Pharmacokinetic Evaluation of New Drugs Using a Multi-Labelling Approach and PET Imaging: Application to a Drug...

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 (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.

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.

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.