A P-gp vesicular transport inhibition assay – Optimization and validation for drug–drug interaction testing

Herédi-Szabó, Krisztina; Palm, Johan; Andersson, Tommy; Pál, Ákos; Méhn, Dóra; Fekete, Zsolt; Beéry, Erzsébet; Jakab, Katalin; Jani, Márton; Krajcsi, Péter

doi:10.1016/j.ejps.2013.04.032

Cited by 29 publications

(13 citation statements)

References 77 publications

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“…For instance, the reported range of IC 50 values for ketoconazole varies by 37-fold and 6.4-fold for the inhibition of P-gp and OATP1B1, respectively (Gnoth et al, 2011;Izumi et al, 2013;Matsson et al, 2013;Mikkaichi et al, 2014). Similarly, IC 50 values for ritonavir vary by 85-fold and 117-fold for the inhibition of P-gp and OATP1B1, respectively (Keogh and Kunta, 2006;Herédi-Szabó et al, 2013;Izumi et al, 2013). Therefore, this work was undertaken to systematically assess the inhibitory effects of ketoconazole, clarithromycin, ritonavir, and itraconazole (and its CYP3A-inhibitory metabolites, hydroxyitraconazole, keto-itraconazole, and N-desalkyl itraconazole) on OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3), OAT1 (SLC22A6), OAT3 (SLC22A8), OCT1 (SLC22A1), OCT2 (SLC22A2), MATE1 (SLC47A1), MATE2-K (SLC47A2), P-gp (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), MRP3 (ABCC3), and BSEP (ABCB11).…”

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confidence: 99%

Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters

Vermeer

Isringhausen

Ogilvie

et al. 2015

Drug Metabolism and Disposition

103

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Ketoconazole is a potent CYP3A4/5 inhibitor and, until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency as a strong CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and European Medicines Agency recommended suspension of ketoconazole use in DDI studies in 2013. The FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of drug transporter activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir, and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto-, and N-desalkyl itraconazole) toward 13 drug transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, P-gp, BCRP, MRP2, MRP3, and BSEP) were systematically assessed in transporterexpressing HEK-293 cell lines or membrane vesicles. In vitro findings were translated into clinical context with the basic static model approaches outlined by the FDA in its 2012 draft guidance on DDIs. The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin, and itraconazole each have unique transporter inhibition profiles. None of the alternatives to ketoconazole provided a clean inhibition profile toward the 13 drug transporters evaluated. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when transporters are potentially involved in a victim drug's pharmacokinetics.

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mentioning

confidence: 99%

Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters

Vermeer

Isringhausen

Ogilvie

et al. 2015

Drug Metabolism and Disposition

103

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“…Breast cancer resistance protein, MDR1 and CTRL + C vesicles were supplemented with cholesterol to enhance transport activity of these transporters or, in the case of CTRL + C, to serve as control for the cholesterol supplemented BCRP and MDR1 vesicles. The method for cholesterol supplementation and the rationale behind it have been previously reported …”

Section: Methodsmentioning

confidence: 99%

“…The method for cholesterol supplementation and the rationale behind it have been previously reported. 19,23,24 Membrane vesicles from human embryonic kidney cells 293 (HEK) expressing MRP1, MRP5 or enhanced yellow fluorescent protein (eYFP, a cytosolic protein, used as a negative control for HEK vesicles) were obtained from PharmTox.…”

Section: Expression Of the Transporters And Preparation Of Inside-omentioning

confidence: 99%

Efflux transport of nicotine, cotinine and trans‐3′‐hydroxycotinine glucuronides by human hepatic transporters

Järvinen

Sjöstedt

Koenderink

et al. 2019

Basic Clin Pharma Tox

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Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans‐3′‐hydroxycotinine (OH‐cotinine) glucuronides. To this end, we tested their transport by multidrug resistance‐associated proteins 1 (MRP1, ABCC1) and MRP3‐6 (ABCC3‐6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P‐gp, ABCB1) that are expressed in the apical membranes of these cells. ATP‐dependent transport of these glucuronides was evaluated in inside‐out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP‐dependent transport was observed only for OH‐cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated Km value of 895 µmol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 µmol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH‐cotinine inhibited MRP2‐4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.

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“…Test chemical interaction with P-gp was measured by dividing NMQ concentrations for all test chemicals by those measured for DMSO to determine % P-gp transport activity. Verapamil and erythromycin, known inhibitors of P-gp, were used as controls (Eberl et al, 2007;Heredi-Szabo et al, 2013). Analyses of P-gp mediated transport of propiconazole (at 1 mM, 10 mM, and 100 mM final concentrations) in membrane vesicles was performed using the same protocol (as with NMQ) with the exception that reaction time varied (3-30 min).…”

Section: Bmentioning

confidence: 99%

P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticide–drug interactions

2015

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A P-gp vesicular transport inhibition assay – Optimization and validation for drug–drug interaction testing

Cited by 29 publications

References 77 publications

Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters

Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters

Efflux transport of nicotine, cotinine and trans‐3′‐hydroxycotinine glucuronides by human hepatic transporters

P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticide–drug interactions

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