Variability in P-Glycoprotein Inhibitory Potency (IC<sub>50</sub>) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug-Drug Interaction Risk Assessment Decision Criteria

Bentz, Joe; O'Connor, Mike; Bednarczyk, Dallas; Coleman, Joann; Lee, Caroline A.; Palm, Johan; Pak, Anne; Perloff, Elke S.; Reyner, Eric L.; Balimane, Praveen; Brännström, Marie; Chu, Xiaoyan; Funk, Christoph; Guo, Ailan; Hanna, Imad; Herédi-Szabó, Krisztina; Hillgren, Kathleen M.; Li, Libin; Hollnack-Pusch, Evelyn; Jamei, Masoud; Lin, Xuena; Mason, Andrew K.; Neuhoff, Sibylle; Patel, Aarti; Podila, Lalitha; Plise, Emile G.; Rajaraman, Ganesh; Salphati, Laurent; Sands, Eric; Taub, Mitchell E.; Taur, Jesse; Weitz, Dietmar; Wortelboer, Heleen M.; Xia, Cindy Q.; Gao, Xiao; Yabut, Jocelyn; Yamagata, Tetsuo; Zhang, Lei K.; Ellens, Harma

doi:10.1124/dmd.112.050500

Cited by 138 publications

(194 citation statements)

References 31 publications

(34 reference statements)

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“…Therefore, IC 50 values may also vary with assay systems and P-gp expression. Furthermore, it was reported that different IC 50 values were given from different laboratories for the same P-gp inhibitor (Bentz et al, 2013). Hence, the cell systems for the determination of in vitro IC 50 values should be taken into consideration also when the DDI likelihood is assessed.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Predictability of Drug-Drug Interaction Likelihood of P-Glycoprotein-Mediated Efflux of Dabigatran Etexilate Based on [I]₂/IC₅₀ Threshold

et al. 2013

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Dabigatran etexilate, an oral, reversible, competitive, and direct thrombin inhibitor, is an in vitro and in vivo substrate of Pglycoprotein (P-gp). Dabigatran etexilate was proposed as an in vivo probe substrate for intestinal P-gp inhibition in a recent guidance on drug-drug interactions (DDI) from the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). We conducted transcellular transport studies across Caco-2 cell monolayers with dabigatran etexilate in the presence of various P-gp inhibitors to examine how well in vitro IC 50 data, in combination with mathematical equations provided by regulatory guidances, predict DDI likelihood. From a set of potential P-gp inhibitors, clarithromycin, cyclosporin A, itraconazole, ketoconazole, quinidine, and ritonavir inhibited P-gp-mediated transport of dabigatran etexilate over a concentration range that may hypothetically occur in the intestine. IC 50 values of P-gp inhibitors for dabigatran etexilate transport were comparable to those of digoxin, a well established in vitro and in vivo P-gp substrate. However, IC 50 values varied depending whether they were calculated from efflux ratios or permeability coefficients. Prediction of DDI likelihood of P-gp inhibitors using IC 50 values, the hypothetical concentration of P-gp inhibitors, and the cut-off value recommended by both the FDA and EMA were in line with the DDI occurrence in clinical studies with dabigatran etexilate. However, it has to be kept in mind that validity of the cut-off criteria proposed by the FDA and EMA depends on in vitro experimental systems and the IC 50 -calculation methods that are employed, as IC 50 values are substantially influenced by these factors.

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Section: Discussionmentioning

confidence: 99%

In Vitro Predictability of Drug-Drug Interaction Likelihood of P-Glycoprotein-Mediated Efflux of Dabigatran Etexilate Based on [I]₂/IC₅₀ Threshold

et al. 2013

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“…In vitro systems including isolated primary hepatocytes and heterogeneous gene overexpressed cell lines have been widely used to investigate transporter-involved drug disposition; however, translation of in vitro kinetics parameters obtained from different systems or different laboratories to in vivo is not always straightforward. For example, through a collaborative effort between 23 pharmaceutical, academic, and contract research laboratories, a minimum of 20-and 24-fold deviation was found to exist between the lowest and highest Pglycoprotein (P-gp/ABCB1) IC 50 values for sertraline and isradipine, respectively, to a maximum of 407-and 796-fold deviation for telmisartan and verapamil (2). The presence of digoxin uptake transporters and their differences in expression in in vitro systems used in different laboratories is considered as the major contributor to the variability of IC 50 values (2,3).…”

Section: Introductionmentioning

confidence: 99%

“…For example, through a collaborative effort between 23 pharmaceutical, academic, and contract research laboratories, a minimum of 20-and 24-fold deviation was found to exist between the lowest and highest Pglycoprotein (P-gp/ABCB1) IC 50 values for sertraline and isradipine, respectively, to a maximum of 407-and 796-fold deviation for telmisartan and verapamil (2). The presence of digoxin uptake transporters and their differences in expression in in vitro systems used in different laboratories is considered as the major contributor to the variability of IC 50 values (2,3). Moreover, IVIVE of biliary elimination that involves drug transporter-mediated processes is not well established from preclinical species to human.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

Qiu

Zhang

Lai

2014

AAPS J

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Abstract. Although global proteomics has shown promise for discovery of many new proteins, biomarkers, protein modifications, and polymorphisms, targeted proteomics is emerging in the proteomics research field as a complement to untargeted shotgun proteomics, particularly when a determined set of low-abundance functional proteins need to be measured. The function and expression of proteins related to drug absorption, distribution, metabolism, and excretion (ADME) such as cytochrome P450 enzymes and membrane transporters are of great interest in biopharmaceutical research. Since the variation in ADME-related protein expression is known to be a major complicating factor encountered during in vitro-in vivo and in vivo-in vivo extrapolations (IVIVE), the accurate quantification of the ADME proteins in complex biological systems becomes a fundamental element in establishing IVIVE for pharmacokinetic predictions. In this review, we provide an overview of relevant methodologies followed by a summary of recent applications encompassing mass spectrometry-based targeted quantifications of membrane transporters.KEY WORDS: drug absorption, distribution, metabolism, and excretion (ADME); drug transporters; in vitro-in vivo and in vivo-in vivo extrapolations (IVIVE); LC-MS/MS; quantitative targeted proteomics.

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“…Caco-2 cells have gradually spread to the transporter community (Bentz et al, 2013). Several reasons for the relatively large interlaboratory differences have been proposed and are under discussion (e.g., different source of Caco-2 cells, equations for estimating inhibition potency, pH gradient in apical-to-basal (AtoB) assay).…”

Section: Introductionmentioning

confidence: 99%

Impact of Endogenous Esterase Activity on In Vitro P-Glycoprotein Profiling of Dabigatran Etexilate in Caco-2 Monolayers

et al. 2013

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Dabigatran etexilate, a double prodrug of dabigatran, is a reversible, competitive, direct thrombin inhibitor that has been approved for use in many countries. A recent guideline from the European Medicines Agency on drug-drug interactions proposed dabigatran etexilate as a sensitive in vivo and in vitro probe substrate for intestinal P-glycoprotein (P-gp) inhibition. We therefore performed a series of in vitro studies to determine the best experimental conditions for evaluation of P-gp involvement on the transport process of dabigatran etexilate across colorectal adenocarcinoma Caco-2 cell monolayers. Experiments using expressed carboxylesterase 1 (CES1) and CES2 bactosomes revealed that dabigatran etexilate was hydrolyzed into BIBR 1087 by CES1 expressed in our Caco-2 cells. The impact of CES1-mediated BIBR 1087 formation during transcellular transport experiments was assessed by comparing several combinations of three experimental approaches: radioactivity detection using [ 14 C]dabigatran etexilate as substrate, liquid chromatographytandem mass spectrometry (LC-MS/MS) quantification of dabigatran etexilate, and in the presence and absence of a CES inhibitor bis(p-nitrophenyl) phosphate (BNPP). The experimental approach that was based on the use of nonlabeled dabigatran etexilate together with LC-MS/MS quantification and the addition of BNPP was selected as the most favorable condition in which to correctly evaluate the permeability coefficient (Papp) of dabigatran etexilate and its transcellular transport by P-gp. The in vitro Caco-2 study at the selected condition revealed that dabigatran etexilate is a P-gp substrate with an efflux ratio of 13.8 and an intrinsic Papp, which is the Papp under the condition of complete blockage of P-gp by P-gp inhibitor, of 29 3 10 26 cm/s.

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Variability in P-Glycoprotein Inhibitory Potency (IC₅₀) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug-Drug Interaction Risk Assessment Decision Criteria

Cited by 138 publications

References 31 publications

In Vitro Predictability of Drug-Drug Interaction Likelihood of P-Glycoprotein-Mediated Efflux of Dabigatran Etexilate Based on [I]₂/IC₅₀ Threshold

In Vitro Predictability of Drug-Drug Interaction Likelihood of P-Glycoprotein-Mediated Efflux of Dabigatran Etexilate Based on [I]₂/IC₅₀ Threshold

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

Impact of Endogenous Esterase Activity on In Vitro P-Glycoprotein Profiling of Dabigatran Etexilate in Caco-2 Monolayers

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Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug-Drug Interaction Risk Assessment Decision Criteria

Cited by 138 publications

References 31 publications

In Vitro Predictability of Drug-Drug Interaction Likelihood of P-Glycoprotein-Mediated Efflux of Dabigatran Etexilate Based on [I]2/IC50 Threshold

In Vitro Predictability of Drug-Drug Interaction Likelihood of P-Glycoprotein-Mediated Efflux of Dabigatran Etexilate Based on [I]2/IC50 Threshold

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

Impact of Endogenous Esterase Activity on In Vitro P-Glycoprotein Profiling of Dabigatran Etexilate in Caco-2 Monolayers

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Variability in P-Glycoprotein Inhibitory Potency (IC₅₀) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug-Drug Interaction Risk Assessment Decision Criteria

In Vitro Predictability of Drug-Drug Interaction Likelihood of P-Glycoprotein-Mediated Efflux of Dabigatran Etexilate Based on [I]₂/IC₅₀ Threshold

In Vitro Predictability of Drug-Drug Interaction Likelihood of P-Glycoprotein-Mediated Efflux of Dabigatran Etexilate Based on [I]₂/IC₅₀ Threshold