KR20210061935A - A pharmaceutical composition for treatment and prevention of acne comprising Zidovudine - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating and preventing acne disease comprising zidovudine. According to the present invention, mitochondrial biosynthesis is reduced by reducing lipid synthesis in sebum cells, inhibiting lipid synthesis-related signal transduction system, reducing the expression of lipid synthesis-related electronic factors, reducing the expression of enzymes involved in lipid synthesis, or inhibiting mitophagy, so the secretion of sebum can be controlled. Therefore, it is possible to be usefully used as a pharmaceutical composition for treating and preventing acne disease or a cosmetic composition for reducing sebum and alleviating acne.

Description

A pharmaceutical composition for treatment and prevention of acne comprising zidovudine as an active ingredient {A pharmaceutical composition for treatment and prevention of acne comprising Zidovudine}

The present invention relates to a pharmaceutical composition for preventing and treating acne diseases, a cosmetic composition for improving acne, or a health functional food composition comprising zidovudine as an active ingredient.

Acne is a chronic inflammatory disease of the hairy sebaceous glands. The lesional causes of acne are known to be excessive proliferation of hair follicles, excessive secretion of sebum, the presence of acne bacteria and inflammatory reactions.

Acne is not a single cause, but multiple causes are involved in a complex manner, and it is known that an increase in sebum secretion is the main cause of acne.

Sebum secreted from sebocytes of the sebaceous gland has a pure function of preventing the penetration of harmful substances outside the skin and regulating oil and moisture. However, when excess sebum is secreted due to changes in adolescent hormones or stress, it causes dysfunction and causes various skin disorders. Seborrhea is a disease or skin troubles of the sebaceous glands characterized by excessive secretion of sebum, causing oily coatings, crusts or scales on the skin. In addition, excessive sebum secretion causes seborrheic dermatitis, also referred to as seborrheic eczema.

On the other hand, the production of sebum is regulated by factors that affect the proliferation and differentiation of sebaceous gland cells. According to reports, it is known that the production of sebum is regulated by factors such as male hormone, female hormone, insulin-like growth factor (IGF-1), and peroxisome proliferator activated receptor (PPAR). Sebum produced by sebaceous gland cells is secreted to the skin surface through the hair follicle tube, and over-produced sebum is known to decrease the skin barrier function by changing the structure of lipids between keratinocytes on the skin surface. For this reason, proper removal of over-produced sebum and suppression of over-produced sebum are important for skin barrier function and maintenance of healthy skin.

There are several treatments that can control this sebum secretion. Among them, there is a drug called Isotretinoin, but there are side effects such as hair loss, itching, atopic dermatitis, cleft dermatitis, dry skin and eyes, and elevated liver levels including ALT.Therefore, the need to develop a new drug that controls sebum secretion is emerging have.

While searching for a new drug that regulates the secretion of sebum, it is a drug that is marketed as a treatment for AIDS, and is a nucleoside reverse transcriptase inhibitor (NRTI) series Zidovudine (3'-azido-). The present invention was completed by confirming the effect of 3'-deoxythymidine, 3'-azido-3'-deoxythymidine; AZT) on controlling the secretion of secretion in acne disease or reducing the amount of lipids in human sebum cells.

An object of the present invention is to provide a pharmaceutical composition for the treatment and prevention of acne disease comprising zidovudine. Another object of the present invention is to provide a cosmetic composition for reducing sebum and improving acne, including zidovudine.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating and preventing acne diseases, including zidovudine.

The acne disease can be selected from the group consisting of excessive proliferation of hair follicles, excessive secretion of sebum, presence of acne bacteria, signs or symptoms of inflammation, and most preferably exerts a remarkable effect when excessive secretion of sebum is caused. .

The zidovudine can reduce lipid synthesis or inhibit the signaling system related to lipid synthesis in sebaceous cells, and the signaling related to lipid synthesis is IGF-1R/mTOR, p-IGF-1R/mTOR, IGF-1R/p-mTOR. It can be selected from the group consisting of.

Zidovudine can reduce the expression of lipid synthesis-related electron factors or lipid synthesis-related enzymes in sebaceous cells, and the lipid synthesis-related electron factors are sterol regulatory element-binding protein-1. , SPEBP-1), sterol regulatory element-binding protein-2 (SPEBP-2), peroxisome proliferator-activated receptor gamma (PPAR-γ), p -SPEBP-1, p-SPEBP-2, n-SPEBP-2, n-SPEBP-1 can be selected from the group consisting of, and the enzymes involved in lipid synthesis are FDFT-1, FASN, SCD, p21, ACC, It can be selected from the group consisting of FADS2.

The zidovudine can inhibit mitophagy in sebaceous cells and reduce mitochondrial biosynthesis.

The pharmaceutical composition of the present invention is preferably a formulation selected from the group consisting of ointments, powders, tablets, capsules, powders, solutions, gels, pastes, patches, and granules, but is not limited thereto.

When formulating the pharmaceutical composition of the present invention, it may be prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In addition, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories are included. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.

In addition, the present invention provides a cosmetic composition for reducing sebum and improving acne, including zidovudine.

The cosmetic may be selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, powders, soaps, surfactant-containing cleansing, oils and sprays.

The cosmetic contains conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

In the case of paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacantha, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or tragacantha, and the like may be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

According to the present invention, by reducing lipid synthesis in sebaceous cells, inhibiting the signaling system related to lipid synthesis, reducing the expression of lipid synthesis-related electron factors, reducing the expression of enzymes involved in lipid synthesis, or inhibiting mitophagy and reducing mitochondrial biosynthesis. , It is possible to provide a pharmaceutical composition for treating and preventing acne diseases, including zidovudine capable of controlling the secretion of sebum.

In addition, it is possible to provide a cosmetic composition for reducing sebum and improving acne, including the zidovudine.

1 shows the results of intracellular lipid inhibition in human sebaceous cells treated with zidovudine IGF-1. (A) Intracellular lipids were analyzed by thin layer chromatography. Intracellular lipids were increased after IGF-1 treatment, but zidovudine decreased intracellular lipids in IGF-1 treated human sebaceous cells. (B) Intracellular lipids were stained with Oil Red O staining. IGF-1 increased the number of lipid droplets in human sebaceous cells, and zidovudine treatment decreased the number of lipid droplets in human sebaceous cells. CTL: control, control; AZT: azidothymidine, zidovudine. Scale bar = 20 μm.
Figure 2 shows the result of suppressing lipid synthesis in human sebaceous cells treated with zidovudine IGF-1. (A) Analysis of sebaceous cell differentiation-related transcription factors in sebaceous cells 12 hours after IGF-1 treatment. (B) Analysis of lipid synthesis-related enzymes in sebum cells 24 hours after IGF-1 treatment. (C) Lipid synthesis-related mRNA analysis in sebum cells 24 hours after IGF-1 treatment. *P<0.05, **P<0.01.
Figure 3 shows the results of Zidovudine suppression of Akt/mTOR signal induced by IGF-1 in human sebaceous cells. IGF-1 increased the phosphorylation of insulin-like growth factor 1 receptor (IGF-1R) and sub-effectors Akt and mTOR. Conversely, zidovudine reduced the phosphorylation of IGF-1R, Akt and mTOR induced by IGF-1.
Figure 4 shows the results of mitochondrial biosynthesis and mitophagy inhibition induced by zidovudine in human sebaceous cells to IGF-1. (A) IGF-1 increased the levels of PGC-1α and mtTFA in human sebaceous cells, indicating that IGF-1 induces mitochondrial biosynthesis. Conversely, zidovudine reduced PGC-1α and mtTFA levels in IGF-1 treated sebaceous cells, indicating the effect of zidovudine reducing IGF-1-induced mitochondrial biosynthesis. (B) IGF-1 increased the levels of mitophagy-related proteins (LC-3, p62 and Parkin), indicating that IGF-1 induces mitophagy in sebaceous cells. However, zidovudine treatment decreased Parkin levels and increased the levels of LC-3 and p62, indicating that zidovudine has the effect of inhibiting PINK1/Parkin-mediated mitophagy induced by IGF-1 in sebaceous cells. .

Hereinafter, examples are presented to aid in the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1> Cell culture

Immortalized human sebaceous cells were used in the experiment. Cell lines were established as described in previous studies. All courses were approved by the institutional committee of Chungnam National University Hospital. Immortalized human sebaceous cells were prepared with 10% fetal bovine serum (Gibco BRL, Rockvile, MD, USA) and 5 ng/mL of recombinant human EGF (Invitrogen, Grand Siland, NY, USA) in sebomed medium (biochrom, Berlin, Germany). Supplemented and used for cell culture.

<Example 2> Thin-layer chromatography (TLC)

For the quantitative analysis of intracellular lipids, in the present invention, the TLC method was used as used in the previous study. Briefly, immortalized human sebaceous cells ^{were cultured in a medium containing 2 μCi of 14} C-acetate and sodium salt (PerkinElmer, Boston, MA, USA), and reacted for 4 hours. Intracellular lipids were extracted with chloroform and methanol (2:1, v/v). The solvent was evaporated and the lipid resuspended in chloroform. TLC (TLC silica gel 60 F ₂₅₄ , Merck KgaA) was used to separate intracellular lipids. After development with hexane and ethyl acetate (6:1, v/v), intracellular lipids were visualized by radioactivity assay.

<Example 3> Oil red O staining

Cells were cultured on a cover glass to measure intracellular lipids in immortalized human sebaceous cells. At the end of the treatment, the cells were washed with PBS and fixed with 10% formaldehyde at room temperature for 1 hour. After washing with distilled water, the fixed cells were stained with a filtered oil red O solution for 30-60 minutes. The cells were washed with distilled water and then counter-stained with hematoxylin.

<Example 4> Western blotting

After collection, the cells were lysed with a protein extraction solution (Intron, Daejeon, Korea). The same amount of protein was loaded and separated by SDA-PAGE, and the protein was transferred to a nitrocellulose membrane (Pall Crop, Port Washington, NY, USA). After blocking with 5% skim milk powder, the membrane was reacted with various primary antibodies. The blot was reacted with a peroxidase-conjugated secondary antibody to visualize a specific protein with ECL (BIOMAX, Seoul, Korea). Primary antibodies used for Western blotting were as follows: actin, SREBP-1 (Santa Cruz, CA), SREBP-2, FDFT-1, PGC-1α, mtTFA, p62, Parkin (Abcam, Cambridge, UK ), FASN, PPAR-γ, IGF-1R, phospho-IGF-1R, Akt, phospho-Akt, mTOR, phospho mTOR (Cell Signaling Technology, Dnavers, MA), SCD (Thermo Scientific, Rockford, IL) and LC3 ( Sigma, St. Louis, MO)

<Example 5> Real-time PCR (RT-PCR)

Total RNA was extracted from immortalized human sebaceous cells using Easy-blue RNA extraction kit (Intron, Daejeon, Korea). Total RNA (2 μg) was reverse transcribed into complementary DNA using M-MLV reverse transcriptase (ELPIS Biotech, Daejeon, Korea) according to the manufacturer's instructions. RT-PCR was performed using a SYBR Green real-time PCR master mix (Applied Biosystems, Waltham, MA). The expression level of each gene was normalized to the GAPDH level and expressed as fold change. Primers for RT-PCR are as follows:

FDFT1: 5'-CATTGGGATGGCAGAGTTTT-3' and 5'-TGGTGCAGTGCATTGGTTAT-3'

FASN: 5'-CCCACCTACGTACTGGCCTA-3' and 5'-CTTGGCCTTGGGTGTGTACT-3'

SCD: 5'-CGACGTGGCTTTTTCTTCTC-3' and 5'-GGGGGCTAATGTTCTTGTCA-3'

GAPDH: 5'-CGACCACTTTGTCAAGCTCA-3' and 5'-AGGGGTCTACATGGCAACTG-3'

<Example 6> Statistical analysis

Results are expressed as mean ± standard deviation. Differences between result sets were analyzed by Student's t- test using SPSS (version 24.0; IBM Corp., Armonk, NY, USA). Statistical significance was P<0.05.

<Test Example 1> Zidovudine reduced intracellular lipid levels in immortalized human sebaceous cells.

The effect of zidovudine on the lipid synthesis induced by IGF-1 was measured using the immortalized human sebaceous cell line established in the previous study. Immortalized human sebaceous cells were treated with zidovudine 10 or 25 μM. After treatment with zidovudine for 1 hour, 50 ng/mL of IGF-1 was treated to sebum cells to induce differentiation and lipid synthesis. From the results of TLC performed 48 hours after IGF-1 treatment, it was confirmed that IGF-1 treatment increased lipid accumulation in immortalized human sebaceous cells, and zidovudine dose-dependently reduced intracellular lipid accumulation (Fig. 1A). . IGF-1 increased the synthesis of intracellular lipids such as cholesterol, triglycerides, wax esters and squalene in immortalized human sebaceous cells. On the other hand, zidovudine treatment reduced IGF-1-induced lipid synthesis. Among the various lipids, the synthesis of squalene and wax esters was significantly reduced by zidovudine. Oil Red O staining confirmed that IGF-1 increased the number of lipid droplets in immortalized human sebaceous cells, and zidovudine treatment reduced the number of lipid droplets in immortalized human sebaceous cells (Fig. 1B).

<Test Example 2> Zidovudine reduced IGF-1-induced lipid synthesis in immortalized human sebaceous cells.

In order to investigate the effect of zidovudine on IGF-1-induced lipid synthesis in immortalized human sebaceous cells, transcription factors and enzymes related to sebaceous cell differentiation and lipid synthesis were investigated. In the group treated with IGF-1 on immortalized human sebaceous cells, the levels of various transcription factors related to sebaceous cell differentiation such as SREBP-1, SREBP-2 and PPAR γ were increased, but zidovudine dose-dependently decreased their levels. (Figure 2A).

The expression of enzymes including FDFT1 for squalene synthesis, FASN and SCD for fatty acid synthesis was examined to see if the result of the transcription factor affects the expression of enzymes related to lipid synthesis. As expected, zidovudine dose-dependently downregulated FDFT1, FASN and SCD increased by IGF-1 treatment (Figure 2B). Moreover, the mRNA expression increased by IGF-1 of these enzymes was downregulated after zidovudine treatment (Figure 2C). These results indicate that zidovudine reduces the lipid synthesis of sebaceous cells induced by IGF-1 through sebaceous cell differentiation and inhibition of lipid synthesis.

In a previous study, as the present inventors reported the role of the Akt/mTOR signaling pathway in sebaceous cells in IGF-1-induced adipose synthesis, we examined whether zidovudine regulates the Akt/mTOR signaling pathway. In the present invention, IGF-1 increased the phosphorylation of IGF-1R and sub-effect factors, Akt and mTOR (Fig. 3). On the other hand, zidovudine significantly dose-dependently reduced the phosphorylation of IGF-1R, Akt and mTOR.

<Test Example 3> Zidovudine reduces mitochondria biogenesis and mitophagy induced by IGF-1.

Since it was known that zidovudine induces mitochondrial dysfunction in adipocytes and reduces autophagy, in the present invention, the role of IGF-1 and zidovudine in mitochondrial biosynthesis and mitophagy in immortalized human sebaceous cells was studied.

As expected, IGF-1 increased PGC-1α, a major mitochondrial biosynthesis regulator (Figure 4A). In addition, the expression of mtTFA, a key regulator of mitochondrial DNA transcription, was upregulated through IGF-1 treatment. On the other hand, zidovudine decreased the expression of PGC-1α and mtTFA in immortalized human sebaceous cells. In order to evaluate mitophagy in immortalized human sebaceous cells, the levels of mitophagy-related proteins were examined, and it was found that IGF-1 increased the expression of LC-3, p62 and Parkin (Fig. 4B). However, zidovudine decreased Parkin while increasing LC-3 and p62 levels, indicating impaired phytophagy regulation of zidovudine in IGF-1 treated immortalized human sebaceous cells, and zidovudine from sebaceous cells to IGF-1. This indicates that there is an effect of inhibiting the induced PINK1/Parkin-mediated mitophagy. The above results indicate that zidovudine inhibits IGF-1-induced mitochondrial biosynthesis and mitophagy in sebaceous cells.

Claims (12)

A pharmaceutical composition for the prevention and treatment of acne diseases containing zidovudine as an active ingredient. The pharmaceutical composition according to claim 1, wherein the acne disease is selected from the group consisting of excessive proliferation of hair follicles, excessive sebum secretion, presence of acne bacteria, signs or symptoms of inflammation. The pharmaceutical composition according to claim 2, wherein the acne disease is caused by excessive sebum secretion. The pharmaceutical composition of claim 1, wherein the zidovudine reduces lipid synthesis or inhibits a signaling system related to lipid synthesis in sebaceous cells. The pharmaceutical composition according to claim 1, wherein the zidovudine decreases the expression of lipid synthesis-related electron factors or decreases the expression of lipid synthesis-related enzymes in sebaceous cells. The pharmaceutical composition of claim 1, wherein the zidovudine reduces mitochondrial biosynthesis by inhibiting mitophagy in sebaceous cells. The pharmaceutical composition of claim 4, wherein the lipid synthesis-related signal transduction is selected from the group consisting of IGF-1R/mTOR, p-IGF-1R/mTOR, and IGF-1R/p-mTOR. The method of claim 5, wherein the lipid synthesis-related electron factors are sterol regulatory element-binding protein-1 (SPEBP-1), sterol regulatory element-binding protein-2 (sterol regulatory element-binding protein- 2, SPEBP-2), Peroxisome proliferator-activated receptor gamma (PPAR-γ), p-SPEBP-1, p-SPEBP-2, n-SPEBP-2, n-SPEBP- Pharmaceutical composition, characterized in that selected from the group consisting of 1. The pharmaceutical composition according to claim 5, wherein the enzyme involved in lipid synthesis is selected from the group consisting of FDFT-1, FASN, SCD, p21, ACC, and FADS2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a formulation selected from the group consisting of ointments, powders, tablets, capsules, powders, solutions, gels, pastes, patches and granules. A cosmetic composition for reducing sebum and improving acne containing zidovudine as an active ingredient. The cosmetic composition of claim 11, wherein the cosmetic composition has a formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a powder, a soap, a surfactant-containing cleansing, an oil and a spray.

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