AU2016102480A4 - New treatment methods utilising lactose compositions - Google Patents

Abstract

Abstract The present invention relates to the use of topical lactose compositions and methods of using said compositions for treating inflammatory disorders/diseases, pain, and disorders of epithelial tissue (skin and mucosa), including skin wounds, hives and rash.

Description

NEW TREATMENT METHODS UTILISING LACTOSE COMPOSITIONS

Field of the Invention

The present invention relates to methods of treating wounds and other conditions of the skin and mucosa.

Background of the Invention

Wound healing is a complex process involving multiple steps. Conventionally, this is broken down into a series of four phases: coagulation, inflammation, proliferation and maturation.

Coagulation, or blood clotting occurs soon after injury. Damage to the endothelium results in collagen being exposed to platelet cells circulating in the blood. The platelets bind directly to the collagen, thereby localising them at the site of the injury. Binding to the collagen results in activated platelets which release the contents of granulocytes which in turn activate further

platelets, forming a platelet plug (primary hemostasis), as well is inducing a series of cascades which result in the formation of strengthening fibrin strands (secondary hemostasis).

The binding of platelets to collagen, as well as epithelial damage triggers the secretion of inflammatory factors which result in the inflammatory phase. In this phase the damaged or dead cells are removed from the injury site by phagocytosis. Growth factors are released which assist in the proliferative phase.

The proliferative phase comprises processes such as angiogenesis (the generation of new

blood vessels), fibroplasia and granulation tissue formation and epithelialisation. Fibroplasia begins with fibroblasts entering the wound site and proliferating as a result of the growth

factors released during the inflammatory phase. The fibroblasts then proceed to lay down the extracellular matrix (ECM) and collagen (initially type III collagen) in the wound site, which then allows epithelialization.

The maturation phase of wound healing occurs as the initial type III collagen is replaced by type I collagen.

It should be understood that while the four steps above are broadly consecutive in nature, there is considerable overlap between the phases. For instance proliferation begins before inflammation is completed.

Fibroblasts are responsible for the generation of collagens, glycosaminoglycans, reticular and elastic fibers and glycoproteins which make up the ECM. Fibroblasts are one of the major cell types present in skin and play a key role in wound healing. The fibroblasts present in the wound during the proliferative phase are thought to primarily migrate from surrounding tissue.

The wound healing process in adult human tissue is the result of collagen deposition and remodelling and thus results in eventual scar formation. Scar-less wound healing only occurs foetally amongst mammals.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art.

At present, intervention in the process of wound healing focusses on rapid wound closure together with minimal aesthetic scarring. Numerous approaches to wound healing have been tried but there remains a need for alternative strategies which are cheaper, more effective, or at least provide the public with a useful choice.

Summary of the Invention

It has now been found that the topical application of lactose to skin wounds and other disorders of epithelial tissue results in surprising improvements in the wound or disorder.

Accordingly, in one broad aspect the invention relates to a method of treating a disorder of epithelial tissue comprising topically administering a therapeutically effective amount of lactose to a subject suffering from the disorder.

In one embodiment the disorder of the epithelial tissue is a wound, preferably a skin wound. The wound can be infected, or in a subject particularly susceptible to infection. In one embodiment the subject is susceptible to reduced wound healing, such as a haemophiliac or diabetic.

In one embodiment the disorder is a burn or other injury to the flesh or skin.

In another embodiment the disorder of the epithelial tissue is a rash, particularly of the skin, preferably as a result of an allergic reaction or other inflammatory condition.

Another broad aspect of the invention relates to a method of treating pain, comprising topically administering a therapeutically effective amount of lactose to a subject suffering from the disorder. In one embodiment the pain is associated with a wound or other disorder of the epithelial tissue. In another embodiment the pain is not associated with a disorder of the epithelial tissue. In one embodiment the pain is a headache.

In a third broad aspect of the invention, there is provided a method of treating an inflammatory disease or condition comprising topically administering a therapeutically effect amount of lactose to a subject suffering from the disease or condition.

The lactose can be applied directly as powder, or alternatively can be applied as a solution. Where the lactose is applied as a solution, it is preferably applied as solution of Img/mL or greater. The lactose may also be applied as a spray, gel, cream, ointment or poultice. In one embodiment the lactose can be incorporated in a device such as a bandage which is for topical application.

The lactose can be applied alone, or in combination with another active ingredient. In certain embodiments the treatment consists of, or consists essentially of topically administering a therapeutically effective amount of lactose.

In another broad aspect the present invention provides for a composition comprising lactose, for topical application in the treatment of a disorder as described herein.

In a still further broad aspect the present invention provides for the use of lactose in the manufacture of a medicament for the treatment of a disorder as described herein.

Brief Description of the Figures

Figure 1 Shows the effect of test sample #1 Lactose A (Smith) at1.0mg/ml and test sample #2 Lactose A (Smith) at 0.25mg/ml) on wound healing in the control culture and the cultures containing the test sample preparation. Each value is the mean ±SEM of triplicate values.

Detailed Description of the Invention Definitions Except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated step or feature but not to preclude the presence or addition of further features in various embodiments of the invention.

The phrase "consists essentially of' and variations thereof, except where context requires otherwise, is used to specify the presence of the stated feature or step and to include other steps which do not materially affect the basic characteristic of the invention. For example, a method which consists essentially of administering a therapeutically effective amount of lactose, may constitute administration with other carriers or aerosols, or even inclusion of lactose in a mechanical aid to wound healing such as a bandage. However, it would not encompass the additional application of another active ingredient.

The phrases "consists of', "consisting of' or other variations are used in an exclusive sense, i.e. to specify the presence of the stated step or feature and no other step, except to the extent that the step or feature would ordinarily be considered to contain variation. For example, a method consisting of administering a therapeutically effective amount of lactose is to be understood as also encompassing lactose in any level of purity (see the definition of lactose below) and thus encompasses the administration of the impurities as well as the lactose.

As used herein the phrase "disorder of epithelial tissue" refers to any condition suffered by epithelial tissue, whether as a result of injury, disease or otherwise. It is intended to include within its scope disorders which are primarily of the epithelial tissue but extend to other tissues. Included within this term are wounds and bums of the skin, or other mucosa such as the eyes, mouth or nose, as well as other diseases whether as a result of external factors such as infection, or as a result of autoimmune disorders. As used herein the term "wound" refers to both open wounds and closed injuries.

The phrase "therapeutically effective amount" refers to an amount of the ingredient sufficient to achieve a therapeutic effect. In the case of wound healing this is, for example, an amount sufficient to result in increased healing, or in better healing, for example reduced scar formation. A therapeutically effective amount may differ depending on how the ingredient is applied, for instance by way of direct application of the powder, or as a solution or poultice.

As used herein the term "topical administration", "administered topically" or other variations thereof should be understood to mean that the compound or composition applied to body surfaces. Included in this term is administration to the skin and administration to other surfaces. The term is not intended to comment on the pharmacodynamic effect of the compound or composition and so is not intended to be limited to situations where the compound or composition has a topical effect.

The term "lactose" is intended to refer to the sugar p-D-galactopyranosyl-(1-4)-D-glucose, however it is obtained and in any purity. Thus the description of applying lactose shall be understood to include the application of a composition or product comprising lactose and other impurities as well as pure lactose. In certain embodiments the lactose is food grade lactose. In other embodiments the lactose is pharmaceutical grade. In particular embodiments the lactose is unmodified by the addition of other side chains.

Lactose, is a disaccharide sugar common in milk. It is one of the major by-products of dairy processing where it can be precipitated from whey using ethanol at relatively high yields. Food or pharmaceutical grade lactose is generally available as a white powder.

Lactose is a common additive to foods such as infant formula, where it is broken down into its constituent monosaccharides, glucose and galactose by the enzyme p-D-galactosidase in

order to produce energy. It is also commonly used as a filler in the pharmaceutical industry, for example in the making of tablets, due to its compressibility and low price.

Topical compositions comprising lactose are also known. In EP 0498532, lactose is used in a composition for debriding wounds. The composition comprises a wound debriding enzyme such as subtilisin A and lactose as excipient and is applied directly to wound site. US 5422103 describes compositions comprising lactose and a collagenase powder again for topical application as a wound debriding composition. Again lactose is used as an excipient.

It has not been previously recognised that lactose itself has therapeutic properties when applied topically to disorders and conditions of the epithelium.

EP1032364 discloses the use of sialyl lactose in regulating inflammatory responses, reducing skin irritation and treating skin reddening. Sialyl lactose is a derivative of lactose whereby a lactose molecule is modified by the addition of a sialyl group. Sialyl lactose does not fall within the scope of the present invention.

Disorders and Conditions Thus it has now surprisingly been found that lactose has a therapeutic effect when applied topically to a wound or other disorder of the epithelial tissue.

One effect of the application of lactose to a wound is a surprisingly rapid healing when compared to natural healing. This has been established across a range of different skin injuries. Moreover it has also been shown that the lactose aids in the healing of injuries to connective tissue such as ligaments.

The invention may be particularly of use in individuals who exhibit slow wound healing, such as haemophiliacs and diabetics, but is also of general use for both human and non human animals.

Without wishing to be bound by any particular theory, it is believed that the lactose has beneficial effects at different stages of the wound healing process. It is thought that in the coagulation stage the lactose aids in the formation of the platelet plug. In addition it appears as though lactose also has an effect on fibroblast proliferation and migration, which are key components of the proliferation stage of wound healing.

Surprisingly the application of lactose also shows a reduction of pain, not only in skin wounds, but more generally, for instance in headaches. This effect could indicate that lactose is also able to reduce inflammation. In fact, lactose has also been shown to have an effect on inflammatory skin conditions such as hives when applied to the affected area.

Thus the present invention also relates to the use of lactose for the treatment of pain and inflammatory conditions by topical administration.

Modes of administration The present invention has shown that lactose is effective when administered topically. Typically where there is an open wound the lactose can be applied in powder form. However it can also be administered as a poultice, for example in combination with water, or be included in a gel, cream, ointment or spray. Where there is no open wound, an application according to one of these means assists in the localisation of the lactose.

Inclusion in a gel or spray will also assist where the site of the injury is not on the skin, for instance in the mouth or nose.

Alternatively the lactose can be incorporated into a product such as a dressing, bandage or patch for application to a particular site on the skin. Inclusion in such a product, especially a dressing has the added benefit of single application of both lactose and the dressing.

Where the lactose is not applied in powder form, a dosage of greater than 0.25mg/mL, more preferably Img/mL is desirable.

It is preferred that the lactose is applied directly to the site of injury. However, the lactose has some intradermal effect, thus it is possible to apply the lactose in a location remote from the injury. In certain cases lactose may also have a systemic effect.

Combination treatments Lactose may be combined with other active ingredients to further aid in the wound healing process. Suitable compounds are those known in the art to have wound healing properties.

The invention will be described further by way of examples below:

Examples In examples 1 to 9, food grade lactose was obtained from Sapa to Cheese USA Inc, sourced from the Miraka Dairy Factory in Taupo New Zealand.

Example ] A subject having diabetes which results in slower than usual healing badly cut his finger on an auger. Application of the lactose powder to the finger resulted in the bleeding stopping instantly. The powder was left on the finger and the finger bandaged. After two days the bandage was removed and the cut had healed.

Example 2 A subject obtained four scratches about 10 inches long and very deep on the arm and hand from a possum. Lactose powder was applied to scratches on the arm but not to two puncture wounds on the hand. After several days the wounds to which the lactose were applied had healed. In contrast the puncture wounds on the hand had become infected. Later application of the lactose to the puncture wounds of the hand treated the infection and resulted in rapid healing.

Example 3 A subject smashed a 10 gauge metal wire post into his left thumb. Application of lactose to the injury resulted in an instant reduction in pain. The injury had almost completely healed within a few days.

The wound was then accidentally re-opened with consequent pain. Licking the thumb and reapplying lactose powder resulted in cessation of pain within 10 seconds.

Example 4 A cut to a dog's right paw resulted in substantial bleeding. Lactose powder was applied to the wound and it was wrapped in a bandage. Within 24 hours the dog was chewing off the bandage and running around without a limp or sign of an injury.

Example 5 Application of powdered lactose to a cut knuckle and tendon resulted in healing within a week. The wound had to be reopened in order to set the tendon in the right position with a physician amazed at the initial healing rate.

Example 6 During skinning of a deer, a 4 inch wide knife slipped and made a very deep cut to the forearm of a subject. The wound initially did not heal properly. Subsequent application of lactose powder resulted in quick healing within a few days.

Example 7 Powdered lactose was mixed into a poultice and applied to an ingrown toe-nail. The ingrown toe-nail healed within approximately a week. This compared to regular medical intervention on another toe nail which took over three months to heal.

Example 8 Contact with horses resulted in the subject presenting with hives over a large portion of her body. Application of lactose powder to the affected area resulted in the welts clearing up quickly.

Example 9 A subject suffering from a headache damped down the region of the head proximal to the pain with water and subsequently applied lactose powder. The headache diminished in seconds.

Example 10 The face and hands of a subject were treated for skin cancers, causing considerable pain and leaving the hands with large lesions akin to third degree burns. Lactose powder was applied the second evening after treatment, resulting in immediate cessation of pain. The next morning the lesions had stopped weeping and that afternoon the wounds had started to dry and harden around the edges. By the evening following the application of the lactose powder, the wounds had started to scab over entirely. By the following morning all treatment areas had completely scabbed and dried.

Example 11 A study was conducted to investigate and compare the effects of lactose on the healing of human skin fibroblasts that have been 'wounded'. The aim of this study was to determine the effects of the 12 test samples at one concentration on wound repair, and in particular the effect on fibroblast mediated steps of wound repair.

Test samples 1 and 2 were lactose obtained from Sapa to Cheese USA Inc, sourced from the Miraka Dairy Factory in Taupo New Zealand. Test sample 1 was diluted to a concentration of lmg/mL in HBSS (Hanks Balanced Salt Solution), while Test sample 2 was diluted to a concentration of 0.25mg/mL.

Description of Test Materials and Test Methods

The methodology used in this study for investigating cell proliferation was a modification of the TBL cell proliferative assay (SOP: TBL-XCP 7.0). Human skin fibroblasts (Detroit 551) were retrieved from cryostorage and cultured to confluence in 24 well plates. Once confluence had been attained, a circular wound was created (6mm in diameter) and the cells removed from within the ring. The cells were then cultured in the presence of the test samples at one concentration, in triplicate with mean values and standard errors expressed graphically. The 'wounds' were photographed at TO, T24, T48 and T72hrs. The wound areas measured digitally and graphed. A bFGF (FGF-2) control and a blank were also run in triplicate.

Characterisation of the Test System 1. Human skin fibroblasts (Detroit 551 ATCC No: CCL110). Stored in liquid N2. 2. Dulbecco's Modified Eagle's Medium (DMEM): GIBCO 12100-038. 10% Foetal Bovine Serum (FBS), 100U/ml Penicillin and 100ig/ml Streptomycin (both are x100 in stock) were added. NOTE: For the assay culturing in the multiwell plates only 2% FBS was used 3. Trypsin-EDTA solution: 0.05% Trypsin/EDTA. SIGMA Cat# 15400054 (xlO in stock). Diluted with PBS. 4. Hanks Balanced Salt Solution (HBBS).

5. Fibroblast Growth Factor (bFGF) (Human): Sigma Cat# F0291. Stored at -80C. Supplied as 25tg lyophilised powder. A stock solution at 10pg/ml in sterile PBS was prepared.

5 Cell Preparation: 1. The frozen stock cell line (D-551 fibroblasts) was removed from liquid nitrogen and immediately thawed in a 37°C water bath. The contents were then transferred to a 250ml (75cm2 ) culture flask containing 20ml DMEM plus 10%FBS (pre-warmed to 37C). 2. The cells were cultured at 37°C in 95% air/5% CO 2 for 24hr. The medium in the flask was then removed and fresh medium added. The cells were then cultured until the cells had reached approximately 80% density/confluence. Cells were examined daily for confluence under the microscope. Once 80% confluence was reached the cell culture was then split according to ATCC guidelines and fresh media added. The new flasks were grown at 37°C in 95% air/5% CO2 until the cells had reached approximately 80% density/confluence. Medium was changed every 3-4 days.

Harvesting the Cells: 1. To detach the adherent cells, the medium was removed from the culture flasks and the adherent cells washed with PBS to remove any traces of FBS. Then 5ml of 0.25% Trypsin/EDTA solution was added and incubated at 37°C for 5 min until all the cells had detached. The trypsin was then neutralised by the addition of an equal volume of pre warmed DMEM and centrifuged at 125g (500rpm) for 7 min at 4°C. 2. The supernatant from each flask was discarded and the cell pellets re-suspended with 10ml of pre-warmed DMEM containing 10% FBS. The cell number was counted and adjusted to 2x10 5/ml.

Cell Culturing: 1. Flat-bottomed well plates for the experiments were used. Three wells were prepared for each sample or control. 2. Prior to adding the cells or medium to the wells, a 6mm diameter circle was drawn on the bottom of each well in the centre of the base of each well using a marker pen. 3. 2.Oml of the cell suspension (2x105cells/ml) was plated into each well except for Control Blanks. 2.Oml of medium was added to each of the Control Blanks.

4. All plates were placed in the incubator to allow the cells to adhere and to reach confluence. 5. When the cells had reached confluence, the media was removed from each well. 6. The cells occupying the 6mm diameter central ring of each well were removed using a pipette. 0 7. Then 1.8ml of culture media was added to each well. A volume of 2 0 pl of the sample and/or controls was added to the appropriate wells as indicated in the diagram below. 8. A photograph of each well was taken immediately to serve as the zero time measurement. 9. The plates were incubated at 37°C in an atmosphere of 95% air/5% CO 2

. 10. At the following timepoints each well was photographed: T24hrs, T48hrs and T72hrs. 11. At each timepoint (T24hrs, T48hrs and T72hrs) 200pl of culture medium was removed from each well and transferred to the wells of a 96 well culture plate and stored at 20'C. 12. The plates were returned to the incubator.

Wound Measurement 1. At each timepoint, each well was photographed and the areas measured in pixels2

, using the Image J 1.48m software. 2. Each area measurement was expressed as a percentage of its original at Time Zero and referenced against the Cells Only Control.

Results and Discussion Presentation of Results and Determinations of Statistical Significance: Wound Healing A summary of the effects of the test samples on the wound healing after 24, 48 and 72 hrs as a mean percentage of the original wound area is presented in the Table 1 below. The measurements for the Cell Control wound areas on all cultures plates have been averaged to obtain a normalised Cell Control so that individual test samples can be compared.

Table 1: Comparison of Wounds: Mean % of Original Wound Area and Standard Error of Mean (SEM)

Sample TO SEM T24 SEM T48 SEM T72 SEM Hour Hours Hours Hours Cells only 100 0 28.13 1.49 11.35 1.88 1.19 0.61 control +HBSS FGF-2 100 0 31.53 2.45 4.15 0.55 0.00 0.00 (250ng/mL) FGF-2 100 0 23.06 3.54 1.22 1.22 0.00 0.00 (50ng/mL) FGF-2 100 0 27.41 0.53 0.00 0.00 0.00 0.00 (1Ong/mL) Lactose 100 0 81.68 5.50 10.57 5.50 0.00 0.00 (1.00mg/mL) Lactose 100 0 94.77 2.00 78.26 10.01 64.48 6.72 (0.25mg/mL)

Figure 1 presents the data in Table 1 in graphical form.

At 1 mg/ml, after 24 hours, the lactose preparation had only reduced the wound size by 18.3%. This was somewhat less than expected. In comparison the untreated wounds were 71.8% closed at this time. The level of closure of this reference culture was greater than for any of the test samples and is unexpectedly high. It is certainly higher than observed in previous studies.

After 48 hours, the wounds treated with this lactose had closed significantly. It was close to being completely closed (it was 89.4% closed). Thus there is a very strong stimulatory effect during this second period.

After a further 24 hours, the wounds treated with this lactose had completely closed. This contrasts with the negative control the wounds had not completely healed at 72 hours.

At the lower concentration of 0.25 mg/ml, this lactose had a slower effect as expected. After 24 hours there was negligible effect. The wound was only 5.2% smaller which is less than the

18.3% achieved with the four times higher concentration. After 48 hours, this lower concentration was still only moderately effective. The wound was 21.7% smaller than the original. This contrasts strongly with the significant effect of the 1 mg/ml concentration. After 3 days, there was only a relatively minor further improvement. The wound was only 35.5% smaller whereas a 1 mg/ml concentration had effected complete closure at this time.

In this study it is obvious that the lactose is effective at promoting wound closure and in particular fibroblast mediated wound closure. The slower initial effect in this model may be advantageous. The cell proliferation and migration is not an early stage of the overall wound healing process. It follows the earlier phases of coagulation and inflammation.

Example 12 (ComparativeExamples)

By way of comparison the above tests were performed for a range of other natural components and other sugars. Samples were prepared in the same manner as above with the exception that the Kawakawa oil was weighed and the concentration adjusted to 1Omg/ml with 15% ethanol in water. To make the 10mg/ml stock solution a volume of110pl was removed and made up to 1ml with the addition of 890tl of 15% ethanol (EtOH). The green tea sample was prepared by suspending 1 gram of dried green tea in 25 ml of distilled water at ambient temperature. This suspension was stirred for 15 min and then centrifuged at 300rpm for 10 min. The supernatant was aspirated and stored frozen at -20°C until required. The precipitate was discarded.

The results obtained are set out in Table 2:

Table 2: Sample TO SEM T24 SEM T48 SEM T72 SEM Hour Hours Hours Hours

Creamed Clover 100 0.00 100.44 7.21 86.42 6.76 76.82 2.51

Honey 0.20mg/mL Rata Honey 100 0.00 90.54 6.08 50.60 3.40 15.22 2.14

0.20mg/ML

Active Manuka 100 0.00 50.84 5.68 21.6. 3.77 2.61 2.61 Honey 2.0 mg/mL Manuka Honey 100 0.00 42.90 1.11 17.64 0.79 4.90 0.71 0.20 mg/mL D-(+)- 100 0.00 39.34 4.17 16.57 0.57 0.94 0.10 Glucose1mg/mL Sucrose 100 0.00 46.18 4.17 17.70 3.56 3.16 0.65 lmg/mL Kawakawa Oil 100 0.00 44.47 3.25 17.23 0.26 2.4 0.20 1mg/mL Green Tea (10% 100 0.00 92.48 2.53 84.22 5.85 80.24 5.61 v/v)

CREAMED CLOVER HONEY

The creamed clover honey sample was tested at a concentration of 0.20 mg/ml. After 24 hours, there was no change to the size of the wounds. After 48 hours, the wound sizes had decreased by only 13.6% and there was a further small decrease of 9.6% after 72 hours. This means that at the end of the 72 hours, the wounds were 76.8% of their original size. So the wound closure effect of this honey is minimal.

As a result, the creamed clover honey was one of the least effective samples in this study at promoting wound healing.

RATA HONEY A sample of Rata Honey was also tested at a concentration of 0.20 mg/ml and was found to be moderately effective at promoting wound healing. After the first 24 hours, the wound sizes had only decreased to 90.5% of their original size (a 9.5% reduction in size). However, after a further 24 hours the wound sizes had decreased to 50.6% of their original size. That is, they were almost half the size of what they were at the start of the study. After 72 hours, there had been an 84.8% reduction in wound size - they were 15.2% of their original size.

So although better than clover honey, rata honey was significantly less effective than lactose.

ACTIVE MANUKA HONEY A The Active Manuka Honey sample was the most effective honey sample in this study. At a concentration of 0.20 mg/ml, the wounds had almost halved in size after 24 hours (49.2% size reduction). After another 24 hours of incubation the wound had almost halved again. That is, the wound areas were only 21.6% of their original size. By 72 hours, the wounds were almost fully closed; they were only 2.6% of the original size.

Thus manuka honey, is initially more effective than lactose at promoting wound closure. With this concentration of honey, the closure after 24 hours was about 2.7 times greater but at the two later times, the rate of closure was similar for both samples.

MANUKA HONEY B Manuka Honey B (supplied by TBL) was the second manuka honey sample tested in this study. It was Airborne Manuka Honey purchased from a retail outlet. Like the Active Manuka Honey A sample, at a concentration of 0.20 mg/ml Manuka Honey B was effective at promoting wound healing however not to the same extent. At both 24 and 48 hours the wound sizes were slightly smaller than those seen for Active Manuka Honey A (42.9% (24 hours) and 17.6% (48 hours) respectively of the original size compared to 50.8% and 21.6% for the former). However, after 72 hours the wound areas for Manuka Honey B were 4.9% of their original size which was only slightly bigger than those seen for Active Manuka Honey A.

As lactose was a common disaccharide, a comparison of the effect of other common low molecular weight sugars was undertaken.

D-(+)-GLUCOSE (Sigma)

D-(+)-glucose was tested at a concentration of 1.00 mg/ml and almost completely closed the wounds after 72 hours. It was the only sample other than lactose to do this.

After 24 hours there was a 60.7% reduction in the size of the wounds meaning that they were 39.3% of the size they were at the start of the experiment. This is a faster initial closure rate than achieved with the lactose. After 48 hours this had decreased to 16.6% of their original size. This is now comparable to the effect of lactose. This is because the latter is really a strong stimulator over this second 24 hour period. After a further 24 hours (72 hours total) the wounds were only 0.9% of their original size. That is, they had almost completely healed. 5 Thus compared with the lactose this sugar was a stronger initial stimulant at the same concentration. However, by 48 hours the effect of both sugars on wound closure was similar and this continued through to 72 hours. At the 72 hour stage it was notable that lactose has caused complete wound closure, while the glucose had not, suggesting the lactose was more effective than the glucose.

SUCROSE (J. T. Baker) Sucrose, at a concentration of 1.00 mg/ml was effective at promoting the healing of "wounds". After 24 hours, the wound sizes had decreased by more than 50% (53.8% size reduction) which was slightly less than that achieved with the same concentration of glucose but certainly better than lactose at the same time. It further decreased in size at both 48 and 72 hours. By 72 hours the wounds were only 3.2% of their original size meaning there had been a 96.8% reduction. Therefore, overall, sucrose was slightly less effective as D-(+) glucose or Lactose.

KAWAKAWA OIL Kawakawa has been traditionally used as a natural treatment of wounds and so was included in this study in the presence of the oil at1 mg/ml. After 24 hours, the wound area had decreased to less than 50% of its original size (55.5%). This indicates that the initial rate of closure is fairly similar to a number of other test samples. The wound was only 17.2% of its original size at 48 hours. By 72 hours, the wound area had decreased to 2.4% of what it was at the start of the experiment. This shows that Kawakawa Oil is effective in the promotion of wound healing. However Lactose at the same concentration was slightly more effective as it closed the wound completely and even after 48 hours it reduced the wound size more than the kawakawa oil.

GREEN TEA The green tea solution (10% v/v) was prepared and tested for its wound healing properties. It was the least effective of all the samples at decreasing the size of the wounds. After 24 hours, the wound areas had only decreased by 7.5% and 15.8% after 48 hours. At the last time point (72 hours) the wounds had only decreased by 19.8%. That is, they were still 80.2% of their original size. Thus it was rather ineffective as an agent for promoting the cell migration and proliferation associated with wound healing.

Summary Over a 72 hour period, lactose compared favourably to all of the samples tested. In fact only the positive control, FGF-2 showed similar properties. Thus the test establishes that lactose has a positive effect on fibroblast growth and proliferation, and consequently wound healing. This effect is particularly noticeable between the 24-48 hour time period, where lactose out performed all of the other samples.

It is to be understood that the scope of the invention is not limited to the described embodiments and therefore that numerous variations and modifications may be made to these embodiments without departing from the scope of the invention as set out in the specification.

Claims (35)

1. What we claim is: 1. A method of treating a disorder of epithelial tissue comprising topically administering a therapeutically effective amount of lactose to a subject suffering from the disorder.
2. 2. A method according to claim 1, wherein the disorder of the epithelial tissue is a wound.
3. 3. A method according to claim 2, wherein the wound is a skin wound.
4. 4. A method according to claim 2 or 3, wherein the wound is infected, or is in a subject particularly susceptible to infection.
5. 5. A method according to any one of claims 2-4, wherein the subject is susceptible to reduced wound healing.
6. 6. A method according to any one of claims 1-5, wherein the subject is a haemophiliac or diabetic.
7. 7. A method according to claim 1, wherein the disorder of the epithelial tissue is a rash.
8. 8. A method according to claim 7, wherein the rash is a rash of the skin.
9. 9. A method according to claim 7 or 8, wherein the rash is a result of an allergic reaction or other inflammatory condition.
10. 10. A method according to any one of claims 7-9, wherein the rash is hives.
11. 11. A method of treating pain comprising topically administering a therapeutically effective amount of lactose to a subject.
12. 12. A method according to claim 11, wherein the pain is associated with a wound or other disorder of the epithelial tissue.
13. 13. A method according to claim 11, wherein the pain is not associated with a disorder of the epithelial tissue.
14. 14. A method according to any one of claims 11 to 13, wherein the pain is a headache.
15. 15. A method of treating an inflammatory disease or condition comprising topically administering a therapeutically effect amount of lactose to a subject suffering from the disease or disorder.
16. 16. A method according to claim 15, wherein the disease or condition is a rash.
17. 17. A method according to claim 16, wherein the rash is hives.
18. 18. A method according to any one of claims I to 17, wherein the lactose is applied directly as powder.
19. 19. A method according to any one of claims 1 to 17, wherein the lactose is applied as a solution.
20. 20. A method according to claim 19, wherein the solution has a concentration oflmg/mL or greater.
21. 21. A method according to any one of claims 1-17, wherein the lactose is applied as a spray, gel, cream, ointment or poultice.
22. 22. A method according to any one of claims 1-17, wherein the lactose can be incorporated in a device for topical application.
23. 23. A method according to claim 22, wherein the device is a bandage or dressing.
24. 24. A method according to any one of claims 1-23, which consists essentially of administering a therapeutically effective amount of lactose.
25. 25. A method according to any one of claims 1-23 which consists of administering a therapeutically effective amount of lactose.
26. 26. A method according to any one of claims 1-23, wherein the lactose is applied alone, or in combination with another active ingredient.
27. 27. A topical composition comprising lactose, for use in the treatment of a disorder of epithelial tissue.
28. 28. A topical composition comprising lactose, for use in the treatment of pain.
29. 29. A topical composition comprising lactose, for use in the treatment of an inflammatory disease or condition.
30. 30. The use of lactose in the manufacture of a medicament for the treatment of a disorder of epithelial tissue.
31. 31. The use of lactose in the manufacture of a medicament for the treatment of pain.
32. 32. The use of lactose in the manufacture of a medicament for the treatment of an inflammatory disease or condition.
33. 33. A method according to any one of claims I to 17, substantially as herein described or exemplified.
34. 34. A composition according to any one of claims 24 to 26, substantially as herein described or exemplified.
35. 35. A use according to any one of claims 28-30, substantially as herein described or exemplified.