Methods of treating musculoskeletal injuries

Abstract

Compositions including metformin and their use for inhibiting or treating soft tissue musculoskeletal injuries, such as tendinopathy, and / or treating or inhibiting scar tissue are disclosed.

Description

[0001] METHODS OF TREATING MUSCULOSKELETAL INJURIES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 534,969, filed August 28, 2023, which is incorporated by reference in its entirety.

[0004] FIELD

[0005] This disclosure relates to methods of treating musculoskeletal soft tissue injuries as well as inhibiting scar tissue formation or treating scar tissue.

[0006] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0007] This invention was made with government support under W81XWH-22-9-0016 awarded by the Defense Health Agency, Medical Research and Development Branch, and AR070340 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Musculoskeletal (MS) injuries, including tendon overuse or tendinopathy, heavily affect military personnel and the general public. Tendinopathy can result in a disability that lasts for several months, and its management is often lengthy and expensive. Indeed, an estimated 30% of consultations for musculoskeletal pain in clinics are related to tendinopathy, with Achilles tendinopathy being the most frequent condition. Current treatments for tendinopathy are largely palliative without long-term benefits. Non-invasive treatments such as RICE (rest, ice, compression, elevation), physiotherapy, non-steroidal antiinflammatory drugs (NSAIDs), and corticosteroids, treat only symptoms, such as pain, which arc only alleviated short-term. There is no clear evidence that NSAIDS and corticosteroids are effective in the treatment of chronic tendinopathy long-term, and such treatments come with a risk of severe side effects, such as internal bleeding, stomach ulcers, and liver / kidney problems. The use of platelet-rich plasma (PRP) to treat tendinopathy is popular, but its efficacy is inconsistent in human studies (Andia et al., Expert Opinion on biological therapies 18:389-398,2018). Exercises like eccentric loading (ECL) or heavy eccentric calf training (HECT) are generally considered to be efficacious, but up to 45% tendinopathy patients are non-responsive. In addition, high quality evidence for the efficacy of ECL treatment for tendinopathy is currently lacking. Thus, existing therapies treat symptoms rather than target the root cause of tendinopathy, and none of the therapeutic options offers satisfactory long-term solutions. Novel therapies targeting the root cause of soft tissue musculoskeletal injuries, such as tendinopathy, are urgently needed to effectively treat, or better yet prevent, injury.

[0010] Skin wounds and impaired wound healing also pose significant challenges to public health. In the United States, chronic wounds affect 6.5 million patients, with annual healthcare costs amounting to approximately $25 billion. The natural process of skin recovery following an injury typically results in scar and society at large. Each year in the United States, about 500,000 individuals receive treatment for burns, many of which result in scarring and painful contractures necessitating extensive surgical intervention. Additionally, around 100 million patients in developed countries develop surgical scars annually. Scars can be painful, disfiguring, and disabling, leading to significant distress. Patients with visible scars, particularly on the face, often face social stigma and experience psychological trauma. Despite this, many commercially available wound healing products are ineffective at promoting skin regeneration and preventing the formation of scar tissue. Thus, novel therapies that promote skin regeneration while preventing formation of scar tissue are needed.

[0011] SUMMARY

[0012] Disclosed are methods of inhibiting or treating a soft tissue musculoskeletal injury in a subject, including administering metformin to the subject. In some aspects, a subject at risk of, or having a soft tissue musculoskeletal injury is selected for treatment with metformin. In some aspects, metformin is administered topically or by injection. In some aspects, metformin is administered directly to the site of the tissue musculoskeletal injury. The soft tissue musculoskeletal injury can be an injury of a tendon, ligament, or muscle. In some aspects, the soft tissue musculoskeletal injury is tendinopathy.

[0013] Further disclosed are methods of inhibiting scar formation and / or treating scar tissue in a subject in need thereof, including administering metformin to the subject. In some aspects, a subject having a wound is selected for treatment with metformin. In some aspects, metformin is administered topically. In some aspects, metformin is administered to a wound site.

[0014] Also disclosed are compositions including metformin, such as pharmaceutical compositions, that are of use in the methods disclosed herein.

[0015] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A-1B: Topical metformin penetrates mouse skin and reaches the Achillies tendon in a timedependent manner (FIG. 1A). In addition, topical application of metformin achieves similar concentrations in the Achillies tendon as IP or local injection (LI) of metformin (FIG. IB).

[0018] FIGS. 2A-2B: Topical metformin reduces inflammation in wounded Achillies tendon. HMGB1 and PGE2, two markers of tissue inflammation, are inhibited by topical application of metformin to a similar extent as IP or local injection of metformin (FIGS. 2A). IP injection or topical application of metformin blocks HMGB1 release in wounded tendon matrices (FIG. 2B).

[0019] FIGS. 3A-3C: Metformin concentration in Achilles tendon tissue over time following local injection with 40 pg (FIG. 3A), 400 pg (FIG. 3B), or 4,000 pg (FIG. 3C) of metformin. concentrations of metformin (0 - 5 mg / ml) in DMEM growth medium. Metformin appears to promote the formation of larger colonies in Met-containing medium as compared to the saline control group (0 pg / ml metformin).

[0020] FIG. 5: Higher doses of metformin ( 1 mg / ml and 5 mg / ml) decrease population doubling time (PDT) of tendon stem cells (TSCs) in culture. Thus, high doses of metformin promote TSC proliferation.

[0021] FIGS. 6A-6B: Topical metformin lotion inhibits ITR-Induced tendinopathy and enthesopathy. FIG. 6A shows H&E staining results which indicate that intensive treadmill running (ITR)-induced tissue structure changes in mouse Achilles tendon and its enthesis after 4 weeks of ITR. Metformin lotion prevents tissue damage from mechanical overuse. ITR-induced tendon degenerative changes are evident in the ITR control, which has thinner collagen fibers, smaller enthesis size, and many round shaped cells. In contrast, 3% and 6% metformin lotion inhibited these changes as evidenced by thicker collagen fibers in the enthesis areas with larger fibrocartilage tissue and fewer round shaped cells in the tendon, which are indicative of chondrogenic differentiation. FIG. 6B shows Histochemical analysis by Safranin O and Fast Green staining of mouse Achilles tendons after 4 weeks of ITR. ITR induces tendon degenerative changes are evident in the ITR control, such as thinner collagen fibers, smaller enthesis size, and many round shaped cells. In contrast, 3% and 6% metformin lotion inhibit these changes as evidenced by the presence of thicker collagen fibers in the enthesis areas showing larger fibrocartilage tissue with heavily stained proteoglycans and less round shaped cells in the tendon.

[0022] FIG. 7A-7C: Application of metformin (Met) lotion treatment reduces the severity of tendinopathy induced by mechanical overuse in mouse Achilles tendons. Tendinopathy by mechanical overuse was induced in mice by intensive treadmill running (ITR) for 4 weeks. Mice were then divided into three treatment groups: (1) no further treatment for three or four weeks (ITR-4w + Cont- 3 / 4w); (2) 50 mg of 3% Met lotion, applied to the skin surface of each hind leg, totaling 100 mg of 3% Met-lotion daily for three / four weeks (ITR-4w + 3% Met-lotion-3 / 4w); or (3) 50 mg of 6% Met lotion, applied to the skin surface of each hind leg, totaling 100 mg of 6% Met-lotion daily for three / four weeks (ITR-4w + 6% Met- lotion- 3 / 4w). FIG. 7A shows H&E staining of tendon tissue sections of mice while FIG. 7B shows safranin O and Fast Green staining on tendon sections of mice. ITR-4w + Cont-4w mouse tissue sections displayed evidence of tendon degenerative changes induced by the 4 weeks of ITR (thinner collagen fibers, reduced enthesis size, and an abundance of round shaped cells), which persisted even after 4 weeks of cage activities. In contrast, ITR-4w + 3% Met lotion-4w and ITR-4w + 6% Met lotion-4w treated mouse tissue sections indicated that Met-lotion treatment effectively mitigated tendon degenerative changes induced by 4 weeks of ITR, as evidenced by thicker collagen fibers in the enthesis areas with larger fibrocartilage tissue size, and a reduced number of round-shaped cells. FIG. 7C shows immunostaining for p-AMPK.

[0023] FIG. 8: H&E staining of mouse Achilles tendons after 12-weeks of ITR. Application of metformin lotion (3% and 6%) prevents ITR-induced tissue damage in the enthesis and tendon. cell nuclei to the extracellular matrix of the mouse Achilles tendon. Tissues were treated with (FIG. 9A) or without (FIG. 9B) Triton®-X-100 nonionic surfactant.

[0024] FIG. 10: Topical metformin lotion inhibits tissue degenerative changes induced by 4 weeks of ITR in the mouse Achilles tendon.

[0025] FIG. 11: Topical metformin lotion inhibits tissue degenerative changes induced by 12 weeks of ITR in the mouse Achilles tendon.

[0026] FIGS. 12A-12B: Histological staining shows that metformin injection decreases aging-induced tendon degeneration (FIG. 12A; scale bar: 25 pm). Western blot analysis confirms that administration of metformin reduces HMGB1 levels in 19M mice (FIG. 12B). 4M = 4 month-old mouse; 19M= 19 month-old mouse; 19M-Met-IP = 19 month-old mouse that received IP injection of metformin for 8 weeks.

[0027] FIG. 13: Masson's trichrome staining of mouse Achilles tendons shows that ITR-induced degenerative changes, characterized by loose collagen fibers, are suppressed by topical metformin application, as indicated by the presence of dense collagen fibers.

[0028] FIGS. 14A and 14B: Piero Sirius Red staining (FIG. 14 A) and polarized light microscopy (FIG. 14B) of mouse tendons shows that topical application of metformin lotion decreases ITR-induced collagen HI formation and enhances dense collagen I formation in mouse Achilles tendons.

[0029] FIG. 15: Immunostaining of mouse Achilles tendons shows that topical application of metformin lotion decreases ITR-induced expression of two chondrocyte markers (collagen II and SOX-9) in mouse Achilles tendon.

[0030] FIG. 16: IR spectrum of metformin hyaluronate (HA-MET), hyaluronic acid (HA), and metformin (MET).

[0031] FIG. 17: Exemplary reaction scheme for producing a metformin hyaluronic acid conjugate.

[0032] FIG. 18: Schematic of metformin lotion skin permeation test using a Franz cell.

[0033] FIGS. 19A-19B: Metformin lotion permeation kinetics in pig ear skin. FIG. 19A. The total amount of 6% Met lotion that permeated through the skin over the time course. FIG. 19B. The percentage of Met lotion that permeated at the same time intervals. It is observed that Met permeation increases in a time-dependent manner. Specifically, at 24 hours, 529 pg or 36.8% of the Met in the lotion permeated through the pig ear skin.

[0034] FIGS. 20A-20B: Met-lotion is anti-inflammatory. FIG. 20A: HMGB1 levels in serum samples from rats with skin wounds treated with either 0% or 6% Met-lotion for 10 days. FIG. 20B: IL- 1 levels under the same conditions. For both HMGB1 and IL-fS 1 , levels in the serum samples from rats treated with 6% Met-lotion were significantly reduced compared to those treated with 0% Met-lotion, indicating a decrease in inflammation (*p < 0.05).

[0035] FIGS. 21A-21I: Application of Met lotion inhibits the release of HMGB1 from cell nuclei into the skin tissue matrix in rats. FIGS. 21A-21D: Skin wounds treated by 0% Met-lotion for 10 days. FIGS. 21E- 21H: Skin wounds treated with 6% Met-lotion for 10 days. FIGS. 21A & 21E: DAPI staining. FIGS. 21B- Merged images of FIGS. 21E-21F. FIG. 2 ID: Enlarged box area in image FIG. 21C. FIG. 21H: enlarged box area in image FIG. 21G. High levels of HMGB1 were found in the skin tissue matrix of the rats treated with 0% Met-lotion (arrows in FIG. 21D). However, in 6% Met-lotion treated group (FIGS. 21E-21H), HMGB1 was positively stained in the cell nuclei (arrows in FIG. 21H). FIG. 211: Semi-quantification results of positive stained cells with HMGB 1 in the nuclei. Semi-quantification results indicated more than 46% of the cells around the wound area of the rat skin treated with 6% Met-lotion kept HMGB1 in the nuclei (FIG. 211), however, less than 3.7% of the cells in the wound area of the rat skin treated with 0% Met- lotion showed HMGB1 in the nuclei (FIG. 211). *p < 0.001 compared to 0% Met-lotion treated group. FIGS. 21A-21C and 21E-21G bars: 200 pm; FIGS. 21D and 21H bars: 50 pm.

[0036] FIGS. 22A-22G: Histological analysis using H&E staining indicates metformin lotion inhibits scar tissue formation in rat skin after 10 days of wound healing. FIGS 22A-22C: Rat skin wounds treated with 0% Met-lotion. FIGS. 22D-22F: Wounds treated with 6% Met-lotion. The results indicate that a dense and thick epidermis formed at the wound area (marked by a dashed line in FIG. 22A) treated with 0% Met-lotion (arrows in FIGS. 22A-22C). In contrast, normal skin-like tissues formed at the wound area (marked by a dashed line in FIG. 22D) treated with 6% Met-lotion (arrows in FIGS. 22D-22F). FIG. 22G: The semiquantification of epidermis thickness in the healed skin tissue at the treated wound areas. Semiquantification shows that epidermis thickness at the wound area treated with 0% Met-lotion was six times thicker than the area treated with 6% Met-lotion (FIG. 22G). There was no significant difference in epidermis thickness between normal skin and skin treated with 6% Met-lotion (FIG. 22G). *p < 0.05 compared to 0% Met-lotion treated wounds. *p < 0.05 compared to normal skin. FIGS. 22A & 22D bars: 1 mm; FIGS. 22B & 22E bars: 500 pm; FIGS. 22C & 22F bars: 200 pm.

[0037] FIGS. 23 -23F: Histological analysis using Trichromc-Masson staining indicates that metformin lotion inhibits scar tissue formation in rat skin after 10 days of wound healing. FIGS. 23A-23C: 0% Met- lotion treated rat skin wounds; FIGS. 23D-23F: 6% Met-lotion treated rat skin wounds. The results reveal a large empty gap (arrows in FIGS. 23A-23C) in the wound area treated with 0% Met-lotion (dashed line area in FIGS. 23A-23C). In contrast, no gap was observed in the wound area treated with 6% Met-lotion (dashed line area in FIGS. 23D-23F). The latter area developed some normal skin-like tissues with hair follicles (arrows in FIG. 23F) and sweat glands (arrows in FIG. 23F). In comparison, very few hair follicles and sweat glands were found in the wound area treated with 0% Met-lotion (FIGS. 23A-23C). Furthermore, a significant amount of fibrosis-like tissues were present in the wound area treated with 0% Met-lotion (dashed line area in FIGS. 23A-23C), while markedly fewer fibrosis-like tissues were found in the wound area treated with 6% Met-lotion (dashed line area in FIGS. 23D-23F). FIGS. 23 A & 23D: 1 mm; FIGS. 23B & 23E bars: 500 pm; FIGS. 23C & 23F bars: 200 pm.

[0038] FIGS. 24A-24E: Piero-Sirius Red staining shows that application of metformin lotion decreases collagen III levels in rat skin after 10 days of wound healing. FIGS. 24A-24B: 0% Met-lotion treated rats; FIGS. 24C-24D: 6% Met-lotion treated rats. FIGS. 24A, 24C: Images obtained using a bright field microscopy, a large gap (arrow in FIG. 24A) was observed in the wound areas treated with 0% Met-lotion (dashed line area in FIG. 24A), while no gap was found in the wound area treated with 6% Met-lotion (dashed line area in FIG. 24C). Under polarized light microscopy, high levels of collagen III were detected in the wound area treated with 0% Met-lotion (FIG. 24B), whereas the tissues in the wound area treated with 6% Met-lotion were positively stained for collagen I (FIG. 24D). FIG. 24E: Semi-quantification of collagen HI expression in skin tissue sections, as determined by a polarized light microscope. Semi-quantitative results showed that more than 55% of the cells in the wounded skin tissues of rats treated with 0% Met- lotion were positive for collagen III, compared to less than 9.1% of the cells in the wounded skin area of rats treated with 6% Met-lotion (FIG. 24E). *p < 0.001 compared to the 0% Met-lotion treated wound. FIGS. 24A-24D bars: 500 pm.

[0039] FIGS. 25A-25I: Immunostaining for collagen III shows that application of metformin lotion decreases collagen III levels in rat skin wound areas after 10 days of healing. FIGS. 25A-25D: 0% Met- lotion treated skin wounds; FIGS. 25E-25H: 6% Met-lotion treated skin wounds. FIGS. 25A, 25E: DAPI staining; FIGS. 25B, 25F: Collagen III staining; FIGS. 25C: Merged images of FIGS. 25A and 25B; FIG. 25G: Merged images of FIG. 25E and 25F; FIG. 25D: Enlarged box area in FIG. 25C. FIG. 25H: Enlarged box area in FIG. 25G. The results indicated that 6% Met-lotion reduced collagen III expression in wounded skin areas (FIGS. 25E-25H). In contrast, wounds treated with the control lotion (0% Met-lotion) showed significantly higher levels of collagen III compared to those treated with 6% Met-lotion (FIGS. 25A-25D). FIG. 251: Semi-quantification of the positively stained cells in the wounded skin area. Semi-quantification revealed that over 68% of the cells in the wound area treated with 0% Met-lotion were positively stained for collagen III, while less than 15.4% of the cells in the wound area treated with 6% Met-lotion showed positive staining for collagen III (FIG. 251). *p < 0.001 compared to the 0% Met-lotion treated wound. FIGS. 25A-25C & 25E-25G bars: 500 pm; FIGS. 25D & 25H bars: 200 pm.

[0040] FIGS. 26A-26I: Immunostaining for a-SMA shows that application of metformin lotion decreases a-SMA levels in rat skin wound areas after 10 days of healing. FIGS. 26A-26D: 0% Met-lotion treated skin wounds; FIGS. 26E-26H: 6% Met-lotion treated skin wounds. FIGS. 26A, 26E: DAPI staining; FIGS. 26B, 26F: a-SMA staining; FIG. 26C: Merged images of FIGS. 26A and 26B; FIG. 26G: Merged images of FIGS. 26E and 26F; FIG. 26D: Enlarged box area in FIG. 26C. FIG. 26H: Enlarged box area in FIG. 26G. FIG. 261: Semi-quantification of a-SMA positively stained cells in the skin tissue sections treated either with 0% Met-lotion or 6% Met-lotion. The results demonstrated that 6% Met-lotion reduced a-SMA expression in wounded skin areas (FIGS. 26E-26H). In contrast, more than 77% of the cells in the skin areas wounded and treated with 0% Met-lotion expressed a-SMA (FIGS. 26A-26D, 261), while approximately 16.7% of the cells in the areas treated with 6% Met-lotion expressed a-SMA (FIGS. 26E-26H, 261). *p < 0.05 compared to the 0% Met-lotion treated wounds. FIGS. 26A-26C & 26E-26G bars: 500 pm; FIGS. 26D & 26H bars: 200 pm. TGF- 1 levels in rat skin wound areas after 10 days of healing. FIGS. 27A-27D: 0% Met-lotion treated skin wounds; FIGS. 27E-27H: 6% Met-lotion treated skin wounds. FIGS. 27 A, 27E: DAPI staining; FIGS. 27B, 27F: TGF-pi staining; FIG. 27C: Merged images of FIGS. 27A and 27B; FIG. 27G: Merged images of FIGS. 27E and 27F; FIG. 27D: Enlarged box area in FIG. 27C. FIG. 27H: Enlarged box area in FIG. 27G. FIG. 271: Semi-quantification of staining cells in the skin tissue sections treated either with 0% or 6% Met- lotion. The results show that 6% Met-lotion inhibited TGF- 1 expression in wounded skin areas (FIGS. 27E-27H). In contrast, the wound treated with control lotion (0% Met-lotion) expressed higher levels of TGF-pi (FIGS. 27A-27D) compared to 6% Met-lotion treated wounds. Semi-quantification results indicated that more than 69% cells in the wounded skin area treated with 0% Met lotion were positively stained with TGF-PI; however, less than 11.2% cells in the wound area treated with 6% Met-lotion expressed TGF-PI (FIG. 271). *p<0.001 compared to the 0% Met-lotion treated wound. FIGS. 27A-27C & 27E-27G bars: 500 pm; FIGS. 27D & 27H bars: 200 pm.

[0041] FIGS. 28A-28I: Immunostaining for phosphorylation of AMPK (p-AMPK) shows that metformin lotion application increases p-AMPK expression in rat skin wound areas after 10 days of healing. FIGS. 28A-28D: 0% Met-lotion treated skin wounds; FIGS. 28E-28H: 6% Met-lotion treated skin wounds. FIGS. 28A, 28E: DAPI staining; FIGS. 28B, 28F: p-AMPK staining; FIG. 28C: Merged images of FIGS. 28A and 28B; FIG. 28G: Merged images of FIGS. 28E and 28F; FIG. 28D: Enlarged box area in FIG. 28C. FIG. 28H: Enlarged box area in FIG. 28G. The results indicated that 6% Met-lotion enhanced p-AMPK expression in wounded skin areas (FIGS. 28E-28H). In contrast, wounds treated with the control lotion (0% Met-lotion) showed significantly lower levels of p-AMPK compared to those treated with 6% Met-lotion (FIGS. 28A-28D). FIG. 281: Semi-quantification of positively stained cells. Semi-quantitative analysis revealed that more than 57% of the cells in the 6% Met-lotion treated wound area expressed p-AMPK, while less than 5% of the cells in the wound area treated with 0% Met-lotion showed p-AMPK expression. *p < 0.001 compared to the 0% Met-lotion treated wound. FIGS. 28A-28C & 28E-28G bars: 500 pm; FIGS. 28D & 28H bars: 200 pm.

[0042] FIGS. 29A-29I: Immunostaining for collagen I shows that metformin lotion application increases collagen I levels in rat skin wound areas after 10 days of healing. FIGS. 29A-29D: 0% Met-lotion treated skin wounds; FIGS. 29E-29H: 6% Met-lotion treated skin wounds. FIGS. 29A, 29E: DAPI staining; FIGS. 29B, 29F: collagen I staining; FIG. 29C: Merged images of FIGS. 29A and 29B; FIG. 29G: Merged images of FIGS. 29E and 29F; FIG. 29D: Enlarged box area in FIG. 29C. FIG. 29H: Enlarged box area in FIG. 29G. The results demonstrated that 6% Met-lotion increased collagen I expression in wounded skin areas (FIGS. 29E-29H). In contrast, wounds treated with the control lotion (0% Met-lotion) showed significantly lower levels of collagen I compared to those treated with 6% Met-lotion (FIGS. 29A-29D). FIG. 291: Semiquantification of collagen I stained cells. Semi-quantitative analysis revealed that more than 50% of the cells in the 6% Met-lotion treated wound area expressed collagen I (FIG. 291), while less than 26% of the to the 0% Met-lotion treated wound. FIGS. 29A-29C & 29E-29G bars: 500 pm; 29D & 29H bars: 200 pm.

[0043] DETAILED DESCRIPTION

[0044] I. Introduction

[0045] Tendons are specialized tissues that enable joint movements by transmitting mechanical forces from muscle to bone. They are relatively hypocellular tissues, predominantly composed of collagen and two major types of cells, tenocytes, and tendon stem cells (TSCs). Under normal conditions, tenocytes are responsible for maintaining tendon homeostasis, whereas TSCs replenish tendon cells by undergoing selfrenewal and differentiation. The multi-differentiation potential of TSCs typically allows these stem cells to differentiate into tenocytes; however, under high stress and injurious conditions, TSCs can undergo aberrant differentiation into adipocytes, chondrocytes, and osteocytes.

[0046] The tendon is not only a load-transmitting tissue, but also a mechanoresponsive live tissue. While proper and normal (moderate) mechanical loading is important for maintaining tendon homeostasis, mechanical overloading / overuse of tendons plays a role in the development of tendinopathy (see, e.g., Zhao et al., PLoS One, 2019, 14(9):e0222369; Scott et al., Arthritis and rheumatism, 2007, 56(3):871-881; Abraham et al., BMC Musculoskelet Disord, 2011, 12:26; Zhang and Wang, J Orthop Res 2010, 28(2): 198- 203; Zhang and Wang, PLoS One, 2013, 8(8):e71740).

[0047] Appropriate moderate exercise has beneficial effects on tendons and can enhance the function of TSCs, resulting in the formation of normal-like tendon tissue at the site of an injury. It is possible that release of low levels of PGEz induced by moderate exercise promotes the sternness of TSCs (see, e.g., Langberg et al. J Physiol 1999, 515 (Pt 3) :919-927). However, high levels of PGE2have been linked to tendon inflammation and degeneration in mechanically overloaded tendon cells and tendons (see, e.g., Wang et al. Connect Tissue Res 2003, 44(3-4): 128-133; Khan et aL, Clin J Sport Med 2005, 15(l):27-33). Moreover, excessive mechanical loading and high levels of PGE2 can induce non-tenocyte differentiation of TSCs (see, e.g., Zhang and Wang, PLoS One 2013, 8(8):e71740; Zhang and Wang, PLoS One 2014, 9(2):e87706). Independently, inflammatory cytokines (e.g., IL-1, and IL-6) and degradative enzymes MMPs (e.g., MMP-1, -3, and -13) can be elevated in tendinopathic tendons (see, e.g., Riley, Scand J Med Sci Sports 2005, 15(4):241-251). A mouse model of tendinopathy due to mechanical overuse / overloading placed on the tendon via long-term intensive treadmill running (ITR) was developed to study tendinopathy. This animal model shows characteristic features of human tendinopathic tendons (see, e.g., Zhao et al. PLoS One 2019, 14(9):e022236; Zhang and Wang, PLoS One 2013, 8(8):e71740; Zhang and Wang, J Orthop Res 2010, 28(5):639-643).

[0048] HMGB1 was identified as a mediator of tendon inflammation and degeneration in response to mechanical overuse, such as ITR (Zhao et al. PLoS One 2019, 14(9):e0222369). HMGB1 is a highly conserved nuclear protein that is expressed by almost all cells, with both intracellular and extracellular roles (Sessa and Bianchi, Gene 2007, 387(1 -2) : 133- 140). Translocation of HMGB1 from the nucleus to the modifies its function and studies have shown that in cells under stress, HMGBl’s release to the ECM triggers pro-inflammatory events (Tang et al., Mol Med 2014, 20:359-362; Venereau el al., J Exp Med 2012, 209(9): 1519-1528).

[0049] Extracellular HMGB1 can promote the recruitment of inflammatory cells to damaged tissues, induce COX expression and PGE2 production, which are traditional targets of drugs, such as NSAIDs. However, HMGB1 can also cause cellular production of other pro-inflammatory cytokines such as IL-6, IL-8, IL-17, TNF etc., which mediate chronic tissue inflammation and degeneration (see, e.g., Akbar et al. Targeting danger molecules in tendinopathy: the HMGB1 / TLR4 axis. RMD Open 2017, 3(2); Zhao et al. PLoS One 2019, 14(9):e0222369; Venereau et al. J Exp Med 2012, 209(9): 1519-1528; Bertheloot and Latz, Cell Mol Immunol 2017, 14(l):43-64). Indeed, using in vitro and in vivo models, it has been shown that excessive mechanical loading in tendons releases HMGB 1 and initiates tendon inflammation and degeneration (see, e.g., Zhao et al. PLoS One 2019, 14(9):e0222369). In addition, recent studies support the pathogenic role of HMGB1 in human tendinopathy by showing that HMGB1 is present in human tendinopathic tendons, HMGB1 is upregulated in tendinopathy patients, and upregulation of HMGB1 is associated with inflammatory responses and ECM disorganization in rat rotator cuff tendon injury model (see, e.g., Akbar et al. Targeting danger molecules in tendinopathy: the HMGB1 / TLR4 axis. RMD Open 2017, 3(2); Mosca et al. BMJ Open Sport Exerc Med 2017, 3(l):e000225; and Thankam et al. Sci Rep 2018, 8(1):8918). These findings support the notion that extracellular HMGB 1 plays an important role in the pathology of tendinopathy.

[0050] Metformin is a specific inhibitor of HMGB1 and binds to the acidic tail of HMGB1 (see, e.g., Horiuchi et al., Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity. J Biol Chem 2017, 292(20): 8436-8446). Metformin has been shown to improve chronic inflammation and have a direct anti-inflammatory effect (Bharath and Nikolajczyk, Am J Physiol Cell Physiol 2021, 320(5):C873-C879; Carvalho et al., Eur J Pain 2021, 25(6) : 1264- 1273). Thus, unlike conventional drugs (such as NSAIDs), which only target COX / PGEi axis-induced inflammation, targeting HMGB1 through metformin may have broader anti-inflammatory effects, for example, on COX / PGEz, pro-inflammatory cytokines (e.g., IL-6, IL-8, IL-17, TNF, etc.), and degrading enzymes (e.g., MMP-3).

[0051] It has recently been shown that IP injection of metformin blocks inflammatory and degenerative changes of tendons in ITR mice (Zhang et al. Foot Ankle Int 2020, 41 (12) : 1455- 1465). IP injection of metformin has also been shown to inhibit tendon degeneration by blocking translocation of HMGB1 and suppressing tendon inflammation and degeneration, and senescence in aging mice, suggesting that the use of metformin not only reduces tendon inflammation and degeneration but can also promote the quality of tendon healing in tendinopathy (Zhang et al., “Metformin Improves Tendon Degeneration by Blocking Translocation of HMGB 1 and Suppressing Tendon Inflammation and Senescence in Aging Mice.” J Orthop Res, 2022). However, current studies have been limited to oral administration of metformin, or in a few cases, administration of metformin by IP injection. Oral administration can lead to off target effects and / or administration outside of a medical office. Thus, a topical formulation / administration of metformin with the aim to reduce / block tendon inflammation / degeneration, and promote healing, repair, and regeneration of injured tendons would be beneficial. However, formulating metformin compositions for topical administration (or local injection) is not straightforward. For example, determining excipients that can facilitate metformin lotion diffusion through human skin and reach the tendon with a sufficient dosage of metformin is a challenge despite what is known of metformin generally. In addition, the PK / PD of a metformin topical composition (e.g., lotion) and LIM (local injection of metformin) must be determined locally and systemically; the safety and toxicity must be evaluated, particularly concerning local skin, tendon, muscles, nerves, etc.,' and the minimum effective doses of metformin must be determined independently for each administration route.

[0052] Data disclosed herein confirms that topical and local administration of metformin is effective, and suggests that metformin lotion may be effective as a post-injury treatment as well as a preventative treatment in high-risk populations of tendinopathy, such as aging individuals (> 65 years), athletes, and military personnel. It was found that local injection or topical administration of metformin was particularly suitable for the treatment of soft tissue musculoskeletal injuries due to the amount of metformin that reaches the tendon via circulation and skin permeation versus an oral administration method, which slowly releases metformin into circulation. Based upon the ability to retain most of the administered metformin in the tissue (e.g., in the tendon), a lower dose can be used than what has previously been shown, for example, with oral administration.

[0053] In addition, it is shown that topical administration of metformin by applying metformin lotion on the skin surface enhances skin wound healing and decreases scar formation. Results disclosed herein demonstrate marked differences between wounds treated with a vehicle control (0% Mct-lotion) and those treated with 6% metformin lotion (6% Met-lotion). Notably, wounds treated with the 6% Met-lotion exhibited accelerated healing and formation of tissue resembling normal skin, as opposed to the slower healing and scar-like tissue formation observed with the control lotion. Topical administration of metformin is advantageous not only because it has higher efficiency at lower doses, but also avoids systemic side effects associated with oral administration.

[0054] II. Summary of Terms

[0055] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin 's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a tendon” includes singular or plural tendons and can be considered equivalent to the phrase “at least one tendon.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0056] To facilitate review of the various aspects of the disclosure, the following explanations of terms are provided:

[0057] About: Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

[0058] Administering or Administration: The introduction of an agent, such as metformin, into a subject by a chosen route. Administration can be local or systemic. Local administration refers to the introduction of an agent directly to a desired a site of action, for example, administering metformin to the bodily location of a soft tissue musculoskeletal injury, for example by local injection or topical administration. Systemic administration refers to the introduction of an agent into the circulatory system. Exemplary routes of administration include, but are not limited to, oral, parenteral (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), local injection, sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes. In some aspects, administration of metformin in a method disclosed herein is transdermal or by local injection.

[0059] Control: A reference standard. A control can be a positive or negative control. In some examples, the control is a measure or sample obtained from a healthy subject. In some examples, the control is a measure or sample obtained from a subject having a soft tissue musculoskeletal injury. In some examples, the control is a measure or sample obtained from a subject prior to metformin administration. In still other examples, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients diagnosed with a disease or condition, for example soft tissue musculoskeletal injuries, that have a known prognosis or outcome, or group of samples that represent baseline or normal values).

[0060] Increase or Decrease: A positive or negative change from a reference value. An increase is a positive change, such as an increase at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500%, as compared to a reference value. A decrease is a negative change, such as a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% decrease as compared to a reference value. In some examples, the reference value is a control. In some examples, the increase or decrease is statistically significant.

[0061] Metformin: A biguanide antidiabetic therapeutic. Metformin reduces blood glucose levels by decreasing glucose production in the liver, decreasing intestinal absorption, and increasing insulin sensitivity. Oral metformin is commonly used to treat type II diabetes mellitus. Metformin also has several non-FDA-approved indications, including gestational diabetes, management of antipsychotic-induced (PCOS).

[0062] Pharmaceutically Acceptable Carriers: The pharmaceutically acceptable carriers of use for the compositions and methods disclosed herein are known. In general, the nature of the carrier depends on the particular mode of administration being employed. For instance, injection formulations may include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. Transdermal formulations may include petroleum jelly, petroleum wax, glycerol, hydrophobic surfactants, etc. Remington’s Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020), describes compositions and formulations suitable for pharmaceutical delivery of metformin, as disclosed herein.

[0063] Soft Tissue Musculoskeletal Injury: An injury of a soft tissue associated with the musculoskeletal system, such as a muscle, tendon, or ligament. Soft-tissue injuries can be acute injuries or chronic injuries. Acute injuries are caused by a sudden trauma, such as a fall, twist, or blow to the body. Examples include sprains, strains, and contusions. Chronic injuries are typically from overuse and / or failed healing responses. A non-limiting example of a soft tissue musculoskeletal injury is tendinopathy, which includes tendinitis (inflammation of a tendon) and tendinosis (degeneration of collagen in a tendon). Other examples of soft tissue musculoskeletal injuries include, but are not limited to enthesopathy, muscle tears or ruptures, bursitis, synovitis, and fasciitis.

[0064] Subject: Living multi-cellular vertebrate organism, a category that includes human and non-human mammals, such as non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, and the like. In some examples, the subject is a mammal. In some examples, the subject is a human. In some examples, the subject is a human or veterinary subject (e.g., dog, cat, or other domestic mammal). In some examples, the subject has a soft tissue musculoskeletal injury and in need of treatment thereof.

[0065] Therapeutically Effective Amount: The amount of an agent, such as metformin, that is sufficient to inhibit (such as prophylaxis), treat, and / or decrease a symptom or underlying cause of a disease or pathological condition, such as a soft tissue musculoskeletal injury. In some examples, a therapeutically effective amount of metformin is an amount that treats a soft tissue musculoskeletal injury, such as decreasing or eliminating one or more symptoms of the soft tissue musculoskeletal injury.

[0066] The exact amount and timing of administration can depend on a number of factors, including the subject being treated, the severity and type of the condition being treated, and the manner of administration. A therapeutically effective amount can be determined by a practitioner through clinical trials. Effective amounts also can be determined through various in vitro, in vivo or in situ assays. A therapeutically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attain a desired response. For example, a therapeutically effective amount of an agent can be administered in a single dose, or in several doses during a course of treatment (e.g., twice daily for two weeks). ameliorates a sign or symptom of a disease or condition (e.g., a soft tissue musculoskeletal injury) after the disease or condition has begun to develop. The term “ameliorating,” with reference to a disease or condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of symptoms in a subject, a reduction in severity of some or all symptoms, a slower progression, an improvement in the quality of life of the subject, or by other parameters that are specific to the particular disease or condition. Treatment may be assessed by objective or subjective parameters, including the results of a physical examination, ultrasound or other imaging, blood or other clinical tests, self-evaluations by the subject, and the like.

[0067] Inhibiting a disease or condition includes reducing the risk of the disease or condition, such as reducing the risk of a soft tissue musculoskeletal injury (e.g., tendinopathy). A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease (e.g., a subject in remission) or exhibits only early signs for the purpose of decreasing the risk of developing a disease or condition. Prophylactic treatment can reduce the risk of a soft tissue musculoskeletal injury. In some examples, the disclosed methods are prophylactic and / or therapeutic. In some examples, the disclosed methods are therapeutic and not prophylactic.

[0068] III. Methods

[0069] Disclosed are methods of inhibiting or treating a soft tissue musculoskeletal injury, including administering metformin to the subject. Also disclosed are methods of inhibiting or treating scar tissue, including administering metformin to the subject. In some aspects, the metformin is a metformin-hyaluronic acid conjugate (e.g., metformin hyaluronate). While any known method of conjugating hyaluronic acid and metformin can be used, such as amide coupling (e.g., see, Farag et al., International Journal of Nanomedicine 16:1005-1019, 2021), Example 8 provides an exemplary process for making a metforminhyaluronic acid conjugate (e.g., metformin hyaluronate).

[0070] Any of the methods disclosed herein can include a step wherein a subject is first selected for treatment with metformin. In some aspects, a subject at risk of, or having, a soft tissue musculoskeletal injury is selected for treatment with metformin. In some aspects, the subject selected for treatment is at risk of, or has, tendinopathy. In some aspects, a subject at risk of a soft tissue musculoskeletal injury is a human subject over the age of 40 (e.g., over the age of 50, over the age of 60, over the age of 65, etc.). The soft tissue musculoskeletal injury can be a chronic injury or an acute injury. In some aspects, the soft tissue musculoskeletal injury is a chronic injury and administration of metformin is topical. In some aspects, the soft tissue musculoskeletal injury is an acute injury and administration of metformin is by local injection.

[0071] In some aspects, a subject at risk of, or having, scar tissue is selected for treatment. In some aspects, the scar tissue is skin tissue, for example, scar tissue of the dermis and / or epidermis. In some aspects, the scar tissue is epidermal scar tissue. In some aspects, the scar tissue is dermal scar tissue. In some aspects, a subject at risk of, or having, scar tissue from a wound (e.g., a lesion, abrasion, laceration, burn, or wound abrasion, laceration, burn, or wound resulting from a surgical procedure) is skin wound, for example, wounded dermal or epidermal tissue. In some aspects, a subject at risk of, or having, scar tissue from a lesion, laceration, or surgical procedure is selected for treatment. In some aspects, the scar tissue is skin tissue and administration of metformin is topical.

[0072] In some aspects, the subject of any of the methods disclosed herein is a human. The subject can be a child or adult human. In some aspects, the subject is a human adult, for example, a human adult at least 18, 21, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old. In particular examples, the subject is a human subject over the age of 40. In further examples, the subject is a human subject over the age of 50. In some aspects, the subject is a human subject over the age of 65. In some aspects, the subject is a human subject between 0 and 110 years of age, for example, 5 to 110, 12 to 110, 16 to 110, 18 to 110, 25 to 110, 30 to 110, 35 to 110, 40 to 110, 45 to 110, 55 to 110, 60 to 110, 65 to 110, 70 to 110, 75 to 110, or 80 to 110. In a nonlimiting example, the subject is a human subject between 45 and 110 years of age. In another non-limiting example, the subject is a human subject between 55 and 110 years of age. In a further non-limiting example, the subject is a human subject between 65 and 110 years of age. In some aspects, the subject is selected for treatment based on age, for example, a human subject at least 40, at least 50, at least 60, at least 65, at least 70, or at least 80 years of age is selected for treatment with metformin. In some aspects, a human subject at least 50 years old is selected for treatment with metformin. In some aspects, a human subject at least 65 years old is selected for treatment with metformin.

[0073] The soft tissue musculoskeletal injury can be any injury of a soft tissue of the musculoskeletal system; non-limiting examples of soft tissues include tendons, ligaments, muscles, bursa, fascia, synovia membrane, etc. In some aspects, the soft tissue musculoskeletal injury is an injury of a tendon, ligament, or muscle. In some aspects, the soft tissue musculoskeletal injury is an injury of a tendon, for example, an Achilles tendon, patellar tendon, rotator cuff tendon, biceps tendon, triceps tendon, quadriceps tendon, hamstring tendon, adductor tendon, extensor tendon, flexor tendon, peroneal tendon, etc. In some aspects, the soft tissue musculoskeletal injury is an injury of a ligament, for example, anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), annular ligament, glenohumeral ligaments, etc. In some aspects, the soft tissue musculoskeletal injury is an injury of a skeletal muscle, for example, quadriceps femoris, hamstrings, gastrocnemius, soleus, rotator cuff muscles, flexor and extensor muscles, etc. In a specific, non-limiting example, the soft tissue musculoskeletal injury is an injury of an Achilles tendon. In another non-limiting example, the soft tissue musculoskeletal injury is a rotator cuff injury. In some aspects, administration of metformin inhibits scar tissue formation.

[0074] In some aspects, administration of metformin inhibits scar tissue formation associated with an injury selected for treatment or treated with metformin, for example, a wound (for example, a lesion, abrasion, laceration, burn, or wound from a surgical procedure), or soft tissue musculoskeletal injury. In some aspects, the injury being treated is a current, unhealed injury (e.g., treating or inhibiting scar tissue a prior, healed injury (e.g., treating scar tissue associated with a past injury that formed scar tissue when it healed). In some aspects, treatment with metformin begins before the injury has healed (e.g., current injury). In some aspects, treatment with metformin begins after the injury is healed (e.g., prior injury).

[0075] In some aspects, the soft tissue musculoskeletal injury is one or more of: tendinopathy, enthesopathy, fasciitis, bursitis, and synovitis. In a non-limiting example, the soft tissue musculoskeletal injury is tendinopathy.

[0076] In some aspects treatment is prophylactic, for example, the subject is at risk of a soft tissue musculoskeletal injury and / or forming scar tissue, and metformin is administered to reduce risk of an injury and / or risk of forming scar tissue. In some aspects the treatment is therapeutic, for example, the subject has a soft tissue musculoskeletal injury and / or scar tissue, and metformin is administered to treat the musculoskeletal injury and / or scar tissue.

[0077] Metformin can be administered by any suitable route. In some aspects, metformin is administered orally, parenterally, topically, intranasally, by inhalation, or systemically. Parenteral administration includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrapleural, and intracisternal injections, or infusion techniques. In a non-limiting example, metformin is administered topically or by a local injection. In a further non-limiting example, metformin is administered topically. The preferred administration route may vary, for example, based on the location or severity of the injury. In some aspects, the preferred administration route for a soft tissue musculoskeletal injury less than 5 cm beneath the skin (for example, less than 4 cm, less than 3 cm, less than 2 cm, or less than 1 cm, beneath the surface of skin) is topical administration. In some aspects, the preferred administration route for a soft tissue musculoskeletal injury more than 5 cm beneath the surface of skin is by local injection.

[0078] In some aspects, the administration route for a soft tissue musculoskeletal injury less than 3 cm below a skin surface of the subject is topical administration. In some aspects, the administration route for a soft tissue musculoskeletal injury more than 3 cm below a skin surface of the subject is by local injection. In some aspects, the administration route for a soft tissue musculoskeletal injury less than 2 cm below a skin surface of the subject is topical administration. In some aspects, the administration route for a soft tissue musculoskeletal injury more than 2 cm below a skin surface of the subject is by local injection. In some aspects, the administration route for a soft tissue musculoskeletal injury less than 1 cm below a skin surface of the subject is topical administration. In some aspects, the administration route for a soft tissue musculoskeletal injury more than 1 cm below a skin surface of the subject is by local injection. In some aspects, the administration route for treating or inhibiting scar tissue is topical administration.

[0079] In some aspects, the administration route for a soft tissue musculoskeletal injury 0 to 5 cm, for example, 0 to 4 cm, 0 to 3 cm, 0 to 2.5 cm, 0 to 2 cm, 0 to 1.5 cm, 0 to 1 cm, 0 to 0.5 cm, 0.1 to 5 cm, 0.1 to 4 cm, 0.1 to 3 cm, 0.1 to 3 cm, 0.1 to 2.5 cm, 0.1 to 2 cm, 0.1 to 1.5 cm, 0.1 to 1 cm, 0.1 to 0.5 cm, 0.2 to 5 cm, 0.2 to 4 cm, 0.2 to 3 cm, 0.2 to 2.5 cm, 0.2 to 2 cm, 0.2 to 1.5 cm, 0.2 to 1 cm, 0.2 to 0.5 cm, 0.5 to 5 cm, 0.5 to 4 cm, 0.5 to 3 cm, 0.5 to 2.5 cm, 0.5 to 2 cm, 0.5 to 1.5 cm, 0.5 to 1 cm, 1 to 5 cm, 1 to 4 cm, 1 to to 5 cm, 2 to 4 cm, 2 to 3 cm, 2.5 to 5 cm, 2.5 to 4 cm, or 2.5 to 3 cm, below a skin surface of the subject is topical administration. In some aspects, the administration route for a soft tissue musculoskeletal injury 0 to 3 cm below a skin surface of the subject is by topical administration. In some aspects, the administration route for a soft tissue musculoskeletal injury 0 to 2 cm below a skin surface of the subject is by topical administration.

[0080] In some aspects, the administration route for a soft tissue musculoskeletal injury 2 to 10 cm, for example, 2 to 9 cm, 2 to 8 cm, 2 to 7 cm, 2 to 6 cm, 2 to 5 cm, 2 to 4 cm, 2 to 3 cm, 3 to 9 cm, 3 to 8 cm, 3 to 7 cm, 3 to 6 cm, 3 to 5 cm, 3 to 4 cm, 4 to 9 cm, 4 to 8 cm, 4 to 7 cm, 4 to 6 cm, 4 to 5 cm, 5 to 9 cm, 5 to 8 cm, 5 to 7 cm, 5 to 6 cm, 6 to 9 cm, 6 to 8 cm, 6 to 7 cm, 7 to 9 cm, 7 to 8 cm, or 8 to 10 cm, below a skin surface of the subject is local injection. In some aspects, the administration route for a soft tissue musculoskeletal injury 1 to 8 cm below a skin surface of the subject is local injection. In some aspects, the administration route for a soft tissue musculoskeletal injury 2 to 8 cm below a skin surface of the subject is by local injection. In some aspects, the administration route for a soft tissue musculoskeletal injury 3 to 8 cm below a skin surface of the subject is by local injection. In some aspects, metformin is not administered systemically. In some aspects, metformin is not administered orally.

[0081] In several aspects, the methods include administering metformin to a site of injury (e.g., a soft tissue musculoskeletal injury, or a wound (e.g., a lesion, an abrasion, a laceration, a burn, or wound from a surgical procedure (e.g., an incision))). In some aspects, the metformin is injected at the injury site (e.g., local injection) or topically applied to the injury or to skin adjacent to the injury (e.g., skin immediately above a soft tissue musculoskeletal injury). In some aspects, skin adjacent to the injury includes skin that is less than 10 cm (e.g., less than 8 cm, less than 6 cm, less than 5 cm, less than 4 cm, less than 3 cm, less than 2 cm, less than 1 cm, etc.) from an injury (e.g., an internal injury or external injury). In some aspects, skin adjacent to the injury includes skin that is 0 to 10 cm from an injury, for example, 0 to 9 cm, 0 to 8 cm, 0 to 7 cm, 0 to 6 cm, 0 to 5 cm, 0 to 4 cm, 0 to 3 cm, 0 to 2 cm, 0 to 1 cm, 0.1 to 10 cm, 0.1 to 9 cm, 0.1 to 8 cm, 0.1 to 7 cm, 0.1 to 6 cm, 0.1 to 5 cm, 0.1 to 4 cm, 0.1 to 3 cm, 0.1 to 2 cm, 0.1 to 1 cm, 0.2 to 10 cm, 0.2 to 9 cm, 0.2 to 8 cm, 0.2 to 7 cm, 0.2 to 6 cm, 0.2 to 5 cm, 0.2 to 4 cm, 0.2 to 3 cm, 0.2 to 2 cm, 0.2 to 1 cm, 0.5 to 10 cm, 0.5 to 9 cm, 0.5 to 8 cm, 0.5 to 7 cm, 0.5 to 6 cm, 0.5 to 5 cm, 0.5 to 4 cm, 0.5 to 3 cm, 0.5 to 2 cm, 0.5 to 1 cm, 1 to 10 cm, 1 to 9 cm, 1 to 8 cm, 1 to 7 cm, 1 to 6 cm, 1 to 5 cm, 1 to 4 cm, 1 to 3 cm, 1 to 2 cm, 2 to 9 cm, 2 to 8 cm, 2 to 7 cm, 2 to 6 cm, 2 to 5 cm, 2 to 4 cm, 2 to 3 cm, 3 to 9 cm, 3 to 8 cm, 3 to 7 cm, 3 to 6 cm, 3 to 5 cm, 3 to 4 cm, 4 to 9 cm, 4 to 8 cm, 4 to 7 cm, 4 to 6 cm, 4 to 5 cm, 5 to 9 cm, 5 to 8 cm, 5 to 7 cm, 5 to 6 cm, 6 to 9 cm, 6 to 8 cm, 6 to 7 cm, 7 to 9 cm, 7 to 8 cm, or 8 to 10 cm from an injury

[0082] (e.g., an internal injury or external injury). In a specific, non-limiting example, the soft tissue musculoskeletal injury is an injury of an Achilles tendon, and metformin is administered topically to skin adjacent to the Achilles tendon.

[0083] In some implementations, metformin is included in a composition, for example, a pharmaceutical composition. The exact formulation of a pharmaceutical composition can depend on a number of factors, and can be determined by a practitioner. Compositions, including pharmaceutical compositions, are further described herein, for example in “IV. Compositions Including Metformin.” Useful formulations are also described, for example, in Remington 's Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier (2020).

[0084] In some aspects, the methods disclosed herein include administering a composition (including a pharmaceutical composition) that includes metformin. In some aspects, the composition includes about 0.1% to about 25% weight by weight (w / w) metformin, for example, 0.1% to 20%, 0.1% to 18%, 0.1% to 15%, 0.1% to 12%, 0.1% to 10%, 0.1% to 8%, 0.1% to 6%, 0.1% to 5%, 0.1% to 4%, 0.1% to 3%, 0.1% to 2%, 0.1% to 1%, 0.2% to 25%, 0.2% to 20%, 0.2% to 18%, 0.2% to 15%, 0.2% to 12%, 0.2% to 10%, 0.2% to 8%, 0.2% to 6%, 0.2% to 5%, 0.2% to 4%, 0.2% to 3%, 0.2% to 2%, 0.2% to 1%, 0.3% to 25%, 0.3% to 20%, 0.3% to 18%, 0.3% to 15%, 0.3% to 12%, 0.3% to 10%, 0.3% to 8%, 0.3% to 6%, 0.3% to 5%, 0.3% to 4%, 0.3% to 3%, 0.3% to 2%, 0.3% to 1%, 0.5% to 25%, 0.5% to 20%, 0.5% to 18%, 0.5% to 15%, 0.5% to 12%, 0.5% to 10%, 0.5% to 8%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2%, 0.5% to 1%, 1% to 20%, 1% to 18%, 1% to 15%, 1% to 12%, 1% to 10%, 1% to 8%, 1% to 6%, 1% to 5%, 2% to 25%, 2% to 20%, 2% to

[0085] 18%, 2% to 15%, 2% to 12%, 2% to 10%, 2% to 8%, 2% to 6%, 2% to 5%, 3% to 25%, 3% to 20%, 3% to

[0086] 18%, 3% to 15%, 3% to 12%, 3% to 10%, 3% to 8%, 3% to 6%, 3% to 5%, 5% to 25%, 5% to 20%, 5% to

[0087] 18%, 5% to 15%, 5% to 12%, 5% to 10%, 5% to 8%, 10% to 25%, 10% to 20%, 10% to 15%, 15% to 25%,

[0088] 15% to 20%, or 20% to 25% weight by weight (w / w) metformin.

[0089] In some aspects, the composition includes about 1% to about 15% weight by weight (w / w) metformin. In some aspects, the composition includes about 1% to about 10% weight by weight (w / w) metformin. In some aspects, the composition includes about 3% to about 6% weight by weight (w / w) metformin. In some aspects, the composition includes about 5% to about 7% weight by weight (w / w) metformin. In some aspects, the composition includes about 5% to about 15% weight by weight (w / w) metformin. In some aspects, the topical composition includes about 5% to about 10% weight by weight (w / w) metformin. In further aspects, the composition includes about 0.1% to 5% weight by weight (w / w) metformin. In some aspects, the composition includes about 0.1% to 2% weight by weight (w / w) metformin. In some aspects, the composition is a topical composition (e.g., a topical pharmaceutical composition). The composition (including a pharmaceutical composition) can be administered by any suitable route, in some aspects the composition is administered topically or by local injection.

[0090] The dose of metformin administered to the subject in the methods provided herein may vary depending on multiple factors, including the timing of administration, the subject being treated, the severity of the condition being treated, and the manner of administration. Generally, an appropriate dose is used that is sufficient to alleviate symptoms of a musculoskeletal soft tissue injury or scar tissue without causing unwanted side effects. An appropriate dose can be determined, for example, by clinical trials.

[0091] Administering metformin includes single or multiple administrations of metformin, for example, metformin can be administered once, or may be administered periodically until a therapeutic result is administered for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, about 12 weeks, or longer. In some aspects, metformin is administered for at least 1 week, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 8 weeks, or longer. In some aspects, metformin is administered no longer than 12 weeks, for example, no longer than 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, or 2 weeks. In some aspects, metformin is administered for about 1 to about 12 weeks, for example, 1 to 10 weeks, 1 to 8 weeks, 1 to 6 weeks, 1 to 4 weeks, 1 to 2 weeks, 2 to 12 weeks, 2 to 10 weeks, 2 to 8 weeks, 2 to 6 weeks, 2 to 4 weeks, 4 to 12 weeks, 4 to 8 weeks, or 6 to 12 weeks. In some aspects, metformin is administered once daily, twice daily, three times a day, four times a day, six times a day, or more. In some aspects, metformin is administered twice daily. In some aspects, metformin is administered four times daily.

[0092] In some aspects, a composition including 1 to 10% metformin (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) is administered twice daily. In some aspects, a composition including 1 to 10% metformin (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) is administered twice daily for at least 2 weeks, for example, at least 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some aspects, a composition including 1 to 10% metformin (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) is administered twice daily for 2 to 12 weeks (e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks). In further aspects, a composition including 2 to 7% metformin is administered twice daily. In some aspects, a composition including 2 to 7% metformin is administered twice daily for at least 2 weeks, for example, at least 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In further aspects, a composition including 2 to 7% metformin (e.g., 2%, 3%, 4%, 5%, 6%, or 7%) is administered twice daily for 2 to 12 weeks (e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks). In a non-limiting example, a composition including 3 to 6% metformin is administered twice daily. In a nonlimiting example, a composition including 3 to 6% metformin is administered twice daily for at least 2 weeks, for example, at least 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In a further non-limiting example, a composition including 3 to 6% metformin (e.g., 3%, 4%, 5%, or 6%) or is administered twice daily for 2 to 12 weeks (e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks). In further aspects, a composition including 5 to 7% metformin (e.g., 5%, 6%, 7%) is topically administered twice daily for at least 2 weeks, for example, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, etc. In a non-limiting example, a composition including 5 to 7% metformin (e.g., 5%, 6%, 7%) is topically administered twice daily for 4 to 12 weeks, for example, 4 to 10, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 weeks.

[0093] In some aspects, metformin is administered while a patient has signs or symptoms of a soft tissue musculoskeletal injury. In some aspects, metformin is administered while a patient has signs or symptoms of scar tissue. or symptoms of a soft tissue musculoskeletal injury or scar tissue. Exemplary signs or symptoms of soft tissue musculoskeletal injury include, but are not limited to: inflammation, pain, swelling, restricted mobility of an affected joint, muscle weakness, and numbness or tingling. In some aspects, administering metformin to the subject decreases inflammation, pain, swelling, restricted mobility of an affected joint, muscle weakness, and / or numbness or tingling relative to a suitable control (e.g., a measurement obtained prior to treatment with metformin). Exemplary signs or symptoms of scar tissue include abnormal skin architecture, for example, the presence of fibrotic tissue, collagen deposits (e.g., abnormal accumulation of collagen I and / or collagen 111), or abnormally thick or dense tissue. In some aspects, administering metformin to the subject decreases tissue fibrosis and / or collagen deposits relative to a suitable control (e.g., a measurement obtained prior to treatment with metformin). In some aspects, administering metformin decreases HMGB1 and / or a-SMA expression relative to a suitable control (e.g., a historical standard or a measurement obtained prior to treatment with metformin). In some aspects, administering metformin decreases TGF-P1 expression and / or increases AMPK phosphorylation relative to a suitable control (e.g., a historical standard or measurement obtained prior to treatment with metformin).

[0094] In some aspects, the methods disclosed herein decrease one or more signs or symptoms of a soft tissue musculoskeletal injury (e.g., inflammation, pain, swelling, restricted mobility of an affected joint, muscle weakness) by at least 10%, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% (elimination of a symptom), as compared to a suitable control. In some aspects, the methods disclosed herein decrease one or more signs or symptoms of a soft tissue musculoskeletal injury by 5% to 100%, for example, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 75% to 100%, 75% to 90%, 75% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, 95% to 100%, or 90% to 95% as compared to a suitable control. In some aspects, administration of metformin eliminates one or more signs or symptoms of the soft tissue musculoskeletal injury in the subject. In some aspects, the control is a measure of a sign or symptom in the subject prior to administration of metformin. A measurement of a sign or symptom can be quantitative (e.g., a clinical measurement) or qualitative (e.g., a patient’s self-reported perception of a symptom, for example, self-reported pain).

[0095] In some aspects, the methods disclosed herein decrease one or more signs or symptoms of scar tissue (e.g., fibrotic tissue, collagen deposits, abnormally thick or dense tissue) at a treatment site by at least 10%, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% (elimination of a symptom), as compared to a suitable control. In some aspects, the methods disclosed herein decrease one or more signs to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 75% to 100%, 75% to 90%, 75% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, 95% to 100%, or 90% to 95% as compared to a suitable control. In some aspects, administration of metformin eliminates one or more signs or symptoms of scar tissue at a treated site in the subject. In some aspects, the control is a measure of a sign or symptom in the subject prior to administration of metformin.

[0096] In some aspects, administration of metformin decreases HMGB 1 and / or a-SMA expression in treated tissue by at least 10%, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% (elimination of a symptom), as compared to a suitable control. In some aspects, the methods disclosed herein decrease HMGB1 and / or a-SMA expression in treated tissue by 5% to 100%, for example, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 75% to 100%, 75% to 90%, 75% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, 95% to 100%, or 90% to 95% as compared to a suitable control. In some aspects, the control is a measure of HMGB1 or a-SMA expression prior to administration of metformin, or is a historical measure or reference standard.

[0097] In some aspects, administration of metformin decreases TGF-pi expression and / or increases AMPK phosphorylation in treated tissue by at least 10%, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% (elimination of a symptom), as compared to a suitable control. In some aspects, the methods disclosed herein decrease TGF-pi expression and / or increase AMPK phosphorylation in treated tissue by 5% to 100%, for example, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 75% to 100%, 75% to 90%, 75% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, 95% to 100%, or 90% to 95% as compared to a suitable control. In some aspects, the control is a measure of TGF-pi expression or AMPK phosphorylation prior to administration of metformin, or is a historical measure or reference standard. tendinopathy or tendon inflammation and / or degeneration, for example, pain, tenderness, swelling, weakness, limited range of motion, thinner collagen fibers, smaller enthesis size, or round shaped tendon cells, in the subject. Thus, in some aspects, treatment of metformin increases the size of collagen fibers, the size of the enthesis, or the number of elongated tendon cells in the subject, or decreases pain, tenderness, swelling, weakness, or limited range of motion in the subject. In a non-limiting example, the methods disclosed herein increase collagen fiber size and / or enthesis size in the subject (relative to the size prior to administration of metformin). In some aspects, the methods disclosed herein decrease one or more signs or symptoms of tendinopathy or tendon degeneration (e.g., thinner collagen fibers, smaller enthesis size, or round shaped tendon cells) by at least 10%, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% (normal tendon physiology), as compared to a suitable control. In some aspects, the methods disclosed herein decrease one or more signs or symptoms of tendinopathy or tendon degeneration by 5% to 100%, for example, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 75% to 100%, 75% to 90%, 75% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, 95% to 100%, or 90% to 95% as compared to a suitable control. In some aspects, administration of metformin eliminates one or more signs or symptoms of tendinopathy or tendon degeneration in the subject. In some aspects, the control is a measure of a sign or symptom of tendinopathy or tendon degeneration in the subject prior to administration of metformin. In some aspects, the control is a standard reference value or range of values obtained from subjects with normal, healthy tendons.

[0098] In some aspects, the methods disclosed herein increase proliferation of tendon stem cells by at least 5%, for example, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or more. In some aspects, the methods disclosed herein increase proliferation of tendon stem cells by 5% to 500%, for example, 5% to 400%, 5% to 300%, 5% to 200%, 5% to 100%, 5% to 75%, 5% to 50%, 10% to 400%, 10% to 300%, 10% to 200%, 10% to 100%, 10% to 75%, 10% to 50%, 25% to 400%, 25% to 300%, 25% to 200%, 25% to 100%, 25% to 75%, 25% to 50%, 50% to 400%, 50% to 300%, 50% to 200%, 50% to 100%, 50% to 75%, 100% to 400%, 100% to 300%, 100% to 200%, or 200% to 500%.

[0099] In some implementations, scar tissue of the soft tissue musculoskeletal injury is removed prior to treatment with metformin. In some aspects, scar tissue (e.g., non-tendinous tissue, such as calcified tissue, or lipid or proteoglycan accumulations), is removed prior to administering metformin to a subject having tendinopathy. In some implementations, a soft tissue musculoskeletal injury is assessed for the presence of the administration of metformin.

[0100] The methods disclosed herein can further include administering to the subject an anti-inflammatory and / or analgesic, such as acetaminophen, aspirin, COX inhibitors, corticosteroids (e.g. cortisone), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen or naproxen), diclofenac, or opioids (e.g., codeine, hydrocodone, oxycodone). In some aspects, the methods further include administering hyaluronic acid. In some aspects, metformin is conjugated to hyaluronic acid (e.g., metformin hyaluronate). While any known method of conjugating hyaluronic acid and metformin can be used, Example 8 provides an exemplary process for making a metformin-hyaluronic acid conjugate (e.g., metformin hyaluronate). The methods disclosed herein can further include administering a cell therapy (e.g., administering stem cells, such as tendon stem cells), platelet rich plasma, or glucose.

[0101] The methods disclosed herein can further include one or more therapy, such as moderate exercise (e.g., moderate treadmill running (MTR)), physiotherapy (e.g., rest, ice, compression, elevation (RICE)), cryotherapy, pressure therapy, extracorporeal shockwave therapy (ESWT), or eccentric loading (ECL). In some aspects, cryotherapy and / or pressure therapy is used to reduce scar tissue thickness or prevent hypertrophic scarring. In some aspects, a corticosteroid cream is used to reduce inflammation, flatten, or soften scar tissue.

[0102] IV. Compositions Including Metformin

[0103] Also disclosed are compositions including metformin. In some aspects, the compositions are pharmaceutical compositions. The compositions (including pharmaceutical compositions) described herein, are of use in the methods disclosed herein.

[0104] The pharmaceutical compositions disclosed herein include metformin and a pharmaceutically acceptable carrier. Many suitable pharmaceutically acceptable carriers have been described, for example, in Remington’s Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier (2020), which describes compositions and formulations suitable for delivery of pharmaceutical compositions in humans. In a specific example, the pharmaceutically acceptable carrier is a carrier for a topical composition, such as a liquid, gel, foam, cream, ointment or lotion. Exemplary suitable carriers for topical application include, but are not limited to, glycerin, CM alcohols, fatty alcohols, fatty ethers, fatty esters, polyols, glycols, vegetable oils, mineral oils, liposomes, laminar lipid materials, silicone oils, water, and combinations thereof. In some aspects, the pharmaceutically acceptable carrier is a carrier for administration of metformin by an injection, such as isotonic salt solutions or oil in water emulsions.

[0105] Pharmaceutical compositions including metformin can be in any suitable form, such as a solid, liquid (including semi-solids), or gas (aerosol). Pharmaceutical compositions including metformin can be formulated such that metformin becomes bioavailable upon administration of the pharmaceutical composition to a subject. Pharmaceutical compositions can take the form of one or more dosage units. For a syringe containing a single unit dose of metformin is also provided.

[0106] In some aspects, a pharmaceutical composition including metformin is in a liquid form, for example, a solution, syrup, or suspension. Such liquid preparations can be prepared by known methods with pharmaceutically acceptable additives, for example, suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous carriers (e.g., almond oil, oily esters, or fractionated vegetable oils), and / or preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid).

[0107] In some aspects, a pharmaceutical composition including metformin is formulated for administration by an injection, such as a local injection at a site of a soft tissue musculoskeletal injury. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, and optionally can include a preservative. A pharmaceutical composition for injection can take forms such as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient (e.g., metformin) can be in powder form for constitution with a suitable carrier, e.g., sterile saline, before use.

[0108] Pharmaceutical compositions can be formulated for controlled release (e.g., delayed release) of an active compound (e.g., metformin), or formulated to improve the half-life of metformin. In some aspects, metformin conjugated to hyaluronic acid (e.g., metformin hyaluronate) provides controlled release of metformin (e.g., into target tissues and / or joints). In some aspects, an injection formulation of metformin further includes glucose and / or platelet rich plasma.

[0109] In some aspects, a pharmaceutical composition including metformin is formulated for transdermal administration. Transdermal administration includes drug delivery through skin penetration. This type of administration can occur in a passive or active manner. Passive administration is characterized by gradient diffusion of a therapeutic (e.g., metformin), while active administration includes artificially inducing penetration of skin, for example, by using an electric field, electroporation, microporation, microneedles, laser ablation, heat, or ultrasound. In some aspects, a pharmaceutical composition including metformin that is formulated for transdermal administration includes one or more excipients, such as petroleum jelly (e.g., Vaseline®), petroleum wax (e.g., paraffin), glycerol, hydrophobic surfactants (e.g., polysorbate) and / or lipid-phase components (e.g., soya phosphatidylcholine). In some aspects, metformin formulated for transdermal administration has superior bioavailability than metformin administered orally or systemically.

[0110] Based on data disclosed herein, topical metformin appears to have higher bioavailability than oral metformin, which allows lower doses (e.g., 10% or less, such as 6% or less, 3% or less, 2% or less, or 1.5% or less) to reach the same therapeutic effect as oral metformin, which minimizes safety and toxicity concerns. Similarly, lower doses can be used for local injection (e.g., 3% or less, such as 2% or less, or 1.5% or less) since it delivers metformin directly to the affected area (e.g., an injured tendon or scar tissue).

[0111] In some aspects, a pharmaceutical composition including metformin is formulated as a topical composition (such as a topical pharmaceutical composition). The topical composition can be any form ointment, paste, plaster, paint, bioadhesive, bandage, spray, suspension, or the like. In some aspects, a topical composition including metformin is a lotion. In some aspects, the topical composition includes an occlusive overlayer to minimize moisture evaporating from the body surface. Actual methods for preparing topical compositions have also been described in detail in, for example, in Remington 's Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020).

[0112] Suitable topical compositions include creams. Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are often water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the “internal” phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.

[0113] Suitable topical compositions include lotions. Lotions are preparations to be applied to the skin surface, generally without friction, and are typically liquid or semiliquid preparations in which particles, including the active agent (e.g., metformin), are present in a water or alcohol base. Lotions are usually suspensions of solids, and preferably, comprise a liquid oily emulsion of the oil-in-water type. Lotions can be used for treating large body areas, because of the ease of applying a more fluid composition. Any insoluble matter in lotion is typically finely divided. Lotions typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethyl-cellulose, or the like.

[0114] Suitable topical compositions include solutions. Solutions are generally homogeneous mixtures prepared by dissolving one or more chemical substances (solutes, e.g., metformin) in a liquid such that the molecules of the dissolved substance arc dispersed among those of the solvent. The solution may contain other pharmaceutically acceptable chemicals to buffer, stabilize and / or preserve the solute. Common examples of solvents used in preparing topical solutions are ethanol, water, propylene glycol or any other acceptable vehicles. These can be applied in any manner, such as spraying them on the skin, painting them on the skin, or wetting a bandage with the solution.

[0115] Suitable topical compositions include gels. Gels are semisolid, suspension-type systems. Singlephase gels contain organic macromolecules distributed substantially uniformly throughout a carrier liquid, which is typically aqueous, but can also contain an alcohol and / or oil. Exemplary suitable gelling agents include, but are not limited to, crosslinked acrylic acid polymers such as the carbomer family of polymers, e.g., carboxypolyalkylenes that are commercially available as CARBOPOL®. Also of use are hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. Dispersing agents, such as alcohol or glycerin, combinations thereof.

[0116] Suitable topical compositions include ointments. Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. Ointment bases are generally inert, stable, non-irritating, and non-sensitizing. Four classes of ointment bases include: oleaginous bases, emulsifiable bases, emulsion bases, and water-soluble bases (see, e.g., Remington 's Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020)). Oleaginous ointment bases include, but are not limited to, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, but are not limited to, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W / O) emulsions or oil-in- water (O / W) emulsions, and include, but are not limited to, acetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight.

[0117] Suitable topical compositions include pastes. Pastes are generally semisolid dosage forms in which the active agent (e.g., metformin) is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from single -phase aqueous gels. The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or the like. The pastes made from single -phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base.

[0118] The topical compositions disclosed herein can include a pharmaceutically acceptable viscosity enhancer, permeation enhancer, and / or film former. A viscosity enhancer increases the viscosity of the formulation to inhibit spread beyond the site of application. Balsam Fir (Oregon) is an example of a pharmaceutically acceptable viscosity enhancer. Permeation enhancers increase tissue permeation of an active ingredient (e.g., metformin) in a subject. Exemplary permeation enhancers include, but arc not limited to, fatty acids and fatty alcohols, alcohols and glycols, terpenes, sulphoxides, laurocapram, pyrrolidones, ionic surfactants, and urea. Specific, non-limiting examples of permeation enhancers include, but are not limited to, sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS), dimethyl sulfoxide (DMSO), polysorbate 80, propylene glycol, N-methylpyrrolidone (NMP), oleic acid, caprylic acid, myristic acid, and isopropyl myristate (IPM). A film former, when it dries, forms a protective film over the site of application. The film inhibits removal of the active ingredient (e.g., metformin) and keeps it in contact with the site being treated. An example of a suitable film former is flexible collodion. Collodions are ethyl ether / ethanol solutions containing pyroxylin (a nitrocellulose) that evaporate to leave a film of pyroxylin. A film former may act additionally as a carrier. Solutions that dry to form a film are sometimes referred to as paints.

[0119] A cream, lotion, gel, ointment, paste or the like may be spread on a target surface of a subject, for example, on skin adjacent to a soft tissue musculoskeletal injury or a wound (such as a lesion, abrasion, laceration, burn, or wound from a surgical procedure). A solution may be applied in the same way, but more also be included as a part of a dressing or a bandage covering a target area.

[0120] The topical compositions disclosed herein can include metformin about 1% to about 25% weight by weight (w / w) metformin, for example, 1% to 20%, 1% to 18%, 1% to 15%, 1% to 12%, 1% to 10%, 1% to 8%, 1% to 6%, 1% to 5%, 2% to 25%, 2% to 20%, 2% to 18%, 2% to 15%, 2% to 12%, 2% to 10%, 2% to 8%, 2% to 7%, 2% to 6%, 2% to 5%, 3% to 25%, 3% to 20%, 3% to 18%, 3% to 15%, 3% to 12%, 3% to 10%, 3% to 7%, 3% to 8%, 3% to 6%, 3% to 5%, 5% to 25%, 5% to 20%, 5% to 18%, 5% to 15%, 5% to 12%, 5% to 10%, 5% to 7%, 5% to 8%, 10% to 25%, 10% to 20%, 10% to 15%, 15% to 25%, 15% to 20%, or 20% to 25% weight by weight (w / w) metformin. In some aspects, the topical compositions include about 1% to about 10% weight by weight (w / w) metformin. In some aspects, the topical compositions include about 2% to about 7% weight by weight (w / w) metformin. In further aspects, the topical compositions include about 3% to about 6% weight by weight (w / w) metformin. In some aspects, the topical compositions include about 5% to about 7% weight by weight (w / w) metformin.

[0121] In some aspects, the topical compositions disclosed herein can include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 18%, about 20%, about 25% or more weight by weight (w / w) metformin. In some aspects, the topical compositions include about 3% weight by weight (w / w) metformin. In some aspects, the topical compositions include about 6% weight by weight (w / w) metformin.

[0122] The disclosed compositions including metformin can further include, for example, fragrances (artificial and / or natural), dyes or coloring agents (e.g., Blue 1, Blue 1 Lake, Red 40, titanium dioxide, D&C blue no. 4, D&C green no. 5, D&C orange no. 4, D&C red no. 17, D&C red no. 33, D&C violet no. 2, D&C yellow no. 10, and D&C yellow no. 11, and iron oxides), adsorbents, lubricants, solvents, moisturizers (including, e.g., emollients, film formers, and agents that affect the natural moisturizing mechanisms of the skin), water-repellants, UV absorbers (physical and chemical absorbers such as avobenzone, titanium dioxide, zinc oxide, etc.), essential oils, trace metals (e.g., zinc, calcium and selenium), anti-irritants (e.g., steroids and non-steroidal anti-inflammatories), botanical extracts (e.g., aloe vera, chamomile, cucumber extract, ginkgo biloba, ginseng, and rosemary), anti-microbial agents, anti-oxidants (e.g., tocopherol), chelating agents (e.g., sodium phytate and trisodium ethylenediamine disuccinate), preservatives (e.g., phenyoxyethanol, caprylyl glycol and chlorphenesin), pH adjusters (e.g., sodium hydroxide and citric acid), absorbents (e.g., aluminum starch octenylsuccinate, kaolin, corn starch, oat starch, cyclodextrin, and zeolite), humectants (e.g., glycerol, sorbitol, urea, and mannitol), exfoliants, waterproofing agents (e.g., magnesium / aluminum hydroxide stearate), and / or skin conditioning agents (e.g., aloe extracts, allantoin, bisabolol, ceramides, dimethicone, hyaluronic acid, and dipotassium glycyrrhizate). In some aspects, the disclosed compositions including metformin further include glucose and / or platelet rich plasma. The disclosed topical compositions including metformin are of use in the methods disclosed herein. [Clause 1] A method of inhibiting or treating a soft tissue musculoskeletal injury, comprising: selecting a subject at risk of or having a soft tissue musculoskeletal injury, and administering metformin to the subject. [Clause 2] The method of clause 1, wherein the soft tissue musculoskeletal injury is an acute or chronic injury.

[0123] [Clause 3] The method of clause 1 or clause 2, wherein administering metformin comprises: i) administering metformin topically to skin adjacent the soft tissue musculoskeletal injury, or ii) injecting metformin at the site of the soft tissue musculoskeletal injury.

[0124] [Clause 4] The method of any one of the prior clauses, comprising administering metformin topically to skin adjacent the soft issue musculoskeletal injury, wherein the soft issue musculoskeletal injury is less than 3 centimeters below a skin surface of the subject.

[0125] [Clause 5] The method of any one of the prior clauses, comprising administering injecting metformin at the site of the soft tissue musculoskeletal injury, wherein the soft issue musculoskeletal injury is greater than 3 centimeters below a skin surface of the subject.

[0126] [Clause 6] The method of any one of the prior clauses, wherein the soft tissue musculoskeletal injury is a tendon, ligament, or muscle injury.

[0127] [Clause 7] The method of any one of the prior clauses, wherein the tendon injury is an Achilles tendon injury.

[0128] [Clause 8] The method of any one of the prior clauses, wherein the subject selected for treatment is at risk of or has tendinopathy.

[0129] [Clause 9] The method of any one of the prior clauses, wherein scar tissue is removed prior to administering metformin.

[0130] [Clause 10] The method of any one of the prior clauses, wherein administering metformin comprises administering a topical pharmaceutical composition comprising metformin.

[0131] [Clause 11] A method of inhibiting or treating scar tissue, comprising: selecting a subject at risk of developing or having scar tissue, and administering metformin to the subject.

[0132] [Clause 12] The method of clause 11, wherein the scar tissue is dermal scar tissue.

[0133] [Clause 13] The method of clause 11 or 12, wherein the scar tissue is epidermal scar tissue.

[0134] [Clause 14] The method of any one of clauses 11 to 13, wherein the scar tissue is from a lesion, abrasion, laceration, burn, or surgical procedure.

[0135] [Clause 15] The method of any one of clauses 11 to 14, wherein administering metformin comprises administering a topical pharmaceutical composition comprising metformin to the scar tissue.

[0136] [Clause 16] The method of any one of the prior clauses, wherein the topical pharmaceutical composition comprises 1% to 15% weight by weight (w / w) metformin.

[0137] [Clause 17] The method of any one of the prior clauses, wherein the topical pharmaceutical composition comprises 3% to 6% w / w metformin. comprises about 3% w / w metformin.

[0138] [Clause 19] The method of any one of the prior clauses, wherein the topical pharmaceutical composition comprises about 6% w / w metformin.

[0139] [Clause 20] The method of any one of the prior clauses, wherein the topical pharmaceutical composition is formulated for transdermal delivery of metformin.

[0140] [Clause 21] The method of any one of the prior clauses, wherein the topical pharmaceutical composition is a lotion, cream, ointment, gel, or paste.

[0141] [Clause 22] The method of any one of the prior clauses, wherein administering metformin comprises local injection of a pharmaceutical composition comprising metformin.

[0142] [Clause 23] The method of any one of the prior clauses, wherein the pharmaceutical composition comprises 0.1% to 5% weight by weight (w / w) metformin.

[0143] [Clause 24] The method of any one of the prior clauses, wherein the pharmaceutical composition comprises 0.5% to 2% w / w metformin.

[0144] [Clause 25] The method of any one of the prior clauses, wherein the pharmaceutical composition further comprises hyaluronic acid, glucose, or platelet rich plasma.

[0145] [Clause 26] The method of any one of the prior clauses, wherein the subject is a human.

[0146] [Clause 27] The method of any one of the prior clauses, wherein the subject is at risk of a soft tissue musculoskeletal injury.

[0147] [Clause 28] The method of any one of the prior clauses, wherein the subject at risk of a soft tissue musculoskeletal injury is a human subject over the age of 65.

[0148] [Clause 29] The method of any one of the prior clauses, wherein the metformin is conjugated to hyaluronic acid.

[0149] EXAMPLES

[0150] The following examples are provided to illustrate particular features or aspects of the disclosure, however the scope of the claims are not limited to those features exemplified.

[0151] Example 1 Materials and Methods

[0152] Metformin Lotion Preparation

[0153] For animal experiments concerning efficacy of topical metformin on tendinopathy (such as FIGS. 6- 11), metformin hydrochloride (3 g or 6 g) was obtained from Sigma (pharmaceutical grade, Cat. #1396342, USP Reference standard) and dissolved in deionized distilled (DD) water (50 ml or 47 ml) in a 100 ml beaker. Subsequently, 5 g of glycerol and 2 g of Span™ 83 sorbitan sesquioleate were added to the metformin-water solution and thoroughly mixed to create solution A. In a separate 200 ml beaker, Vaseline® petroleum jelly (20 g) was heated to 80 °C until it formed a clear solution. Next, 20 g of liquid solution A was carefully poured into solution B (Vaseline-paraffin mixture) while stirring continuously, and the mixture was allowed to cool to room temperature. This process yielded a metformin lotion, with a concentration of either 3% or 6% of metformin. For the control lotion, the same formulation was followed without the addition of metformin (0% concentration).

[0154] Treadmill running protocol for inducing the development of tendinopathy in mice

[0155] C57BL / 6J mice (female, 3 months old) were used for treadmill running experiments. The ITR protocol is as follows: mice ran at 15 meters / min for 3 hours / day, 5 days / week for up to 12 weeks. All mice were sacrificed after 4 or 12 weeks of treatments. After the end of the experiments, the mouse tendons were harvested and processed for histological and ELISA analyses.

[0156] Treadmill running protocol for investigating the effects of Met-lotion on tendon

[0157] C57BL / 6J mice (female, 3 months old) were divided into three groups. The mice in group- 1 were applied on treadmill machine to run the ITR protocol at 14 meters / min for 3 hours / day, 5 days / week for 4 weeks (ITR group). The mice in group-2 ran the same protocol as the ITR group, but with 50 mg of 3% of metformin lotion applied on the skin surface of each hind leg. A total of 100 mg of 3% Met-lotion was given into each mouse (3% Met-lotion group). The mice in group-3 ran the same protocol as the ITR group, but with 50 mg of 6% of metformin lotion smeared on the skin surface of each hind leg, total 100 mg of 6% of Met-lotion was given into each mouse (6% Met-lotion group). The metformin lotion was applied daily during the course of the experiment. All mice were sacrificed after 4 weeks of treatments. The hind legs with Achilles tendon and enthesis tissues were harvested from all mice. The legs were fixed with 4% paraformaldehyde overnight at room temperature and then decalcified with Cal-Ex II™ Fixativc / Dccalcificr (cat. CS511-1D, Fisher Scientific) for one week. The tissue samples were washed 3 times with phosphate - buffered saline (PBS) and soaked in 30% sucrose in PBS at 4°C overnight. Then the tissue samples were embedded with paraffin, and cut into 5 mm tissue section. The mouse tendons were processed for histological (H&E), histochemical (Safranin O / Fast Green), immunohistochemical (IHC), and ELISA analyses.

[0158] Example 2 Topical Metformin Penetrates Skin and Reaches Underlying Musculoskeletal Tissues

[0159] The penetrative and functional abilities of a topical metformin composition (Met lotion as described in Example 1) were assessed. 3% metformin lotion (1.5 mg metformin per 50 mg lotion) was applied daily to the skin right above (adjacent) the Achilles tendons of mice. The mice were sacrificed 24, 48, 72, and 96 hours post-lotion application. Tendons were collected and the concentration of metformin in the tendon tissues was determined by HPLC and UV-VIS spectroscopy and compared to an empty vehicle control. manner after application (FIG. 1A).

[0160] Topical application of metformin was also compared to other delivery routes, such as IP or local injection. In brief, the Achilles tendons of mice (female C57BL / 6J, 8 weeks old, n = 15) were wounded using a 0.5 mm biopsy punch. Immediately after suturing the skin right above the wounded tendon, equal groups of mice were treated as follows: Met-IP (IP injection of Met, 100 pl of 32 mg / ml, which is equivalent to 160 mg / kg body weight, with a mouse body weight 20 g); Met-LI (local injection of metformin into the tendon at 25 pl of 32 mg / ml), and Met-lotion (smearing 50 mg of 3% metformin lotion on the mouse skin right above the wounded Achilles tendon of each leg). After 24 hours, mice were sacrificed and samples were collected. Surprisingly, it was found that topical metformin application achieved a similar concentration of metformin in the Achilles tendon as IP or local injection (FIG. IB).

[0161] Example 3

[0162] Metformin Lotion Reduces HMGB1 and Tendon Inflammation

[0163] Achilles tendons of C57BL / 6J mice (female, 8 weeks old) were wounded using a 0.5 mm biopsy punch. After suturing the skin right above the wounded tendon, 20 mice were divided into the following four equal groups. Group 1 : Wound only; Group 2: Met-IP (IP injection of Met, 100 pl of 32 mg / ml, which is equivalent to 160 mg / kg body weight, with a mouse body weight 20 g); Group 3: Met-LI (local injection of metformin into the tendon at 25 pl of 32 mg / ml), and Group 4: Met-lotion (smearing 50 mg of 3% metformin lotion on the mouse skin right above the wounded Achilles tendon of each leg). After 24 hrs, mice were sacrificed, and the amounts of HMGB1 and PGE2within Achilles tendon tissue was measured. Met-lotion application significantly reduced HMGB1 and PGE2and the results were similar to that obtained by a metformin IP or local injection (FIG. 2A).

[0164] Finally, using immunostaining, HMGB1 was found to be released to the extracellular matrix in a wounded tendon, whereas IP injection of metformin or topical application of metformin lotion blocked HMGB1 release and HMGB1 remained in the nucleus (FIG. 2B).

[0165] These results show that topical application of metformin is equally effective as IP or local injection to deliver metformin to block tendon inflammation and degeneration.

[0166] Example 4 Pharmacokinetics and Cytotoxicity of Metformin

[0167] Metformin at various doses was administered to the Achilles tendon of mice by local injection. Each Achilles tendon from mice (3 months old, 16 / group) was injected with three concentrations of metformin: Group 1, 40 pg metformin in a 5 pl solution (low dose); Group 2, 400 pg of metformin in a 5 pl solution (medium dose); and Group 3, 4,000 pg of metformin in a 10 pl solution (high dose). Two mice from each group were sacrificed at eight time points (0, 1, 2, 3, 4, 5, 6, 18 hours) post-injection. Tendon tissue samples were obtained, weighed, and cut into small pieces. Two Achilles tendons from the same mixture was centrifuged at 15,000g for 15 min. The supernatant was used to measure metformin concentration.

[0168] Pharmacokinetic findings showed that approximately 100% of metformin was within Achilles tendon tissues immediately after a low dose of metformin solution was locally injected (FIG. 3A). However, about 90% and 75% of metformin was found in mouse Achilles tendon tissues immediately after medium and high doses of metformin were locally injected into Achilles tendon (FIGS. 3B-3C). For all three doses, the amount of metformin within Achilles tendon tissue decreased with time. By 18 hours post-injection, metformin was at a minimal level. The data shows that metformin is excreted without accumulation in the tendon over time, thus long-term use of metformin is safe for tendons.

[0169] To assess the possible toxic effects of metformin on tendon cells, the following in vitro experiment was performed. Tendon stem cells (TSCs), which are responsible for tendon repair and regeneration, were isolated from tendons of adult mice and cultured with increasing concentrations of metformin (0 - 5 mg / ml) in DMEM growth medium. Metformin effect on proliferation was assessed by calculating the population doubling time (PDT) of each group. The results show that even exceedingly high concentrations of metformin (e.g., 5 mg / ml) did not negatively affect tendon cell morphology or proliferation. Instead, metformin actually stimulated tendon cell proliferation at high doses of 1 mg / ml and 5 mg / ml (see, FIGS. 4 and 5).

[0170] The results suggest that when present in the Achilles tendon, metformin does not exert negative effects on cells; but rather, promotes TSC proliferation in vivo. Thus, topical metformin may promote repair and regeneration of injured tendons in patients with tendinopathy.

[0171] Example 5

[0172] Topical Metformin Inhibits ITR-Induced Tendinopathy and Enthesopathy

[0173] The effect of metformin lotion (Met lotion) on mouse enthesis and Achilles tendon after intensive treadmill running was studied. C57BL / 6J mice (female, 3 months old) were divided into three groups. The mice in group- 1 ran on treadmill according to intensive treadmill running (ITR) protocol at 14 meters / min for 3 hours / day, 5 days / week for 4 weeks (ITR group). The mice in group-2 ran the same protocol as the ITR group, but with 50 mg of 3% metformin lotion smeared on the skin surface of each hind leg, total 100 mg of 3% metformin lotion was applied to each mouse (3% Met-lotion group) daily before the run. The mice in group-3 ran the same protocol as the ITR group, but with 50 mg of 6% metformin lotion smeared on the skin surface of each hind leg, total 100 mg of 6% metformin lotion was applied to each mouse (6% Met group). All mice were sacrificed after 4 weeks of treatments and the hind legs with Achilles tendon and enthesis tissues were harvested from all mice.

[0174] The legs were fixed with 4% paraformaldehyde overnight at room temperature and then decalcified with Cal-Ex II Fixative / Decalcifier (cat. CS511-1D, Fisher Scientific) for one week. The tissue samples were washed 3 times with phosphate-buffered saline (PBS) and soaked in 30% sucrose in PBS at 4°C slides numbered 10, 15, and 20 in each leg were stained with H & E followed the standard protocol. The staining results were determined under a microscope (FIG. 6A). The results demonstrated that ITR for 4 weeks could induce degenerative changes typical of tendinopathy (thinner collagen fibers, smaller enthesis size, many round shaped cells). Application of 3% and 6% metformin lotion daily before the run could inhibit these degenerative changes as evidenced by thicker collagen fibers, larger fibrocartilage tissue, and a smaller number of round shaped cells.

[0175] Histochemical analysis was also performed on the mouse tendon tissues (FIG. 6B). Histological results also show that degenerative changes (thinner collagen fibers, smaller enthesis size, round shaped cells) induced by 4 weeks ITR were inhibited by daily application of 3% and 6% metformin lotion. Eotion applications resulted in thicker collagen fibers, larger fibrocartilage tissue, and fewer round shaped cells.

[0176] In an experimental repeat, 4 weeks of ITR induced degenerative changes to the Achilles tendon (such as round shaped cells in the tendon and paratenon, tendon tears, and enthesis damage), however, the Achilles tendons of mice treated with topical metformin appeared normal (e.g., tendon cells elongated in shape with fewer round shaped cells, collagen fibers in a normal-like organization, normal enthesis structure, etc.,’ see, FIG. 10). It was further observed that a large number of cells infiltrated into the paratenon and nearby tendon proper in the control (no administration of metformin). The identity of these cells have not yet been determined, but could be inflammatory cells or differentiated tendon stem cells. Metformin administration reduced the number of these infiltrated cells (see, e.g., FIG. 10 top row).

[0177] Similar results were obtained when the experiment was repeated with 12 weeks ITR (FIG. 8). Specifically, in the ITR control group, tendon structure is disorganized with many cells having a round morphology. The Achilles enthesis is also thin and damaged. In contrast, in both the 3% and 6% lotion groups, the Achilles tendon appears normal with highly aligned matrix and elongated cells. FIG. 11 shows additional data from the 12 week ITR experiment. FIG. 11 shows that 12 weeks of ITR induced severe degenerative changes (such as tendon rupture, tendon tears, broken enthesis, and smaller enthesis). Topical application of metformin inhibited degeneration, with treated tendons appearing to be normal young adult tendons (e.g., tendon cells elongated in shape with fewer round shaped cells, collagen fibers in a normal-like organization, larger enthesis, etc. see, FIG. 11). As was observed with 4 weeks ITR, a large number of cells infiltrated into the paratenon and nearby tendon proper in the control (no administration of metformin, FIG. 11). Metformin administration reduced the number of these infiltrated cells.

[0178] The effect of topical metformin on HMGB 1 release was also investigated. It was found that topical metformin (3% and 6%) blocks HMGB1 release (induced by 4 weeks of ITR) from cell nuclei to the extracellular matrix of the mouse Achilles tendon (see, FIGS. 9A and 9B). Without being bound by any particular theory, it is hypothesized that by binding to HMGB1 and preventing its release, topical metformin lotion reduces degenerative changes in the mouse Achilles tendon induced by 4 and 12 weeks of ITR.

[0179] The results collectively show that 3% and 6% metformin lotion inhibits tendinopathy development in a mouse ITR model, which mimics mechanical overuse tendon injury in humans. Interestingly, it was metformin lotion be used to prevent or inhibit a tendon overuse injury (such as tendinopathy), but can also be used as a prophylaxis or treatment option for enthesopathy, namely, tendon-to-bone j unctional injury.

[0180] Example 6 Topical Metformin Improves Recovery After ITR

[0181] Female C57BL / 6J mice, aged 3 months, underwent intensive treadmill running (ITR) at 14 meters / minute for 3 hours per day, 5 days a week, for a total of 4 weeks to induce tendinopathy by mechanical overuse. Subsequently, the mice were divided into three groups, with 5 mice in each group: Group-1: No further treatment for three / four weeks (ITR-4w + Cont- 3 / 4w).

[0182] Group-2: Treated with 50 mg of 3% Met lotion, applied to the skin surface of each hind leg, totaling 100 mg of 3% Met-lotion daily for three / four weeks (ITR-4w + 3% Met-lotion-3 / 4w). Group-3: Treated with 50 mg of 6% Met lotion, applied to the skin surface of each hind leg, totaling 100 mg of 6% Met-lotion daily for three / four weeks (ITR-4w + 6% Met-lotion-3 / 4w). All mice were sacrificed after 7 or 8 weeks of treatments (either ITR-4w+Cont-3 / 4w or ITR-4w + Met-lotion-3 / 4w). The hind legs, including Achilles tendon and enthesis tissues, were harvested, fixed overnight at room temperature with 4% paraformaldehyde, and then decalcified using Cal-Ex II Fixative / Decalcifier for one week. Tissue samples were washed three times with phosphate-buffered saline (PBS) and subsequently soaked in 30% sucrose in PBS at 4°C overnight. The tissue samples were then embedded in paraffin and sliced into 5 pm tissue sections. Three slides from each leg, numbered 10, 15, and 20, were subjected to H&E staining and Safranin O and Fast Green staining following the standard protocols. Additionally, the tissue sections were stained for p-AMPK (phospho-AMPK) to detect if Met lotion treatment stimulated the expression of p-AMPK.

[0183] Hematoxylin and eosin staining (H&E; FIG. 7A) showed that ITR-4w + Cont-4w mouse tissue sections displayed evidence of tendon degenerative changes induced by 4 weeks of ITR, which persisted even after 4 weeks of cage activities, characterized by thinner collagen fibers, reduced enthesis size, and an abundance of round-shaped cells. In contrast, ITR-4w + 3% Met-lotion-4w treated mouse tissue sections indicated that 3% Met-lotion treatment effectively mitigated tendon degenerative changes induced by 4 weeks of ITR, as evidenced by thicker collagen fibers in the enthesis areas with larger fibrocartilage tissue size and a reduced number of round-shaped cells. Similarly, ITR-4w + 6% Met-lotion-4w treated mouse tissue sections demonstrated that 6% Met-lotion treatment also successfully inhibited tendon degenerative changes induced by ITR, as evidenced by thicker collagen fibers in the enthesis areas with larger fibrocartilage tissue size, and fewer round-shaped cells.

[0184] Safranin O and Fast Green staining (FIG. 7B) also revealed signs of tendon degeneration (thinner collagen fibers, reduced enthesis size, and an abundance of round-shaped cells) in ITR-4w + Cont-4w mouse tissue sections. However, treatment with either the 3% or 6% met lotion (ITR-4w + 3% Met lotion-4w or ITR-4w + 6% Met lotion-4w) effectively mitigated tendon degenerative changes, as evidenced by the number of round-shaped cells.

[0185] Immunostaining for p-AMPK was also performed on mouse tendon tissue sections (FIG. 7C). Images of mouse tissue sections from ITR-4w + Cont-3w displayed minimal staining of p-AMPK in both the tendon and Achilles enthesis. In contrast, images of mouse tissue sections from ITR-4w + 3% Met lotion-3w treatment revealed significant p-AMPK staining in both the Achilles tendon and its enthesis. Images of mouse tissue sections from ITR-4w + 6% Met lotion-3w treatment showed more pronounced p- AMPK staining in both the Achilles tendon and its enthesis.

[0186] These results show that topical metformin treatment is effective in the recovery of Achilles enthesis injuries resulting from mechanical overuse. Without being bound by any particular theory, the mechanisms of action may include the inhibition of HMGB 1 and activation of AMPK.

[0187] Example 7 Topical Metformin Inhibits Scar-Like Tissue Formation

[0188] A total of 30 mice were randomly divided equally into three groups: Group 1 : ran on treadmill at 15 meters / min for 3 h / day, 5 days a week for 4 weeks (ITR); Group 2: smeared 3% Met-lotion on the skin surface of the hind legs before ITR daily for 4 weeks (ITR+3% Met-lotion); smeared 6% Met-lotion on the skin surface of the hind legs before ITR daily for 4 weeks (ITR+6% Met-lotion). All mice were sacrificed after 4 weeks ITR, and Achilles tendon tissues were also harvested for histological analysis by Trichrome Masson staining, Piero Sirius Red staining, and immunostaining on collagen II and SOX-9 expression.

[0189] Trichrome Masson staining showed that ITR induced tendon degenerative changes as evidenced by loose collagen fibers stained with blue in ITR tendons (FIG. 13). Topical application of metformin lotion treatment inhibited these ITR-induccd degenerative changes as evidenced by dense collagen fibers being positively stained with red (FIG. 13). Piero Sirius Red staining showed that the collagen fibers in the tendons of ITR-treated mice under bright light microscopy were red, loose, and disorganized (FIG. 14A), and some damaged tissues were found in the enthesis area of the tendon-bone junction areas of the mice treated with ITR. However, the tendons in Met lotion treated groups were dense with very good organization (FIG. 14A). Under polarized light microscopy, those loose collagen fibers in the tendons of ITR mice were positively stained with collagen III (green birefringence in FIG. 14B). The tendons in metformin lotion treated groups showed yellow or orange birefringence, indicating these collagen fibers were collagen type I. Very low levels of collagen III were positively stained in 6% Met lotion treated tendons. The inhibition effect of Met lotion on collagen III formation was in a concentration-dependent manner as evidenced by the expression of collagen III in the tendon matrix of all samples is ITR > ITR+3% Met-lotion > ITR+6% Met-lotion. Immunostaining results indicated that ITR increased collagen II and SOX-9 (FIG. 15) expression in mouse Achilles tendons, however, metformin lotion treatment decreased collagen II and SOX-9 expression induced by ITR, and both levels of collagen II and SOX-9 in 6% Met- lotion treated tendons were lower than 3% Met-lotion. tendinopathy in mouse tendons characterized by the presence of chondrocyte-like cells (increased levels of collagen II and SOX-9) and scar-like tissues (disorganized tendon structure and increased collagen III levels). However, metformin lotion treatment at 3% and 6% inhibited these tendinopathic changes.

[0190] Example 8 Metformin Application Inhibits Age-Induced Tendinopathy

[0191] Three groups of mice (with 6 mice each) were used: 1) 4 months (4M) old young mice for control; 2) 19M old mice that received IP injection of saline; and 3) 19M old mice that received IP injection of metformin (50 mg / kg body weight / day, a much lower amount than that used in previous experiments above). All mice were sacrificed after 8 weeks of treatments. The Achilles tendons were harvested from all mice and used for histological (FIG. 12A) and Western blot analyses (FIG. 12B). It was found that the aging mouse group contained round chondrocyte-like cells, instead of elongated cells in young control tendon. The tendon matrix was also disorganized, in contrast to the highly organized tendon structure in young control group. However, metformin injection increased the number of elongated-shape cells in aging mouse tendons (19M mice) and improved overall tendon organization. Western blot analysis revealed that the levels of total HMGB1 (both isoforms of bands) were markedly higher in aging mouse tendons compared to young counterparts. Metformin injection decreased total HMGB1 levels in aging mouse tendons.

[0192] These results suggest that metformin, by blocking HMGB1 release from the nucleus and / or its activity in the tendon matrix, may be an effective therapy for both prevention and treatment of tendinopathy, particularly in aging individuals, such as individuals 65 or older.

[0193] Example 9 Topical Metformin Permeates Skin

[0194] To evaluate skin permeation, 6% metformin lotion was applied to a pig ear skin sample (0.8 mm thick) on a Franz cell (see, FIG. 18). A 200 pl sample was withdrawn from the water reservoir at multiple time points over a 24 hour period. 200pls ddH2O was immediately replaced following each sampling. HPLC analysis was performed on the samples to detect metformin that had permeated through the skin sample and into the water reservoir (FIGS. 19A-19B). The results show that the 6% metformin lotion formulation effectively diffuses through skin, and therefore is useful for therapeutic delivery of metformin through skin (for example, to treat tendinopathy). Materials and Methods for Example 11

[0195] Materials

[0196] Metformin hydrochloride (Cat. #PHR1084), glycerol (Cat. #15524), Vaseline® petroleum jelly (Cat. #16415), paraffin oil (Cat. #18512), and sorbitan sesquioleate (Span™ 83; Cat. #S3386) were obtained from Millipore Sigma® (Burlington, MA).

[0197] Metformin Lotion Preparation

[0198] Metformin hydrochloride (6 g) was dissolved in 47 ml of deionized distilled (DD) water in a 100 ml beaker. Subsequently, 5 g of glycerol and 2 g of Span™ 83 sorbitan sesquioleate were added to the metformin-water solution and thoroughly mixed to create solution A. In a separate 200 ml beaker, petroleum jelly (20 g) was heated to 80 °C until it formed a clear solution. Next, 20 g of liquid paraffin was incorporated into the petroleum jelly solution and mixed well, resulting in solution B. Finally, solution A was carefully poured into solution B (petroleum jelly -paraffin mixture) while stirring continuously, and the mixture was allowed to cool to room temperature. This process yielded a lotion of 6% of metformin (6% Met). For the control lotion, the same formulation was followed without the addition of metformin (0% concentration). Both the 6% Met and 0% Met (control) formulations produced a smooth, white cream.

[0199] Skin wound healing model

[0200] Ten female Sprague Dawley (SD) rats, aged 3 months, were utilized in this study. A 2 cm incised wound was carefully created on the skin over each Achilles tendon area using a scalpel, after which the wounds were sutured. Subsequently, the wounded rats were divided into two groups and treated daily for 10 days as follows:

[0201] Group 1: Each leg's skin surface received a daily application of 50 mg of control lotion (totaling 100 mg of lotion per rat) containing 0% Met.

[0202] Group 2: Each leg's skin surface was treated daily with 50 mg of 6% Met-lotion (totaling 100 mg of 6% Met-lotion per rat).

[0203] After 10 days post-surgery, the animals were euthanized and blood samples were collected from each rat's heart. These samples were then utilized for inflammation testing using enzyme-linked immunosorbent assay (ELISA). Additionally, skin tissue samples from the Achilles tendon area of the rats were harvested for further analysis. The effect of Met on the healing of wounded skin was assessed through histological examination of rat skin tissue sections.

[0204] Measurement of HMGB1 in rat serum by ELISA kit

[0205] The HMGB 1 concentrations in rat serum samples were measured using an ELISA kit (Cat. #NBP2- 62767; Novus Biologicals®; Centennial, CO, USA) following the manufacturer’s instructions. Blood samples were obtained from the rats' hearts and left at room temperature for 1 hour before being centrifuged cell pellets. If the HMGB1 levels were not immediately determined, the supernatant was stored at -20°C for later analysis.

[0206] Measurement of interleukin-betal (IL- fl ) in rat serum by ELISA kit

[0207] The IL- 1 concentrations in rat serum samples were evaluated using an ELISA kit (Cat. #ab255730; Abeam®; Boston, MA, USA), adhering to the manufacturer’s instructions. Blood samples were drawn from the rats' hearts and left to rest at room temperature for 1 hour before being centrifuged at 1000 x g for 15 minutes. Following centrifugation, the supernatant was carefully isolated from the red blood cell pellets. If the IL-f51 levels were not promptly determined, the supernatant was stored at -20°C for future analysis.

[0208] Histochemical staining for the structural analysis of rat skin tissues

[0209] Skin tissue samples from the Achilles tendon areas of the rat hind legs were carefully dissected and then immersed in 4% paraformaldehyde overnight at 4°C for fixation. The fixed skin tissues were embedded in paraffin and sliced into 5 pm thick sections. These tissue sections were examined for structural analysis with hematoxylin and eosin (H&E), and Masson’s trichrome staining, according to the standard protocols. The stained tissue sections were subsequently examined under a light microscope (Nikon® eclipse, TE2000-U).

[0210] Piero-Sirius red staining and polarized light microscopy of mouse tendon

[0211] The paraffin-embedded rat skin tissue block was sectioned into slices 5 pm thick and stained using a Piero-Sirius Red kit (Cat. #abl50681, Abeam®, Waltham, MA, USA) according to the manufacturer’s instructions. Subsequently, the stained skin tissue sections were analyzed under a polarized light microscope (Nikon®).

[0212] Immuno-fluorescent staining on rat skin tissue sections

[0213] For immunofluorescent staining, the fixed rat skin tissue samples were promptly embedded in O.C.T compound (Sakura Finetek USA® Inc., Torrance, CA) within disposable molds and then frozen at -80°C. Subsequently, cryostat sectioning was carried out at -25°C to obtain approximately 5pm thick tissue sections, which were left to equilibrate to room temperature overnight. Following this, the tissue sections underwent fixation in 4% paraformaldehyde for 15 minutes, followed by three washes with PBS.

[0214] For staining of HMGB1 and AMPK, the fixed tissue sections were treated with 0.1% Triton®-X- 100 nonionic surfactant at room temperature for 30 minutes, then rinsed with PBS three times. The treated tissue sections were then incubated overnight at 4°C with either rabbit anti-HMGBl antibody (1:330; Cat. #ab!8256, Abeam®; Waltham, MA, USA), rabbit anti-AMPK antibody (1:500; Cat. #MA5-15815, ThermoFisher Scientific®; Waltham, MA), rabbit anti-phospho-AMPK antibody (1:500, Cat. #abl33448, Abeam®, Waltham, MA), rabbit anti-a-SMA antibody (1 :500; Cat. #abl24964, Abeam®, Waltham, MA, collagen I antibody (1:500; Cat. #abl38492, Abeam®, Waltham, MA, USA), or rabbit anti-collagen III antibody (1:500; Cat. 184993, Abeam®, Waltham, MA, USA).

[0215] The following morning, the tissue sections were rinsed five times with PBS and then incubated with goat anti-rabbit secondary antibody conjugated with Cy3 (1:500, Cat. #AP132C; Millipore®, Burlington, MA) for 2 hours at room temperature. Finally, the tissue sections were counterstained with DAPI, and the positively stained results were examined using a fluorescent microscope (Nikon®, Eclipse TE2000U).

[0216] Semi-quantitative assessment of positive stained cells in the skin tissue sections

[0217] Three random images were selected from each skin tissue section using a Nikon® Eclipse TE2000- U microscope to generate semi-quantitative staining results. This process yielded a total of nine images analyzed for each group, reflecting three sections from three different rats. Positive staining cells within the tissue sections were manually identified by reviewing the captured images and subsequently analyzed using SPOT imaging software (Diagnostic Instruments®). The percentage of positive staining was calculated using the formula: % of positive staining = (number of positively stained cells I total number of cells stained with DAPI) x 100%. The mean percentage of positive staining for each group was then determined by averaging the resulting values.

[0218] Statistical analysis

[0219] Data were analyzed using One-way ANOVA followed by Fisher’s PLSD for multiple comparisons. A p-value less than 0.05 was considered significantly different between two groups.

[0220] Example 11 Metformin Inhibits Scar Tissue Formation in Rats

[0221] The effect of Met-lotion on skin healing and scar tissue formation was studied. Skin over the Achilles tendon area of rats was wounded using a scalpel. The wounds were then sutured and treated daily for 10 days with either a control lotion (0% metformin) or 6% metformin-enriched lotion. Application of the 6% Met lotion on wounded rat skin demonstrated an anti-inflammatory effect, as confirmed by ELISA assays. Specifically, serum levels of two key inflammatory markers, HMGB1 and IL- 1, were significantly reduced in rats treated with the 6% Met lotion as compared to those treated with the control lotion (FIGS. 20A-20B). IL-01 concentrations dropped from 3.9 pg / ml in the control group to 0.5 pg / ml in the Met- treated group (FIG. 20B). Similarly, HMGB1 levels decreased from 169.6 ng / ml in the control serum to 67.9 ng / ml following treatment with 6% Met lotion (FIG. 20A).

[0222] Immunostaining analyses further supported these findings, showing higher levels of HMGB1 in the skin matrix surrounding the wound in control-treated rats (FIGS. 21A-21D). In contrast, wounds treated with 6% Met lotion exhibited substantially lower HMGB1 levels in the surrounding skin matrix (FIGS. 21E- 21H). Additionally, over 46% of cells in the wound area of rats treated with 6% Met lotion showed nuclear 21C-21D, 211).

[0223] H&E staining results revealed that a dense and thick epidermis formed at the wound area treated with 0% Met-lotion (indicated by dashed lines in FIG. 22A and arrows in FIGS. 22A-22C). In contrast, wounds treated with 6% Met-lotion developed normal skin-like tissues (marked by dashed lines in FIG. 22D and arrows in FIGS. 22D-22F). Semi-quantitative analysis showed that the epidermis at the wound site treated with 6% Met-lotion was six times thinner compared to that treated with 0% Met-lotion (as depicted in FIG. 22G).

[0224] Trichrome Masson staining highlighted a large empty gap (arrows in FIGS. 23A-23C) in the wound area treated with 0% Met-lotion (dashed line area in FIGS. 23A-23C). The majority of the tissue, especially in the area circled with the dotted line, is stained, indicating a high concentration of collagen. This is typical of scar tissue, where collagen is laid down extensively to repair the damage. The staining, visible especially in areas like the one indicated by the arrow, shows blood vessels. Overall, the structure of the tissue within the curved line appears somewhat less organized and denser compared to typical skin tissue, which is characteristic of scar formation where the normal architecture is disrupted and replaced by fibrotic tissue.

[0225] In contrast, no gap was observed in the wound areas treated with 6% Met-lotion (green dashed line area in FIGS. 23D-23F). The dotted circle likely encapsulates an area of skin that includes both the epidermis (the upper layers) and the underlying dermis (stained predominantly due to the presence of collagen). This clear delineation of layers indicates good tissue regeneration. Also within the circled area, the collagen appears well-organized and less dense compared to typical scar tissue, suggesting a healthier and possibly more functional recovery of the dermis. The structure seems more regular and aligned, which is beneficial for the mechanical properties of the skin. The blood vessels (indicated by arrows) are clearly visible and well-defined with staining, showing that there is adequate vascularization supporting the healing tissue. Overall, these areas developed normal skin-like tissues, including hair follicles (arrows in FIG. 23F) and sweat glands (arrows in FIG. 23F). Comparatively, very few hair follicles and sweat glands were present in the wound areas treated with 0% Met-lotion (FIGS. 23A-23C).

[0226] Piero-Sirius Red staining under a bright light microscope revealed a large gap in the wound areas treated with 0% Met-lotion (indicated by a black arrow in FIG. 24A), while no gap was observed in the areas treated with 6% Met-lotion (marked by a blue dashed line in FIG. 24C). Under polarized light, high levels of collagen III were detected in the wound areas treated with 0% Met-lotion (FIG. 24B). In contrast, the tissues in the wound areas treated with 6% Met-lotion exhibited a positive stain for collagen I (FIG. 24D). Semi-quantitative results showed that over 55% of the cells in the wound areas treated with 0% Met-lotion expressed collagen III (FIG. 24E), whereas only about 19.84% of the cells in the areas treated with 6% Met- lotion showed similar expression (FIG. 24E).

[0227] Immunostaining for collagen III expression corroborated the results from the Piero-Sirius Red staining. It showed that more than 68% of the cells in the wound areas of rats treated with 0% Met-lotion the wound areas of rats treated with 6% Met-lotion demonstrated similar staining (FIGS. 25E-25H, 251).

[0228] Additionally, immunostaining for alpha-smooth muscle actin (a-SMA) demonstrated that Met-lotion reduced a-SMA expression in the wounded skin areas (FIGS. 26E-26H). Higher levels of a-SMA were observed in the wound area treated with 0% Met-lotion (FIGS. 26A-26D). Semi-quantitative results showed that more than 77% of the cells in the wound areas of rats treated with 0% Met-lotion expressed a-SMA (FIG. 261), while only about 16.7% of the cells in the wound areas treated with 6% Met-lotion showed a- SMA expression (FIG. 261).

[0229] Further examination revealed that treatment with 6% Met-lotion significantly reduced TGF-01 expression in the wound areas (FIGS. 27E-27H), with more than 69% of the cells in areas treated with 0% Met-lotion expressing TGF-01 (FIGS. 27A-27D, 271). In contrast, less than 11.2% of the cells in areas treated with 6% Met-lotion expressed TGF- 1 (FIGS. 27E-27H, 271).

[0230] Moreover, immunostaining for AMPK phosphorylation indicated that over 57% of the cells in the wound areas treated with 6% Met-lotion were positive for p-AMPK (FIGS. 28E-28I). Conversely, fewer than 5% of the cells in the wound areas treated with 0% Met-lotion showed p-AMPK expression (FIGS. 28A-28D, 281).

[0231] Lastly, more than 50% of the cells in the wound areas that were treated with 6% Met-lotion expressed collagen I (FIGS. 29E-29I). In comparison, about 26% of the cells in the wound areas treated with 0% Met-lotion were positive for collagen I (FIGS. 29A-29D, 291).

[0232] This study introduces a pioneering Met lotion designed for transdermal administration to facilitate reduced scarring or even scarless healing of skin wounds. Metformin is typically administered orally, however, oral administration can lead to side effects impacting multiple organs, including the stomach, liver, and kidneys. In this study, a Met-bascd lotion was designed for topical application directly target skin wounds. It is expected that topical administration of metformin will be effective at lower doses than oral administration, thereby minimizing potential systemic side effects.

[0233] The work disclosed herein demonstrates a marked difference between wounds treated with the control lotion (0% Met-lotion) and those treated with a 6% Met-lotion solution. Notably, wounds treated with the 6% Met-lotion exhibited accelerated healing, resulting in the formation of tissue resembling normal skin, as opposed to the slower healing and scar-like tissue formation observed with the control lotion. ELISA analysis revealed a significant anti-inflammatory effect associated with the 6% Met-lotion, as evidenced by decreased levels of inflammatory markers IL-01 and HMGB1 in the blood samples of rats treated with this formulation compared to those treated with the 0% Met-lotion. Immunostaining further supported these findings, showing that administration of the 6% Met-lotion inhibited the release of HMGB1 from cell nuclei into the tissue matrix. Additionally, the 6% Met-lotion demonstrated anti-fibrotic properties, as indicated by several key observations. Treatment with the 6% Met lotion led to enhanced p- AMPK activity, decreased expression of TGF-01, reduced numbers of a-SMA+ cells, decreased production of collagen III, and increased expression of collagen I. These collective results suggest that the 6% Met- inflammation and fibrosis while facilitating tissue regeneration.

[0234] Application of 6% Met lotion to wounded skin is shown herein to promote wound healing, diminish scar tissue formation, and facilitate the regeneration of normal, functional skin. Without being bound by any particular theory, topical administration of metformin may enhance skin healing by activating AMPK, inhibiting TGF- 1 levels, reducing the number of a-SMA+ cells, thereby diminishing scar tissue formation.

[0235] Example 12 Metformin-Hyaluronic Acid Conjugate

[0236] This provides an exemplary method by which a metformin hyaluronic acid conjugate was produced.

[0237] Sodium hyaluronate (HA: 80 mg, molecular weight 1500 kDa) was dissolved in 8 ml of distilled water to make a HA solution (10 mg / ml), then 4 mg of l-[3-(dimethylamino) propyl]-3 ethylcarbodiimide hydrochloride (EDC) and 5 mg of N-hydroxy succinimide (NHS) were added into the HA solution and stirred on magnetic stirrer for 30 min. After that, 200 mg of metformin hydrochloride was added to the mixture and stirred with a magnetic stirrer at room temperature overnight. The next morning, 8 ml of acetone was added into the reaction mixture, white solid product was removed by centrifugationo at 2000 g for 10 min. The solid was washed with water to remove unreacted starting materials. The HA-conjugated metformin (HA-Met) was then re-dissolved with water for further use.

[0238] The synthesized HA-Met was tested by IR spectrum (FIG. 16). The reaction between HA and metformin, and the structure of HA-conjugated metformin were shown by Scheme I (FIG. 17).

[0239] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of this disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:

1. A method of inhibiting or treating a soft tissue musculoskeletal injury, comprising: selecting a subject at risk of or having a soft tissue musculoskeletal injury, and administering metformin to the subject.

2. The method of claim 1, wherein the soft tissue musculoskeletal injury is an acute or chronic injury.

3. The method of claim 1, wherein administering metformin comprises: i) administering metformin topically to skin adjacent the soft tissue musculoskeletal injury, or ii) injecting metformin at the site of the soft tissue musculoskeletal injury.

4. The method of claim 3, comprising administering metformin topically to skin adjacent the soft issue musculoskeletal injury, wherein the soft issue musculoskeletal injury is less than 3 centimeters below a skin surface of the subject.

5. The method of claim 3, comprising administering injecting metformin at the site of the soft tissue musculoskeletal injury, wherein the soft issue musculoskeletal injury is greater than 3 centimeters below a skin surface of the subject.

6. The method of claim 1, wherein the soft tissue musculoskeletal injury is a tendon, ligament, or muscle injury.

7. The method of claim 6, wherein the tendon injury is an Achilles tendon injury.

8. The method of claim 1, wherein the subject selected for treatment is at risk of or has tendinopathy.

9. The method of claim 1, wherein scar tissue is removed prior to administering metformin.

10. The method of claim 1, wherein administering metformin comprises administering a topical pharmaceutical composition comprising metformin.

11. A method of inhibiting or treating scar tissue, comprising:selecting a subject at risk of developing or having scar tissue, and administering metformin to the subject.

12. The method of claim 11, wherein the scar tissue is dermal scar tissue.

13. The method of claim 12, wherein the scar tissue is epidermal scar tissue.

14. The method of claim 12 or claim 13, wherein the scar tissue is from a lesion, abrasion, laceration, burn, or surgical procedure.

15. The method of claim 11, wherein administering metformin comprises administering a topical pharmaceutical composition comprising metformin to the scar tissue.

16. The method of claim 10 or claim 15, wherein the topical pharmaceutical composition comprises 1% to 15% weight by weight (w / w) metformin.

17. The method of claim 16, wherein the topical pharmaceutical composition comprises 3% to 6% w / w metformin.

18. The method of claim 16, wherein the topical pharmaceutical composition comprises about 3% w / w metformin.

19. The method of claim 16, wherein the topical pharmaceutical composition comprises about 6% w / w metformin.

20. The method of claim 16, wherein the topical pharmaceutical composition is formulated for transdcrmal delivery of metformin.

21. The method of claim 16, wherein the topical pharmaceutical composition is a lotion, cream, ointment, gel, or paste.

22. The method of claim 1, wherein administering metformin comprises local injection of a pharmaceutical composition comprising metformin.

23. The method of claim 22, wherein the pharmaceutical composition comprises 0.1% to 5% weight by weight (w / w) metformin.

24. The method of claim 23, wherein the pharmaceutical composition comprises 0.5% to 2% w / w metformin.

25. The method of claim 22, wherein the pharmaceutical composition further comprises hyaluronic acid, glucose, or platelet rich plasma.

26. The method of claim 1 or claim 11, wherein the subject is a human.

27. The method of claim 1 or 16, wherein the subject is at risk of a soft tissue musculoskeletal injury.

28. The method of claim 27, wherein the subject at risk of a soft tissue musculoskeletal injury is a human subject over the age of 65.

29. The method of claim 1 or claim 11, wherein the metformin is conjugated to hyaluronic acid.

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