Application of p-hydroxybenzoic acid in preparation of medicine for resisting cerebral apoplexy

Abstract

The invention relates to the technical field of drugs for treating stroke, and discloses application of p-hydroxybenzoic acid in preparation of drugs for treating stroke. After the p-hydroxybenzoic acid solution is injected into the subarachnoid space in an intrathecal injection mode, the cerebral infarction volume of the rat can be reduced, and the neurological score of the model rat is improved; the effect of resisting cerebral apoplexy is exerted.

Description

Technical Field

[0001] The present invention relates to the technical field of stroke drugs, and particularly relates to the application of p-hydroxybenzoic acid in the preparation of drugs for anti-stroke. Background Art

[0002] The statements in this section only provide background information related to the disclosure of the present application and may not constitute prior art.

[0003] Stroke is the leading cause of death in China, and ischemic stroke has the highest incidence. Atherosclerosis is the main cause of ischemic stroke, and secondly, it may be caused by cardioembolism, small artery occlusion, etc.

[0004] Currently, the research on drugs for anti-ischemic stroke mainly involves thrombolysis and neuroprotection. t-PA is the only thrombolytic drug approved by the FDA currently and is the most effective drug treatment strategy at present, but it needs to be intravenously used within 4.5 hours after the onset. Exceeding the time window may increase the risk of bleeding. Due to the strict time window limit for drug use, less than 5.2% of patients can benefit from t-PA thrombolytic therapy. After more than 50 years of research on neuroprotective agents, it has been found that drugs such as edaravone, butylphthalide, citicoline, and cerebroprotein hydrolysate have the potential to be used as neuroprotective agents, but so far, no independent neuroprotective agent has been supported by clinical efficacy in the treatment of ischemic stroke. It is extremely important to continue to search for effective treatment strategies and drugs. Summary of the Invention

[0005] The purpose of the present invention is to: in view of the current lack of drugs for the treatment of ischemic stroke, provide the application of p-hydroxybenzoic acid in the preparation of drugs for anti-stroke, and creatively discover that intrathecal injection of p-hydroxybenzoic acid can play a role in anti-ischemic stroke.

[0006] The technical solution of the present invention is as follows:

[0007] On the one hand, the present invention provides the application of p-hydroxybenzoic acid in the preparation of drugs for anti-stroke.

[0008] On the other hand, the present invention provides the application of p-hydroxybenzoic acid in the preparation of drugs for anti-ischemic stroke.

[0009] On the other hand, the present invention provides an application composition of p-hydroxybenzoic acid in the preparation of drugs for anti-stroke, including p-hydroxybenzoic acid.

[0010] According to a preferred embodiment, the pharmaceutical composition is administered by intrathecal injection.

[0011] According to a preferred embodiment, the intrathecal injection is injecting the drug into the subarachnoid space.

[0012] According to a preferred embodiment, the dosage of p-hydroxybenzoic acid in the pharmaceutical composition is: 20 mg / kg.

[0013] According to a preferred embodiment, the drug is designed as an injection.

[0014] According to a preferred embodiment, it further includes pharmaceutical excipients required for the injection.

[0015] According to a preferred embodiment, the pharmaceutical excipients include one or more of an osmotic pressure regulator, a preservative, an antioxidant, a flocculant, a suspending agent, a buffer, or an adsorbent.

[0016] The beneficial effects of the present invention compared with the existing technology are as follows:

[0017] 1. This application provides a new drug for anti-ischemic stroke - p-hydroxybenzoic acid (4-HA), which can reduce the cerebral infarction volume in rats after intrathecal injection and significantly increase the neurological score of the rats in the model group. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Effect of 4-HA on neurological score of MCAO / R rats 24 h after surgery (Note: compared with the sham operation group, ### P < 0.001);

[0019] Figure 2 Effect of intragastric administration of 4-HA on cerebral infarction rate in a rat model of cerebral ischemia-reperfusion injury;

[0020] Figure 3 Effect of 4-HA on neurological score of MCAO / R model rats 24 h after surgery;

[0021] Figure 4 Effect of 4-HA on cerebral infarction volume in rats with cerebral ischemia-reperfusion injury;

[0022] Figure 5 Schematic diagram of the action principle of 4-HA in this application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions made to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase. To better illustrate the present invention, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present invention can be implemented without some specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0025] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0026] The ischemic stroke rat model was administered p-hydroxybenzoic acid through intragastric administration and intrathecal injection respectively. It was found that the mouse model administered through intragastric administration did not show obvious therapeutic effects; while the intrathecal injection method had significant therapeutic effects on the ischemic stroke mouse model; and it improved the volume of cerebral infarction in rats with ischemic reperfusion injury.

[0027] Example 1 Protective effect of intragastric administration of p-hydroxybenzoic acid on rats with cerebral ischemia / reperfusion injury model

[0028] 1. Experimental materials

[0029] 1.1 Experimental animals

[0030] Animal breed and strain: SD rats, grade: SPF grade, weight: 230 - 250 g, sex: male, license number: SCXK(Beijing)2024 - 0001, certificate number: 110324241104041345, purchased from Spf(Beijing) Biotechnology Co., Ltd.

[0031] 1.2 Experimental reagents and materials

[0032]

[0033]

[0034] 1.3 Experimental Instruments

[0035] Instrument Name Model Manufacturer Small Animal Special Inhalation Anesthesia Machine VMR Midmark Group, USA

[0036] Analytical Balance Secura2250 Sartorius, Germany

[0037] 2. Experimental Methods

[0038] 2.1 Reagent Preparation

[0039] Preparation of p-Hydroxybenzoic Acid (4-HA) (10 mg / kg): Accurately weigh 50 mg of 4-HA powder with an electronic balance, dissolve it in double-distilled water using a 50 ml volumetric flask and make up to the mark. Store it in the dark at 4°C for later use.

[0040] Preparation of 4-HA (20 mg / kg): Accurately weigh 100 mg of 4-HA powder with an electronic balance, dissolve it in double-distilled water using a 50 ml volumetric flask and make up to the mark. Store it in the dark at 4°C for later use.

[0041] Preparation of 0.2% TTC Solution: Accurately weigh 0.4 g of TTC with an electronic balance, add 200 mL of 0.1 M PBS buffer, stir until completely dissolved, and use immediately.

[0042] 2.2 Animal Grouping and Drug Administration

[0043] After 3 days of adaptive feeding of SPF-grade male SD rats, rats with a body weight range of 230 - 250 g were selected and randomly divided into a sham operation group, a model group, a 4-HA (10 mg / kg) group, and a 4-HA (20 mg / kg) group, with 8 rats in each group. Intragastric administration was performed at a volume of 1 mL / 100 g for 7 consecutive days. The sham operation group was given an equal volume of solvent by gavage. Starting from the time after gavage on the 7th day, 30 minutes later, the MCAO / R model was replicated. After inserting the suture, timing started. The suture was removed 2 hours later for reperfusion, and the brain was removed under anesthesia 24 hours later for index detection.

[0044] 2.3 Replication of Cerebral Ischemia / Reperfusion Injury (MCAO / R) Model

[0045] Referring to the previously established and stable model replication method of the research group, the rat MCAO / R model was replicated using the suture method. The replication method is as follows:

[0046] SPF male SD rats were fed adaptively for 3 days, during which the diet was controlled to keep the rats' weight within the range of 260-280 g. After inhalation of 5% isoflurane for anesthesia, they were fixed in a supine position on the laboratory operating table. Conventional iodine was applied to the rat's neck, and an incision was made in the middle right of the neck, with a length of about 1.5 cm. The right common carotid artery (CCA) and vagus nerve of the rat were bluntly separated to the bifurcation with forceps. The proximal end of the CCA was ligated with a 3.0 surgical suture, the distal end of the CCA was clamped with an artery clamp, and a slipknot was tied on the CCA with a suture. A "V"-shaped notch was cut 1 cm away from the bifurcation with ophthalmic scissors, and the round head of the thread plug was slowly inserted. Slightly tighten the joint again, loosen the arterial clamp on CCA, and clamp the external carotid artery with the arterial clamp. During this period, adjust the angle of the line so that the suture can successfully reach the anterior cerebral artery (the black mark on the suture reaches the bifurcation of the internal and external carotid arteries), stop inserting the suture, and remove the suture after 2 hours to complete the reperfusion of the middle cerebral artery.

[0047] Relevance of this rat model to human pathology:

[0048] (1) High degree of simulation of pathological mechanisms

[0049] Embolic mechanism: Mechanical occlusion of the middle cerebral artery (MCA) by suture directly simulates large artery occlusive stroke caused by thrombosis or embolism in humans (such as cardiogenic embolism or atherosclerotic thrombosis).

[0050] Ischemic penumbra: Consistent with human stroke, the model can form an ischemic core area and a surrounding penumbra of reversible damage, providing a key basis for studying reperfusion therapy.

[0051] Reperfusion injury: The restoration of blood flow after thrombus removal can trigger oxidative stress, inflammatory response and blood-brain barrier destruction, simulating the reperfusion injury process after clinical thrombolysis or thrombectomy.

[0052] (2) Similarity of clinical manifestations

[0053] Neurological deficits: Model rats experience symptoms similar to human stroke, such as hemiplegia and balance disorders, which can be quantitatively evaluated using standardized scores.

[0054] Imaging characteristics: MRI / CT showed that the infarct lesions were distributed in the cortex and striatum, and the dynamic changes of diffusion-weighted imaging (DWI) were consistent with the evolution of human stroke images.

[0055] Molecular pathological changes: Pathological features at the molecular and cellular levels, such as neuronal necrosis, glial cell activation, and release of inflammatory factors, highly match those of human stroke.

[0056] 2.4 Neurological score

[0057] Neurological scores were evaluated using the Bederson method at 2 h and 24 h after modeling. As shown in Table 1.

[0058] Table 1 Neurological scoring table of the Bederson method

[0059]

[0060] 2.5 TTC staining to evaluate the cerebral infarction rate

[0061] At 24 h after successful modeling, the rats were anesthetized and the brains were removed. The brains were placed on a glass dish and frozen in a -20°C refrigerator for 20 min, then taken out and continuously sectioned coronally with a blade (a total of 5 slices). 0.2% 2,3,5-triphenyltetrazolium chloride (TTC) solution was added in the dark and placed in a pre-set oven at 37°C for 20 min. After 10 min, the slices were turned over to ensure uniform staining on both sides. After 20 min, the slices were taken out, the TTC solution was removed, and 4% paraformaldehyde was added for fixation overnight. The next day, the slices were taken out, arranged, photographed, and the cerebral infarction rate was statistically analyzed using ImageJ to evaluate the cerebral infarction situation of the brain tissue.

[0062] 3. Data processing and analysis

[0063] IBM SPSS Statistics 26 software was used for statistical analysis, and the experimental results were all shown as mean ± standard deviation (Mean ± SD). Homogeneity of variance test was performed for experimental data conforming to normal distribution. The LSD method was used for testing when the variances were homogeneous; the Tamhane's test was used when the variances were inhomogeneous; non-parametric test was used for data not conforming to normal distribution; P < 0.05 represented that the difference was statistically significant, and the obtained results were plotted into charts using Prism software.

[0064] 4. Experimental results

[0065] 4.1 Effects of intragastric administration of 4-HA on neurological scores of MCAO / R model rats

[0066] The results are shown in Table 2 and Figure 1 As shown, compared with the sham operation group, the neurological scores of the model group increased (P < 0.001), and the difference was statistically significant; compared with the model group, there was no significant difference in the cerebral infarction volume between the 4-HA intragastric administration group and the model group, and the difference was not statistically significant.

[0067] Table 2 Effects of 4-HA on neurological scores of MCAO / R model rats at 24 h after operation (Mean ± SD)

[0068]

[0069] Note: Compared with the sham operation group, ###P < 0.001.

[0070] 4.2 TTC staining was used to detect the cerebral infarction rate

[0071] The results are shown in Table 3. Figure 2 . Compared with the sham operation group, the cerebral infarction volume in the model group increased (P < 0.001), and the difference was statistically significant; compared with the model group, there was no significant difference in the cerebral infarction volume between the 4-HA group and the model group, and the difference was not statistically significant.

[0072] Table 3 Protective effect of 4-HA on cerebral infarction rate in rats with cerebral ischemia-reperfusion injury (Mean ± SD, n = 6)

[0073]

[0074] Note: Compared with the sham operation group, ### P < 0.001.

[0075] Example 2 Protective effect of intrathecal administration of 4-hydroxybenzoic acid on rats with MCAO / R model

[0076] 1. Experimental materials

[0077] 1.1 Experimental animals

[0078] Animal breed and strain: SD rats, grade: SPF, body weight: 230 - 250 g, gender: male, license number: SCXK(Beijing)2024 - 0001, certificate number: 110324241104041345, purchased from Spif (Beijing) Biotechnology Co., Ltd.

[0079] 1.2 Experimental reagents and materials

[0080] Same as 1.2 in the first part.

[0081] 1.3 Experimental instruments

[0082] Same as 1.3 in the first part.

[0083] 2. Experimental methods

[0084] 2.1 Reagent preparation

[0085] Preparation of 4-HA (20 mg / kg): Accurately weigh 30 mg of 4-HA powder with an electronic balance, dissolve it in 50 μL of DMSO, and then add 950 μL of normal saline. Prepare it immediately before use.

[0086] Preparation of 0.2% TTC solution: Accurately weigh 0.4 g of TTC with an electronic balance, add 200 mL of 0.1 M PBS buffer, stir until completely dissolved, and prepare it immediately before use.

[0087] 2.2 Animal grouping and drug administration

[0088] After 3 days of adaptive feeding of SPF-grade male SD rats, rats with a body weight range of 230-250 g were selected and randomly divided into a sham operation group, a model group, an intrathecal administration of 4-HA (20 mg / kg) group, and a positive drug (edaravone 6 mg / kg) group, with 10 rats in each group. Intrathecal administration was performed at a volume of 66.67 μL / 100 g. The sham operation group was given an equal volume of solvent. The administration groups were given the first intrathecal administration during model establishment. The MCAO / R model was replicated. Timing started after inserting the suture. Neurological scoring was performed at 2 h, and the suture was removed for reperfusion. The second administration was performed 24 h later, and the brain was removed under anesthesia for index detection 48 h later.

[0089] 2.3 Replication of cerebral ischemia / reperfusion injury (MCAO / R) model

[0090] Same as part 2.3 of Example 1.

[0091] 2.4 Intrathecal administration method

[0092] After the rats were anesthetized, the hair at the injection site was shaved off and the skin surface at the injection site was disinfected. At the injection site, the left thumb and middle finger were used to touch the bilateral iliac crests of the rats and tighten the skin to both sides. The index finger was used to touch the spine of the rats at the same level as the connection line of the bilateral iliac crests. The highest point touched was the L6 spinous process. A sterile micro syringe was inserted vertically into the space between the L5 and L6 vertebral spinous processes. When a sense of cavity appeared, the rat's tail or hind limb would show a slight twitch and a slight blood return was seen when gently aspirating, which proved that the syringe had entered the subarachnoid space. The syringe was slowly tilted from vertical to 45°. 4-HA was injected once according to the body weight. After completion, the syringe was retained for a period of time, and then the micro syringe was slowly removed, and the injection site was gently pressed for 1-2 min.

[0093] 2.5 Neurological scoring

[0094] Same as part 2.4 of Example 1.

[0095] 2.6 Evaluation of cerebral infarction rate by TTC staining

[0096] Same as part 2.5 of Example 1.

[0097] 3. Data processing and analysis

[0098] IBM SPSS Statistics 26 software was used for statistical analysis, and the experimental results were all shown as mean ± standard deviation (Mean±SD). Homogeneity of variance test was performed for experimental data that conformed to normal distribution. The LSD method was used for testing when the variances were homogeneous; the Tamhane's test was used when the variances were heterogeneous; nonparametric test was used for data that did not conform to normal distribution; P < 0.05 represented that the difference was statistically significant, and the obtained results were plotted into charts using Prism software.

[0099] 4. Experimental Results

[0100] 4.1 Effects on Neurological Scores of Rats 48 h after Surgery

[0101] The results are shown in Table 4. Figure 3 . Compared with the sham operation group, obvious neurological deficit symptoms appeared in each model group, and the neurological scores increased significantly ( ### P < 0.001), with statistically significant differences. Compared with the model group, the neurological scores in the 4-HA and edaravone groups increased significantly ( ** P < 0.01), with statistically significant differences.

[0102] Table 4 Effects of 4-HA on Neurological Scores of Rats in the MCAO / R Model 24 h after Secondary Administration (Mean±SD)

[0103]

[0104] Note: Compared with the Sham group, ### P < 0.001; compared with the Model group, ** P < 0.01.

[0105] 4.2 Detection of Cerebral Infarction Rate by TTC Staining

[0106] The results are shown in Table 5 and Figure 4 as follows. Compared with the sham operation group, the cerebral infarction volume in the model group increased significantly ( ### P < 0.001), with statistically significant differences; compared with the model group, the cerebral infarction volume in the 4-HA and edaravone (ED) groups decreased ( ** P < 0.01, ** P < 0.001), with statistically significant differences.

[0107] Table 5 Effects of 4-HA on Cerebral Infarction

[0108] Volume in Rats with Cerebral Ischemia-Reperfusion Injury ( n = 7 - 8)

[0109]

[0110] Note: Compared with the Sham group, ### P < 0.001; compared with the Model group, ** P < 0.01, *** P < 0.001.

[0111] The above-described embodiments merely represent specific implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application.

Claims

1. Application of p-hydroxybenzoic acid in the preparation of anti-stroke drugs.

2. Application of p-hydroxybenzoic acid in the preparation of drugs for anti-ischemic stroke.

3. A pharmaceutical composition for preventing ischemic stroke, characterized in that: Includes parabens.

4. The anti-ischemic stroke drug according to claim 3, characterized in that: Administer via intrathecal injection.

5. The anti-ischemic stroke drug according to claim 4, characterized in that: The intrathecal injection is injected into the subarachnoid space.

6. The anti-ischemic stroke drug according to claim 3, characterized in that: The single dose of the p-hydroxybenzoic acid is 20 mg / kg.

7. The anti-ischemic stroke drug according to claim 3, characterized in that: The drug is designed as an injection.

8. The anti-ischemic stroke drug according to claim 7, characterized in that: It also includes pharmaceutical excipients required for injections.

9. The anti-ischemic stroke drug according to claim 8, characterized in that: The pharmaceutical excipients include one or more of an osmotic pressure regulator, a preservative, an antioxidant, a flocculant, a suspending agent, a buffer or an adsorbent.

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