activity of a calpain, a calpastatin or a myofibrillar protein. For example, the presence or absence of such an aberrant polypeptide in a patient may determined by isolating a biological sample from a patient, contacting said sample with an antigen- binding molecule which binds specifically to said aberrant polypeptide, and detecting a complex comprising said antigen-binding molecule and said aberrant polypeptide in the contacted sample, which indicates the presence of the aberrant polypeptide in the sample.

4.3.2. Screening for aberrant polynucleotides In another embodiment, the invention provides a method of screening a patient for an aberrant polynucleotide linked to a metabolic disorder, comprising the steps of isolating a biological sample from the patient, and detecting a said aberrant polynucleotide by a suitable nucleic acid detection technique. According to the invention, presymptomatic screening of a patient for likelihood of developing a metabolic disorder according to the invention may be possible by detecting an aberrant polynucleotide linked to the disorder. The screening method of the invention allows a presymptomatic diagnosis, including prenatal diagnosis, for the presence of an aberrant gene or transcript encoding a protein selected from the group consisting of a calpain, a myofibrillar protein and calpastatin or encoding modulatory protein or expression product that modulates the level and/or expression of a calpain, a calpastatin or a myofibrillar protein in a patient and thus the basis for an opinion concerning the likelihood that such patient would develop or has developed the disorder or symptoms thereof.

For example, in the method of screening, a tissue sample, preferably a tissue biopsy comprising striated muscle, can be taken from a patient, and screened for the presence of one or more normal polynucleotides encoding said protein (i.e., polynucleotides which are not linked to the disorder). The normal human genes can be characterised based upon, for example, detection of restriction digestion patterns in 'normal' versus the patient's DNA, including Restriction Fragment Length Polymorphism (RFLP) analysis, using nucleic acid probes prepared against the normal gene(s) (or functional fragments thereof). Similarly, mRNA encoding said protein may be characterised and compared to normal mRNA levels and/or size as found in human population not at risk of developing this disorder using similar probes. Alternatively, a nucleic acid extract from the patient may be utilised in concert with oligonucleotide primers corresponding to sense and antisense sequences of an aberrant polynucleotide sequence under test, or flanking sequences thereof, in a nucleic acid amplification reaction such as PCR, or the ligase chain reaction (LCR) as for example described in International Application WO89/09385. Preferably, at least one of said primers is an allele-specific primer specific for the polynucleotide mutant under test. Alternatively, the presence or absence of a restriction endonuclease cleavage site resulting from a mutation in the normal polynucleotide may be taken advantage by subjecting the aberrant polynucleotide to digestion with the restriction endonuclease. Accordingly, the present invention encompasses detecting a polynucleotide mutant by RFLP analysis.

The presence in the biological sample of an aberrant polynucleotide size pattern, such as an aberrant RFLP, and/or aberrant mRNA sizes or levels and/or an aberrant polynucleotide linked to the disorder would indicate that the patient has developed or is at risk of developing the metabolic disorder.

Prenatal diagnosis can be performed when desired, using any known method to obtain foetal cells, including amniocentesis, chorionic villous sampling (CVS), and fetoscopy. Prenatal chromosome analysis can be used to determine if the portion of the respective chromosomes possessing the normal genes is present in a heterozygous state.

5. Method of treatment and therapeutic compositions The invention also encompasses a method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent as broadly described above for a time and under conditions sufficient to modulate the level and or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

The modulatory agent can be-administered to a patient either by itself, or in pharmaceutical compositions where it is mixed with suitable pharmaceutically acceptable carrier.

Depending on the specific conditions being treated, modulatory agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.

The agents can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. The dose of agent administered to a patient should be sufficient to effect a beneficial response in the patient over time such as a reduction in the symptoms associated with the metabolic disorder. The quantity of the agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the agent(s) for administration will depend on the judgement of the practitioner. In determining the effective amount of the agent to be administered in the treatment or prophylaxis of the metabolic disorder, the physician may evaluate tissue levels of a calpain, calpastatin and/or myofibrillar protein, and progression of the disorder. In any event, those of skill in the art may readily determine suitable dosages of the agents of the invention. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymefhyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilisers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as., for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum fragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilising processes.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of active compound doses. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilisers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilisers may be added.

Dosage forms of the modulatory agents of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an agent of the invention may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose, hi addition, controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

Modulating agents of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (e.g., the concentration of a test agent which achieves a half-maximal inhibition of calpain activity). Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et ah, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi).

Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain calpain-inhibitory effects or the calpastatin/myofibrillar protein activating or stabilising effects. Usual patient dosages for systemic administration range from 1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02-3 mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5- 1200 mg/m²/day, commonly from 0.5-150 mg/m²/day, typically from 5-100 mg/m²/day.

Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with muscle-specific antibody. The liposomes will be targeted to and taken up selectively by the muscle.

In cases of local administration or selective uptake, the effective local concentration of the agent may not be related to plasma concentration.

h an alternate embodiment, a polynucleotide encoding a modulatory agent of the invention may be used as a therapeutic or prophylactic composition in the form of a "naked DNA" composition as is known in the art. For example, an expression vector comprising said polynucleotide operably linked to a regulatory polynucleotide (e.g. a promoter, transcriptional terminator, enhancer etc) may be introduced into an animal where it causes production of a modulatory agent or a calpain in vivo, particular in striated muscle tissue,. The step of introducing the expression vector into a target cell will differ depending on the intended use and species, and can involve one or more of non- viral and viral vectors, cationic liposomes, retroviruses, and adenoviruses such as, for example, described in Mulligan, R.C., (1993 Science 260: 926-932. Such methods can include, for example:

A. Local application of the expression vector by injection (Wolff et ah, 1990, Science 247: 1465-1468), surgical implantation, instillation or any other means. This method can also be used in combination with local application by injection, surgical implantation, instillation or any other means, of cells responsive to the protein encoded by the expression vector so as to increase the effectiveness of that treatment. This method can also be used in combination with local application by injection, surgical implantation, instillation or any other means, of another factor or factors required for the activity of said protein.

B. General systemic delivery by injection of DNA, (Calabretta et ah, 1993, Cancer Treat. Rev. 19: 169-179), or RNA, alone or in combination with liposomes (Zhu et al, 1993,

Science 261: 209-212), viral capsids or nanoparticles (Bertling et ah, 1991, Biotech. Appl. Biochem. 13: 390-405) or any other mediator of delivery. Improved targeting might be achieved by linking the polynucleotide/expression vector to a targeting molecule (the so-called "magic bullet" approach employing, for example, an antigen- binding molecule), or by local application by injection, surgical implantation or any other means, of another factor or factors required for the activity of the protein encoded by said expression vector, or of cells responsive to said protein.

C. Injection or implantation or delivery by any means, of cells that have been modified ex vivo by transfection (for example, in the presence of calcium phosphate: Chen et al, 1987, Mole. Cell Biochem. 7: 2745-2752, or of cationic lipids and polyamines: Rose et al, 1991, BioTech. 10: 520-525), infection, injection, electroporation (Shigekawa et al, 1988, BioTech. 6: 742-751) or any other way so as to increase the expression of said polynucleotide in those cells. The modification can be mediated by plasmid, bacteriophage, cosmid, viral (such as adeno viral or retro viral; Mulligan, 1993, Science 260: 926-932; Miller, 1992, Nature 357: 455-460; Salmons et al, 1993, Hum. Gen.

Ther. 4: 129-141) or other vectors, or other agents of modification such as liposomes (Zhu et al, 1993, Science 261- 209-212), viral capsids or nanoparticles (Bertling et al, 1991, Biotech. Appl Biochem. 13 390-405), or any other mediator of modification. The use of cells as a delivery vehicle for genes or gene products has been described by Barr et al, 1991, Science 254: 1507-1512 and by Dhawan et al, 1991, Science 254 1509- 1512. Treated cells can be delivered in combination with any nutrient, growth factor, matrix or other agent that will promote their survival in the treated subject.

The present invention is further described by the following non-limiting example.

EXAMPLE 1

Psammomys obesus is a unique rodent model of obesity, insulin resistance and Type II diabetes. Its natural habitat is the desert regions of the middle east, where it subsists on a diet of salt bush. Under these conditions the animals remain lean and normo glycaemic. However, when housed in laboratory conditions and fed ad libitum chow, a diet on which many other rodent species remain healthy, a range of metabolic responses has been observed (Kalderon et al, 1986, Diabetes 35(6): 717-724; Barnett et al, 1994, In. J. Obes. Relat. Matab. Disord. 4(1-2): 83-99). By 16 weeks of age approximately one third of the animals have normal glucose tolerance, one third are hyperinsulinemic and normoglycemic, and one third develop diabetes (Barnett et al, 1994, supra). Other phenotypes noted include obesity, dyslipidemia and hyperphagia (Barnett et al, 1994, supra); Habito et al, 1995, Acta Diabetol. 32(3): 187-92). The relationship of blood glucose and plasma insulin concentrations forms a continuous inverted U-shaped curve in Psammomys obesus identical to "Starling's curve of the pancreas" in human populations (Zimmet et al, 1979, Diabetes 28(7): 624-8; DeFronzo 1988, Metabolism 37(2): 105-108). This heterogeneous response indicates that Psammomys obesus represents a polygenic animal model of human obesity and type II diabetes.

In further studies of this model, the present inventors discovered that titin gene expression in skeletal muscle of Psammomys obesus was 2-fold higher in lean, insulin-sensitive (Group A) Psammomys obesus when compared to their obese, diabetic (Group C) littermates (Figure 1). hi addition, expression of the titin gene in skeletal muscle was negatively correlated with percentage body fat and plasma insulin concentrations in all animals (Figure 2). The expression of muscle-specific calpain in skeletal muscle of Psammomys obesus tended to be lower in obese, diabetic (Group C) animals (Figure 3). Skeletal muscle p94 gene expression was negatively correlated with blood glucose and plasma insulin concentration, as well as fat mass (Figures 4A and 4B). hi addition, p94 gene expression was positively correlated with carbohydrate oxidation rate and energy expenditure (Figure 4A). hi human subjects, p94 gene expression was negatively correlated with fat mass (Figure 4C) and with plasma glucose concentration in non-obese subjects. Overall, there is considerable evidence from both Psammomys obesus and human subjects to suggest that reduced levels of p94 gene expression are associated with increased fat mass, as well as higher circulating glucose and insulin concentrations indicative of insulin resistance. Calpastatin gene expression in skeletal muscle appeared to be slightly elevated in lean (Group A) animals compared with their obese, diabetic (Group C) littermates (Figure 5).

hi view of the above results, the inventors propose that the levels of titin in skeletal muscle may be regulated by the calpain-calpastatin system. As illustrated in Figure 6, altered activity of skeletal muscle calpains (especially p94) could result in decreased levels of myofibril titin. Not wishing to be bound by any one particular theory or mode of operation, it is believed that different activity levels of calpain could alter or modulate processing of titin. Variation in the skeletal muscle titin content is expected to alter the passive tension and sarcomere formation in the myofibrils, and these two factors could greatly impact on basal and or insulin-stimulated glucose utilisation as well as overall energy expenditure. Thus, it is believed that altered homeostasis in the calpain-calpastatin-titin relationship could be a contributing factor in the pathogenesis of obesity, insulin resistance and type II diabetes. Reduced myofibril titin content could alter sarcomere assembly, passive tension of muscle and glucose uptake and/or utilisation, leading to obesity, insulin resistance and type LT diabetes.

The disclosure of every patent, patent application, and publication cited herein is incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

1. A method for modulating a function of a muscle cell, said function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

2. A method for modulating the energy expenditure of a muscle cell, comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

3. A method for modulating insulin resistance of a muscle cell, comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

4. A method for modulating proteolysis in a muscle cell, comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

5. The method of any one of claims 1 to 4, wherein the calpain is calpain 3.

6. The method of any one of claims 1 to 4, wherein the muscle cell is a striated muscle cell.

7. The method of any one of claims 1 to 4, wherein the myofibrillar protein is titin.

8. A method for modulating a function of a muscle cell, said function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of a calpain.

9. The method of claim 8, wherein the calpain is calpain 3.

10. The method of claim 8, wherein the agent decreases the level and/or functional activity of said calpain.

11. The method of claim 8, wherein the agent increases the level and/or functional activity of said calpain.

12. The method of claim 8, wherein the agent modulates the level and or functional activity of the calpain to a normal reference level and/or functional activity.

13. The method of claim 12, wherein the normal reference level and/or functional activity is other than a level and/or functional activity associated with a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes.

14. The method of claim 12, wherein the agent decreases the level and/or functional activity of the calpain to the normal reference level and/or functional activity.

15. The method of claim 12, wherein the agent increases the level and/or functional activity of the calpain to the normal reference level and/or functional activity.

16. A method for modulating a function of a muscle cell, said function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of a calpastatin.

17. The method of claim 16, wherein the agent increases the level and/or functional activity of the calpastatin.

18. A method for modulating a function of a muscle cell, said function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising contacting said cell with an agent for a time and under conditions sufficient to modulate the level and/or functional activity of a myofibrillar protein.

19. The method of claim 18, wherein the agent increases the level and/or functional activity of the myofibrillar protein.

20. The method of claim 18 or claim 19, wherein the myofibrillar protein is titin.

21. A composition for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said composition comprising an agent which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

22. A composition for treatment and/or prophylaxis of obesity, said composition comprising an agent which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

23. A composition for treatment and/or prophylaxis of insulin resistance, said composition comprising an agent which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

24. A composition for treatment and/or prophylaxis of diabetes, said composition comprising an agent which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

25. The composition of any one of claims 21 to 24, wherein the calpain is calpain 3.

26. The composition of any one of claims 21 to 24, wherein the myofibrillar protein is titin.

27. A composition for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said composition comprising an agent which modulates the level and/or functional activity of a calpain, and optionally together with a pharmaceutically acceptable carrier.

28. The composition of claim 27, wherein the calpain is calpain 3.

29. The composition of claim 27, wherein the agent decreases the level and/or functional activity of said calpain.

30. The composition of claim 27, wherein the agent increases the level and/or functional activity of said calpain.

31. The composition of claim 27, wherein the agent modulates the level and/or functional activity of the calpain to a normal reference level and/or functional activity.

32. The composition of claim 31, wherein the normal reference level and/or functional activity is other than a level and/or functional activity associated with a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes.

33. The composition of claim 31, wherein the agent decreases the level and/or functional activity of the calpain to the normal reference level and/or functional activity.

34. The composition of claim 27, wherein the agent increases the level and/or functional activity of the calpain to the normal reference level and/or functional activity.

35. A composition for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said composition comprising an agent which modulates the level and/or functional activity of a calpastatin, and optionally together with a pharmaceutically acceptable carrier.

36. The composition of claim 35, wherein the agent increases the level and/or functional activity of the calpastatin.

37. A composition for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said composition comprising an agent which modulates the level and/or functional activity of a myofibrillar protein, and optionally together with a pharmaceutically acceptable carrier.

38. The composition of claim 37, wherein the agent increases the level and/or functional activity of the myofibrillar protein.

39. The composition of claim 37 or claim 38, wherein the myofibrillar protein is titin.

40. A method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent, which modulates the level and or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, for a time and under conditions sufficient to modulate the level and/or functional activity of said member.

41. A method for treatment and/or prophylaxis of obesity, comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent, which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, for a time and under conditions sufficient to modulate the level and/or functional activity of said member.

42. A method for treatment and/or prophylaxis of insulin resistance, comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent, which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, for a time and under conditions sufficient to modulate the level and/or functional activity of said member.

43. A method for treatment and/or prophylaxis of diabetes, said method comprising aόtemistering to a patient in need of such treatment a therapeutically effective amount of an agent, which modulates the level and/or functional activity of a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, for a time and under conditions sufficient to modulate the level and/or functional activity of said member.

44. The method of any one of claims 40 to 43, wherein the calpain is calpain 3.

45. The method of any one of claims 40 to 43, wherein the myofibrillar protein is titin.

46. A method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent, which modulates the level and/or functional activity of a calpain, for a time and under conditions sufficient to modulate the level and/or functional activity of said calpain.

47. The method of claim 46, wherein the calpain is calpain 3.

48. The method of claim 46, wherein the agent decreases the level and/or functional activity of said calpain.

49. The method of claim 46, wherein the agent increases the level and/or functional activity of said calpain.

50. The method of claim 46, wherein the agent modulates the level and/or functional activity of the calpain to a normal reference level and/or functional activity.

51. The method of claim 50, wherein the normal reference level and/or functional activity is other than a level and/or functional activity associated with a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes.

52. The method of claim 50, wherein the agent decreases the level and/or functional activity of the calpain to the normal reference level and/or functional activity.

53. The method of claim 50, wherein the agent increases the level and/or functional activity of the calpain to the normal reference level and/or functional activity.

54. A method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent, which modulates the level and/or functional activity of a calpastatin, for a time and under conditions sufficient to modulate the level and/or functional activity of said calpastatin.

55. The method of claim 54, wherein the agent increases the level and/or functional activity of the calpastatin.

56. A method for treatment and/or prophylaxis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of an agent, which modulates the level and or functional activity of a myofibrillar protein, for a time and under conditions sufficient to modulate the level and/or functional activity of said myofibrillar protein.

57. The method of claim 56, wherein the agent increases the level and/or functional activity of the myofibrillar protein.

58. The method of claim 56 or claim 57, wherein the myofibrillar protein is titin.

59. A method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising:

60. A method of screening for an agent which modulates energy expenditure, comprising: - contacting a preparation comprising a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein or a genetic sequence encoding said member with a test agent; and

- detecting a change in trie level and/or functional activity of said member or an expression product of said genetic sequence.

61. A method of screening for an agent which modulates insulin resistance, comprising:

62. A method of screening for an agent which modulates muscle proteolysis, said method comprising:

63. The method of any one of claims 59 to 62, wherein the calpain is calpain 3.

64. The method of any one of claims 59 to 62, wherein the myofibrillar protein is titin.

65. A method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising: - contacting a preparation comprising a calpain or a genetic sequence encoding said calpain with a test agent; and

- detecting a change in the level and/or functional activity of said calpain or an expression product of said genetic sequence.

66. The method of claim 65, wherein the calpain is calpain 3.

67. The method of claim 65, wherein the agent decreases the level and/or functional activity of said calpain.

68. The method of claim 65, wherein the agent increases the level and/or functional activity of said calpain.

69. The method of claim 65, wherein the agent modulates the level and/or functional activity of the calpain to a normal reference level and/or functional activity.

70. The method of claim 69, wherein the normal reference level and/or functional activity is other than a level and/or functional activity associated with a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes.

71. The method of claim 69, wherein the agent decreases the level and/or functional activity of the calpain to the normal reference level and/or functional activity.

72. The method of claim 69, wherein the agent increases the level and/or functional activity of the calpain to the normal reference level and/or functional activity.

73. A method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising:

- contacting a preparation comprising a calpastatin or a genetic sequence encoding said calpastatin with a test agent; and - detecting a change in the level and/or functional activity of said calpastatin or an expression product of said genetic sequence.

74. The method of claim 73, wherein the agent increases the level and/or functional activity of the calpastatin.

75. A method of screening for an agent which modulates a cellular function selected from the group consisting of energy expenditure, insulin resistance and muscle proteolysis, said method comprising:

- contacting a preparation comprising a myofibrillar protein or a genetic sequence encoding said myofibrillar protein with a test agent; and - detecting a change in the level and or functional activity of said myofibrillar protein or an expression product of said genetic sequence.

76. The method of claim 75, wherein the agent increases the level and/or functional activity of the myofibrillar protein.

77. The method of claim 75 or claim 76, wherein the myofibrillar protein is titin.

78. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition to develop or progress said disorder, wherein said disorder is associated with an abenant concentration of at least one member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, said method comprising: - contacting a biological sample with an antigen-binding molecule which binds said at least one member; and

- measuring the concentration of a complex comprising said at least one member and the antigen binding molecule in said contacted sample; and

- relating said measured complex concentration to the concentration of said at least one member in said sample.

79. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition to develop or progress said disorder, wherein said disorder is associated with an aberrant concentration of a calpain, said method comprising:

- contacting a biological sample with an antigen-binding molecule which binds said calpain; and - measuring the concentration of a complex comprising said calpain and the antigen binding molecule in said contacted sample; and

- relating said measured complex concentration to the concentration of said calpain in said sample.

80. The method of claim 79, further comprising comparing the concentration of calpain to a normal concentration of calpain.

81. The method of claim 80, wherein the normal concentration is other than a concentration associated with said metabolic disorder.

82. The method of claim 79, wherein the calpain is calpain 3.

83. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition to develop or progress said disorder, wherein said disorder is associated with an abenant concentration of a calpastatin, said method comprising:

- contacting a biological sample with an antigen-binding molecule which binds said calpastatin; and - measuring the concentration of a complex comprising said calpastatin and the antigen binding molecule in said contacted sample; and

- relating said measured complex concentration to the concentration of said calpastatin in said sample.

84. The method of claim 83, further comprising comparing the concentration of calpastatin to a normal concentration of calpastatin.

85. The method of claim 84, wherein the normal concentration is other than a concentration associated with said metabolic disorder.

86. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and/or diabetes or a predisposition to develop or progress said disorder, wherein said disorder is associated with an abenant concentration of a myofibrillar protein, said method comprising: - contacting a biological sample with an antigen-binding molecule which binds said myofibrillar protein; and

- relating said measured complex concentration to the concentration of said myofibrillar protein in said sample.

87. The method of claim 86, further comprising comparing the concentration of myofibrillar protein to a normal concentration of myofibrillar protein .

88. The method of claim 87, wherein the normal concentration is other than a concentration associated with said metabolic disorder.

89. The method of claim 86, wherein the myofibrillar protein is titin.

90. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising:

- measuring abenant expression in a muscle cell of a polynucleotide encoding a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein.

91. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising: - measuring abenant expression in a muscle cell of a polynucleotide encoding a calpain.

92. The method of claim 91, wherein the calpain is calpain 3.

93. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising:

- measuring abenant expression in a muscle cell of a polynucleotide encoding a calpastatin.

94. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising:

- measuring abenant expression in a muscle cell of a polynucleotide encoding a myofibrillar protein.

95. The method of claim 94, wherein the myofibrillar protein is titin.

96. A method for diagnosis of a metabolic disorder selected from the group consisting of obesity, insulin resistance and diabetes or a predisposition for said disorder, said method comprising: - detecting an abenant genetic sequence encoding a member selected from the group consisting of a calpain, a calpastatin and a myofibrillar protein, or an abenant genetic sequence encoding a modulatory protein that modulates the level and or functional activity of said member or an expression product of a said genetic sequence.