SKIN TREATMENTS
Field of the invention
The invention relates to compositions containing activators of the nuclear liver X receptor (LXR) for use in promoting expression of genes involved in dermal regeneration, such as decorin and fibronectin.
Background to the invention Skin is subject to deterioration through dermatological disorders, environmental abuse (wind, air conditioning, central heating) or through the normal ageing process (chronoageing) which may be accelerated by exposure of skin to sun (photoageing). In recent years the demand for cosmetic methods for improving the appearance and condition and, in particular, for reversing, reducing or preventing the visible signs of wrinkled, aged and/or photodamaged skin has grown enormously.
Collagen, the predominant matrix skin protein is known to impart tensile strength to skin. It is also known in the art that the levels of collagen in skin are significantly reduced with aged and/or photodamaged skin. Many studies have shown that the levels of collagen type I in skin are decreased with age and/or with increased photodamage. The reduction of the levels of collagen in skin is accordingly associated with a decrease in the tensile strength of the skin causing wrinkles and laxity.
The small chondroitin sulphate proteoglycan decorin, co-distributes with collagen fibres and is considered essential to the correct formation of newly synthesised collagen fibrils. Studies have also demonstrated that levels of this protein are significantly reduced in aged or photodamaged skin. Likewise fibronectin, a further important component of the dermal extra-cellular matrix which is associated with collagen fibres also reduces with age.
It is known that activators of the nuclear liver-X-receptor (LXR) can have a beneficial action on skin epidermal barrier function. LXR activators have been shown previously to induce expression of involucrin and transglutaminase (WO98/32444) and filaggrin (WO03/030857), which are proteins involved in the formation of the epidermal barrier, the stratum corneum. However, the majority of research into LXR has focussed on its role in regulating cholesterol and fatty acid metabolism.
Summary of the invention We have now found that LXR activators exhibit a significant effect on the expression of a number of genes involved in dermal regeneration, notably decorin and fibronectin. Further, we have screened a substantial number of plant extracts and identified a number of extracts that have significant LXR agonist activity.
Accordingly, the present invention provides a method of enhancing decorin and/or fibronectin synthesis in the skin of an animal or human which method comprises administering to said animal or human a nuclear liver X receptor (LXR) activating agent.
In one embodiment, the LXR activating agent comprises a compound according to the general formulae;
(A)
or
(B)
wherein;
R represents a hydrogen, a hydroxyl, a keto, an acetyl, a Ci to Cio, substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group.
Ri represents a lower alkyl group, a hydrogen or COR6;
R2 represents a hydrogen, a halogen or hydroxyl group;
R3 represents a hydrogen, a hydroxyl, a halogen, a keto or lower alkyl group;
R4 represents a hydrogen, a hydroxyl, or a keto group;
R5 represents a hydrogen, a hydroxyl, a halogen or lower alkyl group;
R6 represents a lower alkyl group.
X represents a hydrogen, a methyl or a halogen;
Y represents a hydrogen, a hydroxyl, a acetyl or a keto group;
In another embodiment, the LXR activating agent is a plant extract selected from the group consisting of an extract of Dragon's blood resin (Daemorgos draco), an extract of Damar gum, a non-saponified extract of Nettle (Lamium albim), an extract of Breuzihno resin, an extract of red seaweed, an extract of mastic gum, an extract of mountain ash berry, an extract of plantain leaves and mixtures thereof.
The present invention also provides the use of an LXR activating agent in enhancing decorin and/or fibronectin synthesis in the skin of an animal or human.
In a further aspect, the present invention provides a topical composition for enhancing decorin and/or fibronectin synthesis in the skin, said composition comprising;
(a) a plant extract comprising an LXR activating agent, the plant extract being selected from the group consisting of an extract of Dragon's blood resin (Daemorgos draco), an extract of Damar gum, a non-saponified extract of Nettle (Lamium albim), an extract of Breuzihno resin, an extract of red seaweed, an extract of mastic gum, an extract of mountain ash berry, an extract of plantain leaves and mixtures thereof; and
(b) a dermatologically acceptable vehicle.
In a related aspect, the present invention provides a systemic composition for enhancing decorin and/or fibronectin synthesis in the skin, said composition comprising;
(a) a plant extract comprising an LXR activating agent, the plant extract being selected from the group consisting of an extract of Dragon's blood resin (Daemorgos draco), an extract of Damar gum, a non-saponified extract of Nettle (Lamium albim), an extract of Breuzihno resin, an extract of red seaweed, an extract of mastic gum, an extract of mountain ash berry, an extract of plantain leaves and mixtures thereof.
(b) a pharmaceutically acceptable vehicle.
In a further aspect, the LXR activating agents can be used to promote collagen formation in the skin of an animal or human.
Detailed Description of the Invention
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
LXR activating agents
Any reference herein to an activator of LXR includes a reference to an activator of LXRσ and/or of LXR/?, unless specifically stated to the contrary.
The LXR activating agents can be provided as pure or semi-pure compounds or as crude extracts of natural products, such as plant extracts.
One preferred class of LXR activating agents comprises the compounds according to the general formulae;
(A) or
(B)
wherein;
R represents a hydrogen, a hydroxyl, a keto, an acetyl, a Ci to Cι0, substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group;
R-i represents a lower alkyl group, a hydrogen or CORβ; R2 represents a hydrogen, a halogen or a hydroxyl group; R3 represents a hydrogen, a hydroxyl, a halogen, a keto or a lower alkyl group; R4 represents a hydrogen, a hydroxyl, or a keto group;
R5 represents a hydrogen, a halogen, a hydroxyl or lower alkyl group;
R6 represents a lower alkyl group.
X represents a hydrogen, a methyl or a halogen;
Y represents a hydrogen, a hydroxyl, a acetyl or a keto group.
One preferred class of LXR activating compound of formula (A) or (B) is that wherein the R represents -H, -OH, =O, -COCH3, -COHCH3, =CHCH2OH, or -OCOCH3.
Another possible class of compounds of formulae (A) and (B) wherein R is Ci to Cβ alkyl being substituted or unsubstituted, branched or unbranched and saturated or unsaturated with the proviso that when it is C8, it is unsaturated.
In formulae A and B, the R group is linked to the carbon at position 17 will depend on the nature of the R group (indicated by wavy bond). Where R is a hydrogen or a hydroxyl group or acetyl group the bond will be saturated, whereas when R is a keto group the bond will be unsaturated. When R is an alkyl group this group may be linked to the carbon at position 17 via a saturated or unsaturated bond, preferably this is an unsaturated bond.
In one preferred sub-class, R represents a hydroxyl, a keto or an acetyl group.
R may also represent a Ci to C7 (i.e. including C-i, C2, C3, C4, C5, Cβ and C ) substituted or unsubstituted, saturated or unsaturated, branched or unbranched alkyl group. Preferably said Ci to C7 alkyl group comprises at least one substituted group selected from hydroxyl, keto and acetyl groups and R may in particular represent substituted alkyl groups having two and three of said substitutions. More preferably the alkyl groups have undergone substitution with one or more keto or hydroxyl groups. Further preferred an alkyl R group is substituted at one or more positions corresponding or equivalent to C20, C2ι, C22 and C23 shown in figure 7. Where the substitution is with a keto group this is most preferably bonded to C20, whereas when substitution is with a hydroxyl group this is most preferably bonded to a carbon at C2ι and /or C22.
It is preferred that the alkyl R group remains unbranched as this helps to maintain a favoured linear configuration, however in the event that the alkyl group is branch said branches preferably comprise 2 carbons, more preferably 1 carbon.
Where the R group is an alkyl group as described above this will preferably have some degree of unsaturation.
Preferably unsaturation occurs in the form of one or more substituted keto groups.
Where R represents an unsaturated Ci to C8 alkyl group it is most preferred that this group has the formula -C(CH3)(CH2)2C=C(CH3)2.
It appears that the most effective LXR activators of formulae (A) and (B) comprise a small R group. In a preferred embodiment the R group of the LXR activating compound therefore represents a hydrogen, a hydroxyl, a keto or an unsubstituted or, more preferably, substituted Ci to C4 alkyl group. Preferably substitution occurs at C20 or C2ι within the alkyl group. Where the R group is an alkyl group it is preferred that this is forms an unsaturated bond with Cι7 of the ring structure.
In a preferred embodiment R represents a hydrogen, a hydroxyl, a keto or a substituted/unsubstituted Ci to C4 alkyl group. Suitable unsubstituted groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or ter-butyl.
In a particularly preferred embodiment R is selected from the group consisting of -H, -OH, =O, -COCH3, -COHCH3, =CHCH3, =CHCH2OH, -OCOCH3 and C(CH3)(CH2)2C=C(CH3)2.
A "lower alkyl" as employed herein includes both straight and branched chain radicals of up to four carbon atoms, examples of suitable groups are outlined above. In a preferred embodiment Ri is a hydrogen.
R2 represents a hydrogen, a halogen preferably chlorine or a hydroxyl group, preferably R2 represents a hydrogen.
R3 represents a hydrogen, a halogen preferably a fluorine or chlorine, a keto or a lower alkyl group. Preferably R3 is either a keto group or a hydrogen. In a most preferred embodiment R3 is a hydrogen.
Preferably R4 and R5 represent a hydroxyl group or hydrogen, most preferably these represent a hydrogen.
R6 represents a lower alkyl, preferably a methyl group.
X preferably represents a hydrogen, a fluorine or a chlorine, most preferably X is a hydrogen.
Preferably Y represents a hydrogen, a hydroxyl or a keto group.
When Y is a hydrogen, in a compound according to general formula A, a double bond may form between Cι6 and C-ι .
In a compound according to formula B, when Y is hydrogen Ri is a preferably hydrogen or -COR6. Preferably when Y is a keto group the activating molecule conforms to general formula A, whereas, when Y is a hydroxyl group the activating molecule preferably conforms to general formula B.
In a most preferred embodiment the activating compound conforms to formula A wherein Y is a keto group.
Where R is a hydrogen or a hydroxyl group, Y is preferably a keto group in an activating compound according to formula A.
Where R is -COCH3, Y is preferably a hydrogen or a keto group in a activating compound according to either A or B, preferably according to formula A.
Where R is =CHCH3 or -OCOCH3, Y is most preferably a keto group in an activating compound according to general formula A.
Where R is =CHCH2OH, Y is preferably either; a hydrogen in an activating compound according to general formula A, wherein R is preferably a hydroxyl group; or a hydroxyl group in an activating compound according to formula B wherein R-i is a hydrogen.
Where R is C(CH3)(CH2)2C=C(CH3)2, Y is preferably a hydrogen in an activating compound according to formula B wherein Ri is also a hydrogen.
In a preferred embodiment of the use according to the invention the desired activation of LXR is provided by a compound selected from the group consisting of; 4-androsten-3,16-dione, androst-4-ene-3,6,16-trione, 4-androsten-17beta-ol- 3,16-dione acetate, 16-ketotestosterone, 3 ?-acetoxypregna-5,16-dien-20-one, 3 ?-acetoxypregna-5-en-20-one, 3 ?-hydroxypregna-5,16-dien-20-one, 3/?-hydroxy pregna-5-en-20-one, 5,16-dien-pregnane-3,20-diol, 4,16-dienpregna-3,20-dione, 4,17(20)-(cis)-pregnadien-3,16-dione, 4,17(20)-(trans)-pregnadien-3,16-dione, 4-pregnen-3,16,20-trione, 4,17(20)-pregnadien-11 beta,21-diol-3-one, 5,17(20)-
pregnadien-3,16-diol-diacetate, 5,17(20)-pregnadien-3,16-diol, 5-pregnen- 3beta,16alpha,21-triol-20-one, 24-hydroxychol-4-en-3-one, cholesta-5,24-dien-3β- ol, desmosterol, and mixtures thereof.
4,17(20)-(cis)-pregnadien-3,16-dione is particularly preferred.
In one embodiment, dehydroepiandrosterone and its derivatives are specifically excluded.
The preparation of compounds of formulae (A) and (B) has been described in the literature and / or are commercially available e.g. from Sigma Chemical Company.
It is also possible to provide the LXR activator(s) in the form of one or more extracts of natural plant sources, e.g. extracts of one or more of Dragon's blood resin (Daemorgos draco), Damar gum (exudate of Damar tree, Nettle (Lamium albim), red seaweed, Breuzihno resin, mastic gum, mountain ash berry and plantain.
A plant extract differs from the intact plant material in that the various components present in the intact plant material will be present in different amounts in the extract, or substantially absent. Prior to extraction, plant materials may be dried and or mechanically processed, e.g. crushed.
Extracts of plant materials are typically made by solvent extraction. Suitable solvents are those in which LXR activators are soluble. Since LXR activators are typically sterols/steroids, suitable solvents include organic solvents such as hexane, chloroform, benzene, petroleum ether, dichloromethane, acetone, ether, diethyl ether, ethyl acetate and mixtures of the above. Solvents may also include alcohols such as methanol, ethanol and isopropyl alcohol and mixtures thereof, optionally mixtures with water. Preferred solvents are those which are acceptable for use in products destined for human or animal use. Plant materials can also be extracted with supercritical liquid CO2.
Extraction methods include batch extraction and soxhlet extraction at temperatures up to the solvent boiling point. Extraction procedures may therefore include a heating step. Solvent extracted components may be subject to further purification/separation steps such as chromatography or fractional distillation. As used herein, "fraction" means any fractioned part of a solvent containing one or more of the active ingredients described above, e.g. obtained by chromatography or by fractional distillation.
Sterols / steroids represent the unsaponifiable fractions (unsaps) of seed oils and extracts. These are components that cannot be converted to soaps (e.g. non fatty acid / glyceride material). In some extracts e.g. from oils, the unsaps can be enriched by a process of saponification. A suitable method is as follows:
The lipid extract (~1g) from e.g. hexane and methanol extractions is refluxed with 2M potassium hydroxide in ethanol for 1 hour. After cooling the mixture is shaken with diethyl ether. The upper solvent layer containing the unsaponifiable fraction is removed and washed twice with water, dried by passing through a column of sodium sulphate and the solvent removed by evaporation under nitrogen at -70°C. This represents the unsaponifiable material (e.g. sterols).
Formulations
The amount of LXR activator, or mixtures thereof, present in the final composition according to the invention will typically be from 0.001 to 50 wt%, preferably from 0.01 to 10 wt%, and most preferably from 0.1 to 10 % or from 1 to 10 wt% of said composition. The composition is typically formulated for topical application or systemic application.
In one embodiment, the compositions do not contain retinoic acid or a metabolic precursor thereof.
Topical formulations
A dermatologically acceptable vehicle acts as a dilutant, dispersant or carrier for the newly identified activators of LXR in the composition, so as to facilitate its distribution when the composition is topically applied.
Dermatologically acceptable vehicles other than water can include liquid or solid emollients, solvents, humectants, thickeners and powders. Examples of each of these types of vehicle which can be used singly or as mixtures of one or more vehicles, are as follows:
Emollients, such as stearyl alcohol, glycerol monoricinoleate, glycerol monostearate, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl luarate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, eicosanylalcohol, behenyl alcohol, cetyl palmitate, silicone oils such as dimethylpolysiloxane, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, cocoa butter, corn oil, cotton seed oil, tallow, lard, olive oil, palm kernal oil, rapeseed oil, safflower seed oil, evening primrose oil, soybean oil, sunflower seed oil, avocado oil, olive oil, sesame seed oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum jelly, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyloleate, myristyl myristate;
Propellants such as trichlorofluoromethane, dichlorodifluoro- methane, dichlorotetrafluoroethane, monochlorodifluoromethane, trichlorotrifluoroethane, propane, butane isobutanem demethyl ether, carbon dioxide, nitrous oxide;
Solvents such as ethyl alcohol, methylene chloride, isopropanol, acetone, ethylene glycol monoethyl ether, diethlyene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulphoxide, dimethyl formamide, tetrahydrofuran;
Powders, such as chalk, talc, fullers earth, kaolin, starch, gums, colloidal silica sodium polacrylate, tetre alkyl and/or trialkyl aryl ammonium smectites, chemically
modified magnesium aluminium silicate, organically modified montmorillonite clay, hydrated aluminium silicate, fumed silica, carboxyvinyl polmer, sodium carboxymethyl cellulose, ethylene glycol monostearate.
The dermatologically acceptable vehicle will usually form from 10 to 99.99 % wt, preferably from 50 to 99 % of the final composition ready for use by the consumer.
The composition may also comprise water, usually up to 98 % volume, preferably 5 to 80 % volume of said final composition.
A topical or skin composition of the invention can be formulated as a lotion having a viscosity of from 4,000 to 10,000 mPas, a fluid cream having a viscosity of from 10,000 to 20,000 mPas or a cream having a viscosity of from 20,000 to 100,000 mPas or above at a temperature of 20°C. The composition may be packaged in a container to suit its viscosity and intended use by the consumer. For example a lotion or fluid cream can be packaged in a bottle or a roll-ball applicator or a propellant driven aerosol device or a container fitted with a pump suitable for finger operation. When the composition is a cream, it can simply be stored in a non-deformable bottle or a squeeze container, such as a tub or a lidded jar.
As already mentioned, compositions of the invention for topical application include personal wash compositions such as liquid soaps, solid soaps, gels, and oils for washing in either the bath or in a shower or for use as a skin moisturising or conditioning product in shower or bath. The invention accordingly also provides a closed container containing a cosmetically acceptable composition as herein defined.
The present invention relates to methods of enhancing or promoting decorin and/or fibronectin production in the skin of a mammal, typically a human. The present invention also relates to methods of promoting collagen formation as a result of the increases in decorin and/or fibronection expressed resulting from administration of LXR activating agents. In one embodiment, such methods
comprise the administration of a safe and effective amount of a composition of the invention to the skin or regions thereof. The amount of active agent and frequency of application will vary depending on the initial condition of the skin and the desired end result.
A safe and effective amount of active in a topical composition is applied, generally from about 1 μg to about 1 mg per cm2 skin per application, preferably from about 2 g to about 800 μg/cm2 skin per application, more preferably from about 30 μg to about 700 μg/cm2 skin, most preferably from about 75 μg to about 250 μg/cm2 skin. Frequency of application typically ranges from about four times a day to about twice a week, more preferably from about three times a day to about once every other day, more preferably at least twice daily. It is generally preferred that at least one application occurs in the evening.
Systemic formulations
A composition according to the present invention for systemic administration may for example be adapted for oral administration, e.g. in the form of a tablet, lozenge, capsule, liquid (e.g syrup or linctus) or as an injection (e.g. subcutaneous or intramuscular ) or infusion or as a suppository. Typical such formulation techniques and appropriate pharmacologically/pharmaceutically acceptable carriers are well known to those skilled in the art. Suitable compositions for oral administration include those adapted for delayed release and/or for release in the lower gastrointestinal tract.
The amount of the compound administered depends upon the bioavailability of the compound from the composition, in particular where oral administration is used. Typically, however, the LXR activating agents are dosed in an amount of from about 0.01 mg/kg of body weight to about 100 mg/kg, preferably from about 0.1 to about 30 mg/kg of body weight. The amount of the composition depends upon the percent of compound within its formula, which is a function of the amount of the compound required per dose, its stability, release characteristics and other pharmaceutical parameters. The doses are typically administered from once or twice weekly to one or twice daily.
The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular individual.
Another means of systemic dosing comprises dosing any of the aforementioned compositions in a food product which therefore does not necessarily require use of a pharmacologically/pharmaceutically acceptable carrier.
As used herein, the term "food products" includes both food products as such and beverages. Suitable food products as such include spreads, dairy products (including milk and yoghurts), desserts, convenience foods/snacks, breakfast cereals and cereal bars, ready-cook meals, bread and frozen confections such as ice creams, water ices and sorbets and yoghurt ice creams. Food products also include dietary/nutritional supplements. Suitable beverages include tea, tea- flavoured drinks, coffee, soft drinks (e.g. carbonated squashes etc) and fruit juice.
The food products are typically supplemented with the active ingredients of the invention so that they contain higher amounts of the active ingredient(s) than they would normally contain.
The present invention will now be described further with reference to the following examples which are illustrative only and non-limiting.
Example 1 - Gene Expression Analysis of LXR Agonist Effects in Dermal Fibroblasts
Materials and Methods
Cell Culture
For these experiments, skin fibroblasts were isolated from human foreskin. On arrival the skin was stored in holding media DMEM (Dulbecco's Modified Eagles Medium: 2mM L-glutamine, 5 lU/ml penicillin and 5 μg/ml streptomycin, and
nystatin 2 mg/ml) at 4°C for several hours. The skin was then cut into 3 x 3 mm squares and transferred into a sterile petri dish, epidermis side up. A 0.2 % filtered dispase solution is added to cover the skin and then the dish sealed and placed at 4°C overnight.
To isolate the fibroblast cells from the skin, the skin samples are removed from the dispase solution and the epidermis peeled off and discarded. The dermis is cut into 1 x 1 mm squares and transferred into a well of a tissue culture plate. A cover slip is placed over the tissue and 1 ml DMEM containing 10% Fetal Calf Serum (FCS), 2mM L-glutamine, 5 lU/ml penicillin and 5 μg/ml streptomycin is added. This was then incubated at 37°C and the growth media changed at 3-4 day intervals until the fibroblasts are approximately 70% confluent. Sterile forceps were used to lift the cover slips, turning them over and placing them cell side up into spare wells. Fresh growth media is added to all of the wells and the cells cultured until, again, the cells were 70% confluent.
To treat the fibroblasts with ligands of LXR, the fibroblasts were plated out at an approximate seeding density of 7000 cells/cm2 in DMEM with 10% FCS. All cultures were maintained in a humidified incubator with 5% CO2 at 37°C and early passage cultures (l-V) were used throughout. Media was then changed to low serum (-1% FCS) 24 hours post seeding and the LXR agonists, 22(R)-hydroxycholesterol or 4, 17-cis-pregnadien-dione, were added in fresh DMEM (+1 % FCS, 0.1 % ethanol) and incubated on the cells for 24 hours. After the treatment, the media was removed and the cells immediately frozen in an ethanol dry ice bath.
RNA extraction and Analysis
To extract RNA from the cultured fibroblasts, the Qiagen RNeasy methodology was used. Frozen fibroblasts were taken from a -20°C freezer and placed in a Class 1 fume hood, and 350μl of Qiagen RNeasy RLT lysis buffer was applied to each plate well whilst the cells were still frozen. The manufacturer's protocol was then followed, and the RNA eluted from the columns with 2 X 50 μl of RNase free water. The samples were then DNased (Ambion) at 37°C for 1 hour using 6 U of
DNase enzyme and 5 U of SUPERASE (Ambion). The quality of the RNA was then checked on a non-denaturing 1 % agarose gel. Visualisation of the intensity of the 28 S and 18 S ribosomal RNA bands was used to judge RNA degradation (28S being twice as bright for good quality RNA). The RNA was then precipitated 5 using 0.1 volumes of Ammonium Acetate, and 2 volumes of ethanol. Precipitates were then stored at -20°C in 70 % ethanol.
Before using the RNA in a cDNA synthesis reaction, the quantity of total RNA used in each reaction was assessed using the RiboGreen RNA Quantitation o Reagent And Kit (Molecular Probes).
Gene Array
A cDNA array containing 2045 individual clones was used to interrogate the levels of mRNA in the fibroblasts. Standard PCR conditions were used to amplify the 5 cDNA inserts from various plasmid clones. The PCR products were checked on a 1 % agarose ethidium bromide gel and then each one fixed onto Corning Gap II micro-slides in triplicate. Each slide batch was Quality Controlled for spot morphology using SYBR Green II dye (Molecular Probes).
0 Fluorescent labelling and Array Hybridisation
To get fluorescently labelled cDNA from the RNA, the Genisphere 3DNA Array 350 RP™ expression array detection kit was used. The manufacturer's instructions were followed, except that: 1 ) Powerscript (Clontech) was used for reverse transcription. 5 2) Cotl DNA was added at a 0.1 concentration to the total RNA.
3) Washing was carried out for 1 x 10 minute in 2 x SSC, 0.2% SDS at 50°C, 2 x 5 minute 2 x SSC at room temperature, and finally 3 x 5 minute in 0.2 x SSC at room temperature.
0 Array Data Analysis
To get an image and then measure the intensity of fluorescence on each spot, the slides were analysed using a ScanArray 4000XL laser scanner (Packard Biosciences) and the Scan Array image software. Once an image was created, it
was analysed using the software package GenePix™ (Axon), and the intensity scores for each spot saved in excel. The median background values were subtracted from the mean spot values and then these values exported into GeneSpring™ 4.2 (Silicon Genetics) for analysis. The data was normalised using the 50th percentile and the Lowess normalisation algorithm.
Results
The expression of a number of genes changed between the vehicle treated cells (0.1 % ethanol) and the LXR agonist treated cells. In particular the table below outlines the genes known to be involved in dermal regeneration, and, therefore, likely to influence how skin ages. As each cDNA is spotted in triplicate the ranges of the ratios are shown in brackets.
These results show for the first time that LXR activators increase the levels of expression of genes involved in dermal regeneration such as decorin and fibronectin. This would indicate that LXR activators can be used to promote collagen formation.
Example 2 - Screening of plant extracts for LXR agonist activity
Materials and Methods
Nettle (Lamium albim) non-saps extract
This was soxhlet extracted using methanol and the extract was saponified to separate any saps from the unsaps (nonsaps) - as described above.
Dragon's Blood Resin (Daemonorops draco) extract This was an acetone extract of resin purchased from Frontier Natural Products Co-operative, Norway, Indiana, US.
Damar gum extract
This was an ethyl acetate extract of resin purchased from Thew Arnott Ltd., Surrey, UK.
Red seaweed extract
Hexane extracts were prepared of the seaweed material.
Breuzihno resin extract
This was soxhlet extracted using methanol, from a sample purchased from Nahziryah Monastic Community's on-line services, Saint Joe, Arizona, US.
mastic gum extract This was soxhlet extracted using methanol, from a sample of mastic gum purchased commercially. Mastic gum is a natural resin from the Pistacia lentiscus tree (an evergreen shrub from the pistachio tree family), which is found on the Island of Chios in Greece. Available commercially as, for example, Mastika, Natural Chios Mastic Gum.
mountain ash berry extract
This was purchased from Molecular Nature Limited (MNL) who used a soxhlet extraction (dichloromethane) followed by a cleanup to remove excess fats and
chlorophyll. The extract was subjected to normal phase flash chromatography using a hexane:ethyl acetate:methanol gradient system on Flash 75 Biotage columns. The fractions were then sub-fractionated using semi preparative reverse phase chromatography using a gradient of water with increasing amounts of acetonitrile and then washing with acetone.
plantain leaf extract
This was purchased from Molecular Nature Limited (MNL) who used the same soxhlet extraction (dichloromethane) procedure and fractionation method described above for mountain ash berries.
Over 400 compounds or plant extracts were screened for their ability to activate LXR using the LXRσ reporter gene assay described in WO03/030857.
Compounds/extracts which showed a fold-activation over the control of 1.9 or more were selected and are listed below:
The formulation below describes an emulsion cream for use according to the present invention.
Wt %
Example 4
The formulation below describes an oil in water cream suitable for the methods and uses according to the present invention. The percentages indicated are by weight of the composition.
* Brij 56 is cetyl alcohol POE (10) ** Alfol 16RD is cetyl alcohol
Example 5
The formulation below described a soup composition suitable for the methods and uses according to the present invention.
3.4 grams of vegetable fat}
0.5 grams of modified egg yolk} together named "creamer"
6.0 grams of maltodextrin}
1.0 grams of Red Seaweed extract
0.6 grams of maize starch croutons
16.1 grams of dried potato starch
1.0 grams of salt
0.3 grams of onion solids
0.7 grams of onions
0.2 grams of parsley and herb extract
3.2 grams of flavouring agents
The creamer and the other component are mixed in a mixer. The blend obtained is a dried instant onion soup that can be used for making a soup by mixing it with 200 ml of boiling water under stirring.
The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections, as appropriate.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and products of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the relevant fields are intended to be within the scope of the following claims.