Effect of midostaurin on the pharmacokinetics of P-gp, BCRP, and CYP2D6 substrates: assessing potential drug-drug interactions in healthy participants : Brief title: Drug-drug interaction of midostaurin.

PURPOSE: Midostaurin, approved for FLT3-mutated acute myeloid leukemia and advanced systemic mastocytosis, is mainly metabolized by cytochrome P450 (CYP) 3A4. Midostaurin exhibited potential inhibitory effects on P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion-transporting polyprotein 1B1, and CYP2D6 in in vitro studies. This study investigated the pharmacokinetic (PK) effects of midostaurin on P-gp (digoxin), BCRP (rosuvastatin) and CYP2D6 (dextromethorphan) substrates in healthy adults. METHODS: This was an open-label, single-sequence, phase I clinical study evaluating the effect of single-dose midostaurin (100 mg) on the PK of digoxin and rosuvastatin (Arm 1), and dextromethorphan (Arm 2). Participants were followed up for safety 30 days after last dose. In addition, the effect of midostaurin on the PK of dextromethorphan metabolite (dextrorphan) was assessed in participants with functional CYP2D6 genes in Arm 2. RESULTS: The effect of midostaurin on digoxin was minor and resulted in total exposure (AUC) and peak plasma concentration (Cmax) that were only 20% higher. The effect on rosuvastatin was mild and led to an increase in AUCs of approximately 37-48% and of 100% in Cmax. There was no increase in the primary PK parameters (AUCs and Cmax) of dextromethorphan in the presence of midostaurin. The study treatments were very well tolerated with no occurance of severe adverse events (AEs), AEs of grade ≥ 2, or deaths. CONCLUSION: Midostaurin showed only a minor inhibitory effect on P-gp, a mild inhibitory effect on BCRP, and no inhibitory effect on CYP2D6. Study treatments were well tolerated in healthy adults.

Knockout Transporter Cell Lines to Assess Substrate Potential Towards Efflux Transporters.

P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance transporter 2 (MRP2) are efflux transporters involved in the absorption, excretion, and distribution of drugs. Bidirectional cell assays are recognized models for evaluating the potential of new drugs as substrates or inhibitors of efflux transporters. However, the assays are complicated by a lack of selective substrates and/or inhibitors, as well simultaneous expression of several efflux transporters in cell lines used in efflux models. This project aims to evaluate an in vitro efflux cell assay employing model substrates and inhibitors of P-gp, BCRP and MRP2 with knockout (KO) cell lines. The efflux ratios (ER) of P-gp (digoxin, paclitaxel), BCRP (prazosin, rosuvastatin), MRP2 (etoposide, olmesartan) and mixed (methotrexate, mitoxantrone) substrates were determined in wild-type C2BBe1 and KO cells. For digoxin and paclitaxel, the ER decreased to less than 2 in the cell lines lacking P-gp expression. The ER decreased to less than 3 for prazosin and less than 2 for rosuvastatin in the cell lines lacking BCRP expression. For etoposide and olmesartan, the ER decreased to less than 2 in the cell lines lacking MRP2 expression. The ER of methotrexate and mitoxantrone decreased in single- and double-KO cells without BCRP and MRP2 expression. These results show that KO cell lines have the potential to better interpret complex drug-transporter interactions without depending upon multi-targeted inhibitors or overlapping substrates. For drugs that are substrates of multiple transporters, the single- and double-KO cells may be used to assess their affinities for the different transporters.

The role of drug efflux and uptake transporters in the plasma pharmacokinetics and tissue disposition of morphine and its main metabolites.

Morphine is a widely used opioid for the treatment of pain. Differences in drug transporter expression and activity may contribute to variability in morphine pharmacokinetics and response. Using appropriate mouse models, we investigated the impact of the efflux transporters ABCB1 and ABCG2 and the OATP uptake transporters on the pharmacokinetics of morphine, morphine-3-glucuronide (M3G), and M6G. Upon subcutaneous administration of morphine, its plasma exposure in Abcb1a/1b-/-;Abcg2-/--, Abcb1a/1b-/-;Abcg2-/-;Oatp1a/1b-/-;Oatp2b1-/- (Bab12), and Oatp1a/1b-/-;Oatp2b1-/- mice was similar to that found in wild-type mice. Forty minutes after dosing, morphine brain accumulation increased by 2-fold when mouse (m)Abcb1 and mAbcg2 were ablated. Relative recovery of morphine in small intestinal content was significantly reduced in all the knockout strains. In the absence of mOatp1a/1b and mOatp2b1, plasma levels of M3G were markedly increased, suggesting a lower elimination rate. Moreover, Oatp-deficient mice displayed reduced hepatic and intestinal M3G accumulation. Mouse Oatps similarly affected plasma and tissue disposition of subcutaneously administered M6G. Human OATP1B1/1B3 transporters modestly contribute to the liver accumulation of M6G. In summary, mAbcb1, in combination with mAbcg2, limits morphine brain penetration and its net intestinal absorption. Variation in ABCB1 activity due to genetic polymorphisms/mutations and/or environmental factors might, therefore, partially affect morphine tissue exposure in patients. The ablation of mOatp1a/1b increases plasma exposure and decreases the liver and small intestinal disposition of M3G and M6G. Since the contribution of human OATP1B1/1B3 to M6G liver uptake was quite modest, the risks of undesirable drug interactions or interindividual variation related to OATP activity are likely negligible.

The placenta in fetal death: molecular evidence of dysregulation of inflammatory, proliferative, and fetal protective pathways.

BACKGROUND: It is estimated that over 2 million cases of fetal death occur worldwide every year, but, despite the high incidence, several basic and clinical characteristics of this disorder are still unclear. Placenta is suggested to play a central role in fetal death. Placenta produces hormones, cytokines and growth factors that modulate functions of the placental-maternal unit. Fetal death has been correlated with impaired secretion of some of these regulatory factors. OBJECTIVE: The aim of the present study was to evaluate, in placentas collected from fetal death, the gene expression of inflammatory, proliferative and protective factors. STUDY DESIGN: Cases of fetal death in singleton pregnancy were retrospectively selected, excluding pregnancies complicated by fetal anomalies, gestational diabetes, intrauterine growth restriction and moderate to severe maternal diseases. A group of placentas collected from healthy singleton term pregnancies were used as controls. Groups were compared regarding maternal and gestational age, fetal sex and birthweight. Placental messenger RNA expression of inflammatory (interleukin 6), proliferative (activin A, transforming growth factor ß1) and regulatory (vascular endothelial growth factor, vascular endothelial growth factor receptor 2, ATP-binding cassette transporters (ABC) ABCB1 and ABCG2, sphingosine 1-phosphate signaling pathway) markers was conducted using real-time polymerase chain reaction. Statistical analysis and graphical representation of the data were performed using the GraphPad Prism 5 software. For the statistical analysis, Student's t test was used, and P values<.05 were considered significant. RESULTS: Placental mRNA expression of interleukin 6 and vascular endothelial growth factor receptor 2 resulted significantly higher in the fetal death group compared to controls (P<.01), while activin A, ABCB1, and ABCG2 expression resulted significantly lower (P<.01). A significant alteration in the sphingosine 1-phosphate signaling pathway was found in the fetal death group, with an increased expression of the specific receptor isoforms sphingosine 1-phosphate receptor 1, 3, and 4 (sphingosine 1-phosphate1, sphingosine 1-phosphate3, sphingosine 1-phosphate4) and of sphingosine kinase 2, 1 of the enzyme isoforms responsible for sphingosine 1-phosphate synthesis (P<.01). CONCLUSION: The present study confirmed a significantly increased expression of placental interleukin 6 and vascular endothelial growth factor receptor 2 mRNA, and for the first time showed an increased expression of sphingosine 1-phosphate receptors and sphingosine kinase 2 as well as a decreased expression of activin A and of selected ATP-binding cassette transporters, suggesting that multiple inflammatory and protective factors are deranged in placenta of fetal death.

Applicability of MDR1 Overexpressing Abcb1KO-MDCKII Cell Lines for Investigating In Vitro Species Differences and Brain Penetration Prediction.

Implementing the 3R initiative to reduce animal experiments in brain penetration prediction for CNS-targeting drugs requires more predictive in vitro and in silico models. However, animal studies are still indispensable to obtaining brain concentration and determining the prediction performance of in vitro models. To reveal species differences and provide reliable data for IVIVE, in vitro models are required. Systems overexpressing MDR1 and BCRP are widely used to predict BBB penetration, highlighting the impact of the in vitro system on predictive performance. In this study, endogenous Abcb1 knock-out MDCKII cells overexpressing MDR1 of human, mouse, rat or cynomolgus monkey origin were used. Good correlations between ERs of 83 drugs determined in each cell line suggest limited species specificities. All cell lines differentiated CNS-penetrating compounds based on ERs with high efficiency and sensitivity. The correlation between in vivo and predicted Kp,uu,brain was the highest using total ER of human MDR1 and BCRP and optimized scaling factors. MDR1 interactors were tested on all MDR1 orthologs using digoxin and quinidine as substrates. We found several examples of inhibition dependent on either substrate or transporter abundance. In summary, this assay system has the potential for early-stage brain penetration screening. IC50 comparison between orthologs is complex; correlation with transporter abundance data is not necessarily proportional and requires the understanding of modes of transporter inhibition.

In vitro screening model for compound interactions with human and dairy animal BCRP orthologs.

Orthologs of breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) efflux transmembrane transporter, are present in several species. The list of compounds known to interact with BCRP is growing, and many questions remain concerning species-specific variations in substrate specificity and affinity and the potency of inhibitors. As the most abundant efflux transporter known to be present in the blood-milk barrier, BCRP can increase the elimination of certain xenobiotics to milk, posing a risk for suckling offspring and dairy product consumers. Here we developed a model that can be employed to investigate species-specific differences between BCRP substrates and inhibitors. Membrane vesicles were isolated from transiently transduced human embryonic kidney (HEK) 293 cells, overexpressing BCRP, with human, bovine, caprine, and ovine cDNA sequences. To confirm BCRP transport activity in the transduced cells, D-luciferin efflux was measured and to confirm transport activity in the membrane vesicles, [3H] estrone-3-sulfate ([3H]E1S) influx was measured. We also determined the Michaelis-Menten constant (Km) and Vmax of [3H]E1S for each species. We have developed an in vitro transport model to study differences in compound interactions with BCRP orthologs from milk-producing animal species and humans. BCRP transport activity was demonstrated in the species-specific transduced cells by a reduced accumulation of D-luciferin compared with the control cells, indicating BCRP-mediated efflux of D-luciferin. Functionality of the membrane vesicle model was demonstrated by confirming ATP-dependent transport and by quantifying the kinetic parameters, Km and Vmax for the model substrate [3H]E1S. The values were not significantly different between species for the model substrates tested. This model can be insightful for appropriate inter-species extrapolations and risk assessments of xenobiotics in lactating woman and dairy animals.

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.

Hypoxia modulates P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) drug transporters in brain endothelial cells of the developing human blood-brain barrier.

P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) multidrug resistance (MDR) transporters are localized at the luminal surface of the blood-brain barrier (BBB). They confer fetal brain protection against harmful compounds that may be circulating in the peripheral blood. The fetus develops in low oxygen levels; however, some obstetric pathologies such as pre-eclampsia, placenta accreta/previa may result in even greater fetal hypoxic states. We investigated how hypoxia impacts MDR transporters in human fetal brain endothelial cells (hfBECs) derived from early and mid-stages of pregnancy. Hypoxia decreased BCRP protein and activity in hfBECs derived in early pregnancy. In contrast, in hfBECs derived in mid-pregnancy there was an increase in P-gp and BCRP activity following hypoxia. Results suggest a hypoxia-induced reduction in fetal brain protection in early pregnancy, but a potential increase in transporter-mediated protection at the BBB during mid-gestation. This would modify accumulation of various key physiological and pharmacological substrates of P-gp and BCRP in the developing fetal brain and potentially contribute to the pathogenesis of neurodevelopmental disorders commonly associated with in utero hypoxia.

Multidrug resistance transporters P-gp and BCRP limit the efficacy of ATR inhibitor ceralasertib in cancer cells.

The therapeutic effect of chemotherapy and targeted therapy are known to be limited by drug resistance. Substantial evidence has shown that ATP-binding cassette (ABC) transporters P-gp and BCRP are significant contributors to multidrug resistance (MDR) in cancer cells. In this study, we demonstrated that a clinical-staged ATR inhibitor ceralasertib is susceptible to P-gp and BCRP-mediated MDR. The drug resistant cancer cells were less sensitive to ceralasertib compared to the parental cells. Moreover, ceralasertib resistance can be reversed by inhibiting the drug efflux activity of P-gp and BCRP. Interestingly, ceralasertib was able to downregulate the level of P-gp but not BCRP, suggesting a potential regulation between ATR signaling and P-gp expression. Furthermore, computational docking analysis predicted high affinities between ceralasertib and the drug-binding sites of P-gp and BCRP. In summary, overexpression of P-gp and BCRP are sufficient to confer cancer cells resistance to ceralasertib, underscoring their role as biomarkers for therapeutic efficacy.

Exposure to Low-Frequency Radiation Changes the Expression of Nestin, VEGF, BCRP and Apoptosis Markers During Glioma Treatment Strategy: An In Vitro Study.

BACKGROUND: Exposure to physical contamination during chemotherapy, including non-ionizing electromagnetic fields, raises concerns about the widespread sources of exposure to this type of radiation. Glioblastoma multiforme (GBM) is an aggressive central nervous system tumor that is hard to treat due to resistance to drugs such as temozolomide (TMZ). OBJECTIVE: Electromagnetic fields (EMF) and haloperidol (HLP) may have anticancer effects. In this study, we investigated the effects of TMZ, HLP, and EMF on GBM cell lines and analyzed the association between non-ionizing radiation and the risk of change in drug performance. METHODS: Cell viability and reactive oxygen species (ROS) generation were measured by MTT and NBT assay, respectively. Then, the expression levels of breast cancer-resistant protein (BCRP), Bax, Bcl2, Nestin, vascular endothelial growth factor (VEGF) genes, and P53, Bax, and Bcl2 Proteins were evaluated by real-time PCR and western blot. RESULTS: Co-treatment of GBM cells by HLP and TMZ enhanced apoptosis in T-98G and A172 cells by increasing the expression of P53 and Bax and decreasing Bcl-2. Interestingly, exposure of GBM cells to EMF decreased apoptosis in the TMZ+HLP group. CONCLUSION: In conclusion, EMF reduced the synergistic effect of TMZ and HLP. This hypothesis that patients who are treated for brain tumors and suffer from depression should not be exposed to EMF is proposed in the present study. There appears to be an urgent need to reconsider exposure limits for low-frequency magnetic fields, based on experimental and epidemiological research, the relationship between exposure to non-ionizing radiation and adverse human health effects.

The role of membrane transporters in the absorption of atrazine following nasal exposure.

OBJECTIVE: The purpose of these studies was to investigate the uptake of atrazine across the nasal mucosa to determine whether direct transport to the brain through the olfactory epithelium is likely to occur. These studies were undertaken to provide important new information about the potential for the enhanced neurotoxicity of herbicides following nasal inhalation. MATERIALS AND METHODS: Transport of atrazine from aqueous solution and from commercial atrazine-containing herbicide products was assessed using excised nasal mucosal tissues. The permeation rate and the role of membrane transporters in the uptake of atrazine across the nasal mucosa were also investigated. Histological examination of the nasal tissues was conducted to assess the effects of commercial atrazine-containing products on nasal tissue morphology. RESULTS: Atrazine showed high flux across both nasal respiratory and olfactory tissues, and efflux transporters were found to play an essential role in limiting its uptake at low exposure concentrations. Commercial atrazine-containing herbicide products showed remarkably high transfer across the nasal tissues, and histological evaluation showed significant changes in the morphology of the nasal epithelium following exposure to the herbicide products. DISCUSSION: Lipophilic herbicides such as atrazine can freely permeate across the nasal mucosa despite the activity of efflux transporters. The adjuvant compounds in commercial herbicide products disrupt the nasal mucosa's epithelial barrier, resulting in even greater atrazine permeation across the tissues. The properties of the herbicide itself and those of the formulated products play crucial roles in the potential for the enhanced neurotoxicity of herbicides following nasal inhalation.

Effect of Antioxidants in Medicinal Products on Intestinal Drug Transporters.

The presence of mutagenic and carcinogenic N-nitrosamine impurities in medicinal products poses a safety risk. While incorporating antioxidants in formulations is a potential mitigation strategy, concerns arise regarding their interference with drug absorption by inhibiting intestinal drug transporters. Our study screened thirty antioxidants for inhibitory effects on key intestinal transporters-OATP2B1, P-gp, and BCRP in HEK-293 cells (OATP2B1) or membrane vesicles (P-gp, BCRP) using 3H-estrone sulfate, 3H-N-methyl quinidine, and 3H-CCK8 as substrates, respectively. The screen identified that butylated hydroxyanisole (BHA) and carnosic acid inhibited all three transporters (OATP2B1, P-gp, and BCRP), while ascorbyl palmitate (AP) inhibited OATP2B1 by more than 50%. BHA had IC50 values of 71 ± 20 µM, 206 ± 14 µM, and 182 ± 49 µM for OATP2B1, BCRP, and P-gp, respectively. AP exhibited IC50 values of 23 ± 10 µM for OATP2B1. The potency of AP and BHA was tested with valsartan, an OATP2B1 substrate, and revealed IC50 values of 26 ± 17 µM and 19 ± 11 µM, respectively, in HEK-293-OATP2B1 cells. Comparing IC50 values of AP and BHA with estimated intestinal concentrations suggests an unlikely inhibition of intestinal transporters at clinical concentrations of drugs formulated with antioxidants.

One-step synthesis of diaryloxadiazoles as potent inhibitors of BCRP.

Aim: BCRP plays a major role in the efflux of cytotoxic molecules, limiting their antiproliferative activity. We aimed to design and synthesize new BCRP inhibitors to render cancerous tumors more sensitive toward anticancer agents. Materials & methods: Based on our previous work, we conceived potential BCRP inhibitors derived from 1,3,4-oxadiazoles bearing two substituted phenyl rings. Results: Evaluating 19 derivatives, we found that 2,5-diaryl-1,3,4-oxadiazoles possessing methoxy groups were the most active. The highest activity was recorded with derivatives bearing three methoxy groups. The most active compound (3j) was selective in inhibiting BCRP and nontoxic as evidenced by cellular tests. Conclusion: 3j is a promising BCRP inhibitor thanks to its synthetic accessibility and biological profile.

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Membrane-Fusing Vehicles for Re-Sensitizing Transporter-Mediated Multiple-Drug Resistance in Cancer.

Reversing the multiple drug resistance (MDR) arising from the overexpression of the efflux transporters often fails mainly due to the high toxicity or the poor water solubility of the inhibitors of these transporters. Here, we demonstrate the delivery of an inhibitor targeting three ABC transporters (ABCB1, ABCC1 and ABCG2) directly to the cell membrane using membrane-fusing vehicles (MFVs). Three different transfected MDCK II cell lines, along with parental cells, were used to investigate the inhibitory effect of cyclosporine A (CsA) in solution versus direct delivery to the cell membrane. CsA-loaded MFVs successfully reversed MDR for all three investigated efflux transporters at significantly lower concentrations compared with CsA in solution. Results showed a 15-fold decrease in the IC50 value for ABCB1, a 7-fold decrease for ABCC1 and an 11-fold decrease for ABCG2. We observed binding site specificity for ABCB1 and ABCG2 transporters. Lower concentrations of empty MFVs along with CsA contribute to the inhibition of Hoechst 33342 efflux. However, higher concentrations of CsA along with the high amount of MFVs activated transport via the H-binding site. This supports the conclusion that MFVs can be useful beyond their role as delivery systems and also help to elucidate differences between these transporters and their binding sites.

Experimental and Computational Methods to Assess Central Nervous System Penetration of Small Molecules.

In CNS drug discovery, the estimation of brain exposure to lead compounds is critical for their optimization. Compounds need to cross the blood-brain barrier (BBB) to reach the pharmacological targets in the CNS. The BBB is a complex system involving passive and active mechanisms of transport and efflux transporters such as P-glycoproteins (P-gp) and breast cancer resistance protein (BCRP), which play an essential role in CNS penetration of small molecules. Several in vivo, in vitro, and in silico methods are available to estimate human brain penetration. Preclinical species are used as in vivo models to understand unbound brain exposure by deriving the Kp,uu parameter and the brain/plasma ratio of exposure corrected with the plasma and brain free fraction. The MDCK-mdr1 (Madin Darby canine kidney cells transfected with the MDR1 gene encoding for the human P-gp) assay is the commonly used in vitro assay to estimate compound permeability and human efflux. The in silico methods to predict brain exposure, such as CNS MPO, CNS BBB scores, and various machine learning models, help save costs and speed up compound discovery and optimization at all stages. These methods enable the screening of virtual compounds, building of a CNS penetrable compounds library, and optimization of lead molecules for CNS penetration. Therefore, it is crucial to understand the reliability and ability of these methods to predict CNS penetration. We review the in silico, in vitro, and in vivo data and their correlation with each other, as well as assess published experimental and computational approaches to predict the BBB penetrability of compounds.

New Insights into Chemoresistance Mediated by Mdm2 Inhibitors: The Benefits of Targeted Therapy over Common Cytostatics.

The inhibition of the Mdm2-p53 protein-protein interaction is a promising strategy for anticancer therapy. However, the problem of developing secondary chemoresistance in tumors treated with such drugs has not yet been sufficiently studied. In this work, we compared the properties of a drug-resistant cell line obtained during long-term cultivation in the presence of an Mdm2 inhibitor, Nutlin-3a, with a similarly obtained line insensitive to the cytostatic drug paclitaxel. We first confirmed the higher safety levels of Mdm2 inhibitors when compared with cytostatics in terms of the development of secondary chemoresistance. We showed that Nutlin-3a affects both the targeted p53-mediated cellular machinery and the universal ABC-mediated efflux mechanism. While both targeted and general defense mechanisms are activated by the Mdm2 inhibitor, it still increases the susceptibility of tumor cells to other drugs. The results obtained indicate that the risks of developing chemoresistance under the therapy with a targeted agent are fundamentally lower than during cytotoxic therapy.

Roles of breast cancer resistance protein and organic anion transporting polypeptide 2B1 in gastrointestinal toxicity induced by SN-38 under inflammatory conditions.

Drug transporters are among the factors that determine the pharmacokinetic profiles after drug administration. In this study, we investigated the roles of drug transporters involved in transport of SN-38, which is an active metabolite of irinotecan, in the intestine under inflammatory conditions in vitro and determined their functional consequences. The expression alterations of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 2B1 were determined at the mRNA and protein levels, and the subsequent functional alterations were evaluated via an accumulation study with the representative transporter substrates [prazosin and dibromofluorescein (DBF)] and SN-38. We also determined the cytotoxicity of SN-38 under inflammatory conditions. Decreased BCRP expression and increased OATP2B1 expression were observed under inflammatory conditions in vitro, which led to altered accumulation profiles of prazosin, DBF, and SN-38, and the subsequent cytotoxic profiles of SN-38. Treatment with rifampin or novobiocin supported the significant roles of BCRP and OATP2B1 in the transport and cytotoxic profile of SN-38. Collectively, these results suggest that BCRP and OATP2B1 are involved in the increased cytotoxicity of SN-38 under inflammatory conditions in vitro. Further comprehensive research is warranted to completely understand SN-38-induced gastrointestinal cytotoxicity and aid in the successful treatment of cancer with irinotecan.

Evaluation of ABT-751, a novel anti-mitotic agent able to overcome multi-drug resistance, in melanoma cells.

PURPOSE: Drug efflux transporter associated multi-drug resistance (MDR) is a potential limitation in the use of taxane chemotherapies for the treatment of metastatic melanoma. ABT-751 is an orally bioavailable microtubule-binding agent capable of overcoming MDR and proposed as an alternative to taxane-based therapies. METHODS: This study compares ABT-751 to taxanes in vitro, utilizing seven melanoma cell line models, publicly available gene expression and drug sensitivity databases, a lung cancer cell line model of MDR drug efflux transporter overexpression (DLKP-A), and drug efflux transporter ATPase assays. RESULTS: Melanoma cell lines exhibit a low but variable protein and RNA expression of drug efflux transporters P-gp, BCRP, and MDR3. Expression of P-gp and MDR3 correlates with sensitivity to taxanes, but not to ABT-751. The anti-proliferative IC50 profile of ABT-751 was higher than the taxanes docetaxel and paclitaxel in the melanoma cell line panel, but fell within clinically achievable parameters. ABT-751 IC50 was not impacted by P-gp-overexpression in DKLP-A cells, which display strong resistance to the P-gp substrate taxanes compared to DLKP parental controls. The addition of ABT-751 to paclitaxel treatment significantly decreased cell proliferation, suggesting some reversal of MDR. ATPase activity assays suggest that ABT-751 is a potential BCRP substrate, with the ability to inhibit P-gp ATPase activity. CONCLUSION: Our study confirms that ABT-751 is active against melanoma cell lines and models of MDR at physiologically relevant concentrations, it inhibits P-gp ATPase activity, and it may be a BCRP and/or MDR3 substrate. ABT-751 warrants further investigation alone or in tandem with other drug efflux transporter inhibitors for hard-to-treat MDR melanoma.

Enhanced reversal of ABCG2-mediated drug resistance by replacing a phenyl ring in baicalein with a meta-carborane.

Success of chemotherapy is often hampered by multidrug resistance. One mechanism for drug resistance is the elimination of anticancer drugs through drug transporters, such as breast cancer resistance protein (BCRP; also known as ABCG2), and causes a poor 5-year survival rate of human patients. Co-treatment of chemotherapeutics and natural compounds, such as baicalein, is used to prevent chemotherapeutic resistance but is limited by rapid metabolism. Boron-based clusters as meta-carborane are very promising phenyl mimetics to increase target affinity; we therefore investigated the replacement of a phenyl ring in baicalein by a meta-carborane to improve its affinity towards the human ABCG2 efflux transporter. Baicalein strongly inhibited the ABCG2-mediated efflux and caused a fivefold increase in mitoxantrone cytotoxicity. Whereas the baicalein derivative 5,6,7-trimethoxyflavone inhibited ABCG2 efflux activity in a concentration of 5 µm without reversing mitoxantrone resistance, its carborane analogue 5,6,7-trimethoxyborcalein significantly enhanced the inhibitory effects in nanomolar ranges (0.1 µm) and caused a stronger increase in mitoxantrone toxicity reaching similar values as Ko143, a potent ABCG2 inhibitor. Overall, in silico docking and in vitro studies demonstrated that the modification of baicalein with meta-carborane and three methoxy substituents leads to an enhanced reversal of ABCG2-mediated drug resistance. Thus, this seems to be a promising basis for the development of efficient ABCG2 inhibitors.

Breast Cancer Resistance Protein Limits Fetal Transfer of Tadalafil in Mice.

Tadalafil, a phosphodiesterase 5 (PDE5) inhibitor, is a candidate therapeutic agent for fetal growth restriction and hypertensive disorders of pregnancy. In this study, we elucidated the fetal transfer of tadalafil in comparison with that of sildenafil, the first PDE5 inhibitor to be approved. We also examined the contributions of multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) to fetal transfer. Tadalafil or sildenafil was administered to wild-type, Mdr1a/b-double-knockout or Bcrp-knockout pregnant mice by continuous infusion from gestational day (GD) 14.5 to 17.5, and the fetal-to-maternal plasma concentration ratio of unbound drug (unbound F/M ratio) was evaluated at GD 17.5. The values of unbound F/M ratio of tadalafil and sildenafil in wild-type mice were 0.80 and 1.6, respectively. The unbound F/M ratio of tadalafil was increased to 1.1 and 1.7 in Mdr1a/b-knockout and Bcrp-knockout mice, respectively, while the corresponding values for sildenafil were equal to or less than that in wild-type mice, respectively. A transcellular transport study revealed that basal-to-apical transport of both tadalafil and sildenafil was significantly higher than transport in the opposite direction in MDCKII-BCRP cells. Our research reveals that tadalafil is a newly identified substrate of human and mouse BCRP, and it appears that the fetal transfer of tadalafil is, at least in part, attributed to the involvement of BCRP within the placental processes in mice. The transfer of sildenafil to the fetus was not significantly constrained by BCRP, even though sildenafil was indeed a substantial substrate for BCRP.