Use of etomoxir in the preparation of a medicament for treating aortic dissection

Abstract

The application relates to application of Etomoxir in preparation of a drug for treating aortic dissection, and belongs to the technical field of biological medicine. It is found for the first time that Etomoxir has obvious treatment effect on aortic dissection, and it is verified that Etomoxir can reduce the risk of aortic dissection. In a BAPN model group, the incidence of aortic dissection is 71.43%, and after Etomoxir intervention, the incidence of aortic dissection is reduced to 42.86%, the incidence of dissection is greatly reduced, the defects of the current clinical specific anti-aortic dissection small molecule drug are made up, and the current situation that only surgery treatment is relied on is improved. It is found that Etomoxir can significantly up-regulate the expression levels of contraction markers Acta2 and Tagln in vascular smooth muscle cells, and down-regulate the expression level of matrix degradation marker Mmp2 in vascular smooth muscle cells, so that the aortic media destruction is prevented from the cell level, and a new method, a new idea and a new view are provided for the clinical treatment of aortic dissection.

Description

Technical Field

[0001] This invention relates to the application of Etomoxir in the preparation of drugs for treating aortic dissection, and belongs to the field of biomedical technology. Background Technology

[0002] Aortic dissection (AD) is a life-threatening condition typically caused by a tear in the aortic intima or intramural hemorrhage, leading to the separation of the intima, media, and adventitia of the aortic wall, forming a dissection. As blood continues to flow into the aortic wall, the intimal flap can extend proximally and distally from the initial tear or hemorrhage site, potentially affecting collateral arteries. The first two weeks after the onset of aortic dissection are considered the acute phase, during which patients are highly susceptible to life-threatening complications and death. Treatment methods for aortic dissection mainly include drug therapy, interventional therapy, and surgical treatment. Although these treatments have made significant progress, the high difficulty of surgery, postoperative multi-organ dysfunction, and coagulation disorders remain complications that cannot be completely avoided. Furthermore, postoperative vascular degeneration can lead to further degenerative changes, increasing the risk of secondary surgery and rupture. Therefore, preventative and therapeutic drugs for patients with acute aortic dissection remain a hot topic in clinical research.

[0003] The latest research shows that the transformation of vascular smooth muscle cells (VSMCs) from contractile to synthetic phenotype is one of the core pathological pathways in the development of aortic dissection. That is, the phenotype transformation of VSMCs leads to abnormal proliferation and migration of VSMCs, and the secretion of large amounts of matrix metalloproteinases that degrade elastic fibers, resulting in aortic media remodeling and increased fragility of the vessel wall.

[0004] Carnitine palmitoyltransferase 1A (CPT1A) is a key rate-limiting enzyme in the outer mitochondrial membrane, regulating fatty acid oxidation metabolism and mediating lysine succinylation modification, thus regulating mitochondrial homeostasis and cellular metabolic reprogramming. Etomoxir is an irreversible small-molecule inhibitor of CPT1A, initially studied as a treatment for diabetes. Other studies have used it only in disease models such as heart failure, myocardial ischemia, tumors, and atherosclerosis to inhibit fatty acid oxidation and regulate metabolic disorders. There are no reports of targeting etomoxir for aortic dissection. Therefore, exploring the potential application of etomoxir in the prevention and treatment of aortic dissection has significant scientific and social value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of Etomoxir in the preparation of drugs for treating aortic dissection.

[0006] The technical solution of the present invention is as follows: Application of Etomoxir in the preparation of drugs for the prevention and / or treatment of aortic dissection.

[0007] According to a preferred embodiment of the present invention, the application includes: (1) To prepare drugs that reduce the incidence and mortality of aortic dissection; (2) Prepare drugs that restore the contractile phenotype of vascular smooth muscle cells.

[0008] A drug for the prevention and / or treatment of aortic dissection, wherein the active ingredient of the drug includes Etomoxir.

[0009] According to a preferred embodiment of the present invention, Etomoxir is the sole active ingredient in the drug.

[0010] According to a preferred embodiment of the invention, the drug further includes pharmaceutically acceptable excipients.

[0011] According to a preferred embodiment of the present invention, the excipients include at least one of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, or buffer.

[0012] According to a preferred embodiment of the present invention, the dosage form of the drug includes at least one of the following: suspension, granules, capsules, powders, tablets, pills, injections, suppositories, aerosols, or drops.

[0013] The beneficial effects of this invention are as follows: 1. This invention is the first to discover that Etomoxir has a significant therapeutic effect on aortic dissection, and verification experiments have confirmed that Etomoxir can reduce the risk of aortic dissection. In the BAPN model group, the incidence of aortic dissection was 71.43%, and after Etomoxir intervention, the incidence of aortic dissection decreased to 42.86%, significantly reducing the incidence of dissection. This addresses the current clinical deficiency of having no specific small molecule drugs for treating aortic dissection, which necessitates surgical treatment alone.

[0014] 2. The use of Etomoxir, which targets aortic vascular smooth muscle cells, in the prevention and / or treatment of aortic dissection is disclosed for the first time in this invention. It has been found that Etomoxir can significantly upregulate the expression levels of contractile markers Acta2 and Tagln in vascular smooth muscle cells and downregulate the expression level of matrix degradation marker Mmp2 in vascular smooth muscle cells, thereby preventing aortic media destruction at the cellular level. This provides a new method, new ideas, and new perspectives for the clinical treatment of aortic dissection.

[0015] 3. The Etomoxir used in this invention is a commercially available and mature small molecule inhibitor that can be stably procured from the market. Both the injection and tablet formulations use conventional pharmaceutical excipients and general pharmaceutical processes, eliminating the need for complex gene vectors, protein preparations, and other high-cost raw materials. This results in low production barriers and convenient storage and transportation. Compared with gene therapy and peptide drugs, it is easier to industrialize and provides new therapeutic drugs for clinical use, demonstrating good clinical application value and broad application prospects. Attached Figure Description

[0016] Figure 1 shows the in vitro downregulation of Cpt1a expression levels in smooth muscle cells by Etomoxir; In the figure, A shows the expression level of Cpt1a mRNA in VSMC detected by RT-PCR; B shows the expression level of Cpt1a protein in VSMC detected by Western blot; and C shows the expression level of Cpt1a protein in VSMC detected by immunofluorescence. The blue fluorescence represents DAPI, and the red fluorescence represents Cpt1a.

[0017] Figure 2 shows the effect of Etomoxir on reversing the smooth muscle cell phenotype; In the figure, A shows the expression levels of Acta2 mRNA, Tagln mRNA, and Mmp2 mRNA in VSMC detected by RT-PCR; B shows the expression levels of α-Sma protein, Tagln protein, and Mmp2 protein in VSMC detected by Western blot; C shows the expression level of Tagln protein in VSMC detected by immunofluorescence, with blue fluorescence representing DAPI and red fluorescence representing Tagln.

[0018] Figure 3 is a flowchart of the construction and subsequent grouping of aortic dissection model mice.

[0019] Figure 4 This study aimed to demonstrate in vivo, whole-animal levels, that Etomoxir reduces the incidence of aortic dissection, repairs aortic medial injury, inhibits aortic Cpt1a expression, and affects the expression of phenotypic transformation-related markers. In the figure, A is a statistical graph showing the changes in body weight of mice in different groups during the modeling period; B is a statistical graph showing the incidence of aortic dissection in mice in different groups; C is a representative image and statistical graph showing the diameter of the aortic arch in mice in different groups measured by ultrasound; D is the results of EVG staining, HE staining and Masson staining in mice in different groups; E is the expression level of Cpt1a mRNA and Cpt1a protein in the aortic tissue of mice in the BAPN model group and BAPN+Etomoxir group detected by RT-PCR and Western blot; F is the expression level of Mmp2, α-Sma and Tagln mRNA and protein in the aortic tissue of mice in the BAPN model group and BAPN+Etomoxir group detected by RT-PCR and Western blot; G is the expression level of α-Sma protein in the aortic tissue of mice in the BAPN drinking water group detected by immunofluorescence, with blue fluorescence representing DAPI and red fluorescence representing α-Sma. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental operations involved in the embodiments are performed according to conventional procedures; the drugs and reagents involved are all commercially available products unless otherwise specified. Unless otherwise specified, all percentages in the embodiments are mass percentages.

[0021] Etomoxir, molecular formula C 14 H 21 ClO4, CAS No. 124028-86-0; Physicochemical properties: white powder, lipid-soluble, soluble in DMSO, ethanol, and sterile injection solvents; Biological characteristics: specifically binds to the mitochondrial outer membrane Cpt1a protein, irreversibly blocking its enzyme activity. Catalog No. HY-50202, MedChemExpress.

[0022] Sulfobutyl ether-β-cyclodextrin (SBE-β-CD), CAS No. 182410-00-0. Product No. HY-17031, MedChemExpress.

[0023] Etomoxir stock solution: Add 200 mg of Etomoxir to 6.06 mL of dimethyl sulfoxide and stir to mix evenly to obtain Etomoxir stock solution.

[0024] Preparation of Etomoxir injection: 2 g of SBE-β-CD was added to 10 mL of 0.9% saline solution and ultrasonically mixed to obtain SBE-β-CD solution; then, the two were mixed evenly according to the volume ratio of Etomoxir stock solution to SBE-β-CD solution = 1:9, sterilized, and Etomoxir injection was obtained and stored at 20℃ protected from light.

[0025] Example 1: In vitro cell experiment 1. Experimental materials: Primary aortic vascular smooth muscle cells from 4-week-old C57BL / 6J mice; 100 μmol / L Etomoxir solution; DMSO solvent control. The 100 μmol / L Etomoxir solution was obtained by diluting the Etomoxir stock solution.

[0026] 2. Through in vitro cell experiments, we verified that Etomoxir inhibits the expression of Cpt1a in primary mouse aortic vascular smooth muscle cells and reverses the phenotypic transformation of vascular smooth muscle cells. The specific steps are as follows: (1) The aorta of 4-week-old C57BL / 6J mice was isolated and obtained. Primary aortic vascular smooth muscle cells (VSMCs) were extracted from mouse vascular smooth muscle cells digestion solution. The primary aortic vascular smooth muscle cells were then seeded into DMEM medium containing 20% ​​fetal bovine serum and cultured until the cell confluence reached 70%. (2) The aortic vascular smooth muscle cells obtained in step (1) were divided into an experimental group and a control group. The experimental group was given Etomoxir solution to make the final concentration of Etomoxir 100 μmol / L, and the control group was given an equal volume of DMSO. The cells were cultured for 48 h. (3) Collect the cells obtained in step (2) and perform RT-PCR, Western blot, and immunofluorescence experiments according to conventional techniques to detect the expression levels of Cpt1a and vascular smooth muscle phenotypic markers in the cells. The results are as follows: Figures 1-2 As shown; Among them, vascular smooth muscle phenotypic markers include the contractile markers actin Acta2 (α-SMA) and collagen transfer protein Tagln, and the synthetic marker matrix metalloproteinase 2 (Mmp2). RT-PCR was performed using Gapdh as an internal control, and the primers used are shown below: Cpt1a-F: 5'-CTCCGCCTGAGCCATGAAG-3'; Cpt1a-R:5'-CACCAGTGATGATGCCATTCT-3' Acta2-F: 5'-CCCAGACATCAGGGAGTAATGG-3'; Acta2-R: 5'-TCTATCGGATACTTCAGCGTCA-3' Tagln-F: 5'-CCAACAAGGGTCCATCCTACG-3'; Tagln-R:5'-ATCTGGGCGGCCTACATCA-3' Mmp2-F: 5'-GCATCTGCGTTGCATTCTGG-3'; Mmp2-R: 5'-CACTCGATTGTTGTCTGATCCA-3'; Gapdh-F: 5'-AGGTCGGTGTGAACGGATTTG-3'; Gapdh-R: 5'-GGGGTCGTTGATGGCAACA-3' Western blot analysis using β-actin as an internal control: The antibody for detecting Cpt1a is available from Boster (Cat#A00917-3); the antibody for detecting α-SMA is available from Cell Signaling Technology (Cat#19245S); the antibody for detecting MMP2 is available from Proteintech (Cat#10373-2-AP); the antibody for detecting TAGLN is available from Abcam (Cat#ab14106); and the antibody for detecting β-actin is available from Cell Signaling Technology (Cat#3700S).

[0027] Depend on Figure 1 It can be seen that, compared with the control group, the expression levels of Cpt1a mRNA and Cpt1a protein in the experimental group treated with Etomoxir were significantly downregulated, indicating that Etomoxir can effectively inhibit the expression of Cpt1a mRNA and Cpt1a protein in vascular smooth muscle cells.

[0028] Depend on Figure 2 It can be seen that, compared with the control group, the expression levels of contractile markers Acta2 and Tagln in the experimental group treated with Etomoxir were significantly upregulated, while the expression level of synthetic marker Mmp2 was significantly downregulated. This indicates that Etomoxir can effectively promote the expression of Acta2 and Tagln in vascular smooth muscle cells and inhibit the expression of Mmp2.

[0029] In summary, the results show that 100 μmol / L Etomoxir can significantly inhibit the expression of Cpt1a mRNA and Cpt1a protein in vascular smooth muscle cells, thus blocking the transformation of vascular smooth muscle cells into synthetic cells.

[0030] Example 2: In vitro mouse experiment 1. Grouping and Fixed-Point Parameters like Figure 3 As shown, 42 four-week-old male C57BL / 6J mice were divided into a blank control group (Control + Vehicle), a drug-only group (Control + Etomoxir), a BAPN model group (BAPN + Vehicle), and a BAPN + Etomoxir group (BAPN + Etomoxir). The specific treatments are as follows: ① Blank control group (n=7): Normal water intake, daily intraperitoneal injection of physiological saline; ② Single-drug group (n=7): Normal drinking water, Etomoxir injection 15 mg / kg / day intraperitoneally; ③BAPN model group (n=14): BAPN-containing drinking water was administered at a concentration of 1 g / kg / day, while physiological saline was injected intraperitoneally for 4 consecutive weeks. ④BAPN+Etomoxir group (n=14): BAPN-containing drinking water was administered at a concentration of 1 g / kg / day, while Etomoxir was administered intraperitoneally at a concentration of 15 mg / kg / day for 4 consecutive weeks.

[0031] 3. Indicator Testing The body weight of mice in each group was monitored over 4 weeks, and the results were as follows: Figure 4 As shown in Figure A.

[0032] After 4 weeks of culture, mice in each group were administered sodium pentobarbital intraperitoneally at a dose of 150 mg / kg. The aorta was dissected from the body 10 minutes after the initial effect, and the incidence of aortic dissection was calculated based on the aortic width observed under a microscope. The results are as follows: Figure 4 As shown in B.

[0033] After 4 weeks of culture, mice were lightly anesthetized, and the diameter of the aortic arch in each group of mice was measured and recorded using the Vevo 3100LT high-frequency ultrasound imaging system. The results are as follows: Figure 4 As shown in C.

[0034] After 4 weeks of culture, mice in each group were administered sodium pentobarbital intraperitoneally at a dose of 150 mg / kg. After 10 minutes of induction, the mouse aorta was dissected from the body, and the aortic tissue was paraffin-embedded to create the model. EVG staining, HE staining, and Masson staining were then performed. The results are as follows: Figure 4 As shown in D.

[0035] After 4 weeks of culture, mice in the BAPN model group and the BAPN+Etomoxir group were administered sodium pentobarbital intraperitoneally at a dose of 150 mg / kg. After 10 minutes of induction, the aorta of the mice was dissected, and RNA and protein were extracted from the aorta using the TriZol method. The expression levels of Cpt1a mRNA and Cpt1a protein in the BAPN model group and the BAPN+Etomoxir group were then detected. The results are as follows: Figure 4 As shown in E.

[0036] After 4 weeks of culture, mice in the BAPN model group and the BAPN+Etomoxir group were administered sodium pentobarbital intraperitoneally at a dose of 150 mg / kg. After 10 minutes of induction, the aorta of the mice was dissected, and RNA and protein were extracted from the aorta using the TriZol method. The expression levels of mRNA and protein of Mmp2, α-Sma, and Tagln in the BAPN model group and the BAPN+Etomoxir group were detected. The results are as follows: Figure 4 As shown in F.

[0037] After 4 weeks of culture, mice in the BAPN model group and the BAPN+Etomoxir group were administered sodium pentobarbital intraperitoneally at a dose of 150 mg / kg. After 10 minutes of onset, the aorta of the mice was dissected from the body, and the aortic tissue of the model mice was embedded in paraffin. Immunofluorescence detection was then performed, and the results are as follows: Figure 4 As shown in G.

[0038] 3. Experimental Results Depend on Figure 4 As shown in A, there was no significant difference in body weight among the groups of mice.

[0039] Depend on Figure 4 B shows that the incidence of aortic dissection in the BAPN model group was 71.43% (10 / 14), while the incidence of aortic dissection in the BAPN+Etomoxir group decreased to 42.86% (6 / 14).

[0040] Depend on Figure 4 As shown in C, compared with the BAPN model group, the degree of aortic dilation was significantly reduced in the BAPN+Etomoxir group.

[0041] Depend on Figure 4As shown in D, compared with the BAPN model group, the pathological staining of the BAPN+Etomoxir group showed a significant reduction in elastic fiber breakage.

[0042] Depend on Figure 4 E indicates that, compared to the BAPN model group, the expression of Cpt1a in the aortic tissue of the BAPN+Etomoxir group was significantly downregulated.

[0043] Depend on Figure 4 As shown in F, compared with the BAPN model group, the BAPN+Etomoxir group showed increased levels of α-Sma and Tagln, markers of contractile smooth muscle in aortic tissue, and decreased levels of Mmp2, marker of synthetic smooth muscle.

[0044] Depend on Figure 4 G indicates that, compared to the BAPN model group, the BAPN+Etomoxir group showed an increase in the aortic tissue systolic smooth muscle marker Tagln.

[0045] In summary, the results indicate that intraperitoneal injection of 15 mg / kg / day etomoxir effectively alleviates BAPN-induced aortic dissection. Etomoxir demonstrates a significant therapeutic effect on aortic dissection, substantially reducing its incidence and addressing the current lack of specific small-molecule anti-aortic dissection drugs, which previously relied solely on surgical treatment. Furthermore, etomoxir significantly upregulates the expression levels of contractile markers Acta2 and Tagln in vascular smooth muscle cells and downregulates the expression level of Mmp2, a pro-matrix degradation marker, thus preventing aortic media destruction at the cellular level. This provides a new method, new ideas, and new perspectives for the clinical treatment of aortic dissection.

[0046] The above description is only a preferred embodiment of this patent. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of this patent, and these improvements and substitutions should also be considered within the scope of protection of this patent.

Claims

1. The use of Etomoxir in the preparation of drugs for the prevention and / or treatment of aortic dissection.

2. The application as described in claim 1, characterized in that, The applications include: (1) To prepare drugs that reduce the incidence and mortality of aortic dissection; (2) Prepare drugs that restore the contractile phenotype of vascular smooth muscle cells.

3. A drug for the prevention and / or treatment of aortic dissection, characterized in that, The active ingredient of the drug includes Etomoxir.

4. The drug as described in claim 3, characterized in that, Etomoxir is the only active ingredient in the drug.

5. The drug as described in claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The drug as described in claim 5, characterized in that, The excipients include at least one of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, or buffer.

7. The drug as described in claim 3, characterized in that, The dosage form of the drug includes at least one of the following: suspension, granules, capsules, powders, tablets, pills, injections, suppositories, aerosols, or drops.

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