WO2007123848A2 - Therapeutic compositions containing modified class i slrp proteins - Google Patents

Abstract

Therapeutic compositions and methods of treating, and preventing the onset of neuromuscular disorders, conditions or diseases in mammals, especially in humans, including for example the muscular dystrophies and especially DMD and BMD, are provided. The therapeutic compositions include effective amount of one or more non- glycanated Class I SLRP proteins alone or in admixture with a pharmaceutically acceptable vehicle. The therapeutic pharmaceutical compositions of the invention may be administered alone or in combination with each other and/or in combination with other therapeutic compositions.

Description

THERAPEUTIC COMPOSITIONS CONTAINING MODIFIED CLASS I SLRP PROTEINS

FIELD OF THE INVENTION

The invention relates to novel Class I SLRP protein therapeutic compositions. These compositions can be employed in the treatment of dystrophies, sarcoglycanopathies, sarcopaenia, muscle atrophy of indeterminate cause, and biglycanopathies. BACKGROUND OF THE INVENTION

The muscular dystrophies are a group of inherited muscle disorders characterized by progressive skeletal muscle weakness and the death of muscle cells and tissue due, in some cases, to various defects in the genes encoding proteins of the neuromuscular system. Clinically, they are a heterogeneous group of disorders. Patients with Duchenne muscular dystrophy (DMD) exhibit a childhood onset and die in their twenties, usually from respiratory or cardiac failure. Patients with Becker muscular dystrophy (BMD) have moderate muscle weakness typically in adulthood, and live relatively normal life spans. In the limb-girdle muscular dystrophies (LGMD), the initial involvement is in the shoulder and pelvic girdle muscles.

About 30 genes have been implicated in muscular dystrophy. See Durbeej et al. "Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models," Curr Opin Genet Dev. 12: 34 9-61 (2002). DMD and BMD result from a mutation in the gene encoding dystrophin protein, which is part of a complex of proteins (the "dystrophin-associated protein complex" or "DAPC"; also termed the "dystrophin-glycoprotein complex" or "DGC") that connect the muscle fibers of the human cytoskeleton to the surrounding extracellular matrix (ECM). The DAPC is a large, multi-component complex that includes the dystroglycans, biglycan, the sarcoglycans, the anytrophins, the dystrobrevin, nNOS and sarcospan. Mutations in genes encoding components of this complex other than dystrophin are associated with other dystrophies, for example, LGMD. Although it is know that the integrity of the DAPC and its association with the ECM are essential for muscle cell viability, the complete role of the DAPC in normal muscle and how its function is disrupted in dystrophic muscle is still unclear. In addition, mutations in non-DAPC protein encoding genes, such as calpain 3 and caveolin 3 for example, have been shown to cause forms of muscular dystrophy. The proteins decorin (DCN) and biglycan (BGN) are components of the ECM of many connective tissues, including the ECM of muscle fibers. They belong to a family of small leucine-rich repeat proteoglycan (SLRP) proteins each of which possesses 10-12 leucine-rich repeat (LRR) consensus sequences. Class I family members decorin and biglycan, in addition to possessing N-glycosylation, possess chrondroitin sulphate or dermatan sulphate glycosaminoglycan (GAG) side chains (one side chain in the case of decorin, two in the case of biglycan) at their n-terminal domains; Class II family members fibromodulin and lumican possess keratan sulphate side chains at their N-terminal domains. See Roughley et ah, "Presence of pro-forms of decorin and biglycan in human articular cartilage," Biochem. J. 318: 779-84 (1996); Zanotti et al., "Decorin and biglycan expression is differentially altered in several muscular dystrophies," Brain 128: 2546-55 (2005). For a review of the entire superfamily of matrix proteoglycans of which the Class I SLRPs are members, see Iozzo, "Matrix Proteoglycans: From Molecular Design to Cellular Function," Annu. Rev. Biochem. 67: 609-52 (1998). Asporin is another member of the Class I SLRP family; it has gene sequence homology with decorin but lacks any GAG side chains in its native form. See Lorenzo et al., "Identification and characterization of asporin, a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan," J Biol Chem. 276: 12201-11 (2001) and Henry et al., "Expression pattern and gene characterization of asporin: a newly discovered member of the leucine-rich repeat protein family." J Biol Chem. 276: 12212-21 (2001)

Biglycan and decorin are suspected of playing a role in muscular dystrophies. For example, biblycan is over-expressed in an animal model of muscular dystrophy that is characterized by the absence of dystrophin. See U.S. Patent No. 6,864,236; see also Zanotti, supra; Haslett et al., "Gene expression comparison of biopsies from duchenne muscular dystrophy (DMD) and normal skeletal muscle," Proc Natl Acad Sci USA 99: 15000-005 (2002); Bowe et al., "The Small Leucine-rich Repeat Proteoglycan Biglycan Binds to α-dystroglycan and Is Upregulated in Dystrophic Muscle," J. Cell. Biol. 148: 801-10 (2000); Ameye et al., "Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis, osteoarthrisis, Ehlers-Danlos syndrome, muscular dystrophy, and corneal diseases," Glvcobiology 12: 107R-116R (2002); and Mercado et al., "Biglycan targets dystrobrevin, syntrophin and nNOS to the muscle cell membrane," FASEB J. (in press) (2006). The DNA and amino acid sequences of biglycan and decorin from various mammalian and non-mammalian species have been isolated and the recombinant proteins expressed in various expression systems. See for example, Junghans et al., "Purification of a Meningeal Cell-derived Chrondroitin Sulphate Proteoglycan with Neurotopic Activity for Brain Neurons and its Identification as Biglycan," Eur. J. Neuroscience 7: 2341-50 (1995); Johnson et al., "Identification and characterization of glycanated and non-glycanated forms of biglycan and decorin in the human intervertebral disc," Biochem. J. 292: 61-66 (1993); Hocking et al., "Eukaryotic expression of recombinant biglycan: Post transitional processing and the importance of secondary structure for biological activity," J. Biol. Chem. 271: 19571-77 (1996). Also known is that these two LRR family members exist in proteoglycan pro-forms. Biglycan has a 16 amino acid signal sequence and a 21 amino acid propetide sequence. Decorin has a 16 amino acid signal sequence and a 14 amino acid propeptide sequence. Biglycan and decorin also exist in "non-GAG" forms lacking the glycosaminoglycan side chains (either chrondroitin or dermatan sulfate) but including other N-terminal glycosylation and the pro-region.

The different roles and functions of these various forms of these SLRP proteins are unknown and subject to much speculation. For example, U.S. Patent No. 6,864,236 discloses that the non-GAG form of biglycan binds alpha- and gamma- sarcoglycan but does not bind alpha-dystroglycan (see Examples 5-6) and that both the non-GAG and the GAG form of biglycan have a biphasic effect on agrin-induced AChR (acetylcholine receptor) clustering in the neuromuscular junction (see Example 9). However, because the DAPC binding data is incomplete and inconclusive and because the DAPC lacks acetylcholine receptors, these results say nothing about whether either or both forms of the protein could be employed in the therapeutic treatment of, for example, dystrophies, sarcoglycanopathies or biglycanopathies. Indeed, the '236 patent fails to disclose any evidence of an effect of either form of biglycan by itself In cell culture.

In Ameye et al., supra, it is disclosed that biglycan binding appears to occur through the glycosaminoglycan chains and that mice deficient in biglycan display a mild muscular dystrophic phenotype, which suggested to this group of researchers that biglycan (i.e., the glycanated (GAG) form of the protein) may play a role in muscular dystrophies and in the treatment of these pathologies. SUMMARY OF THE INVENTION

It has now been unexpectedly and surprisingly discovered that it is the non- GAG forms of this Class I SLRP (not the glycanated forms) that are the therapeutically relevant forms of the protein efficacious in treating neuromuscular diseases and the conditions in mammals. In addition, other Class I SLRP proteins in their non-GAG forms, by virtue of sharing structural and conformational characteristics and sequence homology with the non-GAG forms of BGN, may be employed in the same manner in treating such diseases and conditions. Although U.S. Patent Publication No. 2005/0059580 published March 17, 2005 discloses various biglycan and decorin proteoglycan compounds (i.e. compounds containing one or more GAG side chains) as therapeutics, there appears to be no recognition or suggestion in that document of a possible therapeutic utility for the non-GAG forms of these proteins.

Accordingly, in one aspect the invention encompasses therapeutic compositions and methods of treating, and preventing, the onset of neuromuscular disorders, conditions, or diseases in mammals, especially in humans, including for example the muscular dystrophies, and especially DMD and BMD. In one aspect, the compositions include therapeutically effective amounts of a non-glycanated (without GAG chains) Class I SLRP protein, for example, BGN⁰*^0", DCN^GAG" or asporin, alone in admixture with a pharmaceutically acceptable vehicle. The compositions of the invention may be administered alone or in combination with each other and/or in combination with other therapeutic compositions. Such other therapeutic compositions for co-administration with one or more Class I SLRP proteins include for example anti-myostatin antibodies (for example MYO-29, Wyeth Pharmaceuticals), other myostatin blocking molecules, calpain inhibitors such as Myodur (Ceptor Corp.), PTC 124 (PTC Therapeutics), 1-arginine, dystrophin, and/or utrophin (via gene therapy for example).

In another aspect, the therapeutic compositions of the invention may be administered as the active ingredient of a cell based therapy or a gene therapy regimen.

The treatment methods include methods of preventing, or ameliorating a dystrophic genetic disease, or one or more symptoms associated with or manifestations of a dystrophic genetic disease, comprising administering to a patient in need thereof an effective amount of a Class I SLRP protein, for example BGN^GAG" protein, DCN^GAG" protein or asporin protein or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, prodrug or poymorph thereof. In addition, the treatment methods include methods of treating acquired (toxic or inflammatory) myopathies.

In a preferred embodiment, the disease or conditions is a dystrophinopathy, a sarcoglycanopathy, a sarcopaenia, a muscle atrophy of indeterminate cause, or a biglycanopathy. Neuromuscular dystrophies within the scope of the invention include myotonic dystrophy (Steinert's disease; DM), spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DME), Becker's muscular dystrophy (BMD), congenital muscular dystrophy (CMD), distal muscular dystrophy, Emery-Dreifus muscular dystrophy, facioscapulohumeral dystrophy, Fukuyama congenital muscular dystrophy (FCMD), limb-girdle muscular dystrophy (LGMD -IA-G; -2A-8), oculopharyngeal muscular dystrophy, scapuloperoneal muscular dystrophy, and severe childhood autosomal recessive muscular dystrophy. Additional diseases and conditions are more fully described in U.S. Patent No. 6,864,236, U.S. Patent Publication 2005/0059580, and on the world wide web at neuro.wustl.edu/neuromuscular/musdist/lg.

In addition to skeletal muscle disorders, the compositions and methods may be employed in the treatment of disorders of smooth and cardiac muscle, for example in the treatment of cardiac myopathies (e.g., hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, welander distal myopathy) of either genetic or environmental origin as is more fully described in U.S. Patent No. 6,864,236. It is known that disruption of the sarcoglycan-sarcospan complex in skeletal and cardiac membranes perturbs vascular function, initiates cardiomyopathy and exacerbates muscular dystrophy, and that alpha, beta, delta and gamma - sarcoglycan null mice exhibit pronounced dystrophic muscle changes and develop cardiomyopathy. Other diseases or conditions that may be treated include those described in U.S. Patent Publication No. 2005/0059580.

The pharmaceutical compositions and treatment methods of the invention are suitable for administration to human patients. It may be most advantageous to administer the compositions and methods when the patient is an infant or child and the disease or condition is in its earliest stages. It is believed that the compositions of the invention function in part as a mechanism that compensates for the lack of the requisite intracellular dystrophin and thereby allows the survival of dystrophin- negative muscle fibers. In yet another aspect, the pharmaceutical compositions of the invention are administered parenterally (including subcutaneously, intramuscularly, by bolus injection, intraarterially. or intravenously); more preferably the compound is administered intraperitoneally (IP), intramuscularly (EVl), or intravenously. In cell based or gene therapies the pharmaceutical compositions of the invention may be administered in the same manner, by surgical exposure of muscle followed by injection or by other suitable routes of administration known to those of skill in the art.

In yet another aspect, the invention includes a method of making non- glycanated forms of Class I SLRP proteins. The method comprises separating the non-glycanated form of a Class I SLRP protein recombinantly produced using anion exchange chromatography with Q-Sepharose followed by elution with a linear gradient of 0.15-2 M NaCl in PBS, 0.2% CHAPS.

BRIEF DESCRIPTION OF THE FIGURES

Figures IA and B are graphical illustrations of the results of BGN^GAG" treatment on agrin-induced AChR clustering in cell culture as detailed in Example 3.

Figures 2A and B are graphical illustrations of the results of low dose and high dose administration of BGN^GAG' on sarcolemma integrity (Fig. 2B) and on myofiber degeneration (Fig. IA) in mice as detailed in Example 4.

Figure 3 is a graphical illustration of the results of low dose administration of BGN^GAG" on utrophin expression in mice as detailed in Example 4.

Figure 4 is a photographic reproduction of the results of the comparison of BGN^GAG" administration versus biglycan administration on four DAPC components in mice as detailed in Example 5.

Figures 5A, B₅ and C are photographic reproductions and graphic illustrations of the results of systemic administration of BGN^GAG" on mononuclear cell infiltration and muscle regeneration in mdx mice as detailed in Example 6.

Figures 6A, B, and C are photographic reproductions and graphic illustrations of the results of systemic administration of BGN^GAG" on utrophin levels in mdx mice as detailed in Example 6.

Figures 7A, a photographic reproduction, and 7B, a graphic illustration, show the results of administration of BGN°^AG" to double knock-out mdx:utr-/- mice. Figures 8A - D are graphic illustrations of the results of BGN^GAG" treatment on muscle function as described in detail in Example 7.

DETAILED DESCRIPTION 1. DEFINITIONS

"GAG" refers to glycosaminoglycan, which is used interchangeable with "mucopolysaccharide", and is a long, unbranched polysaccharide chain composed of repeating disaccharide units. One of the two sugars is always an amino sugar: N- acetyl glucosamine or N-acetylgalactosamine. GAGs are covalently linked via a xylose sugar to a serine residue of a core polypeptide to form a "proteoglycan."

A "glycoprotein" is a protein that contains one or more carbohydrate groups covalently attached to the polypeptide chain. Typically, a glycoprotein contains from 1% to 60% carbohydrate by weight in the form of numerous, relatively short, branched oligosaccharide chains of variable composition. In contrast, "proteoglycans" can be much larger glycoconjugates (up to millions of Daltons), and they can contain 90 to 95% carbohydrate by weight in the form of many long, unbranched, glycosaminoglycan chains.

As used herein, "biglycan" or "BGN" refers to a biglycan protein comprising the amino acid sequence disclosed as SEQ. ID. No. 9 in U.S. Patent No. 6,864,236, and homologs thereof exhibiting at least 80%, preferably 85%, more preferably 90%i and most preferably 95% sequence identity to the human biglycan protein sequence, and at least one of the biological activities of human biglycan disclosed in U.S. Patent No. 6,i64,236. Biglycan is also described in Fisher et al., "Deduced Protein Sequence of Bone Small Protoeoglycan I (Biglycan) shows Homology with Protoglycan II (Decorin) and several Nonconnective Tissue Proteins in a Variety of Species," J. Biol. Chem. 264: 4571-76 (1989) and in U.S. Patent No. 5,340,934, and is deposited with GenBank, Accession No. J04599. Its amino acid and DNA sequences are known. "Biglycan", "BGN" or "BGN^GAG+" refers to one of these protein sequences including one or more of its GAG side chains.

As used herein, "decorin" or "DCN" refers to a decorin protein, at least one of which is disclosed in Krusius, et al, "Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA," PNAS 83: 7683-87 (1986) and in U.S. Patent Publication No. 2003/0032591 published February 13, 2003, and to homologs of the human decorin protein sequence exhibiting at least 80%, preferably 85%, more preferably 90%, and most preferably 95% sequence identity to human decorin protein, and at least one of the biological activities of human decorin disclosed in U.S. Patent Publication No. 2003/032591. "Decorin", DCN, or DCN^GAG+ refers to one of these protein sequences including its GAG side chain

As used herein, "asporin" refers to an asporin protein comprising the amino acid sequence disclosed in Henry et al., "Expression pattern and Gene Characterization of Asporin," J Biol Chem. 276: 12212-21 (2001) and in Lorenzo, et al., "Identification and Characterization of Asporin," J Biol Chem. 276: 12201-11, and to homologs of the human asporin protein sequence exhibiting at least 80%, preferably 85%, more preferably 90%, and most preferably 95% sequence identity to human asporin protein, and at least one of the biological activities of human asporin disclosed in Henry, supra or in Lorenzy, supra.

The term "biglycan core" refers to a biglycan protein that does not include GAG chains. "Biglycan core", non-GAG biglycan, non-GAG BGN, and BGN^GAG" are synonymous, and are used interchangeably to refer to protein forms of biglycan lacking the glycosaminoglycan side chains but including any N-terminal glycosylation and/or pro-region. The polypeptide sequences of biglycan core of various species (human, bovine, avian, mouse, dog, rat) are known to be highly homologous. There is a signal sequence comprising peptide residues 1 through 16 and a propeptide sequence comprising peptide residues 17 through 37. The mature form of the polypeptide begins at residue 38 and the two GAG side chains bind to the serine residues at 42 and 48, keeping the same numbering, or amino acids 5 and 11 in the mature polypeptide (the "SerGly" sequences). The terms "biglycan core", "non- GAG biglycan", "non-GAG BGN" and "BGN⁰^^"" include the mature polypeptide sequence (including N-terminal glycosylation), and either including or excluding the propeptide region.

The term "decorin core" refers to a decorin protein that does not include GAG chains. "Decorin core", non-GAG decorin, non-GAG DCN, and DCN⁰*^0' are synonymous, and used interchangeably to refer to a protein form of decorin lacking the glycosaminoglycan side chains but including any N-terminal glycosylation and/or pro-region.

As used herein, "non-GAG Class I SLRP protein" refers to a member of the Small Leucine-Rich Proteoglycan Class I subfamily of proteins lacking glycosamino- glycan side chains and either with or without the protein's propeptide region, including, for example, a BGN°^AG", DCN^GAG- or an asporin.

A "therapeutically effective amount" means an amount of active ingredient, for example a pharmaceutical composition of the invention, sufficient to provide a therapeutic benefit in the treatment or management of the disease or condition, to delay or minimize symptoms associated with the disease or condition, or to result in the prevention, recurrence, spread or progression of the disease or condition. Such amount may refer to the amount of the active ingredient alone or in combination with other therapies that provides a therapeutic benefit in the treatment or management of the disease. The term encompasses amounts that improve overall therapy, reduce or avoid symptoms or cause of disease, enhance the therapeutic efficacy of another therapeutic agent or synergize with another therapeutic agent.

The terms "manage", "managing", and "management" refer to the beneficial effects that a subject derives from administration of a pharmaceutical composition of the invention, which does not result in a cure of the disease. In some embodiments a subject is administered a compound of the invention in combination with one or more other therapeutic agents to "manage" a disease so as to prevent its progression or worsening.

The terms "prevent", "preventing", and "prevention" refer to the prevention of the onset, recurrence or spread of the disease or condition in a patient resulting from the administration of a pharmaceutical composition of the invention.

The terms "treat", "treating", and "treatment" refer to the eradication or amelioration of the disease or condition or symptoms associated with the disease or condition. In some embodiments, these terms refer to minimizing the spread or worsening of the disease or condition resulting from the administration of one or more therapeutic agents to a patient.

"Pharmaceutically acceptable salts" means salts prepared from pharmaceutically acceptable, non-toxic, acids or bases including inorganic acids and bases, and organic acids and bases. Suitable pharmaceutically acceptable base addition salts include, for example, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, or zinc, or organic salts made from chloroprocaine, choline, diethanolamine, ethylenediamine, lysine, N₅N'- dibenzylethylenediamine, N-methylglucamine or procaine. Suitable exemplary nontoxic acids include, for example, inorganic and organic acids such as acetic, alginic, anthranilic, genzenesulfonic, benzoic, camphorsulfonic, citric, ethensulfonic, formic, fumaric, fiiroic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isothionic, lactic, maleic, malic, mandelic, methansulfonic, mucic, nitric, pamoic, panthothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, or p-toluenesulfonic acid. Specified nontoxic acids include hydrochloric, hydrobromic, methansulfonic, phosphoric, or sulfuric acids. Specific salts include hydrochrloride or esylate salts. Other examples of pharmaceutically acceptable salts are well know to those skilled in the art, see, e.g., Remington's Pharmaceutical Sciences,^ ed., Mack Publishing, Easton, PA (1990).

2. DESCRIPTION AND EXAMPLES

The invention is directed to the in vivo use of one or more modified Class 1 SLRP protein pharmaceutical compositions in the treatment of neuromuscular conditions and disorders including for example a dystrophy, a sarcoglycanopathy or a biglycanopathy. In one aspect such compositions include a therapeutically effective amount of a non-GAG Class I SLRP protein in a pharmaceutically acceptable vehicle. Alternatively, a therapeutically effective amount of non-GAG Class I SLRP protein polymorph, prodrug, salt, clathrate, solvate or hydrate in a pharmaceutically acceptable vehicle may be employed. The non-GAG Class I SLRP protein may be a BGN°^AG-, a DCN^GAG-- or an asporin protein.

The pharmaceutical compositions of the invention may be administered alone or may be co-administered with each other or with another therapeutic agent. Coadministration of two non-GAG Class I SLRP proteins is exemplary, for example coadministration of BGN^GAG" and DCN^GAG" , co-administration of BGN°^AG~ and asporin or co-administration of DCN^GAG' and asporin, but co-administration of one of these Class I SLRP proteins with other therapeutic agents that are not members of the Class I SLRP protein family is preferred. In another preferred embodiment, the composition is administered concurrently with the administration of the other therapeutic agent, which can be part of the composition or can be in a different composition. In yet another embodiment, the composition is administered prior to or subsequent to administration of the other therapeutic agent. Exemplary others therapeutic agents include, for example, anti-myostatin antibodies (for example MYO-029, Wyeth Pharmaceuticals), other myostatϊn blocking molecules, calpain inhibitors such as Myodur (Ceptor Corp,,), PTC 124 (PTC Therapeutics), 1-arginine, dystrophin, and/or utrophin (via gene therapy, for example).

The pharmaceutical compositions of the invention may be administered in the form of, for example, pharmaceutically acceptable salts, or in the form of PEGylated protein compositions. PEGylation of protein therapeutics is well known in the art. PEGylation helps increase the circulation half-life of proteins by reducing their renal clearance rates. Other potential positive effects include enhanced solubility, improved stability, sustained absorption, and reduced immunogenicity, antigenicity and proteolysis. PEGylation may be accomplished by the random conjugation of linear polyethylene glycol chains onto the functional groups along the protein backbone or by the use of branched PEG (polyethylene glycol), or site-specific and controlled PEGylation. PEGylation techniques and the administration of PEGylated protein pharmaceutical compositions are within the level of skill in the art.

Alternatively, the pharmaceutical compositions of the invention may be administered in the form of microspheres. Microsphere-based technologies are known in the art and have been advantageously employed to protect protein therapeutics from proteolytic degradation and to provide sustained release. Techniques for encapsulating the pharmaceutical compositions of the invention into appropriate microspheres for administration in humans are believed to be within the level of skill in the art, as are techniques for their administration.

The pharmaceutical compositions of the invention may also be administered in a cell based therapy or a gene therapy regimen. In cell based therapy (or somatic-cell therapy), autologous, allogeneic, or xenogeneic living somatic cells that have been manipulated to contain the nucleotide sequence encoding a pharmaceutical composition of the invention and operationally linked or associated with regulatory control elements permitting expression of the nucleotide sequence are administered to the patient in need of therapy. The cells may be encapsulated. See United States Patent Publication No. 2006/0251630 entitled Encapsulated Cell Therapy, published November 9, 2006. Like hemophilia, Parkinson's disease, diabetes mellitus and other congenital or acquired diseases and conditions characterized by deficient production of secreted factors, the neuromuscular conditions and disorders included here (dystrophies, sarcoglycanopathies, biglycanopathies) are susceptible to this approach as a form of replacement therapy. In gene therapy, modified or attenuated viruses are employed as vectors to carry the genetic material encoding the pharmaceutical composition operationally linked or associated with a transcriptional promoter capable of directing expression of the sequence and other regulatory elements or sequences necessary or desirable (marker sequences, for example) into the patient's cells. See Hu and Pathak, "Design of Retroviral Vectors and Helper Cells for Gene Therapy," Pharmacological Reviews 52: 493-511 (2000). Other gene therapy approaches include DNA liposome mixtures, directly administered DNA (see for example Nabel et al, "Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biologic activity, and lack of toxicity in humans," Proc Natl Acad Sci USA 90: 11307-311 (1993) and Plautz et al., "Immunotherapy of malignancy by in vivo gene transfer into tumors", Proc Natl Acad Sci USA 90: 4645-49 (1993)) and DNA combined with a targeted delivery system (for example a monoclonal antibody or a cellular receptor targeted ligand-DNA conjugate). Plasmid DNA can be administered directly into the tissue of an organ. The sequence encoding the therapeutic protein is then translated in the tissue. The techniques are known to those of skill in the art.

Other cell and gene therapy techniques are known to those of skill in the art and are described in detail in numerous publications, including United States Patent No. 6,210,963 entitled Recombinant Cells from the Monocyte-macrophage Cell Line for Gene Therapy, issued April 3, 2001; United States Patent No. 5,656,465 entitled Methods of In Vivo Gene Delivery, issued August 12, 1997; and United States Patent No. 5,789,390 entitled Method for Preparing Recombinant Adeno-Associated Viruses (AAV) and Uses, Therefor, issued August 4, 1998.

Cell or tissue based delivery of the pharmaceutical compositions of the invention will comprise the nucleic acid sequence coding for the composition and the necessary and desired regulatory control elements enabling expression of the sequence upon administration. In this circumstance, a regulatable promoter capable of turning expression of the sequence off and on must be employed due to the inverted-U dosage response curve observed upon delivery of BGN°^AG' (see Example 4). Once expression of the sequence has resulted in expression of the pharmaceutical composition of the invention in the desired efficacious dosage, the promoter's regulator can be administered to the patient in order to control the dosage and maintain it in the desired efficacious range. Promoters that can be regulated in this manner are known in the art. See, for example, United States Patent Publication No. 2003/0221203 entitled High Efficiency Regulatable Gene Expression System, published November 27, 2003; Krueger et al., "A gene regulation system with four distinct expression levels," J Gene Med 8: 1037-47 (2006); Agha-Mohammadi et al., "Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness," J Gene Med 6: 817-828 (2004); Zerby et al., "In Vivo Ligand- Inducible Regulation of Gene Expression in a Gutless Adenoviral Vector System," Human Gene Therapy 14: 749-61 (2003); Lu et al., "Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagons and cAMP and down-regulated by insulin," Gene Therapy 5: 888-95 (1998); and Barry et al., "Glucose-Regulated Insulin Expression in Diabetic Rats," Human Gene Therapy 12: 131-39 (2001).

The toxicity and therapeutic efficacy of the protein compounds can be determined by standard pharmaceutical procedures in cell cultures or in experimental animals that are designed to determine the LD50 (the dosage lethal to 50% of the population), the ED₅₀ (the dosage therapeutically effective in 50% of the population), and the therapeutic index (the ratio: LD50/ED50). The data obtained from these studies is used to formulate the range of dosage for use in humans. The dosage lies within a range of circulating concentrations that will include the ED₅0 with little or no toxicity. The dosage can vary within this range depending upon the dosage form used and the route of administration. Initially the therapeutically effective dose can be estimated from cell culture assays. Especially in the case of the Class I SLRPs administered in cell culture, the amounts persisting post-administration decrease little from the initial administered amounts and the levels stably associated with the muscle cells persist for long time periods post-administration. From such data, a dosage can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50, the concentration that achieves half-maximal inhibition of symptoms. This information is then used to more accurately determine useful dosages in humans.

The effective amount of a pharmaceutical composition of the invention will vary depending upon the nature and severity of the disease or condition and the route of administration. It will also vary according to the age, body weight, and response of the individual patient. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors. For the Class I SLRP proteins herein, a therapeutically effective amount of protein is from about 0.10 mg/kg to about 500 mg/kg body weight, preferably from about 1.0 mg/kg to about 300 mg/kg body weight. More specifically, preferred dosage ranges include from about 1.0 mg/kg to about 100 mg/kg, and from about 1.0 mg/kg to about 50 mg/kg. In managing the patient, the therapy should be initiated at a lower dose, perhaps about 1 mg/kg to about 10 mg/kg, and increased if necessary, up to about 20 mg/kg to about 30 mg/kg as either a single dose or divided doses, and of course depending on the patient's response.

Based on the data in mdx mice (see Examples 4 - 6), a single injection of 100 μg (10 mg/kg) of rhBGN^GAG" (recombinant human core biglycan protein) administered interperitoneally induced expression of components of the dystrophin complex including utrophiπ, and alpha, gamma and delta sarcoglycan when assessed three weeks later. Accordingly, dosages should be administered in humans on a weekly (one time per week), bi-weekly, tri-weekly or monthly basis (one time per month), preferably on a bi-weekly or tri-weekly basis (one dosage every two weeks or one dosage every three weeks) for between about one to about ten weeks, preferably between about two to about eight weeks, more preferably between about three to about seven weeks, and even more preferably for four, five or six weeks. It may be necessary to use dosages of the active ingredient outside the ranges disclosed herein or to administer the dosages either more frequently or less frequently than the periods disclosed or for the lengths of time disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. The clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with individual patient response.

In non-cell therapy and non-gene therapy based treatment regimens, the pharmaceutical compositions of the invention may be formulated in the conventional manner for protein therapeutics and will include one or more pharmaceutically acceptable vehicles, for example one or more carriers, excipients or diluents. Suitable carriers, excipients and diluents are well known to those skilled in the art, although non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition depends on factors known in the art including, for example, the way in which the active ingredient will be administered to a patient. The suitability of a particular excipient may also depend on the specific active ingredients. The pharmaceutical compositions of the invention may also include one or more compounds that reduce the rate by which an active ingredient will decompose. Such "stabilizers" include, for example, antioxidants such as ascorbic acid, pH buffers, or salt buffers, and are also known in the art.

The pharmaceutical compositions of the invention may be provided in a form for systemic, i.e. parenteral or depot, administration. The pharmaceutical compositions and dosage forms can be administered parenterally to patients by various routes including but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, or intra-arterial routes in the form of sterile solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, or in the form of emulsions. Solutions or suspensions for parenteral administration can include one or more of the following components: a sterile diluent such as water for injection, saline solutions, fixed oils, polyethylene glycols, propylene glycol, glycerin or other synthetic solvents, antibacterial agents, antioxidants, chelating agents, buffers, tonicity adjusting agents, or pH adjusting agents. Exemplary antibacterial agents suitable for protein therapeutics include benzyl alcohol and methyl parabens. Suitable antioxidants include ascorbic acid and sodium bisulfite. Suitable chelating agents include ehtyleneidaminetetreacetic acid. Suitable buffers include acetates, citrates and phosphates. Tonicity adjusters include sodium chloride and dextrose. pH adjusters include hydrochloric acid and sodium hydroxide. The parenteral formulation can be enclosed in ampoules, disposable syringes, or in multiple dose vials.

The pharmaceutical compounds may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions for water soluble proteins or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, and phosphate buffered saline. In each case, the composition must be sterile and should be fluid. It should be stable under conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms. The proper fluidity can be maintained with surfactants or lecithin. It may be preferable to include one or more isotonicity agents such as mannitol, sorbitol, or sodium chloride, and it may be advantageous to include an agent that delays absorption, for example aluminum monostearate or gelatin. Sterile injectable solutions are prepared by incorporating the required amount of a Class I SLRP protein in an appropriate solvent with one or a combination of carriers and excipients mentioned above, as required, followed by filtered sterilization. Dispersions can be prepared by incorporating the protein into a sterile vehicle that contains a basic dispersion medium and the required other carriers and excipients as chosen from those above-mentioned or already known in the art. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying.

Formulations for injection may be provided in single unit dosage forms, for example, in ampoules, or in multi-dose containers with an added preservative. The composition may be in the form of sterile liquid dosage forms, i.e. suspensions, solutions, colloids, or emulsions, in liposomal, oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents, that are suitable for parenteral administration to a patient. Alternatively, the compositions may take the form of sterile solids (e.g. crystalline or amorphous solids, or lyophilized compositions) that can be reconstituted with a suitable vehicle, such a sterile pyrogen-free water, to provide liquid dosage forms suitable for parenteral administration to a patient. The manner in which specific dosage form may vary and the factors indicative of the need for variance are well within the level of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 18^th ed., Mack Publishing, Easton, PA (1990).

In addition to the formulations described above, the pharmaceutical compositions of the invention may be formulated as depot preparations and administered by subcutaneous or intramuscular implantation or intramuscular injection. In this circumstance, the active ingredient(s) will be formulated with suitable polymeric or hydrophobic materials or ion exchange resins, or as sparingly soluble derivatives.

Additional suitable pharmaceutically acceptable vehicles are well known in the art and include, for example, Water for Injection USP, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or hanks solution. Additional suitable pharmaceutically acceptable vehicles include water-miscible vehicles, such as ethyl alcohol, polyethylene glycol, and polypropylene glycol, or non-aqueous vehicles, such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that increase the solubility of one or more of the active ingredients disclosed can also be incorporated as is known in the art.

In another aspect the invention comprises methods of treating a neuromuscular condition and disorder, including for example a dystrophy, a sarcoglycanopathy, or a biglycanopathy, by administering to a patient in need of treatment an effective amount of one or more of the pharmaceutical compositions of the invention.

These and other aspects of the invention are further described and illustrated by the following non-limiting examples.

Example 1: PRODUCTION OF RECOMBINANT BGN⁰^

Recombinant biglycan and recombinant non-GAG biglycan was produced using a stable transfected 293-EBNA cell line. The cell line was created by transferring a biglycan polyhistidine fusion construct originally created for expression using a vaccinia virus expression system into the pCEP4 (Invitrogen Corporation, Carlsbad, CA) expression vector. See U.S. Patent No. 6,864,236 and U.S. Patent Publication No. 2005/0059580. The resulting plasmid was transfected into 293- EBNA cells and stable expressing cells selected by culturing in the presence of hygromycin B. Following amplification, cells were seeded into a Celligen Plus bioreactor (New Brunswick Scientific, Edision, NJ) containing 100 g Fibra-Cel disks and grown to saturation. Protein production was initiated by replacing the culture media with serum free DMEM. Conditioned media was collected every 48 h with fresh media being added back to the bioreactor. Following concentration of the conditioned media using a Pellicon 2 Tangential Flow system (Millipore Corporation, Bedford, MA), recombinant non-GAG biglycan was purified using nickel chelating chromatography and eluted with a gradient of 0 — 250 mM imidazole in 20 mM Tris/HCL, 500 mM NaCl, 0.2% CHAPS, pH 8.0.

Biglycan polypeptide without the proteoglycan GAG side chains (BGN^GAG") was subsequently separated from biglycan polypeptide with the proteoglycan GAG side chains (BGN°^AG+) following anion exchange chromatography on Q-Sepharose and elution with a linear gradient of 0.15 - 2 M NaCl in PBS, 0.2% CHAPS. Both glycanated and non-glycanated forms were N-glycosylated. Example 2: ACTIVITY OF BGN^GA°- IN MYOTUBE CELL CULTURE SYSTEM

An immortalized line of mouse myoblasts deficient in biglycan expression was created using a temperature-sensitive large T antigen protocol following the method of Morgan et al. as described in "Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue- specific and mutation-specific cell lines," Developmental Biology 162: 486-98 (1994). These cells differentiate to form myotubes. After differentiation, the biglycan expression-deficient myotubes were treated for four (4) hours (i) with medium alone or (ii) with 0.7 mM of the purified recombinant BGN°^AG" produced in Example 1. The myotubes were then labeled and analyzed for nNOS expression and localization. In the medium treated control cells, nNOS is distributed and expressed throughout the cytoplasm. In the BGN^GAG* treated cells, nNOS expression was significantly more highly localized to the plasma membrane. These results demonstrate that BGN^GAG" treatment alone can induce the redistribution of a key DAPC signaling molecule to the myotube surface and demonstrate that BG!Nr ^AG" has agrin-independent signaling capabilities in muscle.

Example 3: ACTIVITY OF rhBGN^GAG" IN CELL CULTURE

A quantitative bioassay to determine the concentration ranges at which BGN^GAG" potentiates agrin-induced AChR clustering was undertaken. Biglycan null chick and mouse cells were grown to about 75% confluence on Perminox chamber slides (Nunc Nalgene, Naperville, IL) and allowed to differentiate into myotubes for 3 - 5 days. The primary chick and mouse myotubes were treated overnight with 1 Unit of recombinant rat agrin 12.4.8 (see Ferns et al., J. Cell. Biol. 132: 937-44 (1996)) and/or purified rhBGN^GΛG" (recombinant human core biglycan) from Example 1 at the concentrations of either 1.4 nM or 140 nM. The myotubes were labeled with rhodamine α-bungarotoxin for 30 minutes at 33° C and the cells were fixed with methanol for 5 min. at -20° C as described in Megeath et al., Neuroscience 122: 659- 68 (1998). AChR clusters greater than 4 μm in length and less than 1000 μm² were counted on a Nikon Eclipse 800 microscope. The results are shown in Figure 1. 1.4 mM BGN^^0' inhibited agrin-induced AChR clustering. See Figure IA. The addition of BGN^GAG' at any concentration tested (1.4 nM or 140 nM) did not increase the number of AChR clusters induced by maximal levels (5 Units) of agrin. See Figure IB.

The activity of biglycan (the proteoglycan including the GAG side chains) produced in the same cell system and purified under the same conditions (as described in Example 1) was also tested. In contrast to BGN^GΛG" , biglycan was inactive at all concentrations tested, from 0.14 nM to 200 nM (data not shown).

Example 4: SYSTEMIC DELIVERY OF BGN^GAG'

In dystrophic muscle, the integrity of the sarcolemma is not maintained due to the absence of dystrophin and the muscle degenerates over time. Increased serum creatine kinase levels indicate increased muscle degeneration and high serum creatine kinase levels are indicative of the muscle cell damage seen in muscular dystrophy.

Fourteen sixteen to nineteen day old mdx CTS/BL/lOScSn-Dmd¹^*/) mice (Jackson Laboratories) were divided into two groups of seven mice each. On postnatal day 19, the mice in Group I were injected intraperitoneally with either 100 μg (10 mg/kg, the "low dose") of BGN^GAG" in 500 μl of CHAPS or 500 μg (50 mg/kg, the "high dose") of rhBGN^GAG" in 500 μl of CHAPS. The mice in Group II were injected intraperitoneally with 500 μl of vehicle (CHAPS) alone as a control.

Two weeks post-administration, blood was collected and serum creatine kinase levels were measured (Units/L). The results are shown in Figure 2B. Administration of the pharmaceutical composition containing BGN^GΛG" reduced serum creatine kinase levels compared to the control by almost 4-fold at a dosage of 10 mg/kg and by almost 2-fold at a dosage of 50 mg/kg. In other words, low doses of BGN°^AG" exhibited a positive therapeutic effect, but that effect was reduced at higher dosage levels. At 50 mg/kg, no positive effect on serum creatine kinase levels was seen. Accordingly and surprisingly, administration of BGN^GAG" displays an inverted- U dosage response curve.

The mice were then euthanized and the diaphragm and quadriceps muscles were harvested and frozen. Frozen sections of the diaphragm muscles were cut and stained with Haemotoxylin and Eosin (Sigma Chemicals) (H&E). Mdx mouse myofibers undergo extensive death and regeneration starting a postnatal day 21 and regenerated myofibers in the mouse remain centrally-nucleated for the life of the animal. Tanabe et al., "Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse," Acta Neuropathol (Berl) 69: 91-95 (1986); Coulton et al., "The mdx mouse skeletal muscle myopathy: 1. A histological, morphometric and biochemical investigation," Neuropathol Appl Neurobiol 14: 53-70 (1988). Because myofibers with centrally-located nuclei are regenerated myofibers (i.e., regenerated following the death of the original myofiber), myofiber degeneration is quantitatively measurable. The results of the administration and analysis are shown in Figure 2A. BGN°^AG' administration decreased the percentage of myofibers with centrally-located nuclei from 15% in the control to 11% in the high dose group and to 5% in the low dose group. These results mirror the inverted-U dosage response curve results seen in the serum creatine kinase results.

Next, utrophin levels were investigated. Frozen quadriceps muscle from the Group I and Group II mice were sectioned, mounted on the same slide and immunostained with anti-utrophin antibodies. Twenty segments of sarcolemma from each group were analyzed. Utrophin levels were quantified with IP lab spectrum software (IP Scanalytics, Inc.) on a Nikon Eclipse 800 microscope in accordance with the manufacturer's instructions. The results of the low dose administration (i.e. at the 10 mg/kg dose) are shown in Figure 3. Utrophin expression at the sarcolemma in the treated mice was approximately 75% higher than in the control mice, indicating the BGN°^AO" treatment upregulates utrophin expression in dystrophin-deficient mice.

Example 5: EFFICACY OF BGN^GAG- COMPARED TO BYGLYCAN

Nine sixteen to nineteen day old mdx C75/BL/10ScSn-Dmd^MDX/j mice (Jackson Laboratories) were divided into three groups. On post-natal day 19, the mice in each group were injected intramuscularly (in the quadriceps) with (i) 50 mg/kg BGN^GAG- in 500 μl of CHAPS (labeled "Bgn core" in Figure 4), (ii) 50 mg/kg biglycan in 500 μl of CHAPS ("Bgn PG") or (iii) 500 μl of CHAPS vehicle alone ("vehicle"). Two weeks post-administration, the mice were euthanized and sacrificed, and the quadriceps muscles were harvested, sectioned, mounted on slides, and immunostained for α-dystrobrevin-1 ("α-DB-1") expression, α-dystrobrevin-2 ("α- DB-2") expression, βl-syntrophin ("β-lSyn") expression, and β2-syntrophin ("β- 2Syn") expression following the protocol described in Example 4 above.

The results are shown in Figure 4A - 4D, respectively. Both BGN^GAG" and biglycan were stably expressed in muscle for at least two weeks. However, only BGN°^AG" was capable or rescuing the expression of α-DB-1, α-DB-2, β-lSyn, and β- 2Syn, the components of the DAPC known to be involved in various dystrophies.

Example 6: SYSTEMIC ADMINISTRATION OF rhBGN^GAG" COUNTERS DYSTROPHIC PATHOLOGY AND UPREGULATES UTROPHIN

Littermate mdx mice were injected interperitoneally (i.p.) with vehicle or with 100 μg rhBGN^GAG" at postnatal day 18 (P18) and harvested at P38. Quadriceps femoris and diaphragms were isolated, frozen in freezing isopentane, mounted, sectioned (10 μm) and either immunostained or stained with H&E. In all experiments, muscle from the littermates was sectioned on the same day and mounted on the same slides. Muscle membrane preparations (30 μg) were run on a 5% SDS- PAGE and transferred to nitrocellulose membranes at IA for 1 h 45 min at 4 ° C. Membranes were washed in dd H₂O and incubated in Sypro Ruby protein blot stain (Invitrogen). Total protein staining was visualized on a Storm Imager (Amersham Bioscience, Piscataway, NJ). Membranes were washed in TBS, 0.1% Tween and blocked in TBS, 0.1% Tween-20, 4% milk, 5% goat serum for 1 h at room temperature. Membranes were incubated with anti-utrophin (N-terminus, VP-U579, Vector, Burlingame, CA) diluted 1:500 for 1 h, washed and incubated with sheep anti- mouse IgG conjugated to horseradish peroxidase (Amersham Bioscineces) diluted 1 :2000 for 1 h. Signal was detected with ECL plus (Amersham) using a Storm Imager. For immunohistochemistry, quadriceps sections were fixed in 1% paraformaldehyde for 5 min at room temperature and blocked in Vecor blocking reagent for 1 h at room temperature. The Vector M.O.M kit was used according to manufacture's protocol. Sections were mounted in Vectashield with DAPI (Vector) to label nuclei. Utrophin immunoreactivty intensity was quantified using Metamorph image analysis software (Universal Imaging Corp, Downingtown, PA). The average pixel intensities of 100 sarcolemmal segments from 2 mice from each condition were measured and the mean background (determined by measuring 100 non-sarcolemmal regions from each condition) was subtracted from them. The average background levels were indistinguishable between conditions.

For scoring the percentage of centrally-nucleated myofibers, all cross- sectioned myofibers greater than or equal tolOμm in diameter in H&E stained sections were counted under a 2OX objective. For morphometry, images were acquired with IP lab Spectrum software and the total muscle fiber area and area of mononuclear infiltration were measured using ImageJ (NIH, Bethesda, MD).

The results are shown in Figures 5 and 6. Figure 5A are photographic reproductions of H&E stained sections of diaphragm from the harvested mice. The right panels display a magnified view of the boxed regions. Extensive areas of regeneration and mononuclear cell infiltration in muscle from vehicle as compared to rhBGN⁰^^" treated mice can be seen whereas muscle from vehicle injected mice display characteristic dystrophic pathology including regenerated myofibers and mononuclear cell infiltration. Scale bars = 50 μm. Figure 5B is a histogram showing the percentages of centrally-nucleated myofibers determined from the H&E stained diaphragm sections. Strikingly, rhBGN^GAG" treated mice had 50% fewer centrally- nucleated myofibers as compared to vehicle-injected mice (n = number of animals; Student's paired t-test, p=0.047). Figure 5C is a histogram showing that muscle from rhBGN°^AG" treated mice showed a greater than 3.5 fold decrease in mononuclear cell infiltration as compared to vehicle-injected littermates (n = number of animals; Student's paired t-test, p = 0.035). This data indicates that systemic administration of rhBGN^GAG" improves myofiber health and counters dystrophic pathology in mdx mice.

As discussed in Example 4 above serum creatine kinase (sCK) levels are a marker of muscle damage. Accordingly, levels were measured from blood collected from anesthetized mice that had received 1 (at P16-19) or 2 (at P19 and 38) i.p. injections of 100 μg rhBGN^GAG" or vehicle as already described. Blood was collected by retro-orbital bleed, clotted, and analyzed for sCK levels by AniLytics (Gaithersburg, MD). The results are shown below in Table 1. Table I.

Number of Analysis age Days post

Experiment Treatment injections (Wk) injection' SCK(LVL)

1 Vehicle 1 5 14 34400

1 rhBGN 1 5 14 6212

2 Vehicle 1 5.5 20 14461

2 rhBGN 1 5.5 20 12776

2 rhBGN 1 5.5 20 6741

2 rhBGN 1 5.5 20 5055

3 Vehicle 1 5.5 23 48145

3 rhBGN 1 5.5 23 40900 4 Vehicle 2 9 25 58315

4 Vehicle 2 9 25 23056

4 rhBGN 2 9 25 22718

4 rhBGN 2 9 25 16732

These results indicate that a single injection resulted in reduced sCK levels up to three weeks post administration.

In both DMD patients and mdx mice, the loss of dystrophin leads to the disruption of its associated protein complex, an ensemble of membrane proteins that is essential for maintaining myofϊber integrity and preventing muscle cell death. The sparing of myofibers and the reduction in serum creatine kinase levels observed in the rhBGN^G animals suggested that this treatment may be working through the stabilization of a related complex. An attractive candidate for such rescue is utrophϊn, the autosomal homolog of dystrophin that can compensate for its loss of function in mdx mice. To confirm the upregulation of utrophin at the biochemical level, mdx mice were given two or three injections of 100 μg of rhBGN^GAG" per injection (in 25 μL 20 mM Tris, 0.5 M NaCl, 0.2% CHAPS) or 100 μg per injection of vehicle (20 mM Tris, 0.5 M NaCl, 0.2% CHAPS) at three week intervals starting at postnatal day 16. Mice were harvested 13-25 days after the final injection. Immunoblot analysis revealed that utrophin expression in muscle membrane fractions was increased in rhBGN°^AG" - injected mice as compared to control mice. Utrophin expression at the sarcolemma in diaphragm and quadriceps of these multiply-dosed animals was also increased as judged by immunostaining. These data also demonstrate that rhBGN°^AG" - induced upregulation of utrophin can be sustained for over two weeks.

Figure 6A are photographic reproductions of utrophin immunostained sectioned quadriceps muscle for littermate mice that received a single i.p. injection of either rhBGN^GAG' or vehicle at Pl 9 as described above. Shown is a region of peripherally-nucleated (i.e. non-regenerated) myofibers. rhBGN treatment increased sarcolemmal utrophin levels in these fibers. Scale bar=25μm. Images of quadriceps sections from mice (treated and stained as above) were acquired and the levels of immunostaining at the sarcolemma (arrows in a.) of non-regenerated fibers were measured. One hundred sarcolemmal segments from each of 4 animals were analyzed (2 littermate pairs, 1 rhBGN- and 1 vehicle- injected animal per pair). As shown in the histogram in Figure 6B, a single administration of rhBGN increased sarcolemmal utrophin expression over 2.5 fold (Student's unpaired t-test, p<0.0001). Figure 6C shown the results of multiple administration. Mdx mice from a single litter were injected at P16 and P38 (top panel) or P16, P38 and P63 (bottom panel) with rhBGN or vehicle. Muscles were harvested 3 weeks after the last injection. Membrane proteins (KCl-washed microsomes, 30μg) were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with utrophin antibody. Loading controls were obtained by SYPRO Ruby total protein stain of the blot.

In sum, the long-acting properties of systemically-delivered rhBGN^GΛG" in mice suggests that this therapeutic strategy could be practical for use in humans where treatment will likely be required for years.

The dependence upon utrophin of rhBGN^GAG" 's ability to counter dystrophic pathology in mdx mice was next tested using mice lacking both dystrophin and utrophin (mdx:utr ^~'^~) bred as described in Deconinck et al., "Utrophin-dystrophin- deficient mice as a model for Duchenne muscular dystrophy," Cell 90: 717-27 (1997). The doubly mutant mice were i.p. injected at P19 with 100 μg (about 12-18 mg.kg) rhBGN°^AG" (in 25 μL 20 mM Tris, 0.5 M NaCl, 0.2% CHAPS) or with 100 μg vehicle ((20 mM Tris, 0.5 M NaCl, 0.2% CHAPS). Diaphragms were isolated 3 weeks later, sectioned and stained with H&E. The characteristic extensive muscle pathology of these double knock out animals — areas of necrosis, mononuclear cell infiltration and centrally-nucleated myofibers — was comparable in rhBGN⁰¹*^0" and vehicle-injected animals. These results are shown in Figure 7. Scale bar = 100 μm.

Example 7: SYSTEMIC ADMINISTRATION OF rhBGN^GAG- IMPROVES MUSCLE FUNCTION IN MDX MICE

This example demonstrates that systemic administration of recombinant human non-GAG BGN (rhBGN^GAG') improves muscle function in mdx mice and is therefore a therapeutically efficacious treatment for DMD (Duchenne's muscular dystrophy), mdx mice were injected intraperitoneally with rhBGN^GAG" (25μg/animal) or vehicle every 3 weeks starting at post-natal day 14 (8 mice per condition) and the physiological properties of the EDL muscles were analyzed ex vivo at 3 months of age as described in Bogdanovich et al., "Functional improvement of dystrophic muscle by myostatin blockade," Nature 420:418-21 (2002) and in Bogdanovich et al., "Myostatin propeptide-mediated amelioration of dystrophic pathophysiology," FASEB J 19:543-9 (2005). The mdx EDL is susceptible to damage caused by lengthening contractions, resulting in diminished force generation after a series of ECCs. See Moens et al., "Increased susceptibility of EDL muscle from mdx mice to damage induced by contractions with stretch," J Muscle Res Cell Motil. 14:446-51 (1993). After the rhBGN^GAG" treatment, a 30% improvement in performance on eccentric contraction measurements was observed when comparing the force drop after the first vs. the fifth ECC (P<0.05; n=16 muscles/condition; Student's t test).

Detailed results are shown in Figure 8A - 5D. Representative first to fifth ECCs of EDL muscles from mdx mice injected with vehicle are shown in panel(a) and rhBGN are shown in panel(b). In panel (c), the average force drop between the first and fifth ECC in rhBGN and vehicle-treated mdx mice is shown (14.9±1.2% vs. 22.2±2.7%, respectively; p<0.05; n=16 muscles in each group; Student's t test). In panel (d), comparisons of ECC force drop between first and second, third, fourth and fifth ECC of control (6.4±1.2%; 12.4±1.9%; 18.4±2.3%; 22.2±2.7%; n=16) and rhBGN treated (3.9±0.3%; 7.5±0.5%; 11.6±0.8%; 14.9±1.2%; n=16) mdx mice are shown, respectively. There is significant (p<0.05; Student's t test) force drop between ECCs of vehicle and rhBGN treated mdx mice on the 3rd, 4th, and 5th contractions. Graphs are presented with ±SEM. These physiological findings, taken together with the histological and sCK measures presented above, demonstrate that rhBGNr ^{A "} is effective in preclinical models of DMD.

SUMMARY OF EXPERIMENTAL RESULTS

The foregoing results demonstrate a novel therapeutic strategy for the treatment of dystrophies based upon the systemic delivery of rhBGN^GΛG\ Several characteristics of this composition indicate that is can be an effective therapy. First, it can be delivered systemically and a single dose is active for at least two weeks. Multiple doses at three week intervals can sustain the response for at least two months. Second, with regard specifically to the treatment of DMD, the compound might selectively target the tissues affected in DMD since it binds to α- and γ- sarcoglycan, which are components of the dystrophin/utrophin protein complex and are expressed exclusively in heart and skeletal muscle. See Hack et al., "Sarcoglycans in muscular dystrophy," Microsc Res Tech 48: 167-80 (2000). Third, endogenous biglycan is expressed in normal and DMD muscle (see Haslett et al., supra, Proc Natl Acad Sci USA 99: 15000-15005 (2002); Zanotti et al., supra. Brain 128: 2546-55 (2005)) and is a highly conserved protein; the composition could thus be expected to elecit a minimal immune response. Fourth, the composition is non-glycanated so it can be readily manufactured in a homogeneous form. Lastly, the composition acts, at least in part, through the upregulation of utrophin, a therapeutic strategy that has been extensively validated in animal studies. See Tinsley et al., Expression of full-length utrophin prevents muscular dystrophy in mdx mice," Nat Med 4: 1441-44 (1998); Ebihara et ah, Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice," Physiol Genomics 3: 133-44 (2000); and Cerletti et al., Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer," Gen Ther 10: 750-57 (2003).

All documents and literature referenced in this specification, including patent and scientific literature, are hereby incorporated by reference for the substance of their disclosure as if they were reproduced in their entirety in this specification.

Claims

We claim:

1. A pharmaceutical composition comprising a therapeutically effective amount of a non-GAG class I SLRP protein in admixture with a pharmaceutically acceptable vehicle.

2. The composition of claim 1 wherein the pharmaceutical composition is an aqueous parenteral composition.

3. The composition of claim 1 wherein the non-GAG Class I SLRP protein is selected from BGN°^AG- , DCN^GAG\ and asporin.

4. The composition of claim 2 wherein the non-GAG Class I SLRP protein is BGN°^AG\

5. The composition of claim 1 wherein the therapeutically effective amount of protein is from about 0.10 mg/kg to about 500 mg/kg body weight.

6. The composition of claim 5 wherein the therapeutically effective amount of protein is from about 1.0 mg/kg to about 100 mg/kg body weight.

7. The composition of claim 6 wherein the therapeutically effective amount of protein is from about 1.0 mg/kg to about 50 mg/kg body weight.

8. A method of managing, treating or preventing a neuromuscular disorder or condition in a mammal comprising administering a therapeutically effective amount of a pharmaceutical composition containing a non-GAG Class I SLRP protein to said mammal.

9. The method of claim 8 wherein the mammal is a human.

10. The method of claim 9 wherein the neuromuscular disorder or condition is selected from a dystrophy, a sarcoglycanopathy or a biglycanopathy.

11. The method of claim 10 wherein the dystrophy is selected from myotonic dystrophy (Steinert's disease; DM), spinal muscular atrophy (SMA), Duchenne muscular dystrophδy (DMD), Becker's muscular dystrophy (BMD), congenital muscular dystrophy (CMD), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, Fukuyama congenital muscular dystrophy (FCMD), limb-girdle muscular dystrophy (LGMD), oculopharyngeal muscular dystrophy, scapuloperoneal muscular dystrophy, and sever childhood autosomal recessive muscular dystrophy.

12. The method of claim 9 wherein the neuromuscular disorder or condition is a cardiac myopathy.

13. The method of claim 9 wherein the neuromuscular disorder or condition is selected form muscle wasting, disuse atrophy, sarcopaenia, and cachexia.

14. The method of claim 9 wherein the non-GAG Class I SLRP protein is selected from BGN⁰*^0" , DCN^GAG\ and asporin.

15. The method of claim 14 wherein the non-GAG Class I SLRP protein is _BGNGAG-

16. The method of claim 11 wherein the non-GAG Class I SLRP protein is selected from BGN^GAG" , DCN^GAG", and asporin.

17. The method of claim 16 wherein the non-GAG Class I SLRP protein is _BGNGAG-

18. The method of claim 8 wherein the pharmaceutical composition containing a non-GAG Class I SLRP protein is co-administered with another therapeutic agent.

19. The method of claim 18 wherein the therapeutic agent is a different non-GAG Class I SLRP protein.

20. The method of claim 18 wherein the therapeutic agent is selected form myostatin, 1-arginine, dystrophin, utrophin and PTC-124.