KR20230041318A - Composition for treating wounds comprising Cell Penetrating Peptide and Medicament - Google Patents

Abstract

The present invention relates to a composition for wound treatment comprising a cell penetrating peptide and a drug, and since the composition has increased cell permeability of the drug, it can be usefully used for wound treatment. An object of the present invention is to provide a pharmaceutical composition for wound treatment comprising a cell penetrating peptide and any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD), and heat shock protein 20 (HSP 20).

Description

Composition for treating wounds comprising a cell penetrating peptide and a drug {Composition for treating wounds comprising Cell Penetrating Peptide and Medicament}

The present invention relates to a composition for wound treatment comprising a cell penetrating peptide and a drug.

Substances known to date that exhibit wound healing effects on the skin include epidermal growth factor (EGF) (Exp. Cell Res., 164, 1-10, 1986), acidic and basic fibroblast growth factors (acid and basic FGF) ( J. surg.Res., 45, 145-153, 1988), transforming factors (TGF-α and TGF-β) (Japanese Patent Laid-Open No. 2-167231, Science, 233:532, 1986) and insulin levels Growth factors such as growth factors (IGF-I and II) (BIO/Technology, 135-140, 1985), adhesion factors such as fibronectin, laminin, vitronectin (Ann. Rev. Biochem, 52:961, 1983), Retinoids and similar compounds (Am. J. OphtHalmol., 95:353, 1983; Ann. Ophthal., 19:175, 1987) and the like are known.

Cytokines are being identified as growth factors related to wound healing, and typical examples are basic fibrogrowth factor, which is produced in keratinocytes and fibroblasts and promotes the growth of epithelial cells, and platelet endothelial tissue. Platelet-derived growth factor (PDGF), which is produced from epidermal growth factor (EGF) and promotes abnormal proliferation of epithelial cells, is produced in fibroblasts and platelets to form connective tissue transforming growth factor-β (TGF-β) that promotes epithelial growth factor-β (TGF-β), epithelial cell growth factor that is produced in salivary gland stimulation glands and promotes the proliferation of epithelial cells, fibroblast growth factor (FGF) and macrophage and interleukin-1, which is produced in epithelial cells and promotes epithelial cell growth and motility. Becaplermin, marketed by Johnson & Johnson as a topical wound treatment under the trade name of Regranex, is a genetically engineered PDGF. European Patent No. 0575484B1 discloses a pharmaceutical composition containing PDGF and dexamethasone for tissue regeneration and treatment of mammals. US Patent No. 5,981,606 discloses a pharmaceutical composition for treating wounds containing TGF-β. International Patent Publication No. WO 96/30038 discloses a pharmaceutical composition for wound treatment containing TGF-β, fibric acid and an antioxidant together. US Patent No. 5183805 discloses a pharmaceutical composition containing EGF and having the effect of regenerating tissues. Japanese Patent No. 05070365 and US Patent No. 6165978 disclose wound healing agents containing FGF.

On the other hand, cell penetrating peptides (CPPs) are cell membrane penetrating peptides composed of about 10-60 short peptides, most of which are protein-transduction domains or membrane-translocating sequences. is derived from Unlike general entry pathways of foreign substances into cells, it is known that CPP moves into cells without damaging the cell membrane and can deliver DNA or proteins that are known to be unable to pass through the cell membrane into cells.

The present inventors completed the present invention by confirming that it can be used for wound treatment as a result of studying a cell penetrating peptide derived from the nucleocapsid protein of HIV (human immunodeficiency virus).

KR10-2020-0000243A

An object of the present invention is a pharmaceutical composition for wound treatment comprising a cell penetrating peptide and any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) is to provide

In addition, another object of the present invention is to provide a method for preparing the pharmaceutical composition for the treatment of wounds.

In order to achieve the above object, one aspect of the present invention is a cell penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, thymosin beta 4 (Thymosin beta 4), thymosin beta 4 actin A pharmaceutical composition for treating wounds comprising any one of a binding domain (T4AD) and heat shock protein 20 (HSP 20) is provided.

As used herein, the term 'Cell Penetrating Peptides (CPP)' is a cell membrane penetrating peptide consisting of about 10 to 60 short peptides, which moves into cells without damaging the cell membrane and passes through the cell membrane. DNA or proteins that cannot be delivered can be delivered into cells.

The cell penetrating peptide comprising any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 of the present invention may be encoded by the polynucleotide sequences of SEQ ID NOs: 4 to 6, respectively (Table 1 ).

ACP1 SEQ ID NO: 1 VKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTE ACP2 SEQ ID NO: 2 VKCFNCGKEGHTARNCRAPRKK ACP4 SEQ ID NO: 3 ARAPRKKGARAPRKKG ACP1 polynucleotide SEQ ID NO: 4 gtgaagtgcttcaattgcggaaaggagggccacacagctagaaactgccgcgcccccagaaagaaaggctgctggaagtgcggcaaggagggccaccagatgaaggactgcacagag ACP2 polynucleotide SEQ ID NO: 5 gtgaagtgcttcaattgcggaaaggagggccacacagctagaaactgccgcgcccccagaaagaaaggc ACP4 polynucleotide SEQ ID NO: 6 gcccgcgcccccagaaagaaaggcgcccgcgcccccagaaagaaaggc

"Thymosin beta 4 (TB4)" in the present invention is a very small protein existing in vivo consisting of 43 amino acids of 4.9 kDa in size, and the above-mentioned thymosin beta 4 (Thymosin beta 4) protein is It is responsible for the function of preventing the formation of F-actin, which is a combined form of G-actin, by combining with a separate form of G-actin. It is a protein that plays a role in altering the structure of the actin cytoskeleton and regulating cell movement by regulating turnover. In particular, the main function of thymosin beta 4 is to promote cell migration by regulating cell migration, which leads to the migration of vascular endothelial cells, migration of cells involved in wound healing, hair myoblasts, and cancer cells. It is involved in blood vessel formation, wound healing, hair growth, and cancer metastasis by promoting the movement of cells. Thymosin proteins in vivo are divided into three main groups by their respective isoelectric points. These are α-thymosin below pH 5.0, β-thymosin between pH 5.0 and pH 7.0, and γ-thymosin above pH 7.0. Thymosin beta 4 belongs to β-thymosin and is a representative factor promoting angiogenesis. Ac(Acetyl)-SDKP peptide, which is expressed in vivo among thymosin beta 4, is formed by prolyl endopeptidase or AspN-like protease using thymosin beta 4 as a precursor, and is used in pluripotent hematopoietic stem cells. It is known to perform the function of suppressing the cell cycle by blocking hematopoietic pluripotent stem cells from going over to S-phase and dividing.

Thymosin beta 4 actin binding domain (T4AD, LKKTETQ) is the main actin binding domain of thymosin beta 4, and is a key domain that has major activities such as wound healing, hair growth, and angiogenesis of thymosin beta 4. . T4AD was found in wound fluid and is produced through natural degradation from thymosin beta 4 and is expected to be involved in the wound healing process. T4AD promotes the expression of regeneration factors by inducing the migration of mast cells to the wound site during the initial wound healing process, and is involved in amplifying the subsequent wound healing process. In addition, it is known to induce angiogenesis by increasing vascular endothelial cell migration and blood vessel formation, and promote wound healing by regulating skin cell mobility. showed the same level of wound healing activity as

The "heat shock protein 20" in the present invention is a type of heat shock protein also expressed as HSP20, and functions as a protein chaperone capable of protecting other proteins from protein denaturation and aggregation caused by heat. The heat shock protein 20 may be the amino acid sequence of SEQ ID NO: 7, which may be encoded by the nucleotide sequence of SEQ ID NO: 8 (Table 2).

HSP20 SEQ ID NO: 7 WLRRASAPLPGLK HSP20 polynucleotide SEQ ID NO: 8 TGGCTGCGCCGCGCCTCGGCCCCGTTGCCCGGACTTAAG

In the present invention, a wound means a state in which a living body is damaged, and a pathology in which a tissue constituting the internal or external surface of a living body, for example, skin, muscle, nerve tissue, bone, soft tissue, internal organ or vascular tissue, is divided or destroyed. Covers enemy status

Examples of wounds include, but are not limited to, non-healing traumatic wounds, destruction of tissue by irradiation, abrasion, bone gangrene, laceration, avulsion, and penetrated wound. , gunshot wound, cut, burn, frostbite, contusion or bruise, skin ulcer, dry skin, keratosis dermatosis, crack, break, dermatitis, pain due to dermatophytosis, surgical wound, vascular disease wound, corneal wound Back wounds, decubitus ulcers, diabetic foot ulcers, conditions related to diabetes and poor circulation such as diabetic skin erosion, chronic ulcers, sutures after plastic surgery, spinal injuries, gynecological wounds, chemical wounds and acne It includes damage to any part of the subject, and specifically, it may be any one of diabetic foot ulcers, pressure sores and burns. In this respect, the formulation according to the present invention can be very useful for repairing, replacing, ameliorating, accelerating, promoting or curing such damaged tissue.

In one embodiment of the present invention, the cell penetrating peptide may be coupled to any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) .

As used herein, the term 'binding' means that a cell-penetrating peptide and a biologically or pharmaceutically active substance are connected through a chemical or physical covalent or non-covalent bond.

In another embodiment of the present invention, the cell penetrating peptide is not bound to any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) can In this case, any one of the cell penetrating peptide, thymosin beta 4, thymosin beta 4 actin binding domain (T4AD), and heat shock protein 20 (HSP 20) is not bound to the wound treatment of the present invention. It may be included in the treatment pharmaceutical composition.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, if necessary, in addition to the fusion protein.

Such pharmaceutically acceptable carriers are commonly used in pharmaceutical preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, and microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like as additives.

The carrier may be included in about 1% to about 99.99% by weight, preferably about 90% to about 99.99% by weight, based on the total weight of the pharmaceutical composition of the present invention, and the additives are about 0.1% by weight. to about 20% by weight.

On the other hand, the pharmaceutical composition of the present invention may be administered orally or parenterally, but may be directly administered to the skin in a topical administration method.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient, or prepared by placing it in a multi-dose container. At this time, the dosage form may be in the form of a solution, suspension or emulsion, or may include an elexir agent, an extract agent, a powder agent, a granule agent, a tablet agent, a tablet agent, an ointment agent, a lotion agent, and an ointment agent.

The daily dosage of the pharmaceutical composition of the present invention is usually in the range of 0.001 to 150 mg/kg body weight, and may be administered once or divided into several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in light of various related factors such as the route of administration, age, sex, weight, and severity of the patient, the dosage is not to limit the scope of the present invention in any aspect. should not be understood

The pharmaceutical composition for wound treatment of the present invention described above includes steps of (a) preparing a cell-penetrating peptide consisting of any one amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and (b) the cell-penetrating peptide and It can be prepared through a step comprising mixing any one of thymosin beta 4, thymosin beta4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20), optionally, Further comprising the step of binding the cell penetrating peptide with any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) after step (b) It can be produced through a manufacturing method.

The pharmaceutical composition for wound treatment of the present invention described above can be usefully used for wound treatment because the cell permeability of the drug is increased.

Another aspect of the present invention is (a) preparing a cell penetrating peptide consisting of any one amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and (b) the cell penetrating peptide and thymosin beta 4 ( Thymosin beta 4), thymosin beta4 actin binding domain (T4AD), and heat shock protein 20 (HSP 20).

Step (a) is a step of preparing a cell penetrating peptide.

Preparation of the cell-penetrating peptide is the synthesis of a cell-penetrating peptide consisting of any one amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 It can be obtained by culturing host cells into which the encoding polynucleotide has been introduced in a culture medium and recovering cell penetrating peptides.

Specifically, the cell-penetrating peptide consisting of any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 can be synthesized or purchased by a known method, and the cell-penetrating peptide SEQ ID NO: 1, SEQ ID NO: 2, and sequence The host cell into which the polynucleotide encoding any one of amino acid sequences of number 3 is introduced is a recombinant vector containing a polynucleotide encoding any one of amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, which are cell penetrating peptides. can be obtained by transforming host cells.

Specifically, the cell-penetrating peptide comprising any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 may be encoded by the polynucleotide sequences of SEQ ID NOs: 4 to 6, respectively.

Alternatively, the polynucleotide may further include a polynucleotide of SEQ ID NO: 8 encoding heat shock protein 20 (HSP20).

As used herein, the term 'polynucleotide' is a polymer of deoxyribonucleotides or ribonucleotides that exist in single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.

The polynucleotides include not only a nucleotide sequence encoding a cell penetrating peptide, but also a sequence complementary to the sequence. The complementary sequences include not only perfectly complementary sequences, but also substantially complementary sequences. In addition, the polynucleotide sequence may be modified, and the modification includes additions, deletions or non-conservative or conservative substitutions of nucleotides.

As used herein, the term 'vector' refers to a means for expressing a target gene in a host cell. For example, it may include, but is not limited to, plasmid vectors, cosmid vectors and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors. Vectors that can be used as the recombinant vector are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, and pUC19, etc.), phages (eg, λgt4λB, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, CMV, SV40, etc.).

The recombinant vector may include the polynucleotide sequence and a promoter operatively linked to the polynucleotide sequence.

As used herein, the term 'operably linked' refers to a functional linkage between a nucleotide expression control sequence (e.g., a promoter sequence) and another nucleotide sequence, whereby the control sequence is linked to the other nucleotide sequence. regulation of transcription and/or translation.

The recombinant vector may include a tag sequence that facilitates purification of the cell-penetrating peptide, for example, a continuous histidine codon, a maltose binding protein codon, and a Myc codon, and the solubility of the fusion protein It may further include a fusion partner (partner) for increasing. In addition, it may include a sequence specifically cleaved by an enzyme to remove unnecessary parts when expressing the fusion protein, an expression control sequence, and a marker or reporter gene sequence for confirming intracellular delivery.

Any host cell known in the art may be used as a host cell capable of stably and continuously cloning or expressing the recombinant vector. Examples of prokaryotic cells include E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, and E. coli W3110. Host cells for transformation into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, plant cells and animal cells, such as SP2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138 , BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines and the like can be used.

As a method of transforming a host cell with the polynucleotide or a recombinant vector containing the polynucleotide, a delivery method widely known in the art may be used. As the delivery method, for example, when the host cell is a prokaryotic cell, a CaCl ₂ method or an electroporation method may be used, and when the host cell is a eukaryotic cell, a microinjection method, a calcium phosphate precipitation method, an electroporation method, Liposome-mediated transfection and gene bombardment may be used, but are not limited thereto.

The method for selecting the transformed host cell can be easily carried out according to a method known in the art using a phenotype expressed by a selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

The host cell culture may be a large-scale cell culture, and a commonly used cell culture method may be used as the cell culture method. For example, the cell culture method is, but is not limited to, batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture , It may be any one or more selected from the group consisting of continuous culture and perfusion culture.

Recovering the cell-penetrating peptide from the culture medium can be performed through various separation and purification methods known in the art. Usually, after centrifugation of the cell lysate to remove cell debris, culture impurities, etc., precipitation, e.g., salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (acetone, ethanol, iso protein fraction precipitation using propyl alcohol, etc.), etc., and dialysis, electrophoresis, various column chromatography, etc. can be performed.

In step (b), the cell penetrating peptide prepared in step (a) and any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) This is the mixing stage. The mixing may be simply mixing the cell penetrating peptide with any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20), optionally, After the step (b), a step of binding the cell penetrating peptide with any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) may be further included. can

Specifically, the binding of the cell penetrating peptide with any one of thymosin beta 4 (Thymosin beta 4), thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) is a chemical and physical covalent or non-covalent bond may be connected.

The pharmaceutical composition for wound treatment can be usefully used for wound treatment because the cell permeability of the drug is increased.

Figure 1 is a photograph showing the results according to Experimental Example 1-3, cell membrane permeability by concentration of ACP2-thymosin beta 4 (Thymosin beta 4) -FITC and ACP2-thymosin beta 4 (Thymosin beta 4) active domain-FITC is the result of checking
Figure 2 is a photograph showing the results according to Experimental Examples 1-4, cell mobility by concentration of ACP2-thymosin beta 4 (Thymosin beta 4) -FITC and ACP2-thymosin beta 4 (Thymosin beta 4) -active domain-FITC is the result of checking
Figure 3 is a photograph showing the results according to Experimental Example 2-3, it can be confirmed the wound healing efficacy of the animal model according to the treatment of EGF, ACP1-EGF, ACP2-TB4 and ACP2-TB4AD.
4 is a graph quantitatively showing the results according to Experimental Example 2-3. ***/** marked if significant compared to G1 (each, p<0.001/p<0.01 )
Figure 5 is a graph showing the results according to Experimental Examples 3-4, it can be confirmed the results of evaluating inflammation (inflammation), angiogenesis (Angiogenesis), fibrosis (fibroplasia) and epithelialization (epithelialization) of the animal model. ***/** marked if significant compared to G1 (each, p<0.001/p<0.01 ).
Figure 6 is a graph showing the results according to Experimental Examples 4-4, and the results of evaluating the wound healing effect and inflammation, angiogenesis, fibroplasia and epithelialization of the animal model over time. You can check. ***/**/* marked if significant compared to G1 (each, p<0.001/p<0.01 / p<0.05 ).
FIG. 7 is a photograph showing the results according to Experimental Examples 1-4, showing cell mobility by concentration of T4AD and ACP4-T4AD (Thymosin beta 4 active domain).
8 is a graph quantitatively showing the results according to Experimental Example 5-4. ***/* marked if significant compared to G1 (each, p<0.001/p<0.05 )
9 is a graph showing the results according to Experimental Examples 5-4, and it can be seen the results of evaluating inflammation, angiogenesis, fibroplasia and epithelialization of the animal model. **/* marked if significant compared to G1 (each, p<0.01/p<0.05 ).
Figure 10 is a photograph showing the results according to Experimental Example 6-4, it can be confirmed the cell absorption capacity.
Figure 11 is a photograph showing the results according to Experimental Example 7-4, it can be confirmed the cell absorption capacity.
12 is a photograph showing the results according to Experimental Example 8-4, and the degree of cell migration by time and concentration of PTD-HSP20 can be confirmed.
13 is a photograph showing the results according to Experimental Example 8-4, and it can be confirmed the degree of cell migration by time and concentration of ACP1-HSP20.
Figure 14 is a photograph showing the results according to Experimental Example 8-4, it can be confirmed the degree of cell migration by time and concentration of ACP1-HSP20.
15 is a graph showing the results according to Experimental Example 8-4, and it can be confirmed the cell migration level for each administration concentration of PTD-HSP20, ACP1-HSP20 and ACP2-HSP20.
16 shows the results according to Experimental Example 8-4, and the expression level of CTGF can be confirmed.
17 is a graph quantitatively showing the results according to Experimental Example 8-4.
18 shows the results according to Experimental Example 8-4, and the expression level of CTGF can be confirmed.
19 is a graph quantitatively showing the results according to Experimental Example 8-4.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Experimental Example 1: Confirmation of cell membrane permeability and cell mobility

1-1. preparation of cells

A431 cells were cultured in RPMI-1640 medium (Hyclone, USA) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. HaCaT cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (HyClone, MA, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA).

1-2. preparation of drugs

ACP2-TB4-FITC, ACP2-TB4AD-FITC, FITC-T4AD, FITC-ACP4-T4AD, T4AD, and ACP4-T4AD were synthesized by FMOC solid-phase method from Chempeptide (Shanghai, China) did The synthesized peptide was purified and analyzed by reverse-phase high performance liquid chromatography (HPLC, HPLC-20AP, Japan) using a C18 analytical RP column (Shiseido capcell pak), and mass spectrometry (SHIMADZU LCMS-2010EV, Japan) Identified using

ACP2-TB4-FITC, ACP2-TB4AD-FITC, FITC-T4AD, FITC-ACP4-T4AD, T4AD, and ACP4-T4AD were dissolved in PBS at respective concentrations immediately before administration and used.

1-3. Confirmation of cell membrane permeability

The drug prepared in 1-2 was administered to the cells prepared in 1-1 above, and cell membrane permeability was confirmed.

A431 cells were treated with ACP2-TB4-FITC or ACP2-TB4AD-FITC at each concentration, and the cells were fixed. The glass to which HeLa cells were attached was removed, placed on a slide glass, and observed under a confocal laser scanning microscope.

Specifically, A431 cells were treated with ACP2-TB4-FITC or ACP2-TB4AD-FITC at each concentration (0.01, 0.1, 1, 10 μM) for the control and experimental groups, respectively, and cell membrane permeability was confirmed. .

As a result, as confirmed in FIG. 1, it was confirmed that the drug was introduced into the cells in proportion to the treatment concentration of the drug, indicating that there is cell membrane permeability.

1-4. Confirmation of cell mobility

After seeding 2x10 ⁵ HaCaT cells in a 12 well cell culture plate, they were cultured in a 37℃ O ₂ incubator for 24 h to form a monolayer. HaCaT cells on the plate seeded the day before were scratched using a sterilized pipette tip and washed three times with 1XPBS. HDF cells were washed three times with 1XPBS after serum starvation for 24 h, followed by scratch induction using a sterilized pipette tip. After processing the prepared test substance, take an image with a microscope and analyze it using the Image J program. After 24, 48, and 72 h, images were taken under a microscope and analyzed using the Image J program.

As a result, as shown in FIGS. 2 and 7 , it can be seen that cell mobility increases in proportion to the treatment concentrations of ACP2-TB4-FITC, ACP2-TB4AD-FITC, and ACP4-T4AD.

Experimental Example 2: Confirmation of wound healing effect

2-1. Preparation of animal models

Minipig (Medicinetic) with an average weight of about 30 kg, temperature 23±3 ℃, relative humidity 55±15%, number of ventilation 10-20 times/hr, lighting time 12 hours (8:00 am to 8:00 pm) Lights off) and illumination 150∼300 Lux, Notus Co., Ltd. Non-rodent Breeding Area No. 4, pigs were bred in a stainless steel breeding box (W 1200 x L 1700 x H 1700 mm) with 1 pig/breeding box, feed and Water was freely available. During the adaptation period, the weights of the mice determined to be healthy were measured and randomly distributed according to the ranked weights so that the average weights of each group were distributed as uniformly as possible. And, for the experiments described later, they were distributed to each group.

Twelve wounds were induced per subject by removing the dermal portion in a size of about 2cmX2cm centered on the spine line of the prepared animal model, and the test substance was applied a total of 14 times at intervals of once/day from the day of wound induction (Day 0). did On Day 0 (the day of induction), 3, 7, 10 and 14, pictures of the wound induction site were taken. Area analysis was performed on the wound area using Image J software (NIH, Bethesda, MD).

On the 14th day after wound induction (Day 14), the test substance application site (skin) of all animals was fixed in 10% neutral buffered formalin solution, and the fixed tissue was general tissue such as trimming, dehydration, paraffin embedding, and Hematoxylin & Eosin staining. After preparing a specimen for histopathological examination through the treatment process, histopathological changes were observed under an optical microscope (Olympus BX53, Japan).

2-2. preparation of drugs

EFG, ACP1-EFG, ACP2-TB4 and ACP2-TB4AD were synthesized by the FMOC solid-phase method from Chempeptide (Shanghai, China). The synthesized peptide was purified and analyzed by reverse-phase high performance liquid chromatography (HPLC, HPLC-20AP, Japan) using a C18 analytical RP column (Shiseido capcell pak), and mass spectrometry (SHIMADZU LCMS-2010EV, Japan) Identified using

EFG, ACP1-EFG, ACP2-TB4 and ACP2-TB4AD were used by dissolving them in PBS at their respective concentrations immediately before administration.

2-3. Administration of drugs and confirmation of wound healing effects

The drug prepared in 2-2 was administered to the animal model prepared in 2-1 above, and the wound healing effect from the start of administration (Day0) to the 14th day of administration was confirmed.

Specifically, the control group administered PBS to the wound site (Vehicle control, G1), the experimental group administered with 2μM EFG (G2), the experimental group administered with 2μM ACP1-EFG (G3), the experimental group administered with 500nM ACP2-TB4 (G4 ) and an experimental group (G5) in which 6 μM of ACP2-TB4AD was administered.

On Day 0 (the day of induction), 3, 7, 10 and 14, pictures of the wound induction site were taken. Area analysis was performed on the wound area using Image J software (NIH, Bethesda, MD).

As a result, as shown in FIGS. 3 and 4, the control group (Vehicle control, G1) administered with PBS and the experimental group (G2) administered with 2 μM of EFG compared to the experimental group (G4) administered with 500 nM of ACP2-TB4 and ACP2-TB4AD A significant wound healing effect was confirmed in the experimental group (G5) administered with 6 μM.

Experimental Example 3: Confirmation of histopathological characteristics

3-1. Preparation of animal models

An animal model was prepared in the same manner as in 2-1.

3-2. preparation of drugs

EFG, ACP1-EFG, ACP2-TB4, ACP2-TB4AD, and ACP1-HSP 20 were synthesized by Chempeptide (Shanghai, China) by the FMOC solid-phase method. The synthesized peptide was purified and analyzed by reverse-phase high performance liquid chromatography (HPLC, HPLC-20AP, Japan) using a C18 analytical RP column (Shiseido capcell pak), and mass spectrometry (SHIMADZU LCMS-2010EV, Japan) Identified using

EFG, ACP1-EFG, ACP2-TB4, ACP2-TB4AD, and ACP1-HSP 20 were used by dissolving them in PBS at respective concentrations immediately before administration.

3-3. administration of drugs

The drug of 3-2 was administered to the animal model prepared in 3-1 above.

Specifically, the control group (Vehicle control, G1) administered PBS to the wound site of the animal model, the experimental group (G2) administered with 2 μM of EFG, the experimental group (G3) administered with 2 μM of ACP1-EFG, the experimental group (G3) administered with 500 nM of ACP2-TB4 They were divided into an experimental group (G4), an experimental group (G5) administered with 6 μM of ACP2-TB4AD, and an experimental group (G9) administered with 50 μM of ACP1-HSP 20.

3-4. Histopathological characterization

The histopathological characteristics of the control group and the experimental group of 3-3 were confirmed.

Specifically, on the 14th day after wound induction (Day 14), the test substance application site (skin) of all animals was fixed in 10% neutral buffered formalin solution, and the fixed tissue was trimmed, dehydrated, paraffin embedded, and stained with Hematoxylin & Eosin. Specimens for histopathological examination were prepared through general tissue processing procedures such as the above, and histopathological changes were observed under an optical microscope (Olympus BX53, Japan). Wound tissue readings were scored based on inflammation, fibrosis, angiogenesis, and epithelialization score criteria, and the results are shown in FIG. 5 .

Regarding inflammation, compared to the control group (Vehicle control, G1) administered with PBS, the experimental group (G4) administered with 500 nM of ACP2-TB4 and the experimental group (G5) administered with 6 μM of ACP2-TB4AD showed a significant improvement in inflammation. there is.

Regarding epithelialization, which is the most important factor in wound healing, ACP2-T4AD TB4 (G4), ACP2-T4AD (G5) and An improved epithelialization effect was confirmed in the experimental group administered with ACP1-HSP 20 (G9).

Experimental Example 4: Confirmation of wound healing effect

4-1. Preparation of animal models

An animal model was prepared in the same manner as in 2-1.

4-2. preparation of drugs

TB4AD, ACP2-TB4AD, ACP2-TB4AD, PTD-HSP ACP1-HSP was synthesized by the FMOC solid-phase method from Chempeptide (Shanghai, China). The synthesized peptide was purified and analyzed by reverse-phase high performance liquid chromatography (HPLC, HPLC-20AP, Japan) using a C18 analytical RP column (Shiseido capcell pak), and mass spectrometry (SHIMADZU LCMS-2010EV, Japan) Identified using

TB4AD, ACP2-TB4AD, ACP2-TB4AD, PTD-HSP and ACP1-HSP were dissolved in PBS at respective concentrations immediately before administration.

4-3. administration of drugs

The drug of 4-2 was administered to the animal model prepared in 4-1 above.

Specifically, a control group in which PBS was administered to the wound site of the animal model (Vehicle control, G1), an experimental group in which 30 μM of TB4AD was administered (G2), an experimental group in which 6 μM of ACP2-TB4AD was administered (G3), and 30 μM of ACP2-TB4AD were administered The experimental group (G4), the experimental group (G5) administered with 50 μM of PTD-HSP, the experimental group (G6) with 10 μM of ACP1-HSP, and the experimental group (G7) with 50 μM of ACP1-HSP were classified.

4-4. Confirmation of wound healing ability and histopathological characteristics

The wound healing ability and histopathological characteristics of the control and experimental groups of 4-3 were confirmed.

On Day 0 (the day of induction), 3, 7, 10 and 14, pictures of the wound induction site were taken. Area analysis was performed on the wound area using Image J software (NIH, Bethesda, MD).

On the 14th day after wound induction (Day 14), the test substance application site (skin) of all animals was fixed in 10% neutral buffered formalin solution, and the fixed tissue was general tissue such as trimming, dehydration, paraffin embedding, and Hematoxylin & Eosin staining. After preparing a specimen for histopathological examination through the treatment process, histopathological changes were observed under an optical microscope (Olympus BX53, Japan). Wound tissue readings were scored based on inflammation, fibrosis, angiogenesis, and epithelialization score criteria, and the results can be seen in FIG. 6 .

Regarding wound healing ability, significant wound healing ability was confirmed only in the experimental group (G6) administered with 10 μM of ACP1-HSP compared to the control group (Vehicle control, G1) administered with PBS on the 3rd day of drug administration, and on the 10th day, the control group administered with PBS (Vehicle control, G1), significant wound healing ability was confirmed only in the experimental group (G6) in which 10 μM of ACP1-HSP was administered, and wound healing ability was also confirmed in other experimental groups. And on the 14th day, significant wound healing ability was confirmed in all experimental groups compared to the control group.

Regarding inflammation, an improvement in inflammation can be seen in all experimental groups (G4) compared to the control group (Vehicle control, G1) administered with PBS.

In addition, in relation to epithelialization, which is the most important factor in wound healing, compared to the control group administered with PBS (Vehicle control, G1), the experimental group administered with 30 μM of ACP2-TB4AD (G4) and the experimental group administered with 50 μM of ACP1-HSP ( The most improved epithelialization effect was confirmed in G7).

Experimental Example 5: Confirmation of wound healing effect

5-1. Preparation of animal models

An animal model was prepared in the same manner as in 2-1.

5-2. preparation of drugs

T4AD and ACP4-T4AD were synthesized by the FMOC solid-phase method from Chempeptide (Shanghai, China). The synthesized peptide was purified and analyzed by reverse-phase high performance liquid chromatography (HPLC, HPLC-20AP, Japan) using a C18 analytical RP column (Shiseido capcell pak), and mass spectrometry (SHIMADZU LCMS-2010EV, Japan) Identified using

T4AD and ACP4-T4AD were dissolved at respective concentrations in PBS immediately before administration and used. Fucidin ointment (manufacture number: J126), a positive control group, was purchased from a pharmacy and used.

5-3. administration of drugs

The drug of 5-2 was administered to the animal model prepared in 5-1 above.

Specifically, a control group in which PBS was administered to the wound site of the animal model (Vehicle control, G1), an experimental group in which fucidin ointment was administered (G2), an experimental group in which 10 μM of ACP4-T4AD was administered (G6), and an experimental group in which 30 μM of ACP4-T4AD were administered (G7) and an experimental group (G8) in which 90 μM of ACP4-T4AD was administered.

5-4. Confirmation of wound healing ability and histopathological characteristics

The wound healing ability and histopathological characteristics of the control and experimental groups of 5-3 were confirmed.

On Day 0 (the day of induction), 3, 7, 10 and 14, pictures of the wound induction site were taken. Area analysis was performed on the wound area using Image J software (NIH, Bethesda, MD), and as a result, as shown in FIG. 8, compared to the control group (Vehicle control, G1) administered with PBS, ACP4-T4AD In the experimental groups (G6, G7, G8) administered with 6μM, a significant wound healing effect equivalent to that of Fucidin, a positive control group, was confirmed.

On the 14th day after wound induction (Day 14), the test substance application site (skin) of all animals was fixed in 10% neutral buffered formalin solution, and the fixed tissue was general tissue such as trimming, dehydration, paraffin embedding, and Hematoxylin & Eosin staining. After preparing a specimen for histopathological examination through the treatment process, histopathological changes were observed under an optical microscope (Olympus BX53, Japan). Wound tissue readings were scored based on inflammation, fibrosis, angiogenesis, and epithelialization score criteria, and the results can be seen in FIG. 9 .

As a result of measuring the wound area, it was found that the level of wound area in all test substance treatment groups was similar from the wound induction date (Day 0) to the 3rd day after the start of test substance administration (Day 3). On Day 7 (Day 7), the wound area level of the ACP4-T4AD 30 μM and 90 μM treated groups was significantly lower than that of the untreated group and the positive control group, and on the 14th day after the start of test substance administration, the ACP4-T4AD The level of wound area in the T4AD (G6-8) treated group was significantly lower than that of the untreated group.

The epithelialization level of the ACP4-T4AD 90 μM treated group tended to be significantly higher than that of the untreated group and the positive control group Fucidin, and the inflammation level tended to be significantly lower than that of the untreated group and the positive control group Fucidin.

Experimental Example 6: Confirmation of cell membrane permeability

6-1. preparation of cells

HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. HeLa cells were cultured in a humidified incubator at 37°C and 5% CO2. The cultured HeLa cells were dispensed at a density of 1x10 ⁵ cells/well in a 12-well plate containing glass and cultured for 24 hours.

6-2. preparation of drugs

ACP-FITC, FITC-PTD-HSP, FITC-ACP1-HSP, and FITC-ACP2-HSP were synthesized by Chempeptide (Shanghai, China) using the FMOC solid-phase method. The synthesized peptide was purified and analyzed by reverse-phase high performance liquid chromatography (HPLC, HPLC-20AP, Japan) using a C18 analytical RP column (Shiseido capcell pak), and mass spectrometry (SHIMADZU LCMS-2010EV, Japan) Identified using

ACP-FITC, FITC-PTD-HSP, FITC-ACP1-HSP, and FITC-ACP2-HSP were used by dissolving them in PBS at respective concentrations immediately before administration.

6-3. Confirmation of cell membrane permeability

The drug prepared in 6-2 was administered to the cells prepared in 6-1 above, and cell membrane permeability was confirmed.

Specifically, HeLA cells were classified into experimental groups treated with 3 μM of ACP-FITC, 100 μM of FITC-PTD-HSP, 100 μM of FITC-ACP1-HSP, and 100 μM of FITC-ACP2-HSP. After treating the cells with each drug, the cells were washed three times with PBS. The washed cells were fixed with 3.7% formaldehyde for 20 minutes, and treated with PBS containing 0.2% Triton X-100 to increase cell membrane permeability. Next, cells were treated with 3% BS, blocked for 1 hour, reacted with tubulin antibody (Santa Cruz Biotechnology, sc-5286) at room temperature for 2 hours, and then washed three times with PBS. After washing, Cy3 secondary antibody was treated and reacted at room temperature for 1 hour, washed twice with PBS, and then stained with DAPI (4', 6-diamidino-2-phenylindol) for 10 minutes. The cell-attached glass was removed, placed on a slide glass, and observed with a confocal laser scanning microscope (LSM 700, Zeiss, Germany).

As a result, as shown in FIG. 10 , the best cell membrane permeability was confirmed in the FITC-ACP1-HSP treatment group.

Experimental Example 7: Evaluation of cell absorption capacity

7-1. preparation of cells

A431 cells were prepared in the same manner as in 1-1 above.

7-2. preparation of drugs

PTD-HSP20, ACP1-HSP20, and ACP2-HSP20 were synthesized by Chempeptide (Shanghai, China) using the FMOC solid-phase method. The synthesized peptide was purified and analyzed by reverse-phase high performance liquid chromatography (HPLC, HPLC-20AP, Japan) using a C18 analytical RP column (Shiseido capcell pak), and mass spectrometry (SHIMADZU LCMS-2010EV, Japan) Identified using

PTD-HSP20, ACP1-HSP20, and ACP2-HSP20 were used by dissolving them in PBS at respective concentrations immediately before administration.

7-3. treatment of drugs

The drug prepared in 7-2 above was treated with the cells of 7-1.

Specifically, the experimental group treated with 100 μM of PTD-HSP2, the experimental group treated with 100 μM of ACP1-HSP20, and the experimental group treated with 100 μM of ACP2-HSP20 were distinguished, and these cells were treated with 10% FBS containing 500 μl of RPMI1640 for 3 hours and 24 hours cultured.

7-4. Evaluation of cell absorption capacity

The experimental groups of 7-3 were cultured for 3 hours and 24 hours, and cell absorption capacity was evaluated.

HeLa cells were treated with PTD-HSP20, ACP1-HSP20, and ACP2-HSP20 at different concentrations, and the cells were fixed and reacted with Cy3 secondary antibody. The glass to which HeLa cells were attached was removed, placed on a slide glass, and observed under a confocal laser scanning microscope.

As a result, as shown in FIG. 11, it can be confirmed that the experimental group treated with 100 μM of ACP1-HSP20 and 100 μM of ACP2-HSP20 compared to the experimental group treated with 100 μM of PTD-HSP2 for 3 hours and 24 hours showed excellent cell absorption capacity. .

Experimental Example 8: Confirmation of wound healing effect

8-1. preparation of cells

A431 cells were prepared in the same manner as in 1-1 above.

8-2. preparation of drugs

The drug was prepared in the same way as in 7-2 above.

8-3. treatment of drugs

The cells prepared in 8-1 were treated with the drug of 8-2.

Specifically, a control group not treated with the drug, an experimental group treated with PTD-HSP2 (15, 30, and 45 μM, respectively), an experimental group treated with ACP1-HSP20 (15, 30, and 45 μM, respectively), and ACP2-HSP20 were classified into experimental groups (15, 30 and 45 μM, respectively) treated with , and these cells were cultured in 1% FBS containing 500 μl of RPMI1640 for 15 hours.

7-4. Check wound healing effect

The wound healing effect of the control group and the experimental group of 7-3 was confirmed.

After seeding A431 cells at 1x10 ⁵ in a 12 well cell culture plate, incubate for 24 h in a 37℃ O ₂ incubator to form a monolayer. A431 cells on the plate seeded the day before were scratched using a sterilized pipette tip and washed three times with 1XPBS. HDF cells were washed three times with 1XPBS after serum starvation for 24 h, followed by scratch induction using a sterilized pipette tip. After processing the prepared test substance, take an image with a microscope and analyze it using the Image J program. After 15 h, images were taken under a microscope and analyzed using the Image J program.

As a result, as shown in FIGS. 12 to 15, immediately after drug treatment (0 hr), it is difficult to confirm the wound healing effect of the control group and the experimental group by concentration, but after 15 hours, all compared to the control group (control) that is not treated with the drug. It was confirmed that the cell proliferation level was improved in the experimental group, and the wound healing effect was found. In addition, it was confirmed that the level of cell proliferation was improved in proportion to the drug treatment concentration in all experimental groups, and in the experimental groups treated with ACP1-HSP20 and ACP2-HSP20 compared to the experimental groups treated with PTD-HSP2 at all concentrations. It was confirmed that the level of cell proliferation was improved.

When this was judged quantitatively, when immediately after drug treatment (0hr) was taken as the standard (100%) (Cont.), the cell proliferation effect of all experimental groups for each concentration could be confirmed at the time point 15 hours after drug treatment, and cell proliferation was concentration-dependent. It can be seen that the effect increases. In particular, the experimental group treated with PTD-HSP2 (15, 30, and 45 μM, respectively), the experimental group treated with ACP1-HSP20 (15, 30, and 45 μM, respectively), and the experimental group treated with ACP2-HSP20 (15, 30, and 45 μM, respectively) ) can confirm an equal or superior cell proliferation effect compared to the experimental group treated with PTD-HSP2 (15, 30 and 45 μM, respectively).

Through these results, it can be seen that the composition of the present invention has an excellent wound healing effect.

Experimental Example 9: Confirmation of protein expression effect

9-1. preparation of cells

A431 cells were cultured in RPMI-1640 medium (Hyclone, USA) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin.

9-2. preparation of drugs

PTD-HSP20, ACP1-HSP20, and ACP2-HSP20 were synthesized by Chempeptide (Shanghai, China) using the FMOC solid-phase method. The synthesized peptide was purified and analyzed by reverse-phase high performance liquid chromatography (HPLC, HPLC-20AP, Japan) using a C18 analytical RP column (Shiseido capcell pak), and mass spectrometry (SHIMADZU LCMS-2010EV, Japan) Identified using

PTD-HSP20, ACP1-HSP20, and ACP2-HSP20 were used by dissolving them in PBS at respective concentrations immediately before administration. TGFβ1 was purchased from R&D Systems and used.

9-3. treatment of drugs

A431 cells prepared in 9-1 were treated with the drug prepared in 9-2.

Specifically, A431 cells were not treated with a negative control group (cont.), TGFβ1-treated experimental groups (1, 3, 5, 10ng each), TGFβ1 or/and HSP-treated experimental groups (TGFβ1 alone 10ng treatment, TGFβ1 10ng + PTD-HSP20 50μM treatment, TGFβ1 10ng + ACP1-HSP20 50μM treatment, TGFβ1 10ng + ACP2-HSP20 50μM treatment).

9-4. Confirmation of protein expression (western blot)

The protein (CTGF, β-actin) expression level of the control and experimental groups in 9-3 was confirmed.

As a result, as confirmed in FIGS. 16 to 19, it was confirmed that the expression level of CTGF increased in proportion to the TGFβ1 treatment concentration in the experimental group (1, 3, 5, and 10 ng each) treated with TGFβ1 compared to the negative control group (cont.). (Figs. 16, 18).

In addition, as shown in FIGS. 17 and 19, TGFβ1 increased in the experimental group treated with 10 ng of TGFβ1 alone compared to the negative control group (cont.). HSP20 50μM treatment, TGFβ1 10ng + ACP2-HSP20 50μM treatment) was confirmed to decrease the CTFG expression level, and among them, it can be confirmed that the CTFG expression level decreased the most when ACP1-HSP20 treatment.

Through these experimental results, it can be seen that the compositions of the present invention have excellent wound healing efficacy by reducing the expression of connective tissue growth factor (CTFG).

Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

<110> AVIXGEN INC. <120> Composition for treating wounds comprising Cell Penetrating Peptides and Medicaments <130> BPN211025 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 39 <212> PRT <213> artificial sequence <220> <223> ACP1 <400> 1 Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Thr Ala Arg Asn Cys 1 5 10 15 Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly His 20 25 30 Gln Met Lys Asp Cys Thr Glu 35 <210> 2 <211> 22 <212> PRT <213> artificial sequence <220> <223> ACP2 <400> 2 Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Thr Ala Arg Asn Cys 1 5 10 15 Arg Ala Pro Arg Lys Lys 20 <210> 3 <211> 16 <212> PRT <213> artificial sequence <220> <223> ACP4 <400> 3 Ala Arg Ala Pro Arg Lys Lys Gly Ala Arg Ala Pro Arg Lys Lys Gly 1 5 10 15 <210> 4 <211> 117 <212> DNA <213> artificial sequence <220> <223> Polynucleotide for ACP1 <400> 4 gtgaagtgct tcaattgcgg aaaggagggc cacacagcta gaaactgccg cgcccccaga 60 aagaaaggct gctggaagtg cggcaaggag ggccaccaga tgaaggactg cacagag 117 <210> 5 <211> 69 <212> DNA <213> artificial sequence <220> <223> Polynucleotide for ACP2 <400> 5 gtgaagtgct tcaattgcgg aaaggagggc cacacagcta gaaactgccg cgcccccaga 60 aagaaaggc 69 <210> 6 <211> 48 <212> DNA <213> artificial sequence <220> <223> Polynucleotide for ACP4 <400> 6 gcccgcgccc ccagaaagaa aggcgcccgc gcccccagaa agaaaggc 48 <210> 7 <211> 13 <212> PRT <213> artificial sequence <220> <223> HSP20 <400> 7 Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu Lys 1 5 10 <210> 8 <211> 39 <212> DNA <213> artificial sequence <220> <223> Polynucleotide for HSP20 <400> 8 tggctgcgcc gcgcctcggc cccgttgccc ggacttaag 39

Claims (6)

A cell penetrating peptide consisting of any one amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and
A pharmaceutical composition for wound treatment comprising any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20).
According to claim 1,
The cell-penetrating peptide is thymosin beta 4 (Thymosin beta 4), thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) binding to any one of the pharmaceutical composition for the treatment of wounds.
According to claim 1,
The wound is any one of diabetic foot ulcers, bedsores and burns, a pharmaceutical composition for treating wounds.
(a) preparing a cell-penetrating peptide consisting of any one amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and
(b) mixing the cell penetrating peptide with any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20);
A method for preparing a pharmaceutical composition for wound treatment.
According to claim 4,
Further comprising the step of binding the cell penetrating peptide with any one of thymosin beta 4, thymosin beta 4 actin binding domain (T4AD) and heat shock protein 20 (HSP 20) after step (b) A method for preparing a pharmaceutical composition for wound treatment.
According to claim 4,
The wound is any one of diabetic foot ulcers, bedsores and burns, a method for producing a pharmaceutical composition for treating wounds.

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