Multivalent Bifunctional Chelator Scaffolds for Gallium-68 Based Positron Emission Tomography Imaging Probe Design: Signal Amplification via Multivalency

Abstract

The role of the multivalent effect has been well recognized in the design of molecular imaging probes towards the desired imaging signal amplification. Recently we reported a bifunctional chelator (BFC) scaffold design, which provides a simple and versatile approach to impart multivalency to radiometal based nuclear imaging probes. In this work, we report a series of BFC scaffolds (tBu3-1-COOH, tBu3-2-(COOH)2 and tBu3-3-(COOH)3) constructed on the framework of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for 68Ga-based PET probe design and signal amplification via multivalent effect. For proof of principle, a known integrin αvβ3 specific ligand (c(RGDyK)) was used to build the corresponding NOTA conjugates (H31, H32, and H33), which present 1 – 3 copies of c(RGDyK) peptide, respectively, in a systematic manner. Using the integrin αvβ3 binding affinities (IC50 values), the enhanced specific binding was observed for multivalent conjugates (H32: 43.9 ± 16.1 nM; H33: 14.7 ± 5.0 nM) as compared to their monovalent counterpart (H31: 171 ± 60 nM) and the intact c(RGDyK) peptide (204 ± 76 nM). The obtained conjugates were efficiently labeled with 68Ga3+ within 30 min at room temperature in high radiochemical yields (> 95%). The in vivo evaluation of the labeled conjugates, 68Ga-1, 68Ga-2 and 68Ga-3, was performed using male severe combined immunodeficiency (SCID) mice bearing integrin αvβ3 positive PC-3 tumor xenografts (n = 3). All 68Ga -labeled conjugates showed high in vivo stability with no detectable metabolites found by radio-HPLC within 2 h post-injection (p.i.). The PET signal amplification in PC-3 tumor by multivalent effect was clearly displayed by the tumor uptake of the 68Ga-labeled conjugates (68Ga-3: 2.55 ± 0.50%ID/g; 68Ga-2: 1.90 ± 0.10 %ID/g; 68Ga-1: 1.66 ± 0.15 %ID/g) at 2 h p.i. In summary, we have designed and synthesized a series of NOTA-based BFC scaffolds with signal amplification properties, which may find potential applications in diagnostic gallium radiopharmaceuticals.

Evaluation of novel 99mTc(I)-labeled homobivalent α-melanocyte-stimulating hormone analogs for melanocortin-1 receptor targeting

“…The high kidney uptake and retention, together with the low tumor uptake led to poor tumor-to-kidney ratios (e.g., 0.08 and 0.025 for 2 and 3, respectively, at 4 h after injection), which are considerably lower than the ratio obtained for the monovalent compound 1 (e.g., 0.94 at 4 h after injection). This behavior is comparable to that reported for other radiolabeled bivalent a-MSH and multivalent RGD derivatives [26,37]. In the former case, the tumor-to-kidney ratios observed for three bivalent 111 In-DOTA-labeled NAPamide analogs ranged between 0.11 and 0.36, which are also considerably lower than the ratio observed for the monovalent 111 In-DOTA-NAPamide (1.67) [37].…”

“…These results, often reported for other radiopeptides, are indicative of pronounced tubular reabsorption, which is likely related to the binding to the multiligand megalin receptor [16,26,37,[49][50][51]. Whether nonspecific uptake by kidneys could have been modulated (e.g., by infusion of basic amino acids) was not studied.…”

“…Such an approach when applied to the design of nuclear probes intends to amplify the imaging signal, a crucial issue for a more effective targeted tumor imaging [1,[23][24][25]. The most successful examples of this approach were achieved with radiolabeled multimeric cyclic RGD peptides, where an increase in peptide multiplicity significantly enhanced the integrin a v b 3 binding affinity with improvement of the tumor-targeting ability [24,[26][27][28][29][30][31].…”

In Vivo PET Imaging of the Cancer Integrin αvβ6 Using 68Ga-Labeled Cyclic RGD Nonapeptides

“…A pronounced hydrophilicity promotes a fast excretion from nontarget tissues via the kidneys and the urinary tract, which is desired for imaging probes. Concerning receptor affinities, it has been shown in numerous studies that tethering multiple copies of receptor ligands, for example, a v b 3 integrin-targeting peptides of the cyclo(RGDXK) type, to a given reporter (e.g., radionuclide or fluorophor) is a reliable method to obtain constructs with increased integrin activity as well as higher uptake in a v b 3 integrin-expressing tumors (36,(47)(48)(49)(50)(51)(52)(53)(54). The same was observed for a5b1 integrin tracers based on a a5b1-specific pseudopeptide (55)(56)(57), suggesting that integrin-targeted radiopharmaceuticals might generally benefit from multimerization.…”

Effects of Chelator Modifications on 68Ga-Labeled [Tyr3]Octreotide Conjugates

“…The NOTA-based scaffolds were synthesized based on previously published procedures [19, 20]. All reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO) and used as received unless otherwise noted.…”