Impact of Multiple Negative Charges on Blood Clearance and Biodistribution Characteristics of ^99mTc-Labeled Dimeric Cyclic RGD Peptides

Abstract

This study sought to evaluate the impact of multiple negative charges on blood clearance kinetics and biodistribution properties of 99mTc-labeled RGD peptide dimers. Bioconjugates HYNIC-P6G-RGD2 and HYNIC-P6D-RGD2 were prepared by reacting P6G-RGD2 and P6D-RGD2, respectively, with excess HYNIC-OSu in the presence of diisopropylethylamine. Their IC50 values were determined to be 31 ± 5 and 41 ± 6 nM, respectively, against 125I-echistatin bound to U87MG glioma cells in a whole-cell displacement assay. Complexes [99mTc(HYNIC-P6G-RGD2)(tricine)(TPPTS)] (99mTc-P6G-RGD2) and [99mTc(HYNIC-P6D-RGD2)(tricine)(TPPTS)] (99mTc-P6D-RGD2) were prepared in high radiochemical purity (RCP > 95%) and specific activity (37–110 GBq/μmol). They were evaluated in athymic nude mice bearing U87MG glioma xenografts for their biodistribution. The most significant difference between 99mTc-P6D-RGD2 and 99mTc-P6G-RGD2 was their blood radioactivity levels and tumor uptake. The initial blood radioactivity level for 99mTc-P6D-RGD2 (4.71 ± 1.00%ID/g) was ∼5× higher than that of 99mTc-P6G-RGD2 (0.88 ± 0.05%ID/g), but this difference disappeared at 60 min p.i. 99mTc-P6D-RGD2 had much lower tumor uptake (2.20–3.11%ID/g) than 99mTc-P6G-RGD2 (7.82–9.27%ID/g) over a 2 h period. Since HYNIC-P6D-RGD2 and HYNIC-P6G-RGD2 shared a similar integrin αvβ3 binding affinity (41 ± 6 nM versus 31 ± 5 nM), the difference in their blood activity and tumor uptake is most likely related to the nine negative charges and high protein binding of 99mTc-P6D-RGD2. Despite its low uptake in U87MG tumors, the tumor uptake of 99mTc-P6D-RGD2 was integrin αvβ3-specific. SPECT/CT studies were performed using 99mTc-P6G-RGD2 in athymic nude mice bearing U87MG glioma and MDA-MB-231 breast cancer xenografts. The SPECT/CT data demonstrated the tumor-targeting capability of 99mTc-P6G-RGD2, and its tumor uptake depends on the integrin αvβ3 expression levels on tumor cells and neovasculature. It was concluded that the multiple negative charges have a significant impact on the blood clearance kinetics and tumor uptake of 99mTc-labeled dimeric cyclic RGD peptides.

Investigation of SP94 Peptide as a Specific Probe for Hepatocellular Carcinoma Imaging and Therapy

“…The binding affinity of HYNIC-SP94 was assessed via a competitive displacement assay with 99m Tc tricine-EDDA/HYNIC-SP94 35 . Briefly, Huh-7 cells (1 × 10 6 ) and 99m Tc tricine-EDDA/HYNIC-SP94 (370 kBq) were added to each test tube in the presence of increasing concentrations of the SP94 peptide (from 0.001 nM to 10000 nM) and incubated for 2 h at 37 °C.…”

Radiolabeled Cyclic RGD Peptide Bioconjugates as Radiotracers Targeting Multiple Integrins

“…49 Protein bonding is generally detrimental because it will reduce the number of radiotracer molecules available for receptor binding, and result in more blood radioactivity (Chart III). 49 Therefore, the protein bonding should be minimized. In addition, hydrophilic radiotracers tend to fast renal excretion, which will lead to lower background radioactivity in the blood pool and normal organs (such as liver, lungs and muscle) with better T/B ratios.…”

“…The blood clearance kinetics of a radiotracer can be measured by collecting blood samples at specific time points over a specific period of time. 49 The blood radioactivity usually expressed as the percentage of its initial radioactivity will be plotted against time. The most important parameter for kinetic studies is the rate of changes in the radiotracer organ uptake.…”

Radiolabeled cyclic RGD peptides as radiotracers for tumor imaging

“…A large number of radiolabeled cyclic RGD (arginine–glycine–aspartic) peptides have been evaluated as SPECT or PET radiotracers for tumor imaging (Liu et al 2005 ; Wu et al 2005 ; Jia et al 2006 ; Liu et al 2006 ; Zhang et al 2006 ; Alves et al 2007 ; Dijkgraaf et al 2007a , b ; Liu et al 2007 ; Wu et al 2007 ; Jia et al 2008 ; Li et al 2008b ; Liu et al 2008a ; Shi et al 2008 ; Wang et al 2008a , b ; Liu et al 2009a , b ; Shi et al 2009a , b , c ; Chakraborty et al 2010 ; Kubas et al 2010 ; Dumont et al 2011 ; Jia et al 2011 ; Shi et al 2011a , b ; Zhou et al 2011b ; Nwe et al 2012 ; Pohle et al 2012 ; Zhou et al 2012 ; Ji et al 2013a , b ; Li et al 2013 ; Simecek et al 2013 ; Tsiapa et al 2013 ; Maschauer et al 2014 ; Yang et al 2014 ; Zheng et al 2015 ). Many excellent review articles have appeared to cover their nuclear medicine applications (D’Andrea et al 2006 ; Liu 2006 ; Meyer et al 2006 ; Beer and Schwaiger 2008 ; Cai and Chen 2008 ; Liu et al 2008b ; Liu 2009 ; Stollman et al 2009 ; Beer and Chen 2010 ; Chakraborty and Liu 2010 ; Dijkgraaf and Boerman 2010 ; Haubner et al 2010 ; Beer et al 2011 ; Michalski and Chen 2011 ; Zhou et al 2011a ; Danhier et al 2012 ; Tateishi et al 2012 .…”