WO2025189809A1 - 泽泻醇b醋酸酯在预防或治疗肥厚型心肌病中的应用 - Google Patents

泽泻醇b醋酸酯在预防或治疗肥厚型心肌病中的应用

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WO2025189809A1
WO2025189809A1 PCT/CN2024/132912 CN2024132912W WO2025189809A1 WO 2025189809 A1 WO2025189809 A1 WO 2025189809A1 CN 2024132912 W CN2024132912 W CN 2024132912W WO 2025189809 A1 WO2025189809 A1 WO 2025189809A1
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hypertrophic cardiomyopathy
ab23a
mice
hypertrophic
cardiomyocytes
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French (fr)
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孙宁
邵晨雯
朱文佳
李杜娟
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Jiangnan University
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to the field of biomedicine, and in particular to application of alismatol B acetate in preventing or treating hypertrophic cardiomyopathy.
  • Hypertrophic cardiomyopathy is an autosomal dominant genetic disease caused by mutations in genes encoding sarcomere-related proteins. It has an insidious onset and a high incidence rate, and is the main cause of malignant arrhythmias and sudden cardiac death. The specific mechanism of hypertrophic cardiomyopathy is still unclear. Although Mavacamten (MYK-461) can partially improve hypertrophic cardiomyopathy caused by outflow tract obstruction by targeted inhibition of cardiac myosin, there is no original specific drug for hypertrophic cardiomyopathy in China. At present, surgical treatment is still the only option for relieving symptoms in critically ill patients, but the prognosis of surgical patients is often poor. Therefore, there is a need to develop a new drug for the treatment of hypertrophic cardiomyopathy.
  • Alisol-B 23-acetate is a triterpenoid compound extracted from the tuber of Alisma plant (Alismatis spp.), a member of the Alismataceae family. It is the primary medicinal component of the traditional Chinese medicine Alismataceae. Its chemical formula is: C 32 H 50 O 5 . Studies have shown that AB23a can promote liver regeneration by activating the farnesoid receptor. It also inhibits IgE/Ag-mediated activation of basophilic leukemia cells and allergic reactions, demonstrating potent anti-inflammatory effects in vitro and in vivo. However, there are currently no reports on the role of AB23a in the development of therapeutics for hereditary hypertrophic cardiomyopathy.
  • the present invention provides the use of alismatol B acetate (AB23a) in the preparation of a drug for preventing or treating hypertrophic cardiomyopathy;
  • the present invention uses AB23a to directly treat cardiomyocytes obtained by directed differentiation of human embryonic stem cells in vitro, and finds that AB23a treatment in vitro can significantly inhibit the hypertrophic phenotype of human embryonic stem cell-cardiomyocytes;
  • feed containing AB23a is used to feed mice with hereditary hypertrophic cardiomyopathy caused by gene mutations, and finds that AB23a significantly alleviates pathological myocardial hypertrophy in mice and improves cardiac function;
  • the present invention proposes for the first time that AB23a can be used to prepare drugs against hypertrophic cardiomyopathy, providing a new approach and means for treating hypertrophic cardiomyopathy.
  • the present invention also provides a pharmaceutical composition for preventing or treating hypertrophic cardiomyopathy and diseases caused by it.
  • the present invention provides the use of alismatol B acetate in the preparation of a medicament for preventing or treating hypertrophic cardiomyopathy and the diseases caused by it.
  • the hypertrophic cardiomyopathy includes diseases caused by hereditary hypertrophic cardiomyopathy and hereditary hypertrophic cardiomyopathy.
  • the hypertrophic cardiomyopathy includes pathological myocardial hypertrophy, increased heart weight or left ventricular weight.
  • the hypertrophic cardiomyopathy includes a significant increase in the expression of hypertrophic markers in myocardial tissue.
  • the hypertrophic cardiomyopathy includes an increase in myocardial cell area and/or an increase in myocardial fibrosis level caused by hereditary hypertrophic cardiomyopathy.
  • the area of myocardial cells derived from patients with hypertrophic cardiomyopathy is increased, and the level of myocardial hypertrophy markers is elevated.
  • the alismatol B acetate is used in the preparation of a drug for preventing or treating hypertrophic cardiomyopathy and the diseases caused by it by inhibiting the hypertrophic phenotype of myocardial cells.
  • the pharmaceutical composition for preventing or treating hypertrophic cardiomyopathy and the diseases caused by it of the present invention contains alismatol B acetate as an active ingredient and a pharmaceutically acceptable carrier.
  • the dosage form of the pharmaceutical composition is capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.
  • the invention relates to an application of the pharmaceutical composition in preparing a drug for preventing or treating hypertrophic cardiomyopathy and the diseases caused by it.
  • the present invention discovers a new medical use for alismatol B acetate (AB23a), which can treat hypertrophic cardiomyopathy and its associated symptoms. Furthermore, alismatol B acetate, the main medicinal component of the traditional Chinese medicine Alisma orientalis, is safer for living organisms and has promising clinical applications.
  • the present invention has the following advantages:
  • the present invention uses human embryonic stem cells to direct differentiation to obtain human cardiomyocytes and establishes an in vitro human cardiomyocyte hypertrophy model.
  • the in vitro human cardiomyocyte hypertrophy model can effectively avoid the situation where the research results do not match the development of human heart disease due to the huge differences between animal-derived cardiomyocytes and human cardiomyocytes.
  • the present invention found that AB23a can significantly inhibit the hypertrophic phenotype of human embryonic stem cell-cardiomyocytes through direct in vitro treatment.
  • the present invention uses a hypertrophic cardiomyopathy mouse model with the Myh6 gene R404Q mutation (Myh6 R404Q ), which has the characteristics of hereditary myocardial hypertrophy, enlarged heart area, and increased heart and left ventricular weight.
  • Myh6 R404Q mice were fed a feed containing AB23a, the symptoms of hereditary pathological myocardial hypertrophy in mice were significantly improved.
  • the present invention uses AB23a to directly treat hypertrophic cardiomyocytes derived from human embryonic stem cells in vitro, significantly reducing cardiomyocyte area and attenuating the increase in expression of cardiac hypertrophy markers induced by norepinephrine/angiotensin II.
  • Feeding Myh6 R404Q mice with a diet containing AB23a reduces left ventricular and cardiac weight, cardiac volume, and diastolic and systolic myocardial wall thickness, and effectively inhibits myocardial fibrosis in mice with hypertrophic cardiomyopathy.
  • the present invention demonstrates that AB23a has significant potential for treating hypertrophic cardiomyopathy and can be developed as a new anti-hypertrophic cardiomyopathy drug, providing a novel approach and method for treating hypertrophic cardiomyopathy. Furthermore, the present invention provides a novel alternative and approach to current anti-hypertrophic cardiomyopathy drugs, broadening the scope of available anti-hypertrophic cardiomyopathy drugs and contributing to the advancement of this technical field.
  • Figure 1 shows the area of human cardiomyocytes after direct treatment with AB23a in vitro; *** indicates p ⁇ 0.001.
  • Figure 2 shows the expression levels of cardiac hypertrophy markers (left: BNP, middle: ANP, right: TNNT2) in human cardiomyocytes after direct treatment with AB23a in vitro; * indicates p ⁇ 0.05; ** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001.
  • FIG3 shows the heart (left) and left ventricular weight (right) of WT and Myh6 R404Q mice after being fed a diet containing AB23a; in the figure, * indicates p ⁇ 0.05; ** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001.
  • Figure 4 shows the results of cardiac ultrasound detection in mice, where (first from the left) is the left ventricular anterior wall thickness during systole, (second from the left) is the left ventricular anterior wall thickness during diastole, (third from the left) is the left ventricular posterior wall thickness during systole, and (fourth from the left) is the left ventricular posterior wall thickness during diastole.
  • * indicates p ⁇ 0.05; ** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001.
  • Figure 5 shows the expression levels of cardiac hypertrophy markers ANP, BNP, and TNNT2 in the hearts of WT and Myh6 R404Q mice fed a diet containing AB23a.
  • ns indicates p >0.05; ** indicates p ⁇ 0.01; **** indicates p ⁇ 0.0001.
  • FIG6 is a diagram showing the results of Masson staining of mouse myocardial tissue; the blue area in the diagram indicates the degree of fibrosis.
  • mice harboring the Myh6 gene R404Q point mutation (Myh6 R404Q ) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the Myh6 gene R404Q point mutation (Myh6 R404Q ) mice were C57BL/6JGpt-Myh6 em1Cin(R404Q) /Gpt, Myh6-p.R404Q
  • Normal mouse feed was purchased from Jiangsu Collaborative Bioengineering Co., Ltd.
  • AB23a compound was purchased from MedChemExpress (HY-N0805, CAS No.: 26575-95-1).
  • the human embryonic stem cell line MYL2 Neo/w -H7 wherein H7 cells were purchased from the WiCell Institute cell bank #WA07, and a stable human embryonic stem cell line MYL2 Neo/w -H7 was obtained after editing using genome-directed modification technology (the construction method refers to Lian X, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A. 2012; 109(27): E1848-E1857.
  • Agonist CHIR-99021 (purchased from MedChemExpress, #HY-10182, CAS No.: 252917-06-9).
  • Pathway inhibitor IWR-1 (purchased from MedChemExpress, #HY-12238, CAS No.: 1127442-82-3).
  • RPMl-1640/B27-no insulin is RPMl-1640 (Gibco) culture medium with B-27 (Thermo Fisher Scientific, Cat#A1895601) added without insulin, where the B-27 content is 2%.
  • the human embryonic stem cell line MYL2 Neo/w -H7 was used to differentiate cardiomyocytes, and then a large number of high-purity human cardiomyocytes were obtained through in vitro screening.
  • the specific steps for differentiating the MYL2 Neo/w -H7 cell line into cardiomyocytes are as follows:
  • the culture medium was replaced with fresh RPM1-1640/B27-no insulin medium and the cells were cultured for 24 h.
  • the cells were washed twice with DPBS and the culture medium was replaced with RPMI1640/B27-no insulin.
  • the culture medium was cultured for 2 days. Beating cardiomyocytes could be observed on the 7th day of differentiation.
  • the culture medium was replaced with RPMl-1640/B27-no insulin medium, and 50 ⁇ g/mL of G418 antibiotic was added to select cardiomyocytes. The selection was continued for 7 days, and the medium and new G418 were replaced every day. Beating cell clusters were observed.
  • This directed differentiation and screening method can be used to obtain a large number of highly pure human cardiomyocytes derived from human embryonic stem cells.
  • Human cardiomyocytes derived from human embryonic stem cell differentiation were uniformly seeded into a 24-well plate containing a cell slide. After 24 hours of adherent growth, the cells were treated with PBS, AngII (purchased from MedChemExpress, #HY-13948, CAS No.: 4474-91-3, 10 ⁇ M, 24 hours), AB23a (2.5 ⁇ M, pre-treated for 2 hours) + AngII (10 ⁇ M, 24 hours), AB23a (5 ⁇ M, pre-treated for 2 hours), and AB23a (5 ⁇ M, pre-treated for 2 hours). Cells were treated with PBS, Ang II, different concentrations of AB23a plus Ang II, and the positive drug MET plus Ang II. After 24 hours of treatment, cardiac troponin (cTNT) immunofluorescence staining was performed on each group.
  • PBS PBS
  • AngII purchased from MedChemExpress, #HY-13948, CAS No.: 4474-91-3
  • cardiac troponin (cTNT) immunofluorescence staining experiment The specific steps of the cardiac troponin (cTNT) immunofluorescence staining experiment are as follows:
  • Human cardiomyocytes were obtained by directed differentiation according to the method in Example 1.
  • the cells were evenly seeded into 12-well plates. After the cells adhered to the wall for 24 hours, they were treated with PBS, AngII (10 ⁇ M, 24 hours), AB23a (1 ⁇ M, pretreatment 2 hours) + AngII (10 ⁇ M, 24 hours), AB23a (2.5 ⁇ M, pretreatment 2 hours) + AngII (10 ⁇ M, 24 hours), AB23a (5 ⁇ M, pretreatment 2 hours) + AngII (10 ⁇ M, 24 hours), and AB23a (10 ⁇ M, pretreatment 2 hours) + AngII (10 ⁇ M, 24 hours) to obtain PBS treatment group, AngII treatment group and different concentrations of AB23a + AngII treatment group.
  • qRT-PCR real-time quantitative polymerase chain reaction
  • RNA precipitate will be visible at the bottom of the tube. Discard the solution and slowly add 1 mL of 75% alcohol along the tube wall to wash the RNA precipitate. Centrifuge for 15 minutes, discard the solution, and invert the tube to remove any excess alcohol.
  • Reverse transcription Prepare a 10 ⁇ L system including 2 ⁇ L reverse transcriptase (5 ⁇ Prime Script RT Mix), 1 ⁇ g RNA, and RNase-free ddH 2 O to make up to 10 ⁇ L.
  • the cDNA sample obtained by reverse transcription was diluted 10-fold with ddH 2 O and used directly for real-time quantitative polymerase chain reaction (qRT-PCR).
  • 6qRT-PCR prepare 10 ⁇ L reaction system: 0.2 ⁇ L PCR Forward Primer (forward primer), 0.2 ⁇ L PCR Reverse Primer (reverse primer), 1 ⁇ L cDNA, 5 ⁇ L SyBR Premix EX Taq (2 ⁇ ), 3.6 ⁇ L ddH 2 O.
  • the experiment used six different batches of differentiated cardiomyocytes, with six replicate in vitro treatments. For qRT-PCR analysis, three replicates were performed for each sample. Data are presented as mean ⁇ standard error (SEM).
  • the ⁇ -ACTIN gene was used to detect the expression of internal reference genes, and cardiac hypertrophy markers primarily include ANP, BNP, and TNNT2.
  • Primer sequences for ⁇ -ACTIN, ANP, BNP, and TNNT2 are shown in Table 1. The results were analyzed using the 2 - ⁇ Ct relative quantitative analysis method, as shown in Figure 2.
  • AngII treatment led to a significant increase in the expression of myocardial hypertrophy markers.
  • Direct treatment with 1 ⁇ M, 2.5 ⁇ M, 5 ⁇ M and 10 ⁇ M AB23a in vitro could significantly and effectively reduce the expression of myocardial hypertrophy markers (p ⁇ 0.05), which was significantly different from the model group, indicating that AB23a has a significant physiological effect against myocardial hypertrophy.
  • mice Effects of feeding a diet containing AB23a on heart and left ventricular weight in mice
  • WT Wild-type mice and mice with hypertrophic cardiomyopathy caused by the R404Q point mutation in the Myh6 gene (Myh6 R404Q ) were randomly divided into five groups. The five groups of mice were then fed a normal diet supplemented with different concentrations of AB23a, resulting in the following: WT, Myh6 R404Q , low-dose AB23a-treated, medium-dose AB23a-treated, and high-dose AB23a-treated groups. Each group had free access to food .
  • WT group wild-type mice (WT) were fed with normal diet;
  • Myh6 R404Q group Myh6 R404Q mice were fed normal chow
  • High-dose AB23a-treated group Myh6 R404Q mice were fed a diet containing AB23a at a drug concentration of 0.1% (1 g AB23a per kg of diet);
  • mice were housed in an SPF animal room for 3 months.
  • mice were bled via eyeballs and then sacrificed by cervical dislocation. The hearts were quickly removed, their surface moisture blotted off, and their weights were measured. RNA was then extracted from the left ventricle for later use. The test results are shown in the left panel of Figure 3. In addition, the mice were observed using echocardiography to measure the weight of their left ventricles. The steps for echocardiography were as follows:
  • Vevo3100 high-resolution in vivo imaging system (Vevo3100LT, Canada) ultrasound detection was performed to quickly locate the heart and record the long-axis and short-axis images and related parameters of the mouse heart in both the B-model and M-model.
  • mice were reared for three months using the method described in Example 3, and then subjected to echocardiography to determine whether feeding the AB23a-containing diet improved the pathological hypertrophic phenotype of the hearts of mice with hypertrophic cardiomyopathy.
  • the echocardiographic procedure was similar to that described in Example 3.
  • the systolic, diastolic, systolic, and diastolic left ventricular anterior wall thicknesses were calculated using the Vevo 3100 system. The results are shown in Figure 4 (from left to right).
  • the left ventricular wall thickness in Myh6 R404Q mice during both diastole and systole was significantly greater than that in WT mice.
  • feeding the Myh6 R404Q mice a diet containing AB23a significantly reduced the left ventricular wall thickness. This suggests that AB23a has a beneficial effect in counteracting the progression of pathological myocardial hypertrophy in hereditary hypertrophic cardiomyopathy.
  • This example investigates the effect of AB23a on the progression of myocardial hypertrophy in vivo by measuring the expression of myocardial hypertrophy markers in the left ventricular tissue of WT and Myh6 R404Q mice fed a diet containing AB23a. Mice were raised using the method described in Example 3, and then RNA was extracted from the left ventricle of the heart. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to measure the expression of myocardial hypertrophy marker mRNA in cardiomyocytes. The specific steps are as follows:
  • mice left ventricular tissue sample Take 50 mg of mouse left ventricular tissue sample and place it in a sterile EP tube. Add 1 mL of Trizol to each tube and then use a tissue disruptor to disrupt the tissue. Centrifuge at 4°C for 5 minutes and collect the supernatant to obtain the mouse myocardial tissue sample, i.e., the mouse myocardial tissue supernatant.
  • RNA precipitate will be visible at the bottom of the tube. Discard the solution and slowly add 1 mL of 75% alcohol along the tube wall to wash the RNA precipitate. Centrifuge for 15 minutes, discard the solution, and invert the tube to remove any excess alcohol.
  • Reverse transcription Prepare a 10 ⁇ L system including: 2 ⁇ L reverse transcriptase (5 ⁇ Prime Script RT Mix), 1 ⁇ g RNA, and fill to 10 ⁇ L with RNase-free ddH 2 O.
  • the cDNA sample obtained by reverse transcription was diluted 10-fold with ddH 2 O and used directly for qRT-PCR.
  • 6qRT-PCR Prepare a 10 ⁇ L reaction system: 0.2 ⁇ L PCR Forward Primer, 0.2 ⁇ L PCR Reverse Primer, 1 ⁇ L cDNA, 5 ⁇ L SyBR Premix EX Taq (2 ⁇ ), and 3.6 ⁇ L ddHO. Set up three replicates for each sample.
  • Mouse myocardial hypertrophy markers primarily include Anp, Bnp, and Tnnt2.
  • the 18S gene was used as a reference gene for detection of expression. Results were analyzed using the 2 - ⁇ Ct relative quantitative analysis method. The analysis results are shown in the table.
  • the primer sequences for the genes tested are shown in Table 5.
  • mice raised for 3 months were sacrificed by cervical dislocation and then rapidly perfused with a 4% paraformaldehyde solution to flush out the residual blood in the myocardial tissue.
  • the mouse heart was removed and placed in 4% paraformaldehyde and fixed overnight.
  • the fixed heart was dehydrated, transparent, and paraffin-embedded. After embedding, paraffin sections (5 ⁇ m) were made. The sections were then stained with Masson's trichrome staining kit (Sevier Bio, G1006) as follows:
  • mice with hypertrophic cardiomyopathy exhibited significantly increased myocardial fibrosis compared to normal mice, and the area of myocardial cells was also significantly increased.
  • AB23a significantly inhibited the progression of myocardial fibrosis in Myh6 R404Q mice and effectively reduced the area of myocardial cells. This suggests that AB23a may have great potential for treating the early stages of hypertrophic cardiomyopathy.
  • AB23a effectively prevents the development and progression of human cardiomyocyte hypertrophy; in vivo studies, AB23a effectively inhibits the progression of hereditary pathological cardiac hypertrophy and the development of myocardial fibrosis.

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Abstract

本发明公开了泽泻醇B醋酸酯在制备预防或治疗肥厚型心肌病及其引起的病症的药物中的应用。本发明首次提出了泽泻醇B醋酸酯(AB23a)预防或治疗肥厚型心肌病，本发明应用AB23a在体外直接处理人类胚胎干细胞定向分化获得的心肌细胞，发现AB23a体外处理可以明显抑制人类胚胎干细胞-心肌细胞肥大表型；应用含AB23a的饲料喂养基因突变导致的遗传性肥厚型心肌病小鼠，发现AB23a显著缓解小鼠病理性心肌肥厚并改善了心功能；AB23a可以用于制备抗遗传性肥厚型心肌病的药物，为治疗肥厚型心肌病提供一种新的途径和手段，AB23a是中药材泽泻中最主要的药用成分，对生物体来说安全，在临床上具有良好的应用前景。

Description

泽泻醇B醋酸酯在预防或治疗肥厚型心肌病中的应用 技术领域
本发明涉及生物医药领域，具体涉及泽泻醇B醋酸酯在预防或治疗肥厚型心肌病中的应用。
背景技术
肥厚型心肌病(Hypertrophic cardiomyopathy)是一种因编码肌小节相关蛋白基因突变导致的常染色体显性遗传病。其起病隐匿、发病率高，是诱发恶性心律失常和心源性猝死的主要原因。肥厚型心肌病的具体机制还不清晰，尽管Mavacamten(MYK-461)可通过靶向抑制心肌肌球蛋白，部分改善因流出道梗阻引起的肥厚型心肌病，但是国内尚无针对肥厚型心肌病的原创特效药。目前，手术治疗仍是重症患者缓解症状的唯一选择，但是手术患者的预后往往不佳。因此，需要研发一种新的用于治疗肥厚型心肌病的药物。
泽泻醇B醋酸酯(Alisol-B 23-acetate，AB23a)是从泽泻科植物泽泻的块茎中提取的三萜类化合物，是中药材泽泻中最主要的药用成分，其化学式为：C32H50O5。研究表明，AB23a能够通过激活法尼醇受体促进受损的肝脏再生；AB23a能够抑制IgE/Ag介导的嗜碱性白血病细胞的活化和过敏反应，具有良好的体内外抗炎作用。但是，目前尚未有报道AB23a在制备遗传性肥厚型心肌病治疗药物中的作用。
发明内容
发明目的：针对现有技术中存在的问题，本发明提供了泽泻醇B醋酸酯(AB23a)在制备预防或者治疗肥厚型心肌病药物中的应用；本发明应用AB23a在体外直接处理人类胚胎干细胞定向分化获得的心肌细胞，发现AB23a体外处理可以明显抑制人类胚胎干细胞-心肌细胞肥大表型；应用含AB23a的饲料喂养基因突变导致的遗传性肥厚型心肌病小鼠，发现AB23a显著缓解小鼠病理性心肌肥厚并改善了心功能；本发明首次提出了AB23a可以用于制备抗肥厚型心肌病的药物，为治疗肥厚型心肌病提供一种新的途径和手段。
本发明还提供一种用于预防或治疗肥厚型心肌病及其引起的病症的药物组合物。
技术方案：为了实现以上技术目的，本发明提供泽泻醇B醋酸酯在制备预防或治疗肥厚型心肌病及其引起的病症的药物中的应用。
其中，所述肥厚型心肌病包括传性肥厚型心肌病以及遗传性肥厚型心肌病引起的病症。
其中，所述肥厚型心肌病包括病理性心肌肥厚、心脏重量或左心室重量增加。
其中，所述肥厚型心肌病包括心肌组织中肥厚标志物的表达显著升高。
其中，所述肥厚型心肌病包括因遗传性肥厚型心肌病导致的心肌细胞面积增大和/或心肌纤维化水平升高。
其中，所述肥厚型心肌病人源心肌细胞面积增大，心肌肥厚标志物水平升高。
其中，所述泽泻醇B醋酸酯通过抑制心肌细胞肥大表型在制备预防或治疗肥厚型心肌病及其引起的病症的药物中的应用。
本发明所述用于预防或治疗肥厚型心肌病及其引起的病症的药物组合物，所述药物组合物其含有泽泻醇B醋酸酯作为活性成分和药学上可接受的载体。
其中，所述的药物组合物的剂型是胶囊剂、散剂、片剂、颗粒剂、丸剂、注射剂、糖浆剂、口服液、吸入剂、软膏剂、栓剂或贴剂。
本发明所述的药物组合物在制备预防或治疗肥厚型心肌病及其引起的病症的药物中的应用。
本发明发现泽泻醇B醋酸酯(AB23a)的新的医药用途，其可以抗肥厚型心肌病以及肥厚型心肌病引起的病症。同时，泽泻醇B醋酸酯是中药材泽泻中最主要的药用成分，对生物体来说更加安全，在临床上具有良好的应用前景。
有益效果：与现有技术相比，本发明具有如下优点：
相比目前采用动物来源的细胞模型进行AB23a的体外研究，本发明利用人类胚胎干细胞定向分化获得人源心肌细胞，并建立了体外人源心肌细胞肥大模型。所述体外人源心肌细胞肥大模型能够有效避免因动物来源的心肌细胞与人类心肌细胞存在的巨大差异导致的研究结果不匹配人类心脏疾病发展的情况，本发明通过AB23a体外直接处理发现其可明显抑制人类胚胎干细胞-心肌细胞肥大表型。此外，本发明利用Myh6基因R404Q突变的肥厚型心肌病小鼠模型(Myh6R404Q)，该模型具有遗传性心肌肥厚、心脏面积增大、心脏及左心室重量增加等特征。Myh6R404Q小鼠喂食含AB23a的饲料后，显著改善小鼠遗传性病理性心肌肥厚的症状。
本发明采用AB23a体外直接处理肥大的人类胚胎干细胞诱导获得的心肌细胞，显著减小心肌细胞面积，并降低去甲肾上腺素/血管紧张素Ⅱ导致的心肌肥厚标志物表达的增高。应用含AB23a的饲料喂养Myh6R404Q小鼠，降低小鼠左心室和心脏重量，减小心脏体积以及舒张期和收缩期心肌壁厚度，并有效抑制肥厚型心肌病小鼠心肌组织纤维化。
本发明通过体外和体内研究发现，AB23a具有治疗肥厚型心肌病的巨大潜能，可以作为新的抗肥厚型心肌病药物进行开发，为治疗肥厚型心肌病提供一种新的途径和手段。同时，本发明为目前抗肥厚型心肌病药物提供了全新的选择和思路，拓宽了抗肥厚型心肌病药物的选择领域，也为该技术领域的发展做出了贡献。
附图说明
图1为AB23a体外直接处理后的人源心肌细胞面积情况图；其中***表示p<0.001。
图2为AB23a体外直接处理后人源心肌细胞中心肌肥厚标志物(左：BNP、中：ANP、右：TNNT2)的表达水平情况图；*表示p<0.05；**表示p<0.01；***表示p<0.001。
图3为喂食含AB23a的饲料后WT和Myh6R404Q小鼠心脏(左)和左心室重量(右)；图中，*表示p<0.05；**表示p<0.01；***表示p<0.001。
图4为小鼠心动超声检测结果，(左一)为收缩期左心室前壁厚度、(左二)为舒张期左心室前壁厚度、(左三)为收缩期左心室后壁厚度、(左四)为舒张期左心室后壁厚度；图中，*表示p<0.05；**表示p<0.01；***表示p<0.001。
图5为喂食含AB23a的饲料后WT和Myh6R404Q小鼠心脏中心肌肥厚标志物ANP、BNP和TNNT2的表达水平。图中，ns表示p>0.05；**表示p<0.01；****表示p<0.0001。
图6为小鼠心肌组织Masson染色结果图；图中蓝色区域表示纤维化程度。
具体实施方式
下面通过实施例具体地说明本发明的内容。在本发明中，以下所述的实施例是为了更好的阐述本发明，并不是用来限制本发明的范围。在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
以下实施例中，所采用的8周龄野生型小鼠(WT)小鼠以及Myh6基因R404Q点突变小鼠(Myh6R404Q)均购于江苏省集萃药康生物科技公司，其中，Myh6基因R404Q点突变小鼠(Myh6R404Q)为C57BL/6JGpt-Myh6em1Cin(R404Q)/Gpt，Myh6-p.R404Q|Strain NO.T051403。小鼠正常饲料购于江苏省协同生物工程有限责任公司。AB23a化合物购自MedChemExpress(HY-N0805，CAS No.:26575-95-1)。
人胚胎干细胞系MYL2Neo/w-H7，其中H7细胞购买于WiCell研究所细胞库#WA07，采用基因组定点修饰技术编辑后获得稳定的人胚胎干细胞系MYL2Neo/w-H7(构建方法参考Lian X,Hsiao C,Wilson G,et al.Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.Proc Natl Acad Sci U S A.2012；109(27):E1848-E1857.doi:10.1073/pnas.1200250109；或者详见专利CN108265029A中说明书0037段：编辑人类多能干细胞(包括人类胚胎干细胞human embryonic stem cells的基因组，将MYL2 drived Neo导入人类多能干细胞基因组并替换相对应的正常基因以及实施例1)。
激动剂CHIR-99021(购买于MedChemExpress，#HY-10182，CAS No.:252917-06-9)。
通路抑制剂IWR-1(购买于MedChemExpress，#HY-12238，CAS No.:1127442-82-3)。
RPMl-1640/B27-no insulin为加入不含胰岛素B-27(ThermoFisherScientifc,Cat#A1895601)的RPMl-1640(Gibco)培养基，其中B-27的含量为2％。
实施例1
人胚胎干细胞定向分化为心肌细胞以及AB23a体外直接处理对心肌细胞面积的影响检测
本实施例利用人胚胎干细胞系MYL2Neo/w-H7分化心肌细胞，再通过体外筛选获得大量高纯度人源心肌细胞。所述MYL2Neo/w-H7细胞系分化为心肌细胞的具体步骤为：
①MYL2Neo/w-H7细胞密度达到85％-90％时，将培养基更换RPMl-1640/B27-no insulin培养基并加入8μM Wnt信号通路激动剂CHIR-99021，连续培养2天；
②第3天将培养基换为新的RPMl-1640/B27-no insulin培养基，培养细胞24h；
③第4天将培养基更换为RPMl-1640/B27-no insulin培养基，并加入5μM的Wnt信号通路抑制剂IWR-1，连续培养2天；
④第6天用DPBS清洗细胞两次后将培养基更换RPMI1640/B27-no insulin，培养2天，在分化第7天开始可以观察到跳动的心肌细胞；
⑤第8天后将培养基更换为RPMl-1640/B27-no insulin培养基，持续培养至14天，每天换液；
⑥第14天时，培养液更换为RPMl-1640/B27-no insulin培养基，并加入50μg/mL的G418抗生素筛选心肌细胞，连续筛选7天，每天换液并更换新的G418，观察到跳动的细胞簇。
通过这种定向分化和筛选方法可以获得大量高纯度的人胚胎干细胞来源的人心肌细胞。
将人胚胎干细胞定向分化获得的人源心肌细胞均匀地种到含有一张细胞爬片的24孔板中，细胞贴壁生长24h后分别给予PBS、AngⅡ(购买于MedChemExpress，#HY-13948，CAS No.:4474-91-3，10μM，24h)、AB23a(2.5μM，预处理2h)+AngⅡ(10μM,24h)、AB23a(5μM，预处理2h)+AngⅡ(10μM,24h)处理细胞，MET(酒石酸美托洛尔，阳性药，购买于MedChemExpress，#HY-17503B，CAS No.:56392-17-7；5μM，预处理2h)+AngⅡ(10μM，24h)，得到PBS处理组、AngⅡ处理组和不同浓度AB23a+AngⅡ处理组以及阳性药MET+AngⅡ处理组。待各组处理24h后，分别对其进行心肌肌钙蛋白(cTNT)免疫荧光染色实验。
其中，心肌肌钙蛋白(cTNT)免疫荧光染色实验具体步骤如下：
①PBS清洗处理后的细胞2次(5min/次)后，用4％多聚甲醛固定细胞20min，弃去固定液后用PBS清洗3次(5min/次)。
②用0.2％ Triton-X100处理①中得到的细胞5min，然后用PBS清洗3次(5min/次)。
③将②中得到的细胞用5％BSA溶液封闭处理2h，封闭完成后用PBS清洗3次(5min/次)。
④孵育cTNT抗体(1:400，PBS稀释)，4℃孵育过夜后，去除抗体，并用PBS清洗3次(5min/次)。
⑤孵育荧光抗兔二抗(1:500，PBS稀释)1h，孵育完成后，去除二抗，用PBS清洗3次(5min/次)。
⑥孵育DAPI(5μg/ml，PBS稀释)20min，孵育完成后，去除DAPI染液，用PBS清洗3次(5min/次)。
⑦最后用抗荧光淬灭剂染进行封片，利用激光共聚焦显微镜(蔡司Carl Zeiss LSM880，德国)拍摄荧光图像。该型号荧光显微镜具有测定荧光染色面积功能，利用该功能可以显示心肌细胞面积。
采用6次不同时间定向分化获得的心肌细胞，每次重复2个复孔，随机拍摄一张cTNT染色的细胞形态图，并分析照片中所有心肌细胞的面积，结果如图1所示。从图1可以看出，利用10μM的AngⅡ处理心肌细胞显著增大细胞面积(AngⅡvs PBS：4432.1±175.9vs 3306.13±119.9)，而AB23a预处理心肌细胞3h后再给予AngⅡ刺激，有效降低AngⅡ导致的细胞面积增大(AB23a+AngⅡvs AngⅡ：2849.4±89vs 4432.1±175.9)。数据以均值±标准误(mean±SEM)表示，PBS组统计了170个心肌细胞，NE组统计了138个心肌细胞，AB23a+AngⅡ组统计了181个心肌细胞。***表示p<0.001。因此，AB23a体外直接处理人源心肌细胞，能够显著抑制AngⅡ诱导的心肌细胞面积增大效应，与空白对照组以及阳性药对照组相比无显著差异。
实施例2
AB23a体外直接处理心肌细胞对细胞中心肌肥厚标志物表达的影响
按照实施例1中方法定向分化获得人源心肌细胞，将细胞均匀地种到12孔板中，细胞贴壁生长24h后分别给予PBS、AngⅡ(10μM，24h)、AB23a(1μM，预处理2h)+AngⅡ(10μM，24h)、AB23a(2.5μM，预处理2h)+AngⅡ(10μM，24h)、AB23a(5μM，预处理2h)+AngⅡ(10μM，24h)、AB23a(10μM，预处理2h,)+AngⅡ(10μM，24h)处理细胞，得到PBS处理组、AngⅡ处理组和不同浓度AB23a+AngⅡ处理组。待各组处理24h后，提取细胞内总RNA，利用实时定量聚合酶链式反应(qRT-PCR)，检测心肌细胞中心肌肥厚标志物mRNA的表达情况。具体步骤如下所示：
①处理后的细胞用4℃预冷的PBS清洗2次。吸干PBS后，每孔中加入1mL的总RNA抽提试剂Trizol，待细胞裂解后室温静置5min，使核酸-蛋白复合物完全分离。静置结束后全部转移至无菌的EP管，4℃高速离心(12,000rpm)5min，取上清液。
②向①步骤得到的上清液中加入200μL氯仿，震荡后4℃高速离心15min，吸取无色水相层(RNA在此层)，转移至新的离心管中。
③向②步骤得到的液体中加入等量异丙醇，震荡混匀，低温离心15min，管底可见明显的RNA沉淀。倒掉液体，沿管壁缓慢加入1mL 75％酒精清洗RNA沉淀，离心15min后弃去液体，将试管倒扣去除多余酒精。
④向RNA沉淀中加入50μL无菌DEPC水，在55℃金属浴中溶解RNA，然后测定RNA浓度，样本直接进行逆转录。
⑤逆转录：配制10μL体系，包括：2μL逆转录酶(5×Prime Script RT Macter Mix)、RNA 1ug、RNase free ddH2O补齐至10μL。逆转得到的cDNA样本，用ddH2O稀释10倍，直接用于实时定量聚合酶链式反应(qRT-PCR)。
⑥qRT-PCR：配制10μL反应体系：0.2μL PCR Forward Primer(正向引物)、0.2μL PCR Reverse Primer(反向引物)、1μL cDNA、5μL SyBR Premix EX Taq(2×)、3.6μL ddH2O。
实验采用了6批不同批次分化的心肌细胞，重复了6次体外处理。qRT-PCR检测时，每个样品设置3个复孔。数据以均值±标准误(mean±SEM)表示。β-ACTIN基因用以检测内参基因的表达，心肌肥厚标志物主要包括ANP、BNP、TNNT2。其中，β-ACTIN、ANP、BNP和TNNT2的引物序列见表1。以2-△△Ct相对定量分析法对结果进行分析，检测结果如图2所示。
表1.人源心肌细胞肥厚标记物的引物序列

从图2中可以看出，AngⅡ处理导致心肌肥厚标志物的表达显著升高，1μM、2.5μM、5μM和10μM AB23a体外直接处理后，均可显著有效降低心肌肥厚标志物的表达(p<0.05)，与模型组比较有显著性差异，表明AB23a具有显著对抗心肌肥厚的生理作用。
实施例3
喂食含AB23a的饲料对小鼠心脏和左心室重量的影响
肥厚型心肌病的疾病进展过程中，由于心脏出现病理性肥厚，心室壁厚度增加，导致心脏重量和左心室重量均会异常增重。本实施例通过检测喂食含AB23a的饲料后WT和Myh6R404Q小鼠心脏和左心室重量来考察体内AB23a对心肌肥厚进展的影响。
选取8周龄野生型小鼠(WT)小鼠以及Myh6基因R404Q点突变导致的肥厚型心肌病小鼠(Myh6R404Q)，并将WT和Myh6R404Q小鼠随机分为五组。然后分别对五组小鼠喂食正常饲养、含不同浓度AB23a的饲料，得到WT组、Myh6R404Q组、低剂量AB23a处理组、中剂量AB23a处理组和高剂量AB23a处理组，各组均自由饮食。
其中，WT组：野生型小鼠(WT)喂食正常饲料；
Myh6R404Q组：Myh6R404Q小鼠喂食正常饲料；
低剂量AB23a处理组：Myh6R404Q小鼠喂食含AB23a的饲料，其中药物浓度为0.025％(每1Kg饲料中含有0.25g AB23a)；
中剂量AB23a处理组：Myh6R404Q小鼠喂食含AB23a的饲料，其中药物浓度为0.05％(每1Kg饲料中含有0.50g AB23a)；
高剂量AB23a处理组：Myh6R404Q小鼠喂食含AB23a的饲料，其中药物浓度为0.1％(每1Kg饲料中含有1g AB23a)；
阳性药处理组：Myh6R404Q小鼠喂食含MET的饲料，其中药物浓度为0.025％(每1Kg饲料中含有0.25g MET)；
小鼠在SPF级动物房中饲养3个月。
饲养3个月后的小鼠通过眼球取血，然后通过脱颈方式处死小鼠，尽快取出心脏，吸干表面水分，称量心脏重量，并提取心脏左心室的组织RNA备用。检测结果见图3的左图。另外，利用小鼠超声心动图观察，以检测小鼠左心室重量。心脏超声心动图检测的步骤如下：
①对小鼠腹部及胸腔进行备毛处理，然后用1.5-2％流量的异氟烷气体麻醉小鼠，控制小鼠心率稳定在430-480次/min内，进行检测。
②使用Vevo3100高分辨率体内成像系统(Vevo3100LT，加拿大)进行超声检测。快速找到心脏位置，分别记录小鼠心脏长轴与短轴在B-Model和M-Model下的图像及相关参数。
③通过Vevo3100系统计算左心室重量，检测结果如图3的右图。
从图3可以看出，与WT小鼠心脏相比，Myh6R404Q小鼠心脏和左心室重量均异常增加，而喂食含AB23a的饲料后显著降低Myh6R404Q小鼠心脏和左心室重量，显著抑制小鼠的心脏肥大。
实施例4
喂食含AB23a的饲料对肥厚型心肌病小鼠左心室室壁厚度的影响
采用实施例3中记载的方法饲养小鼠3个月后对小鼠进行心脏超声心动图，以检测喂食含AB23a的饲料是否改善肥厚型心肌病小鼠心脏的病理性肥厚表型。心脏超声心动图检测的步骤参考实施例3，最后通过Vevo3100系统计算收缩期左心室前壁厚度、舒张期左心室前壁厚度、收缩期左心室后壁厚度、舒张期左心室后壁厚度。检测结果如图4(从左到右)所示。
从图4中可以看出，Myh6R404Q小鼠心脏舒张期和收缩期的左心室室壁厚度均显著高于WT小鼠，而喂食含AB23a的饲料后显著降低Myh6R404Q小鼠的左心室室壁厚度。这说明AB23a具有对抗遗传性肥厚型心肌病病理性心肌肥厚进展的有益效应。
实施例5
喂食含AB23a的饲料对心肌肥厚标志物的影响
本实施例通过检测喂食含AB23a的饲料的WT和Myh6R404Q小鼠左心室组织中心肌肥厚标志物的表达情况来考察体内AB23a对心肌肥厚进展的影响。采用实施例3中记载的方法饲养小鼠，然后提取心脏左心室的组织RNA，利用实时定量聚合酶链式反应(qRT-PCR)，检测心肌细胞中心肌肥厚标志物mRNA的表达情况。具体步骤如下所示：
①取50mg小鼠左心室组织样本至无菌的EP管，每管中加入1mL的Trizol后利用组织破碎震荡仪破碎组织，4℃高速离心5min，取上清得到小鼠心肌组织样本，即小鼠心肌组织上清液。
②向①步骤得到的上清液中加入200μL氯仿，震荡后4℃高速离心15min，吸取无色水相层(RNA在此层)，转移至新的离心管中。
③向②步骤得到的液体中加入等量异丙醇，震荡混匀，低温离心15min，管底可见明显的RNA沉淀。倒掉液体，沿管壁缓慢加入1mL 75％酒精清洗RNA沉淀，离心15min后弃去液体，将试管倒扣去除多余酒精。
④向RNA沉淀中加入50μL无菌DEPC水，在55℃金属浴中溶解RNA，然后测定RNA浓度，样本直接进行逆转录。
⑤逆转录：配制10μL体系，包括：2μL逆转录酶(5×Prime Script RT Macter Mix)、RNA 1ug、RNase free ddH2O补齐至10μL。逆转得到的cDNA样本，用ddH2O稀释10倍，直接用于qRT-PCR。
⑥qRT-PCR：配制10μL反应体系：0.2μL PCR Forward Primer(正向引物)、0.2μL PCR Reverse Primer(反向引物)、1μL cDNA、5μL SyBR Premix EX Taq(2×)、3.6μL ddH O。每个样品设置3个复孔，小鼠心肌组织的肥厚标记物主要包括Anp、Bnp和Tnnt2，18S基因用以检测内参基因的表达情况，以2-△△Ct相对定量分析法对结果进行分析。分析结果如所示。其中，所检测的基因引物序列如表5所示。
表3.小鼠心肌组织的肥厚标记物引物序列

从图5中可以看出，Myh6R404Q小鼠心肌组织中的Anp、Bnp和Tnnt2等肥厚标记物的表达显著高于WT小鼠。而AB23a显著抑制Anp、Bnp和Tnnt2在Myh6R404Q小鼠心脏中的表达，这说明AB23a具有治疗遗传性肥厚型心肌病的巨大潜力。
实施例6
心肌组织Masson染色考察
实施例3中饲养3个月的小鼠脱颈处死后，快速用4％多聚甲醛溶液进行全身灌流，冲去心肌组织中残留的血液。取下小鼠心脏置入4％多聚甲醛中，固定过夜。对固定好的心脏进行组织脱水、透明和石蜡包埋。包埋完成后进行石蜡切片(5μm)。然后使用Masson三色染色试剂盒(赛维尔生物，G1006)对切片进行Masson染色，步骤如下：
①切片进入A液中室温浸泡过夜(约15h)。
②将B液与C液等体积混合(现配现用)，切片入A和B混合液内浸染1min，流水稍洗后经1％盐酸酒精(浓盐酸：无水乙醇＝1：100)分化1min，至细胞核呈灰黑色，背景几乎无色或淡灰色。
③流水稍洗，沥干切片上多余水分，切片入D液浸染6min，此时组织呈现亮红色。切片稍微沥干水分(不能干片)，立即入E液浸泡约1min。此步为分化作用，分化到胶原纤维呈浅红色，纤维呈红色即可，约1-2min。
④切片稍微沥干E液后，不经水洗，直接入F液染色2-30s。
⑤切片经连续三缸1％冰醋酸漂洗分化，毎缸8s左右。然后经连续三缸无水乙醇依次脱水约5s、10s、30s。然后经两缸正丁醇依次脱水30s和2min。
⑥最后经两缸二甲苯透明，每次5min，中性树胶封片，完全晾干后在光学显微镜下拍照，考察结果如图6所示。
从图6中可以看出，肥厚型心肌病小鼠相比于正常小鼠出现明显的心肌纤维化增高现象，且心肌细胞的面积也显著增大，而AB23a显著抑制Myh6R404Q小鼠心肌纤维化的进展，并有效减小心肌细胞的面积。这说明AB23a可能具有治疗肥厚型心肌病早期进展的巨大潜力。
综上所述，本发明在体外实验中发现，AB23a有效阻止人源心肌细胞肥大的发生发展；在体内研究中发现，AB23a有效抑制遗传性病理性心肌肥厚的进展，并有效抑制心肌纤维化的发生。体内体外实验均表明，AB23a能显著改善遗传性肥厚型心肌病小鼠的疾病进展，是治疗肥厚型心肌病的有效靶点，可以作为新的抗肥厚型心肌病药物或者药靶进行开发，为检测和治疗肥厚型心肌病提供了一种新的途径和手段。

Claims (10)

  1. 泽泻醇B醋酸酯在制备预防或治疗肥厚型心肌病及其引起的病症的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述肥厚型心肌病包括传性肥厚型心肌病以及遗传性肥厚型心肌病引起的病症。
  3. 根据权利要求1所述的应用，其特征在于，所述肥厚型心肌病包括病理性心肌肥厚、心脏重量或左心室重量增加。
  4. 根据权利要求1所述的应用，其特征在于，所述肥厚型心肌病包括心肌组织中肥厚标志物的表达显著升高。
  5. 根据权利要求1所述的应用，其特征在于，所述肥厚型心肌病包括因遗传性肥厚型心肌病导致的心肌细胞面积增大和/或心肌纤维化水平升高。
  6. 根据权利要求1所述的应用，其特征在于，所述肥厚型心肌病人源心肌细胞面积增大，心肌肥厚标志物水平升高。
  7. 根据权利要求1所述的应用，其特征在于，所述泽泻醇B醋酸酯通过抑制心肌细胞肥大表型在制备预防或治疗肥厚型心肌病及其引起的病症的药物中的应用。
  8. 一种用于预防或治疗肥厚型心肌病及其引起的病症的药物组合物，其特征在于，所述药物组合物其含有泽泻醇B醋酸酯作为活性成分和药学上可接受的载体。
  9. 根据权利要求8所述的药物组合物，其特征在于，所述的药物组合物的剂型是胶囊剂、散剂、片剂、颗粒剂、丸剂、注射剂、糖浆剂、口服液、吸入剂、软膏剂、栓剂或贴剂。
  10. 一种权利要求8所述的药物组合物在制备预防或治疗肥厚型心肌病及其引起的病症的药物中的应用。
PCT/CN2024/132912 2024-03-12 2024-11-19 泽泻醇b醋酸酯在预防或治疗肥厚型心肌病中的应用 Pending WO2025189809A1 (zh)

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