A pharmaceutical composition and uses thereof

Abstract

The application discloses a kind of pharmaceutical composition and its application, the pharmaceutical composition includes butylphthalide and sedutin, the mass ratio of butylphthalide and sedutin is 1000:1~1:1000.The effect of the pharmaceutical composition of the application in treating cerebrovascular disease is good, butylphthalide and sedutin show synergistic effect, which helps to obtain excellent curative effect, and reduce drug dosage, which helps to improve the safety of clinical drug use.

Description

[0001] This invention claims priority to Chinese invention patent application CN202510869884.2, filed on June 26, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to a pharmaceutical composition and its application, belonging to the field of medicine. Background Technology

[0003] Cerebrovascular disease (CVD) refers to cerebrovascular diseases caused by various factors. Stroke is the main clinical type of CVD, characterized by high incidence, high mortality, high teratogenicity, and high recurrence rate, and is one of the leading causes of disability and death in humans. Ischemic cerebrovascular disease caused by reduced or interrupted blood flow to the brain and hemorrhagic cerebrovascular disease caused by ruptured blood vessels are the two main types of CVD. Ischemic cerebrovascular disease is mainly cerebral infarction (also known as "ischemic stroke"), an acute cerebrovascular disease caused by various factors leading to impaired blood supply to the brain, resulting in local brain tissue ischemia and hypoxic necrosis. This includes cerebral thrombosis, cerebral embolism, and cerebral infarction caused by hemodynamic mechanisms. In addition to cerebral infarction, ischemic cerebrovascular disease also includes transient ischemic attack (TIA), or transient ischemic attack, whose clinical symptoms generally do not last more than 24 hours, also known as mini-stroke. Hemorrhagic cerebrovascular diseases include cerebral hemorrhage and subarachnoid hemorrhage, which are caused by ruptured blood vessels, blood flowing into the brain parenchyma (cerebral hemorrhage, ICH) or blood flowing into the subarachnoid space (subarachnoid hemorrhage, SAH).

[0004] Butylphthalide (3-butyl-1(3H)-isobenzofuranone, 3-n-Butylphthalide, NBP) belongs to the phthalide family of compounds and has neuroprotective effects by improving microcirculatory and energy metabolism disorders in ischemic cerebral areas. Although butylphthalide can reduce the infarct volume and improve neurological deficits in rats with cerebral ischemia / reperfusion, its anti-ischemic stroke effect needs further improvement, and its side effects, such as abnormal liver and kidney function and skin itching, also need to be further reduced.

[0005] Cerdulatinib is an oral multi-target tyrosine kinase inhibitor that inhibits spleen tyrosine kinase (SYK) and Janus kinase (JAK), significantly reducing the cell activity of a subset of non-Hodgkin's lymphoma (NHL) cell lines and inducing apoptosis in NHL cell lines with BCR signaling. It can be used to treat peripheral T-cell lymphoma.

[0006] It is unclear whether the combination of danbutyrate and cedutinib has a synergistic effect in the prevention and / or treatment of stroke, and no relevant reports have been found. Summary of the Invention

[0007] In view of the shortcomings of the prior art, one objective of the present invention is to provide a pharmaceutical composition with excellent efficacy in preventing and / or treating cerebrovascular diseases; another objective of the present invention is to provide the application of the pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of cerebrovascular diseases.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] A pharmaceutical composition comprising butylphthalide and cedutinib, wherein the mass ratio of butylphthalide to cedutinib is 1000:1 to 1:1000.

[0010] Further, the mass ratio of butylphthalide to cedutinib is 100:1 to 1:100, more preferably 50:1 to 1:50, and even more preferably 30:1 to 1:30.

[0011] Further, the mass ratio of butylphthalide to cedutinib is 2-50:1, preferably 4-45:1, more preferably 8-42:1, and even more preferably 16-40:1.

[0012] Optionally, the mass ratio of butylphthalide to celutinib is 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0013] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients, excipients, or carriers.

[0014] Further, the butylphthalide is a butylphthalide compound or a pharmaceutically acceptable salt or ester thereof, and the cedutinib is a cedutinib compound or a pharmaceutically acceptable salt thereof; the pharmaceutically acceptable salt is a commonly used pharmaceutical salt, preferably selected from one or more of the following: acetate, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, benzoate, fumarate, maleate, succinic acid, tartaric acid, citrate, oxalic acid, glyoxylic acid, aspartic acid, tartrate, 2,5-dihydroxybenzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, lecithinylsulfonate, hydroquinone sulfonate, and p-toluenesulfonate, or salts formed with carboxylic acids.

[0015] Optionally, butylphthalide refers to one of the butylphthalide compound (drug) or its pharmaceutically acceptable salt or ester or isomers thereof (including stereoisomers, tautomers, or racemates) or one of their semi-synthetic derivatives or one of their salts (salt of the compound or salt of the semi-synthetic derivative) or one of their esters or one of their ester salts (salt of the ester of the compound or salt of the ester of the semi-synthetic derivative) or a deuterated compound or isotopically labeled compound thereof.

[0016] Optionally, celutinib refers to the celutinib compound (drug) or its pharmaceutically acceptable salt or co-crystal, any stereoisomer, tautomer, hydrate, or solvate thereof.

[0017] Optionally, butylphthalide is a butylphthalide compound. Optionally, cedutinib is a cedutinib compound.

[0018] Generally, the structural formula of butylphthalide compounds is shown in Formula I, and its molecular formula is C. 12 H 14 O2 has a molecular weight of 190.24.

[0019] Formula I.

[0020] Generally, the structural formula of cedutinib compounds is as shown in the formula. As shown, its molecular formula is C 20 H 27 N7O3S has a molecular weight of 445.54.

[0021] Mode .

[0022] Furthermore, the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form according to known techniques. Optionally, the dosage form of the pharmaceutical composition is one or more of the following: suspension, injection, gel, oil, tablet, suppository, powder, capsule, granule, emulsion, polymer, nanoparticle, microsphere, rectal capsule, enema, paste, ointment, cream, plaster, decoction, implant, spray, and aerosol. Preferred dosage forms include injection, capsule, tablet, granule, powder, spray, liposome, oral liquid, and pellet.

[0023] Optionally, when using, administer 1-3 times daily for 1-several months.

[0024] Based on the same inventive concept, the present invention also provides: a pharmaceutical composition comprising butylphthalide and cedutinib, wherein the molar ratio of butylphthalide and cedutinib is 0.01-100:1.

[0025] Further, the molar ratio of butylphthalide to cedutinib is 4-94:1, more preferably 5-50:1, preferably 8-40:1, more preferably 9-38:1, even more preferably 10-35:1, and still more preferably 12-30:1.

[0026] Further, the molar ratio of butylphthalide to cedutinib is 30:1, 28:1, 26:1, 24:1, 22:1, 20:1, 18:1, 16:1, 14:1, 12:1, 10:1, 8:1, 6:1, 5:1 or 4:1.

[0027] Optionally, butylphthalide and cedutinib in the pharmaceutical composition are intended for concurrent, separate, or time-interval use as a medicine, particularly for the prevention and / or treatment of cerebrovascular diseases in individuals. It should be understood that for concurrent use, the compounds present in the pharmaceutical composition containing these compounds may be mixed together or physically separated; for separate or time-interval use, the present compounds must be physically separated.

[0028] According to one embodiment of the present invention, the amount of butylphthalide in the pharmaceutical composition may be from 0.01 mg to 6000 mg / mg cedutinib, preferably from 1 mg to 600 mg (butylphthalide) / mg cedutinib.

[0029] Optionally, butylphthalide and cedutinib in the pharmaceutical composition may be a common dosage form. Also according to the present invention, butylphthalide and cedutinib in the composition may be the same dosage form or different dosage forms.

[0030] Therefore, it should be understood that, according to the present invention, when butylphthalide and cedutinib of the composition are in the same dosage form, they can be administered simultaneously (i.e., at the same time) and via the same route of administration.

[0031] It should also be understood that, according to the present invention, when butylphthalide and cedutinib of the composition are of the same or different dosage forms, they can be administered simultaneously, sequentially, or separately via the same or different routes of administration.

[0032] Preferably, when administered sequentially, butylphthalide and cedutinib are administered at intervals not exceeding about 1 hour (preferably not exceeding about 10 minutes, even more preferably not exceeding about 1 minute).

[0033] According to the present invention, butylphthalide and cedutinib can be used in combination with one or more acceptable excipients or inert carriers (i.e., pharmaceutically inactive and non-toxic excipients) to impart a specific consistency or other specific physical or taste characteristics to the finished product. These include saline solutions, isotonic solutions, physiological solutions, buffer solutions, etc., which are compatible with the pharmaceutical use and are known to those skilled in the art. The pharmaceutical compositions of the present invention may contain one or more reagents or media selected from the group consisting of: solubilizers, dispersants, stabilizers, sweeteners, preservatives, flavoring agents, lubricants, anti-caking agents, disintegrants, adsorbents, etc. In particular, reagents or media (liquid and / or injectable and / or solid) that can be used in formulations include: methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, polysorbate 80, cyclodextrin, gelatin, mannitol, lactose, PEG, animal oils or vegetable oils, etc. The pharmaceutical compositions of the present invention can be formulated as follows: suspensions or ready-to-use or temporary injection solutions, oils, gels, tablets, powders, suppositories, capsules, granules, emulsions, suspensions, polymers, microspheres, nanoparticles, rectal capsules, enemas, pastes, creams, ointments, plasters, implants, decoctions, sprays, aerosols, etc.; optionally, controlled release and / or sustained release can be achieved through dosage forms or devices. For this type of formulation, reagents such as carbonates, cellulose, or starch are advantageously used.

[0034] In one embodiment, the pharmaceutical composition may be formulated into a powder form and reconstituted for intravenous injection.

[0035] Administration may be performed by any method known to those skilled in the art, preferably orally or parenterally. ， For example, administration can be via intramuscular injection, intravenous administration, subcutaneous injection, intra-arterial injection, intraperitoneal injection, intracerebral or intrathecal injection, oral administration, sublingual administration, delivery to the lesion or brain, implantation, or aerosol administration. Intramuscular injection, intravenous administration, subcutaneous injection, oral or sublingual administration are preferred. For long-term treatment, oral, sublingual or transdermal routes of administration are preferred.

[0036] For injection, the compound may be packaged as a suspension or liquid solution and administered using a syringe or infusion set. It should be understood that those skilled in the art can adjust the amount to be administered or the volume and / or rate of injection based on pathology, route of administration, individual circumstances, etc. It should be understood that repeated administration may be performed in combination with other active ingredients and / or any pharmaceutically acceptable carrier (buffer solution, isotonic solution, saline solution, in the presence of stabilizers, etc.).

[0037] According to some aspects, the pharmaceutical compositions of the present invention can be administered before, during, or after cerebral ischemia / reperfusion injury.

[0038] Optionally, as needed, the amount of the drug composition administered daily may be taken as one, two, three, four, five, six or more doses per day, or as multiple sub-dose taken at appropriate intervals throughout the day.

[0039] Optionally, the amount selected may depend on a variety of factors, particularly the route of administration, duration of administration, time of administration, rate of elimination of the compound, different products used in combination with the composition, the individual's age, weight and physical condition, as well as the individual's medical history, the nature of the pathological condition or degenerative process he or she is facing, and any other medically known information.

[0040] Optionally, the doctor may begin with a lower dose than normally used and then gradually increase these doses to better control potential side effects. Preferably, the compositions of the present invention can be administered for a period of 1 day to 20 years, and even more preferably for a period of 1 day to 3 years.

[0041] Based on the same inventive concept, the present invention also provides the use of the pharmaceutical composition described above in the preparation of medicaments for the prevention and / or treatment of cerebrovascular diseases.

[0042] Furthermore, the cerebrovascular disease includes ischemic cerebrovascular disease.

[0043] Furthermore, the cerebrovascular disease includes stroke, which includes one or both of ischemic stroke and hemorrhagic stroke.

[0044] Furthermore, the administration methods in the application include subcutaneous injection, intravenous injection, intramuscular injection, oral administration, sublingual administration, or spray administration.

[0045] The present invention also relates to a treatment method that may include administering a therapeutically effective amount of a pharmaceutical composition comprising butylphthalide and cedutinib to a person or animal in need.

[0046] The present invention also relates to a method for preparing a pharmaceutical composition comprising butylphthalide and cedutinib, wherein the compound of the composition of the present invention is mixed with an acceptable excipient (particularly a pharmaceutically acceptable excipient) according to methods known per se.

[0047] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0048] The pharmaceutical composition of the present invention has excellent efficacy in the prevention and / or treatment of cerebrovascular diseases. The combined use of butylphthalide and cedutinib as active ingredients exhibits a synergistic effect, which helps to achieve better therapeutic effects, reduce drug dosage, and improve clinical drug safety, showing good prospects for clinical application. In addition, the pharmaceutical composition of the present invention is also expected to enrich the types of clinically available drugs in the field of cerebrovascular diseases, so as to better combat problems such as drug resistance and safe drug use. Attached Figure Description

[0049] Figure 1 This is a graph showing the infarct volume measurement of mice in each group in Example 1.

[0050] Figure 2 The graph shows the neurological function scores of mice in each group in Example 1.

[0051] Figure 3 This is a graph showing the infarct volume measurement of mice in each group in Example 2.

[0052] Figure 4 This is a graph showing the neurological function scores of mice in each group in Example 2. Detailed Implementation

[0053] The present invention will be described in detail below with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0054] Application of the combination of butylphthalide and cedutinib in the preparation of drugs for treating cerebrovascular diseases.

[0055] Materials and methods:

[0056] To demonstrate the effects of butylphthalide and cerebrotinib in the treatment of cerebrovascular diseases, the applicant used a mouse model of ischemic stroke (middle cerebral artery occlusion, MCAO) and administered butylphthalide and cerebrotinib at different time points. Neurological function scores and cerebral infarction volume were measured to detect their protective effects against ischemic stroke.

[0057] The experimental reagents used in each example, butylphthalide (compound) and cedutinib (compound), were purchased from MedChemExpress (MCE) and dissolved and prepared according to the company's reagent instructions.

[0058] Example 1

[0059] Validation of the efficacy of drug compositions containing butylphthalide and celidinib in the treatment of ischemic stroke:

[0060] Animal experiments: Using a mouse model of ischemic stroke, the drug combination containing butylphthalide and cedutinib was used to verify its anti-ischemic stroke effect.

[0061] Experimental animals: Seven-week-old male C57BL / 6J mice were purchased from the Department of Experimental Animals, Central South University. All experimental animals were housed in an SPF-grade housing for one week at a temperature of 22℃±2℃, a relative humidity of 45%±15%, with free access to water and following a 12-hour light / dark cycle. Then, they were administered drugs according to the requirements of each experimental group.

[0062] Methods for establishing a mouse model of ischemic stroke: A mouse model of ischemic stroke (cerebral ischemia / reperfusion) was established using the middle cerebral artery occlusion (MCAO) method. The steps are as follows: Eight-week-old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (20 mL / kg). The left common carotid artery (CCA) was isolated, and the left external carotid artery (ECA) and internal carotid artery (ICA) were isolated superiorly. The ECA and ICA were temporarily clamped with ophthalmic forceps, and the proximal end of the CCA was ligated. Then, a knotted spare suture was placed at the distal end of the CCA, and a small slit was cut at the lower end of the suture. The suture was inserted into the internal carotid artery, the arterial clamps on the ECA and ICA were released, and the suture was advanced into the cranium along the ICA. When resistance was encountered, the suture was tightened and fixed. After 1 hour of ischemia, the suture was removed, the skin was sutured, and the animals were treated after 24 hours of reperfusion.

[0063] Model success criteria: The Longa 5-point scale was used to score the neurological deficits in the mouse model of cerebral ischemia / reperfusion injury (neurological function score). The scoring criteria are as follows: 0 points: no neurological deficit symptoms; 1 point: right forelimb cannot be fully extended; 2 points: rotation to the right; 3 points: walking towards the right and falling; 4 points: unable to walk spontaneously, loss of consciousness. Among them, 1-4 points are considered a valid model.

[0064] The methods for TTC staining and infarct volume determination in mouse brains are as follows: After anesthetizing the mice, the brains were quickly removed, the olfactory bulb and hindbrain were removed, and coronal brain slices were cut starting from the frontal pole. The slices were immediately placed in a 1% TTC solution and incubated at 37°C in the dark for 30 minutes. Then, they were fixed by soaking in a 10% paraformaldehyde solution. Infarcted areas appeared white, and non-infarcted areas appeared red. Each group of brain slices was neatly arranged and scanned. ImageJ image processing software was used to measure the area of ​​the infarcted region and the area of ​​the non-infarcted brain tissue (including ipsilateral and contralateral non-infarcted areas) in each brain slice. The corresponding brain tissue volume was calculated using the formula: Corresponding area brain tissue volume = [(Area of ​​corresponding area of ​​brain tissue on the front + Area of ​​corresponding area of ​​brain tissue on the back) / 2] × thickness of each slice. The whole brain volume of the corresponding area was calculated using the same method. The infarct volume (%) was calculated using the formula: Infarct volume (%) = (Brain tissue volume on the contralateral side of the infarction - Brain tissue volume of the non-infarcted area on the infarcted side) / Brain tissue volume on the contralateral side of the infarction × 100%.

[0065] Experimental grouping and administration: Experimental animals were randomly divided into the following groups, with 6 animals in each group. Both butylphthalide and cedutinib were dissolved in a solvent [0.5% (w / v) aqueous solution of sodium carboxymethyl cellulose]. The experimental animals were administered the test drugs by gavage twice, once 1 hour and once 8 hours after reperfusion. The sham-operated group and the model group were administered an equal volume of the solvent by gavage. That is:

[0066] Sham group: The internal and external carotid arteries were separated without obstructing the arteries; 0.5% (w / v) sodium carboxymethyl cellulose solution was administered by gavage 2 hours and 9 hours after artery separation, and the results were measured 24 hours later.

[0067] Cerebral ischemia / reperfusion group (I / R+solvent group): cerebral ischemia for 1 hour and reperfusion for 24 hours; during reperfusion, 0.5% (w / v) sodium carboxymethyl cellulose solution was administered by gavage 1 hour and 8 hours after reperfusion.

[0068] Butylphthalide 1 + Cerebral Ischemia / Reperfusion Group (I / R + Butylphthalide 1): During the above reperfusion period, butylphthalide (20 mg / kg) was administered by gavage 1 h and 8 h after reperfusion, and the results were measured 24 h after reperfusion.

[0069] Butylphthalide 2 + Cerebral Ischemia / Reperfusion Group (I / R + Butylphthalide 2): During the above reperfusion period, butylphthalide (40 mg / kg) was administered by gavage 1 h and 8 h after reperfusion, and the results were measured 24 h after reperfusion.

[0070] Cerebral ischemia / reperfusion group (I / R + Cerebral ischemia / reperfusion group): During the above reperfusion period, cerebral ischemia / reperfusion group (I / R + Cerebral ischemia / reperfusion group) was administered by gavage 1 h and 8 h after reperfusion, and the results were measured 24 h after reperfusion.

[0071] Cerebral ischemia / reperfusion group (I / R + Cerebral ischemia / reperfusion group): During the above reperfusion period, Cerebral ischemia / reperfusion group (I / R + Cerebral ischemia / reperfusion group) was administered by gavage 1 h and 8 h after reperfusion, and the results were measured 24 h after reperfusion.

[0072] Composition 1 containing butylphthalide and cedutinib + cerebral ischemia / reperfusion group (I / R+ Composition 1): During the above reperfusion period, composition 1 (20 mg / kg mouse body weight for butylphthalide and 2.5 mg / kg mouse body weight for cedutinib) was administered by gavage 1 h and 8 h after reperfusion, and the results were measured 24 h after reperfusion.

[0073] Composition 2 containing butylphthalide and cedutinib + cerebral ischemia / reperfusion group (I / R + composition 2): During the above reperfusion period, composition 2 (20 mg / kg mouse body weight for butylphthalide and 5 mg / kg mouse body weight for cedutinib) was administered by gavage 1 h and 8 h after reperfusion, and the results were measured 24 h after reperfusion.

[0074] The drug composition 3 containing butylphthalide and cedutinib + cerebral ischemia / reperfusion group (I / R + composition 3): During the above reperfusion period, drug composition 3 (the dosage of butylphthalide was 40 mg / kg-mouse body weight and the dosage of cedutinib was 2.5 mg / kg-mouse body weight) was administered by gavage 1 h and 8 h after reperfusion, and the results were detected 24 h after reperfusion.

[0075] The neurological function scores of mice and the volume of cerebral infarction were measured to evaluate the drug's efficacy.

[0076] Data statistics: Statistical analysis was performed using GraphPad Prism 9.0 software. All data are expressed as mean ± standard error (±SEM). Independent samples Student's t-test was used to compare means between two groups. One-way ANOVA or two-way ANOVA was used to analyze differences among multiple groups, followed by Tukey's post-hoc test to determine if significant differences existed between groups. Neurological function scores were analyzed using Kruskal-Wallis and Wilcoxon tests. P A value <0.05 was considered statistically significant.

[0077] Experimental results:

[0078] Effects of the combination of butylphthalide and cedutinib on cerebral infarction volume and neurological function in mice.

[0079] like Figure 1-2As shown, mice in the I / R+ solvent group exhibited significantly increased infarct volume and severe neurological deficits. Gavage administration of drug combinations containing butylphthalide and cedutinib [Composition 3 (butylphthalide 40 mg / kg + cedutinib 2.5 mg / kg), Composition 2 (butylphthalide 20 mg / kg + cedutinib 5 mg / kg), Composition 1 (butylphthalide 20 mg / kg + cedutinib 2.5 mg / kg)] significantly reduced cerebral infarct volume and improved neurological function in mice. Compared to butylphthalide or cedutinib monotherapy, the drug combinations containing butylphthalide and cedutinib (butylphthalide 40 mg / kg + cedutinib 2.5 mg / kg, butylphthalide 20 mg / kg + cedutinib 5 mg / kg, butylphthalide 20 mg / kg + cedutinib 2.5 mg / kg) were significantly more effective than either butylphthalide or cedutinib monotherapy. Therefore, the drug combination containing butylphthalide and cedutinib can significantly reduce cerebral ischemia-reperfusion injury and neurological function impairment, and has a therapeutic effect on ischemic stroke. Its effect is superior to that of butylphthalide or cedutinib alone, demonstrating a synergistic effect (data are expressed as mean ± standard error, number of mice per group n=6, **). P <0.01 vs sham surgery group; # P <0.05、 ## P <0.01、 ### P <0.001 vs I / R group; + P <0.05、 ++ P <0.01、 +++ P <0.001 vs I / R + drug alone group).

[0080] Conclusion: The pharmaceutical composition containing butylphthalide and cedutinib of the present invention has a significantly better therapeutic effect than the single drug, exhibiting a synergistic effect. It can significantly reduce the volume of cerebral infarction and neurological deficits, and improve neurological function. It can be used to prepare drugs to alleviate cerebrovascular diseases and to treat ischemic stroke.

[0081] Example 2

[0082] Animal experiments: Using a mouse model of ischemic stroke, the drug combination containing butylphthalide and cedutinib was used to verify its anti-ischemic stroke effect.

[0083] 1. Laboratory animals and methods

[0084] 1.1 Experimental animals: Seven-week-old male C57BL / 6J mice were purchased from the Department of Experimental Animals, Central South University. All experimental animals were housed in an SPF-grade housing for one week at a temperature of 22℃±2℃, relative humidity of 45%±15%, with free access to water and following a 12-hour light / dark cycle. Then, they were administered drugs according to the requirements of each experimental group.

[0085] 1.2 Method for establishing a mouse model of ischemic stroke: A mouse model of ischemic stroke (cerebral ischemia / reperfusion) was established using the middle cerebral artery occlusion (MCAO) method. The steps are as follows: Eight-week-old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (20 mL / kg). The left common carotid artery (CCA) was isolated, and the left external carotid artery (ECA) and internal carotid artery (ICA) were isolated superiorly. The ECA and ICA were temporarily clamped with ophthalmic forceps, and the proximal end of the CCA was ligated. Then, a knotted spare suture was placed at the distal end of the CCA, and a small slit was cut at the lower end of the suture. The suture was inserted into the internal carotid artery, the arterial clamps on the ECA and ICA were released, and the suture was advanced into the cranium along the ICA. When resistance was encountered, the suture was tightened and fixed. After 1 hour of ischemia, the suture was removed, the skin was sutured, and the animals were treated after 24 hours of reperfusion.

[0086] Model success criteria: The Longa 5-point scale was used to score the neurological deficits in the mouse model of cerebral ischemia / reperfusion injury (neurological function score). The scoring criteria are as follows: 0 points: no neurological deficit symptoms; 1 point: right forelimb cannot be fully extended; 2 points: rotation to the right; 3 points: walking towards the right and falling; 4 points: unable to walk spontaneously, loss of consciousness. Among them, 1-4 points are considered a valid model.

[0087] The methods for TTC staining and infarct volume determination in mouse brains are as follows: After anesthetizing the mice, the brains were quickly removed, the olfactory bulb and hindbrain were removed, and coronal brain slices were cut starting from the frontal pole. The slices were immediately placed in a 1% TTC solution and incubated at 37°C in the dark for 30 minutes. Then, they were fixed by soaking in a 10% paraformaldehyde solution. Infarcted areas appeared white, and non-infarcted areas appeared red. Each group of brain slices was neatly arranged and scanned. ImageJ image processing software was used to measure the area of ​​the infarcted region and the area of ​​the non-infarcted brain tissue (including ipsilateral and contralateral non-infarcted areas) in each brain slice. The corresponding brain tissue volume was calculated using the formula: Corresponding area brain tissue volume = [(Area of ​​corresponding area of ​​brain tissue on the front + Area of ​​corresponding area of ​​brain tissue on the back) / 2] × thickness of each slice. The whole brain volume of the corresponding area was calculated using the same method. The infarct volume (%) was calculated using the formula: Infarct volume (%) = (Brain tissue volume on the contralateral side of the infarction - Brain tissue volume of the non-infarcted area on the infarcted side) / Brain tissue volume on the contralateral side of the infarction × 100%.

[0088] 1.3 Experimental Grouping and Administration: Experimental animals were randomly divided into the following groups, with 6 animals in each group. Both butylphthalide and cedutinib were dissolved in a solvent [0.5% (w / v) aqueous solution of sodium carboxymethyl cellulose]. The experimental animals were administered the test drug by gavage 1 hour after reperfusion. The sham-operated group and the model group were administered an equal volume of the solvent by gavage. That is:

[0089] Sham group: The internal and external carotid arteries were separated without obstructing the arteries; 0.5% (w / v) sodium carboxymethyl cellulose solution was administered by gavage 1 hour after artery separation, and the results were measured 24 hours later.

[0090] Cerebral ischemia / reperfusion group (I / R+solvent group): Cerebral ischemia for 1 hour, reperfusion for 24 hours; during reperfusion, 0.5% (w / v) sodium carboxymethyl cellulose solution was administered by gavage 1 hour after reperfusion.

[0091] Butylphthalide 1 + Cerebral Ischemia / Reperfusion Group (I / R + Butylphthalide 1): During the above reperfusion period, butylphthalide (30 mg / kg) was administered by gavage 1 hour after reperfusion, and the results were measured 24 hours after reperfusion.

[0092] Butylphthalide 2 + Cerebral Ischemia / Reperfusion Group (I / R + Butylphthalide 2): During the above reperfusion period, butylphthalide (100 mg / kg) was administered by gavage 1 hour after reperfusion, and the results were measured 24 hours after reperfusion.

[0093] Cerebral ischemia / reperfusion group (I / R + Cerebral ischemia / reperfusion): During the above reperfusion period, Cerebral ischemia / reperfusion group (I / R + Cerebral ischemia / reperfusion group) was administered by gavage 1 hour after reperfusion, and the results were measured 24 hours after reperfusion.

[0094] Composition 1 containing butylphthalide and cedutinib + cerebral ischemia / reperfusion group (I / R + composition 1): During the above reperfusion period, composition 1 (butylphthalide dosage of 30 mg / kg mouse body weight and cedutinib dosage of 2.5 mg / kg mouse body weight) was administered by gavage 1 hour after reperfusion, and the results were detected 24 hours after reperfusion.

[0095] Composition 2 containing butylphthalide and cedutinib + cerebral ischemia / reperfusion group (I / R + composition 2): During the above reperfusion period, composition 2 (butylphthalide dosage of 100 mg / kg mouse body weight and cedutinib dosage of 2.5 mg / kg mouse body weight) was administered by gavage 1 hour after reperfusion, and the results were detected 24 hours after reperfusion.

[0096] The neurological function scores of mice and the volume of cerebral infarction were measured to evaluate the drug's efficacy.

[0097] Data Statistics: Statistical analysis was performed using GraphPad Prism software. All data are expressed as mean ± standard error (±SEM). Independent samples Student's t-test was used to compare means between two groups. One-way ANOVA or two-way ANOVA was used to analyze differences among multiple groups, followed by Tukey's post-hoc test to determine if significant differences existed between groups. Neurological function scores were analyzed using Kruskal-Wallis and Wilcoxon tests. P A value <0.05 was considered statistically significant.

[0098] Experimental results:

[0099] Effects of the combination of butylphthalide and cedutinib on cerebral infarction volume and neurological function in mice.

[0100] like Figure 3-4 As shown, mice in the I / R+ solvent group exhibited significantly increased infarct volume and severe neurological deficits. Gavage administration of drug combinations containing butylphthalide and cedutinib [Combination 2 (butylphthalide 100 mg / kg + cedutinib 2.5 mg / kg), Combination 1 (butylphthalide 30 mg / kg + cedutinib 2.5 mg / kg)] significantly reduced infarct volume and improved neurological function in mice. Compared to butylphthalide and cedutinib monotherapy, the drug combination containing butylphthalide and cedutinib was significantly more effective. Therefore, the drug combination containing butylphthalide and cedutinib can significantly alleviate cerebral ischemia-reperfusion injury and neurological dysfunction, demonstrating a therapeutic effect on ischemic stroke. Its effect is superior to that of butylphthalide and cedutinib monotherapy, exhibiting a synergistic effect (data are expressed as mean ± standard error, number of mice per group n=6, **). P <0.01 vs sham surgery group; # P <0.05、 ## P <0.01、 ### P <0.001 vs I / R group; + P <0.05、 +++ P <0.001 vs I / R + drug alone group).

[0101] Conclusion: The pharmaceutical composition containing butylphthalide and cedutinib of the present invention has a significantly better therapeutic effect than the single drug, exhibiting a synergistic effect. It can significantly reduce the volume of cerebral infarction and neurological deficits, and improve neurological function. It can be used to prepare drugs to alleviate cerebrovascular diseases and to treat ischemic stroke.

[0102] The above embodiments should be understood as being used only to illustrate the present invention more clearly, and not to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims.

Claims

1. A pharmaceutical composition, characterized in that, It includes butylphthalide and cedutinib, wherein the mass ratio of butylphthalide to cedutinib is 8:1 to 40:

1.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of butylphthalide to cedutinib is 10:1 to 40:

1.

3. The pharmaceutical composition according to claim 2, characterized in that, The mass ratio of butylphthalide to cedutinib is 12:1 to 40:

1.

4. The pharmaceutical composition according to claim 3, characterized in that, The mass ratio of butylphthalide to cedutinib is 30:1 to 40:

1.

5. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of butylphthalide to cedutinib is 16:1 to 40:

1.

6. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The pharmaceutical composition may also include pharmaceutically acceptable excipients, excipients, or carriers.

7. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The butylphthalide is a butylphthalide compound or a pharmaceutically acceptable salt or ester thereof, and the cedutinib is a cedutinib compound or a pharmaceutically acceptable salt thereof.

8. The use of the pharmaceutical composition according to any one of claims 1-7 in the preparation of a medicament for the prevention and / or treatment of cerebrovascular diseases, characterized in that, The cerebrovascular disease mentioned is ischemic stroke.

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