A stem cell pharmaceutical composition for improving arthritic microenvironment, protecting cartilage and inhibiting synovial fibrosis, and a preparation method and application thereof

Abstract

The application discloses a stem cell medicine composition for improving arthritic microenvironment, protecting cartilage and inhibiting synovial fibrosis, and a preparation method and application thereof, and belongs to the technical field of cell biology medicine. Active ingredients of the application are P6-P10 generation human umbilical cord mesenchymal stem cells which are directionally domesticated by osteoarticular compound pathological microenvironment, wherein the positive rate of the stem cells transmitted to P15 generation is less than or equal to 7%, and the comprehensive repair activity retention rate is greater than or equal to 93%; the final concentration of cells in the preparation is 1*10^6-2*10^6 pieces / mL, the pH is stable at 7.2-7.4, and injection carriers and low-temperature protection auxiliaries are matched. The stem cells are endowed with joint target repair ability by pathological microenvironment domestication, and multiple target points are synchronously intervened in arthritic inflammation, cartilage damage and synovial fibrosis, so that the industry defects of ordinary stem cells, such as weak targeting, unstable activity and large batch difference, are solved; the preparation has high standardization, can be prepared into liquid injection and freeze-drying storage and transportation dosage forms, is suitable for GMP large-scale production, clinical trials and new drug declaration, and has high clinical conversion value.

Description

[0001] manual Technical Field

[0002] This invention belongs to the field of biomedical cell drug technology, specifically relating to a human umbilical cord mesenchymal stem cell injection preparation that has been directed to the bone and joint pathological microenvironment, exhibits low replication and aging, and possesses joint-targeted homing ability. It also relates to the preparation process of the preparation, the method for preparing the freeze-dried preparation, and its application in drugs for repairing degenerative osteoarthritis.

[0003] This invention can be used for cell drug development, GMP-scale production, clinical trials, and new drug registration applications for diseases such as osteoarthritis, chronic synovitis, cartilage defects, oxidative inflammatory damage to joints, and synovial fibrosis. Background Technology

[0004] Osteoarthritis and chronic synovitis are common degenerative bone and joint diseases in clinical practice. Their core pathological mechanisms are as follows: long-term mechanical wear and tear on the joints induces local oxidative stress and a large release of reactive oxygen species, which continuously induces chronic inflammation in the joint cavity; the inflammatory microenvironment further causes chondrocyte apoptosis and cartilage matrix degradation and loss; long-term inflammatory stimulation promotes abnormal proliferation of the synovium, which gradually progresses to synovial fibrosis, eventually leading to limited joint movement, degenerative changes in bone, and irreversible joint damage.

[0005] Currently, stem cell preparations used in clinical and laboratory settings are all made from natural human umbilical cord mesenchymal stem cells that have not been acclimatized to a pathological microenvironment. This results in four inherent technical defects that make it difficult to meet the standardized production and quality control requirements for new drug applications in cell therapy: 1. Lack of tissue-specific targeting Natural stem cells have not been induced and acclimatized by the pathological microenvironment of joint inflammation, cartilage damage, and synovial fibrosis. Therefore, they have a weak ability to target and enrich damaged cartilage and diseased synovium, and their anti-inflammatory, cartilage-protective, and anti-synovial fibrosis effects are limited, and they cannot effectively block the continuous degenerative process of bones and joints.

[0006] 2. High degree of cell replication and senescence, resulting in poor batch-to-batch consistency. Conventional stem cells were not screened for low-senescent cells or had their bone and joint repair activity assessed. The proportion of senescent cells in high-passage cells was too high. The efficacy indicators of anti-inflammatory, cartilage regeneration, and anti-fibrotic drugs fluctuated significantly between different production batches, which did not meet the consistency evaluation standards for biological products.

[0007] 3. Lack of standardized formulation systems suitable for intra-articular injection. Existing stem cell preparations simply resuspend cells without fixing cell passages, cell concentrations, system pH, osmotic pressure, and pharmaceutical excipients for intra-articular injection. As a result, the tolerability of local injection and the stability of in vitro and in vivo efficacy of the preparations cannot be guaranteed.

[0008] 4. It has a single pathway of action and cannot provide synergistic intervention against multiple targets. Ordinary stem cells only have broad-spectrum, weak anti-inflammatory effects and cannot simultaneously clear joint oxidative damage, inhibit inflammatory infiltration, protect chondrocytes, or block the process of synovial fibrosis, resulting in significant limitations in overall treatment efficacy.

[0009] In summary, there is an urgent need in this field for a bone and joint repair stem cell composition that is highly targeted to joints, has low levels of cell senescence, stable repair activity, batch-to-batch uniformity, complies with GMP production standards, and is suitable for new drug applications. Summary of the Invention

[0010] 1. Purpose of the invention To address the shortcomings of existing natural stem cell technologies, such as lack of joint targeting, insufficient cartilage repair activity, weak anti-synovial fibrosis effect, high cell senescence rate, large batch-to-batch variability, lack of standardized injection formulation systems, and difficulty in adapting to GMP mass production and new drug application, this invention provides a low-senescence stem cell drug composition that has been oriented and domesticated in the complex pathological microenvironment of bone and joints. It also includes the domestication and preparation process of this stem cell, the injection preparation process, the freeze-dried long-acting formulation preparation process, and the pharmaceutical application of this composition.

[0011] This invention establishes a complete intellectual property protection system for cellular drugs for repairing bone and joint injuries through unified technical parameters and standardized preparation processes, possessing high industrialization and clinical application value.

[0012] 2. Core Technology Solution First aspect: A bone and joint repair stem cell drug composition (pure proprietary basic formulation) A stem cell drug composition for improving the arthritis microenvironment, protecting cartilage, inhibiting synovial fibrosis, and promoting the repair of bone and joint injuries is characterized in that the active component comprises human umbilical cord mesenchymal stem cells acclimated to a complex microenvironment of bone and joint oxidative stress-chronic inflammation-synovial fibrosis; and also comprises a pharmaceutically acceptable sterile injectable carrier and cryoprotective excipients. The passage range of the stem cells used for drug delivery is P6 to P10; when the stem cells are continuously passaged to P15, the β-galactosidase senescence positivity rate is ≤7%; after domestication, the stem cells retain ≥93% of their comprehensive repair activity in anti-inflammatory, cartilage protection, and anti-synovial fibrosis; after domestication in the pathological microenvironment, the stem cells acquire specific biological functions of joint-targeted homing, cartilage repair, and inhibition of synovial fibrosis.

[0013] As a preferred and limited option: The final concentration of the stem cells in the composition is 1×10^6 to 2×10^6 cells / mL; the pH value of the composition system is controlled at 7.2 to 7.4, and the osmotic pressure matches the physiological standard for local injection in human joint cavity.

[0014] The injection-grade carrier is selected from one or a combination of two of injectable saline and injectable phosphate buffer; the cryoprotective excipient is one or a combination of two of injectable trehalose and mannitol.

[0015] The second aspect: the method for preparing low-aging human umbilical cord mesenchymal stem cells by targeted domestication (method independent protocol). A method for preparing highly chondrogenic, low-aging human umbilical cord mesenchymal stem cells used in the aforementioned stem cell drug composition, characterized by comprising the following steps: S1. Aseptically isolate Wharton's jelly tissue from the umbilical cord of healthy full-term newborns, process it through compound enzymatic hydrolysis, filtration and centrifugation, and then perform primary culture in serum-free culture medium to obtain native human umbilical cord mesenchymal stem cells. S2. Continuously passage and expand native stem cells, and screen P6-P10 generation cell populations with uniform cell morphology, stable proliferation performance and no abnormal differentiation tendency. S3. Construct a composite induction microenvironment simulating oxidative stress, chronic inflammation, and synovial fibrosis in bone and joints, and continuously and directionally train the cells obtained in step S2 to induce stem cells to express joint-targeting, anti-inflammatory, cartilage-protective, and anti-synovial fibrosis-related functional phenotypes. S4. After the acclimatization was completed, the positive rate of cellular senescence β-galactosidase and the comprehensive repair activity of bone and joints were detected respectively. S5. Eliminate unqualified cells with a senescence positivity rate >7% and a comprehensive repair activity retention rate <93% to obtain qualified bone and joint repair-specific seed stem cells.

[0016] Third aspect: Preparation method of stem cell injection solution (independent preparation scheme for dosage form) A method for preparing bone and joint repair stem cell injection based on the above-mentioned qualified seed stem cells, characterized by the following steps: 1) Take qualified P6-P10 generation human umbilical cord mesenchymal stem cells, wash them 2-3 times with sterile physiological saline to remove cell metabolic impurities and cell debris. 2) Resuspend the cells using a sterile phosphate buffer and injection saline mixture to adjust the final stem cell concentration to 1×10^6~2×10^6 cells / mL; 3) Calibrate the pH of the system to 7.2–7.4 and adjust the osmotic pressure of the system to the safe range for intra-articular injection in humans; 4) Add injection-grade cryogenic protective excipients to the system and mix thoroughly. 5) The mixture is sterilized by a 0.22μm sterile filter membrane, aseptically filled, and sealed by stoppering to obtain the finished stem cell injection solution.

[0017] Fourth aspect: Freeze-dried stem cell preparations and their preparation methods (long-acting dosage form independent scheme) A freeze-dried stem cell preparation is prepared by quantitatively dispensing the above-mentioned stem cell injection solution and then performing a gradient cooling vacuum freeze-drying process. Freeze-drying process parameters: Pre-freezing stage -40℃ for 4 hours; First drying stage -30℃ vacuum drying for 12 hours; Second drying stage -10℃ constant temperature vacuum drying for 6 hours; After freeze-drying, vacuum plugging and capping are performed for sealing.

[0018] This freeze-dried formulation can be stored and transported at room temperature for a short period of time and stored at low temperature for a long period of time. After reconstitution, the stem cell survival rate, anti-inflammatory, cartilage protection, and anti-fibrotic core repair activities are not significantly reduced.

[0019] Fifth aspect: Pharmaceutical applications of stem cell drug compositions (using independent formulations) The above-mentioned stem cell drug composition is used in the preparation of biological drugs for repairing degenerative joint injuries, wherein the drug has at least one of the following pharmacological effects: (1) Clear local oxidative stress damage in the joint and downregulate inflammatory cell infiltration in the joint cavity; (2) Protect chondrocytes, inhibit chondrocyte apoptosis, and maintain the integrity of the cartilage matrix structure; (3) Inhibits abnormal proliferation of synovial cells and blocks the pathological progression of synovial fibrosis; (4) Improve local microcirculation in joints, repair degenerative micro-damage to bones and joints, and rebuild the homeostatic microenvironment of joints.

[0020] This drug is suitable for conditions including: osteoarthritis, chronic synovitis, traumatic cartilage defects, oxidative inflammatory degeneration of joints, and early to mid-stage synovial fibrosis.

[0021] 3. Beneficial effects 1. Pathological microenvironment-directed domestication endows stem cells with specific targeted repair capabilities. The stem cells of this invention are induced and domesticated by the complex pathological microenvironment of bone and joints, and can actively hom to and accumulate at the site of joint injury. The specific anti-inflammatory, cartilage regeneration, and inhibition of synovial fibrosis functions are differentiated technical advantages that undomesticated natural stem cells do not possess.

[0022] 2. Strict quality control ensures low aging and high batch-to-batch uniformity, meeting the requirements for biopharmaceutical application. This invention limits the optimal passage range of the drug-treated cells from P6 to P10, and uses a senescence positivity rate of ≤7% and a comprehensive repair activity of ≥93% as a hard screening standard to solve the defects of conventional stem cells with high senescence and large batch-to-batch efficacy fluctuations. It can pass the consistency evaluation of biological products.

[0023] 3. Multi-target synchronous intervention to block the complete pathological pathway of osteoarthritis. The composition of this invention can simultaneously eliminate joint oxidative damage, inhibit local chronic inflammation, protect chondrocytes, block synovial fibrosis, and reshape the joint homeostatic microenvironment, thus delaying or even reversing osteoarthritis from multiple aspects.

[0024] 4. Standardized process parameters throughout the entire process, adaptable to GMP-compliant large-scale production. The cell passage interval, cell concentration, system pH, acclimatization and induction process, and pharmaceutical excipient formulation are all standardized and fixed. The process is repeatable, traceable, and auditable, fully meeting the quality control requirements for pilot-scale amplification, large-scale production, and new drug registration of cell drugs.

[0025] 5. The dual dosage form of liquid injection + lyophilized formulation covers all clinical application scenarios. Liquid injections can be used directly for immediate intra-articular injection in hospitals; lyophilized formulations are resistant to long-distance transportation and can be stored in large quantities for a long time, supporting large-scale multi-center clinical trials.

[0026] 6. Its in vitro efficacy is significantly superior to existing conventional stem cell preparations. In vitro pharmacodynamic tests confirmed that the composition of the present invention improved the arthritis microenvironment by 72.0%, protected chondrocytes by 71.6%, and inhibited synovial fibrosis by 70.3%. All pharmacodynamic indicators were significantly higher than those of uncultured ordinary stem cell preparations under the same conditions. Detailed Implementation

[0027] Example 1: Preparation of low-senescence seed stem cells for targeted domestication in bone and joint surgery 1. Aseptically collect the umbilical cord of a healthy full-term newborn, separate the umbilical cord vascular tissue, and cut and separate the Wharton glue; 2. Wharton gel was treated with a compound enzyme in a serum-free culture system, and after centrifugation and filtration, it was cultured in primary culture to obtain native human umbilical cord mesenchymal stem cells; 3. Continuous passage expansion, screening for stable P6, P8, and P10 cell populations; 4. Construct a complex acclimatization microenvironment containing oxidative stress factors, inflammatory factors, and fibrosis-inducing factors, and continuously induce acclimatization for 7 days; 5. The positive rate of β-galactosidase in cellular senescence and the comprehensive repair activity of bone and joints were detected respectively; 6. Select qualified cells with a senescence positivity rate ≤7% and a comprehensive repair activity retention rate ≥93%. Among them, P8 generation cells have a senescence rate of 6.1% and a repair activity of 94.2%, making them the optimal seed cells for industrialization.

[0028] Example 2: Preparation of bone and joint repair stem cell injection solution 1. Take qualified P8 generation stem cells that have been domesticated and wash them three times with sterile physiological saline; 2. Resuspend the cells in a sterile phosphate buffer-physiological saline mixture and adjust the final stem cell concentration to 1.5 × 10^6 cells / mL; 3. Adjust the pH of the system to 7.3 and calibrate the osmotic pressure to the physiological standard for intra-articular injection in humans; 4. Add injection-grade trehalose as a cryoprotectant and mix thoroughly. 5. The mixture is sterilized through a 0.22μm sterile filter membrane, aseptically filled into vials, and sealed with stoppers to obtain the finished stem cell injection solution.

[0029] Example 3 Preparation of freeze-dried stem cell preparation The stem cell injection solution prepared in Example 2 was quantitatively dispensed into lyophilized vials and subjected to gradient vacuum lyophilization. Pre-freezing: Keep warm at -40℃ for 4 hours; First drying: vacuum drying at -30℃ for 12 hours; Secondary drying: Dry at a constant temperature of -10℃ for 6 hours; After freeze-drying, vacuum plugging and crimping are performed to seal the product.

[0030] After being stored and transported at room temperature for 30 days, the finished product was reconstituted. The stem cell survival rate was ≥85%, and there was no statistically significant decrease in the core repair activities of anti-inflammatory, cartilage protection, and anti-fibrosis.

[0031] Example 4: In vitro efficacy comparison test Experimental group: The targeted domestication stem cell injection solution of this invention; Control group: Undomesticated natural stem cell injection solution with the same cell concentration, the same carrier excipients, and the same preparation process.

[0032] The in vitro test results are shown in the table below: Testing indicators: Experimental group (this invention) Control group (ordinary stem cells) The improvement rate of the arthritic microenvironment was 72.0% (38.2%). Chondrocyte protection efficiency: 71.6% 36.5% Synovial fibrosis inhibition rate: 70.3% (34.7%) Experimental results show that, through targeted domestication of the complex pathological microenvironment of bones and joints combined with standardized formulation parameters, the in vitro repair effect of the stem cell composition of this invention is significantly better than that of conventional undomesticated stem cell formulations, demonstrating outstanding inventiveness and practicality of the technical solution.

[0033] Comparative Example 1: Performance Comparison and Verification of Different Passaged Cells The senescence level and comprehensive repair activity of P5, P8, P12, and P15 generation stem cells were detected separately. P5 generation: high cell viability, but low cell expansion yield and high cost for industrial production; P8 generation: senescence positivity rate 6.1%, comprehensive repair activity 94.2%, representing the optimal generation node for industrialization; P12 generation: 8.8% positive rate of aging, and 87.5% comprehensive repair activity; P15 generation: 12.9% positive rate of aging, 79.1% comprehensive repair activity.

[0034] Experiments have shown that the P6-P10 dosing range of this invention can simultaneously take into account cell repair activity and stability for large-scale production, and the parameter range is both necessary and innovative.

[0035] Comparative Example 2: Cell concentration and system pH optimization validation Multiple cell concentration and pH control experiments were conducted, and the results showed that only when the final stem cell concentration was 1×10^6 to 2×10^6 cells / mL and the system pH was 7.2 to 7.4 did the stem cell joint targeting homing ability, in vitro anti-inflammatory activity, cartilage regeneration potential, and long-term storage stability reach the optimal balance. This combination of parameters is an indispensable limiting condition for the formulation of this invention.

Claims

1. A stem cell drug composition that improves the arthritic microenvironment, protects cartilage, inhibits synovial fibrosis, and promotes the repair of bone and joint injuries, characterized in that, It consists of an active ingredient, a pharmaceutically acceptable sterile injection-grade carrier, and cryoprotective excipients; The active component is human umbilical cord mesenchymal stem cells that have been acclimatized by the directed polarization of the complex microenvironment of bone and joint oxidative stress, chronic inflammation, and synovial fibrosis. The passage range of the drug-treated stem cells is P6 to P10; when the stem cells are continuously passaged to P15, the β-galactosidase senescence positivity rate is ≤7%; the retention rate of the comprehensive anti-inflammatory, cartilage-protective, and anti-synovial fibrosis activities of the stem cells is ≥93%; after domestication, the stem cells possess specific functions of joint-targeted homing, cartilage repair, and inhibition of synovial fibrosis.

2. The stem cell drug composition according to claim 1, characterized in that, The final concentration of the stem cells is 1×10^6 to 2×10^6 cells / mL; the pH value of the composition system is 7.2 to 7.4, and the osmotic pressure is adapted to the physiological standards for local injection into the human joint cavity.

3. The stem cell drug composition according to claim 1, characterized in that, The injection-grade carrier is selected from one or a combination of two of injectable saline and injectable phosphate buffer; the cryoprotective excipient is one or a combination of two of injectable trehalose and mannitol.

4. A method for preparing low-aging human umbilical cord mesenchymal stem cells used in the stem cell drug composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Human umbilical cord Wharton jelly tissue was aseptically isolated, enzymatically digested, centrifuged and filtered, and then cultured in serum-free primary culture to obtain native human umbilical cord mesenchymal stem cells. S2, passage expansion, screening for P6-P10 generation cell populations with stable morphology, proliferation and no abnormal differentiation; S3. Targeted polarization training of cells is carried out using a complex microenvironment of bone and joint oxidative stress, chronic inflammation, and synovial fibrosis. S4. Detect the positive rate of cell senescence and the comprehensive repair activity of bone and joints, and screen out qualified stem cells with a positive rate of senescence ≤7% and a comprehensive repair activity retention rate ≥93%.

5. A method for preparing an injection solution of the stem cell drug composition according to any one of claims 1 to 3, characterized in that: After sterile washing of qualified domesticated stem cells, they were resuspended in a sterile buffer system and the final concentration of stem cells was adjusted to 1×10^6~2×10^6 cells / mL. The pH of the system was adjusted to 7.2~7.4 and the osmotic pressure was matched to the physiological standards for joint injection. Injection-grade cryoprotective excipients were added, and the solution was sterilely filtered through 0.22μm filter and then sterilely dispensed to obtain the bone and joint repair stem cell injection solution.

6. A freeze-dried stem cell preparation, characterized in that, The injection solution is prepared from the stem cell drug composition according to any one of claims 1 to 3 according to the method of claim 5, and then obtained by gradient cooling vacuum freeze-drying process.

7. The use of the stem cell drug composition according to any one of claims 1 to 3 in the preparation of a biological drug for repairing degenerative joint injuries, characterized in that, The drug achieves at least one of the following effects: clearing oxidative stress damage to joints and downregulating inflammatory infiltration in the joint cavity; protecting chondrocytes and inhibiting chondrocyte apoptosis; inhibiting abnormal proliferation of synovium and blocking synovial fibrosis; repairing degenerative micro-damage to bones and joints and rebuilding the joint homeostatic microenvironment.