Application of taurine in treatment of cerebral apoplexy

Abstract

The invention discloses taurine for treating cerebral arterial thrombosis. The taurine adopts an inhalation administration mode or targeted pulmonary administration, so that the aggregation of the medicine in the lung can be enhanced, the inflammation of the cerebral parenchyma stroke part can be alleviated, the injury of the nervous system of the stroke part can be improved, and the recovery of the damaged nerve function can be promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmacy, in particular to taurine for treating stroke. BACKGROUND

[0002] Stroke, commonly known as apoplexy, is a serious cerebrovascular disease that causes permanent disability and death worldwide. It has high morbidity, high disability rate, high recurrence rate, and poor prognosis, which seriously threatens human life and health. Stroke can be divided into two categories according to pathology: ischemic stroke and hemorrhagic stroke. Ischemic stroke refers to the obstruction of blood supply to the local brain tissue, leading to ischemic and anoxic necrosis of the brain tissue, and further producing the corresponding clinical manifestations of neurological dysfunction. The typical characteristics are abnormal activation of microglial cells, production of inflammatory cytokines, and infiltration of immune cells, which together exacerbate ischemic brain injury and seriously affect the prognosis of patients.

[0003] The prevention and treatment of cerebral ischemia-reperfusion injury is a key and difficult point in the clinical treatment of ischemic stroke. In addition to intravenous drug thrombolysis, mechanical thrombectomy or angioplasty to restore blood reperfusion in the ischemic area as soon as possible, the current treatment drugs for ischemic stroke mainly include the following categories: (1) Antiplatelet drugs: for most patients with acute ischemic stroke, antiplatelet drugs are chosen for single drug therapy. For example, aspirin can be taken orally as soon as possible after the onset, and antiplatelet drugs such as clopidogrel can be chosen for those who cannot tolerate aspirin. (2) Anticoagulants: mainly for the treatment of high coagulation state in patients with ischemic stroke in the early stage of acute cerebral ischemia, including oral anticoagulants such as warfarin, dicumarol, and phenprocoumon, and non-oral anticoagulants such as heparin, among which the bleeding risk of low molecular weight heparin is lower than that of ordinary heparin. (3) Neuroprotective agents: used to prevent and treat sequelae of ischemic stroke and reduce mortality. (4) Thrombolytic drugs: such as tenecteplase, studies have shown that the effectiveness and safety of intravenous tenecteplase and alteplase are similar.

[0004] Taurine is currently only approved for the treatment of cataracts and congestive heart failure, and there is no research on its inhaled administration for treating ischemic stroke. SUMMARY

[0005] The present application proves that taurine can treat ischemic stroke, reduce the infarct area of stroke, reduce inflammation at the site of cerebral infarction, and / or improve neurological function, such as improving neurological damage at the site of stroke and / or promoting recovery of impaired neurological function. The present application proves that the effect of taurine inhaled administration for treating ischemic stroke is better, and the present applicant found that pulmonary administration can enhance the accumulation of drugs in the lungs, reduce the infiltration of immune cells in the brain, reduce inflammation at the site of cerebral infarction, and promote the recovery of impaired neurological function.

[0006] The present application provides taurine preparation, new administration regimen for improving the utilization and efficacy of taurine in treating ischemic stroke. The taurine preparation used contains an effective amount of taurine, which means that taurine as a single drug can achieve the efficacy of treating ischemic stroke.

[0007] The taurine preparation can be a preparation for administration by inhalation or targeting the lung, such as oral inhalation preparation, nasal inhalation preparation, oral and nasal inhalation preparation, which means that inhalation occurs in both the mouth and the nose. The inhalant can be an aerosol, a spray, an inhalation liquid preparation, a powder aerosol or a vaporizable preparation such as a powder or a liquid preparation. To ensure high solubility, taurine can even be provided as its pharmaceutically acceptable salt, such as sodium taurate; or a cosolvent is used. As a further improvement, the inhalant includes an isotonicity regulator such as NaCl, which can be administered by nebulization. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 Fig. 1 is a schematic diagram of the results of 2,3,5-triphenyltetrazolium chloride (TTC) staining and semi-quantitative analysis of different groups in Experiment 1 of the present application;

[0009] Figure 2 Fig. 2 is a body weight change curve of MCAO rats in Experiment 2 of the present application;

[0010] Figure 3 Fig. 3 is the results of neurological function score of MCAO rats in Experiment 2 of the present application (A is the Longa score, B is the water maze, and C is the sticky label test);

[0011] Figure 4 Fig. 4 is the fluorescence staining results of CD45 positive cells (i.e. immune cells) in brain sections of rats after different treatments in Experiment 2 of the present application.

[0012] The grouping of each figure follows a unified principle, the Sham group in this text refers to the sham operation group set up, and the other groups are treatment groups treated with the test substance on MCAO model rats, and the significance mark and its meaning are as follows: *P<0.05, **P<0.01, ***P<0.001, **** represents P<0.0001. DETAILED DESCRIPTION

[0013] The applicant first establishes a rat stroke model by surgery, compares the efficacy by different administration routes, and studies the mechanism. The applicant finds that pulmonary administration is the most optimal: it can enhance the accumulation of drugs in the lungs, significantly reduce the infiltration of immune cells in the brain, reduce inflammation in the brain parenchyma stroke site, and promote the recovery of damaged nerve function. The following takes taurine injection and inhalation administration as examples to confirm its ability to treat ischemic stroke. The experimental methods are conventional methods unless otherwise specified. The following experimental rat models are MCAO models. The experimental materials used in the following experiments are conventional materials or can be purchased from biochemical reagent manufacturers unless otherwise specified.

[0014] Experiment 1 Comparison of the efficacy of different administration groups on SD rat ischemic stroke model

[0015] 1. Experimental materials

[0016] (1) Drug

[0017] Preparation method: dissolve 0.6 mg of taurine in 2 mL of normal saline, ultrasonic for 10 min to form a taurine aqueous solution, and reserve or put into a nebulization administration instrument to form liquid droplets.

[0018] (2) Experimental animals

[0019] SD rats, 6-8 weeks old, male.

[0020] 2. Experimental methods

[0021] The body weight of the animals is controlled between 230-260 g, and the animals are fasted overnight before modeling and allowed to drink water normally.

[0022] (1) Rat anesthesia

[0023] Intraperitoneal injection of 2.6 mL of Avertin (10 uL / g) to anesthetize the rats.

[0024] (2) Surgery

[0025] The skin of the neck is cut, the muscles of the neck are bluntly separated, and the left common carotid artery and the internal and external carotid arteries of the rat are exposed. The common carotid artery is separated, and the external carotid artery, the suboccipital artery, and the internal carotid artery are sequentially separated from the distal end of the common carotid artery. Double lines are prepared on the external carotid artery and the suboccipital artery. The internal carotid artery and the pterygopalatine artery are separated, the pterygopalatine artery is ligated with a vascular clamp, and the internal carotid artery, the only branch of the common carotid artery, is kept open. After ligating the external carotid artery and the suboccipital artery with double lines, the blood vessels are cut, and a loose knot is made on the external carotid artery for standby. The proximal end of the common carotid artery and the internal carotid artery are clamped with an arterial clamp, a 45° small opening is cut on the external carotid artery, a thread is inserted into the internal carotid artery in the direction of the internal carotid artery, and the thread is fixed tightly, and the arterial clamp is loosened. The thread with silicone is carefully inserted from the external carotid artery into the internal carotid artery until the beginning of the middle cerebral artery. The appropriate thread is selected according to the size and head diameter of the experimental animal.

[0026] (3) Reperfusion

[0027] The thread is pulled out, and the head end is withdrawn to the external carotid artery. It is not suitable to pull it out completely to prevent arterial bleeding. The residual end of the external carotid artery is ligated, and the blood flow of the common carotid artery can be reperfused to the middle artery. The wound skin is sutured, and the animal is placed back in the cage after the anesthesia is recovered.

[0028] (4) Postoperative care and vital sign monitoring

[0029] The room temperature is maintained at 25°C, and the animal protection is paid attention to during and after the operation

[0030] (5) Experimental animal grouping

[0031] After the suture is completed, the reperfusion is completed, and the animals are randomly grouped. Immediately after grouping, the drug administration scheme shown in Table 1 is used for injection or administration by using an atomization instrument, and the therapeutic effects of different administration groups are evaluated by TTC staining 24 hours after administration.

[0032] Table 1 Administration grouping of experimental animals

[0033] Note: The difference between the sham operation group (Sham group) and the saline group is that the blood vessels of the rats in the sham operation group are not embolized by the MCAO operation; except for the sham operation group, the other groups are experimental groups in which drugs are treated on the MCAO model rats suffering from cerebral ischemia-reperfusion injury.

[0034] 3. Investigation index: TTC staining evaluation of infarct volume

[0035] 1) decapitation and brain removal: after the rats were anesthetized, they were placed on a rat decapitation table for decapitation, the posterior neck muscles were separated and removed, a tissue scissors was inserted from the foramen magnum and cut open the skull towards the ipsilateral orbit, the rat was cut between the orbits using a bone rongeur, the skull was carefully lifted and the brain tissue was pushed up, the brain nerves were cut, and the complete brain tissue was removed;

[0036] 2) After the brain tissue was washed in physiological saline, it was placed in a -80°C freezer for 10 min;

[0037] 3) After being removed from the freezer, the brain tissue was evenly cut into 6 pieces;

[0038] 4) The brain slices were completely immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution (prepared with PBS), placed in a 37°C incubator in the dark for 20 min, and the container was gently shaken every 5 min to ensure that the brain slices were fully stained;

[0039] 5) The brain slices were removed, arranged in order, photographed, and the TTC staining results are shown in Figure 1 ;

[0040] 6) Image J 1.53 and GraphPad Prism 8.0 were used for image analysis, the infarct area and total area of each piece were measured, and the data were statistically analyzed.

[0041] Experiment 2 Further pharmacodynamic effect study of oral and nasal inhalation taurine preparation on SD rat ischemic stroke model

[0042] 1. Experimental materials

[0043] Each material follows the same principles as Experiment 1, such as the preparation of the inhalation taurine preparation is exactly the same as the aerosol droplets in Experiment 1.

[0044] 2. Experimental method

[0045] Each step follows the same principles as Experiment 1, and the MCAO model is established, the animals are randomly divided into groups, and the specific grouping and dosing are shown in Table 2.

[0046] Table 2 Dosing group of experimental animals

[0047]

[0048] * The rats used in these two groups are also referred to as MCAO rats with reperfusion injury.

[0049] 3. Investigation index:

[0050] (1) The results of the change in rat body weight are shown in Figure 2 .

[0051] (2) The results of the neurological function score are shown inFigure 3

[0052] The neurological function of the rats was scored according to the Longa scoring standard, and the specific scoring standard was as follows:

[0053] 0 points: the rat has no neurological function injury, and the action is normal

[0054] 1 point: after holding the tail of the rat, the contralateral forelimb of the operation cannot be fully stretched

[0055] 2 points: when the rat walks autonomously, it turns to the contralateral side of the operation

[0056] 3 points: when the rat walks autonomously, the body tilts to the contralateral side of the operation

[0057] 4 points: the rat cannot walk autonomously and loses consciousness

[0058] 5 points: death

[0059] After the administration was completed, the rat tail was lifted according to the above scoring standard, the stretching of the contralateral forelimb of the operation was observed, and then it was placed horizontally on the ground, and the turning behavior of the rat was observed. The neurological function of the rat was scored.

[0060] (3) The behavioral evaluation results were as follows: Figure 3

[0061] 1) The behavior of the rat was evaluated by the sticker experiment. First, the rat was trained for the sticker experiment 3 days before the MCAO model was constructed, 3 times a day, and the experiment was performed after the MCAO model was established and administered.

[0062] 2) A 13x13mm 2 sticker was attached to the dorsal side of the rat's paw as a tactile stimulus, and then the rat was returned to the cage. The time when the rat contacted and removed the sticker was recorded.

[0063] (4) Immune cell infiltration in rat brain tissue

[0064] 1) After treatment, the SD rat was euthanized, and the rat brain tissue was washed with PBS solution;

[0065] 2) The brain tissue was soaked in tissue fixative for fixation. After fixation, the brain tissue was paraffin sectioned, and deparaffinization treatment and antigen repair were performed;

[0066] 3) Add blocking solution and incubate at room temperature for 1h to block non-specific binding sites;

[0067] 4) Discard the blocking solution, add CD45 antibody (1:500) diluted as required on the surface of the tissue, and incubate at 4°C in the dark overnight;

[0068] 5) Wash the tissue sample with PBS solution for three times to remove unbound antibodies;

[0069] 6) Add the fluorescently labeled secondary antibody and incubate for 90 min in the dark;

[0070] 7) Add DAPI staining solution dropwise and incubate for 15 min in the dark;

[0071] 8) Add anti-fluorescence quenching agent dropwise to complete the mounting;

[0072] 9) Take pictures and collect images of the tissue sample using a laser scanning confocal microscope, and the results are shown in Figure 4 .

[0073] Experimental results:

[0074] TTC staining was performed on the cerebral infarction area to quantitatively evaluate the brain protection effect of oral-nasal inhalation of taurine, and the specific results are shown in Figure 1 . By comparing intragastric administration, different injection and inhalation administration, it is shown that different administration routes of taurine can reduce the infarction area of ischemic stroke to some extent, but the treatment effect of oral-nasal inhalation of taurine is the most significant. As can be seen from the figure, after oral-nasal inhalation of taurine, the infarction area of MCAO rats decreased significantly lower than the Saline group.

[0075] As can be seen from Figure 2 , the body weight of rats in the Saline group decreased sharply on the first day after MCAO operation. However, the body weight of rats increased after oral-nasal inhalation of taurine, and the body weight was close to that of the Sham group, indicating that oral-nasal inhalation of taurine has good therapeutic effect in vivo.

[0076] The recovery of neural function of rats after treatment was evaluated by neural function score and sticker test, and the specific results are shown in Figure 3 . As can be seen from the figure, compared with the Saline group, the neural function score of MCAO rats after oral-nasal inhalation of taurine decreased significantly, which indicated that inhalation of taurine had a significant effect on improving neural function. In addition, the integration of skin sensitivity and sensory function was evaluated by sticker test, and the time of contact and removal of the sticker in the sticker test of MCAO rats after oral-nasal inhalation of taurine was significantly shortened compared with the Saline group. This indicates that oral-nasal inhalation of taurine after stroke can promote the improvement of sensory motor function of rats.

[0077] The results of immunofluorescence staining are shown in Figure 4 . As can be seen from the figure, compared with the Saline group, the number of immune cells at the stroke site after oral-nasal inhalation of taurine decreased significantly, indicating that oral-nasal inhalation of taurine effectively inhibited the infiltration of immune cells after stroke, thereby reducing the inflammatory response in the brain.

Claims

1. The use of taurine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of ischemic stroke, which targets the lungs or is an inhalant.

2. The application as described in claim 1, characterized in that, The drug can reduce the infarct area of ​​stroke, alleviate inflammation at the stroke site in the brain parenchyma, and / or improve neurological function at the stroke site.

3. The application as described in claim 2, characterized in that, The improvement of neurological function at the stroke site includes improving neurological damage at the stroke site and / or promoting the recovery of damaged nerve function.

4. The application as described in claim 1, characterized in that, The stroke mentioned is an acute ischemic stroke.

5. The application as described in any of the prior claims, wherein, The drug is a spray, powder, aerosol, inhaled liquid preparation, or a preparation that can be converted into vapor.

6. The application as described in any of the prior claims, wherein, The drug is in powder or liquid form.

7. The application as described in any of the prior claims, wherein, The drug contains an isotonic regulator.

8. The application as described in any of the prior claims, wherein, The drug contains NaCl.

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