WO2008037262A1

WO2008037262A1 - Wound healing composition comprising phosphate, iron and copper

Info

Publication number: WO2008037262A1
Application number: PCT/DK2007/050133
Authority: WO
Inventors: Niels Erik Van Wyhe
Original assignee: Stormollen AS
Current assignee: Stormollen AS
Priority date: 2006-09-28
Filing date: 2007-09-28
Publication date: 2008-04-03
Anticipated expiration: 2009-03-28

Abstract

The present invention relates to a pharmaceutical composition for wound healing or for accelerating wound healing, said composition comprising phosphate ions, iron ions, and copper ions. The relative amounts (w/w) of phospor (P), iron (Fe) and copper (Cu), P : Fe : Cu, in said composition are given by the ratios 100 : 15 to 50 : 5 to 15. The invention further relates to methods for making and using the composition.

Description

WOUND HEALING COMPOSITION COMPRISING PHOSPHATE, IRON AND COPPER

This application is a non-provisional of U.S. provisional application Serial No. 60/847,679 filed 28-09-2006, which is hereby incorporated by reference in its entirety.

All patent and non-patent references cited in the above-cited application, or in the present application, are hereby incorporated by reference in their entirety.

Field of invention The present invention relates to a wound healing composition and uses thereof in the treatment of wounds in an individual such as the human or animal body. In one aspect the compositions according to the present invention are used in the treatment of bedsores in sows.

Background of invention

The healing of wounds or related forms of tissue damage generally depends on cellular proliferation and the formation of new connective, endothelial, and epithelial tissue as explained briefly herein below.

Several agents have been reported to favorably influence the cellular processes involved in wound healing, e.g., polypeptid growth factors, allantoin, Vitamin A (and derivatives), zinc, exogenous DNA, and aloe vera preparations. These compounds operate through various poorly defined mechanisms and display varying degrees of effectiveness in particular applications

When an injury occurs, cell damage comes from the precipitating event, such as a cut, resulting in ruptured cells and severed or crushed capillaries and other blood vessels. The interruption of blood flow produces anoxia, causing the death of additional cells. Within 15 minutes of injury the wound is filled with dead and dying cells, extracellular substances collagen, elastic fibers, fat and ground substances, extravasated blood, and possibly bacteria and viruses introduced by the injurious agent. Tissue damage is not restricted to the initial area of injury, it may increase over the next several hours or days as a result of the release of lysomal enzymes from the injured cells or as a consequence of swelling and infection. (See Reese et al., Role of Fibronectin in Wound Healing, the subject matter of which is hereby incorporated by reference). Coagulation, the first phase of the healing process, bridges the gap between the injury and the inflammatory response, the second phase of wound healing. It stops the loss of blood and restores some of the mechanical and physical integrity to the damaged tissue. The proteins of the coagulation cascade are normally confined to the intravascular space but are released into the tissues after blood vessel disruption. Coagulation is initiated by either the intrinsic or extrinsic pathway, both of which must be activated for maximum fibrin formation. The result of the activation of either of the two coagulation pathways is the generation of thrombin, which in turn catalyzes the conversion of fibrinogen to fibrin monomer. Fibrin monomer spontaneously polymerizes to form the clot. Just after polymerization, the fibrin fibers are held together by hydrophobic and ionic forces and are relatively unstable. Fibrin stabilizing factor, which is generated from its proenzyme by thrombin, covalently cross-links the fibrin fibrils by catalyzing a transamination reaction between glutamine and lysine residues in adjacent fibers. The cross-linking of fibers greatly increases the mechanical strength of the clot. Platelets, along with other blood cells, are trapped in the fibrin mesh as the clot forms by fibronectin. The platelet surfaces are heavily coated, and each looks like a nexus with the fibrin fibers radiating out from it.

The second phase of wound repair is the inflammatory response, which is necessary for subsequent phases of healing. It is initiated by the release of histamine and serotonin from platelets and mast cells and by kinins. Histamine and kinins act to increase capillary dilation, opening previously closed capillaries in the area of injury. The increased blood flow through the capillary beds produces two of the characteristics of the inflammatory response: redness and heat. Prostaglandin release within a few hours of injury results in the full development of the inflammatory response, which may last from 3 to 5 days depending on the extent of the injury. The extreme vasodilation produced by the factors just discussed causes a widening of the endothelial cell junctions lining the capillaries. Fluid and macromolecular components of blood escape into the tissues through the gaps, producing swelling, the third characteristic of the inflammatory response. If the swelling is extensive, it may interrupt blood flow, increasing the extent of injury as a result of anoxia. Pain, the final characteristic of inflammation, results from a combination of the kinins as well as the direct effect of lysosomal enzymes and pressure from the swelling on nerve endings. Control of infection at the wound site is of critical importance in successful wound repair. Infections delay healing, enlarge the wound lesion, may lead to systemic infection, and greatly increase the likelihood of disfiguring and physically debilitating scars. Vasodilation of the capillary beds reduces the velocity of blood through the capillaries. This, along with the production of potent chemotactic factors from the complement fixation and the release of chemotactic agents from the damaged tissue, cause the accumulation of polymorphonuclear leukocytes ("PMN's") along the walls of the capillaries which are the host's major cellular defense against infection. The PMN's subsequently pass through the endothelial junctions of the capillary wall into the site of the injury. If bacteria are present in the wound, they may release soluble chemotactic factors and/or activate complement with the subsequent generation of chemotactic fragments. PMN's at the site of an infection or injury release substance that affect the PMNs' mobility, keeping them at the site. Fibronectin facilitates the attachment of the bacterium to the membrane of the phagocyte.

Dead cells, cellular debris, and extracellular proteins must then be removed or readsorbed to allow revascularization and repair to continue. Macrophages are primarily responsible for the clearance of wound debris. Wound macrophages, like wound PMN's, are actively phagocytic. They migrate into the wound using the fibers of the fibrin clot as a scaffold to move within the clot, attaching to the fibers through fibronectin. The macrophages encounter, engulf, and destroy the dead cells trapped in the clot matrix, as well as the damaged cells from the wound margin. The fibrin clot itself is resolved primarily by the activation of the plasminogen that was incorporated into the fibers during their formation. Some of the fibrin fragments are engulfed by macrophages in the area. Since most of the clot fragments are released away from the area of the most intense macrophage activity, many of the fragments are removed by lymphatic drainage and thus enter the circulation. These soluble complexes are removed by the sessile cells of the RES, primarily those of the spleen and liver. Also, PMN's trapped in the clot die as a result of anoxia, releasing their lysosomal contents. These enzymes attack the surrounding clot and dissolve it. Although the release of lysosomal enzymes by PMN's may be considered beneficial to the host in most cases, they may also increase tissue destruction and delay healing. If the PMN's accumulate rapidly within the wound and remain there (as in an infection), their lysosomal enzymes dissolve significant portions of the clot, removing the framework used by the macrophages and fibroblasts to move into the wound and recolonize it. These areas of destruction must eventually be drained or slowly removed by the macrophages. The dissolved portion of the clot is then replaced as part of the chronic inflammatory response.

Repair, or fibroplasia, of the damaged tissue occurs during some of the above stages. Within 12 to 24 hours of injury, fibroblasts, including those at some distance from the wound margins, begin to move toward the area of injury and to proliferate. This response is apparently due to factors released by the injured tissue and platelets and possibly to factors released by the kinin, complement or coagulation cascades. The proliferating fibroblasts derive part of their nutrients from the components of tissue debris and cells released by macrophages. The fibroblast phase may last 2 to 4 weeks in a skin wound, whereas it may persist several months in an injury to the stomach or intestines. Fibroblasts, as the macrophages did, use the fibers of the fibrin clot as a scaffold to move into and within the damages area. The Fibroblasts synthesize and secrete sufficient quantities of fibronectin to promote their own attachment to fibronectin deficient substrates.

Angiogenesis, or revascularization, begins with the growth of capillary beds into the area directly behind the fibroblasts. In the early phases of wound repair, the capillaries are much more numerous than in normal tissue, which probably reflects the high oxygen and nutrient requirements of the rapidly regenerating tissue. The capillaries are very leaky, which facilitates the movement of cells and macromolecules into the wound site. Eventually, the capillaries originating from one side of the wound grow into contact with capillaries originating from the other sides and fuse, reestablishing complete circulation within the wound.

By the end of the fifth day after the injury, fibroblasts begin laying down large quantities of collagen. The collagen molecule is synthesized on the membrane of the endoplastic reticulum. It then undergoes extensive postranslational modification, hydroxylation, glycosylation, and further steps to form the procollagen molecule. The procollagen molecule is then secreted and is further modified to tropocollagen by specific serum peptidases. These activated tropocollagen molecules quickly polymerize to form increasingly large collagen fibers. Thereafter, crosslinking among the collagen fibers occurs. The collagen network in effect replaces the fibrin clot as the major structural element of the wound. This becomes particularly important during the remodeling phase of wound healing.

Reepithelialization begins to occur within a few hours of injury as the attachment of the epithelial cells to the dermis loosened near the margin of the wound, and the cells begin to migrate over the defect, always maintaining contact with the mesenchymal tissue. By 48 hours after the injury, the cells are also beginning to proliferate to replace the lost cells. The epithelial cells continue to divide after the bridge is complete to form a thicker epithelium. Wound contracture aids reepithelialization insofar as it reduces the size of the defect to be reepithelialized by as much as 50%. Contracture is believed to occur as a result of the cellular element of the granulation tissue in the wound~the fibroblasts and myofibroblasts.

Remodeling is the last step of wound healing. Scar tissue continues to gain tensile strength for several months after collagen content stabilizes. This gain in strength comes from the rearrangement of the collagen in the wound and perhaps from increased crosslinking of the collagen. Collagen accumulation is the sum of synthesis and destruction, and both occur simultaneously during the wound healing process. After about 14 days, a balance between collagen synthesis and degradation is reached. The collagenase involved in the remodeling comes from epithelial cells, from fibroblasts encountering new epithelium, and from macrophages that contain collagenase in their lysosomes.

Typical wound healing takes anywhere from 5 to 21 days. This time period is of course longer for the immune compromised patient because such patients are frequently unable to sufficiently stabilize the wound and ward off infection which prevents the proper adherence of fibrin, fibronectin or collagen at an acceptable rate at the locus of the wound. For example, those with vasculitis or other rheumatic or diabetic diseases frequently experience wound healing times far in excess of several weeks. Diabetics frequently develop lesions that take weeks to heal.

Others, such as those with artificial limbs, have continuous injuries at the point of contact between the limb and the point of attachment to the body. Burns also present healing problems insofar as the burned tissue is incapable of timely production of fibrin. Accordingly, there is a great need to shorten the duration of time necessary for wound or burn healing to occur. Summary of invention

The pharmaceutically active compositions according to the present invention are capable of treating and/or stimulating and/or accelerating wound healing in an individual. The compositions have great value in medical and veterinary practice and can be used to improve or speed up the healing of many types of wounds or lesions, including fx. bedsores in animals, such as pigs, in particular sows, cuts in fingers or the forearm resulting from unintentional handling of e.g. kitchen knives, severe sunburns, wounds resulting from burns or cuts with sharp objects in the workplace, surgical incisions, burns induced by heat, chemicals, or irradiation, as well as abrasions, lacerations and amputations.

The pharmaceutically active composition can also be used when wound healing of an individual is impaired, such as in diabetic patients, or in individuals undergoing cancer chemotherapy (e.g. with adriamycin or cyclophosphamide) or corticosteroid treatment, or in patients after therapeutic or accidental exposure to ionizing radiation.

In one aspect the present invention provides pharmaceutically active compositions and pharmaceutical products which can efficiently promote accelerated healing of wounds, ulcers, burns, and other types of lesions in an individual.

The wound healing agents of the pharmaceutically active composition are preferably non-antigenic and preferably they do not carry genetic information.

The effective wound healing agents of the pharmaceutically active compositions according to the present invention are inexpensive, uniform and stable. The wound healing agents of the pharmaceutically active composition can be effectively administered locally. The wound healing agents have no undesirable toxic effects and they can be administered to animals and humans to effectively promote healing of wounds, ulcers, burns, and other types of lesions. The effective wound healing agents are preferably applied locally and/or topically in a variety of delivery vehicles, such as gels and ointments.

The present invention in one aspect is directed to a pharmaceutical composition comprising at least one iron compound, at least one copper compound and at least one phosphor compound, in combination with a physiologically acceptable carrier adapted for local and/or topical administration, wherein said compounds are each present in a concentration so that the composition is capable of promoting wound healing in an individual.

In another aspect there is provided a pharmaceutical composition for wound healing or for accelerating wound healing, said composition comprising

i) phosphate ions, ii) iron ions, and iii) copper ions,

wherein the relative amounts (w/w) of phospor (P), iron (Fe) and copper (Cu), P : Fe : Cu, in said composition are given by the ratios 100 : 15 to 50 : 5 to 15.

Definitions

Wound: The term refers to cuts, incisions, abrasions, lacerations, amputations, burns induced by heat, ionizing radiation, ultraviolet radiation including sunlight, electricity, or chemical substances as well as to other forms of lesions such as ulcers, pressure sores and bedsores.

Individual: The term refers to vertebrates, particular members of the mammalian species, and includes, but is not limited to domestic animals, such as cattle, horses, pigs, sheep, mink, dogs, cats, mice, guinea pigs, rabbits, rats; sports animals, such as horses, poly ponies, dogs, camels, and primates, including humans.

Pharmaceutically active composition: Any composition according to the invention comprising pharmaceutically active compounds, including at least one iron compound, at least one copper compound and at least one phosphor compound in combination with a physiologically acceptable carrier adapted for local and/or topical administration, said composition exerting a physiological effect when administered to an individual. The pharmaceutically active composition can be formulated e.g. as a an ointment or a salve or a creme or a lotion or a gel, in which case the pharmaceutically active composition is mixed with e.g. one or more of an emulsifying agent, a stabilizing agent, a dispersing agent, a suspending agent, a thickening agent and a colouring agent.

Figure legends Figure 1 : Graphic illustration on the grade of shoulder ulcers at the inclusion of the sows and in the end at the weaned/exclusion time for control and Stalosan Salve. Control Weaned/excluded versus start, t=7,8 ^* 10^'5; Stalosan Salve Weaned/excluded versus start, t=0,03; Start Stalosan Salve versus control, t=0,05; Weaned/excluded: Stalosan Salve versus control, t=0,0002.

Detailed description of the invention

One strategy for the administration of the pharmaceutically active compounds according to the present invention is to administer the pharmaceutically active composition according to the invention locally and/or topically e.g. in gels, ointments, solutions, impregnated bandages, liposomes, biodegradable microcapsules comprising the pharmaceutically active compounds according to the present invention.

Compositions or dosage forms for topical application may include solutions, lotions, ointments, cremes, gels, suppositories, sprays, aerosols, suspensions, dusting powder, impregnated bandages and dressings, liposomes and biodegradable polymers.

In one embodiment there is provided a semi-solid pharmaceutical composition for wound healing or for accelerating wound healing, said composition comprising

i) phosphate ions, ii) iron ions, and iii) copper ions,

The iron ions are preferably provided by one or more of iron chloride, iron fluoride, iron nitrate, iron oxide and/or iron sulphate. The copper ions are preferably provided by one or more of copper acetate, copper bromide, copper carbonate, copper chloride, copper fluoborate, copper gluconate, copper nitrate, copper oxide and/or copper sulphate. The phosphate ions are preferably provided by one or more of calcium diphosphate, calcium phosphate, monoammonium phosphate and/or diammonium phosphate .

In embodiments of the present invention, the above-mentioned pharmaceutical composition comprises phosphate ions, iron ions and copper ions, wherein the relative amounts (w/w) of phospor (P) and iron (Fe), P : Fe, in said composition are given by the ratio 100 : 20 to 50; such as 100 : 25 to 50; for example 100 : 30 to 50; such as 100 : 35 to 50; for example 100 : 40 to 50; such as 100 : 45 to 50; for example 100 : 15 to 45; such as 100 : 15 to 40; for example 100 : 15 to 35; such as 100 : 15 to 30; for example 100 : 15 to 25; such as 100 : 15 to 20; for example 100 : 20 to 25; such as 100 : 25 to 30; for example 100 : 30 to 35; such as 100 : 35 to 40; for example 100 : 40 to 45.

In the same or different embodiments of the pharmaceutical composition according to the invention, the relative amounts (w/w) of phospor (P) and copper (Cu), P : Cu, in said composition are given by the ratio 100 : 6 to 15; such as 100 : 7 to 15; for example 100 : 8 to 15; such as 100 : 9 to 15; for example 100 : 10 to 15; such as 100 : 1 1 to 15; for example 100 : 12 to 15; such as 100 : 13 to 15; for example 100 : 14 to 15; such as 100 : 5 to 14; for example 100 : 5 to 13; such as 100 : 5 to 12; for example 100 : 5 to 1 1 ; such as 100 : 5 to 10; for example 100 : 5 to 9; such as 100 : 5 to 8; for example 100 : 5 to 7; such as 100 : 5 to 6; for example 100 : 6 to 7; such as 100 : 7 to 8; for example 100 : 8 to 9; such as 100 : 9 to 10; for example 100 : 10 to 1 1 ; such as 100 : 1 1 to 12; for example 100 : 12 to 13; such as 100 : 13 to 14.

The phosphate ions are preferably provided by calcium diphosphate and calcium phosphate; iron ions are preferably provided by iron sulphate and/or iron oxide; and copper ions are preferably provided by copper sulphate.

In a preferred embodiment the pharmaceutical composition comprises phosphate ions, iron ions and copper ions, wherein the relative amounts A : B : C (w/w of anhydrous compounds) of

A: calcium diphosphate and calcium phosphate; B: iron sulphate and/or iron oxide; and C: copper sulphate; are given by the ratios 100 : 1.5 to 10 : 0.5 to 5.0.

In embodiments of the present invention the above-mentioned pharmaceutical composition comprises phosphate ions, iron ions and copper ions, wherein the relative amounts of A : B (w/w of anhydrous compounds) are given by the ratio 100 : 2.0 to 10; such as 100 : 2.5 to 10; for example 100 : 3.0 : 10; such as 100 : 3.5 to 10; for example 100 : 4.0 to 10; such as 100 : 4.5 to 10; for example 100 : 5.0 to 10; such as 100 : 5.5 to 10; for example 100 : 6.0 to 10; such as 100 : 6.5 to 10; for example 100 : 7.0 to 10; such as 100 : 7.5 to 10; for example 100 : 8.0 to 10; such as 100 : 8.5 to 10; for example 100 : 9.0 to 10; such as 100 : 9.5 to 10; for example 100 : 1 .5 to 9.5; such as 100 : 1 .5 to 9.0; for example 100 : 1.5 to 8.5; such as 100 : 1.5 to 8.0; for example 100 : 1.5 to 7.5; such as 100 : 1 .5 to 7.0; for example 100 : 1 .5 to 6.5; such as 100 : 1 .5 to 6.0; for example 100 : 1.5 to 5.5; such as 100 : 1.5 to 5.0; for example 100 : 1 .5 to 4.5; such as 100 : 1.5 to 4.0; for example 100 : 1.5 to 3.5; such as 100 : 1.5 to 3.0; for example 100 : 1 .5 to 2.5; such as 100 : 1 .5 to 2.0; for example 100 : 2.0 to 2.5; such as 100 : 2.5 to 3.0; for example 100 : 3.0 to 3.5; such as 100 : 3.5 to 4.0; for example 100 : 4.0 to 4.5; such as 100 : 4.5 to 5.0; for example 100 : 5.0 to 5.5; such as 100 : 5.5 to 6.0; for example 100 : 6.0 to 6.5; such as 100 : 6.5 to 7.0; for example 100 : 7.0 to 7.5; such as 100 : 7.5 to 8.0; for example 100 : 8.0 to 8.5; such as 100 : 8.5 to 9.0; for example 100 : 9.0 to 9.5 and wherein B is iron sulphate and/or iron oxide.

In the same or different embodiments of the pharmaceutical composition according to the invention the relative amounts of A : C (w/w of anhydrous compounds) are given by the ratio 100 : 1.0 to 5.0; such as 100 : 1.5 to 5.0; for example 100 : 2.0 to 5.0; such as 100 : 2.5 to 5.0; for example 100 : 3.0 to 5.0; such as 100 : 3.5 to 5.0; for example 100 : 4.0 to 5.0; such as 100 : 4.5 to 5.0; for example 100 : 0.5 to 4.5; such as 100 : 0.5 to 4.0; for example 100 : 0.5 to 3.5; such as 100 : 0.5 to 3.0; for example 100 : 0.5 to 2.5; such as 100 : 0.5 to 2.0; for example 100 : 0.5 to 1 .5; such as 100 : 0.5 to 1.0; for example 100 : 1 .0 to 1.5; such as 100 : 1 .5 to 2.0; for example 100 : 2.0 to 2.5; such as 100 : 2.5 to 3.0; for example 100 : 3.0 to 3.5; such as 100 : 3.5 to 4.0; for example 100 : 4.0 to 4.5.

The concentration of the above-mentioned active compounds in pharmaceutically acceptable carriers can range e.g. from 0.1 to 45%, such as 0.1 to 40%, for example 0.1 to 30%, such as 0.1 to 10%, for example 0.1 to 5%. The dose used in a particular formulation or application will be determined by the requirements of the particular type of tissue lesion and the constraints imposed by the characteristics and capacities of the carrier materials.

In one embodiment the pharmaceutical composition comprises phosphate ions, iron ions and copper ions, wherein the absolute amount of iron ions in said composition is in the range of from 0.25 % to 4.0 % (w/w).

In preferred embodiments the pharmaceutical composition according to the invention comprises phosphate ions, iron ions and copper ions, wherein the absolute amount of iron ions in said composition is in the range of from 0.50 % to 4.0 % (w/w), such as from 1.0 % to 4.0 %, for example from 1.5 % to 4.0 %, such as from 2.0 % to 4.0 %, for example from 2.5 % to 4.0 %, such as from 3.0 % to 4.0 %, for example from 3.5 % to 4.0 %, such as from 0.25 % to 3.5 %, for example from 0.25 % to 3.0 %, such as from 0.25 % to 2.5 %, for example from 0.25 % to 2.0 %, such as from 0.25 % to 1.5 %, for example from 0.25 % to 1 .0 %, such as from 0.25 % to 0.50 %, for example from 0.50 % to 1.0 %, such as from 1.0 % to 1.5 %, for example from 1.5 % to 2.0 %, such as from 2.0 % to 2.5 %, for example from 2.5 % to 3.0 %, such as from 3.0 % to 3.5 %.

In the same or different embodiments of the pharmaceutical composition according to the invention comprises phosphate ions, iron ions and copper ions, wherein the absolute amount of copper ions in said composition is in the range of from 0.05 % to 1 .8 % (w/w).

In preferred embodiments the pharmaceutical composition according to the invention comprises phosphate ions, iron ions and copper ions, wherein the absolute amount of copper ions in said composition is in the range of from 0.1 % to 1.8 %, for example from 0.1 % to 1.5 %; such as from 0.25 % to 1 .5 %, for example from 0.50 % to 1.5 %; such as from 0.75 % to 1.5 %, for example from 1.0 % to 1.5 %; such as from 1.25 % to 1.5 %, for example from 0.05 % to 1.25 %; such as from 0.05 % to 1 .0 %, for example from 0.05 % to 0.75 %; such as from 0.05 % to 0.50 %, for example from 0.05 % to 0.25 %; such as from 0.05 % to 0.1 %; for example from 0.1 % to 0.2 %; such as from 0.2 % to 0.4 %; for example from 0.4 % to 0.6 %; such as from 0.6 % to 0.8 %; for example from 0.8 % to 1.0 %; such as from 1.0 % to 1.2 %; for example from 1.2 % to 1.4 %. The active compounds described herein may be combined or used together or in coordination with e.g. an antibiotic, antifungal, or antiviral substance or substances to accelerate the healing of sores or other infection-damaged tissue simultaneously or sequentially with the treatment of the underlying infection. In addition, the active compounds according to the invention can be combined with or used simultaneously or sequentially with other tissue healing promoters, such as epidermal growth factor, fibroblast growth factor, platelet derived growth factor, transforming growth factor alpha, transforming growth factor beta, and insulin-like growth factor 1 (Brunt, J. V., and Tilansner, A., Biotechnology 6:25-30 (1988)) to promote a more rapid healing of damaged tissue.

It is also useful to co-administer the active compounds according to the invention with topical corticosteroid and anti-inflammatory agents to accelerate the healing of lesions in patients suffering from allergic or inflammatory processes, since steroids are known to slow the healing of wounds.

The pharmaceutically active compositions according to the present invention are particularly useful in conditions in which normal wound healing is impaired. Examples of types of wounds that heal poorly or slowly include venous stasis ulcers, decubitus ulcers, and cutaneous and alimentary tract wounds, or ulcers in patients with diabetes, and in patients subjected to irradiation, cancer chemotherapy (e.g. with adriamycin or cyclophosphamide), and topical or systemic anti-inflammatory glucocorticosteriods.

The following supplementary compounds can be administered simultaneously or sequentially in any order with the pharmaceutically active compositions according to the invention in a method for the treatment of wounds. These supplementary compounds can be used in parallel or in series with the pharmaceutically active composition according to the invention. The supplementary compounds include, but are not limited to: allantoin, retinoic acid, aloe vera, glycine, vitamin A, the B vitamins, especially nicotinamide, vitamins C and E, antibacterial agents (e.g., quaternary ammonium compounds, bacitracin, neomycin and polymyxin), comfrey root preparations, platelets and/or platelet extracts, ribonucleosides, collagen, proline, lysine, elastin, fibronectin, glycosaminoglycans, spermidine (and other polyamines), angiogenic factors, zinc, sucrose, and various peptide growth factors such as the somatomedins, lamin, EGF, the IGF's, PDGF, FGF, EDGF, TGF, CDGF, MDGF, and NGF. The pharmaceutically active compositions of the invention may also be used in conjunction with synthetic skin in treating burns and other wounds, and in supporting the healing of skin or corneal transplants.

The pharmaceutically active composition according to the present invention can be incorporated into any suitable ointment, solution, or insert for the treatment of lesions or ulcers resulting e.g. from chemical damage, surgery, injury, burns, or infection.

Medical or veterinary indications for the use of the invention include, but are not limited to the following situations. The pharmaceutical compositions can be used to accelerate the healing of mechanical wounds or abrasions of the skin or other tissues which are exposed by mechanical injury to the skin or gastrointestinal mucosa of the body. The invention can also be used to accelerate the healing of burns inflicted upon the skin, and any underlying tissues which may be exposed by such injury. The burns may be those caused by heat, ionizing radiation, ultraviolet radiation including sunlight, electricity, or chemical substances.

In one interesting embodiment the pharmaceutical compositions are used for treating bedsores in sows. The pharmaceutical compositions are applied to the area of the wound. This has two effects. Firstly, the pharmaceutical composition is brought into contact with the wounded area, thereby promoting wound healing. Secondly, the application of the pharmaceutical composition to the wounded area prevents or reduces dirt and bacteria from getting acces to the actual wound.

Further, the compositions may be used to accelerate the healing of surgical incisions in any part of the body, external or internal, into which a solution or other carrier composition containing the active compounds of the invention may be introduced. The compositions can also be used to accelerate the healing of ischemic ulcers, pressure sores, bed sores, or ulcers caused by diabetes or other disease processes.

In promoting the healing of skin wounds, including bedsores and surgical incisions, it is best to apply the active compounds according to the present invention locally, either in an ointment or in a wound dressing. A topical antibiotic or antiviral agent might be co- administered. The molar equivalent of about 0.1 to 200 mg of a mixture of active compound(s) according to the present invention can be applied per square cm of wound area, or 0.1 to 100 mg per cm of linear incision.

Formulations of the pharmaceutically active composition The pharmaceutically active composition according to the invention is preferably formulated e.g. as an ointment or a salve or a creme or a lotion or a gel. The aforementioned terms will all form a semi-solid composition capable of being applied to a skin area for topical adiministration of the active compounds according to the invention. Regions for topical administration include the skin surface and also mucous membrane tissues of e.g. the rectum, the nose, the mouth, and the throat. Compositions for topical administration via the skin and mucous membranes should not give rise to signs of irritation, such as swelling or redness.

The topical compositions according to the invention may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of an ointment or a salve or a creme or a lotion or a gel. Additionally, the composition according to the invention may - for example - be in the form of a suspension, a solution, a foam, an aerosol, a spray, a suppository, a syrup or a balm. Methods for preparing such compositions are well known in the pharmaceutical industry.

The composition according to the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions.

Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.

Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.

Cremes, ointments or pastes according to the present invention are semi-solid compositions of the bioactive compounds according to the invention for external application. They may be made by mixing the pharmaceutically active compounds in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic acid together with an alcohol such as propylene glycol or a macrogel.

The pharmaceutically active composition may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

Lotions according to the present invention include those suitable for application to the skin. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.

Besides the pharmaceutically active compounds, the pharmaceutical compositions according to the invention can also include non-toxic, pharmaceutically acceptable carriers, diluents and excipients, suitable for topical application, as are well known, see for example Merck Index, Merck & Co., Rahway, NJ. ; and Gilman et al., (eds) (1996) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8^th Ed., Pergamon Press. For standard dosages of conventional pharmacological agents, see, e.g., Physicians Desk Reference (1997 Edition); and U.S. Pharmacopeia National Formulary (1995) United States Pharmacopeial Convention Inc., Rockville, Md.

Typical pharmaceutically acceptable carriers which make up the for going compositions include alginates, carboxymethylcellulose, methylcellulose, agarose, pectins, gelatins, collagen, vegetable oils, mineral oils, stearic acid, stearyl alcohol, petrolatum, polyethylene glycol, polysorbate, polylactate, polyglycolate, polyanhydrides, phospholipids, polyvinylpyrrolidone, and the like.

Dosage forms for the topical administration of an active composition according to the invention include various mixtures and combinations that can be applied topically and will permit even spreading and absorption into the cutaneous surfaces. Non-limiting examples include sprays, mists, aerosols, lotions, creams, solutions, gels, ointments, pastes, emulsions, foams and suspensions. The active compounds or a composition thereof can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives or buffers which may be required. Topical preparations can be prepared by combining the pharmaceutically active composition with conventional pharmaceutically acceptable carriers commonly used in topical dry, liquid and creme formulations.

Ointment and creams can, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. An exemplary base is water. Thickening agents can be used according to the nature of the base. Lotions can be formulated with an aqueous base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like. Powders can be formed with the aid of any suitable powder base, e.g., talc, lactose starch and the like. Drops can be formulated with an aqueous base or non-aqueous base, and can also include one or more dispersing agents, suspending agents, solubilizing agents, surface active agents and the like.

Ointments, pastes, creams and gels also can contain excipients, such as starch, tragacanth, cellulose derivatives, silicones, bentonites, silicie acid, and talc, or mixtures thereof. Powders and sprays also can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, and calcium silicates, or mixtures of these substances.

Solutions of the pharmaceutically active composition can be converted into aerosols or sprays by any of the known means routinely used for making aerosol pharmaceuticals. In general, such methods comprise pressurizing or providing a means for pressurizing a container of the solution, usually with an inert carrier gas, and passing the pressurized gas through a small orifice. Sprays can additionally contain customary propellants, such inert gases such as nitrogen, carbon dioxide, argon or neon.

Multiple inactive ingredients are generally incorporated in topical formulations to improve cosmetic acceptability, and are optional ingredients in the formulations. Such ingredients are included only in therapeutically acceptable forms and amounts. Examples of such ingredients are emulsifiers, emollients, thickening agents, solvents, hydrating or swelling agents, flavours, sweetening agents, surface active agents, colouring agents, anti-foaming agents, preservatives, fragrances, and fillers may also be added, as is well known in the art; for example, preservatives such as methyl paraben and propyl paraben, texturizing agents, thickeners, anticoagulants such as heparin, β-glucan, hormones, hyaluronic acid, immune potentiating agents such as adjuvants and cytokines such as epidermal growth factor, platelet derived growth factor, transforming growth factor and interleukins, and bone morphogenetic proteins, and the like. Polyvinyl alcohol is a particularly preferred gelling polymer and also acts as a texturizing agent, methyl or propyl parabens are particularly preferred preservatives. These other agents may be included in amounts in the range of 0.1 to 5 wt %.

Surface active agents or foaming agents may be added to the formulations and are particularly advantageous for addition to liquid formulations for use as skin cleansers or for aerosol or foam applications. Surface active agents selected for use should not substantially interfere with the antimicrobial or anti-inflammatory effects of the pharmaceutically active composition.

All agents must be non-toxic and physiologically acceptable for the intended purpose, and must not substantially interfere with the activity of the pharmaceutically active composition so as to deleteriously affect the antimicrobial and anti-inflammatory effect. Ingredients are thus only included in therapeutically acceptable amounts. Ingredients to be generally avoided or limited in the formulations of the present invention, at least in amounts greater than 0.01 wt %, are glycerin, glycerols, chloride salts, aldehydes, ketones, long chain alcohols, and triethanolamine.

The dosage of the pharmaceutically active composition depends upon many factors that are well known to those skilled in the art, for example, the particular form of the active ingredient, the condition being treated, the age, weight, and clinical condition of the recipient patient, and the experience and judgement of the clinician or practitioner administering the therapy. A therapeutically effective amount of the pharmaceutically active composition provides either subjective relief of symptoms, or an objectively identifiable improvement, as noted by the clinician or other qualified observer. The dosing range varies with the pharmaceutically active compositions used, its form, the route of administration and the potency of the particular active compound(s). When the formulation is in the form of a dressing, the dressing can be placed on the affected area of the skin and, depending on the degree of moisture at the membrane, may be further moistened with drops of sterile water, tap water, body fluids such as exudate, or, for example 70% ethanol. The dressing may be then secured in place with an occlusive or semi-occlusive layer, such as an adhesive tape or polyurethane film, which keeps the dressing in a moist environment.

Other forms of formulations, such as gels, pastes, ointments, cremes, emulsions, foams, and liquids can be prepared in stable forms, or prepared fresh from one or more phases, for instance in multicomponent kit form, so as to avoid aging and to maximize the therapeutic effectiveness of the pharmaceutically active composition. Formulations are best used within about 30 days after combining the phases.

Suitable kits or containers are well known for maintaining the phases of formulations separate until the time of use. For instance, the pharmaceutically active composition may be packaged separately from therapeutically acceptable carriers, and possibly other ingredients for mixing at the time of use.

The pharmaceutically active composition may be in dressing or patch form for direct application, or may take other suitable forms to generate liquid formulations and the like, such as a coating on the inside surface of a vial or container, a mesh, or a film. A kit containing e.g. one or more phases, for instance, the pharmaceutically active composition may be packaged separately from therapeutically acceptable carriers, and possibly other ingredients for mixing at the time of use, can further provide a sterile carrier such as water (and other ingredients) in a separate container in dosage specific amounts.

In one embodiment the pharmaceutically active composition is Stalosan F (commercially available from Stormøllen A/S Denmark). Stalosan F can be provided in powdered form and mixed with pharmaceutically acceptable carriers to generate a semi-solid pharmaceutical composition as disclosed herein. Reference is made to Example 1 , panel b). As used herein, the term "kit" refers to packaged formulations, whether the ingredients are in separate phases or mixed, and thus for example, may include a gel in a tube with all ingredients in admixture, or any formulation wherein the ingredients are separated from each other.

Administration as aerosols produces droplets preferably less than 10 μm in size, more preferably less than 5 μm in size, most preferably 1 -3 μm in size. Control of the droplet size is important both to control the dosage delivered and to enhance delivery to the target tissue; thus, depending on the dosage required and the target tissue, it may be important to regulate the droplet size of the aerosol. In this respect, it has been found that droplet size can be regulated, to at least some extent, by the mechanical mister which is used to produce the aerosol. In addition, the aerosol's droplet size can be adjusted, to at least some extent, by modifying the surface tension of the solution. More particularly, the pharmaceutically active composition to be administered in aerosol form typically has water as its solvent, and water has a relatively high surface tension, so it is relatively straightforward to create an aerosol having relatively small droplet size. Surface active agents can be added to the solution so as to reduce the surface tension of the solution, whereby to create an aerosol having a relatively large droplet size. By way of example, such surfactants may comprise sodium alkyl sulfates, sodium laryl sulfate, sodium lauroyl sarconsinate, phospholipids, e.g., lecithin, sphingomyelin, etc.

Depending on the application, solutions generated from powders of the pharmaceutically active composition should avoid inclusion of particulates larger than 2 μm, and more preferably no larger than 1 μm (i.e., submicron) to avoid deleterious immune responses or toxic effects. Larger particulates may be removed by, for example filtration. Particulates may be formed in the liquid and can be removed, for example by filtration.

The aerosol may be created by passing a liquid solution of the active compounds through a mechanical mister (e.g., a nebulizer) and may be applied directly with a pressurized pack (e.g., via a hand inhaler with a propellant such as carbon dioxide or other gas, with a valve metered dosage) or through some other delivery system (e.g., an oxygen tent, etc.). The present invention also provides methods for manufacturing the pharmaceutical composition according to the invention.

In one embodiment the invention provides a method for manufacturing the pharmaceutical composition according to the invention, said method comprising the steps of

i) providing a first composition comprising phosphate ions, iron ions, and copper ions,

wherein the relative amounts (w/w) of phospor (P), iron (Fe) and copper (Cu), P : Fe : Cu, in said composition are given by the ratios 100 : 15 to 50 : 5 to 15,

ii) providing a second composition comprising an aqueous or oily base and optionally a thickening and/or a gelling agent,

iii) mixing said first and second compositions and thereby providing the pharmaceutically active composition according to the invention.

In one embodiment the first composition comprises phosphate, iron and copper, wherein the relative amounts A : B : C (w/w of anhydrous compounds) of

A: phosphate; B: iron; and C: copper;

are given by the ratios 100 : 15 to 50 : 5 to 15.

In embodiments of the invention, the first composition comprises phosphate , iron and copper, wherein the relative amounts of A : B (w/w of anhydrous compounds) are given by the ratio 100 : 20 to 50; such as 100 : 25 to 50; for example 100 : 30 to 50; such as 100 : 35 to 50; for example 100 : 40 to 50; such as 100 : 45 to 50; for example 100 : 15 to 45; such as 100 : 15 to 40; for example 100 : 15 to 35; such as 100 : 15 to 30; for example 100 : 15 to 25; such as 100 : 15 to 20; for example 100 : 20 to 25; such as 100 : 25 to 30; for example 100 : 30 to 35; such as 100 : 35 to 40; for example 100 : 40 to 45.

In further embodiments the first composition comprises phosphate, iron and copper, wherein the relative amounts of A : C (w/w of anhydrous compounds) are given by the ratio 100 : 6 to 15; such as 100 : 7 to 15; for example 100 : 8 to 15; such as 100 : 9 to 15; for example 100 : 10 to 15; such as 100 : 1 1 to 15; for example 100 : 12 to 15; such as 100 : 13 to 15; for example 100 : 14 to 15; such as 100 : 5 to 14; for example 100 : 5 to 13; such as 100 : 5 to 12; for example 100 : 5 to 1 1 ; such as 100 : 5 to 10; for example 100 : 5 to 9; such as 100 : 5 to 8; for example 100 : 5 to 7; such as 100 : 5 to 6; for example 100 : 6 to 7; such as 100 : 7 to 8; for example 100 : 8 to 9; such as 100 : 9 to 10; for example 100 : 10 to 1 1 ; such as 100 : 1 1 to 12; for example 100 : 12 to 13; such as 100 : 13 to 14.

Preferably the first composition comprises phosphate in the form of a phosphate complex or a phosphate compound, iron in the form of inorganic iron and copper in the form of inorganic copper, wherein the absolute amount of phosphate complex and/or phosphate compound is from 80 % to 90 % (w/w), such as 85 % or about 85%.

In embodiments of the present invention, the phosphate complex comprises 5 % to 25 % (w/w) of phosphor, such as 5 % to 20%, for example from 5 % to 15 %, such as from 5 % to 10 %, for example from 7 % to 10 %, such as from 8 % to 10 %, for example from 10 % to 25 %, such as from 15 % to 20 %, for example 20 % to 25 %.

Preferably the phosphate compound of the first composition is selected from the group consisting of, but not limited to, calcium diphosphate, calcium phosphate, diammonium phosphate, mono-ammonium phosphate, disodium phosphate, monosodium phosphate, potassium phosphate, rock phosphate, sodium tripolyphosphate, potassium tripolyphosphate, sodium pyrophosphate and/or potassium pyrophosphate.

The phosphate complex of the first composition preferably comprises calcium diphosphate and/or calcium phosphate.

Preferably the phosphate complex is selected from the group consisting of, but not limited to, superphosphate and/or triple superphosphate. The iron is preferably in the form of iron chloride, iron fluoride, iron nitrate, iron oxide and/or iron sulphate. The copper is preferably in the form of copper acetate, copper bromide, copper carbonate, copper chloride, copper fluoborate, copper gluconate, copper nitrate, copper oxide and/or copper sulphate.

In another embodiment the first composition comprises phosphate, iron and copper, wherein the absolute amount of phosphate in said composition is in the range of from 5.0 % to 9.0 % (w/w).

In embodiments of the invention the first composition comprises phosphate, iron and copper, wherein the absolute amount of phosphate in said composition is in the range of from 5.0 % to 9.0 % (w/w), such as from 5.5 % to 9.0 %, for example from 6.0 % to 9.0 %, such as from 6.5 % to 9.0 %, for example from 7.0 % to 9.0 %, such as from 7.5 % to 9.0 %, for example from 5.0 % to 8.5 %, such as from 5.0 % to 8.0 %, for example from 5.0 % to 7.5 %, such as from 5.0 % to 7.0 %, for example from 6.5 % to 7.5 %, such as from 7.0 % to 6.0 %, for example from 7.2 % of phosphate. Preferably the phosphate is in the form of a phosphate complex. Preferably the phosphate complex comprises calcium diphosphate and/or calcium phosphate. Preferably the phosphate complex is superphosphate.

In further embodiments of the invention the first composition comprises phosphate, iron and copper, wherein the absolute amount of iron in said composition is in the range of from 1.2 % to 9.0 % (w/w).

In embodiments of the invention, the first composition comprises phosphate, iron and copper, wherein the absolute amount of iron in said composition is in the range of from 1.2 % to 9.0 % (w/w), such as from 1 .5 % to 9.0 %, for example from 2.0 % to 9.0 %, such as from 2.5 % to 9.0 %, for example from 3.0 % to 9.0 %, such as from 3.5 % to 9.0 %, for example from 1.2 % to 8.5 %, such as from 1.2 % to 8.0 %, for example from 1 .2 % to 7.5 %, such as from 1 .2 % to 7.0 %, for example from 1.2 % to 6.5 %, such as from 1.2 % to 6.0 %, for example from 1.2 % to 5.5 %, such as from 1.2 % to 5.0 %, for example from 1.2 % to 4.5 %, for example from 1 .5 % to 4.0 %, such as from 2.0 % to 3.5 %, for example from 2.5 % to 3.0 %, such as 3 % iron. Preferably the iron is in the form of inorganic iron. Preferably the iron is iron sulphate and/or iron oxide. In even further embodiments of the invention, the first composition comprises phosphate, iron and copper, wherein the absolute amount of copper in said composition is in the range of from 0.4 % to 4.5 % (w/w).

In embodiments of the invention, the first composition comprises phosphate, iron and copper, wherein the absolute amount of copper in said composition is in the range of from 0.4 % to 4.5 %, for example from 0.4 % to 4.0 %; such as from 0.4 % to 3.5 %, for example from 0.4 % to 3.0 %; such as from 0.4 % to 2.5 %, for example from 0.4 % to 2.0 %; such as from 0.5 % to 1.5 %, for example from 0.5 % to 1.0 %, such as 0.7 % copper. Preferably the copper is in the form of inorganic copper. Preferably the copper is copper sulphate.

Preferably the first composition is Stalosan F (commercially available from Stormøllen A/S, Denmark).

In one embodiment the first and/or second compositions are further mixed with a thickening agent.

In another embodiment the first and/or second compositions are further mixed with one or more of a stabilizing agent, an emulsifying agent, a dispersing agent, a suspending agent, a coloring agent and a perfume.

In yet another embodiment the first and/or second compositions are further mixed with one or more of a starch, a tragacanth, a cellulose derivative, a silicone, a bentonite, a silicic acid, and talc, or mixtures thereof.

In a further embodiment the first and/or second compositions are further mixed with one or more of a lactose, talc, aluminum hydroxide, and calcium silicates, or mixtures of these thereof.

The present invention also provides methods of treatment by administering a therapeutically effective amount of the pharmaceutically active composition, or a solution derived from a nanocrystalline powder of the pharmaceutically active composition, as either a topical formulation, or as a coating on medical dressing, applied to the locally affected area of the skin.

Thus in one aspect the invention relates to a method for healing a wound and/or accelerating wound healing in an individual, said method comprising the steps of contacting the composition according to the invention to said wound, and healing said wound and/or accelerating wound healing as a result of said contacting.

The individual is preferably a mammal. More preferably the individual is a human and/or a domestic animal, such as an animal selected from the group consisting of but not limited to cattle, horses, pigs, sheep, mink, dogs, cats, mice, guinea pigs, rabbits, rats; sports animals, such as horses, poly ponies, dogs, camels, and primates.

In one embodiment the animal is a pig and the wound is a bedsore.

A therapeutically effective amount can be determined by testing formulations containing the pharmaceutically active compositions by in vitro or in vivo testing.

Formulations may be applied one or more times a day. Dressings coated with the pharmaceutically active composition may be changed daily, or even less frequently, and should be kept in a moist condition with the addition of saline, alcohols, or more preferably sterile water, in order to release the pharmaceutically active composition on a sustained basis.

The invention further relates to the use of pharmaceutically active compounds or compositions according to the invention in the manufacture of a medicament for treating a wound or burn in an individual. In one embodiment the composition is Stalosan F (Stormøllen A/S, Denmark).

The invention also relates to the use of pharmaceutically active compounds or compositions according to the invention in the manufacture of a medicament for accelerating wound healing in an individual. In one embodiment the composition is Stalosan F (Stormøllen A/S, Denmark).

In one embodiment the invention relates to the use of a pharmaceutically active composition according to the invention comprising phosphate ions, iron ions, and copper ions, wherein the relative amounts (w/w) of phospor (P), iron (Fe) and copper (Cu), P : Fe : Cu, in said composition are given by the ratios 100 : 15 to 50 : 5 to 15, in the manufacture of a medicament for treating a wound or burn in an individual.

In another embodiment the invention relates to the use of a pharmaceutically active compound according to the invention comprising phosphate ions, iron ions, and copper ions, wherein the relative amounts (w/w) of phospor (P), iron (Fe) and copper (Cu), P : Fe : Cu, in said composition are given by the ratios 100 : 15 to 50 : 5 to 15, in the manufacture of a medicament for accelerating wound healing in an individual.

The individual receiving treatment with the medicament is preferably a mammal, more preferably the individual is a human and/or a domestic animal, such as an animal selected from the group consisting of but not limited to cattle, horses, pigs, sheep, mink, dogs, cats, mice, guinea pigs, rabbits, rats; sports animals, such as horses, poly ponies, dogs, camels, and primates.

In one embodiment the animal is a pig and the wound is a bedsore.

Methods for analyzing the active compounds of the compositions according to the invention

Absolute amount of phosphor, iron and copper

The absolute amount of phosphor, iron and copper can be measured with Inductive Coupled Plasma Atomic Emission Spectrometry (ICO-AES) according to AOAC method 984.27 (Official Methods of Analysis of AOAC INTERNATIONAL (OMA), 18th Edition, William Horwitz; 2005 ). For example, the analysis may be carried out by Steins Laboratorium A/S (Hjaltesvej 8, 7500 Holstebro, Denmark).

Presence of phosphate ions, iron ions and copper ions

Phosphate Ions:

The precipitation usually used to identify phosphate is the formation of yellow ammonium molybdophosphate from ammonium molybdate in acidic solution. Material and Methods

The objectives of this study were to determine if "Stalosan Salve" obtained by the method described in example 2 would be able to provide a useful tool in controlling shoulders ulcers in lactating sows. The study was conducted at a 4200-sow farrow-to-finish swine farm at "Brømdumgard" owned by Esper Goul and located in Sailing, Denmark in August 2007. Sow data were diagnosed and recorded by Gitte Drejer from Danvet every second day from the beginning to the end of the trial. The right and left shoulders of the sows were assigned a score between 0 (normal) and 2 (see below for further information). If a lesion developed, sows were randomly placed into one of two treatment groups. Groups consisted of a control group that received no treatment and a group that was applied with "Stalosan Salve" directly on the ulcer two times a day, morning and evening.

Classification of shoulder lesion The existing Danish classification system on shoulder lesions of sows is created by Lund (2003). His grading scale is based on human grading scales developed on the basis of work done by Shea (1975). These human grading scales are sequential clinical descriptions of soft tissue ulceration caused by pressure and are used to identify and measure the severity of tissue damage (Pedley, 2004). The scale by Lund (2003) was created in order to make standardized registrations on shoulder lesions macroscopically on slaughtered sows and to create the necessary foundation of a forensic decision in cases of animal welfare matters regarding the severity of shoulder lesions of sows. The scale was constructed as a simple scale with five degrees (0-4), all substantiated by histological evidence in order to secure a rapid and precise registration with the different grades reflecting the impact on the animal in question (Lund, 2003). The grading scale is presented in table 1.

Table 1 The morphological grading of shoulder lesion in the shoulder region of sows^* Grade Description

0 No lesions of the skin

1 The lesion is limited to the epidermis, eventually with a moderate crust. 2 The lesion involves the dermis, eventually with extensive crusting. There is little fibrosis and/or granulation tissue.

3 The lesion penetrates to the subcutis. There is intense formation of granulation tissue

4 Ulcer penetrating to the bone or ulcer development with periosteal bony proliferation.

Results

Table 2-16 is the raw data collected from the observations made by veterinarian Gitte

Drejer. The sows are registrated with a number that has been added on the production facilities. Gitte Drejer har observed the lesion grade, and the area of inflammation and ulcer on the shoulder of the sow. All the figures, collected every second day, is expressed in cm² for the inflammation and ulcer and in grades (See above) for the initial and final ulcer diagnosis.

Table 2-7 is control sows that received no treatment and table 8-16 represent sows that received treatment with "Stalosan Salve" twice a day.

Table 2: Control group including sow 1717-3 and 944-3.

Figures are expressed in cm². If the ulcer grade is 0 or 1 , it will be expressed in the column of inflammation and if the ulcer grade is 2 or above, it will be expressed in the column of ulcer.

Both sow 1717-3 and 944-3 leaves the study before time, since the shoulder ulcer is above the legal accepted limit (Grade 2).

Table 3: Control group including sow 1934 and 1651-1.

Sow 1934 leaves the study before time, since the shoulder ulcer is above the legal accepted limit (Grade 2).

Table 4: Control group including sow 2194 and 485.

Both sow 2194 and 485 leaves the study before time, since the shoulder ulcer is above the legal accepted limit (Grade 2).

Table 5: Control group including sow 1322-1 and 558.

Sow 1322-1 leaves the study before time, since the shoulder ulcer is above the legal accepted limit (Grade 2).

Table 6: Control group including sow 1028-3 and 806.

Both Sow 1028-3 and 806 leaves the study before time, since the shoulder ulcer is above the legal accepted limit (Grade 2).

Table 7: Control group including sow 1355 and 1684.

Sow 1355 leaves the study before time, since the shoulder ulcer is above the legal accepted limit (Grade 2).

Table 8: Stalosan Salve group including sow 898 and 1393-3.

Both Sows are weaned with a shoulder ulcer grade below or at the legal accepted limit

(Grade 2).

Table 9: Stalosan Salve group including sow 2230-3 and 1402-3.

(Grade 2).

Table 10: Stalosan Salve group including sow 804-1 and 1636.

(Grade 2).

Table 11: Stalosan Salve group including sow 826 and 1366.

(Grade 2).

Table 12: Stalosan Salve group including sow 1660 and 1339.

(Grade 2).

Table 13: Stalosan Salve group including sow 673 and 803-6.

(Grade 2).

Table 14: Stalosan Salve group including sow 845 and 998.

(Grade 2).

Table 15: Stalosan Salve group including sow 951-3 and 1384-3.

(Grade 2).

Table 16: Stalosan Salve group including sow 961-3.

Sow is weaned with a shoulder ulcer grade below or at the legal accepted limit (Grade

2). The mean grade of shoulder ulcer seen with the included sows is illustrated for the control and Stalosan Salve groups (figure 1 ). It can be calculated that the grade of shoulder wound for the Stalosan Salve group is significantly more decreased compared to control (dark grey columns). Also, it can be calculated that the level of shoulder ulcer is significantly lower after treating with Stalosan Salve at the weaning point compared to before treatment at the start (Stalosan salve columns dark grey and light grey). This is completely opposite in the control group, where the weaning point shows a significantly higher shoulder ulcer grade compared to the start (Control columns dark grey and light grey).

Conclusion

Stalosan Salve has proven a significantly increased healing of shoulder ulcers on sows compared to control/no treatment (see figure 1 ).

The mean shoulder ulcer grade in the weaned group treated with Stalosan Salve was significantly lower compared to the weaned/excluded control group that received no treatment (t=0.0002). At the same time, the grade in the weaned group was significantly lower compared to start (t=0,03). In the control group, the grade of shoulder ulcer in the weaned/excluded group was significantly increased compared to the start in the same group (t=7,8^*10^~5). In all the results, it is important to note that the included sows in the Stalosan Salve group has a higher initial mean grade of shoulder ulcer compared to the included sows in the control group (t=0,05).

In the Stalosan Salve group, all 18 included sows were weaned. In the control group only 4 sows was weaned and 8 sows was excluded due to a shoulder ulcer grade at 3 or higher.

Almost every sow in the control group was excluded after only two weeks, which is in contrast to the Stalosan Salve group, where every sow was keep below grade 3 to the weaning phase (approx. a month). This findings supports the preliminary results that we are receiving at the moment, that Stalosan Salve is very effective against all types of skin ulcers/infections on any mammal.

Claims

1. A pharmaceutical composition comprising

i) phosphate ions, ii) iron ions, and iii) copper ions,

2. The pharmaceutical composition according to claim 1 , wherein the relative amounts (w/w) of phospor (P) and iron (Fe), P : Fe, in said composition are given by the ratio 100 : 20 to 50; such as 100 : 25 to 50; for example 100 : 30 to 50; such as 100 : 35 to 50; for example 100 : 40 to 50; such as 100 : 45 to 50; for example 100 : 15 to

45; such as 100 : 15 to 40; for example 100 : 15 to 35; such as 100 : 15 to 30; for example 100 : 15 to 25; such as 100 : 15 to 20; for example 100 : 20 to 25; such as 100 : 25 to 30; for example 100 : 30 to 35; such as 100 : 35 to 40; for example 100 : 40 to 45.

3. The pharmaceutical composition according to claim 1 , wherein the relative amounts (w/w) of phospor (P) and copper (Cu), P : Cu, in said composition are given by the ratio 100 : 6 to 15; such as 100 : 7 to 15; for example 100 : 8 to 15; such as 100 : 9 to 15; for example 100 : 10 to 15; such as 100 : 1 1 to 15; for example 100 : 12 to 15; such as 100 : 13 to 15; for example 100 : 14 to 15; such as 100 : 5 to 14; for example 100 : 5 to 13; such as 100 : 5 to 12; for example 100 : 5 to 1 1 ; such as 100 : 5 to 10; for example 100 : 5 to 9; such as 100 : 5 to 8; for example 100 : 5 to 7; such as 100 : 5 to 6; for example 100 : 6 to 7; such as 100 : 7 to 8; for example 100 : 8 to 9; such as 100 : 9 to 10; for example 100 : 10 to 1 1 ; such as 100 : 1 1 to 12; for example 100 : 12 to 13; such as 100 : 13 to 14.

4. The pharmaceutical composition according to any of claims 1 to 3, said composition comprising phosphate ions in the form of calcium diphosphate and calcium phosphate; iron ions in the form of iron sulphate and/or iron oxide; and copper ions in the form of copper sulphate.

5. The pharmaceutical composition according to any of claims 1 to 4, wherein the absolute amount of iron ions in said composition is in the range of from 0.25 % to 4.0 % (w/w).

6. The pharmaceutical composition according to claim 5, wherein the absolute amount of iron ions in said composition is in the range of from 0.50 % to 4.0 % (w/w), such as from 1 .0 % to 4.0 %, for example from 1.5 % to 4.0 %, such as from 2.0 % to 4.0 %, for example from 2.5 % to 4.0 %, such as from 3.0 % to 4.0 %, for example from 3.5 % to 4.0 %, such as from 0.25 % to 3.5 %, for example from 0.25 % to 3.0 %, such as from 0.25 % to 2.5 %, for example from 0.25 % to 2.0 %, such as from 0.25 % to 1.5 %, for example from 0.25 % to 1.0 %, such as from 0.25 % to 0.50 %, for example from 0.50 % to 1 .0 %, such as from 1.0 % to 1.5 %, for example from 1.5 % to 2.0 %, such as from 2.0 % to 2.5 %, for example from 2.5 % to 3.0 %, such as from 3.0 % to 3.5 %.

7. The pharmaceutical composition according to any of claims 1 to 4, wherein the absolute amount of copper ions in said composition is in the range of from 0.05 % to 1.8 % (w/w).

8. The pharmaceutical composition according to claim 7, wherein the absolute amount of copper ions in said composition is in the range of from 0.1 % to 1.8 %, for example from 0.1 % to 1.5 %; such as from 0.25 % to 1.5 %, for example from 0.50 % to 1.5 %; such as from 0.75 % to 1 .5 %, for example from 1.0 % to 1.5 %; such as from 1 .25 % to 1.5 %, for example from 0.05 % to 1.25 %; such as from 0.05 % to 1.0 %, for example from 0.05 % to 0.75 %; such as from 0.05 % to 0.50 %, for example from 0.05 % to 0.25 %; such as from 0.05 % to 0.1 %; for example from 0.1 % to 0.2 %; such as from 0.2 % to 0.4 %; for example from 0.4 % to 0.6 %; such as from 0.6 % to 0.8 %; for example from 0.8 % to 1.0 %; such as from 1.0 % to 1.2 %; for example from 1.2 % to 1.4 %.

9. The pharmaceutical composition according to any of claims 1 to 8, said composition further comprising a compound selected from the group consisting of an antibiotic compound, an antifungal compound and an antiviral compound for accelerating the healing infection-damaged tissue.

10. The pharmaceutical composition according to any of claims 1 to 8, said composition further comprising a tissue healing promoter selected from the group consisting of epidermal growth factor, fibroblast growth factor, platelet derived growth factor, transforming growth factor alpha, transforming growth factor beta, and insulin-like growth factor 1 for promoting a more rapid healing of damaged tissue.

1 1 . The pharmaceutical composition according to any of claims 1 to 8, said composition further comprising a topical corticosteroid and/or an anti-inflammatory agent.

12. The pharmaceutical composition according to any of claims 1 to 8, said composition further comprising one or more compounds selected from the group consisting of allantoin, retinoic acid, aloe vera, glycine, vitamin A, the B vitamins, especially nicotinamide, vitamins C and E, antibacterial agents (e.g., quaternary ammonium compounds, bacitracin, neomycin and polymyxin), comfrey root preparations, platelets and/or platelet extracts, ribonucleosides, collagen, proline, lysine, elastin, fibronectin, glycosaminoglycans, spermidine (and other polyamines), angiogenic factors, zinc, sucrose, and various peptide growth factors such as the somatomedins, lamin, EGF, the IGPs, PDGF, FGF, EDGF, TGF, CDGF, MDGF, and NGF.

13. The pharmaceutical composition according to any of claims 1 to 8, wherein said composition is in the form of an ointment.

14. A method for manufacturing the pharmaceutical composition according to any of claims 1 to 8, said method comprising the steps of

wherein the relative amounts (w/w) of phospor (P), iron (Fe) and copper (Cu), P : Fe : Cu, in said composition are given by the ratios 100 : 15 to 50 : 5 to 15, ii) providing a second composition comprising an aqueous or oily base and a thickening and/or a gelling agent,

iii) mixing said first and second compositions and thereby providing the pharmaceutically active composition according to any of claims 1 to 8.

15. The method of claim 14, wherein the first composition comprises phosphate, iron and copper, wherein the relative amounts A : B : C (w/w of anhydrous compounds) of

A: phosphate; B: iron; and C: copper;

are given by the ratios 100 : 15 to 50 : 5 to 15.

16. The method of claim 15, wherein the first composition comprises phosphate, iron and copper, wherein the relative amounts of A : B (w/w of anhydrous compounds) are given by the ratio 100 : 20 to 50; such as 100 : 25 to 50; for example 100 : 30 to 50; such as 100 : 35 to 50; for example 100 : 40 to 50; such as 100 : 45 to 50; for example 100 : 15 to 45; such as 100 : 15 to 40; for example 100 : 15 to 35; such as 100 : 15 to 30; for example 100 : 15 to 25; such as 100 : 15 to 20; for example 100 : 20 to 25; such as 100 : 25 to 30; for example 100 : 30 to 35; such as 100 : 35 to 40; for example 100 : 40 to 45.

17. The method of claim 15, wherein the first composition comprises phosphate, iron and copper, wherein the relative amounts of A : C (w/w of anhydrous compounds) are given by the ratio 100 : 6 to 15; such as 100 : 7 to 15; for example 100 : 8 to 15; such as 100 : 9 to 15; for example 100 : 10 to 15; such as 100 : 1 1 to 15; for example 100 : 12 to 15; such as 100 : 13 to 15; for example 100 : 14 to 15; such as

100 : 5 to 14; for example 100 : 5 to 13; such as 100 : 5 to 12; for example 100 : 5 to 1 1 ; such as 100 : 5 to 10; for example 100 : 5 to 9; such as 100 : 5 to 8; for example 100 : 5 to 7; such as 100 : 5 to 6; for example 100 : 6 to 7; such as 100 : 7 to 8; for example 100 : 8 to 9; such as 100 : 9 to 10; for example 100 : 10 to 1 1 ; such as 100 : 1 1 to 12; for example 100 : 12 to 13; such as 100 : 13 to 14.

18. The method of any of claims 14 to 17, wherein the first composition comprises phosphate, iron and copper, wherein the absolute amount of phosphate in said composition is in the range of from 5.0 % to 9.0 % (w/w).

19. The method of claim 18, wherein the first composition comprises phosphate, iron and copper, wherein the absolute amount of phosphate in said composition is in the range of from 5.0 % to 9.0 % (w/w), such as from 5.5 % to 9.0 %, for example from 6.0 % to 9.0 %, such as from 6.5 % to 9.0 %, for example from 7.0 % to 9.0 %, such as from 7.5 % to 9.0 %, for example from 5.0 % to 8.5 %, such as from 5.0 % to 8.0 %, for example from 5.0 % to 7.5 %, such as from 5.0 % to 7.0 %, for example from 6.5 % to 7.5 %, such as from 7.0 % to 6.0 %, for example from 7.2 % of phosphate.

20. The method of claim 19, wherein the phosphate is in the form of a phosphate complex.

21. The method of claim 20, wherein the phosphate complex comprises calcium diphosphate and calcium phosphate.

22. The method of claim 21 , wherein the phosphate complex is superphosphate.

23. The method of any of claims 14 to 17, wherein the first composition comprises phosphate, iron and copper, wherein the absolute amount of iron in said composition is in the range of from 1 .2 % to 9.0 % (w/w).

24. The method of claim 23, wherein the first composition comprises phosphate, iron and copper, wherein the absolute amount of iron in said composition is in the range of from 1.2 % to 9.0 % (w/w), such as from 1.5 % to 9.0 %, for example from 2.0 % to 9.0 %, such as from 2.5 % to 9.0 %, for example from 3.0 % to 9.0 %, such as from 3.5 % to 9.0 %, for example from 1.2 % to 8.5 %, such as from 1.2 % to 8.0 %, for example from 1 .2 % to 7.5 %, such as from 1.2 % to 7.0 %, for example from 1 .2 % to 6.5 %, such as from 1 .2 % to 6.0 %, for example from 1.2 % to 5.5 %, such as from 1.2 % to 5.0 %, for example from 1.2 % to 4.5 %, for example from

1.5 % to 4.0 %, such as from 2.0 % to 3.5 %, for example from 2.5 % to 3.0 %, such as 3 % iron.

25. The method of claim 24, wherein the iron is in the form of iron sulphate and/or iron oxide.

26. The method of any of claims 14 to 17, wherein the first composition comprises phosphate, iron and copper, wherein the absolute amount of copper in said composition is in the range of from 0.4 % to 4.5 % (w/w).

27. The method of claim 26, wherein the first composition comprises phosphate, iron and copper, wherein the absolute amount of copper in said composition is in the range of from 0.4 % to 4.5 %, for example from 0.4 % to 4.0 %; such as from 0.4 % to 3.5 %, for example from 0.4 % to 3.0 %; such as from 0.4 % to 2.5 %, for example from 0.4 % to 2.0 %; such as from 0.5 % to 1.5 %, for example from 0.5 % to 1.0 %, such as 0.7 % copper.

28. The method of claim 27, wherein the copper is in the form of copper sulphate.

29. The method of any of claims 15 to 28, wherein the first and/or second compositions are further mixed with a thickening agent.

30. The method of any of claims 15 to 28, wherein the first and/or second compositions are further mixed with one or more of a stabilizing agent, an emulsifying agent, a dispersing agent, a suspending agent, a coloring agent and a perfume.

31. The method of any of claims 15 to 28, wherein the first and/or second compositions are further mixed with one or more of a starch, a tragacanth, a cellulose derivative, a silicone, a bentonite, a silicic acid, and talc, or mixtures thereof.

32. The method of any of claims 15 to 28, wherein the first and/or second compositions are further mixed with one or more of a lactose, talc, aluminum hydroxide, and calcium silicates, or mixtures of thereof.

33. A method for healing a wound in an individual, said method comprising the steps of contacting the composition according to any of claims 1 to 13 to said wound, and healing said wound as a result of said contacting.

34. The method of claim 33, wherein the wound is selected from the group consisting of cuts, incisions, abrasions, lacerations, amputations, burns induced by heat, ionizing radiation, ultraviolet radiation including sunlight, electricity, or chemical substances as well as to other forms of lesions such as ulcers, pressure sores and bedsores.

35. The method of any of claims 33 and 34, wherein the individual is a mammal.

36. The method of claim 35, wherein the mammal is a human.

37. The method of claim 35, wherein the individual is a domestic animal.

38. The method of claim 37, wherein the domestic animal is selected from the group cattle, horses, pigs, sheep, mink, dogs, cats, mice, guinea pigs, rabbits, rats; sports animals, such as horses, poly ponies, dogs, camels, and primates.

39. The method of claim 38, wherein the animal is a pig.

40. The method of claim 39, wherein the wound is a bedsore.

41 . A method for accelerating wound healing in an individual, said method comprising the steps of contacting the composition according to any of claims 1 to 13 to said wound, and accelerating wound healing as a result of said contacting.

42. The method of claim 41 , wherein the wound is selected from the group consisting of cuts, incisions, abrasions, lacerations, amputations, burns induced by heat, ionizing radiation, ultraviolet radiation including sunlight, electricity, or chemical substances as well as to other forms of lesions such as ulcers, pressure sores and bedsores.

43. The method of any of claims 41 and 42, wherein the individual is a mammal.

44. The method of claim 43, wherein the mammal is a human.

45. The method of claim 43, wherein the individual is a domestic animal.

46. The method of claim 45, wherein the domestic animal is selected from the group cattle, horses, pigs, sheep, mink, dogs, cats, mice, guinea pigs, rabbits, rats; sports animals, such as horses, poly ponies, dogs, camels, and primates.

47. The method of claim 46, wherein the animal is a pig.

48. The method of claim 47, wherein the wound is a bedsore.

49. Use of a pharmaceutically active compound comprising phosphate ions, iron ions, and copper ions, wherein the relative amounts (w/w) of phospor (P), iron (Fe) and copper (Cu), P : Fe : Cu, in said composition are given by the ratios 100 : 15 to 50 : 5 to 15, in the manufacture of a medicament for treating a wound or burn in an individual.

50. The use of claim 49, wherein the wound is selected from the group consisting of cuts, incisions, abrasions, lacerations, amputations, burns induced by heat, ionizing radiation, ultraviolet radiation including sunlight, electricity, or chemical substances as well as to other forms of lesions such as ulcers, pressure sores and bedsores.

51 . The use of any of claims 49 and 50, wherein the individual is a mammal.

52. The use of claim 51 , wherein the mammal is a human.

53. The use of claim 51 , wherein the individual is a domestic animal.

54. The use of claim 53, wherein the domestic animal is selected from the group cattle, horses, pigs, sheep, mink, dogs, cats, mice, guinea pigs, rabbits, rats; sports animals, such as horses, poly ponies, dogs, camels, and primates.

55. The use of claim 54, wherein the animal is a pig.

56. The use of claim 55, wherein the wound is a bedsore.

57. Use of a pharmaceutically active compound comprising phosphate ions, iron ions, and copper ions, wherein the relative amounts (w/w) of phospor (P), iron (Fe) and copper (Cu), P : Fe : Cu, in said composition are given by the ratios 100 : 15 to 50 : 5 to 15, in the manufacture of a medicament for accelerating wound healing in an individual.

58. The use of claim 57, wherein the wound is selected from the group consisting of cuts, incisions, abrasions, lacerations, amputations, burns induced by heat, ionizing radiation, ultraviolet radiation including sunlight, electricity, or chemical substances as well as to other forms of lesions such as ulcers, pressure sores and bedsores.

59. The use of any of claims 57 and 58, wherein the individual is a mammal.

60. The use of claim 59, wherein the mammal is a human.

61 . The use of claim 59, wherein the individual is a domestic animal.

62. The use of claim 61 , wherein the domestic animal is selected from the group cattle, horses, pigs, sheep, mink, dogs, cats, mice, guinea pigs, rabbits, rats; sports animals, such as horses, poly ponies, dogs, camels, and primates.

63. The use of claim 62, wherein the animal is a pig.

64. The use of claim 63, wherein the wound is a bedsore.

65. The use of any of claims 49 to 64, wherein the pharmaceutically active compound is in a finely-divided or powdered form.

66. The use of any of claims 49 to 65, wherein the pharmaceutically active compound is in solution or suspension in an aqueous or non-aqueous fluid.

67. The use of any of claims 49 to 66, wherein the pharmaceutically active compound is further mixed with a greasy or non-greasy base.

68. The use of any of claims 49 to 67, wherein the medicament is in form of a semi- solid pharmaceutical composition.

69. The use of claim 68, wherein the semi-solid pharmaceutical composition is in form of an ointment or a salve or a creme or a lotion or a gel.

70. The use of claim 69, wherein the semi-solid pharmaceutical composition is in form of an ointment.

71. The use of claim 69, wherein the semi-solid pharmaceutical composition is in form of a salve.

72. The use of claim 69, wherein the semi-solid pharmaceutical composition is in form of a creme.

73. The use of claim 69, wherein the semi-solid pharmaceutical composition is in form of a lotion.

74. The use of claim 69, wherein the semi-solid pharmaceutical composition is in form of a gel.