KR20010108331A - Spingoid Base Derivatives and Uses Thereof - Google Patents

Abstract

The present invention discloses sphingoid base derivatives which are salts of sphingoid bases. The salts sufficiently enhance solubility in an aqueous environment and exhibit improved effects in the composition for topical application.

Description

Spingoid Base Derivatives and Uses Thereof

Sphingoid bases, such as sphingosine, are known as effective regulators of skin cell differentiation and proliferation by interfering with basic biochemical cellular processes (Hannun, Y.A. and Bell, R.N. (1989), Science 243, 500-507). For example, free sphingosine inhibits the activity of protein kinase C, which plays a central role in the regulation of signaling pathways and cell division (Hannun, YA et al. (1986), J. Biol. Chem. 261). , 12604-12609). The action of free sphingosine may be an important factor in regulating epithelial cell proliferation to balance the rate at which cells are lost in the epidermis (Downing, DT (1992), J. Invest. Dermatol. 98, 269-). 273). In addition, other biological activities are known for sphingoid bases, such as antimicrobial whistle (Bibel, D.E. et al., (1992), J. Invest. Dermatol. 98,269-273).

Because of their effects on skin cell differentiation and proliferation and antimicrobial activity, sphingoid bases can be included as active ingredients in various cosmetic compositions. For example, sphingosine is known to treat various skin related disorders and disorders, such as dry skin, dry skin and psoriasis. In addition, sphingosine can protect the skin from various harmful and undesirable effects such as the effects of ultraviolet rays and skin aging. In particular, sphingoid bases have been included in topical compositions such as anti-inflammatory or antimicrobial agents (WO 98/49999).

A disadvantage of sphingoid bases is their very low solubility in aqueous environments. This phenomenon limits the use of these compounds in aqueous formulations. For example, in order to exhibit effective antimicrobial activity, it is important to dissolve the sphingoid base in an aqueous formulation.

The present invention relates to the field of topical application, and more particularly to the topical application of sphingoid base derivatives.

1A is a graph showing the antifungal activity of PS against S. cerevisiae;

1B is a graph showing the antifungal activity of PS against C. albicans;

FIG. 2 is a graph showing the antifungal effect of PS on unnourished and nourished C. albicans;

3A is a graph comparing the antifungal activity of PS and salts thereof against C. albicans;

3B is a graph comparing the effects of hydrochloride and free base on C. albicans;

4A is a graph showing antifungal activity against C. albicans in water;

4B is a graph showing the high effect of PS salt on C. albicans in water;

4C is a graph showing the high effect of PS hydrochloride on C. albicans in water;

5 is a graph showing the antimicrobial effect of phytosphingosine against E. coli; And

6 is a flow chart showing a method of preparing a phytosphingosine containing formulation.

The present invention discloses sphingoid base derivatives having substantially improved solubility in water than their free base controls. As a result, these sphingoid base derivatives show surprisingly improved efficacy when formulated in an aqueous environment.

The sphingoid base derivatives of the present invention are salts of sphingoid bases.

According to the invention, the anion of the sphingoid base salt is derived from a suitable acid. In this respect, a suitable acid is defined as one acid which, when mixed with a sphingoid base in a suitable solvent, itself produces a salt with improved solubility in an aqueous medium relative to the solubility of the sphingoid base.

According to one embodiment of the invention, the acid is itself an acid which may have an effect on topical applications.

According to one embodiment of the invention, the acid is a hydrophilic acid capable of delivering sphingoid base to the aqueous phase of a cosmetic or pharmaceutical composition.

Preferably, the acid is a hydrophilic organic acid or an inorganic acid such as α-hydroxy alkanoic acid, β-hydroxy alkanoic acid, α, β-dihydroxy alkanoic acid, alkanedic acid or the like. More preferred examples of hydrophilic organic acids are lactic acid, glycolic acid, malic acid, pyruvic acid, succinic acid, fumaric acid, citric acid, ascorbic acid, gluconic acid and / or pyroglutamic acid (pyrrolidone carboxylic acid). More preferred examples of inorganic acids are hydrochloric acid, nitric acid and / or phosphoric acid.

According to another embodiment of the invention, the acid is a lipophilic organic acid, the combination with a sphingoid base enhances the efficacy of the sphingoid base as well as the lipophilic acid.

The sphingoid base salt of the present invention is prepared as follows. The sphingoid base is dissolved in a suitable organic solvent and 1 equivalent of at least one suitable acid is added. Typically, the addition of acid will reduce the pH of at least about 3 units. It is recognized that the final pH value will depend on the acid applied. Dissolution of sphingoid bases in organic solvents preferably occurs at elevated temperatures, for example at 50 to 70 ° C. After cooling the mixture, sphingoid base salts will precipitate. The crystallized precipitate can be recovered from the reaction solution by filtration and optionally washed with the same solvent used for the preparation of the solvent, preferably the salt.

Suitable organic solvents are preferably solvents in which the final product, ie the sphingoid base salt, is insoluble. For example, suitable organic solvents are ethanol or methyl isobutyl ketone.

According to one embodiment of the invention, the sphingoid base salt is used as starting compound for the preparation of another sphingoid base salt.

It is essential that the sphingoid base salts of the invention are prepared before they are prepared for their intended use, for example, before they are contained in a topical composition. The inclusion of free sphingoid bases in topical compositions further comprising an anion derived from one or more acids as defined above will not result in improved solubility and / or improved efficacy.

The sphingoid base salt of the present invention is preferably a salt of the sphingoid base, sphingosine, sphinginine or phytosphingosine. More preferably, the sphingoid base salt is a salt of phytosphingosine.

According to one embodiment of the present invention, phytosphingosine is obtained through microbial fermentation. For example, phytosphingosine is obtained from pichia ciferi-derived tetraacetyl-phytosphingosine (TAPS) by appropriate deacetylation reaction. Deacetylation is carried out by chemical methods such as base catalytic hydrolysis or enzymatic methods with potassium hydroxide. After base hydrolysis of TAPS, the resulting phytosphingosine can be isolated pure. The pure separation can be carried out by methods known to those skilled in the art. Enzyme-induced phytosphingosine has the same stereochemical configuration as that of mammalian phytosphingosine, ie, DD-erythro, so that it can be applied to human skin. Same as phytosphingosine present.

The sphingoid base salts of the present invention have solubility in a significantly higher aqueous environment compared to the solubility of the free sphingoid base. In accordance with the present invention, it is further shown that the sphingoid base salts exhibit higher efficacy than the free sphingoid base even in environments where the free sphingoid base is present in dissolved form. The free sphingoid base may be in dissolved form if additional organic solvents and surface active compounds are present in the aqueous medium.

Compositions comprising sphingoid base derivatives of the present invention are suitable for topical application, which includes cosmetic and / or dermatological application to epithelial supports such as skin, hair and mouth, nose, eyes, genitourinary system, and the like. It is understood that

The sphingoid base derivative of the present invention is preferably included in the topical application composition at a predetermined concentration, the concentration being 0.001 to 5% by weight, preferably 0.005 to 5% by weight, more preferably 0.01 to 2.5% by weight. Most preferably from 0.02 to 1% by weight, particularly preferably from 0.02 to 0.5% by weight.

Topical compositions comprising sphingoid base derivatives of the present invention are particularly suitable for the application of various topical sites in which undesirable and / or abnormal conditions associated with inflammation and / or microbial activity occur locally.

Examples of local diseases that cause undesirable and / or abnormal conditions to which topical compositions comprising sphingoid base derivatives of the present invention can be effectively applied include eczema, psoriasis, atopic dermatitis, acne, dandruff, oral cavity and / Or cleft lip infection, mycos, various skin infection diseases or texture. In addition, topical compositions comprising such sphingoid base derivatives are effectively applied to wound healing, for example, normalization of burns and skin bacterial populations.

Because of their antimicrobial activity, the sphingoid base derivatives of the present invention may additionally act as preservatives in cosmetic and dermal compositions to reduce or replace the use of conventional chemical preservatives.

Example 1

PS, Lactate Preparation

A mixture of 50 g of phytosphingosine and 500 ml of absolute ethanol was stirred and heated to 65 ° C. The nearly clear solution was then filtered through paper filter paper in a heated state and placed in 1 l of three flasks.

While stirring, (L) -lactic acid (25.7 g) was added to the filtered solution to reduce the pH from 9.9 to 5.3, with the temperature rising from 66 ° C to 71 ° C. The mixture was stirred and cooled. Crystallization started at ca 45 ° C. and cooling was continued for 3/4 hours to reach 21 ° C.

The precipitate was filtered off and the cake was immersed in 150 ml of ethanol (quick filtration and replacement, total 2 minutes).

The wet cake (110.8 g) was dried under vacuum and 51.2 g of the final product was obtained. NMR analysis showed a purity of 99.3%.

Example 2

PS, Preparation of Glycolate

A mixture of 50 g of phytosphingosine and 500 ml of absolute ethanol was stirred and heated to 65 ° C. The nearly clear solution was then filtered through paper filter paper in a heated state and placed in 1 l of three flasks. Wash with 20 ml hot ethanol. The filtrate was reheated to 65 ° C.

Glycolic acid (13.4 g) was added to the filtered solution while stirring to reduce the pH from 9.9 to 5.6, with the temperature rising from 65 ° C to 68 ° C. The mixture was stirred and cooled. Crystallization started at ca 66 ° C. and cooling continued for 20 minutes to reach 25 ° C.

The precipitate was filtered off and the cake was immersed in 150 ml of ethanol (quick filtration and replacement, total 3 minutes).

The wet cake (87 g) was dried under vacuum overnight to yield 56.6 g of the final product. NMR analysis showed a purity of 98.6%.

Example 3

PS, Preparation of Hydrochloride

A mixture of 50 g of phytosphingosine and 500 ml of ethanol was stirred and heated to 65 ° C. The nearly clear solution was then filtered through paper filter paper in a heated state and placed in 1 L of three flasks. Wash with 20 ml hot ethanol. The filtrate was reheated to 65 ° C.

Hydrochloric acid (36%, ca 13 mL) was added to the filtered solution while stirring to reduce the pH from 10.3 to 0, with the temperature rising from 45 ° C to 50 ° C. The mixture was stirred and cooled. Crystallization started at ca 34 ° C. and cooling continued for 0.5 hours to reach 10 ° C.

The precipitate was filtered off and the cake was immersed in 100 ml of cold ethanol (slow filtration and replacement, 3/4 hours total).

The wet cake (272 g) was dried under vacuum and 48.0 g of final product was obtained. NMR analysis showed a purity of 96.7%.

Example 4

PS, Preparation of Pyroglutamate

A mixture of 25 g of phytosphingosine, 200 ml of methyl isobutyl ketone (MIK) and 2 ml of water was stirred and heated to 66 ° C.

Then 12 g of DL-pyroglutamic acid were added and the pH was changed from 9.4 to 5.8.

A glassy precipitate was obtained. 1 mL of sample was taken which started to crystallize by scratching at 45 ° C. The sample taken was used as flocculation nuclei of the mixture in further cooling.

The mixture was then cooled to 17 ° C., filtered through a glass G3 filter and washed / soaked with fresh 50 ml MIK (quick filtration). The wet cake (57 g) was dried under vacuum to give 34.3 g of the final product.

Example 5

PS, Preparation of Citrate

A mixture of 25 g of phytosphingosine, 200 ml of methyl isobutyl ketone (MIK) and 1 ml of water was stirred and heated to 72 ° C.

Then 18 g citric acid monohydrate were added and the pH was changed from 9.4 to 1.8.

A precipitate was obtained. The mixture was then cooled to 14 ° C., filtered through a glass G3 filter and washed / soaked with fresh 50 ml MIK (quick filtration). The wet cake (84 g) was dried under vacuum to give 39.7 g of final product. NMR analysis showed a purity of 96.4%.

Example 6

Antimicrobial Activity of PS on Yeast

Two different yeast strains were used: Saccharomyces cerevisiae ATCC 9763 and Candida albicans ATCC 10231 in Saccharomyces cerevisiae . All cultures were performed at 30 ° C. ( S. cerevisiae ) or 37 ° C. ( C. albicans ). Both strains were cultured in YEPD2% medium (20 g / l glucose, 10 g / l peptone, 20 g / l yeast extract, pH = 6.0). Incubate overnight, harvest cells by centrifugation in 50 μl of culture, wash with 1 ml sterile buffer (10 mM HEPES (pH = 7.2 with NaOH) +20 g / l glucose), After centrifugation, it was resuspended in 0.5 ml of sterile buffer.

Phytosphingosine (PS) 10 mg / ml stock solution was prepared in a solvent system consisting of 1 volume fraction of ethanol, 2 volume fractions of Tween 20 and 50 volume glycerol in an aqueous solution of 17 volume fractions in water. Each component of the solvent system was added to phytosphingosine in sequence and mixed vigorously after each component was added. After all solvent was added, the mixture was heated at 40 ° C. for 15-30 minutes. If necessary, a dilution of the stock solution was made with ethanol dissolved at 5% in water. All solutions were prepared 24 hours before use and stored at room temperature.

The antifungal effect of the blood toss ping high signal for both types of yeast were investigated by using the Live / Dead yeast viability kit ^{(LIVE / DEAD TM Yeast Viability Kit} L-7009 (Molecular Probes Inc., Oregon, USA)). The kit uses two types of fluorescent stains, FUN-1 ^™ and Calcofluor ^™ White M2R, to distinguish viable and dead cells, which can be observed using a fluorescence microscope with appropriate filters.

For this analysis, the following amounts of fluorescent dye were added to 0.5 ml of yeast suspended in sterile buffer prepared by the method described above: 1 μl FUN-1 ^™ and 2.5 μl Calcofluor ^™ White M2R. After mixing, the suspension was incubated for 30 minutes. Then 50 μl of appropriately diluted phytosphingosine stock solution was added to bring the final concentrations shown in FIGS. 1A and 1B. After mixing, the suspension was incubated and the viable cell fractions and dead cell fractions were counted over time.

Microscopic observations were carried out on an Olifs BHB fluorescence microscope with two different filter sets:

Dichroic mirror blue (B), excitation filter IF490, luminescence filter O530 (surviving cells appear orange, dead cells appear green / orange)

2. Dichroic mirror violet (V), excitation filter U95-B93, luminescence filter Y455 (surviving cells show blue cell wall, no dead cells)

The results are shown in FIGS. 1A and 1B. It is clear that phytosphingosine (PS) kills both yeast strains in a quantity dependent manner.

Example 7

Antifungal Activity of PS on Unnourished Cells

In their natural habitat, microorganisms remain nutrient-free for most of the time. Thus, it was confirmed whether the antimicrobial activity of PS can be applied to the cells in the nutrient supply is cut off.

For this purpose, the method described in Example 6 was slightly modified (unless otherwise noted, all methods and conditions are the same). Cells incubated overnight with Candida albicans ATCC 10231 were harvested by centrifugation. Two different buffers were used for washing and resuspending cells: 10 mM HEPES (pH = 7.2 with NaOH) + 20 g / l glucose and the same buffer removed glucose used in Example 6.

Two kinds of cell suspensions (2.5 mL for each condition) with or without glucose were incubated at 37 ° C. for 10 minutes. Then 125 μl of a properly diluted phytosphingosine stock solution was added to bring the final concentration as shown in FIG. 2. After mixing, the mixture was further incubated and 100 μl of the sample was collected at the designated time. The collected cells were obtained by centrifugation, and resuspended in 100 µl of a buffer solution containing glucose containing the same concentration of fluorescent dye. The assay mixture was further incubated for 10 minutes to infiltrate the dye into the cells, and the counts of viable and dead cells were carried out in the same manner as in Example 6.

As shown in Figure 2, the cells that were cut off nutrition were more sensitive to the antifungal activity of PS than the cells that were fed nutrition. In fact, after the initial slowing step, 250 mg / L of PS, as at 500 mg / L, proved effective in killing unnourished cells. The slowing step is presumed to be due to the presence of intrinsic energy stored in the cells, which shows once again that PS is particularly effective against nutrient cells.

Example 8

Antimicrobial Effects of PS in Cosmetic Products through Agar Diffusion Test

Overnight incubation of Staphylococcus aureus ATCC 14458 was performed in the same manner as in Example 6 (BHI medium (Difco), 37 ° C. incubation).

In order to prepare agar plates used for the diffusion test, 300 ml of BHI medium to which 1% agar and 15% glycerol were added were melted and cooled to 50 ° C. Then 6 ml of sterile glucose solution (50% w / v) and 6 ml of the microbial culture were added. Petri dishes were filled with 12.5 ml of agar medium and the medium solidified.

Test formulations were prepared as shown in FIG. 6 using the lipid phase of octyl dodecyl lactate. The formulations contained 1, 2, or 5 g / l PS.

In order to apply the test sample to the test plate, stainless steel rings (inner diameter 6 mm) were inserted into sterile petri dishes. Two paper discs (6 mm inner diameter) were placed on the bottom surface inside the ring and 50 μl of test formulation was applied to the filter discs. The ring was placed on the surface of the agar plate containing the microorganisms, and the agar plate was stored at 5 ° C. for the time indicated in the table so that the test solution diffused, and then the ring was removed. The agar plates were then incubated at the appropriate temperature (37 ° C.) for the growth of microorganisms. After the microorganism had fully grown in the non-inhibiting region of the agar plate, the extent of inhibition was measured as the non-proliferating region (or reduced proliferating region) extending in two perpendicular directions from the applied region.

Antimicrobial Effects of PS in Cosmetics [PS] (g / ℓ) 5 ℃ 5 days diffusion Diffusion at 5 ° C for 14 days One - - - - - - 2 7 6 5 7/14 7/13 5/11 5 5 6.5 7 7.5 / 14 9.5 / 16 9/15

In the two figures provided, the first represents the non-growth region, while the primary represents the reduced growth region.

As can be seen from Table 1, concentration-dependent growth inhibition by PS was shown.

Example 9

Antifungal Activity of PS and Its Derivatives

It has been found that some derivatives of phytosphingosine have improved solubility in aqueous systems. Thus, the antimicrobial activity of the derivative and the antimicrobial activity of the PS itself was compared. The following derivatives were investigated: glycolates, lactates and hydrochlorides of phytosphingosine. Stock solutions of phytosphingosine salts were prepared using the same method and the same experimental environment as in Example 6 using phytosphingosine.

It was found that all three salts tested had much stronger antifungal activity than the free base. These results are not due to the anions present in the salt solution, since the blanks with the appropriate amount of lactate, hydrochloride or glycolate show no effect (Fig. 3a). 3b shows that the effect of hydrochloride is 2.5 times higher than the effect of PS base.

Example 10

Antifungal Activity of PS and Some Derivatives thereof in Solvent-Free Systems

The solution with demineralized water was prepared in a similar manner to the solvent system (Example 6), including the final heating step.

In Figure 4a, the lack of solvent during the preparation of the sample did not show the antifungal effect of the free PS base, while the effect of the PS salt was not reduced. Indeed, as shown in Figures 4b and 4c, the effect of the PS salt was found to remain high even in the solventless system.

Example 11

Antimicrobial Effects of PS on Bacteria

Two different bacterial strains were used: Staphylococcus aureus ATCC 14458 and Escherichia coli 421. All incubations were performed at 37 ° C. Both strains were incubated by the method of Example 8, the cells contained in 50 μl of the culture were harvested by centrifugation, washed with 1 ml of sterile demineralized water, centrifuged, and then 0.5 ml of Resuspend with sterile demineralized water.

A phytosphingosine (PS) 10 mg / ml stock solution was prepared by the method described above. If necessary, the dilution of the stock solution was made with ethanol dissolved at 5% in water. All solutions were prepared 24 hours before use and stored at room temperature.

Blood antimicrobial effect of the monohydrate ping high signal on the two types of bacteria was investigated by using the Live / Dead yeast viability kit (LIVE / DEAD ^TM BacLight Bacterial Viability Kit ^TM L-7012 (Molecular Probes Inc., Oregon, USA)) . The kit uses two types of fluorescent stains, SYTO 9 and propidium iodide, to distinguish viable and dead cells, which can be observed using a fluorescence microscope with appropriate filters.

The dye solution provided in the kit was mixed 1: 1 before use, and 1.5 μl of the mixed solution was added to the 0.5 ml bacterial suspension. Then 50 μl of appropriately diluted phytosphingosine stock solution was added to bring the final concentration shown in FIG. 5. After mixing, the mixture was incubated and the viable and dead cell fractions were counted over time.

Microscopic observations were performed using an Olifs BHB fluorescence microscope with the following set of filters: dichroic mirror blue (B), excitation filter IF490, luminescence filter O530 (surviving cells appear green, dead cells appear orange / yellow) )

S. aureus was found to be dead. However, this effect could not be quantified, because the killing cells could not be detected due to its degradation activity, and structurally intact killing cells are required for counting with a fluorescent dye. Therefore, the killing effect was demonstrated only by the drastic reduction of the number of viable cells.

E. coli cells were killed very effectively and their dead cell numbers were easily quantified (FIG. 5). It was evident that the cells were killed by PS in a concentration-dependent manner, and that bacteria were more sensitive to this compound than fungi.

Example 12

Antimicrobial and Antifungal Effects of PS Derivatives by Diffusion Test

Test microorganisms were incubated overnight in the methods of Examples 6 and 8 and the samples were applied to test plates by the method of Example 8. Samples were prepared by appropriate dilution of the stock solutions described above. The agar plate was left at 5 ° C. overnight to diffuse the test solution and then remove the sample rings. The agar plates were then incubated at a temperature (37 ° C.) suitable for microbial growth. After the microorganism had fully grown in the non-inhibiting region of the agar plate, the extent of inhibition was measured in mm (in the non-proliferating region (or reduced growth region) extending in two perpendicular directions from the applied region).

Antimicrobial and Antifungal Effects of PS-Derivatives through Agar Diffusion Test derivative (g / ℓ) S. aureus ATCC 14456 C. Albicans ATCC 10231 PS-Lactate 10 12 13 13 9 ^* 9.5 ^* 10.5 ^* 5 12 10.5 12 8 ^* 9 ^* 9 ^* 2 8 8.5 9 ± 5 ^* 5 ^* One 6 5 5 4 ^* 6 ^* 4 ^* PS-glycolate 10 11.5 9.5 11 7.5 5 ^* 6 5 9 9 9 3 3 3 2 7 6 5 - - - One 3 4.5 3 - - - PS-hydrochloride 10 12.5 11 11 7 6 ^* 7 5 9 9 9 2 3 3 2 6 7 5 - - - One 5 5 5 - - -

^* Asterisks indicate inhibited proliferation areas; Unmarked values indicate areas that did not proliferate.

The diffusion test clearly shows that the PS derivatives have sufficient activity against bacteria and fungi, and the effect is concentration dependent.

Claims (12)

Spingoid base derivatives which are salts of sphingoid bases. The sphingoid base derivative according to claim 1, wherein the anion of the salt is derived from a hydrophilic acid. The sphingoid base derivative according to claim 2, wherein the hydrophilic acid is a hydrophilic organic acid or an inorganic acid. 4. The sphingoid base derivative according to claim 3, wherein the hydrophilic acid is selected from the group consisting of lactic acid, glycolic acid, malic acid, paruvic acid, succinic acid, fumaric acid, citric acid, ascorbic acid, gluconic acid and pyroglutamic acid. 4. The sphingoid base derivative according to claim 3, wherein the hydrophilic acid is selected from the group consisting of lactic acid, glycolic acid, pyroglutamic acid, citric acid and hydrochloric acid. The method of claim 1 comprising adding at least one equivalent of acid to the sphingoid base solution of claim 1 in a suitable solvent and recovering the crystalline sphingoid base salt from the reaction solution. Manufacturing method. The method of claim 6, wherein the solvent is ethanol or methyl isobutyl ketone. A composition for topical use comprising the sphingoid base derivative of claim 1. The composition of claim 8, wherein the composition is a cosmetic composition. 10. The method according to claim 8 or 9, wherein the content of the sphingoid base derivative is 0.001 to 5% by weight, preferably 0.005 to 5% by weight, more preferably 0.01 to 2.5% by weight, most preferably 0.02 to 1% by weight, particularly preferably 0.02 to 0.5% by weight of the composition The sphingoid base derivative according to claim 1 for use in medicine. The sphingoid base derivative according to claim 1, which is used for the manufacture of a medicament for antimicrobial treatment and / or anti-inflammatory treatment.