Application of combination of indobufen and butylphthalide in preparation of medicine for preventing and / or treating cerebrovascular diseases

Abstract

The invention provides an application of combination of indobufen and butylphthalide in preparation of a drug for preventing and / or treating cerebrovascular diseases, indobufen and butylphthalide are combined for use, have a synergistic effect, remarkably improve the treatment effect, can be used for preparing the drug for preventing, secondarily preventing or treating the cerebrovascular diseases, and has a wide application prospect. The medicine is especially suitable for treating cerebral apoplexy and transient ischemic attack, and can be used for prophylactically improving cerebral infarction caused by cerebral arterial thrombosis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the use of indobufen in combination with butylphthalide in the preparation of a medicament for preventing or treating cerebrovascular diseases. Background Art

[0002] "Classification of Cerebrovascular Diseases in China 2015" defines cerebrovascular disease (CVD) as transient or permanent neurological deficits caused by one or more cerebrovascular lesions due to various reasons, which is divided into two major categories: ischemic cerebrovascular disease and hemorrhagic cerebrovascular disease. Stroke refers to acute cerebrovascular disease, which is an acute-onset focal vascular neurodeficit syndrome with symptoms lasting for more than 24 hours or death, excluding other non-cerebrovascular disease etiologies. Therefore, the concept of cerebrovascular disease encompasses stroke.

[0003] With the acceleration of social aging and urbanization processes, the prevalence of unhealthy lifestyles among residents, and the widespread exposure of risk factors for cerebrovascular diseases, the burden of stroke diseases in China has shown an explosive growth trend, presenting a rapid increase in the low-income group, obvious gender and regional differences, and a trend of youthfulness. According to the "Report on the Prevention and Treatment of Stroke in China 2019", the standardized incidence rate of first stroke among Chinese residents aged 40 to 74 currently increases by 8.3% on average every year. The standardized prevalence rate of stroke among residents aged ≥40 years has increased from 1.89% in 2012 to 2.32% in 2018. It is estimated that the current number of stroke patients among residents aged ≥40 years is 13.18 million, and more than 1.9 million people die of stroke every year.

[0004] Indobufen is an isoindolinylphenylbutyric acid derivative and is an inhibitor of platelet aggregation.

[0005]

[0006] It is currently the only reversible and selective multi-target anti-thrombotic drug, which effectively blocks thrombus formation by comprehensively inhibiting multiple links and multiple targets during the thrombus formation process. It is used for preventing and treating ischemic cardiovascular lesions, ischemic cerebrovascular lesions, venous thrombosis caused by arteriosclerosis, and preventing thrombus formation during hemodialysis.

[0007] N-butylphthalide (NBP), also known as butylphthalide, has the chemical name of 3-butyl-1(3H)-isobenzofuranone and is commonly known as apiumetin. It is the main component of celery volatile oil and is mainly used to improve the neurological deficits of patients with acute ischemic stroke. Butylphthalide has a dual mechanism of action of opening collateral circulation and protecting mitochondria, and can comprehensively improve the prognosis of recanalized patients by restoring blood perfusion and promoting neurological function remodeling. However, at present, the treatment of indobufen, butylphthalide and cerebrovascular diseases, especially ischemic cerebrovascular diseases, needs to be further studied, and there is an urgent need for more effective drugs for the prevention and treatment of cerebrovascular diseases in clinical practice. Summary of the Invention

[0008] The object of the present invention is to provide a combined drug, including indobufen and butylphthalide. The combined drug is used for the prevention and / or treatment of cerebrovascular diseases. The combined use of drugs has a synergistic effect, which can improve the effects of preventing, secondary preventing and treating cerebrovascular diseases, especially can prophylactically improve the cerebral infarction caused by ischemic stroke.

[0009] Preferably, the cerebrovascular disease is ischemic cerebrovascular disease, and the ischemic cerebrovascular disease is stroke and transient ischemic attack, especially ischemic stroke.

[0010] Indobufen and butylphthalide can be administered simultaneously or sequentially, and the preferred administration method is oral administration.

[0011] Another aspect of the present invention provides a pharmaceutical composition, including indobufen and butylphthalide. When this pharmaceutical composition is used in combination, it has a synergistic effect, which can improve the effects of preventing, secondary preventing and treating cerebrovascular diseases.

[0012] Preferably, the weight ratio of indobufen to butylphthalide is 1:8 to 8:1, and the preferred weight ratios are 1:4 to 8:1, 1:4 to 4:1, 1:2 to 4:1, 2:1 to 4:1.

[0013] Preferably, the weight ratio of indobufen to butylphthalide is 1:1 or 1:2, and preferably 2:1 or 4:1.

[0014] According to the embodiments of the present invention, the human administration dose of indobufen is 1.5 to 12 mg / kg / d, and the human administration dose of butylphthalide is 1.5 to 12 mg / kg / d.

[0015] The above-mentioned pharmaceutical composition is preferably an oral dosage form, and may also contain pharmaceutically acceptable excipients, such as saccharide diluents, cellulose diluents, disintegrants or lubricants.

[0016] Another object of the present invention is to provide an application of the above-mentioned pharmaceutical composition in the preparation of drugs for preventing or treating cerebrovascular diseases.

[0017] Preferably, the above drug composition is used for the preparation of a drug for treating ischemic cerebrovascular diseases.

[0018] Preferably, the above drug composition is used for the preparation of a drug for treating stroke and transient ischemic attack, especially ischemic stroke.

[0019] The combined drug or drug composition provided by the present invention, comprising indobufen and butylphthalide, has a synergistic effect when treating cerebrovascular diseases, can significantly increase the therapeutic effect, can reduce the effective doses of indobufen and butylphthalide, and has low toxicity. Moreover, the drug composition is preferably administered orally, which is convenient to take, has good compliance, is easy to ingest and carry, and has high safety. It is especially suitable for the prevention, secondary prevention and long-term maintenance treatment of stroke and transient ischemic attack, especially for the preventive improvement of cerebral infarction caused by ischemic stroke. Description of the Drawings

[0020] Figure 1 . Effect of continuous 3-day oral gavage (ig.) administration on the balance beam test of stroke mice (Mean±SEM);

[0021] Figure 2 . Effect of continuous 3-day oral gavage (ig.) administration on the grip strength of stroke mice (Mean±SEM);

[0022] Figure 3 . Effect of continuous 3-day oral gavage (ig.) administration on the rotarod test of stroke mice (Mean±SEM);

[0023] Figure 4 . Effect of continuous 3-day oral gavage (ig.) administration on the cerebral blood flow difference of stroke mice (Mean±SEM);

[0024] Figure 5 . Effect of continuous 3-day oral gavage (ig.) administration on the brain volume of stroke mice (Mean±SEM);

[0025] Figure 6 . Effect of continuous 3-day oral gavage (ig.) administration on the cerebral infarction area ratio of stroke mice (Mean±SEM);

[0026] Figure 7 . Effect of continuous 3-day oral gavage (ig.) administration on the brain water content of stroke mice (Mean±SEM);

[0027] Figure 8 . Effects of different administration concentrations (μmol / L) of indobufen and butylphthalide on HUVECS;

[0028] Figure 9Synergyfinder calculations were performed at different administration concentrations (μmol / L) of indobufen and butylphthalide. Detailed implementation manners

[0029] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0030] The present invention proposes the use of a combined drug, indobufen, and the combination of butylphthalide in the preparation of a drug, a pharmaceutical composition, and the use of the pharmaceutical composition in the preparation of a drug, which will be described in detail below respectively.

[0031] Combined drug

[0032] In one aspect of the present invention, the present invention proposes a combined drug. According to an embodiment of the present invention, the combined drug includes: indobufen and butylphthalide. The present invention discovers that by combining indobufen and butylphthalide for drug administration, when treating cerebrovascular diseases, there is a synergistic effect, which can significantly increase the treatment effect.

[0033] As used herein, the term "combined drug" refers to a drug combined for the simultaneous or sequential administration of indobufen and butylphthalide in disease prevention or treatment. The combined drug of the present invention can also be provided in the form of a formulation containing both indobufen and butylphthalide. In the composition containing both indobufen and butylphthalide, preferably, the weight ratio of indobufen to butylphthalide is 1:8 - 8:1, 1:4 - 8:1, 1:4 - 4:1, 1:2 - 4:1, 2:1 - 4:1, more preferably, the weight ratio of indobufen to butylphthalide is 1:1, 1:2, 2:1, 4:1, especially when the weight ratio of indobufen to butylphthalide is 2:1 or 4:1, it can effectively reduce the poor cerebral blood flow after stroke prophylactically and improve the cerebral infarction of stroke. In addition, a preparation containing indobufen and a preparation containing butylphthalide are provided separately, and these preparations can be used simultaneously or sequentially.

[0034] According to an embodiment of the present invention, indobufen is provided in the form of a white powder.

[0035] As used in the text, the term "indobufen" is an isobenzofuranone phenylbutyric acid derivative, and its specific structure and characteristics are described as follows: English name: Indobufen, chemical name: (±)2-[4-(1-oxo-2-isoindolinyl)phenyl]butyric acid, CAS: 63610-08-2, molecular formula: C18H17NO3, molecular weight: 295.34.

[0036] Indobufen is an antiplatelet aggregation drug developed by Farmitalia Carlo Erba Pharmacia (Pharmacia, now Pfizer). The mechanism of action of indobufen is similar to that of aspirin, which reversibly / highly selectively inhibits platelet cyclooxygenase-1 (COX-1) and blocks the synthesis of thromboxane A2 (TXA2). The reversible inhibition hardly affects the synthesis of prostaglandin (PG) in the gastrointestinal mucosa, so there are fewer gastrointestinal reactions and a lower bleeding risk. Indobufen is mainly used clinically for ischemic heart, cerebrovascular and peripheral vascular diseases caused by arteriosclerosis, venous thrombosis, lipid metabolism disorders, etc.; it can also be used to prevent thrombosis during extracorporeal circulation surgery.

[0037] The chemical name of butylphthalide is dl-3-butyl-1(3H)-isobenzofuranone, which is used to improve the neurological deficits of patients with acute ischemic stroke. Butylphthalide is a synthetic racemic n-butylphthalide, which has the same structure as natural levocelery apigenin. Clinically, this product should be administered within 48 hours after the onset of the disease, either by intravenous infusion or orally. Butylphthalide is a light yellow or yellow oily liquid. According to the embodiments of the present invention, indobufen and butylphthalide can further play a synergistic role in improving cerebral infarction caused by stroke and better achieve the therapeutic purpose.

[0038] Use

[0039] In another aspect of the present invention, the present invention proposes the use of indobufen and butylphthalide in combination in the preparation of a drug. According to the embodiments of the present invention, the drug is used for preventing or treating cerebrovascular diseases, especially stroke and transient ischemic attack in ischemic cerebrovascular diseases, especially for prophylactically improving cerebral infarction caused by ischemic stroke.

[0040] As used herein, the term "treatment" refers to obtaining the desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting a disease, such as blocking the development of the disease; or (c) relieving a disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any administration of a drug to an individual to treat, cure, relieve, improve, alleviate or inhibit the disease of the individual, including but not limited to administering the drug described herein to an individual in need.

[0041] According to the embodiments of the present invention, indobufen and butylphthalide are administered simultaneously or sequentially.

[0042] In this text, the term "simultaneous administration" refers to the combined use of indobufen and butylphthalide administered during the same period, which can be administered in the form of a formulated agent, or in the form of a mixture prepared at the time of administration, or other forms of preparations can also be administered during the same period. When used in simultaneous administration, it can be administered through respective routes or the same route, and the dosage forms can be the same or different respectively. In the present invention, when indobufen and butylphthalide are administered separately, the administration order can be to administer butylphthalide after indobufen, or indobufen and butylphthalide can be administered simultaneously, or butylphthalide can be administered after indobufen. In some embodiments, the administration method of indobufen and butylphthalide is simultaneous administration.

[0043] In this text, the term "sequential administration" refers to administering one agent and then another agent, and there is no particular limitation on the administration interval between indobufen and butylphthalide, and factors such as the administration route or dosage form can be considered for setting. In the above drugs, when indobufen and butylphthalide are provided in their respective preparations, the dosage forms of these preparations can be the same dosage form or different dosage forms. For example, both can be one of oral preparations, non-oral preparations, injections, drip infusions, intravenous drip infusions and different dosage forms from each other, and both can also be one of oral preparations, non-oral preparations, injections, drip infusions, intravenous drip infusions and the same dosage form. In addition, more than one different preparation can be combined in the above drugs. In some embodiments, the administration method of indobufen and butylphthalide is oral administration.

[0044] In the present invention, "subject" is not limited and refers to humans or non-human mammals as the administration subjects of the drugs of the present invention, such as mammals including cows, horses, dogs, sheep or cats. The preferred subject is human. The subject includes patients (including humans and non-human mammals).

[0045] The materials, reagents, etc. used in the following examples are all ordinary commercially available products and can be purchased on the market without special instructions.

[0046] The indobufen used therein was self-made by Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd., and its property was white powder; butylphthalide was self-made by Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd., and its property was light yellow or yellow oily liquid.

[0047] The present invention will be further described below through examples, and these descriptions do not further limit the content of the present invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the protection scope of the present invention.

[0048] Example 1 Drug Preparation

[0049] 1.1 Solvent Preparation

[0050] Solvent composition: 5% DMSO + 15% PEG400 + 80% cyclodextrin

[0051] 1.2 Preparation of butylphthalide

[0052] 13.33 mg / mL: Weigh 240 mg of butylphthalide, add 18 mL of solvent, vortex and mix until completely dissolved to obtain Solution ①.

[0053] 6.67 mg / mL: Pipette 4 mL of Solution ①, add 4 mL of solvent, and the final volume is 8 mL to obtain Solution ②.

[0054] 3.33 mg / mL: Pipette 4 mL of Solution ②, add 4 mL of solvent, and the final volume is 8 mL to obtain Solution ③.

[0055] 1.67 mg / mL: Pipette 4 mL of Solution ③, add 4 mL of solvent, and the final volume is 8 mL to obtain Solution ④.

[0056] 1.3 Preparation of indobufen

[0057] 13.3 mg / mL: Weigh 240 mg of indobufen, add 18 mL of solvent, vortex and mix until completely dissolved to obtain Solution ⑤.

[0058] 6.67 mg / mL: Pipette 4 mL of Solution ⑤, add 4 mL of solvent, and the final volume is 8 mL to obtain Solution ⑥.

[0059] 3.33 mg / mL: Pipette 4 mL of Solution ⑥, add 4 mL of solvent, and the final volume is 8 mL to obtain Solution ⑦.

[0060] 1.67 mg / mL: Pipette 4 mL of Solution ⑦, add 4 mL of solvent, and the final volume is 8 mL to obtain Solution ⑧.

[0061] Example 2 Comparison of drug effects

[0062] 1 Materials and methods

[0063] 1.1 Animals Healthy male ICR mice, weighing 25 - 30 g.

[0064] 1.2 Drugs are indobufen and butylphthalide solutions with different concentrations prepared in Example 1 as reagents.

[0065] 1.3 Methods

[0066] 1.3.1 Preparation of the focal cerebral ischemia-reperfusion model. Main steps: Mice were anesthetized by inhaling isoflurane before surgery and fixed in the supine position on a mouse board. The right common carotid artery, external carotid artery, and internal carotid artery were isolated. A nylon suture was slowly advanced through the incision of the main trunk of the right external carotid artery towards the intracranial direction of the internal carotid artery. When a slight resistance was felt during the advancement, the blood supply of the middle cerebral artery was blocked. About 20 minutes after infarction, a Doppler flowmeter was used to observe the cerebral blood flow in the mice to determine the successful establishment of the model and inclusion in the group. The suture was removed 1 hour after infarction to complete the cerebral ischemia-reperfusion injury model. In the sham operation group, only the blood vessels were isolated, and the remaining preparation steps were not carried out in the model group.

[0067] 1.3.2 Animal grouping and drug administration. The experimental animals were randomly divided into 11 groups, with 12 animals in each group, for a total of 132 animals, namely the sham operation group (1 group), the model group (1 group), and the drug groups (9 groups, with the mice in the model group receiving drugs). The drug groups were orally administered the corresponding test drugs with different ratios once a day, and the model group and the sham operation group were given an equal volume of blank solvent in the same way for three consecutive days (surgery for model establishment was performed 1 hour after the last drug administration).

[0068] 1.3.3 Determination of neurological deficit score and cerebral infarction volume

[0069] At the end of the experiment (72 hours after surgery), the changes in cerebral blood flow of all animals were measured using a Doppler flowmeter to evaluate the improvement effect of the test substance on ischemic stroke; behavioral function tests were performed; after the behavioral tests, 12 mice in each group were euthanized, and the brain tissues were collected. Among them, 6 mice in each group were taken, and after collecting the brain tissues, serial coronal sections were made, stained with TTC (2,3,5-triphenyltetrazolium chloride), photographed, and the infarct area was measured using image pro plus 6.0 to calculate the total infarct area, and the percentage of the infarct area in the whole brain area was calculated. For the remaining 6 mice, the brain tissues were collected, weighed, dried, and weighed again to calculate the brain water content.

[0070] Note: Infarct area ratio = infarct area ÷ whole brain area × 100%;

[0071] Infarct improvement rate = (infarct area ratio of the model group - infarct area ratio of the drug administration group) ÷ infarct area ratio of the model group × 100%.

[0072] Brain water content = wet brain weight - brainstem weight.

[0073] 2 Results

[0074] ① Effect on the behavioral function of stroke mice 72 hours after surgery

[0075] Balance beam test: The passing time of the sham operation group mice on the balance beam was 8.06 ± 1.86 sec; the passing time of the model group mice on the balance beam was 60.00 ± 0.00 sec, showing a statistically significant difference compared with the sham operation group (p < 0.01); the passing times of the mice in the butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group on the balance beam were 48.19 ± 20.68 sec and 49.82 ± 15.09 sec respectively. Compared with the model group, the indobufen (133.33 mg / kg) group showed a statistically significant difference (p < 0.05); the passing times of the mice in each group of the butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) group on the balance beam were 49.87 ± 16.52 sec, 59.39 ± 2.11 sec and 58.93 ± 2.50 sec respectively; the passing time of the mice in the butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group on the balance beam was 52.98 ± 9.20 sec, showing a statistically significant difference compared with the model group (p < 0.05); the passing times of the mice in each group of the butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) group on the balance beam were 45.60 ± 18.35 sec, 48.35 ± 16.64 sec and 43.26 ± 18.52 sec respectively, all showing statistical differences compared with the model group (p < 0.05, p < 0.01).

[0076] Table 1. Effects of oral gavage (ig.) administration for 3 consecutive days on the balance beam test of stroke mice (Mean ± SD)

[0077]

[0078] ## p < 0.01 vs. Control; * p < 0.05, ** p < 0.01 vs. Model

[0079] Grip strength test: The grip strength of mice in the sham operation group was 318.43 ± 26.97 g; the grip strength of mice in the model group was 120.67 ± 12.74 g, showing a statistically significant difference compared with the sham operation group (p < 0.01); the grip strengths of mice in the DL - 3 - n - butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group were 167.45 ± 32.36 g and 181.58 ± 29.68 g respectively, showing statistical differences compared with the model group (p < 0.01); the grip strengths of mice in the DL - 3 - n - butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) groups were 176.54 ± 20.02 g, 185.07 ± 29.55 g and 192.75 ± 15.83 g respectively, showing statistical differences compared with the model group (p < 0.01); the grip strength of mice in the DL - 3 - n - butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group was 186.41 ± 20.09 g, showing a statistically significant difference compared with the model group (p < 0.01); the grip strengths of mice in the DL - 3 - n - butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups were 200.00 ± 21.07 g, 190.57 ± 28.06 g and 178.64 ± 24.61 g respectively, showing statistical differences compared with the model group (p < 0.01).

[0080] Table 2. Effects of oral gavage (ig.) administration for 3 consecutive days on the grip strength of stroke rats (Mean ± SD)

[0081]

[0082] ## p < 0.01 vs. Control; * p < 0.05, ** p < 0.01 vs. Model

[0083] The retention time of the sham-operation group mice on the rotarod was 300.00 ± 0.00 sec; the retention time of the model group mice on the rotarod was 54.92 ± 50.77 sec, showing a statistically significant difference compared with the sham-operation group (p < 0.01); the retention times of the mice in the butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group on the rotarod were 78.33 ± 48.78 sec and 71.33 ± 51.76 sec respectively; the retention times of the mice in each group of the butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) group on the rotarod were 85.33 ± 89.91 sec, 71.92 ± 86.56 sec and 57.92 ± 24.96 sec respectively; the retention time of the mice in the butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group on the rotarod was 71.25 ± 43.99 sec; the retention times of the mice in each group of the butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) group on the rotarod were 97.25 ± 45.39 sec, 93.33 ± 85.30 sec and 91.08 ± 59.52 sec respectively. Compared with the model group, the butylphthalide (66.67 mg / kg) + indobufen (133.33 mg / kg) group showed a statistical difference (p < 0.05).

[0084] Table 3. Effects of oral gavage (ig.) administration for 3 consecutive days on the rotarod test of stroke mice (Mean ± SD)

[0085]

[0086]

[0087] ## p < 0.01 vs. Control; * p < 0.05, ** p < 0.01 vs. Model

[0088] ② Effects on cerebral blood flow in stroke mice:

[0089] (1) Twenty minutes after surgery, the cerebral blood flow of mice in each group was observed. The difference in cerebral blood flow of mice in the sham operation group was 0.37 ± 0.30%; the difference in cerebral blood flow of mice in the model group was 54.57 ± 3.41%, showing a statistically significant difference compared with the sham operation group (p < 0.01); the differences in cerebral blood flow of mice in the DL - 3 - n - butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group were 52.24 ± 2.18% and 49.15 ± 7.76% respectively, showing statistical differences compared with the model group (p < 0.05); the differences in cerebral blood flow of mice in the DL - 3 - n - butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) groups were 47.81 ± 6.59%, 46.44 ± 7.43% and 47.61 ± 4.29% respectively, showing statistical differences compared with the model group (p < 0.01); the difference in cerebral blood flow of mice in the DL - 3 - n - butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group was 45.40 ± 5.36%, showing a statistical difference compared with the model group (p < 0.01); the differences in cerebral blood flow of mice in the DL - 3 - n - butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups were 48.01 ± 3.58%, 47.14 ± 8.97% and 48.59 ± 6.79% respectively, showing statistical differences compared with the model group (p < 0.01, p < 0.05).

[0090] (2) The cerebral blood flow of mice in each group was observed 72 h after surgery. The difference in cerebral blood flow of mice in the sham operation group was 0.43 ± 0.21%; the difference in cerebral blood flow of mice in the model group was 47.55 ± 5.04%, showing a statistically significant difference compared with the sham operation group (p < 0.01); the differences in cerebral blood flow of mice in the butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group were 41.89 ± 6.18% and 39.86 ± 11.29% respectively, showing statistical differences compared with the model group (p < 0.05); the differences in cerebral blood flow of mice in the butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) groups were 33.66 ± 14.74%, 39.56 ± 9.17% and 33.47 ± 10.88% respectively, showing statistical differences compared with the model group (p < 0.01, p < 0.05); the difference in cerebral blood flow of mice in the butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group was 33.47 ± 9.42%, showing a statistical difference compared with the model group (p < 0.01); the differences in cerebral blood flow of mice in the butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups were 37.12 ± 10.95%, 38.63 ± 13.21% and 37.04 ± 8.93% respectively, showing statistical differences compared with the model group (p < 0.01, p < 0.05).

[0091] Table 4. Effects of oral gavage (ig.) administration for 3 consecutive days on the difference in cerebral blood flow in stroke mice (Mean ± SD)

[0092]

[0093] ## p < 0.01 vs. Control; * p < 0.05, ** p < 0.01 vs. Model

[0094] ③Effect on brain volume at 72 h after surgery: The brain volume of mice in the sham operation group was 0.350 ± 0.02 mL; the brain volume of mice in the model group was 0.404 ± 0.02 mL, showing a statistically significant difference compared with the sham operation group (p < 0.01); the brain volumes of mice in the butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group were 0.394 ± 0.03 mL and 0.391 ± 0.01 mL, respectively; the brain volumes of mice in the butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) groups were 0.383 ± 0.03 mL, 0.384 ± 0.02 mL, and 0.377 ± 0.01 mL, respectively. Compared with the model group, the butylphthalide (133.33 mg / kg) + indobufen (33.33, 66.67 mg / kg) groups showed statistical differences (p < 0.05, p < 0.01); the brain volume of mice in the butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group was 0.378 ± 0.03 mL, showing a statistical difference compared with the model group (p < 0.05); the brain volumes of mice in the butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups were 0.372 ± 0.03 mL, 0.381 ± 0.04 mL, and 0.383 ± 0.02 mL, respectively. Compared with the model group, the butylphthalide (66.67, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups all showed statistical differences (p < 0.05).

[0095] Table 5. Effect of oral gavage (ig.) administration for 3 consecutive days on brain volume of stroke mice (Mean ± SD)

[0096]

[0097] ## p < 0.01 vs. Control; * p < 0.05, ** p < 0.01 vs. Model

[0098] ④ Cerebral infarction area in mice after stroke at 72 h post-operation: The cerebral infarction area ratio in the sham-operation group was 0.00 ± 0.00%; the cerebral infarction area ratio in the model group was 44.32 ± 3.40%, showing a statistically significant difference compared with the sham-operation group (p < 0.01); the cerebral infarction area ratios in the butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group were 32.90 ± 7.20% and 34.55 ± 3.76% respectively, showing statistical differences compared with the model group (p < 0.01); the cerebral infarction area ratios in the butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) groups were 38.62 ± 6.18%, 32.30 ± 9.65% and 33.20 ± 7.28% respectively, showing statistical differences in the butylphthalide (133.33 mg / kg) + indobufen (33.33, 66.67 mg / kg) groups compared with the model group (p < 0.05); the cerebral infarction area ratio in the butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group was 27.97 ± 9.93%, showing a statistical difference compared with the model group (p < 0.01); the cerebral infarction area ratios in the butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups were 23.75 ± 14.37%, 33.34 ± 11.39% and 35.04 ± 8.27% respectively, showing a statistical difference in the butylphthalide (66.67 mg / kg) + indobufen (133.33 mg / kg) group compared with the model group (p < 0.05).

[0099] At 72 h post-operation, the improvement rate of cerebral infarction in the butylphthalide (133.33 mg / kg) group was 25.76%, the improvement rate of cerebral infarction in the indobufen (133.33 mg / kg) group was 22.05%, the improvement rates of cerebral infarction in the butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) groups were 12.87%, 27.13% and 25.08% respectively, the improvement rate of cerebral infarction in the butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group was 36.89%, and the improvement rates of cerebral infarction in the butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups were 46.41%, 24.78% and 20.94% respectively.

[0100] Table 6. Effects of oral gavage (ig.) administration for 3 consecutive days on the cerebral infarction area ratio in stroke mice (Mean ± SD)

[0101]

[0102] ## p < 0.01 vs. Control; * p < 0.05, ** p < 0.01 vs. Model

[0103] ⑤ Brain water content in stroke mice: The brain water content in the sham operation group was 265.50 ± 13.22 mg; the brain water content in the model group was 324.00 ± 20.51 mg, showing a statistically significant difference compared with the sham operation group (p < 0.01); the brain water contents in the DL - 3 - n - butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group were 300.50 ± 30.34 mg and 298.83 ± 17.20 mg respectively. Compared with the model group, the indobufen (133.33 mg / kg) group showed a statistical difference (p < 0.05); the brain water contents in the DL - 3 - n - butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) groups were 295.50 ± 27.87 mg, 297.17 ± 20.82 mg and 287.33 ± 14.22 mg respectively. Compared with the model group, the DL - 3 - n - butylphthalide (133.33 mg / kg) + indobufen (33.33, 66.67 mg / kg) groups both showed statistical differences (p < 0.05, p < 0.01); the brain water content in the DL - 3 - n - butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group was 293.17 ± 25.54 mg, showing a statistical difference compared with the model group (p < 0.05); the brain water contents in the DL - 3 - n - butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups were 278.83 ± 26.50 mg, 293.17 ± 34.76 mg and 287.67 ± 12.04 mg respectively. Compared with the model group, all showed statistical differences (p < 0.01).

[0104] At 72 h after surgery, the brain water content ratio of mice in the sham operation group was 79.11 ± 0.39%; the brain water content ratio of mice in the model group was 82.44 ± 0.96%, showing a statistically significant difference compared with the sham operation group (p < 0.01); the brain water content ratios of mice in the butylphthalide (133.33 mg / kg) group and indobufen (133.33 mg / kg) group were 81.25 ± 1.21% and 81.57 ± 0.69% respectively. The brain water content ratios of mice in the butylphthalide (133.33 mg / kg) + indobufen (16.67, 33.33, 66.67 mg / kg) groups were 80.96 ± 1.37%, 81.02 ± 1.50% and 80.53 ± 1.45% respectively. Compared with the model group, the butylphthalide (133.33 mg / kg) + indobufen (66.67 mg / kg) group showed a statistical difference (p < 0.05); the brain water content ratio of mice in the butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group was 81.22 ± 1.25%. The brain water content ratios of mice in the butylphthalide (66.67, 33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups were 80.86 ± 1.43%, 80.93 ± 1.29% and 80.71 ± 0.80% respectively. Compared with the model group, the butylphthalide (33.33, 16.67 mg / kg) + indobufen (133.33 mg / kg) groups both showed statistical differences (p < 0.05, p < 0.01).

[0105] Table 7. Effects of oral gavage (ig.) administration for 3 consecutive days on brain water content in stroke mice (Mean ± SD)

[0106]

[0107] 3 Conclusions

[0108] Combined administration of indobufen and butylphthalide can effectively reduce the nerve function injury in stroke mice, improve the neurobehavioral function of grip strength in stroke mice, effectively reduce the poor cerebral blood flow after surgery in stroke mice, and have a protective effect on cerebral infarction in stroke mice, which is superior to the single administration of indobufen or butylphthalide. Among them, the effect of the butylphthalide (133.33 mg / kg) + indobufen (133.33 mg / kg) group is better than that of the butylphthalide (133.33 mg / kg) + indobufen (66.667 mg / kg) group. However, the combined administration groups of butylphthalide (66.67 mg / kg) + indobufen (133.33 mg / kg) and butylphthalide (33.33 mg / kg) + indobufen (133.33 mg / kg), that is, when the ratio of butylphthalide to indobufen is 1:2, 1:4 and between 1:2 - 1:4, have the best improvement effect.

[0109] Example 3 Comparison of in vitro effects of drugs

[0110] 1 Materials and methods

[0111] 1.1 Cells

[0112] Human umbilical vein endothelial cells (HUVECs)

[0113] 1.2 Main reagents

[0114] Complete medium for human umbilical vein endothelial cells; tert-butyl hydroperoxide (t-BHP)

[0115] 2 Experimental methods

[0116] 2.1 Cell culture and grouping treatment

[0117] Culture HUVECs in complete medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The experimental cells are divided into a control group and an experimental group, both of which are intervened with t-BHP (300 μmol / L), and the experimental group is given different concentrations of indobufen and butylphthalide. The cells are grouped and inoculated on a 96-well plate, 100 μL per well, and incubated in the incubator for 24 h. Detect cell viability using a CCK8 kit and measure the absorbance value at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0118] Table 8. Effects of different administration concentrations (μmol / L) of indobufen and butylphthalide on HUVECs

[0119]

[0120] 3 Experimental results

[0121] Input the different concentrations and absorbance values corresponding to indobufen and butylphthalide into synergyfinder for calculation, as shown in the figure below. The results show that different concentration ratios of indobufen and butylphthalide can reduce the oxidative stress damage caused by t-BHP, and further calculate the feasibility of combined use using synergyfinder.

[0122] 4 Conclusion: Administration of indobufen combined with butylphthalide can effectively reduce HUVECs damage. Among them, when the ratio of butylphthalide to indobufen is 1:2, 1:4, and between 1:2 and 1:4, the improvement effect is optimal; the ZIP result calculated by synergyfinder is greater than 10, indicating that the combination of drugs has a synergistic effect.

Claims

1. A combined drug, characterized in that: These include indobufen and butylphthalide.

2. The combined drug according to claim 1, characterized in that: The medicine is used for preventing and / or treating cerebrovascular disease.

3. The cerebrovascular disease as claimed in claim 2 is ischemic cerebrovascular disease.

4. The ischemic cerebrovascular disease as claimed in claim 3 is cerebral stroke and transient ischemic attack.

5. The stroke as claimed in claim 4 is ischemic stroke.

6. The use according to any one of claims 1 to 5, characterized in that: The indobufen and butylphthalide are administered simultaneously or sequentially.

7. The use according to any one of claims 1 to 5, characterized in that: The indobufen and butylphthalide are administered orally.

8. A pharmaceutical composition, characterized in that These include indobufen and butylphthalide.

9. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 1:8 to 8:

1.

10. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 1:4 to 8:

1.

11. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 1:4 to 4:

1.

12. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 1:2 to 4:

1.

13. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 2:1 to 4:

1.

14. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 1:

1.

15. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 1:

2.

16. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 2:

1.

17. The pharmaceutical composition according to claim 8, characterized in that The weight ratio of indobufen to butylphthalide is 4:

1.

18. The pharmaceutical composition according to any one of claims 8 to 17, characterized in that It is an oral dosage form.

19. The pharmaceutical composition according to any one of claims 8 to 17, characterized in that Pharmaceutically acceptable excipients such as sugar diluents, cellulose diluents, disintegrants or lubricants are also included.

20. Use of the pharmaceutical composition according to any one of claims 8 to 17 in the preparation of drugs for preventing and / or treating cerebrovascular diseases. The cerebrovascular disease according to claim 20 is ischemic cerebrovascular disease.

22. The cerebrovascular disease according to claim 21 is cerebral stroke and transient ischemic attack.

23. The cerebral stroke according to claim 22 is ischemic cerebral stroke.

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