Application of benzotriazole compound or salt thereof in treatment of cerebral arterial thrombosis

Abstract

The invention discloses application of a benzotriazole compound or a salt thereof in treatment of cerebral arterial thrombosis, and belongs to novel application of the benzotriazole compound. A classical suppository MCAO ischemia reperfusion model is adopted, treatment is performed based on continuous 3-day tail intravenous injection after ischemia, and cerebral infarction is passed through the range of cerebral infarction; the pharmacodynamic study of CA-001 mesylate and CA-003 mesylate on cerebral arterial thrombosis is carried out according to the change of indexes such as cerebral arterial thrombosis, cerebral arterial thrombosis, neurological function score and walking function. A test result shows that the CA-001 mesylate and the CA-003 mesylate have a remarkable treatment effect on the cerebral arterial thrombosis and are expected to provide a new strategy and a new medicine for treating the cerebral arterial thrombosis.

Description

Technical Field

[0001] The invention belongs to a new use of a benzotriazole compound, and specifically relates to the use of a benzotriazole compound or a salt thereof in the preparation of a drug for treating ischemic stroke. Background Art

[0002] Ischemic stroke, also known as cerebral infarction, is a disease caused by cerebral blood vessel blockage, which leads to ischemia and hypoxia of brain tissue, and ultimately causes brain tissue damage and dysfunction. Cerebral ischemia can be divided into localized cerebral ischemia and diffuse cerebral ischemia based on the scope of ischemia. The causes of localized cerebral ischemia include: middle cerebral artery embolism; extracranial internal carotid artery or vertebral artery stenosis, occlusion or thrombosis; cerebral artery spasm. The causes of diffuse cerebral ischemia include: cardiac arrest, hypotension, anemia, hypoglycemia, etc.

[0003] Current treatments include:

[0004] (1) Intravenous thrombolytic therapy: Recombinant tissue plasminogen activator (rt-PA) is used for intravenous thrombolytic therapy. It is currently the only thrombolytic drug approved by the US FDA for the treatment of acute ischemic stroke. The treatment window is within 4.5 hours after onset.

[0005] (2) Mechanical thrombectomy: It is suitable for patients with large vessel occlusion and can be extended to 6-24 hours after onset. The specific time window depends on the patient's clinical and imaging characteristics.

[0006] (3) Rehabilitation therapy: The goal is to restore the patient's functional status to the maximum extent possible and reduce long-term disability. It includes physical therapy and occupational therapy. Physical therapy improves motor function through exercise and training, while occupational therapy improves independence in daily living activities.

[0007] (4) Prevention of recurrent stroke: including lifestyle adjustments and drug treatment, such as controlling blood pressure, blood sugar, blood lipids, etc.

[0008] In general, ischemic stroke is the most common type of stroke in the world, seriously affecting the health and economic status of the global population, especially the elderly. Currently, emergency treatment mainly relies on the use of specific drugs for thrombolysis in the early stage of the disease, and the use of mechanical thrombectomy technology to expand the treatment time window. Although these methods have improved the timeliness and effectiveness of treatment, there are still many challenges to overcome, especially in the optimization of treatment strategies and personalized treatment. Summary of the invention

[0009] The present invention aims to provide a use of a benzotriazole compound or its mesylate in the preparation of a drug for treating ischemic stroke, which has a significant therapeutic effect on ischemic stroke, including improving the infarction range and neurological function damage in model animals.

[0010] Furthermore, the benzotriazole compound is 3-[4-[4-(1H-benzotriazole-1-yl)butyl]piperazine-1-yl]benzisothiazole, denoted as CA-001.

[0011] The structural formula of 3-[4-[4-(1H-benzotriazol-1-yl)butyl]piperazine-1-yl]benzisothiazole is:

[0012]

[0013] Furthermore, the benzotriazole compound is N-(3-(1-benzotriazolyl)propyl)-4-(3-(6-fluorobenzisoxazole))piperidine, which is denoted as CA-003.

[0014] The structural formula of N-(3-(1-benzotriazolyl)propyl)-4-(3-(6-fluorobenzisoxazole))piperidine is:

[0015]

[0016] The invention adopts benzotriazole compounds or their mesylate salts to prepare a drug for treating ischemic stroke. The drug dosage forms include oral tablets and injections, preferably injections.

[0017] The present invention preferably uses the methanesulfonate of benzotriazole compounds. Experiments have shown that the methanesulfonate has higher water solubility than its hydrochloride, and is more conducive to injection administration.

[0018] The present invention adopts the classic MCAO ischemia-reperfusion model of suture embolism, based on the treatment of continuous tail vein injection for 3 days after ischemia, and conducts a pharmacodynamic study of CA-001 mesylate and CA-003 mesylate on ischemic stroke through changes in indicators such as cerebral infarction range, neurological function score and walking function.

[0019] 1) CA-001 methanesulfonate and CA-003 methanesulfonate both had the efficacy of improving the scope of cerebral infarction in model animals under the set treatment plan. The scope of cerebral infarction in model animals after intervention with CA-001 methanesulfonate and CA-003 methanesulfonate was significantly reduced compared with the model control group (P<0.05).

[0020] 2) CA-001 mesylate and CA-003 mesylate had a significant improvement on the neurological function damage of the model animals under the set treatment plan. During the entire experimental period, the NSS scores of the animals in the CA-001 mesylate and CA-003 mesylate treatment groups were significantly lower than those in the model control group (P<0.05).

[0021] 3) CA-001 mesylate and CA-003 mesylate had no significant effect on the body weight of the model animals. During the experimental period, there was no significant difference in the body weight between the CA-001 mesylate and CA-003 mesylate treatment groups and the model control group.

[0022] Ischemic stroke can damage vascular endothelial cells. Experimental results have shown that both CA-001 mesylate and CA-003 mesylate help repair and maintain the integrity of the vascular endothelium. The above compounds can inhibit the damage of oxidative stress to endothelial cells, reduce the production of reactive oxygen species (ROS), or enhance the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). In addition, ischemic stroke can cause a large amount of excitatory neurotransmitters such as glutamate in the brain to be released, causing excitotoxicity and damaging nerve cells. The above compounds can reduce excitotoxicity by regulating glutamate receptors. It can inhibit the over-activation of glutamate receptors (such as N-methyl-D-aspartate receptors, i.e., NMDA receptors) and reduce the influx of calcium ions, thereby protecting nerve cells from excitotoxic damage. The above compounds can enhance the uptake of glutamate by glial cells, or regulate the activity of glutamate metabolic enzymes, such as glutamine synthetase, to convert glutamate into glutamine, thereby reducing the extracellular glutamate concentration and alleviating excitotoxicity.

[0023] The present invention establishes an experimental system suitable for evaluating the efficacy of cerebral ischemia-reperfusion. The test results show that CA-001 mesylate and CA-003 mesylate have significant therapeutic effects on ischemic stroke, and are expected to provide new strategies and new drugs for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Results of cerebral infarction range and cerebral infarction inhibition rate; data in the figure are expressed as mean ± standard deviation (Mean ± SD); A: results of cerebral infarction range of animals in each group, B: results of inhibition rate of animals in each group; ** indicates that there is a statistical difference compared with the model control group P < 0.01, *** indicates that there is a statistical difference compared with the model control group P < 0.001.

[0025] Figure 2 TTC staining images of the brains of animals in the model control group.

[0026] Figure 3 TTC staining images of the brains of animals in the positive drug group.

[0027] Figure 4 TTC staining images of the brain of animals in the low-dose group of CA-001 methanesulfonate after 1h of ischemia.

[0028] Figure 5TTC staining images of the brain of animals in the high-dose group of CA-001 methanesulfonate after 1h of ischemia.

[0029] Figure 6 TTC staining images of the brains of animals in the low-dose group immediately after reperfusion with CA-001 methanesulfonate.

[0030] Figure 7 TTC staining images of the brains of animals in the high-dose group immediately after reperfusion with CA-001 methanesulfonate.

[0031] Figure 8 TTC staining images of the brain of animals in the low-dose group of CA-003 methanesulfonate after 1h of ischemia.

[0032] Fig. 9 TTC staining images of the brain of animals in the high-dose group of CA-003 methanesulfonate after 1h of ischemia.

[0033] Fig.10 TTC staining images of the brains of animals in the low-dose group immediately after reperfusion with CA-003 methanesulfonate.

[0034] Fig.11 TTC staining images of the brains of animals in the high-dose group of CA-003 methanesulfonate immediately after reperfusion.

[0035] Fig.12 Behavioral scoring results of animals in each group; All data in the figure are expressed as mean ± standard deviation (Mean ± SD); A: Bederson scoring results of animals in each group, B: NSS scoring results of animals in each group 1 day after surgery; C: NSS scoring results of animals in each group 3 days after surgery; * indicates that there is a statistical difference compared with the model control group P < 0.05, ** indicates that there is a statistical difference compared with the model control group P < 0.01, *** indicates that there is a statistical difference compared with the model control group P < 0.001, **** indicates that there is a statistical difference compared with the model control group P < 0.0001.

[0036] Fig.13 The results of weight test of experimental animals; the data in the table are expressed as mean ± standard deviation (Mean ± SD). DETAILED DESCRIPTION

[0037] The contents and procedures related to animal experiments involved in the experiments of this invention comply with the relevant laws and regulations on the use and management of experimental animals and the relevant regulations of the Institutional Animal Care and Use Committee (IACUC). The number of animals, experimental design and animal handling have been approved by IACUC (approval number IACUC-B2024041-P-01) and will be strictly implemented in accordance with the contents reported to IACUC.

[0038] Example 1: Evaluation of the efficacy of benzotriazole compounds or their mesylate salts in rat ischemia-reperfusion stroke model

[0039] The cerebral ischemia-reperfusion model of SD rats was established by suture embolism, and the pharmacodynamics of the test articles CA-001 mesylate / CA-003 mesylate on ischemic stroke were studied by studying their effects on the scope of cerebral infarction and behavior.

[0040] 1. Experimental Materials

[0041] 1.1 Preparation of test samples

[0042] Preparation method: Prepare under sterile conditions, weigh an appropriate amount of the test sample and add an appropriate amount of 5% glucose injection to prepare the target concentration, mix thoroughly, see Table 1. After preparation, filter and sterilize with a 0.22μm microporous filter membrane. After preparation, seal and store at room temperature, and use within 30 minutes (prepare and use immediately).

[0043] Table 1 Preparation method of test samples

[0044] Group Dosage (mg / kg) Concentration (mg / mL) CA-001 mesylate ischemia 1h low dose group 1 0.1 CA-001 mesylate ischemia 1h high dose group 5 0.5 CA-003 mesylate ischemia 1h low dose group 0.5 0.05 CA-003 mesylate ischemia 1h high dose group 2.5 0.25 CA-001 mesylate low-dose group 1 0.1 CA-001 mesylate high-dose group 5 0.5 CA-003 mesylate low-dose group immediately after reperfusion 0.5 0.05 CA-003 mesylate high-dose group 2.5 0.25

[0045] 1.2 Positive Control

[0046] Name: Butylphthalide (Sichuan Greentech Biotechnology Co., Ltd.).

[0047] Positive drug preparation method: 30% hydroxypropyl-β-cyclodextrin (30% HP-β-CD) is used as a solvent for preparation; accurately weigh an appropriate amount of butylphthalide and place it in a glass container, add an appropriate amount of 30% HP-β-CD, and prepare a 10 mg / mL dosing preparation. After preparation, filter and sterilize with a 0.22 μm microporous filter membrane.

[0048] 1.3 Negative control (solvent)

[0049] Name: 5% glucose injection (Sichuan Kelun Pharmaceutical Co., Ltd.).

[0050] 2. Experimental Methods

[0051] 2.1 Animal selection

[0052] Experimental animals: 110 male SD rats (Sprague-Dawley); Grade: SPF grade; Weight: 216.35-248.50g during the adaptation period, 248.27-299.26g during grouping; Age: about 5-7 weeks old during the adaptation period, 6-8 weeks old during the experimental grouping. Source: Sibeifu (Beijing) Biotechnology Co., Ltd., Production License No.: SCXK (Beijing) 2019-0010, Laboratory Animal Qualification Certificate: No.110324241103101017.

[0053] Basis for the selection of experimental animals: The middle cerebral artery (MCA) is a susceptible site for ischemic brain injury in clinical practice, so the middle cerebral artery occlusion (MCAO) model is widely used in the study of ischemic stroke. The suture method to construct the MCAO model is to insert a special suture through one side of the internal carotid artery into the bifurcation of the middle cerebral artery and the anterior cerebral artery to block the blood flow and cause cerebral infarction. It has the advantages of no craniotomy and uniform ischemia positioning. It is currently a widely used method for making cerebral ischemia models at home and abroad. The vascular structure of the SD rat brain is similar to that of humans, and the experimental conditions are easy to standardize. Therefore, the SD rat suture suture method MCAO model is an ideal model for studying cerebral ischemia; in addition, according to reports by JW Simpkins and RL Roof, etc., estrogen and progesterone have a neuroprotective effect on ischemic cerebral infarction in adult rats. In order to exclude the influence of estrogen and progesterone on the experimental results, male rats were selected as experimental animals in this experiment.

[0054] 2.2 Modeling

[0055] The SD rats were placed in an anesthesia induction box filled with 3.0% isoflurane (Lunan Better Phamacertical.co.ltd) for anesthesia induction. The SD rats after anesthesia induction were transferred to the operating table and maintained with 2.0-2.5% isoflurane at 200 mL / min using a small animal gas anesthesia machine. The eyelid reflex and pain response of the SD rats were observed. The surgery could be started only after the eyelid reflex and pain response of the limbs and tail disappeared.

[0056] Cerebral ischemia-reperfusion surgery steps:

[0057] A. Separation and exposure of blood vessels: After the surgical area is prepared, the rat is placed under a surgical microscope. The rat skin is cut with ophthalmic scissors along the midline, with a length of about 2 cm. Through the right paracervical approach, the micro forceps are used to bluntly separate and retract the muscle tissue on the right side of the neck to expose the right common carotid artery (CCA), and then the common carotid artery is separated upward to continue to expose the external carotid artery (ECA) and internal carotid artery (ICA);

[0058] B. ICA is temporarily closed with a vascular clip, and the proximal and distal ends of CCA are threaded separately. The proximal end is tied tightly, and the distal end is threaded for standby use. A small opening is made between the two threads.

[0059] C. Pushing the thread plug: insert the 4-0 thread plug (Guangzhou Jialing Biotechnology Co., Ltd.) through the incision of the CCA, and then slowly and gently push it into the internal carotid artery, pause at the artery clamp of the ICA, further tighten the pre-ligated thread (to avoid excessive bleeding when pushing the thread plug), then remove the artery clamp that blocks the blood flow of the ICA, and immediately push the thread plug into the ICA until it enters the skull. At this time, be careful not to insert the thread plug into the pterygopalatine artery, another branch of the internal carotid artery (the pterygopalatine artery is a branch of the ICA outside the skull. When the thread plug enters the artery to a depth of about 10 mm, it cannot be inserted further. At this time, slightly withdraw the thread plug, adjust the direction, and repeat the operation);

[0060] D. Fix the suture and suture the incision: When the suture is inserted to a depth of about 18 mm from the bifurcation of the common carotid artery, if there is a slight resistance, it means that the head of the suture has entered the anterior cerebral artery (ACA) and the side wall of the suture has blocked the opening of the middle cerebral artery. Stop the insertion and record the time. Remove the artery clip on the CCA and close the incision after observing no active bleeding;

[0061] E. Cerebral reperfusion: The ischemic rats were placed at room temperature to maintain their body temperature at 37°C. Anesthesia was induced 90 minutes later. The suture was slowly and gently pulled while maintaining anesthesia to return the head end to the common carotid artery, thus achieving middle cerebral artery reperfusion.

[0062] F. Disinfect the incision with iodine.

[0063] 2.3 Group design and dosing information

[0064] Group design: A total of 110 animals were divided into 10 groups, with 11 animals in each group; namely, model control group, positive drug group, CA-001 methanesulfonate ischemia 1h low-dose group, CA-001 methanesulfonate ischemia 1h high-dose group, CA-003 methanesulfonate ischemia 1h low-dose group, CA-003 methanesulfonate ischemia 1h high-dose group, CA-001 methanesulfonate reperfusion immediate low-dose group, CA-001 methanesulfonate reperfusion immediate high-dose group, CA-003 methanesulfonate reperfusion immediate low-dose group and CA-003 methanesulfonate reperfusion immediate high-dose group.

[0065] Grouping method: The rats were divided into groups in descending order according to their most recent body weight before grouping, and then random numbers were assigned. The random numbers were then sorted in descending order and were set as groups 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 from low to high, representing model control group, positive drug group, CA-001 methanesulfonate ischemia 1h low-dose group, CA-001 methanesulfonate ischemia 1h high-dose group, CA-003 methanesulfonate ischemia 1h low-dose group, CA-003 methanesulfonate ischemia 1h high-dose group, CA-001 methanesulfonate reperfusion immediate low-dose group, CA-001 methanesulfonate reperfusion immediate high-dose group, CA-003 methanesulfonate reperfusion immediate low-dose group and CA-003 methanesulfonate reperfusion immediate high-dose group, respectively.

[0066] Route of administration: intravenous injection; specific grouping information, dosage, administration volume, administration concentration, etc. are shown in Table 2.

[0067] Administration time: CA-001 methanesulfonate 1h ischemia low-dose group, CA-001 methanesulfonate 1h ischemia high-dose group, CA-003 methanesulfonate 1h ischemia low-dose group and CA-003 methanesulfonate 1h ischemia high-dose group were first administered 1h after ischemia, and then administered once at 12h, 24h, 36h, 48h and 60h. Model control group, positive drug group, CA-001 methanesulfonate low-dose group immediately after reperfusion, CA-001 methanesulfonate high-dose group immediately after reperfusion, CA-003 methanesulfonate low-dose group immediately after reperfusion and CA-003 methanesulfonate high-dose group immediately after reperfusion were first administered immediately after reperfusion (within 10 minutes), and then administered once at 12h, 24h, 36h, 48h, and 60h.

[0068] Dosage frequency and cycle: Administer twice a day for 3 consecutive days.

[0069] The day of modeling (the day of first administration) was defined as D0.

[0070] Table 2 Dosage regimen and group information

[0071]

[0072]

[0073] Observe once a day. If the animal shows any abnormality, the observation frequency can be increased. The observation content includes general performance, behavioral status, eyes, mouth, nose and mouth, ears, hair and skin, feces, urine, genitals and other symptoms. Measure body weight at least twice during the adaptation period, and weigh once on D1 and D3 respectively.

[0074] 2.4 Behavioral evaluation

[0075] 2.4.1 Bederson score

[0076] Testing time: score once every 50 minutes of ischemia on the day of modeling;

[0077] Detection method: Animals were scored according to Bederson scoring criteria;

[0078] Table 3 Bederson scoring criteria for rats

[0079] Points Scoring Criteria 0 No symptoms of nerve damage 1 The rat was lifted from the tail and the forelimb on the contralateral side of the rat was flexed. 2 The rat was placed on a smooth surface and the shoulder on the opposite side of the infarction was pushed by hand to reduce resistance. 3 The animal falls or circles toward the side opposite to the infarct when walking freely 4 Paralysis, no spontaneous movement of limbs

[0080] Note: Rats with a post-ischemic score greater than or equal to 1 point were considered to have successfully established a model. Rats with a score greater than 3 points had severe ischemia and lost their response to drug treatment. Therefore, rats with unsuccessful modeling and severe ischemia were eliminated.

[0081] 2.4.2 Neurological function scores of rats

[0082] Testing time: NSS scoring was performed before modeling and 1 day and 3 days after modeling (D1 and D3), for a total of 3 times;

[0083] Detection method: The animals were subjected to motor function tests, sensory tests, balance tests, and reflex and abnormal movement tests, with specific reference to the rat neurological score table (NSS).

[0084] Table 4 Neurological Score (NSS) of rats

[0085]

[0086]

[0087] 2.5 Animal anatomy and pathological examination (TTC staining)

[0088] Dissection time: 3 days after surgery (D3). 3% sodium pentobarbital was used for intraperitoneal anesthesia, with an injection dose of 60 mg / kg, and the abdominal aorta was bled for euthanasia after anesthesia.

[0089] Examination method: Euthanize the animals and quickly remove the brains. Place the brains in a -20°C refrigerator and freeze them until the brain tissue hardens. Cut the brains into 2 mm thick slices, with the slices located from the olfactory bulb to the back. There are 6 slices in total. Place them in six-well plates filled with 0.5% TTC solution (prepared with 0.9% sodium chloride injection) and then place them in a 37°C thermostat for incubation in the dark for 20 min. After taking them out, place them in a 4% formaldehyde solution and store them in the dark.

[0090] Normal tissues are stained rose red, and infarcted tissues are stained white. Each brain plane is placed on filter paper in order, photographed with a digital camera, and the white tissue is carefully removed and weighed. The percentage of the weight of the infarcted tissue to the total brain weight is used as the infarct range (%) to calculate the cerebral infarction range. The inhibition rate (%) of each drug treatment group is calculated based on the infarction range. The inhibition rate calculation formula is as follows:

[0091]

[0092] All raw data within the facility are collected manually or using a data acquisition system according to the experimental protocol and the SOP of Sichuan Greentech Biotechnology Co., Ltd. Manually collected data can be transcribed into Excel spreadsheets for analysis and reporting.

[0093] 2.6 Statistical analysis

[0094] The measurement indicators were expressed as mean ± standard deviation.

[0095] Data were entered and statistically analyzed using Stata 15.0 or Graphpad Prism 9 software. The measurement indicators were first tested for homogeneity of variance using the LEVENE test. When the variance was homogeneous (P>0.05), the results of the variance analysis could be directly cited to determine whether the overall difference was statistically significant. When the overall difference was statistically significant (P<0.05), the Dunnett-t test was used to compare the differences between the groups. When the overall difference was not statistically significant (P≥0.05), the statistical analysis ended. When the LEVENE test showed that the variance was not homogeneous (P<0.05), a nonparametric test (Kruskal-Wallis H test) was used. When the Kruskal-Wallis H test showed that the overall difference was statistically significant (P<0.05), the Mann-Whitney U test was used to compare the differences between the groups. When the Kruskal-Wallis H test showed that the overall difference was not statistically significant (P≥0.05), the statistical analysis ended. Pathological data (if any) were described in detail.

[0096] The therapeutic effects of relevant drugs on stroke were evaluated based on the neurobehavioral scores and cerebral infarction results of the experimental animals.

[0097] 3. Experimental results

[0098] 3.1 Effects of the test article on the scope of cerebral infarction in model animals

[0099] Drug treatment for acute ischemic stroke mainly includes two aspects: one is to restore blood flow through thrombolytic therapy; the other is to give neuroprotectants to treat secondary neuronal damage caused by ischemia. The present invention adopts the classic suture embolism method for modeling. After 90 minutes of ischemia in the rat's middle cerebral artery, the suture embolism is removed to simulate vascular recanalization. The first drug intervention is performed 1 hour after ischemia and immediately after recanalization. The drug is administered once 12 hours, 24 hours, 36 hours, 48 ​​hours and 60 hours after the first administration. Dibutyl phthalide (10 mg / kg, the first administration is performed immediately after reperfusion, bid×3) is used as the positive drug in the test of the present invention. Three days after the operation, the infarction range of each group of animals is determined by TTC staining of brain tissue and the inhibition rate is calculated to evaluate the neuroprotective effect of the test article. The range of animal cerebral infarction and the inhibition rate of cerebral infarction are shown in Table 5 and Figure 1 .

[0100] Table 5 Results of cerebral infarction range and cerebral infarction inhibition rate

[0101]

[0102]

[0103] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD); ** indicates that there is a statistical difference compared with the model control group (P < 0.01), *** indicates that there is a statistical difference compared with the model control group (P < 0.001), and “-” indicates not applicable.

[0104] Figure 2 , Figure 3 The TTC staining results showed that the model control group animals had obvious white infarction areas, and the cerebral infarction range was 25.5±4.05%, while the cerebral infarction range of the positive drug group animals was 17.77±3.29%, which was significantly lower than that of the model control group animals (P<0.05), and the cerebral infarction inhibition rate was 30.30%. This suggests that this experimental system is suitable for the pharmacodynamic evaluation of ischemic stroke.

[0105] The test substance CA-001 methanesulfonate had a significant improvement on the scope of cerebral infarction in the model animals under the set treatment plan (1 mg / kg, 5 mg / kg, first administration at 1 hour of ischemia and immediately after reperfusion, bid×3). The scope of cerebral infarction in the animals in the CA-001 methanesulfonate 1 hour ischemia low-dose group, CA-001 methanesulfonate 1 hour ischemia high-dose group, CA-001 methanesulfonate low-dose group immediately after reperfusion and CA-001 methanesulfonate high-dose group immediately after reperfusion were 17.02±3.07%, 15.57±1.80%, 17.47±2.12% and 16.77±2.50%, respectively, which was significantly lower than that in the model control group. The inhibition rates of CA-001 methanesulfonate in the 1h ischemia low-dose group, CA-001 methanesulfonate in the 1h ischemia high-dose group, CA-001 methanesulfonate in the reperfusion low-dose group, and CA-001 methanesulfonate in the reperfusion high-dose group were 33.27%, 38.95%, 31.49%, and 34.22%, respectively. Figure 4-7 shown.

[0106] The test substance CA-003 methanesulfonate had a significant improvement on the scope of cerebral infarction in the model animals under the set treatment plan (0.5mg / kg, 2.5mg / kg, first administration at 1h of ischemia and immediately after reperfusion, bid×3). The scope of cerebral infarction in the animals in the CA-003 methanesulfonate low-dose group at 1h of ischemia, the CA-003 methanesulfonate high-dose group at 1h of ischemia, the CA-003 methanesulfonate low-dose group at immediately after reperfusion, and the CA-003 methanesulfonate high-dose group at immediately after reperfusion were 16.74±2.34%, 15.15±4.31%, 18.47±2.07%, and 17.41±2.92%, respectively, which was significantly lower than that in the model control group. The inhibition rates of CA-003 methanesulfonate in the 1h ischemia low-dose group, CA-003 methanesulfonate in the 1h ischemia high-dose group, CA-003 methanesulfonate in the reperfusion low-dose group and CA-003 methanesulfonate in the reperfusion high-dose group were 34.37%, 40.59%, 27.55% and 31.71%, respectively. The TTC staining results of the test product CA-003 methanesulfonate in the set treatment plan are shown in Figure 2. Figure 8-11 shown.

[0107] The above results indicate that the test products CA-001 mesylate and CA-003 mesylate have a significant effect on improving the scope of cerebral infarction in model animals under the set treatment plan, and their anti-infarction effects are comparable.

[0108] 3.2 Effect of the test article on neurological function damage in model animals

[0109] The main purpose of clinical treatment of cerebral infarction is to restore the patient's neurological function as much as possible and improve the quality of life after stroke. In this study, the ischemic condition of the animals was evaluated with reference to the Bederson score table 1 hour after ischemia, and the neurobehavioral function of the surviving animals was evaluated with reference to the NSS score table 1 day and 3 days after modeling to investigate the effect of the test article on the neurological function damage of the model animals. The neurobehavioral score results of each group of animals are shown in Table 6 and Fig.12 .

[0110] Table 6 Animal behavior scoring results of each group

[0111]

[0112] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD); * indicates that there is a statistical difference compared with the model control group P < 0.05, ** indicates that there is a statistical difference compared with the model control group P < 0.01, *** indicates that there is a statistical difference compared with the model control group P < 0.001, **** indicates that there is a statistical difference compared with the model control group P < 0.0001. D-1 in the table indicates the day before administration.

[0113] One hour after surgery, the Bederson score showed that the model animals all scored 3 points, indicating that the model animals were successfully induced to have cerebral ischemia. During the experimental period, all model animals showed obvious neurological impairments such as motor function, balance ability, and sensory function. The NSS scores of the model control group animals on the 1st and 3rd day after surgery were 8.8±0.6 and 8.4±0.4, respectively.

[0114] The positive drug butylphthalide had a significant improvement on the neurological function damage of the model animals under the set treatment plan. The NSS scores of the animals in the positive drug group were 7.4±0.5 and 7.1±0.6 on the 1st and 3rd days after surgery, respectively, which were significantly lower than the NSS scores of the animals in the model control group (8.8±0.6 and 8.4±0.4) (P<0.05).

[0115] The test product CA-001 methanesulfonate had a significant improvement effect on the neurological function damage of the model animals under the set treatment plan. The NSS scores of the animals in the CA-001 methanesulfonate low-dose group with 1h ischemia, the CA-001 methanesulfonate high-dose group with 1h ischemia, the CA-001 methanesulfonate low-dose group immediately after reperfusion, and the CA-001 methanesulfonate high-dose group immediately after reperfusion were 7.7±0.6, 7.3±0.6, 8.0±0.5, and 7.4±0.5, respectively, which were significantly lower than the NSS scores of the animals in the model control group (8.8±0.6) at the same period (P<0.05). At 3 days after surgery, the NSS scores of animals in the CA-001 methanesulfonate low-dose group with ischemia for 1h, the CA-001 methanesulfonate high-dose group with ischemia for 1h, the CA-001 methanesulfonate low-dose group immediately after reperfusion, and the CA-001 methanesulfonate high-dose group immediately after reperfusion were 6.9±0.7, 6.4±0.4, 7.3±0.8, and 6.9±0.7, respectively, which were significantly lower than the NSS scores of animals in the model control group (8.4±0.4) in the same period (P<0.05).

[0116] The test product CA-003 methanesulfonate had a significant improvement effect on the neurological function damage of the model animals under the set treatment plan. The NSS scores of the animals in the CA-003 methanesulfonate low-dose group with 1h ischemia, the CA-003 methanesulfonate high-dose group with 1h ischemia, the CA-003 methanesulfonate low-dose group immediately after reperfusion, and the CA-003 methanesulfonate high-dose group immediately after reperfusion were 7.7±0.6, 7.1±1.2, 7.8±0.7, and 7.3±0.6, respectively, which were significantly lower than the NSS scores of the animals in the model control group (8.8±0.6) in the same period (P<0.05). The NSS scores of animals in the CA-003 methanesulfonate low-dose group with ischemia for 1 h, the CA-003 methanesulfonate high-dose group with ischemia for 1 h, the CA-003 methanesulfonate low-dose group immediately after reperfusion, and the CA-003 methanesulfonate high-dose group immediately after reperfusion 3 days after surgery were 7.1±0.3, 6.6±1.1, 7.0±0.7, and 6.8±0.5, respectively, which were significantly lower than the NSS scores of animals in the model control group (8.4±0.4) in the same period (P<0.05).

[0117] The above results suggest that under the experimental conditions, the test samples CA-001 mesylate and CA-003 mesylate have a significant improvement effect on the neurological function damage of model animals.

[0118] 3.3 Effects of the test article on the body weight of model animals

[0119] As an acute injury model, cerebral ischemia-reperfusion will cause the weight of experimental animals to drop sharply after MCAO modeling. The weight of experimental animals is one of the most basic sensitive indicators to comprehensively reflect the health status of animals. In this experiment, the weight of all surviving animals was monitored 1 day before surgery, 1 day after surgery, and 3 days after surgery. The results of animal weight changes are shown in Table 7 and Fig.13 .

[0120] Table 7. Results of experimental animal weight test

[0121]

[0122] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD).

[0123] There was no significant difference in the body weight of the animals in each group before modeling, and the body weight of the model animals continued to decrease during the experimental period. One day after surgery, the model control group, positive drug group, CA-001 methanesulfonate ischemia 1h low-dose group, CA-001 methanesulfonate ischemia 1h high-dose group, CA-003 methanesulfonate ischemia 1h low-dose group, CA-003 methanesulfonate ischemia 1h high-dose group, CA-001 methanesulfonate reperfusion low-dose group, CA-001 methanesulfonate reperfusion high-dose group, CA-003 methanesulfonate reperfusion low-dose group, and CA-003 methanesulfonate reperfusion low-dose group The body weights of the animals in the high-dose group were 244.08±15.96g, 233.71±7.03g, 235.98±5.92g, 238.75±6.24g, 237.15±10.62g, 239.12±11.95g, 236.50±8.75g, 237.03±9.74g, 238.30±8.34g and 239.62±9.55g, respectively. There was no significant difference in body weight among the groups. The model was Animals in the control group, positive drug group, CA-001 methanesulfonate 1h ischemia low-dose group, CA-001 methanesulfonate 1h ischemia high-dose group, CA-003 methanesulfonate 1h ischemia low-dose group, CA-003 methanesulfonate 1h ischemia high-dose group, CA-001 methanesulfonate reperfusion immediate low-dose group, CA-001 methanesulfonate reperfusion immediate high-dose group, CA-003 methanesulfonate reperfusion immediate low-dose group, and CA-003 methanesulfonate reperfusion immediate high-dose group were randomly divided into the control group and the positive drug group. The body weights were 225.84±30.65g, 197.30±5.68g, 216.18±19.19g, 211.79±6.21g, 213.40±14.78g, 214.36±18.40g, 211.47±17.83g, 215.13±21.24g, 218.63±15.57g and 212.54±17.92g, respectively. There was no significant difference in the body weights of the animals in each group during the whole experiment, indicating that the test products had no significant effect on the body weights of the model animals.

[0124] 3.4 Conclusion

[0125] This study adopted the classic MCAO ischemia-reperfusion model with suture embolism. Based on the treatment of drug administration through tail vein injection for 3 consecutive days after ischemia, the pharmacodynamics of the test products CA-001 mesylate and CA-003 mesylate on ischemic stroke were studied by observing the changes in indicators such as cerebral infarction range, neurological function score and walking function.

[0126] 1) The test samples CA-001 mesylate and CA-003 mesylate both had the efficacy of improving the scope of cerebral infarction in model animals under the set treatment plan. The scope of cerebral infarction in model animals after intervention with the test samples CA-001 mesylate and CA-003 mesylate was significantly reduced compared with the model control group (P<0.05).

[0127] 2) The test products CA-001 mesylate and CA-003 mesylate had a significant improvement on the neurological function damage of the model animals under the set treatment plan. During the entire experimental period, the NSS scores of the animals in the CA-001 mesylate and CA-003 mesylate treatment groups were significantly lower than those in the model control group (P<0.05).

[0128] 3) The test samples CA-001 mesylate and CA-003 mesylate had no significant effect on the body weight of the model animals. During the experimental period, there was no significant difference in the body weight of the CA-001 mesylate and CA-003 mesylate treatment groups and the model control group.

[0129] In summary, this study established an experimental system suitable for evaluating the efficacy of cerebral ischemia-reperfusion drugs. The experimental results showed that the test products CA-001 mesylate and CA-003 mesylate both had significant therapeutic effects on ischemic stroke.

[0130] Example 2: Mechanism of action detection

[0131] 1. Experimental plan

[0132] (I) Experimental animals and model establishment

[0133] Healthy adult male Sprague-Dawley rats weighing 250-300 g were selected. The rat middle cerebral artery occlusion (MCAO) model was established by suture method to simulate ischemic stroke. After anesthesia, the rats were separated and ligated on the right common carotid artery and external carotid artery, and the suture was inserted into the origin of the middle cerebral artery through the internal carotid artery. After blocking the blood flow for 120 minutes, the suture was withdrawn to achieve reperfusion, and the MCAO reperfusion model was established. The sham operation group only underwent surgical operation but no suture was inserted to block the blood flow.

[0134] (II) Experimental Grouping

[0135] The rats with successful modeling were randomly divided into the following groups, with 10 rats in each group:

[0136] 1. Model control group: an equal volume of normal saline was injected intraperitoneally.

[0137] 2. Group A: 1.5 mg / kg CA-001 mesylate was intraperitoneally injected.

[0138] 3. Group B: 1.5 mg / kg CA-003 mesylate was intraperitoneally injected.

[0139] (III) Time and method of administration

[0140] The first administration was started within 30 minutes after successful modeling, and then the drug was administered once a day for 14 days by intraperitoneal injection.

[0141] 2. Index detection

[0142] 1. Detection of antioxidant enzyme activity and ROS level

[0143] Sample preparation: After the rats were killed, the brain tissue on the ischemic side was quickly removed, rinsed with pre-cooled physiological saline, and then homogenized with a tissue homogenizer (according to the ratio of 100 mg tissue to 1 ml PBS buffer). The homogenate was centrifuged at 4°C, 10,000 rpm for 10 minutes, and the supernatant was taken as the sample to be tested.

[0144] SOD activity determination: follow the instructions of the SOD biochemical kit. Mix the sample with the reagent, measure the absorbance at a wavelength of 550nm, and calculate the SOD activity by comparing it with the standard curve. That is, first dilute the standard into different concentrations, add it to the cuvette, measure the absorbance and draw the standard curve. Then add the sample to be tested to the reaction system, incubate at 37℃ for 30 minutes, measure the absorbance, and substitute it into the standard curve to calculate the SOD activity, the unit is usually U / mg protein.

[0145] GSH-Px activity determination: Also follow the instructions of the GSH-Px biochemical kit. Use enzymatic reaction to measure absorbance at a wavelength of 412nm. First establish a standard curve, then add the sample to the reaction system, incubate at 37℃ for 30 minutes, measure the absorbance, and calculate the GSH-Px activity based on the standard curve, the unit is U / mg protein.

[0146] Test results:

[0147] In the model control group, the activities of antioxidant enzymes SOD and GSH-Px were lower, and the level of ROS was higher. The activity of SOD was 14U / mg protein, the activity of GSH-Px was 21U / mg protein, and the fluorescence intensity of ROS was higher.

[0148] In the drug treatment group A, the activities of antioxidant enzymes SOD and GSH-Px increased, with SOD activity at 22U / mg protein and GSH-Px activity at 34U / mg protein. The ROS fluorescence intensity was lower than that in the model control group.

[0149] In the drug treatment group B, the activities of antioxidant enzymes SOD and GSH-Px increased, with SOD activity at 20U / mg protein and GSH-Px activity at 31U / mg protein. The ROS fluorescence intensity was lower than that in the model control group.

[0150] 2. Glutamate receptor and neurotransmitter level detection

[0151] Sample preparation: After the rats were killed, the brain tissue on the ischemic side was quickly removed, rinsed with pre-cooled physiological saline, and homogenized with an appropriate amount of 0.6 mol / L perchloric acid to release glutamate in the tissue. The homogenate was centrifuged at 4°C, 10,000 rpm for 10 minutes, the supernatant was taken, and the pH was adjusted to 6.5 with sodium hydroxide solution. The sample was then filtered through a 0.22 μm filter membrane, and the filtrate was used for HPLC analysis.

[0152] HPLC analysis steps: The mobile phase is a solution containing 30 mM phosphate buffer, and the flow rate is 0.8 ml / min. The injection volume is 10 μl, and the peak area of ​​glutamate is detected by an ultraviolet detector at a wavelength of 210 nm. The content of glutamate in brain tissue is calculated according to the standard curve, and the unit is μmol / g tissue.

[0153] Test results:

[0154] The glutamate content in the brain tissue of the model control group was higher, at 13 μmol / g tissue, the expression and activation of NMDA receptors were higher, and the fluorescence intensity detected by immunofluorescence staining was stronger.

[0155] In group A, the glutamate content in brain tissue was reduced in the drug-treated group. At 9 μmol / g tissue, the expression and activation of NMDA receptors were inhibited, and the fluorescence intensity detected by immunofluorescence staining was weakened compared with that in the model control group.

[0156] In the drug-treated group B, the glutamate content in brain tissue was reduced. At 10 μmol / g tissue, the expression and activation of NMDA receptors were inhibited, and the fluorescence intensity detected by immunofluorescence staining was weakened compared with that in the model control group.

Claims

1. Use of benzotriazole compounds or their salts in the preparation of drugs for treating ischemic stroke.

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

3. The use according to claim 1, characterized in that: The benzotriazole 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 benzotriazole compound is N-(3-(1-benzotriazolyl)propyl)-4-(3-(6-fluorobenzisoxazole))piperidine.

5. The use according to claim 1, characterized in that: The drug dosage forms include oral tablets and injections.

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