Methods for preventing or treating fatty degeneration of skeletal muscle

Abstract

Pharmaceutical compositions comprising selective TGF-β inhibitors, such as TGFβRII-Fc, are provided herein to prevent, reverse, reduce the occurrence of, and / or treat skeletal muscle fatty degeneration. Additionally, selective TGF-β inhibitors can be used to inhibit the differentiation of fibroadipogenic progenitor cells (FAPs) into adipocytes.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 879,009, filed July 26, 2019. The contents of U.S. Provisional Patent Application No. 62 / 879,009 are incorporated herein by reference in their entirety.

[0002] [U.S. Government Rights] This invention was made with government support under GM121499 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present invention relates generally to selective TGF-β inhibitors, pharmaceutical compositions thereof, and uses thereof for preventing and / or treating skeletal muscle fatty degeneration.

[0004] BACKGROUND OF THE INVENTION The background discussion includes information that may be useful in understanding the present disclosure. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] The transforming growth factor beta (TGF-β) family is a well-known family of structurally related proteins that regulate proliferation, differentiation, developmental regulation, and other functions in many cell types. The TGF-β family includes the canonical TGF-β, which forms a group of three isoforms: TGF-β1, TGF-β2, and TGF-β3. The three isoforms, TGF-β1-3, have long been known to have distinct functions. They are differentially expressed in various tissues and at different times during development.

[0006] Fatty degeneration (FD) of muscle tissue can occur following injury, reparative surgery such as rotator cuff (RC) repair, and other conditions. Various approaches (e.g., monoclonal antibodies and small molecules) have been used to attempt to inhibit TGF-β signaling. In particular, targeting the TGF-β pathway with a small molecule inhibitor (SB431542) has been reported to reduce FD in the associated muscles in a mouse model of severe RC rupture. However, SB431542 is unlikely to be effective clinically due to its broad specificity and poor pharmacological properties.

[0007] Additionally, a fusion protein comprising a TGF-β type II receptor linked to a portion of an immunoglobulin constant region ("TGFβRII-Fc") has been reported in published international patent application WO1998 / 048024 and U.S. Pat. No. 9,809,637.

[0008] Thus, although various selective TGF-β inhibitors are known in the art, there are currently no treatments that prevent or alleviate muscle FD in humans. Therefore, there is a need to provide improved compositions and methods for preventing and / or treating FD.

[0009] Summary of the Invention Various compositions and methods for preventing, reversing, reducing the incidence of, and / or treating FD in skeletal muscle using selective TGF-β inhibitors are described herein and include administering a therapeutically effective amount of a selective TGF-β inhibitor to a subject in need thereof.

[0010] In certain embodiments, the selective TGF-β inhibitor is administered to a subject before, during, or after surgery, such as surgery to repair RC.

[0011] In a further embodiment, also described herein is a method for inhibiting differentiation of fibroadipogenic progenitor cells (FAPs) into adipocytes, comprising contacting the intercellular compartment surrounding the FAP cells with an effective amount of a selective TGF-β inhibitor.

[0012] In a specific embodiment, the selective TGF-β inhibitor is TGFβRII-Fc as described herein.

[0013] In a further specific embodiment, the selective TGF-β inhibitor is TGFβRII-Fc derived from an animal species, and the TGFβRII-Fc is used to prevent, reverse, reduce the incidence of, and / or treat FD in the same animal species.

[0014] Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like elements and in which:

[0015] BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1B show two naturally occurring splice forms of TGFβRII: the native short form and the native long form. Figure 1A shows that the long form has a 25 amino acid insertion after amino acid 31, and the amino acid following this insertion changes from Val to Ile (shown in bold). Figure 1B shows the sequences of the extracellular domain (ECD) of both forms of TGFβRII. Uniprot sequence P37173 (SEQ ID NO: 1 herein) is the short form, and Uniprot sequence D2JYI1 (SEQ ID NO: 2 herein) is the long form.

[0016] FIG. 1C is the amino acid sequence of TGFβRII-Fc (SEQ ID NO: 3) used in Examples 1 to 3 herein.

[0017] Figure 2 shows the binding specificity of TGFβRII-Fc. The figure is an image of an SPR-sensorgram showing that TGFβRII-Fc (SEQ ID NO: 3) binds to TGF-β1 and TGF-β3, but not to TGF-β2. See Aykul, S. and Martinez-Hackert, E. Transforming Growth Factor-β family ligands can function as antagonists by competing for type II receptor binding. Journal of Biological Chemistry 291: 10792-10804 (2016). The publication is incorporated herein by reference in its entirety.

[0018] Figures 3A-3M show the inhibition of fibroadipogenic 3T3-L1 progenitor cell differentiation into adipocytes by TGFβRII-Fc. Figure 3A is a schematic diagram showing how fibroadipogenic progenitor cells (FAPs) differentiate into adipocytes. See Aykul, S., Maust, J., Floer, M., and Martinez-Hackert, M. TGF-β Family Inhibitors Blunt Adipogenesis Via Non-Canonical Regulation of SMAD Pathways. bioRxiv. (Published online March 14, 2020). The publication is incorporated herein by reference in its entirety. Figures 3B-3G show immunofluorescence analysis of 3T3-L1 cells grown in adipocyte differentiation medium for 8 days with vehicle control (PBS) or 300 nM TGFβRII-Fc. Figure 3B shows images of cells stained for lipids with Nile Red (green) and nuclei with DAPI (magenta). Figure 3C shows quantification of droplet number, Figure 3D shows average lipid droplet size, Figure 3E shows average lipid area, Figure 3F shows normalized Nile Red fluorescence, and Figure 3G shows lipolytic activity. Hatched bars represent vehicle control, while white bars represent TGFβRII-Fc-treated cells. Figures 3H-3M show gene expression analysis of 3T3-L1 cells grown in differentiation medium for different lengths of time. Induction of adipocyte marker gene expression was analyzed by qRT-PCR in vehicle control (hatched bars) and TGFβRII-Fc-treated cells (white bars) on days 0, 3, and 8. Data are shown as fold induction after normalization to Rpl4; Figure 3H shows Adipoq, Figure 3I shows Cidec, Figure 3J shows Fabp4, Figure 3K shows Lep, Figure 3L shows Plin1, and Figure 3M shows Pparg. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. * p<0.05; ** p<0.01; *** p<0.001;**** p<0.0001).

[0019] Figures 4A-4J show the suppression of fat infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figure 4A shows a photograph of a muscle sectioned and stained with hematoxylin (blue) followed by Oil Red O (ORO). ORO detects intramuscular fat (FI) resulting from lipid droplets formed within muscle fibers, as well as intermyocyte fat (FI) resulting from adipocytes growing between muscle fibers. Figure 4B shows quantification of total fat, Figure 4C shows quantification of intermyocyte fat, and Figure 4D shows quantification of intramyocyte fat from mouse quadriceps muscle (n=3). Figures 4E-4J show gene expression analysis of quadriceps muscle. Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are shown as fold expression relative to diet-fed mice; Figure 4E shows the adipocyte marker gene Adipoq, Figure 4F shows the adipocyte marker gene Cidec, Figure 4G shows the adipocyte marker gene Fabp4, Figure 4H shows the adipocyte marker gene Lep, Figure 4I shows the adipocyte marker gene Plin1, and Figure 4J shows the adipocyte marker gene Pparg. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. * p<0.05; ** p<0.01) Figures 5A-5C show the inhibition of muscle atrophy and FI in shoulder muscles after induced RC injury by TGFβRII-Fc. Figure 5A shows quantification of wet weight loss values ​​for the supraspinatus (SS) and infraspinatus (IS) muscles from appropriate animals (n=6). Muscles on the injured side were compared to muscles on the sham-operated side of the same mice. Data are expressed as the % reduction in muscle weight after induced RC injury by comparing sham-operated muscles to injured (Figure 5B). * p<0.05; ***p<0.001). Figures 5B and 5C show gene expression analysis of shoulder muscles (n=4). Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are presented as gene expression in injured muscles compared to sham-operated muscles; Figure 5B shows Cidec and Figure 5C shows Lep. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. * p<0.05).

[0020] Detailed Description I. Definition The following definitions refer to various terms used above and throughout this disclosure.

[0021] As used herein, the term "selective TGF-β inhibitor or antagonist" refers to a polypeptide that inhibits TGF-β1 and / or TGF-β3 signaling, but does not substantially inhibit TGF-β2 signaling.

[0022] There are two splice forms of TGFβRII, the naturally occurring TGFβRII short form (Uniprot sequence P37173, corresponding to SEQ ID NO: 1 herein), and the naturally occurring TGFβRII long form (Uniprot sequence D2JYI1, corresponding to SEQ ID NO: 2 herein). The long form is a splice variant with a 25 amino acid insertion after amino acid 31, changing the amino acids adjacent to the insertion from Val to Ile (Figure 1).

[0023] As used herein, a "TGFβRII ECD polypeptide fusion" comprises a polypeptide of the extracellular domain ("ECD") of TGFβRII and a heterologous sequence. The TGFβRII portion comprises a polypeptide sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the amino acid sequence set forth as amino acids 23-166 of SEQ ID NO:1; or at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the amino acid sequence set forth as amino acids 23-191 of SEQ ID NO:2, wherein the polypeptide is capable of binding to TGF-β1 and / or TGF-β3, but is substantially incapable of binding to TGF-β2.

[0024] A TGFβRII ECD polypeptide fusion comprises a TGFβRII polypeptide, wherein the TGFβRII comprises a polypeptide sequence beginning at any of positions 23 to 51 of SEQ ID NO: 1 and ending at any of positions 143 to 166 of SEQ ID NO: 1, or a polypeptide sequence beginning at any of positions 23 to 76 of SEQ ID NO: 2 and ending at any of positions 169 to 191 of SEQ ID NO: 2, and the polypeptide is capable of binding to TGF-β1 and / or TGF-β3.

[0025] The heterologous sequence portion of a TGFβRII ECD polypeptide fusion can be the constant domain ("Fc") of human IgG or albumin. When the TGFβRII portion is fused to the Fc of a human immunoglobulin, IgG, the resulting fusion protein is known as "TGFβRII-Fc." In other words, TGFβRII-Fc comprises or consists of the extracellular domain of a TGFβRII receptor, as described above, fused to the Fc of a human IgG. The human IgG portion in TGFβRII-Fc can consist of the Fc domain of human IgG1, IgG2, or IgG4. The TGFβRII portion can be fused to the Fc domain at the N-terminus or C-terminus via a linker having various lengths and amino acid sequences, but is preferably fused without any secondary structure. TGFβRII-Fc can contain either the short or long form of TGFβRII, and fusions containing either splice form have similar binding specificity for TGF-β1 and / or TGF-β3; and they do not substantially bind to TGF-β2.

[0026] TGFβRII-Fc is a known fusion protein. For example, WO1998 / 048024 reports TGFβRII-Fc and a method for producing TGFβRII-Fc, in which a short form of TGFβRII is fused with the Fc of human IgG1. WO1998 / 048024 is incorporated herein by reference in its entirety. Furthermore, U.S. Patent No. 9,809,637 reports TGFβRII-Fc using a long form of TGFβRII. U.S. Patent No. 9,809,637 is also incorporated herein by reference in its entirety. TGFβRII-Fc is an inhibitor of TGF-β1 and / or TGF-β3. However, it does not substantially bind to / inhibit TGF-β2. See Figure 2, adapted from Aykul, S. and Martinez-Hackert, E. Transforming Growth Factor-β family ligands can function as antagonists by competing for type II receptor binding. Journal of Biological Chemistry 291: 10792-10804 (2016) (cited above). Without being bound by theory, it is a so-called "ligand trap" that acts by trapping the ligands TGF-β1 and / or TGF-β3 in the intracellular compartment. This prevents the ligands from interacting with endogenous TGFβRII receptors, thus inhibiting downstream signaling. Therefore, TGFβRII-Fc can be used as a selective antagonist of TGF-β1 and / or TGF-β3, but not substantially as a selective antagonist of TGF-β2.

[0027] In certain embodiments, TGFβRII-Fc has at least 80%, at least 85%, at least 90%, or at least 95% sequence similarity to the amino acid sequence set forth as SEQ ID NO: 3. See Figure 1C. In further specific embodiments, TGFβRII-Fc comprises or consists of the amino acid sequence set forth as SEQ ID NO: 3.

[0028] The terms "treat" and "treatment" refer to a method for reducing, inhibiting, or otherwise ameliorating FD by administering a therapeutically effective amount of a selective TGF-β inhibitor.

[0029] The term "fatty degeneration" ("FD") refers to the abnormal formation of fatty tissue between muscle fibers and associated muscle wasting, which can occur as a result of injury or disease / disorder.

[0030] The term "fatty infiltration" ("FI") refers only to the abnormal formation of adipose tissue.

[0031] The term "administering" refers to both direct and indirect administration of a pharmaceutical composition or drug, where direct administration of a pharmaceutical composition or drug is typically performed by a medical professional (e.g., a doctor, a nurse, etc.), and indirect administration includes providing or making available a pharmaceutical composition or drug to a medical professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.).

[0032] The terms "in combination" or "concomitantly" include administering an agent (e.g., a selective TGF-β inhibitor) in the presence of an additional agent. Concomitant administration in a therapeutic treatment method includes co-administration of a first agent, a second agent, a third agent, or an additional agent. Concomitant administration also includes administration of a first agent or an additional agent in the presence of a second agent or an additional agent, where the second agent or an additional agent may, for example, have been previously administered. A combined therapeutic treatment method may be performed stepwise by different actors. For example, one actor may administer a first agent to a subject, and a second actor may administer a second agent (e.g., a selective TGF-β inhibitor) to the subject, and these administration steps may be performed simultaneously or near simultaneously. The actor and subject may be the same entity (e.g., a human). Thus, the term encompasses both simultaneous administration and substantially simultaneous administration (i.e., near simultaneously).

[0033] The term "sequential administration" means not simultaneously and not nearly simultaneously. For example, a drug (e.g., an active agent) may be taken at one time of day (e.g., in the morning) and another time of day (e.g., in the evening / night), or every other day, etc.

[0034] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or dosage and / or dosing regimen of one or more agents necessary to produce a desired result, e.g., an amount sufficient to prevent FD in a subject, an amount sufficient to reduce the incidence of FD in a subject, and / or an amount sufficient to treat FD in a subject.

[0035] The terms "subject," "individual," and "patient" refer to mammals, preferably humans or non-human primates, as well as domesticated mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cows, pigs, sheep). In various embodiments, a subject can be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) under the care of a physician or other health care professional in a hospital, psychiatric facility, as an outpatient, or in other clinical settings. In certain embodiments, a subject may not be under the care or regimen of a physician or other health care professional.

[0036] The term "surgery" or "surgical procedure" collectively refers to all therapeutic and diagnostic procedures, including those requiring an incision made in a subject, as well as endoscopic procedures.

[0037] II. Pharmaceutical Compositions In some embodiments, a pharmaceutical composition is provided that includes a selective TGF-β inhibitor, such as TGFβRII-Fc, and a pharmaceutically acceptable carrier. For example, TGFβRII-Fc can be formulated with a pharmaceutically acceptable carrier.

[0038] The compositions disclosed herein can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intraspinal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0039] In certain embodiments, the composition is administered subcutaneously, intramuscularly, or intravenously. Pharmaceutical compositions can be formulated according to conventional pharmaceutical practice (see, for example, Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York, which are incorporated herein by reference in their entirety).

[0040] The compositions disclosed herein can be formulated for parenteral administration, where the active ingredient is incorporated into a solution or suspension, or a depot.The solution or suspension can also contain the following components: a sterile diluent (such as water for injection, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents); an antibacterial agent (such as benzyl alcohol or methylparaben); an antioxidant (such as ascorbic acid or sodium bisulfite); a chelating agent (such as ethylenediaminetetraacetic acid); a buffer (such as acetate, citrate, or phosphate); and an agent for adjusting tonicity (such as sodium chloride or dextrose).Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0041] Pharmaceutical forms suitable for injectable use include sterile solutions, dispersions, emulsions, and sterile powders. The final form should be stable under the conditions of manufacture and storage. Furthermore, the final pharmaceutical form should be protected from contamination and thus be capable of inhibiting the growth of microorganisms such as bacteria or fungi. A single dose may be administered subcutaneously. Alternatively, a slow, long-term infusion or multiple short-term daily infusions can be used, typically lasting 1 to 8 days. Dosing every other day or once every few days can also be used.

[0042] Sterile injectable solution can be prepared by incorporating the required amount of compound / complex in one or more suitable solvents, which can be added as needed with other ingredients listed above or known to those skilled in the art.Sterile injectable solution can be prepared by incorporating the required amount of compound into a suitable solvent with various other ingredients as needed.Then, sterilization such as filtration is carried out.Typically, dispersion is prepared by incorporating the compound into a sterile vehicle that also contains dispersion medium and other necessary ingredients as listed above.For sterile powder, specific methods include vacuum drying or freeze-drying, with any necessary ingredients added.

[0043] Suitable pharmaceutical carriers include sterile water; saline; dextrose; dextrose in water or saline; condensation products of castor oil with ethylene oxide (about 30 to about 35 moles of ethylene oxide combined per mole of castor oil); liquid acids; lower alkanols; oils such as corn oil; emulsifiers such as mono- or diglycerides of fatty acids, or peanut oil, sesame oil, etc., with phosphatides (e.g., lecithin); glycols; polyalkylene glycols; aqueous media in the presence of suspending agents (e.g., sodium carboxymethylcellulose); sodium alginate; poly(vinylpyrrolidone), etc., alone or with dispersing agents such as lecithin or polyoxyethylene stearate. The carrier may also contain adjuvants such as preservative stabilizers, wetting agents, emulsifiers, and penetration enhancers. In all cases, the final form, as described, must be sterile and easily passable through an injection device such as a hollow needle. The appropriate viscosity can be achieved and maintained by the appropriate selection of solvents or excipients. Additionally, the use of molecular or particulate coatings such as lecithin, the appropriate selection of particle size in dispersions, or the use of materials with surfactant properties can be utilized.

[0044] U.S. Patent Nos. 5,916,596, 6,506,405, and 6,537,579 teach the preparation of nanoparticles from biocompatible polymers such as albumin. Accordingly, provided herein is a method for producing nanoparticles from oil-in-water emulsions prepared under high shear conditions (e.g., sonication, high-pressure homogenization, etc.) by solvent evaporation techniques.

[0045] III.How to use It has been discovered that selective TGF-β inhibitors, such as TGFβRII-Fc, may be used to inhibit the differentiation of fibroadipogenic progenitor cells (FAPs) into adipocytes. The method comprises providing an effective amount of TGFβRII-Fc to the intercellular compartment surrounding FAP cells.

[0046] Any effective amount for inhibiting adipocyte differentiation can be used, e.g., 250 pM to about 2.5 mM, 100 pM to about 2.0 mM, or 50 pM to about 1.0 mM. In some embodiments, the effective amount is about 250 pM, about 500 pM, about 2.5 nM, about 5 nM, about 25 nM, about 50 nM, about 250 nM, about 500 nM, about 2.5 μM, about 5 μM, about 25 μM, about 50 μM, about 250 μM, about 500 μM, and about 2.5 mM.

[0047] Furthermore, in one embodiment, the method may be performed in vitro. Additionally or alternatively, the method may be performed in vivo.

[0048] In further embodiments, it has now been discovered that by using selective TGF-β inhibitors as defined herein, in particular TGFβRII-Fc, various desired results may be achieved, in particular preventing, reversing, reducing the incidence of, and / or treating FD.

[0049] Additionally or alternatively, a selective TGF-β inhibitor or a composition comprising a selective TGF-β inhibitor can be used to prevent, reverse, reduce the occurrence of, and / or treat muscle atrophy (which may or may not be distinct from FD) in a subject in need thereof.

[0050] The selective TGF-β inhibitor may be administered or otherwise provided in a composition, such as a pharmaceutical composition, further comprising one or more pharmaceutically acceptable excipients as described herein. Additional pharmaceutical therapeutic agents may also be administered simultaneously or sequentially with the selective TGF-β inhibitor.

[0051] In a further embodiment, a therapeutically effective amount of a selective TGF-β inhibitor is an amount that prevents, reverses, reduces the occurrence of, or treats FD. In one embodiment, the therapeutically effective amount of a selective TGF-β inhibitor is in the range of about 50 μg to about 10 mg per kg of subject, or 500 μg to about 3 mg per kg of subject, or 500 μg to about 2 mg per kg of subject, or 500 μg to about 1 mg per kg of subject. In a further embodiment, the therapeutically effective amount of a selective TGF-β inhibitor such as TGF-βRII-Fc is about 0.5 mg to about 500 mg per dose, or about 1 mg to about 500 mg per dose, or about 5 mg to about 500 mg per dose, or about 10 mg to about 500 mg per dose, or about 10 mg to about 400 mg per dose, or about 20 mg to about 200 mg per dose, or about 20 mg to about 100 mg per dose, particularly about 50 mg per dose.

[0052] Alternatively, the selective TGF-β inhibitor may be administered at a dose of about 0.01 to 100 mg / kg body weight, more typically about 0.05 to 10.0 mg / kg body weight, or about 0.1 to 5.0 mg / kg body weight, or about 0.25 to 2.5 mg / kg body weight, or about 0.2 to 2 mg / kg body weight, or about 0.2 to 1.0 mg / kg body weight. Thus, the dosage for a single administration may typically be about 0.5 to 5,000 mg, or about 1 to 500 mg, or about 5 to 250 mg, or about 10 to 200 mg. Of course, the dosage may also be adjusted within the above ranges according to what is best tolerated by the individual.

[0053] A therapeutically effective amount of the selective TGF-β inhibitor or a composition comprising the selective TGF-β inhibitor can be administered once or multiple times depending on the nature and severity of the injury, surgery or condition. For example, the selective TGF-β inhibitor can be administered once a week, twice a week, three or more times a week, less than once a week, once every two weeks, once every three weeks, four or more weeks, every six months, once a month, or once every two months.

[0054] In some embodiments, the selective TGF-β inhibitor or a composition comprising the selective TGF-β inhibitor can be administered to a subject before surgery, during surgery, after surgery, or any combination thereof. For example, the selective TGF-β inhibitor can be administered at least about 1 minute before the start of a surgical procedure, at least about 10 minutes before the start of a surgical procedure, at least about 30 minutes before the start of a surgical procedure, at least about 1 hour before the start of a surgical procedure, at least about 3 hours before the start of a surgical procedure, at least about 6 hours before the start of a surgical procedure, at least about 12 hours before the start of a surgical procedure, at least about 24 hours before the start of a surgical procedure, at least about 48 hours before the start of a surgical procedure, or more than 48 hours before the start of a surgical procedure (e.g., 1 week, 2 weeks, 30 days, 60 days, or 90 days before the start of a surgical procedure); or from about 1 minute to about 24 hours before the start of a surgical procedure, or from about 1 minute to 12 hours before the start of a surgical procedure, or from about 1 minute to about 1 hour before the start of a surgical procedure. Additionally or alternatively, the selective TGF-β inhibitor can be administered less than about 24 hours or the equivalent after the completion of the surgical procedure, less than about 12 hours or the equivalent after the completion of the surgical procedure, less than about 6 hours or the equivalent after the completion of the surgical procedure, less than about 3 hours or the equivalent after the completion of the surgical procedure, less than about 1 hour or the equivalent after the completion of the surgical procedure, less than about 30 minutes or the equivalent after the completion of the surgical procedure, less than about 10 minutes or the equivalent after the completion of the surgical procedure, less than about 1 minute or the equivalent after the completion of the surgical procedure; or from about 1 minute to about 24 hours after the completion of the surgical procedure, from about 1 minute to about 12 hours after the completion of the surgical procedure, or from about 1 minute to about 1 hour after the completion of the surgical procedure, or more than 48 hours after the completion of the surgical procedure (e.g., 1 week, 2 weeks, 30 days, 60 days, or 90 days after the completion of the surgical procedure).

[0055] Additionally or alternatively, the selective TGF-β inhibitor can be administered 90 days before or after surgery, 60 days before or after surgery, 30 days before or after surgery, 25 days before or after surgery, 20 days before or after surgery, 15 days before or after surgery, 10 days before or after surgery, or 5 days before or after surgery.

[0056] The surgery may be a repair surgery, such as tendon, ligament, and / or muscle repair surgery. For example, the repair surgery may be RC surgery, Achilles tendon surgery, Achilles tendon lengthening surgery, gastrocnemius recession surgery, anterior cruciate ligament (ACL) surgery, knee surgery, hip surgery, or spinal surgery. In certain embodiments, the surgery is RC repair surgery.

[0057] Additionally or alternatively, the selective TGF-β inhibitor, or a composition comprising the selective TGF-β inhibitor, can be administered to a subject suffering from or at risk of suffering from osteoarthritis, such as hip and knee osteoarthritis, muscle atrophy, age-related sarcopenia, obesity, metabolic syndrome, diabetes, and / or neurological disorders, such as peripheral neuropathy.

[0058] Additionally or alternatively, a selective TGF-β inhibitor or a composition comprising a selective TGF-β inhibitor can be administered to a subject suffering from or at risk of suffering from a neurodegenerative disease such as amyotrophic lateral sclerosis; a neuromuscular disease such as muscular dystrophy and inclusion body myositis; or chronic obstructive pulmonary disease; or a subject who may be a chronic non-ambulatory stroke patient.

[0059] Also provided herein is a combination therapy.Therefore, a selective TGF-β inhibitor such as TGFβRII-Fc and / or a pharmaceutical composition comprising a selective TGF-β inhibitor and one or more other active agents can be used to prevent, reverse, reduce the occurrence, and / or treat FD.The additional active agent for use with a selective TGF-β inhibitor includes, for example, steroids.The additional active agent can be administered in combination or sequentially as defined herein.

[0060] [Example] The following examples are provided to further illustrate the invention disclosed herein but, of course, should not be construed as limiting its scope. In each example, the TGFβRII-Fc used was a short form of TGFβR fused to human IgG1 (see SEQ ID NO: 3).

[0061] Example 1 - In vivo mechanism of action: inhibition of adipogenic differentiation Mechanistically, muscle-infiltrating adipocytes are generated from muscle-resident fibro-adipogenic progenitor cells (FAPs) that are activated as part of the normal wound-healing response. We found that TGFβRII-Fc inhibited the differentiation of adipocytes from a FAP-like cell line, i.e., 3T3-L1 cells (see Figures 3A-3H). This supports the idea that TGFβRII-Fc acts by preventing the differentiation of FAPs into adipocytes in vivo.

[0062] Cryopreserved mouse 3T3-L1 preadipocytes purchased from ZenBio were thawed and cultured at approximately 10,000 cells / cm. 2Cells were seeded in preadipocyte medium (PM: DMEM, high glucose, pH 7.4 HEPES, 10% bovine calf serum (BCS), and penicillin + streptomycin (PS)) at 37°C in a humidified incubator with 5% CO2 until they reached 100% confluence, which took approximately 4 days. During this time, the growth medium was changed every other day. Two days after reaching confluence, the preadipocyte medium (PM) was replaced with the appropriate volume of differentiation medium (DM: DMEM, high glucose, sodium pyruvate, pH 7.4 HEPES, 10% fetal bovine serum (FBS), 33 μM biotin, 10 μg / mL human insulin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX)) and incubated for 3 days. The differentiation medium was then replaced with adipocyte maintenance medium (MM:DMEM, high glucose, sodium pyruvate, HEPES pH 7.4, 10% FBS, 33 μM biotin, 10 μg / mL human insulin). Cells were maintained for up to 10 days after differentiation, with medium changes every other day.

[0063] In this assay, 3T3-L1 cells were treated with TGFβRII-Fc starting at different stages of differentiation. In one assay, confluent 3T3-L1 cells were grown in PM, differentiated in DM for 3 days, and maintained in MM for up to 5 days. 300 nM TGFβRII-Fc was added on day 0 (beginning of DM treatment), day 3 (end of DM treatment), or day 5 (after 2 days in MM). Cells were maintained under treatment until the end of the experiment on day 8 or 10.

[0064] At the end of the experiment, adipocyte differentiation was measured by immunofluorescence. In this experiment, 10,000 3T3-L1 cells / cm were cultured. 2Cells were plated in 96-well plates in preadipocyte medium. On day 0, cells were treated with TGFβRII-Fc. Treatment continued in the appropriate medium until adipocyte differentiation was allowed to proceed. On day 10, cells were washed twice with PBS and fixed with 10% formalin for 30 minutes at room temperature. Cells were then washed twice with PBS and subsequently stained with 0.01% saponin, 1 μg / mL Nile Red, and 1 μg / mL DAPI prepared in PBS for 15 minutes at room temperature. After staining, cells were washed three times with PBS. Images were taken using an Olympus Fluoview FC1000 confocal laser scanning microscope.

[0065] The results of this experiment are shown in Figures 3A–3M. 3T3-L1 cells were treated with TGFβRII-Fc or vehicle (i.e., PBS) on day 0 and differentiated for 8 days (Figure 3A). The number of new adipocytes was reduced by 95% (Figures 3B–3G). In Figure 3B, green represents Nile Red staining of lipid droplets, and purple represents DAPI staining of nuclei. For quantitative Nile Red and DAPI fluorescence measurements, fluorescence was measured before and after staining according to published protocols. In this experiment, 3T3-L1 cells were treated in quadruplicate in a 96-well plate. Multiple images were taken from each well. The number and size of lipid droplets, average lipid area, normalized Nile Red fluorescence, and lipolytic activity were calculated from two biological replicates using Image J software (Figures 3C–3M).

[0066] In such adipocyte differentiation experiments, the extent of adipocyte formation can also be determined by gene expression analysis. In the experiments resulting in the data shown in Figures 3C–3H, 3T3-L1 cells were treated with TGFβRII-Fc or vehicle on day 0 and differentiated for 8 days. On days 0, 3, and 8, total RNA was isolated from 3T3-L1 cells, and cDNA was synthesized from the RNA using reverse transcriptase using published protocols. To quantify the expression of selected adipocyte marker genes, primer pairs were synthesized to amplify specific regions in these genes, and the cDNA was analyzed using a Lightcycler 480 (Roche). For each experiment, a primer standard curve was created using a four-digit dilution series of genomic DNA isolated from 3T3-L1 cells according to published protocols. The highest concentration of genomic DNA resulted in qRT-PCR amplification around cycle 20 for the majority of primer pairs. Tm curves are also performed as a quality control for each primer pair at the end of each qRT-PCR run to verify that only a single amplicon is generated by each primer pair and that primer dimers are not formed. Data are typically obtained from at least two biological replicates and four technical replicates.

[0067] Example 2 - Diet-induced obesity (DIO) model in mice TGFβRII-Fc inhibits skeletal muscle FI in a mouse DIO model, as shown in Figures 4A-4J. In this model, mice are placed on a high-fat diet (HFD) for several weeks. After 4 weeks, HFD mice show early signs of developing obesity. One of these signs is increased skeletal muscle FI. FI is the first symptom of FD in DIO, followed later by muscle atrophy. New adipocytes are generated by adipogenic differentiation of muscle-resident fibro-adipogenic progenitor cells (FAPs) and can be detected by histopathological and gene expression analysis.

[0068] A total of 20 8-week-old male C57BL / 6 mice obtained from Charles River Laboratories were used in this study. The animals were housed under the supervision of the Michigan State University (MSU) Campus Animal Resources and allowed to acclimate for 7 days prior to the start of the study. Upon arrival, all animals were fed certified rodent chow No. 5CR4 from PMI Nutrition International. At the start of the experiment, 14 mice were placed on a high-fat diet (Research Diet D12492, 60% fat kcal). This diet was available ad libitum for the remainder of the study. Standard chow (certified rodent chow No. 5CR4 from PMI Nutrition International) was provided ad libitum to six control mice throughout the study. Mice on the high-fat diet were divided into two groups: seven mice were treated with TGFβRII-Fc for 4 weeks, and seven mice were treated with vehicle (i.e., PBS) for 4 weeks. TGFβRII-Fc or vehicle control was administered by subcutaneous injection every two weeks. TGFβRII-Fc was provided as a 3 mg / mL stock and administered at 15 mg / kg body weight. TGFβRII-Fc aliquots were kept frozen (-80°C) until use. On the day of administration, aliquots were transferred from -80°C to a room-temperature benchtop at least 1 hour before administration. The thawed samples were gently mixed by inverting the tube to ensure homogeneous distribution, followed by collection of the liquid from the cap by centrifugation. Vortexing and vigorous mixing were avoided to prevent protein denaturation or bubble formation. The vehicle control was administered to the appropriate animals at the same time as TGFβRII-Fc. Experiments were conducted under the supervision of the MSU IACUC to ensure animal safety. At the end of the experiment, mice were sacrificed by CO2 asphyxiation. At necropsy, leg muscles were extracted and flash-frozen in liquid nitrogen.

[0069] The FI of skeletal muscle can be determined in experiments such as histopathological analysis of muscle. For this analysis, muscle can be frozen in liquid nitrogen, then fixed in paraformaldehyde, and subsequently cryoprotected with sucrose. Cryosections of muscle can then be stained with hematoxylin and Oil Red O (ORO). In this assay, ORO detects intermuscular and intramuscular fat. Intermuscular fat is the result of FI, while intramuscular fat is the result of lipid accumulation within muscle fibers. In the DIO model, both intermuscular and intramuscular fat accumulation can be detected.

[0070] In this experiment, shown in Figure 4A, the quadriceps muscles of mice fed a HFD for 4 weeks and treated with TGFβRII-Fc or vehicle control were histopathologically analyzed. The quadriceps muscles were placed in a 15-mL tube containing 8 mL of 4% paraformaldehyde prepared in phosphate buffer (no saline) and maintained at room temperature for 48 hours. The muscles were then transferred to a fresh 15-mL tube containing 8 mL of 30% sucrose prepared in phosphate buffer (no saline) and maintained at 4°C until the muscles sank to the bottom of the tube (i.e., 4 days). The fixed and sucrose-cryoprotected muscles were then embedded in OCT and cryosectioned at 12 μm thickness at -20°C. The sections were stained with ORO and counterstained with hematoxylin. Quantification of ORO was performed using Image J on images acquired using the OlyVIA system at 0.7x magnification. Inhibition of FI by TGFβRII-Fc under these conditions was evidenced by a 70–80% decrease in total ORO staining and a 65–70% decrease in intermyocyte ORO (Figures 4A–4D).

[0071] In this type of experiment, skeletal muscle FI can be determined by gene expression analysis. RNA was isolated from a portion of the appropriate muscle belly (20-25 mg) using a Promega ReliaPrep™ RNA Tissue Miniprep System and a tissue homogenizer. The tissue was placed in a 2 mL screw-cap tube, and two beads and 1.3 mL of TRIzol™ reagent were added. The tissue was homogenized for 25 s using a mixer mill (Retsch). -1 The tissue was homogenized for 2 × 4 minutes at 100 kJ / min. Samples were kept on ice between cycles. 260 μL of chloroform was added, and the sample was vigorously shaken by hand until the solution turned cloudy pink. After centrifugation at 15 kJ / min for 10 min at 4°C, a 750 μL aliquot of the aqueous phase, avoiding the interphase, was transferred to a new tube. 340 μL of ice-cold isopropanol was added, and the sample was mixed by vortexing. The sample was then transferred to a Promega RNA Cell Miniprep column and processed according to the manufacturer's instructions. A DNAse 1 treatment step was performed on the column for 30 minutes. RNA was eluted in 15 μL of RNase-free H2O. The RNA concentration and purity of each sample were determined by Nanodrop. For cDNA synthesis, 1 μg of total isolated RNA from each sample was used and reverse transcribed using the Multiscribe™ Reverse Transcriptase Kit (ThermoFischer). The cDNA was then analyzed as described above using primers for adipocyte markers in a Roche Lightcycler® 480. Data were then normalized to a household gene (Rpl4).

[0072] In this experiment, we analyzed gene expression in the quadriceps muscles of mice fed a HFD for 4 weeks and treated with TGFβRII-Fc or vehicle control. Treatment with TGFβRII-Fc significantly inhibited FI under these conditions, as evidenced by a 5- to 60-fold decrease in the expression of the adipocyte marker genes Adipoq, Cidec, Fabp4, Lep, Plin1, and Pparg (Figures 4E-4J).

[0073] Surprisingly, as shown in Figures 3A-3H and 4A-4J, we found that inhibition of both TGF-β1 and β3 prevented adipogenesis, a counterintuitive result considering that Ignotz, RA, and Massague, J. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8530-4. doi: 10.1073 / pnas.82.24.8530.) previously reported that TGF-β1 itself inhibits adipogenesis. Furthermore, because TGFβRII-Fc does not inhibit TGF-β2 signaling, administration of TGFβRII-Fc (e.g., before, during, and / or after RC repair surgery) can inhibit, prevent, or reduce the onset of muscle FD without interfering with tendon regeneration. Thus, TGFβRII-Fc can also promote tendon regeneration and attachment while inhibiting FD. TGFβRII-Fc may also be used to arrest muscle FD in patients in whom repair surgery for RC is not recommended and conservative treatment is indicated.

[0074] Example 3 - Inhibition of fatty degeneration of skeletal muscle in a mouse model of rotator cuff injury In this experiment, the tendons of two RC muscles, the supraspinatus and infraspinatus, are resected to mimic the RC tendon rupture seen in human patients. This mouse model of RC injury closely mimics human RC injury, as mice also develop severe FD, including FI and muscle atrophy, as well as fibrosis / scarring in the shoulder muscles, after 6 weeks.

[0075] In this experiment, surgery to induce RC injury was performed by RC tenotomy and denervation. Surgical procedures were performed under general anesthesia with 2.5% isoflurane and oxygen. RC surgical interventions were performed on the right shoulder of the mouse, while sham surgery was performed on the left shoulder. For surgery, the mouse was placed on the operating table, and the clavicle and deltoid muscle were exposed through a skin incision. The trapezius muscle was separated to expose the suprascapular nerve, and a 5 mm nerve segment was resected to prevent nerve reattachment and healing. The deltoid muscle was split longitudinally, and the rotator cuff tendons were exposed at the shoulder joint. The supraspinatus and infraspinatus tendons were completely transected by removal of the tendon segments to reduce the chance of spontaneous healing. The deltoid and trapezius muscles, as well as the skin incisions, were then closed. For sham procedures on the contralateral shoulder, skin and muscle incisions were made, the rotator cuff and / or suprascapular nerve were identified, and the muscles and skin were reclosed. Surgery typically lasts approximately 20 minutes per mouse.

[0076] In this experiment, 24 female, 12-week-old C57BL / 6 mice obtained from Jackson Laboratories underwent surgery to induce RC injury. Starting 2 days after surgery, 12 mice were treated with TGFβRII-Fc and 12 mice were treated with vehicle control (i.e., PBS) for 6 weeks. Experiments were performed in the MSU in vivo facility under the supervision of the MSU IACUC to ensure animal safety.

[0077] Initially, animals were housed under the supervision of the MSU Campus Animal Resources. Prior to the start of the study, animals were allowed to acclimate for 7 days. Upon arrival, PMI Nutrition International certified rodent chow No. 5CR4 was provided and fed ad libitum throughout the study. Animals were weighed the day before the study and randomly assigned to two treatment groups in a manner that created cohorts with no significant differences in body weight between groups. After cohorting, animals were housed singly for the study. Mortality / moribundity checks were performed twice daily (once in the morning and once in the evening) for two days after surgery and once daily for the remainder of the study. For postoperative care, the surgical incision was irrigated daily with chlorhexidine solution for two days after surgery. Animals were assessed for limb use daily after surgery, and food intake was assessed by daily weighing. For postoperative pain management, buprenorphine was administered subcutaneously at 1 mg / kg every 12 hours for two days after surgery.

[0078] In this experiment, animals received TGFβRII-Fc or vehicle twice weekly for 6 weeks, starting 2 days after surgery. TGFβRII-Fc or vehicle was administered subcutaneously. TGFβRII-Fc was provided as a 3 mg / mL stock and administered at 15 mg / kg body weight. TGFβRII-Fc aliquots were kept frozen (-80°C) until use. On the day of administration, aliquots were transferred from -80°C to a room-temperature benchtop at least 1 hour prior to administration. The thawed samples were gently mixed by inverting the tubes to ensure homogeneous distribution, followed by collection of the liquid from the cap by centrifugation. Vortexing and vigorous mixing were avoided to prevent protein denaturation or bubble formation. Vehicle control (i.e., PBS) was administered to appropriate animals at the same time as TGFβRII-Fc treatment.

[0079] At the end of the experiment, the experimental animals were sacrificed by CO2 asphyxiation. At autopsy, the shoulder girdle and upper limb were excised in blocks along with muscle and connective tissue. The supraspinatus and infraspinatus muscles on the injured and sham-operated sides were separated from bone and connective tissue and immediately weighed. Wet weights were recorded, and the weight of the operated muscle was calculated as a percentage of that of the sham-operated control. The statistical significance of the difference between muscle weights of TGFβRII-Fc-treated animals and vehicle-treated animals was determined by Student's t-test (n=6).

[0080] In this experiment, treatment with TGFβRII-Fc significantly reduced atrophy of the supraspinatus (SS) and infraspinatus (IS) muscles after RC injury (see Table 1 and Figure 5A). Vehicle-treated animals lost an average of approximately 30% of their SS muscle weight and approximately 55% of their IS muscle weight. In contrast, TGFβRII-Fc-treated animals lost only approximately 10% and 13% of their SS and IS muscle weight, respectively. This corresponds to a 70–80% reduction in muscle atrophy in mice treated with TGFβRII-Fc compared to control mice.

[0081] [Table 1] TIFF0007798356000002.tif81169

[0082] The reduction in FI in shoulder muscles after RC injury by TGFβRII-Fc could also be demonstrated using adipocyte marker gene expression analysis. In this experiment, SS and IS muscles from the injured and sham-operated sides were snap-frozen in liquid nitrogen. RNA was isolated from whole muscles using the Promega ReliaPrep™ RNA Tissue Miniprep System and tissue homogenizer, followed by cDNA synthesis as described above. Treatment with TGFβRII-Fc reduced FI in the SS and infraspinatus (IS) muscles, as indicated by a 2- to 3-fold decrease in gene expression in Cidec and Lep (Figures 5B and 5C). Significance was determined by Student's t-test (n = 4).

[0083] The use of the terms "a," "an," "the," and "at least one," and similar referents in the context of describing the present invention (particularly in the context of the claims below) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items, or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value within the range, and each separate value is incorporated into the specification as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further clarify the invention and does not present a limitation on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0084] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these specific embodiments will be apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect that skilled artisans will adopt such variations as appropriate, and it is intended that the invention be practiced even if not specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context. [Brief explanation of the drawings]

[0085] [Figure 1A] 1A-1B show two naturally occurring splice forms of TGFβRII: the native short form and the native long form. Figure 1A shows that the long form has a 25 amino acid insertion after amino acid 31, and the amino acid following this insertion changes from Val to Ile (shown in bold). [Figure 1B] 1A-1B show two naturally occurring splice forms of TGFβRII: the native short form and the native long form. Figure 1B shows the sequences of the extracellular domains (ECDs) of both forms of TGFβRII. Uniprot sequence P37173 (herein SEQ ID NO: 1) is the short form, and Uniprot sequence D2JYI1 (herein SEQ ID NO: 2) is the long form. [Figure 1C] FIG. 1C is the amino acid sequence of TGFβRII-Fc (SEQ ID NO: 3) used in Examples 1 to 3 herein. [Figure 2]Figure 2 shows the binding specificity of TGFβRII-Fc. The figure is an image of an SPR-sensorgram showing that TGFβRII-Fc (SEQ ID NO: 3) binds to TGF-β1 and TGF-β3, but not to TGF-β2. See Aykul, S. and Martinez-Hackert, E. Transforming Growth Factor-β family ligands can function as antagonists by competing for type II receptor binding. Journal of Biological Chemistry 291: 10792-10804 (2016). The publication is incorporated herein by reference in its entirety. [Figure 3A] Figures 3A-3M show the inhibition of fibroadipogenic 3T3-L1 progenitor cell differentiation into adipocytes by TGFβRII-Fc. Figure 3A is a schematic diagram showing how fibroadipogenic progenitor cells (FAPs) differentiate into adipocytes. See Aykul, S., Maust, J., Floer, M., and Martinez-Hackert, M. TGF-β Family Inhibitors Blunt Adipogenesis Via Non-Canonical Regulation Of SMAD Pathways. bioRxiv. (Published online March 14, 2020). The publication is incorporated herein by reference in its entirety. [Figure 3B] Figures 3A-3M show inhibition of adipogenic differentiation of fibro-adipogenic 3T3-L1 progenitor cells by TGFβRII-Fc. Figures 3B-3G show immunofluorescence analysis of 3T3-L1 cells grown in adipocyte differentiation medium for 8 days with vehicle control (PBS) or 300 nM TGFβRII-Fc. Figure 3B shows images of cells stained with Nile red (green) for fat and DAPI (magenta) for nuclei. [Figure 3C]Figures 3A-3M show the inhibition of adipogenic differentiation of fibro-adipogenic 3T3-L1 progenitor cells by TGFβRII-Fc. Figures 3B-3G show immunofluorescence analysis of 3T3-L1 cells grown in adipocyte differentiation medium for 8 days with vehicle control (PBS) or 300 nM TGFβRII-Fc. Figure 3C shows quantification of droplet numbers. Hatched bars represent vehicle control, and open bars represent TGFβRII-Fc-treated cells. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3D] Figures 3A-3M show the inhibition of adipogenic differentiation of fibro-adipogenic 3T3-L1 progenitor cells by TGFβRII-Fc. Figures 3B-3G show immunofluorescence analysis of 3T3-L1 cells grown in adipocyte differentiation medium for 8 days with vehicle control (PBS) or 300 nM TGFβRII-Fc. Figure 3D shows the average size of lipid droplets. Hatched bars represent vehicle control, and open bars represent TGFβRII-Fc-treated cells. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3E] Figures 3A-3M show the inhibition of adipogenic differentiation of fibro-adipogenic 3T3-L1 progenitor cells by TGFβRII-Fc. Figures 3B-3G show immunofluorescence analysis of 3T3-L1 cells grown in adipocyte differentiation medium for 8 days with vehicle control (PBS) or 300 nM TGFβRII-Fc. Figure 3E shows the average lipid area. Hatched bars represent vehicle control, and open bars represent TGFβRII-Fc-treated cells. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3F] Figures 3A-3M show the inhibition of adipogenic differentiation of fibro-adipogenic 3T3-L1 progenitor cells by TGFβRII-Fc. Figures 3B-3G show immunofluorescence analysis of 3T3-L1 cells grown in adipocyte differentiation medium for 8 days with vehicle control (PBS) or 300 nM TGFβRII-Fc. Figure 3F shows normalized Nile red fluorescence. Hatched bars represent vehicle control, and open bars represent TGFβRII-Fc-treated cells. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3G] Figures 3A-3M show the inhibition of adipogenic differentiation of fibro-adipogenic 3T3-L1 progenitor cells by TGFβRII-Fc. Figures 3B-3G show immunofluorescence analysis of 3T3-L1 cells grown in adipogenic differentiation medium for 8 days with vehicle control (PBS) or 300 nM TGFβRII-Fc. Figure 3G shows lipolytic activity. Hatched bars represent vehicle control, and open bars represent TGFβRII-Fc-treated cells. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3H]Figures 3A-3M show the inhibition of differentiation of fibro-adipogenic 3T3-L1 progenitor cells into adipocytes by TGFβRII-Fc. Figures 3H-3M show gene expression analysis of 3T3-L1 cells grown in differentiation medium for different lengths of time. Induction of adipocyte marker gene expression was analyzed by qRT-PCR in vehicle control (hatched bars) and TGFβRII-Fc-treated cells (white bars) on days 0, 3, and 8. Data are shown as fold induction after normalization to Rpl4, and Figure 3H shows Adipoq. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3I] Figures 3A-3M show the inhibition of differentiation of fibro-adipogenic 3T3-L1 progenitor cells into adipocytes by TGFβRII-Fc. Figures 3H-3M show gene expression analysis of 3T3-L1 cells grown in differentiation medium for different lengths of time. Induction of adipocyte marker gene expression was analyzed by qRT-PCR in vehicle control (hatched bars) and TGFβRII-Fc-treated cells (white bars) on days 0, 3, and 8. Data are presented as fold induction after normalization to Rpl4; Figure 3I shows Cidec. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3J]Figures 3A-3M show the inhibition of differentiation of fibro-adipogenic 3T3-L1 progenitor cells into adipocytes by TGFβRII-Fc. Figures 3H-3M show gene expression analysis of 3T3-L1 cells grown in differentiation medium for different lengths of time. Induction of adipocyte marker gene expression was analyzed by qRT-PCR in vehicle control (hatched bars) and TGFβRII-Fc-treated cells (white bars) on days 0, 3, and 8. Data are shown as fold induction after normalization to Rpl4; Figure 3J shows Fabp4. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3K] Figures 3A-3M show the inhibition of differentiation of fibro-adipogenic 3T3-L1 progenitor cells into adipocytes by TGFβRII-Fc. Figures 3H-3M show gene expression analysis of 3T3-L1 cells grown in differentiation medium for different lengths of time. Induction of adipocyte marker gene expression was analyzed by qRT-PCR in vehicle control (hatched bars) and TGFβRII-Fc-treated cells (white bars) on days 0, 3, and 8. Data are shown as fold induction after normalization to Rpl4, and Figure 3K shows Lep. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3L]Figures 3A-3M show the inhibition of differentiation of fibro-adipogenic 3T3-L1 progenitor cells into adipocytes by TGFβRII-Fc. Figures 3H-3M show gene expression analysis of 3T3-L1 cells grown in differentiation medium for different lengths of time. Induction of adipocyte marker gene expression was analyzed by qRT-PCR in vehicle control (hatched bars) and TGFβRII-Fc-treated cells (white bars) on days 0, 3, and 8. Data are shown as fold induction after normalization to Rpl4, while Figure 3L shows Plin1. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 3M] Figures 3A-3M show the inhibition of differentiation of fibro-adipogenic 3T3-L1 progenitor cells into adipocytes by TGFβRII-Fc. Figures 3H-3M show gene expression analysis of 3T3-L1 cells grown in differentiation medium for different lengths of time. Induction of adipocyte marker gene expression was analyzed by qRT-PCR in vehicle control (hatched bars) and TGFβRII-Fc-treated cells (white bars) on days 0, 3, and 8. Data are presented as fold induction after normalization to Rpl4 and, in Figure 3M, Pparg. Significance was determined by two-way ANOVA and Sidaki or Dunnett's multiple comparison test. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 4A] Figures 4A-4J show the inhibition of fat infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figure 4A shows a photograph of muscle that was sectioned and stained with hematoxylin (blue) followed by Oil Red O (ORO). ORO detects intramuscular fat, which is formed inside muscle fibers, as well as intermuscular fat (FI), which is formed by adipocytes growing between muscle fibers. [Figure 4B]Figures 4A-4J show the inhibition of fat infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figure 4B shows the quantification of total fat (n=3). Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 4C] Figures 4A-4J show the inhibition of fat infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figure 4C shows the quantification of intermuscular fat (n=3). Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 4D] Figures 4A-4J show the inhibition of fat infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figure 4D shows the quantification of intramuscular fat from the quadriceps muscle of mice (n=3). Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 4E] Figures 4A-4J show the suppression of fatty infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figures 4E-4J show analysis of gene expression in quadriceps muscle. Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are presented as fold expression relative to chow-fed mice, with Figure 4E showing the adipocyte marker gene Adipoq. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 4F]Figures 4A-4J show the suppression of fatty infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figures 4E-4J show gene expression analysis in the quadriceps muscle. Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are presented as fold expression relative to chow-fed mice, and Figure 4F shows the data for Cidec. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 4G] Figures 4A-4J show the suppression of fatty infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figures 4E-4J show analysis of gene expression in the quadriceps muscle. Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are presented as fold expression relative to chow-fed mice, with Figure 4G showing Fabp4. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 4H] Figures 4A-4J show the suppression of fatty infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figures 4E-4J show analysis of gene expression in the quadriceps muscle. Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are presented as fold expression relative to chow-fed mice, and Figure 4H shows Lep. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 4I]Figures 4A-4J show the suppression of fatty infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figures 4E-4J show gene expression analysis in the quadriceps muscle. Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are presented as fold expression relative to chow-fed mice, with Figure 4I showing Plin1. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 4J] Figures 4A-4J show the suppression of fatty infiltration ("FI") in the quadriceps muscle of DIO mice by TGFβRII-Fc. Figures 4E-4J show analysis of gene expression in the quadriceps muscle. Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are presented as fold expression relative to chow-fed mice, with Figure 4J showing Pparg. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05; **p<0.01) [Figure 5A] Figures 5A-5C show the inhibition of muscle atrophy and FI in shoulder muscles by TGFβRII-Fc after induced RC injury. Figure 5A shows quantification of wet weight loss values ​​for the supraspinatus (SS) and infraspinatus (IS) muscles from appropriate animals (n=6). Muscles on the injured side were compared with muscles on the sham-operated side of the same mice. Data are expressed as the % reduction in muscle weight after induced RC injury by comparing sham-operated muscles to injured (*p<0.05; ***p<0.001). [Figure 5B] Figures 5A-5C show the inhibition of muscle atrophy and FI by TGFβRII-Fc in shoulder muscles after induced RC injury. Figures 5B and 5C show gene expression analysis of shoulder muscles (n=4). Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are shown as gene expression in injured muscles compared to sham-operated muscles, and Figure 5B shows the expression of Cidec. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05). [Figure 5C] Figures 5A-5C show the inhibition of muscle atrophy and FI by TGFβRII-Fc in shoulder muscles after induced RC injury. Figures 5B and 5C show gene expression analysis of shoulder muscles (n=4). Gene expression was analyzed by qRT-PCR and normalized to the Rpl4 household gene. Data are shown as gene expression in injured muscles compared to sham-operated muscles, and Figure 5C shows Lep. Significance was determined by one-way ANOVA followed by a post-hoc Tukey's test. (*p<0.05).

Claims

1. A pharmaceutical composition for use in a method for preventing, reducing the incidence of, and / or treating skeletal muscle fatty degeneration in a subject in need thereof, said pharmaceutical composition comprising a therapeutically effective amount of TGFβRII-Fc; administering the TGFβRII-Fc to the subject preoperatively, e.g., immediately before surgery to 90 days before surgery; during surgery; or postoperatively, e.g., immediately after surgery to 90 days after surgery; and The pharmaceutical composition, wherein the surgery is a restorative surgery, such as a tendon, ligament and / or muscle surgery.

2. 10. The pharmaceutical composition of claim 1, wherein the repair surgery is rotator cuff (RC) surgery, Achilles tendon surgery, Achilles tendon lengthening surgery, gastrocnemius recession surgery, anterior cruciate ligament (ACL) surgery, knee surgery, hip surgery, or spinal surgery.

3. 3. The pharmaceutical composition of claim 1, wherein the TGFβRII-Fc inhibits fatty degeneration and promotes tendon regeneration and reattachment.

4. 2. The pharmaceutical composition of claim 1, wherein the subject is suffering from or at risk of suffering from osteoarthritis, such as hip and knee osteoarthritis, muscle atrophy, age-related sarcopenia, obesity, metabolic syndrome, diabetes, and / or peripheral neuropathy.

5. 2. The pharmaceutical composition of claim 1, wherein the subject suffers from a neurodegenerative disease such as amyotrophic lateral sclerosis; a neuromuscular disease such as muscular dystrophy and inclusion body myositis; or chronic obstructive pulmonary disease; or the subject is a chronic non-ambulatory stroke patient.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the TGFβRII-Fc is administered at least once a week, once every two weeks, or once every three weeks.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the therapeutically effective amount of TGFβRII-Fc is between about 10 mg and about 500 mg per dose.

8. 8. The pharmaceutical composition of claim 7, wherein the therapeutically effective amount of TGFβRII-Fc is about 50 mg per dose.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the TGFβRII-Fc is administered subcutaneously, intramuscularly, or intravenously.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the subject is a human.

11. The pharmaceutical composition of any one of claims 1 to 9, wherein the subject is a non-human mammal, including a horse, dog, or cat.

12. A composition for inhibiting the differentiation of fibro-adipogenic progenitor (FAP) cells into adipocytes, comprising an effective amount of TGFβRII-Fc.

13. 13. The composition of claim 12, wherein the effective amount is from about 250 pM to about 2.5 mM.

14. 13. The composition of claim 12, wherein the effective amount is selected from the group consisting of about 250 pM, about 500 pM, about 2.5 nM, about 5 nM, about 25 nM, about 50 nM, about 250 nM, about 500 nM, about 2.5 μM, about 5 μM, about 25 μM, about 50 μM, about 250 μM, about 500 μM, and about 2.5 mM.

15. The composition according to any one of claims 12 to 14, wherein the inhibition of differentiation of FAP cells into adipocytes occurs in vitro.

16. The composition according to any one of claims 12 to 14, wherein the inhibition of differentiation of FAP cells into adipocytes occurs in vivo.

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