公开(公告)号：US06197536B1

公开(公告)日：2001-03-06

申请号：US09011961

申请日：1998-02-23

申请人： Christian Steinkühler ,  Antonello Pessi ,  Elisabetta Bianchi ,  Marina Taliani ,  Licia Tomei ,  Andrea Urbani ,  Raffaele De Francesco ,  Frank Narjes

发明人： Christian Steinkühler ,  Antonello Pessi ,  Elisabetta Bianchi ,  Marina Taliani ,  Licia Tomei ,  Andrea Urbani ,  Raffaele De Francesco ,  Frank Narjes

IPC分类号： C12Q137

CPC分类号： C07K14/005 ,  C12N9/506 ,  C12N2770/24222

摘要： The process according to the present invention allows expression and isolation of polypeptides with the proteolytic activity of HCV NS3 protease in a pure, catalytically active form, and in amounts that are sufficient for discovery of NS3 protease inhibitors and for determination of the three-dimensional structure of the NS3 protease. A further subject of the present invention is a procedure that defines the chemical and physical conditions necessary for completion of the proteolytic activity of the above polypeptides. The invention further comprises new compositions of matter (expression vectors) containing nucleotide sequences capable of expressing the above mentioned polypeptides in culture cells. Finally, new compounds of matter are defined, suitable to measure the above proteolytic activity, and useful to develop NS3 protease inhibitors and therefore therapeutic agents for use against HCV. The figure shows the kinetic parameters of HCV NS3 protease using the S3 depsipeptide substrate (SEQ ID NO:45).

摘要翻译： 根据本发明的方法允许以纯的，催化活性形式表达和分离具有HCV NS3蛋白酶的蛋白水解活性的多肽，并且其量足以发现NS3蛋白酶抑制剂和用于确定三维结构 的NS3蛋白酶。 本发明的另一主题是定义完成上述多肽的蛋白水解活性所需的化学和物理条件的方法。 本发明还包括含有能够在培养细胞中表达上述多肽的核苷酸序列的物质组合物（表达载体）。 最后，定义新的物质化合物，适合于测量上述蛋白水解活性，并且可用于开发NS3蛋白酶抑制剂，因此用于抗HCV的治疗剂。 该图显示了使用S3 depipipeptide底物（SEQ ID NO：45）的HCV NS3蛋白酶的动力学参数。

公开(公告)号：US20120074308A1

公开(公告)日：2012-03-29

申请号：US13224910

申请日：2011-09-02

申请人： Andrea Urbani ,  Giorgio Federici

发明人： Andrea Urbani ,  Giorgio Federici

IPC分类号： H01J49/26 ,  G01N33/15

CPC分类号： G01N33/6848 ,  G01N2410/08

摘要： The invention relates to therapeutic drug monitoring (TDM) by mass spectrometry, particularly to the monitoring of immunosuppressant levels in blood of patients with transplanted organs. A liquid phase extraction procedure reproducibly extracts the therapeutic drug molecules from whole blood and mass spectrometric analysis on MALDI instruments, with a matrix substance for highest sensitivity and special sample deposition procedure for a reproducible ionization of the therapeutic drug molecules. Suitable internal standard substances added to the blood in exact amounts ensure a correct absolute quantification. The method is particularly suitable for immunosuppressants belonging to the class of macrocyclic lactones (sirolimus, tacrolimus, everolimus) and cyclic polypeptides (cyclosporin A), and even works as a multiplex method for all four immunosuppressants simultaneously.

摘要翻译： 本发明涉及通过质谱法进行的治疗药物监测（TDM），特别涉及对移植器官患者血液中免疫抑制剂水平的监测。 液相萃取程序从MALDI仪器的全血和质谱分析中重现性提取治疗药物分子，使用最高灵敏度的基质物质和特殊的样品沉积程序，用于治疗药物分子的可重复电离。 合适的内标物质添加到血液中的确切量确保了正确的绝对量化。 该方法特别适用于属于大环内酯类（西罗莫司，他克莫司，依维莫司）和环状多肽（环孢菌素A）类的免疫抑制剂，甚至同时用作所有四种免疫抑制剂的多重方法。