WO2007123848A2 - Préparations thérapeutiques contenant des protéines slrp de classe i modifiées - Google Patents

Préparations thérapeutiques contenant des protéines slrp de classe i modifiées Download PDF

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WO2007123848A2
WO2007123848A2 PCT/US2007/009091 US2007009091W WO2007123848A2 WO 2007123848 A2 WO2007123848 A2 WO 2007123848A2 US 2007009091 W US2007009091 W US 2007009091W WO 2007123848 A2 WO2007123848 A2 WO 2007123848A2
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gag
protein
bgn
class
slrp
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PCT/US2007/009091
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WO2007123848A3 (fr
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Justin R. Fallon
Beth Mckechnie
Alison Rosalind Amenta
Mary Lynn Mercado
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Brown University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • A61K38/1841Transforming growth factor [TGF]

Definitions

  • 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.
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy
  • LGMD limb-girdle muscular dystrophies
  • 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).
  • DAPC distrophin-associated protein complex
  • DGC dystrophin-glycoprotein complex
  • 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.
  • SLRP small leucine-rich repeat proteoglycan
  • 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.
  • Biglycan and decorin are suspected of playing a role in muscular dystrophies.
  • biblycan is over-expressed in an animal model of muscular dystrophy that is characterized by the absence of dystrophin.
  • 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 ⁇ -dystroglycan and Is Upregulated in Dystrophic Muscle," J. Cell. Biol.
  • 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.
  • 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).
  • AChR acetylcholine receptor
  • 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.
  • GAG glycanated
  • 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.
  • the compositions include therapeutically effective amounts of a non-glycanated (without GAG chains) Class I SLRP protein, for example, BGN 0 * 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).
  • 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.
  • the treatment methods include methods of treating acquired (toxic or inflammatory) myopathies.
  • 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.
  • DM myotonic dystrophy
  • SMA spinal
  • 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.
  • cardiac myopathies e.g., hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, welander distal myopathy
  • 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.
  • 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.
  • 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.
  • IP intraperitoneally
  • EVl intramuscularly
  • 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.
  • 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.
  • 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, B 5 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.
  • FIGS 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.
  • 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.”
  • glycoprotein is a protein that contains one or more carbohydrate groups covalently attached to the polypeptide chain.
  • a glycoprotein contains from 1% to 60% carbohydrate by weight in the form of numerous, relatively short, branched oligosaccharide chains of variable composition.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 “BGN 0 ⁇ " " include the mature polypeptide sequence (including N-terminal glycosylation), and either including or excluding the propeptide region.
  • decorin core refers to a decorin protein that does not include GAG chains.
  • Decorin core non-GAG decorin, non-GAG DCN, and DCN 0 * 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.
  • 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.
  • 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.
  • prevent refers 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.
  • treat refers 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, N 5 N'- 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.
  • inorganic and organic acids such as acetic, alginic, anthranilic, genzenesulfonic, benzoic, camphorsulf
  • 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).
  • 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.
  • such compositions include a therapeutically effective amount of a non-GAG Class I SLRP protein in a pharmaceutically acceptable vehicle.
  • 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.
  • 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.
  • 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.
  • 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 myostat ⁇ n blocking molecules, calpain inhibitors such as Myodur (Ceptor Corp,,), PTC 124 (PTC Therapeutics), 1-arginine, dystrophin, and/or utrophin (via gene therapy, for example).
  • anti-myostatin antibodies for example MYO-029, Wyeth Pharmaceuticals
  • other myostat ⁇ n blocking molecules for example, myostat ⁇ n blocking molecules, calpain inhibitors such as Myodur (Ceptor Corp,,), PTC 124 (PTC Therapeutics), 1-arginine, dystrophin, and/or utrophin (via gene therapy, for example).
  • 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.
  • 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.
  • compositions of the invention may also be administered in a cell based therapy or a gene therapy regimen.
  • 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.
  • dystrophies sarcoglycanopathies, biglycanopathies
  • 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).
  • DNA liposome mixtures 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.
  • 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.
  • 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).
  • 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.
  • 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 ED 50 (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 ED 5 0 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • rhBGN GAG recombinant human core biglycan protein
  • 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.
  • 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.
  • compositions of the invention may also include one or more compounds that reduce the rate by which an active ingredient will decompose.
  • stabilizers include, for example, antioxidants such as ascorbic acid, pH buffers, or salt buffers, and are also known in the art.
  • 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.
  • a sterile diluent such as water for injection, saline solutions, fixed oils, polyethylene glycols, propylene glycol, glycerin or other synthetic solvents
  • 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 hydro
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a suitable vehicle such as a sterile pyrogen-free water
  • 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).
  • compositions of the invention may be formulated as depot preparations and administered by subcutaneous or intramuscular implantation or intramuscular injection.
  • 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.
  • 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.
  • a neuromuscular condition and disorder including for example a dystrophy, a sarcoglycanopathy, or a biglycanopathy
  • 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.
  • 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
  • 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.
  • rhBGN G ⁇ G purified rhBGN G ⁇ G" (recombinant human core biglycan) from Example 1 at the concentrations of either 1.4 nM or 140 nM.
  • the myotubes were labeled with rhodamine ⁇ -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 ⁇ m in length and less than 1000 ⁇ m 2 were counted on a Nikon Eclipse 800 microscope. The results are shown in Figure 1.
  • biglycan the proteoglycan including the GAG side chains
  • BGN G ⁇ G the proteoglycan including the GAG side chains
  • mice Fourteen sixteen to nineteen day old mdx CTS/BL/lOScSn-Dmd 1 ⁇ */ 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 ⁇ g (10 mg/kg, the "low dose") of BGN GAG" in 500 ⁇ l of CHAPS or 500 ⁇ g (50 mg/kg, the "high dose") of rhBGN GAG" in 500 ⁇ l of CHAPS. The mice in Group II were injected intraperitoneally with 500 ⁇ l of vehicle (CHAPS) alone as a control.
  • CHAPS vehicle
  • 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.
  • 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.
  • IP Scanalytics, Inc. IP Scanalytics, Inc.
  • mice 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 ⁇ l of CHAPS (labeled "Bgn core” in Figure 4), (ii) 50 mg/kg biglycan in 500 ⁇ l of CHAPS ("Bgn PG") or (iii) 500 ⁇ l of CHAPS vehicle alone ("vehicle").
  • mice Two weeks post-administration, the mice were euthanized and sacrificed, and the quadriceps muscles were harvested, sectioned, mounted on slides, and immunostained for ⁇ -dystrobrevin-1 (" ⁇ -DB-1") expression, ⁇ -dystrobrevin-2 (“ ⁇ - DB-2”) expression, ⁇ l-syntrophin (“ ⁇ -lSyn”) expression, and ⁇ 2-syntrophin (“ ⁇ - 2Syn”) expression following the protocol described in Example 4 above.
  • ⁇ -DB-1 ⁇ -dystrobrevin-1
  • ⁇ -DB-2 ⁇ -dystrobrevin-2
  • ⁇ -lSyntrophin ⁇ -lSyn
  • ⁇ - 2Syn ⁇ 2-syntrophin
  • Littermate mdx mice were injected interperitoneally (i.p.) with vehicle or with 100 ⁇ g rhBGN GAG" at postnatal day 18 (P18) and harvested at P38.
  • Quadriceps femoris and diaphragms were isolated, frozen in freezing isopentane, mounted, sectioned (10 ⁇ m) and either immunostained or stained with H&E.
  • muscle from the littermates was sectioned on the same day and mounted on the same slides.
  • Muscle membrane preparations (30 ⁇ g) 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 H 2 O 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.
  • Figure 5B is a histogram showing the percentages of centrally-nucleated myofibers determined from the H&E stained diaphragm sections.
  • 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 ⁇ g 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.
  • dystrophin leads to the disruption of its associated protein complex, an ensemble of membrane proteins that is essential for maintaining myof ⁇ ber 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 utroph ⁇ n, the autosomal homolog of dystrophin that can compensate for its loss of function in mdx mice.
  • mice were given two or three injections of 100 ⁇ g of rhBGN GAG" per injection (in 25 ⁇ L 20 mM Tris, 0.5 M NaCl, 0.2% CHAPS) or 100 ⁇ g 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.
  • Membrane proteins (KCl-washed microsomes, 30 ⁇ g) 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.
  • mice lacking both dystrophin and utrophin mdx:utr ⁇ ' ⁇ mice 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.
  • Example 7 SYSTEMIC ADMINISTRATION OF rhBGN GAG - IMPROVES MUSCLE FUNCTION IN MDX MICE
  • FIG. 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).
  • composition acts, at least in part, through the upregulation of utrophin, a therapeutic strategy that has been extensively validated in animal studies.
  • utrophin a therapeutic strategy that has been extensively validated in animal studies.
  • 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).

Abstract

L'invention porte sur des préparations thérapeutiques et des méthodes de traitement et prévention des premiers signes de troubles, états ou maladies musculaires spécialement chez l'homme dont par exemple les dystrophies musculaires et en particulier la DMD et la BMD. Lesdites préparations thérapeutiques comportent une quantité efficace d'une ou plusieurs protéines SLRP non glycanées de classe I, seules ou mélangées à un véhicule pharmacocompatible. Les préparations pharmaceutiques thérapeutique de l'invention peuvent s'administrer seule ou associée entre elles et/ou combinées à d'autre préparations thérapeutiques.
PCT/US2007/009091 2006-04-19 2007-04-13 Préparations thérapeutiques contenant des protéines slrp de classe i modifiées WO2007123848A2 (fr)

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WO2010106295A1 (fr) * 2009-03-18 2010-09-23 Genethon Utilisation de la décorine pour augmenter la masse musculaire
WO2011146480A1 (fr) 2010-05-17 2011-11-24 Brown University Mutants de biglycanes et produits thérapeutiques associés et méthodes d'utilisation
US8486982B2 (en) 2003-04-11 2013-07-16 Ptc Therapeutics, Inc. 1,2,4-oxadiazole benzoic acids
US8658596B2 (en) 1999-11-18 2014-02-25 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US8691766B2 (en) 1999-11-18 2014-04-08 Brown University Research Foundation Biglycan and related therapeutics and methods of use
JP2014508920A (ja) * 2010-12-27 2014-04-10 ブラウン ユニバーシティ ビグリカンおよびユートロフィンに関する治療法および診断法
US8822418B2 (en) 2001-08-15 2014-09-02 Brown University Treatment of muscular dystrophies and related disorders
US9873677B2 (en) 2014-03-06 2018-01-23 Ptc Therapeutics, Inc. Pharmaceutical compositions and salts of a 1,2,4-oxadiazole benzoic acid
US10214573B2 (en) 2012-10-10 2019-02-26 Tivorsan Pharmaceuticals, Inc. Biglycan variant polypeptides and methods of use
US10517853B2 (en) 2015-10-30 2019-12-31 Ptc Therapeutics, Inc. Methods for treating epilepsy

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US8658596B2 (en) 1999-11-18 2014-02-25 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US8691766B2 (en) 1999-11-18 2014-04-08 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US8822418B2 (en) 2001-08-15 2014-09-02 Brown University Treatment of muscular dystrophies and related disorders
US10071081B2 (en) 2003-04-11 2018-09-11 Ptc Therapeutics, Inc. Compositions of 1,2,4-oxadiazole benzoic acid compounds and methods for their use
US8486982B2 (en) 2003-04-11 2013-07-16 Ptc Therapeutics, Inc. 1,2,4-oxadiazole benzoic acids
US9861617B2 (en) 2003-04-11 2018-01-09 Ptc Therapeutics, Inc. Compositions of 1,2,4-oxadiazole benzoic acid compounds and methods for their use
US8796322B2 (en) 2003-04-11 2014-08-05 Ptc Therapeutics, Inc. Methods for using 1,2,4-oxadiazole benzoic acid compounds
US8975287B2 (en) 2003-04-11 2015-03-10 Ptc Therapeutics, Inc. Methods for using 1,2,4-Oxadiazole benzoic acid compounds
US9205088B2 (en) 2003-04-11 2015-12-08 Ptc Therapeutics, Inc. Compositions of 1,2,4-oxadiazol benzoic acid compounds and methods for their use
FR2943249A1 (fr) * 2009-03-18 2010-09-24 Genethon Utilisation de la decorine pour augmenter la masse musculaire
WO2010106295A1 (fr) * 2009-03-18 2010-09-23 Genethon Utilisation de la décorine pour augmenter la masse musculaire
US9474782B2 (en) 2009-03-18 2016-10-25 Association Francaise Contre Les Myopathies Use of decorin for increasing muscle mass
EP2571514A4 (fr) * 2010-05-17 2013-11-06 Univ Brown Mutants de biglycanes et produits thérapeutiques associés et méthodes d'utilisation
EP2571514A1 (fr) * 2010-05-17 2013-03-27 Brown University Mutants de biglycanes et produits thérapeutiques associés et méthodes d'utilisation
WO2011146480A1 (fr) 2010-05-17 2011-11-24 Brown University Mutants de biglycanes et produits thérapeutiques associés et méthodes d'utilisation
US9969785B2 (en) 2010-05-17 2018-05-15 Brown University Biglycan mutants and related therapeutics and methods of use
EP2659271A4 (fr) * 2010-12-27 2015-04-08 Univ Brown Méthodes thérapeutiques et diagnostiques impliquant le biglycane et l'utrophine
US9958458B2 (en) 2010-12-27 2018-05-01 Brown University Therapeutic and diagnostic methods involving biglycan and utrophin
EP3026432A3 (fr) * 2010-12-27 2016-07-27 Brown University Procédé de prévision de la réponse du patient au traitement avec biglycan
JP2014508920A (ja) * 2010-12-27 2014-04-10 ブラウン ユニバーシティ ビグリカンおよびユートロフィンに関する治療法および診断法
US10214573B2 (en) 2012-10-10 2019-02-26 Tivorsan Pharmaceuticals, Inc. Biglycan variant polypeptides and methods of use
US9873677B2 (en) 2014-03-06 2018-01-23 Ptc Therapeutics, Inc. Pharmaceutical compositions and salts of a 1,2,4-oxadiazole benzoic acid
US10233161B2 (en) 2014-03-06 2019-03-19 Ptc Therapeutics, Inc. Pharmaceutical compositions and salts of a 1,2,4-oxadiazole benzoic acid
US10618877B2 (en) 2014-03-06 2020-04-14 Ptc Therapeutics, Inc. Pharmaceutical compositions and salts of a 1,2,4-oxadiazole benzoic acid
US10517853B2 (en) 2015-10-30 2019-12-31 Ptc Therapeutics, Inc. Methods for treating epilepsy

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