KR20200056363A - Pharmaceutical composition for preventing hair loss and promoting hair growth comprising isolated mitochondria - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing hair loss and promoting hair growth, comprising isolated mitochondria as an active component. The pharmaceutical composition comprising isolated mitochondria according to the present invention as an active component has an effect of promoting hair growth. Therefore, the pharmaceutical composition is expected to be highly useful as a therapeutic agent for preventing hair loss or promoting hair growth using the same.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTING HAIR LOSS AND PROMOTING HAIR GROWTH COMPRISING ISOLATED MITOCHONDRIA}

The present invention relates to a pharmaceutical composition for preventing hair loss and promoting hair growth, comprising isolated mitochondria as an active ingredient.

Hair loss refers to the condition where there is no hair where the hair should normally be. The human body has about 100,000 to 150,000 hairs, and each hair has a different cycle and grows and falls out through a growth phase, a degenerative phase, and a rest phase. As a result, an average of 50 to 100 hairs per day is normally removed. During these cycles, the growth phase of the hair becomes shorter, and the degenerative or resting period becomes longer, resulting in an increase in the number of hairs that fall off abnormally, resulting in hair loss.

Genetic factors have been known to be the main cause of hair loss, but recently, environmental factors such as environmental pollution, nutritional imbalance due to dietary changes, and hormone secretion, social stress, and the use of indiscriminate chemical agents have also emerged as the cause of hair loss. . Due to these environmental factors, hair loss is also increasing in young men and women in their 20s and 30s, and various studies have been attempted to overcome hair loss as the demand for prevention and treatment increases. As a result of research, dihydrotestosterone theory (DHT), gravity theory, and blood supply theory have been known as general hypotheses for hair loss, but the exact causes and mechanisms of hair loss and hair growth are still unknown. not.

Materials that have been approved by the U.S. FDA and used for the treatment or prevention of hair loss include minoxidil of U.S. Patent No. 3,382,247 and finasteride of Merck mentioned in U.S. Patent No. 5,215,894. However, in the case of minoxidil, side effects that cause sticky feeling and irritation to the skin have been reported. In addition, finasteride is currently used as a formulation for oral administration. However, side effects such as sexual dysfunction have been reported depending on its intake, and there is a side effect that it is effective only when a steady dose is taken for hair loss.

As such, currently used hair loss-related preparations have various problems such as insufficient effect or side effects. Therefore, there is a need to develop a novel agent for preventing or treating hair loss more effectively and safely, and for promoting hair growth or hair growth.

U.S. Patent No. 3,382,247 U.S. Patent No. 5,215,894

Accordingly, the present inventors completed the present invention by confirming that hair growth is promoted when a composition containing mitochondria as an active ingredient is administered to a hair removal site while researching a new material that can replace the existing hair loss-related preparation.

An object of the present invention is to provide a pharmaceutical composition for promoting hair growth comprising isolated mitochondria.

In order to achieve the above object, there is provided a pharmaceutical composition for promoting hair growth comprising isolated mitochondria as an active ingredient.

In addition, it provides a pharmaceutical composition for preventing or treating hair loss comprising the isolated mitochondria as an active ingredient.

The pharmaceutical composition comprising the isolated mitochondria according to the present invention as an active ingredient has the effect of promoting hair growth. Therefore, it is expected to be highly useful as a therapeutic agent for preventing hair loss or promoting hair growth using the same.

1A and 1B show mitochondria labeled with a mitochondrial specific marker (mitotracker green) in mesenchymal stem cells derived from human umbilical cords using a bright-field microscope and a fluorescence microscope.
2A and 2B are observations of mitochondria isolated from human umbilical cord-derived mesenchymal stem cells using a general microscope and a fluorescent microscope.
Figure 3 is observed in 0, 1 and 3 weeks after administration of mitochondria derived from human umbilical cord mesenchymal stem cells to C57BL / 6 mice with hair all over the back of the hair.
FIG. 4A shows the hair length by collecting 10 to 20 hairs from the administered site 2 weeks after administration of human umbilical cord mesenchymal stem cell-derived mitochondria derived from C57BL / 6 mice with hairs all over the back. .
4B and 4C show that after 3 weeks of administration of human umbilical cord mesenchymal stem cell-derived mitochondria to C57BL / 6 mice with hairs all over the back, hairs of 10 to 20 hairs were collected and the length of the hair was collected. It was observed.
5 is 3 weeks after administration of human umbilical cord mesenchymal stem cell-derived mitochondria to C57BL / 6 mice with hairs all over the back, and the density of the hairs at the administered site is analyzed.

In one aspect, the present invention provides a pharmaceutical composition for promoting hair growth, comprising isolated mitochondria as an active ingredient.

As used herein, the term “isolated mitochondria” may be isolated from the same individual to which the mitochondria is administered, but may be isolated from other individuals. At this time, the isolated mitochondria may be obtained from mammals, preferably from humans.

The mitochondria may be obtained from any one selected from the group consisting of stem cells, somatic cells, germ cells, or platelets.

As used herein, the term "stem cell" refers to undifferentiated cells having the ability to differentiate into various types of tissue cells. The stem cells may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, and tissue-derived stem cells. At this time, the mesenchymal stem cells may be any one selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta. In one embodiment of the present invention, mesenchymal stem cells derived from human umbilical cord were used.

In addition, the somatic cell may be one selected from the group consisting of muscle cells, hepatocytes, fibroblasts, epithelial cells, neurons, adipocytes, bone cells, white blood cells, lymphocytes, and mucosal cells. Preferably, it may be obtained from muscle cells or hepatocytes having excellent mitochondrial activity, and may be obtained from autologous or other blood PBMC cells.

Further, the mitochondria may be normal mitochondria obtained from cells in which the biological activity of mitochondria is normal. In addition, the mitochondria may be obtained from autologous or other cells cultured in vitro.

In addition, the mitochondria may be obtained from autologous or other germ cells. The germ cells may be sperm or egg.

In addition, the mitochondria may be obtained from autologous or other platelets.

Meanwhile, when the mitochondria are separated from specific cells, the mitochondria can be separated through various known methods, for example, using a specific buffer solution or using a potential difference and a magnetic field.

The mitochondrial separation can be obtained by crushing and centrifuging the cells in terms of maintaining mitochondrial activity. In one embodiment, the cells are cultured, and the pharmaceutical composition containing these cells is firstly centrifuged to produce pellets, the pellets are resuspended in a buffer solution, homogenized, and the homogenized solution is prepared. It may be performed as a step of preparing a supernatant by secondary centrifugation and purifying the mitochondria by performing a third centrifugation of the supernatant. At this time, it is preferable in terms of maintaining cell activity that the time for the second centrifugation is performed is shorter than the time for the first and third centrifugation, and the third centrifugation in the first centrifugation. You can increase the speed as you go.

Specifically, the first to third centrifugation may be performed at a temperature of 0 to 10 ° C, preferably a temperature of 3 to 5 ° C. In addition, the time at which the centrifugation is performed may be performed for 1 to 50 minutes, and may be appropriately adjusted according to the number of centrifugation and the content of the sample.

In addition, the first centrifugation may be performed at a rate of 100 to 1,000 × g, or 200 to 700 × g, or 300 to 450 × g. In addition, the second centrifugation may be performed at a rate of 1 to 2,000 × g, or 25 to 1,800 × g, or 500 to 1,600 × g. In addition, the third centrifugation may be performed at a rate of 100 to 20,000 × g, or 500 to 18,000 × g, or 800 to 15,000 × g.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating hair loss comprising the isolated mitochondria as an active ingredient. As used in the present invention, the term "hair loss" refers to a state in which there is no hair in a region where hair should normally be present, and generally means that the hair of the scalp falls out.

For the pharmaceutical composition according to the present invention, mitochondria may be included at a concentration of 0.1 to 500 μg / ml, 0.2 to 450 μg / ml or 0.5 to 400 μg / ml.

Meanwhile, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers may be those commonly used in pharmaceutical preparation. The pharmaceutically acceptable carrier may include a sterilized aqueous solution (eg, physiological saline), a non-aqueous solvent, and the like. In addition, if necessary, an appropriate stabilizer, an adjuvant (eg, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.), tonicity agents, preservatives, and the like may be included, but are not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent as an additive.

The carrier may be included from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight based on the total weight of the pharmaceutical composition of the present invention.

In addition, the present invention provides a method for preventing or treating hair loss comprising administering the pharmaceutical composition to an individual. The administration may be administered directly to the affected area via an injection, preferably subcutaneous injection.

The pharmaceutical composition according to the present invention is to be prepared as a physically or chemically very stable injection by adjusting the pH using a buffer solution such as an acidic solution or phosphate, which can be used as an injection, in order to secure product stability according to the distribution of the injection formulation. Can be. The injection may further include a preservative, a painless agent, a solubilizer or a stabilizer.

Specifically, the pharmaceutical composition of the present invention may include water for injection. The water for injection is distilled water made to dissolve a solid injection or to dilute a water-soluble injection, glucose injection, xylitol injection, D-mannitol injection, fructose injection, physiological saline, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactic acid-ringer's solution, or a phosphate buffer solution having a pH ranging from 3.5 to 7.5, or a sodium dihydrogen phosphate-citric acid buffer solution.

The method of administration of the pharmaceutical composition is determined according to the degree of symptoms of hair loss, and a topical administration method is usually preferred. In addition, the dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the degree of disease, the patient's age, sex, and weight, and may be administered once to several times a day.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to them.

Preparation Example 1. Cell preparation

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were obtained and used by Professor Choi Yong-soo of the College of Medicine and Medicine. The cryopreserved cell lines were stored in a 37 ° C. bath until completely thawed. It was then used immediately for mitochondrial delivery. UC-MSCs containing 10% fetal bovine serum (FBS; Hyclone), 100 μg / ml streptomycin and 100 IU / ml penicillin (P / S; Hyclone), and 10 ng / ml basic fibroblasts It was cultured in α-MEM medium (Hyclone Laboratories Inc., Logan, UT) containing growth factors (CHA Meditech, Daejeon, Korea). When cell proliferation reached 80% to 90%, the UC-MSCs in steps # 5 to # 7 were used.

Example 1. Mitochondrial identification and separation

The human umbilical cord mesenchymal stem cells cultured in Preparation Example 1 were stained with a greenish mitochondrial specific marker (Mitotracker Green). In order to confirm whether the green fluorescence was well labeled in the mitochondria in the cell, it was observed using a fluorescence microscope. In order to extract mitochondria, cells were measured using a hemocytometer to recover cells of about 2 × 10 ⁷ cells / ml.

Thereafter, the cell line was subjected to the first centrifugation at a rate of 350 × g for 10 minutes at a temperature of about 4 ° C. At this time, the obtained pellet was recovered, and SHE buffer solution (0.25 M sucrose, 20 mM HEPES (pH 7.4), 2 mM EGFTA, 10 mM KCl, 1.5 mM MgCl ₂ and 0.1%) containing a protease inhibitor (Roche Diagnostics, Mannheim, Germany) BSA (Defatted Bovine Serum Albumin) was homogenized by resuspension using a disposable 1 ml syringe (Syringe).

The composition containing the pellet was subjected to a second centrifugation at a temperature of about 4 ° C. for 3 minutes at a rate of 1,100 × g to obtain a supernatant. Thereafter, the supernatant was centrifuged at a rate of 12,000 × g for 15 minutes at a temperature of about 4 ° C. to separate the mitochondria from the cell line. To identify the separated mitochondria, bright-field microscopes and mitochondria in cells labeled with green fluorescence before separation were taken with a fluorescence microscope, and are shown in FIGS. 1A and 1B, and the separated mitochondria are normal microscopes. And photographed with a fluorescence microscope, and shown in FIGS. 2A and 2B.

As shown in Figures 1a to 2b, it was confirmed that the mitochondria present in the mesenchymal stem cells derived from human umbilical cord were isolated before separation.

Example 2. Hair loss animal model production and administration of mitochondria

Male C57BL / 6 mice 5 to 8 weeks old were purchased from Orient Co., Ltd., Gyeonggi-do, Korea. The purchased mice were subjected to an adaptation period in a clean area and then tested. During the adaptation period, the environment in which the mice lived was formed day and night at 12-hour intervals, and the room temperature of 23 ± 2 ° C. and humidity of 40 to 60% were maintained. After the adjustment period of 7 days, it was put into the experiment.

The thus prepared mouse was applied with 1 g of a wax and cream formulation hair removal agent (Beauty Formulas, United Kingdom) to the entire back of the back to epilate the entire back of the back, and then the mitochondria were administered in a horizontal × vertical size (1 cm × 1 cm). The area to be marked was marked with a square. Experimental groups were prepared by administering 2 µg and 20 µg of the mitochondria isolated according to Example 1 to the labeled regions, respectively, on the upper and lower portions of the labeled regions, and the untreated mice were prepared as controls. From the time when the mitochondria was administered, visual observation and photography were analyzed in 0, 1, and 3 weeks. The results are shown in FIG. 3.

Example 3. Evaluation of hair length and density of mitochondrial-treated mice

In order to evaluate the hair length and density of the mice prepared according to Example 2, 10 to 20 hairs were randomly collected using square tweezers for the hairs of the mitochondrial-administered marker region to analyze length measurement and photography. Did. The results are shown in FIGS. 4A to 4C. In addition, in Example 2, the hair density of mitochondrial-administered mice and untreated mice was analyzed. The results are shown in FIG. 5.

4A to 4C, the hair length of the group administered with the mitochondria derived from human umbilical cord stem cells was observed to be longer than that of the untreated group. In addition, as shown in FIG. 5, the hair density of the group administered with the mitochondria derived from human umbilical cord stem cells was higher than that of the untreated group.

Claims (4)

A pharmaceutical composition for subcutaneous injection for promoting hair growth comprising mitochondria isolated from somatic cells as an active ingredient. According to claim 1,
The somatic cell is characterized in that any one selected from the group consisting of muscle cells, hepatocytes, fibroblasts, epithelial cells, neurons, adipocytes, bone cells, white blood cells, lymphocytes and mucosal cells. A pharmaceutical composition for subcutaneous injection for the prevention or treatment of hair loss comprising mitochondria isolated from somatic cells as an active ingredient. According to claim 3,
The somatic cell is characterized in that any one selected from the group consisting of muscle cells, hepatocytes, fibroblasts, epithelial cells, neurons, adipocytes, bone cells, white blood cells, lymphocytes and mucosal cells.

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