Application of small molecule compound 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 an application of a small molecule compound in preparation of a product for preventing and / or treating pulmonary arterial hypertension and complications thereof. The small molecule compound is 8-[[4-(cyclopropanecarbonyl) piperazine-1-yl] methyl]-1, 3-dimethyl-7-(3-methylbutyl) purine-2, 6-diketone, and the structural formula is as shown in a formula (I): # imgabs0. The small molecule compound and pharmaceutically acceptable salts thereof have a remarkable treatment effect on pulmonary arterial hypertension and complications thereof, and can be used for treating pulmonary arterial hypertension and complications thereof. The effects of preventing and / or treating the pulmonary arterial hypertension include the reduction of right heart hypertrophy, the reduction of right ventricular systolic pressure, right heart enlargement, right heart dysfunction, pulmonary vascular remodeling, pulmonary arterial intima thickness, increase of vascular myelization, pulmonary arterial smooth muscle cell proliferation and migration, the reduction of the right ventricular hypertrophy, the reduction of the right ventricular hypertrophy, the reduction of the right ventricular hypertrophy and the reduction of the right ventricular hypertrophy. The pulmonary arterial intima thickness and the vascular myelization level are improved, pulmonary vascular remodeling is reversed, and pulmonary artery smooth muscle cell proliferation and migration are inhibited.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of a small molecule compound. Background Art

[0002] Pulmonary Arterial Hypertension (PAH) is a serious cardiovascular disease characterized by persistently increased pulmonary artery resistance due to pulmonary vascular remodeling and progressive stenosis. It is characterized by persistently elevated pulmonary artery pressure, ultimately leading to right heart failure and death. The diagnostic criteria for PAH are a mean pulmonary artery pressure greater than 20 mmHg measured by a right cardiac catheter at rest at sea level. The etiology of pulmonary hypertension is complex, and its pathogenesis has not yet been fully elucidated. Patients often have no obvious symptoms in the early stages of the disease, and are usually in the progressive stage when diagnosed, with an extremely poor prognosis. The 5-year survival rate of untreated PAH patients is extremely low, and improving the prognosis of PAH patients has always been a major challenge in clinical practice.

[0003] Existing treatment strategies mainly include prostacyclins, endothelin receptor antagonists, phosphodiesterase-5 inhibitors, soluble guanylate cyclase agonists, and biologics. These drugs relieve patient symptoms by dilating pulmonary artery vessels and reducing pulmonary artery resistance. However, these treatments can only improve hemodynamics and clinical symptoms to a certain extent and cannot reverse the core pathological process of pulmonary vascular remodeling. In addition, existing drugs are often accompanied by significant toxic side effects and adverse reactions, which limit their long-term effectiveness, and the long-term prognosis of patients remains poor. Even with the popularization of targeted drugs, the survival of patients has been extended, but the 5-year survival rate is still far from ideal, especially in the late stage of the disease, when progressive deterioration of cardiopulmonary function is inevitable.

[0004] In recent years, researchers have recognized that reversing or inhibiting pulmonary vascular remodeling is the key to fundamentally improving the prognosis of PAH patients. Therefore, there is an urgent need to develop new therapeutic drugs with novel mechanisms of action. Summary of the Invention

[0005] To address the above technical issues, the present invention proposes the use of a small molecule compound in the preparation of a product for preventing and / or treating pulmonary hypertension and its complications. Products or medicaments containing the small molecule compound of the present invention can inhibit the proliferation and migration of pulmonary arterial smooth muscle cells, thereby preventing or treating vascular remodeling in pulmonary hypertension and improving the thickness of the tunica media and the level of vascular muscularization in pulmonary arterioles.

[0006] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0007] A small molecule compound is used in the preparation of a product for preventing and / or treating pulmonary arterial hypertension and its complications. The small molecule compound is 8-[[4-(cyclopropanecarbonyl)piperazin-1-yl]methyl]-1,3-dimethyl-7-(3-methylbutyl)purine-2,6-dione, referred to as NCT-501, and has the structural formula shown in formula (I):

[0008]

[0009] A use of a pharmaceutically acceptable salt of a small molecule compound in the preparation of a product for preventing and / or treating pulmonary arterial hypertension and its complications, characterized in that the small molecule compound is 8-[[4-(cyclopropanecarbonyl)piperazin-1-yl]methyl]-1,3-dimethyl-7-(3-methylbutyl)purine-2,6-dione, having the structural formula shown in formula (I):

[0010]

[0011] The above-mentioned pharmaceutically acceptable salts are salts prepared from pharmaceutically acceptable non-toxic bases or acids.

[0012] 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.

[0013] Furthermore, the above-mentioned product is a product prepared by compounding the small molecule compound represented by formula (I) or a pharmaceutically acceptable salt thereof with conventional excipients in the art.

[0014] The above products include medicines, food and health products.

[0015] 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.

[0016] Furthermore, the effects of the above-mentioned small molecule compounds and pharmaceutically acceptable salts thereof on the prevention and / or treatment of pulmonary hypertension include dilating the right heart, improving the thickness of the tunica media of pulmonary arterioles, the level of vascular muscularization, improving high-pressure vascular remodeling, and inhibiting the proliferation and migration of pulmonary arterial smooth muscle cells.

[0017] The effective daily dose of the small molecule compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the above product is 3-30 mg / kg body weight.

[0018] The above-mentioned drugs are administered by enteral or parenteral routes, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum.

[0019] The target users of the above products include humans or other mammals.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Furthermore, if necessary, colorants, preservatives, perfumes, flavorings, sweeteners or other materials may be added to the pharmaceutical preparations.

[0026] 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 dosage of the present invention can vary widely. The actual amount of active drug in the final formulation of the pharmaceutical compositions of the present invention can be appropriately adjusted to achieve a therapeutically effective amount.

[0027] The present invention provides a drug for preventing and / or treating pulmonary arterial hypertension, with a dosage of 3-30 mg / kg body weight of a small molecule compound represented by general formula (I) and a pharmaceutically acceptable salt thereof. The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

[0028] The beneficial effects produced by the present invention are:

[0029] 1. The present invention provides a small molecule compound containing 2,6-dicarbonylpurine and a pharmaceutically acceptable salt thereof for use in preventing and / or treating pulmonary hypertension. The research results of the present invention show that in experiments on male mammalian rats, the small molecule compound (abbreviated as NCT-501) and a pharmaceutically acceptable salt thereof can reverse vascular remodeling, significantly improve the thickness of the tunica media of pulmonary arterioles and the level of vascular muscularization, and inhibit the proliferation and migration of pulmonary arterial smooth muscle cells in preventing and / or treating pulmonary hypertension; and have significant effects on the hemodynamic disorder of pulmonary hypertension, and on correcting mean pulmonary artery pressure, right ventricular systolic pressure, right heart dysfunction, and right heart enlargement.

[0030] 2. Compared with the drugs in the prior art, the small molecule compounds provided by the present invention can reverse vascular remodeling, and are the key to breaking the treatment bottleneck and improving the quality of life of patients.

[0031] 3. The above-mentioned drug can produce significant effects in preventing and / or treating pulmonary hypertension within 2 weeks of administration. This drug has the advantages of significant effects in preventing and / or treating pulmonary hypertension, rapid onset of action, low dosage, and minimal side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1 Figure 2: Effects of the small molecule compound NCT-501 on the viability of rat and human PASMCs induced by PDGF-BB. A: NCT-501 structure; B: Effect of NCT-501 on the viability of human PASMCs induced by PDGF; C: Effect of NCT-501 on the viability of rat PASMCs induced by PDGF-BB. All data are mean ± SEM, n = 6; ***p < 0.001.

[0034] Figure 2 Figure 1 shows the inhibition of 5% FBS- or PDGF-BB-induced proliferation of rat PASMCs by the small molecule compound NCT-501. A: Effect of NCT-501 on 5% FBS-induced proliferation of rPASMCs; B: EdU quantitative analysis results; C: Effect of NCT-501 on PDGF-BB-induced proliferation of rPASMCs; D: EdU quantitative analysis results. All data are mean ± SEM, n = 5; ***p < 0.001.

[0035] Figure 3 Figure 1 shows the inhibition of 5% FBS- or PDGF-BB-induced rat PASMC migration by the small molecule compound NCT-501. A: Effect of NCT-501 on 5% FBS-induced rPASMC migration; B: Quantitative analysis of wound wound 24 hours later; C: Effect of NCT-501 on PDGF-BB-induced rPASMC migration; D: Quantitative analysis of wound wound 24 hours later. All data are mean ± SEM, n = 5; ***p < 0.001.

[0036] Figure 4Figure 1 shows the inhibition of 5% FBS- or PDGF-BB-induced rat PASMC migration by the small molecule compound NCT-501. A: Effect of NCT-501 on 5% FBS-induced rPASMC migration; B: Transwell quantitative analysis results; C: Effect of NCT-501 on PDGF-BB-induced rPASMC migration; D: Transwell quantitative analysis results. All data are mean ± SEM, n = 5; ***p < 0.001.

[0037] Figure 5 Figure 1 shows the inhibition of 5% FBS- or PDGF-BB-induced human PASMC cell proliferation by the small molecule compound NCT-501. A: Effect of NCT-501 on 5% FBS-induced hPASMC cell proliferation; B: EdU quantitative analysis results; C: Effect of NCT-501 on PDGF-BB-induced hPASMC cell proliferation; D: EdU quantitative analysis results. All data are mean ± SEM, n = 5; ***p < 0.001.

[0038] Figure 6 Figure 1 shows the inhibition of 5% FBS- or PDGF-BB-induced human PASMC migration by the small molecule compound NCT-501. A: Effect of NCT-501 on 5% FBS-induced hPASMC migration; B: Quantitative analysis of wound wounds 12 hours later; C: Effect of NCT-501 on PDGF-BB-induced hPASMC migration; D: Quantitative analysis of wound wounds 12 hours later. All data are mean ± SEM, n = 5; ***p < 0.001.

[0039] Figure 7 Figure 1 shows the inhibition of 5% FBS- or PDGF-BB-induced human PASMC migration by the small molecule compound NCT-501. A: Effect of NCT-501 on 5% FBS-induced hPASMC migration; B: Transwell quantitative analysis results; C: Effect of NCT-501 on PDGF-BB-induced hPASMC migration; D: Transwell quantitative analysis results. All data are mean ± SEM, n = 5; ***p < 0.001.

[0040] Figure 8 Effects of the small molecule compound NCT-501 on hemodynamics in SuHx-induced PAH mice; A: Animal experiment flow chart; B: Representative RVSP waveforms; C: RVSP statistical results; D: RVHI statistical results. All data are mean ± SEM, n = 10; ***p < 0.001.

[0041] Figure 9 Figure 2: Effects of the small molecule compound NCT-501 on pulmonary vascular remodeling in SuHx-induced PAH mice; A: Representative images of HE staining and EVG staining; B: Data analysis of medial thickness of mouse pulmonary microvessels; All data are expressed as mean ± SEM, n = 4; ***p < 0.001.

[0042] Figure 10 Effects of the small molecule compound NCT-501 on pulmonary vascular muscularization in SuHx-induced PAH mice; A: Representative immunofluorescence double staining images, VWF in green and α-SMA in red. Bar = 50 μm. B: Percentage of mice with vascular muscularization; all data are mean ± SEM, n = 4; ***p < 0.001. DETAILED DESCRIPTION

[0043] 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.

[0044] For the present examples and comparative examples, 6- to 8-week-old male Sprague-Dawley (SD) rats and 6-week-old male C57BL / 6J mice were purchased from Beijing Sprague-Dawley Laboratory Animal Technology Co., Ltd. (SPF). The animal quality certificate number for the SD rats is 110324231107197214; the animal quality certificate number for the C57BL / 6J mice is 110324241102294558. rPASMCs were isolated and cultured from the SD rats (rPASMCs used in this study were no more than passage 8); hPASMCs were purchased from Shanghai Saibaikang Biotechnology Co., Ltd. (hPASMCs used in this study were no more than passage 8).

[0045] The English abbreviations in the embodiments of the present invention are:

[0046] PASMCs cells: pulmonary artery smooth muscle cells;

[0047] PBS: phosphate buffered saline;

[0048] PDGF-BB: platelet-derived factor;

[0049] FBS: fetal bovine serum;

[0050] DMEM: low glucose medium;

[0051] SuHx: SU5416 combined with hypoxia;

[0052] EVG staining: histopathological staining;

[0053] Triton: A commonly used nonionic surfactant and emulsifier, often used in biochemical applications to solubilize proteins. It is considered a relatively mild detergent that does not denature. It is used to lyse cells to extract proteins and organelles, and can also permeabilize living cell membranes for transfection.

[0054] SPF-grade experimental mice: mice that are free of specific pathogens, which means that except for the pathogens that should be excluded by clean mice, they do not carry major potential infections or conditional pathogens and pathogens that interfere with scientific experiments.

[0055] Transwell assay: Cell migration and invasion experiments involve placing a Transwell chamber into a culture plate. The chamber inside the chamber is called the upper chamber, and the plate inside the chamber is called the lower chamber. The upper and lower culture media are separated by a polycarbonate membrane. The cells under study are seeded in the upper chamber. Because the polycarbonate membrane is permeable, components in the lower culture media can affect the cells in the upper chamber. By using polycarbonate membranes with different pore sizes and different treatments, various studies such as co-culture, cell chemotaxis, cell migration, and cell invasion can be conducted.

[0056] EdU is a thymidine analog that can replace T in the period of cell proliferation and be incorporated into the replicating DNA molecule. Specific reaction of fluorescent dyes to detect DNA replication activity;

[0057] CCK-8, short for Cell Counting Kit-8, is a reagent that uses a water-soluble tetrazolium salt, WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate)-2H-tetrazolium monosodium salt). This salt is reduced by cellular dehydrogenases to a highly water-soluble yellow formazan dye under the action of the electron carrier 1-methoxy-5-methylphenazinium methyl sulfate (1-Methoxy PMS). The amount of formazan dye produced is proportional to the number of viable cells. This method is used to assess cell survival and growth.

[0058] The reagents and consumables used in the embodiments of the present invention are shown in Table 1:

[0059] Table 1 Reagents and consumables for the experiment

[0060]

[0061] The main instruments of the embodiment of the present invention are shown in Table 2:

[0062] Table 2 Experimental instruments

[0063]

[0064] Example 1: Effect of CCK-8 Method on Detecting Rat PASMCs Cell Viability 1. Experimental Procedure

[0065] 1.1 Isolation and acquisition of rat PASMCs

[0066] Six-week-old male Sprague-Dawley rats were anesthetized with 1% sodium pentobarbital. The surgical site was disinfected with 75% alcohol, and the rats were sacrificed by thoracotomy. The thoracic cavity was exposed, and the heart and lung tissues were dissected from the white thymus along the spinal column using tissue scissors. The heart and lung tissues were completely removed and immersed in PBS containing 1% double-antibody. After rinsing three times with PBS, the heart was mounted with the apex facing upward. The five lung lobes were fixed to a white foam board using a syringe needle. Connective tissue in the hilar region of the heart and the right upper, right middle, and left lower lobes were removed. Excess lung tissue was trimmed along the blood vessels using ophthalmic scissors to expose the intact left and right pulmonary arteries. The pulmonary arteries were rinsed in PBS until a single intact white vessel was visible. The excised pulmonary artery was transferred to a culture dish containing sterile PBS and then transferred to a clean bench. Using sterile forceps, the artery was transferred to a culture dish containing sterile PBS and rinsed, then transferred to a fresh culture dish containing sterile PBS and rinsed again. After washing again, remove the blood vessel with forceps and place it on the side of the dish to drain excess water. Then cut the pulmonary artery into 1mm 3 After the tissue blocks are attached to the bottom of the culture dish, slowly add DMEM culture medium containing 1% penicillin and streptomycin and 20% FBS, and culture under 5% CO2 and 37℃ conditions.

[0067] 1.2 Detection of cell viability of rat PASMCs or human PASMCs by CCK-8 assay

[0068] Well-growing rPASMCs or hPASMCs were taken and seeded in 96-well plates at 6,000 cells per well. When the cell confluence reached 70%-80%, the old culture medium was removed and replaced with DMEM containing 0.5% FBS for 24 hours to achieve cell cycle synchronization. The cells were then grouped and treated accordingly according to the cell grouping and modeling method. The NCT-501-treated group was incubated with drugs, while the PDGF-BB modeling + NCT-501-treated group was incubated with drugs and 20 ng / mL PDGF-BB was used to construct the cell model. After 24 hours of culture, 10 μL of CCK8 was added to each well and incubated for 1.5 hours. The OD value was measured on a microplate reader (wavelength 450 nm). The culture medium was changed every 3 days. After about 1 week, the cell confluence reached 70-80%, and the cells were digested with 0.25% trypsin to prepare a cell suspension and passaged.

[0069] 2. Experimental Results

[0070] First, the CCK-8 assay was used to detect the effect of the small molecule compound NCT-501 on the cell viability of rat PASMCs and human PASMCs induced by PDGF-BB. The results showed that compared with the control group (Control), PDGF-BB significantly increased the cell viability of rat PASMCs, while NCT-501 (10 μmol / L) treatment significantly reduced cell viability (such as Figure 1 shown).

[0071] Example 2: Effect of Edu method on the proliferation ability of rat PASMCs

[0072] 1. Experimental Procedure

[0073] 1.1 Isolation and acquisition of rat PASMCs

[0074] Six-week-old male Sprague-Dawley rats were anesthetized with 1% sodium pentobarbital. The surgical site was disinfected with 75% alcohol, and the rats were sacrificed by thoracotomy. The thoracic cavity was exposed, and the heart and lung tissues were dissected from the white thymus along the spinal column using tissue scissors. The heart and lung tissues were completely removed and immersed in PBS containing 1% double-antibody. After rinsing three times with PBS, the heart was mounted with the apex facing upward. The five lung lobes were fixed to a white foam board using a syringe needle. Connective tissue in the hilar region of the heart and the right upper, right middle, and left lower lobes were removed. Excess lung tissue was trimmed along the blood vessels using ophthalmic scissors to expose the intact left and right pulmonary arteries. The pulmonary arteries were rinsed in PBS until a single intact white vessel was visible. The excised pulmonary artery was transferred to a culture dish containing sterile PBS and then transferred to a clean bench. Using sterile forceps, the artery was transferred to a culture dish containing sterile PBS and rinsed, then transferred to a fresh culture dish containing sterile PBS and rinsed again. After washing again, remove the blood vessel with forceps and place it on the side of the dish to drain excess water. Then cut the pulmonary artery into 1mm 3 After the tissue blocks are attached to the bottom of the culture dish, slowly add DMEM culture medium containing 1% penicillin and streptomycin and 20% FBS, and culture under 5% CO2 and 37℃ conditions.

[0075] 1.2 Effect of EdU on the proliferation of rat PASMCs

[0076] rPASMCs or hPASMCs in good growth condition were inoculated into 96-well plates with a cell quantity of 6000 cells / well. When the cell confluence reached 70%, the old culture medium was removed and replaced with DMEM containing 0.5% FBS for starvation culture for 24 hours to achieve cell cycle synchronization. The cells were then grouped and treated accordingly according to the cell grouping and modeling methods. The NCT-501 administration group was added with NCT-501 (10 μmol / L) for drug incubation, and the modeling + administration group was incubated with drugs while using 20 ng / mL PDGF-BB or 5% FBS to construct a cell model. After culturing for 24 hours, the cell proliferation detection kit of Ruibo Biotechnology Co., Ltd. was operated according to the instructions. The specific steps are as follows: dilute the EdU solution in the kit with cell culture medium to 50 μmol / L EdU working solution; discard the old culture medium in the 96-well plate, and use 100 μL per well. Incubate with EdU working solution for 2 hours; discard the working solution and wash the cells twice with PBS, 5 minutes each time; add 50 μL of 4% paraformaldehyde fixative to each well, incubate at room temperature for 30 minutes, and then discard the fixative; add 50 μL of 2 mg / mL glycine solution to each well, incubate on a shaker for 5 minutes, discard the glycine solution, and wash the cells with PBS for 5 minutes; add 100 μL of 0.5% Triton X-100 in PBS to each well, incubate on a shaker for 10 minutes, and then wash with PBS for 5 minutes; Add 100 μL of freshly prepared 1× Apollo staining solution to each well and incubate at room temperature in the dark for 30 minutes. Discard the staining solution. Add PBS containing 0.5% Triton X-100 to each well and incubate on a shaker for 10 minutes twice. Discard the waste solution. Dilute the Hochest 33342 reaction solution 100 μL with deionized water at a ratio of 100:1 and add 100 μL to each well. Incubate in the dark for 30 minutes and discard the reaction solution. Discard the reaction solution and wash the cells twice with PBS for 5 minutes each. After adding 100 μL of PBS to each well, photograph using a fluorescence microscope and save the image.

[0077] 2. Experimental Results

[0078] 2.1 NCT-501 inhibits abnormal proliferation of rat PASMCs induced by FBS or PDGF-BB

[0079] Excessive proliferation and migration of PASMCs are the main pathological drivers of pulmonary vascular remodeling. Therefore, the effect of the small molecule compound NCT-501 on the proliferation and migration of rat PASMCs was observed. The EdU experiment was used to detect the effect of NCT-501 on the proliferation of rat PASMCs induced by 5% FBS or 20ng / mL PDGF-BB. The results showed that FBS or PDGF-BB stimulation could induce excessive proliferation of rat PASMCs, while treatment with NCT-501 (10μmol / L) could significantly inhibit the excessive proliferation of rat PASMCs induced by FBS or PDGF-BB stimulation (such as Figure 2 shown).

[0080] 2.2 NCT-501 inhibits abnormal proliferation of human PASMCs induced by FBS or PDGF-BB

[0081] Excessive proliferation and migration of PASMCs are the main pathological drivers of pulmonary vascular remodeling. Therefore, the effect of the small molecule compound NCT-501 on the proliferation and migration of human PASMCs was observed. The EdU experiment was used to detect the effect of NCT-501 on the proliferation of human PASMCs induced by 5% FBS or 20ng / mL PDGF-BB. The results showed that FBS or PDGF-BB stimulation could induce excessive proliferation of human PASMCs, while treatment with NCT-501 (10μmol / L) could significantly inhibit the excessive proliferation of human PASMCs induced by FBS or PDGF-BB stimulation (such as Figure 5 shown).

[0082] Experiment 3: Scratch test and Transwell assay to detect the migration ability of rat PASMCs

[0083] 1. Experimental Procedure

[0084] 1.1 Isolation and acquisition of rat PASMCs

[0085] Six-week-old male Sprague-Dawley rats were anesthetized with 1% sodium pentobarbital. The surgical site was disinfected with 75% alcohol, and the rats were sacrificed by thoracotomy. The thoracic cavity was exposed, and the heart and lung tissues were dissected from the white thymus along the spinal column using tissue scissors. The heart and lung tissues were completely removed and immersed in PBS containing 1% double-antibody. After rinsing three times with PBS, the heart was mounted with the apex facing upward. The five lung lobes were fixed to a white foam board using a syringe needle. Connective tissue in the hilar region of the heart and the right upper, right middle, and left lower lobes were removed. Excess lung tissue was trimmed along the blood vessels using ophthalmic scissors to expose the intact left and right pulmonary arteries. The pulmonary arteries were rinsed in PBS until a single intact white vessel was visible. The excised pulmonary artery was transferred to a culture dish containing sterile PBS and then transferred to a clean bench. Using sterile forceps, the artery was transferred to a culture dish containing sterile PBS and rinsed, then transferred to a fresh culture dish containing sterile PBS and rinsed again. After washing again, remove the blood vessel with forceps and place it on the side of the dish to drain excess water. Then cut the pulmonary artery into 1mm 3 After the tissue blocks are attached to the bottom of the culture dish, slowly add DMEM culture medium containing 1% penicillin and streptomycin and 20% FBS, and culture under 5% CO2 and 37℃ conditions.

[0086] 1.2 Scratch test:

[0087] Take rPASMCs and hPASMCs that are in good growth condition and place them at 3×10 cells per well. 5 Cells were seeded in 6-well plates. The plates were incubated at 37°C in a 5% CO2 incubator. When the cell density in the 6-well plates reached 90%-100%, the old culture medium was removed and replaced with DMEM supplemented with 0.5% FBS for 24 hours to achieve cell cycle synchronization. Subsequently, a vertical line was drawn in the middle of the well using a 200μL yellow pipette tip, and the cells were washed three times with PBS. The cells were then grouped and treated accordingly based on the cell grouping and modeling method. The model group was supplemented with 20ng / mL PDGF-BB or cultured in 5% FBS. After drug treatment, rPASMCs were photographed at 0h and 24h after the scratch; hPASMCs were photographed at 0h and 12h after the scratch. Finally, the scratch area was analyzed using Image J.

[0088] 1.3 Transwell experiment:

[0089] Take rPASMCs and hPASMCs with good growth status, starve the cells in DMEM containing 0.5% FBS for 24 hours, digest with trypsin, and resuspend the cells in serum-free DMEM. 4Cells were seeded in the upper chamber of a Transwell chamber and treated according to experimental group. Induction was performed by adding 20 ng / mL PDGF-BB or 5% FBS to the lower chamber. After 24 hours of culture, the culture medium was discarded, and the chamber was removed and washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature and washed twice with PBS. Next, the cells were stained with crystal violet for 30 minutes. After washing under running water and air-drying at room temperature, randomly selected fields of view were photographed under a microscope.

[0090] 2. Experimental Results

[0091] 2.1 NCT-501 inhibits abnormal migration of rat PASMCs induced by FBS or PDGF-BB

[0092] The scratch test was used to verify the effect of the small molecule compound NCT-501 on the migration of rat PASMCs induced by 5% FBS or 20ng / mL PDGF-BB. Figure 3 As shown in the figure, compared with the normal control group, the number of cells migrated after FBS or PDGF-BB treatment increased significantly; after the addition of NCT-501 (10 μmol / L) intervention, the number of cells migrated was significantly reduced compared with the FBS or PDGF-BB group ( Figure 3 ). This indicates that FBS or PDGF-BB can increase the number of migrating cells and enhance their migration ability; NCT-501 can reduce the cell migration induced by FBS or PDGF-BB.

[0093] To further verify the effect of the small molecule compound NCT-501 on the migration of rat PASMCs induced by FBS or PDGF-BB, the Transwell assay was used to evaluate the migration ability of each group of cells. Figure 4 As shown in the figure, compared with the normal control group, the number of migrating cells treated with FBS or PDGF-BB increased significantly; after intervention with NCT-501 (10 μmol / L), the number of migrating cells decreased significantly compared with the 5% FBS or PDGF-BB group ( Figure 4 ). This indicates that FBS or PDGF-BB can increase the number of migrating cells and enhance their migration ability; NCT-501 can reduce the cell migration induced by FBS or PDGF-BB.

[0094] 2.2 NCT-501 inhibits abnormal migration of human PASMCs induced by FBS or PDGF-BB

[0095] The scratch test was used to verify the effect of the small molecule compound NCT-501 on the migration of human PASMCs induced by 5% FBS or 20ng / mL PDGF-BB. Figure 6As shown in the figure, compared with the normal control group, the number of cells migrated after FBS or PDGF-BB treatment increased significantly; after the addition of NCT-501 (10 μmol / L) intervention, the number of cells migrated was significantly reduced compared with the FBS or PDGF-BB group ( Figure 6 ). This indicates that FBS or PDGF-BB can increase the number of migrating cells and enhance their migration ability; NCT-501 can reduce the cell migration induced by FBS or PDGF-BB.

[0096] To further verify the effect of the small molecule compound NCT-501 on the migration of human PASMCs induced by 5% FBS or 20 ng / mL PDGF-BB, the Transwell assay was used to evaluate the migration ability of each group of cells. Figure 7 As shown in the figure, compared with the normal control group, the number of cells migrated after FBS or PDGF-BB treatment increased significantly; after the addition of NCT-501 (10 μmol / L) intervention, the number of cells migrated was significantly reduced compared with the FBS or PDGF-BB group ( Figure 7 ). This indicates that FBS or PDGF-BB can increase the number of migrating cells and enhance their migration ability; NCT-501 can reduce the cell migration induced by FBS or PDGF-BB.

[0097] Experiment 4: Animal Experiment Grouping Design and Establishment of Pulmonary Hypertension Model

[0098] 1. Experimental Procedure

[0099] 1.1 Experimental Animal Grouping and Treatment

[0100] Six-week-old male C57BL / 6J mice weighing approximately 20g were randomly divided into groups of 10 mice each after one week of acclimation. The SuHx group received a single intraperitoneal injection of Sugen5416 (20mg / kg) on day 1 and were placed in a hypoxic chamber for 21 days to simulate the hypoxic environment (10% oxygen concentration) at an altitude of 5500m. The SuHx + NCT-501 group received a single intraperitoneal injection of Sugen5416 (20mg / kg) on day 1 and were placed in a hypoxic chamber for one week before being administered intraperitoneally at 10mg / kg NCT-501 daily for two weeks. The control group received an equal volume of saline intraperitoneally on day 1 and were housed in a standard environment for 21 days. The NCT-501 group received an equal volume of saline intraperitoneally on day 1 and were housed in a standard environment. Starting on day 8, NCT-501 was administered intraperitoneally at 10mg / kg daily for two weeks. Subsequently, hemodynamics and right ventricular hypertrophy index were assessed. Lung tissues were collected and immediately stored at −80°C or in paraformaldehyde fixative for subsequent analysis.

[0101] 1.2 Right ventricular systolic pressure (RVSP) measurement by right cardiac catheterization

[0102] After the end of drug administration, all C57BL / 6J mice were cannulated with right heart catheters to measure the right ventricular systolic pressure (RVSP). The steps are briefly described as follows: After anesthetizing the mice with sodium pentobarbital, they were placed on a wooden board fixation frame, lying on their backs and then their limbs and heads were fixed. Use vascular clamps to bluntly separate the surrounding tissues and fully expose the right external jugular vein. The external jugular vein is ligated at the distal end, a traction line is reserved, and the proximal end is fixed with an artery clamp. At the proximal end of the right external jugular vein, 1 / 3 of the blood vessel diameter is cut at a 45° angle, and a spare Miller catheter pre-filled with sodium heparin saline is quickly inserted into the blood vessel 0.5-1.0 cm along the direction of the incision. During this period, the direction of the catheter tip is constantly adjusted to ensure smooth passage. Then continue to insert 2-3 cm to reach the right atrium, then slowly pull and rotate the catheter to enter the right ventricle, and record the RVSP after the typical right ventricular waveform appears on the monitor and stabilizes. The data is processed by the Powerlab data acquisition and analysis system. Figure 8 shown.

[0103] 1.3 Determination of right ventricular hypertrophy index (RVHI)

[0104] After right heart catheterization, the mice were killed, the hearts were removed, the left and right atria and the root tissues of the large blood vessels were cut off, and the right ventricular free wall was carefully separated. The separated right ventricular free wall (RV) and the remaining left ventricle and ventricular septum (LV+S) were then dehydrated with filter paper and weighed. The weight was substituted into the formula to calculate the right ventricular hypertrophy index: RVHI=RV / (LV+S).

[0105] 1.4 Lung tissue pathological staining

[0106] 1.4.1 Immunofluorescence staining of lung tissue

[0107] Fresh lung tissue is fixed in paraformaldehyde and stored at room temperature. After fixation, the tissue is trimmed and placed in an embedding frame for subsequent processing. The tissue is then dehydrated in a gradient of alcohol concentrations, ensuring that all water is removed. After dehydration, the tissue is immersed in pre-melted paraffin wax, allowing the wax to fully penetrate the tissue. The wax-soaked tissue block is placed in an embedding machine for embedding. First, melted paraffin is poured into the embedding frame. Before the paraffin solidifies, the tissue is embedded with the cut surface facing down. The embedding frame is then cooled at -20°C. Once the paraffin solidifies, the block is removed from the frame and trimmed. A paraffin microtome is used to cut the block into 4μm sections. After sectioning, the sections are flattened in a 40°C water bath. The flattened sections are removed with slides, and the slides are then baked at 60°C until all water evaporates. The sections are then stored at room temperature. Immerse the dried sections in Dewax Solution I, Solution II, and Solution III, sequentially, for 10 minutes each. Follow with anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and anhydrous ethanol III for 5 minutes. Rinse with distilled water for 1 minute. Immerse the dewaxed sections in antigen retrieval buffer and heat for antigen retrieval. Heat for 10 minutes, then cool for 8 minutes, and finally heat for 7 minutes. Be careful not to dry the sections during heating. After heating, allow the sections to cool to room temperature and rinse with PBS twice for 8 minutes each to remove any remaining buffer. Allow the sections to dry slightly before immersing them in 3% hydrogen peroxide and incubating at room temperature in the dark for 30 minutes. After incubation, wash the sections twice with PBS for 8 minutes each. Discard the PBS and apply 3% BSA in PBS for 30 minutes. Discard the blocking solution and apply the diluted primary antibody to the tissue, incubating overnight at 4°C. Wash the slides twice in PBS for 8 minutes each. After the sections are slightly dry, add an HRP-conjugated secondary antibody solution corresponding to the primary antibody species and incubate at room temperature for 60 minutes. Wash the slides twice in PBS for 8 minutes each. After the sections are slightly dry, add TSA fluorescent solution to the tissues and incubate at room temperature for 10 minutes in the dark. After incubation, wash the slides twice in TBST for 8 minutes each. Immerse the tissue sections in repair buffer and heat for 10 minutes, cool for 8 minutes, and then heat for 7 minutes to remove excess primary and secondary antibodies. Add another primary antibody solution diluted in PBS to the tissues and incubate overnight at 4°C. Wash the slides twice in PBS for 8 minutes each. After the sections are slightly dry, add an HRP-conjugated secondary antibody solution corresponding to the primary antibody species and incubate at room temperature for 60 minutes. Add DAPI stain to the tissues and incubate at room temperature for 15 minutes in the dark. Wash the sections twice in PBS for 8 minutes each. After the sections are dry, mount them with anti-fading mounting medium.

[0108] 1.4.2 HE staining of lung tissue

[0109] Place mouse lung tissue sections in high-definition constant stain pretreatment solution for 1 minute. Stain the sections with hematoxylin solution for 5 minutes and rinse with water for 1 minute to remove excess stain. Differentiate the sections with constant stain solution for 5 seconds and rinse with water for 20 seconds to remove excess differentiation solution. Treat the sections with constant stain bluing solution for 1 minute and rinse with water for 20 seconds to remove excess bluing solution. Dehydrate with 95% ethanol for 1 minute. Stain with eosin solution for 15 seconds. After staining, place the sections in anhydrous ethanol I, II, and III for 2 minutes each, then in n-butanol for 2 minutes, and then in xylene for 2 minutes. Finally, seal the sections with neutral gum.

[0110] 1.4.3 EVG staining of lung tissue

[0111] Prepare a mixed stain solution with a volume ratio of 5:2:2 for stain solution A, stain solution B, and stain solution C. Completely immerse the tissue sections in this solution. Stain for 6 minutes, then rinse with running water to remove excess stain. Differentiation: Prepare a gradient differentiation solution by diluting the stain solution 1:1 with PBS buffer. Follow a dilution, differentiation, and rinse cycle, with each cycle consisting of stain dilution, differentiation, and running water rinsing. Terminate differentiation when elastic fibers exhibit a characteristic black color and a clear background. Counterstaining and Dehydration: Prepare a VG stain solution with a volume ratio of 1:1 for stain solution E: stain solution D. After 2 minutes of counterstaining, rinse with running water to remove excess stain. Rapidly dehydrate by soaking in anhydrous ethanol for 10 seconds. Clearing and Mounting: Pretreat the sections in clean xylene for 20 seconds to remove residual ethanol, then clear in a separate clean xylene bath for 5 minutes. Finally, mount the sections with neutral gum.

[0112] 2. Experimental Results

[0113] One of the important phenotypes of PAH is increased pulmonary vascular resistance, which manifests as abnormal pulmonary hemodynamic indicators. Right cardiac catheterization is an important detection method for measuring pulmonary hemodynamic indicators and one of the main methods for assessing the severity of PAH. In order to comprehensively evaluate the phenotypic relationship of the small molecule compound NCT-501 on SuHx-induced pulmonary hypertension, right cardiac catheterization was first used to detect pulmonary hemodynamic indicators, the main indicators including RVSP, and RVHI was detected after dissection to reflect the changes in right ventricular remodeling in mice. The results showed that compared with the control group, the RVSP and RVHI in the SuHx modeling group were increased, indicating that the model was successfully established. Secondly, after administration of the small molecule compound NCT-501, various pulmonary hemodynamic indicators were alleviated compared with the model group, and the differences were statistically significant ( Figure 8 In summary, the results indicate that the small molecule compound NCT-501 can significantly reduce SuHx-induced elevation of pulmonary hemodynamic indicators and right ventricular hypertrophy.

[0114] The main pathological change of PAH is pulmonary vascular remodeling, especially the changes in pulmonary arterioles and pulmonary microvessels, which manifest as pulmonary vascular intimal proliferation and medial thickening. In severe cases, it can lead to vascular stenosis or even complete occlusion. Pulmonary vascular pathology is the gold standard for evaluating the severity of PAH. Therefore, HE staining and EVG staining of lung tissue sections were used to evaluate the severity of pulmonary vascular remodeling. The results showed that compared with the control group, the vascular wall thickness of the SuHx model group was significantly thickened, while the vascular wall thickness was significantly reduced after treatment with NCT-501 ( Figure 9 ).

[0115] Finally, the lung tissue sections of each group were subjected to immunofluorescence staining for endothelial cell markers (VWF, green) and smooth muscle markers (α-SMA, red). It can be observed that compared with the control group, the endothelial cell layer in the SuHx model group was significantly irregular, the original smooth inner wall disappeared, the endothelial layer was wrapped and thickened by smooth muscle cells, and the vascular lumen became narrowed. To a certain extent, this also caused an increase in right ventricular posterior resistance and increased pulmonary artery pressure. The small molecule compound NCT-501 treatment can improve the above phenomenon ( Figure 10 ).

[0116] 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 a small molecule compound in the preparation of a product for preventing and / or treating pulmonary hypertension and its complications, characterized in that: The small molecule compound is 8-[[4-(cyclopropanecarbonyl)piperazin-1-yl]methyl]-1,3-dimethyl-7-(3-methylbutyl)purine-2,6-dione, and has the structural formula shown in formula (I):

2. Use of a pharmaceutically acceptable salt of a small molecule compound in the preparation of a product for preventing and / or treating pulmonary hypertension and its complications, characterized in that: The small molecule compound is 8-[[4-(cyclopropanecarbonyl)piperazin-1-yl]methyl]-1,3-dimethyl-7-(3-methylbutyl)purine-2,6-dione, and has the structural formula shown in formula (I):

3. Use of a pharmaceutically acceptable salt of a small molecule compound 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 a pharmaceutically acceptable salt of a small molecule compound 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 any one of claims 1 to 4, characterized in that The product is a product prepared by compounding the small molecule compound represented by formula (I) or a pharmaceutically acceptable salt thereof with conventional excipients in the art.

6. The use according to claim 5, characterized in that The products include medicines, foods or health products.

7. The use according to claim 6, 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.

8. The use according to claim 5, characterized in that The effective daily dose of the small molecule compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the product is 3-30 mg / kg body weight.

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

10. The use according to claim 7, characterized in that The product is intended for use by humans or other mammals.

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