A new generation of stem cell therapeutic agent for treating knee arthritis and its preparation process

Abstract

The present invention provides a new generation of stem cell therapeutic agents for treating knee arthritis and their preparation process. The present invention is the first to discover that the therapeutic agent can effectively reduce serum levels of IL-1β, TNF-ɑ, and IL-6 in patients with knee arthritis, inhibit their secretion, alleviate knee arthritis damage, and improve and promote cartilage tissue repair.

Description

Technical Field

[0001] This invention relates to the field of knee osteoarthritis treatment technology, and in particular to a new generation of stem cell therapeutic agent for treating knee osteoarthritis and its preparation process. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell derived from the mesoderm. First isolated from bone marrow, they have shown great promise for clinical applications in immunomodulation and tissue regeneration due to their ease of in vitro expansion and lack of tumorigenicity. MSCs are characterized by two main features: self-renewal and differentiation potential. They also secrete important cytokines, express specific receptors, and can be genetically modified, thus altering their molecular susceptibility to natural behavior. Therefore, MSCs have attracted increasing attention. Many researchers have isolated similar cells from Wharton's jelly and perivascular tissue of the umbilical cord using various methods. Compared to bone marrow mesenchymal stem cells, umbilical cord stem cells have the advantages of being widely available, easy to collect, and capable of being recycled.

[0003] Knee osteoarthritis is a common disease that accompanies and troubles modern people. Normally, the thickness of the knee cartilage is 2.5 cm during adolescence. With the gradual increase in age and the wear and tear of use, the cartilage thickness will become thinner and thinner. Generally, by the age of 55, the cartilage thickness is only 0.5 cm. Moreover, if the knee joint is damaged or subjected to long-term pressure, the cartilage thickness may be even thinner, and in severe cases, bone spurs or ruptures may occur.

[0004] Conventional treatments primarily involve NSAIDs, lubricants, intra-articular injections of corticosteroids, and chondroprotective agents. However, these treatments have limited efficacy and cannot effectively halt disease progression. With the continuous development and popularization of stem cell research, more and more researchers are extending stem cell technology to the treatment of osteoarthritis, effectively addressing the aforementioned problems through stem cell therapy. However, current stem cell drug treatments for knee osteoarthritis still have some issues, such as poor differentiation induction, unstable efficacy of knee joint stem cell drugs, and treatment results that do not yet meet our expectations. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention provides a new generation of stem cell therapeutic agent for treating knee osteoarthritis and its preparation process. This therapeutic agent can effectively reduce the serum levels of IL-1β, TNF-α, and IL-6 in patients with knee osteoarthritis, inhibit the secretion of IL-1β, TNF-α, and IL-6, alleviate inflammatory damage to the knee joint, and improve and promote the repair of damaged cartilage tissue.

[0006] The primary objective of this invention is to provide a new generation of stem cell therapeutic agent for treating knee osteoarthritis, characterized in that the therapeutic agent comprises: 25%-45% bone marrow mesenchymal stem cells, 5-10% chondroitin sulfate, 5-10% anti-CD47 antibody, 0.1-1% Tween-80, 0.1-1% suspending agent, 0.1-1% emulsifier, 0.1-1% stabilizer, 0.2-0.6% pH adjuster, 0.6-2% antioxidant, and the balance being physiological saline.

[0007] Preferably, the therapeutic agent comprises: 25% bone marrow mesenchymal stem cells, 5% chondroitin sulfate, 5% anti-CD47 antibody, 0.1% Tween-80, 0.1% suspending agent, 0.1% emulsifier, 0.1% stabilizer, 0.2% pH adjuster, 0.6% antioxidant, and the balance being physiological saline.

[0008] Preferably, the therapeutic agent comprises: 30% bone marrow mesenchymal stem cells, 8% chondroitin sulfate, 8% anti-CD47 antibody, 0.5% Tween-80, 0.5% suspending agent, 0.5% emulsifier, 0.5% stabilizer, 0.4% pH adjuster, 1% antioxidant, and the balance being physiological saline.

[0009] Preferably, the therapeutic agent comprises: 45% bone marrow mesenchymal stem cells, 10% chondroitin sulfate, 10% anti-CD47 antibody, 1% Tween-80, 1% suspending agent, 1% emulsifier, 1% stabilizer, 0.6% pH adjuster, 2% antioxidant, and the balance being physiological saline.

[0010] More preferably, the suspending agent is selected from one or more of methylcellulose and sodium carboxymethylcellulose.

[0011] More preferably, the emulsifier is selected from polyoxyethylene castor oil, polysorbate 20, polysorbate 40, polysorbate 80, povidone, polyethylene glycol-40, and lecithin.

[0012] More preferably, the stabilizer is selected from disodium ethylenediaminetetraacetate.

[0013] More preferably, the pH adjuster is one or more of the following: phosphoric acid and its salts, boric acid and its salts, citric acid and its salts, acetic acid and its salts, tartaric acid and its salts, hydrochloric acid and its salts, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and aminobutanetriol.

[0014] More preferably, the antioxidant is one or more of vitamin C, sodium sulfite, sodium metabisulfite, and sodium thiosulfate.

[0015] The new generation stem cell therapy formulation of the present invention can be prepared as a sterile injectable solution or suspension; for example, it can be prepared as a solution suitable for administration by means of intra-articular injection, intravenous injection, intramuscular injection, subcutaneous injection or intraperitoneal injection.

[0016] Another object of the present invention is to provide an anti-CD47 antibody, characterized in that the heavy chain variable region sequence of the anti-CD47 antibody is as shown in SEQ ID NO:1, and the light chain variable region sequence is as shown in SEQ ID NO:2.

[0017] Preferably, the heavy chain variable region of the anti-CD47 antibody includes CDR-H1, CDR-H2, and CDR-H3, the sequences of CDR-H1-3 are shown in SEQ ID NO.3-5, and the light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, the sequences of CDR-L1-3 are shown in SEQ ID NO.6-8.

[0018] Furthermore, the present invention also provides the use of anti-CD47 antibody and bone marrow mesenchymal stem cells in combination in the preparation of a new generation of stem cell therapeutic agents for the treatment of knee osteoarthritis.

[0019] Preferably, the dosage form of the therapeutic agent is not limited to an injection.

[0020] More preferably, the therapeutic agent is a sterile injectable solution or suspension;

[0021] The injection solution is a solution administered via intra-articular injection, intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection.

[0022] The advantages of this invention are as follows: This invention provides for the first time a new generation of stem cell therapeutic agent for treating knee osteoarthritis and its preparation process. The therapeutic agent of this invention can effectively reduce the levels of IL-1β, TNF-α and IL-6 in the serum of patients with knee osteoarthritis, inhibit the secretion of IL-1β, TNF-α and IL-6, alleviate inflammatory damage to the knee joint, and improve and promote the repair of damaged cartilage tissue. Attached Figure Description

[0023] Figure 1 Secretion of IL-1β, TNF-α and IL-6 in rat serum under different treatments. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0025] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0026] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0027] Example 1

[0028] An anti-CD47 antibody, characterized in that the heavy chain variable region sequence of the anti-CD47 antibody is as shown in SEQ ID NO:1, and the light chain variable region sequence is as shown in SEQ ID NO:2. The heavy chain variable region of the anti-CD47 antibody includes CDR-H1, CDR-H2, and CDR-H3, the sequences of which are shown in SEQ ID NO:3-5; and the light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, the sequences of which are shown in SEQ ID NO:6-8.

[0029] Example 2

[0030] Under aseptic conditions, bilateral femurs and tibias of rats were collected and placed in low-glucose DMEM medium containing 5% penicillin and streptomycin. The bone marrow cavity was repeatedly washed and repeatedly pipetted until a single-cell suspension was obtained. After washing, the suspension was centrifuged twice to remove impurities and oil. The collected single-cell suspension was resuspended in low-glucose DMEM containing 10% fetal bovine serum and penicillin (final concentration 100 U / mL penicillin, 0.1 mg / mL streptomycin) at a concentration of 1×10⁻⁶. 6 Cells were seeded at a density of 1 / mL in T75 culture flasks and incubated at 37°C in a 5% CO2 incubator. The medium was changed after 2 days to remove non-adherent cells. The medium was then changed every 2 days, and cell growth was observed until cell confluence reached 80% or higher. Cells were then digested with 0.25% trypsin at 37°C for 2–5 minutes until complete detachment. Digestion was terminated with complete culture medium to obtain purified primary bone marrow mesenchymal stem cells. These primary bone marrow mesenchymal stem cells were passaged in twice the volume of DMEM containing 10% fetal bovine serum, with the medium changed every 2 days until cell confluence reached 80% or higher. This process was repeated for 2–5 passages. Cell growth was observed, and cells were digested with trypsin, washed with PBS buffer, centrifuged, collected, freeze-dried, and stored at low temperature.

[0031] Identification was performed using microscopy and flow cytometry. The results showed that bone marrow mesenchymal stem cells could adhere to the surface of the culture flask, and the cell morphology exhibited typical spindle-shaped fibroblast morphology. Flow cytometry analysis showed that the isolated bone marrow mesenchymal stem cells were positive for CD44, CD105, CD73, CD90, and HLA-ABC, with a positive rate >95%; CD45, CD34, and HLA-DR were hardly expressed.

[0032] Example 3

[0033] A new generation stem cell therapy for treating knee osteoarthritis comprises: 25% bone marrow mesenchymal stem cells, 5% chondroitin sulfate, 5% anti-CD47 antibody, 0.1% Tween-80, 0.1% suspending agent, 0.1% emulsifier, 0.1% stabilizer, 0.2% pH adjuster, 0.6% antioxidant, and the balance being physiological saline. The preparation method involves mixing the above raw materials, dissolving them in physiological saline, adjusting the pH to 7.0, and then filtering and sterilizing to obtain the new generation stem cell therapy.

[0034] Example 4

[0035] A new generation stem cell therapy for treating knee osteoarthritis comprises: 30% bone marrow mesenchymal stem cells, 8% chondroitin sulfate, 8% anti-CD47 antibody, 0.5% Tween-80, 0.5% suspending agent, 0.5% emulsifier, 0.5% stabilizer, 0.4% pH adjuster, 1% antioxidant, and the balance being physiological saline. The preparation method involves mixing the above raw materials, dissolving them in physiological saline, adjusting the pH to 7.0, and then filtering and sterilizing to obtain the new generation stem cell therapy.

[0036] Example 5

[0037] A new generation stem cell therapy for treating knee osteoarthritis comprises: 45% bone marrow mesenchymal stem cells, 10% chondroitin sulfate, 10% anti-CD47 antibody, 1% Tween-80, 1% suspending agent, 1% emulsifier, 1% stabilizer, 0.6% pH adjuster, 2% antioxidant, and the balance being physiological saline. The preparation method involves mixing the above raw materials, dissolving them in physiological saline, adjusting the pH to 7.0, and then filtering and sterilizing to obtain the new generation stem cell therapy.

[0038] Example 6

[0039] The rat knee arthritis model was prepared by the following steps: After weighing, each group of rats was anesthetized by intraperitoneal injection of 40 mg / kg 1% sodium pentobarbital. When the rats showed signs of anesthesia, they were fixed on a rat table and kept in a supine position. Before surgery, the hair on the right hind limb knee joint of the rat was clipped, and the knee joint was disinfected with povidone-iodine. Under a microscope, the skin on the medial side of the right knee joint was longitudinally incised with ophthalmic scissors 1 cm proximal to the right knee joint, avoiding the medial transverse and longitudinal arteries, and the medial collateral ligament was severed to open the joint cavity. The joint capsule was incised along the upper edge of the medial collateral ligament. The joint capsule was incised to expose the patella, which was everted and fixed. The joint capsule was incised again until the patella was fully exposed. The right knee joint of the rat was flexed to expose the anterior cruciate ligament and medial meniscus. The anterior cruciate ligament was severed with a scalpel blade, and the meniscus was removed. After the meniscus was removed, the rat was hemostatically controlled, and the joint cavity was cleaned. The patella was then placed in its original position, and each layer of tissue was sutured in sequence. The affected area was bandaged under aseptic conditions. After surgery, the rat was injected intramuscularly with ampicillin (2.0 mg / kg) for 3 consecutive days to prevent incision infection. The rat was then allowed to eat freely.

[0040] Example 7

[0041] The efficacy evaluation of a new generation of stem cell therapy for treating knee osteoarthritis includes the following steps:

[0042] A rat model of knee arthritis was divided into four groups, with five rats in each group. The blank group consisted of normal rats treated only with saline solution throughout the treatment. The control group also received only saline solution treatment. Control group 1 received only anti-CD47 antibody treatment. Control group 2 received only bone marrow mesenchymal stem cell treatment. The treatment groups were treated with the new generation stem cell therapy agent described in Example 3 of this invention. The treatment involved injecting 100 μL of sample into the rat's knee joint cavity. Each treatment course lasted one week, and the injections were repeated for three consecutive weeks. Serum samples were collected from the rats, and the secretion of IL-1β, TNF-α, and IL-6 in the serum was analyzed using an ELISA kit.

[0043] The results are as follows Figure 1As shown, the levels of IL-1β, TNF-α, and IL-6 in the serum of normal rats were significantly low, while the levels of IL-1β, TNF-α, and IL-6 in the serum of the control model group were significantly increased, showing a significant difference compared to the blank group. Furthermore, treatment with anti-CD47 antibody alone did not result in a significant decrease in IL-1β, TNF-α, and IL-6 in the serum of the rat model, demonstrating that anti-CD47 antibody alone is not effective in alleviating knee osteoarthritis. In the group treated with bone marrow mesenchymal stem cells alone, the levels of IL-1β, TNF-α, and IL-6 in the rat serum were somewhat decreased compared to the control model group, but the decrease was not significant. However, unexpectedly, in the rat model treated with the therapeutic agent described in Example 3, the levels of IL-1β, TNF-α, and IL-6 in the serum showed a sharp decrease. This result indicates that the new generation stem cell therapeutic agent of the present invention can inhibit the secretion of IL-1β, TNF-α, and IL-6, alleviate inflammatory damage to the knee joint, and improve and promote the repair of damaged cartilage tissue.

[0044] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A stem cell therapeutic agent for treating knee osteoarthritis, characterized in that, The therapeutic agent comprises: 25%-45% bone marrow mesenchymal stem cells, 5-10% chondroitin sulfate, 5-10% anti-CD47 antibody, 0.1-1% Tween-80, 0.1-1% suspending agent, 0.1-1% emulsifier, 0.1-1% stabilizer, 0.2-0.6% pH adjuster, 0.6-2% antioxidant, with the balance being physiological saline. The heavy chain variable region sequence of the anti-CD47 antibody is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:

2.

2. The therapeutic agent as claimed in claim 1, characterized in that, The suspending agent is selected from one or more of methylcellulose and sodium carboxymethylcellulose.

3. The therapeutic agent as described in claim 1, characterized in that, The emulsifier is selected from polyoxyethylene castor oil, polysorbate 20, polysorbate 40, polysorbate 80, povidone, polyethylene glycol-40, and lecithin.

4. The therapeutic agent as claimed in claim 1, characterized in that, The stabilizer is selected from disodium ethylenediaminetetraacetate.

5. The therapeutic agent as claimed in claim 1, characterized in that, The pH adjuster is one or more of the following: phosphoric acid and its salts, boric acid and its salts, citric acid and its salts, acetic acid and its salts, tartaric acid and its salts, hydrochloric acid and its salts, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and glycerol.

6. The therapeutic agent as claimed in claim 1, characterized in that, The antioxidant is one or more of vitamin C, sodium sulfite, sodium metabisulfite, and sodium thiosulfate.

7. The preparation process of the therapeutic agent according to any one of claims 1-6, characterized in that, The preparation process includes the following steps: mixing the raw materials and dissolving them in physiological saline, adjusting the pH to 7.0, and then filtering and sterilizing to obtain the stem cell therapy agent.

8. An anti-CD47 antibody, characterized in that, The heavy chain variable region sequence of the anti-CD47 antibody is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:

2.

9. The antibody as described in claim 8, characterized in that, The heavy chain variable region of the anti-CD47 antibody includes CDR-H1, CDR-H2, and CDR-H3, the sequences of which are shown in SEQ ID NO.3-5. The light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, the sequences of which are shown in SEQ ID NO.6-8.

10. The use of anti-CD47 antibody and bone marrow mesenchymal stem cells in combination in the preparation of a stem cell therapeutic agent for the treatment of knee osteoarthritis, characterized in that, The heavy chain variable region sequence of the anti-CD47 antibody is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:

2.

11. The use as described in claim 9, characterized in that, The therapeutic agent is a sterile injectable solution or suspension.

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