title: Chemical synthesis of Escherichia coli ST(h) analogues by regioselective disulfide bond formation: biological evaluation of an (111)In-DOTA-Phe(19)-ST(h) analogue for specific targeting of human colon cancers.
abstract: New human Escherichia coli heat-stable peptide (ST(h)) analogues containing a DOTA chelating group were synthesized by sequential and selective formation of disulfides bonds in the peptide. This synthetic approach utilizes three orthogonal thiol-protecting groups, Trt, Acm, and t-Bu, to form three disulfide bonds by successive reactions using 2-PDS, iodine, and silyl chloride-sulfoxide systems. The DOTA-ST(h) conjugates exhibiting high guanylin/guanylate cyclase-C (GC-C) receptor binding affinities were obtained with >98% purity. In vitro competitive binding assays, employing T-84 human colon cancer cells, demonstrated the IC(50) values of <2 nM for GC-C receptor binding suggesting that the new synthetic ST(h) analogues are biologically active. In vitro stability studies of the (111)In-DOTA-Phe(19)-ST(h) conjugate incubated in human serum at 37 degrees C under 5% CO(2) atmosphere revealed that this conjugate is extremely stable with no observable decomposition at 24 h postincubation. HPLC analysis of mouse urine at 1 h pi of the (111)In-DOTA-Phe(19)-ST(h) conjugate showed only about 15% decomposition suggesting that the (111)In-DOTA-Phe(19)-ST(h) conjugate is highly stable, even under in vivo conditions. In vivo pharmacokinetic studies of the (111)In-DOTA-Phe(19)-ST(h) conjugate in T-84 human colon cancer derived xenografts in SCID mice conducted at 1 h pi showed an initial tumor uptake of 2.04 +/- 0.30% ID/g at 1 h pi with efficient clearance from the blood pool (0.23 +/- 0.14% ID/g, 1 h pi) by excretion mainly through the renal/urinary pathway (95.8 +/- 0.2% ID, 1 h pi). High tumor/blood, tumor/muscle, and tumor/liver ratios of approximately 9:1, 68:1, and 26:1, respectively, were achieved at 1 h pi The specific in vitro and in vivo uptake of the radioactivity by human colonic cancer cells highlights the potential of radiometalated-DOTA-ST(h) conjugates as diagnostic/therapeutic radiopharmaceuticals.
authors.affiliation: Department of Radiology, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
authors.forename: Hariprasad
authors.initials: H
authors.lastname: Gali
authors.forename: Gary L
authors.initials: GL
authors.lastname: Sieckman
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Hoffman
authors.forename: Nellie K
authors.initials: NK
authors.lastname: Owen
authors.forename: Dana G
authors.initials: DG
authors.lastname: Mazuru
authors.forename: Leonard R
authors.initials: LR
authors.lastname: Forte
authors.forename: Wynn A
authors.initials: WA
authors.lastname: Volkert
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000138:chemical synthesis* / Q000378:metabolism / Q000493:pharmacokinetics* / Q000627:therapeutic use; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004768:Enterotoxins / Q000138:chemical synthesis* / Q000378:metabolism / Q000493:pharmacokinetics* / Q000627:therapeutic use; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005260:Female; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D007205:Indium Radioisotopes / Q000737:chemistry; D020128:Inhibitory Concentration 50; D051379:Mice; D016513:Mice, SCID; D015394:Molecular Structure; D009368:Neoplasm Transplantation; D019275:Radiopharmaceuticals / Q000138:chemical synthesis* / Q000378:metabolism / Q000493:pharmacokinetics* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured; D000090324:Tumor Protein, Translationally-Controlled 1
chemical_list: D001427:Bacterial Toxins; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D006573:Heterocyclic Compounds, 1-Ring; D007205:Indium Radioisotopes; D019275:Radiopharmaceuticals; C000715648:Tpt1 protein, mouse; D000090324:Tumor Protein, Translationally-Controlled 1; C034151:heat stable toxin (E coli); C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid