KR100668492B1 - New drug formula for melasma - Google Patents

Abstract

The present invention relates to a blemish treatment composition, and more particularly, to a blemish treatment composition containing a corticosteroid and unsaturated fatty acids. The corticosteroid in the present invention is selected from cortisol, cortisone, corticosterone, dioxycorticosterone, aldosterone, prednisolone, triamcinolone, paramethasone, betamethasone and dexamethasone, preferably betamethasone or dexamethasone. In addition, the unsaturated fatty acid in the present invention is selected from linoleic acid, oleic acid and arachidonic acid, preferably linoleic acid. In the present invention, the content of the corticosteroid is 0.001 to 10% by weight, the content of unsaturated fatty acid is 0.1 to 50% by weight. The anti-therapeutic composition containing the corticosteroid and unsaturated fatty acid of the present invention is particularly effective in hypermelanin pigment disease caused by dermal immune system abnormalities, whereas the corticosteroid contained in the present invention suppresses immune dysfunction, while the unsaturated fatty acid is corticosteroid. It has the effect of suppressing the side effects of increased melanin synthesis caused by.

Blemishes, corticosteroids, dexamethasone, betamethasone, gluconate, linoleic acid

Description

Blemish treatment composition {New drug formula for melasma}

1 shows the transcription levels of tyrosinase mRNA in melanoma cells after treatment with dexamethasone, linoleic acid and dexamethasone / linoleic acid.

Figure 2a and Figure 2b is a comparison picture of the prescription (Fig. 2a) and after the prescription (Fig. 2b) of the patient who improves the blemishes by prescribing the skin cream of Example 2 of the present invention for 6 weeks.

Figure 3a and Figure 3b is a comparison picture of the prescription (Fig. 3a) and after the prescription (Fig. 3b) of the patient with a significant improvement in the six-week prescription of the skin cream agent of the present invention Example 2.

Figures 4a and 4b is a comparison picture of the prescription (Fig. 4a) and after the prescription (Fig. 4b) of the patient almost disappeared by prescribing the skin cream of Example 2 of the present invention for 6 weeks.

The present invention relates to a blemish treatment composition, and more particularly, to a blemish treatment composition containing corticosteroids and unsaturated fatty acids, having no side effects and excellent in removing blemishes.

The blemishes are hypermelanin pigmentation on the face, which is a skin disease that occurs gradually or symmetrically on the face, which is primarily exposed to sunlight.

In animals, pigments are derived from melanin, which is synthesized and distributed in the skin, hair roots and eyes, and melanogenesis occurs in melanocytes differentiated from melanocytes, which are non-pigmentary progenitor cells. The enzymes involved in melanin biosynthesis include melanocyte specific enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2). Enzymes are said to be involved in the conversion of tyrosine to melanin. Although these enzymes have similar structures and properties, they are expressed in different genes as well as have specific enzyme properties. Tyrosinase is an enzyme encoded at the albino locus position of the mouse, which converts tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA; dopa) and then converts dopa to dopaquinone. It is known as a rate-controlling enzyme in melanin synthesis that converts to DOPAquinon, and TRP2 is encoded by mouse slaty rocus, which activates dopachrome tautomerase to activate dopachrome (dopachrome tautomerase). dopachrome) is converted to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). TRP1 is located in mouse brown rocus and is said to convert DHICA to indole-5,6-quinone-2-carboxylic acid.

The causes of blemishes are pregnancy, menopause, oral contraceptives, ovarian disease, endocrine system disorders, mental stress, malnutrition, diphenylhydantoin or mesantoin, etc. Irrespective of the species, the exact etiology is not clear, but the most important factors are known as genetic predisposition and UV irradiation.

The therapeutic agents used so far to treat hyperpigmented skin diseases include hydroquinone, hydroquinone monobenzyl ether (MBHH), 4-isopropylcatechol, 4-isopropylcatechol, parabutyl butyl and the like. Strong bleaching effects of phenolic compounds and thio compounds such as amylphenol (paratertiary butyl and amylphenol) and 4-hydroxy anisole have been reported.

However, these agents are not used as effective and safe treatments due to the side effects of skin irritation. In other words, hydroquinone monobenzyl ether is difficult to predict the degree of discoloration and severe irritation, causing a wide range of vitiligo melanoma beyond the application range, phenolic compounds are also known to be severe irritation.

Hydroquinone has been recommended because of its mild side effects and good depigmentation effect.However, when it is used at a high concentration of 4 to 5% for a long time every day, local irritation, allergic contact dermatitis, precocious dysplasia, post-inflammatory hyperpigmentation and ochronosis Side effects occur, and not all bleeding patients are effective in removing blemishes. Hydroquinone is also unstable and easily decomposes.

A new formula for depigmenting human skin.Arch. Dematol., 111: 40-48, 1975 contains 5% hydroquinone, 0.1% dexamethasone and 0.1% tretinoine. It has been reported that the combination cream is effective for black skin, blemish hyperpigmentation disease, and white and oriental people, but these treatments did not show satisfactory effects, and melanin synthesis by corticosteroids The side effect of ascension has always been a problem.

Topical 20% azelaic acid (azelaic acid) has also been reported to be effective for melanin pigmentation such as blemishes, but causes side effects such as skin pruritus, redness, and abortion.

In addition, recent studies on the treatment of blemishes by the combination of tretinoin (retinoic acid) and topical tretinoin and hydroxyanisole, but also had side effects such as skin irritation and skin peeling off.

Therefore, there is an urgent need for the development of a safer, more effective and non-irritating blemish treatment composition.

An object of the present invention is to provide a blemish treatment composition having no side effect and excellent blemish removal effect.

The inventors of the present invention have repeatedly studied to solve the problems of the conventional anti-therapeutic composition, when using a composition containing a corticosteroid and unsaturated fatty acid as a anti-therapeutic agent, immunity in hypermelanin pigment disease caused by abnormal dermal immune system While the above is inhibited by corticosteroids, the present inventors have found that the side effect of increased melanin synthesis caused by corticosteroids is significantly reduced by unsaturated fatty acids, thereby completing the present invention.

The anti-therapeutic composition of the present invention contains a corticosteroid and an unsaturated fatty acid.

Corticosteroids used in the present invention are selected from cortisol, cortisone, corticosterone, deoxycorticosterone, aldosterone, prednisolone, triamcinolone, paramethasone, betamethasone and dexamethasone, preferably betamethasone or dexamethasone.

The content of the corticosteroid used in the anti-therapeutic composition of the present invention is 0.001 to 10% by weight, preferably 0.02 to 0.1% by weight.

In addition, the unsaturated fatty acid used in the present invention is selected from linoleic acid, oleic acid and arachidonic acid, preferably linoleic acid.

The content of unsaturated fatty acid used in the blemish treatment composition of the present invention is 0.1 to 50% by weight, preferably 1 to 10% by weight.

The concentration ratio of linoleic acid to corticosteroid in the anti-therapeutic agent of the present invention is 1: 1 to 1: 1000, preferably 1:10 to 1: 100.

In the present invention, the preparation form of the anti-fog composition is any one of a lotion, a solution, a suspension, an ointment, an aerosol, a spray, a foam, an ointment, a gel, a patch and a skin cream, and preferably an aqueous ointment base is added. It is a skin cream containing.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples, but the present invention is not limited thereto.

Example 1

Dexamethasone and linoleic acid were dissolved in 100% ethanol to prepare 0.001, 0.01, 0.1, 1 and 10 μM dexamethasone and 6.25, 12.5, 25, 50 and 100 μM linoleic acid aesthetic treatment compositions.

Example 2

A skin cream was prepared by mixing 0.02% by weight of betamethasone and 2% by weight of linoleic acid with an aqueous skin base (97.98% by weight).

Experimental Example 1 Inhibitory Effects of Melanoma Cells on Proliferation

The effect of inhibiting the proliferation of melanoma cells after the single treatment of dexamethasone and linoleic acid and the combined treatment of dexamethasone / linoleic acid was examined as follows.

First, white paper B16 melanoma melanoma cells were cells cultured in Minimum Essential Medium (MEM) with 5% fetal calf serum (FBS), 24-well plates (5 × 10 ³ cells / well). -well plate) and dexamethasone, linoleic acid, dexamethasone / linoleic acid from the next day was treated with the same concentrations as Table 1a, Table 1b and Table 1c, respectively, and incubated for 4 days at 5% CO ₂ , 37 ℃. Fresh medium and fresh drug concentrations were adjusted every two days, and cell proliferation was determined by MTT assay after 4 days.

The MTT test is as follows. First, 50 μl of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] (5 mg / ml) dissolved in phosphate buffered saline (PBS) was added, and 4 hours later, dimethyl sulfoxide was added. 200 µl of side (DMSO) and 50 µl of glycine buffer (pH 10.5) were added, followed by stirring for 10 minutes. 200 ul of each well of the stirred plate was transferred to a 96-well plate and the absorbance was measured at 550 nm with a spectrometer (Microwell System Reader 210: Organon, teknika, Austria). Four wells were repeated three times for each treatment group, and statistical analysis was performed using the T-test and the ANOVA test.

The proliferation rate of the cells was expressed as a percentage of the cell proliferation of the drug treatment group when the cell proliferation of the non-pharmaceutical treatment group as the experimental control group was 100.

The experimental results are shown in Table 1a, Table 1b and Table 1c.

Proliferation rate of melanoma cells after dexamethasone treatment. Dexamethasone Concentration (μM) 0 0.001 0.01 0.1 One 10 Cell growth rate (%) 100 89.5 ± 4.8 ^* 84 ± 1.4 ^** 84 ± 1.9 ^** 83 ± 3.1 ^** 81 ± 2.7 ^**

* P <0.05, ** P <0.01

Mean ± Standard Error: Percentage of Control

Proliferation rate of melanoma cells after linoleic acid treatment. Linoleic Acid Concentration (μM) 0 6.25 12.5 25 50 100 Cell growth rate (%) 100 90.5 ± 4.3 92.8 ± 6 92.8 ± 6 95.3 ± 3.0 95.8 ± 5.8

* P <0.05, ** P <0.01

Mean ± Standard Error: Percentage of Control

Proliferation rate of melanoma cells after dexamethasone / linoleic acid combination treatment.  Drug (μM)  Dexamethasone 0.1 0.1 0.1 0.1 0.1 0.1  Linoleic acid 0 6.25 12.5 25 50 100  Cell growth rate (%) 100 93.3 ± 1.7 93.8 ± 3.5 92.3 ± 2.5 92.8 ± 2.8 93 ± 2.7

* P <0.05, ** P <0.01

Mean ± Standard Error: Percentage of Control

When treated with dexamethasone alone, the proliferation of melanoma cells was significantly inhibited in a dose-dependent manner, and the most proliferation of melanoma cells was suppressed by concentrations, especially from 10 μM to 81%.

Treatment with linoleic acid alone did not significantly inhibit the proliferation of melanoma cells.

After fixation of dexamethasone to 0.1 µM, the proliferation of melanoma cells was not significantly inhibited even in the dexamethasone / linoleic acid combination treatment in which linoleic acid was treated by concentration.

Experimental Example 2 Inhibitory Effects of Melanin Synthesis in Melanoma Cells

Dexamethasone and linoleic acid alone treatment or dexamethasone / linoleic acid treatment was performed to investigate the effect of inhibiting melanin synthesis in melanoma cells as follows. The melanin was quantified according to the method of Hosoi et al. (Cancer Res, 45; 1474-1478, 1985).

First, white paper B16 melanoma melanoma cells 2 × 10 ⁴ cells were seeded in 100 mm Petri dish and treated with dexamethasone, linoleic acid, dexamethasone / linoleic acid at the concentrations shown in Tables 2a, 2b and 2c, respectively. Incubated daily. The melanoma cells were harvested and washed twice with phosphate buffered saline (PBS), and then 0.4 ml of phosphate buffered saline solution containing 1% nonidet P-40 was added and stirred thoroughly. Then centrifuged at 13,000 g for 10 minutes. 1N sodium hydroxide and 10% dimethyl sulfoxide (DMSO) were added to the cell precipitate, and the cells were dissolved at 80 ° C. for 2 hours, and then measured at an absorbance (OD) of 475 nm using a spectrometer. Synthetic melanin was subdivided to prepare a standard curve and the melanin was quantified using this. Protein quantification was performed by Lowry et al. (J. Biol. Chem., 193: 265-271, 1951).

The melanin content was calculated by quantifying melanin (㎍) and then dividing by total protein quantifier (mg).

Statistical analysis was performed by T-test and Anova test.

The experimental results are shown in Tables 2a, 2b and 2c below.

Melanin synthesis in melanoma cells after dexamethasone treatment Dexamethasone Concentration (μM) 0 0.001 0.01 0.1 One 10 Melanin content 73.1 ± 3.0 77.9 ± 10.1 116.3 ± 24.5 151.5 ± 24.5 ^* 161 ± 5.7 ^** 180.7 ± 7.3 ^**

Mean ± standard error: melanin (㎍) / total protein (mg)

* P <0.05, ** P <0.01

n = 8

Melanin Intracellular Melanin Synthesis after Linoleic Acid Treatment Linoleic Acid Concentration (μM) 0 6.25 12.5 25 50 100 Melanin content 187 ± 16.5 160 ± 40.2 157.5 ± 27.8 112.5 ± 7.5 ^* 112.5 ± 14.4 ^* 78.3 ± 6.0 ^**

Mean ± standard error: melanin (㎍) / total protein (mg)

* P <0.05, ** P <0.01

n = 8

Melanin synthesis in melanoma cells after dexamethasone / linoleic acid treatment Drug (μM)  Dexamethasone 0.1 0.1 0.1 0.1 0.1 0.1  Linoleic acid 0 6.25 12.5 25 50 100  Melanin content 156.3 ± 8.2 115.5 ± 9.3 112.2 ± 10.9 90.3 ± 14.2 ^* 85.3 ± 6.1 ^* 75.8 ± 10.1 ^**

Mean ± standard error: melanin (㎍) / total protein (mg)

* P <0.05, ** P <0.01

n = 8

Treatment with dexamethasone alone increased melanocyte synthesis in melanoma cells in a dose-dependent manner.

However, treatment with linoleic acid alone inhibited melanin synthesis in melanoma cells in a drug concentration-dependent manner, and most inhibited melanin synthesis in melanoma cells at high concentrations.

In the case of the dexamethasone / linoleic acid combination treatment in which dexamethasone was fixed at 0.1 μM and the linoleic acid was treated by concentration, the synthesis of melanin in melanoma cells was inhibited in a concentration-dependent manner. Therefore, it was confirmed that the synthesis of melanin in melanoma cells is effectively inhibited by linoleic acid even under the influence of dexamethasone.

Experimental Example 3 Effect on Tyrosinase Activity in Melanoma Cells

Dexamethasone and linoleic acid alone or dexamethasone / linoleic acid were combined to examine the effects on melanoma intracellular tyrosinase activity as follows. The measurement of tyrosinase activity of melanoma cells was performed according to the method of Bouchard et al. (J. Exp. Med., 169; 2029-2042, 1989).

First, white paper B16 melanoma melanoma cells 2 × 10 ⁴ cells were seeded in 100 mm Petri dish, and the agents of dexamethasone, linoleic acid, and dexamethasone / linoleic acid were treated at the concentrations shown in Tables 3a, 3b, and 3c, respectively. After culturing for one day, the mixture was washed twice with phosphate buffered saline, and 0.4 ml of phosphate buffered saline containing 1% nonidet P-40 was added and stirred. The supernatant was obtained by centrifugation at 13,000 g for 10 minutes. To a 0.2 ml aliquot of the supernatant, a reaction solution containing 20 μCi / ml of [ ³ H] L-, tyrosine, 2% nonidet P-40, 0.2 mM L-tyrosine, and 2 mM L-dopa was added and left at 37 ° C for 1 hour. After that, 0.2 ml of a charcoal solution (100 mg of charcoal per 1 ml of 0.1 M citric acid) was added to terminate the reaction. The mixture was left on ice for 30 minutes and centrifuged at 2000 g for 5 minutes to obtain a supernatant, and 2 ml of scintillation solution (Scintillation fluis; Aquazol II) was added to 0.2 ml of the aliquot to measure radioactivity. Six petri dishes were repeated at least twice.

Tyrosinase activity was expressed as a percentage of the non-pharmaceutical treatment control, expressed by the following equation.

The calculated results were statistically analyzed by tee test and annova test.

The results are as shown in Tables 3a, 3b and 3c.

Melanoma Intracellular Tyrosinase Activity After Dexamethasone Treatment Dexamethasone Concentration (μM) 0 0.001 0.01 0.1 One 10 Tyrosinase activity 100 116.1 ± 11.5 124.8 ± 8.5 137.3 ± 13.7 147.2 ± 13.7 ^* 150.2 ± 14.5 ^**

Mean ± Standard Error: Percentage of Control

* P <0.05, ** P <0.01

n = 6

Melanoma Intracellular Tyrosinase Activity After Linoleic Acid Treatment Linoleic Acid Concentration (μM) 0 6.25 12.5 25 50 100 Tyrosinase activity 100 119.9 ± 12.5 95.5 ± 10.5 89.6 ± 4.5 83.8 ± 9.4 ^* 61.7 ± 4.9 ^**

Mean ± Standard Error: Percentage of Control

* P <0.05, ** P <0.01

n = 6

Melanoma Intracellular Tyrosinase Activity after Dexamethasone / linoleic Acid Combination Treatment Drug (μM)  Dexamethasone 0.1 0.1 0.1 0.1 0.1 0.1  Linoleic acid 0 6.25 12.5 25 50 100  Tyrosinase activity 100 98.1 ± 4.4 101.9 ± 1.0 79.0 ± 10.6 ^* 69.6 ± 7.0 ^* 59.6 ± 6.6 ^**

Mean ± Standard Error: Percentage of Control

* P <0.05, ** P <0.01

n = 6

Treatment with dexamethasone alone significantly increased tyrosinase activity in melanoma cells.

Treatment with linoleic acid alone reduced drug concentration dependent tyrosinase activity in melanoma cells.

Even under 0.1 μM effect of dexamethasone, linoleic acid decreased the tyrosinase activity in melanoma cells in a dose-dependent manner, and the reduction rate was similar to that of linoleic acid alone. In other words, linoleic acid inhibited tyrosinase activity even under dexamethasone effect.

Experimental Example 4 Effect on Dopachrome Tautomerase Activity in Melanoma Cells

Dexamethasone and linoleic acid alone or dexamethasone / linoleic acid were combined to examine the effects on dopachrome tautomerase activity in melanoma cells. The measurement of dopachrome tautomerase activity in melanoma cells was performed according to Aroca et al. (J. Biochem. Biophys. Meth. 2147-2156,1990).

That is, white paper B16 melanoma melanoma cells 2 x 10 ⁴ cells in a 100 mm Petri dish and treated with dexamethasone, linoleic acid, dexamethasone / linoleic acid at the concentrations shown in Table 4a, Table 4b and Table 4c respectively for 4 days After incubation, the solution was washed twice with phosphate buffered saline, and 0.4 ml of phosphate buffered saline containing 1% nonidet P-40 was added and stirred. The supernatant was obtained by centrifugation at 13,000 g for 10 minutes. Dopachrome was prepared by mixing 0.1 mM L-DOPA and 0.2 mM NaIO ₄ (sodiumperiodate) in 10 mM phosphoric acid (pH 6.0) and 0.1 mM EDTA solution in a 1: 1 ratio. 625 μl of 10 mM phosphate buffer solution was dispensed into a fine centrifuge tube, 250 μl dopachrome solution was added, and 125 μl of cell lysis solution was mixed. Absorbance changes were determined based on blank buffer. The absorbance decrease was measured at 475 nm and the absorbance increase at 308 nm, which was converted to the ratio of the control experiment.

The calculated results were statistically analyzed by tee test and annova test.

The results are as shown in Tables 4a, 4b and 4c.

Dopachrome Tautomerase Activity in Melanoma Cells after Dexamethasone Treatment Dexamethasone Concentration (μM) 0 0.001 0.01 0.1 One 10 Dopachrome Tautomerase Activity 100 113.3 ± 15.6 88.45 ± 8.5 77.6 ± 9.4 ^* 75.6 ± 4.9 ^** 85.8 ± 8.6 ^**

Mean ± Standard Error: Percentage of Control

* P <0.05, ** P <0.01

n = 6

Dopachrome Tautomerase Activity in Melanoma Cells after Linoleic Acid Treatment Linoleic Acid Concentration (μM) 0 6.25 12.5 25 50 100 Dopachrome Tautomerase Activity 100 97.3 ± 8.5 100.0 ± 10.5 92.2 ± 6.6 100.5 ± 10.2 90.5 ± 10.2

Mean ± Standard Error: Percentage of Control

* P <0.05, ** P <0.01

n = 6

Dopachrome Tautomerase Activity in Melanoma Cells after Dexamethasone / linoleic Acid Combination Treatment Drug (μM)  Dexamethasone 0.1 0.1 0.1 0.1 0.1 0.1  Linoleic acid 0 6.25 12.5 25 50 100  Dopachrome Tautomerase Activity 100 111.3 ± 2.4 104.2 ± 6.5 95.5 ± 9.1 108.0 ± 10.5 110.9 ± 9.6

Mean ± Standard Error: Percentage of Control

* P <0.05, ** P <0.01

n = 6

Treatment with dexamethasone alone reduced dopachrome tautomerase activity in melanoma cells (dose-response) (P <0.05)

Linoleic acid alone did not affect dopachrome tautomerase activity in melanoma cells.

In the case of the combination treatment of dexamethasone and linoleic acid, there was no change in dopachrome tautomerase activity in melanoma cells.

Experimental Example 5 Effect on Melonoma Cell Tyrosinase, TRP1 and TRP2 mRNA Transcription

Dexamethasone (0.001 μM, 0.1 μM, 1.0 μM) and linoleic acid (6.25 μM, 25 μM, 100 μM each) or dexamethasone / linoleic acid (0.1 μM of dexamethasone to 6.25 μM, 25 μM, 100 μM) combined with mRNA The impact on transcription was examined through Northern analysis.

First, 1% formaldehyde gel was obtained by extracting mRNA of melanoma cells by oligo (dT) cellulose column chromatography according to Kim et al. (MOL. Cell. Biol., 12; 8, 3636-3643, 1992). Was electrophoresed and transferred to a Gene Screen Plus membrane and fixed with UV light. This membrane was prehybridized and hybridized in treatment buffer, whereby probes were irradiated with mouse MTY811C (Tyrosinase (Tyr), pMT4 (TRP1) and pTRP2a (TRP2) clones. Isotopically labeled and used. The next day, the membrane was washed twice with 2 × SSC / 0.1% SDS solution at 65 ° C. for 20 minutes, and then washed twice with 2 × SSC / 0.1% SDS solution to prepare an autoradiogram.

The mRNA loading degree was compared with the autoradiogram produced by the same method using G3PDH (glyceroaldehyde-3phosphate dehydrogenase) probe.

The results are as shown in Figure 1 below.

As shown in FIG. 1, dexamethasone alone increased Tyr mRNA transcription in melanoma cells depending on drug concentration, but melanoma cells were treated with linoleic acid alone or in combination with dexamethasone / linoleic acid. There was no effect on mRNA transcription of Tyr, TRP1 and TRP2 in. Therefore, the increase in melanin synthesis of dexamethasone is influenced by the increase in Tyr mRNA transcription, but linoleic acid was determined to inhibit melanin synthesis without the regulation of transcription even in the single treatment and in combination with dexamethasone.

Experimental Example 6: Effect on melanoma cells Tyr, TRP1 and TRP2 promoter (Luciferase reporter activity test)

To further confirm that linoleic acid treatment alone and dexamethasone / linoleic acid treatment inhibit melanin synthesis without affecting transcriptional regulation, a promoter of the transcriptional regulatory gene sites of Tyr, TRP1 and TRP2 by the drug was identified by the B16 melanoma cell genome. Amplification by polymerase chain reaction from DNA (pTyr: nucleotide sequence -2237 to +59, pTRP1: nucleotide sequence -1247 to +82, pTRP2: nucleotide sequence -585 to +359), followed by multiplexing of the reporter gene (pGL2B) Recombinant insertion at the cloning site (Sma I-Bgl II) was used.

First, B16 melanoma cells were dispensed at 2 x 10 ⁴ cells / well in 24 well plates and the genes were transferred the next day. In short, 1 µg of plasmids (pMT1.1, pTRP1 and pTRP2) and 300ng of pSV-βGal plasmid were transferred to 200 µl of final volume using 2 µl of lipofectamine, followed by Table 5a, Table 5b, and Table 16. Drug treatment at the concentrations of 5c, Table 6a, Table 6b, Table 6c, Table 7a, Table 7b, and Table 7c, and after 24 hours, 50 μl of cell lysate was added and the activity of luciferase and beta galactase was measured. Measured. The transcription factor microphthalmia gene is a transcription factor that specifically regulates differentiation and melanin synthesis in melanocytes and is known to increase the transcriptional regulation of Tyr, TRP1 and TRP2. In order to observe the effect, 1 µg of microphthalmia expression vector pcDNA3-MIC was simultaneously transferred and measured in the same manner. In this positive control experiment, 10 μM of forskolin, which is known to activate Tyr, TRP1 and TRP2 promoters, was increased by increasing cyclic AMP (cAMP) in melanocytes, and an empty vector was used as a negative control experiment. Phosphorus pGL2B (Promega) was used for gene transfer and stimulation with only medium. Luciferase activity is expressed as a normalization value corrected with beta galactase activity indicating gene transfer efficacy.

The results are as shown in Tables 5a, 5b, 5c, 6a, 6b, 6c, 7a, 7b and 7c.

Effect of Melanoma Cells on Tyr Promoter after Dexamethasone Monotherapy pGL2B Tyr Dexamethasone (μM) 0 0 0.00001 0.001 One Forskolin 10μM MIC + 61 ± 10 5916 ± 834 7168 ± 80 7385 ± 707 ^* 9330 ± 821 ^** 22539 ± 5189 MIC- 59 ± 2 1373 ± 231 1370 ± 6 1514 ± 51 1636 ± 91 6448 ± 235

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

Effect of Linoleic Acid on Melanoma Cells after Tyr Promoter pGL2B Tyr Linoleic Acid (μM) 0 0 6.25 25 100 Forskolin 10μM MIC + 56 ± 1 3724 ± 85 3698 ± 243 3366 ± 398 4621 ± 465 15129 ± 1628 MIC- 59 ± 4 640 ± 24 905 ± 217 989 ± 254 1022 ± 105 3936 ± 264

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

Effect of Melanoma Cells on Tyr Promoter after Dexamethasone / linoleic Acid Combination Treatment pGL2B Tyr Dexamethasone (μM) 0 0.1 0.1 0.1 0.1 Forskolin 10μM Linoleic Acid (μM) 0 0 6.25 25 100 MIC + 61 ± 2 5227 ± 419 6871 ± 401 6864 ± 281 7060 ± 285 17981 ± 1343 MIC- 59 ± 2 1329 ± 103 1305 ± 116 1250 ± 46 1458 ± 182 6881 ± 430

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

Effect of Melanoma Cells on TRP1 Promoter after Dexamethasone Monotherapy pGL2B TRP1 Dexamethasone (μM) 0 0 0.00001 0.001 One Forskolin 10 μM MIC + 303 ± 15 8685 ± 364 8818 ± 34 7965 ± 187 7514 ± 210 30995 ± 1017 MIC- 372 ± 66 6032 ± 345 4630 ± 450 3794 ± 140 4448 ± 104 27586 ± 347

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

Effect of Melanoma Cells on TRP1 Promoter after Linoleic Acid Treatment pGL2B TRP1 Linoleic Acid (μM) 0 0 6.25 25 100 Forskolin 10μM MIC + 173 ± 15 752 ± 35 763 ± 8 772 ± 55 638 ± 12 3314 ± 403 MIC- 252 ± 24 492 ± 50 446 ± 12 456 ± 25 476 ± 4 2795 ± 127

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

Effect of Melanoma Cells on TRP1 Promoter after Dexamethasone / linoleic Acid Combination Treatment pGL2B TRP1 Dexamethasone (μM) 0 0.1 0.1 0.1 0.1 Forskolin 10μM Linoleic Acid (μM) 0 0 6.25 25 100 MIC + 232 ± 16 418 ± 80 437 ± 25 372 ± 31 329 ± 85 1040 ± 28 MIC- 130 ± 6 286 ± 24 296 ± 17 252 ± 2 238 ± 3 1123 ± 77

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

Effect of Melanoma Cells on TRP2 Promoter after Dexamethasone Monotherapy pGL2B TRP2 Dexamethasone (μM) 0 0 0.00001 0.001 One Forskolin 10 μM MIC + 371 ± 20 382 ± 24 340 ± 18 336 ± 48 345 ± 15 954 ± 15 MIC- 205 ± 15 307 ± 26 294 ± 23 330 ± 10 320 ± 10 1438 ± 58

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

Effect of Linoleic Acid on TRP2 Promoter in Melanoma Cells pGL2B TRP2 Linoleic Acid (μM) 0 0 6.25 25 100 Forskolin 10 μM MIC + 945 ± 98 1607 ± 172 1600 ± 51 1279 ± 14 1518 ± 50 2867 ± 93 MIC- 754 ± 74 738 ± 88 1592 ± 83 1058 ± 20 1179 ± 74 2323 ± 25

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

Effect of Melanoma Cells on TRP2 Promoter after Dexamethasone / linoleic Acid Combination Treatment pGL2B TRP2 Dexamethasone (μM) 0 0.1 0.1 0.1 0.1 Forskolin 10μM Linoleic Acid (μM) 0 0 6.25 25 100 MIC + 162 ± 30 199 ± 31 197 ± 7 215 ± 29 261 ± 34 707 ± 43 MIC- 142 ± 18 195 ± 2 200 ± 98 203 ± 38 184 ± 44 397 ± 39

Mean ± Standard Error: Luciferase Activity

* P <0.05, ** P <0.01

n = 6

As can be seen in the table, dexamethasone increased the drug concentration dependently only in the presence of MIC in the measurement of the promoter activity of Tyr showed about twice the activity at 1 μM. Linoleic acid and dexamethasone / linoleic acid showed a tendency to increase slightly, but there was no statistical significance (P <0.2).

In measuring activity against the TRP1 promoter, the presence of MIC was affected and showed a slight decrease in administration of dexamethasone, linoleic acid, or dexamethasone / linoleic acid treatment, but no significant variation (P <0.5).

 In measuring activity against the TRP2 promoter, there was no significant change in administration of dexamethasone, linoleic acid or dexamethasone / linoleic acid treatment regardless of the presence of MIC (P <0.6).

In view of the above, dexamethasone increases melanin synthesis by increasing Tyr activity, which is caused by the regulation of transcription of Tyr mRNA. In addition, dexamethasone decreases dopachrome tautomerase activity, but does not affect TRP2 mRNA and TRP2 promoter activity, and is thought to be involved in posttranscription regulation. In contrast, linoleic acid inhibits melanin synthesis by inhibiting Tyr activity but inhibits melanin synthesis by post-transcriptional regulation that does not affect Tyr mRNA. In the dexamethasone / linoleic acid combined treatment, melanin synthesis inhibitory effect is maintained by the same mechanism as linoleic acid alone. Therefore, linoleic acid was judged to have an anti-mimetic effect by effectively blocking the side effect of increased melanin synthesis by dexamethasone even in the treatment with dexamethasone.

Experimental Example 7: Anti-mimetic effect of betamethasone and linoleic acid skin cream by double blind method

Face coating group of aqueous base (first control group), betamethasone-containing skin cream agent face coating group (second control group), and acetyl acetate betamethasone / linoleic acid-containing skin cream agent face coating group of Example 2 (face of Example 2) Test group), where the patient took and used topical tretinoin, systemic steroid, hydroquinone, tathion, and vitamin (Vit.C) within 6 months. Only patients with no history were included, and underlying diseases that could cause blemishes were identified by questionnaire. The lesions were described in detail by the forehead, nose, etc., under the eyes, cleft lip, wide bone, hair, and described in detail. The wood's lamp was used to distinguish epithelial and mixed forms.

The skin cream was applied once a day after washing and before going to bed without the patient or doctor knowing its contents.

The clinical signs of improvement were finally determined by follow-up for 6 weeks at 2 weeks intervals, according to the classification of K. Jimbow et al. (Arch dermatol, 127: 528-1534, 1991). In other words, N (No effect) is invalid when there is no visible change of the pigment plate, G (Good) is a small arc, and there is a decrease in the visible pigment plate, but a certain boundary of abnormal pigment plate is observed, and F (Fair) is large. Many cases of visible pigmentation decrease due to improvement, but the border of blemishes was visible in the normal area. E (Excellent) is the complete loss of abnormal pigmentation due to complete loss.

Agility: N (No effect); invalidity

         G (Good); Improvement

         F (Fair); Great War

         E (Excellent); Complete loss

         P = 0.0001

Statistical analysis was performed using the Chi-square (Χ ²⁾ test for the non-measuring observations.

The results are shown in Table 8.

Anti-glare effect of Example 2  Drug group Number of patients effect Side Effect N G F E  Base 16 10 6 0 0  1 (itch)  Gil Acetate Betamethasone 16 8 6 2 0  3 (acne, erythema)  Gil Acetic Acid Betamethasone + Linoleic Acid 28 One 14 10 3  1 (erythema soban)

Of the 60 volunteers, 16 patients were followed up for 6 weeks in the aqueous base facial applicator (first control), of which 10 (63%) had no bleeding effect and 6 (27%) had mild improvement. there was. Sixteen patients were followed up in the beta-methasone-only group (the second control group), and two patients (13%) had a great improvement and six patients (38%) improved.

In the beta-methasone / linoleic acid-containing skin cream-treated group (Example 2 facial application group), 28 subjects were followed for 6 weeks. Of these, only one (4%) was ineffective and improved in 14 (50%). There was a great war in 10 (36%) and 3 (11%) were completely lost. By statistical treatment, significant superiority was recognized as compared to the control group only in the group administered with betamethasone / linoleic acid-containing skin cream (P = 0.0003). Examples of improvements (shown in FIGS. 2A and 2B), Great War (shown in FIGS. 3A and 3B), and complete burnout (shown in FIGS. 4A and 4B) are shown in the figures, respectively.

On the other hand, during follow-up, the presence or absence of side effects such as microvascular expansion, itching, redness, skin dropout, and burning sensation was also observed or questioned. There was no difference (P = 0.7).

It can be seen that the acetyl acetate betamethasone / linoleic acid-containing skin cream of Example 2 according to the present invention is safe and has fewer side effects and excellent anti-glare effect.

The composition according to the present invention can be used as a blemish treatment having excellent side effect removal effect with less side effects. In particular, the anti-therapeutic composition containing the corticosteroid and unsaturated fatty acid in the present invention is effective in hypermelanin pigment disease caused by a dermal immune system abnormality. Fatty acids significantly inhibit the side effects of increased melanin synthesis caused by corticosteroids.                     

Claims (11)

A medicinal composition containing a corticosteroid and linoleic acid. The anti-therapeutic composition according to claim 1, wherein the corticosteroid is any one selected from the group consisting of cortisol, cortisone, corticosterone, deoxycorticosterone, aldosterone, prednisolone, triamcinolone, paramethasone, betamethasone and dexamethasone. . The anti-therapeutic composition according to claim 2, wherein the corticosteroid is betamethasone or dexamethasone. delete delete The anti-therapeutic composition according to claim 1, wherein the content of the corticosteroid is 0.001 to 10% by weight. 7. The anti-therapeutic composition according to claim 6, wherein the content of the corticosteroid is 0.02 to 0.1 wt%. The anti-therapeutic composition according to claim 1, wherein the linoleic acid content is 0.1 to 50% by weight. 9. The anti-therapeutic composition according to claim 8, wherein the content of linoleic acid is 1 to 10% by weight. The anti-therapeutic composition according to claim 1, wherein the formulation is any one selected from the group consisting of lotions, solutions, suspensions, ointments, aerosols, sprays, foams, ointments, gels, patches and skin creams. 11. The anti-therapeutic composition according to claim 10, wherein the formulation is a skin cream further containing an aqueous ointment base.

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