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

Abstract

Background 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. Conclusion: 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.

Novel FKBP12 ligand promotes functional improvement in SOD1G93AALS mice

“…In this context, we introduce a novel class of triazole molecules, empirically validated as FKBP12 ligands to facilitate FKBP12-RyR binding. This binding capability serves to stabilize the Ca 2+ flux mediated by RyR, as demonstrated in our previous works (Aizpurua et al, 2021; Passannante et al, 2023). In this study, we introduce MP compounds, novel FKBP12 ligands that exhibit cytosolic calcium modulating activity, with the potential to be used for the treatment of ALS.…”

“…Compounds MP-001 and MP-002, strategically designed to target the interaction site of FKBP12/RyR1, were synthesized using CuAAC “click chemistry” methodologies. Specifically, aryl propargyl sulfides and methyl azidoglycinate were utilized for MP-001, while 2-azidoethyl-N,N-dimethylammonium hydrochloride was employed for MP-002 synthesis (Aizpurua et al, 2021; Passannante et al, 2023). Compound MP-010 was prepared by adding phthaloyl peroxide (Gan et al, 2017) to a solution of MP-002, followed by stirring at room temperature, basification with 7 N NH 3 in MeOH, and evaporation under reduced pressure.…”