Polysorbate 20 increases oral absorption of digoxin in wild-type Sprague Dawley rats, but not in mdr1a(-/-) Sprague Dawley rats

Abstract

The aim was to investigate the ability of polysorbate 20 to alter oral digoxin absorption in vitro and drug exposure in vivo via modulation of transporter mediated efflux. Transport studies were performed in MDCKII-MDR1 and Caco-2 cells using ³H-digoxin. Pharmacokinetic studies were performed in wild type and mdr1a deficient Sprague Dawley rats. ³H-digoxin was quantified using liquid scintillation counting. The results showed an increased absorptive transport and a reduced secretory transport in MDCKII-MDR and Caco-2 cells as a function of polysorbate 20 concentrations. The secretory transport (B–A) of digoxin was reduced by 50% at lower polysorbate 20 concentrations than required to increase the absorptive transport (A-B). In vivo, the oral bioavailability of digoxin in wild type animal was increased by 10–25% (w/v) polysorbate 20. In mdr1a deficient Sprague Dawley rats 25% (w/v) polysorbate 20 did not alter the absorption of digoxin after oral administration, but digoxin exposure was significantly different between wild type and mdr1a deficient rats. In conclusion, polysorbate 20 increased absorptive transport across Caco-2 cell monolayers and in vivo in rats in a concentration dependent manner, most likely via inhibition of P-gp rather than through solubilization of digoxin.

Introduction

ATP-binding cassette (ABC) transporters affect drug absorption, distribution, metabolism, excretion and toxicity (ADMET) properties (Akazawa et al., 2016, Szakacs et al., 2008). The interaction of drugs with ABC-transporters, such as P-glycoprotein (P-gp, MDR1, ABCB1) or breast-cancer resistance protein (BCRP), limits the transport across the intestinal barrier and the blood brain barrier (Glavinas et al., 2004). Furthermore, in cases with polypharmacy were more than one of the administered drugs are substrates for ABC transporters cellular drug concentrations in the kidney and liver are affected (Funk, 2008), which may lead to clinically relevant drug–drug interactions. In the case of intestinal transport, the interaction between a drug substance and P-gp limits transepithelial absorptive permeation, thus limiting the oral bioavailability. This is especially relevant for drug substances with low solubility and high affinity for P-gp (Montanari and Ecker, 2015). In the intestine the expression of P-gp mRNA is increased from the duodenum to the colon in mouse (Mutch et al., 2004), rat (Takara et al., 2003) and human (Zimmermann et al., 2005). At the protein level, P-gp is expressed in the luminal membrane of e.g. Caco-2 cells (Hunter et al., 1993), and the absolute P-gp protein level has also been shown to increase from duodenum to ileum in mouse (Akazawa et al., 2016), and human (Bruyere et al., 2010, Mouly and Paine, 2003). P-gp substrate are likely recognized by the transporter in the inner leaflet of the membrane and either moved to the outer leaflet or the extracellular space using ATP to complete the transport (Sharom, 2014).

Strategies to improve solubility of drug substances and inhibit ABC-transporter mediated cellular efflux in order to increase oral bioavailability are in high demand in drug formulation programs. Several non-ionic surfactants such as polysorbates (tween) and poloxamers have been investigated for their ability to increase in vitro absorption by inhibiting efflux transporters (Bogman et al., 2003, Cornaire et al., 2000, Nerurkar et al., 1996, Nerurkar et al., 1997, Rege et al., 2001, Shono et al., 2004, Takahashi et al., 2002, Wang et al., 2004). This approach originates from findings in cancer cell lines (Woodcock et al., 1992), where Woodcock and co-workers showed that detergents with polyethylene oxide hydrophilic moieties, like cremophor EL, tween 20 and 80, were able to reverse the MDR phenotype in cancer cells in a reversible manner (Woodcock et al., 1992). Despite the many in vitro studies, only few studies have explored the ability of surfactants to increase bioavailability in in vivo models. Furthermore, systematic and mechanistic studies investigating the amount of surfactant needed in vivo to increase absorption are to the best of our knowledge lacking. To distinguish between solubility and transport-mediated effects knock-out animals may provide a powerful tool. To this end, it has been shown that polysorbate 20 and pluronic P85 increase the absorption of topotecan, a BCRP substrate, in wild type mice, without affecting absorption in bcrp (-/-) mice (Yamagata et al., 2007).

We recently showed that even though polysorbate 20 and 80 increased the absorptive doxorubicin transport across Caco-2 cells, this did not translate into a measurable increase in plasma concentrations in vivo after oral administration of doxorubicin and 1–25% (w/v) polysorbate 20 to rats, likely due to hepatic clearance of doxorubicin (Al-Saraf et al., 2016). In contrast, polysorbate 80 has been shown to increase digoxin exposure evaluated as area under the plasma concentration time profile (AUC) and the maximal plasma concentration (C_max) at concentrations of 1 and 10% in rats (Zhang et al., 2003). For further development of drug formulations containing surfactants specifically included to alter efflux transporter function more knowledge about the effect in vivo and knowledge of translation of in vitro to in vivo concentrations are needed. In the present study we have therefore investigated the in vitro and in vivo concentration dependent ability of polysorbate 20 to increase absorption of a low-soluble P-pg marker, i.e.digoxin. Digoxin was chosen as a p-gp marker since it is a low-permeable drug substance, which is a relatively specific substrate for p-gp as compared to BCRP and MRP2. The aim of the present study was thus to investigate the ability of polysorbate 20 to increase digoxin absorption in vitro by inhibiting P-gp mediated cellular efflux, and to investigate the dose of polysorbate 20 required to alter in vivo absorption of digoxin in wild type Sprague Dawley rats, and to use mdr1a (-/-) Sprague Dawley rats to distinguish between transporter and solubility related event during absorption.