Passannante, RossanaGómez Vallejo, VanessaSagartzazu Aizpurua, MaialenVignau Arsuaga, LauraMarco Moreno, PabloAldanondo Aristizabal, GaraziVallejo Illarramendi, AinaraAguiar Fernández, PabloCossío, UnaiMartín, AbrahamBergare, JonasKingston, LeeElmore, Charles S.Morcillo Alonso, Miguel ÁngelFerrón, PabloAizpurua Iparraguirre, Jesus MariLlop, Jordi2025-06-092025-06-092023-01-18Passannante, R., Gómez-Vallejo, V., Sagartzazu-Aizpurua, M., Vignau Arsuaga, L., Marco-Moreno, P., Aldanondo, G., Vallejo-Illarramendi, A., Aguiar, P., Cossío, U., Martín, A., Bergare, J., Kingston, L., Elmore, C. S., Morcillo, M. A., Ferrón, P., Aizpurua, J. M., & Llop, J. (2023). Pharmacokinetic evaluation of new drugs using a multi-labelling approach and PET imaging: application to a drug candidate with potential application in neuromuscular disorders. Biomedicines, 11(2). https://doi.org/10.3390/BIOMEDICINES1102025310.3390/BIOMEDICINES11020253https://hdl.handle.net/20.500.14454/2972Background and objective: The determination of pharmacokinetic properties of new chemical entities is a key step in the process of drug development. Positron emission tomography (PET) is an ideal technique to obtain both biodistribution and pharmacokinetic parameters of new compounds over a wide range of chemical modalities. Here, we use a multi-radionuclide/multi-position labelling approach to investigate distribution, elimination, and metabolism of a triazole-based FKBP12 ligand (AHK2) with potential application in neuromuscular disorders. Methods: Target engagement and stabilizing capacity of the drug candidate (AHK2) towards FKBP12-RyR was evaluated using competitive ligand binding and proximity ligation assays, respectively. Subsequently, AHK2 was labelled either with the positron emitter carbon-11 (11C) via 11C-methylation to yield both [11C]AHK2.1 and [11C]AHK2.2, or by palladium-catalysed reduction of the corresponding 5-iodotriazole derivative using 3H gas to yield [3H]AHK2. Metabolism was first investigated in vitro using liver microsomes. PET imaging studies in rats after intravenous (IV) administration at different doses (1 µg/Kg and 5 mg/Kg) were combined with determination of arterial blood time-activity curves (TACs) and analysis of plasma samples by high performance liquid chromatography (HPLC) to quantify radioactive metabolites. Arterial TACs were obtained in continuous mode by using an in-house developed system that enables extracorporeal blood circulation and continuous measurement of radioactivity in the blood. Pharmacokinetic parameters were determined by non-compartmental modelling of the TACs. Results: In vitro studies indicate that AHK2 binds to FKBP12 at the rapamycin-binding pocket, presenting activity as a FKBP12/RyR stabilizer. [11C]AHK2.1, [11C]AHK2.2 and [3H]AHK2 could be obtained in overall non-decay corrected radiochemical yields of 14 ± 2%, 15 ± 2% and 0.05%, respectively. Molar activities were 60–110 GBq/µmol, 68–122 GBq/µmol and 0.4–0.5 GBq/μmol, respectively. In vitro results showed that oxidation of the thioether group into sulfoxide, demethylation of the CH3O-Ar residue and demethylation of –N(CH3)2 were the main metabolic pathways. Fast metabolism was observed in vivo. Pharmacokinetic parameters obtained from metabolite-corrected arterial blood TACs showed a short half-life (12.6 ± 3.3 min). Dynamic PET imaging showed elimination via urine when [11C]AHK2.2 was administered, probably reflecting the biodistribution of [11C]methanol as the major metabolite. Contrarily, accumulation in the gastrointestinal track was observed after administration of [11C]AKH2.1. Conclusions: AHK2 binds to FKBP12 at the rapamycin-binding pocket, presenting activity as a FKBP12/RyR stabilizer. Studies performed with the 3H- and 11C-labelled FKBP12/RyR stabilizer AHK2 confirm fast blood clearance, linear pharmacokinetics and rapid metabolism involving oxidation of the sulfide and amine moieties and oxidative demethylation of the CH3-O-Ar and tertiary amine groups as the main pathways. PET studies suggest that knowledge about metabolic pathways is paramount to interpret images.eng© 2023 by the authorsPETPharmacokineticsRadiolabellingjournal article2025-06-092227-9059