Evaluation of the peptidomimetic-organotrifluoroborate conjugate, 18F-LLP2A-PEG2-N-Pyr-p-BF3, as an α4β1-specific radiotracer for PET imaging of melanoma

Objectives Over-expression of α4β1 integrin receptors on melanomas, lymphomas and multiple myelomas contributes to angiogenesis, tumor growth, and metastasis. The high-affinity α4β1 specific peptidomimetic, LLP2A, was identified in 2006, with 2 pM (IC50) affinity to this target. We developed a new, highly-stable pyridinyl organotrifluoroborate radioprosthetic (N-Pyr-p-BF3) conjugated to a modified LLP2A derivative, 18F-LLP2A-PEG2-N-Pyr-p-BF3, as radiotracer to visualize α4β1-overexpressing tumors.

Methods The LLP2A peptidomimetic was synthesized using solid phase peptide synthesis (SPPS). After resin cleavage, the modified LLP2A(OtBu)-PEG2-NH2 was obtained and functionalized with an azide group. The alkynyl-N-Pyr-p-BF3 prosthetic group, with a solvolytic t1/2 of ~10,000 min, was then conjugated by click chemistry. 18F-radiolabeling was performed using 80 nmol of precursor in the presence of ~1 Ci 18F in DMF, 40 nmol F- carrier and 1 M pyradazine-HCl (aq) buffer at pH ~2.5. After 20 min of heating at 85 °C, the isotope exchange (IEX) reactions were quenched with 40 mM NH4HCO2 (aq) and the 18F-tracers were HPLC-purified. B16-F10 murine melanoma mouse xenograft models were then used for evaluating the tumor-targeting of 18F-LLP2A-PEG2-N-Pyr-p-BF3 by PET imaging and biodistribution studies.

Results The decay-corrected radiochemical yield was 15.6 ± 5.9 % during synthesis times of 72 ± 8 min (N=4). Analytical HPLC showed an average purity of 96.1 ± 2.5 % (N=4) and a specific activity of 3.29 ± 0.27 Ci/μmol (N=3). For the in vivo studies, 18F-LLP2A-PEG2-N-Pyr-p-BF3 was administered for imaging and/or biodistribution imaging at 1h p.i. (n=6). PET imaging showed excellent tumor-to-background contrast. Tumor uptake was 4.71 ± 0.45 %ID/g with high tumor-to-muscle, -blood, and -kidney ratios of 9.28 ± 1.46, 16.29 ± 3.46 and 5.96 ± 1.01, respectively. Due to endogenous α4β1, there was expected uptake in the bone (3.72 ± 0.46 %ID/g) and spleen (9.53 ± 2.63 %ID/g). Yet, the high uptake in the intestines (57.63 ± 1.13 %ID/g) indicated high hepatobiliary clearance with this LLP2A conjugate.

Conclusions Using facile aqueous-based IEX methods, we produced a new 18F-labeled LLP2A derivative (18F-LLP2A-PEG2-N-Pyr-p-BF3) with good radiochemical yield, excellent radiochemical purity and high specific activity. In vivo PET imaging of B16-F10 murine melanoma models showed good tumor uptake, but hepatobiliary clearance predominated. We are currently investigating alternate BF3 prosthetic groups to modulate tumor uptake and clearance.

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