NO970152L - Askorbylfosforylkolesterol - Google Patents

Description

The present invention relates to the synthesis and use of a new derivative of L-ascorbic acid which is stable, easy to incorporate into cosmetically acceptable carriers and enzymatically bioreversible to its constituent components. Examples of derivatives include 3'-(L-ascorbyl-2-phosphoryl)-cholesterol and 3'-(L-ascorbyl-3-phosphoryl)-cholesterol and salts thereof.

The use of L-ascorbic acid as an antioxidant in food products is known. For example, Steinhart describes the Pro- and Antioxidative Effect of Ascorbic Acid on L-Tryptophan in the System Fe3+/ Ascorbic Acid/ Cb. J. Agric. Food Chem., vol. 41, pages 2275-2277 (1993), the use of L-ascorbic acid as an antioxidant which exerts its function in food by scavenging free radicals and itself undergoes rapid oxidation.

Likewise, free L-ascorbic acid in topical preparations exhibits poor stability and is prone to degradation due to partial oxidative and non-oxidative degradation. The degraded ascorbic acid loses activity and the host product loses its aesthetic appeal by exhibiting a brown color that is unacceptable for commercial cosmetics.

Although cholesterol, especially in its ingested form, is considered unhealthy, the beneficial effects of cholesterol unconjugated with L-ascorbic acid for skin barrier healing are known. For example, Menon describes Structural Basis for the Barrier Abnormality Following Inhibition of HMG CoA Reductase in Murine Epidermis. J. Invest. Dermatol., vol. 98, pages 209-219

(1992), defects detected in the skin barrier repair mechanism when cholesterol synthesis is inhibited by downregulation of HMG CoA reductase.

Mechanical mixing of L-ascorbic acid and cholesterol according to available existing methods results in a product that is also unstable due to the overriding problem of L-ascorbic acid instability. For example, US Patent 4,939,128, in column 3, lines 21-22, describes ascorbic acid in conjunction with a cholestanyl group. The apparent absence of cholesterol and the specific mention of a cholestanyl group reflect a recognition, prior to the present disclosure, that conjugates of L-ascorbic acid and cholesterol were not practical or desired.

Attempts have also been made to conjugate ascorbic acid with a glycyrrhetic group as described in EP patent application 92104149.7; and with a tocopheryl group as disclosed in US Patent 3,151,127. US patents 4,564,686 and 5,306,713 also describe tocopheryl ascorbyl phosphate as an antioxidant having the following structure: Sakamoto, Measurement Method of Efficacv of Antidandruff Cosmetics and Development of the New Active Commercial Product. IFSCC, Yokohama, vol. B206. pages 823-864 (1993), describes the use of tocopheryl linked to L-ascorbic acid. The linked tocopheryl is an antioxidant preservative for the ascorbyl group, but the use of ascorbyl tocopheryl as a skin therapeutic is questionable because tocopheryl, unlike cholesterol, is not a natural substrate for the skin.

The technique needs a method for covalently and bioreversibly linking cholesterol to L-ascorbic acid. The coupled molecule should be stable so that full functional activity is maintained even after uncoupling using naturally occurring acid phosphatases in the skin. The beneficial properties of L-ascorbic acid would be achieved, including increased collagen production and skin lightening, combined with the beneficial effects of freed cholesterol for improved skin elasticity, resistance, color and moisture retention.

An object of the present invention is to provide a method for covalently and bioreversibly linking cholesterol to L-ascorbic acid for stabilization of the resulting molecule.

Another purpose of the present invention is to provide a stable preparation with a number of skin care benefits.

A further object of the present invention is to provide a derivative of L-ascorbic acid which is stable, which can be easily introduced into cosmetic carriers and which is enzymatically bioreversible to free ascorbic acid and a safe cholesterol component.

Yet another object of the present invention is to provide stable cosmetic formulations which exhibit long durability.

These and other purposes will appear from what follows.

The present invention includes a method for linking a molecule of L-ascorbic acid to a molecule of cholesterol through a bioreversible phosphate bond at position 2 or 3 on the ascorbyl group and position 3' on the cholesteryl part. The resulting preparations are also applicable to the present invention. Exemplary compounds include functional or structural homologues of 3'-(L-ascorbyl-2-phosphoryl)-cholesterol (Formula I) such as 3'-(L-ascorbyl-3-phosphoryl)-cholesterol (Formula II). Both formulas are illustrated below.

The conjugated 3'-(L-ascorbyl-2-phosphoryl)-cholesterol (Formula I) was prepared by dissolving cholesterol at -10°C in dry diethyl ether (dried over 4A molecular sieves) containing 1.0 equivalent of triethylamine as a base. Phosphorus oxychloride (1.0 equivalent) was added to provide cholesteryl phosphorodichloridate. The melting point of cholesteryl phosphorodichloridate was measured as 121-122°C and infrared (KBr pellet) analysis showed P=0 absorption at 1298 wavelengths and P-O-C absorption at 1019 wavelengths, without any hydroxyl absorption. Cholesteryl phosphorodichloridate was then reacted for 3 hours at room temperature with 5,6-isopropylidene-L-ascorbic acid in tetrahydrofuran containing 1.0 equivalent of triethylamine. This reaction gave a mixture of cholesteryl 5,6-isopropylidene-2-phosphorochloridate L-ascorbic acid and its isomer cholesteryl 5,6-isopropylidene-3-phosphorochloridate L-ascorbic acid.

The isomeric mixture was hydrolyzed 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 and the final product, 3'-(L-ascorbyl-2-phosphoryl)-cholesterol, was extracted with ethyl acetate and neutralized with one equivalent of KOH. The resulting solution was lyophilized to give the monopotassium salt form.

The new method allows for covalent and bioreversible coupling of cholesterol with L-ascorbic acid, which results in the stabilization of ascorbic acid and increased bioavailability for ascorbic acid and cholesterol. In the ascorbyl-phosphoryl-cholesterol compounds according to the invention, the conjugated ascorbic acid becomes resistant to degradation. The cholesteryl group serves as a carrier moiety and facilitates the delivery of polar ascorbic acid through the non-polar outermost skin protective layer (ie stratum corneum) and increases the bioavailability of the ascorbic acid in the topical application.

Natural enzymes, such as phosphatases found in the skin, cause gradual cleavage of the phosphate bond between cholesterol and ascorbic acid, resulting in the sustained release of free L-ascorbic acid and cholesterol into the stratum corneum. The released cholesterol is a natural substrate for the skin and supplements what is otherwise produced in the body. Topically applied cholesterol improves elasticity, color tone and resistance to drying. A topical formulation according to the present invention can comprise either 3'-(L-ascorbyl-2-phosphoryl)-cholesterol or 3'-(L-ascorbyl-3-phosphoryl)-cholesterol. In addition, ammonium, calcium, lithium, potassium or sodium salts of these compounds are readily incorporated into cosmetically acceptable carriers. A salt with an organic amine such as ethanolamine will also provide the beneficial effects intended by the present invention.

Suitable carriers include conventional lotions, creams or gels. A lotion embodiment may comprise from approx. 0.1 to approx. 20.0% 3'-(L-ascorbyl-2-phosphoryl)-cholesterol or 3'-(L-ascorbyl-3-phosphoryl)-cholesterol, from approx. 0.5 to approx. 6.0% glycerin, from approx. 2.0 to approx. 8.0% propylene glycol dicaprylate/dicaprate, from approx. 1.8 to approx. 4.0% Peg 40 Stearate, from approx. 1.0 to approx. 2.5% Steareth-2, from approx. 0.25 to approx. 0.7% xanthan gum, from approx. 0.25 to approx. 0.7% hydroxyethyl cellulose, from approx. 0.15 to approx. 0.2% disodium EDTA and from approx. 0.20 to approx. 0.25% methylparaben, all areas being expressed as % by weight.

A cream version can include from approx. 0.1 to approx. 20.0% 3'-(L-ascorbyl-2-phosphoryl)-cholesterol or 3'-(L-ascorbyl-3-phosphoryl)-cholesterol, from approx. 0.5 to approx. 4.0% glycerin, from approx. 2.0 to approx. 6.0% propylene glycol dicaprylate/dicaprate, from approx. 1.8 to approx. 3.0% Steareth-20, from approx. 0.8 to approx. 2.0% Steareth-2, from approx. 0.25 to approx. 0.6% xanthan gum, from approx. 0.25 to approx. 0.6% hydroxyethyl cellulose, from approx. 1.0 to approx. 2.5% cetyl alcohol, from approx. 0.9 to approx. 3.5% glycerol monostearate and from approx. 0.15 to approx. 0.2% disodium EDTA.

A gel design can include from approx. 0.1 to approx. 20.0% 3'-(L-ascorbyl-2-phosphoryl)-cholesterol or 3'-(L-ascorbyl-3-phosphoryl)-cholesterol, from approx. 0.15 to approx. 0.2% disodium EDTA, from approx. 2.0 to approx. 6.0% propylene glycol, from approx. 0.4 to approx. 1.5% hydroxyethyl cellulose and from approx. 0.20 to approx. 0.25% methylparaben.

The pH of these formulations can be adjusted to physiologically acceptable levels with sufficient amounts of ammonium hydroxide, calcium hydroxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, ethanolamine, diethanolamine or urea.

The present compounds are generally synthesized by (i) reacting cholesterol with a halogenophosphorylating agent, (ii) coupling the resulting product with 5,6-hydroxyl-protected L-ascorbic acid, (iii) hydrolyzing the product with water, (iv) removing the protecting group with an acidic resin and (v) purifying the product by lyophilization and recrystallization. The derivative is stable in solution, exhibits antioxidant activity and stimulates the production of collagen in fibroblasts.

EXAMPLE 1

Preparation of phosphodiesteric acid and its monopotassium salt

Cholesteryl phosphodichloridate was synthesized using the following procedure. A 250 ml two neck 19/22 ST round bottom flask was selected for the reaction. It included a serum cap (with nitrogen entry needle), a stir bar, and a 19/22 to 24/40 ST expansion adapter containing a 24/40 ST 125 mL dropper funnel equipped with a side arm. This apparatus was flame dried and cooled under a nitrogen purge. The dropping funnel was charged with 4.64 g (12 mmol) Sigma 99+% cholesterol, 75 mL ether (dried over activated 4A molecular sieves) and 1.214 g (12 mmol, 1.672 mL) dry (over KOH) triethylamine.

The flask was charged with 28 mL of dry ether and 1.84 g (12 mmol, 1.118 mL) of phosphorus oxychloride and cooled in an ice/methanol (-10°C) bath. Ether containing the cholesterol-triethylamine was added dropwise rapidly over a period of 20-30 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 the ether in the filtrate had evaporated. Solid precipitate was then removed by filtration through another Buchner funnel and cholesteryl phosphodichloridate was dried in a vacuum desiccator over phosphorus pentoxide. This experiment yielded 3.90 g (65%) of a first solid portion, m.p. 121-122°C and 1.74 g (29%) of another portion of material, m.p. 117-118°C. IR analysis (KBr pellet) showed

(C-H) absorption at 2947 wavelengths, (=C-H) absorption at 2878 wavelengths, (C=C) absorption at 1466 wavelengths, (P=0) absorption at 1298 wavelengths and (P-O-C) absorption at 1019 wavelengths.

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Ascorbic cholesteryl phosphodiester chloride was synthesized by following the procedure outlined below.

A 50 mL three-necked 19/22 ST round bottom flask equipped with a stir bar, serum cap, nitrogen inlet needle, and 50 mL dropping funnel was selected for this experiment. This apparatus was flame dried and cooled under a nitrogen purge. The dropping funnel was filled with 503 mg (1 mmol) of cholesteryl phosphorodichloridate (m.p. 122°C) and 15 ml of dry THF; and the mixture was cooled in an ice/methanol bath (-10°C). To the cooled mixture was added 216 mL (1 mmol) Sigma 5,6-isopropylidene-L-ascorbic acid, 15 mL dry THF and 0.14 mL (101 mg, 1 mmol) dry (KOH) triethylamine. After addition, the mixture was warmed to room temperature and stirred for 3 hours.

A TLC (25% methanol/toluene) analysis showed that the reaction was complete. It also suggested that the product was a mixture of 2-O and 3-O regioisomers. The precipitated triethylamine hydrochloride was removed by filtration through a folded paper. THF was removed by rotary evaporation to give 0.66 g (97%) of impure crystalline ascorbic cholesteryl phosphodiester chloride.

Ascorbic cholesteryl phosphodiesteric acid was prepared using the following procedure. Crude ascorbic cholesteryl phosphodiester chloride (6.76 g, 9.9 mmol) in 60 mL of THF was combined with 30 mL of water and 20 g of wet Amberlyst-15 that had been rinsed in water three times. The resulting mixture was vigorously stirred at room temperature for 55 hours. Amberlyst-15 was removed by filtration through folded paper and was rinsed once with 20 mL of 1:1 THF/water. Most of said THF was removed in a stream of nitrogen to give 53 mL of a thick, cloudy aqueous suspension. 53 ml of THF was added to the suspension giving 106 ml of a 1:1 THF/water solution of crude phosphodiesteric acid which was almost clear. Phosphodiester acid was purified by adding the 1:1 THF/water solution to a column of C-18 reverse phase silica gel (472 g) and eluting with 1:1 THF/water. The THF was removed in a stream of nitrogen and this gave 215 ml of purified phosphodiesteric acid in aqueous suspension. The projected total yield was 1.74 g (28%); and the actual isolated yield was 1.84 g (30%). Reverse phase HPLC analysis indicated 90% purity.

Ascorbic cholesteryl phosphodiester diacid monopotassium salt was prepared by first treating a 1% aqueous solution of the diacid with one equivalent of a standardized potassium hydroxide solution and subsequent lyophilization. The phosphodiester diacid (579 mg, 0.927 mmol) was dissolved in 57.9 mL of water and treated with 9.44 mL of 0.0986 N potassium hydroxide solution (0.931 mmol). The neutralized solution was then lyophilized to remove water to yield 603 mg (98%) of the monopotassium salt as a fluffy white solid.

EXAMPLE 2

Purification by C-18 reverse phase chromatography

C-18 reversed phase silica gel was prepared on a 1 kg scale according to Evans,

Chromatographia, vol. 13, pp. 5-10 (1980). Purification of the phosphodiester acid to a level of 90% was achieved at a 90:1 charge ratio using 1:1 THF/water, followed by THF removal in a stream of nitrogen and water removal by lyophilization. Investigation of other solvent systems by reverse-phase thin-layer chromatography had good potential in terms of (i) improving the purity level, (ii) identifying an effective separation medium that could be removed by rotary evaporation, and (iii) allowing the use of a lower charge ratio. Since C-18 reverse phase silica gel can be reused, the method has good potential for purification of up to 1000 g.

Suitable solvent systems 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 into a non-polar, lipophilic ascorbyl group that is readily absorbed through the stratum corneum. Once past said stratum corneum, the absorbed compound is able to affect underlying fibroblasts. The beneficial effects of bioreversed ascorbic acid and cholesterol have previously been explained. However, the conjugated compound itself surprisingly stimulates collagen synthesis, which promotes the skin's integrity, elasticity and resilience. Further details are given in Example 3.

EXAMPLE 3

Fibroblast studies

This example summarizes a study in which the ability of 3'-(L-ascorbyl-2-phosphoryl)-cholesterol to stimulate collagen production in cultured human skin fibroblasts was demonstrated. A [^Hj-proline incorporation assay recognized in the art was performed with various doses of 3'-(L-ascorbyl-2-phosphoryl)-cholesterol. Juva, Anal. Biochem., vol. 15, pp. 77-83 (1966); Booth, Biochim. Biophys. Acta, vol. 675, pages 117-122 (1981).

Fibroblasts were incubated with 0 µg/ml, 11.3 µg/ml, 22.5 µg/ml and 45 µg/ml of 3'-(L-ascorbyl-2-phosphoryl)-cholesterol for a total of 48 hours. After the first 24 hours, [^H]-labeled proline was added to the culture. After the second 24 hour period, the cells were harvested and prepared for the collagen biosynthesis assay.

Protease inhibitors were added to prevent the breakdown of collagen and other proteins. The cell layer was scraped into a solution containing 0.4 M NaCl and 0.01 M Tris (pH 7.5). Extracts were sonicated to disrupt cell membranes. Separate volumes of the cell-containing solution (1 ml each) were dialyzed overnight against several changes of deionized water. The retentate was removed from dialysis and the hydrolysis in 6 N hydrochloric acid at 120°C overnight. The analysis was carried out using an oxidation process with 2 M chloramine-T. Samples were analyzed for radioactive counts, which represent the amount of newly synthesized [^Hj-hydroxyproline -- an index of new collagen synthesis.

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

Claims (9)

1. Topical formulation, characterized in that it includes a suitable topical carrier and a compound selected from the group consisting of 3'-(L-ascorbyl-2-phosphoryl)-cholesterol, 3'-(L-ascorbyl-3-phosphoryl)-cholesterol and salts thereof. 2. Topical formulation according to claim 1, characterized in that said salt is selected from the group consisting of salts of ammonium, calcium, lithium, potassium, sodium and an organic amine. 3. Topical formulation according to claim 1, characterized in that said carrier is selected from the group consisting of a lotion, cream and gel. 4. 4. Topical formulation, characterized in that it includes: (a) from approx. 0.1 to approx. 20.0% of a compound selected from the group consisting of 3'-(L-ascorbyl-2-phosphoryl)-cholesterol and 3'-(L-ascorbyl-3-phosphoryl)-cholesterol; (b) from approx. 0.5 to approx. 6.0% glycerin; (c) from approx. 2.0 to approx. 8.0% propylene glycol dicaprylate/dicaprate; (d) from approx. 1.8 to approx. 4.0% Peg 40 Stearate; (e) from approx. 1.0 to approx. 2.5% Steareth-2; (f) from approx. 0.25 to approx. 0.7% xanthan gum; (g) from approx. 0.25 to approx. 0.7% hydroxyethyl cellulose; (h) from approx. 0.15 to approx. 0.2% disodium EDTA; and (i) from approx. 0.20 to approx. 0.25% methylparaben. 5. Topical formulation according to claim 4, characterized in that the pH value of the formulation is adjusted to physiologically acceptable levels with sufficient amounts of a compound selected from the group consisting of ammonium hydroxide, calcium hydroxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, ethanolamine, diethanolamine and urea. 6. Topical formulation, characterized in that it includes: (a) from approx. 0.1 to approx. 20.0% of a compound selected from the group consisting of 3'-(L-ascorbyl-2-phosphoryl)-cholesterol and 3'-(L-ascorbyl-3-phosphoryl)-cholesterol; (b) from approx. 0.5 to approx. 4.0% glycerin; (c) from approx. 2.0 to approx. 6.0% propylene glycol dicaprylate/dicaprate; (d) from approx. 1.8 to approx. 3.0% Steareth-20; (e) from approx. 0.8 to approx. 2.0% Steareth-2; (f) from approx. 0.25 to approx. 0.6% xanthan gum; (g) from approx. 0.25 to approx. 0.6% hydroxyethyl cellulose; (h) from approx. 1.0 to approx. 2.5% cetyl alcohol; (i) from approx. 0.9 to approx. 3.5% glycerol monostearate; and 0) from approx. 0.15 to approx. 0.2% disodium EDTA. 7. Topical formulation according to claim 6, characterized in that the formulation's pH value is adjusted to physiologically acceptable levels with sufficient amounts of a compound selected from the group consisting of ammonium hydroxide, calcium hydroxide, lithium hydroxide, potassium hydroxide, sodium hydroxide, ethanolamine, diethanolamine and urea. 8. Topical formulation, characterized in that it includes: (a) from approx. 0.1 to approx. 20.0% of a compound selected from the group consisting of 3'-(L-ascorbyl-2-phosphoryl)-cholesterol and 3'-(L-ascorbyl-3-phosphoryl)-cholesterol; (b) from approx. 0.15 to approx. 0.2% disodium EDTA; (c) from approx. 2.0 to approx. 6.0% propylene glycol; (d) from approx. 0.4 to approx. 1.5% hydroxyethyl cellulose; and (e) from approx. 0.20 to approx. 0.25% methylparaben. 9. Topical formulation according to claim 8, characterized in that the formulation's pH value is adjusted to physiologically acceptable levels with sufficient amounts of a compound selected from the group consisting of ammonium hydroxide, calcium hydroxide, lithium hydroxide, potassium hydroxide, sodium hydroxide and ethanolamine.