Drug for preventing and treating ischemic stroke

Abstract

The present invention provides a drug for preventing and treating ischemic stroke, which belongs to the field of neuromedicine technology. The drug comprises a salmon polypeptide as shown in SEQ ID NO.1 and a pharmaceutically acceptable carrier. Experimental results show that the salmon polypeptide has good safety, can effectively improve brain cell oxygen-glucose deprivation / reoxygenation damage, and significantly reduce neurological deficits and cerebral infarction in ischemic stroke mice. The present invention also discloses a pharmaceutical composition for treating ischemic stroke, comprising salmon polypeptide and edaravone. The composition exhibits a significant synergistic effect, and can more significantly reduce neurological deficits and cerebral infarction compared to the use of salmon polypeptide or edaravone alone, and the actual effect is significantly better than the theoretical superposition effect. Therefore, the present invention provides a new and more effective treatment option for the treatment of ischemic stroke.

Description

Technical Field

[0001] The present invention belongs to the technical field of neuromedicine, and in particular relates to a medicine for preventing and treating ischemic stroke. Background Art

[0002] Cerebrovascular Accident (CVA), also commonly referred to as stroke, is an acute illness resulting from interruption or abnormality of cerebral blood supply, leading to brain dysfunction. Stroke typically causes severe neurological impairment, and as the disease progresses, the severity of brain damage worsens, reducing the likelihood of recovery.

[0003] Stroke can be divided into two major categories: ischemic stroke and hemorrhagic stroke. Among them, ischemic stroke accounts for about 80% of all strokes. Ischemic stroke is caused by vascular stenosis, blockage or thrombosis, which leads to interruption of blood flow to some brain areas, thereby causing brain tissue hypoxia, metabolic disorders, and ultimately brain cell death. At present, although there are some treatments available to relieve the symptoms of ischemic stroke, there is still a great demand for improving treatment effects, reducing side effects, and improving post-illness recovery. Therefore, the development of new drugs and treatment options, especially neuroprotective drugs with higher safety and efficacy, is the focus of current research.

[0004] Salmon polypeptide (amino acid sequence: TPEVHIAVDKF, SEQ ID NO.1) is a bioactive polypeptide that has been successfully extracted from Atlantic salmon by-products through a specific technology. In existing scientific research, it has been clearly confirmed that the polypeptide has a significant hyaluronidase inhibitory effect. This discovery provides an important theoretical basis for its application in the biomedical field. However, based on the current literature, there are no public literature reports on whether the salmon polypeptide TPEVHIAVDKF can play a role in the treatment of stroke and its specific mechanism of action. This also makes its application prospects in the treatment of stroke full of unknowns, and further in-depth research is needed. Summary of the Invention

[0005] The purpose of the present invention is to provide a drug for preventing and treating ischemic stroke and a pharmaceutical composition composed thereof, so as to achieve better therapeutic effects while achieving higher safety.

[0006] In order to achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0007] First, the present invention provides a drug for preventing and treating ischemic stroke, which is composed of a salmon polypeptide and a pharmaceutically acceptable carrier; the amino acid sequence of the salmon polypeptide is shown in SEQ ID NO.1.

[0008] The pharmaceutically acceptable carriers include solvents such as water, physiological saline, and glucose solution; fillers such as starch and microcrystalline cellulose; binders such as starch slurry and hydroxypropyl methylcellulose; and antioxidants such as sodium sulfite, vitamin C, and vitamin E.

[0009] Preferably, the drug is a liquid drug, and the concentration of the salmon polypeptide in the drug is greater than or equal to 80 μg / ml.

[0010] Preferably, in the drug, the concentration of salmon polypeptide is 80 μg / ml-160 μg / ml;

[0011] The pharmaceutically acceptable carrier is physiological saline, and the drug is administered by intravenous injection so that it can quickly pass through the blood circulation to achieve the therapeutic purpose;

[0012] Preferably, in order to enhance the efficacy, the concentration of salmon polypeptide is preferably 160 μg / ml.

[0013] Secondly, the present invention provides a use of a salmon polypeptide in preparing a drug for preventing and treating ischemic stroke, wherein the amino acid sequence of the salmon polypeptide is shown in SEQ ID NO.1.

[0014] Preferably, the drug is a liquid drug prepared from physiological saline, the concentration of the salmon polypeptide in the drug is 80 μg / ml-160 μg / ml, and the drug is administered by intravenous injection.

[0015] Preferably, the concentration of salmon polypeptide in the drug is 160 μg / ml.

[0016] Preferably, the drug can reduce neurological deficits and cerebral infarction caused by ischemic stroke, thereby alleviating ischemic damage, promoting nerve regeneration and repair, and significantly improving the patient's neurological function and quality of life.

[0017] Thirdly, the present invention provides a pharmaceutical composition for treating ischemic stroke. Each 300 μl of the pharmaceutical composition comprises 150 μl of a salmon peptide drug at a concentration of 160 μg / ml and 150 μl of edaravone drug at a concentration of 500 μg / ml. The amino acid sequence of the salmon peptide is shown in SEQ ID NO. 1. Edaravone, as a known free radical scavenger, can effectively reduce oxidative stress and protect nerve cells from damage. The combined use of edaravone and salmon peptide can exert a synergistic effect, thereby improving the therapeutic effect.

[0018] Preferably, the solvent for the salmon polypeptide drug and the edaravone drug is normal saline;

[0019] The pharmaceutical composition is administered by intravenous injection;

[0020] The pharmaceutical composition synergistically reduces neurological impairment and cerebral infarction caused by ischemic stroke.

[0021] Finally, the present invention provides a use of a composition in preparing a drug for treating ischemic stroke, wherein each 300 μl of the composition comprises 150 μl of a salmon polypeptide solution with a concentration of 160 μg / ml and 150 μl of an edaravone solution with a concentration of 500 μg / ml, and the amino acid sequence of the salmon polypeptide is shown in SEQ ID NO.1.

[0022] Preferably, the solvent of the salmon polypeptide solution and the edaravone solution is physiological saline;

[0023] The drug is administered by intravenous injection;

[0024] The drug synergistically reduces neurological impairment and cerebral infarction caused by ischemic stroke through salmon peptide and edaravone.

[0025] The beneficial effects of the present invention are:

[0026] First, the present invention provides a new treatment option for ischemic stroke, disclosing for the first time the use of the salmon polypeptide (amino acid sequence: TPEVHIAVDKF) represented by SEQ ID NO. 1 in the prevention and / or treatment of ischemic stroke. Prior art reports on the use of this salmon polypeptide in stroke treatment have yet to be published. The findings of the present invention provide a novel therapeutic strategy and drug option for the treatment of ischemic stroke, expanding the application area of ​​this polypeptide.

[0027] Secondly, the hemolytic activity and cytotoxicity experimental results of Example 1 show that within the effective concentration range, the salmon polypeptide provided by the present invention has no obvious hemolytic and cytotoxic properties (such as Figure 1 This indicates that it has good safety and can be used for drug preparation, reducing the potential risks of clinical application.

[0028] Finally, the study in Example 3 also found that the combination of salmon peptides and the known free radical scavenger edaravone can produce a significant synergistic effect. Compared with the use of salmon peptides or edaravone alone, the combination therapy can more significantly reduce neurological deficit scores and cerebral infarction percentages (Tables 1 and 2), and the actual effect is significantly better than the theoretical additive effect. This shows that the pharmaceutical composition of the present invention can more effectively intervene in the pathophysiological process of ischemic stroke through a multi-target mechanism of action, thereby significantly improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the result of the drug safety test of salmon peptide;

[0030] in, Figure 1 (a) is the result of hemolytic activity test, Figure 1 (b) is the cytotoxicity test result;

[0031] Figure 2 This is a graph showing the therapeutic effect of salmon peptides on brain cell oxygen-glucose deprivation / reoxygenation damage;

[0032] Figure 1 middle, △△△ =P < 0.001 compared with the control group, * =P < 0.05 compared with the oxygen-glucose deprivation / reoxygenation group, ** =P < 0.01 compared with the oxygen-glucose deprivation / reoxygenation group. DETAILED DESCRIPTION

[0033] Below in detail embodiments of the present invention, the example of described embodiment is shown in the accompanying drawings, wherein identical or similar reference numerals represent identical or similar elements or elements with identical or similar functions throughout. The embodiment described below by reference to the accompanying drawings is exemplary, is intended to be used for explaining the present invention, and is not to be construed as limiting the present invention. In the embodiment, those not indicating specific techniques or conditions are carried out according to the technology or conditions described in the document in this area or according to the product specification. Reagents used or instruments not indicating manufacturers are conventional products that can be obtained commercially.

[0034] Example 1

[0035] Conducting drug safety testing on salmon peptides

[0036] (A) Hemolytic activity assay of salmon peptides

[0037] First, cell grouping is performed as follows:

[0038] In the negative control group, red blood cells were diluted to 1×10 8 / ml, and then 200 μl was added to a 96-well plate;

[0039] In the positive control group, red blood cells were diluted to 1×10 8 / ml, and 200 μl was added to a 96-well plate;

[0040] Treatment group 1 was treated with PBS containing salmon peptide (synthesized by GenScript Biotech Co., Ltd.) at a final concentration of 10 μg / ml. 8 / ml, and then 200 μl was added to a 96-well plate;

[0041] In treatment group 2, erythrocytes were diluted to 1×10 8 After 100 μl was added to a 96-well plate;

[0042] Treatment group 3 was treated with PBS containing salmon peptide at a final concentration of 40 μg / ml. 8 / ml, and then 200 μl was added to a 96-well plate;

[0043] In treatment group 4, erythrocytes were diluted to 1×10 8 / ml, take 200 μl and add it to 96-well plate;

[0044] In treatment group 5, erythrocytes were diluted to 1×10 8 100 μl of the solution was added to a 96-well plate.

[0045] After the grouping and loading, the 96-well plate was placed in a 37°C constant temperature incubator for 30 minutes. After the incubation, the solution was collected and centrifuged at 1000 rpm for 5 minutes. After the centrifugation, 100 μl of supernatant was taken from each well and transferred to a new 96-well plate. Finally, the new 96-well plate was placed in a microplate reader and the absorbance value was measured at OD540. The results were as follows: Figure 1 As shown in (a).

[0046] (B) Cytotoxicity assay

[0047] First, BV2 microglial cells in the logarithmic growth phase were seeded into 96-well plates and cultured in a cell culture incubator. When the cells reached a cell density greater than 90%, the cells were treated according to the following groupings:

[0048] The control group was added with 100 μl of DMEM culture solution;

[0049] Treatment group 1 was added with 100 μl of salmon peptide solution (prepared in DMEM) with a final concentration of 10 μg / ml;

[0050] Treatment group 2 was added with 100 μl of salmon peptide solution (prepared in DMEM) with a final concentration of 20 μg / ml;

[0051] Treatment group 3 was added with 100 μl of salmon peptide solution (prepared in DMEM) with a final concentration of 40 μg / ml;

[0052] Treatment group 4 was added with 100 μl of salmon peptide solution (prepared in DMEM) with a final concentration of 80 μg / ml;

[0053] In treatment group 5, 100 μl of salmon peptide solution (prepared in DMEM) with a final concentration of 160 μg / ml was added.

[0054] After group loading, the 96-well plate was placed in a cell culture incubator at 37°C, 5% CO2 for 24 hours. After incubation, 20 μL of MTT solution (pH 7.4) was added to each well and incubated for another 4 hours at 37°C, 5% CO2. The supernatant was then carefully aspirated and 150 μL of DMSO was added. After shaking for 10 minutes, the plate was placed in a microplate reader and the absorbance was measured at OD570. The results were as follows: Figure 1 (b) shown.

[0055] from Figure 1 (a) and Figure 1 (b) It can be seen that salmon peptides did not cause lysis of red blood cells at different concentrations, proving that they do not produce hemolysis. In addition, through cell culture experiments ( Figure 1 (b) As can be seen, salmon peptides have no significant inhibitory effect on the growth and survival of nerve cells, indicating that they are non-cytotoxic. These results indicate that salmon peptides, as bioactive substances, have good biocompatibility and are suitable for drug preparation.

[0056] Example 2

[0057] Detecting the therapeutic effect of salmon peptides on brain cell oxygen-glucose deprivation / reoxygenation injury

[0058] BV2 cells in the logarithmic growth phase were seeded into 6-well culture plates. When the cell density reached about 80%, subsequent experiments were performed. The cells were grouped as follows:

[0059] Control group: high-glucose DMEM medium was used for normal culture at 37°C and 5% CO2;

[0060] Oxygen-glucose deprivation / reoxygenation group: The cells were cultured in a cell culture incubator at 37°C, 0.3% O2, and 5% CO2 for 6 h, then replaced with high-glucose DMEM medium and placed in a cell culture incubator at 37°C, 21% O2, and 5% CO2 for 24 h.

[0061] Treatment group 1: Cultured in sugar-free DMEM medium at 37°C, 0.3% O2, and 5% CO2 in a cell culture incubator for 6 h, then replaced with high-glucose medium containing 10 μg / ml salmon peptides and placed in a cell culture incubator at 37°C, 21% O2, and 5% CO2 for 24 h.

[0062] Treatment group 2: Cultured in sugar-free DMEM medium at 37°C, 0.3% O2, and 5% CO2 in a cell culture incubator for 6 h, then replaced with high-glucose medium containing 20 μg / ml salmon peptides and placed in a cell culture incubator at 37°C, 21% O2, and 5% CO2 for 24 h.

[0063] Treatment group 3: Cultured in sugar-free DMEM medium at 37°C, 0.3% O2, and 5% CO2 in a cell culture incubator for 6 h, then replaced with high-glucose medium containing 40 μg / ml salmon peptides and placed in a cell culture incubator at 37°C, 21% O2, and 5% CO2 for 24 h.

[0064] Treatment group 4: Cultured in sugar-free DMEM medium at 37°C, 0.3% O2, and 5% CO2 in a cell culture incubator for 6 h, then replaced with high-glucose medium containing 80 μg / ml salmon peptides and placed in a cell culture incubator at 37°C, 21% O2, and 5% CO2 for 24 h.

[0065] Treatment group 5: Cultured in sugar-free DMEM medium at 37°C, 0.3% O2, and 5% CO2 in a cell culture incubator for 6 h, then replaced with high-glucose medium containing 160 μg / ml salmon peptides and placed in a 37°C, 21O2, and 5% CO2 cell culture incubator for re-glucose and reoxygenation for 24 h;

[0066] After incubation, 20 μL of MTT solution (pH 7.4) was added to each well and incubated for another 4 hours at 37°C and 5% CO2. The culture supernatant was then carefully aspirated and 150 μL of DMSO was added. After shaking for 10 minutes, the cells were placed in a microplate reader and the absorbance was measured at OD570. The relative cell viability was then calculated.

[0067] exist Figure 2The relative cell viability of the oxygen-glucose deprivation / reoxygenation group was 69.03%. Among the treatment groups, the relative cell viability of treatment group 1 was 69.59%, the relative cell viability of treatment group 2 was 70.32%, the relative cell viability of treatment group 3 was 71.29%, the relative cell viability of treatment group 4 was 75.99%, and the relative cell viability of treatment group 5 was 80.06%.

[0068] From the above results, it can be observed that the relative cell survival rates of treatment groups 1-3 were improved to a certain extent compared with the OGD / R group, but the differences were not statistically significant. The relative cell survival rates of treatment groups 4 and 5 were higher than those of the OGD / R group, and the differences met the statistical requirements, indicating that the use of 80 or 160 μg / ml salmon peptide to treat OGD / R neurons can effectively improve the recovery ability of cells, and the effect of 160 μg / ml salmon peptide is more significant.

[0069] At the same time, it can be seen that although 160μg / ml salmon peptide can improve the cell recovery ability of the stroke cell model, the effect is only good. This shows that salmon peptide alone can effectively treat stroke, but its effect needs to be further improved.

[0070] Example 3

[0071] Detection of the therapeutic effect of salmon peptide combined with edaravone on the cerebral ischemia-reperfusion mouse model

[0072] Sixty C57BL / 6 mice, approximately 8 months old and weighing approximately 25g, were selected. Half were male and half were female. They were acclimated for 3 days before the experiment. The mice were then randomly divided into five groups, each consisting of 12 mice: a sham-operated group, a model group, a salmon peptide group, an edaravone group, and a combination group (salmon peptide + edaravone). All mice, except those in the sham-operated group, were subjected to a cerebral ischemia-reperfusion model.

[0073] The detailed modeling process is as follows:

[0074] After the mice were anesthetized with sodium pentobarbital, a longitudinal incision was made in the midline of the neck to expose the right common carotid, external carotid and pterygopalatine arteries. The external carotid, pterygopalatine and common carotid arteries were ligated with silk thread at their proximal ends, and the arteries were temporarily clamped near the bifurcation.

[0075] Make a small incision about 4 mm away from the bifurcation of the common carotid artery, insert the nylon thread that has been melted into a ball at the top, remove the artery clamp, and slowly send the thread through the internal carotid artery to the skull to the starting point of the middle cerebral artery. The length is about 9 to 10 mm, and stop when you feel an obstruction.

[0076] The bifurcation was ligated, and the neck incision was sutured, leaving a 4mm stump of the suture. After 1.5 hours of ligation (ischemia), the nylon suture was released to restore blood flow (reperfusion). After MCAO / R surgery, mice were allowed to recover on a heating pad for 2 hours. Model mice that met the experimental requirements were then returned to a warm cage with food and water. In the sham-operated group, no nylon suture was inserted, and all other procedures were the same as for the model mice.

[0077] Two hours after surgery, mice in the sham operation group and the model group were intravenously injected with 150 μl of normal saline twice; the salmon peptide group was injected with 150 μl of normal saline containing 160 μg / ml salmon peptide and 150 μl of normal saline without drug; the edaravone group was injected with 150 μl of normal saline containing 500 μg / ml edaravone and 150 μl of normal saline without drug; the combination group was injected with 150 μl of normal saline containing 160 μg / ml salmon peptide and 150 μl of normal saline containing 500 μg / ml edaravone; the drugs were administered once a day for a total of 7 times.

[0078] (A) Neurological function scores were assessed 7 days after treatment.

[0079] Zea Longa neurological function scoring system:

[0080] Rating 0:

[0081] Normal: The mice do not have any neurological damage and exhibit normal activities and behaviors.

[0082] Rating 1:

[0083] Mild injuries: Mice exhibit mild motor impairments, such as a slight unsteady gait or a slight deviation to one side when walking.

[0084] Rating 2:

[0085] Moderate damage: The mouse shows obvious motor impairment and cannot walk normally. Usually manifested as inability to walk freely, inability to stand in balance or obvious rotational movement.

[0086] Rating 3:

[0087] Severe Injury: The mouse is clearly unable to walk or exhibits severe paralysis. The mouse may have minimal movement or may be unable to move.

[0088] Rating 4:

[0089] Complete Injury: The mouse is completely paralyzed, losing most or all of its voluntary movement. This typically manifests as complete motor paralysis, inability to perform any movements, and possible breathing difficulties or a near-death state.

[0090] The results of the neurological impairment scores of mice are shown in Table 1.

[0091] Table 1 Neurological impairment scores in mice

[0092]

[0093] Note: a indicates P < 0.05 compared with the model group, b indicates P < 0.01 compared with the model group, c indicates P < 0.001 compared with the model group, d indicates P < 0.001 compared with the salmon peptide group, and e indicates P < 0.001 compared with the edaravone group.

[0094] From the results in Table 1, it can be seen that the score of the salmon peptide group is lower than that of the model group, indicating that the use of salmon peptide alone can effectively treat neurological impairment caused by ischemic stroke, further verifying that salmon peptide can be used to treat ischemic stroke.

[0095] Further observations revealed that compared with the use of salmon peptides or edaravone alone, the score of the combined group was significantly reduced, and the magnitude of the decrease was significantly higher than the theoretical decrease of the two combined: compared with the model group, the score of the salmon peptide group decreased by 0.69, the score of the edaravone group decreased by 1.08, and the score of the combined group decreased by 2.7. The theoretical effect of the two combined was a decrease of 1.77 compared with the model group, and the actual combined effect was 1.52 times the theoretical effect. This result shows that the combined use of salmon peptides and edaravone can produce a synergistic treatment for ischemic stroke. The possible reason is that ischemic stroke involves multiple different mechanisms, and salmon peptides and edaravone produce a synergistic effect by acting on different mechanisms.

[0096] (B) TTC staining assay

[0097] After neurological scoring, mice were sacrificed, brain tissue removed and placed in a mouse brain mold. Coronal sections were made every 2 mm starting 2 mm posterior to the bregma at the frontal pole. The brain slices were placed in freshly prepared 1% TTC solution, stained in the dark for 30 minutes, and then fixed in 4% paraformaldehyde. Infarct area was measured and multiplied by thickness to obtain infarct volume. The percentage of infarct in the total cerebral hemisphere was calculated to obtain the infarct percentage.

[0098] The percentage of cerebral infarction in mice is shown in Table 2.

[0099] Table 2 Percentage of cerebral infarction in mice

[0100]

[0101]

[0102] Note: c indicates P < 0.05 compared with the model group, d indicates P < 0.001 compared with the salmon peptide group, and e indicates P < 0.001 compared with the edaravone group.

[0103] As shown in Table 2, compared to the model group, the percentage of cerebral infarction in the salmon peptide group was reduced by 8.33%, the edaravone group by 10.8%, and the combination group by an even more significant 28.97%. This result further demonstrates that drugs containing salmon peptides can significantly reduce the incidence of cerebral infarction in mice with ischemic stroke, thereby effectively alleviating damage caused by cerebral ischemia.

[0104] At the same time, it can be seen that the effect of the combined group is significantly better than the theoretical sum of salmon peptide and edaravone (19.13%). This result shows that the combined use of salmon peptide and edaravone can synergistically reduce the incidence of cerebral infarction in ischemic stroke mice through multi-target treatment.

Claims

1. A use of a salmon polypeptide in the preparation of a drug for preventing and treating ischemic stroke, characterized in that: The amino acid sequence of the salmon polypeptide is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The medicine is a liquid medicine prepared from physiological saline, the concentration of the salmon polypeptide in the medicine is 80 μg / ml-160 μg / ml, and the medicine is administered by intravenous injection.

3. The use according to claim 2, characterized in that The drug can reduce neurological impairment and cerebral infarction caused by ischemic stroke.

4. A pharmaceutical composition for treating ischemic stroke, characterized in that: Each 300 μl of the pharmaceutical composition consists of 150 μl of a salmon polypeptide drug at a concentration of 160 μg / ml and 150 μl of an edaravone drug at a concentration of 500 μg / ml. The amino acid sequence of the salmon polypeptide is shown in SEQ ID NO.

1.

5. The pharmaceutical composition according to claim 4, characterized in that The solvent of the salmon polypeptide drug and the edaravone drug is normal saline; The pharmaceutical composition is administered by intravenous injection; The pharmaceutical composition synergistically reduces neurological impairment and cerebral infarction caused by ischemic stroke.

6. Use of a composition in preparing a drug for treating ischemic stroke, characterized in that: Each 300 μl of the composition consists of 150 μl of a salmon polypeptide solution with a concentration of 160 μg / ml and 150 μl of an edaravone solution with a concentration of 500 μg / ml. The amino acid sequence of the salmon polypeptide is shown in SEQ ID NO.

1.

7. The use according to claim 6, characterized in that The solvent of the salmon polypeptide solution and the edaravone solution is normal saline; The drug is administered by intravenous injection; The drug synergistically reduces neurological impairment and cerebral infarction caused by ischemic stroke through salmon peptide and edaravone.

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