title: Use of different parameters and equations for calculation of IC₅₀ values in efflux assays: potential sources of variability in IC₅₀ determination.
abstract: Drug interactions due to efflux transporters may result in one drug increasing or decreasing the systemic exposure of a second drug. The potential for in vivo drug interactions is estimated through in vitro cell assays. Variability in in vitro parameter determination (e.g., IC₅₀ values) among laboratories may lead to different conclusions in in vivo interaction predictions. The objective of this study was to investigate variability in in vitro inhibition potency determination that may be due to calculation methods. In a Caco-2 cell assay, the absorptive and secretive permeability of digoxin was measured in the presence of spironolactone, itraconazole and vardenafil. From the permeability data, the efflux ratio and net secretory flux where calculated for each inhibitor. IC₅₀ values were then calculated using a variety of equations and software programs. All three drugs decreased the secretory transport of digoxin in a concentration-dependent manner while increasing digoxin's absorption to a lesser extent. The resulting IC₅₀ values varied according to the parameter evaluated, whether percent inhibition or percent control was applied, and the computational IC₅₀ equation. This study has shown that multiple methods used to quantitate the inhibition of drug efflux in a cell assay can result in different IC₅₀ values. The variability in the results in this study points to a need to standardize any transporter assay and calculation methods within a laboratory and to validate the assay with a set of known inhibitors and non-inhibitors against a clinically relevant substrate.
authors: Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, Maryland, 20993-0002, USA, donna.volpe@fda.hhs.gov.
authors: Donna A
authors: DA
authors: Volpe
authors: Salaheldin S
authors: SS
authors: Hamed
authors: Lei K
authors: LK
authors: Zhang
mesh_terms: D000465:Algorithms; D018938:Caco-2 Cells; D002316:Cardiotonic Agents / Q000494:pharmacology*; D004077:Digoxin / Q000494:pharmacology*; D004347:Drug Interactions; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D017964:Itraconazole / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D013148:Spironolactone / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology; D000069058:Vardenafil Dihydrochloride
chemical_list: D002316:Cardiotonic Agents; D007093:Imidazoles; D010879:Piperazines; D013450:Sulfones; D014227:Triazines; D013148:Spironolactone; D017964:Itraconazole; D000069058:Vardenafil Dihydrochloride; D004077:Digoxin