COMPOSITIONS AND MATERIALS COMPRISING COPPER COMPOUNDS

TECHNICAL FIELD

[0001] The present application relates to the field of skin treatment. In particular, the present application relates to the method for reduction and prevention of skin stasis with compositions and materials comprising copper compounds.

BACKGROUND

[0002] Douglas MacG. Jackson in "The diagnosis of the depth of burning", The British Journal of Surgery 40 (1953), pp. 588-596, mentioned that the local response of a skin burn generally can be characterised as resulting in three different injury patterns of varying degrees depending on the severity of the burn and the cause. The point of maximum irreversible damage is referred to as zone of coagulation since there occurs tissue loss due the coagulation of the skin proteins. The zone around the coagulation zone, referred as the zone of stasis, is characterised by decreased tissue perfusion, and depending on the severity of the burn, hypotension, infection, and/or oedema may lead this zone into an area of complete tissue loss. Shehan Hettiaratchy and Peter Dziewulski in "Pathophysiology and types of burns", BMJ 328 (7453) (2004), pp. 1427-1429, showed that around the zone of sepsis, a zone of hyperaemia is characterised by increased perfusion that will invariably recover unless there is severe sepsis or prolonged hypoperfusion. The middle zone of stasis, while initially viable, following the reduced perfusion dies and the initial size of the wound deepens and widens.

[0003] Following sever skin injury of above of 30% of total body surface area, release of cytokines and other inflammatory mediators at the site of injury, may lead to several systemic responses that can be detrimental. These include a) significant increase in capillary permeability, leading to loss of intravascular proteins and fluids into the interstitial compartment, peripheral and splanchnic vasoconstriction and myocardial contractility decrease. These cardiovascular changes coupled with fluid loss from the burn wound, result in systemic hypotension and end organ hypoperfusion; b) bronchoconstriction, and potentially respiratory distress syndrome; c) increase of the basal metabolic rate by up to three times its original rate; and d) non-specific cell mediated and humoral immune responses down regulation. [0004] Ara A. Salibian et al in "Current concepts on burn wound conversion. A review of recent advances in understanding the secondary progressions of bums." , Burns 42(5) (2016), pp. 1025- 1035, suggested that the stasis zone is a therapeutically critical section of burn surface that can be salvaged. Prevention of the progression and expansion of the wound injury into larger and deeper areas may have important local and systemic consequences that may significantly decrease complications and morbidity. Thus, developing therapies that can halt the further deterioration of the zone of stasis following wound burn injury can be an important modality of burn wound treatment after patient stabilization and prior to skin reconstruction.

[0005] Copper is an essential trace element involved in many cellular, metabolic and physiological processes in almost all body tissues. Neena Philips et al in "Stimulation of cell proliferation and expression of matrixmetalloproteinase-1 and interluekin-8 genes in dermal fibroblasts by copper ", Connective Tissue Research 51(3) (2010), pp. 224-229, demonstrated that copper is capable of stimulating dermal fibroblasts proliferation in skin. Further, Neena Philips et al in " Beneficial regulation of fibrillar collagens, heat shock protein-47, elastin fiber components, transforming growth factor-beta 1, vascular endothelial growth factor and oxidative stress effects by copper in dermal fibroblasts" , Connective Tissue Research 53(5) (2012), pp. 373-378, showed that copper in the skin enhances production and secretion of different collagen and elastin types by fibroblasts.

[0006] Also, it has been shown that copper is capable of stabilising the skin extracellular matrix once formed, serves as a cofactor of superoxide dismutase, an antioxidant enzyme present in the skin, important for protection against free radicals, serves as a cofactor of lysyl oxidase, an enzyme that catalyses lysine-derived crosslinks in the skin extracellular matrix, and inhibits cellular oxidative effects such as membrane damage and lipid peroxidation. However, the surprising effect of copper ions on reducing stasis of skin burns has not been known yet. It has now been discovered by the present inventors and constitutes the basis of the present invention.

SUMMARY

[0007] The present application describes embodiments of a method for reduction and prevention of damaged skin stasis comprising applying a composition or material comprising water-soluble or water-insoluble copper compounds to said damaged skin. In some embodiments, composition is an aqueous solution of water-soluble copper compounds or suspension of water-insoluble copper compounds in water, in a solvent or in a mixture thereof. This composition is in a form of a liquid, spray, gel, ointment or powder. [0008] In other embodiments, said material is a woven or non-woven fabric, a foam, a knit fabric, or any type of fabric that is used to make wound dressings, plasters, gauze or the like. This material is impregnated with about 0.1-10% w/w water-insoluble copper particles or coated with about 0.1-10% w/w water-insoluble copper particles.

[0009] In a further embodiment, said water-insoluble copper particles are specifically cuprous iodide (Cul) or cuprous oxide (ί¾0) particles. The exemplary material of the embodiments is a woven fabric impregnated or coated with approximately 1-3% w/w cuprous iodide (Cul) particles or a sterile wound dressing impregnated or coated with approximately 1-3% w/w CU2O particles.

[0010] In yet further embodiment, said composition is in a form of a liquid spray or ointment containing water-soluble copper compounds. The exemplary water-soluble copper compounds of the embodiments are cupric sulphate (CuSOz _t ) or cupric chloride (CuCb).

[0011] In another embodiment, said material is in a form of a polymeric film, fibre, filament or sheath. The exemplary polymeric film of the embodiments comprise polymers selected from the group of polyester, polypropylene, polyethylene, Nylon 66, Nylon 6, polyamide and polyurethane. The polymeric material of the present embodiment comprises water-insoluble particles of copper compounds in a powdered form, embedded directly inside said film, fibre, filament or sheath, wherein a portion of said particles being exposed and protruding from the surface of the film, fibre, filament or sheath, or said polymeric material is coated with water-soluble copper compounds or water-insoluble particles of copper compounds. The exemplary water-insoluble particles of copper compounds of the present embodiment are selected from cuprous iodide, cuprous oxide and cupric oxide, or combinations thereof, in a powdered form.

[0012] Various embodiments may allow various benefits, and may be used in conjunction with various applications. The details of one or more embodiments are set forth in the accompanying figures and the description below. Other features, objects and advantages of the described techniques will be apparent from the description and drawings and from the claims

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Disclosed embodiments will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended figures. The drawings included and described herein are schematic and are not limiting the scope of the disclosure. It is also noted that in the drawings, the size of some elements may be exaggerated and, therefore, not drawn to scale for illustrative purposes. The dimensions and the relative dimensions do not necessarily correspond to actual reductions to practice of the disclosure. [0014] Figs la and lb schematically show a side view and a top view, respectively, of the ex- vivo explant model of the present invention.

[0015] Fig. 2a shows the experimentally generated graph of burn area of the explants at different days following wounding.

[0016] Fig. 2b schematically shows the explant with the burn area having the zone of stasis.

[0017] Fig. 2c shows the photographs of the explants treated with saline only (Control) and with copper ions (0.2 m M of cuprous iodide and 0.2 mM cuprous oxide).

[0018] Figs. 3a, 3c and 3e show the cytokine expression in burn-induced inflammation in human ex vivo skin and comparison between the IL-6, IL-8 and TGF-b secretion, respectively, in the untreated skin explants (Naive Controls) and the wounded skin explants (Burn Controls) following wounding.

[0019] Figs. 3b, 3d and 3f show the effect of addition of copper ions (0.02 mM or 1 mM) on secretion of IL-6, IL-8 and TGF-b, respectively, as compared to Naive Controls and Burn Controls on Day 4 (Figs. 3b and 3d) and on Day 6 (Fig. 3f) after wounding.

[0020] Figs. 4a-4c show histologically stained samples of skin tissue at Days 0, 6 and 27 after wounding, respectively.

DETAILED DESCRIPTION

[0021] In the following description, various aspects of the present application will be described. For purposes of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the present application. However, it will also be apparent to one skilled in the art that the present application may be practiced without the specific details presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the present application.

[0022] The term "comprising", used in the claims, is "open ended" and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. It should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It needs to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression "a material comprising x and z" should not be limited to materials consisting only of compounds x and z. Also, the scope of the expression "a method comprising the steps x and z" should not be limited to methods consisting only of these steps. [0023] Unless specifically stated, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within two standard deviations of the mean. In one embodiment, the term "about" means within 10% of the reported numerical value of the number with which it is being used, preferably within 5% of the reported numerical value. For example, the term "about" can be immediately understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In other embodiments, the term "about" can mean a higher tolerance of variation depending on for instance the experimental technique used. Said variations of a specified value are understood by the skilled person and are within the context of the present invention. As an illustration, a numerical range of "about 1 to about 5" should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges, for example from 1-3, from 2-4, and from 3-5, as well as 1, 2, 3, 4, 5, or 6, individually. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Unless otherwise clear from context, all numerical values provided herein are modified by the term "about". Other similar terms, such as "substantially", "generally", "up to" and the like are to be construed as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skilled in the art. This includes, at very least, the degree of expected experimental error, technical error and instrumental error for a given experiment, technique or an instrument used to measure a value.

[0024] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. Unless otherwise defined, all terms (including 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. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity.

[0025] The present application provides a method for prevention and reduction of damaged skin stasis comprising applying a composition or material comprising copper compounds to said damaged skin. Said composition can be an aqueous solution of water-soluble copper compounds or suspension of water-insoluble copper compounds in water, in a solvent or in a mixture thereof. Said composition can be, for example, in a form of a liquid, spray, gel, ointment or powder. Said material can be a woven or non-woven fabric, a foam, a knit fabric, or any type of fabric that is used to make wound dressings, plasters, gauze or the like.

[0026] In some embodiments, the material is impregnated with about 0.1-10% water-insoluble copper particles. In other embodiments, the material is coated with about 0.1-10% water-insoluble copper particles. Examples of the water-insoluble copper particles used in the material of the present embodiments are cuprous iodide (Cul) and cuprous oxide (ί¾0) particles. In a specific embodiment this material is a sterile wound dressing impregnated with approximately 1-3% weight/weight water-insoluble C¾0 particles. In a specific embodiment, the composition is in a form of a liquid spray or ointment containing water-soluble copper compounds, for example cupric sulphate (CuSOz _t ) or cupric chloride (CuCE).

[0027] In a particular embodiment, the material comprising copper compounds is a polymeric film, fibre, filament or sheath. The polymers used in the polymeric film is selected from polyester, polypropylene, polyethylene, Nylon 66, Nylon 6, polyamide and polyurethane. In a specific embodiment, this polymeric material comprises microscopic water-insoluble particles of copper compounds, such as cuprous oxide (ί¾0) and/or cupric oxide (CuO) in a powdered form, embedded directly inside said film, fibre, filament or sheath, wherein a portion of said particles being exposed and protruding from the surface of the film, fibre, filament or sheath. In yet further specific embodiment, said polymeric material is coated with the water-insoluble particles of copper compounds. The material being embedded or coated with the copper compounds is capable of releasing Cu(I) ions, Cu(II) ions or combination thereof upon contact with the damaged skin.

EXAMPLES

Ex-Vivo Model

[0028] Human skin was obtained from healthy donors undergoing abdominal dermolipectomy after receiving informed consent. A 0.5mm split-thickness skin graft was harvested using a dermatome (Aesculap AG & Co. KG, Tuttlingen, Germany) and was cut into 1 cm pieces. Round burn wounds of with a diameter of 25 mm were inflicted in the 0.7 cm skin pieces by exposure to a soldering iron (95°C, 2 Sec). Triplicate pieces were kept intact as naive controls. The injured and intact skin samples were placed dermis down on a stainless-steel grid and cultured at the air-liquid interface at 37°C with 5% C02 in Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal calf serum (Biological Industries, Beit Ha'emek, Israel), and penicillin/streptomycin (100 IU/mL penicillin, 100 mg/mL streptomycin; Invitrogen). Culture medium was refreshed twice a week. The explants were cultured up to 27 days after wounding. Each individual experiment was performed with skin explants obtained from the same individual and the experiments were repeated at least three times.

[0029] Reference is now made to Figs la and lb schematically showing a side view and a top view, respectively, of this ex-vivo explant model. The skin explants are placed dermis down on a stainless-steel grid. Culture media is added to the bottom of the well, without reaching the epidermal layer, which is exposed to the air. 3 pL of saline only or containing different concentrations of the copper solutions are carefully added on top of the burned area and around it making sure it does not reach the culture media in the bottom of the well.

Copper source and application on the skin explants

[0030] As a source of copper ions, two items were used: woven fabric impregnated with about 1% copper iodide particles and sterile wound dressings impregnated with about 1% weight/weight cuprous oxide particles. Prior to use, the copper iodide impregnated fabric was sterilised using UV light. 0.84 grams of the copper iodide impregnated fabrics and 3.6 grams of the cuprous oxide impregnated wound dressings were immersed in 25 ml of 0.9% saline overnight at 37°C. The resulting concentration of copper ions in the medium was determined by using Aquachek™ copper ions test strips, and these solutions served as copper ions stock solutions, identified hereafter as copper iodide solution and cuprous oxide solution, respectively, to clearly identify the copper ion source. From the stock solutions, solutions of 0.02 m M or 1 mM of copper ions were prepared in saline. Three pL of saline only, 0.02 mM or 1 mM copper solutions were then added on Day 0 and then after every two days onto the skin explants on top of the burn area and the epidermis around the burn area making sure that they do not reach the medium in the chamber but stay on the air interface on top of the skin. Each control and treatment were performed in at least three replicate explants.

Measurement of viability

[0031] To assess the cell viability throughout the culture period a 3-(4,5-dimethylthiazol-2-yl)- 2, 5 -diphenyl tetrazolium bromide (MTT) assay was performed at different days after wounding as follows:

Bioassay

[0032] During harvesting time points, the spent medium from all Test Groups was collected and centrifuged at 1500 x g for 5 minutes to remove particulates. Media were kept at -80 °C until use. The secretion levels of IL-6, IL-8 and TGF-b were measured by commercial Enzyme-Linked Immunosorbent Assay (ELISA) kits according to manufacturer instructions (Biolegend, San Diego, CA). Briefly, plates were coated for 24 hours prior to assay with a specific anti-human capture antibody. At the day of the assay, coated plates were incubated with samples, followed by washes for unbound molecules. Then additional detection antibody was added and detected by Avidin-HRP solution. Finally, wells were incubated with a substrate solution, while absorbance was measured at 570nm.

Statistical Analysis

[0033] The experimental values are presented as average of three replicates and standard errors of the mean (SEM) are provided. Significant differences between values were analysed using the unpaired /-test, while significant results are for p < 0.05.

Experimental Results

[0034] Reference is now made to Fig. 2a showing the experimentally generated graph of burn area of the explants at different days following wounding. The p values between the treatment groups and the untreated Burn Control groups is shown. Fig. 2b schematically shows the explant with the burn area having the zone of stasis. Fig. 2c shows the actual pictures of the explants treated with saline only (Control) or with 0.02 m M copper ions. The differences in size of the wounds between the treatments, especially in the zone of stasis, is clearly visible in Fig. 2c. No significant differences were measured in the metabolic activity, as determined by the MTT assay, between the burned skins explants grown exposed to saline only or exposed to 0.02 mM or 1 mM of copper ions obtained from both test solutions during the ex-vivo explants culture (data not shown).

[0035] As can be seen in Fig. 2a, the burn-wounds treated by saline only increased significantly becoming almost 40% larger three days following wounding and then started to decrease gradually reaching the initial wound size 14 days following wounding. In contrast, the size of the wounds treated with 0.02 mM copper ions obtained from the copper iodide or cuprous oxide particles almost did not change. The size of the wounds treated with saline containing 0.02 mM copper ions was statistically smaller than the wounds treated with saline only three days following wounding and similarly smaller wounds were measured in the copper-treated explants 5- and 10-days following wounding. As clearly seen in Fig. 2c, the skin zone which did not increase in size following burning in the copper-treated explants is the zone of stasis. [0036] In order to further understand how addition of copper ions affected the skin explants, the amounts of Interleukin 6 (IL-6), Interleukin 8 (IL-8) and transforming growth factor beta (TGF-b) found in the medium of the various treated explants were determined. Figs. 3a, 3c and 3e show the cytokine expression in burn-induced inflammation in human ex vivo skin and comparison between the IL-6, IL-8 and TGF-b secretion by the untreated skin explants (Naive Controls) and the wounded skin explants (Burn Controls) following wounding. In these figures, the p values of a /-test between the cytokine expressions per a particular day following wounding are shown.

[0037] Reference is also made to Figs. 3b, 3d and 3f showing the effect of addition of copper ions (0.02 m M or 1 mM) on secretion of IL-6, IL-8 and TGF-b, respectively, as compared to Naive Controls and Burn Controls on Day 4 (treated with saline only) (Figs. 3b and 3d) and on Day 6 (Fig. 3f) after wounding. In the graphs, the star sign indicates the statistically significant difference as compared to the Naive Controls, while the net sign "#" indicates the statistically significant difference as compared to the untreated (treated with saline only) Burn Controls.

[0038] As seen in Fig. 3a, in the Burn Controls, there was a clear increase in IL-6 secretion after one day and four days following wounding as compared to the Naive Controls. Addition of 0.02 mM or 1 mM of copper ions from the Cul stock solution and of 1 mM of copper ions from the C¾0 stock solution to the burned explants abolished the increase in IL-6 secretion by the explants, and actually resulted in even reduced IL-6 secretion as compared to the IL-6 secretion by the Naive Controls (see Fig. 3b). Similarly, the statistically significant increase of IL-8 and TGF-b in the Burn Controls as compared to the Naive Controls on Day 4 after wounding (see Figs. 3c and 3e, respectively) was significantly attenuated or abolished when the burn wounds were exposed to 0.02 mM or 1 mM of copper ions from both copper sources (see Figs. 3d and 3f).

[0039] Reference is now made to Figs. 4a-4c showing histologically stained samples of skin tissue at Days 0, 6 and 27, respectively. At Day 0 (see Fig. 4a), burns were inflicted on skin explants. Upper image in Fig. 4a represents normal healthy skin. Lower image represents damaged skin tissue, immediately following the burn infliction. Epidermis is detached from the dermis (as indicated with the solid arrow).

[0040] At Day 6 (Fig. 4b) and Day 27 (Fig. 4c), old, separated epidermis, following the burn infliction, without or with copper ion treatments, is indicated with the solid arrow. Re-epithelisation is noted on Day 27 (see Fig. 4c) only upon copper-ion treatments, as indicated by the stripe arrows. Samples were fixated on indicated time points and histologically stained by Masson's trichrome dye. Histological analyses revealed that only in the copper-ion treated samples clear re-epithelisation occurred (see Fig. 4c, stripe arrows).

[0041] While certain features of the present application have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will be apparent to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the present application.