In Vitro System To Estimate Renal Brush Border Enzyme-Mediated Cleavage of Peptide Linkages for Designing Radiolabeled Antibody Fragments of Low Renal Radioactivity Levels

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• 作者：Yasushi Fujioka ; Satoshi Satake ; Tomoya Uehara ; Takahiro Mukai ; Hiromichi Akizawa ; Kazuma Ogawa ; Hideo Saji ; Keigo Endo ; and Yasushi Arano
• 刊名：Bioconjugate Chemistry
• 出版年：2005
• 出版时间：November 2005
• 年：2005
• 卷：16
• 期：6
• 页码：1610 - 1616
• 全文大小：122K
• 年卷期：v.16,no.6(November 2005)
• ISSN：1520-4812

文摘

Renal localization of radiolabeled antibody fragments presents a problem in targeted imaging andradiotherapy. We recently reported that Fab fragments labeled with 3'-[¹³¹I]iodohippuryl N-maleoyl-L-lysine (HML) demonstrated markedly low renal radioactivity levels from early postinjection in mice.Previous studies suggested that low renal radioactivity levels were attributable to cleavage of theglycyl-lysine sequence in HML by the action of renal brush border enzymes, followed by urinaryexcretion of the resulting m-iodohippuric acid. In this study, an in vitro system using brush bordermembrane vesicles (BBMVs) isolated from the rat kidney cortex was developed to estimate renal brushborder enzyme(s)-mediated cleavage of the peptide linkage. Low molecular weight HML derivatives,3'-[¹²⁵I]iodohippuryl L-lysine (HL), 3'-[¹²⁵I]iodohippuryl N-tert-butoxycarbonyl-L-lysine (HBL), and theirD-amino acid counterparts, were synthesized and incubated in BBMVs. Both [¹²⁵I]HL and [¹²⁵I]HBLgenerated m-[¹²⁵I]iodohippuric acid after incubation in BBMVs at 37 C while the latter liberatedsignificantly higher amounts of the metabolite. [¹²⁵I]D-HL and [¹²⁵I]D-HBL failed to release themetabolite under similar conditions. The liberation of m-[¹²⁵I]iodohippric acid from [¹²⁵I]HL wassignificantly facilitated or completely inhibited by the addition of an activator or an inhibitor forcarboxypeptidase M. The release of m-[¹²⁵I]iodohippuric acid from [¹²⁵I]HBL increased by the additionof the activator, whereas the inhibitor partially inhibited the release of the metabolite from [¹²⁵I]HBL. The BBMV-mediated release of m-[¹²⁵I]iodohippuric acid from [¹²⁵I]HBL was not impaired bythe addition of inhibitors for neutral endopeptidase or renal dipeptidase. These findings showed thatthe glycyl-L-lysine sequence in HML would be recognized and cleaved by metalloenzymes andnonmetalloenzymes on the renal brush border even when iodine was incorporated into a benzenering and the N-amine residue of lysine was chemically modified, which supported the hypothesisthat low renal radioactivity levels of HML-conjugated Fab fragments would be attributed to the releaseof m-iodohippuric acid by renal brush border enzymes. This study suggested that this in vitro systemusing BBMVs would be useful to estimate radiolabeling reagents of antibody fragments or peptidesdesigned to reduce renal radioactivity with a variety of radionuclides.