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 µm, 2.1 × 50 mm) and a Kinetex® C18 column (5 µm, 2.1 × 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.

Development of a micelle-enhanced high-throughput fluorometric method for determination of niclosamide using a microplate reader.

The present paper describes the development and validation of a simple and sensitive micelle-enhanced high-throughput fluorometric method for the determination of niclosamide (NIC) in 96-microwell plates. The proposed method is based on the reduction of the nitro group of niclosamide to an amino group using Zn/HCl to give a highly fluorescent derivative that was developed simultaneously and measured at λem 444 nm after excitation at λex 275 nm. Tween-80 and carboxymethylcellulose (CMC) have been used as fluorescence enhancers and greatly enhanced the fluorescence by factors of 100-150%. The different experimental conditions affecting the fluorescence reaction were carefully investigated and optimized. The proposed method showed good linearity (r2 ≥ 0.9997) over the concentration ranges of 1-5 and 0.5-5 µg/ml with lower detection limits of 0.01 and 0.008 µg/ml and lower quantification limits of 0.04 and 0.03 µg/ml on using Tween-80 and or CMC, respectively. The developed high-throughput method was successfully applied for the determination of niclosamide in both tablets and spiked plasma. The capability of the method for measuring microvolume samples made it convenient for handling a very large number of samples simultaneously. In addition, it is considered an environmentally friendly method with lower consumption of chemicals and solvents.

Evaluación de la mutagenicidad de la niclosamida en fluidos corporales / Evaluation of utagenicity of niclosamine in body fluids

Se estudió la mutagenicidad de la niclosamida, mediante la evaluación de muestras de sangre y orina de voluntarios tratados con las dosis empleadas en la terapéutica, las cuales fueron ensayadas en cepas de Salmonella typhimurium. Los resultados muestran ausencia de actividad mutagénica para dicha droga en las condiciones experimentales empleadas

Evaluación de la mutagenicidad de la niclosamida en fluidos corporales / Evaluation of mutagenicity of niclosamine in body fluids

Se estudió la mutagenicidad de la niclosamida, mediante la evaluación de muestras de sangre y orina de voluntarios tratados con las dosis empleadas en la terapéutica, las cuales fueron ensayadas en cepas de Salmonella typhimurium. Los resultados muestran ausencia de actividad mutagénica para dicha droga en las condiciones experimentales empleadas