Determination of P-glycoprotein inhibition by excipients and their combinations using an integrated high-throughput process

Excipients often used in pharmaceutical formulations have been reported to have inhibitory effects on P-glycoprotein, an important membrane-associated transport protein. Because inhibition of efflux transporters can have an effect on drug bioavailability, identification of these excipients and their extent of inhibition are therefore important for pharmaceutical development. We have developed an automated and integrated high-throughput process for identifying these excipients and their combinations. Common excipients containing polyethylene glycol (PEG) in the chemical structure were screened using a cytotoxic cell growth assay, and excipients giving inhibition were further combined to identify synergistic effects. Our screens identified excipients previously reported to inhibit P-glycoprotein, such as PEG stearates, PEG fatty acid esters, polysorbates, and poloxamers. We also found new excipients, such as those in the PEG glyceryl fatty acid family, which were among the best inhibitors identified. Dose-response studies of these compounds and of cyclosporin A indicated that the extent of inhibition depended logarithmically on the concentration. This suggests a similar mechanism by which inhibition is obtained, despite widely varying chemical structures. In the particular set of combinatorial studies performed, which involved >20,000 samples, we found that inhibitory effects in binary combinations followed the single-excipient logarithmic trend, rather than being synergistic. These experiments showcased the potential for integrated high-throughput processes that enable combinatorial screens which would otherwise be difficult to perform manually. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.