ABSTRACT: Radiotracers based on the peptide A20FMDV2 selectively target the cell surface receptor integrin ?v?6. This integrin has been identified as a prognostic indicator correlating with the severity of disease for several challenging malignancies. In previous studies of A20FMDV2 peptides labeled with 4-(18)F-fluorobenzoic acid ((18)F-FBA), we have shown that the introduction of poly(ethylene glycol) (PEG) improves pharmacokinetics, including increased uptake in ?v?6-expressing tumors. The present study evaluated the effect of site-specific C-terminal or dual (N- and C-terminal) PEGylation, yielding (18)F-FBA-A20FMDV2-PEG28 (4) and (18)F-FBA-PEG28-A20FMDV2-PEG28 (5), on ?v?6-targeted tumor uptake and pharmacokinetics. The results are compared with (18)F-FBA -labeled A20FMDV2 radiotracers (1- 3) bearing either no PEG or different PEG units at the N terminus.The radiotracers were prepared and radiolabeled on solid phase. Using 3 cell lines, DX3puro?6 (?v?6+), DX3puro (?v?6-), and BxPC-3 (?v?6+), we evaluated the radiotracers in vitro (serum stability; cell binding and internalization) and in vivo in mouse models bearing paired DX3puro?6-DX3puro and, for 5, BxPC-3 xenografts.The size and location of the PEG units significantly affected ?v?6 targeting and pharmacokinetics. Although the C-terminally PEGylated 4 showed some improvements over the un-PEGylated (18)F-FBA-A20FMDV2 (1), it was the bi-terminally PEGylated 5 that displayed the more favorable combination of high ?v?6 affinity, selectivity, and pharmacokinetic profile. In vitro, 5 bound to ?v?6-expressing DX3puro?6 and BxPC-3 cells with 60.5% ± 3.3% and 48.8% ± 8.3%, respectively, with a significant fraction of internalization (37.2% ± 4.0% and 37.6% ± 4.1% of total radioactivity, respectively). By comparison, in the DX3puro control 5: showed only 3.0% ± 0.5% binding and 0.9% ± 0.2% internalization. In vivo, 5: maintained high, ?v?6-directed binding in the paired DX3puro?6-DX3puro model (1 h: DX3puro?6, 2.3 ± 0.2 percentage injected dose per gram [%ID/g]; DX3puro?6/DX3puro ratio, 6.5:1; 4 h: 10.7:1). In the pancreatic BxPC-3 model, uptake was 4.7 ± 0.9 %ID/g (1 h) despite small tumor sizes (20-80 mg).The bi-PEGylated radiotracer 5 showed a greatly improved pharmacokinetic profile, beyond what was predicted from individual N- or C-terminal PEGylation. It appears that the 2 PEG units acted synergistically to result in an improved metabolic profile including high ?v?6+ tumor uptake and retention.
