WO2022150716A1

WO2022150716A1 - Methods of treating wounds and burns

Info

Publication number: WO2022150716A1
Application number: PCT/US2022/011849
Authority: WO
Inventors: John L. Gainer; David G. KALERGIS; Thomas Byrne
Original assignee: Diffusion Pharmaceuticals Llc
Priority date: 2021-01-10
Filing date: 2022-01-10
Publication date: 2022-07-14

Abstract

The subject invention relates to novel methods for the treatment of wounds. Specifically, the invention relates to diffusion enhancing compounds and their use in treatment of wounds, including burns, alone or in conjunction with other procedures.

Description

Methods of Treating Wounds and Burns

This application claims priority to U.S. Provisional Application No. 63/135,737 filed January 10, 2021, and U.S. Provisional Application No. 63/216,481 filed June 29, 2021, each of which is hereby incorporated by reference in its entirety.

Field of the Invention

The subject invention relates to novel methods for the treatment of wounds including burns. Specifically, the invention relates to diffusion enhancing compounds and their use in treatment of wounds and bums, alone or in conjunction with other would healing procedures. Background of the Art

Wound healing is complex, requiring interplay between numerous cell types, cytokines, mediators, and the vascular system.

Wounds

Oxygen is a significant factor in wound healing and plays a substantial role throughout all phases of wound healing. In normal wound healing, the wound may require either conditions of hypoxia or normal levels of oxygen (ie, normoxia) and different conditions may occur in all phases of wound healing.

The effect of oxygen on wound healing varies depending on whether the wound is in a hypoxic, normoxic, or in a hyperoxic state. Oxygen must be tightly governed in all phases of wound healing to produce viable granulation tissue.

Wound Healing Phases

There are three stages in the wound healing process:

1. Inflammatory phase - This phase begins at the time of injury and lasts up to four days.

It includes clotting of platelets and constriction of blood vessels to stop blood loss, in addition to the arrival of white blood cells to kill bacteria and naturally clean the wound site.

2. Proliferative phase - This phase begins about three days after injury and overlaps with the inflammatory phase. It involves cells called fibroblasts that help to produce new collagen, create new blood vessels, and repair the avascular epithelial tissue.

3. Remodeling phase - This phase can continue for six months to one year after injury. Collagen continues to increase and the tissue begins to contract with the help of fibroblasts, both of which add strength to the new tissue. Excessive collagen can cause scar tissue formation.

Oxygen Sensing

Throughout the phases of wound healing, control of oxygen is maintained in a narrow range. This point of normoxia is important because it is used to prevent abnormal periods of hypoxia or hyperoxia, which can create damage to cell membranes. This point of normoxia is the state of oxygenation where the cell or tissue does not report hypoxia nor does it report hyperoxia, which would be oxygen toxicity. If there were a change, the cells or tissue would react by switching on either a hypoxic or hyperoxic response. Depending on the organ of the body, the normoxic set point may be different due to the amount of oxygen required.

Hyperoxia may induce some positive effects, but if it occurs for a period of time exceeding the normoxic set point it can be a risk factor.

In areas where a wound has pockets of hypoxia, the goal is to reestablish normoxia in the areas of hypoxia without exposing the wound to high levels of oxygen, which might cause oxygen toxicity. Wound healing might be delayed in extreme hyperoxia, which can cause growth arrest and cell death by mitochondria apoptosis.

Thermal Burns

Two million people in the U.S. each year are treated for thermal burn injuries. One hundred thousand of these patients require hospitalization. Superficial bums heal spontaneously within the first two weeks. Treatment consists of pain relief and topical wound care to relieve pain and prevent infection. Deeper burns will not heal within 2 weeks and may require surgical intervention and should be cared for by a surgical specialist.

The goal of burn care is to prevent infection and obtain a closed injury site. This may be accomplished in minor wounds by applying topical antibacterial creams. A minor wound may also respond well to non-adherent gauze such as Vaseline impregnated gauze.

When a thermal bum occurs, the initial therapy typically includes cooling of the burn injury site. After that, treatment is rendered by the appropriate family practitioner or regional burn center. Common treatment includes fluid administration (urinary output should be 30 - 50 cc) and a tetanus toxoid booster; 3rd degree bums are treated with topical antibacterial agents and surgical therapy, such as skin grafting, is often contemplated.

Infections in bum patients continue to be the primary source of morbidity and mortality. Topical antimicrobial therapy remains the single most important component of wound care in hospitalized burn patients and in burn care facilities. The goal of prophylactic topical antimicrobial therapy is to control microbial colonization and prevent burn wound infection.

New methods for treating wounds, including bums, to ensure adequate and timely healing are needed.

Summary of the Invention

Provided is a method of treating a mammal having a wound comprising administering a pharmaceutically effective amount of a diffusion enhancing compound to the mammal.

Brief Description of the Drawings

Figure 1 shows a schematic diagram of wound site design.

Figure 2 shows a schematic of a cross section of tissue.

Figure 3 depicts stages of wound healing at sequential time points (Days 0 and 10) for individual sites of each treatment group. Detailed Description of the Invention

The subject invention relates to novel methods for the treatment of wounds including burns. Specifically, the invention relates to diffusion enhancing compounds and their use in treatment of wounds and bums, alone or in conjunction with other procedures.

Compounds and Compositions of the Invention

Diffusion Enhancing Compounds

The diffusion enhancing compounds of the invention include those compounds described in U.S. Pat. 7,759,506, U.S. Pat. 8,030,350, U.S. Pat. 8,901,174 and U.S. Pat 8,206,751, each of which is hereby incorporated by reference in its entirety.

Included are bipolar trans carotenoid compounds having the formula:

YZ-TCRO— ZY where:

• Y=a cation · Z=a polar group which is associated with the cation, and

• TCRO=trans carotenoid skeleton, such as TSC.

More specifically, the subject invention relates to trans carotenoids including trans carotenoid diesters, dialcohols, diketones and diacids, bipolar trans carotenoids (BTC), and bipolar trans carotenoid salts (BTCS) compounds and synthesis of such compounds having the structure:

YZ-TCRO— ZY where: • Y (which can be the same or different at the two ends)=H or a cation other than H, preferably Na⁺ or K+ or Li⁺. Y is advantageously a monovalent metal ion. Y can also be an organic cation, e.g., R.4N⁺, R₂S⁺, where R is H, or C_nH_2+i where n is 1-10, advantageously 1-6. For example, R can be methyl, ethyl, propyl or butyl.

• Z (which can be the same or different at the two ends)=polar group which is associated with H or the cation. Optionally including the terminal carbon on the carotenoid (or carotenoid related compound), this group can be a carboxyl (COO-) group or a CO group (e.g. ester, aldehyde or ketone group), or a hydroxyl group. This group can also be a sulfate group (OSO₃ ) or a monophosphate group (OPO₃ ), (OP(OH)0₂ ), a diphosphate group, triphosphate or combinations thereof. This group can also be an ester group of COOR where the R is C_nH_2n+i.

• TCRO=trans carotenoid or carotenoid related skeleton (advantageously less than 100 carbons) which is linear, has pendant groups (defined below), and typically comprises “conjugated” or alternating carbon-carbon double and single bonds (in one embodiment, the TCRO is not fully conjugated as in a lycopene). The pendant groups (X) are typically methyl groups but can be other groups as discussed below. In an advantageous embodiment, the units of the skeleton are joined in such a manner that their arrangement is reversed at the center of the molecule. The 4 single bonds that surround a carbon- carbon double bond all lie in the same plane. If the pendant groups are on the same side of the carbon-carbon double bond, the groups are designated as cis (also known as “Z”); if they are on the opposite side of the carbon-carbon bond, they are designated as trans (also known as “E”). Throughout this case, the isomers will be referred to as cis and trans.

The compounds of the subject invention are trans. The cis isomer typically is a detriment — and results in the diffusivity not being increased. In one embodiment, a cis isomer can be utilized where the skeleton remains linear. The placement of the pendant groups can be symmetric relative to the central point of the molecule or can be asymmetric so that the left side of the molecule does not look the same as the right side of the molecule either in terms of the type of pendant group or their spatial relationship with respect to the center carbon. The pendant groups X (which can be the same or different) are hydrogen (H) atoms, or a linear or branched hydrocarbon group having 10 or less carbons, advantageously 4 or less, (optionally containing a halogen), or a halogen. X could also be an ester group (COO — ) or an ethoxy/methoxy group. Examples of X are a methyl group (CH₃), an ethyl group (C₂H₅), a phenyl or single aromatic ring structure with or without pendant groups from the ring, a halogen- containing alkyl group (Cl -CIO) such as CH₂C1, or a halogen such as Cl or Br or a methoxy (OCH₃) or ethoxy (OCH₂CH₃). The pendant groups can be the same or different but the pendant groups utilized must maintain the skeleton as linear.

Although many carotenoids exist in nature, carotenoid salts do not. Commonly -owned U.S. Pat. No. 6,060,511 hereby incorporated by reference in its entirety, relates to trans sodium crocetinate (TSC). The TSC was made by reacting naturally occurring saffron with sodium hydroxide followed by extractions that selected primarily for the trans isomer.

The presence of the cis and trans isomers of a carotenoid or carotenoid salt can be determined by looking at the ultraviolet-visible spectrum for the carotenoid sample dissolved in an aqueous solution. Given the spectrum, the value of the absorbence of the highest peak which occurs in the visible wave length range of 380 to 470 nm (the number depending on the solvent used and the chain length of the BTC or BTCS. The addition of pendant groups or differing chain lengths will change this peak absorbance but someone skilled in the art will recognize the existence of an absorbance peak in the visible range corresponding to the conjugated backbone structure of these molecules.) is divided by the absorbency of the peak which occurs in the UV wave length range of 220 to 300 nm can be used to determine the purity level of the trans isomer. When the trans carotenoid diester (TCD) or BTCS is dissolved in water, the highest visible wave length range peak will be at between 380 nm to 470 nm (depending on the exact chemical structure, backbone length and pendant groups) and the UV wave length range peak will be between 220 to 300 nm. According to M. Craw and C. Lambert, Photochemistry and Photobiology, Vol. 38 (2), 241-243 (1983) hereby incorporated by reference in its entirety, the result of the calculation (in that case crocetin was analyzed) was 3.1, which increased to 6.6 after purification.

Performing the Craw and Lambert analysis, using a cuvette designed for UV and visible wavelength ranges, on the trans sodium salt of crocetin of commonly owned U.S. Pat. No. 6,060,511 (TSC made by reacting naturally occurring saffron with sodium hydroxide followed by extractions which selected primarily for the trans isomer), the value obtained averages about 6.8. Performing that test on the synthetic TSC of the subject invention, that ratio is greater than 7.0 (e.g. 7.0 to 8.5), advantageously greater than 7.5 (e.g. 7.5-8.5), most advantageously greater than 8. The synthesized material is a “purer” or highly purified trans isomer.

Methods of Treatment of the Invention

The invention relates to the treatment of the many types of wounds in a mammal, including a human, where enhanced delivery of oxygen (and glucose) is beneficial.

Compromised Skin Grafts and Flaps

Compromised skin grafts and skin flaps are problems involving inadequate oxygen supply to tissue. Skin grafts typically survive as oxygen disperses into them from the original wound bed. Skin grafts can partially or fully fail when there is not enough oxygen supplied. There are a few types of skin grafts which are: full-thickness grafts where all the layers of skin are used, split thickness grafts in which only the top layers and some of the deep layers are used, as well as pedicle grafts where part of the skin remains to the donor site. Factors such as age, nutritional status, smoking, and previous radiation result in an erratic pattern of blood flow to the skin.

Diabetic Foot Ulcers

Neuropathy can contribute to the formation of a diabetic foot ulcer. If left untreated, diabetic foot ulcers can progress to severe infection or gangrene that might require amputation. In fact, diabetic ulcers are one of the most common causes of foot amputation.

Osteomyelitis

Osteomyelitis is an infection in a bone. Infections can reach a bone by traveling through the bloodstream or spreading from nearby tissue. Osteomyelitis can also begin in the bone itself if an injury exposes the bone to germs.

In children, osteomyelitis most commonly affects the long bones of the legs and upper arm, while adults are more likely to develop osteomyelitis in the bones that make up the spine (vertebrae). People who have diabetes may develop osteomyelitis in their feet if they have foot ulcers.

Most people require surgery to remove parts of the bone that have died — followed by strong antibiotics, often delivered intravenously, typically for at least six weeks.

Osteoradionecrosis

Osteoradionecrosis (ORN) is a problem with bone healing that can occur in people who received high doses of radiation, particularly to the jaw. This complication can occur after dental surgery or extraction of teeth. High doses of radiation can decrease the bone’s blood supply. If this happens, the bone gets less oxygen than it needs, resulting in the death (necrosis) of bone tissue. The most commonly affected bone is the jawbone (mandible).

Pressure Ulcer

A pressure ulcer, also known as a bedsore or decubitus ulcer, is a wound of the skin caused by prolonged, unrelieved pressure to that area. Pressure ulcers occur most frequently around bony prominences such as the tailbone, hips, heels, ankles and elbows.

Surgical Wounds

A surgical wound occurs after any type of operation that involves making a cut into skin, including minor procedures carried out by GPs and other doctors, as well as those done by surgeons.

Traumatic Wounds

Traumatic wounds are typically defined as cuts, lacerations or puncture wounds which have caused damage to both the skin and underlying tissues. Acute wounds, cut wounds and penetrating wounds are the three categories that make up traumatic wounds. An acute wound occurs when the skin has been ripped or tom and has a jagged appearance. An acute wound may contain foreign bodies such as gravel, glass, metal or sand.

With acute traumatic wounds, it is not uncommon for layers of tissue to be easily visible along the inside of the cut. A traumatic cut wound is the result of something sharp penetrating the skin and the underlying subcutaneous tissues. Penetrating wounds, however, are considered the deepest and most severe of all traumatic wounds, because they often occur as the result of being stabbed or sustaining a gunshot wound.

Venous Stasis Ulcer

A venous stasis ulcer, also known as a venous insufficiency ulcer, means that there is an impairment or lack of venous blood flow to an area of the skin. These ulcers occur in the lower legs, between the knee and the ankle. The most common place for them to develop is around the ankle.

In most cases, there is a change in the color of the skin before it actually opens (ulcerates), like a red spot or a black and blue bruising. Due to the lack of circulation, which provides the essential nutrients for the skin to survive, the skin begins to die in this spot and opens (ulcerates.) These wounds are typically shallow (do not get very deep.) They are often irregular in shape and tend to elongate (like a run in stockings.) Left untreated, they do not get better and will get worse.

Wounds from Vascular Diseases

Ulcers of the lower extremities, particularly in individuals older than 65 years, are a common cause for visits to the podiatrist, wound care specialist, primary care physician, vascular surgeon, or dermatologist.

The great majority of vascular ulcers are chronic or recurrent. They cause a considerable amount of morbidity among patients with peripheral vascular disease. Additionally, these non-healing ulcers place the patient at much higher risk for lower extremity amputation.

The subject invention relates to methods of treating wounds, including the above mentioned wounds and bums, including thermal burns, comprising administering a therapeutically effective amount of a diffusion enhancing compound, e.g. TSC, that permits delivery of oxygen and glucose to the wound e.g. the stratus corneum, and epidermis of the skin. The patient can be given a diffusion enhancing compound such as TSC, e.g., by IV injection or infusion, IM, or orally, at a dosage in the range of 0.05-5 mg/kg, e.g., 0.05-2.5 mg/kg or 0.1-2 mg/kg or 2.5-5 mg/kg, e.g., 3-5 mg/kg. Typically a compound or composition of the invention is administered 1 time daily at a dose of 0.25-1.5 mg/kg until the damaged skin has healed. As used herein “a therapeutically effective amount” is an amount that enhances the wound healing.

The invention relates to methods of preventing skin infections and minimizing scars while accelerating the healing of wounds. The compounds and compositions of the invention can be used in conjunction with other methods of treating wounds such as treatment with oxygen (including hyperbaric oxygen therapy (HBOT)), treatment with antibiotics, and treatment with other wound healing agents.

Thermal Burns

The compounds and compositions of the invention have been found to be effective in ameliorating skin burns by accelerating the healing processes, and reducing pain and discomfort. The formulations are an effective remedy for minor skin bums (primarily 1^st and 2^nd degree).

The invention also relates to methods of preventing skin infections and minimizing scars while accelerating the healing of burns.

Thermal bums can result in a loss of a large area of skin and the resulting scar will contract, causing the edges of skin to be pulled together, affecting adjacent muscles and tendons and restricting normal movement. These are usually treated with scar removal surgery using a skin flap, or graft and tissue expansion but can also be treated now with a biological skin revival cream.

A thermal bum can also yield abnormal scarring or scars that go beyond the site of injury called keloid scars or it may result in excessively fibrotic scars called hypertrophic scars. Burn-related skin fibrosis leads to loss of tissue function and hypertrophic scar formation with damaging consequences for the burned person. Bum and wound treatment includes keeping the bum clean to prevent infection.

Thermal burns are classified as first, second or third degree burns depending on the amount and depth of tissue damage. 1st Degree Burn is a superficial, reddened area of skin like sunburn. A first-degree burn causes damage to the epidermis, causing pain, redness and some swelling. Typically, this type of burn will heal without scarring.

2nd Degree Bum is a blistered injury site which may heal spontaneously after the blister fluid has been removed. A second-degree bum causes damage to the epidermis and the dermis, and this bum usually results in pain, redness and blistering.

3rd Degree Burn is a burn through the entire skin and will usually require surgical intervention for wound healing. Third degree bums are the most severe because the damage extends past the upper layers of skin to the sensitive subcutaneous tissue, destroying nerves, blood vessels, and other dermal components. Extensive third degree burns can be fatal because the threat of infection is extremely high. In fact, bacterial infection is the leading cause of death in burn victims.

The subject invention relates to methods of treating skin damaged by thermal bum (1^st, 2^nd or 3^rd degree) comprising administering a therapeutically effective amount of a diffusion enhancing compound e.g. TSC, that permits delivery of oxygen and glucose to the stratus corneum, and epidermis of the skin. The patient can be given a diffusion enhancing compound such as TSC, e.g., by IV injection or infusion, IM, or orally, at a dosage in the range of 0.05-5 mg/kg, e.g., 0.05-2.5 mg/kg or 0.1-2 mg/kg or 2.5-5 mg/kg, e.g., 3-5 mg/kg. Typically a compound or composition of the invention is administered 1 time daily at a dose of 0.25-1.5 mg/kg until the damaged skin has healed.

Provided is a method (Method 1) of treating a mammal in need thereof having a wound, wherein the method comprises administering a pharmaceutically effective amount of a diffusion enhancing compound to the mammal. For instance, provided is a method (Method la) of treating a mammal in need thereof having a wound, wherein the method comprises administering a pharmaceutically effective amount of a diffusion enhancing compound to the mammal to prevent the wound from becoming a chronic wound. For instance, provided is a method (Method 1) of treating a wound in a mammal in need thereof, wherein the method comprises administering a pharmaceutically effective amount of a diffusion enhancing compound to the mammal. For instance, provided is a method (Method la) of treating a wound in a mammal in need thereof, wherein the method comprises administering a pharmaceutically effective amount of a diffusion enhancing compound to the mammal to prevent the wound from becoming a chronic wound.

Further provided is any of Method 1, including Method la, as follows:

1.1. Method 1, wherein the diffusion enhancing compound is a bipolar trans carotenoid.

1.2. Method 1 or 1.1, wherein the diffusion enhancing compound is a bipolar trans carotenoid salt.

1.3. Any of Method 1, 1.1, or 1.2, wherein the diffusion enhancing compound is a bipolar trans carotenoid salt having the formula:

YZ-TCRO-ZY, where:

Y = a cation which can be the same or different,

Z = a polar group which can the same or different and which is associated with the cation,

TCRO = a linear trans carotenoid skeleton with conjugated carbon-carbon double bonds and single bonds, and having pendant groups X, wherein the pendant groups X, which can be the same or different, are a linear or branched hydrocarbon group having 10 or less carbon atoms, or a halogen.

1.4. Any one of Methods 1 or 1.1-1.3, wherein the bipolar trans carotenoid is trans crocetin, in acid or pharmaceutically acceptable salt form. For instance, wherein the bipolar trans carotenoid salt is trans sodium crocetinate (TSC) (e.g., synthetic TSC) having the structure shown below:

1.5. Any one of Methods 1 or 1.1-1.4, wherein the absorbency (e.g., in an aqueous solution) of the bipolar trans carotenoid salt (e.g., trans sodium crocetinate) at the highest peak which occurs in the visible wavelength range divided by the absorbency of a peak occurring in the ultraviolet wavelength range is equal to or greater than 7 (e.g., 7 to 8.5), e.g., equal to or greater than 7.5 (e.g., 7.5 to 9, e.g., 7.5 to 8.5), e.g., equal to or greater than 8 (e.g., 8 to 8.8), e.g., greater than 8.5. For instance, wherein the absorbency (e.g., in aqueous solution) of the highest peak which occurs in the visible wavelength range of 380 to 470 nm divided by the absorbency of the peak which occurs in the UV wavelength range of 220 to 300 nm is equal to or greater than 7 (e.g., 7 to 8.5), e.g., equal to or greater than 7.5 (e.g., 7.5 to 9, e.g., 7.5 to 8.5), e.g., equal to or greater than 8 (e.g., 8 to 8.8), e.g., greater than 8.5.

1.6. Any one of Methods 1 or 1.1-1.5, wherein the absorbency (e.g., in an aqueous solution) of TSC at the highest peak which occurs in the visible wavelength range divided by the absorbency of a peak occurring in the ultraviolet wavelength range is equal to or greater than 7 (e.g., 7 to 8.5), e.g., equal to or greater than 7.5 (e.g., 7.5 to 9, e.g., 7.5 to 8.5), e.g., equal to or greater than 8 (e.g., 8 to 8.8), e.g., greater than 8.5. For instance, wherein the absorbency (e.g., in aqueous solution) of TSC at the highest peak which occurs in the visible wavelength range of 380 to 470 nm divided by the absorbency of the peak which occurs in the UV wavelength range of 220 to 300 nm is equal to or greater than 7 (e.g., 7 to 8.5), e.g., equal to or greater than 7.5 (e.g., 7.5 to 9, e.g., 7.5 to 8.5), e.g., equal to or greater than 8 (e.g., 8 to 8.8), e.g., greater than 8.5.

1.7. Any one of Methods 1 or 1.1-1.6, wherein the bipolar trans carotenoid salt (e.g., trans sodium crocetinate) is at least 90% pure as measured by high performance liquid chromatography (HPLC), e.g., > 95% pure as measured by HPLC, e.g., > 96% pure as measured by HPLC.

1.8. Any one of Methods 1 or 1.1-1.7, wherein the TSC is at least 90% pure as measured by high performance liquid chromatography (HPLC), e.g., > 95% pure as measured by HPLC, e.g., > 96% pure as measured by HPLC.

1.9. Any one of Methods 1 or 1.1-1.8, wherein the bipolar trans carotenoid salt is in a composition also comprising a cyclodextrin. For instance, wherein TSC is in a composition also comprising a cyclodextrin (e.g., wherein TSC is in a lyophilized composition with a cyclodextrin).

1.10. Method 1.9, wherein the cyclodextrin is gamma-cyclodextrin. For instance, wherein the bipolar trans carotenoid salt is TSC which is in a composition also comprising gamma- cyclodextrin. Any one of Methods 1 or 1.1-1.10, wherein the composition further comprises mannitol. Any one of Methods 1 or 1.1-1.11, wherein the diffusion enhancing compound is administered intravenously or intramuscularly. For instance, any one of Methods 1 or 1.1-1.11, wherein the diffusion enhancing compound is admixed with sterile water for injection to form an injection. For instance, any one of Methods 1 or 1.1-1.11, wherein TSC is admixed with sterile water for injection to form an injection.

Any one of Methods 1 or 1.1-1.12, wherein the diffusion enhancing compound is administered intravenously.

Any one of Methods 1 or 1.1-1.13, wherein the diffusion enhancing compound is TSC and is administered (e.g., one time daily) at a dose of 0.05-5 mg/kg (e.g., 0.5-5 mg/kg, e.g., 1-5 mg/kg (e.g., 1-2.5 mg/kg), e.g., 1.2-5 mg/kg (e.g., 1.2-2.5 mg/kg)), e.g., 0.05- 2.5 mg/kg, e.g., 2-2.5 mg/kg, or 0.1-2 mg/kg, e.g., 0.2-2 mg/kg or 2.5-5 mg/kg or 3-5 mg/kg. For instance, wherein the diffusion enhancing compound is TSC and is administered (e.g., one time daily) at a dose 0.25-1.5 mg/kg, e.g., 0.5-1.5 mg/kg, e.g., 0.5 or 1.2 mg/kg.

Any one of Methods 1 or 1.1-1.14, wherein the wound is a burn (e.g., a thermal burn, e.g., 1^st, 2^nd or 3^rd degree).

Any one of Methods 1 or 1.1-1.15, wherein the wound is selected from the group consisting of a compromised skin graft or flap, a diabetic foot ulcer, osteomyelitis, osteoradionecrosis, a pressure ulcer, a surgical wound, a traumatic wound, a venous stasis ulcer, and a wound from a vascular disease.

Any one of Methods 1 or 1.1-1.16, wherein the wound is a chronic wound. A chronic wound includes a wound that does not heal in the amount of time expected and/or is recurrent. For instance, a chronic wound includes a wound that does not heal within 3 months or 6 months or 1 year or more.

Any one of Methods 1 or 1.1-1.17, wherein the mammal is also administered oxygen. Any one of Methods 1 or 1.1-1.18, wherein the mammal is also administered hyperbaric oxygen therapy (HBOT).

Any one of Methods 1 or 1.1-1.19, wherein the mammal is also administered an antibiotic.

Any one of Methods 1 or 1.1-1.20, wherein the mammal is a human. 1.22. Any one of Methods 1 or 1.1-1.21, wherein the method comprises improving granulation and/or epithelialization of the wound.

1.23. Any one of Methods 1 or 1.1-1.22, wherein the method comprises reducing erythema, edema, and/or infection of the wound.

1.24. Any one of Methods 1 or 1.1-1.23, wherein the method prevents moderate to severe edema of the wound.

1.25. Any one of Methods 1 or 1.1-1.24, wherein the diffusion enhancing compound is administered for less than a month (e.g., for two weeks, e.g., for 10 days).

Further provided is a diffusion enhancing compound (e.g., a bipolar trans carotenoid salt (e.g., TSC)), e.g., as described in any one of Methods 1, la, or 1.1-1.25, for use in any one of Methods 1, la, or 1.1-1.25.

Further provided is use of a diffusion enhancing compound (e.g., a bipolar trans carotenoid salt (e.g., TSC)), e.g., as described in any one of Methods 1, la, or 1.1-1.25, in the manufacture of a medicament for any one of Methods 1, la, or 1.1-1.25.

Further provided is a pharmaceutical composition comprising an effective amount of a diffusion enhancing compound (e.g., a bipolar trans carotenoid salt (e.g., TSC)), e.g., as described in any one of Methods 1, la, or 1.1-1.25, for use in any one of Methods 1, la, or 1.1-

1.25.

* * *

The present invention will be further understood after careful consideration is given to the following non-limiting examples thereof.

Examples

Example 1 10-Day Full Thickness Wound Healing Treatment with Diabetic Yucatan Miniature Swine The purpose of this study is to evaluate the efficacy of the test article in wound healing in the diabetic miniswine and to determine if there is a dose response effect using two different concentrations of the test article using three diabetic adult male Yucatan miniature swine. On Day 0, four full thickness wounds are surgically-created per animal. Each animal is assigned to one of three different treatment groups that include vehicle control and two different nominal concentrations (0.475 and 1.20 mg/kg) of the test article (TSC). All wounds are periodically observed/evaluated through termination. On Day 10, all animals are euthanized. Wound assessments, including photographs, tracing, scoring (Modified Draize), and collection of a biopsy sample are performed for each wound site. Biopsy samples are shipped to the sponsor for possible histopathology analysis.

Introduction

The objectives for this study are to 1) evaluate the efficacy of the test article in wound healing in the diabetic miniswine and 2) to determine if there is a dose response effect using two different concentrations of the test article.

Experimental Design

This study includes three different treatment groups in three diabetic male Yucatan miniature swine (N = 3; 1 / group). The acclimation period is 10 days. On Day 0, each study animal undergoes a surgical procedure under general anesthesia to create four (two/side) full thickness excision wounds on the dorsal surface. All wound sites (4/animal) are covered with identical dressings. Each animal is administered one of three treatments, including vehicle control (sterile saline) and two different concentrations of TSC. All wounds are periodically observed/evaluated through termination. On Day 10, all animals are euthanized and biopsy samples are collected from all wound sites for possible histopathology analysis. The experimental study design, including treatment groups and variables evaluated with intervals are presented in Table 1 and Table 2, respectively.

Table 1 Details of Treatment Groups

Table 2 Variables Evaluated with Intervals

Materials and Methods Test Article

Test Animals

Test Animals (continued)

Animal Diet and Water Food

Water

Dose Procedure

Pre-Operative Preparation

Animals undergoing anesthesia for surgery and follow-up procedures are food-fasted overnight prior to anesthesia. Atropine sulfate (-0.04 mg/kg, IM) is administered prior to induction for surgery procedures (Day 0).

Induction and Maintenance

On Day 0, anesthesia of the animals is induced with a combination of Telazol (-2.2 mg/kg,

IM) and xylazine (-0.44 mg/kg, IM). Each animal is intubated endotracheally and maintained using isoflurane (0.5 to 5% with 100% oxygen). Surgical Procedure/Dose Administration a. On Day 0, the dorsolateral back area of each animal is closely clipped with electric clippers and/or shaved with a razor to remove hair, if necessary. The surgical wounds are created in a single paraspinal column on each side with efforts made to keep the columns between the crest of the shoulders and the coccygeal tuberosity. Each animal has four (one row of two/side) 5 x 5 cm wound sites created, spaced at least 4 cm apart. Refer to Figure 1. b. Each animal is prepared for surgery using alternating disinfecting scrubs and isopropyl alcohol rinses. The surgical area is draped and a sufficiently large hole(s) is created within the drape(s). c. Eising surgical blade(s), the skin is excised followed by total excision of the subcutaneous tissue down to the muscle fascia layer. Refer to Figure 2. d. Four 5 x 5 cm full-thickness excisional wounds are created (up to the muscle fascia layer). Any excised tissue from each wound site is discarded. e. Direct pressure is utilized to obtain hemostasis. Cautery is discouraged. f. Wound assessments, including photographs, measurements, and scoring (granulation, epithelialization, and general observations), are performed for each wound site. g. Each wound is covered with sterile petrolatum gauze (Tyco Healthcare), sterile gauze, and appropriate dressing (3M Tegaderm™ transparent adhesive dressing). In addition, the entire wound area is covered with a compression dressing (3M Reston™ self-adhering foam pad) and a tear-resistant mesh (McKesson stockinette) to minimize dislodgement of the dressing material. h. Following surgery, each animal is administered with the appropriate amount of vehicle or test article via intravenous (IV) administration through an ear vein. The animal receiving vehicle solution (Group 1) is administered a similar volume to that received by the animal in the high dose group (Group 3). i. Anesthesia is terminated and each animal is given appropriate post operative care. Follow-Up Procedures

On Days 1 through 9, follow-up procedures for dose administration and/or wound dressing changes are performed. Anesthesia of the animals is induced and maintained with direct administration of isoflurane (0.5 to 5% in 100% oxygen) through a nose mask.

Daily (Days 1 through 9), each animal is administered with the appropriate amount of vehicle or test article via intravenous (IV) administration through an ear vein.

Additionally on Days 2, 4, and 7, wound dressing changes are performed for each study animal as follows: a. Wound dressing materials for each animal are carefully removed and discarded. Care is taken not to disturb the wound sites. b. The dorso-lateral back area of each animal is prepared for fresh wound dressing using isopropyl alcohol soaked-gauze and/or dry gauze, as needed. c. Each wound is covered with sterile petrolatum gauze (Tyco Healthcare), sterile gauze, and appropriate dressing (3M Tegaderm™ transparent adhesive dressing). In addition, the entire wound area is covered with a compression dressing (3M Reston™ self-adhering foam pad) and a tear-resistant mesh (McKesson stockinette) to minimize dislodgement of the dressing material.

After each follow-up procedure, anesthesia is terminated and each animal is monitored until it has completely recovered.

Refinement

All study animals are under general anesthesia for surgery and follow-up procedures. On Days 0, 1, and 2, Buprenorphine (target 0.01 mg/kg, IM, BID) is administered to each animal.

Daily Mortality/Morbidity

Daily observations for morbidity/mortality are performed twice daily (AM and PM). Estimated Feed Consumption

All animals are fed at least once a day at a maintenance amount. Animals consume all offered feed during the study.

Physical Examinations All animals are given a physical examination by a veterinarian on Day -4 or Day 0 (pre surgery). Examinations include, but are not limited to, examination of the skin (particularly dose site) and external ears, eyes, abdomen, neurological, behavior, and general body condition.

Body Weight All animals are weighed on Days -1 or 0 (pre-surgery).

Wound Observations and Analyses

Each wound site is visually-observed using a modified Draize scoring system and photographed on Days 0 and 10.

Digital Photographs. High resolution photographs are obtained for each dose site at the designated time points. All photographs contain appropriate identifiers (animal ID, site #, date, reference scale) for identification along with a “color” scale. Each photograph is taken from directly in front of the wound in order to ensure an accurate measurement. The wound, ruler, label, and “color” scale fill the frame, with the ruler positioned as flat as possible.

Epithelial Tracing. Using Vistrak Grid (Smith & Nephew), a tracing of the epithelial border is performed for estimation of wound area.

Wound Scoring: Visual assessments are performed. Wound scoring for eschar, exfoliation, hydration, and infection is performed as per Table 3. In addition, wounds are scored for erythema, edema, granulation, and epithelialization as per Table 4. Table 3 Wound Scoring Scheme

Table 4 Key to Wound Site Evaluations

Biopsy/Histopathology

On Day 10, each study animal is euthanized. No gross necropsy or additional evaluations are performed. Following wound observations, an approximately 1 cm wide strip by sufficient depth to include at least full thickness wound tissue is excised running from medial to lateral through the center of each wound site, including a few millimeters of normal surrounding skin tissue on both ends of the wound site. These central strips of wound tissue are placed into 10% formalin for storage until shipment for possible histopathology analysis. Statistical Analysis

No statistical analysis (other than mean calculations) is performed for this study. Results

Dose Administration

Daily dose solutions are prepared, as per instructions, and all study animals are successfully administered the appropriate vehicle or test article solution. Animals in Groups 1, 2, and 3 are administered with 2.5, 1.0, and 2.3 mL/day or 0, 0.48, and 1.2 mg/kg TSC/day, respectively.

Daily Morbidity/Mortality

One death occurrs during the study. Animal ID 1213 dies following the Day 0 surgical procedures and dosing. A gross necropsy is performed by a veterinarian and the cause of death is attributed to anesthesia complications and not related to the administration of test article. No additional abnormal observations are noted during the conduct of this study.

Estimated Feed Consumption

All animals consume all offered feed during the study.

Blood Glucose Levels Individual animal blood glucose levels are determined to range from 235 to 547 mg/dL during the conduct of this study.

Physical Examinations

All animals have a physical examination prior to study entry during acclimation on Days -4 or 0 (pre-surgery). No abnormalities are determined that preclude study inclusion. Body Weight

All animals are weighed on Days -1 or 0 (pre- surgery). All animals are in the expected weight range for age at the beginning of the study.

Wound Observations

Individual and group mean wound site scores are presented in Table 5 and Table 6. Wound site areas along with changes from Day 0 are presented in Table 7. In general, expected wound healing is observed during this study in all three treatment groups. Table 5 Individual Wound Site Scores by Time Point and Animal

ND = Not determined due to swelling

For wound scoring system refer to Table 3 and Table 4.

Table 6 Mean Wound Site Scores by Time Point and Animal

ND = Not determined due to swelling

For wound scoring system refer to Table 3 and Table 4. Table 7 Wound Area and Wound Area Change from Day 0 by Animal/Dose and

Wound Site

Figure 3 depicts stages of wound healing at sequential time points (Days 0 and 10) for individual sites of each treatment group.

Biopsy/Histopathology

All wound biopsy specimens are obtained at study termination and sent for possible analysis. No histopathology results are presented in this report.

Conclusions

Three diabetic male Yucatan miniature swine successfully undergo wound healing procedures for this study. Four (two/side) approximately 5 cm x 5 cm full thickness excision wounds per animal are evaluated during a 10-day study period. Wound healing is assessed by using standard wound evaluation scoring systems, photographic surveillance, and histopathology. All parameters show evidence of healing for all wound sites. However, based on wound area change and visual assessment of photographs, it appears that there is a direct effect of 1.20 mg TSC/kg on improved wound healing, as compared to vehicle control and 0.48 mg TSC/kg animals in this study. Therefore, higher dosages of TSC may reduce wound area and improve wound healing. Individual Animal Data

Table 8 Individual Animal Information

NOTE: All study animals are castrated male Yucatan miniature swine.

* * * It should be understood that a wide range of changes and modifications could be made to the embodiments described above. It is therefore intended that the foregoing description illustrates rather than limits this invention, and that it is the following claims, including all equivalents, which define this invention.

Claims

What is claimed:

1. A method of treating a human having a wound comprising administering a pharmaceutically effective amount of a diffusion enhancing compound to the human.

2. A method as in claim 1, wherein the diffusion enhancing compound is a bipolar trans carotenoid.

3. A method as in claim 1, wherein said diffusion enhancing compound is a bipolar trans carotenoid salt.

4. A method as in claim 3, wherein said bipolar trans carotenoid salt is formulated with a cyclodextrin.

5. A method as in claim 1, wherein said diffusion enhancing compound is TSC.

6. A method as in any one of claims 1-5, wherein the diffusion enhancing compound is administered IV or IM.

7. A method as in any one of claims 1-6, wherein the diffusion enhancing compound is trans sodium crocetinate (TSC) and is administered at a dose of 0.2-2 mg/kg.

8. A method as in any one of claims 1-7, wherein the wound is a burn.

9. A method as in any one of claims 1-7, wherein the wound is selected from the group consisting of: compromised skin grafts and flaps, diabetic foot ulcers, osteomyelitis, osteoradionecrosis, pressure ulcer, surgical wounds, traumatic wounds, venous stasis ulcer, and wounds from vascular diseases.

10. A method as in any one of claims 1-9, wherein the human is also administered oxygen.

11. A method as in any one of claims 1-10, wherein the human is also administered hyperbaric oxygen therapy (HBOT).

12. A method as in any one of claims 1-11, wherein the human is also administered an antibiotic.