Composition for wound healing

Abstract

The present invention provides a use of a functional thylakoid extract, particularly in combination with a physiologically acceptable carrier, for medical purposes in wound healing. The thylakoid extract can be used to treat wounds in the skin or mucosa, such as open wounds or wounds caused by physical trauma. The thylakoid extract can be used to increase the production or level of GM-CSF growth factor and / or G-CSF growth factor in the skin or mucosa of a subject after wounding (e.g., wounds in the skin, mucosa, or corneal epithelium).

Description

[Technical Field]

[0001] The present invention relates to compositions comprising functional thylakoids, particularly compositions comprising functional thylakoids in specific formulations that ensure the integrity, stability and functionality of the thylakoids (i.e., functional thylakoid extracts), and methods of use for wound healing. [Background technology]

[0002] U.S. Patent No. 5,629,999 discloses antioxidant compositions and methods for their extraction. U.S. Patent No. 5,629,999 discloses their use in combination with other anti-inflammatory compounds. U.S. Patent No. 5,629,999 discloses oral formulations for administration as anti-inflammatory compounds.

[0003] There is a need to develop new compositions and formulations for wound healing. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO 01 / 49305 [Patent Document 2] International Publication No. 03 / 04042 [Patent Document 3] International Publication No. 2005 / 027944 Summary of the Invention

[0005] Thus, there is provided a composition comprising a functional thylakoid extract, or a pharmaceutical formulation thereof, and its use for wound healing.

[0006] Thus, there is also provided a composition comprising a functional thylakoid extract, or a pharmaceutical formulation thereof, and its use for wound healing.

[0007] In a first aspect, there is provided a composition for wound healing in a subject, the composition comprising an effective amount of a functional thylakoid extract, particularly in admixture with a physiologically acceptable carrier.

[0008] In a second aspect, there is provided the use of a functional thylakoid extract in the manufacture of a medicament for wound healing in a subject.

[0009] In a further aspect, there is provided a use of a functional thylakoid extract for wound healing in a subject.

[0010] In a further aspect, there is also provided a method for wound healing in a subject in need thereof, the method comprising topically administering to the subject an effective amount of a functional thylakoid extract, optionally in admixture with a physiologically acceptable carrier.

[0011] In a further aspect, a topical composition is also provided comprising an effective amount of a photosynthetic cell extract, optionally mixed with a physiologically acceptable carrier, for increasing the production or level of GM-CSF growth factor and / or the production or level of G-CSF growth factor in a subject's epithelial tissue (e.g., skin, mucosa, or corneal epithelium) following epithelial wounding compared to an untreated control, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises active thylakoid extract.

[0012] In a further aspect, a topical composition for use in epithelial (e.g., skin, mucosal, or corneal epithelium) wound healing in a subject is also provided, comprising an effective amount of a photosynthetic cell extract, optionally mixed with a physiologically acceptable carrier, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises an active thylakoid extract.

[0013] In a further aspect, there is also provided the use of a topical composition as defined herein for epithelial (e.g., cutaneous, mucosal, or corneal epithelium) wound healing in a subject.

[0014] In a further aspect, there is also provided the use of a topical composition as defined herein to increase the level of GM-CSF growth factor and / or G-CSF growth factor in epithelial tissue (e.g., skin, mucosa, or corneal epithelium) of a subject following epithelial wounding compared to an untreated control, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises an active thylakoid extract.

[0015] In a further aspect, there is also provided a method for epithelial (e.g., skin, mucosal, or corneal epithelium) wound healing in a subject in need thereof, the method comprising administering a topical composition as defined herein.

[0016] In a further aspect, a method is also provided for increasing the level of GM-CSF growth factor and / or G-CSF growth factor in epithelial tissue (e.g., skin, mucosa, or corneal epithelium) of a subject following epithelial wounding compared to an untreated control, the method comprising administering a topical composition as defined herein.

[0017] Other objects, advantages and features of the present specification will become more apparent from a reading of the following non-restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0018] The contents of the documents cited in this disclosure are incorporated herein by reference. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows a flow diagram of the thylakoid extract manufacturing process. [Figure 2] HPLC chromatogram showing the pigment profile of the thylakoid extract. [Figure 3] Diagram showing the progression of wound healing stages in the skin after physical trauma. (Image from Vadivel and Brindha, 2017) [Figure 4A]IFN-γ levels during recovery from mechanical wounds from an in vitro wound healing assay are shown. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 4B] Results from Figure 4A are shown plotted as percent change from untreated controls (ie, cells not treated with thylakoid extract). [Figure 5A] IL-6 levels during recovery from mechanical wounds from an in vitro wound healing assay were shown. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 5B] Results from Figure 5A are shown plotted as percent change from the untreated control (ie, cells not treated with thylakoid extract). [Figure 6A] IL-8 levels during recovery from mechanical wounds from an in vitro wound healing assay are shown. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 6B] Results from Figure 6A are shown plotted as percent change from untreated controls (ie, cells not treated with thylakoid extract). [Figure 7A] IP-10 [CXCL-10] levels during recovery from mechanical wounds from an in vitro wound healing assay are shown. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 7B]Results from Figure 7A are shown plotted as percent change from untreated controls (ie, cells not treated with thylakoid extract). [Figure 8A] Figure 1 shows MCP-1 levels during recovery from mechanical wounds from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 8B] Results from Figure 8A are shown plotted as percent change from untreated controls (ie, cells not treated with thylakoid extract). [Figure 9A] Figure 1 shows RANTES levels during recovery from mechanical wounding from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 9B] Results from Figure 9A are shown plotted as percent change from untreated controls (ie, cells not treated with thylakoid extract). [Figure 10A] Figure 1 shows TNF-α levels during recovery from mechanical wounding from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 10B] Results from FIG. 10A are shown plotted as percent change from the untreated control (ie, cells not treated with thylakoid extract). [Figure 11A]Figure 1 shows basic FGF levels during recovery from mechanical wounds from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 11B] Results from FIG. 11A are shown plotted as percent change from untreated controls (ie, cells not treated with thylakoid extract). [Figure 12A] Figure 1 shows GM-CSF levels during recovery from mechanical wounding from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 12B] Results from FIG. 12A are shown plotted as percent change from the untreated control (ie, cells not treated with thylakoid extract). [Figure 13A] Figure 1 shows G-CSF levels during recovery from mechanical wounds from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 13B] Results from FIG. 13A are shown plotted as percent change from the untreated control (ie, cells not treated with thylakoid extract). [Figure 14A] Figure 1 shows elastin levels during recovery from mechanical wounds from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 14B]Results from FIG. 14A are shown plotted as percent change from the untreated control (ie, cells not treated with thylakoid extract). [Figure 15A] Figure 1 shows procollagen levels during recovery from mechanical wounds from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding under untreated conditions (i.e., direct effect). Statistical significance is assessed below. [Figure 15B] Results from Figure 15A are shown plotted as percent change from the untreated control (ie, cells not treated with thylakoid extract). DETAILED DESCRIPTION OF THE INVENTION

[0020] definition The term "about" as used herein refers to a range of +10% or -10% of the indicated number. For precision, for example, when used in conjunction with about 90%, the term means 90% ± 9%, i.e., 81% to 99%. Alternatively, the term about can refer to +5% or -5% of the indicated number; for example, 90% means 90% ± 4.5%, i.e., 86.5% to 94.5%. Alternatively, the term about can refer to + or -1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of the indicated number. Alternatively, the term about can refer to normal experimental variation for a given device.

[0021] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the culture" includes reference to one or more cultures and equivalents thereof known to those skilled in the art, etc. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless clearly indicated otherwise.

[0022] As used in this specification and claims, the terms "comprising" (and any form of "comprising" such as "comprise" and "comprises"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "including" such as "includes" and "include"), or "containing" (and any form of "containing" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0023] As used herein, the terms "thylakoid," "thylakoid extract," "functional thylakoid extract," or "functional thylakoid," or "active thylakoid / extract" refer to purified functional photosynthetic pigments in a thylakoid membrane environment, particularly in their original thylakoid environment (i.e., in their intact native state such that they are still active or can be activated). More specifically, these terms refer to functional thylakoid membranes extracted by the methods described herein and / or by the procedures disclosed in Bissonnette et al. (2004), WO 01 / 49305, WO 2018 / 081903 A1, WO 2011 / 127559 A1, or WO 2017 / 075698 A1 (all of which are incorporated herein by reference).

[0024] As used herein, the term "wound healing" includes accelerating wound healing (e.g., of the skin, mucosa, or corneal epithelium), reducing scarring, and / or increasing the tensile strength of the skin barrier in a subject compared to an untreated control. According to certain embodiments, the term "wound healing" refers to a measurable or observable reduction in wound size in a treated sample that has undergone treatment compared to an untreated control sample set.

[0025] In particular, with respect to the present thylakoid extract embodiment, the functional quality of a molecular complex can be determined by its ability to respond to light and dissipate its energy (F), as is well known in the art and / or as described in Maxwell (2000). v / F m In one embodiment, the functional thylakoid extract can be measured by fluorescence based on the ratio (F v / F m The ratio is at least 0.7.

[0026] Detailed Description of Specific Embodiments composition According to a particular aspect, the present invention describes a composition for wound healing in a subject, comprising an effective amount of an active thylakoid extract, in particular a spinach thylakoid extract, more particularly extracted from spinach leaves, in admixture with a physiologically acceptable carrier.

[0027] In particular, the composition contains purified functional photosynthetic pigments in a thylakoid membrane environment. More specifically, the extract is dormant and capable of being photosynthetically activated. More specifically, the extract is stabilized (i.e., stable) in its basal state by lacking an electron donor (such as water). The extract can also be in aqueous form.

[0028] Most specifically, the composition is defined as raw organic spinach, activated thylakoid extract, having a ratio of chlorophyll to total pigment or total HPLC content of at least 0.4, specifically at least 0.5, more specifically at least 0.6.

[0029] In particular, the pigments contained in the thylakoid extract are selected from the group consisting of chlorophyll a, chlorophyll b, and carotenoids. More specifically, the pigments contained in the thylakoid extract are selected from the group consisting of chlorophyll a, chlorophyll b, lutein, and, optionally, β-carotene and / or pheophytin. Most specifically, the pigments contained in the thylakoid extract consist essentially of chlorophyll a (greater than 40%), followed by chlorophyll b (about 10-15%), lutein (about 10% or less), β-carotene (about 3%), and pheophytin (less than 1%). In one embodiment, the thylakoid extract has an HPLC profile substantially corresponding to Figure 2. In one embodiment, the thylakoid extract contains chlorophyll a (about 62.5%), chlorophyll b (about 13.1%), lutein (about 9.4%), β-carotene (about 2.98%), and pheophytin (about 0.45%). In one embodiment, the average ratio of chlorophyll a to total peak area response (for all peaks) should not be less than 0.40. In one embodiment, the thylakoid extract contains chlorophyll a (about 62.5%), chlorophyll b (about 13.1%), lutein (about 9.4%), β-carotene (about 2.98%), and pheophytin (about 0.45%). In one embodiment, the average ratio of chlorophyll a to total peak area response should not be less than 0.40. In one embodiment, the thylakoid extract contains 10% (w / w) or less water.

[0030] In particular, the stabilized extract is in solid form, more particularly as a powder. Even more particularly, the extract is in powder form having at least 25 mg of pigment per gram of powder.

[0031] The compositions according to the present invention may be prepared and embodied in all pharmaceutical forms commonly used for topical application. Furthermore, the compositions may contain additives commonly used in the dermatological field, such as fats, emulsifiers and co-emulsifiers, hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active ingredients, preservatives, antioxidants, solvents, fragrances, fillers, hydrophilic and lipophilic filters, dyes, neutralizers, penetration enhancers and polymers.

[0032] The extract can be formulated as a liquid composition (non-lyophilized extract), a lyophilized extract reconstituted in water, saline or any other solution compatible with topical administration, in propylene glycol, or in a solid composition.

[0033] Use and Method According to certain aspects, there is provided use of a thylakoid extract comprising a purified functional photosynthetic pigment in a thylakoid membrane environment in the preparation of a medicament for wound healing in a subject, or use of a thylakoid extract comprising a purified functional photosynthetic pigment in a thylakoid membrane environment for wound healing in a subject.

[0034] According to certain embodiments, the thylakoid extract is for topical application / administration. Topical application / administration refers to application / administration to epithelia, including but not limited to skin, mucous membranes, corneal epithelia, etc. According to certain embodiments, the thylakoid extract is for topical application / administration to skin.

[0035] According to certain embodiments, the wound is an epithelial wound. According to certain embodiments, the wound is a dermal wound. In one embodiment, the wound is an open mechanical wound caused by a mechanical force or a wound caused by physical trauma. In further embodiments, the wound comprises a dermal ulcer (e.g., a neuropathic ulcer, an ischemic ulcer, or a neuroischemic ulcer), an infectious wound, an ischemic wound, a laceration, a surgical wound, a puncture wound, a cut, a puncture wound, a dermal wound from radiation poisoning, or a combination thereof. In further embodiments, the wound comprises a dermal ulcer (e.g., a neuropathic ulcer, an ischemic ulcer, or a neuroischemic ulcer), an infectious wound, an ischemic wound, a surgical wound, a dermal wound from radiation poisoning, or a combination thereof. In some embodiments, the wound penetrates to the skin. In some embodiments, the wound penetrates to the dermis. In some embodiments, the wound penetrates to a lower layer of the skin. In some embodiments, the wound penetrates to the subcutaneous tissue. In some embodiments, the wound penetrates to the muscle. In some embodiments, the thylakoid extract is used to treat the skin wound in combination with other known treatments used to treat skin wounds (e.g., sutures, glue, staples, tape, bandages, antibiotics).

[0036] In some aspects, the thylakoid extract defined herein is used to treat mucosal wounds (e.g., any organ or tissue, such as, but not limited to, the nose, mouth, lungs, anus, ears, genitals, esophagus, bronchi, stomach, uterus, bladder, intestinal mucosa, etc.). For example, mucosal wounds can be caused by surgery (e.g., incision) or lacerations. In some aspects, the thylakoid extract defined herein is used during surgery.

[0037] According to certain embodiments, the wound is not caused by UV A and / or B exposure or an autoimmune disorder or condition such as eczema.

[0038] According to certain aspects, treatment with the composition increases the production or levels of pro-inflammatory cytokines (e.g., IFN-γ, IL-6, IL-8, IP-10 [CXCL-10], MCP-1, RANTES, and / or TNF-α) (e.g., in any layer of the skin or mucosa) compared to untreated controls.

[0039] In certain embodiments, treatment with the composition increases collagen and / or procollagen production or levels (eg, in any layer of the skin or mucosa) compared to an untreated control.

[0040] In certain embodiments, treatment with the composition increases the production or levels of elastin (eg, in any layer of the skin or mucosa) compared to an untreated control.

[0041] According to certain embodiments, treatment with the composition increases the production or levels of basic FGF, G-CSF, and / or GM-CSF (e.g., in any layer of the skin or mucosa) compared to an untreated control.

[0042] In some embodiments, a colloidal effect of the thylakoid extract may be observed. For example, increases in pro-inflammatory cytokine, growth factor, collagen, elastin, and / or procollagen levels may not be significant at certain concentrations (e.g., low and high concentrations), but may be observed at other concentrations (e.g., between low and high concentrations). In some embodiments, a bell-shaped dose response is observed. This may be due to the down- or up-regulation of specific cell surface receptors at different thylakoid extract concentrations.

[0043] According to certain embodiments, there is provided a method for wound healing in a subject in need thereof, the method comprising administering to the subject an effective amount of a thylakoid extract comprising purified functional photosynthetic pigments in a thylakoid membrane environment, particularly mixed with a physiologically acceptable carrier.

[0044] In particular, the present uses and methods may be adapted for the treatment of mammalian subjects, particularly pets or humans, more particularly cats, dogs, horses or humans, most particularly humans.

[0045] formulation According to certain aspects, there is provided a use or method of treatment as defined herein, wherein the composition is formulated for application to epithelial tissue. As used herein, the term "epithelial tissue" includes any epithelial tissue, including, but not limited to, skin, mucous membranes, corneal epithelium, etc.

[0046] Dosage As used herein, the term "effective amount" means a dose sufficient to induce accelerated wound healing and / or reduce scarring and / or increase the tensile strength of the skin barrier.

[0047] According to certain embodiments, the extract is provided in a dosage of about 0.01% to about 0.2%, about 0.01% to about 0.1%, about 0.05% to 0.15%, about 0.075% to 0.125%, or about 0.9% to 0.11%.

[0048] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0049] Embodiment The following embodiments are provided:

[0050] Embodiment 1. A topical composition comprising an effective amount of a photosynthetic cell extract, optionally mixed with a physiologically acceptable carrier, for increasing the production or level of GM-CSF growth factor and / or the production or level of G-CSF growth factor in epithelial tissue of a subject (e.g., skin, mucosa, or corneal epithelium) following an epithelial wound compared to an untreated control, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises active thylakoid extract.

[0051] Embodiment 2. A topical composition for use in epithelial (e.g., skin, mucosal, or corneal epithelium) wound healing in a subject, comprising an effective amount of a photosynthetic cell extract, optionally mixed with a physiologically acceptable carrier, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises an active thylakoid extract.

[0052] Embodiment 3. A topical composition for use according to embodiment 1 or 2, wherein the extract is present in an amount of about 0.01% to about 0.2% based on the total weight of the composition.

[0053] Embodiment 4. A topical composition for use according to any of embodiments 1 to 3, wherein the extract is for administration before a wound is present.

[0054] Embodiment 5. A topical composition for use according to any one of embodiments 1 to 4, wherein the extract is for administration after a wound exists.

[0055] Embodiment 6. The topical composition of any one of embodiments 1-5, wherein the carrier is selected from the group consisting of fats, hydrophilic or lipophilic gelling agents, solvents, and bulking agents.

[0056] Embodiment 7. The topical composition of any one of embodiments 1 to 6, wherein the extract is not freeze-dried.

[0057] Embodiment 8. The topical composition of any one of embodiments 1-7, in the form of a solution, cream, ointment, salve, foam, spray, transdermal patch, or gel.

[0058] Embodiment 9. The topical composition of any one of embodiments 1 to 8, wherein the extract is a lyophilized extract reconstituted in water, saline or any other solution compatible with topical administration, propylene glycol, or a solid composition.

[0059] Embodiment 10. The topical composition of any one of embodiments 1-9, wherein the photosynthetic cell extract is from spinach thylakoids and comprises functional thylakoids.

[0060] Embodiment 11. The topical composition of any one of embodiments 1-10, wherein the active thylakoid extract comprises purified functional photosynthetic pigments in their thylakoid membrane environment.

[0061] Embodiment 12. The topical composition of any one of embodiments 1-11, wherein the wound is an open mechanical wound.

[0062] Embodiment 13. The topical composition of any one of embodiments 1-12, wherein the wound comprises a skin ulcer (e.g., a neuropathic ulcer, an ischemic ulcer, or a neuroischemic ulcer), an infected wound, an ischemic wound, a surgical wound, a skin wound from radiation poisoning, or a combination thereof.

[0063] Embodiment 14. The topical composition of any one of embodiments 1-13, wherein the wound is not caused by UVA and / or UVB exposure or an autoimmune disorder or condition, such as eczema.

[0064] Embodiment 15. A topical composition according to any one of embodiments 1 to 14, wherein treatment with the composition increases collagen production or levels in epithelial tissue compared to an untreated control.

[0065] Embodiment 16. A topical composition according to any one of embodiments 1 to 15, wherein treatment with the composition increases the production or levels of elastin in epithelial tissue compared to an untreated control.

[0066] Embodiment 17. A topical composition according to any one of embodiments 1 to 16, wherein treatment with the composition increases the production or levels of procollagen, collagen and / or elastin in epithelial tissue compared to an untreated control.

[0067] Embodiment 18. The topical composition of any one of embodiments 1-17, wherein treatment with the composition increases the production or levels of basic FGF, G-CSF, and / or GM-CSF in epithelial tissue compared to an untreated control.

[0068] Embodiment 19. The topical composition of any one of embodiments 1-18, wherein treatment with the composition increases the production or levels of pro-inflammatory cytokines (e.g., IFN-γ, IL-6, IL-8, IP-10 [CXCL-10], MCP-1, RANTES, and / or TNF-α) in epithelial tissue compared to untreated controls.

[0069] Embodiment 20. Use of a topical composition according to any one of embodiments 1 to 19 for epithelial (e.g., skin, mucosal, or corneal epithelium) wound healing in a subject.

[0070] Embodiment 21. Use of a topical composition according to any one of embodiments 1 to 19 to increase the level of GM-CSF growth factor and / or G-CSF growth factor in epithelial tissue (e.g., skin, mucosa, or corneal epithelium) of a subject following epithelial wounding compared to an untreated control, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises an active thylakoid extract.

[0071] Embodiment 22. A method for epithelial (e.g., skin, mucosal, or corneal epithelium) wound healing in a subject in need thereof, the method comprising administering a topical composition of any one of embodiments 1-19.

[0072] Embodiment 23. A method for increasing the level of GM-CSF growth factor and / or G-CSF growth factor in epithelial tissue (e.g., skin, mucosa, or corneal epithelium) of a subject following epithelial wounding compared to an untreated control, the method comprising administering a topical composition described in any one of embodiments 1-19. [Example]

[0073] Example 1 - Preparation of thylakoid extract The thylakoid extract is derived from the mesophyll tissue of baby spinach (Spinacia oleracea L.) leaves, which are rich in chloroplasts. The inner membranes of chloroplasts are organized into structures known as thylakoids, which are extracted from baby spinach, concentrated, and stabilized into a solid powder form. The main components of the thylakoid membrane are pigments, proteins, and lipids. The characteristics of the thylakoid extract are shown in Table 1.

[0074] [Table 1]

[0075] Manufacturing method The method for producing thylakoid extract is shown schematically in the flow diagram of FIG.

[0076] The processing steps are carried out under refrigerated conditions with minimal light exposure to preserve maximum activity of photosynthetic pigments, and consist of the following sequence: inspection of spinach leaves and washing with sodium hypochlorite solution, mechanical disruption and homogenization, filtration by centrifugation, freeze-drying, and gamma irradiation.

[0077] Inspection of spinach leaves and washing with sodium hypochlorite solution. After visual inspection to verify size and identity attributes (e.g., leaves are green with no discolored areas or yellowish spots (chlorosis)), spinach leaves are first washed with sodium hypochlorite solution at a predetermined solution to leaf ratio by mass (44 kg:5.4 kg) adjusted to a pH of 7.0-8.0 (target pH: 7.4) to reduce the microbial flora naturally found on fresh produce leaves.

[0078] Mechanical disruption and homogenization. After draining excess sodium hypochlorite solution, the leaves were transferred to a mechanical cutter / mixer with a predetermined volume of tris(hydroxymethyl)aminomethane buffer solution at pH 7.0-8.0 (target pH: 7.4) in a predetermined solution-to-leaf ratio (5.4 kg:3.7 kg) by mass. This process was used to cut and homogenize the leaves into a coarse suspension while liberating fragments of thylakoid membranes derived from chloroplasts.

[0079] Filtration by centrifugation. The suspension is then filtered in a basket centrifuge. Centrifugation is carried out at a target speed of 3100 rpm (range: 2800-3200 rpm). This step allows the removal of fibers, debris, and coarse material retained on the sieve, resulting in a by-product cake that is discarded. The thylakoid extract (active components) is found in the centrate, collected, and kept at a temperature below 10°C for further processing.

[0080] Freeze-drying: The material is then dispensed onto shallow stainless steel plates and frozen in the dark at temperatures below -30°C for at least 2 hours. The plates, maintained at a target temperature of 10°C, are then transferred to a freeze-dryer and the product is freeze-dried.

[0081] Gamma irradiation. A terminal gamma irradiation step is performed. After irradiation, the thylakoid extract is transferred to a bottle with a tightly closed screw cap.

[0082] The material may be filtered after the centrifugation step. For example, size exclusion chromatography can be performed to separate the most active fractions by molecular weight. In particular, crude thylakoids can be further purified by Sephadex G-100 filtration to obtain an F of at least 0.7. v / F m The fractions corresponding to the ratio of

[0083] Dye Composition and Other Features Spinach contains natural antioxidants (e.g., flavonoids) and photosynthetic pigments (chlorophyll and carotenoids). The inner membrane of the chloroplast is organized into structures known as thylakoids. The main components of the thylakoid membrane are pigments, proteins, and lipids.

[0084] Thylakoid extracts are derived from the mesophyll tissue of spinach leaves, which is rich in chloroplasts. To date, the following pigments have been identified in thylakoid extracts using HPLC analysis: lutein, chlorophyll b, chlorophyll a, pheophytin, and β-carotene. A typical chromatogram showing the pigment profile of the thylakoid extract in terms of area % is shown in Figure 2. This analysis indicates that the major component of the thylakoid extract is chlorophyll a (62.5%), followed by chlorophyll b (13.1%), lutein (9.4%), β-carotene (2.98%), and pheophytin (0.45%).

[0085] Preferably, the raw baby spinach is obtained from growers certified according to the National Organic Standards of the United States Department of Agriculture (USDA) to minimize the risk of the presence of potential chemical residues from fertilizers or pesticides in the thylakoid extract.

[0086] Specification Justification Thylakoid extracts are characterized by their pigment content, expressed as milligrams of pigment per gram of powdered extract. Based on processing capacity and taking into account seasonal variations in the herbal starting material, a specification of ≥25 mg pigment / g extract was set. Based on stability data, a shelf-life limit of 80% of the initial pigment content was set.

[0087] The pigment profile also allows the identification of the various pigments present in the thylakoid extract and their ratio in area percent. Considering the profile determined in the batch, it was established that chlorophyll a, chlorophyll b, lutein, and β-carotene should be present and that the average ratio of chlorophyll a to total peak area response should not be less than 0.40.

[0088] Because water is used as the extraction solvent in the manufacturing process, a test was included to determine the water content in the thylakoid extract. To control the water content, a water specification of 10% (w / w) or less was set.

[0089] stability Batches of thylakoid extract drug product sealed in tightly screw-capped bottles are stabilized under the following conditions: 5°C ± 3°C (currently recommended storage conditions). Available data indicate that thylakoid extract is stable after storage under refrigerated conditions for at least 18 months.

[0090] Example 2 - Method Wound healing in vitro assay: Mechanical wound model - Scratch wound healing in dermal fibroblast cultures Scratch wound recovery in skin fibroblast cultures is a gold-standard in vitro assay for mimicking certain aspects of wound healing and, therefore, is related to normal ongoing remodeling to protect the integrity of the skin barrier. Briefly, human primary skin fibroblasts from healthy adult donors were used in the study. Fibroblasts were cultured in flat-bottom 24-well plates until they formed a monolayer at the bottom of each well. To test whether thylakoid extract was applied before wounding, some wells were pretreated with thylakoid extract before mechanical wounding. These same wells were also treated with thylakoid extract after wounding. Using sterile tools, a scratch was created across each well as a model of mechanical wounding. The medium was removed and fresh medium was added to help remove any cell clumps that may have formed during the wounding procedure. This helped to reduce the amount of free-flowing cells that could settle in the wound area and confound analysis of wound repair by cell migration. The cells were then immediately exposed to the thylakoid extract. The concentrations of thylakoid extract used in the assay were selected based on cell viability assays performed on human PBMCs and human skin fibroblasts using the MTT assay.

[0091] Culture plates are allowed to rest for 1 hour to allow for any loose cell clumps that may have settled in the scratched zone to be recorded and not affect the final score for scratch wound recovery. Cultures are examined after 16-24 hours to monitor scratch wound recovery in product-treated cells compared to control cell cultures. Culture supernatants are collected at 24 hours from both wounded and non-wounded cultures and frozen for storage. Culture supernatants are then tested for levels or secreted procollagen (type I) and elastin using commercially available ELISA assays. Culture supernatant cytokines are tested in duplicate for a broad panel of pro- and anti-inflammatory cytokines, antiviral peptides, and regenerative growth factors using Luminex™ magnetic bead arrays and the MagPix™ multiplexing system.

[0092] Example 3 - Effect of thylakoid extract on wound healing The effect of the produced thylakoid extract (as described in Example 1) was evaluated in an in vitro wound healing assay (as described in Example 2). Part of normal wound healing involves the inflammatory, proliferative, and remodeling phases (Figure 3). Freshly wounded skin rapidly enters the inflammatory phase, which is designed to signal acute injury to the skin and underlying tissue and induce the recruitment of fibroblasts and macrophages to aid in the repair process. Fibroblasts enter a proliferative growth phase, which is supported in part by various growth factors. Finally, cell migration is part of the remodeling phase as the skin regains its tactile strength.

[0093] inflammatory phase Figure 4 shows a summary of the pro-inflammatory cytokine multiplex from an in vitro wound-healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (at 0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding. Results are plotted as the percent increase or decrease of pro-inflammatory cytokines (IFN-γ, IL-6, IL-8, IP-10 [CXCL-10], MCP-1, RANTES, or TNF-α) from untreated controls (i.e., cells not treated with thylakoid extract).

[0094] The thylakoid extract significantly increased the levels of many pro-inflammatory cytokines when administered to cells after mechanical wounding, or before and after mechanical wounding, compared with untreated cells in the absence of mechanical wounding or cells treated with the thylakoid extract (Figures 4A, 4B, 5A, 5B, 6A, 6B, 7A, 7B, 8A, 8B, 9A, 9B, 10A, and 10B). Most of the pro-inflammatory cytokines were increased by thylakoid extract treatment, with the most significant changes observed for IL-6, IL-8, MCP-1α, and TNF-α (Figures 5A, 5B, 6A, 6B, 8A, 8B, 10A, and 10B).

[0095] proliferative growth phase Figures 11A, 11B, 12A, 12B, 13A, and 13B show growth factor multiplex results from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding. Results are plotted as the percent increase or decrease in growth factor (basic FGF, GM-CSF, or G-CSF) from untreated controls (i.e., cells not treated with thylakoid extract). GM-CSF growth factor is known to play a role in accelerating wound healing, and G-CSF growth factor also plays a supportive role in healing various types of wounds, including radiation-induced skin injury. G-CSF is further known to mobilize stem cells and help recruit these cells to tissue sites in need of repair. Furthermore, basic FGF induces fibroblast migration as part of the remodeling phase of wound healing.

[0096] Thylakoid extract, when administered to cells before or after mechanical wounding, was shown to significantly increase the levels of three growth factors (basic FGF, GM-CSF, or G-CSF) compared to untreated cells. Interestingly, the increase in basic FGF, GM-CSF, or G-CSF levels was found to be proportional to the thylakoid extract concentration and was therefore greatest at the highest concentration of thylakoid extract (2 mg / L) (Figures 11A, 11B, 12A, 12B, 13A, and 13B).

[0097] Remodeling phase Figures 14A, 14B, 15A, and 15B show elastin and procollagen (type I) results from an in vitro wound healing assay. In this assay, human dermal fibroblasts were treated with thylakoid extract (0.02 mg / L, 0.2 mg / L, or 2 mg / L) either before or after mechanical wounding. Results are plotted as percent increase or decrease in elastin or procollagen from untreated controls (i.e., cells not treated with thylakoid extract).

[0098] Dermal fibroblasts produce elastin and procollagen in culture; these components help cells adhere, migrate, and form an extracellular matrix that maintains an intact cell layer.

[0099] It was shown that thylakoid extract, when administered to cells before, after, or after mechanical wound induction, significantly reduced procollagen levels compared to untreated cells, while elastin levels remained unchanged. However, the reduction in procollagen levels may be due to the timing at which the supernatants were assayed, as procollagen is rapidly converted to collagen during wound healing.

[0100] conclusion These data support the role of thylakoid extract in the natural healing process. Thylakoid extract increases inflammatory cytokine levels, cell growth factors, and remodeling components, strengthening the skin, reducing scarring, and increasing the tensile strength of the skin barrier. Therefore, these data support the therapeutic efficacy of thylakoid extract for treating and healing various types of wounds.

[0101] References Vadivel V and Brindha P.Wound Healing Potential of Ipomoea Carnea Jacq.:An Un-Explored Herb Used in Indian Traditional 000033 System of Medicine.Glob J Pharmaceu Sci.2017;3(1):555601. Maxwell (2000): Chlorophyll fluorescence-a practical guide.Journal of Experimental Botany Vol.51, No.345, pp.659-668-April 2000. Bissonnette E.Y.Proulx L.I.,Turmel V.,Drouin R.,Purcell M.(2004).PCT-233,a novel modulator of pro-and anti-inflammatory cytokine production.Clin.Exp.Immunol.,135:440-447。

Claims

1. A topical composition comprising an effective amount of a photosynthetic cell extract, optionally mixed with a physiologically acceptable carrier, for increasing the production or level of GM-CSF growth factor and / or the production or level of G-CSF growth factor in epithelial tissue of a subject (e.g., skin, mucosa, or corneal epithelium) following an epithelial wound compared to an untreated control, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises an active thylakoid extract.

2. A topical composition for use in epithelial (e.g., skin, mucosal, or corneal epithelium) wound healing in a subject, comprising an effective amount of a photosynthetic cell extract, optionally mixed with a physiologically acceptable carrier, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises an active thylakoid extract.

3. 3. The topical composition for use according to claim 1 or 2, wherein the extract is present in an amount of about 0.01% to about 0.2% based on the total weight of the composition.

4. A topical composition for use according to any one of claims 1 to 3, wherein the extract is for administration before the wound is present.

5. A topical composition for use according to any one of claims 1 to 4, wherein the extract is for administration after the wound exists.

6. The topical composition of any one of claims 1 to 5, wherein the carrier is selected from the group consisting of fats, hydrophilic or lipophilic gelling agents, solvents, and bulking agents.

7. The topical composition of any one of claims 1 to 6, wherein the extract is not freeze-dried.

8. The topical composition of any one of claims 1 to 7, in the form of a solution, cream, ointment, salve, foam, spray, transdermal patch, or gel.

9. 9. The topical composition of any one of claims 1 to 8, wherein the extract is a lyophilized extract reconstituted in water, saline or any other solution compatible with topical administration, propylene glycol, or a solid composition.

10. The topical composition of any one of claims 1 to 9, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises functional thylakoids.

11. The topical composition of any one of claims 1 to 10, wherein the active thylakoid extract comprises purified functional photosynthetic pigments in their thylakoid membrane environment.

12. The topical composition of any one of claims 1 to 11, wherein the wound is an open mechanical wound.

13. 13. The topical composition of any one of claims 1 to 12, wherein the wound comprises a skin ulcer (e.g., a neuropathic ulcer, an ischemic ulcer, or a neuroischemic ulcer), an infected wound, an ischemic wound, a surgical wound, a skin wound from radiation poisoning, or a combination thereof.

14. 14. The topical composition of any one of claims 1 to 13, wherein the wound is not caused by UVA and / or UVB exposure or an autoimmune disorder or condition such as eczema.

15. 15. The topical composition of any one of claims 1 to 14, wherein treatment with the composition increases collagen production or levels in the epithelial tissue compared to an untreated control.

16. 16. The topical composition of any one of claims 1 to 15, wherein treatment with the composition increases the production or levels of elastin in the epithelial tissue compared to an untreated control.

17. 17. The topical composition of any one of claims 1 to 16, wherein treatment with the composition increases the production or levels of procollagen, collagen and / or elastin in the epithelial tissue compared to an untreated control.

18. 18. The topical composition of any one of claims 1 to 17, wherein treatment with the composition increases the production or levels of basic FGF, G-CSF, and / or GM-CSF in the epithelial tissue compared to an untreated control.

19. 19. The topical composition of any one of claims 1-18, wherein treatment with the composition increases the production or levels of pro-inflammatory cytokines (e.g., IFN-γ, IL-6, IL-8, IP-10 [CXCL-10], MCP-1, RANTES, and / or TNF-α) in the epithelial tissue compared to untreated controls.

20. 20. Use of a topical composition according to any one of claims 1 to 19 for epithelial (e.g. skin, mucosal, or corneal epithelium) wound healing in a subject.

21. 20. Use of the topical composition of any one of claims 1 to 19 to increase the level of GM-CSF growth factor and / or G-CSF growth factor in epithelial tissue (e.g., skin, mucosa, or corneal epithelium) of a subject following epithelial wounding compared to an untreated control, wherein the photosynthetic cell extract is derived from spinach thylakoids and comprises an active thylakoid extract.

22. 20. A method for epithelial (e.g., skin, mucosal, or corneal epithelium) wound healing in a subject in need thereof, said method comprising administering a topical composition according to any one of claims 1 to 19.

23. 20. A method for increasing the level of GM-CSF growth factor and / or G-CSF growth factor in epithelial tissue (e.g., skin, mucosa, or corneal epithelium) of a subject following epithelial wounding compared to an untreated control, said method comprising administering a topical composition according to any one of claims 1 to 19.

Images