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Bioconjug Chem. 2019 Apr 17;30(4):1210-1219. doi: 10.1021/acs.bioconjchem.9b00146. Epub 2019 Apr 8.

A Metal-Free DOTA-Conjugated 18F-Labeled Radiotracer: [18F]DOTA-AMBF3-LLP2A for Imaging VLA-4 Over-Expression in Murine Melanoma with Improved Tumor Uptake and Greatly Enhanced Renal Clearance.

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Chemistry Department , University of British Columbia , 2036 Main Mall , Vancouver , British Columbia V6T 1Z1 , Canada.
Molecular Oncology , British Columbia Cancer Agency Research Centre , 675 West 10th Avenue , Vancouver , British Columbia V5Z 1L3 , Canada.


DOTA is commonly used for radiometal chelation in molecular imaging. Yet in the absence of a radiometal, DOTA is hypothesized to promote renal clearance of 18F-labeled peptide tracers. In light of an increasing interest in the use of F18 for PET, here the effect of DOTA is evaluated for the first time with an 18F-labeled tracer and is found to significantly improve the quality of images acquired through positron emission tomography (PET). We chose to image the peptide LLP2A that recognizes the transmembrane protein very-late antigen 4 (VLA-4) that is overexpressed by many cancers. Since it is known that [18F]RBF3-PEG2-LLP2A derivatives gave low tumor uptake values and significant GI tract accumulation, this ligand thus represents an ideal means of testing the additive effects of a DOTA group on clearance while permitting a facile, user-friendly, one-step 18F-labeling. A newly designed RBF3-LLP2A bioconjugate with an appended DOTA moiety increased tumor uptake nearly 3-fold and reduced GI accumulation by more than 10-fold. The DOTA-AMBF3-PEG2-LLP2A was radiolabeled by isotope exchange and was purified by semiprep HPLC and C18 cartridge elution. Male C57BL/6J mice bearing B16-F10 melanoma tumors that overexpress the VLA-4 target were used to evaluate [18F]DOTA-AMBF3-PEG2-LLP2A using a combination of static and dynamic PET scans, biodistribution studies, and blocking controls at 1 h post injection (p.i.). The precursor peptide was synthesized and 18F-labeled to provide formulations with mean (±SD) radiochemical purities of 95.9 ± 1.8%, in radiochemical yields of 4.8 ± 2.9% having molar activities of 131.7 ± 50.3 GBq/μmol. In vivo static PET images of [18F]DOTA-AMBF3-PEG2-LLP2A provided clear tumor visualization, and biodistribution studies showed that tumor uptake was 9.46 ± 2.19% injected dose per gram of tissue (%ID/g) with high tumor/muscle and tumor/blood contrast ratios of ∼8 and ∼10, respectively. Blocking confirmed the specificity of [18F]DOTA-AMBF3-PEG2-LLP2A to VLA-4 in the tumor and the bone marrow. Dynamic PET showed clearance of [18F]DOTA-AMBF3-PEG2-LLP2A mainly via the renal pathway, wherein accumulation in the intestines was reduced 10-fold compared to our previously investigated LLP2A's, while spleen uptake was at levels similar to previously reported LLP2A-chelator radiotracers. [18F]DOTA-AMBF3-PEG2-LLP2A represents a promising VLA-4 radiotracer and provides key evidence as to how a DOTA appendage can significantly reduce GI uptake in favor of urinary excretion. Implications for the development of dual-isotope theranostics that exploit the use fluorine-18 for imaging and DOTA to chelate therapeutic metal cations for therapy are discussed.

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