Pharmacokinetics Studies

Adult male C57BL/6 mice and Sprague Dawley rats were purchased from Charles River Laboratory (Wilmington, MA). PK studies for mice and rats were conducted at NIH animal facility (Bethesda, MD). In-life dog and monkey PK studies were conducted at Absorption System (San Diego, CA) and Frontage Labs (Concord, OH), respectively. Dogs and monkeys were fasted overnight prior to dose administrations. All experimental procedures were reviewed and approved by the Animal Care and Use Committee (ACUC) of the NIH Division of Veterinary Resources (DVR) or CRO labs for in-life studies.

Male C57BL6 mice (n = 3 per time point, 24–40 g) were dosed at 5 mg/kg IV or 10 mg/kg oral gavage. The dosing volume was 3 mL/kg for IV and 10 ml/kg for PO. All dosing solutions were freshly prepared on the day of administration. Blood samples were collected over 24 h.

Male Sprague–Dawley rats (n = 3 or 4 per treatment group, ∼ 343–375 g) were dosed in IV and PO dosing groups: 1 and 5 mg/kg IV, and 10, 30 and 100 mg/kg PO, respectively. Double cannulated rats (one catheter for IV dosing and another one for sampling) were used. Plasma samples were collected from jugular vein over 24 h. In bile duct cannulated (BDC) rat group, a single IV dose of 2 mg/kg was administered to three rats. Rat urine and bile samples were collected for the pre-dose, 0–4 h, 4–8 h, and 8–24 h time periods for BDC rats.

Male Cynomolgus monkeys (n = 3 per treatment group, 3.3–4.3 kg) received IV and PO administration of 2 and 5 mg/kg of GS-441524, respectively. The dosing volumes were 2 mL/kg for IV and 5 mL/kg for PO. Blood samples were obtained from each monkey up to 48 h after IV and PO administration. Urine samples were collected over 48 h for IV group.

Male Beagle dogs (n = 3 per treatment group, 9.0–11.4 kg) received 2 mg/kg IV and 5 mg/kg PO of GS-441524 administration. The dosing volume was 1.0 mL/kg for both groups. Plasma (both IV and PO groups) and urine (IV group) samples were collected over a period of 48 h.

All GS-441524 dosing solutions were prepared in 5% ethanol, 30% PG, 45% PEG-400 and 20% water +1 equivalent of HCl. Blood samples from all groups and species were collected in K₂EDTA tubes and stored on wet ice pending processing. Blood was centrifuged at 2200 g at 4°C for 10 min to isolate plasma. All samples were stored at -80°C until analysis.