WO2010054531A1 - 牛痘疫苗致炎兔皮提取物在制备急性脑血管疾病治疗药物中的用途 - Google Patents

牛痘疫苗致炎兔皮提取物在制备急性脑血管疾病治疗药物中的用途 Download PDF

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WO2010054531A1
WO2010054531A1 PCT/CN2009/001181 CN2009001181W WO2010054531A1 WO 2010054531 A1 WO2010054531 A1 WO 2010054531A1 CN 2009001181 W CN2009001181 W CN 2009001181W WO 2010054531 A1 WO2010054531 A1 WO 2010054531A1
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cerebral
group
cerebrovascular disease
acute
medicament
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PCT/CN2009/001181
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English (en)
French (fr)
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刘万生
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威世药业（如皋）有限公司
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Priority to LTEP09825706.6T priority Critical patent/LT2364711T/lt
Priority to JP2011534986A priority patent/JP6085806B2/ja
Priority to SI200931579A priority patent/SI2364711T1/sl
Priority to KR1020117012994A priority patent/KR101756201B1/ko
Priority to CA2743090A priority patent/CA2743090C/en
Priority to AU2009316168A priority patent/AU2009316168B2/en
Priority to US13/128,254 priority patent/US10265345B2/en
Application filed by 威世药业（如皋）有限公司 filed Critical 威世药业（如皋）有限公司
Priority to EP09825706.6A priority patent/EP2364711B2/en
Priority to DK09825706.6T priority patent/DK2364711T3/en
Priority to ES09825706T priority patent/ES2606051T5/es
Priority to NZ592696A priority patent/NZ592696A/xx
Publication of WO2010054531A1 publication Critical patent/WO2010054531A1/zh
Priority to SM201600477T priority patent/SMT201600477B/it
Priority to CY20171100033T priority patent/CY1118457T1/el

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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 the treatment of acute cerebrovascular diseases, and more particularly to the use of a burdock vaccine-inflamed rabbit skin extract for the preparation of a medicament for the treatment of acute cerebrovascular diseases.
  • Stroke is an acute cerebrovascular disease that is the third leading cause of death and the highest disability in the world. According to the latest epidemiological data, the incidence of cerebrovascular disease in China is 120/100,000-180/100,000, ranking second among the causes of death. Each year, there are 120-1.5 million new cerebrovascular patients, 80-100 million deaths, and about 75% of the survivors are disabling. The recurrence rate is as high as 41% within 5 years, which seriously affects the quality of life of the elderly. The society has a heavy burden and has a tendency to become younger in recent years.
  • Cerebrovascular diseases are mainly divided into two types: hemorrhagic and ischemic, of which the latter account for 60-
  • the drugs currently used clinically for the treatment of cerebral ischemia are mainly calcium antagonists (nimodipine), oxygen free radical scavengers (VitE, SOD), and neurotrophic factors ( Nerve growth factor, neurotrophic factor), excitatory sense acid antagonists, antioxidants, and drugs that improve delayed neuronal damage.
  • These drugs work through different mechanisms of action, but the therapeutic effects are uncertain or not specific or With serious adverse reactions, it is still unable to meet clinical needs.
  • drugs in the domestic market such as Nafflower, flunarizine, karan, ginkgo extract, etc., which are used to improve cerebral circulation, brain metabolism and brain function. Although they have certain characteristics, they can not be used to treat cerebrovascular diseases. sure.
  • Research and development of new type for ischemic cerebral blood Drugs for tube diseases are an important task in the field of pharmacy and pharmacology.
  • vaccinia rabbit skin extract refers to an active substance extracted from the skin of a rabbit infected with vaccinia vaccine, as described in Chinese Patent No. ZL98103220.6, the entire contents of which are incorporated by reference.
  • This vaccinia rabbit skin extract can be obtained commercially, and the product name is Enshishi, which is produced by Wei Shi Pharmaceutical (Rugao) Co., Ltd.
  • the pharmacological effects of Enshiyi include (1) Zhen The painful effect has obvious analgesic effect on the hyperalgesia induced by repeated cold stress, which is achieved by activating the central nervous system descending inhibition system; (2) the effect on cold sense and perceptual abnormality, in vivo and in vitro test It shows that this product has the effect of changing the activity of neurons in the hypothalamus, suggesting that this product has a repairing and regulating effect on the abnormal behavior of sensory neurons.
  • This abnormality is considered to be the cause of neuralgia and perceptual abnormality; (3) The role of good peripheral blood circulation; (4) regulation of autonomic nerves: In vivo and in vitro tests suggest that this product can pass regulation Autonomic nerve activity, improve autonomic dysfunction symptoms; (5) Anti-allergic effects: Animal tests suggest that this product has anti-I-type allergic reaction, inhibits respiratory hypersecretion caused by parasympathetic excitation, and is resistant to nasal mucosa. Up-regulation of body density also has an inhibitory effect; (6) Sedation: Animal tests suggest that this product has a sedative effect on the emotional excitatory state caused by various external stimuli.
  • vaccinia vaccine-induced rabbit skin extracts were also discussed in the following applications: Chinese Patent Application No. 99123485.5, filed on November 12, 1999, Chinese Patent Application No. 96123286.2, filed on December 19, 1996, and January 1998 Chinese patent application 98103914.6 applied for on the 07th.
  • One aspect of the invention provides a method of treating an acute cerebrovascular disease in a mammal, the method comprising administering to a patient in need thereof a vaccinia vaccine-induced rabbit skin extract.
  • Another aspect of the invention provides the use of a vaccinia vaccine-inflamed rabbit skin extract for the preparation of a medicament for the treatment of an acute cerebrovascular disease in a mammal.
  • the acute ischemic cerebrovascular disease described herein is an acute ischemic cerebrovascular disease.
  • Cerebrovascular disease is a neurological impairment caused by abnormal blood supply to the brain. In most countries of the world, cerebrovascular disease is the top three causes of death and can cause adult brain damage. It is the main cause of harm to the health of middle-aged and elderly people, and is the leading cause of death and disability in the elderly in many countries. Ischemic cerebrovascular disease is caused by cerebral vascular occlusion, usually embolism or thrombosis, and its pathophysiological changes are extremely complicated.
  • the acute ischemic cerebrovascular disease described herein includes, but is not limited to, cerebral embolism, transient ischemic attack, cerebral thrombosis, cerebral arteriosclerosis, cerebral arteritis, cerebral arterhesis syndrome, Intracranial sinus and venous thrombosis.
  • Ischemic cerebrovascular disease is caused by a transient or permanent decrease in blood flow in an arterial blood supply area due to embolism, and its pathological process involves complex time and spatial cascades.
  • the direct cause of the disease is that each artery in the brain has its basic blood supply range. After occlusion, the brain tissue of the corresponding area is softened, resulting in a corresponding clinical syndrome. The contralateral limb hemiplegia caused by occlusion of the middle cerebral artery. Equal neurological symptoms are the most common.
  • the middle cerebral artery occlusion also accounts for a large proportion, so the pathological process simulated by the middle cerebral artery occlusion (MCAO) model has great similarity with clinical stroke. .
  • the extent of cerebral infarction was related to the degree of ischemia. After 24 hours of unilateral middle cerebral artery embolization, the TTC staining of the contralateral brain tissue was white, and liquefaction was also seen. The statistical results showed that the volume of cerebral infarction in the sham operation group was similar to that of the solvent group. Ratio, the difference is significant, and the moderate dose The volume of cerebral infarction was significantly lower in the group than in the injured group. Thus, in one embodiment, it is desirable to treat cerebrovascular disease by reducing the extent of cerebral infarction.
  • the brain is the most metabolically active organ with the least energy and oxygen reserves. At rest, brain tissue oxygen consumption accounts for 20% of total systemic oxygen consumption. Neurons are a major component of the oxygen consumption that constitutes the cerebral cortex or the entire brain and are extremely sensitive to ischemia and hypoxia damage. In the absence of a source of fresh oxygen, the tissue can only consume its high-energy phosphate compound reserves and obtain energy by metabolizing the stored glucose and glycogen to MDA. Brain tissue ischemia and hypoxia lead to energy depletion, causing energy pump failure, calcium intracellular overload, increased toxic oxygen free radicals, cellular acidosis and other chain-locking reactions, and the structure and integrity of the cell membrane are destroyed.
  • lactic acid content and MDA of brain tissue were significantly increased after ischemia, and the lactic acid content of brain tissue was significantly decreased.
  • SOD is an important antioxidant enzyme that can effectively inhibit free radical reactions.
  • High SOD activity means strong antioxidant capacity.
  • SOD activity in the brain tissue of rats was significantly reduced, and the ability to scavenge free radicals was reduced.
  • the results showed that the reperfusion of spleen could increase the activity of SOD, indicating that Enshiyi may exert neuroprotective effects by improving the antioxidant capacity of brain tissue.
  • the inventors of the present invention have found that it is possible to exert a protective effect on nerve cell damage.
  • 3 ⁇ 40 2 is an important active oxygen component involved in the pathogenesis of neurological diseases such as cerebral ischemia, trauma, brain aging, and Alzheimer's disease. It can peroxidize membrane lipids, reduce the fluidity of cell membranes, alter intracellular protein components and activity, concentrate chromatin, break DNA, and ultimately lead to cell death. Thus, in one embodiment, it is further useful to improve PC12 cell damage induced by 3 ⁇ 40 2 .
  • Neuronal death is associated with a variety of chronic or acute neuropathy, and excitatory amino acids such as glutamate play an important role in this process.
  • Glutamate can damage nerve cell lines and primary cultured neurons in a dose-dependent manner, which can increase intracellular calcium ions and block
  • the uptake of cystine and the induction of intracellular reduced glutathione (GSH) result in increased production of oxygen free radicals, leading to neuronal cell death. Therefore, the 3 ⁇ 40 2 , glutamate-induced neuronal injury model can be used as a neuroprotective drug screening model.
  • glutamate-induced PC12 cell damage inhibiting cerebral vascular endothelial cell expression or secreting ICAM-1 and/or inhibiting T, ⁇ lymphocyte transformation.
  • lymphocyte transformation experiments are widely used to measure the immune function of individual cells, and can also be used for screening immunomodulators.
  • Enshishi has a certain inhibitory effect on lymphocyte transformation.
  • it is further suitable for inhibiting 1 ⁇ lymphocyte transformation.
  • endothelium composed of endothelial cells constitutes the inner membrane of blood vessels and plays an important role in maintaining vascular homeostasis. Since endothelial cells are in direct contact with blood, their function is easily affected by substances in the blood. Under hypoxic, chronic and acute inflammation, ischemic injury and other pathological conditions can cause endothelial cell activation, and then express some adhesion molecules: ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion) Attached to molecule-1), E-selectin and P-selectin.
  • ICAM-1 intercellular adhesion molecule-1
  • VCAM-1 vascular cell adhesion Attached to molecule-1
  • E-selectin and P-selectin.
  • Adhesion molecules play an important role in vascular endothelium and vascular disease, and ICAM-1 plays a key role in the tight adhesion of leukocytes to the endothelium.
  • it is further suitable for inhibiting the expression or secretion of ICAM-1 by brain vascular endothelial cells.
  • Rats were randomly divided into six groups: sham group, vehicle control, and re-administered group (10 u/kg, 20 u/kg, 40 u/kg), edaravone Group (3 mg/kg). The drug was administered 5 times after 2 hours (2h, 6h, 20h, 24h, 47h). Animals were sacrificed 48 hours after surgery and all indicators were tested.
  • the rat middle cerebral artery occlusion line blocking method was used to prepare the cerebral ischemia model by the insertion method.
  • the rats were fixed in the supine position, the skin was cut in the middle of the neck, and the layers were bluntly separated to expose the left common carotid artery (CCA).
  • CCA common carotid artery
  • the external carotid artery (ECA) after the bifurcation, carefully separate to avoid damage to the vagus nerve and trachea, set the line for use.
  • the ipsilateral external carotid artery was isolated and ligated at approximately 0.8 cm from ECA ⁇ .
  • An arterial clip was placed at the proximal end of the CCA.
  • a "V"-shaped incision with a diameter of about 2 mm was made between the ECA ligation and the bifurcation.
  • the nylon thread was gently inserted into the CCA from the incision, and the artery was clamped through the neck.
  • the external carotid artery bifurcation enters the internal carotid artery, and the nylon thread is slowly pushed into the ICAT intracranial direction.
  • the insertion depth is about 18.5 ⁇ 0.5 mm to the micro-inductance, so that the nylon thread end passes through the MCA start point and reaches a finer point.
  • the anterior cerebral artery at this time, the blood flow obstruction of the left middle cerebral artery is achieved, the ICA is sutured to fix the nylon thread and the bleeding is prevented, and the suture is sutured by the house, and the nylon thread stump remains l cm longer than the skin.
  • the sham operation group only performed preoperative anesthesia and blood vessel separation, and did not ligature and introduce a thread plug.
  • the room temperature was maintained at 24-25 °C during the operation.
  • the limbs are symmetrical downwards.
  • the rats were killed by decapitation.
  • the brain tissue was quickly placed in a refrigerator at -20 °C. After lOmin, the room temperature was set. After the olfactory bulb, cerebellum and low brain stem were removed, 2 mm of the coronal shape was cut at intervals of 2 mm. Cut into 5 consecutive brain coronal sections.
  • the first knife is at the midpoint of the front pole of the brain and the line of intersection; the second knife is at the optic chiasm; the third knife is at the funnel handle; the fourth knife is between the funnel handle and the thick leaf nucleus, and then the brain is quickly.
  • the tablets were immersed in 5 ml TTC solution (containing 4% TTC solution 1.5 ml + 3.4 ml distilled water + 0.1 ml lmol/L K 2 HP0 4 solution), and sealed at 37 ° C for 30 min in a dark water bath. After every 7-8 minutes, the normal brain tissue is rose red, and the infarct tissue is white with clear boundaries.
  • Each group of brain slices was arranged neatly, photographed and saved, and processed by image analysis system software and counted.
  • the infarct area of each brain slice was calculated, multiplied by the thickness of each brain slice by 2 mm, and the infarct area of each brain slice of each animal was multiplied by The thickness is added, which is the volume of cerebral infarction. Infarct volume is expressed as a percentage of the cerebral hemisphere to eliminate the effects of cerebral edema.
  • volume of cerebral infarction (volume of the contralateral hemisphere of the operation - volume of the uninfarcted part of the surgical hemisphere) I volume of the contralateral hemisphere of the operation x l00 %
  • Rats were decapitated and the left and right cerebral hemispheres were separated. After removing the frontal lip 1 mm and the occipital pole 1 mm, the brain tissue was placed in an ice-cold homogenization buffer at a volume ratio of 1:10 (Tris-HCl 50 mmol/L). , NaCl 150 mmol/L, CaCl 5 mmol L, PMSF 0.1 mmol/L, pH 7.4) Medium, minced, 4 °C homogenate, Bradford method for determination of protein concentration [1Q] .
  • the lactate-based shield is catalyzed by lactate dehydrogenase to form pyruvate in the presence of oxidized coenzyme I, and the resulting pyruvic acid reacts with 2,4-dinitrophenylhydrazine to form brown pyruvate dinitrate.
  • Benzoquinone thereby estimating the activity of lactate dehydrogenase by colorimetric determination of pyruvate content.
  • the lactate dehydrogenase activity was measured according to the kit instructions. Add 10 ⁇ of homogenase S ⁇ , 37 to S g'I/ 1 to the buffer base. C incubation for 15 min; then add 0.2 gL -1 of 2, 4-dinitrophenylhydrazine 50 ⁇ , incubate for 15 min at 37 °C; add 0.4 mol/L NaOH 150 ⁇ 1, mix and 440 nm colorimetric, blank Adjust the zero point, read the absorbance value, and make a standard curve with sodium pyruvate standard solution.
  • Superoxide anion radicals are generated by the xanthine and xanthine oxidase reaction system, and the latter oxidizes hydroxylamine to form nitrite, which is purple-red under the action of a color developer.
  • the sample to be tested contains SOD, it has a specific inhibitory effect on superoxide anion radicals, which reduces the formation of diaphorite.
  • the homogenization method is the same as above, and the homogenate is thoroughly mixed, and the SOD activity is detected according to the kit instructions.
  • the value of each milligram of tissue protein in a 1 ml reaction solution with a SOD inhibition rate of 50% corresponds to a nitrite unit.
  • the lactic acid content in the brain tissue of rats increased to 0.98 ⁇ 0.09 mmol/g prot, which was significantly different from the sham operation group (PO.01).
  • the lactic acid content of Enshishi 40u kg was significantly reduced to 0.70 ⁇ 0.07 mmol/g prot was statistically significant compared with the model group ( ⁇ .05); the lactic acid content of the edaravone group was significantly reduced to 0.64 ⁇ 0.08 mmol/g prot, Statistically significant ( ⁇ 0.05) compared to the model group.
  • the results are shown in Table 2.
  • PC 12 cells were purchased from the Institute of Basic Science of Chinese Academy of Medical Sciences; thiazolyl blue (MTT), trypsin, polylysine, standard fetal bovine serum (FBS), 1640 medium, and LDH kit were sourced as before. Other commonly used reagents are domestic analytical pure reagents.
  • PC12 cells were provided by the Institute of Chinese Academy of Medical Sciences in complete 1640 medium (containing 10% horse serum, 5% fetal bovine serum, penicillin 100 U/ml, streptomycin 100 g/ml) at 37 ° C, 5% C02 culture incubator, change every 2-3 days [6].
  • Normal control group PC12 cells were cultured normally in serum-containing DMEM medium
  • H202 model group PC12 cells were plated into a single layer, the original medium was aspirated, and serum-free medium containing H202 at a final concentration of 200 ⁇ L was added to 37. . C, 5%C02 constant temperature incubator for 24 hours
  • Sample treatment group After PC12 cells were layered into a single layer, the original medium was aspirated, and the sample was pretreated for 1 h, then added to the final concentration of 200 mol/L of H202 serum-free. Cultivate for 24 hours.
  • PC12 cells were purchased from the Institute of Basic Science of Chinese Academy of Medical Sciences; thiazole blue (MTT), trypsin, and polylysine were purchased from Sigma. Standard fetal bovine serum (FBS) and 1640 medium were purchased from Gibco. Other commonly used reagents are domestic analytical pure reagents.
  • Fluostar microplate reader BMG, Germany
  • cell culture chamber Sanyo, Japan
  • refrigerated centrifuge DL-4000B Shanghai Anting Scientific Instrument Factory
  • Mirror 1X71 OLEDUS
  • PC12 was cultured in complete 1640 medium (containing 10% horse serum, 5% fetal bovine serum, penicillin 100 U/ml, streptomycin 100 g/ml) in a 37 ° C, 5% CO 2 incubator, every 2 - Change the liquid once in 3 days. 2.
  • serum-free 1640 medium was exchanged and assayed 24 hours later.
  • the administration group was changed to a serum-free 1640 medium containing the drug and incubated with the cells for 24 hours, and then measured.
  • Example 4 The effect of LPS on the expression or secretion of ICAM-1 induced by LPS in cerebral vascular endothelial cells
  • the ICAM-1 ELISA test kit was purchased from Boster. Endothelial growth factor Provided by Roche. Fetal bovine serum Gibco products. Thiazole blue (MTT), trypsin, polylysine, and lipopolysaccharide (LPS) were purchased from Sigma. Standard fetal bovine serum (FBS), 1640 medium was purchased from Gibco.
  • the newborn Wistar rats were taken for 1 ⁇ 5 days, and the cortical tissue was taken from the decapitation.
  • the microvascular segments on the filter surface were collected by slurry filtration.
  • the collagenase was diluted by 0.1%>, and the precipitate was added to contain 15% fetal bovine serum.
  • Vascular endothelial cells were identified by VfflF: Ag immunocytochemistry with a purity of over 95%. In this experiment, the third generation of microvascular endothelial cells were cultured.
  • Brain vascular endothelial cells were distributed in 96-well plates for culture. After cultured until the endothelial cells were spread into a monolayer, LPS (10 ug/ml) was added for stimulation for 24 hours, and then ICAM-1 in the cell supernatant was measured. The ICAM-1 content of each sample was read from a standard curve.
  • 1640 medium (containing 10% calf serum, double antibody, glutamine), double distilled water, double saline, ConA and PMA were purchased from Sigma.
  • mice were purchased from the Institute of Zoology of the Chinese Academy of Medical Sciences.
  • the spleen of Balb/C mice was routinely aseptically taken, and the grinding rod was gently ground, passed through a 200-mesh steel mesh, washed once at 1640, and centrifuged at 2000 rpm x 5 min.
  • Lymphocyte transformation % (sample well OD value - negative control well OD value) / (negative control well OD value - blank control well OD value) xlOO Experimental results

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Description

牛痘疫苗致炎兔皮提取物在制备急性脑血管疾病治疗药物中的用途 技术领域
本发明涉及急性脑血管疾病的治疗， 具体的说， 涉及牛疽疫苗致 炎兔皮提取物在制备急性脑血管疾病治疗药物中的用途。 背景技术
中风， 是一种急性脑血管疾病， 是导致全球人口死亡的第三大原 因和致残率最高的疾病。 根据最新的流行病学资料， 在我国， 脑血管 病的发病率为 120/10万 -180/10万， 在人口死亡原因中位居第二。 每年 新发脑血管患者 120-150万人，死亡 80-100万人，存活者中约 75%致残，' 5年内复发率高达 41%,严重影响着老年人的生活质量，给患者家庭和 社会带来沉重的负担， 且近年来有年轻化的趋势。
脑血管病主要分为出血性和缺血性两大类型， 其中， 后者占 60-
70%, 是脑血管病最常见的类型。 研究缺血性脑血管疾病的病理生理 机制及寻找发挥神经保护作用的药物具有重要意义。
自二十世纪八十年代以来， 对脑缺血病理生理机制的研究一直是 神经科学领域的热点之一， 至今提出了脑缺血的能量代谢、 酸中毒、 过氧化损伤、 兴奋性氨基酸毒性损伤、 钙超载等学说， 其中后两者在 缺血性神经元损伤和死亡中起重要作用。 基于缺血性脑血管病的病理 生理基 ， 目前临床上用于治疗脑缺血的药物主要有钙离子拮抗剂 (尼莫地平） 、 氧自由基清除剂 （VitE、 SOD ) 、 神经营养因子（神 经生长因子、 神经营养因子） 、 兴奋性義基酸拮抗剂、 抗氧化剂和改 善迟发性神经元损伤的药物， 这些药物通过不同的作用机制发挥作 用， 但治疗作用不确定或特异性不强或伴有严重不良反应， 尚不能满 足临床需要。 国内市场上有很多药物如脑复康、 氟桂嗪、 卡兰、 银杏 提取物等用于改善脑循环、 脑代谢和脑功能， 虽都有一定特点， 但用 于治疗脑血管疾病的效果不能肯定。 研究、 开发新型用于缺血性脑血 管疾病的药物， 是药学及药理学领域的重要任务。
本文所述的"牛痘疫苗致炎兔皮提取物，，是指从牛痘疫苗致炎的家 兔皮肤中提取的活性物质， 如中国发明专利 ZL98103220.6中所述， 该 专利的全部内容通过引用结合到本文中。 这种牛痘疫苗致炎兔皮提取 物可市售获得， 商品名为恩再适， 由威世药业（如皋）有限公司生产。 恩再适的药理作用包括（ 1 )镇痛作用， 对反复寒冷应激复合诱导的 痛觉过敏具有明显的镇痛作用， 这是通过激活中枢神经系统下行抑制 系统来实现的； （2 )对冷感、 知觉异常的作用， 体内、 体外试验显 示本品具有改变丘脑下部神经元散发活动的作用， 提示本品对感觉神 经元散发活动方式异常具有修补调节作用， 此种异常被认为是导致神 经痛和知觉异常的原因； （ 3 )？文善末梢血液循环的作用； （ 4 )对植 物神经的调节作用： 体内、 体外试验提示本品能通过调节中枢植物神 经活动， 改善植物神经失调症状; ( 5 )抗变态反应作用： 动物试验 提示本品有抗 I型变态反应的作用， 对副交感神经兴奋导致的呼吸道 分泌亢进具有抑制作用， 对鼻粘膜 M受体密度上调也具有抑制作用； ( 6 )镇静作用： 动物试验提示， 本品对各种外来刺激所引起的情感 性兴奋状态具有镇静作用。
此外 , 牛痘疫苗致炎兔皮提取物还在以下申请中进行了研究讨 论： 1999年 11月 12日申请的中国专利申请 99123485.5 , 1996年 12月 19 日申请的中国专利申请 96123286.2和 1998年 1月 07日申请的中国专利 申请 98103914.6 。
然而， 恩再适对脑缺血损伤的保护作用尚未见报道。 本发明对此 进行了研究， 并发现恩在适对动物模型上的实验性脑缺血具有有利的 影响。 发明内容
本发明的一个方面提供了治疗哺乳动物中的急性脑血管疾病的 方法， 所述方法包括给予有需要的患者牛痘疫苗致炎兔皮提取物。 本发明的另一个方面提供了牛痘疫苗致炎兔皮提取物在制备药 物中的用途， 所述药物用于治疗哺乳动物中的急性脑血管疾病。
在一个实施方案中， 本文所述的急性缺血性脑血管疾病为急性缺 血性脑血管疾病。
脑血管疾病是由脑局部血供异常而引起的神经功能损伤。 在世界 绝大多数国家， 脑血管疾病是引起死亡的前 3位原因， 并可导致成人 脑损伤，是危害中老年人健康的主要原因，是许多国家中老年人致死、 致残的主要原因。 缺血性脑血管疾病多由脑血管闭塞引起， 通常为栓 塞或血栓形成， 其病理生理改变极为复杂。 在一个实施方案中， 本文 所述的急性缺血性脑血管疾病包括但不限于脑栓塞、 短暂性脑缺血发 作、 脑血栓形成、 脑动脉硬化、 脑动脉炎、 脑动脉盗血综合征、 颅内 静脉窦和静脉血栓。
缺血性脑血管疾病是由于某动脉供血区的血流因栓塞而短暂或 永久性的减少所致， 其病理过程涉及复杂的时间和空间级联反应。 近 年来， 对于脑缺血的病理生理机制也有了较深入的认识。 但该病发生 的直接原因在于， 脑内的各个动脉均有其基本的供血范围， 闭塞后引 起相应区域的脑组织软化， 产生相应的临床综合征， 以大脑中动脉闭 塞引起的对侧肢体偏瘫等神经症状学损害最为常见。 而且， 在初次脑 卒中的临床病例中， 大脑中动脉阻塞也占很大比例， 所以动物大脑中 动脉阻塞（ middle cerebral artery occlusion, MCAO )模型模拟的病理过 程与临床脑卒中有着很大的相似性。
Bederson评分和倾斜板实验结果显示， 大鼠脑组织缺血后， 出现 对侧肢体无力、 瘫痪等神经症状学损伤体征。 本发明的发明人发现， 恩再适可明显改善动物的神经症状。 因此， 在一个实施方案中， 恩再 适通过改善神经功能治疗脑血管疾病。
脑梗死灶的范围与缺血程度有关 , 单侧大脑中动脉栓塞手术 24h 后， 对侧脑组织 TTC染色呈白色， 还可见液化灶， 统计学结果显示， 假手术组脑梗塞体积与溶剂组相比， 差异具有显著性， 恩再适中剂量 组脑梗塞体积比损伤組明显降低。 因此， 在一个实施方案中， 恩再适 通过降低脑梗死范围来治疗脑血管疾病。
脑是代谢最活跃的器官， 而自身能量和氧储备最少。 静息状态下 脑組织耗氧量占全身总氧耗的 20%。 神经元是构成大脑皮层或整个脑 的氧耗量的主要部分，对缺血、缺氧损伤极为敏感。在无新鲜氧源时， 组织只能消耗其高能磷酸化合物储备， 并借助于将所贮存的葡萄糖和 糖原代谢为 MDA, 从而获得能量。 脑組织缺血缺氧导致能量耗竭， 引 起能量泵功能衰竭、 神经细胞内钙离子超载、 毒性氧自由基增加、 细 胞酸中毒等一系列链锁反应， 细胞膜的结构和完整性遭到破坏， 从而 引起通透性增加， 细胞毒性水肿程度增加， 细胞内的一些酶大量释放 入血。 结果显示， 脑组织缺血后乳酸含量和 MDA显著升高， 恩再适干 预后脑组织乳酸含量明显降低。 因此， 在一个实施方案中， 恩再适通 过降低脑组织乳酸含量来治疗脑血管疾病。
SOD为重要的抗氧化酶， 可有效地抑制自由基反应， SOD活性高 代表抗氧化能力强。 脑缺血损伤后， 大鼠脑组织 SOD活性显著降低， 清除自由基能力降低。 结果显示， 恩再适干预后可以提高 SOD活性， 说明恩再适可能通过提高脑组织的抗氧化能力发挥神经保护作用。 因 此， 在一个实施方案中， 恩再适提高 SOD活性来治疗脑血管疾病。
在另一个方面， 本发明的发明人发现， 恩再适可发挥神经细胞损 伤的保护作用。
¾02是一种重要的活性氧成分， 参与了脑缺血、 外伤、 脑老化、 阿尔采末氏病等神经系统疾病的发病过程。 它能使膜脂质过氧化， 降 低细胞膜的流动性，改变胞内蛋白质成分和活性，使染色质浓缩， DNA 断裂， 最终导致细胞死亡。 因此， 在一个实施方案中， 恩再适用于改 善 ¾02诱导的 PC12 细胞损伤。
在多种慢性或急性神经病变过程中都伴随有神经元的死亡， 兴奋 性氨基酸如谷氨酸在此过程中起重要作用。 谷氨酸可剂量依赖性地损 伤神经细胞系和原代培养的神经细胞， 可使胞内钙离子升高， 并可阻 断胱氨酸的摄取和诱发胞内还原型谷胱甘肽（GSH )的缺失， 引起氧 自由基产生增多， 导致神经细胞死亡。 因此 ¾02、 谷氨酸致神经细胞 损伤模型可作为神经保护药物筛选模型。 因此， 在一个实施方案中， 恩再适用于改善谷氨酸诱导的 PC12 细胞损伤、 抑制脑血管内皮细胞 表达或分泌 ICAM-1和 /或抑制 T、 Β淋巴细胞转化作用。
Τ淋巴细胞在体外培养时， 受到特异性抗原或非特异性有丝分裂 原刺激后， 可出现细胞体积增大， 代谢旺盛， 蛋白与核酸合成增加， 并能进行分裂淋巴母细胞。 根据淋巴细胞转化率的高低， 可反映个体 的细胞免疫功能， 因此， 淋巴细胞转化实验被广泛应用于测定个体细 胞免疫功能的指标之一，也可用于免疫调节剂的筛选。实脸研究发现， 恩再适对淋巴细胞转化有一定的抑制作用。因此，在一个实施方案中， 恩再适用于抑制1\ Β淋巴细胞转化作用。
由内皮细胞组成的内皮构成血管的内膜， 在维持血管稳态方面起 着重要作用。 由于内皮细胞直接与血液接触， 其功能很容易受到血液 中物质的影响。 在低氧、 慢性和急性炎症、 缺血损伤等病理条件下可 引起内皮细胞的激活，继而表达一些粘附分子： ICAM-1 (细胞间粘附 分子 -1 ) 、 VCAM-1 (血管细胞粘附分子 -1 ) 、 E-选择素和 P-选择素。 粘附分子在血管内皮及血管病变过程中具有重要作用， 其中 ICAM-1 在白细胞对内皮的紧密粘附中起关键作用。因此，在一个实施方案中， 恩再适用于抑制脑血管内皮细胞表达或分泌 ICAM-1。 附图筒述
图 1. 恩再适对永久性 MCAO后 48小时的脑梗死体积的影响。 具体实施方式
除非另有说明， 否则本文使用的所有科学术语都和本领域一般技 术人员通常理解的含义一致。 下文描述了示例方法和材料， 可以使用 其等同物。 本文提及的所有出版物和其它参考文献都通过引用整体结 合到本文中。
提供以下的实施例是为了进一步阐述本发明。 以下实施例无意以 任何理由限制本发明的范围。 实施例 1 恩再适对大鼠急性脑缺血 ( MCAO )的保护作用 实验材料
1. 药品与试剂
恩再适注射液， 规格 lO u/ml, 25ml/瓶 (由威世药业（如皋）有限 公司提供） 。 氯代三苯基四氮唾 ( 2,3,5-Triphenyltetrazolium chloride, TTC )为 Sigma公司 （美国）产品； MDA、 SOD、 乳酸脱氢酶试剂盒 系南京建成生物工程研究所产品。
2. 实验动物及分组
雄性 Wistar大鼠， 体重 280-300g, 由北京维通利华实验动物中心 提供（许可证 SCXK京 2007-0004 )。 手术前、 后常规飼养， 室温保持 23-25 °C , 自由进食、 进水。
大鼠随机分为六组： 假手术组 (sham group)、 损伤模型组 (vehicle control)、 恩再适给药组（10u/kg, 20u/kg, 40u/kg)、 依达拉奉给药组 (3mg/kg)。 手术后 2h后给药 5次（ 2h, 6h, 20h, 24h, 47h )。 术后 48小 时处死动物， 进行各项指标检测。
实验方法
1. 大鼠大脑中动脉栓塞模型制备
参照 Zea 1^01¾&等[5-6]建立的大鼠大脑中动脉内栓线阻断方法 , 采 用插线法制备脑缺血模型。
1.1 尼龙线栓子的制备
在起点及距起点 18.5 mm处作标记， 75% (体积比）酒精清洁后 置 1: 2500单位肝素化生理盐水中备用。
1.2术前麻醉
10 %7j合氯醛溶液 400 mg kg , 腹腔注射。 1.3 手术过程
( 1 )手术视野的准备：
大鼠仰卧位固定， 颈部正中切开皮肤， 钝性分离各层组织， 暴露 左侧颈总动脉 (CCA)。
( 2 )颈部动脉的分离：
分离至颈内动 ( internal carotid artery, ICA )、领夕卜动月永 ( external carotid artery, ECA )分叉后一段，仔细分离避免损伤迷走神经和气管， 置线备用。 分离同侧颈外动脉， 在 ECA^出约 0.8cm处结扎。
( 3 )大脑中动脉的结扎：
于 CCA近心端夹一动脉夹， 在 ECA结扎处与分叉处之间做一直径 约 2mm的" V"形切口， 将尼龙线自切口处轻轻插入 CCA, 开动脉夹， 经颈内、 颈外动脉分叉部进入颈内动脉， 将尼龙线緩慢地向 ICAT颅 内的方向推进， 插入深度约 18.5±0.5mm至微感阻力， 使尼龙线头端通 过 MCA起始处， 到达较细的大脑前动脉， 此时即实现左侧大脑中动脉 的血流阻塞， 缝合 ICA以固定尼龙线和防止出血， 逐屋缝合， 尼龙线 残端留 l cm长于皮外。 假手术组只进行术前麻醉和血管分离术， 不结 扎及导入线栓。 手术过程中室温保持在 24-25 °C。
2.神经行为学检查
2.1 Bederson's评分
动物于处死前进行神经行为学观察， 参照 Bederson[7]和 Belvyev[8] 等的方法， 提鼠尾离开地面约 1尺， 观察两前肢状况； 将大鼠置于水 平地面， 推动其双肩， 观察两侧抵抗力有无差异； 大鼠置于地面， 观 察其行走情况。 对每只动物进行评分。 据其症状严重程度分 3级， 共 10分。根据标准行为学评分，分数越高，说明动物的行为障碍越严重。 具体评分方法如下： 法 症 状 分级 分数
1 + 2 + 3 1 , 2, 3项测试阴性 0级 0
1.提起大鼠尾部， 大鼠离地
损伤后对侧前肢内
一尺左右， 正常大鼠前 1级 1 旋内收
肢对称向下伸。
2. 置动物于一光滑平面， 用 损伤后对侧肌力降
2级 3 手分別挤压体侧。 低， 易向对侧倾倒。
3.置动物于地面观察其行走 动物向一侧旋转 3级 6
3.脑梗死体积的测定
评分后大鼠断头处死， 迅速将取出的脑组织置于 -20 °C冰箱， · l Omin后置室温环境， 切除嗅球、 小脑和低位脑干后按图谱所示间隔 2mm冠状切四刀， 切成 5个大脑连续冠状粗切片。 第一刀在脑前极与 视交叉连线中点处； 第二刀在视交叉处； 第三刀在漏斗柄部位； 第四 刀在漏斗柄与厚叶尾核之间， 然后迅速将脑片浸入 5ml TTC溶液 (含 4%的 TTC溶液 1.5 ml+3.4 ml蒸镏水 +0.1 ml lmol/L K2HP04溶液 ) 中， 37°C避光水浴 30min。每隔 7-8min翻动一次， 经染色后， 正常脑组织呈 玫瑰红色， 而梗塞组织呈白色， 且界限分明。 将每组脑片排列整齐， 拍照保存， 应用图像分析系统软件处理并作统计， 计算每张脑片的梗 塞面积， 乘以每片脑片的厚度 2mm， 每只动物所有脑片梗塞面积乘以 厚度相加， 即为脑梗塞体积。 梗塞体积以所占大脑半球的百分率来表 示， 以消除脑水肿的影响。
脑梗塞体积（％ ) = (手术对侧半球的体积一手术侧半球未梗塞部分 的体积） I手术对侧半球的体积 x l00 %
4.脑组织中生化指标测定
4.1 脑组织匀浆制备
大鼠断头取脑， 将左、 右大脑半球分开， 去除额极 lmm和枕极 lmm后， 将脑組织按 1 :10体积比置于冰冷匀浆緩冲液（ Tris-HCl 50 mmol/L, NaCl 150 mmol/L, CaCl 5 mmol L, PMSF 0.1 mmol/L, pH 7.4 ) 中， 剪碎， 4 °C匀浆， Bradford方法测定蛋白浓度 [1Q]。
4.2 大鼠脑组织中乳酸脱氢酶的测定
( 1 ) 实验原理
在 PH值为 10时， 乳酸基盾在氧化型辅酶 I存在下经乳酸脱氢酶 催化反应生成丙酮酸， 生成的丙酮酸与 2, 4-二硝基苯肼反应生成棕 色的丙酮酸二硝基苯腙， 从而通过比色测定丙酮酸含量推算乳酸脱氢 酶的活力。
( 2 )测定方法
按试剂盒说明书检测乳酸脱氢酶活性。 在緩冲基质液中加入匀浆 10 μΐ, S g'I/1的辅酶 Ι ΙΟ μΙ, 37。C孵育 15 min; 然后加入 0.2 g-L-1 的 2, 4-二硝基苯肼 50 μΐ, 37 °C孵育 15 min;加入 0.4 mol/L NaOH 150 μ1， 混匀后 440 nm比色， 以空白调零点， 读取吸光度值， 以丙酮酸钠 标准液制作标准曲线。
4.3 大鼠脑组织超氧化物歧化酶（ superoxide dismutase, SOD )活性的 测定
( 1 ) 实验原理
通过黄嘌呤及黄嘌呤氧化酶反应系统产生超氧阴离子自由基， 后 者氧化羟胺形成亚硝酸盐， 在显色剂的作用下呈现紫红色。 当被测样 品中含有 SOD时， 对超氧阴离子自由基有专一性的抑制作用， 使形 成的亚肖酸盐减少。
( 2 )检测方法
匀浆方法同上， 匀浆液充分混匀后，按试剂盒说明书进行 SOD活 性检测。 每毫克组织蛋白在 1ml反应液中 SOD抑制率达 50 %时所对 应的值为一个亚硝酸盐单位。
5. 统计学处理
结果以 means士 SD表示， 各组间数据比较采用 t-检验。
实验结果
1. 恩再适对急性脑缺血大鼠神经症状学的影响 脑缺血后， 麻醉大鼠已清醒， 出现不同程度的局灶性神经功能障 碍, 表现为左下肢无力、 伸直行走时向左侧转圈， 严重时向左侧倾倒 甚至不能行走， 乃至出现意识障碍； 提尾时表现为左侧前肢屈曲、 内 收， 后肢伸直并向右侧旋转。 脑缺血后模型组动物出现明显神经损伤 症状， 神经功能评分显著升高 （PO.01 )； 恩再适 40 u/kg明显改善 神经功能症状（PO.05 ) , 10、 20 u kg组无显著改善作用。 依达拉奉 组与模型组相比无显著性差异。 结果见表 1。 表 1. 恩再适对 MCAO大鼠神经症状评分的影响
组别 剂量 动物数 (只） 分数 假手术组 ― 8 0 模型组 ― 8 5.3±2.8 ## 恩再适低剂量组 10 u/kg 8 3.4士 2.5 恩再适中剂量组 20 u/kg 8 3.4±2.8 恩再适高剂量组 40 u/kg 7 2.0±2.6 * 依达拉奉组 3 mg/kg 6 4.3±1.6 数值表示为平均值 ± S.E.M.， 每組 6~8只动物。 #与 手术组相比 P<0.05。 *与模 型组相比 P<0.05。
2. 恩再适对急性脑缺血大鼠脑梗死体积的影响
经染色后正常脑组织呈整个玫瑰红色， 而梗塞組织呈白色， 且界 限分明。除假手术组外，其他各组大鼠大脑皮质均出现明显的梗死灶， 严重的累及到紋状体。 依达拉奉组、 恩再适中剂量组能显著降低脑梗 死范围。 结果见图 1。
3. 恩再适对大鼠急性缺血性脑组织乳酸含量的影响
缺血损伤后， 大鼠脑組织乳酸含量升高至 0.98±0.09 mmol/g prot, 与假手术组相比具有显著性差异 (PO.01); 恩再适 40u kg乳酸含量显 著降低至 0.70±0.07 mmol/g prot , 与模型組相比具有统计学意义 ( ΡΟ.05 )； 依达拉奉組乳酸含量显著降低至 0.64±0.08 mmol/g prot, 与模型组相比具有统计学意义（ <0.05 ) 。 结果见表 2。
表 2.恩再适对永久性 MCAO后 48小时的大鼠脑組织中乳酸含量的影 响
组别 剂量 动物数 (只） 酸含量 (mmol/g prot) 假手术组 -— 4 0.37±0.01 模型组 ― 6 0.98±0.09 # 恩再适低剂量组 10 u/kg 5 0.74±0.14 恩再适中剂量组 20 u/kg 5 0.88士 0.11 恩再适高剂量組 40 u/kg 5 0.70±0.07* 依达拉奉组 3 mg/kg 6 0.64±0.08* 数值表示为平均值 ± S.E.M.， 每组 4~6只动物。 ##与假手术组相比 P<0.01。 *与 模型组相比 P<0.05。
4. 恩再适对大鼠脑组织超氧化物歧化酶活性的影响
缺血损伤后， 大鼠脑组织 SOD含量降低至 165.84±13.14 nmol/g prot,与假手术组相比具有显著性差异 (P<0.01);恩再适 20u/kg、 40u/kg 给药组与模型组相比显著升高 （PO.05 ) ； 与模型组相比， 依达拉奉 组 SOD含量显著升高 （PO.01 ) 。 结果见表 3。
表 3. 恩再适对永久性 MCAO后 48小时的大鼠脑组织中 SOD活性和 MDA含量的影响
组别 剂量 动物数 (只） SOD活性
(U/mg pro) 假手术组 ― 4 354.17±64.26 模型組 ― 6 165.84±13.14 # 恩再适低剂量组 10 u/kg 5 198.96±12.89 恩再适中剂量组 20 u/kg 5 263.46±34.86* 恩再适高剂量组 40 u/kg 5 268.09±32.42* 依达拉奉组 3 mg/kg 6 234.66±15.85 数值表示为平均值 ± S.E.M.， 每組 4~6只动物。 #与假手术组相比 PO.05。 *与模 型组相比 P<0.05。 实施例 2 恩再适对 H202诱导 PC12细胞损伤的影响
实验材料
1. 药品与试剂
PC 12细胞购自中国医学科学院基础所； 噻唑蓝（ MTT ) 、 胰蛋 白酶、 多聚赖氨酸、 标准胎牛血清（FBS ) 、 1640培养基、 LDH试剂 盒来源同前。 其它常用试剂均为国产分析纯试剂。
2.仪器
超净台； Fluostar microplate reader (BMG公司， 德国）； 细胞培养 箱 （三洋公司， 日本）； 冷冻离心机 DL-4000B (上海安亭科学仪器 厂）； 显鼓镜 1X71 ( OLYMPUS ) 。
实验方法
1. PC12细胞培养
PC12细胞由中国医学科学院基础所提供，以完全 1640培养基（含 10%马血清， 5%胎牛血清， 青霉素 100 U/ml, 链霉素 lOO g/ml ) , 于 37°C、 5%C02恒温培养箱中培养， 每 2-3天换液一次 [6]。
2. 细胞处理
正常对照组： PC12细胞在含血清 DMEM培养基中正常培养； H202模型組： PC12细胞铺成单层后， 吸除原培养基， 加入含有终浓 度为 200 μηιοΙ L的 H202的无血清培养基于 37。C、 5%C02恒温培养 箱中培养 24小时； 样品处理组： PC12细胞铺成单层后， 吸除原培养 基， 加入样品预处理 lh后， 加入终浓度为 200 mol/L的 H202无血 清培养 24小时。
3. 细胞活力测定
每孔加入 100 μΐ MTT液， 终浓度为 0.5 mg/ml, 于 37Ό 5% C02 继续培养 4 h, 弃上清， 每孔加入 DMSO ΙΟΟ μΙ, 振荡后于 540 nm处 测定吸光度 OD值。 细胞存活率=八测定孔 /A正常对照孔 100%
实验结果 过氧化氢损伤后， PC12细胞存活率降低至 71.94±3.54 %,与正常 对照組相比具有显著性差异 (P<0.01); 恩再适 0.25、 0.5、 l u/ml给药 组与模型組相比显著升高 （PO.05, ) 。 恩再适对过氧化氢损伤 PC12细胞的保护作用
组别 剂量 细胞存活率（％) 正常对照组 -- 100±4.30
损伤模型组 200 uM H202 71.94±3.54 m
恩再适 1 U/ral 80.17±0.64 *
0.5U/ml 91.53±2.24 ** 恩再适
0.25U/ml 91.57±0.35
恩再适
数值表示为平均值 ± SD, 每组 n=3。 #P<0.05, P<0.01 , 相比正常组。 *P<0.05 **P<0.01 , 相比模型組。 实施例 3 恩再适对谷氨酸损伤神经细胞的保护作用 实验材料
1. 药品与试剂
PC12细胞购自中国医学科学院基础所； 噻唑蓝（MTT ) 、 胰蛋 白酶、 多聚赖氨酸购自 Sigma公司。 标准胎牛血清（FBS ) 、 1640培 养基购自 Gibco公司。 其它常用试剂均为国产分析纯试剂。
2. 仪器
超净台； Fluostar microplate reader (BMG公司， 德国）； 细胞培养 箱 （三洋公司， 日本）； 冷冻离心机 DL-4000B (上海安亭科学仪器 厂）； 显 镜 1X71 ( OLYMPUS ) 。
实验方法
1. PC12细胞培养
PC12以完全 1640培养基 (含 10%马血清， 5%胎牛血清， 青霉 素 100 U/ml, 链霉素 100 g/ml ) , 于 37°C、 5%C02恒温培养箱中培 养， 每 2-3天换液一次。 2. 细胞处理
PC12细胞铺成单层后， 吸除原培养基， 加入无 Mg2+-Earle，s液 ( mmol L: NaCl 142.6, KC1 5.4, CaC12 1.8, NaH2P04 1.0. HEPERS 2.38, Glucose 5.6, pH7.4,含 0.5uM L- gly )， 内含 ImM的 L-谷氨酸，
15分钟后， 换无血清的 1640培养基，24 h后进行测定。给药组换为含 有药物的无血清的 1640培养基与细胞共孵育 24h后进行测定。
3 细色活力测定
每孔加入 100 ul MTT液， 终浓度为 0.5 mg/ml, 于 37 °C 5%C02 继续培养 4 h, 弃上清， 每孔加入 DMSO 100 μΐ , 振荡后于 540 nm处 测定吸光度 OD值。 细胞存活率 A 测定: ft/A正常对照孔 * 100%
实验结果
表 5 恩再适对谷氨酸损伤 PC12细胞的保护作用
谷氨酸损伤后， PC12细胞存活率降低至 74.76±4.86 °/。， 与正常对 照组相比具有显著性差异 (PO.01); 与模型组相比， 恩再适 0.25、 0.5、 l u/ml给药组与模型组相比显著升高 （PO.01 ) 。
组别 剂量 细胞存活率 （％) 正常对照组 100±7.15
损伤模型组 200 uM H202 74.76±4.86 ##
恩再适 1 U/ml 90.88±5.67 **
0.5U/ml 93.42±4.13 **
恩再适
0.25U/ml 91.69±3.61
恩再适
数值表示为平均值 ± SD, 每组 n=4。 #P<0.05, ##P<0.01， 相比正常组。 *P<0.05:
**P<0.01 , 相比模型组。 实施例 4恩再适对 LPS诱导脑血管内皮细胞表达或分泌 ICAM-1的 影响
实验材料
1. 药品与试剂
ICAM-1 ELISA检测试剂盒购自博士德公司。 内皮生长因子由 Roche公司提供。 胎牛血清 Gibco公司产品。 噻唑蓝（MTT ) 、 胰蛋 白酶、 多聚赖氨酸和脂多糖（LPS )均购自 Sigma公司。 标准胎牛血 清（ FBS ) 、 1640培养基购自 Gibco公司。
2. 仪器
超净台； Fluostar microplate reader (BMG公司， 德、国)； 细包培养 箱 （三洋公司， 日本）； 冷冻离心机 DL-4000B (上海安亭科学仪器 厂）；显微镜 1X71( OLYMPUS ); Zenyth200st UV-Vis spectrophotometer (Anthos Co. Austria)
实验方法
1. 大鼠脑血管内皮细胞的培养
取 1 ~ 5 d的新生 Wistar大鼠,断头取皮层组织，勾浆过滤收集滤网上 面的微血管段， 0. 1%>的 W胶原酶消化后离心,沉淀物加入含 15%胎牛 血清的 M199培养液, 37。C, 5%C02孵箱培养， 0. 25%胰酶消化细胞传 代。 血管内皮细胞进行 VfflF: Ag免疫细胞化学方法鉴定，纯度达 95%以 上。 本实验均使用培养第 3代的微血管内皮细胞。
2. ELISA法测定内皮细胞 ICAM- 1的分泌
将脑血管内皮细胞分布于 96孔板中培养。 培养至内皮细胞铺成 单层后， 加入 LPS (10ug/ml)刺激 24小时， 然后测定细胞上清中的 ICAM- 1。 由标准曲线读出每个样品的 ICAM- 1含量。
实验结果
月旨多糖（LPS )损伤后， 脑血管内皮细胞表达或分泌的 ICAM-1 与正常对照组相比具有显著性差异 (P<0.01); 与模型组相比， 恩再适 0.25、 0.5 l u/ml给药组与模型组相比显著升高 ( PO.01 ) 。
恩再适对大鼠脑血管内皮细胞表达或分泌 ICAM- 1的影响 组别 剂量 ICAM-1 (pg/ml) 正常对照組 62.34±4.54
损伤模型組 LPS (lOug/ml) 485.09±12.88冊
恩再适 1 U/ml 205.54±3.21
0.5U/ml 167.73±25.50 ** 恩再适
0.25U/ml 179.55±58.43
恩再适
数值表示为平均值 ± SD， 每组 n=4。 #P<0.05, ##P<0.01 , 相比正常组。 *P<0.05 **P<0.01 , 相比模型组。 实施例 5 恩再适对对淋巴细胞转化作用影响 实验材料
1. 药品与试剂
1640培养液（含 10%小牛血清， 双抗、 谷氨酰胺）、 双蒸水、 双 盐水、 ConA和 PMA均购自 Sigma公司。
2. 动物
Balb/c 小鼠购自中国医学科学院动物所。
3.仪器
超净台； Fluostar microplate reader (BMG公司， 德国)； 细月包培养 箱 （三洋公司， 日本） ； 冷冻离心机 DL-4000B (上海安亭科学仪器 厂 ) ； 显微镜 1X71 ( OLYMPUS )； Zenyth200st UV-Vis
spectrophotometer (Anthos Co. Austria)。
实验方法
1. 常规无菌取 Balb/C小鼠的脾脏， 研磨棒轻轻研磨后， 过 200目钢 网， 1640洗一次， 2000rpmx5min离心。
2. 细包沉淀用双蒸水、 双盐水洗涤破坏红细胞， 离心弃上清， 1640 调细胞浓度至 2xl06/ml, ΙΟΟμΙ/孔， 种于 96孔板， 同时加 ConA (终 浓度 5ug/ml )或 PMA (终浓度 5 ug/ml ) , 90μ1/孔。 加样品或环胞素 Α (终浓度 50ηΜ， 阳性对照） ΙΟμΙ/孔， 另设空白对照孔（^口诱导剂， 未加药） 、 阴性对照孔（加诱导剂， 未加药）。 于 37°C、 5%C02孵 育 72h。
3.培养结束前 4小时， 弃上清， 加 MTT ΙΟΟμΙ (终浓度 0.04% ) , 培 养结束后， 测定 OD540nm。
4.样品对淋巴细胞转化的影响用百分数表示，正数表示促进淋巴细胞 转化， 负数表示抑制淋巴细胞转化。淋巴细胞转化％= (样品孔 OD值 -阴性对照孔 OD值） / (阴性对照孔 OD值 -空白对照孔 OD值） xlOO 实验结果
结果见表 7， 表明恩再适对淋巴细胞转化有一定的抑制作用。
恩再适对淋巴细胞转化作用的影响
组别 剂量 T淋巴细胞转化 ( % ) B淋巴细胞转化（％) 恩再适 0.5U/ml -67.70 -70.66
0.25U -68.62 -19.46
0.125U -71.76 -25.86 参考文献
以下参考文献通过引用整体结合到本文中， 但是并非以任何方式 承认这些参考文献可用作评价本发明的现有技术。
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Claims

权 利 要 求 书
1. 牛痘疫苗致炎兔皮提取物在制备药物中的用途，所述药物用于 治疗哺乳动物中的急性脑血管疾病。
2.权利要求 1的用途，其中所述急性脑血管疾病为急性缺血性脑 血管疾病。
3.权利要求 2的用途，其中所述急性缺血性脑血管疾病选自脑栓 塞、 短暂性脑缺血发作、 脑血栓形成、 脑动脉硬化、 脑动脉炎、 脑动 脉盗血综合征、 颅内静脉窦和静脉血栓。
4.权利要求 1的用途，其中所述药物通过改善神经功能、降低脑 梗死范围、降低脑组织乳酸含量和 /或提高 SOD活性来治疗所述疾病。
5.权利要求 1的用途，其中所述药物通过保护神经细胞来治疗所 述疾病。
6.权利要求 5的用途，其中所述药物可改善 H202诱导的 PC12 细 胞损伤、 改善谷氨酸诱导的 PC12细胞损伤、 抑制脑血管内皮细胞表 达或分泌 ICAM-1和 /或抑制 T、 Β淋巴细胞转化作用。
7.权利要求 1-6中任一项的用途， 其中所述哺乳动物为人。
PCT/CN2009/001181 2008-11-11 2009-10-23 牛痘疫苗致炎兔皮提取物在制备急性脑血管疾病治疗药物中的用途 WO2010054531A1 (zh)

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US13/128,254 US10265345B2 (en) 2008-11-11 2009-10-23 Use of extracts from rabbit skin inflamed by vaccinia virus for the manufacture of a medicament for the treatment of acute cerebrovascular disease
SI200931579A SI2364711T1 (sl) 2008-11-11 2009-10-23 Uporaba ekstraktov iz zajčje kože vnete zaradi virusa vakcinije za izdelavo zdravila za zdravljenje akutne cerebrovaskularne bolezni
KR1020117012994A KR101756201B1 (ko) 2008-11-11 2009-10-23 급성 뇌혈관 질환 치료용 약제 제조에 있어서의 백시니아 바이러스에 의해 염증이 발생된 토끼 피부로부터 유래된 추출물의 용도
CA2743090A CA2743090C (en) 2008-11-11 2009-10-23 Use of extracts from rabbit skin inflamed by vaccinia virus in the manufacture of a medicament for treatment of acute cerebrovascular diseases
AU2009316168A AU2009316168B2 (en) 2008-11-11 2009-10-23 Use of extracts from rabbit skin inflamed by vaccinia virus for the manufacture of a medicament for the treatment of acute cerebrovascular disease
EP09825706.6A EP2364711B2 (en) 2008-11-11 2009-10-23 Use of extracts from rabbit skin inflamed by vaccinia virus for the manufacture of a medicament for the treatment of acute cerebrovascular disease
LTEP09825706.6T LT2364711T (lt) 2008-11-11 2009-10-23 Ekstraktų iš triušio odos, kurioje vakcinijos virusas sukėlė uždegimą, panaudojimas gamybai vaisto, skirto ūmios cerebrovaskuliarinės ligos gydymui
JP2011534986A JP6085806B2 (ja) 2008-11-11 2009-10-23 ワクシニアウイルスによって炎症を起こしたウサギ皮膚由来の抽出物の、急性脳血管疾患の治療用医薬の製造における使用
DK09825706.6T DK2364711T3 (en) 2008-11-11 2009-10-23 Use of extracts from vaccine virus-inflamed rabbit skin for the manufacture of a medicament for the treatment of acute cerebrovascular disease
ES09825706T ES2606051T5 (es) 2008-11-11 2009-10-23 Uso de extractos procedentes de piel de conejo inflamada por el virus de la variolovacuna para la elaboración de un medicamento para el tratamiento de la enfermedad cerebrovascular aguda
SM201600477T SMT201600477B (it) 2008-11-11 2016-12-28 Utilizzo di estratti da pelle di coniglio infiammata con virus del vaccino, per la produzione di un farmaco per il trattamento di malattie cerebrovascolari acute
CY20171100033T CY1118457T1 (el) 2008-11-11 2017-01-11 Χρηση εκχυλισματων απο δερμα κονικλου φλεγμαινοντος εκ του ιου δαμαλιτιδας για την παρασκευη φαρμακου για τη θεραπεια της οξειας αγγειακης εγκεφαλικης νοσου

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WO2020248240A1 (zh) 2019-06-14 2020-12-17 俊熙有限公司 痘苗病毒致炎兔皮提取物治疗癌症的用途

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WO2020248240A1 (zh) 2019-06-14 2020-12-17 俊熙有限公司 痘苗病毒致炎兔皮提取物治疗癌症的用途

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