New use of deuterated colestitinib or its salt in preparing PVNS drugs

Abstract

The present invention relates to a new use of deuterated colesitinib or a salt thereof in preparing a PVNS drug, and specifically provides an application of deuterated colesitinib or a pharmaceutically acceptable salt thereof in preparing a drug for treating and / or relieving pigmented villonodular synovitis, wherein the deuterated colesitinib or a pharmaceutically acceptable salt thereof is used as an active ingredient. The present invention has found through research that the application of deuterated colesitinib to treat cells closely related to PVNS synovial lesions has better indicators of multiple experimental results than the existing drug pecidinib. Related experiments show that deuterated colesitinib has the effect of treating and / or relieving PVNS, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a new use of deuterated corticosteroids or a salt thereof in preparing PVNS drugs. Background Art

[0002] Pigmented villonodullar synovitis (PVNS), also known as diffuse type-giant cell tumor (dTGCT), is a benign intra-articular soft tissue tumor. Its pathological features are hyperplasia of chorionic synovial lesions, accompanied by iron yellow pigmentation and inflammatory cell infiltration, and it is locally invasive. Clinical manifestations include long-term swelling and pain in the joints, limited movement, and bloody synovial fluid after puncture. The incidence rate is 4 / million, and it is more common in the knee joint. At present, the main treatment is surgical resection, but the recurrence rate is as high as 46%, and there is a lack of effective treatment drugs.

[0003] Currently, the only drug approved for the treatment of PVNS is Pexidartinib, and its Phase III clinical trial data show that the efficacy is only 39%, and it is accompanied by adverse reactions such as nausea, fatigue, and liver damage. Therefore, there is an urgent need for PVNS treatment drugs with good efficacy and few adverse reactions. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of deuterated colestitinib or a salt thereof in the preparation of PVNS drugs. Deuterated colestitinib or a salt thereof has the effect of treating and / or alleviating pigmented villonodular synovitis and has good clinical application prospects.

[0005] To this end, in a first aspect, the present invention provides the use of deuterated correxitinib or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or alleviating pigmented villonodular synovitis, wherein the deuterated correxitinib or a pharmaceutically acceptable salt thereof is used as an active ingredient.

[0006] In some embodiments, the medicament comprises an effective dose of deuterated lexitinib or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments, the mechanism of action of deuterated lecitinib or a pharmaceutically acceptable salt thereof as an active ingredient includes any one or more of the following:

[0008] Inhibits TYK2 activation;

[0009] Inhibits proliferation of fibroblast-like synoviocytes in the synovium of pigmented villonodular synovitis;

[0010] Fibroblast-like synoviocyte death in the synovium causing pigmented villonodular synovitis;

[0011] Inhibit the expression of inflammation-related factors.

[0012] In some embodiments, the inflammation-related factors include at least one of IL-6, MCP-1, IL-1β, IL-6 and TNF-α.

[0013] In some embodiments, the drug further comprises other ingredients having the effect of treating and / or alleviating pigmented villonodular synovitis.

[0014] In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

[0015] In some embodiments, the pharmaceutically acceptable excipients include at least one of a filler, a disintegrant, a binder, a surfactant, a suspending agent, a solubilizing agent, a flavoring agent, a preservative, an antioxidant, and a stabilizer.

[0016] In some embodiments, the dosage form of the drug includes at least one of powder, tablet, granule, capsule, solution, emulsion, suspension, injection, aerosol, ointment and drops.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0018] The present invention provides a new use of the drug deuterated colestitinib, and deuterated colestitinib or a pharmaceutically acceptable salt thereof can be used to prepare a drug for treating and / or relieving pigmented villonodular synovitis. The present invention has found through research that the application of deuterated colestitinib to treat cells closely related to PVNS synovial lesions has better indicators than the existing drug pesidatinib in multiple experimental results. Related experiments show that deuterated colestitinib has the effect of treating and / or relieving PVNS, and deuterated colestitinib or a pharmaceutically acceptable salt thereof as a PVNS drug has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0020] Figure 1 : Heat map and FPKM values ​​of JAK family members expression in the synovium of the control (OA) group and PVNS group in the RNA-seq dataset (GSE176133); red indicates up-regulated genes and blue indicates down-regulated genes (n=3);

[0021] Figure 2 : Representative images of immunohistochemical staining of phosphorylated and total TYK2 in synovial tissues of the control (ACL) group and PVNS group; Scale bar: 100 μm;

[0022] Figure 3 : Representative Western blot results and statistical data of phosphorylation and total protein expression levels of TYK2 in synovial tissues of the control (ACL) group and PVNS group (n=6); two-sided unpaired Student's t-test, *p<0.05, **p<0.01;

[0023] Figure 4 : Representative Western blot results and statistical data of phosphorylation and total protein expression levels of TYK2 during 5 min to 12 h of PVNS FLS treatment with IL-6 / sIL-6R (100 ng / mL);

[0024] Figure 5 : Representative Western blot results and statistics of TYK2 phosphorylation and total protein expression levels in PVNS FLS pretreated with deuterated colestitinib (0.3-10 μmol / L) for 1 h, followed by co-incubation with IL-6 / sIL-6R (100 ng / mL) and deuterated colestitinib for 15 min. n = 6, one-way ANOVA with Dunnett's multiple comparisons, *p < 0.05, **p < 0.01 compared with untreated cells (A) or cells stimulated with IL-6 / sIL-6R alone (B, C);

[0025] Figure 6 : PVNS FLS were treated with pesidatinib for 24 h, and cell death was evaluated using cell CCK-8 activity as an indicator and a dose-effect curve was drawn; n = 6;

[0026] Figure 7 PVNS FLS were treated with deuterated corticosteroids for 24 h, and the cell CCK-8 activity was used as an indicator to calculate the IC50 of deuterated corticosteroids-induced PVNS FLS death. 50 Values ​​and draw dose-effect curve; n = 6;

[0027] Figure 8 PVNS FLS were pretreated with deuterated colestitinib for 1 h, and then TNF-α (10 ng / mL) and deuterated colestitinib were used together for 72 h. CCK-8 activity was used as an indicator to evaluate cell viability and calculate the IC value of deuterated colestitinib in inhibiting the proliferation of PVNS FLS 50 Values ​​and draw dose-effect curve; n = 6;

[0028] Fig. 9: PVNS FLS were pretreated with deuterated colecitinib (A, B) or pecidinib (C, D) for 1 h, and then TNF-α (10 ng / mL) and deuterated colecitinib (A, B) or pecidinib (C, D) were added for 24 h, and the mRNA levels of IL-6 (A, C) and MCP-1 (B, D). n = 6, one-way ANOVA with Dunnett's multiple comparisons, **p < 0.01;

[0029] Fig.10 : THP-1-derived macrophages were pretreated with deuterated colecitinib (AC) or pesidatinib (DF) for 1 h, and then LPS (100 ng / mL) and deuterated colecitinib (AC) or pesidatinib (DF) were co-acted for 4 h. The mRNA levels of IL-1β (A, D), IL-6 (B, E), and TNF-α (C, F) were observed. DETAILED DESCRIPTION

[0030] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0031] Deucravacitinib (trade name SOTYKTU TM ) is a highly selective oral inhibitor of tyrosine kinase 2 (TYK2). Deuterated lexitinib works by allosteric inhibition: it binds to the catalytically inactive TYK2 pseudokinase regulatory domain and stabilizes the inhibitory interaction between the regulatory and catalytic domains. Deuterated lexitinib, developed by Bristol-Myers Squibb, is known to be useful in treating a variety of immune-mediated diseases, including psoriasis, psoriatic arthritis, lupus erythematosus, and inflammatory bowel disease.

[0032] The present invention, through database screening and clinical sample verification, found that TYK2 is the only JAK family member whose expression is upregulated in the PVNS synovium. Further experimental studies have found that the expression and activation of TYK2 are both increased in the PVNS synovium. Therefore, we considered evaluating the therapeutic effect of the TYK2 inhibitor, deuterated correxitinib, on PVNS. Through cell experiments, the therapeutic effect of deuterated correxitinib on PVNS was investigated from multiple angles, and the indicators of multiple experimental results were better than those of the existing drug pecidinib. Related experiments show that deuterated correxitinib has the effect of treating and / or alleviating PVNS.

[0033] In some embodiments, there is provided a use of deuterated colestitinib or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or alleviating pigmented villonodular synovitis (also known as diffuse tenosynovial giant cell tumor), wherein the deuterated colestitinib or a pharmaceutically acceptable salt thereof is used as an active ingredient.

[0034] In some embodiments, the medicament comprises an effective dose of deuterated lexitinib or a pharmaceutically acceptable salt thereof.

[0035] The effective dose refers to an amount that can produce a therapeutic effect on humans and / or animals and can be accepted by humans and / or animals. For example, a therapeutically or pharmaceutically effective amount refers to the amount of the drug required to produce the desired therapeutic effect, and the therapeutic effect can be reflected by the results of clinical trials, model animal studies and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the subject of treatment (such as the height, weight, gender, age and medication history of the subject of treatment), and the severity of the disease. In a specific embodiment of the present invention, the effective amount refers to an amount that has an inhibitory or alleviating effect on pigmented villonodular synovitis and can be accepted by the patient.

[0036] In some embodiments, the mechanism of action of deuterated lecitinib or a pharmaceutically acceptable salt thereof as an active ingredient includes any one or more of the following:

[0037] Inhibits TYK2 activation;

[0038] Inhibits proliferation of fibroblast-like synoviocytes in the synovium of pigmented villonodular synovitis;

[0039] Fibroblast-like synoviocyte death in the synovium causing pigmented villonodular synovitis;

[0040] Inhibit the expression of inflammation-related factors.

[0041] In some embodiments, the inflammation-related factors include at least one of IL-6, MCP-1, IL-1β, IL-6 and TNF-α.

[0042] In some embodiments, the drug further comprises other ingredients that have the effect of treating and / or alleviating pigmented villonodular synovitis. For example, the drug may further comprise Pesidatinib.

[0043] In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

[0044] In some embodiments, the pharmaceutically acceptable excipients include at least one of a filler, a disintegrant, a binder, a surfactant, a suspending agent, a solubilizing agent, a flavoring agent, a preservative, an antioxidant, and a stabilizer.

[0045] In a specific embodiment, the specific excipients selected for combination with deuterated corticosteroids or a pharmaceutically acceptable salt thereof are determined according to the desired preparation. When making ordinary preparations, the specific excipients selected are well known to those skilled in the art.

[0046] In some embodiments, the filler can be selected from lactose, dextrin, microcrystalline cellulose, starch, sorbitol, mannitol, pregelatinized starch, etc.; the disintegrant can be selected from cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, hydroxypropyl cellulose, etc.; the binder can be selected from starch slurry, sodium starch slurry, hydroxypropyl methylcellulose, povidone, etc.; the surfactant can be selected from sodium dodecyl sulfate, sodium hexadecyl sulfate, stearic acid, oleic acid, lauric acid, etc.

[0047] In some embodiments, the suspending agent can be selected from gum arabic, tragacanth gum, peach gum, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl cellulose, silica bentonite, etc.; the cosolvent can be selected from hydrochloric acid, phosphoric acid, propionic acid, acetic acid, lactic acid, citric acid, tartaric acid, boric acid, glucuronic acid, gluconic acid, lactobionic acid, malic acid, threonic acid, glucoheptonic acid, 2,5-dihydroxybenzoic acid, acidic amino acids, etc.; the flavoring agent can be selected from sodium saccharin, sodium cyclamate, aspartame, acesulfame K, glycerol, sorbitol, mannitol, sucrose, monosyrup, aromatic syrup, etc.; the preservative can be selected from phenol, cresol, chlorocresol, thymol, benzoic acid and its salts, sorbic acid and its salts, boric acid and its salts, propionic acid, formaldehyde, glutaraldehyde, benzyl alcohol, phenylethyl alcohol, trichlorobutanol, chloroform, chlorhexidine, etc.

[0048] In some embodiments, the antioxidant and stabilizer can be selected from sulfurous acid, sulfites, bisulfites, pyrosulfites, dithionites, thiosulfates, thioureas, glutathione, dimercaprol, thioglycolic acid and salts, thiolactic acid and salts, thiodipropionic acid and salts, gallic acid and salts, caffeic acid or its pharmaceutically acceptable salts, ferulic acid or its pharmaceutically acceptable salts, di-tert-butylparaphenol, 2,5-dihydroxybenzoic acid or its salts, salicylic acid or its salts, ascorbic acid and ascorbate, isoascorbic acid and isoascorbate, niacinamide, tartaric acid, phosphates, pharmaceutically acceptable acetate salts, citrates, EDTA and EDTA salts, etc.

[0049] In some embodiments, the dosage form of the drug includes at least one of powder, tablet, granule, capsule, solution, emulsion, suspension, injection, aerosol, ointment and drops.

[0050] The appropriate dosage of the drug can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, disease state, diet, administration time, administration route, excretion rate and reaction sensitivity.

[0051] In some embodiments, the subject of the drug includes any animal, specifically humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates, sheep, dogs, rodents (such as mice or rats), sheep, pigs, cats, rabbits, cattle, etc.; and non-mammals, such as chickens, amphibians, reptiles, etc. In a specific embodiment of the present invention, the subject of the drug is preferably a human.

[0052] Unless otherwise defined, the technical terms and scientific terms used herein have the same meanings as those commonly used in the field to which the present invention belongs. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0053] Example 1

[0054] This example provides TYK2 as a marker for PVNS. First, TYK2 was obtained by screening an online database, and then verified using clinical samples. According to the results, it is suggested that overexpression and activation of TYK2 may be the main driving force for the pathological progression of PVNS. The specific experiments and analysis are as follows:

[0055] 1. Online database screening:

[0056] RNA-seq dataset (GSE176133) was obtained from GEO database to analyze the gene expression of JAK family members (JAK1, JAK2, JAK3 and TYK2) in synovial membrane of PVNS and control osteoarthritis (OA) groups. The results showed that TYK2 was the only JAK family member upregulated in PVNS synovial membrane (e.g. Figure 1 shown).

[0057] 2. Clinical sample verification and analysis:

[0058] 2.1 Sample acquisition

[0059] The control synovial tissue and PVNS patient synovial tissue were obtained by surgery, wherein the control synovial tissue was taken from the knee joint of a patient with anterior cruciate ligament injury (ACL). The fat, blood vessels and tendons of the synovial tissue were removed, rinsed in pre-cooled PBS, and partly fixed in 4% paraformaldehyde for immunohistochemical staining; partly for protein expression detection; and partly for cell isolation and culture in subsequent examples.

[0060] 2.2 Immunohistochemical staining

[0061] PVNS and control (ACL) synovial tissues were fixed in 4% paraformaldehyde for 48 h, then embedded in paraffin and sliced ​​at 4 μm thickness. The slices were baked at 60°C for 30 min to melt the paraffin, and then immediately placed in xylene. The slices were dewaxed to water in the following order: xylene (Ⅰ, Ⅱ), 5 min each → anhydrous ethanol (Ⅰ, Ⅱ), 5 min each → 95% ethanol, 5 min → 90% ethanol, 5 min → 80% ethanol, 5 min → 70% ethanol, 5 min → PBS solution wash for 5 min → ultrapure water wash once. The slices were placed in 0.01 mol / L citric acid buffer and heated in a 95°C water bath for 30 min for antigen repair. The slices were returned to room temperature and washed 3 times with PBS, 2 min / time, and the tissue range was circled with an immunohistochemical pen. They were incubated with 3% hydrogen peroxide for 15 min to destroy the activity of endogenous peroxidase. They were blocked with 10% goat serum at room temperature for 1 h. Dilute the primary antibody with blocking solution, add the diluted primary antibody to the corresponding position of the tissue, and incubate at 4℃ in the dark overnight. Return the slices to room temperature, wash 3 times with PBS, 2min / time, add secondary antibody, and incubate at 37℃ for 30min. Wash 3 times with PBS, 2min / time, then add DAB solution for color development, and stop staining by immersion in tap water. Record the best color development time and apply it to other slices with the same primary antibody. Counterstain the nucleus with hematoxylin for 5min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 1s, and soak in tap water for 10min to turn blue. Dehydrate the slices in the following order: 70% ethanol, 5min→80% ethanol, 5min→90% ethanol, 5min→95% ethanol, 5min→100% ethanol (I, Ⅱ), 5min each→xylene (I, Ⅱ), 5min each. Seal the slices with neutral gum. When the neutral gum is completely solidified, use a microscope to collect images and analyze the results.

[0062] Immunohistochemical staining results Figure 2 As shown, TYK2 phosphorylation and total protein levels were upregulated in PVNS synovium.

[0063] 2.3 Protein expression detection

[0064] The synovial tissue was weighed, and RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added at a weight-volume ratio of 1:7. The tissue was homogenized with a magnetic bead homogenizer for 20 seconds, and then placed on ice for 30 minutes to fully lyse the tissue. The tissue was centrifuged at 12000g for 10 minutes at 4°C, and the precipitate was discarded and the supernatant was aspirated into a new pre-cooled centrifuge tube. The protein concentration was determined using a BCA protein quantification kit. The protein sample was loaded at 30 μg. According to the measured protein concentration, each sample was diluted to a uniform concentration with RIPA lysis buffer, and a quarter of the total volume of 4× loading buffer was added. The sample was fully mixed and placed in a metal bath and heated at 98°C for 5 minutes to denature it. The prepared tissue protein sample solution was obtained. Gel was prepared (10% separation gel, 5% concentration gel), and the protein sample was added to the corresponding lane for electrophoresis separation of proteins, and then electrophoretically transferred (300 mA, 3 hours) to the PVDF membrane. After the transfer, the PVNS membrane was blocked with 5% skim milk powder at room temperature for 1 hour. After blocking, wash the membrane three times with TBST, 5 minutes each time, cut the band containing the target protein according to the position of the marker, put it into the antibody incubation box, add the diluted primary antibody, and incubate it at 4°C overnight. The next day, recover the primary antibody, wash the membrane three times with TBST, 5 minutes each time, add the diluted HRP-labeled secondary antibody, and incubate it at room temperature for 1 hour. After incubation, wash the membrane three times with TBST, 5 minutes each time. Evenly add ECL luminescent liquid to the membrane, use chemiluminescence developer for imaging, and detect the expression level of the bound protein on the membrane.

[0065] Western blot results Figure 3 As shown, the phosphorylation and total protein levels of TYK2 were upregulated in the PVNS synovium.

[0066] Example 2

[0067] The sample obtained in step 2.1 of Example 1 was taken to separate and culture fibroblast-like synoviocytes (FLS), and the effect of deuterated lecitinib on the FLS was studied.

[0068] The synovium of PVNS joints is mainly composed of fibroblast-like synoviocytes FLS, multinucleated giant cells, lymphocytes and macrophages. Among them, FLS is the main cell type that constitutes the structure of the synovial layer. Normal synovium has only 1 to 2 layers of FLS, while in the PVNS synovium, FLS is abnormally activated and proliferated. Therefore, this example separates and cultures PVNS FLS, and then studies the effect of deuterated corticosteroids on it. The specific steps include:

[0069] 1. Isolation and culture of PVNS FLS

[0070] The sample was cut into pieces and added to a centrifuge tube containing type I collagenase and DNase I (deoxyribonuclease I) (1 mg / mL) in DMEM medium. The centrifuge tube was placed in a shaking constant temperature shaker and digested at 37°C, 250r / min for 40min. The sample was filtered through a 400-mesh screen, the filtrate was collected, and the cells were removed by centrifugation (300g, 5min) to remove the filtrate and collect the cells. The cells were resuspended in red blood cell lysis buffer to remove red blood cells, and the broken red blood cells were removed by centrifugation (300g, 5min). The remaining cells were resuspended in DMEM (high glucose) medium containing 10% FBS, cultured at 37°C, 5% CO2 for 24h, and then the medium was changed to discard the non-adherent cells. The remaining adherent cells were FLS. The culture was continued, and the medium was changed every 2 to 3 days. After the cells were fully grown, they were digested and subcultured with 0.25% trypsin. The 3rd to 7th generation cells were used for subsequent experiments.

[0071] 2. Activation of TYK2 by IL-6 and sIL-6R in PVNS FLS

[0072] PVNS FLS were placed at 5 × 10 5 100 cells / well were inoculated in a 6-well plate and incubated with DMEM (high glucose) medium containing 10% FBS for 24 hours to allow the cells to adhere to the wall. The original culture medium was discarded, and serum-free DMEM medium containing IL-6 (100 ng / mL) and sIL-6R (100 ng / mL) was added to stimulate different time lengths (5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 4 h, 8 h and 12 h). After the cell treatment, the culture medium was discarded, the cells were washed once with PBS, RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added to each well, and the cells were repeatedly blown and aspirated to fully lyse the cells, and the cell lysate was collected in a centrifuge tube, centrifuged at 12000g at 4 ° C for 10 min, the precipitate was discarded, and the supernatant was absorbed in a new pre-cooled centrifuge tube, and the protein concentration was determined using a BCA protein quantification kit to detect the expression level of membrane-bound proteins (see Section 2.3 of Example 1 for specific methods).

[0073] The results are as follows Figure 4 As shown, the results showed that IL-6 / sIL-6R stimulation significantly increased TYK2 activation (phosphorylation level) of PVNS FLS and lasted for at least 12 h.

[0074] 3. Activation of TYK2 in PVNS FLS by deuterated lecitinib

[0075] PVNS FLS were placed at 5 × 10 5100 μmol / L) were inoculated in 6-well plates and incubated for 24 h in DMEM (high glucose) medium containing 10% FBS to allow the cells to adhere to the wall. The original culture medium was discarded, and serum-free DMEM medium containing deuterated corticosteroids (0, 0.3, 1, 3 and 10 μmol / L) was added for pre-incubation for 1 h, and then stimulated with IL-6 (100 ng / mL) and sIL-6R (100 ng / mL) for 15 min. After the cell treatment, the culture medium was discarded, the cells were washed once with PBS, RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added to each well, and the cells were repeatedly blown and aspirated to fully lyse the cells, and the cell lysate was collected in a centrifuge tube, centrifuged at 12000g at 4°C for 10 min, the precipitate was discarded, and the supernatant was absorbed in a new pre-cooled centrifuge tube, and the protein concentration was determined using a BCA protein quantification kit to detect the expression level of the membrane-bound protein (see Section 2.3 of Example 1 for specific methods).

[0076] The results are as follows Figure 5 As shown, the results showed that deuterated lecitinib inhibited TYK2 activation in PVNS FLS in a dose-dependent manner.

[0077] Example 3

[0078] This example detects the effect of deuterated corticosteroids on the survival and proliferation of PVNS FLS, and uses pecidinib as a positive control. The PVNS FLS prepared in step 1 of Example 2 were used to perform the following experiments:

[0079] 1. Pecidinib

[0080] PVNS FLS were placed at 5 × 10 3 Cells were inoculated in 96-well plates and incubated for 24 h in DMEM (high glucose) medium containing 10% FBS to allow the cells to adhere to the wall. The original culture medium was discarded, and serum-free DMEM medium containing different concentrations of pesidatinib (0, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 μmol / L) was added and incubated for 24 h. 10 μL of CCK-8 reagent was added to each well 3 h before the end of incubation, and the 96-well plate was gently shaken horizontally to mix the liquid. After continuing to incubate for 3 h, the absorbance of each well at 450 nm was read using an ELISA reader, and the absorbance of the control well with only culture medium added at 450 nm was subtracted from the absorbance of the well to be tested to calculate the cell viability value of the well to be tested, and then the effect of pesidatinib on PVNS FLS death was evaluated.

[0081] The results are as follows Figure 6 As shown, the results showed that pecidinib caused almost no death in PVNS FLS.

[0082] 2. Deuterated lexitinib

[0083] PVNS FLS were placed at 5 × 103 Cells were inoculated into 96-well plates at 100 μL / well and incubated for 24 h in DMEM (high glucose) medium containing 10% FBS to allow the cells to adhere to the wall. The original culture medium was discarded, and serum-free DMEM medium containing different concentrations of deuterated corticosteroids (0, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 μmol / L) was added and incubated for 24 h. 10 μL of CCK-8 reagent was added to each well 3 h before the end of incubation, and the 96-well plate was gently shaken horizontally to mix the liquid. After incubation for another 3 h, the absorbance of each well at 450 nm was read using an enzyme reader, and the absorbance at 450 nm of the control well with only culture medium added was subtracted from the absorbance at 450 nm of the control well with culture medium added to calculate the cell viability value of the well to be tested, such as Figure 7 As shown, the IC value of deuterated corticosteroids causing PVNS FLS death was calculated. 50 The value was 24.05 μmol / L, which was better than the positive drug pecidinib for treating PVNS.

[0084] 3. Deuterated lecitinib and TNF-α work together

[0085] PVNS FLS were placed at 5 × 10 3 Cells were inoculated into 96-well plates at 100 μL / well and incubated for 24 h with DMEM (high glucose) medium containing 10% FBS to allow the cells to adhere to the wall. The original culture medium was discarded, and serum-free DMEM medium containing different concentrations of deuterated corticosteroids (0, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μmol / L) was added for pre-incubation for 1 h, and then TNF-α (10 ng / mL) was stimulated for 72 h. 10 μL of CCK-8 reagent was added to each well 3 h before the end of incubation, and the 96-well plate was gently shaken horizontally to mix the liquid. After continuing to incubate for 3 h, the absorbance of each well at 450 nm was read using an enzyme reader, and the absorbance at 450 nm of the control well with only culture medium added was subtracted from the absorbance at 450 nm of the control well to calculate the cell viability value of the well to be tested, such as Figure 8 As shown, the IC of deuterated lexitinib for inhibiting the proliferation of PVNS FLS was calculated. 50 The value is 1.47μmol / L.

[0086] Example 4

[0087] This example studies the effect of deuterated corticosteroids on the expression of IL-6 and MCP-1 in PVNS FLS.

[0088] The following experiment was carried out using the PVNS FLS prepared in step 1 of Example 2:

[0089] PVNS FLS were placed at 5 × 10 5Cells were inoculated into 6-well plates and incubated for 24 h in DMEM (high glucose) medium containing 10% FBS to allow the cells to adhere to the wall. The original medium was discarded and different concentrations of deuterated colestitinib (abbreviated as Deuc in the figure, with concentrations of 0.3, 1, 3 and 10 μmol / L) were added ( Fig. 9 A and B) or different concentrations of pexidinib (abbreviated as Pexi in the figure, 1, 3 and 10 μmol / L) ( Fig. 9 C and D) were pre-incubated with serum-free DMEM medium for 1 h, and then stimulated with TNF-α (10 ng / mL) for 24 h. After the cell treatment, total RNA was extracted and reverse transcribed into cDNA, and detected by qPCR and 2 -ΔΔCT Relative quantitative analysis was performed to obtain IL-6 ( Fig. 9 A and C) and MCP-1( Fig. 9 B and D) mRNA expression levels.

[0090] Fig. 9 The results showed that deuterated lecitinib had a significant inhibitory effect on the expression of IL-6 and MCP-1 in PVNS FLS at a concentration of 0.3 μmol / L, while pecidinib had a significant inhibitory effect on the expression of IL-6 in PVNS FLS at a concentration of 10 μmol / L, but had no significant inhibitory effect on MCP-1. The above results suggest that deuterated lecitinib is significantly more effective than pecidinib, a positive drug for the treatment of PVNS, in inhibiting the expression of IL-6 and MCP-1 in PVNS FLS.

[0091] Example 5

[0092] This example studies the effect of deuterated lecitinib on the expression of pro-inflammatory cytokines by THP-1-derived macrophages.

[0093] THP-1 cells were cultured at 6 × 10 5 The cells were inoculated into 12-well plates and incubated for 48 h in RPMI-1640 medium containing PMA (20 ng / mL) and 10% FBS to differentiate them into macrophages. The original medium was discarded and different concentrations of deuterated colestitinib (abbreviated as Deuc in the figure, with concentrations of 0.3, 1, 3 and 10 μmol / L) were added ( Fig.10 AC) or different concentrations of pexidinib (abbreviated as Pexi in the figure, 1, 3 and 10 μmol / L) ( Fig.10 DF) were pre-incubated with serum-free RPMI-1640 medium for 1 h, and then stimulated with LPS (100 ng / mL) for 4 h.

[0094] After the cell treatment, the cell RNA was collected and reverse transcribed into cDNA, and the mRNA expression levels of IL-1β, IL-6 and TNF-α were detected and calculated by qPCR. Fig.10 The results showed that both deuterated lexitinib and pesidatinib had a significant inhibitory effect on the expression of IL-1β by THP-1-derived macrophages at a concentration of 1 μmol / L, deuterated lexitinib had a significant inhibitory effect on the expression of IL-6 and TNF-α by THP-1-derived macrophages at a concentration of 0.3 μmol / L, and pesidatinib had a significant inhibitory effect on the expression of IL-6 only at a concentration of 10 μmol / L. The above results suggest that deuterated lexitinib is better than the positive drug pesidatinib in inhibiting the expression of proinflammatory cytokines by THP-1-derived macrophages.

[0095] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. Use of deuterated colestitinib or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating pigmented villonodular synovitis, wherein the deuterated colestitinib or a pharmaceutically acceptable salt thereof is used as an active ingredient.

2. The use according to claim 1, characterized in that The drug comprises an effective dose of deuterated corticosteroid or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, characterized in that The mechanism of action of deuterated lecitinib or a pharmaceutically acceptable salt thereof as an active ingredient includes: inhibiting TYK2 activation.

4. The use according to claim 1, characterized in that The mechanism of action of deuterated lexitinib or a pharmaceutically acceptable salt thereof as an active ingredient includes: inhibiting the proliferation of fibroblast-like synoviocytes in the synovium of pigmented villonodular synovitis; and / or, Fibroblast-like synoviocyte death in the synovium causes pigmented villonodular synovitis.

5. The use according to claim 1, characterized in that The mechanism of action of deuterated lecitinib or a pharmaceutically acceptable salt thereof as an active ingredient includes: inhibiting the expression of inflammation-related factors.

6. The use according to claim 5, characterized in that The mechanism of action of deuterated lecitinib or a pharmaceutically acceptable salt thereof as an active ingredient includes: the inflammation-related factors include at least one of IL-6, MCP-1, IL-1β, IL-6 and TNF-α.

7. The use according to claim 1, characterized in that The drugs also include Pesidatinib.

8. The use according to any one of claims 1 to 7, characterized in that The drug further comprises pharmaceutically acceptable excipients.

9. The use according to claim 8, characterized in that The pharmaceutically acceptable excipients include at least one of a filler, a disintegrant, a binder, a surfactant, a suspending agent, a solubilizing agent, a flavoring agent, a preservative, an antioxidant and a stabilizer.

10. The use according to claim 8, characterized in that The dosage form of the drug includes at least one of powder, tablet, granule, capsule, solution, emulsion, suspension, injection, aerosol, ointment and drops.

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