Triple therapy medicine composition for treating chronic knee joint injury and preparation method and injection product thereof

Abstract

The invention provides a triple therapy medicine composition for treating chronic knee joint injury and a preparation method and an injection product thereof, and belongs to the technical field of medical biology. The invention provides a triple therapy pharmaceutical composition for treating knee joint injury. The pharmaceutical composition consists of the following components in parts by volume: 2-5 parts of platelet-rich plasma, 2-5 parts of mesenchymal stem cells and 2-5 parts of exosomes. The platelet-rich plasma, the mesenchymal stem cells and the exosome promote each other, so that the effects of relieving pain and promoting cartilage repair are superimposed and strengthened, the symptoms and functions are improved, the disease progression is delayed or partially reversed, and a relatively good effect on treating knee joint injury is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical biotechnology, and particularly relates to a triple therapy drug composition for treating chronic knee joint injury and a preparation method and injection product thereof. BACKGROUND

[0002] Chronic knee joint injury is a common disease with a wide range of influence and a high prevalence rate, including osteoarthritis (OA) and sports-induced injury. Current common treatment methods for OA include basic treatment, physical therapy, drug therapy (including oral and local injection), arthroscopic surgery, etc., which mainly aim at symptom control, delay of disease progression or correction, lack of tissue regeneration and repair capacity, and cannot alleviate the progressive degeneration, and some patients may still need knee replacement. However, some patients cannot adhere to exercise due to long-term failure to recover from knee injury caused by exercise, which causes serious life distress to the patients. Moreover, frequent medical treatment or physiotherapy also causes time and economic pressure on the patients, and seriously affects the survival and life quality of the patients.

[0003] In summary, the traditional treatment has limited effect, and it is necessary to study and try new treatment methods, which can not only alleviate symptoms, but also promote tissue repair, significantly slow down the progressive degeneration or maximize the recovery of motor function, reduce the number of medical visits and medical burden, and ultimately improve the survival or life quality of patients with chronic injury.

[0004] Chinese patent CN116947976A discloses a method for treating cartilage injury by mesenchymal stem cells. The application screens a polypeptide NOS-S2 that specifically targets and inhibits NOS activity through phage library. The polypeptide can effectively inhibit cartilage cell apoptosis after cartilage injury and promote the repair of cartilage injury. The combination of the polypeptide and bone marrow mesenchymal stem cells can significantly improve the treatment effect and has a wide application prospect.

[0005] In the prior art (Preliminary Study on the Role of miR-429 in Repairing Osteoarthritis Cartilage Injury in Human Synovial Mesenchymal Stem Cell Exosomes, Zhou Sunxin, Huo Na, Li Hongkun, et al., Medical Journal of Chinese People's Liberation Army, 2025), exosomes derived from human synovial mesenchymal stem cells in synovial tissue are extracted, differential expression microRNAs are screened by transcriptome sequencing, and the role of the target miRNA in cartilage repair in osteoarthritis is verified. It is found that osteoarthritis patients have a specific miRNA expression profile, and miR-429 is significantly down-regulated. Overexpression of miR-429 has a repairing and inhibiting effect on cartilage cells in an inflammatory microenvironment.

[0006] No related report is found in the prior art on the combination of platelet-rich plasma, mesenchymal stem cells and exosomes for treating knee joint injury. At present, the research on cartilage injury is less and the effect is poor, and it is urgent to provide a drug composition that can effectively treat knee joint injury. SUMMARY

[0007] To solve the above problems, the present application provides a triple therapy pharmaceutical composition for treating chronic knee injury, and a preparation method and an injection product thereof.

[0008] In one aspect, the present application provides a triple therapy pharmaceutical composition for treating knee injury, the pharmaceutical composition consisting of 2-5 parts of platelet-rich plasma, 2-5 parts of mesenchymal stem cells, and 2-5 parts of exosomes by volume.

[0009] According to some embodiments of the present application, the pharmaceutical composition consists of 2 parts of platelet-rich plasma, 2 parts of mesenchymal stem cells, and 2 parts of exosomes by volume.

[0010] According to some embodiments of the present application, the pharmaceutical composition consists of 3 parts of platelet-rich plasma, 3 parts of mesenchymal stem cells, and 3 parts of exosomes by volume.

[0011] According to some embodiments of the present application, the pharmaceutical composition consists of 4 parts of platelet-rich plasma, 4 parts of mesenchymal stem cells, and 4 parts of exosomes by volume.

[0012] According to some embodiments of the present application, the pharmaceutical composition consists of 5 parts of platelet-rich plasma, 5 parts of mesenchymal stem cells, and 5 parts of exosomes by volume.

[0013] Further, the pharmaceutical composition consists of 3 parts of platelet-rich plasma, 3 parts of mesenchymal stem cells, and 3 parts of exosomes by volume.

[0014] According to some embodiments of the present application, the pharmaceutical composition consists of 3 mL of platelet-rich plasma, 3 mL of mesenchymal stem cells, and 3 mL of exosomes.

[0015] Specifically, the pharmaceutical composition includes (5-8) x 10 7 mesenchymal stem cells, (2-4) x 10 7 exosome vesicles.

[0016] According to some embodiments of the present application, the pharmaceutical composition includes 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , or 8 x 10 7 mesenchymal stem cells.

[0017] According to some embodiments of the present application, the pharmaceutical composition includes 2 x 10 7 , 3 x 10 7 , or 4 x 10 7 exosome vesicles.

[0018] Further, the pharmaceutical composition comprises 5x10 7 mesenchymal stem cells, 3x10 7 exosome vesicles.

[0019] Specifically, the mesenchymal stem cells are derived from bone marrow, fat, umbilical cord.

[0020] Further, the mesenchymal stem cells are derived from umbilical cord.

[0021] Specifically, the dosage form of the pharmaceutical composition is tablet, powder, granule, capsule, emulsion, suspension, oil, injection.

[0022] Further, the dosage form of the pharmaceutical composition is injection.

[0023] Still further, the injection includes but is not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracavity injection.

[0024] Specifically, when the pharmaceutical composition is injection, it further comprises water for injection.

[0025] Further, the water for injection is 0.9% sodium chloride injection.

[0026] According to some embodiments of the present application, the preparation method of the platelet-rich plasma is as follows: Peripheral venous blood is collected, centrifuged, and the middle white membrane layer and the upper plasma are sucked and discarded, and the lower red blood cells are discarded, and transferred to a centrifuge tube for secondary centrifugation, and the supernatant is sucked after centrifugation to obtain platelet-rich plasma.

[0027] According to some embodiments of the present application, the preparation method of the exosome is as follows: Take the supernatant of the expanded 3rd generation mesenchymal stem cells, centrifuge at 100 rpm for 10 min, take the supernatant, centrifuge at 10000xg for 10 min, take the supernatant, centrifuge at 120000xg for 60 min to obtain exosomes.

[0028] In another aspect, the present application provides a preparation method of the above-mentioned triple therapy pharmaceutical composition, which comprises mixing platelet-rich plasma, mesenchymal stem cells and exosomes.

[0029] In another aspect, the present application provides an injection product, which comprises the above-mentioned triple therapy pharmaceutical composition.

[0030] In another aspect, the present application provides the use of platelet-rich plasma, mesenchymal stem cells and exosomes in combination in the preparation of a drug for treating knee injury. In another aspect, the present application provides the use of platelet-rich plasma, mesenchymal stem cells and exosomes in combination in the preparation of a drug for treating knee injury.

[0031] Specifically, the knee injury includes osteoarthritis and sports-induced injury.

[0032] Specifically, the medicine includes 2-5 parts of platelet-rich plasma, 2-5 parts of mesenchymal stem cells, and 2-5 parts of exosomes.

[0033] Further, the medicine includes 3 parts of platelet-rich plasma, 3 parts of mesenchymal stem cells, and 3 parts of exosomes.

[0034] According to some embodiments of the present application, the medicine includes 3mL of platelet-rich plasma, 3mL of mesenchymal stem cells, and 3mL of exosomes.

[0035] Specifically, the medicine includes (5-8) x 10 7 mesenchymal stem cells, (2-4) x 10 7 exosome vesicles.

[0036] According to some embodiments of the present application, the medicine composition includes 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 mesenchymal stem cells.

[0037] According to some embodiments of the present application, the medicine composition includes 2 x 10 7 , 3 x 10 7 , 4 x 10 7 exosome vesicles.

[0038] Further, the medicine composition includes 5 x 10 7 mesenchymal stem cells, 3 x 10 7 exosome vesicles.

[0039] Specifically, the mesenchymal stem cells are derived from bone marrow, fat, umbilical cord.

[0040] Further, the mesenchymal stem cells are derived from umbilical cord.

[0041] Specifically, the dosage form of the medicine is tablet, powder, granule, capsule, emulsion, suspension, oil, injection.

[0042] Further, the dosage form of the medicine is injection.

[0043] Further, the injection includes but is not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracavity injection.

[0044] Specifically, when the medicine is an injection, the injection water is further included.

[0045] Further, the injection water is 0.9% sodium chloride injection water.

[0046] Specifically, the medicine can further include a pharmaceutically acceptable carrier.

[0047] The beneficial effects of the present application are: The medicine composition of the present application includes PRP, MSC and exosomes. PRP provides a growth factor environment, reduces inflammatory response, and promotes proliferation and survival of stem cells; MSC provides regenerative capacity and paracrine effect to improve the microenvironment for repair to create the best environment; exosomes promote intercellular signal transmission, regulate immunity, and promote angiogenesis to provide sufficient blood supply, which is beneficial for tissue repair. The three kinds of cells promote each other, and are superimposed and strengthened in relieving pain and promoting cartilage repair, and the final effect is to improve symptoms and function, and delay or partially reverse the progression of the disease. DETAILED DESCRIPTION

[0048] The present application will be further described in detail below in conjunction with specific examples. The following examples are not used to limit the present application, but only to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally performed according to conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0049] Example 1 1. Inclusion criteria: (1) diagnosed as knee osteoarthritis (mild to moderate); (2) no other autoimmune or systemic disease; (3) diagnosed as chronic knee injury (mild to moderate); (4) patients who have not received other intervention treatment; (5) agree not to participate in other studies, and do not receive other osteoarthritis treatment during the participation in the present study.

[0050] 2. Exclusion criteria: (1) severe systemic diseases other than osteoarthritis and sports injuries, including uncontrolled clinically significant urinary, circulatory, respiratory, neurological, mental, digestive, endocrine, etc. system diseases; (2) combined with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, diffuse connective tissue diseases such as polychondritis, ankylosing spondylitis, arthritis related to infectious factors, bone and cartilage diseases with joint manifestations, sarcoidosis, psoriasis, gout, tuberculosis, etc.; (3) patients with malignant tumors or with a history of malignant tumors; (4) Allergic constitution, once have the history of allergy to blood products.

[0051] (5) Without the approval of the hospital ethics committee, without signing the informed consent form.

[0052] 3 Grouping: 90 patients were selected in the invention, and the patients were divided into triple therapy group and traditional PRP+glucosamine oral+physical rehabilitation as control group according to random number table method.

[0053] 4 Treatment method: Control group PRP (n=30): intra-articular injection of PRP 3mL once a week + glucosamine sulfate 0.5g / time, 3 times a day, oral + physical rehabilitation once a week; Triple therapy group (n=30): PRP / 3ml+exosome / 3ml / 30 million vesicles +UC-MSCS / 3ml / 500 million cells, local injection of knee joint in several times, every 2 weeks.

[0054] Comparative example 1 group (n=10): PRP / 3ml+UC-MSCS / 3ml / 500 million cells, local injection of knee joint in several times, every 2 weeks.

[0055] Comparative example 2 group (n=10): exosome / 3ml / 30 million vesicles, local injection of knee joint in several times, every 2 weeks.

[0056] Comparative example 3 group (n=10): PRP / 6ml+exosome / 6ml / 30 million vesicles +UC-MSCS / 6ml / 500 million cells, local injection of knee joint in several times, every 2 weeks.

[0057] (1) Umbilical cord mesenchymal stem cell preparation method: Take fresh umbilical cord, separate out the Wharton's jelly layer, wash with physiological saline, 2000g, 10min, collect the tissue block. Place the tissue block in the CO2 incubator for 8h, then add complete culture medium to the tissue block for culture. Every 3 days, observe whether the cells have crawled out and replace the liquid. When the cells have crawled out and the confluence reaches more than 85%, remove the tissue block. Add PBS for washing; then add trypsin to the cell mass, and after the cells are dispersed, resuspend with complete culture medium, centrifuge at 2000rpm for 5min to obtain the first generation of umbilical cord mesenchymal stem cells.

[0058] (2) Preparation of platelet-rich plasma: Peripheral venous blood 10ml, add 0.2ml anticoagulant, 2500rpm centrifugal 5min, take the middle white membrane layer and the upper layer of plasma, discard the lower layer of red blood cells, transfer to the centrifugal tube for secondary centrifugation, 3500r / min, centrifugal 8min, after centrifugation, take the supernatant, adjust the platelet concentration to 5x10 8

[0059] (3) Preparation of exosomes: Take the supernatant of the expanded 3rd generation mesenchymal stem cells, centrifuge at 100rpm for 10min, take the supernatant, centrifuge at 10000xg for 10min, take the supernatant, centrifuge at 120000xg for 60min, and obtain the exosomes.

[0060] 5. Effect evaluation: WOMAC score (knee function score), MRI evaluation of cartilage thickness change, serum inflammatory factor level (CRP / such as IL-6).

[0061] 5.1 WOMAC score The scoring criteria of the WOMAC score scale are as follows: Pain: a total of 5 items, each item scored 0-4, the score range is 0-20, and the higher the score, the more severe the pain.

[0062] Stiffness: a total of 2 items, each item scored 0-4, the score range is 0-8, and the higher the score, the more severe the stiffness.

[0063] Physical dysfunction: a total of 17 items, each item scored 0-4, the score range is 0-68, and the higher the score, the more severe the dysfunction.

[0064] The scoring results of the WOMAC score scale can be divided into mild (0-20), moderate (21-40) and severe (more than 40).

[0065] 5.2 MRI evaluation of cartilage thickness change 5.2.1 Evaluation method Measure the cartilage thickness: on the MRI image, select the appropriate layer (multiple points), use the built-in professional measurement software, and directly measure; Observe the cartilage morphology: observe whether the cartilage surface is smooth and continuous, whether there is defect or hyperostosis or bone marrow edema, etc.

[0066] 5.2.2 Grading ​Grade 0: normal; Grade 1: focal changes in signal intensity within the cartilage but smooth without thickness changes; Grade 2: thinning of the thickness but not reaching 50% of the whole layer, with wear but not seriously affecting joint function; Grade 3: thickness loss reaching more than 50% but not involving subchondral bone, with pain and limited activity symptoms; Grade 4: global loss, exposing subchondral bone, accompanied by sclerosis or cystic change, and causing joint pain and deformity.

[0067] 5.3 Serum inflammatory factor levels (CRP / IL-6) 5.3.1 Instrument used: Mindray CL8000i chemiluminescence immunoassay analyzer, to detect CRP / IL-6 levels.

[0068] 5.3.2 Judgment criteria: CRP normal <10 mg / L. Mild inflammation 10-50 mg / L, moderate 50-100 mg / L, severe ≥100 mg / L; IL-6 normal 0-7 pg / mL. Mild inflammation 7-35 pg / mL, moderate 35-100 pg / mL, severe ≥100 pg / mL.

[0069] 6 Experimental results Table 1 Comparison of WOMAC scores ±S, score / point)

[0070] Note: compared with the treatment group before treatment, *p<0.05.

[0071] Table 2 Comparison of MRI evaluation of cartilage thickness in patients in the two groups

[0072] Note: compared with the treatment group before treatment, *p<0.05.

[0073] Table 3 Comparison of inflammatory factor levels in patients in the two groups

[0074] Note: compared with the treatment group before treatment, *p<0.05.

[0075] From the above table, it can be seen that PRP, MSC and exosomes can significantly reduce the WOMAC score, increase the cartilage thickness and reduce the levels of inflammatory factors CRP and IL-6, the three components cooperate with each other, synergistically, and play an important role in promoting cartilage repair, but using two components in combination or only using one component cannot effectively repair cartilage damage.

[0076] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A triple therapy pharmaceutical composition for treating knee injury, characterized in that, The pharmaceutical composition consists of 2-5 parts of platelet-rich plasma, 2-5 parts of mesenchymal stem cells and 2-5 parts of exosomes by volume.

2. The triple therapy pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition consists of 3 parts of platelet-rich plasma, 3 parts of mesenchymal stem cells and 3 parts of exosomes by volume.

3. The triple therapy pharmaceutical composition according to any one of claims 1-2, wherein, The pharmaceutical composition includes (5-8) x 10 7 mesenchymal stem cells, (2-4) x 10 7 exosome vesicles.

4. The triple therapy pharmaceutical composition according to claim 3, wherein, The pharmaceutical composition includes 5 x 10 7 mesenchymal stem cells, 3 x 10 7 exosome vesicles.

5. The triple therapy pharmaceutical composition according to claim 1, wherein The mesenchymal stem cells are derived from bone marrow, fat or umbilical cord.

6. The triple therapy pharmaceutical composition according to claim 1, wherein, The dosage form of the pharmaceutical composition is tablet, powder, granule, capsule, emulsion, suspension, oil, injection.

7. The triple therapy pharmaceutical composition according to claim 1, wherein The dosage form of the pharmaceutical composition is injection.

8. A method of preparing a triple therapy pharmaceutical composition according to any one of claims 1 to 7, characterized in that, The preparation method is to mix platelet-rich plasma, mesenchymal stem cells and exosomes.

9. A product for injection, characterized in that The injection product includes the triple therapy pharmaceutical composition of any one of claims 1-7.

10. Application of platelet-rich plasma, mesenchymal stem cells and exosomes in the preparation of a drug for treating knee joint injury.

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