Evaluation of fluorine-18 dipeptide as a marker for overexpressed H⁺/peptide transporters

Objectives: Immunohistochemistry, western blotting and gene expression studies reveal that PepT1 and PepT2 are overexpressed and regulated by different kinases in a variety of human cancer cell lines [1,2], suggesting PepT1 and PepT2 maybe clinically useful target to biomarkers of specific epithelial cancers. H+/Peptide transporters have been shown to be responsible for peptide transport activity in cancer cells [3,4] including pancreatic carcinoma [2]. Previous studies demonstrated that expression of H⁺/peptide transporter PepT is increased in human derived pancreatic cancer cells [1]. In this study we propose to radiolabel dipeptide 1 with fluoride-18. Dipeptide 1 (DP-1) specifically binds to PepT making it an attractive imaging agent for a positron emission tomography (PET) diagnostic of pancreas tumors.

Methods: DP-1 is a modification of the previously known H⁺/peptide transporter imaging agent [¹¹C]-glycylsarcosine [5]. The radiolabeling of DP-1 with fluorine-18 was done in two steps from the corresponding di-protected mesylate. PepT1 expression in ASpc1, Panc1 and BxPC3 human pancreas cancer cell lines were analyzed by Western blot using antibodies specific to PepT1. For cell uptake studies, ASpc1, BxPC3 and Panc1 cells were plated on 24-well plates and incubated with DP-1 for 1h with or without blocking with 100 times excess of unlabeled DP-1. After washing specific activity was read on a γ-counter. For PET imaging studies we inoculated nude mice sc with 1 x 10⁶ pancreatic cancer cells (ASpc1, Panc1 and BxPC3 cell lines). DP-1 was injected iv at 100 μCi/mouse; 30 min after injection static 30 min PET images were acquired. To verify labeling specificity DP-1 activity was blocked by injecting a cold non-labeled DP-1.

Results: The overall synthesis for DP-1 was achieved in two steps. The radiolabeling step was purified through by either solid-phase extraction (SPE) technique or reversed phase high-performance liquid chromatography (HPLC). The acidic deprotection and subsequent formulation provided the desired fluorine-18 dipeptide in excellent radiochemical purity (> 94%). PepT1 transporter was expressed in all studied human pancreatic cancer lines. Cell-uptake studies demonstrated that DP-1 is highly up taken by pancreas cancer cells. This can be blocked by adding an excess amount of the unlabeled DP-1. The highest uptake was achieved at pH of 7.4, with the greatest amount of tracer binding at 80 min. PET imaging of xenograft nude mice inoculated sc with human cancer cells over expressing PepT1 demonstrated specific (tumor / background ratio >2) uptake of DP-1 by the cancer cells. Conclusions: We successfully produced and evaluated DP-1 as a potential imaging agent for tumors overexpressing PepT. In a cell uptake study DP-1 binds to PepT1 expressing human cancer cell lines. PET studies demonstrated a clinical potential for use of DP-1 as an imaging agent for diagnosis of human cancers over expressing PepT1. Acknowledgements: This work was supported by grant NCATS UL1TR001873 (Reilly) Irving Institute/CTSA PCSR Imaging Award and Translational Therapeutics Accelerator. References: [1] W. Tai, et al, Molecular Pharmaceutics 2013, 10, 477-487[2] K. Matsuoka, et al, Eur. J. Pharm. Sci. 2010, 40, 202-208[3] K. Mitsuoka, et al, J. Nucl. Med. 2008, 4, 615-622[4] T. Nakanishi, et al, Cancer Res. 1997, 57, 4118-4122 [5] N. B. Nabulsi, et al, Bioorg. Med. Chem. 2005, 13, 2993-3001

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