Phytosphingosine-1-phosphate derivative, preparation method therefor, and composition for preventing and treating hair loss or for growing hair comprising same

Abstract

The present invention provides: O-cyclic phytospingosine-1-phosphate (O-C-P1P), N-cyclic phytospingosine-1-phosphate (N-C-P1P), a pharmaceutically acceptable salt thereof, or a solvate thereof; a preparation method therefor; a cosmetic composition comprising the same for preventing hair loss or for growing hair; and a pharmaceutical composition for preventing and treating hair loss or for growing hair.

Claims

1. O-cyclic phytospingosine-1-phosphate (O-C-P1P) represented by Formula 1a below or N-cyclic phytospingosine-1-phosphate (N-C-P1P) represented by Formula 1b below, a pharmaceutically acceptable salt of O-C-P1P or N-C-P1P, or a solvate of O-C-P1P or N-C-P1P: ##STR00013##

2. A method of preparing a compound represented by Formula 1a below, the method comprising: deprotecting a protecting group of a compound represented by Formula 4 below by performing a reaction with trifluoroacetic acid or hydrochloric acid (HCl) gas: ##STR00014## wherein, in Formula 4, R.sup.1 is a protecting group.

3. The method of claim 2, wherein the compound of Formula 4 is prepared by performing a reaction between a compound represented by Formula 3 below and POCl.sub.3: ##STR00015## wherein, in Formula 3, R.sup.1 is a protecting group.

4. The method of claim 3, wherein the compound of Formula 3 is prepared by introducing a protecting group to an amino group of D-phytospingosine represented by Formula 2 below: ##STR00016##

5. A method of preparing a compound represented by Formula 1b below, the method comprising: performing a reaction between a compound represented by Formula 5 below and bromotrimethylsilane; and performing another reaction by adding water thereto: ##STR00017## wherein, in Formula 5, R.sup.2 is a protecting group.

6. A cosmetic composition for preventing hair loss or for promoting hair growth, the cosmetic composition comprising the compound of Formulae 1a or 1b, the pharmaceutically acceptable salt of the compound of Formulae 1a or 1b, or the solvate of the compound of Formulae 1a or 1b according to claim 1.

7. A pharmaceutical composition for preventing and treating hair loss or for promoting hair growth, the pharmaceutical composition comprising the compound of Formulae 1a or 1b, the pharmaceutically acceptable salt of the compound of Formulae 1a or 1b, or the solvate of the compound of Formulae 1a or 1b according to claim 1.

8. The composition of claim 6, wherein the composition is a preparation suitable for a topical application to a scalp or the area of skin where hair grows.

9. The composition of claim 8, wherein the composition is in a formulation of liposome, nano-emulsion, shampoo, hair conditioner, or hair lotion.

10. A cosmetic composition for preventing, smoothing, or treating wrinkles, the cosmetic composition comprising the compound of Formulae 1a or 1b, the pharmaceutically acceptable salt of the compound of Formulae 1a or 1b, or the solvate of the compound of Formulae 1a or 1b according to claim 1.

11. The cosmetic composition of claim 10, wherein the composition is a preparation suitable for topical application on wrinkled skin or wrinkle prone skin.

12. The cosmetic composition of claim 11, wherein the composition is in a formulation of liposome or nano-emulsion.

13. The composition of claim 7, wherein the composition is a preparation suitable for a topical application to a scalp or the area of skin where hair grows.

14. The composition of claim 7, wherein the composition is in a formulation of liposome, nano-emulsion, shampoo, hair conditioner, or hair lotion.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a .sup.1H NMR spectrum of a compound represented by Formula 1a (i.e., O-cyclic phytospingosine-1-phosphate) prepared according to an exemplary embodiment of the present invention.

(2) FIG. 2 is a .sup.1H NMR spectrum of a compound represented by Formula 1b (i.e., N-cyclic phytospingosine-1-phosphate) prepared according to an exemplary embodiment of the present invention.

(3) FIG. 3 shows images of the skin treated with the present compounds, a positive (3% minoxidil liquid) control or a negative control. The images were obtained at the 11.sup.th, 14.sup.th, and 18.sup.th days after applying the O-C-P1P or N-C-P1P solution according to the present invention once a day to the shaved flank of C3H mice.

(4) FIG. 4 shows images of the skin treated with the present compound, positive and negative controls, the images were obtained at the 14.sup.th and 18.sup.th days after applying O-C-P1P liposome according to the present invention once a day to the shaved flank of C3H mice.

(5) FIG. 5 shows images of the skin treated with various concentrations of the present compound and negative control, the images were obtained at the 13.sup.th and 17.sup.th days after applying various concentrations of O-C-P1P solution according to the present invention once a day for each concentration to the shaved flank of C3H mice.

(6) FIG. 6 shows images of the skin treated with various salts of the present compound and negative control, the images was obtained at the 14.sup.th and 18.sup.th days after applying an O-C-P1P free base, an HCl salt of O-C-P1P, and a solution containing a disodium salt of O-C-P1P according to the present invention once a day to the shaved flank of C3H mice.

(7) FIGS. 7A and 7B are images of the top of the head treated with the present compound showing the hair growth with time. The images were obtained at the indicated weeks after applying the O-C-P1P liposome according to the present invention once or two times a day to a subject having hair loss in progression

(8) FIG. 8 shows images of the scalp before and 16 weeks after applying the O-C-P1P liposome in various concentrations, the images were obtained in a clinical test performed on the subject having hair loss in progression by applying to the subject the O-C-P1P liposome according to the present invention in an amount of 0.002 weight % (Group A), 0.01 weight % (Group B), and 0.05 weight % (Group C) in a double blind test.

(9) Hereinafter, the present disclosure is further explained in more detail with reference to the following examples. These examples, however, should not be interpreted as limiting the scope of the present invention in any manner.

EXAMPLES

Example 1: Preparation of N-Boc-D-phytospingosine

(10) D-phytospingosine was reacted with (Boc).sub.2O in t-butanol according to the method described in Bull. Korean Chem. Soc., 2003, 24, 267-268, so as to introduce a protecting group (Boc) to an amino group, thereby preparing N-Boc-D-phytospingosine.

Example 2: Preparation of N-Boc-D-phytospingosine-3-O,P-cyclophosphate

(11) 1.05 equivalents of POCl.sub.3 was slowly added dropwise to a pyridine solvent that was cooled to about −30° C. (up to about ⅓ of the total volume), so as to prepare a POCl.sub.3/pyridine solution. Then, the N-Boc-D-phytospingosine (1 equivalent) of Example 1 was dissolved in a pyridine solvent (up to about ⅔ of the total volume), and then, the mixed solution was cooled to a temperature of about −20° C. While maintaining the same temperature, the POCl.sub.3/pyridine solution was added thereto. When the reaction temperature rose to room temperature, the pyridine solvent was removed under reduced pressure, and 6N HCl was used to adjust the solution from which the pyridine solvent was removed to have a pH of 2, and then, the solution having a pH of 2 was extracted with ethyl acetate. Afterwards, moisture was removed from an ethyl acetate extract by using MgSO.sub.4, and the solvent was concentrated under reduced pressure, so as to quantitatively obtain a desired substance, N-Boc-D-phytospingosine-3-O,P-cyclophosphate. N-Boc-D-phytospingosine-3-O,P-cyclophosphate obtained therefrom was used, without performing an additional purification process, as a starting material in Examples 3 and 4.

Example 3: Preparation of O-cyclic phytospingosine-1-phosphate (Formula 1a) (1)

(12) N-Boc-D-phytospingosine-3-O,P-cyclophosphate of Example 2 was dissolved in CH.sub.2Cl.sub.2, and then, the mixed solution was added to a CH.sub.2Cl.sub.2 solvate using trifluoroacetate up to about ⅓ of the total volume of the CH.sub.2Cl.sub.2 solvate. The mixed solution was then stirred at room temperature for 1 hour. Afterwards, the solvate was removed from the mixed solution under reduced pressure, and then, a Conc-HCl solvate was added thereto to be strongly stirred. White solids produced therefrom were filtered, washed subsequently with water and acetone, and then, dried, so as to obtain a final desired product, O-cyclic phytospingosine-1-phosphate (O-C-P1P) in the form of a HCl salt (total yield: 90%).

Example 4: Preparation of O-cyclic phytospingosine-1-phosphate (Formula 1a) (2)

(13) N-Boc-D-phytospingosine-3-0,P-cyclophosphate of Example 2 was dissolved in an ethylacetate solvate, and then, the mixed solution was cooled to a temperature of about 0° C. Then, HCl gas was injected thereto for 1 hour. White solids produced therefrom were filtered, washed with ethyl acetate, and then, dried, so as to obtain a final desired product, O-cyclic phytospingosine-1-phosphate (O-C-P1P) in the form of a HCl salt (total yield: 93%).

(14) Data obtained from .sup.1H NMR (300 MHz, CD.sub.3OD:CD.sub.3COOD=1:1) spectra of the finally obtained product is shown in FIG. 1

(15) The obtained HCl salt was treated with a KOH ethanol solution of the same equivalent with the HCl salt, so as to obtain O-cyclic phytospingosine-1-phosphate from which the HCl salt was removed.

(16) In addition, the obtained O-C-P1P was treated with 3 equivalents of a NaOH aqueous solution, so as to prepare a disodium salt of O-C-P1P.

Example 5: Preparation of N-cyclic phytospingosine-1-phosphate (Formula 1b)

(17) N-Boc-D-phytospingosine-1-phosphoric acid ester was prepared according to the method described in Bull. Korean Chem. Soc., 2003, 24, 267-268. The prepared N-Boc-D-phytospingosine-1-phosphoric acid ester was dissolved in CH.sub.2Cl.sub.2, and then, the mixed solution was cooled to a temperature of about −20° C. Afterwards, 5 equivalents of bromo dimethyl silane were added thereto to be stirred for about 1 hour. Then, water was added thereto to perform heating concentration, and acetone was added to residues obtained from the heating concentration. White solids obtained therefrom were filtered, so as to obtain a title compound (i.e., N-C-P1P) (total yields: 13%).

(18) Data obtained from .sup.1H NMR (300 MHz, CD.sub.3OD:CD.sub.3COOD=1:1) spectra of the finally obtained product is shown in FIG. 2.

Example 6: Preparation of O-C-P1P-Containing Liposome

(19) First, 1 g of 75% soybean phosphatidylcholine (available by Lipoid Company), 0.01 g of O-C-P1P of Example 4, and 0.3 g of vitamin E acetate were dissolved in a mixed solution of 20 g of ethanol and 2 g of ethoxydiglycol, so as to prepare an O-C-P1P ethanol solution. The obtained solution was ultrasonically treated for 5 minutes for dissolution. Next, an aqueous solution in which 0.5 g of menthol, 0.5 g of niacin amide, 3 g of natural organic sulfur, and 0.1 g of hesperidin were dissolved in 71.59 g of distilled water was slowly added to the O-C-P1P ethanol solution. Here, the mixed solution was strongly stirred. After finishing the addition of distilled water, the mixed solution was continuously stirred for 30 minutes. Then, a bath-type sonicator was used to perform ultrasonic treatment thereon for 30 minutes, so that the particles had a particle diameter of about 100 nm, thereby preparing liposome containing O-C-P1P.

(20) Separately, when preparing liposome according to the preparation method described above, as a skin penetration promoter, a mixture of poloxamer+polysorbate 80 (Tween 80) was added, thereby preparing liposome for promoting skin penetration.

Example 7: Preparation of O-C-P1P-Containing Liposome (2)

(21) Liposome containing O-C-P1P was prepared in the same manner as in Example 6, except that in preparing the aqueous solution, 1 g of willow bark extracts, 1 g of green tea extracts, 3 g of C. asiatica extract, 0.5 g of nettle extracts, 0.5 g of sweet flag extracts, 0.5 g of rosemary extracts, 0.5 g of chamomile extracts, and 1 g of fermented soybean were additionally added. Here, the liposome was prepared by using a small amount of distilled water for the amounts of each extract added therein.

Example 8: Preparation of N-C-P1P-Containing Liposome (1)

(22) Liposome containing N-C-P1P 0.01% was prepared in the same manner as in Example 6, except that N-C-P1P of Example 5 was used instead of O-C-P1P.

Example 9: Preparation of N-C-P1P-Containing Liposome (2)

(23) Liposome containing N-C-P1P 0.01% was prepared in the same manner as in Example 7, except that N-C-P1P of Example 5 was used instead of O-C-P1P.

(24) The compositions of the liposomes prepared in Examples 6 to 9 are listed in Table 1 below.

(25) TABLE-US-00001 TABLE 1 Composition of Examples Example 6 Example 7 Example 8 Example 9 O—C-P1P 0.01 0.01 0 0 N—C-P1P 0 0 0.01 0.01 Lecithin 1 1 1 1 Ethanol 20 20 20 20 Ethoxydiglycol 2 2 2 2 Poloxamer 0.5 0.5 0.5 0.5 Tween 80 ® 0.5 0.5 0.5 0.5 Niacin amide 0.5 0.5 0.5 0.5 Menthol 0.5 0.5 0.5 0.5 Natural organic sulfur 3 3 3 3 Hesperidin 0.1 0.1 0.1 0.1 Green tea extracts 0 1 0 1 willow bark extracts 0 1 0 1 Fermented soybean 0 1 0 1 C. asiatica extracts 0 3 0 3 Nettle extracts 0 0.5 0 0.5 Sweet flag extracts 0 0.5 0 0.5 Rosemary extracts 0 0.5 0 0.5 Chamomile extracts 0 0.5 0 0.5 Distilled water 71.89 63.89 71.89 63.89

Experimental Example 1: Hair Growth Test Using C3H Mice (1)

(26) A 6-week C3H mouse was purchased, and the hair on a dorsal part of the mouse was partially removed. Then, hair removal cream was applied to the dorsal part to completely remove the existing hair. A mouse of which the hair was not completely removed was excluded from the test. After finishing the hair removal, 5 mice were randomly bred per cage, and a total of 5 cages were used.

(27) The mice were left one day or so after finishing depilation, and a test compound, which was prepared by dissolving O-C-P1P of Example 4 and N-C-P1P of Example 5 in a mixed solution of ethanol and ethoxydiglycol (9:1, v/v) to a concentration of 0.01 weight %, was applied once a day to the area from which the hair was removed. A negative control group was prepared by applying a mixed solution of ethanol and ethoxydiglycol without using the test compound, and a positive control group was 3% minoxidil preparation (Mynoxyl, Hyundai Pharm). After applying samples to the area from which the hair was removed, images of the area captured at the 14.sup.th and 18.sup.th days are shown in FIG. 3.

(28) Referring to FIG. 3, it was confirmed that hair hardly grew on the area in a non-treated group (i.e., negative control group), resulting in the exposure of the skin to the outside, whereas a rapid growth of hair was made in the O-C-P1P-treated group and the N-C-P1P-treated group. In detail, the effects on faster hair growth were shown better in the O-C-P1P-treated group than in the positive control group. Although the effects on hair growth were quite less in the N-C-P1P-treated group than in the positive control group, the effects were significantly better in the N-C-P1P-treated group than in the negative control group. Therefore, based on the results above, it was clearly confirmed that the compounds according to the present invention were effective to promote hair growth.

Experimental Example 2: Hair Growth Test Using C3H Mice (2)

(29) A hair growth test was performed in the same manner as in Experimental Example 1 by using the liposomes containing O-C-P1P as prepared according to Examples 6 and 7, and the test compounds were continuously applied once a day for 3 weeks. Images of the area from which the hair was removed were captured, and the captured images are shown in FIG. 4.

(30) Referring to FIG. 4, it was confirmed that the O-C-P1P-treated group had significant effects on hair growth compared to those of the negative control group, that the O-C-P1P-treated group had similar effects on hair growth with those of the positive control group.

Experimental Example 3: Hair Growth Test Using Various Concentrations of O-C-P1P (3)

(31) O-C-P1P of Example 4 was dissolved in a mixed solution of ethanol and ethoxydiglycol (9:1, v/v) at three different concentrations, i.e., 0.005 weight %, 0.01 weight %, and 0.02 weight %, so as to prepare three different samples. Then, a hair growth test using these samples was performed in the same manner as in Text Example 1.

(32) After applying the samples to the area from which the hair was removed, images of the area captured at the 13.sup.th and 17.sup.th days are shown in FIG. 5.

(33) Referring to FIG. 5, it was confirmed that the O-C-P1P-treated groups at different concentrations (from 0.005 weight % to 0.02 weight %) showed significant effects on hair growth compared to those of the negative control group. However, there was no significant different in the effects on hair growth among the C-P1P-treated groups at different concentrations (0.005 weight %, 0.01 weight %, and 0.02 weight %). Such results are also similarly shown in a group administered with 3% minoxidil preparations (3 weight %), and accordingly, it was confirmed that O-C-P1P exhibits the same effects on hair growth at a concentration that is about 300 times as low as the concentration of minoxidil.

Experimental Example 4: Hair Growth Test Using Various Salts of O-C-P1P

(34) A hair growth test was performed in the same manner as in Experimental Example 1 by using the salts of O-C-P1P as synthesized in Example 4, O-C-P1P as prepared in Example 4, and the disodium salts of O-C-P1P as synthesized in Example 4.

(35) After the samples to the area from which the hair was removed, images of the area captured at the 14.sup.th and 18.sup.th days are shown in FIG. 6.

(36) Referring to the results of FIG. 6, it was confirmed that the negative control group showed significant effects on hair growth regardless of the types of the salts.

Experimental Example 5: Efficacy of the Present Compound on Hair Growth in Clinical Trials (1)

(37) The liposome containing O-C-P1P as prepared in Example 7 was directly applied to applicants having hair loss in progression, so as to compare hair growth efficacy. The application was performed once or twice a day on two applicants, and more particularly, the liposome was sprayed 5 to 7 times for each application, and then, the liposome-sprayed part was rubbed by hands to prevent the liposome solution from being flown down.

(38) Images of the head captured before applying the sample and images of the head captured with lapse of time are shown in FIGS. 7A and 7B.

(39) Referring to FIGS. 7A and 7B, it was confirmed that the case where the sample was applied with lapse of time showed excellent effects on prevention of hair loss and on hair growth compared to the case where the sample was not applied yet. That is, once directly applied to the human body, excellent effects on prevention of hair loss and on hair grown can be found.

Experimental Example 6: Efficacy of the Present Compound on Hair Growth in Clinical Trials (2)

(40) O-C-P1P liposomes at different concentrations, i.e., 0.002 weight % (Group A), 0.01 weight % (Group B), and 0.05 weight % (Group C), were prepared as described in Example 7, and then, these samples were directly applied to applicants having hair loss in progression, so as to compare hair growth efficacy. Here, the results were compared according to a double blind test not to let a test subject person and an experimenter know the different concentrations of the samples.

(41) For a period of 4, 8, 12, and 16 weeks after applying the samples, changes in density and thickness of the hair were observed, so as to evaluate clinical efficacy according to the different concentrations of O-C-P1P. The results obtained by observing changes in density and thickness of the hair for each concentration are summarized in Tables 2 and 3 below. The evaluation was made in the following manner (unit: evaluation unit for scalp thickness and thickness, 0: no change, 1: improved at a perceptible level, 2: slightly improved, 3: improved, 4: appreciably improved, 5: significantly improved). Images of the hear captured before applying the sample and images of the hear captured at the 16.sup.th weeks after applying the sample were compared according to Groups each having different concentrations, and the comparative results are shown in FIG. 8

(42) TABLE-US-00002 TABLE 2 Changes in hair density during a period of 16 weeks Average 0 week 8 weeks 12 weeks 16 weeks Group A 0 0.17 1.08 1.92* Group B 0 0.22 2.00* 2.78* Group C 0 1.11 1.78* 2.78* *Data having statistical significance

(43) TABLE-US-00003 TABLE 3 Changes in hair thickness during a period of 16 weeks Average 0 week 8 weeks 12 weeks 16 weeks Group A 0 0.00 0.17 0.42 Group B 0 0.11 0.78 1.33* Group C 0 0.11 0.78 1.56* *Data having statistical significance

(44) Referring to Tables 2 and 3 above, it was confirmed that Groups B and C showed statistically significant changes in density and thickness of the hair at the 12.sup.th and 16.sup.th weeks. In addition, referring to FIG. 8, the case where the sample was applied showed increased density of the hair compared to the case where the sample was not applied yet.

Experimental Example 7: Promotion of Cell Proliferation and Collagen Synthesis

(45) To measure anti-wrinkle effects of N-C-P1P and O-C-P1P, effects of human fibroblasts on cell proliferation were measured via an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. To observe collagen synthesis, procollagent type I, which is produced in human fibroblasts (e.g., dermal cells), were measured.

(46) To measure cell proliferation, human fibroblasts were cultured for 24 hours in a Dulbecco's modified eagle medium (DMEM) medium supplemented with 10% FBS and 100 unit/mL of penicillin/streptomycin. The culture circumstances comprising culturing in a 5% CO.sub.2 incubator maintained at a temperature of 37° C. Then, after removing the medium therefrom, N-C-P1P and O-C-P1P were added thereto at different concentrations. The MTT assay was performed to measure effects of N-C-P1P and O-C-P1P on cell proliferation, and the results are summarized in Table 4.

(47) TABLE-US-00004 TABLE 4 Effects on cell proliferation by N-C-P1P and O-C-P1P Concentration Cell Viability (%) Group 0 0.002% 0.004% 0.008% 0.016% N—C-P1P 100 120 110 110 125 O—C-P1P 100 128 135 143 160

(48) To measure collagen synthesis, the human fibroblasts (e.g., dermal cells) were cultured in the same conditions as above for 24 hours. After removing the medium therefrom, a new serum free medium was substituted. N-C-P1P and O-C-P1P (1 uM and 10 uM) that were diluted step by step were added to the new medium, and then, the medium was cultured for another 24 hours. The culture medium was used to measure amounts of synthesized collagen by using a procollagen type I C-peptide EIA kit, and the results measured therefrom are shown in Table 5 below.

(49) TABLE-US-00005 TABLE 5 Effect on the synthesis of collagen by N-C-P1P and O-C-P1P Concentration Collagen synthesis (%) Group 0 0.1 μM 1 μM N—C-P1P 100 110 125 O—C-P1P 100 120 140
Referring to the results of Tables 4 and 5, it was confirmed that N-C-P1P and O-C-P1P were capable of not only promoting cell proliferation, but also promoting synthesis of collagen. Therefore, N-C—P1P and O-C-P1P were found to be applicable to anti-aging and anti-wrinkle products that promote proliferation of human fibroblasts and synthesis of collagen.