WO2008023998A1

WO2008023998A1 - N-acylethanolamines as wound healing agents

Info

Publication number: WO2008023998A1
Application number: PCT/NZ2007/000225
Authority: WO
Inventors: Mikhail Vyssotski; Keryn Dallas Johnson
Original assignee: Industrial Research Limited
Priority date: 2006-08-22
Filing date: 2007-08-22
Publication date: 2008-02-28

Abstract

The use of compounds for promoting the healing of wounds, having the formula 1: where R is a straight or branched chain C1-C30 alkyl which may be substituted by one or more hydroxyl groups or halogen atoms, and may contain one or more epoxy groups. The wounds treatable include chronic wounds, such as cuts, scratches, abrasions, oral ulcers, stomach ulcers, ulcers from cancer therapy, diabetic foot ulcers, venous stasis ulcers, pressure ulcers, and burns, as well as wounds that result from ophthalmic surgery, cosmetic surgery, general surgery, treatment for keloids and hypertrophic scars (anti-scarring treatments).

Description

N-ACYLETHANOLAMINES AS WOUND HEALING AGENTS

TECHNICAL FIELD

This invention relates to the use of N-acylethanolamines as wound healing agents. In particular, the invention relates to the use of N-acylethanolamines for treating chronic wounds such as ulcers and burns.

BACKGROUND

Wounds that do not heal readily through natural healing processes cause major problems. The non-healing of wounds causes significant adverse affects on the quality of life of a patient. A large economic burden is placed on society. Chronic wound care is estimated to cost between $18 billion to $24 billion annually in the United States.

Wounds heal through a process that involves a normal orderly and timely sequence of events and results in the closure of the wound and generation of an effective barrier restoring the function of the skin. The normal sequence of events begins with the formation of a clot to prevent blood loss. This is followed by inflammation where neutrophils enter into the wound and then monocytes which differentiate to macrophages. The inflammatory stage is followed by the proliferative stage of healing involving angiogenesis and fibroblasts production of extracellular matrix proteins. The final stage is wound remodelling resulting in an effective tissue barrier.

Chronic wounds are those which fail to progress through the normal stages of healing and are trapped in an early stage of wound healing with excessive inflammation. Examples of such wounds include diabetic foot ulcers, venous stasis ulcers, pressure ulcers, and burns. The vast majority of chronic wounds lack a sufficient blood supply thereby causing significant delays in healing. Angiogenesis, which is the formation of new blood vessels, is therefore necessary for wound healing. The precursor steps for angiogenesis stimulation are endothelial cell migration and proliferation. Compounds that are shown to promote endothelial cell migration and/or proliferation may promote the healing of these chronic wounds. There are numerous examples of compounds and substances known to promote new blood vessel formation.

Chronic wounds are likely to have significant levels of bacteria. This can result in increased levels of inflammation, and result in the wound failing to heal. At present, such wounds are treated with an antibiotic to reduce inflammation. However, it is not always possible to remedy this problem with antibiotics. Antibiotics are often found to be counter productive because they are toxic to the cells in the wound, and can therefore contribute to delays in healing. There is therefore a need for improved treatment of chronic non-healing wounds. In particular, there is a recognised need for a chronic wound healing agent that is anti-microbial, as well as being non-toxic to eukaryotic cells in the wound.

The development of new wound healing treatments is required for the prevention of scarring in a variety of dermatological, cardiovascular, ophthalmic, central nervous system, Gl/reproductive, fibrotic, and respiratory diseases and conditions.

In addition, there is an increasing demand for over-the-counter nutraceutical treatments for superficial cuts, scratches and grazes that heal wounds without complications and which help to prevent scarring. There is also a need for a product which can be used in the treatment of burn wounds which avoids the problems of administration of treatment products due to pain and sensitivity of touch associated with severe burns.

N-Acylethanolamines (NAEs) are released from lipid membranes upon wounding. The release mechanism involves the N-acylation of phospatidylethanolamine, followed by the action of the phospholipase D-like enzyme. The biosynthesis of anandamide and N-palmitoylethanolamine can occur by sequential actions of phospholipase A2 and lysophospholipase D. Alternative mechanisms of NAEs formation have been suggested, including direct synthesis from ethanolamine and free fatty acids, and formation from partially deacylated N-acyl phosphatidylethanolamine (NAPE), lyso-NAPE, and/or glycerophospho N-acylethanolamine. N- acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) has also been identified as a candidate enzyme involved in the biosynthesis of NAEs. Further studies identified a calcium- independent PLD activity in brains from NAPE-PLD(-/-) mice that accepted multiple NAPEs as substrates, including the anandamide precursor C20:4 NAPE. NAEs are known to be potent as membrane-stabilizing and anti-inflammatory agents.

A range of other derivates or precursors of NAEs are available through exchange of biogenic amines between ethanolamine and compounds such as amino acids histamine, glutamine, taurine, serotonin, and melatonin. Some NAEs are endogenous PPARα receptor binding ligands, which may be part of the immuno/inflammatory axis. The role and importance of this axis in wound healing is unknown. It is known that inflammatory macrophages are able to synthesize NAEs. Macrophages, key immuno modulators of the wound healing cascade, release a range of cytokines and growth factors that orchestrate the healing process. NAEs are processed efficiently by the enzyme fatty acid amino hydrolyase (FAAH). The reaction results in the releasing of a free fatty acid and ethanolamine. Other hydrolases have also been identified that can hydrolyse NAEs.

The inventors have surprisingly found that NAEs or their precursors or breakdown products have the ability to treat wounds. NAEs are both anti-microbial and non-toxic to eukaryotic cells in the wound, and they appear to overcome the problems associated with antibiotics.

It is therefore an object of the present invention to provide an N-acylethanolamine as a wound healing agent, or at least to provide a useful choice.

STATEMENTS OF INVENTION

In a first aspect, the invention provides the use of a compound of formula 1 :

(1) where R is a straight or branched chain C₁-C₃₀ alkyl which may be substituted by one or more hydroxyl groups or halogen atoms, and may contain one or more epoxy groups, for promoting the healing of a wound in a subject.

R may preferably be C₅-C₂₅ alkyl, C₅-C₂! alkyl, C₅-Ci₅ alkyl, C₉-C_2I alkyl, or C₁₃-C₂₃ alkyl. In preferred embodiments of the invention, R is C₁₅-C₂₃ alkyl. R may be saturated or unsaturated. Examples of alkyl groups include: pentadecyl (Ci₅H₃₁) and heptadecenyl (C₁₇H₃₃).

The compound of formula 1 may preferably be selected from N-palmitoylethanolamine, N- oleolyethanolamine, and N-stearoylethanolamine.

The compound of formula 1 may be administered to the subject. Alternatively, a compound that is a precursor compound thereof may be administered to the wound such that the compound of formula 1 forms at the site of the wound. Examples of precursor compounds include N-acyl phosphatidylethanolamines, lyso-N-acyl phosphatidylethanolamines, and glycerophospho N- acylethanolamines.

The wound may be any wound in need of treatment, but is preferably a chronic wound. Such wounds are typically diabetic foot ulcers, venous stasis ulcers, pressure ulcers, or burns. Preferably the compound of formula 1 is obtained by chemical synthesis. Alternatively, the compound of formula 1 may be obtained from a natural source.

The compound of formula 1 , or a precursor compound thereof, is preferably administered with one or more of a pharmaceutically acceptable excipient, adjuvant, carrier, buffer, and stabiliser.

In a preferred embodiment the compound of formula 1 , or a precursor compound thereof, is administered as an oil, a solution, an emulsion, a wax, or a spray.

In another aspect, the invention provides the use of a compound of formula 1, or a precursor compound thereof, in the manufacture of an agent for promoting the healing of a wound.

DETAILED DESCRIPTION

The term "alkyl" means any saturated or unsaturated hydrocarbon radical having up to 30 carbon atoms and includes C₅-C₂₅ alkyl, C₅-C₂₀ alkyl, C₅-C₁₅ alkyl, C₉-C₂₁ alkyl, or C₁₃-C₂₁ alkyl, and is intended to include both straight- and branched-chain alkyl groups.

The term "precursor compound" means, with reference to a compound of formula 1 , any compound that may be broken down or metabolised to a compound of formula 1 following administration to the site of a wound. Examples include N-acyl phosphatidylethanolamines, lyso-N-acyl phosphatidylethanolamines, and glycerophospho N-acylethanolamines.

The term "wound" means an external or internal break in the normal tissue architecture, any break in the skin or an organ caused by violence or surgical incision, one in which the skin or another external surface is torn, pierced, cut, or otherwise broken.

The term "chronic wound" is defined by the Wound Healing Society as a wound that fails to progress through a normal, orderly and timely sequence of repair or a wound that passes through the repair process without restoring anatomic and functional results.

The term "burn" is defined as damage to the skin or other body parts caused by extreme heat (thermal) burn, flame, contact with heated objects, or chemicals, or frost, or electrical sources. Burn depth is generally categorised as first, second, or third degree. The treatment of burns depends on the depth, area, and location of the burn, as well as additional factors, such as material that may be burned onto or into the skin. Treatment options range from simply applying a cold pack to emergency treatment to skin grafts. The underlying reasons why chronic wounds fail to heal are diverse and range from issues related to age, weight e.g. obesity, nutritional status, dehydration, inadequate blood supply to the wound site, immune response e.g. immuno suppressed patient with HIV allowing increased infection, chronic disease e.g. diabetes and radiation therapy. Chronic wounds also differ to acute wounds with respect to pH, protease levels, bacterial burden, and are often recalcitrant to growth factor therapy because of previously mentioned reasons. Each chronic wound type requires an understanding of the underlying biology to be able to address its non-healing nature before the correct treatment can be used to restore the wound to a healing outcome.

The therapeutic application of NAEs to wounds is alleged to either cause no effect or an antiinflammatory effect depending on what fatty acid chain is present in the molecule. Antiinflammatory properties of NAEs have been observed for palmitoylethanolamide and oieoylethanolamide.

Addition of the appropriate NAEs enables recruitment of inflammatory cells. Monocytes and macrophages, and coordination of the wound-healing cascade by macrophages, enables acceleration of healing. This is speculated to be due to the recruitment of appropriate inflammatory cells, which produce the appropriate cytokines and growth factors that co-ordinate healing. The regulation of the interplay between cells, their extracellular matrix and signalling molecules controls the wound healing cascade and leads to wound closure. This process is regulated by neurons, pain and loss of the electrical circuits in the body around the wound site. NAEs play a role in the interplay between the nervous system and inflammatory system (immuno inflammatory axis).

The wound may be any wound in need of treatment, but the invention is more suited to a chronic wound. Such wounds are typically diabetic foot ulcers, venous stasis ulcers, pressure ulcers, or burns. Other relevant wounds include wounds from ophthalmic surgery, cosmetic surgery, general surgery, and treatments for keloids and hypertrophic scars (anti-scarring treatments), cuts, scratches and abrasions, oral ulcers, stomach ulcers or ulcers that develop due to cancer therapy (mucositis in oral, esophageal and gastrointestinal tract). The invention has use for scar prevention and treatment, in dermatology - prevention and reduction of scarring following elective surgery or acute injury, for cardiovascular procedures - restenosis following coronary surgery, catheterisation, peripheral vascular surgery, for eye treatments and ophthalmics - prevention of scarring following injury to cornea or re-sculpturing laser surgery, for CNS procedures - neurosurgery and nerve trauma, spinal adhesions, Gl/reproductive - prevention of adhesions following internal surgery - cardiac, abdominal, pelvic, for fibrotic disorders - chronic kidney fibrosis, liver cirrhosis, glomerulonephritis, uterine fibrosis and diffuse scleroderma, and respiratory function - pulmonary fibrosis, COPD.

The inventors hypothesise that NAEs re-initiate the wound-healing cascade and have an overarching role in the co-ordination of the healing process. NAEs are naturally synthesized at the moment of injury and are important signalling molecules. NAEs may not be formed at appropriate levels in chronic wounds.

Preferably, the present invention relates to a mixture of NAEs synthesised from oils with long chain fatty acids C₆ to C₂₄. The long chain fatty acids can be either saturated or unsaturated.

The invention relates to pure synthetic NAEs, containing one of the following fatty acids: palmitic, stearic, oleic, linoleic, linolenic or any other natural saturated, monounsaturated or polyunsaturated fatty acid

Another aspect of the invention relates to mixtures of NAEs, which are produced semi- synthetically from naturally existing lipid mixtures. Advantages of using a mixture of NAEs include a lower melting point, and improved solubility in skin-compatible substances and solvents. However a major disadvantage of using mixtures is that the raw material for their production (natural lipid mixtures) may vary in fatty acid compositions. This may cause variation in wound-healing activity. Mixtures of NAEs, produced semi-synthetically from naturally existing lipid mixtures, may contain small quantities of other naturally existing compounds. This may affect the healing process.

A further aspect of the present invention relates to a pharmaceutical which contains the NAEs active compound, as well as an excipient. The excipient allows a sustained release of the pharmaceutical compound. The inventors have found that sustained delivery of NAEs overcomes fatty acid amino hydrolyase (FAAH), which is the enzyme that degrades NAEs, to enable a pharmaceutical effect to occur.

Vaseline has been found to facilitate the application of lipophilic NAEs. A number of different excipients could be used in either an emulsion, as in sunscreens, or mixed into Vaseline. Suitable excipients include: carboxymethylcellulose, cyclodextrins, isopropyl myristate, magnesium stearate gels, polysaccharides such as cellulose acetate phthalate, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxypropylethylcellulose, ethylcellulose, methylcellulose, microcrystalline cellulose and carrageenan, alginate, xylose, fucose, anionic and cationic polymers of methacrylic acid, copolymers of methacrylates, copolymers of acrylates and methacrylates, copolymers of ethacrylate and methylmethacrylate, dibutyl sebacate, dextrose, lauryl lactate, maltodextrin, polyvinyl acetate phthalate, povidone, sodium starch glycolate, glycogen, sorbitol, crystalline, starch DC, sugar spheres, triacetin, triethylcitrate, xanthan gum, xylitol DC, aspartame, lactose, acetyltri-n-butyl citrate, and acetyltriethyl citrate.

Penetration enhancers including: DMSO, azones (e.g. laurocapram), pyrrolidones (for example 2-pyrrolidone, 2P), alcohols and alkanols (ethanol, or decanol), glycols (for example propylene glycol, PG, a common excipient in topically applied dosage forms), surfactants (also common in dosage forms), and terpenes.

A carrier is essential for pure crystalline NAEs with high melting points. Examples include: N- palmitoylethanolamine (m.p. 98°C) and N-stearoylethanolamine (m.p. 102⁰C).

If the surface of the skin/wound is not warm enough then the NAE will stay crystalline. The NAEs will not be able to penetrate the skin or enter the wound and carry out their function.

If oil is used as the starting point for synthesis, the oil can also act as the carrier or excipient. In such a formulation, application to skin is not a problem as the NAEs are not in the crystalline form.

Another aspect of the invention relates to a spray on formulation which may be best suited for burn wounds. This overcomes some administration problems of the compound which arise due to the pain and sensitivity of touch associated with severe burns.

Histological analysis has shown an increased number of inflammatory cells and an overall acceleration of wound healing compared to the placebo treated control.

Stimulation of wound healing has been seen in a delayed healing model, rabbit ear ulcer model. The delayed healing is due to tissue ischaemia induced by ligation of two of the three arteries in the rabbit's ear.

Promotion of wound healing in the rabbit ear ulcer model gives strong evidence that compounds of this nature will promote wound healing in human chronic wounds. This is the first known instance of NAEs being used in delayed wound healing models, implicating their promise as effective therapeutics for human chronic wounds. EXAMPLES

Example 1 - General NAEs Synthesis Method

Syntheses of N-palmitoylethanolamine, N-stearoylethanolamine and N-oleoylethanolamine were performed essentially as described by Roe et al. (Roe, E. T., J.T.; Scanlan, J.T.; Swern, D. 1949. Fatty Acid Amides. I. Preparation of Amides of Oleic and the 9,10-Dihydroxystearic Acids. J. Am. Chem. Soc. 71 : 2215-2218). To prepare the natural oils-based mixtures of NAEs, the method of Young & Rubinstein (1946, US 2,394,833) was used, with a modification to eliminate residual ethanolamine by adding an excess of triacylglycerols. Freshly distilled ethanolamine (3 g) was added to a screw-capped glass vial, and 15.7 g of either vegetable or fish oil (containing 99% of triacylglycerols) was added. The vial was flushed with argon or nitrogen, closed, and placed for 4 hours in an oven preheated to 130⁰C. At room temperature, the product was a wax-like white, beige or light-brown substance, melting within a temperature range of 45-70⁰C. The product was assessed by TLC in methanol-water-NH₄OH (7:3:1, by v/v/v) and hexane- ether-acetone-acetic acid (30:40:20:1 , by v/v/v/v). No residual ethanolamine was detected with ninhydrin spray. Small quantities of monoacylglycerols and free fatty acids were present as byproducts (detected by gentle charring with 5% sulphuric acid in ethanol and plate development in a hexane-ether-acetone-acetic acid system).

Product assessment was also performed by GLC of TMS-derivatives, produced by reaction of up to 2 mg of the NAEs mixture with 20 μl of BSTFA (N,O-bis-trimethylsilylacetamide) at 50- 60⁰C for 30 min. For GLC analysis, 0.2 to 0.5 μl of this solution was injected into GC (quartz capillary column with BP-5 phase (30m, 0.25 mm i.d., 0.25 μ film), injector temperature 300⁰C, and oven temperature increased from 200⁰C at a rate 2°C/min to 280⁰C and was held at this temperature for 10 min, FID). GC-MS runs were performed with HP-5MS capillary column (30m, 0.25 mm i.d., 0.25 μ film) with the following temperature program: starting temperature 200C for 6 min, then temperature increased at 2C/min to 280C and kept for 10 min at the final temperature. N-palmitoylethanolamine and N-stearoylethanolamine were used as NAE standards.

Example 2 - Rabbit Ear Ulcer Model Methodology

The rabbit ear ulcer model produces maximum ischaemia and maximum congestion, with complete survival of the ear by selective division of two of the three ear arteries. The wound created has an avascular cartilagenous base which is unable to close by contraction. This property differentiates this wound from most other small animal models and provides a greater resemblance to human wound closure. The model can also be modified by induction of ischemia through partial ligation of two of three arteries in the rabbit's ear. Those products which can promote earlier and faster healing will reduce the secondary infection rate and scarification often associated with skin damage.

The ear wound surgically created in the rabbits was amenable to recurrent treatment and dressing changes. This model offered a variety of measurable parameters such as morphometric measurements of wound closure and wound filling; non-invasive measurement of tissue perfusion, and histological measurement of epithelialisation, granulation and angiogenesis.

Wound healing scores were documented for each individual rabbit twice daily. Particular attention was paid to immunogenic, vasodilator/ or necrotising changes of the treatment area. Post Mortem histological examinations of the wound sites were performed on all animals at the end of the study.

The animal phase commenced with identification and randomisation of 15 female rabbits into five treatment groups of three animals each between Day -5 and Day -1. The five males were placed into a single treatment group of three animals and one negative control group of 2 animals. Animals were numbered sequentially with a non-toxic indelible marker, written on the dorsal region. Each pen was labelled with an appropriate card according to the treatment group it contained. The animals were weighed at the onset of the test and every day for the first week in order to determine the overall effect of the surgical procedure on the animals. Animals were then weighed on Day 14 prior to termination.

On Day 0 the animals were administered an anaesthetic combination through IM injection. Rabbits were given an initial injection of acepromazine at 1.0 mg/kg IM, which sedated the animal in approximately 15 minutes. This was followed by intramuscular injections of Ketamine HCL and Xylazine at 35 mg/kg and 5 mg/kg respectively. The lateral arteries were ligated on each ear by use of an external loop suture at approximately 5-8 cm from the base, which limited the perfusion of the distal ear to the medial artery only. Two 0.4 cm ulcers were made to the depth of the cartilage in each ear, using a 0.4 cm circular dermatologic punch device. The wound base was scraped exposing the cartilaginous surface. The particular test substance for that group was applied to the surgical lesions in both ears. For the purpose of this experiment, the treatment unit was the individual ear of each rabbit, providing six units per treatment group with each unit having two lesions to be measured.

The appropriate test substance was applied to the wound area by means of either filling or irrigating the ulcer with the test substance according to the nature of its viscosity as a direct superficial application. Following test substance application, the wounds were covered accordingly with an adhesive bandage in order to ensure the retention of the test substance at the site. Compounds were applied according to the following schedule.

Test mixture I, a mixture of N-acylethanolamides derived from Hoki oil fatty acids was applied on Day 1 only.

Test mixture II, a mixture of N-acylethanolamides derived from olive oil fatty acids was applied on Days 1 , 3, 5 and 7.

Test mixture III, (10%) in Vaseline, a mixture of N-acylethanolamides derived from olive oil fatty acids was applied on Days 1 , 3, 5 and 7.

The healing process was evaluated at 24 hours, days 3, 5, 7, 9, 12 and 14 following surgery and treatment application. The surgical site was examined and photographed on. days 1 , 3, 5, 7, 9, 12 and 14. The healing process was assessed according to measurable parameters such as morphometric measurements of wound closure and wound filling; non-invasive measurement of tissue perfusion, and histological measurement of epithelialization, granulation and angiogenesis.

On Day 1 to 7 the animals were weighed daily. Animals were weighed again on Day 14. On Day 14 the treated ulcers of the six treatment groups and the untreated ulcers of the negative control group were removed and examined histologically, with emphasis on the presence of new vessels, epithelialization, leukocyte infiltration and fibrosis.

All animals were anaesthetised on Day 0 through a combination injection administered intramuscularly. Rabbits were given an initial injection of acepromazine at 1.0 mg/kg IM, which sedated the animal in approximately 15 minutes. This was followed by intramuscular injections of Ketamine HCL and Xylazine at 35 mg/kg and 5 mg/kg respectively. Following the creation of the full depth ulcers, two of which were made in each ear using a 0.4 cm circular dermatologic punch device, the wound base was scraped, and the appropriate treatment for that group was applied by either filling or irrigating the ulcer. Treatment was repeated as per the schedule.

Test mixture I: Group 1 Test mixture II: Group 2 Test mixture III: Group 3

Post surgical procedure, all animals were observed twice daily. Examination of the wound, photographs, and an assessment of healing was made for all animals at 24 hours, 72 hours, 120 hours, Day 7, Day 9, Day 12 and Day 14. Photographs were taken of all animals on Day 7 and Day 14.

Table 1 - Wound Healin Scores

All animals tested remained healthy with none showing any abnormalities according to the daily health score record.

Example 3 - Wound Healing Scores

NAEs were tested for their effect on wound healing according to the methodology of Example 2. Analysis of wound healing scores was performed to gauge the effectiveness of NAEs for promoting healing. The most significant variable between the test substance groups that could be observed visually was the degree of healing evidenced by the decreasing diameter of the punch biopsy wound created on Day 0.

Table 5 ! - Mean Wound Healing Scores

Wound Wound Wound Wound Wound Wound

Wound Diam. Diam. Avg. Diam. Avg. Diam. Avg. Diam Avg. Diam Avg. Diam Avg.

Group Avg Day 1 Day 3 Day 5 day 7 day 9 day 12 day 14

1 3.3 3.0 2.7 2.7 2.7 2.7 1.7

2 3.3 2.8 2.8 2.5 2.5 2.5 1.7

3 4.0 3.8 3.8 3.2 3.2 3.0 2.5

C 4.0 3.5 3.3 3.3 3.3 3.3 3.0

As can be seen from Table 2, the two lipid products, Text mixture I and Test mixture Il (Groups 1 and 2 respectively) appeared to have the largest immediate impact on decrease in wound size as compared to the control group. All products compared to the Controls demonstrated an enhancement to wound healing. The controls behaved properly as a model of chronic wound healing due to decreased perfusion, in which the decrease in wound size from day 0 was minimal and probably more the result of wound contraction than the actual healing process. Table 3 - Percentage of Wound Healing over Test Duration

Healing % Healing % Healing % Healing % Healing % Healing % Healing %

Group Day1 Day 3 Day 5 Day 7 Day 9 Day 12 Day 14

1 0.17 0.25 0.33 0.33 0.33 0.33 0.58

2 0.17 0.29 0.29 0.38 0.38 0.38 0.58

3 0.00 0.04 0.04 0.21 0.21 0.25 0.38

C 0.00 0.13 0.19 0.19 0.19 0.19 0.25

Wound healing as measured by decreased wound diameter was most significant at onset of treatment in Groups 1 and 2 (Test mixture I and Test mixture Il respectively). However, both these products continued to promote excellent healing throughout the fourteen day period (58% by Day 14).

Example 4 - Body Weights

NAEs were tested for their effects on rabbit body weights according to the methodology of Example 2. The rabbit body weight is an indicator of the influence of NAEs on subclinical infection, pain, and irritation, which lead to reduced appetite. The change in body weight over the duration of the test for each test group is shown in Table 4. Although weight gain is not a direct measure of wound healing, processes that occur during healing such as inflammation, infection, pain and irritation directly influence normal weight gain, and therefore the animals showing the greatest weight gain were also those undergoing the best healing process. In this regard, Group 2 (Test mixture II) demonstrated the maximum weight gain compared with other groups.

Table 4 - Individual Weights

Group ID DayO Day1 Day 3 Day 7 Day 14

1 13 2166 2174 2263 2392 2586 1 12 2168 2187 2257 2315 2568 1 2 1618 1627 1698 1799 2045

2 8 2003 2019 2061 2175 2710 2 7 2051 2082 2157 2291 2476 2 5 2362 2404 2442 2539 2753

3 4 2705 2690 2442 2683 2920 3 15 2058 2056 2148 2217 2396 3 9 2408 2407 2487 2598 2789

C 20 2153 2176 2203 2340 2500 C 19 2240 2186 2236 2389 2529

The most noticeable increase in weight was by the Group 2 animals, which went from a 252 g differential between Group 2 and Group 3 on Day 0 to only a 55 g differential on Day 14. This indicated that the Group 2 animals fared far better than any of the other groups. The increase in body weight of approximately 0.5 kg suggested that those factors which reduced appetite amongst the other groups, such as subclinical infection, pain, irritation, were not present to the same degree in Group 2. This was a further indication that the animals that received the Test mixture Il product containing NAEs from olive oil performed much better in the entire healing process than any of the other groups on test. The NAE is predominantly N-oleolyethanolamine when produced from olive oil.

Example 5 - Histopathology

Histopathology was used to gauge a number of morphometric parameters including the degree of epithelialisation, granulation and vascularisation, as well as inflammatory cell types present within the wound. Histopathology was examined using the same animals of Example 2. On day 14 all animals were euthanased via an intravenous overdose of barbiturates and the sections from each ear where the biopsy punches were performed were removed and placed in

10% formalin. Each wound was examined as an independent lesion by a boarded histopathologist, thereby providing four lesions per animal, and twelve lesions per group, except for the controls which only had a total of eight lesions examined.

In total, forty ears were examined histologically and each ear had its two healing biopsy sites which were referred to as 'a' and 'b' within the following tables. Photomicrographs were taken and morphometric software was used to analyse the sections. Variables assessed included the total area of the granulation tissue, the distance between the surface epithelium and the deep edge of the granulation tissue (height of granulation), and the distance between the two lateral margins of granulation tissue (length of granulation). In addition, the height of the epidermis that was hyperplastic was also measured. Any ulceration within the sample was also measured. Using light microscopy, the extent of angiogenesis and inflammation with the samples was assessed using four 4OX microscopic fields. The first field was immediately underlying the epidermis in the area of maximum height of granulation tissue. The second was immediately superficial to the deep margin of the granulation tissue in the area of maximum height of granulation tissue. The third and fourth fields were at the lateral margins of the granulation tissue. The total number of blood vessels was recorded. The numbers of each of the inflammatory cells were graded between 0 and 4 in each of the four fields. The number recorded in the Tables is the total score for the cell types out of a possible sixteen.

Ideal healing involved the minimisation of granulation with the maximisation of epithelialisation. This required a high infiltration of blood vessels into the lesion site accompanied by those circulating blood cells responsible for eliminating necrotic debris and providing a controlled immune response such as neutrophils, lymphocytes and plasma cells. Similarly, a decrease in the number of macrophages reduces the degree of granulation. The mean histopathological results are shown in Table 5.

Table 5 - Mean Histopathological Results by Groups

Table 5 demonstrates the differences between treatment groups based on the histopathological analysis of the ulcer sites. According to the histological evidence, Group 2 animals treated with NAEs prepared from olive oil, rich in N-oleolyethanolamine, demonstrated reduced granulation with maximal epithelialisation, accompanied by the most appropriate cellular infiltration of the wound site to promote healing. The N-oleolyethanolamine rich preparation had the greatest overall healing process. Although the length of granulation for this group was not dissimilar from the others, the lower height of granulation was the more significant granulation factor as height correlates more closely to the level of scar tissue produced.

Olive oil NAEs, rich in N-oleolyethanolamine had the best combination of the desired factors required for healing of the dermal ulcer with the greatest length of epithelialisation of the test product groups, one of the highest numbers of blood vessel infiltrations, the highest level of polymorphic neutrophils, and lymphocytes, as well as the second highest level of plasma cells.

Statistical examination of the area of granulation was declared significant, p = 0.029, when the combined values of all groups are examined but when each group is compared individually against the Control group, then the hypotheses are not rejected and there were no significant differences. Similarly, when the height of granulation is examined for the combined groups, it was declared significant at p = 0.046. When the length of granulation was examined as combined groups, it was declared significant at p = 0.0004, but was also significant in direct group comparisons to the control at p = 0.048 for Group 1 , p = 0.042 for Group 2, and p = 0.001 for Group 3. The probability that the median heights of epithelialisation of all groups are equal was rejected at p = 0.008, but when each group is compared individually to the Control, the hypothesis is accepted and no significant differences are found. It is when the length of the epithelialisation process is examined that the difference between groups is demonstrated. As a combined comparison, p = 0.319, the hypothesis is accepted, but on a direct comparison between individual groups and the Control, then p = 0.044 for Group 2, demonstrating that it was significantly different from the Control, and thereby to the other groups as well, none of the others having demonstrated any significant difference.

The same is true for neutrophil infiltration of the wound site in that as a combined comparison of groups, p = 0.211 , but on direct comparison between individual groups and the Control then p = 0.029 for Group 2, demonstrating that it was significantly different from the Control and thereby to the other groups as well, none of the others having demonstrated any significant difference.

Similarly for lymphocyte infiltration of the wound site in that as a combined comparison of groups, p = 0.062, but on direct comparison between individual groups and the Control then p = 0.007 for Group 2, demonstrating that these two groups were significantly different from the Control and thereby to the other groups as well, none of the others having demonstrated any significant difference.

Therefore, in support of the clinical observations, the statistical analysis would indicate that by three of the critical criteria analysed, Group 2 did demonstrate significant differences.

Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in the specification.

INDUSTRIAL APPLICATION

The N-acylethanolamines of formula (1) are useful for treating chronic wounds, such as cuts, scratches, abrasions, oral ulcers, stomach ulcers, ulcers from cancer therapy, diabetic foot ulcers, venous stasis ulcers, pressure ulcers, and burns. The N-acylethanolamines are also useful for treating wounds that result from ophthalmic surgery, cosmetic surgery, general surgery, treatment for keloids and hypertrophic scars (anti-scarring treatments).

Claims

1. The use of a compound of formula 1 :

(1) where R is C₁-C₃₀ alkyl which may be substituted by one or more hydroxyl groups or halogen atoms, and may contain one or more epoxy groups, for promoting the healing of a wound in a subject.

2. The use as claim in claim 1 where R is C₁-C₂₃ alkyl, C₁-C₂₁ alkyl, C₁-C₁₉ alkyl, C₁-C₁₅ alkyl or C₁-C₉ alkyl.

3. The use as claimed in claim 2 where R is C₁₃-C₂₃ alkyl.

4. The use as claimed in any one of claims 1 to 3 where R is unsaturated.

5. The use as claimed in claim 4 where R has 1 , 2 or 3 double bonds.

6. The use as claimed in any one of claims 1 to 5 where the compound of formula 1 is selected from: a. N-palmitoylethanolamine; b. N-oleolyethanolamine; and c. N-stearoylethanolamine.

7. The use as claimed in any one of claims 1 to 6 where the compound of formula 1 is administered to the subject.

8. The use as claimed in any one of claims 1 to 6 where the compound of formula 1 is formed from a precursor compound thereof following administration to the subject of the precursor compound.

9. The use as claimed in claim 8 where the precursor compound is an N-acyl phosphatidylethanolamine, a lyso-N-acyl phosphatidylethanolamine, or a glycerophospho N-acylethanolamine.

10. The use as claimed in any one of claims 1 to 9 where the wound is a chronic wound.

11. The use as claimed in any one of claims 1 to 10 where the wound is selected from the group comprising cut scratch, abrasion, oral ulcer, stomach ulcer, ulcer from cancer therapy, diabetic foot ulcer, venous stasis ulcer, pressure ulcer, and burn.

12. The use as claimed in any one of claims 1 to 11 where the wound results from ophthalmic surgery, cosmetic surgery, general surgery, treatment for keloids and hypertrophic scars (anti-scarring treatments).

13. The use as claimed in any one of claims 1 to 12 where the compound of formula 1 is obtained by chemical synthesis.

14. The use as claimed in any one of claims 1 to 12 where the compound of formula 1 may be obtained from a natural source.

15. The use as claimed in any one of claims 1 to 14 where the compound of formula 1 , or a precursor compound thereof, is administered in the form of a composition containing a pharmaceutically acceptable excipient, adjuvant, carrier, buffer, or stabiliser.

16. The use as claimed in any one of claims 1 to 14 where the compound of formula 1 , or a precursor compound thereof, is administered as an oil or together with an oil.

17. The use of a compound of formula 1 as defined in claim 1 , or a precursor compound thereof, in the manufacture of an agent for promoting the healing of a wound.