After high speed centrifugation 100 mL of supernatant was added to 200 mL of a compensation solution and directly injected onto the LC-MS. The second extraction method was a liquid-liquid extraction method. Plasma samples were diluted 1:1 with methanol containing internal standard and three volumes of water added. These mixtures were vortexed and the entire volume transferred to the extraction plate. Minimal vacuum was applied to load the samples and then allowed to stand for 5 minutes. Methyl tertiary-butyl ether was added to all wells and eluted with minimal vacuum. The solvent was evaporated to dryness under nitrogen. The samples were reconstituted by gently vortexing the plate afterwards. The antiviral efficacy of ST-246 against poxviruses has been demonstrated after oral administration in mice, rabbits, ground squirrels, prairie dogs, and NHP. The AZ 960 JAK inhibitor pharmacokinetics of ST-246 after oral administration has been thoroughly characterized in mice, NHP and humans, with limited information in rabbits, rats, and dogs. A complete understanding of the pharmacokinetics is important in species in which the efficacy is also being evaluated, as the selection of the human therapeutic dose will necessarily be chosen based on the animal PK/PD relationship, due to the lack of evaluable orthopox GSK1363089 disease in humans. The similarity of the plasma concentration time profiles after oral and IV administration demonstrated that IV administration of a dose of ST-246 should provide efficacy against orthopoxviruses, assuming the administration is slow enough to avoid what appeared to be a Cmax-related toxicity. Oral administration of 100 mg/kg provided optimal efficacy in mice against ectromelia virus. Exposure after the oral 100 mg/kg doses was close to that measured after the 10 mg/kg IV slow push administration , indicating a reasonable dose at which to start to evaluate antiviral activity with the IV formulation. Elimination in mice appeared to be mono-exponential after oral administration, but appeared to have a very short and rapid distribution phase after IV administration. Oral administration of ST-246 in mice had not elicited any dose-limiting toxicity at doses of up to 2000 mg/kg, although this might have been due to the fact that absorption after oral administration appeared to be saturated and higher doses in particular did not result in concomitantly higher peak plasma concentrations and exposure. The observed dose-limiting toxicity of unsteady gait and disequilibria after IV administration in mice, which was observed briefly at the end of the IV infusion, and that resolved within an hour, suggested that the toxicity might be related to the maximum plasma concentration.