Pharmacokinetics analysis in mice

All in vivo experiments were carried out in eight-week-old female 129×1/SvJ mice (Jackson Laboratory); as a minimum, animals were monitored once daily. Single-dose PK studies were carried out after i.v. and oral substance administration. For i.v. dosing, GHP-88309 was formulated in 28% PEG200, 5% dimethylacetamide, 67% 30% HPB cyclodextrin in sterile PBS at 1.25 mg/ml. For oral dosing, compound was formulated in 1% methylcellulose in water for a homogeneous suspension of up to 18.76 mg/ml. Mice were administered either 5 mg/kg via tail vein injection, or 50 mg/kg or 150 mg/kg via oral gavage, followed by blood collection after 0, 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours (i.v. group) or 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours (per oral group). Plasma was prepared from whole blood (2000×g, 5 minutes, 4°C) within 15 minutes of blood collection and samples stored at −80°C until analysis by LC-MS/MS. Calibration curve range was 10–10,000 ng/ml, samples above quantitation limit were diluted with commercial CD-1 mouse plasma and retested. Quality control samples were interspersed throughout the run, and no chromatographic interferences were detected in blank CD-1 or 129×1/SvJ mouse plasma. For multi-dose PK studies, mice received GHP-88309 orally at 150 mg/kg dose concentration in a b.i.d. regimen for five days. In the first 4 days of dosing, blood samples were collected 0.5 hours (C_max) and 11.5 hours (trough) after the morning dose. On day 5, a full PK study (lacking the 6-hour time point) was performed after the morning dose as outlined. Blood samples were processed and analyzed as above. To determine GHP-88309 organ distribution, mice were administered a single oral dose of GHP-88309 at 150 mg/kg. Selected organs (brain, lung, spleen, kidney, liver, and heart) were extracted 90 minutes after dosing, flash frozen in liquid nitrogen, and stored at −80°C until further analysis by LC-MS/MS as described.