AU740577B2 - Ascorbyl-phosphoryl-cholesterol - Google Patents

Description

.11W S&FRef: 364391D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Avon Products, Inc.

1251 Avenue of the Americas New York New York 11020-1196 United States of America Dmitri Ptchelintsev Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Ascorbyl-Phosphoryl-Cholesterol The following statement is a full description of this invention, including the best method of performing it known to me/us:- IP Australia Documents ro o'n 0 3 DEC 1999 5845c Ascorbyl-Phosphoryl-Cholesterol Background of the Invention Field of the Invention The present invention relates to the synthesis and use of a novel derivative of L-ascorbic acid.

This derivative of L-ascorbic acid includes cholesterol. The resultant product is stable, easily incorporated into cosmetically acceptable vehicles and enzymatically bioreversible.

Description of the Prior Art The use of L-ascorbic acid as an antioxidant in food preparations is known. For example, Steinhart, Pro- and Antioxidative Effect of Ascorbic Acid on L-Tryptophan in the Fe 3 /Ascorbic Acid/O J. Agric. Food Chem., Vol. 41, pages 2275-2277 (1993) describes the use of L-ascorbic acid as an antioxidant that functions in food to remove free radicals and undergoing rapid oxidation.

Similarly, free L-ascorbic acid in topical preparations demonstrates poor stability and tends to break down due to partially oxidative and non-oxidative degradation. The degraded ascorbic acid loses activity and the resultant product loses aesthetic appeal since it exhibits a cosmetically undesired brown colour.

While cholesterol is considered unhealthy especially when ingested, the benefits of cholesterol, necessitated with L-ascorbic acid, for skin barrier repair are known. For example, Menon, Structural Basis for the Barrier Abnormality Following Inhabitations of HMG CoA Reductase in Murine Epidermis, J. Invest. Dermatol., Vol. 98, pages 209-219 (1992), describes deficiencies in the skin 20 barrier repair mechanism when cholesterol synthesis is inhibited by regulation of HMG CoA reductase.

Presently, mechanical mixing of L-ascorbic acid and cholesterol results in an unstable product S.due to the instability of L-ascorbic acid.

For example, US. 4 939 128 is directed to the use of phosphoric acid esters of ascorbic acid for 25 the treatment of diseases, not for cosmetics, topical dermatological or skin uses, and teaches that *certain phosphoric acid esters of ascorbic acid display improved oxygen scavenging properties. One of the phosphoric acid esters in the patent is substituted with a cholestanyl group. The conspicuous absence of cholesterol and the specific mention of a cholesteryl group recognises that conjugates of L-ascorbic acid and cholesterol were then not practical or desired.

Attempts have been made to conjugate ascorbic acid with a glycyrrhetic group as described in EP. 921 04149.7; and with a tocopheryl group as indicated by US. 3 151 127. US. 4 564 686 and 306 713 disclose tocopheryl ascorbyl phosphate as an anti-oxidant having the following structure:

CH

3 HO 0 O O 3 C O CH3 HO O1 'OO C H 3 C H3 C H 3 HO OH

CH

3 Also, Sakamoto, Measurement Method of Efficacy of Antidandruff Cosmetics and Development of the New Active Commercial Product, IFSCC, Yokohama, Vol. B206, pages 823-864 (1993) describes the use of tocopheryl coupled to L-ascorbic acid. The coupled tocopheryl is an antioxidant C07320 preservative for the ascorbyl group, but the use of the ascorbyl-tocopheryl as a skin therapeutic is questionable since, unlike cholesterol, tocopheryl is not a natural substrate for the skin.

Heretofore, there has been needed a stable product having cholesterol coupled to L-ascorbic acid, which product retained full functional activity even after decoupling by naturally occurring acidic phosphatases in the skin. This product would provide the beneficial properties of L-ascorbic acid, including increased collagen production and skin-lightening, combined with the benefits of released cholesterol, namely improved elasticity, resistance, tone and moisture retention of the skin.

Accordingly, there has been needed a method for covalently and bioreversibly effecting the coupling of cholesterol to L-ascorbic acid.

Summary of the Invention It is an object of the present invention such a stable composition that is a novel derivative of Lascorbic acid that includes cholesterol.

It is another object of the present invention to provide to provide such a stable composition of cholesterol coupled to L-ascorbic acid for use in cosmetic products.

15 It is still another object of the present invention is to provide such a stable composition having multiple skin care benefits.

It is a further object of the present invention is to provide such a stable composition that is easily carried in cosmetic vehicles, enzymatically bioreversible, and demonstrates extended shelf-life.

It is yet a further object of the present invention is to provide a method for covalently and 20 bioreversibly coupling cholesterol to L-ascorbic acid for stabilisation of the resulting molecule.

To accomplish the forgoing objects and advantages, the present is invention, in brief summary, is a derivative of L-ascorbic acid that includes cholesterol. Such derivatives are, for example, ascorbyl-2-o-phosphoryl)-cholesterol and the salts thereof.

These and other objects of the present invention will become evident from the invention 25 described below.

Detailed Description of the Preferred Embodiment The present invention includes a novel derivative of L-ascorbic acid. The derivative is formed by a coupling of L-ascorbic acid and cholesterol. The novel derivative, that can be easily included in a suitable topical vehicle, is selected from the group consisting of 3'-(L-ascorbyl-2-o-phosphoryl)cholesterol, 3'-(L-ascorbyl-3-o-phosphoryl)-cholesterol and the salts thereof. The exemplary compounds include functional or structural homologues of 3'-(L-ascorbyl-2-o-phosphoryl)-cholesterol (Formula I) such as 3'-(L-ascorbyl-3-o-phosphoryl)-cholesterol (Formula II). Both formulas are illustrated below.

HO oo 0 HO om 1 s7

H

O OH Formula I C07320 HO Formula II The L-ascorbic acid is covalently bounded to the cholesterol by phosphoryl or phosphates so that the L-ascorbic acid derivative set forth above is also called ascorbyl-phosphoryl-cholesterol.

In the ascorbyl-phosphoryl-cholesterol compounds of the present invention, the conjugated ascorbic acid becomes resistant to degradation. The cholesteryl group serves as a carrier moiety and facilitates delivery of polar ascorbic acid through the non-polar outermost protective layer of skin (ie., the stratum corneum) and increases the bioavailability of the ascorbic acid in the topical application.

Natural enzymes, such as phosphatases present in the skin, gradually cleave the phosphoryl or phosphate linkage between cholesterol and ascorbic acid, resulting in sustained release of free Lascorbic acid and cholesterol into the stratum comeum. The released cholesterol is a natural substrate for skin and supplements that otherwise produced by the body. Topically applied cholesterol improves elasticity, tone and resistance to drying.

The basic topical formula may comprise from about 0.0001 to about 100, with all ranges set forth in wt%, of the L-ascorbic acid derivative. In a preferred embodiment, about 0.05 to about of the L-ascorbic acid derivative is in a cosmetically acceptable vehicle. In a more preferred embodiment, about 0.10 to about 20wt% of the L-ascorbic acid derivative is combined with a cosmetically acceptable vehicle, and in an even more preferred embodiment about 1.0 to about 10wt%. Salts of the L-ascorbic acid derivative, namely ammonium, calcium, lithium, potassium or sodium can be incorporated with the L-ascorbic derivative into a cosmetically acceptable vehicle. A 20 salt with an organic amine, such as ethanolamine, may also be used in combination with the Lascorbic acid derivative.

Suitable vehicles include conventional lotions, creams or gels. A "physiologically acceptable vehicle" or a "suitable vehicle" means drugs, cosmetics, medicaments or inert ingredients that are suitable for use in direct contact with human tissues without undue toxicity.

25 A first or more basic lotion comprises about 0.10 to about 20.0wt% of the L-ascorbic acid derivative, and the remainder is or includes water. Most preferably, the L-ascorbic acid derivative is 3'- (L-ascorbyl-2-o-phosphoryl)-cholesterol (Formula I) or 3'-(L-ascorbyl-3-o-phosphoryl)-cholesterol (Formula II) and, preferably, the L-ascorbic acid derivative is a homologue and/or salt thereof. A second lotion has about 0.10 to about 20.0wt% L-ascorbic acid derivative, about 0.001 to about 1.5wt% thickener or thickening agent, and the remainder is or includes water. The second lotion may also include up to about 1.Owt% fragrance.

Examples of thickening agents suitable for use with the L-ascorbic acid derivative include xanthene gum, xanthene gum brine tolerant, hydroxypropylcellulose, hydroxyethylcellulose, carbopol and gum acacia, Sepigel 305 (available from Seppic Co., France), vee-gum or magnesium aluminium silicate or combination thereof. The thickening agent is preferably xanthene gum or hydroxyethylcellulose or a combination thereof.

C07320 A third lotion has, besides about 0.10 to about 20.0wt% L-ascorbic acid derivative, about 0.50 to about 1.40wt% of a thickening agent, about 0.50 to about 6.0wt% of an emollient, about 4.8 to about 14.5wt% of an emulsifier, and the remainder is or includes water. It may also include about 0.35 to about 0.45wt% of a preservative.

In the third lotion, the thickening agent is preferably about 0.25 to about 0.70wt% of xanthene gum, and about 0.25 to about 0.70wt% of hydroxyethylcellulose. The emollient, which can be a humectant, preferably is glycerin. The emulsifier is preferably a combination of emulsifiers, namely about 2.0 to about 8.0wt% of propylene glycol decapitate, about 1.8 to about 4.0wt% of Peg Stearate, and about 1.0 to about 2.5wt% of Steareth-2. The preservative is preferably about 0.15 to about 0.20wt% of disodium EDTA or EDTA salt, and about 0.20 to about 0.25wt% of methylparaben.

A second cosmetic vehicle, a cream, comprises about 0.10 to about 20.0wt% of the L-ascorbic acid derivative, about 0.1 to about 1.20wt% of a thickening agent; about 0.1 to about 15wt% of an emulsifier, and the remainder is or includes water. It may also include up to about lwt% of fragrance.

A second, less preferable, cream has about 0.5 to about 4.0wt% of an emollient, preferably glycerin; about 2.0 to about 6.0wt% of an emollient/humectant, preferably propylene glycol; emulsifiers, preferably about 1.8 to about 3.0wt% Steareth-20, about 0.8 to about 2.0wt% Steareth-2, about 1.0 to about 2.5wt% cetyl alcohol, and about 0.9 to about 3.5wt% glycerol mono-stearate; thickening agents, such as about 0.25 to about 0.6wt% xanthene gum and about 0.25 to about 0.6wt% hydroxyethylcellulose; and a preservative, preferably about 0.15 to about 0.2wt% disodium EDTA or EDTA salt. While such lotions or creams can be made by conventional homogenisation methods, such lotions and creams can also be made by a process of microfluidisation that involves co-mixing the aqueous phase and the oil phase of such creams and lotions in a high-pressure homogeniser that reduces the emulsion particle size dramatically to about 1/400 t the size of those in creams and lotions prepared without applying high pressure. Microfluidisation permits the preparation of elegant stable creams and lotions containing effective amounts of the L-ascorbic acid derivative without the use of traditional emulsifiers and surfactants.

With respect to the L-ascorbic acid derivative in a gel vehicle, a first or preferred gel has about 0.10 to about 20wt% L-ascorbic acid derivative, about 0.30 to about 2.0wt% thickening agent, and the remainder includes water. A second or less preferred gel has about 0.10 to about 20.0wt% L-ascorbic acid derivative; about 2.0 to about 6.0wt% of an emollient/humectant, preferably propylene glycol; about 0.4 to about 1.5wt% of a thickening agent, preferably hydroxyethylcellulose; and a preservative, preferably about 0.15 to about 0.20wt% disodium EDTA or EDTA salt and about 0.20 to about 0.25wt% methylparaben.

The pH of the lotion, cream or gel formulas can be adjusted to physiologically acceptable levels with sufficient amounts (preferably about 3.0 to about 7.5wt%) of ammonium hydroxide, calcium hydroxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, ethanolamine, diethanolamine or urea.

As stated above, an emollient used in the above lotion, cream and gel formulas is glycerin and an emollient/humectant is propylene glycol. Besides such emollients, the L-ascorbic acid derivative or the lotion, cream or gel formulas can also be combined with most other conventional emollients, such C07320 as mineral oil, petrolatum paraffin, ceresin, ozokerite, microcrystalline wax, perhydrosqualene, dimethylpolysiloxanes, methylphenylpolysiloxanes, silicone-glycol copolymers, triglyceride esters, acetylated monoglycerides, ethoxylated glycerides, alkyl esters of fatty acids, fatty acids and alcohols, lanolin and lanolin derivatives, polyhydric alcohol esters, sterols, beeswax derivatives, polyhydric alcohols and polyethers, and amides of fatty acids. Other suitable emollients can be found in Sagarin, Cosmetics. Science and Technology, 2nd Ed., vol. 1, pp. 32-43 (1972), the contents of which are incorporated by reference herein.

In the above formulas, the emulsifiers can be cationic, anionic, non-ionic, amphoteric, or a combination thereof. A non-ionic emulsifier is preferred. As set forth above, the non-ionic emulsifiers propylene glycol decapitate, PEG 40 Stearate, Steareth-20, Steareth-2 and cetyl alcohol are used in various formulas. Examples of other non-ionic emulsifiers are commercially available sorbitans, alkoxylated fatty alcohols and alkyl polyglycosides. Anionic emulsifiers may include soaps, alkyl sulfates, monoalkyl and dialkyl phosphates, alkyl sulfonates and acyl isethionates. Other suitable emulsifiers can be found in McCutcheon, Detergents and Emulsifiers, North American Edition, pp.

317-324 (1986), the contents of which are incorporated herein by reference.

Other suitable preservatives, besides Distoma EDTA and methylparaben set forth above, include alkanols, especially ethanol and benzyl alcohol, parabens, sorbates, urea derivatives and isothiazolinones.

Suitable humectants include urea, PCA, amino acids, certain polyols and other compounds with hygroscopic properties.

S* The present invention includes topically applying an effective amount in a physiologically acceptable vehicle to a skin area, normally once or twice daily. The effective amount and the frequency of application will vary depending on the particular skin, the age and physical condition of S. the person, and like factors within the knowledge and expertise of those skilled in the art.

The L-ascorbic acid derivative in an amount about 0.05 to about 10wt%, and more preferably about 0.05 to about 5wt%, can be in topical compositions alongside keratolytic agents and skin lightening agents. The keratolytic agents may include salicylic acid and benzoyl peroxide. The skin lightening agents may include kojic acid, benzoquinone, liquorice derivatives, magnesium ascorbyl phosphate, glycerhetinic acid and its derivatives.

30 The L-ascorbic acid derivative in an amount about 0.001 to about 25wt% can also be used with organic and inorganic sunscreens, such cinnamic acid derivatives (menthyl, octyl, 2-ethylhexyl, benzyl, a-phenyl cinnamonitrile, and butyl cinnamoyl pyruvate), titanium dioxide, zinc oxide, benzylidene camphor, anthranilates, and naphtholsulfonates. The cinnamic acid derivatives are preferred. About 0.001 to about 10wt%, and more preferably about 0.001 to 5wt% of the L-ascorbic acid derivative can be co-formulated with retinoids, hormonal compounds, a-hydroxyacids or polyhydroxy a-hydroxy acids, or a-keto acids.

The retinoids include, for example, retinol, retinoic acid, retinyl palmitate, retinyl propionate, retinyl acetate, isotretinoin and synthetic retinoid mimics. The hormonal compounds include, for example, estriol, estradiol, estrone or conjugated estrogens. The a-hydroxyacids or polyhydroxy ahydroxy acids include, for example, glycolic acid, lactic acid, tartaric acid, gulonic acid and other C07320 carboxylic acids and their monomeric, polymeric, cyclic or acyclic derivatives. The cc-keto acids include, for example, pyruvic acid, 2-oxopropanoic acid, 2-oxobutanoic acid, 2-oxopentanoic acid and the like.

The L-ascorbic acid derivative can be used for additional benefits in topical formulations that include the following ingredients: vitamins, enzyme co-actors such as vitamin B6, vitamin B12, vitamin D3, 1,25-dihydroxy vitamin D3, vitamin B1, riboflavin, vitamin K, vitamin E, tocotrienols and their derivatives, nicotinic acid and its esters, pantothenic acid and its esters, panthenol, folic acid and its derivatives, choline, camitine and substances without formal vitamin status or "pseudo-vitamins" such as vitamin F or cis,cis-linoleic acid, vitamin M or pteroylglutamic acid, vitamins B10 and B11, sesame seed factor, termitin, penicin, insectine, hypomycin and mycoine, vitamin L or anthranilic acid, vitamin L2 or adenylthiomethyl-pentose, myoinositol or cis-1,2,3,5-trans-4-6-cyclohexanehexol and its esters, especially phytic acid, laetrile or 1-mandelo-nitrile-P-glucuronic acid, amygdalin, vitamin 815 or pangamic acid, vitamin 813 or orotic acid, vitamin H3 or procaine hydrochloride, vitamin U or methylsulfonium salts of methionine and pyrroloquinoline quinone, or effective amounts of antifungal agents such as clotrimazole, ketoconazole, miconazole, naftifine, tolnaftate, amphotericin B, nystatin, fluorocytosine, griseofulvin, haloprogin, of which tolnaftate, haloprogin and miconazole are most preferred. In formulas that include one or both of the preferred, the L-ascorbic acid derivative is present in an amount from about 0.001 to about 10 and, more preferably, about 0.001 to about About 0.001 to about 20wt% of the L-ascorbic acid derivative can be used with one or more of: self-tanning agents, such as dihydroxyacelone and lawsone, with the former one being most preferred; anti-mycobacterial agents, such as erythromycin, tetracyclin and related compounds, especially doxycyclin and methacyclin, cephalosporine, penicillins, macrolides, peptide compounds selected from the group consisting of novobiocin, vancomycin, oleandomycin, paromomycin, 25 leucomycine, amphomycin with macrolide molecules preferred over the polypeptide compounds, quinolone derivatives, and other compounds which interfere with bacterial cell wall synthesis, :membrane function, RNA metabolism, puline, pyrimidine and protein synthesis, respiration or phosphorylation; topical analgesics, such as lidocaine, benzocaine, butamben, butacaine, tetracaine, clove 30 oil, eugenol, with lidocaine and benzocaine being most preferred; lipidic compounds essential for the skin's barrier function such as ceramides, essential fatty acids and their esters, especially glycerides, co-hydroxy fatty acids and their esters derived with alkanols through carboxylic hydroxyl or with other fatty acids at the o-hydroxy, the latter type being most preferred, with phospholipids. The lipidic compounds can be added to a topical composition either as singular molecular entities or as a complex mixture of lipids derived from either synthetic, animal or plant sources; antiallergenic agents and H1 and/or H2 antihistamines, such as diphenylhydramine, clemizole, antazoline, thenaldine, phenyltoloxamine citrate, tricyclic antiallergenics, such as ketotifene, dithiadene and 3-thienylsulfide of thiadene, H2-receptor blockers, especially burimamide, metiamide and cimetidien, cromolic acid and its salts; C07320 the L-ascorbic acid derivative can be used with topical anti-inflammatory agents that can reduce inflammation. These agents are at a concentration from about 0.001% to about preferably, about 0.5 to about with the concentration of the anti-inflammatory adjusted up or down depending upon the potency of the utilised agents. Examples of steroidal anti-inflammatories that can be used with the L-ascorbic acid derivative include hydrocortisone, hydroxytriamcilone aomethyldexamethasone, dexamethasone phosphate, beclamethasone dipropionate, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, prednisolone, prednisone, and mixtures thereof, with the most preferred being prednisolone and hydrocortisone; non-steroidal anti-inflammatories can also be employed, is such as described in Rainsford, to Antiinflammatory and Anti-Rheumatic Drugs, Vols. I-III, CRC Press, Boca Raton, Florida (1985).

Specific examples of suitable non-steroidal anti-inflammatories include oxicams (eg. piroxicam, isoxicam), fenamic acid derivatives, meclofenamic acid derivatives (eg. sodium meclofenamate), flufenamic acid derivatives, mefenamic acid derivatives, propionic acid esters, such as ibuprofen, naproxen, benoxaprofen, flubiprofen, ketoprofen, suprofen, with ibuprofen being most preferred; pyrazolidinediones, with phenylbutazone being most preferred; the acetic acid derivatives, such as diclofenec, fenclofenac, indomethacin, sulindac, with indomethacin being most preferred; salicylic acid derivatives, such as aspirin, disalacid, benorylate, with aspirin and disalacid being most preferred.

The compositions of the present invention may also include safe anti-inflammatory products of natural origin shown to possess anti-inflammatory activity such as aloe vera extracts, extracts from genus Rubis (Rubia Cordifolio), extracts from genus Commiphom (Commiphora Mukul), Willow bark, matricarria flowers, arnica flower, comfrey root, fenugreek seed and the like known to those skilled in he art.

About 0.001 to about 20wt% of the L-ascorbic acid derivative can be used in formulas that contain anti-oxidants with phenolic hydroxy functions, such as gallic acid derivatives (eg. propyl 25 gallate), bio-flavonoids (eg. quercetin, rutin, daidzein,, genistein), ferrulic acid derivatives (eg. ethyl ferrulate, sodium ferrulate), 6-hydroxy-1,2,5,7-tetramethylchroman-2-carboxylic acid. The compositions may also contain effective concentrations of water soluble anti-oxidants such as, for example, uric acid, reductic acid, tannic acid, rosmarinic acid and catechins. Also the L-ascorbic acid derivative can be co-formulated with nitric oxide synthase inhibitors to reduce skin redness, 30 vasodilation and inflammatory reactions, especially in response to electromagnetic and ionising radiation or to the action of chemically or biochemically aggressive compounds. The nitric oxide synthase inhibitors can be added at concentrations from about 0.05% to about 10%, most preferably from about 1% to about The nitric oxide synthase inhibitors are selected from the group consisting of guanidine derivatives, especially monoaminoguianidine and methylguanidine, L-arginine derivatives, especially NG-nitro-L-arginine and its esters, NG-monomethyl-L-arginine, 2iminopiperidines and other 2-iminoazaheterocycles.

Other possible anti-oxidants that the derivative may contain are those that have one or more thiol functions in either reduced or non-reduced form such as glutathione, lipoic acid, thioglycolic acid and other sulfhydryl compounds. The levels of sulfhydryl anti-oxidants should not exceed for cosmetic uses of the composition, but may be higher for pharmaceutical uses as dictated by the C07320 considerations of efficacy. The composition may also include inorganic anti-oxidants, such as sulfites, bisulfites, metabisulfites or other inorganic salts and acids containing sulfur in oxidation state The preferred level of inorganic sulfur-containing anti-oxidants is about 0.01 to about 0.5 with the most preferred level about 0.1 to about 0.4 by wt%.

The L-ascorbic acid derivative may be used with about 0.025% to about preferably about to about 3wt%, and most preferably about 0.5 to about 1wt%, of compounds known to be electron spin-traps, such as nitrones, N-tert-butylnitrone and a-[4-pyridyl-1-oxide]-N-tertbutyl nitrone or other compounds known to form free radicals with half-life time of more than 1 min.

About 0.001 to about 50wt% of the L-ascorbic acid derivative can also be used in compositions that contain insect repellents such as aliphatic, cyclic or aromatic amides, citronella oil, terpineol, cineole, neem oil and terephthalic acid and its esters. Other suitable insect repellents can be found in Technical Bulletin No. 1549 from the U.S. Department of Agriculture or in their Agricultural Handbook Nos. 69, 340 and 461.

The about 0.001 to about 50wt% L-ascorbic acid derivative is also suitable for topical compositions that contain skin cooling compounds such as, for example, menthol, menthyl glycerol, asymmetrical carbonates, thiocarbonates and urethanes, N-substituted carboxamides, ureas or phosphine oxides such as described in J. Cosmet. Chain., vol. 29, p. 185 (1978), menthyl lactate and menthone glycerine acetal.

The L-ascorbic acid derivative can be used with other cosmetic and pharmaceutical actives and exponents, such as, for example, antifungals, antiallergenic agents, depigmenting agents, antiinflammatory agents, anaesthetics, surfactants, moisturisers, exfolients, stabilisers, antiseptics, i* lubricants, chelating agents and skin penetration enhancers. When used with these ingredients, the Lascorbic acid derivative may provide additional dermatological and/or cosmetic benefits.

The L-ascorbic acid derivative can also be formulated in the form of micro-emulsions. The 25 micro-emulsion system would typically contain an effective amount of the L-ascorbic acid derivative, up to 18% of a hydrocarbon, up to 40% of an oil, up to 25% of a fatty alcohol, up to 30% of a non-ionic surfactant, and up to 30% of water.

The L-ascorbic acid derivative is suitable and convenient for use in topical products formulated in the form of oil-in-water or water-in-oil emulsions, ointments, sticks, sprays, tapes, patches, as 30 multiphase emulsion compositions, such as water-in-oil-in-water type as disclosed in U.S. Patent No.

4,254,105, incorporated herein by reference. The L-ascorbic acid derivative can also be formulated as triple emulsions of the oil-in-water-silicone fluid type as disclosed in US. 4 960 764 incorporated herein by reference.

The L-ascorbic acid derivative can also be made as a liposomal formulation, for example, according to the methods described in Mezei, J. Pharmaceut. Pharmacol., vol. 34, pp. 473-474 (1982) or modification thereof. In such compositions, droplets of the L-ascorbic acid derivative can be entrapped inside the liposomal vesicles and then incorporated into the final formula with the shell of the liposome being a phospholipid but which can be replaced with other suitable lipids skin lipids). The liposomes can then be added to any carrier system described above according, for example, to the preparation modes, uses and compositions of topical liposomes as described in C07320 Mezei, Topics in Pharmaceutical Sciences, Breimer et al. Eds., pp. 345-358, Elsevier Science Publishers BV, New York (1985), incorporated herein by reference, or according to the reverse-phase evaporation method described in Szoka et al., Proc. Nat. Acad. Sciences, vol. 75, pp. 4194-4198 (1978), and also in Diploses et al., J. Soc. Cosmetic Chemists, vol. 43, pp. 93-1 00 (1992), all s incorporated herein by reference, is The L-ascorbic acid derivative can also be entrapped in polymeric vesicles with a shell consisting of a suitable polymeric material, such as gelatin, cross-linked gelatin, polyamide, poylacrylates and the like. These vehicles can then be incorporated into any composition set forth herein.

The general activity and mildness to skin can also be enhanced by neutralisation to pH3.5 to 8.0, most preferably from pH3.7 to 5.6, with one or more amphoteric and pseudoamphoteric compounds selected from a group including, but not limited to, glycine, alanine, valine, serine, thionine, methionine, leucine, asparagine, histidine, glutamic acid, glutamine, lysine, cystine, cystein, tryptophan, seine, phenylalanine, citrulline, creatine, proline, 3- or 4-hydroxyproline, omithine and its derivatives, 3-aminopropanoic acid and other aminocarboxylic acids, canavanine, canaline, homoarginine, taurine, aminoaldonic acids and aminosugars, aminouronic acid, aminoaldaric acid, deacetylated hyaluronic acid, hyalobiuronic acid, chondrosine, desulfated heparin, neuraminic or sialic acid, methionine sulfone, glycylglycine, chondroitin, D,L-sphingosine, sphingomyelin, ophidine, glucagon, homocarnosine, phosphatidyl serine, cocoamphoglycine, phosphatidyl ethanolamine, cysteinesulfinic acid, glutathione, amphoteric inorganic oxides, polyamidoamines, polyamidoamines-based dendrimers, sodium hydroxymethylglycinate and polyethylene amine.

When about 0.001 to about 20wt% of L-ascorbic acid derivative is used with certain chelating agents, the utility and mildness of the composition can also be enhanced. The chelating agents should be from about 0.01 to about 25, more preferably from about 0.5 to about 10, and most 25 preferably from about 1 to about 5wt%. Suitable examples of chelating agents include those that have a high affinity for zinc, calcium, magnesium, iron and/or copper ions, such as ethylene-diamine-tetraacetic acid (ethylenedioxy)-diethylene-dinitrilo-tetra-acetic acid, salicylaldoxime, quinolinol, diaminocyclohexane-tetra-acetic acid, diethylene-triaminopenta-acetic acid, dirnethylglyoxime, benzoin oxime, triethylenetetramine, desferrioxamine or mixtures thereof.

30 The L-ascorbic acid derivative has been unexpectedly and surprisingly found to be useful as active agent in topical preparations for treating signs of dermatological aging, both photoageing and intrinsic aging, including skin wrinkles such as fine wrinkling in the eye areas or "crows feet," or fine wrinkles around the mouth area, irregular pigmentation, sallowness, loss of skin resilience and elasticity. The present compound is also useful for treating disorders associated with the nails, cuticles and hair such as ingrown hair, folliculitis and Pseudofolliculitis barbae. It has been discovered that the present compounds soften hair and promotes the elimination of hair ingrowths, and are particularly useful for shaving.

The L-ascorbic acid derivative also enhances protection against UV provided by known sunscreen formulations.

C07320 The present invention also relates to a method for coupling a molecule of L-ascorbic acid to a molecule of cholesterol. The coupling preferably occurs through a bioreversible phosphate linkage at position 2 or 3 on the ascorbyl group and position 3' on the cholesteryl moiety. Resulting compositions are also contemplated by this invention.

Formula I was formed by preparing the conjugated 3'-(L-ascorbyl-2-o-phosphoryl)-cholesterol by dissolving cholesterol at -10 0 C in dry diethyl ether (dried with 4A molecular sieves) containing equivalent of triethylamine as a base. Phosphorous oxychloride (1.0 equivalent) was added to provide cholesteryl phosphorodichloridate.

The melting point of the cholesteryl phosphorodichloridate was measured as 121-122 0 C and infrared (KBr pellet) analysis showed P=O absorption at 1298 wavelengths and P-O-C absorption at 1019 wavelengths, with no hydroxy absorption. Cholesteryl phosphorodichlondate was subsequently reacted for 3h at room temperature with 5,6-isopropylidene-L-ascorbic acid in tetrahydrofuran containing 1.0 equivalent of triethylamine. This reaction yielded a mixture of cholesteryl 5,6 isopropylidene-2-phosphorochlondate L-ascorbic acid and its isomer cholesteryl 5,6-isopropylidene-3phosphorochloridate L-ascorbic acid.

The isomeric moisture was hydrolysed in an aqueous solution of THF and stirred for several hours at room temperature with Amberlyst-15, a strongly acidic sulfonic acid ion exchange resin. THF and water were then removed. The final product, 3'-(L-ascorbyl-2-o-phosphoryl)-cholesterol, was extracted with ethyl acetate and neutralised with an KOH equivalent. The resulting solution was lyophilised to obtain the monopotassium salt form.

This novel method permits covalent and bioreversible coupling of cholesterol with L-ascorbic acid resulting in the stabilisation of ascorbic acid, and increased bioavailability for ascorbic acid and S cholesterol.

The compounds of the present invention are generally synthesised by reacting cholesterol with 25 a halogenophosphorelating agent, coupling the resulting product with 5,6-hydroxy protected Lascorbic acid, hydrolysing the product with water,, stripping the protective group with an acidic resin and purifying the product with lyophilisation and recrystallisation. The derivative is stable in solution, exhibits anti-oxidant activity and stimulates production of collagen in fibroblasts.

Example 1 Preparation of Phosphodiester Acid and its Mono Potassium Salt Cholesteryl phosphodichloridate was synthesised using the following procedure. A 250mL two neck 19/22 ST round bottom flask was selected for the reaction. It included a serum cap (with nitrogen inlet needle), a stirring bar and a 19/22 to 24/40 ST 12:5mL dropping funnel equipped with a side arm.

This apparatus was flame dried and cooled under a nitrogen sweep. The dropping funnel was charged with 4.64g (12mmole) of Sigma 99+% cholesterol, 75mL of ether (dried over activated 4A molecular sieves) and 1.214g (12mole, 1.672mL) of dry (over KOH) triethylamine.

The flask was charged with 28mL of dry ether and 1.84g (12mole, 1.118mL) of phosphorous oxychloride and cooled in an ice/methanol (-10 0 C) bath. Ether containing the cholesterol-triethylamine C07320 was added dropwise at a brisk rate over a period of 20 to 30 minutes. The solution was warmed to room temperature and stirred for 2.5 hours.

Precipitated solids were filtered off on a Buchner funnel and washed three times in water with thorough stirring. Air was introduced through the Buchner funnel until all of the ether in the filtrate evaporated. Solid precipitate was then removed by filtration through a second Buchner funnel and cholesteryl phosphodichloridate was dried in a vacuum desiccator over phosphorous pentoxide. This experiment yielded 3.90g of first crop solid, mp 121-122°C and 1.74g of second crop material, mp 117-118°C. IR analysis (KBr pellet) showed absorption at 2947 wavelengths, H) absorption at 2878 wavelengths, (CC) absorption at 1466 wavelengths, absorption at 1298 wavelengths and absorption at 1019 wavelengths.

Ascorbic cholesteryl phosphodiester chiondate was synthesised following the procedure as outlined below.

A 50mL three neck 19/22 ST round bottom flask fitted with a stirring bar, serum cap, nitrogen inlet needle and 50mL dropping funnel was selected for this experiment. This apparatus was flame dried and cooled under a nitrogen sweep. The dropping funnel was charged with 503mg (Immole of cholesteryl phosphorodichloridate (mp 122°C) and 15mL of dry THF, and the mixture was cooled in an ice/methanol bath To the cooled mixture was added 216mg (Immole) of Sigma 5,6isopropylidene-L-ascorbic acid, 15mL of dry THF and 0.14mL (101mg, Immole) of dry (KOH) triethylamine. After addition, the mixture was warmed to room temperature and stirred for 3h.

A TLC (25% methanol/toluene) analysis indicated the reaction was complete. It also suggested that the product was a mixture of 2-0 and 3-0 regioisomers. The precipitated triethylamine hydrochloride was removed by filtration through fluted paper. THF was removed by rotary evaporation *'to provide 0.66g of crude crystalline ascorbic cholesteryl phosphodiester chioridate.

Ascorbic cholesteryl phosphodiester acid was prepared utilising the following procedure. Crude 25 ascorbic cholesteryl phosphodiester chloridate (6.76g, 9.9mmole) in 60mL of THF was combined with of water and 20g of wet Amberlyst-15 that had been rinsed in water three times. The resulting mixture was stirred vigorously at room temperature for 55h. Amberlyst-15 was removed by filtration :through fluted paper and was rinsed once with 20mL of 1:1 THF/water.

Most of the THF was removed in a stream of nitrogen to provide 53mL of a thick cloudy 30 aqueous suspension.

Fifty three (53)mL of THF was added to the suspension to yield 106mL of 1:1 THF/water solution of crude phosphodiester acid that was nearly clear. Phosphodiester acid was purified by adding the 1:1 THF/water solution to a column of C-18 reverse phase silica gel (472g) and eluting with 1:1 THF/water. THF was removed in a stream of nitrogen to give 215mL of purified phosphodiester acid in aqueous suspension. The projected total yield was 1.74g and the actual isolated yield was 1.84g Reverse phase HPLC analysis indicated 90% purity.

Ascorbic cholesteryl phosphodiester diacid mono potassium salt was made by first treating a 1% aqueous solution of the diacid with one equivalent of a standardised potassium hydroxide solution and subsequent lyophilisation. The phosphodiester diacid (579mg, 0.927mmole) was dissolved in 57.9mL of water and treated with 9.44mL of 0.0986N potassium hydroxide solution (0.931mmole).

C07320 The neutralised solution was then lyophilised to remove water and yield 603mg of mono potassium salt as a fluffy white solid.

Example 2 Purification by Reverse Phase C-18 Chromatography s Reverse Phase C-18 silica gel was prepared on a 1kg scale according to Evans, Chromatographia, Vol. 13, pages 5-10 (1980). Purification of the phosphodiester acid to a level of was achieved at a 90:1 load ratio using 1:1 THF/water, followed by THF removal in a stream of nitrogen and water removal by lyophilisation. Investigation of other solvent systems by reverse phase thin layer chromatography has good potential to improve the level of purity, (ii) identify an effective separation medium that could be removed by rotary evaporation, and (iii) allow the use of a lower load ratio. Since the reverse phase C-18 silica gel is reusable, the method has good potential for purification up to 1000g.

Solvent systems that are suitable include THF/methanol, THF/ethanol, THF/isopropanol, dioxane/methanol, dioxane/ethanol, dioxane/isopropanol, ether/methanol, ether/ethanol, ether/isopropanol, ethyl acetate/methanol, ethyl acetate/ethanol, ethyl acetate/isopropanol, methylene chloride/ethanol, methylene chloride/methanol, methylene chloride/isopropanol, DME/methanol, DME/ethanol and DME/isopropanol.

Conjugation with cholesterol converts the polar ascorbic acid to a more non-polar lipophilic ascorbyl group that is readily absorbed through the stratum corneum. Once past the stratum corneum, the absorbed compound is able to effect underlying fibroblasts. The benefits of bioreversed ascorbic acid and cholesterol have been previously explained. Surprisingly, the conjugated compound itself stimulates collagen synthesis which enhances the integrity, elasticity and resiliency of skin. Additional details are provided in Example 3.

Example 3 Fibroblast Studies This example summarises a study in which the ability of 3'-(L-ascorbyl-2-o-phosphoryl)cholesterol to stimulate collagen production in cultured human skin fibroblasts is demonstrated. An art-recognised 3 H]-Proline Incorporation Assay was performed with different doses of 3'-(L-ascorbyl- 2-o-phosphoryl)-cholesterol. Juva, Anal. Biochem., Vol. 15, pages 77-83 (1966); Booth, Biochem.

30 Biophys. Acta, Vol. 675, pages 117-122 (1981).

Fibroblasts were incubated with Opjg/mL, 11.3pg/mL, 22.5lg/mL and 451ig/mL of ascorbyl-2-o-phosphoryl)-cholesterol for a total of 48h. After the first 24h 3 H]-labelled proline was added to the culture. Following the second 24h period, the cells are harvested and prepared for the collagen biosynthesis assay.

Protease inhibitors are added to prevent degradation of collagen and other proteins. The cell layer is scraped into a solution containing 0.4M NaCI and 0.01M Tris (pH7.5). Extracts are sonicated to disrupt cell membranes. Separate volumes of the cell-containing solution (1mL each) are dialysed overnight against several changes of deionised water. The retentate is removed from dialysis and hydrolysed in 6N hydrochloric acid at 120°C overnight. The assay is performed using an oxidation C07320 process with 2M chloramine-T. Samples are analysed for radioactive counts, which represent the amount of newly synthesised 3 H]-hydroxyproline an index for new collagen synthesis.

It was discovered that 3'-(L-ascorbyl-2-o-phosphoryl)-cholesterol increased production of new collagen by human skin fibroblasts in a dose dependent manner as illustrated in Figure 1.

Various modifications and alterations to the present invention may be appreciated based on a review of this disclosure. These changes and additions are intended to be within the scope and spirit of this invention as defined by the following claims.

e a.

C07320

Claims (41)

1. A topical formulation comprising a suitable topical vehicle and a compound selected from the group consisting of 3'-(L-ascorbyl-2-o-phosphoryl)-cholesterol, 3'-(L-ascorbyl-3-o-phosphoryl)- cholesterol, an isomer thereof, or salt thereof and a mixture thereof.

2. The topical formulation of claim 1, wherein said topical vehicle is a cream, a lotion or a gel.

3. The topical formulation of claim 1 or claim 2, wherein said vehicle further comprises at least one component selected from a fragrance, an emulsifier, a thickening agent or an emollient.

4. The topical formulation of any one of claims 1 to 3, wherein said salt is ammonium, o1 calcium, lithium, potassium, sodium or an organic amine. The topical formulation of any one of claims 1 to 4, wherein said compound is ascorbyl-2-o-phosphoryl)-cholesterol or 3'-(L-ascorbyl-3-o-phosphoryl)-cholesterol.

6. The topical formulation of any one of claims 1 to 5, wherein said compound is about 0.0001 to about 100wt%.

7. The topical formulation of any one of claims 1 to 5, wherein said compound is about 0.05 to about St 8. The topical formulation of any one of claims 1 to 5, wherein said compound is about 0.10 to about

9. The topical formulation of any one of claims 1 to 5, wherein said compound is about to about

10. The topical formulation of any one of claims 1 to 9, wherein the pH of the topical formula is adjusted to physiologically acceptable levels with sufficient amounts of ammonium hydroxide, calcium hydroxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, ethanolamine, diethanolamine or urea. 25 11. The topical formulation of any one of claims 1 to 10, further comprising an a-hydroxy acid, wherein the topical formula comprises about 0.001wt% to about 10wt% of said compound.

12. The topical formulation of any one of claims 1 to 11, further comprising a retinoid, is wherein the topical formula comprises from about 0.001wt% to about 10wt% of said compound.

13. The topical formulation of claim 12, wherein said retinoid is retinol.

14. The topical formula of any one of claims 1 to 13, further comprising a sunscreen, wherein the topical formula comprises from about 0.001wt% to about 25wt% of said compound. The topical formulation of claim 14, wherein said sunscreen is cinnamic acid derivatives (menthyl, octyl, 2-ethylhexyl, benzyl, a-phenyl cinnamonitrile, and butyl cinnamoyl pyruvate), titanium dioxide, zinc oxide, benzylidene camphor, anthranilates, or naphtholsulphonates.

16. The topical formulation of any one of claims 1 to 15, further comprising an antioxidant.

17. The topical formulation of claim 16, wherein said antioxidant is sulfites, bisulfites, metabisulfites or other inorganic salts and acids containing sulfur in oxidation state +4.

18. The topical formulation of claim 17, wherein said antioxidant is a is sulfite. C07320

19. The topical formulation of any one of claims 1 to 18, further comprising a hormonal compound, wherein the topical formula comprises from about 0.001wt% to about 10wt% of said compound. The topical formulation of claim 19, wherein said hormonal compound is estriol, estradiol, estrone or a conjugated estrogen.

21. The topical formulation of any one of claims 1 to 20, further comprising a self-tanning agent, wherein the topical formula comprises from about 0.001wt% to about 20wt% of said compound.

22. The topical formulation of claim 21, wherein said self-tanning agent is dihydroxyacetone or lawsone.

23. The topical formulation of any one of claims 1 to 22, wherein said vehicle further comprises at least one component selected from a fragrance, an emulsifier, a thickening agent or an emollient.

24. A topical formulation, substantially as hereinbefore described with reference to any one of the examples. 1i 25. A topical formulation being a lotion comprising about 0.10 to about 20.0wt% of ascorbyl-2-o-phosphoryl)-cholesterol, an isomer or salt thereof, and water.

26. The topical formulation of claim 25, further comprising from about 0.001 to about thickening agent.

27. The topical formulation of claim 26, wherein said thickening agent is xanthan gum, hydroxyethylcellulose or a combination thereof.

28. A topical formulation comprising: about 0.10 to about 20.0wt% of 3-(L-ascorbyl-2-o- phosphoryl)-cholesterol, an isomer or salt thereof; about 0.5 to about 6.0wt% emollient; about 4.8 to about 14.5wt% emulsifier; and, about 0.5 to about 1.4wt% thickening agent.

29. The topical formulation of claim 28, further comprising about 0.35 to about 0.45wt% 25 preservative.

30. The topical formulation of claim 29, wherein the emollient is glycerin; and wherein the emulsifier is a combination of about 2.0 to about 8.0wt% of propylene glycol decapitate, about 1.8 to about 4.0wt% of Peg 40 Stearate, and about 1.0 to about 2.5wt% of Steareth-2.

31. The topical formulation of claim 30, wherein the thickening agent is about 0.25 to about 30 0.70wt% of xanthan gum and about 0.25 to about 0.70wt% of hydroxyethylcellulose.

32. The topical formulation of claim 33, wherein the preservative is about 0.15 to about 0.20wt% of an EDTA salt, and about 0.20 to about 0.25wt% of methylparaben.

33. A topical formulation being a cream comprising: about 0.10 to about 20.0wt% of Sascorbyl-2-o-phosphoryl)-cholesterol, an isomer or salt thereof; about 0.1 to about 1.2wt% of a 35 thickening agent; about 0.1 to about 15wt% of an emulsifier; and water.

34. The topical formulation of claim 33, further comprising up to about 1wt% of fragrance. A topical formulation being a cream comprising: about 0.1 to about 20.0wt% of ascorbyl-2.-o-phosphoryl)-cholesterol, an isomer, homologue or salt thereof; about 0.5 to about f 4.0wt% emollient; about 2.0 to about 6.0wt% emollient/humectant; about 4.5 to about 11.Owt% C07320 emulsifier; about 0.5 to about 1.2wt% thickening agent; and, about 0.15 to about 0.2wt% preservative.

36. The topical formulation of claim 35, wherein the emollient is glycerin; and wherein the emollient/humectant is propylene glycol decapitate; and wherein the emulsifier is about 1.8 to about 3.0wt% Steareth-20, about 0.8 to about 2.0wt% Steareth-2, about 1.0 to about 2.5wt% cetyl alcohol, and about 0.9 to about 3.5wt% glycerol mono-stearate; and wherein the thickening agent is about 0.25 to about 0.6wt% xanthan gum and about 0.25 to about 0.6wt% hydroxyethylcellulose.

37. The topical formulation of claim 36, wherein the preservative is an EDTA salt.

38. A topical formulation being a gel comprising: about 0.10 to about 20wt% of ascorbyl-2-o-phosphoryl)-cholesterol, an isomer or salt thereof; about 0.30 to about 2.0wt% thickening agent; and water.

39. A topical formulation being a gel comprising: about 0.10 to about 20.0wt% of ascorbyl-2-o-phosphoryl)-cholesterol, an isomer or salt thereof; about 2.0 to about emollient/humectant; about 0.4 to about 1.5wt% thickening agent; and, about 0.35 to about 0.45wt% preservative. The topical formulation of claim 39, wherein the emollient/humectant is propylene glycol; wherein the preservative is about 0.20 to about 0.25wt% methylparaben, and about 0.15 to about 0.20wt% an EDTA salt; and wherein the thickening agent is xanthene gum, xanthan gum, hydroxypropylcellulose, hydroxyethylcellulose, carbopol or gum acacia, Sepigel 305, vee-gum or magnesium aluminium silicate.

41. The topical formulation of claim 40, wherein said thickening agent is hydroxyethylcellulose.

42. A method for stimulating production of collagen in human skin fibroblasts, which method S. includes or consists of administering to said skin an effective amount of a formulation according to any S 25 one of claims 1 to 41.

43. A formulation according to any one of claims 1 to 41, when used in stimulating production of collagen in human skin fibroblasts.

44. A formulation according to any one of claims 1 to 41, for use in stimulating production of collagen in human skin fibroblasts.

45. The use of a formulation according to any one of claims 1 to 41, for the manufacture of a medicament for stimulating production of collagen in human skin fibroblasts.

46. A method of softening hair, promoting the elimination of hair ingrowths, or treating folliculitis and/or pseudofolliculitis barbae, the method comprising the step of applying a formulation according to any one of claims 1 to 41 to said hair or to the base of said hair. 35 47. A formulation according to any one of claims 1 to 41, when used in softening hair, promoting the elimination of hair ingrowths, or treating folliculitis and/or pseudofolliculitis barbae.

48. A formulation according to any one of claims 1 to 41, for use in softening hair, promoting /I.ji imination of hair ingrowths, or treating folliculitis and/or pseudofolliculitis barbae. C07320

49. The use of a formulation according to any one of claims 1 to 41,for the manufacture of a medicament for softening hair, promoting the elimination of hair ingrowths, or treating folliculitis and/or pseudofolliculitis barbae. Dated 3 September 2001 AVON PRODUCTS, INC. Patent Attorneys for the ApplicantlNominated Person SPRUSON FERGUSON 6*e*OS 0 S 0* S S000 0 SeE,. S *.SS S. 55 S S S S S. S S S 0 *055*5 "S. S S. S C ~~0

55.5 S 0505 a 0 'St. S S 0 *55V C07320