CN111184725B

CN111184725B - Medicinal preparation for preventing and treating cerebral infarction and preparation method thereof

Info

Publication number: CN111184725B
Application number: CN202010124089.8A
Authority: CN
Inventors: 邬芬赞; 魏晓捷; 陈再丰; 闻路通; 许信龙
Original assignee: Cixi People's Hospital Medical And Health Group Cixi People's Hospital
Current assignee: Cixi People's Hospital Medical And Health Group Cixi People's Hospital
Priority date: 2020-02-27
Filing date: 2020-02-27
Publication date: 2020-12-01
Anticipated expiration: 2040-02-27
Also published as: CN111184725A

Abstract

The invention discloses a pharmaceutical preparation for preventing and treating cerebral infarction and a preparation method thereof, wherein the pharmaceutical preparation is prepared from a compound A shown in the following structural formula, optional other drugs for preventing and treating cerebral infarction and a pharmaceutically acceptable carrier. The invention also provides application of the compound A in preparing a medicine for preventing and treating cerebral infarction.

Description

Medicinal preparation for preventing and treating cerebral infarction and preparation method thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a pharmaceutical preparation for preventing and treating cerebral infarction and a preparation method thereof.

Background

Cerebral infarction (also known as cerebral infarction) and ischemic stroke (cerebral ischemic stroke) is an acute cerebrovascular disease with extremely high fatality rate and disability rate and seriously threatening human life and health, and usually refers to ischemic necrosis or softening of local brain tissue caused by ischemia and hypoxia caused by cerebral blood flow supply disorder. Cerebral infarction is a sudden brain disease, has rapid onset of disease, mostly has no obvious prodromal symptoms, and is mainly characterized by speech disturbance, hemiplegia, sudden syncope and unconsciousness in clinical manifestation.

Cerebral infarction may be caused by the gradual formation of thrombus in the lumen of an artery, which eventually blocks the artery due to arterial stenosis. Another common cause of cerebral infarction is the detachment of tiny emboli on the inner wall of cerebral vessels or mural thrombus of heart valves, which flow to cerebral arteries and cause embolism. No matter what kind of reason causes the cerebral infarction, all can take place in any age bracket, and have the trend of gradually rising along with the age, is mostly seen in the middle-aged and old people of 45 ~ 70 years old. Cerebral infarction not only poses a great threat to human health and life, but also brings great pain and heavy burden to patients, families and society.

Cerebral infarction belongs to difficult diseases, effective treatment measures are always lacked in medicine at present, only few kinds of thrombolytic drugs, surgical operations and the like are available in clinical treatment of cerebral infarction, and the clinical use time window is very short. In view of the serious hazard of cerebral infarction, there is an unmet need in the art for a drug for preventing cerebral infarction.

Disclosure of Invention

The invention aims to solve the technical problem of finding an effective medicament for preventing and treating cerebral infarction and a preparation method thereof.

The inventors of the present invention have completed the present invention based on the following unexpected findings: n- [ (4aR,7aS) -6- [ 5-fluoro-4- (1-hydroxy-1-methyl-ethyl) -6-methyl-pyrimidin-2-yl ] -7 a-phenyl-4, 4a,5, 7-tetrahydropyrrolo [3,4-d ] [1,3] thiazin-2-yl ] benzamide hydrochloride (hereinafter referred to aS Compound A) having the following structural formula

Has the application of preventing and/or treating cerebral infarction. Based on this, the inventors studied and developed a pharmaceutical preparation suitable for the prevention and treatment of cerebral infarction and a method for producing the same.

To this end, in one aspect, the present invention provides a pharmaceutical preparation for the prevention and treatment of cerebral infarction, wherein the pharmaceutical preparation is prepared from compound a, optionally other drugs for the prevention and treatment of cerebral infarction, and a pharmaceutically acceptable carrier.

In one aspect, the pharmaceutical preparation contains other drugs for preventing and treating cerebral infarction, wherein the drugs are selected from one or more of urokinase, heparin sodium, nimodipine, aspirin and tanshinone IIA.

In one aspect, the pharmaceutical formulation is free of other drugs for preventing and treating cerebral infarction.

In one aspect, the pharmaceutical formulation is in the form of an emulsion suitable for intravenous injection, and the pharmaceutically acceptable carrier consists of ethyl oleate, lecithin, poloxamer 188, tween 80, propylene glycol, and water for injection.

In one aspect, per 100ml of the emulsion comprises:

the rest is water for injection.

In one aspect, per 100ml of the emulsion comprises:

the rest is water for injection.

In one aspect, the method of preparing the pharmaceutical formulation comprises the steps of:

(1) fully mixing the compound A, lecithin, poloxamer 188, tween 80, propylene glycol and water for injection accounting for 50% -70% of the total volume until the mixture is completely dissolved under the condition of heating and stirring to form a water phase;

(2) slowly dripping ethyl oleate into the water phase under the condition that the water phase is kept under heating and stirring, keeping for 10-20 minutes under the condition that the water phase is heated and stirred after mixing is finished, and then adding the rest of water for injection to a constant volume to obtain primary emulsion;

(3) emulsifying the primary emulsion for 2-4 times by high pressure homogenizing machine, filtering with 0.8 micrometer and 0.22 micrometer microporous filter membranes, packaging in appropriate container, charging inert gas, and sealing.

In one aspect, the conditions of the heating and stirring in the steps (1) and (2) refer to conditions of a temperature of 50-70 ℃ and a stirring speed of 4000-.

The compounds A used in the present invention are previously described in patent document WO2013/151832A 1. This patent document relates generally to novel tetrahydropyrrolothiazine compounds that are potent inhibitors of beta-site amyloid precursor protein cleaving enzyme (BACE), which are believed to be useful for inhibiting BACE and inhibiting the production of a β peptide, and thus for treating alzheimer's disease or for preventing progression from mild cognitive impairment to alzheimer's disease. In this patent document, the chemical formula, structural formula and synthesis method of compound a are disclosed in example 22. However, this patent document does not mention that the compound has any pharmacological activity in preventing and treating cerebral infarction, which constitutes an unexpected finding of the present invention.

The invention also provides application of the compound A in preparing a medicament for preventing and treating cerebral infarction.

In one aspect, the medicament is a pharmaceutical formulation as described above.

Detailed Description

Preferred embodiments of the present invention and effects thereof will be described in further detail with reference to specific examples. It should be understood, however, that these preferred embodiments are provided to further illustrate the features and advantages of the present invention and are not to be construed as limiting the claims in any way.

Example 1

The components and the dosage of the pharmaceutical preparation for preventing and treating cerebral infarction in the embodiment are shown in the following table:

components	Dosage of
		Compound A	2.5g
Oleic acid ethyl ester	20g
		Lecithin	1.2g
Poloxamer 188	1.2g
		Tween 80	5g
Propylene glycol	2g
		Water for injection	Adding to 100ml

The preparation method of the pharmaceutical preparation comprises the following steps:

(1) fully mixing the compound A, lecithin, poloxamer 188, tween 80, propylene glycol and 60mL of water for injection at the temperature of 60 ℃ and the stirring speed of 6000 rpm until the compound A, the lecithin, the poloxamer 188, the tween 80 and the propylene glycol are completely dissolved to form a water phase;

(2) slowly dripping ethyl oleate into the water phase under the conditions that the temperature of the water phase is kept at 60 ℃ and the stirring speed is 6000 rpm, keeping the water phase at the temperature of 60 ℃ and the stirring speed is 6000 rpm for 15 minutes after mixing, and adding the rest of water for injection to a constant volume to obtain primary emulsion;

(3) emulsifying the primary emulsion for 3 times by high pressure homogenizing machine, filtering with 0.8 micrometer and 0.22 micrometer microporous filter membrane, subpackaging in 10mL ampoule, charging nitrogen gas, and sealing.

Example 2

components	Dosage of
		Compound A	5g
Oleic acid ethyl ester	20g
		Lecithin	1.2g
Poloxamer 188	1.2g
		Tween 80	5g
Propylene glycol	2g
		Water for injection	Adding to 100ml

Example 3

components	Dosage of
		Compound A	10g
Oleic acid ethyl ester	20g
		Lecithin	1.2g
Poloxamer 188	1.2g
		Tween 80	5g
Propylene glycol	2g
		Water for injection	Adding to 100ml

Example 4

The purpose of this experiment was to investigate the neuronal protective effect of compound a of the present invention using an ex vivo cerebral ischemia model of glucose and glucose deprivation (OGD).

The experimental method of this experiment is as follows:

with reference to the methods disclosed in "establishment of model of persistent glucose deprivation in PC12 cells", military medicine ", vol.35, 3 rd, p.189-192, 3.2011, PC12 cells were used to establish model of persistent glucose deprivation isolated cerebral ischemia. Specifically, PC12 in logarithmic growth phaseCells were as per 6X 10⁵The cells/ml were seeded at a density of 100. mu.l/well in sterile 96-well plates, incubated in a humidified incubator for 24h, the plates were removed and washed 2 times (5 min/time) with 37 ℃ pre-warmed PBS to remove glucose. Then, a solution containing 5X 10 of the active ingredient was added to each well of a 96-well plate⁴U/ml penicillin and streptomycin in sugar-free serum-free medium (100. mu.l/well with or without test drugs as shown in Table 1 below); as a control group, a control group containing glucose (1.0g/L), 10% fetal bovine serum and 5X 10 cells was added to each well of a 96-well plate⁴U/ml penicillin and streptomycin medium (100. mu.l/well). Finally, the 96-well plate was incubated in a 37 ℃ incubator (the 96-well plate of the oxygen-sugar deprived group was placed in a hypoxia chamber filled with a mixed gas of 95% nitrogen and 5% carbon dioxide) for 30 minutes. After the incubation was completed, the medium was removed, 10. mu.L of MTT (5 mg/mL PBS solution) was added to each well of the 96-well plate and returned to the incubator for further incubation for 3 hours, then the medium was discarded, 100. mu.L of DMSO was added, the mixture was shaken in a water bath at 37 ℃ for 30 minutes, and after the purple crystals were completely dissolved, the absorbance value (OD) at a wavelength of 570 nm was measured with a microplate reader₅₇₀) And OD of sugar oxygen deprived group₅₇₀The value is OD of the control group₅₇₀The percentage of values reflects the survival of PC12 cells.

Statistics of survival for this experiment are presented in table 1.

TABLE 1 survival of PC12 cells treated with different test drugs

Kind of drug	Drug concentration	Survival rate (%)
			-	-	26.3±4.3
Nimodipine	5mg/ml	49.9±6.1
			Tanshinone IIA	5mg/ml	57.2±5.9
Aspirin	5mg/ml	43.2±7.2
			Compound A	5mg/ml	68.2±9.7

Note: each test drug is provided with 12 compound holes; results are expressed as mean ± standard deviation.

Generally speaking, the higher the survival rate of PC12 cells after treatment with a drug in a model of sugar-oxygen deprivation isolated cerebral ischemia reflects that the drug has a better protective effect on nerve cell damage caused by cerebral infarction. From the above results, compound a has a significant neuroprotective effect on sugar oxygen deprived PC12 cells, and this neuroprotective effect is superior to the other test drugs of the reference at the same concentration.

Example 5

The purpose of the experiment is to use a rat cerebral ischemia-reperfusion injury model to investigate the pharmacological activity of the pharmaceutical preparation of the invention on cerebral infarction.

1. Laboratory animal

Male adult SD rats weighing 240 g. + -.10 g were used in this experiment. Animals were acclimated for two days at room temperature 23-25 ℃, 50% relative humidity and switching light and dark every 12 hours. The rats were provided with freely accessible clean food and drinking water.

2. Establishment of rat cerebral ischemia reperfusion injury model

Rats were anesthetized with intraperitoneal injection of 25% urethane solution. The rat was then mounted on the operating table in the lateral decubitus position, a median cervical incision was made, the common carotid artery was exposed, and the internal carotid artery was isolated. Then, the common carotid artery and the internal carotid artery are clamped, and the nylon wire is slowly pushed towards the intracranial direction of the internal carotid artery through the main incision of the external carotid artery to reach the thinner anterior cerebral artery, so that the middle cerebral artery is blocked to completely block the local blood flow. After 90 minutes, the nylon thread is pulled out, the artery stump is tied tightly, the wound is sutured, and the establishment of the cerebral ischemia reperfusion injury model of the rat is completed.

3. Dosing regimens

The molded rats were randomly divided into 20 groups, namely, a model control group, an example 1 group, an example 2 group and an example 3 group. Another 20 rats that did not occlude the middle cerebral artery after anesthesia were used as a sham group.

The dosing schedule for each experimental group was as follows:

example 1, example 2, example 3 groups: immediately after the molding was completed, the pharmaceutical preparations of example 1, example 2 and example 3 were each administered by tail intravenous injection at a dose of 1.0mL/kg body weight, followed by administration 1 time per day in the same manner for 3 consecutive days.

Model control group: after completion of molding, physiological saline was immediately administered by tail intravenous injection, followed by administration 1 time per day in the same manner for 3 days continuously.

The sham operation group: the same model as the control group.

4. Observation index

(1) Bederson neurological deficit score:

on the day of experiment completion, evaluation of the model and degradation of a neurological evolution, Stroke, 1986 was used by Bederson JB et al, Rat middle carbohydrate array occlusion; 17(3) 472 + 476, scoring the behavioral deficits of the rats by neurofunctional deficits:

(2) measurement of cerebral ischemic infarct volume and water content:

finally, the rats in each group except the sham group were sacrificed by decapitation. Randomly selecting 5 rats, taking the whole brain, weighing the wet weight of the brain, and slicing. The section is placed in a 2% triphenyltetrazolium chloride (TFC) solution for dyeing for 30min, fixed, and separated into a pale area (an infarct area) and a non-pale area (a normal area), and the weight percentage of the infarct area range to the weight of the brain is taken as the cerebral infarction range.

Another 5 rats were randomly selected, whole brains were taken, brain tissues were dried in an oven at 110 ℃ to constant weight, the dry weight was weighed, and brain water content was calculated. Brain water content (brain tissue wet weight-brain tissue dry weight)/brain tissue wet weight × 100%.

5. Results

The experimental statistics are shown in the following tables 1-2.

TABLE 1 Bederson neurological deficit scores for groups of rats

Group of	Scoring
		Artificial operation group	0
Model control group	3.78±0.53
		EXAMPLE 1 group	2.96±0.68
EXAMPLE 2 group	2.27±0.61*
		EXAMPLE 3 group	1.39±0.46*

Note: results are expressed as mean ± standard deviation, p <0.05 compared to model control.

TABLE 2 cerebral ischemic infarct volume and water content of each group of rats

Note: results are expressed as mean ± standard deviation, p <0.05 compared to model control group.

The above results suggest that the pharmaceutical preparation of the present invention can significantly improve the neurological deficit of rats with cerebral ischemia-reperfusion injury models, and reduce the volume and water content of cerebral ischemia infarctions, and the improvement is most significant with the effect of example 3, which indicates that the pharmaceutical preparation of the present invention has a positive correlation between the pharmacological activity of cerebral infarction and the content of compound a in the pharmaceutical preparation. In conclusion, the above results indicate that the pharmaceutical preparation of the present invention can be used for preventing and treating cerebral infarction.

Claims

1. The application of the compound A in preparing a medicinal preparation for preventing and treating cerebral infarction, wherein the medicinal preparation is prepared from the compound A and a pharmaceutically acceptable carrier, wherein the compound A is N- [ (4aR,7aS) -6- [ 5-fluoro-4- (1-hydroxy-1-methyl-ethyl) -6-methyl-pyrimidin-2-yl ] -7 a-phenyl-4, 4a,5, 7-tetrahydropyrrolo [3,4-d ] [1,3] thiazin-2-yl ] benzamide hydrochloride shown in the structural formula

，

And wherein the pharmaceutical formulation is in the form of an emulsion suitable for intravenous injection and the pharmaceutically acceptable carrier consists of ethyl oleate, lecithin, poloxamer 188, tween 80, propylene glycol and water for injection, and wherein the pharmaceutical formulation comprises, per 100ml of the emulsion:

the balance of water for injection, and wherein the preparation method of the pharmaceutical preparation comprises the following steps:

(3) emulsifying the primary emulsion for 2-4 times by a high-pressure emulsion homogenizer, filtering by 0.8 micron and 0.22 micron microporous filter membranes in sequence, subpackaging in a proper container, filling inert gas, and sealing to obtain the final product, wherein the conditions of heating and stirring in the steps (1) and (2) refer to the conditions of the temperature of 50-70 ℃, the stirring speed of 4000-.