WO2013037207A1 - N-(3-pyridine formoxyl)- 3,5-dimethyl-1- amantadine or its pharmaceutical salt for treating vascular dementia - Google Patents

N-(3-pyridine formoxyl)- 3,5-dimethyl-1- amantadine or its pharmaceutical salt for treating vascular dementia Download PDF

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WO2013037207A1
WO2013037207A1 PCT/CN2012/074351 CN2012074351W WO2013037207A1 WO 2013037207 A1 WO2013037207 A1 WO 2013037207A1 CN 2012074351 W CN2012074351 W CN 2012074351W WO 2013037207 A1 WO2013037207 A1 WO 2013037207A1
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熊晓云
苏建英
唐柳
张敬乐
赵艳菊
赵艳丽
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西安利君制药有限责任公司
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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
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds
    • 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 present invention relates to a novel indication of N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine or a pharmaceutically acceptable salt thereof, that is, in the treatment of vascular dementia application.
  • Vascular dementia is caused by dementia caused by cerebrovascular disease, such as multiple infarction and cerebral hemorrhage. It is mainly caused by cerebral arteriosclerosis, cerebral vascular stenosis, and insufficient oxygen supply to the brain.
  • MID multi-infarct dementia
  • VD vascular dementia
  • MID multi-infarct dementia
  • VD vascular dementia
  • VD VD ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • Vascular dementia is very similar to the symptoms of Alzheimer's disease. It is often mixed in clinical practice. Alzheimer's disease (Alzheimer's disease, AD) is a progressively developing lethal nerve. Degenerative diseases, clinical manifestations of cognitive and memory function deterioration, progressive decline in daily living ability, and a variety of neuropsychiatric symptoms and behavioral disorders. There are differences between the two: 1. Different causes: Vascular dementia is caused by atherosclerosis in the brain arteries of patients, causing slow blood flow in the brain. Caused by brain tissue ischemia, hypoxia or even cerebral infarction; the cause of senile dementia is still not very clear, clinically it is believed that the disease is caused by degeneration and necrosis of the brain cells of the patient. 2.
  • vascular dementia patients often has volatility, and its condition is light and heavy, showing a trend of step-type progression, and relative relief can occur; patients with the disease can be treated after treatment. Significant relief.
  • the course of senile dementia tends to be a slow development trend, and there is no relative remission period. The symptoms are relatively fixed and there is no obvious volatility.
  • vascular dementia patients often have high blood pressure, cerebrovascular, atherosclerosis and other medical history; and senile dementia patients have a history of hypertension and cerebrovascular disease are not many people.
  • vascular dementia is often more acute, the family members of the patient can generally accurately describe the time of onset; and most of the senile dementia is relatively insidious, no obvious time limit, the patient is often unable to Accurately say the specific time of its onset.
  • clinical manifestations are slightly different: patients with vascular dementia may appear emotional instability, temper tantrums, prone to depression and fear of anxiety in the early stages of the disease, in the late stage, there will be emotional out of control; senile dementia patients in the onset After the symptoms can appear, apathy, slow response and other symptoms, a small number of patients may have smirk for unknown reasons. 6.
  • vascular dementia The principle of treating vascular dementia is to improve cerebral blood flow, prevent cerebral infarction, and promote brain metabolism to prevent the disease from developing and improving symptoms.
  • the following drugs can be mainly used to treat various symptoms caused by vascular dementia: 1.
  • Vasodilators These drugs can mainly increase cerebral blood flow, such as brain sputum, ring palm butter, flunar chloride ⁇ (Sibiling) and so on;
  • brain metabolism regulating drugs These drugs can mainly improve cognitive functions, such as hydrogenated ergot (Xid Dezhen), Duxie, Naofukang, resuscitation, chloroester awake, Huperzine A has different degrees of improvement in intelligence.
  • anticoagulant drugs heparin, dicoumarin, new anti-coagulation tablets.
  • thrombolytic drugs streptokinase, urokinase and so on. 5.
  • Drugs for improving psychotic symptoms Anti-anxiety and anti-depressant drugs, such as sulpiride, Jiajing Anding, Luola, and Duoyi; When using the above drugs, due to slow absorption and excretion of drugs, accumulation of poisoning and side effects are prone to occur, and only Can improve a certain aspect of the symptoms.
  • drugs currently used to treat the disease such as a small variety of targets, a single target, and poor long-term efficacy. It is urgently needed for research and development with new features, effective and safe. VD treatment drugs.
  • the present invention resides in the use of the compound (3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine or a pharmaceutically acceptable salt thereof, which is disclosed in Patent No.: 200710130249. 4, Patent Name: N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine or a pharmaceutically acceptable salt thereof for the treatment of Alzheimer's disease.
  • the research of the present invention shows that the dementia model induced by chronic hypoperfusion injury in rats has the effect of delaying the progression of the disease and improving the memory; reducing the degree of neuropathy in the model of multiple infarction dementia, and protecting the normal function of the neurons; the focal cerebral ischemia in rats Reperfusion injury can reduce the protective effect; can significantly improve the acquired memory impairment in mice induced by scopolamine; increase the cellular activity of glutamate-injured neurons and increase the growth factor (NGF) and synaptophysin (SYN) in culture medium Levels reduce the intracellular calcium concentration and increase the length of the synapse; there is an increase in the cell activity of the 0 2 injured neurons.
  • NTF growth factor
  • SYN synaptophysin
  • the object of the present invention is to provide N-(3-pyridine formyloxy)-3, 5
  • NGF nerve growth factor
  • SYN synaptophysin
  • the pharmaceutically acceptable salt is selected from the group consisting of: hydrochloride, nitrate, sulfate, hydrobromide, phosphate, formate, acetate, benzoate, malate, Maleate or citrate.
  • the pharmaceutical preparation form is selected from the group consisting of: a tablet, a capsule, an oral solution, a granule or a powder.
  • the application of the invention has a clear effect in improving the ability of learning and memory, regulating the level of neurotransmitters, and regulating the expression of NMDA receptors; and has a certain effect on anti-inflammatory;
  • the compound of the present invention is a compound in which nicotinic acid and memantine hydrochloride are coupled by an amide bond.
  • the compound can not only avoid the side effects of niacin oral administration, but also synergistically exert the multi-target dementia prevention and treatment effect of nicotinic acid and memantine; and can also make it easier to pass blood by increasing the fat solubility and metabolic stability of memantine hydrochloride.
  • the brain barrier increases the concentration and duration of action in the cerebrospinal fluid, thereby achieving the goal of reducing peripheral adverse reactions of memantine hydrochloride.
  • MeN061016_l has a novel mechanism of action. In addition to treating Alzheimer's disease, it can also find a new treatment plan for clinical treatment of vascular dementia, with great social value and clinical value.
  • the compounds of the present invention are also superior in therapeutic efficacy to the prior art, such as superior to niacin and memantine hydrochloride.
  • the beneficial effects of the present invention are further illustrated by experimental data, which is referred to as MEN061016-11 or MEN in the experiment.
  • Rat hydrated chloral aldehyde anesthesia (350mg/kg), midline incision in the neck, bilateral common carotid arteries were isolated and ligated (surgical group only separated without ligating the common carotid artery), sutured and returned to the word, penicillin against infection for 4 days.
  • Chronic cerebral ischemia is a common pathological process in the development of various diseases such as vascular dementia (VaD), Alzheimer's disease (AD) and Binswanger disease.
  • VaD vascular dementia
  • AD Alzheimer's disease
  • Binswanger disease vascular dementia
  • Studies have confirmed that blood flow can continue to decrease after bilateral common carotid artery ligation (2VO) in rats, chronic phase after 3 weeks, and still lower than normal group in 1 month; neuronal atrophy and degeneration involved in learning and memory in cortex and hippocampus Loss was progressively aggravated; the binding strength of M-type acetylcholine receptors in cortex and hippocampus decreased 3 months after 2VO; behavioral behaviors such as learning and memory showed significant obstacles.
  • 2VO bilateral common carotid artery ligation
  • NR1, NR2A and NR2B receptors in the five dose groups were significantly inhibited.
  • Further in situ hybridization analysis of NMDA receptor subunit mRNA in rat prefrontal cortex showed that high expression of NR1 mRNA was significantly inhibited in the 5-40 mg/kg dose group, and high expression of NR2A mRNA and NR2B mRNA was also significantly inhibited in the 2.5-40 mg/kg dose group.
  • Pathological examination showed that the neurons in the anterior cortex of the model group were sparsely arranged, the nucleus was shrunk, the nucleoli were small, deep stained, the surrounding space was visible, the nucleolus disappeared, the neuron degeneration and necrosis were severe, and there was obvious softening lesion.
  • MEN 2.5-40mg/kg group The precortical cells are arranged in order, the intercellular structure is tight, the nuclear membrane is full, and the neuropathy is reduced to some extent.
  • MEN061016 can significantly improve the ability of learning and memory, its mechanism may be related to inhibiting NMDA receptor, reducing GLU/GABA ratio, protecting damaged nerve cells, thereby indirectly increasing hippocampal ACh levels, but at the same time, 40mg/kg is observed to be irritating and nervous. Phenomenon, the specific reasons need further investigation.
  • the external carotid artery was ligated, the common carotid artery was clamped by the artery, and O.lmL KMG was injected from the external carotid artery into the internal carotid artery with an injector.
  • the artery clamp was loosened, the external carotid artery was ligated, the wound was sutured, and the cage was replanted.
  • the successful models were divided into 8 groups: sham operation group, model group, memantine hydrochloride 20 mg/kg group, niacin 20 mg/kg group, memantine hydrochloride + niacin equimolar concentration (Meijing just 13.5 mg + niacin 7.7 mg/ Kg) group, MEN 2.00mg/kg group, MEN 6.3mg/kg group, MEN 20.00mg/kg group.
  • the intragastric administration was started 10 days after the operation, and the dose was 2 ml/kg.
  • the sham operation group and the model group were intragastrically administrated with the same volume of distilled water once a day for 60 days.
  • the method of injecting the internal carotid artery into the internal carotid artery caused a multi-infarct dementia (MID) model in rats, and MEN061016 was administered by gavage for 2 months to observe the therapeutic effect of the drug on MID.
  • the results showed that the model group showed learning and memory impairment, the MORRIS water maze latency and the path length of the search platform were significantly prolonged, the cortical and hippocampal GLU, GABA levels were significantly decreased, NMDA receptors NR1, NR2A, NR2B over-expression, cortical tissue homogenate The CHAT activity was significantly reduced and the TNF level was significantly increased. It indicated that the model group had degeneration and necrosis of neurons after ischemia, and the amino acid level and acetylcholine synthesis ability related to learning and memory decreased, and the learning and memory ability decreased.
  • MID multi-infarct dementia
  • memantine hydrochloride inhibited NMDA receptors
  • increased brain GLU levels and CHAT activity were slightly stronger than hydrochloric acid, and the ability to learn and memory and inhibit inflammatory reactions was weaker than that of hydrochloric acid.
  • the learning and memory ability of MEN group at the same dose was better than that of memantine hydrochloride group and niacin group.
  • the increase of GLU level in cortex and hippocampus was smaller than that in memantine group greater than niacin group, and MEN inhibited the high expression of NMDA receptor NR1 and NR2A.
  • the intensity was weaker than that of memantine hydrochloride, which was stronger than that of hydrochloric acid.
  • MEN increased the CHAT activity of brain tissue more strongly than the two and was significantly different from the niacin group. MEN inhibits TNF levels in brain tissue more strongly than memantine hydrochloride, which is comparable to hydrochloric acid.
  • MEN can improve the learning and memory ability of MID rats, significantly increase the level of GLU in cortex and hippocampus, significantly inhibit the expression of NMDA receptors NR1, NR2A and NR2B, significantly enhance cortical CHAT activity, and significantly reduce cortical TNF levels. It suggests that MEN can protect the normal function of neurons, inhibit the overexpression of NMDA receptors in damaged brain tissue, maintain the balance of amino acid release, increase the ability of acetylcholine synthesis and inhibit inflammatory reaction. This may be the role of MEN in the treatment of multiple infarct dementia in rats. way. Compared with the reference drug, memantine and niacin, the different indicators have different strengths and weaknesses.
  • model group memantine hydrochloride 20 mg/kg group, niacin 20 mg/kg group; memantine hydrochloride + niacin equimolar concentration (Meijing just 13.5 mg+ Niacin 7.7mg/kg) group, MEN 2.00 mg/kg group, MEN 6.3 mg/kg group, MEN061016-1 20.00 mg/kg group; intragastric administration in 2 mL/kg volume.
  • sham operation group only the CCA was ligated with ECA, and the nylon thread was not inserted, and distilled water was administered in a volume of 2 mL/kg.
  • NMDA receptors NR1, NR2A and NR2B were observed by immunohistochemistry through dehydration, waxing, embedding, sectioning, dewaxing to water and antigen retrieval. 2mm at the back of the cardia, a knife was cut in the crown, and the two parts were loaded into the cryotube, and the liquid nitrogen was solidified.
  • the latter part of the brain area was decocted with 0.15M HCL0 4 (lOOmg brain weight; 3 ⁇ 4lmL) 14000xg and centrifuged at 4°C for 20min. The supernatant was removed and filtered through a 0.22 ⁇ filter. The autosampler was added 50 ⁇ l of the derivatized reagent to the 25 ⁇ l sample, mixed 3 times, allowed to stand for 1 min, and injected 20 ⁇ l. The anterior part of the brain was made into 10% homogenate with physiological saline and centrifuged at 3000 rpm for 30 min. The activity of SOD and GSH-PX and the content of MDA in the ischemic brain tissue were measured. The NOS activity and NO content were determined by plasma.
  • the rat model of focal cerebral ischemia was induced by suture method. After 1 hour, it was reperfused and administered intragastrically the next day after reperfusion for 7 days.
  • the analysis may be caused by a large amount of peroxide and inflammatory reaction after cerebral ischemia, and the compensatory enhancement of antioxidant SOD by brain tissue.
  • GSH-PX activity decreased levels of GLU and GAB A in the brain, overexpression of NMDA receptor subtypes NR1, NR2A and NR2B, decreased CHAT activity; analysis is caused by neuronal apoptosis or necrosis caused by focal cerebral ischemia .
  • MEN061016-1 has a protective effect on focal cerebral ischemia-reperfusion injury in rats.
  • the experiment was divided into 7 groups: model group; memantine hydrochloride 20 mg/kg group; niacin group; memantine hydrochloride + niacin equimolar group (memantine 13.5 mg + niacin 7.7 mg/kg); MEN 2 mg/kg Group; MEN 6.32 mg/kg group; MEN 20 mg/kg group.
  • the ventilator parameters were respiratory rate 70 beats / min, breath ratio 1:2, and tidal volume 9 ml.
  • the medical oxygen flow rate was 900 ml/min and the isoflurane concentration was 3%.
  • the laser Doppler flowmeter was connected to record the cerebral blood flow (rCBF). When the blood flow was stable for 10 min, the CCA silk suture was ablated, the ICA was clamped with an arterial clip, and the incision was cut at the ECA to insert a diameter of 0.24 mm.
  • the nylon thread has obvious resistance, and the insertion depth is 20 ⁇ 2 mm (from the bifurcation of the common carotid artery), which causes the middle cerebral artery to block the ischemia.
  • the rCBF is reduced by more than 70% as the model success index, and the standard is not discarded. .
  • Duodenal administration was carried out after the blood flow of the model was reduced to a standard.
  • the volume of the drug was 2 mL/kg.
  • the laser Doppler flowmeter was used to record the rCBF of the rat before ischemia to 120 min after ischemia.
  • the nylon thread is released, the CCA slip is released, reperfusion is performed, and the observation is continued for 60 min.
  • the set temperature of the thermal blanket during the experiment was 37.0 ⁇ 0.5 °C.
  • the rat model of cerebral ischemia-reperfusion injury was induced by suture method.
  • the ischemic immediate doep-duodenal administration was performed.
  • Laser Doppler flowmetry recorded 120 minutes before ischemia and 120 minutes after reperfusion.
  • the results showed that compared with the model group, the rCBF of the control niacin group increased significantly after administration, and continued to 120 minutes after ischemia, and the blood flow continued to increase after reperfusion; the comparator drug memantine group, memantine + tobacco There was no significant increase in rCBF in the acid equimolar group and the MEN three dose groups. There was no significant difference in rCBF after reperfusion.
  • the effect of blood re-recovery (reperfusion) on brain tissue after focal ischemic cerebral ischemia is affected by ischemic time and reperfusion blood flow.
  • ⁇ ⁇ Ischemia after a long period of time a large number of reflows will form reperfusion Sexual injury further aggravates brain tissue damage.
  • drugs can increase blood supply to brain tissue during ischemia, and reduce damage caused by hypoxia in brain tissue. When the blood flow is restored, the blood supply is obtained from the brain tissue that has not reached the level of irreversible damage. Increased blood flow, brain tissue damage and infarct size shrink. The drug does not improve the ischemia-induced hypoxia during the ischemic phase.
  • Animals were divided into 7 groups: blank control group, model group, memantine hydrochloride 30 mg/kg group, niacin 30 mg/kg group, memantine hydrochloride + niacin equimolar group (Memantine 20.2 mg + niacin) 11.52mg/kg), ⁇ 3.0mg/kg group, MEN 9.5mg/kg group, MEN 30.0mg/kg group.
  • mice were intragastrically administered daily at a dose of 20 mL/kg, and the blank control group and the model group were given distilled water once daily for 15 consecutive days. On the 14th day, 50 minutes after the administration, the blank control group was intraperitoneally injected with physiological saline at a volume of 10 mL/kg, and the other groups were injected with scopolamine (6 mg/kg). After lOmin, the mice were placed in the automatic control system of the platform for 3 minutes, then energized, and the mice were subjected to electric shock for 5 times. When the mice were shocked, they jumped onto the platform, escaped the electric shock, and trained for 5 minutes to obtain memory.
  • each group of animals was placed in a platform automatic control instrument to measure the number of times (the number of errors) and the time of occurrence (latency) of the mice that were subjected to the electric shock from the platform. The results were statistically processed (t-test).
  • mice M-receptor blocker scopolamine before training can cause a decrease in acetylcholine content in the brain and formation of acquired memory defects.
  • the platform test was carried out on the basis of this chemical damage, and the influence of MEN061016-1 on the model was observed by using the error number and latency of the mouse within 5 minutes.
  • the results showed that MEN061016-1 significantly improved scopolamine-induced memory impairment in mice, suggesting that the drug may have an effect on increasing acetylcholine content in the brain. See Table 21.
  • MEN061016-1 Effects of MEN061016-1 on neurons, glial cells and brain microvascular endothelial cells
  • the third generation cells 90% confluent cultured brain microvascular endothelial cells were digested and passaged, and inoculated in 2% gelatin coating.
  • the 96-well plate the number of viable cells in the trypan blue, adjusted the pass density to 1*10 5 ⁇ cm 2 .
  • dosing After 37 hours of incubation at 37 ° C, 5 % C0 2 , dosing, and 2 ⁇ glutamic acid treatment for 2 h after 24 h , measuring MTT.
  • the above tissue fluid was collected in a centrifuge tube, centrifuged at 2000 rpm for 5 min, the supernatant was removed, the complete culture solution was added to terminate the digestion, and the cells were repeatedly gently blown with a fine-diameter pipette until a uniform cell suspension was formed. It was then filtered through a 200 mesh stainless steel mesh to form a single cell suspension. The cells were counted by a hemocytometer, adjusted to a cell density of 5. OX 10 5 /ml, and the cells were seeded in a sterile plastic flask previously immersed in 0.001% polylysine, 5 ml per bottle, and then placed. In a 37 ° C, 5% C0 2 incubator, the culture medium was changed after 24 h. Thereafter, the culture solution was changed once every 2-3 days, and 1/2 culture solution was replaced each time. Passage was performed when monolayer cells were grown and fused into a sheet.
  • the supernatant was removed, and the whole culture solution was blown to re-disperse the cells into a cell suspension.
  • the cell concentration was adjusted to 1.0 X 10 Vml, and the cells were seeded in a 96-well plate coated with polylysine. Incubate inside. After 24 hours, all the culture medium was changed, and the third half was changed for the experiment.
  • the pre-dosing was carried out for 24 hours. After 24 hours, 2 ⁇ glutamic acid was treated for 2 hours, and MTT was measured. There are three kinds of pre-dosing, post-dosing and normal dosing. Each drug has 3 doses, which are 200nM, 400nM and 800nM respectively. Repeat each experiment. 3 results
  • glutamate modeling was used to simulate brain damage. Glutamate is an excitatory amino acid in the brain, and its increase is an intermediate link in many brain injuries. Inhibition of glutamate damage can lead to the conclusion of brain cell protection.
  • the drugs used in this experiment are all finished products.
  • the molar number is calculated according to the molecular weight.
  • the molar concentration is the same.
  • each group is compared with the model group.
  • the MEN is in the star glue.
  • the effect of cytoplasmic cell administration for 48 hours was significant, which was significantly better than that of memantine and niacin groups. Pre-dosing also performed well, and the difference was significant compared with the model group. ⁇ 0. 01, has a good protective effect, but there is no significant difference compared with the memantine group.
  • the high dose group of memantine showed strong toxicity, and MEN had no obvious toxicity.
  • Niacin also has brain protection. The protective effect on brain microvascular endothelial cells is prominent in memantine; MEN is not prominent.
  • MEN061016-1 has a significant protective effect on glutamate-damaged astrocytes and is slightly superior to memantine at 48 hours of administration. It has a certain enhancement effect on normal brain microvascular endothelial cells and glutamate damage to brain microvascular endothelial cells. The experiment also found that MEN061016-1 showed no significant toxicity to astrocytes and brain microvascular endothelial cells at 800 nM.
  • MEN061016-1 reverses 1-methyl-4-phenylpyridinium ion (MPP+) inhibits glial cell activity
  • the whole brain of the newborn Id SD rats was removed under aseptic conditions, and the meninges and blood vessels were removed, and the cell suspension was digested with 0.25% trypsin, and then inoculated with polylysine 0.11 g/L- 1. After 24 hours of treatment, the medium was changed after 24 hours, and the medium was replaced 3 days later. At d 7 , put it in a constant temperature shaker at 100 r/min -1 for 5 h, discard the medium, digest it with 0.5% trypsin, inoculate it in the culture plate, and grow into a continuous sheet. After the layers, they were administered in groups according to the test requirements, and 6 replicate wells in each group were continued for 48 h.
  • group III mGluRs agonist L-AP4100 ⁇ can reverse ⁇ + 150 ⁇ , which can be inhibited by 200uM group mGluRs antagonist MSOP, while L- ⁇ 4 10 ⁇ and MSOP 50uM have no effect.
  • the results of the test 1 determined the concentration of the drug in the next experiment.
  • Glu is the major excitatory neurotransmitter in the central nervous system. Since there is no enzyme system in the extracellular fluid that inactivates Glu, the main pathway for Glu clearance in the synaptic cleft is the active uptake of Glu by high-affinity Glu transporters (GluTs) on glial cells.
  • GluTs high-affinity Glu transporters
  • 1 ⁇ Methyl ⁇ 4 ⁇ Phenylpyridinium ion 1 ⁇ methyl ⁇ 4 ⁇ phenylpyridinium, MPP + ) can inhibit Glu uptake by glial cells and is considered to be an important mechanism for MPP+ indirect damage to neurons.
  • glutamate receptors can be divided into two categories: ionotropic glutamate recep " tors (iGluRs) and metabotropic glutamate receptors (mGluRs).
  • mGluRs are a class of G-protein coupled receptors widely distributed in the central nervous system. They are divided into three groups according to amino acid sequence homology, signal transduction mechanism and pharmacological properties.
  • Group I mGluRs include mGluRl and mGluR5.
  • Group II mGluRs include mGluR2 and mGluR3, and group III mGluRs include mGluR4, mGluR6, mGluR7 and mGluR8. Studies have shown that each group of mGluRs is distributed on astrocytes.
  • mGluRs on astrocytes can release a variety of Nerve growth factor, including transforming growth factor, nerve growth factor (NGF), etc., exerts neuroprotective effects; in addition, activation of mGluRs on astrocytes can also alter glutathione Metabolism of (glutathione, GSH).
  • GSH glutathione Metabolism of (glutathione, GSH).
  • MPP+ inhibition of astrocyte uptake of Glu may be associated with a direct effect on the function of glutamate transporters (GluTs), using a metabotropic glutamate receptor agonist in groups II and III to activate astrocytes.
  • Group II and III mGluRs can exert neuroprotective effects by promoting GluTs uptake of Glu and further reducing the Glu concentration of extracellular fluid.
  • MEN061016-1 reverses the effect of 1-methyl-4-phenylpyridinium (MPP+) on inhibition of glutamate uptake
  • MPP+ was incubated with astrocytes for 48 h and the ability of astrocytes to uptake [3 ⁇ 4] D, L-Glu was determined. It was found that MPP+ 150 uM significantly inhibited the uptake of Glu by astrocytes. Pre-administration of MEN for 1 h can reverse this inhibition.
  • the mGLU receptor agonists DCG IV and LAP40 100 uM have no additive effect with MEN061016-1MEN. It is speculated that MEN061016-1 has a metabotropic glutamate receptor agonism similar to DCG IV and LAP40.
  • Glu is the main excitatory neurotransmitter in the central nervous system, but excitotoxicity occurs when the extracellular Glu concentration is too high. Glu's neurotoxicity is mediated through NMDA and non-NMDA receptors, but in most pathological conditions, especially in chronic progressive diseases of the central nervous system, NMDA receptors are overexcited to mediate neuronal delay Denaturation or necrosis plays an important role.
  • MPP+ is a neurotoxin commonly used in the study of glutamate toxicity, and it exerts neurotoxic effects mainly by inhibiting mitochondrial complex I.
  • astrocytes were selected as the tool cells of the study. The results showed that MPP+ can significantly inhibit the uptake of Glu without affecting the concentration of astrocyte activity.
  • this study demonstrates that MEN061016-1 can reverse MMP + inhibition of glutamate uptake, and may have similar metabotropic glutamate receptor agonism.
  • Radioligand binding assay Total reaction volume was 200 L 0. 100 L of cortical receptor suspension was added to each tube, different concentrations of drug and 3 ⁇ 4-MK-801. The non-specific binding tube was added with non-labeled MK-801, and the final concentration was 1 mol/L at 0 37 °C for 30 min. The reaction was terminated with 3 m L of ice cold 50 mmol/L Tris-HCl (pH 7. 7). The Whatman filter paper was suction filtered, and the filter was rinsed twice with 3 mL of ice-cold Tris-HC1, and the filter was taken out and dried at 80 °C for 20 min. Put in a scintillation bottle and count with a liquid scintillator
  • the drug or 3 ⁇ 4-MK-801 are all configured with 50 mmol/L Tris-HC1 (pH 7.7).
  • the drug concentration is based on the final concentration and is grouped as follows: (1) lOOul cerebral cortex suspension + 3 H-MK-801 (10nM)
  • Memantine H ydrochlo ride is a novel, low- and moderate-intensity, voltage-dependent, non-competitive NMDA (N-methyl-Daspastate) receptor antagonist that significantly improves Alzheimer's disease (Alzheiner) 's disease, AD) vascular dementia (VaD) patients with cognitive impairment, lack of psychomotor drive, depression, dyskinesia, improve their daily living ability and social activities.
  • NMDA N-methyl-Daspastate
  • AD Alzheimer's disease
  • VaD vascular dementia
  • Glutamate the most common excitatory neurotransmitter in the brain, is closely related to the death of nerve cells in the brain of dementia patients.
  • Increased glutamate concentration or increased sensitivity of ganglia to glutamate can increase receptor NMDA, AMPA and glutamate binding, causing voltage-regulated calcium channels to open or activate the phosphoinositide loop, causing intracellular calcium overload, Lead to neuronal apoptosis.
  • Memantine hydrochloride can non-competitively block NMDA receptors, reduce excessive excitability of NMDA receptors induced by glutamate, prevent apoptosis, thereby improving memory and enhancing cognitive function in patients.
  • memantine hydrochloride Since memantine hydrochloride has low and moderate affinity for NMDA receptors, it blocks the glutamate excitotoxicity and does not prevent glutamate from participating in the physiological functions of normal learning and memory.
  • This study compared the binding affinity of MEN and memantine to NMDA receptors by comparing drug-interventional binding of NMDA receptors in the cerebral cortex to the 3 ⁇ 4-labeled non-competitive NMDA receptor antagonist MK-801.
  • NMDA receptors At present, the molecular structure of NMDA receptors is not fully understood. There are at least six independent sites in pharmacology, which are NMDA/glutamate independent sites, glycine (gly) sites, and Zn 2+ sites on the receptor surface. Point; Mg 2+ site, PCP site located in the ion channel.
  • NMDA receptor antagonists can be divided into competitive and non-competitive antagonists, which in turn can be divided into ion channel blockers, Gly and PA site antagonists.
  • non-competitive NMDA receptor antagonists that have entered the clinic include MK801 and DX.
  • m P ⁇ 0.01 compared with model group: *P ⁇ ft05, K0.01; Table 11 for MID rat hippocampal Glu, GABA levels Impact ( ⁇ s) Qiliang Glu GABA delete ⁇ ⁇ g/g brain weight) ( gg brain weight)
  • Cerebral cortex suspension + memantine 500 ⁇ +3 ⁇ 4- ⁇ -801 ( ⁇ ) 195. 46 + 26. 94
  • Figure 1-2 shows the effect of MEN061016-1 on regional cerebral blood flow in ischemic rats.
  • Figure 3-14 shows the effect of MEN061016-1 on neurons, astrocytes and brain microvascular endothelial cells.
  • Figure 15-17 shows MEN061016- A pair of metabotropic and ionotropic glutamate receptor mechanisms
  • Fig.1 Effect of MEN061016-1 on regional cerebral blood flow (rCBF) in rats with cerebral ischemia/reperfusion
  • Fig.2 Effect of MEN061016-1 on regional cerebral blood flow (rCBF) in rats with cerebral ischemia
  • Figure 14 Pre-dosing of brain microvascular endothelial cells 24h glutamate treatment 2h after adding 120hMTT
  • Figure 15 Effect of agonists and antagonists on MPP+ inhibition of astrocyte MTT
  • IR NaCl crystal smear
  • cm- 1 The absorption peak at cm- 1 is the skeleton vibration of the pyridine ring, which is conjugated to the carbonyl group and displaced to a low wave number.
  • the absorption peak at 1317 cm- 1 is the stretching vibration absorption peak of CN, the carbonyl absorption peak of amide at 1645 cm- 1 , and the stretching vibration peak of NH at 3309 cm- 1 .
  • N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride is pulverized and sieved through a 100 mesh sieve, and weighed, and after passing through a 100 mesh sieve.
  • Pre-gelatinized starch calcium hydrogen phosphate and lactose are mixed, 10% starch slurry is added to make soft material, 16 mesh nylon sieve is used for granulation, wet granules are dried at 40 ° C, and dry granules are mixed with magnesium stearate.
  • the tablet weight is calculated, and a 6 mm dimple or flat die is selected. Oral dosage is recommended for adults 1 tablet / time, 2 times / day.
  • N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride is pulverized and passed through a 100 mesh sieve, weighed, and passed through a 100 mesh sieve.
  • the starch and microcrystalline cellulose are mixed and hooked.
  • Adults recommend oral dosage 1 capsule / time, 2 times / day.

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Abstract

The invention relates to the application of N-(3-pyridine formoxyl)- 3,5-dimethyl-1- amantadine or its pharmaceutical salt in treating vascular dementia.

Description

N- (3-吡啶甲酰氧基) -3, 5-二甲基 -1-金刚烷胺或其可药用盐治疗血管性痴呆 技术领域：  N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine or a pharmaceutically acceptable salt thereof for treating vascular dementia
本发明涉及 N- (3-吡啶甲酰氧基 ) -3, 5-二甲基 -1-金刚烷胺或其可药用盐的 一种新适应症， 即其在治疗血管性痴呆方面的应用。  The present invention relates to a novel indication of N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine or a pharmaceutically acceptable salt thereof, that is, in the treatment of vascular dementia application.
背景技术： Background technique:
血管性痴呆是由于脑血管病变引起的痴呆， 如多发性梗塞以及脑出血等， 主 要是由于脑动脉硬化后， 脑血管变窄堵塞， 脑部氧及血液供给不足造成的。 既往 称多发性梗塞性痴呆 （multi-infarct dement ia, 简称 MID)， 新出版的诊断分 类***都已改称为血管性痴呆 （vascular dementia, 简称 VD)。 VD多见于 60岁 以上伴动脉硬化的老年人， 男性多于女性。血管性痴呆发病率很高， 约 1/4的脑 中风存活者可能发生。 有数据显示， 中国 65岁以上人群痴呆发病率为 3. 9%， 其 中血管性痴呆占半数以上， 严重地影响着老年人的生活质量。  Vascular dementia is caused by dementia caused by cerebrovascular disease, such as multiple infarction and cerebral hemorrhage. It is mainly caused by cerebral arteriosclerosis, cerebral vascular stenosis, and insufficient oxygen supply to the brain. Previously referred to as multi-infarct dementia (MID), the newly published diagnostic classification system has been renamed vascular dementia (VD). VD is more common in older people over 60 years of age with arteriosclerosis, more men than women. The incidence of vascular dementia is high, and about 1/4 of stroke survivors may occur. According to statistics, the incidence of dementia in Chinese people over 65 years old is 3.9%, and vascular dementia accounts for more than half of them, which seriously affects the quality of life of the elderly.
VD 的起病相对较急， 一般是在某次卒中后痴呆症状变得明显。 病程多呈阶 梯式加重。 少数慢性起病的患者， 开始表现为情绪改变， 其后才有记忆和智能损 害。 临床表现主要有以下几方面： 1、 全面的认知功能下降， 包括记忆力、 语言 功能、 视空间能力、 认知功能（计算、 理解、 判断、 抽象思维、 学习能力等）； 2、 脑卒中的症状与体征， 多灶梗塞性痴呆患者多有两次或两次以上的卒中史； 多发 腔隙性； 脑梗死患者常有轻微脑卒中史； 3、 脑卒中与痴呆在时间上有相关性： 卒中后 3个月内发生的痴呆， 认知功能呈突然或阶梯性恶化； 4、 常有强哭、 强 笑及假性球***表现； 5、常有精神行为异常， 如情绪激动、暴躁、精神混乱、 骂人、 虚构等， 但人格相对保持良好； 6、 常合并有抑郁。  The onset of VD is relatively acute, and the symptoms of dementia become apparent after a certain stroke. The course of the disease is mostly stepped and the weight is increased. A small number of patients with chronic onset begin to show emotional changes, followed by memory and intelligence impairment. The clinical manifestations mainly include the following aspects: 1. Comprehensive cognitive decline, including memory, language function, visual spatial ability, cognitive function (computation, understanding, judgment, abstract thinking, learning ability, etc.); Symptoms and signs, patients with multi-infarct dementia have two or more strokes; multiple lacunar; cerebral infarction patients often have a history of mild stroke; 3, stroke and dementia are related in time: Dementia occurring within 3 months after stroke, cognitive function suddenly or stepwise deterioration; 4, often have strong crying, strong laughter and pseudo-ball paralysis; 5, often have mental behavior abnormalities, such as emotional excitement, irritability , mental confusion, deafness, fiction, etc., but the personality is relatively good; 6, often combined with depression.
血管性痴呆与老年性痴呆症状极其相似， 临床上常常将二者混为一谈， 老年 性痴呆， 又称阿尔茨海默病， （Alzheimer' s disease, AD), 是一种进行性发展 的致死性神经退行性疾病， 临床表现为认知和记忆功能不断恶化， 日常生活能力 进行性减退， 并有各种神经精神症状和行为障碍。 二者是有区别的： 1、 病因不 同： 血管性痴呆是由于患者的脑动脉出现了粥样硬化， 引起脑部血液流动缓慢， 导致脑组织缺血、缺氧甚至脑梗塞造成的；老年性痴呆的病因目前尚不十分清楚， 临床上多认为该病是由于患者的脑神经细胞变性坏死所致。 2、 病程不同： 血管 性痴呆患者的病程往往具有波动性， 其病情时轻时重， 呈现出一种阶梯型进展的 趋势， 并可出现相对缓解的时候； 该病患者在接受治疗后病情可明显缓解。 而老 年性痴呆的病程往往呈缓慢发展的趋势， 没有相对的缓解期， 其症状相对比较固 定， 无明显的波动性。 3、 以住病史不同： 血管性痴呆患者往往都有高血压、 脑 血管， 粥样硬化等病史； 而老年性痴呆患者有高血压和脑血管病病史的人不多。 4、 发病时的情况不同： 血管性痴呆往往发病较急， 患者家属一般都可以准确地 说出其发病的时间； 而老年性痴呆大多起病比较隐匿， 无明显的时间界限， 患者 隶属常常无法准确地说出其发病的具体时间。 5、 临床表现略有不同： 血管性痴 呆患者在发病的早期可出现情感不稳定、爱发脾气、容易出现情绪低落和恐惧不 安等症状， 到了晚期则会出现情绪失控； 老年性痴呆患者在发病后可出现情， 感 淡漠、 反应迟钝等症状， 少数患者可出现不明原因的傻笑等。 6、 患者对所患疾 病的态度不同： 血管性痴呆患者对自己出现的记忆力下降、 智力下降、情绪变化 以及身体不适等症状存在着一定的认识能力， 希望得到治疗； 而老年性痴呆患者 则对自己所患的疾病缺乏分析、判断的能力， 有的患者甚至还认为自己的身体很 好， 不需要治疗。 7、 进行辅助检查后的结果不同： 对血管性痴呆患者进行 CT、 核磁共振等辅助检查时可发现他们中多数人患有脑血管疾病：对老年性痴呆患者 进行上述检查后可发现他们中的多数人患有弥漫性脑萎缩。 Vascular dementia is very similar to the symptoms of Alzheimer's disease. It is often mixed in clinical practice. Alzheimer's disease (Alzheimer's disease, AD) is a progressively developing lethal nerve. Degenerative diseases, clinical manifestations of cognitive and memory function deterioration, progressive decline in daily living ability, and a variety of neuropsychiatric symptoms and behavioral disorders. There are differences between the two: 1. Different causes: Vascular dementia is caused by atherosclerosis in the brain arteries of patients, causing slow blood flow in the brain. Caused by brain tissue ischemia, hypoxia or even cerebral infarction; the cause of senile dementia is still not very clear, clinically it is believed that the disease is caused by degeneration and necrosis of the brain cells of the patient. 2. Different course of disease: The course of vascular dementia patients often has volatility, and its condition is light and heavy, showing a trend of step-type progression, and relative relief can occur; patients with the disease can be treated after treatment. Significant relief. The course of senile dementia tends to be a slow development trend, and there is no relative remission period. The symptoms are relatively fixed and there is no obvious volatility. 3, to live a different history: vascular dementia patients often have high blood pressure, cerebrovascular, atherosclerosis and other medical history; and senile dementia patients have a history of hypertension and cerebrovascular disease are not many people. 4, the situation is different at the time of onset: vascular dementia is often more acute, the family members of the patient can generally accurately describe the time of onset; and most of the senile dementia is relatively insidious, no obvious time limit, the patient is often unable to Accurately say the specific time of its onset. 5, clinical manifestations are slightly different: patients with vascular dementia may appear emotional instability, temper tantrums, prone to depression and fear of anxiety in the early stages of the disease, in the late stage, there will be emotional out of control; senile dementia patients in the onset After the symptoms can appear, apathy, slow response and other symptoms, a small number of patients may have smirk for unknown reasons. 6. Patients have different attitudes toward the disease: Patients with vascular dementia have a certain ability to recognize their own memory loss, mental decline, emotional changes and physical discomfort, and hope to get treatment; while patients with senile dementia The disease that one suffers from lacks the ability to analyze and judge. Some patients even think that their body is very good and do not need treatment. 7. The results after the auxiliary examination are different: When CT, MRI and other auxiliary examinations are performed on patients with vascular dementia, most of them may have cerebrovascular diseases: after performing the above examination on patients with senile dementia, they may find them. Most people suffer from diffuse brain atrophy.
治疗血管性痴呆的原则是改善脑血流、预防脑梗塞、促进大脑代谢以达到阻 止病情发展和改善症状的目的。根据治疗原则， 目前临床上主要可以选用下列药 物治疗血管性痴呆引起的各种症状： 1、 血管扩张药： 这类药物主要能增加脑血 流量， 如脑益嗉、 环扁桃脂、 盐酸氟桂嗉 （西比灵） 等； 2、 大脑代谢调节药： 这类药物主要能改善认知功能， 如氢化麦角碱 （喜得镇）、 都可喜、 脑复康、 脑 复新、 氯酯醒、 石杉碱甲等都有不同程度的改善智能作用。 3、 抗凝血药物： 肝 素、 双香豆素、 新抗凝片等。 4、 溶血栓药物： 链激酶、 尿激酶等。 5、 改善精 神症状药物： 抗焦虑及抗抑郁药物， 如舒乐安定、 ***、 罗拉、 多虑平等； 使用上述药物时， 因药物吸收与***慢， 容易发生蓄积中毒与副作用， 且仅能改 善某一方面症状。另外目前用于治疗该疾病的药物存在种类较少、作用靶点单一、 长期疗效欠佳等问题， 临床上迫切需要研究开发具有新型作用特点、有效、 安全 的 VD治疗药物。 The principle of treating vascular dementia is to improve cerebral blood flow, prevent cerebral infarction, and promote brain metabolism to prevent the disease from developing and improving symptoms. According to the principle of treatment, at present, the following drugs can be mainly used to treat various symptoms caused by vascular dementia: 1. Vasodilators: These drugs can mainly increase cerebral blood flow, such as brain sputum, ring palm butter, flunar chloride嗉 (Sibiling) and so on; 2, brain metabolism regulating drugs: These drugs can mainly improve cognitive functions, such as hydrogenated ergot (Xid Dezhen), Duxie, Naofukang, resuscitation, chloroester awake, Huperzine A has different degrees of improvement in intelligence. 3, anticoagulant drugs: heparin, dicoumarin, new anti-coagulation tablets. 4, thrombolytic drugs: streptokinase, urokinase and so on. 5. Drugs for improving psychotic symptoms: Anti-anxiety and anti-depressant drugs, such as sulpiride, Jiajing Anding, Luola, and Duoyi; When using the above drugs, due to slow absorption and excretion of drugs, accumulation of poisoning and side effects are prone to occur, and only Can improve a certain aspect of the symptoms. In addition, there are many types of drugs currently used to treat the disease, such as a small variety of targets, a single target, and poor long-term efficacy. It is urgently needed for research and development with new features, effective and safe. VD treatment drugs.
本发明在于采用化合物 (3-吡啶甲酰氧基 ) -3, 5-二甲基 -1-金刚烷胺或其 可药用盐， 该化合物公开在专利号： 200710130249. 4， 专利名称： 用于老年痴呆 症治疗的 N- (3-吡啶甲酰氧基) -3, 5-二甲基 -1-金刚烷胺或其可药用盐中。本发明 经过研究显示，对大鼠慢性低灌注损伤致痴呆模型有延缓病程发展改善记忆的作 用； 减轻多发梗塞性痴呆模型神经病变程度， 保护神经元正常功能； 对大鼠局灶 性脑缺血再灌注损伤有减轻保护作用；可明显改善东莨菪碱所致小鼠获得性记忆 障碍； 有增加谷氨酸损伤神经元的细胞活性并增加培养液中神经生长因子 (NGF) 和突触素（SYN)水平而减少细胞内钙浓度以及增加突触长度的作用； 有增加 02 损伤神经元的细胞活性作用。其作用机理与抑制受损脑组织 NMDA受体过表达， 维 持氨基酸释放平衡， 增加乙酰胆碱合成能力以及抑制炎性反应等作用有关。提示 本化合物在治疗血管性痴呆有显著作用。 The present invention resides in the use of the compound (3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine or a pharmaceutically acceptable salt thereof, which is disclosed in Patent No.: 200710130249. 4, Patent Name: N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine or a pharmaceutically acceptable salt thereof for the treatment of Alzheimer's disease. The research of the present invention shows that the dementia model induced by chronic hypoperfusion injury in rats has the effect of delaying the progression of the disease and improving the memory; reducing the degree of neuropathy in the model of multiple infarction dementia, and protecting the normal function of the neurons; the focal cerebral ischemia in rats Reperfusion injury can reduce the protective effect; can significantly improve the acquired memory impairment in mice induced by scopolamine; increase the cellular activity of glutamate-injured neurons and increase the growth factor (NGF) and synaptophysin (SYN) in culture medium Levels reduce the intracellular calcium concentration and increase the length of the synapse; there is an increase in the cell activity of the 0 2 injured neurons. Its mechanism of action is related to inhibiting the overexpression of NMDA receptors in damaged brain tissue, maintaining the balance of amino acid release, increasing the ability to synthesize acetylcholine and inhibiting inflammatory responses. This compound is suggested to have a significant effect in the treatment of vascular dementia.
发明内容: Summary of the invention:
本发明目的在于提供 N- (3-吡啶甲酰氧基 ) -3, 5  The object of the present invention is to provide N-(3-pyridine formyloxy)-3, 5
治疗血管性痴呆的药物中的应用。 Application in the treatment of drugs for vascular dementia.
本发明的应用， 其中 (3-吡啶甲酰氧基 ) -3, 5 Application of the invention, wherein (3-pyridine formyloxy) -3, 5
Figure imgf000005_0001
Figure imgf000005_0001
本发明的应用，其中所述应用针对血管性痴呆病程中延缓病程发展和改善记 忆的应用。 The use of the invention, wherein the application is directed to delaying the progression of the disease and improving the memory in the course of vascular dementia.
本发明的应用， 其中所述应用针对减轻多发梗塞性痴呆神经病变程度， 保护 神经元正常功能。  The use of the invention, wherein the application is directed to alleviating the degree of neuropathy in multiple infarct dementia and protecting the normal function of the neuron.
本发明的应用， 其中所述应用针对缺血再灌注损伤的保护作用。  Use of the invention, wherein the application is directed to the protection of ischemia-reperfusion injury.
本发明的应用，其中所述应用针对增加谷氨酸损伤神经元的细胞活性并增加 培养液中神经生长因子（NGF)和突触素（SYN)水平而减少细胞内钙浓度以及增 加突触长度的作用。 The use of the invention, wherein the application is directed to increasing the cellular activity of glutamate-damaged neurons and increasing The effects of nerve growth factor (NGF) and synaptophysin (SYN) levels in culture fluids reduce intracellular calcium concentration and increase synaptic length.
本发明的应用， 其中所述应用针对增加 02损伤神经元的细胞活性作用。 本发明的应用， 其中所述可药用盐选自： 盐酸盐、 硝酸盐、 硫酸盐、 氢溴酸 盐、 磷酸盐、 甲酸盐、 乙酸盐、 苯甲酸盐、 苹果酸盐、 马来酸盐或柠檬酸盐。 The use of the invention, wherein the application is directed to increasing the cellular activity of the 0 2 injured neurons. The use of the present invention, wherein the pharmaceutically acceptable salt is selected from the group consisting of: hydrochloride, nitrate, sulfate, hydrobromide, phosphate, formate, acetate, benzoate, malate, Maleate or citrate.
本发明的应用， 其中所述药物是是任何一种药物制剂形式。  The use of the invention, wherein the medicament is in the form of any pharmaceutical preparation.
本发明的应用， 其中所述药物制剂形式选自： 片剂、 胶囊、 口服液、 颗粒剂 或粉剂。  The use of the invention, wherein the pharmaceutical preparation form is selected from the group consisting of: a tablet, a capsule, an oral solution, a granule or a powder.
本发明的应用， 在改善学***、 调节 NMDA受 体表达方面有明确的效果； 在抗炎方面有一定的作用；  The application of the invention has a clear effect in improving the ability of learning and memory, regulating the level of neurotransmitters, and regulating the expression of NMDA receptors; and has a certain effect on anti-inflammatory;
本发明的化合物为烟酸与盐酸美金刚胺通过酰胺键偶合的化合物。该化合物 不仅可避免烟酸口服的副作用，而且可协同发挥烟酸和美金刚胺的多靶点痴呆防 治作用； 并且还可通过增加盐酸美金刚胺的脂溶性和代谢稳定性， 使其更易通过 血脑屏障， 增加其在脑脊液的分布浓度和作用时间， 从而达到降低盐酸美金刚胺 外周不良反应的目的。 MeN061016_l作用机制新颖， 除能治疗老年痴呆外， 还可 为血管性痴呆临床治疗药物找到全新的治疗方案，具有极大的社会价值和临床价 值。  The compound of the present invention is a compound in which nicotinic acid and memantine hydrochloride are coupled by an amide bond. The compound can not only avoid the side effects of niacin oral administration, but also synergistically exert the multi-target dementia prevention and treatment effect of nicotinic acid and memantine; and can also make it easier to pass blood by increasing the fat solubility and metabolic stability of memantine hydrochloride. The brain barrier increases the concentration and duration of action in the cerebrospinal fluid, thereby achieving the goal of reducing peripheral adverse reactions of memantine hydrochloride. MeN061016_l has a novel mechanism of action. In addition to treating Alzheimer's disease, it can also find a new treatment plan for clinical treatment of vascular dementia, with great social value and clinical value.
本发明的化合物在治疗效果上也优于现有技术， 如优于烟酸和盐酸美金刚。 以下通过实验数据进一步说明本发明的有益效果， 实验中本发明药物称为 MEN061016-11或 MEN。  The compounds of the present invention are also superior in therapeutic efficacy to the prior art, such as superior to niacin and memantine hydrochloride. The beneficial effects of the present invention are further illustrated by experimental data, which is referred to as MEN061016-11 or MEN in the experiment.
MEN061016-11对大鼠慢性低灌注损伤致痴呆模型的影响  Effect of MEN061016-11 on dementia model induced by chronic hypoperfusion injury in rats
1、造模雄 1. Modeling
大鼠水合氯醛麻醉 （350mg/kg)， 颈部正中切口， 分离双侧颈总动脉并结扎 (假手术组只分离不结扎颈总动脉）， 缝合后回笼词养， 青霉素抗感染 4天。  Rat hydrated chloral aldehyde anesthesia (350mg/kg), midline incision in the neck, bilateral common carotid arteries were isolated and ligated (surgical group only separated without ligating the common carotid artery), sutured and returned to the word, penicillin against infection for 4 days.
2、分 ^¾给药  2, divided into ^3⁄4 dose
1个月后进行 MORRIS水迷宫游泳试验，以持续时间为指标筛选出现学习记 忆障碍趋势的大鼠， 随机分组： 模型组， 盐酸美金刚 20mg/kg组， 烟酸 20mg/kg 组， MEN 2.5mg/kg组， MEN 5mg/kg组， MEN 10mg/kg组， MEN 20mg/kg组， MEN6 40mg/kg组。 每日经口给药一次 （2mL/kg)， 连续 3个月。 假手术组和模 型组给予等体积蒸馏水。 One month later, the MORRIS water maze swimming test was conducted, and the rats with the tendency of learning and memory disorder were screened by duration. The model group, model group, memantine hydrochloride 20 mg/kg group, niacin 20 mg/kg group, MEN 2.5 mg /kg group, MEN 5mg/kg group, MEN 10mg/kg group, MEN 20mg/kg group, MEN6 40mg/kg group. Oral administration once daily (2 mL/kg) for 3 consecutive months. Sham operation group and model The group was given an equal volume of distilled water.
3、 结果  3, the results
慢性脑缺血是血管性痴呆 （VaD)、 Alzheimer病 （AD) 及 Binswanger病等 多种疾病发展过程中的一个共同病理过程。 研究证实， 大鼠双侧颈总动脉结扎 (2VO) 后血流可持续下降， 3周后为慢性期， 1个月仍低于正常组； 皮质、 海 马等参与学习记忆的神经元萎缩、 变性、 脱失呈进行性加重； 2VO后 3个月皮 质和海马 M型乙酰胆碱受体结合力下降； 学习记忆等行为学出现明显障碍。  Chronic cerebral ischemia is a common pathological process in the development of various diseases such as vascular dementia (VaD), Alzheimer's disease (AD) and Binswanger disease. Studies have confirmed that blood flow can continue to decrease after bilateral common carotid artery ligation (2VO) in rats, chronic phase after 3 weeks, and still lower than normal group in 1 month; neuronal atrophy and degeneration involved in learning and memory in cortex and hippocampus Loss was progressively aggravated; the binding strength of M-type acetylcholine receptors in cortex and hippocampus decreased 3 months after 2VO; behavioral behaviors such as learning and memory showed significant obstacles.
许多研究表明， 脑缺血后可引起兴奋性氨基酸过度释放， 与抑制性氨基酸 GABA的比例失衡， 导致 NMDA受体过度激活， 细胞内 Ca2 + 超载及氧化应激等 一系列反应， 从而引起线粒体损害， 直至神经元病变或坏死， 功能破坏。 大脑前 额叶和海马的神经元对缺血十分敏感，而这些区域是与学习记忆能力密切相关的 脑区。脑缺血后这些脑区神经元功能和结构的损害会造成与学习记忆相关的神经 递质紊乱， 引起学习记忆能力的下降， 甚至痴呆。 Many studies have shown that after cerebral ischemia can cause excessive release of excitatory amino acids, amino acid imbalance in the ratio of the inhibitory GABA, resulting in excessive activation of NMDA receptors, Ca 2 + overload and oxidative stress in a series of reactions within the cell, causing the mitochondria Damage, until neuronal lesions or necrosis, functional disruption. Neurons in the prefrontal and hippocampus of the brain are very sensitive to ischemia, and these areas are brain regions closely related to learning and memory. Damage to neuronal function and structure in these brain regions after cerebral ischemia can cause neurotransmitter disorders associated with learning and memory, causing a decline in learning and memory, and even dementia.
本试验以慢性低灌注损伤致痴呆模型证实了上述生化、病理学及行为学变化 过程，并观察到 MEN以 2.5-40mg/kg剂量经口给药 3个月，可观察到大鼠 MORRIS 水迷宫寻找平台时间及路径长度显著缩短。 以 HPLC-ECD检测脑内神经递质的 结果表明， 2.5-20mg/kg剂量组前皮层的 GABA水平升高， GLU/GABA降低； 海马 ACh水平升高。 用免疫组化检测大鼠前皮层 NMDA受体表达的结果发现， 给药的五个剂量组 NR1、 NR2A、 NR2B受体高表达均有显著抑制。 进一步的原 位杂交对大鼠前皮层 NMDA受体亚基 mRNA的分析表明， 5-40mg/kg剂量组 NRlmRNA高表达均有显著抑制， 2.5-40mg/kg剂量组 NR2AmRNA、NR2BmRNA 高表达也有显著抑制。病理学检查表明： 模型组前皮层区神经元排列稀疏， 细胞 核可见皱缩， 核仁较小， 深染， 周边可见空隙， 核仁消失， 神经元变性坏死严重， 有明显的软化灶， 还可见间质炎性细胞及胶质细胞增生。 MEN 2.5-40mg/kg组 前皮层细胞排列有序，细胞间结构紧密，核膜较饱满，神经病变有不同程度减轻。 MEN061016可明显提高学***有关， 但同 时观察到 40mg/kg有激惹和紧张的现象， 具体原因有待进一步考察。  In this study, the above-mentioned biochemical, pathological and behavioral changes were confirmed by a model of dementia induced by chronic hypoperfusion injury, and it was observed that MEN was orally administered at a dose of 2.5-40 mg/kg for 3 months, and a rat MORRIS water maze was observed. Looking for platform time and path length is significantly shorter. The results of HPLC-ECD detection of neurotransmitters in the brain showed that the level of GABA in the anterior cortex was increased, GLU/GABA was decreased, and the level of ACh in hippocampus was increased in the 2.5-20 mg/kg dose group. Immunohistochemistry was used to detect the expression of NMDA receptor in rat anterior cortex. The high expression of NR1, NR2A and NR2B receptors in the five dose groups were significantly inhibited. Further in situ hybridization analysis of NMDA receptor subunit mRNA in rat prefrontal cortex showed that high expression of NR1 mRNA was significantly inhibited in the 5-40 mg/kg dose group, and high expression of NR2A mRNA and NR2B mRNA was also significantly inhibited in the 2.5-40 mg/kg dose group. . Pathological examination showed that the neurons in the anterior cortex of the model group were sparsely arranged, the nucleus was shrunk, the nucleoli were small, deep stained, the surrounding space was visible, the nucleolus disappeared, the neuron degeneration and necrosis were severe, and there was obvious softening lesion. Interstitial inflammatory cells and glial cells proliferate. MEN 2.5-40mg/kg group The precortical cells are arranged in order, the intercellular structure is tight, the nuclear membrane is full, and the neuropathy is reduced to some extent. MEN061016 can significantly improve the ability of learning and memory, its mechanism may be related to inhibiting NMDA receptor, reducing GLU/GABA ratio, protecting damaged nerve cells, thereby indirectly increasing hippocampal ACh levels, but at the same time, 40mg/kg is observed to be irritating and nervous. Phenomenon, the specific reasons need further investigation.
结果见表 1-7。 MEN061016-1对多发梗塞性痴呆模型大鼠的影响 The results are shown in Table 1-7. Effect of MEN061016-1 on rats with multiple infarction dementia
1造模雄 1 model male
大鼠水合氯醛腹腔麻醉 （400mg/kg)， 颈部备毛、 消毒， 正中切口， 分离右 侧颈总动脉、 颈内动脉及颈外动脉。 结扎颈外动脉， 动脉夹夹闭颈总动脉， 以注 射器从颈外动脉向颈内动脉注入 O.lmL KMG， 松开动脉夹， 结扎颈外动脉， 缝 合伤口， 回笼词养。 假手术组只分离颈总动脉、 颈内动脉及颈外动脉， 向颈内动 脉注射等体积葡萄糖生理盐水。注射用青霉素钠抗感染四天。手术次日观察动物 行为表现， 以前肢有行为学改变者判定为模型成立 (否则弃去不用)。  Peritoneal anesthesia with chloral hydrate (400mg/kg), hair preparation in the neck, disinfection, median incision, separation of the right common carotid artery, internal carotid artery and external carotid artery. The external carotid artery was ligated, the common carotid artery was clamped by the artery, and O.lmL KMG was injected from the external carotid artery into the internal carotid artery with an injector. The artery clamp was loosened, the external carotid artery was ligated, the wound was sutured, and the cage was replanted. In the sham operation group, only the common carotid artery, the internal carotid artery, and the external carotid artery were separated, and an equal volume of glucose physiological saline was injected into the internal carotid artery. Penicillin sodium for injection was anti-infected for four days. The behavior of the animals was observed on the next day of surgery, and the behavioral changes in the previous limbs were determined to be valid (or discarded).
2分组及给药 2 grouping and administration
将模型成功者分为 8组： 假手术组， 模型组， 盐酸美金刚 20mg/kg组， 烟酸 20mg/kg组， 盐酸美金刚 +烟酸等摩尔浓度 （美金刚 13.5mg+烟酸 7.7mg/kg) 组， MEN 2.00mg/kg组， MEN 6.3mg/kg组， MEN 20.00mg/kg组。 手术 10天后开始 灌胃给药， 给药量 2 ml/kg， 假手术组和模型组均灌胃等体积蒸馏水， 每日一次， 连续 60天。  The successful models were divided into 8 groups: sham operation group, model group, memantine hydrochloride 20 mg/kg group, niacin 20 mg/kg group, memantine hydrochloride + niacin equimolar concentration (Meijing just 13.5 mg + niacin 7.7 mg/ Kg) group, MEN 2.00mg/kg group, MEN 6.3mg/kg group, MEN 20.00mg/kg group. The intragastric administration was started 10 days after the operation, and the dose was 2 ml/kg. The sham operation group and the model group were intragastrically administrated with the same volume of distilled water once a day for 60 days.
3、结果 3, the results
研究认为脑梗塞是否引起痴呆主要与梗塞灶的大小、 多少及部位有关。 调 查表明， 梗死灶体积大于 50mL可以合并痴呆， 大于 lOOmL则经常合并痴呆； 也有调查发现， VaD患者中大面积梗死占 11.2%， 小面积梗死灶占 88.8%， 多发 病灶占 97.6%， 提示病灶体积小， 也可发生痴呆， 尤其是梗死灶数目越多， 痴呆 发生率越高。 也有研究认为， 脑梗死的部位是导致痴呆的关键性因素； 多数报道 指出， CT 上发现有脑室旁白质病变改变者， 痴呆发生率显著增高。 因而基于 MID进行相关研究对判断药物治疗 VaD的有效性有指导作用。  Studies have shown that whether cerebral infarction causes dementia is mainly related to the size, number and location of the infarct. Investigations have shown that infarct volume greater than 50mL can be combined with dementia, more than lOOmL often combined with dementia; also found that VaD patients accounted for 11.2% of large area infarction, small area infarct accounted for 88.8%, multi-lesion accounted for 97.6%, suggesting lesion volume Small, dementia can also occur, especially the greater the number of infarcts, the higher the incidence of dementia. Some studies have also suggested that the location of cerebral infarction is a key factor leading to dementia; most reports indicate that the incidence of dementia is significantly increased in patients with altered white matter lesions on the CT. Therefore, relevant research based on MID has a guiding role in judging the effectiveness of drug treatment VaD.
试验以生物微球注入颈内动脉的方法， 造成大鼠多发梗塞性痴呆（MID)模 型， 灌胃给予 MEN061016 2个月， 观察药物对 MID的治疗作用。 结果显示， 模 型组动物出现学***台的路径长度显著 延长， 皮层和海马 GLU、 GABA水平均显著降低， NMDA受体 NR1、 NR2A、 NR2B过表达， 皮层组织均浆中 CHAT活性显著降低， TNF水平显著升高。表明 模型组大鼠缺血后神经元变性坏死，与学***及乙酰胆碱合 成能力下降， 学习记忆能力降低。  In the test, the method of injecting the internal carotid artery into the internal carotid artery caused a multi-infarct dementia (MID) model in rats, and MEN061016 was administered by gavage for 2 months to observe the therapeutic effect of the drug on MID. The results showed that the model group showed learning and memory impairment, the MORRIS water maze latency and the path length of the search platform were significantly prolonged, the cortical and hippocampal GLU, GABA levels were significantly decreased, NMDA receptors NR1, NR2A, NR2B over-expression, cortical tissue homogenate The CHAT activity was significantly reduced and the TNF level was significantly increased. It indicated that the model group had degeneration and necrosis of neurons after ischemia, and the amino acid level and acetylcholine synthesis ability related to learning and memory decreased, and the learning and memory ability decreased.
给药 4周， 与模型组比较， 烟酸组大鼠 MORRIS水迷宫潜伏期和游泳路径 长度均显著缩短，其他组没有显著性差异;给药 8周第 1天，所有给药组 MORRIS 水迷宫潜伏期均显著缩短， 除美金刚组外其余各组路径长度均显著缩短； 给药 8 周第 5天， 烟酸组和 MEN组潜伏期和路径长度显著缩短， 美金刚及美金刚 +盐 酸组没有显著性差异。 表明各组均有改善记忆能力的作用， 烟酸和 MEN尚有改 善学习能力的作用， 且烟酸起效最快， 而美金刚组学习能力较弱。 4 weeks after administration, compared with the model group, the MORRIS water maze latency and swimming path in the niacin group The lengths were significantly shortened, and there was no significant difference in other groups. On the first day of 8 weeks of administration, the latency of MORRIS water maze was significantly shortened in all drug-administered groups, and the path lengths of all groups except the memantine group were significantly shortened; On the fifth day, the latency and path length of the niacin group and the MEN group were significantly shortened, and there was no significant difference between the memantine and memantine + hydrochloric acid groups. It indicates that each group has the effect of improving memory ability. Niacin and MEN have the effect of improving learning ability, and niacin has the fastest effect, while the memantine group has weak learning ability.
给药 8周后， 盐酸美金刚抑制 NMDA受体、升高脑组织 GLU水平及 CHAT 活性的作用均略强于盐酸， 学***的升高幅度小于盐酸美金刚组大于烟酸组， MEN 抑制 NMDA受体 NR1、 NR2A高表达的强度弱于盐酸美金刚强于盐酸， 抑制 NR2B高表达的强度高于两者， 但组间比较均无显著性差异。 MEN升高脑组织 CHAT活性的作用强于两者且与烟酸组比较有显著性差异。 MEN抑制脑组织中 TNF水平的作用强于盐酸美金刚， 与盐酸相当。  After 8 weeks of administration, memantine hydrochloride inhibited NMDA receptors, increased brain GLU levels and CHAT activity were slightly stronger than hydrochloric acid, and the ability to learn and memory and inhibit inflammatory reactions was weaker than that of hydrochloric acid. There was no significant difference. The learning and memory ability of MEN group at the same dose was better than that of memantine hydrochloride group and niacin group. The increase of GLU level in cortex and hippocampus was smaller than that in memantine group greater than niacin group, and MEN inhibited the high expression of NMDA receptor NR1 and NR2A. The intensity was weaker than that of memantine hydrochloride, which was stronger than that of hydrochloric acid. The intensity of inhibition of high expression of NR2B was higher than that of the two, but there was no significant difference between the groups. MEN increased the CHAT activity of brain tissue more strongly than the two and was significantly different from the niacin group. MEN inhibits TNF levels in brain tissue more strongly than memantine hydrochloride, which is comparable to hydrochloric acid.
MEN可使 MID大鼠提高学***， 显著抑制 NMDA受体 NR1、 NR2A、 NR2B表达， 显著增强皮层 CHAT活性， 显著降低皮层 TNF水平。提示 MEN具有保护神经元正常功能，抑制受损脑组织 NMDA受体过表达， 维持氨基酸释放平衡， 增加乙酰胆碱合成能力以及抑制炎 性反应等作用， 这可能是其治疗大鼠多发梗塞性痴呆的作用途径。与对照药盐酸 美金刚和烟酸比较， 不同的指标作用强弱有所不同。  MEN can improve the learning and memory ability of MID rats, significantly increase the level of GLU in cortex and hippocampus, significantly inhibit the expression of NMDA receptors NR1, NR2A and NR2B, significantly enhance cortical CHAT activity, and significantly reduce cortical TNF levels. It suggests that MEN can protect the normal function of neurons, inhibit the overexpression of NMDA receptors in damaged brain tissue, maintain the balance of amino acid release, increase the ability of acetylcholine synthesis and inhibit inflammatory reaction. This may be the role of MEN in the treatment of multiple infarct dementia in rats. way. Compared with the reference drug, memantine and niacin, the different indicators have different strengths and weaknesses.
结果纖 844。  The result is 844.
MEN061016-1对缺血再灌注损伤恢复期大鼠的影响  Effect of MEN061016-1 on rats with recovery from ischemia-reperfusion injury
1、造模雄 1. Modeling
动物 4.0%水合氯醛腹腔注射麻醉 (400 mg/kg体重)， 保温毯上仰卧位固定， 分离右侧颈总动脉 (CCA；)、 颈内动脉 (ICA)及颈外动脉 (ECA),结扎 ECA并剪断， CCA用丝线打活结暂时阻断， 用动脉夹夹闭 ICA后,于 ECA剪开切口,***直径 为 0.24 mm的尼龙线至有明显阻力，***深度 20±2 mm (从颈总动脉分叉处计）， 致使大脑中动脉阻塞缺血； lh后抽出尼龙线， 放开 CCA丝线， 实现再灌注。 实验过程中保温毯设定温度 37 °C至 37.5 °C， 监测大鼠肛温。 再灌后次日以行为 学障碍判定阳性为模型成功， 随机分组： 模型组， 盐酸美金刚 20mg/kg组， 烟酸 20mg/kg组； 盐酸美金刚 +烟酸等摩尔浓度 （美金刚 13.5mg+烟酸 7.7mg/kg) 组， MEN 2.00mg/kg组， MEN 6.3mg/kg组， MEN061016-1 20.00mg/kg组； 以 2mL/kg 体积灌胃给药。 假手术组只分离 CCA结扎 ECA， 不插尼龙线， 按 2 mL/kg体积 给予蒸馏水。 Animals 4.0% chloral hydrate was intraperitoneally injected (400 mg/kg body weight), placed on the blanket in the supine position, and the right common carotid artery (CCA;), internal carotid artery (ICA) and external carotid artery (ECA) were isolated and ligated. ECA was cut, CCA was temporarily blocked by silk suture, after clamping ICA with arterial clip, the incision was cut at ECA, and a nylon wire with a diameter of 0.24 mm was inserted until there was significant resistance. The insertion depth was 20±2 mm (from the neck total The arterial bifurcation is caused by obstruction of the middle cerebral artery; after lh, the nylon thread is withdrawn, and the CCA thread is released to achieve reperfusion. During the experiment, the temperature of the insulation blanket was set at 37 °C to 37.5 °C, and the anus temperature of the rats was monitored. The model was successfully tested on the next day after reperfusion, and was randomly grouped: model group, memantine hydrochloride 20 mg/kg group, niacin 20 mg/kg group; memantine hydrochloride + niacin equimolar concentration (Meijing just 13.5 mg+ Niacin 7.7mg/kg) group, MEN 2.00 mg/kg group, MEN 6.3 mg/kg group, MEN061016-1 20.00 mg/kg group; intragastric administration in 2 mL/kg volume. In the sham operation group, only the CCA was ligated with ECA, and the nylon thread was not inserted, and distilled water was administered in a volume of 2 mL/kg.
2、 给药及取材  2, drug delivery and materials
连续给药 10d，动物经腹主动脉取血分离血浆，断头取脑，在囟门后 4.3-4.5mm 处冠状切一刀， 将右侧半脑（缺血侧）分离为前后半脑， 后面部分中性*** 固定， 3日后， 经脱水、 浸蜡、 包埋、 切片、 脱蜡至水、 抗原修复等程序， 进行 免疫组化观察 NMDA受体 NR1、 NR2A、 NR2B表达。 在囟门后 2mm处冠状切一 刀，分别将前后两部分装入冷冻管，液氮固化，后部分脑区冰浴中以 0.15M HCL04 勾浆（lOOmg脑重； ¾lmL) 14000xg 4°C离心 20min，取上清液， 0.22μηι滤膜过滤， 自动进样器将 50μ1衍生试剂加入到 25μ1样品中， 混合 3次， 静置 lmin， 进样 20μ1。 前部分脑区以生理盐水制成 10%均浆， 3000rpm离心 30min， 分别测定缺血侧脑组 织 SOD及 GSH-PX活性， MDA含量； 血浆测定 NOS活性及 NO含量。 After continuous administration for 10 days, the animals were separated from the blood by abdominal aorta, and the brain was decapitated. The brain was cut at 4.3-4.5 mm after the cardia, and the right hemisphere (ischemic side) was separated into the anterior and posterior hemispheres. Neutral formalin was fixed. After 3 days, the expression of NMDA receptors NR1, NR2A and NR2B was observed by immunohistochemistry through dehydration, waxing, embedding, sectioning, dewaxing to water and antigen retrieval. 2mm at the back of the cardia, a knife was cut in the crown, and the two parts were loaded into the cryotube, and the liquid nitrogen was solidified. The latter part of the brain area was decocted with 0.15M HCL0 4 (lOOmg brain weight; 3⁄4lmL) 14000xg and centrifuged at 4°C for 20min. The supernatant was removed and filtered through a 0.22 μηι filter. The autosampler was added 50 μl of the derivatized reagent to the 25 μl sample, mixed 3 times, allowed to stand for 1 min, and injected 20 μl. The anterior part of the brain was made into 10% homogenate with physiological saline and centrifuged at 3000 rpm for 30 min. The activity of SOD and GSH-PX and the content of MDA in the ischemic brain tissue were measured. The NOS activity and NO content were determined by plasma.
3、 结果 3, the results
试验用线栓法造成大鼠局灶性脑缺血模型， lh后再灌注， 于再灌注后次日 灌胃给药， 连续 7d。  The rat model of focal cerebral ischemia was induced by suture method. After 1 hour, it was reperfused and administered intragastrically the next day after reperfusion for 7 days.
试验观察到模型组大鼠脑内 MDA水平及 SOD、 GSH-PX活性显著升高， 分 析可能是脑缺血后过氧化物大量产生并发生炎性反应，脑组织代偿性的增强抗氧 化 SOD、 GSH-PX活性； 脑内 GLU及 GAB A水平降低， NMDA受体亚型 NR1、 NR2A及 NR2B过表达， CHAT活性降低； 分析是由于局灶性脑缺血致使神经元 凋亡或坏死造成的。  It was observed that the MDA level and the activity of SOD and GSH-PX in the brain of the model group were significantly increased. The analysis may be caused by a large amount of peroxide and inflammatory reaction after cerebral ischemia, and the compensatory enhancement of antioxidant SOD by brain tissue. , GSH-PX activity; decreased levels of GLU and GAB A in the brain, overexpression of NMDA receptor subtypes NR1, NR2A and NR2B, decreased CHAT activity; analysis is caused by neuronal apoptosis or necrosis caused by focal cerebral ischemia .
观察 MEN061016-1 在改善脑内 GLU、GABA等神经递质异常释放及 NMDA 受体表达、 抗氧化等方面的神经保护作用。 结果表明， 与模型组比较， MEN061016-1三个剂量组脑内 MDA水平显著降低， 20.0mg/kg组血浆 NO水平 显著下降， GLU水平显著升高， NMDA受体 NR1、 NR2A、 NR2B缺血后过表 达显著抑制； 皮层神经元变性程度， 软化灶， 胶质细胞增生以及海马锥体细胞坏 死程度等病变均得到抑制和改善。 提示， MEN061016-1 对大鼠局灶性脑缺血再 灌注损伤有一定的保护作用。  To observe the neuroprotective effects of MEN061016-1 on the abnormal release of neurotransmitters such as GLU and GABA, NMDA receptor expression and anti-oxidation. The results showed that compared with the model group, MDA levels in the brain of MEN061016-1 three groups were significantly decreased, plasma NO levels in the 20.0 mg/kg group were significantly decreased, GLU levels were significantly increased, and NMDA receptors NR1, NR2A, NR2B were isolated. Significant inhibition of overexpression; cortical neuron degeneration, softening, gliosis and hippocampal pyramidal necrosis were inhibited and improved. It is suggested that MEN061016-1 has a protective effect on focal cerebral ischemia-reperfusion injury in rats.
结果见表 15-19 MEN061016-1对缺血大鼠局部脑血流量的影响 The results are shown in Table 15-19. Effect of MEN061016-1 on regional cerebral blood flow in ischemic rats
1、造模雄与分组  1. Modeling and grouping
实验分为 7组： 模型组； 盐酸美金刚 20 mg/kg组； 烟酸组； 盐酸美金刚 +烟 酸等摩尔浓度组 （美金刚 13.5mg+烟酸 7.7mg/kg); MEN 2 mg/kg组； MEN 6.32 mg/kg组； MEN 20 mg/kg组。  The experiment was divided into 7 groups: model group; memantine hydrochloride 20 mg/kg group; niacin group; memantine hydrochloride + niacin equimolar group (memantine 13.5 mg + niacin 7.7 mg/kg); MEN 2 mg/kg Group; MEN 6.32 mg/kg group; MEN 20 mg/kg group.
动物 4.0 %水合氯醛腹腔注射麻醉 (400 mg/kg体重)， 保温毯上仰卧位固定， 分离右侧颈总动脉 (CCA；)、颈内动脉 (ICA) 及颈外动脉 (ECA),结扎 ECA并剪断备 用。 切开大鼠头顶部皮肤， 于囟门后 2 mm， 右 5 mm处电钻磨薄颅骨， 医用胶 固定电极固定座， *** PROBE 407-1 KIT 多用途表面电极， 气管切开， ***三 通， 连接呼吸机及麻醉机维持麻醉深度。 呼吸机参数为呼吸频率 70次 /min， 吸 呼比 1:2.， 潮气量 9ml。 医用氧流量为 900ml/min， 异氟烷浓度为 3%。 大鼠右侧 肋下备皮， 开一 1cm切口， 小心找出十二指肠， 穿线后放回。 连接激光多普勒 血流测定仪， 记录大鼠脑血流量 （rCBF)， 当血流稳定 lOmin后 CCA丝线系活 结， 用动脉夹夹闭 ICA,于 ECA剪开切口， ***直径为 0.24 mm的尼龙线至有明 显阻力， ***深度 20±2 mm (从颈总动脉分叉处计），致使大脑中动脉阻塞缺血， 以 rCBF下降 70 %以上为模型成功指标， 没达到标准的弃去不用。在造模血流下 降达到标准后十二指肠给药， 给药体积为 2 mL/kg, 激光多普勒血流仪记录缺血 前至缺血 120 min大鼠 rCBF， 缺血 2 h拔出尼龙线， 松开 CCA活结， 实现再灌 注， 继续观察 60 min。 实验期间保温毯设定温度为 37.0±0.5 °C。  Animals 4.0% chloral hydrate was intraperitoneally injected (400 mg/kg body weight), placed on the blanket in the supine position, and the right common carotid artery (CCA;), internal carotid artery (ICA) and external carotid artery (ECA) were isolated and ligated. ECA and cut off the spare. Cut the top of the head of the rat, 2 mm behind the cardia, 5 mm right, drill the thin skull, medical glue fixed electrode holder, insert PROBE 407-1 KIT multi-purpose surface electrode, tracheotomy, insert tee, connect The ventilator and anesthesia machine maintain the depth of anesthesia. The ventilator parameters were respiratory rate 70 beats / min, breath ratio 1:2, and tidal volume 9 ml. The medical oxygen flow rate was 900 ml/min and the isoflurane concentration was 3%. Prepare the skin under the ribs on the right side of the rat, open a 1 cm incision, carefully find the duodenum, and put it back after threading. The laser Doppler flowmeter was connected to record the cerebral blood flow (rCBF). When the blood flow was stable for 10 min, the CCA silk suture was ablated, the ICA was clamped with an arterial clip, and the incision was cut at the ECA to insert a diameter of 0.24 mm. The nylon thread has obvious resistance, and the insertion depth is 20±2 mm (from the bifurcation of the common carotid artery), which causes the middle cerebral artery to block the ischemia. The rCBF is reduced by more than 70% as the model success index, and the standard is not discarded. . Duodenal administration was carried out after the blood flow of the model was reduced to a standard. The volume of the drug was 2 mL/kg. The laser Doppler flowmeter was used to record the rCBF of the rat before ischemia to 120 min after ischemia. The nylon thread is released, the CCA slip is released, reperfusion is performed, and the observation is continued for 60 min. The set temperature of the thermal blanket during the experiment was 37.0 ± 0.5 °C.
2、 结果 2, the result
试验用线栓法造成大鼠脑缺血再灌注损伤模型，缺血即刻一次性十二指肠给 药，激光多普勒血流仪记录缺血前至缺血 120 min和再灌后 60 min大鼠局部脑血 流量 （rCBF)。 结果表明， 与模型组比较， 对照药烟酸组大鼠给药后 rCBF显著 增加， 并可持续至缺血后 120min， 再灌后血流量继续升高； 对照药美金刚组、 美金刚 +烟酸等摩尔组及 MEN三个剂量组 rCBF均未见显著增加， 再灌后 rCBF 亦无统计学差异。  The rat model of cerebral ischemia-reperfusion injury was induced by suture method. The ischemic immediate doep-duodenal administration was performed. Laser Doppler flowmetry recorded 120 minutes before ischemia and 120 minutes after reperfusion. Regional cerebral blood flow (rCBF) in rats. The results showed that compared with the model group, the rCBF of the control niacin group increased significantly after administration, and continued to 120 minutes after ischemia, and the blood flow continued to increase after reperfusion; the comparator drug memantine group, memantine + tobacco There was no significant increase in rCBF in the acid equimolar group and the MEN three dose groups. There was no significant difference in rCBF after reperfusion.
大脑局灶性缺血后血液再恢复（再灌注）对脑组织的影响受到缺血时间和再 灌注血流量的影响， ω·缺血较长时间后大量的血流复灌， 会形成再灌注性损伤， 使脑组织损伤进一步加重。如药物在缺血期就能增加对脑组织的供血， 减少脑组 织缺氧造成的损害。血流恢复时，未达到不可逆损伤程度的脑组织获得血流供应， 血流量增加， 脑组织损伤及梗死面积缩小。药物在缺血期对缺血组织缺氧改善不 明显， 未形成有效保护时， 则在血流恢复期适当缓慢增加再灌血流量亦对缺血组 织有一定的益处。 实验仅观察到对照药烟酸有显著增加缺血大鼠 rCBF的作用， 对照药美金刚和受试药 MEN061016-1则无此作用。 提示 MEN061016-1—次性十 二指肠给药， 在缺血 120 min内及再灌注 60 min内无增加缺血大鼠局部脑血流量 的作用。 The effect of blood re-recovery (reperfusion) on brain tissue after focal ischemic cerebral ischemia is affected by ischemic time and reperfusion blood flow. ω · Ischemia after a long period of time, a large number of reflows will form reperfusion Sexual injury further aggravates brain tissue damage. For example, drugs can increase blood supply to brain tissue during ischemia, and reduce damage caused by hypoxia in brain tissue. When the blood flow is restored, the blood supply is obtained from the brain tissue that has not reached the level of irreversible damage. Increased blood flow, brain tissue damage and infarct size shrink. The drug does not improve the ischemia-induced hypoxia during the ischemic phase. When the effective protection is not formed, the appropriate slow increase in the blood flow recovery period and the reperfusion flow also have certain benefits for the ischemic tissue. The experiment only observed that the control drug niacin significantly increased the rCBF of ischemic rats, and the control drug memantine and the test drug MEN061016-1 did not. It is suggested that MEN061016-1-subduodenal administration has no effect on increasing regional cerebral blood flow in ischemic rats within 120 min of ischemia and 60 min after reperfusion.
结果见附表 20， 图 1、 图 2。  The results are shown in Schedule 20, Figure 1, Figure 2.
MEN061016-1对东莨菪碱所致小鼠获得性记忆障碍的影响 Effect of MEN061016-1 on scopolamine-induced acquired memory impairment in mice
1、造模雄  1. Modeling
动物雌雄各半， 随机分为 7组： 空白对照组， 模型组， 盐酸美金刚 30mg/kg 组， 烟酸 30mg/kg 组， 盐酸美金刚 +烟酸等摩尔浓度组 （美金刚 20.2mg+烟酸 11.52mg/kg) ,ΜΕΝ 3.0mg/kg组, MEN 9.5mg/kg组, MEN 30.0mg/kg组。  Animals were divided into 7 groups: blank control group, model group, memantine hydrochloride 30 mg/kg group, niacin 30 mg/kg group, memantine hydrochloride + niacin equimolar group (Memantine 20.2 mg + niacin) 11.52mg/kg), ΜΕΝ 3.0mg/kg group, MEN 9.5mg/kg group, MEN 30.0mg/kg group.
动物每日按 20mL/kg体积灌胃给药，空白对照组及模型组动物给予蒸馏水， 每日一次， 连续 15 日。 第 14天给药后 50min按 lOmL/kg体积给予空白对照组 小鼠腹腔注射生理盐水， 其余各组动物均注射东莨菪碱 （6mg/kg)。 lOmin后将 小鼠放入跳台程序自动控制仪内适应 3min， 然后通电， 使小鼠遭受电击剌激 5 次， 小鼠遭到电击时跳上跳台， 逃避电击， 训练 5min， 使获得记忆。 第 15日给 药后 60min， 将各组动物放入跳台程序自动控制仪中， 测定小鼠离开跳台遭受电 击的次数 （错误次数） 及发生时间 （潜伏期）。 结果进行统计学处理 （t检验）。  Animals were intragastrically administered daily at a dose of 20 mL/kg, and the blank control group and the model group were given distilled water once daily for 15 consecutive days. On the 14th day, 50 minutes after the administration, the blank control group was intraperitoneally injected with physiological saline at a volume of 10 mL/kg, and the other groups were injected with scopolamine (6 mg/kg). After lOmin, the mice were placed in the automatic control system of the platform for 3 minutes, then energized, and the mice were subjected to electric shock for 5 times. When the mice were shocked, they jumped onto the platform, escaped the electric shock, and trained for 5 minutes to obtain memory. 60 minutes after the drug was given on the 15th day, each group of animals was placed in a platform automatic control instrument to measure the number of times (the number of errors) and the time of occurrence (latency) of the mice that were subjected to the electric shock from the platform. The results were statistically processed (t-test).
2、结果  2, the results
训练前给予小鼠 M-受体阻断剂东莨菪碱， 可造成脑内乙酰胆碱含量降低， 形成获得性记忆缺损。 本试验在此化学损伤基础上进行跳台试验， 以小鼠 5min 内错误次数、 潜伏期为指标， 观察 MEN061016-1 对该模型的影响。 结果表明 MEN061016-1 显著改善东莨菪碱所致小鼠获得性记忆障碍， 提示该药可能有增 加脑内乙酰胆碱含量的作用。 见表 21。  Administration of mouse M-receptor blocker scopolamine before training can cause a decrease in acetylcholine content in the brain and formation of acquired memory defects. In this test, the platform test was carried out on the basis of this chemical damage, and the influence of MEN061016-1 on the model was observed by using the error number and latency of the mouse within 5 minutes. The results showed that MEN061016-1 significantly improved scopolamine-induced memory impairment in mice, suggesting that the drug may have an effect on increasing acetylcholine content in the brain. See Table 21.
MEN061016-1对神经元、 胶质细胞及脑微血管内皮细胞的影响  Effects of MEN061016-1 on neurons, glial cells and brain microvascular endothelial cells
1 内皮细胞培养旅及分组 1 Endothelial cell culture brigade and grouping
雄性 SD大鼠 12只， 体重 60-80g， 无菌条件下取出双侧大脑半球， 置预冷的 冲洗液中， 剔除软脑膜、 表面血管及白质， 收集皮质。 剪碎成 lmm3的小块， 置手 动玻璃勾浆器，冰上操作上下勾浆约 25次。依次通过 80目和 200目尼龙网过滤， 收集 200 目网上微血管段。 用 0. 1%胶原酶， 37°C消化 30分钟。 冲洗液冲洗两 遍， 1000r/min离心 5min， 将微血管段重悬于完全培养基中， 接种于 2%明胶预先 包被的 25cm2培养瓶中， 共接种 2瓶。 37°C、 5%(：02培养箱静置培养 48小时后， 第一次换液。 以后 2〜3d更换一次完全培养液， 约 6〜7d细胞基本融合成片， 进 行消化传代， 消化液为 0. 125%的胰蛋白酶 _0. 02%EDTA (1 : 1)。 本实验用第三代 细胞， 90%汇合培养的脑微血管内皮细胞经消化后传代， 接种于 2%明胶包被的 96 孔板， 台盼蓝计活细胞数， 调整传代密度为 1*105 · cm2。 37°C、 5 %C02培养 24小 时后， 加药， 24h后进行 2μΜ谷氨酸处理 2h， 测 MTT。 分预加药， 后加药和正常加 药三种， 每种药物设个计量， 分别为 200nM、 400nM、 800nM。 Twelve male Sprague-Dawley rats weighing 60-80 g were used to remove the bilateral cerebral hemispheres under sterile conditions. The pre-cooled irrigating solution was used to remove the pia mater, surface blood vessels and white matter, and the cortex was collected. Cut into small pieces of lmm 3 , set a manual glass grout, and operate on the ice for about 25 times. Filter through 80 mesh and 200 mesh nylon mesh in turn. Collect 200 mesh online microvascular segments. Digested with 0.1% collagenase at 37 ° C for 30 minutes. Rinse the rinsing solution twice, centrifuge at 1000r/min for 5min, resuspend the microvascular segment in complete medium, inoculate 2ml gelatin pre-coated 25cm 2 culture flask, and inoculate 2 bottles. 37 ° C, 5% (: 0 2 incubator for 48 hours after standing culture, the first time to change the liquid. After 2 to 3 days, replace the complete culture solution, about 6 ~ 7d cells are basically fused into a piece, for digestion and passage, digestion The solution is 0. 125% trypsin_0. 02% EDTA (1: 1). In this experiment, the third generation cells, 90% confluent cultured brain microvascular endothelial cells were digested and passaged, and inoculated in 2% gelatin coating. The 96-well plate, the number of viable cells in the trypan blue, adjusted the pass density to 1*10 5 · cm 2 . After 37 hours of incubation at 37 ° C, 5 % C0 2 , dosing, and 2 μ glutamic acid treatment for 2 h after 24 h , measuring MTT. Pre-dosing, post-dosing and normal dosing three, each drug is set to measure, respectively, 200nM, 400nM, 800nM.
2星形胶质细胞培养纯化 2 astrocyte culture and purification
1 )初接种： 取出生 2— 3d内的大鼠， -20°C预冻 lOmin左右取出，以 75%的洒精 消毒两次， 无菌条件下断头取脑，并置盛有解剖液的培养皿内。 剥除脑膜， 取下 大脑皮质， 剪成碎块后， 用初接种消化液， 在 37°C 5 %C02培养箱消化 30min,中 间振摇 1一 2次。 收集上述组织液于离心管中， 2000rpm离心 5min， 去上清液， 加 入完全培养液终止消化， 并用细口径吸管反复轻轻吹打， 直到形成均勾的细胞悬 液。 再用 200目不锈钢网过滤使成单细胞悬液。 用血球计数板计数细胞， 调节细 胞密度为 5. O X 105个 /ml， 将细胞接种于预先经 0. 001%多聚赖氨酸浸泡的无菌塑 料培养瓶中，每瓶 5ml， 而后置于 37°C、 5%的 C02培养箱内， 24h后全部更换培养液。 此后每 2-3d换培养液一次， 每次更换 1/2培养液。 当单层细胞生长且融合成片状 时进行传代。 1) Initial inoculation: Take out the rats within 2 to 3 days, take out at -20 °C for about 10 minutes, and disinfect with 25% of the sprinkle. Under the sterile conditions, take off the brain and set the anatomy. Inside the petri dish. The meninges were removed, and the cerebral cortex was removed. After cutting into pieces, the digestive juice was inoculated with the initial inoculation, and digested in a 5 ° C0 2 incubator at 37 ° C for 30 min, shaking 1 or 2 times in the middle. The above tissue fluid was collected in a centrifuge tube, centrifuged at 2000 rpm for 5 min, the supernatant was removed, the complete culture solution was added to terminate the digestion, and the cells were repeatedly gently blown with a fine-diameter pipette until a uniform cell suspension was formed. It was then filtered through a 200 mesh stainless steel mesh to form a single cell suspension. The cells were counted by a hemocytometer, adjusted to a cell density of 5. OX 10 5 /ml, and the cells were seeded in a sterile plastic flask previously immersed in 0.001% polylysine, 5 ml per bottle, and then placed. In a 37 ° C, 5% C0 2 incubator, the culture medium was changed after 24 h. Thereafter, the culture solution was changed once every 2-3 days, and 1/2 culture solution was replaced each time. Passage was performed when monolayer cells were grown and fused into a sheet.
2) 转接种： 当 Ast培养至第 8d (细胞融合成片状） 时， 全换培养液， 培养 箱内孵育 2-3h后， 置于 37°C恒温摇床内， 240rpm振荡 18h。 去除全部培养液， 用预温的解剖液洗 2— 3次， 每个培养瓶内加入转接种消化液 1ml进行消化， 镜 下观察， 见 50%— 70%的细胞回缩、 脱落时立即终止消化。 用吸管反复吹打使细 胞从瓶壁脱落， 收集细胞悬液， 2000rpm离心 5min。去上清， 加完全培养液吹打， 使细胞再分散成细胞悬液， 调节细胞浓度为 1. 0 X 10 Vml， 转接种于铺被有多 聚赖氨酸的 96孔板， 置培养箱内孵育。 24h后全部更换培养液， 第 3d换半液， 用于实验， 预加药 24h， 24h后进行 2μΜ谷氨酸处理 2h， 测 MTT。 分预加药， 后加 药和正常加药三种， 每种药物设 3个剂量， 分别为 200nM、 400nM、 800nM。 每个 实验重复一次。 3结果 2) Trans-inoculation: When Ast was cultured until the 8th day (cell fusion into flakes), the culture medium was completely changed, incubated for 2-3 hours in the incubator, placed in a 37 ° C constant temperature shaker, and shaken at 240 rpm for 18 h. Remove all the culture solution, wash it with pre-warmed anatomical solution 2-3 times, add 1 ml of the inoculum digestive solution to each culture flask for digestion, observe under the microscope, see 50% - 70% of the cells retract, immediately fall off Stop digestion. The cells were detached from the wall by repeated pipetting with a pipette, and the cell suspension was collected and centrifuged at 2000 rpm for 5 min. The supernatant was removed, and the whole culture solution was blown to re-disperse the cells into a cell suspension. The cell concentration was adjusted to 1.0 X 10 Vml, and the cells were seeded in a 96-well plate coated with polylysine. Incubate inside. After 24 hours, all the culture medium was changed, and the third half was changed for the experiment. The pre-dosing was carried out for 24 hours. After 24 hours, 2μ glutamic acid was treated for 2 hours, and MTT was measured. There are three kinds of pre-dosing, post-dosing and normal dosing. Each drug has 3 doses, which are 200nM, 400nM and 800nM respectively. Repeat each experiment. 3 results
本实验选用谷氨酸造模模拟了脑损伤。谷氨酸是脑内兴奋性氨基酸， 它的增 多是众多脑损伤的中间环节。 阻抑谷氨酸损伤， 可以得到脑细胞保护的结论。  In this experiment, glutamate modeling was used to simulate brain damage. Glutamate is an excitatory amino acid in the brain, and its increase is an intermediate link in many brain injuries. Inhibition of glutamate damage can lead to the conclusion of brain cell protection.
本实验所用药物均为成品， 按分子量计算出摩尔数， 所加摩尔浓度相同， 同 摩尔浓度之间比较， 各组再分别与模型组比较， 正常加药与正常组比较， MEN 在星形胶质细胞造模后再给药 48小时效果显著， 明显优于美金刚和烟酸组， 预 加药也表现很好， 与模型组比较差异显著？< 0. 01， 具有很好的保护作用， 但与 美金刚组比较无显著差异。美金刚大剂量组表现出较强毒性， MEN没有明显毒性。 烟酸也有脑保护作用。 对脑微血管内皮细胞的保护作用美金刚表现突出； MEN则 表现不突出。  The drugs used in this experiment are all finished products. The molar number is calculated according to the molecular weight. The molar concentration is the same. Compared with the molar concentration, each group is compared with the model group. Compared with the normal group, the MEN is in the star glue. The effect of cytoplasmic cell administration for 48 hours was significant, which was significantly better than that of memantine and niacin groups. Pre-dosing also performed well, and the difference was significant compared with the model group. < 0. 01, has a good protective effect, but there is no significant difference compared with the memantine group. The high dose group of memantine showed strong toxicity, and MEN had no obvious toxicity. Niacin also has brain protection. The protective effect on brain microvascular endothelial cells is prominent in memantine; MEN is not prominent.
MEN061016-1 对谷氨酸损伤的星形胶质细胞有显著的保护作用， 并在给药 48h作用略优于美金刚。对正常脑微血管内皮细胞及谷氨酸损伤脑微血管内皮细 胞活性有一定的增强作用。实验还发现 MEN061016-1在 800nM浓度下对星形胶质 细胞及脑微血管内皮细胞试验中没有表现出明显毒性。  MEN061016-1 has a significant protective effect on glutamate-damaged astrocytes and is slightly superior to memantine at 48 hours of administration. It has a certain enhancement effect on normal brain microvascular endothelial cells and glutamate damage to brain microvascular endothelial cells. The experiment also found that MEN061016-1 showed no significant toxicity to astrocytes and brain microvascular endothelial cells at 800 nM.
从以上结果可以看出， 2μΜ的谷氨酸 2h模型是成功的，造成了细胞的可逆 性损伤， 利于药效的观察， 且比较灵敏。脑微血管内皮细胞和星形胶质细胞都是 脑内神经元的支持细胞， 是神经血管单元的组成部分， 是药物作用到神经元的必 由之路。  From the above results, it can be seen that the 2 μΜ glutamic acid 2 h model is successful, resulting in reversible damage of the cells, which is advantageous for observation of drug efficacy and is relatively sensitive. Both brain microvascular endothelial cells and astrocytes are supporting cells of neurons in the brain, which are components of neurovascular units and are the only way for drugs to act on neurons.
结果见图 3-14。  The results are shown in Figure 3-14.
MEN061016-1对亲代谢型和离子型谷氨酸受体机制研究 Study on the mechanism of metabotropic and ionotropic glutamate receptors in MEN061016-1
1、 MEN061016-1逆转 1-甲基- 4-苯基吡啶离子（MPP+)抑制 胶质细胞活性 1. MEN061016-1 reverses 1-methyl-4-phenylpyridinium ion (MPP+) inhibits glial cell activity
①星形胶质细胞培养 1 astrocyte culture
无菌条件下取出新生 Id SD 大鼠的全脑， 去除脑膜和血管， 用 0. 25%胰蛋白 酶消化成细胞悬液后， 接种到预先用多聚赖氨酸 0. 1 g/L—1处理 24 h 的培养瓶内， 24 h 后更换培养基， 以后 3 d 更换一次培养基。 d 7 时， 放入恒温摇床内 100 r/min— 1 振摇 5 h， 弃去培养基， 用 0. 5% 的胰蛋白酶消化吹勾后， 接种于培养 板中， 待长成连续单层后， 按试验需要分组给药， 每组 6个复孔， 继续培养 48 h。The whole brain of the newborn Id SD rats was removed under aseptic conditions, and the meninges and blood vessels were removed, and the cell suspension was digested with 0.25% trypsin, and then inoculated with polylysine 0.11 g/L- 1. After 24 hours of treatment, the medium was changed after 24 hours, and the medium was replaced 3 days later. At d 7 , put it in a constant temperature shaker at 100 r/min -1 for 5 h, discard the medium, digest it with 0.5% trypsin, inoculate it in the culture plate, and grow into a continuous sheet. After the layers, they were administered in groups according to the test requirements, and 6 replicate wells in each group were continued for 48 h.
②结果 2 results
试验一、 II组 mGluRs激动剂 DCG_IV、 III 组 mGluRs 激动剂 L一 AP4， II组 mGluRs 拮抗剂 APICA和 III组 mGluRs拮抗剂 MSOP对 MPP+抑制星形胶质细胞 MTT的影响 本实验结果发现， ΜΡΡ+150 μ Μ可明显抑制星形胶质细胞增殖。 II组 mGluR4 激动剂 DCG-IV100 μ Μ可逆转 ΜΡΡ+150 μ Μ作用，该作用可被 200uMII组 mGluRs 拮抗 剂 APICA抑制， 而 DCG- ΐν ΐΟ μ Μ、 APICA 50uM 没有作用。 同样， III 组 mGluRs 激 动剂 L一 AP4100 μ Μ可逆转 ΜΡΡ+150 μ Μ作用，该作用可被 200uMIII组 mGluRs拮抗剂 MS0P抑制， 而 L一 ΑΡ4 10 μ Μ、 MSOP 50uM 没有作用。 试验一结果确定了下一步实 验的工具药浓度。 Test I, group II mGluRs agonist DCG_IV, group III mGluRs agonist L-AP4, group II mGluRs Antagonist APICA and group III mGluRs antagonist MSOP on MPP + inhibition of astrocyte MTT The results of this experiment found that ΜΡΡ + 150 μ Μ can significantly inhibit astrocyte proliferation. Group II mGluR4 agonist DCG-IV100 μ Μ reversible ΜΡΡ + 150 μ Μ, this effect can be inhibited by 200uMII group mGluRs antagonist APICA, while DCG- ΐν ΐΟ μ Μ, APICA 50uM has no effect. Similarly, group III mGluRs agonist L-AP4100 μΜ can reverse ΜΡΡ + 150 μΜ, which can be inhibited by 200uM group mGluRs antagonist MSOP, while L-ΑΡ4 10 μΜ and MSOP 50uM have no effect. The results of the test 1 determined the concentration of the drug in the next experiment.
试验二、 MEN061016-1对 MPP+抑制星形胶质 MTT的影响 Experiment 2: Effect of MEN061016-1 on MPP+ inhibition of astrocyte MTT
本实验测定了 MPP+对星形胶质细胞活性的影响。 结果发现， ΜΡΡ+150 μ Μ可以 抑制星形胶质细胞活力， ΜΕΝ50、 500ηΜ均不同程度可逆转 ΜΡΡ作用， 推测 MEN可能 有 II、 III组 mGluRs受体激动作用或 GluTs激活作用。 进一步研究发现 I I、 III组 mGluRs受体激动剂 DCG_IV、 L一 AP4与 Men无叠加作用， II组 mGluRs 拮抗齐 [JAPICA 和 III组 mGluRs拮抗剂 MS0P可逆转 Men作用， 推测 MEN具有部分 II、 III组 mGluRs 受体激动作用。 This experiment determined the effect of MPP+ on astrocyte activity. It was found that ΜΡΡ + 150 μΜ can inhibit the astrocyte viability, and ΜΕΝ50 and 500ηΜ can reverse the sputum effect to different degrees. It is speculated that MEN may have group II and III mGluRs receptor agonism or GluTs activation. Further studies found that group II and III mGluRs receptor agonists DCG_IV, L-AP4 and Men have no additive effect, group II mGluRs antagonize Qi [JAPICA and group III mGluRs antagonist MSOP can reverse the effect of Men, speculation that MEN has some groups II and III mGluRs receptor agonism.
结果见图 15-16。  The results are shown in Figure 15-16.
③结论 3 conclusions
Glu 是中枢神经***中主要的兴奋性神经递质。 由于细胞外液中没有使 Glu 灭活的酶***，突触间隙中 Glu 清除的主要途径是通过胶质细胞上高亲和力 Glu 转运体（ glutamate transporters, GluTs) 对 Glu 的主动摄取。 1■ 甲基■ 4■ 苯基吡啶离子（ 1■ methyl■ 4■ phenylpyridinium, MPP+)可以抑制神经胶质细 胞摄取 Glu， 被认为是 MPP+ 间接损伤神经元的一个重要机制。  Glu is the major excitatory neurotransmitter in the central nervous system. Since there is no enzyme system in the extracellular fluid that inactivates Glu, the main pathway for Glu clearance in the synaptic cleft is the active uptake of Glu by high-affinity Glu transporters (GluTs) on glial cells. 1 ■ Methyl ■ 4 ■ Phenylpyridinium ion ( 1 ■ methyl ■ 4 ■ phenylpyridinium, MPP + ) can inhibit Glu uptake by glial cells and is considered to be an important mechanism for MPP+ indirect damage to neurons.
根据信号转导途径可以把谷氨酸受体分为两大类： 亲离子型谷氨酸受体 ( ionotropic glutamate recep " tors , iGluRs)禾口亲代谢型谷氨酸受体 ( metabotropicglutamate receptors , mGluRs)。 mGluRs 是广泛分布于中枢神 经***的一类 G 蛋白耦联受体， 根据氨基酸序列同源性、信号转导机制和药理学 特性将其分为三组， I 组 mGluRs 包括 mGluRl 和 mGluR5， II组 mGluRs 包括 mGluR2 和 mGluR3， III组 mGluRs 包括 mGluR4、 mGluR6、 mGluR7 和 mGluR8。 研究发现， 各组 mGluRs 在星形胶质细胞上均有分布。激活星形胶质细胞上的 mGluRs 可以释 放多种神经生长因子， 包括转化生长因子、神经生长因子 (NGF)等， 而发挥神经 保护作用； 另外， 激活星形胶质细胞上的 mGluRs 也可以改变谷胱甘肽 (glutathione, GSH) 的代谢。这些研究都表明激活星形胶质细胞上 mGluRs 有神 经保护作用。研究表明 MPP+抑制星形胶质细胞摄取 Glu 可能与直接影响谷氨酸转 运体（GluTs) 的功能有关， 采用 II、 III组亲代谢型谷氨酸受体激动剂激活星形 胶质细胞上的 I I、 III组 mGluRs 可以通过促进 GluTs 摄取 Glu、 进而降低细胞外 液的 Glu 浓度而发挥神经保护作用。 According to the signal transduction pathway, glutamate receptors can be divided into two categories: ionotropic glutamate recep " tors (iGluRs) and metabotropic glutamate receptors (mGluRs). mGluRs are a class of G-protein coupled receptors widely distributed in the central nervous system. They are divided into three groups according to amino acid sequence homology, signal transduction mechanism and pharmacological properties. Group I mGluRs include mGluRl and mGluR5. Group II mGluRs include mGluR2 and mGluR3, and group III mGluRs include mGluR4, mGluR6, mGluR7 and mGluR8. Studies have shown that each group of mGluRs is distributed on astrocytes. Activation of mGluRs on astrocytes can release a variety of Nerve growth factor, including transforming growth factor, nerve growth factor (NGF), etc., exerts neuroprotective effects; in addition, activation of mGluRs on astrocytes can also alter glutathione Metabolism of (glutathione, GSH). These studies have shown that activation of mGluRs on astrocytes is neuroprotective. Studies have shown that MPP+ inhibition of astrocyte uptake of Glu may be associated with a direct effect on the function of glutamate transporters (GluTs), using a metabotropic glutamate receptor agonist in groups II and III to activate astrocytes. Group II and III mGluRs can exert neuroprotective effects by promoting GluTs uptake of Glu and further reducing the Glu concentration of extracellular fluid.
本实验测定了 MEN061016-1对星形胶质细胞活性的影响。结果发现，给予 MPP+ 可抑制星形胶质细胞活力， MEN可逆转 MPP作用， MEN的这一作用可被 II、 III组 mGluRs拮抗剂阻断， 推测 MEN有 II、 III组 mGluRs受体激动作用激活作用。  This experiment determined the effect of MEN061016-1 on astrocyte activity. It was found that administration of MPP+ inhibited astrocyte viability, and MEN reversed MPP. This effect of MEN was blocked by group II and III mGluRs antagonists. It is speculated that MEN has group II and III mGluRs receptor agonistic activation. effect.
MEN061016-1逆转 1-甲基 -4-苯基吡啶离子 (MPP+)抑制谷氨酸摄入的影响MEN061016-1 reverses the effect of 1-methyl-4-phenylpyridinium (MPP+) on inhibition of glutamate uptake
①星形胶质细胞培养 1 astrocyte culture
无菌条件下取出新生 1 d SD 大鼠的全脑， 去除脑膜和血管， 用 0. 25%胰 蛋白酶消化成细胞悬液后， 接种到预先用多聚赖氨酸 0. 1 g/L— 1处理 24 h 的培 养瓶内， 24 h 后更换培养基， 以后 3 d 更换一次培养基。 d 7 时， 放入恒温摇 床内 100 r/min— 1 振摇 5 h， 弃去培养基， 用 0. 5% 的胰蛋白酶消化吹勾后， 接种 于培养板中， 待长成连续单层后， 按试验需要分组给药， 每组 6 个复孔， 继续培 养 48 h o The whole brain of the newborn 1 d SD rats was removed under aseptic conditions, and the meninges and blood vessels were removed, and the cell suspension was digested with 0.25% trypsin, and then inoculated with polylysine 0.11 g/L. 1 Treat the culture flask for 24 h, change the medium after 24 h, and replace the medium 3 d later. At d 7 , put it in a constant temperature shaker at 100 r/min -1 for 5 h, discard the medium, digest it with 0.5% trypsin, inoculate it in the culture plate, and grow into a continuous sheet. After the layer, grouped according to the test needs, 6 groups of each hole, continue to culture for 48 ho
② [ ] - D， L-Glu摄取的测定  2 [ ] - D, determination of L-Glu uptake
在星形胶质细胞培养基中加入 [¾] -D， L-Glu (终浓度 0. 19 μ mol · L— ， 共 同孵育 15 min 后， 用冰冷的 0. 9% NaCl 溶液终止反应并冲洗 3 遍， 随即加入 200 μ 1 NaOH溶液 0. 3 mol · L— 1裂解细胞， 最后离心， 收集上清液加入闪烁液， 使用 液闪计数仪测定 L- [¾] -Glu 摄取量 (nmol · ιιΰη— 1 * g— ^rot) 。 考马斯亮兰法测定 每孔细胞蛋白质含量。 Add [3⁄4] -D, L-Glu (final concentration 0. 19 μ mol · L-) to the astrocyte culture medium for 15 min, then stop the reaction with ice-cold 0.9% NaCl solution and rinse. 3 times, then add 200 μl NaOH solution 0. 3 mol · L- 1 to lyse the cells, finally centrifuge, collect the supernatant and add the scintillation solution, and measure the L-[3⁄4]-Glu intake by liquid flash counter (nmol · Ιιΰη— 1 * g—^rot) The Coomassie Brilliant Blue method measures the protein content of each well.
③结果  3 results
将 MPP+ 与星形胶质细胞共同孵育 48 h 然后测定星形胶质细胞对 [¾] D， L-Glu 的摄取能力。结果发现， MPP+ 150 uM 能明显抑制星形胶质细胞摄取 Glu。 而预先给予 MEN孵育 1 h可以逆转这种抑制作用。 mGLU受体激动剂 DCG IV 和 LAP40 100 uM与 MEN061016-1MEN无叠加作用。 推测 MEN061016-1 有类似于 DCG IV和 LAP40的亲代谢型谷氨酸受体激动作用。进一步研究发现 II组 mGluRs 拮抗剂 APICA和 III组 mGluRs拮抗剂 MS0P可逆转 MEN061016-1 作用， 推测 MEN061016-1具有部分 II、 III组 mGluRs受体激动作用。 结果见图 17。 ④结论 MPP+ was incubated with astrocytes for 48 h and the ability of astrocytes to uptake [3⁄4] D, L-Glu was determined. It was found that MPP+ 150 uM significantly inhibited the uptake of Glu by astrocytes. Pre-administration of MEN for 1 h can reverse this inhibition. The mGLU receptor agonists DCG IV and LAP40 100 uM have no additive effect with MEN061016-1MEN. It is speculated that MEN061016-1 has a metabotropic glutamate receptor agonism similar to DCG IV and LAP40. Further studies found that group II mGluRs antagonist APICA and group III mGluRs antagonist MSOP can reverse the effect of MEN061016-1, suggesting that MEN061016-1 has partial group II and III mGluRs receptor agonism. The results are shown in Figure 17. 4 conclusions
Glu 是中枢神经***主要的兴奋性神经递质，但是细胞外 Glu 浓度过高时， 会产生兴奋毒性。 Glu 的神经毒性是通过 NMDA 和非 NMDA 受体介导的， 但在大 多数病理情况下， 尤其是在中枢神经***慢性进行性疾病中， NMDA 受体在过度 兴奋介导的神经细胞迟发性变性或坏死起重要作用。 MPP+ 是目前研究谷氨酸毒 性常用的神经毒素， 它主要通过抑制线粒体复合物 I 发挥神经毒性作用。本文工 作选择星形胶质细胞作为研究的工具细胞，研究结果表明， 在不影响星形胶质细 胞活性的浓度时， MPP+ 即可显著抑制 Glu 的摄取能力。 同时本研究证明 MEN061016-1可以逆转 MMP+抑制谷氨酸摄入作用，可能有类似亲代谢型谷氨酸受 体激动作用。 Glu is the main excitatory neurotransmitter in the central nervous system, but excitotoxicity occurs when the extracellular Glu concentration is too high. Glu's neurotoxicity is mediated through NMDA and non-NMDA receptors, but in most pathological conditions, especially in chronic progressive diseases of the central nervous system, NMDA receptors are overexcited to mediate neuronal delay Denaturation or necrosis plays an important role. MPP+ is a neurotoxin commonly used in the study of glutamate toxicity, and it exerts neurotoxic effects mainly by inhibiting mitochondrial complex I. In this paper, astrocytes were selected as the tool cells of the study. The results showed that MPP+ can significantly inhibit the uptake of Glu without affecting the concentration of astrocyte activity. At the same time, this study demonstrates that MEN061016-1 can reverse MMP + inhibition of glutamate uptake, and may have similar metabotropic glutamate receptor agonism.
本实验测定了 MEN061016-1对星形胶质细胞谷氨酸摄入的影响。 结果发现， 给予 MPP+ 可抑制星形胶质细胞谷氨酸摄入， MEN061016-1可逆转 MPP作用， MEN 的这一作用可被 II III组 mGluRs拮抗剂阻断， 推测 MEN有 II III组 mGluRs受体 激动作用。  This experiment determined the effect of MEN061016-1 on glutamate uptake by astrocytes. It was found that administration of MPP+ inhibited astrocytic glutamate uptake, and MEN061016-1 reversed MPP. This effect of MEN was blocked by II III mGluRs antagonists. It is speculated that MEN has II III mGluRs. Body agonism.
3 MEN061016-1对大鼠脑皮质 MDA受体与其配体结合的影响  Effects of MEN061016-1 on the binding of MDA receptors to their ligands in rat cerebral cortex
①方法  1 method
脑皮质受体悬液的制备： 放射配基结合检测当天， SD大鼠断头处死， 每个 Preparation of cerebral cortical receptor suspension: On the day of radioligand binding assay, SD rats were decapitated, each
1. 5 mL Eppendorf 离心管中加入脑皮质 0. 1 g， 每只鼠共取 6管， 每管加 1 mL 0. 32 mol/L冰冷蔗糖溶液， 用 1 mL—次性注射器反复抽吸脑组织勾浆。 900 g 低温离 心 10 min， 取上清， 11500 g 低温离心 20 min， 取沉淀， 加冰冷 50 ol/L Tris- HC1 (pH 7. 7) 0. 5 mL,悬浮， 11500 g 低温离心 20 min,取沉淀，每管加 Tris-HCl 0. 5 mL, 混勾， 6管混合， 加 50 mmol/L Tris-HCl 5 mL, 考马斯亮兰法测定蛋白 浓度， 用 Tris-HCl调为 0. 4 g/L， 备用。 以上操作均在冰浴下进行。 1. Add 5 g of cerebral cortex to a 5 mL Eppendorf centrifuge tube, and take 6 tubes per mouse. Add 1 mL of 0.32 mol/L ice-cold sucrose solution to each tube and repeatedly aspirate the brain with 1 mL-secondary syringe. Organize the pulping. Centrifuge at 900 g for 10 min, take the supernatant, centrifuge at 11500 g for 20 min, take the precipitate, add 50 ol/L Tris-HC1 (pH 7. 7) 0.5 ml, suspend, and centrifuge at 11500 g for 20 min. 4 g。 Tris-HCl was adjusted to 0. 4 g. Tris-HCl was adjusted to 0. 4 g. Tris-HCl was adjusted to 0. 4 g. /L, spare. The above operations were all carried out under an ice bath.
放射配基结合分析： 总反应体积为 200 L0 每管内加入 100 L 脑皮质受 体悬液， 不同浓度药物及 ¾-MK-801。 非特异性结合管加入非标记 MK-801， 使其 终浓度为 1 mol/L0 37 °C反应 30min，力口 3 m L冰冷 50 mmol/L Tris- HCl (pH 7. 7) 终止反应， 用 Whatman滤纸抽滤， 用 3 mL冰冷 Tris- HC1冲洗滤片 2次， 取出滤膜 于 80 °C烘干 20 min。 放入闪烁瓶中， 用液闪仪计数 Radioligand binding assay: Total reaction volume was 200 L 0. 100 L of cortical receptor suspension was added to each tube, different concentrations of drug and 3⁄4-MK-801. The non-specific binding tube was added with non-labeled MK-801, and the final concentration was 1 mol/L at 0 37 °C for 30 min. The reaction was terminated with 3 m L of ice cold 50 mmol/L Tris-HCl (pH 7. 7). The Whatman filter paper was suction filtered, and the filter was rinsed twice with 3 mL of ice-cold Tris-HC1, and the filter was taken out and dried at 80 °C for 20 min. Put in a scintillation bottle and count with a liquid scintillator
②分组及药物配制： 药物或 ¾-MK-801均以 50 mmol/L Tris- HC1 (pH 7. 7) 配 置， 药物浓度以终浓度计， 分组如下： ( 1 ) lOOul脑皮质悬液 +3H-MK-801 (10nM) 2 Grouping and drug preparation: The drug or 3⁄4-MK-801 are all configured with 50 mmol/L Tris-HC1 (pH 7.7). The drug concentration is based on the final concentration and is grouped as follows: (1) lOOul cerebral cortex suspension + 3 H-MK-801 (10nM)
( 2) lOOul脑皮质悬液+美金刚 500nM+¾-MK-801 (ΙΟηΜ)  (2) lOOul cerebral cortex suspension + memantine 500nM+3⁄4-MK-801 (ΙΟηΜ)
( 3) lOOul脑皮质悬液+美金刚 100nM+¾-MK-801 (ΙΟηΜ)  (3) lOOul cerebral cortex suspension + memantine 100nM+3⁄4-MK-801 (ΙΟηΜ)
(4) lOOul脑皮质悬液 +Men 500nM+¾-MK-801 (ΙΟηΜ)  (4) lOOul cerebral cortex suspension +Men 500nM+3⁄4-MK-801 (ΙΟηΜ)
( 5) lOOul脑皮质悬液 +Men 100nM+¾-MK-801 (ΙΟηΜ)  (5) lOOul cerebral cortex suspension +Men 100nM+3⁄4-MK-801 (ΙΟηΜ)
③结果及结论 3 results and conclusions
盐酸美金刚（Memantine H ydrochlo ride) 是一种新型、 低、 中度亲和力、 电压依赖、 非竞争性 NMDA ( N- methyl- Daspastate)受体拮抗剂， 能显著改善阿 尔茨海默氏病 (Alzheiner' s disease, AD) 禾口血管性痴呆 (vascular dementia, VaD) 患者认知障碍、 精神运动驱动缺乏、 抑郁程度、 运动障碍， 提高其日常生 活能力和社交活动。 分别于 2002 年和 2003 年被欧洲 CPMP 和美国 FDA批准用于 治疗中、 重度 AD。  Memantine H ydrochlo ride is a novel, low- and moderate-intensity, voltage-dependent, non-competitive NMDA (N-methyl-Daspastate) receptor antagonist that significantly improves Alzheimer's disease (Alzheiner) 's disease, AD) vascular dementia (VaD) patients with cognitive impairment, lack of psychomotor drive, depression, dyskinesia, improve their daily living ability and social activities. In 2002 and 2003, it was approved by European CPMP and US FDA for the treatment of moderate to severe AD.
谷氨酸作为脑中最常见的兴奋性神经递质，与痴呆患者脑中神经细胞死亡密 切相关。谷氨酸浓度增加或神经节对谷氨酸敏感性增强均可增加受体 NMDA、 AMPA 与谷氨酸结合， 引起电压调控性钙通道开放或激活磷酸肌醇环路， 使细胞内钙超 载， 导致神经细胞凋亡。 盐酸美金刚可非竞争性阻断 NMDA 受体， 降低谷氨酸引 起的 NMDA 受体过度兴奋， 防止细胞凋亡， 从而改善记忆， 增强患者认知功能。 由于盐酸美金刚与 NMDA 受体亲和力呈低、 中度， 因此， 在阻断谷氨酸兴奋性毒 性的同时， 不妨碍谷氨酸参与正常学习和记忆的生理作用。本研究通过比较药物 干预脑皮质 NMDA受体与 ¾标记非竞争性 NMDA受体拮抗剂 MK-801的结合， 来比较 MEN和美金刚对 NMDA受体亲和力的大小。  Glutamate, the most common excitatory neurotransmitter in the brain, is closely related to the death of nerve cells in the brain of dementia patients. Increased glutamate concentration or increased sensitivity of ganglia to glutamate can increase receptor NMDA, AMPA and glutamate binding, causing voltage-regulated calcium channels to open or activate the phosphoinositide loop, causing intracellular calcium overload, Lead to neuronal apoptosis. Memantine hydrochloride can non-competitively block NMDA receptors, reduce excessive excitability of NMDA receptors induced by glutamate, prevent apoptosis, thereby improving memory and enhancing cognitive function in patients. Since memantine hydrochloride has low and moderate affinity for NMDA receptors, it blocks the glutamate excitotoxicity and does not prevent glutamate from participating in the physiological functions of normal learning and memory. This study compared the binding affinity of MEN and memantine to NMDA receptors by comparing drug-interventional binding of NMDA receptors in the cerebral cortex to the 3⁄4-labeled non-competitive NMDA receptor antagonist MK-801.
目前 NMDA受体分子结构还未充分认识， 在药理学上至少有 6个独立的位点， 分别是受体表面的 NMDA/谷氨酸独立位点、 甘氨酸 （gly) 位点、 Zn2+位点； 位于 离子通道内的 Mg2+位点、 PCP位点。 NMDA受体拮抗剂可分为竞争性和非竞争性拮 抗剂， 后者又可分为离子通道阻滞剂、 Gly和 PA部位拮抗剂。 目前已进入临床的 非竞争性 NMDA受体拮抗剂有 MK801和 DX等。 At present, the molecular structure of NMDA receptors is not fully understood. There are at least six independent sites in pharmacology, which are NMDA/glutamate independent sites, glycine (gly) sites, and Zn 2+ sites on the receptor surface. Point; Mg 2+ site, PCP site located in the ion channel. NMDA receptor antagonists can be divided into competitive and non-competitive antagonists, which in turn can be divided into ion channel blockers, Gly and PA site antagonists. Currently, non-competitive NMDA receptor antagonists that have entered the clinic include MK801 and DX.
本实验观测到美金刚可拮抗 NMDA受体与其配体 ¾-MK 801的结合， 且有剂量- 效应对应关系， 提示美金刚可抑制兴奋性氨基酸毒性作用， 从而发挥对脑损伤的 保护作用。 MEN061016-1对丽 DA受体与其配体 ¾-MK 801的也有一定的结合， 说 明对 NMDAR受体有弱抑制作用。 结果见表 22。 In this experiment, it was observed that memantine can antagonize the binding of NMDA receptor to its ligand 3⁄4-MK 801, and has a dose-response relationship, suggesting that memantine can inhibit the excitatory amino acid toxicity and thus protect against brain damage. MEN061016-1 also has a certain binding to the RDA receptor and its ligand 3⁄4-MK 801, indicating a weak inhibitory effect on the NMDAR receptor. The results are shown in Table 22.
以下为表格:  The following is a table:
表 1 - 对 VaD大鼠 MORRIS水迷宫潜伏期的影响 ( X ±s ) 剂量 潜伏期 （s)  Table 1 - Effect on latency of MORRIS water maze in VaD rats (X ± s ) Dose latency (s)
分组 n  Grouping n
(mg/kg) 给药 2个月 给药 3个月 假手术组 12 4.30+1.43 3.78±1.05 模型组 10 17.03+8.74## 11.10+5.84## 美金刚组 20 10 10.64+7.62 5.64+3.33* 烟酸组 20 11 10.67+6.69 6.81+2.41*  (mg/kg) 2 months administration 2 months sham operation group 12 4.30+1.43 3.78±1.05 model group 10 17.03+8.74## 11.10+5.84## memantine group 20 10 10.64+7.62 5.64+3.33* Niacin group 20 11 10.67+6.69 6.81+2.41*
2.5 10 11.40+4.72 8.93±4.94 2.5 10 11.40+4.72 8.93±4.94
5 10 9.73±6.59 6.44+3.81*5 10 9.73±6.59 6.44+3.81*
MEN组 10 9 8.34+5.45* 5.82±1.66* MEN group 10 9 8.34+5.45* 5.82±1.66*
20 10 7.78±5.78* 5.81+2.21* 20 10 7.78±5.78* 5.81+2.21*
40 6 6.12+3.22** 4.33+1.90* 注： 与假手术组比较： ##Ρ<0.01', 与模型组比较: *P<0.05, **尸<請。 表 2 对 VaD大鼠 MORRIS水迷宫路径长度的影响 ( X ±s ) 剂量 路径长度 （cm) 分组 n 40 6 6.12+3.22** 4.33+1.90* Note: Compared with the sham group: ## Ρ<0.01', compared with the model group: *P<0.05, ** corpse <please. Table 2 Effect on path length of MORRIS water maze in VaD rats (X ± s ) Dose path length (cm) Group n
(mg/kg) 给药 2个月 给药 3个月 假手术组 12 54.76+19.00 58.96±15.02 模型组 10 218.98+135.58## 163.36+99.16## 美金刚组 20 10 142.51+107.56 139.53+109.66 烟酸组 20 11 141.53+90.51 120.87+56.20  (mg/kg) administration 2 months administration 3 months sham operation group 12 54.76+19.00 58.96±15.02 model group 10 218.98+135.58## 163.36+99.16## memangan group 20 10 142.51+107.56 139.53+109.66 Acid group 20 11 141.53+90.51 120.87+56.20
2.5 10 124.20+71.69 156.29+133.74 2.5 10 124.20+71.69 156.29+133.74
5 10 121.46+88.08 91.94+57.445 10 121.46+88.08 91.94+57.44
MEN组 10 9 105.54+54.06* 97.90±48.91 MEN group 10 9 105.54+54.06* 97.90±48.91
20 10 95.95+66.52* 93.22+29.10* 20 10 95.95+66.52* 93.22+29.10*
40 6 59.86+31.66** 64.37+38.41* 注： 与假手术组比较： ##Ρ<0.01', 与模型组比较: *P<0.05, **尸<請。 表 3 对 VaD大鼠前皮层 Glu、 GABA水平及 Glu/GABA的影响 （ ±s) 齐糧 Glu GABA 40 6 59.86+31.66** 64.37+38.41* Note: Compared with the sham group: ## Ρ<0.01', compared with the model group: *P<0.05, ** corpse <please. Table 3 Effects of Glu, GABA levels and Glu/GABA on the cortex of VaD rats (±s) Qiliang Glu GABA
删 (mg^ n ^g/g脑重） ^g/g脑重） Glu/GABA  Delete (mg^ n ^g/g brain weight) ^g/g brain weight) Glu/GABA
)  )
12 181.5±68.2 25.7±13.7 8.66+4.36 灘且 9 99.5±63.7## 8.3±6.5## 13.64±6.98# 12 181.5±68.2 25.7±13.7 8.66+4.36 Beach and 9 99.5±63.7 ## 8.3±6.5 ## 13.64±6.98 #
20 9 22.3+11.8** 3.8±2.8 7.86+4.55* 髓且 20 9 77.5+30.4ΛΛ 12.8±8.5ΔΔ 8.34+5.57*20 9 22.3+11.8** 3.8±2.8 7.86+4.55* Pulp and 20 9 77.5+30.4 ΛΛ 12.8±8.5 ΔΔ 8.34+5.57*
2.5 10 151.4+76.7ΛΛ Α 24.4+11.9*ΛΛ Α 7.92+5.07*2.5 10 151.4+76.7 ΛΛ Α 24.4+11.9* ΛΛ 7.9 7.92+5.07*
5 9 158.6+79.3ΛΛ Α Α 23.1+11.3**ΛΛ Α 8.46+4.97*5 9 158.6+79.3 ΛΛ Α Α 23.1+11.3** ΛΛ Α 8.46+4.97*
10 171.3+27.8**ΛΛ Α 28.7+15.3**ΛΛ Α 10 171.3+27.8** ΛΛ Α 28.7+15.3** ΛΛ Α
ME 组 9 ▲ 7.96+4.78*  ME group 9 ▲ 7.96+4.78*
20 10 155.6+81.0ΛΛ Α 17.6+9.2*ΛΛ 11.96+9.1420 10 155.6 +81.0 ΛΛ Α 17.6+9.2* ΛΛ 11.96+9.14
40 6 32.2+12.8*Α Α 2.5±0.8Α 13.25+4.00' 注：与假手术组比较： ** Ρ<0.01 与模型组比较： *Ρ<0.05, **Ρ<0.01;与盐酸美金刚组比较： Ρ<0.01 ; 与烟酸组比较： P<0.05， K KP<0.01 表 4 对 VaD大鼠海马 ACh水平的影响 （ X ） 40 6 32.2+12.8* Α Α 2.5±0.8 Α 13.25+4.00' Note: Compared with sham operation group: ** Ρ<0.01 compared with model group: *Ρ<0.05, **Ρ<0.01; with memantine hydrochloride group Comparison: Ρ<0.01; compared with niacin group: P<0.05, KK P<0.01 Table 4 Effect on hippocampal ACh levels in VaD rats (X)
齐糧 ACh  Grain ACh
删 η (ng/g脑重） Delete η (ng/g brain weight)
Figure imgf000020_0001
Figure imgf000020_0001
灘且 9 917.1+263.5## Beach and 9 917.1+263.5 ##
20 9 1543.2+630.3*  20 9 1543.2+630.3*
髓且 20 10 1790.0+482.0**  Pulp and 20 10 1790.0+482.0**
2.5 10 1573.9+671.5**  2.5 10 1573.9+671.5**
5 7 1757.1+302.1**  5 7 1757.1+302.1**
ME 组 10 7 163.23+58.17**  ME group 10 7 163.23+58.17**
20 7 1138.3+709.9  20 7 1138.3+709.9
40 6 1051.2+279.2  40 6 1051.2+279.2
注： 与假手术组比较： ** P<O.OI 与模型组比较： *ρ<αο5, **ρ<ο.οι. 表 5 对 VaD大鼠前皮层 NMDA受体 NR1、 NR2A、 NR2B表达的影响 （ ) ~~ S 积分灰度值 （IOD )  Note: Compared with the sham operation group: ** P<O.OI compared with the model group: *ρ<αο5, **ρ<ο.οι. Table 5 Expression of NMDA receptors NR1, NR2A, NR2B in the anterior cortex of VaD rats Effect ( ) ~~ S integral gray value (IOD)
(mg/kg) n Rl NR2A NR2B m 5 923.5+106.7 742.4+156.3 800.6+120.1 灘且 5 3987.3+452.8## 4309.8+271.7## 4540.1±287.8## (mg/kg) n Rl NR2A NR2B m 5 923.5+106.7 742.4+156.3 800.6+120.1 Beach and 5 3987.3+452.8 ## 4309.8+271.7 ## 4540.1±287.8 ##
20 5 2030.0+207.4** 1804.7+180.8** 2334.1+396.3** 髓且 20 5 1924.7+207.8** 2028.1+114.7** 1683.8+150.2**  20 5 2030.0+207.4** 1804.7+180.8** 2334.1+396.3** Pulp and 20 5 1924.7+207.8** 2028.1+114.7** 1683.8+150.2**
2.5 5 2473.1+260.5** 3048.7±272.8** 3649.0+427.3** 2.5 5 2473.1+260.5** 3048.7±272.8** 3649.0+427.3**
5 5 1994.8+246.7** 2555.4+312.4** 2852.8+183.5**5 5 1994.8+246.7** 2555.4+312.4** 2852.8+183.5**
ME 组 10 5 1931.5+61.9** 2053.2+226.8** 2105.8+269.1** ME group 10 5 1931.5+61.9** 2053.2+226.8** 2105.8+269.1**
20 5 1900.6+101.5** 2255.2±217.6** 2064.0+124.9** 20 5 1900.6+101.5** 2255.2±217.6** 2064.0+124.9**
40 5 1504.0+278.6**' 2036.3+105.5**A 1953.9+154.3** 注： 与假手术组比较： M P<0.01 与模型组比较： **Ρ<0.01 ·' 与盐酸美金刚组比较： ΔΡ<0.05·' 与烟酸 组比较： P<0.05, "P<0.01 表 6对 VaD大鼠前皮层 NMDA受体 NRl、 NR2A、 NR2B mRNA表达的影响 （ ±s) 剂量 积分灰度值 （IOD ) 40 5 1504.0+278.6**' 2036.3+105.5** A 1953.9+154.3** Note: Compared with sham group: M P<0.01 compared with model group: **Ρ<0.01 ·' Compared with memantine hydrochloride group: Δ Ρ<0.05·' compared with niacin group: P<0.05, "P<0.01 Table 6 Effect on NMDA receptor NR1, NR2A, NR2B mRNA expression in VaD rat prefrontal cortex (±s) Dose-integrated gray value ( IOD)
组别 Π  Group Π
(mg/kg) NRlmRNA NR2A mRNA NR2B mRNA  (mg/kg) NR1 mRNA NR2A mRNA NR2B mRNA
5 854.9+137.0 894.6+118.5 904.4+94.6 灘且 5 4171.3±960.2## 4681.6±336.6## 3938.1+560.5## 2098.3+216.8** 2018.3+185.6** 2276.9+300.2** 5 854.9+137.0 894.6+118.5 904.4+94.6 Beach and 5 4171.3±960.2 ## 4681.6±336.6 ## 3938.1+560.5 ## 2098.3+216.8** 2018.3+185.6** 2276.9+300.2**
1604.9+130.1** 1899.6+336.9** 2091.4+207.2** 1604.9+130.1** 1899.6+336.9** 2091.4+207.2**
3583.9+168.0 3053.6+406.8** 3266.5+225.3*3583.9+168.0 3053.6+406.8** 3266.5+225.3*
2726.0+227.8* 3009.2+431.4** 2931.2+330.7**2726.0+227.8* 3009.2+431.4** 2931.2+330.7**
ME 组 2558.0+376.3** 2524.4+87.1** 2568.6+338.3** ME group 2558.0+376.3** 2524.4+87.1** 2568.6+338.3**
1647.6+137.5** 1780.5+161.6** 2361.5+346.0** 1647.6+137.5** 1780.5+161.6** 2361.5+346.0**
1504.5+241.1** 1898.2+273.6** 2452.7+294.2** 注： 与假手术组比较： m P<0.01 与模型组比较： *P<a05， **P<0.0L 表 7对 VaD大鼠血桨 SS水平的影响 （； C ±s) 1504.5+241.1** 1898.2+273.6** 2452.7+294.2** Note: Compared with the sham group: m P<0.01 compared with the model group: *P<a05, **P<0.0L Table 7 for VaD rat blood Effect of paddle SS level (; C ± s)
齐糧 SS  Qiliang SS
删 η (pg/mL脑重） Delete η (pg/mL brain weight)
Figure imgf000021_0001
Figure imgf000021_0001
灘且 9 228.98+32.80## Beach and 9 228.98+32.80 ##
20 9 235.21+46.33  20 9 235.21+46.33
髓且 20 9 312.43+68.17**  Pulp and 20 9 312.43 + 68.17**
2.5 10 265.58+23.93**  2.5 10 265.58+23.93**
5 9 260.55+56.58  5 9 260.55+56.58
ME 组 10 9 279.55+35.10**  ME group 10 9 279.55+35.10**
20 10 269.26+36.29*  20 10 269.26+36.29*
40 6 248.88+43.10  40 6 248.88+43.10
注： 与假手术组比较： ** P<0.01 与模型组比较： <0.05, **P<0.01。 表 8 对 MID大鼠 MORRIS水迷宫潜伏期的影响（ ±s) 剂量 给药 4周 给药 8周 给药 8周 组别 动物数  Note: Compared with the sham operation group: ** P < 0.01 compared with the model group: <0.05, **P <0.01. Table 8 Effect of MID rat MORRIS water maze latency (±s) Dosing 4 weeks Administration 8 weeks Administration 8 weeks Group Number of animals
(mg/kg) 第 5天 （s) 第 1天 （s) 第 5天 （s) 假手术组 11 17.15±9.77 37.16±23.86 12.50+9.09 模型组 11 68.11±69.15# 91.86±73.33# 45.23+36.41## 美金刚组 20.0 12 49.78+48.24 42.92+30.33* 24.09+25.74 烟酸组 20.0 12 16.03+13.94* 21.89±22.52** 13.64+9.22* 美 +烟等摩尔组 13.5+7.7 12 45.46+65.91 25.07+15.44* 22.67+25.67 (mg/kg) Day 5 (s) Day 1 (s) Day 5 (s) Sham group 11 17.15 ± 9.77 37.16 ± 23.86 12.50 + 9.09 Model group 11 68.11 ± 69.15 # 91.86 ± 73.33 # 45.23 + 36.41 ## 美金刚组20.0 12 49.78+48.24 42.92+30.33* 24.09+25.74 Niacin group 20.0 12 16.03+13.94* 21.89±22.52** 13.64+9.22* US+ smoke equimolar group 13.5+7.7 12 45.46+65.91 25.07+ 15.44* 22.67+25.67
MEN组 2.0 11 39.53±49.91 31.53+28.21* 19.60+14.94*MEN group 2.0 11 39.53±49.91 31.53+28.21* 19.60+14.94*
MEN组 6.3 12 36.56+34.12 23.97+21.27** 17.33+10.45*MEN group 6.3 12 36.56+34.12 23.97+21.27** 17.33+10.45*
MEN组 20.0 10 35.00+42.07 20.98+12.59** 10.67+4.84** 注： 与假手术组比较： * P<0.05, mP<0.01; 与模型组比较： *P<0.05, **P<0.01。 MEN group 20.0 10 35.00+42.07 20.98+12.59** 10.67+4.84** Note: Compared with sham operation group: * P<0.05, m P<0.01; compared with model group: *P<0.05, **P<0.01 .
表 9 对 MID大鼠 MORRIS水迷宫路径长度的影响 （x ±s) 剂量 给药 4周 给药 8周 给药 8周 组别 动物数 Table 9 Effect on path length of MID rat MORRIS water maze (x ± s) Dosing for 4 weeks, 8 weeks, 8 weeks, group of animals
(mg/kg) 第 5天 （s) 第 1天 （s) 第 5天 （s) 假手术组 11 220.7±135.02 384.4+317.8 139.5+99.5 模型组 13 914.0+950.5# 1052.6+918.0# 597.8+455.7## 美金刚组 20.0 12 765.4± 783.9 553.3+489.4 326.7+370.5 烟酸组 20.0 12 214.0+185.9* 294.7+401.7* 168.5+123.0** 美 +烟等摩尔组 13.5+7.7 12 568.4+837.8 284.8+180.7* 303.0+392.8 (mg/kg) Day 5 (s) Day 1 (s) Day 5 (s) Sham group 11 220.7 ± 135.02 384.4 + 317.8 139.5 + 99.5 Model group 13 914.0 + 950.5 # 1052.6 + 918.0 # 597.8 + 455.7 ## 美金刚组20.0 12 765.4± 783.9 553.3+489.4 326.7+370.5 Niacin group 20.0 12 214.0+185.9* 294.7+401.7* 168.5+123.0** US + smoke equimolar group 13.5+7.7 12 568.4+837.8 284.8+180.7 * 303.0+392.8
MEN组 2.0 11 465.9+545.9 351.2+277.9* 293.7+269.6MEN group 2.0 11 465.9+545.9 351.2+277.9* 293.7+269.6
MEN组 6.3 12 516.0+433.6 327.5+339.9* 223.0+145.8*MEN group 6.3 12 516.0+433.6 327.5+339.9* 223.0+145.8*
MEN组 20.0 10 445.1+510.0 235.6+140.9** 125.3+66.5** 注： 与假手术组比较： * P<0.05， **Ρ<0.01; 与模型组比较： *P<0.05, **P<0.0 表 10对 MID大鼠皮层 Glu、 GABA水平的影响 （ ±s) 齐糧 Glu GABA 删 η ^g/g脑重） ( g g脑重） MEN group 20.0 10 445.1+510.0 235.6+140.9** 125.3+66.5** Note: Compared with sham operation group: * P<0.05, **Ρ<0.01; compared with model group: *P<0.05, **P< 0.0 Table 10 Effect on Glu and GABA levels in MID rat cortex (±s) Qiliang Glu GABA deletion η ^g/g brain weight) ( gg brain weight)
11 123.88+56.73 18.93±10.14 灘且 13 75.33±14.06## 12.70±5.28# 11 123.88+56.73 18.93±10.14 Beach and 13 75.33±14.06## 12.70±5.28#
m 20.0 12 153.58+108.39* 22.84+17.52* 髓且 20.0 12 105.48+36.17* 11.24+6.19 鋼 瞧 13.5+7.7 12 136.22+43.49** 18.69+5.81**  m 20.0 12 153.58+108.39* 22.84+17.52* pith and 20.0 12 105.48+36.17* 11.24+6.19 steel 瞧 13.5+7.7 12 136.22+43.49** 18.69+5.81**
ME 组 2.0 11 123.14+26.98** 12.27+5.65  ME group 2.0 11 123.14+26.98** 12.27+5.65
ME 组 6.3 12 128.81+78.66* 12.20+4.77  ME group 6.3 12 128.81+78.66* 12.20+4.77
ME 组 20.0 10 135.76+44.99** 12.59±8.12 注： 与假手术组比较： m P<0.01 与模型组比较： *P<ft05， K0.01; 表 11 对 MID大鼠海马 Glu、 GABA水平的影响 （ ±s) 齐糧 Glu GABA 删 η ^g/g脑重） ( g g脑重） ME group 20.0 10 135.76+44.99** 12.59±8.12 Note: Compared with sham operation group: m P<0.01 compared with model group: *P<ft05, K0.01; Table 11 for MID rat hippocampal Glu, GABA levels Impact (±s) Qiliang Glu GABA delete η ^g/g brain weight) ( gg brain weight)
11 109.43+52.82 24.21+17.34 灘且 13 59.44±23.99## 16.93±7.24  11 109.43+52.82 24.21+17.34 Beach and 13 59.44±23.99## 16.93±7.24
20.0 12 136.48+46.81** 18.21+10.76 髓且 20.0 12 96.85±21.39 23.21+8.76 鋼 瞧 13.5+7.7 12 129.17+39.82** 26.44+7.23** 20.0 12 136.48+46.81** 18.21+10.76 Pulp and 20.0 12 96.85±21.39 23.21+8.76 Steel 瞧 13.5+7.7 12 129.17+39.82** 26.44+7.23**
ME 组 2.0 11 94.22+32.66** 14.87+7.56 ME group 2.0 11 94.22+32.66** 14.87+7.56
ME 组 6.3 12 97.98+40.28** 18.46+7.66  ME group 6.3 12 97.98+40.28** 18.46+7.66
ME 组 20.0 10 128.74±23.26**A A 27.23+10.41* 注： 与假手术组比较： ** P<0.01 与模型组比较： *P<0.05, **P<0.01; 与烟酸组比较： "P<0.01 表 12对 MID大鼠 NMDA受体 NR1、 NR2A、 NR2B表达的影响 （ ±s ) 剂量 积分灰度值 （IOD ) 组别 (mg/kg) n ME group 20.0 10 128.74±23.26** AA 27.23+10.41* Note: Compared with sham operation group: ** P<0.01 compared with model group: *P<0.05, **P<0.01; compared with niacin group: P<0.01 Table 12 Effect on the expression of NMDA receptors NR1, NR2A, NR2B in MID rats (±s) Dose-integrated gray value (IOD) group (mg/kg) n
NR1 NR2A NR2B NR1 NR2A NR2B
5 831.65+212.64 732.65+119.08 877.15+115.13 灘且 5 2064.28+437.70## 1818.72±329.14## 1888.36±141.46## 5 831.65+212.64 732.65+119.08 877.15+115.13 Beach and 5 2064.28+437.70## 1818.72±329.14## 1888.36±141.46##
20.0 5 1326.90+186.81** 1554.22+114.68* 1508.02+154.48** 髓且 20.0 5 1517.65+171.02** 1651.53+229.23 1595.31+159.21** 鋼 瞧 13.5+7.7 5 1469.54+178.27** 1387.73+105.17** 1434.71+192.85** 20.0 5 1326.90+186.81** 1554.22+114.68* 1508.02+154.48** Pulp and 20.0 5 1517.65+171.02** 1651.53+229.23 1595.31+159.21** Steel 瞧 13.5+7.7 5 1469.54+178.27** 1387.73+105.17** 1434.71 +192.85**
ME 组 2.0 5 1732.63+135.23* 1751.45+127.31 1624.62+132.82**ME group 2.0 5 1732.63+135.23* 1751.45+127.31 1624.62+132.82**
ME 组 6.3 5 1574.39±87.47* 1617.23+167.62 1580.58+159.85**ME group 6.3 5 1574.39±87.47* 1617.23+167.62 1580.58+159.85**
ME 组 20.0 5 1467.63+62.82** 1629.81+152.46 1484.43+202.03** 注： 与假手术组比较： ** P<0.01 与模型组比较： <0.05, **P<0.01; 表 13 对 MID大鼠脑组织 CHAT及 ACHE活性的影响 （； ）
Figure imgf000023_0001
ME group 20.0 5 1467.63+62.82** 1629.81+152.46 1484.43+202.03** Note: Compared with sham operation group: ** P<0.01 compared with model group: <0.05, **P<0.01; Table 13 for MID rats Effects of CHAT and ACHE activity in brain tissue (; )
Figure imgf000023_0001
灘且 13 251.03±31.91# 3.332+0.886  Beach and 13 251.03±31.91# 3.332+0.886
^m m 20.0 14 312.72+23.18* 2.977+0.407 髓且 20.0 13 267.79+58.83 2.930+0.553 鋼 瞧 13.5+7.7 11 266.96+33.67 0.977+0.891** ^m m 20.0 14 312.72+23.18* 2.977+0.407 medulla and 20.0 13 267.79+58.83 2.930+0.553 steel 瞧 13.5+7.7 11 266.96+33.67 0.977+0.891**
ME 组 2.0 11 284.83+31.82* 3.340±1.038 ME group 2.0 11 284.83+31.82* 3.340±1.038
ME 组 6.3 11 290.67+47.66* 2.600+0.431*  ME group 6.3 11 290.67+47.66* 2.600+0.431*
ME 组 20.0 11 357.51+71.21**A A 3.299+0.448 注： 与假手术组比较： # P<0.05 与模型组比较： *P<0.05, **P<0.01; 与烟酸组比较： A AP<0.01 表 14对 MID大鼠脑组织 TNF水平的影响 （ X ）
Figure imgf000023_0002
ME group 20.0 11 357.51+71.21** AA 3.299+0.448 Note: Compared with sham operation group: # P<0.05 Compared with model group: *P<0.05, **P<0.01; compared with niacin group: AA P< 0.01 Table 14 Effect on TNF levels in brain tissue of MID rats (X)
Figure imgf000023_0002
11 1.97士1.00  11 1.97 1.00
灘且 13 3.97士 0.94#  Beach and 13 3.97士 0.94#
20.0 14 3.52士 0.70  20.0 14 3.52士 0.70
髓且 20.0 13 2.29士 0.46*  Pulp and 20.0 13 2.29 ± 0.46*
鋼 瞧 13.5+7.7 11 2.52士 0.73*  Steel 瞧 13.5+7.7 11 2.52士 0.73*
ME 组 2.0 11 3.21士 1.29  ME group 2.0 11 3.21士 1.29
ME 组 6.3 11 2.74士 0.80  ME group 6.3 11 2.74士 0.80
ME 组 20.0 11 2.30士 0.53*  ME group 20.0 11 2.30士 0.53*
注： 与假手术组比较： * P<0.05 与模型组比较： *P<0.05, **P<0.01 表 15 对 MCAO大鼠脑组织 SOD及 GSH-PX活性的影响 （ 士
Figure imgf000023_0003
灘且 13 367.57±116.90# 130.02+47.48# Note: Compared with sham operation group: * P<0.05 compared with model group: *P<0.05, **P<0.01 Table 15 Effect on SOD and GSH-PX activity in brain tissue of MCAO rats
Figure imgf000023_0003
Beach and 13 367.57±116.90# 130.02+47.48#
20.0 10 337.42+89.60 128.37+49.62 髓且 20.0 14 371.97+89.89 153.05+45.23 鋼 瞧 13.5+7.7 13 350.08+51.13 120.43+24.63 20.0 10 337.42+89.60 128.37+49.62 Pulp and 20.0 14 371.97+89.89 153.05+45.23 Steel 瞧 13.5+7.7 13 350.08+51.13 120.43+24.63
ME 组 2.0 11 326.47+81.22 127.79+39.10ME group 2.0 11 326.47+81.22 127.79+39.10
ME 组 6.3 11 356.54+33.93 137.93+42.17ME group 6.3 11 356.54+33.93 137.93+42.17
ME 组 20.0 11 366.54+33.93 123.39+25.35 注： 与假手术组比较： * P<0.05 对 MCAO大鼠脑组织 MDA及血桨 NO水平的影响 （ ±s)ME group 20.0 11 366.54+33.93 123.39+25.35 Note: Compared with sham operation group: *P<0.05 effect on brain tissue MDA and blood plasma NO level in MCAO rats (±s)
L)
Figure imgf000024_0001
L)
Figure imgf000024_0001
11 24.96+3.1 7.98±0.86 灘且 13 33.67+12.6# 7.64+2.71  11 24.96+3.1 7.98±0.86 beach and 13 33.67+12.6# 7.64+2.71
20.0 10 24.91+6.3 6.20+1.85** 髓且 20.0 14 26.12+6.0 5.12+1.60** 鋼 瞧 13.5+7.7 13 24.01+2.3* 5.07+1.38** 20.0 10 24.91+6.3 6.20+1.85** Pulp and 20.0 14 26.12+6.0 5.12+1.60** Steel 瞧 13.5+7.7 13 24.01+2.3* 5.07+1.38**
ME 组 2.0 11 23.96+5.0* 6.19+2.93ME group 2.0 11 23.96+5.0* 6.19+2.93
ME 组 6.3 11 24.27+3.2* 5.96+1.49ME group 6.3 11 24.27+3.2* 5.96+1.49
ME 组 20.0 11 24.66+4.6* 5.52+1.47* 注： 与假手术组比较： * P<0.05与模型组比较： *P<0.05, **P<ftW。 表 17对 MCAO大鼠脑组织 Glu、 GABA水平的影响 （ ±s) 齐糧 Glu GABA 删 n ^g/g脑重） ( g g脑重） ME group 20.0 11 24.66+4.6* 5.52+1.47* Note: Compared with sham operation group: * P<0.05 compared with model group: *P<0.05, **P<ftW. Table 17 Effect on Glu and GABA levels in brain tissue of MCAO rats (±s) Qiliang Glu GABA deletion n ^g/g brain weight) ( g g brain weight)
11 156.16+64.54 28.63+8.25 灘且 13 35.33±25.08## 9.84±5.62## 11 156.16+64.54 28.63+8.25 Beach and 13 35.33±25.08## 9.84±5.62##
^m m 20.0 10 123.11+77.71** 23.20+16.25* 髓且 20.0 13 53.27+22.88 12.46+7.00 鋼 瞧 13.5+7.7 12 96.53+32.21** 21.48+11.62**^m m 20.0 10 123.11+77.71** 23.20+16.25* medulla and 20.0 13 53.27+22.88 12.46+7.00 steel 瞧 13.5+7.7 12 96.53+32.21** 21.48+11.62**
ME 组 2.0 11 101.28+57.92** 17.19+9.31 ME group 2.0 11 101.28+57.92** 17.19+9.31
ME 组 6.3 11 108.73+46.88** 16.18+9.32  ME group 6.3 11 108.73+46.88** 16.18+9.32
ME 组 20.0 10 107.8+48.00** 20.66±10.15 注： 与假手术组比较： m P<0.01与模型组比较： *P<ft05， **P<0.01 表 18 对 MCAO大鼠皮层 NMDA受体 NR1、 NR2A、 NR2B表达的影响 （ ±s) 剂量 积分灰度值 （IOD ) ME group 20.0 10 107.8+48.00** 20.66±10.15 Note: Compared with sham operation group: m P<0.01 compared with model group: *P<ft05, **P<0.01 Table 18 for MCAO rat cortical NMDA receptor NR1 , NR2A, NR2B expression effects (±s) dose integral gray value (IOD)
组别 (mg/kg) n  Group (mg/kg) n
NR1 NR2A NR2B NR1 NR2A NR2B
5 1020.3±138.1 702.0+48.6 952.9+69.2 灘且 5 1798.2+329.0## 2077.7+167.1## 1984.5+82.8## m 20.0 5 1449.9+233.0 1719.9+204.6** 1601.0+264.7** 髓且 20.0 5 1552.6+207.0 1672.0+325.3^ ' 1783.6+48.7** 鋼 瞧 13.5+7.7 5 1625.4+165.2 1683.8+116.9=· :* 1588.0±223.5** ME 组 2.0 5 1666.0+162.2 1640.3+177.6** 1953.7+140.1**5 1020.3±138.1 702.0+48.6 952.9+69.2 Beach and 5 1798.2+329.0## 2077.7+167.1## 1984.5+82.8## m 20.0 5 1449.9+233.0 1719.9+204.6** 1601.0+264.7** Pulp and 20.0 5 1552.6+ 207.0 1672.0+325.3^ ' 1783.6+48.7** Steel 瞧 13.5+7.7 5 1625.4+165.2 1683.8+116.9=· : * 1588.0±223.5** ME group 2 .0 5 1666.0+162.2 1640.3+177.6** 1953.7+140.1**
ME 组 6.3 5 1541.0±94.1 1577.8+183.4** 1489.1+102.5**ME group 6. 3 5 1541.0±94.1 1577.8+183.4** 1489.1+102.5**
ME 组 20.Q 5 1382.7+246.3* 1332.4+192.3** 1427.2+152.8**ME group 20 . Q 5 1382.7+246.3* 1332.4+192.3** 1427.2+152.8**
~~注： 与假手术组比较： ## P<0.01与模型组比较： K0.01 ; ~~ Note: Compared with the sham operation group: ## P<0.01 compared with the model group: K0.01;
19 对 MCAO大鼠脑组织 CHAT活性的影响 （； C ±s) 19 Effects on CHAT activity in brain tissue of MCAO rats (; C ± s)
齐糧 CHAT  Qiliang CHAT
删 η (U/g) Delete η (U/g)
Figure imgf000025_0001
Figure imgf000025_0001
灘且 13 103.12+47.27##  Beach and 13 103.12+47.27##
20.0 10 119.96+49.14  20.0 10 119.96+49.14
髓且 20.0 14 98.28+32.52  Pulp and 20.0 14 98.28+32.52
鋼 瞧 13.5+7.7 13 98.08+40.76  Steel 瞧 13.5+7.7 13 98.08+40.76
ME 组 2.0 11 121.26+31.43  ME group 2.0 11 121.26+31.43
ME 组 6.3 11 125.77+30.94  ME group 6.3 11 125.77+30.94
ME 组 20.0 11 128.89+30.12  ME group 20.0 11 128.89+30.12
注： 与假手术组比较： ** P<0.01 Note: Compared with sham operation group: ** P<0.01
表 20 对大鼠 rCBF的影响 （； c ±s) Table 20 Effect on rat rCBF (; c ± s)
iCBF (%) 娜 J n 再數  iCBF (%) Na J n
(/kg)  (/kg)
0 15 30 45 60 75 90 105 120 135 150 165  0 15 30 45 60 75 90 105 120 135 150 165
16.7 23.3 23.1 18.7 16.9 19.4 17.5 19.3 57.4 72.8 69.9 68.8 模型组 10 100 16.7 23.3 23.1 18.7 16.9 19.4 17.5 19.3 57.4 72.8 69.9 68.8 Model Group 10 100
±2.8 ±3.2 ±3.3 ±3.5 ±3.6 ±3.5 ±3.5 ±3.6 ±26.2 ±27.3 ±27.2 ±28.8 ±2.8 ±3.2 ±3.3 ±3.5 ±3.6 ±3.5 ±3.5 ±3.6 ±26.2 ±27.3 ±27.2 ±28.8
20.9 24.2 19.1 18.7 17.8 16.6 16.3 18.4 49.2 82.0 82.6 86.7 美金刚组 20.0 10 100 20.9 24.2 19.1 18.7 17.8 16.6 16.3 18.4 49.2 82.0 82.6 86.7 Memantine Group 20.0 10 100
±3.1 ±4.7 ±3.3 ±3.3 ±3.2 ±2.7 ±2.8 ±3.8 ±12.7 ±32.8 ±32.8 ±33. ±3.1 ±4.7 ±3.3 ±3.3 ±3.2 ±2.7 ±2.8 ±3.8 ±12.7 ±32.8 ±32.8 ±33.
16.3 54.4 45.8 54.9 55.5 54.3 51.8 51.4 107.2 112.7 115.4 119.9 烟酸组 20.0 10 100 16.3 54.4 45.8 54.9 55.5 54.3 51.8 51.4 107.2 112.7 115.4 119.9 Niacin Group 20.0 10 100
±3.2 +9.4** ±8.2* +7 9*^ ' ±10.5** ±10.6** ±10.4** ±10.3** ±34.4 ±30.6 ±29.3 ±30.±3.2 +9.4** ±8.2* + 7 9*^ ' ±10.5** ±10.6** ±10.4** ±10.3** ±34.4 ±30.6 ±29.3 ±30.
18.9 24.5 24.3 24.2 25.4 26.4 26.5 26.8 20.4 21.9 23.1 21.4 美金刚 +烟酸组 13.5+7.7 10 100 18.9 24.5 24.3 24.2 25.4 26.4 26.5 26.8 20.4 21.9 23.1 21.4 Memantine + Niacin Group 13.5+7.7 10 100
±2.3 ±3.0 ±2.4 ±3.2 ±5.4 ±7.3 ±7.7 ±8.3 ±8.1 ±9.7 ±10.3 ±8.6 ±2.3 ±3.0 ±2.4 ±3.2 ±5.4 ±7.3 ±7.7 ±8.3 ±8.1 ±9.7 ±10.3 ±8.6
13.3 21.6 28.0 27.2 27.6 25.5 22.6 20.9 18.8 20.1 20.1 20.713.3 21.6 28.0 27.2 27.6 25.5 22.6 20.9 18.8 20.1 20.1 20.7
MEN组 2.0 10 100 MEN group 2.0 10 100
±2.1 ±4.2 ±5.8 ±5.2 ±5.5 ±4.8 ±4.8 ±3.3 ±8.9 ±7.4 ±6.3 ±7.2 ±2.1 ±4.2 ±5.8 ±5.2 ±5.5 ±4.8 ±4.8 ±3.3 ±8.9 ±7.4 ±6.3 ±7.2
15.7 24.1 30.2 25.8 27.0 27.7 25.8 25.0 34.4 41.0 47.2 42.215.7 24.1 30.2 25.8 27.0 27.7 25.8 25.0 34.4 41.0 47.2 42.2
MEN组 6.3 10 100 MEN group 6.3 10 100
±2.8 ±4.1 ±4.2 ±4.4 ±3.5 ±3.5 ±3.7 ±3.9 ±9.1 ±8.1 ±8.9 ±7.1 ±2.8 ±4.1 ±4.2 ±4.4 ±3.5 ±3.5 ±3.7 ±3.9 ±9.1 ±8.1 ±8.9 ±7.1
13.3 22.5 28.4 30.4 29.3 27.0 25.9 25.9 28.8 29.2 30.3 31.013.3 22.5 28.4 30.4 29.3 27.0 25.9 25.9 28.8 29.2 30.3 31.0
MEN组 20.0 10 100 MEN group 20.0 10 100
±3.4 ±5.1 ±6.5 ±6.8 ±7.2 ±6.9 ±7.7 ±8.2 ±9.5 ±9.2 ±9.4 ±10. 注： 与模型组比较： *Ρ<0.05， **尸<0.01 ±3.4 ±5.1 ±6.5 ±6.8 ±7.2 ±6.9 ±7.7 ±8.2 ±9.5 ±9.2 ±9.4 ±10. Note: Compared with the model group: *Ρ<0.05, **尸<0.01
表 21 对东莨菪碱所致小鼠获得性记忆障碍的影响 （x ±s) Table 21 Effect of scopolamine-induced acquired memory impairment in mice (x ± s)
剂量 5min内  Dosage within 5min
组别 n  Group n
(mg/kg) 错误次数 潜伏期 （S) 空白对照组 16 0.7士 0.8 173.8士132.9 模型组 15 1.7士1.7# 71.0士100.3# 美金刚组 30.0 16 1.6士1.2 141.5士107.8 烟酸组 30.0 16 1.4±1.4 146.1±130.3 美金刚 +烟酸组 20.2+11.52 16 1.1士1.3 177.3士118.8*(mg/kg) error latency (S) blank control group 16 0.7 ± 0.8 173.8 ± 132.9 model group 15 1.7 1.7 1.7 # 71.0 士 100.3 #美金刚组30.0 16 1.6士1.2 141.5士 107.8 niacin group 30.0 16 1.4± 1.4 146.1±130.3 Memant + niacin group 20.2+11.52 16 1.1 ± 1.3 177.3 ± 118.8 *
MEN组 3.0 14 1.4士1.7 150.2士124.8MEN group 3.0 14 1.4 1.7 150.2 ± 124.8
MEN1组 9.5 14 0.9士 1.1 171.4士 134.6*MEN1 group 9.5 14 0.9士 1.1 171.4士 134.6*
MEN组 30.0 14 0.9士 0.8 176.4士 119.1* 注： 与空白对照组比较： ff P<0.05 ; 与模型组比较： *P<0.05。 不同浓度 MEN061016-1和美金刚对 N DA受体与3 H-MK801结合的影响 MEN group 30.0 14 0.9 ± 0.8 176.4 ± 119.1 * Note: Compared with the blank control group: ff P < 0.05 ; compared with the model group: * P < 0.05. Effects of different concentrations of MEN061016-1 and memantine on the binding of N DA receptor to 3 H-MK801
组别 丽 DA受体与 ¾-MK-801结合量 脑皮质悬液 +¾-ΜΚ-801 (ΙΟηΜ) (空白对照组） 447. 96 ±66. 00  Group DA receptor and 3⁄4-MK-801 binding amount Cerebral cortical suspension +3⁄4-ΜΚ-801 (ΙΟηΜ) (blank control group) 447. 96 ±66. 00
脑皮质悬液+美金刚 500ηΜ+¾-ΜΚ-801 (ΙΟηΜ) 195. 46 + 26. 94 Cerebral cortex suspension + memantine 500ηΜ+3⁄4-ΜΚ-801 (ΙΟηΜ) 195. 46 + 26. 94
lOOul脑皮质悬液+美金刚 100nM+3H-MK-801 (10nM) 343. 58 + 35. 89 *** lOOul脑皮质悬液 +Men 500nM+¾-MK-801 (10nM) 405. 29 ± 33. 78 lOOul cerebral cortex suspension + memantine 100nM+ 3 H-MK-801 (10nM) 343. 58 + 35. 89 *** lOOul cerebral cortex suspension +Men 500nM+3⁄4-MK-801 (10nM) 405. 29 ± 33 . 78
lOOul脑皮质悬液 +Men 100nM+¾-MK-801 (ΙΟηΜ) 397. 58 ±62. 29 * lOOul cerebral cortex suspension +Men 100nM+3⁄4-MK-801 (ΙΟηΜ) 397. 58 ±62. 29 *
注： *** p<0. 001 , * p<0. 5vs空白对照组;  Note: *** p<0. 001 , * p<0. 5vs blank control group;
附图说明： BRIEF DESCRIPTION OF THE DRAWINGS:
图 1-2为 MEN061016-1对缺血大鼠局部脑血流量的影响 图 3-14为 MEN061016-1对神经元、 星形胶质细胞及脑微血管内皮细胞的影响 图 15-17为 MEN061016-1对亲代谢型和离子型谷氨酸受体机制研究  Figure 1-2 shows the effect of MEN061016-1 on regional cerebral blood flow in ischemic rats. Figure 3-14 shows the effect of MEN061016-1 on neurons, astrocytes and brain microvascular endothelial cells. Figure 15-17 shows MEN061016- A pair of metabotropic and ionotropic glutamate receptor mechanisms
图 1、 MEN061016-1对大鼠脑缺血 /再灌注局部脑血流量 （rCBF) 的影响 图 2、 MEN061016-1对大鼠脑缺血时局部脑血流量 （rCBF) 的影响  Fig.1 Effect of MEN061016-1 on regional cerebral blood flow (rCBF) in rats with cerebral ischemia/reperfusionFig.2 Effect of MEN061016-1 on regional cerebral blood flow (rCBF) in rats with cerebral ischemia
图 3、 正常星形胶质细胞加药 48小时 MTT值  Figure 3. Normal astrocyte dosing 48 hours MTT value
图 4、 正常星形胶质细胞加药 48小时 MTT值 （重复）  Figure 4. Normal astrocyte dosing 48 hours MTT value (repeated)
图 5、 星形胶质细胞预加药 24h谷氨酸处理 2hMTT值  Figure 5. Astrocyte pre-dosing 24h glutamate treatment 2hMTT value
图 6、 星形胶质细胞预加药 24h谷氨酸处理 2hMTT值 （重复） 图 7、 星形胶质细胞预加药 24h谷氨酸处理 2h后再加药 48h测 MTT值 Figure 6. Astrocyte pre-dosing 24h glutamate treatment 2hMTT value (repeated) Figure 7. Astrocyte pre-dosing 24h glutamate treatment for 2h and then add for 48h to measure MTT value
图 8、 星形胶质细胞预加药 24h谷氨酸处理 2h后再加药 48h测 MTT值 （重复） 图 9、 正常脑微血管内皮细胞加药 72hMTT值 Figure 8. Astrocyte pre-dosing 24h glutamate treatment 2h and then add medicine 48h to measure MTT value (repeated) Figure 9. Normal brain microvascular endothelial cell dosing 72hMTT value
图 10、 正常脑微血管内皮细胞加药 72MTT值 （重复） Figure 10. Normal brain microvascular endothelial cell dosing 72MTT value (repeated)
图 11、 脑微血管内皮细胞预加药 24h谷氨酸处理 2h后再加药 24hMTT Figure 11. Pre-dosing of brain microvascular endothelial cells 24h glutamate treatment 2h after adding 24hMTT
图 12、 脑微血管内皮细胞预加药 24h谷氨酸处理 2h后再加药 24hMTT (重复） 图 13、 脑微血管内皮细胞预加药 24h谷氨酸处理 2h后再加药 96hMTT Figure 12. Pre-dosing of brain microvascular endothelial cells 24h glutamate treatment 2h after adding 24hMTT (repeated) Figure 13. Pre-dosing of brain microvascular endothelial cells 24h glutamate treatment 2h after adding 96hMTT
图 14、 脑微血管内皮细胞预加药 24h谷氨酸处理 2h后再加药 120hMTT 图 15 激动剂及拮抗剂对 MPP+ 抑制星形胶质细胞 MTT的影响 Figure 14. Pre-dosing of brain microvascular endothelial cells 24h glutamate treatment 2h after adding 120hMTT Figure 15 Effect of agonists and antagonists on MPP+ inhibition of astrocyte MTT
图 16 MEN061016-1对 MPP+ 抑制星形胶质 MTT的影响 Figure 16 Effect of MEN061016-1 on MPP+ inhibition of star-shaped colloidal MTT
图 17 MEN061016-1逆转 1_甲基 _4_苯基吡啶离子 (MPP + )抑制谷氨酸摄入的影响 具体实 式 Figure 17 MEN061016-1 reversal 1_methyl _4_phenylpyridinium ion (MPP + ) inhibits the effects of glutamate uptake
以下列举具体实例以进一步阐述本发明，应理解实例并非用于限制本发明的保护 范围。 The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
实施例 1 Example 1
DCC/DMAP法 (活性酯法)制备 N-(3-吡啶甲酰氧基）_3，5-二甲基 -1-金刚烷胺  Preparation of N-(3-pyridineformyloxy)_3,5-dimethyl-1-adamantanamine by DCC/DMAP method (active ester method)
2.46g (0.02mol)烟酸和 4.32g (0.02mol) 盐酸美金刚胺室温下溶于干燥的 120 mL DMF中， 滴加 2.30 mL无水 TEA后加入 0.30g (0.002mol) DMAP, 搅拌均勾。 于 0-5°C滴加 6.19g (0.03mol) DCC的50mL DMF溶液， 室温搅拌过夜， 过滤， 减压 浓缩， 加入适量乙酸乙酯， 过滤， 依次用水、 0.1N HC1、 饱和 NaHC03、 饱和食 盐水洗 3次， 无水硫酸镁干燥， 减压浓缩， 快速柱层析 (硅胶 H， 洗涤液： 石油醚- 丙酮)， 得 N-(3-吡啶甲酰氧基 ) -3,5-二甲基 -1-金刚烷胺， 无色粘稠状物质, 3.58 g， 产率 63%。 2.46g (0.02mol) of nicotinic acid and 4.32g (0.02mol) memantine hydrochloride were dissolved in dry 120 mL DMF at room temperature, 2.30 mL of anhydrous TEA was added dropwise, 0.30 g (0.002 mol) of DMAP was added, and the mixture was stirred. . At 0-5 ° C was added dropwise 6.19g (0.03mol) DCC in 50mL DMF was stirred at room temperature overnight, filtered, and concentrated under reduced pressure, adding an appropriate amount of ethyl acetate, filtered, washed with water, 0.1N HC1, saturated NaHC0 3, saturated Washed with brine 3 times, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography ( silica gel H, washing: petroleum ether-acetone) to give N-(3-pyridoyloxy) -3,5- Methyl-1-adamantanamine, colorless viscous material, 3.58 g, yield 63%.
MS(EI) , m/z:284(M+)， 269(M+-C¾), 178(C12H2。NO+)， 106(C6H4NO+)， 78(C5H4N+)。 MS (EI), m / z : 284 (M +), 269 (M + -C¾), 178 (C 12 H 2 .NO +), 106 (C 6 H 4 NO +), 78 (C 5 H 4 N +).
1HNMR(400MHz, CDC13)， (400MHz, D20+CDC13)， 8(ppm):
Figure imgf000029_0001
 1 H NMR (400 MHz, CDC1 3 ), (400 MHz, D 2 0+CDC1 3 ), 8 (ppm):
Figure imgf000029_0001
0.86(6H,s,美金刚胺两个甲基上的 H);l.l〜2.4(13H,m,金刚烷上各碳原子上 的 H);5.86(lH,s,N-H,氘代后积分面积减少）； 7.33(lH,m,5-H); 8.02(lH,d, J=8Hz, 4-H); 8.65(lH,d, J=4Hz,6-H); 8.67(lH,s,2-H)。 0.86 (6H, s, H on two methyl groups of memantine; ll~2.4 (13H, m, H on each carbon atom on adamantane); 5.86 (lH, s, NH, integrated area after deuteration) Reduction); 7.33 (lH, m, 5-H) ; 8.02 (lH, d, J = 8 Hz, 4-H); 8.65 (lH, d, J = 4 Hz, 6-H) ; 8.67 (lH, s, 2-H).
IR(NaCl晶体涂抹)： 1417 cm"1, 1473
Figure imgf000029_0002
cm—1处的吸收峰为 吡啶环的骨架振动,其与羰基共轭，向低波数位移。 1317 cm— 1处的吸收峰为 C-N的 伸缩振动吸收峰， 1645 cm— 1处为酰胺的羰基吸收峰， 3309 cm— 1处为酰胺的 N-H的 伸缩振动峰。 IR (NaCl crystal smear): 1417 cm" 1 , 1473
Figure imgf000029_0002
The absorption peak at cm- 1 is the skeleton vibration of the pyridine ring, which is conjugated to the carbonyl group and displaced to a low wave number. The absorption peak at 1317 cm- 1 is the stretching vibration absorption peak of CN, the carbonyl absorption peak of amide at 1645 cm- 1 , and the stretching vibration peak of NH at 3309 cm- 1 .
实施例 2 Example 2
CDI/DMAP法 (活性酯法)制备 N-(3-吡啶甲酰氧基) _3，5-二甲基 -1-金刚烷胺  Preparation by CDI/DMAP method (active ester method) N-(3-pyridine formyloxy)_3,5-dimethyl-1-adamantanamine
0.62g (5 mmol)烟酸溶于干燥的 25 mL DMF溶液中，快速加入 0.81g (0.002mol) CDI和 0.08g (0.5 mmol)DMAP， 于 0-5°C搅拌反应 1 h。滴加 1.08g (5 mmol) 盐酸美 金刚胺的 2.30 mL无水 TEA和 25mL DMF溶液的混合溶液， 室温搅拌过夜， 减压 浓缩， 加入适量乙酸乙酯， 过滤， 依次用水、 0.1N HC1、 饱和 NaHC03、 饱和食 盐水洗 3次， 无水硫酸镁干燥， 减压浓缩， 快速柱层析 (硅胶 H， 洗涤液： 石油醚- 丙酮)， 得 N-(3-吡啶甲酰氧基 ) -3,5-二甲基 -1-金刚烷胺， 无色粘稠状物质, 0.99 g， 产率 70%。 0.62 g (5 mmol) of nicotinic acid was dissolved in dry 25 mL of DMF solution, and 0.81 g (0.002 mol) of CDI and 0.08 g (0.5 mmol) of DMAP were quickly added, and the reaction was stirred at 0-5 ° C for 1 h. A mixed solution of 1.08 g (5 mmol) of memantine hydrochloride in 2.30 mL of anhydrous TEA and 25 mL of DMF solution was added dropwise, stirred at room temperature overnight, concentrated under reduced pressure, added with ethyl acetate, filtered, and then water, 0.1 N HCl, sat. NaHC0 3 and saturated brine were washed three times, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure, and purified by column chromatography (silica gel H, washing liquid: petroleum ether-acetone) to give N-(3-pyridinecarbonyloxy) -3 , 5-dimethyl-1-adamantanamine, colorless viscous material, 0.99 g, yield 70%.
实施例 3 Example 3
混合酸酐法制备 N-(3-吡啶甲酰氧基) -3,5-二甲基 -1-金刚烷胺 Preparation of N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine by mixed acid anhydride method
冰盐浴下将 0.32 g (2.60mmol)烟酸溶于适量无水 CH2C12中， 依次缓慢滴加 1 mL (7.18 mmol) TEA和 0.4 mL (3.13 mmol) 无水苯磺酰氯， 搅拌反应 1 h， 力口 入 0.40 g ( 1.86 mmol)美金刚胺盐酸盐和 TEA的 CH2C12混合溶液， 搅拌反应 3 h， 减压浓缩， 加入适量乙酸乙酯， 过滤， 依次用水、 0.1 N HC1 、 饱和 NaHC03、 饱和食盐水洗 3次， 无水硫酸镁干燥， 减压浓缩， 得红色油状物， 柱层析分离纯 化 (硅胶 H, 洗脱液：石油醚-丙酮混合溶剂)得 N-(3-吡啶甲酰氧基） -3,5-二甲基 -1- 金刚烷胺， 无色粘稠状物质, 0.41 g， 产率 77%。 实施例 4 0.32 g (2.60 mmol) of nicotinic acid was dissolved in an appropriate amount of anhydrous CH 2 C1 2 under ice-salt bath, and 1 mL (7.18 mmol) of TEA and 0.4 mL (3.13 mmol) of anhydrous benzenesulfonyl chloride were slowly added dropwise, and the reaction was stirred. 1 h, a mixture of 0.40 g ( 1.86 mmol) of memantine hydrochloride and TEA in CH 2 C1 2 was added , stirred for 3 h, concentrated under reduced pressure, added with aq. ethyl acetate, filtered, sequentially water, 0.1 N After washing with HCl, saturated NaHC0 3 and saturated brine, dried over anhydrous magnesium sulfate, and evaporated, evaporated, evaporated, evaporated. (3-Pyridoyloxy)-3,5-dimethyl-1-adamantanamine, colorless viscous material, 0.41 g, yield 77%. Example 4
酰氯法制备 N-(3-吡啶甲酰氧基 ) _3，5-二甲基 -1-金刚烷胺 Preparation Method chloride N- (3-pyridyl formyloxy) 3, 5 - dimethyl-1-adamantanamine
在干燥无水条件下， 装好干燥装置及气体吸收装置， 将 10 mL氯化亚砜加 入反应瓶中，用冰水浴冷却到 0°C左右，搅拌， 向其中加入 3.0 g(24.37mmol)烟酸， 并滴入 5-7滴无水 DMF， 油浴缓慢升温至 77°C左右回流， 3 h后减压蒸除过量氯化 亚砜， 得烟酰氯盐酸盐 (浅黄色固体)。 冰盐浴下向干燥的反应瓶中加入适量无水 丙酮， 强烈搅拌， 滴加 2.10 mL无水 TEA,加入盐酸美金刚胺 0.50 g C2.32 mmol),撤 去冰浴，换为油浴， 加热回流反应 2 h。 停止反应， 滤过， 滤液减压浓缩后加入适 量乙酸乙酯， 依次用水、 0.1N HC1、 饱和 NaHC03、 饱和食盐水洗至中性， 无水 硫酸镁干燥， 过滤， 减压浓缩， 快速柱层析 (硅胶 H， 洗涤液： 石油醚-丙酮)， 得 N-(3-吡啶甲酰氧基 ) -3,5-二甲基 -1-金刚烷胺，无色粘稠状物质, 0.64 g，产率 97%。 实施例 5 Under dry and anhydrous conditions, the drying device and the gas absorption device were installed, 10 mL of thionyl chloride was added to the reaction flask, cooled to about 0 ° C with an ice water bath, stirred, and 3.0 g (24.37 mmol) of tobacco was added thereto. Acid, and 5-7 drops of anhydrous DMF were added dropwise, and the oil bath was slowly heated to reflux at about 77 ° C. After 3 hours, excess thionyl chloride was distilled off under reduced pressure to give nicotinyl chloride hydrochloride (yellow solid). Add an appropriate amount of anhydrous acetone to the dry reaction flask under ice salt bath, stir vigorously, add 2.10 mL of anhydrous TEA, add memantine hydrochloride 0.50 g C2.32 mmol), remove the ice bath, and change to oil bath, heat. The reaction was refluxed for 2 h. The reaction was stopped by adding an appropriate amount of ethyl acetate. After filtration, the filtrate was concentrated under reduced pressure, washed with water, 0.1N HC1, saturated NaHC0 3, washed with brine to neutral, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure, flash column (silica gel H, washing liquid: petroleum ether-acetone) to give N-(3-pyridinecarbonyloxy)-3,5-dimethyl-1-adamantanamine as colorless viscous material, 0.64 g The yield was 97%. Example 5
N-(3-吡啶甲酰氧基 ) _3，5-二甲基 -1-金刚烷胺盐酸盐的制备  Preparation of N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride
将 1 g (3.5 mmol)N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺溶于 30ml*** 和 30ml异丙醇中， 缓缓滴加适量重量浓度为 14%的盐酸溶液， 析出白色固体， 过 滤， 异丙醇 -水重结晶， 干燥得 N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺盐酸 盐 1 g， 经 HPLC分析， 含量 98%。  Dissolve 1 g (3.5 mmol) of N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantamine in 30 ml of ether and 30 ml of isopropanol, and slowly add appropriate weight concentration. As a 14% hydrochloric acid solution, a white solid precipitated, filtered, recrystallized from isopropanol-water, and dried to give N-(3-pyridoyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride. 1 g of salt, analyzed by HPLC, content 98%.
实施例 ό Example ό
Ν-(3-吡啶甲酰氧基 ) _3，5-二甲基 -1-金刚烷胺硫酸盐的制备  Preparation of Ν-(3-pyridinecarbonyloxy) _3,5-dimethyl-1-adamantanamine sulfate
将 1 g (3.5 mmol)N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺溶于 30ml*** 和 30ml丙酮中，缓缓滴加适量重量浓度为 20%的硫酸溶液，析出白色固体，过滤， 丙酮 -水重结晶，干燥得 N-(3-吡啶甲酰氧基 ) -3,5-二甲基小金刚烷胺硫酸盐 1.2 g， 经 HPLC分析， 含量 98%。  Dissolve 1 g (3.5 mmol) of N-(3-picoyloxy)-3,5-dimethyl-1-adamantamine in 30 ml of ether and 30 ml of acetone, and slowly add an appropriate amount of 20 by weight. % sulfuric acid solution, white solid precipitated, filtered, recrystallized from acetone-water, dried to give N-(3-pyridineformyloxy)-3,5-dimethyl-diamantanamine sulfate 1.2 g, analyzed by HPLC , the content is 98%.
实施例 7 Example 7
N-(3-吡啶甲酰氧基 ) _3，5-二甲基 -1-金刚烷胺柠檬酸盐的制备  Preparation of N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine citrate
将 1 g (3.5 mmol)N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺溶于 40 ml石油 醚和 20 ml丙酮中， 加入 0.7 g(3.5 mmol)柠檬酸， 回流 1 h， 冷却， 减压浓缩析出白 色固体， 丙酮 -水重结晶， 干燥得 N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺柠 檬盐 1.3 g， 经 HPLC分析， 含量 98%。 实施例 8 1 g (3.5 mmol) of N-(3-picoyloxy)-3,5-dimethyl-1-adamantamine was dissolved in 40 ml of petroleum ether and 20 ml of acetone, and 0.7 g (3.5 mmol) was added. Citric acid, refluxed for 1 h, cooled, concentrated under reduced pressure to give a white solid, recrystallized from acetone-water and dried to give N-(3-pyridoyloxy) -3,5-dimethyl-1-adamantamine Lemon salt 1.3 g, analyzed by HPLC, content 98%. Example 8
N-(3-吡啶甲酰氧基 ) _3，5-二甲基 -1-金刚烷胺盐酸盐片剂的制备 Preparation of dimethyl-1-adamantanamine hydrochloride tablets - N- (3- pyridin-formyloxy) 3, 5
处方 ( 10000片)  Prescription (10,000 tablets)
Figure imgf000031_0001
Figure imgf000031_0001
制备方法： 将 N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺盐酸盐粉碎并过 100 目筛， 称重后， 与已过 100目筛的预胶化淀粉、 磷酸氢钙及乳糖混合均勾， 加入 10%淀粉浆制成软材， 以 16目尼龙筛制粒， 湿粒于 40°C干燥， 干颗粒与硬脂酸镁 混合均勾， 再经 16目筛整粒， 经含量测定后， 计算出片重， 选择 6mm浅凹或平冲 模压片即得。 成人推荐口服用量 1 片 /次， 2次 /日。  Preparation method: N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride is pulverized and sieved through a 100 mesh sieve, and weighed, and after passing through a 100 mesh sieve. Pre-gelatinized starch, calcium hydrogen phosphate and lactose are mixed, 10% starch slurry is added to make soft material, 16 mesh nylon sieve is used for granulation, wet granules are dried at 40 ° C, and dry granules are mixed with magnesium stearate. After granulating through a 16-mesh sieve, after the content is determined, the tablet weight is calculated, and a 6 mm dimple or flat die is selected. Oral dosage is recommended for adults 1 tablet / time, 2 times / day.
实施例 9  Example 9
N-(3-吡啶甲酰氧基 ) _3，5-二甲基 -1-金刚烷胺盐酸盐胶囊剂的制备  Preparation of N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride capsule
处方 ( 10000粒)  Prescription (10,000 capsules)
Figure imgf000031_0002
Figure imgf000031_0002
制备方法： 将 N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺盐酸盐粉碎并过 100目 筛， 称重后， 与已过 100目筛的淀粉、 微晶纤维素混合均勾， 加入 10%淀粉浆制 成软材， 以 16目尼龙筛制粒， 湿粒于 40°C干燥， 干颗粒再经 16目筛整粒， 分装于 3号硬胶囊壳， 盖上节， 压平， 打光即可。 成人推荐口服用量 1粒 /次， 2次 /日。 实施例 10 Preparation method: N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride is pulverized and passed through a 100 mesh sieve, weighed, and passed through a 100 mesh sieve. The starch and microcrystalline cellulose are mixed and hooked. Add 10% starch slurry to make soft material, granulate with 16 mesh nylon sieve, wet the pellet at 40 °C, dry granules and then sieve through 16 mesh sieve, and dispense into 3 No. Hard capsule shell, cover the knot, flatten, and light. Adults recommend oral dosage 1 capsule / time, 2 times / day. Example 10
N-(3-吡啶甲酰氧基 ) _3，5-二甲基 -1-金刚烷胺马来酸盐的制备  Preparation of N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine maleate
将 1 g (3.5 mmol)N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺溶于 40 ml石油 醚和 20 ml乙酸乙酯中， 加入 0.4 g(3.5 mmol)马来酸的乙酸乙酯溶液， 回流 1 h， 冷 却，减压浓缩析出白色固体，异丙醇 -水重结晶，干燥得 N-(3-吡啶甲酰氧基 ) -3,5- 二甲基 -1-金刚烷胺马来酸盐 1.1 g， 经 HPLC分析， 含量 98%。  1 g (3.5 mmol) of N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine was dissolved in 40 ml of petroleum ether and 20 ml of ethyl acetate, and 0.4 g ( 3.5 mmol) of a solution of maleic acid in ethyl acetate, refluxed for 1 h, cooled, concentrated under reduced pressure to give a white solid, crystallised from isopropyl alcohol-water and dried to give N-(3-pyridoyloxy) -3,5 - Dimethyl-1-adamantanamine maleate 1.1 g, analyzed by HPLC, content 98%.
实施例 11  Example 11
N-(3-吡啶甲酰氧基 ) _3，5-二甲基 -1-金刚烷胺盐酸盐口服液的制备  Preparation of N-(3-pyridineformyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride oral solution
处方 ( 10000 ml)  Prescription (10000 ml)
Figure imgf000032_0001
Figure imgf000032_0001
制备方法： 分别称取 N-(3-吡啶甲酰氧基） -3,5-二甲基 -1-金刚烷胺盐酸盐、 山梨酸钾，溶于适量蒸馏水中，加入果味香精后添加蒸馏水至全量，搅勾，过滤， 滤液分装于 10 ml棕色瓶中， 于 100°C流通蒸汽灭菌 30min即得。成人推荐口服 用量 10 ml/次， 2次 /日  Preparation method: N-(3-picoyloxy)-3,5-dimethyl-1-adamantanamine hydrochloride, potassium sorbate, and dissolved in an appropriate amount of distilled water, after adding fruit flavor Distilled water was added to the whole amount, the hook was stirred, and the filtrate was dispensed into a 10 ml brown bottle and steam-sterilized at 100 ° C for 30 min. Adult recommended oral dosage 10 ml / time, 2 times / day

Claims

权利要求书 Claim
1、 K3-吡啶甲酰氧基 ) -3, 5-二甲基 -1-金刚烷胺或其可药用盐在制备治疗血管 性痴呆的药物中的应用。 Use of K3-picoyloxy)-3,5-dimethyl-1-adamantanamine or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating vascular dementia.
2、 权利要求 1的应用， 其特征在于， 其中 Κ3-吡啶甲酰氧基 ) -3, 5-二甲基-l- 金刚烷胺如式 I所示  The use of claim 1 wherein Κ3-pyridine formyloxy) -3,5-dimethyl-l-adamantanamine is as shown in formula I
Figure imgf000033_0001
式 I
Figure imgf000033_0001
Formula I
3、权利要求 1的应用， 其特征在于， 所述应用针对血管性痴呆病程中延缓病程发 展和改善记忆的应用。  3. Use according to claim 1, characterized in that the application is directed to the application of delaying the progression of the disease and improving memory in the course of vascular dementia.
4、权利要求 1的应用， 其特征在于， 所述应用针对减轻多发梗塞性痴呆神经病变 程度， 保护神经元正常功能。  4. The use of claim 1 wherein said application is directed to reducing the extent of neuropathy in multiple infarct dementia and protecting normal function of the neuron.
5、 权利要求 1的应用， 其特征在于， 所述应用针对缺血再灌注损伤的保护作用。  5. Use according to claim 1, characterized in that said application is directed to the protective effect of ischemia-reperfusion injury.
6、权利要求 1的应用， 其特征在于， 所述应用针对增加谷氨酸损伤神经元的细胞 活性并增加培养液中神经生长因子 (NGF)和突触素（SYN)水平而减少细胞内钙 浓度以及增加突触长度的作用。  6. The use according to claim 1, characterized in that the application is directed to increasing the cellular activity of glutamate-injured neurons and increasing the levels of nerve growth factor (NGF) and synaptophysin (SYN) in the culture medium to reduce intracellular calcium. Concentration and the effect of increasing synaptic length.
7、 权利要求 1的应用， 其特征在于， 所述应用针对增加 02损伤神经元的细胞活 性作用。 7. Use according to claim 1, characterized in that said application is directed to increasing the cellular activity of the 0 2 injured neurons.
8、 权利要求 1的应用， 其特征在于， 所述可药用盐选自： 盐酸盐、 硝酸盐、 硫酸 盐、 氢溴酸盐、 磷酸盐、 甲酸盐、 乙酸盐、 苯甲酸盐、 苹果酸盐、 马来酸盐或柠 檬酸盐。  8. The use of claim 1 wherein said pharmaceutically acceptable salt is selected from the group consisting of: hydrochloride, nitrate, sulfate, hydrobromide, phosphate, formate, acetate, benzoic acid Salt, malate, maleate or citrate.
9、 权利要求 1的应用， 其特征在于， 所述药物是是任何一种药物制剂形式。  9. Use according to claim 1, characterized in that the medicament is in the form of any pharmaceutical preparation.
10、 权利要求 1的应用， 其特征在于， 所述药物制剂形式选自： 片剂、 胶囊、 口 服液、 颗粒剂或粉剂。  10. Use according to claim 1, characterized in that the pharmaceutical preparation form is selected from the group consisting of: tablets, capsules, oral solutions, granules or powders.
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