Medical use of baloxavir marboxil or baloxavir acid as a VISTA agonist and its pharmaceutical composition

Abstract

The present invention discloses the medical use of baloxavir disoproxil or baloxavir acid as a VISTA agonist and its pharmaceutical composition. The present invention proposes for the first time that baloxavir disoproxil or baloxavir acid can be used to treat autoimmune diseases, especially autoimmune diseases such as lupus erythematosus, asthma and psoriasis, and can be used to prepare drugs for preventing or treating autoimmune diseases. The present invention provides the use of baloxavir disoproxil or baloxavir acid as an agonist of the immune checkpoint VISTA, and specifically relates to the use of baloxavir disoproxil or baloxavir acid to stimulate the immune checkpoint VISTA to treat autoimmune diseases such as lupus erythematosus, asthma and psoriasis.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technologies, and particularly to the medical use of baloxavir marboxil or baloxavir acid as a VISTA agonist and its pharmaceutical composition. Background Art

[0002] The T cell activation inhibitor immunoglobulin variable domain (VISTA, V domain immunoglobulin suppressor of T-cell activation) is an immune checkpoint protein of the B7 family and plays various roles in regulating peripheral tolerance, autoimmunity, inflammation, and regulation.

[0003] VISTA inhibits T cells, dendritic cells, macrophages, and TCRγδ T cells and negatively regulates immune function in various autoimmune diseases. Chen Lieping et al. found that VISTA is highly expressed in immune cells in the lesions of human systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE), and the skin lesions of lupus mouse models (MRL / lpr mice), and found that VISTA is a key factor in the occurrence and development of lupus. Activating VISTA can effectively treat systemic and cutaneous lupus. (Sci Transl Med. 2019, 11: 522 - 536). In the experimental autoimmune encephalomyelitis (EAE) model, the application of the VISTA blocking antibody (13F3) significantly accelerates disease progression and exacerbates disease severity (J Exp Med. 2011, 208: 577 - 592). In the ear inflammation model, VISTA acts through CD4 +T cells play an immunosuppressive role. VISTA can regulate the IL-23 / IL-17 inflammatory axis and plays an important role in the development of psoriasis (Sci Rep. 2017:1486). In 2020, the research group of the present inventors reported that VISTA deficiency could exacerbate psoriasis-like skin inflammation, and the numbers of T cells and dendritic cells increased significantly (Theranostics, 2020, 10:10483-10497). In a murine experimental asthma model, VISTA deficiency promoted the massive accumulation of eosinophils in the lungs, leading to increased production of airway inflammatory cytokines such as innate cytokines (IL-6, MCP-1 and TNF-α) and Th2 cytokines (IL-5 and IL-13). Treatment with a VISTA agonist monoclonal antibody could reduce the severity of asthma and alleviate lung inflammation (CELL MOL IMMUNOL 2017, 15(9)). In a murine collagen antibody-induced arthritis (CAIA) model, it was found that the expression of VISTA on myeloid cells could induce arthritis. After VISTA knockout or treatment with an anti-VISTA monoclonal antibody (8G8), the joint damage in mice was alleviated (Arthritis Res The 2017, 19(1):270.). Therefore, the important negative regulatory role of VISTA in innate and adaptive immunity enables it to play an important role in autoimmune diseases and inflammatory responses.

[0004] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that commonly affects women of childbearing age and severely impacts people's daily lives. Its clinical manifestations can involve all systems and tissues of the body, including the skin and mucosa, joints and muscles, kidneys, blood, nerves, etc. [The pathogenesis of SLE is not fully understood yet. Most scholars generally believe that this disease has genetic susceptibility and is affected by multiple factors such as the environment, hormones, and immune levels. If not treated promptly, it may cause irreversible damage to tissues and organs, and in severe cases, it can lead to the death of patients. Currently, SLE cannot be cured clinically. Minimizing the damage of the disease to various organ systems to the greatest extent, delaying the rapid progression of the disease, effectively improving the expected outcome, and preventing the recurrence of the disease are the goals that scholars have been pursuing. Currently, the main treatments include glucocorticoids, immunosuppressants, and antimalarial drugs, etc. The obvious side effects of taking them for a long time often trouble people's lives. Therefore, new drugs for treating SLE need to be developed to meet social needs.

[0005] Asthma is a chronic bronchial inflammatory disease. Allergic asthma is the most common form of asthma and is an airway inflammatory disease caused by the interaction of genetic factors and environmental antigens. Its clinical features include wheezing, shortness of breath, chest tightness, and coughing; pathological features include pulmonary eosinophilia, goblet cell metaplasia, hyperplasia, increased epithelial mucin, airway hyperresponsiveness, and elevated serum allergen-specific IgE concentration. Currently, the main drugs for treating asthma are β2-agonists, leukotriene receptor antagonists, and inhaled corticosteroids. However, short-acting β2-agonists do not reduce the risk of attacks, and the compliance of adults and children with inhaled corticosteroids is very poor. Therefore, in the past decade or so, scientists have been committed to developing some monoclonal antibodies against asthma-related cytokines and studying new targets.

[0006] Psoriasis vulgaris, commonly known as psoriasis, is a common chronic inflammatory autoimmune disease in dermatology, mainly manifested by erythema and scales, with a global prevalence of 2%-3%. The disease has a long course, is difficult to treat and prone to recurrence, seriously affecting the quality of life of patients. The pathogenesis of psoriasis involves genetic, environmental, and immune factors. Pathological phenomena such as excessive proliferation of epidermal cells, accompanied by incomplete keratinization and infiltration of dermal lymphocytes occur during the disease process.

[0007] Baloxavir marboxil is a class of small molecule inhibitors of the polymerase acidic (PA) protein subunit of the influenza virus polymerase complex. After oral administration, baloxavir marboxil is metabolized into the active form baloxavir acid; therefore, baloxavir marboxil is a prodrug of baloxavir. Baloxavir blocks influenza virus replication by inhibiting the cap-dependent endonuclease activity of the PA protein. In preclinical in vitro studies, baloxavir marboxil inhibited viral RNA transcription and replication. Baloxavir marboxil is effective against both influenza A and B viruses, including oseltamivir-resistant strains and avian influenza virus strains (H7N9, H5N1). It has now been approved by the US Food and Drug Administration (FDA) for the treatment of acute uncomplicated influenza in patients 12 years of age and older who have had symptoms for no more than 48 hours.

[0008]

[0009] So far, no literature has reported the effect of baloxavir marboxil on autoimmune diseases such as lupus erythematosus, asthma, and psoriasis through the VISTA target. Summary of the Invention

[0010] Object of the Invention: To solve the problem that there is currently a lack of effective drugs for treating autoimmune diseases such as lupus erythematosus, asthma, and psoriasis in clinical practice, the present invention provides a new use of baloxavir marboxil as a VISTA agonist in the prevention or treatment of autoimmune diseases such as lupus erythematosus, asthma, and psoriasis.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases such as lupus erythematosus, asthma, and psoriasis.

[0012] Technical Solution: In order to achieve the above technical objects, the present invention provides the use of baloxavir marboxil or baloxavir acid or a pharmaceutically acceptable salt thereof in the preparation of a VISTA agonist.

[0013] The use of baloxavir marboxil or baloxavir acid or a pharmaceutically acceptable salt thereof as a VISTA agonist in the preparation of a drug for preventing or treating diseases mediated by VISTA.

[0014] Among them, the diseases mediated by VISTA are autoimmune diseases.

[0015] The use of baloxavir marboxil or baloxavir acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating autoimmune diseases.

[0016] Among them, the autoimmune diseases include lupus erythematosus, asthma, psoriasis, autoimmune encephalomyelitis, autoimmune hepatitis, or rheumatoid arthritis.

[0017] Among them, the autoimmune diseases are lupus erythematosus, asthma, or psoriasis.

[0018] Furthermore, the lupus erythematosus includes: cutaneous lupus erythematosus (malar erythema, bullous lupus, papular rash, photosensitivity-like lupus, psoriasis-like rash, annular polymorphic rash-like, verrucous lupus erythematosus, tumid lupus erythematosus, deep lupus erythematosus, chilblain-like lupus erythematosus, discoid lupus, lupus erythematosus and lichen planus overlap syndrome), systemic lupus erythematosus (discoid erythema, oral and nasal ulcers, antiphospholipid syndrome, Raynaud's phenomenon, alopecia, hemolytic anemia, lupus nephritis, lupus neurological lesions, lupus arthritis, pericarditis, and pleurisy), or drug-induced lupus erythematosus; the asthma includes: allergic asthma, non-allergic asthma, late-onset asthma, asthma with fixed airflow limitation, severe asthma, refractory asthma, or obesity-related asthma; the psoriasis is plaque psoriasis, generalized pustular psoriasis, erythrodermic psoriasis, or psoriatic arthritis.

[0019] Use of the pharmaceutical composition of the present invention in the preparation of a medicament for preventing or treating autoimmune diseases, wherein the pharmaceutical composition contains baloxavir marboxil or baloxavir acid or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

[0020] Among them, the autoimmune diseases include lupus erythematosus, asthma, psoriasis, autoimmune encephalomyelitis, autoimmune hepatitis or rheumatoid arthritis.

[0021] Among them, the dosage form of the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

[0022] The present invention first preliminarily screens out compounds with good affinity for human VISTA protein in the selleck compound library by surface plasmon resonance (SPR) technology, and then for the first time discovers through cell-level experiments that both baloxavir marboxil or baloxavir acid and their in vivo active metabolite baloxavir acid can significantly bind to VISTA protein and activate its downstream signaling pathway, thereby inhibiting the release of inflammatory factors such as IFN-γ, TNF-α and IL-2. Therefore, baloxavir marboxil can be used to prepare a VISTA agonist.

[0023] For the systemic lupus erythematosus described above, the present invention uses cGVHD-like systemic lupus erythematosus mice as experimental subjects. At the level of mouse animal experiments, the dosage of baloxavir marboxil is 50 mg / kg, and the compound can be purchased through commercial channels. The inventors of the present invention found that baloxavir marboxil effectively reduced urinary protein, blood urea nitrogen, creatinine, anti-dsDNA antibody in peripheral blood, inflammatory factors IL-6 / MCP-1 / TNF-α / TGF-β / IL-1β in the kidney, and deposition of immune complex IgG in the kidney of lupus mice, and alleviated the pathological damage of the kidney in lupus mice; for the asthma described above, the present invention established an asthma model by inducing Balb / c mice with ovalbumin (OVA). At the level of mouse animal experiments, the dosage of baloxavir marboxil is 50 mg / kg, and the compound can be purchased through commercial channels. The inventors of the present invention found that baloxavir marboxil can reduce the content of IgE in the serum of mice, improve the infiltration of inflammatory cells in the lungs of mice, reduce the hyperplasia of pulmonary mucus and goblet cells, and reduce the secretion of inflammatory cytokine IL-4 in bronchoalveolar lavage fluid; for the psoriasis described above, the present invention uses imiquimod-induced psoriasis mice as experimental subjects. At the level of mouse animal experiments, the dosage of baloxavir marboxil is 50 mg / kg, and the compound can be purchased through commercial channels. The inventors of the present invention found that baloxavir marboxil effectively reduced the ear thickness of psoriasis mice and the degree of skin and ear damage. At the same time, after oral administration, baloxavir marboxil was metabolized into the active form baloxavir acid (Clin Drug Investig, 2018, 38(12):1189-1196). The present invention proves that baloxavir acid can also be used as an agonist targeting VISTA for the treatment of various autoimmune diseases.

[0024] Beneficial effects: Compared with the prior art, baloxavir marboxil can effectively improve the condition of lupus mice, and the specific manifestations are as follows: (1) The urinary protein level of lupus mice significantly decreased after treatment with baloxavir marboxil; (2) Baloxavir marboxil can reduce the levels of blood urea nitrogen, creatinine, and anti-dsDNA antibody in the peripheral blood of lupus mice; (3) After intervention with baloxavir marboxil, the pathological damage of the kidney in lupus mice was significantly alleviated, and the deposition of inflammatory factors IL-6 / MCP-1 / TNF-α / TGF-β / KIM-1 and immune complex IgG in the kidney also decreased. At the same time, baloxavir marboxil can effectively improve the condition of asthma mice, and the specific manifestations are as follows: (1) Baloxavir marboxil can reduce the content of IgE in the serum of mice at the animal level; (2) Baloxavir marboxil can improve the infiltration of inflammatory cells in the lungs of mice, reduce the hyperplasia of pulmonary mucus and goblet cells; (3) Reduce the secretion of inflammatory cytokine IL-4 in bronchoalveolar lavage fluid. On the other hand, baloxavir marboxil can effectively improve the condition of psoriasis mice, and the specific manifestations are as follows: The ear thickness, skin and ear damage degree of psoriasis mice decreased after treatment with baloxavir marboxil.

[0025] The present invention for the first time provides the use of baloxavir marboxil as an immune checkpoint VISTA agonist, specifically relating to the use that baloxavir marboxil can activate the immune checkpoint VISTA, thereby exerting the use for treating autoimmune diseases such as lupus erythematosus, asthma and psoriasis. In addition, baloxavir marboxil is an antiviral drug that has been applied clinically, and its safety and side effects have passed the clinical test. The present invention has verified through in vitro and in vivo experiments that it can be used as a VISTA agonist to treat or prevent the progression of lupus erythematosus, asthma and psoriasis, thereby accelerating the clinical research of the drug and promoting clinical medication to solve the problems of lack of treatment drugs, large side effects and high prices for existing autoimmune diseases such as systemic lupus erythematosus, asthma and psoriasis.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0027] The present invention for the first time provides the use of baloxavir marboxil or baloxavir acid as an immune checkpoint VISTA agonist, specifically relating to the use that baloxavir marboxil or baloxavir acid can activate the immune checkpoint VISTA, thereby exerting the use for treating autoimmune diseases such as lupus erythematosus, asthma and psoriasis. In addition, baloxavir marboxil is an antiviral drug that has been applied clinically, and its safety and side effects have passed the clinical test. The present invention has verified through in vitro and in vivo experiments that it can be used as a VISTA agonist to treat or prevent the progression of lupus erythematosus, asthma and psoriasis, thereby accelerating the clinical research of the drug and promoting clinical medication to solve the problems of lack of treatment drugs, large side effects and high prices for existing autoimmune diseases such as systemic lupus erythematosus, asthma and psoriasis.

[0028] The present invention firstly proposes the use of baloxavir marboxil or baloxavir acid in the preparation of drugs for preventing or treating autoimmune diseases, including lupus erythematosus, asthma and psoriasis. Through in-vivo experiments, the present invention can effectively improve the condition of lupus mice, and the specific manifestations are as follows: (1) After treatment with baloxavir marboxil, the urine protein level of lupus mice decreased significantly; (2) Baloxavir marboxil can reduce the levels of blood urea nitrogen, creatinine and anti-dsDNA antibody in the peripheral blood of lupus mice; (3) After intervention with baloxavir marboxil, the pathological damage of the kidneys of lupus mice was significantly reduced, and the deposition of inflammatory factors IL-6 / MCP-1 / TNF-α / TGF-β / KIM-1 and immune complex IgG in the kidneys also decreased. At the same time, baloxavir marboxil can effectively improve the condition of asthmatic mice, and the specific manifestations are as follows: (1) Baloxavir marboxil can reduce the content of IgE in the serum of mice at the animal level; (2) Baloxavir marboxil can improve the infiltration of inflammatory cells in the lungs of mice, and reduce the hyperplasia of pulmonary mucus and goblet cells; (3) Reduce the secretion of inflammatory cytokine IL-4 in bronchoalveolar lavage fluid. On the other hand, baloxavir marboxil can effectively improve the condition of psoriasis mice, and the specific manifestations are as follows: After treatment with baloxavir marboxil, the ear thickness, skin and ear damage degree of psoriasis mice decreased.

[0029] Therefore, the present invention firstly discovers that baloxavir marboxil or baloxavir acid or their pharmaceutically acceptable salts can be used as a targeting immune checkpoint VISTA agonist, which can efficiently and specifically activate VISTA, and thus can be used in the preparation of drugs for preventing or treating diseases mediated by VISTA. Description of the Drawings

[0030] Figure 1 Results of pH pre-enrichment and protein coupling amount of VISTA protein in surface plasmon resonance (SPR);

[0031] Figure 2 Results of affinity test of compound baloxavir marboxil with human VISTA protein detected by surface plasmon resonance (SPR);

[0032] Figure 3 Effect diagram of CCK-8 evaluation of the toxicity of compound baloxavir marboxil to Jurkat and PBMC cells;

[0033] Figure 4 Effect diagram of human VISTA protein inhibiting IL-2 secretion of Jurkat cells;

[0034] Figure 5 Effect diagram of compound baloxavir marboxil on the effect of human VISTA protein on IL-2 secretion of Jurkat cells;

[0035] Figure 6Effect diagram of human VISTA protein inhibiting the secretion of IFN-γ, TNF-α and IL-2 in PBMC cell supernatant;

[0036] Figure 7 Effect diagram of the compound baloxavir marboxil affecting the effect of human VISTA protein on the secretion of IFN-γ, TNF-α and IL-2 in PBMC cell supernatant;

[0037] Figure 8 Effect diagram of establishing a PBMC activation model of human VISTA protein by CFSE experiment;

[0038] Figure 9 Effect diagram of CFSE detecting the proliferation effect of the compound baloxavir marboxil on PBMC cells;

[0039] Figure 10 Effect diagram of the effect of the compound baloxavir marboxil on the inhibition rate of IL-2 in Jurkat-EV / FL cells;

[0040] Figure 11 Effect diagram of the purity of CD4 + T cells extracted from the spleens of wild type (WT) and VISTA knockout (KO) mice;

[0041] Figure 12 Effect of the compound baloxavir marboxil on the inhibition rate of IL-2 in CD4 + T cells of wild type (WT) and VISTA knockout (KO) mice;

[0042] Figure 13 Test results of the affinity of the compound baloxavir acid to human VISTA protein detected by surface plasmon resonance (SPR);

[0043] Figure 14 Effect diagram of CCK-8 evaluating the toxicity of the compound baloxavir acid to Jurkat and PBMC cells;

[0044] Figure 15 Effect diagram of the compound baloxavir acid affecting the effect of human VISTA protein on the secretion of IL-2 in Jurkat cells;

[0045] Figure 16 Effect diagram of the compound baloxavir acid affecting the effect of human VISTA protein on the secretion of IFN-γ, TNF-α and IL-2 in PBMC cell supernatant; Effect of CFSE detecting the proliferation effect of the compound baloxavir acid on PBMC cells;

[0046] Figure 17Effect diagram of the inhibitory rate of baloxavir acid on IL-2 in Jurkat-EV / FL cells;

[0047] Figure 18 Effect of baloxavir acid on the inhibitory rate of IL-2 in CD4 + T cells of wild type (WT) and VISTA knockout (KO) mice;

[0048] Figure 19 Effect diagram of baloxavir marboxil on the levels of anti-dsDNA antibody, blood urea nitrogen and creatinine in the serum of cGVHD-like SLE mice;

[0049] Figure 20 Effect diagram of baloxavir marboxil on the level of urinary protein in the urine of cGVHD-like SLE mice;

[0050] Figure 21 Effect diagram of baloxavir marboxil on the spleen weight of cGVHD-like SLE mice;

[0051] Figure 22 Effect diagram of baloxavir marboxil on the mRNA levels of IL-6, MCP-1, TNF-α, TGF-β and KIM-1 in cGVHD-like SLE mice;

[0052] Figure 23 HE staining diagram of the kidneys of cGVHD-like SLE mice treated with baloxavir marboxil;

[0053] Figure 24 Effect diagram of the effect of baloxavir marboxil on the deposition of immune complexes in the kidneys of cGVHD-like SLE mice;

[0054] Figure 25 Observation of the pathological changes of the lung tissue of asthmatic mice by HE staining after treatment with baloxavir marboxil;

[0055] Figure 26 Observation of the pathological changes of the lung tissue of asthmatic mice by PAS staining after treatment with baloxavir marboxil;

[0056] Figure 27 Comparison diagram of the effect of baloxavir marboxil on the level of immunoglobulin IgE detected by enzyme-linked immunosorbent assay;

[0057] Figure 28 Effect of baloxavir marboxil on the levels of cytokines in the BALF of asthmatic mice detected by enzyme-linked immunosorbent assay; among them, (A) effect diagram of baloxavir marboxil on the secretion of IL-4 in BALF, (B) effect diagram of baloxavir marboxil on the secretion of IFN-γ in BALF, (C) effect diagram of baloxavir marboxil on the secretion of IL-10 in BALF;

[0058] Figure 29 To collect mouse lung tissues, after extracting total RNA, the expression levels of IL-6, MCP-1, Arg-1 and Ym-1 mRNA were determined by qRT-PCR; among them, (A) the effect diagram of baloxavir marboxil on IL-6 mRNA in lung tissues, (B) the effect diagram of baloxavir marboxil on MCP-1 mRNA in lung tissues, (C) the effect diagram of baloxavir marboxil on Arg-1 mRNA in lung tissues, (D) the effect diagram of baloxavir marboxil on Ym1 mRNA in lung tissues;

[0059] Figure 30 To collect mouse lung tissues, after extracting total protein, the expression levels of pSTAT3 and STAT3 were determined by Western blotting;

[0060] Figure 31 Effect diagram of compound baloxavir marboxil on the ear thickness and skin redness degree score of psoriasis model mice;

[0061] Figure 32 HE staining diagrams of the skin and ears of psoriasis model mice treated with compound baloxavir marboxil. Detailed implementation manners

[0062] The content of the present invention will be specifically described below through examples. In the present invention, the following examples are for better explaining the present invention and are not used to limit the scope of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0063] The content of the present invention will be specifically described below through examples. In the present invention, the following examples are for better explaining the present invention and are not used to limit the scope of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0064] Baloxavir marboxil was purchased from Shanghai sellcek Company, and its CAS number is 1985606-14-1.

[0065] Baloxavir acid was purchased from Shanghai sellcek Company, and its CAS number is 1985605-59-1.

[0066] Example 1

[0067] Surface plasmon resonance (SPR) was used for primary screening of the affinity of 30 old drugs (selleck compound library) with human VISTA protein

[0068] Surface Plasmon Resonance (SPR) is an optical phenomenon. When incident light enters the interface of two media with different refractive indices at the critical angle, it can cause the resonance of free electrons in the metal. Due to resonance, the electrons absorb the light energy, resulting in a significant attenuation of the reflected light within a certain angle. When the surface plasmon wave resonates with the evanescent wave, a significant attenuation of the detected reflected light energy occurs. At this time, the corresponding incident light wavelength is the resonance wavelength, and the corresponding incident angle is the resonance angle, that is, the SPR angle. SPR changes with the change of the surface refractive index, and the change of the refractive index is proportional to the mass of the biomolecules bound to the metal surface. Therefore, by obtaining the dynamic change of the SPR angle during the biological reaction process, specific signals of the interaction between biomolecules can be obtained. In the SPR experiment, human VISTA protein (RD system, Cat#7126-B7) was immobilized on a CM5 chip (GE Healthcare, Cat#BR-1005-30) by amino coupling. Using a BiacoreT200 (GE Healthcare) instrument, the response values of different concentrations of the compound flowing through the surface of the CM5 chip were detected at 25°C.

[0069] ① pH pre-enrichment of VISTA protein: The chip used in the experiment was CM5. Human VISTA protein was diluted to 10 μg / ml with sodium acetate at pH 4.5, 5.0, and 5.5 respectively. 100 μl of each was prepared with a flow rate of 10 μl / min and injected successively for 180 s. 50 mM sodium hydroxide was used as the eluent, and 1x PBS-P+ (GE Healthcare, Cat#28-9950-84) was used as the buffer solution for the entire flow path system. Through the pre-enrichment experiment, it was determined that the condition of pH = 5.0 was the optimal coupling condition. Therefore, VISTA protein was diluted to 10 μg / ml with sodium acetate at pH = 5.0, and 200 μl was used for the formal coupling operation. The results are as Figure 1 .

[0070] ② Ligand coupling of VISTA protein: Human VISTA protein was diluted to 20 μg / ml with sodium acetate solution at the optimal pH = 5.0 and coupled to the Flow cell 2 sample channel of the CM5 chip by amino coupling. Flow cell 1 was a blank channel used for background subtraction. The maximum target coupling amount of VISTA protein was set to 10000 RU. 1x PBS-P+ was used as the buffer solution for the entire flow path system. After mixing EDC and NHS in a 1:1 ratio, it was injected into the system to activate the surface of the chip. VISTA protein flowed through at a flow rate of 10 μl / min for 600 s to fully bind to the surface of the CMS chip. Finally, ethanolamine was injected into the flow path to block the remaining ester groups on the surface of the chip. The results are as Figure 1As shown, the coupling on the surface of the display chip is 10375RU. The target coupling amount has been reached, that is, the result of successful protein coupling.

[0071] ③ Solvent correction: The running buffer for small molecule samples is 1xPBS-P+ containing 5% DMSO (Sigma, Cat#d8418). Mix the 4.5% and 5.8% DMSO stock solutions as shown in Table 1 below to prepare a 5% DMSO concentration calibration curve.

[0072] Table 1 Running buffer formulation

[0073] Serial number 1 2 3 4 5 6 7 8 4.5% DMSO (μl) 0 100 200 300 400 500 600 700 5.8% DMSO (μl) 700 600 500 400 300 200 100 0

[0074] ④ Affinity determination: Dilute the 0.4 mM small molecule stock solution 20-fold with 1.05x PBS-P+ to obtain a 20 μM small molecule in 5% DMSO, and 20 μM is used as the highest injection concentration. Then, use the prepared 5% DMSO running buffer to serially dilute the analyte concentration by half at least 5 concentration gradients (adjust the concentration gradient according to the actual sample affinity), and add a 0 concentration as a blank control. Inject the 1x PBS-P+ running buffer containing 5% DMSO at a flow rate of 10 μl / min, and set both the sample binding time and dissociation time to 60 s. Detect and process the data using the Biacore T200 Evaluation software. The experimental results show (as shown in Figure 2 and Figure 13 ), that baloxavir marboxil and its active metabolite baloxavir acid have a strong binding ability to human VISTA protein.

[0075] Example 2

[0076] CCK-8 evaluation of the cytotoxic effect of compounds on PBMC and Jurkat cells

[0077] Cell Counting Kit-8 (abbreviated as CCK-8) (Beyotime, Cat#C0038) is a kit that is widely used for rapid and highly sensitive detection of cell proliferation and cytotoxicity based on WST-8. The CCK-8 reagent contains WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt), which is a compound similar to MTT. In the presence of the electron-coupling reagent 1-methoxy-5-methylphenazinium sulfate dimethyl ester (1-Methoxy PMS), it can be reduced by dehydrogenases in mitochondria to formazan, an orange-yellow water-soluble product. The amount of formazan generated is proportional to the number of live cells. Therefore, this property can be used directly for cell proliferation and toxicity analysis. The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color.

[0078] When using the CCK-8 kit for operation, first adjust the density of the revived PBMC cells (Allcells, Cat#PB003F-C-10M) to 1x10 6 cells / ml, and adjust the density of Jurkat cells to 2x10 5 cells / ml. Inoculate 100 μl / well into a 96-well plate. At the same time, add 100 μl of the compound to each well to treat the cells. There are 3 replicate wells for each concentration. Control the final concentrations of baloxavir marboxil to be 0.78125 μM, 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM, and control the final concentrations of baloxavir acid to be 0.09765625 μM, 0.1953125 μM, 0.390625 μM, 0.78125 μM, 1.5625 μM, 3.125 μM, and 6.25 μM. After culturing the cells in a 37°C incubator for 48 h, discard the culture medium. Add 100 μl of fresh medium containing 10% CCK-8 to each well in the dark and continue culturing for 4 hours. Then shake on a microplate shaker for 5 minutes, and read the absorbance value at 450 nm with an enzyme-linked immunosorbent assay reader. The results are as Figure 3 shown that the compound baloxavir marboxil has no toxicity at 0 - 50 μM, as Figure 14 shown that the compound baloxavir acid has no toxicity at 0 - 6.25 μM.

[0079] Example 3

[0080] Establishment of a Jurkat T cell activation model for identifying the cell activity of compounds

[0081] PMA (Phorbol-12-myristate-13-acetate) and phytohemagglutinin (PHA), as a kind of phorbol ester, can jointly activate Jurkat T cells, stimulate the proliferation of T cells and the secretion of interleukin-2. When the system has inhibitory signals produced by VISTA protein during cell activation, the activation effect will be weakened. During the construction of the cell model, an agonist and VISTA protein are given simultaneously, and the activation of T cells is judged by analyzing the secretion of cytokines in the cell supernatant.

[0082] ① Jurkat cell activation experiment

[0083] Day 1: Coat the human VISTA protein solution in a 96-well plate. Dilute human VISTA protein (RDsystem, Cat#7126-B7) to 5 μg / ml and 10 μg / ml respectively using PBS. Add 100 μl of the protein solution to each well and coat overnight at 4°C.

[0084] Day 2: Aspirate the coated protein solution in the wells, wash twice with PBS, 200 μl per well. Add 100 μl of Jurkat cells (Cell Bank of the Chinese Academy of Sciences) to each well, 2 x 10 5 cells per well. Then add 100 μl of the mixture of PMA (Beyotime, Cat#S1819) and PHA (Sigma, Cat#L1668) to each well, controlling the final concentration of PMA to be 1 ng / ml and the final concentration of PHA to be 6 μg / ml. Place the 96-well plate in a 37°C cell culture incubator, and after 48 h, collect the cell supernatant and detect the secretion amount of IL-2 using an ELISA kit (Biolegend, Cat#431804) (the group settings are shown in Table 2). The results are as Figure 4 shown. Both 5 μg / ml and 10 μg / ml of human VISTA protein can inhibit the secretion of IL-2 by PMA-PHA-induced Jurkat T cells.

[0085] Table 2 Group settings for T cell activation experiment

[0086]

[0087] ② Compound activity detection experiment

[0088] Day 1: Coat the human VISTA protein solution in a 96-well plate. Dilute human VISTA protein to 5 μg / ml using PBS. Add 100 μl of the protein solution to each well and coat overnight at 4°C.

[0089] Day 2: Aspirate the coating protein solution in the wells, wash twice with PBS, 200 μL per well. First add 100 μL of Jurkat cells to each well, 2x10 5 cells per well. Add 100 μL of the mixture of PMA and PHA containing the test compound to the compound detection group, and at the same time control the final concentrations of baloxavir marboxil to be 10 μM, 25 μM and 50 μM. The final concentrations of baloxavir acid are 3.125 μM and 6.25 μM. Add 100 μL of the mixture of PMA and PHA to the control group. Add 100 μL of complete medium to the blank group. Control the final concentration of PMA to be 1 ng / ml and the final concentration of PHA to be 6 μg / ml among groups. Place the 96-well plate in a 37 °C cell culture incubator, and collect the cell supernatant after 48 h to detect the secretion amount of IL-2 (the group settings are shown in Table 3). The results are as Figure 5 shown. Baloxavir marboxil can reduce the secretion of IL-2 in PMA-PHA-induced Jurkat cells at 5 μM, 20 μM and 50 μM and shows a dose-dependence, as Figure 15 shown. Baloxavir acid can reduce the secretion of IL-2 in PMA-PHA-induced Jurkat cells at 3.125 μM and 6.25 μM and shows a dose-dependence, indicating that baloxavir marboxil and baloxavir acid promote the inhibitory effect of VISTA protein and are agonists of VISTA protein.

[0090] Table 3 Group settings for the experimental groups of compound cell activity

[0091]

[0092] Example 4

[0093] Establishment of a PBMC cell activation model for identifying the cell activity of compounds

[0094] The binding of Anti-CD3 to the T cell receptor (TCR) on T cells activates the TCR-related signaling pathway, and CD28, as a co-stimulatory molecule, can strengthen the TCR signal stimulation after binding to B7 of antigen-presenting cells (APCs). Therefore, anti-CD28 mimics the role of APCs, binds to CD28 and then enhances the TCR signal, thereby stimulating the proliferation of T cells and the secretion of cytokines. At the same time of cell activation, if there is an inhibitory signal generated by human VISTA protein in the system, the activation effect will be weakened. During the construction of the cell model, an agonist and VISTA protein are given simultaneously, and the activation status of T cells is judged by analyzing the secretion of cytokines in the cell supernatant.

[0095] ① PBMC cell activation experiment

[0096] Day 1: Coat the blank group with PBS in a 96-well plate. In the agonist group, dilute anti-human-CD-3 antibody (Biolegend, Cat#317326) and anti-human-CD28 antibody (Biolegend, Cat#302934) to 2.5 μg / ml with PBS. Keep the concentrations of anti-human-CD-3 antibody and anti-human-CD28 antibody unchanged in the inhibitor group. At the same time, set the human VISTA protein concentration to 2.5 μg / ml,

[0097] 5 μg / ml, and 10 μg / ml. When coating, add 100 μl of protein mixed solution (proteins are all diluted with PBS) to each well and coat at 4°C

[0098] for 18 hours.

[0099] Day 2: Aspirate the coated protein solution in the wells and wash twice with PBS, 200 μl per well. Add 100 μl of complete cell culture medium to both the control group and the blank group and pre-treat in an incubator at 37°C for 30 min. Then add 100 μl of resuscitated PBMC (Allcells, Cat#PB003F-C-10M) to each well, with 1 x 10 5 cells per well (group settings are shown in Table 4). Place the 96-well plate in a cell culture incubator at 37°C. After 48 hours, collect the cell supernatant and detect the secretion levels of IFN-γ, IL-2, and TNF-α using an ELISA kit (Biolegend). The results are as Figure 6 shown in A, B, and C. It shows that human VISTA protein concentrations of 2.5 μg / ml, 5 μg / ml, and 10 μg / ml can all inhibit the secretion of IFN-γ, IL-2, and TNF-α by PBMC cells induced by anti-human-CD-3 antibody and anti-human-CD28 antibody.

[0100] Table 4 Group settings for T cell activation experiment

[0101]

[0102] ② Compound activity detection experiment

[0103] Day 1: Coat the blank group with PBS in a 96-well plate. In the agonist group, dilute anti-human-CD-3 antibody and anti-human-CD28 antibody to 2.5 μg / ml with PBS. Keep the concentrations of anti-human-CD-3 antibody and anti-human-CD28 antibody unchanged in the inhibitor group and the compound group. When coating with a human VISTA protein concentration of 2.5 μg / ml, add 100 μl of protein mixed solution to each well and coat at 4°C for 18 hours.

[0104] Day 2: Aspirate the coating protein solution in the wells and wash twice with PBS, 200 μl per well. Add 100 μl of the test compound to each well in the compound detection group, and simultaneously control the final concentrations of baloxavir marboxil to be 10 μM,

[0105] 25 μM and 50 μM. The final concentrations of baloxavir acid are 3.125 μM and 6.25 μM. Add 100 μl of complete cell culture medium to both the control group and the blank group, and pre-treat in an incubator at 37 °C for 30 min. Then add 100 μl of the resuscitated PBMC (Allcells, Cat#PB003F-C-10M) cells to each well, with 1 x 10 5 cells per well (the group settings are shown in Table 5). Place the 96-well plate in a cell culture incubator at 37 °C. After 48 hours, collect the cell supernatant and detect the secretion levels of IFN-γ, IL-2, and TNF-α using an ELISA kit (Biolegend). The results are as Figure 7 shown in A, B, and C. Baloxavir marboxil can reduce the secretion of IFN-γ, IL-2, and TNF-α by PBMC cells induced by anti-human-CD-3 antibody and anti-human-CD28 antibody and shows a dose-dependent manner, indicating that the compound baloxavir marboxil promotes the inhibitory effect of VISTA protein and is an agonist of VISTA protein.

[0106] As Figure 16 shown in A, B, and C. Baloxavir acid can reduce the secretion of IFN-γ, IL-2, and TNF-α by PBMC cells induced by anti-human-CD-3 antibody and anti-human-CD28 antibody and shows a dose-dependent manner, indicating that the compound baloxavir acid promotes the inhibitory effect of VISTA protein and is an agonist of VISTA protein.

[0107] Table 5 Group settings for the compound cell activity experiment

[0108]

[0109]

[0110] Example 5

[0111] Establish a PBMC agonist model of human VISTA protein using the CFSE experiment to identify the effect of the compound on the proliferation of PBMC cells

[0112] The fluorescent dye CFSE is a new type of dye that can fluorescently label live cells. It can label live somatic cells. After CFSE enters live cells, it can covalently bind to intracellular proteins and release green fluorescence after hydrolysis.

[0113] CFSE is evenly distributed to daughter cells during cell division, resulting in a gradual decrease in fluorescence intensity. Based on this property, it can be used to detect cell proliferation viability by flow cytometry.

[0114] ① Establish a PBMC activation model of human VISTA protein with the CFSE experiment

[0115] Day 1: Coat the blank group with PBS in a 96-well plate. Dilute the anti-human-CD-3 antibody (Biolegend, Cat#317326) to 1 μg / ml with PBS in the activation group, and keep the concentration of the anti-human-CD-3 antibody unchanged in the inhibition group. Set the concentrations of human VISTA protein to 2.5 μg / ml, 5 μg / ml, and 10 μg / ml. The proteins are all diluted with PBS, and 100 μl of the protein mixture solution is added to each well during coating, and coated at 4°C for 18 hours (the group settings are shown in Table 6).

[0116] Day 2: First, prepare the CFSE dye stock solution. Take a tube of CFSE solid dye (5 mM, Biolegend, Cat#423801) and add 36 μl of anhydrous DMSO to prepare a stock solution with a concentration of 5 mM. After dissolution, mix well, aliquot, 2 μl per tube, and store in the dark at -20°C for later use; then perform cell staining. Take 1 μl of the CFSE dye stock solution, add PBS to 1 ml to prepare a CFSE dye working solution with a final concentration of 5 μM. Then take the resuscitated PBMC, centrifuge at 200x g at room temperature for 15 minutes, and discard the supernatant. Add 0.2 ml of the CFSE dye working solution to the cells, incubate at 37°C in the dark for 10 minutes, centrifuge at 200x g at room temperature for 15 minutes, and discard the supernatant. Resuspend the precipitate with 2 ml of fresh 1640 complete medium, incubate at room temperature in the dark for 10 minutes, adjust the cell density to 1x10 6 cells / ml for later use. Discard the solution in the plate from the previous day, wash twice with PBS, then add 100 μL of CFSE-stained PBMC to each well, and then add 100 μL of the medium, and then place it in a cell culture incubator for 120 hours. After culturing for 120 hours, collect the samples in 1.5 mL centrifuge tubes, centrifuge at 800x g at room temperature for 5 minutes, and discard the supernatant; resuspend the cells with 200 μL of flow cytometry staining solution, centrifuge at 800x g at room temperature for 5 minutes, and discard the supernatant, and repeat the washing twice. After resuspending the cells with 200 μL of flow cytometry staining solution (PBS + 2% FBS), perform flow cytometry using a BD C6 flow cytometer and perform data processing with FlowJo-v10. The experimental results are as Figure 8 shown. Human VISTA protein at 2.5 μg / mL, 5 μg / mL, or 10 μg / mL can inhibit the proliferation of PBMC

[0117] Table 6 Experimental group settings for activating PBMCs expressing human VISTA protein using CFSE

[0118]

[0119] ②Compound activity detection experiment

[0120] Day 1: Coat the blank group with PBS in a 96-well plate. Dilute anti-human-CD-3 antibody (Biolegend, Cat#317326) to 1 μg / ml with PBS for the agonist group, and keep the concentration of anti-human-CD-3 antibody unchanged for the inhibitor group. Set the concentrations of human VISTA protein at 2.5 μg / ml, 5 μg / ml, and 10 μg / ml. Dilute the protein with PBS and add 100 μl of the protein mixture solution to each well for coating at 4°C for 18 hours.

[0121] anti-human-CD-3 antibody (Biolegend, Cat#317326) to 1 μg / ml, and keep the concentration of anti-human-CD-3 antibody unchanged for the inhibitor group. Set the concentrations of human VISTA protein at 2.5 μg / ml, 5

[0122] μg / ml, 10 μg / ml. Dilute the protein with PBS and add 100 μl of the protein mixture solution to each well for coating at 4°C for 18 hours.

[0123] μg / ml, 10 μg / ml. Dilute the protein with PBS and add 100 μl of the protein mixture solution to each well. Coat at 4°C for 18 hours.

[0124] Day 2: First, prepare the CFSE stock solution. Add a tube of solid CFSE dye to 36 μl of anhydrous DMSO to prepare a stock solution with a concentration of 5 mM. Mix well after dissolution, aliquot 2 μl into each tube, and store in the dark at -20°C

[0125] protected from light for later use. Then, perform cell staining. Take 1 μl of the CFSE stock solution and add PBS to 1 ml to prepare a CFSE dye working solution with a final concentration of 5 μM. Take the resuscitated PBMCs, centrifuge at 200x g at room temperature for 15 minutes, and discard the supernatant. Add 0.2 ml of the CFSE dye working solution to the cells, incubate at 37°C in the dark for 10 minutes, centrifuge at 200x g at room temperature for 15 minutes, and discard the supernatant. Resuspend the pellet in 2 mL of fresh complete 1640 medium, incubate at room temperature in the dark for 10 minutes, count the cells, and adjust the cell density to 1x 10 6Reserve at [X] cells / ml. Discard the solution in the plate from the previous day, wash twice with PBS, add 100 μl of baloxavir marboxil at the specified concentration to each well, and control the final concentration of the compound to be 20 μM and 50 μM. Incubate at 37 °C for 30 minutes (group settings are shown in Table 7). Then add 100 μl of CFSE-stained PBMC to each well and culture it in a 37 °C cell culture incubator for 120 hours. After culturing for 120 hours, collect the samples in 1.5 mL centrifuge tubes, centrifuge at 800 x g at room temperature for 5 minutes, and discard the supernatant; resuspend the cells with 200 μl of flow cytometry staining solution, centrifuge at 800 x g at room temperature for 5 minutes, discard the supernatant, and repeat the washing twice. After resuspending the cells with 200 μL of flow cytometry staining solution, perform flow cytometry using a BD C6 flow cytometer and process the data with FlowJo-v10. The experimental results are as Figure 9 and Figure 16 shown. Baloxavir marboxil and baloxavir acid will exacerbate the inhibitory effect of 2.5 μg / mL of human VISTA protein on the proliferation of PBMC. It is shown that baloxavir marboxil and baloxavir acid are agonists of VISTA.

[0126] Table 7 Group settings for the experimental groups of compound cell activity

[0127]

[0128] Example 6

[0129] Establishment of an agonist model of Jurkat cells overexpressing full-length VISTA for identifying the targeting of compounds

[0130] First, Jurkat cells overexpressing full-length VISTA (Jurkat-VISTA-FL) and empty vector (Jurkat-EV) cells were constructed by lentiviral transfection (Br J Pharmacol, 2021, 178: 1445 - 1458). First, adjust the density of the two types of cells to 2 x 10 5 cells / ml and seed 100 μl / well in a 96-well plate. Add 100 μl of a mixture of PMA and PHA containing the compound to be tested to the compound detection group, and control the final concentration of baloxavir marboxil to be 10 μM, 25 μM, and 50 μM. The final concentration of baloxavir acid is 1.5625 μM, 3.125 μM, and 6.25 μM. Add 100 μl of a mixture of PMA and PHA to the control group. Add 100 μl of complete medium to the blank group. Control the final concentration of PMA to be 1 ng / ml and the final concentration of PHA to be 6 μg / ml among groups (group settings are shown in Table 8). Place the 96-well plate in a 37 °C cell culture incubator and collect the cell supernatant after 48 h to detect the inhibition rate of the compound on IL-2 in Jurkat-VISTA-FL and Jurkat-EV cells. The experimental results are asFigure 10 and Figure 17 As shown, it indicates that baloxavir marboxil and baloxavir acid are agonists targeting VISTA.

[0131] Table 8 Experimental group settings for compound cell targeting

[0132]

[0133] Example 7

[0134] Effect of the compound on the inhibition rate of IL-2 in CD4 + T cells of BALB / c wild-type (WT) and BALB / c VISTA knockout (KO) mice for identifying the targeting of the compound

[0135] Day 1: Coat anti-mouse-CD-3 antibody in 96-well plates respectively. The concentration of anti-mouse-CD-3 antibody in the agonist group and the compound experimental group is 2.5 μg / ml. The control group does not add anti-mouse-CD-3 antibody and only adds an equal volume of PBS (the group settings are shown in Table 9). The antibody is diluted with PBS, and 100 μl of anti-mouse-CD-3 solution is added to each well during coating, and it is coated overnight at 4°C.

[0136] Day 2: First, use the EasySep Mouse CD4 + T Cell Isolation Kit to isolate total mouse CD4 + T cells from the spleens of BALB / C wild-type (wildtype, WT, Shanghai Model Organisms Center, Inc.) and BALB / C VISTA knockout (knockout, KO, Shanghai Model Organisms Center, Inc.) mice. It is required that the purity of CD4 + T cells reaches more than 90% before it can be used for the experiment. The cell purity results are as Figure 11 shown. Then aspirate the coated anti-mouse-CD-3 antibody in the wells, wash 2 times with PBS, 200 μl per well. Then adjust the density of the extracted CD4 + T cells to 1x10 6 cells / ml, and inoculate 100 μl / well into 96-well plates. Then put the cells into a 37°C cell incubator and incubate for 30 min, and then add the compound. Control the final concentration of the compound to be 10 μM. After adding the compound, put the 96-well plates into a 37°C cell incubator. After 72 h, collect the supernatant to detect the inhibition rate of the compound on WT CD4 + T and VISTA KO CD4 + T cell IL-2, and the results are asFigure 12 and Figure 18 As shown, it indicates that baloxavir marboxil and baloxavir acid are agonists targeting VISTA.

[0137] Table 9 Experimental Group Settings for Compound Cell Targeting

[0138]

[0139] Example 8

[0140] Therapeutic Effect of Baloxavir Marboxil on Mice with cGVHD-like Systemic Lupus Erythematosus

[0141] ① Test animals: CB6F1 mice and BALA / c mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., female, 48 - 56 days old; raised in a conventional environment with free access to food and water.

[0142] ② Drug preparation: The administration dose of baloxavir marboxil was 50 mg / kg, dissolved in an aqueous solution of 0.5% sodium carboxymethylcellulose, and cyclophosphamide (CTX) was used as the positive control group, with CTX dissolved in physiological saline to prepare a concentration of 25 mg / kg.

[0143] ③ Animal grouping, model establishment and drug administration: The mice were randomly divided into a blank control group (Control), a model group (Model) and a compound group. Then, the mice in the model group and the compound group were established as cGVHD-like lupus mouse models. Female BALB / c mice at 6 - 8 weeks old and (C57BL / 6J×BALB / c)F1 hybrid female mice were selected. Under sterile conditions, the spleen, lymph nodes (mesenteric, inguinal, cervical) and thymus of BALB / c mice were removed to prepare lymphocyte suspensions. The cell density was adjusted to 1x10 8 cells / ml, and 200 μl was injected into the Fl generation mice through the tail vein each time, at 1, 4, 7, and 10 days respectively. The control group was injected with physiological saline to induce the lupus model. After 6 weeks, the control group and the model group were given an aqueous solution of 0.05% sodium carboxymethylcellulose, and the compound group was given baloxavir marboxil by gavage twice a day (50 mg / kg) for a dosing period of four weeks.

[0144] ④ Determination of serum anti-dsDNA antibody, blood urea nitrogen and creatinine levels: The ELISA kit was used to determine the anti-dsDNA antibody in the serum of each group of mice; the blood urea nitrogen and creatinine detection kits (Nanjing Jiancheng) were used to measure the blood urea nitrogen and creatinine levels in the serum of mice. The results are as Figure 19 shown. The levels of anti-dsDNA antibody, blood urea nitrogen and creatinine in the model group were higher than those in the control group, and the levels of anti-dsDNA antibody, blood urea nitrogen and creatinine in the baloxavir marboxil group were significantly lower than those in the model group.

[0145] ⑤ Urinary protein content determination: The urinary protein content in the urine of mice in each group was measured using a urinary protein detection kit. The results are as Figure 20 shown. The urinary protein in the model group was higher than that in the control group, and the urinary protein level in the baloxavir marboxil group was significantly lower than that in the model group.

[0146] ⑥ Spleen weight measurement: After the administration was completed, the spleens of the mice were taken and weighed. As shown in Figure 21, the spleen weight of the mice in the model group was higher than that in the control group, and the spleen weight level in the baloxavir marboxil group was significantly lower than that in the model group.

[0147] ⑦ RT-PCR: Total RNA was extracted using TRIzol reagent, and mRNA was transcribed into cDNA using PrimeScript RT Master Mix. RT-PCR analysis was used to measure the mRNA levels of IL-6, MCP-1, TNF-α, TGF-β, and KIM-1. The results are as Figure 22 shown. The levels of IL-6, MCP-1, TNF-α, TGF-β, and KIM-1 in the model group were higher than those in the control group, and the levels of IL-6, MCP-1, TNF-α, TGF-β, and KIM-1 in the baloxavir marboxil group were significantly lower than those in the model group.

[0148] ⑧ HE staining: After the administration of the mice was completed, the kidneys of the mice were taken and immersed in 4% paraformaldehyde, and then paraffin-embedded. Staining was performed with hematoxylin and eosin (HE). The HE staining procedure is as follows: Deparaffinization of paraffin sections to water: The sections were successively placed in xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and then washed with tap water. Hematoxylin staining: The sections were immersed in hematoxylin staining solution for 3 - 5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with bluing solution, and rinsed with running water. Eosin staining: The sections were successively dehydrated in 85% and 95% gradient ethanol for 5 min each, and then stained in eosin staining solution for 5 min. Dehydration and mounting: The sections were successively placed in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and then mounted with neutral gum. Microscopic examination and image acquisition and analysis were performed. The HE results are as Figure 23 shown. The results showed that the degree of kidney injury and the degree of inflammatory cell infiltration in the model group were greater than those in the control group, and there were significant differences. The degree of kidney injury and the degree of inflammatory cell infiltration in the baloxavir marboxil group were significantly improved compared with those in the model group.

[0149] ⑨ Detection of the deposition of immune complexes in the kidneys by immunofluorescence: After the mice were administered the drugs, their kidneys were taken and made into frozen sections with a thickness of 5 - 7 μm, and fixed with acetone. The deposition of IgG in the kidney tissues was detected by direct immunofluorescence. First, the frozen sections were taken out and dried at room temperature for 30 min, then blocked with 5% BSA for 1 hour, and then rinsed 3 times with PBST for 5 min each time; the sections were incubated overnight at 4°C with goat anti-mouse IgG conjugated with Alexa Fluor 488 (1:1000), and then the next day, they were rinsed 3 times with PBST for 5 min each time to wash away the unbound goat anti-mouse IgG. The sections were stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min, rinsed with PBST again, and observed under a fluorescence inverted microscope after mounting. The immunofluorescence results are as Figure 24 shown. The results showed that there was a large amount of deposition of immune complex IgG in the glomeruli and mesangium of the kidneys in the model group, while the deposition of immune complex IgG was significantly reduced in the baloxavir marboxil group.

[0150] ⑩ Statistical analysis: Data such as body weight and urinary protein were processed using GraphPad Prism software, and one-way analysis of variance (One-Way ANOVA) for comparing the means of multiple samples was used.

[0151] In summary, after treatment with the compound baloxavir marboxil, the urinary protein level of lupus mice decreased significantly, and the levels of blood urea nitrogen, creatinine, and anti-dsDNA antibody in peripheral blood also decreased significantly. At the same time, the pathological damage of the kidneys in lupus mice was significantly reduced, and the deposition of inflammatory factors IL-6 / MCP-1 / TNF-α / TGF-β / KIM-1 and immune complex IgG in the kidneys also decreased. It indicates that the compound baloxavir marboxil has a significant effect in the treatment of systemic lupus erythematosus such as lupus nephritis.

[0152] Example 9

[0153] Effect of baloxavir marboxil on a mouse asthma model induced by OVA

[0154] ① Experimental animals: BALA / c mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., female, 48 - 56 days old; they were raised in a conventional environment with free access to food and water.

[0155] ② Drug preparation: The administration dose of baloxavir marboxil was 50 mg / kg, dissolved in an aqueous solution of 0.5% sodium carboxymethylcellulose, and dexamethasone was used as the positive control group. Dexamethasone was dissolved in physiological saline to prepare a concentration of 1 mg / kg.

[0156] ③ Animal grouping, model establishment, and drug administration: Mice were randomly divided into a normal control group (Control), a model group (Model), a baloxavir marboxil group, and a dexamethasone group. Except for the normal group, asthma models were established in the other groups. On days 0 and 5 of the experiment, mice were sensitized by intraperitoneal injection of 0.2 mL of the sensitizing solution (containing 20 μg of OVA and 4 mg of aluminum hydroxide adjuvant). Mice in the normal group were injected with normal saline instead of the sensitizing solution, with the same injection method, injection site, and dose. From days 12 to 18, the mice were placed in a closed nebulizer and challenged with a 5% ovalbumin solution for 30 min. During the challenge, normal saline was used instead of the challenge solution for the normal group. Starting from day 12 for 7 consecutive days, 0.2 mL of the drug at a dose of 50 mg / kg was administered by gavage to the mice in the baloxavir marboxil group 60 min before atomization, and baloxavir marboxil was administered again 6 hours after atomization. Mice in the dexamethasone group were injected intraperitoneally with 0.2 mL of the drug at a dose of 1 mg / kg. Mice in the normal group and the model group were given the same dose of 0.5% CMC-Na by gavage. The mice were sacrificed 24 hours after the last challenge to study the anti-asthmatic effect of baloxavir marboxil.

[0157] ④ Collection of blood and bronchoalveolar lavage fluid

[0158] Twenty-four hours after challenge, the mice were weighed, sacrificed by eye blood collection, and left to stand at room temperature for 2 h. Then, they were centrifuged at 3000 rpm for 15 min at 4°C to extract serum, which was aliquoted and stored at -80°C for the determination of IgE. The chest and abdominal cavities were disinfected with 75% alcohol cotton balls, the chest cavity was exposed, the mice were cut open in the middle of the abdomen and then cut all the way up to the neck along the middle. The right lung was ligated with a thin thread, the neck tissue was carefully separated, the tissue around the trachea was separated, and a pair of forceps was passed under the trachea. A 1 mL syringe was used to slowly inject pre-cooled normal saline solution into the lung for a total of 3 light lavages. After standing for 30 s, it was aspirated back and forth 3 times slowly, and the lavage fluid was collected in a 1.5 mL EP tube. The bronchoalveolar lavage fluid was collected. The lavage fluid was placed on ice. The lavage fluid was centrifuged in a 4°C centrifuge, and the samples were centrifuged at 1200 r / min at 4°C for 7 min. After centrifugation, the supernatant was aliquoted and stored in a -80°C refrigerator for the determination of cytokines IL-4, IL-10, and IFN-γ.

[0159] ⑤ Preparation of lung tissue pathological sections

[0160] After bronchoalveolar lavage, the right upper lobe of the lung was excised and placed in paraformaldehyde fixative for HE and PAS staining. The HE staining procedure is as follows: Deparaffinize the paraffin sections to water: sequentially place the sections in xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water. Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 - 5 min, wash with tap water, differentiate with differentiating solution, wash with tap water, blue with blueing solution, and rinse with running water. Eosin staining: Immerse the sections in gradient alcohols of 85% and 95% for dehydration for 5 min each, and stain in eosin staining solution for 5 min. Dehydration and mounting: sequentially place the sections in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and mount with neutral balsam. Microscopic examination and image acquisition and analysis.

[0161] The PAS staining procedure is as follows: Deparaffinize the paraffin sections to water: sequentially place the sections in xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water; Immerse the sections in PAS staining solution B for 10 - 15 min, wash with tap water, and wash twice with distilled water; Immerse the sections in PAS staining solution A for impregnation for 25 - 30 min, protected from light, and rinse with running water for 5 min; Immerse the sections in PAS staining solution C for 30 s, wash with tap water, differentiate with hydrochloric acid aqueous solution, wash with tap water, blue with ammonia water, and rinse with running water; Dehydration and mounting: sequentially place the sections in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene for 5 min - xylene II for 5 min for clearing, and mount with neutral balsam. Microscopic examination and image acquisition and analysis. It can be seen from Figure 25 that the HE staining results of lung tissue showed that in the control group, the bronchial and alveolar structures were clear, the bronchial wall was not thickened or edematous, and there was no inflammatory infiltration around the bronchi; in the asthma model group, the alveolar structure was damaged, the bronchial wall was significantly thickened, showing edema, bronchial stenosis, and a large number of inflammatory cell infiltrations around, mainly eosinophils; in the dexamethasone group, there were occasional damaged cavities in the bronchi and alveoli, and the conditions of bronchial wall thickening and edema and inflammatory infiltration were alleviated compared with the model group; in the baloxavir marboxil group, the damaged phenomena of the bronchi and alveoli were alleviated, the conditions of bronchial wall thickening and edema were significantly alleviated compared with the model group, and the airway stenosis and inflammatory infiltration conditions were effectively improved. It can be seen from Figure 26 that the PAS staining results of lung tissue showed that in the control group, there was no goblet cell hyperplasia and mucus production, in the model group, there was a large amount of goblet cell hyperplasia and mucus production, in the dexamethasone group, there was less goblet cell hyperplasia and mucus production, and in the baloxavir marboxil group, there was a small amount of goblet cell hyperplasia and mucus production.

[0162] ⑥ Enzyme-linked immunosorbent assay was used to detect IgE, IL-4, IFN-γ and IL-10

[0163] According to the instructions of the Biolegend ELISA kit, the expressions of immunoglobulin Ig-E in the sera of mice in each group and IL-4, IFN-γ, and IL-10 in the bronchoalveolar lavage fluid were detected. The specific experimental steps are as follows: On the first day, 100 μL of diluted capture antibody was incubated overnight at 4 °C on the coated plate; on the second day, after washing 4 times, 200 μL of blocking solution was added and incubated for 1 hour. After washing the plate 4 times, 100 μL of the sample to be tested or standards of different concentrations was added and incubated at 37 °C for 120 min. The plate was washed 4 times, 100 μL of biotinylated antibody working solution was added, and incubated at 37 °C for 60 min. The plate was washed 5 times, 100 μL of enzyme conjugate working solution was added, and incubated in the dark at 37 °C for 30 min. The plate was washed 5 times, 100 μL of chromogenic substrate was added, and incubated in the dark at 37 °C for 15 - 20 min. Finally, 100 μL of stop solution was added, and then the absorbance values of each well at wavelengths of 450 nm and 570 nm were measured immediately. The concentrations of each cytokine were calculated using the plotted standard curve. IgE is a type of immunoglobulin, mainly related to allergic diseases, and is produced by Figure 27 It can be seen that the IgE in the model group was significantly higher than that in the control group, while the dexamethasone group and the baloxavir marboxil group could significantly reduce the content of IgE in the sera of mice. IL-4 can drive IgE synthesis. Interferon-γ (IFN-γ) produced by T helper cell 1 (Th1) cells has an inhibitory effect on the production of IgE. From Figure 28 it can be seen that the IL-4 in the model group was significantly higher than that in the control group, while the dexamethasone group and the baloxavir marboxil group could significantly reduce the content of IL-4 in the sera of mice. The IFN-γ and IL-10 in the model group were significantly lower than those in the control group, while the dexamethasone group and the baloxavir marboxil group could significantly increase the content of IFN-γ and IL-10 in the sera of mice. In summary, baloxavir marboxil at 50 mg / kg could significantly reduce the level of IL-4 and increase the levels of IFN-γ and IL-10.

[0164] ⑦ Detection of IL-6, MCP-1, Arg-1, and Ym-1 by RT-qPCR

[0165] Extraction of total RNA: Take the frozen lung tissue sample and cut it on an ice box. Take 15 mg of lung tissue, cut it into pieces with a small knife and put it into a 2 mL centrifuge tube. Add 1 ml of TotalI RNA Extractor, 2 small magnetic beads and 1 large magnetic bead to the taken sample, place it on ice, and process it with a homogenizer at 60 Hz for 60 s, twice. Make it pulverized and lysed. Place the lysed sample on ice for 10 min to completely separate the nuclear protein from the nucleic acid. Add 0.2 ml of chloroform, shake vigorously for 15 sec, place it on ice for 10 min, and centrifuge at 12000 rpm at 4℃ for 10 min. Aspirate 300 uL of the upper aqueous phase and transfer it to a clean 1.5 mL centrifuge tube. Add an equal volume of pre-cooled isopropanol, mix well with a machine oscillator, and place it on ice for 10 min. Centrifuge at 12,000 rpm at 4℃ for 10 min, discard the supernatant, and a white precipitate can be seen. Add 1 ml of 75% ethanol (anhydrous ethanol: DEPC water = 3:1) to wash the precipitate. Centrifuge at 12,000 rpm at 4℃ for 10 min. Discard the supernatant. After a short centrifugation, aspirate the supernatant with a pipette gun, invert the EP tube on the absorbent paper and air-dry it for 5 - 10 min. Add 20 uL of RNase-free ddH2O (DEPC water) to fully dissolve the RNA, and measure the concentration of the RNA sample and A260 / A280 with nona.

[0166] Reverse transcription: After measuring the concentration of the RNA sample, dilute it to a loading amount of 2 μg. First, add RNA, DEPC water and μL of 4×gDNA wiper Mix, centrifuge briefly to mix evenly, place it in a reverse transcription instrument at 42℃ for 2 min, take out the enzyme-free 8-well tube and add 5×HiScript II qRT SuperMix IIa solution, centrifuge briefly and mix evenly with a pipette gun and place it in the reverse transcription instrument, select 50℃ for 15 min, and then react at 85℃ for 5 s to obtain cDNA. It can be used immediately or stored at -20℃. For long-term storage, it should be aliquoted and stored at -80℃.

[0167] Real-time quantitative PCR: Prepare the following mixed solution in a 96-well tube, seal it with a sealing film, and the reaction system is 10 uL.

[0168] Table 7 Reaction system

[0169]

[0170] Figure 29 It can be seen that baloxavir marboxil can significantly reduce the mRNA expression of IL-6, MCP-1, Arg-1 and Ym-1.

[0171] ⑧ Detection of pSTAT3 and STAT3 protein expression by Western blotting

[0172] Total protein extraction: Weigh 15 mg of lung tissue into a 2 mL centrifuge tube, add 2 small steel beads and 1 large steel bead, then add 300 μL of RIPA lysis buffer and grind twice (60 HZ, 60 s), lyse on ice for 20 min, centrifuge at 12,000 rpm at 4 °C for 15 min, aspirate the supernatant into a 1.5 mL centrifuge tube, take 2.4 μL of the sample for protein quantification, and mix the remaining sample with loading buffer and place it in a 95 °C metal bath for 10 min to denature it. Centrifuge at 12,000 rpm for 1 min. Calculate the loading volume according to the concentration, with each sample containing 40 μg of protein.

[0173] Western Blot: Prepare separating gel and stacking gel with corresponding concentrations. After adding sufficient Running Buffer, add protein samples. Use low-voltage constant-voltage electrophoresis (60 V, 30 min) when running on the upper gel, and use high-voltage constant-voltage electrophoresis (about 120 V, 1 h) when the loading buffer enters the lower gel. Stop electrophoresis when the bromophenol blue reaches near the bottom of the gel and proceed with membrane transfer. Set the membrane transfer current to 200 mA and the membrane transfer time to 120 minutes. After membrane transfer is completed, immediately place the membrane into the pre-prepared TBST to wash off the membrane transfer solution on the membrane. Add TBST to the glass plate, cut the bands of the required proteins, place the cut bands into a blocking box, add blocking solution, and slowly shake on a shaker to block at room temperature for 1 h. Aspirate all the blocking solution, immediately add the diluted primary antibody, and slowly shake and incubate overnight at 4 °C. Recover the primary antibody, add TBST to wash the membrane, and then dilute the secondary antibody with BSA dilution solution. Aspirate all the washing solution, immediately add the diluted secondary antibody, and slowly shake and incubate at room temperature on a side-swing shaker for one hour. Recover the secondary antibody, add TBST, and wash the membrane. Place the band on the exposure machine, use a pipette to add the exposure solution dropwise onto the band, and wait for 1 min before exposure. Figure 30 It can be seen that the expression of p-STAT3 protein in the model group was significantly higher than that in the control group, and baloxavir marboxil could significantly reduce the expression of p-STAT3 protein. It is indicated that baloxavir marboxil may regulate murine asthma by affecting the expression of STAT3 protein.

[0174] In summary, after treatment with the compound baloxavir marboxil, the pathological conditions of the lungs of asthmatic mice were significantly improved, the levels of IgE and IL-4 in the serum of mice were significantly reduced, the levels of IFN-γ and IL-10 were increased, the mRNA expressions of IL-6, MCP-1, Arg-1, and Ym-1 in the lungs could be significantly reduced, and the expression of p-STAT3 protein could be significantly reduced, thus alleviating the symptoms of murine asthma.

[0175] Example 10

[0176] Therapeutic effect of the compound baloxavir marboxil on a murine psoriasis model

[0177] ① Selection of experimental animals: BALB / C mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., female, 48 - 56 days old; raised in a conventional environment with free access to food and water.

[0178] ② Drug preparation: The administration dose of baloxavir marboxil was 50 mg / kg, dissolved in an aqueous solution of 0.5% sodium carboxymethylcellulose.

[0179] ③ Animal grouping, modeling and administration: The mice were randomly divided into a blank control group (Control), a model group (Model), and a compound group according to body weight. The back hair was removed with a hair clipper to expose a skin area of 2 cm × 3 cm. 5% imiquimod (IMQ) cream (local dose 62.5 mg) was administered to the right ear and back every day. Toll-like receptors play an important role in the occurrence and development of psoriasis. Imiquimod is a Toll-like receptor agonist and can be used for psoriasis modeling. Modeling was performed in the morning and administration in the afternoon, and the experimental period was 7 days. The mice were weighed, photographed, the thickness of the right ear of each mouse was measured, and the right ear and back of the mice were observed every day. In order to score the severity of skin inflammation in mice, an objective scoring system (Psoriasis Area and Severity Index; PASI) was developed based on the clinical psoriasis area and severity index. Erythema and scaling were independently scored from 0 to 4 (0: asymptomatic; 1: mild; 2: moderate; 3: severe; 4: extremely severe). The results were as Figure 31 shown. Compared with the ear thickness and degree of erythema in the model group, the ear thickness and degree of erythema in the control group and the administration group were significantly reduced.

[0180] ④ HE staining: Starting from the application of imiquimod cream for modeling, the mice were photographed and the right ear and back of the mice were observed every day, and the thickness of the right ear of each mouse was measured every day. The measurement results of ear thickness were as Figure 21 shown. The skin specimens of the back and right ear of the mice were immersed in 4% paraformaldehyde and embedded in paraffin; stained with hematoxylin and eosin (HE); the quantitative results of HE were as Figure 32 shown. The results showed that the acanthosis thickness in the model group was greater than that in the control group, and there was a significant difference (P < 0.05). The acanthosis thickness in the compound group was significantly reduced compared with that in the model group.

[0181] ⑤ Statistical analysis: Data such as HE staining and right ear thickness were processed using GraphPad Prism software, and one-way analysis of variance (One-Way ANOVA) was used for multiple groups.

[0182] In summary, after treatment with baloxavir marboxil, the ear thickness, skin and ear damage degree of psoriasis mice were significantly reduced. It indicates that the compound baloxavir marboxil has a significant therapeutic effect on psoriasis.

Claims

1. Use of baloxavir marboxil or baloxavir acid or a pharmaceutically acceptable salt thereof as a VISTA agonist in the preparation of a medicament for preventing or treating an autoimmune disease; the autoimmune disease is lupus erythematosus, asthma or psoriasis.

2. The use according to claim 1, characterized in that the lupus erythematosus includes: cutaneous lupus erythematosus, systemic lupus erythematosus or drug-induced lupus erythematosus; the asthma includes: allergic asthma, non-allergic asthma, late-onset asthma, asthma with fixed airflow limitation, severe asthma, refractory asthma or obesity-related asthma; the psoriasis is plaque psoriasis, generalized pustular psoriasis, erythrodermic psoriasis or psoriatic arthritis.

3. Use of a pharmaceutical composition in the preparation of a medicament for preventing or treating an autoimmune disease, the pharmaceutical composition containing baloxavir marboxil or baloxavir acid or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient and a pharmaceutically acceptable carrier; the autoimmune disease is lupus erythematosus, asthma or psoriasis.

4. The use according to claim 3, characterized in that the dosage form of the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

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