Application of HGF receptor inhibitor in preparation of medicine for treating / preventing myasthenia gravis

Abstract

The invention relates to the technical field of biomedicine, discloses application of an HGF receptor inhibitor in preparation of a medicine for treating / preventing myasthenia gravis, provides a novel treatment medicine for the myasthenia gravis, and innovatively discovers that the HGF receptor inhibitor can be used for treating / preventing the myasthenia gravis by regulating body inflammation and relieving the progress, clinical phenotype and pathological phenotype of MG diseases. The myasthenia gravis disease is treated.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of HGF receptor inhibitors in the preparation of drugs for the treatment / prevention of myasthenia gravis. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Myasthenia gravis (MG) is an autoimmune disease whose core pathological mechanism involves impaired binding of autoantibodies produced by abnormal activation of autoreactive B cells to acetylcholine receptors or function-related molecules on the postsynaptic membrane of the neuromuscular junction (NMJ). Although it remains a rare disease, its incidence is increasing annually, and approximately 20% of patients progress to myasthenic crisis within two years, which is life-threatening.

[0004] Current treatments for myasthenia gravis involve immunomodulatory drugs, but these drugs (such as glucocorticoids and cyclophosphamide) have limited efficacy and significant side effects. Therapies targeting B cell markers such as CD20 / CD19 cannot cover progenitor cells, some plasmablasts, and long-lived plasma cells, and are insufficient for patients with AChR antibody positivity. Complement inhibitors mainly act on downstream processes, failing to eliminate abnormally activated B cells or correct immune imbalances, while also increasing the risk of infection.

[0005] Therefore, new therapeutic drugs are urgently needed. Summary of the Invention

[0006] The purpose of this invention is to provide a new treatment for myasthenia gravis, addressing the current lack of such treatments.

[0007] The technical solution of the present invention is as follows: This invention provides the use of HGF receptor inhibitors in the preparation of drugs for the treatment / prevention of myasthenia gravis.

[0008] According to a preferred embodiment, the HGF receptor inhibitor includes PF04217903, SU11274, Crizotinib, Cabozantinib, ARQ197, XL880, INC280, MGCD265, or ASP3026.

[0009] According to a preferred embodiment, the drug is an injection, tablet, powder, granule, pill, capsule, oral liquid, ointment, cream, or spray.

[0010] According to a preferred embodiment, the dosage of the HGF receptor inhibitor is 40 mg / kg.

[0011] According to a preferred embodiment, the drug further includes one or more pharmaceutically acceptable excipients.

[0012] According to a preferred embodiment, the excipients include diluents, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, and sustained-release agents commonly used in the pharmaceutical field.

[0013] Compared with existing technologies, the advantages of this invention are: 1. Application of HGF receptor inhibitors in the preparation of drugs for the treatment / prevention of myasthenia gravis: This invention, through oral administration of the HGF receptor inhibitor PF04217903 to an EAMG rat model, found that HGF inhibitors can alleviate the progression, clinical phenotype, and pathological phenotype of MG disease by regulating inflammation in the body; and treat myasthenia gravis. Attached Figure Description

[0014] Figure 1 In the middle section, A represents the relationship between protein expression and the onset of myasthenia gravis (MG) as shown by the unadjusted Cox proportional hazards regression model; B represents the relationship between protein expression and the onset of myasthenia gravis (MG) as shown by the age- and sex-adjusted Cox proportional hazards regression model. Figure 2 A in the diagram is a Venn diagram visualizing the intersection between disease-related proteins and druggable target proteins; B is a Venn diagram visualized using the Clump method (r 2 =0.2) Perform Mendelian randomization analysis; Figure 3 In the middle, A represents the use of the Clump method (r 2 =0.001) was used for Mendelian randomization analysis; B is a forest plot showing the relationship between hepatocyte growth factor (HGF) and myasthenia gravis, based on the results of the UK Biobank cohort study and the Mendelian randomization study (in the UK Biobank Cox proportional hazards model, the effect size is expressed as the hazard ratio (HR), reflecting the correlation of the event occurrence time; in the MR analysis of drug targets, the effect size is expressed as the odds ratio (OR), reflecting the causal relationship under the MR assumption). Figure 4The middle section validates plasma HGF levels in myasthenia gravis (MG) patients in a cross-sectional cohort and explores the relationship between HGF and MG prognosis. A represents plasma HGF levels in healthy controls, ocular MG, and extensive MG patients; B represents plasma HGF levels in healthy controls, MG patients with thymic abnormalities, and MG patients without thymic abnormalities; C represents the ROC curve and AUC value of HGF plasma levels used to distinguish MG from healthy controls; D represents the diagnostic performance of HGF plasma levels in MG diagnosis, compared to healthy controls; E represents the correlation between HGF plasma levels and MG-specific activities of daily living (ADL) scores; F represents the correlation between HGF plasma levels and MG comprehensive score (MGC); G represents the relationship between high / low plasma HGF levels and future all-cause mortality in MG patients; H is a forest plot illustrating the relationship between plasma HGF levels and future all-cause mortality in MG, calculated using a multiple Cox proportional hazards regression model (Model 1: unadjusted; Model 2: adjusted for age and sex; Model 3: adjusted for age, sex, height, and weight); I is a schematic diagram illustrating the relationship between HGF and the onset and progression of MG. p<0.05, p<0.01, p<0.001; Data are expressed as mean ± standard deviation and compared by one-way ANOVA; Figure 5 The figures show the effects of PF04217903 intervention on EAMG; Figure A shows the changes in rat body weight in each group; B shows the time course of clinical severity scores in each group; C shows the bar chart of clinical scores in each group 28 days after the second immunization; D shows the bar chart of grip strength assessment in each group 28 days after the second immunization; E shows muscle MAC immunofluorescence staining; F shows the statistical analysis of AChR antibody titers; G shows the statistical analysis of C5b-9 fluorescence intensity; H shows the flow cytometry analysis of Th1 cells (CD4+IFNγ+); I shows the quantitative analysis of Th1 cell flow cytometry results; J shows the flow cytometry analysis of Th17 cells (CD4+IL17+); and K shows the quantitative analysis of Th17 cell flow cytometry results. All experiments were performed using 6 biological replicates and 3 technical replicates. p<0.05, p<0.01, p<0.001; data are expressed as mean ± standard deviation and compared by one-way ANOVA. Detailed Implementation

[0015] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0017] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0018] Example 1: Screening for novel biomarkers and therapeutic targets for myasthenia gravis (MG) (1) Research Overview: To develop novel biomarkers and therapeutic targets for myasthenia gravis (MG), we combined prospective cohort studies, Mendelian randomization analysis of drug targets, and cross-sectional cohort studies to identify and validate potential pathogenic molecules and therapeutic targets for MG, discovering the potential of HGF as a diagnostic biomarker for MG. We validated the potential of HGF inhibitors as therapeutic agents for MG by intervening with an experimental autoimmune myasthenia gravis (EAMG) model.

[0019] (2) Prospective proteomics cohort and Mendelian randomization revealed a significant correlation between HGF and MG. This prospective cohort study utilized data from the UK Biobank (UKB), a large-scale population-based biobank containing over 500,000 participants. Participants were aged 40 to 69 years and recruited from 22 recruitment centers in the UK between 2006 and 2010. The UKB study received ethical approval from the Northwest Multicenter Research Ethics Committee (application number 103677). All participants provided written informed consent at baseline. Diagnosis of myasthenia gravis was recorded using the International Classification of Diseases, 10th Revision (ICD-10) coding system, with the diagnostic code G70.0. In the analysis of myasthenia gravis onset, follow-up was up to the earliest date of diagnosis, death, or last available hospital / GP record (March 1, 2023). In the analysis of myasthenia gravis prognosis, follow-up was up to the date of death, crisis, or last available hospital / GP record (March 1, 2023).

[0020] In the UK Biobank (UKB), 53,021 participants had proteomics data (2,923 proteins) at baseline. After excluding 14 individuals with myasthenia gravis (MG) at baseline, 6,704 individuals with more than 50% missing data at baseline, and 12 individuals with more than 20% missing data, 46,303 participants (2,911 proteins) met the analysis criteria. During a median follow-up of 14.1 years, 43 participants developed MG.

[0021] Measuring HGF levels in each individual presents significant challenges. We developed a computational modeling approach utilizing routine blood test parameters (including complete blood cell counts and biochemical markers) from 46,303 participants in the UK Biobank. Predictive algorithms were constructed using various machine learning techniques to estimate HGF expression levels. We used a total of 63 data points, including 31 blood cell count data points (e.g., white blood cell count, red blood cell count, etc., from Biosample > Blood Tests > Blood Cell Counts), 28 blood biochemistry data points (e.g., blood calcium, cholesterol, etc., from Biosample > Blood Tests > Blood Biochemistry), and age, sex, height, and weight data from the UK Biobank. After median-based imputation using the "zoo" package (version 1.8-12), data standardization, and normalization were performed, and the data were split into a 70% training set and a 30% validation set for computation. We constructed several machine learning models to predict plasma HGF levels, including a linear model, an xgboost model (eta: 0.1, 0.3; gamma: 0.1, 0.2, 0.3), a GBM model (Nhidden: 3, 5, 10), an SVM linear model (cost: 1, 3, 5, 10; gamma: 0.01, 0.1, 1), and a decision tree model. All models were constructed using the "lm" function, xgboost (version 1.7.7.1), gbm (version 2.2.2), e1071 (version 1.7-14), and rpart (version 4.1.23) packages. When calculating the predictive performance of the models, the mean squared error (MSE) and the correlation coefficient between predicted and true values ​​were used. A smaller MSE value and a higher correlation coefficient indicate better regression model performance.

[0022] To explore the relationship between plasma protein expression levels and future myasthenia gravis (MG) incidence, we analyzed standardized protein expression levels (2911 plasma proteins) and the likelihood of future MG using a Cox proportional hazards regression model. We constructed an unadjusted model (Model 1), an age- and sex-adjusted model (Model 2), and a model adjusted for age, sex, height, and weight (Model 3).

[0023] Cox proportional hazards regression analysis was performed using the coxph function in the survival package (version 3.5-8) for a population that included individuals without myasthenia gravis at baseline; 43 out of 46,303 individuals developed myasthenia gravis during follow-up. Follow-up was from baseline to the date of first onset of myasthenia gravis, death, or last follow-up (March 1, 2023), with a median follow-up time of 14.1 years. Univariate and multivariate Cox regression analyses (adjusted for age and sex) were performed, and the q-value adjusted for false discovery rate (FDR) was calculated using the fdrtool package (version 1.2.18).

[0024] In Model 1, after FDR correction, we identified 134 proteins associated with MG pathogenesis, including 123 risk proteins and 11 protective proteins. In the further adjusted Model 2, we found 37 risk proteins whose expression levels were positively correlated with the risk of future MG development. The three plasma proteins with the highest risk were LEP (HR: 2.207, CI: 1.495-3.258, FDR: 0.0294), HAVCR2 (HR: 1.824, CI: 1.471-2.262, FDR: 0.0001), and CTSZ (HR: 1.713, CI: 1.308-2.241, FDR: 0.0163). In addition, we identified three protective proteins whose expression levels were negatively correlated with the future risk of mycoplasma leukemia (MG): FASLG (HR: 0.600, CI: 0.458–0.787, FDR: 0.0151), ECM1 (HR: 0.653, CI: 0.531–0.804, FDR: 0.0140), and IGDCC4 (HR: 0.696, CI: 0.587–0.826, FDR: 0.0099). Figure 1 A, B).

[0025] We performed Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on proteins significantly associated with MG pathogenesis. We found that these proteins were significantly enriched in biological processes (BP) related to neutrophil functions, mainly located in the "extracellular plasma membrane" of the cellular composition (CC), and primarily enriched in molecular functions (MF) and cytokine interactions and ligand-receptor binding-related functions as enriched in KEGG.

[0026] To further investigate the core proteins among 40 proteins (age and sex adjusted, FDR corrected p<0.05), we constructed a protein-protein interaction (PPI) network based on the STRING database and used the CytoHubba module to identify key core proteins. We found that HGF, CCL11, and IL2RA were the top three core proteins.

[0027] To better eliminate potential confounding factors and identify proteins with therapeutic potential, we performed Mendelian randomization analysis of drug targets.

[0028] GWAS summary data for plasma protein quantitative trait loci (pQTLs) were obtained from two recently published studies: Pietzner et al. (measuring 4,775 plasma proteins in 10,708 participants) and Ferkingstad et al. (measuring 4,907 plasma proteins in 35,559 participants). Potential druggable targets were obtained from the DrugBank database (https: / / go.drugbank.com / ). GWAS summary data for myasthenia gravis (ebi-a-GCST90093061) were obtained from openGWAS (https: / / gwas.mrcieu.ac.uk / ), including 38,243 individuals of European descent (nCase = 1,873, nControl = 36,370), with a total of 23,679,120 single nucleotide polymorphisms (SNPs). Instrumental variable (IV) selection criteria: IVs should meet the following criteria: (1) Exposure-associated SNPs are genome-wide significant (p<5e-8); (2) SNPs with the lowest p-value are retained in a linkage disequilibrium (LD) block (r²<0.2 or R²<0.001, EU 1000G reference panel, cluster window of 10,000 kb); (3) If the IVs are not present in the outcome GWAS, surrogate SNPs are used (lowest LD R-squared value: 0.8), and all palindromic SNPs are removed. We used plasma proteins as exposures and myasthenia gravis as outcomes, and performed Mendelian randomization analysis using “TwoSampleMR” (https: / / github.com / MRCIEU / TwoSampleMR). If a protein has only one pQTL data, the Wald ratio is used; if there are two or more genetic instrumental variables, the inverse variance weighted Mendelian randomization (MR-IVW) method is used for analysis.

[0029] Of the 40 proteins associated with the pathogenesis of myocardial infarction (MG), we found 27 with therapeutic potential. Figure 2A). We performed Mendelian randomization analysis on these 27 proteins to identify drug targets, using clustering r. 2 =0.2 and clustering r 2 =0.001 was used as the threshold for calculation. We found that when cluster r 2 At a mean value of 0.2, RNASET2, TGFA, CTSO, and HGF were significantly associated with the pathogenesis of myasthenia gravis (MG), with HGF showing the largest effect size (OR: 2.96, P: 0.045). Figure 2 B). When clustering r 2 At a mean value of 0.001, IL2RA and HGF were significantly associated with the pathogenesis of myasthenia gravis (MG), followed by HGF, which had the largest effect size (OR: 4.01, P: 0.034). Figure 3 A). In both threshold conditions of Mendelian randomization analysis of drug targets and in prospective cohort studies, we observed a significant association between elevated plasma HGF levels and the pathogenesis of MG. Figure 3 B).

[0030] Therefore, combining prospective cohort data with Mendelian randomization results for drug targets, we conclude that elevated plasma HGF levels promote the pathogenesis of MG, and that HGF may be a potential therapeutic target.

[0031] (3) Plasma HGF levels were significantly correlated with disease scores and future all-cause mortality. To further explore the application value of HGF in myasthenia gravis (MG), we collected plasma samples from 56 MG patients who were AChR antibody positive and had not received steroid or immunosuppressant treatment, using the myasthenia gravis cohort of the Department of Neurology, First Affiliated Hospital of Chongqing Medical University. HGF levels were then measured in a healthy control group matched for age and sex.

[0032] A cohort study of myasthenia gravis in the Department of Neurology, First Affiliated Hospital of Chongqing Medical University, included 56 patients and 59 age- and sex-matched healthy controls. This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Ethics Approval No.: 2020-382), and written informed consent was obtained from all participants. Inclusion criteria were: (a) age over 18 years; (b) positive serum acetylcholine receptor (AChR) antibody; (c) no use of immunosuppressants or steroids in the past three months; and (d) no other autoimmune diseases, inflammatory or infectious diseases, pregnancy, or metabolic diseases. After blood samples were collected, they were allowed to stand at room temperature for two hours and then centrifuged at 2500 rpm for 10 minutes. Plasma was stored at -80°C.

[0033] We found that HGF levels were significantly elevated in MG patients ( Figure 4A, B). HGF levels demonstrated excellent diagnostic performance in distinguishing MG from healthy controls, with an AUC of 0.847. Other diagnostic parameters also reflected its excellent diagnostic ability, including accuracy of 0.824, sensitivity of 0.746, and specificity of 0.903. Figure 4 CD). Furthermore, we found a significant positive correlation between HGF levels and Activities of Daily Living (ADL) scores and Myasthenia Gravis Complex (MGC) scores. Figure 4 EF). High HGF levels reduce the patient's survival probability. Figure 4 G). High HGF levels are a risk factor for all-cause mortality in MG patients, and this association remained stable after adjusting for age and sex (Model 2) and further adjusting for age, sex, height, and weight (Model 3). Figure 4 H). Through multiple population-based studies, we have found that plasma HGF levels are closely related to the onset, progression, and disease activity of MG (Hypergillus oryzae). Figure 4 I).

[0034] (4) HGF receptor inhibitor (PF04217903) relieves EAMG symptoms Construction of the EAMG rat model: T-AChR protein was purified from the electric organ of the electric eel. Antigen emulsions were prepared using a high-speed homogenizer to ensure emulsification. Each rat was injected with a total volume of 200 μL, comprising 75 μg of purified T-AChR protein, mixed with 100 μL of PBS, followed by the addition of 100 μL of complete Freund's adjuvant (CFA) and 1 mg of tuberculosis mycoplasma (H37Ra), and then emulsified. The emulsified mixture was subcutaneously injected into the bilateral shoulders and hind paw pads of the rats. Control rats received the same volume of emulsion (PBS emulsified with adjuvant, without AChR). Four weeks after the first immunization, rats received a second immunization, either injected with a solution containing CFA and AChR (model group) or a solution of CFA and PBS (control group). After the second immunization, disease scores were assessed and body weight was recorded. Clinical scores of rats were assessed according to the Lennon scoring system (0 = normal strength; 1 = mild (reduced activity, weak grip, fatigue); 2 = weakness, hunched posture at rest, weight loss, tremor; 3 = severe (widespread weakness, significant weight loss, near death); 4 = death). Grip strength was measured using a grip dynamometer (Sansbio, Jiangsu, China). Rats were held by their tails and allowed to grasp a horizontal bar with their forepaws. Once a rat had grasped the bar with both forepaws, it was pulled off the bar until it released it. When rats exhibited significant muscle weakness (score of grade 2 or higher) within 28 days of their second immunization, blood was collected from the orbital venous plexus under anesthesia, and serum samples were retained according to human serum sample processing procedures.

[0035] The criteria for successful establishment of the EAMG model are as follows: 1) Clinical score ≥1; 2) Positive neostigmine test; 3) Positive serum AChR-Ab; 4) Deposition of immune complexes in muscle. If criterion 1) is met, and any two of criteria 2), 3), or 4) are satisfied, the EAMG model is considered to have been successfully established.

[0036] Pharmacological intervention: PF04217903 is a highly selective c-Met inhibitor (HGF receptor), with a selectivity for c-Met exceeding that of other kinases by more than 1000 times. PF04217903 (40 mg / kg) was dissolved in 10% DMSO, 5% Tween 80, 40% PEG300, and 45% saline. The control solution contained 10% DMSO, 5% Tween 80, 40% PEG300, and 45% saline. In the PF04217903 intervention experiment, rats were randomly assigned to two groups (n = 6 per group), receiving PF04217903 (40 mg / kg, 3 ml) orally once daily via gastric tube (EAMG + PF04217903 group) or only the corresponding control solution (3 ml) orally for 28 consecutive days, starting after the second immunization.

[0037] The following molecular biological assays were performed on the two groups of rats: (1) Immunofluorescence To evaluate the effect of the HGF inhibitor PF04217903 on the EAMG model, anterior tibialis muscle was perfused, dehydrated, and embedded in an OCT. The tissue was then serially sectioned, each 10 μm thick. After equilibration with room temperature, the sections were washed three times with PBS. Subsequently, the sections were permeabilized in a solution containing 0.4% Triton X-100 for 15 minutes and blocked with PBS containing 10% goat serum for 1 hour. The sections were then incubated overnight at 4°C with primary antibody. The primary antibody used was C5b-9 antibody (Santa Cruz, USA, Cat#: sc-66190). After washing three times with PBS, the sections were incubated at room temperature with secondary antibody for 1 hour. The secondary antibodies used were CF 488-labeled α-snake venom (α-BTX) (Biotium, USA, Cat#: 00005-100ug) and Dylight 594-labeled goat anti-mouse IgG (Abbkine, USA, Cat#: A23410). Following a series of reactions, sections were stained with DAPI (Servicebio, China, Cat#: G1012) and imaged using an Olympus VS200 microscope (Nikon, Japan). The mean fluorescence intensity (MFI) of different markers was analyzed using ImageJ software.

[0038] (2) Western Blot Proteins were extracted from rat tissues using RIPA lysis buffer (Beyotime, China, Cat#: P0013B) and benzoyl sulfonyl fluoride (PMSF) (Beyotime, China, Cat#: ST506), followed by sonication. Tissues were lysed on ice for 30 min, and samples were centrifuged at 12,000 g for 16 min at 4 °C, collecting the protein-containing supernatant. Protein concentration was determined according to the manufacturer's instructions using the BCA Protein Assay Kit (Beyotime, China, Cat#: P0012). An equal volume of protein (30 µg) was then denatured by heating to 100 °C for 5 min in SDS-PAGE sample loading buffer (Beyotime, China, Cat#: P0015).

[0039] Proteins were separated using a Tris-glycine electrophoresis system on a 10% SDS-PAGE gel (Epizyme Biotech, China, Cat#: PG112). After electrophoresis, the proteins were transferred to a 0.22 μm PVDF membrane (Merck Millipore, USA, Cat#: ISEQ00010) using a wet transfer system for 80 minutes. Following transfer, the membrane was blocked with QuickBlock™ Western blocking buffer (Beyotime, China, Cat#: P0252) to prevent nonspecific binding. After blocking, the membrane was incubated overnight at 4°C with primary antibodies (anti-rabbit HGF antibody, 1:700, ABclonal, China, Cat#: A1193; β-actin antibody, 1:500, Proteintech, China, Cat#: 81115-1-RR).

[0040] The next day, the membrane was washed with PBST to remove unbound primary antibody and incubated for 1 hour at room temperature with HRP-labeled anti-rabbit secondary antibody (1:10,000, Proteintech, China, Cat#: SA00001-7H). After multiple washes, protein bands were visualized on a Bio-Rad imaging system using a highly sensitive ECL chemiluminescent substrate (ThermoFisher, USA, Cat#: 34580).

[0041] (3) ELISA According to the manufacturer's instructions, plasma HGF levels in the MG group and healthy control group were determined using a human and rat HGF (hepatocyte growth factor) ELISA kit (Elabscience, China, Cat#: E-EL-H0084, E-EL-R0496) via a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). HGF levels were detected by measuring the optical density (OD) of each well at 450 nm using a microplate reader (ThermoFisher, USA). Since there is a positive correlation between HGF concentration and the measured OD450 value, the concentration of HGF in each sample could be accurately calculated by plotting a standard curve and comparing the OD450 values ​​of the samples. Neutrophil degranulation was assessed by measuring the release of neutrophil elastase (NE) and myeloperoxidase (MPO) levels using a rat neutrophil elastase ELISA kit (CUSABIO, China, Cat#: CSB-E08847r) and a rat myeloperoxidase (MPO) ELISA kit (CUSABIO, China, Cat#: CSB-E08722r). Following B cell stimulation, IgG secretion was quantified using a rat IgG ELISA kit (CUSABIO, China, Cat#: CSB-E07981r).

[0042] (4) Anti-AChR IgG antibody assay Blood was collected via orbital puncture and centrifuged at 3000 rpm for 10 minutes. The supernatant was used as the sample. Purified AChR solution (2 µg / mL) was coated onto a 96-well plate overnight and incubated at 4°C. After washing three times with PBS-T, the plate was blocked with 10% fetal bovine serum at room temperature for 2 hours. Plasma was then diluted (1:1000, 100 µL / well) and added to the wells, incubated at room temperature for 2 hours. After washing, HRP-labeled goat anti-rat IgG (1:500, Beyotime, China, Cat#: A0192) was added, and the plate was incubated at 37°C for 1 hour. After washing three times with PBST, TMB substrate solution was added, and the plate was incubated in the dark for 15 minutes. After adding stop solution, the OD value at 492 nm was measured within 15 minutes.

[0043] (5) Cell isolation and cell culture Neutrophils were isolated from rat peripheral blood mononuclear cells (PBMCs) and bone marrow using a rat peripheral blood neutrophil isolation kit (Solarbio, China, Cat#: P9200) and a rat bone marrow neutrophil isolation kit (Solarbio, China, Cat#: P2610). To select the optimal concentration and time, neutrophils were co-cultured with recombinant rat HGF (0, 1, 10, 100, 1000 ng / ml, Abclonal, China, Cat#: RP01776) for 24 hours. After determining the optimal concentration, the optimal concentration (100 ng / mL) was selected to stimulate neutrophils or HL60 cells at different time points (6 hours, 12 hours, 24 hours, 48 ​​hours). Neutrophils or HL60 cells were divided into four groups and cultured in vitro at the optimal concentration and time (100 ng / mL, 24 hours): a control group (NC group) with only solvent, a group stimulated only with recombinant rat HGF (HGF group), a group treated with recombinant rat HGF and PF-04217903 (100 nM, ApexBio, USA, Cat#: A5096) (HGF + PF-04217903 group), and a group treated with recombinant rat HGF and rapamycin (20 nM, MCE, Cat#: HY-10219) (HGF + rapamycin group). The HGF + PF-04217903 group was pretreated with PF-04217903 (100 nM) for 20 minutes before the addition of 100 ng / mL recombinant HGF. CD4+ T cells were isolated from rat spleen cells using a rat CD4+ T cell isolation kit (StemCell, Canada, Cat#: 19642) and stimulated with anti-CD3 / CD28 antibody (eBioscience, USA, Cat#: 14-0030-82, 16-0280-81) for 24 or 72 hours, then co-cultured with recombinant rat HGF (100 ng / mL) or a solvent. CD19+ B cells were isolated from rat spleen cells using a rat B cell isolation kit (StemCell, Canada, Cat#: 19644) and stimulated with R848 (1 μg / mL, MCE, HY-13740) and recombinant rat IL-2 protein (10 ng / mL, Abclonal, China, Cat#: RP01769) for 48 hours, then co-cultured with recombinant rat HGF (100 ng / mL) or a solvent. Macrophages (Mo&Mac) were obtained by culturing rat spleen cells at 37°C for 8 hours, followed by washing twice with PBS to remove the upper layer of cells, leaving the remaining adherent cells as macrophages. Mo&Mac cells were then co-cultured with recombinant rat HGF (100 ng / mL) or a solvent for 24 hours.All cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0044] (6) Cell staining and flow cytometry To detect Th1 and Th17 cells, cells were co-cultured with a cell-activating cocktail (containing Brefeldin A) (2 μL / mL, BioLegend, USA, Cat#: 423303) at 37°C for 5 hours. The cell-stimulating cocktail should not be added before staining FOXP3. Cells were then stained using different antibody combinations to label the corresponding cell populations. Cells were washed twice with PBS and stained with surface antibodies anti-CD4-FITC (Elabscience, China, Cat#: E-AB-F1105C) and anti-CD3-APC (Elabscience, China, Cat#: E-AB-F1228E). Then, after permeabilization using a Fixation / permeabilization solution kit (eBioscience, USA, Cat#: 00-5521-00), staining was performed with anti-IFNγ-PE (Biolegend, USA, Cat#: 507806), anti-IL-17-PE-Cy7 (eBioscience, USA, Cat#: 25-7177-82), and anti-Foxp3-PE-Cy7 (eBioscience, USA, Cat#: 25-5773-82), and incubated at 4°C for 1 hour. To detect B cell subsets, isolated CD19+ B cells were stained with anti-rat CD45R-PE-Cy7 (eBioscience, USA, Cat#: 25-0460-82), CD27-BV-421 (Biolegend, USA, Cat#: 124223), anti-rat IgG-FITC (Biolegend, USA, Cat#: 405404), and Igκ-PE (Biolegend, USA, Cat#: 407805). To detect mTOR and phosphorylated mTOR, HL60 cells were stained with mTOR(7C10) rabbit monoclonal antibody (labeled with AlexaFluor® 488) (Cell Signaling Technology, USA, Cat#: 5043) and p-mTOR(pS2448)-PE (BD Biosciences, USA, Cat#: 563489) flow cytometry antibodies.Neutrophils were immunolabeled using recombinant mTOR antibody (Proteintech, China, Cat#: 81670-1-RR) and recombinant phosphorylated mTOR (Ser2448) antibody (Proteintech, China, Cat#: 80596-1-RR), followed by permeabilization using a Fixation / permeabilization solution kit, and then incubated with the secondary antibody CoraLite488-labeled goat anti-rabbit IgG (H+L) (Proteintech, China, Cat#: SA00013-2). Cell analysis was performed using a flow cytometer (SA3800 spectrometer, Sony Biotechnology, Tokyo, Japan), and data were analyzed using a Sony SA300 and FlowJo software (version 10.8.1, FlowJo, Ashland, Oregon, USA). Apoptosis was detected by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, China, Cat#: C1062S). The percentage of apoptosis was calculated using Annexin-V+ / PI- (early apoptosis) and Annexin-V+ / PI+ (late apoptosis).

[0045] We found that, compared to the EAMG group, the weight loss trend was suppressed in the PF04217903 intervention group. Figure 5 A), and disease progression was significantly suppressed ( Figure 5 B). 28 days after the second immunization, we observed significant improvements in clinical scores and grip strength in the PF04217903 intervention group compared to the EAMG group. Figure 5 C, D). Muscle pathological staining showed a significant and typical reduction in membrane attack complex (MAC) deposition in the EAMG group compared to the intervention group. Figure 5 E). Following PF04217903 intervention, the titer level of AChR antibody in peripheral blood was significantly reduced, and the mean fluorescence intensity of C5b-9 in muscle was also significantly decreased. Figure 5 F, G). In addition, the inflammatory T cell subset Th17 (IL-17+ T cells, Figure 5 J, Figure 5 K) and Th1 (IFNγ+ T cells, Figure 5 H, Figure 5 I) The T-cell-mediated inflammation was significantly reduced after PF04217903 intervention, indicating that the inflammation was alleviated.

[0046] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. Application of HGF receptor inhibitors in the preparation of drugs for the treatment / prevention of myasthenia gravis.

2. The application according to claim 1, characterized in that, The HGF receptor inhibitors include PF04217903, SU11274, Crizotinib, Cabozantinib, ARQ197, XL880, INC280, MGCD265, or ASP3026.

3. The application according to claim 1, characterized in that, The dosage of the HGF receptor inhibitor is 40 mg / kg.

4. The application according to claim 1, characterized in that, The drug is in the form of injection, tablet, powder, granule, pill, capsule, oral liquid, ointment, cream or spray.

5. The application according to claim 1, characterized in that, The drug also includes one or more pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The excipients include diluents, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, and sustained-release agents commonly used in the pharmaceutical field.

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