Pharmaceutical composition for targeted removal of osteoarthritis articular cavity apoptotic cells and application thereof

Abstract

The invention discloses a pharmaceutical composition for targeted removal of osteoarthritis articular cavity apoptotic cells and application, and belongs to the technical field of biological medicines. The pharmaceutical composition comprises a targeting ligand, an apoptotic cell scavenger and a drug carrier, the targeting ligand is selected from an anti-phosphatidylserine monoclonal antibody or Annexin V protein, and can be specifically combined with phosphatidylserine on the surface of an apoptotic cell; the apoptotic cell scavenger is selected from demethoxycurcumin, andrographolide and the like, and can promote apoptotic cell phagocytosis and inhibit inflammatory factor release; and the drug carrier is hyaluronic acid-polylactic acid copolymer nanoparticles, and has good articular cavity retention property and drug sustained release effect. The pharmaceutical composition disclosed by the invention can be used for accurately targeting apoptotic cells in osteoarthritis articular cavities, efficiently removing the apoptotic cells, inhibiting inflammatory response and delaying cartilage degeneration, is good in biological safety and provides a new effective means for treating osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a pharmaceutical composition and its application that targets and eliminates apoptotic cells in the joint cavity of osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease characterized by degenerative changes in articular cartilage, osteophyte formation at the joint margins, and inflammatory responses in the joint cavity. Its core pathological mechanisms involve chondrocyte apoptosis, abnormal activation of synovial cells, cytokine storms, and impaired clearance of apoptotic cells. With the increasing aging of the population, the incidence of OA is rising year by year. Statistics show that the global prevalence of OA in people over 60 years of age exceeds 30%. Osteoarthritis severely impacts patients' quality of life and places a heavy burden on social healthcare systems.

[0003] Currently, clinical treatment for osteoarthritis (OA) is mainly symptomatic, including nonsteroidal anti-inflammatory drugs (NSAIDs) such as celecoxib to relieve pain, intra-articular injection of sodium hyaluronate to improve joint lubrication, and short-term control of acute inflammation with glucocorticoids. However, these methods cannot reverse articular cartilage damage, nor can they fundamentally address the core pathological issue of OA—the accumulation of apoptotic cells in the joint cavity. Studies have shown that when apoptotic cells (including chondrocytes, synovial cells, and inflammatory cells) in the joint cavity cannot be effectively cleared, they release large amounts of damage-associated molecular patterns (DAMPs, such as ATP and HMGB1) and inflammatory factors (such as TNF-α and IL-1β), activating the pro-inflammatory phenotype of synovial macrophages, forming a vicious cycle of apoptotic cell accumulation-inflammatory amplification-further apoptosis, accelerating cartilage degradation and joint destruction.

[0004] Current strategies for clearing apoptotic cells mainly rely on enhancing the function of endogenous phagocytes, such as using macrophage polarization regulators. However, these drugs lack specificity and are prone to interfering with the systemic immune system. Drugs that directly clear apoptotic cells, on the other hand, cannot precisely target joint lesions, resulting in insufficient local drug concentrations, limited efficacy, and a high risk of side effects. Therefore, developing a drug composition that can specifically target apoptotic cells in the joint cavity of osteoarthritis, efficiently clear apoptotic cells within the lesion, and inhibit the inflammatory response has become an urgent need in the field of OA treatment. Summary of the Invention

[0005] To address the limitations of existing osteoarthritis treatments, which fail to target and eliminate apoptotic cells in the joint cavity and have limited efficacy, this invention provides a pharmaceutical composition that targets and eliminates apoptotic cells in the joint cavity of osteoarthritis. This composition is characterized by strong targeting, high elimination efficiency, significant anti-inflammatory effects, and good biocompatibility. The invention also provides a method for preparing this pharmaceutical composition and its application in the treatment of osteoarthritis.

[0006] To achieve the above objectives, the present invention provides a pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis. The active ingredients include a targeting ligand, an apoptotic cell scavenger, and a drug carrier. The targeting ligand is a biomolecule that specifically binds to molecules exposed on the surface of apoptotic cells. The apoptotic cell scavenger is an active substance that promotes phagocytosis of apoptotic cells or directly induces degradation of apoptotic cells. The drug carrier is a biocompatible carrier with joint cavity retention capability.

[0007] Preferably, the targeting ligand is selected from one or more of antiphosphatidylserine (PS) monoclonal antibody, Annexin V protein or its functional fragments; wherein the titer of the antiphosphatidylserine monoclonal antibody is 1:1000-1:5000 and the purity of the Annexin V protein is ≥95%.

[0008] The most typical characteristic molecule on the surface of apoptotic cells is phosphatidylserine (PS), which is normally located on the inner side of the cell membrane. During apoptosis, it everts to the cell membrane surface, becoming a "recognition tag" for apoptotic cells. Anti-PS monoclonal antibodies can specifically bind to PS on the surface of apoptotic cells. Annexin V protein is a calcium-dependent phospholipid-binding protein with extremely high affinity for PS. Both can serve as targeting ligands to achieve precise localization of apoptotic cells.

[0009] Preferably, the apoptotic cell scavenger is selected from one or more of curcumin derivatives, andrographolide, or recombinant human macrophage colony-stimulating factor (rhM-CSF); wherein the curcumin derivative is demethoxycurcumin with a purity ≥98%; the andrographolide has a purity ≥97%; and the specific activity of rhM-CSF is ≥1×10⁻⁶. 7 U / mg.

[0010] Demethoxycurcumin, a natural derivative of curcumin, not only retains the anti-inflammatory activity of curcumin but also has stronger water solubility and cell penetration. It can promote the phagocytosis of apoptotic cells by activating macrophage phagocytic receptors (such as CD36) while inhibiting the release of inflammatory factors from apoptotic cells. Andrographolide can inhibit the inflammatory response by regulating the NF-κB signaling pathway and enhance the ability of synovial macrophages to clear apoptotic cells. rhM-CSF can specifically stimulate the proliferation, differentiation, and activation of macrophages, and improve their efficiency in phagocytizing apoptotic cells. The synergistic effect of the three can achieve an integrated effect of targeted localization, efficient clearance, and inflammation suppression.

[0011] Preferably, the drug carrier is modified hyaluronic acid-polylactic acid copolymer (HA-PLA) nanoparticles, wherein the molecular weight of hyaluronic acid is 100-500kDa, the molecular weight of polylactic acid is 5-20kDa, the mass ratio of HA to PLA is 1:2-1:5, the particle size of the nanoparticles is 100-300nm, and the zeta potential is -10 to -20mV.

[0012] Hyaluronic acid (HA) has good adhesion and biocompatibility to the joint cavity and is a major component of synovial fluid, which can prolong the retention time of drugs in the joint cavity (the retention time can reach 7-14 days). Polylactic acid (PLA) is a biodegradable polymer material. When copolymerized with HA, the nanoparticle carrier can achieve a sustained-release effect of drugs and avoid drug degradation in a short time. At the same time, HA can bind to CD44 receptors on the surface of synovial cells, further enhancing the accumulation of drugs in the lesion area of ​​the joint cavity.

[0013] Preferably, the mass fractions of each active ingredient in the pharmaceutical composition are: 0.5-5 parts of the targeting ligand, 5-20 parts of the apoptosis cell scavenger, and 75-94.5 parts of the drug carrier; more preferably, the mass fractions are: 1-3 parts of the targeting ligand, 8-15 parts of the apoptosis cell scavenger, and 82-91 parts of the drug carrier.

[0014] Preferably, the pharmaceutical composition further comprises pharmaceutical excipients, which are selected from one or more of a suspending agent, a stabilizer, and a pH adjuster; wherein the suspending agent is polysorbate 80, and the amount used is 0.1-1% of the total mass of the pharmaceutical composition; the stabilizer is mannitol, and the amount used is 1-5% of the total mass of the pharmaceutical composition; the pH adjuster is phosphate buffer, which adjusts the pH value of the pharmaceutical composition to 6.5-7.5.

[0015] A method for preparing a pharmaceutical composition that targets and eliminates apoptotic cells in the joint cavity of osteoarthritis includes the following steps: S1. Preparation of HA-PLA nanoparticle carrier: Hyaluronic acid and polylactic acid were dissolved in dichloromethane at a mass ratio of 1:2-1:5. Polysorbate 80 was added as an emulsifier. The mixture was stirred and emulsified at 37℃ and 500-800rpm for 30-60min. Then, phosphate buffer (pH 7.0) was added for reverse emulsification. Dichloromethane was removed by vacuum evaporation. The precipitate was collected by centrifugation and freeze-dried to obtain HA-PLA nanoparticle carrier. S2, Loading apoptotic cell scavenger: Dissolve the apoptotic cell scavenger in anhydrous ethanol, add the HA-PLA nanoparticle carrier prepared in S1, and incubate at 25℃ and 100-200rpm for 2-4h to allow the scavenger to be loaded into the nanoparticles through hydrophobic interactions. Centrifuge to remove the unloaded scavenger and obtain nanoparticles loaded with the scavenger. S3, Coupling the Targeting Ligand: The targeting ligand was dissolved in phosphate buffer (pH 6.8), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added as a coupling agent. After activation at room temperature for 30 min, nanoparticles loaded with the scavenger obtained in S2 were added, and the reaction was carried out at 4 °C for 8-12 h. The uncoupled targeting ligand and coupling agent were removed by dialysis to obtain the crude drug composition. S4. Formulation: Mannitol is added to the crude drug composition obtained in S3 as a stabilizer, the pH is adjusted to 6.5-7.5 with phosphate buffer, and after volume adjustment, it is filtered through a 0.22μm filter membrane for sterilization to obtain the final drug composition, which can be made into injections or gels.

[0016] This invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating osteoarthritis. The pharmaceutical composition is administered via intra-articular injection, targeting the ligand to specifically bind to PS on the surface of apoptotic cells, guiding the drug carrier to accumulate around the apoptotic cells. The drug carrier slowly releases an apoptotic cell scavenger, which on the one hand activates the phagocytic function of synovial macrophages, promoting the phagocytic degradation of apoptotic cells, and on the other hand directly inhibits the release of inflammatory factors from apoptotic cells, breaking the vicious cycle of apoptotic cell accumulation and amplified inflammation, thereby delaying cartilage degeneration and reducing joint inflammation.

[0017] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0018] The technical solution of the present invention will be further described below through embodiments.

[0019] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention are clearly and completely described below through embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanations of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0020] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0021] Example 1 A method for preparing a pharmaceutical composition that targets and eliminates apoptotic cells in the joint cavity of osteoarthritis includes the following steps: Preparation of S1 and HA-PLA nanoparticle carriers: 10 mg of hyaluronic acid (molecular weight 300 kDa) and 30 mg of polylactic acid (molecular weight 10 kDa) were dissolved in 5 mL of dichloromethane. 2 mg of polysorbate 80 was added, and the mixture was stirred and emulsified at 37 °C and 600 rpm for 45 min. Subsequently, 10 mL of phosphate buffer (pH 7.0) was slowly added, and stirring was continued for 30 min. The dichloromethane was removed by vacuum evaporation, and the precipitate was collected by centrifugation at 8000 rpm for 20 min. After freeze-drying, approximately 35 mg of HA-PLA nanoparticle carriers were obtained.

[0022] S2, loading demethoxycurcumin: Take 10 mg of 98% pure demethoxycurcumin, dissolve it in 2 mL of anhydrous ethanol, add 35 mg of HA-PLA nanoparticle carrier prepared in S1, incubate at 25℃ and 150 rpm for 3 h, centrifuge at 10000 rpm for 15 min to remove unloaded demethoxycurcumin, and obtain nanoparticles loaded with scavenging agent.

[0023] S3. Conjugation of anti-PS monoclonal antibody: Dissolve 2 mg of anti-PS monoclonal antibody with a titer of 1:2000 in 2 mL of phosphate buffer (pH 6.8), add 0.5 mg of EDC, and activate at room temperature for 30 min. Add the activated antibody solution to nanoparticles loaded with scavenging agent and react at 4 °C for 10 h; then dialyze through a dialysis bag with a molecular weight cutoff of 100 kDa for 24 h to remove unconjugated antibody and EDC, obtaining the crude drug composition.

[0024] S4. Formulation: Add 15 mg of mannitol to the crude product, adjust the pH to 7.0 with phosphate buffer, bring the volume to 5 mL, filter and sterilize with a 0.22 μm filter membrane to obtain the injectable drug composition, wherein the mass fractions of each component are: 2 parts of anti-PS monoclonal antibody, 10 parts of demethoxycurcumin, and 88 parts of HA-PLA nanoparticle carrier.

[0025] Example 2 A method for preparing a pharmaceutical composition that targets and eliminates apoptotic cells in the joint cavity of osteoarthritis includes the following steps: Preparation of S1 and HA-PLA nanoparticle carriers: 8 mg of hyaluronic acid (molecular weight 200 kDa) and 32 mg of polylactic acid (molecular weight 15 kDa) were dissolved in 4 mL of dichloromethane. 1.5 mg of polysorbate 80 was added, and the mixture was stirred and emulsified at 37℃ and 700 rpm for 50 min. 8 mL of phosphate buffer (pH 7.0) was added for reverse emulsification. After removing the dichloromethane under reduced pressure, the precipitate was collected by centrifugation at 9000 rpm for 18 min and freeze-dried to obtain approximately 36 mg of HA-PLA nanoparticle carrier.

[0026] S2, Andrographolide loading: Take 8 mg of 97% pure andrographolide, dissolve it in 1.5 mL of anhydrous ethanol, add 36 mg of HA-PLA nanoparticle carrier, incubate at 25℃ and 180 rpm for 2.5 h, and centrifuge at 12000 rpm for 12 min to remove free drug.

[0027] S3. Conjugation with Annexin V protein: Take 1 mg of Annexin V protein with a purity of 95%, dissolve it in 1.5 mL of phosphate buffer at pH 6.8, add 0.4 mg of EDC to activate for 25 min, mix with drug-loaded nanoparticles, react at 4 °C for 9 h, and dialysis to purify to obtain crude product.

[0028] S4. Formulation: Add 12mg of mannitol, adjust the pH to 6.8, bring the volume to 4mL, filter and sterilize to obtain a gel drug composition. The mass fractions of each component are: 1 part Annexin V protein, 8 parts andrographolide, and 91 parts HA-PLA nanoparticle carrier.

[0029] Example 3 In vitro efficacy validation of the drug composition.

[0030] Experimental materials: rat chondrocyte cell line (ATCC CRL-2846), rat synovial macrophage cell line (RAW 264.7), drug composition prepared in Example 1 (experimental group), drug composition without targeting ligand (control group 1), HA-PLA nanoparticles alone (control group 2), and physiological saline (blank group).

[0031] Experimental methods: (1) Induction of chondrocyte apoptosis: Chondrocytes were treated with 10 μM TNF-α for 24 h to establish an apoptotic cell model, and the apoptosis rate was detected by flow cytometry (ensuring the apoptosis rate ≥60%).

[0032] (2) Group treatment: Apoptotic chondrocytes and synovial macrophages were co-cultured at a ratio of 1:2. The experimental group, control group 1, and control group 2 were respectively given drugs (final drug concentration of 100 μg / mL) and physiological saline. Each group was set up with 3 replicates and cultured for 24 h.

[0033] Detection indicators: ① Flow cytometry to detect the apoptotic cell clearance rate; ② ELISA to detect the content of TNF-α and IL-1β in cell culture supernatant.

[0034] The test results are shown in Table 1 below: Table 1 Test results of test indicators

[0035] Note: P <0.01, the difference was extremely significant compared with the experimental group.

[0036] As shown in Table 1, the apoptotic cell clearance rate in the experimental group was significantly higher than that in each control group. P <0.01), while the levels of inflammatory factors TNF-α and IL-1β were significantly lower than those in the control groups ( P <0.01), indicating that the pharmaceutical composition of the present invention can efficiently eliminate apoptotic cells and inhibit inflammatory responses through the action of targeted ligands.

[0037] Example 4 Sixty male SPF-grade SD rats, aged 8 weeks and weighing 200-220g, were randomly divided into experimental group, control group 1, control group 2, and blank group, with 15 rats in each group. Anterior cruciate ligament transection (ACLT) was used to establish a rat model of osteoarthritis. Successful modeling was confirmed 4 weeks after surgery (increased joint effusion and rough cartilage surface).

[0038] The experimental group received intra-articular injection of the drug composition prepared in Example 1 (0.2 mL / animal, concentration 100 μg / mL), the control group 1 received the drug composition without the targeting ligand, the control group 2 received HA-PLA nanoparticles alone, and the blank group received saline. The drugs were administered once a week for 4 consecutive weeks.

[0039] Testing indicators: (1) Joint function score: The Lysholm knee joint function score was used to evaluate the knee joint function of rats 4 weeks after administration.

[0040] (2) Pathological examination: After the rats were sacrificed, the knee joint cartilage tissue was taken and stained with HE and Safranin O-Fix Green. The cartilage structure was observed and the degree of cartilage damage was assessed by the Mankin score. (3) Detection of inflammatory factors: Collect joint effusion and detect the levels of TNF-α and IL-1β by ELISA.

[0041] The results are shown in Table 2 below: Table 2. Results of joint function score, cartilage pathology score, and inflammatory factor content detection.

[0042] Note: P <0.01, the difference was extremely significant compared with the experimental group.

[0043] As shown in Table 2, the Lysholm joint function scores of the control groups were significantly lower than those of the experimental groups, while the Mankin cartilage pathology scores and the levels of TNF-α and IL-1β in the joint effusion were significantly higher in the control groups than in the experimental groups, with all differences being statistically significant. This indicates that the pharmaceutical composition of the present invention can effectively improve knee joint function in rats with osteoarthritis, significantly reduce the degree of cartilage damage and joint inflammation, and has excellent and stable in vivo therapeutic effects in the treatment of osteoarthritis.

[0044] Example 5 Acute toxicity and long-term toxicity tests were conducted on the pharmaceutical composition prepared in Example 1: (1) Acute toxicity test: The drug composition was injected intra-articularly into SD rats at a dose of 500 mg / kg. The rats were observed for 14 consecutive days. No rats died, their diet and activity were normal, and there were no significant differences in liver and kidney function indicators (ALT, AST, BUN, Cr) compared with the blank control group. P >0.05).

[0045] The results of liver and kidney function tests are shown in Table 3 below: Table 3 Results of Liver and Kidney Function Indicators

[0046] Note: There were no significant differences in any indicators between the two groups compared with the blank control group (ns, P >0.05), which is within the normal reference range for liver and kidney function in rats.

[0047] (2) Long-term toxicity test: The drug composition was injected into the joint cavity of SD rats once a week at a dose of 100 mg / kg for 12 consecutive weeks. The rats had normal weight gain, no obvious adhesion or fibrosis in the joint cavity, and no abnormalities in the pathological sections of the liver, kidney, spleen and other major organs.

[0048] The above results indicate that the pharmaceutical composition of the present invention has good biocompatibility.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis, characterized in that: It includes targeted ligands, apoptotic cell scavengers, and drug carriers; the targeted ligands are biomolecules that specifically bind to molecules exposed on the surface of apoptotic cells, the apoptotic cell scavengers are active substances that promote phagocytosis of apoptotic cells or directly induce degradation of apoptotic cells, and the drug carriers are biocompatible carriers with joint cavity retention capabilities.

2. The pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis according to claim 1, characterized in that: The targeting ligand is selected from one or more of the following: antiphosphatidylserine monoclonal antibody, Annexin V protein, or functional fragments thereof; the titer of the antiphosphatidylserine monoclonal antibody is 1:1000-1:5000; the purity of the Annexin V protein is ≥95%.

3. The pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis according to claim 1, characterized in that: The apoptotic cell scavenger is selected from one or more of curcumin derivatives, andrographolide, or recombinant human macrophage colony-stimulating factor; the curcumin derivative is demethoxycurcumin with a purity ≥98%; Andrographolide purity ≥97%; Specific activity of recombinant human macrophage colony-stimulating factor ≥1×10 7 U / mg.

4. The pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis according to claim 1, characterized in that: The drug carrier is modified hyaluronic acid-polylactic acid copolymer nanoparticles. The molecular weight of hyaluronic acid is 100-500 kDa, the molecular weight of polylactic acid is 5-20 kDa, and the mass ratio of hyaluronic acid to polylactic acid is 1:2-1:

5. The particle size of the nanoparticles is 100-300 nm, and the zeta potential is -10 to -20 mV.

5. The pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis according to claim 1, characterized in that: By weight, it includes 0.5-5 parts of the targeting ligand, 5-20 parts of the apoptosis scavenger, and 75-94.5 parts of the drug carrier.

6. The pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis according to claim 1, characterized in that: Based on mass fractions, it includes 1-3 parts of the targeting ligand, 8-15 parts of the apoptosis cell scavenger, and 82-91 parts of the drug carrier.

7. The pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis according to claim 1, characterized in that: It also includes pharmaceutical excipients, which are selected from one or more of the following: suspending agents, stabilizers, and pH adjusters; the suspending agent is polysorbate 80, and the amount used is 0.1-1% of the total mass of the pharmaceutical composition; the stabilizer is mannitol, and the amount used is 1-5% of the total mass of the pharmaceutical composition; the pH adjuster is phosphate buffer, which adjusts the pH value of the pharmaceutical composition to 6.5-7.

5.

8. A method for preparing a pharmaceutical composition for targeting and eliminating apoptotic cells in the joint cavity of osteoarthritis as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Preparation of hyaluronic acid-polylactic acid copolymer nanoparticle carrier; S2. Load the apoptotic cell scavenger onto the nanoparticle carrier; S3. Couple the targeting ligand to the surface of the nanoparticles loaded with the scavenger; S4. Add pharmaceutical excipients, adjust the pH value, sterilize, and prepare the formulation.

9. The use of a pharmaceutical composition according to any one of claims 1-7 in the preparation of a product for treating osteoarthritis.

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