Automated synthesis of [(18)F]DCFPyL via direct radiofluorination and validation in preclinical prostate cancer models

Abstract

Background: Prostate-specific membrane antigen (PSMA) is frequently overexpressed and upregulated in prostate cancer. To date, various (18)F- and (68)Ga-labeled urea-based radiotracers for PET imaging of PSMA have been developed and entered clinical trials. Here, we describe an automated synthesis of [(18)F]DCFPyL via direct radiofluorination and validation in preclinical models of prostate cancer.

Methods: [(18)F]DCFPyL was synthesized via direct nucleophilic heteroaromatic substitution reaction in a single reactor TRACERlab FXFN automated synthesis unit. Radiopharmacological evaluation of [(18)F]DCFPyL involved internalization experiments, dynamic PET imaging in LNCaP (PSMA+) and PC3 (PSMA-) tumor-bearing BALB/c nude mice, biodistribution studies, and metabolic profiling. In addition, reversible two-tissue compartmental model analysis was used to quantify pharmacokinetics of [(18)F]DCFPyL in LNCaP and PC3 tumor models.

Results: Automated radiosynthesis afforded radiotracer [(18)F]DCFPyL in decay-corrected radiochemical yields of 23 ± 5 % (n = 10) within 55 min, including HPLC purification. Dynamic PET analysis revealed rapid and high uptake of radioactivity (SUV5min 0.95) in LNCaP tumors which increased over time (SUV60min 1.1). Radioactivity uptake in LNCaP tumors was blocked in the presence of nonradioactive DCFPyL (SUV60min 0.22). The muscle as reference tissue showed rapid and continuous clearance over time (SUV60min 0.06). Fast blood clearance of radioactivity resulted in tumor-blood ratios of 1.0 after 10 min and 8.3 after 60 min. PC3 tumors also showed continuous clearance of radioactivity over time (SUV60min 0.11). Kinetic analysis of PET data revealed the two-tissue compartmental model as best fit with K 1 = 0.12, k 2 = 0.18, k 3 = 0.08, and k 4 = 0.004 min(-1), confirming molecular trapping of [(18)F]DCFPyL in PSMA+ LNCaP cells.

Conclusions: [(18)F]DCFPyL can be prepared for clinical applications simply and in good radiochemical yields via a direct radiofluorination synthesis route in a single reactor automated synthesis unit. Radiopharmacological evaluation of [(18)F]DCFPyL confirmed high PSMA-mediated tumor uptake combined with superior clearance parameters. Compartmental model analysis points to a two-step molecular trapping mechanism based on PSMA binding and subsequent internalization leading to retention of radioactivity in PSMA+ LNCaP tumors.

Keywords: 18FDCFPyL; Automated radiosynthesis; Positron emission tomography (PET); Prostate cancer; Prostate-specific membrane antigen (PSMA).

Fig. 1

Structure of ¹⁸F-labeled small-molecule PSMA inhibitors

Fig. 2

Synthesis of lysine-urea-glutamate peptidomimetics

Fig. 3

Automated synthesis unit for the radiosynthesis of [¹⁸F]DCFPyL

Fig. 4

Radiosynthesis of [¹⁸F]DCFPyL

Fig. 5

a HPLC traces for confirmation of identity after co-injection of [¹⁸F]DCFPyL with reference compound DCFPyL and b quality control of the final product. Detectors are connected in series

Fig. 6

Cell-specific internalization experiments of [¹⁸F]DCFPyL in PSMA+ LNCaP and PSMA− PC3 cells 60 min after incubation with the radiotracer at 37 °C

Fig. 7

PET images of [¹⁸F]DCFPyL after 60 min p.i. into LNCaP (left) and PC3 (right) tumor-bearing BALB/c nude mice. Middle: Time-activity curves for radioactivity uptake in LNCaP and PC3 tumor

Fig. 8

PET images of [¹⁸F]DCFPyL after 60 min p.i. into a LNCaP tumor-bearing BALB/c nude mouse. Left: control; Middle: time-activity curves for uptake and blocking in LNCaP tumors in the absence and presence of nonradioactive DCFPyL; Right: blocked

Fig. 9

Distribution of [¹⁸F]DCFPyL in the blood (top) revealing only minimal binding to plasma proteins and therefore good bioavailability and evaluation of the metabolic stability of [¹⁸F]DCFPyL (bottom) following injection in BALB/c mice (n = 3)

Fig. 10

Tracer kinetic analysis of [¹⁸F]DCFPyL in PSMA+ LNCaP and PSMA− PC3 tumor-bearing Balb/c mice