CN110664799B

CN110664799B - Pharmaceutical composition for treating cerebral ischemia and application thereof

Info

Publication number: CN110664799B
Application number: CN201910972736.8A
Authority: CN
Inventors: 董超; 侯谦; 张世岩; 苏彤; 董楠
Original assignee: Second Hospital of Hebei Medical University
Current assignee: Second Hospital of Hebei Medical University
Priority date: 2019-10-14
Filing date: 2019-10-14
Publication date: 2020-06-19
Anticipated expiration: 2039-10-14
Also published as: CN110664799A

Abstract

The invention provides a novel pharmaceutical composition for treating cerebral ischemia, which comprises the following components of salvinorin A and an ALDH2 activator, wherein the ALDH2 activator is a substituted phenolic compound containing a 3, 4-dihydroxy benzene substituent; the experimental result shows that the combined use of the salvinorin A and the ALDH2 activator produces good synergistic effect in the process of treating cerebral ischemia, and has the advantages of small dosage, small potential risk of side effect and strong clinical application prospect.

Description

Pharmaceutical composition for treating cerebral ischemia and application thereof

Technical Field

The invention relates to the field of pharmacy, in particular to a pharmaceutical composition for treating cerebral ischemia and application thereof.

Background

Ischemic cerebrovascular disease (ischenical cerebral vascular disease) is one of the common clinical diseases, and is a disease with corresponding nervous system symptoms caused by cerebral blood supply insufficiency due to various reasons, wherein chronic cerebral ischemia is a common pathological state and is accompanied by Vascular Dementia (VD), senile dementia (AD) and other nervous system diseases, and mainly comprises chronic cerebral blood supply insufficiency (CCCI), white matter damage caused by ischemia or subcortical arteriosclerotic encephalopathy and encephalatrophy. The mechanisms of pathological injury following ischemia are complex, and especially long-term pathological injury may lead to progressive or persistent cognitive and neurological dysfunction. The incidence of cerebral ischemia is rising with age, and is one of the leading causes of death and disability in adults in various countries around the world. At present, with the increasing aging of the population structure of China, the number of cerebrovascular diseases is obviously increased. Therefore, prevention and treatment of cerebral ischemia is a social concern, and how to prevent and treat cerebral ischemia diseases and brain injuries caused by the cerebral ischemia diseases has attracted extensive attention of relevant scholars at home and abroad.

Currently, in the research of drugs for treating cerebral ischemia, two strategies are mainly used, one of which is that the research on the function of blood vessels, which is performed for the purpose of improving blood supply, is hoped to restore the blood supply in cerebral ischemia areas through thrombolysis, blood vessel dilation or blood vessel reconstruction. The medicine for improving blood supply is commonly used clinically at present; for example, t-PA, which has excellent thrombolytic effect, can restore blood supply to cerebral ischemic patients in a short time, but t-PA is applied with a strict time window limit because the ischemic area, if too long, can cause ischemia reperfusion injury once blood supply is restored, thus accelerating death of patients.

Secondly, the development of neuroprotective agents for the purpose of protecting nerve cell functions is aimed at protecting and restoring nerve functions in ischemic regions by blocking the cascade of nerve cell death by drugs. The intervention of the nerve protective agent can block different links of cerebral ischemia waterfall reaction, protect still viable neurons, prolong the cerebral cell ischemia tolerance time and treatment window, reverse the penumbra and reduce the infarct volume. Because the nerve protective agent is based on the intervention of the biochemical cascade of cell pathology, the nerve protective agent can relieve ischemic brain injury and promote the recovery of nerve function by reducing the overload of intracellular calcium, antagonizing the toxicity of excitatory amino acid, eliminating oxygen free radicals, inhibiting inflammatory reaction and the like.

Salvinorin A (salvinorin A) is an active ingredient of Salvia mexicana (Salvia divairum), a perennial herb of the Labiatae (Mentha) family, indigenous to Mexico. Studies have shown that salvinorin a is the most potent, naturally occurring non-peptide and the only non-nitrogenous Kappa Opioid Receptor (KOR) agonist. Unlike other KOR agonists, salvinorin A does not produce significant hallucinogenic effects nor is it irritating, and has rapid onset, shortness, no respiratory depression, and the like.

ALDH2 is an enzyme present in mitochondria, and is involved in the metabolic process of ethanol, and promotes the conversion of acetaldehyde (produced by the catalysis of dehydrogenase) as a toxic intermediate into acetic acid, thereby exerting alcohol detoxification. In addition to acetaldehyde, ALDH2 clearly detoxifies other toxic aldehydes produced in the body, including 4-hydroxy-2-nonenal (4-HNE). Research shows that the increased ALDH2 content or activity can reduce ischemic brain damage, and the mechanism may eliminate excessive toxic metabolic aldehyde 4-HNE, etc. produced in brain to play neuroprotective role. Therefore, the development of the ALDH2 activator is beneficial to preventing and treating the ischemic injury of the heart and brain and is also beneficial to preventing and treating other diseases related to ALDH 2. Protocatechualdehyde, tanshinol, salvianolic acid B and salvianolic acid A belong to salvianolic acid compounds.

The applicant surprisingly discovers through long-term research that the combined use of the nerve drug salvinorin A and the ALDH2 activator of the salvianolic acid compound has excellent synergistic activity for treating cerebral ischemia diseases, small dosage and small potential risk of side effect, and is expected to become a clinically effective new drug for treating cerebral ischemia.

The present invention is directed to solving the above technical problems.

Disclosure of Invention

One of the purposes of the invention is to provide a novel pharmaceutical composition for treating cerebral ischemia based on the prior art, which has the advantages of small dosage, small potential risk of side effect and good treatment effect. To achieve this object, the present invention provides a pharmaceutical composition comprising the following components salvinorin a and an ALDH2 activator, said ALDH2 activator being a substituted phenolic compound comprising a 3, 4-dihydroxybenzene substituent.

Further, the ALDH2 activator is preferably danshensu, salvianolic acid a, salvianolic acid B, or protocatechualdehyde, or a combination thereof; particularly preferred is tanshinol.

In a further embodiment, the molar ratio of salvinorin a and ALDH2 activator is 1:3-5, preferably 1: 3-4.

Furthermore, the pharmaceutical composition also comprises bamboo leaf flavone, and the molar ratio of the salvinorin A, ALDH2 activator to the bamboo leaf flavone is 1:3-5:0.2-1, preferably 1:3-4: 0.5-0.8.

In another embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, for example, the excipients may be selected from one or more of the following in combination: cellulose derivatives, polyvinylpyrrolidone, polyethylene glycol, mannitol, xylitol, starch, cyclodextrin, magnesium stearate, ethyl p-hydroxybenzoate; particularly preferred are cellulose derivatives, and examples of the cellulose derivatives include microcrystalline cellulose, hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose, croscarmellose sodium, and the like.

Further, the pharmaceutical composition is prepared into an oral dosage form or an injection dosage form; preferably an oral dosage form such as a tablet, pill, capsule, oral liquid, etc., preferably a tablet or capsule.

The invention also aims to provide application of the pharmaceutical composition in preparing a medicament for treating cerebral ischemia.

In the above medical applications, the pharmaceutical composition can be prepared into suitable pharmaceutical preparations according to the conditions of the animals and the application sites for convenient administration, and the administration time and the administration frequency of the pharmaceutical composition of the present invention are determined according to the specific diagnosis results of the conditions of the animals, which is within the technical scope of the skilled in the art. For example, it will be apparent to one of ordinary skill in the art that the effective dose of all drugs to humans can be converted to the effective dose of the drug to mice when the therapeutic regimen for mice is applied to humans.

Detailed Description

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art and are intended to be within the scope of the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned in this application are herein incorporated by reference.

EXAMPLE 1 protective Effect of pharmaceutical composition on model of hypoxia and hypoglycaemia of neural cells

1. Experimental Material

Cultured PC-12 cells, azomethiazole blue (MTT) purchased from Guangzhou chemical industries; enzyme-linked immunosorbent assay (ELISA) instrument is purchased from Thermo company; LDH assay kit: nanjing is built into a product of a bioengineering research institute.

2. Experimental methods

2.1 the experiment was divided into model groups, and different pharmaceutical compositions, salvinorin A, salvianic acid A, nimodipine positive control group and normal group were used to make cell oxygen deprivation reperfusion model group: the PC-12 cells are fully washed by sugar-free Earle's balanced salt solution for several times at the 3 rd culture stage to make the final concentration of glucose less than 1mol/L, EBSS solution is added, and the mixture is placed into an anoxic box, and N is continuously and slowly introduced₂Taking out the mixed gas for 4h, absorbing and removing the balanced salt buffer solution, replacing the complete culture medium with CO₂Incubating in an incubator, simulating the process of ischemia reperfusion in vivo, and incubating for 24 h; establishing a sugar-deficient and anoxic model by using sugar-free EBSS liquid, adding different medicines before the reperfusion time point, and performing other treatments on the same-oxygen sugar-deprivation reperfusion model group; nimodipine positive control group: replacing sugar-free EBSS liquid to establish an oxygen sugar deprivation model, adding nimodipine before the reperfusion time point, and performing other treatments with the oxygen sugar deprivation reperfusion model group; normal group: the cells were not treated at all.

2.2 Effect of different drugs on the cell viability of sugar-deficient hypoxia reperfusion injury PC-12 cells: after the cells treated with different drugs were cultured for 24 hours under normal conditions of oxygen deprivation and oxygen recovery, 20. mu.L of 5mg/mL MTT solution was added, and the culture was terminated after further incubation for 4 hours. Carefully absorbing the supernatant, adding 150. mu.L of dimethyl sulfoxide DMSO into each well, mixing, selecting 490nm wavelength to measure the light absorption value of each well, and recording the result. Cell viability (%) ═ experimental light absorption/control light absorption × 100%.

And 2.3, detecting the LDH content of the supernatant of each hole by adopting an LDH kit.

3, results:

table 1: effect of different drugs on survival of PC-12 cells after oxygen deprivation

The results in table 1 show that the combination of salvinorin a and ALDH2 activator selected from salvianic acid A, salvianic acid B or protocatechuic aldehyde significantly reduces the rate of PC-12 cell damage after oxygen deprivation reperfusion, and compared to the single use of the above drugs, a synergistic effect is obtained.

Table 2: LDH content in cell supernatant of each group is detected by adopting LDH biochemical kit

Example 2 protective Effect of pharmaceutical compositions on rat MCAO model

Preparation of the model

One day prior to the experiment, rats (200-250g) were fasted overnight with only water available. On the same day of the experiment, isoflurane gas is used for anesthesia, the dorsal position is fixed, the skin is cut along the median cervical line, the right common carotid artery is exposed, the nerves and fascia around the blood vessel of the common carotid artery which is forked to the skull base are carefully separated, the external carotid artery branch occipital artery, the superior thyroid artery, the lingual artery and the maxillary artery in the palate are separated, and the arteries are cut off after being ligated. The method is characterized in that a silk thread with the diameter of 0.2mm is inserted from the free end of an external carotid artery, a nylon thread is introduced into an internal carotid artery from the distal end of the external carotid artery and is inserted into the middle cerebral artery of Willis to effectively block the middle cerebral artery, and the length of the inserted nylon thread is 18-20 mm away from the bifurcation of a common carotid artery. The free end of the external carotid artery was then ligated along with the intraluminal nylon thread to prevent bleeding. The subcutaneous fascia and skin are sutured layer by layer, and penicillin is injected intramuscularly to prevent infection. Only the internal carotid artery was isolated from the normal group of animals. The rats were scored for neurological deficits 10min after awakening, and those with obvious neurological deficits (>8 points) were successfully sculpted.

Grouping and administration: the method specifically comprises a normal group, a model group and an administration group. After 1 hour of molding, the drug was administered by tail vein infusion, and the administration volume was calculated as the maximum administration volume, i.e., 1.0ml/100g (rat body weight) and the infusion time was 35 min. The normal group and the model group were given an equal amount of physiological saline, and the rats were sacrificed 24 hours after molding for the relevant examination.

1. Mortality and TTC infarct size test: before each group of rats is sacrificed, the mortality of each group is counted, the brains of the rats are taken out by using a bone forceps and cut into slices with the thickness of 2mm along the coronal plane, the half brain slices are taken at intervals and put into 2 percent TTC staining solution, the half brain slices are incubated for 10min at 37 ℃ in a dark place for staining, image analysis software of a computer is adopted to measure the area of the infarcted area and the area of the whole brain, and the cerebral infarction range (the area of the infarcted area accounts for the area of the whole brain) is calculated, and the specific results are as follows:

table 3: protective Effect of different drugs on rat MCAO model

The results in table 3 show that the combination of salvinorin A and ALDH2 activator significantly reduces the cerebral infarction area of rats and the death rate of rats, and compared with the single use of the above drugs, the synergistic effect is obviously achieved.

Table 4: ELISA detection method is adopted to detect the contents of LDH, VEGF and TNF-a in the serum of each group of rats

Examples 3-6 tablet preparation of pharmaceutical compositions of the invention

Weighing the medicines of the components and the auxiliary materials except the magnesium stearate according to the mass in the following table, grinding and uniformly mixing, granulating by using 70% ethanol, sieving, adding the magnesium stearate, uniformly mixing, and tabletting to obtain the traditional Chinese medicine.

Tablets were prepared containing the following ingredients (in mg):

table 5: examples of tablet Components of the pharmaceutical compositions of the present invention

	Example 3	Example 4	Example 5	Example 6
					Salvianic acid A	1	1	1	1
Salvianic acid A	3	-	-	-
					Monophenolic acid A	-	3.5	-	-
Salvianolic acid B	-	-	4	-
					Protocatechualdehyde	-	-		5
Mannitol	55	62	59	60
					Microcrystalline cellulose	25	18	22	10
Hydroxymethyl cellulose	6	10.5	8	16
					Magnesium stearate	10	5	6	8

EXAMPLE 7 preparation of an injection of the pharmaceutical composition of the present invention

Weighing 0.1g of salvinorin A, 0.4g of salvianolic acid/0.3 g of salvianolic acid A/0.3g of salvianolic acid B/0.4g of protocatechualdehyde, 0.7-1.0 g of polyethylene glycol and 0.6-0.9 g of NaCl, adding 1L of water for injection, heating to dissolve, adjusting the pH value to 7-7.5, sterilizing and filling to obtain the salvinorin A.

This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.

Claims

1. A pharmaceutical composition for treating cerebral ischemia, comprising salvinorin A and ALDH2 activator, wherein the ALDH2 activator is selected from salvianic acid A, salvianolic acid B or protocatechualdehyde, or a combination thereof; the structural formula of the salvinorin A is as follows:

wherein the molar ratio of the salvinorin A to the ALDH2 activator is 1: 3-5.

2. The pharmaceutical composition of claim 1, wherein: the ALDH2 activator is danshensu.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that: the pharmaceutical composition also comprises bamboo leaf flavone, and the molar ratio of the salvinorin A, ALDH2 activator to the bamboo leaf flavone is 1:3-5: 0.2-1.

4. The pharmaceutical composition according to claim 1 or 2, characterized in that: the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials, and the auxiliary materials are selected from one or more of the following combinations: cellulose derivatives, polyvinylpyrrolidone, polyethylene glycol, mannitol, xylitol, starch, cyclodextrin, magnesium stearate, and ethyl p-hydroxybenzoate.

5. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is prepared as an oral dosage form or an injectable dosage form.

6. Use of a pharmaceutical composition according to claim 1 or 2 for the preparation of a medicament for the treatment of cerebral ischemia.