Synthesis and in vitro evaluation of an 111In-labeled ST-peptide enterotoxin (ST) analogue for specific targeting of guanylin receptors on human colonic cancers.

BACKGROUND: Human colonic cancer cells are known to express guanylate cyclase C (GC-C) receptors for guanylin and uroguanylin. E. coli ST is a peptide with high metabolic stability that specifically binds to GC-C receptors. An in vitro evaluation of a new synthetic indium-111 labeled ST conjugate for specific targeting of human colonic cancers that express GC-C receptors was performed. MATERIALS AND METHODS: A DOTA conjugated ST analogue DOTA-NCS-6-Ahx-Phe19-ST[1-19] (DOTA-NCS-ST) was synthesized and labeled with indium-111. The non-radioactive indium analogue (In-DOTA-NCS-ST) was also prepared in macroscopic quantities. 111In-DOTA-NCS-ST was produced as a single species (>80% RCP) and purified by HPLC. Human colon cancer CaCO-2 and T-84 cells were used to evaluate the in vitro IC50 values for GC-C receptor binding and determine the cell uptake and retention of radioactivity. RESULTS: The DOTA-NCS-ST and In-DOTA-NCS-ST conjugates exhibit high in vitro binding affinity for GC-C receptors with IC50 values <10 nM. The in vitro cell binding studies with the 111In-DOTA-NCS-ST conjugate demonstrated that 111In-label ST internalizes in human colon cancer cells and exhibits long-term retention. CONCLUSION: The combination of radiolabeling efficacy and specific in vitro cell uptake and retention suggests that the DOTA-NCS-ST construct holds potential for the development of diagnostic or therapeutic radiopharmaceuticals labeled with trivalent radiometals for specific targeting of human colonic cancers.

In vivo evaluation of an 111In-labeled ST-peptide analog for specific-targeting of human colon cancers.

In vitro competitive binding studies of In-DOTA-NCS-6-Ahx-Phe(19)-ST[1-19] vs. 125I-Tyr(5)-6-Ahx-Phe(19)-ST[1-19] with guanylate cyclase -C (GC-C) receptors on human colon cancer LS-180 cells revealed an IC(50) value of 7.7 +/- 0.1.6 nM. The in vitro cellular residualization studies of the 111In-DOTA-NCS-ST peptide and GC-C receptor mediated stimulated cGMP production with LS-180 cells demonstrates that this peptide selectively binds to LS-180 cells in an agonistic fashion. In vivo biodistribution studies in LS-180 tumor bearing SCID mice demonstrates that the 111In-DOTA-NCS-ST peptide targets the tumor with a specific uptake of 0.94 +/- 0.31%ID/g at 1 hr p.i. and approximately 23% was retained by the tumor at 4 hrs p.i. The radioactivity cleared rapidly from the blood stream with 84.5 +/- 3.4%ID at 1h p.i. found in the urine. High activity in urine and kidney, and minimal activity in liver and intestines, demonstrates preferential clearance of the radioactivity through the renal/urinary pathway. The specific in vitro and in vivo accumulation of the radioactivity by LS-180 human colonic cancer cells highlights the potential of radiometallated-DOTA-ST analogs as diagnostic/therapeutic radiopharmaceuticals.

Synthesis, characterization, and labeling with 99mTc/188Re of peptide conjugates containing a dithia-bisphosphine chelating agent.

Radiolabeling of small receptor-avid peptides at specific predetermined chelation sites with radioactive metals has been an effective approach for production of target-specific radiopharmaceuticals for diagnosis and therapy of diseases. Among various electron-donating groups found on chelator frameworks, phosphines are unique because they display versatile coordination chemistry with a wide range of transition metals. We have recently reported the utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to form 99mTc-labeled receptor-avid peptides by the preconjugation approach. Here we report a novel strategy for labeling small peptides with both 99mTc and 188Re using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)propylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approach. The first step in this approach involves the coupling of the corresponding (PH2)2S2-COOH intermediate to the N-terminus of the peptide(s). Formylation of P-H bonds with aqueous formaldehyde in the presence of HCl in ethanol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjugates in the form of an oxidatively stable phosphonium salt. The P2S2-peptide conjugates are generated (where the PH2 groups are converted to P(CH2OH)2 groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M sodium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with 99mTc is achieved by direct reduction with Sn(II) tartarate to yield the 99mTc-P2S2-peptide conjugate in near quantitative yields. Complexation of the BFCA conjugates with 188Re is achieved by transchelation with 188Re citrate in yields of >/=90%. In this study, (PH2)2S2-COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)2S2-COOH BFCA conjugate was converted to the hydroxymethylene phosphine form and complexed with 99mTc to produce the 99mTcO2-P2S2-GlyGlyOEt conjugate 8 in RCPs of >/=95%. This singular 99mTc product is stable over 24 h in aqueous solution as confirmed by HPLC. Identical retention times of the 99mTcO2-P2S2-GlyGlyOEt complex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The utility of this postconjugation strategy was further demonstrated by synthesizing a P2S2-D-Lys6-LHRH conjugate and producing its corresponding 99mTc complex in RCPs of >/=88%. Finally, the P2S2-5-Ava-BBN[7-14]NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converted to P(CH2OH)2 groups and subsequently labeled with 188Re to yield a 188Re-labeled bombesin analogue with a RCP of >/=90%. The biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)2S2-COOH BFCA can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (99mTc) and therapeutic (188Re) radionuclides via the postconjugation approach in high yields.

99mTc-labeling and in vivo studies of a bombesin analogue with a novel water-soluble dithiadiphosphine-based bifunctional chelating agent.

Recent progress in the synthesis of water-soluble phosphine ligand systems and their corresponding 99mTc complexes prompted the development of a new bifunctional chelating agent (BFCA) based on a tetradentate dithiadiphosphine framework (P2S2-COOH). The detailed synthesis of this new BFCA is described here. The corresponding 99mTc complex, 99mTc-P2S2-COOH, can be formed in >95% yield. To demonstrate the potential of this chelate to efficiently label peptides, 99mTc-P2S2-COOH was coupled to the N-terminal region of the truncated nine-amino acid bombesin analogue, 5-Ava-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 [BBN(7-14)], to form 99mTc-P2S2-BBN(7-14). Conjugation to the peptide was performed in borate buffer (pH 8.5) by applying the prelabeling approach in yields of >60%. In competitive binding assays, using Swiss 3T3 mouse fibroblast cells against [125I-Tyr4]bombesin, Re-P2S2-BBN(7-14) exhibited an IC50 value of 0.8 +/- 0.4 nM. The pharmacokinetic studies of 99mTc-P2S2-BBN(7-14) and its ability to target tissue expressing gastrin-releasing peptide (GRP) receptors were performed in normal mice. The 99mTc-P2S2-BBN(7-14) exhibited fast and efficient clearance from the blood pool (0.6 +/- 0.1% ID, 4 h postinjection) and excretion through the renal and hepatobiliary pathways (56.4 +/- 8.2 and 28.1 +/- 7.9% ID, 4 h postinjection, respectively). Significant uptake in the pancreas was observed (pancreatic acini cells express bombesin/GRP receptors), producing pancreas:blood and pancreas:muscle ratios of ca. 22 and 80, respectively, at 4 h postinjection.