Breast Cancer Resistance Protein Substrate Research Articles

ABCG2 regulatory single-nucleotide polymorphisms alter in vivo enhancer activity and expression.

The expression and activity of the breast cancer resistance protein (ABCG2) contributes toward the pharmacokinetics of endogenous and xenobiotic substrates. The effect of genetic variation on the activity of cis-regulatory elements and nuclear response elements in the ABCG2 locus and their contribution toward ABCG2 expression have not been investigated systematically. In this study, the effect of genetic variation on the in vitro and in vivo enhancer activity of six previously identified liver enhancers in the ABCG2 locus was examined. Reference and variant liver enhancers were tested for their ability to alter luciferase activity in vitro in HepG2 and HEK293T cell lines and in vivo using a hydrodynamic tail vein assay. Positive in vivo single-nucleotide polymorphisms (SNPs) were tested for association with gene expression and for altered protein binding in electrophoretic mobility shift assays. Multiple SNPs were found to alter enhancer activity in vitro. Four of these variants (rs9999111, rs12508471, ABCG2RE1*2, and rs149713212) decreased and one (rs2725263) increased enhancer activity in vivo. In addition, rs9999111 and rs12508471 were associated with ABCG2 expression in lymphoblastoid cell lines, lymphocytes, and T cells, and showed increased HepG2 nuclear protein binding. This study identifies SNPs within regulatory regions of the ABCG2 locus that alter enhancer activity in vitro and in vivo. Several of these SNPs correlate with tissue-specific ABCG2 expression and alter DNA/protein binding. These SNPs could contribute toward reported tissue-specific variability in ABCG2 expression and may influence the correlation between ABCG2 expression and disease risk or the pharmacokinetics and pharmacodynamics of breast cancer resistance protein substrates.

The phytoestrogens daidzein and equol inhibit the drug transporter BCRP/ABCG2 in breast cancer cells: potential chemosensitizing effect.

The soy isoflavone genistein has been described to up-regulate breast cancer resistance protein (BCRP) and, thus, enhance chemoresistance in breast cancer cells. The aim of this work was to assess the effect of long- and short-term incubation with daidzein, the second most abundant soy isoflavone and its metabolite equol on the expression and activity of P-glycoprotein, multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) and BCRP in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with phytoestrogen concentrations within the range achieved in individuals with a high isoflavone intake. Transporter expression was evaluated at protein and mRNA level through western blot and qRT-PCR, respectively. Transporter activity was determined using doxorubicin, mitoxantrone and carboxy-dichlorofluorescein as substrates. Daidzein (5µM) up-regulated MRP2- and down-regulated MRP1 protein expressions in MCF-7 and MDA-MB-231 cells, respectively. Both effects were ER-dependent, as determined using the antagonist ICI 182,780. The decrease in MRP1 mRNA in MDA-MB-231 cells indicates a transcriptional mechanism. On the contrary, MRP2 induction in MCF-7 cells takes place post-transcriptionally. Whereas changes in the transporter expression had a minor effect on the transporter activity, acute incubation with daidzein, R-equol and S-equol led to a strong inhibition of BCRP activity and an increase in the IC50 of BCRP substrates. In contrast to previous reports for genistein, daidzein and equol do not provoke a major up-regulation of the transporter expression but instead an inhibition of BCRP activity and sensitization to BCRP substrates.

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. Kp,uu,brain and Kp,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, Kp,uu,brain for the BCRP substrates was low. In contrast, Kp,uu,CSF for both BCRP substrates was close to unity, resulting in Kp,uu,CSF/Kp,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

Elucidation of the Impact of P-glycoprotein and Breast Cancer Resistance Protein on the Brain Distribution of Catechol-O-Methyltransferase Inhibitors.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are clinically important efflux transporters that act cooperatively at the blood-brain barrier, limiting the entry of several drugs into the central nervous system (CNS) and affecting their pharmacokinetics, therapeutic efficacy, and safety. In the present study, the interactions of catechol-O-methyltransferase (COMT) inhibitors (BIA 9-1059, BIA 9-1079, entacapone, nebicapone, opicapone, and tolcapone) with P-gp and BCRP were investigated to determine the contribution of these transporters in their access to the brain. In vitro cellular accumulation and bidirectional transport assays were conducted in Madin-Darby canine kidney (MDCK) II, MDCK-MDR1, and MDCK-BCRP cells. In vivo pharmacokinetic studies were carried out for tolcapone and BIA 9-1079 in rats, with and without elacridar, a well-known P-gp and BCRP modulator. The results suggest that BIA 9-1079, nebicapone, and tolcapone inhibit BCRP in a concentration-dependent manner. Moreover, with net flux ratios higher than 2 and decreased over 50% in the presence of verapamil or Ko143, BIA 9-1079 was identified as a P-gp substrate while BIA 9-1059, entacapone, opicapone, and nebicapone were revealed to be BCRP substrates. In vivo, brain exposure was limited for tolcapone and BIA 9-1079, although tolcapone crossed the blood-brain barrier at a greater rate and to a greater extent than BIA 9-1079. The extent of brain distribution of both compounds was significantly increased in the presence of elacridar, attesting to the involvement of efflux transporters. These findings provide relevant information and improve the understanding of the mechanisms that govern the access of these COMT inhibitors to the CNS.

Development and Validation of a Computational Model Ensemble for the Early Detection of BCRP/ABCG2 Substrates during the Drug Design Stage.

Breast Cancer Resistance Protein (BCRP) is an ATP-dependent efflux transporter linked to the multidrug resistance phenomenon in many diseases such as epilepsy and cancer and a potential source of drug interactions. For these reasons, the early identification of substrates and nonsubstrates of this transporter during the drug discovery stage is of great interest. We have developed a computational nonlinear model ensemble based on conformational independent molecular descriptors using a combined strategy of genetic algorithms, J48 decision tree classifiers, and data fusion. The best model ensemble consists in averaging the ranking of the 12 decision trees that showed the best performance on the training set, which also demonstrated a good performance for the test set. It was experimentally validated using the ex vivo everted rat intestinal sac model. Five anticonvulsant drugs classified as nonsubstrates for BRCP by the model ensemble were experimentally evaluated, and none of them proved to be a BCRP substrate under the experimental conditions used, thus confirming the predictive ability of the model ensemble. The model ensemble reported here is a potentially valuable tool to be used as an in silico ADME filter in computer-aided drug discovery campaigns intended to overcome BCRP-mediated multidrug resistance issues and to prevent drug-drug interactions.

Abstract 1192: Regorafenib (Stivarga®) reverses BCRP-mediated multidrug resistance in vitro and in vivo

Abstract Overexpression of breast cancer resistance protein (BCRP) has been shown to produce multidrug resistance (MDR) in various kinds of cancers, such as breast, colon, lung and ovarian cancers. Regorafenib, an oral multi-kinase inhibitor, was found to have inhibitory effects on BCRP-mediated MDR both in vitro and in vivo. Regorafenib significantly sensitized BCRP-overexpressing cancerous cells to BCRP substrates by increasing their intracellular accumulation. There are no significant changes in the expression level or the subcellular distribution of BCRP in the cells exposed to regorafenib. Our mechanism studies revealed that regorafenib inhibited the ATP-driven efflux function of BCRP. Our induced-fit docking and molecular dynamics simulations suggested the existence of strong and stable interactions between regorafenib and BCRP protein. Animal study revealed that the combination of regorafenib and topotecan resulted in great inhibitory effects on the growth of S1-M1-80 xenograft tumors. Regorafenib significantly increased the intratumoral concentration of topotecan but had no significant effects on topotecan plasma concentration. In conclusion, our study indicated that regorafenib could be useful in combating multidrug resistance in cancer treatments. Citation Format: Yun-Kai Zhang, Yi-Jun Wang, Guan-Nan Zhang, Wen-Ji Zhang, Meng-Ning Wei, Zhi Shi, Zhe-Sheng Chen. Regorafenib (Stivarga®) reverses BCRP-mediated multidrug resistance in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1192. doi:10.1158/1538-7445.AM2017-1192

Integrated safety analysis of rolapitant with coadministered drugs from phase II/III trials: an assessment of CYP2D6 or BCRP inhibition by rolapitant

BackgroundRolapitant, a long-acting neurokinin (NK)1 receptor antagonist (RA), has demonstrated efficacy in prevention of chemotherapy-induced nausea and vomiting in patients administered moderately or highly emetogenic chemotherapy. Unlike other NK1 RAs, rolapitant does not inhibit or induce cytochrome P450 (CYP) 3A4, but it does inhibit CYP2D6 and breast cancer resistance protein (BCRP). To analyze potential drug–drug interactions between rolapitant and concomitant medications, this integrated safety analysis of four double-blind, randomized phase II or III studies of rolapitant examined adverse events (AEs) by use versus non-use of drug substrates of CYP2D6 or BCRP.Patients and methodsPatients were randomized to receive either 180 mg oral rolapitant or placebo ∼1–2 h before chemotherapy in combination with a 5-hydroxytryptamine type 3 RA and dexamethasone. Data for treatment-emergent AEs (TEAEs) and treatment-emergent serious AEs (TESAEs) during cycle 1 were pooled across the four studies and summarized in the overall population and by concomitant use/non-use of CYP2D6 or BCRP substrate drugs.ResultsIn the integrated safety population, 828 of 1294 patients (64%) in the rolapitant group and 840 of 1301 patients (65%) in the control group experienced at least one TEAE. Frequencies of common TEAEs were similar in the rolapitant and control populations. Overall, 53% of patients received CYP2D6 substrate drugs, none of which had a narrow therapeutic index (like thioridazine or pimozide), and 63% received BCRP substrate drugs. When grouped by concomitant use versus non-use of CYP2D6 or BCRP substrate drugs, TEAEs and TESAEs occurred with similar frequency in the rolapitant and control populations.ConclusionsThe results of this study support the safety of rolapitant as part of an antiemetic triple-drug regimen in patients receiving emetogenic chemotherapy, including those administered concomitant medications that are substrates of CYP2D6 or BCRP, such as ondansetron, docetaxel, or irinotecan.

Effects of Catechins and Their Related Compounds on Cellular Accumulation and Efflux Transport of Mitoxantrone in Caco-2 Cell Monolayers.

The ability of catechins and their related compounds to inhibit breast cancer resistance protein (BCRP) function in Caco-2 cell monolayers was investigated with mitoxantrone as a BCRP substrate. The gallate or pyrogallol moiety on the catechin structure seemed to promote increased cellular accumulation and inhibit efflux transport of mitoxantrone. The ability of gallate catechins such as (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) to increase cellular accumulation and inhibit efflux transport of mitoxantrone was greater than that of nongallate catechins. Gallic acid octyl ester (GAO) also increased intracellular mitoxantrone accumulation. Experiments using GAO derivatives indicated that the gallate moiety required the presence of a long carbon chain for BCRP inhibition. Cellular accumulation and reduced efflux transport of mitoxantrone were greater with epigallocatechin 3-(3″-O-butyl) gallate than with EGCG. EGCG inhibition of BCRP seemed to be restricted by hydrophobicity. The co-administration of catechins, particularly EGCG and related compounds, with greater hydrophobicity may increase the therapeutic activities of BCRP substrates such as mitoxantrone.

In Vitro Interactions of Epacadostat and its Major Metabolites with Human Efflux and Uptake Transporters: Implications for Pharmacokinetics and Drug Interactions.

Epacadostat (EPAC) is a first-in-class, orally active inhibitor of the enzyme indoleamine 2,3-dioxygenase 1 and has demonstrated promising clinical activity. In humans, three major plasma metabolites have been identified: M9 (a glucuronide-conjugate), M11 (a gut microbiota metabolite), and M12 (a secondary metabolite formed from M11). It is proposed, based on the human pharmacokinetics of EPAC, that the biliary excretion of M9, the most abundant metabolite, leads to the enterohepatic circulation of EPAC. Using various in vitro systems, we evaluated in the present study the vitro interactions of EPAC and its major metabolites with major drug transporters involved in drug absorption and disposition. EPAC is a substrate for efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), but it is not a substrate for hepatic uptake transporters [organic anion transporting polypeptides OATP1B1 and OATP1B3]. The low permeability of M9 suggests an essential role for transporters in its disposition. M9 is likely excreted from hepatocytes into bile via multidrug resistance-associated protein 2 (MRP2) and BCRP, excreted into blood via MRP3, and transported from blood back into hepatocytes via OATP1B1 and OATP1B3. M11 and M12 are not substrates for P-gp, OATP1B1 or OATP1B3, and M11, but not M12, is a substrate for BCRP. With respect to inhibition of drug transporters, the potential of EPAC, M9, M11, and M12 to cause clinical drug-drug interactions via inhibition of P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, or organic cation transporter 2 was estimated to be low. The current investigation underlines the importance of metabolite-transporter interactions in the disposition of clinically relevant metabolites, which may have implications for the pharmacokinetics and drug interactions of parent drugs.

Correlation between Membrane Protein Expression Levels and Transcellular Transport Activity for Breast Cancer Resistance Protein.

Emerging evidence indicates an important role for the breast cancer resistance protein (BCRP) in limiting brain penetration of substrate drugs. While in vitro transwell assays can provide an indication of BCRP substrate potential, the predictability of these assays in relation to in vivo brain penetration is still under debate. The present study examined the correlation of BCRP membrane protein expression level and transcellular transport activity across Madin-Darby canine kidney (MDCK) II monolayers. We expressed human BCRP or murine BCRP1 in MDCKII wild-type cells using BacMam2 virus transduction. The selective P-glycoprotein (P-gp) inhibitor LY335979 (1 μM) was included in the transport medium to measure BCRP-mediated transcellular transport for P-gp and BCRP cosubstrates. The BCRP levels in membrane extracts from MDCKII-BCRP or MDCKII-Bcrp1 cells were quantified by liquid chromatography-tandem mass spectrometry. The results are summarized as follows: 1) the membrane protein expression levels correlate with the corrected efflux ratios of substrates for human BCRP and murine BCRP1 within the efflux ratios investigated; 2) we demonstrate good concordance in rank order between the BCRP and BCRP1-mediated efflux ratios for 12 drugs; and 3) we propose an approach to contextualize in vitro BCRP transport data of discovery compounds by comparing them to the in vitro and in vivo transport data of the reference drug dantrolene and taking into account interbatch variation in BCRP expression. This approach correctly predicted compromised brain penetration for 25 discovery compounds in rodents, which were BCRP substrates but not P-gp or weak P-gp substrates. These results suggest that BCRP-expressing MDCKII cells are useful in predicting the in vivo role of BCRP in brain penetration.

R- and S-Warfarin Were Transported by Breast Cancer Resistance Protein: From In Vitro to Pharmacokinetic-Pharmacodynamic Studies

Warfarin, a racemate of R- and S-warfarin, is an important oral anticoagulant with narrow therapeutic window. Being an acidic drug, warfarin (pKa = 4.94) exists mainly as anion under physiological pH. We hypothesized that the transport of warfarin anion across cell membrane was mediated by breast cancer resistance protein (BCRP), an efflux transporter having a variety of acidic substrates. This study aimed at verifying that warfarin was a substrate of BCRP. Cell lines and mice were used for transport assay and pharmacokinetic-pharmacodynamic studies, respectively. The concentrations of R- and S-warfarin were simultaneously determined by liquid chromatography-mass spectrometry method. Transport assay showed that the intracellular concentrations of R- and S-warfarin in MDCKII-BCRP were significantly lower than those in MDCKII. In addition, Ko143, a potent BCRP inhibitor, significantly inhibited the efflux transport of R- and S-warfarin in MDCKII-BCRP, but not in MDCKII. Pharmacokinetic study showed that the plasma concentrations of R- and S-warfarin in Bcrp-/- mice were significantly higher than those in wild-type mice at 6 h after dosing. Anticoagulation measurement showed that the international normalized ratio in Bcrp-/- mice was significantly higher than that in wild-type mice at 24 h after dosing. In conclusion, R- and S-warfarin were transported by BCRP.

Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

A new mechanism for increasing the oral bioavailability of scutellarin with Cremophor EL: Activation of MRP3 with concurrent inhibition of MRP2 and BCRP

Efflux transporters are extensively distributed and expressed in the intestinal epithelium and contribute to the low oral bioavailability of flavonoids and flavonoid glucuronides by pumping these compounds back into intestinal lumen. Our previous study has shown the inhibitory effect of Cremophor EL, a non-ionic surfactant, on efflux transporter multidrug resistance-associated protein (MRP) 2. In the current study, by using membranes overexpressing several common ATP-binding cassette (ABC) transporters including P-glycoprotein (P-gp), MRP1, MRP2, MRP3 and breast cancer resistance protein (BCRP), scutellarin, a poorly water-soluble flavonoid, was identified as the substrate of MPR2, MRP3 and BCRP. The effects of Cremophor EL on the transmembrane transportation of scutellarin by MRP2, BCRP, and MRP3 were investigated with inside-out Sf9 vesicles. Results showed that at nontoxic concentrations, Cremophor EL enhanced the transportation of scutellarin by MRP3 and inhibited the efflux transportation of scutellarin by MRP2 and BCRP concurrently. The relations between Cremophor EL and these transporters were explored using MDCK II-MRP2, MDCK II-BCRP, and MDCK II-MRP3 cell models. Compared with the control group, 5μg/ml Cremophor EL decreased the Papp(BL-AP) of scutellarin in MDCK II-MRP2 cell monolayers by >4 fold (from 13.57±0.76×10−7 to 2.90±0.14×10−7cm/s), and the Papp(BL-AP) in MDCK II-BCRP cell monolayers decreased from 9.12±0.15×10−7 to 6.34±0.08×10−7cm/s. On MDCK II-MRP3 cell monolayers, 5μg/ml Cremophor EL increased the Papp(AP-BL) of scutellarin by 3.5 fold (from 7.88±0.43×10−7 to 2.79±1.61×10−6cm/s), and caused an over 5-fold increase in Papp(AP-BL)/Papp(BL-AP). These findings suggested that Cremophor EL possesses the potent ability of inhibiting MRP2 and BCRP, as well as activating MRP3 effectively. In vivo pharmacokinetic research in rats further confirmed the improvement of oral absorption of scutellarin by Cremophor EL. In summary, our present study has identified a new mechanism for increasing the oral absorption and bioavailability of poorly absorbed drugs in which Cremophor EL increased MRP3 mediated transport but reduced MRP2 and BCRP mediated efflux concurrently, thereby enhancing the entry of drugs from enterocytes into the blood circulation.

Tariquidar Is an Inhibitor and Not a Substrate of Human and Mouse P-glycoprotein.

Since its development, tariquidar (TQR; XR9576; N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide) has been widely regarded as one of the more potent inhibitors of P-glycoprotein (P-gp), an efflux transporter of the ATP-binding cassette (ABC) transporter family. A third-generation inhibitor, TQR exhibits high affinity for P-gp, although it is also a substrate of another ABC transporter, breast cancer resistance protein (BCRP). Recently, several studies have questioned the mechanism by which TQR interfaces with P-gp, suggesting that TQR is a substrate for P-gp instead of a noncompetitive inhibitor. We investigated TQR and its interaction with human and mouse P-gp to determine if TQR is a substrate of P-gp in vitro. To address these questions, we used multiple in vitro transporter assays, including cytotoxicity, flow cytometry, accumulation, ATPase, and transwell assays. A newly generated BCRP cell line was used as a positive control that demonstrates TQR-mediated transport. Based on our results, we conclude that TQR is a potent inhibitor of both human and mouse P-gp and shows no signs of being a substrate at the concentrations tested. These in vitro data further support our position that the in vivo uptake of [(11)C]TQR into the brain can be explained by its high-affinity binding to P-gp and by it being a substrate of BCRP, followed by amplification of the brain signal by ionic trapping in acidic lysosomes.

Abstract C63: Effects of rolapitant on the pharmacokinetics of digoxin and sulfasalazine in healthy subjects

Abstract Introduction: Rolapitant is a selective and long acting NK-1 receptor antagonist for the prevention of chemotherapy-induced nausea and vomiting (CINV). In vitro results indicated that rolapitant inhibited P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), the efflux transporters that play important roles in drug disposition and distribution, with an IC50 of 7.4 μM and 4.6 μM, respectively. The major metabolite SCH720881 did not inhibit these transporters. The aims of these studies were to 1) evaluate the effects of rolapitant on the pharmacokinetics (PK) and pharmacodynamics (PD) of a P-gp substrate (digoxin), and on the PK of a BCRP substrate (sulfasalazine); and 2) evaluate the safety and tolerability of rolapitant co-administered with these substrates in healthy subjects. Methods: These were open-label, drug-drug interaction studies of orally administered substrate (0.5 mg of digoxin or 500 mg sulfasalazine, respectively) in the absence and presence of a single oral dose of 180 mg rolapitant. Cohorts consisted of 16 and 20 subjects, respectively. For the P-gp study, blood samples for digoxin PK were collected up to 96 hours post digoxin dose in both Period 1 (digoxin alone) and Period 2 (digoxin + a single dose of rolapitant). For PD evaluation, ECGs were performed at specified time points for both Periods 1 and 2. For the BCRP study, blood samples were collected up to 30 hours after Day 1 (sulfasalazine alone), Day 3 (sulfasalazine + rolapitant) and Day 10 (sulfasalazine alone) dosing. All samples were used to evaluate the impact of rolapitant and its major metabolite, SCH720881, on the PK of the relevant substrates. Results: Co-administration of digoxin with rolapitant increased mean digoxin Cmax value by 71% and mean AUC by 30%; trough concentrations were not impacted. There is no consistent pattern or trend to indicate a different PD profile of digoxin when administered with rolapitant. Digoxin administered either alone or in the presence of rolapitant was well tolerated. Rolapitant inhibited BCRP resulting in an increase in exposure of sulfasalazine. Geometric mean ratios were 2.4 and 1.2 for Cmax; 2.3 and 1.3 for AUC for the comparison of Day3/Day1 and Day10/Day1, respectively. There were no noteworthy adverse events or laboratory findings in this study. Conclusions: Rolapitant was well tolerated when co-administered with either the P-gp or BCRP substrate. Co-administration of rolapitant (180 mg) and digoxin (0.5 mg) had no clinically meaningful effect on PD response when compared with digoxin alone. Co-administration of rolapitant (180 mg) increased the exposure of sulfasalazine (500 mg) by approximately 2-fold. Monitoring for adverse reactions related to P-gp or BCRP substrates with a narrow therapeutic index may be necessary if rolapitant is concomitantly administered. Citation Format: Xiaodong Wang, Zhi-Yi Zhang, Sujata Arora, Lorraine Hughes, Jing Wang, Sharon Lu, Jennifer Christensen, Vikram Kansra. Effects of rolapitant on the pharmacokinetics of digoxin and sulfasalazine in healthy subjects. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C63.

Effect of Ursolic Acid on Breast Cancer Resistance Protein-mediated Transport of Rosuvastatin In Vivo and Vitro.

To evaluate whether ursolic acid can inhibit breast cancer resistance protein (BCRP)-mediated transport of rosuvastatin in vivo and in vitro. Firstly, we explored the pharmacokinetics of 5-fluorouracil (5-FU, a substrate of BCRP) in rats in the presence or absence of ursolic acid. Secondly, we studied the pharmacokinetics of rosuvastatin in rats in the presence or absence of ursolic acid or Ko143 (inhibitor of BCRP). Finially, the concentration-dependent transport of rosuvastatin and the inhibitory effects of ursolic acid and Ko143 were examined in Madin-Darby Canine Kidney (MDCK) 2-BCRP421CC (wild type) cells and MDCK2-BCRP421AA (mutant type) cells. As a result, significant changes in pharmacokinetics parameters of 5-FU were observed in rats following pretreatment with ursolic acid. Both ursolic acid and Ko143 could significantly affect the pharmacokinetics of rosuvastatin. The rosuvastatin transport in the BCRP overexpressing system was increased in a concentration-dependent manner. However, there was no statistical difference in BCRP-mediated transport of rosuvastatin betweent the wild type cells and mutant cells. The same as Ko143, ursolic acid inhibited BCRP-mediated transport of rosuvastatin in vitro. Ursolic acid appears to be a potent modulator of BCRP that affects the pharmacokinetic of rosuvastatin in vivo and inhibits the transport of rosuvastatin in vitro.

Neochamaejasmin B increases the bioavailability of chamaechromone coexisting in Stellera chamaejasme L. via inhibition of MRP2 and BCRP

Chamaechromone and neochamaejasmin B (NCB) are the most abundant components in the dried roots of the toxic perennial herb Stellera chamaejasme L. and have pharmacological activities. The objective of this study was to investigate the transport mechanism of these two components in vivo and in vitro. The transport and cellular accumulation studies in Madin–Darby canine kidney (MDCK) cells overexpressing human multidrug resistance protein 2 (MRP2) or P-gp and LLC-PK1 cells overexpressing human breast cancer resistance protein (BCRP) were performed. The results showed that chamaechromone was a good substrate of MRP2 and BCRP but not a substrate of P-gp. NCB was found to be a MRP2 inhibitor in transfected cells and significantly enhanced the cellular accumulation of chamaechromone in MDCK cells overexpressing MRP2. Similar results were obtained in LLC-PK1-BCRP cells. In addition, the influence of NCB on the bioavailability of chamaechromone following their co-administration was also determined in rats. The results showed that the area under the plasma concentration–time curve and maximal plasma concentration of chamaechromone in rats were increased by 48.9% and 81.9%, respectively. The mechanism of improving the oral bioavailability of chamaechromone was attributable to the inhibition of the BCRP and MRP2-mediated efflux of chamaechromone by NCB.

Quantitative Analysis of the ABCG2 c.421C > A Polymorphism Effect on In Vivo Transport Activity of Breast Cancer Resistance Protein (BCRP) Using an Intestinal Absorption Model

ABCG2 c.421C>A is one of the most frequent polymorphisms in ABCG2, which encodes the breast cancer resistance protein (BCRP). Clinical pharmacogenetic studies have shown that the plasma area under the concentration-time curve (AUC) values after oral administration of BCRP substrate drugs are significantly higher in subjects homozygous for the c.421C>A polymorphism (421AA) than in wild-type subjects (421CC). The aim of this study was to quantitatively estimate the in vivo decrease of BCRP function caused by the c.421C>A polymorphism based on clinical pharmacokinetic data. Assuming that the pharmacokinetic alteration is accounted for by intestinal BCRP, the ratio of the transport activity of the mutated BCRP to that of the wild-type was optimized by comparing calculations from an intestinal absorption model and clinical pharmacokinetic data. In conclusion, the in vivo intestinal BCRP transport activity in 421AA subjects is estimated to be approximately 23% of that in the 421CC subjects.

Obatoclax as a perpetrator in drug-drug interactions and its efficacy in multidrug resistance cell lines.

Obatoclax is a pan-Bcl-2 inhibitor with promising efficacy, especially when combined with other antineoplastic agents. Pharmacokinetic drug-drug interactions can occur systemically and at the level of the tumour cell. Thus, this study scrutinised the interaction potential of obatoclax in vitro. Obatoclax was screened for P-gp inhibition by calcein assay, for breast cancer resistance protein (BCRP) inhibition by pheophorbide A assay and for inhibition of cytochrome P450 isoenzymes (CYPs) by commercial kits. Induction of mRNA of drug-metabolising enzymes and drug transporters was quantified in LS180 cells via real-time polymerase chain reaction and involvement of nuclear receptors was assessed by reporter gene assays. Proliferation assays were used to assess whether obatoclax retains its efficacy in cell lines overexpressing BCRP, P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (MRP2). Obatoclax induced the mRNA expression of several genes (e.g. CYP1A1, CYP1A2 and ABCG2 (five to seven-fold) through activation of the aryl hydrocarbon receptor in the nanomolar range. Obatoclax inhibits P-gp, BCRP and some CYPs at concentrations exceeding plasma levels. P-gp, MPR2 or BCRP overexpression did not influence the efficacy of obatoclax. Obatoclax retains its efficacy in cells overexpressing P-gp, MRP2 or BCRP and might act as a perpetrator drug in interactions with drugs, for example being substrates of CYP1A2 or BCRP.

Downregulation of BCRP and anti-apoptotic proteins by proadifen (SKF-525A) is responsible for the enhanced mitoxantrone accumulation and toxicity in mitoxantrone-resistant human promyelocytic leukemia cells.

Multidrug resistance caused by the overexpression of ABC transporter proteins in cancer cells remains a major obstacle limiting chemotherapy efficacy. Drugs inhibiting these transporters have been shown to increase the anti-proliferative properties of chemotherapeutics. As we previously described, proadifen, a P450 monooxygenase inhibitor, might also be able to inhibit some ABC transporters, including breast cancer resistance protein (BCRP). Because mitoxantrone (MTX) is a strong BCRP substrate and is often used in the treatment of leukemia, we investigated the effect of 24 h proadifen pre-treatment on the cytotoxicity of MTX in leukemic cell lines that are sensitive to MTX (HL-60) and MTX-resistant ABCG2-overexpressing subclone (cBCRP). We show for the first time that proadifen is able to enhance the cytotoxic properties of MTX in cBCRP cells, particularly through the inhibition of BCRP expression and activity. This proadifen-MTX synergism was also mediated by the inhibition of various cellular proteins engaged in apoptosis, including Mc-1, Bcl-xL, survivin and activation of procaspase-3. Proadifen also decreased the expression of γH2AX, which is involved in the recruitment of reparation proteins. Moreover, the inhibition of DNA damage repair proteins Ku86 and B23 after proadifen treatment indicate a possible role of proadifen in DNA repair blockage, thus suppressing the reparation rate of MTX-induced DSBs.