Pharmacokinetic Evaluation of New Drugs Using a Multi-Labelling Approach and PET Imaging: Application to a Drug Candidate with Potential Application in Neuromuscular Disorders

• Published in: Biomedicines Vol. 11; no. 2; p. 253
• Main Authors: Passannante, Rossana, Gómez-Vallejo, Vanessa, Sagartzazu-Aizpurua, Maialen, Vignau Arsuaga, Laura, Marco-Moreno, Pablo, Aldanondo, Garazi, Vallejo-Illarramendi, Ainara, Aguiar, Pablo, Cossío, Unai, Martín, Abraham, Bergare, Jonas, Kingston, Lee, Elmore, Charles S, Morcillo, Miguel Angel, Ferrón, Pablo, Aizpurua, Jesus M, Llop, Jordi
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 18.01.2023; MDPI
• Subjects: Affinity labeling; Blood circulation; Chloride; Chromatography; Demethylation; Drug development; Evaluation; Gamma rays; Half-life; High-performance liquid chromatography; Homeostasis; Labeling; Metabolic pathways; Metabolism; Metabolites; Methods; Methylation; Microsomes; Neuromuscular diseases; Palladium; Permeability; PET; PET imaging; Pharmacokinetics; Positron emission tomography; Potash; Potassium; Proteins; Radioactivity; radiolabelling; Rapamycin; Ryanodine receptors; Sodium; Spain; pharmacokinetics; PET; radiolabelling
• ISSN: 2227-9059; 2227-9059
• DOI: 10.3390/biomedicines11020253

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. 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 ( C) via C-methylation to yield both [ C]AHK2.1 and [ C]AHK2.2, or by palladium-catalysed reduction of the corresponding 5-iodotriazole derivative using H gas to yield [ H]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. In vitro studies indicate that AHK2 binds to FKBP12 at the rapamycin-binding pocket, presenting activity as a FKBP12/RyR stabilizer. [ C]AHK2.1, [ C]AHK2.2 and [ H]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 CH O-Ar residue and demethylation of -N(CH ) 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 [ C]AHK2.2 was administered, probably reflecting the biodistribution of [ C]methanol as the major metabolite. Contrarily, accumulation in the gastrointestinal track was observed after administration of [ C]AKH2.1. AHK2 binds to FKBP12 at the rapamycin-binding pocket, presenting activity as a FKBP12/RyR stabilizer. Studies performed with the H- and C-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 CH -O-Ar and tertiary amine groups as the main pathways. PET studies suggest that knowledge about metabolic pathways is paramount to interpret images.