Radiosynthesis and initial in vitro evaluation of [18F]F-PEG6-IPQA--a novel PET radiotracer for imaging EGFR expression-activity in lung carcinomas

Abstract

Introduction: Epidermal growth factor receptor (EGFR)-targeted therapies with antibodies and small molecular EGFR kinase inhibitors have shown poor efficacy in unselected populations of patients with advanced non-small cell lung carcinomas (NSCLC). In contrast, patients with overexpression of EGFR and activating mutations in EGFR kinase domain demonstrated improved responses to EGFR kinase inhibitors. Therefore, we have developed a novel radiotracer, [(18)F]F-PEG(6)-IPQA for PET imaging of EGFR expression-activity in NSCLC, and have described its radiosynthesis and in vitro evaluation in two NSCLC cell lines with wild-type and L858R active mutant EGFR.

Methods: A mesylate precursor was synthesized in multiple steps and radiofluorinated using K(18)F/Kryptofix. The fluorinated intermediate compound was reduced to an amino derivative then treated with acryloyl isobutyl carbonate, followed by purification by HPLC to obtain the desired product.

Results: Decay-corrected radiochemical yields of [(18)F]F-PEG(6)-IPQA were 3.9-17.6%, with an average of 9.0% (n = 11). Radiochemical purity was >97% with specific activity of 34 GBq/μmol (mean value, n = 10) at the end of synthesis. The accumulation of [(18)F]F-PEG(6)-IPQA in H3255 cells was ten-fold higher than in H441 cells, despite a two-fold lower level of activated phospho-EGFR expression in H3255 cells compared with H441 cells. The accumulation of [(18)F]F-PEG(6)-IPQA in both cell lines was significantly decreased in the presence of a small molecular EGFR kinase inhibitor, Iressa, at 100 μM concentration in culture medium.

Conclusion: We have synthesized [(18)F]F-PEG(6)-IPQA and demonstrated its highly selective accumulation in active mutant L858R EGFR-expressing NSCLC cells in vitro. Further in vivo studies are warranted to assess the ability of PET imaging with [(18)F]F-PEG(6)-IPQA to discriminate the active mutant L858R EGFR-expressing NSCLC that are sensitive to therapy with EGFR kinase inhibitors vs NSCLC that express wild-type EGFR.

Fig. 1

Analytical radio-HPLC chromatogram of [¹⁸F]F-PEG₆-IPQA 14 after formulation. The UV trace showed a small amount of unknown impurity.

Fig. 2

Time-dependent accumulation of [¹⁸F]F-PEG₆-IPQA 14 in H441 and H3255 tumor cells in vitro, before and after the addition of Iressa (100 μM) into the culture medium.

Fig. 3

Western blot analysis of total EGFR and phosphotyrosine 845 of EGFR expression in H441 and H3255 tumor cells. The autoradiogram of protein electrophoresis membrane demonstrates preferential irreversible and covalent binding of [¹⁸F]F-PEG₆-IPQA to active mutant L858R EGFR kinase domain.

Scheme 1

Synthetic schemes for preparation of the non-radioactive compound 4-[(3-iodophenyl)amino]-7-[2-{2-(2-[2-{2-(2-fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide 1. a Synthesis of 1. b Preparation of 7. c Preparation of precursor 9.

Scheme 2

Radiosynthesis scheme for the preparation of [¹⁸F]F-PEG₆-IPQA 14.