CN112402418B - 一种单胺氧化酶b(mao-b)抑制剂及其应用 - Google Patents
一种单胺氧化酶b(mao-b)抑制剂及其应用 Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/465—Nicotine; Derivatives thereof
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- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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Abstract
本发明涉及尼古丁(Nicotine，又名烟碱)作为单胺氧化酶B(MAO‑B)抑制剂及其应用，属于神经生物学领域。本发明通过Docking理论模拟证明尼古丁具有与MAO‑B活性空腔结合的潜力，通过Amplex Red探针检测荧光法验证了尼古丁在体外对MAO‑B纯蛋白的抑制作用。基于尼古丁可作为MAO‑B抑制剂的作用，将其用于过表达MAO‑B的神经细胞系和帕金森疾病果蝇模型的治疗中，得到保护效果。尼古丁作为一种MAO‑B抑制剂，可用于其对抗帕金森疾病、用于治疗和预防可通过单胺氧化酶B抑制剂进行调节的疾病。
Description
技术领域
本发明涉及尼古丁(Nicotine)可以作为一种MAO-B抑制剂，更具体地，本发明涉及尼古丁作为MAO-B抑制剂可用于对抗帕金森疾病的应用潜力。
背景技术
单胺氧化酶-B(Monoamine Oxidase B,MAO-B)是机体内降解多巴胺的关键酶，该蛋白位于线粒体膜上，其辅酶FAD的活性中心的半胱氨酸残基可以与多巴胺产生反应最终产生醛类和双氧水。MAO-B的过表达或活性增加是帕金森疾病(PD)发病的重要因素，脑组织中所含MAO-B活性增加是脑功能衰退指标之一。因此MAO-B抑制剂(如Pargyline、Resagyline等)被用作治疗帕金森疾病的常用药物。
已有大量研究表明，尼古丁具有对抗PD的作用。目前主要的观点认为，尼古丁是通过结合烟碱型乙酰胆碱受体发挥其作用，主要的分子通路为尼古丁结合到受体后，激活细胞内钙离子释放，后者又激活下游的众多信号分子，包括蛋白激酶A(PKA)、细胞分裂原活化蛋白激酶(ERK)、钙调蛋白(CaM)、磷脂酰肌醇-3-激酶(PI3K)等，从而减少了神经元的凋亡，起到对多巴胺神经元的保护作用。
但目前，尼古丁作为MAO-B抑制剂仍未被报道。因此其作为MAO-B抑制剂的作用可补充说明尼古丁对抗PD的分子机制。
发明内容
本发明提出了尼古丁作为一种MAO-B抑制剂，可用于其对抗帕金森疾病、用于治疗和预防可通过单胺氧化酶B抑制剂进行调节的疾病。
本发明解决的技术问题是：在体外检测尼古丁对MAO-B蛋白的活性抑制率，并且在细胞和果蝇实验中对其作为MAO-B抑制剂产生的对抗PD的效果进行初步验证。
为了解决上述技术问题，本发明提出的技术方案是：一种单胺氧化酶B(MAO-B)抑制剂，为尼古丁(Nicotine，又名烟碱)，其结构式如下式1。
优选的，包括活性成分为式1所示的尼古丁，以及药学上可接收的药用辅料。
优选的，包括活性成分为式(1)所示的尼古丁，以及药学上可接受的载体制备成片剂、胶囊剂、注射剂、粉针剂、颗粒剂、脂肪乳剂、微囊、滴丸、软膏剂或透皮控释贴剂剂型的药物。
优选的，尼古丁具有抑制MAO-B蛋白活性的作用，通过体外小分子-蛋白对接Docking理论模型计算模拟得出尼古丁具有与MAO-B活性空腔结合的可能性，尼古丁分子可以进入MAO-B蛋白的活性空腔，其活性N位点可以接近MAO-B蛋白辅酶FAD的活性位点。
优选的，1μM浓度下尼古丁即可用于保护神经细胞SH-SY5Y由于过表达MAO-B造成的神经毒性易感性，并可影响细胞代谢和凋亡相关因子的变化。
尼古丁细胞给药后0.5h即可到达线粒体，且线粒体中尼古丁含量在6h内保持稳定。本发明首次报道尼古丁细胞给药后可以到达线粒体，区别于尼古丁关于对抗帕金森症的基于细胞膜上受体作用的现有研究结果，可补充完善尼古丁的作用机制。
为了解决上述技术问题，本发明提出的技术方案是：所述的单胺氧化酶B(MAO-B)抑制剂的应用，可用于制备用于治疗和预防可通过单胺氧化酶B抑制剂进行调节的疾病中药物的制备。
优选的，可用于治疗帕金森症状药物的制备。
优选的，所述疾病包括中枢神经系统病症、精神病学病症、人格障碍、物质相关病症、解离性障碍、进食障碍、睡眠障碍、发育障碍、神经退行性病症、创伤相关病症、疼痛病症和认知障碍。
通过Docking理论模拟计算出尼古丁与单胺氧化酶B(MAO-B)的活性位点互作潜力，为尼古丁作为一种MAO-B抑制剂提供理论基础。本发明通过体外蛋白活性检测实验(Amplex Red Kit)，检测尼古丁在体外对MAO-B的活性抑制率。在体外蛋白活性测试中，尼古丁具有抑制MAO-B的效果，且在0-0.1μM之间其抑制效果优于商业抑制剂pargyline，浓度大于0.1μM时保持80％左右的抑制率。
进一步地，通过细胞活性检测，细胞凋亡检测及蛋白检测考察尼古丁作为一种MAO-B抑制剂，在神经细胞SH-SY5Y中，起到对抗其由于过表达MAO-B与MPTP反应引起的细胞损伤和凋亡。
进一步地，在PD果蝇模型中，验证尼古丁作为一种MAO-B抑制剂，起到对抗PD果蝇症状使PD果蝇恢复大脑多巴胺含量的作用。
所属领域技术人员可根据实际情况容易地确定尼古丁的药物浓度如：细胞实验中给药浓度为1μM，小鼠及其他动物实验给药剂量如2mg/kg体重。
本发明的有益效果:
本发明涉及尼古丁(Nicotine)，又名烟碱，用于改善帕金森症(PD)的一种新认识，属于神经生物学领域。流行病学显示吸烟与PD呈负相关关系，目前研究已证明烟草中的尼古丁可通过与细胞膜上的乙酰胆碱受体结合，引起钙离子内流从而激活一系列信号通路达到保护神经元的作用。但是当抑制尼古丁与乙酰胆碱受体结合后，仍具有一定的保护作用，此机制仍未阐明。单胺氧化酶B(MAO-B)是位于线粒体外膜的催化多种胺类神经递质的酶，是帕金森病发病的重要因子。
我们的研究首次发现：①通过蛋白Docking模拟发现尼古丁具有与MAO-B活性空腔结合的潜力；②通过Amplex Red试剂盒与U1探针发现尼古丁在体外具有抑制MAO-B蛋白活性的作用；③在细胞层面验证尼古丁可达到商业抑制剂Pagyline的保护效果。基于上述发现，本发明区别于已有研究的特点是：尼古丁通过抑制单胺氧化酶B(MAO-B)的活性，减弱其对神经递质多巴胺等的降解作用，从而产生保护神经元的作用。相较于此前已发现的尼古丁通过受体通路发挥对抗帕金森疾病的作用，其通过抑制MAO-B活性的非受体通路可以补充说明其对抗PD的机制，使之更加完善。
尼古丁是烟草的重要成分，其分子量较小，容易进入MAO-B蛋白活性空腔。尼古丁作为一种MAO-B抑制剂与其他商业抑制剂相比优势在于：其在低浓度0.1μM时对MAO-B纯蛋白抑制率达到70％，而同浓度下商业MAO-B抑制剂Pargyline的抑制率为50％，可见在低浓度下尼古丁的抑制效率高于商业抑制剂。并且已有文献报道，尼古丁吸入后可利用转运体通过血脑屏障到达脑内发挥作用，尼古丁相比于其他递质对于相关转运体更具有竞争性结合能力(Tega Y,Yamazaki Y,et al.Impact of Nicotine Transport across the Blood-Brain Barrier.Biol Pharm Bull,2018.)，这得益于尼古丁的极性较小，因此在脑部疾病的治疗方面，尼古丁相比于其他MAO-B抑制剂具有更快透过血脑屏障的优势。
尼古丁作为一种MAO-B抑制剂，其特征在于，尼古丁具有抑制MAO-B蛋白活性的作用，通过体外小分子-蛋白对接Docking理论模型计算模拟得出尼古丁具有与MAO-B活性空腔结合的可能性，尼古丁分子可以进入MAO-B蛋白的活性空腔，其活性N位点可以接近MAO-B蛋白辅酶FAD的活性位点。
在体外蛋白活性测试中，尼古丁具有抑制MAO-B的效果，且在0.1μM浓度下抑制效果优于商业抑制剂pargyline。
1μM尼古丁给药可用于保护神经细胞SH-SY5Y由于过表达MAO-B造成的神经毒性易感性，并可影响细胞代谢和凋亡相关因子的变化。尼古丁细胞给药后0.5h即可到达线粒体，且线粒体中尼古丁含量6h内保持稳定。
10μM尼古丁给药可用于缓解果蝇由于帕金森症状引起的大脑多巴胺含量下降。
附图说明
下面结合附图对本发明的作进一步说明。
图1：尼古丁/Pargyline与MAO-B蛋白结合Docking模拟构象
图2：尼古丁作为一种MAO-B抑制剂对体外MAO-B纯蛋白的抑制作用
图3：尼古丁作为一种MAO-B抑制剂对过表达MAO-B神经细胞的保护作用
图4：尼古丁进入细胞后在不同组分中的含量分布
图5：尼古丁作为一种MAO-B抑制剂对PD果蝇的影响
图6：尼古丁作为一种MAO-B抑制剂起到对抗帕金森疾病的机理示意图
具体实施方式
实施例1Docking理论计算
利用Grid软件构建MAO-B蛋白具有FAD活性空腔的除水蛋白模型，蛋白氨基酸序列来自于PDB官网的human MAO-B。利用Autodock软件将尼古丁和Pargyline结构导入作为配体。将蛋白模型和配体结构均处理为.pdbqt格式，确定MAO-B辅酶FAD活性空腔的box长宽高数值以及相对位置，同时确保配体的所有单键均可旋转。在Pymol软件中确定结合空间，进行cmd演算，计算出配体对蛋白结合的能量值，能量最低的为最稳定构象如图1所示。尼古丁与MAO-B结合能量最低的构象中，其反应位点间距为
而Pargyline与MAO-B的反应位点间距为
说明尼古丁与MAO-B的反应位点间距短于Pargyline，为尼古丁可作为一种MAO-B抑制剂提供了理论基础。
实施例2体外纯蛋白抑制率检测
将尼古丁和Pargyline分别均匀稀释于PBS溶液中分别配制成0.1μM，1μM，10μM，100μM，500μM，1000μM的溶液待用。配制MAO-B(来自Thermofisher公司，人源MAO-B纯蛋白)50μg/mL的PBS溶液待用。根据Amplex Red Monoamine Oxidase Assay Kit(Invitrogen)提供的检测试剂和检测方法，将MAO-B底物benzylamine与马血清以及amplex red探针配制成工作液待用。在buffer溶液中分别加入尼古丁/Pargyline，MAO-B蛋白，使尼古丁/Pargyline的最终浓度分别为0.01μM，0.1μM，1μM，10μM，50μM，100μM，MAO-B的最终浓度为10μg/mL，将混合溶液置于37℃的摇床上振荡1h后，等体积加入工作液，继续在37℃摇床振荡1h。反应结束后将溶液转移至黑色384孔板中，每组设三个复孔。利用酶标仪(Synergy HTX)检测各组溶液的荧光强度。检测条件为激发光波长542nm，接收发射波长570-700nm。该试剂盒检测原理为MAO-B与其底物benzylamine反应产生过氧化氢，amplex red探针可以检测过氧化氢含量而转化为荧光信号。当溶液中含有抑制MAO-B活性的物质存在时，产生的过氧化氢减少，荧光信号减弱。因此可以通过反应后溶液的荧光强度间接判断MAO-B抑制剂的抑制效率。
抑制率检测结果如表1和图2所示，当抑制剂浓度为0.01μM时，尼古丁可达到72％的抑制率，而同样的浓度下Pargyline的抑制率仅为50％。但当浓度逐渐增大至1μM以上时，尼古丁的抑制效率达到饱和，接近于80％，而pargyline的饱和抑制效率达到90％以上。
表1：尼古丁和Pargyline在不同浓度下分别对MAO-B的抑制率比较
实施例3MTT法检测药物毒性和细胞活力
将3×104个细胞均匀接种于96孔板中，共6行11列，每列设为一组。置于37℃，5％CO2培养箱中培养。当细胞密度达到60％左右时，每组加入含有不同浓度神经毒性药物MPTP的培养基，最终浓度分别为0，0.1，1，10，25，50，100，250，500，1000μM，另设一列为空白组。每组所含DMSO含量均为0.1％，孵育24h后，每孔加入5mg/mL MTT 20μL,避光孵育4h，出现蓝紫色结晶。轻轻吸去培养液，每孔加入200μL DMSO溶液，利用酶标仪(Synergy HTX)振荡1min使结晶完全溶解分散均匀后检测波长452nm处的吸光度，按以下公式计算每组的细胞存活率：
细胞存活率＝(A实验组-A空白组)/(A对照组-A空白组)
实验重复三次。
通过细胞活力检测结果如图3(1)(2)所示，MPTP使用浓度为100μM时，过表达MAO-B的神经细胞比普通神经细胞的活力显著降低。当同时给与100μM的MPTP和梯度浓度的尼古丁药物后，过表达MAO-B的神经细胞活力有较明显的恢复，从75％的活力提升至90％左右。说明尼古丁可对抗MAO-B过表达引起的神经细胞对MPTP的毒性刺激。
实施例4细胞中NAD+/NADH水平检测
将1.2×106个细胞均匀接种在6孔板中，当细胞密度达到60％左右时，每孔加入含有相同浓度MPTP(100μM)，不同浓度尼古丁的培养基，最终浓度分别为0，0.1，1，10，100μM，Pargyline作为阳性对照药物浓度为10μM。24h后，收集各组细胞，用NAD+/NADH检测试剂盒(碧云天)提供的试剂和方法处理细胞。具体为，每孔用200μL酸性提取液在冰上裂解细胞，10000g 4℃离心10min，取上清，加入等体积碱性提取液，充分混合后10000g 4℃离心10min，取上清10μL用于蛋白浓度测定(BCA蛋白浓度试剂盒)，另取50μL上清分别按序加入试剂盒提供的检测试剂，混匀后，用酶标仪检测570nm处的吸光度，按以下公式计算每组细胞中NAD+含量：
NAD+含量(nmol/mg)＝(A测定-A对照-0.099)×V50/(V10×Cprotein)
实验重复三次。
通过NAD+/NADH水平检测，结果如图3(4)所示，1μM尼古丁给药作用下，过表达MAO-B的神经细胞中，NAD+水平有显著增加达到5.5nmol/L。但继续增加尼古丁浓度，其水平又有所下降，说明尼古丁浓度增加时或产生毒性。因此由此实验结果可知，尼古丁在细胞实验中最佳给药浓度为1μM。
实施例5细胞中ATP水平检测
细胞接种及给药方式同实施例4，每孔用200μL ATP裂解液冰上充分裂解细胞后12000g 4℃离心15min，取上清10μL检测蛋白浓度，收集剩余得到样品液。将ATP标准品分别稀释成5μM，1μM，0.5μM，0.1μM，0.05μM，0.01μM。将ATP标准液和样品液加入白色96孔板中，每孔20μL，每组设三个复孔。每孔加入100μL检测工作液，迅速用酶标仪通过化学发光通道检测RLU值。绘制标准曲线，计算出各样品组的ATP含量以及蛋白浓度，通过比例可以算出每mg蛋白中所含有ATP的量。
通过ATP含量检测，结果如图3(5)所示，当给与MPTP刺激，过表达MAO-B的细胞表现出更明显的ATP水平下降，给与1μM尼古丁后，ATP得以恢复至8.5nmol/L左右，接近于对照组水平。而在普通的神经细胞中也有一定的保护作用但是保护作用弱于过表达MAO-B的细胞系。说明尼古丁产生的保护作用有一部分来自于抑制过表达的MAO-B。
实施例6Western-Blot检测细胞中凋亡相关蛋白水平
细胞接种及给药方式同实施例4，每孔用200μL含有1％蛋白酶抑制剂(PMSF)的细胞裂解液(RIPA)冰上充分裂解细胞后12000g 4℃离心5min，取上清10μL检测蛋白浓度，另取160μL上清加入40μL 5×上样缓冲液(loading buffer)，充分混匀后100℃加热10min，得到样品液。根据各组样品的蛋白浓度换算出各组上样量(电泳蛋白上样量为30μg)，公式为：
v＝1.25×30/c(对应蛋白浓度)
配制合适浓度的SDS-PAGE凝胶，根据计算出的上样量将样品加入各凝胶泳道中，用1×上样缓冲液补齐每一泳道的样品总体积。加满电泳液，80V恒压电泳15min，120V恒压电泳约60min使目标条带分子量完全分开。取出胶块，用甲醇活化过的PVDF膜进行转膜。转膜条件为恒流300mA 45min，冰浴。将PVDF膜置于5％BSA封闭液中室温摇床封闭1h，用目标蛋白的一抗稀释液在4℃下摇床孵育过夜。TBST洗膜三次，每次10min。用一抗相同种属的二抗稀释液室温下孵育1h，TBST洗膜三次，每次10min。将配制好的ECL显色工作液均匀铺满PVDF膜，用凝胶成像仪进行曝光成像，并用Image J软件分析条带灰度值。
通过蛋白印迹实验，结果如图3(6)所示，MPTP给药组的细胞中凋亡相关蛋白P-JNK和C-PARP表达上升，而给与1μM和10μM尼古丁后，相关蛋白表达有所下调。说明尼古丁可缓解由于过表达MAO-B和MPTP产生的细胞凋亡。
实施例7流式细胞术检测细胞凋亡
细胞处理步骤同实施例4，胰酶消化，离心收集细胞后，以缓冲液：Annexin V：PI＝100：1：1的比例配制工作液，重悬细胞，避光37℃孵育30min后PBS洗涤三次，以PBS重悬细胞，用流式细胞仪(贝德曼C6)检测细胞凋亡情况。
通过流式细胞术实验，结果如图3(7)所示，MPTP给药组的细胞早凋比例为84.7％，相比于对照组的77.4％，早期凋亡比例增加了近7％。而给与1μM尼古丁和1μM Pargyline的组别，细胞晚凋比例分别下降至77.5％和81.1％，说明两者都缓解了MPTP造成的细胞凋亡，并且1μM浓度下尼古丁的保护作用优于Pargyline。
实施例8HPLC检测不同细胞组分中尼古丁含量
利用细胞线粒体分离试剂盒(碧云天)对细胞(前处理同实施例4)进行线粒体提取和分离。对分离后的组分进行蛋白印迹实验检测组分纯度。各组分的细胞裂解液取200μL，加入1mL乙酸乙酯，振荡混匀10min，离心后取800μL上清，挥干，用8：2甲醇水200μL复溶，离心取180μL上清，得到待测样品溶液。配制浓度分别为2，5，10，20，50，100ng/mL的尼古丁标准溶液，选用对乙酰氨基酚为内标物质，建立标准曲线。用相同条件和方法检测样品溶液，得到样品中尼古丁的含量。高效液相色谱分析实验条件如下：
色谱柱型号：Thermo C18 100×2.1mm；
流动相：10mmoL醋酸铵：甲醇＝15：85
通过HPLC检测细胞中不同组分尼古丁的含量，结果如图4所示，当尼古丁给药浓度为1μM时如图4(1)，给药后0.5h，胞质中浓度达到8ng/Ml，2h后降至1.5ng/mL，而线粒体中，从给药后0.5h至6h，浓度均在2.5ng/mL左右,无明显改变。当尼古丁给药浓度为10μmol/L时如图4(2)，给药后0.5h，胞质中浓度达到8.5ng/mL，2h后降至3ng/mL，6h时，胞质浓度又上升至6.5ng/mL，而在线粒体组分中，浓度仍维持在2.5ng/mL左右,略有下降但无明显变化。这些结果提示，尼古丁的确可以到达线粒体，这也为其抑制位于线粒体外膜上的MAO-B提供了空间基础。
实施例9PD果蝇给药后其行为学检测和大脑多巴胺含量检测
选取150只一日龄的野生型(Wild type)雄性果蝇为对照组，选取150只一日龄的帕金森疾病模型(Parkin null)雄性果蝇为疾病组，选取150只一日龄的帕金森疾病模型(Parkin null)雄性果蝇为治疗组。对照组与疾病组均给予正常食物，治疗组给予含有10μM尼古丁的食物。每一组果蝇分为三管平行组，每小组50只。每天更换食物并记录生存率。
果蝇行为学检测：各组果蝇培养到50天时，进行爬行实验。将每组果蝇随机取出30只存活果蝇，转移至带有刻度的透明长管中，两头用棉花塞紧，饥饿处理2h后，将管子竖直放置，果蝇从底端开始往上爬行，记录1min内能爬过20cm高度的果蝇数量并记录。每组果蝇重复爬行三次，每次爬行后间隔5min。最后计算出各组果蝇的爬行率。每组取10只果蝇用CO2麻醉，在显微镜下用手术级镊子剥离出完整的果蝇大脑，置于120μL PBS中，用超声细胞粉碎仪破碎组织(冰浴，超声2S,暂停2S，共15次)，10000g 4℃离心15min，取100μL上清即为待测样品溶液。使用多巴胺检测试剂盒(金霖生物)提供的检测试剂和方法，配制一系列浓度梯度(1000pg/mL，333.33pg/mL，111.11pg/mL，37.04pg/mL，12.35pg/mL)的多巴胺标准品，把标准品和待测样品溶液加入试剂盒提供的96孔板中，每孔50μL，先后用试剂盒提供的检测工作液A、B于37℃各孵育1h后，吸去孔中溶液，每孔加入90μL底物溶液，37℃避光显色后加入50μL终止液。随即用酶标仪检测450nm波长处的吸光度，建立标准曲线，计算出各组果蝇大脑中多巴胺含量。
在果蝇实验中，尼古丁的给药浓度为10μM。如图5(1)所示，Parkin null果蝇老龄时翅膀呈现无法收拢的状态，而Wild type果蝇翅膀可以正常收拢，喂食尼古丁的Parkinnull果蝇翅膀形态有所改善。如图5(2)(3)(4)所示，Wild type果蝇的爬行率，大脑多巴胺含量，肌肉组织ATP含量均显著高于Parkin null果蝇，而喂食尼古丁的Parkin null果蝇相应指标也有所改善。
实施例10
片剂的制备
处方(以1000片的处方量计)：
尼古丁纯品100g；
蔗糖60g；
玉米淀粉80g；
硬脂酸镁2g。
制备方法：将活性成分与蔗糖、玉米淀粉混合，加水湿润，搅拌均匀，干燥，粉碎过筛，加入硬脂酸镁，混合均匀，压片。平均片重242mg/片，活性成分含量为100mg。
实施例11
注射液的制备
处方：(以1000支的处方量计)
尼古丁纯品100g；
丙二醇100g；
注射用水加至1000ml。
将处方量的尼古丁纯品溶解于丙二醇中，加注射用水至1000ml，混合均匀，过滤，将所获得的溶液在无菌条件下分装于安瓿瓶中，制成1ml/瓶注射液，活性成分含量为100mg/mL。
本发明的不局限于上述实施例所述的具体技术方案，凡采用等同替换形成的技术方案均为本发明要求的保护范围。
Claims (1)
1.一种尼古丁用于制备治疗帕金森症状药物中单胺氧化酶B（MAO-B）抑制剂的应用，其特征在于: 尼古丁作为制备治疗帕金森症状药物中单胺氧化酶B抑制剂，用于治疗和预防可通过单胺氧化酶B抑制剂进行调节的帕金森疾病。
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