CN109912685B - Eye protection peptide and composition thereof and use of the same - Google Patents

Abstract

The present invention relates to eye protection peptides, compositions thereof and uses of the peptides. Specifically, the present invention provides a synthetic peptide, which is composed of the amino acid sequence of the sequence ArgAsnProLeuGluGluThr (SEQ ID NO: 1). At the same time, a pharmaceutical composition containing the peptide and helpful for eye protection or eye health and its use are provided.

Description

Eye protection peptide and composition thereof and use of the same

technical field

The present invention relates broadly to peptides effective in protecting eye health, compositions thereof and uses of the peptides.

Background technique

Without eyes, we cannot operate computers, watch television, read newspapers, drive vehicles and play chess. Recently, most people spend a lot of time watching movies through non-mobile or mobile devices, such as televisions, computers, desktop computers, laptops, tablets, mobile phones, etc., resulting in visual impairment or a series of symptoms such as dry eyes, discomfort, irritation , burning, redness, excessive tearing, blurred vision, fatigue, soreness, nervousness, photophobia, myopia, astigmatism.

To prevent any damage to the eyes, there are many methods developed in different ways. For example, eye fatigue can be easily relieved by sleeping, dozing off, closing eyes, exercising, music, farsightedness, massaging the eyes, wearing sunglasses, taking vitamins, eye drops, etc. There are other ways to reduce eye irritation.

Medications such as vitamins or herbs or combinations thereof have been proposed to meet the needs of eye health. But so far, drugs that benefit eye health are uncertain.

We still hope to find or develop a non-toxic, non-antigenic and inexpensive eye health agent for eye health.

SUMMARY OF THE INVENTION

Unexpectedly in the present invention, the peptide having the amino acid sequence of ArgAsnProLeuGluGluThr (SEQ ID NO: 1) has an effect of enhancing the migration of corneal epithelial cells, which is beneficial for eye protection or eye health.

Therefore, the present invention provides, in one aspect, a synthetic peptide comprising the amino acid sequence of SEQ ID NO: 1, which is named peptide No. 12. This peptide provides an effect of enhancing the migration of corneal epithelial cells.

In another aspect, the present invention provides a pharmaceutical composition beneficial to eye protection or eye health, comprising an effective amount of SEQ ID NO: 1 and a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the composition is formulated for systemic or topical application. In another specific embodiment, the composition is formulated for topical administration, such as eye drops.

In another aspect, the present invention provides a method of eye protection for preventing eye damage, comprising administering to an individual in need thereof a peptide having the amino acid sequence of SEQ ID NO: 1 in an amount effective to enhance migration of corneal epithelial cells amount of utility.

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

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

Description of drawings

The foregoing summary and the following detailed description of the invention are better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred.

In the attached image:

Figure 1A and Figure 1B show that peptide No. 12 enhances the migration of corneal epithelial cell HCECs at 24 hours; wherein Figure 1A provides a representative graph of 24 hours of culture with different concentrations of peptide No. 12 (scale bar, 1 mm) using a transwell migration assay. . Figure IB provides the results of statistical analysis of the transpore migration assay at 24 hours (*P<0.05).

Figures 2A and 2B show that peptide No. 12 enhances the migration of corneal epithelial cell HCECs at 48 hours; wherein Figure 2A provides a representative graph of 48 hours of culture with different concentrations of peptide No. 12 using transwell migration assay (scale bar, 1 mm). . Figure 2B provides the results of statistical analysis of the transpore migration assay at 48 hours (**P<0.01).

Figure 3 shows that peptide No. 12 has no significant effect on the viability of HCEC cells at 24 hours, wherein HCEC cells were cultured at different concentrations of peptide No. 12 for 24 hours by MTT assay.

Figure 4 shows that peptide No. 12 had no significant effect on the viability of HCEC cells at 48 hours, wherein HCEC cells were cultured at different concentrations of peptide No. 12 for 48 hours by MTT assay.

Detailed ways

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

The term "a" or "an" when used with the term "comprising" in the claims and/or specification can mean "an," but it can 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 sense, i.e. a polymer whose monomers are amino acids, linked to each other by amide bonds, alternatively it refers to a polypeptide (polypeptide). When the amino acid is an α-amino acid, either the L-optical isomer or the D-optical isomer can be used. In addition, unnatural amino acids such as beta-alanine, phenylglycine, and homoarginine may also be included. Standard abbreviations for amino acids are used.

The term "individual" as used herein refers to a vertebrate, preferably a mammal, especially a human. In the following, a person as an individual is specifically referred to as a "human individual".

As used herein, the term "carrier" refers to a material typically used to formulate pharmaceutical or cosmetic compositions that enhance 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, eg, water, buffer, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. The composition may contain physiologically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, solution tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, chlorine Sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The terms "systemic" or "systemically" herein refer to the route of administration of a drug or other substance to the circulatory system such that the entire body of an individual is affected by the administration. The administration can be by enteral administration (absorption of the drug or other substance through the gastrointestinal tract) or parenteral administration such as injection, infusion or implantation.

The terms "topical" or "topical" are used herein in their conventional sense to refer to a location that may be in or on any part of the body, including but not limited to the epidermis, any other dermis, or any other body tissue. Topically applying or administering means bringing the peptide into direct contact with tissue, such as skin or membranes containing melanin-producing cells.

The term "effective amount" as used herein refers to a sufficient amount of the peptide to provide the desired therapeutic or cosmetic effect or to induce a particular type of response in accordance with the present invention. The effective amount required varies among individuals, depending on the state of the disease, physical condition, age, sex, type and weight of the individual, and the like. 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 peptides 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, saline, buffered saline, dextrose, water, glycerol, ethanol, propylene glycol, polyoxyethylene castor oil, nanoparticles, liposomes, polymers, and combinations thereof. In addition to standard carriers, the pharmaceutical compositions of the present invention are comprised 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 Examples, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 can be synthesized by standard methods or in any manner commonly used or known to those skilled in the art. It has been shown to be effective in enhancing keratinocyte expression and fibroblast migration. Therefore, the present invention also provides a method for promoting wound healing, which comprises administering to an individual in need a peptide having the amino acid sequence of SEQ ID NO: 1 (also named as peptide No. 12) in an amount effective to improve corneal epithelium The amount of cell migration.

In another aspect, the present invention provides a pharmaceutical or health protection composition that is beneficial to eye protection or eye health using the peptide of the invention.

In addition, the present invention provides a composition or pharmaceutical composition that is helpful for eye protection or eye health, comprising an effective amount of SEQ ID NO: 1 and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may be administered by one or more pharmaceutically acceptable carriers in a manner suitable for the chosen mode of administration, including systemic or topical administration, by enteral or parenteral administration such as injection, infusion or Implant, oral, transdermal or topical administration. Some embodiments of the present invention, whose compositions are prepared as pharmaceutically or cosmetically acceptable carriers, include liquids, ointments, gels, serums, creams, lotions, powders and latexes and any administrable form. In some embodiments, the formulation is administered as drops in one eye.

The invention is further illustrated by the following examples, which are provided for the purpose of illustration and not limitation.

Example

Example 1: Preparation of No. 12 peptide

The peptide of SEQ ID NO: 1 (sequence: ArgAsnProLeuGluGluThr) was synthesized by MDBio, Inc. (Taipei, Taiwan, China), and confirmed by high performance liquid chromatography (HPLC) and mass spectrometry. Purity and composition of peptides. 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: Efficacy experiment

Materials and Methods

cell culture

Human corneal epithelial cells HCEC were cultured in serum-free medium containing keratinocytes, 5ng/ml human recombinant EGF, 0.05mg/ml bovine pituitary extract, 0.005mg/ml at 37°C with 5% _CO2 Insulin and 500ng/ml of cortisol.

Through-hole migration assay

Cells (3.5×10 ⁴ ) in 0.25 ml serum-free medium of keratinocytes were seeded in the upper chamber with 8 μm pore size membranes (Corning, USA). Serum-free KSF with or without peptide number 12 was loaded into the lower chamber of the wells of a 24-well plate. After 24 or 48 hours of culture, cells were fixed and stained with 0.5% (w/v) crystal violet (Sigma) containing 20% (v/v) methanol. The number of migrated cells from 5 random fields was counted under a phase contrast microscope. The results were analyzed by Student's t-test and plotted as mean ± standard deviation. The results were obtained from two independent experiments.

MTT detection

结晶。以分光亮度计测定在550和630nm的双波长下的所得光密度。Cells ( ² x 104) in complete KSF were seeded in 12-well plates with or without peptide #12. 70 μl of 5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide solution (MTT; Sigma) was added to each well, and incubated at 37°C for 3 hours. After that, add 700 μl of 10% SDS in 0.01 N HCl to dissolve the MTT formazan

crystallization. The resulting optical density was measured spectrophotometrically at dual wavelengths of 550 and 630 nm.

result

Human corneal epithelial cell HCECs were treated with peptide number 12 at concentrations of 5, 10, 25, 50 μg/ml for 24 and 48 hours and then analyzed using a transwell migration assay. As shown in Figure 1A, Figure 1B, Figure 2A, Figure 2B, peptide No. 12 enhanced the migration of corneal epithelial cell HCEC. However, Figures 3 and 4 show that peptide No. 12 had no significant effect on the viability of HCEC cells at concentrations of 5, 10, 25, 50 μg/ml, 24 and 48 hours.

In view of the above, it is inferred that the peptide of SEQ ID NO: 1 enhances the migration of corneal epithelial cells, but not the viability of corneal epithelial cells.

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

sequence listing

<110> Sanfan Biotechnology R&D Co., Ltd.

<120> Eye protection peptide and composition thereof and use of the same

<130> ASB0004WO

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 7

<212> PRT

<213> Artificial sequences

<400> 1

Arg Asn Pro Leu Glu Glu Thr

1 5

Claims (9)

What is claimed is: 1. A synthetic polypeptide consisting of the amino acid sequence of ArgAsnProLeuGluGluThr (SEQ ID NO: 1). 2. A composition or pharmaceutical composition useful for eye protection or eye health, comprising an effective amount of the synthetic polypeptide of claim 1, the effective amount being an amount effective to promote migration of corneal epithelial cells, and A pharmaceutically acceptable carrier. 3. The eye protection or eye health benefit composition or pharmaceutical composition of claim 2 formulated into a systemic or topical formulation. 4. The eye protection or eye health benefit composition or pharmaceutical composition of claim 2 formulated into a topical formulation. 5. The eye protection or eye health benefit composition or pharmaceutical composition of claim 3, wherein the topical formulation is in the form of eye drops. 6. Use of a synthetic polypeptide as claimed in claim 1 for the preparation of a medicament for eye protection to prevent eye damage. 7. The use of claim 6, wherein the drug is administered systemically or topically to an individual. 8. The use of claim 7, wherein the medicament is administered topically to the individual. 9. The use according to claim 7, wherein the medicament is formulated as eye drops.

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