US20040043044A1 - Skin conditioning compositions containing compounds for mimicking the effect on skin of retinoic acid - Google Patents

Skin conditioning compositions containing compounds for mimicking the effect on skin of retinoic acid Download PDF

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US20040043044A1
US20040043044A1 US10/312,659 US31265903A US2004043044A1 US 20040043044 A1 US20040043044 A1 US 20040043044A1 US 31265903 A US31265903 A US 31265903A US 2004043044 A1 US2004043044 A1 US 2004043044A1
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acid
boosters
phosphatidyl
retinol
choline
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US10/312,659
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Stewart Granger
Ian Scott
Robert Donovan
Susanne Iobst
Lisa Licameli
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Unilever Home and Personal Care USA
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Unilever Home and Personal Care USA
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Assigned to UNILEVER HOME & PERSONAL CARE USA, DIVISION OF CONOPCO, INC. reassignment UNILEVER HOME & PERSONAL CARE USA, DIVISION OF CONOPCO, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GRANGER, STEWART PATON, LICAMELI, LISA, DONOVAN, ROBERT MARK, IOBST, SUSANNE TEKLITS, SCOTT, IAN RICHARD
Publication of US20040043044A1 publication Critical patent/US20040043044A1/en
Priority to US12/045,339 priority Critical patent/US7959913B2/en
Priority to US13/097,142 priority patent/US8226933B2/en
Priority to US13/529,004 priority patent/US8409550B2/en
Priority to US13/764,090 priority patent/US20140050676A1/en
Abandoned legal-status Critical Current

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67Vitamins
    • A61K8/671Vitamin A; Derivatives thereof, e.g. ester of vitamin A acid, ester of retinol, retinol, retinal
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34Alcohols
    • A61K8/347Phenols
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36Carboxylic acids; Salts or anhydrides thereof
    • A61K8/361Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/494Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
    • A61K8/4946Imidazoles or their condensed derivatives, e.g. benzimidazoles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • A61K8/498Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67Vitamins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/06Antipsoriatics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/02Preparations for care of the skin for chemically bleaching or whitening the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/08Anti-ageing preparations

Definitions

  • the present invention relates to cosmetic skin conditioning compositions containing certain compounds which mimic the effect on skin of retinoic acid.
  • Retinol 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.
  • 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 ( C ellular R etinoic A cid B inding P rotein II) binding of retinoic acid (B4) and inhibiting cytochrome P450 dependent retinoic acid oxidation (B5).
  • ARAT/LRAT AcylCoenzymeA(CoA): retinol acyl transferase/Lecithin: retinol acyl transferase activity
  • B1 enhancing retinol dehydrogenase activity
  • B3 inhibiting retinal reductase activity
  • 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.
  • a retinoid e.g. retinol, retinyl esters, retinal, retinoic acid
  • the preferred compositions include a retinoid in the composition, co-present with a booster or a combination of boosters, to optimise performance.
  • 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.
  • 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.
  • 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
  • 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.
  • boosters or combinations thereof included in the inventive compositions are selected from the group consisting of:
  • a booster selected from the group consisting of B2; B3; B4;
  • 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.
  • conditioning 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 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.
  • 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-11-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 C 1 -C 30 esters of retinol, preferably C 2 -C 20 esters, and most preferably C 2 , C 3 , and C 16 esters because they are more commonly available.
  • 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, retiny
  • 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
  • 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.
  • 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 PO 4 /5 mM DTT, pH 7 buffer using a Heat Systems Ultrasonics, Inc.
  • 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.
  • the pellet was sonicated in ⁇ 5 mL of 0.1M PO4/0.1 mM EDTA/5 mM MgCl 2 , pH 7.4 buffer as described above and stored as aliquots at ⁇ 70° C. Protein concentrations were determined as described above.
  • 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
  • the incubation solution contains 40 ⁇ M acyl donor, 100 ⁇ M or less inhibitor, 10 ⁇ M retinol, approximately 30 ⁇ g/mL microsomal protein, and nearly 0.1M PO 4 /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.
  • test compound 40 mM test compound in appropriate solvent (water, buffer, ethanol, chloroform or DMSO).
  • 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.
  • 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).
  • 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 MnCl 2 , 2.5 mM phenylmethylsulfonyl fluoride, 2.5 mM benzamidine, 6 ⁇ g/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.
  • step a 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.
  • CRABPII Gel filtration fractions containing CRABPII were loaded onto a quaternary amine anion-exchange column FPLC (Fast Protein Liquid Chromatography) type monoQ (Pharmacia).
  • 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.
  • 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 ( ⁇ fraction (1/1000) ⁇ dilution of mouse anticlonal 5-CRA-B3) in the same buffer at 4° C. overnight.
  • 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.
  • ECLTM peroxidase conjugated anti-mouse antibody
  • 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 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 PO 4 /0.1 mM EDTA/5 mM MgCl 2 , pH 7.4.
  • 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.
  • retinoic acid 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 ⁇ M or less inhibitor, 10 ⁇ M retinoic acid, approximately 4 mg/mL rat liver microsomal protein and nearly 0.1M PO 4 /0.1 mM EDTA/5 mM MgCl 2 .
  • Samples for retinoid quantitation by HPLC were prepared by dissolving the residue in each vial with 100 ⁇ L of methanol. The solution was transferred to a 150 ⁇ L glass conical tube within a 1 mL shell vial, capped tightly, and placed inside a Waters 715 Autosampler. Aliquots of 60 ⁇ L 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.
  • 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 325 ex/480 em.
  • the boosters suitable for use in the present invention include but are not limited to the boosters listed in Tables B 1 through to B 5 below.
  • the table below gives the booster class (B 1 - B 5 ), 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 B 2 , retinaldehyde inhibation for B3, CRABP is binding for B 4 and retinoic acid oxidation inhibition for B 5 .
  • ARAT/LRAT Inhibitors (B1) % Inhibition % Inhibition % Inhibition % Inhibition % Inhibition Overall Overall ARAT ARAT LRAT LRAT Class Compound TG(-ROH/RE) TG (IC 50) (10 jm) (100 jm) (10 jm) (100 jm) 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
  • Retinaldehyde Reductase Inhibitors (B3) % Inhibition Overall Retinal Class Compound TG(IC 50) 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 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%
  • Tgase transglutaminase
  • 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 N ⁇ -( ⁇ -glutamyl) lysine isodipeptide bonds catalysed by the action of at least two different transglutaminases (TGases) expressed in the epidermis.
  • TGases transglutaminases
  • TGase I is a useful marker of epidermal keratinocyte differentiation with high TGase I levels indicating a more differentiated state.
  • Keratinocytes (cultured as described above) were plated in 96 well plates at a density of 4,000-5,000 cells per well in 200 ⁇ l 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 ⁇ l of 1 ⁇ PBS. The cells were incubated for one hour at room temperature (R/T) with TBS/5% BSA (wash buffer, bovine serum albumin).
  • R/T room temperature
  • BSA wash buffer, bovine serum albumin
  • TGase primary antibody 50 ⁇ l 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 ⁇ l 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.
  • secondary antibody Fab fragment, peroxidase conjugated anti-mouse IgG obtaining from Amersham
  • the cells were incubated with 100 ⁇ l substrate solution (4 mg o-phenylenediamine and 3.3 ⁇ l 30% H 2 O 2 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 ⁇ l 4N H 2 SO 4 . 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.
  • A. Reagents Cells Human Keratinocytes Neonatal Human foreskin (P2 in T75 flasks; P3 in 96 well assay plates) Primary Antibody: TGm specific Biogenesis (Cat # 5560-6006) monoclonal Ab
  • B.C1 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 ⁇ l of 30% H 2 0 2 Sigma H-1909
  • a suspension of keratinocytes was prepared at a concentration of 3000 cells/200 ⁇ l/well in KGM medium (Used 3 ⁇ 10 5 cells /12 ml media in each microtitre plate)
  • Aspirate blocking buffer Incubated with 100 ⁇ l/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.
  • 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.
  • retinoid boosters include: inhibitors of ARAT/LRAT (B1boosters), 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.
  • a retinoid e.g. retinol, retinyl ester, retinal, retinoic acid
  • the preferred compositions 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.
  • boosters included in the inventive compositions are selected from the group consisting of:
  • Two boosters wherein both are selected from the group consisting of B2, B3 and B4;
  • 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
  • boosters of different classes 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 ⁇ M
  • 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.
  • a retinoid e.g.retinol, retinyl ester, and retinaldehyde
  • 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 preferred 100:1 to 1:100 1 mM-100 nM booster; 0.01-1% retinoid
  • 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.
  • 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.
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • HLB hydrophilic-lipophilic balance
  • 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.
  • octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone also known as oxybenzone
  • 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.
  • EFAs essential fatty acids
  • the essential fatty acids are preferably chosen from linoleic acid, ⁇ -linolenic acid, homo- ⁇ -linolenic acid, columbinic acid, eicosa-(n-6,9,13)-trienoic acid, arachidonic acid, ⁇ -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 cococaprylate/caprate (a blend of coco-caprylate and cococaprate), 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.
  • polyols which may serve as emollients are linear and branched chain alkyl polyhydroxyl compounds.
  • propylene glycol, sorbitol and glycerin are preferred.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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

  • The present invention relates to cosmetic skin conditioning compositions containing certain compounds which mimic the effect on skin of retinoic acid. [0001]
  • 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., [0002] 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: [0003]
  • Retinol metabolism in the epidermis: [0004]
  • enzyme names [0005]
    Figure US20040043044A1-20040304-C00001
  • 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 ([0006] 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. [0007]
  • 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. No. 5,028,628; U.S. Pat. No. 5,037,829; U.S. Pat. No. 5,151,421; U.S. Pat. No. 476,852; U.S. Pat. No. 5,500,435; U.S. Pat. No. 5,583,136; U.S. Pat. No. 5,612,354). [0008]
  • 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. No. 5,665,367, U.S. Pat. No. 5,747,049, U.S. Pat. No. 5,853,705, U.S. Pat. No. 5,766,575, and U.S. Pat. No. 5,849,310. [0009]
  • 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. [0010]
  • 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. [0011]
  • Best Groups of Boosters
  • B1 Compounds [0012]
    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 [0013]
    1. Phosphatidyl choline Most preferred as most active
    activator of Retinol
    Dehydrogenase
    2. Sphingomyelin
  • B3 Compounds [0014]
    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 [0015]
    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 [0016]
    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. [0017]
  • The boosters or combinations thereof included in the inventive compositions are selected from the group consisting of: [0018]
  • (a) a booster selected from the group consisting of B2; B3; B4; [0019]
  • (b) binary combinations of boosters selected from the group consisting of: [0020]
  • B1/B2; B1/B3; B1/B4; B1/B5; B2/B3, B2/B4; B2/B5, B3/B4; B3/B5; B4/B5 [0021]
  • (c) ternary combinations of boosters selected from the group consisting of: [0022]
  • 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 [0023]
  • (d) quaternary combinations of boosters selected from the group consisting of: [0024]
  • B1/B2/B3/B4; B1/B2/B3/B5; B1/B2/B4/B5; B1/B3/B4/B5; B2/B3/B4/B5; and [0025]
  • (e) a combination of five groups of boosters: [0026]
  • B1/B2/B3/B4/B5. [0027]
  • The preferred compositions include from about 0.001% to about 10%, by weight of the composition of a retinoid.[0028]
  • 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. [0029]
  • 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. [0030]
  • The presence of the selected compounds in the inventive product substantially improves the performance of a retinoid. [0031]
  • 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-11-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. [0032]
  • Retinyl ester is an ester of retinol. The term “retinol” has been defined above. Retinyl esters suitable for use in the present invention are C[0033] 1-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 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. [0034]
  • 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%. [0035]
  • 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. [0036]
  • Section A: Identification of Booster
  • [0037]
    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: [0038]
  • 1. Materials [0039]
  • 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[0040] 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 [0041]
  • 2.1 Isolation of RPE Microsomes (Modified from (1)) [0042]
  • 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. [0043]
  • 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 PO[0044] 4/5 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) [0045]
  • 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 ultracentrifuged for 80 min. at 105,000 g. The pellet was sonicated in ˜5 mL of 0.1M PO4/0.1 mM EDTA/5 mM MgCl[0046] 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 B1) [0047]
  • 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 PO[0048] 4/5 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: PO[0049] 4/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 PO[0050] 4/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) [0051]
  • The following stock solutions were prepared: [0052]
  • [0053] 50 mM KH2PO4, pH 7.4 buffer, sterile filtered.
  • 10 mM all trans Retinol (Sigma R7632) in DMSO. [0054]
  • 200 mM Nicotinamide adenine dinucleotide phosphate, sodium salt (NADP) (Sigma N0505) in sterile water. [0055]
  • 40 mM test compound in appropriate solvent (water, buffer, ethanol, chloroform or DMSO). [0056]
  • 1:10 dilution of rat liver Microsomes in 50 mM KH2PO4, pH 7.4 buffer (4 μg/μl). [0057]
  • In a two-dram glass vial with screw cap, the following were added in order: [0058]
  • Buffer to give a final volume of 400 μl [0059]
  • 25 μl diluted Microsomes (final=100 μg)—boiled Microsomes were used for controls and regular Microsomes for test samples. [0060]
  • 4 μl of 200 mM NADP (final=2 mM) [0061]
  • 1 μl of 40 mM test compound (final=100 μM) [0062]
  • 8 μl of 10 mM retinol (final=200 μM) [0063]
  • 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. [0064]
  • 2.5 Assay for Retinal Reductase Activity (To Identify B3) [0065]
  • All stock solution were prepared as above with the following substitutions: [0066]
  • 10 mM all trans Retinaldehyde (Sigma R2500) in DMSO—instead of retinol. [0067]
  • 200 mM, Nicotinamide adenine dinucleotide phosphate, reduced form, tetrasodium salt (NADPH) (Sigma N7505) in sterile water—instead of NADP. [0068]
  • In a two-dram glass vial with screw cap, add the following in order: [0069]
  • Buffer to give a final volume of 400 μl [0070]
  • 25 μl diluted Microsomes (final=100 μg)—use boiled Microsomes for controls and regular Microsomes for test samples. [0071]
  • 4 μl of 200 mM NADPH (final=2 mM) [0072]
  • 1 μl of 40 mM test compound (final=100 μM) [0073]
  • 3 μl of 10 mM retinaldehyde (final=75 μM) [0074]
  • Follow the same incubation and extraction procedure as detailed above. [0075]
  • 2.6 Assay for CRABPII Antagonists (To Identify B4) [0076]
  • 2.6.1. Synthesis of CRABPII [0077]
  • a. System of expression [0078]
  • 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 [0079] 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 [0080] E. coli BLR(DE3)pLysS cells by transformation according to the manufacturer protocol (Novagen).
  • b. Induction [0081]
  • 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. [0082]
  • 2.6.2. Purification [0083]
  • Purification was performed according to the method described in Norris and Li, 1997. [0084]
  • a. Lysis [0085]
  • 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 MnCl[0086] 2, 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 [0087]
  • 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. [0088]
  • c. Anion-exchange Chromatography [0089]
  • 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. [0090]
  • d. Detection of the Presence of CRABPII [0091]
  • 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). [0092]
  • 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 ({fraction (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). [0093]
  • The concentration of purified CRABPII was determined using BSA kit (Pierce). [0094]
  • 2.6.3. Radioactive Binding Assay [0095]
  • 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. [0096]
  • 2.7 Assay for NADPH Dependent Retinoic Acid Oxidation (To Identify B5) [0097]
  • 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 PO[0098] 4/0.1 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. [0099]
  • 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 O[0100] 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 td 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 PO[0101] 4/0.1 mM EDTA/5 mM 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. [0102]
  • 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 325 ex/480 em. [0103]
  • 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. [0104]
  • 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. [0105]
  • 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). [0106]
  • 2 J. C. Saari & D. L. Bredberg, “ARAT & LRAT Activities of Bovine Retinal Pigment Epithelial Microsomes”, Methods Enzymol. 190, 156-163 (1990). [0107]
  • 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). [0108]
  • 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). [0109]
  • 5 A. B. Barua, “Analysis of Water-Soluble Compounds: Glucuronides”, Methods Enzymol. 189, 136-145 (1990). [0110]
  • The boosters suitable for use in the present invention include but are not limited to the boosters listed in Tables B[0111] 1 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 % Inhibition % Inhibition % Inhibition % Inhibition
    Overall Overall ARAT ARAT LRAT LRAT
    Class Compound TG(-ROH/RE) TG (IC 50) (10 jm) (100 jm) (10 jm) (100 jm)
    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 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% 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%
  • [0112]
    Retinol Dehydrogenase Activators (B2)
    Class Compound % Increase Retinol Dehydrogenase
    Phospholipid Phosphatidyl Choline 21% increase
    Phospholipid Sphingomyelin 26% increase
  • [0113]
    Retinaldehyde Reductase Inhibitors (B3)
    % Inhibition
    Overall Retinal
    Class Compound TG(IC 50) 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 ethanolamine 37%
  • [0114]
    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%
  • [0115]
    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 & Other Lauryl Hydroxyethylimidazoline 4.67E−07
    Surfactants
    Fatty Acid Amides & Other Oleyl Hydroxyethylimidazoline 3.02E−05 54% 80%
    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. [0116]
  • 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. [0117]
  • 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 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 3× with PBS. For colourimetric development, the cells were incubated with 100 μl substrate solution (4 mg o-phenylenediamine and 3.3 μl 30% H[0118] 2O2 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 of Tgase Assay [0119]
  • 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: [0120]
  • A. Reagents [0121]
    Cells: Human Keratinocytes Neonatal Human foreskin
    (P2 in T75 flasks; P3 in 96
    well assay plates)
    Primary Antibody: TGm specific Biogenesis (Cat # 5560-6006)
    monoclonal Ab B.C1
    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 [0122]
    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 0.2 M dibasic
    sodium phosphate and 0.1 M Sigma (Cat # C-1909)
    citric acid
    4 N H2SO4
  • C. Culture Ware [0123]
    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 [0124]
    Biotek Model EL 340 Microplate Bio-tek Instuments Inc.
    reader
    Multiprobe II Packard
  • E: Cell Culture Procedure [0125]
  • Seeding of Keratinocytes in 96 Well Plates [0126]
  • 1. A suspension of keratinocytes was prepared at a concentration of 3000 cells/200 μl/well in KGM medium (Used 3×10[0127] 5 cells /12 ml media in each microtitre plate)
  • 2. 200 μl of the keratinocyte suspension was transferred into each of the inner 60 wells only. [0128]
  • 3. 200 μl of KGM media was pipetted into the outer wells (to maintain thermal equilibrium). [0129]
  • 4. Each plate was incubated at 37° C. and 5% CO[0130] 2 for 3 days or until cells are ˜50% confluent.
  • Treatment of Keratinocytes with Samples [0131]
  • 5. Stock solutions of the samples were prepared in DMSO. [0132]
  • 6. The samples were diluted to desired concentration with the final assay concentration of DMSO being 0.1%. [0133]
  • 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. [0134]
  • 8. Plates were incubated at 37° C. and 5% CO[0135] 2 for 72 hours.
  • 9. Media were completely removed from each well. [0136]
  • 10. Wells were rinsed with 2× with 200 μl of 1×PBS [0137]
  • 11. Finally they were frozen for at least 1.5 hours at −70° C. [0138]
  • F: Transglutaminase Assay [0139]
  • 1. Block: [0140]
  • Incubate plates at room temperature with 200 μl/well of blocking buffer for 1 hour. [0141]
  • 2. Primary Antibody: [0142]
  • 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. [0143]
  • 3. Rinsed wells 6× with washing buffer. [0144]
  • 4. Secondary Antibody: [0145]
  • Incubated with 100 μl/well peroxidase labeled antimouse IgF(ab)2 fragment (diluted 1:4000 in washing buffer) at 37° C. for 2 hours. [0146]
  • 5. Rinsed wells 3× with washing buffer (added 200 μl) and aspirated after each rinse. [0147]
  • 6. Rinsed wells 3× with PBS w/o Tween. [0148]
  • 7. Incubated with 100 μl/well substrate solution at room temperature for exactly 5 minutes. [0149]
  • 8. Stopped reaction with 50 μl/well 4N H[0150] 2SO4.
  • 9. Read absorbance at 492 nm in the Bio-tek plate reader. [0151]
  • I. Optimization Studies [0152]
  • a. Time Course of Transglutaminase Production [0153]
  • 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[0154] 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 [0155]
  • 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. [0156]
  • C: Dose Response Curves: Retinoic Acid and Retinol [0157]
  • 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. [0158]
  • D: Final Conditions Used to Test Boosters or Combination of Boosters [0159]
    Days of incubation of keratinocytes with 3 days
    retinol and boosters
    Final DMSO concentration less than 0.1%
    Retinol concentration 10-7 M (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. [0160]
  • Transglutaminase levels were determined and expressed in the Tables B1 through B5 either as: [0161]
  • (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 [0162]
  • (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[0163] −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 (B1boosters), 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. [0164]
  • 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. [0165]
  • 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. [0166]
  • The boosters included in the inventive compositions are selected from the group consisting of: [0167]
  • a. Two boosters, wherein both are selected from the group consisting of B2, B3 and B4; [0168]
  • 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 [0169]
  • 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 [0170]
  • 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 [0171]
  • e. A combination of five groups of boosters B1/B2/B3/B4/B5. Booster to Booster Ratios [0172]
  • 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%: [0173]
    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. [0174]
  • For optimum performance, the concentration of retinoid to booster should be present in the composition in ratios as given below: [0175]
    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 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[0176] 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 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. [0177]
  • 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. [0178]
    Con. Used for synergy
    (binary,
    Booster tertiary,
    Class Compound Name IC50 IC20 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−06 to 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
  • 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. [0179]
  • B2 Class Boosters Phosphatidyl Choline (B2)
  • [0180]
    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)
  • [0181]
    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)
  • [0182]
    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)
  • [0183]
    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
  • [0184]
    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)
  • [0185]
    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)
  • [0186]
    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)
  • [0187]
    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)
  • [0188]
    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)
  • [0189]
    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)
  • [0190]
    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)
  • [0191]
    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)
  • [0192]
    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)
  • [0193]
    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)
  • [0194]
    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)
  • [0195]
    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)
  • [0196]
    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 [0197]
  • 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. IC[0198] 20). 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. [0199]
    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
    glycera-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 Oleoyl 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 [0200]
  • 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[0201] 20). 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 teritiary 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-Damascone 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 Benzotriazole 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. IC[0202] 20).
  • 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; [0203]
  • 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. [0204]
    Tertiary
    Quarternary (1-3
    TG combo; Difference
    Compound 1 Compound 2 Compound 3 Compound 4 (% C) TG % C) (<30% = synergy)
    B1/B2/B3/B4
    Combination:
    Castor oil Methyl Phosphatidyl Glycyrrhetinic 12-Hydroxystearic 21 64 42
    Ester Acid (MEA) Choline Acid acid
    Naringenin Phosphatidyl Glycyrrhetinic 12-Hydroxystearic 15 57 41
    Choline Acid acid
    Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic 12-Hydroxystearic −3 40 43
    Monoethanolamide sn-glycero-3- Acid acid
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic Isostearic acid 5 40 35
    Monoethanolamide sn-glycero-3- Acid
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl 12-Hydroxystearic −3 42 45
    Monoethanolamide sn-glycero-3- acid
    (LAMEA) phosphoethanol-
    amide
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Elaidic acid 8 42 34
    Monoethanolamide sn-glycero-3-
    (LAMEA) phosphoethanolamide
    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
    phosphoethanolamide
    Cocoyl Phosphatidyl Arachidonic Acid 2-Hydroxystearic −1 37 38
    hydroxyethylimidazoline Choline (Na+ salt) acid
    Cocoyl 1,2-dioctanoyl- Glycyrrhetinic Linseed oil −2 45 47
    hydroxyethylimidazoline sn-glycero-3- Acid
    phosphoethanolamide
    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-
    phosphoethanolamide
    Geraniol 1,2-dioctanoyl- Amsacrine-HCl Oleoyl 3 47 44
    sn-glycero-3- hydroxyethlimidazoline
    phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic Climbazole 2 40 37
    Monoethanolamide sn-glycero-3- Acid
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic Miconazole 5 40 35
    Monoethanolamide sn-glycero-3- Acid
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic Ketoconazole 0 40 40
    Monoethanolamide sn-glycero-3- Acid
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic Lauryl −2 40 41
    Monoethanolamide sn-glycero-3- Acid hydroxyethylimidazoline
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic Oleoyl 5 40 35
    Monoethanolamide sn-glycero-3- Acid hydroxyethlimidazoline
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic 7H-Benzimidazo[2,1- 1 40 39
    Monoethanolamide sn-glycero-3- Acid a]Benz[de]-isoquinolin-
    (LAMEA) phosphoethanolamide 7-one
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Climbazole 7 42 35
    Monoethanolamide sn-glycero-3-
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Clotrimazole 10 42 32
    Monoethanolamide sn-glycero-3-
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Miconazole 5 42 37
    Monoethanolamide sn-glycero-3-
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Ketoconazole 11 42 32
    Monoethanolamide sn-glycero-3-
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Lauryl −4 42 46
    Monoethanolamide sn-glycero-3- hydroxyethylimidazoline
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Oleoyl 5 42 37
    Monoethanolamide sn-glycero-3- hydroxyethlimidazoline
    (LAMEA) phosphoethanolamide
    Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl 7H-Benzimidazo[2,1- 8 42 35
    Monoethanolamide sn-glycero-3 a]Benz[de]-isoquinolin-
    (LAMEA) phosphoethanolamide 7-one
    Palmitamide 1,2-dioctanoyl- Amsacrine-HCl Miconazole 13 43 30
    Monoethanolamide sn-glycero-3-
    phosphoethanolamide
    Palmitamide 1,2-dioctanoyl- Amsacrine-HCl Oleoyl 3 43 40
    Monoethanolamide sn-glycero-3- hydroxyethlimidazoline
    phosphoethanolamide
    Alpha-Damascone 1,2-dioctanoyl- Amsacrine-HCl Miconazole 11 48 37
    sn-glycero-3-
    phosphoethanolamide
    Alpha-Damascone 1,2-dioctanoyl- Amsacrine-HCl Ketoconazole 13 48 34
    sn-glycero-3-
    phosphoethanolamide
    Alpha-Damascone 1,2-dioctanoyl- Amsacrine-HCl Lauryl 15 48 33
    sn-glycero-3- hydroxyethylimidazoline
    phosphoethanolamide
    Alpha-Damascone 1,2-dioctanoyl- Amsacrine-HCl Oleoyl hydroxyethlimidazoline 3 48 45
    sn-glycero-3-
    phosphoethanolamide
    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
    phosphoethanolamide
    1,2-dioctanoyl-sn- Glycyrrhetinic Isostearic acid Oleoyl 6 37 31
    glycero-3- Acid hydroxyethlimidazoline
    phosphoethanolamide
    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
    phosphoethanolamide
    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. [0205]
  • 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. [0206]
  • 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. [0207]
  • 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. [0208]
  • 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. [0209]
  • The exact amount of sunscreen employed in the emulsions can vary depending upon the degree of protection desired from the sun's UV radiation. [0210]
  • 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. [0211]
  • 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. [0212]
  • 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 cococaprylate/caprate (a blend of coco-caprylate and cococaprate), propylene glycol myristyl ether acetate, diisopropyl adipate and cetyl octanoate. [0213]
  • 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. [0214]
  • 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. [0215]
  • 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. [0216]
  • 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. [0217]
  • 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. [0218]
  • 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. [0219]
  • 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. [0220]
  • 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. [0221]
  • 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. [0222]
  • The invention accordingly also provides a closed container containing a cosmetically acceptable composition as herein defined. [0223]

Claims (7)

1. 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 B1 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 B1 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 B1 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 classes of boosters belonging to the classes B1 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 B1 to B5 in an almount 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.
US10/312,659 2000-06-30 2001-06-25 Skin conditioning compositions containing compounds for mimicking the effect on skin of retinoic acid Abandoned US20040043044A1 (en)

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US12/045,339 US7959913B2 (en) 2000-06-30 2008-03-10 Skin conditioning compositions containing compounds for mimicking the effect on skin of retinoic acid
US13/097,142 US8226933B2 (en) 2000-06-30 2011-04-29 Method for treating skin with booster compositions
US13/529,004 US8409550B2 (en) 2000-06-30 2012-06-21 Method for treating skin with retinoids and retinoid boosters
US13/764,090 US20140050676A1 (en) 2000-06-30 2013-02-11 Method for Treating Skin with Retinoids and Retinoid Boosters

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US13/097,142 Expired - Lifetime US8226933B2 (en) 2000-06-30 2011-04-29 Method for treating skin with booster compositions
US13/529,004 Expired - Fee Related US8409550B2 (en) 2000-06-30 2012-06-21 Method for treating skin with retinoids and retinoid boosters
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US13/529,004 Expired - Fee Related US8409550B2 (en) 2000-06-30 2012-06-21 Method for treating skin with retinoids and retinoid boosters
US13/764,090 Abandoned US20140050676A1 (en) 2000-06-30 2013-02-11 Method for Treating Skin with Retinoids and Retinoid Boosters

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