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.

Description: SKIN CONDITIONING COMPOSITIONS CONTAINING COMPOUNDS FOR MIMICKING THE EFFECT ON SKIN OF ETINOIC ACID 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, CN. 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: Retinol metabolism in the epidermis: enzyme names Retinyl Esters Retinol aded ucts RBP- Retinol (systemic) 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 Bl 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 (Bl) , 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 (US patent numbers: 5759556, 5756109, 5747051,5716627, 5811110, 5536740, 5747051, 5599548, 5955092, 5885595, 5759556, 5693330). The boosters described in these patents are restricted to class Bl 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. 5028628; U.S. 5037829; U.S. 5151421; U.S. 476852; U.S. 5500435; U.S. 5583136; U.S. 5612354) . 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 US 5,665,367, US 5747049, US 5853705, US 5766575, and US 5849310. 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 Bl through to B5. Best Groups of Boosters Bl Compounds B2 Compounds B3 Compounds B4 Compounds B5 Compounds 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 desqua ation 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-ll-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-C 3 0 esters of retinol, preferably C2- C 2 0 esters, and most preferably C2 r C3, and Cig esters because they are more commonly available. Examples of retinyl esters include but are not limited to: retinyl palmitate, 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 CRABPII Antagonist Assay (To identify B4 boosters) Retinoic Acid Oxidation Assay (To iden tif B5 boosters) 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 N 2 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 . L. Lawson Co., Lincoln, NE, USA. The eyes were thawed overnight and the colored iridescent membrane was removed by peeling with forceps. Each eyecup was washed with 2x 0.5mL cold buffer (0.1M P04 / ImM 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 20,000g (Sorvaal model RC-5B centrifuge with an SS34 rotor in 2.5x10cm tubes at 14,000 RPM) . The resulting supernatant was subjected to further centrifugation for 60 min. at 150,000g (Beckman model L80 Ultracentrifuge with an S 50.1 rotor in 13x51mm tubes at 40,000 RPM). The resulting pellets were dispersed into ~5mL 0. IM P0 4 / 5mM 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. IM tris / 0.1M KC1 / ImM 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 ho ogenate was successively centrifuged for 30 min. at 10,000g, 30 min. at 20,000g, and 15 min. at 30,000g, and the resulting supernatant was ultra- centrifuged for 80 min. at 105,000g. The pellet was sonicated in ~5mL of 0.1M P04 / 0. ImM EDTA / 5mM MgCl 2 , 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 Bl) 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. IM P0 4 / 5mM dithiothreitol, pH 7.0 (PO 4 /DTT) . On the day of the assay, 2mg BSA per mL of buffer was added to give a P0 4 / DTT / BSA working buffer. ImM retinol substrate was prepared in acetonitrile and stored in amber bottles under nitrogen gas at -20°C. Solutions of 4mM Palmitoyl-CoA in working buffer (stored in aliquots) and 4mM dilauroyl phosphatidyl choline in ethanol were prepared and stored at -20 °C. Inhibitors were prepared as lOmM stock solutions in H 2 0, ethanol, acetonitrile or DMSO. The quench solution was prepared using pure ethanol containing 50μg/mL butylated hydroxytoluene (BHT) , and a hexane solution containing 50μg/mL BHT was used for the extractions. To a 2 dram glass vial, the following - were added in order: P0 4 / DTT / BSA buffer to give a total volume of 500μL, 5μL acyl donor (4mM palmitoyl-CoA and/or dilauroyl phosphatidyl choline) , 5μL inhibitor or solvent blank (lOmM stock or further dilutions) followed by approximately 15μg of RPE microsomal protein (approximately 15μL of a ~lmg/mL microsomal protein aliquot) . The mixture was incubated for 5 min. at 37 °C to equilibrate the reaction temperature and then 5μL ImM 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 0.5mL ethanol/BHT. The retinoids were extracted by adding 3mL 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 3mL 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 40μM acyl donor, lOOμM or less inhibitor, lOμM retinol, approximately 30μg/mL microsomal protein, and nearly 0. IM P0 4 / pH 7 / 5mM DTT / 2mg/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: 50mM KH2P04, pH 7.4 buffer, sterile filtered. lOmM all trans Retinol (Sigma R7632) in DMSO. 200mM Nicotinamide adenine dinucleotide phosphate, sodium salt (NADP) (Sigma N0505) in sterile water. 40mM test compound in appropriate solvent (water, buffer, ethanol, chloroform or DMSO) . 1:10 dilution of rat liver Microsomes in 50mM KH2P04, pH 7.4 buffer (4μg/μl) . In a two-dram glass vial with screw cap, the following were added in order: Buffer to give a final volume of 400μl 25μl diluted Microsomes (final = lOOμg) - boiled Microsomes were used for controls and regular Microsomes for test samples . 4μl of 200mM NADP (final = 2mM) Iμl of 40mM test compound (final = lOOμM) 8μl of lOmM retinol (final = 200μM) The vials were incubated in a 37°C shaking water bath for 45 minutes. 500μl ice-cold ethanol was added to each vial to quench the reaction. The retinoids were extracted twice with ice cold hexane (2.7ml per extraction). Retinyl acetate (5μl of a 900μM 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 lOOOrpm, 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 : lOmM all trans Retinaldehyde (Sigma R2500) in DMSO - instead of retinol. 200mM, 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 400μl 25μl diluted Microsomes (final = lOOμg) - use boiled Microsomes for controls and regular Microsomes for test samples . 4μl of 200mM NADPH (final = 2mM) lμl of 40mM test compound (final = lOOμM) 3μl of lOmM retinaldehyde (final = 75μM) 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 2xYT containing 50 μg/mL kanamycin and 25μg/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 ImM and the culture was incubated for an additional two hours. The cells were harvested by centrifugation at 5,000g 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- HC1, pH 8, 10% (w/v) sucrose, 1 mM EDTA, 0.05% (w/v) sodium azide, 0.5 mM DTT, 10 mM MnCl 2 , 2.5 mM phenylmethylsulfonyl fluoride, 2.5 mM benzamidine, 6μg/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 2.5x100 cm column of sephacryl S-300 (Pharmacia) at room temperature. The elution buffer was 20 mM Tris-HCl, pH 8, 0.5mM DTT, 0.05% sodium azide (buffer A) . The flow rate was 2mL/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 L of gel filtration fractions containing CRABPII were loaded onto a quaternary amine anion-exchange column FPLC (Fast Protein Liquid Chromatography) type onoQ (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 2X loading buffer (100 mM Tris-HCl pH6.8, 4% SDS, 0.2% BPB, 20% glycerol, ImM DTT) and denatured by heating (2 mins . at 80°C) . The samples were loaded onto the gel that was immersed in a IX 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 IX 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 IX 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 lxPBS (3x5 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 70μL. 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 6kD 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. IM P0 4 / 0. ImM EDTA / 5mM MgCl 2 , pH 7.4. On the day of the assay, a 60mM NADPH solution in buffer was prepared. Inhibitor stocks, acidified ethanol / BHT quench solution, and hexane / BHT were prepared as described above. A working ImM retinoic acid solution was prepared by dilution of a 15mM stock (in DMSO) with ethanol. To a 2 dram vial, the following were added in order: assay buffer to give a final volume of 500μL, 20μL 60mM NADPH, 5μL inhibitor or solvent blank, followed by approximately 2mg of rat liver microsomal protein. The mixture was incubated for 5 mins . at 37 °C, then 5μL working ImM retinoic acid solution was added. Incubation was continued for 60mins. at 37 °C - the vials were not capped, since the oxidation process required molecular 0 2 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 2.4mM NADPH, lOOμM or less inhibitor, lOμM retinoic acid, approximately 4mg/mL rat liver microsomal protein and nearly 0. IM P0 / 0. ImM EDTA / 5mM MgCl 2 . HPLC analysis of individual retinoids Samples for retinoid quantitation by HPLC were prepared by dissolving the residue in each .vial with lOOμL of methanol. The solution was transferred to a 150μL glass conical tube within a lmL shell vial, capped tightly, and placed inside a Waters 715 Autosampler. Aliquots of 60μL 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 3.9x300mm 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 lmL/min. for 10 min. The eluate was monitored for absorbance at 325nm and fluorescence at 325ex/480em. A shorter Waters 3.9x150mm 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 lOmM ammonium acetate to 4:l(v/v) methanol: dichloromethane followed by a 5 mins. hold at a flow rate of lmL/min. The column eluate was monitored from 300nm to 400nm. 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- 400nm) 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 E p i t h e l i a l 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 Bi through to B 5 below. The table below gives the booster class (Bi - B5) , the chemical name of the compound, and the results from the appropriate assays used to identify the booster (i.e. ARAT/LRAT for Bl, retinol dehydrogenase for B2, retinaldehyde inhibation for B3, CRABP is binding for B4 and retinoic acid oxidation inhibition for B 5 . ARAT/LRAT Inhibitors (Bl) %Inhibitiσn Class Compound Overall Overall %Inhibition %Inhibition ^Inhibition %Inhibition TG(-ROH/RE) TG (IC 50) ARAT(lOjm) ARAT LRAT (lOj ) LRAT (100.1m) (lOOjrα) Carotenoid Crocetin 3 . 75E-05 15% 34 % 0 15% Fatty Acid & Other Acetyl Sphingosine 6. 78E-06 19%+/-12 62%+/-ll 10%+/-10 50%+/-18 Surfactants Fatty Acid Amides 5 C13 Beta-Hydroxy Acid/ 17% 281 25% Other Surfactants Amide Fatty Acid Amides s Castor Oil MEA 3 . 25E-05 Other Surfactants Fatty Acid Amides & Coca idopropyl Betaine 25% Other Surfactants Fatty Acid Amides Coco Hydroxyethyl- 2 . 84E-07 68 % 65% Other Surfactants imidazoline Fatty Acid Amides Cocoamide-MEA (or 111 13% 34% Other Surfactants Cocoyl Monoethanol- amide) Fatty Acid Amides Glycerol-PCA-Oleate 41%+/-6 58%+/-2 Other Surfactants 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<j 12%+/-3 43%+/-3 11% + /- -9 51%+/-15 Other Surfactants inoleoyl Diethanolamide) Fatty Acid Amides & inoleamide-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 S Melinamide 64%+/-15 43%+/-21 Other Surfactants Fatty Acid Amides & Myristoyl Sarcosine 4 + /-14 11%+/-11 o ro I I I dp 1 tip tiP dp o\° tiP o\° dp tip vo o\° o\° o\° tip dP dp o\° o\° ti dP ro o + r- L cM Lo σi Co σϊ ^ oo L 'xi cM rH C c ro r H co ro o +\° dp r- ( θ , ι r~ r-- cri ι-l ' o ^f r-H n r~ ιH ^r r < τ ) csι o M n ro 1 dP CM cn -^ LO so r- en ro r- dp tiP tip tip dp r- ro O rH r~ i-i O • f M < -< "* CM CM CM ^c r- 1 1 o + + ro oo tip tiP so r- r-H so LO ^r SΩ o o o o O O I 1 1 1 1 H H W o r- r- r- 00 ro CM tiP rH CM 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%+/-l Phospholipid Phosphatidyl ethanolamine 37% 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) Overall % Inhibition % Inhibition TG(IC 50) Retinoic Retinoic Class Compound Acid (lOμM) Acid(lθθμ ) 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 & Other Lauryl Hydroxyethylimidazolme 4.67E-07 Surfactants Fatty Acid Amides & Other Oleyl Hydroxyethylimidazoline 3.02E-05 54% 80% Surfactants Flavanoids Quercet 6.29E-05 40% 74% Coumann Couma m Qumolme (7H-Benz mιdazo [2,l-a]Benz 8.59E-07 [de] -Isoqumolm-7-one Qumolme Hydroxyqumoline (Carbostyril) 3.64E-04 Qumolme Metyrapone (2~Methyl-l,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 15nm 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 200μl 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 200μl of lxPBS . 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: 50μl 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 6x with wash buffer. Cells were then incubated with 50μl 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 3x with PBS. For colourimetric development, the cells were incubated with lOOμl substrate solution (4 mg o- phenylenediamine and 3.3 μl 30% H 2 O 2 in 10ml O.lM citrate buffer pH 5.0) for exactly five minutes, R/T, in darkness (under aluminum foil) . The reaction was stopped by the addition of 50μl 4N H 2 SO 4 . The absorbance of samples was read at 492nm 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 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 : TG specific Biogenesis (Cat# 5560 monoclonal Ab B . Cl -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% H 2 0 2 Sigma H-1909 B. Media/Buffers Keratinocyte Growth Media (KGM) Clonetics (Cat# 3111; Phosphate Buffered Saline; Dulbecco's without Ca/MgCl2) Life Technology (Cat # 14200-075 Tris Buffered Saline Blocking buffer (lxTBS + 5% dry milk) BioRad (Cat #170-6404) Washing buffer (1% dry milk in TBS + 0.05% Tween 20) Sigma (Cat # P-7949) Phosphate citrate buffer: 1:1 mixture of 0.2M dibasic Sigma (Cat # S-9763) sodium phosphate and O.l citric acid Sigma (Cat # C-1909) 4 N H 2 SO 4 C. Culture ware 96-well polypropylene microtitre plate Costar (Cat # 3595] 96-well polypropylene U-bottom plate Costar (Cat # 3794) T75- vent cap Costar (Cat # 3376) D: Instrumentation/Equipment Biotek Model EL 340 Microplate reader Bio-tek Instuments Inc. Multiprobe II Packard 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 3x10 cells /12 ml media in each microtitre plate) 2.200μl of the keratinocyte suspension was transferred into each of the inner 60 wells only. 3.200μl of KGM media was pipetted into the outer wells (to maintain thermal equilibrium) . 4. Each plate was incubated at 37°C and 5% CO 2 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 μi • , ~ \ 8. Plates were incubated at 37°C and 5% C0 2 for 72 hours. 9. Media were completely removed from each well. 10. Wells were rinsed with 2x with 200 μl of lxPBS 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 6x 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 3X with washing buffer (added 200μl) and aspirated after each rinse. 6. Rinsed wells ' 3X 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 H 2 SO 4 . 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 CaCl 2 - 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 retinol and boosters - 3 days Final DMSO concentration - less than 0.1% Retinol concentration - 10-7M (O.lμM) 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 Bl 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 -7 constant retinol concentration of 10 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 (Bl 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 Bl 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 Bl/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 (Bl 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: Invention Ratios of boosters Concentrations to retinoids 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 preferred 100:1 to 1:100 1 mM-100 nM booster; 0.01-1% 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 (IC 20 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 IC5 0 and IC 20 (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. 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) Sphingomyelin (B2) TCC (B2) ,2 dioctanoyl-sn-glycero-3-phopshoethanolamide (B2) B3 Class boosters Amscrine B3 Carbenoxolone (B3) Glyrrhetinic acid (B3) Linoleic acid (B3) Linolenic acid (B3) Myristic acid (B3) Vanillin (B3 ) B4 Class boosters Hexadecanedioic acid (B4) 12-hydroxysteric acid (B4) Elaidic acid (B4) Linseed Oil (B4) Isosteric acid (B4) 2-hydroxysteric acid (B4) 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. 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. IC 2 o) • 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; Bl/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 teritiary combinations of boosters given in the following table synergistically inhibited Tgase in the presence of 10-7M retinol. Compound 1 Compound 2 Compound 3 TG as % TG as % TG as % TG as C C C % C Compd 1 Compd 2 Compd 3 Combo B1/B2/B3 combinations : Phosphatidyl Choline Glycyrrhetinic Castor oil Methyl 91 85 53 Acid Ester Acid (MEA) Phosphatidyl Choline Glycyrrhetinic Echinacea 91 119 52 Acid Phosphatidyl Choline Glycyrrhetinic Naringenin 91 94 52 Acid Phosphatidyl Choline Glycyrrhetinic Acetyl sphingosine 91 99 58 Acid (C2 Ceramide) Phosphatidyl Choline Glycyrrhetinic Farnesol 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 94 66 glycero-3- Choline phosphoethanolamide 1, 2-dioctanoyl-sn- Amsacrine-HCl Linoleoyl 79 127 60 glycero- 3 - Monoethanolamide phosphoethanolamide (LAMEA) 1,2-dioctanoyl-sn- Amsacrine-HCl Palmitamide 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 91 94 75 glycero-3- Acid phosphoethanolamide 1, 2-dioctanoyl-sn- Glycyrrhetinic Echinacea 81 91 119 77 glycero-3- Acid phosphoethanola ide 1, 2-dioctanoyl-sn- Glycyrrhetinic Dimethyl 81 91 87 67 glycero-3- Acid i idazolinone 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 84 119 75 Phosphatidyl Choline Climbazole Naringenin 84 94 83 Phosphatidyl Choline Climbazole Geraniol 84 105 76 Phosphatidyl Choline Climbazole Famesol 88 84 118 82 Phosphatidyl Choline Climbazole Acetyl sphingosine 84 99 82 (C2 Ceramide) Phosphatidyl Choline Miconazole a-Ionone 92 88 70 Phosphatidyl Choline Miconazole Castor oil Methyl 92 85 72 Ester Acid (MEA) B1/B3/B4 Combinations : Amsacrine-HCl Dimethyl Elaidic acid 79 87 93 0 imidazolinone D-Ionone Ams acrine-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 Combinations : Amsacrine-HCl Dimethyl 2- 79 87 95 32 imidazolinone Hydroxyquinoline (C arbostyril) Amsacrine-HCl Dimethyl Lauryl 79 87 52 -13 imidazolinone hydroxyethylimidaz oline Amsacrine-HCl Dimethyl Quercetin 79 87 92 -24 imidazolinone Amsacrine-HCl Dimethyl Oleoyl 79 87 76 39 imidazolinone hydroxyethlimidazo line 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 hydroxyethlimidazo Ceramide ) line Hexanoyl 3, 4,-Dihydro- Vanillin 104 90 134 62 sphingosine (C6 2(1H)- Ceramide) quinolinone (Hyd rocarbostyril) 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) 0-Ionone Amsacrine-HCl 7H- 68 79 94 54 Benzimidazo [2 , 1- aJ Benz [de] - isoquinolin-7-one Utrecht-2 Carbenoxolone Quercetin 76 92 74 Utrecht-2 Carbenoxolone Oleoyl \ 1 76 88 76 69 hydroxyethlimidazo line ϋtrecht-2 Carbenoxolone 7H- 76 94 73 Benzimidazo [2,1- a]Benz [de] - isoquinolin-7-one Utrecht-2 Carbenoxolone 3, 4,-Dihydro- 76 90 70 2(1H)- quinolinone (Hydroc arbostyril) Myristic Acid Climbazole Geraniol 79 84 105 74 Myristic Acid Climbazole G-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 (Hyd rocarbostyril) 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-Damascone 93 95 89 80 (Carbostyril) Elaidic acid 2-Hydroxyquinoline Famesol .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 91 103 62 glycero-3- Acid phosphoethanolamide 1, 2-dioctanoyl-sn- Glycyrrhetinic Elaidic acid 91 93 75 glycero-3- Acid phosphoethanoland.de Phosphatidyl Choline 2-Hydroxystearic Arachidonic Acid 83 78 60 acid (Na+ salt) B2/B3/B5 Combinations : Phosphatidyl Choline Climbazole Linolenic Acid 84 208 84 Phosphatidyl Choline Climbazole Arachidonic Acid 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- 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 (Hydroc arbostyril) 1, 2-dioctanoyl-sn- Glycyrrhetinic 2- 81 91 95 75 glycero-3- Acid Hydroxyquinoline (C phosphoethanolamide arbostyril) 1, 2-dioctanoyl-sn- Glycyrrhetinic Amino 81 91 105 72 glycero-3- Acid Benzotriazole phosphoethanolamide 1, 2-dioctanoyl-sn- Glycyrrhetinic Lauryl 91 52 79 glycero-3- Acid hydroxyethylimidaz phosphoethanolamide oline 1, 2-dioctanoyl-sn- Glycyrrhetinic Quercetin 91 92 73 glycero-3- Acid phosphoethanolamide 1,2-dioctanoyl-sn- Glycyrrhetinic Climbazole 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 Benzotriazole 83 105 77 acid Phosphatidyl Choline 2-Hydroxystearic Lauryl 83 52 74 acid hydroxyethylimidazoline Phosphatidyl Choline 2-Hydroxystearic Quercetin 88 83 92 69 acid Phosphatidyl Choline 2-Hydroxystearic Oleoyl 83 76 75 acid hydroxyethlimidazoline Phosphatidyl Choline 2-Hydroxystearic 7H-Benzimidazo[2, 1- 83 94 79 acid a] Benz [de] -isoquinolin-7-one Phosphatidyl Choline Climbazole Elaidic acid 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. Compound 1 Compound 2 Compound 3 Compound 4 Quarter- Tertiary Differ- nary TG (1-3 ence (%C) combo ; (<30%=sγ TG %C) nergy) B1/B2/B3/B4 Combination: Castor oil Methyl Phosphatidyl Glycyrrhetinic l 2 -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 3 5 Monoethanolamide sn-glycero-3- Acid (LAMEA) phosphoethanol- a ide Linoleoyl 1 , 2 -dioctanoyl- Amsacrine-HCI 12-Hydroxy- -3 42 45 Monoethanolamide sn-glycero-3- stearic acid (LAMEA) phosphoethanol- amide Linoleoyl 1, 2-dioctanoyl- Amsacrine-HCl Elaidic acid 8 42 3 Monoethanolamide sn-glycero-3- (LAMEA) phosphoethanol- amide Hexanoyl TCC Glycyrrhetinic Isostearic acid 7 54 47 sphingosine (C6 Acid Ceramide) Lyrial TCC Vanilin Hexadecan10 48 38 edioic acid Cocoyl 1, 2-dioctanoyl- Glycyrrhetinic Isostearic acid 0 37 37 hydroxyethylimid- sn-glycero-3- Acid azoline phosphoethanol- amide Cocoyl Phosphatidyl Arachidonic Acid 2-Hydroxy-1 37 38 hydroxyethylimid- Choline (Na+ salt) stearic acid azoline Cocoyl 1,2-dioctanoyl- Glycyrrhetinic Linseed oil -2 45 47 hydroxyethylimid- sn-glycero-3- Acid azoline 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-Benzimidaz 1 o[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- a ide Geraniol 1, 2-dioctanoyl- Amsacrine-HCI 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, -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, ?-dioctanoyl- Amsacrine-HCl Miconazole 13 43 30 Monoethanolamide sn-glycero-3- phosphoethanol- a ide Palmitamide 1 , 2-dioctanoyl- Amsacrine-HCl Oleoyl 43 40 Monoethanolamide sn-glycero-3- hydroxyethlimidazoline phosphoethanol- a ide 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- Amsac ine-HCI Oleoyl hydroxyethlimid48 45 sn-glycero-3- azoline 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 hydroxyethylimi4 55 51 Ester Acid (MEA) Choline dazoline 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 CC Amsacrine-HCl Quercetin 12 44 32 imidazolinone Dimethyl CC Amsacrine-HCl Coumarin 14 58 44 imidazolinone Hexanoyl TCC Glycyrrhetinic Amino Benzotriazole 48 40 sphingosinei (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 (Carbost yril) Hexanoyl Glycyrrhetinic 12-Hydroxystearic Amino Benzotriazole 14 48 33 sphingosine (C6 Acid acid Ceramide) Dimethyl Phosphatidyl 2-Hydroxystearic 7H-Benzimidazo [2,1- 44 42 imidazolinone Choline acid a] Benz [de] -isoquinolin- 7-one Melinamide Phosphatidyl 2-Hydroxystearic 7H-Benzimidazo[2, 1- 44 39 Choline acid a] Benz [de] -isoquinolin- 7-one Geranyl geraniol Phosphatidyl 2-Hydroxystearic 7H-Benzimidazo [2, 1- 44 35 Choline acid a] Benz [de] -isoquinolin- 7-one Cocoyl Phosphatidyl 2-Hydroxystearic 7H-Benzimidazo [2, 1- 44 52 hydroxyethylimidaz Choline acid a] Benz [de] -isoquinolin- oline 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- 44 40 [de] -isoquinolin- Cocoyl Phosphatidyl Elaidic acid Climbazole 30 34 hydroxyethylimidaz Choline oline 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 54 48 sphingosine (C6 Acid hydroxyethlimidazoline Ceramide) Crocetin Linoleic Acid Elaidic acid -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 hydroxyethylimid(Carbostyril) azoline B2/B3/B4/B5 Combination : 1, 2-dioctanoyl-sn- Glycyrrhetinic Isostearic acid Ketoconazole 37 33 glycero-3- Acid phosphoethanol- amide 1, 2-dioctanoyl-sn- Glycyrrhetinic Isostearic acid Oleoyl 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 phosphoethanolamid e 1, 2-dioctanoyl-sn- Glycyrrhetinic Linseed oil Oleoyl 45 38 glycero-3- Acid hydroxyethlimidazoline phosphoethanol- amide Phosphatidyl Carbenoxolone 2-Hydroxystearic 7H-Benzimidazo[2, 1- 44 36 Choline acid a] Benz [de] -isoquinolin- 7-one Phosphatidyl Linoleic Acid 2-Hydroxystearic 7H-Benzimidazo[2, 1- 44 47 Choline acid a] Benz [de] -isoquinolin- 7-one Phosphatidyl Glycyrrhetinic Elaidic acid Climbazole 30 33 Choline Acid Phosphatidyl Linoleic Acid Elaidic acid Climbazole 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-β, 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, fume ' d 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 " -reventing or reducing the appearance of wrinkled or aged skin. In use, a small quantity of the composition, for example from 1 to 5ml, 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: CLAIMS l.A skin care composition comprising: a. from 0.001% to 10% of a retinoid; b. a combination of at least 2 retinoid boosters belonging to classes Bl to B5 in an amount of from 0.0001% to 50% where the ratios of the two boosters to each other in the range of is 1:1000 to 1000:1; c. a cosmetically acceptable vehicle. 2. The skin care composition of claim 1 where the combination of boosters comprises at least three boosters belonging to the classes Bl to B5 in an amount of from 0.0001% to 50%. 3. The skin care composition of claim 1 or claim 2 where the second composition has a combination of at least 4 boosters belonging to the classes -Bl to B5 in an amount of from 0.0001% to 50%. 4. The skin care composition of any of the preceding claims where the second composition has a combination of all the 5 classes of boosters belonging to the classes Bl to B5. 5. A cosmetic method of conditioning skin, the method comprising applying topically to the skin the product of any one of claims 1 through to 5. 6. A cosmetic method of mimicking the effect on skin or retinoic acid, the method comprising applying to the skin the product of any one of claims 1-5. 7. A skin care composition comprising: a. a combination of at least 2 retinoid boosters belonging to classes Bl to B5 in an amount of from 0.0001% to 50% where the ratios of the two boosters to each other in the range of is 1:1000 to 1000:1; b. a cosmetically acceptable vehicle.

Inventors: GRANGER STEWART PATON, SCOTT IAN RICHARD, GRANGER, STEWART, PATON, SCOTT, IAN, RICHARD
Applicants: GRANGER STEWART PATON, LEVER HINDUSTAN LTD, SCOTT IAN RICHARD, UNILEVER NV, UNILEVER PLC, GRANGER, STEWART, PATON, HINDUSTAN LEVER LIMITED, SCOTT, IAN, RICHARD
Application year: 2001
Main classification: A61K7
IPC codes: A61K8, A61Q5, A61Q19