WO2022099574A1 - 哌唑嗪用于制备治疗和/或预防脑血管疾病药物中的应用 - Google Patents

哌唑嗪用于制备治疗和/或预防脑血管疾病药物中的应用 Download PDF

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WO2022099574A1
WO2022099574A1 PCT/CN2020/128506 CN2020128506W WO2022099574A1 WO 2022099574 A1 WO2022099574 A1 WO 2022099574A1 CN 2020128506 W CN2020128506 W CN 2020128506W WO 2022099574 A1 WO2022099574 A1 WO 2022099574A1
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prazosin
treating
application
stroke
preparation
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PCT/CN2020/128506
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French (fr)
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陈新平
刘菁菁
赵文阳
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兰州大学
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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • 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

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  • the invention relates to the technical field of medicine, in particular to the application of prazosin for preparing medicines for treating and/or preventing cerebrovascular diseases.
  • Stroke also known as cerebrovascular accident, is an acute cerebrovascular disease. It is a disease caused by cerebral vascular rupture or vascular obstruction caused by stroke injury. It is one of the three major fatal diseases in my country. High rates, high mortality rates and high disability rates are a serious threat to people's health. In addition to threatening the health of patients, stroke also poses a heavy economic burden. As many as 1.9 million people die from stroke every year in China, and with the aging of the population, the incidence of ischemic stroke is increasing year by year. Stroke is divided into hemorrhagic stroke and ischemic stroke, among which ischemic stroke accounts for 80% of all strokes. The most common cause of ischemic stroke is a small embolus on the inner wall of the blood vessels in the brain.
  • Drugs for the treatment of ischemic stroke include antifibrillar drugs, anticoagulant drugs, antiplatelet aggregation drugs and neuroprotective drugs.
  • antifibrillar drugs include anticoagulant drugs, antiplatelet aggregation drugs and neuroprotective drugs.
  • anticoagulant drugs include antiplatelet aggregation drugs and neuroprotective drugs.
  • antiplatelet aggregation drugs include neuroprotective drugs.
  • neuroprotective drugs due to their own limitations such as single component and single target, they may lead to side effects such as hemorrhage and cerebral edema. So far, there is no drug that has been recognized in clinical practice.
  • Cerebral ischemia-reperfusion injury has a complex mechanism.
  • cerebral ischemia-reperfusion injury is mainly related to oxidative stress injury, inflammatory response, excitatory amino acids, calcium overload, cerebral edema and cell death.
  • oxidative stress plays an important role in the pathogenesis of ischemia-reperfusion injury.
  • Ischemia-reperfusion injury is an intensification of free radical chain reactions, which can lead to a rapid increase in the level of lipid peroxides in brain tissue and serum. Clinical symptoms were significantly aggravated, resulting in severe delayed neurological impairment.
  • Prazosin is an oral vasodilator that antagonizes alpha-adrenergic receptors and has been used for the treatment of hypertension since 1976.
  • the hemodynamic effects of prazosin in patients with hypertension and congestive heart failure are due to its peripheral vasodilatory effects.
  • the effects of prazosin on the vasculature begin with direct effects on vascular smooth muscle distal to ⁇ -adrenergic receptors, inhibition of phosphodiesterase, or neurotransmitter inhibition due to inhibition of dopamine ⁇ -hydroxylase. Synthesis is reduced. However, these effects occurred at prazosin concentrations of 10-3 to 10-4 M, whereas its effective plasma concentration in patients was only 10-7 to 10-8 M.
  • prazosin When prazosin is at an appropriate clinical concentration, its mechanism of action is mainly the competitive blockade of alpha blood adrenergic receptors in vascular postsynaptic blood vessels (see Colucci WS, Zhu Ruizhen, Wu Baiming. Baiming adrenergic receptors) The blocker prazosin in the treatment of hypertension and heart failure and its potential new uses [J]. Foreign Medicine. Cardiovascular Diseases, 1983 (04): 205-209.). Hu Guojun and other studies found that prazosin also has a certain curative effect on variant angina pectoris (Hu Guojun. Pharmacology and clinical application of prazosin [J].
  • Trishenning study found that prazosin Zosin can also be used as a preventive treatment for urination in elderly patients after major surgery, but it cannot be used in cases where urinary catheters have been used (Trishenning. New uses of prazosin [J]. Foreign Medicine. Synthetic Drugs . Biochemical Drugs. Preparations Volume, 1989(01): 62-63.). In addition, prazosin is used to treat enlarged prostate.
  • prazosin has a significant curative effect on stroke, especially for ischemic stroke;
  • prazosin Zine can improve the contents of MDA, MPO, GSH and GSSG after ischemia-reperfusion and make them close to normal, indicating that prazosin mainly achieves anti-cerebral ischemia by reducing oxidative stress injury, inflammatory response and inhibiting cell death. The role of reperfusion injury.
  • the present invention provides new uses of prazosin and pharmaceutically acceptable salts thereof, and specifically provides prazosin and pharmaceutically acceptable salts thereof for preparing medicines for treating and/or preventing cerebrovascular diseases applications in .
  • cerebrovascular disease is specifically stroke.
  • the stroke includes hemorrhagic stroke and/or ischemic stroke.
  • the stroke is ischemic stroke.
  • the present invention also provides the application of prazosin and its pharmaceutically acceptable salts for preparing neuroprotective drugs.
  • the present invention also provides the application of prazosin and its pharmaceutically acceptable salts for preparing medicines for treating and/or preventing nerve damage.
  • the present invention also provides the application of prazosin and its pharmaceutically acceptable salts for preparing medicines for treating and/or preventing cerebral ischemia-reperfusion injury.
  • the present invention also provides the application of the composition containing prazosin and pharmaceutically acceptable salts thereof in the preparation of medicines for treating and/or preventing cerebrovascular diseases
  • the prazosin and its pharmaceutically acceptable salts can be drop pills, controlled-release tablets, sustained-release tablets, suppositories or cyclodextrin inclusion complexes.
  • the present invention provides a new use of prazosin, specifically provides the application of prazosin in the preparation of medicines for treating and/or preventing cerebrovascular diseases, by adopting MCAO cerebral ischemia-reperfusion injury animals
  • the model evaluates the therapeutic effect of prazosin on ischemic stroke, confirming that (1) prazosin has the effect of treating ischemic stroke, and can improve cerebral infarction to a greater extent than clinical drug butylphthalide
  • Prazosin mainly achieves the effect of anti-cerebral ischemia-reperfusion injury by reducing the degree of oxidative stress injury, improving inflammatory response and inhibiting cell death.
  • Fig. 1 is a histogram of neurological function scores of rats in each administration group provided by the present invention.
  • SHAM is the sham operation group
  • IR is the ischemia-reperfusion model control group
  • NBP is the positive drug butylphthalide control group
  • PZ is the prazosin group.
  • Fig. 2 accompanying drawing is the TTC staining diagram of each administration group rat of the present invention.
  • Fig. 3 is the evaluation result of cerebral infarction volume of rats in each administration group provided by the present invention.
  • Figure 4 is a diagram for measuring the content of oxidative stress-related indexes in rats of each administration group provided by the present invention
  • the TTC staining in the present invention refers to 2,3,5-triphenyltetrazolium chloride staining.
  • butylphthalide is mainly used to treat mild and moderate acute ischemic stroke.
  • butylphthalide is used as a positive drug.
  • the inventors used prazosin small molecule compound to perform neurological function score, cerebral infarction volume and TTC staining on SD rats under different feeding conditions, as well as determine the contents of oxidative stress-related indicators MDA, MPO, GSH and GSSG, It is confirmed that prazosin has the effect of treating ischemic stroke and its mechanism of action.
  • the MPO described in the following embodiments of the present invention refers to myeloperoxidase, which is a heme protein rich in neutrophils, synthesized in the bone marrow by granulocytes before entering the circulation and stored in azurine granules Inside. External stimuli can cause neutrophils to aggregate and release myeloperoxidase.
  • the relative molecular weight of MPO is 150kDa. It is a tetramer formed by covalent bonding of two subunits. Each subunit has a heavy chain ⁇ (relative molecular weight 60kDa) and a light chain ⁇ chain (relative molecular weight 15kDa) constitute.
  • the MDA described in the following examples of the present invention refers to malondialdehyde, and the MDA content is an important parameter reflecting the body's antioxidant potential, which can reflect the body's lipid peroxidation rate and intensity, and can also indirectly reflect the degree of tissue peroxidative damage.
  • the GSH described in the following examples of the present invention refers to reduced glutathione.
  • the GSSG described in the following examples of the present invention refers to oxidized glutathione.
  • the CMC-Na described in the following examples of the present invention refers to sodium carboxymethyl cellulose.
  • Prazosin (Prazosin, PZ, HPLC ⁇ 99%), purchased from McLean Biotechnology Co., Ltd., batch number: C10884181, molecular weight 419.86;
  • Butylphthalide soft capsules (NBP, NBP), purchased from NBP Pharmaceutical Co., Ltd. of CSPC;
  • TTC 2,3,5-Triphenyltetrazolium chloride
  • Sham operation group (SHAM, orally administered an equal volume of 0.5% CMC-Na);
  • Model control group (IR, orally administered an equal volume of 0.5% CMC-Na);
  • NBP negative drug group
  • Prazosin group (PZ, oral dose of 20 mg/kg/d).
  • the administration time was one week after adaptive feeding. All SD rats were administered for 2 days before modeling, and then administered for 24 h after modeling, once a day.
  • the sham operation group was the same as the MCAO model operation group, except that no suture was inserted.
  • Recovered rats were scored according to Zea Longa 5, 0: no neurological deficit; 1: unable to fully extend the forepaw on the opposite side of the lesion; 2: spontaneous rotation to the opposite side of the lesion; 3: to the lesion Contralateral tipping; 4 points: no spontaneous walking and loss of consciousness, select the rats with a score greater than or equal to 2 as the successful model establishment, discard the unsuccessful model establishment rats in each group, select the successful model establishment rats and press
  • the above doses were administered by gavage, and after 24 hours of drug treatment, the brain tissue was separated for TTC staining.
  • the rats in each group were decapitated, the brain tissue was separated on ice, the olfactory bulb and brain stem were removed, rinsed with normal saline, and immediately placed in -20 °C for 15 min, then taken out, and the brain was uniformly sliced along the optic chiasm plane to the pituitary plane coronally.
  • 3 coronal sections were immersed in 1.5% TTC solution, incubated in a constant temperature water bath at 37°C for 45 min for staining (protected from light), and turned every 15 min to make the staining uniform.
  • the reaction with deoxygenase in normal tissue was red, and the ischemic area was white. That is, the unstained area is the infarct area, and the infarct volume of the animal brain tissue was determined according to the percentage of the total brain volume in each coronal section of the brain tissue in the total brain volume.
  • the neuroprotective effect of prazosin (56.08%) is stronger than the neuroprotective effect of butylphthalide (44.12%), that is, the prazosin of the present invention is better than the clinical drug butylphthalide in the treatment of ischemia.
  • the role of neurological damage in stroke is better than the clinical drug butylphthalide in the treatment of ischemia.
  • the results of the cerebral infarction volume of the prazosin group showed that compared with the results of the butylphthalide positive drug group, prazosin could reduce the size of the cerebral infarction.
  • the volume of cerebral infarction is improved to a greater extent, indicating that the neuroprotective effect of prazosin is better than that of butylphthalide, that is, the prazosin of the present invention has a better effect on the treatment of ischemic stroke nerve damage .
  • prazosin can significantly down-regulate the ratio of GSSG and GSH (**p ⁇ 0.01), which further indicates that prazosin plays an anti-ischemia effect through antioxidant; The potential early activation signal of apoptosis, and the subsequent generation of oxygen free radicals promotes cell apoptosis.
  • prazosin can up-regulate the content of GSH in stroke serum samples.
  • prazosin can inhibit cell death by inhibiting cell death. To achieve the role of stroke prevention.
  • the present invention evaluates the therapeutic effect of prazosin on ischemic stroke by using the MCAO cerebral ischemia-reperfusion injury animal model, confirming that (1) prazosin has the effect of treating ischemic stroke, and Compared with clinical drug butylphthalide, it can improve cerebral infarction to a greater extent; (2) Prazosin mainly achieves anti-cerebral ischemia-regeneration resistance by reducing the degree of oxidative stress injury, improving inflammatory response and inhibiting cell death. The role of perfusion injury.

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Abstract

一种哌唑嗪用于制备治疗和/或预防脑血管疾病药物中的应用，尤其是在制备治疗缺血性脑卒中药物中的应用，其抗缺血的主要机制是通过采用MCAO脑缺血再灌注损伤动物模型评价哌唑嗪的对缺血性脑卒中治疗效果。实验结果表明：哌唑嗪具有治疗缺血性脑卒中的作用，且与临床用药丁苯酞相比，能更大程度的改善脑梗死灶；哌唑嗪主要通过减轻氧化应激损伤的程度、改善炎症反应及抑制细胞死亡等途径达到抗脑缺血再灌注损伤的作用。

Description

哌唑嗪用于制备治疗和/或预防脑血管疾病药物中的应用 技术领域
本发明涉及医药技术领域，具体涉及哌唑嗪用于制备治疗和/或预防脑血管疾病药物中的应用。
背景技术
脑卒中又称脑血管意外，是一种急性脑血管疾病，是由于脑部血管破裂或因血管阻塞而引起脑卒中损伤的一种疾病，是引起我国三大死亡疾病之一，其特点是发病率高、死亡率高和致残率高，严重威胁着人民的身体健康。脑卒中除了威胁患者的健康，还造成了沉重的经济负担。国内每年死于脑卒中人数高达190万，而且随着人口老龄化的加剧，缺血性脑卒中的发病率逐年上升。脑卒中分为出血性脑卒中和缺血性脑卒中两大类，其中缺血性脑卒中占全部脑卒中的80％。缺血性脑卒中发生的最常见原因是脑部血管内壁上有小栓子，脱落后导致动脉-动脉栓塞而发生卒中，也可由颈内动脉或椎动脉狭窄和闭塞而形成梗死灶。脑缺血发生时十分危险，即使度过危险期也会留下各种各样的后遗症，严重者可致残。在有效时间窗内重新恢复或开通病变血管，实现缺血脑组织的再灌注是缺血性脑卒中急性期治疗的重要措施。目前临床上最常用的治疗方法是溶栓治疗，最终目的是开通血管，实现缺血脑组织再灌注。但是其缺点是受溶栓时间窗的限制，对于来不及接受溶栓治疗的患者来说，只能选择药物治疗来降低其神经功能损伤。缺血性脑卒中的治疗药物包括降纤药物、抗凝药物、抗血小板聚集药物和神经保护药物，但由于其单成分、单靶点等自身局限性会导致出血、脑水肿等副作用的产生，目前为止临床上还没有一种药物取得公认的确切疗效。
脑缺血再灌注损伤具有复杂的发生机制，大量研究表明，脑缺血再灌注损伤主要与氧化应激损伤、炎症反应、兴奋性氨基酸、钙超载、脑水肿和细胞死亡等有关。其中氧化应激在缺血再灌注损伤的发病机制中起着重要的作用，缺血再灌注损伤是自由基连锁反应激化，可导致脑组织和血清中脂质过氧化物水平的迅速升高和临床症状明显加重，造成严重的迟发性神经功能损害。
哌唑嗪是为拮抗α-肾上腺素能受体的口服血管扩张剂，1976年起用于治疗高血压。哌唑嗪在高血压和充血性心力衰竭患者中的血流动力学效应是由于其周围血管的扩张作用。哌唑嗪在血管系统的作用，开始时是对α-肾上腺素能受体远端血管平滑肌的直接影响、磷酸二酯酶的抑制、或由于多巴胺β羟基化酶的抑制而导致神经递质的合成减少。然而这些效应是在哌唑嗪浓度为10 -3至10 -4M时出现，而其在患者中的有效血浆浓度仅为10 -7至10 -8M。哌唑嗪在合适的临床浓度时，其作用机制主要是血管突触后α血肾上腺素能受体的竞争性阻滞(参见文献Colucci WS，朱瑞珍，吾柏铭.柏铭肾上腺素能受体阻滞剂哌唑嗪治疗高血压和心力衰 竭及其潜在的新用途[J].国外医学.心血管疾病分册，1983(04):205-209.)。胡国钧等研究发现哌唑嗪对变异型心绞痛也有一定的疗效(胡国钧.哌唑嗪的药理及临床应用[J].新药与临床，1985(02):83-85.)曲申宁研究发现哌唑嗪也可以作为对重大手术后老年病人尿闭症的预防性治疗，但不能用于已采用导尿管的病例(曲申宁.哌唑嗪的新用途[J].国外医药.合成药.生化药.制剂分册，1989(01):62-63.)。另外，哌唑嗪还被用于治疗前列腺肥大症。
但是，现有技术并没有公开哌唑嗪具有治疗脑血管疾病的用途，发明人也不能从现有技术中得到任何的技术启示。发明人所在的课题组一直致力于研究脑缺血再灌注引起的损伤机制，开发具有防治和治疗脑缺血再灌注损伤的药物，进而降低缺血性脑卒中的死亡率和致残率，这也是当前科学研究的一项重大课题，在研究过程中意外地发现，(1)哌唑嗪对于脑卒中具有显著的疗效，尤其是缺血性脑卒中的疗效更为显著；(2)哌唑嗪能够改善缺血再灌注后MDA、MPO、GSH和GSSG的含量，并使之趋近于正常，表明哌唑嗪主要通过减轻氧化应激损伤、炎症反应和抑制细胞死亡来达到抗脑缺血再灌注损伤的作用。
发明内容
针对上述技术问题，本发明提供了哌唑嗪及其药学上可接受的盐的新用途，具体提供了哌唑嗪及其药学上可接受的盐用于制备治疗和/或预防脑血管疾病药物中的应用。
进一步的，所述的脑血管疾病具体为脑卒中。
进一步的，所述的脑卒中包括出血性脑卒中和/或缺血性脑卒中。
进一步的，所述的脑卒中为缺血性脑卒中。
本发明还提供了哌唑嗪及其药学上可接受的盐用于制备神经保护药物中的应用。
本发明还提供了哌唑嗪及其药学上可接受的盐用于制备治疗和/或预防神经损伤药物中的应用。
本发明还提供了哌唑嗪及其药学上可接受的盐用于制备治疗和/或预防脑缺血再灌注损伤药物中的应用。
本发明还提供了含有哌唑嗪及其药学上可接受的盐的组合物在制备治疗和/或预防脑血管疾病药物中的应用
进一步的，所述的哌唑嗪及其药学上可接受的盐可以为滴丸、控释片、缓释片、栓剂或环糊精包合物。
本发明的有益效果是：本发明提供了哌唑嗪的新用途，具体提供了哌唑嗪用于制备治疗和/或预防脑血管疾病药物中的应用，通过采用MCAO脑缺血再灌注损伤动物模型评价哌唑嗪的对缺血性脑卒中治疗效果，证实(1)哌唑嗪具有治疗缺血性脑卒中的作用，且与临床用药丁苯酞相比，能更大程度的改善脑梗死灶；(2)哌唑嗪主要通过减轻氧化应激损伤的程度、 改善炎症反应及抑制细胞死亡等途径达到抗脑缺血再灌注损伤的作用。
附图说明
图1为本发明提供的各给药组大鼠神经功能评分柱状图；
其中“SHAM”为假手术组，“IR”为缺血再灌注模型对照组，“NBP”为阳性药物丁苯酞对照组，“PZ”为哌唑嗪组。
与假手术组“SHAM”相比， ###p﹤0.001；与“IR”相比，***p<0.001
图2附图为本发明的各给药组大鼠的TTC染色图；
图3为本发明提供的各给药组大鼠脑梗死体积评价结果；
与假手术组“SHAM”相比， ###p﹤0.001；与“IR”相比，**p<0.01和***p<0.001
图4为本发明提供的各给药组大鼠氧化应激相关指标含量测定图；
与假手术组“SHAM”相比， #p﹤0.05、 ##p﹤0.01和 ###p﹤0.001；与“IR”相比，*p<0.05、**p<0.01和***p<0.001
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚完整的描述，很显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，同时保护哌唑嗪在所有脑血管疾病中的治疗效果，包括不同剂型、剂量等。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动力前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明所述的TTC染色是指2，3，5-氯化三苯基四氮唑染色。
本发明以下实施例中，丁苯酞主要用于治疗轻、中度急性缺血性脑卒中。本发明实施例将丁苯酞作为阳性药使用。
发明人用哌唑嗪小分子化合物，在不同饲喂条件下，对SD大鼠进行神经功能评分、脑梗死体积及TTC染色，以及测定氧化应激相关指标MDA、MPO、GSH和GSSG的含量，证实了哌唑嗪具有治疗缺血性脑卒中的作用及其作用机制。
本发明以下实施例所述的MPO是指髓过氧化物酶，是一种血红素蛋白，富含于中性粒细胞中，由粒细胞进入循环之前在骨髓内合成并存储于噬天青颗粒内。外界刺激可导致中性粒细胞聚集，从而释放髓过氧化物酶。MPO的相对分子量为150kDa，是由两个亚单位通过共价结合形成的四聚体，每个亚单位又有一条重链α(相对分子量60kDa)和一条轻链β链(相对分子量为15kDa)构成。
本发明以下实施例所述的MDA是指丙二醛，MDA含量是反映机体抗氧化潜在能力的重要参数，可以反映机体脂质过氧化速率和强度，也能间接反映组织过氧化损伤程度。
本发明以下实施例所述的GSH是指还原型谷胱甘肽。
本发明以下实施例所述的GSSG是指氧化型谷胱甘肽。
本发明以下实施例所述的CMC-Na是指羧甲基纤维素钠。
实施例一、哌唑嗪治疗缺血性脑卒中的效果研究
1.实验动物
SPF级健康雄性SD大鼠，体重220-280g，实验前未用过任何药物，由中国农业科学院兰州兽医研究所提供。实验动物于温度为24～26℃，12h/12h的白昼规律交替的环境中适应饲养一周，给予动物饮食并自由饮水，然后分组进行实验。
2.药物及试剂
哌唑嗪(Prazosin，PZ，HPLC≥99％)，购自麦克林生物科技有限公司，批号：C10884181，分子量为419.86；
丁苯酞软胶囊(恩必普，NBP)，购自石药集团恩必普药业有限公司；
2，3，5-氯化三苯基四氮唑(TTC)，购自Sigma公司。
3.实验分组和灌胃给药剂量
将雄性3月龄SD大鼠按体重随机分为4组，每组10只，分组和给药剂量如下：
假手术组(SHAM，口服给予等体积0.5％CMC-Na)；
模型对照组(IR，口服给予等体积0.5％CMC-Na)；
阳性药组(NBP，口服给予丁苯酞，20mg/kg/d)；
哌唑嗪组(PZ，口服剂量为20mg/kg/d)。
给药时间为适应性饲养一周后，所有的SD大鼠在造模前给药2天，造模结束后再给药24h，每天给药一次。
4.缺血性脑卒中模型组的制备
(1)将成功麻醉的大鼠仰卧位固定，颈部正中皮肤去毛，碘伏消毒局部，取长度约1.5cm颈部正中切口，沿胸锁乳突肌内缘分离肌肉和筋膜，切记勿伤气管两侧甲状腺及其外上方甲状旁腺，在见到搏动的颈总动脉后仔细分离颈总动脉表面膜组织，且应避免过度牵拉血管以防血管过度刺激或离断；
(2)成功分离颈总动脉后，在颈总动脉近心端置一丝线备用，继续向远心端分离血管以及与血管伴行的神经，注意动作轻柔避免损伤迷走神经，在甲状腺水平可见颈总动脉分出颈外动脉和颈内动脉，然后在近心端分离出长度3-4mm颈外动脉，备一丝线用于结扎；
(3)沿颈内动脉继续向颅底方向分离，可见血管在到达鼓泡处略为膨大并发出一较大分支即翼腭动脉，此时结扎位于颈外动脉近心端的丝线，使用微小动脉夹暂时夹闭颈内、颈总 动脉；
(4)在颈外动脉上用显微手术剪剪一小口，测量该切口至颈内动脉与颈外动脉分叉处的距离(一般为3-4mm)，将制备好的栓线，头端呈球形，距头端1.8-2.0cm处做一标记插入，在颈外动脉剪口处打一松结以阻断颈内动脉的血液反流，去除颈内动脉的微动脉夹，将线从颈外动脉插入，进入颈内动脉的颅内段，进线长度约为距颈总动脉分叉18.5±0.5mm，此时进线有轻微阻力，栓线正好插至颅内的大脑前动脉，堵住大脑中动脉的开口。
(5)阻断2h后，拔出尼龙线栓约1cm，扎紧动脉残端，消毒后逐层缝合皮下组织和皮肤。为避免感染，缝合前在手术局部使用青霉素，缝合后碘伏消毒颈部皮肤，完成脑缺血再灌注损伤模型，术中及术后用保温垫以维持其肛温在37±1℃，直到恢复活动。
(6)假手术组与MCAO模型操作组相同，只是不需要插入线栓。
5.检测指标
(1)神经功能缺损评分
按照Zea Longa 5级对恢复活动的大鼠进行评分，0分：无神经功能缺损；1分：病灶对侧前爪不能完全伸展；2分：向病灶对侧自发性旋转；3分：向病灶对侧倾倒；4分：无自发性行走且出现意识丧失，选取评分大于等于2分的为模型制作成功大鼠，舍弃各组中造模不成功的大鼠，选择模型制作成功的大鼠按上述剂量进行灌胃给药，药物处理24h后，分离脑组织，进行TTC染色。
(2)TTC染色
取每组大鼠，断头，冰上分离脑组织，去除嗅球和脑干，生理盐水冲洗后立即放入-20℃冷冻15min，取出，将大脑沿视交叉平面至垂体平面冠状切均匀切成3片冠状切片，浸入1.5％TTC溶液，37℃恒温水浴中孵育染色(避光)45min，每15min翻一次，使染色均匀，与正常组织中的脱氧酶反应呈红色，缺血区呈白色，即未染色的区域为梗死区域，根据脑组织各冠状切片梗死体积总和占全脑体积百分比测定动物脑组织梗死体积。
(3)血清生化指标的测定
上述每组大鼠(n≥6)处死后，分离血清，按照MPO、MDA、GSH和GSSG相关试剂盒测定说明书进行相关指标的测定，并对数据进行统计分析。
6.实验结果
(1)神经功能缺损评分评价
SD大鼠预防连续给药2天，MCAO模型缺血2h后，再次进行药物治疗，再灌注24h后，各组大鼠神经功能行为学评分见附图1。如图1所示，与假手术组相比，模型组的神经功能评分显著升高( ###p﹤0.001)，表明MCAO模型成功构建。与模型组相比，哌唑嗪与阳 性药丁苯酞均能显著的降低神经功能缺损评分(***p<0.001)，表明哌唑嗪与丁苯酞一样具有对脑卒中大鼠的神经保护作用，且哌唑嗪的神经保护作用(56.08％)强于丁苯酞的神经保护作用(44.12％)，即本发明哌唑嗪相较于临床用药丁苯酞具备更佳的治疗缺血性脑卒中神经损伤的作用。
(2)TTC染色和脑梗死体积评价
TTC染色结果和脑梗死体积结果见图2和图3。由图2可知，假手术组脑组织TTC染色呈均匀的红色，未见缺血白色梗死灶，而模型组可见大区域的白色梗死灶，表明大鼠MCAO模型构建成功。与模型组相比，阳性药组的脑梗死区域显著缩小，且梗死区域分界呈不清晰状态，有显著性差异(**p<0.01)；与模型组相比，哌唑嗪组的脑梗死区域显著缩小，且梗死区域分界呈不清晰状态，有显著性差异(**p<0.001)，哌唑嗪组脑梗死体积结果表明，与丁苯酞阳性药组结果相比，哌唑嗪能更大程度的改善脑梗死体积，说明哌唑嗪的神经保护作用优于丁苯酞的神经保护作用，即本发明所述的哌唑嗪具备更佳的治疗缺血性脑卒中神经损伤的作用。
(3)哌唑嗪对脑卒中大鼠血清MPO、MDA、GSH和GSSG含量的影响
MPO、MDA、GSH和GSSG含量测定结果见图4。由图4可知，(1)与假手术组相比，模型组MPO的含量显著升高(#p<0.05)；与模型组相比，哌唑嗪给药组能显著的降低MPO的含量(*p<0.05)，表明哌唑嗪可抑制中性粒细胞浸润、粘附及释放细胞毒性物质，可以起到较少脑组织损伤的作用，我们进一步推测哌唑嗪可通过抑制炎症反应来达到抗缺血的作用；(2)与假手术组相比，模型组MDA的含量显著升高(###p<0.001)，其脂质过氧化反应增强；与模型组相比，哌唑嗪给药组能显著的降低MDA的含量(***p<0.001)，表明哌唑嗪可通过抗氧化发挥其防治脑卒中的作用；(3)血清中还原型和氧化型谷胱甘肽(GSH和GSSG)含量变化，是机体氧化物牵累的重要指标。本实验中哌唑嗪能显著的下调GSSG和GSH的比例(**p<0.01)，进一步表明了哌唑嗪是通过抗氧化起到抗缺血的作用；同时，GSH含量的降低时一种潜在的凋亡早期激活信号，随后产生的氧自由基促使细胞发生凋亡，由图4可知，哌唑嗪可上调脑卒中血清样本中GSH的含量，我们推断哌唑嗪可通过抑制细胞死亡来达到防治脑卒中的作用。
7.实验结论
综上所述，本发明通过采用MCAO脑缺血再灌注损伤动物模型评价哌唑嗪的对缺血性脑卒中治疗效果，证实(1)哌唑嗪具有治疗缺血性脑卒中的作用，且与临床用药丁苯酞相比，能更大程度的改善脑梗死灶；(2)哌唑嗪主要通过减轻氧化应激损伤的程度、改善炎症反应及抑制细胞死亡等途径达到抗脑缺血再灌注损伤的作用。
本发明的保护范围不限于实施例。凡技术人员按照本发明的思路在现有技术上通过逻辑分析、推断和实验得出的技术方案，均属于权利要求书中保护的范围。

Claims (9)

  1. 哌唑嗪及其药学上可接受的盐用于制备治疗和/或预防脑血管疾病药物中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述的脑血管疾病具体为脑卒中。
  3. 如权利要求2所述的应用，其特征在于，所述的脑卒中包括出血性脑卒中和/或缺血性脑卒中。
  4. 如权利要求3所述的应用，其特征在于，所述的脑卒中为缺血性脑卒中。
  5. 哌唑嗪及其药学上可接受的盐用于制备神经保护药物中的应用。
  6. 哌唑嗪及其药学上可接受的盐用于制备治疗和/或预防神经损伤药物中的应用。
  7. 哌唑嗪及其药学上可接受的盐用于制备治疗和/或预防脑缺血再灌注损伤药物中的应用。
  8. 含有哌唑嗪及其药学上可接受的盐的组合物在制备治疗和/或预防脑血管疾病药物中的应用。
  9. 如权利要求1-8任一项所述的应用，其特征在于，所述的哌唑嗪及其药学上可接受的盐可以为滴丸、控释片、缓释片、栓剂或环糊精包合物。
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