Application of (5R)-5-hydroxytriptolide in medicine preparation

Abstract

The invention discloses an application of (5R)-5-hydroxytriptolide in preparation of a medicine, in particular to an application in preparation of a medicine for treating and / or preventing abnormal immune activation of AIDS (acquired immune deficiency syndrome) or immune reconstitution insufficiency related to the abnormal immune activation of the AIDS. Experiments carried out by using (5R)-5-hydroxytriptolide (T8) show that the (5R)-5-hydroxytriptolide (T8) has immunosuppressive activity in abnormal immune activation of AIDS or immune reconstitution insufficiency related to the abnormal immune activation of AIDS, and has the advantages of high efficiency, low toxicity and very good safe therapeutic index.

Description

[0001] This application is a divisional application of a Chinese patent application with national application number CN201880089248.6 and invention-creation name "Application of (5R)-5-hydroxy triptolide in the preparation of drugs". The patent application with application number CN201880089248.6 is an application in the Chinese national phase of a PCT application with an international filing date of December 14, 2018 and international application number PCT / CN2018 / 121201. This application claims the priority of a priority date of February 13, 2018 and priority number 201810149149.4. Technical Field

[0002] The present invention belongs to the field of chemical pharmaceuticals, and relates to the application of (5R)-5-hydroxy triptolide in the preparation of drugs, in particular to the application of (5R)-5-hydroxy triptolide in the preparation of drugs for treating and / or preventing abnormal immune activation or immune reconstitution deficiency in AIDS. Background Art

[0003] Tripterygium wilfordii is a woody vine of the genus Tripterygium in the family Celastraceae. It grows in shady and humid places at the edge of mountain forests and is distributed in various places south of the Yangtze River Basin and southwestern regions. Its chemical components are mainly diterpenes, triterpenes, sesquiterpenes, alkaloids, etc. Research in the past two decades has shown that Tripterygium wilfordii has anti-inflammatory, immunosuppressive, anti-fertility, anti-tumor, antibacterial and other activities. Especially in terms of immunosuppressive effects, triptolide in Tripterygium wilfordii has strong pharmacological activities. However, the toxic and side effects of triptolide limit its clinical application. The inventor of Patent CN1223595C, through painstaking research on the structure-activity relationship of triptolide, modified and decorated the structure of triptolide, and disclosed a batch of new low-toxic triptolide derivatives, which can be used as anti-inflammatory immunosuppressants for the prevention and treatment of autoimmune diseases, infectious diseases, etc. Especially (5R)-5-hydroxy triptolide (LLDT-8, abbreviated as T8; its structural formula is as follows), has been confirmed to have extensive anti-inflammatory immunosuppressive activities in the research of various in vitro test systems and in vivo experimental animal models, showing high efficiency and low toxicity, and having a good safety therapeutic index.

[0004]

[0005] Acquired immunodeficiency syndrome (AIDS) is an infectious disease caused by human immunodeficiency virus (HIV) infection, which is characterized by the specific attack of HIV virus on helper T lymphocytes (CD4 +), causing progressive damage to the human immune system and eventually leading to the occurrence of various opportunistic infections and corresponding tumors. Since the 1990s, highly active antiretroviral therapy (HAART) has been used, which can significantly reduce the plasma HIV load and effectively increase the CD4 + T cell count, enabling patients to undergo immune reconstitution, thereby reducing the incidence and mortality of AIDS-related diseases. However, the latest research suggests that HIV virus infection is a chronic inflammatory disease, and continuous activation of the immune system is the most important immunopathological change in AIDS, accompanying the entire process of HIV infection, including the reduction of CD4 + T cells, the formation and long-term maintenance of viral reservoirs, and the reconstitution barrier. In particular, the complications and relatively high mortality of patients with long-term successful antiviral treatment are also considered to be directly or indirectly related to abnormal immune activation. Immune reconstitution refers to the process of the recovery of the immune system function in AIDS patients, bone marrow transplantation patients, or other patients with severe immunosuppression. When the immune reconstitution process is impaired, it is called immune reconstitution failure. Immune reconstitution failure mainly occurs in AIDS patients, and these patients are also called immune non-responders (INRs). For AIDS patients, immune non-response means that after 2 years of regular antiviral treatment, the viral load is below the detection limit, but the CD4 cell count does not increase (less than 250 / μL). Such patients are prone to various opportunistic infections, tumors, etc., and have a very high mortality rate; Abnormal immune activation refers to the long-term abnormally increased activation state of the immune system. Based on the important role of abnormal immune activation in the pathogenesis of AIDS, the effect of antiviral treatment, and the occurrence of complications, in recent years, immune activation has been studied as a potential therapeutic target for AIDS. Currently, there is no drug available at home and abroad that can effectively treat or prevent abnormal immune activation or immune reconstitution failure. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defect in the prior art of the lack of drugs for abnormal immune activation in AIDS and immune reconstitution failure in AIDS, and to provide an application of (5R)-5-hydroxytriptolide in the preparation of drugs, especially in the preparation of drugs for treating and / or preventing abnormal immune activation in AIDS or immune reconstitution failure in AIDS.

[0007] The immune activation caused by HIV is different from the immune response and immune activation generated by the body after traditional viral infections. Current research shows that the main mechanisms are as follows:

[0008] 1. Direct pathway: HIV gene products (such as gp120) directly activate lymphocytes and macrophages;

[0009] 2. Indirect pathway: During HIV infection, due to the immune deficiency state caused by HIV infection, some pathogens with persistent infection in the body, such as cytomegalovirus (CMV) and Epstein-Barr virus, will be reactivated and replicated, thus causing immune activation;

[0010] 3. Intestinal flora translocation: The loss of a large number of CD4 + T cells in mucosal lymphoid tissue will cause damage to the immune components in the intestinal mucosal barrier, leading to intestinal flora translocation and then immune activation.

[0011] 4. Reduction of regulatory T cells (Treg): Treg is of great significance for maintaining the homeostasis of the body's immune system. Its function is to down-regulate the immune activation state. Its activation and transformation are closely related to both immune activation and immune reconstitution. Studies have shown that in long-term non-progressing HIV-infected individuals, CD4+CD25+Treg is significantly more than that in typical progressors, and the proportion of Treg is negatively correlated with the activated subset of CD8 + T cells.

[0012] 5. CD4 + T cell pyroptosis: A study published by Gilad Doitsh et al. in the journal Nature in 2014 showed that the vast majority (95%) of CD4 + T cell deaths caused by HIV virus are accompanied by the release of inflammatory factors such as IL-1β and cell contents. This caspase-1-mediated cell pyroptosis further promotes the aggregation, infection and pyroptosis of uninfected CD4 + T cells in the body, thus forming a vicious cycle of "pyroptosis - inducing aggregation of uninfected CD4 + T cells - infecting new CD4 + T cells - new pyroptosis" in the body. In 2015, a study by Galloway et al. was published in Cell Reports. They proved through a series of experiments that the direct contact (forming synaptic connections) between HIV-infected CD4 + T cells and uninfected CD4 + T cells is a necessary condition for triggering virus transmission and cell pyroptosis. Free HIV virus, even with a high load, cannot directly cause cell pyroptosis.

[0013] Persistent immune activation can produce a series of serious consequences, including the continuous replication of HIV and immune reconstitution disorders, especially increasing the mortality rate of AIDS patients. Even long-term effective antiviral treatment cannot completely eliminate this impact. Its impacts mainly include the following aspects:

[0014] 1. Viral replication: The direct result of T cell activation is the production of intracellular NF-κB, which increases the transcription of viral genes integrated into the host genome, generating new viral particles, thus falling into a vicious cycle.

[0015] 2. Apoptosis: For activated T cells, immune activation means that they will differentiate, transform or even undergo apoptosis. Apoptosis related to immune activation is also considered an important cause of the reduction of peripheral CD4 + T cells.

[0016] 3. Immunosenescence and exhaustion: In the acquired immune response, naive T cells are activated after contacting antigen-presenting cells and proliferate and differentiate into effector T cells and memory T cells. However, continuous antigen stimulation or other mechanisms leading to continuous immune activation will make effector T cells lose their functions, and this phenomenon is called "immune exhaustion". In addition, the thymus of infected individuals also undergoes atrophy disproportionate to age, which means that T cells developed in the thymus cannot timely supplement the peripherally consumed T cells. Immune activation also leads to a decline in bone marrow hematopoiesis function, lymphoid tissue fibrosis, etc.

[0017] 4. Immunological reconstitution impairment: In immunologically non-responsive individuals, although antiviral therapy is effective and plasma viral replication is basically inhibited, there are still residual viruses, which may come from viral reservoirs (mainly memory T cells) and continuous low-level viral replication. And these residual viruses and their low-level replication are related to continuous immune activation and incomplete immunological reconstitution.

[0018] 5. Other inflammation-related abnormalities: Such as osteoporosis, atherosclerosis, neurocognitive decline, aging, etc., are now also considered to be related to immune activation, leading to a significant increase in the mortality rate of patients.

[0019] The abnormal immune activation and inflammation caused by HIV infection are important pathogenic mechanisms of non-AIDS-related diseases and incomplete immunological reconstitution. Inhibiting excessive immune activation and inflammation may promote the recovery of CD4 + T lymphocytes in AIDS patients and reduce the occurrence of non-AIDS-related diseases; however, excessive inhibition may lead to the aggravation of incomplete immunological reconstitution. Therefore, it is crucial to seek a drug that can both inhibit abnormal immune activation and not aggravate incomplete immunological reconstitution; and as a drug, in addition to the therapeutic effect, its drug safety issues also need to be considered. The inventor of the present invention creatively studied and found that (5R)-5-hydroxytriptolide (T8) has an inhibitory effect on the excessive immune activation and inflammation of AIDS patients, opening up a new therapeutic drug for the treatment of abnormal immune activation or incomplete immunological reconstitution and non-AIDS-related diseases in AIDS patients.

[0020] In addition, the activation of the NF-κB pathway is crucial for the replication of HIV and the reactivation of latency, and the NF-κB pathway controls the expression of various inflammatory factors in cells.

[0021] The present invention mechanistically elucidates that (5R)-5-hydroxytriptolide (T8) can inhibit cellular immune activation by inhibiting NF-κB activity, and it is found that (5R)-5-hydroxytriptolide (T8) inhibits the activation and proliferation of CD4 + T cell subsets.

[0022] The present invention mainly solves the above technical problems by the following technical means:

[0023] The present invention provides the use of (5R)-5-hydroxytriptolide in the preparation of a drug for treating and / or preventing abnormal immune activation in AIDS or immune reconstitution deficiency related to abnormal immune activation in AIDS. Among them, abnormal immune activation in AIDS is related to immune reconstitution deficiency in AIDS (Yuchao Liu, Taisheng Li. Immune activation and immune reconstitution deficiency in chronic human immunodeficiency virus-infected patients, Chinese Journal of Peking Union Medical College Hospital, Vol.8, No.4-5, p100-104, 2017.).

[0024] The abnormal immune activation in AIDS described in the present invention is preferably NF-κB-related abnormal immune activation in AIDS.

[0025] The abnormal immune activation in AIDS described in the present invention is preferably chronic abnormal immune activation in AIDS.

[0026] In the present invention, the form of the drug is not particularly limited and can be various forms such as solid tablets, liquids, gels, semi-liquids or aerosols, etc., and preferably solid tablets.

[0027] In the present invention, the triptolide derivative is preferably one of the active ingredients or the only active ingredient of the drug.

[0028] The present invention also provides the use of (5R)-5-hydroxytriptolide in the preparation of an NF-κB signaling pathway inhibitor. The (5R)-5-hydroxytriptolide is preferably one of the active ingredients or the only active ingredient of the drug.

[0029] The present invention also provides the use of (5R)-5-hydroxytriptolide in the preparation of a T lymphocyte activity inhibitor. The (5R)-5-hydroxytriptolide is preferably one of the active ingredients or the only active ingredient of the drug.

[0030] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0031] The reagents and raw materials used in the present invention are all commercially available.

[0032] The positive and progressive effects of the present invention are as follows:

[0033] After conducting experiments with (5R)-5-hydroxytriptolide (T8), it was found that it has immunosuppressive activity in abnormal immune activation in AIDS or immune reconstitution insufficiency related to abnormal immune activation in AIDS, and shows high efficiency and low toxicity, with a very good safety therapeutic index. Description of the Drawings

[0034] Figure 1 It is a detection result graph of the inhibitory effect of (5R)-5-hydroxytriptolide on NF-κB activity.

[0035] Figure 2 It is a result graph of the inhibitory effect of (5R)-5-hydroxytriptolide on the proliferation of CD4 + T cells.

[0036] Figure 3 It is the inhibitory effect of (5R)-5-hydroxytriptolide on the activation of CD4 + T cell subsets.

[0037] Figure 4 It is the cytotoxicity detection of (5R)-5-hydroxytriptolide (T8) and its control (triptonide).

[0038] Figure 5 It is the detection of the inhibitory effect of (5R)-5-hydroxytriptolide (T8) and its control (triptonide) on the activity of the NF-κB signaling pathway.

[0039] Figure 6 is the detection of the inhibitory effect of (5R)-5-hydroxytriptolide (T8) and its control (triptonide) on the immune activation of CD4 + T cell subsets. Freshly isolated healthy human peripheral blood cells PBMCs or magnetic beads further coated with anti-CD4 antibody were used to isolate CD4 + T cells by positive selection. The cells were stimulated with PMA (100 nM) and Ionomycin (1 μM) or magnetic beads coated with anti-CD3 / CD28 antibody for 72 hours to activate the cells, and at the same time, T8 or triptonide (100 ng / mL) was added; the cells were collected, and anti-CD4-PE antibody was used for staining to select CD4 + T cells, and flow cytometry was used to detect the expression of CD38 and HLA-DR on the cell surface. Figure 6A It is the result of multiple completely independent repeated flow cytometry detections; Figure 6B It is the statistical result of flow cytometry detection. The positive cell rate was statistically analyzed respectively and the mean fluorescence intensity (MFI) was calculated. The paired student t-test was used to analyze the significant differences.

[0040] Figure 7 Inhibition of CD4 by T8 or jatrorrhizine + Detection of T cell proliferation. PBMCs were stained with CFSE; PHA-P (5 μg / mL) was added to stimulate cell proliferation, and at the same time, the compound to be detected, T8 or jatrorrhizine (100 ng / mL), was added; after 72 hours, the cells were collected, and anti-CD4-PE antibody was used for staining to select CD4 + T cells, and CFSE was analyzed by flow cytometry to detect cell proliferation. (A) Results of three completely independent repeated flow cytometry detections; (B) Statistical results of flow cytometry detection. Paired student t-test was used to analyze the significant differences. Detailed implementation manners

[0041] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, conventional methods and conditions are followed, or they are selected according to the product instructions.

[0042] Example 1 Detection of the inhibition of NF-κB activity by T8

[0043] In HEK293T cells (purchased from ATCC), a plasmid expressing luciferase driven by NF-κB (p3κB-luc, 100 ng) and an internal reference plasmid pBgal (purchased from Bioonline www.bioon.com.cn) (pSV-β-Galactosidase Control Vector, β-galactosidase expression plasmid, 5 ng) were transfected; after 24 hours, the cells were treated with TNF-alpha (20 ng / ml) or not, and at the same time, T8 compound (100 nM) was added or not; after another 24 hours, the cells were collected and lysed with reporterlysis buffer (purchased from Promega); Luciferase assay system (Promega) was used to detect the luciferase activity, and Beta-Glo Asaay system (Promega) was used to detect the β-Galactosidase activity to analyze the inhibitory effect of T8 on NF-κB activity.

[0044] The detection results are as Figure 1 shown. The test results show that (5R)-5-hydroxytriptolide inhibits NF-κB activity, which can not only effectively inhibit the basal NF-κB activity (without TNF-alpha treatment), but also effectively inhibit the NF-κB activity activated by TNF-alpha.

[0045] Example 2 Inhibitory effect of T8 on CD4 + Inhibition of T cell proliferation

[0046] Healthy human peripheral blood mononuclear cells (PBMC) (purchased from ATCC) were cultured in a medium containing RPMI-1640 / 10% FBS serum with IL-2 (20 IU), stained with CFSE (carboxyfluorescein succinimidyl ester) (10 nM) at 37 °C for 10 minutes, washed twice with the above medium, and then stimulated with PHA-P (phytohemagglutinin P) (5 μg / ml) to promote cell proliferation. T8 compound (100 nM) was added or not added simultaneously. After 48 hours of culture, the cells were collected, stained with anti-human CD4 flow antibody (purchased from Invitrogen) (4 °C, 30 min), and CD4 + T cells were detected for CFSE staining intensity to analyze cell proliferation.

[0047] The detection results are as Figure 2 shown. The detection results indicate that (5R)-5-hydroxytriptolide inhibits PHA-stimulated CD4 + T cell proliferation (cell proliferation decreased from 16.6% to the background proliferation of 1.9%).

[0048] Example 3 Inhibitory effect of T8 on the activation of CD4 + T cell subsets

[0049] 1×10 healthy human peripheral blood mononuclear cells (PBMC) 7 were stimulated with PMA (propylene glycol monomethyl ether acetate) (20 nM) and Ionomycin (1 μM) at 37 °C, and T8 compound (100 nM) was added or not added simultaneously; after 48 hours, anti-human CD4, CD38, HLA-DR cell flow antibodies (purchased from Invitrogen) were added for staining, and CD4 + T cells were detected for the expression of cell surface molecules CD38 and HLA-DR.

[0050] The detection results are as Figure 3 shown. The detection results indicate that (5R)-5-hydroxytriptolide inhibits cell activation induced by PMA + Ionomycin (CD38 decreased from 41.6% to 31%; HLA-DR decreased from 36.3% to 3.5%).

[0051] Comparative Example 1 Cytotoxicity detection of (5R)-5-hydroxytriptolide (T8) and its reference substance (triptolide, i.e., triptolide)

[0052] 1. Experimental materials

[0053] 1) Compounds to be detected

[0054] (5R)-5-Hydroxytriptolide (T8) and its reference substance (triptolide) were provided by Shanghai Pharmaceutical Group; white powder, purity > 99%, stored at 4 °C for later use;

[0055] Preparation method: Prepare the stock solution with DMSO and the working solution with the culture medium (RPMI 1640, Gibco). When culturing cells, the final concentration of DMSO is < 0.02%, and this DMSO concentration has no effect on cell growth.

[0056] 2) Cells and reagents

[0057] HEK293T cells were cultured in complete DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin;

[0058] DMEM medium and fetal bovine serum were purchased from Gibco; penicillin and streptomycin were purchased from Invitrogen;

[0059] MTT [3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide], SDS (Sodium dodecyl sulfate), and DMF (N,N-Dimethyl formamine) were all purchased from sigma;

[0060] MTT (Formazan) dissolution solution (100 mL): 10 g of SDS (Sodium dodecyl sulfate); 50 mL of DMF (N,N`-Dimethyl formamine); 50 mL of H2O, heated and stirred evenly, stored at 4 °C.

[0061] 2. Experimental methods

[0062] The cytotoxicity was detected by MTT colorimetric method: HEK293T cells were seeded into 96-well plates (1×10 4Into (100 μL / well), add 100 μL of compounds at different concentrations (compound concentrations are shown in the legend). There are three replicate wells for each dilution, and control wells without compounds are set simultaneously. Incubate in a 37 °C, 5% CO₂ cell culture incubator for 72 hours; aspirate 100 μL of the supernatant from each well, add 20 μL of MTT solution (5 mg / mL), incubate in a 37 °C, 5% CO₂ cell culture incubator for 4 hours, then add 100 μL of Formazan solubilization solution to each well and incubate overnight in the incubator until all Formazan is dissolved as observed under an ordinary optical microscope, and measure the absorbance at 595 nm. Calculate the CC 50 value (50% Cytotoxic Concentration), that is, the experimental drug concentration when it is toxic to 50% of HEK293T cells.

[0063] 3. Experimental results

[0064] Using HEK293T cells to detect the cytotoxicity of two compounds, it was found that: The CC 50 (50% Cytotoxic Concentration) value of (5R)-5-hydroxytriptolide (T8) for HEK293T cells is approximately 435 ng / ml, while that of the reference substance (triptonide) for HEK293T cells is 50 approximately 17 ng / ml. It can be seen that compared with triptonide, T8 shows lower cytotoxicity (see details in Figure 4 ).

[0065] Comparative Example 2 Detection of the inhibitory activity of (5R)-5-hydroxytriptolide (T8) and its reference substance (triptonide) on the NF-κB signaling pathway

[0066] 1. Experimental materials

[0067] 1) The compounds to be detected are the same as above

[0068] 2) Cells and reagents

[0069] HEK293T cells are cultured in complete DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin;

[0070] DMEM medium, fetal bovine serum, and OPTI-MEM were purchased from Gibco; penicillin and streptomycin were purchased from Invitrogen; TNF-α was purchased from R&D; lipofectamine 2000 Transfection Reagent was purchased from Life Technologies; Luciferase assay system and Glo Asaay system were purchased from Promega;

[0071] The pSV-β-Galactosidase Control Vector plasmid was from Promega (CATALOG#E1081).

[0072] 2. Experimental methods

[0073] The NF-κB reporter gene plasmid p3κB-luc was transfected into HEK293T cells for expression. At the same time, the pSV-β-Galactosidase Control Vector was co-transfected as a transfection internal reference. The activation of NF-κB was induced by TNF-α treatment. T8 and Jiaosu were added, and the expression of the NF-κB reporter gene was indicated by detecting the luciferase activity to evaluate the effects of T8 and Jiaosu on the activation of NF-κB induced by TNF-α;

[0074] Cell transfection: HEK293T cells were transfected using lipofectamine 2000 Transfection Reagent according to the reagent instructions. 1 μL of the transfection reagent was used to transfect 100 ng of plasmid per well in a 24-well cell culture plate; the plasmid and the transfection reagent were diluted and mixed with 50 μL of OPTI-MEM medium respectively; after the mixture was allowed to stand at room temperature for 5 minutes, the mixture was added to each well of the cells, gently mixed, and then returned to the 37°C incubator for continued culture until the specified time;

[0075] Luciferase activity detection: HEK293T cells were transfected with the NF-κB reporter gene plasmid p3κB-luc plasmid (100 ng) and the internal reference plasmid pSV-β-Galactosidase Control Vector (5 ng). After 24 hours, (or not) TNF-α (20 ng / mL) was added for stimulation for 24 hours. The cells were collected and lysed using reporter lysis buffer, and then the luciferase activity was detected using the Luciferaseassay system, and the β-Galactosidase activity was detected using the Beta-Glo Asaay system.

[0076] 3. Experimental results

[0077] According to Figure 5 It can be seen that both (5R)-5-hydroxytriptolide (T8) and its reference substance (triptolide) can effectively inhibit the activity of the basal NF-κB signaling pathway, and their activities are equivalent. The P value for the significant difference analysis of inhibiting the basal NF-κB activity is 0.1, indicating no significant difference; both (5R)-5-hydroxytriptolide (T8) and its reference substance (triptolide) can effectively inhibit the activity of the NF-κB signaling pathway activated by TNF-α, and their activities are equivalent. The P value for the significant difference analysis of inhibiting the NF-κB activity activated by TNF-α is 0.056, indicating no significant difference.

[0078] Comparative Example 3 Detection of the inhibition of immunocyte activation by (5R)-5-hydroxytriptolide (T8) and its reference substance (triptolide)

[0079] 1. Experimental materials

[0080] 1) The compounds to be detected are the same as above

[0081] 2) Cells and reagents:

[0082] Peripheral blood mononuclear cells (PBMCs) were purchased from Changhai Hospital, Shanghai. The cells were cultured in complete RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 20 IU / mL IL-2 (R&D).

[0083] RPMI-1640 medium and fetal bovine serum were purchased from Gibco; penicillin and streptomycin were purchased from Invitrogen; IL-2 was purchased from R&D; phorbol 12-myristate 13-acetate (PMA) and ionomycin were purchased from Sigma; anti-CD3 / CD28 antibody and CD4 antibody-coated microbeads were purchased from MACS; flow antibodies CD3, CD4, CD38, and HLA-DR were purchased from eBioscience.

[0084] 2. Experimental method:

[0085] Freshly isolated healthy human peripheral blood cells PBMC (3×10 7Cultured in cell medium containing IL-2 (20 IU / mL); stimulated with PMA (100 nM) and Ionomycin (1 μM) for 72 hours, and at the same time, the compound to be detected (100 ng / mL) was added; washed twice with FACS buffer, and anti-CD3-PerCP, CD4-PE, CD38-PE-Cy7, HLA-DR-APC antibodies were added, and stained at 4°C for 30 minutes; washed twice with FACS buffer, and the expression of the above molecules was detected by FACS.

[0086] Freshly isolated healthy human peripheral blood cells PBMCs, CD4 was isolated by positive selection using magnetic beads coated with anti-CD4 antibody + T cells, cultured in medium containing IL-2 (20 IU / mL); stimulated with magnetic beads coated with anti-CD3 / CD28 antibodies to activate the cells for 72 hours, and at the same time, the compound to be detected was added; the cells were collected, washed twice with FACS buffer, and anti-CD3-PerCP, CD4-PE, CD38-PE-Cy7, HLA-DR-APC antibodies were added, and stained at 4°C for 30 minutes; washed twice with FACS buffer, and the cells were collected for flow cytometry analysis of the expression of the above molecules.

[0087] 3. Experimental results

[0088] By detecting the expression of cell activation markers CD38 and HLA-DR, it was proved that (5R)-5-hydroxytriptolide (T8) and its reference substance (triptonide) could significantly inhibit the immune activation of CD4 + T cell subsets;

[0089] (5R)-5-hydroxytriptolide (T8) and its reference substance (triptonide) inhibited CD4 + The effects of T cell subset immune activation were equivalent (see Figure 6 for details).

[0090] Comparative Example 4

[0091] 1. Experimental materials:

[0092] 1) The compound to be detected was the same as above

[0093] 2) Cells and reagents:

[0094] Peripheral blood mononuclear cells (PBMCs) were purchased from Changhai Hospital of Shanghai. The cells were cultured in complete RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 20 IU / mL IL-2 (R&D). The cells were cultured in complete medium containing 5 μg / mL phytohemagglutinin-P (PHA-P) for 72 hours to induce CD4 + T cell proliferation.

[0095] RPMI-1640 medium and fetal bovine serum were purchased from Gibco; penicillin and streptomycin were purchased from Invitrogen; IL-2 was purchased from R&D; PHA-P was purchased from Sigma; CFSE dye was purchased from abcam; anti-CD4-PE antibody was purchased from eBioscience.

[0096] 2. Experimental methods:

[0097] Freshly isolated healthy human peripheral blood cells PBMC (1×10 7 ) were cultured in cell medium containing IL-2 (20 IU / mL); CFSE (10 nM), stained at 37°C for 10 minutes; after washing twice with the medium, PHA-P (5 μg / mL) was added, and at the same time, the test compound T8 or triptolide (100 ng / mL each) was added; at 37°C, after 72 hours, the cells were collected, stained with anti-CD4-PE antibody at 4°C for 30 minutes, washed twice with FACS buffer, and the cell proliferation was analyzed by flow cytometry.

[0098] 3. Experimental results

[0099] According to Figure 7 it can be seen that (5R)-5-hydroxytriptolide (T8) and its reference substance (triptolide) can significantly inhibit the proliferation of CD4 + T cells in healthy human peripheral blood, and the effects on inhibiting the proliferation of CD4 + T cells in healthy human peripheral blood are quite equivalent.

[0100] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples. Without departing from the principles and essence of the present invention, various changes or modifications can be made to these embodiments. Therefore, the protection scope of the present invention is defined by the appended claims.

Claims

1. Use of (5R)-5-hydroxytriptolide in the preparation of a drug for treating and / or preventing abnormal immune activation in AIDS or immune reconstitution failure related to abnormal immune activation in AIDS.

2. The application according to claim 1, characterized in that, The abnormal immune activation in AIDS mentioned above is NF-κB-related abnormal immune activation in AIDS.

3. The application according to claim 1 or 2, characterized in that, The abnormal immune activation in AIDS mentioned above is chronic abnormal immune activation in AIDS.

4. The application according to claim 1, characterized in that The form of the drug mentioned above is tablets.

5. The application according to any one of claims 1 to 4, characterized in that The (5R)-5-hydroxytriptolide mentioned above is one of the active ingredients or the only active ingredient of the drug.

6. Use of (5R)-5-hydroxytriptolide in the preparation of an NF-κB signaling pathway inhibitor.

7. The application according to claim 6, characterized in that, The (5R)-5-hydroxytriptolide mentioned above is one of the active ingredients or the only active ingredient of the drug.

8. Use of (5R)-5-hydroxytriptolide in the preparation of a T lymphocyte activity inhibitor.

9. The application according to claim 8, wherein The (5R)-5-hydroxytriptolide mentioned above is one of the active ingredients or the only active ingredient of the drug.

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