Development, validation and utility of an in vitro technique for assessment of potential clinical drug-drug interactions involving P-glycoprotein.
Regulatory interest is increasing for drug transporters generally and P-glycoprotein (Pgp) in particular, primarily in the area of drug-drug interactions. To aid in both identifying and discharging the potential liabilities associated with drug-transporter interactions, the pharmaceutical industry has a growing requirement for routine and robust non-clinical assays. An assay was designed, optimised and validated to determine the in vitro inhibitory potency of new chemical entities (NCEs) towards human Pgp-mediated transport. [3H]-Digoxin was established as a suitable probe substrate by investigating its characteristics in the in vitro system (MDCKII-MDR1 cells grown in 24-multiwell inserts). The inhibitory potencies (apparent IC50) of known Pgp inhibitors astemizole, GF120918, ketoconazole, itraconazole, quinidine, verapamil and quinine were determined over at least a 1000-fold concentration range. Validation was carried out using manual and automatic techniques. [3H]-Digoxin was found to be stable and have good mass balance in the system. In contrast to [A-->B] transport, [3H]-digoxin [B-->A] transport rates were readily measured with good reproducibility. There was no evidence of saturation of transport up to 10 microM digoxin and 30 nM digoxin was selected for routine assay use, reflecting clinical therapeutic concentrations. IC50 values ranged over approximately 100-fold with excellent reproducibility. Results from manual and automated versions were in close agreement. This method is suitable for routine use to assess the in vitro inhibitory potency of NCEs on Pgp-mediated digoxin transport. Comparison of IC50 values against clinical interaction profiles for the probe inhibitors indicated the in vitro assay is predictive of clinical digoxin-drug interactions mediated via Pgp.
Keogh
John P
JP
Preclinical Drug Metabolism and Pharmacokinectics, GlaxoSmithKline, The Frythe, UK.
Kunta
Jeevan R
JR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016589:Astemizole / Q000494:pharmacology; D001331:Automation; D002460:Cell Line; D004077:Digoxin / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004347:Drug Interactions; D007654:Ketoconazole / Q000494:pharmacology; D011802:Quinidine / Q000494:pharmacology; D015203:Reproducibility of Results; D014162:Transfection; D014316:Tritium
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014316:Tritium; D004077:Digoxin; D016589:Astemizole; D011802:Quinidine; D007654:Ketoconazole