CN118994360A - Small molecule active peptide, composition and application thereof - Google Patents

Abstract

The invention provides a small molecule active peptide, which has an amino acid sequence shown as a formula (I), leu-Lys-Lys-Asn-Gly-Ser-Cys-Lys-Arg-Gly (I). The small molecular active peptide provided by the invention is derived from a functional core structural domain of fibroblast growth factor 1, has higher biocompatibility, directly acts on fibroblasts, promotes proliferation, differentiation and migration of the fibroblasts by stimulating a Fibroblast Growth Factor (FGF) receptor (FGFR) signal channel, and directly stimulates endogenous collagen generation; the small molecule active peptide is composed of only 10 amino acids, is favorable for skin absorption, and has higher medical value and commercial value.

Description

Small molecule active peptide, composition and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a small molecule active peptide, a composition and application thereof.

Background

Bioactive peptides are generic term for peptide compounds that are beneficial or physiologically active to the vital activity of a living organism. Is a bioactive substance capable of regulating cell functions, and amino acid molecules are combined to form an amino acid chain, which is the most important active substance and nutrient element in human body, and generally less than 50 amino acids. Wherein, the small molecular protein composed of 2-10 amino acids is called oligopeptide with molecular weight of 50-2000Da, the oligopeptide can penetrate through the stratum corneum space of the skin to enter into the dermis to play a role, and the small molecular protein has higher medical and commercial values.

Bioactive peptides, tretinoin and vitronectin are also known as anti-aging three-giant-head. Wherein, tretinoin is high-efficient but has irritation, and the vitriol causes mild but has long effect time, and the bioactive peptide between tretinoin and vitriol causes has more comprehensive effect. Has high safety and activity diversity, has the functions of anti-inflammatory, antioxidant and whitening and spot-lightening effects while repairing and improving skin, and has the advantages of safety, stability, easy absorption, good effect and the like. Therefore, bioactive peptides are the most popular research subject and functional factors with great development prospects.

Fibroblasts (fibroblastic), also known as fibroblasts, are the major cellular component of loose connective tissue, differentiated from mesenchymal cells in embryonic stages. The fibroblasts are large and well-defined, and mostly have a protruding spindle-shaped or star-shaped flat structure, and the nuclei are regular oval and large and obvious. Accounting for more than 95 percent of all cells in the dermis. The maintenance of structure and function in the whole dermis layer plays a major role, thereby maintaining the firmness, elasticity and moisture of the skin. Fibroblasts are "factories" of synthetic proteins (collagen, elastin) and glycosaminoglycans (hyaluronic acid). The number and quality and activity of fibroblasts in the dermis layer directly determine the degree of aging of the skin. Therefore, the proliferation, differentiation and migration of the fibroblasts are promoted, the activity of the fibroblasts is improved, and the method becomes an important research direction for beautifying and caring skin, resisting aging and removing wrinkles and healing wound tissues.

Currently, peptides applied to the field of medical and cosmetic skin care mainly belong to the following categories: 1. signal peptide (SIGNAL PEPTIDES) -palmitoyl pentapeptide-3, palmitoyl oligopeptide, palmitoyl hexapeptide, palmitoyl tripeptide-5, hexapeptide-9, myristyl pentapeptide-11, and the like; 2. neurotransmitter-rejecting peptides (Neurotransmitter-inhibiting peptides) -acetyl hexapeptide-3, acetyl octapeptide-1, pentapeptide-3, dipeptide snake toxins and pentapeptide-3; 3. a carrier peptide (CARRIED PEPTIDES) -such as copper peptide (marker peptide). Peptides applied to medical and aesthetic skin care in the market belong to cell matrix protein fragments, and are used for inhibiting nerve activity or metal ion carriers, and lack bioactive peptides directly acting on skin cells, particularly skin fibroblasts.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a small molecule active peptide, a composition and application thereof. The small molecule active peptide provided by the invention directly acts on fibroblasts, promotes proliferation, differentiation and migration of the fibroblasts, and directly stimulates endogenous collagen to generate.

According to one technical scheme, the small molecule active peptide has an amino acid sequence shown in a formula (I), and Leu-Lys-Lys-Asn-Gly-Ser-Cys-Lys-Arg-Gly (I).

Preferably, the functional core domain W122-G136 derived from fibroblast growth factor 1.

Preferably, it is obtained by chemical synthesis.

Preferably, the recombinant expression is obtained by a genetic engineering recombinant expression method.

In another aspect of the invention, a composition comprises an effective amount of the small molecule active peptide described above, together with at least one excipient and optionally an adjuvant.

In another aspect of the invention, a delivery system or sustained release system comprises an effective amount of the above-described small molecule active peptide; or a combination of the above.

According to another technical scheme, the skin care product comprises an effective amount of the small molecule active peptide; or a combination thereof; or a delivery system or a slow release system as described above.

Preferably, the skin care product is in a dosage form comprising cream, emulsion, gel, powder, tablet, film, aerosol, spray or implant.

In another aspect of the invention, the use of the small molecule active peptide, or composition, or delivery system or sustained release system described above, for the preparation of a composition for use in cosmetic products.

Preferably, the cosmetic product is used for skin anti-aging, moisturizing, skin or mucous membrane repair, skin soothing.

The term "salt" of the peptide of formula (I) as used herein refers to a salt approved for use in animals, and particularly humans, and may be prepared according to conventional methods known in the art.

By "effective amount" as used herein is meant an amount of the peptide of the invention that is non-toxic but sufficient to provide the desired effect, in the range of 10 to 2000ppm.

The term "delivery system" as used herein refers to a diluent, adjuvant, excipient or carrier with which the peptides of the present invention are administered, and diluents, adjuvants, excipients or carriers known in the art may be employed.

By "sustained release system" as used herein is meant a delivery system that provides gradual release of a drug over a period of time, and is not limited to having a relatively constant level of drug release over the entire period of time.

The beneficial effects are that:

The small molecule active peptide provided by the invention is derived from a functional core structural domain of fibroblast growth factor 1, has higher biocompatibility and directly acts on fibroblasts. By stimulating Fibroblast Growth Factor (FGF) receptor (FGFR) signaling pathway, promoting fibroblast proliferation, differentiation and migration, directly stimulating endogenous collagen production; the small molecule active peptide is composed of only 10 amino acids, is favorable for skin absorption, and has higher medical value and commercial value.

The small molecular active peptide provided by the invention has good skin care activity and high transdermal activity, can slow down skin cell aging, stimulate endogenous collagen regeneration, accelerate wound healing and tissue regeneration, and can be effectively applied to the fields of cosmetics, medical skin care, implants, anti-aging, regenerative medicine and the like.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.

FIG. 1 is a graph showing the results of liquid chromatography (HPLC) analysis of the small molecule active peptide prepared in example 1 of the present invention;

FIG. 2 is a graph showing the results of Mass Spectrometry (MS) analysis of the small molecule active peptide prepared in example 1 of the present invention;

FIG. 3 is an experiment of the small molecule active peptide prepared in example 1 of the present invention to promote in vitro cell proliferation;

FIG. 4 shows the effect of the small molecule peptides of example 1 on in vitro cell proliferation at various doses;

FIG. 5 shows the experimental results of the small molecule active peptide prepared in example 1 of the present invention for promoting in vitro cell migration

FIG. 6 shows the adhesion test results of the small molecule active peptide prepared in example 1 of the present invention.

Detailed Description

The application will be described in detail below with reference to the drawings in connection with embodiments. The principles and features of the present application are described below with reference to the drawings, and it should be noted that embodiments of the present application and features of the embodiments may be combined with each other without conflict. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the application.

The experimental reagents and materials used in the present specification are all commercially available products, except for the specific descriptions.

The small molecule active peptide of the invention is derived from a natural short peptide of a functional core domain W122-G136 of fibroblast growth factor 1 (FGF-1) and consists of 10 amino acids.

The invention uses bioinformatics method, artificial intelligence, molecular design screening model and sequence comparison, such as T-coffee software, to analyze the homology of the region, and obtain the amino acid sequence. One skilled in the art can determine suitable parameters for aligning sequences, including, for example, any algorithm required to achieve a superior alignment or optimal alignment for the full length of the compared sequences, and any algorithm required to achieve a superior alignment or optimal alignment for the parts of the compared sequences.

The amino acids described in the present invention may be any amino acids, not limited to natural and alpha, beta and gamma amino acids. The amino acids of the present invention may be naturally occurring L-or D-forms such that the peptide formed consists of L-amino acids, D-amino acids, or a mixture of L-and D-forms.

In the present invention, the small molecule active peptide may be modified, for example, by pegylation, lipidation, glycosylation and/or acetylation, hydroxylation, N-methylation, phosphorylation, myristoylation and palmitoylation of amino acids. Including structural modifications such as circularization.

The small molecule active peptides of the invention can be prepared by conventional synthetic methods of the prior art, recombinant DNA techniques, enzymatic cleavage of full-length proteins from which the peptide sequences are derived, or a combination of these methods.

For example, by solid phase synthesis, comprising the steps of:

1. Selection and pretreatment of resin: first, it is necessary to select a resin suitable for the target polypeptide, such as a polystyrene resin, a polyacrylamide resin, etc. The choice of resin depends largely on the sequence and scale of synthesis of the polypeptide of interest. The resin needs to be pretreated before use, such as removing protecting groups, activating functional groups, etc.

2. Protection of amino acids: during the synthesis of polypeptides, protection of the amino and carboxyl groups of amino acids is required to prevent side reactions. Typical amino protecting groups are Fmoc (fluorenylmethoxycarbonyl) and Boc (t-butoxycarbonyl), and the carboxyl protecting group is an ester or amide group.

3. Condensation reaction: the first protected amino acid is reacted with the reactive functional group on the resin to form a first peptide bond of the peptide chain. This step is usually carried out using a carbodiimide-based condensing agent such as DIC (N, N ' -diisopropylcarbodiimide) or HBTU (O-benzotriazol-1-yl-N, N, N ', N ' -tetramethyluronium hexafluorophosphate) or the like.

4. Deprotection: after completion of the condensation of one amino acid, the amino protecting group needs to be removed in order to proceed with the condensation of the next amino acid. For Fmoc protecting groups, deprotection is typically performed using a weakly basic solution (e.g., 20% piperidine/DMF); for Boc protecting groups, deprotection is typically performed using an acidic solution (e.g., TFA/DCM).

5. Repeated condensation and deprotection: according to the amino acid sequence of the target polypeptide, the protecting amino acid is condensed onto the peptide chain in turn, and deprotection is performed after each condensation. This process may be performed manually or by an automated polypeptide synthesizer.

6. Cleavage and removal of side chain protecting groups: after all amino acids have condensed onto the peptide chain, the polypeptide needs to be cleaved from the resin and the side chain protecting groups removed. This step is typically performed using a strong acid (e.g., HF) or a strong base (e.g., sodium hydroxide) to cleave the side chain protecting group.

7. Purification and analysis of the polypeptide: the cleaved polypeptides are purified by reverse phase high performance liquid chromatography (RP-HPLC) and analyzed for structure and purity by means of Mass Spectrometry (MS), ultraviolet absorption spectroscopy (UV), and the like.

The small molecule active peptide of the invention can promote proliferation, survival, adhesion and migration of skin cells, in particular skin fibroblasts, by stimulating fibroblast growth factor receptor signaling pathways. Has good skin care activity and high transdermal activity, in particular has good biocompatibility, can slow down skin cell aging, stimulates endogenous collagen regeneration, accelerates the capability of wound healing and tissue regeneration, and can be effectively applied to the fields of medical and aesthetic skin care, three medical instrument implants, anti-aging, regenerative medicine and the like.

The composition of the invention comprises the effective amount of the small molecule peptide, at least one excipient and optional adjuvant, and can be one or more selected from glycerol, mannitol, trehalose and stem cell exosomes.

The delivery system or sustained release system of the present invention comprises the above-described effective amount of a small molecule peptide, or the above-described composition, and further comprises a lipid carrier or hydrogel.

The skin care product of the invention comprises the effective amount of the small molecule peptide, or the composition, or the delivery system or the slow release system.

Example 1

The embodiment is a small molecule active peptide, which is a natural short peptide derived from a functional core domain W122-G136 of fibroblast growth factor 1 (FGF-1), and consists of 10 amino acids, and has a molecular weight of 1000Da. The amino acid sequence was obtained by analysis of the homology of this region by artificial intelligence, molecular design screening model and T-coffee software using bioinformatics method and named XZtide1. The amino acid sequence is shown as a formula (I):

Leu-Lys-Lys-Asn-Gly-Ser-Cys-Lys-Arg-Gly (Ⅰ)；

Wherein Leu is leucine, lys is lysine, asn is asparagine, gly is glycine, ser is serine, cys is cysteine, arg is arginine.

The structural formula is shown as formula (II):

the small molecule active peptide of the embodiment is prepared by adopting a solid phase synthesis method, and the specific steps are as follows:

1. Swelling resin

0.6G of Cl-Gly resin (SD=0.35) was added to the reactor, and DCM (6 mL) was added and swollen by shaking for 5min.

2. Deprotection of resins

The swelling reagent was dried under vacuum, 6mL of 20% pip/DMF was added and the mixture was shaken at room temperature for 20min, and after the deprotection reaction was completed, the reaction solution was removed.

3. Deprotection wash

The deprotected reagent was dried under vacuum and washed 5 times with DMF (6 mL) for 0.5min each and dried for 50s.

4. Deprotection detection

And (3) putting 20 resin particles into a detection test tube, adding 1mL of ninhydrin detection reagent, putting the detection test tube into a metal bath at 100 ℃ for 2min, taking out and observing the color of the resin, and ensuring that the color of the resin becomes dark positive, thereby indicating that the deprotection is successful.

5. Condensation of the second amino acid

5ML (5 eq) of a pre-prepared 0.2M Fmoc-Arg (pbf) -OH solution containing Oxyma was taken, 3eq DIC was added thereto, and the reaction was allowed to shake at room temperature for 35min.

6. Reaction detection and washing

Putting 20 resin particles into a detection test tube, adding 1mL of ninhydrin detection reagent, putting the detection test tube into a metal bath at 100 ℃ for 2min, taking out and observing the color of the resin, and ensuring that the resin color has no obvious change and the condensation is successful. After the condensation was successful, the reaction mixture was washed 3 times with DMF (6 mL) each for 0.5min and drained for 50s.

7. Peptide chain extension

The procedure of steps 2 to 6 was repeated, condensing the amino acids in the sequence in sequence from right to left.

8. Resin pumping

After the end of the deprotection of the last amino acid, DMF (6 mL) was washed 3 times and the resin was washed 3 times with methanol (6 mL) and dried for cleavage.

9. Cleavage of

Add 6mL of lysate: TFA (92.5%) TIS (2.5%) EDT (2.5%) was cleaved at room temperature for 3h, 2 tubes of reaction solution were filtered out, 3mL of each tube was slowly added to 30mL of glacial ethyl ether, the precipitate was centrifuged, washed 3 times with glacial ethyl ether, the rotational speed was set at 3000R/min in the centrifuge, and the centrifugation time was 2min, yielding crude polypeptide solid to be purified.

10. Crude product central control detection

The chromatographic column is a C18 reverse phase silica gel column; column temperature: 40 ℃; detection wavelength: 220nm and 254nm; flow rate: 1.0mL/min; sample injection amount of automatic sample injection needle: 5 μl; mobile phase a: aqueous 0.065% tfa; mobile phase B: acetonitrile solution of 0.05% tfa. Referring to fig. 1 and 2, the HPLC analysis results and mass spectrometry analysis results of the small molecule active peptides prepared in this example are shown.

11. Crude purification

Taking crude peptide, adding acetonitrile/pure water mixed reagent for ultrasonic dissolution, and filtering by a vacuum filter and loading after a sample is clear and transparent. C18 gradient elution is carried out according to the arrangement, the peak type is judged by 220nm wavelength, the liquid is collected by the tube to be replaced with obvious turns, and the tube is replaced properly according to the situation without obvious turns.

12. Freeze-drying split charging

The wall-hung bottle is frozen by liquid nitrogen, and is placed on a freeze dryer for wall-hanging freeze-drying after being frozen. And after the freeze-drying is finished, taking down the freeze-drying bottle, weighing and split charging the polypeptide sample, and taking a small sample for QC detection.

The purity of the small molecule active peptide sample obtained in the embodiment is more than 95%, and the mass-to-charge ratio (M/z) of [ M+H ] ⁺ excimer ion peak is 1090.31.

Test of the small molecule active peptide (XZtide 1) prepared in this example

1. In vitro cell proliferation assay

1. NIH/3T3 (mouse embryo fibroblasts) and HacaT (human epidermal cell line cells) in logarithmic growth phase were taken, pancreatin digested, counted, and cell density was adjusted to 4X 10 ⁴ to prepare 100. Mu.L of cell suspension in 96-well plates. The plates were pre-incubated in an incubator for 24 hours at 37℃with 5% CO ₂.

2. To the plates 10. Mu.L of XZtide to be tested 1 were added and incubated in the incubator for 72 h.

3. To each well 10. Mu.L of CCK-8 solution was added.

4. The culture plate is incubated in an incubator for 1-4 hours.

5. The absorbance at 450nm was measured with a microplate reader.

And (3) activity calculation: .

Cell viability (%) = [ a (dosing) -a (blank) ]/[ a (0 dosing) -a (blank) ]x100;

Wherein, a (dosing): absorbance of wells with cells, CCK-8 solution and XZtide a drug solution;

A (blank): absorbance of wells with medium and CCK-8 solution without cells;

A (non-dosed): absorbance of wells with cells, CCK-8 solution without XZtide a drug solution.

Referring to FIG. 3, the effect of XZtide1 on mouse embryonic fibroblasts NIH/3T3 is shown, and compared to the blank, the experimental group clearly shows that XZtide1 promotes proliferation of mouse embryonic fibroblasts NIH/3T3 cells. Referring to FIG. 4, XZtide1 experimental group had the highest effect on cells at concentrations of 0.1nm, 1nm, 2nm, 4nm, 6nm, and 10nm, respectively, and it was seen that proliferation was highest at a concentration of 2 nm.

2. In vitro cell migration experiments

1. Cell plate coating the experimental group, which was diluted XZtide with coating buffer, and the reference group, which was FGF1 as reference, were brought to different concentrations (1 nM-10 μm), 1ml was added to each well, overnight at 4 ℃, or water bath at 37 ℃ for 1 hour. The coating solution was discarded, and PBS was washed twice for further use.

2. Cell culture

Marking three horizontal and longitudinal marking lines of each hole behind the 6-hole plate by using a marker pen, sowing cells according to about 5 multiplied by 10 ⁵ of each hole, and aiming at reaching a confluence state of 95-100% after culturing for 24 hours; each group had 3 duplicate wells.

3. Scratch test

Drawing 3 transverse lines on the bottom of the pore plate by using a marker pen compared with a ruler, aligning the pore plate by using a 10 mu L gun head compared with the ruler after culturing the cells for 24 hours, lightly pushing down the longitudinal streaks to form scratches, rinsing the cells for 3 times by using PBS, removing the scratched cells, and adding 2mL of serum-free culture medium into an experimental group and a reference group respectively; a blank control group was set.

After culturing in a 5% CO ₂ incubator at 37℃for 0h, 24h, 48h, taking photographs under a 20-fold microscope with the intersection of transverse and longitudinal streaks as the core, obtaining photographs of 9 sites per well, and combining 27 data.

4. Data processing

The scratch area was measured and the cell mobility of each group was calculated by dividing the total area of the stationary scratch area transitional cells by the initial area of the stationary scratch area.

Referring to fig. 5, the effect of XZtide on human epidermal cell line HacaT cell migration is shown, with XZtide being evident that human epidermal cell line HacaT cell migration is promoted by XZtide compared to the placebo group.

3. Adhesion test

1. Cell preparation

NIH/3T3 cells were cultured at 37℃in a 5% CO ₂ incubator, and the cell density and status were observed under an inverted microscope every day. When the cells grow to 80% -90% of the culture flask, the cells are passaged. Cells were diluted to 5X 10 ⁴/mL using complete medium that had been pre-mixed with Hoechst33342 fluorescent stain (10%). mu.L of cells were added to the wells, covered with aluminum foil, and incubated for 1h at 37℃in a 5% CO ₂ incubator. 3 replicates were measured and well 4 was used to adjust microscope parameters.

2. Detection of

Using a relative centrifugal force of 350g, a fluorescent tile was captured for each of the 3 wells using an inverted microscope. Each well was filled with D-PBS to form a "reverse meniscus", the bubbles were purged and covered with a sealing film. Centrifuge (placed upside down) at 22℃for 5min with optimal relative centrifugal force. After centrifugation, the sealing film was discarded, and the supernatant was removed from the well. After washing 1 time with D-PBS, 100. Mu. L D-PBS was added. Shooting was performed for each of the 3 wells.

3. Result calculation

The number of fluorescently labeled nuclei was counted using an automatic cell count procedure and the number of cells before and after centrifugation was determined. The percentage adhesion is calculated according to formula (a):

V=nt/nc×100% (a), where:

V-percentage adhesion;

Cell number after Nt-centrifugation;

nc-number of cells before centrifugation;

The relative percentage adhesion is calculated according to formula (b):

p=v1/V2 (b), wherein:

P-relative cell adhesion ratio;

v1-average value of adhesion percentage of each compound hole of the sample;

average percent adhesion for each duplicate well of V2-negative control.

Referring to FIG. 6, the effect of XZtide1 on mouse embryonic fibroblasts NIH/3T3 is shown by fluorescent staining experiments, and XZtide1 experiments clearly showed an effect of promoting the adhesion of mouse embryonic fibroblasts NIH/3T3 cells compared to the blank.

Example 2

The embodiment is a composition, which comprises the small molecular active peptide prepared in the embodiment 1, and glycerin and water are added into the small molecular active peptide product according to the prescription amount, and the small molecular active peptide is completely dissolved.

Example 3

This example is a delivery system comprising the small molecule active peptide prepared in example 1, wherein the carrier is a lipid carrier or hydrogel and can be prepared in a manner conventional in the art.

Example 4

The embodiment is a moisturizing and anti-aging cosmetic liquid, which comprises the small molecular active peptide of the embodiment 1 or the composition of the embodiment 2, and the dosage form of the skin care product can be selected from cream, emulsion, gel, powder, tablet, film, aerosol, spray or implant, and can be prepared according to the conventional operation mode in the field.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (10)

1. The small molecule active peptide is characterized by having an amino acid sequence shown in a formula (I), wherein Leu-Lys-Lys-Asn-Gly-Ser-Cys-Lys-Arg-Gly (I).

2. The peptide of claim 1, wherein the functional core domain W122-G136 is derived from fibroblast growth factor 1.

3. The peptide according to claim 1, characterized in that it is obtained by chemical synthesis.

4. The peptide according to claim 1, characterized in that it is obtained by means of recombinant expression by genetic engineering.

5. A composition comprising an effective amount of the peptide of claim 1, and at least one excipient and optionally an adjuvant.

6. A delivery system or sustained release system comprising an effective amount of the peptide of claim 1; or the composition of claim 5.

7. A skin care product comprising an effective amount of the peptide of claim 1; or the composition of claim 5; or the delivery system or sustained release system of claim 6.

8. The skin care product according to claim 8, wherein the formulation of the skin care product comprises cream, emulsion, gel, powder, tablet, film, aerosol, spray or implant.

9. Use of the peptide of claim 1, or the composition of claim 6, or the delivery system or sustained release system of claim 7, in the preparation of a composition for use in a cosmetic product.

10. The use according to claim 10, wherein the cosmetic product is for skin anti-ageing, moisturizing, skin or mucous membrane repair, skin soothing.