Chemical Synthesis of Escherichia Coli STh Analogues by Regioselective Disulfide Bond Formation: Biological Evaluation of an 111In-DOTA−Phe19-STh Analogue for Specific Targeting of Human Colon Cancers

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Abstract
New human Escherichia coli heat-stable peptide (STh) 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−STh 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 IC50 values of h analogues are biologically active. In vitro stability studies of the 111In-DOTA−Phe19-STh conjugate incubated in human serum at 37 °C under 5% CO2 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 111In-DOTA−Phe19-STh conjugate showed only about 15% decomposition suggesting that the 111In-DOTA−Phe19-STh conjugate is highly stable, even under in vivo conditions. In vivo pharmacokinetic studies of the 111In-DOTA−Phe19-STh 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−STh conjugates as diagnostic/therapeutic radiopharmaceuticals.