Paclitaxel

The unbound fraction was determined by equilibrium dialysis using a rapid equilibrium dialysis device (Thermo Fisher Scientific, Waltham, MA, USA). Paclitaxel or [³H(G)]digoxin ([³H] digoxin, 39.8 Ci/mmol; PerkinElmer, Boston, MA, USA) was spiked into maternal or fetal plasma at a final concentration of 26 or 13 nM. Spiked plasma (100 μL) was loaded into the sample chambers of the device, and the buffer chambers were filled with 350 μL of phosphate-buffered saline. The device was covered with sealing tape and incubated for 12 h at 37°C on an orbital shaker running at 250 rpm. Following incubation, aliquots from both chambers were taken for measurement of Paclitaxel concentration or radioactivity of [³H]digoxin using LC-MS/MS or liquid scintillation counting, respectively. The unbound fraction in the maternal and fetal plasma (f_u,mp and f_u,fp) was calculated as the PBS-to-plasma concentration (radioactivity) ratio. Albumin concentration in maternal and fetal plasma was determined using an LBIS Mouse Albumin ELISA Kit (Fujifilm Wako Shibayagi, Shibukawa, Japan) according to the manufacturer’s protocol.

Doxorubicin content was measured fluorimetrically as detailed previously by us [34 (link)]. Vinblastine and paclitaxel accumulation were measured by labelling cells with 1 μCi [³H]-vinblastine sulphate (PerkinElmer, Waltham, MA) and [³H]-paclitaxel (Moravek Inc., Brea, CA). Cells were washed twice with PBS, detached with trypsin and sonicated. The intracellular drug content was measured by liquid scintillation. The results were expressed as nmol drug/mg cell proteins, according to titration curves previously set.