Use of annexin in aging resisting

Abstract

The present invention relates to the use of an annexin in aging resisting, and particularly provides the use of the annexin in the preparation of an anti-aging pharmaceutical composition or health-care product composition.

Description

Application of annexin in anti-aging Technical Field

[0001] The present invention relates to the use of annexin in anti-aging. Specifically, the present invention provides the use of annexin in the preparation of anti-aging pharmaceutical compositions or health care product compositions. Background Art

[0002] Aging is a natural and inevitable process that occurs over time. It is a complex natural phenomenon characterized by structural degeneration and functional decline, ultimately leading to the end of life. While the universality, intrinsic nature, progressiveness, and harmfulness of aging are widely accepted as defining characteristics of aging, various theories regarding its causes, such as the somatic mutation theory, the free radical theory, and the natural cross-linking theory of biomolecules, have yet to be supported by experimental research. Current research suggests that aging is the result of a combination of factors, including stem cell decline, DNA degradation, dietary and psychological factors, and activation of aging genes. However, a unified theory of aging remains elusive. Despite this, the pursuit of anti-aging continues, with many researchers focusing on systemic anti-aging approaches such as stem cells and exosomes. However, the limitations of cell therapy have severely limited their clinical application, and reliable anti-aging methods remain unavailable. In-depth research into the molecular mechanisms of aging and the screening and discovery of novel anti-aging agents provide important theoretical and scientific foundations for proactively delaying aging, preventing and treating age-related diseases, and improving the quality of life of the elderly.

[0003] Therefore, there is a need in the art to provide more drugs with anti-aging activity.

[0004] Summary of the Invention

[0005] The object of the present invention is to provide the use of annexin in the preparation of anti-aging pharmaceutical compositions or health care product compositions.

[0006] In a first aspect, the present invention provides the use of annexin in the preparation of an anti-aging pharmaceutical composition or a health product composition.

[0007] In another preferred embodiment, the anti-aging effect includes delaying systemic aging.

[0008] In another preferred embodiment, the anti-aging effect includes delaying the aging of skin, muscles, organs, bones, joints, and brain.

[0009] In another preferred embodiment, the anti-aging includes prolonging lifespan.

[0010] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0011] In another preferred embodiment, the annexin is annexin A5.

[0012] In another preferred embodiment, the annexin is human annexin or non-human mammal annexin.

[0013] In another preferred embodiment, the non-human mammal includes pigs, monkeys, cows, sheep, rats, mice or rabbits.

[0014] In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier, a healthcare product acceptable carrier, or a cosmetically acceptable carrier.

[0015] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, solid dosage form.

[0016] In another preferred embodiment, the dosage form of the composition is selected from the following group: tablets, capsules, microcapsules, powder injections, oral solutions, and injections.

[0017] The second aspect of the present invention provides a composition, characterized in that the composition comprises:

[0018] (a) Annexin;

[0019] (b) another anti-aging drug selected from the group consisting of rapamycin, metformin, resveratrol, NAD+, or a combination thereof; and

[0020] (c) a pharmaceutically or health-care acceptable carrier.

[0021] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0022] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, solid dosage form.

[0023] In another preferred embodiment, the dosage form of the composition is selected from the following group: tablets, capsules, microcapsules, powder injections, oral solutions, and injections.

[0024] In a third aspect, the present invention provides a method for extending the lifespan of nematodes or delaying aging of nematodes in vitro, comprising the steps of adding annexin to a nematode culture medium and culturing the nematodes, thereby extending the lifespan of the nematodes or delaying aging of the nematodes.

[0025] In another preferred embodiment, the concentration of the culture medium is >0.002 mg / ml, preferably >0.0022 mg / ml, such as 0.005 mg / ml 0.01 mg / ml or 0.02 mg / ml, more preferably 0.0022 mg / ml-0.011 mg / ml.

[0026] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0027] In another preferred embodiment, the method is in vitro, non-diagnostic and non-therapeutic.

[0028] In a fourth aspect, the present invention provides an anti-aging method, comprising the steps of administering to a subject in need thereof a pharmaceutical composition or a health product composition comprising annexin as an active ingredient, thereby achieving anti-aging.

[0029] In another preferred embodiment, the subjects are subjects of various age groups.

[0030] In another preferred embodiment, the subject is a subject that has aged, is aging, or will age.

[0031] In another preferred embodiment, the subject is a human or a non-human mammal.

[0032] In another preferred embodiment, the non-human mammal includes pigs, monkeys, cows, sheep, rats, mice or rabbits.

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows the effect of Annexin A5 on nematode lifespan; DETAILED DESCRIPTION

[0035] After extensive and in-depth research, numerous screenings, and testing, the inventors discovered for the first time that annexin can be used to significantly delay systemic aging and prolong lifespan, and can be used to prepare anti-aging pharmaceutical compositions or health supplement compositions. Based on this, the present invention was completed.

[0036] the term

[0037] 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.

[0038] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0039] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0040] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0041] Annexin

[0042] Annexins are a widely distributed class of calcium-dependent phospholipid-binding proteins. Based on molecular phylogenetic and comparative genomic analyses, this protein family has been uniformly designated "annexin" (abbreviated as Anx), followed by capital letters representing species: A, B, C, D, and E, representing vertebrates, invertebrates, fungi and myxomycetes, plants, and protists, respectively. Currently, the Anx A family has 12 members: A1-A11 and A13 (A12 is unassigned).

[0043] Typically, the annexin includes annexin A5. Annexin A5 (AnxA5) is widely distributed in various tissue cells of the body. In vitro studies have found that AnxA5 has multiple functions such as anti-phospholipase, anti-coagulation, and anti-kinase, but its specific in vivo function is not clear.

[0044] The annexins that can be used in the present invention are not particularly limited and can be annexins derived from any organism, preferably from mammals (such as primates), more preferably from humans, monkeys, rats or mice. The annexins of the present invention can also be functional analogs thereof, such as proteins having 50%, 60%, 70%, 75% or more, such as 80%, 85% or more, 90% or more, or even more preferably 95% or 99% or more identity with human annexin; it should be understood that "annexin" includes wild-type or mutant (including truncated) annexins, as long as the mutant annexins retain or maintain the detoxification activity of the wild-type annexins, they can also be used in the present invention. In addition, the annexins can also be multimers of natural annexins, fusion proteins, or chemically modified variants thereof (such as PEG-modified), provided that these variants have the activity of the wild-type annexins.

[0045] The human annexin A5 sequence is as follows:

[0046] The present invention provides a use of the annexin as an active ingredient in anti-aging.

[0047] Anti-aging

[0048] The anti-aging mentioned in the present invention refers to the process of "preventing", "delaying", "resisting" or "improving" the aging of the body, such as local aging (such as skin, muscles, organs, bones, joints, brain, etc.), especially systemic aging. Biologically speaking, aging is a spontaneous and inevitable process of organisms over time. It is a complex natural phenomenon, manifested as degenerative changes in structure and decline in function, and decreased adaptability and resistance. In the present invention, the aging can be physiological aging or pathological aging, such as natural aging, aging induced by chemotherapy drugs, aging induced by oncogenes, aging induced by radiation, replicative aging, oxidative stress-induced aging, DNA damage-induced aging, or a combination thereof. One aspect of the anti-aging mentioned in the present invention is the extension of lifespan.

[0049] The terms "preventing," "delaying," "resisting," or "improving" aging as described herein include delaying or alleviating one or more indicators or aspects related to aging of the body to a certain extent, but do not necessarily require 100% delay of aging. In some embodiments, compared to the absence of the active ingredient of the present invention, the active ingredient of the present invention or a composition comprising the same delays or alleviates one or more indicators related to aging of the subject by, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 30%; or the lifespan of the subject is extended by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 30%.

[0050] Composition

[0051] The present invention also provides a pharmaceutical composition or a health product composition that can be used for anti-aging, which comprises the above-mentioned annexin as an active ingredient and a pharmaceutically or health product acceptable carrier.

[0052] The term "pharmaceutically or nutraceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gels suitable for human use, of sufficient purity, and with sufficiently low toxicity. "Compatibility" in this context refers to the ability of the components of the composition to blend with the active ingredient of the invention, and with each other, without significantly reducing the compound's efficacy. Such carriers include, but are not limited to, saline, buffered solutions, dextrose, water, glycerol, ethanol, and combinations thereof. The dosage form should be compatible with the route of administration.

[0053] The composition according to the present invention can be used for: parenteral, intravenous, intra-arterial, intraperitoneal, intramuscular or subcutaneous administration. Can be by the active ingredient of the required amount in a suitable solvent and various other ingredients (if necessary) as listed above merged, then by sterilization to make a sterile injectable solution. It is best to use the above composition in the form of a sterile aqueous solution, the sterile aqueous solution can contain other substances, such as enough salt or glucose, so that the solution and blood are isotonic. If necessary, the aqueous solution is appropriately buffered (preferably pH between 3-9). Standard pharmaceutical techniques well known to those skilled in the art can be utilized to easily prepare suitable pharmaceutical formulations under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight-about 50 mg / kg body weight every day.

[0054] The active ingredient of the present invention can be used alone or in combination with other drugs for anti-aging and / or prevention or treatment of related diseases caused by aging. Other drugs for anti-aging and / or prevention or treatment of related diseases caused by aging can be selected from the following group: rapamycin, metformin, resveratrol, NAD+, or a combination thereof.

[0055] The precise amount of active ingredient provided to an individual for a therapeutically effective amount will depend on the mode of administration, the type and severity of the disease and / or condition, and the characteristics of the individual, such as general health, age, sex, weight, and tolerance to the drug. One of ordinary skill in the art will be able to determine an appropriate dosage based on these and other factors. When administered in combination with other therapeutic agents, the "therapeutically effective amount" of any other therapeutic agent will depend on the type of drug used. Suitable dosages are known for approved therapeutic agents and can be adjusted by one of ordinary skill in the art based on the individual's condition, the type of condition being treated, and the amount of the compound of the present invention used below, for example, as reported in the literature and recommended in the Physician's Desk Reference (57th Edition, 2003). Preferably, the composition should be formulated so that an inhibitor dose of 0.01-100 mg / kg body weight / day can be administered to patients receiving these compositions. In certain embodiments, the compositions of the present invention provide a dosage of 0.01 mg to 50 mg. In other embodiments, a dosage of 0.1 mg to 25 mg or 5 mg to 40 mg is provided.

[0056] The present invention also provides an anti-aging method, comprising the steps of administering a pharmaceutical composition or a health product composition comprising annexin as an active ingredient to a subject in need, thereby achieving anti-aging.

[0057] Examples of subjects to whom the active ingredient of the present invention or a pharmaceutical composition comprising the same can be administered include mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.).

[0058] The main advantages of the present invention include:

[0059] The inventors discovered for the first time that annexin can significantly delay aging and prolong lifespan.

[0060] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0061] Annexin A5: extracted and purified from human adipose tissue, accession number: P08758.

[0062] Example 1

[0063] 1. Experimental content

[0064] 1.1 Model preparation

[0065] Synchronized Caenorhabditis elegans (C. elegans) N2 strain: Fertilized eggs of C. elegans were incubated and cultured on NGM solid medium for 3 days and developed into young adults for use in experiments.

[0066] 1.2 Culture conditions

[0067] The first wild-type C. elegans N2 was provided by the Caenorhabditis Genetics Center (CGC) at the University of Minnesota, U.S.A. The nematodes were cultured on NGM (Nematode Growth Medium) solid medium containing Escherichia coli OP50 at a temperature of 20°C and a humidity of 40-60%.

[0068] 1.3 Drug preparation

[0069] Annexin A5, with a stock concentration of 0.55 mg / ml, was diluted with ddH2O to two concentrations, each dissolved in NGM liquid medium (containing FUDR) at a 1 / 10 volume ratio. All drugs and NGM liquid medium were thoroughly mixed in a 3 cm dish and dispensed into 24-well plates, 480 μL per well.

[0070] 2. Experimental Methods

[0071] Wild-type N2 nematodes were synchronized at 20°C (by transferring 20 adult worms to a 6 cm NGM culture plate containing Escherichia coli OP50, and after laying eggs for 2 hours, the adults were removed, leaving only the eggs). After developing into adults, they were picked into 24-well plates containing 480 μL of NGM liquid medium per well. The medium contained an appropriate concentration of sample, with 8-10 nematodes per well. Starting from the 10th day, the survival rate of the nematodes was counted every 1-2 days [2] until all the nematodes died. FUDR (400 μL) was added to the culture medium to inhibit the nematodes from producing offspring.

[0072] 3. Data Analysis

[0073] Data were analyzed using GraphPad.Prism.v5.0. “*” p<0.05, “**” p<0.01, “***” p<0.001.

[0074] 4. Experimental Results

[0075] 4.1 The first life test

[0076] Results: As shown in Table 1, when nematodes were cultured in liquid culture medium containing 0.0022 mg / ml and 0.011 mg / ml (5-fold dilution) of Annexin A5, drug concentrations of 0.0022 mg / ml and 0.011 mg / ml increased nematode lifespan. 50 mM metformin served as a positive control.

[0077] Table 1 Effects of drugs on the survival rate of nematodes lifespan

[0078] 4.2 Second life test

[0079] A second lifespan assay was performed using the same method as the first lifespan assay, but with different concentrations of Annexin A5.

[0080] Results: As shown in Figure 1 and Table 2, when nematodes were cultured in liquid culture medium containing 0.00044 mg / ml, 0.0022 mg / ml, and 0.011 mg / ml (5-fold dilutions), drug concentrations of 0.0022 mg / ml and 0.011 mg / ml increased nematode lifespan, while 0.00044 mg / ml had no significant effect on nematode lifespan compared to the negative control. 50 mM metformin served as a positive control.

[0081] Table 2 Effects of drugs on the survival rate of nematodes lifespan

[0082] 5. Conclusion

[0083] The use of Caenorhabditis elegans to study aging and aging-related diseases has a history of nearly 30 years. The short life cycle of Caenorhabditis elegans, from L4 larvae to adult death, only takes 2-3 weeks, making it an ideal animal for screening anti-aging drugs.

[0084] Experiments of the present invention have shown that when Caenorhabditis elegans is cultured in samples with different concentrations of Annexin A5, concentrations of 0.0022 mg / ml and 0.011 mg / ml of Annexin A5 significantly prolong the lifespan of the nematodes, and there is a clear dose-dependent effect, which indicates that Annexin A5 has significant anti-aging activity, thereby prolonging the survival period of Caenorhabditis elegans.

[0085] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Application of annexin in the preparation of anti-aging pharmaceutical compositions or health care product compositions.

2. The use according to claim 1, characterized in that The anti-aging effect includes delaying systemic aging of the whole body.

3. The use according to claim 1, characterized in that The anti-aging includes delaying the aging of skin, muscles, organs, bones, joints and / or brain.

4. The use according to claim 1, characterized in that The anti-aging effect includes extending lifespan.

5. The use according to claim 1, characterized in that The annexin is annexin A5.

6. The use according to claim 1, characterized in that The annexin is human annexin or non-human mammal annexin.

7. The use according to claim 1, characterized in that The composition further comprises a pharmaceutically acceptable carrier, a health product acceptable carrier, or a cosmetically acceptable carrier.

8. The use according to claim 1, characterized in that The dosage form of the composition is selected from the following group: liquid dosage form, solid dosage form.

9. A composition for anti-aging, characterized in that: The composition comprises: (a) Annexin; (b) another anti-aging drug selected from the group consisting of rapamycin, metformin, resveratrol, NAD+, or a combination thereof; and (c) a pharmaceutically or nutraceutically acceptable carrier.

10. The composition according to claim 9, characterized in that The annexin is annexin A5.

11. An anti-aging method, comprising the steps of: administering a pharmaceutical composition or a health product composition comprising annexin as an active ingredient to a subject in need, thereby achieving anti-aging.

12. The method according to claim 11, characterized in that The annexin is annexin A5.

13. A method for extending the life span of nematodes or delaying the aging of nematodes in vitro, comprising the steps of: adding annexin to a nematode culture medium and culturing the nematodes, thereby extending the life span of the nematodes or delaying the aging of the nematodes.

14. The composition according to claim 13, characterized in that The annexin is annexin A5.

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