Design and Evaluation of Albumin Mutants for Clinical Application

临床应用白蛋白突变体的设计和评价

• 批准号: 11694298
• 负责人: OTAGIRI Masaki
• 金额: $ 4.67万
• 依托单位: KUMAMOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694298/
• 关键词: Albumin preparation; Site-directed mutagenesis; Molecular design; Artificial blood; Functional evaluation; Blood preparation; Clinical application

International collaboration work for reducing the rate of albumin preparations usage was undertaken to develop a safe and effective recombinant albumins. The results obtained are as follow :1) Two single-mutants (K199A, K525A) were produced. The recombinant albumins were correctly folded, as evidenced by the fact that they showed similar patterns as the native albumin in the far- and near-UV CD spectrum as well as reactivity towards polyclonal anti-serum raised against native albumin.2) Thermal denaturation experiments by using GdnCl and DSC revealed no significant difference in conformational stability for native and mutant albumins.3) The ligand binding properties and esterase-like activity of the albumin mutants were nearly the same as those for the native albumin.4) The plasma levels of the albumin mutants labeled with ^<125>I after intravenous bolus injections to rats showed two-phase profiles but their half-lives were shorten than that for the native albumin.5) Dr.Kragh-Hansen of Aarhus University visited Kumamoto University twice (Oct.23-Nov.7, 1999 ; May 5-Oct.29, 2000) to discuss about pharmacokinetic and biological properties of the albumin mutants. Dr.Curry of Imperial College also visited Kumamoto University (Sep.27-Oct.26, 2000) to discuss about structural characterization of the albumin mutants.

为降低白蛋白制剂的使用率，开展了国际合作工作，以开发安全有效的重组白蛋白。1）获得了两个单突变体（K199 A、K525 A）。重组白蛋白正确折叠，这一事实证明，它们在远-和近-UV CD光谱中显示出与天然白蛋白相似的模式，以及对针对天然白蛋白产生的多克隆抗血清的反应性。2）通过使用GdnCl和DSC的热变性实验显示天然和突变白蛋白的构象稳定性没有显著差异。3）配体结合性质和酯酶-白蛋白突变体的类似活性与天然白蛋白的活性几乎相同。4）静脉推注给大鼠后，用^ I标记的白蛋白突变体的血浆水平<125>显示出双相曲线，但其半衰期比天然白蛋白短。5）Aarhus大学的Kragh-Hansen博士两次访问熊本大学（1999年10月23日至11月7日; 2000年5月5日至10月29日），讨论白蛋白突变体的药代动力学和生物学特性。帝国理工学院的咖喱博士还访问了熊本大学（2000年9月27日至10月26日），讨论了白蛋白突变体的结构特征.

项目成果

Y.Tsutsumi: "Decreased bilirubin-binding capacity in uremicserum caused by an accumulation of furan dicarboxylic acid"Nephrom. 84・(3)(in press). (2000)

Y. Tsutsumi：“呋喃二羧酸积累导致尿毒症血清胆红素结合能力降低”Nephrom 84・(3)（出版中）。

Sakai T, et al.: "Interaction mechanism between indoxyl sulfate, a typical uremic toxin bound to site II, and ligands bound to site I of human serum albumin."Pharm Res.. (in press). (2001)

Sakai T 等人：“硫酸吲哚酚（一种与位点 II 结合的典型尿毒症毒素）与与人血清白蛋白位点 I 结合的配体之间的相互作用机制。”Pharm Res..（出版中）。

V.T.G.Chuang et al: "Helix of subdomain IIIA of HSA is the region primainly photolabelet by betoproten"Biochimica Biophysica Acta. 1434. 18-30 (1999)

V.T.G.Chuang 等人：“HSA 子结构域 IIIA 的螺旋是主要由 Betoproten 进行光标记的区域”Biochimica Biophysicala Acta。

T.Sakai et al: "Interaction between indoxyl sulfate, a typical uremic toxin bound to site II, and ligands bound to site I of HSA"Pharmaceutical Research. 18(4)(in press). (2001)

T.Sakai 等人：“硫酸吲哚酚（一种与位点 II 结合的典型尿毒症毒素）与与 HSA 位点 I 结合的配体之间的相互作用”药物研究。

丸山徹,小田切優樹(分担)沢田康文 他(編集): "医薬品の適正使用のための臨床薬物動態"じほう. 834 (2000)

Toru Maruyama、Yuki Odagiri（撰稿人）、Yasufumi Sawada 等人（编辑）：“正确使用药物的临床药代动力学”Jiho 834 (2000)。