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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.
AuthorsHariprasad Gali, Gary L Sieckman, Timothy J Hoffman, Nellie K Owen, Dana G Mazuru, Leonard R Forte, Wynn A Volkert
JournalBioconjugate chemistry (Bioconjug Chem) 2002 Mar-Apr Vol. 13 Issue 2 Pg. 224-31 ISSN: 1043-1802 [Print] United States
PMID11906259 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)
Chemical References
  • Bacterial Toxins
  • Disulfides
  • Enterotoxins
  • Escherichia coli Proteins
  • Heterocyclic Compounds, 1-Ring
  • Indium Radioisotopes
  • Radiopharmaceuticals
  • Tpt1 protein, mouse
  • Tumor Protein, Translationally-Controlled 1
  • heat stable toxin (E coli)
  • 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid
Topics
  • Animals
  • Bacterial Toxins (chemical synthesis, metabolism, pharmacokinetics, therapeutic use)
  • Binding, Competitive
  • Chromatography, High Pressure Liquid
  • Colonic Neoplasms (drug therapy, metabolism)
  • Disulfides (metabolism)
  • Enterotoxins (chemical synthesis, metabolism, pharmacokinetics, therapeutic use)
  • Escherichia coli (chemistry)
  • Escherichia coli Proteins
  • Female
  • Heterocyclic Compounds, 1-Ring (chemistry)
  • Humans
  • Indium Radioisotopes (chemistry)
  • Inhibitory Concentration 50
  • Mice
  • Mice, SCID
  • Molecular Structure
  • Neoplasm Transplantation
  • Radiopharmaceuticals (chemical synthesis, metabolism, pharmacokinetics, therapeutic use)
  • Tumor Cells, Cultured
  • Tumor Protein, Translationally-Controlled 1

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