WO2012003802A1 - 5α-雄甾（烷）-3β，5，6β-三醇在制备神经元保护药物中的应用 - Google Patents

5α-雄甾（烷）-3β，5，6β-三醇在制备神经元保护药物中的应用 Download PDF

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WO2012003802A1
WO2012003802A1 PCT/CN2011/076967 CN2011076967W WO2012003802A1 WO 2012003802 A1 WO2012003802 A1 WO 2012003802A1 CN 2011076967 W CN2011076967 W CN 2011076967W WO 2012003802 A1 WO2012003802 A1 WO 2012003802A1
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neurons
spinal cord
ischemia
glutamic acid
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PCT/CN2011/076967
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English (en)
French (fr)
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颜光美
胡海燕
冷田东
桑韩飞
张静夏
邱鹏新
周树佳
陈婕思
游秀华
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中山大学
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Priority to BR112012031861A priority Critical patent/BR112012031861B1/pt
Priority to SI201131126A priority patent/SI2591785T1/sl
Application filed by 中山大学 filed Critical 中山大学
Priority to SG2012088688A priority patent/SG186135A1/en
Priority to CA2802133A priority patent/CA2802133C/en
Priority to RU2012151400/15A priority patent/RU2541093C2/ru
Priority to AU2011276800A priority patent/AU2011276800B2/en
Priority to RS20170319A priority patent/RS55845B1/sr
Priority to JP2013516989A priority patent/JP5681931B2/ja
Priority to US13/805,436 priority patent/US8809309B2/en
Priority to KR1020127032524A priority patent/KR101463477B1/ko
Priority to LTEP11803155.8T priority patent/LT2591785T/lt
Priority to ES11803155.8T priority patent/ES2620754T3/es
Priority to DK11803155.8T priority patent/DK2591785T3/en
Priority to EP11803155.8A priority patent/EP2591785B1/en
Publication of WO2012003802A1 publication Critical patent/WO2012003802A1/zh
Priority to US14/332,338 priority patent/US9320743B2/en
Priority to HRP20170418TT priority patent/HRP20170418T1/hr
Priority to CY20171100375T priority patent/CY1118789T1/el

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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • C07J1/0003—Androstane derivatives
    • C07J1/0007—Androstane derivatives not substituted in position 17

Definitions

  • the present invention relates to a novel pharmaceutical use of the compound 5 ⁇ -androst( ⁇ :)-3 ⁇ ,5,6 ⁇ -triol (abbreviated as YC-6). Background technique
  • the treatment of acute ischemic stroke mainly through two ways: one is to dissolve thrombus, and the other is neuroprotection.
  • the latter refers to the prevention of a series of pathological changes in brain tissue caused by ischemia in the treatment of acute ischemic stroke. Biochemical reactions, interfere with all aspects of the ischemic waterfall response, and prolong the survival of drugs or measures of neurons.
  • neuroprotective therapy for acute ischemic stroke has become a research hotspot in the treatment of stroke.
  • Many neuroprotective agents are currently undergoing clinical development experiments, and their mechanism of action is to block various harmful pathological processes caused by ischemia. Occurs, thereby preventing or limiting brain damage caused by ischemia, reducing brain tissue death and promoting functional recovery.
  • neuroprotective agents can reduce the area of cerebral infarction, do not cause bleeding, no bleeding complications during thrombolysis and anticoagulant therapy, no detailed diagnosis of the cause is required before use, making early treatment possible, thus neuroprotective agents The treatment effect and prospects are encouraging.
  • CBs calcium channel blockers
  • GABA gamma-aminobutyric acid
  • neuroactive bodies have a widespread effect on neuroprotection and are receiving increasing attention. These levels of neuroactive steroids are associated with the development of central nervous system (CNS) diseases, and have significant regulatory effects on neuronal damage, death, and various central nervous system diseases.
  • CNS central nervous system
  • NAS neuroactive steroids
  • Estrogen is one of the most known neuroprotective NASs. In menopausal women, ovaries stop producing estrogen, which can cause beta-amyloid protein ( ⁇ ) deposition, leading to Alzheimer's disease (Alzheimer s disease).
  • estrogen to AD can significantly reduce the level of ⁇ in the brain.
  • Clinical use of estrogen for the treatment of AD has received good results.
  • 5 ⁇ - ⁇ :) -3 ⁇ ,5,6 ⁇ -triol (YC-6) is a neuroprotective compound we found during neurosteroid studies. Its chemical structure is shown in the figure. According to our literature search, to date, no reports of pharmacological effects of YC-6 have been reported, and no studies have reported that YC-6 has active or neuroprotective effects on nervous tissues.
  • An object of the present invention is to provide a use of 5 ⁇ -androstidine-3 ⁇ ,5,6 ⁇ -triol for the preparation of a neuroprotective drug, thereby providing a novel drug for the treatment of neuronal diseases.
  • YC-6 has neuroprotective effects in vivo
  • focal cerebral ischemia model and 2) blocked spinal aorta-induced spinal cord injury model, and studied YC-6 on rat cerebral ischemia.
  • YC-6 has obvious protective effect on brain neuron injury.
  • YC-6 has protective effects on neuronal damage caused by cerebral ischemia, spinal cord ischemia and hypoxia. To date, there have been no reports of YC-6 activity or neuroprotective effects on neural tissue. DRAWINGS
  • Figure 1 is a protective effect of YC-6 on glutamate-induced cerebellar granule neurons, spinal motor neurons, and cerebral cortical neurons excitatory damage, in which A is cerebellar granule neurons and B is spinal motor neurons, C For cerebral cortical neurons, D is the release rate of LDH, E is the survival rate of neurons; * and ** indicate that there is a significant difference between the YC-6 group and the glutamate (Glu) group of cerebellar granule neurons, *P ⁇ 0.05 , ** ⁇ 0.01; # ⁇ ## respectively indicate that there is a significant difference between the YC-6 group and the glutamate (Glu) group in spinal motor neurons, #P ⁇ 0.05, ## ⁇ 0.01; $ and $ $ indicates that there is a significant difference between the cerebral cortical neurons YC-6 group and the glutamic acid (Glu) group, ⁇ O.OS , ⁇ P O.OL Figure 2 is a protective effect of YC-6 on hypoxia-induced cortical
  • Figure 3 is a neuroprotective effect of YC-6 on rabbit spinal cord ischemia induced by abdominal aortic clipping, in which A is a neurological function score, B is a histopathological section, and C is a normal spinal motor neuron count.
  • Figure 4 shows the neuroprotective effect of YC-6 on focal cerebral ischemia model, in which A is the neurological function score, B is the cerebral infarction slice, and C is the cerebral infarct volume comparison.
  • SD rats were isolated from the spinal cord for 15 days of pregnancy to remove the meninges and blood membranes.
  • the fetal rat spinal cord tissue was digested with 0.125% trypsin, centrifuged, and the middle layer containing abundant motor neurons was removed. After centrifugation to remove the cell debris, the differential wall was attached. 1 h, select suspended spinal motor neurons with slow adherence. After 24 h of inoculation, cytarabine was added, and the whole amount was changed on the third day, and the culture was continued with L-15 serum-free medium, and the medium was changed every half 2-3 days. Experiments were performed on days 3-5.
  • the cortex of the meninges and blood vessels of the rats in which the Id was taken out was removed, and after digesting with 0.25 g/L trypsin, the cells were blown into a single cell suspension by a 0.05 g/L DNase I blowing solution, centrifuged, and a precipitate was taken. Diluted with DMEM-F12 medium containing 5% (v/v) FBS and 2% B27 and seeded in polylysine pre-coated petri dishes. After 24 hours of inoculation, lO M Ara-C was added to inhibit the growth and proliferation of non-neuronal cells, and then changed 2-3 times a week. Experiment on the 10th day.
  • the cerebellar granule neurons were cultured for 8 days, and the normal control group, the glutamic acid group, the MK801+ glutamic acid, and the YC-6+ glutamic acid group were respectively set.
  • the normal control group did not do any treatment.
  • the glutamic acid group was added with 200 ⁇ glutamic acid.
  • the other groups were pre-added with MK801 ( ⁇ ) and different concentrations of YC-6. After incubation at 37 ° C for 30 minutes, glutamic acid was added and used for 24 h.
  • the morphology of the nerve cells was observed by phase contrast microscopy; the cells were counted by FDA staining, inverted fluorescence microscopy, and the survival rate of neurons was calculated. The activity of each group of lactate dehydrogenase was measured.
  • YC-6 has a concentration dependence, see Figure 1-A, D, E. In the above dose range, YC-6 had no effect on the survival rate of normal nerve cells.
  • the cortical neurons were cultured for 10 days, and the normal control group, the glutamic acid group, the MK801+ glutamic acid, and the YC-6+ glutamic acid group were respectively set.
  • the normal control group did not do any treatment.
  • the glutamic acid group was added with 200 ⁇ M glutamic acid.
  • the other groups were pre-charged with solvent, MK801 ( ⁇ ) and different concentrations of YC-6. After incubation at 37 ° C for 30 minutes, glutamic acid was added. After h, the morphology of the nerve cells was observed by phase contrast microscope, stained with FDA, inverted fluorescence microscope, cell count, and the survival rate of neurons was calculated. The activity of each group of lactate dehydrogenase was measured.
  • MTT assay showed that hypoxia treatment significantly reduced neuronal survival (P ⁇ 0.05), while YC-6 showed a high concentration-dependent increase in neuron survival due to hypoxia damage (Fig. 2B).
  • the LDH release results were consistent with the MTT method, and the YC-6 pretreatment group reduced the neuronal damage caused by hypoxia in a concentration-dependent manner (Fig. 2-C, ⁇ 0.05).
  • Control group made rabbit spinal cord ischemia model
  • YC-6 group received 2 mg.Kg_EH YC-6 via ear vein 30 minutes before spinal cord ischemia
  • Vehicle group the same volume of hydroxypropyl cyclodextrin (lml.Kg- 1 ) was injected in the same manner 30 min before spinal cord ischemia; the Sham group only exposed the abdominal aorta without blocking.
  • the physiological parameters between the groups were measured immediately before ischemia, 10 min after ischemia and 20 min after reperfusion.
  • the functional scores of rabbits in each group were scored by Talov score [5]: 0 points, complete paralysis of hind limbs; 1 point, joint movement of hind limbs can be detected; 2 points, hind limbs can move freely but can not stand; 3 points, can stand but can not Walking; 4 points, the hindlimb motor function is fully restored and can walk normally.
  • the lumbar spinal cord tissue (L 5 -L 7 ) was taken under anesthesia, and the paraffin was embedded and sectioned, HE staining, and the pathology of the spinal cord was observed under light microscope by an observer who did not understand the grouping. Change and count normal motor neurons in the anterior horn of the spinal cord. The normal neurons in the anterior horn of the spinal cord of each animal were counted as the mean of 3 slice counts.
  • YC-6 has a significant protective effect on spinal cord ischemia.
  • MCAO middle cerebral artery occlusion
  • the rats were sacrificed by decapitation, and the brains of the rats were quickly taken out, sliced, and quickly stained with TTC solution for 30 min, and then fixed with paraformaldehyde buffer. After 24 hours, take a photo with a digital camera, input the computer, and calculate the infarct area with image processing software (ADOBE, PHOTOSHOP 8. 0) (the pink area is normal brain tissue, and the white area is infarct area:). In order to correct the deviation of the infarct volume caused by cerebral edema, the infarct volume is expressed as a percentage of the normal volume of the contralateral side.
  • image processing software ADOBE, PHOTOSHOP 8. 0
  • Infarct volume percentage (contralateral normal tissue volume - volume of ipsilateral normal tissue:) I contralateral normal tissue volume x l 00%.
  • NNS neurobehavioral score
  • YC-6 ie, male ⁇ ( ⁇ ) -3 ⁇ , 5 ⁇ , 6 ⁇ -triol has protective effects on neuronal damage caused by hypoxia, cerebral ischemia and spinal cord ischemia.

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Abstract

本发明公开了5α-雄甾（烷）-3β，5，6β-三醇在制备神经元保护药物中的应用。所述化合物对脑缺血、脊髓缺血及缺氧引起的神经元损伤具有明显的保护作用，且在有效剂量下未见明显的毒副反应。

Description

说 明 书
5α-雄甾 (垸: )-3β,5,6β-三醇在制备神经元保护药物中的应用 技术领域
本发明涉及化合物 5α-雄 (垸:) -3β,5,6β-三醇 （简称 YC-6) 的新药物用途。 背景技术
急性缺血性脑卒中的治疗主要通过两个途径： 一为溶解血栓， 二为神经保 护， 后者指在急性缺血性脑卒中的治疗中， 阻止缺血引起的脑组织一系列的病 理及生化反应， 干扰缺血瀑布反应的各个环节， 延长神经元存活的药物或措施。
当前， 急性缺血性卒中的神经保护剂治疗已成为脑卒中治疗的研究热点， 许多神经保护剂目前正在临床开发试验中， 其作用机制在于通过阻断由缺血所 致各种有害病理过程的发生， 从而防止或局限缺血所引起的脑损害， 减少脑组 织死亡和促进功能恢复。 由于神经保护剂可减少脑梗死面积， 不引发出血， 无 溶栓、 抗凝治疗过程中出现的出血并发症， 用前无需进行详细的病因鉴别诊断， 使得早期治疗成为可能， 因而神经保护剂的治疗效果和前景， 是令人鼓舞的。
然而， 迄今为止还没有发现已证明为安全有效的神经保护剂， 目前正在进 行临床试验的、 具有潜在临床应用前景的药物有钙通道阻滞剂（calcium channel blocker, CCB ) 、 钙通道调节剂、 谷氨酸释放抑制剂、 γ-氨基丁酸（GABA) 受 体激动剂、 自由基清除剂、 抗细胞间黏附分子抗体等。
在众多化合物中， 神经活性 体对神经保护具有广泛效应而日益受到关注。 这些神经活性甾体水平与中枢神经系统（ central nervous system, CNS )疾病的发 生发展相关， 对神经元损伤、 死亡以及多种中枢神经系统疾病有显著调节作用。 上世纪 80年代， 科学家们开始把这些天然存在或人工合成的， 在神经组织具有 活性的甾体激素命名为神经活性甾体（neuroactive steroids, NAS )。 目前临床上 已将这些 体激素作为替代疗法运用于临床。 雌激素是已知神经保护作用最强 的 NAS之一， 绝经妇女卵巢停止产生雌激素， 可引起 β-淀粉样蛋（beta-amyloid protein, Αβ) 沉积， 导致阿尔茨海默病 (Alzheimer s disease, AD 而给予雌激 素能明显降低大脑的 Αβ水平， 临床上将雌激素用于 AD的治疗， 收到良好的效 果。 有研究表明， 别孕烯醇酮可以保护由缺氧和谷氨酸引起的体外培养海马神 经元不可逆的神经毒性损伤。 5α-雄^垸:) -3β,5,6β-三醇 （YC-6 ) 是我们在神经 甾体研究过程中发现的具有神经保护作用的化合物， 其化学结构如图如示。 据 我们的文献检索， 到目前为此， 未见 YC-6药理作用的报导， 也没有 YC-6对神 经组织具有活性或具有神经保护作用的研究报导。
Figure imgf000003_0001
5α-雄甾 (垸) -3β,5,6β-三醇的结构式
发明内容
本发明的目的在于提供 5α-雄 (垸) -3β,5,6β-三醇在制备神经元保护药物中 的应用， 从而为神经元疾病的治疗提供一种新药物。
我们的研究表明， 5α-雄甾 (垸: )-3β,5,6β-三醇 （简称 YC-6) 可明显抑制谷氨 酸诱导的小脑颗粒神经元、 皮层神经元和脊髓运动神经元的兴奋性毒性损伤， 提高神经元的存活率， 降低乳酸脱氢酶的释放， 并呈剂量依赖关系， 最低有效 浓度为 1μΜ。 YC-6可明显抑制缺氧引起的大脑皮层神经元损伤， 呈剂量依赖关 系， 最低有效浓度为 2.5μΜ。
为了证实 YC-6在体内有无神经保护作用， 我们采用 1 ) 局灶性大脑缺血模 型及 2 ) 阻断腹主动脉引起的脊髓损伤模型， 研究了 YC-6对大鼠大脑缺血及兔 脊髓缺血引起的神经元损伤的保护作用。
YC-6组在大鼠脑缺血前 30分钟经尾静脉静脉注射 lmg.K^ YC^ ,其神经 功能评分明显高于未处理的对照组， 脑梗死体积明显小于未处理的对照组。 说 明 YC-6对脑神经元损伤有明显的保护作用。
施行脊髓缺血前 30分钟预先注射 mg.Kg^YC^ ， 其神经功能评分明显高 于未处理的对照组， 且无动物出现瘫痪。 而对照组全部动物呈现瘫痪。 组织病 理学显示， YC-6组的正常脊髓前角运动神经元的数量明显多于对照组， 进一歩 说明 YC-6对脊髓神经元损伤有明显的保护作用。
综上， YC-6对脑缺血、 脊髓缺血及缺氧引起的神经元损伤有保护作用。 到 目前为此， 还没有 YC-6对神经组织具有活性或具有神经保护作用的研究报导。 附图说明
图 1为 YC-6对谷氨酸诱导的小脑颗粒神经元、脊髓运动神经元、大脑皮层 神经元兴奋性损伤的保护作用图， 其中 A为小脑颗粒神经元 ， B为脊髓运动 神经元， C为大脑皮层神经元， D为 LDH释放率， E为神经元存活率； *与 ** 分别表示小脑颗粒神经元 YC-6组与谷氨酸 (Glu)组相比存在显著性差别， *P < 0.05 , ** < 0.01 ; #与##分别表示脊髓运动神经元 YC-6组与谷氨酸 (Glu)组相比 存在显著性差别， #P < 0.05， ## < 0.01 ； $与$$分别表示大脑皮层神经元 YC-6 组与谷氨酸 (Glu)组相比存在显著性差别， ^ O.OS , ^P O.OL 图 2为 YC-6对缺氧诱导的皮层神经元损伤的保护作用图， 其中 A为相差 显微镜结果， B为神经元存活率， C为 LDH释放值； ##表示缺氧组与 control组 相比有显著性差别 ^ < 0.01 ; *与 **分别表示 YC-6组与缺氧组相比有显著性差别 *尸< 0.05， **尸< 0.01。
图 3为 YC-6对腹主动脉夹闭致兔脊髓缺血模型的神经保护作用图， 其中 A 为神经功能评分， B为组织病理切片， C为正常脊髓运动神经元记数。
图 4为 YC-6对局灶性大鼠大脑缺血模型的神经保护作用图， 其中 A为神 经功能评分， B为大脑梗死切片图， C为大脑梗死容积比较。 具体实施方式
下面通过具体实施例进一歩解释本发明， 但是本发明不仅限于实施例中。 实施例 1.神经元的培养
1.大鼠小脑颗粒神经元
取出生 7〜8d， 体重约 15〜20g大鼠的去除脑膜和血管的小脑， 用 0.25 g/L胰酶 消化后， 用 0.05 g/L DNase I 的吹散液将细胞吹散为单细胞悬液， 离心， 取沉淀， 用含 10% vA FBS 和 25 mM KCl 的 BME 培养基稀释并接种于经多聚赖氨酸预 包被的培养皿中。 接种 24 小时后加入 10 M Ara-C 以抑制非神经元细胞的生长 和增殖，使小脑颗粒神经元的纯度在 95%以上。培养时加入葡萄糖补充细胞代谢 所需能量。 第 8天进行实验。
2.大鼠脊髓运动神经元
孕 15天 SD大鼠分离脊髓，去除脊膜和血膜，取胎鼠脊髓组织用 0.125%胰酶消 化后， 离心， 取含有丰富运动神经元的中层， 离心去掉细胞碎片后， 差速贴壁 1 h， 选取贴壁速度慢的， 悬浮的脊髓运动神经元。 接种 24 h后加入阿糖胞苷， 第 3 天全量换液， 以 L-15无血清培养基继续培养， 每 2-3天半量换液。 第 3-5天进行实 验。
3.大鼠皮层神经元
取出生 Id 的大鼠的去除脑膜和血管的皮层， 用 0.25 g/L胰酶消化后， 用 0.05 g/L DNase I 的吹散液将细胞吹散为单细胞悬液，离心，取沉淀，用含 5%(v/v) FBS 和 2% B27 的 DMEM-F12 培养基稀释并接种于经多聚赖氨酸预包被的培养皿 中。 接种 24 小时后加入 lO M Ara-C 以抑制非神经元细胞的生长和增殖， 以后 每周 2-3 次半量换液。 第 10 天进行实验。
实施例 2. YC-6对原代培养神经元细胞的保护作用
1. YC-6对谷氨酸诱导的小脑颗粒神经元兴奋性损伤的保护作用
培养 8天的小脑颗粒神经元， 分别设正常对照组、 谷氨酸组、 MK801+谷氨 酸、 YC-6+谷氨酸组。 正常对照组不做任何处理， 谷氨酸组加入 200μΜ谷氨酸， 其它组预先加入 MK801 ( ΙΟμΜ)和不同浓度的 YC-6, 37°C孵育 30分钟后加入谷 氨酸， 24 h后用相差显微镜观察神经细胞形态； 用 FDA染色， 倒置荧光显微镜 观察， 进行细胞计数， 计算神经元的存活率。 并测定各组乳酸脱氢酶的活性。
存活率 =各组活细胞数 /对照组活细胞数 X 100%。
结果发现， YC-6+谷氨酸组和 MK801+谷氨酸组大部分小脑颗粒神经元能保 持胞体和突起的完整， 细胞存活率增加， LDH释放值降低， 与谷氨酸组比较呈 现明显差异，且 YC-6存在浓度依赖关系，见图 1-A,D,E。而在上述剂量范围 YC-6 对正常神经细胞的存活率无影响。
2. YC-6对谷氨酸诱导的脊髓运动神经元兴奋性损伤的保护作用 培养 5天的脊髓运动神经元， 分别设正常对照组、 谷氨酸组、 MK801+谷氨 酸、 YC-6+谷氨酸组。 正常对照组不做任何处理， 谷氨酸组加入 200 μΜ谷氨酸， 其它组预先加入 MK801 ( ΙΟμΜ)和不同浓度的 YC-6, 37°C孵育 30分钟后再加入 谷氨酸， 24 h后用相差显微镜神经细胞形态， 用 FDA染色， 倒置荧光显微镜进 行细胞计数， 计算神经元的存活率。 并测定各组乳酸脱氢酶的活性。
存活率 =各组活细胞数 /对照组活细胞数 X 100%。
结果发现， 倒置相差显微镜下观察， 对照组存活的脊髓运动神经元数目多， 胞体丰满， 呈三角形、 多角形、 立体感强， 有光暈可见分枝和突起； 谷氨酸组 存活脊髓运动神经元数少， 但有突起形成， 细胞严重受损； 而 YC-6+谷氨酸和 MK801+谷氨酸组脊髓运动神经元数目明显增多， 并有很多突起， 但仍有少数死 亡细胞。 与对照组比， 其余三组的脊髓运动神经元存活率均有不同程度下降， 与谷氨酸组相比， YC-6+谷氨酸组存活率有明显提高， 且与 YC-6存在浓度依 赖关系， 见图 1-B,D,E。 而在上述剂量范围 YC-6对正常神经细胞的存活率无影 响。
3. YC-6对谷氨酸诱导的皮层神经元兴奋性损伤的保护作用
培养 10天的皮层神经元， 分别设正常对照组、 谷氨酸组、 MK801+谷氨酸、 YC-6+谷氨酸组。 正常对照组不做任何处理， 谷氨酸组加入 200 μΜ谷氨酸， 其 它组预先加入溶剂、 MK801 ( ΙΟμΜ)和不同浓度的 YC-6, 37°C孵育 30分钟后加 入谷氨酸， 24 h后用相差显微镜神经细胞形态， 用 FDA染色， 倒置荧光显微镜 观察， 进行细胞计数， 计算神经元的存活率。 并测定各组乳酸脱氢酶的活性。
存活率 =各组活细胞数 /对照组活细胞数 X 100%。
结果发现， YC-6+谷氨酸组和 MK801+谷氨酸组大部分皮层神经元能保持胞 体和突起的完整， 细胞存活率增加， LDH释放值降低， 与谷氨酸组比较呈现明 显差异，且与 YC-6存在浓度依赖关系， 见图 1-C,D,E。而在上述剂量范围 YC-6 对正常神经细胞的存活率无影响。
4. YC-6对缺氧诱导的皮层神经元兴奋性损伤的保护作用
培养到 10天的原代皮层神经元正常对照组、缺氧组、 MK801+缺氧组、 YC-6+ 缺氧组。 每组设置副孔 3 个， 正常对照组置于二氧化碳箱正常孵育， 缺氧组置 于缺氧工作站 (；氧气浓度 1%)。 MK801+缺氧组、 YC-6+缺氧组则在置入缺氧工作 站 (氧气浓度 1%)前 30 min用 MK801 ( ΙΟμΜ) 和不同浓度的 YC-6预处理。 12 小时后于相差显微镜下观察并拍照。
在 96孔板中如上分组以及处理， 然后加入 MTT储存液 200 μ1， 作用 4 h， 存活的细胞中形成紫蓝色结晶。 去除各孔中的液体， 加入 DMSO 150 μΐ, 溶解 紫蓝色结晶。 半小时后， 待结晶完全溶解， 用酶联仪 570 nm波长测定 OD值。 损伤组与对照组各时间点留取培养液 50 依试剂盒说明书检测各孔培养液 LDH含量。 各组数据以 mean ± SD表示， 采用多个样本均数间方差分析及两两 比较的 t检验检验进行统计学分析。 参考文献 [1]和 [2]。 [1] Brewer G.J. Isolation and culture of aldut rat hippocampal neurons. J. Neurosci. Meth. 1997, 71 : 143-155.
[2] Lee M.M., Hseih M.T. Magnolol protects cortical neuronal cells from chemical hypoxia in rats. Neuroreport 1998, 9: 3451-3456.
结果表明， 大鼠皮层神经元培养 10天后， 细胞呈锥形或多极形， 胞体清晰 明亮， 边界清楚， 有很强的折光性， 神经元突起相互交织成网， 细胞核明显。 缺氧处理的皮层神经元完整性受到破坏， 折光性降低， 突触断裂、 消失， 胞质 颗粒性变， 部分胞体肿胀甚至消失。 MK801预处理组与 YC-6预处理组与正常 对照组相比， 皮层神经元基本无形态学差异。神经元保护作用与 YC-6呈浓度依 赖关系（图 2 A)。 MTT法结果表明， 缺氧处理明显减低了神经元的存活率 P < 0.05 )， 而 YC-6呈浓度依赖性提高高了缺氧损害造成的神经元存活率（图 2 B)。 LDH释放结果与 MTT法相符， YC-6预处理组浓度依赖性地降低了缺氧造成的 神经元损伤 (图 2-C， < 0.05
实施例 3. YC-6对腹主动脉夹闭致兔脊髓缺血模型的神经保护作用
40只雄性新西兰大白兔分为 4组（η = 10 ) ： Control组制作兔脊髓缺血模型； YC-6组在脊髓缺血前 30分钟经耳缘静脉注射 2mg.Kg_ E H本 YC-6; Vehicle组在脊 髓缺血前 30 min 同样方式注射等容量羟丙基环糊精（lml.Kg—1 ) ; Sham组仅仅暴 露腹主动脉， 不实施阻断。
兔脊髓缺血模型制作参照文献报道 [3,4] 和我们先前的研究 [5]。 [3] Celik M.， et al. Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proc Natl Acad Sci U S A, 2002, 99: 2258-2263. [4] Johnson SH, Kraimer J.M., Graeber G.M. Effects of flunarizine on neurological recovery and spinal cord blood flow in experimental spinal cord ischemia in rabbits. Stroke, 1993, 24: 1547-1553. [5] Sang H.， Cao L.， Qiu P., Xiong L., Wang R.， Yan G. Isoflurane produces delayed preconditioning against spinal cord ischemic injury via release of free radicals in rabbits. Anesthesiology, 2006,105: 953-960.
缺血前即刻， 缺血后 lOmin及再灌注后 20 min测定组间各生理学参数。 用 Talov评分法 [5]进行各组兔的功能评分： 0分， 后肢完全瘫痪； 1分， 可以觉察 的后肢关节运动； 2分， 后肢可以自由运动但无法站立； 3分， 可以站立但无法 行走； 4分， 后肢运动功能完全恢复， 可以正常行走。
神经功能评分完毕后， 在麻醉状态下取腰段脊髓组织（L5-L7)， 行石腊包埋 后切片， HE染色， 光镜下由一不了解分组情况的观察者观察脊髓病理学改变并 对脊髓前角正常运动神经元计数。 每只动物脊髓前角正常神经元计数为 3 张切 片计数的均值。
结果表明， 缺血前即刻， 缺血后 10 min及再灌注后 20 min测定各组间生理 学参数无明显差异 （Ρ>0.05 ) 。 各组动物神经功能评分结果详见图 3-Α。 假手术 组兔的后肢神经功能在整个观察期间完全正常 （4分）； 而 Control和 Vehicle组中 没有一只兔能够站立； YC-6组中有 7只兔能够站立 (大于或等于 3分：)。 YC-6组和 Sham组的神经功能评分均明显高于 Control和 Vehicle组 （PO.OS
在 Control和 Vehicle组， 腰段脊髓有严重的损伤， 表现为正常运动神经元基 本消失、 大量空泡变性； 而在 YC-6组， 腰段脊髓损伤明显减轻， 可见正常的运 动神经元， 图 3-B。 YC-6组和 Sham组的脊髓前角正常神经细胞数明显增多 <0.05)o 见图 3-C。
综上， YC-6对脊髓缺血具明显的保护作用。
实施例 4. YC-6对大鼠局灶性脑缺血模型 （MCAO) 的神经保护作用
30只雄性 SD大鼠随机均分为 3组（n=10): Control组制作大鼠局灶性脑缺 血模型； YC-6组在脑缺血前 30分钟经尾静脉静脉注射 lmg.Kg^YC ; Vehicle 组在脊髓缺血前 30钟同样方式注射等容量的羟丙基环糊精 Qml.Kg^
术前禁食 12 h， 自由饮水。 采用线栓法制作大脑中动脉阻闭 （MCAO)模型 [6]。
[6] Wang Q., Peng Y" Chen S., Gou X., Hu B.， Du J., Lu Y.， Xiong L. Pretreatment with electroacupuncture induces rapid tolerance to focal cerebral ischemia through regulation of endocannabinoid system. Stroke, 2009, 40(6): 2157-2164. 阻闭 120min 后抽出尼龙线， 恢复再灌注。 激光多普勒血流仪监测局部脑血流。 术毕麻醉苏 醒后， 将动物放回鼠笼， 自由饮食。 脑缺血再灌注后 72 h， 由一不了解分组情 况的观察者根据 Longa评分法 [7]。 [7] Longa E.Z., Weinstein P.R., Carlson S., Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989, 20(1): 84-91.评估并记录神经功能评分： 0级， 无功能障碍； 1级， 不能伸展左侧前肢； 2级， 向左侧旋转； 3级， 向左侧倾倒； 4级， 无自主活动 伴意识抑制； 5级， 死亡。
神经功能评分完成后， 断头处死大鼠， 迅速取出鼠脑， 切片后迅速用 TTC 溶液染色 30 min， 然后用多聚甲醛缓冲液固定。 24 h后用数码相机拍照， 输入 计算机， 用图像处理软件 (ADOBE, PHOTOSHOP 8. 0)计算梗死面积 (粉红色区为 正常脑组织,白色区为梗死区：)。 为校正脑水肿带来梗死容积的偏差， 用占对侧 正常容积的百分比表示梗死容积。
梗死容积百分比= (对侧正常组织容积 -同侧正常组织的容积：） I对侧正常组 织容积 x l 00%。
神经行为学评分 （NBS ) 采用非参 （Kruskal-Wallis ) 检验， 若存在组间差 异， 以 Mann- Whitney U检验 Bonferroni校正进行两两比较。 梗死容积和生理学 参数采用均数 ±标准差 （mean±SD) 表示， 采用单因素方差分析 （ANOVA) , 多 组间两两比较采用 SNK (post hoc Student-Newman-Keuls ) 检验。 pO.OS表示 有显著的统计学差异。
各组动物神经功能评分结果详见图 5。与 Control和 Vehicle组相比， YC-6组神 经功能明显改善， 脑梗死容积明显减少 P O.OS 综上， YC-6, 即雄甾 (垸) -3β,5α,6β-三醇对低氧、 脑缺血、 脊髓缺血引起的 神经元损伤有保护作用。

Claims

权 利 要 求 书
1.5α-雄 (垸： )-3β,5,6β-三醇在制备神经元保护药物中的应用。
2.根据权利要求 1 所述的应用， 其特征在于所述神经元保护; ί为 治疗脑缺血;
；.根据权利要求 1 所述的应用， 其特征在于所述神经元保护药物为 治疗脊髓缺血药物。
4.根据权利要求 1 所述用途， 其特征在于所述神经元保护药物为治 疗由于缺氧引起的神经元损伤药物。
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Cited By (5)

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JP2015511587A (ja) * 2012-03-08 2015-04-20 グァンチョウ セルプロテック ファーマシューティカル カンパニー リミテッド アンドロスタン−３β，５α，６β−トリオールの結晶形化合物及びその製造方法
JP2015514731A (ja) * 2012-04-19 2015-05-21 ユニベルシテ・ド・リエージュＵｎｉｖｅｒｓｉｔｅ Ｄｅ Ｌｉｅｇｅ 神経障害の処置における使用のためのエストロゲン成分
US9320743B2 (en) 2010-07-09 2016-04-26 Guangzhou Cellprotek Pharmaceutical Ltd. Use of 5α-androstane-3β,5,6β-triol in preparation of neuroprotective drugs
JP2016514729A (ja) * 2013-03-28 2016-05-23 グァンチョウ セルプロテック ファーマシューティカル カンパニー リミテッド ２β，３α，５α−トリヒドロキシ−アンドロスタ−６−オン、その製造方法及びその用途
RU2672264C2 (ru) * 2014-04-25 2018-11-13 Гуанчжоу Селпротек Фармасьютикал Ко., Лтд. Нейропротективные агенты и их применение

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101961311B (zh) * 2010-09-21 2012-11-21 中山大学 一种5α-雄甾(烷)-3β,5,6β-三醇注射剂及其制备方法
CN109985049B (zh) 2017-12-29 2022-02-22 广州市赛普特医药科技股份有限公司 5α-雄甾-3β,5,6β-三醇在制备治疗脑小血管病的药物中的应用
CN109985047B (zh) * 2017-12-29 2021-07-27 广州市赛普特医药科技股份有限公司 5α-雄甾-3β,5,6β-三醇在制备治疗出血性脑卒中药物中的应用
US11516173B1 (en) * 2018-12-26 2022-11-29 Snap Inc. Message composition interface

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008155534A2 (en) * 2007-06-20 2008-12-24 Bionature E.A. Ltd. Neurosteroid compounds
CN101884638A (zh) * 2010-07-09 2010-11-17 中山大学 5α-雄甾(烷)-3β,5,6β-三醇在制备神经元保护药物中的应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2191576A (en) * 1936-11-21 1940-02-27 Soc Of Chemical Ind 3,5,6-trihydroxy androstane and pregnane compounds
US6046185A (en) * 1996-07-11 2000-04-04 Inflazyme Pharmaceuticals Ltd. 6,7-oxygenated steroids and uses related thereto
US20030060425A1 (en) * 1998-11-24 2003-03-27 Ahlem Clarence N. Immune modulation method using steroid compounds
US20070099880A1 (en) * 2003-11-24 2007-05-03 Blizzard Timothy A Estrogen receptor modulators
CN1557829A (zh) * 2004-01-19 2004-12-29 复旦大学 化合物5α-雄甾烷-1α,3β,17β-三醇17-醋酸酯和衍生物,及其制备方法
US8354396B2 (en) * 2006-11-17 2013-01-15 Harbor Therapeutics, Inc. Drug identification and treatment method
CN101683348B (zh) * 2008-09-23 2012-07-04 中山大学 胆甾烷-3β,5α,6β-三醇在制备神经元保护药物中的应用
CN101961311B (zh) * 2010-09-21 2012-11-21 中山大学 一种5α-雄甾(烷)-3β,5,6β-三醇注射剂及其制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008155534A2 (en) * 2007-06-20 2008-12-24 Bionature E.A. Ltd. Neurosteroid compounds
CN101884638A (zh) * 2010-07-09 2010-11-17 中山大学 5α-雄甾(烷)-3β,5,6β-三醇在制备神经元保护药物中的应用

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BREWER G.J.: "Isolation and culture of aldut rat hippocampal neurons", J. NEUROSCI. METH., vol. 71, 1997, pages 143 - 155
CELIK M. ET AL.: "Erythropoietin prevents motor neuron apopotosis and neurologic disability in experimental spinal cord ischemic injury", PROC NATL ACAD SCI USA, vol. 99, 2002, pages 2258 - 2263
CHEN, JINGBO ET AL.: "Studies on the Hemisuccination of Hydroxysterols", CHINESE JOURNAL OF SYNTHETIC CHEMISTRY, vol. 8, no. 5, 2000, pages 466 - 468 *
JOHNSON SH; KRAIMER J.M.; GRAEBER G.M.: "Effects of flunarizine on neurological recovery and spinal cord blood flow in experimental spinal cord ischemia in rabbits", STROKE, vol. 24, 1993, pages 1547 - 1553
LEE M.M.; HSEIH M.T.: "Magnolol protects cortical neuronal cells from chemical hypoxia in rats", NEUROREPORT, vol. 9, 1998, pages 3451 - 3456
LI, QUN ET AL.: "Pregnane-3p,5a,6p-triol for Inhibiting Hypopotassemia Induced Apoptosis of Cerebellar Granule Neurons", CHINESE JOURNAL OF PHARMACOLOGY AND TOXICOLOGY, vol. 15, no. 5, October 2001 (2001-10-01), pages 337 - 341 *
LONGA E.Z.; WEINSTEIN P.R.; CARLSON S.; CUMMINS R.: "Reversible middle cerebral artery occlusion without craniectomy in rats", STROKE, vol. 20, no. 1, 1989, pages 84 - 91
SANG H.; CAO L.; QIU P.; XIONG L.; WANG R.; YAN G.: "Isoflurane produces delayed preconditioning against spinal cord ischemic injury via release of free radicals in rabbits", ANESTHESIOLOGY, vol. 105, 2006, pages 953 - 960
See also references of EP2591785A4 *
WANG Q.; PENG Y.; CHEN S.; GOU X.; HU B.; DU J.; LU Y.; XIONG L.: "Pretreatment with electroacupuncture induces rapid tolerance to focal cerebral ischemia through regulation of endocannabinoid system", STROKE, vol. 40, no. 6, 2009, pages 2157 - 2164

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* Cited by examiner, † Cited by third party
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US9320743B2 (en) 2010-07-09 2016-04-26 Guangzhou Cellprotek Pharmaceutical Ltd. Use of 5α-androstane-3β,5,6β-triol in preparation of neuroprotective drugs
EP3112374A1 (en) * 2012-03-08 2017-01-04 Guangzhou Celprotek Pharmaceutical Co. Ltd. Crystalline forms of 5alpha-androstane-3beta,5,6beta-triol and preparation methods therefor
EP2889303A4 (en) * 2012-03-08 2016-02-17 Guangzhou Celprotek Pharmaceutical Co Ltd CONNECTION OF A CRYSTAL FORM OF ANDROSTA-3B, 5A, 6B-TRIOL AND METHOD FOR THE PRODUCTION THEREOF
JP2015511587A (ja) * 2012-03-08 2015-04-20 グァンチョウ セルプロテック ファーマシューティカル カンパニー リミテッド アンドロスタン−３β，５α，６β−トリオールの結晶形化合物及びその製造方法
RU2608894C2 (ru) * 2012-03-08 2017-01-26 Гуанчжоу Селлпротек Фармасьютикал Ко., Лтд. КРИСТАЛЛИЧЕСКИЕ ФОРМЫ 5α-АНДРОСТАН-3β,5,6β-ТРИОЛА И СПОСОБЫ ИХ ПОЛУЧЕНИЯ
US9688716B2 (en) 2012-03-08 2017-06-27 Guangzhou Cellprotek Pharmaceutical Co., Ltd. Crystalline forms of 5α-androstane-3β,5,6β-triol and preparation methods therefor
US9944673B2 (en) 2012-03-08 2018-04-17 Guangzhou Cellprotek Pharmaceutical Co., Ltd. Crystalline forms of 5α-androstane-3β,5,6β-triol and preparation methods therefor
US9944672B2 (en) 2012-03-08 2018-04-17 Guangzhou Cellprotek Pharmaceutical Co., Ltd. Crystalline forms of 5α-androstane-3β,5,6β-triol and preparation methods therefor
JP2015514731A (ja) * 2012-04-19 2015-05-21 ユニベルシテ・ド・リエージュＵｎｉｖｅｒｓｉｔｅ Ｄｅ Ｌｉｅｇｅ 神経障害の処置における使用のためのエストロゲン成分
JP2016514729A (ja) * 2013-03-28 2016-05-23 グァンチョウ セルプロテック ファーマシューティカル カンパニー リミテッド ２β，３α，５α−トリヒドロキシ−アンドロスタ−６−オン、その製造方法及びその用途
EP2980096A4 (en) * 2013-03-28 2016-11-16 Guangzhou Cellprotek Pharm Co Ltd 2,3,5-trihydroxy-androst-6-one, as well as method of preparation and use thereof
KR101771192B1 (ko) 2013-03-28 2017-08-24 광저우 셀프로텍 파마슈티컬 컴퍼니 리미티드 2β,3α,5α-트리하이드록시-안드로스트-6-원 및 이의 제조 방법 및 이의 용도
RU2672264C2 (ru) * 2014-04-25 2018-11-13 Гуанчжоу Селпротек Фармасьютикал Ко., Лтд. Нейропротективные агенты и их применение
US10357500B2 (en) 2014-04-25 2019-07-23 Guangzhou Cellprotek Pharmaceutical Co., Ltd. Neuro-protective agents and uses thereof

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RS55845B1 (sr) 2017-08-31
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SI2591785T1 (sl) 2017-07-31
SG186135A1 (en) 2013-01-30
CN101884638A (zh) 2010-11-17
KR101463477B1 (ko) 2014-11-19
RU2541093C2 (ru) 2015-02-10
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US20140329790A1 (en) 2014-11-06
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EP2591785A4 (en) 2013-12-11
US8809309B2 (en) 2014-08-19
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US20130157993A1 (en) 2013-06-20
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