CN1147853A - 免疫抑制剂的定量法 - Google Patents
免疫抑制剂的定量法 Download PDFInfo
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- CN1147853A CN1147853A CN95192949.6A CN95192949A CN1147853A CN 1147853 A CN1147853 A CN 1147853A CN 95192949 A CN95192949 A CN 95192949A CN 1147853 A CN1147853 A CN 1147853A
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- calcineurin
- compound
- immunophilin
- antibody
- hydroxyl
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Abstract
本发明涉及一种测定具有钙调神经磷酸酶(Calcineurin)抑制活性的免疫抑制剂的方法及试剂盒,它是通过测定一种含免疫亲和素(immunophilin),钙调神经磷酸酶,钙调素,钙离子以及一种具有钙调神经磷酸酶抑制活性的免疫抑制剂的复合物来完成的。根据本发明,在测定血液中的具有钙调神经磷酸酶抑制活性的免疫抑制剂如FK506或环孢菌素A的水平时就有可能更准确的确定具有真正具有免疫抑制剂效果的物质的浓度。
Description
技术领域
FK506和环孢菌素A是一类能够阻止钙调神经磷酸酶(Calcineurin)的作用的免疫抑制剂。本发明即是有关这类免疫抑制剂的正确定量方法的,能应用于医学领域。
背景技术
以下面的结构式和化学名称表示FK506或称FR-900506,这种化合物有很强的免疫抑制作用,可用于器官移植的排斥反应或自体免疫疾病的预防药物和治疗药物,这是众所周知的(如EP-0184162-A2)。
化学名称:17-烯丙基-1,14-二羟基-12-[2-(4-羟基-3-甲氧基环己基)-1-甲代乙烯基]-23,25-二甲氧基-13,19,21,27-四甲基-11,28-二氧杂-4-氮杂三环[22.3.1.04,9]二十八碳-18-烯-2,3,10,16-四酮。
对FK506免疫抑制作用机理进行研究后认为,FK506与细胞质內的FK506结合蛋白FKBP-12结合后,同钙调素和钙离子等一起与钙调神经磷酸酶形成复合物(FK506∶FKBP-12∶钙调神经磷酸酶=1∶1∶1)。结果,钙调神经磷酸酶的磷酸酶活性失去,这种磷酸酶失去活性便导致活性T细胞的核因子(NFAT)的活化受到抑制,阻止了IL2的产生,最终起到了免疫抑制作用。
另外,人们认为显示相同免疫抑制作用的环孢菌素A也形成同样的复合物而具有免疫抑制作用的。(如参考生物化学与生物物理研究通信,192(3),1388-1394(1993)和Angew,Chem,Int.Ed Engl,31(1992)384-400)。
作为免疫抑制剂,FK506的可用性得到了较好的研究,在日本特别作为肝移植时的排斥反应抑制剂已经成为商品出售。
但是,由于其抑制作用极强,所以最佳给药剂量便成了重要问题。适宜的给药剂量在避免副作用发生及发挥有效的免疫抑制活性作用方面都是极为重要的。
因此,人们提出了几种测定方法(如参考Clin,Chem.38/7,1307-1310(1992))。如利用能识别FK506中抗原决定簇的抗体的酶免疫测定法(如EP-0293892-A2)、上述酶免疫测定法和HPLC联合法以及利用FK506结合蛋白(FKBP-12)的放射受体法等。
一方面,由代谢机理可知,FK506在生物体内进行代谢,其代谢产物中有某些物质混在其中,如仍具有免疫抑制活性的物质或与FK506相对的能与单克隆抗体结合的物质等,这种物质的免疫抑制活性极弱(如药物代谢与处置,21(6),971-977(1993))。
另外,还有与FK506结合蛋白(FKBP)结合的、不具有免疫抑制活性的物质存在(如506BD)。(如Angew,Chem,Int,Ed,Eng1.31(1992)384-400)。
因此,能识别FK506抗原决定簇的抗体与FK506的结合、FKBP和FK506的结合,仅仅以此为指标的过去的那些测定方法,很难说是正确测定了实际的免疫抑制状态。可望能开发一种正确测定包括具有免疫抑制活性的代谢产物在內的活性药物总浓度的方法。
发明内容
本专利的发明者们基于FK506或环孢菌素A这一类的免疫抑制剂与免疫亲和素(immunophilin)(与FKBP-12或环啡林(Cyclophilin)这类免疫抑制剂相结合的蛋白)结合后,与钙调神经磷酸酶(Calcineurin)、钙调素、钙离子形成复合物可阻断钙调神经磷酸酶的活性这一事实,利用它们的复合物形成,成功确定了免疫抑制剂的定量法。
即本发明提供了由(1)免疫亲和素、(2)钙调神经磷酸酶、(3)钙调素、(4)钙离子及(5)对具有阻断钙调神经磷酸酶作用的由免疫抑制剂组成的复合物进行定量发析,由复合物的定量而得具有阻断钙调神经磷酸酶作用的免疫抑制剂的定量法。
下面就本发明范围中包含的各种定义及成功的实例子以详细说明。
“免疫亲和素”是指与免疫抑制剂结合的细胞质內的受体蛋白。例如,分子量约为12K的FK506结合蛋白中具有PPIase活性的FKBP-12、环孢菌素A的细胞受体中同样具有肽基脯氨酰反式-顺式异构酶(PPIase)活性的、分子量约17K的免疫亲和素等。较为理想的是牛和人类哺乳动物产生的FKBP-12和环啡林。
这是众所周知的。在美国化学联合会杂志113,1409-1411(1991)及美国国家科学院院刊88,6229-6233(1991);自然346,671-674(1991);WO92/01052;WO91/17439;自然,337,473-475,476-478(1989);特开平2-209897等资料均有同样说明。
钙调神经磷酸酶是钙离子和钙调素赖以存在的丝氨酸/苏氨酸磷酸酶这是众所周知的,可由鼠、牛和人类哺乳动物获得并使用。例如大鼠和牛的钙调神经磷酸酶是由A、B两种亚基形成的杂二聚体,而A亚基又有Aα和Aβ两种,这可由大鼠脑中分离、纯化而得(如参考神经化学杂志58,1643-1651(1992))。另外,牛的钙调神经磷酸酶参照酶学进展61,149-200(1989)或酶学方法102,244-256(1983)即可获得。另外Upstate Biotechonology有限公司和Sigma公司以蛋白钙调神经磷酸酶等商品在市场上销售,这些均可使用。
“钙调素”作为钙结合蛋白是一种众所周知的物质,在钙离子存在下,它可活化前述的钙调神经磷酸酶等的各种酶。牛和人类哺乳动物的钙调素即可使用。如牛的钙调素与生物化学杂志87,1313-1320(1980)同样承制,即可获得。也可以使用Upstate Biotechnology有限公司或Sigma公司的销售产品。
本发明中要定量的“具有抑制钙调神经磷酸酶作用的免疫抑制剂”,是指由免疫亲和素、免疫亲和素钙调神经磷酸酶、钙调素和钙离子共同形成复合物,使钙调神经磷酸酶的磷酸酶活性失去,从而具有免疫抑制活性的化合物。较为理想的例子,如下所示的化合物: [I]式中,R1为羟基或被保护的羟基;R2为氢、羟基或被保护的羟基;R3为甲基、乙基、丙基或烯丙基;R4为羟基或甲氧基;R5为氢或R4、R5一起形成氧代;n为1或2的整数,实线和虚线表示的符号是指单键或双键。但R4为羟基同时R5为氢,或R4、R5一起形成氧代时(R2不是被保护的羟基)。
上述通式〔I〕中使用的符号说明及后记说明中使用的“低级”一词,如无特别指明,就是指碳原子数为1~6个。
“被保护的羟基”中合适的保护基团如下例示:甲硫甲基、乙硫甲基、丙硫甲基、异丙基硫甲基、丁硫甲基、异丁基硫甲基、己硫甲基等低级烷基硫甲基一类的l-(低级烷基硫)(低级)烷基、优选的为C1~C4的烷基硫甲基、最优选的是甲硫甲基;
三甲基甲硅烷基、三乙基甲硅烷基、三丁基甲硅烷基、叔丁基-二甲基甲硅烷基、三叔丁基甲硅烷基等三(低级)烷基甲硅烷基;甲基二苯基甲硅烷基、乙基二苯基甲硅烷基、丙基二苯基甲硅烷基、叔丁基二苯基甲硅烷基等低级烷基-二芳基甲硅烷基,象这些三取代甲硅烷基中,较好的是三(C1~C4)烷基甲硅烷基及C1~C4烷基二苯基甲硅烷基,最好的是叔丁基二甲基甲硅烷基和叔丁基二苯基甲硅烷基;还有由羧酸、磺酸和氨基甲酸衍生的脂肪酰基、芳香酰基以及以芳基取代的脂肪酰基等。
关于上述酰基,举出更为详细的具体实例进行如下说明。
脂肪酰基如甲酰基、乙酰基、丙酰基、丁酰基、异丁酰基、戊酰基、异戊酰基、三甲基乙酰基、己酰基、羧乙酰基、羧丙酰基、羧丁酰基、羧己酰基等可含有一个以上羧基之类适当取代基的低级链烷酰基;如环丙氧乙酰基、环丁氧丙酰基、环庚氧丁酰基、氧乙酰基、氧丙酰基、氧丁酰基、氧戊酰基、氧己酰基等可含有一个以上被低级烷基这种适当的取代基所取代的环基(低级)烷氧基(低级)链烷酰基、樟脑磺酰基如羧甲基氨基甲酰基、羧乙基氨基甲酰基、羧丙基氨基甲酰基、羧丁基氨基甲酰基、羧戊基氨基甲酰基、羧己基氨基甲酰基等羧基(低级)烷基氨基甲酰基;另外如三甲基甲硅烷基甲氧羰基乙基氨基甲酰基、三甲基甲硅烷基乙氧羰基丙基氨基甲酰基、三乙基甲硅烷基乙氧羰基丙基氨基甲酰基、叔丁基二甲基甲硅烷基乙氧羰基丙基氨基甲酰基、三甲基甲硅烷基丙氧羰基丁基氨基甲酰基等三(低级)烷基甲硅烷基(低级)烷氧羰基(低级)烷氨甲酰基之类保护羧基(低级)烷基氨基甲酰基的含有一个以上羧基或保护羧基取代的烷基氨基甲酰基。
芳酰基如苯甲酰、甲苯酰、二甲苯甲酰、萘酰、硝基苯甲酰、二硝基苯甲酰、硝基萘甲酰等可含有一个以上为硝基等适当基团取代的芳酰基,如苯磺酰、甲苯磺酰、二甲苯磺酰、萘磺酰、氟代苯磺酰、氯代苯磺酰、溴代苯磺酰、碘代苯磺酰等可含有一个以上卤素之类适当取代基的芳磺酰基。
以芳基取代的脂肪酰基如苯乙酰、苯丙酰、苯丁酰、2-三氟甲基-2-甲氧基-2-苯乙酰基、2-乙基-2-三氟甲基-2-苯乙酰基、2-三氟甲基-2-丙氧基-2-苯乙酰基等可含有一个以上(低级)烷氧基和三卤化(低级)烷基一类适当取代的链烷酰基。
上述酰基中,较好的酰基是:可含羧基的C1~C4的链烷酰基,环烷基部分含有2个(C1~C4)烷基的环(C5~C6)烷氧基(C1~C4)链烷酰基、樟脑磺酰基、羧基(C1~C4)烷基氨基甲酰基、三(C1~C4)烷基甲硅烷基(C1~C4)烷氧羰基(C1~C4)烷基氨基甲酰基,可含一、二个硝基的苯酰基、含有卤素的苯磺酰基、含有C1~C4烷氧基和三卤代(C1~C4)烷基的苯(C1~C4)链烷酰基,其中最为理想的是乙酰基、羧丙酰基、氧乙酰基、樟脑磺酰基、苯酰基、硝基苯酰基、二硝基苯酰基、碘代苯磺酰及2-三氟甲基-2-甲氧基-2-苯乙酰基等。
前述一般式〔I〕所表示的化合物及其合成法已周知,如参照EP-0184162-A2或EP-0353678-A2的方法可得。特别是表示为ER900506(=FK506)、FR900520、FR900523及FR900525的化合物,其获得可参考EP-0184162-A2中记载的链霉菌属,特别是Streptomyces tsukubaensis No.9993 (FERM BP 927号)或吸水链霉菌yakushimaensis亚种NO.7238(FERM BP-928号)
另外,在美国专利4,117,118、4,215,199、4,228,431、4,388,307、Helv.Chim.Acta 60,1568(1977)和65,1655(1982)、Transplant.Proc.17,1362(1985)中可知,如环孢菌素A、B、C、D、E、F、G等的环孢菌素或其衍生物也可与环孢亲和素、钙调神经磷酸酶、钙调素和钙离子形成复合物,成为具有阻断钙调神经磷酸酶作用的免疫抑制剂,因而可利用本发明的定量法进行测定。
本发明中免疫亲和素、钙调神经磷酸酶、钙调素、钙离子及对钙调神经磷酸酶具有阻断作用的免疫抑制剂的复合物,其制得方法没有特别的特点,只是将免疫亲和素、钙调神经磷酸酶、钙调素和钙离子同含有对钙调神经磷酸酶具有阻断作用的免疫抑制剂的试样在适当的溶剂中利用常规方法进行反应而得的。
例如将各成分在适当的缓冲溶液中加热至30~40℃,放置数小时即可。
另外,在医疗实际应用该定量法时,所谓“含有具有对钙调神经磷酸酶起阻断作用的免疫抑制剂的试样”就是指接受对钙调神经磷酸酶起阻断作用的免疫抑制剂的患者的血液或血浆,如按EP-0293892-A2所示进行SepPak处理、或用二氯甲烷、甲醇等提取液进行预处理将是更为理想的。
所得到的复合物,用适当的方法分离后,再以放射性同位素或酶标记各成分,或者利用识别复合物某个部位的抗体,用这种常规方法定量测定复合物,从而能够定量测定目标物免疫抑制剂。
本发明以如下的具体方法实施。另外,以下将“具有对钙调神经磷酸酶有阻断作用的免疫抑制剂”简记为“免疫抑制剂”。(1)将复合物中某一适当的成分以常规的方法固定在金属板、试管或有孔玻璃珠等固相上,这样将复合物固定在固相上,复合物制得后很容易地将其从含有不需要成分的反应液中分离出来。
固定在固相上的,复合物组成成分中适当的成分是指免疫亲和素、钙调神经磷酸酶和钙调素。(1-1)例如固定在固相上的过剩的免疫亲和素和过剩的钙调素、钙离子及含有用来测定钙调神经磷酸酶的免疫抑制剂的试样共同反应,生成同所含免疫抑制剂相当量的复合物。然后,通过反应液的吸滤及适当的缓冲液淋洗,将复合物分离出来。再利用能识别钙调神经磷酸酶或钙调素的适当的酶标记抗体,即可对复合物即形成复合物的免疫抑制剂进行定量测定。
所谓的“识别钙调神经磷酸酶或钙调素的酶标记抗体”,即是利用常规方法将通常用于酶免疫测法的酶(如过氧物酶,β-D-半乳糖苷酶、葡萄糖氧化酶、碱性磷酸酶、乙酰胆碱脂酶、葡糖-6-磷酸脱氢酶、苹果酸脱氢酶或脲酶等)结合起来的抗钙调神经磷酸酶抗体或抗钙调素抗体。
上述酶标记抗体发生作用后,可以通过EP-0293892-A2中所述的常规方法测定标记用的酶的活性,从而定量测定复合物的量。如标记所用酶为过氧物酶时,以0-苯基二胺和过氧化氢水溶液为酶低物溶液;酶为碱性磷酸酶时,以磷酸4-甲基 繖形酮 酯为酶底物溶液,分别测定其显色度,从而可知复合物量。(1-2)(1-1)中的“识别钙调神经磷酸酶或钙调素的酶标记抗体”可用识别钙调神经磷酸酶或钙调素抗体(第一抗体)及识别第一抗体的酶标记抗体(第二抗体)代替。
上述第一抗体及(1-1)中使用的抗钙调神经磷酸酶抗体或抗钙调素抗体,可用以钙调神经磷酸酶或钙调素为抗原而制得的多克隆抗体或单克隆抗体(如神经化学杂志58(5)、1643-1651(1992)),同样可使用由Upstate Biotechnology公司销售的抗钙调神经磷酸酶抗体(=抗蛋白钙调神经磷酸酶)或抗钙调素抗体。这些抗体的类型没有特别限定,但IgG较好、可利用小鼠体免疫或类似的方法所得抗体。
上述的第二抗体,可以用能识别第一抗体的多克隆抗体或单克隆抗体,也可使用将(1-1)中所述的酶以常规方法结合起来的抗体。例如,以碱性磷酸酶标记的抗免IgG市场有售,可从美国的VECTORLabs.研究所购买使用。(1-3)另外,将过量的免疫亲和素固定于固相,如(1-1)所述,制得复合物时,在使用一般的钙调神经磷酸酶或钙调素之前,先利用如前述的酶或放射性同位素(如125I等)标记的钙调神经磷酸酶或钙调素,将生成的与所含免疫抑制剂等量的复合物以酶活性或放射性剂量为指标进行常规测定。(1-4)不仅是免疫亲和素,也可以将过量的钙调神经磷酸酶或钙调素固定于固相。这是加入过量的免疫亲和素钙离子和试样,如前述同样操作可制得并分离复合物,然后利用以常规方法制得的“适当的酶标记的、能识别免疫亲和素的抗体”(酶标记抗免疫亲和素抗体)定量测定复合物量。另外,还可使用如(1-2)中所述的识别免疫亲和素的第一抗体及能识别第一抗体的酶标记第二抗体。(2)组成复合物的成分不固定也可以分离制得的复合物。即:使用能识别复合物某一组成成分的抗体,进行沉淀,分离即可。(2-1)如使用识别钙调神经磷酸酶或钙调素的抗体可将复合物沉淀、分离,这时以含有一定量的用3H一类放射性元素标记的免疫抑制剂来定量测定。(2-2)也可如国际申请公开WO94/04700一样,利用识别制得的免疫亲和素的多克隆和单克隆抗体将复合物沉淀,分离出来。这时将生成复合物时所利用的钙调神经磷酸酶和钙调素与(1-3)同样,以适当的酶或放射性同位素(如125I)标记,然后用常规方法定量测定复合物。
另外,上述(2-2)、(1-3)及(1-4)中用来标记的适当的酶,与(1-1)中所叙述的一样。
前面我们介绍了各种测定方法,较好的是:固定于固相的过量免疫亲和素与过量钙调神经磷酸酶、钙调素、钙离子及样品中的免疫抑制剂反应,生成与免疫抑制剂对应量的复合物,分离处理后,以酶免疫测定法定量测定构成复合物的钙调神经磷酸酶的量,从而定量测定免疫抑制剂。这时,作为酶免疫测定法,最好用过氧物酶或碱性磷酸酶等常用酶标记的抗钙调神经磷酸酶抗体或两种抗体即抗钙调神经磷酸酶抗体和上述酶标记的抗IgG抗体。
本发明的定量法,用自动或非自动(手动)方法均可实行。
另外在应用本定量法时,可提供方便的试剂盒。试剂盒可含有免疫亲和素、钙离子、钙调素、钙调神经磷酸酶和全部或部分作为标准品的免疫抑制剂。也可以含有相应放射性同位素标记的免疫抑制剂、适当的酶或放射性同位素标记的钙调神经磷酸酶或钙调素、适当的酶标记的抗免疫亲和素抗体或抗钙调神经磷酸酶抗体及抗钙调素抗体、抗免疫亲和素抗体、抗钙调神经磷酸酶抗体或抗钙调素抗体等。
另外,在生成复合物时,通常使用钙离子,但其它可与钙调神经磷酸酶、钙调素和免疫抑制剂共同形成复合物的离子也可以使用。如生物化学与生物物理研究通信192(3),1388-1394(1993)用锰离子同样形成复合物并得以确证。
还有,只要对生成的复合物的分离、定量没有影响,可以向由免疫亲和素、钙调神经磷酸酶、钙调素、钙离子和具有阻断钙调神经磷酸酶作用的免疫抑制剂形成的复合物中加入其它成分。
下面由实施例说明本发明。制备例1:FKBP-12的制备
以自然,346,671-674(1990)中哈佛大学S.LSchreiber等报道了DNA序列为基础。由此利用Applied Biosystem公司的DNA合成仪合成了FKBP-12C末端相应的DNA48聚体
5′-CCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATGA-3’
将该48聚体用25P标记并用做探针。对一种T细胞cDNA文库HL1016b 的500,000个菌斑进行筛选,从中得到一个阳性菌斑。从该菌斑中,一个含FKBP-12cDNA亚克隆[pUC-23(Ec)]。该pUC-23(Ec)的测序发现对应于该DNA序列的N末端的第1~32核苷酸缺失。在补齐该缺陷位点之后,该亚克隆与一个约80b.p的富含AT的同义突变的合成的为增强其在大肠杆菌中的表达的合成的N-末端DNA一起插入,该插入是做为EcoRI-BamHI位点插入到一个能在色氨酸启动子控制下进行基因表达的质粒中的,其制备如生物化学杂志101,123-134(1987),中所述,从中可得到一个表达载体,KBP333。使用该载体,将其转入大肠杆菌HB101 中并产生一个表达细胞系,HB101/pFKBP(AT)311。将细胞在L-氨苄青霉素培养基中培养19小时并加入IAA(吲哚乙酸)致终浓度90μg/ml以诱导蛋白合成。收集大肠杆菌细胞并在50mM Tris-HCl,2mM β-ME,2mM CaCl2,10mM MgCl2和5%甘油中用French Press破碎细胞,接着进行离心(4℃,6,000×g,30分钟)。上清在60℃热处理15分钟并且接着离心(4℃,6,000×g,45分钟+4℃,18,000×g,20分钟2次),透析[20mM Tris-HCl(pH7.4,4℃,过夜]并进行DEAE-Toyo Pearl,650M反相HPLC(C4),以纯化FKBP-12。
使用这样得到的FKBP-12和含下述组合物的无钙、镁离子的磷酸缓冲液,制得溶于磷酸缓冲液中的FKBP-12(50μg/ml)。磷酸缓冲液(pH7.4)组合物
氯化钠 8.0g
氯化钾 0.2g
无水磷酸氢二钠(Na2HPO4) 1.15g
无水磷酸二氢钾(KH2PO4) 0.2g
加入蒸馏水至 1000ml。实施例1 FK506的测定法
(1)将制备例1所得磷酸缓冲液FKBP-12(50μg/ml)100μl加到免疫微型滴定板中,将其在4℃下振荡过夜,然后用制备例1同样的磷酸缓冲液洗三次,将FKBP-12固定在孔中。向各孔中加入下述组成的测定缓冲液(300μl),15分钟后去除溶液,各孔中剩余的蛋白结合位点被阻断。
测定缓冲组合物
tris-盐酸盐 (50mM,pH 7.5)
牛血清白蛋白 (5mg/ml)
Triton (0.001%)
DTT (0.5mM)
氯化钙 (1mM)
(2)然后,将遵从下述要点制得的牛钙调神经磷酸酶粗提液(50μl)、牛钙调素液(50μl)及FK506溶液(100μl)的稀释液分别加到各孔中,将板在37℃下放置1小时,使其形成复合物。
牛钙调神经磷酸酶粗提液
将以常规方法配制的溶于20mM tris-盐酸盐(pH=7.0)溶液(5mg/ml)的粗产品用前述测定缓冲液稀释40倍。
牛钙调素溶液
将市场销售的牛钙调素以前述测定缓冲液适当稀释(配制为孔中最终浓度95nM)。
FK506溶液
将FK506的甲醇溶液(1mg/ml)以前述测定缓冲液适当稀释。(3)以不含牛血清白蛋白的前述测定缓冲液洗各孔(4次),然后以众所周知的方法如神经化学杂志58,1643-1651(1992),加入以测定缓冲液适当稀释的识别钙调神经磷酸酶Aα链的兔多克隆抗体(100μl),室温下反应一小时。(4)以不含牛血清白蛋白的前述测定缓冲液洗各孔(4次),然后加入测定缓冲液稀释的以碱性磷酸酶标记的抗兔IgG(100μl)(VectorLaboratories)。(5)以不含牛血清白蛋白的测定缓冲液洗各孔(4次),然后各加200μl以下列方法制得的4-甲基繖形酮磷酸酯(以下简记为4-MU)底物溶液(1mM),室温下经20分钟后利用荧光计2350(商标Millipore<美国>有限公司制造),进行荧光分析(激发波长360nm,发射波长460nm)。结果如表1所示。
表1 FK506及其代谢物的定量测定FK506及其 荧光强度(平均值±标准误差(n=3))代谢物浓度
(ng/ml) FK506 M-II M-III
0 762.8±192.3 885.3±34.6 948.3±16.3
3.9 1302.3±4.9 1318.8±9.9 962.3±12.0
7.8 1591.3±41.7 1639.8±36.8 967.8±43.8
15.6 1933.3±112.4 1883.3±98.3 936.8±0.0
31.3 2131.3±119.5 2188.8±0.0 892.8±134.4
62.5 2753.8±67.9 2472.3±55.9 1033.3±16.3
125 2967.3±47.4 2701.8±96.2 1147.8±193.7
250 3148.3±224.2 2867.3±30.4 1034.8±99.0
500 3392.8±0.0 3040.8±24.0 978.8±4.2
1000 3519.3±17.7 3192.8±8.5 1150.3±55.94-MU底物溶液
用水将4-MU(Sigma公司产品)配成100mM的溶液后,用二乙醇胺(0.7ml/l)和六水合氯化镁(0.1g/l)组成的缓冲液(10mM,pH=10.0)稀释成1mM的4-MU底物溶液。实施例2 FK506的代谢物的测定方法
将FK506用大鼠肝微粒体处理时所得代谢物,例如可由DrugMetabolism and Disposition 21(6),971-977等得知。用代谢物M-II和M-III同实施例一样进行测定。M-IIM-III结果列于表1
表1结果显示,FK506和M-II的浓度与荧光强度呈对应关系,而M-III浓度和强度值并不对应。实施例3
不仅与FKBP-12结合,为了确认能够测定实际具有免疫作用的FK506及其代谢物的血中浓度,将FK506及其代谢物(M-II、M-III)同FKBP-12的结合活性及免疫抑制活性分别进行如下测定。(1)与FKBP-12的结合活性的测定,同临床化学38/7,1307-1310(1992)中记录的测定法相同,利用3H-二氢-FK506、FKBP-12、附着活性炭的右旋糖酐(Dexstran charcoled)等进行。
(2)另外,免疫抑制活性的测定由欧洲专利公报EP-0184162-A267页中记录的广为人知的体外 混合淋巴细胞反应(MLR)试验进行。结果列于表2:
表2
FKBP结合活性 免疫抑制活性 复合物形成活性
(相对值%) (MLP) (相对值%)
(相对值%)
FK506 100 100 100
M-II 14.2 100 79.7
M-III 116.0 0 0
表2中的复合物形成活性(%)是由表1的值求得的。
由表2可知,该复合物测量法不能检测出与FKBP-12有结合活性而无免疫活性的如M-III之类的代谢物(即复合物形成活性(%)为0),只能检测象FK506及其代谢物(M-II)那样具有免疫抑制活性的物质。实施例4 环孢菌素A的测定方法(1)代替FKBP-12,将从Sigma公司购买的环啡林与实施例1(1)相同固定于各孔中。(2)然后,将与实施例1(2)相同的牛钙调神经磷酸酶粗提液,牛钙调素液及下述的环孢菌素A溶液在各孔中反应,形成复合物。
环孢菌素A溶液
环孢菌素A的甲醇溶液(1mg/ml)以与实施例1(1)相同的测定缓冲液适当稀释。(3)然后与实施例1(3)相同,用不含牛血清白蛋白的测定缓冲液洗涤后,向各孔中加入100μ1以前述分析缓冲液稀释的、Upstate Biotecnology
-公司购买的与钙调神经磷酸酶(calcineurin)的β亚基相对的小鼠单克隆抗体(α-CN β-MoAb)溶液(1μl/ml),反应一小时。
然后与实施例1(4)和(5)相同进行荧光分析从而定量测定与环孢菌素A的浓度呈对应关系的所形成的复合物。结果如表3所示。
表3 环孢菌素A的定量测定
环孢菌素A的浓度 | 荧光强度 |
0 | 289 |
15.6 | 470 |
31.3 | 540 |
62.5 | 672 |
125 | 948 |
250 | 1297 |
500 | 1613 |
1000 | 2060 |
2000 | 2207 |
4000 | 2408 |
使用本发明的定量法,将对钙调神经磷酸酶有阻断作用的免疫抑制剂,特别是以FK506为代表的化合物[I]作为治疗或预防药物给药于有如以下症状或疾病的患者时,能够正确地定量测定血中浓度。
心脏、肾脏、肝脏、骨髓、皮肤、角膜、肺、脾脏、小肠、手脚、肌肉、神经、椎间盘、气管等脏器或组织移植时的排斥反应;骨髓移植引起的移植物对宿主反应;慢性风湿性关节炎;系统性红斑狼疮、桥本甲状腺炎、多发性硬化、重度肌无力、原发性粘膜水肿、自体免疫性萎缩性胃炎、早发闭经、男性不育症、青少年糖尿病、寻常天疱疮、类天疱疮、交感性眼炎、水晶性葡萄膜炎、特发性白血球减少、活动性慢性肝炎、特发性肝硬化、圆片状红斑性狼疮、I型糖尿病等自身免疫疾病;及病原性微生物(如烟曲霉、尖孢镰孢、星形发癣菌)等引起的感染:
炎性及增生元进性皮肤病及免疫学的中介皮肤病(如牛皮癣、特异性皮炎、接触性皮炎、湿疹性皮炎、脂漏性皮炎、扁平苔癣、天疱疮、水疱疮类天疱疮、表皮水疱症、荨麻疹、血管性水肿、脉管炎、红斑、皮肤嗜曙红细增多症、红斑性狼疮、痤疮及圆形脱发症);自体免疫病的眼病(如角结膜炎、春季结膜炎、贝切特氏症关联葡萄膜炎、角膜炎、疱疹性角膜炎、圆椎形角膜炎、角膜上皮营养不良、角膜白斑、眼天疱疮、负伦氏溃疡、强膜炎、克雷布氏眼疾、福格特-小抑 原田综合症、类肉瘤等)可逆的闭塞性气道疾病(哮喘(如支气管哮喘、变应性哮喘、內因性哮喘、外因性哮喘及尘埃性哮喘)、特慢性或难愈性哮喘(如迟发性哮喘及气道反应性元进)及支气管炎等);
粘膜及血管炎症(如胃溃疡、缺血及血栓引起的气管损伤、缺血性肠病、肠炎、坏死性全肠炎、由烧伤引起的肠损伤、白细胞三烯B4-相关症等);
肠炎/变态反应(如腹腔疾病、直肠炎、嗜酸性胃肠炎、细胞肥大症、Crohn′s病及溃疡性大肠炎);
远隔胃肠道部位症候性症状出现食物关联变态反应疾病(如偏头痛、鼻炎及湿疹);
肾病(如间质性肾炎、Good pasture′s综合症、溶血性尿毒综合症及糖尿病性肾病);
神经性疾病(如多发性肌炎、格一巴二氏综合症、梅尼埃尔氏病、多发性神经炎、单发性神经炎及神经根病);
內分泌疾病(如甲状腺功能亢进及巴塞壮氏病);
血液疾病(如巨成红细胞病、再生不良性贫血、形成不良性贫血、特发性血小板减少性紫斑病、自体免疫性溶血性贫血、粒细胞缺乏症、恶性贫血、巨成红细胞性贫血及红细胞缺乏症);
骨疾病(如骨质疏松症);
呼吸系统疾病(如类肉瘤病、肺纤维病及特发性间质性肺炎);
皮肤病(如皮肤肌炎、寻常性白斑病、寻常性鱼鳞癣、光线过敏病及皮肤T细胞淋巴瘤);
循环系统病(如动脉硬化、动脉粥样硬化、大动脉炎综合症、结节性多发性动脉炎及心肌炎);
胶原病(如硬皮症、韦格纳肉芽肿病、肖格伦综合症);
脂肪过多病;
嗜曙红细胞性肌膜炎;
牙周病(如牙龈、牙周、牙槽骨、牙骨质损伤);
肾综合症(如肾小球性肾炎);
男性型秃发或老人性秃发病;
肌营养不良;
脓皮病及Sézary′s综合症;
阿狄森氏病;
染色体异常病(如唐恩综合症);
活泼氧中介病〔如脏器损伤(保持、移植或缺血性疾病(血栓病、心肌梗塞等)时产生的(心、肝、肾、消化器等的)脏器的缺血性血流损伤)(如血栓形成,心脏梗塞):肠病(如內毒素休克、伪膜性大肠炎、药物或放射性引起的大肠炎):肾病(如缺血性急性肾功能衰竭、慢性肾功能衰竭):肺病〔如肺內氧或药物(如对草快(paracort)、博莱霉素)引起的中毒、肺癌、肺气肿):眼病[如白內障、铁质沉着、网膜炎、色素沉着病、老年性斑点变质、玻璃体瘢痕、碱烧伤角膜]:皮炎(如多形性红斑、线状免疫球蛋白A皮炎、骨质性皮炎):及其它疾病〔如牙龈炎、牙周炎、败血症、脾炎、或环境污染(如大气污染)、老龄化、致癌物质、癌转移、高山病等引起的疾病〕〕;
游离的组胺及白细胞三烯C4引起的疾病。
Claims (8)
1.一种测定具有钙调神经磷酸酶(Calcineurin)抑制活性免疫抑制剂的方法,其中包括形成包含(1)一种免疫亲和素(immunophilin),(2)钙调神经磷酸酶,(3)钙调素,(4)钙离子和(5)一种具有钙调神经磷酸酶抑制活性的免疫抑制剂的复合物,然后测定该复合物。
2.权利要求1的测定方法,其中的免疫亲和素是FKBP-12或环啡林(Cyclophilin)。
3.权利要求2的测定方法,其中免疫亲和素是FKBP-12并且具有钙调神经磷酸酶抑制活性的免疫抑制剂是由下面通式所表示的化合物〔1〕 [I]
(其中R1是羟基或被保护羟基,R2是氢原子,羟基或被保护的羟基,R3是甲基,乙基,丙基或烯丙基,R4是羟基或甲氧基,R5是氢原子,或R4和R5一起形成氧基,n是一个1或2的整数,含实线或虚线的符号指一个单键或一个双键,条件是R4是羟基以及R5是氢原子或当R4和R5一起形成氧基,R2不是被保护的羟基)。
4.权利要求3的测定方法,其中化合物〔1〕是17-烯丙基-1,14-二羟基-12-〔2-(4-羟基-3-甲氧基环己基)-1-甲基乙烯基〕-23,25-二甲氧基-13,19,21,27-四甲基-11,28-二氧杂-4-氮杂三环〔22.3.1.04,9〕二十八碳-18-烯-2,3,10,16-四酮。
5.权利要求2的测定方法,其中的免疫亲和素是环啡林并且具有钙调神经磷酸酶抑制活性的免疫抑制剂是环孢菌素A或其衍生物。
6.权利要求1,2,3,4或5的测定方法,其中的免疫亲和素是结合在固相上的。
7.权利要求6的测定方法,其中组成该复合物的钙调神经磷酸酶是用一种酶免疫测定技术测定的。
8.权利要求7的测定方法,其中钙调神经磷酸酶的测定是用一种识别钙调神经磷酸酶的抗钙调神经磷酸酶的抗体,以及一种识别所述的抗钙调神经磷酸酶抗体的酶标记的抗IgG抗体进行的。
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CN104164444A (zh) * | 2014-07-31 | 2014-11-26 | 宜昌美光硅谷生命科技有限公司 | 一种融合蛋白质、制备方法及其应用 |
CN104749009B (zh) * | 2015-03-30 | 2018-05-04 | 上海云泽生物科技有限公司 | 用于免疫分析的免疫抑制剂药物提取试剂 |
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Publication number | Priority date | Publication date | Assignee | Title |
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EP0976823A1 (de) | 1998-07-22 | 2000-02-02 | Helge Dr. Völkel | Rekombinantes Expressions System und Hoch-Durchsatz BioAssay für die therapeutische Anwendung von Calcineurin-A-Alpha, Calcineurin-A-Beta, Calcineurin-A-Gamma, Calcineurin-B und Copper/Zinc-Superoxide Dismutase bzw. zur Identifizierung von Pharmazeutika |
US7186518B2 (en) * | 2003-11-21 | 2007-03-06 | Dade Behring Inc. | Method and composition useful for determining FK 506 |
DE102004003362A1 (de) * | 2004-01-22 | 2005-08-11 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Verfahren zur Identifizierung und Herstellung von Effektoren Calmodulin-abhängiger Peptidyl-Prolyl cis/trans Isomerasen |
WO2006108130A2 (en) | 2005-04-06 | 2006-10-12 | Abbott Laboratories | Methods to measure immunosuppressive tacrolimus, sirolimus, and cyclosporin a complexes in a blood sample |
EP1810675A1 (en) | 2006-01-18 | 2007-07-25 | Institut Curie | Method for treating Huntington's disease by inhibiting dephosphorylation of huntingtin at S421 |
US7642338B2 (en) * | 2006-06-20 | 2010-01-05 | Siemens Healthcare Diagnostics Inc. | Tacrolimus standard and methods of using same |
US20080081379A1 (en) * | 2006-07-13 | 2008-04-03 | Sigler Gerald F | Homogeneous double receptor agglutination assay for immunosuppressant drugs |
US20110039789A1 (en) * | 2009-08-14 | 2011-02-17 | Institut Curie | Use of Huntingtin Protein for the Diagnosis and the Treatment of Cancer |
ES2576927T3 (es) | 2010-10-22 | 2016-07-12 | T2 Biosystems, Inc. | Sistemas de RMN y métodos para la detección rápida de analitos |
US8563298B2 (en) | 2010-10-22 | 2013-10-22 | T2 Biosystems, Inc. | NMR systems and methods for the rapid detection of analytes |
US8409807B2 (en) | 2010-10-22 | 2013-04-02 | T2 Biosystems, Inc. | NMR systems and methods for the rapid detection of analytes |
US9562271B2 (en) | 2012-04-20 | 2017-02-07 | T2 Biosystems, Inc. | Compositions and methods for detection of Candida species |
EP3405479A4 (en) | 2016-01-21 | 2019-08-21 | T2 Biosystems, Inc. | NMR METHOD AND SYSTEMS FOR THE FAST DETECTION OF BACTERIA |
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US5362629A (en) * | 1991-08-05 | 1994-11-08 | President And Fellows Of Harvard College | Detection of immunosuppressants |
US5457182A (en) | 1994-02-15 | 1995-10-10 | Merck & Co., Inc. | FK-506 cytosolic binding protein, FKBP12.6 |
US5639852A (en) | 1994-09-01 | 1997-06-17 | Wisconsin Alumni Research Foundation | Immunostimulatory agents |
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CN104164444A (zh) * | 2014-07-31 | 2014-11-26 | 宜昌美光硅谷生命科技有限公司 | 一种融合蛋白质、制备方法及其应用 |
CN104749009B (zh) * | 2015-03-30 | 2018-05-04 | 上海云泽生物科技有限公司 | 用于免疫分析的免疫抑制剂药物提取试剂 |
US11573223B2 (en) | 2015-03-30 | 2023-02-07 | Shanghai Inzex Biotechnology Co., Ltd. | Extraction reagent of immunosuppressant drug for immunoassays |
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JP3551431B2 (ja) | 2004-08-04 |
EP0750193B1 (en) | 2002-11-27 |
EP0750193A1 (en) | 1996-12-27 |
AU1861795A (en) | 1995-09-25 |
WO1995024645A1 (fr) | 1995-09-14 |
NZ281717A (en) | 1997-03-24 |
AU686762B2 (en) | 1998-02-12 |
DE69528968D1 (de) | 2003-01-09 |
US6338946B1 (en) | 2002-01-15 |
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