Medicine for treating myasthenia gravis and application

Abstract

The invention relates to a medicine for treating myasthenia gravis and application thereof, and belongs to the technical field of medical biology. The invention relates to a medicament for treating myasthenia gravis (MG), which is characterized in that the medicament contains a Bruton's tyrosine kinase (BTK) inhibitor Pirtobrutinib, and the medicament can be used for treating myasthenia gravis (MG). The Pirtobrutinib is a third generation of BTK (brutinib) inhibitor with high selectivity and a non-covalent (reversible) binding mode. The Pirtobrutinib can be used for effectively treating the MG, and meanwhile, the serious safety defect of similar medicines in the prior art can be avoided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical biotechnology, and particularly relates to the medical use of Bruton's Tyrosine Kinase (BTK) inhibitors, in particular the application of a highly selective, non-covalent (reversible) BTK inhibitor Pirtobrutinib in the preparation of a medicament for treating Myasthenia Gravis (MG). BACKGROUND

[0002] Myasthenia Gravis (MG) is a chronic autoimmune disease mediated by pathogenic autoantibodies. Its core pathology lies in the immune system attacking key proteins at the neuromuscular junction (NMJ), mainly acetylcholine receptor AChR, leading to impaired neuromuscular signal transmission, and clinical manifestations of fluctuating weakness and easy fatigue of skeletal muscles. B lymphocytes play a key role in this process, which differentiate into plasma cells and produce pathogenic antibodies.

[0003] Current standard therapies for MG, such as acetylcholinesterase inhibitors and broad-spectrum immunosuppressants (such as corticosteroids), have problems such as poor efficacy and significant side effects. Emerging targeted therapies, such as complement inhibitors and neonatal Fc receptor (FcRn) antagonists, have made progress, but still face limitations such as inconvenient administration, high cost, and not effective for all patients.

[0004] Bruton's Tyrosine Kinase (BTK) is a key node in the B-cell receptor (BCR) signaling pathway. Therefore, inhibiting BTK is a potential strategy for treating B-cell mediated autoimmune diseases such as MG. However, the history of clinical development in this field reveals major challenges, particularly that other BTK inhibitors have shown significant risk of liver toxicity when used for neurological autoimmune diseases, which has posed a technical obstacle in the field. For example, the covalent BTK inhibitor Tolebrutinib developed by Sanofi, its phase III clinical trial (URSA, NCT05132569) for MG was suspended by the US FDA due to multiple cases of drug-induced liver injury (DILI), and was eventually terminated. Similarly, the BTK inhibitor Fenebrutinib developed by Roche also faced partial clinical suspension in its phase III clinical trial for multiple sclerosis (MS) due to cases of DILI. These published failure cases have shown to those skilled in the art that BTK inhibitors in this class of drugs may be associated with a significant risk of liver damage when used to treat neurological autoimmune diseases, which is generally considered a "class effect". This technical status hinders further exploration in this direction.

[0005] Therefore, there is a clinical need for a safe, effective, well-tolerated and convenient (especially oral) long-term intervention. SUMMARY

[0006] The present application aims at providing a drug capable of effectively treating MG, which, as a BTK inhibitor, can avoid the serious safety defects exhibited by similar drugs in the prior art when used to treat MG, and has a lower risk of hepatotoxicity compared to other BTK inhibitor therapies (such as Tolebrutinib).

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows: The present application first protects the use of Pirtobrutinib in the preparation of a drug for reducing the number of antibody subpopulation secreting cells in patients with myasthenia gravis (MG). The drug can reduce the number of antibody subpopulation secreting cells in myasthenia gravis patients in the peripheral blood, spleen, and lymph nodes.

[0008] Pirtobrutinib is a Bruton's Tyrosine Kinase (BTK) inhibitor, and in particular, Pirtobrutinib is a third-generation BTK inhibitor with high selectivity and non-covalent (reversible) binding mode.

[0009] Further, the present application also protects the use of Pirtobrutinib in the preparation of a drug for reducing the activation phenotype of B cells in myasthenia gravis patients. The drug reduces the activation phenotype of B cells in myasthenia gravis patients in the peripheral blood, spleen, and lymph nodes.

[0010] Further, the present application also protects the use of Pirtobrutinib in the preparation of a drug for reducing serum AChR antibody levels in myasthenia gravis patients.

[0011] Further, the present application also protects the use of Pirtobrutinib in the preparation of a drug for inhibiting BTK signaling in myasthenia gravis patients.

[0012] Further, the present application also protects the use of Pirtobrutinib in the preparation of a drug for improving clinical symptoms in myasthenia gravis patients, including weight gain, clinical score, and grip strength recovery.

[0013] Further, the present application also protects the use of Pirtobrutinib in the preparation of a drug for improving nerve-muscle junction pathology in myasthenia gravis patients.

[0014] Preferably, in the above-mentioned applications, the dosage form of the Pirtobrutinib drug is an oral preparation.

[0015] Preferably, in the above-mentioned applications, the administration dose of Pirtobrutinib is 5 mg / kg-25 mg / kg (specifically 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, etc.), twice a day.

[0016] Further, the present application also protects a drug for treating myasthenia gravis, for use in the above-mentioned applications.

[0017] Further, in the drug for treating myasthenia gravis, the BTK inhibitor Pirtobrutinib is used as a single component or in combination with other pharmaceutically acceptable components to prepare a drug for treating myasthenia gravis.

[0018] Further, in the drug for treating myasthenia gravis, the other pharmaceutically acceptable components can be drugs that have no antagonistic effect with the BTK inhibitor Pirtobrutinib, or any one or more excipients (carriers) allowed in pharmacy.

[0019] In the above-mentioned applications, the administration of Pirtobrutinib can significantly improve the clinical symptoms of the disease, reduce the level of pathogenic autoantibodies, and protect the structural integrity of the neuromuscular junction. Through research in an experimental autoimmune myasthenia gravis (EAMG) rat model of myasthenia gravis, the present application first confirms that Pirtobrutinib has a significant therapeutic effect on myasthenia gravis.

[0020] Further, the clinical symptoms include but are not limited to weight gain, reduction of clinical score, recovery of grip strength.

[0021] Further, the protection of the structural integrity of the neuromuscular junction includes but is not limited to increasing AChR density and reducing complement deposition.

[0022] The present technical solution combines the known good safety characteristics of Pirtobrutinib with the newly discovered therapeutic effectiveness of Pirtobrutinib in the field of MG, solving the problem in the prior art that efficacy and safety cannot be achieved simultaneously.

[0023] Compared with the prior art, the present application has the following beneficial effects: (1) Breakthrough in technical effect: The present application provides a drug that can effectively treat myasthenia gravis, while successfully avoiding the serious liver toxicity risk of similar BTK inhibitors in this treatment field. Since the prior art has shown that BTK inhibitors are not suitable for MG treatment due to liver toxicity, the present application achieves safe and effective treatment by selecting Pirtobrutinib as a specific inhibitor. This result is contrary to the technical expectations established by the prior art, providing a new approach for the treatment of MG that was previously considered unfeasible. Compared with other BTK inhibitors that have failed in clinical development in the field of neuroimmunological diseases, Pirtobrutinib has significantly different safety characteristics, especially its very low risk of liver toxicity.

[0024] (2) Clear therapeutic effect: In the EAMG rat model, Pirtobrutinib can effectively improve clinical symptoms (increased body weight, decreased clinical score, recovered grip strength), significantly reduce the level of pathogenic autoantibodies (anti-AChR antibodies), protect the structural integrity of the neuromuscular junction (increase AChR density, reduce complement deposition), and effectively inhibit the activation and proliferation of pathogenic B cells, demonstrating its exact in vivo efficacy.

[0025] (3) Convenience of oral administration: Pirtobrutinib is an oral drug, which improves the convenience of medication and the quality of life of patients compared with biologics that need to be injected or infused, and is conducive to the long-term treatment compliance of chronic diseases.

[0026] (4) Good overall tolerability: The high selectivity of Pirtobrutinib makes it have a better side effect profile compared to the first generation of BTK inhibitors, such as lower cardiac toxicity and bleeding risk, making it more suitable as a long-term maintenance therapy drug for MG. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure for single-cell transcriptomic analysis of public data revealing the activation of B cells and upregulation of BTK signaling pathway in myasthenia gravis; Figure 2 Figure for Pirtobrutinib improving the disease progression of EAMG; Figure 3 Figure for Pirtobrutinib reducing the proportion, activation and antigen presentation function of B cells in peripheral blood, and changing the expression of antibody subgroup secreting cells; Figure 4 Figure for Pirtobrutinib reducing the proportion, activation and antigen presentation function of B cells in the spleen, and changing the expression of antibody subgroup secreting cells; Figure 5Pirtobrutinib reduces the proportion of B cells in lymph nodes, activation and antigen presentation function, and changes the expression profile of antibody subpopulation secreting cells; Figure 6 Pirtobrutinib reduces germinal center B cell (GCB) population profile in EAMG model. DETAILED DESCRIPTION

[0028] The specific embodiments of the present application are further described in conjunction with the following examples. The following examples are intended to illustrate the present application and are not intended to limit the scope of the present application. The present application encompasses all possible alternatives, modifications and equivalents within the scope of the claims. The specific techniques or conditions not specifically recited in the examples below are routine techniques or conditions, or are as described in the literature, or are as per the product instructions.

[0029] Example 1 In this study, an experimental autoimmune myasthenia gravis (EAMG) rat model was used.

[0030] Sources of experimental materials 1. Experimental animals: female Lewis rats, 6-8 weeks old, weighing 90-150 grams, purchased from Vital River Laboratories, Beijing, China. All rats were raised in a SPF (specific pathogen free) level environment, with temperature controlled at 22±1°C, using a 12 hour light / dark cycle, free access to autoclaved feed and drinking water. All experimental protocols were approved by the institutional ethics committee.

[0031] 2. Including key compounds and reagents, flow cytometry antibodies, immunofluorescence antibodies Key compounds and reagents: Pirtobrutinib: purchased from MedChemExpress (MCE) company, item number HY-131328 Acetylcholine receptor (AChR): purified from torpedo tissue Complete Freund's adjuvant (CFA) and inactivated Mycobacterium tuberculosis: purchased from BD company, item number 231141 Solvent: polyethylene glycol 300 (PEG 300) and 0.9% normal saline Red blood cell lysis solution: purchased from Solarbio company Intracellular fixation and membrane rupture buffer: purchased from BD company, item number 554714 HRP-conjugated goat anti-rat IgG antibody: purchased from Beyotime Antibodies: Flow cytometry antibodies: anti-rat CD45R-PE-Cy7 (ThermoFisher, #25-0460-82), CD80-PE (Biolegend, #200205), CD86-AF-647 (Biolegend, #200314), MHCII-PerCP-e710 (ThermoFisher, #46-0463-82), CD27-BV-421 (Biolegend, #124223), IgG-FITC (Biolegend, #405404), IgK-PE (Biolegend, #407805). Peanut agglutinin (PNA)-FITC labeling (Vector Laboratories, #FL-1071-5); Immunofluorescence antibodies: mouse anti-rat C5b-9 antibody (Santa Cruz), CF488 labeled a-bungarotoxin (a-BTX) (Biotium, #00013), Dylight 488 / 594 labeled secondary antibody (Abbkine).

[0032] Experimental methods 1. Data source and processing The single-cell RNA sequencing (scRNA-seq) data used in this analysis was obtained from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI) with accession number GSE227835. This dataset contains peripheral blood mononuclear cells (PBMCs) from myasthenia gravis (MG) patients and healthy control (HC) individuals 2. Bioinformatics analysis methods The raw gene expression matrix was processed using the Seurat R package. First, quality control was performed, and low-quality cells were filtered out according to the criteria of the number of unique genes detected, total molecules, and percentage of mitochondrial gene expression. After filtering, the data was normalized by log transformation. Then, principal component analysis (PCA) was performed on the standardized data, and the top-ranked principal components were used for graph-based clustering to identify different cell populations. Visualization was performed using the uniform manifold approximation and projection (UMAP) algorithm. According to the expression of classical marker genes, the cell types were manually annotated, among which B cells were specifically identified by high expression of CD19, CD79B, JCHAIN, and MS4A1. Wilcoxon rank-sum test was used to analyze the differential gene expression of B cells between MG patients and healthy controls. To identify dysregulated biological pathways, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the list of significantly up-regulated genes.

[0033] 3. EAMG model induction and drug preparation and use Model induction: AChR protein purified from Torpedo was dissolved in PBS and emulsified with complete Freund's adjuvant (CFA) supplemented with inactivated Mycobacterium tuberculosis at a volume ratio of 1:1. Each rat was injected subcutaneously with 200 microliters of emulsion at multiple points (bilateral hind footpads and back), so that each rat received 75 micrograms of AChR. On day 28 after immunization, a booster immunization was performed using the same dose of AChR dissolved in incomplete Freund's adjuvant (IFA).

[0034] Drug preparation and use: Pirtobrutinib powder was dissolved in a 1:1 mixture of PEG 300 and 0.9% normal saline, and freshly prepared daily. The successfully modeled EAMG rats were randomly divided into three groups (n=6 per group): EAMG model group (given solvent control), Pirtobrutinib low-dose group (given 5 mg / kg Pirtobrutinib), and Pirtobrutinib high-dose group (given 25 mg / kg Pirtobrutinib). From the booster immunization on day 28, the drug was administered twice daily by oral gavage, for a continuous treatment of 14 days, until the end of day 42.

[0035] 4. Clinical symptom evaluation During the experiment, the body weight and clinical scores of rats in each group were recorded every three days. The clinical scoring criteria (Lennon score) are as follows: Grade 0, no obvious muscle weakness; Grade 1, slight decrease in activity, weak grasping or squeaking, easy fatigue; Grade 2, arched back, drooping head, bent forelimbs, occasional tremors, and incoordination; Grade 3, severe muscle weakness, persistent tremors, and a near-death state; Grade 4, death. For individuals with mild symptoms, a fatigue test (having the rat repeatedly grasp the cage mesh for 30 seconds) was performed to assist in the scoring.

[0036] 5. Serum anti-AChR antibody level detection (ELISA) An in-house developed indirect ELISA method was used for detection. In short, 96-well plates were coated overnight with AChR protein (2 μg / mL). The following day, after washing, non-specific sites were blocked with 10% fetal bovine serum. Rat serum samples diluted 1:1000 were added and incubated at room temperature for 2 hours. After washing, HRP-conjugated goat anti-rat IgG secondary antibody was added, and the plate was incubated at 37°C for 1 hour. Finally, TMB substrate was added for color development, and the reaction was terminated with 2N sulfuric acid. The absorbance was read at 492 nm.

[0037] 6. Immunofluorescence analysis of the neuromuscular junction Sections were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then blocked with 5% BSA. Subsequently, they were incubated with primary antibody (anti-C5b-9 antibody), fluorescently labeled secondary antibody, and α-BTX (α-Blocked krait venom, used to label AChR). Images were acquired using a Nikon microscope, and the mean fluorescence intensity of AChR and C5b-9 was analyzed using ImageJ software.

[0038] 7. Flow cytometry analysis Single-cell suspensions were prepared from peripheral blood, spleen, and lymph nodes, respectively. After removing erythrocytes using erythrocyte lysis buffer, cell surface staining was performed with a predetermined concentration of fluorescent antibody combination for the analysis of B cells (B220, CD80, CD86, MHCII, CD27) and germinal center B cells (PNA). For intracellular staining (IgG, Igκ), cells were treated with fixation and permeabilization buffer after surface staining, followed by incubation with intracellular antibodies. Data were acquired using a Sony SA3800 flow cytometer (>50,000 cells per sample) and analyzed using FlowJo v10.9 software.

[0039] 8. Statistical Analysis All data are presented as mean ± SD. Statistical analysis was performed using SPSS 26.0 and GraphPad Prism 10 software. Unpaired Student's t test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups. P < 0.05 was considered statistically significant.

[0040] Drug preparation and administration regimen 1. Grouping: Rats successfully induced EAMG model were randomly divided into three groups: EAMG model group (solvent control), Pirtobrutinib low-dose treatment group, Pirtobrutinib high-dose treatment group.

[0041] Drug: Pirtobrutinib.

[0042] 2. Administration regimen: The low-dose group was 5 mg / kg, and the high-dose group was 25 mg / kg, twice a day, oral gavage, continuous treatment for 14 days.

[0043] Solvent: Polyethylene glycol 300 (PEG 300) mixed with 0.9% normal saline at a volume ratio of 1:1.

[0044] 3. Efficacy and safety evaluation Efficacy endpoints: include clinical scores, body weight, grip strength, serum anti-AChR IgG antibody levels, AChR density and C5b-9 deposition at NMJ, and analysis of B cell subsets in peripheral blood, spleen, and lymph nodes. See Figures 1 to 6 for specific results.

[0045] Figure 1 Single-cell transcriptomic analysis of public data revealed activation of B cells and upregulation of BTK signaling pathway in MG patients. The data used in this analysis were derived from the Gene Expression Omnibus (GEO) with accession number GSE227835. This dataset contains peripheral blood mononuclear cells (PBMCs) from clinically standard diagnosed MG patients and age- and sex-matched healthy controls (HCs). (a) Uniform Manifold Approximation and Projection (UMAP) plot of all analyzed cells, colored by individual sample (left), group (middle), and unsupervised clustering result (right, 21 cell clusters were identified). (b) Dot plot showing the expression levels of classical marker genes in the 21 identified cell clusters. The size of the dot represents the percentage of cells in the cluster expressing the gene, and the color intensity indicates the average expression level. (c) UMAP feature plot showing key B cell lineage marker genes used to identify B cell populations (CD19, CD27, CD38, CD45, CD69, CD80, CD86, CD95, CD103, CD138, CXCR4, FcεRI, IgD, IgM, IL-21R, and MHCII). CD19 , CD79B , JCHAIN andMS4A1 ) expression. (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes between B cells of clinically confirmed MG patients and age- and sex-matched healthy control HC individuals. Bar plots represent the highest degree of enrichment of pathways ordered by their corrected p-value. (e) Statistical significance plot of differentially expressed genes within the B cell receptor signaling pathway, where key pathway components (including BTK ) have been highlighted. (f) Violin plots comparing the normalized expression levels of BTK in B cells of MG patients and HC, showing a significant increase in expression in the MG cohort.

[0046] Figure 2 Figure 17. Graph showing the improvement of Pirtobrutinib in the disease progression of EAMG. Data were derived from the EAMG model of female Lewis rats induced with tAChR. The groups shown in the figure were three groups in total, namely: EAMG model group (solvent control group), Pirtobrutinib low-dose group (5 mg / kg, twice a day), and Pirtobrutinib high-dose group (25 mg / kg, twice a day). The sample size of each group was n=6. (a) Body weight of EAMG group, low-dose (5 mg / kg) and high-dose (25 mg / kg) BTKi treatment group rats was recorded every three days (n=6 per group; single experiment). The arrows indicate the start day of Pirtobrutinib treatment and booster immunization. (b) Forelimb grip strength was measured on day 44 after the first immunization (n=6). (c) Clinical scores of the three groups were evaluated every three days. (d) Anti-AChR IgG antibody levels in serum on day 44 were quantitatively detected by ELISA (n=6). (e) Confocal immunofluorescence images of nerve-muscle junction (NMJ) in quadriceps and tibialis anterior muscle sections, with staining targets of AChR (green) and C5b-9 (red); scale bar = 100 µm. (f) Quantitative analysis of C5b-9 aggregation density (relative fluorescence intensity) in each group (n=6). (g) Quantitative analysis of AChR deposition (relative fluorescence intensity) in each group (n=6). Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the unpaired Student's t-test. ns indicates no significant difference; * indicates comparison between EAMG group and EAMG+5 mg / kg BTKi group; # indicates comparison between EAMG group and EAMG+25 mg / kg BTKi group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; #p<0.05, ##p<0.01, ####p<0.0001. Data are representative results of three independent experiments.

[0047] Figure 3To reduce the proportion of B cells, their activation and antigen-presenting function, and to change the expression profile of antibody-subset-secreting cells in the peripheral blood for Pirtobrutinib. Samples were obtained from Figure 2 The EAMG rat model described above, with the same grouping as Figure 2 (solvent control group, 5 mg / kg Pirtobrutinib group, 25 mg / kg Pirtobrutinib group), n = 6 in each group. (a) Representative flow cytometry plots showing the expression of B220, CD80, CD86, MHCII and CD27 on peripheral blood (PB) B cells of EAMG rats in the three groups. (b-f) Quantitative analysis of the percentage of B220 + B cells (b), and B220 + CD80 + (c), CD86 + (d), MHCII + (e) and CD27 + (f) cells in the B cell population. (g) Representative flow cytometry plots showing the expression of B220, IgG and IgK on peripheral blood B cells. (h-j) Quantitative analysis of the percentage of B220 + B cells (h), and B220 + IgG + (i) and IgK + (j) cells in the B cell population. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using unpaired Student's t test. ns indicates no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are representative results of three independent experiments.

[0048] Figure 4 To reduce the proportion of B cells, their activation and antigen-presenting function, and to change the expression profile of antibody-subset-secreting cells in the spleen for Pirtobrutinib. Samples were obtained from Figure 2 The EAMG rat model described above, with the same grouping as Figure 2 (solvent control group, 5 mg / kg Pirtobrutinib group, 25 mg / kg Pirtobrutinib group), n = 6 in each group. (a) Representative flow cytometry plots showing the expression of B220, CD80, CD86, MHCII and CD27 on spleen (SP) B cells of EAMG rats in the three groups. (b-f) Quantitative analysis of the percentage of B220 + B cells (b), and B220 + CD80 + (c), CD86+ (d) MHCII + (e) and CD27 + (f) quantification of cell percentage. (g) Representative flow cytometry plots showing the expression of B220, IgG and IgK on splenic B cells. (h-j) Total number of B220 + IgG + (i) and IgK + (j) quantification of cell percentage. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using unpaired Student's t test. ns, not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are representative of three independent experiments.

[0049] Figure 5 Pirtobrutinib reduced the proportion of B cells, activation and antigen-presenting function in lymph nodes, and changed the expression profile of antibody- secreting cells of B cell subsets. Samples were from Figure 2 EAMG rats as described in Figure 2 (solvent control group, 5 mg / kg Pirtobrutinib group, 25 mg / kg Pirtobrutinib group), n = 6 in each group. (a) Representative flow cytometry plots showing the expression of B220, CD80, CD86, MHCII and CD27 on LN B cells of EAMG rats in three groups. (b-f) Proportion of B220 + B cells (b), and B220 + CD80 + (c), CD86 + (d), MHCII + (e) and CD27 + (f) quantification of cell percentage. (g) Representative flow cytometry plots showing the expression of B220, IgG and IgK on LN B cells. (h-j) Total number of B220 + B cells (h), and B220 + IgG + (i) and IgK + (j) quantification of cell percentage. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using unpaired Student's t test. ns, not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are representative of three independent experiments.

[0050] Figure 6 A plot of germinal center B cell (GCB) population in the EAMG model reduced by Pirtobrutinib. Samples from... Figure 2 The EAMG rat model described above was grouped in the same way. Figure 2 (Solvent control group, 5 mg / kg Pirtobrutinib group, 25 mg / kg Pirtobrutinib group), n=6 in each group. (a) Representative flow cytometry plots showing B220 from spleen (SP, top row) and lymph nodes (LN, bottom row). + PNA in B cell population + Gating strategy of GCB cells. Samples were obtained from three groups of EAMG rats. Numbers within the gates represent PNA. + Cells in B220 + Percentage of B cells. (b, c) B220 in spleen (b) and lymph nodes (c) of each treatment group. + PNA in B cell population + Quantitative analysis of the percentage of GCBs. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using unpaired Student's t-test. ns indicates no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data represent the results of three independent experiments.

[0051] In conclusion, we can see that: 1. Specific selection and characteristics of the drug in this application: The BTK inhibitor selected in this invention, Pirtobrutinib, is a third-generation BTK inhibitor with a highly selective and non-covalently reversible binding mode. Published clinical data (e.g., the BRUIN study in oncology and the FDA-approved drug label) show that Pirtobrutinib has a significantly different safety profile compared to other BTK inhibitors that have failed in clinical development in the field of neuroimmunology, particularly with its extremely low risk of hepatotoxicity. According to the LiverTox database of the National Institutes of Health (NIH), Pirtobrutinib "has not been associated with clinically significant cases of acute liver injury."

[0052] 2. Newly established therapeutic effects: This invention, through a study in an experimental autoimmune myasthenia gravis (EAMG) rat model, demonstrates for the first time the significant therapeutic effect of pirtobrutinib on myasthenia gravis. Experimental results show that administration of pirtobrutinib significantly improves clinical symptoms, reduces pathogenic autoantibody levels, and protects the structural integrity of the neuromuscular junction.

[0053] The technical solution combines the known good safety characteristics of Pirtobrutinib with the newly discovered therapeutic effectiveness of Pirtobrutinib in the field of MG, solving the problem in the prior art that efficacy and safety cannot be achieved simultaneously.

[0054] Safety endpoint: [Information clarification]: The core of this study is to verify the therapeutic efficacy of Pirtobrutinib in the MG model. Although this study does not directly include detection of liver function biochemical indicators (such as ALT, AST) or liver histopathology of experimental animals, the proposed scheme of the present application is based on the good safety record of Pirtobrutinib, which has been proven to be superior to other BTK inhibitors in a large number of published preclinical and clinical studies. The success of this study confirms that this "safer option" is also an "effective option".

[0055] Those skilled in the art should understand that the method described in the present application is not limited to the examples described in the specific embodiments, and the above specific description is only for the purpose of explaining the present application and is not intended to limit the present application. Those skilled in the art can derive other embodiments from the technical solution of the present application, which also belong to the technical innovation range of the present application, and the protection scope of the present application is defined by the claims and their equivalents.

Claims

1. The use of Pirtobrutinib in the preparation of drugs for patients with reduced antibody subset secretory cell numbers in myasthenia gravis.

2. The use of Pirtobrutinib in the preparation of drugs for reducing B cell activation phenotype in patients with myasthenia gravis.

3. The use of Pirtobrutinib in the preparation of drugs for reducing serum AChR antibody levels in patients with myasthenia gravis.

4. Application of Pirtobrutinib in the preparation of drugs for inhibiting BTK signaling in patients with myasthenia gravis.

5. Use of Pirtobrutinib in the preparation of a medicine for improving clinical symptoms in patients with myasthenia gravis, including weight gain, clinical score, and recovery of grip strength.

6. The use of Pirtobrutinib in the preparation of drugs for improving neuromuscular junction pathology in patients with myasthenia gravis.

7. The application as described in claim 1, characterized in that: In patients with myasthenia gravis, the number of antibody subset secretory cells is reduced in the peripheral blood, spleen, and lymph nodes.

8. The application as described in claim 2, characterized in that: In patients with myasthenia gravis, the sites of decreased B-cell activation phenotype are peripheral blood, spleen, and lymph nodes.

9. The application as described in any one of claims 1-8, characterized in that: The drug is in oral dosage form. The dosage of Pirtobrutinib is 5 mg / kg to 25 mg / kg.

10. A drug for treating myasthenia gravis, characterized in that: For use in any of the applications described in claims 1-8.

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