In this pilot study, four groups of animals were used. The first group was the control group with a standard intravenous tracer injection. The other three groups received an oral administration of [18F]MC225, each group with a different protocol.

The intravenous group, G1 (n = 3, body weight of 309 ± 8 g), was first anaesthetized with a mixture of 0.02 mL/g of ketamine and dexmedetomidine. This anesthetic combination was used because it has been shown not to alter intestinal motility compared with isoflurane, propofol, and pentobarbital [21, 22]. The animals were intravenously injected with 28.0 ± 8.0 MBq in a tail vein, while positioned in the PET scanner, using an infusion pump at a rate of 1 mL/min with a total administered volume of 1 mL.

The oral administration methodology was based on a previous scoping review [8], which guided the selection of acquisition protocols to optimize tracer administration. Previous studies indicated that oral routes typically require approximately 40–70% of the radioactivity used for i.v. injections to reduce scattering and prevent high tracer concentrations in localized areas (e.g., stomach) [9, 10, 23, 24]. The gavage technique was selected for oral administration. This method delivers the tracer near the gastro-esophageal sphincter, allowing gravity to facilitate its entry into the stomach [25].

Tracer production for oral administration followed the same method as for i.v. administration [18, 19]. [18F]MC225, diluted in 1 mL saline, was administrated using disposable needles. For G2, the gavage needle used was Cadence Science 9918 (18G), from Fisher Scientific (Landsmeer, the Netherlands). However, close inspection indicated that changing the needle type could facilitate tracer administration more effectively, prompting a switch of needles. For one animal in G3 and the entire G4, the needles were substituted with FTP-15–78-SAM from Instech Laboratories (Plymouth, USA). Animals were awake during administration to monitor whether there were any complications. Following oral administration, 0.5 mL of saline was administered to flush the esophagus, ensuring that the tracer reached the stomach.

In the second group, G2 (n = 3 body weight 324 ± 4 g), the tracer was administered orally via gavage with an activity of 8.8 ± 1.7 MBq. Subsequently, animals were anesthetized using the same combination of anesthetics as for G1.

The third group, G3 (n = 3, body weight 302 ± 11 g), received the tracer orally via gavage as well, but with a higher activity of 17.0 ± 0.7 MBq. In addition, these animals underwent a fasting period of 12–14 h prior to the scan. The protocol modifications between G3 and G2 were introduced to assess whether higher radioactivity uptake, better image quality and more accurate quantification could be obtained.

To estimate whether oral administration could also detect any changes in P-gp function, the P-gp inhibitor tariquidar was administered in the fourth group. Previous studies using [18F]MC225 have indicated that doses higher than 3 mg/kg of tariquidar could significantly impact brain P-gp uptake in rats [15, 19]. To avoid the need for anesthesia, which would be needed for i.v. administration, tariquidar was administered intraperitoneally (i.p.). Previous studies have shown that the effects of tariquidar in rats remain constant and comparable to those after i.v. injection [26].

Tariquidar was prepared in a vehicle solution of 5% dimethyl sulfoxide (DMSO), 10% TWEEN 20, 25% polyethylene glycol 400 (PEG400), and 65% H2O, following the same protocol as for i.v. administration [15]. A dose of 10 mg/kg, selected as an intermediate value based on previous studies, was administered [15]. Group G4 (n = 3, body weight 286 ± 30 g) received a dose of 10.1 ± 1.6 mg/kg of tariquidar, with an average volume of 0.86 ± 0.21 mL (Bio-Techne Ltd., Abingdon, UK), 1 h before tracer administration. This timing was chosen to ensure that tariquidar’s peak concentration, which occurs 2 h after i.p. injection, coincided with tracer acquisition [26].

The tracer, with an activity of 16.6 ± 3.2 MBq, was administered via oral gavage following a 12–14 h fasting period prior to the scan. Table 1 summarizes the groups, body weights, administered activities and routes, and time between tracer administration and start of scan.