*4.1. Synthesis of [18F]FACH*

The radiosynthesis of [18F]FACH has recently been published [38]. Briefly, [18F]FACH is produced via a one-step radiosynthesis approach by using a mesylate precursor bearing an unprotected carboxylic acid function and the Kryptofix 2.2.2/K2CO3/[ 18F]F-complex system. Isolation of [18F]FACH was performed by semi-preparative HPLC (Reprosil-Pur C18-AQ column, 250 × 10 mm, 10 μm, 50% CH3CN/20 mM NH4HCO2 (aq.), pH = 4–4.5, flow 3.5 mL/min). The tracer (chemical structure shown in Figure 4) was finally purified via solid-phase extraction (Sep-Pak® C18 light cartridge) and formulated in 10% EtOH/saline solution with molar activities in the range of 50–120 GBq/μmol (*n* = 8, at the end of synthesis) using starting activities of 1–3 GBq.

**Figure 4.** Chemical structure of [18F]FACH.

#### *4.2. Preclinical Dosimetry Studies—In Vivo PET*/*CT Imaging in Pigs*

All animal experiments were approved by the responsible institutional and federal state authorities (Landesdirektion Leipzig; TVV 18/18, Reference Number DD24.1-5131/446/19).

Three piglets (age: ~6 weeks, weight: ~13–15 kg) were fasted on the day of imaging and received an intranuscular. injection of 1 mL azaperone and 4 mL ketamine to introduce anesthesia. After 15 min, 2 mL of ketamine and 1 mL of midazolam (5 mg/mL) were iv injected (ear vein, V. auricularis), followed by 5 mL of G40, 3 mL of ketamine, and 1.5 mL of midazolam in 50 mL of NaCl 0.9% with an infusion pump at a flow rate of 37.5 mL/h to maintain the narcosis throughout the entire investigation time. During narcosis, the animals maintained spontaneous respiration and no mandatory ventilation was applied. The subjects were sequentially imaged after an iv injection (contralateral ear vein) of <sup>156</sup> <sup>±</sup> 54 MBq [18F]FACH (0.63 <sup>±</sup> 0.49 <sup>μ</sup>g) in a PET/CT system (Biograph16, SIEMENS, Erlangen, Germany). The piglets were positioned prone with legs alongside the body on a custom-made plastic trough including a piglet head-holder (Figure 5). The PET acquisition was divided into a sequential (4 × 9 min, 3 × 12 min) and a static part (1 × 24 min, 1 × 30 min, 1 × 36 min), each of which was preceded by a low-dose CT to acquire structural data for attenuation correction (AC) and anatomical orientation (Figure 6). Post mortem, the urine was collected by bladder punctuation, weighted, and divided into three 1 mL samples for activity measurements in a gamma-counter (Packard Cobra II 5003 Auto Gamma Counting System, GMI, Ramsey, MN, USA).

**Figure 5.** The animals were placed in a plastic trough and a special head rest to guarantee reproducible positioning and avoid the movement of artefacts.

**Figure 6.** PET/CT imaging protocol comprising a dynamic and static part with increasing duration per bed position (BP) to compensate for decay and, thus, decreasing count statistics, preceded by a low-dose CT (LD-CT) for attenuation correction and anatomical orientation, respectively. Positioning of the animal in the PET field of view.

PET data reconstruction was done using low-dose CT attenuation correction (AC) and an iterative OSEM algorithm with 4 iterations and 8 subsets. As the PET/CT system is in daily clinical use, it is periodically subjected to detector normalization and activity calibration. Furthermore, all peripheral activity-measuring devices to be used for the investigation (dose calibrator, gamma counter) are cross-calibrated in terms of timing and a radioactivity adjustment.
