Application of artemisinin derivatives TPN10467 and TPN10476 as drugs for preventing and treating autoimmune diseases

Abstract

The invention relates to the field of biological medicines, in particular to application of artemisinin derivatives TPN10467 and TPN10476 as medicines for preventing and treating autoimmune diseases. According to the invention, in an in-vitro CD4 + T cell differentiation experiment, it is observed that the two artemisinin derivatives TPN10467 and TPN10476 can both reduce the differentiation proportion of Th1 and Th17 cells, wherein the differentiation inhibition effect of TPN10467 is better than that of artemisinin. According to the application, an EAE mouse model is selected for in-vivo experiment research, and in the administration treatment of an EAE mouse, the two novel artemisinin derivatives TPN10467 and TPN10476 have a relatively good relieving effect on the clinical phenotype of the EAE, and the infiltration number and demyelination proportion of inflammatory cells in a central nervous system are reduced. In a word, the data of the invention shows that the TPN10467 and the TPN10476 can alleviate the symptoms of the autoimmune diseases, and a new potential drug choice can be provided for the treatment of the autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological medicines, and in particular to the use of artemisinin derivatives TPN10467 and TPN10476 as medicines for preventing and treating autoimmune diseases. Background Art

[0002] Autoimmune disease refers to a disease in which the body's immune response to its own antigens causes damage to its own tissues. Multiple sclerosis (MS) is a chronic demyelinating disease caused by damage to the nerves by immune cells, leading to inflammatory lesions of the central nervous system, resulting in tissue damage and disability symptoms. EAE is an animal disease model with similar clinical, histological and pathological similarities to MS. Since samples from MS patients are not easy to obtain, EAE is an important model for studying the pathological process and pathogenesis of MS, and is of great significance in the study of clinical neuroimmunology. CD4 + The role of T cells in multiple sclerosis has been widely supported by research: Th1 and Th17 cells are directly involved in the pathological process of multiple sclerosis, and two types of CD4 + After T cells are activated in the peripheral immune system and migrate into the CNS, the cytokines they secrete can cause persistent neurological damage.

[0003] As a widely used antimalarial drug, artemisinin therapy has the advantages of good patient tolerance and low price. In addition to its good therapeutic effect on malaria, previous studies have shown that artemisinin also has a variety of anti-inflammatory and immunomodulatory effects. Artemisinin can achieve anti-inflammatory and immunomodulatory functions by inhibiting T cell proliferation and activation, inhibiting B cell activation and antibody production, increasing regulatory T cells, and reducing the release of inflammatory cytokines. Summary of the invention

[0004] In response to the problems of the prior art, the present invention discloses for the first time the therapeutic effects of two newly synthesized artemisinin derivatives on autoimmune diseases.

[0005] In vitro experiments showed that artemisinin derivatives TPN10467 and TPN10476 reduced CD4 + The final ratio of T cells to Th1 and Th17 cells.

[0006] Furthermore, the EAE disease model study found that oral administration of TPN10467 and TPN10476 could alleviate the clinical symptoms of EAE mice.

[0007] Furthermore, the test found that the total number of inflammatory cells in the CNS of mice treated with TPN10467 was significantly reduced, and spinal cord injury was also significantly alleviated.

[0008] In summary, the present work suggests that TPN10467 and TPN10476 are potential drugs for the treatment of autoimmune diseases.

[0009] Beneficial effects of the present invention: Th1 and Th17 cells are key pro-inflammatory subsets of CD4+ T cells, playing an important role in the onset of various autoimmune diseases and inflammation. In the in vitro CD4+ T cell differentiation experiment, the present invention observed that two artemisinin derivatives, TPN10467 and TPN10476, can reduce the differentiation ratio of Th1 and Th17 cells, among which TPN10467 has a better effect in inhibiting differentiation than artemisinin. Multiple sclerosis (MS) is a type of chronic inflammatory neurodegenerative disease, and experimental autoimmune encephalomyelitis (EAE) is the main disease model for studying MS. Studies have shown that damage to neuronal axons in MS patients is the main cause of movement disorders and other disabilities. During the onset of multiple sclerosis, after Th1 and Th17 cells enter the CNS, they establish and maintain an inflammatory environment with other inflammatory cells such as CD8+ effector T cells, leading to the occurrence of demyelination and ultimately loss of neuronal function. Based on the cell experiment, the present invention selected the EAE mouse model for in vivo experimental research. In the drug administration treatment of EAE mice, the present invention found that two new artemisinin derivatives, TPN10467 and TPN10476, had a good alleviating effect on the clinical phenotype of EAE and reduced the number of inflammatory cell infiltration and the proportion of demyelination in the central nervous system. In short, the data of the present invention show that TPN10467 and TPN10476 can alleviate the symptoms of autoimmune diseases and provide new potential drug options for the treatment of autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of TPN10467 and TPN10476 inhibiting Th1 and Th17 cell differentiation in vitro; Figure 2 Schematic diagram of TPN10467 and TPN10476 suppressing clinical symptoms of the disease during the entire course of EAE disease progression; Figure 3 Schematic diagram showing that TPN10467 and TPN10476 can alleviate spinal cord injury in EAE mice; Figure 4Schematic diagram of TPN10467 suppressing clinical symptoms of EAE mice during therapeutic administration; Figure 5 Schematic diagram showing that therapeutic administration of TPN10467 and TPN10476 can alleviate spinal cord injury in EAE mice. DETAILED DESCRIPTION

[0011] The technical solution provided by the present application will be further described below in conjunction with specific embodiments and accompanying drawings. The advantages and features of the present application will become more apparent with the following description.

[0012] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0013] Materials and Methods animal Male C57BL / 6 mice were purchased from Jicui Yaokang (Nanjing, China). All mice were kept under SPF clean conditions. Male mice aged 8–10 weeks were used for animal experiments. All animal experiments were approved by the Animal Ethics Committee of Tongji University.

[0014] EAE induction and treatment Male C57BL / 6 mice (8-10 weeks) were immunized with complete Freund's adjuvant containing inactivated Mycobacterium tuberculosis (H37Ra strain, 5 mg / ml, BD Diagnostics) and myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide (ChinaPeptides), and pertussis toxin (200 ng / mouse, Millipore) was injected on day 0 and day 2 of immunization. The clinical symptoms of the disease in mice were examined every day and scored on a scale of 0-5, as follows: 0, no clinical symptoms; 1, tail paralysis; 2, mild paralysis (unilateral or bilateral hind limb weakness, incomplete paralysis); 3, paraplegia (complete paralysis of bilateral hind limbs); 4, paraplegia and forelimb weakness or paralysis; 5, moribund state or death. In terms of drug treatment, TPN10467 and TPN10476 were dissolved in 0.5% sodium carboxymethylcellulose (CMC-Na) solution, and mice were gavaged with 10, 30, or 100 mg / kg body weight of the drug (200 μl / mouse) every day starting from day 0 after immunization until the end of the experiment. The drug-treated group was given an equal volume of 0.5% CMC-Na solution.

[0015] Histopathological and immunohistochemical analysis Each mouse was deeply anesthetized with 200 μL of 10% chloral hydrate, and then cardiac perfusion was performed with 0.9% NaCl solution to remove peripheral blood from each organ. Tissue samples from the lumbar cord were taken, immersed in 4% paraformaldehyde and fixed overnight at 4°C. The fixed spinal cord tissue was dehydrated, paraffin-embedded and sectioned, and HE staining was used to observe the infiltration of inflammatory cells in the spinal cord, and LFB staining was used to observe the demyelination of the spinal cord. Finally, the pictures taken under the microscope were statistically analyzed using Image-Pro software.

[0016] Cell staining and flow cytometry analysis CD4 collection + T cells were stimulated with PMA (50 ng / ml, Sigma-Aldrich), ionomycin (750 ng / ml, Sigma-Aldrich), and brefeldin A (5 μg / ml, Sigma-Aldrich) at 37°C for 5 hours. Cell surface marker CD4 (1994141, 1:100 dilution, Invitrogen) was stained with antibodies for 30 minutes at 4°C in the dark, and then the cells were resuspended in fixation permeabilization buffer and stained with flow cytometry antibodies for intracellular IL-17A (BioLegend, 506904, 1:100) and IFN-γ (BioLegend, 505810, 1:100 dilution), respectively. Flow cytometry was performed using the BD FACS Verse system and analyzed using Flow Jo V10 software. Isolation of CNS-infiltrating cells The brain and spinal cord were removed from EAE mice, ground into tissue homogenate, filtered through a 70 μm cell strainer and collected into a 50 ml centrifuge tube. The cells were centrifuged at 500 g for 10 min at 4 °C to obtain the cells. The cells were then resuspended in 8 ml of 37% Percoll solution, and 4 ml of 70% Percoll solution was gently added dropwise. Density gradient centrifugation was performed at 780 g for 25 min at 25 °C. The cells at the 37 / 70% Percoll interface were collected for subsequent analysis.

[0017] CD4 + T cell isolation and in vitro differentiation CD4 + T cells, 4×10 5Cells were seeded into 96-well plates at a density of 1.54 μg / well and cultured in 200 μL of 1640 complete medium containing 10% FBS, 2 mM L-glutamine, and 50 μM β-mercaptoethanol. Anti-CD3 (2 μg / ml, BD Pharmingen) and anti-CD28 (2 μg / ml, BD Pharmingen) antibodies were added to activate the cells, and IL-12 (10 ng / ml, Peprotech) and anti-IL-4 (10 μg / ml, BD Pharmingen) were added to induce Th1 cell differentiation. For Th17 cell differentiation, in addition to anti-IL-4 (10 μg / ml) and anti-IFN-γ (10 μg / ml, BD Bioscience, 551216) antibodies, a mixture of Th17 cell differentiation factors, including IL-6 (30 ng / ml), TGF-β1 (3 ng / ml), TNF-α (10 ng / ml) and IL-1β (10 ng / ml), was added. Different concentrations of TPN10467 or TPN10476 were added together with the cytokines, and the culture supernatant and cells were collected after 3 days of culture to detect their effects on CD4 + Effects of T cell subtype differentiation.

[0018] ELISA to detect cytokine levels The culture supernatant or serum was diluted at a certain ratio, and the concentration of cytokines in the culture supernatant or serum was detected using relevant ELISA kits (Invitrogen) according to the manufacturer's instructions.

[0019] Statistical analysis The statistical differences among the EAE mouse treatment groups were analyzed using two-way ANOVA test, and other statistical differences were analyzed using Student's t test. The statistical graphs are expressed as mean±SEM, and P<0.05 was considered statistically significant.

[0020] result Embodiment 1: TPN10467 and TPN10476 inhibit Th1 and Th17 cell differentiation in vitro A large number of studies have confirmed that artemisinin and its derivatives, which also have a sesquiterpene lactone structure, have a certain therapeutic effect on some acute and chronic inflammatory diseases. The present invention is expected to combine artemisinin with different anti-inflammatory molecules, retain the sesquiterpene lactone structure and introduce new groups to achieve better therapeutic effects. The synthesis process of TPN10467 and TPN10476 and the chemical structure are as follows Figure 1As shown in A: The present invention selects two small molecules, 4-trifluoromethoxyphenyl isocyanate and lipoic acid, for synthesizing TPN10467 and TPN10476 with artemisinin respectively. 4-Trifluoromethoxyphenyl isocyanate is often used as an intermediate for synthesizing drugs. The bicyclic heteroarylamine drugs developed with this molecule can be used to treat pain, inflammation and traumatic injuries; and lipoic acid, an antioxidant, is an important cofactor of mitochondrial enzyme complexes. In previous studies, lipoic acid can inhibit MMP-9 activity and the activity and migration ability of T cells, and reduce the incidence of EAE through anti-oxidation.

[0021] CD4 + T cells play a key role in the pathogenesis of multiple sclerosis, among which Th1 cells that secrete IFN-γ and Th17 cells that secrete IL-17A promote spinal cord damage and thus aggravate the development of the disease. + The in vitro differentiation experiment of T cells was used to preliminarily evaluate the effects of the two types of artemisinin derivatives. The spleen of 7-8 week old C57BL / 6 mice was taken and CD4 + T cell sorting to obtain undifferentiated naive CD4 + T cells were cultured under the conditions of Th1 and Th17 cell differentiation, and different concentrations of artemisinin, TPN10467 or TPN10476 (300 nM, 1 μM, dissolved in DMSO) were given in the culture medium. The control group was added with an equal volume of DMSO. After 3 days of differentiation, flow cytometry analysis showed that the proportion of Th1 and Th17 cells in the TPN10467-treated group and the TPN10476-treated group was significantly reduced compared with the control group. Further analysis showed that the higher the concentration of artemisinin derivatives, the more obvious the degree of inhibition of T cell differentiation into Th1 and Th17 cells. Figure 1 Medium B and Figure 1 As shown in C and D. At the same time, after comparing with the statistical results of the artemisinin treatment group, the present invention found that TPN10467 had an effect on CD4 + The inhibitory effect on T cell differentiation in vitro is more significant than that of artemisinin.

[0022] like Figure 1 : A: Synthesis reactions and structural formulas of TPN10467 and TPN10476.

[0023] B: Naive CD4 isolated from the spleen of 7-8 week old C57BL / 6 mice +T cells were induced to differentiate into Th1 and Th17 cells in vitro under different differentiation conditions, and different concentrations (300 nM, 1 μM) of artemisinin, TPN10467, TPN10476 or DMSO as a control were added and cultured for 3 days. Th1 and Th17 cells were detected and analyzed by intracellular IFN-γ and IL-17A staining by FACS. IFN-γ + (C) and IL-17A + (D) Statistical analysis of T cell percentage and cytokine levels in differentiation medium (E, F). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t test). like Figure 1 ZhongE and Figure 1 In Figure F, after detecting the levels of IFN-γ and IL17A in the supernatant of cell culture, the results obtained by the present invention are also consistent with the flow cytometry results.

[0024] Embodiment 2: TPN10467 and TPN10476 alleviate clinical symptoms of EAE The onset of multiple sclerosis is closely related to Th1 and Th17 cells. The results of the present invention show that TPN10467 and TPN10476 can effectively inhibit the differentiation of Th1 and Th17 cells in vitro. Therefore, the present invention further evaluates whether TPN10467 and TPN10476 have a certain therapeutic effect on multiple sclerosis. First, 8-week-old C57BL / 6 male mice were treated with MOG 35-55 The EAE model was induced by immunization with pertussis toxin, and then different doses of TPN10467 or TPN10476 (10 mg / kg, 30 mg / kg, 100 mg / kg) were administered by gavage on the day of immunization. The control group was given the same volume (200 μL) of 0.5% CMC-Na solution. The process was as follows: Figure 2 As shown in A. like Figure 2 : A: Schematic diagram of EAE modeling and oral administration of TPN10467 and TPN10476. B, C: EAE mice were intragastrically administered with TPN10467 or TPN10476 at doses of 10 mg / kg, 30 mg / kg, and 100 mg / kg body weight. The same volume of 0.5% CMC-Na solution was used as a control. The EAE clinical scores were recorded. The data are expressed as mean ± SEM (n = 5) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-way ANOVA test).

[0025] The clinical scores of EAE mice were evaluated every day until the end of the experiment, and a clinical score curve was drawn as shown in FIG. Figure 2 Medium B and Figure 2 As shown in C, the results indicate that both TPN10467 and TPN10476 can intervene in the pathogenesis of EAE.

[0026] Embodiment 3: The present invention selected the TPN10467 (100 mg / kg) administration group and the TPN10476 (30 mg / kg) administration group that showed significant differences in statistical analysis to perform histological analysis on the spinal cord of EAE mice to detect the infiltration of leukocytes in the central nervous system and myelin damage, such as Figure 3 As shown in A, HE staining results showed that the number of inflammatory cell infiltration in the spinal cord of the TPN10467 and TPN10476 groups was significantly reduced compared with the control group. Figure 3 As shown in B, the LFB staining results also showed that TPN10467 and TPN10476 administration can reduce the degree of spinal cord demyelination in mice. Statistical analysis was performed using Image-Pro software. Figure 3 C and Figure 3 D, The reduction of inflammatory cell infiltration and alleviation of demyelination in the spinal cord in the TPN10467 and TPN10476 treatment groups were significantly different from those in the control group.

[0027] In conclusion, the research data indicate that TPN10467 and TPN10476 have certain therapeutic and protective effects against EAE.

[0028] Explanation of the attached figure, Figure 3 middle: A, B: HE and LFB staining sections of the spinal cord in the control, TPN10467 (100 mg / kg), and TPN10476 (30 mg / kg) groups. Scale bars, 200 μm.

[0029] C, D: Statistical analysis of inflammatory cell infiltration and demyelination in the spinal cord of mice treated with the drug throughout the course. 10-20 sections were randomly selected for pathological statistics. Data are mean ± SEM (n = 10), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t test).

[0030] Embodiment 4: TPN10467 and TPN10476 exert EAE-alleviating effects when administered therapeutically The above results show that TPN10467 and TPN10476 alleviated the symptoms of EAE within about 1 month of administration. Considering the clinical situation of patients, the present invention hopes to further explore whether the administration of artemisinin derivatives after the onset of the disease, that is, therapeutic administration, will work. Figure 4 As shown in A, mice were fed according to normal procedures from the beginning of EAE modeling to the onset of EAE. After all mice developed EAE, the present invention divided the mice into three groups, and made the initial average clinical score of each group the same: the control group and the TPN10467 (100 mg / kg) and TPN10476 (30 mg / kg) groups. The drug-treated group was gavaged with 200 μL of drug solution every day, and the control group was given the same volume of 0.5% CMC-Na solution.

[0031] from Figure 4 In the clinical score curve of B, the present invention found that TPN10467 still had a significant alleviating effect on EAE symptoms during the therapeutic administration process; while the clinical score of the TPN10476 group decreased slightly, but there was no significant difference compared with the control group. The present invention took out the brain and spinal cord of EAE mice to isolate immune cells, and performed flow cytometry and pathological section analysis.

[0032] After separating immune cells from CNS homogenate, the present invention uses a hemocytometer to count the total number of cells. Figure 4 Middle C and Figure 4 The flow cytometry results of D showed that CD4 + The proportion of T cell subsets did not show any difference. However, the calculated lymphocytes, CD4 + After comparing the number of T cells and Th1 and Th17 cell subsets, the immune cells in the CNS of mice in the TPN10467 group were significantly reduced compared with the control group, while there was no significant difference between the TPN10476 group and the control group. Figure 4 As shown in E.

[0033] Explanation of the attached figure, Figure 4 middle: A: Schematic diagram of EAE modeling and therapeutic dosing process of TPN10467 and TPN10476. B: EAE mice were intragastrically administered with TPN10467 or TPN10476 at the corresponding dose after onset of the disease. The same volume of 0.5% CMC-Na solution was used as a control. The EAE clinical scores were recorded. The data are expressed as mean ± SEM (n = 5) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-way ANOVA test).

[0034] C, D: Flow cytometry analysis of CD4 + The proportion of T cells and Th1 and Th17 cell subsets.

[0035] E: Lymphocytes in CNS, CD4 + The absolute numbers of T cells and Th1 and Th17 cells. Data are mean ± SEM (n=5), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t test).

[0036] Embodiment 5: The spinal cord samples of EAE mice in the treatment and administration experiment were made into pathological sections for observation and statistics. Figure 5 As shown in the HE staining results of Figure A, the leukocyte infiltration in the spinal cord of the TPN10467 and TPN10476 treatment groups was significantly reduced compared with the control group. Figure 5 The LFB staining results in B also showed that the degree of demyelination in the treatment group was significantly reduced. Figure 5 Middle C and Figure 5 In the statistical results of Figure D, the present invention found that both TPN10467 and TPN10476 can reduce the number of inflammatory cell infiltrations and demyelination injury rate in the spinal cord of EAE mice.

[0037] Explanation of the attached figure, Figure 5 middle: A, B: HE and LFB staining sections of the spinal cord in the control, TPN10467 (100 mg / kg), and TPN10476 (30 mg / kg) groups. Scale bars, 200 μm. C, D: Statistical analysis of inflammatory cell infiltration and demyelination in the spinal cord of EAE mice treated with therapeutic drugs. 10-20 sections were randomly selected for pathological statistics. Data are mean ± SEM (n = 10), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t test).

[0038] discuss Autoimmune diseases refer to a wide range of clinical conditions that result from the destruction of self-immune tolerance, leading to an immune response against one's own targets, which in turn results in tissue lesions and clinical symptoms. Multiple sclerosis is a chronic inflammation of the central nervous system. Studies have shown that damage to axons in multiple sclerosis patients is the main cause of movement disorders and other disabilities. Past studies have shown that artemisinin can alleviate autoimmune diseases in a variety of ways, but the toxicity caused by long-term use makes it difficult to enter clinical use.

[0039] The present invention studies the therapeutic effects of two novel artemisinin derivatives, TPN10467 and TPN10476, on multiple sclerosis. + The T cell subsets are Th1 and Th17 cells. These cells enter the central nervous system and interact with other inflammatory cells such as CD8 + T cells establish and maintain an inflammatory environment. IFN-γ secreted by Th1 cells can directly damage oligodendrocytes. IL-17A secreted by Th17 can promote the expression of proinflammatory cytokines IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor α (TNF-α). These cytokines lead to neuronal demyelination and ultimately loss of normal neural signaling function. + In the in vitro differentiation experiment of T cells, the present invention observed that both TPN10467 and TPN10476 could reduce the differentiation rates of Th1 and Th17 cells, and the inhibitory effect of TPN10467 was better than that of artemisinin.

[0040] Next, the present invention uses the EAE mouse model that simulates the onset of MS to report the effects of TPN10467 and TPN10476 on the disease phenotype. Both TPN10467 and TPN10476 can alleviate the clinical symptoms during the EAE mouse modeling period and reduce the infiltration of inflammatory cells and demyelination in the central nervous system during the full administration and therapeutic administration. These results suggest that TPN10467 and TPN10476 have a neuroprotective effect on EAE mice and have potential therapeutic application value for autoimmune diseases.

[0041] The above description of the embodiments is to facilitate those skilled in the art to understand and use the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

Claims

1. Use of artemisinin derivatives TPN10467 and TPN10476 as drugs for preventing and treating autoimmune diseases, characterized in that: Artemisinin derivatives TPN10467 and TPN10476 reduce CD4 in a dose-dependent manner + The final ratio of T cells to Th1 and Th17 cells.

2. The use according to claim 1, characterized in that Intragastric administration of TPN10467 and TPN10476 can alleviate the clinical symptoms of EAE mice.

3. The use according to claim 1, characterized in that TPN10467 can reduce the total number of inflammatory cells in the CNS of mice and alleviate spinal cord injury.

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