WO2006067379A1

WO2006067379A1 - Use of essential fatty acid molecules

Info

Publication number: WO2006067379A1
Application number: PCT/GB2005/004799
Authority: WO
Inventors: Reto Muggli
Original assignee: Efamol Ltd
Current assignee: Efamol Ltd
Priority date: 2004-12-23
Filing date: 2005-12-20
Publication date: 2006-06-29
Anticipated expiration: 2007-06-23
Also published as: GB0428166D0

Abstract

The invention provides compositions containing essential fatty acid molecules, in particular, gamma-linolenic acid (GLA) and dihomo-gamma-linolenic acid (DGLA), for use in improving the condition and appearance of skin. The invention further provides use of gamma-linolenic acid (GLA) or a functional derivative thereof for the manufacture of a medicament or a nutraceutical for rejuvenating the skin.

Description

USE OF ESSENTIAL FATTY ACID MOLECULES

The present invention relates to compositions containing essential fatty acid molecules, in particular, gamma-linolenic acid (GLA) and dihomo-gamma-linolenic acid (DGLA), for use in improving the condition and appearance of skin.

The condition and general appearance of human skin is important to both sexes. For example, the smoothness of human skin is widely regarded as a desirable characteristic, particularly in women, hi addition, it is a desirable characteristic for many people to have improved physical properties of skin, such as for example, firmness, elasticity and fatigue strength, which govern the overall condition and appearance of the skin, and therefore play a role in the production of wrinkles, and accordingly, the visible aging of the skin. In addition, the softness, suppleness, fine lines, signs of fatigue, and complexion of the skin, are also determined by the physical properties of the skin.

Hence, it is an aim of embodiments of the present invention to address the problems with the prior art, and to provide a composition for use in improving the physical properties of skin.

According to a first aspect of the present invention, there is provided use of gamma-linolenic acid (GLA) or a functional derivative thereof for the manufacture of a medicament or a nutraceutical for rejuvenating the skin.

According to a second aspect of the present invention, there is provided a method of treating a subject, the method comprising administering to a subject in need of such treatment, a composition comprising gamma-linolenic acid (GLA) or a functional derivative thereof, wherein the composition is for rejuvenating the skin.

The inventors conducted a series of experiments on test subjects, in order to investigate the effect of the essential fatty acid, GLA (18:3, n-6), on a number of different parameters of skin, with a view to investigating it's effect on the development of wrinkles in skin, and ultimately on aging. The parameters that were investigated in this study included skin roughness, moisture, redness, Trans Epidermal Water Loss (TEWL), firmness, elasticity, and fatigue strength. One set of test subjects had normal healthy skin, and a second set of subjects had experimentally irritated skin. The inventors observed that GLA had little or no effect on skin rednesss over the course of the experiments. However, following analysis of their results, to their surprise, administration of GLA considerably and significantly improved the firmness, elasticity, and also fatigue strength of the skin in each of the subjects tested.

It will be appreciated that the overall health, structure and therefore appearance of the skin including the softness, suppleness, signs of fatigue, complexion, and development of wrinkles, and therefore aging of the skin, are governed by it's physical or biomechanical properties. As the biomechanical properties change over the course of time, they cause the skin to degenerate, which in turn causes wrinkles and ultimately, aging in the skin. For example, the firmness, elasticity and fatigue strength of the skin each play a role in the speed of development, and also extent (size, depth, length) of wrinkles and lines in the skin. It will be appreciated that wrinkles are more likely to develop if the skin is less firm, or less elastic, or has a lower fatigue strength. Accordingly, as more wrinkles form in the skin, and as the extent of the wrinkles worsens, then the skin becomes more visibly aged.

Accordingly, it is especially preferred that the medicament or nutraceutical according to the invention is adapted to improve the physical or biomechanical properties of the skin, i.e. it is used to treat skin in which the physical or biomechanical properties of skin have substantially deteriorated. By the term

"biomechanical properties of skin", we mean the firmness of the skin, the elasticity of the skin and/or the fatigue strength of the skin. By the term "nutraceutical", we mean a food or dietary substance that is nutritionally enhanced or supplemented with nutrients, vitamins, or herbal supplements, for example, GLA or a functional derivative thereof. The medicament or nutraceutical may improve the physical or biomechanical properties of the skin, by reversing the course of age-related physical properties of skin.

It is preferred that the medicament or nutraceutical increases the firmness of the skin. It is also preferred that the medicament or nutraceutical increases the elasticity of the skin. It is also preferred that the medicament or nutraceutical increases the fatigue strength of the skin. Advantageously, as the medicament treats these biomechanical properties of the skin, it results in delaying the onset, and minimising the extent of wrinkling in the skin. Because wrinkling is a visible indicator that the skin is ageing, caused because the biomechanical properties of the skin are deteriorating, improving such properties may be used to delay the visible aspect of aging of the skin. In addition, the medicament or nutraceutical may also be used to improve the softness, suppleness, fine lines, signs of fatigue, and complexion of the skin, each of which are governed by the firmness, elasticity and fatigue strength thereof.

The medicament or nutraceutical may be used under the supervision of a physician. Alternatively, or additionally, the medicament or nutraceutical may be a cosmetic composition, which may be used and administered at home by individuals wishing to treat themselves.

Each of the biomechanical properties of the skin (i.e. skin firmness, elasticity and fatigue strength) may be assessed using a Cutometer, and measurements may be based on the vacuum-suction principle, which is well known to the skilled technician (A. O. Barel, W. Courage, P. Clarys, "Suction Method for Measurement of Skin Mechanical Properties: The Cutometer ®", Handbook of Non-Invasive Methods and the Skin, J. Serup and G.B.E. Jemec, eds. CRC Press, Ann Arbor, (1995).

For example, by applying a constant negative pressure for a given time period, skin may be drawn into a hollow tube with an orifice of, for example, 2 mm in diameter. Then, at normal air pressure, the skin may be allowed to retract. The penetration depth of the skin into the tube may be recorded optically without friction, and without mechanical influence. A number of standardised parameters may be calculated from the resulting penetration depth curve.

Most of the parameters are a function of skin thickness and thus cannot be simply compared between subjects and regions. Therefore, in order to increase the accuracy of the measurements taken, and to capture information on the properties of skin under repeated external stress, it is preferred that the cycle may be repeated several times and parameters selected for evaluation are based on areas rather than individual measurement points. The delineation of the areas is based on the fitted logarithmic envelope curves of the minimum and maximum extensions as described in Example 4, and as illustrated in Figure 5, where parameter F2 is equivalent to the area of the graph above the maximum envelope; parameter F3 is equivalent to the area of the graph between the maximum and minimum envelopes; and parameter F4 is equivalent to the area of the graph below the maximum envelope.

In Example 4 the suction-release cycle is repeated consecutively 20 times in cycles of 1 second suction (450 mbar) and 1 second retraction. The delineation of the areas is based on envelope curves through the minimum and maximum amplitudes of the 20 cycles and fitting the equation: y = (In x + b)/ a (x = time , y = maximum or minimum amplitudes).

Three area parameters were selected to assess changes in the biomechanical properties of the skin:- 1) skin firmness (defined by the area below the maximum envelope curve (F4); 2) skin elasticity (defined by the ratio F3/F4); and 3) skin fatigue strength (defined by F2). The calculation of these three parameters was conducted by

WinCT (Courage & Khazaka GmbH, Cologne, Germany)

It will be appreciated that skin firmness may be assessed by determining the value of the parameter, F4, i.e. the area below the approximated envelope function of the maximum extensions. Accordingly, a decrease in the F4 parameter corresponds to an increase in skin firmness, which is a preferred feature of the medicament. Skin firmness is defined as the property of the skin to resist shape change under stress. It will be appreciated that skin elasticity may be assessed by determining the value of the ratio of parameters F3/F4. The larger F3 in comparison to F4, the larger are the restoring forces and the smaller is the remaining residual deformation. Hence, the closer the resulting value is to 1, then the more elastic the skin, which is a preferred feature of the medicament. Skin elasticity is defined as the property of the skin to return to normal after being stretched or compressed.

It will also be appreciated that skin fatigue strength may be assessed by determining the value of parameter, F2. F2 is the area between the final extension and the approximated envelope function of the maximum extensions. Hence, a decrease in F2 corresponds to an increase in fatigue resistance, which is also a preferred feature of the medicament. Skin fatigue strength is defined as the property of skin to resist structural change that occurs when skin is subjected to fluctuating stress and strain.

Therefore, preferably the medicament or nutraceutical is adapted to increase the firmness of the skin, and preferably, the elasticity of the skin. Preferably, the medicament or nutraceutical is adapted to increase the fatigue strength of the skin.

The two common series of essential fatty acids (EFAs) are: (i) the n-6 series (or omega-6 fatty acids), which are metabolites of linoleic acid, and (ii) the n-3 series, which are metabolites of alpha-linolenic acid. Essential fatty acids (EFAs) of the linoleic acid series are known to be required for normal skin structure and function.

When they are eliminated from the diet, the skin becomes structurally and functionally abnormal in many ways. The parent EFA, linoleic acid (LA), in order to fulfill its full biological functions in the skin, must in part be metabolised within the body for example to GLA, or dihomo-gamma-linolenic acid (DGLA). The conversion of linoleic acid to GLA, cannot take place in the skin, whereas the conversion of GLA to DGLA can occur in the skin. Therefore, the formation of GLA from linoleic acid

(LA), must take place in other body tissues, notably the liver, from -which the required fatty acids are transported to the skin.

The conversion of linoleic acid to GLA is a slow step. It can be further inhibited by a variety of factors including diabetes, aging, zinc deficiency, excess alcohol consumption, high levels of cholesterol, certain viral infections and catecholamines released during stress. Furthermore, people who have inherited an atopic disposition (atopies), which makes them liable to develop eczema, asthma, allergic rhinitis or other allergies, are less able to convert linoleic acid to GLA than other people. Thus, insufficient amounts of GLA may be produced by the body even in the presence of adequate LA. The skin is particularly sensitive to sub-optimal GLA formation as it lacks the enzyme, which transforms LA to GLA, which may have profound effects on skin structural and functional properties. The skin is an organ that seems to depend on the supply of pre-formed GLA, i.e. GLA is a conditional essential fatty acid for skin. Hence, there is a need to provide new compositions, which improve the delivery of GLA and/or DGLA to the skin for improving its biomechanical properties. The medicament and the nutraceutical according to the invention addresses this problem.

The metabolism of linoleic acid from which the omega-6 fatty acids are produced is well known. It will be appreciated that linoleic acid is converted to GLA (18:3 delta-6, 9, 12) by the enzyme delta-6 desaturase; and GLA is converted to DGLA (20:3 delta-8, 11, 14) by a process of elongation. Hence, by the term "functional derivatives of GLA", we mean dihomo-gamma-linolenic acid (DGLA).

Currently, there are only a few naturally occurring sources of oils having a high concentration of GLA, and some functional derivatives thereof, and there are no known natural sources of DGLA. One source of GLA or a functional derivative thereof is the seed of the Evening Primrose species, for example, Oenothera biennis and Oenothera lamarckiana. The Evening Primrose Oil (EPO) extract from these species contains about 9% - 10% GLA.

The EPO is extracted from the seeds by techniques well known to the skilled technician. Fractionation of a typical sample of EPO in the form of methyl esters contains: about 6.15% palmitate; about 1.6% stearate; about 10.15% oleate; about 72.6% linoleate; and about 8.9% gammalinolenate (the active ingredient). A preferred species of Evening Primrose is the plant variety known as RIGEL, which is protected under the New Zealand Plant Variety Right No. 1085. EPO oil extracted from Rigel is a clear green-yellow liquid, free from any extraneous matter, and has the following chemical and physical properties:-

The fatty acid profile of a sample of EPO extracted from the Rigel plant variety contains:-

A preservative, for example, alpha-tocopherol may be added to the EPO in a concentration of about 0.1% to produce the final composition.

The composition according to the invention may take a number of different forms depending, in particular, on the manner in which it is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid. It is most preferred that the composition is orally administered, for example, in the form of a capsule or tablet. Most surprisingly, the inventors have found that when the composition is administered orally, i.e. the active ingredient of the GLA or functional derivative thereof, is taken systemically, there is a marked improvement to the biomechanical properties of the skin, and results in a decrease in the onset and extent of wrinkling, and therefore aging of the skin.

However, in some embodiments, the composition may be in the form of an ointment, cream, gel, hydrogel, aerosol, spray, micelle, liposome or any other suitable form that may be administered to a person or animal. It will be appreciated that the composition comprising the active ingredient, GLA or functional derivative thereof, should be one which is well tolerated by the subject to whom it is given and enables delivery of the active ingredient to the target tissue, i.e. the skin.

The agents may be used in a number of ways. For instance, systemic administration may be preferred in which case the GLA or functional derivative thereof may be contained within a composition, which may, for example, be ingested orally in the form of a tablet, capsule or liquid. Alternatively, the composition may be administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion). The composition may also be administered by inhalation (e.g. intranasally).

The composition may also be incorporated within a slow or delayed release device. Such devices may, for example, be inserted under the skin and the composition may be released over weeks or even months. The devices may be particularly advantageous if the composition normally requires frequent administration (e.g. at least daily ingestion of a tablet or daily injection). The composition according to the invention may be initially dissolved in solvents such as DMSO before dilution in aqueous solution for the preparation of liquid medicaments.

The composition may be topically applied to the skin in the form or a cream or lotion. It is preferred that the composition is thus applied directly to sites, which particularly require treatment.

However, as discussed herein, it is especially preferred that the composition is orally administered, for example, in the form of a capsule or tablet. The inventors were astounded to find that when the composition is administered orally, i.e. the active ingredient of the GLA or functional derivative thereof, is taken systemically, there is a marked improvement to the biomechanical properties of the skin, and results in a decrease in the onset and extent of wrinkling, and therefore aging of the skin. The inventors believe that they are the first to report the efficacy of oral (systemic) administration of the essential fatty acid GLA or functional derivative thereof for the treatment of aging, and in particular to improve the biomechanical properties of skin.

The composition is very easy to administer to subjects who wish to improve the quality (wrinkles and aging) of their skin. Oral administration is less invasive than administering by injection, and is also easier than using creams or lotions, which are topically applied directly to the skin. Hence, orally administrable medicaments are advantageous and are therefore the especially preferred mode of administration. This is particularly surprising, as one would have thought that oral administration would result in a certain amount of the active ingredient (i.e. GLA or derivative thereof) being degraded, for example, in the lining of the stomach. However, the inventors surprisingly found that no such degradation occurred, and the active ingredient imparted its effect on the subject being treated with a very high efficiency.

It will be appreciated that the composition may be administered either to a subject who has irritated or unhealthy skin, or to a subject who has normal, healthy skin, or at least superficially normal, healthy skin. The inventors were surprised to find that the composition is effective at improving the biomechanical properties of irritated skin. However, they found that surprisingly, the composition is particularly effective at improving those of normal, healthy skin. The inventors believe that they are the first to report the efficacy of oral (systemic) administration of the essential fatty acid GLA or functional derivative thereof to minimise or prevent wrinkling, and therefore to treat aging in healthy, or at least superficially healthy skin. Hence, the medicament or nutraceutical may be used as a prophylactic, or as a cosmetic in order to delay the onset of wrinkling, and therefore the visible aging in the skin.

It will be appreciated that the amount of the composition required is determined by it's biological activity, and bioavailability, which in turn depends on the mode of administration and the physicochemical properties of the GLA or functional derivatives thereof. The frequency of administration will also be influenced by the abovementioned factors and particularly the half-life of the active agents within the subject being treated. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc), may be used to establish specific formulations of GLA or functional derivative thereof, and precise therapeutic regimes (such as daily doses and the frequency of administration). Generally, a daily dose of between O.lmg/kg of body weight and 50mg/kg of body weight of GLA or functional derivative thereof may be used for improving the biomechanical properties of the skin. Preferably, the daily dose of GLA or functional derivative thereof is between 0.5mg/kg of body weight and 25mg/kg of body weight, more preferably, between lmg/kg of body weight and lOmg/kg of body weight, and yet still more preferably, between 2mg/kg of body weight and 8mg/kg of body weight, and most preferably, between 4mg/kg of body weight and 6mg/kg of body weight.

Assuming a body weight of 70kg, it is preferred that about lmg to 1Og of GLA or functional derivative thereof is used for improving the biomechanical properties of skin. It is especially preferred that about lOmg to Ig is used per daily treatment. For example, about lOOmg to 500mg, or preferably between about 300mg to 400mg may be used per daily treatment. In a preferred embodiment, about 340mg to 350mg are used per day.

Purely by way of example, suitable doses of the GLA or functional derivative thereof, according to the invention, may be between 200mg-400mg, and preferably between 320mg and 370mg per day.

Daily doses may be given as a single administration (e.g. a daily tablet for oral consumption or as a single daily injection or a single application of a cream or lotion to the skin). Alternatively, the composition may require administration twice or more times during a day. A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime), or for example, at 3 or 4 hourly intervals thereafter. By way of example only, the composition may comprise a 500mg capsule, containing about 55-60mg GLA; and about 5mg Vitamin E, encapsulated by a shell comprising gelatin, glycerine, and water. Three capsules may be taken twice daily (in the morning and evening).

Alternatively, a slow release device may be used to provide optimal doses to a patient without the need to administer repeated doses. A preferred route of administration may be by intravenous infusion. Administration may be over several hours or even days. The Examples describe the administration of GLA to test subjects over the course of 12 weeks. Hence, it is preferred that the composition according to the invention is administered every day for at least a week, and preferably, over periods of more than at least a week. Preferably, the composition is administered for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, or more. It is especially preferred that the GLA or functional derivative thereof is administered for at least 4 weeks, and preferably, longer.

The composition may be administered to a subject which may be any animal of veterinary interest, for instance, cats, dogs, horses etc. However, it is especially preferred that the composition is administered to a mammal, such as a human, e.g. male or female.

The inventors believe that the effects of the composition according to the second aspect, on age-related structural and functional skin changes in the subject being treated, may be linked with differences in sex steroids between men and women. Examples of sex steroids include oestrogen, progesterone and testosterone, the concentrations of which vary between the sexes, and also between individuals of the same sex. It will be appreciated that the levels of hormones in men (in particular, testosterone) and women (in particular, oestrogen and progesterone) also vary during aging. In addition, the concentrations of sex steroids vary during a woman's menstrual cycle.

During normal aging, the skin becomes thinner, looser, less elastic etc. hi men this is a fairly gradual effect, which accelerates after the age of about 45. However, in women, the abrupt hormonal changes at menopause are more sudden and drastic. The decrease in oestrogen levels can cause a thinning and drying of the skin, which can produce more wrinkles. Accordingly, it is preferred that the composition may be administered to subjects who are suffering from an imbalance in at least one sex steroid. Examples of such conditions suffered by women include PMS, (see http://www.drmirkin.com/women/8211.html; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=A bstract&list_uids=12762829; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=A bstract&list_uids=l 1535422; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db^:=pubmed&dopt=^:A bstract&list_uids=l 1705091 ; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=A bstract&list_uids=2013350)); http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=A bstract&list_uids=12955259)

Hence, it is believed that administration of the composition according to the second aspect would complement traditional treatment used by women, to treat for example, PMS, hormonal dysbalance etc.

It is believed that oestrogen levels below about 75pg/ml cause changes in the connective tissue in the skin and contribute to the formation of wrinkles. Hence, it is preferred that the composition is administered to subjects having an oestrogen concentration of below about 75pg/ml, more preferably, below 60pg/ml, even more preferably, below about 45pg/ml, and most preferably, below about 30pg/ml.

In a preferred embodiment, the treatment comprises administering by mouth three capsules, each one containing about 60mg GLA, to subjects at least twice a day. The inventors were surprised to find that after about 12 weeks of administration to subjects having healthy skin, a marked improvement to the biomechanical properties of their skin was observed. The onset of wrinkles was minimised thereby resulting in younger looking skin.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:-

Figure 1 is a graph showing skin moisture values;

Figure 2 is a graph showing increase in skin moisture in %; Figure 3 is a graph showing skin redness values;

Figure 4 is a graph showing TEWL values;

Figure 5 is a graph showing cutometer parameters;

Figure 6 is a graph showing F4 values relating to skin firmness;

Figure 7 is a graph showing increase in skin firmness in %; Figure 8 is a graph showing F3/F4 values relating to skin elasticity;

Figure 9 is a graph showing increase in skin elasticity in %;

Figure 10 is a graph showing F2 values relating to skin fatigue strength;

Figure 11 is a graph showing increase in skin fatigue strength;

Figure 12 is a graph showing skin roughness (Rz) values; Figure 13 is a graph showing decrease in skin roughness in %; and

Figure 14a-14g shows graphs showing percentage change in the skin parameters examined.

A series of experiments, which are described in detail below, were carried out on a group of test subjects in order to investigate the effect of the essential fatty acid,

GLA, on a number of different parameters of the skin, i.e. moisture, redness, Trans

Epidermal Water Loss (TEWL), firmness, elasticity, fatigue strength, and skin roughness.

Performance of Test

The subjects were selected from the Derma Consult GmbH database. They were informed about the importance and meaning of the study, and written informed consent was obtained from all the subjects prior to entry into the trial. The following criteria were used for the selection of the subjects :- (a) Included in study: -

- Female and male (> 18 years of age)

- clinically healthy - dry, rough skin (healthy) according to volunteer and dermatologist assessment

(b) Excluded from study: -

- skin diseases according to dermatologist assessment

- pregnancy

The subjects of this study were between 32-56 years of age (average: panel 1:

43,7; panel 2: 44,2). This means that the mean age of the GLA group was comparable to that of the placebo group. This is important as the biomechanical properties of skin are age-dependent. The volunteers could withdraw from the study at any time without giving any reason. The subjects were instructed not to use any topical preparations on the test areas starting from seven days prior to testing and until the end of the test. For cleansing, water or a mild syndet was used (Eubos flϋssig - blau; manufacturer: Dr. Hobein, D-53340 Meckenheim-Merl, Germany) was allowed only (for the whole study including the run-in phase). Syndet or SYNthetic DETergents, are soaps that are fabricated from synthetically produced wash-active substances, and are also known as tensides. hi their liquid form, tensides serve as raw materials for numerous cosmetic products, such as shampoos, shower gels and so on. Although they are strictly based on synthetic materials, syndet soaps are still for the great part made from naturally occuring raw materials, as the raw material basis of tensides are in turn fatty acids and fatty alcohols.

Prior to the first treatment, measurements were taken at clearly defined sites of the inner sides of the forearms. Further measuring was performed after 28 and 84 days of treatment (adaptation time: 30 min, room temperature: 21±1 C, relative humidity: 50± 5%).

Product

Identically looking 500 mg soft gelatine capsules were manufactured for the evening primrose and the placebo group. Evening primrose oil (Rigel® quality) was obtained from Efamol Ltd, Brackenholme, UK. Medium-chain triglycerides (Miglyol 812 N (R), SYNOPHARM GmbH & Co. KG, D-22885 Barsbϋttel, Germany) served as placebo. Both oils were stabilized with 8 mg of dl-alpha-tocopherol acetate. Each 500mg capsule consisted of: 55-60mg GLA; 5mg Vitamin E, encapsulated by a shell comprising gelatin, glycerine, and water. The GLA label claim per capsule was 57.5 mg.

One panel was treated with a product A (an Evening Primrose Oil capsule containing active ingredient GLA), and the other panel was treated with a product B (placebo capsule). Product A contained Evening Primrose Oil, which had been derived from the RIGEL variety of the species Oenothera biennis. A typical sample of the EPO consisted of about 6.0% palmitate; about 2.1% stearate; about 6.4% oleate; about 70.4% linoleate; and about 13.6% gammalinolenate (the active ingredient).

The subjects used the test product (active A or placebo B) orally (3x500mg capsules) twice daily (in the morning and evening).

Biometry

The products A (active) or B (placebo) were administered to each subject in the test every day (3 capsules taken in the morning, and 3 capsules taken in the evening) over the course of 12 weeks. The parameters moisture, redness, Trans Epidermal Water Loss (TEWL), firmness, elasticity, fatigue strength, and skin roughness, were measured after 4 weeks and 12 weeks of treatment. Measured data was automatically saved by a computer, and after a validity check and quality assurance, was stored centrally in a database. Evaluation of the data was done using the software NAG Statistical Add-Ins for Excel - NAG Ltd., United Kingdom. The two treatment groups were compared by means of the Wilcoxon signed-rank test on differences. The 0.05 (two-sided) confidence level was selected as the point of minimal acceptance of statistical significance.

Example 1 - Measurement of Skin Moisture/Humidity (Corneometrv)

A Corneometer CM 825 PC (Courage and Khazaka, Cologne, Germany) registers the electrical capacitance of the skin surface. The capacitance was expressed digitally in arbitrary units (a.u.). A probe head (7x7 mm) consisting of a condenser was applied to the skin surface at a constant pressure (3.5 N). The measuring principle is based on distinctly different dielectric constants of water (approximately 81) and most other materials (less than seven). Three separate measurements were performed on each test area and the mean was used to define the moisture state of the stratum comeum.

Referring to Figure 1, there are shown changes in the moisture values in group 1 treated with product A in comparison to group 2 treated with product B. An increase in the measurement value corresponds to an increase in skin moisture. After 84 days (12 weeks) of treatment a statistically significant (p=0.012) increase in skin moisture was observed in the panel 1 treated with sample A as compared to panel 2 treated with sample B. The respective changes in skin moisture as percentages relative to the initial conditions and between treatments are shown in Figure 2 and Figure 14a, respectively. As illustrated in Figures 2 and 14a, after 12 weeks of treatment with product A containing the active ingredient GLA, increases in skin moisture of about 13% was seen. (Bettinger, J., Gloor, M., Vollert, A., Kleesz, P., Fluhr, J. and Gehring, W. Skin Res. Technol. 5, 21-27 (1999).

Example 2 - Measurement of Skin Redness (Chromameter) The skin colour was measured with a Minolta Chromameter CR 300 (Minolta,

Japan) in compliance with the Commission Internationale de l'Eclairage (CIE) system, according to which the registration of colour is adjusted to the non-linear colour sensitivity of human eye. A colour is expressed in a three-dimensional coordinate system with green-red (a*), yellow-blue (b*) and L* axes (lightness). The skin surface is illuminated by a Xenon flashlight and remitted light registered and analysed by a photoreceiver. Chromametry is sensitive and accurate for the characterization of skin colour. In reddened skin, a positive change on the a* axis is observed. Each value is the average of three recordings. Before each measuring series, the instrument was calibrated against a standard white tile. Measurements were performed according to the guidelines of the Standardisation Group of the European Society of Contact Dermatitis (Fullerton et al., Contact Dermatitis, 1996, 35, 1-10).

Referring to Figure 3, there are shown changes in the redness values of group 1 treated with product A (active) in comparison to group 2 treated with product B (placebo). A decrease in the measurement value corresponds to a decrease in skin redness. As shown in Figure 14b, no statistically significant decrease in skin redness was observed in either panel, so the treatment with the two samples had little or no influence on skin redness.

Example 3 - Measurement of Transepidermal Water Loss (TEWU)

Measurements of TEWL were performed with the Tewameter TM 210

(Courage & Khazaka, Cologne, Germany). The Tewameter is a device for measurement of water evaporation on skin surfaces based on the diffusion principle discovered by A. Fick in 1885. The TEWL was calculated automatically and expressed digitally in g/m^h. Measurements were carried out in accordance with the guidelines of the standardisation group of the European Contact Dermatitis Society (Pinnagoda et al. Contact Dermatitis 1990: 22: 164-178). Each value was the average of three separate measurements.

Referring to Figures 4 and 14c, there are shown changes in the TEWL values in group 1 treated with product A (active) in comparison to group 2 treated with product B (placebo). A decrease in the measurement value corresponds to a decrease in TEWL and in a normalisation of the skin barrier function. After 84 days of treatment a statistically significant (p=0.034) decrease in TEWL was observed in the panel 1 treated with sample A as compared to panel 2 treated with sample B.

Example 4 - Measurement of Biomechanical Properties (Elasticity, Firmness, Fatigue strength)

The biomechanical properties of the skin were assessed using the Cutometer SEM 575 (Courage + Khazaka Electronic GmbH, Cologne). The measurement is based on the vacuum-suction principle. By applying a constant negative pressure for a given time period, skin is drawn into a hollow tube with an orifice of 2 mm in diameter. Then, at normal air pressure, the skin is allowed to retract. The penetration depth of the skin into the tube is recorded optically without friction and without mechanical influence. A number of standardized parameters can be calculated from the resulting penetration depth curve. Most of the parameters are a function of skin thickness and thus cannot be simply compared between subjects and regions.

In order to increase the accuracy and to capture information on the properties of skin under repeated external stress, the cycle was repeated several times and parameters selected for evaluation were based on areas rather than individual measurement points. The delineation of the areas is based on the fitted logarithmic envelope curves of the minimum and maximum extensions according to the equation:-

\n x + b y = a

(x =time, y = max. amplitude or min amplitude).

The parameters can be illustrated as shown in Figure 5, where parameter F2 is equivalent to the area of the graph above the maximum envelope; parameter F3 is equivalent to the area of the graph between the maximum and minimum envelopes; and parameter F4 is equivalent to the area of the graph below the maximum envelope.

The study was conducted with 20 successive measurement cycles, 1 second suction, 1 second retraction, with a 450 mbar vacuum. The following parameters were selected to assess changes in the biomechanical properties of the skin:-

(i) Skin firmness - skin firmness was assessed by the parameter F4, the area below the approximated envelope function of the maximum extensions. A decrease in F4 corresponds to an increase in skin firmness.

(ii) Skin elasticity - skin elasticity was assessed by the ratio of F3 / F4. The larger F3 in comparison to F4, the larger are the restoring forces and the smaller is the remaining residual deformation. The closer the resulting value is to 1, then the more elastic the skin.

(iii) Skin fatigue strength - Skin fatigue strength was assessed by the parameter F2. F2 is the area between the final extension and the approximated envelope function of the maximum extensions. A decrease in F2 corresponds to an increase in fatigue strength.

The calculation of the parameters was performed with the software WinCT (Courage & Khazaka GmbH, Cologne - Germany)

(i) Skin Firmness

Referring to Figure 6, there are shown changes in the F4 values in group 1 treated with product A (active) in comparison to group 2 treated with product B

(placebo). A decrease in the measurement value corresponds to an increase in skin firmness. After 84 days of treatment a statistically highly significant (p=0.001) increase in skin firmness was observed in the panel 1 treated with sample A as compared to panel 2 treated with sample B. The respective changes in skin firmness as percentages relative to the initial conditions and between treatments are shown in

Figure 7 and 14d, respectively. As illustrated in Figures 7 and 14d, after 12 weeks of treatment with product A containing the active ingredient GLA, about a 17% increase in skin firmness was seen.

(ii) Skin Elasticity

Referring to Figure 8, there are shown changes in the ratio F3/F4 values in group 1 treated with product A (active) in comparison to group 2 treated with product B (placebo). An increase in the measurement value corresponds to an increase in skin elasticity. After 84 days of treatment a statistically highly significant (p=0.002) increase in skin elasticity was observed in the panel 1 treated with sample A as compared to panel 2 treated with sample B. The respective changes in skin elasticity as percentages relative to the initial conditions and between treatments are shown in Figure 9 and Figuren 14e, respectively. As illustrated in Figures 9 and 14e, after 12 weeks of treatment with product A containing the active ingredient GLA, about a 5% increase in skin elasticity was seen.

(iii) Skin Fatigue strength

Referring to Figure 10, there are shown changes in the F2 values in group 1 treated with product A (active) in comparison to group 2 treated with product B (placebo). A decrease in the measurement value corresponds to an increase in skin fatigue strength. After 84 days of treatment a statistically significant (p=0.036) increase in skin fatigue strength was observed in the panel 1 treated with sample A as compared to panel 2 treated with sample B. The respective changes in skin fatigue strength as percentages relative to the initial conditions and between treatments are shown in Figure 11 and Figure 14f, respectively. As illustrated in Figures 11 and 14f, after 12 weeks of treatment with product A containing the active ingredient GLA, about a 14% increase in skin fatigue strength was seen.

Example 5 - Measurement of Roughness/Smoothness

PRIMOS (Phase-Shifting rapid in vivo measurement of skin) is a non-contact measurement device, which allows for real-time three-dimensional in vivo measurement of the microtopography of human skin based on the technology of active image triangulation. The measurement head consists of a digital micromirror device as projection unit and a CCD-camera as recording unit, mounted onto an adjustable rack. For active image triangulation, an intensity encoded point M is projected onto the surface under investigation. Its image on the surface is recorded by the CCD-camera from a specific angle. The height information of the structured surface is coded in the distorted intensity pattern, which is recorded. The resolution and accuracy depends on the optical and topographical characteristics of the measured surface and on the noise characteristics of the measurement system. For accurate in vivo measurements of human skin, depending on the measured part of the human body (inner forearm, forehead, eye zone), different parameters of effective wavelength and amplification factor should be used.

To regard the differences of human skin and avoid undesired distortions by movements, the fast phase-shift technique was used for the measurement. Distortions in the topography due to hairs can be digitally removed by the PRIMOS software. Skin roughness was assessed by means of the parameter Rz (mean depth of roughness). To mitigate potential directional effects, the evaluation was conducted using the arithmetic average of Rz from 32 different radial cuts.

The mean depth of roughness is defined as:-

Λz - -∑ Λ_Λ where n is the number of equal segments into which the scan length, 1, has been divided into and Rzi is the maximum peak to valley depth within each of the segments. In accordance with the German Standard Din 4768/1, Rz was calculated using 5 segments of equal length.

Referring to Figure 12, there are shown changes in the roughness values (Rz) in group 1 treated with product A (active) in comparison to group 2 treated with product B (placebo). A decrease in the measurement value corresponds to a decrease in skin roughness. After 84 days of treatment a statistically significant (p=0.040) decrease in skin roughness was observed in the panel 1 treated with sample A as compared to panel 2 treated with sample B. The respective changes in skin fatigue strength as percentages relative to the initial conditions and between treatments are shown in Figure 13 and Figure 14g, respectively. As illustrated in Figures 11 and 14f, after 12 weeks of treatment with product A containing the active ingredient GLA, an about 22% decrease skin roughness was seen.

Summary

The supplements were well tolerated by the test subjects, and none of the volunteers withdrew from the study. The complete set of planned measurements could be obtained from all volunteers. The data are presented as percent change between the two treatments (Figures 14a to 14g).

The mean values of the two treatment groups at baseline did not differ significantly from each other and were very similar, with the exception of the roughness values. This indicates that the EPO and the placebo group were highly comparable with respect to their skin properties. None of the mean values of the placebo group at week 4 and 12 differed significantly from the pre-treatment value. The values after EPO treatment at week 4 did not differ significantly from either the corresponding baseline values or from those of the placebo group. However, the picture was completely different 8 weeks later. Moreover, at week 12, the values of all skin parameters were significantly different for EPO compared to placebo, with the exception of skin redness. Moisture, TEWL, elasticity, firmness, fatigue strength and roughness had significantly improved by 13.8%, 7.7%, 4.7%, 16.7%, 14.2% and 21.7%, respectively. The levels of statistical significances ranged between P=O.034 and 0.001.

The results of the various experiments are summarised below for the effects of GLA on (i) normal, healthy skin, and on (ii) experimentally induced irritated skin.

(i) Normal Skin - significances with respect to placebo The results for (i) healthy skin are given in Table 1.

Table 1 - Effects of Evening Primrose Oil on healthy skin (n = 20)

Means±SD." Wilcoxon signed-rank test on differences between evening primrose oil and placebo.

A summary of the statistical significance for the results of treating healthy skin with EPO are given in Table 2. Table 2 - Statistical significance of treating healthy skin with EPO

(ii) Irritated skin - significances with respect to placebo

The study design was the same as for the previous experiments. Prior to the first treatment, measurements were taken at clearly defined sites of the inner sides of the forearms. After the initial measurements, skin irritation was induced in the test sites (inner sides of forearms) by applying Sodium Dodecyl Sulphate (SDS, an experimentally used irritant) 2% in distilled water under occlusion for 24h. After 24h hours, occlusion was removed and the skin was wiped with a soft paper towel to remove remaining solution, rinsed with tape water and gently dried with a soft paper towel. Four hours later moisture, redness, TEWL, firmness, elasticity, fatigue strength, and roughness, were recorded (day 1). Further measuring was performed after 4 and 12 weeks of treatment. A summary of the statistical significance for the results of treating irritated skin with EPO are given in Table 3.

Table 3 - Statistical significance of treating irritated skin with EPO

Hence, it will be appreciated that the active component in the capsules, i.e.

GLA had an improving effect on a number of different parameters relating to the health of skin. Most surprisingly, was the improvement to the biomechanical properties of healthy skin, i.e. firmness, elasticity, and fatigue strength, when GLA was administered systemically.

The present study shows that the regular intake of evening primrose oil, an oil with a high GLA content, demonstrably strenghens the physiological skin barrier function, improves the biomechanical properties and smoothens the surface profile of skin. Lending credence to the findings was the observation that it took longer than 4 weeks for the effects to show which is in line with the findings that it takes about 2 months for dietary fatty aids to reach a new steady-state equilibrium. Even though the exact mechanism of activity is unknown the combined evidence suggests that GLA is a conditionally essential fatty acid for skin and that many people should consider using it as a skin care preparation containing GLA or as a GLA supplement.

Claims

1. Use of gamma-linolenic acid (GLA) or a functional derivative thereof for the manufacture of a medicament or a nutraceutical for rejuvenating the skin.

2. Use according to claim 1, wherein the medicament or nutraceutical is adapted to improve the physical or biomechanical properties of the skin.

3. Use according to either claim 1 or claim 2, wherein the medicament or nutraceutical is adapted to increase the firmness of the skin.

4. Use according to any preceding claim, wherein the medicament or nutraceutical is adapted to increase the elasticity of the skin.

5. Use according to any preceding claim, wherein the medicament or nutraceutical is adapted to increase the fatigue strength of the skin.

6. Use according to any preceding claim, wherein the medicament is adapted to delay the onset, and minimise the extent of wrinkling in the skin.

7. Use according to any preceding claim, wherein the medicament or nutraceutical is adapted to improve the softness, suppleness, fine lines, signs of fatigue, and/or complexion of the skin, each of which are governed by the firmness, elasticity and fatigue strength thereof.

8. Use according to any preceding claim, wherein the medicament or nutraceutical is for treating aging.

9. Use according to any preceding claim, wherein the medicament or nutraceutical is a cosmetic composition.

10. Use according to any preceding claim, wherein the functional derivative of GLA comprises dihomo-gamma-linolenic acid (DGLA).

11. Use according to any preceding claim, wherein the source of GLA or a functional derivative thereof is the seed of the Evening Primrose species, for example, Oenothera biennis and Oenothera lamarckiana.

12. Use according to claim 11, wherein the species of Evening Primrose is the plant variety known as RIGEL, which is protected under the New Zealand Plant Variety Right No. 1085.

13. Use according to any preceding claim, wherein the medicament or neutraceutical is in the form of a powder, tablet, capsule, or liquid.

14. Use according to any preceding claim, wherein the medicament or neutraceutical is administered orally.

15. Use according to any preceding claim, wherein a suitable dose of the GLA or functional derivative thereof is between 200mg-400mg per day.

16. A method of treating a subject, the method comprising administering to a subject in need of such treatment, a composition comprising gamma-linolenic acid (GLA) or a functional derivative thereof, wherein the composition is for rejuvenating the skin.

17. A method according to claim 16, wherein the composition is in the form or a medicament or neutraceutical as defined in any one of claims 1-15.

18. A method according to either claim 16 or claim 17, wherein the composition is administered to a subject who is suffering from an imbalance in at least one sex steroid.

19. A method according to any one of claims 16-18, wherein the administration of the composition would complement traditional treatment used by women, to treat for example, PMS, hormonal dysbalance etc.

20. A method according to any one of claims 16-19, wherein the treatment comprises administering by mouth three capsules, each one containing about 60mg GLA, to subjects at least twice a day.