CN86108593A - 蛇毒液生长抑制肽 - Google Patents
蛇毒液生长抑制肽 Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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Abstract
新的细胞毒性剂是作为与得自大响尾蛇之低分子量肽有关的小的多肽被提供的。该化合物本身或与其它试剂(如抗体)结合被用于抑制细胞生长。
Description
毒剂具有多种用途。特别是其中细胞毒性剂对特殊的菌株或细胞类型是特异的，或可被修饰成为特异的，故可使用细胞毒性剂从包含许多不同类型细胞的培养物或组织中除去特殊菌株或细胞，如在恶性肿瘤的情况下，可望能特异地消除恶性细胞，而对正常或健康细胞则无严重的致死性作用。
毒素的一个来源是蛇毒液，其中许多毒素是已知的。然而仍有兴趣寻找另外的毒素以增加可作特殊应用之毒素的来源。毒素可因宿主对毒素的免疫应答能力、其与其它试剂偶联的难易以及毒素的作用部位等而有所不同。为从蛇毒液中找到新的毒素，须建立分析其效能、彻底提取、纯化及分析方法，以确定其纯度及氨基酸序列。
已分离、描述了从几个种属响尾蛇之毒液中分离的细胞毒性肽、並分析了其氨基酸序列。肌毒素是从大草原响尾蛇（Crotalus）（Fox等，Biochemistry（1979）18：678-83）的毒液中制取的;响尾蛇毒蛋白是从南美洲响尾蛇（Crotalus durissus terrificus）（Lauree and Hoppe-Seyler，Physiol.Chem.（1975）256：213-15）的毒液中分离的;而毒性肽C则是从南太平洋响尾蛇（Crotalus Viridis helleri）（Maeda et al.Toxicon（1978）16：431-41）的毒液中制得的。
本发明所提供的新的细胞毒性肽或细胞生长抑制肽与从西部棱纹背响尾蛇（rotalus atrox）的毒液分离的肽有关联。这些肽与特异的结合部分如配基和受体所形成的结合物可用于由细胞混合物中选择性地除去某些细胞。
所提供的细胞毒素包括一由大响尾蛇（Crotalus atrox）的毒液中制得的分子量约6千道尔顿（KD）的肽、其细胞毒性类似物以及细胞毒素与肽，特别是可特异地结合于某互补结合部位的配基与受体形成的结合物。活性部分至少有约15个氨基酸，通常是约25个，更常见是至少约35个且不多于60个，最常见的是不多于50个氨基酸。这些活性部分可与下面将述及的许多种其它化合物结合。
本发明的肽具有如下的结构式：
PP1QC1aa4aa5aa6GGaa9C2aa11aa12aa13aa14C3aa16aa17aa18aa19SDaa22GKaa25aa26C4aa28aa29aa30WKC5C6Kaa36aa37aa38aa39PP2
其中：
PP1是N末端。且可以是氢、单个氨基酸，特别是极性氨基酸，更特别是羟基取代的氨基酸或碱性氨基酸，或者是含2-20个氨基酸、一般是2-10个氨基酸的多肽，其可贡献多种功能，如作为连接基团、修饰基团等;
PP2是C末端，且可以是末端羟基、单个氨基酸，特别是极性氨基酸，更特别是含羧酰胺基的氨基酸或含2-20个氨基酸，更一般是含2-10个氨基酸的多肽，其亦可贡献如PP1的功能。
作为一字母氨基酸密码的部分，各个字母均有其标准含义，现指出如下：
多达5个、通常不超过大约3个氨基酸可作为结合体，以至从结构中被删除。
倘存在半胱氨酸桥时，则C1与C5、C2与C4以及C3与C4形成半胱氨酸桥。
aa4是脂族酸性或芳族氨基酸，特别是D、E、H;
aa5是一脂族极性或碱性氨基酸，特别是羧酰胺基取代的或碱性氨基酸，尤其是N、Q、K、R;
aa6是一脂族带电荷氨基酸，可以是酸性的或是碱性的，特别是D、E、K或R;
aa9是芳香族氨基酸，特别是F、H、W、Y;
aa11是脂族带电荷氨基酸，特别是碱性氨基酸，更特别是K、R;
aa12是芳族氨基酸或脂族极性氨基酸或带电荷的氨基酸，特别是酸性或中性的，更特别是羟基取代的中性氨基酸，包括F、H、W、Y、D、E、S、T;
aa13是一脂族非极性或带电荷氨基酸，特别是非极性或碱性的，更特别是含4-6个碳原子的氨基酸，包括L、I、V、K、R;
aa14是脂族非极性氨基酸，特别是含4-6个碳原子者，即L、I、V;
aa16是脂族非极性氨基酸並有4-6个碳原子，特别是P、I、L、V;
aa17是有3-5个碳原子的脂族非极性或极性氨基酸，特别是羟基取代的，更特别是S、T、A、P、V;
aa18是脂族带电荷的或非极性的氨基酸，特别是当其带电时为碱性的，並有3-6个、通常是4-6个碳原子，特别是P、I、L、V、K、R;
aa19是有3-4个碳原子的脂族极性氨基酸，特别是羟基取代的，更特别是S、T;
aa22是脂族非极性或芳族氨基酸，当其为脂族氨基酸时含5-6个碳原子，特别是F、I、L、V;
aa25是脂族中性氨基酸且含3-6个碳原子，特别是含4-6个碳原子的极性或非极性氨基酸，当为极性氨基酸时则含有硫原子，更特别是M、I、L、V;
aa26是脂族带电荷的或非极性氨基酸，当为极性者时则特别是含2-6个、通常是含2-4个碳原子的酸性氨基酸，更特别是G、A、P、D、E;
aa28是含脂族带电荷且有4-6个碳原子的氨基酸，其可以是碱性或酸性的，特别是D、E、K、R;
aa29是脂族或芳族氨基酸，当其为脂族时则含3-5个碳原子，特别是A、P、V、F、H、W、Y;
aa30是脂族中性非极性或碱性氨基酸，当为中性非极性者时则含4-6个，通常是5-6个碳原子，特别是I、L、V、K、R;
aa36是脂族碱性氨基酸，特别是K、R;
aa37是脂族或芳族氨基酸，当为脂族氨基酸时则含有2-3个碳原子，特别是G、A、F、H、W、Y;
aa38是含2-4个碳原子的脂族中性极性或非极性氨基酸，当为极性的时则特别是羟基取代的，更特别是G、A、P、S、T;
aa39是含2-6个碳原子，特别是含2-3个碳原子的脂族非极性氨基酸，特别是G、A、P、I、L、V。
各种氨基酸按取代基分类及一字母表示法如下：
脂族
中性
非极性 G、A、P、V、I、L
极性 S、T、M、C、N、Q
带电荷
酸性 D、E
碱性 K、R
芳族 F、H、W、Y
G-甘氨酸 N-天冬酰胺
A-丙氨酸 Q-谷氨酰胺
P-脯氨酸 D-天冬氨酸
V-缬氨酸 E-谷氨酸
I-异亮氨酸 K-赖氨酸
L-亮氨酸 R-精氨酸
S-丝氨酸 F-苯丙氨酸
T-苏氨酸 H-组氨酸
M-蛋氨酸 W-色氨酸
C-半胱氨酸 Y-酪氨酸
特别有用的是有下列结构式的肽：
SQCEQEGGF HCR KFLL VI
CP LSRT SSDI LGKL MGCEPLWKCCK
R KWGG
其中：
当在同一位点上标示有两个氨基酸时，则两者均可能存在于该位点上，但优选的情况是上边一列是同时出现的，而且下边的一列也是一起存在的。
已进一步了解到，保留置换是可以允许的。借助保留置换，下列在同一列上的氨基酸可互相置换。
G，A
V，I，L
D，E
K，R
S，T
F，H，W，Y
肽的N末端可以是被封闭或未封闭的，封闭的方法通常包括使一脂族酸（如甲酸或乙酸）产生烷基化等。在本文实验部分所述的试验程序下，已发现主题化合物对热和酸是稳定的。
其中特别重要的是天然存在的生长抑制肽，其至少有90%、较好是95%、更好是99%的纯度。其可单独使用或与其它毒素联合使用。
可依惯用技术，使本主题细胞毒性或生长抑制化合物与各种各样的配基及受体结合。参予连结的功能基可以是酸性基团，如羰基、磺酰基和磷酰基、硫羟与烯烃的结合物、二硫代、醛、偶氮基、重氮基等。其中大部分是使用双功能化合物，其可分步反应，但亦可使用能同时反应的双功能化合物。可作举例说明的试剂包括戊二醛、甲醛、对位马来酰亚胺苯甲酸、甲基二硫代乙酸、重氮苯甲酸等。对于将细胞毒性部分与特异性结合部分结合的特定方式，本发明並不作严格限定。
当特异性结合部分提供与特殊靶的特异结合时，特异性结合部分应是配基和受体。例如，细胞正常情况下具有表面抗原以及由特殊细胞类型所限定的特异性受体。因此，使用适当的配基或受体结合毒剂，毒剂即可选择性地针对具有相对应的特异性结合部分的细胞发挥作用。
可用作配基的各种化合物包括类固醇、低密度脂蛋白、生长因子、病毒蛋白等。相反，如果受体是针对特异性表面抗原的，则可使用天然受体或优选的免疫球蛋白或它们的片段。有用的免疫球蛋白包括IgA、IgD、IgM、IgE和IgG，其中最好是包括任一亚型的IgG。免疫球蛋白可得自任何方便的来源，特别是小鼠或人。免疫球蛋白亦可得自杂交瘤细胞，或被转化了的细胞如EBV转化的淋巴细胞，或者通过DNA重组技术制得。特别是，小鼠可变区可与人或其它宿主恒定区结合以提供具有较低抗原性的嵌合免疫球蛋白。另外，可不必使完整的免疫球蛋白，而是用其片段，如Fab、F（ab′）2、Fv等片段。
可借助键使配基与毒素结合，这样的键可以是稳定的，或者在靶细胞中是不稳定的，以使毒素与靶试剂能在细胞中被分离。可以期望毒素被内吞至细胞内，以便在细胞内发挥其作用。如靶试剂对毒素的毒性无不利影响，则不一定要有可裂解的键。如要求用可裂解的键，则比较方便的是利用二硫键，其在宿主细胞内可被还原。
结合物之靶试剂、特异性结合部分和毒素间可有各种不同的比例。一般说来，每分子靶试剂有至少一分子毒素，但每0.5千道尔顿（KD）靶试剂不多于约1分子毒素。通常是每100KD靶试剂至少1分子毒素，特别是每50KD靶试剂至少1分子毒素。如果使用小分子靶试剂如低分子量半抗原，则每分子毒素可有2个或2个以上的配基，通常是每分子毒素有约5个以下的配基分子。
本主题化合物可于体外或体内使用。如在体外使用，它们可用于破坏培养物内或组织内可区别于其它细胞的某些细胞。可将本主题化合物以足以破坏预清除之细胞的量加到培养基内。例如，在检测特殊的组织相容性抗原中，可将含有对该特殊组织相容性抗原特异之抗体的试剂加入培养基内。经加入只能被死细胞吸附的染料，细胞中存在颜料即指示出特殊的组织相容性类型。击中靶试剂之毒素的量可有很大差异，並应使之最适化以达适于体外使用的特定状况。击中靶试剂的毒素不应使用过多，以防导致非特异结合及假阳性结果;但同时亦不应过少，否则将因结合水平低不易检测而出现假阴性结果。
当作体内使用时，该结合物可经胃肠道外途经或注射给药，特别是静脉内给药。根据将被杀死之细胞的性质、细胞群体的范围、细胞对结合物的抗性、结合物的效力等，所用结合物的量可有很大程度的变异。当在特异位点以外给药时，所用蛋白的量为每公斤宿主体重约1μg至10mg，通常达2mg。如在特异位点给药时，所用蛋白的量应是上述浓度范围的较低部分。可将结合物加在生理上可接受的基质如磷酸盐缓冲盐水、盐水或其它适用的载体内使用。作为粉末，可将结合物与其它物质组合，以使所得组合物被引向特定器官並能迟缓释放等。将蛋白组合物引入宿主的方法很多，且为本领域内所熟知的，在此不作更详细的讨论。
作为靶细胞，特别引人关注的是肿瘤细胞和病原微生物。而其中更突出的是广泛出现的肿瘤如肺癌、结肠和直肠癌、乳腺癌、子宫癌、前列腺癌、膀胱癌和胃癌、淋巴瘤、白血病及何杰金氏病的癌细胞。
可作举例说明的病原微生物是原生动物，如Plasmodium vivas、P.maleriae、P.ovale和P.falciparum、革兰氏阴性细菌如假单胞菌属、克雷氏菌属和萘瑟氏菌属、以及革兰氏阳性细菌等。
实验部分
材料和方法
由大响尾蛇（Crotalus atrox）毒液中纯化生长抑制肽
提取大响尾蛇毒液得到粗制肽化合物，並于冷干前经低速离心澄清之。将冻干的毒液溶解于1M醋酸内並经低速离心除去不溶性物质。
将7.5ml样品加于用1M醋酸予平衡的Bio-Gel P10柱上层析分离，收集各管洗脱部分（3.5ml）並除去等分部分，之后冻干完成初步纯化。将各等分部分加于A549人肺癌细胞培养物内以试验其对DNA合成的抑制作用。简单地说，即是将4×104个A549细胞接种于96孔板内，附着后用适当的柱洗脱部分处理细胞。5天后加125Ⅰ脱氧尿苷于各孔内並测定脉冲数，根据125Ⅰ-脱氧尿苷掺入的多少，与对照孔比较测定洗脱部分对细胞DNA合成的影响。可见到两个主要的抑制作用峰。用高压液相层析法（HPLC）进一步纯化在接近6000Mr胰岛素标志物处洗脱的低分子量抑制活性之第一个峰值部分。
冻干样品並重新悬浮于0.05%三氟醋酸（TFA）（溶液用HPLC级纯化水〔Water associates〕配制）内。离心除去不溶性物质並将样品注入C18-Novapak柱内。用溶于0.05%TFA的20至60%乙腈线性梯度洗脱样品，温度22℃，流速1ml/分。分别收集每份A214吸附肽並试验各等分部分的DNA合成抑制作用。54%乙腈洗脱的肽抑制了A549细胞的DNA合成，但没有看到柱上洗脱的其它部分有抑制作用。这一活性部分的SDS-PAGE显示出一单一的银染色带;但凝胶上没有看到其它带，此提示肽已被纯化为均一物质。
被抑制细胞的形态学
证明被抑制的细胞经用定名为“生长抑制肽”（GAP）的肽处理24小时后出现一明显的形态学改变。在这一检测试验中，人肿瘤细胞和未转化的成纤维细胞均受到浓度为大约100ng/ml之肽的抑制。经处理后细胞变为园形，而且大多数树枝样隆起均已显而易见地消失，但仍粘附在平板上。当用洗液洗处理之细胞后这一效应呈现为不可逆转的，而且用含10%血清之新鲜培养基重新培养时亦不能使细胞恢复其原来的表型。
GAP的化学结构
用微序列分析法测定GAP的氨基酸序列，该肽是用（1）蛋白内切酶赖氨酸-C;（2）TPCK-胰蛋白酶（L-（1-甲基磺酰胺-2-苯基-乙基氯甲基酮）;和（3）金黄色葡萄球菌V8酶促消化已还原的和S-羧酰胺甲基化的GAP得到的。使用挥发性溶剂经反相HPLC纯化肽片段。将氨基末端封闭的肽在12NHCI中于室温下保温16小时。之后经冻干以使样品干燥。样品在470A型气相蛋白序列仪（Applied Biosystems，Inc.）中经受自动Edman降解。以rpHPLH法分析乙内酰苯硫脲氨基酸。
GAP的氨基酸序列如下：
该结构是经微序列分析法测定的。简单地说，先用二硫苏糖醇还原GAP，用碘乙酰胺使之S-羧酰胺甲基化，並取10%等份部分作自动Edman降解处理。既使经几个周期的Edman降解后亦未检测出N末端氨基酸，此提示GAP的末端氨基酸已被封闭。
用赖氨酸-C酶消化45%等份的S-羧酰胺甲基化的GAP。经rpHPLC分离肽K-24和K25-32。肽K33-35和K36-39未被柱保留而以混合物的形式被洗脱。没有试图去纯化这些肽。分别取各50%等份的K1-24和K25-32作自动Edman降解。测定K1-24的氨基酸序列时没有检测到N末端氨基酸。继后用TPCK-胰蛋白酶消化其余50%等份的K1-24。经rpHPLC分离用胰蛋白酶消化的肽T1-11、T12-18和T19-24，並进行Edman降解。在测定50%等份之肽T1-11的氨基酸序列时没有检测到N末端氨基酸，此提示T1-11是K1-24的N末端肽。继后用12NHCI处理使其余50%等份之T1-11去封闭，並测定酸处理之T1-11的氨基酸序列。T12-18含有一C末端精氨酸残基，而K1-24含有一C末端赖氨酸残基，因此假想其为K1-24的羧基末端肽。所提供的数据支持所提出之GAP的自残基1-32的氨基酸序列。
用金黄色葡萄球菌V8消化其余45%等份的S-羧酰胺甲基化的GAP。经rpHPLC分离肽E1-6、E7-21和E22-39。取50等份的肽E1-6、E7-21和E22-39作自动Edman降解。结果肽E1-6没有检测到N末端氨基酸。E7-21和E22-39的完整序列扩展了所提出的GAP的从残基1-39的结构，並进一步证实了对肽T12-18、T19-24和K25-32的序列推断。之后用12NHCI处理50%等份的肽E1-6使之去封闭，此可能通过Ser-1位上某酸催化的N-O乙酰基转移达到。测定酸处理之E1-6的序列。因为未经羧基肽酶处理GAP以肯定Gly-39是C末端氨基酸，所以在残基Gly-39后面的结构中可能存在有另外的肽。然而，GAP和响尾蛇毒蛋白（Crotamine）之间、肌毒素A和毒性肽C之间的广泛的序列相符却为假定的结构提供了有力的证据。
从结构上说，GAP属于响尾蛇毒素族，为一类通过损伤内质网（作为初级靶结构）引起肌肉变性的多肽。与储存于蛋白序列数据库（PIR Release 5.0，1985年5月）中的所有蛋白质序列比较，没有发现GAP与其它已知序列间有任何广泛的相符。在GAP的残基10和11之间插入两个缺失，可将GAP和响尾蛇毒素的序列连成一线相对排列，从而使半胱氨酸残基展示有相似位置。在GAP中存在有六个“半”一胱氨酸残基，提示多肽中有三个二硫桥键。就氨基酸序列来说，上面指出的氨基酸序列可有广泛的变异，同时保留有相一致的结构和细胞毒活性。
按照已知的方法学，可用本主题毒素代替蓖麻蛋白、白喉毒素、相思豆毒素等。（如见Jansen等，Recept.-Mediated Biding Intern.Toxins Horm.〔Proc.Symp.〕1980（1981年出版）351-356（C.A.95：126185μ）;Masuho等，Biochem.Biophys.Res.Comm.（1979）90：320-326;美国专利4340535号）。
虽然为清楚了解起见已通过图解和实施例在一定程度上详细地描述了前述发明，但应明确地是，实践中可在不超出待批权利要求之范围的前提下进行某些改变和改动。
Claims (13)
1、有下列结构式的毒性肽：
PP1QC1aa4aa5aa6GGaa9C2aa11aa12aa13aa14C3aa16aa17aa18aa19SDaa22GKaa25aa26C4aa28aa29aa30WKC5C6Kaa36aa37aa38aa39PP2
其中：
PP1是N末端，且是氢或有1-20个氨基酸的氨基酸链；
PP2是C末端且可以是羟基、或是有1-20个氨基酸的氨基酸链；
在一字母氨基酸密码中所提供的各个字母均有其标准的意义；
多达5个氨基酸指定作为结合体；
当存在半胱氨酸桥时，则C1与C5、C2和C4、C3和C6配对结合；
aa4是脂族酸性或芳族氨基酸；
aa5是脂族极性或碱性氨基酸；
aa6是脂族带电荷氨基酸；
aa9是芳族氨基酸；
aa11是脂族碱性氨基酸；
aa12是芳族氨基酸或脂族极性或带电荷氨基酸；
aa13是脂族非极性或带电荷氨基酸；
aa14是脂族非极性氨基酸；
aa16是有4至6个碳原子的脂族非极性氨基酸；
aa17是有3至5个碳原子的脂族非极性或极性氨基酸；
aa18是有3至6个碳原子的脂族非极性或碱性氨基酸；
aa19是有3至4个碳原子的脂族极性氨基酸；
aa22是脂族非极性或芳族氨基酸；
aa25是有3至6个碳原子的脂族中性氨基酸；
aa26是有2至5个碳原子的脂族带电荷的或非极性氨基酸；
aa28是有4至6个碳原子的脂族带电荷氨基酸；
aa29是有3至5个碳原子的脂族带电荷氨基酸或是芳族氨基酸；
aa30是有4至6个碳原子的脂族非极性或碱性氨基酸；
aa36是脂族碱性氨基酸；
aa37是有2至3个碳原子的脂族氨基酸或是芳族氨基酸；
aa38是有2至4个碳原子的脂族中性氨基酸；且
aa39是脂族非极性氨基酸，其中N末端可以是被封闭的或未封闭的。
2、根据权利要求1的毒性肽，其中所说的N末端是末被封闭的。
3、根据权利要求1的毒性肽，其中PP1是氢且PP2是羟基。
4、根据权利要求3的毒性肽，其中N末是被封闭的。
5、有下列结构式的毒性肽：
SQCEQEGGF HCR KFLL VI
CP LSST SSDI LGKL MGCEPLWKCCK
R KWGG
其中当在同一位点指出有两个氨基酸时，可存在有任一个氨基酸。
6、根据权利要求5的毒性肽，其结构中只存在上面的那些氨基酸。
7、根据权利要求5的毒性肽，其结构中只存在有底下的那些氨基酸。
8、一种包含根据权利要求1之毒性肽的毒性肽结合物，其中毒性肽共价结合于特异性结合对之一者。
9、根据权利要求8的毒性肽结合物，其中所说的特异性结合对之一是配基。
10、根据权利要求8的毒性肽结合物，其中所说的特异性结合对之一是受体。
11、根据权利要求10的毒性肽结合物，其中所说的受体是一种抗体。
12、一种包含共价结合于一特异性结合对之一者的得自大响尾蛇之毒素的毒性肽结合物。
13、一种抑制细胞混合物内予定细胞群之生长的方法，其包括：
使所说的细胞的混合物与根据权利要求10的毒性肽结合物结合，其中所说的受体对细胞的所说群体之表面上的抗原是特异的，具有足以抑制所说的细胞生长的量，从而使所说的细胞之生长受到抑制。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/801,019 US4731439A (en) | 1985-11-22 | 1985-11-22 | Snake venom growth arresting peptide |
| US801,019 | 1985-11-22 |
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| Publication Number | Publication Date |
|---|---|
| CN86108593A true CN86108593A (zh) | 1987-09-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN198686108593A Pending CN86108593A (zh) | 1985-11-22 | 1986-11-22 | 蛇毒液生长抑制肽 |
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| Country | Link |
|---|---|
| US (1) | US4731439A (zh) |
| KR (1) | KR900006575B1 (zh) |
| CN (1) | CN86108593A (zh) |
| AU (3) | AU6556386A (zh) |
| BE (1) | BE905790A (zh) |
| CH (1) | CH670451A5 (zh) |
| DE (1) | DE3639796A1 (zh) |
| DK (1) | DK560986A (zh) |
| ES (1) | ES2002915A6 (zh) |
| FI (1) | FI864715A (zh) |
| FR (1) | FR2590576A1 (zh) |
| GB (1) | GB2185023B (zh) |
| GR (1) | GR862788B (zh) |
| IT (1) | IT1207580B (zh) |
| LU (1) | LU86683A1 (zh) |
| NL (1) | NL8602939A (zh) |
| PT (1) | PT83790B (zh) |
| SE (1) | SE8604991L (zh) |
| YU (1) | YU199586A (zh) |
| ZA (1) | ZA868849B (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU5939890A (en) * | 1989-06-07 | 1991-01-07 | Genentech Inc. | Platelet aggregation inhibitors and related molecules |
| WO1991001740A1 (en) * | 1989-08-05 | 1991-02-21 | Varánusz Gm | Process for producing pharmacosmetics |
| US5232911A (en) * | 1990-01-03 | 1993-08-03 | Ventech Research Inc. | Mixture of a non-covalent heterodimer complex and a basic amphiphatic peptide as cytotoxic agent |
| US5766897A (en) * | 1990-06-21 | 1998-06-16 | Incyte Pharmaceuticals, Inc. | Cysteine-pegylated proteins |
| DE4433890C2 (de) * | 1994-09-22 | 1999-02-18 | Deutsches Krebsforsch | Konjugat aus einem Wirkstoff und einem nicht als körperfremd angesehenen, nativen Protein |
| SG129211A1 (en) * | 1997-11-06 | 2007-02-26 | Univ Singapore | Therapeutic molecules |
| BR0101088A (pt) * | 2001-03-19 | 2003-03-18 | Biolab Sanus Farmaceutica Ltda | Processo de isolamento e purificação de peptìdeos inibidores das vasopeptidases, com especificidade para o sìtio carboxìlico da enzima conversora da angiotensina, secretados pelas glândulas do veneno de serpentes (bpps), particularmente bothrops jararaca, ou produzidos endogenamente (evasins) possuindo ação vasodilatadora e anti-hipertensiva; processo de determinação da sequência de amido-ácidos dos peptìdios inibidores secretados pela glândula de veneno de serpentes (bpps) ou endógenos (evasins); processo de determinação da sequência de aminoácidos dos bpps por dedução do cdna dos precursores dessas moléculas expressos em tecidos de serpentes, especificamente bothrops jararaca. processo de determinação da sequência de aminoácidos dos evasins por dedução do cdna dos precursores dessas moléculas expressos em tecidos de serpentes, especificamente bothrops jararaca, processo de amplificação do cdna a partir das bibliotecas de cdna de pâncreas e/ou cérebro de serpentes, especificamente bothrops jararaca; processo de sìntese em fase sólida de peptìdeos inibidores das vasopeptidases com ação vasodilatadora e anti-hipertensiva, peptìdeos inibidores das vasopeptidases com ação anti-hipertensiva; utilização dos peptìdeos inibidores das vaso peptidases com ação vasodilatadora e anti-hipertensiva na obtenção de composições farmacêuticas; processo de determinação da atividade inibitória sobre as vasopeptidases e de atividade biológica sobre músculo liso, sistema cardiovascular e microcirculatório. |
| BRPI0501037B8 (pt) * | 2005-03-18 | 2021-07-27 | Fund De Amparo A Pesquisa Do Estado De Sao Paulo | uso de crotamina e composição |
| US20060234934A1 (en) * | 2005-04-15 | 2006-10-19 | Kilmon Joseph A | Composition and Method for Selective Cytostasis |
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| JPS5836308B2 (ja) * | 1977-02-10 | 1983-08-08 | 大塚製薬株式会社 | 抗体の製造方法 |
| US4220722A (en) * | 1978-02-10 | 1980-09-02 | Syva Company | Method for conjugating to polyamino compounds employing haloacyl groups and compositions prepared thereby |
| FR2437213A1 (fr) * | 1978-09-28 | 1980-04-25 | Cm Ind | Produits cytotoxiques formes par liaison covalente de la chaine a de la ricine avec un anticorps et leur procede de preparation |
| US4500637A (en) * | 1982-07-19 | 1985-02-19 | The United States Of America As Represented By The Department Of Health And Human Services | Prevention of graft versus host disease following bone marrow transplantation |
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1985
- 1985-11-22 US US06/801,019 patent/US4731439A/en not_active Expired - Fee Related
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1986
- 1986-11-19 FI FI864715A patent/FI864715A/fi not_active Application Discontinuation
- 1986-11-19 NL NL8602939A patent/NL8602939A/nl not_active Application Discontinuation
- 1986-11-19 BE BE0/217441A patent/BE905790A/fr not_active IP Right Cessation
- 1986-11-20 GB GB8627710A patent/GB2185023B/en not_active Expired - Fee Related
- 1986-11-21 DE DE19863639796 patent/DE3639796A1/de not_active Ceased
- 1986-11-21 ES ES8603138A patent/ES2002915A6/es not_active Expired
- 1986-11-21 SE SE8604991A patent/SE8604991L/xx not_active Application Discontinuation
- 1986-11-21 IT IT8622425A patent/IT1207580B/it active
- 1986-11-21 CH CH4672/86A patent/CH670451A5/de not_active IP Right Cessation
- 1986-11-21 YU YU01995/86A patent/YU199586A/xx unknown
- 1986-11-21 AU AU65563/86A patent/AU6556386A/en not_active Abandoned
- 1986-11-21 FR FR8616270A patent/FR2590576A1/fr not_active Withdrawn
- 1986-11-21 GR GR862788A patent/GR862788B/el unknown
- 1986-11-21 LU LU86683A patent/LU86683A1/fr unknown
- 1986-11-21 ZA ZA868849A patent/ZA868849B/xx unknown
- 1986-11-21 PT PT83790A patent/PT83790B/pt not_active IP Right Cessation
- 1986-11-21 DK DK560986A patent/DK560986A/da not_active Application Discontinuation
- 1986-11-22 CN CN198686108593A patent/CN86108593A/zh active Pending
- 1986-11-22 KR KR1019860009886A patent/KR900006575B1/ko not_active IP Right Cessation
- 1986-11-24 AU AU65609/86A patent/AU6560986A/en not_active Abandoned
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1990
- 1990-11-16 AU AU66675/90A patent/AU6667590A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| BE905790A (fr) | 1987-05-19 |
| FI864715A0 (fi) | 1986-11-19 |
| NL8602939A (nl) | 1987-06-16 |
| AU6556386A (en) | 1987-05-28 |
| US4731439A (en) | 1988-03-15 |
| FI864715A (fi) | 1987-05-23 |
| IT8622425A0 (it) | 1986-11-21 |
| AU6667590A (en) | 1991-03-28 |
| FR2590576A1 (fr) | 1987-05-29 |
| GB8627710D0 (en) | 1986-12-17 |
| KR900006575B1 (ko) | 1990-09-13 |
| GB2185023A (en) | 1987-07-08 |
| ES2002915A6 (es) | 1988-10-01 |
| GB2185023B (en) | 1990-08-29 |
| DE3639796A1 (de) | 1987-07-09 |
| KR870005014A (ko) | 1987-06-04 |
| IT1207580B (it) | 1989-05-25 |
| GR862788B (en) | 1987-03-26 |
| CH670451A5 (zh) | 1989-06-15 |
| SE8604991D0 (sv) | 1986-11-21 |
| ZA868849B (en) | 1987-07-29 |
| LU86683A1 (fr) | 1987-06-26 |
| PT83790B (pt) | 1989-09-14 |
| YU199586A (en) | 1988-06-30 |
| AU6560986A (en) | 1987-05-28 |
| PT83790A (en) | 1986-12-01 |
| DK560986D0 (da) | 1986-11-21 |
| DK560986A (da) | 1987-05-23 |
| SE8604991L (sv) | 1987-05-23 |
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