New medical uses of azvudine or its pharmaceutically acceptable salts or their use in combination with HIV inhibitors

Abstract

The new medical use of azvudine, in particular, the use of azvudine or its pharmaceutically acceptable salt, or a pharmaceutical composition comprising the same in the preparation of a drug for treating human immunodeficiency virus infection and / or AIDS caused by poor immune function reconstruction; or the use of azvudine or its pharmaceutically acceptable salt, or a pharmaceutical composition comprising the same in the preparation of a drug for treating human immunodeficiency virus infection and / or AIDS with poor immune function reconstruction. Optionally, the pharmaceutical composition comprises azvudine or its pharmaceutically acceptable salt and other HIV inhibitors. Azvudine has a significant therapeutic effect on human immunodeficiency virus infection and / or AIDS caused by poor immune function reconstruction.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to the use of azvudine or a pharmaceutically acceptable salt thereof, or in combination with HIV inhibitors, in the preparation of medicaments for treating human immunodeficiency virus infection and / or AIDS with impaired immune reconstitution, or in the preparation of medicaments for treating human immunodeficiency virus infection and / or AIDS with impaired immune reconstitution. Background Technology

[0002] Human immunodeficiency virus (HIV) infection, or AIDS, is a major infectious disease that seriously threatens human health. Antiviral therapy (ART) is currently the most effective treatment and is widely used in clinical practice, transforming AIDS into a chronic and manageable disease. However, it is undeniable that ART can lead to adverse reactions, such as poor immune reconstitution, drug toxicity, and viral resistance. Among these, poor immune reconstitution has always been a key focus and challenge in AIDS research, widely considered a difficult problem. Therefore, exploring new strategies and methods to promote immune reconstitution in HIV / AIDS patients after ART treatment is imperative.

[0003] Azvudine is a broad-spectrum RNA virus inhibitor. As a synthetic nucleoside analog of viral RNA-dependent RNA polymerase (RdRp), it is metabolized in cells into an antiviral 5'-triphosphate metabolite (azvudine triphosphate), which specifically targets the SARS-CoV-2 polymerase (RdRp). Its target is the viral RdRp, and it can block the synthesis and replication of RNA chains in host cells by inhibiting the activity of RdRp.

[0004] Patent document CN201010506595.X discloses the use of azvudine for the treatment of tumors, such as colon cancer, liver cancer, stomach cancer, esophageal cancer, lung cancer, breast cancer, cervical cancer, leukemia, and lymphoma. It was found that azvudine has a significant inhibitory effect on various human cancer cells and transplanted tumors in animals. Summary of the Invention

[0005] Retroviral therapy (ART) is a widely used clinical treatment for HIV / AIDS. In most patients, CD4+ T lymphocytes increase rapidly within 3-6 months after ART initiation. However, some patients, despite long-term HIV suppression, exhibit significantly low CD4+ T cell counts, indicating poor immune reconstitution. CD8+ T lymphocytes play a crucial role in fighting and clearing HIV infection; therefore, detecting CD8+ T lymphocytes helps assess the efficacy of immune reconstitution after treatment. If the CD4 / CD8 ratio is below normal (1.4–2.0) after ART treatment, incomplete immune reconstitution is considered.

[0006] Through extensive research and screening, the inventors discovered that, in addition to being used as a broad-spectrum antiviral and antitumor drug, azvudine has a significant therapeutic effect on improving immune function reconstruction caused by human immunodeficiency virus infection or AIDS.

[0007] In view of this, a first aspect of the present invention provides the use of azvudine or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the thereof, in the preparation of a medicament for treating impaired immune function reconstitution caused by human immunodeficiency virus infection and / or AIDS.

[0008] A second aspect of the invention further provides the use of azvudine or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the thereof, in the preparation of a medicament for treating human immunodeficiency virus infection and / or AIDS with impaired immune reconstitution.

[0009] The pharmaceutical composition further includes other HIV inhibitors; optionally, the HIV inhibitor is selected from one or more of zidovudine, lamivudine, tenofovir, tenofovir disoproxil fumarate, tenofovir alafenamide, abacavir, nevirapine, efavirenz, rilpivirine, lopinavir / ritonavir, darunavir, atazanavir, dolutegravir, raltegravir, epovitamide, enfuvirtide, malaviro, and their pharmaceutically acceptable salts; optionally, the HIV inhibitor is selected from one or more of tenofovir disoproxil fumarate, nevirapine, efavirenz, and their pharmaceutically acceptable salts. Optionally, in the pharmaceutical composition, the molar ratio of azvudine or its pharmaceutically acceptable salt to the HIV inhibitor is 1:7.5-1:2177, optionally 1:7.5-1:540, further optionally 1:18-1:860, and even further optionally 1:45-1:2177.

[0010] Optionally, the active pharmaceutical ingredient, azvudine or a pharmaceutically acceptable salt thereof, and the HIV inhibitor are either mixed together in the same formulation or separated into two formulations.

[0011] Optionally, the active ingredients of the pharmaceutical composition include azvudine, tenofovir disoproxil fumarate, and nevirapine, wherein the molar ratio of azvudine to tenofovir disoproxil fumarate is 1:7.5-1:540, preferably 1:23-1:135, more preferably 1:11-1:45, and the molar ratio of azvudine to nevirapine is 1:18-1:860, preferably 1:36-1:215, more preferably 1:18-1:72; Optionally, the active ingredients of the pharmaceutical composition include azvudine, tenofovir disoproxil fumarate, and efavirenz, wherein the molar ratio of azvudine to tenofovir disoproxil fumarate is 1:7.5-1:540, preferably 1:23-1:135, more preferably 1:11-1:45, and the molar ratio of azvudine to efavirenz is 1:45-1:2177, preferably 1:91-1:544, more preferably 1:45-1:91.

[0012] Optionally, the active pharmaceutical ingredient of the pharmaceutical composition comprises azvudine, tenofovir disoproxil fumarate, and nevirapine.

[0013] Optionally, the active pharmaceutical ingredient of the pharmaceutical composition comprises azvudine, tenofovir disoproxil fumarate, and efavirenz.

[0014] In a preferred embodiment of the present invention, the pharmaceutically acceptable salt of azvudine comprises salts formed by azvudine with the following acids: hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, mandelic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, ascorbic acid palmitic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, phthalic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, or leucine.

[0015] In a preferred embodiment of the present invention, the pharmaceutically acceptable salt of azvudine is a salt formed by azvudine with hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, or phthalic acid. In another preferred embodiment of the present invention, poor immune reconstitution refers to a CD4+ T lymphocyte count that does not reach 350 / μL after ART treatment, and / or a peripheral blood human immunodeficiency virus load that is below the detection limit (VL < 50 copies / mL) after ART treatment.

[0016] In a preferred embodiment of the present invention, the poor immune function reconstruction may also refer to other causes that lead to a CD4+ T lymphocyte count that is consistently below 350 / μL, such as known types of immunodeficiency or immunodeficiency, chronic viral infection, hematologic malignancies, long-term use of immunosuppressive drugs, etc.

[0017] In another aspect, the present invention also provides a method for treating impaired immune function reconstruction caused by human immunodeficiency virus infection and / or AIDS, comprising administering to a subject in need (e.g., a living human) a therapeutically effective amount of azvudine or a pharmaceutically acceptable salt thereof, or administering to a subject in need (e.g., a living human) a therapeutically effective amount of a pharmaceutical composition comprising azvudine or a pharmaceutically acceptable salt thereof and the other HIV inhibitors described above.

[0018] In another aspect, the present invention also provides a method for treating human immunodeficiency virus infection and / or AIDS with poor immune reconstitution, comprising administering to a subject in need (e.g., a human animal) a therapeutically effective amount of azvudine or a pharmaceutically acceptable salt thereof, or administering to a therapeutically effective amount of a pharmaceutical composition comprising azvudine or a pharmaceutically acceptable salt thereof, or administering to a therapeutically effective amount of a pharmaceutical composition comprising azvudine or a pharmaceutically acceptable salt thereof and the other HIV inhibitors described above.

[0019] In this invention, the active ingredient, azvudine or a pharmaceutically acceptable salt thereof, and HIV inhibitors or pharmaceutically acceptable salts thereof, can be their isotope labels, prodrugs, stereoisomers, and various physical forms, including crystals, amorphous substances, and solvates such as hydrates. In this document, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometrical isomers (cis / trans) isomers, and transisomers.

[0020] The term "isotope-labeled compound" refers to a compound whose structure differs only in the presence of one or more isotope-enriched atoms. Examples of isotopes used in the azvine of this invention can include isotopes of hydrogen, such as... 2 H(D) and 3 H(T), isotopes of carbon, such as 11 C 13 C and 14 C, isotopes of fluorine, such as 18 F, such as 13 N and 15 N, an isotope of oxygen, such as 15 O、 17 O and 18 O.

[0021] In this invention, after two months of treatment with azvudine, the CD4+ T lymphocytes and CD8+ T lymphocytes of the subjects were significantly increased, suggesting that azvudine has a significant therapeutic effect on immune function reconstruction caused by human immunodeficiency virus infection or AIDS. Attached Figure Description

[0022] Figure 1 This is a bar chart showing the CD4+T and CD8+T counts of subjects in Clinical Trial 1 before and after treatment with azvudine.

[0023] Figure 2 This is a line graph showing the CD4+T / CD8+T count ratio before and after subjects in Clinical Trial 1 received azvudine treatment.

[0024] Figure 3 This is a bar chart showing the CD4+T and CD8+T counts of subjects in Clinical Trial 2 before and after treatment with azvudine.

[0025] Figure 4 This is a line graph showing the CD4+T / CD8+T count ratio before and after azvudine treatment in subjects of clinical trial 2.

[0026] Figure 5 This is the morphological result of the differentiation of MDSC cells from three different donors under different concentrations of the test compound in Experiment Example 3.

[0027] Figure 6 This is a schematic diagram of the gating strategy for detecting MDSC cell differentiation by flow cytometry in Experiment Example 3.

[0028] Figure 7 In Experiment 3, the cell viability of MDSC cells under different concentrations of the test compound is given. The data are expressed as mean ± standard error (n=3).

[0029] Figure 8-1 Examples 8-2, 8-3, and 8-4 show the effects of different concentrations of the test compound on CD11b in MDSC cells during Experiment 3. + CD33 + and CD11b + CD33 + Data are expressed as mean ± standard error (n=3). **: p<0.01, ***: p<0.001, ****: p<0.0001, ns: no significant difference.

[0030] Figure 9 This refers to the secretion of IL-10 during MDSC cell differentiation in Experiment 3. Data are expressed as mean ± standard error (n=3). *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, ns: no significant difference.

[0031] Figure 10 This is a schematic diagram of the gating strategy used by flow cytometry to detect the inhibition of T cell proliferation by MDSCs in Experiment Example 3.

[0032] Figure 11 The data represents the viability of T cells co-cultured with MDSCs in Experiment 3. The data are expressed as mean ± standard error (n=3).

[0033] Figure 12 This refers to the proliferation of T cells co-cultured with MDSCs in Experiment 3. Data are expressed as mean ± standard error (n=3). p <0.01, ***: p <0.001, ****: p <0.0001, ns: no significant difference.

[0034] Figure 13 In Experiment 3, IFN-γ secretion data of T cells co-cultured with MDSCs are expressed as mean ± standard error (n=3). *: p<0.05, **: p <0.01, ***: p <0.001, ****: p <0.0001, ns: no significant difference. Detailed Implementation

[0035] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental Example 1 Clinical Trial 1 Subject: A 62-year-old male diagnosed with HIV infection, transmitted through blood product transfusion.

[0036] Prior to enrollment in this clinical trial, the subjects received the following treatment regimen: lamivudine + tenofovir disoproxil fumarate + nevirapine, i.e., 3TC + TDF + NVP.

[0037] For 3-4 years after the subjects received the above treatment regimen and before enrollment in this clinical study, the HIV viral load was 0 and the CD4+ T lymphocyte count was 293 / μL (i.e. the subjects' baseline CD4+ T lymphocyte count).

[0038] The treatment regimen for subjects enrolled in this clinical trial was: Azvudine 3mg + Tenofovir disoproxil fumarate 300mg + Nevirapine 200mg, i.e., FNC + TDF + NVP.

[0039] In this clinical trial, CD4+ T lymphocyte counts and CD8+ T lymphocyte counts were measured for the first and second time at weeks 8 (approximately 2 months) and weeks 12 (approximately 3 months) of treatment administration. Specific test results and comparisons are shown in Table 1 below.

[0040]

[0041] Table 1 Figure 1 and Figure 2 The results showed that the subjects' baseline CD4+ T lymphocyte count was 293 / μL. After treatment with the study regimen containing azvudine, the first follow-up test showed a CD4+ T lymphocyte count of 474 / μL, an increase of 61.77% from baseline; the second follow-up test (December 4, 2024) showed a CD4+ T lymphocyte count of 462 / μL, an increase of 57.68% from baseline. The subjects' baseline CD8+ T lymphocyte count was 536 / μL. After treatment with the study regimen containing azvudine, the first follow-up test showed a CD8 / μL, an increase of 15.30% from baseline; the second follow-up test showed a CD8 / μL, an increase of 16.79% from baseline. The baseline CD4 / CD8 ratio was 0.55. After treatment with the study regimen containing azvudine, the first follow-up test showed an increase of 40.31% from baseline, and the second follow-up test showed an increase of 35.01% from baseline.

[0042] This indicates that the subjects experienced poor immune function reconstruction after ART, which was significantly improved after treatment with azvudine.

[0043] Clinical Trial 2 Subject: 53-year-old male, diagnosed with HIV infection, the route of infection was non-marital commercial heterosexual transmission.

[0044] Prior to enrollment in this clinical trial, the subjects received the following treatment regimen: lamivudine + tenofovir disoproxil fumarate + efavirenz, i.e., 3TC + TDF + EFV.

[0045] For 3-4 years after the subjects received the above treatment regimen and before enrollment in this clinical study, the HIV viral load was 0 and the CD4+ T lymphocyte count was 245 / μL (i.e. the subjects' baseline CD4+ T lymphocyte count).

[0046] The treatment regimen for subjects enrolled in this clinical trial was: 3 mg azvudine + 300 mg tenofovir disoproxil fumarate + 600 mg efavirenz, i.e., FNC + TDF + EFV.

[0047] In this clinical trial, CD4+ T lymphocyte counts and CD8+ T lymphocyte counts were measured for the first and second time at weeks 8 (approximately 2 months) and weeks 16 (approximately 4 months) of treatment administration. Specific test results and comparisons are shown in Table 2 below.

[0048]

[0049] Table 1 Figure 1 and Figure 2 The results showed that the baseline CD4+ T lymphocyte count of the subjects was 245 / μL. After treatment with the study regimen containing azvudine, the first retest showed a CD4+ T lymphocyte count of 334 / μL, an increase of 36.33% from baseline; the second retest showed a CD4+ T lymphocyte count of 390 / μL, an increase of 59.18% from baseline. The baseline CD8+ T lymphocyte count was 1053 / μL. After treatment with the study regimen containing azvudine, the first retest showed a count of 1345 / μL, an increase of 27.73% from baseline; the second retest showed a count of 1448 / μL, an increase of 37.51% from baseline. The baseline CD4 / CD8 ratio was 0.23. After treatment with the study regimen containing azvudine, the first retest showed an increase of 6.73% from baseline, and the second retest showed an increase of 15.76% from baseline.

[0050] This indicates that the subjects experienced poor immune function reconstruction after ART, which was significantly improved after treatment with azvudine.

[0051] Experimental Example 3 1. Experimental Design Human peripheral blood mononuclear cells from three donors were used in the experiments. In donor 1, only the effect of the test compound on MDSC cell differentiation was studied. In donors 2 and 3, the effects of the test compound on MDSC cell differentiation and MDSC cell inhibition of T cell activation were studied. MDSC cell differentiation and T cell activation inhibition assays were performed using flow cytometry and enzyme-linked immunosorbent assay (ELISA). The experimental design for flow cytometry is shown in Tables 3 and 4. The ELISA evaluated the effects of the test compound by detecting IL-10 secretion after MDSC cell differentiation and IFN-γ secretion in the MDSC cell inhibition of T cell activation assay.

[0052]

[0053] 2. Experimental Materials and Instruments 2.1 Information on the compound to be tested The compound to be tested was azvudine FNC (ZSSW2102001-01), supplied by Henan Zhenshi Biotechnology Co., Ltd., with a molecular weight of 286.22 g / mol and a storage concentration of 10 mM, stored at -80℃.

[0054] 2.2 Instruments, Materials, and Reagents The reagents and materials are shown in Table 5.

[0055] Table 5 Reagents and Materials

[0056] The instrument information is shown in Table 6.

[0057]

[0058] 3. Experimental Methods 3.1 Preparation of cell culture medium and staining buffer Complete culture medium (CM): RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin solution. Magnetic bead sorting solution: Phosphate buffer containing 2% fetal bovine serum and 1 mM EDTA. Staining buffer: Phosphate buffer containing 2% fetal bovine serum.

[0059] 3.2 On day 0, primary mononuclear cells were isolated from PBMCs. PBMCs from three different donors (batch numbers 22075198 (donor 1), 21069025, and 21069645) were retrieved from liquid nitrogen, thawed in a 37°C water bath, washed once with magnetic bead sorting solution, and cell counts were performed. The results are shown in Table 7.

[0060] Centrifuge at 350 × g for 5 minutes at room temperature, discarding the supernatant. Every 10... 7Each cell was resuspended in 80 µL of buffer. Every 10 cells... 7 Add 7 µL of CD14 microspheres to each cell. Mix well and incubate at 4°C for 15 minutes. Repeat for every 10 cells. 7 Wash cells with 1-2 mL of buffer per cell, centrifuge at 350 × g for 10 minutes. Discard the supernatant completely. Repeat for every 10 cells. 8 Each cell was resuspended in 500 µL of buffer. The separation column was placed on a suitable MACS separator magnetic rack and rinsed with 3 mL of buffer. The cell suspension was injected into the separation column, and CD14 cells were collected using centrifuge tubes. - Cells were washed three times with 3 mL of buffer. The separation column was removed from the magnetic rack and placed on a 50 mL centrifuge tube. 5 mL of buffer was added to the separation column. The plunger was pushed firmly into the column to immediately flush out the magnetically labeled cells (CD14). + (Cells). Centrifuge at 350 × g for 5 min. Discard the supernatant. Resuspend the cells in complete culture medium and count them. The number and viability of monocytes are shown in Table 7.

[0061] Monocytes were seeded into 96-well plates. For donor 1: Monocytes were diluted to 2 M / mL, 1 M / mL, and 0.5 M / mL with complete culture medium and seeded at 50 μL / well (0.1 M, 0.05 M, and 0.025 M / well) into 96-well plates. For donor 2 and donor 3: Monocytes were diluted to 2 M / mL with complete culture medium and seeded at 50 μL / well (0.1 M / well) into 96-well plates. Then, 50 μL of the test compound (4×) or DMSO (4×, 0.4%) (preparation process shown in Tables 10 and 11) and 100 μL of a cytokine mixture (2×) (preparation process shown in Tables 8 and 9) were added to the corresponding wells.

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] 3.3 Day 4, half-change of fluid Centrifuge the 96-well plate at 350× g for 5 minutes and discard 100 μL of supernatant. Then add 50 μL of cytokine mixture (2×) (preparation process is shown in Tables 12 and 13) and 50 μL of the test compound (2×) (preparation process is shown in Tables 14 and 15).

[0068]

[0069]

[0070]

[0071]

[0072] 3.4 Day 7: MDSC cell differentiation assay Seven days after MDSC cell differentiation, the morphology of the differentiated cells was photographed under a microscope. 150 μL of the supernatant was collected and human IL-10 secretion was detected using ELISA. Cell expression of CD11b and CD33 on the cell surface was detected using flow cytometry.

[0073] 3.4.1 Flow cytometry detection: Wash the cells once with PBS, then add 50 μL of TrypLE to each well and digest at 37°C for 15 minutes.

[0074] Add 100 μL of complete culture medium to stop digestion and transfer all cells to a 96-well V plate.

[0075] Add 150 μL of staining buffer to each well to wash the cells once.

[0076] Resuspend cells in 20 μL of phosphate buffer (1:500 dilution) containing live / dead dye, incubate at room temperature for 15 minutes, and wash cells once with 150 μL of staining buffer per well.

[0077] Resuspend the cells in 20 μL of buffer containing Fc receptor blocking solution (1 μL of Fc receptor blocking solution per well) and incubate at room temperature for 10 minutes. Add 20 μL of antibody mixture (containing 1 μL of CD11b and CD33 per well) or isotype control antibody / FMO to each well and mix well. Incubate at 4°C in the dark for 30 minutes; Wash cells twice with 150 μL staining buffer; Cells were resuspended in 150 μL of staining buffer and analyzed by flow cytometry.

[0078] 3.4.2 ELISA detection of human IL-10: Before use, mix all reagents thoroughly to avoid foaming. Antibodies must be centrifuged before use to collect the liquid at the bottom of the tube. The required number of strips is determined based on the number of test wells (blank and standard). Both the test sample (including standard) wells and the blank wells should be duplicated. Dilute the supernatant by 2 times; Sample loading: Add 100 μL of diluted cytokine standard to each well of the standard. Add 100 μL of the sample to be tested to each well of the sample plate. Seal the plate with sealing film and incubate at room temperature for 2 hours. Washing the plate: Remove the liquid from the wells, add 300 μL of washing solution to each well, let stand for 1 minute, then discard the liquid from the wells. Repeat 3 times, patting dry on filter paper each time. Add detection antibody: Add 100 μL of biotin-labeled antibody working solution to each well, seal with sealing film and incubate at room temperature for 1 hour; Wash the plate: Repeat step E; Add enzyme: Add 100 μL of horseradish peroxidase-conjugated streptavidin working solution to each well, seal the plate with sealing film, and incubate at room temperature for 30 minutes. Wash the plate: Repeat step E; Color development: Add 100 μL of the chromogenic substrate to each well. For donor 1 plates, incubate at room temperature in the dark for 8 minutes; for donor 2 and donor 3 plates, incubate at room temperature in the dark for 4 minutes. The reaction terminates as indicated by the intensity of the color (dark blue) in the wells. To terminate the reaction, add 100 μL of stop solution to each well; Plate reading: Within 10 minutes after termination, measure the OD value using a microplate reader, set to 450 nm and 610 nm (as a reference).

[0079] Day 7: T cells were isolated from PBMCs and labeled with CellTrace Violet. Human peripheral blood mononuclear cells (batch numbers 21069025 and 21069645) from two different donors were thawed and resuscitated in a 37°C water bath, and washed once with magnetic bead sorting buffer. Cell count and viability were assessed using a cell counter, and the results are shown in Table 16. Cells were centrifuged at 350 × g for 5 minutes at room temperature. The supernatant was discarded, and the cells were resuspended in magnetic bead sorting buffer to a final volume of 5 × 10⁶ cells / mL. 7 / mL; Add the cell resuspension to the specified size flow cytometry tube, then add the separation antibody (50 μL per mL of cell suspension); mix well and incubate at room temperature for 5 minutes; vortex to separate the magnetic beads for 30 seconds, then add to the cell suspension (40 μL per mL of cell suspension) and mix well; add magnetic bead sorting buffer to make a total volume of 5 mL, and pipette 2-3 times to mix. Place the flow cytometry tube (without cap) on a magnetic rack and incubate at room temperature for 5 minutes; transfer the cell suspension to a new flow cytometry tube, place it on a magnetic rack, and incubate at room temperature for 5 minutes; transfer the cell suspension to a 50 mL centrifuge tube containing complete culture medium and centrifuge at 350 × g for 5 minutes at room temperature; resuspend the cells in complete culture medium and count the cells using a cell counter, the counting results are shown in Table 16; centrifuge at 350 × g for 5 minutes at room temperature. Discard the supernatant and resuspend the T cells in phosphate-buffered saline (0.1% BSA) containing 0.1% bovine serum albumin, then dilute the cells to 2 × 10⁶ cells / mL. 7 / mL; Add CellTraceViolet (1:500) to 0.1% BSA, and add 0.1% BSA containing CellTraceViolet to the T cell resuspension. Each 1 μL of CellTraceViolet can label 1×10 7 Count T cells; incubate at 37°C in a 5% CO2 incubator for 18 minutes; add 10 mL of complete culture medium to terminate the reaction, and continue to incubate for 5 minutes; centrifuge at 350 × g for 5 minutes at room temperature. Discard the supernatant and resuspend the cells in 5 mL of complete culture medium; use a cell counter to detect the cell number and viability of the labeled T cells (results shown in 16), centrifuge at 350 × g for 5 minutes at room temperature; discard the supernatant and dilute the labeled T cells to 2 M / mL.

[0080]

[0081] 3.6 Day 7: Co-culture of T cells and MDSC cells Prepare CD3 / CD28 immunomagnetic beads by washing them twice with PBS, then diluting them to 2 M / mL (immunomagnetic beads:T cells = 1:1), as shown in Table 17. Centrifuge the MDSC cell plate at 350 × g for 5 minutes at room temperature and discard all supernatant. Add 100 μL of complete culture medium to each well. Add 50 μL of T cells (0.1 M / well) and 50 μL of CD3 / CD28 immunomagnetic beads (0.1 M / well) to each well of the corresponding MDSC cell plate. Incubate at 37°C and 5% CO2 for 3 days.

[0082]

[0083] 3.7 Day 10: T cell proliferation and cytokine secretion detection After co-incubating MDSCs and T cells for 3 days, the cells were pipetted and transferred to 96-well U-plates. The plates were placed on a magnetic plate rack for 2 minutes, and the CD3 / CD28 immunomagnetic beads were removed. The cell suspension was then transferred to 96-well V-plates and centrifuged at 350 × g for 5 minutes at room temperature. 150 μL of the supernatant was collected and IFN-γ secretion was detected by ELISA. T cells were collected and CD4+ was detected by flow cytometry. + T cells and CD8 + T cell proliferation.

[0084] 3.7.1 Flow cytometry detection A. Wash cells once with 150 μL of staining buffer per well; Resuspend the cells in 20 μL of buffer containing Fc receptor blocking solution (1 μL of Fc receptor blocking solution per well) and incubate at room temperature for 10 minutes. C. Add 20 μL of antibody mixture (containing 2 μL of CD4 and CD8 per well) or isotype control antibody to each well and mix well; D. Incubate at 4℃ in the dark for 30 minutes; Wash cells twice with 150 μL staining buffer; F. Discard the supernatant and resuspend the cells in each well with 50 μL of staining buffer; G. Before flow cytometry, add 50 μL of staining buffer containing 7-AAD to the cell suspension (50 μL / well contains 1 μL of 7-AAD, total volume is 100 μL / well), incubate at room temperature for 5 minutes, and then use an LSR Fortessa™ X-20 cell analyzer for detection.

[0085] 3.7.2 ELISA detection of human IFN-γ Before use, mix all reagents thoroughly to avoid foaming. Antibodies must be centrifuged before use to collect the liquid at the bottom of the tube. The required number of strips is determined based on the number of test wells (blank and standard). Both the test sample (including standard) wells and the blank wells should be duplicated. The supernatants of donor 2 and donor 3 were diluted 50 times. Sample loading: Add 100 μL of diluted cytokine standard to each well of the standard. Add 100 μL of the sample to be tested to each well of the sample plate. Seal the plate with sealing film and incubate at room temperature for 2 hours. Washing the plate: Remove the liquid from the wells, add 300 μL of washing solution to each well, let stand for 1 minute, then discard the liquid from the wells. Repeat 3 times, patting dry on filter paper each time. Add detection antibody: Add 100 μL of biotin-labeled antibody working solution to each well, seal with sealing film and incubate at room temperature for 1 hour; Wash the plate: Repeat step E; Add enzyme: Add 100 μL of horseradish peroxidase-conjugated streptavidin working solution to each well, seal the plate with sealing film, and incubate at room temperature for 30 minutes. Wash the plate: Repeat step E; Color development: Add 100 μL of the chromogenic substrate to each well. For donor 2 and donor 3 plates, incubate at room temperature in the dark for 4 minutes. The reaction terminates as indicated by the intensity of the color (dark blue) in the wells. To terminate the reaction, add 100 μL of stop solution to each well; Plate reading: Within 10 minutes after termination, measure the OD value using a microplate reader, set to 450 nm and 610 nm (as a reference).

[0086] 3.8 Data Analysis Flow cytometry data were analyzed using Flowjo V10 and Graphpad Prism 10 software. ELISA data were analyzed using Graphpad Prism 10 software. Data analysis included: MDSC cell viability; The positive rates and median fluorescence intensity of CD11b and CD33 in MDSC cells, the double positive rate of CD11b and CD33 in MDSC cells, and the number of double positive cells.

[0087] T cell viability of donor 2 and donor 3; CD4 from donors 2 and 3 + T cells and CD8 + T cell proliferation; The secretion of human IL-10 during MDSC cell differentiation, and the secretion of human IFN-γ during the proliferation of donor 2 and donor 3 T cells; Results are expressed as mean ± standard error. Statistical significance was determined using a t-test (non-parametric test) in GraphPad Prism 10 software.

[0088] 4. Results Analysis 4.1 Morphological results of MDSC differentiation To investigate the effect of the test compound on human MDSC cell differentiation, CD14 was first isolated from human PBMCs from three donors. + Monocytes, then CD14 +Monocytes differentiated for 7 days in the presence of cytokines (GM-CSF and IL-6) and the test compound. After differentiation, morphological changes in the cells were observed under a microscope to assess the effect of the test compound on MDSC cell differentiation.

[0089] like Figure 5 As shown, compared with the Vehicle group, in the three different donors, the cells treated with medium and high concentrations of the drug had irregular outlines, small areas, and were even incomplete granular, similar to the morphology of lysed cells. The differentiation morphology of MDSC cells was significantly inhibited, indicating that the test compound ZSSW2102001 significantly inhibited the differentiation of MDSC cells in a dose-dependent manner.

[0090] 4.2 Flow cytometry results of MDSC differentiation Cells were collected 7 days after differentiation, and the expression of CD11b and CD33 was detected by flow cytometry. The proportion and relative number of CD11b and CD33 double-positive cells were counted to evaluate the effect of the test compound on MDSC cell differentiation. The gating strategy was as follows: Figure 6 As shown.

[0091] 4.2.1 MDSC cell viability Monocytes from different donors were differentiated for 7 days in the presence of the test compound. Cell viability was determined by flow cytometry. Figure 7 The results show the cell viability of MDSC cells from three different donors under different concentrations of the test compound. The data are expressed as mean ± standard error (n=3).

[0092] like Figure 7 As shown, for donor 1 and donor 2, when the concentration of the test compound reached 1.11 μM or higher, the cell viability was significantly reduced compared with the control group. For donor 3, when the concentration of the test compound reached 0.37 μM or higher, the cell viability was significantly reduced, indicating that high concentrations of ZSSW2102001 have significant drug toxicity to MDSC cells.

[0093] 4.2.2 CD11b in MDSC cells + CD33 + and CD11b + CD33 + The expression situation The presence of the test compound in CD11b in MDSC cells 7 days after differentiation was detected by flow cytometry. + CD33 + and CD11b + CD33 + The expression. CD11b + CD33+ Statistical differences were analyzed between the percentage and cell number of the Vehicle group and the various concentrations of the test compound. Figure 8-1 Figures 8-2, 8-3, and 8-4 show the CD11b levels in MDSC cells from three different donors under different concentrations of the test compound. + CD33 + and CD11b + CD33 + The expression of .

[0094] like Figure 8-1 As shown in Figures 8-2, 8-3, and 8-4, in donor 1, compared to the Vehicle group, the test compound ZSSW2102001 reduced CD11b in a dose-dependent manner during the differentiation of different numbers of monocytes into MDSC cells. + CD33 + The number of MDSC cells. In donors 2 and 3, the test compound ZSSW2102001 also reduced CD11b in a dose-dependent manner. + CD33 + The number of MDSC cells. However, in all three different donors, ZSSW2102001 significantly inhibited the expression of CD11b, CD33, and CD11b starting from 1.1 μM. + CD33 + The percentage of cells.

[0095] 4.3 ELISA results of MDSC differentiation The secretion of IL-10 in the supernatant of MDSC cells 7 days after subdifferentiation was detected using ELISA in the presence of the test compound. Statistical differences were analyzed between the Vehicle group and each concentration of the test compound. Figure 9 The results show the secretion of IL-10 cytokine in MDSC cells from three different donors under the action of different concentrations of the test compound.

[0096] like Figure 9 As shown, in donor 1, compared with the Vehicle group, the test compound ZSSW2102001 significantly inhibited the secretion of human IL-10 cytokine in a dose-dependent manner during the differentiation of different numbers of monocytes into MDSC cells. In donors 2 and 3, compared with the Vehicle group, ZSSW2102001 also significantly inhibited the secretion of human IL-10 cytokine in MDSC cells in a dose-dependent manner.

[0097] 4.4 Flow cytometry results of MDSCs inhibiting T cell activation To investigate the inhibitory function of the test compound in MDSC cells, CD14 cells derived from donors 2 and 3 were used. + Monocytes were induced to differentiate with cytokines in the presence of the test compound for 7 days. After the test compound was removed, the monocytes were co-cultured with T cells from the same donor for 3 days under CD3 / CD28 immunomagnetic bead activation conditions. CD4 counts were then performed by flow cytometry. + T and CD8 + T cell proliferation, such as Figure 11 and Figure 12 Encirclement strategy, such as Figure 10 As shown.

[0098] like Figure 11 As shown, compared with the Vehicle group, the test compound had no significant effect on the viability of T cells from the two donors.

[0099] like Figure 12 As shown, compared to the Vehicle group, donor 2's CD4 + T cells and CD8 + T cell proliferation significantly increased with decreasing MDSC cell count, but the increase decreased after ZSSW2102001 concentration exceeded 1.1 μM; in donor 3, compared with the Vehicle group, CD4 + T cells and CD8 + T cell proliferation also increased significantly as the number of MDSC cells decreased, but the increase in proliferation decreased when the concentration of ZSSW2102001 was greater than 0.37 μM.

[0100] 4.5 ELISA results of MDSC inhibiting T cell activation IFN-γ secretion in the supernatant of T cells co-cultured with MDSC cells for 3 days was detected using ELISA. Statistical differences were analyzed between the Vehicle group and each concentration of the test sample. Results are as follows: Figure 13 As shown.

[0101] like Figure 13 As shown, during the T cell proliferation of donors 2 and 3, compared with the Vehicle group, the secretion of IFN-γ increased with the decrease of MDSC cell number, but the degree of promotion of IFN-γ secretion decreased when the ZSSW2102001 concentration was greater than 1.1 μM.

[0102] Summary and Discussion The purpose of this experiment was to evaluate the effects of the test compound ZSSW2102001 on the differentiation and inhibitory function of MDSC cells. The differentiation results of MDSC cells indicated that the test compound ZSSW2102001 could dose-dependently inhibit CD11b. + CD33+ The differentiation of MDSCs was consistent with the morphological observations under the microscope. The inhibitory effect of MDSCs on T cell activation indicated that when the test compound ZSSW2102001 dose-dependently inhibited MDSC cell differentiation, CD4+ activation was reduced. + T cells and CD8 + Both T cell proliferation and IFN-γ secretion increased in a dose-dependent manner.

[0103] The result that FNC inhibits MDSC and thereby activates T cell proliferation and function suggests that in the human microenvironment, FNC can improve the activity of T cells in the corresponding environment by inhibiting immunosuppressive MDSC, thereby promoting the reconstruction of immune cell function.

[0104] Experiment 4: A multi-parameter flow cytometry analysis of rat peripheral blood lymphocyte phenotypes 1. Experiment Introduction Immunophenotyping utilizes the specific binding of antibodies to specific antigens on the surface of each cell subset to differentiate and assess specific cell subsets. Immunophenotyping can be used to monitor changes in cell subsets. This flow cytometry method employs a combined immunophenotypic analysis to determine the immunophenotype of peripheral blood from rats treated for 28 consecutive days. The immunophenotypes included total T cells (CD45+CD3+); helper T cells (CD45+CD3+CD4+CD8a-); and cytotoxic T cells (CD45+CD3+CD4-CD8a+).

[0105] 2. Instruments and Materials 2.1 Information on the compound to be tested The compound to be tested is azvudine FNC (ZSSW2102001-01), supplied by Henan Zhenshi Biotechnology Co., Ltd. The drug preparation (colorless solution) should be stored in a refrigerator at 2°C to 8°C and used within 10 days.

[0106] 2.2 Instruments and Materials

[0107]

[0108]

[0109]

[0110] 2.3 Reagent Preparation 1× Pyrolysis Buffer (Catalog No.: 349202) Add 50 mL of BD FACS lysis buffer to 450 mL of ultrapure water and mix well. Store at 2°C to 8°C for 1 month.

[0111] 2.4 Immunophenotypic Analysis of Peripheral Blood Lymphocyte Subsets in Rats SD rats were orally administered the drug for 28 consecutive days, once 24±1 hours after administration on day 28 and once on day 57 (calculated from the start of administration) after drug withdrawal. Blood was collected from the jugular vein of all experimental animals for lymphocyte immunophenotypic analysis. 0.8 mL of blood was collected and placed in a blood collection tube containing K2EDTA as an anticoagulant. The tube was gently inverted several times to ensure thorough mixing, placed on moist ice, and immediately transferred to the immunology laboratory.

[0112] The complete flow cytometry procedure is as follows: 1) Gently invert the blood collection tube 3-6 times to ensure thorough mixing, and draw 50 μL of blood (using the aspiration method) to the bottom of each tube.

[0113] 2) Add 1 mL of Duchenne phosphate buffer (DPBS) to each flow cytometry tube. Centrifuge at approximately 400 × g for 5 minutes at room temperature. Aspirate the supernatant and gently vortex.

[0114] 3) For blank control, skip this step and proceed to step 4. For other controls (such as compensation control or FMO and isotype control) and test samples, add surface antibody or mixed surface-labeled antibody solution.

[0115] 4) Vortex and avoid direct white light exposure at room temperature, incubate on a shaker (set to about 500 rpm) for 30 minutes.

[0116] 5) Add 1 mL of 1× lysis buffer (catalog number: 349202) to each tube and incubate at room temperature away from direct white light for 10-12 minutes. Centrifuge at approximately 400×g for 5 minutes at room temperature, discard the supernatant, and gently vortex.

[0117] 6) Add 1 mL of DPBS to each tube, centrifuge at approximately 400 × g for 5 minutes at room temperature, and discard the supernatant. 7) Resuspend the cells in 200-350 μL of DPBS per tube.

[0118] 8) Circle lymphocytes as AmCyan (CD45) positive and side-scattered (SSC) or front-scattered (FSC) and SSC.

[0119] 9) Cell collection was performed using flow cytometry. Approximately 10,000 lymphocytes were collected.

[0120] 2.5 Data Analysis The method for lymphocyte subset analysis is as follows: 1) Lymphocyte phylogenetics were obtained using forward scattering (FSC) and side scattering (SSC) phylogenetics, and lymphocyte populations were obtained using AmCyan (CD45) positive events and 90° side scattering (SSC).

[0121] 2) CD45+CD3+ represents total T cells. In the CD45+ lymphocyte phylum, a histogram is plotted, with the right side representing CD45+CD3+. The statistical results show the % Parent or #Event values ​​for CD45+CD3+.

[0122] 3) CD45+CD3+CD4+CD8a- are helper T cells. Within the CD45+CD3+ lymphocyte phylum, a scatter plot of CD4 and CD8a cells is created. The CD45+CD3+CD4+CD8a- cell population is located in the upper left quadrant (Q1) of the scatter plot. The statistical results show the %Grandparent or #Event value for CD45+CD3+CD4+CD8a-.

[0123] 4) CD45+CD3+CD4-CD8a+ are cytotoxic T cells. Within the CD45+CD3+ lymphocyte phylum, a scatter plot of CD4 and CD8a is created. The CD45+CD3+CD4-CD8a+ cell population is located in the lower right quadrant (Q4) of the scatter plot. The statistical results show the %Grandparent or #Event value for CD45+CD3+CD4-CD8a+.

[0124] Lymphocyte subset percentages, mean values, and SDs will be expressed as percentiles (two decimal places). %CV and %difference will be expressed as one decimal place.

[0125] NOT (CD3+) is equivalent to CD3-.

[0126] 2.6 Results Analysis 2.6.1 Total T cells (CD45+CD3+) The average percentage of total T cell subsets (%) in each group of animals is shown in Table 21 below.

[0127] Statistical results showed that, compared with the control group, the percentage of total T cells in the drug-treated group was significantly increased at 24 hours on day 28, demonstrating a dose-response relationship. No significant change in total T cells was observed in the drug-treated group after recovery on day 57. Therefore, all animals showed an increase in total T cell subsets related to the test substance.

[0128]

[0129] 2.6.2 Helper T cells (CD45+CD3+CD4+CD8a-) The average percentage (%) of helper T cell subsets in each group of animals is shown in Table 22 below.

[0130] Statistical results showed that, compared with the control group, the percentage of helper T cells in the drug-treated group was significantly increased at 24 hours on day 28, demonstrating a dose-response relationship. No significant changes in helper T cells were observed in the drug-treated group after recovery on day 57. Therefore, all animals showed an increase in helper T cell subset indicators related to the test substance.

[0131]

[0132] 2.6.3 Cytotoxic T cells (CD45+CD3+CD4+CD8a-) The average percentage (%) of cytotoxic T cell subsets in each group of animals is shown in Table 23 below.

[0133] Statistical results showed that, compared with the control group, the percentage of cytotoxic T cells in the drug-treated group was significantly increased at 24 hours on day 28, demonstrating a dose-response relationship. No significant changes in cytotoxic T cells were observed in the drug-treated group after recovery on day 57. Therefore, all animals showed an increase in cytotoxic T cell subsets related to the test substance.

[0134]

[0135] 2.6.4 Percentage ratio of helper T cells to cytotoxic T cells The average percentage (%) of cytotoxic T cell subsets in each group of animals is shown in Table 24 below.

[0136] Statistical results showed no statistically significant differences in the percentage ratios of helper T cells and cytotoxic T cells between the treatment group and the control group. No significant changes in the percentage ratios of helper T cells and cytotoxic T cells were observed at the end of the recovery period. Therefore, no changes in the percentage ratios of helper T cells and cytotoxic T cells related to the test substance were observed in any of the animals.

[0137]

[0138] in conclusion SD rats were administered FNC at doses of 1.5, 5.0, and 7.5 mg / kg / day orally for 28 days, a total of 28 doses (once daily). Following a 28-day recovery period, the treated rats showed a significant increase in the percentages of total T cells, helper T cells, and cytotoxic T cells associated with FNC at 24 hours on day 28, which returned to control levels by day 57. No changes were observed in the percentage ratios of helper T cells and cytotoxic T cells.

Claims

1. Use of azvudine or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising them, in the preparation of a medicament for treating impaired immune reconstitution due to human immunodeficiency virus infection and / or AIDS.

2. Use of azvudine or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising them, in the preparation of a medicament for the treatment of human immunodeficiency virus infection and / or AIDS with impaired immune reconstitution.

3. The use according to claim 1 or 2, characterized in that, The pharmaceutical composition also includes other HIV inhibitors; Optionally, the HIV inhibitor is selected from one or more of zidovudine, lamivudine, tenofovir, tenofovir disoproxil fumarate, tenofovir alafenamide, abacavir, nevirapine, efavirenz, rilpivirine, lopinavir / ritonavir, darunavir, atazanavir, dolutegravir, raltegravir, epovitamide, enfuvirtide, malaviro, and their pharmaceutically acceptable salts; Optionally, the HIV inhibitor is selected from one or more of tenofovir disoproxil fumarate, nevirapine, efavirenz, and their pharmaceutically acceptable salts; Optionally, the active pharmaceutical ingredient, azvudine or a pharmaceutically acceptable salt thereof, and the HIV inhibitor are either mixed together in the same formulation or separated into two formulations.

4. The use according to any one of claims 1-3, characterized in that, The active ingredients of the pharmaceutical composition include azvudine, tenofovir disoproxil fumarate, and nevirapine. The molar ratio of azvudine to tenofovir disoproxil fumarate is 1:7.5-1:540, preferably 1:23-1:135, more preferably 1:11-1:45, and the molar ratio of azvudine to nevirapine is 1:18-1:860, preferably 1:36-1:215, and more preferably 1:18-1:

72. Alternatively, the active ingredients of the pharmaceutical composition include azvudine, tenofovir disoproxil fumarate, and efavirenz, wherein the molar ratio of azvudine to tenofovir disoproxil fumarate is 1:7.5-1:540, preferably 1:23-1:135, more preferably 1:11-1:45, and the molar ratio of azvudine to efavirenz is 1:45-1:2177, preferably 1:91-1:544, more preferably 1:91-1:

181.

5. The use according to claim 4, characterized in that, The active pharmaceutical ingredient of the pharmaceutical composition consists of azvudine, tenofovir disoproxil fumarate, and nevirapine.

6. The use according to claim 4, characterized in that, The active pharmaceutical ingredient consists of azvudine, tenofovir disoproxil fumarate, and efavirenz.

7. The use according to any one of claims 1-6, characterized in that, Pharmaceutically acceptable salts of azvudine include salts formed by azvudine with the following acids: hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, mandelic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, ascorbic acid palmitic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, phthalic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, or leucine.

8. The use according to claim 7, characterized in that, The pharmaceutically acceptable salts of azvudine are those formed by azvudine with hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, or phthalic acid.

9. The use according to any one of claims 1-8, characterized in that, The corresponding human immunodeficiency virus load for poor immune function reconstitution is <50 copies / mL.

10. The use according to any one of claims 1-9, characterized in that, The corresponding CD4+ T lymphocyte count for poor immune function reconstruction is <350 cells / μL.

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