Artemisinin derivative and application thereof in medicine for treating multiple sclerosis

Abstract

The invention relates to the field of biological medicine, in particular to an artemisinin derivative and application of the artemisinin derivative in medicine for treating multiple sclerosis. The artemisinin derivative is characterized in that the structural formula of the artemisinin derivative is as follows: # imgabs0 #. The traditional Chinese medicine composition can become an effective medicine for curing or relieving multiple sclerosis patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an artemisinin derivative and its application in a drug for treating multiple sclerosis. Background Art

[0002] Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination of the central nervous system. Approximately 2.8 million people worldwide suffer from MS, and it is also one of the leading causes of non-traumatic disability in young and middle-aged people. As people's understanding of MS continues to deepen, effective treatments such as immunomodulation, antibody therapy and stem cell transplantation have been used clinically. However, current treatments still have some limitations and challenges, including issues such as tolerability, safety and durability of efficacy. In addition, since the clinical phenotypes of MS vary, clinical treatment must take into account the characteristics and severity of the disease and the individual needs of patients. Therefore, the development of new therapeutic drugs can help fill the gaps in existing treatments and provide more options and more effective treatment options.

[0003] Given the central role of immune responses in the disease, immunotherapy drugs are a promising approach for treating multiple sclerosis. CD4+T helper (Th) cells, especially IL-17A-producing Th17 cells and IFN-γ-producing Th1 cells, are closely associated with the pathological changes of multiple sclerosis. In the early stages of the onset of multiple sclerosis, T cells in the peripheral immune system are abnormally activated and produce immune responses to self-antigens. These autoreactive Th1 and Th17 cells migrate to the central nervous system and disrupt the blood-brain barrier under the action of cytokines, chemokines, and integrins. Infiltrating T cells activate macrophages and microglia in the central nervous system, triggering an inflammatory cascade in the central nervous system, ultimately leading to demyelination and neurodegeneration. Therefore, intervening and regulating immune responses by targeting T cells is an effective strategy for treating multiple sclerosis.

[0004] Artemisinin is a sesquiterpene lactone compound with a peroxide bridge structure obtained from the Asteraceae plant Artemisia annua L. It does not contain a nitrogen heterocyclic structure. The structural modification of artemisinin is carried out on the basis of retaining the peroxide bridge. Artemisinin and its derivatives are safe and effective drugs for the treatment of malaria. In addition to antimalarial effects, artemisinin and its derivatives also have strong immunomodulatory properties and have potential in the treatment of autoimmune diseases. It has been reported that artemisinin can improve the disease symptoms of multiple sclerosis mouse models by regulating the balance of Th1 and Th2. However, artemisinin has low oral bioavailability and short half-life, which limits its therapeutic effect in chronic diseases. In order to improve its absorption and metabolic properties and enhance drug efficacy, people have obtained a series of derivatives through structural modification. Artemisinin derivatives such as dihydroartemisinin, artesunate and SM934 have been used to treat various experimental autoimmune and inflammatory models. In view of the potential of artemisinin and its derivatives in autoimmune diseases, further optimization based on the structure of artemisinin can provide new options for the treatment of autoimmune diseases. Summary of the invention

[0005] In order to overcome the defects of the prior art, an effective biological drug component that can be used to cure or alleviate multiple sclerosis patients is provided. The present invention discloses an artemisinin derivative and its use in drugs for treating multiple sclerosis.

[0006] The present invention achieves the purpose of the invention through the following technical solutions:

[0007] An artemisinin derivative TPN10519, characterized in that: the structural formula is as shown in formula (A):

[0008]

[0009] The use of the artemisinin derivative in drugs for treating multiple sclerosis is characterized in that when used, the artemisinin derivative is dissolved in a sodium carboxymethyl cellulose solution (ie, CMC-Na) with a mass percentage concentration of 0.5% to form a derivative combination solution.

[0010] The use of the artemisinin derivative TPN10519 in the drug for treating multiple sclerosis is characterized in that when used, the dosage of the derivative combination solution is 20 mg to 200 mg per kilogram of body weight.

[0011] In order to effectively improve the physicochemical properties and drug-making ability of artemisinin, the present invention has modified artemisinin on the basis of retaining the peroxide bridge structure, and generated the artemisinin derivative TPN10519. TPN10519 can significantly alleviate the disease symptoms of multiple sclerosis mouse model. The technical scheme of the present invention shows that TPN10519 can reduce the number of CD4+T cells and their subgroups Th1 and Th17 cells infiltrating the central nervous system. In addition, flow cytometry and transcriptome analysis showed that TPN10519 can inhibit the activation of the peripheral immune system of mice in the early stage of the disease. In vitro differentiation confirmed that TPN10519 can effectively inhibit the differentiation of inflammatory Th1 and Th17 cells without affecting anti-inflammatory Treg cells. In summary, TPN10519 disclosed in the present invention can become an effective drug for curing or alleviating patients with multiple sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a comparative schematic diagram showing that TPN10519 treatment can alleviate the clinical symptoms of EAE.

[0013] Figure 2 This is a comparative diagram showing that TPN10519 can reduce T cells infiltrating the central nervous system of EAE mice.

[0014] Figure 3 This is a comparative diagram showing that TPN10519 can inhibit the activation of Th1 and Th17 cells in the spleen of EAE mice in the early stage of the disease.

[0015] Figure 4 This is a comparative diagram of the effect of TPN10519 on the activation of T cells in the lymph nodes of early EAE mice.

[0016] Figure 5 This is a comparative schematic diagram of TPN10519 inhibiting the activation of spleen inflammatory pathways in early EAE mice.

[0017] Figure 6 This is a comparative diagram showing that TPN10519 does not affect the survival and activation of T cells.

[0018] Figure 7 This is a comparative schematic diagram of TPN10519 inhibiting the in vitro differentiation of Th1 and Th17 cells. DETAILED DESCRIPTION

[0019] The present invention is further described below by means of specific examples.

[0020] Example 1

[0021] An artemisinin derivative TPN10519, the structural formula of which is shown in formula (A):

[0022]

[0023] When this embodiment is applied to the drug for treating multiple sclerosis, the artemisinin derivative TPN10519 is dissolved in a sodium carboxymethyl cellulose solution (ie, CMC-Na) with a mass percentage concentration of 0.5% to form a derivative combination solution, and the dosage of the derivative combination solution is 20 mg to 200 mg per kilogram of body weight.

[0024] The specific application process is as follows:

[0025] I. Mice:

[0026] Wild-type C57BL / 6 mice (male, 6-8 weeks old) were purchased from Nanjing Jicui Pharmaceutical Co., Ltd. Mice were housed in a specific pathogen-free facility at the Animal Health Center of Tongji University. All animal experimental protocols were approved by the Animal Ethics Committee of Tongji University.

[0027] II. EAE induction:

[0028] On day 0, each mouse was subcutaneously injected with 200ul of 200μg MOG35-55

[0029] Emulsion of (MEVGWYRSPFSRVVVHLYRNGK) peptide and complete Freund's adjuvant (containing 5 mg / mL heat-killed Mycobacterium tuberculosis H37Ra). On days 0 and 2, each mouse was injected intraperitoneally with 200 mg of pertussis toxin. Disease scores and body weights were recorded daily. EAE clinical scores were evaluated as follows: 0: no disease symptoms; 1: tail weakness; 2: tail weakness and unilateral hindlimb weakness; 3: bilateral hindlimb paralysis; 4: hindlimb paralysis and forelimb weakness; 5: moribund or dead.

[0030] II. TPN10519 Treatment:

[0031] TPN10519 was dissolved in 0.5% sodium carboxymethylcellulose (CMC-Na). From the beginning of immunization to the end of the experiment, the oral doses were 20, 60 and 200 mg / kg, respectively. The control group was given an equal volume of 0.5% CMC-Na. III. Histopathological analysis:

[0032] The mice were anesthetized and perfused with 0.9% sodium chloride solution to remove residual blood. The spinal cord was then perfused with 4% paraformaldehyde to fix the spinal cord. The lumbar cord of the mice was isolated and placed in 4% paraformaldehyde at 4°C overnight. The tissue was then embedded in paraffin, sectioned, and stained with H&E and LFB.

[0033] Isolation of CNS-infiltrating T cells

[0034] Mice were perfused with 0.9% sodium chloride solution to remove residual blood. The brain and spinal cord were separated and collected for grinding and filtered through a 70 micron cell strainer on ice. The obtained cells were centrifuged at 500g and 4°C for 10 minutes and then resuspended in 8mL37% percoll. 4mL70% percoll was slowly added to the cell suspension to avoid damaging the interface layer. Subsequently, centrifuged at 780g (acceleration and deceleration were both 0) for 25 minutes at 25°C. Cells at the 37%-70% percoll interface were collected and analyzed using a flow cytometer.

[0035] IV. Flow Cytometry:

[0036] For intracellular cytokine staining, cells were stimulated for 4 h in advance with 50 ng / mL phorbol 12-myristate13-acetate (PMA), 750 ng / mL ionomycin, and 5 μg / mL brefeldinA (all from Sigma Aldrich). Cells were surface stained with anti-mouse CD4 monoclonal antibody (PE-Cyanine7, eBioscience) and anti-mouse CD8 monoclonal antibody (FITC, eBioscience) according to the manufacturer's recommendations. After surface staining, the cells were permeabilized / fixed and then subjected to intracellular staining. Intracellular staining was performed using anti-mouse IL-17 monoclonal antibody (PE, eBioscience) and anti-mouse IFN-γ monoclonal antibody (APC, Biolegend). Data provided by BD FACSVerse TM Flow cytometry was used to acquire and analyze the data.

[0037] V. Enzyme-linked immunosorbent assay (ELISA):

[0038] On the 10th day of EAE modeling, mouse splenocytes were restimulated with MOG35-55 peptide for 48 hours and the supernatant was collected. The IFN-γ, IL-17A, TNF-α, GM-CSF and IL-6 content in the supernatant was determined by ELISA kit (eBioscience) according to the manufacturer's instructions. The absorbance of the sample was read and the concentration of the analyzed protein was calculated according to the standard curve.

[0039] VI. RNA Sequencing Analysis:

[0040] On the 10th day of EAE modeling, total spleen RNA was isolated using TRIzol reagent. RNA extraction, library construction and quality control were completed by Beijing Tiangen Biotechnology Co., Ltd. The constructed RNA-seq library was sequenced using an illumina sequencer. Gene expression is expressed in FPKM (Fragments Per Kilobase per Million). Genes with a significance threshold of p<0.05 and a change of at least 2 times were selected as differentially expressed genes. GO functional analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes. Bubble plots and GSEA enrichment analysis were drawn using the data analysis and visualization online platform (https: / / www.bioinformatics.com.cn). VII. In vitro differentiation of CD4+T cells:

[0041] Naive CD4+ T cells were isolated from the spleens of 7-week-old wild-type C57BL / 6 mice and isolated using a magnetic bead sorting kit ( Untouched TMThe cells were purified using Mouse CD4 Cells Isolation Kit (Invitrogen) and cultured in complete RPMI1640 medium containing 10% FBS, 50 μM β-mercaptoethanol and 100 U / mL penicillin / streptomycin (Biosharp). Different Th cell differentiation requires the addition of corresponding cytokines and blocking antibodies: Th1 cell differentiation requires 2μg / mL anti-mouse CD3 antibody (Bio X Cell), 2μg / mL anti-mouse CD28 antibody (Bio X Cell), 10μg / mL anti-mouse IL-4 antibody (Bio X Cell) and 10ng / mL IL-12 (PeproTech); Th17 cell differentiation requires 2μg / mL anti-mouse CD3 antibody (Bio X Cell), 2μg / mL anti-mouse CD28 (Bio X Cell), 10μg / mL anti-mouse IFN-γ antibody (Bio X Cell), 10μg / mL anti-mouse IL-4 antibody (Bio X Cell), 3ng / mL TGF-β1 (PeproTech), 30ng / mL IL-6 (PeproTech), 10ng / mL IL-1β (PeproTech), 10ng / mL TNF-α (PeproTech) and 10ng / mL IL-β (Biolegend). 23 (Biolegend); Treg cell differentiation requires 2 μg / mL anti-mouse CD3 antibody (Bio X Cell), 2 μg / mL anti-mouse CD28 (Bio X Cell) antibody, 10 μg / mL anti-mouse IFN-γ antibody (Bio X Cell), 5 ng / mL TGF-β1 (PeproTech) and 10 ng / mL IL-2 (PeproTech). Cells were cultured under appropriate differentiation conditions for 72 hours.

[0042] VIII. Statistical Analysis:

[0043] Statistical analysis was performed using GraphPad PRISM 8.0. Data are presented as mean ± SEM. Comparisons between two groups were performed using Student's t-test. One-way ANOVA was used for comparisons between three or more groups. Comparisons of EAE scores and body weight were performed using two-way ANOVA. p < 0.05 was considered statistically significant.

[0044] IX. Results:

[0045] IX.1TPN10519 can alleviate clinical symptoms and pathological changes in EAE mice:

[0046] Artemisinin and its derivatives have been found to have therapeutic potential in multiple sclerosis and other autoimmune disease models. In order to further improve its drug activity and efficacy, this example generates a new artemisinin derivative TPN10519, formula (A). EAE is the most commonly used mouse model for multiple sclerosis, providing a reliable basis for disease research and treatment. Therefore, this example uses the EAE model to verify the pharmacological efficacy of TPN10519 on multiple sclerosis. To induce EAE, MOG35-55 peptide was injected subcutaneously on the back of mice on day 0, and PTX ( Figure 1 B). Mice were orally administered TPN10519 from day 0, and CMC-Na was used as a vehicle control ( Figure 1 B). Compared with the vehicle group, 60 mg / kg and 200 mg / kg TPN10519 significantly slowed down the weight loss and alleviated the clinical symptoms of EAE mice ( Figure 1 C and 1D). In the final stage of EAE, inflammatory cells infiltrate the central nervous system and cause demyelination. Therefore, in this example, the spinal cord of mice was removed on the 21st day for pathological staining to observe the infiltration of inflammatory cells and demyelination. The results of H&E staining showed that 60mg / kg TPN10519 could reduce the inflammatory infiltration of the central nervous system ( Figure 1 E and 1F). Luxor Fast Blue (LFB) histological staining showed that TPN10519 limited the extent of demyelination in EAE mice ( Figure 1 G, 1H). Taken together, these data indicate that TPN10519 treatment can alleviate clinical symptoms and reduce the infiltration of inflammatory cells and the extent of demyelination in EAE mice.

[0047] Figure 1 This is a schematic diagram of the index control coordinates showing that TPN10519 treatment can alleviate the clinical symptoms of EAE. Figure 1 middle:

[0048] B: Schematic diagram of EAE mouse model and experimental design;

[0049] C: Body weight of EAE mice;

[0050] D: Clinical scores of EAE mice: vehicle (0.5% CMC-Na), 20 mg, 60 mg, or 200 mg / kg TPN10519 were orally administered daily;

[0051] E: Representative H&E staining results of spinal cord sections of control (0.5% CMC-Na) and 60 mg / kg treated EAE mice (day 21), scale bar 100 μm;

[0052] F: Image Pro plus6.0 was used to quantify the infiltrating inflammatory cells;

[0053] G: Representative spinal cord section LFB staining results of EAE mice in the control group (0.5% CMC-Na) and the 60 mg / kg treatment group (day 21), scale bar 100 μm;

[0054] H: The demyelinated fraction was quantified using Image Pro plus 6.0, and the data are expressed as mean ± SEM.

[0055] Statistical analysis was performed using two-way ANOVA (C and D) and unpaired Student’s t-test (H and N), *p<0.05, **p<0.01, ***p<0.001.

[0056] TPN1051 reduces Th1 and Th17 cells infiltrating the central nervous system of EAE mice:

[0057] Th1 and Th17 cells are subsets of CD4+T cells and are one of the main driving factors in the pathological process of multiple sclerosis and EAE. It has been reported that Th1 and Th17 cells infiltrate the central nervous system of EAE mice and induce inflammation. Therefore, this example isolated central nervous system infiltrating lymphocytes on the 21st day of EAE modeling and performed flow cytometry analysis. Through this example, it can be found that:

[0058] The total number of CNS-infiltrating lymphocytes was significantly reduced in EAE mice treated with TPN10519. Figure 2 As shown in B;

[0059] The number of CD4+T cells in the TPN10519 treatment group was significantly reduced. Figure 2 As shown in A and C;

[0060] In addition, the number of Th1 cells in the TPN10519-treated group was reduced compared with the vehicle group, as shown in Figure 2 As shown in A, and C to E;

[0061] The Th17 cells in the TPN10519 group also tended to decrease, e.g. Figure 2 As shown in D and F.

[0062] These results suggest that TPN10519 treatment reduces the number of lymphocytes infiltrating the CNS of EAE mice.

[0063] Figure 2 middle:

[0064] A: Representative flow cytometry of CD4+ cells isolated from the central nervous system of EAE mice (day 21);

[0065] B: Total number of infiltrating lymphocytes isolated from the central nervous system of EAE mice (day 21);

[0066] C: Total number of CD4+ lymphocytes isolated from the central nervous system of EAE mice (day 21);

[0067] D: Representative flow cytometry of IL-17A+ and IFN-γ+ cells isolated from the CNS of EAE mice (day 21);

[0068] F: Statistical analysis of the percentage of IL-17A+ and IFN-γ+ cells.

[0069] Data are presented as mean ± SEM. Unpaired Student's t-test (BF) was used for statistical analysis. *P<0.05. IX.2 TPN10519 restricts the activation of Th1 and Th17 cells in the spleen in the early stage of EAE.

[0070] In the early stages of EAE, autoreactive T cells are activated in the peripheral immune system and migrate to the central nervous system through the blood circulation. Dynamic studies of T cells in EAE mice found that the proportion of CD4+T cells in the blood reached a peak 10 days after EAE induction.

[0071] In addition, in this example, the spleen of mice was extracted on the 10th day of EAE induction and flow cytometry analysis was performed to observe the activation of immune cells, such as Figure 3 As shown in A;

[0072] The results of flow cytometry analysis showed that TPN10519 did not affect the ratio of CD4+ and CD8+ cells. Figure 3 As shown in B;

[0073] However, TPN10519 significantly reduced the proportion of Th1 and Th17 cells in the spleen, as Figure 3 As shown in C;

[0074] Th1-like Th17 cells that secrete both IL-17A and IFN-γ are considered to be one of the key pathogenic factors of EAE.

[0075] It can be seen from this example that TPN10519 also reduces the proportion of IL-17A+IFN-γ+ double positive cells, such as Figure 3 As shown in C;

[0076] Inflammatory cytokines produced by mouse spleen, such as IL-6, TNF-α and GM-CSF, promote the activation and proliferation of peripheral T lymphocytes. In this example, MOG35-55 peptide was used to re-stimulate isolated splenocytes, and the supernatant was collected to detect the expression of inflammatory factors.

[0077] Consistent with the flow cytometry results, the expression of IL-17A and IFN-γ in the TPN10519 group was significantly decreased. Figure 3 As shown in D and E;

[0078] In addition, TPN10519 can also reduce the expression of inflammatory mediators such as IL-6, TNF-α and GM-CSF in the spleen cells of EAE mice. Figure 3 As shown in F and G;.

[0079] In conclusion, TPN10519 reduced the activation of Th1 and Th17 cells and decreased the expression of inflammatory factors in the spleen of EAE mice.

[0080] Figure 3 middle:

[0081] A: Schematic diagram of early EAE mouse model and experimental design;

[0082] B: Flow cytometry detection of the ratio of CD4+ and CD8+ cells in the spleen of EAE mice (day 10). Representative flow cytometry images (left). Cell ratio (right);

[0083] C: Flow cytometry detection of the proportion of IL-17A+ and IFN-γ+ cells in the spleen of EAE mice (day 10). Representative flow cytometric images (left). Cell proportion (right);

[0084] D~H: The spleen of EAE mice (day 10) was restimulated with MOG35-55 peptide for 48 hours, and the cell supernatant was collected and the expression levels of D~H were detected by ELISA:

[0085] D: IFN-γ,

[0086] E:IL-17A,

[0087] F: TNF-α,

[0088] G:GM-CSF,

[0089] H:IL-6.

[0090] Data are presented as mean ± SEM. Statistical analysis was performed using unpaired Student's T test (BH). *p<0.05, **p<0.01, ***p<0.001.

[0091] IX.3TPN10519 inhibits the development of immune cells in lymph nodes:

[0092] In the early stages of EAE, T cells in peripheral lymph nodes will also migrate to the central nervous system after antigen activation. Therefore, this example further observed the effect of TPN10519 on T cells in lymph nodes of EAE mice. Figure 4 As shown in A.

[0093] Consistent with the flow cytometry results of the spleen, TPN10519 did not affect the ratio of CD4 and CD8+ T cells in DLN, as Figure 4 As shown in B. Compared with the control group, the proportion of Th1 and Th17 cells in DLN of the TPN10519-treated group decreased, as shown in Figure 4 As shown in C. Mesenteric lymph nodes are also the site of immune cell activation and proliferation. Unlike the spleen and DLN, TPN10519 downregulated the proportion of CD4+T cells but did not affect the proportion of Th1 and Th17 cells, as shown in Figure 4 As shown in D and E. These results indicate that TPN10519 affects the activation of immune cells in lymph nodes in the early stage of EAE.

[0094] Figure 4 middle:

[0095] A: Schematic diagram of the early EAE mouse model and experimental design;

[0096] B: Flow cytometry detection of the ratio of CD4 and CD8 cells in DLN of EAE mice (day 10), representative flow cytometry images (left), cell frequency (right);

[0097] C: Flow cytometry was used to detect the proportion of IL-17A+ and IFN-γ+ cells in DLN of EAE mice (day 10), representative flow cytometric images (left), and cell frequency (right);

[0098] D: Flow cytometry detection of the ratio of CD4 and CD8 cells in MLN of EAE mice (day 10), representative flow cytometric images (left), cell frequency (right);

[0099] E: Flow cytometry analysis of the proportion of IL-17A+ and IFN-γ+ cells in MLN of EAE mice (day 10), representative flow cytometric images (left), and cell frequency (right).

[0100] Data are presented as mean ± SEM. Statistical analysis was performed using unpaired Student's T test (BH). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0101] IX4.TPN10519 inhibits the activation of inflammatory pathways in early EAE:

[0102] In order to further explore the specific mechanism of TPN10519 in treating EAE, this example performed transcriptome sequencing on the spleen of mice in the early stage of EAE. Compared with the control group, this example shows that 1308 genes were downregulated and 990 genes were upregulated in the TPN10519 group, such as Figure 5 As shown in A.

[0103] Among them, inflammatory genes such as Il17a, Il1b, and Irf4, which have been reported to be associated with the pathogenesis of multiple sclerosis, were downregulated in the TPN10519 group, e.g. Figure 5 As shown in A.

[0104] The results of KEGG and GO enrichment analysis of differentially expressed genes showed that the inflammation-related signaling pathway was one of the most enriched pathways, such as Figure 5 As shown in B and D.

[0105] Subsequently, this example performed GSEA enrichment analysis on the signal pathways with significant differences.

[0106] The results showed that cytokine-cytokine receptor interactions and MAPK signaling pathways were negatively correlated with TPN10519 treatment, which were reported to control T cell activation and effector function, such as Figure 5 As shown in C and E.

[0107] Regulatory T (Treg) cells are primarily responsible for maintaining immune tolerance and preventing excessive immune responses. Unlike effector Th1 and Th17 cells, Treg cells rely primarily on oxidative phosphorylation rather than glycolysis to maintain their immunosuppressive function.

[0108] This example shows that TPN10519 administration is positively correlated with oxidative phosphorylation, which suggests that it may enhance the immunosuppressive function of early EAE, such as Figure 5 As shown in F.

[0109] In addition, GO enrichment analysis showed that the regulation of immune effector processes was positively correlated with TPN10519, e.g. Figure 5 As shown in G in .

[0110] In summary, the transcriptomic results indicate that TPN10519 exerts a therapeutic effect by inhibiting the activation of immune responses in the early stage of EAE, which is consistent with previous wet experiment results.

[0111] Figure 5 Shown is the extraction of total RNA from spleens of control and TPN10519-treated (200 mg / kg) EAE mice (day 10) for RNA-seq analysis. Figure 5 middle:

[0112] A: Volcano plot of gene expression differences;

[0113] B: KEGG enrichment analysis of differentially expressed genes between the TPN10510 treatment group and the control group;

[0114] D: GO enrichment analysis;

[0115] GSEA plots of significant enrichment in KEGG pathways;

[0116] C: Cytokine-cytokine receptor interaction;

[0117] E: MAPK signaling pathway;

[0118] F: oxidative phosphorylation;

[0119] GSEA plot of significantly enriched GO biological processes, regulation of immune effector processes.

[0120] IX4.TPN10519 inhibits the differentiation of Th1 and Th17 cells in vitro:

[0121] Previous experiments and transcriptional results confirmed that TPN10519 can alleviate disease symptoms by inhibiting early inflammatory activation in EAE. In the early stages of multiple sclerosis and EAE models, T cell survival, activation, and differentiation are key factors leading to inflammation. Therefore, we tested the effects of TPN10519 on the above key nodes in vitro. PI staining results showed that TPN10519 did not affect T cell survival, such as Figure 6 As shown in A in Figure 1. CD69 and CD25 are markers of early and late T cell activation, respectively. After 24 hours of anti-CD3 and anti-CD28 stimulation, it can be observed that T cells are significantly activated, and the proportion of CD25+ and CD69+ cells is upregulated, as shown in Figure 1. Figure 6 However, TPN10519 treatment did not affect T cell activation, as shown in B. Figure 6 As shown in B. Subsequently, this example observed the effect of TPN10519 on T cell differentiation. The spleen of 7-week-old mice was isolated and naive CD4+ T cells were sorted using magnetic beads. Cytokines and antibodies corresponding to different Th cells were added to the cells and cultured for 72 hours. Figure 7 As shown in A in FIG. The cells were collected and subjected to flow cytometry analysis. The flow cytometry analysis results showed that TPN10519 inhibited the differentiation of Th1 and Th17 cells in a dose-dependent manner. Figure 7 In addition, TPN10519 also reduced the protein levels of IL-17A and IFN-γ in the supernatant, as shown in Figure 2B and D. Figure 7 As shown in C and E. It should be noted that TPN10519 does not affect the differentiation of Treg cells responsible for maintaining immune balance, such as Figure 7 As shown in F.

[0122] In summary, the in vitro differentiation results suggest that TPN10519 may play a therapeutic role in autoimmune diseases by affecting the differentiation of effector T cells rather than their survival and activation.

[0123] Figure 6 Shown are C57BL / 6 mouse splenocytes stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours. Figure 6 middle:

[0124] A: PI staining was used to detect cell survival;

[0125] B: Flow cytometry detection of CD4+CD25+ and CD4+CD69+ cells, representative flow cytometric images (left), cell frequencies (right).

[0126] Data are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (AB). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns (not significant).

[0127] Figure 7 middle:

[0128] A: Schematic diagram of CD4+T cell differentiation in vitro. In the presence of solvent (CMC-Na) and TPN10519 (10nM, 100nM, 1000nM), naiveCD4 cells differentiate into different T cell subsets in vitro.

[0129] B: Flow cytometry detection of in vitro differentiated Th1 cells (IFN-γ+), representative flow cytometric images (left), frequency of IFN-γ+ cells (right);

[0130] C: IFN-γ in the supernatant of in vitro differentiated Th1 cells was detected by ELISA;

[0131] D: Flow cytometry was used to detect the frequency of IL-17A+ cells (right) differentiated Th17 cells in vitro;

[0132] E: IL-17A in the supernatant of in vitro differentiated Th17 cells was detected by ELISA;

[0133] F: Flow cytometry detection of Treg cells differentiated in vitro (Foxp3+);

[0134] Representative flow cytometry images (left), frequency of Foxp3+ cells (right).

[0135] Data are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (BF). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0136] IX.4 Summary:

[0137] Multiple sclerosis is an autoimmune disease of the nervous system mediated by inflammation, and artemisinin and its derivatives are reported to have therapeutic potential in autoimmune diseases. Here, it can be seen from this example that a new artemisinin derivative TPN10519 can effectively reduce infiltrating inflammatory cells in the central nervous system, thereby alleviating the clinical symptoms of EAE. Subsequently, experiments and transcriptomes showed that TPN10519 treatment limited the activation of the peripheral immune system in the early stages of EAE. Finally, it can be seen from this example that TPN10519 may affect the activation of the peripheral immune system by affecting T cell differentiation. These results show the therapeutic potential of TPN10519 for multiple sclerosis and provide new options for the future treatment of autoimmune diseases.

[0138] In the pathological process of multiple sclerosis or EAE, the activation of peripheral T cells is essential for initiating and maintaining autoimmune responses against myelin proteins in the central nervous system. Activated Th1 cells activate macrophages and microglia by producing proinflammatory cytokines such as IFN-γ and TNF-α, thereby exacerbating local immune responses. Th17 cells not only enhance the inflammatory response of the central nervous system, but also directly target oligodendrocytes to accelerate the process of myelin shedding. In the study of this embodiment, it can be seen that TPN10519 can inhibit the activation of peripheral Th1 and Th17 cells during early EAE. In addition, the results of in vitro differentiation also show the inhibitory effect of TPN10519 on Th1 and Th17 cells. These results indicate that TPN10519 may improve the disease severity of EAE mainly by inhibiting Th1 and Th17 cells.

[0139] As one of the most widely used antimalarial drugs, artemisinin's unique peroxide bridge structure has inspired the development of a variety of derivatives, aiming to improve pharmacokinetic properties, enhance therapeutic effects or reduce side effects. In addition to combating malaria, artemisinin and its derivatives have also shown potential value in the treatment of autoimmune diseases, which has been verified in previous studies. Recent scientific studies have further revealed the mechanism of action of artemisinin and its derivatives in regulating a variety of biological processes. It is particularly noteworthy that these compounds can selectively kill cancer cells by inducing ferroptosis pathways. Ferroptosis is a unique form of programmed cell death driven by lipid peroxidation, accompanied by changes in mitochondrial energy metabolism, oxidative phosphorylation, and ATP generation rates. Consistent with this finding, this example observed through analysis of RNA-Seq data that in cells treated with TPN10519, differentially expressed genes were significantly enriched in ferroptosis-related signaling pathways; at the same time, upregulated genes were mainly concentrated in oxidative phosphorylation-related pathways. Th1 and Th17 cells are overactive immune cells. These mechanisms indicate that TPN10519 can control the development of autoimmune diseases by regulating iron allergy and oxidative phosphorylation, providing a theoretical basis for the development of new treatments.

[0140] In summary, the results of this example show that TPN10519 has shown excellent potential in the treatment of multiple sclerosis and provides a more effective option for the treatment of autoimmune diseases. In addition, the study of the mechanism of action of TPN10519 in multiple sclerosis not only enhances the understanding of the pathological process of the disease, but also lays the foundation for the development of more precise treatment strategies.

Claims

1. An artemisinin derivative, characterized in that: The structural formula is shown in formula (A): ——(A)。 2. The use of the artemisinin derivative as claimed in claim 1 in a drug for treating multiple sclerosis, characterized in that: When in use, the artemisinin derivative is dissolved in a sodium carboxymethyl cellulose solution with a mass percentage concentration of 0.5% to form a derivative combination solution.

3. The use of the artemisinin derivative TPN10519 as claimed in claim 2 in a drug for treating multiple sclerosis, characterized in that: When used, the dosage of the derivative combination solution is 20 mg to 200 mg per kilogram of body weight.

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