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1.  Use of Different Parameters and Equations for Calculation of IC50 Values in Efflux Assays: Potential Sources of Variability in IC50 Determination 
The AAPS Journal  2013;16(1):172-180.
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.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-013-9554-7) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-013-9554-7
PMCID: PMC3889528  PMID: 24338112
Caco-2; drug interaction; IC50; inhibition; P-glycoprotein
2.  Application of Method Suitability for Drug Permeability Classification 
The AAPS Journal  2010;12(4):670-678.
Experimental models of permeability in animals, excised tissues, cell monolayers, and artificial membranes are important during drug discovery and development as permeability is one of several factors affecting the intestinal absorption of oral drug products. The utility of these models is demonstrated by their ability to predict a drug’s in vivo intestinal absorption. Within the various permeability models, there are differences in the performance of the assays, along with variability in animal species, tissue sources, and cell types, resulting in a variety of experimental permeability values for the same drug among laboratories. This has led to a need for assay standardization within laboratories to ensure applicability in the drug development process. Method suitability provides a generalized approach to standardize and validate a permeability model within a laboratory. First, assay methodology is optimized and validated for its various experimental parameters along with acceptance criteria for the assay. Second, the suitability of the model is demonstrated by a rank order relationship between experimental permeability values and human extent of absorption of known model compounds. Lastly, standard compounds are employed to classify a test drug’s intestinal permeability and ensure assay reproducibility and quality. This review will provide examples of the different aspects method suitability for in situ (intestinal perfusions), ex vivo (everted intestinal sacs, diffusion chambers), and in vitro (cell monolayers, artificial membranes) experimental permeability models. Through assay standardization, reference standards, and acceptance criteria, method suitability assures the dependability of experimental data to predict a drug’s intestinal permeability during discovery, development, and regulatory application.
doi:10.1208/s12248-010-9227-8
PMCID: PMC2976984  PMID: 20811966
artificial membranes; drug permeability; ex vivo perfusion; in situ perfusion; in vitro cell monolayers; method suitability
3.  Effect of Ethanol on Opioid Drug Permeability Through Caco-2 Cell Monolayers 
The AAPS Journal  2008;10(2):360-362.
doi:10.1208/s12248-008-9046-3
PMCID: PMC2751396  PMID: 18592381
Caco-2; ethanol; hydromorphone; oxycodone; oxymorphone; permeability
4.  Permeability classification of representative fluoroquinolones by a cell culture method 
AAPS PharmSci  2004;6(2):1-6.
This study was undertaken to categorize representative fluoroquinolone drug substance permeability based on the methods outlined in the Food and Drug Administration's biopharmaceutic classification system (BCS) Guidance for Industry. The permeability of ciprofloxacin, levofloxacin, lomefloxacin, and ofloxacin was measured in an in vitro Caco-2 assay with previously demonstrated method suitability. The permeability class and efflux potential were ascertained by comparing test drug results with standard compounds (metoprolol, atenolol, labetalol, and rhodamine-123). All 4 quinolones drugs demonstrated concentration-dependent permeability, indicating active drug transport. In comparing absorptive versus secretive in vitro transport, the tested fluoroquinolones were found to be subject to efflux in varying degrees (ciprofloxacin > lomefloxacin > rhodamine 123 > levofloxacin > ofloxacin). Based on comparison to labetalol, the high permeability internal standard, ciprofloxacin was classified as a low permeability drug, whereas lomefloxacin, levofloxacin, and ofloxacin were classified as high permeability drugs. The in vitro permeability results matched human in vivo data based on absolute bioavailabilities. This laboratory exercise demonstrated the applicability of an in vitro permeability method for classifying drugs as outlined in the BCS Guidance.
doi:10.1208/ps060213
PMCID: PMC2751005  PMID: 18465265
permeability; Caco-2; biopharmaceutics classification system; fluoroquinolones

Results 1-4 (4)