Pharmacokinetic mechanism involved in the prolonged high retention of laninamivir in mouse respiratory tissues after intranasal administration of its prodrug laninamivir octanoate.

Laninamivir octanoate (LO) (Inavir; Daiichi Sankyo, Japan) is an ester prodrug of the neuraminidase inhibitor laninamivir. We previously reported that a prolonged high retention of laninamivir in mouse respiratory tissues was achieved by intranasal administration of LO. In this study, we evaluated intrapulmonary pharmacokinetics both in vivo and in vitro to investigate the potential mechanism involved in such a preferable retention. After intranasal administration of LO to mice (0.5 µmol/kg), the drug was distributed from the airway space into the lungs, and laninamivir remained in the lung at 24 hours postdose (2680 pmol/g), with a higher concentration than that in the epithelial lining fluid. The laninamivir was localized mainly on the epithelial cells of airway tracts, determined by microautoradiography using (14)C-labeled LO. In mouse airway epithelial cells, the cellular uptake and hydrolysis of LO were observed over incubation time without any apparent saturation at the highest concentration tested (1000 µM). Furthermore, after additional incubation in drug-free medium, the intracellular laninamivir was released very slowly into the medium with an estimate rate constant of 0.0707 h(-1), which was regarded as a rate-limiting step in the cellular retention. These results demonstrated that the prolonged high retention of laninamivir in the respiratory tissues was attributed to a consecutive series of three steps: uptake of LO into the airway epithelial cells, hydrolysis of LO into laninamivir by intracellular esterase(s), and limited efflux of the generated laninamivir due to its poor membrane permeability. This prodrug approach could be useful for lung-targeting drug delivery.

Aqueous normal phase liquid chromatography coupled with tandem time-of-flight quadrupole mass spectrometry for determination of zanamivir in human serum.

An aqueous normal phase (ANP) liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (ANP-LC-micrOTOFQ) method was used for the determination of zanamivir in human serum. Zanamivir was extracted with methanol from protein-precipitated human serum samples and further purified with SCX solid-phase extraction cartridges. Scherzo SM-C18, Agilent Zorbax SB-Aq, Cogent Diamond Hydride, Cogent Bidentate and Luna HILIC columns were compared and optimized for the retention and separation of zanamivir and the Luna HILIC and Diamond Hydride columns exhibited the best retention of zanamivir. The former provided a shorter retention time, a sharper peak and relatively high sensitivity, whereas the latter exhibited a longer retention time and less matrix interference. The analytical range of the calibration curve was between 5 and 1000 ng/mL.

Quantification of the anti-influenza drug zanamivir in plasma using high-throughput HILIC-MS/MS.

BACKGROUND: parenteral zanamivir is a promising drug for the treatment of severe influenza. However, quantification of this polar drug in biological matrices has traditionally been difficult and the methods developed have been relatively insensitive. RESULTS: a high-throughput bioanalytical method for the analysis of zanamivir in human plasma using SPE in the 96-well plate format and LC coupled to positive MS/MS has been developed and validated according to US FDA guidelines. The method uses 50 microl of plasma and covers a large working range from 1-50, 000 ng/ml with a LOD of 0.50 ng/ml. CONCLUSION: this new LC-MS/MS assay is more sensitive than previous methods despite using a small plasma volume sample. It is particularly suitable for clinical studies on both parenteral and inhaled zanamivir.

Determination of zanamivir in rat and monkey plasma by positive ion hydrophilic interaction chromatography (HILIC)/tandem mass spectrometry.

A hydrophilic interaction chromatography (HILIC)/mass spectrometric assay was developed for the determination of zanamivir, a neuraminidase inhibitor used to treat influenza, in rat and monkey plasma. An organic solvent with hydrophilic properties, methanol, was used to precipitate proteins in plasma to assure the highly polar zanamivir of staying in solution. Chromatographic separation was obtained using a HILIC silica column with multiple reaction monitoring turboionspray positive ion detection. The stable label of zanamivir, [(13)C(1)(15)N(2)] GR121167C, was used as the internal standard. The assay was validated for the determination of zanamivir in rat and monkey plasma. The lower and upper limits of quantitation were 2 and 10000 ng/mL, using 0.05 mL plasma aliquot, respectively. The signal to noise ratio of a typical 2 ng/mL was approximately 5:1. The inter-day precision (relative standard deviation) and accuracy (relative error) in rat plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 6 to 10% and -6.5 to 0.2%, respectively. The inter-day precision (relative standard deviation) and accuracy (relative error) in monkey plasma, derived from the analysis of validation samples at five concentrations, ranged from 2 to 8% and -2.3 to 2.1%, respectively. Zanamivir was found to be stable for at least 5 days at approximately -80 degrees C and at room temperature in plasma. This assay incorporates a simple protein precipitation with methanol and hydrophilic interaction chromatography which is sensitive, accurate, precise, and is being used to support oral formulation and toxicokinetic studies in rat and monkey, respectively.