CN112755015A

CN112755015A - Application of PT2385 in preparation of medicine for preventing and treating pulmonary hypertension

Info

Publication number: CN112755015A
Application number: CN202110136271.XA
Authority: CN
Inventors: 高钰琪; 陈德伟; 王授衔; 徐刚
Original assignee: Army Medical University
Current assignee: Army Medical University
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2021-05-07

Abstract

The invention relates to an application of PT2385 in preparation of a medicine for preventing and treating pulmonary hypertension, and belongs to the technical field of biomedicine. The invention provides a new medicinal application of PT2385, namely an application of PT2385 in preparing a medicament for preventing and treating pulmonary hypertension. The medicine prepared by utilizing the PT2385 can well prevent and treat pulmonary hypertension, and has no obvious adverse reaction.

Description

Application of PT2385 in preparation of medicine for preventing and treating pulmonary hypertension

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of PT2385 in preparing a medicine for preventing and treating pulmonary hypertension.

Background

Pulmonary Arterial Hypertension (PAH) refers to a hemodynamic and pathophysiological state in which Pulmonary arterial pressure rises above the normal range, and may be an independent disease, or a complication, or a syndrome. Pulmonary hypertension is mainly characterized by: short breath, hypodynamia, dizziness, chest pain, palpitation, syncope and edema of lower limbs after exercise. The diagnostic standard of hemodynamics is that the average pulmonary artery pressure measured by a right heart catheter is more than 25mmHg under the resting state of sea level. The pulmonary vascular disease treatment method is mainly characterized by persistent pulmonary vasoconstriction and pulmonary vascular reconstruction, which are characterized in that the pulmonary vascular wall is thickened, the pulmonary vascular is narrowed, the pulmonary vascular is blocked, and the pulmonary vascular resistance is progressively increased, so that the right heart load is aggravated, further the right heart dysfunction and the structural reconstruction are caused, and finally the right heart failure and even death are caused. Therefore, pulmonary hypertension is also the central link of the onset of pulmonary heart disease and high altitude heart disease, and the proper and effective reduction of pulmonary artery pressure is also the key measure for delaying the onset of pulmonary heart disease and high altitude heart disease.

The pathogenesis of the pulmonary hypertension is complex, and the pulmonary hypertension has comprehensive effects in the pathogenesis process without environmental, genetic and pulmonary vasculitis reactions. According to the pathogenesis characteristics, pulmonary hypertension can be classified into 5 types: 1. idiopathic and hereditary pulmonary hypertension; 2. pulmonary hypertension due to left heart disease; 3. pulmonary hypertension due to pulmonary disease or hypoxia; 4. pulmonary hypertension due to thromboembolic disease; 5. pulmonary hypertension is caused by other causes. However, pulmonary hypertension caused by different reasons can cause pulmonary hemodynamic abnormality, finally cause pulmonary insufficiency and cardiac dysfunction, cause hypoxia to occur to the body, and further promote pulmonary arterial pressure rise and right heart failure to form vicious circle due to hypoxia; the oxygen inhalation therapy is also an important measure for reducing the pulmonary artery pressure and delaying the development of the disease. Therefore, the pulmonary hypertension animal model reproduced by chronic hypoxia treatment is an important and representative pulmonary hypertension animal model, can well reflect the pathophysiological characteristics of pulmonary hypertension, and is also a common model for drug efficacy research of pulmonary hypertension.

At present, oxygen therapy, vasodilators (such as prostaglandin, endothelin receptor antagonist and the like) and the like are mainly used for treating pulmonary hypertension, so that the survival rate of a patient is improved to a certain extent, but the occurrence of pulmonary vascular remodeling cannot be effectively inhibited and reversed. At the same time, there is also a lack of drugs that are directed against the inflammatory response of pulmonary vessels to inhibit or reverse pulmonary vascular remodeling. In recent years, it has been found that pulmonary vasculitis response is an important mechanism for promoting pulmonary vascular remodeling. Adhesion molecules secreted by endothelial cells, such as intercellular adhesion molecule 1 (ICAM 1), are important adhesion molecules that mediate adhesion of circulating inflammatory immune cells to the vascular wall. The research shows that chronic hypoxia can induce the adhesion molecules such as ICAM1, CAM-1, E-selectin and the like in the lung vascular endothelial cells to be obviously and highly expressed, further promote the adhesion and aggregation of inflammatory cells around blood vessels and secrete a large amount of inflammatory mediators to promote the reaction of pulmonary vasculitis, and further promote the pulmonary vascular remodeling. Research suggests that inhibition of the expression of lung vascular endothelial cell adhesion molecules may serve as a target for pulmonary hypertension therapy.

PT2385 is a specific inhibitor of HIF-2 alpha, and can inhibit the interaction between HIF-2 alpha and ARNT, and further inhibit the expression of HIF-2 alpha downstream gene. The medicine has good safety and effectiveness in animal models and clinical experiments of the renal cell carcinoma.

Disclosure of Invention

The invention aims to provide application of PT2385 in preparation of a medicine for preventing and treating pulmonary hypertension. The medicine prepared by the application of the invention can well prevent and treat pulmonary hypertension, and has no obvious adverse reaction.

The invention provides application of PT2385 in preparation of a medicine for preventing and treating pulmonary hypertension.

The invention also provides application of PT2385 in preparing a medicine for reducing right ventricular systolic pressure.

The invention also provides application of PT2385 in preparing a medicine for inhibiting right ventricular hypertrophy.

The invention also provides application of PT2385 in preparing a medicament for inhibiting pulmonary vascular remodeling.

The invention also provides application of PT2385 in preparing a medicament for preventing and treating pulmonary heart disease.

The invention also provides application of PT2385 in preparing a medicament for preventing and treating the plateau heart disease.

The invention also provides application of PT2385 in preparation of a medicine for inhibiting high expression of hypoxia-induced cell adhesion molecules.

The invention also provides application of PT2385 in preparation of a medicament for inhibiting hypoxia-induced inflammatory cell adhesion to pulmonary vascular endothelial cells.

The invention also provides a medicine for preventing and treating pulmonary hypertension, which comprises PT2385 and a pharmaceutically acceptable carrier or auxiliary material; the auxiliary materials comprise one or more of dimethyl sulfoxide, sodium carboxymethylcellulose, tween 80 and physiological saline.

Preferably, the medicament dosage form comprises injection, powder injection, capsules, granules, spray, microcapsules, tablets or transdermal controlled release patches.

The invention provides application of PT2385 in preparation of a medicine for preventing and treating pulmonary hypertension. Experimental research shows that the PT2385 is used for preventing and treating pulmonary hypertension, can obviously reduce pulmonary artery pressure, improve pulmonary vascular remodeling, relieve pressure load of right ventricle, improve myocardial hypertrophy and ventricular remodeling, delay the occurrence of heart failure, show good effect of preventing and treating pulmonary hypertension, have no obvious adverse reaction in the experimental process, and is expected to be developed into a new generation of safe and effective medicament for preventing and treating pulmonary hypertension.

Drawings

FIG. 1 is a graph showing the effect of PT2385 provided by the present invention on the right ventricular systolic pressure of a mouse model of chronic hypoxic-induced Hypoxic Pulmonary Hypertension (HPH);

FIG. 2 is a graph showing the effect of PT2385 provided by the present invention on the right ventricular hypertrophy index (Hermann Wilson index) in a mouse model of HPH;

FIG. 3 is a section staining of the effects of PT2385 provided by the present invention on the reconstruction of pulmonary vascular structures of different sizes of HPH mouse models;

FIG. 4 is a graph showing the effect of PT2385 provided by the present invention on the wall thickness of pulmonary aorta, middle artery and arteriole vessels of an HPH mouse model;

FIG. 5 is a graph showing the effect of PT2385 provided by the present invention on the mRNA expression of the adhesion molecule ICAM1 in hypoxia-treated HPAEC;

FIG. 6 is a graph showing the effect of PT2385 provided by the present invention on protein expression of adhesion molecule ICAM1 in hypoxia-treated HPAEC;

FIG. 7 is a graph showing the effect of PT2385 provided by the present invention on HPAEC adhesion THP-1 after hypoxia treatment.

Detailed Description

The invention provides application of PT2385 in preparation of a medicine for preventing and treating pulmonary hypertension. The source of the PT2385 is not particularly limited in the present invention, and conventional PT2385 commercially available products known to those skilled in the art may be used, and in the specific embodiment of the present invention, PT2385 is preferably available from mce (mcchemexpress), cat #: HY-12867. PT2385 has a molecular formula of C₁₇H₁₂F₃NO₄S, CAS No.1672665-49-4, the structural formula is shown in formula I:

the PT2385 shows good safety and effectiveness in large-scale population clinical experiments, and the results of the embodiment of the invention show that the PT2385 prepared into the medicament for preventing and treating pulmonary hypertension can obviously reduce the pulmonary arterial pressure rise induced by hypoxia, improve pulmonary vascular remodeling caused by chronic hypoxia, relieve the pressure load of the right ventricle, improve myocardial hypertrophy and ventricular remodeling and delay the progress of heart failure.

The invention also provides application of PT2385 in preparing a medicine for reducing right ventricular systolic pressure. Test results show that compared with the normal pressure and normal oxygen group, the right ventricular systolic pressure of mice in the hypoxic pulmonary hypertension model group is obviously increased; compared with the hypoxic pulmonary hypertension model group, the right ventricular systolic pressure of the mice in the PT2385 treatment group is obviously reduced, which shows that the PT2385 can obviously reduce the right ventricular systolic pressure.

The invention also provides application of PT2385 in preparing a medicine for inhibiting right ventricular hypertrophy. Test results show that PT2385 can obviously inhibit right ventricular hypertrophy and improve right ventricular function.

The invention also provides application of PT2385 in preparing a medicament for inhibiting pulmonary vascular hypertrophy. Test results show that PT2385 can obviously inhibit the thickening of pulmonary vessel walls and improve pulmonary vessel remodeling.

The invention also provides application of PT2385 in preparing a medicament for preventing and treating pulmonary heart disease. As the pulmonary hypertension is a central link of the onset of the pulmonary heart disease, the test result shows that the PT2385 can obviously improve pulmonary vascular remodeling, reduce right ventricular systolic pressure and inhibit right ventricular hypertrophy, and can also effectively delay the progress of the pulmonary heart disease.

The invention also provides application of PT2385 in preparing a medicament for preventing and treating the plateau heart disease. In view of the fact that pulmonary hypertension is a central link of the onset of the plateau heart disease, experimental results show that PT2385 can significantly improve pulmonary vascular remodeling induced by the plateau hypoxia, reduce right ventricular systolic pressure and inhibit right ventricular hypertrophy, and can also effectively delay the progress of the plateau heart disease.

The invention also provides application of PT2385 in preparation of a medicine for inhibiting high expression of hypoxia-induced cell adhesion molecules. The test result shows that PT2385 can obviously reduce the expression of mRNA and protein of ICAM1 in pulmonary artery endothelial cells increased by hypoxia induction, thereby relieving the occurrence of pulmonary vasculitis.

The invention also provides application of PT2385 in preparation of a medicament for inhibiting hypoxia-induced inflammatory cell adhesion to pulmonary vascular endothelial cells. The test result shows that PT2385 can obviously reduce the number of monocyte THP-1 cells adhered to the surface of pulmonary artery endothelial cells after hypoxia treatment.

PT2385 can inhibit endothelial cells from secreting cell adhesion molecules, inhibit adhesion of the cells to circulating inflammatory cells, and reduce pulmonary vasculitis, so as to reduce pulmonary artery pressure and right ventricular pressure, delay right heart failure, and improve survival rate of patients with pulmonary hypertension.

The invention also provides a medicine for treating and preventing pulmonary hypertension, which comprises PT2385 and a pharmaceutically acceptable carrier or auxiliary material; the auxiliary materials comprise one or more of dimethyl sulfoxide, sodium carboxymethylcellulose, tween 80 and physiological saline. In the invention, the PT2385 content in the medicine is preferably 0.1-1% by mass, and more preferably 0.2% by mass. In the invention, when the auxiliary material is dimethyl sulfoxide, PT2385 is preferably prepared into 1mM stock solution by using dimethyl sulfoxide as a solvent, and is preferably diluted to a final concentration of 10 μ M before use. In the invention, when the auxiliary materials are sodium carboxymethylcellulose, tween 80, dimethyl sulfoxide and normal saline, the mass percentage content of the sodium carboxymethylcellulose in the medicine is preferably 0.4-0.8%, and more preferably 0.5%; the volume percentage content of the Tween 80 in the medicine is preferably 2-3%, and more preferably 2.5%; the volume percentage content of the dimethyl sulfoxide in the medicine is preferably 2-3%, more preferably 2.5%, and the normal saline is a solvent.

The application of PT2385 in the preparation of a medicament for treating hypoxic pulmonary hypertension is described in further detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.

Example 1

PT2385 Observation of protective Effect in hypoxia-induced pulmonary hypertension model

PT2385 purchased from MCE, cat #: HY-12867.

The preparation method comprises the following steps: PT2385 is dissolved in 0.5% (by weight) sodium carboxymethylcellulose, 2.5% (by volume) Tween 80, and 2.5% (by volume) dimethyl sulfoxide in physiological saline, and then the mixture is subjected to intragastric administration (20mg/kg) for 2 days by using C57BL/6 mice.

Experimental methods

SPF male C57BL/6 mice (purchased from Schleickzeda laboratory animals Co., Ltd., the same below) were selected for 6-8 weeks and randomly divided into three groups: 7 normoxic groups (C), 6 hypoxic groups (H), and 8 hypoxic + PT groups (H + PT-2385, 20mg/kg/2d, gavage). A hypoxic pulmonary arterial hypertension (HPH) mouse model is replicated by adopting a persistent hypoxic-hypoxic method, namely hypoxic mice are placed in a plateau hypoxic chamber with a simulated altitude of 5000m for continuous hypoxic treatment for 28 days. Then weighing the mouse, inhaling isoflurane (1-3%) for anesthesia, fixing the mouse on an operating table in a supine position, making a median incision on the neck, dissociating the external jugular vein on the right side, inserting a silicone tube anticoagulated by heparin into the right ventricle through the right atrium, connecting the pressure transducer with a Power Lab signal acquisition system, and recording the pressure of the right ventricle of the mouse. After the hemodynamic index is collected, the mouse is killed by a blood-letting method, the chest cavity is opened, the trachea part is clamped, the lung and the heart are separated together and placed on ice, the heart is taken down, the atrium and the surrounding vascular tissues are carefully cut off, the position of the pulmonary artery outlet is found, Right Ventricular tissue (Right Ventricular, RV) is separated from the pulmonary artery outlet along the Ventricular septum, and the rest tissue is the Left ventricle and the Ventricular septum (LV + S). Respectively weighing and calculating RV/(LV + S), namely the right ventricular hypertrophy index, namely Hermann Wilson index; RV/BW (body weight) is the right ventricular body weight index (RVWI).

After the heart is dissociated, the left upper lung is dissociated and placed in a wax plate on ice, the lung tip part is removed by a blade, the middle part of the lung tip part is transversely cut into a tissue block with the thickness of about 0.5cm, the tissue block is placed in 4% paraformaldehyde for fixation for 1 to 3 days, the tissue block is continuously shaken in the process, the vacuum pumping is used for promoting the gas in the lung to be discharged, after the lung tissue is completely sunk to the bottom of a glass bottle, the lung tissue is placed in 70% ethanol, and paraffin embedding slicing, dyeing, microscopic observation and photographing are further carried out. And respectively measuring the outer diameter and the inner diameter of the pulmonary artery blood vessel with the tube diameter of less than 50 microns, 50-100 microns and more than 100 microns, and respectively calculating the thickness (WT) of the blood vessel wall.

The results are shown in tables 1 and 2 and FIGS. 1 to 4. Wherein, figure 1 is the effect of PT2385 on right ventricular systolic blood pressure of a mouse model of chronic hypoxic-induced Hypoxic Pulmonary Hypertension (HPH); FIG. 2 is a graph of the effect of PT2385 on the Hermann Wilson index mouse model of HPH; FIG. 3 is a section staining of PT2385 on the effect of different size pulmonary vascular structure reconstruction in HPH mouse model; figure 4 is a graph of the effect of PT2385 on the wall thickness of pulmonary aorta, middle artery and arteriole vessels in a mouse model of HPH.

As can be seen from table 1 and fig. 1, right ventricular systolic pressure of mice in the hypoxic pulmonary hypertension model group was significantly increased compared to that in the normbaric normoxic group; compared with the hypoxic pulmonary hypertension model group, the PT2385 treatment group mice have significantly reduced right ventricular systolic pressure.

TABLE 1 Effect of PT2385 on Right ventricular systolic pressure in a mouse model of chronic hypoxic-induced Hypoxic Pulmonary Hypertension (HPH)

As can be seen from table 2 and fig. 2 (since one mouse per group was used for fixation, no value was measured for the mice for fixation), right ventricular hypertrophy index (i.e., Hermann Wilson index) was significantly increased in the hypoxic pulmonary hypertension model group compared to the normotensive group, thereby demonstrating that hypoxia for 28 days significantly affects right ventricular function and significantly thickens the right ventricle; compared with the hypoxic pulmonary hypertension model group, the right ventricular hypertrophy index (namely Hermann Wilson index) of the mice in the PT2385 treatment group is remarkably reduced, so that the PT2385 can remarkably inhibit the right ventricular hypertrophy and improve the right ventricular function.

TABLE 2 influence of PT2385 on the HPH mouse model Hermann Wilson index

As can be seen from fig. 3 and 4, compared with the normal pressure and normal oxygen group, the wall thickness of each level of pulmonary blood vessel (<50 μm, 50-100 μm, >100 μm) of the mouse in the hypoxic pulmonary artery hypertension model group is significantly increased, and the structure reconstruction is obvious; compared with the hypoxic pulmonary hypertension model group, the thickness of each level pulmonary vessel (<50 μm, 50-100 μm, >100 μm) wall of the mice in the PT2385 treated group is obviously reduced, so that the PT2385 can obviously inhibit the pulmonary vessel hypertrophy and improve the pulmonary vessel reconstruction.

Example 2

Regulation of endothelial cell adhesion potential under chronic hypoxia PT2385

PT2385 purchased from MCE, cat #: HY-12867.

The preparation method comprises the following steps: PT2385 dimethyl sulfoxide was prepared as a 1mM stock solution, which was diluted to a final concentration of 10. mu.M in endothelial cell culture medium immediately before use.

The experimental method comprises the following steps:

human primary pulmonary artery endothelial cell (Human pulmony endothelial cell en)dothelial cells, HPAEC) were purchased from Sciencell, cultured in HPAEC-specific medium, and the cells were incubated at 37 ℃ with 5% CO₂Culturing in a cell culture box. THP-1 cells were purchased from ATCC, passaging was initiated when the cells grew to 90% confluence, and cells in logarithmic growth phase were selected for the experiment. When the cells are subjected to hypoxia treatment, the inoculated cells are placed in a culture medium containing N₂And CO₂In a three-gas culture box, and adding O₂Concentration was set to 1% (5% CO)₂，94％N₂) And taking out the culture medium after corresponding culture time for subsequent experiments. PT2385 treatment cells were added at a dose concentration of 10. mu.M. Extracting RNA extracted from cells according to the kit instructions, and performing RT-PCR to check the content of mRNA; extracting total cell protein, and detecting protein content by using a Western blot method.

Experimental method for THP-1 adhesion endothelial cells

THP-1 cell markers

The THP-1 cells were labeled with DiI (cell membrane red fluorescent probe). Light protection, Dil stock: 10mg/ml, adding PBS according to the proportion of 1:1000 to stain THP-1, 5-20 min (generally 10min), washing with PBS (800g × 5min) for 3 times, and resuspending with PBS for later use.

Interaction of THP-1 with endothelial cells

After removing the endothelial cells after the hypoxia treatment (for example, 6-well plate), the residual solution was aspirated by washing 1 to 3 times with PBS at 37 ℃. 1ml of Dil-labeled THP-1 cells resuspended in PBS were added, incubated at 37 ℃ for 30min, then insufficiently adhered THP-1 cells were carefully washed off with PBS, and after digestion with trypsin without EDTA, digestion was stopped with complete medium.

3. Flow cytometry detection

Washing with PBS for 3 times, and collecting appropriate amount (300ul) to test the amount of adhered THP-1.

The results are shown in FIGS. 5-7, and FIG. 5 shows the effect of PT2385 provided by the present invention on the mRNA of ICAM1 in HPAEC treated with hypoxia. FIG. 6 shows the effect of PT2385 provided by the present invention on ICAM1 protein in hypoxic-treated HPAEC. FIG. 7 is a graph showing the effect of PT2385 provided by the present invention on the amount of HPAEC-adhered THP-1 after hypoxia treatment.

As can be seen from fig. 5, after hypoxia treatment, the mRNA content of ICAM1 in HPAEC can be significantly increased, and after PT2385 treatment, the mRNA content of ICAM1 in HPAEC can be significantly decreased, thereby demonstrating that PT2385 treatment can significantly decrease hypoxia-induced ICAM1 expression increase, and further alleviate pulmonary vasculitis.

As can be seen from fig. 6, PT2385 treatment significantly reduced the reduction in hypoxia-induced ICAM1 protein content, thereby reducing endothelial cell adhesion capacity and alleviating the occurrence of pulmonary vasculitis.

As shown in FIG. 7, under the hypoxia treatment, the number of the THP-1 cells adhered to the pulmonary artery endothelial cells was significantly increased, while the PT2385 treatment significantly reduced the number of the THP-1 cells adhered to the pulmonary artery endothelial cells.

The above results indicate that PT2385 can significantly reduce the expression of ICAM1 mRNA and protein in the lung artery endothelial cells induced by hypoxia, and significantly reduce the amount of HPAEC adhered THP-1 after hypoxia treatment. Reduce pulmonary vasculitis reaction, thereby reducing pulmonary artery pressure, further delaying right heart failure, and improving survival rate of patients with pulmonary hypertension.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

The application of PT2385 in preparing medicine for preventing and treating pulmonary hypertension is disclosed.
Use of PT2385 in the manufacture of a medicament for lowering right ventricular systolic pressure.
The application of PT2385 in preparing medicine for inhibiting right ventricular hypertrophy.
Use of PT2385 in the manufacture of a medicament for inhibiting pulmonary vascular remodeling.
The application of PT2385 in preparing medicine for preventing and treating pulmonary heart disease is provided.
The application of PT2385 in preparing medicine for preventing and treating high altitude heart disease is disclosed.
Application of PT2385 in preparing medicine for inhibiting high expression of cell adhesion molecule induced by hypoxia.
Use of PT2385 in the manufacture of a medicament for inhibiting hypoxia-induced inflammatory cell adhesion to vascular endothelial cells.
9. The medicine for treating pulmonary hypertension is characterized by comprising PT2385 and a pharmaceutically acceptable carrier or auxiliary material; the auxiliary materials comprise one or more of dimethyl sulfoxide, sodium carboxymethylcellulose, tween 80 and physiological saline.
10. The medicament of claim 9, wherein the medicament is in a dosage form of injection, powder injection, capsule, granule, spray, microcapsule, tablet or transdermal controlled release patch.