WO2009115007A1

WO2009115007A1 - Use of tat-n25 polypeptide in treating dermatosis relative to abnormal growth of cell and psoriasis

Info

Publication number: WO2009115007A1
Application number: PCT/CN2009/070653
Authority: WO
Inventors: 夏献民
Original assignee: Xia Xianmin
Priority date: 2008-03-21
Filing date: 2009-03-05
Publication date: 2009-09-24
Also published as: CN101244258B; CN101244258A

Abstract

Use of TAT-N25 polypeptide in treating dermatosis relative to abnormal growth of cell and psoriasis is provided.

Description

Use of TAT-N25 polypeptide in the treatment of abnormal cell growth diseases such as psoriasis

The invention relates to a use of a fusion polypeptide in the treatment of abnormal cell growth diseases such as psoriasis, and belongs to the field of medical bioengineering. Background technique

Psoriasis, commonly known as "psoriasis", is one of the most common and refractory skin diseases in dermatology. According to the clinical and pathological features of psoriasis, it can be generally divided into six types: vulgaris, arthritis, pustular, palmoplantar pustulosis, erythrodermic, and continuous acral dermatitis. Because the disease is stubborn and difficult to treat, it is listed as an important research topic in the field of dermatology in the world today.

The etiology of psoriasis is unknown. The main pathological features are abnormal proliferation and parakeratosis of epidermal cells. The specific pathological features are as follows: 1. Excessive proliferation and differentiation of epidermal keratinocytes; 2. Microvascular dilation of the dermis; 3. Inflammation reaction.

Clinically, many cytotoxic anticancer drugs such as white blood, ethylenimine, bismorpholine, cyclophosphamide, dexamethasone, prednisone, betamethasone and other hormonal drugs, immunosuppressants and epidermal cells The differentiation promoting agent is treated with retinoic acid and the like. Because the above drugs and various preparations thereof can only act on a single pathological change of psoriasis and have serious damage to the liver, kidney and immune function of the body; some patients have certain short-term effects after use, but stop the drug. After the disease rebounded to aggravate the worsening side effects, many patients have been deeply affected.

An effective method of curing or comprehensively improving the condition of psoriasis is highly desirable. This method should ideally control abnormal proliferation and parakeratosis of epidermal cells and inhibit inflammation in the patient's area. The fusion polypeptides described in the present invention satisfy this need and have the above advantages. Summary of the invention

The object of the present invention is to provide a fusion polypeptide comprising: a polypeptide which inhibits cell growth, promotes cell differentiation, a polypeptide or a molecule which penetrates a cell membrane, and a polypeptide which penetrates a cell membrane. Or the molecule carries a polypeptide that inhibits cell growth into the cell. It has been proved by experiments that this fusion polypeptide has the ability to inhibit epidermal cell growth and promote epidermal cell differentiation. In addition, the fusion polypeptide can effectively inhibit the pathological changes of patients with psoriasis and achieve significant therapeutic effects.

The fusion polypeptide has been disclosed in patent application 200710051286.6, and the fusion polypeptide comprises: a polypeptide that inhibits cell growth, promotes cell differentiation, and a polypeptide or molecule that penetrates the cell membrane, and the polypeptide that inhibits cell growth and the polypeptide or molecule that penetrates the cell membrane are chemically linked. The polypeptide that inhibits cell growth and the polypeptide that penetrates the cell membrane are expressed in the cell as a fusion polypeptide.

The polypeptide that inhibits cell growth of the fusion polypeptide is linked to a tagged protein that purifies the fusion polypeptide. The polypeptide which inhibits cell growth and promotes cell differentiation is a Ν-25 polypeptide consisting of 25 amino acid residues at the amino terminus of the phosphatidylinositol-3-kinase regulatory subunit p55PIK, the sequence of which is: methionine-aspartate Acid-arginine-aspartate-aspartate-alanine-aspartate-tryptophan-arginine-glutamate-valine-methionine-methionine -proline-tyrosine-serine-threonine-glutamate-leucine-isoleucine-phenylalanine-tyrosine-isoleucine-glutamate-methionine (MDRDDADWREVMMPYSTELIFYIEM), or one of the polypeptides obtained by consecutively or intermittently deleting or substituting any number of amino acid residues in 1-12 from the amino terminus of the polypeptide, or passing the sequence of the polypeptide through one or several amino acid residues One of the polypeptides having the activity of inhibiting cell growth obtained by the addition.

The polypeptide that penetrates the cell membrane is a polypeptide derived from HIV virus prion protein, and its sequence is: tyrosine-glycine-arginine-lysine-lysine-arginine-arginine-glutamine-spermine Acid-arginine-arginine (YGRKKRRQRRR).

The role of the ΤΑΤ polypeptide in the ΤΑΤ~Ν25 fusion polypeptide is to help the Ν25 polypeptide enter the cell and exert the pharmacological effect of Ν25. In addition to sputum, other polypeptides are known, such as the TDT polypeptide from X protein, which has a sequence of xxxxx and similar functions to help the polypeptide penetrate the cell membrane. Other molecules such as polylysine, some liposomes can also help N25 or other peptides penetrate the cell membrane. I'm expecting the above molecules and Ν24 to form new compounds by covalent or non-covalent associations should also have similar efficacy to ΤΑΤ-Ν25.

The tagged protein used to purify the fusion polypeptide is an oligohistidine tag in which the number of histidines is Any number from 5-10.

The use of a N25 polypeptide that inhibits cell growth and a TAT polypeptide or molecule that penetrates the cell membrane by chemical covalent linkage to a fusion polypeptide (TAT-N25 polypeptide) for the treatment of diseases caused by psoriasis and other abnormalities of skin cell proliferation and differentiation.

A TAT-N25 polypeptide drug for treating dysplasia-related skin diseases and psoriasis, a polypeptide ointment prepared by formulating a TAT-N25 polypeptide and a pharmaceutically acceptable agent.

TI^N25 polypeptide ointment, characterized by: by weight percent: stearyl alcohol: 3%; glyceryl monostearate: 6%; glycerol: 10%; liquid paraffin: 10%; white petrolatum: 3%; Ethanolamine: 0.1%; sodium dodecyl gramide: 0.2%; hydroxyphenylethyl ester: 0.1%; TAT-N25 polypeptide: 0.2%; water: 60%; urea: 7.4%.

The fusion polypeptide drug should include an internal and external drug containing or using the fusion polypeptide in combination with a pharmaceutically acceptable agent.

N25 polypeptides can also be covalently or non-covalently linked to other peptides or other molecules that help N25 enter the cell membrane to form new drugs for treating cell growth abnormalities associated with skin diseases and psoriasis.

Fusion polypeptide drugs for the treatment of related skin diseases: skin inflammation, immunological diseases of cell abnormalities in the context of polygenic inheritance, use of epidermal hyperproliferation and differentiation abnormalities and diseases associated with abnormal cell growth such as psoriasis, and application of the drug by different means The route to treat these diseases.

The mechanism of TAT-N25 peptide ointment for the treatment of psoriasis is:

1. inhibit epithelial cell proliferation;

2. Promote epithelial cell differentiation;

3. inhibiting neovascularization;

4. Inhibit lymphocyte division and reduce the synthesis and secretion of inflammatory factors.

The method of using the peptide ointment is extremely convenient: the ointment is smeared on the patient's skin for 3 times, and the course of treatment is 10 days to 1 month.

Compared with the prior art, the invention has the advantages of high cure rate, small safety side effects and the like, and provides an ideal medicine for patients with psoriasis. detailed description

The invention is illustrated by the following specific experiments and the results of the data, but it should be noted that these examples are not intended to limit the invention.

The TAT-N25 polypeptide demonstrates its effectiveness in the treatment of psoriasis in the following experiments, including but not limited to the following experiments:

Example 1 TAT-N25 polypeptide inhibits keratinocyte growth and DNA synthesis

The effect of TAT-N25 fusion polypeptide on cell growth was examined using human keratinocyte line HACAT cells. HACAT cells were cultured in 10 cm cell culture medium in a cell culture medium (DMEM) containing 10% fetal calf serum at 37 ° C, 5% CO ₂ /95% air culture, and the cells were cultured to logarithmic growth phase. The polypeptide was added to a final concentration of 15 μg/ml, and the blank group and the urea group were cultured for control, and the cells were cultured for 24 hours, and the cell cycle distribution of these cells was analyzed by flow cytometry. The TAT-N25 polypeptide stock solution is dissolved in 6M urea, and the 6M urea refers to 6 moles of urea per liter of water. The following tubes are called 6M urea. The results showed that the expression of TAT-N25 fusion polypeptide had no significant effect on apoptosis at this dose, and increased the number of G0/G1 phase cells. The effect is shown in Table 1 below. Table 1: Effect of TAT-N25 on cell cycle

DNA synthesis is a hallmark of cell proliferation. In the next experiment, we examined the effect of TAT-N25 on cellular DNA synthesis. Because BrdU is a thymine analog, it can be incorporated into DNA in the S phase of DNA synthesis. After immunostaining of BrdU incorporated into DNA, the synthesis of DNA is analyzed by flow cytometry by detecting changes in fluorescence intensity. The blank group and the urea group were used for comparison. The results showed that the number of cells in the S phase of DNA synthesis in tumor cells did not change much after the addition of TAT-N25 polypeptide, but the average BrdU incorporation was significantly reduced compared with the blank group, as shown in Table 2, indicating inhibition of fusion polypeptide. The synthesis of DNA reduces the proliferation of tumor cells. Table 2: TAT-N25 inhibits cellular DNA synthesis

Example 2 TAT-N25 polypeptide inhibits vaginal epithelial cell layer proliferation in mice

Female mice were pre-injected with estrogens such as diethylstilbestrol for 7 days to place them in the estrogen physiological cycle. 0.2 (wt)% of TAT-N25 polypeptide ointment was administered vaginally for 7 days, and colchicine was intraperitoneally injected after the last administration to terminate cell division. The vaginal tissue of the mice was taken, fixed, stained, and the mitotic index of the vaginal epithelial cells was observed. 0.2 (wt)% TAT-N25 polypeptide ointment is: Percent by weight: stearyl alcohol: 3%; glyceryl monostearate: 6%; glycerol: 10%; liquid paraffin: 10%; white petrolatum: 3% Triethanolamine: 0.1%; sodium lauryl sulfate: 0.2%; hydroxyethyl ester: 0.1%; TAT-N25 polypeptide: 0.2%; water: 60%; urea: 7.4%.

The test results are shown in Table 3, indicating that the TAT-N25 polypeptide is effective in inhibiting mitosis of vaginal epithelial cells. Table 3: TAT-N25 inhibits mitosis of vaginal epithelial cells

Example 3 Effect of TAT-N25 polypeptide on the production of epidermal cells in mouse tail scales. Mice were taken. 0.2 (wt)% TAT-N25 polypeptide ointment was directly applied to the tail for 7 days, 3 times a day, and the tail root was taken. Skin, fixation, staining, measurement of rat tail epidermal thickness and particle formation.

The test results are shown in Table 4, indicating that the TAT-N25 polypeptide ointment has a role in promoting the formation of the rat tail particle layer. Table 4: TAT-N25 polypeptide promotes granule formation in rat tail cells

Example 4 TAT-N25 polypeptide inhibits inflammatory factor secretion

The concentration of 30 μg/ml TAT-N25 polypeptide culture solution was incubated for 1 day in 1 liter, and the contents of IL-1, 6, 8, TNF- and IFN- in the culture solution were determined.

The results indicate that the TAT-N25 polypeptide inhibits the secretion of inflammatory factors by lymphocytes.

Experiment 5 TAT-N25 peptide ointment for the treatment of patients with psoriasis

0.2 0% of TAT-N25 peptide ointment was applied to the skin of patients with psoriasis 3 times a day. The results are shown in Table 5, indicating that TAT-N25 peptide has obvious effects in treating psoriasis. Table 5: Summary of results of TAT-N25 peptide ointment for patients with psoriasis

Claims

Claim

1. Use of a TAT-N25 polypeptide for the treatment of a skin disorder associated with abnormal cell growth.

2. The use of TAT-N25 polypeptide in the treatment of psoriasis.

3. A TAT-N25 polypeptide drug, characterized in that it comprises a therapeutic drug comprising or combining a TAT-N25 polypeptide and a pharmaceutically acceptable agent.

4. A TAT-N25 polypeptide drug for treating a skin disease abnormality-related skin disease, comprising: a polypeptide ointment prepared by formulating a TAT-N25 polypeptide and a pharmaceutically acceptable agent.

5. A TAT-N25 polypeptide drug for treating psoriasis, which is characterized in that: a polypeptide ointment prepared by formulating a TAT-N25 polypeptide and a pharmaceutically acceptable agent.

The TAT-N25 polypeptide drug according to claim 4 or 5, which is characterized by: octadecyl alcohol: 3%; glyceryl monostearate: 6%; glycerin: 10%; liquid paraffin : 10%; white petrolatum: 3%; triethanolamine: 0.1%; sodium lauryl sulfate: 0.2%; hydroxyethyl ester: 0.1%; TAT-N25 polypeptide: 0.2%; water: 60%; urea: 7.4 %.

7. N25 peptides and other peptides or other molecules that help N25 enter the cell membrane through covalents.