Use of fingolimod in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke

Abstract

The present invention relates to the use of fingolimod in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, belonging to the field of pharmaceutical technology. The use of fingolimod in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, the chemical structural formula of the fingolimod is as follows. The present invention adopts the administration method of oral and nasal inhalation, uses fingolimod to treat ischemic stroke, enhances the aggregation of the drug in the lungs, inhibits the migration of pulmonary immune cells to the brain, thereby reducing the inflammation at the stroke site in the brain parenchyma, improving the damage to the nervous system at the stroke site, and promoting the recovery of damaged nerve function. Moreover, the oral and nasal inhalation administration method avoids the side effects such as cardiac conduction block and bradycardia that may be caused when fingolimod is taken orally, and provides a new type of safe and effective administration route for patients with cerebral infarction, especially those with heart disease.

Description

Technical Field

[0001] The present invention relates to the use of fingolimod in the preparation of an oral and nasal inhalation preparation for the treatment of ischemic stroke, and belongs to the field of pharmaceutical technology. Background Art

[0002] Stroke, commonly known as apoplexy, is a serious cerebrovascular disease that causes permanent disability and death worldwide. It is mainly caused by the sudden rupture of blood vessels in the brain or the blockage of blood vessels, resulting in the inability of blood to flow into the brain and causing damage to brain tissue. Stroke has the characteristics of high incidence, high disability rate, high recurrence rate, and poor prognosis, seriously threatening human life and health. Stroke can be divided into two major categories according to pathology: ischemic stroke and hemorrhagic stroke. The incidence of ischemic stroke is higher than that of hemorrhagic stroke, accounting for about 80% of the total number of strokes. Ischemic stroke refers to the blood supply disorder in a local brain tissue area, resulting in ischemic and hypoxic necrosis of brain tissue, and then producing corresponding neurological deficits clinically. The typical characteristics are the abnormal activation of microglia, the production of inflammatory cytokines, and the infiltration of peripheral blood immune cells (such as neutrophils, macrophages). These mechanisms together lead to the aggravation of ischemic brain injury and seriously affect the prognosis of patients.

[0003] The prevention and treatment of cerebral ischemia-reperfusion injury are the key points and difficulties in the clinical treatment of ischemic stroke. In addition to treatment methods such as intravenous drug thrombolysis, mechanical thrombectomy, or angioplasty to quickly restore blood reperfusion in the ischemic area, the current treatment drugs for ischemic stroke mainly include the following categories: (1) Antiplatelet drugs: For most patients with acute ischemic stroke, single-drug antiplatelet therapy is selected. For example, aspirin can be given orally as early as possible after the onset. For those who cannot tolerate aspirin, antiplatelet drugs such as clopidogrel can be selected. (2) Anticoagulants: Mainly used for the hypercoagulable state of ischemic stroke patients in the early treatment of acute cerebral ischemia, including oral anticoagulants such as warfarin, dicumarol, phenprocoumon, etc., and non-oral anticoagulants such as heparin. Among them, 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. Some studies have shown that the effectiveness and safety of intravenous tenecteplase are similar to those of alteplase.

[0004] Currently, the drugs studied for neuroprotection mainly include glutamate receptor antagonists, free radical scavengers, calcium channel antagonists, γ-aminobutyric acid (GABA) agonists, citicoline, inflammatory response inhibitors, estrogen, erythropoietin (EPO), etc. Some experiments have proved that these drugs have certain neuroprotective effects, but the clinical effects still need further research. Therefore, deeply exploring the pathological mechanism of stroke is crucial for finding new, safe, and reliable targeted treatment drugs.

[0005] Fingolimod (FTY720) has a strong immunosuppressive effect in the central nervous system and was approved by the FDA in 2010 as the first oral immunosuppressant for the treatment of relapsing-remitting multiple sclerosis. FTY720 is a sphingosine analogue and a high-affinity agonist of sphingosinol-1-phosphate receptor (S1PR). S1PR is a series of G protein-coupled receptors widely distributed in the central nervous system, among which S1PR1 plays a more prominent role, mainly expressed in endothelial cells and playing an important role in regulating angiogenesis, inflammatory cell infiltration and immune response.

[0006] The pulmonary circulation has the largest and most dynamic intravascular leukocyte pool, and there is no intermediate microvascular bed between the cerebral blood vessels and the pulmonary blood vessels. These leukocytes can quickly respond to local and remote danger signals (such as cytokines, exosomes, damage associated molecular patterns (DAMPs)) from damaged brain tissue. Therefore, it becomes possible to deliver drugs to the lungs through oral and nasal inhalation for the treatment of ischemic stroke. Currently, there is no research on using oral and nasal inhalation of fingolimod to treat ischemic stroke in the existing technology. Summary of the Invention

[0007] The purpose of the present invention is to provide the application of fingolimod in the preparation of an oral and nasal inhalation preparation for the treatment of ischemic stroke, and to provide a new dosing regimen for ischemic stroke to extend the treatment time window.

[0008] In order to achieve the above purpose, the technical solution of the application of fingolimod in the preparation of an oral and nasal inhalation preparation for the treatment of ischemic stroke in the present invention is:

[0009] The application of fingolimod in the preparation of an oral and nasal inhalation preparation for the treatment of ischemic stroke.

[0010] The beneficial effect of the above technical solution is that the application of fingolimod in the preparation of an oral and nasal inhalation preparation for the treatment of ischemic stroke is an invention of a new use of a known product. The inventors of the present invention found through repeated research and experiments that FTY720 can reduce the mortality rate after cerebral ischemia. The present invention first constructs a rat ischemic stroke model (cerebral ischemia-reperfusion model (MCAO model)) and treats it by oral and nasal inhalation of the same dose of edaravone, butylphthalide or fingolimod. It is found that the curative effects of commonly used clinical edaravone and butylphthalide are not as good as those of oral and nasal inhalation of fingolimod preparation. At the same time, the influence of different dosing methods of fingolimod on its curative effect is also compared, and it is found that the treatment effect of oral and nasal inhalation of fingolimod is the most significant compared with intraperitoneal injection and tail vein administration. This fully shows that fingolimod can be prepared into an oral and nasal inhalation preparation for the treatment of ischemic stroke.

[0011] Further research has revealed that oral and nasal inhalation of fingolimod for the treatment of ischemic stroke effectively enhances drug accumulation in the lungs and inhibits the migration of pulmonary immune cells to the brain, thereby reducing inflammation in the brain parenchyma at the stroke site, improving neurological damage at the stroke site, and promoting recovery of damaged neurological function. The present invention utilizes oral and nasal inhalation of fingolimod for the treatment of ischemic stroke, demonstrating significant efficacy, low toxicity, good stability, and high safety, and holds great promise for the development of new immune cell inhibitors for the treatment of brain diseases. In addition, oral and nasal administration has the following advantages: (1) Patients with cerebral infarction are prone to fatigue and their swallowing centers may be damaged, which may cause difficulty in swallowing and choking when drinking water. Some patients may be bedridden. Nebulization treatment can solve the problem of drug administration; (2) The key to the treatment of acute ischemic stroke (AIS) is ultra-early thrombolytic therapy, which helps to restore the patient's cerebral blood supply and save neurological function, thereby reducing mortality and improving prognosis. However, the effective treatment time window for thrombolytic therapy is strictly limited to 3 to 4.5 hours. Whether the patient can go to the hospital for treatment within this golden time is the key to the success of thrombolysis. Nebulization administration is convenient and fast, and patients have better compliance and can take the drug independently, thereby extending the treatment time window for acute ischemic stroke patients to go to the hospital for treatment.

[0012] Specifically, the pulmonary circulation has the largest and most dynamic pool of intravascular leukocytes, and there is no intermediate microvascular bed between the cerebral and pulmonary vasculature. These leukocytes can rapidly respond to local and remote danger signals (such as cytokines, exosomes, and damage-associated molecular patterns (DAMPs)) from damaged brain tissue. FTY720 is a high-affinity agonist of the S1P receptor (sphingosinol-1-phosphate receptor, S1PR1). Analysis found that FTY720 can induce the sustained internalization and degradation of S1PR1 on the surface of lymphocytes, inhibiting the migration of T and B lymphocytes and thus causing T and B lymphocytes to remain in lymphoid organs, reducing their distribution in the blood, lymph, and mucosa. In addition, FTY720 can also execute functional agonist mechanisms through S1P receptors expressed on the surface of various cells, including neurons, astrocytes, microglia, and vascular endothelial cells. These functions may play an important role in regulating anti-apoptotic systems, angiogenesis, inflammatory cell infiltration, and immune responses.

[0013] More specifically, the drug targets for the conventional treatment of ischemic stroke are in the brain, while FTY720 targets immune cells (white blood cells). The lungs are the natural and largest white blood cell pool. Therefore, we deliver the drug to the lungs by nebulized administration, and provide a new solution for extending the treatment time window of ischemic stroke by inhibiting the migration of white blood cells in the lungs to the brain.

[0014] Specifically, the chemical structural formula of the fingolimod is

[0015] As a further improvement, the oral and nasal inhalation preparation comprises fingolimod or a pharmaceutically acceptable salt thereof.

[0016] As a further improvement, the oral and nasal inhalation preparation is a preparation for reducing the infarct area of ischemic stroke.

[0017] As a further improvement, the oral and nasal inhalation preparation is a preparation for improving nerve function.

[0018] As a further improvement, the preparation for improving nerve function is a preparation for reducing inflammation in the brain parenchyma stroke site, improving the nervous system damage at the stroke site, and promoting the recovery of damaged nerve function.

[0019] As a further improvement, the oral and nasal inhalation preparation is a spray or powder inhaler.

[0020] As a further improvement, the oral and nasal inhalation preparation is in a liquid dosage form.

[0021] As a further improvement, the oral and nasal inhalation preparation comprises an isotonicity regulator.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. Compared with the injection route, the oral and nasal inhalation administration method has better patient compliance, can be self-administered, and avoids side effects such as cardiac conduction block and bradycardia that may be caused by fingolimod during oral administration. Moreover, the inhalation solution preparation fills the domestic market gap and provides a new type of safe and effective administration route for cerebral infarction patients, especially those with heart disease.

[0024] 2. Preparing fingolimod into an oral and nasal inhalation preparation enables patients to directly inhale through the mouth and nose, which not only enhances the accumulation of the drug in the lungs, but also greatly improves the curative effect, can effectively inhibit the infiltration of brain immune cells, and has many advantages such as high bioavailability, low adverse reactions, and low invasiveness.

[0025] 3. The existing formulations of fingolimod have poor fluidity, while the fingolimod liquid formulation for oral and nasal inhalation avoids the problem of affecting the long-term stability of the formulation due to easy moisture absorption. Moreover, by comparing different administration methods, the present invention finds that the effect of the fingolimod formulation by aerosol inhalation is significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the TTC staining results of different treatment groups in Example 1 of the present invention;

[0027] Figure 2 It is the body weight change curve of MCAO rats in Example 2 of the present invention;

[0028] Figure 3 It is the neurological function score results of MCAO rats in Example 2 of the present invention (where A is the Longa score, B is the water maze, C is the sticky label test, **** represents P<0.0001);

[0029] Figure 4 It is the TTC staining image and semi-quantitative analysis of brain slices of MCAO rats in different treatment groups in Example 2 of the present invention (where **** represents P<0.0001);

[0030] Figure 5 It is the HE staining and Nissl staining images of brain slices of MCAO rats in Example 2 of the present invention;

[0031] Figure 6 It is the immunofluorescence staining results of NeuN and TUNEL in Example 2 of the present invention;

[0032] Figure 7 It is the immunofluorescence staining results of ROS in Example 2 of the present invention;

[0033] Figure 8 It is the HE staining of major organs of rats in each group after different treatments in Example 2 of the present invention;

[0034] Figure 9 It is the expression of TNF-α, IL-6, Arg-1, and IL-10 in the stroke site after different treatments in Example 2 of the present invention (where * represents P<0.05, **** represents P<0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0035] The invention first established a rat stroke model through surgery and treated it by inhaling equal doses of edaravone, butylphthalide, or fingolimod through the nose and mouth. It was found that the curative effects of commonly used clinical drugs, edaravone and butylphthalide, were inferior to those of inhaled fingolimod preparation through the nose and mouth. At the same time, the effects of different administration methods of fingolimod on its curative effect were also compared. It was found that compared with intraperitoneal injection and tail vein administration, the therapeutic effect of inhaled fingolimod through the nose and mouth was the most significant, which fully demonstrated that fingolimod could be prepared into an inhaled preparation through the nose and mouth for the treatment of ischemic stroke.

[0036] Then further research found that inhaled fingolimod through the nose and mouth could enhance the accumulation of the drug in the lungs, inhibit the migration of pulmonary immune cells to the brain, thereby reducing the inflammation in the brain parenchyma stroke site, improving the nervous system damage at the stroke site, and promoting the recovery of damaged nerve function. The invention proves that inhaled fingolimod through the nose and mouth for the treatment of ischemic stroke has the advantages of significant effect, low toxicity, and high safety. At the same time, the invention also reveals the fact that immune cells are involved in the pathological damage of ischemic stroke, and has outstanding promotion prospects for the development of new immune cell inhibitors for the treatment of brain diseases.

[0037] The following further describes the present invention in combination with specific embodiments, but the protection scope of the present invention is not limited thereto; however, these embodiments are only examples and do not constitute any limitation to the scope of the present invention. Modifications or substitutions can be made to the details and forms of the present invention without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all obtained from conventional biochemical reagent manufacturers unless otherwise specified.

[0038] The cerebral ischemia-reperfusion model (MCAO model) used in the following embodiments is a conventional ischemic stroke model in the art.

[0039] Specific embodiments of the application of fingolimod in the preparation of an inhaled preparation through the nose and mouth for the treatment of ischemic stroke:

[0040] The present invention uses a cerebral ischemia-reperfusion model (MCAO model) to verify whether inhaled fingolimod through the nose and mouth can be used to treat ischemic stroke and conducts in-depth research on its mechanism of action. The specific implementation operations are as follows:

[0041] Example 1 Comparison of the curative effects of different administration groups on the ischemic stroke model of SD rats

[0042] 1. Experimental drugs and preparations

[0043] Take 20 mg of fingolimod hydrochloride, edaravone, and dl-3-n-butylphthalide respectively and dissolve them in 2 mL of physiological saline. Ultrasonicate for 10 min to form an aqueous solution of fingolimod hydrochloride, edaravone, and dl-3-n-butylphthalide, and put it into an atomization drug delivery device to form atomized droplets.

[0044] 2. Experimental animals

[0045] SD rats, 6 - 8 weeks old, male, were purchased from Beijing SPF Biotechnology Co., Ltd., with the production license number: SCXK(Beijing)2019 - 0010; the license number for the experimental unit to use: SYCK(He'nan)2023 - 0004. They were raised in an SPF environment, in a positive pressure purification and ventilation animal house, at a room temperature of 23 ± 2 °C, a humidity of 30 - 70%, with artificial lighting simulating day and night changes, and free access to food and water. The body weights of the animals were all controlled between 230 - 260 g. They were fasted overnight before model establishment and had normal access to water.

[0046] 3. Experimental procedures

[0047] (1) Anesthetizing the rats

[0048] First, adjust the flow rate of the anesthetic gas to 300 - 500 mL / min, adjust the induction concentration of isoflurane to 3 - 4%, anesthetize the SD rats, and adjust the maintenance concentration to 1.5 - 2% during the operation.

[0049] (2) Surgery

[0050] Disinfect with iodophor, cut open the skin of the neck, bluntly separate the neck muscles, and expose the left common carotid artery, internal carotid artery, and external carotid artery of the rat. Separate the common carotid artery, and sequentially separate the external carotid artery and internal carotid artery along the common carotid artery towards the distal end. Thread two lines through the common carotid artery and the external carotid artery for later use. Separate the internal carotid artery and the pterygopalatine artery, ligate the pterygopalatine artery with a vascular clip, and keep the internal carotid artery, the only branch of the common carotid artery, open. Make a loose knot on the common carotid artery for later use. Clamp the proximal end of the common carotid artery and the internal carotid artery with an artery clip, make a 45° small incision on the common carotid artery, insert a ligature thread towards the internal carotid direction, tighten and fix the thread, and then release the artery clip. Carefully insert the thread embolization wrapped with silica gel from the common carotid artery into the internal carotid artery until the origin of the middle cerebral artery. Select a suitable thread embolization according to the size and head diameter of the experimental animals.

[0051] (3) Reperfusion

[0052] Pull out the thread embolization so that the head end retreats to the external carotid artery. Do not pull it out completely to avoid bleeding from the arterial rupture. Ligate the stump of the external carotid artery, and the blood flow of the common carotid artery can then reperfuse into the middle artery. Suture the skin of the wound, and put the animal back into the cage after it wakes up from anesthesia.

[0053] (4) Postoperative care and monitoring of vital signs

[0054] It is advisable to maintain the room temperature at 25°C, and animal protection should be noted during and after the operation, and postoperative care should be paid attention to.

[0055] (5) Grouping of experimental animals for drug administration

[0056] After the suture is completed, that is, after reperfusion is completed, the animals are randomly grouped. Immediately after grouping, a nebulizer is used for drug administration. After 24 hours of drug administration, TTC staining is used to evaluate the therapeutic effects of different drug administration groups. The specific grouping and drug dosage are shown in Table 1.

[0057] Table 1 Grouping of experimental animals for drug administration

[0058]

[0059] Note: The Sham group is the control group cultured normally, while the other groups are the experimental groups treated with drugs on the cerebral ischemia-reperfusion model (MCAO model).

[0060] (6) Evaluation of infarct volume by TTC staining

[0061] 1) Decapitation to obtain the brain: After anesthetizing the rats, place them on the rat decapitation table for decapitation. Separate and remove the muscles at the back of the neck. Insert the tissue scissors deep into the foramen magnum, cut the skull in the direction of the ipsilateral orbit, use a rongeur to cut between the orbits of the rats, carefully lift the skull, push the brain tissue upward, cut the cranial nerves, and take out the complete brain tissue;

[0062] 2) After washing the brain tissue in physiological saline, place it in a -80°C refrigerator and freeze it for 10 minutes;

[0063] 3) After taking out the frozen tissue, cut the brain tissue evenly into 6 slices;

[0064] 4) Completely immerse the brain slices in 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution (prepared with PBS), place them in a 37°C incubator in the dark for 20 minutes, and gently shake the container every 5 minutes to fully stain the brain slices;

[0065] 5) Take out the brain slices, arrange them neatly in sequence, take pictures and record. The TTC staining results are as Figure 1 shown.

[0066] 4. Experimental results

[0067] The treatment methods for stroke are very limited. Recombinant tissue plasminogen activator (rt-PA) is the only thrombolytic agent approved by the FDA, aiming to dissolve vascular occlusion and rapidly restore cerebral blood flow. Although rt-PA-mediated thrombolysis has been used as the standard treatment for ischemic stroke, this drug must be injected within 4.5 hours after the onset of stroke symptoms, and only a small number of patients (2-7%) can benefit from it. Therefore, it is crucial to develop new treatment strategies to treat the majority of ischemic stroke patients who miss the rt-PA treatment time window.

[0068] Edaravone (EDV), as a small molecule ROS scavenger, was approved in Japan in 2001 for the treatment of patients with ischemic stroke. EDV reduces the size of ischemic lesions in patients by reducing oxidative stress, inhibiting lipid peroxidation, and alleviating long-term inflammation. Studies have shown that although EDV should be administered in the ultra-early stage to maximize its neuroprotective effect, due to the short half-life of EDV (5.4 minutes), frequent administration (twice / day / person) is required, which leads to complications such as renal dysfunction.

[0069] Butylphthalide was initially extracted from celery seeds. Its isomer DL-3-N-butylphthalide (DL-NBP), also known as apiumetin, is a new type of drug independently developed in China. In addition to its mitochondrial protection effect, it can also inhibit cellular oxidative stress, improve collateral circulation and microcirculation of cerebral blood vessels, protect the blood-brain barrier, and thus improve the neurological function and short-term clinical prognosis of stroke patients. Previous clinical trials of butylphthalide in the treatment of acute ischemic cerebral infarction found that oral and intravenous use of butylphthalide occasionally caused abnormal liver function and gastrointestinal discomfort.

[0070] In this example, equal doses of edaravone and butylphthalide were inhaled through the mouth and nose first, and it was found that their curative effects were inferior to those of fingolimod preparations inhaled through the mouth and nose. At the same time, we also compared intraperitoneal injection and tail vein administration. The results showed that different administration routes of fingolimod could reduce the infarct area of ischemic stroke to a certain extent, but the therapeutic effect of fingolimod inhaled through the mouth and nose was the most significant. Moreover, conventional oral administration and tail vein injection of edaravone were also carried out. Compared with the atomization administration method, oral administration of edaravone reduced the infarct area of ischemic stroke, but its effect was only equivalent to that of intraperitoneal injection of fingolimod.

[0071] Study on the main pharmacodynamic effects of fingolimod preparation for oral and nasal inhalation on the ischemic stroke model of SD rats

[0072] 1. Experimental animals

[0073] The same as in Example 1.

[0074] 2. The experimental steps (1) - (4) are the same as those described in the experimental steps (1) - (4) of Example 1.

[0075] (5) Grouping of experimental animals for drug administration

[0076] After the suture is completed, that is, after reperfusion is completed, the animals are randomly grouped. Immediately after grouping, a nebulizer is used for drug administration. After drug administration, the changes in the body weight and survival rate of the rats are examined. The specific grouping and drug dosage are shown in Table 2.

[0077] Table 2 Grouping of experimental animals for drug administration

[0078]

[0079] (6) Examination of changes in body weight and survival rate of rats

[0080] 1) Neurological function score

[0081] The neurological function of the rats is scored according to the Longa scoring standard. The specific scoring standard is as follows:

[0082] 0 points: The rat has no neurological function damage and moves normally.

[0083] 1 point: After lifting the rat's tail by hand, the contralateral forelimb of the surgical side cannot be fully extended.

[0084] 2 points: When the rat walks autonomously, it circles to the contralateral side of the surgery.

[0085] 3 points: When the rat walks autonomously, its body topples to the contralateral side of the surgery.

[0086] 4 points: The rat cannot walk spontaneously and loses consciousness.

[0087] 5 points: Death

[0088] After the drug administration is completed, according to the above scoring standard, lift the rat's tail and pay attention to observing the extension of the contralateral forelimb of the rat's surgery. Then place it on the horizontal ground and observe whether it has circling behavior when walking autonomously, and score the rat's neurological function.

[0089] 2) Behavioral evaluation

[0090] a. The behavior of the rats is evaluated through the sticky note experiment and the Morris water maze experiment. First, the rats are trained in the sticky note experiment and the Morris water maze experiment 3 days before constructing the MCAO model, 3 times a day. The experiments are carried out on the 3rd, 5th, and 7th days after establishing the MCAO model.

[0091] b. Stick a 13×13mm sticker on the dorsal side of the rat's paw 2The rats were placed back into their cages and the time it took for the rats to touch and remove the sticky sticks was recorded.

[0092] c. Morris water maze was used to evaluate the memory function of rats. Rats were placed in a water maze with a diameter of 210 cm and an escape platform area of 12 cm. 2 The rats were placed in a pool with a water temperature of 20℃, and their swimming trajectories, time spent finding the hidden platform, and swimming distance were recorded.

[0093] (7) TTC staining to evaluate infarct volume

[0094] 1) Decapitation and brain removal: After anesthetizing the rat, place it on a rat guillotine and decapitate. Remove the posterior neck muscles. Use tissue scissors to penetrate the foramen magnum and cut the skull toward the ipsilateral orbit. Use rongeurs to cut between the rat's orbits. Carefully lift the skull, push the brain upward, sever the cranial nerves, and remove the intact brain tissue.

[0095] 2) Wash the brain tissue in normal saline and freeze it in a -80°C freezer for 10 minutes;

[0096] 3) After freezing, cut the brain tissue into 6 even slices;

[0097] 4) Completely immerse the brain sections in 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution (prepared in PBS) and incubate in a 37°C incubator in the dark for 20 minutes. Gently shake the container every 5 minutes to ensure adequate staining.

[0098] 5) Remove the brain slices, arrange them neatly in order, and take photos;

[0099] 6) Image J 1.53 and GraphPad Prism 8.0 were used for image analysis, the infarct area and total area of each slice were measured, and the data were statistically analyzed.

[0100] (8) Pathological changes in rat brain tissue

[0101] After the treatment, the SD rats were euthanized, and the brain tissues were fixed with 4% paraformaldehyde for 24 h, dehydrated, paraffin-embedded, sliced, and dewaxed. The tissue sections were processed with hematoxylin-eosin (HE) and Nissl staining to observe the cell morphology of each group and analyze the pathological changes.

[0102] (9) Detection of rat brain tissue apoptosis and neuronal function recovery

[0103] After the treatment, the SD rats were euthanized, and the rat brain tissues were fixed with 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, sectioned, and dewaxed. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method and the incubation of NeuN antibody method were used to detect the cell apoptosis and the recovery of neuron function in each group.

[0104] (10) Biological safety evaluation of VIN

[0105] After 7 days, the rats were sacrificed, and the main organs (heart, liver, spleen, lung, and kidney) of each group were taken out. After being fixed with 4% paraformaldehyde, they were paraffin-embedded, sectioned, and HE-stained. The morphological changes of the tissues in each group were observed under a microscope and photographed for analysis.

[0106] (11) Detection of inflammatory factors in rat brain tissue

[0107] The brain tissues were collected, and the brain tissues on the injured side were homogenized in PBS. Then, they were centrifuged (12000 rpm, 4 °C, 15 min) to collect the supernatant, and the contents of related factors were measured using an ELISA kit.

[0108] 3. Experimental results

[0109] It can be seen from Figure 2 that on the first day after MCAO surgery, the body weight of the rats in the Saline group decreased sharply. However, after treatment with fingolimod inhaled through the nose and mouth, the body weight of the rats gradually increased, and their body weight was close to that of the Sham group on the 7th day, indicating that inhaled fingolimod through the nose and mouth has good therapeutic effects in vivo.

[0110] The recovery of nerve function in the rats after treatment was evaluated by neurological function scoring, Morris water maze, and adhesive removal test. The specific results are as Figure 3 shown. It can be seen from the figure that compared with the Saline group, the neurological function score of the MCAO rats after treatment with inhaled fingolimod through the nose and mouth was significantly reduced, indicating that inhaled fingolimod by atomization has significant curative effects in improving nerve function. The Morris water maze was used to further evaluate and test the spatial cognitive ability of the MCAO rats. After treatment in each group, the rats were placed in the Morris water maze, and their swimming trajectories were recorded. Compared with the Saline group, the path length and escape latency of the rats treated with inhaled fingolimod through the nose and mouth were significantly decreased, indicating that the learning and memory abilities of the rats in the inhaled fingolimod through the nose and mouth treatment group were improved. In addition, the skin sensitivity and the integration of sensory function were evaluated through the adhesive removal test. Compared with the Saline group, the time for the MCAO rats after treatment with inhaled fingolimod through the nose and mouth to contact and remove the adhesive in the adhesive removal test was significantly shortened. This indicates that inhaled fingolimod through the nose and mouth after stroke can promote the improvement of the sensorimotor function of the rats.

[0111] The cerebral infarction area was stained with TTC to quantitatively evaluate the brain protection effect of fingolimod by nasal and oral inhalation. The specific results are as Figure 4 shown. It can be seen from the figure that after treatment with nasal and oral inhalation of fingolimod, the infarction area of MCAO rats decreased, which was significantly lower than that of the Saline group.

[0112] The results of HE staining and Nissl staining in pathological examination are as Figure 5 shown. It can be seen from the figure that compared with the rats treated with the Saline group, nasal and oral inhalation of fingolimod played an obvious neuroprotective role after ischemic stroke.

[0113] From Figure 6 it can be seen that compared with the Saline group, the increase in NeuN-positive cells in the nasal and oral inhalation of fingolimod treatment group was the most significant, and correspondingly, the apoptosis of TUNEL cells decreased, indicating that nasal and oral inhalation of fingolimod treatment effectively inhibited MCAO-induced neuronal loss and apoptosis.

[0114] From Figure 7 it can be seen that compared with the Saline group, the content of ROS in the stroke area was significantly reduced after treatment with nasal and oral inhalation of fingolimod, indicating that oxidative stress in the stroke area was significantly inhibited after treatment with nasal and oral inhalation of fingolimod, and thus effectively improved the nervous system damage in the stroke area.

[0115] The histological HE staining analysis of the main organs (heart, liver, spleen, lung and kidney) of rats was used to evaluate the biosafety. The specific results are as Figure 8 shown. It can be seen from the figure that compared with the Sham group, there were no obvious pathological toxicity and adverse reactions in each treatment group.

[0116] The inflammatory factors in the rat brain tissue were detected by ELISA method. The specific results are as Figure 9 shown. It can be seen from the figure that after treatment with nasal and oral inhalation of fingolimod, the expressions of pro-inflammatory cytokines TNF-α and IL-6 decreased, while the expressions of anti-inflammatory cytokines Agr-1 and IL-10 increased. This indicates that after treatment with nasal and oral inhalation of fingolimod, neutrophil recruitment, oxidative stress, neuronal loss and apoptosis were reduced, thereby reducing the brain parenchymal inflammation in the stroke area of rats and promoting the recovery of the damaged nerve function of rats.

[0117] Based on the above results, it can be seen that in this invention, a rat stroke model was first established by surgery, and the rats were treated by nasal and oral inhalation of the same dose of edaravone, butylphthalide or fingolimod. It was found that the curative effects of clinically commonly used edaravone and butylphthalide were not as good as those of nasal and oral inhalation of fingolimod preparations. At the same time, the effects of different administration methods of fingolimod on its curative effect were also compared. It was found that compared with intraperitoneal injection and tail vein administration, the therapeutic effect of nasal and oral inhalation of fingolimod was the most significant. This fully shows that fingolimod can be prepared into a nasal and oral inhalation preparation for the treatment of ischemic stroke.

[0118] Further research found that intranasal inhalation of fingolimod can enhance the accumulation of the drug in the lungs, inhibit the migration of lung immune cells to the brain, thereby reducing the inflammation at the stroke site in the brain parenchyma, improving the nervous system damage at the stroke site, and promoting the recovery of damaged nerve function.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of fingolimod in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke.

2. Use of fingolimod according to claim 1 in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, characterized in that: The oral and nasal inhalation preparation comprises fingolimod or a pharmaceutically acceptable salt thereof.

3. Use of fingolimod according to claim 1 or 2 in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, characterized in that: The oral and nasal inhalation preparation is a preparation for reducing the infarct area of ischemic stroke.

4. Use of fingolimod according to claim 1 or 2 in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, characterized in that: The oral and nasal inhalation preparation is a preparation for improving nerve function.

5. Use of fingolimod according to claim 4 in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, characterized in that: The preparation for improving nerve function is a preparation for reducing inflammation at the stroke site in the brain parenchyma, improving nervous system damage at the stroke site, and promoting the recovery of damaged nerve function.

6. Use of fingolimod according to claim 1 in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, characterized in that: The oral and nasal inhalation preparation is a spray or powder aerosol.

7. Use of fingolimod according to claim 1 in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, characterized in that: The oral and nasal inhalation preparation is in a liquid dosage form.

8. Use of fingolimod according to claim 7 in the preparation of an oral and nasal inhalation preparation for treating ischemic stroke, characterized in that: The oral and nasal inhalation preparation comprises an isotonicity regulator.

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