Transporters Breast Cancer Resistance Protein Research Articles

Bisphenol A Inhibits the Transporter Function of the Blood-Brain Barrier by Directly Interacting with the ABC Transporter Breast Cancer Resistance Protein (BCRP).

The breast cancer resistance protein (BCRP) is an important efflux transporter in the blood-brain barrier (BBB), protecting the brain from a wide range of substances. In this study, we investigated if BCRP function is affected by bisphenol A (BPA), a high production volume chemical used in common consumer products, as well as by bisphenol F (BPF) and bisphenol S (BPS), which are used to substitute BPA. We employed a transwell-based in vitro cell model of iPSC-derived brain microvascular endothelial cells, where BCRP function was assessed by measuring the intracellular accumulation of its substrate Hoechst 33342. Additionally, we used in silico modelling to predict if the bisphenols could directly interact with BCRP. Our results showed that BPA significantly inhibits the transport function of BCRP. Additionally, BPA was predicted to bind to the cavity that is targeted by known BCRP inhibitors. Taken together, our findings demonstrate that BPA inhibits BCRP function in vitro, probably by direct interaction with the transporter. This effect might contribute to BPA’s known impact on neurodevelopment.

Influence of ABC transporters on the excretion of ciprofloxacin assessed with PET imaging in mice

Ciprofloxacin is a commonly prescribed fluoroquinolone antibiotic which is cleared by active tubular secretion and intestinal excretion. Ciprofloxacin is a known substrate of the ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4). In this work, we used positron emission tomography (PET) imaging to investigate the influence of BCRP, MRP4, MRP2 and P-glycoprotein (P-gp) on the excretion of [18F]ciprofloxacin in mice. Dynamic 90-min PET scans were performed after intravenous injection of [18F]ciprofloxacin in wild-type mice without and with pre-treatment with the broad-spectrum MRP inhibitor MK571. Moreover, [18F]ciprofloxacin PET scans were performed in Abcc4(-/-), Abcc2(-/-), Abcc4(-/-)Abcg2(-/-) and Abcb1a/b(-/-)Abcg2(-/-) mice. In addition to non-compartmental pharmacokinetic (PK) analysis, a novel three-compartment PK model was developed for a detailed assessment of the renal disposition of [18F]ciprofloxacin. In MK571 pre-treated mice, a significant increase in the blood exposure to [18F]ciprofloxacin was observed along with a significant reduction in the renal and intestinal clearances. PK modelling revealed a significant reduction in renal radioactivity uptake (CL1) and in the rate constants for transfer of radioactivity from the corticomedullary renal region into blood (k2) and urine (k3), respectively, after MK571 administration. No changes in the renal clearance or in the estimated kidney PK model parameters were observed in any of the studied knockout models, while a significant reduction in the intestinal clearance was observed in Abcc2(-/-) and Abcc4(-/-)Abcg2(-/-) mice. Our data failed to reveal a role of any of the studied ABC transporters in the tubular secretion of ciprofloxacin. This may indicate that ciprofloxacin is handled in the kidneys by more than one transporter family, most likely with a great degree of mutual functional redundancy. Our study highlights the potential of PET imaging for an assessment of transporter-mediated renal excretion of radiolabelled drugs.

A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity.

TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.

Sulfasalazine alleviates neuropathic pain hypersensitivity in mice through inhibition of SGK-1 in the spinal cord

Diurnal variations in pain hypersensitivity are common in chronic pain disorders. Temporal exacerbation of neuropathic pain hypersensitivity is dependent on diurnal variations in glucocorticoid secretion from the adrenal glands. We previously demonstrated that spinal expression of serum- and glucocorticoid-inducible kinase-1 (SGK-1) is associated with glucocorticoid- induced exacerbation of pain hypersensitivity, but there are no available strategies to inhibit SGK-1 in the spinal cord. By screening a clinically approved drug library (more than 1,200 drugs), we found that sulfasalazine (SSZ) has inhibitory effects on SGK-1. SSZ is a prodrug composed of 5-aminosalicylic acid and sulfapyridine linked by NN bond, which is therapeutically effective for inflammatory bowel diseases. However, the NN bond in SSZ was necessary for its inhibitory action against SGK-1. Although intrathecal injection of SSZ to nerve-injured mice significantly alleviated mechanical pain hypersensitivity, no significant anti- neuropathic pain effects of SSZ were detected after oral administration due to its low bioavailability and limited spinal distribution, which were associated with efflux by the xenobiotic transporter breast cancer resistance protein (BCRP). Concomitant oral administration of SSZ with febuxostat (FBX), which is an approved drug to inhibit BCRP, improved the distribution of SSZ to the spinal cord. The concomitant oral administration with FBX also increased the anti-neuropathic pain effects of SSZ. Our study revealed a previously unrecognized pharmacological effect of SSZ to alleviate SGK-1-induced painful peripheral neuropathy, and concomitant oral administration of SSZ with FBX may also be a preventative option for diurnal exacerbation of neuropathic pain hypersensitivity.

Examination of Physiologically-Based Pharmacokinetic Models of Rosuvastatin.

Physiologically‐based pharmacokinetic (PBPK) modeling is increasingly used to predict drug disposition and drug–drug interactions (DDIs). However, accurately predicting the pharmacokinetics of transporter substrates and transporter‐mediated DDIs (tDDIs) is still challenging. Rosuvastatin is a commonly used substrate probe in DDI risk assessment for new molecular entities (NMEs) that are potential organic anion transporting polypeptide 1B or breast cancer resistance protein transporter inhibitors, and as such, several rosuvastatin PBPK models have been developed to try to predict the clinical DDI and support NME drug labeling. In this review, we examine five representative PBPK rosuvastatin models, discuss common challenges that the models have come across, and note remaining gaps. These shared learnings will help with the continuing efforts of rosuvastatin model validation, provide more information to understand transporter‐mediated drug disposition, and increase confidence in tDDI prediction.

Interactions of the major effective components in Shengmai formula with breast cancer resistance protein at the cellular and vesicular levels

Shengmai Formula (SMF) is one of the traditional Chinese medicine representative formulas and is widely used for the treatment of cardio- and cerebrovascular disease. Previous studies demonstrated that the major effective ingredients in SMF can interact with each other based on some uptake transporters. However, the role of the efflux transporter breast cancer resistance protein (BCRP) in these interactions involving SMF remains unclear. The purpose of this study was to investigate the interactions of the major active components of SMF with BCRP and the compatibility mechanism of these complex components in SMF based on BCRP. We selected 4 main fractions, including ginseng total saponins (GTS), ophiopogon total saponins (OTS), ophiopogon total flavonoids (OTF), and fructus schisandrae total lignans (STL), and 12 bioactive components, including ginsenosides Re, Rd, Rb1, and Rg1, ophiopogonins D and Dˊ, methylophiopogonanones A and B, schizandrins A and B, and schizandrols A and B to explore the interactions of SMF with BCRP in LLC-PK1 and LLC-PK1/BCRP cells and BCRP membrane vesicles. The results showed that ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A can be transported by BCRP into LLC-PK1/BCRP cells. Schisandrol B exhibited a markedly inhibitory effect on the transport function of BCRP and can significantly inhibit the uptake of methylophiopogonanone B and schizandrin A into LLC-PK1/BCRP cells. In “Inside-Out” BCRP membrane vesicles, BCRP mediated the transport of ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A, with Km values of 111.9 ± 31.26 μM, 82.01 ± 16.72 μM, 57.06 ± 8.789 μM, and 37.19 ± 6.512 μM, respectively. GTS, STL, ginsenosides Rd and Rb1, and schisandrol B were potent inhibitors of BCRP and showed different degrees of inhibition on the transport of ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A via BCRP. In conclusion, GTS, STL, ginsenosides Rd and Rb1, and schizandrol B are potential inhibitors of BCRP. Ginsenosides Re and Rg1, methylophiopogonanone B, and schizandrin A are potential substrates of BCRP, and their transport, which is mediated by BCRP, may be inhibited by potential inhibitors in SMF. There are potential interactions of these main effective components of SMF at the cellular and vesicular levels that are mediated by BCRP. The interplay of these bioactive components based on BCRP may be an important compatibility mechanism in SMF.

Induced expression of P-gp and BCRP transporters on brain endothelial cells using transferrin functionalized nanostructured lipid carriers: A first step of a potential strategy for the treatment of Alzheimer's disease.

P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two transporters expressed in human neural stem/progenitor cells and at the Blood-Brain Barrier (BBB) level with decreased activity in the early stage of Alzheimer's disease (AD). Both proteins, have a protective role for the embryonic stem cells in the early developmental step, maintaining them in an undifferentiated state, and limit the access of exogenous and endogenous agents to the brain. Recently, MC111 selected from a P-gp/BCRP ligands library was investigated as multitarget strategy for AD treatment, considering its ability to induce the expression and activity of both proteins. However, MC111 clinical use could be limited for the ubiquitous physiological expression of efflux transporters and its moderate toxicity towards endothelial cells. Therefore, a selective MC111 delivery system based on nanostructured lipid carriers (NLC) functionalized with transferrin were developed. The results proved the formation of NLC with average size about 120nm and high drug encapsulation efficiency (EE% greater than 50). In vitro studies on hCMEC/D3 cells revealed that the MC111 was selectively released by NLC at BBB level and then inducing the activity and expression of BCRP and P-gp, involved in the clearance of amyloid β peptide on brain endothelial cells.

Inhibition of Breast Cancer Resistance Protein (BCRP) by Ko143 Can Affect Pharmacokinetics of Enrofloxacin in Exopalaemon carinicauda

Adenosine triphosphate-binding cassette transporter breast cancer resistance protein (BCRP) exists highly in the apical membranes of epithelia, and is involved in drug availability. Ko143 is a typical inhibitor of BCRP in rodents. The synthetic antibacterial agent enrofloxacin (ENRO) is a fluoroquinolone employed as veterinary and aquatic medicine, and also a substrate for BCRP. BCRP gene highly expressed in the hepatopancreas and intestine of Exopalaemon carinicauda as was determined with real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) method. The effects of Ko143 on the abundance of BCRP mRNA and ENRO pharmacokinetics in E. carinicauda were studied. The mRNA abundance of BCRP decreased significantly in hepatopancreas and intestine (P <0.05) after Ko143 treatment. Co-administration of Ko143 significantly changed the pharmacokinetics of orally administered enrofloxacin, which was supported by higher distribution half-life (t1/2α), elimination half-life (t1/2β), area under the curve up to the last measurable concentration (AUC0-t), peak concentration (Cmax) and lower clearance (CL/F). These findings revealed that Ko143 downregulates BCRP expression in hepatopancreas and intestine, thus affects the pharmacokinetics of orally administered enrofloxacin in E. carinicauda. The drug-drug interaction can be caused by the change in BCRP activity if ENRO is used in combination with other drugs in shrimp.

Abstract 6321: BEND3 modulates sensitivity to the UBA1 inhibitor TAK-243 by regulating expression of the multidrug transporter BCRP

Abstract TAK-243 (MLN7243) is a first-in-class inhibitor of the ubiquitin-activating enzyme (UBA1) that catalyzes the first step in the ubiquitylation cascade whereby proteins are tagged with mono- or poly-ubiquitin to induce their degradation or modify their functions. Based on its preclinical efficacy and tolerability, TAK-243 has entered phase 1 clinical trials in advanced malignancies. However, the determinants of sensitivity to TAK-243 remain largely unknown. Therefore, we conducted a positive-selection, genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with lethal TAK-243 concentrations to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout conferred resistance to TAK-243 (FDR = 0.0012). BEND3-targeting gRNAs were enriched up to 10,000-fold after selection with the drug. To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells and confirmed the resistance phenotype. In vivo, tumors of BEND3-knockout cells were resistant to TAK243 (20 mg/kg twice weekly) as opposed to control tumors that showed dramatic reductions in tumor growth rate. As assessed by immunoblotting, BEND3 knockout dampened TAK-243 effects on ubiquitylation, proteotoxic stress and DNA damage response. Mechanistically, BEND3 knockout upregulated the ABC efflux transporter breast cancer resistance protein (BCRP; ABCG2), and decreased intracellular levels of TAK-243. It also conferred partial cross-resistance to pevonedistat and TAK-981–related selective inhibitors of the NEDD8-activating enzyme (NAE) and the SUMO-activating enzyme (SAE), respectively, as well as known substrates of BCRP (mitoxantrone and doxorubicin). Finally, TAK-243 sensitivity strongly correlated with BCRP expression in a panel of 30 cancer cell lines of different origin, and chemical inhibition of BCRP but not P-gp sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity. Citation Format: Samir H. Barghout, Ahmed Aman, Zachary Blatman, Karen Arevalo, Geethu Thomas, Neil MacLean, Xiaoming Wang, Rose Hurren, Troy Ketela, Moustafa Abohawya, Taira Kiyota, Rima Al-Awar, Aaron D. Schimmer. BEND3 modulates sensitivity to the UBA1 inhibitor TAK-243 by regulating expression of the multidrug transporter BCRP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6321.

Effects of Chrysin and Its Major Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide on Cytochrome P450 Enzymes and on OATP, P-gp, BCRP, and MRP2 Transporters.

Chrysin is an abundant flavonoid in nature, and it is also contained by several dietary supplements. Chrysin is highly biotransformed in the body, during which conjugated metabolites chrysin-7-sulfate and chrysin-7-glucuronide are formed. These conjugates appear at considerably higher concentrations in the circulation than the parent compound. Based on previous studies, chrysin can interact with biotransformation enzymes and transporters; however, the interactions of its metabolites have been barely examined. In this in vitro study, the effects of chrysin, chrysin-7-sulfate, and chrysin-7-glucuronide on cytochrome P450 enzymes (2C9, 2C19, 3A4, and 2D6) as well as on organic anion-transporting polypeptides (OATPs; 1A2, 1B1, 1B3, and 2B1) and ATP binding cassette [P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein (BCRP)] transporters were investigated. Our observations revealed that chrysin conjugates are strong inhibitors of certain biotransformation enzymes (e.g., CYP2C9) and transporters (e.g., OATP1B1, OATP1B3, OATP2B1, and BCRP) examined. Therefore, the simultaneous administration of chrysin-containing dietary supplements with medications needs to be carefully considered due to the possible development of pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Chrysin-7-sulfate and chrysin-7-glucuronide are the major metabolites of flavonoid chrysin. In this study, we examined the effects of chrysin and its conjugates on cytochrome P450 enzymes and on organic anion-transporting polypeptides and ATP binding cassette transporters (P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2). Our results demonstrate that chrysin and/or its conjugates can significantly inhibit some of these proteins. Since chrysin is also contained by dietary supplements, high intake of chrysin may interrupt the transport and/or the biotransformation of drugs.

Effect of rutin on pharmacokinetic modulation of diclofenac in rats

Diclofenac is an extensively used nonsteroidal anti-inflammatory drug, but gastrointestinal liabilities and cardiovascular complications take the shine away from such a widely prescribed drug. On the other hand, rutin, a dietary bioflavonoid, has quite a few pharmacological attributes to improve the efficacy and reduce the dose-related toxicities of diclofenac through the intended food-drug/herb-drug interaction. The aim of the present research work was to investigate the role of rutin on pharmacokinetic modulation and the consequent efficacy of diclofenac. At first, pharmacodynamics and pharmacokinetics of diclofenac as alone and in the presence of rutin were investigated orally in a rat model. Then, mechanistic studies were performed to explain the effect of rutin on improvement in oral exposure as well as the efficacy of diclofenac using a battery of in-vitro/in-situ/in-vivo studies. Results displayed that rutin enhanced efficacy as well as oral bioavailability of diclofenac in rats. A marked increase in permeability of diclofenac by rutin was displayed that is linked to inhibition of Breast Cancer Resistance Protein (BCRP) transporters. There was no significant effect of rutin on the modulation of intestinal transit, CYP2C9 inhibition in human liver microsomes, and CYP2C9/CYP2C11 expression in rat liver tissues to boost the oral exposure of diclofenac. Rutin is found to be an inhibitor for BCRP transporters and can act as an oral bioavailability enhancer for a drug like diclofenac.

Comparative Pharmacokinetics of Δ9-Tetrahydrocannabinol in Adolescent and Adult Male Mice.

We investigated the pharmacokinetic properties of Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive constituent of cannabis, in adolescent and adult male mice. The drug was administered at logarithmically ascending doses (0.5, 1.6, and 5 mg/kg, i.p.) to pubertal adolescent (37-day-old) and adult (70-day-old) mice. Δ9-THC and its first-pass metabolites-11-hydroxy-Δ9-THC and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC)-were quantified in plasma, brain, and white adipose tissue (WAT) using a validated isotope-dilution liquid chromatography/tandem mass spectrometry assay. Δ9-THC (5 mg/kg) reached 50% higher circulating concentration in adolescent mice than in adult mice. A similar age-dependent difference was observed in WAT. Conversely, 40%-60% lower brain concentrations and brain-to-plasma ratios for Δ9-THC and 50%-70% higher brain concentrations for Δ9-THC metabolites were measured in adolescent animals relative to adult animals. Liver microsomes from adolescent mice converted Δ9-THC into 11-COOH-THC twice as fast as adult microsomes. Moreover, the brains of adolescent mice contained higher mRNA levels of the multidrug transporter breast cancer resistance protein, which may extrude Δ9-THC from the brain, and higher mRNA levels of claudin-5, a protein that contributes to blood-brain barrier integrity. Finally, administration of Δ9-THC (5 mg/kg) reduced spontaneous locomotor activity in adult, but not adolescent, animals. The results reveal the existence of multiple differences in the distribution and metabolism of Δ9-THC between adolescent and adult male mice, which might influence the pharmacological response to the drug. SIGNIFICANCE STATEMENT: Animal studies suggest that adolescent exposure to Δ9-tetrahydrocannabinol (Δ9-THC), the intoxicating constituent of cannabis, causes persistent changes in brain function. These studies generally overlook the impact that age-dependent changes in the distribution and metabolism of the drug might exert on its pharmacological effects. This report provides a comparative analysis of the pharmacokinetic properties of Δ9-THC in adolescent and adult male mice and outlines multiple functionally significant dissimilarities in the distribution and metabolism of Δ9-THC between these two age groups.

A Combination of Clioquinol, Zinc and Copper Increases the Abundance and Function of Breast Cancer Resistance Protein in Human Brain Microvascular Endothelial Cells

Modulating the abundance of the blood-brain barrier (BBB) efflux transporter breast cancer resistance protein (BCRP) has the potential to impact brain levels of drugs and endogenous substrates. Studies have demonstrated that the metal ionophore clioquinol (CQ) increases BBB abundance of P-glycoprotein (P-gp), an effect associated with increased endothelial cell levels of Cu2+. This study therefore assessed whether human brain endothelial (hCMEC/D3) cell abundance and function of BCRP is modulated by CQ. hCMEC/D3 cells were treated with CQ, Zn2+ and Cu2+ (CZC) (0.5 μM, 0.5 μM, 0.1 μM, respectively) for 24 h and BCRP mRNA and protein abundance was determined by Western blot and qPCR, respectively. After a series of optimisation studies assessing specificity of bodipy prazosin (BP) and Ko143 as a substrate and inhibitor of BCRP, respectively, the impact of CZC on BP uptake was assessed. While CZC did not increase mRNA expression of BCRP, BCRP abundance was increased 1.8 ± 0.1-fold; this was associated with a 68.1 ± 3.3% reduction in accumulation of BP in hCMEC/D3 cells. This is the first study to demonstrate that augmenting metal ion availability enhances protein abundance and function of BCRP at the BBB, which may be exploited to modulate CNS access of therapeutics and endogenous substrates.

Epigenetic Regulation of Multidrug Resistance Protein 1 and Breast Cancer Resistance Protein Transporters by Histone Deacetylase Inhibition.

Multidrug resistance protein 1 (MDR1, ABCB1, P-glycoprotein) and breast cancer resistance protein (BCRP, ABCG2) are key efflux transporters that mediate the extrusion of drugs and toxicants in cancer cells and healthy tissues, including the liver, kidneys, and the brain. Altering the expression and activity of MDR1 and BCRP influences the disposition, pharmacodynamics, and toxicity of chemicals, including a number of commonly prescribed medications. Histone acetylation is an epigenetic modification that can regulate gene expression by changing the accessibility of the genome to transcriptional regulators and transcriptional machinery. Recently, studies have suggested that pharmacological inhibition of histone deacetylases (HDACs) modulates the expression and function of MDR1 and BCRP transporters as a result of enhanced histone acetylation. This review addresses the ability of HDAC inhibitors to modulate the expression and the function of MDR1 and BCRP transporters and explores the molecular mechanisms by which HDAC inhibition regulates these transporters. While the majority of studies have focused on histone regulation of MDR1 and BCRP in drug-resistant and drug-sensitive cancer cells, emerging data point to similar responses in nonmalignant cells and tissues. Elucidating epigenetic mechanisms regulating MDR1 and BCRP is important to expand our understanding of the basic biology of these two key transporters and subsequent consequences on chemoresistance as well as tissue exposure and responses to drugs and toxicants. SIGNIFICANCE STATEMENT: Histone deacetylase inhibitors alter the expression of key efflux transporters multidrug resistance protein 1 and breast cancer resistance protein in healthy and malignant cells.

Effect of GenX on P-Glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance-Associated Protein 2 at the Blood-Brain Barrier.

Background:Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX) is a replacement for perfluorooctanoic acid in the production of fluoropolymers used in a variety of consumer products. GenX alters fetal development and antibody production and elicits toxic responses in the livers and kidneys of rodents. The GenX effect on the blood–brain barrier (BBB) is unknown. The BBB protects the brain from xenobiotic neurotoxicants and harmful endogenous metabolites.Objectives:We aimed to investigate the effects of GenX on the transport activity and expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance–associated protein 2 (MRP2) at the BBB.Methods:Transporter activities were measured in isolated rat brain capillaries by a confocal microscopy–based method. ATPase (enzymatic hydrolysis of adenosine triphosphate to inorganic phosphate) levels were measured in vitro. Western blotting determined P-gp and BCRP protein levels. Cell survival after GenX exposure was determined for two human cell lines.Results:Nanomolar levels of GenX inhibited P-gp and BCRP but not MRP2 transport activities in male and female rat brain capillaries. P-gp transport activity returned to control levels after GenX removal. GenX did not reduce P-gp- or BCRP-associated ATPase activity in an in vitro transport assay system. Reductions of P-gp but not BCRP transport activity were blocked by a peroxisome proliferator–activated receptor () antagonist. GenX reduced P-gp and BCRP transport activity in human cells.Conclusion:In rats, GenX at rapidly (in 1–2 h) inhibited P-gp and BCRP transport activities at the BBB through different mechanisms. was required for the GenX effects on P-gp but not BCRP transport activity. https://doi.org/10.1289/EHP5884

Drug-drug interaction (DDI) potential of oral napabucasin in healthy adults.

142 Background: Napabucasin is an NQO1-bioactivatable investigational agent hypothesized to affect multiple oncogenic cellular pathways including pSTAT3 through the generation of reactive oxygen species. This phase 1 open-label study evaluated the DDI potential of napabucasin and its major metabolite (M1) with respect to 7 major human drug cytochrome P450 (CYP) enzymes and the breast cancer resistance protein (BCRP) transporter. Methods: Healthy adult subjects who initially demonstrated they could tolerate administration of 240 or 480 mg twice daily (BID) napabucasin over 2-days (D) received single doses of the CYP and transporter substrates, followed by ≥7-day washout. In the DDI portion, subjects received napabucasin 240 mg BID on D1–11 with the phenotyping cocktail administered on D6 (omeprazole [CYP2C19] 20 mg, caffeine [CYP1A2] 100 mg, flurbiprofen [CYP2C9] 50 mg, bupropion [CYP2B6] 150 mg, dextromethorphan [CYP2D6] 30 mg, and oral midazolam [CYP3A] 2 mg), intravenous (IV) midazolam 2 mg on D7, repaglinide (CYP2C8) 0.25 mg on D8, and rosuvastatin (BRCP transporter) 10 mg on D9. Results: DDI potential was evaluated in 17 subjects. Exposure to omeprazole, flurbiprofen, and oral midazolam with (test) or without (reference) napabucasin 240 mg BID were similar. Napabucasin increased exposure (area under the curve to infinity) to caffeine (124%), IV midazolam (118%), repaglinide (127%), and rosuvastatin (213%); and decreased exposure to bupropion (79%) and dextromethorphan (71%). None of these changes were expected to be clinically meaningful. The exposure of the major metabolites of the probe drugs with or without napabucasin or M1 were similar. Of the 17 subjects, 12 (70.6%) reported adverse events (AEs); 58.8% reported gastrointestinal disorders. One patient had a grade 3 AE (neutrophil count low); no serious AEs were observed. Conclusions: The data suggest minimal in vivo DDI potential for napabucasin with respect to 7 major human drug CYP enzymes and the BCRP transporter. Napabucasin 240 mg BID was generally tolerable in healthy subjects. Co-administration of napabucasin with CYP and transporter substrates was safe and tolerable. Clinical trial information: NCT03411122.

Proton Pump Inhibitors Associated with Higher Incidence of Mucositis in Patients Receiving Methotrexate for Graft Vs Host Disease Prophylaxis

<h3>Introduction</h3> Graft-versus-host disease (GVHD) is a major complicating factor in determining morbidity and mortality following allogeneic stem cell transplant. To prevent GVHD, our institution administers calcineurin inhibitors and low doses of methotrexate (MTX) (5-15 mg/m²) on days +1, +3, +6, +11 for GVHD prophylaxis. Proton pump inhibitors (PPIs) are used for the treatment of gastroesophageal reflux disease, pyrosis, and/or peptic ulcer disease. PPIs have been shown to inhibit the ATP-binding cassette drug transporter Breast Cancer Resistance Protein's (BCRP/ABCG2) ability to excrete methotrexate into the urine in a concentration-dependent manner. Delayed excretion of methotrexate can lead to adverse events such as renal impairment, increased severity of mucositis, and increased infection rates. <h3>Objectives</h3> The purpose of this retrospective, single center study is to assess the clinical outcomes of patients receiving MTX and PPIs compared to MTX and no PPI for GVHD prophylaxis. <h3>Methods</h3> A single-site retrospective analysis of patients 18 years of age or older who underwent allogeneic stem cell transplantation from 2013 to 2018. Only patients that received methotrexate for GVHD prophylaxis were included. Cases were identified from clinical databases and were divided into two cohorts, those on PPIs, and those not on PPIs (no-PPI). The primary endpoint was grade of mucositis in each cohort. Secondary endpoints were total parenteral nutrition (TPN) use, need for patient controlled analgesia (PCA), time to engraftment of absolute neutrophil count (ANC) and hospital length of stay (LOS). <h3>Results</h3> 284 cases were identified, 119 (42%) patients were on a PPI at the time of MTX administration. The median age of patients on PPI was younger than those not on PPI (60 vs 54 p <0.0001). There were no differences in sex, BSA, conditioning regimen intent, dose of MTX, degree of match, or stem cell source. Patients on PPI had a higher incidence of grade 1-4 mucositis compared to no-PPI (Table 1) and a higher percentage of grade 3-4 mucositis (Figure 1). Patients on PPIs had a higher incidence of TPN use and need for PCA. Use of PPI did not increase the time to ANC engraftment or LOS. <h3>Conclusions</h3> The concurrent use of PPIs and MTX was associated with a higher incidence and severity of mucositis with increased need for PCA, and TPN. This study was limited by retrospective review. Grading of mucositis was based on clinician assessment and documentation. This study raises the question if PPIs should be discontinued or patients transitioned to a histamine blocker prior to MTX administration to improve severity of mucositis and reduce costs associated with TPN/PCA use.

Evaluation of the effect of P-glycoprotein inhibition and induction on talazoparib disposition in patients with advanced solid tumours.

AimsIn vitro data show that talazoparib is a substrate for P‐glycoprotein (P‐gp) and breast cancer resistance protein transporters. This open‐label, 2‐arm, drug–drug interaction Phase 1 study in patients with advanced solid tumours assessed the effect of a P‐gp inhibitor (itraconazole) and a P‐gp inducer (rifampicin) on the pharmacokinetics of a single dose of talazoparib. The safety and tolerability of a single dose of talazoparib with and without itraconazole or rifampicin were also assessed.MethodsThirty‐six patients were enrolled (Arm A [itraconazole], n = 19; Arm B [rifampicin], n = 17). Patients in both arms received 2 single oral doses of talazoparib (0.5 mg, Arm A; 1 mg, Arm B) alone and with multiple daily oral doses of itraconazole (Arm A) or rifampicin (Arm B).ResultsCoadministration of itraconazole and talazoparib increased talazoparib area under the plasma concentration–time profile from time 0 extrapolated to infinity by ~56% and maximum observed plasma concentration by ~40% relative to talazoparib alone. Coadministration of rifampicin and talazoparib increased talazoparib maximum observed plasma concentration by approximately 37% (geometric mean ratio 136.6% [90% confidence interval 103.2–180.9]); area under the curve was not affected relative to talazoparib alone (geometric mean ratio 102.0% [90% confidence interval 94.0–110.7]). Talazoparib had an overall safety profile consistent with that observed in prior studies in which talazoparib was administered as a single dose.ConclusionCoadministration of itraconazole increased talazoparib plasma exposure compared to talazoparib alone. A reduced talazoparib dose is recommended if coadministration of potent P‐gp inhibitors cannot be avoided. Similar exposure was observed when talazoparib was administered alone and with rifampicin suggesting that the effect of rifampicin on talazoparib exposure is limited.

Abundant Expression of OCT2, MATE1, OAT1, OAT3, PEPT2, BCRP, MDR1, and xCT Transporters in Blood-Arachnoid Barrier of Pig and Polarized Localizations at CSF- and Blood-Facing Plasma Membranes.

The physiologic and pharmacologic roles of the blood-arachnoid barrier (BAB) remain unclear. Therefore, the purpose of the present study was to comprehensively evaluate and compare the absolute protein expression levels of transporters in the leptomeninges and plexus per cerebrum, and to determine the localizations of transporters at the cerebrospinal fluid (CSF)-facing and blood (dura)-facing plasma membranes of the BAB in pig. Using multidrug resistance protein 1 (MDR1) and organic anion transporter (OAT) 1 as blood (dura)-facing and CSF-facing plasma membrane marker proteins, respectively, we established that breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP) 4, organic anion-transporting polypeptide (OATP) 2B1, multidrug and toxin extrusion protein 1 (MATE1), and glucose transporter 1 (GLUT1) are localized at the blood-facing plasma membrane, and OAT3, peptide transporter (PEPT) 2, MRP3, organic cation transporter (OCT) 2, xCT, monocarboxylate transporter (MCT) 1, MCT4, and MCT8 are localized at the CSF-facing plasma membrane of the BAB. The absolute protein expression levels of OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc in the whole BAB surrounding the entire cerebrum were much larger than those in the total of the choroid plexuses forming the blood-cerebrospinal fluid barrier (BCSFB). Although MRP4, OATP2B1, MCT8, GLUT1, and MCT1 were also statistically significantly more abundant in the BAB than in the choroid plexuses per porcine cerebrum, these transporters were nevertheless almost equally distributed between the two barriers. In contrast, OATP1A2, MRP1, OATP3A1, and OCTN2 were specifically expressed in the choroid plexus. These results should be helpful in understanding the relative overall importance of transport at the BAB compared with that at the BCSFB, as well as the rank order of transport capacities among different transporters at the BAB, and the directions of transport mediated by individual transporters. SIGNIFICANCE STATEMENT: We found that BCRP, MRP4, OATP2B1, MATE1, and GLUT1 localize at the blood-facing plasma membrane of the blood-arachnoid barrier (BAB), while OAT3, PEPT2, MRP3, OCT2, xCT, MCT1, MCT4, and MCT8 localize at the CSF-facing plasma membrane. 4F2hc is expressed in both membranes. For OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc, the absolute protein expression levels in the whole BAB surrounding the entire cerebrum are much greater than the total amounts in the choroid plexuses.

Proton Pump Inhibitors Associated with Higher Incidence of Mucositis in Patients Receiving Methotrexate for Graft Vs Host Disease Prophylaxis

Introduction- Graft-versus-host disease (GVHD) is a major complicating factor in determining morbidity and mortality following allogeneic stem cell transplant. To prevent GVHD, our institution administers calcineurin inhibitors and low doses of methotrexate (MTX) (5-15 mg/m2) on days +1, +3, +6, +11 for GVHD prophylaxis. While MTX is an efficacious agent in preventing GVHD, one of the undesirable adverse effects, which can be a dose limiting toxicity, is mucositis. Proton pump inhibitors (PPIs) are used for the treatment of gastroesophageal reflux disease, pyrosis, and/or peptic ulcer disease. PPIs have been shown to inhibit the ATP-binding cassette drug transporter Breast Cancer Resistance Protein's (BCRP/ABCG2) ability to excrete methotrexate into the urine in a concentration-dependent manner. Delayed excretion of methotrexate can lead to adverse events such as renal impairment, increased severity of mucositis, and increased infection rates. High dose MTX, defined as 500mg/m2 or more, has been shown to have delayed excretion when administered concomitantly with PPIs. The purpose of this retrospective, single center study is to assess the clinical outcomes of patients receiving low dose MTX and PPIs compared to MTX and no PPI for GVHD prophylaxis. Patients and Methods- A single-site institutional review board exempt retrospective analysis of patients 18 years of age or older who underwent allogeneic stem cell transplantation from 2013 to 2018 at our institution. Only patients that received methotrexate for GVHD prophylaxis were included. Cases were identified from clinical databases at our institution and were divided into two cohorts, those on PPIs, and those not on PPIs (no-PPI). Chart review was performed for the clinical variables of interest. The primary endpoint was grade of mucositis in each cohort. Secondary endpoints were total parenteral nutrition (TPN) use, need for patient controlled analgesia (PCA), leucovorin use, time to engraftment of absolute neutrophil count (ANC) and hospital length of stay (LOS). Lab values for analysis were assessed at admission and days +1/+5/+15. Results- 284 cases were identified, 119 (42%) patients were on a PPI at the time of MTX administration. The median age of patients on PPI was younger than those not on PPI (60 vs 54 p &lt;0.0001). There were no differences in sex, BSA, conditioning regimen intent (ie myeloablative vs reduced intensity), dose of MTX, degree of match, or stem cell source. Elevation in serum creatinine and liver function tests were similar in both cohorts prior to and during MTX administration. Patients on PPI had a higher incidence of grade 1-4 mucositis compared to no-PPI (Table 1) and a higher percentage of grade 3-4 mucositis (Figure 1). Patients on PPIs had a higher incidence of TPN use, need for PCA, but no increase in hospital LOS. Patients on PPI had higher use of leucovorin than those not on PPI (81.2% vs 58.2% p &lt;0.0001). Use of PPI did not increase the time of ANC engraftment. Conclusions- The concurrent use of PPIs and MTX was associated with a higher incidence and severity of mucositis. PPIs were also shown to increase the need for PCA, and TPN. PPI use did not increase the average length of hospitalization. This study was limited by retrospective review. Grading of mucositis was based on clinician assessment and documentation. While the indications for PPI were not readily known, this study raises the question if PPIs should be discontinued or patients transitioned to a histamine blocker prior to MTX administration to improve severity of mucositis and reduce costs associated with TPN/PCA use. Additional studies are needed in order to further characterize the association of PPI with MTX and its impact on clinical outcomes. Disclosures No relevant conflicts of interest to declare.