TWI674276B - Peptide for promoting wound healing, its composition and method of using the same - Google Patents

Abstract

The disclosure is a synthetic peptide composed of an amino acid sequence of the sequence HisThrSerThrGluAlaLys (SEQ ID NO: 1). A pharmaceutical composition containing the peptide for effectively promoting wound healing and a method for promoting wound healing using the peptide are also provided.

Description

Peptide for promoting wound healing, composition thereof and method of using the same

The present invention relates to an effective peptide for promoting wound healing, a composition thereof, and a method for using the same.

Wound healing requires the cooperation of several cell types including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. This process includes cell proliferation and migration, collagen deposition and remodeling, wound contraction and angiogenesis. Fibroblasts are the most important cells for manufacturing and rebuilding the extracellular matrix, and their proliferation and migration are also important roles in the formation of granulation tissue, which subsequently heals the wound. Cell migration involves multiple cyclic steps necessary for wound healing. The basic mode of migration requires the extension of the protrusion, a stable connection leading to the edge of the protrusion, and the forward movement of the cell body, the release of adhesion, and the backward retraction of the cells (Lauffenburger and Horwitz. Cell migration: a physically integrated molecular process. Cell 84: 359–369, 1996.). Because fibroblast migration is important for wound healing, the effects of wound healing can be explored in vitro.

Beneficial is that it is non-toxic, non-antigenic and an inexpensive wound healing agent, with the ability to promote wound healing and to heal unhealed wounds.

Unexpectedly in the present invention, the peptide having the amino acid sequence of HisThrSerThrGluAlaLys (SEQ ID NO: 1) has the effect of promoting fibroblast migration, which makes it have the potential to promote wound healing.

Therefore, in one aspect, the present invention provides a synthetic peptide consisting of SEQ ID NO: 1, which consists of the amino acid sequence of SEQ ID NO: 1, and is named as peptide No.9. This peptide provides an effect to promote wound healing.

In another aspect, the present invention provides a cosmetic or medicinal composition for promoting wound healing, which comprises an effective amount of No. 9 peptide and a cosmetically or pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method for promoting wound healing, which comprises administering a peptide having the amino acid sequence of SEQ ID NO: 1 to an individual in need thereof in an amount effective to promote migration of fibroblasts The amount.

According to one embodiment of the method of the invention, the peptide is administered topically to the individual.

It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The terms "a" or "an" when used with the term "comprising" in the scope and / or specification of a patent application may mean "a", but they may also be used in conjunction with "one or more", "at least one", and "One or more than one" has the same meaning.

The term "peptide" is used herein in its conventional meaning, that is, a polymer whose monomers are amino acids and are linked to each other by an amidine bond. Alternatively, the Refers to polypeptide. When the amino acid is an α-amino acid, an L-optical isomer or a D-optical isomer may be used. In addition, non-natural amino acids such as β-alanine, phenylglycine, and spermine can also be included. Standard abbreviations for amino acids are used.

The term "individual" as used herein refers to a vertebrate, preferably a mammal, and most preferably a human. In the following, people as individuals are particularly referred to as "human individuals."

The term "carrier" as used herein refers to a material that is generally used to formulate a pharmaceutical or cosmetic composition that improves stability, sterility, and delivery. When the peptide delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffers, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. The composition may contain physiologically acceptable auxiliary substances, such as pH adjusting and buffering agents, solution tonicity adjusting agents, wetting agents, and the like, which can access physiological conditions as needed, such as sodium acetate, sodium lactate, chlorine Sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The term "systematic" or "systemically" refers herein to the route by which drugs or other substances are administered to the circulatory system such that the entire body of the individual is affected by the administration. The administration can be performed by enteral administration (absorption of drugs or other substances through the gastrointestinal tract) or parenteral administration such as injection, infusion or implantation.

The terms "topical" or "locally" are used herein in their conventional meaning and refer to locations that can be in or on any part of the body, including but not limited to the epidermis, any other dermis, or any other Body tissue. Local administration or administration means that the peptide is brought into direct contact with a tissue, such as the skin or membrane containing melanin-producing cells.

The term "effective amount" refers herein to a sufficient amount of a peptide that provides a desired therapeutic or cosmetic effect or induces a particular type of response according to the present invention. The demand for the effective amount varies among individuals, depending on the state of the disease, physical condition, age, gender, type and weight of the individual. However, a suitable effective amount can be synthesized by standard methods or in any manner commonly used or known to those skilled in the art. For example, the peptide according to the invention can be administered systemically, transdermally or topically.

The term "pharmaceutically acceptable carrier" as used herein refers to any standard pharmaceutical carrier. The carrier includes, but is not limited to, physiological saline, buffered saline, glucose, water, glycerol, ethanol, propylene glycol, polyoxyethylene castor oil, nano particles, liposomes, polymers, and combinations thereof. In addition to the standard carriers, the pharmaceutical compositions of the present invention consist of one or more excipients commonly used in common standard formulations, such as surfactants, solubilizers, stabilizers, emulsifiers, thickeners and preservatives. Such excipients are well known to those skilled in the art.

As shown in the example, the peptide having the amino acid sequence of SEQ ID NO: 1 can be synthesized by standard methods or in any manner generally used or known to those skilled in the art. It has been shown to be effective in enhancing the expression of keratinocytes and the migration of fibroblasts. Therefore, the present invention also provides a method for promoting wound healing, which comprises administering a peptide having the amino acid sequence of SEQ ID NO: 1 to an individual in need, the amount of which is used to effectively promote collagen or elastin in fibroblasts The expression or amount of fibroblast migration.

The pharmaceutical composition of the present invention may be administered from one or more pharmaceutically acceptable carriers in a manner suitable for the chosen mode of administration, including systemic or local administration by parenteral or parenteral administration such as injection, infusion or implantation. Oral, oral, transdermal or topical administration. In some embodiments of the present invention, the composition is prepared as a pharmaceutically or cosmetically acceptable carrier. The preparation includes a liquid, an ointment, a gel, a slurry, a cream, an emulsion, a powder, and a latex, and any form that can be administered. . In some embodiments, the formulation is administered through a spray device, dressing, or patch.

The invention is further illustrated by the following examples, which are provided for illustrative purposes and are not limiting.

Examples

Example 1: Preparation of the peptide of SEQ ID NO: 1

The peptide of SEQ ID NO: 1 (sequence: HisThrSerThrGluAlaLys) was synthesized by MDBio, Inc. (Taipei, Taiwan) and confirmed by high performance liquid chromatography (HPLC) and mass spectrometry The purity and composition of the peptide. After dissolving 10 mg of lyophilized peptide powder in 250 μl of double deionized water (ddH2O), the peptide raw material was stored at -20 ° C.

Example 2: Cell culture

Human keratinocyte HaCaT and fibroblast CCD-966SK were cultured at 37 ° C and 5% CO ₂ in DMEM (Dulbecco's) containing 10% (v / v) FBS and penicillin / streptomycin (100IU / 50μg / ml). Modified Eagle Medium), in 5% CO 2.

Example 3: Through-hole migration detection

Human keratinocytes HaCaT (5 × 10 ⁴ ) and fibroblasts CCD-966SK (5 × 10 ³ ) in 0.5 ml serum-free DMEM were planted in the upper chamber with a membrane (Corning, USA) with an aperture of 8 μm. Serum-free DMEM (0.5 ml) with or without 50 μg / ml peptide NO. 9 was loaded into the lower chamber in the well of a 24-well culture plate. After 24 hours of incubation, the cells were fixed and stained with 0.5% (w / v) crystal violet (Sigma) containing 20% (v / v) methanol. The number of cells from 5 random migrating cells was counted under a phase contrast microscope. The results were analyzed by Student's t-test and plotted as mean ± SD.

The results are shown in Figures 1A and 1B. FIG. 1A shows a representative diagram of cell migration of human keratinocyte HaCaT after 24 hours incubation of peptide No. 9 at 50 μg / ml. FIG. 1B provides the results of statistical analysis. The effect of peptide NO. 9 on promoting human keratinocyte HaCaT cell migration (P <0.05).

Figures 2A and 2B depict the peptide NO. 9 enhanced cell migration of fibroblast CCD966SK. FIG. 2A shows a representative diagram of cell migration of fibroblast CCD-966SK in a perforated migration assay after 50 hours of culture of 50 μg / ml peptide NO.9. FIG. 2B shows the effect of peptide 9 on the fibroblast CCD-966SK cell migration (P <0.01).

Example 4: MTT detection

Human keratinocyte HaCaT (3 × 10 ³ ) in 100 μl of complete DMEM was seeded in a 96-well plate with or without 50 μg / ml of peptide NO.9. Add 10 microliters of 5mg / ml 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazole bromide solution (MTT; Sigma) to each well, And cultured at 37 ° C for 3 hours. After that, 100 μl of 10% SDS in 0.01N HCl was added to dissolve the MTT formazan crystals. The resulting optical density was measured spectrophotometrically at two wavelengths of 550 and 630 nm.

HaCaT of human keratinocytes was cultured at 50 μg / ml No. 9 peptide for 24 hours and analyzed for cell viability by MTT assay. The results are shown in Figure 3A (24 hours), Figure 3B (48 hours), and Figure 3C (72 hours) Peptide NO.9 had no significant effect on the viability of HaCaT cells.

In the same manner, FIG. 4A shows the cell viability of CCD996SK fibroblasts cultured at 50 μg / ml of No. 9 peptide for 24 hours using the MTT test, showing that No. 9 peptide has no significant effect on the viability of CCD996SK fibroblasts.

FIG. 4A shows the cell survival rate of CCD996SK fibroblast cells cultured at 50 μg / ml of No. 9 peptide for 48 hours using the MTT test, showing that No. 9 peptide has no significant effect on the viability of CCD996SK fibroblasts.

FIG. 4A shows the cell viability of CCD996SK fibroblasts cultured at 50 μg / ml of No. 9 peptide for 72 hours using the MTT test, showing that No. 9 peptide has no significant effect on the viability of CCD996SK fibroblasts.

In view of the above, it is inferred that the peptide of SEQ ID NO: 1 provides anti-aging effects by enhancing the expression of collagen and / or elastin in fibroblasts and the migration of fibroblasts, rather than by the fibroblasts. Viability.

Those skilled in the art will understand that the above specific embodiments may be modified without departing from the broad inventive concept of the present invention. Therefore, it can be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined by the scope of the appended patent applications.

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The previous summary of the invention and the following detailed description are better understood when read in conjunction with the accompanying drawings. To illustrate the invention, the presently preferred embodiments are shown in the drawings.

In the illustration:

FIG. 1A provides a representative image of HaCaT keratinocytes cultured at 50 μg / ml of No. 9 peptide for 24 hours (scale bar, 1 mm) using a transwell migration assay, showing that No. 9 peptide enhances HaCaT keratinocyte migration.

FIG. 1B provides a statistical analysis result of HaCaT keratinocytes cultured at 50 μg / ml of No. 9 peptide for 24 hours (* P &lt; 0.05), showing that No. 9 peptide provides an effect of enhancing HaCaT keratinocyte migration.

FIG. 2A provides a representative image of CCD996SK fibroblasts cultured at 50 μg / ml of No. 9 peptide for 24 hours using a transwell migration assay, showing that No. 9 peptide enhances the migration of CCD996SK fibroblasts.

Figure 2B provides the results of statistical analysis of CCD996SK fibroblasts cultured at 50 μg / ml for 9 hours using peptide migration assay (* P <0.05), showing that 9 peptides provide an enhanced migration of CCD996SK fibroblasts. effect.

Figure 3A provides the cell viability of HaCaT keratinocytes cultured at 50 μg / ml of No. 9 peptide for 24 hours using MTT assay, showing that No. 9 peptide has no significant effect on the viability of HaCaT keratinocytes.

Figure 3B provides the cell viability of HaCaT keratinocytes cultured at 50 μg / ml of No. 9 peptide for 48 hours using the MTT test, showing that No. 9 peptide has no significant effect on the viability of HaCaT keratinocytes.

Figure 3C provides a 72-hour cell survival rate of HaCaT keratinocytes cultured at 50 μg / ml of peptide 9 using MTT assay, showing that peptide 9 has no significant effect on the viability of HaCaT keratinocytes.

FIG. 4A shows the cell survival rate of CCD996SK fibroblast cells cultured at 50 μg / ml of No. 9 peptide for 24 hours using the MTT test, showing that No. 9 peptide has no significant effect on the viability of CCD996SK fibroblasts.

FIG. 4B shows the cell survival rate of CCD996SK fibroblast cells cultured at 50 μg / ml of No. 9 peptide for 48 hours using the MTT test, showing that No. 9 peptide has no significant effect on the viability of CCD996SK fibroblasts.

The CCD996SK fibroblasts were cultured for 72 hours at 50 μg / ml of 9-peptide using the MTT test. It was shown that the 9-peptide had no significant effect on the viability of CCD996SK fibroblasts.

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Claims (6)

A pharmaceutical composition for wound healing, comprising a synthetic peptide such as SEQ ID NO: 1 and a pharmaceutically acceptable carrier, wherein the amount of the synthetic peptide used is an amount effective to enhance the migration of fibroblasts. The pharmaceutical composition according to claim 1, which is formulated as a systemic, transdermal or topical preparation. The pharmaceutical composition of claim 2, wherein the topical preparation comprises an ointment, a gel, and an emulsion. The pharmaceutical composition according to claim 3, which is administered topically. The pharmaceutical composition according to claim 3, which is administered through a spray device, dressing or patch. Use of a pharmaceutical composition according to any one of claims 1 to 5 for the preparation of a medicament for promoting wound healing.