The previously reported radiosynthesis of 18F-FPIA was adapted to prepare GMP grade radiopharmaceutical using the GE FASTLab™ automated radiosynthesis platform [11, 13]. A detailed description of the automated radiosynthesis is described in the supplementary information (Supplementary material and methods S1). In brief, the precursor methyl 2,2-dimethyl-3-[(4-methylbenzenesulfonyl)oxy] propanoate  was radiolabelled by displacement of the tosylate group with dry 18F-fluoride to produce intermediate 18F-2, the methyl ester of 18F-FPIA (Scheme S1). Compound 18F2 was hydrolysed under basic conditions to give 18F-FPIA which was purified by semi-preparative HPLC using biocompatible solvents (15% EtOH, 85% sodium dihydrogen phosphate buffer, pH 4.5). The fraction containing 18F-FPIA was diluted in water and passed through a sterile filter into a sterile vial for clinical use. The identity and purity (chemical and radiochemical purity) of the final product were determined by HPLC; other quality control tests were performed, according to European Pharmacopoeia guidelines (Table S1).
Twenty-four healthy volunteers (12 men, 12 women), were categorised into 2 groups (fed vs fasted). Six men and 6 women were enrolled in the fed group and 6 men and 6 women in the fasted group. Mean age ± SD (59 ± 6.14y); age range, 51–71 y; weight, 76 kg; weight range, 55.3–103.7 kg) were enrolled. In both groups, subjects were asked to fast (water only) from midnight and the fed group were given a light lunch at least 1 h prior to scanning. The fasted group remained fasted until after the scan (approximately 16–18 h). Any subjects taking medications were asked to proceed as normal. Inclusion criteria were age above 50 y, ability to provide written informed consent, and a normal medical history (including physical examination, electrocardiogram, haematology, and biochemistry). Exclusion criteria were pregnancy, lactation, subjects diagnosed with diabetes mellitus, and those with abnormal raised blood lipid levels at baseline as deemed unsuitable for study by clinical investigator. In addition, any chronic illness that would preclude brief discontinuation of medication or musculoskeletal condition that would not allow comfortable performance for the duration of the scan. Subjects receiving any investigational therapy within 14 days or 5 half-lives of a drug prior to the first dose of 18F-FPIA injection were also excluded as well as those undergoing monitoring of occupational ionising radiation exposure.
Subjects taking medications (substrates of Cytochrome P450 enzymes-CYP3A4, CYP2C8, and CYP2D6) that may cause any interference were advised to be cautious for at least 7 days and up to 15 days after the last dose of 18F-FPIA, due to the potential for alterations in the pharmacologic effects of these medications or an increased risk for serious or life threatening adverse events associated with such medications secondary to the possible inhibition of specific CYP enzymes, by 18F-FPIA, as it is metabolised in the liver. Ethical approval for the study was granted by the London-Brent Research Ethics Committee. All volunteers gave written informed consent to participate in the study, according to the Declaration of Helsinki. The administration of radioactivity was approved by the Administration of Radioactive Substances Advisory Committee, U.K.
Safety, image acquisition, analysis, and Dosimetry
Safety data was obtained during and 24 h after radiotracer administration. Data recorded included vital signs (heart rate, blood pressure, respiratory rate, and body temperature), physical examination, and laboratory parameters (serum biochemistry, haematology, coagulation, and urinalysis). Any adverse events were recorded using the common toxicity criteria (version 5.0: https://ctep.cancer.gov/ protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quick_Reference_5x7.pdf).
Images were acquired on a Biograph 6 TruePoint PET/CT scanner (with TrueV; extended field of view [Siemens]) with 21.6-cm axial and 60.5-cm transaxial fields of view. An attenuation CT scan of each patient was obtained before administration of 18F-FPIA, from the vertex to the mid-thigh (CT settings: tube potential, 130 kV; exposure, 15 effective mAs; pitch, 1.5; slice thickness, 5 mm; rotation time, 0.6 s; resulting in an effective dose of 2.5 mSv). This scan was then followed by a multi-bed whole-body PET scanning protocol on 6 occasions within a 4 h period. A break after the fourth PET scan allowed voiding to enhance radiotracer clearance and was followed by a second CT and the last 2 multi-bed whole-body PET scans (Table 1). All emission scans were reconstructed using the ordered-subsets expectation maximization algorithm (3 iterations and 21 subsets) with corrections for dead time, scatter, attenuation, and radioactive decay. Regions of interest (ROIs) for as many of the possible International Commission on Radiological Protection (ICRP), 103 source organs were outlined , manually on screen using a circular paint brush of fixed diameter and width on Hermes (Hermes Diagnostics, Stockholm, Sweden) by a single investigator (SD) to avoid any interobserver variation. The bladder was treated separately due to the fact that it increases in volume over the course of scanning. The full bladder volume was outlined in a ROI for each PET scan by a single investigator (NK) and the mean activity concentration and volume recorded to give the total activity in the bladder for each scan.
Blood and urine activity measurements and metabolite analysis
Discrete venous bloods and plasma (at 5, 10, 15, 30, 60, and 120 min post injection (p.i.) were obtained for radioactivity counting and metabolite analysis, as previously described . Urine, ≥ 1 mL, was collected for HPLC measurements of 18F-FPIA and metabolites after the 4th and before the 5th and 6th PET scan. Urine (500 μL) was diluted in 500 μL of mobile phase (80:20, sodium phosphate monobasic (0.25 M) + 0.5% tetrabutyl ammonium hydroxide (pH 4.5): methanol), and filtered using 0.22 μm sterile syringe filters; run time 20 min.
Non-esterified fatty acids and acylcarnitine measurements
As non-esterified fatty acids (NEFAs) or carnitine levels could potentially modify radiotracer uptake, blood samples were taken for measuring NEFA and carnitine profiles at 3 time-points; at baseline prior to scanning, 60 min p.i. of 18F-FPIA, and the end of scan. All samples were centrifuged (1942 g, room temp, 5 min), within 30 min of collection, and stored at −80 °C until transfer to laboratories for analysis.
Data analysis, biodistribution and dosimetry
The mean non–decay corrected 18F activity concentration was obtained for the source organ ROIs at each whole-body scan time, resulting in time-activity curves (TACs). The activity measured in each bladder ROI was divided by the reference man bladder volume, to give a nominal activity concentration for each point on bladder TAC [15,16,17,18].The curves were decay corrected to the mid-point of each whole-body scan to most closely represent the average activity distribution for the scan. The curves were converted to activity per organ using the volume of organs in ICRP 23 reference man  and normalized by the injected activity to give the fractional uptake in each organ as a function of time. These TACs were trapezoidally integrated to generate organ residence times (τ); the total number of disintegrations in each source organ per unit injected activity. To account for the activity remaining in the body at the end of the scan protocol, the time-activity curves were extrapolated from the last whole-body scan with the simplification that radioactive decay would be the only significant change. The ED was calculated using firstly the mean residence time over all subjects for each organ and secondly using residence times for individual subjects with OLINDA/EXM v1.1, which uses the organ weighting factors from ICRP 60 .