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WO2009155777A1 - Utilisation et procédé de composé de fasudil et composition pharmaceutique pour ceux-ci - Google Patents

Utilisation et procédé de composé de fasudil et composition pharmaceutique pour ceux-ci Download PDF

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Publication number
WO2009155777A1
WO2009155777A1 PCT/CN2009/000509 CN2009000509W WO2009155777A1 WO 2009155777 A1 WO2009155777 A1 WO 2009155777A1 CN 2009000509 W CN2009000509 W CN 2009000509W WO 2009155777 A1 WO2009155777 A1 WO 2009155777A1
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Prior art keywords
fasudil
compound
effective amount
group
disease
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PCT/CN2009/000509
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English (en)
Chinese (zh)
Inventor
肖保国
丁晶
吕传真
姚小青
孙长海
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Tianjin Chase Sun Pharmaceutical Co Ltd
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Tianjin Chase Sun Pharmaceutical Co Ltd
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Priority to CN2009801229640A priority Critical patent/CN102026643B/zh
Publication of WO2009155777A1 publication Critical patent/WO2009155777A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • 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:
  • fasudil hydrochlorides Common salts of fasudil are hydrochlorides, nitrates, sulfates, sulfonium sulfonates, the most commonly used of which is fasudil hydrochloride.
  • 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.
  • 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.
  • people have been actively seeking effective treatments.
  • 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.
  • 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.
  • 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.
  • bone marrow-derived cells such as bone marrow mesenchymal stem cells
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the adult brain neuron is an endogenous neural cell.
  • 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.
  • 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.
  • cerebral ischemia e.g., cerebral embolism and cerebral infarction
  • chronic inflammatory neurodegenerative disease e.g., chronic inflammatory neurodegenerative disease.
  • 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.
  • 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.
  • 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
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the use of fasudil 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.
  • 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 combat 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.
  • 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).
  • SVZ subventricular zone
  • 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.
  • Example 1 Fasudil promotes brain neural stem cell proliferation in vitro
  • 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.
  • MAP-2 microtubule-associated protein-2
  • MAP-2 positive cells were red, suggesting neurons, and blue was HOCHEST staining nuclei.
  • the identified neuronal cells were used to establish an oxygen-glucose deprivation (ODD) model (an in vitro model of human cerebral ischemia).
  • ODD oxygen-glucose deprivation
  • 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.
  • 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.
  • BrdU (purchased from Roche, Switzerland) (1 ug/ml) was added on days 3 and 4.
  • 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.
  • 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.
  • mice 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.
  • 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.
  • 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.
  • 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 purchasedd from Invitrogen, USA
  • the immunoblot map was taken by a digital camera and processed by Image-pro Plus 5.0 image analysis software with ⁇ -actin as an internal reference.
  • 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 ⁇ m, 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.
  • Rho kinase inhibitors provide the basis.
  • test cells of this example were prepared using the test cells of Example 1 and the methods described in the preparation thereof.
  • 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.
  • Example 1 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.
  • LDH lactate dehydrogenase
  • 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 Propidine iodide
  • test animals of this example were prepared using the test cells of Example 2 and the methods described in the preparation thereof.
  • mice 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.
  • 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.
  • G-CSF granulocyte colony-stimulating factor
  • VEGF vascular endothelial growth factor
  • 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.
  • FIG. 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.
  • Fasudil promotes differentiation of endogenous neural stem cells into cholinergic functional neurons 1.
  • test animals of this example were prepared using the test cells of Example 2 and the methods described in the preparation thereof.
  • mice 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.
  • 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.
  • the brain was taken for liquid nitrogen and then stored at -80 °C for detection 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.

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Abstract

La présente invention concerne une nouvelle utilisation du fasudil dans l’induction de la régénération de cellules souches neurales de cerveau adulte et/ou la protection des fonctions neurologiques, et dans la prévention et/ou le traitement de maladies associées aux dommages et/ou à la mort des neurones. L’utilisation du fasudil dans la présente invention comprend l’utilisation dans la fabrication de médicaments pour induire la régénération de cellules souches neurales de cerveau adulte et/ou la protection des fonctions neurologiques. De plus, l’utilisation de fasudil dans la fabrication de médicaments pour prévenir et/ou traiter des maladies associées à des dommages et/ou à la mort des neurones.
PCT/CN2009/000509 2008-06-26 2009-05-11 Utilisation et procédé de composé de fasudil et composition pharmaceutique pour ceux-ci Ceased WO2009155777A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2009801229640A CN102026643B (zh) 2008-06-26 2009-05-11 法舒地尔化合物的用途、方法及其药物组合物

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CN200810126351A CN101612157A (zh) 2008-06-26 2008-06-26 法舒地尔诱导成年脑内源性神经干细胞再生的用途
CN200810126351.1 2008-06-26

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WO2013135596A1 (fr) 2012-03-12 2013-09-19 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts, Universitätsmedizin Inhibiteurs de kinase rho s'utilisant dans le traitement de la sclérose latérale amyotrophique
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CN102973571A (zh) * 2012-12-12 2013-03-20 天津红日药业股份有限公司 法舒地尔的新用途
CN104983704A (zh) * 2015-08-13 2015-10-21 青岛蓝盛洋医药生物科技有限责任公司 一种改善脑血管痉挛的药物盐酸法舒地尔组合物片剂
CN105055332A (zh) * 2015-09-10 2015-11-18 青岛蓝盛洋医药生物科技有限责任公司 一种治疗缺血性脑血管疾病的药物盐酸法舒地尔组合物干混悬剂
US11666583B2 (en) 2020-01-09 2023-06-06 Woolsey Pharmaceuticals, Inc. Methods of treating cortical dementia associated wandering
EP4087564A4 (fr) * 2020-01-09 2024-01-24 Woolsey Pharmaceuticals, Inc. Procédés de traitement des comportements de déambulations (wandering) associés à la démence corticale
US11642352B2 (en) 2020-03-25 2023-05-09 Woolsey Pharmaceuticals, Inc. Methods of treating wandering in Lewy dody dementia
EP4181925A4 (fr) * 2020-07-14 2024-07-31 Woolsey Pharmaceuticals, Inc. Méthodes de traitement de protéinopathies
US11311553B1 (en) 2020-10-22 2022-04-26 Woolsey Pharmaceuticals, Inc. Methods of treating 4-repeat tauopathies
US11865119B2 (en) 2021-11-29 2024-01-09 Woolsey Pharmaceuticals, Inc. Methods of treating agitation and other dementia-associated behavioral symptoms

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