WO2009155777A1 - 法舒地尔化合物的用途、方法及其药物组合物 - Google Patents

法舒地尔化合物的用途、方法及其药物组合物 Download PDF

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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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fasudil
compound
effective amount
group
disease
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English (en)
French (fr)
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肖保国
丁晶
吕传真
姚小青
孙长海
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天津红日药业股份有限公司
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Priority to CN2009801229640A priority Critical patent/CN102026643B/zh
Publication of WO2009155777A1 publication Critical patent/WO2009155777A1/zh

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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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Description

法舒地尔化合物的用途、 方法及其药物组合物 技术领域
本发明属于医药技术领域。 本发明涉及法舒地尔的新用途， 具体地， 本发明涉及法舒地尔在制备用于诱导成年脑神经千细胞再生和 /或保护神 经功能的药物中的用途， 以及相应的神经***疾病的可能应用前景； 法舒 地尔在制备用于预防和 /或治疗与神经元损伤和 /或死亡相关疾病的药物中 的用途； 诱导成年脑神经干细胞再生和 /或保护神经功能的方法； 以及用于 诱导成年脑神经千细胞再生和 /或保护神经功能的药物组合物。 背景技术
法舒地尔 （Fasudil ) 为异喹啉磺胺衍生物， 其化学名称为六氢 -1- ( 5- 异喹啉磺酰基） -1H-1 , 4-二氮杂卓 [hexahydro-1- ( 5-isoquino lylsulfonyl ) -1H-1 , 4-diazepime]或 1- ( 5-异喹啉磺酰基） 高哌嗪 [1- ( 5-Isoquinolinesulfonyl ) homopiperazine] , 分子式为 C14H17N3O2S , 结构 式如下：
Figure imgf000002_0001
法舒地尔的常用盐有盐酸盐、 硝酸盐、 硫酸盐、 曱磺酸盐， 其中最常用的 是盐酸法舒地尔。
2002年， 法舒地尔在中国上市， 其适应症为预防和治疗^ ^蛛网膜下 腔出血后脑血管痉挛等引起的缺血性脑血管疾病症状。 蛛网膜下腔出血是 指脑内的血管破裂出血后血液流入蛛网膜下腔， 其为出血性脑血管病的一 种类型， 分为原发性和继发性两种， 主要症状是头痛、 呕吐和颈项强直。 脑血管痉挛是指动脉在一段时间内的异常收缩状态， 常见于颅内动脉瘤破 裂的蛛网膜下腔出血病人， 成为蛛网膜下腔出血的致残与死亡的主要原 因。 目前已有许多临床报告证实盐酸法舒地尔治疗蛛网膜下腔出血后引起 的脑血管痉挛的有效性和安全性。 体内试验对盐酸法舒地尔药代动力学和 代谢活性产物进行的研究结果表明， 盐酸法舒地尔进入体内很快转变为具 有相同活性的代谢产物 -羟基法舒地尔 （ hydroxyfasudil ) , 其浓度可达母 药的 86 % , 并具有较长的半衰期 （盐酸法舒地尔半衰期 0.78小时， 羟基 法舒地尔半衰期 4.66小时） 。 羟基法舒地尔的 12小时峰谷值为 0.077M, 并且具有与盐酸法舒地尔相同的生物活性， 可在有效浓度范围内 （0.025 _ 1.6 M ) 抑制 Rho激酶。 因此， 盐酸法舒地尔作为一种新型、 高效的血 管扩张药 （其为一种蛋白激酶抑制剂， 即细胞内钙离子拮抗剂） ， 能有效 緩解脑血管痉挛， 改善蛛网膜下腔出血后引起的脑血管痉挛。
脑缺血和慢性炎性神经变性性疾病如阿尔茨海默病、 帕金森病、 多发 性硬化， 视网膜疾病等神经***疾病在其发生和发展过程中会导致神经元 的损伤和死亡。对于这组疾病，人们一直都在积极地寻求有效的治疗药物。 过去 20年来， 人们研究和观察了大量的神经保护剂， 试图可以预防和治 疗这些神经***疾病的发生和发展， 包括钙通道拮抗剂、 自由基清除剂、 谷氨酸拮抗剂、 细胞膜稳定剂和神经营养因子等， 但由于脑损伤后多种因 素同时或先后参与了神经元的损伤与死亡， 单纯作用于单个靶点的药物难 以取得令人满意的疗效， 上述神经保护剂的临床治疗效果并不理想。
神经***损伤后的再生修复问题一直是神经科学领域关注的重点。 随 着对神经千细胞研究的不断深入， 人们尝试利用神经干细胞修复受损神经 元成为近年来神经科学领域研究的热点之一。 神经干细胞在修复受损神经 组织中发挥重要作用， 是修复和代替受损组织的有效方法， 能够重建部分 环路和功能。 目前， 研究较广泛的移植物主要还是胚胎干细胞和神经干细 胞， 但因来源有限、 伦理障碍及免疫排斥等问题， 这些细胞的实用性受到 极大限制。 近来研究表明， 骨髓间充质干细胞具有分化为神经元和神经胶 质细胞的能力。 但是， 骨髓源细胞如骨髓间充质干细胞有较高的病毒感染 危险， 且随着其来源个体的年龄增长其细胞数量和增殖、 分化能力也显著 下降， 较难满足临床的需求。 1992年， Reynolds等首先从成年鼠纹状体中 分离出神经千细胞， 从而突破了成年组织不可再生的观点。 该实验充分证 明成年神经千细胞产生的新生神经元不仅在形态上与成熟神经元相同， 而 且对突触刺激有动作电位反应， 并且可以有效地整合到神经环路中， 从而 为临床应用神经千细胞奠定了基础， 为我们利用成年神经干细胞进行脑修 复治疗指明了一个方向。 但是， 到目前为止， 该研究领域仍然没有找到一种能够有效地在体内 作用于成年神经千细胞， 诱导其再生， 从而修复受损神经细胞以达到治疗 神经***疾病的药物。 因此， 寻找一种能够自由通过血脑屏障的、 相对稳 定的、 可以诱导内源性成年脑神经干细胞再生的物质一直是人们努力寻找 的方向。 发明内容
本发明人经过长期认真地研究， 出人意料地发现法舒地尔 具有神经 保护功能和具有能够促进成年脑神经千细胞增生或分化、 诱导成年脑神经 干细胞再生的功能， 从而完成了本发明。
因此， 本发明的一个目的在于， 提供法舒地尔在诱导成年脑神经干细 胞再增生和 /或保护神经功能中的新用途。
本发明的另一个目的在于，提供法舒地尔在预防和 /或治疗与神经元损 伤和 /或死亡相关疾病中的新用途。
本发明的另一个目的在于，提供诱导成年脑神经干细胞再生和 /或保护 神经功能的方法。
本发明的另一个目的在于， 提供用于诱导成年脑神经千细胞再生和 / 或保护神经功能的药物组合物。
本发明的目的是采用以下技术方案来实现的。
一方面， 本发明提供了法舒地尔在制备用于诱导成年脑神经干细胞再 生和 /或保护神经功能的药物中的用途。
优选地， 其中所述的成年脑神经千细胞为内源性神经千细胞。
另一方面，本发明提供了法舒地尔在制备用于预防和 /或治疗与神经元 损伤和 /或死亡相关疾病的应用前景药物中的用途。
优选地，其中所述的与神经元损伤和 /或死亡相关的疾病为神经***疾 病， 主要包括脑缺血 （如脑栓塞和脑梗塞） 和慢性炎性神经变性性疾病。
优选地， 其中所述的慢性炎性神经变性性疾病主要包含但不限于： 阿 尔茨海默病、 帕金森病、 月几萎缩性侧索硬化、 亨廷顿舞蹈病、 多发性硬化。
优选地， 前述的法舒地尔包括药学上可接受的盐 结晶或水合物等药 学上可接受的形式， 其中药学上可接受的盐优选选自盐酸盐、 硝酸盐、硫酸 盐、 磷酸盐、 氢溴酸盐、 曱磺酸盐和柠檬酸盐、 更 选选自盐酸盐。
优选地， 前述的法舒地尔有效量为 0.5-8mg/kg, 优选 l-6mg/kg, 更优 选 3-5mg/kg, 最优选 5mg/kg„
又一方面，本发明提供了一种诱导成年脑神经干细胞再生和 /或保护神 经功能的方法， 包括法舒地尔给药治疗有效量的法舒地尔或包含法舒地尔 的药物组合物。
再一方面， 本发明提供了一种预防和 /或治疗与神经元损伤和 /或死亡 相关疾病的方法， 包括给药治疗有效量的法舒地尔或包含法舒地尔的药物 组合物。
优选地， 前述的法舒地尔或包含法舒地尔的药物组合物的治疗有效量 为 0.5-8mg/kg, 优选有效量为 l-6mg/kg， 更优选有效量为 3-5mg/kg， 最优 选有效量为 5mg/kg。
还一方面，本发明提供了一种用于诱导成年脑神经干细胞再生和 /或保 护神经功能的药物组合物， 所述的组合物包含法舒地尔化合物以及药学上 可接受的载体； 所述药物组合物的剂型选自： 片剂、 胶囊、 口服液、 注射 剂和粉针剂， 优选为注射剂， 更优选为静脉注射剂。
又一方面， 本发明提供了一种用于预防和 /或治疗与神经元损伤和 /或 死亡相关疾病的的药物组合物， 所述的组合物包含法舒地尔化合物以及药 学上可接受的载体； 所述药物组合物的剂型选自： 片剂、 胶嚢、 口服液、 注射剂和粉针剂， 优选为注射剂， 更优选为静脉注射剂。
优选地， 前述的法舒地尔或包含法舒地尔的药物组合物的给药剂型包 括片剂、 胶嚢、 口服液、 注射剂或粉针剂等适于给药的肠道或非肠道给药 剂型， 优选注射剂， 更优选静脉注射剂。
优选地， 前述的法舒地尔或包含法舒地尔的药物组合物的给药途径包 括口服、 静脉注射、 肌肉注射、 腹腔注射和皮下注射等本领域已知的任何 给药途径， 优选腹腔注射。
综上所述， 本发明所提供的法舒地尔的用途包括以下几个方面： 保护 神经功能、 诱导成年脑神经千细胞再生 （即诱导成年脑内源性神经千细 胞）、治疗与神经元损伤和死亡相关的疾病的用途（即治疗神经***疾病）、 制备用于诱导成年脑神经千细胞再生的药物中的用途、 制备用作神经保护 剂的药物中的用途、 制备用于治疗神经***疾病的药物中的用途。
此外， 本发明提供一种治疗神经***疾病的药物组合物， 其包含治疗 有效量的法舒地尔或盐酸法舒地尔。 本发明所述的法舒地尔， 包括其盐酸 盐、 硝酸盐、 硫酸盐、 磷酸盐、 氢溴酸盐、 曱磺酸盐、 柠檬酸盐等药学上 可接受的盐形式， 其可以为任意形式， 包括结晶形式 （包括带有结晶水的 和不带有结晶水的） 、 水合物形式等药学上可接受的形式， 优选法舒地尔 盐酸盐。 这些盐及不同的形式可以按照本领域熟知的方法制备。
本发明所述的有效量为法舒地尔在用于神经保护或治疗神经***疾 病中所可能产生医学上认可的功效的量。 本发明人经多次研究发现， 适于 诱导成年脑神经千细胞再生， 即适于可能治疗神经***疾病的法舒地尔的 有效量为 0.5-8mg/kg, 优选 l-6mg/kg, 更优选 3-5mg/kg, 最优选 5mg/kg„ 对于上述法舒地尔的各种盐及包含法舒地尔的药物组合物的用量， 以相当 于能提供上述法舒地尔有效量的相应法舒地尔盐的量即为相应盐的有效 量。
可以按照本领域已知的常规技术， 将本发明所述的法舒地尔或包含有 效量的法舒地尔的药物组合物制成适于给药的任何剂型， 包括但不限于肠 道给药制剂或非肠道给药制剂， 例如片剂、 胶囊、 口服液、 注射剂、 粉针 剂等， 优选注射剂， 更优选静脉注射剂。 本发明所述制剂的给药途径可以 是本领域已知的任何给药途径， 优选腹腔注射。 本发明所述制剂可以每天 给药 3次， 每次给药量为法舒地尔可以治疗所述疾病的有效量， 即上述给 出的有效量。
本发明所述的与神经元损伤和死亡相关的疾病， 即所述神经***疾病 包括， 但不限于脑缺血、 脑损伤、 阿尔茨海默病、 帕金森病、 多发性硬化 等。 附图的简要说明
以下， 结合附图来详细说明本发明的实施例， 其中：
图 1为显示体外原代培养的海马神经元 MAP-2和 HOCHEST双染色 荧光显微镜结果图， 其中红色为 MAP-2神经元 , 而蓝色为 HOCHEST核 染色。
图 2为显示体外原代神经元培养后和 PBS对照的 Brdu阳性细胞荧光 显微镜结果图， 其中 A为法舒地尔组， B为 PBS对照组。
图 3为显示小鼠在正常氧和脑缺氧（ 8 %氧） 6小时后， 复氧 72小时 后海马不同部位神经元染色结果图， 其中 CA1、 CA2和 CA3分别代表海 马 CA1、 CA2和 CA3区。
图 4为显示小鼠在脑缺氧（ 8 %氧） 不同时间 （O h, 2 h, 6 h, 8h )脑 组织匀浆后免疫印染法测定 Rockll蛋白的表达结果图。
图 5为显示小鼠在脑缺氧（ 8 %氧） 不同时间 （O h, 2 h, 6 h, 8h )脑 组织切片免疫组化测定 ROCK II蛋白的表达结果图，箭头均为表达的绿色 荧光 ROCK II。
图 6为显示小鼠脑缺氧（ 8 %氧） 6小时后注射生理盐水（A、 C和 E ) 和法舒地尔（ B、 D和 F ) , 24小时后侧脑室下区（ SVZ )后角 （ A和 B ) , 前角 （ C和 D ) 和海马 DG区 （ E和 F ) Brdu阳性细胞结果图。
图 7为显示小鼠脑缺氧（8 %氧） 6小时后注射法舒地尔， 24小时后 侧脑室下区 （ SVZ )后角 DCX和 Brdu双阳性细胞荧光显微镜结果图， 其 中 A )红色为 Brdu阳性细胞， B )绿色为 DCX阳性细胞， C )二种颜色的 重叠复染细胞 （增生的神经千细胞） 。
图 8为显示法舒地尔抑制缺氧后神经元死亡而导致培养上清液 LDH 释放减少和 PI阳性细胞数减少的柱状图， 其中 A为 LDH测定， B为 PI 测定。
图 9为显示的小鼠脑组织匀浆上清液 G-CSF和 VEGF浓度水平的柱 状图， 其中 A为 G-CSF浓度， B为 VEGF浓度。
图 10为显示星形胶质细胞标志 GFAP和 G-CSF的免疫双染结果图， 其中 A是生理盐水对照组， B是法舒地尔组。
图 11为显示多克隆 ChAT 抗体检测胆碱能神经元的结果图， 其中左 侧脑图中的蓝色小方块表示右侧所示的脑区位置， A为生理盐水对照组， B为法舒地尔组。 实施发明的最佳方式
本发明将结合下述实施例或实施例做进一步详细阐述， 但应当理解， 下述实施例仅仅用于阐述和解释本发明， 而并不限制本发明的范围。
应当理解， 对于本发明中提及但没有详细说明的方法、 步骤、 装置、 仪器、 材料等， 普通技术人员可以采用本领域熟知的相应方法、 步骤、 装 置、 仪器、 材料等， 或者按照本领域的常规知识和技术获得。 实施例 1 : 法舒地尔体外促进脑神经干细胞增生试验
1. 试验细胞及其制备
将孕 17 - 18天昆明 （KM )孕鼠（购自上海斯莱克实验动物有限责任 公司）使用脱颈法处死， 75%酒精消毒皮肤。 在无菌条件下剥出胎鼠， 置 于冰预冷的 DMEM培养液（购自美国 GIBCO公司 ) 中。 仔细分离胎鼠脑 组织， 剥除脑膜及血管， 去除小脑、 脑干和基底节， 取出海马组织， 置于 水预冷的 DMEM中轻轻吹打， 使其形成单细胞悬浮液。 收集细胞悬液， 在 4°C下， 以 1000转 /分钟离心 5分钟。 弃去上清液， 用新鲜配制的神经 元培养液（购自美国 GIBCO公司） 重新悬浮。 计数细胞， 计算细胞悬液 中细胞的密度。 以 2xl05/cm2种植在多聚赖氨酸包被的玻片上， 在 37°C下、 5 % C02培养箱内培养， Ί天后更换原先体积 1/2神经元培养液。 14天后进 行神经元特异性标记： 微管相关蛋白 -2 (MAP-2, 购自美国 Chemicon公 司）染色鉴定， 同时用荧光染色剂 HOCHEST33342 购自美国 Sigma公司） 标记细胞核。 荧光显微镜 ( Olympus BX- 60F, 购自日本 Olympus公司） 下观测， 结果如图 1所示， 其中 MAP-2阳性细胞为红色， 提示为神经元， 蓝色为 HOCHEST染色细胞核。
2. 缺糖缺氧细胞损伤模型
使用上述鉴定后的神经元细胞来建立缺糖缺氧损伤 （ oxygen-glucose deprivation, OGD )模型 （人类脑缺血的体外模型） 。 无糖型 DMEM培养 液， 用前通入 5%CO2、 10%H2、 85%N 合气体饱和 15分钟， 去除培养 液中的氧气， 制备缺糖缺氧神经元培养液。 取培养 10天左右， 生长良好 的神经元， 弃去培养液， 用 PBS液冲洗 3 次，加入无糖型 DMEM培养液。 将培养板置于厌氧培养箱内， 向箱内通入 5%C02、 10%H2、 85%N 合 气体， 将培养箱密封保持 37°C, 并使箱内氧浓度低于 1%。 缺氧 6小时后 将培养板取出，吸除无糖培养液，分别加入原神经元培养液，放置于 37°C、 5%C02 和 95%空气的培养箱中复氧培养。
3. 给药
向上述建立的缺糖缺氧神经元给予法舒地尔注射液（购自天津红曰药 业股份有限公司） （母药浓度为 30mg/2ml = 15mg/ml ) 。 实验浓度设定为 低 ( l g/ml) , 中 ( lO g/ml) 和高 ( lOO g/ml) 三个浓度。 分别在当天、 第 2天和第 3天分三次给药， 第 5天收集细胞和上清液做进一步检测用。
4. 对照试险
实验模型确定后， 采用加入相同体积的 PBS代替法舒地尔注射液， 在 完全相同的实验条件下作为法舒地尔的对照。 正常氧状态下培养的细胞作 为缺氧细胞的基线。 5. 申经元 BrdU检测
加入法舒地尔第 3天后， 分别在第 3天和第 4天加 BrdU (购自瑞士 Roche公司 ) ( 1 ug/ml ) 。 第 5天收集细胞， 用 PBS漂洗细胞 2次， 每次 10分钟， 4%多聚曱醛室温固定 20分钟， l xPBS漂洗细胞 2次， 每次 10 分钟， 3 %羊血清室温封闭 15分钟。 BrdU—抗（ 1 : 200, 美国 Lab Version 公司） 稀释于含 1 羊血清 PBS中， 4°C , 过夜。 羊抗小鼠 CY3标记二抗 ( 1 : 100,购自美国 Sigma公司 ）稀释于 PBS中， 室温避光 1小时。 l xPBS 漂洗细胞 3次， 每次 5分钟， 50 %甘油封片， 荧光显微镜下观测结果。
6. 结果和分析
体外原代神经元培养后， Brdu阳性细胞荧光显微镜结果图， 如图 2所 示， 其中 A为法舒地尔组， B为 PBS对照组。 从图中可以看出， 与 PBS 对照组相比， 加入法舒地尔组的神经元形成较为明显的神经球， Brdu阳性 的增生神经元细胞明显多于 PBS对照。 经多次重复都得到相同的结杲， 清 楚地表明法舒地尔可在体外促进脑神经千细胞增生。 实施例 2: 法舒地尔体内促进脑神经千细胞增生试验
1. 试-险动物
KM小鼠 （购自上海斯莱克实验动物有限责任公司 ） 。 动物到达实验 室后给与清洁级饲养， 自由饮食和饮水， 静养 5 - 7天。
2. 小鼠缺氧模型
将 32只 KM小鼠分成 4组， 每组 8只， 在缺氧***中 （8 %氧气）缺 氧 2小时、 4小时、 6小时、 8小时。 在缺氧箱中放置缺氧仪， 动态检测缺 氧箱中氧浓度， 保证小鼠缺氧程度的可控性。 箱体可容纳 30 - 40只小鼠， 将 32只小鼠一次放入可以保证缺氧条件的可比性。 复氧 72小时后生理盐 水灌注， 取脑液氮速冻后 _ 80°C保存， 待免疫印染和免疫组化时脑组织切 片后， 海马神经元和 ROCK II染色。
3. 给药
法舒地尔注射液， 母药浓度为 30mg/2ml = 15mg/ml。 依据体外实验的 结果， 体内试验浓度设定为小鼠在缺氧***中缺氧 6小时后腹腔注射盐酸 法舒地尔 （ 5 mg/kg ) 和 Brdu ( 50 mg/kg ) , 24小时后再重复注射一次； 对照组注射相同体积生理盐水和 Brdu。 法舒地尔组 8只鼠， 而生理盐水对 照组 7只鼠。 复氧 24小时后生理盐水灌注， 取脑液氮速冻后 - 80°C保存， 待免疫印染和免疫组化时脑组织切片后， 进行 Brdu (购自瑞士 Roche公 司 ) 和 DCX (购自美国 BD Bioscience公司） 染色。
4. 对照试验：
进行实验模型的同时， 采用注射相同体积的生理盐水代替法舒地尔注 射液， 在完全相同的实验条件下作为法舒地尔的对照。 另外设定正常氧动 物 8只在完全相同的条件下进行作为对照。
5. 免疫印染测定
经 10% SDS-PAGE不连续凝胶电泳分离后， 转膜于 PVDF膜上 15-30 分钟， 转移后的硝酸纤维膜置于丽春红染色液中显色， 标记蛋白质 Marker 及谱带位置后， 转移緩冲液洗至脱色。 封闭硝酸纤维膜置于膜封闭液中， 摇床上緩慢摇动， 室温封闭 1小时。抗 ROCK II抗体（购自美国 Bioscience 公司）用 5%的小牛血清稀释（ 1 : 500 ) 4°C过夜。 采用 Alexa Fluor 488 抗 小鼠二抗 （购自美国 Invitrogen公司） 1 : 1000 稀释， 室温孵育 1小时。 用化学发光法显色， X光片显影。 免疫印迹的图谱由数码相机拍照， 经 Image-pro Plus 5.0 图像分析软件处理， 以 β-actin为内参。
6. 免疫组化测定
小鼠断头后沿中线切开头皮并沿矢状缝剪开颅骨， 取出大脑， 浸入 20%蔗糖中 4°C脱水， 沉底后再置于 30%蔗糖中 4°C脱水至组织下沉。用滤 纸吸千脑组织表面的蔗糖溶液， 用 OCT 在液氮中包埋， 以 8μηι的厚度作 冠状位冰冻连续切片， 按照先后顺序存放备用。 免疫荧光染色用 5 %羊血 清室温下封闭 10分钟， 抗 ROCK II抗体， 抗 Brdu抗体和抗 DCX抗体作 为一抗 4°C孵育 24 小时后， Alexa Fluor 488 标记的抗小鼠抗体（购自美 国 Invitrogen公司） 和 Rhodamine 标 i己抗豚鼠抗体 （购自美国 Chemicon 公司 ) 室温避光作用 1 小时， l xPBS 洗涤 3 次， 每次 5分钟后， 50 %甘 油封片， 荧光显微镜下观察、 拍照。
7. 结果和分析
结果显示， 所建立的缺氧模型可以引起海马不同区域， 特别是 CA1 和 CA2区内神经元的丟失， 如图 3所示。 用免疫印染法 （参见图 4 )和免 疫组化法 （参见图 5， 其中箭头均为表达的绿色荧光 ROCK II ) 证明脑缺 氧 6小时后 Rho激酶的亚型 Rockll蛋白的表达增加， 为使用 Rho激酶抑 制剂提供了依据。
缺氧后注射法舒地尔可以明显增加脑 SVZ区前角和后角位置 Brdu阳 性的细胞再生， 结果如图 6所示， 其中 A、 C和 E为对照生理盐水组， B、 D和 F为法舒地尔组， A和 B为侧脑室下区（SVZ )后角， C和 D为前角， E和 F为海马 DG区。
进一步的研究发现， 这些 Brdu阳性的增生细胞同时表达神经干细胞 标志 DCX (参见图 7, 其中 A ) 红色为 Brdu阳性细胞， B ) 绿色为 DCX 阳性细胞， C ) 二种颜色的重叠复染细胞， 即增生的神经千细胞） ， 表明 法舒地尔诱导的 SVZ区增生细胞是神经干细胞。 实旄例 3: 法舒地尔对神经元的保护作用
1. 试验细胞
采用实施例 1中试验细胞及其制备中所述的方法制备本实施例的试验 细胞。
2. 缺糖缺氧损伤模型
采用实施例 1中缺糖缺氧损伤模型所述的方法制备本实施例的缺糖缺 氧损伤模型。
3. 给药
根据实施例 1中法舒地尔不同浓度比较预实验， 在本实施例中选择了 中间的浓度， 即 l(^g/ml。 其余方法与实施例 1的给药方法完全相同。
4. 对照试险
试验模型确定后，采用加入相同体积的 PBS在完全相同的实验条件下 作为法舒地尔的对照。
5. 细胞 LDH释放率测定
为确定给予神经元不同时间处理后， 在不同的复糖复氧时间内神经元 损伤的情况， 通过测定乳酸脱氢酶（LDH )检测细胞损伤的程度。 神经元 受到损伤后细胞内的 LDH将释放到细胞培养液内。 因而检测损伤后细胞 上清液内的 LDH值， 反应了细胞受损情况。 将正常细胞给予反复冻溶， 其上清液内为全部细胞死亡后释放的 LDH量。 将所检测的细胞上清液 LDH值与细胞完全死亡总释放的 LDH值相比， 得到细胞 LDH释放率。 LDH测定依据 LDH试剂盒（购自美国 Promega公司）说明书操作。 在酶 标仪 ( Thermo Multiskan MK3， 购自美国 Thermo公司）上选用波长 490nm 检测每孔的 OD 值。 LDH释放率以检测组 OD 值 /细胞总释放的 OD 值 xl 00 %来表示。 6. PI染色流式细胞仪检测
捵化丙啶（propidine iodide, PI ) 是一种核酸染料， 它不能透过完整 的细胞膜， 但在凋亡中晚期的细胞和死细胞， PI能够透过细胞膜而使细胞 核红染。 因此， 利用流式细胞仪（ Coulter Epics XL, 购自美国 Beckman 公司）检测 PI阳性细胞代表死亡神经元细胞数。
7. 结果和分析
用 LDH试剂盒检测发现法舒地尔处理可以减少神经元细胞培养上清 液中 LDH浓度（ p<0.05 ) , 用流式细胞仪检测发现法舒地尔处理可以减少 PI细胞数（ p<0.01 ) , 结果如图 8所示， 其中 A为 LDH测定， B为 PI测 定。 以上结果表明， 采用不同的方法（ LDH释放和流式细胞仪检测）均证 实法舒地尔可以保护缺氧后神经元的损伤。 实施例 4: 法舒地尔诱导神经干细胞的作用机理
I .法舒地尔增加脑组织中 G-CSF浓度
1. 试验动物
采用实施例 2中试验细胞及其制备中所述的方法制备本实施例中的试 验动物。
2. 小鼠缺氧模型
16只小鼠分别在缺氧***中 （8 %氧气） 缺氧 6小时。 在缺氧箱中放 置缺氧仪， 动态检测缺氧箱中氧浓度， 保证小鼠缺氧程度的可控性。 法舒 地尔组和生理盐水组分别为 8只。
3. 给药
法舒地尔注射液（母药浓度为 30mg/2ml = 15mg/ml ) 。 依据体外实验 的结果， 体内试验浓度设定为小鼠在缺氧***中缺氧 6小时后腹腔注射盐 酸法舒地尔 （ 5 mg/kg )和 Brdu ( 50 mg/kg ) , 24小时后再重复注射一次， 对照组注射相同体积生理盐水和 Brdu。 法舒地尔组 8只鼠， 生理盐水对照 组 8只鼠。 复氧 24小时后生理盐水灌注， 取脑液氮速冻后 - 80°C保存， 进行脑组织匀浆上清液细胞因子测定。
4. 对照试险：
进行实验模型的同时， 采用注射相同体积的生理盐水在完全相同的实 验条件下作为法舒地尔的对照。
5. 细胞因子测定 为深入研究法舒地尔诱导内源性神经千细胞的细胞分子机理， 分别采 用试剂盒检测脑组织匀浆上清液中粒细胞集落刺激因子 （G-CSF ) (试剂 盒购自美国 PeproTech公司 ） 和血管内皮生长因子 （ VEGF ) ( 试剂盒购 自美国 BioSource公司 ) 浓度， 检测步骤参照说明书。
6. 星形胶质细胞 G-CSF表达
采用原位免疫组化的方法进一步确认法舒地尔产生 G-CSF的细胞来 源， 既中枢神经***中什么细胞在法舒地尔的作用下产生 G-CSF。 方法筒 述如下： 小鼠脑片经抗小鼠 G-CSF抗体 4°C过夜后， 加荧光素标记的第二 抗体室温 2小时。 然后， 脑片再加抗小鼠 GFAP抗体 （购自美国 Thermo Scientific公司） 4°C过夜， 最后加荧光素标记的第二抗体室温 2小时。 充 分洗涤后用荧光显微镜观察。
7. 结果和分析
试验结果显示缺氧后法舒地尔处理显著地增加了脑组织中 G-CSF的 浓度（ρθ.01 ) ， 结果如图 9所示， 其中 A为 G-CSF浓度， B为 VEGF 浓度。 法舒地尔处理鼠脑星形胶质细胞表达 G-CSF的程度明显高于对照 鼠。 这些结果表明法舒地尔诱导内源性神经千细胞可能与 G-CSF增加有 关。
进一步采用原位免疫组化的方法， 证实法舒地尔可以促进星形胶质细 胞， 而不是小胶质细胞表达 G-CSF。 星形胶质细胞标志 GFAP和 G-CSF 的免疫双染， 结果见图 10, 其中 A是生理盐水对照组， GFAP染色的红色 星形胶质细胞很少表达 G-CSF, B是法舒地尔组， 其星形胶质细胞表达 G-CSF的程度明显增加， 表现为大量黄色的细胞。
II . 法舒地尔促进内源性神经干细胞向胆碱能功能性神经元分化 1. 试验动物
采用实施例 2中试验细胞及其制备中所述的方法制备本实施例中的试 验动物。
2. 小鼠缺氧模型
16只小鼠分别在缺氧***中 （8 %氧气） 缺氧 6小时。 在缺氧箱中放 置缺氧仪， 动态检测缺氧箱中氧浓度， 保证小鼠缺氧程度的可控性。 法舒 地尔组和生理盐水组分别为 6只。
3. 给药 法舒地尔注射液（母药浓度为 30mg/2ml = 15mg/ml ) 。 依据体外实验 的结果， 体内试验浓度设定为小鼠在缺氧***中缺氧 6小时后腹腔注射盐 酸法舒地尔 （ 5 mg/kg )和 Brdu ( 50 mg/kg ) , 对照组注射相同体积生理盐 水和 Brdu。 法舒地尔组 6只鼠， 生理盐水对照组 6只鼠。 法舒地尔给与 30天后， 取脑置液氮速冻后 - 80°C保存用于检测胆碱能神经元。
4. 对照试险：
进行实验模型的同时， 采用注射相同体积的生理盐水在完全相同的实 验条件下作为法舒地尔的对照。
5. 胆碱能神经元测定
采用原位免疫组化的方法进一步观察胆碱能神经元的分化。 方法筒述 如下： 小鼠脑片经抗小鼠 ChAT 抗体（ 1 : 500; 购自美国 Chemicon公司） 4°C过夜后， 加荧光素标记的第二抗体室温 2小时。 然后， 脑片再加抗小 鼠 MAP-2抗体（购自美国 Chemicon公司 ) 4°C过夜， 最后加荧光素标记 的第二抗体室温 2小时。 充分洗涤后用荧光显微镜观察。
6. 结果和分析
胆碱能神经元用多克隆 ChAT 抗体（ 1 :500; 购自美国 Chemicon公司） 测定的结果如图 11所示， 其中左侧脑图中的蓝色小方块表示右侧所示的 脑区位置， A为生理盐水对照组， B为法舒地尔组， 其中 Brdu代表 Brdu 染色， ChAT代表 ChAT染色， Merge代表 Brdu/ChAT染色重叠。 结果显 示， Brdu阳性细胞在三个脑区都明显增加， ChAT阳性的胆碱能神经元也 明显增加（重叠后黄色）， 说明法舒地尔不仅可以动员内源性神经干细胞， 还可以促进这些细胞向胆碱能功能性神经元分化。
本发明已经描述了作为参考的优选实施方案。 通过以上对本发明的详 细描述， 本发明的变化和改进对于所属领域技术人员将是显而易见的。 所 有这些变化和改进都包含在本发明的保护范围之内。

Claims

权 利 要 求
1. 法舒地尔化合物在制备用于诱导成年脑神经干细胞再生和 /或保护 神经功能的药物中的用途。
2. 根据权利要求 1所述的用途， 其特征在于， 其中所述的成年脑神 经千细包为内源性神经千细包。 病的药物中的用途； 优选地， 其中所述的与神经元损伤和 /或死亡相关疾病 主要包括脑缺血和慢性炎性神经变性性疾病。
4. 根据权利要求 3所述的用途， 其特征在于， 其中所述的与神经元损 伤和 /或死亡相关疾病选自脑缺血和慢性炎性神经变性性疾病， 优选地， 所 述慢性炎性神经变性性疾病选自以下一种或几种： 阿尔茨海默病、 帕金森 病、 /L萎缩性侧索硬化、 亨廷顿舞蹈病和多发性硬化。
5. 根据权利要求 1至 4中任一项所述的用途， 其特征在于， 其中所述 的法舒地尔化合物选自： 法舒地尔、 其药学上可接受的盐、 结晶或水合物 等药学上可接受的形式， 其中药学上可接受的盐 选选自盐酸盐、 硝酸盐、 硫酸盐、 磷酸盐、 氢溴酸盐、 曱磺酸盐和柠檬酸盐， 更拔选为盐酸盐。
6. 根据权利要求 1至 5中任一项所述的用途， 其特征在于， 其中所述 的法舒地尔化合物的治疗有效量为 0.5-8mg/kg, 优选有效量为 l-6mg/kg, 更优选有效量为 3-5mg/kg, 最优选有效量为 5mg/kg。
7. 一种诱导成年脑神经干细胞再生和 /或保护神经功能的方法， 其特 征在于， 所述方法包括向有需要的受试者给予治疗有效量的法舒地尔化合 物或包含法舒地尔化合物的药物组合物。
8. 一种预防和 /或治疗与神经元损伤和 /或死亡相关疾病的方法， 其特 征在于， 所述方法包括向有需要的受试者给予治疗有效量的法舒地尔化合 物或包含法舒地尔化合物的药物组合物。
9. 根据权利要求 8所述的方法， 其特征在于， 所述的与神经元损伤和 /或死亡相关疾病选自脑缺血和慢性炎性神经变性性疾病， 优选地， 所述慢 性炎性神经变性性疾病选自： 阿尔茨海默病、 帕金森病、 肌萎缩性侧索硬 化、 亨廷顿舞蹈病和多发性硬化。
10. 根据权利要求 7至 9中任一项所述的方法， 其特征在于， 其中所 述的法舒地尔化合物选自： 法舒地尔、 其药学上可接受的盐， 结晶或水合 物等药学上可接受的形式，其中药学上可接受的益优选选自盐酸盐、硝酸盐、 硫酸盐、 磷酸盐、 氢溴酸盐、 曱磺酸盐和柠檬酸盐， 更 选为盐酸盐。
1 1. 根据权利要求 7至 10中任一项所述的方法， 其特征在于， 其中所 述的法舒地尔化合物或包含法舒地尔化合物的药物组合物的治疗有效量 为 0.5-8mg/kg, 优选有效量为 l-6mg/kg, 更优选有效量为 3-5mg/kg, 最优 选有效量为 5mg/kg。
12. 根据权利要求 7至 12中任一项所述的方法， 其特征在于， 其中所 述的法舒地尔化合物或包含法舒地尔化合物的药物组合物的给药途径包 括口服、 静脉注射、 肌肉注射、 腹腔注射、 皮下注射等本领域已知的任何 给药途径， 优选腹腔注射。
13. 一种用于诱导成年脑神经干细胞再生和 /或保护神经功能的药物 组合物， 其特征在于， 所述的组合物包含法舒地尔化合物以及药学上可接 受的载体； 所述药物组合物的剂型选自： 片剂、 胶嚢、 口服液、 注射剂和 粉针剂， 优选为注射剂， 更优选为静脉注射剂。
14. 一种用于预防和 /或治疗与神经元损伤和 /或死亡相关疾病的的药 物组合物， 其特征在于， 所述的组合物包含法舒地尔化合物以及药学上可 接受的载体； 所述药物组合物的剂型选自： 片剂、 胶嚢、 口服液、 注射剂 和粉针剂， 优选为注射剂， 更优选为静脉注射剂。
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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
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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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