Application of benzotriazolyl piperazine compound or salt thereof in preparation of pulmonary arterial hypertension treatment medicine

Abstract

The invention discloses an application of a benzotriazolyl piperazine compound or a salt thereof in preparation of a medicine for treating pulmonary arterial hypertension, and belongs to a new application of the benzotriazolyl piperazine compound. 3-[4-[4-(1H-benzotriazole-1-yl) butyl] piperazine-1-yl] benzisothiazole hydrochloride (PH-001) is subjected to an in-vivo animal experiment; mainly based on pharmacological experiments in a rat pulmonary arterial hypertension model induced by monocrotaline, it is found that the right ventricular systolic pressure (RVSP), the right ventricular hypertrophy index (RVHI) and the pulmonary arterial wall thickness (PAWT) of a rat are all remarkably reduced after intervention of a test sample PH-001, and the technical scheme opens up a new way for clinical application in the field of pulmonary arterial hypertension treatment. Therefore, PH symptoms and disease course development are effectively relieved, a theoretical basis is provided for clinical development, and the traditional Chinese medicine composition is expected to become a new effective treatment strategy.

Description

Technical Field

[0001] The present invention relates to a new use of benzotriazolyl piperazine compounds, and in particular to the use of benzotriazolyl piperazine compounds or salts thereof in the preparation of drugs for treating pulmonary hypertension. Background Art

[0002] Pulmonary hypertension (PH) refers to an abnormal hemodynamic state and pathophysiological syndrome caused by structural and / or functional changes in the pulmonary vascular bed due to various reasons, leading to increased pulmonary artery pressure, right heart dilation, right heart failure and even death. According to the "Chinese Clinical Pathway for the Diagnosis and Treatment of Pulmonary Hypertension (2023)" and the "2022 ESC / ERS Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension", its hemodynamic definition refers to: at sea level and resting state, the mean pulmonary artery pressure (mPAP) measured by right heart catheterization (RHC) is >20mmHg (1mmHg=0.1333kPa).

[0003] According to pulmonary artery wedge pressure (PAWP) and pulmonary vascular resistance (PVR), PH is divided into precapillary PH, simple postcapillary PH and mixed postcapillary PH. According to the cause, PH can be clinically divided into five categories: the first category is arterial pulmonary hypertension, the second category is PH caused by left heart disease, the third category is PH caused by lung disease and / or hypoxia, the fourth category is PH caused by pulmonary artery obstruction, and the fifth category is PH caused by unknown mechanism and / or multiple factors. Among them, the second category is PH caused by left heart disease, which is the most common, accounting for 65% to 80% of the PH population, and is postcapillary PH. The first, third and fourth categories are precapillary PH; the fifth category is relatively rare and can be precapillary PH or postcapillary PH.

[0004] The clinical symptoms of PH lack specificity, mainly manifested as symptoms related to progressive right heart failure, which are often induced by exertion, manifested as fatigue, dyspnea, chest tightness, chest pain and syncope. Some patients may also experience dry cough and exercise-induced nausea and vomiting. Late-stage patients may have symptom attacks at rest. As right heart failure worsens, edema of the ankles, lower limbs, and even the abdomen and body may occur. The underlying disease or concomitant disease that causes PH will also have corresponding clinical manifestations. The clinical manifestations of some patients are related to the complications of PH and abnormal distribution of pulmonary blood flow, including hemoptysis, hoarseness, chest pain, etc. Severe pulmonary artery dilatation can cause pulmonary artery rupture or dissection. The incidence of PH is on the rise, but the timeliness of treatment and prognosis are still not optimistic.

[0005] Different types of PH have different treatment strategies. Before treatment, the cause and nature of PH need to be clarified to determine the treatment plan. General measures include rehabilitation exercises under the guidance of professional physicians, contraception for women of childbearing age, vaccination, social and psychological support, and avoiding travel to high altitude areas. Basic treatment includes oxygen inhalation, anticoagulation, diuretics, and cardiotonic drugs. Specific treatment includes the use of calcium antagonists and targeted drugs. In addition, appropriate treatment should be carried out according to PH complications.

[0006] According to the etiology of PH, there are many treatment methods and drugs. The drugs for the treatment of pulmonary hypertension that have been marketed are mainly calcium antagonists and targeted drugs. Among them, calcium antagonists include diltiazem, nifedipine and amlodipine; targeted drugs include iloprost (inhalation), treprostinil (subcutaneous injection / intravenous administration), selexipag (oral), macitentan, bosentan, ambrisentan, sildenafil, tadalafil and riociguat.

[0007] The above drugs have the following main limitations:

[0008] (1) Limited efficacy: Although there are some drugs available for the treatment of pulmonary hypertension, they are not effective for all patients. Even with the combined use of multiple drugs, some patients still have difficulty in effectively controlling their condition.

[0009] (2) Obvious side effects: Existing drugs may cause some serious side effects, such as liver damage, hypotension, facial flushing, etc., which limit their clinical application.

[0010] (3) Expensive: Some targeted therapeutic drugs for pulmonary arterial hypertension are expensive, which many patients cannot afford, resulting in poor treatment compliance. Currently, the commonly used targeted drugs iloprost (inhaled), treprostinil (intravenous), selexipag (oral) and macitentan are all under patent and expensive.

[0011] Therefore, there is an urgent unmet need for the clinical treatment of pulmonary hypertension. Summary of the invention

[0012] Based on the numerous limitations of the above-mentioned drugs for treating pulmonary hypertension, the present invention is intended to provide a use of a benzotriazolyl piperazine compound or its hydrochloride in the preparation of a drug for treating pulmonary hypertension. The benzotriazolyl piperazine compound or its hydrochloride is used to prepare a drug for treating pulmonary hypertension, which has the therapeutic potential to improve PH symptoms, inhibit pulmonary vasoconstriction and the progression of the disease.

[0013] Furthermore, the benzotriazolyl piperazine compound is 3-[4-[4-(1H-benzotriazole-1-yl)butyl]piperazine-1-yl]benzisothiazole.

[0014] 3-[4-[4-(1H-benzotriazole-1-yl)butyl]piperazine-1-yl]benzisothiazole hydrochloride, denoted as PH-001, has the structural formula:

[0015]

[0016] The pharmaceutical dosage forms include oral tablets, injections, and inhalants.

[0017] The present invention conducted an in vivo animal experiment on 3-[4-[4-(1H-benzotriazole-1-yl)butyl]piperazine-1-yl]benzisothiazole hydrochloride (PH-001), mainly based on the efficacy experiment in the rat pulmonary hypertension model induced by monocrotaline, and found that after the intervention of the test sample PH-001, the right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) and pulmonary artery wall thickness (PAWT) of the rats were significantly reduced.

[0018] During the onset of pulmonary hypertension, pulmonary artery smooth muscle cells will undergo abnormal proliferation. Experimental results have confirmed that PH-001 can interfere with the signal transduction pathways associated with cell proliferation and inhibit the activity of certain growth factor receptors, such as platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), etc. These growth factor receptors play a key role in the proliferation of smooth muscle cells. In addition, inflammatory response plays an important role in the pathophysiological process of pulmonary hypertension. Experimental results have confirmed that PH-001 can also inhibit the activation and aggregation of inflammatory cells (such as macrophages, lymphocytes, etc.). It has a therapeutic effect on pulmonary hypertension.

[0019] In summary, the technical solution of the present invention opens up a new path for clinical application in the field of pulmonary hypertension treatment, thereby effectively alleviating PH symptoms and disease progression, providing a theoretical basis for clinical development, and is expected to become a new and effective treatment strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Body weight changes of rats (Mean±SEM).

[0021] Figure 2 Effect of the test substance PH-001 on right ventricular systolic pressure (RVSP) (Mean±SEM); compared with the normal control group (Control), **** p<0.0001; compared with the model group (Model), # p<0.05, ## p<0.01.

[0022] Figure 3 Effect of the test substance PH-001 on the right heart hypertrophy index (RV / LV+S) (Mean±SEM); compared with the normal control group (Control), **** p<0.0001; compared with the model group (Model), # p<0.05, ## p<0.01.

[0023] Figure 4 Effect of the test substance PH-001 on the right heart body weight ratio (RV / BW) (Mean±SEM); compared with the normal control group (Control), **** p<0.0001; compared with the model group (Model), # p<0.05.

[0024] Figure 5 Effect of the test substance PH-001 on the thickness of the right ventricular free wall (RV thickness) (Mean±SEM); compared with the normal control group (Control), **** p<0.0001; compared with the model group (Model), ## p<0.01.

[0025] Figure 6 Effect of the test substance PH-001 on the medial thickening (MT%) of pulmonary arterioles (Mean±SEM); compared with the normal control group (Control), **** p<0.0001; compared with the model group (Model), # p<0.05, ## p<0.01. DETAILED DESCRIPTION

[0026] Example 1: Study on the efficacy of PH-001 in the monocrotaline-induced pulmonary hypertension model in rats

[0027] All animal experimental operations involved in the implementation process are carried out in accordance with the standard laboratory animal operation procedures stipulated by the (IACUC), the IACUC laboratory animal care and use guidelines, and the animal welfare requirements stipulated in the ethical review guidelines for laboratory animal welfare. This guideline is in line with the principles of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0028] 1. Experimental Materials

[0029] 3-[4-[4-(1H-Benzotriazol-1-yl)butyl]piperazin-1-yl]benzisothiazole hydrochloride (PH-001): Original Drug Port Life Sciences (Liaoning) Group Co., Ltd.;

[0030] Sildenafil citrate tablets (Sildenafil): Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd.;

[0031] Hydroxypropyl methylcellulose (HPMC): Sigma;

[0032] Monocrotaline (MCT): Sigma;

[0033] Experimental animals: Male SD rats, about 6-8 weeks old when grouped, weighing 224.64-276.51 g. Animal source: Sibeifu (Beijing) Biotechnology Co., Ltd., animal certificate number NO.110324241104821654, 70 animals in the group.

[0034] 2. Experimental Procedure

[0035] After the rats were adaptively fed, they were randomly divided into a normal control group, a model control group, a PH-001 low-dose group (1 mg / kg), a PH-001 medium-dose group (5 mg / kg), a PH-001 high-dose group (15 mg / kg), and a positive control group of sildenafil (25 mg / kg) according to their body weight. There were 10 animals in the normal control group and 12 animals in each of the other groups. Pulmonary hypertension was induced by a single intraperitoneal injection of monocrotaline (MCT, 60 mg / kg) to establish a rat pulmonary hypertension model. The drug was administered on the second day of model induction, and the administration method was oral gavage, twice a day, 7-8 hours apart, for a total of 27 days (54 times). The weight of the animals was collected twice a week during the administration period. After the administration period, the right ventricular systolic pressure (RVSP) of the rats was measured. After the right ventricular pressure test was completed, the animals were euthanized, the weight of the right ventricular free wall (RV), the weight of the left ventricle + ventricular septum (LV+S) and the fasting body weight (BW) of the animals were weighed, and the RV / (LV+S) and RV / BW ratios were calculated. Then, the right ventricular free wall and the left lung lobe were fixed and embedded, and the thickness of the right ventricular free wall was measured by HE staining, and the percentage of the pulmonary arteriolar media thickness (MT%) was calculated. According to the changes in the above indicators, the efficacy of the test product PH-001 in treating pulmonary hypertension was evaluated. The experimental grouping and administration information are shown in Table 1. The solvent / control substance / drug preparation information is shown in Table 2.

[0036] Table 1 Experimental animal grouping and drug administration information

[0037] Group Treatment factors Dosage (mg / kg) Normal control group Solvent (1%HPMC) - Model control group Solvent (1%HPMC) - PH-001 low dose group PH-001 1 PH-001 medium dose group PH-001 5 PH-001 high dose group PH-001 15 Positive control group Sildenafil 25

[0038] Table 2 Drug preparation information

[0039]

[0040]

[0041] 2.1 Right ventricular systolic pressure (RVSP) measurement

[0042] On the second day after the last administration, the rats were deeply anesthetized (2% sodium pentobarbital, 60 mg / kg), fixed on the operating table in the supine position, and the right common jugular vein was bluntly separated. An intravenous catheter was inserted into the right jugular vein and entered the right ventricle through the subclavian vein and right atrial appendage. Data was collected by the biological signal acquisition system, and a stable RVSP waveform was recorded when it appeared, and the average value was calculated.

[0043] 2.2 Cardiac tissue collection and calculation of right ventricular hypertrophy index (RV / LS+S)

[0044] Before dissection, the fasting body weight (BW) of the rats was measured. The rats were euthanized, the hearts were dissected, the blood vessel roots and atrial appendages were removed, the free wall of the right ventricle was cut along the entrance of the pulmonary artery, and the left ventricle plus the ventricular septum was cut open, cleaned and dried. The right ventricle (RV) and left ventricle + ventricular septum (LV + S) were weighed. The right heart hypertrophy index and right heart-to-body weight index were calculated according to the formula;

[0045] Calculation formula:

[0046]

[0047] 2.3 Histopathological examination

[0048] The rat lungs were removed and weighed, and then the left lung was perfused with 10% neutral buffered formalin fixative through the trachea, and the lungs together with the right ventricular free wall were preserved in neutral buffered formalin fixative, embedded in paraffin, and sliced ​​for HE staining.

[0049] Right ventricular free wall: Select three different positions under light microscopy to measure the thickness of the right ventricular free wall, and take the average value as the thickness of the right ventricular free wall of the animal;

[0050] Left lung lobe: Under a light microscope, 10-20 relatively round pulmonary arteriolar vessels were measured for each rat. The inner and outer diameters of the lumen were measured, and the ratio of the medial wall thickness (MT) to the outer diameter (ED) of the pulmonary artery vessel wall was calculated. The medial wall thickness percentage was used as an indicator of pulmonary vascular reconstruction, MT% = (2MT / ED) × 100%.

[0051] 2.4 Statistical analysis

[0052] The experimental data of each group of animals were expressed as mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) was used for comparison between multiple groups that met normal distribution and had equal variance, and Tukey's HSD test was used for further multiple comparisons between groups; Kruskal-Wallis H test (KW method) was used for analysis if normal distribution was not met or variance was unequal, and Dunn's method was used for multiple comparisons between groups. p < 0.05 was considered statistically significant.

[0053] 3. Experimental results

[0054] 3.1 Weight gain and mortality

[0055] Compared with the model control group during the same period, the body weight of the animals in the model control group was significantly reduced (p < 0.01), and 1, 5 and 15 mg / kg of PH-001 had no significant effect on the body weight gain of the model animals (see Figure 1, Table 3). During the test period, the mortality rate of the model control group was 25.00%. PH-001 had a certain improvement on the mortality of the model animals. The mortality rates of the animals in the PH-0015mg / kg and 15mg / kg dose groups were 16.67% and 8.33%, respectively, which were lower than those in the model control group during the same period. The mortality rate of the PH-0011mg / kg dose group was 25.00%, which was the same as the model control group. The mortality rate of the positive control group was 16.67%.

[0056] Table 3 Effects of the test articles on the weight gain of model animals (Mean±SEM)

[0057]

[0058] Note: D0 is the day of grouping of the experimental model, D28 is the end point of the experiment (last medication), and the body weight gain from D0 to D28 is calculated. * Compared with the normal control group; ** : p<0.01; **** :p<0.0001.

[0059] 3.2 Right ventricular systolic pressure (RVSP)

[0060] The right ventricular systolic pressure of the animals in the model control group was significantly higher than that in the normal control group (p<0.0001). Compared with the model control, the right ventricular systolic pressure of the positive control group was significantly lower (p<0.05). There was no significant change in the RVSP of the PH-0011mg / kg dose group, while the RVSP of the PH-0015mg / kg and 15mg / kg dose groups decreased to varying degrees. The RVSP of the PH-00115mg / kg dose group improved significantly (p<0.01). See for details. Figure 2 , Table 4.

[0061] Table 4 Effect of the test sample PH-001 on RVSP (Mean±SEM)

[0062] Group RVSP (mmHg) Normal control group 28.49±3.02 Model control group <![CDATA[103.94±12.55 **** ]]> PH-0011mg / kg <![CDATA[104.89±14.72 & ]]> PH-0015mg / kg 89.54±16.49 PH-00115mg / kg <![CDATA[75.85±15.74 ## ]]> Positive control group <![CDATA[79.86±16.44 # ]]>

[0063] Note: * Compared with the normal control group; **** :p<0.0001. # indicates compared with the model control group; # :p<0.05, ## :p<0.01. & Compared with the positive control group; & :p<0.05.

[0064] 3.3 Right ventricular hypertrophy index (RV / LV+S)

[0065] Compared with the normal control group, the RV / LV+S of the model control group animals was significantly increased (p<0.0001). Compared with the model control group, the positive control had a certain improvement effect on the increase of RV / LV+S in the model animals. The RV / LV+S of the PH-0015mg / kg and 15mg / kg dose groups showed different degrees of decrease compared with the model control group during the same period. Among them, the RV / LV+S of the 15mg / kg dose group showed the most obvious improvement, which was significantly lower than the model control group (p<0.05). For details, see Figure 3 , Table 5.

[0066] Table 5 Statistical analysis results of right heart hypertrophy index (RV / LV+S) (Mean±SEM)

[0067] Group RV / (LV+S) Normal control group 0.28±0.02 Model control group <![CDATA[0.56±0.06 **** ]]> PH-0011mg / kg 0.56±0.06 PH-0015mg / kg 0.51±0.06 PH-00115mg / kg <![CDATA[0.48±0.05 # ]]> Positive control group 0.49±0.06

[0068] Note: * Compared with the normal control group; **** :p<0.0001. # indicates compared with the model control group; # :p<0.05.

[0069] 3.4 Right heart to body weight ratio (RV / BW)

[0070] Compared with the normal control group, the RV / BW of the animals in the model control group was significantly increased (p<0.0001). The positive control had a significant improvement effect on the increase in RV / BW of the model animals (p<0.05). The RV / BW of the PH-001 1mg / kg, 5mg / kg and 15mg / kg dose groups were all reduced to varying degrees compared with the model control group during the same period, among which the improvement in the 15mg / kg dose group was obvious, which was significantly lower than that of the model control group (p<0.05). Figure 4 , Table 6.

[0071] Table 6 Statistical analysis results of right heart weight ratio (RV / BW) (Mean±SEM)

[0072] Group RV / BW(g / kg) Normal control group 0.58±0.05 Model control group <![CDATA[1.35±0.21 **** ]]> PH-0011mg / kg 1.27±0.16 PH-0015mg / kg 1.25±0.20 PH-00115mg / kg <![CDATA[1.08±0.14 # ]]> Positive control group <![CDATA[1.08±0.11 # ]]>

[0073] Note: * Compared with the normal control group; **** :p<0.0001. # indicates compared with the model control group; # :p<0.05.

[0074] 3.5 Right ventricular free wall thickness (RVthickness)

[0075] Compared with the normal control group, the RVthickness of the animals in the model control group was significantly increased (p<0.0001). The positive control substance had a certain degree of improvement on the increase of RV thickness in the model animals. The RVthickness of the PH-00115mg / kg dose group was significantly reduced compared with the model control group (p<0.01). Figure 5 , Table 7.

[0076] Table 7 Statistical analysis results of right ventricular free wall thickness (RVthickness) (Mean±SEM)

[0077] Group RVThickness(mm) Normal control group 1.09±0.08 Model control group <![CDATA[1.69±0.12 **** ]]> PH-0011mg / kg 1.71±0.15 PH-0015mg / kg 1.67±0.14 PH-00115mg / kg <![CDATA[1.46±0.11 ## ]]> Positive control group 1.53±0.13

[0078] Note: * Compared with the normal control group; **** :p<0.0001. # indicates compared with the model control group; ## :p<0.01.

[0079] 3.6 Pulmonary arteriolar media thickening (MT%)

[0080] Compared with the normal control group, the MT% in the model control group increased significantly (P<0.0001). The positive control had a significant improvement effect on the changes in MT% in the model animals (p<0.05). The MT% in the PH-0015mg / kg and 15mg / kg dose groups decreased to varying degrees compared with the model control group during the same period, among which the improvement in the 15mg / kg dose group was obvious, which was significantly lower than that in the model control group (p<0.01). Figure 6 , Table 8.

[0081] Table 8 Effects of the test articles on medial thickening of pulmonary arterioles in model animals (Mean±SEM)

[0082]

[0083]

[0084] Note: * Compared with the normal control group; **** :p<0.0001. # indicates compared with the model control group; # :p<0.05, ## :p<0.01.

[0085] In the above experiment, a rat model of pulmonary hypertension that meets the clinical characteristics of the disease was successfully established. The results of the efficacy study in this model showed that PH-001 has a good therapeutic effect on pulmonary hypertension induced by monocrotaline. The onset dose is 5 mg / kg and the effective dose is 15 mg / kg.

[0086] Example 2: Mechanism of action detection

[0087] 1. Experimental plan

[0088] (I) Experimental animals and model establishment

[0089] Healthy adult male Sprague-Dawley rats weighing 250-300g were selected. The pulmonary hypertension model was induced by intraperitoneal injection of monocrotaline (MCT). After one week of adaptive feeding, the rats were intraperitoneally injected with MCT at a dose of 60 mg / kg and fed normally after injection. The sham operation group was only injected with an equal volume of normal saline.

[0090] (II) Experimental Grouping

[0091] The rats with successful modeling were randomly divided into the following groups, with 10 rats in each group:

[0092] 1. Model control group: an equal volume of normal saline was injected intraperitoneally.

[0093] 2. Drug treatment group: PH-001, 2 mg / kg, intraperitoneal injection.

[0094] (III) Time and method of administration

[0095] The drug was administered one week after the injection of MCT and was administered once a day for 4 weeks. The drug was administered by intraperitoneal injection to ensure that the drug was evenly distributed in the body.

[0096] 2. Index detection

[0097] 1. Growth factor receptor expression detection (immunohistochemical staining)

[0098] Sample preparation: After the rats were killed, the pulmonary artery tissue was quickly removed, rinsed with precooled saline, and then fixed in 4% paraformaldehyde for 24 hours. The fixed tissue was dehydrated with gradient alcohol (70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, 100% ethanol for 1 hour × 2 times), then transparentized with xylene (xylene for 1 hour × 2 times), and finally the tissue was embedded in paraffin. 5μm thick pulmonary artery tissue sections were made, attached to poly-lysine-treated slides, and baked in a 60°C oven for 1 hour.

[0099] Immunohistochemical staining steps:

[0100] Dewaxing and hydration: The sections were dewaxed in xylene for 10 min × 2 times, and then hydrated to distilled water through gradient alcohol (100% ethanol 5 min × 2 times, 95% ethanol 5 min, 90% ethanol 5 min, 80% ethanol 5 min, 70% ethanol 5 min).

[0101] Antigen retrieval: Place the slices in citric acid buffer (pH 6.0), heat in a microwave oven at high heat until boiling, then maintain medium heat for 15 minutes and cool naturally to room temperature.

[0102] Endogenous peroxidase blocking: The sections were incubated with 3% hydrogen peroxide solution for 10 minutes to block endogenous peroxidase activity, and then rinsed with PBS buffer (pH 7.4) three times, 5 minutes each time.

[0103] Primary antibody incubation: Add diluted PDGFR and FGFR primary antibodies (dilution ratio 1:400) and incubate at 4°C overnight.

[0104] Secondary antibody incubation: rinse with PBS buffer three times, 5 minutes each time, add biotin-labeled secondary antibody (dilution ratio 1:400) and incubate at room temperature for 30 minutes.

[0105] Color development: rinse with PBS buffer for 3 times, 5 minutes each time, add horseradish peroxidase-labeled streptavidin, incubate at room temperature for 30 minutes, then develop with DAB color development kit, observe the color development under a microscope, and stop the color development after 10 minutes.

[0106] Counterstaining: Counterstain the cell nucleus with hematoxylin for 2 minutes, then differentiate with hydrochloric acid alcohol (1% hydrochloric acid in 70% alcohol), and rinse with water to return to blue.

[0107] Dehydration, transparentization and sealing: The sections were dehydrated with gradient alcohol (70% ethanol for 5 minutes, 80% ethanol for 5 minutes, 90% ethanol for 5 minutes, 95% ethanol for 5 minutes, 100% ethanol for 5 minutes × 2 times), transparentized with xylene (xylene for 5 minutes × 2 times), and finally sealed with neutral gum abs9177.

[0108] Detection and analysis: Pulmonary artery tissue sections were observed under a microscope, 10 fields of view were randomly selected, and the average optical density values ​​of PDGFR and FGFR positive expression areas were measured using image analysis software (Image-Pro Plus) to reflect their expression levels.

[0109] The results showed that the expression levels of PDGFR and FGFR growth factor receptors were higher in the model control group, while the expression levels in the drug treatment group were lower than those in the model control group due to the effects of the drugs.

[0110] 2. Observation of inflammatory cell infiltration (immunohistochemical staining)

[0111] Sample preparation is the same as that in the growth factor receptor expression assay.

[0112] Immunohistochemical staining steps: dewaxing and hydration, antigen retrieval, and endogenous peroxidase blocking steps are the same as the corresponding steps in the growth factor receptor expression detection.

[0113] Primary antibody incubation: Add diluted CD68 antibody (to mark macrophages, dilution ratio 1:400) and CD3 antibody (to mark lymphocytes, dilution ratio 1:400) respectively, and incubate at 4°C overnight.

[0114] The steps of secondary antibody incubation, color development, counterstaining, dehydration, transparency and sealing are the same as those in the growth factor receptor expression detection.

[0115] Detection and analysis: Pulmonary artery tissue sections were observed under a microscope, and 10 fields of view were randomly selected to count the number of CD68-positive macrophages and CD3-positive lymphocytes, and the number of positive cells per unit area was calculated.

[0116] The results showed that in the model control group, there were 98 macrophages (CD68 positive) per square millimeter. This is because under inflammatory stimulation, monocytes will migrate into tissues and differentiate into macrophages, participating in the inflammatory process and tissue repair. The pathological state caused by MCT will continue to stimulate the aggregation of macrophages. Lymphocytes (CD3 positive) have 56 per square millimeter. Lymphocytes play a role in immune response. They are recruited to the site of inflammation by chemokines to help fight inflammation or participate in immunopathological processes.

[0117] In the drug treatment group, there were 37 macrophages (CD68 positive) per square millimeter. The drug may reduce the number of macrophages in the pulmonary artery wall by inhibiting inflammatory signaling pathways, reducing the differentiation of monocytes into macrophages and the chemotaxis of macrophages. There were 22 lymphocytes (CD3 positive) per square millimeter. The drug may interfere with the recruitment and activation process of lymphocytes, resulting in a decrease in the number of lymphocytes at the site of inflammation.

Claims

1. Use of benzotriazolyl piperazine compounds or their salts in the preparation of drugs for the treatment of pulmonary arterial hypertension.

2. The use according to claim 1, characterized in that: The salt refers to the hydrochloride of benzotriazolylpiperazine compounds.

3. The use according to claim 1, characterized in that: The benzotriazolyl piperazine compound is 3-[4-[4-(1H-benzotriazole-1-yl)butyl]piperazine-1-yl]benzisothiazole.

4. The use according to claim 1, characterized in that: The pharmaceutical dosage forms include oral tablets, injections, and inhalants.

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