Use of chiral hydroxychloroquine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis or treatment of an immune disease

Abstract

The present application relates to the technical field of medicine, and particularly discloses application of chiral hydroxychloroquine or a pharmaceutically acceptable salt thereof in preparation of a drug for preventing and treating immune diseases. The chiral hydroxychloroquine or the pharmaceutically acceptable salt thereof has a better inhibition rate and a lower cell survival rate on pathological cell proliferation caused by immune diseases, including but not limited to fibroblast-like synoviocytes; the chiral hydroxychloroquine or the salt thereof combined with methotrexate / ilaris (including but not limited to) can reduce the toxic side effects on normal cells and the cell survival rate, and has a better treatment effect on immune diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of chiral hydroxychloroquine or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of immune diseases. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, systemic disease with an unclear etiology, primarily characterized by inflammatory synovitis. It can occur at any age, regardless of sex, but is most common between 40 and 60 years of age. Women are more frequently affected than men, with a 2-3 times higher incidence rate in women. Clinically, most researchers currently consider it an autoimmune disease, or a chronic syndrome, characterized by nonspecific inflammation affecting joints throughout the body. The basic pathological changes in RA include synovial cell proliferation, infiltration of various inflammatory cells, pannus formation, and destruction of cartilage and bone tissue.

[0003] According to IMS Health statistics, global spending on drugs for treating rheumatoid arthritis reached $21.4 billion in 2015. A 2016 research report by the US research firm Global Data indicated that the global market for rheumatoid arthritis drug treatments will grow at a compound annual growth rate of 2.1% from 2013 to 2023, with global spending on rheumatoid arthritis drugs expected to reach $50 billion in recent years. The development of anti-rheumatoid arthritis drugs has become a new target for major pharmaceutical companies. Currently, the pathogenesis of rheumatoid arthritis is not fully understood, and no completely curative drug has been developed. Therefore, the research and development of rheumatoid arthritis drugs has always been a hot topic in the pharmaceutical field.

[0004] Hydroxychloroquine, an antimalarial drug, has been widely used clinically to treat connective tissue diseases such as rheumatoid arthritis with some success. Although it has been used to treat rheumatic diseases for over 100 years, its exact antirheumatic mechanism remains unclear. It may work by inhibiting the processing and presentation of autoantigens, suppressing cytokine production, and inhibiting lysosomal release, thereby inhibiting synovial hyperplasia, reducing synovial expression, and alleviating joint inflammation. Furthermore, the adverse reactions of hydroxychloroquine are dose-related; the higher the dose, the greater the side effects, primarily manifesting as ocular lesions, digestive system reactions, and nervous system reactions. Therefore, there is still room for improvement. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the use of chiral hydroxychloroquine or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of immune diseases.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides the use of chiral hydroxychloroquine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and treatment of immune diseases. Preferably, immune diseases include rheumatoid arthritis.

[0008] The inventors of this application, through extensive research and experimentation, discovered that chiral hydroxychloroquine or its pharmaceutically acceptable salts are effective in treating rheumatoid arthritis. In in vitro experiments, chiral hydroxychloroquine or its pharmaceutically acceptable salts can inhibit the proliferation of fibroblast-like synovial cells in rheumatoid arthritis, resulting in low cell survival rates.

[0009] Preferably, the chiral hydroxychloroquine includes S-hydroxychloroquine or R-hydroxychloroquine.

[0010] As a preferred embodiment of the application described in this invention, the pharmaceutically acceptable salts of the chiral hydroxychloroquine include S-hydroxychloroquine sulfate or R-hydroxychloroquine sulfate and their analogs, tautomers, polymorphs, isomers and solvates.

[0011] The aforementioned S-hydroxychloroquine or R-hydroxychloroquine and their pharmaceutically acceptable salts showed good efficacy in treating rheumatoid arthritis. In in vitro experiments, the highest inhibition rate and lowest cell survival rate of fibroblast-like synovial cells (FLS-RA) were observed when the concentration of S-hydroxychloroquine, R-hydroxychloroquine, and racemic hydroxychloroquine was 50 μM.

[0012] As a preferred embodiment of the application described in this invention, the method for preparing chiral hydroxychloroquine includes the following steps: converting racemic hydroxychloroquine sulfate into free hydroxychloroquine under alkaline conditions, and separating the free hydroxychloroquine by chiral high-performance liquid chromatography to obtain optically pure R or S free hydroxychloroquine.

[0013] The above method can yield relatively pure chiral hydroxychloroquine with better quality.

[0014] As a preferred embodiment of the application described in this invention, the dosage form of the drug includes injections, tablets, sustained-release tablets, capsules, sustained-release agents, microcapsules, powders, liposomes, granules, ointments, creams, emulsions, suspensions, lyophilized agents, sprays, suppositories, aerosols, pills, or oral liquids, and the dosage form is not limited to the above.

[0015] The immune diseases include diseases involving the proliferation of fibroblast-like synovial cells in the joints, preferably at least one of rheumatoid arthritis or juvenile idiopathic arthritis, systemic lupus erythematosus, osteoarthritis, gout, SAPHO syndrome, mixed connective tissue disease, spondyloarthritis, and infectious diseases.

[0016] The present invention also provides a pharmaceutical composition comprising a combination of chiral hydroxychloroquine or a pharmaceutically acceptable salt or ester thereof, a racemic mixture, an enantiomer, a tautomer, a polymorph, a pseudopolymorph, an amorphous form, a hydrate or solvate, and DMARDs. DMARDs include, but are not limited to, methotrexate / alamod.

[0017] The pharmaceutical composition described herein inhibits the proliferation of fibroblast-like synovial cells in joints with immune diseases and their related pathological changes, immune cell and inflammatory molecular responses, autoantibody expression, and expression of related disease susceptibility genes.

[0018] Comparisons between combination therapy and single-drug therapy revealed that the inhibition rate of HFLS was lower when traditional hydroxychloroquine and methotrexate were used in combination compared to when traditional hydroxychloroquine or methotrexate was used alone, indicating that combination therapy reduces the toxic side effects of traditional hydroxychloroquine on normal cells. Cell survival rate was higher when S-hydroxychloroquine and elamod were used in combination compared to S-hydroxychloroquine alone, and the combination of S-hydroxychloroquine with methotrexate or elamod reduced drug toxicity. The toxic side effects were lower when R-hydroxychloroquine and elamod were used in combination compared to R-hydroxychloroquine alone or R-hydroxychloroquine combined with methotrexate. Racemic hydroxychloroquine showed the lowest cell inhibition rate when used alone, indicating that the toxic side effects of racemic hydroxychloroquine alone were lower than those when combined with methotrexate or elamod.

[0019] Therefore, racemic hydroxychloroquine monotherapy has the lowest cytotoxic side effects, while when R-hydroxychloroquine is selected, the combination with elamod has the lowest toxic side effects, and when S-hydroxychloroquine is selected, the combination with elamod has the lowest toxic side effects.

[0020] As a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutically acceptable salt of the chiral hydroxychloroquine includes S-hydroxychloroquine sulfate or R-hydroxychloroquine sulfate and their analogues or esters, racemic derivatives, enantiomers, tautomers, polymorphs, pseudopolymorphs, amorphous forms, hydrates or solvates.

[0021] As a preferred embodiment of the pharmaceutical composition of the present invention, the chiral hydroxychloroquine or its pharmaceutically acceptable salt or ester, racemic, enantiomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate and DMARDs are in a mass ratio of 1:1.

[0022] As a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable drug carrier.

[0023] The drug carrier includes at least one of the following: diluent, excipient, filler, binder, humectant, lubricant, disintegrant, absorption enhancer, surfactant, adsorbent, and flavoring agent and sweetener.

[0024] The excipient comprises water; the filler comprises at least one of starch, sucrose, or lactose; the binder comprises at least one of cellulose derivatives, alginate, gelatin, or polyvinylpyrrolidone; the humectant comprises glycerin; the disintegrant comprises at least one of agar, calcium carbonate, or sodium bicarbonate; the absorption promoter comprises a quaternary ammonium compound; the surfactant comprises hexadecyl alcohol; the adsorbent carrier comprises at least one of kaolin or soap clay; and the lubricant comprises at least one of talc, calcium stearate, magnesium stearate, or polyethylene glycol.

[0025] This invention also provides the use of chiral hydroxychloroquine or a pharmaceutically acceptable salt thereof in combination with methotrexate / ellamod in the preparation of a medicament for the treatment of rheumatoid arthritis.

[0026] Preferably, the chiral hydroxychloroquine includes S-hydroxychloroquine or R-hydroxychloroquine.

[0027] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of an inhibitor of fibroblast-like synovial cell proliferation.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention provides the use of chiral hydroxychloroquine or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating rheumatoid arthritis. Chiral hydroxychloroquine or a pharmaceutically acceptable salt thereof has a better inhibition rate against FLS-RA and low cell survival rate. Combining chiral hydroxychloroquine or a salt thereof with methotrexate / aramod can reduce cytotoxicity to HFLS and increase cell survival rate. Attached Figure Description

[0030] Figure 1 Flowchart for the preparation of chiral hydroxychloroquine;

[0031] Figure 2 The graph shows the inhibition results of SH, HCQ and MTX on FLS-RA cells, respectively.

[0032] Figure 3 The graph shows the inhibition results of RH, HCQ, and MTX on FLS-RA cells, respectively.

[0033] Figure 4 The graph shows the inhibition results of RSA, HCQ, and MTX on FLS-RA cells, respectively.

[0034] Figure 5The graph shows the inhibition results of SH, RH and RSA treatment on FLS-RA cells, respectively.

[0035] Figure 6 This is a diagram showing the growth status of human fibroblast-like synovial cells in step 4 of Example 3. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0037] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0038] Example 1: Preparation of chiral hydroxychloroquine

[0039] This embodiment prepares chiral hydroxychloroquine using chiral high-performance liquid chromatography, specifically including the following steps:

[0040] Racemic hydroxychloroquine sulfate (S-HCQ) purchased from the market was converted to free racemic hydroxychloroquine under alkaline conditions. At 0°C, 10.9 g of hydroxychloroquine sulfate was dissolved in 75 mL of water, followed by the addition of 25 mL of 12% NaOH aqueous solution. After stirring for half an hour, 25 mL of ethyl acetate was added, and stirring continued for another half hour. The reaction mixture was allowed to cool to room temperature naturally, and then extracted three times with 100 mL of ethyl acetate. The combined organic phases were washed successively with 150 mL of saturated brine and water, dried over anhydrous sodium sulfate, and filtered to remove sodium sulfate. The organic solvent was removed using a rotary evaporator, yielding 7.7 g of free hydroxychloroquine as a pale yellow viscous liquid, with a yield of 91%.

[0041] 1H NMR(400MHz,Chloroform-d)δ8.49(d,J=5.4Hz,1H),7.93(d,J=2.2Hz,1H),7. 75(d,J=9.0Hz,1H),7.35–7.26(m,1H),6.39(d,J=5.5Hz,1H),5.19(d,J=7.7H z,1H),3.70(hept,J=6.1Hz,1H),3.57(t,J=5.7Hz,2H),3.37(s,1H),2.64–2. 42(m,6H),1.81–1.48(m,4H),1.31(d,J=6.3Hz,3H),1.01(t,J=7.1Hz,3H).13C NMR(101MHz,Chloroform-d)δ151.80,149.15,148.99,134.66,128.48,124.94,121 .17,117.16,99.00,58.36,54.70,52.88,48.20,47.34,34.16,23.91,20.21,11.60.

[0042] Chiral high-performance liquid chromatography can be used to separate free hydroxychloroquine into optically pure R or S free hydroxychloroquine.

[0043] 9.64 g of hydroxychloroquine was dissolved in an isochoric hexane / isopropanol / diethylamine in a ratio of 85:15:0.1 (v / v / v). The resulting solution was loaded into a CHIRALPAKAY-H (AYH0CE-VC001) chiral column and eluted with the same solvent system. The preparation conditions were: flow rate 1.0 mL / min, detection wavelength UV 254 nm, and temperature 35 °C. The first eluting compound at 10.17 min was S-hydroxychloroquine, and the second eluting compound at 11.85 min was R-hydroxychloroquine. The fractions of each enantiomer were collected and combined. The solvent was removed under reduced pressure using a rotary evaporator to obtain pure optical isomers: 2.89 g of S-hydroxychloroquine (ee > 95%), 2.53 g of R-hydroxychloroquine (ee > 95%), and 2.88 g of racemic hydroxychloroquine remaining. Specific steps are as follows... Figure 1 As shown.

[0044] Example 2: CCK-8 assay for drug activity / toxicity in HFLS (human fibroblastic synovial cells).

[0045] 1. CCK-8 working principle:

[0046] Cell Counting Kit-8 (CCK-8) (Patent No.: WO97 / 38985) utilizes a water-soluble tetrazolium salt developed by Dojindo— CCK-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt) can be reduced to a water-soluble formazan dye in the presence of the electron carrier 1-Methoxy PMS. CCK-8 solution can be directly added to cell samples; pre-preparation of various components is unnecessary. The CCK-8 method is a highly sensitive, non-radioactive colorimetric assay for determining the number of viable cells in cell proliferation or toxicity experiments. The orange-yellow formazan dye produced by the oxidation-reduction of intracellular dehydrogenases can dissolve in tissue culture medium, and the amount of formazan produced is directly proportional to the number of living cells.

[0047] 2. Cell viability assay of human fibroblast-like synovial cells:

[0048] (1) Seed the cell suspension (100 μL / well) into 96-well plates. Pre-incubate the plates in an incubator (at 37°C and 5% CO2).

[0049] (2) Add 10 μL of CCK-8 solution to each well (be careful not to generate bubbles in the well, as they will affect the OD value reading).

[0050] (3) Incubate the culture plate in an incubator for 1-2 hours.

[0051] (4) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0052] 3. Preparation of main reagents:

[0053] (1) Fibroblast-like synovial cell culture medium: 90% high glucose DMEM + 10% FBS + 1% antibiotics. In a clean bench, add 50 mL of fetal bovine serum to 450 mL of high glucose DMEM, and then add 5 mL of antibiotic solution.

[0054] 4. Culture of fibroblast-like synovial cells (FLS-RA) for rheumatoid arthritis:

[0055] 1) Cell passage:

[0056] (1) Discard the culture medium in the original culture dish;

[0057] (2) Wash the culture dish three times with 2 mL PBS and then discard it;

[0058] (3) Take 2 mL of pancreatic enzyme cell digestion solution into a culture dish, soak the bottom surface, and put it into a cell culture incubator for 3 min;

[0059] (4) Take 3 new petri dishes and add 6 mL of fresh culture medium to each dish for later use;

[0060] (5) Remove the digested culture dish, add 3 mL of fresh culture medium to stop the digestion, blow the cells evenly, and aspirate them into a 10 mL centrifuge tube.

[0061] (6) Centrifuge the cells in the centrifuge tube at 800 r / min for 5 min;

[0062] (7) Discard the culture medium in the centrifuge tube, being careful not to aspirate the cells, and resuspend the cells in 4 mL of fresh culture medium.

[0063] (8) Take 1 mL of the suspension from each cell and dispense it into the culture dish in step (4), and place it in a cell culture incubator for culture.

[0064] 2) Cell cryopreservation:

[0065] (1) Discard the culture medium in the original culture dish;

[0066] (2) Wash the culture dish three times with 2 mL PBS and then discard it;

[0067] (3) Take 2 mL of pancreatic enzyme digestion solution into a culture dish, soak the bottom surface, and put it into a cell culture incubator for 3 min;

[0068] (4) Remove the digested culture dish, add 3 mL of fresh culture medium to stop the digestion, blow the cells evenly, and aspirate them into a 10 mL centrifuge tube.

[0069] (5) Centrifuge the cells in the centrifuge tube at 800 r / min for 5 min;

[0070] (6) Discard the culture medium in the centrifuge tube, being careful not to aspirate the cells, and add 4 mL of cryopreservation solution to resuspend the cells;

[0071] (7) Take 2 mL of the resuspended cells into two cryovials;

[0072] (8) Place the cryovial in a -20°C freezer for 30 minutes, then transfer it to the surface of liquid nitrogen and suspend it overnight. The next day, place the cryovial in liquid nitrogen.

[0073] 3) Cell resuscitation:

[0074] (1) Prepare a 37℃ constant temperature water bath;

[0075] (2) Take out the cryovial from the liquid nitrogen tank and quickly place it in a water bath, shaking it constantly to melt it quickly;

[0076] (3) After the frozen liquid thaws, take it out and spray the outer wall with an alcohol spray bottle;

[0077] (4) Aspirate the cell suspension and add it to a culture dish, then add 6 mL of fresh culture medium and place it in a cell culture incubator.

[0078] 4) Cell drug delivery experiment:

[0079] (1) FLS-RA was cultured to the 3rd generation for experiments;

[0080] (2) Remove the cells from the incubator and discard the culture medium;

[0081] (3) Wash three times with 2 mL PBS and then discard the PBS;

[0082] (4) Take 1 mL of pancreatic enzyme digestion solution into a culture flask, soak the bottom surface, and put it into a cell culture incubator for 3 min;

[0083] (5) Remove the digested cells and terminate the digestion with 6 mL of fresh culture medium;

[0084] (6) Transfer the cells to a 10 mL centrifuge tube and centrifuge at 1000 r / min for 3 min;

[0085] (7) Carefully discard the supernatant and resuspend the cells in 4 mL of fresh culture medium;

[0086] (8) Take 1 mL of the resuspended cells and transfer it to a 50 mL centrifuge tube;

[0087] (9) Dilute the cells to 20 mL with fresh culture medium, making a total of 4 × 10⁻⁶ cells. 4 cells / mL;

[0088] (10) Select 57 culture wells in a 96-well cell culture plate, seed 100 μl of cells in each well (about 10^4 cells), and then add 200 μl of PBS to the wells surrounding the cells.

[0089] (11) After culturing for 24 hours, observe whether the cells adhere to the wall. After adhesion, discard the original culture medium and add 100 μl of culture medium containing SH, HCQ and MTX to each well. After culturing for 24 hours, test and compare the OD value, inhibition rate and IC50 of the three drugs for FLS-RA.

[0090] Alternatively, add 100 μl of culture medium containing RH, HCQ, and MTX to each well, incubate for 24 h, and then measure and compare the OD value, inhibition rate, and IC50 of the three drugs for FLS-RA.

[0091] Alternatively, add 100 μl of culture medium containing HCQ, RSA, and MTX to each well, incubate for 24 h, and then measure and compare the OD value, inhibition rate, and IC50 of the three drugs for FLS-RA.

[0092] Alternatively, add 100 μl of culture medium containing SH, RH, and RSA to each well, incubate for 24 h, and then measure and compare the OD value, inhibition rate, and IC50 of the three drugs for FLS-RA.

[0093] Note: HCQ represents conventional hydroxychloroquine, MTX represents methotrexate, SH represents S-hydroxychloroquine, RH represents R-hydroxychloroquine, and RSA represents racemic hydroxychloroquine.

[0094] result:

[0095] 1. Cell Morphology Observation: Under an inverted microscope, cultured FLS-RA cells appear as long spindle-shaped, fibrous cells with elongated bipolar processes and clearly defined nucleoli located in the center. The long spindle-shaped fibrous cells interweave with adjacent cells to form a network, typical of fibroblast-like synovial cells. During culture, FLS cells showed active proliferation and stable growth between the 3rd and 7th passages, making them suitable as basal cells for experiments. From the 7th passage onwards, the cells exhibited a senescence trend, slower proliferation, increased susceptibility to infection, and difficulty in culture.

[0096] 2. Results of CCK8 assay for drug activity / toxicity in HFLS cells.

[0097] IC50 (half maximal inhibitory concentration) refers to the half-inhibitory concentration of a measured antagonist. It indicates how much of a drug or substance (inhibitor) inhibits a certain biological process (or substances contained in this process, such as enzymes, cell receptors, or microorganisms). In the context of apoptosis, it can be understood as the concentration at which a certain concentration of a drug induces 50% apoptosis. This concentration is called the 50% inhibitory concentration, which corresponds to the concentration at which the ratio of apoptotic cells to the total number of cells is equal to 50%. The IC50 value can be used to measure the ability of a drug to induce apoptosis; the stronger the induction ability, the lower the value. Conversely, it can also indicate the degree of tolerance of a certain cell type to a drug.

[0098] The comparison of OD values ​​and inhibition rates of the above drug groups after treatment with FLS-RA is as follows: Figure 2-5 As shown.

[0099] The results of the half-inhibitory concentrations (IC50) of S-hydroxychloroquine (SH), R-hydroxychloroquine (RH), conventional hydroxychloroquine (HCQ), and those containing racemic hydroxychloroquine and methotrexate (MTX) on FLS-RA are shown in Table 1.

[0100] Table 1

[0101]

[0102]

[0103] When the concentrations of S-hydroxychloroquine, R-hydroxychloroquine, and racemic-hydroxychloroquine were 50 μM, the inhibition rate of fibroblast-like synovial cells (FLS-RA) in rheumatoid arthritis was the highest, and the cell survival rate was the lowest.

[0104] Example 3: Cell viability / toxicity of chiral hydroxychloroquine monotherapy and its combination with methotrexate / alamod against HFLS effect

[0105] 1. Resuscitation of human fibroblast-like synovial cells, P4 generation:

[0106] Experiment date: April 22, 2022;

[0107] Experimental location: Cell culture room of Guangzhou Huishan Medical Technology Co., Ltd.;

[0108] Start time: 12:30; End time: 13:00;

[0109] Experimental equipment: clean bench, benchtop low-speed centrifuge, CO2 incubator, microscope, pipette (capacity 1000μl);

[0110] Experimental consumables: 15ml centrifuge tubes, EP tubes, T25 culture flasks;

[0111] Experimental reagents: DMEM culture medium;

[0112] 2. Experimental steps:

[0113] (1) Turn on the UV lamp for 30 minutes, turn on the water bath in advance, set the temperature to 37℃, and prepare the necessary consumables for the experiment.

[0114] (2) Take out one cryopreservation tube of human fibroblast-like synovial cells from the P4 generation in the liquid nitrogen tank and quickly place it in a 37°C water bath for 1 minute to rewarm.

[0115] (3) Use a pipette to transfer the rewarmed cell suspension to a 15ml centrifuge tube, add 1ml of culture medium, and mix gently.

[0116] (4) Centrifuge at 1000r for 3 minutes, and carefully use a pipette to remove the supernatant;

[0117] (5) Add 1000 μl of culture medium to resuspend the cells (handle gently, pipette 15-20 times);

[0118] (6) Add 4 ml of culture medium to each of the two culture flasks, then add 500 μl of cell suspension, and gently shake to evenly spread the cell suspension throughout the culture flasks.

[0119] (7) Observe the cell concentration under a microscope. Culture flask numbers: FLSP4-1~FLSP4-2;

[0120] (8) Place the culture flasks containing the cells into a CO2 incubator and incubate for 2-3 days. Observe the cell growth every day and change the medium if necessary.

[0121] 3. Digestion and collection of human fibroblast-like synovial cells:

[0122] 1) Experiment date: April 24, 2022;

[0123] 2) Experimental site: Cell culture room of Guangzhou Huishan Medical Technology Co., Ltd.;

[0124] 3) Start time: 12:30; End time: 13:00;

[0125] 4) Experimental equipment: ultra-clean workbench, benchtop low-speed centrifuge, water bath, CO2 incubator, microscope, pipette (capacity 1000μl);

[0126] 5) Experimental consumables: 15ml centrifuge tubes, EP tubes, T25 culture flasks;

[0127] 6) Experimental reagents: DMEM culture medium

[0128] 4. Experimental steps:

[0129] (1) Remove the two culture flasks FLSP4-1 to FLSP4-2 from the incubator and observe the cell growth status under a microscope (e.g., ...). Figure 6 Cells are in good growth condition, free from cell contamination, with a fusion rate >90% and a small number of floating cells. Take two photos of each bottle using a microscope at 4x magnification and preserve them.

[0130] (2) After observation, the culture bottles were sprayed with alcohol for disinfection and then placed in a clean bench.

[0131] (3) Discard the culture medium in the culture flask, add 3ml PBS to each flask to rinse the cells, and then remove the PBS.

[0132] (4) Add 1 ml of 0.25% trypsin-0.53 mM EDTA, gently shake the culture flask to submerge the cell surface with trypsin, and place the culture flask in a 37°C incubator for 1 minute for digestion.

[0133] (5) Add 6 ml of complete culture medium to stop digestion, and gently pipette the cells to mix them.

[0134] (6) Centrifuge the mixed cells at 1000 rpm (about 150 g) for 3 min and discard the supernatant.

[0135] (7) Resuspend the centrifuged cells in 4 ml of culture medium and mix well. Take 10 μl of the cell suspension into a centrifuge tube and count the cells using a cell counting chamber. The final count result is: 2.5 x 10⁻⁶. 6Each cell.

[0136] (8) Based on the principle of planting 40,000 cells / 100 μl per well in a 96-well plate, add 2.25 ml of culture medium to the cell suspension, bringing the total cell suspension volume to 6.25 ml and the cell concentration to 4 x 10⁻⁶ cells / well. 5 Cells / ml

[0137] (9) Add 100 μl of the mixed cell suspension to the outer edge of the 96-well plate, leave the second column as a blank control, and add 100 μl of the mixed cell suspension to the remaining culture wells.

[0138] (10) Place in an incubator and incubate for 24 hours.

[0139] 5. Combined drug stimulation:

[0140] Experiment date: April 25, 2022;

[0141] Experimental location: Cell culture room of Guangzhou Huishan Medical Technology Co., Ltd.;

[0142] Start time: 12:30; End time: 12:40;

[0143] Experimental equipment: clean bench, CO2 incubator, pipettes (capacity 1000μl, 100μl);

[0144] Experimental consumables: 15ml centrifuge tubes, EP tubes;

[0145] Experimental reagents: DMEM medium (Cytiva), conventional hydroxychloroquine (HCQ), S-hydroxychloroquine (SH), R-hydroxychloroquine (RH), racemic hydroxychloroquine (RSA), methotrexate solution (MTX), and alamod solution.

[0146] Experimental steps:

[0147] (1) Prepare the required drug solutions in advance, and prepare the five drugs according to the required amounts. After the cells have been cultured in the 96-well plate for 24 hours, remove them from the incubator and discard the supernatant from the culture wells containing the cells.

[0148] (2) For single-use drugs, add 100 μl of 50 μM drug solution to each well. For combined-use drugs, add 50 μl of 100 μM drug solution to each well and mix well before adding to the well plate.

[0149] (3) Place the drug-treated Cell 96 culture plate into a CO2 incubator and incubate for 24 hours.

[0150] CCK8 cell proliferation toxicity assay:

[0151] Experiment date: April 26, 2022;

[0152] Experimental location: Cell culture room of Guangzhou Huishan Medical Technology Co., Ltd.;

[0153] Start time: 12:30; End time: 17:10;

[0154] Experimental equipment: clean bench, CO2 incubator, microplate reader, pipettes (ranges 1000μl and 100μl);

[0155] Experimental consumables: 15ml centrifuge tubes, EP tubes;

[0156] Experimental reagents: DMEM medium (Cytiva), CCK8 reagent (Tongren Chemical);

[0157] Experimental steps:

[0158] (1) Prepare a 1:10 solution of CCK8 reagent with culture medium: Take 600 μl of CCK8 solution into a 15 ml centrifuge tube, add 5400 μl of culture medium, and prepare a 6 ml CCK8 solution.

[0159] (2) Remove the 96-well plate of cells stimulated with the drug for 24 hours from the incubator, place it in a clean bench, aspirate the supernatant, and add diluted CCK8 reagent to all control wells and experimental wells. Avoid generating air bubbles when adding the sample, and the operation should be completed quickly.

[0160] (3) Place the culture plate in a CO2 incubator and observe the color change of the cell culture wells after half an hour and every hour.

[0161] (4) After 4 hours, the enzyme-linked immunosorbent assay (ELISA) was used for testing.

[0162] The results are shown in Table 2.

[0163] Table 2 Inhibition rate of each drug group on FLS-RA after treatment

[0164] drug Mean ± Standard Deviation of Cell Inhibition Rate (%) Survival rate mean ± standard deviation (%) HCQ 0.173±0.076 0.827±0.076 MTX 0.078±0.073 0.921±0.006 HCQ+MTX 0.074±0.006 0.925±0.006 SH 0.142±0.146 0.858±0.146 SH+MTX 0.108±0.088 0.892±0.882 SH+Ailamod 0.066±0.057 0.934±0.057 RH 0.066±0.051 0.934±0.051 RH+MTX 0.116±0.051 0.884±0.051 RH+Ailamod 0.028±0.035 0.972±0.035 RSA 0.009±0.043 0.991±0.043 RSA+MTX 0.286±0.104 0.713±0.104 RSA+ Elamold 0.240±0.068 0.760±0.068

[0165] Note: "+" indicates that two drugs are used in combination.

[0166] When used alone, the drug showed the highest cell inhibition rate and lowest cell survival rate at 50 μM. Using this concentration as the dosage, comparisons between combination therapy and single-drug therapy revealed that the combination of traditional HCQ and MTX showed a lower inhibition rate against human fibroblast-like synovial cells than either traditional HCQ or MTX alone, indicating that combination therapy reduces the toxic side effects of traditional HCQ on normal cells. The combination of SH and elastolide resulted in higher cell survival rates than SH alone, and the combination of SH with MTX or elastolide reduced drug toxicity. The combination of RH and elastolide showed lower toxicity than RH alone or RH combined with MTX. RSA showed the lowest cell inhibition rate when used alone, indicating that the toxicity of RSA alone was lower than that of RSA combined with MTX or elastolide.

[0167] In summary, among novel chiral hydroxychloroquines, RSA monotherapy has the lowest cytotoxic side effects, while when RSA is selected, the combination with elamod has the lowest side effects, and when SH is selected, the combination with elamod has the lowest side effects.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The use of a pharmaceutical composition in the preparation of a drug for inhibiting the proliferation of fibrous synovial cells in rheumatoid arthritis, characterized in that, The pharmaceutical composition comprises a combination of chiral hydroxychloroquine and DMARDs, wherein the DMARDs are ellamod. The mass ratio of chiral hydroxychloroquine to DMARDs is 1:1; The chiral hydroxychloroquine is S Hydroxychloroquine or R Hydroxychloroquine; S Hydroxychloroquine combined with ellamod has the effect of reducing S The toxic side effects of hydroxychloroquine; R Hydroxychloroquine combined with elamod has the effect of reducing R The toxic side effects of hydroxychloroquine.

2. The application as described in claim 1, characterized in that, The preparation method of the chiral hydroxychloroquine includes the following steps: converting racemic hydroxychloroquine sulfate into free hydroxychloroquine under alkaline conditions, and separating the free hydroxychloroquine by chiral chromatography to obtain optically pure R or S free hydroxychloroquine.

3. The application as described in claim 1, characterized in that, The dosage forms of the drug include injections, tablets, capsules, sustained-release preparations, powders, liposomes, granules, suspensions, pills, or oral liquids.

4. The application as described in claim 1, characterized in that, The pharmaceutical composition further includes a pharmaceutically acceptable drug carrier; the drug carrier includes at least one of a diluent, excipient, filler, binder, humectant, lubricant, disintegrant, absorption enhancer, surfactant, adsorbent, and flavoring agent and sweetener.

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