The effects of commonly used polyethoxylated excipients on P-glycoprotein activity in vitro

Earlier studies in our laboratory revealed that Cremophor EL and Tween 80 caused inhibition of P-glycoprotein (or P-gp) activity in Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. P-gp, a 170 kD apically polarized efflux protein encoded by the human MDR1 gene, is responsible for the elimination of structurally diverse drugs in tumors and normal tissues of the body. Therefore, the objective of the present study was to examine the effects of commonly used polyethoxylated excipients [e.g., polyethylene glycol (PEG)-300, Cremophor EL, and Tween 80] in various cell culture models representative of the intestinal mucosa in attempt to probe this inhibition mechanism in greater detail.; Transport studies of [3H]-taxol, a model P-gp substrate, across Caco-2 and MDR1-MDCK cell monolayers in the presence and absence of various concentrations of PEG-300, Cremophor EL, and Tween 80 revealed that only PEG-300 caused complete inhibition of P-gp activity in vitro. Cremophor EL and Tween 80 caused partial inhibition of P-gp activity in Caco-2 cell monolayers, whereas incomplete inhibition was observed by these excipients in MDR1-MDCK cell monolayers. Since only PEG-300 caused complete inhibition of P-gp activity, it was selected as a model excipient for use in further studies.; An automated cellular transport assay was developed to screen for excipient effects in vitro. In this study, a TECAN robot was used to automate transport studies of AcPhe(NMePhe)₂NH₂, a P-gp substrate, across Caco-2, MDR1-MDCK, and wild-type MDCK cell monolayers in the presence and absence of various concentrations of PEG-300. The data generated from these studies confirmed PEG-induced inhibition of P-gp activity. However, disruption in MDR1- and wild-type-MDCK cellular tight junction integrity was observed at 10% (v/v) PEG-300 and above. Therefore, only kinetic modeling studies were feasible with the Caco-2 cell data in the presence of PEG.; In conclusion, the exact mechanism for PEG-induced inhibition of P-gP activity remains inconclusive, based on kinetic modeling studies alone. However, it is quite evident from membrane fluidity studies with these excipients that excipient-induced changes in the fluidity of the cell membrane are critically linked to the activity of membrane-spanning efflux transport proteins such as P-gp.