Use of a gtse1 inhibitor in the manufacture of a medicament for treating hypoxic pulmonary hypertension

Abstract

The application discloses an application of a GTSE1 inhibitor in preparation of a medicine for treating hypoxic pulmonary arterial hypertension, and belongs to the field of medicines. It is found for the first time that GTSE1-IN-1 can effectively intervene in the pathological process of hypoxic pulmonary arterial hypertension, so that GTSE1-IN-1 is used for treating hypoxic pulmonary arterial hypertension or other forms of pulmonary arterial hypertension. By using GTSE1-IN-1, abnormal proliferation and migration of pulmonary arteriole smooth muscles are inhibited, pulmonary arteriole remodeling is inhibited, and a brand-new disease modification strategy independent of vasodilation is provided for hypoxic pulmonary arterial hypertension.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the use of a GTSE1 inhibitor (GTSE1-IN-1) in the preparation of a drug for treating hypoxic pulmonary hypertension. Background Technology

[0002] GTSE1 (G2 / S phase response-related protein 1) is a key protein in tumor cells that resists DNA damage and maintains the integrity of the G2 / M checkpoint. GTSE1 inhibitors are a class of anticancer compounds that interfere with tumor cell cycle regulation and DNA damage repair by inhibiting the function of the GTSE1 protein. Among them, GTSE1-IN-1 is an orally active GTSE1 inhibitor that has been shown to inhibit the transcription and expression of GTSE1. Its structural formula is as follows: .

[0003] GTSE1-IN-1 can inhibit cancer cell proliferation by inducing cell cycle arrest and cellular senescence through persistent DNA damage, and has been used in cancer research, such as for colon and lung cancer.

[0004] Hypoxic pulmonary hypertension (HPH) is a syndrome caused by prolonged hypoxia, leading to increased pulmonary vascular resistance and pulmonary microvascular remodeling, resulting in a sustained increase in pulmonary artery pressure. It can ultimately lead to right heart failure and is one of the most common complications of various chronic lung diseases. Currently, clinical treatment for HPH primarily involves oxygen therapy, but this only relieves hypoxic symptoms and cannot reverse pulmonary vascular remodeling. Furthermore, although targeted therapies for pulmonary hypertension are available, current clinical evidence does not support their efficacy and safety in patients with hypoxic pulmonary hypertension; on the contrary, these drugs may increase the risk of pulmonary edema. Based on the above, international expert consensus clearly recommends that targeted therapies for pulmonary hypertension should not be routinely used to treat hypoxic pulmonary hypertension. Therefore, in-depth research into the mechanisms of pulmonary vascular remodeling in hypoxic pulmonary hypertension and the discovery of potential therapeutic targets are of significant clinical importance. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, the present invention provides the application of a GTSE1 inhibitor in the preparation of a drug for treating hypoxic pulmonary hypertension.

[0006] In a first aspect, the present invention provides the use of a reagent for reducing the expression level of the GTSE1 gene or protein, which is achieved by the following technical solution.

[0007] The use of a reagent that reduces the expression level of the GTSE1 gene or protein in the preparation of drugs for the prevention and / or treatment of hypoxic pulmonary hypertension.

[0008] Furthermore, agents that reduce the expression level of the GTSE1 gene or protein include GTSE1 inhibitors.

[0009] Specifically, the GTSE1 inhibitor is GTSE1-IN-1.

[0010] Secondly, the present invention provides a drug for treating hypoxic pulmonary hypertension, which is achieved by the following technical solution.

[0011] A drug for treating hypoxic pulmonary hypertension includes agents that reduce the expression level of the GTSE1 gene or protein.

[0012] Furthermore, agents that reduce the expression level of the GTSE1 gene or protein include GTSE1 inhibitors.

[0013] Specifically, the GTSE1 inhibitor is GTSE1-IN-1.

[0014] In one embodiment of this application, the drug further includes a pharmaceutically acceptable salt, solvate, or hydrate of GTSE1-IN-1.

[0015] This application has the following beneficial effects: 1. This invention discloses for the first time a novel pharmaceutical use of GTSE1-IN-1. In the prior art, GTSE1-IN-1 has only been reported as an orally active GTSE1 inhibitor used to inhibit the proliferation of cancer cells, including colon and lung cancer. Currently, there are no technological indications or literature reports on the use of GTSE1-IN-1 to treat HPH or other forms of pulmonary hypertension. This invention is the first to discover that GTSE1-IN-1 can effectively intervene in the pathological process of HPH.

[0016] 2. This invention solves a long-standing technical problem in the treatment of HPH. The core pathological mechanism of HPH is hypoxia-induced pulmonary vascular remodeling, and there is a lack of drugs clinically capable of reversing vascular wall thickening, fibrosis, and myofibrosis. Conventional pulmonary vasodilators (such as prostacyclin analogs and endothelin receptor antagonists) not only have unclear efficacy in HPH, but may also exacerbate ventilation / perfusion mismatch and increase the risk of pulmonary edema. International consensus clearly states that the use of the above-mentioned targeted drugs is not recommended. This invention, by using GTSE1-IN-1, inhibits the abnormal proliferation of pulmonary arteriolar smooth muscle, providing a novel disease modification strategy for HPH that is independent of vasodilation.

[0017] 3. This invention is not obvious. First, GTSE1 (G2 / S phase response-related protein 1) has traditionally been considered a key protein for tumor cells to resist DNA damage and maintain the integrity of the G2 / M checkpoint, and those skilled in the art have no motivation to use tumor cell cycle inhibitors to treat hypoxic pulmonary vascular disease. Second, hypoxia itself induces cellular DNA damage, and the expression changes of GTSE1 under hypoxic conditions and its regulation of vascular wall cell fate (proliferation, senescence, apoptosis) were previously unclear. This invention, through inventive experiments, reveals for the first time the crucial role of GTSE1 in the pathogenesis of HPH and demonstrates the therapeutic effect of its specific inhibitor, GTSE1-IN-1, a finding that cannot be directly derived from existing anticancer applications. Attached Figure Description

[0018] Figure 1 This is a diagram showing the results of the GTSE1-IN-1 method reducing RVSP in mice according to the present invention. In the diagram, A. Immunofluorescence staining shows the expression of GTSE1 (green) and α-SMA (red) in the pulmonary arteries of the three groups of mice; B. Mean right ventricular systolic pressure measured by right heart catheterization;

[0019] Figure 2 This is a graph showing the results of the GTSE1-IN-1 method of the present invention reducing the medial thickness of pulmonary arterioles and the right ventricular hypertrophy index in mice. A. HE staining shows the thickness of the pulmonary vessel wall; B. vessel wall thickness; C. right ventricular hypertrophy index (RVHI), ***=P<0.001, ****=P<0.0001, n=10;

[0020] Figure 3 This is a graph showing the results of the GTSE1-IN-1 inhibitory effect on the proliferation and migration of PASMCs in this invention. A. Western blot analysis showed that the inhibitor GTSE1-IN-1 could reduce the expression of GTSE1; B. CCK8 assay was used to determine cell proliferation capacity; C. Scratch assay was used to determine cell migration activity. *=P<0.05, ***=P<0.001, n=3.

[0021] Figure 4 This is a graph showing the results of the GTSE1-IN-1 of the present invention having no effect on liver and kidney function in mice. In the graph, A represents the creatinine level in mouse plasma; B represents the aspartate aminotransferase level in mouse plasma; and C represents the alanine aminotransferase level in mouse plasma.

[0022] Figure 5This is a graph showing the effects of the GTSE1-IN-1 of the present invention on blood inflammation and erythrocyte-related indicators in mice. In this graph, AE represents the number of white blood cells, neutrophils, lymphocytes, monocytes, and basophils, respectively; and FJ represents the number of red blood cells, hemoglobin content, hematocrit, mean erythrocyte volume, and mean erythrocyte hemoglobin concentration, respectively.

[0023] Figure 6 This is a graph showing the effect of the GTSE1-IN-1 of the present invention on platelet-related indicators in mouse blood, where AD represents platelet count, platelet distribution width, mean platelet volume, and platelet hematocrit, respectively. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials used in the preparation process in the following embodiments have not undergone further processing and are all commercially available.

[0025] I. Experimental Procedure 1. Experimental Materials and Reagents

[0026] The GTSE1-IN-1 used in this invention was purchased from MedChemExpress (catalog number HY-162808), CAS number 3002034-34-3, with a molecular weight of 393.46 and a molecular formula of C. 21 H 24 FN7. GTSE1-IN-1 is a powdered solid. Store at -20°C in a dry, light-protected environment. Prepare a working solution of the required concentration before use.

[0027] Laboratory animals: Healthy 7-week-old SPF-grade male C57 / BL mice. All animals were housed in an SPF-grade animal facility at a temperature maintained at 22±2℃, relative humidity at 50%±10%, with a 12-hour / 12-hour circadian rhythm, and free access to food and water. All animal experimental protocols were approved by the institution's laboratory animal ethics committee.

[0028] 2. Establishment of an animal model of hypoxic pulmonary hypertension (HPH) This invention establishes an HPH animal model using hypoxia combined with SU5416. The specific steps are as follows:

[0029] (1) Animal grouping: After 1 week of acclimatization, C57 / BL mice were randomly divided into the following three groups using a random number table: ① Normal control group (noroxic + control solvent), ② Model group (SU5416 / hypoxia + control solvent), ③ GTSE1-IN-1 treatment group (SU5416 / hypoxia model + GTSE1-IN-1 administration).

[0030] (2) Model preparation conditions: Animals in the model group and treatment group were placed in a fully automated low-pressure hypoxic chamber. The oxygen concentration in the hypoxic chamber was maintained at 10%±0.5%. The temperature inside the chamber was maintained at 22-25℃. A blower circulation device was used to ensure uniform gas mixing inside the chamber, and the carbon dioxide concentration was controlled to be below 0.5% by a soda lime absorption device. During the modeling period, the changes in animal weight and general behavioral performance were recorded daily.

[0031] 3. Experimental Intervention Protocol GTSE1-IN-1 Dosage Regimen: The dosage of GTSE1-IN-1 is 30 mg / kg. The specific preparation method is as follows: Based on a dose of 30 mg / kg, use DMSO as the solvent and PEG300 and Tween-80 as co-solvents to prepare the drug solution. For example, if 10 mice weighing 20g each need to be injected with the drug, a total of 6mg of drug is required, dissolved in 200μL of DMSO (ultrasonic dissolution) to prepare the stock solution. Working solution formulation: 200μL stock solution + 800μL PEG300 + 100μL Tween-80 + 900μL physiological saline. Administer 200μL of GTSE1-IN-1 working solution intraperitoneally to each mouse in the treatment group every other day.

[0032] Normal control group (normative oxygen + control solvent, represented by "Nor" in the attached figure) dosing regimen: Mice were placed in a normoxic (21% O2) environment and injected intraperitoneally with 200 μL of control solvent (1% DMSO) every two days while the mice were observed. They were fed a free diet for 28 days.

[0033] The model group (SU5416 / hypoxia + control solvent, represented by "Su / Hyp+DMSO" in the attached figure) was treated as follows: Mice were placed in a hypoxic (10%±0.5%O2) incubator and injected intraperitoneally with 200 μL of control solvent (1% DMSO) every two days. Su5416 (Semaxanib, 20 mg / kg) was injected subcutaneously every week, and the mice were observed. They were fed a free diet for 28 days.

[0034] The GTSE1-IN-1 treatment group (SU5416 / hypoxia + GTSE1-IN-1, represented by "Su / Hyp + GTSE1-IN-1" in the attached figure) was administered the following regimen: Mice were placed in a hypoxic environment (10% ± 0.5% O2), and 200 μL of GTSE1-IN-1 (30 mg / kg) drug solution was injected intraperitoneally every two days. Su5416 (20 mg / kg) was injected subcutaneously every week, and the mice were observed. They were fed a free diet for 28 days.

[0035] (2) Treatment period: The entire experimental period was 30 days. During the experiment, the animals' weight changes, general condition, feeding and activity were recorded daily. All animals underwent specimen collection and various indicator tests after the experiment.

[0036] 4. Pharmacodynamic evaluation indicators and detection methods 4.1 Determination of hemodynamic parameters by right heart catheterization

[0037] To evaluate the effects of GTSE1-IN-1 on pulmonary artery pressure and right ventricular function in HPH model rats, this invention employs right ventricular catheterization for invasive hemodynamic testing.

[0038] Detailed operation steps: 1) Experimental animals were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg); the animals were fixed in a supine position on the operating table, the skin of the neck was cut along the midline, the right external jugular vein was separated and exposed to a length of about 3 cm, and the distal end was ligated with silk thread; a Millar catheter filled with heparinized saline (250 IU / mL) was inserted into the right external jugular vein and entered the right ventricle through the right atrium;

[0039] 2) Use a PowerLab multi-channel physiological recorder (ADInstruments, Australia) to continuously record pressure waveforms. After the waveforms stabilize, continue recording for 5 minutes. Determine the catheter tip position based on the morphological characteristics and pressure values ​​of the pressure waveforms (right ventricular pressure waveforms are high-amplitude, narrow waveforms with a peak pressure of approximately 30 / 0 mmHg; pulmonary artery waveforms are low-amplitude, flat waveforms with a peak pressure of approximately 25 / 10 mmHg). Record the following parameters: mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP). After recording, slowly withdraw the catheter, ligate the external jugular vein stump, and suture the skin layer by layer.

[0040] 4.2 Determination of the right ventricular hypertrophy index After the experiment, the heart was removed via thoracotomy. Atrial tissue was excised along the atrioventricular groove, and the right ventricle (RV) and left ventricle plus interventricular septum (LV+S) were separated along the edge of the interventricular septum. After blotting off surface moisture with filter paper, each ventricle was weighed on a precision electronic balance. The right ventricular hypertrophy index (RVHI) was calculated: RVHI = RV / (LV+S). This index reflects the long-term changes in right ventricular afterload and is one of the core pathological indicators for assessing the severity of pulmonary hypertension.

[0041] 4.5 Observation of pulmonary vascular morphological changes by HE staining of lung tissue Tissue from the right middle lobe of rat lung was fixed in 4% paraformaldehyde, followed by routine gradient ethanol dehydration, xylene clearing, and paraffin embedding to prepare 4 μm thick tissue sections. After routine dewaxing and hydration, the sections underwent hematoxylin-eosin (HE) staining: hematoxylin staining for 5-10 minutes, differentiation with hydrochloric acid alcohol for a few seconds, eosin staining for 3-5 minutes, followed by gradient ethanol dehydration, xylene clearing, and mounting with neutral resin. The morphology of pulmonary vessels and surrounding tissues was observed under an optical microscope (200× and 400× magnification), and the following indicators were evaluated:

[0042] Pulmonary arteriole wall thickening: wall area percentage WA% = (total vessel area − lumen area) / total vessel area.

[0043] 4.6 Western blot detection of GTSE1 protein expression in lung tissue Rat lung tissue was collected and homogenized in RIPA lysis buffer (containing 1% PMSF). The homogenate was centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA method. Equal amounts of protein (30 μg / sample) were separated by 10% SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk powder for 1 hour, and incubated with GTSE1 primary antibody (1:1,000 dilution, Abcam) and GAPDH internal control antibody overnight at 4°C. The next day, HRP-labeled secondary antibody was incubated for 1 hour, and the membrane was developed using ECL chemiluminescence. Band gray values ​​were analyzed using QuantityOne software. The relative expression level of GTSE1 protein was calculated as (target protein band gray value - background gray value) / (internal control protein band gray value - background gray value). The downregulation effect of GTSE1-IN-1 on GTSE1 expression in HPH model lung tissue was verified by comparing the expression differences between the treatment group, model group, and control group.

[0044] 5. In vitro experiments to verify the inhibitory effect of GTSE1-IN-1 on the proliferation of pulmonary artery smooth muscle cells under hypoxic conditions.

[0045] To further verify the target and cellular mechanism of GTSE1-IN-1, this invention also constructed a hypoxic culture model of primary pulmonary artery smooth muscle cells (PASMCs) for in vitro verification experiments.

[0046] 5.1 Isolation and Culture of Primary PASMCs The pulmonary artery trunk and branches were harvested from healthy SD rats (8-10 weeks old, weighing 200-250 g). Under aseptic conditions, the thoracic cavity was opened, the pulmonary artery trunk was dissected down to the secondary branches, and the adventitia was peeled off. The smooth muscle layer of the media was then digested with collagenase II (2 mg / mL). Cell clusters from the digestion solution were collected and seeded in DMEM / F12 medium containing 20% ​​fetal bovine serum (FBS) and cultured in a cell culture incubator at 37°C and 5% CO2. α-SMA expression was identified by immunofluorescence staining, and cells with a purity >95% were used for subsequent experiments. Cells from passages 3-5 were used in this study.

[0047] 5.2 Hypoxia Cell Culture Model Cells in the model group and treatment group were placed in a hypoxic incubator, maintaining an oxygen concentration of 1% (5% CO2, 94% N2) and a temperature of 37℃, and cultured for 24-72 hours. The normal control group was cultured for the same amount of time under normoxic conditions (21% O2, 5% CO2).

[0048] 5.3 Edu incorporation assay to detect cell proliferation activity PASMCs were seeded at 5 × 10³ / well in 96-well plates. After 48 hours of hypoxia treatment, Edu working solution (final concentration 10 μmol / L) was added, and incubation continued for 2 hours. After fixation with 4% paraformaldehyde, permeabilization with 0.5% Triton X-100, and staining with Apollo staining solution in the dark for 30 minutes, the cell nuclei were counterstained with DAPI. Five fields of view (200×) were randomly photographed from each well under a confocal microscope, and the proportion of Edu-positive cells (proliferating cells) to DAPI-positive cells (total cells) was counted using ImageJ software.

[0049] 6. GTSE1-IN-1 treatment for 30 days did not affect the levels of creatinine (CR), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in mouse plasma. Mice were treated with GTSE1-IN-1 for 30 days, then fasted for 4 hours. Blood was collected from the orbital vein and centrifuged at 4°C for 10 minutes to separate the plasma. The levels of creatinine (CR), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the mouse plasma were detected using a Hitachi 3100 fully automated biochemical analyzer.

[0050] 7. Effects of GTSE1-IN-1 treatment for 30 days on complete blood counts in mice Mice were treated with GTSE1-IN-1 for 30 days, then fasted for 4 hours. Blood was collected from the orbital vein, and the blood was thoroughly mixed with the anticoagulant and diluent immediately after collection before analysis. Mouse blood samples were analyzed using a Sysmex XN-1000V complete blood count analyzer.

[0051] II. Experimental Results 1. GTSE1-IN-1 significantly reduces pulmonary artery pressure and improves hemodynamics.

[0052] In an animal model of hypoxic pulmonary hypertension (HPH), treatment with GTSE1-IN-1 (30 mg / kg, intraperitoneal injection every other day for 30 consecutive days) significantly reduced right ventricular systolic pressure (RVSP) in the model animals. This effect indicates that GTSE1-IN-1 can effectively alleviate hypoxia-induced pulmonary vascular resistance and restore hemodynamic homeostasis. Figure 1 ).

[0053] 2. Inhibits pulmonary arteriolar myoplasia and remodeling, reversing vascular pathological changes. Through α-SMA immunohistochemical staining and morphological quantitative analysis, this invention confirms that GTSE1-IN-1 can significantly inhibit hypoxia-induced pulmonary arteriolar media thickness (PAL) reduction. Figure 2 ).

[0054] 3. GTSE1-IN-1 reverses right ventricular hypertrophy and reduces cardiac afterload. GTSE1-IN-1 treatment significantly reduced the right ventricular hypertrophy index (RVHI) in HPH model rats, indicating that the drug can long-term alleviate compensatory right ventricular hypertrophy, delay or prevent the onset of right heart failure, and improve overall cardiac function. Figure 2 ).

[0055] 4. Downregulate GTSE1 protein expression to block key pathological signals. Western blot results showed that GTSE1-IN-1 significantly reduced the expression level of GTSE1 protein in the lung tissue of HPH model rats, verifying the targeted inhibitory effect of the drug in vivo. Figure 3 ).

[0056] 5. Inhibits abnormal proliferation of pulmonary artery smooth muscle cells in vitro In a primary pulmonary artery smooth muscle cell (PASMC) hypoxia (1% O2) culture model, GTSE1-IN-1 inhibited cell proliferation and migration, further demonstrating its direct anti-vascular remodeling cellular mechanism. Figure 3 ).

[0057] 6. GTSE1-IN-1 treatment for 30 days did not affect the levels of creatinine (CR), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in mouse plasma. The results showed that GTSE1-IN-1 had no significant effect on CR, ALT, and AST levels in mice, suggesting that it does not produce hepatotoxicity or nephrotoxicity. Figure 4 ).

[0058] 7. Effects of GTSE1-IN-1 treatment for 30 days on complete blood counts in mice The results showed that GTSE1-IN-1 treatment had no significant effect on inflammation-related indicators (white blood cell count, neutrophil count, lymphocyte count, monocyte count, basophil count) in mouse blood; and no significant effect on erythrocyte-related indicators (erythrocyte count, hemoglobin content, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration) in mouse blood. Figure 5 It had no significant effect on platelet-related parameters in mouse blood (platelet count, platelet distribution width, mean platelet volume, and plateletcrit). Figure 6 ).

[0059] Compared with existing simple oxygen therapy (which only relieves hypoxemia and cannot reverse vascular remodeling) and conventional targeted drugs for pulmonary hypertension (such as endothelin receptor antagonists and prostacyclin analogs, whose efficacy in HPH is unclear and may increase the risk of pulmonary edema), the GTSE1-IN-1 provided by this invention offers a novel treatment strategy for HPH that can be taken orally or injected, can reverse disease progression, and has good safety by inhibiting GTSE1-mediated pulmonary arteriolar myoplasia and remodeling.

[0060] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The use of an agent that reduces the expression level of the GTSE1 gene or protein in the preparation of drugs for the prevention and / or treatment of hypoxic pulmonary hypertension, characterized in that: The reagent that reduces the expression level of the GTSE1 gene or protein is a GTSE1 inhibitor, specifically GTSE1-IN-1.

Images