In Vitro and in Vivo Comparison of Human Escherichia coli Heat-Stable Peptide Analogues Incorporating the ¹¹¹In-DOTA Group and Distinct Linker Moieties

Abstract

Three human Escherichia coli heat-stable peptide (STh) analogues, each containing a DOTA chelating group, were synthesized by SPPS and oxidative refolding and compared in in vitro and in vivo systems. One analogue, DOTA-F19-STh(1-19), contains an N-terminal DOTA group attached via an amide bond linkage to an STh moiety which is essentially wild-type except for a Tyr to Phe alteration at position 19 of the molecule. A second analogue, DOTA-R1,4,F19-STh(1-19), differs from the first in that asparagine residues in positions 1 and 4 have been altered to arginine residues in order to examine the effect of positively charged groups in the linker domain. A third analogue, DOTA-11AUN-F19-STh(1-19), differs from the first in that it incorporates an 11-aminoundecanoic acid spacer group between the DOTA group and the first asparagine residue. In vitro competitive binding assays utilizing T-84 human colon cancer cells demonstrated that significant alterations to the N-terminal region of the STh molecule were well tolerated and did not significantly affect binding affinity of STh for the guanylyl cyclase C (GC-C) receptor. Internalization and efflux studies of the indium-labeled species demonstrated that inclusion of positive charge in the linker moiety inhibits internalization of the compound within tumor cells. The characteristics of the three analogues were compared in an in vivo model utilizing T-84 human colon cancer cell xenografts in SCID mice. Clearance of all analogues was rapid, primarily via renal excretion into the urine, with >89% ID excreted into the urine at 1 h pi for all analogues. The 111In-DOTA-R1,4,F19-STh(1-19) and 111In-DOTA-11AUN-F19-STh(1-19) analogues both had longer residence times in the blood than did the 111In-DOTA-F19-STh(1-19) analogue, probably accounting for increased %ID/g values for tumors and nontarget tissues at 1 h pi. At 4 h pi, significant differences between analogues were only seen with respect to metabolic routes of excretion, indicating that increased blood residence time did not result in increased tumor residualization. Reduction of hepatic uptake of these compounds, however, could have significance in the development of agents for the imaging of hepatic metastases. The ability to manipulate in vivo pharmacodynamics and tumor uptake of radiolabeled STh peptides through modification of linker moieties is under continuing investigation in order to produce optimal imaging and therapeutic radiopharmaceuticals.

Comparative Evaluation of Three64Cu-LabeledE. coliHeat-Stable Enterotoxin Analogues for PET Imaging of Colorectal Cancer

“…At 1 h pi, uptake in tumor for each construct was higher than for all other normal tissues with the exception of kidney (Table 1). Blocking studies, carried out by coadministration of unlabeled F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), reduced accumulation of the NOTA conjugate in tumor tissue by ∼42% (p < 0.05), comparable to previous results obtained for this class of peptides (17,18). Kidney uptake values at 1 h pi for the NOTA conjugate (3.7 ( 1.6%ID/g) were lower than for either the DOTA (6.5 ( 2.7%ID/g) or TETA (11.4 ( 2.9%ID/g) constructs (Table 1, Figure 5).…”

“…Several differences exist between in vivo biodistribution patterns of 64 Cu-DOTA-F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and the previously described 111 In-complex of this DOTA-peptide (18). At 1 h pi, tumor uptake of 64 Cu-DOTA-F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) was significantly lower than that of 111 In-DOTA-F 19 -STh(2-19) (1.33 ( 0.52%ID/g vs 2.35 ( 0.43%ID/g, respectively). Simultaneously, the liver uptake of the 64 Cu-labeled peptide was higher than for the 111 Incomplex (1.21 ( 0.65 vs 0.2 ( 0.09, respectively).…”

“…The polyaminocarboxylate chelating moieties DOTA, TETA, and NOTA were appended to the N-terminus of F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) via NHS ester activation. The addition of DOTA was carried out using a commercially available NHS ester of DOTA, while TETA and NOTA were appended via in situ NHS ester activation using a 100-fold excess of sulfo NHS and EDC in aqueous solution.…”

Comparative Evaluation of Three64Cu-LabeledE. coliHeat-Stable Enterotoxin Analogues for PET Imaging of Colorectal Cancer

“…At 1 h pi, uptake in tumor for each construct was higher than for all other normal tissues with the exception of kidney (Table 1). Blocking studies, carried out by coadministration of unlabeled F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), reduced accumulation of the NOTA conjugate in tumor tissue by ∼42% (p < 0.05), comparable to previous results obtained for this class of peptides (17,18). Kidney uptake values at 1 h pi for the NOTA conjugate (3.7 ( 1.6%ID/g) were lower than for either the DOTA (6.5 ( 2.7%ID/g) or TETA (11.4 ( 2.9%ID/g) constructs (Table 1, Figure 5).…”

“…Several differences exist between in vivo biodistribution patterns of 64 Cu-DOTA-F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and the previously described 111 In-complex of this DOTA-peptide (18). At 1 h pi, tumor uptake of 64 Cu-DOTA-F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) was significantly lower than that of 111 In-DOTA-F 19 -STh(2-19) (1.33 ( 0.52%ID/g vs 2.35 ( 0.43%ID/g, respectively). Simultaneously, the liver uptake of the 64 Cu-labeled peptide was higher than for the 111 Incomplex (1.21 ( 0.65 vs 0.2 ( 0.09, respectively).…”

“…The polyaminocarboxylate chelating moieties DOTA, TETA, and NOTA were appended to the N-terminus of F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) via NHS ester activation. The addition of DOTA was carried out using a commercially available NHS ester of DOTA, while TETA and NOTA were appended via in situ NHS ester activation using a 100-fold excess of sulfo NHS and EDC in aqueous solution.…”

Synthesis and Characterization of an111In-Labeled Peptide for the in Vivo Localization of Human Cancers Expressing the Urokinase-Type Plasminogen Activator Receptor (uPAR)

“…125 I-labeled ATF uptake was higher than (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla) 2 -K-K( 111 In-DOTA)-NH 2 uptake in all normal tissues at these time points, with the exception of liver and kidney, which demonstrated higher levels of 111 In-peptide. Clearance of the 111 In-peptide occurs through both hepatobiliary and renal/urinary routes, and liver and kidney retention at 4 h pi was higher than is usually observed for peptides in this molecular weight range (17)(18)(19)(20)(21)(22)(23). Increased liver and kidney retention is still observed for the 111 In-labeled branched peptide relative to 125 I-ATF at 24 h pi, suggesting a structural mechanism of retention, perhaps due to the branched nature of this construct, distinct from normal residualization of 111 In-DOTA-amino acid conjugates.…”

Challenges in Optimizing a Prostate Carcinoma Binding Peptide, Identified through the Phage Display Technology

“…The kidney radioactivity at 15 min was about 8% ID/g for the iodinated peptide [14], while it was measured to be 13.5% for the DOTA-conjugated molecule. The fast renal clearance is caused by the hydrophilic chelator DOTA [33].…”

Comparative Evaluation of Three64Cu-LabeledE. coliHeat-Stable Enterotoxin Analogues for PET Imaging of Colorectal Cancer

“…At 1 h pi, uptake in tumor for each construct was higher than for all other normal tissues with the exception of kidney (Table 1). Blocking studies, carried out by coadministration of unlabeled F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), reduced accumulation of the NOTA conjugate in tumor tissue by ∼42% (p < 0.05), comparable to previous results obtained for this class of peptides (17,18). Kidney uptake values at 1 h pi for the NOTA conjugate (3.7 ( 1.6%ID/g) were lower than for either the DOTA (6.5 ( 2.7%ID/g) or TETA (11.4 ( 2.9%ID/g) constructs (Table 1, Figure 5).…”

“…Several differences exist between in vivo biodistribution patterns of 64 Cu-DOTA-F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and the previously described 111 In-complex of this DOTA-peptide (18). At 1 h pi, tumor uptake of 64 Cu-DOTA-F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) was significantly lower than that of 111 In-DOTA-F 19 -STh(2-19) (1.33 ( 0.52%ID/g vs 2.35 ( 0.43%ID/g, respectively). Simultaneously, the liver uptake of the 64 Cu-labeled peptide was higher than for the 111 Incomplex (1.21 ( 0.65 vs 0.2 ( 0.09, respectively).…”

“…The polyaminocarboxylate chelating moieties DOTA, TETA, and NOTA were appended to the N-terminus of F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) via NHS ester activation. The addition of DOTA was carried out using a commercially available NHS ester of DOTA, while TETA and NOTA were appended via in situ NHS ester activation using a 100-fold excess of sulfo NHS and EDC in aqueous solution.…”

Synthesis and Characterization of an111In-Labeled Peptide for the in Vivo Localization of Human Cancers Expressing the Urokinase-Type Plasminogen Activator Receptor (uPAR)

“…125 I-labeled ATF uptake was higher than (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla) 2 -K-K( 111 In-DOTA)-NH 2 uptake in all normal tissues at these time points, with the exception of liver and kidney, which demonstrated higher levels of 111 In-peptide. Clearance of the 111 In-peptide occurs through both hepatobiliary and renal/urinary routes, and liver and kidney retention at 4 h pi was higher than is usually observed for peptides in this molecular weight range (17)(18)(19)(20)(21)(22)(23). Increased liver and kidney retention is still observed for the 111 In-labeled branched peptide relative to 125 I-ATF at 24 h pi, suggesting a structural mechanism of retention, perhaps due to the branched nature of this construct, distinct from normal residualization of 111 In-DOTA-amino acid conjugates.…”

Challenges in Optimizing a Prostate Carcinoma Binding Peptide, Identified through the Phage Display Technology

“…The kidney radioactivity at 15 min was about 8% ID/g for the iodinated peptide [14], while it was measured to be 13.5% for the DOTA-conjugated molecule. The fast renal clearance is caused by the hydrophilic chelator DOTA [33].…”

Comparative Evaluation of Three64Cu-LabeledE. coliHeat-Stable Enterotoxin Analogues for PET Imaging of Colorectal Cancer

“…At 1 h pi, uptake in tumor for each construct was higher than for all other normal tissues with the exception of kidney (Table 1). Blocking studies, carried out by coadministration of unlabeled F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), reduced accumulation of the NOTA conjugate in tumor tissue by ∼42% (p < 0.05), comparable to previous results obtained for this class of peptides (17,18). Kidney uptake values at 1 h pi for the NOTA conjugate (3.7 ( 1.6%ID/g) were lower than for either the DOTA (6.5 ( 2.7%ID/g) or TETA (11.4 ( 2.9%ID/g) constructs (Table 1, Figure 5).…”

“…Several differences exist between in vivo biodistribution patterns of 64 Cu-DOTA-F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and the previously described 111 In-complex of this DOTA-peptide (18). At 1 h pi, tumor uptake of 64 Cu-DOTA-F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) was significantly lower than that of 111 In-DOTA-F 19 -STh(2-19) (1.33 ( 0.52%ID/g vs 2.35 ( 0.43%ID/g, respectively). Simultaneously, the liver uptake of the 64 Cu-labeled peptide was higher than for the 111 Incomplex (1.21 ( 0.65 vs 0.2 ( 0.09, respectively).…”

“…The polyaminocarboxylate chelating moieties DOTA, TETA, and NOTA were appended to the N-terminus of F 19 -STh (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) via NHS ester activation. The addition of DOTA was carried out using a commercially available NHS ester of DOTA, while TETA and NOTA were appended via in situ NHS ester activation using a 100-fold excess of sulfo NHS and EDC in aqueous solution.…”

Synthesis and Characterization of an111In-Labeled Peptide for the in Vivo Localization of Human Cancers Expressing the Urokinase-Type Plasminogen Activator Receptor (uPAR)

“…125 I-labeled ATF uptake was higher than (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla) 2 -K-K( 111 In-DOTA)-NH 2 uptake in all normal tissues at these time points, with the exception of liver and kidney, which demonstrated higher levels of 111 In-peptide. Clearance of the 111 In-peptide occurs through both hepatobiliary and renal/urinary routes, and liver and kidney retention at 4 h pi was higher than is usually observed for peptides in this molecular weight range (17)(18)(19)(20)(21)(22)(23). Increased liver and kidney retention is still observed for the 111 In-labeled branched peptide relative to 125 I-ATF at 24 h pi, suggesting a structural mechanism of retention, perhaps due to the branched nature of this construct, distinct from normal residualization of 111 In-DOTA-amino acid conjugates.…”

Challenges in Optimizing a Prostate Carcinoma Binding Peptide, Identified through the Phage Display Technology

“…The kidney radioactivity at 15 min was about 8% ID/g for the iodinated peptide [14], while it was measured to be 13.5% for the DOTA-conjugated molecule. The fast renal clearance is caused by the hydrophilic chelator DOTA [33].…”