Development and validation of an ultrahigh-performance liquid chromatography-tandem mass spectrometry method to investigate the plasma pharmacokinetics of a KCa 2.2/KCa 2.3 positive allosteric modulator in mice

Abstract

Rationale: There is currently no treatment for spinocerebellar ataxias (SCAs), which are a group of genetic disorders that often cause a lack of coordination, difficulty walking, slurred speech, tremors, and eventually death. Activation of K_Ca 2.2/K_Ca 2.3 channels reportedly exerts beneficial effects in SCAs. Here, we report the development and validation of an analytical method for quantitating a recently developed positive allosteric modulator of K_Ca 2.2/K_Ca 2.3 channels (compound 2q) in mouse plasma.

Methods: Mouse plasma samples (10 μL) containing various concentrations of 2q were subjected to protein precipitation in the presence of a structurally similar internal standard (IS). Subsequently, the analytes were separated on a C₁₈ ultrahigh-performance liquid chromatography column and detected by a tandem mass spectrometer. The method was validated using US Food and Drug Administration (FDA) guidelines. Finally, the validated assay was applied to the measurement of the plasma concentrations of 2q in plasma samples taken from mice after single intravenous doses of 2 mg/kg of 2q, and the pharmacokinetic parameters of 2q were determined.

Results: The calibration standards were linear (r² ≥ 0.99) in the range of 1.56-200 nM of 2q with intra- and inter-run accuracy and precision values within the FDA guidelines. The lower limit of quantitation of the assay was 1.56 nM (0.258 pg on the column). The recoveries of 2q and IS from plasma were >94%, with no appreciable matrix effect. The assay showed no significant carryover, and the plasma samples stored at -80°C or the processed samples stored in the autosampler at 10°C were stable for at least 3 weeks and 36 h, respectively. After intravenous injection, 2q showed a bi-exponential decline pattern in the mouse plasma, with a clearance of 30 mL/min/kg, a terminal volume of distribution of 1.93 mL/kg, and a terminal half-life of 45 min.

Conclusions: The developed assay is suitable for preclinical pharmacokinetic-pharmacodynamic studies of 2q as a potential drug candidate for ataxias.