Application of combination of teneplase and deoxyribonuclease I in preparation of medicine for treating acute ischemic stroke

Abstract

The invention belongs to the technical field of medicine. The invention relates to the field of medicine, in particular to application of combination of teneplase and deoxyribonuclease I in preparation of medicine for treating acute ischemic stroke. According to the research of the invention, the combined application of the teneplase and the deoxyribonuclease I to the treatment of acute ischemic stroke has the advantages that compared with the single use of the teneplase, the composition can significantly improve the effects of dissolving thrombus and recovering cerebral blood flow in in-vivo and in-vitro tests, and more importantly, the composition can be applied to the treatment of acute ischemic stroke. The infarct area can be obviously reduced, and the neurological impairment is improved. Therefore, the combined therapy of the teneplase and the deoxyribonuclease I has a very good clinical value for AIS treatment, and the invention provides a new direction for AIS treatment.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology. More specifically, it relates to the application of tenecteplase combined with deoxyribonuclease Ι in the preparation of a medicament for treating acute ischemic stroke. Background Art

[0002] Acute ischemic stroke (AIS) is a disease caused by the occlusion of cerebral arteries, resulting in ischemia and hypoxia of brain tissue, and then triggering local brain tissue damage and infarction. Along with the damage of neurons, astrocytes, and oligodendrocytes, it is one of the main causes of death and disability worldwide, and is the leading cause of death and disability in Chinese adults, with the five characteristics of high incidence, high disability rate, high mortality rate, high recurrence rate, and high economic burden. The key to the acute-phase treatment lies in opening the occluded blood vessels as early as possible, restoring blood flow, and rescuing the ischemic penumbra; as well as the treatment for improving the infarct area and neurological function.

[0003] Currently, the recanalization methods include drug thrombolytic therapy and endovascular mechanical thrombectomy. The latter is costly and requires certain medical technologies and equipment. Drug thrombolytic therapy is still the main way to open the occluded blood vessels of AIS at present. Existing drug thrombolytics include urokinase, tissue-type plasminogen activator, alteplase, tenecteplase (TNK), etc. Among them, recombinant tissue-type plasminogen activator (rt-PA) is the most widely used in thrombolytic therapy.

[0004] However, the main problems of existing drug thrombolysis are the relatively low thrombolytic recanalization rate, and the low effect on improving the infarct area and neurological function, with limited therapeutic effect on AIS. Research (Stroke. 2019; 50: 00 - 00. DOI: 10.1161 / STROKEAHA.119.026848.) explored the pharmacological regulation of neutrophil extracellular traps to reverse rt-PA resistance and thus improve the thrombolytic recanalization rate. The research results showed that deoxyribonuclease Ι (DNase-1) combined with rt-PA could reverse the resistance of rt-PA to a certain extent and improve its thrombolytic efficiency; however, further research showed that this combined regimen could not effectively affect the therapeutic effect of stroke mice. For example, the animal experiment results in this literature showed that in terms of reducing the infarct area and improving neurological deficits in mice, the group treated with alteplase alone did not improve, the group treated with DNase-1 alone had obvious improvement, while the group treated with alteplase and DNase-1 did not have obvious improvement; that is, in terms of improving the infarct area and neurological function, the combined treatment effect was worse than that of the group treated with DNase-1 alone.

[0005] Therefore, it is of great clinical significance and urgent need to explore and develop AIS treatment drugs and treatment plans that have high thrombolytic recanalization rates and, more importantly, can significantly reduce the infarct area, improve neurological deficits, and have clinical therapeutic value. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of existing clinical AIS treatment drugs and develop a new direction for AIS treatment.

[0007] The present invention aims to provide the use of deoxyribonuclease I in the preparation of a synergist for tenecteplase in the treatment of acute ischemic stroke.

[0008] Another object of the present invention is to provide the use of tenecteplase combined with deoxyribonuclease I in the preparation of a drug for treating acute ischemic stroke.

[0009] A drug for the treatment of acute ischemic stroke containing tenecteplase and deoxyribonuclease I.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] After a lot of research and exploration, the present invention has obtained a combined treatment scheme of tenecteplase and deoxyribonuclease I for AIS, which not only significantly improves the thrombolytic effect, but also significantly reduces the infarct area and improves neurological deficits. Specifically, we used three samples of AIS thrombus, artificial blood clot and acute ischemic stroke animal model, and both in vitro and in vivo experimental studies showed that the combined use of tenecteplase and deoxyribonuclease I, compared with the use of tenecteplase alone, not only has a better thrombolytic effect, can more effectively restore cerebral blood flow, but also, very importantly, can significantly reduce the infarct area, improve neurological deficits, and has a very positive clinical treatment synergistic effect. Therefore, the combined use of tenecteplase and deoxyribonuclease I is a new direction for the treatment of AIS.

[0012] Therefore, the present invention claims protection for:

[0013] Application of deoxyribonuclease I in the preparation of a synergist for tenecteplase in the treatment of acute ischemic stroke.

[0014] Application of tenecteplase combined with deoxyribonuclease I in the preparation of drugs for the treatment of acute ischemic stroke.

[0015] A drug for treating acute ischemic stroke, the drug comprising tenecteplase and deoxyribonuclease I.

[0016] Further optionally, the drug may also include pharmaceutically acceptable excipients or carriers.

[0017] Further optionally, the drug can be prepared into an injection form.

[0018] Based on the above solution, the present invention further provides a medicine box, and the medicine box contains a drug comprising tenecteplase and deoxyribonuclease Ι. Further, the drug may further include a pharmaceutically acceptable excipient or carrier.

[0019] Further, the medicine box further includes a container for containing the drug.

[0020] The present invention has the following beneficial effects:

[0021] According to the research of the present invention, when tenecteplase and deoxyribonuclease Ι are combined and applied to the treatment of acute ischemic stroke, compared with the use of tenecteplase alone, this composition can significantly improve the effects of dissolving thrombus and restoring cerebral blood flow in in vivo and in vitro tests. And importantly, it can also significantly reduce the infarct area and improve neurological deficits. Therefore, the combined therapy of tenecteplase and deoxyribonuclease Ι of the present invention has good clinical value for the treatment of AIS, and the present invention provides a new direction for the treatment of AIS. Description of the Drawings

[0022] Figure 1 It is a physical picture of the AIS thrombus in Example 1 and a statistical chart of the thrombolysis rate during the in vitro thrombolysis test; in the figure, TNK is tenecteplase, Glu-plasminogen is plasminogen, and DNase-1 is deoxyribonuclease Ι.

[0023] Figure 2 It is a physical picture of the artificial blood clot in Example 2 and a statistical chart of the thrombolysis rate during the in vitro thrombolysis test; in the figure, TNK is tenecteplase, Glu-plasminogen is plasminogen, and DNase-1 is deoxyribonuclease Ι.

[0024] Figure 3 It is a flow chart of the animal experiment in the present invention.

[0025] Figure 4 It is a statistical chart of the cerebral blood flow recovery situation and blood flow values observed by a laser speckle flowmeter in the animal experiment of the present invention for each group.

[0026] Figure 5 It is a representative diagram of cerebral TTC staining and a statistical chart of the infarct area for each group of mice after modeling for 24 hours in the animal experiment of the present invention.

[0027] Figure 6 It is a statistical chart of the neurological deficit assessment (Longa score) for each group of mice 24 hours after modeling in the animal experiment of the present invention. Detailed Embodiments

[0028] The following further illustrates the present invention in conjunction with the drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form.

[0029] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0031] The present invention uses thrombi from AIS patients and artificial blood clots to explore the thrombolytic efficiency of tenecteplase and deoxyribonuclease Ι through in vitro thrombolysis experiments, and further uses AIS animal model experiments to study the therapeutic effects of the combination of tenecteplase and deoxyribonuclease Ι on AIS, including the effects of reducing infarct area and improving neurological deficits. The main contents of the research are as follows: (1) In vitro experiment: By collecting fresh thrombi from AIS patients after endovascular thrombectomy and preparing artificial blood clots, tenecteplase is used for in vitro thrombolysis with and without deoxyribonuclease Ι, and the thrombolysis rate is calculated; (2) In vivo experiment: An AIS mouse model is used to observe the therapeutic effects of tenecteplase + / - deoxyribonuclease Ι.

[0032] In the in vitro experiments of Examples 1 and 2, plasminogen (Glu-plasminogen) was added simultaneously to simulate the in vivo environment (in animals). Both plasminogen and tenecteplase used in the examples are commercially available.

[0033] The deoxyribonuclease Ι used in the following examples is commercially available and is produced by Roche Group, Switzerland.

[0034] Example 1 In vitro thrombolysis experiment of thrombus samples from AIS patients

[0035] 1. Test method

[0036] After collecting fresh thrombi from AIS patients who received endovascular interventional thrombectomy in the First Affiliated Hospital of Jinan University, each thrombus sample was transversely cut into two equal parts, and the two halves of the thrombus were treated as follows:

[0037] (1) One half of the thrombus + tenecteplase (1 μg / mL) + plasminogen (1 μM), incubated at 37°C;

[0038] (2) The other half of the thrombus + tenecteplase (1 μg / mL) + deoxyribonuclease Ι (100 IE / mL) + plasminogen (1 μM), incubated at 37°C;

[0039] Then, an ultra-precision balance was used to measure the weight of each half thrombus sample at 0, 20, 40, and 60 minutes of incubation. The thrombolysis rate was calculated as the percentage of the thrombus weight at each time point relative to the initial weight before incubation.

[0040] 2. Test results

[0041] The results are as Figure 1 shown. Compared with using tenecteplase alone, adding deoxyribonuclease Ι accelerated the dissolution of thrombus, and the weight of thrombus at 60 minutes of treatment was lower than that in the group using tenecteplase alone, indicating a higher thrombolysis rate. At 60 minutes, the thrombus weight in the tenecteplase group was 48% of the initial weight, while in the group combined with deoxyribonuclease Ι it was 31%.

[0042] Example 2 In vitro thrombolysis test of artificial blood clots

[0043] 1. Test method

[0044] Peripheral blood of healthy volunteers was collected using sodium citrate anticoagulant tubes. 16.6 mM anhydrous calcium chloride was added to 250 μL of blood, and it was incubated at 37 °C for 30 minutes for calcification to form a blood clot core. After the remaining blood was centrifuged at 120 g for 15 minutes to obtain leukocyte-rich and platelet-rich plasma, the blood clot core was transferred into it and incubated at 37 °C for 3.5 hours to form artificial blood clots.

[0045] Then, the thrombolysis rate of the artificial blood clots was calculated using the same method for treating AIS thrombus samples in Example 1. That is: the artificial blood clots were treated as follows:

[0046] (1) Blood clot + tenecteplase (1 μg / mL) + plasminogen (1 μM), incubated at 37 °C;

[0047] (2) Blood clot + tenecteplase (1 μg / mL) + deoxyribonuclease Ι (100 IE / mL) + plasminogen (1 μM), incubated at 37 °C;

[0048] Then, the weight of each blood clot sample was measured using an ultra-precise balance at 0, 20, 40, and 60 minutes of incubation. The thrombolysis rate was calculated as the percentage of the blood clot weight at each time point relative to the initial weight before incubation.

[0049] 2. Test results

[0050] The results are as Figure 2 shown. The results of thrombolysis of artificial blood clots were similar to those of thrombus in Example 1. Compared with using tenecteplase alone, adding deoxyribonuclease Ι accelerated the dissolution of blood clots, and the weight of blood clots at 60 minutes of treatment was lower than that in the group using tenecteplase alone, indicating a higher thrombolysis rate. At 60 minutes, the blood clot weight in the tenecteplase group was 53% of the initial, and the remaining weight of the blood clot at 60 minutes after adding deoxyribonuclease Ι was 32% of the initial.

[0051] Example 3 Treatment test of photochemical AIS mouse model

[0052] 1. Test method

[0053] Eight-week-old male C57BL / 6 mice (23 - 26 g) were randomly divided into four groups: sham operation group and operation group. The operation group was further divided into saline control group, tenecteplase group, and tenecteplase + deoxyribonuclease Ι group. The specific experimental protocol is as Figure 3 shown.

[0054] Sham operation group: After anesthetizing the mice with intraperitoneal injection of 1% sodium pentobarbital, rose bengal solution (50 mg / kg) was injected via the tail vein. The exposed bone window of the left middle cerebral artery was not irradiated with a laser beam.

[0055] Operation group: After anesthetizing the mice with intraperitoneal injection of 1% sodium pentobarbital, rose bengal solution (50 mg / kg) was injected via the tail vein. Five minutes later, a laser beam (532 nm, 5 mW) was placed at the exposed bone window of the left middle cerebral artery and irradiated for 16 minutes and 30 seconds. Two hours after vascular occlusion, each group was injected via the tail vein with:

[0056] Saline control group: 250 μL of saline,

[0057] Tenecteplase group: 250 μL of tenecteplase solution (the dose of tenecteplase was 2.5 mg / kg based on the body weight of the mice),

[0058] Tenecteplase + deoxyribonuclease Ι group: 250 μL of tenecteplase + deoxyribonuclease Ι solution (the dose of tenecteplase was 2.5 mg / kg based on the body weight of the mice; the dose of deoxyribonuclease I was 50 μg per mouse).

[0059] Laser speckle flow imaging was used to measure the cerebral blood flow images of the mice before modeling and at 0, 20, 40, and 60 minutes after modeling. Twenty-four hours later, neurological deficit was evaluated using the Longa score, and then the brain was taken for TTC staining to observe the infarction area.

[0060] 2. Experimental results

[0061] The experimental results of the AIS mouse model are as Figures 4-6 shown, and the results indicate that: compared with the use of tenecteplase alone, the combined treatment with tenecteplase and deoxyribonuclease Ι is more effective:

[0062] Recanalization effect of occluded blood vessels: The cerebral blood flow recovery of the mice in the tenecteplase + deoxyribonuclease Ι group was more significant at 60 minutes (tenecteplase group vs tenecteplase + deoxyribonuclease Ι group: 27.5% vs 66.0%, p < 0.001).

[0063] Improvement effect of neurological deficit: The neurological deficit of mice was significantly improved after 24 hours (Longa score, tenecteplase group vs tenecteplase + deoxyribonuclease Ι group: 3 vs 1, p = 0.003; saline group vs tenecteplase + deoxyribonuclease Ι group: 3 vs 1, p = 0.001).

[0064] Effect of reducing cerebral infarction area: Observed by TTC staining, the cerebral infarction area in the tenecteplase + deoxyribonuclease Ι group was also significantly reduced compared with the tenecteplase alone group (tenecteplase group vs tenecteplase + deoxyribonuclease Ι group: 14.6% vs 11.2%, p < 0.001).

[0065] In summary, the test results show that the combination of tenecteplase and deoxyribonuclease Ι can improve the phenomenon of fibrinolytic resistance. In in vitro tests, compared with using tenecteplase alone, the thrombolysis in the combination treatment group was more sufficient, and the cerebral blood flow recovery in mice was more obvious in in vivo tests; in addition, the combination of tenecteplase and deoxyribonuclease Ι treatment had a smaller infarction area and more significant improvement in neurological deficit. The results indicate that the combination of tenecteplase and deoxyribonuclease Ι can significantly improve the treatment effect of AIS.

[0066] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of deoxyribonuclease I in the preparation of a synergist of tenecteplase for the treatment of acute ischemic stroke.

2. Application of tenecteplase combined with deoxyribonuclease I in the preparation of drugs for the treatment of acute ischemic stroke.

3. The use according to claim 1 or 2, characterized in that: The drug also includes pharmaceutically acceptable excipients or carriers.

4. The use according to claim 3, characterized in that: The dosage form of the drug is injection.

5. A drug for treating acute ischemic stroke, characterized in that: Contains tenecteplase and deoxyribonuclease I.

6. The drug according to claim 5, characterized in that Pharmaceutically acceptable excipients or carriers are also included.

7. The drug according to claim 5 or 6, characterized in that The drug dosage form is injection.

8. A medicine box, characterized in that: The kit includes a drug containing tenecteplase and deoxyribonuclease I.

9. The medicine box according to claim 9, characterized in that: The drug also includes pharmaceutically acceptable excipients or carriers.

10. The medicine box according to claim 8 or 9, characterized in that: The medicine kit also includes a container for containing the medicine.

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