NC2459, an alpha, beta --unsaturated ketone, is currently in the preclinical phase for the treatment of cutaneous leishmaniasis caused by Leishmania major. Advancement of this compound requires a bioanalytical assay for quantification, evaluation of physio-chemical properties and development of suitable drug delivery systems for determination of pharmacokinetic properties in vivo. These preliminary studies will serve as reference to future preclinical and clinical progression of NC2459. This work reports the development of bioanalytical assays for NC2459, preformulation studies for the characterization of physiochemical properties, and development of simple and advanced drug delivery systems for the determination of pharmacokinetic parameters in an animal model.

An LC-MS/MS system equipped with an ACERTM Excel SuperC18 UHPLC column for separation and a 4000 QTRAP for chromatographic analysis was used to develop LC-MS/MS methods for NC2459 in solution, urine and plasma. The assays were validated according to FDA specifications. Octanol-water partition coefficient (log P), stability, and solubility in different solvents were determined for the compound. Ultracentri-fugation was used to evaluate in vitro protein binding in rat plasma. Co-solvent systems comprised of various components and ratios were evaluated for aqueous stability. A solid lipid nanoparticle for oral delivery was prepared for NC2459. A pharmacokinetic study in male SD rats (body weights 0.3-0.33 kg) was conducted using optimal formulations. Concentrations of NC2459 were analyzed via LC-MS/MS methods in blood and urine samples collected at predetermined time points. Pharmacokinetic parameters were estimated using Phoenix WinNonlin RTM software.

Standard curves of NC2459 in solution, plasma and urine were linear in the concentration range of 10 -- 1000 ng/mL. Intra- and inter-day accuracy (%RE) and precision (%CV) were within the 15% acceptance range per FDA guidance. NC2459 was highly lipophilic (log P 6.28 +/- 0.34), practically insoluble in water (16.63 +/- 2.34 mug/mL), and is highly bound to plasma protein (89.7 +/- 3.34 > %). These studies will help guide future preclinical and clinical evaluations of this compound.