In vivo pharmacokinetic studies in rats

Three Sprague Dawley rats (males, 276–300 g; Charles River Laboratories, Wilmington, MA, USA) used in the study received standard care in compliance with an approved protocol by the Institutional Animal Care and Use Committee at the University of North Texas Health Science Center. All procedures were followed according to the Guide for the Care and Use of Laboratory Animals and referred to the Animal Welfare Act. Prior to conduction of the experiment, the rats were kept on a 12-hour light/dark cycle with unlimited access to food and water. RTV ISNP granules and RTV oral solution were administered to the rats by oral gavage at 10 mg/kg of RTV. After administration, 250 μL of blood was collected through the tail vein at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 24 hours, and centrifuged at 3,400 rpm for 5 minutes at 4°C to obtain plasma. Plasma was harvested and stored at −20°C until further analysis by an LC–mass spectrometry method previously reported.27

Plasma concentrations of RTV were analyzed by noncompartmental methods using Phoenix WinNonlin pharmacokinetic software (version 6.4; Certara, St Louis, MO, USA). Maximum plasma concentration (C_max) and time to reach C_max (T_max) were obtained by direct inspection of the plasma concentration–time profile. The area under the plasma concentration–time curve from time 0 to the last measured sample (AUC_0–last) was determined using the log-linear trapezoidal rule.