Application of phosphodiesterase 4D inhibitor in preparation of preparation for treating arthritis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a phosphodiesterase 4D inhibitor in preparation of a preparation for treating arthritis. According to the application disclosed by the invention, in gouty arthritis diseases, BPN14770 can inhibit activation of NLRP3 inflammasomes, so that MSU-induced gouty arthritis symptoms can be inhibited in physiological indexes and pathological experiments. In a mouse with gouty arthritis induced by MSU, BPN14770 inhibits the activation of NLRP3 inflammasome by inhibiting the activity of PDE4D, influences the release of IL-1beta inflammatory factors, and improves arthritis. On the basis, the invention provides a compound capable of being used for preparing a compound for treating the MSU-induced gouty arthritis, and the compound has good social benefits and economic benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a phosphodiesterase 4D inhibitor in the preparation of an arthritis treatment preparation. Background Art

[0002] Arthritis includes gouty arthritis, calcium pyrophosphate deposition disease, hydroxyapatite deposition disease, rheumatoid arthritis, osteoarthritis, etc. Among them, gouty arthritis is an inflammatory disease caused by monosodium urate crystals deposited in joint tissues. Its typical feature is sudden onset, especially at night. Patients often feel severe joint pain when waking up in the early morning. This pain is closely related to the swelling, heat and erythema of the joint and surrounding tissues, resulting in obvious functional impairment. During an acute gout attack, usually one or more joints are involved. The first attack is mostly monoarticular and lasts for several days to several weeks, with self-limiting nature, but patients are prone to recurrent attacks.

[0003] During a gouty arthritis attack, MSU activates the innate immune system, leading to an inflammatory cascade reaction, accompanied by the recruitment of monocytes and neutrophils. As a pathogenic component of innate immune effector cells of the human immune system, tissue-resident macrophages activate the assembly of the NLRP3 inflammasome by recognizing the deposited MSU crystals. The NLRP3 inflammasome is a multi-protein oligomeric complex formed by the cytoplasmic proteins NLRP3, ASC, and Caspase-1, which can sense danger signals such as mitochondrial ROS release, potassium efflux, or lysosomal protease leakage, and integrate these signals into the inflammasome complex, thereby activating Caspase-1. The activated Caspase-1 then cleaves pro-IL-1β into its biologically active and secreted form, namely mature IL-1β, and promotes the polarization of macrophages into a pro-inflammatory M1-like phenotype, ultimately triggering the release of IL-1β.

[0004] Macrophages are one of the important cell types in the immune system and play a key role in the occurrence and development of acute gout, mediating the entire process of acute gout. The interaction between MSU crystals and resident macrophages is considered the main cause of gouty arthritis. For a long time, it has been recognized that resident macrophages and monocyte-derived macrophages are different subsets, and attempts have been made to study their roles in acute gout separately. Previous studies have shown that resident macrophages initiate and drive inflammation, while monocyte-derived macrophages differentiate into M1-like macrophages in response to MSU crystals. IL-1β released by macrophages can activate other cells through the IL-1 receptor IL-1R, inducing pro-inflammatory cytokines and chemokines, leading to local inflammation and sometimes systemic reactions, such as chills and fever manifested clinically. It has been reported that macrophages respond to MSU crystals through the action of multiple receptors, organelles, and signaling pathways to produce and release inflammatory mediators, thereby triggering gouty inflammation. MSU crystals activate resident mononuclear phagocytes to release IL-1β as a trigger for joint inflammation.

[0005] Currently, the management of gouty arthritis mainly focuses on improving lifestyle, reducing uric acid levels, and anti-inflammatory treatment. Reducing alcohol and purine intake and controlling body weight play a positive role in preventing hyperuricemia. Clinically, urate-lowering drugs that increase uric acid excretion, such as probenecid, sulfinpyrazone, and benzbromarone, are usually combined with uric acid production inhibitors such as allopurinol and febuxostat for the treatment of gouty arthritis. Urate-lowering therapy plays a key role in the prevention and management of hyperuricemia. Anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs, colchicine, glucocorticoids, and IL-1 antagonists, are the first choice for the treatment of acute gouty arthritis. However, commonly used drugs may cause various side effects, such as nephrotoxicity and cardiovascular risks, or cannot be used in patients with cardiovascular diseases, diabetes, or kidney diseases.

[0006] In summary, there is an urgent need to provide a new strategy for the treatment of gouty arthritis. Summary of the Invention

[0007] The object of the present invention is to provide an application of a phosphodiesterase 4D inhibitor in the preparation of an arthritis treatment preparation, solving the problems existing in the prior art.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides an application of a phosphodiesterase 4D inhibitor in the preparation of an arthritis treatment preparation.

[0010] Preferably, the phosphodiesterase 4D includes at least one of BPN14770, D159687, and CP671305.

[0011] Preferably, the arthritis includes one or more of gouty arthritis, calcium pyrophosphate deposition disease, hydroxyapatite deposition disease, rheumatoid arthritis, and osteoarthritis.

[0012] Preferably, the arthritis treatment preparation uses a PDE4D inhibitor as the active ingredient and is formulated with pharmaceutically acceptable excipients.

[0013] Preferably, the pharmaceutically acceptable excipients are one or several of diluents, disintegrants, precipitation inhibitors, glidants, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, and stabilizers.

[0014] Preferably, the diluent includes any one of starch, lactose, sucrose, and mannitol.

[0015] Preferably, the disintegrant includes any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0016] Preferably, the precipitation inhibitor includes any one of sodium dodecyl sulfate, Tween-80, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.

[0017] Preferably, the glidant includes any one of cationic polyacrylamide, poly(diallyldimethylammonium chloride), and cationic starch.

[0018] Preferably, the binder includes any one of starch paste, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.

[0019] Preferably, the dispersant includes any one of sodium dodecyl sulfate, polyvinylpyrrolidone, and sodium carboxymethylcellulose.

[0020] Preferably, the suspending agent includes any one of gum arabic, tragacanth, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose.

[0021] Preferably, the isotonic agent includes any one of sodium chloride, glucose, and mannitol.

[0022] Preferably, the thickener includes any one of gum arabic, xanthan gum, and sodium carboxymethylcellulose.

[0023] Preferably, the emulsifier includes any one of sodium dodecyl sulfate, benzalkonium chloride, and sorbitan fatty acid esters.

[0024] Preferably, the preservative includes any one of benzoic acid, sorbic acid, methyl paraben, and benzalkonium bromide.

[0025] Preferably, the stabilizer includes any one of sodium sulfite, sodium bisulfite, tocopherol, and disodium edetate.

[0026] Preferably, the arthritis treatment preparation further comprises a uric acid-lowering drug or a uric acid production inhibitor.

[0027] Preferably, the uric acid-lowering drug comprises one or more of probenecid, sulfinpyrazone and benzbromarone.

[0028] Preferably, the uric acid production inhibitor comprises one or more of allopurinol and febuxostat.

[0029] Preferably, the acceptable dosage forms of the preparation include tablets, capsules, granules, injections, pills, powders, ointments or oral liquids.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] The present invention provides an application of a phosphodiesterase 4D inhibitor in the preparation of an arthritis treatment preparation. The present invention discovers that in gouty arthritis, BPN14770 can inhibit the activation of NLRP3 inflammasome, thereby showing inhibition of MSU-induced gouty arthritis symptoms in physiological indexes and pathological experiments. In MSU-induced gouty arthritis mice, BPN14770 inhibits the activation of NLRP3 inflammasome by inhibiting the activity of PDE4D, affects the release of IL-1β inflammatory factor, and improves joint inflammation. Based on this, the present invention provides a compound that can be used to prepare a treatment for MSU-induced gouty arthritis, which has good social and economic benefits.

[0032] Therefore, the purpose of the present invention is to provide a novel compound that can improve gouty arthritis by inhibiting the activity of PDE4D and further inhibiting the activation of NLRP3 inflammasome. The present invention verifies the therapeutic effect of the PDE4D inhibitor BPN14770 on gouty arthritis mice through in vivo experiments. Description of the Drawings

[0033] Figure 1 For the detection results of toe swelling degree and pain degree of mice in each group, the paw photos of the normal group, model group, BPN14770 group and colchicine group are shown as A - D in sequence; E: pain threshold of mice in each group; F: toe swelling degree of mice in each group.

[0034] Figure 2 For the secretion level of IL-1β in the joint cavity of mice in each group.

[0035] Figure 3 For the macrophage infiltration in the joint tissues of mice in each group, A: detection result by flow cytometry; B: corresponding statistical result of A.

[0036] Figure 4HE staining results of joint tissues of mice in each group. A: Results of two biological replicates in the normal group; B: Results of two biological replicates in the model group; C: Results of two biological replicates in the BPN14770 group; D: Results of two biological replicates in the colchicine group.

[0037] Figure 5 Expression of PDE4D in joint tissues of mice in each group. A: Fluorescence detection results; B: Statistical results corresponding to A.

[0038] Figure 6 Expression of F4 / 80 in joint tissues of mice in each group. A: Fluorescence detection results; B: Statistical results corresponding to A.

[0039] Figure 7 Effect of PDE4D on the co-expression of PDE4D and F4 / 80 in joint tissues of mice with monosodium urate (MSU)-induced gouty arthritis. A: Fluorescence detection results; B: Statistical results corresponding to A.

[0040] Figure 8 Effect of BPN14770 on the expression of PDE4D and NLRP3 inflammasome-related proteins in joint tissues of mice with MSU-induced gouty arthritis.

[0041] Figure 9 Structural formula of BPN14770. Detailed implementation manners

[0042] The present invention will be further described below through specific examples, but the scope of the present invention is not limited thereby. Modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and these modifications or substitutions all fall within the protection scope of the present invention.

[0043] The inventive concept of the present invention is as follows:

[0044] Currently, the management of gouty arthritis mainly focuses on improving lifestyle, reducing uric acid levels, and anti-inflammatory treatment. Reducing alcohol and purine intake and controlling body weight play a positive role in preventing hyperuricemia. Clinically, drugs that increase urate excretion, such as probenecid, sulfinpyrazone, and benzbromarone, are usually combined with uric acid production inhibitors, such as allopurinol and febuxostat, for the treatment of gouty arthritis. Urate-lowering therapy plays a key role in the prevention and management of hyperuricemia. Anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs, colchicine, glucocorticoids, and IL-1 antagonists, are the first choice for the treatment of acute gouty arthritis. However, commonly used drugs may cause various side effects, such as nephrotoxicity and cardiovascular risks, or cannot be used in patients with cardiovascular diseases, diabetes, or kidney diseases.

[0045] Based on this, the present invention proposes an application of a phosphodiesterase 4D inhibitor in the preparation of an arthritis treatment preparation. The present invention takes the PDE4D inhibitor BPN14770 as an example for the following experiments. BPN14770, D159687, and CP671305 all belong to phosphodiesterase 4D inhibitors and can produce technical effects similar to those of BPN14770 in the preparation of arthritis treatment preparations.

[0046] BPN14770, also known as Zatolmilast, is a selective allosteric inhibitor of phosphodiesterase 4D, with the chemical formula C 21 H 15 ClF3NO2, a molecular weight of 405.80, a CAS number of 1606974-33-7, and the structural formula is shown in Figure 9 .

[0047] Phosphodiesterases are a class of enzymes encoded by multiple genes that break down the secondary messengers cAMP and cGMP into AMP and GMP, respectively. The PDE4 subtype was the first variant to be identified. As a member of the PDE4 family, PDE4D is widely distributed in various tissues such as the brain, heart, bladder, muscle tissue, and thyroid, as well as in immune system cells such as monocytes, macrophages, and neutrophils, and is involved in the occurrence of diseases such as neurodegenerative diseases, depression, stroke, multiple sclerosis, and chronic obstructive pulmonary disease, cardiovascular diseases, etc. Currently, PDE4D inhibitors are mainly used in clinical practice to treat diseases such as Alzheimer's disease and fragile X syndrome. One of the PDE4D inhibitors, BPN14770, shows potential for improving memory and cognitive function in the treatment of Alzheimer's disease, and has relatively few side effects, with good clinical application prospects.

[0048] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following further illustrates the present invention with specific examples. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0049] The abbreviations used in the present invention are shown in Table 1.

[0050] Table 1 Abbreviation Table

[0051]

[0052] Example 1

[0053] The application of a phosphodiesterase 4D inhibitor in the preparation of an arthritis treatment preparation is as follows:

[0054] 1. Experimental method.

[0055] Eight-week-old wild-type C57BL / 6 mice were raised in a specific pathogen-free (SPF) environment with normal light, and given food and water normally. They were randomly divided into 4 groups, namely the Normal group, the MSU model group, the BPN14770 + MSU group, and the colchicine + MSU group, which were denoted as the normal group, the model group, the BPN14770 group, and the colchicine group in sequence, with 10 mice in each group. The normal group and the model group were given distilled water, the colchicine group was given 1 mg / kg colchicine, and the BPN14770 group was given 0.3 mg / kg BPN14770. They were continuously gavaged for 4 days respectively. One hour after drug administration on the fourth day, an MSU solution was aspirated with a sterilized syringe and injected into the mice at a dose of 50 mg / kg. For the mice in the model group, the BPN14770 group, and the colchicine group, the solution was injected into the ankle joint along the 45-degree direction on the dorsal side of the right ankle joint of the mice, with the injection standard being the swelling on the contralateral side, to induce an acute gouty arthritis model in the mice. The normal group was injected with an equal volume of normal saline.

[0056] 2. Evaluation indicators.

[0057] 2.1 Measurement of ankle joint swelling in mice.

[0058] Before and at 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, 24 h, 36 h, and 48 h after modeling, the right ankle joint diameter of each group of mice was measured with a vernier caliper to calculate the swelling degree. The calculation formula was: Ankle joint swelling degree = Ankle joint diameter at the measurement point - Ankle joint diameter before modeling. The units of the ankle joint swelling degree, the ankle joint diameter at the measurement point, and the ankle joint diameter before modeling were all mm.

[0059] 2.2 Measurement of mechanical pain.

[0060] The mice were placed in a transparent resin glass cage with a metal grid at the bottom to adapt to the environment for 30 min. The plantar skin surface was vertically stimulated with a Von Frey filament from below the sole, and each stimulation lasted for 6 s. The force gradients of the Von Frey filaments were 0.008 g, 0.020 g, 0.040 g, 0.070 g, 0.160 g, 0.400 g, 0.600 g, 1.000 g, 1.400 g, 2.000 g, and 4.000 g respectively. A rapid paw withdrawal or flinching behavior of the mouse in response to the stimulation was considered a positive reaction. When it was a positive reaction, the adjacent lower-level force stimulation was used until no positive reaction occurred. The lowest force required to cause a positive reaction was recorded as the paw withdrawal threshold (PWT).

[0061] 2.3 Flow cytometry detection of the number of macrophages in the joint cavity fluid.

[0062] Collect 0.5 mL of synovial fluid and rinse it with 0.9% NaCl solution by mass percentage. First, collect the cells, centrifuge at 2500 rpm / min for 5 min, separate the supernatant and the precipitate. The precipitate is the cells, which are used for infiltrating immunocyte flow cytometry analysis to detect immunocytes CD45, CD11b, and double-positive macrophages of F4 / 80.

[0063] 2.4. Determination of inflammatory cytokines in mouse synovial fluid.

[0064] Centrifuge the supernatant obtained in 2.3 again at 3000 rpm for 10 min, and take the supernatant for ELISA detection of the IL-1β inflammatory index.

[0065] 2.5. Histopathological observation of mouse joint tissues.

[0066] After the experiment, sacrifice the mice, separate the mouse feet, soak and fix them in 4% paraformaldehyde solution by volume percentage for 48 h, perform decalcification treatment with EDTA decalcifying solution for several weeks to several months, dehydrate step by step, embed in paraffin and section, and the section thickness is 4 μm. Observe the pathological changes of mouse joint tissues by HE staining under the tissue in-situ cell scanning analysis system.

[0067] 2.6. Immunofluorescence observation of the expression of macrophage markers F4 / 80 and PDE4D in mouse joint tissues and their co-localization.

[0068] Soak and fix each mouse joint tissue in 4% paraformaldehyde solution by volume percentage for 48 h, perform decalcification treatment with EDTA decalcifying solution, dehydrate step by step, embed in paraffin and section. After dewaxing and antigen repair, label the immunofluorescence-labeled antibodies PDE4D and F4 / 80, and observe under the tissue in-situ cell scanning analysis system after sealing the slices, and analyze the expression and co-localization of PDE4D and F4 / 80 in each group of joint tissues.

[0069] 2.7. Detection of the expression of PDE4D, Caspase-1, and IL-1β proteins in mouse joint tissues by Western blot method.

[0070] Extract the protein of each group of mouse joint tissues for Western blot, and analyze the obtained bands using the software Image J.

[0071] 3. Experimental results.

[0072] 3.1. BPN14770 alleviated the symptoms of MSU-induced gouty arthritis in mice.

[0073] After the modeling, measure the ankle swelling degree and mechanical pain of the mice. Figure 1The results showed that after MSU induction, the joint swelling degree and pain degree of mice were high, indicating the successful establishment of the model. After treatment with BPN14770, the swelling degree and pain degree of mice were significantly alleviated.

[0074] 3.2. Detection of the secretion level of inflammatory cytokines by ELISA.

[0075] As Figure 2 shown, compared with the normal group of mice, the secretion of IL-1β in the joint cavity of gouty arthritis mice induced by MSU increased. Compared with the MSU group of mice, the secretion of IL-1β in the joint cavity of mice in the BPN14770 treatment group decreased significantly. This indicates that BPN14770 can alleviate the inflammatory response of gouty arthritis mice induced by MSU.

[0076] 3.3. Detection of the infiltration of joint macrophages by flow cytometry.

[0077] As Figure 3 shown, compared with the normal group of mice, the infiltration of joint macrophages in gouty arthritis mice induced by MSU increased; compared with the MSU group of mice, the infiltration of joint macrophages in the BPN14770 treatment group decreased significantly, indicating that BPN14770 can reduce the infiltration of macrophages in gouty arthritis mice induced by MSU.

[0078] 3.4. Histopathological observation of mouse joint tissues - HE staining.

[0079] The functional change of gouty arthritis is the infiltration of inflammatory cells. As Figure 4 shown, compared with the normal group of mice, the infiltration of inflammatory cells in the joint tissues of gouty arthritis mice induced by MSU increased; compared with the MSU group of mice, the infiltration of inflammatory cells in the arthritis of mice in the BPN14770 treatment group decreased significantly, indicating that BPN14770 can improve the pathological manifestations of gouty arthritis mice induced by MSU.

[0080] 3.5. Immunofluorescence detection of the expression and co-localization of PDE4D and NLRP3 in mouse joint tissues.

[0081] As Figures 5 to 7 shown, compared with normal mice, the expression of PDE4D and the macrophage marker F4 / 80 in the joint tissues of the model group mice increased significantly, and their co-localization also increased significantly. Compared with the model group mice, the BPN14770 treatment group could significantly reduce the expression of PDE4D, F4 / 80 and their co-localization.

[0082] 3.6. Detection of the expression of related proteins by Western blot.

[0083] As Figure 8It was shown that, compared with the normal group of mice, the expression of PDE4D and NLRP3-dependent inflammatory cytokine IL-1β in the joint tissues of the model group of mice was up-regulated. At the same time, the activation of caspase-1 in the joint tissues was detected, and the activated caspase-1 was significantly increased. Compared with the model group of mice, after administration of BPN14770, the expression of PDE4D in the joint tissues of mice was significantly decreased, and the expressions of IL-1β and activated caspase-1 were also significantly decreased, indicating that BPN14770 can inhibit the expression of NLRP3 inflammasome-related proteins.

[0084] The present invention also provides a specific formulation of the arthritis treatment preparation as shown in Table 2.

[0085] Table 2 Formulation of the arthritis treatment preparation of the present invention

[0086]

[0087] In Table 2, "-" indicates that there is no such item.

[0088] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0089] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims

1. Use of phosphodiesterase 4D inhibitors in the preparation of agents for treating arthritis.

2. The application according to claim 1, wherein The phosphodiesterase 4D inhibitors include at least one of BPN14770, D159687, and CP671305.

3. The application according to claim 1, characterized in that, The arthritis includes one or more of gouty arthritis, calcium pyrophosphate deposition disease, hydroxyapatite deposition disease, rheumatoid arthritis, and osteoarthritis.

4. The application according to claim 1, characterized in that, The agent for treating arthritis uses a PDE4D inhibitor as the active ingredient and is formulated with pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients are one or several of diluents, disintegrants, precipitation inhibitors, glidants, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, and stabilizers.

6. The application according to claim 1, characterized in that, The agent for treating arthritis further includes a uric acid-lowering drug or a uric acid production inhibitor.

7. The application according to claim 6, characterized in that, The uric acid-lowering drugs include one or more of probenecid, sulfinpyrazone, and benzbromarone.

8. The application according to claim 6, wherein The uric acid production inhibitors include one or more of allopurinol and febuxostat.

9. The application according to claim 1, characterized in that, The acceptable dosage forms of the agent include tablets, capsules, granules, injections, pills, powders, ointments, or oral liquids.

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