Methods and compositions for treating muscle diseases and disorders

Abstract

The present invention relates to methods and compositions for treating muscle diseases and disorders. The invention provides methods for treating muscle myopathies, including muscular dystrophy and cardiomyopathy, by administering stable, long-acting therapeutic agents for vasoactive intestinal peptides. These agents include one or more elastin-like peptides and can be administered at low doses.

Description

[0001] This application is a divisional application of the invention application filed on February 9, 2016, with Chinese application number 201680020418.6 and entitled "Method and Composition for Treating Muscle Diseases and Symptoms".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 113,943, filed February 9, 2015; U.S. Provisional Application No. 62 / 145,770, filed April 10, 2015; and U.S. Provisional Application No. 62 / 150,679, filed April 21, 2015, the contents of which are incorporated herein by reference in their entirety.

[0004] Instructions for electronically submitted text files

[0005] The contents of the electronically submitted text file are incorporated herein by reference in their entirety: a computer-readable copy of the sequence list (filename: PHAS_032 / 04WO__SeqList_ST25.txt, date of record: February 9, 2016, file size: 32 kilobytes). Background Technology

[0006] Duchenne and Beck muscular dystrophy (DMD and BMD) represent the most common neuromuscular diseases in humans, with an incidence of 1 / 6000 to 1 / 3500 in males at birth, depending on the study population (Bushby et al. (2010)). DMD and BMD are allelic disorders caused by mutations in the dystrophin gene. In DMD, functional dystrophin is completely absent in the muscles, while in BMD, although some dystrophin is present, the amount is insufficient for normal muscle function. In addition to skeletal muscle weakness, dystrophin deficiency in the myocardium also leads to progressive cardiomyopathy.

[0007] Currently, there are no approved specific treatments for DMD / BMD. High-dose corticosteroids are frequently used to treat myasthenia gravis and maintain walking as much as possible, but they involve unacceptable side effects and / or adverse responses. It is also unclear whether these treatments help or hinder cardiac function. Other treatment options are needed to address both the skeletal and cardiac abnormalities associated with muscular dystrophy. Invention Overview

[0008] This invention provides a long-acting vasoactive intestinal peptide (VIP) therapy for treating, delaying, preventing, or improving muscle diseases. Myopathy arises from repeated muscle contractions, which are damaged but lack adequate self-repair capabilities, leading to defects such as fibrosis. These defects often inhibit muscle function by impairing its ability to contract. Preventing, delaying, or improving the development of these defects can treat myopathy in patients. The VIP therapeutic agents disclosed herein can also improve cardiac function in patients with myopathy.

[0009] In some aspects, the present invention provides a method for treating muscle diseases, comprising administering to a patient in need a pharmaceutical composition containing vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP).

[0010] In some respects, the present invention provides a method for preventing muscle contraction-induced injury in patients in need, comprising administering a pharmaceutical composition containing a vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP).

[0011] In some aspects, the present invention provides a method for slowing the progression of cardiomyopathy, comprising administering to a patient in need a pharmaceutical composition containing a vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP).

[0012] In some aspects, the present invention provides a method for treating cardiomyopathy, comprising administering to a patient in need a pharmaceutical composition containing vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP).

[0013] In some respects, the pharmaceutical composition comprises the amino acid sequence of SEQ ID NO:15.

[0014] This invention also includes the following:

[0015] 1. A method for treating myopathy, comprising administering to a patient in need a pharmaceutical composition comprising a vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP).

[0016] 2. A method for preventing muscle contraction-induced injury in patients in need, comprising administering a pharmaceutical composition containing a vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP).

[0017] 3. A method for slowing the progression of cardiomyopathy, comprising administering to a patient in need a pharmaceutical composition containing a vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP).

[0018] 4. A method for treating cardiomyopathy, comprising administering to a patient in need a pharmaceutical composition containing a vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP).

[0019] 5. The method of any one of items 1-4, wherein the pharmaceutical composition reduces muscle fibrosis.

[0020] 6. The method of any of the preceding items, wherein the ELP comprises a repeating unit of any of SEQ ID NO:1-13, or a combination thereof.

[0021] 7. The method of any of the preceding items, wherein the ELP comprises a repeating unit of VPGXG (SEQ ID NO:3).

[0022] 8. The method of item 7, wherein the ELP comprises 120 repeating units of VPGXG, wherein X is independently selected from Val, Ala and Gly.

[0023] 9. The method of item 8, wherein X is independently selected from Val, Ala and Gly in a ratio of approximately 5:2:3.

[0024] 10. The method of any one of items 1-4, wherein the VIP peptide has a relative binding preference for VPAC2 compared to VPAC1.

[0025] 11. The method of any one of claims 1-4, wherein the pharmaceutical composition is formulated for subcutaneous, intramuscular or intravenous administration.

[0026] 12. The method of item 11, wherein the pharmaceutical composition is administered subcutaneously.

[0027] 13. The method of any one of claims 1-4, wherein the pharmaceutical composition is administered in a low dose.

[0028] 14. The method of item 13, wherein the dose is from 0.1 mg / kg daily to 10 mg / kg daily.

[0029] 15. The method of any one of claims 1-4, wherein the pharmaceutical composition is administered daily.

[0030] 16. The method of any one of items 1-4, wherein the pharmaceutical composition is applied 1-3 times per week.

[0031] 17. The method of any one of items 1-4, wherein the pharmaceutical composition is administered weekly.

[0032] 18. The method of any one of items 1-4, wherein the pharmaceutical composition is applied once or twice a month.

[0033] 19. The method of item 5, wherein the muscle fibrosis is reduced by approximately 5%, 10%, 20%, 30%, 40%, or 50% compared to untreated patients.

[0034] 20. The method of item 5, wherein the muscle fibrosis is delayed by approximately 1 month, 6 months, 1 year or 5 years compared to untreated patients.

[0035] 21. The method of any one of items 1-4, wherein muscle contractility is maintained in the patient.

[0036] 22. The method of item 21, wherein the muscle contractility is maintained in the patient at approximately 90%, 80%, 70%, 60%, or 50% compared to a healthy subject.

[0037] 23. The method of any one of items 1-4, wherein muscle strength is maintained in the patient.

[0038] 24. The method of item 23, wherein the muscle strength is maintained in the patient at approximately 90%, 80%, 70%, 60%, or 50% compared to a healthy subject.

[0039] 25. The method of any one of items 19-24, wherein the muscle is skeletal muscle.

[0040] 26. The method of any one of items 19-24, wherein the muscle is cardiac muscle.

[0041] 27. The method of any one of claims 1-4, wherein the pharmaceutical composition causes the muscle cells to remain shortened compared to untreated muscle cells.

[0042] 28. The method of any one of claims 1-4, wherein the pharmaceutical composition causes the muscle cells to maintain a re-elongation rate compared to untreated muscle cells.

[0043] 29. The method of any one of claims 1-4, wherein the pharmaceutical composition maintains the contractility of the muscle cells compared to untreated muscle cells.

[0044] 30. The method of any one of claims 1-4, wherein the pharmaceutical composition keeps the muscle cells relaxed compared to untreated muscle cells.

[0045] 31. The method of any one of items 27-30, wherein the muscle cells are cardiomyocytes.

[0046] 32. The method of any one of items 27-30, wherein the muscle cell is a skeletal muscle cell.

[0047] 33. The method of any one of items 1-4, wherein the patient has muscular dystrophy.

[0048] 34. The method of item 33, wherein the muscular dystrophy is selected from the group consisting of: myotonic dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, limb girdle muscular dystrophy, facial scapular muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, peripheral muscular dystrophy, and Edwin muscular dystrophy.

[0049] 35. The method of any one of items 1-4, wherein the patient has inflammatory myopathy.

[0050] 36. The method of item 35, wherein the inflammatory myopathy is selected from the group consisting of polymyositis, dermatomyositis and inclusion body myositis.

[0051] 37. The method of any one of items 3-4, wherein the cardiomyopathy is caused by muscular dystrophy.

[0052] 38. The method of item 37, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, or X-linked dilated cardiomyopathy.

[0053] 39. The method of any one of claims 1-4, wherein the pharmaceutical composition comprises SEQ ID NO:15 or SEQ ID NO:20.

[0054] 40. The method of item 39, wherein the pharmaceutical composition is administered subcutaneously to a subject.

[0055] 41. The method of item 40, wherein the pharmaceutical composition is administered to the subject once or twice a month.

[0056] 42. The method of item 41, wherein the pharmaceutical composition is administered in a low dose.

[0057] 43. The method of item 42, wherein the pharmaceutical composition is administered at a dose of 1 mg / kg daily to 9 mg / kg daily.

[0058] 44. The method of item 43, wherein the subject has muscular dystrophy.

[0059] 45. The method of item 44, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, or X-linked dilated cardiomyopathy.

[0060] 46. ​​The method of item 45, wherein the subject has cardiomyopathy.

[0061] 47. The method of any one of claims 1-4, wherein the pharmaceutical composition comprising vasoactive intestinal peptide (VIP) and one or more elastin-like peptides (ELP) is formulated for sustained release.

[0062] 48. The method of any one of claims 3-4, wherein administration of the pharmaceutical composition maintains the shortening fraction compared to untreated myopathy subjects.

[0063] 49. The method of item 48, wherein the shortening score is maintained at approximately 20%–50% compared to untreated myopathy subjects.

[0064] 50. The method of any one of claims 3-4, wherein administration of the pharmaceutical composition increases the ventricular filling rate compared to untreated myopathy subjects.

[0065] 51. The method of item 50, wherein the ventricular filling velocity is increased by approximately 10-50% compared to untreated myopathy subjects.

[0066] 52. The method of any one of claims 3-4, wherein the administration of the pharmaceutical composition increases the maximum rate of pressure rise compared to an untreated myopathy subject.

[0067] 53. The method of 52, wherein the maximum rate of increase of said pressure is about 20% to about 50% compared to untreated myopathy subjects.

[0068] 54. The method of any one of claims 3-4, wherein administration of the pharmaceutical composition increases the relaxation Tau constant compared to untreated myopathy subjects.

[0069] 55. The method of item 54, wherein the relaxed Tau constant is increased by about 10% to about 50% compared to untreated myopathy subjects.

[0070] 56. The method of any one of claims 1-4, wherein the administration of the pharmaceutical composition reduces the collagen content in the muscle compared to an untreated myopathy subject.

[0071] 57. The method of 56, wherein the collagen content in the muscle is reduced by about 20% to about 50% compared to an untreated myopathy subject.

[0072] 58. The method of any one of claims 1-4, wherein administration of the pharmaceutical composition reduces the count of immune cells in the muscle compared to an untreated myopathy subject.

[0073] 59. The method of item 58, wherein the immune cell is a macrophage.

[0074] 60. The method of 59, wherein the number of macrophages in the muscle is reduced by approximately 20-50% compared to untreated myopathy subjects.

[0075] 61. The method of any one of items 56-60, wherein the muscle is the gastrocnemius, quadriceps femoris, diaphragm, tibialis anterior, and / or cardiac muscle.

[0076] 62. A method for treating myopathy, comprising administering to a patient in need a pharmaceutical composition comprising the amino acid sequence of SEQ ID NO:15.

[0077] 63. A method for preventing muscle contraction-induced injury, comprising administering to a subject in need a pharmaceutical composition containing the amino acid sequence of SEQ ID NO:15.

[0078] 64. A method for slowing the progression of cardiomyopathy, comprising administering to a subject in need a pharmaceutical composition containing the amino acid sequence of SEQ ID NO:15.

[0079] 65. A method for treating cardiomyopathy, comprising administering to a patient in need a pharmaceutical composition containing the amino acid sequence of SEQ ID NO:15.

[0080] 66. The method of any one of claims 62-65, wherein the pharmaceutical composition reduces muscle fibrosis. Brief description of the attached diagram

[0081] Figure 1A -B shows cardiac function as monitored by echocardiography: n = 10⁻¹¹, *P < 0.05 vs. control. Inset A shows fractional area shortening in MDX (muscular dystrophy-deficient mice). Inset B shows the E / A ratio in MDX mice. PB1046 treatment maintained fractional area shortening after 32 weeks of treatment. PB1046 administration before the development of cardiomyopathy in MDX and double knockout mice maintained the duration of QRS complexes and slowed the deterioration of cardiac function (fractional area shortening and E / A ratio) in MDX mice.

[0082] Figure 2A -E displays the in vivo assessment of left ventricular function in MDX mice during the terminal step. n = 4-5 (MDX) and n = 2 (DKO). * P < 0.05 vs. control. Both mdx and double knockout treated animals showed preserved dP / dt. max (Contraction) and faster Tau (relaxation).

[0083] Figure 3A -B shows the results of skeletal muscle physiological assessment in the extensor digitorum longus isolated from MDX mice. n = 4–16. Treatment with PB1046 maintained eccentric contraction compared to untreated mice, indicating that the muscles in the treatment group were more tolerant to contraction-induced damage.

[0084] Figure 4A -C shows collagen content in MDX mice as measured using Sirius Red staining (inset AB). Macrophage recognition was determined by immunohistochemistry (inset C) n = 4-7.* P<0.05 vs. control. As determined by collagen deposition, the degree of fibrosis was reduced in both skeletal muscle (gastrocnemius) and cardiac muscle. Invention Details

[0085] Musculomyopathy, which can affect skeletal and cardiac muscles, is a condition in which muscle fibers no longer function properly, leading to muscle weakness. Muscle weakness can cause muscle atrophy, paralysis, and even death. Cardiac dysfunction is a common manifestation of many musculomyopathy conditions and a common cause of death in individuals with these conditions.

[0086] Often, in hereditary myopathy such as muscular dystrophy, patients exhibit mutations in the dystrophin gene. The dystrophin gene plays both a structural and regulatory role in muscle contraction. Dystrophin is part of the larger transmembrane complex, the dystrophin glycoprotein complex (DGC) (Lapidos et al. (2004)), and its absence directly affects contractility. In many patients with muscle diseases (e.g., Duchenne or Beck muscular dystrophy, or dystrophin-associated cardiomyopathy), dystrophin may be completely absent or only partially functional. The absence of dystrophin increases intracellular calcium and leads to excessive nitric oxide production, which triggers protein degradation, fibrosis, necrosis, macrophage activation, and ultimately skeletal muscle and cardiomyopathy (Townsend et al. (2011); Judge et al. (2011)).

[0087] In cardiomyocytes, these pathological consequences are partly mediated by increased calcium permeability and increased myocyte calcium concentration, which subsequently initiate a cascade of events, including the expression of intracellular inflammatory cytokines and the inflammatory cell response to myocyte necrosis (Zhou et al. (2010); Klinger et al. (2012)). Furthermore, disruption of the intracellular cyclic guanosine monophosphate (cGMP) signaling pathway directly contributes to muscle dysfunction (Lapidos et al. (2004)); Townsend et al. (2011); Byers et al. (1991)).

[0088] In addition to the direct impact on muscle function, the loss of dystrophin and the corresponding changes in nitric oxide synthase activity lead to mitochondrial and metabolic stress, which stimulates cytokine production and apoptosis. These events result in further loss of muscle mass, loss of muscle function, and ultimately fibrosis. Specifically, increased cardiomyocyte stress leads to the production of IL-6 and TGF-β, which stimulate fibroblasts, collagen synthesis, and macrophage infiltration, all of which contribute to increased fibrosis.

[0089] In healthy muscle, following acute tissue injury, infiltrating inflammatory cells and intrinsic stem cells restore tissue homeostasis. However, in long-term tissue injury processes such as muscular dystrophy, inflammatory cell infiltration and fibroblast activation persist, while the repair capacity of stem cells (e.g., satellite cells) diminishes. In various malnutritions, muscle undergoes a constant cycle of fibrosis degradation associated with chronic inflammation. In DMD, the satellite cell population responsible for repairing muscle damage depletes over time or loses its ability to mediate repair, and muscle tissue is gradually replaced by adipose and fibrous tissue. Both fibrosis and loss of muscle tissue in muscular dystrophy reduce motor and contractile functions.

[0090] Myofibrosis is the excessive formation of fibrous scar tissue bands between muscle fibers. Although fibrosis can develop in any organ, skeletal muscle fibrosis and cardiac muscle fibrosis are the only known types of muscle fibrosis. Fibrous scar tissue develops after muscle injury to fill the open spaces in the damaged muscle, providing more surface area for regenerating muscle fibers to adhere to it. The connective tissue cells containing the scar tissue cannot contract and relax to allow movement. Once excessive fibrous scar tissue begins to form, the muscle gradually weakens.

[0091] Vasoactive intestinal peptide (VIP) plays a role in the development of fibrosis. Among other activities, VIP acts through VPAC1 and VPAC2 receptors to increase cAMP and cGMP levels. Importantly, in murine macrophages, VIP has been shown to reduce the production of TGF-β (an important regulator of cardiac fibrosis in DMD) (Ameen et al. (2010); Burks et al. (2011); Bujak et al. (2007)). VIP also stimulates regulatory T cells (T cells) that suppress muscle inflammation and damage in muscular dystrophy. reg (Villata et al. (2014)).

[0092] This invention provides a method for preventing, delaying, or improving the onset of symptoms (including the development of muscle fibrosis) in patients with myopathy by administering a stable, long-acting vasoactive intestinal peptide (VIP) therapeutic agent.

[0093] Vasoactive intestinal peptides

[0094] Vasoactive intestinal peptide (VIP) is a neuropeptide that binds to both VPAC1 and VPAC2 receptors. VIP and its functional and structural analogues are known to have a variety of physiological functions, including smooth muscle relaxation (bronchodilation, intestinal peristalsis) and regulation of various immune functions (anti-inflammation, immune cell protection) (Hinkle et al. (2005)).

[0095] Mature VIP has 28 amino acid residues and has the following sequence: HSDAVFTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 17). VIP is derived from the processing of a 170-amino acid precursor molecule, prepro-VIP. The structure of VIP and exemplary analogues have been described in U.S. Patents 4,835,252, 4,939,224, 5,141,924, 4,734,400, 4,605,641, 6,080,837, 6,316,593, 5,677,419, 5,972,883, 6,489,297, 7,094,755, and 6,608,174.

[0096] In some aspects, the present invention provides therapeutic compositions comprising one or more VIP peptides, variants, or analogs. In some embodiments, the VIP peptide is a variant. In some embodiments, the VIP peptide is an analog. In some embodiments, the VIP peptide is a mature VIP (e.g., SEQ ID NO:17). In some embodiments, the VIP peptide is modified compared to a mature VIP (e.g., SEQ ID NO:17). In some embodiments, the modified VIP peptide is a variant compared to a mature VIP (e.g., SEQ ID NO:17). In some embodiments, the modified VIP peptide is a functional variant compared to a mature VIP (e.g., SEQ ID NO:17). In some embodiments, the modified VIP peptide is a functional analog compared to a mature VIP (e.g., SEQ ID NO:17).

[0097] In some embodiments, the modified VIP peptide contains one or more amino acid substitutions compared to the amino acid sequence of the mature VIP (e.g., SEQ ID NO: 17). In some embodiments, 1-20 amino acids are substituted compared to the amino acid sequence of the mature VIP (SEQ ID NO: 17). In some embodiments, the modified VIP peptide contains about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions compared to the amino acid sequence of the mature VIP (SEQ ID NO: 17).

[0098] In some embodiments, the modified VIP peptide contains one or more amino acid deletions compared to the mature VIP amino acid sequence (SEQ ID NO:17). In some embodiments, 1-20 amino acids are deleted compared to the mature VIP amino acid sequence (SEQ ID NO:17). In some embodiments, the modified VIP peptide has about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid deletions compared to the mature VIP amino acid sequence (SEQ ID NO:17). In some embodiments, 1-10 amino acids are deleted at either end compared to the mature VIP amino acid sequence (SEQ ID NO:17). In some embodiments, 1-10 amino acids are deleted at both ends compared to the mature VIP amino acid sequence (SEQ ID NO:17). In some embodiments, the amino acid sequence of the modified VIP peptide is at least about 70% identical to the mature VIP amino acid sequence (SEQ ID NO:17). In some embodiments, the modified VIP peptide has an amino acid sequence that is approximately 70%, 80%, 85%, 90%, 95%, 96%, or 97% identical to the mature VIP amino acid sequence (SEQ ID NO:17). Percentage identity can be calculated using the alignment program ClustalW2, available at http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / . The following default parameters can be used for pairwise alignment: protein weight matrix = BLOSUM62, nick opening = 10; nick extension = 0.1.

[0099] In various aspects, the present invention provides modified VIP peptides that have a relative receptor preference for VPAC2 or VPAC1 compared to mature VIP (i.e., SEQ ID NO:17). For example, the modified VIP peptide may have a relative binding preference for VPAC2 that is at least about 2:1, about 5:1, about 10:1, about 25:1, about 50:1, about 100:1, about 500:1 or more than that for VPAC1. In other embodiments, the modified VIP peptide may have a relative binding preference for VPAC1 that is at least about 2:1, about 5:1, about 10:1, about 25:1, about 50:1, about 100:1, about 500:1 or more than that for VPAC2. For example, in some embodiments, the modified VIP peptide activates the VPAC2 receptor with an EC50 that is about 2-4 times higher than that of mature human VIP (SEQ ID NO:17). However, in some embodiments, the same modified VIP peptide is 50 or 100 times less potent than the mature, unmodified human VIP peptide (SEQ ID NO:17) in the activated VPAC1 receptor.

[0100] In some embodiments, the modified VIP peptide contains additional amino acid residues compared to the mature VIP (SEQ ID NO:17). In some embodiments, the modified VIP peptide contains one or more amino acids added to the N- and / or C-terminus compared to the mature VIP (SEQ ID NO:17). Such a modified VIP peptide may contain a modified N-terminal region, such as by adding 1 to about 500 amino acids to the N-terminal histidine of the VIP, the added amino acids possibly comprising amino acid sequences from heterologous mammals (e.g., non-humans). The additional sequence added to the N-terminus of the VIP can be any sequence, including biologically active and biologically inert sequences of 1 to about 100, 1 to about 50, 1 to about 20, 1 to about 10, and 1 to about 5 amino acids. For example, the modified VIP may contain a single methionine residue at the N-terminus of the native N-terminal histidine of the mature VIP. Although methionine can sometimes be removed by methionine aminopeptidase (MA) in bacterial expression systems, histidine (H) is the least favorable residue at position 2 for MA. In some embodiments, the modified VIP peptide is SEQ ID NO:14. This modified VIP peptide containing an N-terminal methionine can be prepared in *E. coli* or other bacterial or yeast expression systems because methionine will not be removed by *E. coli* when the adjacent amino acid is histidine. Alternatively, the N-terminal amino acid can be any naturally occurring amino acid, namely alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine, and proline. In other embodiments, the VIP peptide can be activated by a peptidase or protease, such as an endogenous peptidase or protease. Such an activatable sequence is described, for example, in International Application No. PCT / US2009 / 068656. As used herein, the terms "peptidase" and "protease" are used interchangeably. For example, the VIP peptide can be designed to be activated by a dipeptidyl peptidase. Exemplary dipeptidyl peptidases include dipeptidyl peptidase-1 (DPP-I), dipeptidyl peptidase-3 (DPP-III), dipeptidyl peptidase-4 (DPP-IV), dipeptidyl peptidase-6 (DPP-VI), dipeptidyl peptidase-7 (DPP-VII), dipeptidyl peptidase-8 (DPP-VIII), dipeptidyl peptidase-9 (DPP-IX), and dipeptidyl peptidase-10 (DPP-X). The substrate sequences of these dipeptidases are known.

[0101] In some embodiments, the N-terminus of the activatable VIP peptide may have a structure ZN, where Z is a substrate of a dipeptidase (e.g., Z is removed by exposure to the dipeptidase), and N is the N-terminus of the VIP. The activatable VIP peptide may have an N-terminal sequence of the formula MXN, where M is methionine, X is Pro, Ala, or Ser, and N is the N-terminus of the VIP or a VIP analogue. In this manner, M and X will be sensitive to and removed by the host cell (e.g., *E. coli*) and / or a subsequent dipeptidase (e.g., DPP-IV). Alternatively, the N-terminal sequence of the activatable VIP may be X1-X2-N, where X1 is Gly, Ala, Ser, Cys, Thr, Val, or Pro; X2 is Pro, Ala, or Ser; and N is the N-terminus of the VIP. X1-X2 is a substrate of a dipeptidase (e.g., DPP-IV), and dipeptidase digestion will expose N, which is the expected N-terminus of the VIP or a VIP analogue. In such embodiments, the VIP peptide can be produced by expressing a construct encoding M-X1-X2-N (where M is methionine) in a host cell (e.g., *E. coli*), because Gly, Ala, Ser, Cys, Thr, Val, or Pro at the second position will transmit a signal to remove Met, thereby leaving X1-X2 at the N-terminus, which can be activated in vivo by a dipeptidase (e.g., DPP-IV). In some embodiments, the peptidase may be present in the body and can act on the activated VIP peptide after injection. In some embodiments, the activated VIP peptide comprises an amino acid sequence MAA added to the N-terminus compared to the mature VIP (e.g., SEQ ID NO: 17). In some embodiments, the activated VIP peptide is SEQ ID NO: 18.

[0102] In other embodiments, the N-terminus of the modified activatable VIP peptide has the structure MZN, where M is methionine, Z is a substrate of a dipeptidase (e.g., Z is removed by exposure to the dipeptidase), and N is the non-His N-terminus of the activatable VIP. For example, the modified activatable VIP peptide may have an N-terminal sequence of the formula MXN, where M is methionine; X is Pro, Ala, or Ser; and N is the non-His N-terminus of the activatable VIP. In this manner, M and X will be sensitive to and removed by the host cell (e.g., *E. coli*) and / or a subsequent dipeptidase (e.g., DPP-IV). Alternatively, the N-terminal sequence of the activatable VIP peptide may be X1-X2-N, where X1 is Gly, Ala, Ser, Cys, Thr, Val, or Pro; X2 is Pro, Ala, or Ser; and N is the non-His N-terminus of the activatable VIP. X1-X2 are substrates for dipeptidases (e.g., DPP-IV), and dipeptidase digestion will expose N, which is the N-terminus of the non-His conjugate VIP.

[0103] In other embodiments, the N-terminus of the activatable VIP peptide has the structure MZSN, where M is methionine; Z is a substrate of a dipeptidase (e.g., Z is removed by exposure to the dipeptidase); N is the N-terminus (His) of the mature VIP; and S is one or more amino acids that will be exposed after dipeptidase digestion, providing the activatable VIP as described above. For example, the activatable VIP peptide may have an N-terminal sequence of the formula MXSN, where M is methionine, X is Pro, Ala, or Ser; N is the N-terminus of the mature VIP (e.g., SEQ ID NO: 17); and S is one or more amino acids that will be exposed after dipeptidase digestion, providing receptor preference. Alternatively, the N-terminal sequence of the activatable VIP peptide may be X1-X2-SN, where X1 is Gly, Ala, Ser, Cys, Thr, Val, or Pro; X2 is Pro, Ala, or Ser; N is the non-His N-terminus of the VIP; and S is one or more amino acids that will be exposed after dipeptidase digestion. X1-X2 are substrates for dipeptidases (e.g., DPP-IV), and dipeptidase digestion will expose S.

[0104] In other embodiments, the VIP peptide is modified by fusion with a mammalian heteroprotein, such as a mammalian protein effective for extending the half-life of a therapeutic molecule. Such sequences can be mammalian sequences, such as albumin, transferrin, or antibody Fc sequences. Such sequences are described in U.S. Patent Nos. 7,238,667 (particularly for albumin fusions), 7,176,278 (particularly for transferrin fusions), and 5,766,883. In some embodiments, the VIP peptide is modified by fusion with a mammalian heteroprotein at the N-terminus. In some embodiments, the VIP peptide is modified by fusion with a mammalian heteroprotein at the C-terminus. In some embodiments, the VIP peptide is modified by fusion with a mammalian heteroprotein at both the N-terminus and C-terminus.

[0105] In some embodiments, N-terminal chemical modifications to the N-terminus of the VIP peptide provide receptor preference. Chemical modifications of proteins and methods thereof are known in the art. Non-limiting exemplary chemical modifications include PEGylation, methylglyoxylation, reductive alkylation, performic acid oxidation, succinylation, aminoethylation, and lipoylation (Clifton, New Protein Techniques, New Jersey: Humana Press, 1985. ISBX. 0-89603-126-8. Volume 3 of. Methods in Molecular Biology). Chemical groups such as PEGylation can be attached to previously described cysteine, methionine, histidine, lysine, arginine, tryptophan, tyrosine, and carboxyl modifications (see Lundblad, Techniques in Protein Modification, CRC Press, 1995).

[0106] In other embodiments, the VIP peptide is modified by fusion with a protein containing a repeating amino acid sequence, such as a sequence containing proline, alanine, and serine (e.g., PASylation (Schlapschy, M. et al. (2013)) or an XTEN sequence (Schellenberger, V. et al. (2009)).

[0107] elastin-like peptides

[0108] In some aspects, the present invention provides therapeutic compositions comprising a vasoactive intestinal peptide and one or more elastin-like peptides (ELPs). In some embodiments, the VIP peptide and one or more ELPs are fused together. In some embodiments, the VIP peptide and one or more ELPs are generated as a recombinant fusion polypeptide. In some embodiments, the therapeutic composition comprises a vasoactive intestinal peptide and one or more ELPs as separate molecules. In other embodiments, the composition comprises a VIP-ELP fusion protein and ELPs as separate molecules. In some embodiments, the composition comprises SEQ ID NO:15. In some embodiments, the composition comprises SEQ ID NO:19. In some embodiments, the composition comprises SEQ ID NO:16.

[0109] The ELP sequence comprises a structural peptide unit or sequence that is associated with or is an elastin mimic. The ELP sequence is constructed from structural units of 3 to 20 amino acids, or in some embodiments, from structural units of 4 to 10 amino acids, such as 4, 5, or 6 amino acids. The length of a single structural unit can be varied or can be uniform. For example, the structural units include those defined by SEQ ID NO:1-13, which may be used as repeating structural units, including tandem repeats, or may be used in some combinations. Thus, the ELP comprises a basic structural unit selected from SEQ ID NO:1-13.

[0110] In some embodiments, the amino acid sequence of the ELP unit is about 1 to about 500 structural units, or in some embodiments about 9 to about 200 structural units, or in some embodiments about 10 to about 200 structural units, or in some embodiments about 50 to about 200 structural units, or in some embodiments about 80 to about 200 structural units, or about 80 to about 150 structural units, as defined by one or a combination of units defined by SEQ ID NO:1-13. Thus, the structural unit may uniformly have a length of about 50 to about 2000 amino acid residues, or about 100 to about 800 amino acid residues, or about 200 to about 700 amino acid residues, or about 400 to about 600 amino acid residues. In an exemplary embodiment, the amino acid sequence of the ELP structural unit comprises about 3 structural units, about 7 structural units, about 9 structural units, about 10 structural units, about 15 structural units, about 20 structural units, about 40 structural units, about 80 structural units, about 90 structural units, about 100 structural units, about 120 structural units, about 140 structural units, about 144 structural units, about 160 structural units, about 180 structural units, about 200 structural units, or about 500 structural units. In an exemplary embodiment, the structural unit may uniformly have a length of about 45 amino acid residues, about 90 amino acid residues, about 100 amino acid residues, about 200 amino acid residues, about 300 amino acid residues, about 400 amino acid residues, about 500 amino acid residues, about 600 amino acid residues, about 700 amino acid residues, about 720 amino acid residues, about 800 amino acid residues, or about 1000 amino acid residues.

[0111] Using selected formulations, the ELP amino acid sequence exhibits a visible and reversible inverse phase transition. That is, the ELP amino acid sequence can be structurally disordered and highly soluble in formulations below the transition temperature (Tt), but exhibits a rapid (2-3°C range) disorder-to-order phase transition as the formulation temperature rises above Tt. Besides temperature, the length of the amino acid polymer, amino acid composition, ionic strength, pH, pressure, temperature, selected solvent, presence of organic solutes, and protein concentration can also affect the nature of the transition, and these can be adjusted in the solvent for a desirable absorption profile. The absorption profile can be readily tested by determining the plasma concentration and activity of the active agent over time.

[0112] In some implementations, the ELP component may be formed from polypeptide structural units (e.g., tetrapeptides, pentapeptides, hexapeptides, octapeptides, or nonapeptides), including but not limited to:

[0113] (a) Tetrapeptide Val-Pro-Gly-Gly, or VPGG (SEQ ID NO:1);

[0114] (b) Tetrapeptide Ile-Pro-Gly-Gly, or IPGG (SEQ ID NO:2);

[0115] (c) Pentapeptide Val-Pro-Gly-X-Gly (SEQ ID NO:3) or VPGXG, wherein X is any natural or non-natural amino acid residue, and wherein X is optionally varied in polymer or oligomeric repeats;

[0116] (d) Pentapeptide Ala-Val-Gly-Val-Pro or AVGVP (SEQ ID NO:4);

[0117] (e) Pentapeptide Ile-Pro-Gly-X-Gly or IPGXG (SEQ ID NO:5), wherein X is any natural or non-natural amino acid residue, and wherein X may optionally vary in a polymer or oligomeric repeat.

[0118] (e) Pentapeptide Ile-Pro-Gly-Val-Gly or IPGVG (SEQ ID NO:6);

[0119] (f) Pentapeptide Leu-Pro-Gly-X-Gly or LPGXG (SEQ ID NO:7), wherein X is any natural or non-natural amino acid residue, and wherein X is optionally varied in polymer or oligomeric repeat;

[0120] (g) Pentapeptide Leu-Pro-Gly-Val-Gly or LPGVG (SEQ ID NO:8);

[0121] (h) Hexapeptide Val-Ala-Pro-Gly-Val-Gly or VAPGVG (SEQ ID NO:9);

[0122] (i) Octapeptide Gly-Val-Gly-Val-Pro-Gly-Val-Gly or GVGVPGVG (SEQ ID NO:10);

[0123] (j) Nonapeptide Val-Pro-Gly-Phe-Gly-Val-Gly-Ala-Gly or VPGFGVGAG (SEQ ID NO:11);

[0124] (k) nonapeptide Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Gly or VPGVGVPGG (SEQ ID NO:12); and

[0125] (l) Pentapeptide Xaa-Pro-Gly-Val-Gly or XPGVG (SEQ ID NO:13), wherein X is any natural or non-natural amino acid residue, and wherein X is optionally varied in polymer or oligomeric repeat.

[0126] The polypeptide structural units defined in SEQ ID NO:1-13 form elastin-like peptide assemblies, or may be used in combination to form ELPs. In some embodiments, the ELP comprises more than one structural unit. In some embodiments, the ELP comprises any two or more structural units of SEQ ID NO:1-13, which may be combined in any manner. In some embodiments, the two or more structural units are identical and repeated in tandem. In some embodiments, the two or more structural units are different and repeated alternately. In some embodiments, the ELP comprises structural units that are repeated in tandem in one or more portions of the sequence, and units of different structures that are repeated alternately in other portions of the sequence. In some embodiments, the ELP assembly is entirely (or almost entirely) formed of one or a combination of structural units selected from SEQ ID NO:1-13 (e.g., 2, 3, or 4). In other embodiments, at least 75% or at least 80% or at least 90% of the ELP assembly is formed of one or a combination of structural units selected from SEQ ID NO:1-13. In some embodiments, the ELP comprises repeating units containing tandem repeating units of Val-Pro-Gly-X-Gly (SEQ ID NO:3), wherein X is as defined above, and the percentage of Val-Pro-Gly-X-Gly units used is greater than about 50%, or greater than about 75%, or greater than about 85%, or greater than about 95% of the entire ELP assembly (which may contain structural units other than VPGXG). The ELP may comprise a motif of 5-15 structural units (e.g., about 10 structural units) of SEQ ID NO:3, wherein the guest residue X differs in at least two or at least three units of the motif. The guest residue may be independently selected from, for example, nonpolar or hydrophobic residues, such as amino acids V, I, L, A, G, and W (and may be selected to retain desirable inverse phase transition properties). In some embodiments, the guest residue is selected from V, G, and A. In some embodiments, the ELP comprises the ELP 1 series (VPGXG:V5A2G3). In some embodiments, the ELP comprises the amino acid sequence of SEQ ID NO:21. In some embodiments, the ELP comprises the ELP 4 series (VPGXG:V-5). In some embodiments, the ELP comprises a combination of the ELP 1 and ELP 4 series. Without being theoretically limited, the differences in hydrophobicity of the ELP polymers are determined by the guest residues and their ratios, wherein the ELP 4 series is more hydrophobic than the ELP 1 series.

[0127] In some implementations, ELP is the ELP-1 series, which includes [VPGXG]. mWhere m is any number from 1 to 200, each X is selected from V, G, and A, and the ratio of V:G:A can be approximately 5:3:2. In some embodiments, ELP comprises [VPGXG]. 90 Each X is selected from V, G, and A, and the ratio of V:G:A can be approximately 5:3:2. In some embodiments, ELP comprises [VPGXG]. 120 Each X is selected from V, G, and A, and the ratio of V:G:A can be approximately 5:3:2.

[0128] In some implementations, ELP includes [VPGXG] 144 Each X is selected from V, G, and A, and the ratio of V:G:A can be approximately 7:2:0. In some embodiments, ELP comprises [VPGXG]. 144 Each X is selected from V, G, and A, and the ratio of V:G:A can be approximately 7:0:2. In some embodiments, ELP comprises [VPGXG]. 144 Each X is selected from V, G, and A, and the ratio of V:G:A can be approximately 6:0:3. In some embodiments, ELP comprises [VPGXG]. 144 Each X is selected from V, G, and A, and the ratio of V:G:A can be approximately 5:2:2.

[0129] In some implementations, ELP includes [XPGVG] m , where m is any number from 1 to 200, and each X is selected from V, G, and A. In some implementations, ELP contains [XPGVG] 144 Where m is any number from 1 to 200, and each X is selected from V, G, and A, where the ratio of V:G:A is approximately 5:0:4. In some implementations, ELP contains [XPGVG]. 144 Each X is selected from V, G, and A, and the ratio of V:G:A is approximately 5:0:4.

[0130] Alternatively, ELP includes [VPGVG]. 90 Or [VPGVG] 120 The ELP-4 series. This ELP's 120 structural units can provide a transition temperature for proteins ranging from approximately 0.005 to approximately 0.05 mg / ml (e.g., approximately 0.01 mg / ml) at approximately 37°C. Alternatively, the ELP contains [VPGXG]. 144 Or [XPGVG] 144 For example, the 144 structural units of these ELPs can provide a transition temperature of approximately 28°C to 35°C.

[0131] In some embodiments, the ELP comprises repeating units comprising tandem repeating units of Xaa-Pro-Gly-Val-Gly (SEQ ID NO: 13), wherein X is as defined above, and wherein the percentage of the Xaa-Pro-Gly-Val-Gly unit relative to the complete ELP assembly (which may include structural units other than XPGVG) is greater than about 50%, or greater than about 75%, or greater than about 85%, or greater than about 95% of the ELP. The ELP may comprise a motif of 5-15 structural units (e.g., about 9 structural units) of SEQ ID NO: 13, wherein guest residue X varies between at least 2 or at least 3 units in the motif. Guest residues may be independently selected from, for example, nonpolar or hydrophobic residues, such as amino acids V, I, L, A, G, and W (and may be selected to retain desirable inverse phase transition properties). In some embodiments, guest residues are selected from V and A.

[0132] In some embodiments, the ELP comprises repeating units comprising tandem repeating units of any one or a combination of SEQ ID NO:1-13. In one embodiment, the ELP comprises repeats of any two or more combinations of SEQ ID NO:1-13. In some embodiments, the ELP comprises repeats of SEQ ID NO:3 and SEQ ID NO:13. In some embodiments, the ELP comprises repeats of SEQ ID NO:3 and SEQ ID NO:13, wherein the guest residues are independently selected from, for example, nonpolar or hydrophobic residues, such as amino acids V, I, L, A, G, and W (and are selected to retain desirable inverse phase transition properties). In some embodiments, the guest residues are selected from V and A. In some embodiments, the ELP comprises a decimer containing five copies of the pentapeptide disclosed herein. In some embodiments, the ELP comprises a decimer containing any combination of SEQ ID NO:3 and 13. In some embodiments, the ELP comprises alternating sequences between SEQ ID NO:3 and 13. In some embodiments, the ELP comprises a decimer containing nine copies of one or more ELP structural units disclosed herein. In some embodiments, the ELP comprises a 9-netamer containing nine copies of the pentapeptide disclosed herein. In some embodiments, the ELP comprises a 9-netamer of any combination of SEQ ID NO:3 and 13. In some embodiments, the ELP comprises alternating sequences between SEQ ID NO:3 and 13. ELPs with different numbers of 9-netamers can be combined to produce ELPs having, for example, 18, 27, 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, 153, 162, 171, or 180 copies of the 9-netamer. In some embodiments, the ELP comprises the amino acid sequence of SEQ ID NO:20.

[0133] In some embodiments, the ELP comprises a 9-mer containing SEQ ID NO:3, wherein the guest residues are selected from V, G, and A. In some embodiments, the ELP comprises a 9-mer containing SEQ ID NO:3, wherein the ratio of V, G, and A is 7:2:0 (α). In some embodiments, the ELP comprises a 9-mer containing SEQ ID NO:3, wherein the ratio of V, G, and A is 7:0:2 (βv1). In some embodiments, the ELP comprises a 9-mer containing SEQ ID NO:3, wherein the ratio of V, G, and A is 6:0:3 (βv2). In some embodiments, the ELP comprises a 9-mer containing SEQ ID NO:3, wherein the ratio of V, G, and A is 5:2:2 (γ). In some embodiments, the ELP comprises a 9-mer containing SEQ ID NO:13, wherein the guest residues are selected from V, G, and A. In some embodiments, the ELP comprises a 9-mer containing SEQ ID NO:13, wherein the ratio of V, G, and A is 5:0:4 (δ).

[0134] In some embodiments, the ELP comprises a combination of α, βv1, βv2, and / or δ9 polymers. For example, a γELP is constructed by alternating copies of 16 α9 polymers and βv1 9 polymers up to a total of 144 polymers. In some embodiments, the ELP comprises a combination of α and βv1 9 polymers. In some embodiments, the ELP comprises a combination of α and βv2 9 polymers. In some embodiments, the ELP comprises a combination of α and δ9 polymers. In some embodiments, the ELP comprises a combination of βv1 and βv2 9 polymers. In some embodiments, the ELP comprises a combination of βv1 and δ9 polymers. In some embodiments, the ELP comprises a combination of βv2 and δ9 polymers. In some embodiments, the ELP comprises a combination of α, βv1, and βv2 9 polymers. In some embodiments, the ELP comprises a combination of α, βv1, and δ9 polymers. In some embodiments, the ELP comprises a combination of α, βv2, and δ9 polymers. For example, in a specific arrangement, ELPβv2 may contain guest residues in its structural units that iterate in the following sequence: AVAVVAVAV. The iterative sequence in the ELP may be repeated sequentially about 10, about 15, about 16, about 20, about 25, about 30, or about 35 or more times. In some aspects, the ELP contains about 10 to about 20 iterations of the sequence. In other aspects, the ELP contains about 15 to 20 iterations of the sequence. In some aspects, the ELP contains about 16 iterations of the sequence.

[0135] In some embodiments, the ELP comprises a decameric unit containing 10 copies of one or more ELP structural units disclosed herein. In some embodiments, the ELP comprises a decameric unit containing 10 copies of the pentapeptide disclosed herein. In some embodiments, the ELP comprises a decameric unit containing any combination of SEQ ID NO:3 and 13, and in some embodiments, the ELP comprises alternating sequences between SEQ ID NO:3 and 13. Different numbers of decameric ELPs can be combined to produce ELPs having, for example, 20, 30, 40, 60, 90, 100, 120, 150, 160, or 200 copies of the decameric unit.

[0136] In some embodiments, the ELP can form a β-turn structure. Exemplary peptide sequences suitable for generating β-turn structures are described in International Patent Application PCT / US96 / 05186. For example, the fourth residue (X) in the sequence VPGXG can be altered without eliminating the formation of the β-turn.

[0137] An exemplary ELP structure can be represented using the notation ELPk[X]. i Y j The description is given by -n], where k refers to a specific ELP repeating unit, the uppercase letters in parentheses are single-letter amino acid codes and their corresponding subscripts indicate the relative proportion of each guest residue X in the structural unit (if applicable), and n describes the total length of the ELP in terms of the number of structural repeats. For example, ELP1[V5A2G3-10] refers to an ELP component containing 10 pentapeptide VPGXG repeat units, wherein X is valine, alanine, and glycine in a relative ratio of approximately 5:2:3; ELP1[K1V2F1-4] refers to an ELP component containing 4 pentapeptide VPGXG repeat units, wherein X is lysine, valine, and phenylalanine in a relative ratio of approximately 1:2:1; ELP1[K1V7F1-9] refers to a polypeptide containing 9 pentapeptide VPGXG repeat units, wherein X is lysine, valine, and phenylalanine in a relative ratio of approximately 1:7:1; ELP1[V-5] refers to a polypeptide containing 5 pentapeptide VPGXG repeat units, wherein X is valine; ELP1[V-20] refers to a polypeptide containing 20 pentapeptide VPGXG repeat units, wherein X is valine; ELP2[5] refers to a polypeptide containing 5 pentapeptide AVGVP repeat units (SEQ ID). NO:4), ELP3[V-5] refers to a polypeptide containing 5 pentapeptide IPGXG repeat units (SEQ ID NO:5), where X is valine; ELP4[V-5] refers to a polypeptide containing 5 pentapeptide LPGXG repeat units (SEQ ID NO:7), where X is valine.

[0138] For ELPs, Tt is a function of the hydrophobicity of the guest residues. Therefore, ELPs can be synthesized by altering the identity and mole fraction of the guest residues, exhibiting inverse conversion over a wide range. Thus, the Tt for a given ELP length can be reduced by incorporating a larger fraction of hydrophobic guest residues into the ELP sequence. Examples of suitable hydrophobic guest residues include valine, leucine, isoleucine, phenylalanine, tryptophan, and methionine. Moderately hydrophobic tyrosine can also be used. Conversely, Tt can be increased by incorporating residues selected from the following: glutamic acid, cysteine, lysine, aspartic acid, alanine, asparagine, serine, threonine, glycine, arginine, and glutamine.

[0139] For peptides with a molecular weight >100,000 Da, the hydrophobicity measure disclosed in PCT / US96 / 05186 provides a way to predict the approximate Tt of a specific ELP sequence. For peptides with a molecular weight <100,000 Da, Tt can be predicted or determined by the following quadratic function: Tt = M0 + M1X + M2X², where X is the molecular weight (MW) of the fusion protein, and M0 = 116.21; M1 = -1.7499; M2 = 0.010349.

[0140] In some embodiments, the ELP is selected or designed to provide a Tt ranging from about 10 to about 37°C under preparation conditions, such as about 20 to about 37°C, or about 25 to about 37°C. In some embodiments, the transition temperature under physiological conditions (e.g., 0.9% saline) is about 34 to 36°C, taking into account slightly lower ambient temperatures.

[0141] Elastin-like peptide (ELP) protein polymers and recombinant fusion proteins can be prepared as described in U.S. Patent Publication No. 2010 / 0022455. In some embodiments, ELPs are constructed via recursive ligation to rapidly clone highly repetitive polypeptides of any sequence and specified length across a wide range of molecular weights. In a single cycle, the two halves of a parental plasmid containing copies of oligomers are ligated together, thereby dimerizing the oligomers and reconstructing the functional plasmid. This process is repeated recursively to assemble oligomeric genes with the desired number of repetitive sequences. For example, an ELP structural subunit (e.g., a pentapeptide or a 9-mer of a pentapeptide) is inserted into a vector. The vector is digested, and another ELP structural unit (e.g., a pentapeptide or a 9-mer of a pentapeptide) is inserted. Each subsequent digestion and ligation cycle doubles the number of ELP structural units contained in the resulting vector until the ELP polymer is of the desired length.

[0142] In other embodiments, the ELP comprises a random coil or non-spherical extended structure. For example, the ELP comprises the amino acid sequences disclosed in U.S. Patent Publication No. 2008 / 0286808, WIPO Patent Publication No. 2008 / 155134, and U.S. Patent Publication No. 2011 / 0123487.

[0143] For example, in some embodiments, the ELP amino acid sequence comprises a non-structural recombinant polymer of at least 40 amino acids. For instance, a non-structural polymer can be defined where the sum of glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) constitutes more than about 80% of the total amino acids. In some embodiments, at least 50% of the amino acids lack secondary structure, as determined by the Chou-Fasman algorithm. The non-structural polymer comprises more than about 100, 150, 200, or more consecutive amino acids. In some embodiments, the amino acid sequence forms a random coil domain. In particular, peptides or amino acid polymers having or forming a “random coil conformation” substantially lack defined secondary and tertiary structures.

[0144] In several embodiments, the intended subject is a human with a body temperature of about 37°C, and the therapeutic agent is designed to provide sustained release at or near this temperature (e.g., between about 28°C and about 37°C). Even if the body temperature remains constant, the slow release into circulation, with reversed hydrogen bonding and / or hydrophobic interactions, is driven by a decrease in concentration as the product diffuses at the injection site. In other embodiments, the subject is a non-human mammal, and the therapeutic agent is designed to exhibit sustained release at mammalian body temperatures (which in some embodiments may be about 30 to about 40°C, such as for some domesticated pets (e.g., dogs or cats)) or livestock (e.g., cattle, horses, sheep, or pigs)). Generally, the Tt is higher than the storage conditions of the formulation (which may be about 2°C to about 30°C, or about 10°C to about 25°C, or about 15°C to about 22°C, or about 2°C to about 8°C), thus retaining the therapeutic agent in an injectable solution. Alternatively, the therapeutic agent may be frozen, for example, at about -80°C to about -20°C.

[0145] In some embodiments, the ELP can provide a transition temperature in the range of 27°C to 36°C. In some embodiments, the ELP can provide a transition temperature in the range of 28°C to 35°C. In some embodiments, the ELP can provide a transition temperature in the range of 29°C to 34°C. In some embodiments, the ELP can provide a transition temperature in the range of 27°C to 33°C. In some embodiments, the ELP can provide a transition temperature in the range of 30°C to 33°C. In some embodiments, the ELP can provide a transition temperature in the range of 31°C to 31°C. In some embodiments, the ELP can provide a transition temperature in the range of 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or 36°C. In some embodiments, when the protein concentration in 110 mM NaCl is 10 mg / mL, the ELP can provide a transition temperature in the range of 28°C to 35°C.

[0146] In some implementations, ELP protein polymers are constructed via recursive ligation to rapidly clone DNA encoding highly repetitive polypeptides of any sequence and specified length across a wide range of molecular weights. In a single cycle, the two halves of a parental plasmid, each containing an oligomeric copy, are ligated together, thereby dimerizing the oligomer and reconstructing the functional plasmid. This process is repeated recursively to assemble oligomeric genes with the desired number of repetitive sequences. For example, an ELP structural subunit (e.g., a pentapeptide or a 9-mer of a pentapeptide) is inserted into a vector. The vector is digested, and another ELP structural unit (e.g., a pentapeptide or a 9-mer of a pentapeptide) is inserted. Each subsequent digestion and ligation cycle doubles the number of ELP structural units contained in the resulting vector until the ELP polymer is of the desired length. Different lengths of ELPs can be easily constructed by varying the number of pentapeptides in the initial structural units. Alternative construction methods (i.e., methods other than recursive ligation) can be used to generate ELPs of alternative lengths.

[0147] In some embodiments, the vector contains one or more additional amino acid or ELP structural unit repeats. For example, the vector may add an additional pentamer repeat sequence to the N-terminus of the ELP having a valine residue at the guest site and an additional pentamer to the C-terminus having a tryptophan residue at the guest site. Tryptophan can be used to increase the molecular extinction coefficient, which allows for better absorption measurement, for example, at 280 nm, which can be used to determine protein concentration or to monitor protein content during purification. The pentamer added to either end can also be designed to encode DNA containing restriction enzyme recognition sites for cloning fusion couplers to either end of the ELP coding sequence.

[0148] In some embodiments, the therapeutic composition comprises an active agent and one or more ELPs. In some embodiments, the therapeutic composition comprises an active agent having one or more ELPs at the N- or C-terminus. In some embodiments, the therapeutic composition comprises an active agent having one or more ELPs at both the N- and C-termini. In some embodiments, the ELPs are substantially the same size. In some embodiments, the ELPs are of different sizes. In some embodiments, the ELP at one end is larger than the ELP at the other end. In some embodiments, the N-terminal ELP is larger than the C-terminal ELP. In some embodiments, the C-terminal ELP is larger than the N-terminal ELP.

[0149] Myopathy and Treatment

[0150] Myopathy is a neuromuscular disease in which muscle fibers fail to function, leading to muscle weakness. Phenotypic, these diseases are characterized by inflammation of muscle tissue, atrophy of skeletal muscle, muscle loss, and fibrosis that can cause premature death through respiratory and cardiac failure. Myopathy can affect any type of muscle, including skeletal muscle, cardiac muscle, and / or smooth muscle. In some embodiments, myopathy is characterized by increased muscle fibrosis. In some embodiments, myopathy is characterized by decreased muscle strength and / or contractility. In some embodiments, myopathy is characterized by reduced muscle cell shortening. In some embodiments, myopathy is characterized by decreased muscle cell re-elongation rate. In some embodiments, myopathy is characterized by reduced muscle cell relaxation. In some embodiments, the muscle cells are skeletal muscle cells. In some embodiments, the muscle cells are cardiac muscle cells.

[0151] In some aspects, this disclosure provides methods for treating muscular myopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. This disclosure provides methods for preventing muscular myopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. This disclosure provides methods for delaying muscular myopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, this disclosure provides methods for alleviating muscular myopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, this disclosure provides methods for improving muscular myopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need.

[0152] The types of myopathy that can be treated, prevented, delayed, or improved by the pharmaceutical compositions disclosed herein include, but are not limited to, muscular dystrophy, myotonia, neurosarcoma, congenital myopathy (e.g., fibromyopathy, multinucleated / micronucleated myopathy, central nucleus myopathy), mitochondrial myopathy, familial periodic paralysis, inflammatory myopathy, metabolic myopathy (e.g., glycogen storage disease, lipid storage disease); acquired myopathy (e.g., drug-induced myopathy, glucocorticoid myopathy, alcoholic myopathy), dermatomyositis, polymyositis, inclusion body myositis, ossifying myositis, rhabdomyolysis, and myoglobinuria.

[0153] The types of muscular dystrophy that can be treated, prevented, delayed, or improved by the pharmaceutical compositions disclosed herein include, but are not limited to, BMD (Becker's muscular dystrophy), DMD (Duchenne muscular dystrophy), ankylosing dystrophy, LGMD (limb girdle muscular dystrophy), oculopharyngeal muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Landouzy-Dejerine muscular dystrophy, Emery-Dreifuss muscular dystrophy, and facioscapulohumeral muscular dystrophy.

[0154] Cardiomyopathy is a measurable deterioration of the heart's contractile ability, leading to heart failure. The disease progresses over time and is characterized by variable episodes of arrhythmias and ventricular dysfunction. Electrocardiographic abnormalities can be detected early in the disease and progress with age. The development of cardiomyopathy is characterized by initial diastolic dysfunction, followed by eccentric hypertrophy.

[0155] In some aspects, this disclosure provides methods for treating cardiomyopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, this disclosure provides methods for preventing cardiomyopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, this disclosure provides methods for delaying cardiomyopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, this disclosure provides methods for slowing the progression of cardiomyopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, this disclosure provides methods for improving cardiomyopathy, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need.

[0156] The types of cardiomyopathy that can be treated, prevented, delayed, or improved by the pharmaceutical compositions disclosed herein include, but are not limited to, hereditary cardiomyopathy (e.g., hypertrophic cardiomyopathy (HCM or HOCM); arrhythmogenic right ventricular cardiomyopathy (ARVC); isolated ventricular noncompaction; mitochondrial myopathy), dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), acquired cardiomyopathy (e.g., peripartum cardiomyopathy; Takotsubo cardiomyopathy; Loeffler endocarditis), metabolic / storage cardiomyopathy (e.g., amyloidosis, hemochromatosis), inflammatory cardiomyopathy (e.g., "viral myocarditis"; cardiomyopathy caused by Chagas disease), endocrine cardiomyopathy (e.g., diabetic cardiomyopathy; cardiomyopathy caused by hyperthyroidism; acromegaly), toxic cardiomyopathy (e.g., chemotherapy cardiomyopathy, alcoholic cardiomyopathy), X-linked dilated cardiomyopathy, and / or neuromuscular cardiomyopathy (e.g., muscular dystrophy). In some preferred embodiments, the cardiomyopathy is caused by muscular dystrophy. In some embodiments, the cardiomyopathy is caused by Duchenne muscular dystrophy. In some embodiments, the cardiomyopathy is caused by Becker muscular dystrophy.

[0157] Treatment, prevention, delay, or improvement of cardiomyopathy symptoms can be measured by any means known in the art. For example, assessment may include echocardiographic evaluation, cardiac magnetic resonance imaging (MRI), and cardiac MRI with late gadolinium enhancement. Specifically, LV size, thickness, volume, EF, and myocardial scar / inflammatory burden, as well as strain, can be assessed. Changes in strain, LV size and volume, and scar burden are key measurements.

[0158] Treatment, prevention, delay, or improvement of myopathy symptoms can be measured in any manner known in the art. For example, tests used to assess patients with myopathy include, but are not limited to, creatine kinase (CK) levels with isoenzymes, electrolyte levels, calcium and magnesium, serum myoglobin levels, serum creatinine and blood urea nitrogen levels, urinalysis (e.g., myoglobinuria indicated by a positive urinalysis showing few red blood cells on microscopic evaluation), complete blood count, erythrocyte sedimentation rate, thyroid function tests, aspartate aminotransferase levels, electrocardiogram, antinuclear antibody levels, electromyography, magnetic resonance imaging, and / or muscle biopsy. The effects of administration of the pharmaceutical compositions disclosed herein can be measured in any relevant muscle, including but not limited to skeletal muscle, cardiac muscle, gastrocnemius muscle, quadriceps femoris muscle, diaphragmatic muscle, and / or tibialis anterior muscle.

[0159] In some aspects, the present invention provides methods for preventing cardiac deterioration, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for delaying cardiac deterioration, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for slowing the progression of cardiac deterioration, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for improving cardiac deterioration, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some embodiments, cardiac deterioration is prevented, delayed, or improved for approximately 1 week, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 8 months, approximately 1 year, approximately 2 years, approximately 5 years, and / or approximately 10 years, compared to cardiac deterioration in untreated myopathy subjects. In some embodiments, cardiac deterioration is prevented, delayed, or improved by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to untreated myopathy subjects. In some embodiments, prevention, delay, or improvement of cardiac deterioration is observed at the time points disclosed herein. For example, cardiac deterioration may be delayed by about 20% at week 32. In some embodiments, cardiac deterioration is a reduction in contractility. In some embodiments, cardiac deterioration is a reduction in relaxation. In some embodiments, cardiac deterioration is thickening of the cardiac muscle. In some embodiments, cardiac deterioration is cardiac hypertrophy.

[0160] In some embodiments, administration of the pharmaceutical composition disclosed herein improves cardiac function in subjects compared to untreated myopathy subjects. In some embodiments, cardiac function improves for approximately 1 week, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 8 months, approximately 1 year, approximately 2 years, approximately 5 years, and / or approximately 10 years compared to untreated myopathy subjects. In some embodiments, cardiac function improves for approximately 1%, approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90% compared to untreated myopathy subjects. In some embodiments, the improvement in cardiac function is observed at the time points disclosed herein. In some embodiments, the cardiac improvement is an increase in contractility. In some embodiments, the cardiac improvement is an increase in relaxation. In some embodiments, the cardiac improvement is less thickening of the cardiac muscle compared to cardiomyopathy subjects. In some embodiments, the cardiac improvement is less cardiac hypertrophy compared to cardiomyopathy subjects.

[0161] In some aspects, the present invention provides methods for preventing the development of fibrosis, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for delaying the development of fibrosis, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for slowing the progression of fibrosis, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for improving the development of fibrosis, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some embodiments, the development of fibrosis is prevented, delayed, or improved for approximately 1 week, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 8 months, approximately 1 year, approximately 2 years, approximately 5 years, and / or approximately 10 years, compared to the development of fibrosis in untreated myopathy subjects. In some embodiments, the development of fibrosis is prevented, delayed, or improved by approximately 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the development of fibrosis in untreated myopathy subjects. In some embodiments, prevention, delay, or improvement of fibrosis is observed at the time points disclosed herein. In some embodiments, fibrosis is measured in a biopsy using histological techniques (e.g., hematoxylin and eosin (HE) and trichrome staining), wherein fibrosis can be assessed by determining the percentage of collagen present relative to the total tissue area.

[0162] In some aspects, the present invention provides a method for reducing collagen production, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides a method for delaying collagen production, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides a method for improving collagen production, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some embodiments, collagen production is reduced, delayed, or improved for about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 1 year, about 2 years, about 5 years, or about 10 years compared to collagen production in untreated myopathy subjects. In some embodiments, collagen production is reduced, delayed, or improved by approximately 1%, approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90% compared to collagen production in untreated myopathy subjects. In some embodiments, this reduction, delay, or improvement in collagen production is observed at the time points disclosed herein. For example, the amount of collagen produced in myopathy patients may be reduced by approximately 25% at week 32.

[0163] In some embodiments, administration of the pharmaceutical composition disclosed herein maintains muscle contractility and / or contractile strength in a subject. In some embodiments, muscle contractility and / or contractile strength are maintained at approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the muscle contractility and / or contractile strength of a healthy subject. In some embodiments, administration of the pharmaceutical composition disclosed herein maintains muscle contractility and / or contractile strength in a subject for approximately 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 1 year, 2 years, 5 years, and / or 10 years, compared to the muscle contractility and / or contractile strength of a healthy subject. In some embodiments, the degree to which muscle contractility and / or contractile strength is maintained in a subject at the time points disclosed herein. A healthy subject is defined as a subject who does not suffer from muscular dystrophy or other muscle atrophy diseases or conditions.

[0164] In some embodiments, administration of the pharmaceutical composition disclosed herein maintains muscle strength in a subject. In some embodiments, muscle strength is maintained at approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, or approximately 99% of the muscle strength of a healthy subject. In some embodiments, administration of the pharmaceutical composition disclosed herein maintains muscle strength in a subject for approximately 1 week, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 8 months, approximately 1 year, approximately 2 years, approximately 5 years, and / or approximately 10 years, compared to the muscle strength of a healthy subject. In some embodiments, the degree to which muscle strength is maintained in a subject at the time points disclosed herein. Muscle function can be measured at the overall level, at the organ level, and / or at the muscle cell level. Skeletal muscle strength can be measured by any means known in the art, and for example, by manual muscle testing and / or using a dynamometer. Myocardial strength can be measured by any means known in the art, and for example by using electrocardiography, echocardiography, magnetic resonance imaging (MRI), pressure-volume assessments (e.g., ejection fraction, fractional shortening, end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), myocardial strain, end-diastolic pressure (EDP), end-systolic pressure (ESP)). For example, in some embodiments, the fractional area retained by the drug composition is shortened compared to untreated myopathic subjects. In some embodiments, the fractional ejection fraction is retained compared to untreated myopathic subjects. In some embodiments, the ventricular filling rate is increased compared to untreated myopathic subjects. In some embodiments, the maximum rate of pressure rise is increased compared to untreated myopathic subjects. In other embodiments, the relaxation Tau constant is increased compared to untreated myopathic subjects.

[0165] In some embodiments, administration of the pharmaceutical compositions disclosed herein prevents, delays, or improves contraction-induced muscle injury in subjects. In some embodiments, administration of the pharmaceutical compositions disclosed herein prevents, delays, or improves contraction-induced muscle injury by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and / or about 95% compared to untreated myopathy subjects. In some embodiments, administration of the pharmaceutical compositions disclosed herein prevents, delays, or improves contraction-induced muscle injury in subjects for about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 1 year, about 2 years, about 5 years, and / or about 10 years compared to untreated myopathy subjects. In some embodiments, the degree of prevention, delay, or improvement of contraction-induced muscle injury is observed in subjects at the time points disclosed herein. For example, administration of the pharmaceutical compositions disclosed herein may reduce contraction-induced muscle injury by 25% at week 10. In some embodiments, administration of the pharmaceutical compositions disclosed herein prevents, delays, or improves contraction-induced muscle injury in a subject without altering muscle strength. In some embodiments, administration of the pharmaceutical compositions disclosed herein prevents contraction-induced muscle injury, delays the progression of contraction-induced muscle injury, or improves contraction-induced muscle injury and improves muscle strength in a subject.

[0166] In some embodiments, administration of the pharmaceutical compositions disclosed herein maintains myocyte function. In some embodiments, administration of the pharmaceutical compositions disclosed herein maintains myocyte shortening, myocyte re-elongation, relaxation, and / or myocyte contractility. In some embodiments, myocyte shortening, myocyte re-elongation, relaxation, and / or myocyte contractility are maintained at approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, and / or approximately 99% of the myocyte shortening, myocyte re-elongation, relaxation, and / or myocyte contractility in approximately healthy subjects. In some embodiments, administration of the pharmaceutical composition disclosed herein maintains muscle cell shortening, re-elongation, relaxation, and / or contractility in subjects for approximately 1 week, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 8 months, approximately 1 year, approximately 2 years, approximately 5 years, and / or approximately 10 years, compared to muscle cell shortening, re-elongation, relaxation, and / or contractility in healthy subjects. In some embodiments, the degree of maintenance of muscle cell shortening, re-elongation, relaxation, and / or contractility is observed in subjects at the time points disclosed herein. Muscle cell function can be determined by any means known in the art, including, for example, measuring biomarkers related to function and / or injury, and / or directly testing muscle cells. In some embodiments, the muscle cells are skeletal muscle cells. In some embodiments, the muscle cells are cardiomyocytes.

[0167] In some embodiments, administration of the pharmaceutical composition disclosed herein affects the count of immune cells in muscle. In some embodiments, administration of the pharmaceutical composition disclosed herein reduces the count of immune cells in muscle. In some embodiments, administration of the pharmaceutical composition disclosed herein reduces the count of macrophages in muscle. In some embodiments, the immune cell count is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the immune cell count in the muscle of an untreated myopathy subject. In some embodiments, administration of the pharmaceutical composition disclosed herein reduces the immune cell count in the muscle of a subject for about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 1 year, about 2 years, about 5 years, and / or about 10 years, compared to the immune cell count in the muscle of an untreated myopathy subject. In some embodiments, this reduction in the immune cell count in the muscle of the subject is observed at the time points disclosed herein. For example, administration of the pharmaceutical composition disclosed herein can reduce the count of immune cells in muscle by approximately 25% at week 32.

[0168] In some aspects, the present invention provides methods for preventing inflammation, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a patient in need. In some aspects, the present invention provides methods for delaying inflammation, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for slowing the progression of inflammation, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for improving inflammation, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some aspects, the present invention provides methods for reducing inflammation, comprising administering a pharmaceutical composition of a vasoactive intestinal peptide and one or more ELPs to a subject in need. In some embodiments, inflammation is prevented, delayed, reduced, or improved for about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 1 year, about 2 years, about 5 years, and / or about 10 years, compared to inflammation in untreated myopathy subjects. In some embodiments, inflammation is prevented, delayed, reduced, or improved by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to inflammation in untreated myopathy subjects. In some embodiments, prevention, delay, reduction, or improvement of inflammation is observed at the time points disclosed herein. In some embodiments, administration of the pharmaceutical composition disclosed herein prevents, delays, reduces, or improves inflammation by inhibiting the expression or activity of pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are any pro-inflammatory cytokines, including but not limited to OPN, LTβ4, TNF-α, interleukin-6 (IL-6), and soluble tumor necrosis factor-α receptor (sTNFR).

[0169] Pharmaceutical Compositions and Administration

[0170] The present invention provides a pharmaceutical composition comprising a vasoactive intestinal peptide and one or more ELPs, as well as one or more pharmaceutically acceptable excipients and / or diluents.

[0171] This invention provides sustained-release formulations comprising the therapeutic agents disclosed herein and one or more pharmaceutically acceptable excipients and / or diluents. For example, such excipients include salts and other excipients that can act to stabilize hydrogen bonds. Any suitable excipients known in the art can be used. Exemplary excipients include, but are not limited to, amino acids such as histidine, glycine, or arginine; glycerol; sugars such as sucrose; surfactants such as polysorbate 20 and polysorbate 80; citric acid; sodium citrate; antioxidants; salts including alkaline earth metal salts such as sodium, potassium, and calcium; counterions such as chlorides and phosphates; preservatives; sugar alcohols (e.g., mannitol, sorbitol); and buffers. Exemplary salts include sodium chloride, potassium chloride, magnesium chloride, calcium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. In some embodiments, the pharmaceutical compositions disclosed herein have enhanced potency, bioavailability, therapeutic half-life, persistence, and resistance to degradation.

[0172] In some embodiments, the formulation may contain about 5 mM histidine to about 100 mM histidine. In some embodiments, the formulation may contain about 50 mM histidine, about 40 mM histidine, about 30 mM histidine, about 25 mM histidine, about 20 mM histidine, or about 15 mM histidine. In some embodiments, the formulation may contain about 10 mM sodium chloride to about 200 mM sodium chloride. In some embodiments, the formulation contains about 20 mM sodium chloride, about 40 mM sodium chloride, about 60 mM sodium chloride, about 75 mM sodium chloride, about 100 mM sodium chloride, about 120 mM sodium chloride, or about 150 mM sodium chloride. In some embodiments, the formulation may contain about 10 mM histidine to about 30 mM histidine and about 60 mM sodium chloride to about 80 mM sodium chloride. In some embodiments, the formulation may contain about 20 mM histidine and about 75 mM sodium chloride.

[0173] The pharmaceutical composition is formulated with excipients of sufficient pH and ionic strength to allow matrix formation at body temperature (e.g., 37°C or, in some embodiments, 34 to 36°C) and generally used. The pharmaceutical composition is generally prepared so that it does not form a matrix under storage conditions. The formulation can be stored frozen, refrigerated, or at room temperature. Storage conditions may be below freezing point, such as below about -10°C, or below about -20°C, or below about -40°C, or below about -70°C. Storage conditions are generally below the transition temperature of the formulation, such as below about 32°C, or below about 30°C, or below about 27°C, or below about 25°C, or below about 20°C, or below about 15°C. In some embodiments, the formulation is stored at 2°-8°C. For example, the formulation may be isotonic with blood or have an ionic strength that mimics physiological conditions. For example, the formulation may have the following ionic strengths: at least 25 mM sodium chloride, at least 30 mM sodium chloride, at least 40 mM sodium chloride, at least 50 mM sodium chloride, at least 75 mM sodium chloride, or at least 100 mM sodium chloride, or at least 150 mM sodium chloride. In some embodiments, the formulation has an ionic strength equivalent to that of 0.9% brine (154 mM sodium chloride).

[0174] In some embodiments, the formulation is prepared at a physiological pH. In some embodiments, the formulation is prepared in a pH range of about 5.5 to about 8.5. In some embodiments, the formulation is prepared in a pH range of about 6.0 to about 8.0. In some embodiments, the formulation is prepared in a pH range of about 6.5 to about 7.5. In some embodiments, the formulation is prepared at pH 7.5. In some embodiments, the formulation with a lower pH exhibits improved formulation stability compared to the formulation at a higher pH. In some embodiments, the formulation with a higher pH exhibits improved formulation stability compared to the formulation at a lower pH.

[0175] In some embodiments, the formulation is stable under storage conditions. Stability can be measured using any suitable method in the art. Generally, stable formulations are those in which the increase in degradation products or impurities is less than 5%. In some embodiments, the formulation is stable under storage conditions for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 1 year, or at least about 2 years or longer. In some embodiments, the formulation is stable at 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or at least about 1 year or longer.

[0176] The protein concentration in the formulation is adjusted to allow agglomerates to form at the application temperature. For example, higher protein concentrations help drive agglomerate formation, and the protein concentration required for this purpose varies depending on the ELP series used. For example, in some embodiments using ELP1-120 or an ELP with a comparable transition temperature, the protein is present in the range of about 1 mg / mL to about 200 mg / mL, or in the range of about 5 mg / mL to about 125 mg / mL. In embodiments using ELP4-120 or an amino acid sequence with a comparable transition temperature, the protein is present in the range of about 0.005 mg / mL to about 10 mg / mL, or in the range of about 0.01 mg / mL to about 5 mg / mL.

[0177] In exemplary embodiments, the present invention provides a sustained-release pharmaceutical composition comprising a vasoactive intestinal peptide disclosed herein (e.g., having an N-terminal portion such as methionine) and one or more containing [VPGXG]. 90 [VPGXG] 120 [VPGXG] 160 Or [VPGXG] 180 The amino acid sequence, wherein each X is selected from V, G, and A. V, G, and A may be present in ratios of approximately 5:3:2, approximately 7:2:0, approximately 7:0:2, approximately 6:0:3, or approximately 5:2:2. Alternatively, the amino acid sequence may contain [VPGVG]. 90 Or [VPGVG] 120 In exemplary embodiments, this document provides sustained-release pharmaceutical compositions comprising a vasoactive intestinal peptide or a derivative thereof (e.g., having an N-terminal portion such as methionine) and one or more ingredients containing [XPGVG]. 144 The amino acid sequence, where each X is selected from V, G, and A. V, G, and A may be present in a ratio of approximately 5:0:4. Alternatively, the amino acid sequence contains [XPGVG]. 144 The formulation further comprises one or more pharmaceutically acceptable excipients and / or diluents for forming a reversible matrix from an aqueous form when administered to a human subject. VIP and its derivatives are disclosed in U.S. Patent Publication No. 2011 / 0178017.

[0178] On the other hand, this document provides a method for formulating a sustained release of the vasoactive intestinal peptide disclosed herein. The method includes administering the pharmaceutical composition described herein to a subject in need, wherein the pharmaceutical composition is administered about once to about eight times per month. In some embodiments, the pharmaceutical composition is administered about once, about twice, about three times, and / or about four times per month. In some embodiments, the pharmaceutical composition is administered weekly. In some embodiments, the pharmaceutical composition is administered daily. In some embodiments, the pharmaceutical composition is administered 1 to 3 times per week. In some embodiments, the pharmaceutical composition is administered once every two weeks. In some embodiments, the pharmaceutical composition is administered 1 to 2 times per month. In a specific embodiment, the pharmaceutical composition is administered about once per month. In some embodiments, the pharmaceutical composition is administered about once every 2 months, about once every 3 months, about once every 4 months, about once every 5 months, and / or about once every 6 months. In some embodiments, the VIP may have an additional portion, such as methionine, at its N-terminus to alter the receptor binding profile, as described in U.S. Patent Publication No. 2011 / 0178017. In some embodiments, VIP is fused with ELP1 (having about 90 to about 180 ELP units). In some embodiments, VIP is fused with ELP4 (having about 90 to about 180 ELP units). In some embodiments, VIP is fused with ELPβv2 (having about 90 to about 180 ELP units). The pharmaceutical composition may be packaged in the form of a pre-filled pen or syringe for once-weekly, twice-weekly, or once to eight times-monthly administration, or alternatively filled in conventional vials, etc.

[0179] Advantageously, the composition provides prolonged pharmacokinetic exposure due to the sustained release of the active agent. In a specific aspect, the maximum exposure level may be achieved at approximately 10 hours, 24 hours, 48 ​​hours, or 72 hours after administration; typically, the maximum exposure level is achieved at approximately 10 hours to approximately 48 hours after administration. After achieving the maximum exposure level, the composition can achieve a sustained release level, thereby obtaining a substantial percentage of the maximum level over a period of time. For example, the sustained level may be approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 100%. Exemplary time periods for maintaining the sustained rate are approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 1 week, approximately 2 weeks, approximately 4 weeks, approximately 6 weeks, or approximately 8 weeks after achieving the maximum exposure rate. Subsequently, the sustained level may be reduced to a reduced exposure level. This reduced exposure rate may be approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, or approximately 60%.

[0180] In some embodiments, the pharmaceutical compositions disclosed herein are applied for an extended period. In some embodiments, the pharmaceutical compositions disclosed herein are applied for about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, or longer. The pharmaceutical compositions may be applied at any desired dose and / or frequency disclosed herein.

[0181] In some embodiments, the pharmaceutical composition disclosed herein is administered until myopathic symptoms improve. In some embodiments, the pharmaceutical composition disclosed herein is administered until myopathic symptoms improve. In some embodiments, the pharmaceutical composition disclosed herein is administered until myopathic symptoms are delayed. In some embodiments, the pharmaceutical composition disclosed herein is administered until myopathic symptoms are cured.

[0182] In some embodiments, the pharmaceutical composition disclosed herein is administered before the patient begins to present with one or more myopathic symptoms. In some embodiments, the pharmaceutical composition disclosed herein is administered when myopathic symptoms occur. In some embodiments, the pharmaceutical composition disclosed herein is administered before the patient begins to present with cardiomyopathy. In some embodiments, the pharmaceutical composition disclosed herein is administered when cardiomyopathy symptoms occur.

[0183] In some embodiments, the pharmaceutical composition is administered to a subject identified as having muscle atrophy. In some embodiments, the pharmaceutical composition is administered to a subject identified as having myopathy. In some embodiments, an increased level of circulating muscle protein in the blood of the subject is determined compared to a non-myopathy subject. In some embodiments, an increased level of circulating creatine kinase (CK) in the blood of the subject is determined compared to a non-myopathy subject. In some embodiments, an increased level of circulating lactate dehydrogenase (LDH) in the blood of the subject is determined compared to a non-myopathy subject. In some embodiments, an increased level of circulating pyruvate kinase (PK) in the blood of the subject is determined compared to a non-myopathy subject. Muscle protein levels can be determined using a serum enzyme test.

[0184] In some implementations, the altered levels of electrolytes in the subject's urine are determined. In some implementations, the increased levels of calcium in the subject's urine are determined compared to non-myopathy subjects. In some implementations, the increased levels of magnesium in the subject's urine are determined compared to non-myopathy subjects. Urinary electrolyte levels can be determined using a urine test.

[0185] In some embodiments, the pharmaceutical composition is administered to a subject identified as having inflammatory myopathy. In some embodiments, the subject is identified as having increased levels of circulating antibodies in the blood compared to a non-myopathy subject. In some embodiments, the subject is identified as having increased levels of circulating myositis-associated antibodies in the blood compared to a non-myopathy subject. In some embodiments, the myositis-associated antibodies include, but are not limited to, Jo-1, PL-7, PL-12, EK, OJ, KS, Zo, and Ha. In some embodiments, the myositis-associated antibodies bind to antigens, including but not limited to those bound by antibodies Jo-1, PL-7, PL-12, EK, OJ, KS, Zo, and Ha. In some embodiments, the antibodies include, but are not limited to, SRP, Mi-2, PMS1, P155, p140, CADM-140, MJ(p140), MU, SAE, core proteoglycan, KU, KJ, HMGCR, Mup44, cortical proteins, nuclear pores, FHL1, PM-Scl, 56kD, SSA / ro, U1-nRNP, U2-NRNP, Fer, MAS, and mitochondria. In some embodiments, the antibodies bind to antigens, including but not limited to those bound by SRP, Mi-2, PMS1, P155, p140, CADM-140, MJ(p140), MU, SAE, core proteoglycan, KU, KJ, HMGCR, Mup44, cortical proteins, nuclear pores, FHL1, PM-Scl, 56kD, SSA / ro, U1-nRNP, U2-NRNP, Fer, MAS, and mitochondria antibodies.

[0186] In some implementations, subjects are identified as having abnormal echocardiograms compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having changes in cardiac performance compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having increased PR intervals compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having increased U waves compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having wide QRS complexes compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having nonspecific ST-T changes compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having sinus arrhythmias compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having deep Q waves and elevated R waves in the precordial region compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having abnormal tetanic contractions (TC) compared to non-cardiomyopathy subjects. In some implementations, tetanic contraction originates from a period of calcium imbalance, manifested as sustained ion-driven myocyte contraction resulting in echocardiographic findings of left ventricular "unfilling" (Su et al. (2015)). In some implementations, subjects are identified as having arrhythmias compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having sinus tachycardia compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having systolic dysfunction compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having diastolic dysfunction compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having reduced mitral systolic wave velocity compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having wall motion abnormalities compared to non-cardiomyopathy subjects. Cardiac function and / or characteristics can be measured using electrocardiography.

[0187] In some implementations, subjects are identified as having increased expression of pro-inflammatory cytokines compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having increased activity of pro-inflammatory cytokines compared to non-cardiomyopathy subjects. In some implementations, pro-inflammatory cytokines are any pro-inflammatory cytokines, including but not limited to OPN, LTβ4, TNF-α, interleukin-6 (IL-6), and soluble tumor necrosis factor-α receptor (sTNFR). The expression or activity of pro-inflammatory cytokines in subjects can be measured by cytokine assay.

[0188] In some implementations, subjects are identified as having reduced nNOS protein expression compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having reduced nNOS activity compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having altered nNOS protein accumulation compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having increased nNOS protein accumulation in the skeletal muscle cytoplasm compared to non-cardiomyopathy subjects. In some implementations, subjects are identified as having reduced nNOS protein accumulation in the skeletal muscle sarcolemma compared to non-cardiomyopathy subjects. Protein accumulation can be determined by histological techniques. Protein expression can be determined by immunoprecipitation techniques. Protein activity can be measured by NOS catalysis assays.

[0189] Pharmaceutical compositions are generally intended for “systemic delivery,” meaning that the agent is not delivered locally to a pathological site or site of action. Instead, the agent is absorbed into the bloodstream from the injection site, whereby the agent acts systemically or is transported to the site of action via circulation. Therapeutic agents can be administered via any known route, such as, for example, oral, intravenous, intramuscular, intranasal, subcutaneous, intravaginal, and rectal. In one embodiment, the formulation is generally intended for subcutaneous administration. In one embodiment, subcutaneous administration of the agent prolongs pharmacokinetic (PK) parameters. In one embodiment, the half-life of the fusion protein is prolonged. In one embodiment, subcutaneous administration of the agent prolongs PK parameters compared to administration via other methods (e.g., intravenous). In one embodiment, subcutaneous administration of the agent prolongs the storage of the agent compared to administration via other methods (e.g., intravenous).

[0190] In some embodiments, the formulation is administered approximately monthly, and may be administered subcutaneously or intramuscularly. In some embodiments, the formulation is administered approximately weekly, and may be administered subcutaneously or intramuscularly. In some embodiments, the application site is not a pathological site, such as a site not intended for treatment.

[0191] In several embodiments, during multiple administrations (e.g., at least 2 times, at least about 5 times, at least about 10 times), the plasma concentration of the active agent does not change by more than about 10 times, about 5 times, or about 3 times. The administration is substantially uniform, such as, for example, about daily, or about once a week, or once to five times a month, or about once every two months, or about once every three months.

[0192] The pharmaceutical compositions disclosed herein can be administered at lower doses and / or less frequently compared to their unfused or unconjugated counterparts. While those skilled in the art can determine the desired dose for each case, the appropriate dose of the therapeutic agent to achieve therapeutic benefit can range, for example, from 1 microgram (μg) to about 100 milligrams (mg) per kilogram of the recipient's body weight per day, preferably from about 10 μg to about 50 mg per kilogram of body weight per day, and most preferably from about 100 μg to about 10 mg per kilogram of body weight per day. In some embodiments, the pharmaceutical composition is administered at a low dose. In some embodiments, the pharmaceutical composition is administered at a dose between 1 mg per kilogram of body weight per day and about 9 mg per kilogram of body weight per day. In some embodiments, the pharmaceutical composition is administered at about 1 mg per kilogram of body weight per day, about 3 mg per kilogram of body weight per day, and / or about 9 mg per kilogram of body weight per day. A desired dose can be expressed as a single dose or two or more sub-dose administered at appropriate time intervals throughout the day. These sub-dose can be administered in unit dosage forms, for example, per unit dosage form containing about 10 μg to about 1000 mg, preferably about 50 μg to about 500 mg, and most preferably about 50 μg to about 250 mg of active agent. Alternatively, if the recipient's condition requires it, the dose can be administered as a continuous infusion.

[0193] In some implementations, the subject is a human, but in other implementations it can be a non-human mammal, such as a domesticated pet (e.g., a dog or cat), or a livestock or farm animal (e.g., a horse, cow, sheep, or pig).

[0194] Combination therapy

[0195] The compositions disclosed herein can be administered in conjunction with a variety of therapies for the treatment, prevention, delay, or improvement of myopathy, muscular dystrophy, and / or cardiomyopathy, including but not limited to physical therapy, respiratory therapy, speech therapy, occupational therapy, corrective surgery, and / or therapeutic agents. The pharmaceutical compositions disclosed herein can be used alone or in combination with one or more therapeutic agents. These one or more therapeutic agents can be any compound, molecule, or substance that exerts a therapeutic effect on a subject in need.

[0196] The one or more therapeutic agents may be administered as a single pharmaceutical composition or mixed in a single pharmaceutical composition for "co-administration," i.e., co-administered to the subject in a coordinated manner. Through "co-administration," the one or more therapeutic agents may also be administered simultaneously with or separately from the pharmaceutical compositions of the present invention, including administration at different times and frequencies. The one or more therapeutic agents may be administered via any known route, such as oral, intravenous, intramuscular, intranasal, subcutaneous, intravaginal, rectal, etc.; and the therapeutic agents may also be administered via any conventional route. In several embodiments, at least one therapeutic agent may be administered subcutaneously.

[0197] These one or more therapeutic agents include, but are not limited to, glucocorticoids; steroids; phenytoin; procainamide; quinine; glucocorticoids (e.g., deflazacort, VBP15, prednisone triamcinolone, systemic methylprednisolone). Systemic drugs, betamethasone, budesonide, prednisolone, hydrocortisone, dexamethasone, and / or cortisone; anticonvulsants; immunosuppressants (e.g., cyclosporine, tacrolimus, prednisolone, hydrocortisone, sirolimus, everolimus, azathioprine, mycophenolic acid, methotrexate, basiliximab, daclizumab, rituximab, anti-thymocyte globulin, anti-lymphocyte globulin). Globulin; penicillins (e.g., penicillin and amoxicillin); cephalosporins (e.g., cephalexin); macrolides (e.g., erythromycin, clarithromycin, and azithromycin); fluoroquinolones (e.g., ofloxacin, levofloxacin, and ofloxacin); sulfonamides (e.g., co-trimoxazole and trimethoprim). rim); Tetracyclines (e.g., tetracycline and doxycycline); Aminoglycosides (e.g., gentamicin and tobramycin); ACE inhibitors (e.g., perindopril and enalapril); Angiotensin II receptor blockers; Beta blockers; Calcium channel blockers; Digoxin; Antiarrhythmic drugs;Anticoagulants; antibiotics; diuretics (e.g., spironolactone, eplerenone); exon skipping therapy (e.g., eteplirsen, drisapersen); anti-myosin antibodies (e.g., PF-06252616); anti-connective tissue growth factor antibodies (e.g., FG-3019); PDE5 inhibitors (e.g., tadalafil, sildenafil); PDE9 inhibitors; NF-κB inhibitors; stop codon reading drugs (e.g., ataluren); myotrophic protein modulators (e.g., SMT C1100, SMT022357); antifibrotic agents (e.g., halofuginone, angiotensin [1-7]); coenzyme Q; 10 Synthetic analogues (e.g., idebenone); allogeneic cardiac cell therapy (e.g., CAP-1002); Toll-like receptor antagonists (e.g., IMO-8400); mineralocorticoid receptor antagonists; β-adrenergic receptor antagonists; resveratrol; SIRT1 activators.

[0198] When two or more therapeutic agents are used in combination, the dosage of each agent is generally the same as when used alone. However, if a therapeutic agent interferes with the metabolism of other therapeutic agents, the dosage of each agent should be adjusted accordingly. Alternatively, when two or more therapeutic agents exhibit a synergistic effect, the dosage of one or more may be reduced. Each therapeutic agent may be administered simultaneously or separately at appropriate time intervals.

[0199] It should be understood that, for convenience, the singular forms such as “a,” “an,” and “described” are used throughout this application; however, unless the context or explicit statement indicates otherwise, the singular forms are intended to include the plural forms as well. The entire range of numbers should be understood to include the numerical points within each and every numerical range, and should be understood to describe each and every numerical point individually. The endpoints of the entire range of the same component or feature are inclusive and intended to be independently combinable.

[0200] When used in conjunction with a reference numerical indication, the term “about” means the reference numerical indication plus or minus 10% of the reference numerical indication. For example, the expression “about 50” covers the range of 45-55.

[0201] As used herein, the term "comprising" and its variations are not intended to be limiting, and the list of items thus enumerated does not exclude other items that may be used in the materials, compositions, apparatus, and methods of this technology. Similarly, the terms "capable" and "may" and their variations are not intended to be limiting, and the description of embodiments that are capable of or may include certain elements or features does not exclude other embodiments of this technology that do not include those elements or features. Although the open-ended term "comprising" is used herein to describe and claim the disclosure as synonymous with terms including, containing, or having, the technology of the present invention or embodiments thereof may alternatively be described using more restrictive terms such as "consisting of the described ingredients" or "consisting substantially of the described ingredients."

[0202] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used to practice or test the invention, preferred methods and materials are described herein.

[0203] This document is further illustrated by the following non-limiting embodiments.

[0204] Example 1

[0205] Example 1 - Effects of PB1046 on cardiomyopathy and musculoskeletal myopathy in patients with muscular dystrophy

[0206] Clinical trials were conducted in patients with Duchenne muscular dystrophy and Beck muscular dystrophy to test the efficacy of PB1046 treatment in delaying the onset of skeletal and cardiac symptoms.

[0207] Design: This is a randomized (2:1), double-blind, placebo-controlled trial. Initially, patients aged 18 years or older will be evaluated, but the study is also open to adolescents aged 12 years or older. To assess dose response, at least two dose levels will be evaluated, for example, doses between 0.4 mg / kg and 3.2 mg / kg, such as 0.8 mg / kg and 1.6 mg / kg. Higher or lower doses may also be evaluated for safety reasons. At least six patients will be treated at each dose level.

[0208] The primary treatment endpoint is efficacy, measured as the change from baseline in overall cardiac / regional functional characteristics 12 weeks after treatment. This function will be additionally assessed at week 24. Echocardiography and cardiac MRI with late gadolinium enhancement will be used for evaluation. The effect of treatment on left ventricular peridiastolic strain will be measured. Left ventricular end-diastolic volume (LVEDV), right ventricular end-diastolic volume (RVEDV), enhancement as a measure of cardiac fibrosis, fractional shortening (FS), and / or cardiac output (CO) will be measured. LV size, thickness, volume, ejection fraction, and myocardial scar / inflammatory burden, strain, and changes in LV size and volume and scar burden are key measurements.

[0209] Secondary efficacy endpoints were respiratory function, quantitative muscle strength, and functional and quality assessments.

[0210] Once the study is complete, continuous open-label therapy will be made available to patients based on the recommendations of the Data Safety Monitoring Board.

[0211] Example 2 - Long-term treatment with PB1046 (a stable and long-acting vasoactive intestinal peptide receptor agonist) improved cardiac and musculoskeletal function in Duchenne muscular dystrophy mice.

[0212] Materials and Methods: MDX (C57BL10 / ScSnDMDmdx, dystrophin deficient, n=21) and DKO (double knockout) mdx / utrn- / - (dystrophin / utrophin-associated protein deficient, n=13) mice were administered subcutaneously three times weekly using PB1046 (1.5 mg / kg) or 0.9% NaCl saline (control) for the duration of the study (32 weeks for MDX and up to 4 weeks for DKO). Changes in left ventricular function (via echocardiography; ECHO) and electrocardiogram (ECG) were monitored during routine examinations. Endpoint assessments included assessment of systemic / left ventricular hemodynamics or skeletal muscle strength in the extensor digitorum longus (EDL) muscle (MDX only) in animals prepared for anesthesia using a pressure catheter. Tissue samples were rapidly frozen or fixed in formalin for histological evaluation (Sirius staining and macrophage and CD counts).

[0213] The most commonly used ventricular echocardiographic parameter is the fractional shortening (FS%), which is used to estimate the ratio of the left ventricular diameter at end-diastole to its diameter at end-systole. Fractional area shortening (FAS) and fractional shortening were measured. To calculate FAS, the left ventricular region was visualized in the short axis at the papillary level using sectional imaging, and the boundaries of the endocardium at end-diastole and systole were manually traced. FAS was determined in animals before and during treatment. The rate of increase in left ventricular pressure (LV) during early systole (dP / dt) was also measured. maxThe overall contractility of the left ventricle (LV) is measured. The greater the contractile force, the higher the rate of increase in left ventricular pressure. This rate can be measured invasively (e.g., using a high-fidelity microcatheter within the LV) as well as non-invasively (continuous-wave Doppler ultrasound from mitral regurgitation). Tau, representing the exponential decay of ventricular pressure during isovolumetric relaxation, is also measured. A longer Tau indicates cardiac dysfunction.

[0214] The extensor digitorum longus (EDL) muscles from both legs of mice were dissected at the root tendon and placed in Krebs-Henselet (KH) buffer. The muscles were analyzed with some adjustments, as published by Heller et al. (2013) and Rodino-Klapac et al. (2007). Briefly, one root tendon was strapped to the lever arm of a force sensor, and the other root tendon to a linear servo motor. Once the muscle was stabilized, the optimal length was set to 1 gram and preheated with three 1 Hz twitches every 30 seconds followed by three 150 Hz twitches per minute. After a 3-minute rest period, the EDL was stimulated at 50, 100, 150, and 200 Hz, with a 1-minute rest period between each stimulation to determine maximum tetanic tension. Muscle length was measured after stimulation. After a 5-minute rest period, the sensitivity of the EDL muscle to contraction-induced injury was assessed. After 500 ms of stimulation, the muscle was stretched to 10% of its optimal length and stimulated at 150 Hz for 700 ms. After this stimulation, the muscle returned to its optimal length. This cycle repeats 10 times per minute.

[0215] Specific force is calculated by dividing the maximum tetanic tension by the cross-sectional area of ​​the EDL muscle. For comparison purposes, all force measurements are in terms of contractile material per unit cross-sectional area (CSA) (normalized isometric contractile force or tension, mN / mm). 2 () represents. Calculate CSA using the following equation:

[0216]

[0217] In addition, the extent of collagen deposition, a marker of fibrosis, was determined by analyzing tissue samples using Sirius red staining.

[0218] Results: Long-term PB1046 administration slowed cardiac deterioration in MDX mice. Fractional area shortening (contraction function index) was maintained throughout the study duration (P<0.05). Figure 1A Ventricular filling velocity (E / A ratio – diastolic function index) tended to be faster during the study, but no statistically significant difference was achieved. Figure 1B The shortening score (FS) was also maintained throughout the study period (data not shown).

[0219] Electrocardiograms were also assessed at these time points. Throughout the study, PB1046-treated MDX mice were prone to shorter QRS durations (preserved ventricular conduction) when compared to placebo-treated animals, but this did not achieve statistical significance (data not shown). A similar trend was observed in DKO mice, where PB1046 treatment masked the QRS prolongation observed in the control group (data not shown).

[0220] In vivo analysis showed that in MDX mice, treatment with PB1046 increased the maximum rate of pressure rise (dP / dt). max -The contractile function index) was significantly larger ( Figure 2A -B) and the relaxed Tau constant is faster (P<0.05) Figure 2C -E).

[0221] In skeletal muscle function assessment, PB1046 treatment prevented contraction-induced damage in isolated EDL muscles without altering specificity in MDX mice. Figure 3A -B).

[0222] Consistent with increased bone and cardiac function, PB1046 significantly reduced the degree of fibrosis (collagen content) in the gastrocnemius muscle of MDX mice (no statistical significance in the quadriceps, diaphragm, or tibialis anterior muscle, although the trend was evident). Figure 4A Furthermore, there is a trend of decreased collagen content in the heart muscle of MDX patients. Figure 4B Although n was small, a similar trend of fibrosis was observed in the hearts of DKO mice (data not shown).

[0223] A significant reduction in total macrophage count was observed after treatment with PB1046. Figure 4C However, no differences were observed in other immune cells (CD3, CD4, CD8; data not shown).

[0224] Conclusion: Long-term treatment with the novel VIP receptor agonist PB1046 alleviates DMD by slowing cardiac deterioration and preventing contraction-induced damage in skeletal muscle. In addition to its positive inotropic and diastolic effects on cardiac function, reduced fibrosis (collagen content) may also contribute to the positive effects of PB1046 on both the heart and skeletal muscles.

[0225] By incorporating references

[0226] All publications, patents, and patent publications cited are incorporated herein by reference in their entirety.

[0227] For all purposes, this application is incorporated in its entirety by reference to the following publications: US 2001 / 0034050; US 2009 / 0220455; US 8,334,257; US 2013 / 0310538; US 2013 / 0172274; US 2011 / 0236384; US 6,582,926; US 7,429,458; US 7,364,859; US 8,178,495; US 2013 / 0079277; US 2013 / 0085099; US 2013 / 0143802; US 2014 / 0024600; US 2011 / 0178017; US 7,709,227; US US 2011 / 0123487; US 8,729,018; US 2014 / 0171370; US 2013 / 0150291; WO / 2014 / 113434; US 2014 / 0213516; and US application number 62 / 082,945, filed on November 21, 2014.

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Arnold, Susan Ballance, David James <120> Methods and compositions for treating muscle diseases and conditions <130> PHAS‑032 / 04WO 309646‑309646 <150> 62 / 113,943 <151> 2015-02-09 <150> 62 / 145,770 <151> 2015-04-10 <150> 62 / 150,679 <151> 2015-04-21 <160> twenty one <170> PatentIn version 3.5 <210> 1 <211> 4 <212> PRT <213> Artificial sequence <220> <223> ELP component sequence <400> 1 Val Pro Gly Gly 1 <210> 2 <211> 4 <212> PRT <213> Artificial sequence <220> <223> ELP component sequence <400> 2 Ile Pro Gly Gly 1 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> ELP component sequence <220> <221> misc_feature <222> (4)..(4) <223> Xaa can be any naturally occurring or non-natural amino acid. <400> 3 Val Pro Gly Xaa Gly 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> ELP component sequence <400> 4 Ala Val Gly Val Pro 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> ELP component sequence <220> <221> misc_feature <222> (4)..(4) 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<213> artificial sequence <220> <223> MAA-VIP <400> 18 Met Ala Ala His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu 1 5 10 15 Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20 25 30 <210> 19 <211> 633 <212> PRT <213> artificial sequence <220> <223> VIP ELP1‑120 <400> 19 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn Val Pro Gly Val 20 25 30 Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly 35 40 45 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 50 55 60 Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro 65 70 75 80 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly 85 90 95 Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 100 105 110 Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly 115 120 125 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val 130 135 140 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 145 150 155 160 Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly 165 170 175 Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly 180 185 190 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 195 200 205 Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val 210 215 220 Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 225 230 235 240 Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 245 250 255 Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala 260 265 270 Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 275 280 285 Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 290 295 300 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro 305 310 315 320 Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly 325 330 335 Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val 340 345 350 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly 355 360 365 Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val 370 375 380 Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro 385 390 395 400 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 405 410 415 Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val 420 425 430 Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly 435 440 445 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 450 455 460 Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro 465 470 475 480 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly 485 490 495 Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 500 505 510 Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly 515 520 525 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val 530 535 540 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 545 550 555 560 Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly 565 570 575 Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly 580 585 590 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 595 600 605 Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val 610 615 620 Pro Gly Gly Gly Val Pro Gly Trp Pro 625 630 <210> 20 <211> 730 <212> PRT <213> Artificial Sequence <220> <223> ELPβV2-144 <400> 20 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val 1 5 10 15 Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro 20 25 30 Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 35 40 45 Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val 50 55 60 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 65 70 75 80 Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val 85 90 95 Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro 100 105 110 Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 115 120 125 Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val 130 135 140 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 145 150 155 160 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val 165 170 175 Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro 180 185 190 Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly 195 200 205 Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val 210 215 220 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 225 230 235 240 Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 245 250 255 Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro 260 265 270 Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly 275 280 285 Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val 290 295 300 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 305 310 315 320 Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 325 330 335 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 340 345 350 Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly 355 360 365 Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala 370 375 380 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 385 390 395 400 Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 405 410 415 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro 420 425 430 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly 435 440 445 Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala 450 455 460 Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly 465 470 475 480 Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 485 490 495 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro 500 505 510 Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 515 520 525 Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala 530 535 540 Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly 545 550 555 560 Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 565 570 575 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro 580 585 590 Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 595 600 605 Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala 610 615 620 Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly 625 630 635 640 Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val 645 650 655 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro 660 665 670 Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 675 680 685 Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val 690 695 700 Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly 705 710 715 720 Val Pro Gly Ala Gly Val Pro Gly Val Gly 725 730 <210> 21 <211> 605 <212> PRT <213> Artificial Sequence <220> <223> ELP1-120 <400> 21 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val 1 5 10 15 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 20 25 30 Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly 35 40 45 Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly 50 55 60 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 65 70 75 80 Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val 85 90 95 Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 100 105 110 Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 115 120 125 Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala 130 135 140 Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 145 150 155 160 Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 165 170 175 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro 180 185 190 Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly 195 200 205 Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val 210 215 220 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly 225 230 235 240 Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val 245 250 255 Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro 260 265 270 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 275 280 285 Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val 290 295 300 Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly 305 310 315 320 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 325 330 335 Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro 340 345 350 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly 355 360 365 Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 370 375 380 Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly 385 390 395 400 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val 405 410 415 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 420 425 430 Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly 435 440 445 Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly 450 455 460 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 465 470 475 480 Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val 485 490 495 Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 500 505 510 Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 515 520 525 Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala 530 535 540 Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 545 550 555 560 Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 565 570 575 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro 580 585 590 Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Trp Pro 595 600 605

Claims

1. Use of a composition in the preparation of a medicament for treating skeletal muscle deterioration in patients with muscular dystrophy, the composition comprising a fusion protein having the amino acid sequence shown in SEQ ID NO: 15, wherein the fusion protein is present in the range of 1 mg / mL to 200 mg / mL.

2. The use according to claim 1, wherein the composition further comprises sodium chloride present in the range of 25 mM to 150 mM.

3. The use according to claim 1, wherein the composition further comprises histidine in the range of 10 mM to 20 mM.

4. The use according to any one of claims 1-3, wherein the application of the composition reduces gastrocnemius muscle fibrosis, maintains muscle contractility, or maintains muscle strength in patients.

5. The use according to any one of claims 1-3, wherein the composition is formulated for subcutaneous, intramuscular or intravenous administration.

6. The use according to claim 5, wherein the composition is administered subcutaneously.

7. The use according to any one of claims 1-3, wherein the dosage of said composition is from 0.1 mg / kg to 10 mg / kg daily.

8. The use according to any one of claims 1-3, wherein the composition is applied daily.

9. The use according to any one of claims 1-3, wherein the composition is applied 1-3 times per week.

10. The use according to any one of claims 1-3, wherein the composition is applied weekly.

11. The use according to any one of claims 1-3, wherein the composition is applied 1-2 times per month.

12. The use according to any one of claims 1-3, wherein muscle contractility is maintained in the patient.

13. The use according to claim 12, wherein the muscle is skeletal muscle.

14. The use according to any one of claims 1-3, wherein the application of the composition maintains the contractility of the muscle cells compared to untreated muscle cells.

15. The use according to any one of claims 1-3, wherein the application of the composition keeps the muscle cells relaxed compared to untreated muscle cells.

16. The use according to claim 14, wherein the muscle cell is a skeletal muscle cell.

17. The use according to claim 15, wherein the muscle cell is a skeletal muscle cell.

18. The use according to any one of claims 1-3, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

19. The use according to any one of claims 1-3, wherein the composition is formulated for sustained release.

20. The use according to any one of claims 1-3, wherein the composition comprises 75 mM sodium chloride, 20 mM histidine, and 100 mg / mL of the fusion protein.

21. The use according to any one of claims 1-3, wherein the application of the composition in a patient prevents muscle contraction-induced injury.

22. The use according to any one of claims 1-3, wherein: (a) Compared to untreated muscle cells, application of the composition resulted in muscle cells maintaining a fraction of shortening; (b) Compared to untreated muscle cells, application of the composition maintains the contractility of the muscle cells; (c) Compared to untreated muscle cells, application of the composition maintains the rate of muscle cell re-elongation; or (d) Compared to untreated muscle cells, the application of the composition keeps the muscle cells relaxed.

23. The use according to any one of claims 1-3, wherein: (a) Compared to untreated myopathy subjects, administration of the composition reduced collagen content in muscles; or (b) Compared to untreated myopathy subjects, administration of the composition reduced the macrophage count in muscles.

24. The use according to any one of claims 1-3, wherein the dosage of said composition is from 0.4 mg / kg to 3.2 mg / kg.

25. Use of a composition in the preparation of a medicament for treating skeletal muscle deterioration in patients with muscular dystrophy in need, said composition comprising a protein with the amino acid sequence shown in SEQ ID NO: 15.

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