MBD2 activators and their use in prevention and treatment of pulmonary arterial hypertension diseases

Abstract

The invention discloses an MBD2 activator and application thereof in prevention and treatment of pulmonary arterial hypertension disease, the MBD2 activator affects the expression level or / and function of MBD2 gene, and the MBD2 activator is used for prevention and / or treatment of individual pulmonary arterial hypertension disease; uses include preparation of pharmaceutical compositions. The MBD2 activator inhibits abnormal proliferation and migration of hypoxia-induced pulmonary artery smooth muscle cells so as to slow down the pulmonary vascular remodeling process, provides a novel MBD2 targeting treatment strategy for the pulmonary hypertension disease, further lays a foundation for developing novel high-safety medicines capable of effectively treating the pulmonary hypertension disease, and has a wide application prospect. The popularization and use are convenient.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, specifically relating to an MBD2 activator and its use in the prevention and treatment of pulmonary hypertension. Background Technology

[0002] The role of epigenetic regulation in the pathogenesis of pulmonary hypertension is receiving increasing attention. Methyl-CpG binding domain protein 2 (MBD2), as an important epigenetic reader, can recognize methylated DNA and recruit other repressive complexes, thereby regulating gene expression (Desai et al. NucleicAcids Res. 2015 Mar 31;43(6):3100-13). Dysregulation of MBD2 expression is associated with various pathological conditions, including autoimmune diseases (Wardowska et al. Int Immunopharmacol. 2020;83:106451) and cancer (Nishiyama et al. Trends Genet. 2021;37(11):1012-1027), but its role in the pathogenesis of pulmonary hypertension remains unclear with current technology.

[0003] Pulmonary arterial hypertension (PAH) is a progressive and fatal vascular disease characterized by pulmonary endothelial cell dysfunction, abnormal proliferation, smooth muscle cell hypertrophy, and extracellular matrix remodeling, ultimately leading to persistently elevated pulmonary vascular resistance, increased right ventricular load, and right heart failure (Mocumbi A, et al. Nat Rev Dis Primers, 2024, 10(1): 1). Pulmonary artery remodeling caused by hypoxia is a crucial and irreversible pathological basis for PAH, with abnormal changes in the biological behavior of pulmonary artery smooth muscle cells, such as proliferation and migration, being key factors. However, the pathogenesis remains incompletely understood (Shlobin et al. Eur Respir J, 2024, 64(4)). Although targeted drug therapy for PAH, such as endothelin receptor antagonists, prostacyclin analogs, and PDE5 inhibitors, has made some progress in recent years, it still cannot effectively reverse the structural lesions of the pulmonary vessels, resulting in poor long-term prognosis and a persistently high mortality rate. Therefore, exploring new therapeutic targets and intervention strategies to delay or even reverse pulmonary vascular remodeling has become an urgent need in this field of research.

[0004] Current treatment methods mainly include calcium channel blockers, vasodilators, and antifibrotic drugs. These drugs cannot reverse the process of pulmonary vascular remodeling, and their clinical efficacy and safety are insufficient to meet treatment needs. Therefore, there is an urgent need to conduct in-depth research on the mechanisms of pulmonary vascular remodeling, identify new therapeutic targets, and fully integrate innovative pharmaceutical development trends to develop novel drugs that can effectively treat pulmonary hypertension with high safety. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing an MBD2 activator and its use in the prevention and treatment of pulmonary hypertension. The MBD2 activator slows down the process of pulmonary vascular remodeling by inhibiting the abnormal proliferation and migration of pulmonary artery smooth muscle cells induced by hypoxia, thus providing a new MBD2-targeted treatment strategy for pulmonary hypertension. This further lays the foundation for the development of novel drugs that can effectively treat pulmonary hypertension and have high safety, and facilitates their widespread use.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: An MBD2 activator that affects the expression level and / or function of the MBD2 gene, said MBD2 activator for the prevention and / or treatment of individual pulmonary hypertension.

[0007] The MBD2 activator mentioned above is Branaplam.

[0008] The MBD2 activator mentioned above is a small molecule splicing regulator.

[0009] The aforementioned MBD2 activator simultaneously targets the splicing process of the pre-mRNA of the motor neuron survival gene 2 SMN2 and the Huntington's disease-associated neurofilament light chain NfL biomarker.

[0010] The aforementioned MBD2 activators upregulate the expression level of the MBD2 gene.

[0011] The aforementioned MBD2 activators slow down the process of pulmonary vascular remodeling by inhibiting the abnormal proliferation and migration of pulmonary artery smooth muscle cells induced by hypoxia.

[0012] The present invention also discloses the use of an MBD2 activator in the prevention and treatment of pulmonary hypertension, wherein the MBD2 activator is the MBD2 activator described above, and the use includes the preparation of pharmaceutical compositions for the prevention and / or treatment of individual pulmonary hypertension.

[0013] The use of the aforementioned MBD2 activator in the prevention and treatment of pulmonary hypertension, wherein the individuals are mammals.

[0014] The use of the above-mentioned MBD2 activator in the prevention and treatment of pulmonary hypertension, wherein the pharmaceutical composition includes injectable, capsule, tablet, nasal spray and aerosol formulations.

[0015] Compared with the prior art, the present invention has the following advantages: The present invention explores how MBD2 activators can slow down the process of pulmonary vascular remodeling by inhibiting the abnormal proliferation and migration of pulmonary artery smooth muscle cells induced by hypoxia, thus providing a new MBD2-targeted treatment strategy for pulmonary hypertension and laying the foundation for the development of novel drugs that can effectively treat pulmonary hypertension and have high safety. Attached Figure Description

[0016] Figure 1 Tricuspid annular systolic shift (TAPSE) was measured in wild-type mice, high-dose branaplam wild-type mice, hypoxic mice, low-dose branaplam hypoxic mice, and high-dose branaplam hypoxic mice, with **p<0.01; ****p<0.0001; ####p<0.0001.

[0017] Figure 2 Right ventricular systolic pressure (RVSP) was measured in wild-type mice, high-dose branaplam wild-type mice, hypoxic mice, low-dose branaplam hypoxic mice, and high-dose branaplam hypoxic mice, with *p<0.05; ***p<0.001; ****p<0.0001; ####p<0.0001.

[0018] Figure 3 The Fulton index was used to compare wild-type mice, high-dose branaplam wild-type mice, hypoxic mice, low-dose branaplam hypoxic mice, and high-dose branaplam hypoxic mice. For each pair of mice, #p < 0.05, **p < 0.01, and ns were not statistically significant.

[0019] Figure 4 Representative images of H&E staining, α-SMA staining, and EVG staining of pulmonary arterioles in wild-type mice, high-dose Brapalam wild-type mice, hypoxic mice, low-dose Brapalam hypoxic mice, and high-dose Brapalam hypoxic mice.

[0020] Figure 5 Quantitative results of pulmonary artery wall thickness in wild-type mice, high-dose branaplam wild-type mice, hypoxic mice, low-dose branaplam hypoxic mice, and high-dose branaplam hypoxic mice; where *p<0.05; **p<0.01; ****p<0.0001; scale bar = 100 mm.

[0021] Figure 6 Quantitative results of the proportion of myovascularization in wild-type mice, high-dose branaplam wild-type mice, hypoxic mice, low-dose branaplam hypoxic mice, and high-dose branaplam hypoxic mice; where *p<0.05; **p<0.01; ****p<0.0001; ###p<0.001; ####p<0.0001; scale bar = 100 mm.

[0022] Figure 7 Western blot and quantitative analysis results of MBD2 in lung tissues of patients with pulmonary hypertension and healthy controls, western blot and quantitative analysis results of MBD2 in lung tissues of control group and hypoxic pulmonary hypertension mice, and western blot and quantitative analysis results of MBD2 in pulmonary artery smooth muscle cells at different hypoxic time points; where *p<0.05; **p<0.01; ns had no statistical significance.

[0023] Figure 8 The proliferation of pulmonary artery smooth muscle cells after treatment with Branaplam under normoxic and hypoxic conditions is shown. *p<0.05; **p<0.01, scale bar = 100 mm.

[0024] Figure 9 The migration of pulmonary artery smooth muscle cells after treatment with Branaplam under normoxic and hypoxic conditions is shown. ***p<0.001; ****p<0.0001, scale bar = 100 mm.

[0025] Figure 10 Representative H&E staining images of heart, liver, spleen, and kidney tissues from wild-type mice and high-dose Branaplam wild-type mice, scale bar = 100 mm. Detailed Implementation

[0026] This invention discloses an MBD2 activator that affects the expression level and / or function of the MBD2 gene. The MBD2 activator is used to prevent and / or treat individual pulmonary hypertension. The MBD2 activator is Branaplam; it is a small molecule splicing regulator; it simultaneously targets the splicing process of the motor neuron survival gene 2 (SMN2) pre-mRNA and the Huntington's disease-associated neurofilament light chain (NfL) biomarker; it upregulates the expression level of the MBD2 gene; and it slows down pulmonary vascular remodeling by inhibiting hypoxia-induced abnormal proliferation and migration of pulmonary artery smooth muscle cells.

[0027] The use of the MBD2 activator of the present invention in the prevention and treatment of pulmonary hypertension, wherein the MBD2 activator is described herein, and the use includes preparing pharmaceutical compositions for the prevention and / or treatment of pulmonary hypertension in an individual; wherein the individual is a mammal; and wherein the pharmaceutical compositions include injectable, capsule, tablet, nasal spray, and aerosol formulations.

[0028] Definitions and usage of terms: Pulmonary hypertension: Pulmonary hypertension is defined as a mean pulmonary artery pressure (mPAP) ≥ 20 mmHg as measured by right heart catheterization (RHC) at sea level and at rest.

[0029] The core pathological features of pulmonary hypertension are abnormal vasoconstriction, progressive remodeling, and occlusive lesions of the pulmonary vessels. At its core lies endothelial dysfunction in the pulmonary vessels, leading to a decrease in vasodilators (such as nitric oxide and prostacyclin) and an increase in vasoconstrictors (such as endothelin-1), causing persistent vasoconstriction. Subsequently, abnormal proliferation, inhibited apoptosis, and extracellular matrix deposition occur in cells of all layers of the pulmonary artery (intima, media, and adventitia), including endothelial cells, smooth muscle cells, and fibroblasts, resulting in significant thickening of the vessel wall and narrowing of the lumen. Some patients exhibit characteristic plexiform lesions and thrombus organization, further obstructing the vessels. These changes collectively lead to a progressive and irreversible increase in pulmonary vascular resistance, ultimately increasing right ventricular afterload and causing right heart failure.

[0030] MBD2 activator: In this invention, MBD2 activator refers to Branaplam, a small molecule splicing regulator that targets the splicing process of pre-messenger RNA (pre-mRNA) of the survival gene of motor neuron 2 (SMN2) and the neurofilament light chain (NfL) biomarker associated with Huntington's disease.

[0031] Individual: In this invention, an individual refers to a mammal, including but not limited to rats, mice, non-human primates, humans, dogs, cats, horses, cattle, sheep, pigs, and goats, preferably humans or mice.

[0032] Prevention and treatment: In this invention, prevention refers to preventing or reducing the occurrence of pulmonary vascular remodeling after use in the presence of possible pro-pulmonary vascular remodeling factors. In this invention, treatment refers to reducing the degree of pulmonary vascular remodeling, or curing pulmonary vascular remodeling to normalize it, or slowing down the process of pulmonary vascular remodeling.

[0033] Pharmaceutical composition: In this invention, a pharmaceutical composition refers to a composition comprising the above-mentioned MBD2 activator and a pharmaceutically acceptable carrier. The pharmaceutical composition may be an injectable form, capsule, tablet, nasal spray, or aerosol, etc., and may be administered to the test subject in an individually acceptable manner, such as injection, oral administration, nasal spray, etc.

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Example 1: Effects of oral administration of MBD2 activator on hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling; Laboratory animals and materials: Laboratory animals: The experimental animals were wild-type mice (WT, C57BL / 6) bred in the animal facility of Tongji Medical College; the breeding age was 8-10 weeks. Experimental methods Step A1: Divide the mice into groups: Normoxia group: normoxic control; Normoxia Branaplam-High group: normoxia + 24 mg / kg Branaplam by gavage; Hypoxia group: hypoxia exposure (10% O2, 4 weeks); Hypoxia Branaplam-Low group: hypoxia + 12 mg / kg Branaplam by gavage; Hypoxia Branaplam-High group: hypoxia + 24 mg / kg Branaplam by gavage.

[0035] Step A2: Prepare the medication. Branaplam (MCE) was dissolved in 100% DMSO, with a stock solution concentration of 10 mg / mL.

[0036] Step A3, Model Construction and Drug Administration: Mice in the Hypoxia group were placed in a 10% O2 chamber for 4 weeks; Branaplam was administered by gavage every other day for 2 weeks; and the Normoxia group was given an equal volume of solvent.

[0037] Experimental results: Tricuspid annular systolic displacement (TAPSE): such as Figure 1 As shown, compared with the Normoxia group, the TAPSE in the Hypoxia group was significantly lower, and after Branaplam treatment, the RVSP in the treatment group was significantly higher than that in the Hypoxia group.

[0038] Right ventricular systolic pressure (RVSP): such as Figure 2 As shown, RVSP was significantly higher in the Hypoxia group compared to the Normoxia group, but significantly lower in the treatment group after Branaplam treatment compared to the Hypoxia group.

[0039] Right ventricular hypertrophy index (Fulton index): such as Figure 3 As shown, the Fulton index was significantly higher in the Hypoxia group compared to the Normoxia group. After high-dose Branaxam treatment, the Fulton index was significantly lower in the treatment group compared to the Hypoxia group.

[0040] H&E staining of pulmonary arterioles: such as Figure 4 As shown, compared with the Normoxia group, the pulmonary small vessel wall thickness was significantly increased in the Hypoxia group. After treatment with Branaplam, the pulmonary small vessel wall thickness was significantly reduced in the treatment group compared with the Hypoxia group.

[0041] α-SMA staining: such as Figure 5 As shown, compared with the Normoxia group, the Hypoxia group showed a significant increase in α-SMA expression. After treatment with Branaplam, compared with the Hypoxia group, the treatment group showed a significant decrease in α-SMA expression.

[0042] EVG staining of pulmonary arterioles: as shown Figure 6 As shown, the proportion of myovascularized vessels was significantly higher in the Hypoxia group compared to the Normoxia group. After treatment with Branaplam, the proportion of myovascularized vessels was significantly lower in the treatment group compared to the Hypoxia group.

[0043] Based on the above experimental results, gavage administration of Branaplam can significantly improve hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling.

[0044] Example 2: Effect of Branaplam on MBD2 protein levels; Experimental methods: The protein level of MBD2 was detected by Western blot in the lung tissues of patients with hypoxic pulmonary hypertension and healthy controls, and the protein level of MBD2 was detected by Western blot in the lung tissues of mice in the control group and the hypoxic pulmonary hypertension group.

[0045] Experimental results: such as Figure 7As shown, compared with the control group, the level of MBD2 protein in the lung tissue of patients with hypoxic pulmonary hypertension was significantly reduced; compared with the control group, the level of MBD2 protein in the lung tissue of mice with hypoxic pulmonary hypertension was significantly reduced; compared with the control group, the expression of MBD2 protein in pulmonary artery smooth muscle cells was significantly reduced with prolonged hypoxia time.

[0046] Example 3: Effects of Branaplam on hypoxia-induced proliferation and migration of pulmonary artery smooth muscle cells; Pulmonary artery remodeling induced by hypoxia is a crucial pathological basis for irreversible pulmonary hypertension, with abnormal changes in the biological behaviors of pulmonary artery smooth muscle cells, such as proliferation and migration, being a key factor. Therefore, further research is needed on the effects of branaplam on the proliferation and migration of pulmonary artery smooth muscle cells after hypoxia stimulation.

[0047] Laboratory animals and materials: The experimental cells were derived from immortalized pulmonary artery smooth muscle cells; Experimental methods: Step B1: Divide the experimental cells into groups: Normoxia group: normoxic control; Normoxia + Branaplam group: normoxic + 100 nM Branaplam; Hypoxia group: hypoxia exposure (1% O2, 48h); Hypoxia + Branaplam group: hypoxia + 100 nM Branaplam.

[0048] Step B2: Prepare the medication. Prepare fresh culture medium immediately before use (final concentration 100 nM). Step B3, Model Construction and Drug Administration: Normoxia cells were placed in a 1% O2 hypoxic incubator for 48 hours, while the control group was given an equal volume of solvent.

[0049] Experimental results: EdU: such as Figure 8 As shown, compared with the Normoxia group, Branaplam significantly reduced the proliferative activity of pulmonary artery smooth muscle cells in the Normoxia group. Compared with the Normoxia group, hypoxia significantly promoted the proliferation of pulmonary artery smooth muscle cells. Branaplam can significantly inhibit hypoxia-induced excessive proliferation of pulmonary artery smooth muscle cells.

[0050] Transwell: such as Figure 9As shown, compared with the Normoxia group, Branaplam significantly reduced migration in Normoxia mice. Compared with the Normoxia group, hypoxia significantly promoted pulmonary artery smooth muscle cell migration. Branaplam can significantly inhibit hypoxia-induced excessive migration of pulmonary artery smooth muscle cells.

[0051] Example 4: Multi-organ toxicity assessment of Branaplam; Laboratory animals: The experimental animals were wild-type mice (WT, C57BL / 6) bred in the animal facility of Tongji Medical College; the breeding age was 8-10 weeks. Experimental methods: Step C1: Divide the mice into groups: Normoxia group: normoxic control; Normoxia Fimepinostat-High group: normoxia + 24 mg / kg Branaplam by gavage; Step C2: Prepare the medication. Branaplam (MCE) was dissolved in 100% DMSO, with a stock solution concentration of 10 mg / mL.

[0052] Step C3, Model Construction and Drug Administration: Branaplam was administered by gavage every other day for two weeks, while the control group received an equal volume of solvent.

[0053] Experimental results: Multi-organ toxicity: such as Figure 10 As shown, compared with the Normoxia group, the Branaplam-High dose resulted in normal tissue structure of the heart, liver, spleen, kidneys and intestines under normoxic conditions, with no pathological changes, confirming the good safety of the drug at this dose.

[0054] The above examples demonstrate that upregulating MBD2 expression in PASMCs can significantly inhibit hypoxia-induced abnormal proliferation and migration of PASMCs, which play a crucial role in the pathogenesis of pulmonary hypertension. The data indicate that inhibiting the MBD2 activator Branaplam can suppress hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling. Since pulmonary vascular remodeling plays a vital role in the development and progression of pulmonary hypertension, inhibiting pulmonary vascular remodeling can slow the progression of pulmonary hypertension, providing a new therapeutic target.

[0055] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An MBD2 activator, characterized in that, The MBD2 activator affects the expression level and / or function of the MBD2 gene, and is used to prevent and / or treat individual pulmonary hypertension.

2. The MBD2 activator according to claim 1, characterized in that, The MBD2 activator is Branaplam.

3. The MBD2 activator according to claim 1, characterized in that, The MBD2 activator is a small molecule splicing regulator.

4. The MBD2 activator according to claim 1, characterized in that, The MBD2 activator simultaneously targets the splicing process of the pre-mRNA of the motor neuron survival gene 2 SMN2 and the Huntington's disease-associated neurofilament light chain NfL biomarker.

5. The MBD2 activator according to claim 1, characterized in that, The MBD2 activator upregulates the expression level of the MBD2 gene.

6. The MBD2 activator according to claim 1, characterized in that, The MBD2 activator slows down the process of pulmonary vascular remodeling by inhibiting the abnormal proliferation and migration of pulmonary artery smooth muscle cells induced by hypoxia.

7. The use of an MBD2 activator in the prevention and treatment of pulmonary hypertension, characterized in that, The MBD2 activator is the MBD2 activator as described in claims 1-6, and the use includes preparing pharmaceutical compositions for the prevention and / or treatment of individual pulmonary hypertension.

8. The use of the MBD2 activator according to claim 7 in the prevention and treatment of pulmonary hypertension, characterized in that, The individual in question is a mammal.

9. The use of the MBD2 activator according to claim 7 in the prevention and treatment of pulmonary hypertension, characterized in that, The pharmaceutical compositions include injectable, capsule, tablet, nasal spray, and aerosol formulations.

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