Treatment of stroke

Abstract

Disclosed is a method for treating ischemic stroke, comprising inhibiting the migration of lung immune cells to the brain. The mode of administration of the method is inhalation or lung-targeted administration.

Description

Treatment of stroke TECHNICAL FIELD

[0001] This application claims priority to the Chinese patent application No. 202410821154.0, filed on June 24, 2024, with the State Intellectual Property Office of China, the content of which is incorporated herein by reference in its entirety. BACKGROUND

[0002] Stroke is a serious cerebrovascular disease that causes permanent disability and death, which is mainly caused by the sudden rupture of the blood vessels in the brain or the blockage of the blood vessels, resulting in damage to the brain tissue. Stroke 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 nerve function loss. The typical characteristics are abnormal activation of microglial cells, production of inflammatory cytokines, and infiltration of immune cells, which collectively exacerbate ischemic brain injury and seriously affect the prognosis of patients.

[0003] The prevention and treatment of cerebral ischemia-reperfusion injury is a key point and difficulty in the clinical treatment of ischemic stroke. Currently, one of the molecules under research for ischemic stroke is a neuroprotective agent: used to prevent and treat sequelae of ischemic stroke and reduce mortality. Some experiments have shown that some drugs have certain neuroprotective effects, but the clinical effect needs further study.

[0004] S1PR (sphingosinol-1-phosphate receptor) is a series of G protein-coupled receptors, widely distributed in the central nervous system, one of which is S1PR1. The marketed S1PR1 modulators include fingolimod (FTY720), siponimod, and ozanimod, among which fingolimod is approved for the treatment of multiple sclerosis and is an oral immunosuppressant. SUMMARY

[0005] The first object of the present application is to treat ischemic stroke with S1PR modulators, which provides an inhalation or lung-targeted preparation thereof, providing an administration scheme for extending the treatment time window for ischemic stroke. The S1PR1 modulator can be selected from fingolimod, siponimod, and ozanimod.

[0006] The present inventors have found that FTY720 can reduce mortality after cerebral ischemia through repeated research and experiments. Some embodiments of the present application construct a rat ischemic stroke model (a cerebral ischemia-reperfusion model (MCAO model)), and find that the efficacy of edaravone and butylphthalide is not as good as that of fingolimod by inhaling the same dose of edaravone, butylphthalide or fingolimod. The effects of different administration methods of fingolimod are also compared, and it is found that the treatment effect of fingolimod inhaler is the most significant compared with intraperitoneal injection and tail vein administration. The preparation of fingolimod into an inhaler not only enhances the accumulation of the drug in the lungs, but also greatly improves the efficacy, effectively inhibits the infiltration of immune cells in the brain, and has high bioavailability, low adverse reactions, low trauma and other advantages.

[0007] Some embodiments of the present application find that the use of fingolimod inhalation for the treatment of ischemic stroke can effectively enhance the accumulation of fingolimod in the lungs, inhibit the migration of immune cells in the lungs to the brain, reduce inflammation in the parenchymal stroke site, improve the nervous system damage in the stroke site, and promote the recovery of damaged nerve function. The use of oral and nasal inhalation of fingolimod for the treatment of ischemic stroke has the advantages of significant effect, low toxicity, good stability, high safety, and has outstanding popularization prospects for the development of new immune cell inhibitors for the treatment of brain diseases.

[0008] Some embodiments of the present application find that FTY720 inhaler targets immune cells (white blood cells), such as white blood cells in the lungs, delivers the drug to the lungs, and can inhibit the migration of white blood cells in the lungs to the brain, providing a new solution for extending the treatment time window of ischemic stroke.

[0009] Further, acute ischemic stroke (AIS) refers to the early stage of ischemic stroke, and the key to AIS treatment is super-early thrombolytic therapy, which can help restore blood supply to the brain of patients, save nerve function, and thus reduce mortality and improve prognosis. However, the effective treatment time window for thrombolysis is strictly limited to 3-4.5 h, and whether the patient can be treated in hospital within this golden time is the key to successful thrombolysis. Some embodiments of the present application find that inhalation administration is convenient and fast, and has better patient compliance, and can be self-administered, thereby extending the treatment time window for acute ischemic stroke patients to hospital treatment.

[0010] Some embodiments of the present application provide a fingolimod preparation and a method for treating ischemic stroke, and the fingolimod is targeted for pulmonary administration or is an inhaler, such as oral, nasal or pulmonary administration, which can be atomized for administration.

[0011] As an example, the preparation includes an effective amount of fingolimod or a pharmaceutically acceptable salt thereof, such as a hydrochloride salt.

[0012] As an example, the formulation can reduce infarct size in ischemic stroke, reduce inflammation at the site of the stroke in the brain parenchyma, and / or improve stroke-related neurological function, including sensory and / or motor dysfunction, as further examples, improving neurological function includes improving neurological damage at the site of the stroke and promoting recovery of impaired neurological function.

[0013] As an example, the stroke is acute ischemic stroke.

[0014] As an example, the formulation is an oral inhalation formulation, a nasal inhalation formulation, or an oral-nasal inhalation formulation, which means that inhalation occurs through both the mouth and the nose.

[0015] As an example, the inhalation formulation is in the form of a powder or a liquid.

[0016] As further examples, the inhalation formulation is in the form of a spray, a powder mist, an aerosol, an inhalation liquid formulation, or a formulation that can be converted to a vapor.

[0017] As further examples, the inhalation formulation is in the form of a liquid.

[0018] As further examples, the inhalation formulation includes an isotonicity adjusting agent, such as NaCl.

[0019] As an example, the formulation further includes a pharmaceutically acceptable carrier.

[0020] A second object of the present application is to provide a new method for treating ischemic stroke, which includes inhibiting the migration of immune cells in the lung to the brain, and the drug can target the lung or be in the form of an inhalation. The immune cells can be leukocytes, such as neutrophils. The drug can be an immunosuppressant, such as fingolimod, and the route of administration, the form of administration, the form of the formulation, and the efficacy of the drug can be as described above for the first object.

[0021] Some embodiments of the present application find that the immune cells infiltrating the brain in patients with ischemic stroke are mainly derived from the lung tissue, and are mainly leukocytes, especially neutrophils. The immune cells in the lung are involved in the process of inflammation and tissue repair. After administration of an immunosuppressant, such as fingolimod, the infiltration of the immune cells in the lung to the site of the stroke in the brain parenchyma is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the embodiments or mechanisms of the present application, the drawings required to be used will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments or mechanisms of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0023] Fig. 1 is a black and white image (white area is cerebral infarction area) converted from the TTC staining results of different groups in Example 1 of the present application;

[0024] Fig. 2 is a body weight change curve of MCAO rats in Example 2 of the present application;

[0025] Fig. 3 is the neurological function score results of MCAO rats in Example 2 of the present application (A is the Longa score, B is the water maze, and C is the sticker test);

[0026] Fig. 4 is a semi-quantitative analysis of TTC staining images of brain sections of MCAO rats in different groups in Example 2 of the present application;

[0027] Fig. 5 is the expression level of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), arginase-1 (Arg-1), and interleukin-10 (IL-10) in the stroke site after different treatments;

[0028] Fig. 6 is a semi-quantitative analysis of CD45 positive cells (i.e. immune cells) in brain tissue flow cytometry results at different periods after ischemic stroke;

[0029] Fig. 7 is a semi-quantitative analysis of CD45 positive cells in lung tissue flow cytometry results at different periods after ischemic stroke;

[0030] Fig. 8 is a pie chart of the proportion of cell populations in lung tissue of rats in different treatment groups analyzed by single cell sequencing.

[0031] The grouping identification of each figure follows a unified principle. The Sham group is the sham operation group, FTY720 represents the fingolimod treatment group, and the significance identification and its meaning are as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and **** represents P < 0.0001. DETAILED DESCRIPTION

[0032] The present application will be further described in conjunction with the specific embodiments, but the protection scope of the present application is not limited to this; various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the spirit of the present application, all fall within the protection scope determined by the claims of the present application. The following experimental methods are conventional methods unless otherwise specified; the following experimental materials used are conventional materials or can be purchased from biochemical reagent manufacturers unless otherwise specified:

[0033] The cerebral ischemia-reperfusion model (MCAO model) used in the following experiments is a conventional ischemic stroke model in the art. The purchased SD rats are 6-8 weeks old male, and the body weight is controlled between 230-260 g after feeding. The rats are fasted overnight before modeling, and normal water is provided. After anesthesia, the operation is performed to establish the model, and then reperfusion is performed to cause injury.

[0034] For example, TTC staining is also a conventional method for evaluating infarct volume, and in the embodiments of the present application, it specifically includes:

[0035] 1) Brain removal: the rats are sacrificed after anesthesia, and the whole brain tissue is removed;

[0036] 2) After washing the brain tissue in physiological saline, it is frozen;

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

[0038] 4) The brain slices are completely immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution (prepared with PBS), and incubated to allow the brain slices to be fully stained;

[0039] 5) The brain slices are removed, photographed, and analyzed as shown in the accompanying drawings.

[0040] I. Examples:

[0041] Example 1: Therapeutic effect of different drug administration groups on ischemic stroke model

[0042] 1. Experimental drugs and preparation

[0043] Take 20 mg of fingolimod hydrochloride, edaravone, and butylphthalide respectively, and dissolve them in 2 mL of normal saline. Ultrasonic for 10 min to form an aqueous solution of each drug. Part of it is for injection, and the other part is put into the atomization drug administration instrument to form atomized droplets.

[0044] 2. Experiment

[0045] Establish the MCAO model, and group the experimental animals for drug administration. TTC staining is performed 24 h after drug administration to evaluate the therapeutic effect of different drug administration groups. The specific grouping and drug administration doses are shown in Table 1.

[0046] Table 1: Drug administration grouping of experimental animals

[0047] Group No. Group Animal number Test substance dose (mg / kg) 1 Sham group 5 Normal saline 2 Saline group 5 Normal saline 3 Atomization inhalation group 5 Butylphthalide 4 Atomization inhalation group 5 Edaravone 5 Atomization inhalation group 5 Fingolimod 6 Oral administration group 5 Edaravone 7 Intraperitoneal injection group 5 Fingolimod 8 Tail vein group 5 Edaravone 9 Tail vein group 5 Fingolimod

[0048] Note: The control group is set in accordance with the practice in the art. The difference between the sham group and the saline group is that the blood vessels of the rats in the sham group are not embolized by the MCAO operation. The rats in the other groups are MCAO model rats (with cerebral ischemia-reperfusion injury) except for the sham group.

[0049] Evaluation of infarct volume by TTC staining: The TTC staining method is as previously described. The TTC staining results are recorded according to the group number as shown in FIG. 1. The results show that:

[0050] The therapeutic effects of inhaled edaravone and inhaled fengolmod are not as good as inhaled fengolmod. Meanwhile, the results of intraperitoneal injection and tail vein administration are compared, and it is shown that different administration routes of fengolmod can reduce the infarct area of ischemic stroke to some extent, but the therapeutic effect of inhaled fengolmod is the most significant.

[0051] Example 2: More pharmacodynamic effect research of the oral-nasal inhalation fengolmod preparation on ischemic stroke model

[0052] The same MCAO model is established according to the same principles as in Example 1. The preparation of the fengolmod aerosol is also the same as in Example 1. After aerosol administration, the rats are investigated. The specific grouping and administration dose are shown in Table 2.

[0053] Table 2: Administration grouping of experimental animals

[0054] Group number Group Animal number Test substance dose (mg / kg) 1 Sham group 5 physiological saline 12 Saline group 5 physiological saline 13 aerosol inhalation group 5 fengolmod

[0055] Investigation method and results:

[0056] (1) The change in rat body weight is investigated as shown in FIG. 2. The body weight of the rats after fengolmod treatment gradually increases.

[0057] (2) The results of neurological function and behavior evaluation are shown in FIG. 3. The neurological function score of the rats after fengolmod treatment significantly decreases. The swimming path length and escape latency significantly decrease. The time for touching and removing the sticky note significantly shortens. This indicates that oral-nasal inhalation of fengolmod after stroke can promote the improvement of sensory motor function and improve the neurological function of rats. The specific investigation method is as follows:

[0058] 1) Neurological function score

[0059] The neurological function of rats is scored according to the Longa scoring standard. The specific scoring criteria are as follows:

[0060] 0 points: The rat has no neurological injury and moves normally

[0061] 1 point: After holding the rat's tail, the contralateral forelimb of the rat cannot be fully stretched

[0062] 2 points: the rat turns to the side opposite to the operation when walking autonomously

[0063] 3 points: the rat falls to the side opposite to the operation when walking autonomously

[0064] 4 points: the rat cannot walk autonomously and loses consciousness

[0065] 5 points: death

[0066] After the end of administration, the tail of the rat is lifted, the stretching of the forelimb on the side opposite to the operation is observed, and then the rat is placed on the ground, and the turning behavior of the rat is observed, and the neurological function of the rat is scored according to the above scoring standard.

[0067] 2) Behavioral evaluation

[0068] a. The behavior of the rat is evaluated by the sticker test and the Morris water maze test. First, the rat is trained for the sticker test and the Morris water maze test 3 days before the MCAO model is constructed, 3 times a day, and the experiment is performed on the 3rd, 5th and 7th days after the MCAO model is constructed;

[0069] b. A 13x13mm 2 sticker is attached to the dorsal side of the rat's paw as a tactile stimulus, and then the rat is returned to the cage, and the time for the rat to touch and remove the sticker is recorded;

[0070] c. The memory function of the rat is evaluated by the Morris water maze. The rat is placed in a water tank with a diameter of 210 cm, an escape platform area of 12 cm 2 , and a water temperature of 20℃, and the swimming trajectory, search time for the hidden platform and swimming distance of the rat are recorded.

[0071] (3) TTC staining to evaluate the infarct volume

[0072] TTC staining is performed as in Example 1. The infarct area and total area of each photo are measured, and the data are statistically analyzed. As shown in Figure 4, the infarct area of the MCAO rat is significantly reduced after treatment with fingolimod.

[0073] (4) Histopathological changes in rat brain tissue

[0074] After treatment, the rats are euthanized, and the rat brain tissue is fixed with 4% paraformaldehyde for 24 h, dehydrated, paraffin-embedded, sectioned, and deparaffinized. The tissue sections are treated with hematoxylin-eosin (HE) staining and Nissl staining to observe the cell morphology and analyze the pathological changes. The results show that fingolimod has a significant neuroprotective effect after ischemic stroke.

[0075] (5) Rat brain tissue apoptosis and neuron function recovery detection

[0076] After the end of treatment, the rats were euthanized, and the rat brain tissue was fixed with 4% paraformaldehyde for 24 h, dehydrated, paraffin-embedded, sectioned, and deparaffinized. The TUNEL method and NeuN antibody incubation method were used to detect cell apoptosis and neuron function recovery in each group. The results showed that the NeuN positive cells in the fingolimod treatment group increased most significantly, and the TUNEL cell apoptosis decreased, thereby inhibiting the MCAO-induced loss and apoptosis of neurons.

[0077] In addition, immunofluorescence staining was used to detect the content of reactive oxygen species (ROS) at the stroke site. The results showed that compared with the saline group, the content of ROS at the stroke site was significantly reduced after fingolimod treatment.

[0078] (6) Rat brain tissue inflammatory factor detection

[0079] The brain tissue was collected, and the injured side brain tissue was homogenized in PBS and centrifuged to collect the supernatant. Then, the rat brain tissue inflammatory factors were detected by ELISA. The specific results are shown in Figure 5. As can be seen from the figure, after fingolimod treatment, the expression of pro-inflammatory cytokines TNF-a and IL-6 decreased, while the expression of anti-inflammatory cytokines Agr-1 and IL-10 increased. This indicates that after fingolimod treatment, oxidative stress, neuron loss and apoptosis are reduced, thereby reducing the inflammation of the brain parenchyma at the stroke site in rats and promoting the recovery of the damaged nerve function in rats.

[0080] II. Confirmation of the mechanism of treating ischemic stroke by inhibiting the migration of lung immune cells to the brain

[0081] 1. The brain-infiltrating immune cells mainly come from the lung, and fingolimod inhibits the migration of lung immune cells to the brain

[0082] (1) The same MCAO model was established following the principles consistent with Example 1. Single cell suspensions were prepared by collecting brain tissues at different times, and flow cytometry and immunofluorescence experiments were used to investigate the proportion of immune cells in brain tissues at different times. The results showed that, as shown in Figure 6, after the occurrence of stroke, immune cells gradually infiltrated into the brain tissue, and the degree of infiltration showed a continuous upward trend with the passage of time, reaching a peak at 48 hours, and then decreasing.

[0083] To trace the source of immune cells in the brain after ischemic stroke, the same MCAO model was established, and blood, liver, spleen, kidney and lung tissues at different time periods were collected to prepare single cell suspensions, and the proportion of immune cells in each tissue at different time periods was investigated. The results showed that after ischemic stroke, the proportion of immune cells in the tissue changed most significantly in the lung, and the proportion of immune cells in the lung showed a sharp increase and decrease, as shown in Figure 7: Within 0.5 to 3 hours after ischemic stroke, the proportion of immune cells increased rapidly, then maintained at a high level at 6 hours, and then gradually decreased.

[0084] (2) The lung immune cells of rats were specifically labeled by oropharyngeal method, and the MCAO model was established. The drug administration groups were the same as in Example 2. The distribution characteristics of lung-derived immune cells in the brain tissue were tracked 24 hours after administration combined with immunofluorescence experiments. The results showed that compared with the Sham group, the FITC green fluorescence signal in the lung tissue of the MCAO group was significantly weakened, and the FITC green fluorescence signal in the brain tissue was significantly enhanced, indicating that lung immune cells can migrate to the brain in large quantities after ischemic stroke; and compared with the MCAO group, the FITC green fluorescence signal in the brain tissue was significantly weakened after FTY720 treatment, indicating that FTY720 can inhibit the migration of lung immune cells to the brain.

[0085] 2. Subgroup analysis of immune cells migrating from the lung to the brain

[0086] Using single cell sequencing technology, the lung tissue was analyzed at high resolution to further reveal the dynamic changes of lung immune cells after ischemic stroke. The specific results are shown in Figure 8: After ischemic stroke, the proportion of neutrophils increased significantly from 10.24% in the Sham group to 30%, while the proportion of T cells, B cells, monocytes and NK cells changed relatively little. As an important part of the innate immune system, neutrophils recruit in large numbers after ischemic stroke, indicating that they play a key role in brain inflammation.

Claims

1. The use of an inhibitor that inhibits the migration of pulmonary immune cells to the brain in the preparation of a medicament for treating ischemic stroke, the medicament being targeted to the lungs or being an inhaler, wherein the inhibitor is optionally an S1PR modulator, preferably an S1PR1 modulator, such as fingolimod, sinimod, ozamod or a pharmaceutically acceptable salt thereof. Alternatively, the immune cells may be white blood cells, such as neutrophils.

2. Use of fingolimod or a pharmaceutically acceptable salt thereof in the preparation of inhaled medications for the treatment of ischemic stroke or medications targeting the lungs.

3. The application as described in claim 1 or 2, wherein, The drug can reduce mortality after cerebral ischemia, reduce the infarct area of ​​stroke, alleviate inflammation at the stroke site in the brain parenchyma, and / or improve stroke-related neurological function.

4. The application as described in the preceding claim, wherein, The improvement of stroke-related neurological function includes improving neurological damage at the stroke site and / or promoting the recovery of damaged nerve function.

5. The application as described in any of the prior claims, wherein, The drug is an oral inhalation preparation, a nasal inhalation preparation, or an oral-nasal inhalation preparation.

6. The application as described in any of the prior claims, wherein, The stroke mentioned is an acute ischemic stroke.

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

8. 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.

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

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

11. The application as described in any of the prior claims, wherein, The pharmaceutically acceptable salt is a hydrochloride salt.

12. The application as described in any of the prior claims, wherein, The drug contains a pharmaceutically usable carrier.

Images