title: Design, synthesis, and evaluation of [188Re]organorhenium-labeled antibody fragments with renal enzyme-cleavable linkage for low renal radioactivity levels.
abstract: Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. We have reported that Fab fragments labeled with 3'-[131I]iodohippuryl Nepsilon-maleoyl-lysine (HML) showed markedly low renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[131I]iodohippuric acid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallic radionuclides, [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was conjugated with Nepsilon-tert-butoxycarbonyl-glycyl-lysine or Nepsilon-maleoyl-glycyl-lysine to prepare [188Re]CpTR-GK-Boc or [188Re]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [188Re]CpTR-COOH ([188Re]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid. The hydrolysis rate of the peptide bond in [188Re]CpTR-GK-Boc was compared with that in 3'-[125I]iodohippuryl Nepsilon-Boc-lysine ([125I]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [188Re]CpTR-GK was conjugated to thiolated Fab fragments to prepare [188Re]CpTR-GK-Fab. The biodistribution of radioactivity after injection of [188Re]CpTR-GK-Fab was compared with that of [125I]HML-Fab and [188Re]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [188Re]CpTR-COOH with antibody fragments. While [188Re]CpTR-GK-Boc liberated [188Re]CpTR-Gly in BBMVs, [125I]HL-Boc liberated m-[125I]iodohippuric acid at a much faster rate. In addition, although [125I]HL-Boc was hydrolyzed by both metalloenzymes and nonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [188Re]CpTR-GK-Boc. In biodistribution studies, [188Re]CpTR-GK-Fab exhibited significantly lower renal radioactivity levels than did [188Re]CpTR-Fab. However, the renal radioactivity levels of [188Re]CpTR-GK-Fab were slightly higher than those of [125I]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [188Re]CpTR-GK-Fab showed that [188Re]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [188Re]CpTR-GK-Fab significantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findings indicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelate of high inertness and by designing a radiometabolite of high urinary excretion when released from antibody fragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemical structure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysis reaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brush border membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeled antibody fragments that exhibit renal radioactivity levels similar to those of [131I]HML-labeled ones. The in vitro system using BBMVs might be useful for selecting an appropriate peptide linkage.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Uehara
authors.forename: Miho
authors.initials: M
authors.lastname: Koike
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakata
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hanaoka
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Iida
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Akizawa
authors.forename: Keigo
authors.initials: K
authors.lastname: Endo
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Arano
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology*; D007554:Isotopes; D007668:Kidney / Q000201:enzymology* / Q000276:immunology; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008871:Microvilli / Q000201:enzymology; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology* / Q000493:pharmacokinetics; D011863:Radioimmunoassay; D051381:Rats; D012211:Rhenium / Q000737:chemistry*
chemical_list: D007140:Immunoglobulin Fab Fragments; D007554:Isotopes; D009942:Organometallic Compounds; D010446:Peptide Fragments; C517413:tricarbonyl(cyclopentadienylcarbonate)rhenium; D012211:Rhenium; D008239:Lysine