WO2009155777A1 - The use and method of the compound of fasudil and the pharmaceutical composition thereof - Google Patents

Abstract

A new use of fasudil in inducing the regeneration of adult brain neural stem cells and /or protecting the neurological functions, and in preventing and/or treating diseases related to damages and/or death of neuron. The use of fasudil in the present invention includes the use in manufacturing medicants for inducing the regeneration of adult brain neural stem cells and /or protecting the neurological functions. Moreover, the use of fasudil in manufacturing medicants for preventing and/or treating diseases related to damages and/or death of neuron.

Description

 Use of fasudil compound, method and pharmaceutical composition thereof

 The invention belongs to the field of medical technology. The present invention relates to a novel use of fasudil, in particular, the present invention relates to the use of fasudil for the preparation of a medicament for inducing regeneration of adult cranial nerve cells and/or for protecting nerve function, and corresponding neurological diseases Possible application prospects; use of fasudil for the preparation of a medicament for preventing and/or treating diseases associated with neuronal damage and/or death; a method of inducing regeneration of adult brain neural stem cells and/or protecting nerve function; A pharmaceutical composition for inducing regeneration of adult cranial nerve cells and/or protecting nerve function. Background technique

Fasudil is an isoquinoline sulfonamide derivative whose chemical name is hexahydro-1-(5-isoquinolinesulfonyl)-1H-1, 4-diazepine [hexahydro-1- ( 5-isoquino lylsulfonyl ) -1H-1 , 4-diazepime] or 1-( 5-isoquinolinesulfonyl) homopiperazine, having a molecular formula of C ₁₄ H ₁₇ N ₃ O2S , The structure is as follows:

Common salts of fasudil are hydrochlorides, nitrates, sulfates, sulfonium sulfonates, the most commonly used of which is fasudil hydrochloride.

In 2002, fasudil was marketed in China, and its indications were the prevention and treatment of ischemic cerebrovascular disease caused by cerebral vasospasm after subarachnoid hemorrhage. Subarachnoid hemorrhage refers to the blood flow into the subarachnoid space after rupture of blood vessels in the brain. It is a type of hemorrhagic cerebrovascular disease, which is divided into primary and secondary. The main symptoms are headache. Vomiting and neck stiffness. Cerebral vasospasm refers to the abnormal contraction of the artery over a period of time. It is common in patients with subarachnoid hemorrhage of intracranial aneurysm rupture, which is the main cause of disability and death of subarachnoid hemorrhage. There have been many clinical reports confirming that fasudil hydrochloride is caused by subarachnoid hemorrhage The effectiveness and safety of cerebral vasospasm. In vivo experiments on the pharmacokinetics and metabolically active products of fasudil hydrochloride showed that fasudil hydrochloride enters the body and quickly transforms into a metabolite with the same activity, hydroxyfasudil. The concentration can reach 86% of the parent drug and has a long half-life (fasudil hydrochloride half-life 0.78 hours, hydroxyfasudil half-life 4.66 hours). Hydroxyfasudil has a 12-hour peak-to-valley value of 0.077 M and has the same biological activity as fasudil hydrochloride, and inhibits Rho kinase in an effective concentration range (0.025 _ 1.6 M). Therefore, fasudil hydrochloride is a new and highly effective vasodilator (a protein kinase inhibitor, an intracellular calcium antagonist) that can effectively relieve cerebral vasospasm and improve subarachnoid hemorrhage. Caused by cerebral vasospasm.

 Neurological diseases such as cerebral ischemia and chronic inflammatory neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and retinal diseases cause neuronal damage and death during their development and progression. For this group of diseases, people have been actively seeking effective treatments. Over the past 20 years, a large number of neuroprotective agents have been studied and observed in an attempt to prevent and treat the development and progression of these neurological diseases, including calcium channel antagonists, free radical scavengers, glutamate antagonists, cell membrane stabilizers, and Neurotrophic factors, etc., but because of various factors after brain injury, they are involved in the damage and death of neurons at the same time or in succession. It is difficult to obtain satisfactory therapeutic effects on drugs that act on a single target. The clinical therapeutic effect of the above neuroprotective agents is not ideal.

The problem of regeneration and repair after nervous system injury has always been the focus of neuroscience. With the deepening of research on neural cells, attempts to repair damaged neurons by using neural stem cells have become one of the hotspots in the field of neuroscience in recent years. Neural stem cells play an important role in repairing damaged nerve tissue and are an effective way to repair and replace damaged tissue, and can reconstruct part of the loop and function. At present, the most widely studied grafts are mainly embryonic stem cells and neural stem cells, but the practicality of these cells is greatly limited due to limited sources, ethical disorders and immune rejection. Recent studies have shown that bone marrow mesenchymal stem cells have the ability to differentiate into neurons and glial cells. However, bone marrow-derived cells, such as bone marrow mesenchymal stem cells, are at higher risk of viral infection, and their cell number, proliferation, and differentiation capacity are also significantly decreased as the age of the individual is increased, making it difficult to meet clinical needs. In 1992, Reynolds et al. first isolated neural cells from the striatum of adult rats, thus breaking through the view that adult tissues are not regenerable. This experiment fully proves that the newborn neurons produced by adult neural cells are not only morphologically identical to mature neurons, but also have action potential responses to synaptic stimuli, and can be effectively integrated into the neural circuit. The cells laid the foundation and pointed us in a direction for the use of adult neural stem cells for brain repair treatment. However, to date, the research field has yet to find a drug that can effectively act on adult neural cells in the body, induce its regeneration, and repair damaged nerve cells to treat diseases of the nervous system. Therefore, the search for a relatively stable substance that can freely pass through the blood-brain barrier and induce the regeneration of endogenous adult brain neural stem cells has been the direction that people are striving for. Summary of the invention

 The inventors have unexpectedly studied for a long time and have unexpectedly found that fasudil has a neuroprotective function and a function capable of promoting adult brain neuronal cell proliferation or differentiation and inducing regeneration of adult brain neural stem cells, thereby completing the present invention.

 Accordingly, it is an object of the present invention to provide a novel use of fasudil for inducing re-proliferation of adult brain neural stem cells and/or protecting nerve function.

 Another object of the present invention is to provide a novel use of fasudil for preventing and/or treating diseases associated with neuronal damage and/or death.

 Another object of the present invention is to provide a method of inducing regeneration and/or protecting nerve function of adult brain neural stem cells.

 Another object of the present invention is to provide a pharmaceutical composition for inducing regeneration of adult cranial nerve cells and/or protecting nerve function.

 The object of the present invention is achieved by the following technical solutions.

 In one aspect, the invention provides the use of fasudil for the manufacture of a medicament for inducing regeneration and/or protecting neurological function of adult brain neural stem cells.

 Preferably, wherein the adult brain neuron is an endogenous neural cell.

 In another aspect, the invention provides the use of fasudil for the preparation of a prophylactic medicament for the prevention and/or treatment of diseases associated with neuronal damage and/or death.

 Preferably, the disease associated with neuronal damage and/or death is a neurological disease, mainly including cerebral ischemia (e.g., cerebral embolism and cerebral infarction) and chronic inflammatory neurodegenerative disease.

 Preferably, the chronic inflammatory neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, atrophic lateral sclerosis, Huntington's disease, multiple sclerosis.

 Preferably, the aforementioned fasudil comprises a pharmaceutically acceptable salt such as a pharmaceutically acceptable salt crystal or hydrate, wherein the pharmaceutically acceptable salt is preferably selected from the group consisting of a hydrochloride, a nitrate, a sulfate, and a phosphate. And hydrobromide, sulfonate and citrate, more preferably selected from the hydrochloride.

Preferably, the aforementioned fasudil effective amount is 0.5-8 mg/kg, preferably l-6 mg/kg, more preferably Choose 3-5mg/kg, most preferably 5mg/kg

 In still another aspect, the present invention provides a method of inducing regeneration and/or protection of neural function of adult brain neural stem cells, comprising administering fasudil to a therapeutically effective amount of fasudil or a pharmaceutical composition comprising fasudil .

 In a further aspect, the invention provides a method of preventing and/or treating a disease associated with neuronal damage and/or death comprising administering a therapeutically effective amount of fasudil or a pharmaceutical composition comprising fasudil.

 Preferably, the aforementioned fasudil or a pharmaceutical composition comprising fasudil is a therapeutically effective amount of 0.5-8 mg/kg, preferably an effective amount of 1-6 mg/kg, more preferably an effective amount of 3-5 mg/kg. The most preferred effective amount is 5 mg/kg.

 In still another aspect, the present invention provides a pharmaceutical composition for inducing regeneration and/or protection of nerve function of adult brain neural stem cells, the composition comprising a fasudil compound and a pharmaceutically acceptable carrier; The dosage form of the pharmaceutical composition is selected from the group consisting of a tablet, a capsule, an oral solution, an injection, and a powder injection, preferably an injection, more preferably an intravenous injection.

 In still another aspect, the present invention provides a pharmaceutical composition for preventing and/or treating a disease associated with neuronal damage and/or death, the composition comprising a fasudil compound and a pharmaceutically acceptable The carrier; the dosage form of the pharmaceutical composition is selected from the group consisting of: a tablet, a capsule, an oral solution, an injection, and a powder injection, preferably an injection, more preferably an intravenous injection.

 Preferably, the aforementioned fasudil or a pharmaceutical composition comprising fasudil comprises a tablet, a capsule, an oral solution, an injection or a powder injection, and the like, which is suitable for administration intestine or parenteral administration. The pharmaceutical form, preferably an injection, is more preferably an intravenous injection.

 Preferably, the aforementioned route of administration of fasudil or a pharmaceutical composition comprising fasudil includes oral, intravenous, intramuscular, intraperitoneal and subcutaneous injection, and any administration route known in the art, preferably abdominal cavity injection.

 In summary, the use of fasudil provided by the present invention includes the following aspects: protecting nerve function, inducing regeneration of adult brain nerve cells (ie, inducing endogenous neural cells in adult brain), treatment and neurons Use of a disease associated with injury and death (ie, treatment of a nervous system disease), use in the preparation of a medicament for inducing regeneration of adult brain neuronal cells, use in the preparation of a medicament for use as a neuroprotective agent, preparation for the treatment of the nervous system Use in medicines for diseases.

Further, the present invention provides a pharmaceutical composition for treating a nervous system disease comprising a therapeutically effective amount of fasudil or fasudil hydrochloride. The fasudil of the present invention includes physicochemicals such as hydrochloride, nitrate, sulfate, phosphate, hydrobromide, sulfonate, citrate and the like. Acceptable salt forms, which may be in any form, including crystalline forms (including water with and without water of crystallization), hydrated forms, and the like, preferably fasudil hydrochloride. These salts and the different forms can be prepared according to methods well known in the art.

 An effective amount of the present invention is an amount of fasudil that may produce a medically recognized effect in neuroprotection or treatment of a nervous system disorder. The present inventors have found through repeated studies that it is suitable for inducing adult brain neuronal cell regeneration, that is, an effective amount of fasudil suitable for treating nervous system diseases is 0.5-8 mg/kg, preferably l-6 mg/kg, more Preferably, 3-5 mg/kg, most preferably 5 mg/kg „ for each of the above fasudil salts and a pharmaceutical composition comprising fasudil, corresponding to an amount effective to provide the above-mentioned fasudil The amount of fasudil salt is the effective amount of the corresponding salt.

 The fasudil of the present invention or a pharmaceutical composition comprising an effective amount of fasudil can be formulated into any dosage form suitable for administration according to conventional techniques known in the art, including but not limited to intestinal administration. A pharmaceutical preparation or a parenteral preparation such as a tablet, a capsule, an oral solution, an injection, a powder injection or the like is preferably an injection, more preferably an intravenous injection. The route of administration of the formulations of the invention may be any route of administration known in the art, preferably by intraperitoneal injection. The preparation of the present invention can be administered three times a day, each dose being an effective amount of fasudil which can treat the disease, i.e., an effective amount as described above.

 The diseases associated with neuronal damage and death according to the present invention, i.e., the diseases of the nervous system include, but are not limited to, cerebral ischemia, brain damage, Alzheimer's disease, Parkinson's disease, multiple sclerosis and the like. BRIEF DESCRIPTION OF THE DRAWINGS

 Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

 Figure 1 is a graph showing the results of double-stained fluorescence microscopy of MAP-2 and HOCHEST in primary cultured hippocampal neurons in vitro, in which red is MAP-2 neurons and blue is HOCHEST nuclear staining.

 Fig. 2 is a graph showing the results of fluorescence microscopy of Brdu-positive cells after primary neuron culture in vitro and PBS control, wherein A is a fasudil group and B is a PBS control group.

 Figure 3 is a graph showing the results of neuronal staining in different parts of the hippocampus after reoxygenation for 72 hours in normal oxygen and cerebral hypoxia (8% oxygen) for 6 hours, in which CA1, CA2 and CA3 represent hippocampal CA1, CA2 and CA3, respectively. Area.

Figure 4 shows the brain in mice with hypoxia (8 % oxygen) at different times (O h, 2 h, 6 h, 8 h) After tissue homogenization, the expression of Rockll protein was determined by immunoblotting.

 Figure 5 is a graph showing the expression of ROCK II protein in brain tissue sections of mice with hypoxia (8 % oxygen) at different times (O h, 2 h, 6 h, 8 h). Arrows are expressed in green. Fluorescent ROCK II.

 Figure 6 shows the injection of normal saline (A, C and E) and fasudil (B, D and F) after 6 hours of hypoxia (8 % oxygen) in the brain of mice, and the subventricular zone ( SVZ ) after 24 hours. Images of Brdu positive cells in the posterior horn (A and B), anterior horn (C and D) and hippocampal DG (E and F).

 Figure 7 is a graph showing the results of fluorescence microscopy of DCX and Brdu double positive cells after injection of fasudil for 6 hours after hypoxia (8 % oxygen) in mice, and A) red after 24 hours of subventricular zone (SVZ) For Brdu-positive cells, B) green for DCX-positive cells, C) for overlapping counterstained cells of two colors (proliferating neural thousand cells).

 Figure 8 is a bar graph showing that fasudil inhibits neuronal death after hypoxia, resulting in a decrease in LDH release and a decrease in PI positive cells in culture supernatant, where A is the LDH assay and B is the PI assay.

 Figure 9 is a bar graph showing the concentration levels of G-CSF and VEGF in mouse brain homogenate supernatant, where A is the concentration of G-CSF and B is the concentration of VEGF.

 Figure 10 is a graph showing the results of immunostaining of astrocyte markers GFAP and G-CSF, wherein A is a saline control group and B is a fasudil group.

 Figure 11 is a graph showing the results of detecting a cholinergic neuron by a polyclonal ChAT antibody, wherein a small blue square in the left brain map indicates the position of the brain region shown on the right side, A is a saline control group, and B is a Fashu Dier group. The best way to implement the invention

 The present invention will be further described in detail with reference to the accompanying claims, the accompanying claims

 It should be understood that, for a method, a step, a device, an instrument, a material, etc., which are mentioned in the present invention but are not described in detail, a person skilled in the art may employ a corresponding method, step, device, instrument, material, etc., which is well known in the art, or according to the field. Regular knowledge and technology gained. Example 1 : Fasudil promotes brain neural stem cell proliferation in vitro

 1. Test cells and preparation thereof

Pregnancy 17 - 18 days Kunming (KM) pregnant rats (purchased from Shanghai Slack Laboratory Animals Limited Liability Company) killed by cervical dislocation, 75% alcohol disinfected skin. The fetal rats were peeled off under aseptic conditions and placed in ice-cold DMEM medium (purchased from GIBCO, USA). The fetal rat brain tissue was carefully separated, the meninges and blood vessels were removed, the cerebellum, brain stem and basal ganglia were removed, and the hippocampus tissues were taken out and gently pipetted in water-pre-cooled DMEM to form a single cell suspension. The cell suspension was collected and centrifuged at 1000 rpm for 5 minutes at 4 °C. The supernatant was discarded and resuspended with freshly prepared neuronal culture (purchased from GIBCO, USA). Count the cells and calculate the density of the cells in the cell suspension. The cells were cultured on a polylysine coated slide at ² x ^{10 5} /cm ² , cultured at 37 ° C in a 5 % C0 ₂ incubator, and the original volume 1/2 neuron culture was replaced after sputum. Neuron-specific labeling was performed 14 days later: microtubule-associated protein-2 (MAP-2, purchased from Chemicon, USA) was stained and identified, and fluorescent nuclei HOCHEST33342 was purchased from Sigma, USA to label the nuclei. The fluorescence microscope (Olympus BX-60F, purchased from Olympus, Japan) was observed, and the results are shown in Fig. 1. Among them, MAP-2 positive cells were red, suggesting neurons, and blue was HOCHEST staining nuclei.

 2. Hypoglycemia and hypoxia cell injury model

The identified neuronal cells were used to establish an oxygen-glucose deprivation (ODD) model (an in vitro model of human cerebral ischemia). The sugar-free DMEM culture solution was incubated with 5% CO ₂ , 10% H ₂ , and 85% N gas for 15 minutes, and the oxygen in the culture solution was removed to prepare a hypoglycemic and hypoxic neuron culture solution. The cultured neurons were cultured for about 10 days, the culture solution was discarded, and the cells were washed three times with PBS solution, and a non-sugar DMEM culture solution was added. The culture plate was placed in an anaerobic incubator, and 5% C0 ₂ , 10% H ₂ , 85% N gas was introduced into the tank, and the incubator was sealed at 37 ° C, and the oxygen concentration in the tank was lower than 1 %. After 6 hours of hypoxia, the culture plate was taken out, the sugar-free culture solution was aspirated, and the original neuron culture solution was separately added, and placed in an incubator at 37 ° C, 5% CO ₂ and 95% air for reoxygenation.

 3. Administration

 Fasudil injection (purchased from Tianjin Hongyu Pharmaceutical Co., Ltd.) was administered to the above-mentioned hypoglycemic and hypoxic neurons (the concentration of the parent drug was 30 mg/2 ml = 15 mg/ml). The experimental concentrations were set to low (l g/ml), medium (10 g/ml) and high (100 g/ml) concentrations. Three doses were administered on the day, day 2, and day 3, respectively, and cells and supernatants were collected on day 5 for further testing.

 4. Test the test

After the experimental model was determined, the same volume of PBS was added instead of fasudil injection, which served as a control for fasudil under the exact same experimental conditions. Cells cultured under normal oxygen conditions serve as a baseline for hypoxic cells. 5. Shen Yuan Yuan BrdU detection

 On the third day after the addition of fasudil, BrdU (purchased from Roche, Switzerland) (1 ug/ml) was added on days 3 and 4. On the 5th day, the cells were collected, and the cells were rinsed twice with PBS for 10 minutes, 4% polyacetal was fixed at room temperature for 20 minutes, and the cells were rinsed twice with l xPBS for 10 minutes each time, and 3% sheep serum was blocked at room temperature for 15 minutes. BrdU-anti (1:200, American Lab Version) was diluted in PBS containing 1 goat serum at 4 ° C overnight. The goat anti-mouse CY3 labeled secondary antibody (1:100, purchased from Sigma, USA) was diluted in PBS and protected from light for 1 hour at room temperature. l XPBS rinse the cells 3 times, 5 minutes each time, 50% glycerol seals, and observe under fluorescent microscope.

 6. Results and analysis

 The fluorescence microscopy results of Brdu-positive cells after in vitro primary neuron culture are shown in Figure 2, where A is the fasudil group and B is the PBS control group. As can be seen from the figure, compared with the PBS control group, the neurons added to the fasudil group formed more obvious neurospheres, and the Brdu-positive proliferating neurons were significantly more than the PBS control. The same scab was obtained after repeated repetitions, clearly indicating that fasudil can promote brain cell proliferation in vitro. Example 2: Fascidil promotes brain cell proliferation

 Test-risk animal

 KM mice (purchased from Shanghai Slack Laboratory Animals LLC). Animals arrive at the laboratory and are given clean grades, free diet and water, and rest for 5-7 days.

 2. Mouse hypoxia model

 Thirty-two KM mice were divided into 4 groups, 8 in each group, and hypoxia in the anoxic system (8% oxygen) for 2 hours, 4 hours, 6 hours, and 8 hours. An anoxic meter is placed in the hypoxia chamber to dynamically detect the oxygen concentration in the anoxic chamber to ensure controllability of the degree of hypoxia in the mouse. The chamber can hold 30 to 40 mice, and 32 mice can be placed in one time to ensure the compensability of hypoxic conditions. After 72 hours of reoxygenation, the physiological saline was perfused, and the brain fluid was stored at _80 °C after rapid freezing. After the brain tissue was cut by immunoblotting and immunohistochemistry, hippocampal neurons and ROCK II were stained.

 3. Administration

Fasudil injection, the concentration of the parent drug is 30mg/2ml = 15mg/ml. Based on the results of in vitro experiments, the in vivo concentration was set to intraperitoneal injection of fasudil hydrochloride (5 mg/kg) and Brdu (50 mg/kg) in mice after hypoxia for 6 hours, after 24 hours. The injection was repeated once; the control group was injected with the same volume of normal saline and Brdu. There were 8 rats in the fasudil group and 7 rats in the saline control group. After reoxygenation for 24 hours, the saline was perfused, and the brain fluid was frozen at -80 °C after freezing. After brain sections were sectioned for immunoblotting and immunohistochemistry, Brdu (purchased from Roche, Switzerland) and DCX (purchased from BD Bioscience, USA) were stained.

 4. Control experiment:

 At the same time as the experimental model, the same volume of physiological saline was injected instead of the fasudil injection, and it was used as a control for fasudil under the same experimental conditions. In addition, normal oxygenator 8 was set to be used as a control under exactly the same conditions.

 5. Immunostaining assay

 After separation by 10% SDS-PAGE discontinuous gel electrophoresis, the membrane was transferred to PVDF membrane for 15-30 minutes. The transferred nitrocellulose membrane was stained in Ponceau staining solution, and labeled Marker and band position. , Transfer buffer to wash to discolor. The closed nitrocellulose membrane was placed in a membrane blocking solution, shaken slowly on a shaker, and blocked at room temperature for 1 hour. Anti-ROCK II antibody (purchased from Bioscience, USA) was diluted with 5% calf serum (1:500) overnight at 4 °C. Alexa Fluor 488 anti-mouse secondary antibody (purchased from Invitrogen, USA) was diluted 1 : 1000 and incubated for 1 hour at room temperature. Color development by chemiluminescence, X-ray film development. The immunoblot map was taken by a digital camera and processed by Image-pro Plus 5.0 image analysis software with β-actin as an internal reference.

 6. Immunohistochemistry

 After the mouse was decapitated, the epidermis was cut along the midline and the skull was cut along the sagittal suture. The brain was removed, immersed in 20% sucrose and dehydrated at 4 °C, and then placed in 30% sucrose and dehydrated at 4 °C until the tissue sinks. . The sucrose solution on the surface of the brain tissue was sucked with filter paper, embedded in liquid nitrogen by OCT, and serially sliced in a coronal position at a thickness of 8 μm, and stored in the order of use. Immunofluorescence staining was blocked with 5% sheep serum for 10 minutes at room temperature, anti-ROCK II antibody, anti-Brdu antibody and anti-DCX antibody were incubated as a primary antibody at 4 ° C for 24 hours, Alexa Fluor 488 labeled anti-mouse antibody (purchased from the United States) Invitrogen and Rhodamine anti-guinea pig antibody (purchased from Chemicon, USA) were washed at room temperature for 1 hour, l xPBS for 3 times, and after 5 minutes, 50% glycerol was mounted, photographed under a fluorescence microscope.

 7. Results and analysis

 The results show that the established hypoxia model can cause loss of neurons in different regions of the hippocampus, especially in CA1 and CA2, as shown in Figure 3. The expression of Rho kinase subtype Rockll protein was increased after 6 hours of cerebral hypoxia by immunoblotting (see Figure 4) and immunohistochemistry (see Figure 5, where the arrows are all expressed green fluorescent ROCK II). Rho kinase inhibitors provide the basis.

Infusion of fasudil after hypoxia can significantly increase the position of the anterior and posterior horns of the SVZ region of the brain. Sexual cell regeneration, the results are shown in Figure 6, wherein A, C and E are the control saline group, B, D and F are the fasudil group, and A and B are the posterior angle of the lateral ventricle (SVZ). C and D are anterior horns, and E and F are hippocampal DG regions.

 Further studies have found that these Brdu-positive proliferating cells simultaneously express the neural stem cell marker DCX (see Figure 7, where A) red is Brdu-positive cells, B) green is DCX-positive cells, and C) two color overlapping counterstained cells, That is, hyperplastic neural cells), indicating that fasudil-induced SVZ-proliferating cells are neural stem cells. Example 3: Protective effect of fasudil on neurons

 Test cell

 The test cells of this example were prepared using the test cells of Example 1 and the methods described in the preparation thereof.

 2. Hypoglycemia and hypoxia injury model

 The glucose-deficient and hypoxia-injury model of this example was prepared by the method described in the model of glucose deficiency and hypoxia injury in Example 1.

 3. Administration

 According to the preliminary experiments of different concentrations of fasudil in Example 1, the intermediate concentration, i.e., l (g/ml), was selected in the present example. The other methods were identical to those of Example 1.

 4. Test the test

 After the experimental model was determined, the same volume of PBS was added as a control for fasudil under the exact same experimental conditions.

 5. Cell LDH release rate determination

To determine the extent of neuronal damage in different glycosidic reoxygenation times after treatment of neurons at different times, the extent of cellular damage was determined by measuring lactate dehydrogenase (LDH). After the neurons are damaged, the LDH in the cells will be released into the cell culture medium. Thus, the LDH value in the supernatant of the cells after the injury was detected, which reflected the damage of the cells. Normal cells were repeatedly thawed, and the supernatant was the amount of LDH released after all cells died. The LDH release rate of the cell supernatant was compared with the LDH value of total cell death total release. The LDH assay was performed according to the instructions of the LDH kit (purchased from Promega, USA). The OD value of each well was measured at a wavelength of 490 nm on a microplate reader ( Thermo Multiskan MK3, available from Thermo Corporation, USA). The LDH release rate is expressed as the OD value of the test group/the total OD value of the cells x l 00 %. 6. PI staining flow cytometry

 Propidine iodide (PI) is a nucleic acid dye that does not penetrate intact cell membranes. However, in advanced and late apoptotic cells, PI can pass through the cell membrane and cause nuclear red staining. Therefore, the number of dead neuronal cells was determined by flow cytometry (Coulter Epics XL, available from Beckman, USA).

 7. Results and analysis

 Using LDH kit to detect fasudil treatment can reduce the concentration of LDH in neuronal cell culture supernatant (p<0.05). Flow cytometry showed that fasudil treatment can reduce the number of PI cells (p<0.01). The results are shown in Figure 8, where A is the LDH assay and B is the PI assay. The above results indicate that different methods (LDH release and flow cytometry) have demonstrated that fasudil can protect neurons from hypoxia. Example 4: Mechanism of action of fasudil on neural stem cells

 I. Fasudil increases the concentration of G-CSF in brain tissue

 Test animal

 The test animals of this example were prepared using the test cells of Example 2 and the methods described in the preparation thereof.

 2. Mouse hypoxia model

 Sixteen mice were hypoxic for 6 hours in an anoxic system (8% oxygen). The hypoxia meter is placed in the hypoxia chamber to dynamically detect the oxygen concentration in the hypoxia chamber to ensure the controllability of the degree of hypoxia in mice. There were 8 fasudil group and saline group, respectively.

 3. Administration

 Fasudil injection (the concentration of the parent drug is 30mg/2ml = 15mg/ml). According to the results of in vitro experiments, the in vivo concentration was set to intraperitoneal injection of fasudil hydrochloride (5 mg/kg) and Brdu (50 mg/kg) in mice after hypoxia for 6 hours, 24 hours later. The injection was repeated once, and the control group was injected with the same volume of normal saline and Brdu. Eight rats in the fasudil group and 8 rats in the saline control group. After reoxygenation for 24 hours, the saline was perfused, and the brain fluid was stored at -80 ° C after the rapid freezing of nitrogen, and the cytokine of the brain homogenate supernatant was determined.

 4. Check the test:

 While carrying out the experimental model, the same volume of physiological saline was injected as a control for fasudil under exactly the same experimental conditions.

5. Cytokine determination In order to further study the cellular molecular mechanism of fasudil-induced endogenous neural cells, the kit was used to detect granulocyte colony-stimulating factor (G-CSF) in brain tissue homogenate supernatant (the kit was purchased from PeproTech, USA). And vascular endothelial growth factor (VEGF) (kit purchased from BioSource, USA) concentration, the detection procedure refers to the instructions.

 6. Astrocyte G-CSF expression

 In situ immunohistochemistry was used to further confirm that fasudil produced a cellular source of G-CSF, and that both cells in the central nervous system produced G-CSF under the action of fasudil. The method was as follows: Mouse brain slices were incubated with anti-mouse G-CSF antibody at 4 ° C overnight, and fluorescein-labeled secondary antibody was added for 2 hours at room temperature. Then, the brain slices were further added with anti-mouse GFAP antibody (purchased from Thermo Scientific, USA) at 4 ° C overnight, and finally fluorescein-labeled secondary antibody was added at room temperature for 2 hours. After thorough washing, it was observed with a fluorescence microscope.

 7. Results and analysis

 The results showed that fasudil treatment significantly increased the concentration of G-CSF in brain tissue (ρθ.01) after hypoxia. The results are shown in Figure 9, where A is the concentration of G-CSF and B is the concentration of VEGF. Fascidil treatment of rat brain astrocytes expressed G-CSF significantly higher than control mice. These results indicate that fasudil induced endogenous neuronal cells may be associated with increased G-CSF.

 Further, in situ immunohistochemistry was used to confirm that fasudil can promote astrocytes, but not microglia to express G-CSF. The astrocyte markers GFAP and G-CSF were double-stained. The results are shown in Figure 10. A is a saline control group. GFAP-stained red astrocytes rarely express G-CSF, and B is Fashudi. In the group, the degree of expression of G-CSF by astrocytes was significantly increased, showing a large number of yellow cells.

II. Fasudil promotes differentiation of endogenous neural stem cells into cholinergic functional neurons 1. Test animals

 The test animals of this example were prepared using the test cells of Example 2 and the methods described in the preparation thereof.

 2. Mouse hypoxia model

 Sixteen mice were hypoxic for 6 hours in an anoxic system (8% oxygen). The hypoxia meter is placed in the hypoxia chamber to dynamically detect the oxygen concentration in the hypoxia chamber to ensure the controllability of the degree of hypoxia in mice. There were 6 fasudil group and saline group, respectively.

3. Administration Fasudil injection (the concentration of the parent drug is 30mg/2ml = 15mg/ml). According to the results of in vitro experiments, the in vivo test concentration was set to intraperitoneal injection of fasudil hydrochloride (5 mg/kg) and Brdu (50 mg/kg) in the hypoxic system for 6 hours after hypoxia. Volume saline and Brdu. Six rats in the fasudil group and 6 rats in the saline control group. After 30 days of fasudil administration, the brain was taken for liquid nitrogen and then stored at -80 °C for detection of cholinergic neurons.

 4. Check the test:

 While carrying out the experimental model, the same volume of physiological saline was injected as a control for fasudil under exactly the same experimental conditions.

 5. Determination of cholinergic neurons

 The differentiation of cholinergic neurons was further observed by in situ immunohistochemistry. The procedure is as follows: Mouse brain slices were treated with anti-mouse ChAT antibody (1:500; purchased from Chemicon, USA) overnight at 4 ° C, and fluorescein-labeled secondary antibody was added for 2 hours at room temperature. Then, the brain piece was further added with an anti-mouse MAP-2 antibody (purchased from Chemicon, USA) at 4 ° C overnight, and finally a fluorescein-labeled secondary antibody was added at room temperature for 2 hours. After washing thoroughly, it was observed with a fluorescence microscope.

 6. Results and analysis

 The results of determination of cholinergic neurons using polyclonal ChAT antibody (1:500; purchased from Chemicon, USA) are shown in Figure 11, where the small blue squares in the left brain map indicate the location of the brain regions shown on the right. A is a saline control group, and B is a fasudil group, wherein Brdu represents Brdu staining, ChAT represents ChAT staining, and Merge represents Brdu/ChAT stain overlap. The results showed that Brdu-positive cells were significantly increased in all three brain regions, and ChAT-positive cholinergic neurons were also significantly increased (yellow after overlapping), indicating that fasudil can not only mobilize endogenous neural stem cells, but also promote these The cells differentiate into cholinergic functional neurons.

 The invention has been described as a preferred embodiment for reference. Variations and modifications of the present invention will be apparent to those skilled in the art from the detailed description of the invention. All such changes and modifications are intended to be included within the scope of the present invention.

Claims

 Rights request Use of a fasudil compound for the preparation of a medicament for inducing regeneration of neural stem cells and/or protecting nerve function in adult brain.  2. The use according to claim 1, wherein the adult brain neuron is an endogenous neuronal fine packet. Use in a diseased drug; preferably, the diseases associated with neuronal damage and/or death mainly include cerebral ischemia and chronic inflammatory neurodegenerative diseases.  The use according to claim 3, wherein the disease associated with neuronal damage and/or death is selected from the group consisting of cerebral ischemia and chronic inflammatory neurodegenerative diseases, preferably, the chronic inflammation The neurodegenerative disease is selected from one or more of the following: Alzheimer's disease, Parkinson's disease, /L atrophic lateral sclerosis, Huntington's disease, and multiple sclerosis.  The use according to any one of claims 1 to 4, wherein the fasudil compound is selected from the group consisting of: fasudil, a pharmaceutically acceptable salt thereof, a crystal or a hydrate And a pharmaceutically acceptable form, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochlorides, nitrates, sulfates, phosphates, hydrobromides, sulfonates, and citrates, more preferably selected as salts Acid salt.  The use according to any one of claims 1 to 5, wherein the therapeutically effective amount of the fasudil compound is 0.5-8 mg/kg, preferably the effective amount is 1-6 mg/kg. More preferably, the effective amount is 3-5 mg/kg, and the most preferred effective amount is 5 mg/kg.  7. A method of inducing regeneration and/or protection of neural function in adult brain neural stem cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of a fasudil compound or comprising fasudil A pharmaceutical composition of a compound.  A method for preventing and/or treating a disease associated with neuronal damage and/or death, characterized in that the method comprises administering to a subject in need thereof a therapeutically effective amount of a fasudil compound or inclusion method. A pharmaceutical composition of a sultil compound.  9. The method according to claim 8, wherein the disease associated with neuronal damage and/or death is selected from the group consisting of cerebral ischemia and chronic inflammatory neurodegenerative diseases, preferably, said chronic inflammatory Neurodegenerative diseases are selected from the group consisting of: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and multiple sclerosis. The method according to any one of claims 7 to 9, wherein the fasudil compound is selected from the group consisting of: fasudil, a pharmaceutically acceptable salt thereof, crystal or hydrate Or a pharmaceutically acceptable form, wherein the pharmaceutically acceptable benefit is preferably selected from the group consisting of hydrochlorides, nitrates, Sulfate, phosphate, hydrobromide, sulfonate and citrate, more preferably hydrochloride. 1 1. The method according to any one of claims 7 to 10, wherein the therapeutically effective amount of the fasudil compound or the pharmaceutical composition comprising the fasudil compound is 0.5-8 mg The /kg, preferably an effective amount is 1-6 mg/kg, more preferably the effective amount is 3-5 mg/kg, and most preferably the effective amount is 5 mg/kg.  The method according to any one of claims 7 to 12, wherein the administration route of the fasudil compound or the pharmaceutical composition comprising the fasudil compound comprises oral or intravenous injection Any route of administration known in the art, such as intramuscular, intraperitoneal, subcutaneous, and the like, is preferably intraperitoneal.  A pharmaceutical composition for inducing regeneration and/or protection of nerve function of adult brain neural stem cells, characterized in that the composition comprises a fasudil compound and a pharmaceutically acceptable carrier; The dosage form is selected from the group consisting of a tablet, a capsule, an oral solution, an injection, and a powder injection, preferably an injection, more preferably an intravenous injection.  A pharmaceutical composition for preventing and/or treating a disease associated with neuronal damage and/or death, characterized in that the composition comprises a fasudil compound and a pharmaceutically acceptable carrier; The pharmaceutical composition is selected from the group consisting of a tablet, a capsule, an oral solution, an injection, and a powder injection, preferably an injection, more preferably an intravenous injection.