WO2020052629A1

WO2020052629A1 - Application of p300 activator ctpb and derivative thereof in improving collagen col17a1 expression

Info

Publication number: WO2020052629A1
Application number: PCT/CN2019/105587
Authority: WO
Inventors: 苏锦熙
Original assignee: 潘治忠
Priority date: 2018-09-13
Filing date: 2019-09-12
Publication date: 2020-03-19
Also published as: CN109045001A

Abstract

An application of a p300 activator CTPB and a preparation thereof in preparing a hair restorer and/or a hair loss treatment agent and for preparing a medication for treating hair loss/promoting hair restoration.

Description

Use of p300 activator CTPB and its derivative in increasing collagen Col17A1 expression

Technical field

The present invention relates to the field of pharmaceuticals and toiletries, and more specifically, the present invention discloses the use of a CTPB activator.

Background technique

Hair follicle stem cells promote hair follicle regeneration and hair growth, and also repair damaged epidermis. The differentiation of hair follicle stem cells is regulated by a variety of pseudogenes, including enhancers and the marker histone H3 (H3K27Ac). Enhancers are short DNA sequences that regulate the expression of a target gene. When the function of the enhancer is affected, it can lead to various diseases, such as autoimmune diseases and cancer. At present, there are different methods to identify enhancers, such as ATAC-seq and immunoprecipitation combined with high-throughput sequencing chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), which can find out how a fixed target is connected in the genome Such as histone markers, transcription factors or co-acting factors. We can find the enhancers in a certain tissue by performing the active histone labeling H3K27ac or acetyltransferase p300 ChIP-seq, and ChIP-seq integrating multiple transcription factors. But generally speaking, there are often thousands of enhancers identified through the whole genome, which makes it extremely difficult to identify true functional enhancers.

Therefore, we need a more accurate method to find functional enhancers. In recent years, Super-enhancer (SE) has attracted much attention. Super enhancers are a unique group of active enhancers. They are dense enhancers and have a particularly strong active histone marker H3K27ac. Studies have shown that SE has important functions for stem cell activation and differentiation. Generally speaking, there are only hundreds of SEs in the cell, which greatly increases the accuracy of identifying functional enhancers. So far, SE has been identified in a variety of stem cells, including superenhancers in hair stem cells. In terms of biotechnology development, SE is an excellent target for epigenetic regulation of cell function. Various drugs have been tested in clinical trials, and their purpose is to target activated SE in cancer cells to treat cancer.

During the aging process, most organs will become smaller, and at the same time, their DNA will be damaged, resulting in reduced function and regeneration capacity. Studies have found that during the aging process of mouse and human hair follicle stem cells, neutrophil DNA will also be damaged, and the protease neutrophil elastase will be released, thereby digesting collagen Col17A1 outside the hair follicle stem cells. The digestion of Col17A1 will cause the regeneration function of hair follicle stem cells to decline and cause hair loss. Studies have also shown that reducing the expression of Col17A1 induces alopecia, and that over-expressing Col17A1 in aging mice can prevent hair loss. Therefore, being able to find the regulatory mechanism of the Col17A1 gene is very important to treat the phenomenon of aging in human aging.

Summary of the Invention

The first object of the present invention is to provide a use of a p300 activator CTPB to increase the expression of collagen Col17A1, which is completed based on the following research by the inventors: by analyzing a variety of super-enhancers in cells, including immune cells , Cerebellum, cerebral cortex, heart, liver, spleen, thyroid, intestine, and hair follicle stem cells, the inventors found that the super enhancer that regulates the Col17A1 gene is a super enhancer specific to hair follicle stem cells, so it can be a target to observe How various compounds and pseudogenetic mechanisms regulate the expression of Col17A1 gene. The inventors found that CTPB increased the expression of Col17A1 in a concentration gradient in fibroblasts, indicating that the apparent regulatory protein p300 is closely related to the differentiation of fibroblasts into hair follicle cells. Hair loss began to occur in 20-month-old C57 mice, and was applied to the skin with p300 activator CTPB (apply daily at a concentration of 200 nM in PEG-400 / ethanol / water at a ratio of 40% / 20% / 40%), mice weighed 35g / one, and treated for 14 days), compared with the control group (PEG-400 / ethanol / water only, the ratio was 40% / 20% / 40%), CTPB could increase Col17A1 Express and improve dissection. The above findings contributed to the present invention.

The terms of the present invention have the following meanings:

Enhancers / super-enhancers, hundreds of super-enhancers control most of the key genes that confer properties and functions to each cell. Changes in cell identity may begin and end with these superenhancers, which are very powerful, but also extremely sensitive to environmental changes.

p300 is a member of the p300-CBP transcription coactivator family. It is involved in the transcriptional regulation of multiple transcription factors in different physiological processes, and has attracted much attention. Although P300 and CBP are encoded by different genes, their structures and functions are very similar, so p300 and CBP are classified into the same protein family. With a molecular weight of 300 kDa, p300 has a fairly high homology to CBP and is highly conserved among mammals. There are many functions of p300, including various physiological processes such as cell cycle regulation and cell differentiation. As a histone acetyltransferase (HAT), p300 can acetylate four core histones. p300 catalyzes the acetyl group in acetyl-CoA and transfers it to the ε-amino group of the N-terminal lysine residue of histone, neutralizing the positive charge above and weakening the interaction between DNA and histone, thereby opening The highly ordered chromatin structure facilitates the binding of transcription factors and RNA polymerase II to DNA, thereby facilitating transcription.

CTPB, as a small molecule compound, is an activator of the p300-CBP protein family and acts on p300. ECM, extracellular matrix; Fibroblast, fibroblasts are cells that synthesize extracellular matrix and collagen, and play an important role in animal wound healing.

The first object of the present invention is the use of the p300 activator CTPB to increase the expression of collagen Col17A1;

Preferably, the use of the p300 activator CTPB in the preparation of a drug for treating hair loss;

Preferably, the use of the p300 activator CTPB in the preparation of a medicament for promoting hair hyperplasia;

Preferably, the molecular formula of CTPB of p300 activator is

Another object of the present invention is to disclose a preparation which is a solution containing CTPB, PEG-400, ethanol, and water, wherein the concentration of CTPB and its derivatives is 50 nM-4 μM.

Preferably, the concentration of CTPB and its derivatives in the above formulation is 50 nM-4 μM.

A final object of the present invention is the use of the p300 activator CTPB in the preparation of hair restorers and / or treatments for hair loss.

The invention has at least the following advantages:

CTPB is a p300 protein activator in the prior art, which can promote the proliferation of nerve cells to improve Parkinson's disease (PD). The inventors of the present invention have found that CT300, a p300 activator, can increase the expression of collagen Col17A1. When hair follicle stem cells age, collagen Col17A1 is digested by proteases, which promotes hair follicle stem cells to apoptosis, and at the same time enhances the inflammatory response, which eventually leads to hair loss. Histone acetylation is an important part of epigenetic research. P300 regulates the transcriptional expression of genes by binding with acetylated lysine, and is a driving factor for hair follicle stem cell differentiation, while the small molecule compound CTPB is its activator. The present invention With p300 as a target, CTPB and its derivatives, a small molecule compound for treating hair loss or promoting hair hyperplasia, are provided, which can be used for the early prevention and treatment of the disease. At the same time, it can also be used as a hair growth agent or hair loss treatment in shampoo products.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The results of the action of the super enhancer related to the Col17A1 gene on hair follicle stem cells, indicating that the super enhancer of COL17A1 is found only in hair follicle stem cells;

Figure 2 Example 1 qRT-PCR test results of the expression level of the Col17A1 gene in the skin of young and aging mice;

FIG. 3 is a comparison diagram of CTPB applied to the skin for treatment of hair loss or promotion of hair growth in Example 2;

Figure 4 Example 1 qRT-PCR test results of CTPB on Col17A1 expression level in fibroblast 10T 1/2;

Fig. 5 Example 2 qRT-PCR results of CTPB on Col17A1 expression level in skin of C57 mice at 20 months.

detailed description

The following further describes the technical solution of the present invention through specific embodiments. It should be understood that the following is only an exemplary description of the present invention and is not intended to limit the protection scope of the claims of the present invention.

The mouse in the test example of the present invention is a C57BL / 6J mouse, which is from The Jackson Laboratory in the United States. Other raw materials are commercially available unless otherwise specified.

Example 1: The effect of CTPB (Santa Cruz, USA) on the expression of Col17A gene in fibroblast 10T1 / 2 was investigated.

Tsinghua University's Bioinformatics Group released a super enhancer database (http://asntech.org/dbsuper/index.php), using the Detailed Search function, the parameters are as follows: mouse (mm9), select all cell / tissue type , Gene name Col17A1. We found that the superenhancer that regulates the Col17A1 gene is specific to hair follicle stem cells. This superenhancer has no activity in the other twenty-two cells. The results are shown in Figure 1. Because hair follicle cells can also be differentiated from fibroblasts, we used fibroblasts to study the effect of CTPB on the expression of the Col17A1 gene. In addition, the expression level of Col17A1 gene also decreased during the skin aging process in mice. The results are shown in Figure 2.

In addition, a C57BL / 6J mouse experimental model was used to extract 1 cm2 of skin tissue from six- and twenty-month-old mice, and then RNA was extracted using Trizol (Life Technolodies, USA), and Superscript IV Reverse Transcriptase ( Life (Technologies) for reverse transcription, and finally qRT-PCR with SYBR Green to detect the expression level of Col17A1 gene, and use GAPDH expression as an internal reference. The expression level of Col17A1 gene also decreases during skin aging in mice. The results are shown in Figure 2.

10T 1/2 cells (CCL-226, ATCC, USA), a batch of cells inoculated into 6-well plates and treated with CTPB (0nM-500nM) separately were used to quantitatively detect Col17A1 by qRT-PCR (GAPDH is a universal internal reference, not a test 200nM CTPB increases the expression of Col17A1, and the effect of CTPB is positively related to its increase in Col17A1, indicating that CTPB plays an important role in 10T1 / 2 cells. The reason for choosing CTPB for hair loss is super enhancement of p300 and Col17A1. Son is the target of treatment. GAPDH is widely used as an internal reference for qRT-PCR cDNA standardization. The target cDNA content of each sample is divided by the GAPDH content of this sample to obtain the relative content of the target cDNA of each sample. Compare. The same results are shown in Figure 4. The qRT-PCR results showed that CTPB at 200nM and higher concentrations significantly increased the expression of Col17A1 after 16 hours, while CTPB increased the expression of Col17A1 in a gradient-dependent manner, of which 200nM inhibited JQ1. It is sufficiently significant that its role in activating p300 is positively correlated with Col17A1 expression, confirming the role of epigenetic regulatory protein p300 in 10T1 / 2 cells.

Example 2: The effect of exogenously added CTPB on treating hair loss or promoting hair hyperplasia was studied.

1. The establishment of mouse hair loss model and the effect of CTPB on hair regeneration.

Six senile (20 months) C57BL / 6 mice were randomly divided into the control group and the experimental group, and 100 μl of CTPB (concentration 200nM, dissolved in PEG-400 / ethanol / water ratio 40%) were applied to the skin every day. / 20% / 40%), and the mouse weight was 35g / head) as the treatment group (PEG-400 / ethanol / water, the ratio was 40% / 20% / 40% as the control group). After treatment, photographs were taken to observe the treatment of hair loss. The results are shown in Fig. 3. As shown in Fig. 3, compared with the 20-month-old mice, the 20-month-old mice showed significant hair loss. The CTPB 14-day treatment group had significantly improved hair loss compared with the control group.

2. Effect of CTPB on the expression of Col17A1 in mouse hair removal model

The skin of the treated C57BL / 6 mice was taken, RNA was extracted with Trizol (Life Technologies, USA), and then reverse transcription was performed with Superscript IV Reverse Transcriptase (Life Technologies). Finally, the expression level of Col17A1 was detected by qRT-PCR with SYBR Green Using GAPDH expression as an internal reference, the results are shown in Figure 5 and show that the expression of Col17A1 increased after CTPB was applied.

The above is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made. These improvements and retouches also It should be regarded as the protection scope of the present invention.

Claims

The use of p300 activator CTPB to increase the expression of collagen Col17A1.
The use according to claim 1, characterized in that the use of p300 activator CTPB in the preparation of a medicine for treating hair loss.
The use according to claim 1, characterized in that the use of the p300 activator CTPB in the preparation of a medicament for promoting hair hyperplasia.
The use according to any one of claims 1 to 3, wherein the molecular formula of the CT300 p300 activator is:
A preparation is a solution containing p300 activator CTPB, PEG-400, ethanol, and water.
The formulation of claim 5 wherein the p300 activator CTPB concentration is 50 nM-4 μM.
Use of the preparation according to claim 5 or 6 to increase the expression of collagen Col17A1.
Use of the preparation according to claim 5 or 6 in the preparation of a medicine for treating hair loss.
Use of the preparation according to claim 5 or 6 in the preparation of a medicament for promoting hair growth.
The use according to claim 1, characterized in that the use of p300 activator CTPB in the preparation of hair restorer and / or treatment of hair loss.