Breast Cancer Resistance Protein Inhibitor Research Articles

Ticagrelor and Statins: Dangerous Liaisons?

Polypharmacy is often necessary in complex, chronic, comorbid and cardiovascular patients and is a known risk factor for potential drug-drug interaction (DDI) that can cause adverse reactions (toxicity or therapeutic failure). Anti-thrombotic drugs (largely low-dose aspirin and a platelet P2Y12 receptor inhibitor) and statins are among the most co-administered drugs in cardiovascular patients. Ticagrelor is a selective antagonist of the platelet P2Y12-receptor, highly effective in inhibiting platelet aggregation and bio-transformed by the CYP3A4 and substrate of transporters, such as the breast cancer resistance protein (BCRP). Statins have different pharmacokinetic profiles; some undergo CYP3A4-mediated metabolism; rosuvastatin is primarily metabolized by the CYP2C9; and they have different affinities for drug transporters. Rhabdomyolysis is a very rare but severe adverse event, which is specific for statins which can be triggered by DDIs that increase statin's concentrations through blockade of their biotransformation and/or elimination. Large pharmacovigilance and small observational studies reported increased rhabdomyolysis in patients treated with some statins and ticagrelor but not aspirin, clopidogrel or prasugrel. Recent studies in vitro, pharmacokinetic trials and in silico drug modelling identified and validated the BCRP inhibition by ticagrelor, as a mechanism contributing to the DDI with statins, as 'victim' drugs, leading to increased rhabdomyolysis. While the clinical impact of this DDI deserves further investigation, a careful evaluation should be advised when ticagrelor is co-prescribed with some statins.

Clinical Assessment of Breast Cancer Resistance Protein (BCRP)-Mediated Drug-Drug Interactions of Sepiapterin with Curcumin and Rosuvastatin in Healthy Volunteers.

Sepiapterin, also known as PTC923 and CNSA-001, is a synthetic form of endogenous sepiapterin being developed as a novel oral treatment for phenylketonuria. Sepiapterin is a natural precursor of tetrahydrobiopterin (BH4) and, when orally administered, is converted to BH4 via the pterin salvage pathway. In vitro studies have demonstrated that both sepiapterin and BH4 are both substrates and inhibitors of the breast cancer resistance protein (BCRP) transporter. This phase I study investigated BCRP-mediated drug-drug interactions of sepiapterin as a victim and as a perpetrator. An open-label, fixed-sequence, four-period, crossover, single-dose study was conducted in adult male and female healthy volunteers (18-55 years of age). In a given treatment period, subjects received a single oral dose of sepiapterin (20 mg/kg), sepiapterin (20 mg/kg) plus curcumin (2 g), rosuvastatin (10 mg), or rosuvastatin (10 mg) plus sepiapterin (60 mg/kg). The pharmacokinetics of sepiapterin, its metabolite BH4, and rosuvastatin were studied, and geometric mean ratios of exposures in the presence and absence of the BCRP inhibitor curcumin or sepiapterin were estimated. The presence of the BCRP c.421C>A polymorphism was evaluated in all subjects. A total of 29 subjects were enrolled and included in the safety analysis. Among them, 26 subjects were included in the pharmacokinetic and drug-drug interaction analyses. Following oral administration20 mg/kg sepiapterin, sepiapterin was rapidly and extensively converted to BH4, and BH4 maximum observed concentration (415.0 ng/mL) was observed 4.95 h (time to maximum observed concentration) post-dose. Sepiapterin maximum observed concentration and area under the concentration-time curve from time 0 to time of the last quantifiable measurement or the last sample collection time (AUClast) were <1% of BH4 values. Coadministration of the BCRP inhibitor curcumin (2 g) increased BH4 maximum observed concentration, AUClast, and area under the concentration-time curve from time 0 extrapolated to infinity by 24%, 21%, and 20%, respectively. When sepiapterin was coadministered with the BCRP substrate rosuvastatin, there was no effect on the pharmacokinetics of rosuvastatin. BCRP c.421C/A carriers (n = 4) had higher plasma exposures of BH4 (1.39 × for AUClast) and rosuvastatin (1.61 × for AUClast) than c.421C/C carriers (n = 22). Greater increases in BH4 exposures (1.33 vs 1.18 for AUClast) were observed in c.421C/A carriers compared with c.421C/C carriers when sepiapterin was coadministered with curcumin. All treatments were well tolerated during the study. Oral coadministration of the BCRP inhibitor curcumin slightly increased the plasma exposure of sepiapterin and its metabolite BH4 in healthy volunteers. This modest increase was deemed not clinically meaningful. Sepiapterin did not alter the pharmacokinetics of the BCRP substrate rosuvastatin.

The role of efflux transporters in cytotoxicity and intracellular concentration of chlorpyrifos and chlorpyrifos oxon in human cell lines

In this study, we investigated the role of two efflux transporters, p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in the cytotoxicity and intracellular accumulation of the organophosphate pesticide chlorpyrifos (CPF) and its active metabolite, CPF-oxon (CPFO), in a human-derived liver cell line (HepG2) and kidney epithelial cell line (HK−2). The cytotoxicity to CPF and CPFO differed between cell lines where HK-2 had lower IC50 values which could be attributed to lower basal expression and inducibility of metabolizing enzymes, transporters, and nuclear receptors in HK-2 cells. In HepG2 cells, co-exposure of CPF with a specific inhibitor of either P-gp or BCRP enhanced the cytotoxicity of CPF while co-exposure of CPFO with VRP enhanced the cytotoxicity of CPFO, suggesting the role of these transporters in the elimination CPF and CPFO. Inhibition of efflux transporters did not affect the cytotoxicity of CPF and CPFO in HK-2 cells. Co-incubation of CPF with P-gp and BCRP inhibitors increased the intracellular concentration of CPF in HepG2 cells suggesting that both transporters play a role in limiting the cellular accumulation of CPF in HepG2 cells. Our results provide evidence that inhibition of efflux transporters can enhance CPF-induced toxicity through enhanced cellular accumulation and raises additional questions regarding how pesticide-transporter interactions may influence toxicity of mixtures containing pesticides and other environmental chemicals.

Coproporphyrin-I as a Selective OATP1B Biomarker Can Be Used to Delineate the Mechanisms of Complex Drug-Drug Interactions: Cedirogant Case Study.

Cedirogant is an inverse agonist of retinoic acid-related orphan receptor gamma thymus developed for the treatment of chronic plaque psoriasis. Cedirogant induces cytochrome P450 (CYP) 3A4 while inhibiting P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, and OATP1B3 in vitro. Static drug-drug interactions (DDIs) predictions suggested possible clinical induction of CYP3A4, and inhibition of P-gp, BCRP, and OATP1B1, leading to challenges in interpreting DDI studies between cedirogant and substrates of CYP3A, P-gp, BCRP, and OATP1B1/3. Here the effects of cedirogant on the pharmacokinetics of two statin drugs were investigated in healthy participants. Coproporphyrin-I (CP-I), a selective endogenous OATP1B biomarker, was used to assess the impact of cedirogant on OATP1B. Cedirogant (375 mg once daily) increased rosuvastatin maximum plasma concentration (Cmax) and area under the plasma concentration curve (AUCtau) by 141% and 55%, respectively when co-administered, whereas atorvastatin Cmax increased by 40% with no effect on its AUCtau compared with administration of rosuvastatin/atorvastatin alone. Cedirogant did not increase CP-I exposures, indicating no clinical OATP1B inhibition. The increased rosuvastatin exposure and minimal change in atorvastatin exposure with co-administration of cedirogant is attributed to BCRP inhibition and interplay between P-gp/BCRP inhibition and CYP3A induction, respectively. Correlation analysis with data from two investigational drugs (glecaprevir and flubentylosin) demonstrated that OATP1B1 R-value of > 1.5 and [Cmax,u]/[OATP1B1 IC50] of > 0.1 are associated with > 1.25-fold increase in CP-I Cmax ratio. This demonstrates the utility of CP-I in disentangling mechanisms underlying a complex DDI involving multiple transporters and enzymes and proposes refined criteria for static OATP1B inhibition predictions.

Breast Cancer Resistance Protein: Structure, Localization, Functions, Significance for Rational Pharmacotherapy

INTRODUCTION: Currently, investigation of efflux transport systems of an organism is an important scientific and practical task permitting to more deeply investigate the pharmacokinetics of medical drugs and to optimize pharmacotherapy of a number of diseases. The super family of ABC transporters plays a significant role in transport of biobiotics, the processes of absorption, distribution and excretion of medical drugs from an organism, in realization of undesired drug-drug interactions and development of pharmacoresistance. An important representative of this super family is breast cancer resistance protein (BCRP). AIM: Systematization of the data on BCRP structure, localization, functions, substrates and modulators of its activity. This literature review presents modern data on the molecular and spatial structure of BCRP, its localization in the cell, in organs and tissues. The data from studies of the functions of BCRP in an organism, of its role in the development of undesired drug-drug interactions at different stages of pharmacokinetics are summarized. An up-to-date list of medicinal drugs that are substrates and inhibitors of BCRP is given. Modern approaches to testing medical drugs for belonging to BCRP substrates or modulators of its activity are disclosed. CONCLUSION: The significance of BCRP consists in the existence of a wide range of drugs that are its substrates or modulators of its activity. Upon that, of increasing significance is the investigation both of new and long-known medicinal substances for their belonging to substrates, inducers or inhibitors of breast cancer resistance protein, in order to increase the effectiveness of pharmacotherapy and reduce the risk of undesired drug interactions. Search for non-drug substrates and modulators of BCRP activity permits to obtain new markers for conducting effective studies of safe pharmacokinetics both in vitro and in vivo.

The ATP-Binding Cassette Transporter-Mediated Efflux Transport of Ganciclovir at the Blood-Brain Barrier.

Recent studies have highlighted the key role of the ATP-binding cassette (ABC) transporters, including theP-glycoprotein (P-gp), the breast cancer resistance protein (BCRP), and the multi-drug resistance protein 4 (MRP4) in limiting the brain distribution of several antiviral agents. In this study, we investigated whether the inhibition of these transporters increases the permeability of the blood-brain barrier (BBB) to ganciclovir. A microdialysis and high-performance liquid chromatographic method was developed to monitor the concentrations of unbound ganciclovir in the brain interstitial fluid and plasma, with and without the administration of ABC transporter inhibitors. Pharmacokinetic parameters, including the area under the plasma concentration-time curve from time 0 to time of the last measurable analyte concentration (AUC0-t,plasma), the area under the brain interstitial fluid concentration-time curve from time 0 to time of the last measurable analyte concentration (AUC0-t,brain), and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) were calculated. The mean AUC0-t,plasma, AUC0-t,brain, and Kp,uu,brain in rats who received ganciclovir (30 mg/kg, intraperitoneal) alone were 1090 min·µg/mL, 150 min·µg/mL, and 14%, respectively. After the administration of tariquidar (inhibitor of P-gp), Ko143 (inhibitor of BCRP), or MK-571 (inhibitor of MRP4), the Kp,uu,brain of ganciclovir increased to 31 ± 2.1%, 26 ± 1.3%, and 32 ± 2.0%, respectively. The findings of this studysuggest that ABC transporters P-gp, BCRP, and MRP4 mediate the efflux of ganciclovir at the BBB and that the inhibition of these transporters facilitates the penetration of the BBB by ganciclovir.

Minimal Involvement of P-gp and BCRP in Oral Absorption of Ensitrelvir, An Oral SARS-CoV-2 3C-like Protease Inhibitor, in a Non-Clinical Investigation

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are important transporters causing drug-drug interaction (DDI). Here, we investigated the involvement of P-gp and BCRP in the oral absorption of ensitrelvir in non-clinical studies and estimated the DDI risk mediated by P-gp and BCRP inhibition in humans. Although ensitrelvir is an in vitro P-gp and BCRP substrate, it demonstrated high bioavailability in rats and monkeys after oral administration. Plasma exposures of ensitrelvir following oral administration were comparable in wild type (WT) and Bcrp (-/-) mice. On the other hand, the area under the plasma concentration-time curve (AUC) ratio of ensitrelvir in the Mdr1a/1b (-/-) mice to the WT mice was 1.92, indicating that P-gp, but not BCRP, was involved in the oral absorption of ensitrelvir. Based on our previous retrospective analyses, such a low AUC ratio (<3) in the Mdr1a/1b (-/-) mice indicates a minimal impact of P-gp on the oral absorption in humans. In conclusion, our studies demonstrate that the involvement of both P-gp and BCRP in the oral absorption of ensitrelvir is minimal, and suggest that ensitrelvir has a low risk for DDIs mediated by P-gp and BCRP inhibition in humans.

Clinical Analysis and in Vitro Correlation of BCRP-Mediated Drug-Drug Interaction in the Gastrointestinal Tract.

Breast cancer resistance protein (BCRP) is a drug efflux transporter expressed on the epithelial cells of the small intestine and on the lateral membrane of the bile duct in the liver; and is involved in the efflux of substrate drugs into the gastrointestinal lumen and secretion into bile. Recently, the area under the plasma concentration-time curve (AUC) of rosuvastatin (ROS), a BCRP substrate drug, has been reported to be increased by BCRP inhibitors, and BCRP-mediated drug-drug interaction (DDI) has attracted attention. In this study, we performed a ROS uptake study using human colon cancer-derived Caco-2 cells and confirmed that BCRP inhibitors significantly increased the intracellular accumulation of ROS. The correlation between the cell to medium (C/M) ratio of ROS obtained by the in vitro study and the absorption rate constant (ka) ratio obtained by clinical analysis was examined, and a significant positive correlation was observed. Therefore, it is suggested that the in vitro study using Caco-2 cells could be used to quantitatively estimate BCRP-mediated DDI with ROS in the gastrointestinal tract.

High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The KD of xanthotoxin, bergapten, and naringenin were 5.14 μM, 4.57 μM, and 3.72 μM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.

A Pilot Study To Assess the Suitability of Riboflavin As a Surrogate Marker of Breast Cancer Resistance Protein in Healthy Participants.

We recently showed that riboflavin is a selected substrate of breast cancer resistance protein (BCRP) over P-glycoprotein (P-gp) and demonstrated its prediction performance in preclinical drug-drug interaction (DDI) studies. The aim of this study was to investigate the suitability of riboflavin to assess BCRP inhibition in humans. First, we assessed the substrate potential of riboflavin toward other major drug transporters using established transfected cell systems. Riboflavin is a substrate for organic anion transporter (OAT)1, OAT3, and multidrug and toxin extrusion protein (MATE)2-K, with uptake ratios ranging from 2.69 to 11.6, but riboflavin is not a substrate of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)2, and MATE1. The effects of BMS-986371, a potent in vitro inhibitor of BCRP (IC 50 0.40 μM), on the pharmacokinetics of riboflavin, isobutyryl carnitine, and arginine were then examined in healthy male adults (N = 14 or 16) after oral administration of methotrexate (MTX) (7.5 mg) and enteric-coated (EC) sulfasalazine (SSZ) (1000 mg) alone or in combination with BMS-986371 (150 mg). Oral administration of BMS-986371 increased the area under the plasma concentration-time curves (AUCs) of rosuvastatin and immediate-release (IR) SSZ to 1.38- and 1.51-fold, respectively, and significantly increased AUC(0-4h), AUC(0-24h), and C max of riboflavin by 1.25-, 1.14-, and 1.11-fold (P-values of 0.003, 0.009, and 0.025, respectively) compared with the MTX/SSZ EC alone group. In contrast, BMS-986371 did not significantly influence the AUC(0-24h) and C max values of isobutyryl carnitine and arginine (0.96- to 1.07-fold, respectively; P > 0.05). Overall, these data indicate that plasma riboflavin is a promising biomarker of BCRP that may offer a possibility to assess drug candidate as a BCRP modulator in early drug development. SIGNIFICANCE STATEMENT: Endogenous compounds that serve as biomarkers for clinical inhibition of breast cancer resistance protein (BCRP) are not currently available. This study provides the initial evidence that riboflavin is a promising BCRP biomarker in humans. For the first time, the value of leveraging the substrate of BCRP with acceptable prediction performance in clinical studies is shown. Additional clinical investigations with known BCRP inhibitors are needed to fully validate and showcase the utility of this biomarker.

Improving brain penetration using elacridara P-glycoprotein and BCRP inhibitor

The central nervous system (CNS) is a site for a myriad of disorders and diseases, such as schizophrenia, Alzheimers disease, and Parkinsons disease. Many medications targeting these illnesses remain challenged due to efflux transporters forming a blood-brain barrier (BBB). To combat such challenges, elacridar shown promise at inhibiting such transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), therefore improving brain penetration. However, as an early clinical candidate, many remain unknown about elacridars pharmacokinetic (PK) properties in the human body. This paper aims to obtain a better understanding of elacridars pharmacokinetic profile and predict the optimal dose and dose interval for its application in the clinic. To begin with, a series of basic PK models were created using fundamental PK equations and the models were fit to elacridar clinical data to obtain elacridar-specific PK parameters. Next, elacridar preclinical PK as well as in vitro inhibition assay were used to determine the therapeutic window. Our final, multiple-dose extravascular PK model reveals that the most convenient and effective dose regimen is 900 mg BID (bis in die; twice per day). With this elacridar PK model, researchers can leverage elacridar human PK to improve clinical outcomes.

Carborane-Based ABCG2-Inhibitors Sensitize ABC-(Over)Expressing Cancer Cell Lines for Doxorubicin and Cisplatin.

The ABCG2 transporter protein, as part of several known mechanisms involved in multidrug resistance, has the ability to transport a broad spectrum of substrates out of the cell and is, therefore, considered as a potential target to improve cancer therapies or as an approach to combat drug resistance in cancer. We have previously reported carborane-functionalized quinazoline derivatives as potent inhibitors of human ABCG2 which effectively reversed breast cancer resistance protein (BCRP)-mediated mitoxantrone resistance. In this work, we present the evaluation of our most promising carboranyl BCRP inhibitors regarding their toxicity towards ABCG2-expressing cancer cell lines (MCF-7, doxorubicin-resistant MCF-7 or MCF-7 Doxo, HT29, and SW480) and, consequently, with the co-administration of an inhibitor and therapeutic agent, their ability to increase the efficacy of therapeutics with the successful inhibition of ABCG2. The results obtained revealed synergistic effects of several inhibitors in combination with doxorubicin or cisplatin. Compounds DMQCa, DMQCc, and DMQCd showed a decrease in IC50 value in ABCB1- and ABCG2-expressing SW480 cells, suggesting a possible targeting of both transporters. In an HT29 cell line, with the highest expression of ABCG2 among the tested cell lines, using co-treatment of doxorubicin and DMQCd, the effective inhibitory concentration of the antineoplastic agent could be reduced by half. Interestingly, co-treatment of compound QCe with cisplatin, which is not an ABCG2 substrate, showed synergistic effects in MCF-7 Doxo and HT29 cells (IC50 values halved or reduced by 20%, respectively). However, a literature-known upregulation of cisplatin-effluxing ABC transporters and their effective inhibition by the carborane derivatives emerges as a possible reason.

Poor Correlation of Rivaroxaban Concentration with the Routine Coagulation Screening Test in Chinese Patients with Atrial Fibrillation

Aims. The aim of this study is to assess the relationship between rivaroxaban plasma concentration quantified by the gold standard and anticoagulant activities measured by routine coagulation assays in Chinese atrial fibrillation (AF) patients. Whether the normal results of these tests were reliable to rule out clinically relevant rivaroxaban levels at various thresholds was also explored. The effect of clinical drug-drug interactions (DDIs) on the exposure and anticoagulant effect of rivaroxaban were further evaluated. Methods. 116 patients receiving rivaroxaban for the management of nonvalvular AF were recruited. Rivaroxaban concentrations and coagulation tests were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and a blood coagulation analyzer, respectively. Results. The correlation of trough concentration (Ctrough) and prothrombin time (PT) or international normalized ratio (INR) was moderate with Spearman’s correlation coefficient of 0.495 and 0.506, respectively. A normal PT/INR was unable to rule out Ctrough levels of &gt;30 ng/mL and &gt;50 ng/mL, but the negative predictive value reached 100% to exclude Ctrough of &gt;100 ng/mL. Ctrough showed a small correlation with activated partial thromboplastin time (aPTT) (Spearman’s correlation coefficient: 0.241) and no correlation with thrombin time (TT) (Spearman’s correlation coefficient: 0.074). Neither aPTT nor TT accurately predicted Ctrough at any concentration. Peak concentration (Cpeak) did not correlate with any coagulation parameters. The presence of digoxin and febuxostat significantly increased rivaroxaban Ctrough by 2.18 fold and prolonged PT and INR by 44.16% and 43.60%, respectively. Conclusions. Normal routine coagulation assays were insufficient to monitor therapy with rivaroxaban. Poor correlations between rivaroxaban concentration and routine coagulation assays were observed in Chinese AF patients. The use of digoxin/febuxostat alone had no effect on rivaroxaban concentrations; however, combined strong breast cancer resistance protein inhibitor (febuxostat) and P-glycoprotein probe (digoxin) in patients with renal impairment is likely to cause clinically significant DDI with rivaroxaban. More studies are needed to establish routine therapeutic drug monitoring of rivaroxaban in clinical practice.

Dissimilar Effect of P-Glycoprotein and Breast Cancer Resistance Protein Inhibition on the Distribution of Erlotinib to the Retina and Brain in Humans and Mice.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [11C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 μg) of [11C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp (n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg, n = 17) to saturate erlotinib transport. In addition, transport of [3H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [11C]erlotinib total volume of distribution (VT) in the human retina by -25 ± 8% (p ≤ 0.05) and -41 ± 16% (p ≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [11C]erlotinib VT in the human brain (+20 ± 16%, p ≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [11C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution.

Inhibition of efflux transporters by poly ADP-ribose polymerase inhibitors.

Poly ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of various cancers. They share a similar mechanism of action but have differences in pharmacokinetic characteristics and potential for drug-drug interactions (DDI). This study evaluated the potential ATP-binding cassette transporter-mediated interactions between PARP inhibitors (niraparib, olaparib and rucaparib) and statins (atorvastatin and rosuvastatin). We studied the inhibitory activity of PARP inhibitors on breast cancer resistance protein (BCRP), multidrug resistance-associated protein 3 (MRP3) and P-glycoprotein (P-gp) using vesicular transport assays and determined the concentrations required for 50% inhibition (IC50 ). Then, we predicted the increase of statin exposure followed by the administration of PARP inhibitors using a mechanistic static model. Rucaparib was the strongest inhibitor of BCRP-mediated rosuvastatin transport (IC50 13.7 μM), followed by niraparib (42.6 μM) and olaparib (216 μM). PARP inhibitors did not affect MRP3. While niraparib appeared to inhibit P-gp, the inhibition showed large variability. The inhibition of intestinal BCRP by rucaparib, niraparib and olaparib was predicted to elevate rosuvastatin exposure by 52%, 37% and 24%, respectively. The interactions between PARP inhibitors and rosuvastatin are probably of minor clinical significance alone, but combined with other predisposing factors, they may increase the risk of rosuvastatin-associated adverse effects.

Interactions of organophosphate flame retardants with human drug transporters

Organophosphate flame retardants (OPFRs) are environmental pollutants of increasing interest, widely distributed in the environment and exerting possible deleterious effects towards the human health. The present study investigates in vitro their possible interactions with human drug transporters, which are targets for environmental chemicals and actors of their toxicokinetics. Some OPFRs, i.e., tris(2-butoxyethyl) phosphate (TBOEP), tris(1,3-dichloroisopropyl) phosphate (TDCPP), tri-o-cresyl phosphate (TOCP) and triphenyl phosphate (TPHP), were found to inhibit activities of some transporters, such as organic anion transporter 3 (OAT3), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2 (OCT2) or breast cancer resistance protein (BCRP). These effects were concentration-dependent, with IC50 values ranging from 6.1 µM (for TDCPP-mediated inhibition of OCT2) to 51.4 µM (for TOCP-mediated inhibition of BCRP). OPFRs also blocked the transporter-dependent membrane passage of endogenous substrates, notably that of hormones. OAT3 however failed to transport TBOEP and TPHP. OPFRs additionally repressed mRNA expressions of some transporters in cultured human hepatic HepaRG cells, especially those of OAT2 and OCT1 in response to TOCP, with IC50 values of 2.3 µM and 2.5 µM, respectively. These data therefore add OPFRs to the expanding list of pollutants interacting with drug transporters, even if OPFR concentrations required to impact transporters, in the 2–50 µM range, are rather higher than those observed in humans environmentally or dietarily exposed to these chemicals.

Evaluation of Encequidar as An Intestinal P-gp and BCRP Specific Inhibitor to Assess the Role of Intestinal P-gp and BCRP in Drug-Drug Interactions.

The differences between intestinal and systemic (hepatic and renal) P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) roles in drug disposition are difficult to define. Accordingly, we characterized Encequidar (ECD) as an intestinal P-gp and BCRP specific inhibitor to evaluate their role in drug disposition. We assessed the in vitro and in vivo inhibition potential of ECD towards human and animal P-gp and BCRP. ECD is a potent inhibitor with a high degree of selectivity in inhibiting human P-gp (hP-gp) over human BCRP (hBCRP) (IC50s of 0.0058 ± 0.0006 vs. > 10µM, respectively). In contrast, ECD is a potent inhibitor of rat and cynomolgus monkey BCRP (IC50 ranged from 0.059 to 0.18µM). While the AUC of IV paclitaxel (PTX) was significantly increased by elacridar (ELD) (P < 0.05) but not ECD in rats (15mg/kg; PO) (2.55- vs. 0.93-fold), that of PO PTX was significantly elevated to a similar extent between the inhibitors (39.5- vs. 33.5-fold). Similarly, the AUC of PO sulfasalazine (SFZ) was dramatically increased by ELD and ECD (16.6- vs. 3.04-fold) although that of IV SFZ was not significantly affected by ELD and ECD in rats (1.18- vs. 1.06-fold). Finally, a comparable ECD-induced increase of the AUC of PO talinolol in cynomolgus monkeys was observed compared with ELD (2.14- vs. 2.12-fold). ECD may allow an in-depth appraisal of the role of intestinal efflux transporter(s) in drug disposition in animals and humans through local intestinal drug interactions.

Method for Testing of Drugs Belonging to Substrates and Inhibitors of the Transporter Protein BCRP on Caco-2 Cells

Introduction. Breast cancer resistance protein (BCRP) is an efflux membrane transporter that controls the pharmacokinetics of a large number of drugs. Its activity may change when taking some endo- and exogenous substances, thus making it a link in drug interactions.Aim. The aim of the study was to develop a method for testing of drugs for belonging to BCRP substrates and inhibitors in vitro.Materials and methods. The work was performed on Caco-2 cells overexpressing BCRP, the cultivation was performed in a transwell-system consisting of the apical and basolateral chambers. Cells were seeded at the bottom of the apical chamber, which is a semipermeable membrane. Primarily, the transport of BCRP substrates: methotrexate, mitoxantrone and quercetin was evaluated in the concentration range of 1, 5, 10, and 50 μM in the direction from the basal chamber to the apical one (Papp b-a) and in the opposite direction (Papp a-b). The ratio Papp b-a / Papp a-b more than «2» characterizes the participation of transporter proteins in the transcellular transport of substances. To confirm the participation of BCRP in their transport the experiment was carried out with the addition of a transporter inhibitor, reserpine, to the transport medium at a concentration of 50 μM. The concentration of substrates in the chambers was analyzed by HPLC-MS/MS.Results and their discussion. The addition of methotrexate (1 μM), mitoxantrone (1 μM), and quercetin (1–10 μM) to both the apical or basolateral chambers of the transwell-system, their content in the recipient chamber was not detected. When methotrexate concentration became 5 μM the Papp b-a / Papp a-b ratio was 3.38 ± 0.08, which indicates the involvement of transporters in its transfer. The addition of methotrexate to the donor chamber at concentrations of 10 and 50 μM, Papp b-a / Papp a-b decreased to values below «2». At mitoxantrone concentration of 5 μM Papp b-a / Papp a-b was 2.72 ± 0.16. An increase in the concentration to 10 μM led to an increase in Papp b-a / Papp a-b to 6.18 ± 0.08. With a substance content of 50 μM the indicator decreased but remained above the value «2». In the quercetin concentration of 50 microns, Papp b-a / Papp was below "2". Reserpine reduced Papp b-a / Papp a-b of methotrexate by 3.31 times (p = 0.0002), which indicates the elimination of asymmetry in the transport of the substance. At a mitoxantrone concentration of 10 microns, reserpine reduced its Papp b-a / Papp a-b by 3.36 times (p &lt; 0.0001). The results indicate the participation of BCRP in the control of the transfer of both substances through the cellular monolayer.Conclusion. A method of testing drugs belonging to BCRP substrates and inhibitors using methotrexate (5 μM) and mitoxantrone (10 μM) as marker substrates and reserpine (50 μM) as inhibitor was developed and tested on Caco-2 cells.

Two curcumin analogs inhibited the function and protein expression of breast cancer resistance protein: in vitro and in vivo studies

1. Two curcumin analogs, (1E,6E)-1,7-bis(3,5-diethyl-4-hydroxyphenyl)hepta-1,6-diene-3,5- dione (N17) and its prodrug ((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2,6-diethyl-4,1- phenylene)bis(3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate) (N17′), were evaluated as breast cancer resistance protein (BCRP) inhibitors. 2. MDCKII-BCRP and MDCKII-WT were used to evaluate the modulation effects of N17 and N17′ on BCRP and to explore the relevant mechanism. Sprague-Dawley rats were orally administered rosuvastatin (ROS), a probe substrate of BCRP, without and with N17′ (100 mg/kg) to investigate the effect of N17′ on ROS pharmacokinetics. 3. In cell studies, N17 and N17′ were substrates of BCRP, and they decreased the activity and protein expression of BCRP. In rat study, N17′ increased the systemic exposure of ROS by 218% (p = 0.058). 4. N17 and N17′ are potential BCRP inhibitors and will be promising candidates for overcoming the BCRP-mediated multidrug resistance.

Transporter and metabolic enzyme-mediated intra-enteric circulation of SN-38, an active metabolite of irinotecan: A new concept

SN-38, an active metabolite of irinotecan (CPT-11), is thought to circulate enterohepatically via organic anion-transporting polypeptides (OATPs), UDP-glucuronyl transferases (UGTs), multidrug resistance-related protein 2 (MRP2), and breast cancer resistance protein (BCRP). These transporters and enzymes are expressed in not only hepatocytes but also enterocytes. Therefore, we hypothesized that SN-38 circulates between the intestinal lumen and the enterocytes via these transporters and metabolic enzymes. To test this hypothesis, metabolic and transport studies of SN-38 and its glucuronide (SN-38G) were conducted in Caco-2 cells. The mRNA levels of UGTs, MRP2, BCRP, and OATP2B1 were confirmed in Caco-2 cells. SN-38 was converted to SN-38G in Caco-2 cells. The efflux of intracellularly generated SN-38G across the apical (digestive tract) membranes was significantly higher than the efflux across the basolateral (blood, portal vein) membranes of Caco-2 cells cultured on polycarbonate membranes. SN-38G efflux to the apical side was significantly reduced in the presence of MRP2 and BCRP inhibitors, suggesting that SN-38G is transported across the apical membrane by MRP2 and BCRP. Treatment of Caco-2 cells with OATP2B1 siRNA increased the SN-38 residue on the apical side, confirming that OATP2B1 is involved in the uptake of SN-38 into enterocytes. No SN-38 was detected on the basolateral side with or without siRNA treatment, suggesting that the enterohepatic circulation of SN-38 is limited, contrary to previous reports. These results suggest that SN-38 is absorbed into the enterocytes via OATP2B1, glucuronidated by UGTs to SN-38G, and excreted into the digestive tract lumen by MRP2 and BCRP. SN-38G can be deconjugated by β-glucuronidase from intestinal bacteria in the digestive tract lumen to regenerate SN-38. We named this new concept of local drug circulation “intra-enteric circulation.” This mechanism may allow SN-38 to circulate in the intestine and cause the development of delayed diarrhea, a serious side effect of CPT-11.