Bioanalysis of niclosamide in plasma using liquid chromatography-tandem mass and application to pharmacokinetics in rats and dogs

Niclosamide, which is an anti-tapeworm drug, was developed in 1958. However, recent studies have demonstrated the antiviral effects of niclosamide against the SARS-CoV-2 virus, which causes COVID-19. In this study, we developed and validated a quantitative analysis method for the determination of niclosamide in rat and dog plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and used this method for pharmacokinetic studies. Biological samples were prepared using the protein precipitation method with acetonitrile. Ibuprofen was used as an internal standard. The mobile phase used to quantify niclosamide in rat or dog plasma consisted of 10 mM ammonium formate in distilled water-acetonitrile (30:70, v/v) or 5 mM ammonium acetate-methanol (30:70, v/v). An XDB-phenyl column (5 mu m, 2.1 x 50 mm) and a Kinetex (R) C18 column (5 mu m, 2.1 x 500 mm) were used as reverse-phase liquid chromatography columns for rat and dog plasma analyses, respectively. Niclosamide and ibuprofen were detected under multiple reaction monitoring conditions using the electrospray ionization interface running in the negative ionization mode. Niclosamide presented linearity in the concentration ranges of 1-3000 ng/mL (r = 0.9967) and 1-1000 ng/mL (r = 0.9941) in rat and dog plasma, respectively. The intra- and inter-day precision values were < 7.40% and < 6.35%, respectively, for rat plasma, and < 3.95% and < 4.01%, respectively, for dog plasma. The intra- and inter-day accuracy values were < 4.59% and < 6.63%, respectively, for rat plasma, and < 12.1% and < 10.9%, respectively, for dog plasma. In addition, the recoveries of niclosamide ranged between 87.8 and 99.6% and 102-104% for rat and dog plasma, respectively. Niclosamide was stable during storage under various conditions (three freeze-thaw cycles, 6 h at room temperature, long-term, and processed samples). A reliable LC-MS/MS method for niclosamide detection was successfully used to perform pharmacokinetic studies in rats and dogs. Niclosamide presented dose-independent pharmacokinetics in the dose range of 0.3-3 mg/kg after intravenous administration, and drug exposure in rats and dogs after oral administration was very low. Additionally, niclosamide presented high plasma protein binding (>99.8%) and low metabolic stability. These results can be helpful for further developing and understanding the pharmacokinetic characteristics of niclosamide to expand its clinical use.