Fitting the Elementary Rate Constants of the P-gp Transporter Network in the hMDR1-MDCK Confluent Cell Monolayer Using a Particle Swarm Algorithm

P-glycoprotein, a human multidrug resistance transporter, has been extensively studied due to its importance to human health and disease. In order to understand transport kinetics via P-gp, confluent cell monolayers overexpressing P-gp are widely used. The purpose of this study is to obtain the mass action elementary rate constants for P-gp's transport and to functionally characterize members of P-gp's network, i.e., other transporters that transport P-gp substrates in hMDR1-MDCKII confluent cell monolayers and are essential to the net substrate flux. Transport of a range of concentrations of amprenavir, loperamide, quinidine and digoxin across the confluent monolayer of cells was measured in both directions, apical to basolateral and basolateral to apical. We developed a global optimization algorithm using the Particle Swarm method that can simultaneously fit all datasets to yield accurate and exhaustive fits of these elementary rate constants. The statistical sensitivity of the fitted values was determined by using 24 identical replicate fits, yielding simple averages and standard deviations for all of the kinetic parameters, including the efflux active P-gp surface density. Digoxin required additional basolateral and apical transporters, while loperamide required just a basolateral tranporter. The data were better fit by assuming bidirectional transporters, rather than active importers, suggesting that they are not MRP or active OATP transporters. The P-gp efflux rate constants for quinidine and digoxin were about 3-fold smaller than reported ATP hydrolysis rate constants from P-gp proteoliposomes. This suggests a roughly 3∶1 stoichiometry between ATP hydrolysis and P-gp transport for these two drugs. The fitted values of the elementary rate constants for these P-gp substrates support the hypotheses that the selective pressures on P-gp are to maintain a broad substrate range and to keep xenobiotics out of the cytosol, but not out of the apical membrane

Prediction of the Transporter-Mediated Drug-Drug Interaction Potential of Dabrafenib and Its Major Circulating Metabolites RUNNING TITLE: Risk for transporter-mediated drug-drug interactions for dabrafenib and metabolites

Abbreviations: AB, Apical to basolateral; BA, Basolateral to apical; BA/AB ratio, P app BA /P app AB ; BCRP, breast cancer resistance protein; CHO, Chinese hamster ovary cell line; DDI, drug-drug interaction; DMEM, Dulbecco's Modified Eagle Medium; DMF, dimethyl formamide; DMSO, dimethyl sulphoxide; DPBS, Dulbecco's Phosphate Buffered Saline; ITC, International Transporter Consortium; K m , affinity constant; MDCK = Madin Darby canine kidney cells; MDR1, multi-drug resistance protein 1; OATP, organic anion transporting polypeptide; OAT, organic anion transporter; OCT, organic cation transporter; P-gp, P-glycoprotein; P app , apparent permeability; V max , maximum uptake rate. ABSTRACT The BRAF inhibitor dabrafenib was recently approved for the treatment of certain BRAF V600 mutation-positive tumors, either alone or in combination therapy with the MEK inhibitor trametinib. This article presents the dabrafenib transporter-mediated drug-drug interaction risk assessment, which is currently an important part of drug development, regulatory submission and drug registration. Dabrafenib and its major circulating metabolites (hydroxy-, carboxy-and desmethyl-dabrafenib) were investigated as inhibitors of the clinically relevant transporters Pgp, BCRP, OATP1B1, OATP1B3, OCT2, OAT1 and OAT3. The DDI Guidance risk assessment decision criteria for inhibition of BCRP, OATP1B1 and OAT3 were slightly exceeded and therefore a minor DDI effect resulting from inhibition of these transporters remained possible. Biliary secretion is the major excretion pathway of dabrafenib-related material (71.1% of orally administered radiolabeled dose recovered in feces), while urinary excretion was observed as well (22.7% of the dose). In vitro uptake into human hepatocytes of the dabrafenib metabolites, but not of dabrafenib parent compound, was mediated, at least in part, by hepatic uptake transporters. The transporters responsible for uptake of the pharmacologically active hydroxy-and desmethyl dabrafenib could not be identified, whereas carboxy-dabrafenib was a substrate of several OATPs. Dabrafenib, hydroxyand desmethyl dabrafenib were substrates of P-gp and BCRP, while carboxy-dabrafenib was not. While a small increase in exposure to carboxy-dabrafenib upon inhibition of OATPs and an increase in exposure to desmethyl-dabrafenib upon inhibition of P-gp or BCRP cannot be excluded, the clinical significance of such increases is likely to be low

Inhibition of Intestinal OATP2B1 by the Calcium Receptor Antagonist Ronacaleret Results in a Significant Drug-Drug Interaction by Causing a 2-Fold Decrease in Exposure of Rosuvastatin

ABSTRACT Rosuvastatin is a widely prescribed antihyperlipidemic which undergoes limited metabolism, but is an in vitro substrate of multiple transporters [organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP1A2, OATP2B1, sodium-taurocholate cotransporting polypeptide, breast cancer resistance protein (BCRP), multidrug resistance protein 2 (MRP2), MRP4, organic anion transporter 3]. It is therefore frequently used as a probe substrate in clinical drug-drug interaction (DDI) studies to investigate transporter inhibition. Although each of these transporters is believed to play a role in rosuvastatin disposition, multiple pharmacogenetic studies confirm that OATP1B1 and BCRP play an important role in vivo. Ronacaleret, a drugdevelopment candidate for treatment of osteoporosis (now terminated), was shown to inhibit OATP1B1 in vitro (IC 50 = 11 mM), whereas it did not inhibit BCRP. Since a DDI risk through inhibition of OATP1B1 could not be discharged, a clinical DDI study was performed with rosuvastatin before initiation of phase II trials. Unexpectedly, coadministration with ronacaleret decreased rosuvastatin exposure by approximately 50%, whereas time of maximal plasma concentration and terminal half-life remained unchanged, suggesting decreased absorption and/or enhanced first-pass elimination of rosuvastatin. Of the potential in vivo rosuvastatin transporter pathways, two might explain the observed results: intestinal OATP2B1 and hepatic MRP4. Further investigations revealed that ronacaleret inhibited OATP2B1 (in vitro IC 50 = 12 mM), indicating a DDI risk through inhibition of absorption. Ronacaleret did not inhibit MRP4, discharging the possibility of enhanced first-pass elimination of rosuvastatin (reduced basolateral secretion from hepatocytes into blood). Therefore, a likely mechanism of the observed DDI is inhibition of intestinal OATP2B1, demonstrating the in vivo importance of this transporter in rosuvastatin absorption in humans

Prediction of the Transporter-Mediated Drug-Drug Interaction Potential of Dabrafenib and Its Major Circulating Metabolites s

ABSTRACT The BRAF inhibitor dabrafenib was recently approved for the treatment of certain BRAF V600 mutation-positive tumors, either alone or in combination therapy with the mitogen-activated extracellular signal regulated kinase 1 (MEK1) and MEK2 inhibitor, trametinib. This article presents the dabrafenib transporter-mediated drug-drug interaction (DDI) risk assessment, which is currently an important part of drug development, regulatory submission, and drug registration. Dabrafenib and its major circulating metabolites (hydroxy-, carboxy-, and desmethyl-dabrafenib) were investigated as inhibitors of the clinically relevant transporters P-gp, BCRP, OATP1B1, OATP1B3, OCT2, OAT1, and OAT3. The DDI Guidance risk assessment decision criteria for inhibition of BCRP, OATP1B1 and OAT3 were slightly exceeded and therefore a minor DDI effect resulting from inhibition of these transporters remained possible. Biliary secretion is the major excretion pathway of dabrafenib-related material (71.1% of orally administered radiolabeled dose recovered in feces), whereas urinary excretion was observed as well (22.7% of the dose). In vitro uptake into human hepatocytes of the dabrafenib metabolites, but not of dabrafenib parent compound, was mediated, at least in part, by hepatic uptake transporters. The transporters responsible for uptake of the pharmacologically active hydroxy-and desmethyl dabrafenib could not be identified, whereas carboxy-dabrafenib was a substrate of several OATPs. Dabrafenib, hydroxy-, and desmethyl-dabrafenib were substrates of P-gp and BCRP, whereas carboxy-dabrafenib was not. Although a small increase in exposure to carboxy-dabrafenib upon inhibition of OATPs and an increase in exposure to desmethyldabrafenib upon inhibition of P-gp or BCRP cannot be excluded, the clinical significance of such increases is likely to be low