Application of WZ3146 in preparation of product for preventing and / or treating pulmonary arterial hypertension and complications thereof

Abstract

The invention belongs to the technical field of medicines, and discloses application of WZ3146 in preparation of products for preventing and / or treating pulmonary arterial hypertension and complications thereof. The WZ3146 and the pharmaceutically acceptable salt thereof have a remarkable treatment effect on pulmonary arterial hypertension and complications thereof, such as mean pulmonary arterial pressure, high right ventricular systolic pressure, right heart enlargement, right heart dysfunction, pulmonary vascular remodeling, pulmonary arterial intima thickness, increase of vascular myelization, pulmonary artery smooth muscle cell proliferation and migration and the like; the effects of preventing and / or treating pulmonary arterial hypertension comprise expanding the right heart, improving the pulmonary arterial intima thickness and the vascular myelization level, reversing pulmonary vascular remodeling and inhibiting pulmonary artery smooth muscle cell proliferation and migration.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a method for preparing a medicine for preventing and / or treating pulmonary hypertension. Background Art

[0002] Pulmonary arterial hypertension (PH) is a disease characterized by a progressive increase in pulmonary arterial pressure. Its clinical diagnostic criteria are a resting mean pulmonary artery pressure (mPAP) ≥ 20 mmHg, as measured by right cardiac catheterization. The incidence of PH has been increasing in recent years, and it is a disease with high disability and mortality rates. Unlike cardiovascular diseases such as hypertension, PH has a complex pathogenesis. Drugs commonly used to treat hypertension cannot be used to lower PH pressure, and existing treatments do not fully meet clinical needs. PH is associated with a poor prognosis and an extremely low 5-year survival rate, making it a serious disease involving both the cardiovascular and respiratory systems.

[0003] When pulmonary hypertension develops, patients experience symptoms such as dyspnea, palpitations, chest pain, hemoptysis, and syncope. In late stages, they may develop hypoxia, right ventricular hypertrophy, and even right ventricular failure. In severe cases, death may occur. Currently, there is a severe lack of treatment options and medications for this disease, and the prognosis is poor. Only 20% of patients can be effectively controlled, and most patients may die within 2-3 years of diagnosis. Currently, bosentan is the first-line drug for the clinical treatment of pulmonary hypertension, but its therapeutic effect is unsatisfactory. Developing new drugs that can effectively treat pulmonary hypertension is a very important and urgent task.

[0004] Currently, clinical treatments for pulmonary hypertension mainly include vasodilators, endothelin receptor antagonists, prostacyclins, phosphodiesterase-5 inhibitors, and icariin (CN102247398A). Although these drugs can relieve symptoms and delay disease progression to a certain extent, they often have many limitations. For example, vasodilators may cause serious adverse reactions such as a sudden drop in blood pressure; endothelin receptor antagonists may develop drug resistance after long-term use, and some patients do not respond well to them; prostacyclins need to be administered through special routes (such as intravenous injection, subcutaneous injection, etc.), which brings inconvenience to patients and also has certain risks such as bleeding; phosphodiesterase-5 inhibitors may cause fatal hypotension in certain circumstances (such as when used in combination with certain nitrates) and have limited therapeutic effects on patients with advanced pulmonary hypertension.

[0005] In the process of exploring new treatments, people are constantly searching for compounds with superior efficacy, greater safety, and more convenient administration methods. As an emerging compound, WZ3146 has attracted the attention of researchers due to its unique chemical structure and potential biological activity. Studies have shown that WZ3146 has certain therapeutic potential in other related disease areas, such as anti-inflammatory, antioxidant, and regulation of cell proliferation and apoptosis. However, there are currently no reports on the use of WZ3146 in the prevention and / or treatment of pulmonary arterial hypertension and its complications. Summary of the Invention

[0006] In response to the above technical problems, the present invention proposes the use of WZ3146 in the preparation of products for preventing and / or treating pulmonary hypertension and its complications.

[0007] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0008] Use of WZ3146 or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and / or treating pulmonary hypertension and its complications; wherein the structural formula of WZ3146 is as follows:

[0009] Furthermore, the above-mentioned pharmaceutically acceptable salts are salts prepared from pharmaceutically acceptable non-toxic bases or acids.

[0010] The pharmaceutically acceptable salts are salts formed with the following acids: one or more of phosphoric acid, carbonic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, succinic acid, tartaric acid and p-toluenesulfonic acid.

[0011] The above-mentioned pulmonary hypertension and its complications have one or more of the following indications: increased mean pulmonary artery pressure and right ventricular systolic pressure, right heart enlargement and right heart dysfunction, pulmonary vascular remodeling, increased pulmonary arteriolar media thickness and vascular muscularization, and pulmonary artery smooth muscle cell proliferation and migration.

[0012] The above products include medicines, food or health products

[0013] The above-mentioned drugs are administered by enteral or parenteral routes, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum.

[0014] The above products are intended for human beings or other mammals.

[0015] A pharmaceutical composition for preventing and / or treating pulmonary hypertension and its complications, comprising WZ3146 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0016] The pharmaceutical composition is in the form of a solution, suspension, freeze-dried powder injection, emulsion, pill, capsule, powder, controlled release, sustained release preparation and microsome delivery system.

[0017] In order to prepare unit dosage forms into tablets, a wide variety of carriers well known in the art can be used. In the carrier: diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, methylcellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0018] To prepare the dosing unit into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, gelucire, kaolin, and talc; binders, such as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants, such as agar powder, dry starch, alginate, sodium lauryl sulfate, methylcellulose, and ethylcellulose.

[0019] In order to prepare the administration unit into a suppository, various carriers known in the art can be widely used, such as polyethylene glycol, lecithin, cocoa butter, higher alcohols, enzymes of higher alcohols, gelatin, semi-synthetic glycerol enzymes, etc.

[0020] To prepare a dosing unit as a capsule, the active ingredient is mixed with the various carriers described above, and the resulting mixture is placed in a hard gelatin capsule or soft capsule. Alternatively, the active ingredient can be formulated into microcapsules and suspended in an aqueous medium to form a suspension, which can then be encapsulated in a hard capsule or formulated as an injection.

[0021] For example, the composition of the present invention is prepared into an injectable preparation, such as a solution, suspension solution, emulsion, or freeze-dried powder injection. This preparation can be aqueous or non-aqueous and can contain one or more pharmacologically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, or dispersants. For example, the diluent can be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitol fatty acid enzyme, and the like. In addition, to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injectable preparation. In addition, conventional cosolvents, buffers, pH adjusters, and the like can also be added. These excipients are commonly used in the art.

[0022] Furthermore, if necessary, colorants, preservatives, perfumes, flavorings, sweeteners or other materials may be added to the pharmaceutical preparations.

[0023] The dosage of the pharmaceutical compositions of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, and the number of doses. Therefore, the therapeutic dose of the present invention can vary widely. The amount of the effective drug in the final formulation of the pharmaceutical composition of the present invention can be appropriately adjusted to achieve a therapeutically effective dose. For example, the effective daily dose of WZ3146 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.1-10 mg / kg body weight.

[0024] The beneficial effects produced by the present invention are:

[0025] This invention proposes for the first time the use of WZ3146 and its pharmaceutically acceptable salts for the prevention and / or treatment of pulmonary hypertension. Research results in this invention demonstrate that, in experiments with male rats, WZ3146 can effectively reduce mean pulmonary artery pressure and pulmonary artery systolic pressure, improving hemodynamics; lower right ventricular hypertrophy index and improve right ventricular function; reduce pulmonary arteriolar media thickness and vascular muscularization, improve pulmonary vascular remodeling; and enhance activity tolerance and daily activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1Effects of WZ3146 on the viability of hPASMCs / rPASMCs induced with 5% FBS. (A) WZ3146 structure; (B) Effect of WZ3146 on the viability of hPASMCs induced with 5% FBS; (C) Effect of WZ3146 on the viability of rPASMCs induced with 5% FBS. n = 6, all data are mean ± SEM; **P < 0.01, ***P < 0.001.

[0028] Figure 2 Effects of WZ3146 on the phenotype of hPASMCs induced by 5% FBS. (A) (D) Wound wound assay results and quantitative analysis; (B) (E) Transwell assay results and quantitative analysis; (C) (F) EdU assay results and quantitative analysis. All values are expressed as mean ± SEM. n = 6. ns, no statistically significant difference; ***P < 0.001.

[0029] Figure 3 Effects of WZ3146 on the PDGF-BB-induced hPASMC phenotype. (A) (D) Wound wound assay results and quantitative analysis; (B) (E) Transwell assay results and quantitative analysis; (C) (F) EdU assay results and quantitative analysis. All values are expressed as mean ± SEM. n = 6. ns, no statistically significant difference; ***P < 0.001.

[0030] Figure 4 Effects of WZ3146 on the phenotype of rPASMCs induced by 5% FBS. (A) (D) Wound wound assay results and quantitative analysis; (B) (E) Transwell assay results and quantitative analysis; (C) (F) EdU assay results and quantitative analysis. All values are expressed as mean ± standard error (SEM). n = 6. ns, no statistically significant difference; ***P < 0.001.

[0031] Figure 5 Effects of WZ3146 on the phenotype of rPASMCs induced by PDGF-BB. (A) (D) Wound wound assay results and quantitative analysis; (B) (E) Transwell assay results and quantitative analysis; (C) (F) EdU assay results and quantitative analysis. All values are expressed as mean ± SEM. n = 6. ns, no statistically significant difference; ***P < 0.001.

[0032] Figure 6WZ3146 improves hemodynamic parameters in MCT-induced PAH rats. (A) Representative image of RVSP in experimental rats; (B) RVSP data analysis; (C) mPAP data analysis; (D) Fulton index analysis, n = 10. All data are expressed as mean ± SEM, n.s., indicating no statistically significant differences; *P < 0.05; ***P < 0.001.

[0033] Figure 7 WZ3146 improves hemodynamic parameters in SuHx-induced PAH mice. (A) Representative images of RVSP in experimental mice; (B) RVSP data analysis; (C) Fulton index analysis. n = 10, all data are mean ± SEM, n.s., no statistically significant differences; ***P < 0.001.

[0034] Figure 8 WZ3146 ameliorates pulmonary vascular remodeling in MCT-induced PAH rats. (A) Representative images of H&E staining, EVG, and immunofluorescence staining in MCT-induced PAH rats; (B) Medial thickness analysis, n = 6. (C) Quantitative analysis of vascular muscularization ratio, n = 4. All data are expressed as mean ± SEM, n.s., with no statistically significant differences; **P < 0.01, ***P < 0.001.

[0035] Figure 9 WZ3146 ameliorates pulmonary vascular remodeling in SuHx-induced PAH mice. (A) Representative images of H&E staining, EVG, and immunofluorescence staining in SuHx-induced PAH mice; (B) Medial thickness analysis, n = 6. (C) Quantitative analysis of vascular muscularization ratio, n = 4. All data are expressed as mean ± SEM, n.s., with no statistically significant differences; **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0037] The SPF-grade male Sprague-Dawley (SD) rats of 6-7 weeks old used in the examples of the present invention were purchased from Beijing Sprague-Dawley Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2024-0001, animal quality certificate number: No. 110324241105443173. All experimental rats were kept in a barrier environment, and the indoor temperature was kept constant at 25°C and the relative humidity was 55%. Rats had free access to regular feed and drinking water, and the breeding environment adopted a 12-hour light and 12-hour dark alternating cycle. The animal experiments involved in this study strictly followed the principles of experimental animal protection of Henan University, met the ethical requirements of medical experimental animals, and were approved by the Ethics Committee of Henan University (approval number).

[0038] No. HUSOM-2024-379, April 15, 2024).

[0039] SPF-grade male C57BL / 6J mice, 6–7 weeks old, were purchased from Beijing Sibeifu Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2019-0010, and animal quality certificate number: No. 11032424110166185.

[0040] The experimental instruments used in the embodiments of the present invention are shown in Table 1:

[0041] Table 1 Experimental instruments

[0042]

[0043] The experimental reagents used in the examples of the present invention are shown in Table 2:

[0044] Table 2 Experimental reagents

[0045]

[0046] The method for isolating and obtaining rat PASMCs in the following examples is as follows:

[0047] Six to seven-week-old SD rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg), and their skin was disinfected with 75% alcohol. After being sacrificed, the thorax was opened, the heart and lungs were removed, and the pulmonary artery was carefully cut from the right heart and lungs. The pulmonary artery was placed in sterile PBS and gently rinsed to completely remove any remaining blood. After flushing, transfer the pulmonary artery to another sterile culture dish containing PBS; carefully remove the fat and connective tissue adhering to the blood vessel in the culture dish containing PBS until a complete white blood vessel is visible to the naked eye, and then transfer it to another sterile PBS culture dish; spray the culture dish with 75% ethanol for disinfection and quickly transfer it to a clean bench; use sterile tweezers to transfer the blood vessel to a culture dish containing sterile PBS prepared in advance on the workbench and rinse it repeatedly to ensure that impurities and other attachments remaining on the surface of the blood vessel are removed; remove the rinsed blood vessel with tweezers and place it on the side wall of a new sterile culture dish, ensuring that the blood vessel is in a tilted state so that excess PBS can flow naturally and dry; use scissors to cut the blood vessel into pieces, avoiding excessive squeezing or damaging the blood vessel tissue during the operation, and then use tweezers to divide it into small pieces and spread them flat on the bottom of the culture dish; let it stand at room temperature until the moisture around the tissue evaporates naturally and sticks to the bottom of the culture dish, then slowly add 10mL of DMEM culture medium containing 20% FBS and place it in an incubator for continued culture. After 3-4 days, fresh culture medium is replaced and subculture is performed after the cells crawl out from around the tissue block.

[0048] Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from Shanghai Saibaikang Biotechnology Co., Ltd. (the hPASMCs used in this study were no more than passage 8).

[0049] Example 1: Effect of WZ3146 on the viability of PASMCs induced by 5% FBS

[0050] Purchased hPASMCs or cultured rat PASMCs (rPASMCs) suspension were inoculated into 96-well plates, 8×10 3 Cells were cultured at 37°C, 5% CO₂ in a 37°C, 5% CO₂ incubator. After reaching 70-80% confluence, serum was withdrawn and cells were starved for 24 hours to synchronize. Cells were then pretreated with chlorpheniramine WZ3146 (1, 3, and 10 μmol / L) or corresponding controls for 1 hour. 5% FBS and corresponding controls were then added for 24 hours. PASMC viability was assessed using the CCK-8 assay.

[0051] Data analysis was performed using GraphPad Prism 8 software. All experimental data were presented as mean ± standard error (SEM) to intuitively demonstrate the central tendency and dispersion of the data. For experimental data with three or more groups, one-way ANOVA analysis of variance was used to analyze the data to explore whether the differences between the groups were statistically significant. After confirming the existence of inter-group differences, the Kruskal-Wallis multiple comparison test was used to further determine the specific differences between any two groups. If the P value obtained by the analysis was less than 0.05, the difference between the groups was considered statistically significant.

[0052] The optimal in vitro doses screened by CCK-8 assay in hPASMCs and rPASMCs were 1 μmol / L and 3 μmol / L, respectively. At these two concentrations, WZ3146 could significantly inhibit the malignant proliferation of PASMCs induced by 5% FBS. At the same time, the cytotoxicity test results showed that the drug had no significant toxic effect on cells at these concentrations ( Figure 1 ).

[0053] Example 2: Effect of WZ3146 on the phenotype of PASMCs induced by 5% FBS / PDGF-BB

[0054] The cell scratch test and Transwell assay were used to investigate the migration ability of PASMCs cells. The specific process is as follows.

[0055] Transwell assay: Starved cells (24 h in DMEM supplemented with 0.5% FBS) were collected into a centrifuge tube and centrifuged at 800 rpm for 5 min. Resuspend the cells in 1 mL of culture medium. Count the cells using a counting chamber. Seed 200 μL of a suspension of PASMCs (at a cell density of 5 × 10⁴ / well) pretreated with WZ3146 (1 or 3 μmol / L), 5% FBS, or PDGF-BB (20 ng / mL), or a control (pretreated) into the upper transwell insert. Add 600 μL of DMEM supplemented with 5% FBS to the lower insert, and incubate the plate at 37°C, 5% CO₂ for 24 h. Subsequently, a cotton swab was used to remove excess cells in the upper embedded chamber, and the lower layer of the chamber was fixed with 4% paraformaldehyde for 30 minutes and washed twice with PBS. Then, the cells on the lower surface of the chamber were stained with 0.1% crystal violet solution for 30 minutes and washed twice with PBS. The chamber was naturally air-dried at room temperature overnight. The next day, photos were taken under an inverted microscope and the migration rate of PASMCs in each treatment group was calculated.

[0056] Scratch test: hPASMCs / rPASMCs suspension was seeded into 6-well plates, 5×10 5Cells were cultured at 37°C in a 5% CO2 incubator. When cell confluence reached 70-80%, serum was withdrawn and the cells were starved for 24 hours to synchronize them. wz3146 (1 or 3 μmol / L) or corresponding controls were added for 1 hour. Cells were scratched with a 200 μL pipette tip, and photographs were taken at 0 hours. Cells were then induced with 5% FBS or PDGF-BB for 24 hours, and photographs were taken at 24 and 48 hours.

[0057] EdU method was used to detect the proliferation of PASMCs: rPASMCs / hPASMCs cell suspension was inoculated into 96-well plates, and 8×10 3 Cells were cultured at 37°C in a 5% CO2 incubator. When cell confluence reached 70-80%, serum was withdrawn and the cells were starved for 24 hours to synchronize them. WZ3146 (1 or 3 μmol / L) or corresponding controls were added for pretreatment for 1 hour. Then, 5% FBS or PDGF-BB (20 ng / mL) or corresponding controls were added for 24 hours.

[0058] After treatment, dilute the EdU solution (reagent A) with complete cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50μM EdU culture medium and continue incubating the cells for 4 hours. Discard the culture medium and wash with PBS 1-2 times, 5 minutes each time; add 50μL of 4% paraformaldehyde to each well and incubate at room temperature for 30 minutes, then discard the fixative; add 50μL of 2mg / mL glycine and incubate on a decolorizing shaker for 5 minutes, then discard the glycine solution and add 100μL PBS, decolorize and wash on a shaker for 5 minutes, discard PBS; add 100 μL of permeabilization agent (0.5% TritonX-100 in PBS) and incubate on a decolorization shaker for 10 minutes, wash once with PBS for 5 minutes; add 100 μL of 1×Apollo staining reaction solution, incubate in the dark at room temperature on a decolorization shaker for 30 minutes, and discard the staining reaction solution; add 100 μL of permeabilization agent (0.5% TritonX-100 in PBS) and decolorize and wash on a shaker 2-3 times, each time for 10 minutes, discard the permeabilization agent, and wash with PBS for 5 minutes; dilute F at a ratio of 100:1, prepare an appropriate amount of 1×Hoechst, 100 μL per well, incubate in the dark for 30 minutes, wash once with PBS for 5 minutes; add 100 μL of PBS, take pictures using a fluorescence microscope, and use image J software to calculate the percentage of Edu staining positive in each image.

[0059] (1) Effects on hPASMCs phenotype

[0060] Through cell scratch, Transwell, EdU and other experiments, it was found that after administration of 1 μmol / L WZ3146, the malignant proliferation and migration ability of hPASMCs induced by 5% FBS were significantly inhibited ( Figure 2 ).

[0061] Simultaneously, PDGF-BB was used to establish the model, and the results showed that WZ3146 could significantly inhibit the malignant proliferation and migration of hPASMCs induced by PDGF-BB ( Figure 3 ).

[0062] (2) Effects on rPASMCs phenotype

[0063] Administration of WZ3146 (3 μmol / L) significantly inhibited the abnormal proliferation and migration of rPASMCs induced by 5% FBS ( Figure 4 ). It can also inhibit the abnormal proliferation and migration of rPASMCs induced by PDGF-BB ( Figure 5 ).

[0064] Example 3: Effect of WZ3146 on hemodynamics in MCT / SuHx-induced PAH rats

[0065] 1. Effects of WZ3146 on hemodynamics in rats with MCT-induced PAH

[0066] (1) Establishment of MCT-induced PAH rat model

[0067] After one week of adaptive feeding, male SD rats weighing 200-250g were randomly divided into five groups (10 rats per group): control group, WZ3146-treated group, MCT model group, MCT+WZ3146 group, and MCT+Ambrisentan group. Model group rats were subcutaneously injected with 60mg / kg MCT (Day 0), and their body weight and mental status were recorded daily. Two weeks after the model group rats were subcutaneously injected with 60mg / kg MCT, the WZ3146, MCT+WZ3146, and MCT+Ambrisentan groups were intraperitoneally injected with 1mg / kg of WZ3146 or Ambrisentan daily on Day 14. On Day 28, the animals were anesthetized with sodium pentobarbital, immobilized, and underwent right cardiac catheterization for hemodynamic analysis.

[0068] (2) Right cardiac catheterization method for measuring RVSP and mPAP

[0069] A PE-50 catheter with a length of about 13 cm was selected to prepare a catheter with an arc-shaped end, with an arc length of about 1 cm and an arc of 120°. After completing the initial plasticization, it was placed in water at about 80°C and soaked for 4 minutes. After being taken out, the arc was stretched appropriately and cooled to form. After preparation, a mark was made at a distance of 7 cm to determine the position during intubation. The rat was weighed and anesthetized with an intraperitoneal injection of 50 mg / kg of prepared sodium pentobarbital. After the injection, the rat's condition was closely observed. When the rat's limbs were found to be limp and the muscle tension was significantly reduced, it was determined that the anesthesia effect had been achieved and the rat was fixed on the operating table.

[0070] Connect the instrument, turn on the multichannel physiological recorder and control interface, connect the pressure transducer to the homemade PE-50 catheter with a curved end, fill the pressure transducer and catheter with heparinized saline, ensure there are no bubbles in the pressure transducer and catheter, and select the appropriate channel for use.

[0071] Make a longitudinal incision in the rat's neck, slightly to the right midline. After opening the skin, carefully expose the external jugular vein using blunt dissection. Once the external jugular vein is located, ligate its distal end with surgical suture to block blood flow. Next, use ophthalmic scissors to make an oblique cut in the upper middle third of the external jugular vein. After the cut is complete, gently lift the proximal edge of the incision with forceps to avoid additional damage to the vein. Next, slowly insert the prepared right cardiac catheter into the external jugular vein along the raised incision. Continue advancing the catheter. Slowly advance the catheter approximately 2 cm, at which point it will enter the superior vena cava. Once the catheter enters the superior vena cava, gently rotate the catheter clockwise while continuing to advance it slowly. This will facilitate smooth passage through the superior vena cava and into the right atrium. Continue advancing the catheter until it reaches a depth of approximately 4 cm, allowing it to enter the right ventricle. Once the right cardiac catheter has successfully entered the right ventricle, maintain its position and pause briefly. Due to the effects of intracardiac hemodynamics, the catheter will naturally follow the direction of blood flow toward the pulmonary artery. During this process, pay close attention to the changes in pressure values and waveforms on the multichannel physiological recorder.

[0072] After right heart catheterization, the rats were killed, the hearts were removed, the left and right atria and the root tissues of the great blood vessels were cut off, the right ventricular free wall was carefully separated, and the separated right ventricular free wall and the remaining left ventricle and ventricular septum were then absorbed with filter paper to remove moisture and weighed. The right ventricular hypertrophy index was calculated according to the formula: RVHI = RV / (LV + S).

[0073] One of the important phenotypes of PAH is increased pulmonary vascular resistance, which manifests as abnormal pulmonary hemodynamic indicators. The results of right cardiac catheterization showed that the RVSP of rats in the MCT-induced PAH model group was significantly higher than that in the normal control group, and the RVSP of animals was reduced after administration of WZ3146 ( Figure 6A), quantitative results are as follows ( Figure 6 B). The mPAP of the model group rats was significantly higher than that of the control group, but decreased after drug administration. The quantitative results showed that there was no significant difference between the MCT+WZ3146 group and the MCT+Ambrisentan group ( Figure 6 C). The results of the cardiac Fulton index are as follows ( Figure 6 D) This indicates that the MCT-induced PAH rat model was successful, and WZ3146 administration significantly improved the hemodynamic parameters of MCT-induced PAH rats.

[0074] Effects of WZ3146 on hemodynamics in rats with SuHx-induced PAH

[0075] (1) Establishment of SuHx-induced PAH model in mice

[0076] The hypoxia chamber was set to a preset oxygen concentration of 10%. Desiccant, moisture absorbers, and other dehumidifying items were placed inside to maintain normal humidity and CO2 concentration. Mice were also ensured to have adequate ventilation during the experiment and to have adequate food and water. A control group of mice was housed in a normoxic housing room, with all other conditions except oxygen levels being the same. After one week of acclimatization, male C57BL / 6J mice weighing 20-25g were randomly divided into five groups (10 mice per group): normoxia, WZ3146-treated, SuHx-treated, SuHx+WZ3146, and SuHx+Ambrisentan. This was designated Day 0. Mice in the SuHx and SuHx-treated groups were housed in the hypoxia chamber throughout the experiment. Sugen 5416 (20 mg / kg) was injected intraperitoneally on Days 7, 14, and 21 to establish a more stable hypoxic mouse model. From Day 7 to Day 27, the WZ3146, SuHx+WZ3146, and SuHx+Ambrisentan groups received a daily intraperitoneal injection of 2 mg / kg of WZ3146 or Ambrisentan. The hypoxia chamber door was closed promptly after administration. On Day 28, the animals were anesthetized with sodium pentobarbital and immobilized before right cardiac catheterization for hemodynamic analysis.

[0077] (2) The specific process of the right cardiac catheter hemodynamic test is the same as above.

[0078] Compared with normoxic mice, the RVSP and right ventricular hypertrophy index (RVHI) of mice under SuHx conditions were significantly increased, indicating that the SU5416 combined with hypoxia-induced pulmonary hypertension mouse model was successfully established. Mice in the SuHx+WZ3146 group showed improved hemodynamic indicators, including reduced RVSP and RVHI ( Figure 7 ).

[0079] Example 4: WZ3146 inhibits MCT / SuHx-induced pulmonary vascular remodeling in PAH rats

[0080] In order to further observe the pathological changes of rat pulmonary arterioles, the rat lung tissue was paraffin-embedded and then subjected to H&E, EVG and immunofluorescence staining. The specific process is as follows:

[0081] (1) Paraffin embedding

[0082] Fresh lung tissue was fixed in 4% paraformaldehyde for at least 48 hours. The tissue was removed from the fixative and placed in a dehydration box under running water overnight. The next day, the tissue was washed three times with PBS on a decolorizing shaker for 15 minutes each. The dehydration box was then placed in a gradient of alcohol for dehydration. The concentrations of the alcohol in the dehydration box increased in sequence: 50%, 60%, 70%, 80%, 90%, 95% I, 95% II, 100% I, and 100% II, for 0.5 hours each. After dehydration, the lung tissue was cleared in xylene I and II for 10 minutes. Once the lung tissue was fully cleared, it was then immersed in paraffin wax: soft wax for 2 hours, hard wax I for 1.5 hours, and hard wax II for 1.5 hours each. The wax-soaked lung tissue was placed in an embedding mold and embedded in paraffin. After the paraffin solidified, the wax block was removed from the embedding mold and trimmed with a knife.

[0083] (2) H&E staining, EVG and immunofluorescence staining

[0084] H&E staining: Lung tissue was cut into 5 μm sections using a paraffin slicer. The sections were fully expanded in a 42°C water bath and adhered to disposable slides after expansion. Lung tissue sections were baked in a 65°C electric forced-air drying oven for 1.5 h to remove moisture. The sections were then placed in xylene I and II for 20 min each to deparaffinize the sections. The sections were then placed in 100% ethanol I, 100% ethanol II, 95% ethanol I, and 95% ethanol II for 5 min, followed by 3 min in 80% ethanol, 60% ethanol, and distilled water for rehydration. The slides were wiped dry with filter paper strips. An appropriate amount of hematoxylin was added to the lung tissue for staining (approximately 3 min). The staining was terminated by washing with water to stain the cell nuclei. The sections were then placed in 1% hydrochloric acid in ethanol for approximately 5 min to differentiate the nuclei into blue. Dehydrate the sections in 60%, 70%, and 80% ethanol solutions for 2 minutes, wipe the slides dry with filter paper, and stain the lung tissue with an appropriate amount of eosin (approximately 2 minutes). End the staining with water washing to stain the cytoplasm. Dehydrate the slides in 100% ethanol for 5 minutes, clear them in xylene for 5 minutes, and then mount them with neutral gum. Finally, photograph and record the slides using an upright microscope.

[0085] EVG staining: Place sections in environmentally friendly dewaxing solution I for 20 minutes, then environmentally friendly dewaxing solution II for 20 minutes, then anhydrous ethanol I for 5 minutes, then anhydrous ethanol II for 5 minutes, then 75% alcohol for 5 minutes. Rinse with tap water. Then, mix EVG stain solution A:EVG stain solution B:EVG stain solution C in a ratio of 5:2:2 (prepare 2 days in advance) and stain the sections in EVG stain solution for 5 minutes. Rinse with tap water. Dilute EVG stain solution B 100% and allow to differentiate briefly. Rinse with tap water. Repeat this process, monitoring differentiation under a microscope until the elastic fibers appear purple-black and the background appears off-white, nearly colorless. Add EVG stain solution E to 1 mL of EVG stain solution D (prepare the desired amount for use) and stain for 1-3 minutes. (The staining time depends on the elastic fiber composition of the tissue. Too short a staining time will result in a pale collagen color, while too long a staining time will cause the elastic fibers to fade.) Rinse quickly with water and dehydrate quickly in three cylinders of anhydrous ethanol. Finally, use two cylinders of clean xylene for 20 seconds and 5 minutes each (xylene is dedicated to this method and should not be used with other xylenes), wet-seal with neutral gum, and examine under a microscope for image acquisition and analysis.

[0086] Immunofluorescence staining:

[0087] (a) Section Pretreatment: Remove sections from the preservative solution after previous experimental treatments and rinse three times with PBS (phosphate-buffered saline) for 5 minutes each time to remove residual reagents. Paraffin sections should first be dewaxed and hydrated using the same dewaxing and hydration procedures as previously described, sequentially treating with xylene, then varying concentrations of ethanol, followed by a final rinse with PBS.

[0088] (b) Antigen retrieval: Place the sections in a retrieval box containing antigen retrieval solution (e.g., citrate buffer) and heat in a microwave or pressure cooker until boiling. Keep the temperature for a certain period of time (usually 10-15 minutes in a microwave and 2-3 minutes in a pressure cooker). Then cool the sections naturally to room temperature and rinse them with PBS three times for 5 minutes each time.

[0089] (c) Blocking: To reduce nonspecific staining, immerse the sections in a blocking solution, typically 5%-10% bovine serum albumin (BSA) or goat serum, and incubate at room temperature for 30-60 minutes.

[0090] (d) Primary Antibody Incubation: Discard the blocking solution and, without washing, add an appropriately diluted primary antibody (determine the dilution ratio according to the antibody instructions), ensuring that the primary antibody evenly covers the sections. Place in a humidified chamber and incubate overnight at 4°C. The primary antibody specifically recognizes and binds to the target antigen.

[0091] (e) Washing: The next day, sections were removed and rinsed three times with PBS for 5 minutes each time to remove unbound primary antibody.

[0092] (f) Secondary Antibody Incubation: Add the fluorescently labeled secondary antibody corresponding to the primary antibody. Refer to the secondary antibody dilution instructions for the dilution ratio. Incubate at room temperature in the dark for 30-60 minutes. The secondary antibody binds to the primary antibody and carries a fluorescent group, causing the target antigen to fluoresce.

[0093] (g) Nuclear counterstaining: Counterstain the nuclei with a nuclear staining reagent such as DAPI (4',6-diamidino-2-phenylindole). Incubate at room temperature for 5-10 minutes to make the nuclei appear blue, facilitating localization and observation. Rinse with PBS three times for 5 minutes each.

[0094] (h) Mounting: Place an appropriate amount of anti-fluorescence quenching mounting medium on a glass slide. Remove the slice from PBS, absorb excess water with absorbent paper, and place the slice face down on the mounting medium to avoid bubbles.

[0095] (i) Microscopic observation: After the sealing medium solidifies, it can be observed under a fluorescence microscope.

[0096] H&E and EVG results showed that the model group had significantly thickened pulmonary arteriolar walls, smooth muscle cells and collagen fiber proliferation compared with the control group. The vascular morphology of the MCT+WZ3146 group was improved, which produced similar effects compared with MCT+Ambrisentan. In order to further evaluate the degree of muscularization of pulmonary small blood vessels, anti-vWF and anti-α-SMA double immunofluorescence staining experiments were performed, and about 80 blood vessels with a diameter of less than 100μm were analyzed for each rat to quantify the degree of muscularization. The classification criteria are: non-muscularization (no muscular component, only green fluorescence labeled with anti-vWF is visible), partial muscularization (the blood vessels have both green fluorescence labeled with anti-vWF and red fluorescence labeled with anti-α-SMA, but the red fluorescence does not form a complete circumference) or complete muscularization (the blood vessels have both green fluorescence labeled with anti-vWF and red fluorescence labeled with anti-α-SMA ( Figure 8 Microscopic scanning revealed a decrease in the number of non-muscularized small vessels and an increase in the number of fully muscularized small vessels in the MCT group compared to the control group. The degree of vascular muscularization was significantly improved in the MCT + WZ3146 group after administration. Therefore, histological results suggest that WZ3146 can inhibit MCT-induced fibrosis and protect the pulmonary arteries in PAH rats.

[0097] Mice in the SuHx+WZ3146 group showed attenuated pulmonary vascular remodeling. H&E and EVG results showed that the thickness of the pulmonary vascular media of SuHx-induced PAH mice was significantly increased. Treatment with WZ3146 in the SuHx group alleviated the thickening of the pulmonary vascular media and the narrowing of the vascular lumen. vWF immunofluorescence results showed that the level of pulmonary vascular muscularization in SuHx-induced PAH mice was also significantly increased. After WZ3146 administration, the number of fully muscularized blood vessels in mice decreased, while the number of non-muscularized blood vessels increased ( Figure 9). This indicates that the administration has a certain therapeutic effect and improves the vascular remodeling in the lungs of model mice.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of WZ3146 in the preparation of a product for preventing and / or treating pulmonary hypertension and its complications, characterized in that: The structural formula of WZ3146 is as follows:

2. Use of a pharmaceutically acceptable salt of WZ3146 in the preparation of a product for preventing and / or treating pulmonary hypertension and its complications, characterized in that: The structural formula of WZ3146 is as follows:

3. Use of the pharmaceutically acceptable salt of WZ3146 according to claim 2 in the preparation of a product for preventing and / or treating pulmonary hypertension and its complications, characterized in that: The pharmaceutically acceptable salts are salts prepared from pharmaceutically acceptable non-toxic bases or acids.

4. Use of the pharmaceutically acceptable salt of WZ3146 according to claim 3 in the preparation of a product for preventing and / or treating pulmonary hypertension and its complications, characterized in that: The pharmaceutically acceptable salt is a salt formed with the following acids: one or more of phosphoric acid, carbonic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, succinic acid, tartaric acid and p-toluenesulfonic acid.

5. The use according to claim 1 or 2, characterized in that: The pulmonary hypertension and its complications have one or more of the following indicators: increased mean pulmonary artery pressure and right ventricular systolic pressure, right heart enlargement and right heart dysfunction, pulmonary vascular remodeling, increased pulmonary arteriolar media thickness and vascular muscularization, and pulmonary artery smooth muscle cell proliferation and migration.

6. The use according to claim 1 or 2, characterized in that The products include medicines, foods or health products.

7. The use according to claim 6, characterized in that The drug is administered enterally or parenterally.

8. The use according to claim 6, characterized in that The product is used by humans or other mammals.

9. A pharmaceutical composition for preventing and / or treating pulmonary hypertension and its complications, characterized in that: The pharmaceutical composition comprises WZ3146 or a pharmaceutically acceptable salt thereof according to claim 1, and pharmaceutically acceptable excipients.

10. The pharmaceutical composition for preventing and / or treating pulmonary hypertension and its complications according to claim 9, characterized in that: The pharmaceutical composition is selected from any one of the following dosage forms: solution, suspension, lyophilized powder injection, emulsion, pill, capsule, powder, controlled release, sustained release preparation and microsome delivery system.

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