Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers

Abstract

The objectives of this study were (1) to investigate the transporter inhibition activity of three nonionic surfactants on P-glycoprotein, the human intestinal peptide transporter, and the monocarboxylic acid transporter in Caco-2 cell monolayers, and (2) to evaluate the role of membrane fluidity and protein kinase C in surfactant-induced transporter inhibition. All three surfactants inhibited P-glycoprotein (P-gp). Over a range from 0 to 1 mM, Tween 80 and Cremophor EL increased apical-to-basolateral permeability (AP-BL) and decreased basolateral-to-apical (BL-AP) permeability of the P-gp substrate rhodamine 123. Vitamin E TPGS’s effect was equally large, but essentially only reduced the BL-AP permeability of rhodamine 123, and did so at a vitamin E TPGS concentration of only 0.025 mM. These P-gp inhibition effects would appear to be related to these excipients’ modulation of membrane fluidity, where Tween 80 and Cremophor EL fluidized cell lipid bilayers, while vitamin E TPGS rigidized lipid bilayers. However, among the three surfactants, only Tween 80 inhibited the peptide transporter, as measured by glycyl sarcosine permeability. Likewise, only Cremophor EL inhibited the monocarboxylic acid transporter, as measured by benzoic acid permeability. Nevertheless, at least one of these three surfactants inhibited each P-gp, the human intestinal peptide transporter, and the monocarboxylic acid transporter. A common functional feature of these three surfactants was their ability to modulate fluidity, although results indicate that even strong membrane fluidity modulation alone was not sufficient to reduce transporter activity. N-octyl glucoside, a nonionic surfactant that did not modulate membrane fluidity, did not affect transporter functioning. Protein kinase C inhibitors failed to affect rhodamine 123 and glycyl sarcosine permeability, suggesting protein kinase C inhibition was not the mechanism of transporter inhibition. These results suggest that surfactants can inhibit multiple transporters but that changes in membrane fluidity may not be a generalized mechanism to reduce transporter activity.

Introduction

Surfactants are extensively used in pharmaceutical formulations as wetting agents to improve dissolution and absorption of poorly soluble drugs. Low molecular weight ionic surfactants like sodium lauryl sulfate, in concentrations that are not toxic to the intestinal mucosa, are probably the most commonly used surfactants for this purpose. Nonionic surfactants have been shown to be even less toxic than ionic surfactants to biological membranes (Davis et al., 1970). Additionally, being more hydrophobic than ionic surfactants, nonionic surfactants also possess greater capacity to dissolve poorly soluble drugs, are very efficient emulsifiers, and can be used in self-emulsifying drug delivery systems.

Several nonionic surfactants have been shown to inhibit transporters (Rege et al., 2001, Koga et al., 2000, Miller et al., 1999, Nerurkar et al., 1996, Woodcock et al., 1992). Most reports of surfactant-induced inhibition of membrane transporters have focused on P-glycoprotein (P-gp). P-gp is expressed extensively in the GIT and potentially contributes to reduced oral absorption of drugs. Many nonionic surfactants effectively inhibit P-gp, although the mechanism of inhibition remains unclear. These nonionic surfactants have potential to increase oral absorption of P-gp substrates. Examples of nonionic surfactant with activity on various efflux pumps include Tweens, Spans, Cremphors (EL and RH40), Pluronic block copolymers, and vitamin E TPGS.

Numerous drugs are substrates for active influx transport systems, which facilitate absorption. Examples include the human apical sodium-dependent bile acid transporter (hASBT), the human intestinal peptide transporter (hPepT-1), and the monocarboxylic acid transporter (MCT). Inhibition of the peptide transporter by nonionic surfactants has been observed (Koga et al., 1998, Koga et al., 1999a, Koga et al., 1999b, Koga et al., 2000). This effect of influx transporters would make surfactants potentially undesirable formulation additives, as these surfactants may reduce drug absorption.

It has been suggested that surfactants can alter membrane fluidity, thereby changing the conformation of membrane bound transporters (Dudeja et al., 1995, Woodcock et al., 1992). This conformation change is associated with inhibition of P-gp’s ATPase activity. It has also been suggested that such surfactants inhibit protein kinase C (PKC), which is involved in the functioning of many transporters like P-gp (Zhao et al., 1989). PKC phosphorylates several membrane transporters, such that transporter function can be modulated by PKC inhibition or activation. Some of these surfactants have also been shown to inhibit metabolizing enzymes like cytochrome P450 (Mountfield et al., 2000). It has also been reported that only surfactant monomers active in transporter inhibition, such that inhibitory activity essentially plateaus above the critical micelle concentration (cmc) (Nerurkar et al., 1997).

The first objective of this study was to evaluate the influence of three nonionic surfactants on three membrane transporters, namely P-gp, PepT-1, and MCT. The three evaluated surfactants were Tween 80, Cremophor EL, and vitamin E TPGS (α-tocopheryl polyethylene glycol 800 succinate), each of which contains poly(ethylene oxide) groups. N-octyl glucoside, a nonionic surfactant without poly(ethylene oxide) groups, was also evaluated. Additionally, the concentration dependence of surfactant-induced transporter inhibition was determined. Specifically, we evaluated whether micelles were active species, like the monomers, and aimed to compare the transporter inhibition potency of the three surfactants. The second objective was to evaluate the role of membrane fluidity and PKC in the surfactant-induced inhibition of these membrane transporters.