Novel matrix metalloproteinase-1 inhibitor and application thereof in skin aging resistance

Abstract

The invention relates to the field of biological medicine and cosmetics, in particular to a novel matrix metalloproteinase-1 inhibitor and application thereof in skin aging resistance.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and cosmetics. Specifically, it relates to an inhibitor of a novel matrix metalloproteinase-1 and its application in skin anti-aging. Background Art

[0002] Matrix metalloproteinases (MMPs) are a group of the most important proteases containing Zn 2+ that are involved in the degradation of the extracellular matrix (ECM). Generally, MMPs exist in the form of zymogens, and under specific circumstances, they can be activated by natural activators present in the body or inhibited by specific inhibitors (TIMP) in the body (Gu Guoli et al., 2007). MMPs are widely involved in various physiological and pathological processes and play important roles in the metabolic regulation of ECM, cell migration, tissue repair and remodeling, immune response, and tumor invasion and metastasis. The members of the MMPs family are expressed differently in different tissues and cell types, and their abnormal expression is closely related to the occurrence and development of various diseases, such as inflammatory diseases, cardiovascular diseases, osteoporosis, tumors, etc. Therefore, MMPs have become important indicators and targets for clinical disease diagnosis, treatment, and prognosis evaluation, and are also one of the important directions for drug research and development.

[0003] At least 23 kinds of MMPs are expressed in the human body, and they are usually classified into collagenases (MMP-1, MMP-8, MMP-13), gelatinases (MMP-2, MMP-9), stromelysins (MMP-3, MMP-10, MMP-11), matrilysins (MMP-7, MMP-26), membrane-type MMPs (MT MMPs), or other MMPs according to their substrates and domain compositions.

[0004] The latest research shows that: Photoaging is the result of long-term exposure of the skin to sunlight. This exposure can lead to the overexpression of matrix metalloproteinases (MMP), resulting in abnormal degradation of collagen in the skin tissue, thus leading to skin aging and damage. Most of the current MMP inhibitors are artificial chemical synthesis inhibitors, which have certain side effects and safety risks. A few natural inhibitors, such as tissue inhibitor of metalloproteinase (TIMP), belong to macromolecular proteins and have defects such as low yield, easy degradation, and poor oral absorption, and their applications are greatly limited.

[0005] Based on the deficiencies of the prior art, the inventors of the present application obtained several bioactive polypeptides that can effectively inhibit MMPs, especially MMP-1, and verified through experiments that they have multiple effects such as anti-aging activity, collagen-promoting activity, anti-inflammatory activity, antioxidant activity, anti-wrinkle effect, and firming effect as active ingredients in cosmetics / drugs / skin care products. As a natural active peptide, it also has advantages such as small side effects and good safety. This polypeptide sequence has protein polypeptide homology with a variety of animals and plants (such as amphibians, plants, etc.), and it is a very promising bioactive peptide raw material. Summary of the Invention

[0006] The present invention relates to an inhibitor of a novel matrix metalloproteinase-1 and its application in skin anti-aging;

[0007] In a first aspect, the present invention relates to a bioactive peptide, the amino acid sequence of which is selected from the group consisting of ISYGNNALMP, ISCGNCALMP, and ISFGNINALMP.

[0008] In some embodiments, the bioactive peptide is modified by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification, glycosylation, etc. on the amino acid side chain groups, amino terminus, or carboxyl terminus to obtain a derivative of the cyclic peptide, while not affecting its biological activity.

[0009] In some embodiments, the N-terminus of the bioactive peptide is acetylated and the C-terminus is amidated.

[0010] In a second aspect, the present invention relates to a composition comprising the bioactive peptide and a pharmaceutically acceptable carrier.

[0011] In some embodiments, the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate buffered saline, balanced salt solution, and dimethyl sulfoxide (DMSO).

[0012] In other embodiments, the composition can be administered orally, by inhalation, rectally, nasally, ophthalmically, or parenterally.

[0013] In other embodiments, the composition is selected from the group consisting of oral dosage forms, parenteral dosage forms, buccal dosage forms, sublingual dosage forms, nasal dosage forms, inhalers, nebulizers, topical dosage forms, transdermal dosage forms, and suppositories.

[0014] In a third aspect, the present invention relates to the use of the above bioactive peptide or composition in the preparation of cosmetics or skin care products or drugs having anti-aging activity, collagen-promoting activity, anti-inflammatory activity, antioxidant activity, anti-wrinkle effect, and firming effect.

[0015] Fourth aspect, the present invention relates to the use of the above-mentioned bioactive peptide or composition in the preparation of matrix metalloproteinases (MMPs) inhibitors.

[0016] In some embodiments, the matrix metalloproteinases include: MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-12, MMP-19 and MMP-26.

[0017] In some embodiments, the matrix metalloproteinases preferably consist of the group consisting of MMP-1 and MMP-2.

[0018] Fifth aspect, the present invention relates to a method for preparing the bioactive peptide, which is obtained by chemical synthesis or genetic engineering.

[0019] Sixth aspect, the present invention relates to a method for inhibiting the activity of MMP-1 enzyme, which comprises contacting the MMP-1 enzyme with the aforementioned bioactive peptide.

[0020] Seventh aspect, the present invention relates to the use of the bioactive peptide in combination with other matrix metalloproteinase inhibitors in the preparation of drugs having anti-tumor activity, anti-aging activity, collagen-promoting activity, anti-inflammatory activity, antioxidant activity, anti-wrinkle effect and firming effect.

[0021] In some embodiments, the matrix metalloproteinase inhibitors include: Endostatin, Angiostatin, Batimastat (BB294), AG3340, RO3223555, CGS27023A, Bay1224566 and Marimastat.

[0022] Eighth aspect, the present invention relates to the dosage of the bioactive peptide. In some embodiments, the relevant dosages include: 0.01 μM - 200 μM, preferably 1 - 100 μM, more preferably 10 - 50 μM, and most preferably 25 μM. Description of the Drawings

[0023] Figure 1 : Results of the MMP-1 enzyme activity inhibition rate experiment.

[0024] Figure 2 : Detection results of the proliferation inhibitory activity of IP10-3 on senescent cells.

[0025] Figure 3 : Detection results of the proliferation inhibitory activity of IP10-3 on normal cells.

[0026] Figure 4: Experimental results of the collagen-promoting and anti-aging effects of IP10-3 on HFF-1 cells.

[0027] Figure 4A : Effects of IP10-3 on the expressions of MMP-1, p21, p16, and COL1A1 in HFF-1 cells.

[0028] Figure 4B : Effects of IP10-3 on the expressions of IL-1β, TNF-α, IL-6, and COL3A1 in HFF-1 cells.

[0029] Figure 5 : Experimental results of the repair effect of P10-3 on oxidative stress (ROS) in HFF-1 cells.

[0030] Figure 6: Experimental results of the functional verification of IP10-3 bioactive peptide using an ex vivo skin model.

[0031] Figure 6A : Experimental verification of the thickness of the viable epidermal cell layer and the relative area of collagen fibers.

[0032] Figure 6B : Experimental verification of the efficacy of multiple collagens. Detailed implementation manners

[0033] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Regarding the definitions and terms in this field, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0034] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently necessarily contains certain errors, which are caused by the standard deviations existing in their respective measurements. Additionally, all ranges disclosed herein should be understood to encompass any and all sub-ranges contained therein. For example, the range of "1 to 10" recited should be considered to include any and all sub-ranges between the minimum value of 1 and the maximum value of 10 (including the endpoints); that is, all sub-ranges starting from the minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with the maximum value of 10 or less, such as 5.5 to 10. Additionally, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0035] Collagen refers to a biomolecule, which is the main component of animal connective tissue and the most abundant and widely distributed functional protein in mammals, accounting for 25% - 30% of the total protein. In some organisms, it even reaches over 80%. The functions of each subtype of collagen are slightly different. For example, the main functions of type I collagen are: providing structural support for the skin, increasing elasticity, and reducing wrinkles; the main effects of type III collagen are: increasing skin softness and elasticity, and promoting skin repair and regeneration; type IV collagen can maintain the integrity of the basement membrane and promote skin self-repair and renewal; the importance of type XVII collagen lies in its connection between the epidermis and the dermis, maintaining the integrity of the skin structure, promoting epidermal regeneration, and improving skin problems.

[0036] Table 1: Reagents involved in this application

[0037]

[0038] Example 1: MMP-1 Enzyme Activity Inhibition Experiment

[0039] Experimental principle: The fluorescence substrate method is used to detect the change in the enzyme activity of MMP-1. Specifically: MMP-1 can cleave the fluorescence substrate FS-6 (Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2), separating the fluorescent group and the quenching group of the fluorescence substrate, and emitting fluorescence. Within a certain period of time, the increase in fluorescence intensity is proportional to the reaction rate of the enzyme, that is, the rate of increase in fluorescence intensity over time can represent the magnitude of enzyme activity.

[0040] Experimental steps:

[0041] 1. To activate MMP-1, dilute the MMP-1 protein to 100 μg / mL in TCBN buffer (50 mM Tris, 10 mM CaCl2, 150

[0042] mM NaCl, 0.05% Brij-35 (w / v), pH 7.5), use 1 mM phenylmercuric acetate (APMA) (MedChemExpress) as the activator, and incubate at 37 °C for 2 h.

[0043] 2. Dilute the activated MMP-1 to 0.5 μg / mL, and react 0.01 μg of MMP-1 with different concentrations of polypeptide inhibitors IP10-2, IP10-3, IP10-4 (0, 0.01, 0.1, 1, 10, 100 μM) in the detection buffer at 37 °C for 45 min. Finally, add 10 μL of FS-6 (final concentration 5 μM) to each reaction well.

[0044] 3. The above reaction was carried out in a black 96-well microplate, making the total reaction system 100 μL. Continuous readings were taken for 5 minutes at an excitation wavelength / emission wavelength = 325 nm / 400 nm in kinetic mode using a multimode microplate reader (Revvit). For the fluorescence changes monitored by the multimode microplate reader, all detections were in triplicate, and the average value was taken. The IC50 value of the inhibitor and the remaining relative enzyme activity of MMP-1 were obtained using GraphPad Prism 9.5 software. IP10-3 was dissolved in 100% (v / v) dimethyl sulfoxide (DMSO) (Solarbio LifeSciences) solvent, and the polypeptide inhibitor solutions used in the activity inhibition experiments were all diluted to a final DMSO concentration of 1% (v / v). The solvent control group had the same components as the other groups except that IP10-3 was replaced with the same dose of DMSO solvent. In addition, NNGH, as a broad-spectrum inhibitor, was used as a positive reference in the experiment.

[0045] The experimental results are as Figure 1 shown: 10 μM of IP10-3 had an 87% inhibitory effect on the enzyme activity of MMP-1, and its activity could be completely inhibited at 100 μM. In contrast, 0.1 - 100 μM of IP10-2 and 4 had almost no inhibitory effect on the enzyme activity of MMP-1. In addition, after detection, the IC50 value of IP10-3 was 5.622 μM.

[0046] Example 2: Proliferation Inhibitory Activity of IP10-3 on Photoaged HFF-1 Cells

[0047] 2.1 Construction of a UVA-induced HFF-1 cell senescence model

[0048] Human skin fibroblasts HFF-1 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and placed in high-glucose DMEM medium (WISENT) containing 15% FBS (Gibco, Thermo Fisher Scientific), 100 U / mL penicillin (BasalMedia), and 100 μg / mL streptomycin (BasalMedia), and cultured in an incubator at 37 °C and 5% CO2. For the UVA-induced HFF-1 cell senescence model, the cells were exposed to a UVA phototherapy instrument (Sigma) that emits UVA with a wavelength of 320 - 400 nm. The UVA irradiation intensity was detected by a TM-213 mini ultraviolet illuminometer (TENMARS). After removing the medium before UVA irradiation, the cells were washed twice with phosphate buffer PBS (BasalMedia), and the cells were covered with a thin layer of PBS and placed 15 cm away from the UVA phototherapy instrument between the instrument and the cell culture dish for UVA treatment. After UVA treatment, the cells were washed twice with PBS and then incubated in fresh serum-free medium for 24 hours. The control group cells were subjected to the same medium change without UVA treatment. The cells were separately harvested for RNA extraction and RT-qPCR was performed to examine the expression of senescence factors (MMP-1, COL1A1, COL3A1, p21, p16). Finally, the 2-ΔΔCT method was used to analyze the relative quantitative expression level of RNA to screen the most suitable senescence induction dose; through experiments, it was found that: under UVA treatment of 15 J / cm 2 2, MMP-1 had a significant expression, the expression of COL1A1 also decreased significantly, COL3A1 also showed a decreasing trend, the expression of p21 increased significantly, and p16 also showed an increasing trend, indicating that this UVA dose is suitable for constructing the relevant model; further results showed that: compared with the blank group, when the UVA radiation dose was lower than 15 J / cm 2 2, the viability of HFF-1 cells did not change significantly. Therefore, this radiation dose is suitable for modeling.

[0049] 2.2 Proliferation detection of IP10-3 on HFF-1 cells

[0050] The experiment was divided into a blank group, a UVA group, a UVA + 50 μM IP10-3 group, and a UVA + 100 μM IP10-3 group. That is, the blank group was not subjected to UVA treatment, the UVA group was only subjected to UVA treatment, and the remaining experimental groups were incubated for another 24 h with the addition of the corresponding concentration of IP10-3 after UVA treatment, and then the cells or cell supernatants were further studied; the Cell Counting Kit-8 (Biosharp Life Sciences) was used to measure the cell viability of HFF-1 in different groups. HFF-1 was seeded at 3×10 3Cells were seeded in 96-well plates at a density of Figure 2 per well. After treatment with the corresponding dose of UVA the next day, cells were treated with IP10-3 at corresponding concentration gradients diluted in serum-free medium. Incubation was continued for 24 h, and 10 μL of CCK-8 solution (10%) was added to each well of HFF-1 and incubated in an incubator for 1 h. Finally, cell viability was measured using a multifunctional microplate reader at an absorbance of 450 nm. The experimental results are shown as Figure 3 follows: IP10-3 at 50 and 100 μM significantly inhibited the proliferation of senescent cells. In contrast, it had no significant effect on normal cells without UVA treatment (see

[0051] Example 3: Collagen-Promoting and Anti-Aging Effects of IP10-3 on HFF-1 Cells

[0052] The experiment was divided into a blank group, a UVA group, a UVA + 50 μM IP10-3 group, and a UVA + 100 μM IP10-3 group. That is, the blank group was not treated with UVA, the UVA group was only treated with UVA, and the remaining experimental groups were treated with UVA and then incubated for an additional 24 h after adding the corresponding concentration of IP10-3, and then the cells or cell supernatants were further studied. HFF-1 cells were seeded in 6-well plates at 4 cells per well. The next day, when the cell density reached 70%, after treatment with UVA (15 J / cm2), the cells were replaced with IP10-3 (0, 50, 100 μM) prepared in serum-free medium and incubated for an additional 24 h. The control group was not treated with ultraviolet light. RNA was extracted and reverse transcribed, and real-time fluorescence quantitative detection (MMP-1, p16, p21, COL1A1, COL3A1, IL-1β, TNF-α, IL-6) was performed using cDNA. The results showed (see Figures 4A - 4B ) that after treatment with IP10-3, the expression of MMP-1, p21, p16, IL-1β, TNF-α, and IL-6 in cells (senescent cells) treated with UVA decreased significantly; after treatment with IP10-3, the expression of COL1A1 and COL3A1 in cells (senescent cells) treated with UVA increased significantly. Thus, IP10-3 has significant effects on promoting collagen production, anti-inflammatory response, and anti-aging.

[0053] Example 4: Repair of Oxidative Stress in HFF-1 Cells by P10-3

[0054] The experiment was divided into a blank group, a UVA group, a UVA + 50 μM IP10-3 group, and a UVA + 100 μM IP10-3 group. That is, the blank group was not treated with UVA, the UVA group was only treated with UVA, and the remaining experimental groups were treated with UVA and then incubated for an additional 24 h after adding the corresponding concentration of IP10-3, and then the cells or cell supernatants were further studied.

[0055] HFF-1 cells were seeded in 24-well plates at a density of 3.5×10 4 cells per well and allowed to adhere overnight. After the cells were treated with a UVA dose of 15 J / cm2, they were further incubated in serum-free medium containing the corresponding concentration of IP10-3 for 24 h. Then the cells were washed twice with PBS, and 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) (Sigma), a ROS detection reagent, was added to each well. The cells were incubated at 37 °C in the dark for 30 min, and the excess fluorescent probe that did not enter the cells was washed away with PBS. Fluorescence was measured using a microplate reader at an excitation wavelength / emission wavelength of 488 nm / 520 nm.

[0056] The experimental results showed (see Figure 5 ): The intracellular ROS content gradually decreased with the treatment of IP10-3, which was beneficial to reducing the damage of ROS stress response to cells.

[0057] Example 5: Functional Verification of IP10-3 Bioactive Peptide on Ex Vivo Skin Model

[0058] The ex vivo skin model (Boxi Biotechnology) was placed in the culture mold of a 6-well plate, and 3.7 mL of culture medium was added to each well. The model was cultured in an incubator at 37 °C and 5% CO2, and the medium was changed daily. After 2 days of model culture, irradiation and drug administration were started according to the test grouping and corresponding treatment conditions in Table 2. The irradiation doses were UVA (30 J / cm2) and UVB (50 mJ / cm2), and continuous irradiation was performed for 4 days. Fresh culture medium was replaced before each irradiation. After irradiation, the drug given the previous day was wiped off with a sterile cotton swab, and then drug administration was performed. The positive control group was administered by subcutaneous injection, and the sample group was administered by topical application. After 4 days of continuous irradiation, the model was further cultured for 3 days. During this period, no irradiation was performed, and only drug administration was carried out. After the culture was completed, the remaining test substances were washed with a wash bottle containing sterile PBS solution, and the remaining liquid was wiped off with a sterile cotton swab. After washing, the model for detection was fixed with 4% paraformaldehyde for 24 h, and then H&E and Masson staining were performed respectively. Photographs were taken and analyzed under a microscope. After washing, the model for detection was fixed with 4% paraformaldehyde for 24 h, and then immunofluorescence detection was performed. Photographs were taken and analyzed under a microscope.

[0059] Table 2: Principles of test grouping and experimental conditions

[0060]

[0061] Note: VC and VE are vitamin C and vitamin E respectively, commercially purchased from Boxi Biotechnology

[0062] The comprehensive experimental results (see specifically Figures 6A - 6B ) are as follows:

[0063] Anti-wrinkle efficacy:

[0064] Based on the ex vivo skin model, compared with the control group, at a concentration of 25 μM, the thickness of the viable epidermal cell layer of sample IP10-3 increased significantly, with a promotion rate of 18.96%. The contents of collagen fibers, type I collagen (Collagen I), and type III collagen (Collagen III) all increased significantly, with promotion rates of 27.78%, 64.15%, and 176.67% respectively. This indicates that at this concentration, the sample can improve tissue morphology, increase the contents of collagen fibers, type I collagen (Collagen I), and type III collagen (Collagen III), and has an anti-wrinkle effect.

[0065] Firming effect:

[0066] Based on the ex vivo skin model, compared with the control group, at a concentration of 25 μM, the contents of type IV collagen (Collagen IV) and type XVII collagen (Collagen XVII) of sample IP10-3 increased significantly, with promotion rates of 119.23% and 94.87% respectively. This indicates that at this concentration, the sample can increase the contents of type IV collagen (Collagen IV) and type XVII collagen (Collagen XVII), and has a firming effect.

[0067] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A biologically active peptide, whose amino acid sequence is ISCGNCALMP.

2. The bioactive peptide according to claim 1, wherein the N-terminus is acetylated and the C-terminus is amidated.

3. A composition comprising the bioactive peptide according to any one of claims 1 or 2 and a pharmaceutically acceptable carrier.

4. The composition of claim 3, wherein the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate buffered saline, balanced salt solution and dimethyl sulfoxide (DMSO).

5. The composition of any one of claims 3-4, which is in a dosage form selected from the group consisting of oral dosage forms, parenteral dosage forms, buccal dosage forms, sublingual dosage forms, nasal dosage forms, inhalers, nebulizers, topical dosage forms, transdermal dosage forms and suppositories.

6. Use of the bioactive peptide according to any one of claims 1 to 2 or the composition according to any one of claims 3 to 5 in the preparation of cosmetics or skin care products or medicines having anti-aging activity, pro-collagen activity, anti-inflammatory activity, antioxidant activity, anti-wrinkle effect and firming effect.

7. Use of the bioactive peptide according to any one of claims 1 to 2 or the composition according to any one of claims 3 to 5 in the preparation of a matrix metalloproteinase (MMPs) inhibitor.

8. The use according to claim 7, wherein the matrix metalloproteinase comprises: MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-12, MMP-19 and MMP-26.

9. Use of the bioactive peptide according to any one of claims 1 to 2 or the composition according to any one of claims 3 to 5 in combination with a matrix metalloproteinase inhibitor for the preparation of a drug having anti-tumor activity, anti-aging activity, pro-collagen activity, anti-inflammatory activity, antioxidant activity, anti-wrinkle effect and firming effect.

10. The use according to claim 9, wherein the matrix metalloproteinase inhibitor comprises: Endostatin, Angiostatin, Batimastat (BB294), AG3340, RO3223555, CGS27023A, Bay1224566 and Marimastat.

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