Plasma analysis

Approximately 30–35 arterial blood samples (each 1 mL) were collected p.i. at the following time points: as fast as possible over 1.5 min; every 30 s between 1.5–3 min; every 2 min between 3–5 min; and every 5 min between 5 min to the end of the emission scan. Plasma was isolated by centrifugation at 4 °C (5 min at 2200 g) and radioactivity was counted using a 2480 WIZARD² gamma counter (PerkinElmer, Waltham, MA).

Larger (~4–6 mL) blood samples for measurement of parent [¹⁸F]FNDP and metabolites occurred at the following time points: before [¹⁸F]FNDP injection for background measure, and p.i. at 5, 10, 20, 30, 45, 60, 75, and 90 min. Additional blood samples were obtained at 105, 120, 150, and 180 min p.i. for emission scans lasting 180 min (N=3). Parent [¹⁸F]FNDP and metabolites were analyzed using a high performance liquid chromatography (HPLC) system from Agilent Technologies consisting of a 1260 Infinity quaternary pump, a 1260 Infinity column compartment module, a 1260 Infinity UV detector, and Raytest GABI Star radiation detectors controlled by OpenLab CDS EZChrom (A.01.04) software. The HPLC system was first standardized using [¹⁸F]FNDP and a non-radioactive analog of the tracer prior to analysis. Plasma samples were loaded onto a 2 mL Rheodyne injector loop and directed to a capture column (packed with Phenomenex Strata-X 33μm polymeric reversed phase sorbent) with 1% acetonitrile and 99% water mobile phase at 2 mL/min. The effluent from the capture column contained polar metabolites measured by a detector. After 2 min of elution, an analytical mobile phase (65% acetonitrile, 35% aqueous 0.06 M ammonium formate) was applied to direct the trapped non-polar metabolites and [¹⁸F]FNDP to an analytical column (4.6 × 250 mm, XBridge Column, 5 μm) at 2 mL/min, which were then measured by a detector. HPLC chromatograms were integrated to provide percentage of parent [¹⁸F]FNDP relative to radiometabolite peaks at each time point. Metabolite-corrected plasma time-activity curves (TACs) were obtained by applying the percent parent [¹⁸F]FNDP time-profiles from HPLC to the total plasma TACs after linear interpolation using PMOD (v3.7, PMOD Technologies Ltd, Zurich, Switzerland).

Plasma free fraction (f_P) of [¹⁸F]FNDP was assessed using plasma isolated from blood sampling prior to radiotracer injection. Plasma (1 mL) was incubated with 0.37 MBq of [¹⁸F]FNDP and incubated (5 min) at room temperature. Then three separate 150 uL aliquots of the mixture were applied to Centrifree membrane filters (Millipore, Burlington, MA) and centrifuged (20 min, 26000 g, 25 °C). To calculate f_P, 50 uL samples of elute and three 50 uL samples of plasma incubated with [¹⁸F]FNDP were counted on an automated gamma counter, with f_P = counts in ultrafiltrate relative to plasma.