2.5. In vivo studies

The developed method was applied to an in vivo experiment examining the pharmacokinetics of orally-administered talazoparib in mice, using 4–5 animals per group. As previously described [16], pharmacokinetic studies were performed by administering talazoparib (0.5 mg/kg; p.o.) to adult female mice (12–20 weeks old) from an inbred wild-type FVB strain or mice lacking ABCB1A, ABCB1B, and ABCG2 on the same background strain [ABCB1/ABCG2(−/−) mice] (Taconic Biosciences, Cambridge City, IN, USA) [17]. The dose of 0.5 mg/kg talazoparib was based on previous research [18], and the drug was formulated in a mixture of dimethylacetamide (10%), Solutol HS (5%), and phosphate-buffered saline (85%). The final concentration of talazoparib in the oral dosing solution was 0.1 mg/mL. In select experiments, novobiocin (formulated in 5% Dextrose Injection, USP) or vehicle was administered by oral gavage 30 min before talazoparib at a dose of 50 mg/kg.

After administration of talazoparib, approximate 30-μL of each whole blood was collected from each mouse at specific time points: 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h. During the time interval of 5–30 min, samples were collected using a sterile 5 mm Goldenrod animal lancet (Braintree Scientific, Braintree, MA, USA) from a submandibular vein and a heparinized capillary tube (Thermo Fisher Scientific). Samples collected at 1 h and 2 h were obtained from the retro-orbital venous plexus using capillary tubes after mice were anesthetized using 2% isoflurane. The final sample was collected by cardiac puncture. For all samples, plasma was obtained by collecting the supernatant from whole blood samples after centrifugation at 13,000×g immediately after collection. Plasma was immediately placed on dry ice and stored at −80 °C until analysis. The pharmacokinetic experiments conducted at The Ohio State University was approved by the University Laboratory Animal Resources (ULAR) Animal Care and Use Committee. All used mice were housed with free access to water and a standard diet in a temperature- and light-controlled environment and were fasted for 2 h on the day of experiment before administration of talazoparib.

Protein precipitation was performed with following procedure. Prior to analysis, 5-μL aliquots of plasma samples thawed at room temperature, were transferred into 0.5-mL Eppendorf tubes followed by 4 μL of 500 ng/mL internal standard working solution and 61 μL of methanol and then vortex-mixed for 30s The mixture in tubes were subjected to centrifugation at 15,000×g for 10 min at 4 °C. Subsequently, 55-μL of the resulting supernatant were carefully transferred to non-coated plastic microplates (96-well format, Thermo Fisher Scientific). A precise volume of 3 μL was injected into the LC-MS/MS system for analysis.

Phoenix WinNonlin version 8.3.4.295 (Certara, Princeton, NJ, USA) was used to conduct non-compartmental analysis and derive pharmacokinetic parameters for talazoparib. Peak plasma concentration (C_max) was determined by visually examining the log concentration vs time curve. The area under the plasma concentration-time curve between time zero and the final collection point with detectable levels of talazoparib (AUC) was obtained using the log-linear trapezoidal rule. An unpaired Student's t-test was used to compare pharmacokinetic parameters of talazoparib between wild-type and ABCB1/ABCG2(−/−) mice or between treatments with and without novobiocin.