Abstract 4205: Intracellular pharmacokinetics of 5FU and palbociclib: Uptake and efflux in disaggregated cells and 3D models
Authors: Jove Maria , Loadman Paul , Spencer Jade , Sulayman Lava , Wicks Jo , Race Amanda , Twelves Chris 
Published: July 2017

Introduction:

Pre-clinical drug development does not routinely assess drug penetration beyond use of the human intestinal Caco-2 cell line as an in vitro model of the intestinal barrier to evaluate potential drug absorption. Detailed in vivo tumour pharmacokinetic (PK) studies are unusual. However, the ability of a drug to reach its target plays a key role in drug efficacy.

Here we present a methodology to study drug PK in disaggregated cells and in a 3D cell culture model (spheroids) using 5FU and palbociclib. The comparison of these 2 drugs is of interest taking into account their different mechanisms and chemical properties, logP 0.58 and 2.12 respectively. Knowledge of cellular uptake and efflux in disaggregated cells and 3D models together with intracellular binding characteristics is vital to optimise a better drug delivery.

Experimental procedures:

MCF-7 and DLD-1 cells were used for cellular and spheroid experiments. Spheroid growth was optimized to produce a spheroid of 300-400µm diameter at day 4. Drug uptake: 1x106 cells or 30 spheroids/time point were exposed to 10µM 5FU or 0.1µM of palbociclib for different time durations. For 5FU, drug uptake at 100µM was also monitored to detect 5FU metabolites. Drug efflux: 1x106 cells were exposed first to 100µM 5FU or 0.1µM of palbociclib for 60min then, drug media removed, substituted by fresh media and sample over a 4h period. Drug concentration inside cells and in the fresh media was measured. Drug concentrations were measured using HPLC-Tandem Mass Spectrometry. Each experiment was carried out in triplicate.

Results:

5FU achieves intracellular steady state within 5-10min in cells and spheroids with equal concentrations inside and outside the cell/spheroids. Intracellular metabolites of 5FU were only seen with 100µM treatment and had a similar PK characteristics to 5FU. 5FU drug efflux experiments showed that efflux occurred rapidly within 5min achieving a new equilibrium with 30% of 5FU remaining inside the cells. 5FUrd was also detected extracellularly after 1h in the fresh media, showing that cells have both 5FU and metabolite efflux. In contrast, palbociclib cellular uptake had an initial intracellular drug concentration peak at 5-10min which then plateaued. Interestingly, total intracellular concentrations of palbociclib were over 30 times higher compared to the external media drug at steady state. Spheroid and efflux experiments of palbociclib are on-going and will be presented.

Conclusions:

Our results suggest that both drugs penetrate quickly in cancer cells. Palbociclib intracellular concentrations were 30 times higher than the external media suggesting excessive intracellular binding while 5FU achieve equal concentrations between the inside and outside of the cell. These data and methodology may be useful to allow generation of mathematical models to improve drug delivery design.

Citation Format: Maria Jove, Paul Loadman, Jade Spencer, Lava Sulayman, Jo Wicks, Amanda Race, Chris Twelves. Intracellular pharmacokinetics of 5FU and palbociclib: Uptake and efflux in disaggregated cells and 3D models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2017-4205