Application of alpha ketoglutaric acid prodrug in preparation of medicine for treating myelodysplastic syndrome

Abstract

The invention discloses application of an alpha-ketoglutaric acid prodrug in preparation of a medicine for treating myelodysplastic syndrome, and belongs to the technical field of medicines, and particularly, the alpha-ketoglutaric acid prodrug can select dimethyl 2-ketoglutarate, the dimethyl 2-ketoglutarate is metabolized to generate alpha-ketoglutaric acid, and experiments prove that the alpha-ketoglutaric acid prodrug can be used for preparing a medicine for treating myelodysplastic syndrome. Alpha-ketoglutaric acid prodrugs are metabolized to generate alpha-ketoglutaric acid, the alpha-ketoglutaric acid weakens methylation of myeloid differentiation related genes, enhances the myeloid differentiation proportion, reduces the gonorrhea differentiation proportion and improves pathological hematopoiesis, and the alpha-ketoglutaric acid has wide application prospects in preparation of drugs for treating myelodysplastic syndromes and can avoid adverse reactions of chemotherapeutic drugs.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and in particular relates to the application of alpha-ketoglutaric acid prodrug in the preparation of a drug for treating myelodysplastic syndrome. Background Art

[0002] Hematopoietic stem cells self-renew and differentiate in the bone marrow, maintaining all the blood cells needed by the body throughout the life cycle. Under healthy conditions, hematopoietic stem cells differentiate into myeloid and lymphoid lines to maintain a relatively constant ratio of peripheral mature blood cells. Myeloid and lymphoid differentiation abnormalities and myeloid bias are common in blood system diseases. Myelodysplastic syndromes (MDS) are malignant blood system diseases and a group of clonal diseases originating from hematopoietic stem cells (hematopoietic stem / progenitor cells, HSC), characterized by blood cell reduction, abnormal development of one or more myeloid cell lines, ineffective hematopoiesis, and an increased risk of evolving into acute myeloid leukemia. The occurrence and evolution of MDS is a multi-step pathological process involving multiple genes.

[0003] Current treatments for MDS include supportive therapy, hematopoiesis promotion, demethylating drugs (azacitidine, decitabine), combined chemotherapy (cytarabine, anthracyclines), hematopoietic stem cell transplantation, etc. However, supportive therapy is not effective, chemotherapy has adverse reactions that affect the patient's quality of life, and hematopoietic stem cell transplantation may cause complications.

[0004] A Chinese patent document with publication number CN104127543A discloses a Chinese medicine composition for treating myelodysplastic syndrome and its application. The Chinese medicine composition comprises: 10 to 50 parts of Lygodium japonicum, 10 to 50 parts of Eucommia ulmoides leaves, and 40 to 90 parts of Hedyotis diffusa. The medicinal ingredients of the Chinese medicine composition can inhibit the proliferation of MDS cell line Skm-1 cells, and can simultaneously activate intrinsic and exogenous pathways (death receptor pathway and mitochondrial pathway) to induce apoptosis of Skm-1 cells; can block Skm-1 cells in the G0 / G1 phase and induce cell apoptosis; can cause cell apoptosis through the PI3K / Akt signaling pathway and the NF-κB signaling pathway, and reduce drug resistance. A Chinese patent document with publication number CN103655857A discloses a Chinese medicine composition for treating myelodysplastic syndrome. The Chinese medicine composition is made of the following raw materials in parts by weight: 1500-1700 parts of Gardenia, 3100-3300 parts of Tangerine Peel, 900-1100 parts of Stir-fried Phellodendron, 2600-2800 parts of Radix Pseudostellariae, 700-900 parts of Honey-roasted Licorice, 1500-1700 parts of Coptis chinensis, 3100-3300 parts of Rhizoma Cyperi, 300-500 parts of Borneolum, 700-900 parts of Stir-fried Atractylodes macrocephala with bran, 1500-1700 parts of Artificial Bezoar, 0.7-0.9 parts of Musk, and 3100-3300 parts of Radix Scutellariae. The invention verifies through clinical experiments that the corresponding Chinese medicine composition can increase the hemoglobin concentration in peripheral blood and improve the quality of life of patients. However, the traditional Chinese medicine treatment method has the defects of unclear pharmacological research and slow drug effect, and is not suitable for treating patients with rapidly progressive diseases.

[0005] A Chinese patent document with publication number CN112168829A discloses the use of an antiviral drug in the preparation of a drug for treating myelodysplastic syndrome, wherein the antiviral drug is famciclovir (FCV). The invention finds that the use of FCV in the treatment of MDS can play a dual role: on the one hand, it can resist viral infection, and on the other hand, it can reduce abnormally proliferating blood cells in MDS. However, famciclovir has side effects on the nervous system or the digestive system.

[0006] In summary, it is necessary to find effective and economical drugs for the treatment of MDS. Summary of the invention

[0007] The present invention provides application of alpha-ketoglutarate prodrug in preparing medicine for treating myelodysplastic syndrome.

[0008] The specific technical solutions adopted are as follows:

[0009] Application of alpha-ketoglutaric acid prodrug in preparing medicine for treating myelodysplastic syndrome.

[0010] Optionally, the α-ketoglutaric acid prodrug is dimethyl 2-ketoglutarate, and the structural formula is shown below:

[0011]

[0012] Direct supplementation of α-ketoglutarate cannot enter the cell membrane, but injection or oral administration of dimethyl 2-ketoglutarate, or cell culture with dimethyl 2-ketoglutarate added to the culture medium, can supplement α-ketoglutarate in vivo or in cells. That is, α-ketoglutarate prodrug is metabolized to produce α-ketoglutarate, which weakens the methylation of myeloid differentiation-related genes (TET2, etc.), enhances the myeloid differentiation ratio (promotes the myeloid differentiation of hematopoietic stem cells), and reduces the lymphoid differentiation ratio, thereby treating myelodysplastic syndrome.

[0013] Preferably, the administration concentration of dimethyl 2-ketoglutarate is 10-100 mg kg -1 / d.

[0014] The present invention also provides a medicine for treating myelodysplastic syndrome, comprising dimethyl 2-ketoglutarate.

[0015] Optionally, the drug for treating myelodysplastic syndrome further comprises a pharmaceutically acceptable carrier or diluent to prepare a pharmaceutically acceptable dosage form.

[0016] Specifically, the drug for treating myelodysplastic syndrome can be taken orally or injected, and the dosage forms include tablets, granules, capsules, powders or injections.

[0017] Preferably, the drug for treating myelodysplastic syndrome is in the form of an injection, which is administered intraperitoneally.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention supplements α-ketoglutarate in vivo by supplementing α-ketoglutarate prodrugs such as 2-ketoglutarate dimethyl ester. 2-ketoglutarate dimethyl ester and other α-ketoglutarate prodrugs are metabolized to generate α-ketoglutarate. Alpha-ketoglutarate weakens the methylation of myeloid differentiation-related genes (TET2, etc.), enhances myeloid differentiation, reduces the lymphoid differentiation ratio, and significantly reduces HSC function in transplantation experiments, improves pathological hematopoiesis, and has broad application prospects in the treatment of myelodysplastic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the effect of in vivo administration of dimethyl 2-ketoglutarate on the number of HSCs;

[0021] Figure 2 This is a graph showing the effect of in vitro administration of dimethyl 2-ketoglutarate on the proportion of myeloid cells in the bone marrow;

[0022] Figure 3 This is a graph showing the effect of in vitro administration of dimethyl 2-ketoglutarate on the number of bone marrow myeloid cells;

[0023] Figure 4 This is a graph showing the effect of in vivo administration of dimethyl 2-ketoglutarate on the proportion of M cells, T and B lymphocytes in the bone marrow;

[0024] Figure 5 This is a graph showing the effect of 2-ketoglutarate dimethyl ester administration on reducing the peripheral blood T cell chimerism rate after LT-HSC transplantation;

[0025] Figure 6 This is a graph showing the effect of 2-ketoglutarate dimethyl ester administration on reducing the chimerism rate of peripheral blood B cells after LT-HSC transplantation;

[0026] Figure 7 This is a graph showing the effect of 2-ketoglutarate dimethyl ester administration on reducing the peripheral blood M cell chimerism rate after LT-HSC transplantation;

[0027] Figure 8 This is a graph showing the effect of 2-ketoglutarate dimethyl ester administration on reducing the total peripheral blood cell chimerism rate after LT-HSC transplantation;

[0028] Fig. 9 This is a statistical graph showing that administration of dimethyl 2-ketoglutarate reduces the self-renewal ability of LT-HSC after transplantation;

[0029] Fig.10 Administration of dimethyl 2-ketoglutarate reduces Mll PTD / WT / Runx1 Δ / Δ Statistical graph of proliferation and differentiation ability of BM cells;

[0030] Fig.11 Dimethyl 2-ketoglutarate was administered to inhibit Mll PTD / WT / Runx1 Δ / Δ Effect diagram of BM cells differentiating into mature granulocytes;

[0031] Fig.12 This is the survival curve of MDS transplanted mice administered with dimethyl 2-oxoglutarate.

[0032] In the accompanying drawings, the results of significance analysis are *p<0.05, **p<0.01, ****p<0.0001. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the examples and accompanying drawings. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods of the following examples that do not specify specific conditions are usually performed under normal conditions or under conditions recommended by the manufacturer. The content that is not described in detail in this specification sheet belongs to the prior art known to professionals in the field. The experimental materials used in the following examples, if not otherwise specified, can be purchased by conventional biochemical reagent companies.

[0034] In the following examples, the SPF-grade C57BL / 6 mice used were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and kept in the clean animal room of the Experimental Animal Center of the International Health Medical Research Institute of Zhejiang University. The mice were 8-12 weeks old during the experiment. Throughout the in vivo experiment, all operations complied with the "Regulations on the Use of Experimental Animals". 2-Ketoglutarate dimethyl ester was purchased from Sigma-Aldrich, USA, and the α-ketoglutarate quantitative detection kit was purchased from BioVision, USA. All flow antibodies used, unless otherwise specified, were purchased from eBioscience or BD Biosiences, USA. All experimental results were obtained from three independent repeated experiments, and the data were analyzed using an unpaired t-test. The data are expressed as mean ± standard error of the mean SEM (n = 3), *p < 0.05, **p < 0.01, ****p < 0.0001.

[0035] Example 1

[0036] Mice of appropriate age were divided into an intraperitoneal injection group (2-ketoglutarate dimethyl ester DMKG, intraperitoneal injection, 100 mg / kg) and a control group. DMKG was metabolized to produce α-ketoglutarate. After the administration, bone marrow cells were obtained from the bone tissue for further analysis.

[0037] (1) Based on cell surface markers and in vitro cell culture experiments, hematopoietic stem cells (HSCs) at different stages of differentiation can be grouped and sorted. By analyzing the different groups of HSCs, such as long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs), the effect of α-ketoglutaric acid on HSC self-renewal can be determined. The results are as follows: Figure 1 As shown, Figure 1 This indicates that administration of dimethyl 2-ketoglutarate can reduce the number of hematopoietic stem cells.

[0038] (2) Further analysis of the downstream progenitor cell populations of HSC differentiation showed that the progenitor cells were mainly divided into myeloid progenitors (GMP), which can mainly differentiate into granulocytes, and common lymphoid progenitors (CLP), which can mainly differentiate into T cells, B cells, and natural killer cells. The effect of α-ketoglutarate on the differentiation tendency of HSC was further determined. CD16 / 32 + and CD11b + It is a surface marker of mouse bone marrow myeloid cells. The detection of the above markers shows that α-ketoglutarate can increase the number and proportion of LT-HSC to myeloid cells ( Figure 2-Figure 3 ).

[0039] (3) Further analysis of the terminal differentiated cell populations downstream of progenitor cell differentiation, including T cells, B cells, and M cells. Figure 4 To determine the effect of α-ketoglutarate on the differentiation tendency of HSC, Figure 4 This indicates that α-ketoglutarate causes a deviation in the ratio of myeloid cells to T and B lymphocytes in the bone marrow.

[0040] Example 2

[0041] Due to the complex in vivo environment, it is impossible to determine whether the in vivo intervention of α-ketoglutarate causes changes in differentiation tendency by regulating HSC or directly changes the differentiated population of progenitor cells. HSC in vivo transplantation experiments can clarify this issue. Continuous HSC transplantation experiments can evaluate HSC self-renewal ability, long-term multi-lineage hematopoietic reconstruction ability and differentiation tendency. We will focus our research on LT-HSC, the group with the most stem cell characteristics. The effect of α-ketoglutarate on HSC self-renewal will be determined by evaluating the self-renewal ability of LT-HSC after transplantation.

[0042] In this transplantation experiment, 500 LT-HSCs were sorted from the mice in each group after administration and added into 5*10 5 The competitor's whole bone marrow nucleated cells (flow cytometry, CD45.1 cells) were transplanted into recipient mice, and the peripheral blood B cells, T cells, M cells and total (T, B, M) chimerism rates were detected 1, 2, and 3 months after transplantation. The recipient mice were sacrificed 3 months after transplantation, the bone marrow was removed, and the HSC chimerism rate was detected by flow cytometry. This experiment is the most important experiment that can illustrate the function of LT-HSC.

[0043] From the analysis results, it can be seen that the total chimerism rate in the peripheral blood of mice showed a significant downward trend after administration of dimethyl 2-ketoglutarate ( Figure 8 ), while the chimerism rates of T cells, B cells, and M cells all showed a significant downward trend ( Figure 5-Figure 7 ). The results of HSC chimerism analysis showed that α-ketoglutarate reduced the self-renewal efficiency of LT-HSC, and the peripheral blood chimerism rate showed that the increase in α-ketoglutarate content directly reduced the directional differentiation of HSC ( Figure 5-Figure 9 ).

[0044] In vivo administration of dimethyl 2-ketoglutarate inhibits the number of HSCs, promotes the expansion of myeloid cells, increases myeloid progenitor cells, decreases lymphoid progenitor cells, causes a deviation in the ratio of myeloid cells to T and B lymphocytes in the bone marrow, and reduces the self-renewal function of LT-HSCs after transplantation.

[0045] Example 3

[0046] From Examples 1 and 2, it can be seen that the α-ketoglutarate generated by metabolism can promote HSC differentiation into myeloid lineage by demethylation. Correspondingly, under the condition of increasing the level of intracellular α-ketoglutarate step by step, the number of bone marrow pathological cells can be seen to decrease. In the colony forming unit test, bone marrow cells were extracted from mouse bone tissue in the same way as above, single cell suspensions were prepared, dispersed into culture dishes, and analyzed and counted by flow cytometry after 7 days of culture. We can also prove that pathological cells are sensitive to dimethyl 2-ketoglutarate and show dose dependence ( Fig.10 ). After administration, CD11b and Gr-1 double-negative pathological cells were selectively killed, and myeloid differentiation was reduced accordingly.

[0047] In vivo and in vitro conditions, we verified that α-ketoglutarate produced by the metabolism of dimethyl 2-ketoglutarate can reduce the proliferation, differentiation and maturation of bone marrow cells in model mice: Mll PTD / WT / Run △ / △ It is a mouse model of myelodysplastic syndrome (MDS), in which the DNA and histones of its bone marrow cells have high methylation levels. After intraperitoneal administration, the methylation levels of its DNA and histones decrease.

[0048] The colony-forming cells were collected and stained with Giemsa. The size, number and color depth of the myeloid granules in the pathological cells of the experimental group and the control group were counted respectively to determine the differentiation of myeloid cells in each group. The results showed that dimethyl 2-ketoglutarate could significantly inhibit Mll PTD / WT / Run △ / △ The myeloid cells in the mouse bone marrow differentiated into mature stages, and the number of immature myeloid cells decreased ( Fig.11 ).

[0049] The recipient mice transplanted with MDS pathological cells were treated with 2-ketoglutarate dimethyl ester, and a control group was set up to observe the survival rate of the recipient mice: Fig.12As shown, the survival rate of recipient mice administered with dimethyl 2-oxoglutarate was significantly prolonged, indicating that it has the effect of treating myelodysplastic syndrome.

[0050] The content of α-ketoglutarate in bone marrow pathological cells of human MDS patients was further detected and treated with dimethyl 2-ketoglutarate to observe the changes in the chimerism rate of bone marrow pathological cells: after human bone marrow pathological cells treated with dimethyl 2-ketoglutarate were transplanted into model mice, the chimerism rate of pathological cells in the bone marrow and spleen was significantly reduced: compared with the control group, dimethyl 2-ketoglutarate significantly reduced the chimerism rate of pathological cells at low concentrations, and the chimerism rate further decreased at higher concentrations. The above results prove that dimethyl 2-ketoglutarate also has the same therapeutic effect on humans.

[0051] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of α-ketoglutarate prodrug in the preparation of drugs for the treatment of myelodysplastic syndrome.

2. Use of the α-ketoglutarate prodrug according to claim 1 in the preparation of a drug for treating myelodysplastic syndrome, characterized in that: The alpha-ketoglutarate prodrug is dimethyl 2-ketoglutarate.

3. Use of the α-ketoglutarate prodrug according to claim 2 in the preparation of a drug for treating myelodysplastic syndrome, characterized in that: The dosage concentration of the dimethyl 2-ketoglutarate is 10-100 mg kg -1 / d.

4. Use of the α-ketoglutarate prodrug according to claim 1 in the preparation of a drug for treating myelodysplastic syndrome, characterized in that: α-ketoglutarate prodrug is metabolized to generate α-ketoglutarate, which weakens the methylation of myeloid differentiation-related genes, enhances the myeloid differentiation ratio, and reduces the lymphoid differentiation ratio.

5. A drug for treating myelodysplastic syndrome, characterized in that: Includes dimethyl 2-ketoglutarate.

6. The drug for treating myelodysplastic syndrome according to claim 5, characterized in that: The medicine for treating myelodysplastic syndrome also includes a pharmaceutically acceptable carrier or diluent to prepare a pharmaceutically acceptable dosage form.

7. The drug for treating myelodysplastic syndrome according to claim 5, characterized in that: The dosage form of the medicine for treating myelodysplastic syndrome includes tablets, granules, capsules, powders or injections.

8. The drug for treating myelodysplastic syndrome according to claim 5, characterized in that: The dosage form of the medicine for treating myelodysplastic syndrome is injection, which is administered through the abdominal cavity.

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