Nanometer polymer for specific targeted degradation of COX-2, and preparation method and application thereof
By constructing nanopolymers that specifically target COX-2, the problem of insufficient stability and targeting in vivo PROTAC technology is solved, and the continuous degradation of COX-2 is achieved, and the safety and effectiveness of osteoarthritis treatment is improved.
Patent Information
- Application Number
- CN202510547205.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-07-11
AI Technical Summary
The existing PROTAC technology has poor stability in vivo, insufficient water solubility, and limited tissue targeting, making it difficult to effectively target COX-2, resulting in side effects and drug resistance problems in traditional non-steroidal anti-inflammatory drugs.
The nanopolymers that specifically target the degradation of COX-2 are constructed through RAFT polymerization, combined with PROTAC technology and nanodrug delivery systems, and continuously targeted degradation of COX-2 is achieved using ROS-responsive polymers and EPR effects. The nanopolymers include functional monomers that target the degradation of COX-2 and ligand structural monomers that recruit E3 ligases.
It achieves continuous and stable degradation of COX-2, reduces systemic exposure, enhances local efficacy, avoids the side effects and drug resistance of traditional drugs, and provides an efficient, safe and long-lasting treatment plan for osteoarthritis.
Smart Images
Figure CN120289706A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a nanopolymer for specifically targeting and degrading COX-2, a preparation method thereof, and uses thereof. Background Art
[0002] Osteoarthritis (OA), as a chronic inflammatory disease centered around the degenerative lesions of articular cartilage, its pathogenesis is closely linked to the overproduction of inflammatory mediators. Among them, prostaglandin E2 (PGE2) is a key inflammatory mediator, and cyclooxygenase-2 (COX-2) is a crucial rate-limiting enzyme in the synthesis process of PGE2. Therefore, COX-2 has become a key target for regulating the inflammatory response in OA.
[0003] In past treatment methods, traditional non-steroidal anti-inflammatory drugs (NSAIDs) alleviated the pain and inflammatory symptoms of patients to a certain extent by inhibiting the activity of COX-2. However, such drugs have obvious limitations. On the one hand, they are prone to cause side effects such as gastrointestinal and cardiovascular problems, bringing additional physical burdens to patients; on the other hand, long-term use may also lead to the generation of drug tolerance, greatly reducing the treatment effect. In addition, even when using such drugs, it is still difficult to avoid incomplete inhibition of COX-2 enzyme activity or the occurrence of compensatory upregulation of COX-2, thereby leading to fluctuating curative effects and potential toxicity risks. Therefore, there is an urgent need to develop a new treatment strategy that can achieve continuous degradation of COX-2 protein rather than just transient activity inhibition, which is of great significance for the clinical treatment of OA.
[0004] In recent years, the proteolysis targeting chimera (PROTAC) technology, as a highly innovative drug modality, has shown great potential. It recruits E3 ubiquitin ligases to ubiquitinate target proteins, and then achieves the specific degradation of target proteins through the ubiquitin-proteasome system (UPS). Compared with traditional inhibitors, the PROTAC technology has many significant advantages, such as long-lasting effects, the ability to target "undruggable" proteins that are difficult to target by traditional methods, and the effective avoidance of drug resistance.
[0005] However, the PROTAC technology also faces some challenges in practical applications. Among them, PROTAC molecules generally have poor water solubility, which leads to insufficient stability in vivo and difficulty in maintaining effective drug concentrations. At the same time, their tissue targeting is limited and they cannot accurately reach the lesion site to play a role. These problems severely restrict the clinical application of the PROTAC technology. Summary of the Invention
[0006] In view of the above deficiencies in the prior art, the present invention discloses a nano-polymer for specifically and continuously targeting the degradation of COX-2, a preparation method and uses thereof. By integrating the PROTAC technology with a nano-drug delivery system, the present invention first proposes a strategy of a nano-polymer (nano-prodrug polymer) for specifically and continuously targeting the degradation of COX-2, providing an innovative solution with high efficiency, safety and durability for the treatment of osteoarthritis.
[0007] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is: The object of the present invention is to provide a nano-polymer for specifically and continuously targeting the degradation of COX-2, which is constructed in one step by RAFT polymerization of poly(ethylene glycol) methylacrylate with special surface modification, a functional monomer for targeting the degradation of COX-2, and a ligand structure monomer for recruiting E3 ligase.
[0008] Further, poly(ethylene glycol) methylacrylate with special surface modification: an active oxygen-responsive monomer capable of specifically binding to the microenvironment of the inflammatory site is introduced during the polymerization of poly(ethylene glycol) methylacrylate.
[0009] Further, the functional monomer for targeting the degradation of COX-2: a methacryloyl group is introduced into the molecular structure of the COX-2 inhibitor to endow it with polymerizable activity. The methacryloyl group undergoes an acylation reaction with the amino group in the COX-2 inhibitor molecule, introducing methacryloyl chloride to form an amide bond, thereby obtaining a prodrug monomer with polymerization ability.
[0010] Further, the ligand structure monomer for recruiting E3 ligase: a methacryloyl group is introduced into the molecular structure of the E3 ligase ligand to endow it with polymerizable activity. The methacryloyl group undergoes an acylation reaction with the amino group in the E3 ligase ligand molecule, introducing methacryloyl chloride to form an amide bond, thereby obtaining a prodrug monomer with polymerization ability.
[0011] Another object of the present invention is to provide a method for preparing the above nano-polymer for specifically and continuously targeting the degradation of COX-2, specifically: The ROS-responsive polyethylene glycol methacrylate, the functional monomer targeting the degradation of COX-2, and the ligand structural monomer recruiting the E3 ligase are mixed and dissolved, and a RAFT polymerization reaction occurs under the action of a catalyst, a chain transfer agent, and an initiator, thereby obtaining a polymer (PKP@NP) that specifically targets the degradation of COX-2 based on PROATC.
[0012] Furthermore, the mass ratio of the ROS-responsive polyethylene glycol methacrylate, the functional monomer targeting the degradation of COX-2, and the ligand structural monomer recruiting the E3 ligase is 8:2~3:2~3.
[0013] Furthermore, the PKP@NP polymer is self-assembled to obtain the PKP@NP nanopolymer.
[0014] Furthermore, the self-assembly method is to drop the PKP@NP polymer solution into deionized water stirred on a magnetic stirrer, and after 10 minutes, dialysis and filtration are carried out to obtain the PKP@NP nanopolymer.
[0015] Furthermore, the KP@NP nanopolymer is administered by intravenous injection.
[0016] Furthermore, the solvent used is an organic solvent, preferably N,N-dimethylformamide.
[0017] Furthermore, the reaction is carried out in an anaerobic environment, and nitrogen is used to remove oxygen. The reaction conditions are an oil bath, and the temperature is 60~70 °C.
[0018] Furthermore, the catalyst is DIPEA.
[0019] Furthermore, the chain transfer agent is CTA.
[0020] Furthermore, the initiator is AIBN.
[0021] Another object of the present invention is to provide the use of the above-mentioned nanopolymer that specifically and continuously targets the degradation of COX-2 in the preparation of drugs for treating chronic inflammatory diseases.
[0022] Furthermore, the chronic inflammatory disease is osteoarthritis.
[0023] Another object of the present invention is to provide a drug for treating osteoarthritis, which comprises the above-mentioned nanopolymer that specifically and continuously targets the degradation of COX-2.
[0024] The beneficial effects of the present invention: 1. The present invention innovatively couples a functional monomer that targets and degrades COX-2 with a ligand structural monomer that recruits E3 ligase through a rationally designed linker (Linker) to construct a novel COX-2-targeted PROTAC molecule. Further, to solve the delivery problem of PROTAC molecules, the present invention synthesizes a nano-polymer that specifically and continuously targets and degrades COX-2 through RAFT polymerization. This nano-polymer system not only solves the water solubility and biological stability problems of PROTAC, but also enables precise delivery to the joint cavity through inflammation microenvironment response and enhanced permeability and retention (EPR) effect, thereby reducing systemic exposure and enhancing local efficacy.
[0025] 2. Aiming at the disadvantages of traditional non-steroidal anti-inflammatory drugs in the treatment of osteoarthritis, such as gastrointestinal and cardiovascular side effects, easy generation of drug tolerance, fluctuating efficacy and potential toxicity, the present invention utilizes a novel nano-polymer system that targets and continuously degrades COX-2 to improve the stability and therapeutic effect of the drug at the target site. This nano-polymer delivery system can protect the functional monomer that targets and degrades COX-2 and the ligand structural monomer that recruits E3 ligase from the influence of factors such as oxidation and enzymatic hydrolysis in the internal environment, showing good stability; the nano-polymer can enable the prodrug to be continuously and stably released at the osteoarthritis site, and the ligand structural monomer that recruits E3 ligase avoids the problem of short-lived efficacy caused by the rapid metabolism of traditional drugs, ensuring the continuous degradation effect on COX-2 protein.
[0026] 3. The present invention also confirms that the prepared PKP@NP nano-polymer has more excellent therapeutic performance compared with traditional NSAIDs drugs. The PKP@NP nano-polymer can not only improve the stability of PROTAC molecules, specifically and continuously degrade COX-2 protein, inhibit chondrocyte apoptosis and promote cartilage tissue repair; but also accurately aggregate in the joint cavity by means of inflammation microenvironment response and EPR effect, prolong the in vivo retention time, relieve joint inflammation, repair cartilage damage and have no obvious side effects. It provides an efficient, safe and long-lasting solution for the treatment of osteoarthritis, opening up new ideas for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 1H NMR spectrum of the functional monomer that targets and degrades COX-2 prepared in the present invention; Figure 2 1H NMR spectrum of the ligand structural monomer that recruits E3 ligase prepared in the present invention; Figure 3 1H NMR spectrum of the nano-polymer that specifically and continuously targets COX-2 degradation prepared in the present invention; Figure 4Electron microscopy morphology diagram of the nano-polymer PKP@NP with specific targeting of COX-2 degradation prepared by the present invention; Figure 5 Figure showing the effect of PKP@NP on primary chondrocytes detected by Alcian blue staining; Figure 6 Figure showing the effect of PKP@NP on the continuous degradation of COX-2 in primary chondrocytes detected by western blot experiment; Figure 7 Target response diagram of PKP@NP for knee arthritis in vivo; Figure 8 Three-dimensional reconstruction of knee joint X-ray micro-CT images to detect the efficacy of PKP@NP on knee arthritis in vivo; Figure 9 Figure showing the degradation effect of PMKTP@NP on COX-2 in knee arthritis in vivo detected by knee joint immunohistochemistry. Detailed implementation manners
[0028] The following describes the detailed implementation manners of the present invention to facilitate the understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.
[0029] Example 1 Preparation of PKP@NP nano-polymer 1. Synthesize a functional monomer for targeted degradation of COX-2, and its preparation route is as follows: ; The specific process is as follows: 2-(3-Benzoylphenyl)propionic acid (2.5 g, 1 mmol), N-(3-aminopropyl)methacrylamide (1.62 g, 1 mmol), catalyst DIPEA (9.77 μL, 6 mmol), condensing agent HOBT (1.73 g, 1.3 mmol) and HBTU (4.85 g, 1.3 mmol) are dissolved in 200 mL of DMF solution, reacted at room temperature for 24 hours, the reaction process is monitored by thin layer chromatography, after the reaction is completed, a large amount of ethyl acetate is added, and it is extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride respectively, dried with anhydrous sodium sulfate, the liquid is taken after filtration, and purified by flash column chromatography to obtain ketoprofen monomer (nuclear magnetic resonance hydrogen spectrum is as Figure 1 shown).
[0030] 2. Synthesize a ligand structure monomer for recruiting E3 ligase, and its preparation route is as follows: ; The specific process is as follows: 4 - ((6 - Aminohexyl) amino) - 2 - (2,6 - dioxopiperidin - 3 - yl) isoindoline - 1,3 - dione (5 g, 1 mmol), methacryloyl chloride (4.392 mL, 1.3 mmol), triethylamine (7.359 mL, 1.5 mmol) were dissolved in 150 mL of DMF solution and reacted at room temperature for 24 hours. The reaction progress was monitored by thin - layer chromatography. After the reaction was completed, a large amount of ethyl acetate was added, and the mixture was extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride respectively, dried over anhydrous sodium sulfate, filtered, and the liquid was taken. It was purified by flash column chromatography to obtain the pomalidomide monomer (the proton nuclear magnetic resonance spectrum is as Figure 2 shown).
[0031] 3. Synthesis of PKP@NP nanopolymer, and its preparation route is as follows: ; The specific process is as follows: The functional monomer for targeted degradation of COX - 2 (0.2 g), the ligand - structure monomer for recruiting E3 ligase (0.1 g), the ROS - responsive monomer (0.1 g), polyethylene glycol methacrylate (0.8 g), catalyst DIPEA (0.5 g, 3.85 mmol), chain transfer agent CTA (27.97 mg) and initiator AIBN (5.75 mg) were dissolved in 100 mL of DMF solution. The reaction was carried out in an anaerobic environment, and nitrogen was used to remove oxygen. The reaction conditions were an oil bath with a temperature of 60 - 70 °C, and the preferred reaction temperature was 65 °C. After the reaction was completed, dialysis was carried out for 24 hours, and the nanopolymer with specific and continuous targeted degradation of COX - 2 was obtained by rotary evaporation and vacuum pumping with an oil pump (the proton nuclear magnetic resonance spectrum is as Figure 3 shown).
[0032] The obtained nanopolymer was prepared into a TEM sample, and the morphology and size of the PKP@NP nanopolymer were observed by transmission electron microscopy. The results are shown in Figure 4 .
[0033] As Figure 4 shown, the PKP@NP nanopolymer prepared by the present invention is circular, has uniform size, and the particle size is about 100 nm.
[0034] Example 2 Influence of PKP@NP Nanopolymer on Primary Chondrocytes 1. Detection by Alcian blue staining Primary chondrocytes were isolated from the knee joint cartilage of neonatal rats using type II collagenase and cultured in DMEM / F12 medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin. The cells were cultured in a carbon dioxide incubator at 37 °C with 95% air, 5% CO2, and saturated humidity, and the cell growth pattern was adherent growth.
[0035] Twenty-four hours after seeding the primary chondrocytes in a 6-well plate, 5 μg / mL of the PKP@NP nanopolymer was added and the cells were treated for 24 h. The cell clusters were fixed with paraformaldehyde, stained with Alcian blue for 30 minutes, and the different groups were observed using an upright microscope and the results were saved. The integrated optical density (IOD) values of each micro-mass were calculated using Image J software, and the results are shown in Figure 5 .
[0036] As Figure 5 shown, the PKP@NP nanopolymer prepared in the present invention can better maintain the chondrocyte morphology than the group with IL-1β alone, and the IOD value of Alcian blue also increases by 60% compared with the group with IL-1β alone according to the duration.
[0037] 2. Effect on COX-2 in primary chondrocytes Twenty-four hours after seeding the primary chondrocytes in a 6-well plate, 5 μg / mL of the PKP@NP nanopolymer was added and the cells were treated for 24 h. The proteins in the 6-well plate were collected for western blot experiments, and the results are shown in Figure 6 .
[0038] As Figure 6 shown, the PKP@NP nanopolymer prepared in the present invention can continuously degrade the COX-2 protein compared with the group with IL-1β alone.
[0039] Example 3 Animal experiment 1. Response to the in vivo inflammatory microenvironment The nanopolymer specifically targeting imaging and degrading COX-2 of the present invention has the ability to specifically respond to the in vivo inflammatory microenvironment. The above response characteristics were verified through in vivo imaging experiments: the nanopolymer was injected into the constructed osteoarthritis animal model through the tail vein, and 24 hours after injection, the animals were observed using an in vivo imaging system. The results are shown in Figure 7 .
[0040] As Figure 7As shown, compared with the normal tissue area, the fluorescent signal in the osteoarthritis inflammatory joint area is significantly enhanced. This fluorescent signal comes from the markers carried by the nanopolymer, thus confirming that the nanopolymer can respond to the inflammatory microenvironment and specifically accumulate in the osteoarthritis inflammatory site, so as to ensure that the functional monomer targeting the degradation of COX-2 and the ligand structure monomer recruiting the E3 ligase can effectively play their roles in the lesion area, achieving the therapeutic purposes of continuously degrading COX-2 protein, inhibiting chondrocyte apoptosis, and repairing cartilage tissue damage.
[0041] 2. Effects on OA in vivo Fifteen male mice were used to complete the in vivo experiment. For the post-traumatic arthritis animal model, an OA model was established by medial meniscus destabilization (DMM) surgery. The mice were randomly divided into three groups: the control group, the OA model group, and the PKP@NP nanopolymer treatment group. The control group and the OA model group were injected with PBS via the tail vein, and the PKP@NP nanopolymer treatment group was injected with the PKP@NP nanopolymer via the tail vein once a week for a total of 4 weeks. One week after the end, the knee joint cartilage tissues were collected. The changes in the microscopic structure of the bones of the mice were analyzed by X-ray, micro-CT reconstruction, and subchondral bone reconstruction of the bones of the mice. And the effect of targeted degradation of COX-2 protein in vivo was analyzed by immunohistochemistry. The results are shown in Figures 8 - 9 .
[0042] According to Figure 8 the detection results, it can be known that the mice treated with the PMKTP@NP nanopolymer prepared by the present invention had less osteophyte formation; and the PKP@NP nanopolymer could significantly reduce synovitis and cartilage wear compared with the OA model group.
[0043] According to Figure 9 the detection results, it can be known that the PKP@NP nanopolymer prepared by the present invention could significantly reduce the expression of COX-2 compared with the OA model group, indicating that the PMKTP@NP nanopolymer could significantly reduce the catabolism of the knee joint. Based on the above detection results, it can be known that the specifically targeted nanopolymer for COX-2 degradation prepared by the present invention can repair the osteoarthritis caused by DMM.
[0044] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.
Claims
1. A nanopolymer that specifically targets and degrades COX-2, characterized in that, It includes poly(ethylene glycol) methacrylate with ROS responsiveness, a functional monomer for targeted degradation of COX-2, and a ligand structural monomer for recruiting E3 ligase.
2. The nanopolymer for specifically targeting and degrading COX-2 according to claim 1, wherein The preparation method of the functional monomer for targeted degradation of COX-2 is as follows: It is prepared by introducing a methacryloyl group into the COX-2 inhibitor molecule and carrying out an acylation reaction with the amino group in the COX-2 inhibitor molecule.
3. The nano-polymer for specifically targeting and degrading COX-2 according to claim 1, characterized in that, The preparation method of the ligand structural monomer for recruiting E3 ligase is as follows: It is prepared by introducing a methacryloyl group into the ligand molecular structure of E3 ligase and carrying out an acylation reaction with the amino group in the ligand molecule of E3 ligase.
4. A nanopolymer for specifically targeting and degrading COX-2 according to any one of claims 1 to 3, characterized in that, The poly(ethylene glycol) methacrylate with ROS responsiveness, the functional monomer for targeted degradation of COX-2, and the ligand structural monomer for recruiting E3 ligase are mixed and dissolved, and a RAFT polymerization reaction occurs under the action of a catalyst, a chain transfer agent, and an initiator, thereby obtaining the polymer PKP@NP for targeted degradation of COX-2.
5. The method according to claim 4, characterized in that The mass ratio of the poly(ethylene glycol) methacrylate with ROS responsiveness, the functional monomer for targeted degradation of COX-2, and the ligand structural monomer for recruiting E3 ligase is 8:2 to 3:2 to 3.
6. The method according to claim 4, characterized in that The PKP@NP polymer obtains the PKP@NP nanopolymer through self-assembly.
7. The method according to claim 4, wherein The reaction is carried out in an anaerobic environment, and the reaction temperature is 60 - 70 °C.
8. Use of the nanopolymer for specifically targeted degradation of COX-2 according to any one of claims 1 to 3 in the preparation of a drug for treating chronic inflammatory diseases.
9. The use according to claim 8, wherein, The chronic inflammatory disease is osteoarthritis.
10. A drug for treating osteoarthritis, characterized in that, It includes the nanopolymer for specifically targeted degradation of COX-2 according to any one of claims 1 to 3.