Application of TP receptor inhibitors in the preparation of drugs for treating cardiac fibrosis

Abstract

This invention provides the application of TP receptor inhibitors in the preparation of drugs for treating cardiac fibrosis, relating to the field of biomedical technology. This application clarifies the role of TP receptors in the cardiac Gli1... + This study identifies the key role of TP receptors in cells and establishes them as specific drug targets for anti-fibrosis, overcoming the limitations of insufficient targeting in existing therapies. By inhibiting TP receptors, the study effectively activates the AMPK signaling pathway, thereby promoting the ubiquitination and degradation of Gli1 protein, a key factor in pro-fibrosis, thus inhibiting the generation and activation of myofibroblasts at the source and precisely intervening in the fibrosis process.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of TP receptor inhibitors in the preparation of drugs for treating cardiac fibrosis. Background Technology

[0002] Cardiac fibrosis is a common pathological feature of the end-stage of many cardiovascular diseases. It is characterized by the overactivation of cardiac fibroblasts into myofibroblasts, accompanied by abnormal deposition of extracellular matrix, ultimately leading to cardiac structural remodeling and functional failure. Currently, clinical treatments for cardiac fibrosis are very limited, mainly relying on non-specific drugs such as renin-angiotensin-aldosterone system inhibitors. While these therapies can slow disease progression to some extent, they cannot reverse or specifically inhibit the core mechanisms of fibrosis, and long-term use may be accompanied by numerous side effects.

[0003] Recent studies have suggested that cell populations positive for the Hedgehog signaling pathway transcription factor Gli1 (Gli1) + Gli1 cells play a crucial role in cardiac fibrosis and may be an important source of myofibroblasts. However, the driving force behind Gli1... + The key upstream receptor targets for cell activation and transformation, and their specific molecular mechanisms, remain unclear. Current techniques have failed to elucidate whether a specific, druggable target exists that can directly and effectively regulate Gli1. + The lack of knowledge regarding the fibrotic phenotypic transformation of cells has created a significant bottleneck in developing highly effective and low-toxicity anti-cardiac fibrosis-specific therapies targeting this cell population. Therefore, there is an urgent need to identify new targets and elucidate their regulatory networks to provide a theoretical basis and new intervention pathways for developing targeted therapeutic strategies.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of TP receptor inhibitors in the preparation of drugs for treating cardiac fibrosis, offering a novel treatment strategy for cardiac fibrosis.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides the use of TP receptor inhibitors in the preparation of medicaments for treating cardiac fibrosis, wherein the TP receptor inhibitors are selected from SQ29548, NTP42, or pharmaceutically acceptable salts or derivatives thereof.

[0007] Furthermore, the effective dose of the TP receptor inhibitor is 0.1 mg / kg body weight / day to 20 mg / kg body weight / day.

[0008] Furthermore, the effective dose of the TP receptor inhibitor is 0.5 mg / kg body weight / day to 5 mg / kg body weight / day.

[0009] Furthermore, the cardiac fibrosis includes any one or more of the following: myocardial hypertrophy, dilated cardiomyopathy, hypertrophic cardiomyopathy, heart failure, ischemic myocardial injury, or drug-induced myocardial fibrosis.

[0010] Furthermore, the medication for treating cardiac fibrosis also includes any one of AMPK activators, TGF-β inhibitors, or anti-inflammatory drugs.

[0011] Furthermore, the TP receptor inhibitor is used to inhibit the stability of Gli1 protein and / or promote the ubiquitination and degradation of Gli1 protein.

[0012] The present invention also provides a pharmaceutical composition for treating cardiac fibrosis, the pharmaceutical composition comprising: a TP receptor inhibitor and a pharmaceutically acceptable carrier or excipient.

[0013] Furthermore, the pharmaceutical composition also includes any one of an AMPK activator, a TGF-β inhibitor, or an anti-inflammatory drug.

[0014] Furthermore, the AMPK activator is either metformin or AICAR.

[0015] Furthermore, the dosage form of the pharmaceutical composition is any one of an oral formulation, an injection, or a sustained-release formulation.

[0016] Furthermore, the mass ratio of the TP receptor inhibitor to the AMPK activator is (1:100) to (100:1).

[0017] Furthermore, the mass ratio of the TP receptor inhibitor to the AMPK activator is (1:10) to (10:1).

[0018] The present invention has the following technical effects: This application discloses the application of TP receptor inhibitors in the preparation of drugs for treating cardiac fibrosis, demonstrating significant innovation and application potential. This application clarifies the role of the TP receptor in the cardiac Gli1... +This invention identifies key TP receptors within cells and establishes them as specific drug targets for anti-fibrosis, overcoming the limitations of insufficient targeting in existing therapies. By inhibiting TP receptors, the AMPK signaling pathway can be effectively activated, thereby promoting the ubiquitination and degradation of the key pro-fibrotic factor Gli1 protein, thus inhibiting the generation and activation of myofibroblasts at the source and precisely intervening in the fibrosis process. Animal experiments have confirmed that this strategy can significantly improve cardiac function, reduce myocardial hypertrophy and inflammatory response, and effectively reduce collagen deposition. Furthermore, the TP receptor inhibitors (such as SQ29548 and NTP42) provided by this invention can be used in combination with AMPK activators, exhibiting synergistic effects and laying the foundation for the development of highly effective and low-toxicity compound preparations. This approach has a clear mechanism and strong targeting, providing a new direction for drug development and treatment options for the clinical treatment of various cardiac fibrosis-related diseases such as myocardial hypertrophy and heart failure. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 A mouse model of cardiac fibrosis using the heart Gli1 + The co-expression results of TP, POSTN, α-SMA, Col1α1, and Gli1 in cells are shown below. A is the UMAP diagram of cardiac cell subsets from healthy controls and patients with hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM); B is a bubble diagram of gene expression in different cell populations; C is the expression density of GLi1 in all cardiac cells; D is the expression density of ACTA2, POSTN, COL1A1, GLi1, and TP in cardiac fibroblasts; E is the Western blot analysis of Gli1 expression in isoproterenol-induced mouse hearts. + Cellular protein levels of TP, Postn, α-SMA, Col1a1, and Gli1; ​​F represents Western blot analysis of Gli1 levels in mouse hearts after aortic arch constriction surgery. + Cellular protein levels of TP, Postn, α-SMA, Col1α1, and Gli1; Figure 2 Expression levels of Gli1 and TP in different cell lines, where A represents the expression levels of Gli1 in the heart as analyzed by Western blot. +Gli1 protein expression level in cells, LX-2 cells, NIH-3T3 cells and BMSCs; B represents the expression levels of COX-1, TxAS and TP genes in NIH-3T3 cells and BMSCs (n=3). Figure 3 : Cre-mediated gene recombination in the heart Gli1 + The TP gene was knocked out in cells, where A represents genomic DNA extracted from tail tissue and analyzed using PCR for Gli1. CreERT2 Rosa-RFP / TP F / F Genotyping analysis was performed on mice; B represents Gli1. CreERT2 Rosa-RFP / TP F / F mouse heart Gli1 + TP gene expression level in cells (n=5-6; Mann-Whitney U test; **P<0.01 vs. control group); Figure 4 Gli1 + Knockout of TP in cells alleviated isoproterenol-induced myocardial hypertrophy and inflammation in mice, where A represents Gli1 after isoproterenol induction. CreERT2 Rosa26-RFP / TP F / F and Gli1 CreERT2 Rosa26-RFP mice cardiac PDGFR + Quantitative analysis of cell number (n=5-6; two-way ANOVA with Tukey multiple comparison test; ***P<0.001); B represents Gli1 after isoproterenol stimulation. CreERT2 Rosa26-RFP / TP F / F Mice and Gli1 CreERT2 Rosa26-RFP control mouse heart Gli1 + Quantitative analysis of cell number (n=6; two-way ANOVA with Tukey multiple comparison test; ***P<0.001) C represents Gli1 cells induced by isoproterenol. CreERT2 Rosa26-RFP / TP F / F Hematoxylin and eosin (HE) staining of heart tissues from mice and their littermates were performed. Blue scale bars were 2 mm long, and black scale bars were 60 μm long. D represents Gli1 treated with isoproterenol. CreERT2 Rosa26-RFP / TP F / FWGA staining of heart tissues from mice and their littermates, scale bar = 20 μm; E represents the quantitative analysis of cardiomyocyte size in D (n=7; two-way ANOVA using Tukey's multiple comparison test; **P<0.01, ***P<0.001); F represents the gene expression level of IL-1β; G represents the gene expression level of IL-6; H represents the gene expression level of TNF-α; I represents the gene expression level of Gli1 after isoproterenol stimulation. CreERT2 Rosa26-RFP / TP F / F Systolic blood pressure in mice and their littermate controls; J represents the prostaglandin level in mouse heart tissue as determined by isoproterenol test (n=8-10; two-tailed Student's t-test; *P<0.05, **P<0.01). Figure 5 Gli1 + Knockout of TP in cells alleviated myocardial hypertrophy and inflammation induced by aortic arch coarctation in mice. Among them, A was the Gli1 inhibitor induced by aortic arch coarctation. CreERT2 Rosa26-RFP / TP F / F Mice and Gli1 CreERT2 Rosa26-RFP control mice cardiac PDGFRα + Quantitative analysis of cell count (n=5-6; Tukey two-way ANOVA; ***P<0.001); B is the same group as A, representing cardiac Gli1 cells. + Cell number quantification (n=6; two-way ANOVA using Tukey's multiple comparison test; ***P<0.001); C represents Gli1 CreERT2 Rosa26-RFP / TP F / F Hematoxylin and eosin (HE) staining of cardiac tissues from mice and their littermates after aortic arch coarctation surgery; blue scale bars = 2 mm, black scale bars = 60 μm; D represents Gli1 CreERT2 Rosa26-RFP / TP F / F WGA staining of cardiac tissues from mice and their littermates after aortic arch constriction surgery, scale bar = 20 μm; E represents the quantitative analysis of cardiomyocyte size in cardiac tissue induced by aortic arch constriction surgery in D (n=7; two-way ANOVA with Tukey multiple comparison test; **P<0.01, ***P<0.001), F represents Gli1 CreERT2 Rosa26-RFP / TP F / F Gene expression levels of IL-1β in cardiac tissues of mice and their littermates after aortic arch coarctation surgery; G represents Gli1 CreERT2 Rosa26-RFP / TP F / F Gene expression levels of IL-6 in cardiac tissues of mice and their littermates after aortic arch coarctation surgery, with H representing Gli1. CreERT2Rosa26-RFP / TP F / F Gene expression levels of TNFα in cardiac tissues of mice and their littermates after aortic arch coarctation (n=6-8; two-way ANOVA, Tukey multiple comparison test; *P<0.05, **P<0.01, ***P<0.001); I was the expression level of TNFα in cardiac tissues after aortic arch coarctation. CreERT2 Changes in systolic blood pressure in Rosa26-RFP / TPF / F mice and their littermate controls; J: prostaglandin levels in cardiac tissue of mice with aortic arch constriction (n=9-12; two-tailed Student's t-test; *P<0.05, **P<0.01). Figure 6 The diagram illustrates the antifibrotic effect of AMPK inhibition in reversing TP deficiency in mice. A represents a schematic diagram of the tamoxifen-induced isoproterenol and compound C dosing regimen in mice, and B represents the Gli1 inhibitory effect with or without isoproterenol administration. CreERT2 Rosa-RFP / TP F / F Echocardiography of mice; C represents the cardiac ejection fraction of mice identical to B; D represents the cardiac shortening fraction of mice identical to B (n=7; one-way ANOVA using Dunnett's multiple comparison test; **P<0.01, ***P<0.001); E represents representative images of masson trichrome staining of mouse heart tissue treated with isoproterenol with or without compound C, with blue scale bars = 1 mm and black scale bars = 100 μm; F represents the quantitative analysis of fibrotic areas in E (n=5-7; one-way ANOVA using Kruskal-Wallis test and Dunn's multiple comparison test; *P<0.05, **P<0.01); G represents Western spectroscopy. Blot analysis of Fn1 and α-SMA protein levels in isoproterenol-treated mouse heart tissue with and without compound C; H represents the quantitative levels of fibrosis proteins in isoproterenol-treated mouse heart tissue with and without compound C (n=7; one-way ANOVA with Dunnett's multiple comparison test; *P<0.05, ***P<0.001); I represents representative immunofluorescence images observed in isoproterenol-treated mouse heart tissue with and without compound C; J represents Gli1... + Statistical analysis of cell activation rate, scale bar = 20 μm (n = 6-7); one-way ANOVA with Dunnett's multiple comparison test; **P < 0.01, ***P < 0.001; Figure 7 CaMKKβ and LKB1 in Gli1 of TP-deficient myocardium + Cellular activity, control and TP-deficient myocardial Gli1 +Western blot analysis of cell pLKB1, LKB1, pCAMKKβ, and CAMKKβ; Figure 8 TP deficiency can attenuate TGF-β1-induced fibrinogen activity in Gli1 + The results of expression in cells are shown in Figure A, where Fn1 represents the fibrin level in the quantitative graph; and n-SMA represents the fibrin level in the quantitative graph (two-way ANOVA with Tukey multiple comparison test was used; *P<0.05, ***P<0.001). Figure 9 : Control group and TP-deficient Gli1 + Comparable expression of Hedgehog-related genes in cells, from Gli1 CreERT2 RosaRFP / TP F / F Gli1 heart isolated from mice and their littermates + Relative expression levels of Ptch1, N-myc, and Smo mRNA in cells (n=7-8; two-tailed Student's t-test). Figure 10 U46619 significantly slowed down the degradation of Gli1 protein in NIH-3T3 cells after treatment with cycloheximine (CHX). Western blot analysis was performed on the Gli1 protein level in NIH-3T3 cells treated with U46619 in the presence or absence of CHX. Figure 11 Inhibition of AMPK can eliminate TP receptor inhibitor-induced Gli1 degradation. In this study, A represents the effect of SQ29548 (10 μM) on Gli1 phosphorylation in NIH-3T3 cells with or without compound C, as analyzed by Western blot. B represents the Gli1 ubiquitination in NIH-3T3 cells treated with SQ29548 with or without compound C, as analyzed by Western blot. C represents the levels of Fn and α-SMA proteins in NIH-3T3 cells treated with TGF-β1 and / or compound C, as analyzed by Western blot. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0022] In a first aspect, the present invention provides the use of TP receptor inhibitors in the preparation of medicaments for treating cardiac fibrosis, wherein the TP receptor inhibitors are selected from SQ29548, NTP42, or pharmaceutically acceptable salts or derivatives thereof.

[0023] In some embodiments, the effective amount of the TP receptor inhibitor is 0.1 mg / kg body weight / day to 20 mg / kg body weight / day.

[0024] In some embodiments, the effective amount of the TP receptor inhibitor is 0.5 mg / kg body weight / day to 5 mg / kg body weight / day.

[0025] In some embodiments, the cardiac fibrosis includes any one or more of the following: myocardial hypertrophy, dilated cardiomyopathy, hypertrophic cardiomyopathy, heart failure, ischemic myocardial injury, or drug-induced myocardial fibrosis.

[0026] In some embodiments, the medication for treating cardiac fibrosis also includes any one of AMPK activators, TGF-β inhibitors, or anti-inflammatory drugs.

[0027] In some embodiments, the TP receptor inhibitor is used to inhibit the stability of the Gli1 protein and / or promote the ubiquitination and degradation of the Gli1 protein.

[0028] In a second aspect, the present invention provides a pharmaceutical composition for treating cardiac fibrosis, the pharmaceutical composition comprising: a TP receptor inhibitor and a pharmaceutically acceptable carrier or excipient.

[0029] In some embodiments, the pharmaceutical composition further includes any one of an AMPK activator, a TGF-β inhibitor, or an anti-inflammatory drug.

[0030] In some embodiments, the AMPK activator is either metformin or AICAR.

[0031] In some embodiments, the dosage form of the pharmaceutical composition is any one of an oral formulation, an injection, or a sustained-release formulation.

[0032] In some embodiments, the mass ratio of the TP receptor inhibitor to the AMPK activator is (1:100) to (100:1).

[0033] In some embodiments, the mass ratio of the TP receptor inhibitor to the AMPK activator is (1:10) to (10:1).

[0034] The following is a detailed explanation using specific embodiments: Example 1: Detection of cardiac Gli1 + Expression of TP receptor in cells The part number and source of SQ29548 in this application are: SQ29548 (#19025) Cayman Chemical Company (Ann Arbor, MI, USA).

[0035] Compound C catalog number and source: Compound C (#S7306) Selleck Chemicals (Houston, TX, USA). This substance is an AMPK inhibitor used to inhibit the AMPK signaling pathway.

[0036] NTP42 part number and source: NTP42 (#HY-129851) MedChemExpress.

[0037] Experimental procedure: After induction with tamoxifen, Gli1 cells from the heart of mice were sorted out. + Cells were stimulated in vitro to detect the expression of genes or proteins such as TP receptor, Gli1, α-SMA, and Collagen I in cardiac tissue.

[0038] Experimental results are as follows Figure 1 As shown, from Figures 1-2 The experimental results show that TP receptors and fibrosis markers are involved in cardiac fibrosis, and Gli1 + It is specifically and highly co-expressed in cells.

[0039] Example 2: Gene knockout animal model experiment Experimental content: After modeling by isoproterenol or aortic arch coarctation, cardiac function was assessed by echocardiography, the area of ​​cardiac fibrosis was displayed by Masson staining, the expression level of inflammatory factors in cardiac tissue was detected by qPCR, and changes in cardiomyocyte size were observed and statistically analyzed by WGA staining.

[0040] Experimental results are as follows Figures 3-6 As shown in the above experiment, it can be seen that AMPK inhibits the anti-fibrotic effect of reversible TP deficiency in mice.

[0041] Example 3: In vivo experiments of pharmacological inhibitors Experimental content: The isoproterenol model mice were treated with the TP receptor inhibitor NTP42. The cardiac fibrosis phenotype of the mice was identified by cardiac ultrasound and tissue staining, and the efficacy was evaluated accordingly.

[0042] The experimental results are shown in Table 1 and Figure 7 As shown.

[0043] Table 1: Gli1 treated with isoproterenol CreERT2 Echocardiographic analysis of the pharmacological inhibitory effect of NTP42 on TP in Rosa26-RFP mice Data are expressed as mean ± standard error. One-way ANOVA and Dunnett's multiple comparison test were used. *P<0.05, **P<0.01, ***P<0.001 vs. Saline, #P<0.05 vs. isoproterenol.

[0044] The experimental results above show that systemic administration of TP receptor inhibitors can improve cardiac function and reduce fibrosis.

[0045] Example 4: In vitro cell mechanism experiment Experimental procedures: The TP receptor inhibitor SQ29548 was added to TGF-β1-treated cells, and the expression of fibrin was detected; the effects of SQ29548 on Gli1 protein stability, ubiquitination level and AMPK activity were detected; the reversal of the TP inhibition effect was observed using an AMPK inhibitor.

[0046] Experimental results are as follows Figures 8-11 As shown in the experimental results, it can be seen that TP receptor inhibitors promote the ubiquitination and degradation of Gli1 protein by activating the AMPK signaling pathway, thereby inhibiting the expression of fibrotic proteins.

[0047] Example 5: Combined Drug Trial Experimental Procedure: A mouse model of cardiac fibrosis induced by isoproterenol was established. Patients were randomly divided into a normal control group, a model control group, an SQ29548 monotherapy group, a metformin monotherapy group, and a combination therapy group (SQ29548 and metformin). In the combination therapy group, the two drugs were administered at a 1:1 mass ratio. The synergistic protective effect on the heart was observed. The experimental results are shown in Table 2.

[0048] Table 2: Synergistic therapeutic effect of SQ29548 and metformin on a mouse model of cardiac fibrosis. Note: Data are expressed as mean ± standard deviation, *P<0.05 vs model control group; # P<0.05 vs SQ29548 single-drug group; † P<0.05 vs metformin monotherapy group (based on one-way ANOVA and Tukey post-hoc test).

[0049] The above experiments demonstrate that the AMPK pathway is a key downstream pathway for TP inhibition to exert its therapeutic effect, and the combined use of TP receptor inhibitors and AMPK activators can produce synergistic or enhanced effects. Based on this mechanism, it can be seen that TP receptor inhibitors can be used in combination with AMPK activators (such as metformin) to potentially enhance the anti-cardiac fibrosis efficacy. Specific combination regimens and ratios can be determined through routine experimental optimization.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. The application of TP receptor inhibitors in the preparation of drugs for treating cardiac fibrosis, characterized in that, The TP receptor inhibitor is selected from SQ29548; The medication for treating cardiac fibrosis also includes an AMPK activator, wherein the AMPK activator is metformin; The mass ratio of metformin to SQ29548 is 1:1; The TP receptor inhibitor is used to inhibit the stability of Gli1 protein and / or promote the ubiquitination and degradation of Gli1 protein.

2. The use of the TP receptor inhibitor according to claim 1 in the preparation of a drug for treating cardiac fibrosis, characterized in that, The effective dose of the TP receptor inhibitor is 0.1 mg / kg body weight / day to 20 mg / kg body weight / day.

3. The use of the TP receptor inhibitor according to claim 1 in the preparation of a drug for treating cardiac fibrosis, characterized in that, The cardiac fibrosis includes any one or more of the following: myocardial hypertrophy, dilated cardiomyopathy, hypertrophic cardiomyopathy, heart failure, ischemic myocardial injury, or drug-induced myocardial fibrosis.

4. A pharmaceutical composition for treating cardiac fibrosis, characterized in that, The pharmaceutical composition comprises: a TP receptor inhibitor and a pharmaceutically acceptable carrier or excipient; The pharmaceutical composition also includes an AMPK activator; The AMPK activator is metformin; The TP receptor inhibitor is SQ29548; The mass ratio of SQ29548 to metformin is 1:

1.

5. The pharmaceutical composition according to claim 4, characterized in that, The dosage form of the pharmaceutical composition is any one of an oral preparation, an injection, or a sustained-release preparation.

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