A drug for treating osteoarthritis

Abstract

The present invention provides a drug for treating osteoarthritis. Specifically, the present invention for the first time provides the use of annexin A2 in the preparation of a pharmaceutical composition for preventing or treating osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a drug for treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is the most common joint disease characterized by degeneration of articular cartilage. OA mainly affects the major weight-bearing joints, including the hip and knee joints. The clinical manifestations of OA are joint pain, swelling, limited mobility, and dysfunction. The changes in the intra-articular tissues of OA are manifested as destruction and reduction of cartilage matrix, osteophyte formation, subchondral bone sclerosis, and aseptic synovitis. The occurrence of OA is affected by multiple mechanisms, among which the main factors are genetics, trauma, age, and obesity. However, despite the increasing incidence and severity of OA, due to insufficient understanding of the pathogenesis of the disease, there is currently no treatment or effective disease-modifying therapy. The current medical treatment for OA, such as corticosteroids, hyaluronic acid, and opioids, is mainly used to relieve symptoms that can neither prevent nor reverse cartilage degeneration. In addition, these drugs have side effects and toxicity. Due to the irreversible disease progression, most patients eventually require joint replacement. These limitations require the development of new therapies that can improve cartilage structure and reverse cartilage damage in addition to anti-inflammatory and analgesic effects to improve overall joint function while reducing side effects. However, such a treatment method does not currently exist. Therefore, there is an urgent need to find effective drugs to delay the progression of OA and study its potential molecular mechanisms. Summary of the Invention

[0003] The object of the present invention is to provide a drug for treating osteoarthritis.

[0004] In a first aspect of the present invention, the use of annexin A2 in the preparation of a pharmaceutical composition for preventing or treating osteoarthritis is provided.

[0005] In another preferred embodiment, the osteoarthritis includes joint cartilage degeneration and injury, and reactive hyperplasia of the joint margin and / or subchondral bone caused by aging, obesity, strain, trauma, and / or congenital joint abnormalities.

[0006] In another preferred embodiment, the osteoarthritis is selected from the group consisting of cervical osteoarthritis, lumbar osteoarthritis, knee osteoarthritis, hip osteoarthritis, or a combination thereof.

[0007] In another preferred embodiment, the osteoarthritis includes knee osteoarthritis. Knee osteoarthritis is a chronic osteoarthropathy mainly characterized by degenerative changes of knee joint cartilage.

[0008] In another preferred embodiment, the prevention or treatment of arthritis is carried out by one or more of the following methods selected from the group:

[0009] (a) Reduce cartilage damage;

[0010] (b) Promote chondrocyte proliferation;

[0011] (c) Alleviate joint (including synovium) inflammatory response;

[0012] (d) Alleviate joint pain.

[0013] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0014] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, a topical preparation or an injection preparation.

[0015] In another preferred embodiment, the injection preparation is an intravenous injection or an intramuscular injection.

[0016] In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as tablets, capsules, microcapsules, powder injections, oral liquids, injections, solutions, gels, emulsions, ointments, creams, pastes, powders, patches or lozenges.

[0017] In another preferred embodiment, the composition or preparation is administered by topical, local, or subcutaneous injection.

[0018] In a second aspect of the present invention, there is provided a pharmaceutical composition, the composition comprising (a) annexin A2; and (b) a pharmaceutically acceptable carrier.

[0019] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0020] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, a topical preparation or an injection preparation.

[0021] In another preferred embodiment, the injection preparation is an intravenous injection or an intramuscular injection.

[0022] In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as tablets, capsules, microcapsules, powder injections, oral liquids, injections, solutions, gels, emulsions, ointments, creams, pastes, powders, patches or lozenges.

[0023] In another preferred embodiment, the composition or preparation is administered by topical, local, or subcutaneous injection.

[0024] In another preferred embodiment, in the pharmaceutical composition, the mass percentage of annexin A2 is 0.01 wt% - 99 wt%, preferably 0.1 - 50 wt%, 1 - 20 wt%, based on the total weight of the composition.

[0025] In the third aspect of the present invention, there is provided a method for preventing or treating osteoarthritis, which comprises administering annexin A2 or the composition of the second aspect of the present invention to a subject in need thereof.

[0026] In another preferred embodiment, the subject is a human or non-human mammal.

[0027] In another preferred embodiment, the non-human mammal includes rodents such as rats and mice.

[0028] 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 specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Promotion of chondrocyte proliferation by annexin A2.

[0030] Figure 2 Protective effect of annexin A2 on chondrocytes.

[0031] Figure 3 Treatment with annexin A2 alleviates joint pain symptoms.

[0032] Figure 4 Annexin A2 inhibits synovial inflammation in joints.

[0033] Figure 5 Annexin A2 protects against cartilage damage. DETAILED DESCRIPTION OF THE INVENTION

[0034] Through extensive and in-depth research, and through a large number of screenings and tests, the present inventors have for the first time provided the use of annexin A2 in preventing or treating osteoarthritis. On this basis, the present invention has been completed.

[0035] TERMS

[0036] Unless otherwise defined, 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.

[0037] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of...", or "consisting of...".

[0038] Annexin A2

[0039] Annexin is a soluble protein that exists in eukaryotes and is absent in yeast and prokaryotes. Vertebrates have a total of 12 annexins, named Annexin. Annexins have the property of binding to membranes and expose negatively charged phosphatidylserine in a calcium-dependent manner. Annexin A2 is a 39 kDa member of the annexin family, expressed in most cells and tissues and binds to many ligands. Annexin A2 is a pleiotropic protein closely related to functions such as cell transmembrane transport, cytoskeletal dynamics, extracellular receptor activity, and signal transduction, promoting pathological processes such as invasion and migration of various tumor cells, proliferation and survival of vascular endothelial cells, migration of skin fibroblasts, and repair after intestinal mucosal epithelial injury. Currently, there is a lack of corresponding treatment methods for cartilage damage in osteoarthritis, and annexin A2, as a new active ingredient, plays a role in promoting chondrocyte regeneration and has great application potential.

[0040] The annexin A2 that can be used in the present invention can be annexin A2 derived from any organism, preferably from mammals (such as primates), and more preferably from humans, monkeys, rats, or mice. The annexin A2 of the present invention can also be its functional analogs, such as proteins having 50%, 60%, 70%, more than 75%, such as 80%, more than 85%, more than 90%, or even more preferably more than 95% or 99% identity with human annexin A2; it should be understood that "annexin A2" includes wild-type or mutant (including truncated) annexin A2, as long as the mutant annexin A2 retains or maintains the activity of wild-type annexin A2, it can also be used in the present invention. In addition, annexin A2 can also be a multimer, fusion protein, or chemically modified variant (such as PEG modification) of natural annexin A2, provided that these variants possess the activity of wild-type annexin A2.

[0041] The human-derived annexin A2 sequence is shown as SEQ ID No:1 below:

[0042] 1gctcagcatt tggggacgct ctcagctctc ggcgcacggc ccagcttcct tcaaaatgtc

[0043] 61tactgttcac gaaatcctgt gcaagctcag cttggagggt gatcactcta cacccccaag

[0044] 121tgcatatggg tctgtcaaag cctatactaa ctttgatgct gagcgggatg ctttgaacat

[0045] 181tgaaacagcc atcaagacca aaggtgtgga tgaggtcacc attgtcaaca ttttgaccaa

[0046] 241ccgcagcaat gcacagagac aggatattgc cttcgcctac cagagaagga ccaaaaagga

[0047] 301acttgcatca gcactgaagt cagccttatc tggccacctg gagacggtga ttttgggcct

[0048] 361attgaagaca cctgctcagt atgacgcttc tgagctaaaa gcttccatga aggggctggg

[0049] 421aaccgacgag gactctctca ttgagatcat ctgctccaga accaaccagg agctgcagga

[0050] 481aattaacaga gtctacaagg aaatgtacaa gactgatctg gagaaggaca ttatttcgga

[0051] 541cacatctggt gacttccgca agctgatggt tgccctggca aagggtagaa gagcagagga

[0052] 601tggctctgtc attgattatg aactgattga ccaagatgct cgggatctct atgacgctgg

[0053] 661agtgaagagg aaaggaactg atgttcccaa gtggatcagc atcatgaccg agcggagcgt

[0054] 721gccccacctc cagaaagtat ttgataggta caagagttac agcccttatg acatgttgga

[0055] 781aagcatcagg aaagaggtta aaggagacct ggaaaatgct ttcctgaacc tggttcagtg

[0056] 841cattcagaac aagcccctgt attttgctga tcggctgtat gactccatga agggcaaggg

[0057] 901gacgcgagat aaggtcctga tcagaatcat ggtctcccgc agtgaagtgg acatgttgaa

[0058] 961aattaggtct gaattcaaga gaaagtacgg caagtccctg tactattata tccagcaaga

[0059] 1021cactaagggc gactaccaga aagcgctgct gtacctgtgt ggtggagatg actgaagccc

[0060] 1081gacacggcct gagcgtccag aaatggtgct caccatgctt ccagctaaca ggtctagaaa

[0061] 1141accagcttgc gaataacagt ccccgtggcc atccctgtga gggtgacgtt agcattaccc

[0062] 1201ccaacctcat tttagttgcc taagcattgc ctggccttcc tgtctagtct ctcctgtaag

[0063] 1261ccaaagaaat gaacattcca aggagttgga agtgaagtct atgatgtgaa acactttgcc

[0064] 1321tcctgtgtac tgtgtcataa acagatgaat aaactgaatt tgtactttag aaacacgtac

[0065] 1381tttgtggccc tgctttcaac tgaattgttt gaaaattaaa cgtgcttggg gttcagctgg

[0066] 1441tgaggctgtc cctgtaggaa gaaagctctg ggactgagct gtacagtatg gttgccccta

[0067] 1501tccaagtgtc gctatttaag ttaaatttaa atgaaataaa ataaaataaa atca

[0068] Osteoarthritis and Its Symptoms

[0069] Osteoarthritis (OA) is a cartilage degenerative disease that begins in articular cartilage and gradually erodes into subchondral bone and surrounding tissues, leading to focal and erosive joint lesions, thereby causing symptoms such as joint pain, joint stiffness, joint swelling, movement disorders, and deformities.

[0070] In the present invention, there are many inducing factors for osteoarthritis, and the etiology is not yet fully clear. It may be related to factors such as advanced age, obesity, drugs, and occupational overuse.

[0071] In the present invention, the occurrence site of the osteoarthritis is not particularly limited. For example, it can be weight-bearing joints and joints with a relatively large amount of activity, such as arthritis in the cervical vertebrae, lumbar vertebrae, knee joints, hip joints, etc.

[0072] The present invention first discovers that annexin A2 can be used as an active ingredient for preventing or treating osteoarthritis.

[0073] Terms such as "prevent", "delay", "treat", "resist", or "improve" in the present invention include delaying or alleviating one or more indicators or aspects related to osteoarthritis to a certain extent, but do not necessarily require 100% cure. In some embodiments, compared with the absence of the active ingredient of the present invention, the active ingredient of the present invention or a composition containing it delays or alleviates one or more indicators related to arthritis 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%.

[0074] Compositions and Administration

[0075] The compositions of the present invention include (but are not limited to): annexin A2 and a pharmaceutically acceptable carrier.

[0076] The term "pharmaceutically or nutraceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatible" herein means that the components in the composition can be admixed with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the compound. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical dosage form should be matched with the mode of administration.

[0077] Representatively, the annexin A2 of the present invention can be formulated into pharmaceutical compositions in dosage forms such as tablets, capsules, powders, microparticles, solutions, lozenges, gels, cream preparations, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical compositions can be prepared by commonly known preparation techniques, and suitable pharmaceutical additives can be added to the drugs.

[0078] The compositions according to the present invention can be used for: parenteral, intravenous, intra-arterial, intraperitoneal, intramuscular or subcutaneous administration. A sterile injectable solution can be prepared by combining the required amount of the active ingredient in a suitable solvent with the various other ingredients (if required) listed above and then sterilizing. It is preferred to use the above compositions in the form of a sterile aqueous solution, which may contain other substances, such as sufficient salts or glucose, to make the solution isotonic with blood. If necessary, the aqueous solution is appropriately buffered (preferably with a pH between 3 and 9). Suitable pharmaceutical formulations can be easily prepared under sterile conditions using standard pharmaceutical techniques well known to those skilled in the art. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight per day to about 50 milligrams per kilogram of body weight.

[0079] The active ingredient of the present invention can be used alone or in combination with other drugs, such as anti-inflammatory and analgesic drugs.

[0080] The precise amount of the active ingredient to be administered to an individual in 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. A person 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. Appropriate dosages are known for approved therapeutic agents and can be adjusted by a person of ordinary skill in the art based on the condition of the individual, the type of condition being treated, and the amount of the compound of the present invention used hereinafter, for example, the dosages reported in the literature and recommended in the Physician’s Desk Reference (57th Edition, 2003). Preferably, the composition should be formulated such that an inhibitor dosage 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, dosages of 0.1 mg - 25 mg or 5 mg - 40 mg are provided.

[0081] The present invention also provides a method for preventing or treating osteoarthritis, comprising administering an annexin A2 or the composition of the second aspect of the present invention to a subject in need thereof, thereby preventing or treating osteoarthritis.

[0082] Examples of subjects to whom the active ingredient of the present invention or the pharmaceutical composition containing the same is administered include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.).

[0083] The main advantages of the present invention include:

[0084] 1. The present invention discovers for the first time the use of annexin A2 in preventing or treating osteoarthritis.

[0085] 2. The present invention provides a new drug for preventing or treating osteoarthritis.

[0086] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0087] Example 1

[0088] (1) Chondrocyte experiment

[0089] 1. Rat knee joint chondrocytes were cultured in vitro, and different concentrations of Annexin A2 (SEQ ID No: 1) were added during the culture process. After 72 hours of culture, cell proliferation was detected by CCK8. The cell proliferation rate was calculated compared with the control group without drugs.

[0090] 2. Rat knee joint chondrocytes were cultured in vitro, and a cell injury model was established by adding sodium iodoacetate (final concentration: 2 μM) during the culture process. Different concentrations of Annexin A2 were added simultaneously. After 72 hours of culture, cell proliferation was detected by CCK8. The cell proliferation rate was calculated compared with the control group without drugs.

[0091] Experimental results

[0092] 1. As Figure 1 shown, the cell proliferation ability of rat chondrocytes was enhanced under the treatment of Annexin A2. Among them, the effect of the concentration of 0.25 μg / ml was better than that of 0.1 μg / ml, showing a dose-dependent effect.

[0093] 2. As Figure 2 shown, the cell proliferation of rat chondrocytes was inhibited in the presence of sodium iodoacetate, and the cell proliferation ability was protected in the presence of Annexin A2.

[0094] Example 2

[0095] (2) Animal experiment for arthritis

[0096] 1. SPF-grade male SD rats (8 weeks old), weighing 280 - 320 g. They had free access to water and food. The room temperature in the animal house was (25 ± 2) °C, and the relative humidity was 65% ± 3%. There was a 12-hour light / dark cycle every day. They were adaptively fed for 2 weeks.

[0097] 2. Animal grouping: control group, model group, and model + Annexin A2 low (0.1 μg / ml), high (1 μg / ml) dose groups, with 5 animals in each group.

[0098] 3. Rats were anesthetized by inhaling isoflurane (concentration: 2 - 3% for induction, 1.5 - 2% for maintenance). A rat knee osteoarthritis model was prepared by intra-articular injection of sodium iodoacetate. The injection dose was 1 mg of sodium iodoacetate (20 mg / ml) per joint, and the injection volume was 0.05 ml.

[0099] 4. Treatment: Drug treatment was given 7 days after the injection of sodium iodoacetate. According to the specified dose, Annexin A2 was intra-articularly injected, with an injection volume of 0.1 ml, once a week for six consecutive weeks. The model group was injected with 0.1 ml of normal saline. The pain threshold of the affected limb was detected weekly during this period.

[0100] At the 7th week after treatment, the animals were sacrificed, and the knee joints of the rats were taken. They were fixed with 4% paraformaldehyde for 24 h, decalcified with EDTA decalcifying solution for 4 weeks, dehydrated, embedded in paraffin, sectioned, and stained with HE.

[0101] Experimental results

[0102] 1. As Figure 3 shown, intra-articular injection of sodium iodoacetate can cause joint pain and shorten the pain threshold time. In the annexin A2 treatment group, the pain was improved and gradually returned to normal with the increase of treatment times, and the higher the concentration, the better the effect; while the model group recovered slowly.

[0103] 2. As Figure 4 shown, histological analysis of the synovial tissue of the joint showed that there were a large number of inflammatory cell infiltrations in the synovium of the model group, and the inflammatory cell infiltrations in the annexin A2 treatment group were not obvious, and the higher the concentration, the better the effect.

[0104] 3. As Figure 5 shown, histological analysis of the articular cartilage showed degeneration and destruction of the articular cartilage in the model group, and the articular cartilage in the annexin A2 treatment group was intact, with no obvious difference from the normal control group.

[0105] In summary, the above results prove that annexin A2 has the effects of reducing cartilage damage, promoting chondrocyte proliferation, reducing joint inflammation, and alleviating joint pain, thus treating osteoarthritis.

[0106] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of annexin A2 in the preparation of a pharmaceutical composition for preventing or treating osteoarthritis, wherein the prevention or treatment of osteoarthritis is one or more selected from the following groups: (a) Reducing cartilage damage; (b) Promoting chondrocyte proliferation; (c) Alleviating joint inflammatory response; and (d) Alleviating joint pain; and the annexin A2 is human wild-type annexin A2.

2. The use according to claim 1, wherein, the osteoarthritis includes degenerative damage of articular cartilage, reactive hyperplasia of joint margin and / or subchondral bone caused by aging, obesity, strain, trauma and / or congenital joint abnormalities.

3. The use according to claim 1, wherein, the osteoarthritis is selected from the following groups: cervical osteoarthritis, lumbar osteoarthritis, knee osteoarthritis, hip osteoarthritis, or a combination thereof.

4. The use according to claim 1, wherein, the prevention or treatment of osteoarthritis is to promote chondrocyte proliferation and / or alleviate joint inflammatory response.

5. The use according to claim 1, wherein, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

6. The use according to claim 1, wherein, the dosage form of the pharmaceutical composition is an injection preparation.

7. The use according to claim 6, wherein, the dosage form of the pharmaceutical composition is a solid dosage form or a liquid dosage form.

8. The use according to claim 6, wherein, the injection preparation is an intravenous injection or an intramuscular injection.

9. The use according to claim 1, wherein, the gene sequence of the annexin A2 is as shown in SEQ ID No: 1.

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