Application of methyl α-D-glucopyranoside in the preparation of thalassemia treatment drugs

Abstract

The present invention discloses the use of methyl α-D-glucopyranoside in the preparation of thalassemia therapeutic drugs. Comprehensive analysis shows that when methyl α-D-glucopyranoside was intraperitoneally injected into β-thalassemia mice, it was found that methyl α-D-glucopyranoside was one of the metabolites with the largest down-regulation amplitude, and had multiple beneficial effects on Th3 / + mice, including increasing the total red blood cell count, hemoglobin level and hematocrit, and reducing the coefficient of variation of the red blood cell volume distribution width; reducing the iron content in the liver, splenomegaly and iron content in the spleen; increasing the GSH / GSSG ratio, reducing the ROS level, reducing the apoptosis rate of peripheral blood cells, and increasing the lifespan of red blood cells. After GP treatment, the ineffective hematopoiesis, anemia and iron overload of thalassemia mice were effectively alleviated without obvious toxic side effects. The treatment is convenient to implement, and GP is cheap and easy to store.

Description

Technical Field

[0001] The present invention relates to a new application of methyl α-D-glucopyranoside, and in particular to an application of methyl α-D-glucopyranoside in the preparation of a drug for treating thalassemia, belonging to the field of medical technology. Background Art

[0002] β-thalassemia (β-TH) is an inherited blood disorder characterized by reduced or abnormal production of hemoglobin β chains. The disease occurs widely, with approximately 68,000 newborns born annually in tropical and subtropical regions with various thalassemia syndromes. Globally, there are reportedly 80 to 90 million carriers, representing 1.5% of the global population.

[0003] Despite significant progress in the treatment of β-TH, currently available traditional approaches, including iron chelation therapy, blood transfusions, splenectomy, hydroxyurea, and hematopoietic stem cell transplantation (HSCT), still present many challenges and limitations. For example, lifelong iron chelation and blood transfusions can lead to serious complications that affect overall survival. HSCT is limited by the inherent risk of graft-versus-host disease and the availability of human leukocyte antigen-matched donors. In recent years, several new treatments have been attempted to control β-TH. These newly introduced therapies can be divided into three major categories: targeting ineffective erythropoiesis (IE), correcting globin chain imbalances, and iron metabolism. Despite the promising results achieved by the aforementioned therapies, there is currently no inexpensive universal treatment for several forms of this disease. Therefore, the search for new therapeutic approaches for clinical treatment remains urgent.

[0004] The applicant intends to discover new strategies for treating thalassemia through research, such as targeting metabolites in thalassemia metabolomics, which can effectively improve anemia, alleviate secondary iron overload, reduce possible complications, reduce the financial burden on patients' families, and increase the universality of treatment for patients.

[0005] Based on the defects of the main methods for treating thalassemia patients at this stage, finding a more economical, reliable and universally treatable strategy is a new direction for thalassemia treatment.

[0006] There are currently no reports on the use of methyl α-D-glucopyranoside in the treatment of thalassemia. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the use of methyl α-D-pyranoglucoside in the preparation of a drug for treating thalassemia.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] 1. Use of methyl α-D-glucopyranoside (GP) in the preparation of a drug for treating thalassemia, wherein the structural formula of methyl α-D-glucopyranoside is as follows:

[0010]

[0011] As one of the preferred technical solutions, the types of thalassemia can be divided into α type, β type, δβ type, δ type, εβγδ type, but are not limited to: α type, β type, δβ type, δ type, εβγδ type.

[0012] As one of the preferred technical solutions, the dose of GP for treating thalassemia mice is 0.25 g / kg, injected intraperitoneally 3 times a week for 4 weeks.

[0013] 2. A pharmaceutical composition comprising methyl α-D-glucopyranoside.

[0014] 3. Use of the aforementioned pharmaceutical composition in the preparation of a drug for treating thalassemia.

[0015] 4. A drug for treating thalassemia, the active ingredient of which is methyl α-D-pyranoglucoside.

[0016] As one of the preferred technical solutions, the drug further comprises pharmaceutically acceptable excipients.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides the use of methyl α-D-pyranoglucoside in the preparation of a drug for treating thalassemia. Macrogenomic analysis showed that the functions of some metabolism-related genes in the feces of thalassemia mice (Th3 / +) were enhanced. Metabolomics analysis showed that there were significant metabolic differences between peripheral blood (PB) cells and plasma of Th3 / + mice and wild-type mice (Wt). Comprehensive analysis showed that when methyl α-D-pyranoglucoside was intraperitoneally injected into β-thalassemia mice, it was found that methyl α-D-pyranoglucoside was one of the metabolites with the largest downregulation, and had multiple beneficial effects on Th3 / + mice, including increasing the total red blood cell count, hemoglobin level and hematocrit, reducing the coefficient of variation of red blood cell volume distribution width; reducing liver iron content and splenomegaly and spleen iron content; increasing the GSH / GSSG ratio, reducing ROS level, reducing the apoptosis rate of peripheral blood cells, and improving red blood cell lifespan.

[0019] After GP treatment, the ineffective hematopoiesis, anemia and iron overload of thalassemia mice were effectively alleviated without obvious toxic side effects. The treatment is easy to implement, GP is cheap and easy to store. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The present invention used routine blood tests to examine the effect of GP on peripheral blood anemia indicators in mice: A is red blood cell count (RBC), B is hemoglobin concentration (HGB), C is hematocrit (HCT%), and D is the coefficient of variation of red blood cell volume distribution width (RDW-CV%). *p < 0.05; **p < 0.01; ***p < 0.001.

[0021] Figure 2 The effects of GP treatment on the spleen and liver of mice: A is spleen appearance, B is spleen weight, C is spleen index, D is liver Prussian blue staining, E is liver iron content, F is spleen Prussian blue staining, and G is spleen iron content. *p < 0.05; **p < 0.01; ***p < 0.001.

[0022] Figure 3 The figure shows the effects of GP treatment on GSH / GSSG and ROS levels in K562 cells in the present invention; wherein, A is GSH / GSSG, B is ROS level, GP 0.5 represents an intervention concentration of GP of 0.5 μM; GP1 represents an intervention concentration of GP of 1 μM, *p<0.05; **p<0.01; ***p<0.001.

[0023] Figure 4 The figure shows the effect of GP treatment on the apoptosis rate of peripheral blood cells in mice. *p<0.05; **p<0.01; ***p<0.001.

[0024] Figure 5 The present invention used flow cytometry to detect the effect of GP on the half-life of peripheral blood red blood cells in thalassemia mice. *p<0.05; **p<0.01; ***p<0.001. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0026] Example 1: Effect of GP on peripheral blood anemia in mice using blood routine test

[0027] (1) Male wild-type mice (purchased from Hunan Slake Jingda Company) and β-thalassemia mice aged 6-8 weeks were divided into two groups: one untreated group served as the control group, and one group received an intraperitoneal injection of 100 μL of GP and served as the experimental group. The dose of GP treatment was 0.25 g / kg, intraperitoneally injected three times a week for 4 weeks.

[0028] β-thalassemia mice were constructed using Th3 / + mice (purchased from Jackson Laboratory) with reference to the following literature: B Yang, S Kirby, J Lewis, PJ Detloff, N Maeda, O Smithies. A mouse model for beta 0-thalassemia. Proc Natl Acad Sci USA, 92 (1995), pp. 11608-11612.

[0029] (2) Collect peripheral blood from mice treated with vehicle / GP for 2-6 weeks using anticoagulant centrifuge tubes.

[0030] (3) According to the instrument usage rules, the sample was diluted with diluent for blood cell analysis (DCL) to 6 times the volume of peripheral blood, loaded into a blood analyzer (Sysmex, Japan) and the data was read.

[0031] The experimental results are as follows Figure 1 As shown: GP treatment for 4-6 weeks, the number of red blood cells in the peripheral blood of thalassemia mice ( Figure 1 A) and hemoglobin levels ( Figure 1 Middle B) and hematocrit ( Figure 1 In addition, the coefficient of variation of the volume distribution width of peripheral blood red blood cells in thalassemia mice ( Figure 1 Middle D) showed a significant decrease after GP treatment, indicating that GP treatment can effectively improve the peripheral blood anemia indicators of thalassemia mice.

[0032] Example 2: Observation of the effect of GP treatment on the spleen of mice

[0033] Experimental steps:

[0034] (1) The mice were grouped and treated as in Example 1;

[0035] (2) Weigh the mice and record the weight, dissect the mice, remove the intact spleen, weigh the spleen and record the weight;

[0036] (3) Arrange the mouse spleens on a paper with a clear background, take photos, process the data, perform statistical analysis, and calculate the spleen weight index (ratio of spleen index to body weight).

[0037] (4) Prussian blue iron staining kit (Solarbio, Beijing, China) was used to measure the iron content of mouse spleen, and Dab enhanced Prussian blue staining kit (Solarbio, Beijing, China) was used to measure the iron content of mouse liver. The iron index (the percentage of cells positive for iron particles) was calculated.

[0038] The experimental results are as follows Figure 2 The results showed that after 4 weeks of GP treatment, the spleen shape of thalassemia mice ( Figure 2 A), weight ( Figure 2 Middle B) and spleen index ( Figure 2 C) and other factors were significantly improved, and the iron content of liver and spleen was significantly reduced ( Figure 2 These results indicate that GP can effectively alleviate splenomegaly and iron deposition in thalassemia mice.

[0039] Example 3: Observation of the effect of GP treatment on GSH / GSSG and ROS in K562 cells

[0040] Experimental steps:

[0041] (1) The mice were grouped and treated as in Example 1;

[0042] (2) The GSH / GSSG ratio in mouse peripheral blood cells (PB cells) and K562 cells was measured using the Bio-Tech GSH and GSSG Detection Kit (S0053). The reactive oxygen species (ROS) in K562 cells was measured using the Bio-Tech Reactive Oxygen Species Detection Kit (S0033S).

[0043] (3) Experimental results are as follows Figure 3 The results showed that after GP treatment, the GSH / GSSG ratio in peripheral blood cells and K562 cells of thalassemia increased ( Figure 3 ROS( Figure 3 This indicates that GP can enhance the antioxidant capacity of cells and reduce the level of ROS in cells.

[0044] Example 4: Observation of the effect of GP treatment on apoptosis of peripheral blood cells in mice

[0045] Experimental steps:

[0046] (1) The mice were grouped and treated as in Example 1;

[0047] (2) The vazyme Annexin V-FITC / PIApoptosis Detection Kit (A211) was used to measure the effect of GP on the apoptosis rate of mouse peripheral blood cells.

[0048] (3) Experimental results are as follows Figure 4 As shown, the results showed that the apoptosis rate of peripheral blood cells in mice was significantly reduced after GP treatment.

[0049] Example 5: Flow cytometry was used to detect the effect of GP on the half-life of biotin-labeled mouse peripheral blood erythrocytes. Experimental steps:

[0050] (1) The mice were grouped and treated as in Example 1;

[0051] (2) 200 μL of peripheral blood from mice in the vehicle / GP treatment group was collected in an anticoagulant centrifuge tube;

[0052] (3) Add 300 μM biotin labeling solution (Thermo Fisher, USA), mix gently, and incubate at 37°C in the dark for 60 min;

[0053] (4) After labeling, the cells were washed once and the labeled red blood cells were injected into wild-type recipient mice via the orbital vein;

[0054] (5) Starting from 24 hours after the cells were transfused into the recipient mice, 2-3 μL of peripheral blood was collected every 7 days, and phycoerythrin (PE)-coupled streptavidin (BioLegend, California, USA) was added. After incubation in the dark for half an hour, the proportion of PE-positive cells was detected by upflow cytometry, and the results were analyzed and statistically analyzed.

[0055] The experimental results are as follows Figure 5 As shown in the data, the decay of red blood cells in the thalassemia mouse-GP-treated group was significantly slower than that in the control group, and the survival time of red blood cells was prolonged, indicating that GP treatment can effectively increase and prolong the half-life of red blood cells in thalassemia mice.

[0056] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. Use of methyl α-D-glucopyranoside in the preparation of a drug for treating thalassemia, characterized in that: The thalassemia is β-thalassemia.

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