Alfatide[18F]

Integrin α6 has been considered a promising biomarker, is overexpressed in many tumors, and plays a vital role in tumor formation, recurrence, and metastasis. In this study, we identified a novel high-affinity integrin α6-targeted peptide named RD₂ (Arg-Trp-Tyr-Asp-PEG4)₂-Lys-Lys and developed a ¹⁸F-radiolabeled peptide tracer ([¹⁸F]-AlF-NOTA-RD₂) and evaluated its potential application in positron emission tomography (PET) imaging of pancreatic cancer. [¹⁸F]-AlF-NOTA-RD₂ was produced using GMP (Good Manufacturing Practice of Medical Products)-compliant automatic radiosynthesis on a single GE FASTLab2 cassette-type synthesis module. The stability of [¹⁸F]-AlF-NOTA-RD₂ was analyzed in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). The cell uptake assay of the tracer was assessed using PANC-1 cells. In addition, small-animal PET imaging and biodistribution studies of [¹⁸F]-AlF-NOTA-RD₂ were performed in pancreatic cancer subcutaneous tumor-bearing mice. The PET tracer [¹⁸F]-AlF-NOTA-RD₂ was obtained with a radiochemical yield of 23.7 ± 4.7%, radiochemical purity of >99%, and molar activity of 165.7 ± 59.1 GBq/μmol. [¹⁸F]-AlF-NOTA-RD₂ exhibited good in vitro stability in PBS and FBS. LogP octanol water value for the tracer was -2.28 ± 0.05 (n = 3). The binding affinity of RD₂ to the integrin α6 protein (K_d = 0.13 ± 3.65 μM, n = 3) was significantly higher than that of the RWY (CRWYDENAC) (K_d = 6.97 ± 1.44 μM, n = 3). Small-animal PET imaging and biodistribution also revealed that [¹⁸F]-AlF-NOTA-RD₂ displayed rapid and good tumor uptake and lower liver background uptake in PANC-1 tumor-bearing mice. [¹⁸F]-AlF-NOTA-RD₂ showed significant radioactivity accumulation in tumors and was successfully blocked by NOTA-RD₂. Compared with [¹⁸F]-FDG, [¹⁸F]-AlF-NOTA-RD₂ PET imaging and biodistribution studies in PANC-1 xenograft tumor-bearing mice confirmed a good tumor-to-muscle ratio (8.69 ± 2.03 vs 1.41 ± 0.23, respectively) at 0.5 h and (2.99 ± 3.02 vs 1.43 ± 0.17, respectively) at 1 h post injection. Autoradiography of human pancreatic cancer tumor tissues further confirmed high accumulation of [¹⁸F]-AlF-NOTA-RD₂. In summary, we developed an optimized integrin α6-targeted imaging tracer and obtained high radioactivity products with a cassette-type synthesis module; moreover, the tracer exhibited good binding affinity with integrin α6 and good target specificity for PANC-1 cells in xenograft pancreatic tumor-bearing mice, demonstrating its promising application as a noninvasive PET radiotracer of integrin α6 expression in pancreatic cancer.

For the further study of [19F]‐alfatide, the development of highly sensitive analytical methods for its determination is an urgent issue. In this paper, a method for simultaneously determining [19F]‐alfatide and alfatide using liquid chromatography/tandem mass spectrometry was created and validated. The plasma samples were pretreated using the protein precipitation method. Poroshell 120 EC‐C18 (4.6 × 50 mm, 2.7 μm, Agilent) was used for the separation of the analytes to be measured. The mobile phase, consisting of acetonitrile and water (0.1% acetic acid, 50:50, v/v), was delivered at a flow rate of 0.60 ml/min for sample analysis. Positive electrospray ionization was performed using multiple‐reaction monitoring with transitions of m/z 715.2 → 636.6 for [19F]‐alfatide, the ion pairs 700.3 → 851.5 for alfatide and the ion pairs 237.1 → 194.2 for carbamazepine (internal standard). According to the results, the method had high specificity, precision and accuracy as well as an extended linear range. The matrix effect and extraction recovery were also acceptable. The method was successfully applied to the pharmacokinetic study of [19F]‐alfatide in rats.