AU2017380471B2

AU2017380471B2 - Topical formulation comprising green lipped mussel and honey

Info

Publication number: AU2017380471B2
Application number: AU2017380471A
Authority: AU
Inventors: Keith Nicholas Bowhill
Original assignee: Mm Health Ltd
Current assignee: Mm Health Ltd
Priority date: 2016-12-23
Filing date: 2017-12-20
Publication date: 2023-12-21
Anticipated expiration: 2037-12-20
Also published as: AU2017380471A1; WO2018117869A1

Abstract

The invention relates to a combination which is typically a composition such as a medicament and which is particularly suited for topical administration. The combination is useful in the prophylaxis and/or treatment of a range of diseases, including skin diseases such as psoriasis, eczema, dermatitis, as well as other diseases including gum diseases, shingles, joint pain. In particular the combination of the invention includes an extract of green lipped mussel and honey.

Description

Topical Formulation Comprising Green Lipped Mussel and Honey

RELATED APPLICATION

This application claims priority to NZ727858 the contents of which are incorporated herein by reference.

TECHNICAL FIELD

SUMMARY OF THE INVENTION

In one aspect the invention provides a pharmaceutical composition including:

a) an extract of green lipped mussel; and

b) honey.

In another aspect the invention provides a method of preventing or treating a disease, the method including the step of administering a therapeutically effective amount of a pharmaceutical composition including an extract of green lipped mussel and honey to a subject in need thereof.

In a further aspect the invention provides the use of an extract of green lipped mussel and honey in the preparation of a medicament for the prevention or treatment of a disease.

In a still further aspect the invention provides a pharmaceutical combination including:

a) an extract of green lipped mussel; and

b) honey;

for separate, sequential or simultaneous administration.

The combination may further include instructions for use in the prevention or treatment of a disease. Without wishing to be bound by theory, it is believed that the extract of green lipped mussel and the honey may act synergistically to provide a therapeutic and/or prophylactic effect. Evidence is provided that the action may be selective against psoriatic keratinocytes over non-psoriatic keratinocytes.

In a further aspect the invention provides a method of stimulating non-psoriatic keratinocytes including the step of administering to the non-psoriatic keratinocytes: a) an extract of green lipped mussel; and

b) honey.

In a further aspect the invention provides a use of:

a) an extract of green lipped mussel; and

b) honey

to stimulate non-psoriatic keratinocytes.

In a further aspect the invention provides a method of inhibiting psoriatic keratinocytes including the step of administering to the psoriatic keratinocytes:

a) an extract of green lipped mussel; and

b) honey.

In a further aspect the invention provides a use of:

a) an extract of green lipped mussel; and

b) honey

to inhibit psoriatic keratinocytes.

In a further aspect the invention provides a method of selectively inhibiting psoriatic keratinocytes over non-psoriatic keratinocytes including the step of administering to the psoriatic keratinocytes and non- psoriatic keratinocytes:

a) an extract of green lipped mussel; and

b) honey.

In a further aspect the invention provides a use of:

a) an extract of green lipped mussel; and

b) honey

to selectively inhibit psoriatic keratinocytes over non-psoriatic keratinocytes.

In a further aspect the invention provides a method of inhibiting the formation of Elafin/SKALP in psoriatic keratinocytes including the step of administering to the psoriatic keratinocytes:

a) an extract of green lipped mussel; and

b) honey.

In a further aspect the invention provides a use of: a) an extract of green lipped mussel; and

b) honey

to inhibit the formation of Elafin/SKALP in psoriatic keratinocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a comparison of skin discolouration on the left wrist and left hand of Subject S in

example 4 before and 7 days after treatment with the inventive composition;

Figures 2A-2C show a time course assay of skin discolouration (example 4) with right hand as untreated control, the inventive composition being applied only to the left hand which contains a scab of dried skin which had not healed in over two years. The final photo from day 23 was taken after 5 days of no application from day 10 to 14 and shows a rose bush scratch on day 21;

Figures 3A-3C show a time course assay of skin discolouration (example 4) of the right side of face treated with the inventive composition;

Figures 4A-4C show a time course assay of skin discolouration (example 4) of the left side of face

treated with the inventive composition;

Figures 5A-C show a time course assay of skin discolouration (example 4) of the front of face treated with the inventive composition;

Figures 6A-6E show a before/after treatment assay of psoriasis on Subject T;

Figure 7 shows a time course treatment assay of psoriasis on Subject U;

Figure 8 shows a time course treatment assay of eczema on Subject S;

Figure 9 shows a before/after treatment assay of eczema on Subject V;

Figure 10 shows a graph of proliferation of undifferentiated human keratinocytes in the control cultures and the cultures containing the test sample preparations. The proliferation is expressed as the absorbance at 570 nm following reaction with MTT. Each value is the mean ± SEM of triplicate values; Figure 11 shows the effect of the test sample preparations on the proliferation of undifferentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values;

Figure 12 shows the effect of the test sample preparations on the proliferation of undifferentiated human keratinocytes, expressed as a percentage inhibition of the control culture. Each value is the mean of triplicate values; Figure 13 shows proliferation of differentiated human keratinocytes in the control cultures and the cultures containing the test sample preparations. The proliferation is expressed as the absorbance at 570 nm following reaction with MTT. Each value is the mean ± SEM of triplicate values;

Figure 14 shows the effect of the test sample preparations on the proliferation of differentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values;

Figure 15 shows the effect of the test sample preparations on the proliferation of differentiated human keratinocytes, expressed as a percentage inhibition of the control culture. Each value is the mean of triplicate values; Figure 16 shows the concentration of Elafin/SKALP in cultures of undifferentiated keratinocytes in the presence of the test samples. The values are the mean ± SEM (n=3);

Figure 17 shows the effect of the test sample preparations on the production of elafin/SKALP by the undifferentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values; Figure 18 shows the effect of the test sample preparations on the production of elafin/SKALP by the undifferentiated human keratinocytes, expressed as a percentage inhibition of the control culture. Each value is the mean of triplicate values;

Figure 19 shows the concentration of Elafin/SKALP in cultures of differentiated keratinocytes in the presence of the test samples. The values are the mean ± SEM (n=3); Figure 20 shows the effect of the test sample preparations on the production of elafin/SKALP by the differentiated human keratinocytes, expressed as a percentage inhibition of the control culture. Each value is the mean of triplicate values;

Figure 21 shows the concentration of cytokeratin 10 in cultures of undifferentiated keratinocytes in the presence of the test samples. The values are the mean ± SEM (n=3); Figure 22 shows the effect of the test sample preparations on the production of cytokeratin 10 by the undifferentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values;

Figure 23 shows the effect of the test sample preparations on the production of cytokeratin 10 by the undifferentiated human keratinocytes, expressed as a percentage inhibition of the control culture. Each value is the mean of triplicate values; Figure 24 shows the concentration of cytokeratin 10 in cultures of differentiated keratinocytes in the presence of the test samples. The values are the mean ± SEM (n=3);

Figure 25 shows the effect of the test sample preparations on the production of cytokeratin 10 by the differentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values;

Figure 26 shows the concentration of PGE₂ in cultures of undifferentiated keratinocytes in the presence of the test samples. The values are the mean ± SEM (n=3);

Figure 27 shows the effect of the test sample preparations on the production of PGE₂ by the undifferentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values;

Figure 28 shows the effect of the test sample preparations on the production of PGE₂ by the undifferentiated human keratinocytes, expressed as a percentage inhibition of the control culture. Each value is the mean of triplicate values;

Figure 29 shows the concentration of PGE₂ in cultures of differentiated keratinocytes in the presence of the test samples. The values are the mean ± SEM (n=3);

Figure 30 shows the effect of the test sample preparations on the production of PGE₂ by the differentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values;

Figure 31 shows the concentration of LTB4 in cultures of undifferentiated keratinocytes in the presence of the test samples. The values are the mean + SEM (n=3);

Figure 32 shows the effect of the test sample preparations on the production of LTB₄ by the undifferentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values;

Figure 33 shows the effect of the test sample preparations on the production of LTB₄ by the undifferentiated human keratinocytes, expressed as a percentage inhibition of the control culture. Each value is the mean of triplicate values;

Figure 34 shows the concentration of LTB₄ in cultures of differentiated keratinocytes in the presence of the test samples. The values are the mean ± SEM (n=3);

Figure 35 shows the effect of the test sample preparations on the production of LTB₄ by the differentiated human keratinocytes, expressed as a percentage stimulation of the control culture. Each value is the mean of triplicate values; and

Figure 36 shows the effect of the test sample preparations on the production of LTB₄ by the differentiated human keratinocytes, expressed as a percentage inhibition of the control culture. Each value is the mean of triplicate values. DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention provides a pharmaceutical composition including:

a) an extract of green lipped mussel; and

b) honey.

a) an extract of green lipped mussel; and

b) honey;

for separate, sequential or simultaneous administration.

Perna viridis is a species of "green mussel" which, despite being widely eaten throughout the Indo- Pacific region, is noted for containing saxitoxin which is produced by dinoflagellates that it feeds upon, as well as a high heavy metal content. Perna canaliculus, on the other hand, is known as "green lipped mussel" and is endemic to New Zealand and is a prized food source.

As referred to herein, "an extract of green lipped mussel" refers to an extract of Perna canaliculus. In its broadest form, the "extract" refers to less than the whole green lipped mussel, and includes the whole (such as fresh) meat product of the green lipped mussel and also the dried meat product of the green lipped mussel.

As used herein the expression "extract of green lipped mussel" refers to any extract of green lipped mussel, and includes those materials referred to generally in the industry as "green lipped mussel extract" (GLME).

There are a number of further extraction processes that may be performed on the whole and/or dried meat products of the green lipped mussel, including GLME, which result in the fractionation of the chemical components of the mussel. These further extraction processes typically fractionate the whole and/or dried meat products of the green lipped mussel on the basis of the polarity of the chemical components. Green lipped mussel typically contains a range of inorganic compounds, proteins, carbohydrates, phospholipids, fatty acid esters (including triglycerides and sterol esters) and free fatty acids. One method of fractionation involves the use of one or more solvents to separate soluble components from those components that are not soluble (or only sparingly soluble) in the solvent. The composition of the extract so fractionated will depend on the nature of the solvent(s) used, however each of these fractionated extracts are typically referred to in the industry as "green lipped mussel oil" (GLMO). For example, the use of a non-polar solvent (such as supercritical carbon dioxide - sC0₂) will typically dissolve non-polar organic components from the whole and/or dried meat products, leaving the polar components as residues. Typically this extraction process will leave the phospholipid component as residue.

By way of further example, the use of a polar solvent such as an alcohol (such as ethanol) as the extraction solvent will typically dissolve polar organic components from the whole and/or dried meat products. Typically this extraction process will solubilise the phospholipid component.

By way of further example, further single solvent systems or also combinations of solvents may also be used including acetone, tert-butyl methyl ether (TBME), hexane, ethanol and/or isopropyl alcohol which may allow for the extraction of a mixture of non-polar and polar organic components. Preferably this extraction process will solubilise the phospholipid component. Prior disclosures of the use of this dual solvent system for extracting lipids using hexane/isopropyl alcohol is found in Anal. Biochem. 1978, 90, 420 and Mar Drugs. 2015 Oct; 13(10): 6453-6471. Hexane/isopropyl alcohol is a preferred solvent extraction system.

By way of further example, WO2005/073354 (the whole contents of which is incorporated by reference) discloses the use of acetone, hexane or ethyl acetate to dissolve at least part of the organic fraction of green lipped mussel, before subjecting that solution to nanofiltration and further rotary evaporation, so as to yield a particular PUFA oil which is suitable for use as "an extract of green lipped mussel" referred to herein. As referred to in WO2005/073354, the nanofiltration technique can be used to partition organic soluble components according to size based on the pore size of the membrane used.

In preferred embodiments, in the composition of the invention the extract of green lipped mussel will be GLME, and more preferably will be GLMO. In more preferred embodiments the GLMO will have been obtained by the use of a fractionation process that retains the phospholipid component, such as a dual-solvent fractionation process. Most preferably the GLMO will have been obtained by the use of a dual-solvent fractionation process using isopropyl alcohol and hexane.

Honey is a complex natural product, however its major component is carbohydrate (about 82% - primarily the monosaccharides fructose and glucose) together with a minor amount of water. Honey has been used therapeutically for thousands of years, however despite this use its mode of action remains unclear. In more recent times, the honey produced by bees foraging on trees that are endemic to Australia and New Zealand, has been shown to have an improved therapeutic profile over honey derived from other flowering species. In particular, honey derived from:

• trees of the genus Leptospermum, particularly those of the species Leptospermum scoparium (Manuka); and

• trees of the genus Kunzea, particularly those of the species Kunzea ericoides (Kanuka);

have been shown to provide increased antibacterial activity over honey produced from clover, for example. Preferred honeys for use in the present invention are referred to as Manuka honey and Kanuka honey. Still more preferably, honeys having a Unique Manuka Factor (UMF) of at least 5 such as at least 10 or at least 15 are most preferred. The source of the bactericidal activity of Manuka honey has not been definitively shown, but is believed to be due at least in part to the presence of hydrogen peroxide, methylglyoxal, low water content and/or high osmolarity. To that end, the present invention also contemplates the preferred use of non-Manuka/Kanuka honey that has been fortified by the addition of hydrogen peroxide and/or methylglyoxal, such as clover honey that has been fortified by the addition of methylglyoxal.

Without wishing to be bound by theory, it is believed that the extract of green lipped mussel and the honey may act synergistically to provide a therapeutic and/or prophylactic effect. As used herein, the term "synergistic" means that the effect achieved with the compositions and combinations of the invention is greater than the sum of the effects that result from using the extract of green lipped mussel and the honey each as a monotherapy. Advantageously, such synergy provides greater efficacy at the same doses, and provides an effect where otherwise there would be no discernible effect.

Without wishing to be bound by theory it is also believed that synergy can be provided at very low loadings of each of the active components. In some embodiments the extract of green lipped mussel and the honey are each present, independently, at concentrations less than 10% wt/wt. For example the extract of green lipped mussel and the honey are each present, independently, at a concentration of less than 5% wt/wt. By way of further example, the extract of green lipped mussel and the honey are each present at approximately 1% wt/wt. By way of further example still, the extract of green lipped mussel may be present between 0.1% wt/wt and 2% wt/wt, such as between 0.5% wt/wt and 1.0% wt/wt. The honey may be present between 0.1% wt/wt and 2% wt/wt, such as between 0.5% wt/wt and 1.0% wt/wt.

Without wishing to be bound by theory it is believed that the ratio of the extract of green lipped mussel and the honey can be advantageously optimized to provide synergy. The ratio of the extract of green lipped mussel to honey may be between 10:1 and 1:10, such as between 5:1 and 1:5, such as from 1:2 to 2:1, such as from 0.5:1 to 1:0.5.

The term "composition" is intended to include the formulation of the extract of green lipped mussel and honey with carrier, to give for example a gel.

While the extract of green lipped mussel and honey may be the only two active ingredients administered to the subject, the administration of other active ingredient(s) with the extract of green lipped mussel and honey is within the scope of the invention. For example, the extract of green lipped mussel and honey could be administered with one or more therapeutic agents in combination. The combination may allow for separate, sequential or simultaneous administration of the extract of green lipped mussel and honey as hereinbefore described, optionally with separate, sequential or simultaneous administration of the other active ingredient(s). The combination may be provided in the form of a pharmaceutical composition.

As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the animal (preferably mammal) to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the extract of green lipped mussel and honey care should be taken to ensure that the activity of the extract of green lipped mussel and honey is not destroyed in the process and that the extract of green lipped mussel and honey is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the extract of green lipped mussel and honey by means known in the art, such as, for example, micro encapsulation. Similarly the route of administration chosen should be such that the extract of green lipped mussel and honey reaches its site of action.

Those skilled in the art may readily determine appropriate formulations for the extract of green lipped mussel and honey of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including carotenoids such as astaxanthin, phenolic compounds such as BHT or vitamin E

(a-tocopherol), reducing agents such as methionine or sulphite, and metal chelators such as EDTA.

Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Advantageously, the composition of the invention may be provided as an aqueous gel. In such embodiments, an alcohol (such as ethanol) can be used as a co- solvent. Glycerin can also be used. Surfactants such as polysorbate 20 can be used.

Supplementary active ingredients can also be incorporated into the compositions. One such ingredient is menthol, which is believed to act as a local anaesthetic and have counterirritant properties.

The term "therapeutically effective amount" refers to that amount which is sufficient to effect treatment, as defined below, when administered to an animal, preferably a mammal, more preferably a human in need of such treatment. The therapeutically effective amount will vary depending on the subject and nature of the disease being treated, the severity of the infection and the manner of administration, and may be determined routinely by one of ordinary skill in the art.

The pharmaceutical composition may comprise an amount of the extract of green lipped mussel and honey, sufficient to ameliorate and/or alleviate at least in part the disease that is subject to the prophylaxis and/or treatment in the animal (preferably human).

While the extract of green lipped mussel and honey is intended primarily for use in the treatment or prevention of a disease in a human, the extract of green lipped mussel and honey may also be used on other animals, preferably domesticated animals, such as horses, cows, sheep, goats, pigs and household pets including dogs, cats, rabbits, mice, rats, birds.

The terms "treatment" and "treating" as used herein cover any treatment of a condition or disease in an animal, preferably a human, and includes: (i) inhibiting the disease; (ii) relieving the disease; or (iii) relieving the conditions caused by the disease, eg symptoms of the disease.

The terms "prevention" and preventing" as used herein cover the prevention or prophylaxis of a disease in an animal, preferably a human and includes preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed with the disease.

The combination and composition of the present invention is preferably formulated for topical administration. Preferably, the extract of green lipped mussel and honey of the invention will be applied topically such as in a cream, lotion or gel. The term "topical", as used herein, refers to a composition meant for application to the skin, nail, or mucosal tissue (such as gum). The combination and composition of the present invention is believed to be particularly effective in the treatment and/or prevention of a range of skin, nail and mucosal disorders.

It has been found that the inventive composition is particularly well-suited to address diseases associated with inflammation and/or bacterial infection and/or where penetration/softening skin is an issue, including: • pain and joint stiffness (preferably acute rather than chronic);

• open wounds;

• gum disease;

• redness/discolouration;

• eczema;

• psoriasis;

• shingles.

It is believed that the inventive composition absorbs into the skin quickly; softens the skin and is easily spread. In preferred embodiments the inventive composition can be provided with a fragrance to mask any undesirable odour associated with any one or more of the other components of the composition.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

EXAMPLES

Example 1 - compositions

Further exemplary compositions according to the invention are as follows:

Example 2 - Method of manufacture

1. Non-heat sensitive aqueous phase ingredients combined with heating;

2. Non-heat sensitive organic phase ingredients combined;

3. Combined aqueous ingredients with organic phase ingredients and blend two phases together with heating;

4. Emulsion left to cool;

5. Heat sensitive ingredients (eg perfume) added to cooled emulsion;

6. pH adjusted to suitable level

7. Product packaged and labelled.

Example 3 - Pain Trial

Overview

This is a review of an informal consumer trial of 20 people with a variety of pain related problems.

The preliminary results have been written up in this report, and suggest that the KNZ Cream is very effective in the reduction of activity induced pain and stiffness, as well as inflammatory skin conditions. Within this trial, there were some who had skin problems where they applied the cream to beneficial effect.

Also in a separate write up, there is a more detailed photographic study of a 65 year old man with severe skin problems that appear to have improved markedly through the application of KNZ cream. Of course, this is very much an informal consumer trial, and the results are nothing more than a collection of anecdotal stories.

Nevertheless, there is some very convincing supporting evidence that there are some profound benefits that have accrued to most of those on this trial.

It seems from that there could be an accumulating beneficial effect of the application of the gel to helping mitigate pain levels

Subject A - age 62

Subject A has recently retired and has been very active in improving the house and landscaping the garden in the last year and also enjoys kayaking and walking.

His hand and fingers are usually quite stiff after working with his hands and they can also become painful and sometimes his fingers "lock up". At rest, there is no pain as such, but rather general stiffness, but after physical activities, which is part and parcel of his life the more specific pain and stiffness occurs.

Subject A has used the inventive composition for a few days with the following results:

1. After working in the garden, he had severe cramp in some of his fingers to the point that they "locked up" and has a pain score of 6. Within c. 20 seconds of applying the gel the fingers free up and the pain disappeared.

2. Generally, his fingers are always stiff, even without exercise, and although there is no pain after applying the gel they become supple and with easy movements.

3. After working particularly hard in the garden one day, where he had been wheel-barrowing and moving rocks he was very tired and retired early to bed. When he lay down he had extreme cramps in the tops of both his thighs, where the pain was very intense that he almost was screaming in pain (score 8). His wife applied the gel and within 10 seconds the pain had completely gone, and he was then able to go straight off to sleep.

Subject B - age 70

Subject B has stiff fingers all the time, after years of gardening. After rubbing in the inventive composition she can feel her fingers loosen within 2 minutes and the effect lasts 2 days. Whilst discussing the outcome of the gel trial, Subject B had a couple of sand-fly bites, which are very irritating for her and where the irritation lasts 3 hours or so. She applied a little amount of the inventive composition to the bites and the irritation was immediately relieved.

As a former nurse, Subject B said that she was amazed at the speed and effectiveness of the inventive composition, where compared to Voltaren, as it is far faster in acting and the effect lasts far longer. She was amazed how quickly it worked on her husband's acute cramping and never seen anything as effective in a topical application.

Subject C - age 72

Subject C is a retired DoC worker, and is still very active in the garden and enjoys hiking. Over the last 18 months or so, he has become to experience back pains where an M I scan shows arthritis and degeneration.

When sitting and resting, Subject C does not suffer any pain, but standing makes his back hurt. Also, when walking the pain in his back comes on gradually, so that after 20 minutes or so walking uphill the pain increases from a zero to a 5 or so and he has to rest for 10 minutes before continuing.

Subject C was an avid hiker, and has hiked the 1000 km Bibbulman Track from near Perth to Albany, and several c. 3 day hikes around NZ, but now can only go for very short walks due to the pain in his back coming on so quickly once he starts the walk.

His local walk is probably about 80 minutes with a few hundred metre gradual ascent to the top.

On Monday 7^th November, he applied the inventive composition before the start of the walk, and then was able to walk to the top of the hill without stopping which was unusual. There was a minimal pain of 0.5 at the top of the hill, which allowed him to continue without stopping and by the time he returned home the pain was gone.

The next day, Tuesday 8^th, he went for another walk, but this time he did not apply any inventive composition as his back still felt so good from the day before. He went for a longer walk where the track was also much steeper. This time there was some pain, but at a 1.5 level it was much less pain than a 5, and so he was able to complete the walk without stopping.

He believes that the positive effect from the inventive composition lasted over 24 hours.

On 10^th November, Subject C was standing for a few hours and ended up with a pain of 3 in his back. As soon as he got home he put the inventive composition on his back and within 10 minutes the pain had gone.

Subject C now feels that with the inventive composition, and with appropriate training beforehand, he would be able to go on extensive hikes again. Subject D - age 65

Subject D is a plumber by trade, and still works part time. He had an operation on his right knee some years ago, and has arthritis in his knees as well problems with his right hip, spine and both shoulders through general wear and tear of hard physical work over the years.

His right hip, shoulder and back cause the most severe problems and after work the evening's pain score can be a 7 or 8 out of 10.

He applied the inventive composition to his right hip and lower back and right shoulder twice daily, which are the most painful areas.

After 2 days the following results were reported:

On Saturday 12^th, after 7 days of application, Subject D reported that his hips were now a 1, his back a 1 and his shoulder a 1. He had spent quite a lot of the previous day bending over and weeding his garden and his back had not suffered any pain. He said that he almost wouldn't put on the inventive composition every day.

Also, his eczema wound was definitely continuing to improve and heal which it hadn't done for many years. He will continue to monitor this.

Subject E - age 62

Subject E has had a hard, physical working life, initially working for years as a shearer, and then as a DoC worker. He is also an avid hunter and walks miles in the bush carrying the meat back to his truck. He has had a hip replacement to the right hip, and tolerates pain exceptionally well. Currently he has pains and cramps to his left leg which are exacerbated by exercise. After a long hike, Subject E has severe pain in the 6/10 range, and after applying the inventive composition the pain level drops to 2 or less within 5 minutes of application of the composition.

Subject E also commented that the inventive composition is much better than Voltaren in effectiveness. Subject F - age 57

Subject F is a big man and a diesel mechanic by trade, who has to work with heavy machinery and equipment, and though pretty robust is now suffering from joint pains. He has been taking capsules containing GLM oil, and more recently magnesium tablets and these have relieved some of the pain and cramps. However, when he has been standing for some time, or working on machinery he gets a very painful left knee.

He also has had a rash on his left thigh area for some time which he finds very irritating with the desire to scratch it.

He put the first application of the inventive composition on his knee on the night of Friday 4th, and the next day Subject F said that he felt that the pain was much reduced and gave him greater flexibility the next morning.

On the Sunday, he reported that the rash on his leg looked better.

On the Tuesday, his knee felt good all day at work and had no real pain.

At a follow meeting after his work up on Wednesday, Subject F said that he thought there was some continuing improvement in his knee and that after it seems to be a bit easier to get up after he has been working on large vehicles and equipment. Also, the itchiness of his rash was much reduced. He felt much better and more mobile where it was easier to get up from under big machinery that he was working on.

He had used up all of the sample composition by the Wednesday, and on Thursday he was OK at work without the inventive composition, but by the Friday he had a small ache in his knees again. On the Saturday, after going into work for a few hours he said his knees were very stiff again and he couldn't easily get up from under a truck without holding on to something to pull himself up.

Subject G - age 74

Subject G is a very keen model maker and works for many hours on intricate parts for his model boats. This work involves dextrous use of the thumbs and index fingers. He takes GLM capsules and these have helped the general stiffness in the hands, but when he is working on his model boats his thumbs and index fingers get so painful and stiff and he is forced to stop when the pain gets too much.

Normally Subject G has to have a week's rest between bouts of working on his models.

On 5^th November, Subject G was working on his model making for about 5 hours and had to stop when the pain became too great (6/10). He applied the inventive composition and within 15 minutes the pain began to ease and within 90 minutes the pain had gone (0).

The next day, 6^th November, Subject G was able to recommence work on his boat, however, this time he applied the inventive composition before commencing work. After 6 hours he was completely pain free and his fingers were still supple. Subject G thinks that the inventive composition is a great product, providing great relief - and acts incredibly quickly.

Subject H

Some years ago, Subject H was in a car accident and suffered whip-lash which has left her with a very sore neck and left shoulder, and where the pain often keeps her awake at night. She also gets stiffness and aches in her fingers from gardening, which she does most days.

Subject H started applying the inventive composition on her shoulders and neck from the evening of 7^th November and also inadvertently to her hands.

There has been a positive result to her hands, but not the neck or shoulder.

"As I have been gardening most of these days, my hands are usually sore and often ache the following day. I usually wear fingerless gloves to keep them warm and lower the throb but the gel appears to have relieved this pain to the point that I forgot to wear my gloves to work! The pain is usually a nagging 4. After applying the gel to my neck area, I rub my hands together so as to not waste any and so inadvertently apply to my fingers and hands."

Subject I - fingers, knees and others, suffers severe problems

Subject I suffers from genetic hemochromatosis (compound) and is in constant and severe pain. He takes GLM capsules as well as strong doses of Ibuprofen. He tried the inventive composition on his hands, knees, elbows and feet. The inventive composition was effective on his hands, but not the other joints:

Subject K - hips

Subject K had a hip operation a year ago, where the surgeon moved the sciatic nerve in her right hip out of its normal place! This has resulted in Subject K now having to have a brace for her feet and she now walks very badly with an exaggerated movement to bring her feet up. This in turn has had the consequence of giving her pain in both hips when she walks, to the extent that she is now quite limited in how far she can walk and what she can do.

She has applied the inventive composition to the hips when she feels the pain (3) coming on, and finds that the pain subsides to a 0 within 10 minutes or so. She uses the inventive composition as a preventative and finds it lasts over 24 hours.

"Magic" is how she describes the gel, and thinks that she will be able to walk further again. Also, she feels it works much better than Voltaren.

Subject L

Subject L has: 1) degenerative lower back discs; 2) 10 months post arthritis scraping operation; and 3) arthritis? in her fingers. She works part time at a refuse centre, but can now only work for one day on at a time, for a total of 3 days a week.

Her comments: The back, knee and fingers all much improved. Her back is usually very sore after work, but not now. The finger joint is much better, and her knee is improved. She applied the inventive composition "before vacuuming and her back didn't do its usual spasm."

It feels pretty good and she used it on the sore bits after showering and seems to prevent any worsening.

She was first given the inventive composition in the morning of Monday 7^th at her work, and two hours later she said that the results were "spooky, what is in this stuff that it works so quickly?" No details were provided.

Subject M

Subject M is an engineer, and has problems with his right elbow, which has been operated on to remove bone fragments and he has also two nerve operations on it.

The right elbow gives restricted movement and, as an engineer, he has to use his arms a lot in order to perform work tasks. By the end of the day his elbow becomes quite painful. Subject M first applied the inventive composition on Monday evening, 7^th November, after a day's work and subsequently applied the inventive composition just in the evening after work. On Wednesday evening, 9^th November, (just 3 applications) he said that he felt that there was a more relaxed movement in the elbow and the pain was a little less.

From Thursday 10^th November he increased the applications to morning and evening.

On the Saturday 12^th, he reported that he definitely felt that he had more movement in the elbow, and that simple tasks had become easier again e.g. holding the telephone in this right hand to his ear for a conversation he then realized wasn't hurting and he hadn't switched hands.

He felt that there was some continuing improvement with mobility and pain from his elbow.

Subject N - shoulder pain

Subject N has had shoulder pain since a teenager, and has been to the doctor several times.

Subject Q- gums, following wisdom teeth extraction

Recently Subject Q, had her two lower wisdom teeth surgically removed. After a week the pain was unbearable and she had an infection in the gums which were swollen with ulcers. She had been on antibiotics, but the course had finished. On Friday 4^th she tried the inventive composition on her gums. "It didn't taste the nicest but to my surprise within one day the/ swelling had gone down. After two days the pain had been reduced to a small niggle and the fat red rawness of my mouth was just about back to normal."

Subject R - suffers from arthritis in the ankles

Before using the inventive composition the pain in the subject's knee was 8 out of 10. Following the initial application, the subject applied the inventive composition on her knee morning and night and found that it eased the pain significantly. After 10 days, the pain level had dropped to 4 out of 10 and the subject is now able to enjoy a short walk gain.

Example 4 - skin discolouration

Subject S is a 65 years old man, who had surgery many years ago to remove a brain tumour and had extensive radiotherapy.

His skin is extremely red, blotchy, with lesions and sores some of which are pre-cancerous scabs etc. and he has had skin cancer removed as well. It has been like this for many years.

Subject S has been applying the inventive composition twice a day for just over a week now, and attached are some photographs of his left hand at day 0 before application and also at day 7.

The improvement seems to me to be remarkable and in such a short time.

Day to day pictures (days 0 to day 8) of: 1) left side of his face; 2) right side of his face 3) face from front; 4 hands.

The pictures show that the redness almost completely gone, with wounds and discolouration substantially improved.

Subject S reports that he is staggered with the improvement in such a short time and after so many years and his skin feels much smoother.

Example 5 - psoriasis

A 67 year old man (Subject T) with psoriasis applied the inventive composition to wounds on his left shin, left and right knees and left and right elbows. The open wounds have begun to heal, the redness is much reduced and the scabs are reduced in size.

Example 6 - psoriasis

Subject U applied the inventive composition to psoriasis wounds, with sores healing up in a few days with redness decreasing, and then a gradual improvement over the next couple of weeks. Subject U's elbows at day 0 and day 11 are shown in Figure 4. Example 7 - eczema

There have been three people who have used the inventive composition and again the lesions dry up quickly and healing begins.

Subject V has had eczema on the left side of his face by his eye for several years that has continuously weeping and had never healed.

This year he has had 3 shots of liquid nitrogen applied to it and it still did not heal, remaining open and weeping. He put some of the inventive composition on this lesion on Saturday evening 5^th November, and after 2 days, Monday 7^th 2pm, i.e. less than 2 days later, the wound had started to scab.

The photos of day 7 shows that the lesion had begun to heal and the redness had decreased compared with the start.

By day 26 the redness has gone completely and the area around the lesion appears to be clearing up and healing.

Example 8 - shingles

The inventive composition has been used by a 94 year old woman on her shingles which is above her right eye brow , and where she has had constant pain for several (8?) years.

She has been to the doctor's and tried all sorts of creams but has been in constant pain for all this time, where the pain level varies in intensity and where sometimes it is a constant ache and other times is extremely painful. In addition she has shooting/stabbing pains.

Over the last two weeks she has been applying the cream to her right brow and the swelling there has been substantially reduced (it may have gone completely, her daughter will report back in the next week).

The constant ache and the more intense pain in her brow has now gone completely.

Unfortunately she still gets stabbing pains for a couple of seconds from time to time, sometimes 3 times a day - sometimes none. Interestingly, now she is free of the constant pain to her brow she is now able to differentiate where the stabbing pain originates so that she can now tell that it comes from a specific point in her head, rather than just having an intense migraine headache and being unaware of the origin of the intense stabbing pain.

It seems as if the inventive composition has made a profound difference to the quality of life for this elderly lady - and its effect has been immediate - where all other treatments have made little to no difference.

Example 9 - in vitro testing SUMMARY The project aimed to investigate the effects of 11 samples on psoriasis using an established and recognised in vitro model based on the culturing of keratinocytes. The effect of the samples on the psoriatic keratinocytes and on normal unaffected keratinocytes were determined by measuring the changes in the concentration of elafin/SKALP. Additionally, the effects on the viability and the normal metabolism of keratinocytes were investigated by observing changes in the cell concentration and the cytokeratin 10 concentration.

Because considerable inflammation is associated with psoriasis, the effects of the 11 samples on the activities of the COX-2 and lipoxygenase pathways of inflammation were also investigated. These were measured by changes in the concentration of prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄).

Cell Viability ■ Several of the samples affected the viability of the keratinocytes although the effects were sometimes different when the cells were cultured in growth medium (psoriasis) compared with basal medium (normal skin).

^■ In the basal medium which simulates the healthy cells, Sample 8 (GLMO:Manuka honey (0.5:1)) (170.2%), Sample 7 (GLMO:Manuka honey (1:1)) (97.3%), Sample 9 (GLMO:Manuka honey (1:0.5)) (48.8%) and Sample 6 (Clover honey) (34.6%) stimulated the growth of the cells.

^■ In contrast, in the growth medium which simulates the psoriasis cells, none of the samples had any stimulatory effect on the viability of the cells.

^■ In the basal medium four samples inhibited the growth of the cells. The strongest were Sample 10 (Lotion including GLMO (dual solvent extraction):Manuka Honey (1:1)) (74.3%), Sample 4 (GLMO oil-Lyprinol) (75.8%), Sample 3 (GLMO oil (Bio-Mer)) (48.3%) and Sample 11 (EgoPsoryl)

(37.9%). That Sample 10 inhibited the growth of healthy cells, but Samples 7-9 stimulated the growth of healthy cells suggests that the other components of the lotion may have played a deleterious role in the growth of healthy cells.

^■ For the cells in the psoriasis culture, most of the test samples inhibited the survival of the cells.

The strongest were Sample 8 (GLMO:Manuka honey (0.5:1)) (91.4%), Sample 9 (GLMO:Manuka honey (1:0.5)) (90.3%) and Sample 7 (GLMOrManuka honey (1:1)) (85.0%). Also Sample 2 (GLMO Dual solvent extraction) (94.5%) and Sample 3 (GLMO oil (Bio-Mer)) (85.0%) were very effective at reducing the number of cells.

^■ Retinoic acid at 20 μΜ reduced the number of healthy cells by 66.2% but only reduced the psoriatic cells by 19.9%.

Elafin/SKALP

^■ This compound is the marker of the state of psoriasis. A product that is potentially beneficial would be expected to reduce the concentration of elafin/SKALP in the cultures grown in the growth medium.

^■ The majority of the samples were strong inhibitors of elafin/SKALP in the keratinocytes in the psoriasis environment. Almost complete inhibition was achieved with Sample 7 (GLMO:Manuka honey (1:1)) (89.8%), Sample 8 (GLMO:Manuka honey (0.5:1)) (91.2%) and Sample 9 (GLMO:Manuka honey (1:0.5)) (89.1%).

^■ In the basal medium, the keratinocytes produced much lower levels of elafin/SKALP as expected. It was nearly six times lower. With the exception of the two honey samples (Sample 5 (Manuka honey) and Sample 6 (Clover honey) all the other samples were very strong inhibitors of this low level of elafin/SKALP in normal cells.

^■ Sample 2 (GLMO Dual solvent extraction) and Sample 3 (GLMO oil (Bio-Mer)) were also strong antagonists of elafin/SKALP production by the psoriatic keratinocytes. The reductions were 64.3% and 69.0% respectively.

^■ The honey samples had negligible effect on the elafin/SKALP production by the cells in the psoriasis medium. The Manuka honey (Sample 5) reduced it by 4.6% and the Clover honey (Sample 6) reduced it by 16.4% - both effects were insignificant.

^■ The normal healthy keratinocytes were stimulated by the honeys to produce more elafin/SKALP. Sample 5 (Manuka honey) increased it by 60.9% and Sample 6 (Clover honey) by 42.6%.

Cytokeratin 10

^■ Cytokeratin 10 is a major product of keratinocytes and so is a marker of the metabolic activity of the cells.

^■ In the psoriatic keratinocytes, all of the test samples stimulated the production of cytokeratin 10. In the control cultures, the concentration was quite low but several samples stimulated it to the extent that its concentration was greater than the highest level of detection. These included Sample 1 (Lotion) Sample 10 (Lotion including GLMO (dual solvent extraction):Manuka Honey (1:1)) and Sample 11 (EgoPsoryl).

^■ The least effect was produced by Sample 9 (GLMO:Manuka honey (1:0.5)) (6.4%).

^■ In normal healthy keratinocytes, the production of cytokeratin 10 was also quite low. Several samples stimulated it, the strongest being Sample 4 (GLMO oil (Lyprinol)) (186.2%), Sample 2 (GLMO Dual solvent extraction) (113.1%), and Sample 5 (Manuka honey) (84.8%).

^■ Four of the test samples inhibited the synthesis of Cytokeratin 10 by normal keratinocytes. The strongest were Sample 7 (GLMO:Manuka honey (1:1)) and Sample 9 (GLMO:Manuka honey (1:0.5)) which reduced it to below the detection level. Sample 3 (GLMO oil (Bio-Mer)) (75.4%) and Sample 8 (GLMO:Manuka honey (0.5:1)) (28.5%) were also significant inhibitors.

PGE₂

^■ One of the possible promoters of the inflammation associated with psoriasis is prostaglandin E₂ (PGE₂) which is a product of the COX-2 pathway. Inhibition of this enzyme implies an antiinflammatory effect.

^■ Surprisingly none of the test samples inhibited the production of PGE₂ by the cells grown in the growth medium. In fact all stimulated with Sample 11 (EgoPsoryl) (725.3%) and Sample 9 (GLMO:Manuka honey (1:0.5)) (451.4%) being the strongest.

^■ In the growth medium, the least stimulation was produced by Sample 5 (Manuka honey) (27.6%) and Sample 7 (GLMO:Manuka honey (1:1)) (31.8%).

^■ As anticipated the concentration of PGE₂ in the culture of normal healthy keratinocytes was negligible. Consequently the increases in the PGE₂ concentration resulting from inclusion of the test samples appear to be very high. In this medium all samples except Sample 7 (GLMO:Manuka honey) (1:1)) were stimulatory.

^■ One of the possible promoters of the inflammation is leukotriene B4 (LTB4) which is a product of the lipoxygenase pathway.

^■ The production of LTB₄ by the psoriatic keratinocytes is about 26 times higher than by normal healthy cells indicating the inflammatory state of psoriasis cells. ^■ Two of the samples were potent antagonists of LTB₄ synthesis. They were completely inhibitory in both the normal and the psoriatic cell cultures. They are Sample 3 (GLMO oil (Bio-Mer)) and Sample 8 (GLMO:Manuka honey (0.5:1)).

^■ Three of the test samples significantly stimulated the production of LTB₄ under the conditions of psoriasis. These were Sample 9 (GLMO:Manuka honey (1:0.5)) (256.6%), Sample 2 (GLMO Dual solvent extraction) (110.8%) and Sample 4 (GLMO Lyprinol) (94.2%).

^■ The healthy cells produced little LTB₄ as expected. However Sample 3 (GLMO oil (Bio-Mer)) and Sample 8 (GLMO:Manuka honey (0.5:1)) appeared to completely inhibit the production.

^■ Other samples stimulated the lipoxygenase activity under the basal conditions with the exception of Sample 5 (Manuka honey). The strongest stimulators were Sample 7

(GLMO:Manuka honey (1:1)), Sample 9 (GLMO:Manuka honey (1:0.5)) and Sample 2 (GLMO Dual solvent extraction).

Based on the effects of the GLMO:Manuka honey mixes on the production of elafin/SKALP by the psoriatic cells and their reduced effect on normal keratinoytes, it seems that they are quite specific for inhibiting the diseased cells.

It is also observed that these levels of inhibition of the psoriasis phenotype by these highly active samples are almost identical to the loss of viability of these cells. Again this appears to be specific to the diseased keratinocytes. This would suggest that the mechanism of action of GLMO and GLMO: Manuka honey samples is through the destruction of these cell exclusively. The investigation of the effects on the inflammatory pathways has produced surprising data. The levels of activity of both COX-2 and lipoxygenase in keratinocytes in both normal and psoriatic cells are not high. However, there is little evidence for these pathways being important in the inflammation associated with psoriasis. Other mechanisms are more relevant. Therefore it is arguable that the data acquired in these elements of the project should be given less weight because it may have reduced relevance.

1. Objective

The effects of 11 test samples on the growth and differentiation of human keratinocytes was investigated. The levels of Cytokeratin 10 synthesised and Elafin/SKALP released by the keratinocytes were determined. The ability of the samples to affect the expression of these markers would be an indicator of their potential use in the treatment of skin psoriasis. Additionally, the levels of the inflammatory markers PGE₂ and LTB₄ were assessed to determine any potential anti-inflammatory effect of the test samples.

The effects of the test samples in both normal and psoriatic cells were determined in triplicate for each of these five parameters.

2. Test Items The test samples 1-11 (Table 1) were used as follows:

Table 1. Test samples used in this study

3. Reference Item Retinoic Acid

4. Description of Test Materials and Test Methods The methodology used in this investigation was based on that described in the following paper:

Amigo, M, Schalkwijk, J, Olthuis, D, De Rosa, S, Paya, M, Terencio, MC, Lamme, E (2006). Identification of avarol derivatives as potential antipsoriatic drugs using an in vitro model for keratinocyte growth and differentiation. Life Sciences 79: 2395 - 2404.

The human keratinocyte cell line KerTr was retrieved from cryostorage and cultured in keratinocyte basal medium (KBM) and in keratinocyte growth medium (KGM). The cells were then incubated with the test samples for 72 hours after which the supernatant was removed and used for determining the levels of Elafin/SKALP, Cytokeratin 10, Prostaglandin E₂ (PGE₂) and Leukotriene B, (LTB4). The adhered cells in the plates were used in a MTT assay to determine cell viability. Each sample was assayed in triplicate. Retinoic acid was included as a positive control and untreated cells were used as a baseline control.

The percentage standard error of the mean was assessed and extreme outliers were removed if the SEM% >15%. Preliminary statistical significance was assessed with an independent Student t-test at a 0.05 (without outliers). Graphical representations of averages +/- standard errors are presented. i. Sample Preparation

The test samples were dissolved in Hanks Buffered Salt Solution (HBSS) as stock solutions at the concentrations shown in Table 2. Samples 3 and 4 were dissolved in 15% EtOH/HBSS (Deviation 1).

Table 2. Concentrations of the test samples used in this project. ii. Experimental Procedures Characterisation of the Test System

1. Human keratinocyte cell line (KerTr CCD1106 ATCC No. CRL-2309). Stored in liquid N₂.

2. Phosphate buffered saline (PBS). Freshly prepared.

3. Hanks' Balanced Salt Solution (HBSS). Freshly prepared.

4. 0.05% Trypsin-EDTA solution: Trypsin/EDTA (Sigma, Cat. No. 15400054) diluted with PBS.

5. Keratinocyte basal medium (KBM) (Gibco, Cat. No. 1072401). Supplemented with 100 U/ml penicillin - 100 μg/ml streptomycin (Sigma, Cat. No. P0871).

6. Keratinocyte basal medium supplement (EGF Human Recombinant & Bovine Pituitary Extract) (Gibco, Cat. No. 3700001). Keratinocyte growth medium (KGM) was made by combining Keratinocyte basal medium (KBM) and keratinocyte basal medium supplement as described by the manufacturer of the medium and the supplement.

7. Foetal calf serum (FCS) (Gibco, Cat. No.10091-148).

8. Retinoic acid (Sigma, Cat. No. R-2625) Stored at -20°C. This was dissolved in PBS at 200 μΜ

9. MTT Reagent: 100 mg/vial, (Sigma, Cat. No. M-2128) dissolved in PBS at 10 mg/ml and stored at -20⁹C. 5 mg/ml MTT solution was prepared in PBS and stored at 4°C as the working solution.

10. MTT lysis buffer: 10% sodium dodecyl sulphate (SDS)/45% dimethyl formamide (20 g SDS was dissolved in 100 ml of double-distilled water (DDW), and 90 ml of dimethyl formamide was added to the SDS solution). The pH was adjusted to 4.7 with glacial acetic acid, and DDW added up to a final volume of 200 ml.

11. Human Elafin/SKALP ELISA kit (Abeam, Cat. No. abl00658).

12. Cytokeratin 10 ELISA kit (Mybiosource, Cat. No.MBS766016) (Amendment 1).

13. Prostaglandin E₂ ELISA Kit (R&D Systems, Cat. No. KGE004B). Stored at -20°C.

14. Leukotriene B₄ ELISA Kit (R&D Systems, Cat. No. KGE006B). Stored at -20°C. Cell Preparation, Harvesting and Culturing of Human Keratinocytes (KerTr)

1. The frozen stock cell line (human keratinocytes cell line KerTr CCD1106) was removed from liquid nitrogen and immediately thawed in a 37°C water bath. The contents were then transferred to a 250 ml (75 cm²) culture flask containing 20 ml KGM. 2. The cells were cultured at 37°C in 95% air/5% CO2 for 24 hours. The medium in the flask was then removed and fresh medium added. The cells were subsequently cultured until they had reached approximately 80% density/confluence. Cells were examined daily for confluence under the microscope. The 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. Then 5 ml of 0.05% Trypsin/EDTA solution was added and incubated at 37°C for 5 min until all the cells were detached. The trypsin was then neutralised by adding an equal volume of pre-warmed DMEM and the suspension transferred to tubes and centrifuged at 125 g (500 rpm) for 7 min at 4°C. The re-suspended cells were centrifuged in 2 tubes. 2. The supernatant from the tubes was discarded and the cell pellet in tube #1 re-suspended with pre-warmed DMEM containing 100 U/ml penicillin and 100 μg/ml streptomycin.

3. The cell pellet in tube #2 was re-suspended in KGM as described in section above plus 5% FCS.

4. For each medium type, the cell number was counted and adjusted to 2.5 x 104 cells/ml. Cell Culturing: 1. Flat-bottomed 96-well plates, treated for cell adhesion, were used. Four plates were set up. Plate 1A and 2A contained cells in KBM (i.e. no supplement). Plate IB and 2B contained cells in KGM supplemented with 5% FCS (see plate layouts below).

2. For the assays, 190 μΙ of the appropriate cell suspension (2.5 x 10⁴ cells/ml) were plated into wells A-F 1-6 and G 1-3 of each plate giving approximately 4.75 xlO³ cells/well. 190 μΙ of medium only was added to wells A-F 7-12 and G 7-9 of each plate. PLATE LAYOUTS

3. The plates were placed in the incubator (37°C, 5% CC>2/95% air) for 3 hours to allow the cells to adhere to the wells.

4. After 3 hours the plates were removed from the incubator. To all of the plates, the various samples and controls were added to the appropriate wells as indicated in the plate layout above. 5. To the wells labelled "Cells Only + HBSS" and "Medium Only + HBSS", 20 μΙ of HBSS were added.

6. The plates were incubated at 37°C in 95% Air/5% C0₂ for a total of 72 hours.

7. At the time of termination, the culture medium was aspirated from the wells and transferred to the wells of new sterile 96 well plates (non-treated) and stored at -20°C until required for the assays.

8. After the removal of the supernatants (See Step 7, Cell Culturing above), 175 μΙ of fresh medium was added to each well of plates 1A and IB. a. Elafin/SKALP Concentration:

Aliquots of supernatant (See Step 7, Cell Culturing above) were used for determining the concentration of Elafin/SKALP. These were measured by following the instructions from the manufacturer of the kit. b. Cytokeratin 10 Determination:

Aliquots of the supernatants from Plates 1A and IB (See Step 7, Cell Culturing above) were used for determining the concentration of cytokeratin 10. These were measured by following the instructions from the manufacturer of the kit. c. PGE₂ and LTB₄ Determination:

Aliquots of supernatant from each well of Plates 2A and 2B (See Step 7, Cell Culturing above) were used for determining the concentrations of PGE₂ and LTB₄. These were measured by following the instructions from the manufacturers of the ELISA kits for these two compounds d. MTT Determination:

1. After fresh medium had been added to each well, 20 μΙ of MTT working solution (5 mg/ml) was added to each well on plates 2A and 2B and incubated for an additional 4 hours at 37⁹C.

2. 100 μΙ of MTT lysis buffer was then added to each of these wells and the plate incubated overnight at 37°C.

3. The absorbance of each well was read using the VersaMax microplate reader at 570 nm. 7. Results and Discussion i. Summary Cell Viability

• Several of the samples affected the viability of the keratinocytes although the effects were sometimes different when the cells were cultured in growth medium (psoriasis) compared with basal medium (normal skin).

• In the basal medium which simulates the healthy cells, Sample 8 (GLMO:Manuka honey (0.5:1)) (170.2%), Sample 7 (GLMO:Manuka honey (1:1)) (97.3%), Sample 9 (GLMO:Manuka honey (1:0.5)) (48.8%) and Sample 6 (Clover honey) (34.6%) stimulated the growth of the cells.

• In contrast, in the growth medium which simulates the psoriasis cells, none of the samples had any stimulatory effect on the viability of the cells.

• In the basal medium four samples inhibited the growth of the cells. The strongest were Sample 10 (Lotion including GLMO (dual solvent extraction):Manuka Honey (1:1)) (74.3%), Sample 4 (GLMO oil-Lyprinol) (75.8%), Sample 3 (GLMO oil (Bio-Mer)) (48.3%) and Sample 11 (EgoPsoryl) (37.9%).

• For the cells in the psoriasis culture, most of the test samples inhibited the survival of the cells. The strongest were Sample 8 (GLMO:Manuka honey (0.5:1)) (91.4%), Sample 9 (GLMO:Manuka honey (1:0.5)) (90.3%) and Sample 7 (GLMO:Manuka honey (1:1)) (85.0%). Also Sample 2 (GLMO Dual solvent extraction) (94.5%) and Sample 3 (GLMO oil (Bio-Mer)) (85.0%) were very effective at reducing the number of cells.

• Retinoic acid at 20 μΜ reduced the number of healthy cells by 66.2% but only reduced the psoriatic cells by 19.9%.

Elafin/SKALP

• This compound is the marker of the state of psoriasis. A product that is potentially beneficial would be expected to reduce the concentration of elafin/SKALP in the cultures grown in the growth medium.

• The majority of the samples were strong inhibitors of elafin/SKALP in the keratinocytes in the psoriasis environment. Almost complete inhibition was achieved with Sample 7 (GLMO:Manuka honey (1:1)) (89.8%), Sample 8 (GLMO:Manuka honey (0.5:1)) (91.2%) and Sample 9 (GLMO:Manuka honey (1:0.5)) (89.1%).

• In the basal medium, the keratinocytes produced much lower levels of elafin/SKALP as expected. It was nearly six times lower. With the exception of the two honey samples (Sample 5 (Manuka honey) and Sample 6 (Clover honey) all the other samples were very strong inhibitors of this low level of elafin/SKALP in normal cells. · Sample 2 (GLMO Dual solvent extraction) and Sample 3 (GLMO oil (Bio-Mer)) were also strong antagonists of elafin/SKALP production by the psoriatic keratinocytes. The reductions were 64.3% and 69.0% respectively.

• The honey samples had negligible effect on the elafin/SKALP production by the cells in the psoriasis medium. The Manuka honey (Sample 5) reduced it by 4.6% and the Clover honey (Sample 6) reduced it by 16.4% - both effects were insignificant.

• The normal healthy keratinocytes were stimulated by the honeys to produce more elafin/SKALP.

Sample 5 (Manuka honey) increased it by 60.9% and Sample 6 (Clover honey) by 42.6%.

Cytokeratin 10

• Cytokeratin 10 is a major product of keratinocytes and so is a marker of the metabolic activity of the cells.

• In the psoriatic keratinocytes, all of the test samples stimulated the production of cytokeratin 10. In the control cultures, the concentration was quite low but several samples stimulated it to the extent that its concentration was greater than the highest level of detection. These included Sample 1 (Lotion) Sample 10 (Lotion plus GLMOrManuka honey (1:1)) and Sample 11 (EgoPsoryl). · The least effect was produced by Sample 9 (GLMOrManuka honey (1:0.5)) (6.4%).

• In normal healthy keratinocytes, the production of cytokeratin 10 was also quite low. Several samples stimulated it, the strongest being Sample 4 (GLMO (Lyprinol)) (186.2%), Sample 2 (GLMO Dual solvent extraction) (113.1%), and Sample 5 (Manuka honey) (84.8%).

• Four of the test samples inhibited the synthesis of Cytokeratin 10 by normal keratinocytes. The strongest were Sample 7 (GLMOrManuka honey (1:1)) and Sample 9 (GLMO:Manuka honey (1:0.5)) which reduced it to below the detection level. Sample 3 (GLMO (Bio-Mer)) (75.4%) and Sample 8 (GLMO:Manuka honey (0.5:1)) (28.5%) were also significant inhibitors.

PGE₂

• One of the possible promoters of the inflammation associated with psoriasis is prostaglandin E₂ (PGE₂) which is a product of the COX-2 pathway. Inhibition of this enzyme implies an antiinflammatory effect.

• Surprisingly none of the test samples inhibited the production of PGE2 by the cells grown in the growth medium. In fact all stimulated with Sample 11 (EgoPsoryl) (725.3%) and Sample 9 (GLMO:Manuka honey (1:0.5)) (451.4%) being the strongest. • In the growth medium, the least stimulation was produced by Sample 5 (Manuka honey) (27.6%) and Sample 7 (GLMO:Manuka honey (1:1)) (31.8%).

• As anticipated the concentration of PGE₂ in the culture of normal healthy keratinocytes was negligible. Consequently the increases in the PGE₂ concentration resulting from inclusion of the test samples appear to be very high. In this medium all samples except Sample 7 (GLMO:Manuka honey) (1:1)) were stimulatory.

LTBq

• One of the possible promoters of the inflammation is leukotriene B₄ (LTB₄) which is a product of the lipoxygenase pathway. · The production of LTB₄ by the psoriatic keratinocytes is about 26 times higher than by normal healthy cells indicating the inflammatory state of psoriasis cells.

• Two of the samples were potent antagonists of LTB₄ synthesis. They were completely inhibitory in both the normal and the psoriatic cell cultures. They are Sample 3 (GLMO oil (Bio-Mer)) and Sample 8 (GLMO:Manuka honey (0.5:1)). · Three of the test samples significantly stimulated the production of LTB under the conditions of psoriasis. These were Sample 9 (GLMO:Manuka honey (1:0.5)) (256.6%), Sample 2 (GLMO Dual solvent extraction) (110.8%) and Sample 4 (GLMO Lyprinol) (94.2%).

• The healthy cells produced little LTB as expected. However Sample 3 (GLMO oil (Bio-Mer)) and Sample 8 (GLMO:Manuka honey (0.5:1)) appeared to completely inhibit the production. · Other samples stimulated the lipoxygenase activity under the basal conditions with the exception of Sample 5 (Manuka honey). The strongest stimulators were Sample 7 (GLMO:Manuka honey (1:1)), Sample 9 (GLMO:Manuka honey (1:0.5)) and Sample 2 (GLMO Dual solvent extraction). ii. Presentation of Results and Determinations of Statistical Significance

MTT

The effects of the test samples on the viability and proliferation of the human keratinocytes (KerTr cells) in the basal medium and in the growth and differentiation media are presented in Tables 3 and below and in Figures 10-15.

Table 3: The effects of the test samples on the proliferation of undifferentiated human keratinocytes.

Each absorbance is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean.

Table 4: The effects of the test samples on the proliferation of differentiated human keratinocytes. Each absorbance is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean. Elafin/SKALP

A summary of the effects of test samples on the production of elafin/SKALP by KerTr cells in both KBM and KGM is presented in Tables 5 and 6 below and Figures 16 to 20.

Table 5: The effect of the test samples on the production of elafin/SKALP by undifferentiated human keratinocytes. Each concentration (pg/ml) is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean.

Table 6: The effect of the test samples on the production of elafin/SKALP by differentiated human keratinocytes. Each concentration (pg/ml) is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean. Cytokeratin 10

A summary of the effects of test samples on the production of cytokeratin 10 by KerTr cells is presented in Tables 7 and 8 below and Figures 21-25.

Table 7: The effect of the test samples on the production of cytokeratin 10 by undifferentiated human keratinocytes. Each concentration (pg/ml) is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean.

Table 8: The effect of the test samples on the production of cytokeratin 10 by differentiated human keratinocytes. Each concentration (pg/ml) is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean.

PGE₂

A summary of the effects of test samples on the production of PGE₂ by KerTr cells is presented in Tables 9 and 10 below and Figures 26-30.

Table 9: The effect of the test samples on the production of PGE₂ by undifferentiated human keratinocytes. Each concentration (pg/ml) is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean.

Table 10: The effect of the test samples on the production of PGE₂ by differentiated human keratinocytes. Each concentration (pg/ml) is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean.

LTB,

A summary of the effects of test samples on the production of LTB4 by KerTr cells is presented in Tables 11 and 12 below and Figures 31-36.

Table 11: The effect of the test samples on the production of LTB₄ by undifferentiated human keratinocytes. Each concentration (pg/ml) is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean.

Table 12: The effect of the test samples on the production of LTB₄ by differentiated human keratinocytes. Each concentration (pg/ml) is the mean of triplicates. The percentage stimulation or inhibition of each sample relative to the negative control (cells only + HBSS) is presented. NS = Not significant; SEM = Standard error of the mean. iii) Discussion

Retinoic acid

Retinoic acid is included in this study as a positive control and is expected to have little effect on Cytokeratin production and to inhibit the production of Elafin/SKALP by the differentiated cells. When the level of cytokeratin 10 was measured in the supernatant from cells incubated with 20 μΜ retinoic acid in basal medium, there was a 68.1% decrease when compared to the cell control. This was statistically significant. However, as the growth of the keratinocytes was 66.2% less than untreated cells, there would appear to be very little change in cytokeratin 10 production. Likewise, when the keratinocytes in growth medium were incubated with retinoic acid, there was a retardation of cellular growth (19.9% inhibition). There was 17.8% stimulation in the production of cytokeratin 10 which was not significant statistically.

There was a statistically significant 94.2% inhibition of elastin/SKALP with retinoic acid at 20 μΜ but as the growth of keratinocytes was also inhibited the level of inhibition is probably not as high. 39.8% inhibition was seen with the keratinocytes in growth medium which was significant statistically. After taking into account the almost 20% inhibition of cell growth, the inhibition of elafin production is likely to be less.

Retinoic acid led to a 1465.8% increase in the inflammatory marker PGE₂ produced by keratinocytes in basal medium. This was one of the lowest levels of stimulation but was still a large increase. There was also stimulation of PGE₂ in the culture of keratinocytes in growth medium with a 115.1% increase which was much less than in the basal medium. As with cytokeratin 10, retinoic acid led to a stimulation of lipoxygenase activity by the normal keratinocytes. There was a non-significant 233.2% increase in the amount of LTB₄ present. Although there was also stimulatory effect on psoriatic keratinocytes, this was less at 57.7%.

Sample 1 Lotion (control)

With both the basal medium (KBM) and supplemented medium (KGM) there was a small stimulation of keratinocyte growth when Sample 1 was included in the medium. Although this was statistically significant for both, the level of it means that Sample 1 has negligible effect on cell growth.

When cytokeratin 10 levels were measured for cells cultured with Sample 1 in both medium types, the amount detected was greater than the level of detection of the assay kit. This suggests that Sample 1 has a significant stimulatory effect on the metabolic activity of the keratinocytes in both psoriatic and normal environments although it did not significantly affect the cell viability.

The level of elafin/SKALP in the supernatant was able to be detected in both medium types. There was a statistically significant 85.5% decrease in the basal medium and 39.3% in KGM.

From these results it looks like Sample 1 slightly increases cell proliferation whilst decreasing elafin. However, this was more inhibitory in KBM and the levels of cytokeratin 10 appear to be elevated. As expected the elafin/SKALP concentration was much higher in the cells cultured in the growth medium (about 5.9 times) than in basal medium. This is because it is the marker of psoriasis. So the more important result is the effect on the concentration in the growth medium. In this medium Sample 1 reduced it by 39.8% while in the basal medium the already low level was reduced by 94.2% to a barely detectable level.

Sample 1 (Lotion) induced the normal keratinocytes to produce 40 times more PGE₂ than in the control culture of cells with HBSS. This was statistically significant. However it should be noted that in the basal medium the inflammatory level would be expected to be quite low. The concentration of PGE₂ is barely detectable (0.48 pg/ml). As with retinoic acid, there was also PGE₂ stimulation by the psoriatic keratinocytes but to a much lower level than in the "normal" conditions. This effect of Sample 1 is somewhat unexpected on these cells although it is of the same order as that produced by 20 μΜ retinoic acid.

When the levels of LTB₄ were measured, the effect of Sample 1 was similar for both healthy and psoriatic keratinocytes. For both conditions, there was an approximately 15% increase in LTE production. However this was not significant statistically. This would indicate that Sample 1 has very little effect on the production of LTB₄ by both normal and psoriatic keratinocytes. Sample 2 GLMO (Dual Solvent Extraction)

Sample 2 had very little effect on the growth of keratinocytes in basal medium - there was an insignificant 1.5% increase in cells compared to the control. In contrast, there was almost complete inhibition of cell viability with Sample 2 in the growth medium (94.5% decrease) which indicates that it is strongly cytotoxic for the psoriatic keratinocytes specifically but has no effect on normal keratinocytes.

In both types of medium, there was a stimulation of cytokeratin 10 when this sample was present - 113.1% increase in KBM and 1085.2% increase in KGM. This is not easily explained. This suggests that the metabolic activity of the normal keratinocytes is significantly stimulated by Sample 2 and is even more strongly increased in the psoriasis cells. This latter is the more unusual considering the cytotoxic effect of this sample on these cells.

Elafin/SKALP decreased in the presence of Sample 2. For the unsupplemented cells this was a significant 98.1% decrease and for the supplemented cells it was 64.3% inhibition. However when the 94.5% decrease in cells is taken into account for the KGM cells, it would seem that this effect on psoriasis by Sample 2 is largely due to the killing of the cells by the sample. Sample 2 increased PGE₂ production when it was included with the unsupplemented cells.

Although the percentage increase appears to be exceptionally high, this is a distortion because the reference concentration of PGE₂ (cells only) is virtually undetectable. This effect was a 205.6% stimulation when the cells were supplemented with the growth medium. This is not indicative of an anti-inflammatory effect. PGE2 and the COX-2 enzyme are not expected to be a major source of inflammation in psoriasis. Nor are keratinocytes classified as having significant COX-2 activity.

Under both cell conditions, the level of LTB₄ produced by the cells incubated with Sample 2 increased considerably. With the normal keratinocytes, there appeared to be a very large increase in lipoxygenase activity when Sample 2 was included in the culture. Even though the basal concentration was quite low, this stimulation of inflammation is quite unexpected and would need further investigation in order to understand it. Under psoriatic conditions, Sample 2 increased the level of LTE by a statistically significant 110.8%. This was the second highest stimulation seen with psoriatic cells and also should be investigated further in order to understand.

Sample 3 GLMO oil (Bio-Mer)

When the keratinocytes were incubated with Sample 3 there was a decrease in the cell viability. For the cells under "normal" conditions, this was an inhibition of 48.3%. However this was insignificant statistically. With the supplemented medium there was a statistically significant 85.0% decrease in cell viability. So it would seem that Sample 3 is very strongly toxic for the psoriatic cells specifically.

The level of cytokeratin 10 present in the medium of unsupplemented cells incubated with Sample 3 was 75.4% less than what was in the medium of the cell control. This reduction in the cytokeratin 10 concentration is only slightly greater than the loss of cells suggesting that much of the reduction is a consequence of the decrease in cells. However, in the supplemented medium there was an 81.7% increase in cytokeratin detected. This was unexpected as the viability of the keratinocytes was significantly inhibited in the presence of Sample 3. An implication of these observations is that the surviving cells have become much more metabolically active as a consequence of the inclusion of

Sample 3 in the medium.

For both medium types, when the keratinocytes were incubated with Sample 3 there was a statistically significant decrease in the production of elafin/SKALP. For the cells in basal medium there was an almost complete inhibition of elafin/SKALP (92.6%) and for those in the growth medium the elafin decreased by 67.0%. The effects of this sample on elafin/SKALP production by both the normal and the psoriatic keratinocytes are very similar to that observed for Sample 2. That is, they both reduced the production of the psoriatic features largely by lowering the cell number. There was a very strong stimulation of PGE₂ production by the cells grown in basal medium with

Sample 3 present when compared to the cell control (76.1 pg/ml compared with 0.462 pg/ml). It is noted again that the PGE₂ concentration in the control cells was almost undetectable which results in the percentage increase in the presence of Sample 3 being very large. Although there were also raised PGE2 levels in the supernatant of cells grown in growth medium with this sample, this was a 155.3% increase (94.5 pg/ml PGE₂). This increase was rather similar to that recorded when Sample 2 was added to these cells. Sample 3 was one of two samples that were potent LTB₄ antagonists. There was no LTB₄ detected in either the basal medium or in the growth medium of keratinocytes incubated with Sample 3. This means that, unlike most of the other GLMO containing samples, Sample 3 is a potent inhibitor of lipoxygenase activity.

Sample 4 GLMO oil (Lyprinol) When the keratinocytes and Sample 4 were incubated together in KBM there was a 75.8% decrease in cell viability. In contrast, the cells with Sample 4 in KGM were slightly stimulated (12.8% increase). This was statistically significant. With the psoriatic cells, all the other GLMO containing test samples were significant inhibitors of their viability and would seem to be the possible reason for much of GLMO's effect as an anti-psoriatic agent. However Sample 4 would appear to be an exception and is not toxic although it did reduce the cell concentration in the normal cells.

There was an unexpected 186.2% increase (statistically significant) in cytokeratin 10 in the

unsupplemented cell supernatant. An increase in cytokeratin 10 levels was also evident in the supernatant from the supplemented cells (70.5% stimulation) but was not significant statistically. Sample 4 would seem to be a stimulator of the metabolism of keratinocytes under normal and psoriasis conditions. This is particularly so for the healthy cells as their concentration was significantly reduced by this sample.

Elafin/SKALP levels decreased in both conditions. As with the cells incubated with Sample 3, there was almost complete inhibition in the cells incubated with Sample 4 grown in KBM (95.0%). The level of this mark of psoriasis is low in this culture as expected and so the strong inhibition is not highly relevant clinically. Under psoriatic conditions, the decrease in the amount of elafin was a statistically significant 29.0%. As there was an increase in cell viability with KGM, this suggests Sample 4 has the ability to inhibit the production of elafin/SKALP. Therefore this sample does have an effect on the psoriasis although it is not particularly strong especially as there is an increase in the cell concentration. This would suggest that this product, unlike other examples tested, does not exert its effect by killing the psoriatic cells.

Sample 4 caused one of the greatest increases in PGE₂ production by the cells in non-psoriatic conditions. This stimulation was similar to what was seen for both Sample 5 and Sample 6. This stimulation was on cells that normally have almost undetectable levels of this inflammatory compound. There was a statistically significant 315.7% stimulation by the psoriatic cells incubated with this sample. Interestingly, this was the same level as that seen for Sample 10.

There was a large increase in LTB₄ produced by the healthy keratinocytes when Sample 4 was tested which was significant statistically. This means that Sample 4 (Lyprinol) is a stimulator of the lipoxygenase pathway when added to normal keratinocytes. This contrasts with the inhibitory effect of Sample 3. Likewise, the psoriatic keratinocytes were stimulated to produce more LTB₄ by this sample (94.3% increase) which also is the opposite to the effect of Sample 3.

Sample 5 Manuka honey (UMF15) There was an insignificant 5.9% increase in the proliferation of keratinocytes in KBM incubated with Sample 5. With KGM there was an apparent 8.2% decrease in cell viability. These are both negligible indicating that manuka honey does not affect either normal or psoriatic keratinocytes.

When cytokeratin 10 was measured, there was a statistically significant increase for both medium types when Sample 5 (Manuka honey) was included in the culture media. There was an 84.9% increase when compared to keratinocytes incubated on their own in basal medium which was almost double the increase seen for cells incubated with Sample 5 in KGM. As the cell viability did not change noticeably when compared to the cell control, it appears Sample 5 stimulated the production of cytokeratin 10, a marker of the cell's metabolic activity.

There was also an increase in the amount of elafin/SKALP present in the KBM culture (60.9%). However there was little change in the culture of supplemented cells with Sample 5 (an insignificant

4.6% reduction). So the deduction is that Sample 5 has no effect on the psoriasis. The increase observed with normal keratinocytes reaches a level that is only 27.4% of the concentration in unsupplemented psoriatic keratinocytes.

As with Sample 4, healthy cells incubated with Sample 5 stimulated the production of the inflammatory marker PGE2 significantly. Although there was also stimulation for the psoriatic cells, this was a 27.6% increase which was the lowest seen for the samples alongside that observed with Sample 7. Therefore, compared with other test samples, Sample 5 had the lowest stimulation on the COX-2 enzyme in the diseased keratinocytes.

Sample 5 was one of three samples to inhibit the production of LTB₄ by keratinocytes grown in basal medium. Whilst Sample 3 and Sample 8 completely inhibited the production of this inflammatory marker, Sample 5 caused a 38.6% decrease which was not significant statistically. In the growth medium, there was a small increase in LTB₄ (16.6%). However this was also not significant. Sample 6 Clover honey

Sample 6 stimulated the proliferation of keratinocytes in KBM by 34.6%. It was slightly inhibitory for cells grown in KGM with a 12.5% decrease evident. Neither of these effects can be considered as significant. In both medium conditions, the presence of Sample 6 increased cytokeratin 10 levels. There was a 28.2% increase for the cells incubated with basal medium and a 35.1% increase in the growth medium. However, as the number of keratinocytes increased in the basal medium, there was little change in cytokeratin 10 levels when this is taken into account. In the growth medium, there was an apparent relatively minor stimulation of the keratinocytes. There was almost a 50% increase in the level of elafin detected in the basal medium of keratinocytes incubated with Sample 6 although this was largely offset by the 34.6% increase in cell proliferation thus indicating it had relatively little effect on this marker. Likewise the non-significant 16.4% decrease in elafin/SKALP production by the supplemented cells was mirrored by a 12.5% decrease in cell viability indicating its ineffectiveness on the features of psoriasis. The other sample to show a large stimulation of PGE₂ by the normal cells was Sample 6. Once again this stimulation is on cells that were showing negligible COX-2 activity. However there was 139.2% increase in the psoriatic cell supernatant which was a similar increase as that seen for Sample 3. The importance of this in relation to modulating inflammation in psoriasis is questionable.

Sample 6 stimulated LTB₄ production by normal keratinocytes by 131.1%. In contrast, there was very little change in LTB₄ production when the psoriatic cells incubated with this sample were compared with the cell control. It seems that in these cells the lipoxygenase activity is unaffected by this sample.

Sample 7 GLMO:Manuka honey (1:1)

Incubating keratinocytes with Sample 7 had different outcomes when basal and growth media were used. When compared to keratinocytes on their own, there was a significant 97.3% increase in cell viability when Sample 7 was added to the cells in basal medium. In contrast, there was a statistically significant 85.0% decrease in the cell proliferation in the growth medium. This combination of GLMO and Manuka honey has quite different effects on normal and psoriasis cells. It promotes the proliferation of normal keratinocytes but is strongly cytotoxic for those that are characteristic of psoriasis. This is very similar to the effect of Sample 2 which is the GLMO alone and reduced the cell concentration by 94.5%.

There was the strongest inhibition of cytokeratin 10 production by undifferentiated keratinocytes when Sample 7 was included in the culture. There was 81.6% reduction which was the same level of inhibition as that seen with Sample 9. In contrast, the cytokeratin 10 levels were increased by 54.15% with differentiated cells. This was statistically significant. The deduction from this is that this combination of GLMO and Manuka honey in equal proportions activates the metabolic activity of the psoriasis cells. This is despite the strong killing of these cells by the sample. Consequently the surviving cells have become very highly active.

There was almost complete inhibition of elafin/SKALP production by undifferentiated cells with this sample (statistically significant 96.5% inhibition). A similar level of inhibition was seen in the differentiated cell culture (89.8%). This was one of the strongest inhibitors of elafin/SKALP in these conditions. The production of this marker is relatively low in normal keratinocytes and so the consequence of this reduction is clinically of minor importance. However with the diseased cells, this combination is highly effective as an anti-psoriatic agent. It seems that this combination achieves its effect by killing these cells.

Sample 7 was the only sample to have an inhibitory effect on PGE₂ production. It was a complete inhibitor with no PGE₂ detected in the supernatant of the healthy cells. There was a stimulation with the psoriatic cells but this was low (31.8%) and statistically insignificant. This was similar to the effect seen with Sample 5. It would seem that this combination does have some degree of anti-inflammatory effect through the COX-2 pathway but as discussed above, the relevance of this to the inflammation associated with psoriasis is uncertain. Sample 7 was the strongest stimulator of lipoxygenase activity. There was a highly significant increase in level of LTB₄ in the normal keratinocytes from 123.7 pg/ml to 5510.0 pg/ml. With the psoriatic keratinocytes this was an 89.2% increase but was not significant statistically.

Sample 8 GLMO:Manuka honey (0.5:1)

When compared to the cell culture control (cells + HBSS), there was a significant 170.2% increase in the proliferation of the keratinocytes in basal medium with the addition of Sample 8. This was the strongest level of stimulation out of all the samples tested. However, for the cells in supplemented medium the complete opposite effect was seen. There was 91.4% inhibition of cell viability. So, as with Sample 8 which is also a mix of GLMO and Manuka honey, Sample 9 had a proliferative effect on normal keratinocytes but was highly toxic for the cells that were psoriatic. The level of effect was similar to Sample 8 which has a high proportion of Manuka honey and also to

Sample 2 which has no honey in it.

For both the undifferentiated and differentiated cells there was an inhibition of cytokeratin 10 when Sample 8 was present. This reduction was 28.5% in the basal medium and 61.6% for the growth medium conditions. In both cases, these results were statistically significant. The reduction in cytokeratin 10 production indicates an elevated metabolic activity by the remaining viable ones. With the diseased cells, the drop in this activity parallels the decrease in the cell concentration. The level of elafin/SKALP produced by the keratinocytes was similar in both undifferentiated and differentiated cells with the addition of Sample 8. That is, there was 96.3% inhibition and 91.2% respectively. This changing of the ratio of GLMO to Manuka honey has no effect on the anti-psoriasis activity. That the effects of both Sample 7 and Sample 8 (and also Sample 9 (see below)) are virtually identical and are approaching full inhibition would suggest that the concentrations of these samples used in the test should be reduced in order to discriminate their relative activities. They would seem to be more potent as antagonists of psoriasis than anticipated when the study was designed.

PGE2 was increased by 980.9% when Sample 8 was incubated with the keratinocytes grown under normal conditions. This decreased to a 239.2% stimulation under psoriatic conditions with 125.6 pg/ml of PGE₂ detected. As discussed with other samples above, the clinical relevance of this is debatable.

Sample 8 behaved in the same way as Sample 3 in terms of its effect on LTB₄ production. Under both cell conditions, there was complete inhibition of this marker of lipoxygenase activity.

Sample 9 GLMO:Manuka honey (1:0.5)

Sample 9 was another sample that increased proliferation of undifferentiated keratinocytes. This was almost 50% more than the cell control. This was not significant statistically. There was a 90.3% inhibition of the differentiated cells which was also very similar to that seen with a number of samples. This sample which contained a mix of GLMO and Manuka honey, had an almost identical effect on keratinocyte viability and proliferation as did the other two which were mixes (Sample 7 and Sample 8).

There was a significant 81.6% decrease in the amount of cytokeratin 10 detected in the culture of undifferentiated keratinocytes with Sample 9. A different result was seen with the differentiated keratinocytes; there was very little difference between the cell control and cells with this sample (an insignificant 6.5% increase). This implies again that the general cell activity was significantly stimulated in the surviving cells.

As with Sample 8, there was very little difference in the level of inhibition of elafin/SKALP production. For the undifferentiated cells there was a statistically significant 97.1% decrease in the production of this protein and for the differentiated cells this was 89.1%. So the comments made about the anti-psoriatic activity of Sample 7 and Sample 8 are applicable to this sample. All of these mixtures are strong potent inhibitors of the psoriatic keratinocytes primarily through killing of the diseased cells. In order to determine the relative efficacies of the different ratios of GLMO and Manuka honey, lower concentrations should be tested. The results for Sample 10 (see below) indicate that this could be a worthwhile approach. The highest level of stimulation of PGE₂ production by psoriatic cells was seen with Sample 9.

There was a statistically significant 451.4% increase when compared to control cells. The importance of this stimulation is doubtful as the majority of the inflammation associated with psoriasis originates from invasive cells such as dendritic cells and neutrophils rather than from the resident keratinocytes. As well, inflammation produced by the activity of COX-2 appears to be of minor importance in psoriasis. Sample 9 stimulated lipoxygenase activity in the healthy keratinocytes to an almost identical level as that seen with Sample 2. It also significantly stimulated the production of LTB₄ under psoriatic conditions with 256.6% stimulation. This was the greatest stimulation by any of the samples. Similar comments to those made for the other samples apply here also.

Sample 10 Lotion including GLMO (dual solvent extraction):Manuka Honey (1:1) Sample 10 includes GLMO (dual solvent extraction):Manuka Honey (1:1) at a 50-fold dilution to

Sample 7.

As was seen with a number of other samples, there was a decrease in the viability of undifferentiated cells with incubation with Sample 10. This was a 74.3% decrease and was highly significant. For the differentiated cells there was very little change in the viability. That is, there was 2.6% stimulation but was not statistically significant. This lack of effect on the survival of the psoriatic cells contrasts with the 85% decrease observed with Sample 7. This difference may be explained by Sample 7 being 50 times more concentrated.

For both cell conditions, the amount of cytokeratin 10 produced when the cells were incubated with Sample 10 was greater than the detection capability of the assay kit. There was more than 170 pg/ml of cytokeratin 10 produced. Because of this, it is not possible to determine whether there was a difference in the amount produced in the two conditions. However, the reduced viability of the cells in the basal medium containing this sample means that this sample is a very strong activator of the cells that remain. In the psoriasis conditions, however, this increase in cellular activity occurs without any change in the level of cell survival. The production of elafin/SKALP was almost completely inhibited by Sample 10 for the keratinocytes grown in basal medium. There was a statistically significant 96.2% inhibition. In the growth medium, there was still inhibition however this was slightly less at 35.2%. Bearing in mind that Sample 10 is a 50 fold dilution of Sample 7, this level of inhibition is less than the 89.8% for the latter. This difference may be readily explained by the difference in concentration. As well, because the cell concentration is unchanged, the mechanism of anti-psoriasis may not be the same as for Sample 7 which appeared to be primarily due to specific cytotoxic activity. Sample 10 had a stimulatory effect on the production of PGE₂ by healthy keratinocytes with a statistically significant increase observed. An increase in PGE₂ was also seen with the psoriatic keratinocytes (315.5% stimulation) which was also significant statistically. This was the same level of stimulation as seen for Sample 4. In the basal medium, this stimulatory effect should be compared with the complete inhibitory effect of the higher concentration (Sample 7) on the COX-2 activity. In psoriasis, the high concentration stimulated the COX-2 by an insignificant 31.8% but at a 50 times lower concentration this stimulation was much higher (315.5%).

Although the production of LTE was increased in the psoriatic cells when Sample 10 was tested (55.5% increase) this was ten times less than the level of stimulation seen under normal healthy conditions. This was a significant 555.7% stimulation. The comments made above regarding the lipoxygenase pathway are applicable here.

Sample 11 EgoPsoryl

Sample 11 was one of four samples that inhibited keratinocytes viability in the basal medium. This was a statistically significant 37.9% decrease in viability. The psoriatic cells were also inhibited but this was minimal. There was a 4.8% inhibitory effect which was not significant. This effect on the psoriatic keratinocytes contrasts with the effects of the GLMO based samples. This would suggest that the mechanism of action on psoriasis is different for this product.

Sample 11 had a similar effect on the production of cytokeratin 10 as Sample 10. In both medium conditions the level of cytokeratin 10 present was higher than the level of detection of the kit. There did appear to be higher levels of this protein in the growth medium than in the basal medium but as these were so high, it is not possible to conclusively say this is significant. Therefore it would seem that Sample 11 stimulates the biochemical activity of both normal and diseased keratinocytes.

When elafin/SKALP was measured, there appeared to be almost complete inhibition of its production under normal conditions (93.4% decrease compared to the control). This was the result seen with most of the samples tested in this experiment. There was a decrease in elafin production by the psoriatic cells with Sample 11, which was what was seen for all the other samples. However this was one of the smaller levels of inhibition at 23.8% and so would seem to be a weaker antagonist than the GLMO samples. The mechanism of inhibition with this sample is different from the others as the cell viability was retained in the presence of it.

The highest level of PGE₂ stimulation with the psoriatic cells was seen with Sample 11. There was a 725.3% increase which was significant statistically. It also was a very strong stimulator of COX-2 in the normal cells. Sample 11 stimulated the production of LTB₄ by keratinocytes grown in basal medium by almost 50%. There was also stimulation observed by the cells grown in growth medium however this was not as high (29.1% increase).

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

Throughout this specification, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

Claims

Claims:

1. A pharmaceutical composition including:

a) an extract of green lipped mussel; and

b) honey.

2. The pharmaceutical composition according to claim 1 wherein the extract of green lipped mussel is green lipped mussel oil.

3. The pharmaceutical composition according to claim 2 wherein the green lipped mussel oil is obtained from green lipped mussels by the use of a fractionation process that retains the phospholipid component in the oil.

4. The pharmaceutical composition according to claim 3 wherein the green lipped mussel oil is obtained from green lipped mussels by a dual-solvent fractionation process.

5. The pharmaceutical composition according to any one of claims 1 to 4 wherein the honey is honey derived from:

a) trees of the genus Leptospermum;

b) trees of the genus Kunzea; and/or

c) clover.

6. The pharmaceutical composition according to any one of claims 1 to 5 wherein the honey is manuka honey.

7. The pharmaceutical composition according to any one of claims 1 to 6 wherein the ratio of the extract of green lipped mussel and honey is between 10:1 and 1:10.

8. The pharmaceutical composition according to any one of claims 1 to 7 wherein the ratio of the extract of green lipped mussel and honey is between 5:1 and 1:5.

9. The pharmaceutical composition according to any one of claims 1 to 8 wherein the ratio of the extract of green lipped mussel and honey is from 1:2 to 2:1.

10. The pharmaceutical composition according to any one of claims 1 to 9 wherein the ratio of the extract of green lipped mussel and honey is from 0.5:1 to 1:0.5.

11. The pharmaceutical composition according to any one of claims 1 to 10 formulated for topical application.

12. The pharmaceutical composition according to any one of claims 1 to 11 formulated as a gel.

13. The pharmaceutical composition according to any one of claims 1 to 12 wherein the extract of green lipped mussel is present between 0.1% wt/wt and 2% wt/wt.

14. The pharmaceutical composition according to any one of claims 1 to 13 wherein the extract of green lipped mussel is present between 0.5% wt/wt and 1.0% wt/wt.

15. The pharmaceutical composition according to any one of claims 1 to 12 wherein the honey is present between 0.1% wt/wt and 2% wt/wt.

16. The pharmaceutical composition according to any one of claims 1 to 13 wherein the honey is present between 0.5% wt/wt and 1.0% wt/wt.

17. A method of preventing or treating a disease, the method including the step of administering a therapeutically effective amount of a pharmaceutical composition including an extract of green lipped mussel and honey to a subject in need thereof.

18. The method according to claim 17 wherein the pharmaceutical composition is as defined in any one of claims 2 to 16.

19. Use of an extract of green lipped mussel and honey in the preparation of a medicament for the prevention or treatment of a disease or condition.

20. Use according to claim 19 wherein the medicament is a pharmaceutical composition as defined in any one of claims 1 to 16.

21. A pharmaceutical combination including:

a) an extract of green lipped mussel; and

b) honey;

for separate, sequential or simultaneous administration.

22. The combination according to claim 21 further including instructions for use in the prevention or treatment of a disease.

23. Method of stimulating non-psoriatic keratinocytes including the step of administering to the non-psoriatic keratinocytes:

a) an extract of green lipped mussel; and

b) honey.

24. Use of:

a) an extract of green lipped mussel; and

b) honey to stimulate non-psoriatic keratinocytes.

25. Method of inhibiting psoriatic keratinocytes including the step of administering to the non- psoriatic keratinocytes:

a) an extract of green lipped mussel; and

b) honey.

26. Use of:

a) an extract of green lipped mussel; and

b) honey

to inhibit psoriatic keratinocytes.

27. Method of selectively inhibiting psoriatic keratinocytes over non-psoriatic keratinocytes including the step of administering to the psoriatic keratinocytes and non-psoriatic keratinocytes: a) an extract of green lipped mussel; and

b) honey.

28. Use of:

a) an extract of green lipped mussel; and

b) honey

29. Method according to claim 27 or use according claim 28 wherein the selectivity provided is greater than the selectivity provided by the action of retinoic acid on psoriatic keratinocytes and non- psoriatic keratinocytes.

30. Method of inhibiting the formation of Elafin/SKALP in psoriatic keratinocytes including the step of administering to the psoriatic keratinocytes:

a) an extract of green lipped mussel; and

b) honey.

31. Use of:

a) an extract of green lipped mussel; and

b) honey

to inhibit the formation of Elafin/SKALP in psoriatic keratinocytes.

32. Method according to any one of claims 23, 25, 27, 29 or 30 wherein the extract of green lipped mussel and the honey are administered simultaneously in combination.

33. Use according to any one of claims 24, 26, 28 or 31 wherein the extract of green lipped mussel and the honey are prepared in a medicament.