CN103054866A - Application of chloroxine in preparing anti-angiogenesis medicine - Google Patents

Application of chloroxine in preparing anti-angiogenesis medicine Download PDF

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CN103054866A
CN103054866A CN2013100314447A CN201310031444A CN103054866A CN 103054866 A CN103054866 A CN 103054866A CN 2013100314447 A CN2013100314447 A CN 2013100314447A CN 201310031444 A CN201310031444 A CN 201310031444A CN 103054866 A CN103054866 A CN 103054866A
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chloro
angiogenesis
zebrafish
oxine
anti
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CN2013100314447A
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李春启
朱晓宇
朱凤
刘洪翠
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杭州雷索药业有限公司
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Abstract

The invention relates to an application of chloroxine in preparing anti-angiogenesis medicine. Until now no relevant reports about the anti-angiogenesis activity of the chloroxine are available. The invention provides the application of chloroxine in preparing anti-angiogenesis medicines, anti-tumor medicines and medicines for preventing wet age-related macular degeneration. The in-vivo pharmacodynamic experiment using a zebra fish angiogenesis model shows that the chloroxine can remarkably suppress zebra fish angiogenesis, remarkably suppress the growth of transplanted human tumor cells and has a therapeutic effect to wet age-related macular degeneration. Therefore, the chloroxine can be used for preparing anti-angiogenesis medicines, anti-tumor medicines and medicines for preventing wet age-related macular degeneration.

Description

氯喔星在制备抗血管生成类药物中的应用技术领域 TECHNICAL FIELD chloro oxine in the manufacture of anti-angiogenic drugs in

[0001] 本发明涉及医药技术领域,具体地说是氯喔星在制备抗血管生成类药物中的应用。 [0001] The present invention relates to the technical field of medicine, particularly use in the manufacture of chlorine oxinoid antiangiogenic drugs in.

背景技术 Background technique

[0002] 血管生成(angiogenesis)与人类多种重大疾病高度相关,如恶性肿瘤,老年视黄斑变性(Age-related macular degeneration, AMD),动脉粥样硬化(Atherosclerosis),风湿性关节炎(Rheumatoid arthritis),糖尿病视网膜变性(Diabetic retinopathy)以及肿瘤转移(Tumor metastasis)等[1]。 [0002] angiogenesis (angiogenesis) is highly correlated with a variety of major human diseases, such as cancer, age-related macular degeneration view (Age-related macular degeneration, AMD), atherosclerosis (Atherosclerosis), rheumatoid arthritis (Rheumatoid arthritis ), diabetic retinal degeneration (diabetic retinopathy) and tumor metastasis (tumor metastasis) [1]. 随着中国人口老龄化逐渐加剧,目前这些重大疾病已严重危害我国人民群众的生命健康。 With China's aging population gradually increased and currently these major diseases has seriously endanger life and health of the people of our country.

[0003] 1971年,哈佛大学Judah Folkman教授首次提出抗肿瘤血管疗法,他认为实体肿瘤的生长和扩散依赖于肿瘤内新血管的形成,并通过新生血管获取养分;新血管的形成和生长,促进了肿瘤细胞的转移[2_4]。 [0003] In 1971, Harvard University Professor Judah Folkman first proposed anti-tumor angiogenesis therapy, he believes that the growth and spread of solid tumors depends on the formation of tumor new blood vessels, and obtain nutrients by neovascularization; formation and growth of new blood vessels, promote the metastasis of tumor cells [2_4]. 经过40年大量的基础理论研究,目前基于这一疗法,已有多个重磅专利新药上市,如罗氏的Bevacizumab (商品名Avastin, 2010年全球销售额67亿美元),拜耳的Sorafenib (商品名Nexavar, 2010年全球销售额9. 94亿美元),辉瑞的Sunitinib (商品名Sutent, 2010年全球销售额10. 7亿美元)[卜6]。 After a lot of fundamental research for 40 years, currently based on this therapy, there are a number of heavy patent new drugs, such as Roche's Bevacizumab (trade name Avastin, 2010 global sales of $ 6.7 billion), Bayer's Sorafenib (trade name Nexavar, 2010 global sales of US $ 994 million), Pfizer Sunitinib (trade name Sutent, 2010 global sales of US $ 1.07 billion) [BU 6]. 但这些药物价格十分昂贵,均为外企医药巨头垄断。 However, these drugs are very expensive, both foreign pharmaceutical giant monopoly. 国内目前只有I支真正意义的抗肿瘤血管生成药物通过SFDA批准上市(恩度,先声药业,2005年上市)m,但年销售额很少(2010年销售额仅 China currently only true sense I support the generation of anti-tumor angiogenesis drugs approved for marketing by the SFDA (Endostar, Simcere, listed in 2005) m, but few sales (sales in 2010 only

2. 5亿人民币),尚无法参与全球竞争。 250 million yuan), still unable to compete globally. 其次,由于恩度(重组人血管内皮抑制素注射液,Recombinant Human Endostatin Injection)为大分子蛋白质类药物,生产这类高纯度安全制剂需要很高的技术门槛,而且此类型的药物在体内的半衰期较短,从而限制了这些活性蛋白的临床应用[7]。 Second, since Endostar (recombinant human endostatin injection, Recombinant Human Endostatin Injection) is a macromolecular protein drugs, the production of such high purity safety formulations require high technical threshold, and this type of drug half-life in vivo short, thereby limiting the clinical application of these active proteins [7].

[0004]老年视黄斑变性(Age-re`lated macular degeneration,简称AMD)是一种累及眼底视网膜黄斑区的变性眼疾。 [0004] Depending senile macular degeneration (Age-re`lated macular degeneration, referred AMD) is a degenerative eye disease involving the macular area of ​​retina. 它因年龄增长而产生黄斑区变性,可引起中心视力急剧下降。 It is due to age-related macular degeneration produce area, can cause a sharp decline in central vision. 据统计,全世界超过3000万人罹患此病。 According to statistics, more than 3,000 million people worldwide suffer from the disease. 黄斑变性分为干性和湿性两种。 Macular degeneration is divided into dry and wet two kinds. 湿性老年视黄斑变性主要因为脉络膜血管异常生成,新生的无效微血管出现渗漏,血管渗漏的液体进而破坏黄斑,导致中心视力显著下降,影响生活质量,甚至变盲。 Wet age-related macular degeneration depends mainly due to choroidal vascular abnormalities generated, newborn invalid microvascular leakage in liquid vascular leakage and thus damage the macula, resulting in central vision decreased significantly affect the quality of life, and even become blind. 湿性老年视黄斑变性(AMD)已经成为65岁以上老年人失明的罪魁祸首[8_9]。 Depending wet age-related macular degeneration (AMD) has become the blindness in the elderly over 65 years of culprit [8_9].

[0005] 治疗湿性AMD的方法主要有光动力疗法和抗血管药物疗法I9]。 [0005] The method of treatment of wet AMD is the main light photodynamic therapy and antiangiogenic drug therapy I9]. 光动力疗法主要通过静脉注入光敏药物,继而采用特定波长的非热能激光照射脉络膜新生血管病灶,将光敏药物活化。 Photodynamic therapy is mainly photosensitive drug by intravenous injection, followed by non-thermal laser irradiation of choroidal neovascularization lesions of a specific wavelength, the photosensitive drug activation. 用光动力疗法治疗湿性AMD,只能稳定或降低湿性AMD视力下降的风险,并非对因治疗,不能阻止复发的可能。 Photodynamic therapy for wet AMD, can only stabilize or reduce the risk of vision loss of wet AMD, not due to treatment, not prevent possible recurrence. 一般需要多次治疗。 Generally require multiple treatments. 而且治疗后要避光48小时,以免发生光敏反应,造成皮肤灼伤,因此,给患者带来很多痛苦。 And after dark treatment for 48 hours in order to avoid photosensitive reactions, causing skin burns, therefore, bring patients a lot of pain. 目前治疗湿性AMD,已上市的药物主要有:辉瑞的哌加他尼钠(Pegaptanib,商品名Macugen),诺华的兰尼单抗(Ranibizumab,商品名Lucentis),拜耳的艾力亚(VEGF_Trap_eye,商品名Eylea),这些药物的价格非常昂贵,一般需要每月经玻璃体内注射给药,这种冗长的给药过程很难被患者接受。 Currently the treatment of wet AMD, marketed drugs are: Pfizer pegaptanib sodium (Pegaptanib, trade name Macugen), Novartis ranibizumab (Ranibizumab, trade name Lucentis), Bayer's Levitra Asia (VEGF_Trap_eye, commodity name Eylea), the price of these drugs are very expensive, typically monthly intravitreal injections required by this process is difficult to be lengthy patient receiving administration. 因此,开发新型廉价的滴眼液来治疗老年黄斑变性是未来的发展趋势。 Therefore, the development of new low-cost eye drops to treat age-related macular degeneration is the future trend of development.

[0006] 综上所述,在小分子化合物中寻求新的血管生成抑制剂来有效治疗上述疾病已成为研发热点,开发具有自主知识产权的专利靶向抗血管生成小分子药物迫在眉睫。 [0006] In summary, the search for new inhibitors of angiogenesis effective treatment of these diseases have become hot topics in small molecule compounds, the development of proprietary patented anti-angiogenic targeting small molecule drug is imminent.

[0007] 氯喔星,英文通用名=Chloroxine ;中文名称:氯喔星;化学名称:5,7_ 二氯_8_轻基喹啉;商品名称=Capitrol ;分子量:214. 05。 [0007] oxine chloro, English common name = Chloroxine; Chinese name: oxine-chloro; Chemical Name: 5,7_-dichloro _8_ light quinoline; trade name = Capitrol; molecular weight: 21,405. 氯喔星是一种用于治疗头皮屑和脂溢性皮炎的抗菌药[1°]。 Chloro oxine is a method for the treatment of dandruff and seborrheic dermatitis antibacterials [1 °]. 氯喔星用于治疗头皮屑的机制还不清楚,通常被认为是减慢头皮屑弓I 起的有丝分裂活性增强的结果(http://www.druglib.com /drug info/ capitrol/description pharmacology/)。 Mechanism of chlorine used to treat dandruff oxine is not clear, generally considered to be slowed down since I bow dandruff mitotic activity enhancement results (http://www.druglib.com / drug info / capitrol / description pharmacology / ). 氯喔星的化学结构式如下: Chlorine oxine the following chemical structure:

Figure CN103054866AD00041

尚未见有关氯喔星抗血管生成(ant1-angiogenesis)活性的相关报道。 It has not been related to chlorine oxine anti-angiogenic (ant1-angiogenesis) activity reports.

[0008] [0008]

发明内容 SUMMARY

[0009] 本申请的发明人意外发现,氯喔星具有抗血管生成的作用。 [0009] The present inventors have surprisingly found that application, chloro oxine having anti-angiogenic effect.

[0010] 因此,本发明提供氯喔星在制备抗血管生成类药物中的应用。 [0010] Accordingly, the present invention provides chloro dioxines in the preparation of anti-angiogenic drugs in.

[0011 ] 本发明还提供氯喔星在制备抗肿瘤药物中的应用,所述的氯喔星通过抑制肿瘤内新血管的生成来预防或治疗肿瘤。 [0011] The present invention further provides use in the manufacture of chlorine oxinoid antineoplastic, a chlorine oxinoid by suppressing the formation of new blood vessels within the tumor to prevent or treat cancer.

[0012] 本发明还提供氯喔星在制备抗湿性老年视黄斑变性药物中的应用,所述的氯喔星通过抑制脉络膜血管异常增生来预防或治疗湿性老年视黄斑变性。 [0012] The present invention further provides the preparation of anti-chloro-oxine view wet age-related macular degeneration medicament application, the chlorine oxinoid choroidal neovascularization by inhibiting the abnormal proliferation of prevention or treatment of wet age-related macular degeneration view.

[0013] 上述药物为口服给药剂型、注射给药剂型、粘膜给药剂型或经皮给药剂型,更具体讲,如片剂、胶囊剂、颗粒剂、口服液、注射液、贴剂或凝胶剂形式。 [0013] The pharmaceutical oral dosage forms, injection forms, transmucosal administration or transdermal dosage form, and more specifically, such as tablets, capsules, granules, oral liquid, injection, or patch gel forms. 可以通过本领域已知的方法制备药物制剂。 Pharmaceutical formulations can be prepared by methods known in the art.

[0014] 本发明利用斑马鱼血管生成模型进行氯喔星抗血管药效学实验。 [0014] The present invention chloro-oxine using the pharmacodynamics of an anti-angiogenic zebrafish model of angiogenesis. 和传统的血管研究模型(啮齿类的老鼠和鸡胚尿囊模)相比,目前大量的研究证实,斑马鱼是最理想的血管生物学以及抗血管生成药物评价模型[11_2°]。 Model traditional vessels (mice and rodents chorioallantoic die) compared to the current number of studies confirmed that zebrafish vascular biology and is the best anti-angiogenic drugs evaluation model [11_2 °]. 啮齿类的老鼠和鸡胚尿囊模存在各自的缺点[14_15]。 Rodents and mice die chorioallantoic their shortcomings [14_15]. 通过利用斑马鱼血管生成模型进行药效学评价和药物新靶点验证,已有多支抗癌药物进入临床前实验(Pre-clinical Trial)或者临床试验(Clinical Trial)阶段(包括已获得FDA 批准上市的药物),如Vatalanib (Novartis) [16]>Compound 6 (TargeGen) [17]、Rosuvastatin[18]>Solenopsin (Eli Lilly) [19]等;主要基于斑马鱼抗血管生成模型发现的一种治疗恶性肿瘤的老药新用药物——反应停(Thalidomide)已获FDA批准上市[2°]。 By and pharmacodynamic evaluation of new drug target validation using zebrafish model of angiogenesis, existing anticancer drugs into the multi-branching preclinical (Pre-clinical Trial) or clinical trial (Clinical Trial) phase (including FDA-approved listed drugs), such as Vatalanib (Novartis) [16]> Compound 6 (TargeGen) [17], Rosuvastatin [18]> Solenopsin (Eli Lilly) [19]; ​​major anti-angiogenic based zebrafish model found a treatment of malignant tumors of old drugs with new drugs - thalidomide (thalidomide) has been approved by the FDA to market [2 °].

[0015] 经斑马鱼体内血管模型证实,氯喔星对斑马鱼肠下静脉血管(Subintestinalvein, SIV)有显著抑制的功能,并呈现一定的剂量依耐性,因此,可用于制备抗血管生成抑制剂。 [0015] zebrafish vessel model was confirmed, oxine-chloro significant inhibition of the function of intestine zebra vein (Subintestinalvein, SIV), and in a dose-depending resistance, it is useful for the preparation of an anti-angiogenesis inhibitor .

[0016] 国内外在斑马鱼作为癌症移植模型和湿性视黄斑变性模型方面也有大量研究[21_26]。 [0016] at home and abroad in terms of zebrafish as a model for cancer and transplantation, as the wet macular degeneration model also has a large number of studies [21_26]. 经斑马鱼人类胃癌(SGC-7901)移植模型证实,氯喔星醇能够显著抑制胃癌(SGC-7901)的生长;经斑马鱼视黄斑变性模型证实,氯喔星醇对湿性老年视黄斑变性具有显著的治疗效果。 By zebrafish human gastric cancer (SGC-7901) Transplantation model validation, chloro oxinoid alcohol can be significantly inhibited the growth of gastric cancer (SGC-7901); and confirmed by zebrafish view macular degeneration model, chloro oxinoid alcohol having wet age-view-related macular degeneration significant therapeutic effect. 因此,氯喔星醇可用于抗肿瘤及治疗湿性老年视黄斑变性。 Thus, oxine-chloro alcohols useful antitumor therapy and wet age-related macular degeneration view.

[0017] 本发明氯喔星安全性高、原料来源广泛,辅以药学上可接受的辅料,采用常规制剂技术即可制成各种口服、注射、外用制剂,具有良好的开发前景。 [0017] The present invention oxine-chloro-safe, widely available raw materials, combined with pharmaceutically acceptable excipients, using conventional formulation techniques can be made into various oral, injection, external preparation, with good prospects for development.

附图说明 BRIEF DESCRIPTION

[0018] 图1为本发明受精后48h(48hpf)血管转基因荧光斑马鱼体节间血管(ISV)模型。 [0018] Figure 1 is a fertilized invention 48h (48hpf) vascular fluorescence transgenic zebrafish section between vessels (ISV) model. 框定区域为斑马鱼体节血管网络局部放大观察的部位。 Zebrafish framed area enlarged partial section of the vascular network observation site. 箭头指示体节间血管(ISV,intersegmental vessel)。 Arrows indicate blood vessels between the body segments (ISV, intersegmental vessel).

[0019] 图2为本发明定性观察氯喔星对斑马鱼体节间血管生成(angiogenesis)的抑制效果。 [0019] FIG 2 is observed qualitatively the effect of suppressing the inter-chloro-oxine section zebrafish angiogenesis (angiogenesis) of the present invention. 图ac,受精后23hpf的斑马鱼经过药物处理24h,观察时相为48hpf。 FIG ac, 23hpf zebrafish 24h after drug treatment after fertilization, when viewed with respect to 48hpf. 图a为阴性对照(O. 1%DMS0),图b为氯喔星处理组,图c为阳性对照(10 μ M洛伐他汀)。 Figure a negative control (O. 1% DMS0), b is chloro FIG oxine treated group, c is a positive control, FIG. (10 μ M lovastatin). 与阴性对照相t匕,10 μ M氯喔星能够完全抑制斑马鱼体节间血管(ISV)的生成。 T dagger and a negative control, 10 μ M completely inhibited chloro oxinoid generate vascular section between zebrafish (ISV) is.

[0020] 图3为本发明氯喔星对斑马鱼体节间血管(ISV)生成的抑制率。 [0020] Figure 3 is chloro inhibition rate of oxine vascular section between zebrafish (ISV) generated by the invention. 氯喔星对斑马鱼体节间血管(ISV)生成的抑制率随着浓度的上升而呈现梯度增加,各浓度氯喔星组对斑马鱼体节间血管(ISV)生成的抑制率分别为:0. 25μΜ(0%),0· 5μΜ(7%),1μΜ(20%),2· 5μΜ(43%),5μΜ (78%), 10 μ M (92%), 25 μ M (100%)。 Oxine-chloro-section between zebrafish vessels (ISV) generation inhibition rate presented as the concentration gradient increases, the concentration of each of the group of chloro dioxines vascular section between zebrafish (ISV) inhibition rate were generated: 0. 25μΜ (0%), 0 · 5μΜ (7%), 1μΜ (20%), 2 · 5μΜ (43%), 5μΜ (78%), 10 μ M (92%), 25 μ M (100% ).

[0021] 图4为本发明受精后72小时(72hpf)血管转基因荧光斑马鱼肠下血管(SIV)模型。 (72hpf) vascular transgenic zebrafish intestine vessel under fluorescence (SIV) model 72 hours after fertilization [0021] FIG. 4 of the present invention. 框定区域为肠下血管(SIV)网络局部放大观察的部位。 Frame the blood vessel region (SIV) network intestinal partially enlarged observation site. 箭头指示肠下血管(SIV,subintestinal vessel)。 Arrows indicate blood vessels (SIV, subintestinal vessel) intestinal.

[0022] 图5为本发明定量观察氯喔星对斑马鱼肠下血管(SIV)的抑制效果。 [0022] FIG. 5-chloro-oxine observed inhibitory effects on vascular zebra intestine (SIV) in the present invention is quantitative. 图ac,受精后48hpf的斑马鱼经过药物处理24h,观察时相为72hpf。 FIG ac, 48hpf zebrafish 24h after drug treatment after fertilization, when viewed with respect to 72hpf. 图a为阴性对照(O. 1%DMS0),图b为氯喔星处理组,图c为阳性对照(ΙΟμΜ洛伐他汀)。 Figure a negative control (O. 1% DMS0), b is chloro FIG oxine treated group, c is a positive control, FIG. (ΙΟμΜ lovastatin). 虚线所示区域为肠下血管(SIV)面积放大观察的部位。 Magnifying observation of a blood vessel (SIV) area shown in phantom at intestinal part region. 与阴性对照相比,氯喔星能够显著抑制斑马鱼肠下血管(SIV)的生成,表现为肠下血管面积减小。 Compared to negative control, chloro oxinoid significantly inhibited angiogenesis (SIV) in zebra intestine, showed reduced intestinal vascular area.

[0023] 图6为本发明氯喔星对斑马鱼肠下血管(SIV)生成的抑制率。 [0023] 6-chloro-oxine inhibition ratio of the vascular zebra intestine (SIV) generated under the present invention. 氯喔星对斑马鱼肠下血管(SIV)生成的抑制率随着浓度的上升而呈现梯度增加,各浓度氯喔星组对斑马鱼肠下血管(SIV)生成的抑制率分别为:0. 25 μ M (23%),0·5μΜ (25%),1μΜ (38%),2·5μΜ(43%),5μΜ (64%), 10 μ M (98%), 25 μ M (100%)。 Oxine-chloro zebra intestine Arteries (SIV) generation inhibition rate presented as the concentration gradient increases, the concentration of each of the group of chloro dioxines zebra intestine vessel (SIV) the inhibition rates were generated: 0. 25 μ M (23%), 0 · 5μΜ (25%), 1μΜ (38%), 2 · 5μΜ (43%), 5μΜ (64%), 10 μ M (98%), 25 μ M (100% ).

[0024] 图7为本发明斑马鱼人类胃癌(SGC-7901)移植模型评价氯喔星的抗肿瘤药效。 [0024] FIG. 7 zebrafish human gastric cancer (SGC-7901) transplantation model to evaluate the anti-tumor efficacy of oxine chlorine present invention. 图af,移植人类胃癌(SGC-7901)后2dpf的斑马鱼经过药物处理4d,观察时相为6dpf。 FIG af, after transplantation of human gastric cancer (SGC-7901) 2dpf zebrafish after drug treatment 4d, phase 6dpf viewed. 图a为空白对照,图b为阴性对照(O. 1%DMS0),图df为不同浓度的氯喔星处理组,图c为阳性对照(1000 μ M5-FU)。 FIG is a blank control, a negative control panel b (O. 1% DMS0), FIG df different concentrations of chlorine oxine treated group, c is a positive control, FIG. (1000 μ M5-FU).

[0025] 图8为本发明氯喔星对移植癌细胞的生长抑制率。 [0025] Figure 8 is chloro oxinoid growth inhibition of cancer cells transplanted invention. 氯喔星对人类移植癌细胞生长的抑制率随着浓度的上升而呈现梯度增加,三个浓度氯喔星组抑制率分别为:1 μ Μ(6. 4%), Oxine-chloro-human transplantation, the inhibition rate of cancer cell growth with increasing concentration gradient of increasing presentation, three chlorine concentration oxine inhibition rates were set: 1 μ Μ (. 6 4%),

2. 5μΜ (27. 6%), 10 μ M (35.9%)。 2. 5μΜ (27. 6%), 10 μ M (35.9%). [0026] 图9为本发明定量评价氯喔星对视黄斑变性的治疗作用。 [0026] FIG. 9 degeneration quantitatively evaluated the therapeutic effect in the eye macula chloro dioxines present invention. 图ae,受精后Idpf的斑马鱼经过药物处理4d,观察时相为5dpf。 FIG ae, Idpf after fertilization zebrafish after drug treatment 4d, phase 5dpf viewed. 虚线所示圆形区域内为脉络膜血管。 Shown in phantom circular area of ​​choroidal neovascularization. 图a为阴性对照(O. 1%DMS0),图b为模型组(lmg/ml氯化钴),图c_e为不同剂量的氯喔星处理组。 Figure a negative control (O. 1% DMS0), panel b is a model group (lmg / ml cobalt chloride), different doses of FIG c_e chloro oxine treated group.

[0027] 图10为本发明氯喔星对脉络膜血管异常增生的抑制率。 [0027] FIG. 10 is the rate of inhibition of chloro dioxines proliferation of abnormal choroidal vessels invention. 氯喔星对脉络膜血管异常增生的抑制率随着注射剂量的上升而呈现梯度增加,三个氯喔星剂量组抑制率分别为:O. 21 μ g (3. 8%), O. 71 μ g (24. 5%), 2. 14 μ g (31.9%)。 Oxine-chloro abnormal proliferation of choroidal neovascularization inhibition rate of the injected dose with increasing gradient of increasing presented, three-chloro-oxine dose inhibition rates were:. O 21 μ g (3. 8%), O. 71 μ g (24. 5%), 2. 14 μ g (31.9%).

具体实施方式 Detailed ways

[0028] 下面结合说明书附图和实施例对本发明作进一步阐述,但本发明的保护范围并不限于此。 [0028] the following description in conjunction with the accompanying drawings and embodiments of the present invention is further illustrated, but the scope of the present invention is not limited thereto.

[0029] 斑马鱼相关缩略词 [0029] Zebrafish related acronyms

受精后小时数:hpf-hours postfertilization After several hours of fertilization: hpf-hours postfertilization

背长轴血管:DLAV-dorsal longitudinal anastomotic vessel Back long axis of the vessel: DLAV-dorsal longitudinal anastomotic vessel

体节间血管:ISV_intersegmental vessel Intermediate Section vessels: ISV_intersegmental vessel

背主动脉:DA_dorsal aorta Dorsal aorta: DA_dorsal aorta

后主静脉:PCV- posterior cardinal vessel Posterior cardinal vein: PCV- posterior cardinal vessel

肠下静脉血管:SIV_subintest inal vessel Intestinal vein: SIV_subintest inal vessel

绿色突光蛋白:GFP-green fluorescent protein Sudden green light protein: GFP-green fluorescent protein

实施例1定性观察氯喔星对斑马鱼体节间血管(ISV)生成模型的抑制效果斑马鱼: Inhibition effect on the model of the vascular section between zebrafish (ISV) Example 1 zebrafish qualitative observation chloro oxine embodiment:

本实施例使用的斑马鱼为血管转基因绿色荧光斑马鱼(一种由斑马鱼内皮细胞特异表达的基因作为驱动子驱动绿色荧光蛋白在斑马鱼血管内皮细胞特异表达)(图1),饲养和使用标准严格参照美国实验动物管理和使用委员会(IACUC)的要求进行。 Zebrafish present embodiment uses vascular transgenic green fluorescent zebrafish (one gene expressed by fish endothelial cell specific zebra as driving sub driving GFP specific expression in zebrafish endothelial cells) (FIG. 1), care and use standard closely follows the requirements of the United States animal Care and use Committee (IACUC) were.

[0030]养鱼水(Fish water): [0030] water fish (Fish water):

配制方法:IL 反渗透水(reverse osmosis (RO) water)加入0. 3g 海盐(InstantOcean salts)。 Preparation method: IL RO water (reverse osmosis (RO) water) was added 0. 3g salt (InstantOcean salts).

[0031] 二甲基亚砜(DMS0,分析纯): [0031] dimethyl sulfoxide (DMSO, analytical grade):

购买于阿拉丁(货号#1095515,批号#30573)。 Purchased from Aladdin (Item # 1095515, Lot # 30573). 0. 1% DMSO溶液(阴性对照)配制:使用时,用养鱼水配制成浓度为0. 1%的工作液,现配现用。 0.1% DMSO solution (negative control) formulation: When used with fish water were formulated as 0.1% of the working fluid, now with the current.

[0032] 洛伐他汀(阳性对照): [0032] Lovastatin (positive control):

购买于大连美仑,纯度大于98%(HPLC法)。 Dalian melengestrol later, purity of greater than 98% (HPLC method). 使用时,用0. 1% DMSO溶液配制成实验所需的浓度,本实验中阳性对照药的使用浓度为10 μ M0 When used with a 0. 1% DMSO solution was prepared to a concentration required for the experiment, this experiment a positive control drug concentration is 10 μ M0

[0033]氯喔星(Closantel): [0033] Chloro-oxine (Closantel):

购买于Sigma-Aldrich 公司(货号#57808-65-8,批号# SZBA292XV),使用时用0. 1%DMSO溶液配制成不同浓度的氯喔星溶液,使用浓度为10 μ M0 Purchased from Sigma-Aldrich (cat # 57808-65-8, Lot # SZBA292XV), formulated in different concentrations of chlorine oxine solution 0. 1% DMSO solution, when used with a concentration of 10 μ M0

[0034] 实验方法: [0034] Experimental method:

(I)实验分组及胚胎处理:取45只发育良好的斑马鱼胚胎,胚胎发育时相为受精后23hpf (hour-postfertilization,hpf),随机分为3组(阴性对照组,药物处理组,阳性对照组),每组胚胎数量为15只。 (I) and experimental groups treated embryos: Take 45 good zebrafish embryos, embryonic development 23hpf phase after fertilization (hour-postfertilization, hpf), were randomly divided into 3 groups (a negative control group, drug treatment group, positive control group), the number of embryos in each group was 15. 操作时将胚胎均匀分配至48孔细胞培养平板(Greiner,德国)中,每孔15只胚胎,每孔胚胎饲养用水1ml。 Embryos operation evenly distributed to 48-well cell culture plates (Greiner, Germany), 15 embryos per well, each well embryonic feeder water 1ml.

[0035] (2)药物处理:用移液器(量程100〜ΙΟΟΟμΙ,Eppendorf)迅速将预先配制好的药液加入48孔细胞培养平板对应的孔中,每孔lml。 [0035] (2) Drug treatment: Pipette (range 100~ΙΟΟΟμΙ, Eppendorf) pre-formulated liquid quickly added to 48-well cell culture plates corresponding holes, each hole lml. 加药液之前,用移液器(量程10〜ΙΟΟΟμΙ,Eppendorf)将48孔板中孵育胚胎的饲养用水尽力移出,此操作需在短时间内预先完成,以防止胚胎干燥。 Before adding liquid, with a pipette (range 10~ΙΟΟΟμΙ, Eppendorf) 48 well plates were incubated embryo rearing water tried out, this need to complete in a short time in advance, the embryo to prevent drying. 实验环境温度控制在28. 5°C左右,相对湿度40〜70%。 Experimental ambient temperature was controlled at about 28. 5 ° C, a relative humidity of 40~70%. 然后用锡箔纸将48孔板包裹好,做好实验标记,迅速放置于斑马鱼培养箱中继续培养24h (培养箱温度控制在28. 5±0. 5°C )。 The plate 48 is then wrapped with foil, and do experiment tags, zebrafish quickly placed in the incubator and cultured 24h (the incubator temperature control 28. 5 ± 0. 5 ° C).

[0036] (3)表型观察及统计:在体式显微镜下观察各孔胚胎的表型,观察指标:观察药物对胚胎发育、血液循环,心脏跳动等方面的影响。 [0036] (3) phenotype was observed and statistics: observation of each well at embryonic phenotype stereo microscope, OUTCOME MEASURES: The effects of drugs on aspects of embryonic development, blood circulation, the heart beat or the like. 然后,将血液循环受到影响的胚胎置于体式荧光显微镜(Nikon AZlOO体式荧光显微镜)下进一步观察拍照,拍照时相为48hpf,以确认血管生成抑制表型。 Then, the blood circulation is affected embryos were placed in the style of a fluorescence microscope (Nikon AZlOO style fluorescent microscope) observation photograph Further, when the phase 48hpf photographed to confirm angiostatic phenotype.

[0037] 实验结果见图2 :10 μ M氯喔星显著抑制斑马鱼节间血管(ISV)的生成,表现为节间血管缺失。 [0037] The results shown in Figure 2: 10 μ M significantly inhibited chloro oxinoid generation vascular section between zebrafish (ISV), the performance of vessels deletion internodes.

[0038] [0038]

实施例2定量评价氯喔星对斑马鱼体节间血管(ISV)生成模型的抑制效果斑马鱼血管内皮细胞出芽从受精后20hpf开始,30-31hpf左右形成主要的体节间血管网络,如背长轴血管(DLAV)和体节间血管(ISV),48hpf基本上形成完整的体轴血管网络 Example 2 Quantitative Evaluation chloro oxine generated model of the vascular section between zebrafish (ISV) inhibitory effect zebrafish endothelial cells sprouting from 20hpf begins after fertilization, formed between the main body section network of blood vessels around 30-31hpf, such as back between the major axis of the blood vessel (DLAV) and the body segment blood vessel (ISV), 48hpf body axis substantially form a complete network of blood vessels

[27],此时清晰可见完整的体节间血管(ISV)。 [27] In this case clearly visible between the full section of the vessel body (ISV). 完整的体节间血管主要指连接背主动脉(DA)和背长轴血管之间(DLAV)之间的那一段血管,见图l(48hpf血管转基因荧光斑马鱼体节间血管模型)。 Full body segments between the main blood vessel refers to that period between the vessel (DLAV) connected between the dorsal aorta (DA) and the major axis of the blood vessel back, see Fig. L (48hpf vascular fluorescence transgenic zebrafish section between the vessel model). 48hpf的斑马鱼一共有28对完整的体节间血管(ISVs)。 48hpf a zebrafish with a total of 28 pairs of somites intact blood vessels (ISVs). 实验方法如下: Experimental methods are as follows:

(I)实验分组及胚胎处理:取270只发育良好的斑马鱼胚胎,胚胎发育时相为受精后23hpf (hour-postfertilization, hpf),随机分为9 组,见下表: (I) and experimental groups treated embryos: Take 270 well developed zebrafish embryos after fertilization embryonic development phase 23hpf (hour-postfertilization, hpf), were randomly divided into 9 groups as follows:

Figure CN103054866AD00071

每组斑马鱼胚胎数量30只。 Number of embryos in each group 30 zebrafish. 操作时将胚胎均匀分配至48孔细胞培养平板(Greiner,德国)中,每孔15只胚胎,每个药物浓度处理30只胚胎,每孔胚胎饲养用水lml。 Embryos operation evenly distributed to 48-well cell culture plates (Greiner, Germany), 15 embryos per well, treatment of each drug concentration 30 embryos per well embryonic feeder water lml. (2)药物处理:见实施例1中的实验方法操作步骤(2)。 (2) Drug treatment: see Experimental Procedure The method of Example 1 (2) embodiment.

[0039] (3)表型观察及定量统计:将各药物浓度处理后的胚胎在体式荧光显微镜(NikonAZ100体式荧光显微镜)下进行观察拍照,拍照时相为48hpf,以分析各药物浓度对斑马鱼体节间血管(ISV)生成的影响。 [0039] (3) phenotype was observed and quantified Statistics: embryos will each drug concentrations observed photographed under pose a fluorescence microscope (NikonAZ100 style fluorescence microscopy), phase 48hpf when photographed to analyze each drug concentration zebrafish PRODUCTION intermediate section of blood vessel (ISV). 从各实验组随机取10只胚胎进行定量统计,统计指标如下: Statistics from each experimental group were randomly selected 10 embryos quantitative statistical indicators are as follows:

①完整的体节间血管(ISVs)数量:连接背主动脉(DA)和背长轴血管之间(DLAV) ① The full vessels somites (ISVs) Number: connecting the dorsal aorta (DA) and the major axis of the blood vessel between the back (DLAV)

之间的血管 Between vessels

Figure CN103054866AD00081

利用GraphPad Prism软件进行统计作图,并计算氯喔星抑制斑马鱼体节间血管(ISV)生成的IC5tl。 Using GraphPad Prism software for statistical plotted, and calculates inter-chloro oxinoid inhibition IC5tl zebrafish vessel section (ISV) generated. 实验结果见图3 :氯喔星对斑马鱼体节间血管(ISV)生成的抑制率随着浓度的上升而呈现梯度增加,各浓度氯喔星组对斑马鱼体节间血管(ISV)生成的抑制率分别为: The results shown in Figure 3: inhibition rate chloro oxine between zebrafish vessel section (ISV) generated presented as the concentration gradient increases, the concentration of each of the group of chloro dioxines vascular section between zebrafish (ISV) to generate the inhibition rates were as follows:

O. 25 μ M (0%),0·5μΜ (7%),1μΜ (20%),2·5μΜ (43%),5μΜ (78%),10 μ M (92%), 25 μ M(100%), IC50 为2. 73 μ Mo O. 25 μ M (0%), 0 · 5μΜ (7%), 1μΜ (20%), 2 · 5μΜ (43%), 5μΜ (78%), 10 μ M (92%), 25 μ M ( 100%), IC50 of 2. 73 μ Mo

[0040] 实施例3定性观察氯喔星对斑马鱼肠下血管(SIV)生成模型的抑制效果 Model inhibitory effect [0040] Example 3-chloro-oxine qualitative observation generates zebra intestine vessel (SIV) under

斑马鱼肠下血管(SIV, subintestinal vessel)生长在卵黄囊两侧,其形状似一个篮子,肠下血管(SIV)由体节腹侧向下延伸的长度约为50〜100 μ m[15_16]。 Zebra lower intestine vessel (SIV, subintestinal vessel) grown on both sides of the yolk sac, which is shaped like a basket, vascular (SIV) intestinal ventral section extending downward from the body length of about 50~100 μ m [15_16] . 见图4(72hpf血管转基因荧光斑马鱼肠下血管模型)。 Figure 4 (72hpf fluorescent transgenic zebrafish vascular vessel model intestine). 实验方法如下: Experimental methods are as follows:

(I)实验分组及胚胎处理:取45只发育良好的斑马鱼胚胎,胚胎发育时相为受精后48hpf (hour-postfertilization, hpf),随机分为3组(阴性对照组,药物组,阳性对照组),每组胚胎数量为15只。 (I) and experimental groups treated embryos: Take 45 good zebrafish embryos, embryonic development phase 48hpf after fertilization (hour-postfertilization, hpf), were randomly divided into 3 groups (a negative control group, drug group, positive control group), the number of embryos in each group was 15. 操作时将胚胎均匀分配至48孔细胞培养平板(Greiner,德国)中,每孔15只胚胎,每孔胚胎饲养用水lml。 Embryos operation evenly distributed to 48-well cell culture plates (Greiner, Germany), 15 embryos per well, each well embryonic feeder water lml.

[0041] (2)药物处理:见实施例1中的实验方法操作步骤⑵。 [0041] (2) Drug treatment: see Example 1, Experimental Procedures Procedure ⑵ embodiment.

[0042] (3)表型观察及统计:在体式显微镜下观察各孔胚胎的表型,观察指标:观察药物对胚胎发育、血液循环,心脏跳动等方面的影响。 [0042] (3) phenotype was observed and statistics: observation of each well at embryonic phenotype stereo microscope, OUTCOME MEASURES: The effects of drugs on aspects of embryonic development, blood circulation, the heart beat or the like. 然后,将血液循环受到影响的胚胎置于体式荧光显微镜(Nikon AZ100体式荧光显微镜)下进一步观察拍照,拍照时相为72hpf,以确认血管生成抑制表型。 Then, the blood circulation is further affected embryos were placed under observation camera style fluorescent microscope (Nikon AZ100 style fluorescent microscope), when taking phase 72hpf to confirm angiostatic phenotype.

[0043] 实验结果见图5 :10 μ M氯喔星显著抑制斑马鱼肠下血管(SIV)的生成,表现为肠下血管面积减小。 [0043] The results shown in Figure 5: 10 μ M significantly inhibited chloro oxinoid angiogenic (SIV) in zebra intestine, showed reduced intestinal vascular area.

[0044] 实施例4定量评价氯喔星对斑马鱼肠下血管(SIV)生成模型的抑制效果 Inhibitory effect model [0044] Quantitative evaluation of Example 4-chloro-oxine on angiogenesis (SIV) lower intestine Zebra

(I)实验分组及胚胎处理:取270只发育良好的斑马鱼胚胎,胚胎发育时相为受精后48hpf (hour-postfertilization, hpf),随机分为9 组,见下表: (I) and experimental groups treated embryos: Take 270 well developed zebrafish embryos after fertilization embryonic development phase 48hpf (hour-postfertilization, hpf), were randomly divided into 9 groups as follows:

Figure CN103054866AD00091

每组斑马鱼胚胎数量30只。 Number of embryos in each group 30 zebrafish. 操作时将胚胎均匀分配至48孔细胞培养平板(Greiner,德国)中,每孔15只胚胎,每个药物浓度处理30只胚胎,每孔胚胎饲养用水lml。 Embryos operation evenly distributed to 48-well cell culture plates (Greiner, Germany), 15 embryos per well, treatment of each drug concentration 30 embryos per well embryonic feeder water lml.

[0045] (2)药物处理:见实施例1中的实验方法操作步骤⑵。 [0045] (2) Drug treatment: see Example 1, Experimental Procedures Procedure ⑵ embodiment.

[0046] (3)表型观察及定量统计:将各药物浓度处理后的胚胎在体式荧光显微镜(NikonAZ100体式荧光显微镜)下进行观察拍照,拍照时相为72hpf,以分析各药物浓度对斑马鱼肠下血管(SIV)生成的影响。 [0046] (3) phenotype was observed and quantified Statistics: embryos will each drug concentrations observed photographed under pose a fluorescence microscope (NikonAZ100 style fluorescence microscopy), phase 72hpf when photographed to analyze each drug concentration zebrafish vascular (SIV) produced lower intestine. 从各实验组随机取10只胚胎进行定量统计,统计指标如下: Statistics from each experimental group were randomly selected 10 embryos quantitative statistical indicators are as follows:

①肠下血管面积(SIV area):利用Nikon AZ100体式突光显微镜配置的NIS-Elements 3.1软件进行计算 ① intestinal vessel area (SIV area): using NIS-Elements 3.1 software configuration projecting light microscope Nikon AZ100 style calculated

Figure CN103054866AD00092

利用GraphPad Prism软件进行统计作图,实验结果见图6 :氯喔星对斑马鱼肠下血管(SIV)生成的抑制率随着浓度的上升而呈现梯度增加,各浓度氯喔星组对斑马鱼肠下血管(SIV)生成的抑制率分别为:0. 25μΜ (23%),0·5μΜ (25%), ΙμΜ (38%),2·5μΜ (43%),5μΜ (64%), 10 μ M (98%), 25 μ M (100%),IC50 为4· 61 μ Μ。 Using GraphPad Prism software for statistical plotted, results shown in Figure 6: oxine-chloro zebra intestine vessel (SIV) generated under the inhibition rate presented as the concentration gradient increases, the concentration of chlorine in each group oxine zebrafish intestinal blood vessels (SIV) generated under inhibition rates were:. 0 25μΜ (23%), 0 · 5μΜ (25%), ΙμΜ (38%), 2 · 5μΜ (43%), 5μΜ (64%), 10 μ M (98%), 25 μ M (100%), IC50 of 4 · 61 μ Μ.

[0047] [0047]

实施例5斑马鱼人类胃癌(SGC-7901)移植模型评价氯喔星的抗肿瘤药效实体肿瘤的生长和扩散依赖于肿瘤内新血管的形成,并通过新生血管获取养分;新血管的形成和生长,促进了肿瘤细胞的转移。 Growth and spread of solid tumors dependent anti-tumor efficacy of Example 5 zebrafish human gastric cancer (SGC-7901) Evaluation of chlorine oxine transplant model of new blood vessels within the tumor, and angiogenesis by acquiring nutrient; new blood vessel formation and growth, promoting the metastasis of tumor cells. 本实施例用于说明氯喔星能够抑制肿瘤的生长和迁移。 Examples for describing the present embodiment can suppress chlorine oxinoid tumor growth and migration. 实验方法如下: Experimental methods are as follows:

(I)实验分组及胚胎处理:取150只移植有人类胃癌(SGC-7901)细胞的斑马鱼胚胎,胚胎发育时相为受精后2dpf (day-postfertilization, dpf),随机分为5组,见下表: (I) and experimental groups treated embryos: 150 transplanted human gastric zebrafish embryos (SGC-7901) cells, embryonic development phase after fertilization 2dpf (day-postfertilization, dpf), were randomly divided into 5 groups, see The following table:

Figure CN103054866AD00101

每组斑马鱼胚胎数量30只。 Number of embryos in each group 30 zebrafish. 操作时将胚胎均匀分配至6孔细胞培养平板(Greiner,德国)中,每孔30只胚胎,每个药物浓度处理30只胚胎,每孔胚胎饲养用水3ml。 Embryos operation evenly distributed into 6-well cell culture plates (Greiner, Germany), 30 embryos per well, treatment of each drug concentration 30 embryos per well embryonic feeder water 3ml.

(2)药物处理:用移液器迅速将预先配制好的药液加入6孔细胞培养平板对应的孔中,每孔3ml。 (2) Drug treatment: rapidly pipetted good liquid previously prepared was added 6-well cell culture plates corresponding holes, each hole 3ml. 然后用锡箔纸将6孔板包裹好,做好实验标记,放置于斑马鱼培养箱中继续培养4d (培养箱温度控制在35. 5±0. 5°C )。 The 6-well plate was then wrapped with aluminum foil, and do experiment tags placed in the incubator and cultured zebrafish 4d (the incubator temperature control 35. 5 ± 0. 5 ° C).

[0048] (3)表型观察及定量统计:将各浓度药物处理后的胚胎在体式荧光显微镜(NikonAZ100体式荧光显微镜)下进行观察拍照,拍照时相为6dpf,以分析各药物浓度对斑马鱼人类胃癌(SGC-7901)移植模型的抑制作用。 [0048] (3) phenotype was observed and quantified Statistics: embryos after each concentration of drug treatment were observed photographed under pose a fluorescence microscope (NikonAZ100 style fluorescence microscopy), phase 6dpf when photographed to analyze each drug concentration zebrafish inhibition of human gastric cancer (SGC-7901) transplantation model. 从各实验组随机取10只胚胎进行定量统计,统计指标如下: Statistics from each experimental group were randomly selected 10 embryos quantitative statistical indicators are as follows:

①定性评价氯喔星对肿瘤转移的抑制作用; ① Inhibition of chloro dioxines qualitative evaluation of tumor metastasis;

②定量评价氯喔星对肿瘤生长的抑制作用:利用尼康NIS-Elements 3.1软件计算肿瘤细胞荧光强度(S),统计学处理结果以mean±SE表示;氯喔星对肿瘤生长的抑制效果计算公式如下: ② Quantitative evaluation chloro oxinoid tumor growth inhibition: using Nikon NIS-Elements 3.1 software to calculate the fluorescence intensity of tumor cells (S), statistical results are expressed as mean ± SE; chloro oxinoid formula inhibitory effect on tumor growth as follows:

臟长抑制率(削ι-ϋΙϋΙ^)韻 Dirty growth inhibition rate (cut ι-ϋΙϋΙ ^) rhyme

利用GraphPad Prism软件进行统计作图,实验结果见图7〜图8 :氯喔星对人类移植癌细胞生长的抑制率随着浓度的上升而呈现梯度增加,三个浓度氯喔星组抑制率分别为:ΙμΜ (6·4%),2·5μΜ (27.6%),10 μ M (35.9%)。 Using GraphPad Prism software for statistical mapping, the experimental results are shown in FIG. 8 July to: oxine-chloro inhibition rate of growth of human cancer cell transplantation presented as the concentration gradient increases, the concentration of chlorine three oxine group inhibition rate is: ΙμΜ (6 · 4%), 2 · 5μΜ (27.6%), 10 μ M (35.9%).

[0049] 实施例6定量评价氯喔星对湿性老年视黄斑变性的治疗作用 Example 6 Therapeutic effect quantitative evaluation of wet age-chloro-oxine view macular degeneration [0049] Embodiment

湿性老年视黄斑变性主要因为脉络膜血管异常生成,新生的无效微血管出现渗漏,血管渗漏的液体进而破坏黄斑。 Wet age-related macular degeneration depends mainly generated because of choroidal vascular abnormalities, microvascular leakage in newborn invalid, thereby destroying the liquid macular vascular leakage. 氯化钴能够诱导斑马鱼视网膜脉络丛血管增生、视细胞变性,类似于人类湿性老年视黄斑变性的改变[28]。 Cobalt chloride zebrafish capable of inducing the choroid plexus of retinal vessel proliferation, degeneration of visual cells, similar to the view change human wet age-related macular degeneration [28]. 本实施例用于说明氯喔星对湿性老年视黄斑变性具有治疗效果。 Examples for describing the present embodiment Chloro oxinoid a therapeutic effect on visual wet age-related macular degeneration. 实验方法如下: Experimental methods are as follows:

(I)实验分组及胚胎处理:取150只发育良好的斑马鱼胚胎,胚胎发育时相为受精后Idpf (day-postfertilization, dpf),随机分为5 组,见下表: (I) and experimental groups treated embryos: Take 150 good zebrafish embryos fertilized with the Idpf (day-postfertilization, dpf), were randomly divided into 5 groups embryonic development, as follows:

Figure CN103054866AD00102

每组斑马鱼胚胎数量30只。 Number of embryos in each group 30 zebrafish. 操作时将胚胎均匀分配至6孔细胞培养平板(Greiner,德国)中,每孔30只胚胎,每孔胚胎饲养用水3ml。 Embryos operation evenly distributed into 6-well cell culture plates (Greiner, Germany), 30 embryos per well, each well embryonic feeder water 3ml.

(2)药物处理:阴性对照组中加入DMSO,使其终浓度为O. 1% ;模型组中加入氯化钴,使其终浓度为I mg/ml ;氯喔星通过显微注射方式给药,每组均注射30只胚胎,注射后将胚胎按组分别放入3ml含有I mg/ml氯化钴的饲养用水中。 (2) Drug treatment: negative control group, DMSO was added to a final concentration of O. 1%; cobalt chloride was added to the model group, to give a final concentration of I mg / ml; chloro dioxines by microinjection manner to drugs, each group 30 injected embryos, embryos after injection in groups are placed in the rearing water containing 3ml I mg / ml of cobalt chloride.

[0050] (3)表型观察及定量统计:将各剂量药物处理后的胚胎在体式荧光显微镜(NikonAZ100体式荧光显微镜)下进行观察拍照,拍照时相为5dpf,以分析各药物剂量对斑马鱼眼部脉络膜异常增生血管的抑制作用。 [0050] (3) phenotype was observed and quantified Statistics: embryos after each dose of drug treatment were observed photographed under pose a fluorescence microscope (NikonAZ100 style fluorescence microscopy), phase 5dpf taking pictures, to analyze each drug dose zebrafish inhibition of choroidal vascular dysplasia eye. 从各实验组随机取10只胚胎进行定量统计,统计指标如下: Statistics from each experimental group were randomly selected 10 embryos quantitative statistical indicators are as follows:

①定性评价氯喔星对眼部脉络膜异常增生血管的抑制作用; ① Inhibition qualitative evaluation of ocular choroidal chloro oxinoid abnormal proliferation of blood vessels;

②定量评价氯喔星对脉络膜异常增生血管的抑制作用:利用NIS-ElementS 3.1软件计算脉络膜异常增生血管荧光强度(S),统计学处理结果以mean±SE表示;氯喔星对脉络膜异常增生血管的抑制效果计算公式如下: ② Quantitative Evaluation of inhibition of choroidal chloro oxinoid abnormal proliferation of blood vessels: Calculated choroidal vascular dysplasia fluorescence intensity (S) using NIS-ElementS 3.1 software, statistical results are expressed as mean ± SE; abnormal proliferation of blood vessels chloro oxinoid choroidal the inhibitory effect is calculated as follows:

Figure CN103054866AD00111

利用GraphPad Prism软件进行统计作图,实验结果见图9〜图10 :氯喔星对脉络膜血管异常增生的抑制率随着剂量的上升而呈现梯度增加,三个氯喔星剂量组抑制率分别为: Using GraphPad Prism software for statistical mapping, the experimental results are shown in FIG. 10 9~: oxine-chloro abnormal proliferation of choroidal neovascularization inhibition rate presented with increasing gradient of increasing doses, dose three chloro oxinoid inhibition rates were :

O. 21 ug (3. 8%),0. 71ug (24·5%),2· 14μ g (31.9%)。 O. 21 ug (3. 8%), 0. 71ug (24 · 5%), 2 · 14μ g (31.9%).

[0051] [0051]

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Claims (9)

1.氯喔星在制备抗血管生成类药物中的应用。 1. Application of chlorine in the manufacture of oxine antiangiogenic drugs in.
2.根据权利要求1所述的应用,其特征在于,所述药物为口服给药剂型、注射给药剂型、粘膜给药剂型或经皮给药剂型。 The use according to claim 1, wherein the medicament is an oral dosage form, injectable dosage forms, transmucosal administration or transdermal administration dosage forms.
3.根据权利要求1所述的应用,其特征在于,所述药物为片剂、胶囊剂、颗粒剂、口服液、注射液、贴剂或凝胶剂形式。 3. Use according to claim 1, wherein the medicament is a tablet, capsule, granules, oral liquid, injection, in the form of a patch or gels.
4.氯喔星在制备抗肿瘤药物中的应用。 4. Application of chloro dioxines in the preparation of antitumor drugs.
5.根据权利要求4所述的应用,其特征在于,所述的氯喔星通过抑制肿瘤内新血管的生成来预防或治疗肿瘤。 5. Use as claimed in claim 4, wherein said chlorine oxinoid preventing or treating cancer by generating new blood vessels within the tumor suppression.
6.根据权利要求4所述的应用,其特征在于,所述药物为片剂、胶囊剂、颗粒剂、口服液、注射液、贴剂或凝胶剂形式。 6. The use as claimed in claim 4, wherein the medicament is a tablet, capsule, granules, oral liquid, injection, in the form of a patch or gels.
7.氯喔星在制备抗湿性老年视黄斑变性药物中的应用。 7. Preparation of Anti-chloro-oxine in view of wet age-related macular degeneration medicament application.
8.根据权利要求7所述的应用,其特征在于,所述的氯喔星通过抑制脉络膜血管异常增生来预防或治疗湿性老年视黄斑变性。 The use according to claim 7, wherein said chlorine oxinoid choroidal neovascularization by inhibiting the abnormal proliferation of prevention or treatment of wet age-related macular degeneration view.
9.根据权利要求7所述的应用,其特征在于,所述药物为片剂、胶囊剂、颗粒剂、口服液、注射液、贴剂或凝胶剂形式。 9. Use according to claim 7, wherein the medicament is a tablet, capsule, granules, oral liquid, injection, in the form of a patch or gels.
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