EP4731230A1 - Bicarbonate for wound healing - Google Patents

Abstract

A method of treating a wound is provided comprising the administration of a therapeutically effective amount of a bicarbonate to a wound. Bicarbonate has been found to facilitate wound healing by promoting at least one of cell proliferation, collagen production and/or fibronectin production, or treating undesirable inflammation, within a wound.

Description

BICARBONATE FOR WOUND HEALING

Field of the Invention

[0001 ] The present invention relates to wound healing, and in particular, to compositions and methods useful for wound healing.

Background

[0002] Wound management in patients remains a challenge for clinicians. Overall, in the United States, ~2% of the total population are estimated to be affected by chronic wounds. Nonhealing wounds, when left untreated and improperly managed, can result in significant medical issues, including infection, sepsis, amputation, and even death.

[0003] The wound healing process involves a number of stages which are required in order to achieve healing in an orderly and well-timed manner. Ultimately, the goal is to restore dermo-epidermal integrity and function. The four stages of the healing process include hemostasis, inflammation, proliferation, and remodeling, and all four phases must occur in the proper sequence. Wounds that exhibit impaired healing, including delayed acute wounds and chronic wounds, have generally failed to progress through the normal stages of healing.

[0004] Hemostasis involves vasoconstriction, platelet activation, and formation of a fibrin clot within the wound which is a temporary matrix that supports cell migration. Next, inflammation occurs which is characterized by the sequential infiltration into the wound of neutrophils, macrophages, and lymphocytes. Neutrophils play a role in the clearance of invading microbes and cellular debris from the wound area. Macrophages release cytokines that promote the inflammatory response by recruiting and activating additional leukocytes and are also responsible for inducing and clearing apoptotic cells, paving the way for the resolution of inflammation. Lymphocytes migrate into the wound following the inflammatory cells and macrophages, and peak during the late-proliferative/early-remodeling phase. The next stage of the healing process is cell proliferation which involves re-epithelialization, angiogenesis and granulation tissue formation, the second temporary matrix formed which is rich in fibroblasts and macrophages. At this stage, the fibroblasts synthesize collagen and myofibroblasts initiate the process of wound edge contraction. Following robust proliferation and synthesis of the extracellular matrix, remodelling and maturation are the final processes of wound healing in which the temporary matrix is replaced with the final epithelial matrix, organized and rich in mature collagen.

[0001] In lung infections, such as those seen in cystic fibrosis (CF) and other lung diseases, chronic inflammation is a critical factor driving tissue damage. In CF, thick, sticky mucus accumulates in the lungs, creating an ideal environment for persistent bacterial infections. These infections trigger ongoing inflammatory responses, which are initially meant to contain infection and promote repair, much like inflammation around a wound. However, when inflammation becomes chronic, it can severely disrupt healing, damaging healthy tissues and preventing effective repair. Neutrophils and other immune cells continuously release enzymes and inflammatory molecules to fight infection, but in doing so, they also harm surrounding lung tissue. This resembles a poorly healing wound in which ongoing inflammation and infection prevent closure and recovery, leading to scarring or tissue degradation instead of regeneration. Each cycle of infection and inflammation in the lungs of a CF patient gradually erodes lung function, as damaged tissue is replaced by scar tissue or fibrotic areas that lack elasticity. This progressive loss of functional lung tissue can lead to respiratory failure over time. Targeted therapies that control inflammation could help prevent this cycle, preserving lung tissue and slowing disease progression. Anti-inflammatory therapies hold promise for maintaining lung function and improving quality of life for individuals with CF and other respiratory diseases.

[0005] Given the prevalence of chronic wounds, it would be desirable to develop a novel treatment that is effective to facilitate the healing of wounds.

Summary

[0006] It has now been determined that bicarbonate facilitates wound healing by stimulating cell proliferation, growth and differentiation.

[0007] Thus, in one aspect of the invention, a method of treating a wound is provided comprising the administration of a therapeutically effective amount of a bicarbonate to a wound. [0008] In another aspect, a method of promoting at least one of cell proliferation, collagen production and/or fibronectin production within a wound is provided, comprising administering to a wound an effective amount of a bicarbonate.

[0009] In a further aspect, use of a bicarbonate to promote at least one of cell proliferation, collagen production and/or fibronectin wound repair within a wound is provided.

[0010] A composition comprising bicarbonate together with a pharmaceutically acceptable carrier or adjuvant is provided in another aspect.

[0011] In another embodiment, a wound dressing or biocompatible matrix coated with or impregnated with bicarbonate is provided.

[0012] In a further embodiment, a kit comprising an inhaler and bicarbonate is provided.

[0013] These and other aspects of the invention are described herein in the detailed description that follows by reference to the following figures.

Brief Description of the Figures

[0014] Figure 1 illustrates the results of an in vitro scratch assay conducted on human primary fibroblasts treated with A) control buffer, HEPES, and B) bicarbonate treatment at varying doses. Images were taken at 24 and 48 hours after wounding.

[0015] Figure 2 is a schematic of the biopsy punch model.

[0016] Figure 3 provides images showing wound measurements of A) untreated wounds and B) bicarbonate-treated wounds on day 8 following wounding by biopsy punching.

[0017] Figure 4 provides histology results showing bicarbonate treatment led to enhanced healing of wounds as evidenced by: A) hematoxylin-eosin staining; B) enhanced collagen production as evidenced by trichrome staining with trichrome; C) enhanced proliferation as evidenced by higher levels of CD31; D) enhanced levels of proliferation as determined by marker by Ki67; E) reduced inflammation as determined by IL-1 marker; and F) increased fibronectin production. [0018] Figure 5 provides histology results comparing A) vehicle control treatment and

B) bicarbonate treatment of damaged lung tissue in which the bicarbonate treatment led to reduced inflammation as determined by IL-1 marker.

Detailed Description of the Invention

[0019] A method of wound treatment is provided comprising administration to a wound of a therapeutically effective amount of a bicarbonate. The method has been determined to promote at least one of cell proliferation, collagen production and/or fibronectin production, or reduction of inflammation, within a wound.

[0020] The term "wound" is used herein to refer to any injury of mammalian tissue

Tissue is defined herein as an assembly of similar cells and their extracellular matrix from the same embryonic origin that together carry out a specific function. Types of tissue include connective, muscle, nervous, and epithelial. Tissue wounds, thus, include both external and internal wounds. Tissue wounds may be open or closed wounds, resulting from an incision, cut, laceration, abrasion, puncture, penetration, blunt force trauma, burn, chemical exposure, inflammation, disease or other causes. Wounds to mucosal surfaces such as oral, ocular and vaginal surfaces are encompassed, as well as chronic wounds associated with connective, muscle, nervous or epithelial tissue including skin ulcers such as pressure, arterial, venous and diabetic ulcers, and damage to the tissue of an organ such as, but not limited to, the lung, heart, liver, kidney and pancreas that results in scar tissue or fibrotic areas (for example, that may result from disease such as respiratory disease, e.g. cystic fibrosis or chronic obstructive pulmonary disease (COPD), liver disease such as cirrhosis of the liver, chronic pancreatitis, and nephritis). The wound may or may not result from an infection by a microorganism or may or may not be characterized by the prsence of a microorganism.

[0021] The term "treat” or “treatment” as used herein with respect to a wound refers to the amelioration or healing of a wound. Wound healing may be measured based on parameters such as lesion size, granulation tissue, inflammation, inflammatory mediators such as IL-1, mineralization, scab formation, tissue regeneration, collagen and/or fibronectin production depth of lesion, structural and functional integrity and re-epithelialization, and thus may be evident by the extent of improvement in one or more of these parameters, including the extent of wound closure. Thus, an improvement in one or more of these parameters of at least about 10%, or wound closure of at least about 10%, is indicative of wound healing. The term “improvement” refers to a change in a given parameter that is indicative of wound healing, e.g. a reduction in lesion size, inflammation, inflammatory mediators or depth of lesion, or an increase in granulation tissue, mineralization, tissue regeneration, structural and functional integrity, re-epithelialization, scab formation, collagen production or fibronectin production. A cumulative score of these parameters visually observed (wherein 0 is healthy, 1 is mild, 2 is moderate and 3 is severe) may also be used to determine wound healing. Thus, a decrease in the cumulative score of these parameters in a wound is indicative of wound healing, e.g. a decrease in the cumulative score of about 10%, preferably 20% and more preferably 30% or greater, is indicative of wound healing.

[0022] The term "mammal" is used herein to encompass both human and non-human mammals such as cats, dogs, rodents, horses, cows, goats, sheep, pigs and the like.

[0023] The term “bicarbonate” as used herein refers to a compound of the formula

XHCO3, wherein X is a suitable cation. In some embodiments, “bicarbonate” refers to the anion HCO3- together with a cation. In some embodiments, the cation is an alkali metal cation. For example, the cation may be sodium, lithium or potassium. In some embodiments, the cation is an alkaline earth metal cation. For example, the cation may be magnesium or calcium. In other embodiments, the cation is ammonium or zinc. Thus, bicarbonate as used herein may be sodium bicarbonate, lithium bicarbonate, potassium bicarbonate, magnesium bicarbonate, calcium bicarbonate, ammonium bicarbonate or zinc bicarbonate.

[0024] The present method comprises administration of a therapeutically effective amount of a bicarbonate to a wound. The term "therapeutically effective" as it is used herein refers to a dosage of bicarbonate that is effective to treat a given wound without causing unacceptable adverse side effects. The term “administered” refers to any appropriate means of providing the bicarbonate to a wound to be treated. For example, the bicarbonate may be administered parenterally by injection or infusion, mucosally or topically, including via inhalation or intranasal administration, as will be described in more detail.

[0025] Therapeutically effective dosages according to the present method will generally be an amount sufficient to yield an improvement in at least one parameter of the wound such as lesion size, depth of the lesion, granulation tissue, inflammation, mineralization, scab formation, collagen and/or fibronectin production and wound closure. In one embodiment, a dosage of bicarbonate in the range of about 1-900 mM is effective to treat a wound. In some embodiments, the composition comprises bicarbonate at a concentration of about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM or about 150 mM. In some embodiments, the composition comprises bicarbonate at a concentration of greater than 150 mM, such as about 175 mM to about 900 mM. In some embodiments, the composition comprises bicarbonate at a concentration of about 175 mM to about 225 mM, about 200 mM to about 300 mM, about 300 mM to about 400 mM, about 400 mM to about 500 mM, about 500 mM to about 600 mM, about 600 mM to about 700 mM, about 700 mM to about 800 mM or about 800 mM to about 900 mM. In other embodiments, a dosage of bicarbonate is used that provides a concentration that is greater than a physiological concentration. Thus, the dosage or amount of the bicarbonate is greater than about 25 mM of bicarbonate, for example, about 30 mM to about 500 mM. For example, the bicarbonate dosage may be about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 120 mM, about 130 - 150 mM, about 175 mM to about 225 mM, about 200 mM to about 300 mM, about 300 mM to about 400 mM, or about 400 mM to about 500 mM. Where the composition comprises bicarbonate at a particular M or mM concentration, the M or mM concentration is moles or millimoles, respectively, of bicarbonate per liter of water.

[0026] In other embodiments, a dosage of bicarbonate in the range of about 0.01 wt% to about 10 wt% of a composition is effective to treat a wound. In some embodiments, the composition comprises bicarbonate in an amount of about 0.01 wt% to about 1.0 wt%, or about 0.20 wt% to about 0.5 wt% of the composition. In other embodiments, the composition comprises bicarbonate in a composition in an amount of about 1.0 wt% to about 8.4 wt% of the composition.

[0027] The bicarbonate may be administered in the treatment of a wound alone or in a composition combined with a pharmaceutically acceptable adjuvant or carrier. The expression "pharmaceutically acceptable" means acceptable for use in the pharmaceutical arts, i.e. not being unacceptably toxic, or otherwise unsuitable for administration to a mammal. Examples of pharmaceutically acceptable adjuvants include, but are not limited to, diluents, excipients and the like. Reference may be made to "Remington's: The Science and Practice of Pharmacy", 21st Ed., Lippincott Williams & Wilkins, 2005, for guidance on drug formulations generally. The selection of adjuvant depends on the intended mode of administration of the composition. In one embodiment of the invention, the compounds are formulated for administration by infusion, or by injection either subcutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic. Thus, the compounds may be administered in distilled water or, more desirably, in saline, phosphate-buffered saline or 5% dextrose solution. In another embodiment, the bicarbonate is formulated for application topically as a cream, lotion or ointment. For such topical application, the bicarbonate is combined with an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent and other cosmetic additives such as skin softeners and the like as well as fragrance. Aerosol formulations for application to a wound may also be prepared in which suitable propellant adjuvants are used. Compositions of the present invention may also be administered as a paste. Compositions for mucosal administration are also encompassed, including oral, nasal, rectal or vaginal administration for the treatment of wounds in these areas. Such compositions generally include one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax, a salicylate or other suitable carriers. Other adjuvants may also be added to the composition regardless of how it is to be administered which, for example, may aid to extend the shelf-life thereof.

[0028] In particular embodiments, bicarbonate may be topically applied to a wound via a biocompatible wound dressing such as a film, gauze, bandage, foam, hydrocolloid or hydrogel. Thus, in another aspect, such a wound dressing is provided comprising bicarbonate applied thereto, or is provided in kit form comprising the wound dressing and the bicarbonate not applied to the wound dressing. An example of a suitable film dressing is a polyurethane film. Suitable foam dressings include polyurethane or silicone foams. Examples of suitable hydrogels include, but are not limited to polyvinyl alcohol, polysaccharide-based natural hydrogels such as chitosan, collagen, alginate, dextran, and hyaluronic acid, gelatin-based hydrogels, and agar. Modified hydrogels may also be used such as bioadhesive hydrogels modified with a polyphenol derived moiety such as catechol, dopamine, gallic acid, or tannic acid, methacrylate-modified hydrogels, or blended hydrogels to provide a hydrogel with desired biodegradation properties or a hydrogel which enhances granulation, migration or neoangiogenesis during wound healing.

[0029] A suitable matrix or polymer mesh, e.g. artificial or non-artificial skin grafts, may alternatively be impregnated with bicarbonate for application to a wound to permit slow-release of the bicarbonate for continuous treatment of the wound over a period of time.

[0030] In another embodiment, a kit comprising an inhaler and bicarbonate is provided for use to deliver bicarbonate to the lungs of a patient. The inhaler includes any suitable inhaler selected from a dry powder inhaler, a metered dose inhaler, a mist inhaler and a nebulizer. The bicarbonate may be provided in powder form, or in solution or suspension form suitable for use with the selected inhaler.

[0031] The present method results in promotion of at least one of cell proliferation, collagen production and/or fibronectin production within a wound. Cell proliferation refers to the combination of cell growth and division to produce cells of uniform size within a population. These parameters of wound repair may be measured using various established methodologies. For example, cell proliferation may be identified by hematoxylin-eosin staining. Cell proliferation may also be identified by measuring the levels of biomarkers such as CD31 and Ki67.

[0032] Collagen helps to stimulate new tissue growth, while encouraging autolytic debridement, angiogenesis and re-epithelialization. Collagen production may be determined by staining with trichrome. In a standard Masson's Trichrome procedure, collagen is stained blue, nuclei are stained dark brown, muscle tissue is stained red, and cytoplasm is stained pink.

[0033] Fibronectin is an important factor during wound healing. It promotes the spreading of platelets at the site of injury, the adhesion and migration of neutrophils, monocytes, fibroblasts, and endothelial cells into the wound region, and the migration of epidermal cells through the granulation tissue. Fibronectin may be detected in a wound by immunohistochemical staining pattern. [0034] The present method may also result in a reduction of undesirable inflammation within a wound, e.g. chronic inflammation, which disrupts the healing process and damages healthy tissues preventing or hampering effective wound repair.

[0035] In another aspect, the bicarbonate may be administered to a mammal in need of wound treatment in combination with one or more additional therapeutic agents, including for example, a wound healing agent such as a growth factor, e.g. epidermal growth factor, bFCF, PDGF; platelets, dermal fibroblasts and keratinocytes. In this regard, the bicarbonate may be administered to a mammal in the treatment of a wound either individually in separate formulations, simultaneously or at different times, or together in a combined formulation.

[0036] Bicarbonate may be further utilized in a combination therapy in which laser therapy, for example, is applied to the wound site with repeated applications of bicarbonate to effect wound healing.

[0037] Embodiments of the invention are described in the following specific example which is not to be construed as limiting.

Example 1

[0038] The efficacy of bicarbonate to heal wounds was tested using an in vitro assay that reports on cell migration as a proxy for wound healing, as well as utilizing both in vivo animal bum and skin-punch biopsy models.

[0039] In vitro wound scratch assay - The scratch assay is typically utilized to quantify cellular migration on two-dimensional surfaces over time upon different treatments. It is one of the most commonly used in vitro wound-healing assays. Human primary fibroblasts were grown to confluency in a monolayer, and a scratch was made with a pipette tip to create an incision-like “wounded” gap. The ‘wounded’ area was photographed immediately after wounding and at 24 and 48 hours thereafter, and cell migration was observed as representative of closure/healing of the scratch area. The cells were treated with bicarbonate (25 mM solution of sodium bicarbonate). HEPES buffer was used as a control. Treatment with bicarbonate induced fibroblast cell migration in a time-dependent manner observed as increased scratch closure compared to the control. [0040] Excisional wound model using a biopsy punch - Mice were anesthetized with 4% isofluorane, and eye lubricant was applied, nails were trimmed, and blood glucose was measured using a OneTouch Verio Reflect (the tail of a mouse was pricked with a lancet to draw blood). The dorsal surface of the mouse was shaved, and the area was treated with an ethanol wipe. Mice were rotated on their side, and their skin was pinched along their midline. The skin was folded over a metal surface, and a 4 mm biopsy punch was created. Immediately after, 10 pL of treatment was applied to each wound (either solvent control (sterile water) or 50 mM sodium bicarbonate. Mice recovered on a heating pad until they were alert and singly caged. Treatment was applied every 24 hours. Fig. 2 provides a schematic of the protocol used.

[0041] Wound measurements were determined on day 8 following treatment. As shown in Fig. 3, bicarbonate-treated wounds exhibited more complete healing based on wound closure as indicated by wound measurements than the untreated controls.

[0042] Histological analysis - Prior to being sacrificed, mice were anesthetized and vertically dislocated. Tissue was resected from mouse where injury happened. Tissue was cut in half and placed in histology cassettes. Cassettes were labeled and placed in formalin for 48 hrs. After 48 hrs, cassettes were moved to 70% ethanol.

[0043] Tissue was submitted for embedding and sections were cut and stained. Stains and markers used in the study included hematoxylin-eosin (allows visualisation of the structure, distribution of cells and morphological changes within a tissue sample); trichrome (allows visualization of connective tissues, particularly collagen); Ki-67 (marker of cell proliferation); CD31 (marker of endothelial cells); IL-1 (marker of inflammation); and fibronectin (stains extracellular matrix for tissue repair).

[0044] Histopathological evaluation (see images Figs. 4A-F) demonstrated that treatment with bicarbonate (50 mM) led to enhanced collagen production over vehicle control, as evidenced by staining with trichrome (Fig. 4B); enhanced proliferation as evidenced by higher levels of CD31 (Fig. 4C) which is a marker of endothelial cells; enhanced levels of proliferation as determined by marker by Ki67 (Fig. 4D), and reduced inflammation as compared to vehicle control as determined by IL-1 marker (Fig. 4E). Fibronectin shows more advanced tissue repair with bicarbonate treatment than with vehicle (Fig. 4F), and full wound healing is evidenced by Hematoxylin-Eosin staining (Fig. 4A).

[0045] In all, the beneficial effects of bicarbonate on wound healing is demonstrated.

Bicarbonate promotes more rapid healing and closure of wounds. This discovery highlights the power of formulating wound treatments with bicarbonate. Indeed, use of bicarbonate in wound healing products or alone improves therapeutic outcomes.

Example 2

[0046] A lung infection model was used to determine the efficacy of bicarbonate to heal a lung tissue wound associated with inflammation caused by bacterial infection.

[0047] Mice were infected intranasally with P. aeruginosa and treated intranasally with vehicle or 7.5% bicarbonate solution. Mice were sacrificed and the lungs removed and processed for histological analysis.

[0048] Histopathological evaluation of the lung tissue demonstrated that treatment with bicarbonate led to reduced inflammation (Fig. 5A) as compared to vehicle control (Fig. 5B) as determined by IL-1 marker.

Claims

1. A method of treating a wound in a mammal comprising the administration of a therapeutically effective amount of a bicarbonate to a wound.

2. The method of claim 1, wherein the bicarbonate is sodium bicarbonate, lithium bicarbonate, potassium bicarbonate, magnesium bicarbonate, calcium bicarbonate, ammonium bicarbonate or zinc bicarbonate.

3. The method of claim 1 or 2, wherein the bicarbonate is administered at a concentration of 1-900 mM.

4. The method of any one of claims 1-3, wherein the treatment results in a reduction of inflammation in the wound.

5. The method of any one of claims 1-4, wherein the treatment promotes at least one of cell proliferation, collagen production and/or fibronectin production within the wound.

6. The method of any one of claims 1-5, wherein the wound is an epithelial wound.

7. The method of any one of claims 1-6, wherein the wound is in organ tissue.

8. The method of claim 7, wherein the tissue is lung tissue.

9. The method of any one of claims 1 -8, wherein the wound is as a result of inflammation.

10. The method of claim 1, wherein the bicarbonate is administered topically.

11. The method of claim 1, wherein the bicarbonate is administered subcutaneously.

12. The method of claim 1, wherein the bicarbonate is administered intranasally or by inhalation.

13. The method of claim 11 or 12, wherein the bicarbonate is administered at a concentration of about 0.01 wt% to about 10 wt%.

14. The method of claim 1, wherein the mammal has a respiratory disease.

15. The method of claim 1, wherein the bicarbonate is administered to a lung.

16. A composition for wound treatment comprising a bicarbonate in combination with a pharmaceutically acceptable carrier.

17. The composition of claim 16, comprising a wound healing agent selected form epidermal growth factor, bFCF, PDGF; platelets, dermal fibroblasts and keratin ocytes.

18. Use of a composition as defined in claim 16 or 17 to treat a wound in a mammal.

19. The use of claim 18 in combination with a wound treating therapy.

20. The use of claim 18, wherein the wound is as a result of chronic inflammation.

21. A wound dressing or biocompatible matrix coated with or impregnated with bicarbonate or a composition as defined in claim 16 or 17.

22. A kit comprising an inhaler and bicarbonate.

23. The kit of claim 22, wherein the inhaler is selected from a dry powder inhaler, a metered dose inhaler, a mist inhaler and a nebulizer.