Application of IL-36R (Interleukin-36R) inhibitor in preparation of medicines for treating bone joints

Abstract

The invention relates to application of an IL-36R inhibitor in preparation of medicines for treating bone joints. Experiments prove that the IL-36R inhibitor can slow down chondroproteoglycan degradation and fibrosis, so that cartilage degradation is slowed down, occurrence and development of osteoarthritis are relieved and improved, and the IL-36R inhibitor has extremely high application value and clinical significance. Meanwhile, transdermal drug delivery of the IL-36R inhibitor is achieved through the microneedle product, and it is found that compared with a drug delivery method of direct injection of a traditional injection, the microneedle has a more remarkable treatment effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of an IL-36R inhibitor in the preparation of drugs for treating bone and joint diseases. Background Art

[0002] Osteoarthritis (OA) is the most common degenerative bone and joint disease in the elderly and is also the main cause of chronic disability in people over middle age. The pathogenesis of OA has not been fully elucidated, and effective drugs are still lacking, and there is a lack of precise treatment. End-stage OA patients can only rely on artificial joint replacement. There are already some treatment options clinically, such as delaying the progression of OA through pharmaceutical compositions, physical therapy, or biological agents. However, due to the chronicity and complexity of OA itself, many treatment means still have obvious defects. For example, some in the pharmaceutical composition rely on calcium intake to fill the cavitary damage of bone tissue, but long-term extra calcium intake may lead to calcium metabolism imbalance and elevated blood calcium levels. Another example is that the stem cell and gene therapies in biological agents have low efficiency of stem cell directed differentiation, and gene editing has the risk of off-target. Therefore, it is urgent to explore a stable, safe, and effective treatment means to solve the impact of OA on human health. Summary of the Invention

[0003] The purpose of the present invention is to disclose the application of an IL-36R inhibitor in the preparation of drugs for treating bone and joint diseases, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0004] The first aspect of the present invention lies in providing a new application direction of an IL-36R inhibitor.

[0005] The second aspect of the present invention lies in providing a microneedle for treating osteoarthritis.

[0006] The application described in the first aspect of the present invention is to use an IL-36R inhibitor to prepare drugs for treating osteoarthritis.

[0007] In a further application embodiment, the IL-36R inhibitor slows down the degradation and / or fibrosis of cartilage proteoglycan.

[0008] In a further application embodiment, the IL-36R inhibitor slows down cartilage degradation.

[0009] In a further application embodiment, the osteoarthritis includes knee osteoarthritis, elbow osteoarthritis, shoulder osteoarthritis, hip osteoarthritis, hip bone osteoarthritis, spondylosis, cervical osteoarthritis, or lumbar osteoarthritis.

[0010] In a further application embodiment, the IL-36R inhibitor is a monoclonal antibody.

[0011] In a further application embodiment, the monoclonal antibody includes spesolimab and / or imsidolimab. Spesolimab is a monoclonal antibody targeting IL-36R, which can specifically inhibit IL-36 signal transduction to improve generalized pustular psoriasis. The prior art's understanding of the IL-36R inhibitor mainly focuses on its use in treating dermatitis.

[0012] In a further application embodiment, the drug further includes pharmaceutically acceptable excipients.

[0013] In a further application embodiment, the pharmaceutically acceptable excipients include at least one of diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, solubilizers, cosolvents, preservatives, pH regulators, osmotic pressure regulators, surfactants, coating materials, antioxidants, bacteriostatic agents, or buffers.

[0014] In a further application embodiment, the dosage form of the drug includes at least one of suspensions, granules, capsules, powders, tablets, emulsions, solutions, dripping pills, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0015] In a further application embodiment, the administration route of the drug includes at least one of nasal administration, transdermal administration, intraspinal administration, rectal administration, intra-articular administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, or sublingual administration.

[0016] The microneedle described in the second aspect of the present invention is solidified and formed by mixing the IL-36R inhibitor in a microneedle carrier.

[0017] In a further application embodiment, the microneedle is a gel transdermal microneedle (Microneedle, MN). MN integrates the advantages of microneedle technology and drug gel formulations. It can penetrate the epidermal layer of the skin and even directly reach deeper skin layers, causing almost no pain, while effectively reducing the risk of infection. Compared with traditional injection methods, MN provides a non-invasive and convenient drug delivery method, which is suitable for long-term treatment and multiple-dose scenarios. The gel formulation carrying the drug can be designed to respond to specific physiological conditions, such as changes in pH value or temperature, to precisely control the drug release rate, thereby achieving continuous and stable drug release.

[0018] In a further application embodiment, the concentration of the IL-36R inhibitor in the microneedle is 200 - 2000 ng / mL.

[0019] In a further application embodiment, the microneedle carrier comprises methacrylated gelatin (GelMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (photoinitiator LAP), and polyethylene glycol diacrylate (PEGDA).

[0020] The beneficial effects of the present invention are as follows: Through experiments, the present invention verifies that the IL-36R inhibitor can slow down the degradation and fibrosis of cartilage proteoglycan, thereby slowing down cartilage degradation, and further alleviating and improving the occurrence and development of osteoarthritis, with extremely high application value and clinical significance. At the same time, the percutaneous administration of the IL-36R inhibitor is achieved through a microneedle product, and it is found that it has a more significant therapeutic effect compared with the traditional injection method of direct injection of a needle agent. Description of the Drawings

[0021] Figure 1 It is a diagram of the experimental results of the cartilage explants of joint replacement in OA patients in Example 1; Figure 2 It is a diagram of the experimental results of the cell and animal models of OA mice in Examples 2 and 3; Figure 3 It is a schematic diagram of MN-SPE in Example 4; Figure 4 It is a schematic diagram for verifying the therapeutic effect through different administration methods in Example 5. Detailed Embodiments

[0022] The following examples further illustrate the content of the present invention, but should not be construed as a limitation to the present invention. Modifications and substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all fall within the scope of the present invention.

[0023] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0024] Experimental Materials: Spesolimab is produced by Wuhan PuJian Biotechnology Co., Ltd. (AtaGenix), Cat No.: ATAD00573, CAS No.: 2097104-58-8. According to the product manual, a working solution with a corresponding concentration is prepared, aliquoted, and stored in a -80°C ultra-low temperature refrigerator to avoid drug inactivation caused by repeated freezing and thawing. The concentration of the Spesolimab solution for in vitro experiments is 200 ng / mL; the concentration of the Spesolimab solution for explant experiments is 2 μg / mL; the concentration of the Spesolimab injection for in vivo intra-articular injection is 0.1 mg / mL.

[0025] C57BL / 6J mice were all purchased from Gempharmatech Co., Ltd, strain: C57BL / 6JGpt, number: N000013. The mice were housed in a specific pathogen-free (SPF) animal room, maintaining the room temperature at 20 - 26 °C, humidity at 50 - 60%, and a 12-hour light / dark cycle. The experimental rodent and rabbit maintenance diet (1022) was purchased from Beijing Huafukang Bioscience Co., Ltd, product batch number 2310220724, execution standard: GB 14924 - 2010. All food and water were fed to the mice after autoclaving treatment.

[0026] Main experimental reagents: Gelatin Methacryloyl (GelMA, EFL; CAS No.: EF-GM-90), Lithium Phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP) (Sigma Aldrich; CAS No.: 85073-19-4), PEGDA (Sigma Alorick; CAS No.: 26570-48-9); Fluorescein-5-isothiocyanate; FITC; DMEM / F-12 basal medium (Gibco; product number: C11330500BT). Fetal bovine serum, Australia (Gibco; product number: 10099-141C). Penicillin-streptomycin double antibody 100× solution (Corning; product number: 30-002-CI). 0.25% Trypsin-EDTA (Gibco; product number 25200-072). 0.1% collagenase type II (Sigma-Aldrich; product number C-BIOC). Bone tissue Safranine O and Fast Green staining solution (Sigma Aldrich). Alcian blue staining solution (Sigma Aldrich). Protease inhibitor (Thermo Fisher; product number 87785), Ripa cell lysate (ThermoFisher; product number 89901) protein marker (Thermo Fisher; product number 26616). 30% polyacrylamide-N,N'-methylenebisacrylamide / Acr-Bis (Beijing Leagene Biotechnology). Non-fat milk powder (Inner Mongolia Yili). Sensitive ECL luminescent solution (MedChemExress; product number HY-K1005). PVDF membrane (Sigma Aldrich; product number ISEQ00010). 0.3% hydrogen peroxide (Guangzhou Sanxun). 4% paraformaldehyde (Beijing LanJieKe). Neutral gum. DMSO (MedChemExress).

[0027] The WB antibodies are shown in the following table.

[0028]

[0029] Example 1: In vitro culture of cartilage explants from joint replacements of OA patients.

[0030] Collect joint tissues from OA patients who have undergone knee joint replacement, and exclude samples of patients with malignant tumors, diabetes, and some other serious chronic diseases within five years to ensure that the selected OA samples are all caused by degenerative osteoarthropathy.

[0031] (1)Cut the knee joint lesion cartilage samples of OA patients who have received total knee arthroplasty and have fresh tissue into pieces about 1 cm 3 in size and wash them 3 times with PBS buffer containing 1% penicillin and streptomycin.

[0032] (2)Transfer the cartilage pieces to a 12-well plate and add 1.5 mL of DMEM / F12 complete medium containing 10% fetal bovine serum and 1% double antibody.

[0033] (3)Add Spesolimab to the wells of the experimental group for incubation, and add DMSO to the wells of the control group. Incubate in a cell culture incubator at 37°C with 5% CO 2 for 14 days, changing the medium every other day and supplementing the drug at the same time.

[0034] (4)After culturing in the 12-well plate for 14 days, fix with 4% paraformaldehyde, dehydrate, embed, and section.

[0035] (5)Perform safranin O-fast green staining: Immerse the dewaxed and hydrated tissue sections in PBS for 5 minutes, stain with the prepared 1% fast green staining solution for 60 seconds, fix with 3% acetic acid fixing solution for 3 seconds, then wash off the fixing solution, and then stain with 0.5% safranin O staining solution for 30 seconds. Wash with deionized water to remove the floating color. Finally, dehydrate, make transparent and seal with neutral gum.

[0036] Collect images with an Olympus upright microscope. Two experienced researchers used the Osteoarthritis Research Society International (OARSI) grading system (0 - 6 points) to blindly score the severity of OA in articular cartilage. The experimental results are as Figure 1 shown. The experimental group (labeled "Spesolimab") significantly inhibited cartilage proteoglycan degradation and fibrosis. It shows that Spesolimab has clinical treatment potential.

[0037] Example 2: Cell experiment to verify the improvement effect of Spesolimab on OA cartilage degradation.

[0038] (1)Extract primary mouse chondrocytes.

[0039] Take 7 - 10 neonatal mice within 3 days after birth. After anesthesia with tribromoethanol at a standard of 10 μL / g, decapitate them, place them in 75% alcohol for disinfection for 15 minutes, and transfer the disinfected neonatal mice to a sterile large dish. Use autoclaved tissue scissors and forceps to isolate the knee joint. After thoroughly separating the skin, ligaments, muscles, and fat around the knee joint, carefully peel off the cartilage caps at the top of the tibia and femur and place them in a sterile small Ep tube. Add 0.25% trypsin and incubate in a 37°C incubator for 30 minutes. Then, use micro forceps to try to strip off the fascia and fibers attached to the surface of the cartilage pieces as cleanly as possible for the second time. Put the separated cartilage pieces into a centrifuge tube containing DMEM / F12 complete medium with 0.1% type II collagenase and 10% FBS, and place it on a 37°C shaker for digestion for 12 hours. When the cartilage pieces are digested until they are invisible to the naked eye, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add DMEM / F12 complete medium containing penicillin / streptomycin double antibody and 10% fetal bovine serum to resuspend the chondrocytes, and then seed them into a 10 cm cell culture dish (this is the P0 generation), at 37°C, 5% CO 2 culture.

[0040] (2) Set up a blank group (labeled "Control"), a control group (labeled "IL - 1β"), and an experimental group (labeled "IL - 1β + Spesolimab"). The blank group is primary mouse chondrocytes; the control group is constructed by adding IL - 1β with a final concentration of 10 ng / mL to primary mouse chondrocytes and stimulating for 24 hours; the experimental group is formed by adding both IL - 1β and Spesolimab to primary mouse chondrocytes and treating for 24 hours.

[0041] (3) Perform Western blot (WB) detection and Alcian Blue staining on the cells of the three groups respectively.

[0042] WB detection: Use Ripa cell lysis buffer and protease inhibitor to extract cell proteins on ice. Then use 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate the lysates, and transfer the separated proteins to a 0.22 μm PVDF membrane by wet transfer method. Block the PVDF membrane with 5% skim milk on a slow - shaking decolorizer at room temperature for 1 hour, and then incubate with the primary antibody at 4°C for 12 - 16 hours. The next day, incubate the PVDF membrane with the secondary antibody at room temperature for 1 hour. Finally, image and collect protein band images in a chemiluminescence imager (Tanon, model 5200CE) with sensitive ECL chemiluminescent solution.

[0043] The results of WB detection are as Figure 2As shown in (a) in the figure. The expression level of type II collagen (Col2a1) in the cells of the control group decreased significantly under the induction of IL-1β, and the catabolic enzyme (ADAMTS5) increased significantly. In the cells of the experimental group, under the intervention of Spesolimab, the expression level of Col2a1 was close to that of the blank group, and the increase of ADAMTS5 induced by IL-1β was significantly inhibited.

[0044] Alcian Blue staining: Select chondrocytes in good condition and inoculate them into a 6-well plate, and place them in an incubator at 37°C with 5% CO 2 until 95% confluence. Digest the cells with trypsin and transfer them to a 15 mL centrifuge tube, then centrifuge and remove the supernatant. Resuspend the cells with 100 μL of complete medium. Use a sterile pipette with a 100 μL range to aspirate the high-concentration cell suspension and drop the cells onto the 6-well plate in a dropwise manner to make the chondrocytes closely connected into a chondrocyte cluster with a diameter of 0.5 cm. Then carefully transfer the 6-well plate to a 37°C cell incubator. After 6 hours, when the cell cluster is completely adherent, complete medium can be added. After the treatment is completed, remove the medium, wash it once with PBS, add 4% paraformaldehyde to fix the cells for 10 minutes, wash the fixing solution, add Alcian Blue staining solution and stain for 15 - 20 minutes, and then wash the staining solution. Finally, place the well plate into a scanner to collect images.

[0045] The results of Alcian Blue staining are as shown in Figure 2 As shown in (a) in the figure. Spesolimab restored the cartilage matrix degradation induced by IL-1β. This indicates that Spesolimab can counteract the catabolic effect of inflammatory factors on chondrocytes.

[0046] Example 3: Animal experiments were conducted to verify the improvement effect of Spesolimab on OA cartilage degradation.

[0047] (1) A mouse OA model was constructed by DMM surgery.

[0048] DMM-OA, that is, an osteoarthritis model induced by medial meniscus instability. Select 10-week-old C57BL / 6J wild-type mice and perform surgery on the right posterior knee joint: After anesthetizing the mice, perform skin preparation and disinfection. Cut through the medial skin of the joint and then separate the patellar ligament along the medial side of the knee joint to expose the joint cavity. Cut the tibial ligament of the medial meniscus and free the medial meniscus to cause knee joint instability. After completion, close the joint cavity and suture the skin. Sham-operated group mice: Only cut through the skin of the right knee joint and suture it. After 8 weeks, collect knee joint specimens. The severity of OA was evaluated using the OARSI (Osteoarthritis Research Society International) scoring standard (0 - 6 points).

[0049] (2)The experiment established a blank group (labeled "Control"), a control group (labeled "OA-8W"), and an experimental group (labeled "OA-8W+Spesolimab i.a").

[0050] The experimental procedure was as shown in Figure 2 b in the figure. For the mouse OA model, Spesolimab was injected into the joint cavity (intra-articular, i.a) once a week, 10 μL each time. Samples were collected in the eighth week, and the samples were treated with safranin O-fast green staining.

[0051] The experimental results were as shown in Figure 2 c in the figure. In the control group, obvious degeneration and degradation of articular cartilage occurred at the 8th week after surgery, while the articular cartilage in the experimental group was smoother and the cartilage degradation was significantly reduced. This indicated that Spesollimab could alleviate the progression of experimental OA in mice, demonstrating the potential of Spesolimab as a therapeutic drug for OA.

[0052] Example 4: Preparation of gel transdermal microneedles loaded with Spesolimab.

[0053] To solve the problems of low oral drug delivery efficiency and poor compliance with intra-articular injection in clinical practice, it was decided to attempt to prepare a gel transdermal microneedle (MN-SPE) delivery system loaded with Spesolimab. Through orthogonal experiments, 15%, 20%, and 25% (w / v) of GelMA, 0.25% (w / v) of LAP, and 3%, 4%, and 5% (w / v) of PEGDA were respectively mixed and dissolved in deionized water, and mixed into a sol in a 37°C water bath environment. Subsequently, the mixture was poured into a polydimethylsiloxane (PDMS) mold and subjected to ultraviolet polymerization. Among them, the structure of the microneedles with a concentration ratio of 15% (w / v) GelMA, 0.25% (w / v) LAP, and 5% (w / v) PEGDA was stable, and obvious cracks and looseness occurred in the microneedles with the other ratios to varying degrees (as shown in Figure 3 a in the figure).

[0054] After determining the raw material ratio concentration of the microneedle carrier, Spesolimab was then mixed with the fluorescent substance FITC in the above sol before solidification and drying. Whether the drug in the microneedles was evenly distributed in the matrix was observed through a fluorescence microscope. We found that there was uneven distribution of Spesolimab drug inside the microneedles. Therefore, we used ultrasonic-assisted dissolution of Spesolimab drug and adjusted the mixing order and stirring method to ensure that the drug could be evenly dissolved and distributed in the microneedles (as shown in Figure 3 b in the figure).

[0055] After determining the conditions, MN-SPE was synthesized next. First, 15% (w / v) GelMA, 0.25% (w / v) LAP, and 5% (w) PEGDA were dissolved in deionized water and mixed into a sol in a 37 °C water bath environment. Then, Spesolimab was dissolved and mixed with the sol. The content of Spesolimab in each MN-SPE was 0.1 mg. Subsequently, the sol containing Spesolimab was poured into the PDMS mold to cover the pinhole part, and the mold was placed in a vacuum dryer to remove air bubbles, and then photocured by ultraviolet light for 30 seconds. Subsequently, the mixed sol without Spesolimab was continuously added to the surface of the mold as a substrate, and then continuously dried and photocured until the MN-SPE was demolded (the preparation process is as shown at c in Figure 3 . The image of MN-SPE under a scanning electron microscope is as shown at d in Figure 3 .

[0056] The in vitro drug release curve (as shown at e in Figure 3 ) showed that the drug release rate of Spesolimab was 30.11% within 24 hours and 81.43% within 168 hours, indicating that MN-SPE had a long-acting sustained release effect.

[0057] Example 5, Animal experiment of MN-SPE.

[0058] A mouse OA model was constructed according to the method provided in Example 3.

[0059] The experimental groups included a control group (labeled "Ctrl"), an experimental group (labeled "MN-SPE"), an injection control group (labeled "Spesolimab i.a"), and an oral control group (labeled "Spesolimab oral").

[0060] The hair on the back skin of the mice in the experimental group with an area of 4×5 cm 2 was shaved off, MN-SPE was applied, covered with a moist gauze, and fixed with a bandage to prevent MN-SPE from moving and detaching, and it was kept replaced once a week.

[0061] The mice in the injection control group were injected with Spesolimab into the joint cavity once a week, 10 μL each time.

[0062] The mice in the oral control group were orally administered tablets once a week, with a net content of 100 μg of Spesolimab each time.

[0063] All groups were sampled and sectioned at the eighth week of OA, and the results of safranin O-fast green are as shown in Figure 4As shown at a in the figure, the cartilage surface of the control group was severely worn at the eighth week, and the other three groups all improved cartilage degradation and wear to varying degrees. Among them, the articular cartilage surface of the mice in the experimental group was smoother than that of the mice in the oral control group or the injection control group, and the degradation of the cartilage matrix was significantly reduced (see Figure 4 as shown at b in the figure).

[0064] In the treatment of OA, the clearance of drugs injected into the joint at the joint site, the risk of infection, and the pain caused by frequent injections make this route have extremely poor compliance for patients. Example 5 confirmed that the MN-SPE route is superior to Spesolimab through intra-articular injection or oral route in improving experimental OA in mice. MN-SPE is a method that can create hundreds of microchannels in the skin to maintain the drug supply of Spesolimab painlessly to exert its therapeutic effect on OA, providing a longer duration of Spesolimab drug and expanding the route of Spesolimab in the clinical treatment of OA. This drug delivery system is very likely to be applied as an important OA treatment route in future clinical diagnosis and treatment, bringing good news to the vast number of OA patients.

[0065] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

Claims

1. Application of IL-36R inhibitors in the preparation of drugs for the treatment of osteoarthritis.

2. The use according to claim 1, characterized in that: The IL-36R inhibitor slows cartilage proteoglycan degradation and / or fibrosis.

3. The application according to claim 1, characterized in that: The IL-36R inhibitor slows cartilage degradation.

4. The use according to claim 1, characterized in that: The osteoarthritis includes knee osteoarthritis, elbow osteoarthritis, shoulder osteoarthritis, hip osteoarthritis, hip osteoarthritis, spondyloarthropathies, cervical osteoarthritis or lumbar osteoarthritis.

5. The use according to claim 1, characterized in that: The IL-36R inhibitor is a monoclonal antibody.

6. The use according to claim 5, characterized in that: The monoclonal antibodies include spesolimab and / or imidorimab.

7. The use according to any one of claims 1 to 6, characterized in that: The drug also includes pharmaceutically acceptable excipients; preferably, the pharmaceutically acceptable excipients include: at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubilizer, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, an antibacterial agent or a buffer.

8. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, oral preparations, suppositories, enemas, aerosols, patches or drops.

9. The use according to any one of claims 1 to 6, characterized in that: The administration route of the drug includes at least one of nasal administration, transdermal administration, intraspinal administration, rectal administration, intraarticular administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and sublingual administration.

10. A microneedle for treating osteoarthritis, characterized in that: The IL-36R inhibitor is mixed in the microneedle carrier and solidified.

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