Use of naphthoquine phosphate salts in the manufacture of medicaments for the treatment of autoimmune diseases

Abstract

The present invention provides a use of naphthoquine phosphate in the manufacture of a medicament for the treatment of autoimmune diseases. In a mouse rheumatoid arthritis model, intragastric administration of naphthoquine phosphate has been shown to significantly improve symptoms such as joint redness, swelling, and deformity, and to reduce antigen-specific antibody levels. In a mouse systemic lupus erythematosus model, intragastric administration of naphthoquine has been shown to significantly reduce anti-double-stranded DNA antibody and antinuclear antibody levels. Pharmacological studies have demonstrated that naphthoquine phosphate has excellent immunosuppressive activity and can be used in the manufacture of a medicament for the treatment of autoimmune diseases. The present invention also provides a use of naphthoquine phosphate as a medicament for the treatment and adjunctive treatment of rheumatoid arthritis and systemic lupus erythematosus. The medicament is a pharmaceutical composition containing a therapeutically effective amount of naphthoquine phosphate as an active ingredient and pharmaceutical excipients.

Description

[Technical Field]

[0001] The present invention relates to a new indication for a medicine, in particular to the use of naphthoquine phosphate, an antimalarial drug, in the manufacture of a medicine for the treatment of a new indication, and more particularly to the use of naphthoquine phosphate in the manufacture of a medicine for the treatment of an autoimmune disease. [Background technology]

[0002] Rheumatoid arthritis (RA) is a chronic, progressive, systemic, and disabling autoimmune disease characterized by primary inflammatory synovitis, cartilage destruction, and bone erosion. Common clinical symptoms are joint stiffness, swelling, and pain, and as the disease progresses, destruction of articular cartilage and bone tissue occurs, ultimately leading to joint deformity and impaired mobility. The pathology of RA is complex, and clinical treatment remains focused on medication, aiming to suppress disease symptoms, mitigate disease progression, prevent bone and joint damage, reduce disability rates, improve prognosis, and enhance patients' quality of life (see references). [1] Ref. Conventional RA treatments mainly include nonsteroidal anti-inflammatory drugs, glucocorticoids, disease-modifying antirheumatic drugs, biologics, and small molecule inhibitors. However, in clinical treatment, many patients have low or no response to current therapeutic measures, so the search for novel therapeutic measures remains an urgent need for RA treatment.

[0003] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which pathogenic autoantibodies and immune complexes are formed, mediating organ and tissue damage. Clinically, symptoms are present across multiple systems, and various autoantibodies, such as antinuclear antibodies, are present in the serum. The complex pathogenesis of SLE makes pharmaceutical research and development difficult. Currently, the main therapeutic agents for SLE are nonsteroidal anti-inflammatory drugs, corticoids, antimalarials, immunomodulators, and biologics (see references). [2] reference).

[0004] Antimalarial drugs such as hydroxychloroquine and chloroquine are widely used clinically to treat rheumatoid arthritis, systemic lupus erythematosus, and other inflammatory rheumatoid arthritis. Hydroxychloroquine is currently the most commonly used antimalarial drug for the treatment of autoimmune diseases. It can reduce disease activity in patients with systemic lupus erythematosus, reduce the risk of organ damage and blood clots, improve blood lipid levels, improve survival rates, and improve metabolic levels in patients with RA, reducing the occurrence of cardiovascular events. [3] Naphthoquine phosphate belongs to the quinoline derivatives, along with quinine, chloroquine, and hydroxychloroquine, and is used to treat malignant malaria. Naphthoquine phosphate has the following structural formula:

[0005] [ka]

[0006] There have been no reports of naphthoquine phosphate being used to treat rheumatoid arthritis or systemic lupus erythematosus. Using a collagen-induced mouse arthritis model, the inventors found that naphthoquine phosphate significantly improved the clinical score of arthritis, reduced serum collagen-specific antibody levels, and inhibited lymphocyte proliferation. Using an adjuvant-induced rat arthritis model, they found that administration of naphthoquine phosphate significantly reduced the clinical score of arthritis and improved paw swelling in experimental rats. In an experimental mouse model of spontaneous systemic lupus erythematosus, intragastric administration of naphthoquine significantly reduced serum anti-dsDNA and antinuclear antibody levels. This finding is of great significance as it could be developed as a therapeutic or adjuvant-supported treatment for rheumatoid arthritis and systemic lupus erythematosus. Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to further research and develop new uses of naphthoquinone phosphates in medicine, particularly in the manufacture of medicines for the treatment or adjunctive treatment of rheumatoid arthritis and systemic lupus erythematosus. [Means for solving the problem]

[0008] The present invention provides the use of a naphthoquinone phosphate in the manufacture of a medicament for the treatment of an autoimmune disease. In the present invention, it has been demonstrated that intragastric administration of naphthoquine phosphate significantly improves symptoms such as joint redness, swelling, and deformity in a mouse rheumatoid arthritis model, and can reduce the level of antigen-specific antibodies. In a mouse systemic lupus erythematosus model, it has been demonstrated that intragastric administration of naphthoquine significantly reduces the levels of anti-double-stranded DNA antibodies and antinuclear antibodies. Pharmacological studies have demonstrated that naphthoquine phosphate has ideal immunosuppressive activity and can be used in the manufacture of medicines for the treatment of autoimmune diseases.

[0009] In this study, we used a collagen-induced mouse arthritis model and found that naphthoquine phosphate significantly improved the clinical score of arthritis in experimental mice, reduced serum collagen-specific antibody levels, and inhibited lymphocyte proliferation. Using an adjuvant-induced rat arthritis model, we found that administration of naphthoquine phosphate significantly reduced the clinical score of arthritis in experimental rats and improved paw swelling. In an experimental mouse model of spontaneous systemic lupus erythematosus, intragastric administration of naphthoquine significantly reduced serum anti-double-stranded DNA antibody and antinuclear antibody levels. This compound may be developed as a therapeutic or adjunctive treatment for rheumatoid arthritis and systemic lupus erythematosus.

[0010] In the present invention, an experiment in which naphthoquine phosphate was administered intragastrically to mice to significantly improve arthritis induced by bovine type II collagen demonstrated that naphthoquine phosphate can improve the clinical score of arthritis and reduce the level of serum pathogenic antibodies, and can be used to manufacture medicines for the treatment or adjuvant treatment of rheumatoid arthritis and systemic lupus erythematosus.

[0011] Thus, the present invention further provides the use of a naphthoquinone phosphate in the manufacture of a medicament for ameliorating the symptoms of arthritic disease and reducing the level of lupus pathogenic antibodies.

[0012] The present invention provides use of a naphthoquinone phosphate in the manufacture of a medicament for the treatment of an autoimmune disease, wherein the medicament is a pharmaceutical composition containing a therapeutically effective amount of naphthoquinone phosphate as an active ingredient and pharmaceutical additives.

[0013] The present invention provides the use of naphthoquinone phosphate in the manufacture of a medicament for the treatment or adjunctive treatment of rheumatoid arthritis and systemic lupus erythematosus.

[0014] The present invention provides the use of naphthoquinone phosphate in the manufacture of a medicament for the treatment or adjunctive treatment of rheumatoid arthritis and systemic lupus erythematosus.

[0015] The medicine of the present invention is a pharmaceutical composition containing a therapeutically effective amount of naphthoquinone phosphate as an active ingredient and pharmaceutical additives.

[0016] The pharmaceutical composition may be in the form of tablets, capsules, powders, granules, oral liquid preparations, or intravenous or intramuscular injections.

[0017] The present invention further provides a method for treating or adjunctively treating rheumatoid arthritis and systemic lupus erythematosus, comprising administering a therapeutically effective amount of naphthoquine phosphate to a subject in need thereof.

[0018] The present invention further provides a method for ameliorating the symptoms of arthritis disease and reducing the level of lupus pathogenic antibodies, comprising administering a therapeutically effective amount of naphthoquinone phosphate to a subject in need thereof.

[0019] As used herein, the term "therapeutically effective amount" refers to an amount having a therapeutic effect that can be used to prevent or treat a specific disease, illness, or condition described herein. For example, a "therapeutically effective amount" can be the amount necessary to provide a therapeutic or desired effect to the individual being treated. As known to those skilled in the art, a therapeutically effective amount will vary depending on the route of administration, the use of excipients, and the possibility of co-administration with other therapies. [Effects of the Invention]

[0020] The present invention provides the use of naphthoquinone phosphate in the manufacture of a medicament for the treatment of autoimmune diseases. Experiments using naphthoquinone phosphate in a mouse rheumatoid arthritis model have demonstrated that intragastric administration of naphthoquinone phosphate significantly improves symptoms such as joint redness, swelling, and deformity, and reduces the level of antigen-specific antibodies. In a mouse systemic lupus erythematosus model, intragastric administration of naphthoquinone significantly reduces the levels of anti-double-stranded DNA antibodies and antinuclear antibodies. Pharmacological studies have demonstrated that naphthoquinone phosphate has ideal immunosuppressive activity and can be used to manufacture a medicament for the treatment of autoimmune diseases.

[0021] In the present invention, using a collagen-induced mouse arthritis model, research has found that naphthoquinone phosphate can significantly improve the clinical score of arthritis in experimental mice, reduce the serum collagen-specific antibody level, and inhibit lymphocyte proliferation.Using an adjuvant-induced rat arthritis model, research has found that administration of naphthoquinone phosphate can significantly reduce the clinical score of arthritis in experimental rats and improve their paw swelling.In an experimental mouse model of spontaneous systemic lupus erythematosus, intragastric administration of naphthoquinone can significantly reduce the serum anti-dsDNA antibody and antinuclear antibody levels of model mice, further providing the use of naphthoquinone phosphate in medicine for the treatment and adjuvant treatment of rheumatoid arthritis and systemic lupus erythematosus, showing good clinical application prospects and great social benefits. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram showing the results of intragastric administration of naphthoquinone phosphate significantly improving the clinical score of mouse arthritis induced by bovine type II collagen. [Figure 2] FIG. 1 is a schematic diagram showing the results of suppressing swelling in both hind paws of arthritic mice by intragastric administration of naphthoquine phosphate. [Figure 3] FIG. 1 is a schematic diagram showing the results of inhibiting the proliferation of splenic lymph node lymphocytes in arthritic mice by intragastric administration of naphthoquine phosphate. [Figure 4] FIG. 1 is a schematic diagram showing the results of intragastric administration of naphthoquinone phosphate to reduce the level of collagen-specific antibodies in the serum of arthritic mice. [Figure 5] 1 shows representative photographs showing that intragastric administration of naphthoquine phosphate ameliorates paw swelling and inhibits bone erosion in arthritic mice. [Figure 6] FIG. 1 is a schematic diagram showing the results that intragastric administration of naphthoquine phosphate significantly inhibits the activation of spleen-purified B cells from arthritic mice mediated by Toll-like receptor signaling. [Figure 7]FIG. 1 is a schematic diagram showing the results of intragastric administration of naphthoquinone phosphate significantly improving the symptoms of adjuvant-induced arthritis in rats. [Figure 8] FIG. 1 is a schematic diagram showing the results of intragastric administration of naphthoquine phosphate significantly reducing serum pathogenic antibody levels in spontaneous systemic lupus erythematosus experimental mice. DETAILED DESCRIPTION OF THE INVENTION

[0023] Example 1. In vitro inhibition of mitogen-induced mouse intact splenic lymphocyte proliferative responses by naphthoquinone phosphate

[0024] 1. Main experimental materials and where to obtain them (1) Experimental animals: SPF-grade BALB / c mice, female, approximately 6 to 8 weeks old, purchased from Beijing Huafukang Biotechnology Co., Ltd., with the certificate number 110322211100631475.

[0025] (2) Main experimental drugs: Naphthoquinone phosphate, pale yellow powder, elemental analysis (C 24 H 34 ClN3O9P2), provided by Shanghai Pharmaceutical Industry Co., Ltd.; Hydroxychloroquine sulfate, white powder, elemental analysis (C 18 H 26 ClN3O·H2SO4), provided by Sanwei Pharmaceutical Co., Ltd. or purchased from Sigma; chloroquine, white powder, elemental analysis (C 18 H 26 ClN3) was purchased from Sigma.

[0026] (3) Reagents: RPMI 1640 liquid medium was purchased from GibcoBRL; fetal bovine serum was purchased from Hyclone; bacterial lipopolysaccharides (LPS) were purchased from Sigma; concanavalin A (ConA) was purchased from Sigma; hydroxychloroquine sulfate was provided by Sanwei Pharmaceutical Co. or purchased from Sigma; thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma; and dimethyl sulfoxide (DMSO) was purchased from Sinopharm. 3 H-thymidine ( 3 H-Thymidine: 3 H-TdR), and liquid scintillation cocktail were purchased from Perkin Elmer.

[0027] 2. Experimental Method BALB / c mice were euthanized by cervical dislocation, and their spleens were aseptically removed to prepare whole spleen lymphocytes. The compounds were diluted in a four-fold gradient starting from 250 μM to 10 concentrations: 250, 62.5, 15.625, 3.906, 0.977, 0.244, 0.061, 0.015, 0.004, and 0.001 μM.

[0028] (1) Detection of nonspecific cytotoxicity: Mouse spleen lymphocyte suspension: 8 x 10 5MTT cells were seeded into a 96-well plate at 1 / well and the 10 compounds were added at a gradient concentration. Separately, corresponding cell controls (i.e., compound-free spleen lymphocyte cultures) and RPMI 1640 liquid medium background controls (blank liquid medium controls) were set up, and the cells were cultured for 48 hours in an incubator at 37°C and 5% CO2. Four hours before the end of the culture, 20 μl of 5 mg / ml MTT solution was added, and the incubation continued until the end of the culture (i.e., 48 hours). The supernatant was then aspirated and discarded, and 200 μl of DMSO was added to each well to dissolve the crystals. Mouse spleen lymphocyte suspensions were obtained, and the absorbance at 570 nm was measured using a microplate reader (Spectra Max 190, Molecular Devices).

[0029] (2) Detection of lymphocyte proliferation: Mouse spleen lymphocyte suspension: 5 x 10 5 The cells were seeded in a 96-well plate at 1 / well, and ConA (final concentration 1 μg / ml) or LPS (final concentration 10 μg / ml) and the above 10 compounds were added at a concentration gradient. Separately, control wells without ConA or LPS and control wells with stimulation or no drug were set up, and the cells were cultured for 48 hours in an incubator at 37°C and 5% CO2. 8 hours before the end of the culture, 3 H-thymidine was added, and the incubation was continued until the end of the experiment (i.e., 48 hours). The cells were harvested onto a glass fiber membrane using a cell harvester, and 5 ml of liquid scintillation cocktail was added. The radioactivity count per minute was then read using a beta counter (2450 Microplate Counter, PerkinElmer) (model number).

[0030] 3. Experimental Results ConA and LPS, as mitogens, stimulate the proliferation and differentiation of T and B lymphocytes, respectively. This process resembles the in vivo lymphocyte activation process. Therefore, mitogen-induced lymphocyte proliferation is often used as an index to evaluate lymphocyte function. As shown in Table 1, naphthoquine phosphate exhibited significant inhibitory activity against both ConA-induced T cell activation and proliferation and LPS-induced B cell activation and proliferation, and its bioactivity selectivity index (SI) was superior to that of chloroquine and hydroxychloroquine.

[0031] Table 1. In vitro inhibition of mitogen-induced mouse splenic lymphocyte proliferation by naphthoquinone phosphates.

[0032] [Table 1]

[0033] Note: CC50 is the compound concentration at which cell viability is 50%; IC50 is the compound concentration at which cell proliferation is reduced by 50%; SI=CC50 / IC50.

[0034] As is clear from the above results, naphthoquine phosphate significantly inhibits the activation of splenic lymphocytes induced by mitogens, and in particular has excellent inhibitory activity against the activation and proliferation of B cells, and its immunosuppressive activity is higher than that of chloroquine and hydroxychloroquine sulfate.

[0035] Example 2. Inhibition of LPS-induced macrophage inflammatory cytokine production by naphthoquinone phosphate

[0036] 1. Main experimental materials and where to obtain them (1) Cells: Mouse macrophages RAW264.7 were purchased from ATCC, USA.

[0037] (2) Main experimental chemical: naphthoquinone phosphate, pale yellow powder, elemental analysis (C 24 H 34 ClN3O9P2), provided by Shanghai Pharmaceutical Industry Co., Ltd.; Hydroxychloroquine sulfate, white powder, elemental analysis (C 18 H 26 ClN3O·H2SO4), provided by Sanwei Pharmaceutical Co., Ltd.; chloroquine, white powder, elemental analysis (C 18 H 26 ClN3), purchased from Sigma.

[0038] (3) Reagents: DMEM high-carbohydrate medium was purchased from GibcoBRL; fetal bovine serum was purchased from Hyclone; LPS was purchased from Sigma-Aldrich; TNF-α and IL-6 ELISA kits were purchased from BD USA; mouse IL-1β cytokine ELISA detection kit was purchased from Invitrogen; MTT was purchased from Sigma, and DMSO was purchased from China Pharmaceutical Group.

[0039] 2. Experimental Method (1) Detection of nonspecific cytotoxicity: 1 x 10 RAW264.7 cells 5Cells were seeded into 96-well plates at 1 / well and cultured in a 37°C, 5% CO2 incubator for 6 hours. Compounds were diluted in 10 concentrations starting from 250 μM in a 4-fold gradient. After cell monolayer culture, gradient-diluted compounds (compound concentrations were diluted in 10 concentrations starting from 250 μM in a 4-fold gradient: 250, 62.5, 15.625, 3.906, 0.977, 0.244, 0.061, 0.015, 0.004, and 0.001 μM) were added. Corresponding solvent controls (cell controls) and liquid medium background controls (blanks) were also prepared. Cells were cultured in a 37°C, 5% CO2 incubator for 48 hours. 15 min before the end of the incubation, 20 μl of 5 mg / ml MTT was added and allowed to react until the end of the incubation. The supernatant was then aspirated and discarded, and 200 μl of DMSO was added to dissolve the crystals. The absorbance at 570 nm was measured using a microplate reader (Spectra Max 190, Molecular Devices).

[0040] (2) Detection of cytokine production levels: 1 x 10 RAW264.7 cells 5 Cells were seeded at 1 / well in a 96-well plate and cultured in a monolayer at 37°C in a 5% CO2 incubator for 6 hours. Compounds were diluted in a 4-fold gradient starting from 250 μM to 10 concentrations: 250, 62.5, 15.625, 3.906, 0.977, 0.244, 0.061, 0.015, 0.004, and 0.001 μM. After cell monolayer culture, the stimulant LPS (final concentration 10 μg / ml) and the compounds diluted in the above gradient were added. Separate stimulated and unstimulated controls were also set up. After incubation, the cell culture supernatants were collected and the contents of TNF-α, IL-1β, and IL-6 were assayed by ELISA.

[0041] 3. Experimental Results LPS is an important biological pro-inflammatory cytokine and can induce the massive release of inflammatory cytokines by activating nuclear factor kappa-B (NF-κB). Therefore, we evaluated the effects of compounds on the production of inflammatory cytokines using the LPS-induced macrophage RAW264.7 system. As shown in Table 2, naphthoquinone phosphate, hydroxychloroquine sulfate, and chloroquine all significantly inhibited LPS-induced IL-1β production, but had no significant effect on TNF-α and IL-6 production.

[0042] Table 2. Effect of naphthoquinone phosphate on LPS-induced inflammatory cytokine production in RAW264.7 cells

[0043] [Table 2]

[0044] Note: CC50 is the compound concentration at which cell viability is 50%; IC50 is the compound concentration at which cytokine secretion is reduced by 50%; SI=CC50 / IC50.

[0045] As is clear from the above results, naphthoquinone phosphate has a selective inhibitory effect on the secretion of inflammatory cytokines from mouse macrophage RAW264.7 cells induced by LPS.

[0046] Example 3. In vitro inhibition of TLR-L-stimulated mouse splenic lymphocyte proliferation and antibody and cytokine secretion by naphthoquinone phosphate

[0047] 1. Main experimental materials and where to obtain them (1) Experimental animals: SPF-grade BALB / C mice, female, approximately 6 to 8 weeks old, purchased from Beijing Huafukang Biotechnology Co., Ltd., with the certificate number 110322211100631475.

[0048] (2) Main experimental chemical: naphthoquinone phosphate, pale yellow powder, elemental analysis (C 24 H 34 ClN3O9P2), provided by Shanghai Pharmaceutical Industry Co., Ltd.; Hydroxychloroquine sulfate, white powder, elemental analysis (C 18 H 26 ClN3O·H2SO4), provided by Sanwei Pharmaceutical Co., Ltd.

[0049] (3) Reagents: RPMI 1640 liquid medium was purchased from GibcoBRL; fetal bovine serum was purchased from Hyclone; TLR4, 7, and 9 agonists (TLRs Ligand: TLRs-L) were purchased from Invitrogen; mouse IL-6, TNF-α, and IL-10 cytokine ELISA detection kits were purchased from BD; mouse IL-1β cytokine ELISA detection kits were purchased from Invitrogen; and antibody detection kits were purchased from Invitrogen.

[0050] 2. Experimental Method BALB / c mice were euthanized by cervical dislocation, and the spleens were aseptically removed to prepare whole spleen lymphocytes. The cells were collected at a concentration of 5 × 10 6 Hydroxychloroquine sulfate was diluted in a 3-fold gradient from 30 μM to three concentrations of 30, 10, and 3 μM, and naphthoquine phosphate was diluted in a 3-fold gradient from 10 μM to three concentrations of 10, 3, and 1 μM.

[0051] (1) Detection of lymphocyte proliferation: Mouse splenic lymphocyte suspensions were seeded in 96-well plates at 100 μl / well, and stimulators TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM) and compounds diluted in the above concentration gradient were added. Corresponding stimulated controls and cell controls (unstimulated) were also prepared separately. The plates were cultured for 48 hours in an incubator at 37°C with 5% CO2. 8 hours before the end of the culture period, 3After adding 3H-TdR, the cells were incubated until the end of the experiment. The cells were harvested onto a glass fiber membrane using a cell harvester, and 5 ml of liquid scintillation cocktail was added. The radioactivity counts per minute were measured using a beta counter (2450 Microplate Counter, PerkinElmer).

[0052] (2) Cytokine detection Mouse splenic lymphocytes were seeded in a 96-well plate at 100 μl per well and treated with the stimulators TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM) and the compounds diluted in the above concentration gradient. The corresponding stimulated controls and unstimulated cell controls were also prepared separately. The cells were cultured for 48 hours in an incubator at 37°C with 5% CO2. After incubation, the cell culture supernatants were collected and the contents of IL-1β, IL-6, and IL-10 were assayed by ELISA.

[0053] (3) Detection of antibody levels Mouse splenic lymphocytes were seeded in a 96-well plate at 100 μl per well and stimulatory agents TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM) and the compounds diluted in the above concentration gradient were added. Corresponding stimulated controls and unstimulated cell controls were also prepared. The cells were cultured for 120 hours in an incubator at 37°C and 5% CO2. After incubation, the cell culture supernatants were collected and the IgG and IgM contents were assayed by ELISA.

[0054] 3. Experimental Results Toll-like receptors (TLRs) belong to the pattern recognition receptor family and are expressed on various immune cells. They can recognize various pathogen-associated molecular patterns, mediate the differentiation and maturation of antigen-presenting cells, and play important roles in inflammation, immune cell regulation, survival, and proliferation. As shown in Tables 3 and 4, TLRs-L can mediate the proliferation and activation of splenic lymphocytes, and naphthoquinone phosphate significantly inhibited the proliferation and cytokine and antibody secretion mediated by specific TLRs-L.

[0055] Table 3. Effects of naphthoquinone phosphate on TLRs-L-stimulated mouse whole spleen lymphocyte proliferation and antibody secretion

[0056] [Table 3]

[0057] Note: CC50 is the compound concentration at which cell viability is 50%; IC50 is the compound concentration at which cell proliferation and antibody secretion are reduced by 50%; SI=CC50 / IC50.

[0058] Table 4. Effect of naphthoquinone phosphate on cytokine secretion by intact mouse spleen lymphocytes stimulated by TLRs-L.

[0059] [Table 4]

[0060] Note: CC50 is the compound concentration at which cell viability is 50%; IC50 is the compound concentration at which cytokine secretion is reduced by 50%; SI=CC50 / IC50.

[0061] As is clear from the above results, naphthoquinone phosphate can inhibit the activation of splenic lymphocytes induced by specific TLR signals.

[0062] Example 4. In vitro inhibition by naphthoquinone phosphate of the activation of purified mouse B cells stimulated by TLR agonists

[0063] 1. Main experimental materials and where to obtain them (1) Experimental animals: SPF-grade BALB / C mice, female, approximately 6 to 8 weeks old, purchased from Beijing Huafukang Biotechnology Co., Ltd., with the certificate number 110322211102061763.

[0064] (2) Main experimental chemical: naphthoquinone phosphate, pale yellow powder, elemental analysis (C 24 H 34 ClN3O9P2), provided by Shanghai Pharmaceutical Industry Co., Ltd.; Hydroxychloroquine sulfate, white powder, elemental analysis (C 18 H 26 ClN3O·H2SO4), provided by Sanwei Pharmaceutical Co., Ltd.

[0065] (3) Reagents: RPMI 1640 liquid medium was purchased from GibcoBRL; fetal bovine serum was purchased from Hyclone; PE-CD19 was purchased from BD; Anti-PE beads were purchased from Miltenyi; TLRs-L was purchased from Invitrogen; mouse IL-6 antibody detection kit was purchased from Invitrogen; MTT was purchased from Sigma, and DMSO was purchased from China Pharmaceutical Group.

[0066] 2. Experimental Method (1) Preparation of purified B cells: BALB / c mice were euthanized by cervical dislocation, and the spleens were aseptically removed. Spleen cells were prepared by dissolving the cells in 1 × 10 8 The cells were blocked with 2.4G2 (final concentration: 10 μg / ml) for 20 minutes, incubated with PE-CD19 (final concentration: 5 μg / ml) for 20 minutes, washed with MACS buffer, centrifuged at 300 G for 10 minutes at 4°C, and then collected at 1 × 10 cells / ml. 8Add 100 μl of Anti-PE beads and 900 μl of MACS buffer per cell, incubate for 15 minutes, wash with MACS buffer, and centrifuge at 4°C and 300 G for 10 minutes. Discard the supernatant and collect 1 × 10 cells. 8 500 μl of MACS buffer was added per cell to resuspend the cells, and the cells were separated using a MACS column. The magnetically labeled cells were retained on the column and eluted after removing the MACS column from the magnetic field.

[0067] (2) Detection of nonspecific cytotoxicity: Hydroxychloroquine sulfate was diluted in a two-fold gradient from 8 μM to three concentrations: 8, 4, and 2 μM. Naphthoquine phosphate was diluted in a two-fold gradient from 0.5 μM to three concentrations: 0.5, 0.25, and 0.125 μM. Purified mouse B cells were cultured at 1 × 10 6 The cells were seeded at 1 / well in a 96-well plate, and gradient-diluted compounds were added. Separate blanks and cell controls were also prepared. The cells were cultured for 48 hours in an incubator at 37°C with 5% CO2. Four hours before the end of the culture, MTT solution was added and the reaction was continued until the end of the culture. The supernatant was then aspirated and discarded, and 200 μl of DMSO was added to each well to dissolve the crystals. The absorbance at 570 nm was measured using a microplate reader (Spectra Max 190, Molecular Devices).

[0068] (3) Detection of B cell proliferation: Hydroxychloroquine sulfate was diluted in a two-fold gradient from 8 μM to three concentrations: 8, 4, and 2 μM. Naphthoquine phosphate was diluted in a two-fold gradient from 0.5 μM to three concentrations: 0.5, 0.25, and 0.125 μM. A mouse purified B cell suspension was added to 4 × 10 5 The cells were seeded in a 96-well plate at 100x100 / well, and stimulators TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM) and gradient diluted compounds were added. Corresponding stimulated controls and cell controls (unstimulated) were also prepared separately, and the cells were cultured in an incubator at 37°C and 5% CO2 for 48 hours. 8 hours before the end of the culture, 3After adding 3H-TdR, the cells were incubated until the end of the experiment. The cells were harvested onto a glass fiber membrane using a cell harvester, and 5 ml of liquid scintillation cocktail was added. The radioactivity counts per minute were measured using a beta counter (2450 Microplate Counter, PerkinElmer).

[0069] (4) Cytokine detection 4 x 10 mouse purified B cell suspension 5 Cells were seeded in a 96-well plate at 1 / well. Hydroxychloroquine sulfate was diluted in a two-fold gradient starting from 8 μM to three concentrations: 8, 4, and 2 μM. Naphthoquine phosphate was diluted in a two-fold gradient starting from 0.5 μM to three concentrations: 0.5, 0.25, and 0.125 μM. The stimulators TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM) and the diluted compounds were added. Separately, corresponding stimulated controls and unstimulated controls were also included. The cells were incubated for 48 hours at 37°C in a 5% CO2 incubator. After incubation, the cell culture supernatants were collected and the contents of TNF-α, IL-1β, and IL-6 were assayed by ELISA.

[0070] (5) Detection of antibody levels 4 x 10 mouse purified B cell suspension 5 Cells were seeded at 1 / well in a 96-well plate. Hydroxychloroquine sulfate was diluted in a two-fold gradient starting from 8 μM to three concentrations: 8, 4, and 2 μM. Naphthoquine phosphate was diluted in a two-fold gradient starting from 0.5 μM to three concentrations: 0.5, 0.25, and 0.125 μM. The stimulators TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM) and the diluted compounds were added. Separately, corresponding stimulated controls and unstimulated cell controls were also included. The cells were incubated for 120 hours at 37°C in a 5% CO2 incubator. After incubation, the cell culture supernatants were collected and IgG and IgM contents were assayed by ELISA.

[0071] 3. Experimental Results As shown in Table 5, both hydroxychloroquine sulfate and naphthoquine phosphate could inhibit the proliferation of purified mouse splenic B cells and the production of inflammatory cytokines and antibodies induced by specific TLR signals, but the inhibitory effect of naphthoquine phosphate was superior to that of hydroxychloroquine sulfate.

[0072] Table 5. In vitro inhibition of TLRs-L-stimulated mouse purified B cell activation by naphthoquinone phosphates.

[0073] [Table 5]

[0074] Note: CC50 is the compound concentration at which cell viability is 50%; IC50 is the compound concentration at which cytokine secretion is reduced by 50%; SI=CC50 / IC50.

[0075] Example 5. Significant improvement of bovine type II collagen-induced arthritis in mice by intragastric administration of naphthoquinone phosphate

[0076] 1. Main experimental materials and where to obtain them (1) Animals: SPF grade DBA / 1 mice, male, 70 mice, approximately 4 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., with the certificate number 110011211101563086.

[0077] (2) Main experimental chemical: naphthoquinone phosphate, pale yellow powder, elemental analysis (C 24 H 34 ClN3O9P2), provided by Shanghai Pharmaceutical Industry Co., Ltd.; Hydroxychloroquine sulfate, white powder, elemental analysis (C 18 H 26 ClN3O·H2SO4) was provided by Sanwei Pharmaceutical Co., Ltd.; dexamethasone was purchased from Chenxin Pharmaceutical Co., Ltd.

[0078] (3) Reagents: Bovine type II collagen (product number: 20021) was purchased from Chondrex, Inc.; acetic acid (glacial acetic acid) (product number: 10000218) was purchased from China Pharmaceutical Group Chemical Reagents; Freund's complete adjuvant and incomplete adjuvant, LPS, and ConA were purchased from Sigma; RPMI 1640 liquid medium was purchased from GibcoBRL; fetal bovine serum was purchased from Hyclone; LPS was purchased from Sigma; ConA was purchased from Sigma; purified NA / LE Hamster Anti-Mouse CD3e was purchased from BD; and HRP-rabbit anti-mouse IgG (H+L), IgG1, IgG2a, and IgG2b were all purchased from Invitrogen.

[0079] 2. Experimental Method (1) Experimental grouping: normal group, model group, positive drug dexamethasone 1 mg / kg group, naphthoquine phosphate (25, 50, 100 mg / kg) group, hydroxychloroquine sulfate 100 mg / kg group.

[0080] (2) Model construction: 10 mg of type II collagen (CII) was swollen in 2.5 ml of 0.1 mol / L glacial acetic acid to prepare a 4 mg / ml CII solution, which was thoroughly emulsified with an equal volume of CFA and injected subcutaneously in 50 μl into the base of the mouse tail. 21 days later, 4 mg / ml CII solution was thoroughly emulsified with an equal volume of IFA and injected subcutaneously in 50 μl into the base of the mouse tail to enhance immunization.

[0081] (3) Indicator detection: 1) Arthritis score: The severity of arthritis was scored by observing the lesions in the limb joints of the mice. Detectable arthritis accompanied by redness in one or more fingers was scored as 1 point, moderate redness and swelling from the ankle joint to the midfoot was scored as 2 points, severe redness and swelling from the ankle joint to the fingers was scored as 3 points, and severe swelling accompanied by ankylosis was scored as 4 points. At the same time as scoring, the thickness of both hind paws was measured.

[0082] 2) 3Detection of lymphocyte proliferation status in each group by H-TdR uptake method: Splenic lymph nodes of mice from each group were isolated, lymphocyte suspensions were prepared, and the suspensions were seeded into each well of a 96-well plate. ConA (final concentration 5 μg / ml) and LPS (final concentration 10 μg / ml) were added, and the plates were cultured for 48 hours in an incubator at 37°C and 5% CO2. Eight hours before the end of the culture, 25 μl of PBS was added to each well. 3 H-TdR; Anti-CD3 (final concentration 5 μg / ml, pre-incubated overnight) was added and the cells were cultured in an incubator at 37°C, 5% CO2 for 72 hours; CII (final concentration 10 μg / ml) was added and the cells were cultured in an incubator at 37°C, 5% CO2 for 96 hours. Eight hours before the end of the culture, 25 μl of 3 H-TdR was added. A corresponding cell blank was set up for each group, and the culture was continued until the end of the experiment. The cells were harvested onto a glass fiber membrane using a cell harvester, and 5 ml of liquid scintillation cocktail was added. The radioactivity counts per minute were read using a beta counter (2450 Microplate Counter, PerkinElmer).

[0083] 3) Detection of anti-CII antibody levels in serum by ELISA: 50 μl of CII solution (final concentration 50 μg / ml) was added to each well of a 96-well plate for overnight coating, and the plate was washed four times with 200 μl of washing solution (1 L PBS + 500 μl Tween 20) per well; 100 μl of 1% BSA was added to each well for 1 hour of blocking, and the plate was washed four times with 200 μl of washing solution per well; 50 μl of diluted mouse serum samples from each group were added to each well for 2 hours of incubation, and the plate was washed four times with 200 μl of washing solution per well; and horseradish peroxidase (Horseradish) was added to each well for 2 hours of incubation. IgG secondary antibody coupled with peroxidase (HRP) was added and incubated for 1 hour. The plate was washed five times with 200 μl of washing solution per well. 50 μl of tetramethylbenzidine (TMB) substrate was added, and depending on the color development, 30 μl of 1 M sulfuric acid solution was added to each well to terminate the color development. The absorbance values at 450 nm / 570 nm were measured using a microplate reader (Spectra Max 190, Molecular Devices).

[0084] 4) Bone morphological analysis using micro-computed tomography (Micro-CT): Three-dimensional reconstruction was performed using Micro-CT technology, and bone morphological analysis was performed using specialized analysis software.

[0085] 3. Experimental Results (1) Intragastric administration of naphthoquinone phosphate significantly improved clinical scores and paw swelling in arthritic mice. As shown in Figure 1, severe joint lesions occurred in the model group mice, and administration of naphthoquine phosphate significantly reduced the arthritis score of the model mice, and the therapeutic effect was superior to that of hydroxychloroquine sulfate at the same dose. (Note: Compared to the model group, the naphthoquine phosphate 100 mg / kg group * P value < 0.05, ** P value < 0.01, *** P value < 0.001; naphthoquinone phosphate 50 mg / kg group compared with the model group #P value < 0.05, ## P value < 0.01, ### P value <0.001.

[0086] As shown in Figure 2, administration of naphthoquinone phosphate can improve the swelling of the paws of arthritic mice. (Note: Compared with the dexamethasone control group and the model group, * P value < 0.05, ** P value < 0.01, *** P value < 0.001; comparing the normal group with the model group # P value < 0.05, ## P value < 0.01, ### P value < 0.001; comparing naphthoquinone phosphate 25 mg / kg group with the model group. § P value < 0.05, §§ P value < 0.01, §§§ P value <0.001.

[0087] (2) Intragastric administration of naphthoquinone phosphate markedly inhibited the proliferation of splenic and lymph node lymphocytes in arthritic mice. As shown in Figure 3, at the end of the experiment, the spleen and lymph nodes of the mice were collected to prepare single cell suspensions, and their proliferation was stimulated using different stimulants. The administration of naphthoquinone phosphate significantly inhibited the proliferation of spleen and lymph node lymphocytes in arthritic mice. (Note: Compared to the model group, * P value < 0.05, ** P value < 0.01, *** P value <0.001.

[0088] (3) Intragastric administration of naphthoquinone phosphate significantly reduced the level of collagen-specific antibodies in arthritic mice. As shown in Figure 4, at the end of the experiment, the eyeballs were removed, blood was collected, the serum was separated, and the level of collagen-specific antibodies in the serum of the experimental mice was detected by ELISA. The administration of naphthoquinone phosphate significantly reduced the level of collagen-specific antibodies in the serum of arthritic mice. (Note: Compared to the model group, * P value < 0.05, ** P value < 0.01, ***P value <0.001.

[0089] (4) Intragastric administration of naphthoquinone phosphate significantly improved bone damage caused by bone erosion in the joints of arthritic mice. At the end of the experiment, the hind limbs of the mice in each group were photographed and recorded, and the left hind limb was isolated and subjected to Micro-CT scanning analysis. As shown in Figure 5, the mice in the model group exhibited significant joint swelling and deformation, and severe bone erosion occurred. Administration of naphthoquine phosphate inhibited bone damage in the model mice and demonstrated a certain bone-protecting effect.

[0090] Example 6. Intragastric administration of naphthoquinone phosphate markedly inhibits TLRs-L-induced activation of spleen-purified B cells from arthritic mice

[0091] 1. Main experimental materials and where to obtain them (1) Animals: SPF grade DBA / 1 mice, male, 70 mice, approximately 4 weeks old, purchased from Beijing Huafukang Biotechnology Co., Ltd., with the certificate number 110322211101922685.

[0092] (2) Main experimental chemical: naphthoquinone phosphate, pale yellow powder, elemental analysis (C 24 H 34 ClN3O9P2), provided by Shanghai Pharmaceutical Industry Co., Ltd.; Hydroxychloroquine sulfate, white powder, elemental analysis (C 18 H 26 ClN3O·H2SO4), provided by Sanwei Pharmaceutical Co., Ltd.

[0093] (3) Reagents: Bovine type II collagen (product number: 20021) was purchased from Chondrex, Inc.; acetic acid (glacial acetic acid) (product number: 10000218) was purchased from China Pharmaceutical Group Chemical Reagents; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; RPMI 1640 liquid medium was purchased from Gibco BRL; fetal bovine serum was purchased from Hyclone; rat anti-mouse CD16 / CD32 (2.4G2), PE rat anti-mouse CD19, and mouse IL-6 ELISA detection kits were purchased from BD; anti-PE beads, MACS columns, and MACS buffer were purchased from Miltenyi; TLRs-L was purchased from Invitrogen; antibody detection kit was purchased from Invitrogen; and antibodies for flow cytometry were all purchased from BD. 3 H-TdR and liquid scintillation cocktail were purchased from PerkinElmer.

[0094] 2. Experimental Method (1) Experimental grouping: normal group, model group, positive drug methotrexate 1 mg / kg group, naphthoquine phosphate (50, 100 mg / kg) group, hydroxychloroquine sulfate 100 mg / kg group.

[0095] (2) Model construction: Same as in Example 5.

[0096] (3) Detection of indicators: At the end of the experiment, the spleens of the mice in each group were collected, and splenic B cells were purified in the same manner as in Example 4. They were stimulated with different TLRs-L, 3 The proliferation status was detected by H-TDR uptake assay, the secretion levels of IL-6 and antibodies were detected by ELISA, and the expression of CD80, CD86, and CD69 was detected by flow cytometry.

[0097] 1) Detection of B cell proliferation: 4 x 10 mouse purified B cell suspensions in each group 5The cells were seeded in a 96-well plate at 1 / well and treated with the stimuli TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM). A corresponding blank was set up for each group. The cells were cultured for 48 hours in an incubator at 37°C with 5% CO2. 8 hours before the end of the culture period, 3 After adding 3H-TdR, the cells were incubated until the end of the experiment. The cells were harvested onto a glass fiber membrane using a cell harvester, and 5 ml of liquid scintillation cocktail was added. The radioactivity counts per minute were measured using a beta counter (2450 Microplate Counter, PerkinElmer).

[0098] 2) Cytokine detection 4 x 10 mouse purified B cell suspensions in each group 5 The cells were seeded at 1 / well in a 96-well plate and treated with the stimuli TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM). A corresponding blank was also prepared for each group. The cells were cultured for 48 hours in an incubator at 37°C and 5% CO2. After the culture, the cell culture supernatant was collected and the IL-6 content was measured by ELISA.

[0099] 3) Detection of antibody levels: 4 x 10 mouse purified B cell suspension 5 The cells were seeded at 1 / well in a 96-well plate and treated with the stimuli TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM). A corresponding blank was also included in each group. The cells were cultured for 120 hours in an incubator at 37°C with 5% CO2. After incubation, the cell culture supernatants were collected and the IgG and IgM contents were assayed using ELISA.

[0100] 4) Detection of CD86, CD80, and CD69 expression by flow cytometry: 4 x 10 mouse purified B cell suspensions in each group 5The cells were seeded in a 96-well plate at 1 / well and treated with the stimuli TLR4-L (final concentration 10 μg / ml), TLR7-L (final concentration 5 μg / ml), or TLR9-L (final concentration 1 μM). A corresponding blank was also prepared for each group. The cells were cultured for 48 hours in an incubator at 37°C with 5% CO. The cells were collected in 4 ml of FACS wash buffer (prescribed) and centrifuged at 500 G at 4°C for 6 min; 5 μl of 2.4G 2 was added to each tube for 20 min of blocking, with shaking once every 10 min; 10 μl of flow cytometry antibody dye was added to each tube for 20 min of incubation, with shaking once every 10 min; 4 ml of FACS wash buffer was added to each tube and centrifuged at 500 G at 4°C for 6 min; 300 μl of FACS wash buffer was added to each tube to resuspend the cells, and the expression of CD86, CD80, and CD69 was detected using a flow cytometer (LSR FORTESSA, BD).

[0101] 3. Experimental Results As shown in Figure 6, administration of naphthoquinone phosphate significantly inhibited B cell activation, cytokine secretion, and antibody production mediated by TLRs signals, suggesting that blocking TLRs signals may inhibit the pathological B cell immune response in RA. (Note: Compared to the model group, * P value < 0.05, ** P value < 0.01, *** P value <0.001.

[0102] Example 7. Significant improvement of disease symptoms in adjuvant-induced arthritis in rats by intragastric administration of naphthoquine phosphate

[0103] 1. Main experimental materials and where to obtain them (1) Animals: 38 male SD rats weighing approximately 150 g were purchased from Beijing Huafukang Biotechnology Co., Ltd., with the certificate number 110322211101936668.

[0104] (2) Main experimental drugs: Naphthoquinone phosphate, pale yellow powder, elemental analysis (C24 H 34 ClN3O9P2), provided by Shanghai Pharmaceutical Industry Co., Ltd.; Hydroxychloroquine sulfate, white powder, elemental analysis (C 18 H 26 ClN3O·H2SO4) was provided by Sanwei Pharmaceutical Co., Ltd.; injectable methotrexate was purchased from Horen Pharmaceutical Co., Ltd.

[0105] (3) Reagents: BCG vaccine was purchased from DIFCO; lanolin was purchased from China National Pharmaceutical Group Chemical Reagents; and paraffin oil was purchased from China National Pharmaceutical Group Chemical Reagents.

[0106] 2. Experimental Method (1) Experimental grouping: normal group, model group, positive drug methotrexate 1 mg / kg group, naphthoquine phosphate (25, 50, 100 mg / kg) group, hydroxychloroquine sulfate 100 mg / kg group.

[0107] (2) Model construction: An appropriate amount of lanolin and paraffin oil were sterilized and placed on ice. One bottle of BCG vaccine (100 mg) and 1.5 ml of PBS were ground using a glass homogenizer until no granules were present. After thorough grinding, 3.5 ml of PBS was added and placed on ice. The sterilized lanolin was heated to a liquid state in a 56°C water bath. 675 μl of paraffin oil, 225 μl of lanolin, and 900 μl of thoroughly ground BCG vaccine-containing PBS were dispensed into an emulsifying tube and thoroughly emulsified. 0.1 ml of the mixture was injected intradermally into the left hind footpad of each rat.

[0108] (3) Indicator detection: 1) Arthritis score: The severity of arthritis was scored by observing the lesions in the rat's ankle joints. Detectable arthritis accompanied by redness of one or more fingers was scored as 1 point, moderate redness and swelling from the ankle to the midfoot as 2 points, severe redness and swelling from the ankle to the toes as 3 points, and severe swelling accompanied by ankylosis as 4 points. At the same time as scoring, the thickness of both hind paws was measured.

[0109] 2) Pathological analysis of model animal joints: Bone and joint tissues were analyzed by H&E staining.

[0110] 3) Bone morphological analysis using micro-computed tomography (Micro-CT): Three-dimensional reconstruction was performed using Micro-CT technology, and bone morphological analysis was performed using specialized analysis software.

[0111] 3. Experimental Results As shown in Figure 7, the rats in the model group developed severe swelling and deformation in the ankle joints and toe joints. Administration of naphthoquine phosphate improved the clinical score of arthritis in the experimental rats, suppressed the degree of swelling in their feet, and had a superior therapeutic effect to that of hydroxychloroquine sulfate at the same dose. (Note: Compared to the model group, * P value < 0.05, ** P value < 0.01, *** P value <0.001.

[0112] Example 8. Significant reduction of spontaneous systemic lupus erythematosus in experimental mice by intragastric administration of naphthoquine phosphate

[0113] 1. Main experimental materials and where to obtain them (1) Animals: SPF-grade MRL / lpr mice, female, approximately 6 to 8 weeks old, purchased from Shanghai Lingcheng Biotechnology Co., Ltd., with a certificate number of 20180003016877.

[0114] (2) Main experimental drugs: Naphthoquinone phosphate, pale yellow powder, elemental analysis (C 24 H 34 ClN3O9P2), provided by Shanghai Pharmaceutical Industry Co., Ltd.; Hydroxychloroquine sulfate, white powder, elemental analysis (C 18 H 26 ClN3O·H2SO4) was provided by Sanwei Pharmaceutical Co., Ltd.; prednisolone was purchased from Shanghai Jinsu Biotechnology Co., Ltd.

[0115] (3) Reagents: Coomassie brilliant blue stain was purchased from BIO-RAD; creatinine detection kit was purchased from Abcam; mouse antinuclear antibody (ANA) kit was purchased from Alpha Diagnostic International; HRP-conjugated goat anti-mouse IgG (H+L) was purchased from Beyotime; and calf thymus DNA was purchased from Sigma.

[0116] 2. Experimental Method (1) Experimental grouping: normal group, model group, positive drug prednisolone 1 mg / kg group, naphthoquine phosphate (25, 50 mg / kg) group, hydroxychloroquine sulfate 100 mg / kg group.

[0117] (2) Measurement of proteinuria and urinary creatinine: After weighing the mice, 20 μl of urine was collected per mouse. The protein content of the urine samples was measured using Coomassie brilliant blue, and the urinary creatinine level was measured using a creatinine kit.

[0118] (3) Detection of anti-double-stranded DNA antibodies: 50 μl of double-stranded DNA solution (final concentration 50 μg / ml) was added to each well of a 96-well plate, and the plate was coated overnight. The plate was then washed four times with 200 μl of washing solution (1 L of PBS + 500 μl of Tween 20) per well. 100 μl of 1% BSA was added to each well for 1 hour of blocking, and the plate was then washed four times with 200 μl of washing solution per well. 50 μl of diluted mouse serum samples from each group were added to each well, and the plate was then incubated for 2 hours, and the plate was then washed four times with 200 μl of washing solution per well. Horseradish peroxidase (Horseradish) was added to each well. An IgG (H+L) secondary antibody coupled with HRP (Peroxidase) was added and incubated for 1 hour. The plate was then washed five times with 200 μl of washing solution per well. 50 μl of tetramethylbenzidine (TMB) substrate was added, and depending on the color development, 30 μl of 1 M sulfuric acid solution was added to each well to terminate the color development. The absorbance values at 450 nm / 570 nm were measured using a microplate reader (Spectra Max 190, Molecular Devices).

[0119] 3. Experimental Results At the end of the experiment, the eyes were removed and blood was collected. The levels of anti-dsDNA antibodies in the serum of the experimental mice were detected using the ELISA method, and the levels of antinuclear antibodies were detected using an antinuclear antibody kit. As shown in Figure 8, administration of naphthoquinone phosphate significantly reduced the levels of anti-dsDNA antibodies and antinuclear antibodies in the serum of the experimental mice. (Note: Compared to the model group, * P value < 0.05, ** P value < 0.01, *** P value <0.001.

[0120] As is clear from the above results, administration of naphthoquinone phosphate significantly improved the disease symptoms in arthritic mice, reduced the levels of serum collagen-specific antibodies, and inhibited lymphocyte proliferation; significantly improved the clinical scores and suppressed secondary paw swelling in arthritic rats; reduced the levels of pathogenic antibodies in the serum of lupus model mice; and inhibited the activation of B cells mediated by TLRs signals, exerting therapeutic effects on autoimmune diseases and potentially being developed as a medicine for the treatment of autoimmune diseases. [Prior art documents] [Non-patent literature]

[0121] [Non-Patent Document 1] [1] Chang YJ, Liu KS, Wang JJ, et al. Antimalarial primaquine for skin infiltration analgesia in rats[J]. J Pharm Pharmacol, 2021, 73(2):206-11. [Non-patent document 2] [2] Tsokos G C. Systemic lupus erythematosus[J]. N Engl J Med, 2011, 365(22):2110-21. [Non-patent document 3] [3] Lee SJ, Silverman E, Bargman J M. The role of antimalarial agents in the treatment of SLE and lupus nephritis[J]. Nat Rev Nephrol, 2011, 7(12):718-29.

Claims

1. Use of naphthoquinone phosphate in the manufacture of a medicament for the treatment of an autoimmune disease.

2. 2. The use according to claim 1, wherein the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus.

3. 2. The use according to claim 1, wherein the use is a naphthoquinone phosphate salt in the manufacture of a medicament for the treatment or adjunctive treatment of rheumatoid arthritis.

4. 2. The use according to claim 1, wherein the use is naphthoquinone phosphate in the manufacture of a medicament for the treatment or adjunctive treatment of systemic lupus erythematosus.

5. 5. The use according to claim 1, 3 or 4, wherein the medicine is a pharmaceutical composition containing a therapeutically effective amount of naphthoquinone phosphate as an active ingredient and pharmaceutical additives.

6. The use according to claim 5, characterized in that the pharmaceutical composition is in the form of an oral or injectable preparation.

7. The use according to claim 6, characterized in that the oral administration dosage form is a tablet, capsule, powder / granule or oral liquid preparation, and the injection is an intravenous or intramuscular injection.

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