# Patent

## Title
Skin conditioning compositions containing compounds for mimicking the effect on skin of retinoic acid

## Abstract

A skin care product comprising from about 0.001% to about 10% of a retinoid, in combination with 0.0001% to about 50% of a combination of retinoid boosters.

## Background
The present invention relates to cosmetic skin conditioning compositions containing certain compounds which mimic the effect on skin of retinoic acid.
Retinol (vitamin A) is an endogenous compound which occurs naturally in the human body, and is essential for normal epithelial cell differentiation. Natural and synthetic vitamin A derivatives have been used extensively in the treatment of a variety of skin disorders and have been used as skin repair or renewal agents. Retinoic acid has been employed to treat a variety of skin conditions, e.g., acne, wrinkles, psoriasis, age spots and discoloration. See e.g., Vahlquist, A. et al., J. Invest. Dermatol., Vol. 94, Holland D. B. and Cunliffe, W. J. (1990), pp. 496-498; Ellis, C. N. et al., “Pharmacology of Retinols in Skin”, Vasel, Karger, Vol. 3, (1989), pp. 249-252; Lowe, N. J. et al., “Pharmacology of Retinols in Skin”, Vol. 3, (1989), pp. 240-248; PCT Patent Application No. WO 93/19743.
It is believed that retinol esters and retinol are enzymatically converted in the skin into retinoic acid according to the following mechanism:




The present invention is based on the discovery that certain compounds enhance the conversion of retinyl esters and retinol to retinoic acid. The compounds are collectively termed “boosters” and are coded as groups B1 to B5 according to the boosting mechanism of the particular compound. The mechanism of the booster groups is as follows: inhibiting ARAT/LRAT (AcylCoenzymeA (CoA): retinol acyl transferase/Lecithin: retinol acyl transferase) activity (B1), enhancing retinol dehydrogenase activity (B2), inhibiting retinal reductase activity (B3), antagonising CRABP-II (Cellular Retinoic Acid Binding Protein II) binding of retinoic acid (B4) and inhibiting cytochrome P450 dependent retinoic acid oxidation (B5).
The boosters alone or in combination with each other potentiate the action of retinoids by increasing the conversion of the retinoids to retinoic acid and preventing the degradation of retinoic acid. The boosters act in conjunction with a retinoid (e.g. retinol, retinyl esters, retinal, retinoic acid), the latter being present endogenously in the skin. The preferred compositions, however, include a retinoid in the composition, co-present with a booster or a combination of boosters, to optimise performance.
Several patents by Granger et al describe the use of retinoid boosters in cosmetic compositions to improve the efficacy of retinol and retinyl esters (U.S. Pat. Nos. 5,759,556, 5,756,109, 5,747,051, 5,716,627, 5,811,110, 5,536,740, 5,747,051, 5,599,548, 5,955,092, 5,885,595, 5,759,556, 5,693,330). The boosters described in these patents are restricted to class B1 and B5. Furthermore Johnson & Johnson have a series of patents which describe the use of molecules which fall into class 5 booster molecules (U.S. Pat. Nos. 5,028,628; 5,037,829; 5,151,421; 0,476,852; 5,500,435; 5,583,136; 5,612,354).
The molecules which act as retinoid boosters are common ingredients in cosmetic products. There is considerable prior art describing their use in cosmetic compositions. There is substantial prior art describing the use of two or more of these molecules in the same composition. Some of the examples of the prior art are as in U.S. Pat. Nos. 5,665,367, 5,747,049, 5,853,705, 5,766,575, and 5,849,310.
However, the prior art does not describe synergy arising from combinations of booster molecules. This observation of a synergistic boosting of retinoid activity from combinations of booster molecules was an unexpected finding. The prior art does not describe optimal concentrations or ratios of booster molecules or ratios of booster molecules to that of retinoids. Thus, the present invention is novel in that by combining cosmetic retinoids with booster molecules, at the most appropriate concentrations or ratios, a substantial improvement in efficacy of the retinoids is obtained.
The classes of boosters suitable for use in the present invention include but are not limited to the boosters listed in Tables B1 through to B5.
Best Groups of Boosters










B1 Compounds


















1. Fatty Acid Amides
These are readily commercially




available and have the added




advantage of being surfactants and




thus help generate emulsions




suitable for cosmetic preparations.



2. Ceramides
These can additionally act as




precursors of stratum corneum




barrier ceramides.



3. Carotenoids
These can offer some UV protection




and act as natural colorants.



4. Flavanoids
Natural antioxidants.



5. Cyclic fragrances
These are readily commercially




available and additionally can be




used to fragrance the product.



6. Non-cyclic
These can be used to fragrance the



fragrances
product.



7. Phospholipid
These can be utilised by skin cells



analogues
to nourish the generation of




barrier components.



8. Ureas
These are readily commercially




available and can also act as




preservatives for the product.




















B2 Compounds


















1. Phosphatidyl choline
Most preferred as most active




activator of Retinol




Dehydrogenase



2. Sphingomyelin




















B3 Compounds


















Arachidonic Acid
Fatty Acids which can be



Linoleic Acid
useful in maintaining stratum



Linolenic Acid
corneum barrier



Myristic Acid



Linoleic Acid
Essential Fatty Acids



Linolenic Acid



Arachidonic Acid
Non-essential fatty acids



Myristic Acid



Glycyrrhetinic Acid
Polycyclic triterpene




carboxylic acid which is




readily obtained from plant




sources.



Phosphatidyl ethanolamine
Can be incorporated into




cellular membranes.




















B4 Compounds


















Hexadecanedioic acid
Saturated fatty acids.



12-hydroxystearic acid



Isostearic acid



Linseed oil
Unsaturated fatty acids



Elaidic acid



Elaidic acid
Solid at room temperature



Isostearic acid



Hexadecanedioic acid



Linseed oil
Liquid at room temperature



12-hydroxystearic acid




















B5 Compounds


















Bifonazole
Antimicotics



Climbazole



Clotrimazole



Econazole



Ketoconazole



Miconazole



Climbazole
Readily commercially available



Lauryl
Compounds which are readily



hydroxyethylimidazoline
commercially available and have




the added advantage of being




surfactants and thus help




generate emulsions suitable for




cosmetic preparations.



Quercetin
Naturally occuring flavanoid




which has antioxidant




properties.



Coumarin
Natural colorant



Quinolines



Isoquinolines



Metyrapone










The present invention includes, in part, a skin conditioning composition containing from about 0.0001% to about 50%, preferably from 0.001% to 10%, most preferably from 0.001% to 5% by weight of the composition of a booster or combination of boosters and a cosmetically acceptable vehicle.
The boosters or combinations thereof included in the inventive compositions are selected from the group consisting of:

    
    
        (a) a booster selected from the group consisting of B2; B3; B4;
        (b) binary combinations of boosters selected from the group consisting of:
        
            B1/B2; B1/B3; B1/B4; B1/B5; B2/B3, B2/B4; B2/B5, B3/B4; B3/B5; B4/B5
        
        
        (c) ternary combinations of boosters selected from the group consisting of:
        
            B1/B2/B3; B1/B2/B4; B1/B2/B5; B1/B3/B4; B1/B3/B5; B1/B4/B5; B2/B3/B4; B2/B3/B5; B2/B4/B5; B3/B4/B5
        
        
        (d) quaternary combinations of boosters selected from the group consisting of:
        
            B1/B2/B3/B4; B1/B2/B3/B5; B1/B2/B4/B5; B1/B3/B4/B5; B2/B3/B4/B5; and
        
        
        (e) a combination of five groups of boosters:
        
            B1/B2/B3/B4/B5.
        
        
    
    


The preferred compositions include from about 0.001% to about 10%, by weight of the composition of a retinoid.
The compounds included in the present invention as boosters are selected based on the ability of such compounds to pass, at a certain concentration listed in Table A, in-vitro Assays for a specific enzymes as described below under sections 2.1 through to 2.7. Such a booster is included in the present invention even if it is not explicitly mentioned herein. Put another way, if a compound inhibits or enhances sufficiently an enzyme in an assay described below, it will act in combination with a retinoid to mimic the effect on keratinocytes (skin cells) of retinoic acid, and thus it is included within the scope of the present invention.
The term “conditioning” as used herein means prevention and treatment of dry skin, acne, photo-damaged skin, appearance of wrinkles, age spots, aged skin, increasing stratum corneum flexibility, lightening skin colour, controlling sebum excretion and generally increasing the quality of skin. The composition may be used to improve skin desquamation and epidermal differentiation.
The presence of the selected compounds in the inventive product substantially improves the performance of a retinoid.
The inventive compositions contain, as a preferred ingredient, a retinoid, which is selected from retinyl esters, retinol, retinal and retinoic acid, preferably retinol or retinyl ester. The term “retinol” includes the following isomers of retinol: all-trans-retinol, 13-cis-retinol, 11-cis-retinol, 9-cis-retinol, 3,4-didehydro-retinol, 3,4-didehydro-13-cis-retinol; 3,4-didehydro-1′-cis-retinol; 3,4-didehydro-9-cis-retinol. Preferred isomers are all-trans-retinol, 13-cis-retinol, 3,4-didehydro-retinol, 9-cis-retinol. Most preferred is all-trans-retinol, due to its wide commercial availability.
Retinyl ester is an ester of retinol. The term “retinol” has been defined above. Retinyl esters suitable for use in the present invention are C1-C30 esters of retinol, preferably C2-C20 esters, and most preferably C2, C3, and C16 esters because they are more commonly available. Examples of retinyl esters include but are not limited to: retinyl palmitater retinyl formate, retinyl acetate, retinyl propionate, retinyl butyrate, retinyl valerate, retinyl isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate, retinyl decanoate, retinyl undecandate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadeconoate, retinyl stearate, retinyl isostearate, retinyl nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate, and retinyl oleate.
The preferred ester for use in the present invention is selected from retinyl palmitate, retinyl acetate and retinyl propionate, because these are the most commercially available and therefore the cheapest. Retinyl linoleate and retinyl oleate are also preferred due to their efficacy.
Retinol or retinyl ester is employed in the inventive composition in an amount of from about 0.001% to about 10%, preferably in an amount of from about 0.01% to about 1%, most preferably in an amount of from about 0.01% to about 0.5%.
The essential ingredient of the inventive compositions is a compound which passes in vitro Assays described below in sections 2.1 through to 2.7. A compound suitable for use in the present invention inhibits or enhances at a concentration listed in Table A an enzyme to at least a broad % listed in Table A.


SECTION A
Identification of Booster
Table A
Booster Test Concentrations and % Inhibition/Increase










ARAT/LRAT Assay (To identify B1 boosters)












Compound




Invention
Concentration
% Inhibition







Broad
100 μM
>10%



Preferred
100 μM
>25%



Most Preferred
100 μM
>40%



Optimum
100 μM
>50%




















Retinol Dehydrogenase Assay (To identify B2 boosters)












Compound




Invention
Concentration
% Increase







Broad
100 μM
>10%



Preferred
100 μM
>15%



Most Preferred
100 μM
>20%



Optimum
100 μM
>25%




















Retinal Reductase Assay (To identify B3 boosters)












Compound




Invention
Concentration
% Inhibition







Broad
100 μM
 >5%



Preferred
100 μM
>10%



Most Preferred
100 μM
>20%



Optimum
100 μM
>35%




















CRABPII Antagonist Assay (To identify B4 boosters)












Compound:Retinoic




Invention
acid Ratio
% Inhibition







Broad
7000:1
>25%



Preferred
7000:1
>50%



Most Preferred
 70:1
>25%



Optimum
 70:1
>50%




















Retinoic Acid Oxidation Assay(To identify B5 boosters)












Compound




Invention
Concentration
% Inhibition







Broad
100 μM
>25%



Preferred
100 μM
>45%



Most Preferred
100 μM
>70%



Optimum
100 μM
>80%










The in vitro Microsomal Assays employed for determining the suitability of the inclusion of the compound in the inventive compositions are as follows:
1. Materials
All-trans-retinol, all-trans-retinoic acid, palmitoyl-CoA, dilauroyl phosphatidyl choline, NAD, and NADPH were purchased from Sigma Chemical Company. Stock solutions of retinoids for the microsomal assays were made up in HPLC grade acetonitrile. All retinoid standard stock solutions for HPLC analysis were prepared in ethanol, stored under atmosphere of N2 at −70° C. and maintained on ice under amber lighting when out of storage. Other chemicals and the inhibitors were commercially available from cosmetic material suppliers or chemical companies such as Aldrich or International Flavours and Fragrances.
2. Methods
2.1 Isolation of RPE Microsomes (Modified from (1))
50 frozen hemisected bovine eyecups, with the retina and aqueous humor removed were obtained from W. L. Lawson Co., Lincoln, Nebr., USA. The eyes were thawed overnight and the colored iridescent membrane was removed by peeling with forceps. Each eyecup was washed with 2×0.5 mL cold buffer (0.1M PO4/1 mM DTT/0.25M sucrose, pH 7) by rubbing the darkly pigmented cells with an artist's brush or a rubber policeman. The cell suspension was added to the iridescent membranes and the suspension was stirred for several minutes in a beaker with a Teflon stir bar. The suspension was filtered through a coarse filter (Spectra/Por 925μ pore size polyethylene mesh) to remove large particles, and the resulting darkly colored suspension was homogenized using a Glas-Col with a motor driven Teflon homogenizer.
The cell homogenate was centrifuged for 30 min. at 20,000 g (Sorvaal model RC-5B centrifuge with an SS34 rotor in 2.5×10 cm tubes at 14,000 RPM). The resulting supernatant was subjected to further centrifugation for 60 min. at 150,000 g (Beckman model L80 Ultracentrifuge with an SW50.1 rotor in 13×51 mm tubes at 40,000 RPM). The resulting pellets were dispersed into  ̃5 mL 0.1M PO4/5 mM DTT, pH 7 buffer using a Heat Systems Ultrasonics, Inc. model W185D Sonifier Cell Disruptor, and the resulting microsomal dispersion was aliquoted into small tubes and stored at −70° C. The protein concentrations of the microsomes were determined using the BioRad Dye binding assay, using BSA as a standard.
2.2 Isolation of Rat Liver Microsomes (4)
Approximately 6 grams of frozen rat liver (obtained from Harlan Sprague Dawley rats from Accurate Chemical and Scientific Corp.) was homogenized in 3 volumes of 0.1M tris/0.1M KCl/1 mM EDTA/0.25M sucrose, pH 7.4 buffer using a Brinkmann Polytron. The resulting tissue suspension was further homogenized in the motor driven Teflon homogenizer described above. The resulting homogenate was successively centrifuged for 30 min. at 10,000 g, 30 min. at 20,000 g, and 15 min. at 30,000 g, and the resulting supernatant was ultra-centrifuged for 80 min. at 105,000 g. The pellet was sonicated in  ̃5 mL of 0.1M PO4/0.1 mM EDTA/5 mM MgCl2, pH 7.4 buffer as described above and stored as aliquots at −70° C. Protein concentrations were determined as described above.
2.3 Assay for ARAT and LRAT Activity (To Identify B1)
The procedure below was a modification of a method described in the literature (2). The following buffer was prepared and stored at 4° C., 0.1M PO4/5 mM dithiothreitol, pH 7.0 (PO4/DTT). On the day of the assay, 2 mg BSA per mL of buffer was added to give a PO4/DTT/BSA working buffer. 1 mM retinol substrate was prepared in acetonitrile and stored in amber bottles under nitrogen gas at −20° C. Solutions of 4 mM Palmitoyl-CoA in working buffer (stored in aliquots) and 4 mM dilauroyl phosphatidyl choline in ethanol were prepared and stored at −20° C. Inhibitors were prepared as 10 mM stock solutions in H2O, ethanol, acetonitrile or DMSO. The quench solution was prepared using pure ethanol containing 50 μg/mL butylated hydroxytoluene (BHT), and a hexane solution containing 50 μg/mL BHT was used for the extractions.
To a 2 dram glass vial, the following were added in order: PO4/DTT/BSA buffer to give a total volume of 500 μL, 5 μL acyl donor (4 mM palmitoyl-CoA and/or dilauroyl phosphatidyl choline), 5 μL inhibitor or solvent blank (10 mM stock or further dilutions) followed by approximately 15 μg of RPE microsomal protein (approximately 15 μL of a  ̃1 mg/mL microsomal protein aliquot). The mixture was incubated for 5 min. at 37° C. to equilibrate the reaction temperature and then 5 μL 1 mM retinol was added. The vials were capped, vortexed for 5 seconds and incubated for 30-90 minutes at 37° C. The reaction was quenched by adding 0.5 mL ethanol/BHT. The retinoids were extracted by adding 3 mL hexane/BHT, vortexing the tubes for several seconds several times and centrifuging the tubes at low speed for 5 min. to quickly separate the layers. The upper hexane layer was removed into a clean vial, and the aqueous layer re-extracted with another 3 mL hexane/BHT, as described above. The hexane layers were combined, and the hexane evaporated by drying at 37° C. under a stream of nitrogen gas on a heated aluminum block. The dried residue was stored at −20° C. until HPLC analysis. The amount of retinyl palmitate and retinyl laurate was quantitated for ARAT and LRAT activity, respectively, by integration of the HPLC signal as described below.
Note that the incubation solution contains 40 μM acyl donor, 100 μM or less inhibitor, 10 μM retinol, approximately 30 μg/mL microsomal protein, and nearly 0.1M PO4/pH 7/5 mM DTT/2 mg/mL BSA. All steps subsequent to the addition of retinol were done in the dark or under amber lights.
2.4 Assay for Retinol Dehydrogenase Activity (To Identify B2)
The following stock solutions were prepared:

    50 mM KH2PO4, pH 7.4 buffer, sterile filtered.
    10 mM all trans Retinol (Sigma R7632) in DMSO
    200 mM Nicotinamide adenine dinucleotide phosphate, sodium salt (NADP) (Sigma N0505) in sterile water.
    40 mM test compound in appropriate solvent (water, buffer, ethanol, chloroform or DMSO).
    1:10 dilution of rat liver Microsomes in 50 mM KH2PO4, pH 7.4 buffer (4 μg/μl).


In a two-dram glass vial with screw cap, the following were added in order:

    Buffer to give a final volume of 400 μl
    25 μl diluted Microsomes (final=100 μg)—boiled Microsomes were used for controls and regular Microsomes for test samples.
    4 μl of 200 mM NADP (final=2 mM)
    1 μl of 40 mM test compound (final=100 μM)
    8 μl of 10 mM retinol (final=200 μM)


The vials were incubated in a 37° C. shaking water bath for 45 minutes 500 μl ice-cold ethanol was added to each vial to quench the reaction. The retinoids were extracted twice with ice cold hexane (2.7 ml per extraction). Retinyl acetate (5 μl of a 900 μM stock) was added to each vial during the first extraction as a means of monitoring the extraction efficiency in each sample. Samples were vortexed for ten seconds before gently centrifuging for five minutes at 1000 rpm, 5° C. in a Beckman GS-6R centrifuge. The top hexane layer containing the retinoids was removed from the aqueous layer after each extraction to a clean two-dram vial. The hexane was evaporated off under a gentle stream of nitrogen gas. The dried residue was then stored at −20° C. until HPLC analysis.
2.5 Assay for Retinal Reductase Activity (To identify B3)
All stock solution were prepared as above with the following substitutions:

    10 mM all trans Retinaldehyde (Sigma R2500) in DMSO-instead of retinol.
    200 mM, Nicotinamide adenine dinucleotide phosphate, reduced form, tetrasodium salt (NADPH) (Sigma N7505) in sterile water—instead of NADP.


In a two-dram glass vial with screw cap, add the following in order:

    Buffer to give a final volume of 400 μl
    25 μl diluted Microsomes (final=100 μg)—use boiled Microsomes for controls and regular Microsomes for test samples.
    4 μl of 200 mM NADPH (final=2 mM)
    1 μl of 40 mM test compound (final=100 μM)
    3 μl of 10 mM retinaldehyde (final=75 μM)


Follow the same incubation and extraction procedure as detailed above.
2.6 Assay for CRABPII Antagonists (To Identify B4)
2.6.1. Synthesis of CRABPII
a. System of Expression
The gene CRABPII was cloned in pET 29a-c(+) plasmid (Novagen). The cloned gene was under control of strong bacteriophage T7 transcription and translation signals. The source of T7 polymerase was provided by the host cell E. coli BLR(DE3)pLysS (Novagen). The latter has a chromosomal copy of T7 polymerase under lacUV5 control, induced by the presence of IPTG.
The plasmid was transferred into E. coli BLR(DE3)pLysS cells by transformation according to the manufacturer protocol (Novagen).
b. Induction
An overnight culture of the transformed cells was diluted 1:100 into 2×YT containing 50 μg/mL kanamycin and 25 μg/mL chloramphenicol. The cells grew while shaking at 37° C. until the OD at 600 nm reached 0.6-0.8. Then IPTG was added to a final concentration of 1 mM and the culture was incubated for an additional two hours. The cells were harvested by centrifugation at 5,000 g for 10 minutes at room temperature. The pellet was stored at −20° C.
2.6.2. Purification
Purification was performed according to the method described in Norris and Li, 1997.
a. Lysis
The frozen pellet was thawed at RT and resuspended in 1-2 pellet volumes of freshly prepared lysis buffer (50 mM Tris-HCl, pH 8, 10% (w/v) sucrose, 1 mM EDTA, 0.05% (w/v) sodium azide, 0.5 mM DTT, 10 mM MnCl2, 2.5 mM phenylmethylsulfonyl fluoride, 2.5 mM benzamidine, 6 μg/mL DNase). The lysate was incubated for 30 mins. at room temperature. Further lysis was accomplished by sonication (six 30-sec bursts at 10,000 psi alternated with five 30-sec delay on ice). The insoluble fraction of the lysate was removed by centrifugation at 15,000 rpm 1 hour at 4° C. and the supernatant is stored at −20° C.
b. Gel Filtration on Sephacryl S300
The supernatant from step a. was loaded onto a 2.5×100 cm column of sephacryl S-300 (Pharmacia) at room temperature. The elution buffer was 20 mM Tris-HCl, pH 8, 0.5 mM DTT, 0.05% sodium azide (buffer A). The flow rate was 2 mL/min. Collected 2-mL fractions were checked for ultraviolet absorbance at 280 nm. The fractions representing the peaks were examined by SDS-page for the presence of CRABPII.
c. Anion-exchange Chromatography.
2 mL of gel filtration fractions containing CRABPII were loaded onto a quaternary amine anion-exchange column FPLC (Fast Protein Liquid Chromatography) type monoQ (Pharmacia). CRABPII was eluted using a gradient buffer from 100% buffer A to 30% buffer B (100% buffer B=buffer A+250 mM NaCl) over a 20-min period at room temperature. 1 mL-fractions were collected every minute. Once more, the presence of CRABPII was checked by SDS page. CRABPII was stored at 4° C. before freeze-drying using a Micromodulyo 1.5K with vial platform attachment (Edwards High Vacuum International). The desiccated samples were stored at room temperature until their use in the binding assay.
d. Detection of the Presence of CRABPII
The expression and purification of CRABPII was validated using denaturing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on a 7-15% polyacrylamide gel (Biorad). 10 μL samples were mixed with 10 μL of 2× loading buffer (100 mM Tris-HCl pH6.8, 4% SDS, 0.2% BPB, 20% glycerol, 1 mM DTT) and denatured by heating (2 mins. at 80° C.). The samples were loaded onto the gel that was immersed in a 1× Tris-glycine buffer (Biorad) and a constant current (25 mA) was applied for 1 hour at room temperature. After Coomassie blue staining, the protein was identified according to its molecular weight as determined with the Benchmark pre-stained protein ladder (Gibco BRL).
A western blot was used to confirm the presence of CRABPII. The proteins separated on the SDS-PAGE were transferred on an Immobilon-P transfer membrane (Millipore) using a Biorad cassette. The transfer occurred in 1× Tris-glycine buffer (Biorad)+10% methanol. An electrical currant (60 mA) was applied for 3 hours to allow the protein to migrate through the membrane. Afterwards, the membrane was blocked with 5% dry milk in 1×TBS for one hour at room temperature and probed with primary antibodies to CRABPII (1/1000 dilution of mouse anticlonal 5-CRA-B3) in the same buffer at 4° C. overnight. The following day, the membrane was washed with PBS (3×5 minutes) and then incubated with 1:2000 dilution of the secondary antibody, peroxidase conjugated anti-mouse antibody (ECLTM, Amersham), for 1 hour at room temperature. The membrane was washed with 1×PBS (3×5 minutes) and the protein was detected using ECL detection kit according to the manufacturer instruction (Amersham).
The concentration of purified CRABPII was determined using BSA kit (Pierce).
2.6.3. Radioactive Binding Assay
220 pmol of CRABPII was incubated in 20 mM Tris-HCl buffer pH 7.4 with 15 pmol of radioactive all trans retinoic acid (NEN) in a total volume of 70 μL. For the competitive assay, another ligand in excess (6670:1, 670:1 or 70:1) was added to the mix. The reaction occurred for one hour at room temperature in the dark. In order to separate the unbound all-trans retinoic acid from the bound all-trans retinoic acid, a 6 kD cut-off minichromatography column (Biorad) was used. The storage buffer was discarded using a Microplex manifold for according to the manufacturer instruction (Pharmacia). The samples were loaded onto the column and the separation occurred by gravity over a 30-min period. Retinoic acid (“RA”) bound to CRABPII appeared in the filtrate while free RA remained in the column. The radioactivity of the filtrate was measured by scintillation counter.
2.7 Assay for NADPH Dependent Retinoic Acid Oxidation (To Identify B5)
The procedure below is a modification of a method described in the literature (4). The following assay buffer was prepared and stored at 4° C.: 0.1M PO4/0.1 mM EDTA/5 mM MgCl2, pH 7.4. On the day of the assay, a 60 mM NADPH solution in buffer was prepared. Inhibitor stocks, acidified ethanol/BHT quench solution, and hexane/BHT were prepared as described above. A working 1 mM retinoic acid solution was prepared by dilution of a 15 mM stock (in DMSO) with ethanol.
To a 2 dram vial, the following were added in order: assay buffer to give a final volume of 500 μL, 20 μL 60 mM NADPH, 5 μL inhibitor or solvent blank, followed by approximately 2 mg of rat liver microsomal protein.
The mixture was incubated for 5 mins. at 37° C., then 5 μL working 1 mM retinoic acid solution was added. Incubation was continued for 60 mins. at 37° C.—the vials were not capped, since the oxidation process required molecular O2 in addition to NADPH. Quenching was carried out with acidified ethanol/BHT and extraction was carried out with hexane/BHT as described above. Quantitation of the quickly eluting polar retinoic acid metabolites (presumed to be 4-oxo retinoic acid) was carried out by integration of the HPLC signal as described below.
All steps subsequent to the addition of retinoic acid were done in the dark or under amber lights. The final incubation solution contained 2.4 mM NADPH, 100 μM or less inhibitor, 10 μM retinoic acid, approximately 4 mg/mL rat liver microsomal protein and nearly 0.1M PO4/0.1 mM EDTA/5M MgCl2.
HPLC Analysis of Individual Retinoids
Samples for retinoid quantitation by HPLC were prepared by dissolving the residue in each vial with 100 μL of methanol. The solution was transferred to a 150 μL glass conical tube within a 1 mL shell vial, capped tightly, and placed inside a Waters 715 Autosampler. Aliquots of 60 μL were injected immediately and analysed for retinoid content.
The chromatography instrumentation consisted of a Waters 600 gradient controller/pump, a Waters 996 Photodiode Array detector and a Waters 474 Scanning Fluorescence detector. Two HPLC protocols were used for retinoid analysis. For the ARAT and LRAT assay, the separation of retinol and retinol esters was performed with a Waters 3.9×300 mm C18 Novapak reverse-phase analytical column and Waters Sentry NovaPak C18 guard column with an 80:20 (v/v) methanol/THF isocratic mobile phase adjusted to a flow rate of 1 mL/min. for 10 min. The eluate was monitored for absorbance at 325 nm and fluorescence at 325ex/480em.
A shorter Waters 3.9×150 mm C18 Novapak reverse-phase analytical column and Waters Sentry NovaPak C18 guard column were used to separate retinoid acids and alcohols for the retinol and retinoic acid oxidation assays utilising a modification of a gradient system described by Barua (5). This system consisted of a 20 mins. linear gradient from 68:32 (v/v) methanol/water containing 10 mM ammonium acetate to 4:1 (v/v) methanol:dichloromethane followed by a 5 mins. hold at a flow rate of 1 mL/min. The column eluate was monitored from 300 nm to 400 nm.
These protocols were selected based on their ability to clearly resolve pertinent retinoid acids, alcohols, aldehydes, and/or esters for each assay and relative quickness of separation. Identification of individual retinoids by HPLC was based on an exact match of the retention time of unknown peaks with that of available authentic retinoid standards and UV spectra analysis (300-400 nm) of unknown peaks against available authentic retinoids.
REFERENCES


    1 J. C. Saari & D. L. Bredberg, “CoA and Non-CoA Dependent Retinol Esterification in Retinal Pigment Epithelium”, J. Bill. Chem. 263, 8084-8090 (1988).
    2 J. C. Saari & D. L. Bredberg, “ARAT & LRAT Activities of Bovine Retinal Pigment Epithelial Microsomes”, Methods Enzymol. 190, 156-163 (1990).
    3 J. L. Napoli & K. R. Race, “The Biosynthesis of Retinoic Acid from Retinol by Rat Tissues in vitro”, Archives Biochem. Biophys. 255, 95-101 (1987).
    4 R. Martini & M. Murray, “Participation of P450 3A Enzymes in Rat Hepatic Microsomal Retinoic Acid 4-Hydroxylation”, Archives Biochem. Biophys. 303, 57-66 (1993).
    5 A. B. Barua, “Analysis of Water-Soluble Compounds: Glucuronides”, Methods Enzymol. 189, 136-145 (1990).


The boosters suitable for use in the present invention include but are not limited to the boosters listed in Tables B1 through to B5 below. The table below gives the booster class (B1-B5), the chemical name of the compound, and the results from the appropriate assays used to identify the booster (i.e. ARAT/LRAT for B1, retinol dehydrogenase for B2, retinaldehyde inhibation for B3, CRABP is binding for B4 and retinoic acid oxidation inhibition for B5.










ARAT/LRAT Inhibitors (B1)
















% Inhibition
Overall

% Inhibition
% Inhibition
% Inhibition




Overall
TG
% Inhibition
ARAT
LRAT
LRAT


Class
Compound
TG(-ROH/RE)
(IC 50)
ARAT (10jm)
(100jm)
(10jm)
(100jm)





Carotenoid
Crocetin

3.75E−05
15%
34%
0
15%


Fatty Acid & Other
Acetyl Sphingosine

6.78E−06
19% +/− 12
62% +/− 11
10% +/− 10
50% +/− 18


Surfactants


Fatty Acid Amides &
C13 Beta-Hydroxy Acid/
17%


28%

25%


Other Surfactants
Amide


Fatty Acid Amides &
Castor Oil MEA

3.25E−05


Other Surfactants


Fatty Acid Amides &
Cocamidopropyl Betaine



25%


Other Surfactants


Fatty Acid Amides &
Coco Hydroxyethyl-

2.84E−07

68%

65%


Other Surfactants
imidazoline


Fatty Acid Amides &
Cocoamide-MEA (or
11%


13%

34%


Other Surfactants
Cocoyl Monoethanol-



amide)


Fatty Acid Amides &
Glycerol-PCA-Oleate



41% +/− 6

58% +/− 2


Other Surfactans


Fatty Acid Amides &
Hexanoamide



20%


Other Surfactants


Fatty Acid Amides &
Hexanoyl Sphingosine

9.99E−05

28% +/− 4

37% +/− 9


Other Surfactants


Fatty Acid Amides &
Hydroxyethyl-2-

3.29E−05

35%

35%


Other Surfactants
Hydroxy-C12 Amide


Fatty Acid Amides &
Hydroxyethyl-2-



25%

30%


Other Surfactants
Hydroxy-C16 Amide


Fatty Acid Amides &
Lauroyl Sarcosine



20%


Other Surfactants


Fatty Acid Amides &
Lidocaine



12%

0


Other Surfactants


Fatty Acid Amides &
Linoleamide-DEA (or
59%

12% +/− 3
43% +/− 3
11% +/− 9
51% +/− 15


Other Surfactants
Linoleoyl



Diethanolamide)


Fatty Acid Amides &
Linoleamide-MEA (or

1.61E−05
14%
35%
20% +/− 8
35%


Other Surfactants
Linoleoyl Monoethanol-



amide)


Fatty Acid Amides &
Linoleamidopropyl



69% +/− 18

75% +/− 4


Other Surfactants
Dimethylamine


Fatty Acid Amides &
Melinamide



64% +/− 15

43% +/− 21


Other Surfactants


Fatty Acid Amides &
Myristoyl Sarcosine



41% +/− 14

11% +/− 11


Other Surfactants


Fatty Acid Amides &
Oleyl Betaine

2.80E−05

47%


Other Surfactants


Fatty Acid Amides &
Palmitamide-MEA


 6%
23%
12%
33%


Other Surfactants


Fatty Acid Amides &
Stearylhydroxyamide



10%

10%


Other Surfactants


Fatty Acid Amides &
Utrecht-1
21%

43%
54%
51%
48% +/− 6


Other Surfactants


Fatty Acid Amides &
Utrecht-2

3.47E−06
42%
83% +/− 9
51%
92% +/− 3


Other Surfactants


Flavanoids
Naringenin



33%

14%


Fragrances
Allyl Alpha-Ionone


16% +/− 14
22% +/− 23
17% +/− 10
36%/−7


Fragrances
Alpha-Damascone

3.35E−04
67% +/− 27
83% +/− 12
87% +/− 6
98% +/− 1


Fragrances
Alpha-Ionone

9.27E−04

45% +/− 27

49% +/− 30


Fragrances
Alpha-Methyl Ionone



67%

77%


Fragrances
Alpha-Terpineol



26%

25%


Fragrances
Beta-Damascone


45%
84%
52%
92%


Fragrances
Brahmanol



70%

75%


Fragrances
Damascenone


23%
70%
29%
79%


Fragrances
Delta-Damascone


58%
87%
64%
95%


Fragrances
Dihydro Alpha-Ionone



13%

18%


Fragrances
Ethyl Saffranate



51%

49%


Fragrances
Fenchyl Alcohol



12%

 4%


Fragrances
Gamma-Methyl Ionone



21%

38%


Fragrances
Isobutyl Ionone



 8%

45%


Fragrances
Isocyclogeraniol



18%

16%


Fragrances
Isodamascone



80%

92%


Fragrances
Lyral

1.27E−04

76%

71%


Fragrances
Santalone



23%

12%


Fragrances
Santanol



15%

43%


Fragrances
Timberol



34%

33%


Fragrances
Tonalid



50%

33%


Fragrances
Traseolide



41%

21%


Miscellaneous
Coco Trimethyl-



27%



ammonium Cl-


Miscellaneous
Urosolic Acid

1.46E−06

21%

28%


Noncyclic
Citral



20%


Fragrances


Noncyclic
Citronellol



30%

0


Fragrances


Noncyclic
Farnesol

9.35E−05
23% +/− 18
53% +/− 18
10% +/− 7
53% +/− 19


Fragrances


Noncyclic
Geraniol

7.83E−03
13%
32%


Fragrances


Noncyclic
Geranyl Geraniol


38% +/− 12
81% +/− 6
16% +/− 9
77% +/− 13


Fragrances


Noncyclic
Linalool



28%

0


Fragrances


Noncyclic
Nonadieneal



20%


Fragrances


Noncyclic
Pseudoionone



12%

37%


Fragrances


Phospholipid
Dioctylphosphatidyl


23%
50% +/− 2
0
17% +/− 17



Ethanolamine


Urea
Dimethyl
22%



Imidazolidinone


Urea
Imidazolidinyl Urea
35%



















Retinol Dehydrogenase Activators (B2)









Class
Compound
% Increase Retinol Dehydrogenase





Phospholipid
Phosphatidyl Choline
21% increase


Phospholipid
Sphingomyelin
26% increase



















Retinaldehyde Reductase Inhibitors (B3)












Overall
% Inhibition


Class
Compound
TG (IC 50)
Retinal Reductase





Aldehyde
Vanillin
9.70E−03
 6%


Fatty Acid
Arachidic Acid

20%


Fatty Acid
Arachidonic Acid

49%


Fatty Acid
Linoleic Acid
1.63E−04
62% +/− 2


Fatty Acid
Linolenic Acid
1.34E−04
54% +/− 16


Fatty Acid
Myristic Acid
1.72E−05
26%


Miscellaneous
Amsacrine
6.26E−06
22% +/− 8


Miscellaneous
Carbenoxolone
3.61E−07
26% +/− 2


Miscellaneous
Glycyrretinic Acid
8.64E−06
38% +/− 1


Phospholipid
Phosphatidyl

37%



ethanolamine



















CRABPII Antagonists (B4)












Overall
% Inhibition


Class
Compound
TG(IC 50)
CRABPII





Fatty Acid
Elaidic Acid
6.50E−05
>50%


Fatty Acid
Hexadecanedioic Acid
1.30E−04
>50%


Fatty Acid
12-Hydroxystearic Acid
2.91E−05
>50%


Fatty Acid
Isostearic Acid
6.88E−05
>50%


Fatty Acids
Linseed Oil

>50%



















Retinoic Acid Oxidation Inhibitors (B5)














% Inhibition
% Inhibition




Overall
Retinoic
Retinoic


Class
Compound
TG (IC 50)
Acid (10 μM)
Acid (100 μM)














Imidazole
Bifonazole

89%
100%


Imidazole
Climbazole
4.47E−06
80%
92%


Imidazole
Clotrimazole

76%
85%


Imidazole
Econazole

88%
100%


Imidazole
Ketoconazole
1.85E−07
84%
84%


Imidazole
Miconazole
2.78E−07
74%
86%


Fatty Acid Amides
Lauryl Hydroxyethylimidazoline
4.67E−07


& Other Surfactants


Fatty Acid Amides
Oleyl Hydroxyethylimidazoline
3.02E−05
54%
80%


& Other Surfactants


Flavanoids
Quercetin
6.29E−05
40%
74%


Coumarin
Coumarin


Quinoline
(7H-Benzimidazo [2,1-a]Benz
8.59E−07



[de]-Isoquinolin-7-one


Quinoline
Hydroxyquinoline (Carbostyril)
3.64E−04


Quinoline
Metyrapone (2-Methyl-1,2-di-3-


47%



Pyridyl-1-Propane)









SECTION B
Effects of Booster Combinations
In order to assess the effect of combinations of booster molecules an assay is required which encompasses the effect of each of the five booster classes. A single enzyme assay is not suitable for this purpose, as it will be specific only for one class of booster molecule. An assay which reflects retinoid concentration in keratinocytes is necessary to relate the effects of single booster molecules with combination of booster molecules. For this reason, a transglutaminase (Tgase) assay was utilised. Tgases are calcium dependent enzymes that catalyse the formation of covalent cross-links in proteins. Several Tgase enzymes are membrane bound in keratinocytes which is important for epidermal cell maturation. This enzyme is inhibited by retinoic acid. The higher the concentration of retinoic acid, the greater the inhibition of Tgase expression. Hence Tgase is a good marker of both keratinocyte differentiation and of the retinoid effect on keratinocytes.
Transglutaminase as a Marker of Skin Differentiation
During the process of terminal differentiation in the epidermis, a 15 nm thick layer of protein, known as the cornified envelope (CE) is formed on the inner surface of the cell periphery. The CE is composed of numerous distinct proteins which have been cross-linked together by the formation of NΣ-(γ-glutamyl) lysine isodipeptide bonds catalysed by the action of at least two different transglutaminases (TGases) expressed in the epidermis. TGase I is expressed in abundance in the differentiated layers of the epidermis, especially the granular layer, but is absent in the undifferentiated basal epidermis. Thus TGase I is a useful marker of epidermal keratinocyte differentiation with high TGase I levels indicating a more differentiated state. An ELISA based TGase I assay, using a TGase I antibody, was used to assess the state of differentiation of the cultured keratinocytes in the examples that follow.
Keratinocytes (cultured as described above) were plated in 96 well plates at a density of 4,000-5,000 cells per well in 200 μl media. After incubation for two to three days, or until cells are  ̃50% confluent, the media was changed to media containing test compounds (five replicates per test). The cells were cultured for a further 96 hours after which time the media was aspirated and the plates stored at −70° C. Plates were removed from the freezer, and the cells were washed twice with 200 μl of 1×PBS. The cells were incubated for one hour at room temperature (R/T) with TBS/5% BSA (wash buffer, bovine serum albumin). Next the TGase primary antibody was added: 50 μl of monoclonal anti-Tgase I Ab B.C. diluted 1:2000 in wash buffer. The primary antibody was incubated for 2 hours at 37° C. and then rinsed 6× with wash buffer. Cells were then incubated with 50 μl of secondary antibody (Fab fragment, peroxidase conjugated anti-mouse IgG obtaining from Amersham) diluted 1:4,000 in wash buffer for two hours at 37° C., then rinsed three times with wash buffer. Following the rinse with washing buffer, the cells were rinsed 3× with PBS. For colourimetric development, the cells were incubated with 100 μl substrate solution (4 mg o-phenylenediamine and 3.3 μl 30% H2O2 in 10 ml 0.1M citrate buffer pH 5.0) for exactly five minutes, R/T, in darkness (under aluminum foil). The reaction was stopped by the addition of 50 μl 4N H2SO4. The absorbance of samples was read at 492 nm in a 96 well plate UV spectrophotometer. Out of the five replicates, four were treated with both antibodies, the fifth one was use as a Tgase background control. TGase levels were determined and expressed as percentage control.
Details of Tgase Assay:
Prior to initiating experiments, to determine the effects of combinations of booster molecules standard Tgase assay conditions were investigated. A fully validated Tgase assay was established as follows:










A. Reagents


















Cells: Human Keratinocytes
Neonatal Human foreskin



(P2 in T75 flasks; P3 in 96



well assay plates)



Primary Antibody: TGm specific
Biogenesis (Cat # 5560-



monoclonal Ab B.C1
6006)



Secondary Ab: Peroxidase
Amersham (Cat # NA9310)



labeled antimouse Ig F(ab)2



Substrate solution: For 10 ml



phosphate citrate buffer



4.0 mg o-phenylenediamine
Sigma P-7288



3.3 μl of 30% H202
Sigma H-1909




















B. Media/Buffers


















Keratinocyte Growth Media (KGM)
Clonetics (Cat # 3111)



Phosphate Buffered Saline;
Life Technology



Dulbecco's without Ca/MgCl2)
(Cat # 14200-075)



Tris Buffered Saline



Blocking buffer (1xTBS + 5%
BioRad (Cat # 170-6404)



dry milk)



Washing buffer (1% dry milk in
Sigma (Cat # P-7949)



TBS + 0.05% Tween 20)



Phosphate citrate buffer: 1:1
Sigma (Cat # S-9763)



mixture of 0.2M dibasic



sodium phosphate and 0.1 M
Sigma (Cat # C-1909)



citric acid



4 N H2SO4




















C. Culture ware


















96-well polypropylene microtitre
Costar (Cat # 3595)



plate



96-well polypropylene U-bottom
Costar (Cat # 3794)



plate



T75-vent cap
Costar (Cat # 3376)




















D: Instrumentation/Equipment


















Biotek Model EL 340 Microplate
Bio-tek Instuments Inc.



reader
Packard



Multiprobe II










E: Cell Culture Procedure

Seeding of Keratinocytes in 96 Well Plates

    1. A suspension of keratinocytes was prepared at a concentration of 3000 cells/200 μl/well in KGM medium (Used 3×105 cells/12 ml media in each microtiter plate)
    2. 200 μl of the keratinocyte suspension was transferred into each of the inner 60 wells only.
    3. 200 μl of KGM media was pipetted into the outer wells (To maintain thermal equilibrium).
    4. Each plate was incubated at 37° C. and 5% CO2 for 3 days or until cells are  ̃50% confluent.

Treatment of Keratinocytes with Samples.

    5. Stock solutions of the samples were prepared in DMSO.
    6. The samples were diluted to desired concentration with the final assay concentration of DMSO being 0.1%.
    7. 20 μl of the sample was transferred into wells and 180 μl of KGM medium added to give a final assay volume of 200 μl.
    8. Plates were incubated at 37° C. and 5% CO2 for 72 hours.
    9. Media were completely removed from each well.
    10. Wells were rinsed with 2× with 200 μl of 1×PBS
    11. Finally they were frozen for at least 1.5 hours at −70° C.

F: Transglutaminase Assay

    1. Block:
    
        Incubate plates at room temperature with 200 μl/well of blocking buffer for 1 hour.
    
    
    2. Primary Antibody:
    
        Aspirate blocking buffer. Incubated with 100 μl/well of TGm-specific monoclonal antibody B.C1 (diluted 1:2000 in washing buffer) at 37° C. for at least 2 hours. The primary antibody was not added in background control wells.
    
    
    3. Rinsed wells 6× with washing buffer.
    4. Secondary Antibody:
    
        Incubated with 100 μl/well peroxidase labeled anti-mouse IgF(ab)2 fragment (diluted 1:4000 in washing buffer) at 37° C. for 2 hours.
    
    
    5. Rinsed wells 3× with washing buffer (added 200 μl) and aspirated after each rinse.
    6. Rinsed wells 3× with PBS w/o Tween.
    7. Incubated with 100 μl/well substrate solution at room temperature for exactly 5 minutes.
    8. Stopped reaction with 50 μl/well 4N H2SO4.
    9. Read absorbance at 492 nm in the Bio-tek plate reader.

I. Optimization Studies

a. Time Course of Transglutaminase Production



A time course experiment was conducted to determine the optimal incubation time for transglutaminase production in keratinocytes grown in 96-well plates (4000 cells/well). This time course study was conducted with multiple variables including dose response analyses of retinoic acid and retinol as well as incubation in the presence of 1.2 mM CaCl2. Although the transglutaminase production in the control cells (0.1% DMSO) was not altered, both retinoic acid and retinol exhibited a dose dependent inhibition of transglutaminase production over the five day incubation period. The most pronounced retinoid effect was observed on day 2 and day 3. The maximal inhibition was observed on day 2 with the transglutaminase production being inhibited by 85% and 55% in the presence of the highest concentration (1 μM) of retinoic acid and retinol respectively. The same experiment was also conducted with varying cell density (3000 cells/well and 5000 cells/well) and comparable results were observed.
B: DMSO Sensitivity
Various concentrations of DMSO ranging from 0-2% were tested for the effect on transglutaminase production in keratinocytes. The assay was sensitive to DMSO concentration with significant inhibition of activity, above 0.5% DMSO. Hence, a final assay concentration of 0.1% was selected for subsequent sample concentration studies.
C: Dose Response Curves: Retinoic Acid and Retinol
Based on the data, day 3 was selected as the optimal time and 0.1% DMSO was selected as the concentration to be used for further testing. An additional dose response experiment was carried out with retinoic acid and retinol in the presence of 0.1% DMSO, with the transglutaminase production being assayed on day 3. A good dose response was observed for Tgase inhibition by retinoic acid and retinol. 10-7M retinol gave an inhibition of Tgase in the linear range of concentration. Therefore, this concentration of retinol was chosen to evaluate the booster combinations.










D: Final conditions used to test boosters or combination


of boosters


















Days of incubation of keratinocytes with
3 days



retinol and boosters



Final DMSO concentration
less than 0.1%



Retinol concentration
10-7M (0.1 μM)



Booster concentrations
10 mM to 0.1 nM










Using the above conditions, dose response for all the different boosters (B1-B5) were tested to identify the best concentration of booster to test in combinations.
Transglutaminase levels were determined and expressed in the Tables B1 through B5 either as:

    (i) % (booster+retinol inhibition/control inhibition)−% (ROH inhibition/control inhibition), which measures the added effect of booster+retinol induced TGase inhibition over retinol alone, or
    (ii) as an IC50 value when the inhibitory effect of multiple booster concentrations was examined—this provides the concentration of booster which, in combination with a constant retinol concentration of 10−7 M, inhibits TGase by 50%.

Booster Combinations and Booster Ratios:



It has been discovered surprisingly that certain compounds increase the endogenous levels of retinoic acid formation from retinol or retinyl esters by different mechanisms. These compounds are collectively called here as “retinoid boosters”. These include: inhibitors of ARAT/LRAT (B1 boosters), inhibitors of retinaldehyde reductase (B3 boosters), inhibitors of retinoic acid binding to CRABP-2 (B4 boosters) and inhibitors of retinoic acid oxidation catalysed by cytochrome P450 enzymes (B5 boosters), or certain other compounds which enhance or activate retinol dehydrogenase (B2 boosters). These boosters are coded as groups B1 through to B5, as seen in chart 1 herein above.
The boosters alone or in combination with each other, potentiate the action of a retinoid by increasing the amount of retinol available for conversion to retinoic acid and inhibiting the degradation of retinoic, acid. The boosters act in conjunction with a retinoid (e.g. retinol, retinyl ester, retinal, retinoic acid) the latter being present endogenously in the skin. The preferred compositions, however, include a retinoid in the composition, co-present with a booster, to optimise performance.
The present invention includes, in part, a second composition containing from about 0.0001% to about 50%, preferably from 0.001% to 10%, most preferably from 0.001% to 5% by weight of the composition of at least one booster compound, or a combination of binary, tertiary, quaternary or 5 booster combinations. The combined concentration of the booster combinations of 0.001% to 5% in specified ratios as shown below, inhibit transglutaminase in an in vitro transglutaminase assay to more than 50%, and a cosmetically acceptable vehicle.
The boosters included in the inventive compositions are selected from the group consisting of:

    a. Two boosters, wherein both are selected from the group consisting of B2, B3 and B4;
    b. Binary combinations of boosters selected from the group consisting of B1/B2; B1/B3, B1/B4; B1/B5; B2/B3, B2/B4; B2/B5; B3/B4, B3/B5; B4/B5
    c. Ternary combinations of boosters selected from the group consisting of B1/B2/B3; B1/B2/B4; B1/B2/B5; B1/B3/B4; B1/B3/B5; B1/B4/B5; B2/B3/B4; B2/B3/B5; B2/B4/B5; B3/B4/B5
    d. Quaternary combinations of boosters selected from the group consisting of B1/B2/B3/B4; B1/B2/B3/B5; B1/B2/B4/B5; B1/B3/B4/B5; B2/B3/B4/B5; and
    e. A combination of five groups of boosters B1/B2/B3/B4/B5.

Booster to Booster Ratios:



The boosters of different classes (B1 to B5) in combinations as shown above have an optimal concentration of between 0.001% to 5% in a cosmetic product at specific ratios as shown below for inhibition of Tgase activity to at least below 50%:












Invention
Ratios of boosters to boosters
Concentrations







Broad
1:10,000 to 10,000:1
100 mM to 1 nM 


Preferred
1:1000 to 1000:1
10 mM to 10 nM


Most preferred
1:100 to 100:1
 1 mM to 100 nM


Optimum
1:10 to 10:1
0.1 mM to 1 μM  









Retinoid to Booster Ratios:

The preferred composition includes a retinoid (e.g. retinol, retinyl ester, and retinaldehyde) in the composition, co-present with a booster or a combination of the boosters, to optimise performance.
For optimum performance, the concentration of retinoid to booster should be present in the composition in ratios as given below:













Ratios of boosters



Invention
to retinoids
Concentrations







Broad
10,000:1 to 1:10,000
100 mM-1 nM booster; 0.001-10%




retinoids


Preferred
1000:1 to 1:1000
10 mM-10 nM booster; 0.001-10%




retinoid


Most
100:1 to 1:100
1 mM-100 nM booster; 0.01-1%


preferred

retinoid









Concentrations of Individual Boosters Used in the Examples:

Since the objective is to establish synergistic inhibition of transglutaminase expression by combinations of the active compounds with retinol, it was essential to determine the dose response profiles (IC20 and IC50 values) of the active compounds, when tested individually in the presence of retinol. The detailed dose response of boosters belonging to B2-B4 is given in the tables following the IC50 and IC 20 table below. This data was used to identify an appropriate sub-maximal inhibitory concentration of each active compound, to eventually make it possible to identify putative synergistic effects of the mixtures of the active compounds in the presence of retinol. The data in the following table represents the IC50 and IC20 (80% of control) values and the concentrations used when testing synergies with combinations of boosters.
In order to demonstrate synergy of two compounds, it is essential to select concentrations to test that are at most IC20, in other words, a compound concentration that individually boosts the retinol inhibition of Tgase expression by 20%. Two such compounds should have an additive inhibition of 40%. Using this strategy to determine concentrations leaves a window of 40-100% for further inhibition for detecting synergy of the two compounds under examination.
A more challenging concentration criterion would be selecting concentrations of compounds which alone showed no inhibition effect, but in combination show inhibition. In this study however, we chose an even more challenging criteria. We selected concentrations of compounds that were 10 to 1000 fold lower than the minimally effective Tgase inhibiting concentration. Identification of synergistic combinations using such very low concentrations would mean that the most effective synergistic combinations were identified.


















Con. Used for


Booster



synergy (binary,


Class
Compound Name
IC50
IC20
tertiary, quaternary)







B1
LinoleoylMonoethanolamide
1.61E−05
1.48E−05
1E−05 to 1E−09



(LAMEA)



Palmitamide Monoethanolamide
ND
ND
1E−06 to 1E−10



Oleyl Betaine
2.80E−05
1.08E−05
1E−05 to 1E−8



Naringenin
ND
ND
1E−05 to 1E−09



Echinacea
ND
ND
1E−05 to 1E−09



Dimethyl imidazolinone
ND
ND
1E−05 to 1E−09



Melinamide
ND
ND
1E−05 to 1E−09



Geranyl geraniol
ND
ND
1E−05 to 1E−09



Farnesol
9.35E−05
7.82E−05
1E−06 to 1E−09



Geraniol
7.83E−03
4.72E−03
1E−03 to 1E−07



α-Damascone
3.35E−04
1.69E−04
1E−04 to 1E−08



α-Ionone
9.27E−04
1.42E−04
1E−04 to 1E−08



Castor oil Methyl Ester Acid
3.25E−05
9.38-E06    
1E−06 to 1E−09



(MEA)



Ursolic Acid
1.46E−06
5.94-E07    
1E−06 to 1E−09



Utrecht-2
3.47-E06    
3.30-E06    
1E−06 to 1E−09



Cocoyl
2.84E−07
9.21E−08
1E−08 to 1E−11



hydroxyethylimidazoline



Acetyl sphingosine (C2
6.78E−06
5.15E−06
1E−06 to 1E−09



Ceramide)



Hexanoyl sphingosine (C6
9.99E−05
6.94E−05
1E−05 to 1E−09



Ceramide)



Crocetin
3.75E−05
2.52E−05
1E−05 to 1E−09



Lyrial
1.27E−04
4.00E−05
1E−05 to 1E−09



N-Hydroxyethyl-2-
3.29E−05
2.40E−05
1E−05 to 1E−09



hydroxydodecyl amide


B2
Phosphatidyl Choline
ND
ND
1E−05 to 1E−09



Sphingomyelin
ND
ND
1E−05 to 1E−09



TCC
9.64E−07
6.18-E07    
1E−07 to 1E−10



1,2-dioctanoyl-sn-glycero-3-
ND
ND
1E−05 to 1E−09



phosphoethanolamide


B3
Amsacrine-HCl
6.26E−06
3.30E−06
1E−06 to 1E−09



Carbenoxolone
3.61E−07
2.00E−07
1E−07 to 1E−10



Glycyrrhetinic Acid
8.64E−06
5.96E−06
1E−06to 1E−09



Linoleic Acid
1.63E−04
8.95E−05
1E−05 to 1E−09



Linolenic Acid
1.34E−04
1.21E−04
1E−05 to 1E−09



Arachidonic Acid (Na+ salt)
ND
ND
1E−05 to 1E−09



Myristic Acid
1.72E−05
1.05E−05
1E−05 to 1E−09



Vanilin
9.70E−03
8.47E−03
1E−03 to 1E−06


B4
Hexadecanedioic acid
1.30E−04
8.40E−05
1E−05 to 1E−09



12-Hydroxystearic acid
2.91E−05
1.45E−05
1E−05 to 1E−09



Elaidic acid
6.50E−05
5.88E−05
1E−05 to 1E−09



Linseed oil
ND
ND
1E−05 to 1E−09



Isostearic acid
6.88E−05
6.23E−05
1E−05 to 1E−09



2-Hydroxystearic acid
ND
ND
1E−05 to 1E−09


B5
Climbazole
4.47E−06
2.45E−07
1E−07 to 1E−10



Clotrimazole
ND
ND
1E−05 to 1E−09



Miconazole
2.78E−07
8.42E−08
1E−08 to 1E−11



Coumarin
ND
ND
1E−05 to 1E−09



Ketoconazole
1.85E−07
5.52E−08
1E−08 to 1E−11



3,4,-Dihydro-2(1H)-
ND
ND
1E−05 to 1E−09



quinolinone(Hydrocarbostyril)



2-
3.64E−04
1.70E−04
1E−04 to 1E−08



Hydroxyquinoline(Carbostyril)



Amino Benzotriazole
ND
ND
1E−05 to 1E−09



Lauryl
4.67E−07
2.69E−07
1E−07 to 1E−10



hydroxyethylimidazoline



Quercetin
6.29E−05
5.11E−05
1E−05 to 1E−09



Oleoyl hydroxyethlimidazoline
3.02E−05
5.65E−06
1E−06 to 1E−09



7H-Benzimidazo[2,1-
8.59E−07
4.69E−07
1E−07 to 1E−09



a]Benz[de]-isoquinolin-7-one





ND: Not determined or a clear dose response was not observed. For synergies, a wide range of concentration (4 orders of magnitude 10-5 to 10-9M) was tested.






Dose Response for Boosters Class B2 to B4

The following tables include the data on the dose response of boosters belonging to class B2 to B4. Concentration of boosters are given in Molar; mean Tgase level and Standard deviation of 4 replicates is expressed as % of control (0.1% DMSO and 10-7M retinol). Higher numbers (close to 100 or above 100) indicate no inhibition of Tgase. The lower the number, the more potent the inhibitor is at that concentration. The IC50 and IC20 values were calculated from this dose response table and expressed in the above table.
B2 Class Boosters










Phosphatidyl choline (B2)










Tgase levels



Concentration
(Mean)
Tgase (SD)












4.4E−05
90.9
0.01


1.47E−05 
120.3
10.6


4.89E−06 
70.1
11.4


1.63E−06 
98.8
0.00


5.43E−07 
86.7
6.19


1.8E−07
75.9
20.5


6.0E−08
87.8
3.9


1.2E−08
159
42.3


2.4E−09
85.5
0.39



















Sphingomyelin (B2)










Tgase levels



Concentration
(Mean)
Tgase (SD)












 3.0E−05
45
3.21


 1.0E−05
77.8
25.5


3.33E−06
76.4
7.55


 1.1E−06
98.8
0.00


3.73E−07
91.6
14.9


1.23E−07
70.0
3.63


4.10E−08
74.6
4.19


 8.2E−08
115.2
1.02


1.65E−09
68.4
2.03


3.29E−10
69.2
2.1



















TCC (B2)










Tgase levels



Concentration
(Mean)
Tgase (SD)












1.14E−03
36.3
4.6


 3.8E−04
3.8
0.96


3.31.23E−04  
−3.2
0.91


4.22E−05
−11.2
0


1.41E−06
−.3
4.88


4.69E−07
15.9
3.52


6.26E−08
18.9
3.12


1.25E−08
100.2
23.3


 6.9E−09
77.6
21.2


 1.0E−09
54.4
11.23



















1,2 dioctanoyl-sn-glycero-3-phopshoethanolamide (B2)










Tgase levels



Concentration
(Mean)
Tgase (SD)












 1.6E−04
58.1
2.08


5.33E−05
95.4
21.3


1.78E−05
104
4.01


5.93E−06
129
0.0


1.98E−06
110
8.74


6.58E−07
92.8
15.78


2.19E−09
88.6
12.3


4.39E−08
127.3
3.39


8.78E−09
119
21.1


1.79E−9 
82
15.6



















B3 Class boosters


Amscrine B3










Tgase levels



Concentration
(Mean)
Tgase (SD)












 3.0E−05
−10
3.29


 1.0E−05
1.8
7.45


3.33E−06
64
4.2


 1.1E−06
84
0


3.73E−07
109
6.2


1.23E−07
65
15.8


4.10E−08
110
10.5


 8.2E−08
131
27


1.65E−09
113
18


3.29E−10
92
8.9



















Carbenoxolone (B3)










Tgase levels



Concentration
(Mean)
Tgase (SD)












 3.0E−06
−7.1
0


 1.0E−06
27.3
1.15


3.33E−07
51.7
0


 1.1E−07
158
0


3.73E−08
126
4.67


1.23E−08
81
29


4.10E−09
135
6.88


 8.2E−10
112
32


1.65E−10
77.8
10.6


3.29E−11
64
49



















Glyrrhetinic acid (B3)










Tgase levels



Concentration
(Mean)
Tgase (SD)












 3.0E−04
−0.3
3.9


 1.0E−05
0.7
3.55


3.33E−05
2.5
2.1


 1.1E−06
96.4
0.00


3.73E−06
120
33.2


1.23E−07
112
38


4.10E−07
93
11


 8.2E−08
225
108


1.65E−08
103
11


3.29E−9 
100
6.2



















Linoleic acid (B3)










Tgase levels



Concentration
(Mean)
Tgase (SD)












 9.0E−03
−6
3.06


 3.0E−03
0.1
2.01


  1E−03
−16.4
16.3


 1.1E−04
4.4
0


3.73E−04
79.2
0


1.23E−05
62.6
6.2


4.10E−05
76.8
3.69


 8.2E−06
146
44.2


1.65E−07
106
20.2


3.29E−07
60.2
2.3



















Linolenic acid (B3)










Tgase levels



Concentration
(Mean)
Tgase (SD)












 9.0E−03
−11
8.7


 3.0E−03
−5.7
0.74


  1E−03
−7.5
7.8


 1.1E−04
−23
0


3.73E−04
68
0.57


1.23E−05
94.9
17.2


4.10E−05
65.9
0.03


 8.2E−06
119
1.6


1.65E−07
77
8.5


3.29E−07
98
7.0



















Myristic acid (B3)










Tgase levels



Concentration
(Mean)
Tgase (SD)












  1E−03
−2
4.1


 1.1E−04
−8
2.3


3.73E−04
−6
1.16


1.23E−05


4.10E−05
75.1
1.06


 8.2E−06
74.2
10.0


1.65E−07
88.9
8.4


3.29E−07
101
4.47


 5.0E−08


 1.1E−08



















Vanillin (B3)










Tgase levels



Concentration
(Mean)
Tgase (SD)












 1.4E−02
21.5
24.2


 4.8E−03
93.8
1.7


  1E−03
124
15.6


 1.1E−04


3.73E−04
101
14.3


1.23E−05
82
14.6


4.10E−05
98
2.4


 8.2E−06
109
22


1.65E−07
80
4


3.29E−07
93
41



















B4 Class boosters


Hexadecanedioic acid (B4)










Tgase levels



Concentration
(Mean)
Tgase (SD)












  1E−03




 1.1E−04
14.2
2.7


3.73E−04
43.4
8.4


1.23E−05
130
0


4.10E−05
105
14


 8.2E−06
114
12


1.65E−07
95
1.9


3.29E−07


 5.0E−08
74
6.7


 1.1E−08
70
10.4



















12-hydroxysteric acid (B4)










Tgase levels



Concentration
(Mean)
Tgase (SD)












3.73E−04




1.23E−05
−5.2
2.3


4.10E−05
32.4
5.3


 8.2E−06
97.6
0


1.65E−07
90.2
11


3.29E−07
82
28


 5.0E−08
81
3.8


 1.1E−08
98
24


 2.0E−08
118
28


 4.3E−09
71
2.3



















Elaidic acid (B4)










Tgase levels



Concentration
(Mean)
Tgase (SD)












  1E−03
12.8
12.1


 1.1E−04
8
0.45


3.73E−04
13.8
1.92


1.23E−05
80.9
0


4.10E−05
58.2
8.8


 8.2E−06


1.65E−07
58
0.13


3.29E−07
69
44


 5.0E−08
50.5
3.8


 1.1E−08



















Linseed Oil (B4)










Tgase levels



Concentration
(Mean)
Tgase (SD)












  1E−04
138
15


3.73E−05
145
2.5


1.23E−05
88
12


4.10E−06
113
0


 8.2E−06
113
13


1.65E−07
96
18


3.29E−07
106
10


 5.0E−08
134
22


 1.1E−09
83
13


 9.9E−10
73
15



















Isosteric acid (B4)










Tgase levels



Concentration
(Mean)
Tgase (SD)












  1E−03
−8.6
3.4


 1.1E−04
1.2
3.0


3.73E−04
−5.3
1.1


1.23E−05
80
00


4.10E−05
67
7.9


 8.2E−06
103
12.3


1.65E−07
95
5.5


3.29E−07
123
0.5


 5.0E−08
78
12.2


 1.1E−08
78
29



















2-hydroxysteric acid (B4)










Tgase levels



Concentration
(Mean)
Tgase (SD)












 9.1E−04
46.6
6.2


3.73E−04
69.3
8.3


1.23E−04
51
8.8


3.10E−05
96.0
0.0


 1.2E−05
105
30


3.65E−06
63
8.0


1.29E−06
80
4.7


 2.0E−07
142
34


 5.1E−08
64
20


 1.0E−08
58
17









Synergy of Tgase Inhibition with Binary Combinations of Boosters

To investigate synergistic inhibition of Tgase expression by combinations of 2 different classes of boosters with retinol, selected combinations of compounds were tested at concentrations given in the above table. The concentrations tested were one log order of magnitude less than the concentration required for minimal inhibition of Tgase activity (i.e. IC20). The compounds were tested alone and in combination and the % inhibition of Tgase is given for each compound and the combination.
The following examples give the synergistic combinations in all possible binary combinations (B1/B2; B1/B3, B1/B4; B1/B5; B2/B3, B2/B4; B2/B5; B3/B4, B3/B5; B4/B5). When the % inhibition of the combination is more than the inhibition of each compound added together, it indicates synergy (i.e. Inhibition by combination is greater than inhibition by compound 1+compound 2). All the binary combination examples given in the following table synergistically inhibited Tgase.


















TG as
TG as



Binary


% C
% C
TG % C


combinations
Compound 1
Compound 2
Compd 1
Compd 2
Combination




















B1/B2
Dimethyl
Phosphatidylcholine
99
97
84



imidazolidinone


B1/B2
Alpha-demascone
Phospahtidylcholine
95
97
86


B1/B2
Hexanoyl sphingosine
Phospahtidylcholine
109
97
86


B1/B2
Alpha-ionone
Sphingomyelin
101
98
76


B1/B2
1,2 dioctanoyl-sn-
Phosphatidyl choline
106
98
78



glycero-3-



phosphoethanolamide


B1/B2
Alpha-demascone
Sphingomyelin
95
84
67


B1/B3
1,2 dioctanoyl-sn-
Amsacrine
123
134
75



glycero-3-



phosphoethanolamide


B1/B3
1,2 dioctanoyl-sn-
Carbenoxelone
123
164
96



glycero-3-



phosphoethanolamide


B1/B3
Caster oil MEA
Carbenoxelone
96
164
67


B1/B3
Utrecht-2
Amsacrine
102
98
86


B1/B3
Utrecht-2
Carbenoxelone
102
164
91


B1/B3
Hexanoyl sphingosine
Carbenoxelone
122
164
78


B1/B3
Lyral
Carbenoxelone
120
164
82


B1/B3
Castor oil MEA
Carbenoxelone
110
164
78


B1/B3
Hexanoyl sphingosine
Amsacrine
122
134
92


B1/B3
Hexanoyl sphingosine
Eliadic acid
122
144
85


B1/B3
Alpha ionone
Amsacrine
101
134
78


B1/B3
1,2 dioctanoyl-sn-
Glyccyrrhetinic acid
95
92
69



glycero-3-



phosphoethanolamide


B1/B4
Naringenin
2-hydroxy steric acid
95
112
78


B1/B4
Hexanoyl sphingosine
2-hydroxy steric acid
99.3
112
77


B1/B4
Lyral
Hexadecanoic acid
120
95
69


B1/B4
Castor oil MEA
Hexadecanedioic acid
110
125
82


B1/B4
Hexanoyl sphingosine
Isostearic acid
122
146
93


B1/B4
Oleoyl betaine
Hexadecanedioic acid
99.5
125
80


B1/B5
Hexanoyl sphingosine
Cocoyl
99
102
68




hydorxyethylimidazoline


B1/B5
Farnesol
Ketokonazole
98
111
84


B1/B5
Hexanoyl sphingosine
Miconazole
99
101
56


B1/B5
Hexanoyl sphingosine
Ketoconazole
99
99
65


B1/B5
Hexanoyl sphingosine
Lauryl
99
98
51




hydroxyethylimiazoline


B1/B5
Utrecht-2
Amino benzotriazole
122
105
83


B1/B5
Hexanoyl sphingosine
3,4-dihydro-2
122
102
89




quinolinone


B1/B5
Hexanoyl sphingosine
Amino benzotriazole
122
126
85


B1/B5
Castor oil MEA
Lauryl
110
98
56




hydroxyethylimiazoline


B1/B5
Hexanoyl sphingosine
Climbazole
122
98
83


B1/B5
Hexanoyl sphingosine
Miconazole
122
99
78


B1/B5
Hexanoyl sphingosine
Ketoconazole
122
110
90


B1/B5
Olecyl beatine
ketoconazole
96
116
81


B1/B5
Utrecht-2
Lauryl
122
98
57




hydroxyethylimiazoline


B1/B5
Alpha-demascone
Oleoyl
112
73
76




hydroxyethylimiazoline


B1/B5
Alpha-ionone
Lauryl
101
98
49




hydroxyethylimiazoline


B1/B5
Alpha-ionone
Oleoyl
101
73
75




hydroxyethylimiazoline


B2/B3
Phosphatidyl choline
Glycyrrhetinic acid
98
92
73


B2/B4
Phosphatidyl choline
2-hydroxy steric acid
98
82
70


B2/B5
Phosphatidyl choline
Climbazole
98
102
82


B2/B5
Phosphatidyl choline
Miconazole
98
111
92


B2/B5
Phosphatidyl choline
Ketoconazole
98
101
89


B2/B5
Phosphatidyl choline
Lauryl
98
106
82




hydorxyimidazoline


B3/B4
Amscarine
2-hydroxy steric acid
102
82
75


B3/B4
Myristic acid
2-hydroxy steric acid
110
82
78


B3/B5
Amscarine
Aminobenzotriazole
102
98
84


B3/B5
Amscarine
Dimethyl imidazoline
102
112
94


B3/B5
Myristic acid
Climbazole
110
102
82


B4/B5
Linseed oil
Lauryl hydroxyethyl
98
73
57




imidazoline


B4/B5
2-hydroxystearic acid
Ketaconazole
92
109
77


B4/B5
Linseed oil
Oleoyl
98
92
75




hydorxyethylimdazoline


B4/B5
2-hydroxystearic acid
Coumarin
92
96
70









Synergy of Tgase Inhibition with Tertiary Combinations of Boosters

To investigate synergistic inhibition of Tgase expression by combinations of 3 different classes of boosters with retinol, selected combinations of compounds were tested. The concentrations tested were one log order of magnitude less than the concentration required for minimal inhibition of Tgase activity (i.e. IC20). The compounds were tested alone and in combination and the % inhibition of Tgase is given for each compound and the combination. The following examples give the synergistic combinations in all possible tertiary combinations (B1/B2/B3; B1/B2/B4; B1/B2/B5; B1/B3/B4; B1/B3/B5; B1/B4/B5; B2/B3/B4; B2/B3/B5; B2/B4/B5; B3/B4/B5). The % inhibition of the combination is more than the inhibition of each compound added together, which indicates synergy (i.e. Inhibition by combination is greater than inhibition by compound 1+compound 2+compound 3). All the examples of tertiary combinations of boosters given in the following table synergistically inhibited Tgase in the presence of 10-7M retinol





















TG as
TG as





TG as % C
TG as % C
% C
% C


Compound 1
Compound 2
Compound 3
Compd 1
Compd 2
Compd 3
Combo















B1/B2/B3 combinations:













Phosphatidyl Choline
Glycyrrhetinic
Castor oil Methyl
88
91
85
53



Acid
Ester Acid (MEA)


Phosphatidyl Choline
Glycyrrhetinic
Echinacea
88
91
119
52



Acid


Phosphatidyl Choline
Glycyrrhetinic
Naringenin
88
91
94
52



Acid


Phosphatidyl Choline
Glycyrrhetinic
Acetyl sphingosine
88
91
99
58



Acid
(C2 Ceramide)


Phosphatidyl Choline
Glycyrrhetinic
Farnesol
88
91
118
49



Acid


1,2-dioctanoyl-sn-
Glycyrrhetinic
a-Damascone
81
91
89
58


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Phosphatidyl
Naringenin
81
88
94
66


glycero-3-
Choline


phosphoethanolamide


1,2-dioctanoyl-sn-
Amsacrine-HCl
Linoleoyl
81
79
127
60


glycero-3-

Monoethanolamide


phosphoethanolamide

(LAMEA)


1,2-dioctanoyl-sn-
Amsacrine-HCl
Palmitamide
81
79
95
63


glycero-3-

Monoethanolamide


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
a-Damascone
81
91
89
58


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Naringenin
81
91
94
75


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Echinacea
81
91
119
77


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Dimethyl
81
91
87
67


glycero-3-
Acid
imidazolinone


phosphoethanolamide


Castor oil Methyl
Carbenoxelone
Phosphatidyl
85
95
88
63


Ester Acid (MEA)

Choline







B1/B2/B4


Combinations:


B1/B2/B5


Combinations:













Phosphatidyl Choline
Climbazole
Echinacea
88
84
119
75


Phosphatidyl Choline
Climbazole
Naringenin
88
84
94
83


Phosphatidyl Choline
Climbazole
Geraniol
88
84
105
76


Phosphatidyl Choline
Climbazole
Farnesol
88
84
118
82


Phosphatidyl Choline
Climbazole
Acetyl sphingosine
88
84
99
82




(C2 Ceramide)


Phosphatidyl Choline
Miconazole
a-Ionone
88
92
88
70


Phosphatidyl Choline
Miconazole
Castor oil Methyl
88
92
85
72




Ester Acid (MEA)







B1/B3/B4


Combinations:













Amsacrine-HCl
Dimethyl
Elaidic acid
79
87
93
0



imidazolinone


□-Ionone
Amsacrine-HCl
12-Hydroxystearic
68
79
95
62




acid


Lyrial
Hexadecanedioic
Vanillin
97
90
134
81



acid


Hexanoyl sphingosine
Isostearic acid
Glycyrrhetinic
104
87
91
58


(C6 Ceramide)

Acid







B1/B3/B5


Combinations:













Amsacrine-HCl
Dimethyl
2-
79
87
95
32



imidazolinone
Hydroxyquinoline(Carbostyril)


Amsacrine-HCl
Dimethyl
Lauryl
79
87
52
−13



imidazolinone
hydroxyethylimidazoline


Amsacrine-HCl
Dimethyl
Quercetin
79
87
92
−24



imidazolinone


Amsacrine-HCl
Dimethyl
Oleoyl
79
87
76
39



imidazolinone
hydroxyethlimidazoline


Amsacrine-HCl
Dimethyl
7H-
79
87
94
32



imidazolinone
Benzimidazo[2,1-




a]Benz[de]-




isoquinolin-7-one


Amsacrine-HCl
Dimethyl
Coumarin
79
87
80
30



imidazolinone


Hexanoyl
Carbenoxolone
Oleoyl
104
88
76
64


sphingosine (C6

hydroxyethlimidazoline


Ceramide)


Hexanoyl
3,4,-Dihydro-
Vanillin
104
90
134
62


sphingosine (C6
2(1H)-


Ceramide)
quinolinone(Hydrocarbostyril)


Amsacrine-HCl
Amino
Echinacea
79
105
119
48



Benzotriazole


Hexanoyl
Amino
Sphingomyelin
104
105
60
69


sphingosine (C6
Benzotriazole


Ceramide)


Amsacrine-HCl
Amino
Acetyl sphingosine
79
105
99
−7



Benzotriazole
(C2 Ceramide)


□-Ionone
Amsacrine-HCl
7H-
68
79
94
54




Benzimidazo[2,1-




a]Benz[de]-




isoquinolin-7-one


Utrecht-2
Carbenoxolone
Quercetin
76
88
92
74


Utrecht-2
Carbenoxolone
Oleoyl
76
88
76
69




hydroxyethlimidazoline


Utrecht-2
Carbenoxolone
7H-
76
88
94
73




Benzimidazo[2,1-




a]Benz[de]-




isoquinolin-7-one


Utrecht-2
Carbenoxolone
3,4,-Dihydro-
76
88
90
70




2(1H)-




quinolinone(Hydrocarbostyril)


Myristic Acid
Climbazole
Geraniol
79
84
105
74


Myristic Acid
Climbazole
□-Damascone
79
84
89
73


Myristic Acid
Climbazole
Acetyl sphingosine
79
84
99
70




(C2 Ceramide)


Oleyl Betaine
Ketoconazole
Carbenoxolone
62
85
88
78


Oleyl Betaine
Ketoconazole
Glycyrrhetinic
62
85
91
71




Acid


Oleyl Betaine
Ketoconazole
Linoleic Acid
62
85
11
83


Oleyl Betaine
Ketoconazole
Linolenic Acid
62
85
208
80


Hexanoyl sphingosine
3,4,-Dihydro-
Vanillin
104
90
134
62


(C6 Ceramide)
2(1H)-



quinolinone(Hydrocarbostyril)







B1/B4/B5


Combinations:













Elaidic acid
2-Hydroxyquinoline
Castor oil Methyl
93
95
85
75



(Carbostyril)
Ester Acid (MEA)


Elaidic acid
2-Hydroxyquinoline
Naringenin
93
95
94
86



(Carbostyril)


Elaidic acid
2-Hydroxyquinoline
a-Demascone
93
95
89
80



(Carbostyril)


Elaidic acid
2-Hydroxyquinoline
Farnesol
93
95
118
82



(Carbostyril)


Elaidic acid
2-Hydroxyquinoline
Crocetin
93
95
90
78



(Carbostyril)







B2/B3/B4


Combinations:













1,2-dioctanoyl-sn-
Glycyrrhetinic
12-Hydroxystearic
81
91
95
57


glycero-3-
Acid
acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Linseed oil
81
91
103
62


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Elaidic acid
81
91
93
75


glycero-3-
Acid


phosphoethanolamide


Phosphatidyl Choline
2-Hydroxystearic
Arachidonic Acid
88
83
78
60



acid
(Na+ salt)







B2/B3/B5


Combinations:













Phosphatidyl Choline
Climbazole
Linolenic Acid
88
84
208
84


Phosphatidyl Choline
Climbazole
Arachidonic Acid
88
84
78
83




(Na+ salt)


1,2-dioctanoyl-sn-
Amsacrine-HCl
Climbazole
81
79
84
58


glycero-3-


phosphoethanolamide


1,2-dioctanoyl-sn-
Amsacrine-HCl
7H-
81
79
94
59


glycero-3-

Benzimidazo[2,1-


phosphoethanolamide

a]Benz[de]-




isoquinolin-7-one


1,2-dioctanoyl-sn-
Glycyrrhetinic
3,4,-Dihydro-
81
91
90
56


glycero-3-
Acid
2(1H)-


phosphoethanolamide

quinolinone(Hydrocarbostyril)


1,2-dioctanoyl-sn-
Glycyrrhetinic
2-
81
91
95
75


glycero-3-
Acid
Hydroxyquinoline(Carbostyril)


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Amino
81
91
105
72


glycero-3-
Acid
Benzotriazole


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Lauryl
81
91
52
79


glycero-3-
Acid
hydroxyethylimidazoline


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Quercetin
81
91
92
73


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Climbazole
81
91
84
54


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Clotrimazole
81
91
79
42


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Miconazole
81
91
82
43


glycero-3-
Acid


phosphoethanolamide







B2/B4/B5


Combinations:













Phosphatidyl Choline
2-Hydroxystearic
Amino Beozotriazole
88
83
105
77



acid


Phosphatidyl Choline
2-Hydroxystearic
Lauryl
88
83
52
74



acid
hydroxyethylimidazoline


Phosphatidyl Choline
2-Hydroxystearic
Quercetin
88
83
92
69



acid


Phosphatidyl Choline
2-Hydroxystearic
Oleoyl
88
83
76
75



acid
hydroxyethlimidazoline


Phosphatidyl Choline
2-Hydroxystearic
7H-Benzimidazo[2,1-
88
83
94
79



acid
a]Benz[de]-isoquinolin-7-one


Phosphatidyl Choline
Climbazole
Elaidic acid
88
84
93
81







B3/B4/B5


Combinations:













Elaidic acid
2-Hydroxyquinoline
Carbenoxolone
93
95
88
69



(Carbostyril)


Elaidic acid
2-Hydroxyquinoline
Vanillin
93
95
134
81



(Carbostyril)


Amsacrine-HCl
Amino
Linseed oil
79
105
103
45



Benzotriazole


Myristic Acid
Climbazole
12-Hydroxystearic acid
79
84
95
81


Myristic Acid
Climbazole
Linseed oil
79
84
103
81


Elaidic acid
2-Hydroxyquinoline
Arachidonic Acid (Na+ salt)
93
95
78
63



(Carbostyril)









Synergy of Tgase Inhibition with Quaternary Combinations of Boosters

To investigate synergistic inhibition of Tgase expression by combinations of 4 different classes of boosters with retinol, selected combinations of compounds were tested. The concentrations tested were one log order of magnitude less than the concentration required for minimal inhibition of Tgase activity (i.e. IC20).
The compounds were tested alone and in combination and the % inhibition of Tgase is given for each compound and the combination. The following examples give the synergistic combinations in all possible quaternary combinations (B1/B2/B3/B4; B1/B2/B3/B5; B1/B2/B4/B5; B1/B3/B4/B5; B2/B3/B4/B5). Synergy was confirmed if the difference in % inhibition of the combination (of 4 boosters) is more than 30% that of the inhibition by 3 booster combinations (i.e. % inhibition of 4 booster combo is equal to or greater than % inhibition of 3 booster combo+30%). All the quaternary combinations of boosters shown in the table given below showed synergy.





















Tertiary







Quarternary
(1-3
Difference






TG
combo;
(<30% =


Compound 1
Compound 2
Compound 3
Compound 4
(% C)
TG % C)
synergy)















B1/B2/B3/B4 Combination:













Castor oil Methyl
Phosphatidyl
Glycyrrhetinic
12-Hydroxy-
21
64
42


Ester Acid (MEA)
Choline
Acid
stearic acid


Naringenin
Phosphatidyl
Glycyrrhetinic
12-Hydroxy-
15
57
41



Choline
Acid
stearic acid


Linoleoyl
1,2-dioctanoyl-
Glycyrrhetinic
12-Hydroxy-
−3
40
43


Monoethanolamide
sn-glycero-3-
Acid
stearic acid


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Glycyrrhetinic
Isostearic acid
5
40
35


Monoethanolamide
sn-glycero-3-
Acid


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
12-Hydroxy-
−3
42
45


Monoethanolamide
sn-glycero-3-

stearic acid


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
Elaidic acid
8
42
34


Monoethanolamide
sn-glycero-3-


(LAMEA)
phosphoethanol-



amide


Hexanoyl
TCC
Glycyrrhetinic
Isostearic acid
7
54
47


sphingosine (C6

Acid


Ceramide)


Lyrial
TCC
Vanilin
Hexadecanedioic
10
48
38





acid


Cocoyl
1,2-dioctanoyl-
Glycyrrhetinic
Isostearic acid
0
37
37


hydroxyethylimidazoline
sn-glycero-3-
Acid



phosphoethanol-



amide


Cocoyl
Phosphatidyl
Arachidonic Acid
2-Hydroxy-
−1
37
38


hydroxyethylimidazoline
Choline
(Na+ salt)
stearic acid


Cocoyl
1,2-dioctanoyl-
Glycyrrhetinic
Linseed oil
−2
45
47


hydroxyethylimidazoline
sn-glycero-3-
Acid



phosphoethanol-



amide







B1/B2/B3/B5


Combination:













Castor oil Methyl
Phosphatidyl
Glycyrrhetinic
Climbazole
20
64
44


Ester Acid (MEA)
Choline
Acid


Castor oil Methyl
Phosphatidyl
Glycyrrhetinic
Clotrimazole
26
64
38


Ester Acid (MEA)
Choline
Acid


Castor oil Methyl
Phosphatidyl
Glycyrrhetinic
Miconazole
9
64
55


Ester Acid (MEA)
Choline
Acid


Castor oil Methyl
Phosphatidyl
Glycyrrhetinic
Ketoconazole
5
64
59


Ester Acid (MEA)
Choline
Acid


Castor oil Methyl
Phosphatidyl
Glycyrrhetinic
Lauryl
15
64
49


Ester Acid (MEA)
Choline
Acid
hydroxyethylimidazoline


Castor oil Methyl
Phosphatidyl
Glycyrrhetinic
Oleoyl
2
64
61


Ester Acid (MEA)
Choline
Acid
hydroxyethlimidazoline


Castor oil Methyl
Phosphatidyl
Glycyrrhetinic
7H-Benzimidazo[2,1-
25
64
39


Ester Acid (MEA)
Choline
Acid
a]Benz[de]-isoquinolin-





7-one


Echinacea
Phosphatidyl
Glycyrrhetinic
12-Hydroxystearic acid
18
62
44



Choline
Acid


Echinacea
Phosphatidyl
Glycyrrhetinic
Climbazole
22
62
40



Choline
Acid


Echinacea
Phosphatidyl
Glycyrrhetinic
Clotrimazole
24
62
38



Choline
Acid


Echinacea
Phosphatidyl
Glycyrrhetinic
Miconazole
13
62
50



Choline
Acid


Echinacea
Phosphatidyl
Glycyrrhetinic
Ketoconazole
12
62
50



Choline
Acid


Echinacea
Phosphatidyl
Glycyrrhetinic
Lauryl
14
62
49



Choline
Acid
hydroxyethylimidazoline


Echinacea
Phosphatidyl
Glycyrrhetinic
Oleoyl
3
62
59



Choline
Acid
hydroxyethlimidazoline


Echinacea
Phosphatidyl
Glycyrrhetinic
7H-Benzimidazo[2,1-
24
62
39



Choline
Acid
a]Benz[de]-isoquinolin-





7-one


Naringenin
Phosphatidyl
Glycyrrhetinic
Miconazole
1
57
56



Choline
Acid


Naringenin
Phosphatidyl
Glycyrrhetinic
Ketoconazole
22
57
34



Choline
Acid


Naringenin
Phosphatidyl
Glycyrrhetinic
Lauryl
10
57
46



Choline
Acid
hydroxyethylimidazoline


Naringenin
Phosphatidyl
Glycyrrhetinic
Oleoyl
2
57
54



Choline
Acid
hydroxyethlimidazoline


Naringenin
Phosphatidyl
Glycyrrhetinic
7H-Benzimidazo[2,1-
15
57
42



Choline
Acid
a]Benz[de]-isoquinolin-





7-one


Palmitamide
Phosphatidyl
Glycyrrhetinic
Miconazole
−2
39
41


Monoethanolamide
Choline
Acid


Palmitamide
Phosphatidyl
Glycyrrhetinic
Oleoyl
6
39
33


Monoethanolamide
Choline
Acid
hydroxyethlimidazoline


Farnesol
Phosphatidyl
Glycyrrhetinic
Miconazole
3
43
40



Choline
Acid


Farnesol
Phosphatidyl
Glycyrrhetinic
Oleoyl
6
43
37



Choline
Acid
hydroxyethlimidazoline


Geraniol
1,2-dioctanoyl-
Amsacrine-HCl
Miconazole
11
47
36



sn-glycero-3-



phosphoethanol-



amide


Geraniol
1,2-dioctanoyl-
Amsacrine-HCl
Oleoyl
3
47
44



sn-glycero-3-

hydroxyethlimidazoline



phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Glycyrrhetinic
Climbazole
2
40
37


Monoethanolamide
sn-glycero-3-
Acid


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Glycyrrhetinic
Miconazole
5
40
35


Monoethanolamide
sn-glycero-3-
Acid


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Glycyrrhetinic
Ketoconazole
0
40
40


Monoethanolamide
sn-glycero-3-
Acid


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Glycyrrhetinic
Lauryl
−2
40
41


Monoethanolamide
sn-glycero-3-
Acid
hydroxyethylimidazoline


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Glycyrrhetinic
Oleoyl
5
40
35


Monoethanolamide
sn-glycero-3-
Acid
hydroxyethlimidazoline


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Glycyrrhetinic
7H-Benzimidazo[2,1-
1
40
39


Monoethanolamide
sn-glycero-3-
Acid
a]Benz[de]-isoquinolin-


(LAMEA)
phosphoethanol-

7-one



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
Climbazole
7
42
35


Monoethanolamide
sn-glycero-3-


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
Clotrimazole
10
42
32


Monoethanolamide
sn-glycero-3-


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
Miconazole
5
42
37


Monoethanolamide
sn-glycero-3-


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
Ketoconazole
11
42
32


Monoethanolamide
sn-glycero-3-


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
Lauryl
−4
42
46


Monoethanolamide
sn-glycero-3-

hydroxyethylimidazoline


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
Oleoyl
5
42
37


Monoethanolamide
sn-glycero-3-

hydroxyethlimidazoline


(LAMEA)
phosphoethanol-



amide


Linoleoyl
1,2-dioctanoyl-
Amsacrine-HCl
7H-Benzimidazo[2,1-
8
42
35


Monoethanolamide
sn-glycero-3-

a]Benz[de]-isoquinolin-


(LAMEA)
phosphoethanol-

7-one



amide


Palmitamide
1,2-dioctanoyl-
Amsacrine-HCl
Miconazole
13
43
30


Monoethanolamide
sn-glycero-3-



phosphoethanol-



amide


Palmitamide
1,2-dioctanoyl-
Amsacrine-HCl
Oleoyl
3
43
40


Monoethanolamide
sn-glycero-3-

hydroxyethlimidazoline



phosphoethanol-



amide


Alpha-Damascone
1,2-dioctanoyl-
Amsacrine-HCl
Miconazole
11
48
37



sn-glycero-3-



phosphoethanol-



amide


Alpha-Damascone
1,2-dioctanoyl-
Amsacrine-HCl
Ketoconazole
13
48
34



sn-glycero-3-



phosphoethanol-



amide


Alpha-Damascone
1,2-dioctanoyl-
Amsacrine-HCl
Lauryl
15
48
33



sn-glycero-3-

hydroxyethylimidazoline



phosphoethanol-



amide


Alpha-Damascone
1,2-dioctanoyl-
Amsacrine-HCl
Oleoyl hydroxyethlimidazoline
3
48
45



sn-glycero-3-



phosphoethanol-



amide


Castor oil Methyl
Phosphatidyl
Carbenoxolone
12-Hydroxystearic acid
3
55
52


Ester Acid (MEA)
Choline


Castor oil Methyl
Phosphatidyl
Carbenoxolone
Climbazole
6
55
49


Ester Acid (MEA)
Choline


Castor oil Methyl
Phosphatidyl
Carbenoxolone
Miconazole
−2
55
57


Ester Acid (MEA)
Choline


Castor oil Methyl
Phosphatidyl
Carbenoxolone
Ketoconazole
1
55
54


Ester Acid (MEA)
Choline


Castor oil Methyl
Phosphatidyl
Carbenoxolone
Lauryl hydroxyethylimidazoline
4
55
51


Ester Acid (MEA)
Choline


Castor oil Methyl
Phosphatidyl
Carbenoxolone
Oleoyl
3
55
52


Ester Acid (MEA)
Choline

hydroxyethlimidazoline


Castor oil Methyl
Phosphatidyl
Carbenoxolone
7H-Benzimidazo[2,1-
11
55
44


Ester Acid (MEA)
Choline

a]Benz[de]-isoquinolin-





7-one


Naringenin
Phosphatidyl
Linoleic Acid
Climbazole
−1
45
46



Choline


Geraniol
Phosphatidyl
Linoleic Acid
Climbazole
1
40
39



Choline


Acetyl sphingosine
Phosphatidyl
Linoleic Acid
Climbazole
0
40
40


(C2 Ceramide)
Choline


Acetyl sphingosine
Phosphatidyl
Linolenic Acid
Climbazole
10
40
30


(C2 Ceramide)
Choline


Dimethyl
TCC
Amsacrine-HCl
Elaidic acid
14
47
33


imidazolinone


Dimethyl
TCC
Amsacrine-HCl
Quercetin
12
44
32


imidazolinone


Dimethyl
TCC
Amsacrine-HCl
Coumarin
14
58
44


imidazolinone


Hexanoyl
TCC
Glycyrrhetinic
Amino Benzotriazole
8
48
40


sphingosine (C6

Acid


Ceramide)


Alpha-Damascone
TCC
Myristic Acid
Climbazole
10
44
34







B1/B2/B4/B5


Combination:













Lyrial
Vanilin
Hexadecanedioic
Miconazole
12
48
36




acid


Lyrial
Vanilin
Hexadecanedioic
Oleoyl
4
48
45




acid
hydroxyethlimidazoline


Crocetin
TCC
Elaidic acid
2-
11
48
37





Hydroxyquinoline(Carbostyril)


Hexanoyl
Glycyrrhetinic
12-Hydroxystearic
Amino Benzotriazole
14
48
33


sphingosine (C6
Acid
acid


Ceramide)


Dimethyl
Phosphatidyl
2-Hydroxystearic
7H-Benzimidazo[2,1-
2
44
42


imidazolinone
Choline
acid
a]Benz[de]-isoquinolin-





7-one


Melinamide
Phosphatidyl
2-Hydroxystearic
7H-Benzimidazo[2,1-
5
44
39



Choline
acid
a]Benz[de]-isoquinolin-





7-one


Geranyl geraniol
Phosphatidyl
2-Hydroxystearic
7H-Benzimidazo[2,1-
9
44
35



Choline
acid
a]Benz[de]-isoquinolin-





7-one


Cocoyl
Phosphatidyl
2-Hydroxystearic
7H-Benzimidazo[2,1-
−8
44
52


hydroxyethylimidazoline
Choline
acid
a]Benz[de]-isoquinolin-





7-one


Acetyl sphingosine
Phosphatidyl
2-Hydroxystearic
7H-Benzimidazo[2,1-
10
44
34


(C2 Ceramide)
Choline
acid
a]Benz[de]-isoquinolin-





7-one


Crocetin
Phosphatidyl
2-Hydroxystearic
7H-Benzimidazo[2,1-
10
44
34



Choline
acid
a]Benz[de]-isoquinolin-





7-one


N,N-Diethyl
Phosphatidyl
2-Hydroxystearic
7H-Benzimidazo[2,1-
4
44
40


Cocamide(Cocamide
Choline
acid
a]Benz[de]-isoquinolin-


DEA)


7-one


Cocoyl
Phosphatidyl
Elaidic acid
Climbazole
−4
30
34


hydroxyethylimidazoline
Choline







B1/B3/B4/B5


Combination:













Dimethyl
Amsacrine-HCl
Elaidic acid
Miconazole
7
47
40


imidazolinone


Dimethyl
Amsacrine-HCl
Elaidic acid
Ketoconazole
6
47
41


imidazolinone


Dimethyl
Amsacrine-HCl
Elaidic acid
Oleoyl
3
47
44


imidazolinone


hydroxyethlimidazoline


Hexanoyl
Glycyrrhetinic
Isostearic acid
Clotrimazole
20
54
34


sphingosine (C6
Acid


Ceramide)


Hexanoyl
Glycyrrhetinic
Isostearic acid
Miconazole
10
54
43


sphingosine (C6
Acid


Ceramide)


Hexanoyl
Glycyrrhetinic
Isostearic acid
Lauryl
20
54
33


sphingosine (C6
Acid

hydroxyethylimidazoline


Ceramide)


Hexanoyl
Glycyrrhetinic
Isostearic acid
Oleoyl
5
54
48


sphingosine (C6
Acid

hydroxyethlimidazoline


Ceramide)


Crocetin
Linoleic Acid
Elaidic acid
2-Hydroxyquinoline
0
48
48





(Carbostyril)


Crocetin
Linolenic Acid
Elaidic acid
2-Hydroxyquinoline
−2
48
50





(Carbostyril)


Castor oil Methyl
Linoleic Acid
Elaidic acid
2-Hydroxyquinoline
−1
31
32


Ester Acid (MEA)


(Carbostyril)


Cocoyl
Carbenoxolone
Elaidic acid
2-Hydroxyquinoline
−6
28
34


hydroxyethylimidazoline


(Carbostyril)







B2/B3/B4/B5


Combination:













1,2-dioctanoyl-sn-
Glycyrrhetinic
Isostearic acid
Ketoconazole
4
37
33


glycero-3-
Acid


phosphoethanol-


amide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Isostearic acid
Oleoyl
6
37
31


glycero-3-
Acid

hydroxyethlimidazoline


phosphoethanol-


amide


Phosphatidyl
Arachidonic
2-Hydroxystearic
Miconazole
6
37
31


Choline
Acid (Na+ salt)
acid


Phosphatidyl
Arachidonic
2-Hydroxystearic
Oleoyl
5
37
32


Choline
Acid (Na+ salt)
acid
hydroxyethlimidazoline


1,2-dioctanoyl-sn-
Glycyrrhetinic
Linseed oil
Miconazole
−1
45
47


glycero-3-
Acid


phosphoethanolamide


1,2-dioctanoyl-sn-
Glycyrrhetinic
Linseed oil
Oleoyl
7
45
38


glycero-3-
Acid

hydroxyethlimidazoline


phosphoethanol-


amide


Phosphatidyl
Carbenoxolone
2-Hydroxystearic
7H-Benzimidazo[2,1-
8
44
36


Choline

acid
a]Benz[de]-isoquinolin-





7-one


Phosphatidyl
Linoleic Acid
2-Hydroxystearic
7H-Benzimidazo[2,1-
−3
44
47


Choline

acid
a]Benz[de]-isoquinolin-





7-one


Phosphatidyl
Glycyrrhetinic
Elaidic acid
Climbazole
−3
30
33


Choline
Acid


Phosphatidyl
Linoleic Acid
Elaidic acid
Climbazole
−2
30
32


Choline









Cosmetically Acceptable Vehicle

The composition according to the invention also comprises a cosmetically acceptable vehicle to act as a dilutant, dispersant or carrier for the active components in the composition, so as to facilitate their distribution when the composition is applied to the skin.
Vehicles other than or in addition to water can include liquid or solid emollients, solvents, humectants, thickeners and powders. An especially preferred non-aqueous carrier is a polydimethyl siloxane and/or a polydimethyl phenyl siloxane. Silicones of this invention may be those with viscosities ranging anywhere from about 10 to 10,000,000 centistokes at 25° C. Especially desirable are mixtures of low and high viscosity silicones. These silicones are available from the General Electric Company under trademarks Vicasil, SE and SF and from the Dow Corning Company under the 200 and 550 Series. Amounts of silicone which can be utilised in the compositions of this invention range anywhere from 5 to 95%, preferably from 25 to 90% by weight of the composition.
Optional Skin Benefit Materials and Cosmetic Adjuncts
An oil or oily material may be present, together with an emulsifier to provide either a water-in-oil emulsion or an oil-in-water emulsion, depending largely on the average hydrophilic-lipophilic balance (HLB) of the emulsifier employed.
Various types of active ingredients may be present in cosmetic compositions of the present invention. Various types of active ingredients may be present in cosmetic compositions of the present invention. Actives are defined as skin or hair benefit agents other than emollients and other than ingredients that merely improve the physical characteristics of the composition. Although not limited to this category, general examples include sunscreens, skin lightening agents, and tanning agents.
Sunscreens include those materials commonly employed to block ultraviolet light. Illustrative compounds are the derivatives of PABA, cinnamate and salicylate. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone (also known as oxybenzone) can be used. Octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commercially available under the trademarks, Parsol MCX and Benzophenone-3, respectively.
The exact amount of sunscreen employed in the emulsions can vary depending upon the degree of protection desired from the sun's UV radiation.
Another preferred optional ingredient is selected from essential fatty acids (EFAs), i.e., those fatty acids which are essential for the plasma membrane formation of all cells, in keratinocytes EFA deficiency makes cells hyperproliferative. Supplementation of EFA corrects this. EFA's also enhance lipid biosynthesis of epidermis and provide lipids for the barrier formation of the epidermis. The essential fatty acids are preferably chosen from linoleic acid, γ-linolenic acid, homo-γ-linolenic acid, columbinic acid, eicosa-(n-6,9,13)-trienoic acid, arachidonic acid, γ-linolenic acid, timnodonic acid, hexaenoic acid and mixtures thereof.
Emollients are often incorporated into cosmetic compositions of the present invention. Levels of such emollients may range from about 0.5% to about 50%, preferably between about 5% and 30% by weight of the total composition. Emollients may be classified under such general chemical categories as esters, fatty acids and alcohols, polyols and hydrocarbons.
Esters may be mono- or di-esters. Acceptable examples of fatty di-esters include dibutyl adipate, diethyl sebacate, diisopropyl dimerate, and dioctyl succinate. Acceptable branched chain fatty esters include 2-ethyl-hexyl myristate, isopropyl stearate and isostearyl palmitate. Acceptable tribasic acid esters include triisopropyl trilinoleate and trilauryl citrate. Acceptable straight chain fatty esters include lauryl palmitate, myristyl lactate, oleyl eurcate and stearyl oleate. Preferred esters include coco-caprylate/caprate (a blend of coco-caprylate and coco-caprate), propylene glycol myristyl ether acetate, diisopropyl adipate and cetyl octanoate.
Suitable fatty alcohols and acids include those compounds having from 10 to 20 carbon atoms. Especially preferred are such compounds such as cetyl, myristyl, palmitic and stearyl alcohols and acids.
Among the polyols which may serve as emollients are linear and branched chain alkyl polyhydroxyl compounds. For example, propylene glycol, sorbitol and glycerin are preferred. Also useful may be polymeric polyols such as polypropylene glycol and polyethylene glycol. Butylene and propylene glycol are also especially preferred as penetration enhancers.
Exemplary hydrocarbons which may serve as emollients are those having hydrocarbon chains anywhere from 12 to 30 carbon atoms. Specific examples include mineral oil, petroleum jelly, squalene and isoparaffins.
Another category of functional ingredients within the cosmetic compositions of the present invention are thickeners. A thickener will usually be present in amounts anywhere from 0.1 to 20% by weight, preferably from about 0.5% to 10% by weight of the composition. Exemplary thickeners are cross-linked polyacrylate materials available under the trademark Carbopol from the B.F. Goodrich Company. Gums may be employed such as xanthan, carrageenan, gelatin, karaya, pectin and locust beans gum. Under certain circumstances the thickening function may be accomplished by a material also serving as a silicone or emollient. For instance, silicone gums in excess of 10 centistokes and esters such as glycerol stearate have dual functionality.
Powders may be incorporated into the cosmetic composition of the invention. These powders include chalk, talc, Fullers earth, kaolin, starch, smectite clays, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, aluminum starch octenyl succinate and mixtures thereof.
Other adjunct minor components may also be incorporated into the cosmetic compositions. These ingredients may include coloring agents, opacifiers and perfumes. Amounts of these materials may range anywhere from 0.001% up to 20% by weight of the composition.
Use of the Composition
The composition according to the invention is intended primarily as a product for topical application to human skin, especially as an agent for conditioning and smoothening the skin, and preventing or reducing the appearance of wrinkled or aged skin.
In use, a small quantity of the composition, for example from 1 to 5 ml, is applied to exposed areas of the skin, from a suitable container or applicator and, if necessary, it is then spread over and/or rubbed into the skin using the hand or fingers or a suitable device.
Product Form and Packaging
The topical skin treatment composition of the invention can be formulated as a lotion, a fluid cream, a cream or a gel. The composition can be packaged in a suitable 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 capsule, 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 squeeze container, such as a tube or a lidded jar.
The invention accordingly also provides a closed container containing a cosmetically acceptable composition as herein defined.

## Claims

1. A method for treating skin comprising the steps of:
(i) applying to the skin a composition comprising:
(a) from 0.01% to 10% of a retinoid;
(b) at least one compound that will enhance conversion of retinoid to retinoic acid and prevent the degradation of retinoic acid;
(c) a cosmetically acceptable vehicle; and
(d) fatty alcohol or fatty acid or both,
(e) the compound or compounds that will enhance conversion of retinoids to retinoic acid and prevent the degradation of retinoic acid comprise an antimicotic agent; and

(ii) treating the skin for wrinkles, psoriasis, age spots or discoloration.

2. The method according to claim 1 wherein the antimicotic agent is bifonazole, climbazole, or a mixture thereof.

3. The method according to claim 1 wherein the antimicotic agent is climbazole.

4. The method according to claim 1 wherein the skin care composition further comprises a fatty alcohol and the fatty alcohol is cetyl alcohol.

5. The method according to claim 1 where the compound or compounds that will enhance conversion of retinoids to retinoic acid and prevent the degradation of retinoic acid make up from about 0.0001% to 50% of the skin care composition.

6. The method according to claim 1 wherein the compound or compounds that will enhance conversion of retinoids to retinoic acid and prevent the degradation of retinoic acid make up form about 0.0001% to 50% of the skin care composition.

7. The method according to claim 1 wherein the compound or compounds will potentiate the action of retinoids by increasing the conversion of retinoid to retinoic acid and further wherein the compound or compounds will cause an action which is an inhibition of ARAT/LRAT activity, an enhancement of retinol dehydrogenase activity, an inhibition of retinal reductase activity, antagonistic to CRABP-II binding of retinoic acid and/or an inhibition of cytochrome P450 dependent retinoic acid oxidation.

8. The method according to claim 1 wherein the composition comprises climbazole and cetyl alcohol.

9. The method according to claim 1 wherein the composition comprises bifonazole and cetyl alcohol.

10. The method according to claim 1 wherein the composition comprises bifonazole, climbazole and cetyl alcohol.

11. The method according to claim 1 wherein the composition further comprises a sunscreen and skin lightening agent.

12. The method according to claim 2 wherein the composition further comprises linoleoyl monoethanolamide, palmitamide monoethanolamide, castor oil methyl ester acid or a mixture thereof.

13. The method according to claim 5 wherein the compound or compounds that will enhance conversion of retinoids to retinoic acid and prevent the degradation of retinoic acid make up from about 0.001% to 10% of the skin care composition.

14. The method according to claim 6 wherein the compound or compounds that will enhance conversion of retinoids to retinoic acid and prevent the degradation of retinoic acid make up from about 0.001% to 10% of the skin care composition.