Methods for Treating Peripheral Nerve Fibrosis and Hypoxia-Related Injury - Patent application

Abstract

Described herein are methods and compositions for treating and preventing peripheral nerve injury, more particularly hypoxia-related tissue injury, such as peripheral nerve fibrosis. The methods described include administering a therapeutically effective amount of a source of glutamine to a peripheral nerve or one or more tissues surrounding a peripheral nerve.

Description

[Technical field]

[0001] The present invention relates to the treatment of hypoxia-related tissue damage in a subject. More particularly, the method relates to the treatment of hypoxia-related tissue damage in a subject during a surgical procedure according to a specific dosing regimen. [Background technology]

[0002] The peripheral nervous system is a network of 43 pairs of motor and sensory nerves that connect the brain and spinal cord (the central nervous system) to the entire body. These nerves control sensory, motor, and motor coordination functions. The nerves are fragile, and peripheral nerve injury ("PNI") can be caused by trauma or disease. Peripheral nerve fibrosis, accompanied by chronic inflammation, perineural adhesions, and scarring, is often reported in patients with PNI.

[0003] The severity of PNI can be influenced by the presence of various pathophysiological conditions, including hypoxia. Chronic hypoxia and periods of hypoxia have been reported to induce peripheral polyneuropathy. At the molecular level, the response to hypoxia is orchestrated by hypoxia-inducible factors (HIFs), which consist of an oxygen-dependent α-subunit (HIF-1α, HIF-2α) and an oxygen-dependent β-subunit. HIF-α-subunits are constitutively expressed and are rapidly degraded in normoxia. However, under hypoxia, HIF-1α and HIF-2α are stabilized and form an active transcriptional complex. The complex binds to hypoxia response elements (HREs) in the promoter regions of numerous downstream target genes, collectively initiating an adaptive response to hypoxia. Under hypoxic conditions, the α-subunit translocates to the nucleus and forms a heterodimer with the β-subunit. The HIF heterodimer binds to hypoxia-responsive factors and further associates with other coactivators to regulate gene expression. Research in recent years has shown that HIF1α signaling is involved in promoting fibrogenesis through excessive ECM activity and production. Excessive deposition of ECM factors such as collagen, fibronectin, and proteoglycans is a hallmark of tissue fibrosis, and HIF1α has been found to be involved in the upregulation of ECM factors under hypoxic conditions. Moreover, overexpression of HIF1α in fibrotic diseases further indicates the involvement of HIF1α in the pathogenesis of fibrosis. Summary of the Invention

[0004] The present invention relates to the need for compounds and methods for treating and preventing peripheral nerve injury, and more particularly hypoxia-related tissue injury, such as peripheral nerve fibrosis, comprising administering a therapeutically effective amount of a source of glutamine to a peripheral nerve or one or more tissues surrounding the peripheral nerve.

[0005] One aspect is a method of treating or preventing peripheral nerve fibrosis in a subject, the method comprising administering to one or more peripheral nerves or one or more tissues surrounding one or more peripheral nerves, a source of glutamine is administered before, during or after surgery involving one or more peripheral nerves. In some embodiments, one or more additional doses of the source of glutamine are administered to the subject at a certain time period after the first administration, at a certain time interval after the first administration, and / or immediately before closing all surgical incisions. In some embodiments, the source of glutamine is administered during surgery involving one or more peripheral nerves. In some embodiments, the source of glutamine is an oligopeptide comprising glutamine. In some embodiments, the oligopeptide is a dipeptide. In some embodiments, the dipeptide is L-alanyl-L-glutamine.

[0006] Another aspect is a method of treating or preventing adhesions in one or more peripheral nerves in a subject undergoing a surgical procedure, the method comprising administering to a peripheral nerve or tissue surrounding one or more peripheral nerves, the source of glutamine being administered before, during, or after surgery involving one or more peripheral nerves.

[0007] Another aspect is a method of treating or preventing hypoxic tissue-related damage to one or more peripheral nerves in a subject, the method comprising administering a therapeutically effective amount of a glutamine source to tissue that is subject to hypoxia-related tissue damage in a subject during a surgical procedure, wherein the source of glutamine is administered before, during, or after surgery involving one or more peripheral nerves.

[0008] Another aspect is a method of prophylactically reducing the incidence of fibrosis to one or more peripheral nerves in a subject, comprising administering an effective amount of glutamine to one or more peripheral nerves or one or more tissues surrounding said peripheral nerves.

[0009] Another aspect is a method of reducing HIF-1α in peripheral nerves suffering from hypoxia-related damage, the method comprising administering to one or more peripheral nerves or one or more tissues surrounding one or more peripheral nerves, the source of glutamine comprising administering the source of glutamine before, during, or after surgery involving one or more peripheral nerves.

[0010] Another aspect is a kit for treating or reducing peripheral nerve adhesions or fibrosis in a subject comprising an effective amount of a source of glutamine, wherein the source of glutamine is formulated as a dosage form selected from the group consisting of a surgically implantable film, an injectable material, a topical cream, and a dosage form suitable for iontophoresis. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1A is a schematic diagram of the isolation of primary rat fibroblasts. [Figure 1B] FIG. 1B shows that the morphological and phenotypic characteristics of the isolated cells were identified and confirmed by ICC imaging for specific markers, including α-SMA, HSP47, and Vimentin. [Figure 1C] FIG. 1C shows the primary fibroblasts that, after phenotypic characterization, were used in the experiments under three different experimental conditions. [Diagram 2] 2A-B show the results of cell proliferation and cell death of hypoxic primary rat fibroblasts treated with an embodiment of the invention. [Diagram 3] 3A and 3B show the reduction in expression of HIF-1α and pro-fibrotic factors in primary fibroblasts treated with an embodiment of the invention. [Figure 4] 4A-4E show the reduction of expression of pro-fibrotic markers in an in-vitro chronic hypoxia model according to an embodiment of the invention. [Diagram 5] 5A-5E show the reduction in expression of pro-fibrotic markers in an in-vitro chronic hypoxia model according to an embodiment of the invention. [Figure 6]6A and 6B show the reduction in expression of FBN and other pro-fibrotic factors associated with peripheral nerve fibrosis, according to an embodiment of the invention. [Figure 7] 7A-7C show reduced expression of pro-fibrotic and adhesion biomarkers in hypoxic primary rat fibroblasts according to an embodiment of the invention. [Figure 8] FIG. 8 shows modulation of signaling pathways through control of specific pro-fibrotic and adhesion biomarkers in hypoxic primary rat fibroblasts according to an embodiment of the invention. [Figure 9] 9A and 9B show the reduction of expression of pro-fibrotic markers in an in-vitro chronic hypoxia model, according to an embodiment of the invention. [Figure 10] 10A and 10B show the reduction of expression of pro-fibrotic markers in an in-vitro chronic hypoxia model, according to an embodiment of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] DETAILED DESCRIPTION OF THE DRAWINGS AND PRESENTLY PREFERRED EMBODIMENTS The following paragraphs more particularly define the embodiments of the invention described herein. The following embodiments are not meant to limit the invention or narrow its scope, as it is readily apparent to those skilled in the art that suitable modifications and adaptations can be made without departing from the scope of the invention, embodiments, or specific aspects described herein. All patents and publications cited herein are incorporated herein by reference in their entirety.

[0013] The term "glutamine source" or "source of glutamine" includes glutamine and its physiologically acceptable salts, as well as glutamine conjugates and peptides containing glutamine, as described herein. The terms "dosage" or "dose" or "dosage form" as used herein refer to any form or formulation of a glutamine source that contains an amount sufficient to produce a therapeutic effect in a single or multiple administrations. The term "surgical incision," as used herein, means a wound made by a cutting instrument, such as a scalpel, laser, or other cutting instrument, before or during a surgical procedure, including an incision or entry point made for a laparoscopic or other minimally invasive surgical technique. The term "prevention of," as used herein, includes the meaning of reducing. The amount of reduction can be from about 0.001% to about 100%. The term "effective amount" is an amount that achieves a desired effect. For example, in the present invention, an effective amount of one or more glutamine sources is an amount that prevents or reduces hypoxia-related tissue damage after one or more administrations. The term "drug loading," as used herein, refers to the weight percent of the glutamine source relative to the total weight of the dosage form. The term "formulation" or "composition" as used herein refers to a glutamine source combined with one or more pharma- ceutically acceptable diluents and / or excipients. The term "extended release" or "sustained release," as used herein, refers to a composition that releases an active ingredient according to a desired profile over an extended period of time under physiological conditions or in an in vitro assay. By "extended period of time" is meant a continuous period of at least about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, or even longer, specifically over a period of about 18 hours under physiological conditions or in an in vitro assay. The term "delayed release", as used herein, refers to a composition that releases the active ingredient after a period of, for example, days, minutes, or hours, such that the active ingredient is not released immediately. A delayed release composition can provide, for example, release of a drug or active ingredient from a dosage form after a certain period of time under physiological conditions or in an in vitro test.

[0014] The term "treatment" refers to administering a therapy in an amount, manner, or mode effective to ameliorate a condition, symptom, disorder, or parameter associated with a disorder, or the likelihood of occurrence thereof (e.g., a therapeutic effect). In some embodiments, the term "treatment" refers to treating or preventing adhesion formation following surgery. In other embodiments, the term "treatment" refers to treating or preventing hypoxia-related tissue damage in a patient undergoing surgery. The term "prophylactically treating" refers to administering a therapy in an amount prior to incurring a condition, symptom, disorder, or parameter associated with a disorder, or reducing the likelihood of its occurrence. The term "subject" refers to any mammal, including animals and humans. The subject may be a medical patient in need of treatment. In one embodiment, the subject is a human. The terms "prevent" or "reduce" refer to preventing or reducing the progression of a disorder such as adhesions or fibrosis, either to a statistically significant degree or to a degree detectable by one of skill in the art. The term "substantially" as used herein means to a significant or significant degree, but not completely. "A" or "an" as used herein means one or more, unless otherwise specified. The terms "include," "including," "contain," "containing," and "having" mean "comprising."

[0015] Described herein is a method for treating hypoxia-related tissue damage, and in particular a method for preventing or treating peripheral nerve injury and peripheral nerve fibrous adhesions by administering a glutamine source at a specific time.Described herein is a method comprising administering a glutamine source to a subject at least once within a specific period.In certain embodiments, the glutamine source is administered to a subject one or more times.For example, a subject receives a first dose of a glutamine source within a specified period, and then receives one or more additional doses of a glutamine source at a specified period, at a certain time interval, and / or immediately before closing all surgical incisions, and / or after the completion of surgery.

[0016] Some embodiments described herein are methods of treating or reducing hypoxia-related tissue damage or fibrosis to peripheral nerves. Fibrosis is characterized by excessive collagen deposition, ECM matrix contraction, tissue damage and abnormal wound healing. Scarring and tissue fibrosis are caused by the overactivation of effector cells, including fibroblasts. The overactivation of fibroblasts and their differentiation into myofibroblasts results in differential amounts of collagen formation and scar tissue formation. Excessive collagen deposition in peripheral nerves inhibits axonal regeneration at the injury site. Furthermore, collagen cross-linking induces tissue destruction that is resistant to protease degradation. Tissue fibrosis due to chronic injury involves a series of biochemical signaling of several intracellular and extracellular factors. In addition, the presence of a hypoxic microenvironment can promote tissue damage and peripheral neuropathy.

[0017] HIF-1 acts on fibroblasts and regulates the ECM matrix under hypoxic conditions, making it a key regulator of tissue architecture and fibrosis. Together, compelling evidence suggests that HIF1α regulates complex fibrosis in various organs, and that HIF1α signaling is clinically significant. Its role on the pathological outcome of fibroblasts and macrophages also makes it a suitable target for pharmacological and genetic inhibition in PNI. In addition, several components of the HIF-1 signaling cascade control the biochemical signaling of various extracellular growth factors, further indicating its diverse role in tissue remodeling.

[0018] Glutamine is a conditionally essential amino acid that is well absorbed, promotes wound healing, and regulates the function of neutrophils, macrophages, and lymphocytes. It also serves as a substrate for the production of antioxidant glutathione. During catabolic stress (trauma, sepsis, burn), glutamine is released from muscle stores into serum, and intracellular levels of glutamine in muscle decrease. The tissues, especially visceral organs, rapidly take up glutamine, and serum concentrations of glutamine subsequently decrease. Previous studies have demonstrated the efficacy of glutamine-containing solutions in preventing peritoneal adhesion formation in rat models. See U.S. Patent No. 9,011,883.

[0019] Furthermore, glutamine is safe, well absorbed, and has no reported side effects. Glutamine is known to promote wound healing. Glutamine and its dipeptides have been used as parenteral and enteral supplements in critically ill patients and other clinical settings. The first use of glutamine sources has been employed either enterally (e.g., food supplements) or parenterally via the intravenous route. At least one study described the administration of glutamine to highly vascularized areas such as the peritoneum, where it is readily absorbed into the vasculature. At these sites, L-glutamine prevented loss of vascularization due to peritoneal sutures that cause devascularization and adhesion formation. Thus, it was believed that glutamine could prevent fibrosis and adhesion formation by being incorporated into the vasculature and preventing loss of vascularization. As described herein, it has been discovered that application of a glutamine source to peripheral nerves or cells surrounding said peripheral nerves significantly reduces fibrosis and associated adhesions.

[0020] Some embodiments described herein are methods for treating or reducing adhesion formation, fibrosis, or hypoxia-related tissue damage in a subject undergoing a surgical procedure.Exemplary and non-limiting surgical procedures contemplated herein include surgery and procedures that affect the head or neck of a subject, the pelvic cavity, the abdominal cavity, the thoracic cavity, or one or more of the subject's extremities.Examples of surgery that affect the pelvic cavity include, but are not limited to, myoma precipitation, oophorectomy, hysterectomy, removal of endometriosis, tubal ligation, that is, any laparoscopic, laparotomy, or open surgery or procedure that involves any of the reproductive organs, including, but not limited to, the uterus, ovaries, tubes, prostate, urethra, and the bladder, pelvic colon, and rectum (including intravaginal procedures of the uterus such as dilation and curettage, IVF, etc.), or any surgery that involves the major arteries, veins, muscles, and nerves, membranes, ligaments, or viscera of the pelvic cavity. Examples of surgeries affecting the abdominal cavity include, but are not limited to, cholecystectomy, liver resection, lap band surgery, colonic anastomosis, appendectomy, i.e., any surgery involving the stomach, liver, pancreas, spleen, gallbladder, kidneys, and most of the small and large intestines, any surgery involving the major arteries, veins, muscles, and nerves, membranes, ligaments, or viscera of the abdominal cavity. Examples of surgeries affecting the thoracic cavity include, but are not limited to, laparoscopic or laparotomy or open chest cardiovascular surgeries and procedures, lung surgery, liver surgery, gallbladder surgery, any surgery involving the major arteries, veins, muscles, and nerves, membranes, ligaments, bones, or viscera of the thoracic cavity. Examples of surgeries affecting one or more of a subject's extremities include, but are not limited to, laparoscopic or laparotomy or open surgeries involving the arms, legs, elbows, shoulders, spine, including but not limited to, any surgery involving the major arteries, veins, muscles, and nerves, membranes, ligaments, bones, or internal organs, and examples of surgeries affecting the head and neck include, but are not limited to, laparoscopic or laparotomy or open surgeries and procedures including brain-ophthalmic surgeries or procedures, ear, nasopharynx, teeth, gums and jaw surgeries or procedures, cosmetic reconstructive surgeries and procedures of the face, head and neck, cosmetic procedures of the teeth, gums and jaw, any surgery involving the major arteries, veins, muscles, and nerves, membranes, ligaments, bones, or internal organs of the head and neck.

[0021] In one embodiment, a source of glutamine is administered to a subject before, at the same time, or after surgery involving one or more peripheral nerves.Pre-administration protects one or more peripheral nerves from undergoing fibrosis and eventual adhesion.In some cases, pre-administration of a source of glutamine can further shorten or treat the arthritis and post-operative recovery time of a subject.In another embodiment, a source of glutamine is administered to a subject after undergoing surgery involving one or more peripheral nerves.Similarly, one or more peripheral nerves are protected from further fibrosis and adhesion.It is believed that administration of a source of glutamine can reduce adhesion.

[0022] In another embodiment, the source of glutamine is administered to the subject after one or more peripheral nerves are injured.The peripheral nerve injury may result from accidents, falls, or sports that cause the nerve to be stretched, compressed, crushed, or severed; medical conditions such as diabetes, Guillain-Barre syndrome, and carpal tunnel syndrome; autoimmune diseases including lupus, rheumatoid arthritis, and Sjogren's syndrome; and other causes including arterial narrowing, hormone imbalance, and cancer treatments including tumors, chemotherapy, or radiation therapy.

[0023] In some embodiments, a subject has previously undergone surgery or injury to one or more peripheral nerves, and is administered a source of glutamine, and has a reduction in the amount of fibrosis and / or adhesions in the peripheral nerves.In some embodiments, the amount of reduction in fibrosis and adhesions in the subject's peripheral nerves is at least 10% in the subject after administration of a source of glutamine.In some embodiments, the amount of reduction in adhesions in a subject with previous surgery or injury is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or even 100%.

[0024] In some embodiments, one or more pathological conditions of one or more peripheral nerves or one or more tissues adjacent to or surrounding a peripheral nerve, including fibrosis, inflammation, angiogenesis, immune cell infiltration, scarring, decreased joint flexibility, joint pain, or a combination of these conditions, are alleviated by administering a source of glutamine to one or more peripheral nerves or one or more tissues surrounding one or more peripheral nerves of a subject.

[0025] In some embodiments, the amount of reduction in one or more pathological conditions of the peripheral nerves is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or even 100%. These pathological conditions of the peripheral nerves can be assessed by clinical tests known in the art.

[0026] In some embodiments, a source of glutamine is administered to one or more peripheral nerves in a manner that can indicate that the residence time of glutamine is increased in peripheral nerves or one or more tissues surrounding peripheral nerves.Without being bound by any theory, it is believed that increasing the time that glutamine is in contact with peripheral nerves or tissues that are susceptible to adhesion or fibrosis events can reduce the degree of adhesion or fibrosis to a greater extent.Glutamate has a high turnover rate in nature and is rapidly absorbed by immune cells, including lymphocytes and macrophages.

[0027] In some embodiments, a source of glutamine is administered in a single dose to one or more peripheral nerves and an effective amount of glutamine is retained at or near the administration site for at least about 30 minutes to about 48 hours. In some embodiments, an effective amount of glutamine following a single dose is retained at or near the administration site for at least about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, or about 48 hours.

[0028] In some embodiments, an effective amount of the source of glutamine for reducing adhesions or one or more pathological conditions of peripheral nerves described herein is administered in a single dose. In some embodiments, the amount of the source of glutamine administered in a single dose ranges from about 0.0001 g / kg to about 5 g / kg of the subject's body weight. In some embodiments, the amount of the source of glutamine administered in a single dose is about 0.0001 g / kg, about 0.001 g / kg, about 0.01 g / kg, about 0.05 g / kg, about 1 g / kg, about 1.5 g / kg, about 2 g / kg, about 2.5 g / kg, about 3 g / kg, about 3.5 g / kg, about 4 g / kg, about 4.5 g / kg, about 5 g / kg. The exact amount of the source of glutamine administered to a subject will vary depending on the surgery or injury that the peripheral nerve being treated has undergone, as well as the amount and medical history of the subject's existing fibrosis or adhesions. Glutamine is a non-toxic amino acid and therefore may be administered in large amounts without any expected side effects.

[0029] Some embodiments described herein are pharmaceutical dosage forms for reducing peripheral nerve fibrosis and adhesions in a subject. Some embodiments are methods for treating a subject in need thereof with the pharmaceutical dosage forms described herein. Suitable pharmaceutical dosage forms include a source of glutamine and other pharma- ceutical acceptable carriers and excipients for administration to tissue or tissues surrounding peripheral nerves.

[0030] In some embodiments, the glutamine source comprises glutamine or any pharma- ceutically acceptable salt thereof.In some embodiments, the glutamine source comprises or is D-glutamine, or comprises or is L-glutamine.L-glutamine is known to have relatively low water solubility (i.e., about 40 g / L at room temperature) and low stability during storage.

[0031] Thus, the source of glutamine may also include additional carrier amino acids, or glutamine may be incorporated as part of an oligopeptide to increase one or both of the solubility and stability of L-glutamine. The oligopeptide may include any naturally occurring or non-naturally occurring amino acid. A suitable oligopeptide includes L-glutamine and can be metabolized to provide L-glutamine. Preferably, such peptides show increased water solubility and increased stability of L-glutamine. In many cases, such peptides also show increased resistance to degradation during sterilization and storage. The oligopeptide containing L-glutamine may further include a cleavable linker peptide moiety known in the art. These cleavable linkers may be utilized for attachment to various scaffolds and implants, as well as for the administration methods described herein. In some embodiments, the source of glutamine is L-glutamine incorporated as part of a dipeptide. Two such dipeptides that may be used are dipeptides that include L-glutamine and either L-alanine or glycine. The dipeptide alanyl-glutamine (glutamine residue at the C-terminus) has high solubility in water (568 g / L). Glycyl-glutamine (glutamine at the C-terminus) also has high solubility in water (154 g / L) compared to glutamine. Each of the latter dipeptides is stable enough to withstand heat sterilization and long-term storage, so they have been previously employed in total parenteral nutrition formulations for intravenous injection. Thus, in one embodiment, the source of glutamine comprises L-alanyl-L-glutamine. In another embodiment, the source of glutamine comprises D-glutamine incorporated as part of a dipeptide. Two such peptides that may be used are dipeptides that contain D-glutamine and either D-alanine or glycine. Thus, in one embodiment, the source of glutamine comprises D-alanyl-D-glutamine. In some embodiments, the source of glutamine is provided as part of an oligopeptide that includes glutamine, which has greater water solubility and stability in aqueous solutions than glutamine alone.In another embodiment, the glutamine source is a glutamine conjugate, and at least one glutamine residue is linked to the compound via an aminoester bond.Examples of such glutamine sources include, but are not limited to, dichloroacetylglutamine, acetylglutamine, butyrylglutamine, pyruvylglutamine, and glutamine linked to any other suitable organic acid via an aminoester bond.

[0032] L-alanyl-L-glutamine is a dipeptide known for its ability to promote wound healing, immune regulation, and regulation of ECM matrix and peritoneal fibrosis. We believe that hypoxia in fibrotic tissues can be chronic and acute, depending on the extent of injury and tissue damage. Chronic hypoxia is hypothesized to be present at the exact site of injury, which we have expressed as consistent or continuous hypoxia. On the other hand, acute hypoxia can be present at the distal end from the exact site of injury, as represented by irregular or episodic hypoxia. Without being bound to any particular theory, we surprisingly found that administration of a glutamine source, such as L-alanyl-L-glutamine, to hypoxic primary rat fibroblasts dramatically downregulated HIF1-α. Furthermore, since HIF1-α regulates other profibrotic factors, including SMAD signaling, we found that a glutamine source can downregulate these factors as well. Consequently, the present inventors have observed that administering a source of glutamine leads to the conclusion that L-alanyl-L-glutamine modulates both modes of hypoxic injury (chronic and transient).

[0033] Pharmaceutically acceptable preparations of L-glutamine and L-glutamine including peptides (including L-alanyl-L-glutamine) are commercially available.In addition, L-glutamine including peptides for use in the methods described herein can also be synthesized according to known methodologies and purified and sterilized for pharmaceutical use.Other pharmacologic acceptable preparations can be prepared according to embodiments of the present invention, including, but not limited to, D-glutamine and D-glutamine including peptides (including D-alanyl-D-glutamine) preparations.

[0034] In some embodiments, the source of glutamine is administered with one or more additional active pharmaceutical ingredients. The additional active pharmaceutical ingredients can be administered in the same manner as the source of glutamine or in a different suitable parenteral or enteral manner. In some embodiments, the additional active pharmaceutical ingredients are administered with the source of glutamine by the same administration route before, simultaneously, or after the administration of the source of glutamine. In other embodiments, the additional active pharmaceutical ingredients are administered with the source of glutamine by a different administration route before, simultaneously, or after the administration of the source of glutamine. Suitable active pharmaceutical ingredients include any medicine or drug used to treat joint pain, inflammation, or adhesions. Exemplary and non-limiting pharmaceutical agents include the nonsteroidal anti-inflammatory drugs (NSAIDs) aceclofenac, acemetacin, amoxiprin, aspirin, azapropazone, benorylate, bromfenac, carprofen, celecoxib, choline magnesium salicylate, diclofenac, diflunisal, etodolac, etoricoxib, faislamine, fenbufen, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, lornoxicam, loxoprofen, meloxicam, meclofenamic acid, mefenamic acid. , meloxicam, metamizole, methyl salicylate, magnesium salicylate, nabumetone, naproxen, nimesulide, oxyphenbutazone, parecoxib, phenylbutazone, piroxicam, salicylate, sulindac, sulfinpyrazone, suprofen, tenoxicam, tiaprofenic acid, tolmetin, or valdecoxib; selective COX-2 inhibitors such as celecoxib, rofecoxib, prostaglandins; anti-arthritic supplements such as hyaluronic acid, chondroitin sulfate, and glucosamine; corticosteroids such as betamethasone, methylprednisolone, and triamcinolone.The source of glutamine may be further administered with anti-autoimmune disease medications such as disease-modifying antirheumatic drugs (DMARDs), including but not limited to anti-folates, methotrexate, leflunomide, mycophenolate mofetil, sulfasalazine, apremilast, tofamitinib; biologics such as tocilizumab, certolizumab, etanercept, adalimumab, anakinra, abatacept, infliximab, and rituximab.

[0035] In some embodiments, the source of glutamine is administered by intra-articular injection, topically, transdermally, iontophoresis, or surgically. In some embodiments, the glutamine source is formulated for delivery to peripheral nerves or tissues surrounding peripheral nerves by injection, including intra-articular injection methods known in the art.In one embodiment, the glutamine source (e.g., L-alanyl-L-glutamine) can be formulated as part of an aqueous solution with sterilized distilled water, sterilized isotonic solution, sterilized saline, or a pharma- ceutically acceptable carrier such as a dry buffer and / or salt mixture or concentration that forms such a solution when diluted.

[0036] In some embodiments, the formulation may be a liquid, paste, microparticle (including but not limited to liposomes and other vesicles), or gel, containing a glutamine source dissolved in an aqueous phase. The compositions described herein may be such formulations, or may be compositions intended to produce such formulations when hydrated. In its simplest form, the formulation for use in the present invention consists of a glutamine source dissolved in a sterile aqueous liquid vehicle, suitable for injection into peripheral nerves during surgery or for intra-articular injection performed thereafter. The formulation may be injected through a port created during laparoscopic surgery or laminectomy.

[0037] In some embodiments, the source of glutamine is formulated as part of a gel or hydrogel. Hydrogel formulations allow for long-term delivery of suitable drugs, including, for example, L-glutamine or dipeptides or tripeptides that contain at least one glutamine residue. Hydrogels typically form a depot that further allows for high-concentration application of drugs. The hydrogels containing the source of glutamine may be generated prior to administration and impregnated with the source of glutamine for further implantation. Alternatively, hydrogels that form in situ after environmental changes, including pH and temperature, may be utilized for intra-articular injection. Various physical and chemical crosslinking polymers for in situ hydrogel formation are known in the art. Exemplary and non-exemplary hydrogels may include copolymers that include blocks of polyethylene oxide (PEO) propylene oxide (PPO), poly(lactic-co-glycolic acid) (PLGA), poly(N-isopropylacrylamide), poly(propylene fumarate), poly(caprolactone), and the like. Suitable hydrogels may also be produced from naturally occurring proteins and peptides. The particular hydrogel formulation may be determined by the site of application or the particular joint being treated.

[0038] In some embodiments, the glutamine source is formulated as part of an implantable film that includes the glutamine source. The glutamine source can be applied or impregnated into a surgically implantable film or other surgical implant. For example, the glutamine source can be formulated as part of a gel and attached to the exterior of an implant. Implants made of materials such as woven resorbable cellulose, available from Ethicon as INTERCEED®, can be impregnated with the liquid or gel formulation of the present invention. Other contemplated films include polyesteramide-based films (PEA-III) (see, for example, PCT Publication WO 2014 / 053542 A1). In some other embodiments, the glutamine source is formulated for topical, transdermal, or iontophoretic administration.

[0039] In some embodiments, the drug loading of the source of glutamine in the dosage form is about 2% to about 90%, including each integer within the specified range. In some embodiments, the drug loading is about 10% to about 80%. In some embodiments, the drug loading is about 20% to about 60%. In some embodiments, the drug loading is about 20% to about 50%. In some embodiments, the drug loading is about 20% to about 40%. In some embodiments, the drug loading is about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%.

[0040] In some embodiments, the drug is impregnated into the implantable film. In some embodiments, the drug loading in the implantable film is about 2% to about 90%, including each integer within the specified range. In some embodiments, the drug loading in the implantable film is about 10% to about 80%. In some embodiments, the drug loading in the implantable film is about 20% to about 60%. In some embodiments, the drug loading in the implantable film is about 20% to about 50%. In some embodiments, the drug loading in the implantable film is about 20% to about 40%. In some embodiments, the drug loading in the implantable film is about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%. In one embodiment, the drug loading in the implantable film is about 30%. In another embodiment, the drug loading in the implantable film is about 50%.

[0041] In some embodiments, the dosage form comprising the source of glutamine is stable for months or years. In some embodiments, the pharmaceutical dosage form of the source of glutamine described herein is stable for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 10 months, about 11 months, about 12 months, or even longer at 25° C. and 60% relative humidity (RH).

[0042] Some embodiments described herein are kits for preventing or reducing peripheral nerve fibrosis or adhesions in a subject. The kits described herein include a source of glutamine (e.g., L-alanyl-L-glutamine) in a suitable dosage form as described herein. The kits described herein further include instructions and a suitable administration schedule depending on the peripheral nerve to be treated. In one embodiment, the kit includes a dosage form that is delivered as an injectable material. In another embodiment, the dosage form includes a dosage form that is a film impregnated with the source of glutamine. In another embodiment, the kit includes a dosage form that is a topical cream. In another embodiment, the kit includes a dosage form suitable for iontophoretic delivery. In some embodiments, the kit includes multiple dosage forms for administration to a subject in need thereof. EXAMPLES

[0043] The study investigated the ability of L-alanyl-L-glutamine to regulate the HIF1α-mediated profibrotic response in hypoxic primary fibroblasts isolated and purified from rat sciatic nerves.

[0044] (Isolation and culture of rat fibroblasts) The local veterinary board and state veterinary office approved our study and animal protocols, including euthanasia, harvesting of the sciatic nerve, and further use for isolation of primary fibroblasts. Adult Sprague-Dawley rats were cultured at 4°C for 12 h at 20°C for 1 h at 20°C. 2The animals were euthanized using a gas chamber, and the sciatic nerves were immediately dissected under sterile conditions and placed in ice-cold Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich). Each adult sciatic nerve was cleaned of excess tissue, including muscle, fat, and blood vessels, while working under a Stereo dissection microscope (Carl Zeiss Miscroscopy GmbH, Germany). The epineurium was then peeled off using fine forceps. The nerve segments were subsequently dissected into small fragments (2-5 nm) and 5-6 segments were placed in 6-well plates supplemented with a medium consisting of Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS) (BI) and 1% penicillin / streptomycin (pen / strep) (Gibco) for 12-14 days. The medium was usually changed every 2-3 days. Primary fibroblasts were generated from neurons (Figure 1A), and when the cells approached confluency, tissue segments were removed from 6-well plates using forceps. Cells were washed twice with ice-cold PBS to remove tissue debris, trypsinized with 0.25% trypsin (Gibco), and subsequently centrifuged at 1800 rpm for 5 min. Cells were counted using trypan blue and plated in T25 / T75 culture flasks (Corning) until further experiments. Prior to experiments, all isolated cells were stained with α-SMA, HSP47, and Vimentin to confirm the phenotype of these cells (Figure 1B). All experiments with primary rat fibroblasts were performed from the first passage to the fifth passage.

[0045] (Hypoxia and L-alanyl-L-glutamine treatment) To mimic a hypoxic microenvironment, primary rat fibroblasts were exposed to hypoxia. Cells were exposed to two separate conditions: continuous hypoxia and transient hypoxia. For continuous hypoxia, cells were placed in a hypoxic chamber (2% O 2 ) for 48 hours and treated with or without L-alanyl-L-glutamine, as shown in Figure 1C, and not returned to normoxia. Cells were then harvested and processed for experiments. For transient hypoxia, cells were incubated in a hypoxic chamber (2% O 2 ) for 2 hours and then returned to normoxia for 48 hours, at which point the cells were treated with or without L-alanyl-L-glutamine as shown in Figure 1C. Cells were then harvested 48 hours later and processed for experiments.

[0046] (Proliferation assay) The proliferation of primary fibroblasts was determined by Alamar Blue assay (Invitrogen). Briefly, cells were plated in 96-well plates treated with or without L-alanyl-L-glutamine for 48 hours under hypoxic conditions as described above. The interaction of Alamar Blue with cells was analyzed for 4 hours by measuring the fluorescence at 540 nm and 585 nm, respectively. Proliferation was also detected using a flow cytometry-based EdU assay using EdU-Click488 (baseclick).

[0047] Cell death assay The effect of L-alanyl-L-glutamine on hypoxic fibroblast cell death was determined with Annexin V / 7-AAD (BD Pharmingen) using flow cytometry. For cell death assay, adherent fibroblast cells were cultured under hypoxic conditions and treated with or without L-alanyl-L-glutamine at various concentrations. Cells were harvested after 48 hours and washed and stained with Annexin V and 7-AAD and analyzed using flow cytometry.

[0048] (Western Blotting) Protein samples were prepared by resuspending cells growing in 6-well plates in RIPA lysis and extraction buffer (Thermo) containing protease and phosphatase inhibitors (Roche). Protein concentration was quantified using protein assay dye reagent (Bio-Rad). Samples were mixed with 4X LDS sample buffer (Thermo) and denatured by boiling at 95°C for 6 min. After estimation of protein concentration, equal protein samples containing 25 μg total protein were loaded and separated on a 4-15% mini protein TGX gel (Bio-Rad). Fractionated proteins were transferred to 0.2 μm nitrocellulose membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Membranes were blocked with 5% skim milk (Non-Fat dry milk) and incubated overnight with primary antibodies.

[0049] During the overnight incubation, the following primary antibodies were used at the respective concentrations: anti-collagen I (1:500; ab34710) and anti-collagen IV (1:500, ab6586) were purchased from Abcam. Anti-heat shock protein 47 (HSP-47) (1:400; sc-13150) and anti-fibronectin (1:500; sc-8422) were ordered from Santa Cruz Biotechnology. Other antibodies, including anti-SMAD2 / 3 (1:500; 85865S), anti-GAPDH (1:5000; 97166S) and anti-HIF-1α (1:750; 14179S), were obtained from Cell Signaling Technology. Horseradish peroxidase-labeled secondary antibodies were used at a concentration of 1:5000 for the immunoreaction, and protein bands were detected using Pierce™ ECL Plus Western Blotting Substrate (Thermo) and ChemiDoc Imaging Systems (Bio-Rad).

[0050] (Immunofluorescence staining) For immunofluorescence microscopy, cells were cultured in 4-well plate chamber slides (IBIDI) under appropriate hypoxic conditions with or without L-alanyl-L-glutamine treatment. After washing with ice-cold PBS, cells were fixed with 4% paraformaldehyde. Fixed cells were permeabilized with 0.5% Triton X-100 and incubated with blocking reagent (1% BSA in PBS) for 30 min, followed by overnight incubation with appropriate primary antibodies. After washing steps, the medium was labeled and incubated with secondary antibodies, and nuclei were stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI, Sigma).

[0051] Fluorescence microscopy image analysis was performed using a NIKON Ti2 inverted microscope equipped with a Photometrics Prime 95B camera for fluorescence imaging. All images were digitized and further refined for all quantification and presentation using NIS-Elements Advanced Research analysis software. For quantitative analysis, pictures of random fields of independent wells were taken with 10x and 40x magnification objectives, and the total protein expression of each marker was determined relative to the total number of cells based on DAPI quantification.

[0052] For immunofluorescence, primary antibodies and their respective concentrations are as follows: anti-collagen I (1:500; ab34710), anti-collagen IV (1:500; ab6586), and anti-α-SMA (1:500; ab124964) were purchased from Abcam. Anti-HSP-47 (1:250; sc-13150), anti-fibronectin (1:250; sc-8422), and anti-HIF-1α (1:500; sc-13515) were ordered from Santa Cruz Biotechnology. Anti-SMAD2 / 3 (1:500; 85865S) was obtained from Cell Signaling Technology. Additionally, the following secondary antibodies were obtained from Life Technologies: goat anti-mouse Alexa Flour (AF) 488 (A11029), goat anti-mouse AF546 (A11030), goat anti-rabbit AF488 (A11034), and goat anti-mouse AF546 (A11010). DAPI (D9564) from Sigma was used at a concentration of 1:1000.

[0053] (Sample preparation for phosphoproteomics analysis) After isolation of fibroblasts from rat sciatic nerves (8E10 primary fibroblasts), they were lysed by sonication (Bioruptor, 10 cycles, 30 sec on / off, Diagenode, Belgium) in 80 μL of 8 M urea, 0.1 M ammonium bicarbonate, phosphatase inhibitor (Sigma P5726&P0044), and proteins were digested as previously described (PMID:27345528). Briefly, proteins were reduced with 5 mM TCEP for 60 min at 37°C and alkylated with 10 mM chloroacetamide for 30 min at 37°C. After diluting the samples with 100 mM ammonium carbonate buffer to a final urea concentration of 1.6 M, proteins were digested by incubation with sequencing grade modified trypsin (1 / 50, w / w; Promega, Madison, Wisconsin) for 12 h at 37°C. After acidification with 5% TFA, peptides were desalted using C18 reverse-phase spin columns (Macrospin, Harvard Apparatus) according to the manufacturer's instructions, dried under vacuum, and stored at -20°C until further use.

[0054] Peptide samples were enriched for phosphopeptides using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform as described recently (PMID: 28107008). Phosphoenriched peptides were resuspended in 0.1% aqueous formic acid and subjected to LC-MS / MS analysis using an EASY-nLC1000 and a Q-Exactive plus mass spectrometer (both Thermo Fisher Scientific) equipped with a custom-made column set at 60°C. Peptides were separated using a home-made RP-HPLC column (75 μm × 30 cm) packed with C18 resin (ReproSil-Pur C18-AQ, 1.9 μm resin; Dr. Maisch GmbH) at a flow rate of 0.2 μL / min. The following gradient was used for peptide separation: 5% B to 8% B in 5 min, 20% B in 45 min, 25% B in 15 min, 30% B in 10 min, 35% B in 7 min, 42% B in 5 min, 50% B in 3 min, 95% B in 2 min, then 95% B for 18 min. Buffer A was 0.1% formic acid in water and buffer B was 80% acetonitrile, 0.1% formic acid in water.

[0055] Mass spectrometry was performed on a Q-Exactive plus mass spectrometer equipped with a nanoelectrospray ion source (both Thermo Fisher Scientific). Each MS1 scan was followed by high-collision-dissociation (HCD) of the 10 most abundant precursor ions for 20 seconds with dynamic exclusion. The total cycle time was approximately 1 second. In MS1, 3e6 ions were accumulated in the Orbitrap cell for a maximum time of 100 ms and scanned at a resolution of 70,000 FWHM (200 m / z). MS2 scans were acquired with a target setting of 1e5 ions, an accumulation time of 50 ms and a resolution of 17,500 FWHM (200 m / z). Singly charged ions and ions with unassigned charge states were excluded from triggering MS2 events. The normalized collision energy was set to 27%, the mass isolation window was set to 1.4 m / z, and microscans were acquired for each spectrum.

[0056] The obtained raw files were imported into Progenesis QI software (v2.0, Nonlinear Dynamics Limited) and peptide precursor ion intensities of all samples were extracted using default parameters. The generated mgf-files were searched using MASCOT against normal and reversed sequences of predicted (Proteomes) Uniprot entries of Rattus norvegicus (www.uniprot.org, release date 2020 / 03 / 10) generated using the SequenceReverser tool of MaxQuant software (Version 1.0.13.13), as well as a decoy database containing commonly observed contaminants (60,690 sequences in total). The search criteria were as follows: complete trypsin specificity was required (cleavage after lysine or arginine residues, except when followed by proline); three missed cleavages were allowed; carbamidomethylation (C) was set as a fixed modification; oxidation (M) and phosphorylation (STY) were applied as variable modifications; and mass tolerances of 10 ppm (precursor) and 0.02 Da (fragment) were set. Database search results were filtered using ion scores based on the number of reverse protein sequence hits in the dataset, with a false discovery rate (FDR) set at 1% at the peptide and protein levels, respectively. Results of quantitative analysis from label-free quantification were processed using the SafeQuant R package v.2.3.2. (PMID: 27345528, https: / / github.com / eahrne / SafeQuant / ) to obtain peptide relative amounts. The analysis involved global data normalization by equalizing total peak / reporter areas across all LC-MS runs, data imputation using the knn algorithm, summing peak areas per LC-MS / MS run, and then calculating peptide abundance ratios. Only isoform-specific peptide ion signals were included for quantification. Summary peptide expression values ​​were used for statistical testing of differentially abundant peptides between conditions.Here, empirical Bayes adjusted t-tests were applied as implemented in the R / Bioconductor limma package (http: / / bioconductor.org / packages / release / bioc / html / limma.html). The resulting comparison p-values ​​for each protein and condition were adjusted for multiple testing using the Benjamini-Hochberg method.

[0057] (statistical analysis) Statistical analysis was performed using Prism 7.0 software. Experimental data and graph plots are expressed as the mean ± standard deviation of a particular experiment from at least three independent experiments with three biological replicates, unless otherwise stated. Statistical significance of various conditions was characterized by Student's t-test or one-way ANOVA. For multiple comparisons, Sidak's multiple comparison test was used, and a P value of <0.05 was considered statistically significant. Statistically significant values ​​were expressed as the degree of significance, * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001).

[0058] (result) L-Alanyl-L-glutamine does not affect cell proliferation and viability of hypoxic primary fibroblasts Since hypoxia is the major microenvironment in fibrotic tissues, all in vitro analyses were performed with fibroblasts derived from the perineurium of primary rats exposed to hypoxic conditions. To investigate whether L-alanyl-L-glutamine treatment affects primary rat fibroblasts, the viability was investigated for 48 h using the Alamar Blue assay. In vitro analyses showed that L-alanyl-L-glutamine did not affect the viability and cell proliferation of hypoxic primary rat fibroblasts. None of the concentrations, including 1 mM, 10 mM, and 100 mM, caused any significant changes in cell viability and cell proliferation. As shown in Figures 2A-2D, compared to untreated controls, primary rat fibroblasts exposed to continuous (48 h) and transient (2 h) hypoxia showed no changes in cell proliferation when treated with L-alanyl-L-glutamine at various concentrations.

[0059] L-Alanyl-L-glutamine downregulates fibrous protein expression in primary fibroblasts exposed to continuous hypoxia Hypoxia increases the expression of profibrotic and adhesion biomarkers in fibrotic diseases. To investigate whether L-alanyl-L-glutamine induces any changes in the hypoxic microenvironment, the expression of HIF1-α was examined under the treatment of various concentrations of L-alanyl-L-glutamine. As shown in Figure 3A, Western blotting of whole cell lysates of hypoxic primary fibroblasts showed that L-alanyl-L-glutamine significantly downregulated markers involved in the induction of hypoxic microenvironment, HIF1-α and SMAD2 / 3. HIF1-α was significantly downregulated upon treatment with L-alanyl-L-glutamine at concentrations of 1 mM, 10 mM, and 100 mM. SMAD2 / 3 was also significantly downregulated only at 100 mM L-alanyl-L-glutamine in hypoxic primary fibroblasts, as shown in Figure 3A.

[0060] In addition, we further investigated the effect of L-alanyl-L-glutamine on other profibrotic factors. We observed that continuous hypoxia for 48 h induced the expression of profibrotic markers including collagen-IV, collagen-I, and fibronectin, compared with normoxic untreated controls. As shown in Figure 3B, treatment with 1 mM, 10 mM, and 100 mM L-alanyl-L-glutamine significantly reduced the expression of these fibrotic proteins in a dose-dependent manner. Since HYP47 is another important profibrotic factor responsible for collagen biosynthesis, we tested our hypothesis about HSP47, and as shown in Figure 3B, L-alanyl-L-glutamine reduced the expression of HSP47, but no significant downregulation was observed.

[0061] Immunofluorescence analysis of hypoxic primary fibroblasts treated with various concentrations of L-alanyl-L-glutamine or without L-alanyl-L-glutamine was also performed. In vitro immunofluorescence analysis revealed that L-alanyl-L-glutamine treatment resulted in a significant decrease in factors responsible for the cellular response to hypoxia, including HIF1-α and SMAD2 / 3, as shown in Figures 4A, 4B, 4C, and 9A. HIF1-α and SMAD2 / 3 were stained in the nucleus, and both were downregulated upon treatment with L-alanyl-L-glutamine.

[0062] Cytoplasmic staining of profibrotic factors including fibronectin, collagen-IV, collagen-I and HSP47 was also performed. As shown in Figures 4A, 4B, 4D, 4E and 9B, these targets are accumulated in hypoxic primary fibroblasts and correspondingly significantly downregulated in L-alanyl-L-glutamine-treated fibroblasts. Our statistical analysis revealed a dose-dependent downregulation of HIF1-α, SMAD2 / 3, collagen-IV and HSP47 in L-alanyl-L-glutamine-treated fibroblasts (Figures 4A, 4C, 4D and 4E), whereas fibronectin and collagen-I were significantly decreased in a dose-independent manner (Figures 4A and 4B). Further experiments to understand this regulation need to be performed to understand this behavior of L-alanyl-L-glutamine decreasing factors involved in collagen biosynthesis.

[0063] L-Alanyl-L-glutamine downregulates fibrous protein expression in primary fibroblasts exposed to transient hypoxia Overexpression of HIF1-α in fibrotic tissues results in chronic or acute hypoxic microenvironments. The effect of L-alanyl-L-glutamine on profibrotic factors in primary rat fibroblasts under transient hypoxia was examined. The effect of L-alanyl-L-glutamine in hypoxic primary fibroblasts exposed to transient hypoxia for 2 h and then treated with or without L-alanyl-L-glutamine for 48 h was investigated.

[0064] Intracellular staining of specific targets responsible for fibrogenic potential as well as proteins regulated by HIF1-α signaling was performed. Immunofluorescence analysis showed that L-alanyl-L-glutamine effectively reduced the expression of profibrotic proteins including fibronectin, collagen-I, collagen-IV and HSP47, as shown in Figures 5A, 5B, 5D, 5E and 10B. A significant downregulation of downstream targets of HIF1-α signaling pathway, namely HIF1-α and SMAD2 / 3 in L-alanyl-L-glutamine, was also observed as shown in Figures 5A, 5B, 5C and 10A. After immunocytochemistry, the average intensity per cell was calculated for these proteins in L-alanyl-L-glutamine treated samples and compared with untreated hypoxic samples. L-alanyl-L-glutamine significantly reduced the expression of adhesion and fibrotic proteins in a dose-dependent manner. All the above factors as shown in Figures 5B-5E were significantly increased in hypoxic samples (Figures 5B-5E). We observed a dose-dependent decrease in the expression of all proteins except collagen-I, which was significantly decreased at 1 mM L-alanyl-L-glutamine but remained constant thereafter (Figures 5A, 5B; Supplementary Note 3B).

[0065] Western blotting of total cell lysates of transiently hypoxic primary fibroblasts was also performed. In contrast to immunocytochemistry, Western analysis did not show any dose-dependent regulation of the desired targets. HIF1-α signaling factors and profibrotic targets were significantly increased in hypoxic samples compared to L-alanyl-L-glutamine-treated samples. As shown in Figure 6B, there was no significant downregulation across various concentrations of L-alanyl-L-glutamine. Interestingly, in hypoxic fibroblasts treated with 1 mM L-alanyl-L-glutamine shown in Figure 6, we found a significant downregulation of COL-IV, which was slightly different from the immunofluorescence analysis, as shown in Figure 5E.

[0066] Identification of key proteins and pathway analysis in hypoxic fibroblasts treated with L-alanyl-L-glutamine This study is the first attempt to compile a phosphoproteome profile of L-alanyl-L-glutamine treated under chronic hypoxic conditions treated with different L-alanyl-L-glutamine concentrations. A total of 5110 proteins were profiled in all four conditions (0 mM, 1 mM, 10 mM, and 100 mM). 1322 proteins were found to be significantly regulated between treatment groups (1 mM, 10 mM, and 100 mM) when compared to the 0 mM hypoxic sample. Of these 1322 proteins, 607 proteins were significantly downregulated and 715 were significantly upregulated in L-alanyl-L-glutamine. Biological triplicates of each condition were used, and a pooled analysis of all biological triplicates was performed between the treatment groups and the untreated 0 mM condition. Positive values ​​of the log_2 ratio indicate upregulation of the relevant proteins in the L-alanyl-L-glutamine treated group, while negative values ​​indicate downregulation. Further analysis to identify significant factors and associated pathways was performed using DAVID statistical software and KEGG pathway analysis as previously described. The analysis focused on downregulated targets and identified proteins and associated pathways that were restricted by L-alanyl-L-glutamine and reduced the fibrotic phenotype. Pathway analysis showed that the significantly downregulated proteins in the treated group were associated with mTOR, PI3k, cell adhesion, and focal adhesion.

[0067] As shown in Figures 7A-7C, L-alanyl-L-glutamine effectively down-regulated key factors involved in cell adhesion and fibrosis progression, and we were also able to identify a dose-dependent effect of L-alanyl-L-glutamine on the expression of these factors. Several targets related to HIF1-α, ECM-interaction, ERBb, FOXO and TGF-β signaling pathways were also found. Importantly, as shown in Figure 8, key profibrotic factors including Fndc1, Fndc3b, Col1a1 and Akt1s1, as well as Pxn, were significantly down-regulated in L-alanyl-L-glutamine-treated samples compared to untreated chronic hypoxia samples, consistent with the in-vitro findings by Western blotting and immunofluorescence imaging.

[0068] Furthermore, protein analysis found that they were significantly upregulated in primary fibroblasts treated with L-alanyl-L-glutamine. Pathway analysis indicated that these proteins were associated with the upregulation of GnRH signaling, glutamatergic guidance signaling, and axon guidance signaling. Figures 7A-7C summarize the findings of the upregulated factors.

[0069] While particular embodiments of the invention have been illustrated and described, it will be apparent that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the invention, as set forth in the claims that follow.

Claims

1. 1. A composition for treating or preventing peripheral nerve fibrosis in a subject, the composition comprising a source of glutamine, and the treating or preventing comprising administering the composition to one or more peripheral nerves or one or more tissues surrounding one or more peripheral nerves, and administering the composition before, during or after surgery involving one or more peripheral nerves.

2. The composition of claim 1, wherein the treatment or prevention comprises administering one or more additional doses of a glutamine source to the subject for a specified period of time after an initial administration, at certain time intervals after the initial administration, and / or immediately prior to closure of any surgical incisions.

3. The composition of claim 1, wherein the treatment or prevention comprises administering the composition during surgery involving one or more peripheral nerves.

4. The method of claim 3 , wherein the surgical procedure affects the abdominal cavity of the subject.

5. The method of claim 3 , wherein the surgical procedure affects the subject's thoracic cavity.

6. The method of claim 3 , wherein the surgical procedure affects the subject's head, neck, or spine.

7. The method of claim 3 , wherein the surgical procedure affects one or more limbs of the subject.

8. 2. The composition of claim 1, wherein the source of glutamine is an oligopeptide containing glutamine.

9. The composition of claim 8 , wherein the oligopeptide is a dipeptide.

10. The composition of claim 9 , wherein the dipeptide is L-alanyl-L-glutamine.

11. 2. The composition of claim 1, wherein the source of glutamine comprises L-glutamine.

12. 2. The composition of claim 1, wherein the source of glutamine comprises D-glutamine.

13. 13. The composition of claim 1 for use in combination with the source of glutamine and one or more additional active pharmaceutical ingredients.

14. The composition of claim 1, wherein the treatment or prevention comprises administering the composition by injection, topical administration, transdermal administration, or iontophoresis.

15. A composition for treating or preventing adhesions in one or more peripheral nerves in a subject undergoing a surgical procedure, the composition comprising a source of glutamine, and the treating or preventing comprising administering the composition to a peripheral nerve or tissue surrounding one or more peripheral nerves, and administering the composition before, during or after surgery involving one or more peripheral nerves.

16. 16. The composition of claim 15, wherein the source of glutamine is an oligopeptide containing glutamine.

17. The composition of claim 16 , wherein the oligopeptide is a dipeptide.

18. 18. The composition of claim 17, wherein the dipeptide is L-alanyl-L-glutamine.

19. A composition for treating or preventing hypoxia-related damage to one or more peripheral nerves in a subject, the composition comprising a therapeutically effective amount of a glutamine source for tissues that are subject to hypoxia-related tissue damage, the treating or preventing comprising administering the composition to a subject during a surgical procedure, and administering the composition before, during or after surgery involving one or more peripheral nerves.

20. The composition of claim 19 , wherein the surgical procedure affects the abdominal cavity of the subject.

21. 20. The composition of claim 19, wherein the surgical procedure affects the subject's thoracic cavity.

22. 20. The method of claim 19, wherein the surgical procedure affects the subject's head, neck, or spine.

23. 20. The method of claim 19, wherein the surgical procedure affects one or more limbs of the subject.

24. 20. The composition of claim 19, wherein the glutamine in the glutamine source is L-glutamine.

25. 20. The composition of claim 19, wherein the glutamine in the glutamine source is D-glutamine.

26. A composition for a treatment to prophylactically reduce the incidence of fibrosis in one or more peripheral nerves of a subject, the composition comprising an effective amount of glutamine for administration to one or more peripheral nerves or one or more tissues surrounding a peripheral nerve, the treatment comprising administering the composition to one or more peripheral nerves or one or more tissues surrounding a peripheral nerve.

27. A composition for treatment to reduce HIF-1α in peripheral nerves that have suffered hypoxia-related damage, the composition comprising a source of glutamine, the treatment comprising administering the composition to one or more peripheral nerves or one or more tissues surrounding one or more peripheral nerves, and administering the composition before, during, or after surgery involving one or more peripheral nerves.

28. 1. A kit for treating or alleviating peripheral nerve adhesions or fibrosis in a subject comprising an effective amount of a source of glutamine, wherein the source of glutamine is formulated in a dosage form selected from the group consisting of a surgically implantable film, an injectable material, a topical cream, and a dosage form suitable for iontophoresis.

29. 30. The kit of claim 28, further comprising a pre-filled syringe containing the source of glutamine and a biologically acceptable carrier.

30. 30. The kit of claim 28, further comprising a surgical film impregnated with the source of glutamine.

31. 30. The kit of claim 28, wherein the source of glutamine is from about 10% to about 50% by weight.

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