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. 2011 Aug 17;22(8):1682-9.
doi: 10.1021/bc200252j. Epub 2011 Jul 20.

Synthesis and Evaluation of Novel Gonadotropin-Releasing Hormone Receptor-Targeting Peptides

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Free PMC article

Synthesis and Evaluation of Novel Gonadotropin-Releasing Hormone Receptor-Targeting Peptides

Haixun Guo et al. Bioconjug Chem. .
Free PMC article

Abstract

The purpose of this study was to develop novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor-targeting peptides for breast cancer imaging. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated GnRH peptides were designed and synthesized. The radiometal chelator DOTA was conjugated to the epsilon or alpha amino group of D-lysine, or the epsilon amino group of L-lysine via an Ahx {aminohexanoic acid} linker to generate DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3), respectively. The conjugation of the DOTA to the epsilon amino group of D-lysine (rather than alpha amino group of D-lysine nor epsilon amino group of L-lysine) maintained the nanomolar GnRH receptor binding affinity. The IC(50) values of DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3) were 36.1 nM, 10.6 mM and 4.3 mM, respectively. Since only DOTA-Ahx-(D-Lys(6)-GnRH1) displayed nanomolar receptor binding affinity, the specific GnRH receptor binding of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) was determined in human GnRH receptor membrane preparations. Furthermore, the biodistribution and tumor imaging properties of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) were examined in MDA-MB-231 human breast cancer-xenografted nude mice. (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) exhibited specific GnRH receptor binding and rapid tumor uptake (1.76 ± 0.58% ID/g at 0.5 h postinjection) coupled with fast whole-body clearance through the urinary system. The MDA-MB-231 human breast cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 1 h postinjection of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1). The profound impact of DOTA position on the binding affinity of the GnRH peptide provided a new insight into the design of novel radiolabeled GnRH peptides. The successful imaging of MDA-MB-231 human breast cancer-xenografted tumor lesions using (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) suggested its potential as a novel imaging probe for human breast cancer imaging.

Figures

Figure 1
Figure 1
Synthetic schemes of three novel GnRH peptides.
Figure 2
Figure 2
The competitive binding curves of the GnRH peptides. The IC50 values of DOTA-Ahx-(D- Lys6-GnRH1), DOTA-Ahx-(D-Lys6-GnRH2) and DOTA-Ahx-(L-Lys6-GnRH3) were 36.1 nM, 10.6 mM and 4.3 mM, respectively.
Figure 3
Figure 3
Radioactive HPLC profiles of 111In-DOTA-Ahx-(D-Lys6-GnRH1) (A, T=0) and its mouse serum stability (B, T=2 h) after 2 h incubation at 37°C. The retention time of 111In-DOTA-Ahx-(D-Lys6-GnRH1) was 12.0 min; Binding of 111In-DOTA-Ahx-(D-Lys6-GnRH1) on human GnRH receptor membrane preparations (C) with (□) or without (■) the presence of 1 μM of DOTA-Ahx-(D-Lys6-GnRH1). *P<0.05.
Figure 4
Figure 4
Immunohistochemistry staining of GnRH receptor expressions in MDA-MB-231 human breast cancer-xenografted tumor (A, ×400). The MDA-MB-231 xenografted tumor exhibited strong brown cytoplasmic staining. As a comparison, the MDA-MB-231 xenografted tumor were stained without primary goat anti-human GnRH antibody (B, ×400).
Figure 5
Figure 5
Three-dimensional (A), coronal (B) and transversal (C) SPECT/CT images of MDA-MB-231 human breast cancer-xenografted tumor at 1 h post-injection of 5.6 MBq of 111In-DOTA-Ahx-(D-Lys6-GnRH1). Flank breast cancer lesions (T) were highlighted with arrows on the images.

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