To detect transporter specific effects of excipients an in vitro experimental setup was designed based on the Caco-2 monolayer system, focusing on two apical efflux transporters ABCB1 (P-glycoprotein, MDR1) and ABCG2 (BCRP, MXR) which are held to play an important role on drug disposition. Model Excipients (TWEEN 80, Cremophor EL, Vitamin E TPGS) with reported data on transporter function were selected based upon literature data.2,3,4 Initial concentrations were set to their reported Critical Micelle Forming concentrations in aqueous solutions.4
Specific probe substrates for the two major efflux transporters (ABCB1 and ABCG2) were selected. Digoxin is a well known ABCB1 reference substrate in in vitro transport experiments. Estrone-3-sulfate is efficiently transported by ABCG2 but is not a substrate of ABCB1, therefore a suitable ABCG2 selective probe in systems like Caco-2 where both transporters are present. Specific small molecule inhibitors (PSC833 of ABCB1 and Ko143 for ABCG2) were also used to demonstrate the transporter specific inhibitory effect. Passive permeability controls with no known transporter interactions (Antipyrine and Lucifer Yellow) were applied to serve as an interim quality control and to ensure that the inactive component in detected transport values is within normal range. Na-Caprate a known permeation enhancer was applied as a positive control for paracellular transport. All Excipients were able to increase the A-B transport and decrease the B-A transport of Digoxin, markedly reducing the efflux ratio. All Excipients were able to significantly reduce B-A permeability of Estrone-3-sulfate, but have a more modest effect on A-B permeability. Results generated however show that the excipients do not show real selectivity between ABCB1 and ABCG2 as all compounds were able to cross inhibit the other transporter as well. Transporter specificity have to be covered with using transporter specific probe substrates and small molecule inhibitors, or transfected cell lines expressing one specific transporter in future testing of excipients. The observed effect of model excipients on the permeability of passive permeability controls corresponded with previous data and expectations. This experimental setup can serve as a reliable in vitro platform for testing effect of excipients on transporters and passive permeation.
- Nururkar MM, Burton PS, Borchardt RT (1996) The use of surfactants to enhance the permeability of peptides through Caco-2 cells by inhibition of an apically polarized efflux system. Pharm.Res.13:528–534.
- Rege BD, Kao JPY, Polli JE (2002) Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers. Eur. J. Pharm. Sci. 16 (4–5):237–246
- Hugger ED, Novak BL, Burton PS, Audus KL, Borchardt RT (2002) A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro. J. Pharm. Sci. 91 (9):1991–2002
- Yamagata T, Kusuhara H, Morishita M, Takayama K, Benameur H (2007) Effect of excipients on breast cancer resistance protein substrate uptake activity. J. Control. Release 124:1–5