Application of TP receptor inhibitor in preparation of medicine for treating cardiac fibrosis

Abstract

The invention provides application of a TP receptor inhibitor in preparation of a medicine for treating cardiac fibrosis, and relates to the technical field of biomedicine. According to the application, the key effect of the TP receptor in heart Gli1 + cells is defined, the TP receptor is determined as an anti-fibrosis specific drug target, and the limitation of insufficient targeting of the existing therapy is broken through. By inhibiting a TP receptor, an AMPK signal channel can be effectively activated, and then ubiquitination degradation of a fibrosis-promoting key factor Gli1 protein is promoted, so that generation and activation of myofibroblasts are inhibited from the source, and the fibrosis process is accurately intervened.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to application of TP receptor inhibitor in preparation of a drug for treating cardiac fibrosis. BACKGROUND

[0002] Cardiac fibrosis is a common pathological feature of the end stage of various cardiovascular diseases, which is characterized by excessive activation of cardiac fibroblasts into myofibroblasts and abnormal deposition of extracellular matrix, eventually leading to cardiac structural remodeling and functional failure. At present, the treatment of cardiac fibrosis in the clinic is very limited, mainly relying on non-specific drugs such as renin-angiotensin-aldosterone system inhibitors. Although these therapies can delay the progression of the disease to some extent, they cannot reverse or specifically inhibit the core mechanism of fibrosis, and long-term use may be accompanied by many side effects.

[0003] In recent years, studies have shown that Gli1 positive cell group (Gli1 + cells) plays an important role in the process of cardiac fibrosis, and may be an important source of myofibroblasts. However, the upstream key receptor target that drives the activation and transformation of Gli1 + cells is not clear. The existing technology cannot clarify whether there is a specific target that can be intervened by drugs, which can directly and effectively regulate the fibrosis phenotype transformation of Gli1 + cells. The lack of this key knowledge leads to a major bottleneck in developing efficient and low-toxicity specific therapies for cardiac fibrosis. Therefore, it is urgent to identify new target points and reveal their regulatory network to provide a theoretical basis and intervention for developing targeted treatment strategies.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] In order to solve the above technical problems, the present application provides application of TP receptor inhibitor in preparation of a drug for treating cardiac fibrosis, which provides a new treatment strategy for the treatment of cardiac fibrosis.

[0006] In order to achieve the purpose of the present application, the following technical solutions are adopted: The present application provides application of TP receptor inhibitor in preparation of a drug for treating cardiac fibrosis, wherein the TP receptor inhibitor is selected from SQ29548, NTP42 or a pharmaceutically acceptable salt or derivative thereof.

[0007] Further, the effective amount of the TP receptor inhibitor is 0.1 mg / kg body weight / day-20 mg / kg body weight / day.

[0008] Further, the effective amount of the TP receptor inhibitor is 0.5 mg / kg body weight / day-5 mg / kg body weight / day.

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

[0010] Further, the drug for treating cardiac fibrosis further includes any one of an AMPK activator, a TGF-β inhibitor or an anti-inflammatory drug.

[0011] Further, the TP receptor inhibitor is used for inhibiting the stability of Gli1 protein and / or promoting the ubiquitination degradation of Gli1 protein.

[0012] The application further provides a pharmaceutical composition for treating cardiac fibrosis, which comprises a TP receptor inhibitor and a pharmaceutically acceptable carrier or excipient.

[0013] Further, the pharmaceutical composition further comprises any one of an AMPK activator, a TGF-β inhibitor or an anti-inflammatory drug.

[0014] Further, the AMPK activator is any one of metformin or AICAR.

[0015] Further, the dosage form of the pharmaceutical composition is any one of oral preparation, injection or sustained-release preparation.

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

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

[0018] The application has the following technical effects: The application provides application of a TP receptor inhibitor in preparation of a drug for treating cardiac fibrosis, and has significant innovation and application potential. +The key role in cells and its establishment as a specific drug target for anti-fibrosis breaks through the limitations of the existing therapy targeting deficiency. By inhibiting the TP receptor, the AMPK signaling pathway can be effectively activated, and then the ubiquitination degradation of the key fibrosis-promoting factor Gli1 protein is promoted, so as to inhibit the generation and activation of myofibroblasts from the source, and precisely intervene in the fibrosis process. Animal experiments prove that this strategy can significantly improve heart function, reduce myocardial hypertrophy and inflammatory response, and effectively reduce collagen deposition. In addition, the TP receptor inhibitor (such as SQ29548, NTP42) provided by the application can be used in combination with the AMPK activator, showing a synergistic effect, laying a foundation for the development of high-efficiency and low-toxicity compound preparations. The scheme has a clear mechanism and strong targeting, and provides a new drug development direction and treatment selection for the clinical treatment of myocardial hypertrophy, heart failure and other various heart fibrosis-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0020] Figure 1 : Gli1 in the heart of a murine model of cardiac fibrosis + Co-expression results of TP, POSTN, a-SMA, Col1a1 and Gli1 in cells, wherein A is a UMAP plot of heart cell subgroups of healthy controls and patients with hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM); B is a gene expression bubble plot of different cell groups; C is the expression density of GLi1 in all heart cells; D is the expression density of ACTA2, POSTN, COL1A1, GLi1 and TP in heart fibroblasts; E is the Western blot analysis of Gli1 + protein level in cells; F is the Western blot analysis of Gli1 + protein level in cells; F is the Western blot analysis of Gli1 Figure 2 : Expression levels of Gli1 and TP between different cell lines, wherein A is the Western blot analysis of Gli1 +Gli1 protein expression levels in cells, LX-2 cells, NIH-3T3 cells and BMSCs; B is the COX-1, TxAS and TP gene expression levels in NIH-3T3 cells and BMSCs (n=3); Figure 3 : Knockout of TP gene in Gli1 + cells of the heart of the mouse by Cre-mediated gene recombination, wherein A is the genomic DNA extracted from the tail tissue, and the Gli1 CreERT2 Rosa-RFP / TP F / F mouse is genotyped; B is the Gli1 CreERT2 Rosa-RFP / TP F / F mouse heart Gli1 + expression levels in cells (n=5-6; Mann-Whitney U test; **P<0.01 vs. control group); Figure 4 : Gli1 + knockout of TP in Gli1 CreERT2 Rosa26-RFP / TP F / F and Gli1 CreERT2 Rosa26-RFP mouse heart PDGFR + quantitative analysis of cell number (n=5-6; two-way ANOVA with Tukey's multiple comparison test; ***P<0.001); B is the Gli1 CreERT2 Rosa26-RFP / TP F / F mouse and Gli1 CreERT2 Rosa26-RFP control mouse heart Gli1 + quantitative analysis of cell number (n=6; two-way ANOVA with Tukey's multiple comparison test; ***P<0.001) C is the Gli1 CreERT2 Rosa26-RFP / TP F / F mouse and its littermate control mouse heart tissue hematoxylin and eosin (HE) staining, blue scale bar=2mm, black scale bar=60μm; D is the Gli1 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. Use of a TP receptor inhibitor for the manufacture of a medicament for the treatment of cardiac fibrosis, characterized in that, The TP receptor inhibitor is selected from the group consisting of SQ29548, NTP42, or pharmaceutically acceptable salts or derivatives thereof.

2. Use of a TP receptor inhibitor according to claim 1 for the manufacture of a medicament for the treatment of cardiac fibrosis, characterized in that, The effective amount of the TP receptor inhibitor is 0.1 mg / kg body weight / day-20 mg / kg body weight / day.

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

4. Use of a TP receptor inhibitor according to claim 1 for the manufacture of a medicament for the treatment of cardiac fibrosis, characterized in that, The drug for treating cardiac fibrosis further includes any one of AMPK activator, TGF-β inhibitor, or anti-inflammatory drug.

5. Use of a TP receptor inhibitor according to claim 1 for the manufacture of a medicament for the treatment of cardiac fibrosis, characterized in that, The TP receptor inhibitor is used for inhibiting the stability of Gli1 protein and / or promoting the ubiquitination degradation of Gli1 protein.

6. A pharmaceutical composition for treating cardiac fibrosis, characterized by, The pharmaceutical composition includes TP receptor inhibitor and pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition for use in the treatment of cardiac fibrosis according to claim 6, characterized in that, The pharmaceutical composition further includes any one of AMPK activator, TGF-β inhibitor, or anti-inflammatory drug.

8. The pharmaceutical composition for use in the treatment of cardiac fibrosis according to claim 7, characterized in that, The AMPK activator is any one of metformin or AICAR.

9. The pharmaceutical composition of claim 6, wherein, The dosage form of the pharmaceutical composition is any one of oral preparation, injection, or sustained release preparation.

10. The pharmaceutical composition according to any one of claims 6-7, characterized in that, The mass ratio of the TP receptor inhibitor to the AMPK activator is (1:100)-(100:1).

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