CN115645525B - Application of CTGF (cytotoxic T-lymphocyte) neutralizing antibody in preparation of medicine for preventing and treating postmenopausal osteoarthritis - Google Patents
Application of CTGF (cytotoxic T-lymphocyte) neutralizing antibody in preparation of medicine for preventing and treating postmenopausal osteoarthritis Download PDFInfo
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- CN115645525B CN115645525B CN202211600221.3A CN202211600221A CN115645525B CN 115645525 B CN115645525 B CN 115645525B CN 202211600221 A CN202211600221 A CN 202211600221A CN 115645525 B CN115645525 B CN 115645525B
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Abstract
The invention discloses application of a CTGF neutralizing antibody in preparation of a medicament for preventing and treating postmenopausal osteoarthritis, wherein the CTGF neutralizing antibody is applied to subchondral bone of the postmenopausal osteoarthritis once and takes effect in a short time. The CTGF neutralizing antibody can be prepared into a pharmaceutical composition with other medically acceptable auxiliary materials or carriers. The CTGF neutralizing antibody provided by the invention can reduce catabolism indexes, increase the expression of ACAN and Collagen-II, and protect cartilage homeostasis so as to inhibit cartilage degeneration; in addition, the composition can increase the amount of subchondral bone, increase bone density, and improve the microstructure of subchondral bone, thereby inhibiting the subchondral bone loss caused by estrogen deficiency. And the single injection can last long-term curative effect, avoids the problem of repeated administration of other osteoarthritis treatment medicines, enhances the adherence and convenience of administration, and has better effect.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to application of a CTGF neutralizing antibody in preparation of a medicament for preventing and treating postmenopausal osteoarthritis.
Background
Osteoarthritis (OA) is a common degenerative disease of the joints, placing a heavy burden on patients, families, and society. The pathological changes are manifested as cartilage degeneration, subchondral bone sclerosis, osteophyte formation. Over 3 hundred million OA patients are reported worldwide, with women having a higher incidence than men. With the increasing aging of the population, OA will become an important problem affecting the quality of life of people, and the market demand for osteoarthritis drugs will continue to expand.
According to the Chinese osteoarthritis diagnosis and treatment guideline 2021 edition, the current OA treatment drugs include: analgesic, joint cavity injection, slow-acting medicine for relieving OA symptoms, anxiolytic and Chinese patent medicine. However, these drugs have certain disadvantages, such as (1) vigilance of gastrointestinal and cardiovascular adverse events when used to treat OA, such as non-selective nsaids and selective cyclooxygenase 2 inhibitors. (2) Such as glucosamine, chondroitin sulfate, etc., generally have slow effect and take effect after several months of treatment. Therefore, the development of a novel osteoarthritis treatment drug which is good in safety, quick in curative effect and prominent in curative effect has important clinical significance.
Chondrocytes are the only cell type in cartilage Tissue, and chondrocyte homeostasis is regulated by a variety of cytokines, including Connective Tissue Growth Factor (CTGF). The concentration of CTGF is obviously increased when OA is carried out, and the concentration of CTGF in joint synovial fluid and serum is in direct proportion to the severity of OA.
At present, no document reports the new application of the CTGF neutralizing antibody as a medicament for preventing and treating postmenopausal osteoarthritis.
Disclosure of Invention
The invention aims to solve the technical problem of providing an application of a CTGF neutralizing antibody in preparing a medicament for preventing and treating postmenopausal osteoarthritis, wherein the CTGF neutralizing antibody is applied to subchondral bone of the postmenopausal osteoarthritis for a single time and takes effect in a short time. It is manifested by increased subchondral bone mass, increased bone density, and alleviation of cartilage extracellular matrix loss.
The technical problem to be solved by the invention is realized by the following technical scheme:
application of CTGF neutralizing antibody in preparing medicine for preventing and treating postmenopausal osteoarthritis.
A pharmaceutical composition for preventing or treating post-menopausal osteoarthritis, comprising an effective amount of a CTGF neutralizing antibody, and a pharmaceutically acceptable adjuvant or carrier.
Preferably, the adjuvant comprises one or more of a diluent, a solvent, an excipient, an absorbent, a wetting agent, an adhesive, a disintegrating agent, a lubricant, a solubilizer, an emulsifier, a suspending agent, a surfactant, a film-forming agent, a propellant, an antioxidant, a fragrance, a bactericide or a preservative.
Preferably, the pharmaceutical composition further comprises a bioengineered material comprising plastids, nanoparticle materials, microspheres, gels, and bioscaffolds.
Preferably, the effective amount of the CTGF neutralizing antibody in the pharmaceutical composition is 10-20 mu g/ml, the injection amount is 100 mu l, and the injection site is the knee joint subchondral bone in a single injection.
Preferably, the CTGF neutralizing antibody is prepared from serum of a rabbit preimmunized with recombinant hCTGF having a purity of more than 98%, and the anti-human CTGF specific antibody is purified by an immobilized hCTGF matrix and affinity chromatography.
Preferably, the dosage form of the pharmaceutical composition comprises oral agents, tablets, granules, pills, powders, capsules, dispersing agents and suspending agents.
The technical scheme of the invention has the following beneficial effects:
the invention provides a CTGF neutralizing antibody which can reduce catabolism indexes (MMP 3, MMP-13, ADAMTT-1 and ADAMTT-5), increase the expression of ACAN and Collagen-II, protect the cartilage homeostasis and inhibit cartilage degeneration; in addition, the composition can increase the bone mass of subchondral bone, increase bone density and improve the microstructure of subchondral bone so as to inhibit the subchondral bone loss caused by estrogen deficiency. And the single injection can last long-term curative effect, avoids the problem of repeated administration of other osteoarthritis treatment medicines, enhances the adherence and convenience of administration, and has better effect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
Fig. 1 is a Micro CT scan and subchondral bone structure analysis (a is a Micro-CT image of subchondral bone of each group of tibial plateau, B is trabecular bone volume, C is trabecular bone gap, D is trabecular bone thickness, and E is trabecular bone number).
FIG. 2 is a graph of safranin fast green staining and cartilage scores (A is the safranin fast green staining image of each knee joint group and B is the cartilage score of each group).
FIG. 3 is a graph showing the results of improvement of cartilage degeneration and cartilage-related gene expression using a CTGF neutralizing antibody according to example of the present invention.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise.
Example 1 acquisition of CTGF neutralizing antibodies
The CTGF neutralizing antibody used as the reagent in this example was as follows: reagent production company: peproTech; reagent cargo number: 500-P252-100.
Example 2 Desovariorthritis rat subchondral bone local single injection CTGF neutralizing antibody
Following standard procedures, 6-month-old female SD rats were acclimatized for at least 1 week, fasted the day prior to surgery, and weighed and randomly grouped on the day of surgery. Anesthetic (3 ml/kg) was injected intraperitoneally and the back hair was carefully shaved with a shaver after the anesthesia had been effective. Exposing ovaries at two sides of the left and right sides of the back of the animal by small incisions, cutting off the ovaries at two sides, and suturing the wounds layer by layer; after 9 weeks the rats were again anesthetized, the hairs near the knee joint were removed, fixed on a sterile microsurgical table and sterilized with alcohol. The knee joint skin is opened, and the subcutaneous fascia is separated bluntly by forceps, so that the blood vessel is prevented from being injured. The tibialis anterolateral muscles are then separated layer by layer until the tibialis anterolateral border is exposed. The length and position of the needle were determined by X-ray and inserted from the anterior-inferior side of the tibial plateau using a 1ml syringe, and 1. Mu.g/ml, 10. Mu.g/ml, 20. Mu.g/ml of CTGF neutralizing antibody and 100. Mu.l of lgG-unloaded protein were injected into the left and right knee joints respectively using a micro-syringe along the channel created by the 1ml syringe, and the puncture hole was closed with bone wax. The experimental group is on the left and the blank control group is on the right. One month after injection, the lethal dose of the anesthetic is intraperitoneally injected to cause death, and the knee joint of a rat is soaked in 4% paraformaldehyde for fixation and detection.
Example 3 MicroCT scanning
And fixing the knee joint sample on a Micro-CT scanning plate, placing the knee joint sample at the initial position of a scanning table, and closing the cabin door. The bone tissue microstructure was subjected to a micro-ct test using inversion Research Workplace (version 3.0, siemens Medical Solutions USA, inc, IL, USA), a three-dimensional reconstruction of the bone tissue microstructure, and automatic calculation of parameters such as Trabecular volume/Total tissue volume, BV/TV, trabecular thickness (tb.th), trabecular number (tb.n), trabecular spacing (tb.sp), and the like according to software provided in the Research workstation. The results are shown in FIG. 1 (FIG. 1, graph A shows the Micro-CT image of subchondral bone of each tibial plateau, graph B shows trabecular volume, graph C shows trabecular space, graph D shows trabecular thickness, and graph E shows trabecular number).
Results and analysis:
compared to the OVX group, the medium and high dose (10 μ g/μ l and 20 μ g/μ l, respectively) CTGF neutralizing antibody injection group showed significant inhibition of estrogen-deficient OA progression as evidenced by trabecular bone volume, trabecular bone thickness and number significantly higher than the OVX group, and trabecular gap significantly lower than the OVX group ([ P ] for P <0.01, [ P ] for P <0.001, [ P ] for P < 0.0001).
Example 4 tissue specimen treatment and safranin fast Green staining
Placing the fixed knee joint in 10% EDTA decalcification solution for decalcification for 6 weeks, changing the solution every 3 days, embedding, slicing coronal plane with thickness of 5 μm, performing safranin fast green staining according to staining standard, and observing change of tissue morphology under normal light microscope; the sections were scored for degenerative changes (Cartilage degeneration, osteophytes, cartilage calcification and subchondral bone damage number and extent, number of synovial inflammation) by different investigators, with the scoring criteria referenced to Cartilage oxidation scoring system. The results are shown in FIG. 2 (in FIG. 2, A is a safranine fast green staining image of each knee joint group, and B is a cartilage score of each group).
Results and analysis:
compared with the OVX group, the CTGF neutralizing antibody injection group with medium and high doses (10 mug/ml and 20 mug/ml respectively) shows obvious effect of inhibiting the progress of the estrogen-deficiency OA, inhibition of polysaccharide loss was shown by significantly lower cartilage defect scores in the high dose groups than in the OVX groups (. Beta. Represents P <0.01,. Beta. Represents P <0.001,. Beta. Represents P < 0.0001).
Example 5 Primary chondrocyte extraction, culture and intervention
After 9 weeks of operation in 6-month-old female SD rats, lethal dose of anesthetic was intraperitoneally injected, and the carcass was immersed in 75% alcohol for 15 min after sacrifice. Then the rat is transferred into a clean bench, fixed in a supine position, the skin of the leg is cut, the knee is opened, the cartilage of the knee joint at two sides is cut under the aseptic condition, the rat is placed into a small aseptic beaker, the solution of PBS containing the double antibody is used for repeatedly washing for 2-3 times, and then the tissue block is transferred into another small aseptic beaker. The tissue pieces were minced with an ophthalmic scissors (about 10-20 min), then 10 ml of 0.3% collagenase II solution was added and digested for 4-6 h in a 37 ℃ incubator. Digestion was then stopped by adding complete medium and the cell suspension was transferred to a 15 ml centrifuge tube and centrifuged at 800 rpm for 5 min at room temperature. Discarding the supernatant, suspending chondrocytes in a complete culture medium, and blowing, beating and uniformly mixing. Inoculating the cell suspension into a culture dish of 10 cm, performing static culture in a constant-temperature incubator at 37 ℃ containing 5% CO2, observing the cell adherence condition after 3 days, performing first liquid change, performing liquid change every 2-3 days, and performing passage or cryopreservation when the cell fusion reaches 70-80%.
Note that: the subsequent experiments used P1 generation chondrocytes.
Digesting Sham group and OVX group with cartilage cells according to 1' \ 10005a/10 5 Density per well was seeded in 6-well plates. Primary OVX chondrocytes were stem-pre-cultured with complete medium and complete medium of CTGF neutralizing antibody at a concentration of 100ng/ml, respectively.
Example 6 RT-PCR
(1) RNA extraction
The cell culture medium was aspirated and washed 3 times with PBS solution for 5 min each time. Adding 350 μ l Trizol into each well, repeatedly blowing, fully lysing cells, adding 350 μ l absolute ethyl alcohol, blowing, mixing, transferring the liquid to a column (upper tube + lower tube), centrifuging at 12000 rpm for 1 min, removing the lower tube, and replacing with a new lower tube. Add 400. Mu.l PreWash (160 ml stock solution plus 40 ml absolute ethanol) to the tube, centrifuge at 12000 rpm for 1 min, discard the solution and do not change the tube. This step is repeated once. 700 mul Wash Buffer was added to the upper tube, centrifuged at 12000 rpm for 2 min, and the solution was discarded without changing the tube. To completely remove the Wash Buffer, the centrifugation was repeated once (in this case, the Wash Buffer was not added), the tube was discarded, and the 1.5 ml RNase-free EP tube was replaced. The tubes were loaded with 20. Mu.l DEPC water, centrifuged at 12000 rpm for 2 min, the EP tubes were collected and placed on ice, at which time the RNA had dissolved in 20. Mu.l water in the EP tubes. Taking 2 mul RNA to measure the concentration and purity on an ultraviolet spectrophotometer, when the ratio of OD260/OD280 is 1.8-2.0, the extracted RNA is relatively pure and has no DNA and protein residues.
(2) Reverse transcription
Reverse transcription reaction system (20 μ l):
adding the reaction system into 0.2 μ l EP tube, shaking, mixing, gently separating, heating at 65 deg.C for 5 min, and cooling on ice for 2-5 min.
Preparing a reverse transcription reagent:
preparing a reverse transcription reagent mixed solution (8 mu l), shaking, uniformly mixing, carrying out light separation, and then adding into the reaction system in the step 1. Setting a program: 5 min at 25 ℃, 60 min at 42 ℃ and 15 min at 70 ℃. After the reaction was completed, the cDNA was stored in a low temperature refrigerator at-20 ℃ for use.
(3) RT-PCR reaction
The total reaction system was 15. Mu.l:
the system preparation method comprises the steps of firstly, uniformly mixing SYBR Select Master Mix and primers according to a proportion, and then, adding ddH 2 Mixing the O and cDNA templates at a certain proportion, adding the eight rows in turn,each sample has three multiple holes, and the sample is separated slightly before being loaded on a machine and is fully and uniformly mixed. The primer sequences are detailed in Table 1.
Setting a reaction program: denaturation at 94 ℃ for 5 min, then carrying out denaturation, annealing and extension circulation under the conditions of: at 94 deg.C for 3 min, at 94 deg.C for 45 s, at 60 deg.C for 30 s, and at 72 deg.C for 45 s, for 40 cycles, and at last at 72 deg.C for 8 min.
TABLE 1 primer sequences of rats
The results are shown in FIG. 3: CTGF neutralizing antibody group significantly upregulated chondrocytesAcanmRNA expression is simultaneously down-regulatedAdamts1、Adamts5、Mmp3、Mmp13Expression of mRNA. Therefore, the CTGF neutralizing antibody of the present application has a superior therapeutic effect.
Although the present invention has been disclosed in the context of embodiments and examples, it should be understood that the invention is not limited to those embodiments and that various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention.
Claims (4)
- Application of CTGF neutralizing antibody in preparing medicine for preventing and treating postmenopausal osteoarthritis.
- 2. The use of a CTGF neutralizing antibody according to claim 1, wherein the medicament is a pharmaceutical composition which comprises an effective amount of the CTGF neutralizing antibody and pharmaceutically acceptable excipients or carriers, and the effective amount of the CTGF neutralizing antibody in the pharmaceutical composition is 10-20 μ g/ml, the injection amount is 100 μ l, and the injection is a single injection.
- 3. The use of a CTGF neutralizing antibody according to claim 2, wherein the injection site of the pharmaceutical composition is the subchondral bone of the knee joint in the preparation of a medicament for the prevention and treatment of post-menopausal osteoarthritis.
- 4. The use of a CTGF neutralizing antibody in the manufacture of a medicament for the prevention and treatment of post-menopausal osteoarthritis according to claim 2 wherein, said CTGF neutralizing antibody is prepared from serum from rabbit preimmunized with recombinant hCTGF having a purity greater than 98%, and from immobilized hCTGF matrix and purified anti-human CTGF-specific antibody by affinity chromatography.
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| WO1999033878A1 (en) * | 1997-12-25 | 1999-07-08 | Japan Tobacco Inc. | Monoclonal antibody against connective tissue growth factor and medicinal uses thereof |
| US7405274B2 (en) * | 2003-06-04 | 2008-07-29 | Fibrogen, Inc. | Connective tissue growth factor antibodies |
| TW202110880A (en) * | 2019-06-04 | 2021-03-16 | 大陸商江蘇恆瑞醫藥股份有限公司 | Anti-ctgf antibody and use thereof |
| CN116391041A (en) * | 2020-12-03 | 2023-07-04 | 江苏恒瑞医药股份有限公司 | Pharmaceutical composition containing anti-connective tissue growth factor antibody |
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| Zihuan Yang等.CTGF as a multifunctional molecule for cartilage and a potential drug for osteoarthritis.《Frontiers in Endocrinology》.2022,第13卷全文. * |
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