Application of compound DMT207 in preparation of beta-globin disease treatment medicine
Compound DMT207 induces fetal hemoglobin expression in a β-globinopathy model and specifically inhibits DNA methyltransferase 1, thus solving the problems of high toxicity and off-target effects of existing drugs and achieving low-toxicity and high-efficiency treatment for β-globinopathy.
Patent Information
- Application Number
- CN202511292831.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-11
- Publication Date
- 2025-12-16
AI Technical Summary
There is a lack of drugs with high specificity and low toxicity for the treatment of β-globinopathy. Existing treatments such as hydroxyurea have poor efficacy, and decitabine causes cellular and genomic damage and off-target effects.
The compound DMT207 was used to induce fetal hemoglobin expression and activate HBG expression in human umbilical cord blood-derived erythropoietin cell lines and human primary hematopoietic stem cells, and specifically inhibit the catalytic activity of DNA methyltransferase 1, avoiding off-target effects and exhibiting lower toxicity than decitabine.
DMT207 activates fetal hemoglobin expression, alleviates splenomegaly, promotes erythrocyte maturation, and relieves symptoms of β-thalassemia and sickle cell anemia without affecting body weight. It has less toxicity than decitabine and its therapeutic effect is superior to existing drugs.
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Figure CN121129843A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, in particular to an application of a compound DMT207 in preparation of a drug for treating β-thalassemia. BACKGROUND
[0002] β-thalassemia includes β-thalassemia and sickle cell anemia. β-thalassemia (β-thalassemia) is also known as β-globin production disorder anemia, which is a single gene recessive genetic disease prevalent worldwide. The disease is prevalent in the southern region of China, and the pathogenic gene carrying rate in some provinces is more than 9%, much higher than the world average (1.5%). β-thalassemia patients usually have symptoms such as anemia, jaundice, splenomegaly, iron overload, poor quality of life, and severe patients usually die before the age of 5. Intermediate patients usually live to adulthood, but need lifelong blood transfusion to maintain. Sickle cell anemia is also a disease caused by mutation of β-globin gene. The 6th amino acid of β-globin is mutated from glutamic acid to valine, resulting in abnormal polymerization of hemoglobin structure, and then forming sickle-shaped red blood cells. Sickle cell anemia patients have symptoms such as blood vessel obstruction, organ ischemia, and early splenic function, which seriously affect the quality of life of patients. At present, the treatment for these two diseases is mostly limited to symptom relief, such as blood transfusion, pain relief, splenectomy (β-thalassemia), and iron removal therapy (β-thalassemia). The existing radical treatment methods only include hematopoietic stem cell transplantation and gene therapy based on CRISPR / Cas9 system, but these treatment methods belong to highly customized solutions and cannot be carried out on a large scale.
[0003] β-thalassemia patients usually cannot normally produce adult hemoglobin (HbA), and some studies focus on compensating for defects by activating fetal hemoglobin (HbF). So far, hydroxyurea is the only approved drug for treating sickle cell anemia that can activate fetal hemoglobin expression, but its efficacy is poor. In addition, it has also been reported that DNA methylation inhibitors, pomalidomide and other drugs can activate fetal hemoglobin expression, but these drugs have not been approved for the treatment of these two diseases.
[0004] DNA methylation is a classic epigenetic modification that plays an important role in various biological processes such as development and cell differentiation. It has been reported that knocking out DNA methyltransferase 1 (DNMT1) alone is sufficient to activate fetal hemoglobin expression in red blood cells. DNA methylation inhibitor decitabine (5-aza-2'-deoxycytidine) has been shown in preclinical studies and clinical trials to alleviate the symptoms of β-thalassemia. However, decitabine has limitations: it can cause genomic damage, has off-target effects (inhibits three DNA methyltransferases), and has strong toxicity. Therefore, there is an urgent need for a β-thalassemia treatment drug with high specificity and low toxicity. SUMMARY
[0005] To this end, the technical problem to be solved by the present application is to overcome the problem of lack of a drug for treating beta-globinopathy with high specificity and low toxicity in the prior art.
[0006] To solve the above technical problem, the present application provides the use of a compound DMT207 in the preparation of a drug for treating beta-globinopathy. The present application finds that the compound (hereinafter referred to as "DMT207", the structural formula of which is shown in formula I) exhibits a lower toxicity than decitabine in human cord blood-derived erythroid progenitor cell line (HUDEP)-2 and human primary hematopoietic stem cells, can induce high expression of fetal hemoglobin while maintaining healthy cell viability, and its induction effect is much higher than that of decitabine. In a beta-thalassemia model mouse, the compound of the present application activates fetal hemoglobin expression without affecting body weight, relieves splenomegaly, promotes red blood cell maturation, relieves beta-thalassemia symptoms, and can specifically inhibit the catalytic activity of DNA methyltransferase 1, avoiding off-target effects.
[0007] The first object of the present application is to provide the use of a compound in the preparation of a drug for treating beta-globinopathy, the structural formula of the compound being shown in formula I:
[0008]
[0009] Further, the beta-globinopathy includes beta-thalassemia and sickle cell anemia.
[0010] Further, the compound DMT207 of the present application activates HBG expression, and the activation of HBG expression can relieve beta-thalassemia and sickle cell anemia.
[0011] Further, the preparation method of the compound DMT207 is disclosed in the patent document with patent application number 2024104035592.
[0012] The second object of the present application is to provide a drug for treating beta-globinopathy, which comprises the above-mentioned compound.
[0013] Further, the beta-globinopathy includes beta-thalassemia and sickle cell anemia.
[0014] Further, the drug for treating beta-globinopathy further comprises a pharmaceutically acceptable carrier.
[0015] Further, the drug for treating beta-globinopathy is a tablet, a capsule, a pill, a preparation for injection, a sustained-release or controlled-release preparation.
[0016] A third object of the present application is to provide a product for inducing fetal hemoglobin expression, which comprises the above-mentioned compound.
[0017] Further, the fetal hemoglobin is derived from a red progenitor cell line or a primary hematopoietic stem cell.
[0018] A fourth object of the present application is to provide a DNA methyltransferase inhibiting product, which comprises the above-mentioned compound.
[0019] Further, the DNA methyltransferase comprises DNA methyltransferase I.
[0020] Advantages of the present application:
[0021] The compound DMT207 according to the present application can activate fetal hemoglobin expression, relieve splenomegaly, promote red blood cell maturation, and relieve β-thalassemia symptoms. Therefore, the application of the compound DMT207 according to the present application to β-thalassemia can specifically inhibit the catalytic activity of DNA methyltransferase 1, avoid DNA damage, has less toxicity than decitabine, has better treatment effect than existing β-thalassemia treatment drugs, and has broad application prospects. BRIEF DESCRIPTION OF DRAWINGS
[0022] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which
[0023] Figure 1 is a graph showing the fetal hemoglobin (γ-globin) expression activated by DMT207 in a human-derived red progenitor cell line (HUDEP)-2;
[0024] Figure 2 is a graph showing the fetal hemoglobin (γ-globin) expression activated by DMT207 in a human primary hematopoietic stem cell, in which HBA represents globin alpha chain, HBB represents globin beta chain, HBG represents globin gamma chain, and GATA1 represents GATA-binding factor 1 GATA binding element 1;
[0025] Figure 3 is a graph showing the fetal hemoglobin (HbF, fetal hemoglobin) expression activated by DMT207 in a hematopoietic stem cell derived from a β-thalassemia patient, and AHSP represents hemopexin;
[0026] Figure 4Figure 3 is a chart showing the activation of fetal hemoglobin expression by DMT207 in a beta-thalassemia model mouse, wherein WT Vehicle indicates solvent injection in wild-type mice, Bth Vehicle indicates solvent injection in beta-thalassemia mice, and Bth DMT207 indicates DMT207 injection in beta-thalassemia mice;
[0027] Figure 5 Figure 4 is a chart showing the specificity detection of DMT207;
[0028] Figure 6 Figure 5 is a chart showing the effect comparison between decitabine and DMT207. DETAILED DESCRIPTION
[0029] The present application will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand the present application and implement it. The examples are not intended to limit the present application.
[0030] The detection methods involved in the following examples are as follows:
[0031] Cell viability detection: ApexBio CCK-8 kit was used for testing, and Thermo Varioskan LUX enzyme labeler was used for absorbance reading.
[0032] Real-time fluorescent quantitative reverse transcription PCR (RT-qPCR) for mRNA level determination: Takara MiniBEST Universal RNA Extraction Kit was used to extract total RNA, Takara RR047A kit was used for reverse transcription to obtain cDNA, Roche FastStart Universal SYBR Green Master (Rox) was used for qPCR testing, and Applied Biosystems 7500 real-time fluorescent quantitative PCR system was used for instrument.
[0033] Western blot reaction: Thermo RIPA lysis buffer was used to extract protein, Millipore NC membrane was used for transfer, corresponding antibodies were used for incubation, Thermo SuperSignal West Atto developing solution was used for development, Tian Neng EPS-300 digital display stable voltage and current electrophoresis apparatus was used for electrophoresis and transfer, and Bio-Rad ChemiDoc system was used for development.
[0034] Flow analysis uses Thermo Attune system.
[0035] Example 1: DMT207 activates fetal hemoglobin (γ-globin) expression in human erythroid progenitor cell line (HUDEP)-2
[0036] HUDEP-2 cells were induced to differentiate for 4 days in IMDM medium (supplemented with necessary cytokines), at the beginning of differentiation, cells were treated with four inhibitors (DMT207, decitabine, RG108 and SGI-1027) respectively at serial dilution concentrations, at the fourth day, cell viability was detected using CCK-8 (Cell counting kit-8, Apexbio) kit. Then, we selected series of concentrations for four inhibitors that can maintain cells in a relatively healthy state, detected HBG transcription by RT-qPCR, results as Figure 1 .
[0037] When treated with 100 nM of each of the four inhibitors, DMT207 can maintain cell viability at about 80%, decitabine at about 30% and 50% respectively. Therefore, the toxicity of compound DMT207 to cells is less than that of decitabine (DAC) Figure 1 A-B). At the highest test concentration, DMT207 can induce mRNA levels of γ-globin to increase by 45-fold and 5-fold, decitabine by 30-fold and 0.5-fold respectively. Therefore, under the drug concentration that ensures cell viability as much as possible, DMT207 can activate higher fetal hemoglobin (γ-globin) expression Figure 1 B-C). DMT207 was also compared with two other developed DNA methylation inhibitors, RG108 (CAS number 48208-26-0) and SGI-1027 (CAS number 1020149-73-8). The compound DMT207 of the present application can better activate fetal hemoglobin (γ-globin) expression under the premise of low toxicity.
[0038] Example 2: DMT207 activates fetal hemoglobin (γ-globin) expression in human primary hematopoietic stem cells
[0039] The culture of human primary hematopoietic stem cells adopts a three-stage differentiation culture method, related cytokines (including interleukin-3 (IL3), stem cell factor (SCF), erythropoietin (EPO)) are added according to Figure 2DMT207 and DAC were used to treat cells at different concentrations for 3 days. Cell viability was tested by CCK-8. The results showed that DMT207 was less toxic than DAC, allowing us to use higher concentrations to treat cells. Then, we treated cells continuously with three-day medium and drug changes until day 14. Cells were collected on day 16 for testing.
[0040] Compound DMT207 showed less toxicity than decitabine (DAC) during the in vitro differentiation and culture of human primary hematopoietic stem cells Figure 2 A-B), which was consistent with the results in Example 1. Under the premise of ensuring cell viability as much as possible, DMT207 was able to activate higher fetal hemoglobin (γ-globin) expression without affecting erythroid differentiation (GATA1) Figure 2 C).
[0041] Example 3: DMT207 activates fetal hemoglobin (γ-globin) expression in β-thalassemia patient-derived hematopoietic stem cells
[0042] After obtaining hematopoietic stem cells, the experiment was still carried out using the differentiation and culture method shown in Figure 2 A, but the drug treatment was changed to day 5 to day 9. Cells were collected on day 16 for testing.
[0043] In β-thalassemia patient-derived hematopoietic stem cells (genotype of the patient as shown in Figure 3 A), compound DMT207 (200 nM) was selected to activate fetal hemoglobin (γ-globin) expression, and the activation effect was better than that of decitabine (DAC 50 nM). See Figure 3 B-D, the cell viability was worse when DAC was used at 50 nM, and the induction effect was weaker than that of DMT207. Therefore, it can be shown that DMT207 can induce higher fold of γ-globin expression with lower toxicity.
[0044] Example 4: DMT207 activates fetal hemoglobin expression in β-thalassemia model mice and relieves β-thalassemia symptoms
[0045] Ten β-thalassemia model mice (knockout of each one copy of mouse two Hbb-b1 and Hbb-b2 adult globin genes, while replaced by human β-thalassemia mutant gene, HBB gene containing IVSII-654 mutation, resulting in HBB unable to be normally expressed) were randomly divided into two groups according to body weight (Vehicle group (control group, solvent injection), DMT207 group (experimental group, DMT207 injection)). The experimental period was two weeks, and only on days 1-5 and 8-10, 50 mg / kg was administered by intraperitoneal injection. During the experiment, we weighed the mice and found that the body weight was not significantly affected at this dose. We collected mouse bone marrow cells for RT-qPCR detection of globin expression, and observed the mouse spleen. We also observed the splenomegaly symptoms of β-thalassemia mice, which were consistent with the phenomena observed in β-thalassemia patients. After administration, the splenomegaly symptoms of the experimental group mice were relieved.
[0046] Figure 4 In D, Ter119+ is a marker of erythroid differentiation, Ter119+ represents cells at the erythroid differentiation stage (including red blood cells at different differentiation stages), Cd71 is transferrin receptor 1, which is a marker of erythroid maturation, and during differentiation, Cd71 expression first increases and then decreases, Ter119+ Cd71+ represents immature red blood cells, Ter119+ Cd71lo represents mature red blood cells, and Ter119+ Cd71- represents highly mature red blood cells.
[0047] In β-thalassemia model mice, compound DMT207 had no significant effect on mouse body weight after two weeks of intraperitoneal injection (only on days 1-5 and 8-10) Figure 4 A) by collecting bone marrow cells to detect mouse fetal hemoglobin gene expression, the expression of two subunits εy and βh1 (equivalent to human γ-globin) was increased (mRNA level increased by 7 times) Figure 4 B), while the splenomegaly symptoms were relieved Figure 4 C). Collect mouse peripheral blood and detect peripheral blood red blood cell maturation by two erythroid maturation markers Cd71 and Ter119, β-thalassemia mouse red blood cells are only 15% mature, and after DMT207 treatment, mature red blood cells rise to about 30%, relieving anemia symptoms Figure 4 D).
[0048] Example 5: Specificity of DMT207
[0049] The specificity of DMT207 was evaluated by administering different concentrations of DMT207 to K562 cell lines and detecting the expression levels of DNMT1, DNMT3A and DNMT3B, and the results are as followsFigure 5 As shown, DMT207 was able to specifically degrade DNMT1 without affecting the protein levels of DNMT3A / 3B.
[0050] Comparative Example: Effect comparison between decitabine and DMT207
[0051] One of the DAC toxicities is DNA damage caused by integration into the genome, which induces cells to repair DNA damage, and the marker thereof is γ-H2AX. We selected a series of concentrations to treat HUDEP-2 cells for two days, and after the experiment, we collected cell lysates to run polyacrylamide gel electrophoresis and WB to detect γ-H2AX.
[0052] The results are shown in Figure 6 As shown, Histone H2A.X (also known as H2AX) is a histone that belongs to the Histone H2A family and plays an important role in maintaining genome stability and DNA damage repair. Gamma H2A.X is a phosphorylated form of Histone H2A.X protein, and the appearance of γ-H2AX is closely related to DNA double-strand breaks. One of the reasons for the cytotoxicity of decitabine (DAC) is to trigger the genome damage repair mechanism, and the marker γ-H2AX accumulates with the increase of decitabine concentration. DMT207 does not produce this phenomenon, indicating that it does not cause genome damage.
[0053] Obviously, the above embodiments are only examples for the sake of clarity, and are not a limitation on the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.
Claims
1. The use of a compound in the preparation of a therapeutic drug for β-globinopathy, characterized in that, The compound is shown in Formula I:
2. The application according to claim 1, characterized in that, The β-globinopathies include β-thalassemia and sickle cell anemia.
3. A drug for treating β-globinopathy, characterized in that, The β-globinopathic treatment includes the compound of claim 1.
4. The β-globinopathic treatment drug according to claim 3, characterized in that, The β-globinopathies include β-thalassemia and sickle cell anemia.
5. The β-globinopathic treatment drug according to claim 3, characterized in that, The β-globinopathic treatment also includes a pharmaceutically acceptable carrier.
6. The β-globinopathic treatment drug according to claim 3, characterized in that, The β-globinopathic disease treatment drugs are tablets, capsules, pills, injectable preparations, sustained-release or controlled-release preparations.
7. A product for inducing fetal hemoglobin expression, characterized in that, The product includes the compound of claim 1.
8. The product according to claim 7, characterized in that, The fetal hemoglobin is derived from the erythroblast cell line or primary hematopoietic stem cells.
9. A DNA methyltransferase inhibitor product, characterized in that, The DNA methyltransferase inhibitor product includes the compound of claim 1.
10. The DNA methyltransferase inhibitor product according to claim 9, characterized in that, The DNA methyltransferase includes DNA methyltransferase I.