Pgp Substrate Research Articles

A novel delocalized lipophilic cation-chlorambucil conjugate inhibits P-glycoprotein in HepG2/ADM cells

Multidrug resistance (MDR) limits the application of a large number of cancer-fighting agents in clinical therapy. One reason is that P-glycoprotein (Pgp) efflux pumps are usually overexpressed and lead to drug efflux in the cancer cells, which limits the viability of many chemotherapeutics. Current available inhibitors which block the Pgp pump efflux are usually not widely used in clinical practice, because they change other drug pharmacokinetic profiles or increase side effects. Here, through covalent linkage of cancer-targeting delocalized lipophilic cation FF and DNA-damaging drug nitrogen mustard chlorambucil (CLB), we rationally designed and synthesized a tumor-targeting anticancer agent FFCLB. And we found and proved that the FFCLB was capable of reducing the outflow of Pgp substrates efficiently. This conjugate selectively improves adriamycin uptake and toxicity through reducing MDR1 mRNA and Pgp protein expression. Based on molecular targeted strategy, this study can facilitate the discovery of superior MDR reducing agents to provide a more effective and safer way of resensitizing MDR.

ELENBECESTAT PHARMACOKINETIC DRUG-DRUG INTERACTIONS INDICATED NO DOSAGE ADJUSTMENTS REQUIRED FOR MOST CONCOMITANT TREATMENTS

Elenbecestat (E2609) is a BACE inhibitor primarily metabolized by carboxylesterase 2 (CES2) and to lesser extent by CYP3A. Drug-drug interaction studies assessed the effects of itraconazole (strong CYP3A and CES2 inhibitor), rifampin (strong CYP3A inducer), and donepezil on elenbecestat pharmacokinetics, as well as the effects of elenbecestat on digoxin (Pgp substrate) pharmacokinetics. This open label study in healthy adult subjects was conducted in 3 parts. Part A: subjects received a single oral dose of elenbecestat 50 mg on day 1; from day 6 to 20, subjects received daily doses of either itraconazole 200 mg or rifampin 600 mg; with a second dose of elenbecestat 50 mg on day 14. Part B: subjects received digoxin 0.25 mg on day 1, elenbecestat 50 mg/day from day 7 to 20, and a second digoxin dose at day 14. Part C: a 3-way crossover study in which each subject received a single dose of elenbecestat 50 mg, elenbecestat 50 mg in combination with donepezil 10 mg, and elenbecestat 50 mg dosed 2 hours before donepezil 10 mg, according to a randomized treatment sequence with ≥21-day washout between treatments. Pharmacokinetic assessments of drugs, including elenbecestat and metabolites M1, M2, and M5, were conducted. Co-administration of elenbecestat and itraconazole resulted in increases in elenbecestat Cmax, AUC(0-inf), and t1/2 compared with administration of elenbecestat alone (Figures). The Cmax of all elenbecestat metabolites decreased with itraconazole co-administration. Co-administration of elenbecestat and rifampin resulted in relatively no change in Cmax or t1/2, a minor decrease in AUC(0-inf), along with pronounced decreases in M2 and M5. In Part B, elenbecestat and digoxin co-administration increased digoxin Cmax slightly, but no changes in overall systemic exposure (AUC) were observed. Co-administration of elenbecestat and donepezil at the same time resulted in non-clinically meaningful increases in Cmax and AUC(0-inf). However, no notable changes in elenbecestat exposure occurred when administered 2 hours before donepezil. Results indicate that itraconazole's interaction was primarily due to CES2 inhibition, therefore, no restrictions or dose adjustments are warranted for elenbecestat when co-administered with CYP3A inhibitors. Furthermore, no dose adjustments are necessary for elenbecestat when co-administered with Pgp substrates or donepezil. The effect of co-administered drugs on elenbecestat pharmacokinetics (expressed as 90% CI of geomean). Donepezil administered * concomitantly or **2 h postdose with elenbecestat Horizontal axis shows fold change in Cmax and AUC relative to control (elenbecestat alone). Note: Generic name elenbecestat is not fixed at this time. The effect of elenbecestat on pharmacokinetics of digoxin (expressed as 90% CI of geomean).

Effects of a novel photoactivated photosensitizer on MDR1 over-expressing human breast cancer cells

Multidrug resistance (MDR) was the main reason of cancer chemotherapy failure. Photodynamic therapy (PDT) has been applied to the treatment of tumor and considered as a strategy for the overcoming of MDR phenomenon. Present study focused on a novel porphyrin-based photosensitizer DTP (meso-5-[p-diethylene triamine pentaacetic acid-aminophenyl]-10,15,20-triphenyl-porphyrin)-mediated photocytotoxicity on MDR1 highly expressing human breast cancer cell line MCF-7/ADR (adriamycin resistant) and the parental MCF-7 cell line. Experimental results indicated that DTP-PDT induced significant photocytotoxicity on MDR1 highly expressing MCF-7/ADR cell line, in spite of slightly weaker than on MCF-7 cell line, which was due to the relatively lower level of intracellular DTP in resistant MCF-7/ADR cells. Furthermore, intracellular DTP level in resistant MCF-7/ADR cells could not be altered with a Pgp inhibitor, verapamil and this indicated that DTP was not a possible substrate for the multidrug transporter Pgp. More importantly, photoactivated DTP could significantly reduce the expression of MDR1 gene at all the levels of mRNA, protein and function. The combined treatment with DTP-PDT and adriamycin was found to be more effective than adriamycin or DTP-PDT alone. In conclusion, our data demonstrated that DTP probably will be a potential photosensitizer in combating MDR phenomenon during the treatment of human breast cancer.

A Prediction Method for P-glycoprotein–Mediated Drug–Drug Interactions at the Human Blood–Brain Barrier From Blood Concentration–Time Profiles, Validated With PET Data

The purpose of this study was to establish physiologically based pharmacokinetic models to predict in humans the brain concentration–time profiles and P-glycoprotein (Pgp)–mediated brain drug–drug interactions between the model Pgp substrate (R)-[11C]verapamil (VPM), the model dual Pgp/breast cancer resistance protein (BCRP) substrate [11C]tariquidar (TQD), and the Pgp inhibitor tariquidar. The model predictions were validated with results from positron emission tomography studies in humans. Using these physiologically based pharmacokinetic models, the differences between predicted and observed areas under the concentration–time curves (AUC) of VPM and TQD in the brain were within a 1.2-fold and 2.5-fold range, respectively. Also, brain AUC increases of VPM and TQD after Pgp inhibitor administration were predicted with 2.5-fold accuracy when in vitro inhibition constant or half-maximum inhibitory concentration values of tariquidar were used. The predicted rank order of the magnitude of AUC increases reflected the results of the clinical positron emission tomography studies. Our results suggest that the established models can predict brain exposure from the respective blood concentration–time profiles and rank the magnitude of the Pgp-mediated brain drug–drug interaction potential for both Pgp and Pgp/BCRP substrates in humans.

Oxidative Stress Promotes Doxorubicin-Induced Pgp and BCRP Expression in Colon Cancer Cells Under Hypoxic Conditions.

P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are ATP binding cassette (ABC) transporters that are overexpressed in different drug-resistant cancer cell lines. In this study, we investigated whether doxorubicin promotes Pgp and/or BCRP expression to induce drug resistance in colon cancer cells under hypoxic conditions. We analyzed HIF-1α activity via ELISA, Pgp, and BCRP expression by qRT-PCR and the relationship between doxorubicin uptake and ABC transporter expression via confocal microscopy in HT-29WT and HT-29 doxorubicin-resistant colon cancer cells (HT-29DxR). These cells were treated with doxorubicin and/or CoCl2 (chemical hypoxia), and reactive oxygen species inductors. We found that the combination of chemically induced hypoxia and doxorubicin promoted Pgp mRNA expression within 24 h in HT-29WT and HT-29DxR cells. Both doxorubicin and CoCl2 alone or in combination induced Pgp and BCRP expression, as demonstrated via confocal microscopy in each of the above two cell lines. Thus, we surmised that Pgp and BCRP expression may result from synergistic effects exerted by the combination of doxorubicin-induced ROS production and HIF-1α activity under hypoxic conditions. However, HIF-1α activity disruption via the administration of E3330, an APE-1 inhibitor, downregulated Pgp expression and increased doxorubicin delivery to HT-29 cells, where it served as a substrate for Pgp, indicating the existence of an indirect relationship between Pgp expression and doxorubicin accumulation. Thus, we concluded that Pgp and BCRP expression can be regulated via cross-talk between doxorubicin and hypoxia, promoting drug resistance in HT-29 WT, and HT-29DxR cells and that this process may be ROS dependent. J. Cell. Biochem. 118: 1868-1878, 2017. © 2017 Wiley Periodicals, Inc.

Molecular Mimics of Classic P-Glycoprotein Inhibitors as Multidrug Resistance Suppressors and Their Synergistic Effect on Paclitaxel.

P-glycoprotein (Pgp) is a membrane bound efflux pump spread in a variety of tumor cells and considered as a main component of multidrug resistance (MDR) to chemotherapies. In this work, three groups of compounds (imidazolone, oxazolone and vinyl dipeptide derivatives) were synthesized aiming to develop a molecular framework that effectively suppresses MDR. When tested for their influence on Pgp activity, four compounds coded Cur1-01, Cur1-12V, Curox-1 and Curox-3 significantly decreased remaining ATP concentration indicating Pgp substrate site blocking. On the other hand, Cur-3 and Cur-10 significantly increased remaining ATP concentration, which is indicative of Pgp ATPase inhibition. The cytotoxicity of synthesized compounds was examined against Pgp expressing/highly resistant colorectal cancer cell lines (LS-174T). Compounds Cur-1 and Cur-3 showed considerable cytotoxicity with IC50 values of 7.6 and 8.9 μM, respectively. Equitoxic combination (at IC50 concentrations) of PTX and Cur-3 greatly diminished resistant cell clone from 45.7% to 2.5%, albeit with some drop in potency from IC50 of 7.9 nM to IC50 of 23.8 nM. On the other hand, combination of PTX and the non-cytotoxic Cur1-12V (10 μM) significantly decreased the IC50 of PTX to 3.8 nM as well as the resistant fraction to 16.2%. The combination test was confirmed using the same protocol but on another resistant CRC cell line (HCT-116) as we obtained similar results. Both Cur-3 and Cur1-12V (10 μM) significantly increased the cellular entrapment of Pgp probe (doxorubicin) elevating its intracellular concentration from 1.9 pmole/cell to 3.0 and 2.9 pmole/cell, respectively.

Cardiovascular Ion Channel Inhibitor Drug-Drug Interactions with P-glycoprotein.

P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that plays a major role in cardiovascular drug disposition by effluxing a chemically and structurally diverse range of cardiovascular therapeutics. Unfortunately, drug-drug interactions (DDIs) with the transporter have become a major roadblock to effective cardiovascular drug administration because they can cause adverse drug reactions (ADRs) or reduce the efficacy of drugs. Cardiovascular ion channel inhibitors are particularly susceptible to DDIs and ADRs with Pgp because they often have low therapeutic indexes and are commonly coadministered with other drugs that are also Pgp substrates. DDIs from cardiovascular ion channel inhibitors with the transporter occur because of inhibition or induction of the transporter and the transporter's tissue and cellular localization. Inhibiting Pgp can increase absorption and reduce excretion of drugs, leading to elevated drug plasma concentrations and drug toxicity. In contrast, inducing Pgp can have the opposite effect by reducing the drug plasma concentration and its efficacy. A number of in vitro and in vivo studies have already demonstrated DDIs from several cardiovascular ion channel inhibitors with human Pgp and its animal analogs, including verapamil, digoxin, and amiodarone. In this review, Pgp-mediated DDIs and their effects on pharmacokinetics for different categories of cardiovascular ion channel inhibitors are discussed. This information is essential for improving pharmacokinetic predictions of cardiovascular therapeutics, for safer cardiovascular drug administration and for mitigating ADRs emanating from Pgp.

ABC Transporters and Drug Resistance in Patients with Epilepsy.

Resistance to antiepileptic drugs (AED) remains a major problem in clinical epileptology. This pharmacoresistance is independent of the choice of AEDs. Different hypotheses have been proposed to explain the neurobiological basis for pharmacoresistance in epilepsy. The transporter hypothesis is the mostly investigated theory. Hereby, overexpression of multidrug efflux transporters, such as P-glycoprotein (Pgp), at the blood-brain-barrier (BBB) is thought to be involved in pharmacoresistance in epilepsy by extruding AEDs from their target site. Accumulating evidence supports an overexpression of Pgp in pharmacoresistant epilepsy. Molecular Imaging studies provide unique opportunities for the in-vivo study of the transporter hypothesis in the central nervous system (CNS). Several studies demonstrated that positron emission tomography (PET) with [11C]-radiolabled Pgp substrates is a promising tool for in vivo investigation of Pgp function at the rat, monkey and human BBB. Quantification of Pgp over activity in epilepsy patients by in vivo imaging could be highly useful because altered treatment strategies or novel AED could then be applied.

Using the Variable-Nearest Neighbor Method To Identify P-Glycoprotein Substrates and Inhibitors.

Permeability glycoprotein (Pgp) is an essential membrane-bound transporter that efficiently extracts compounds from a cell. As such, it is a critical determinant of the pharmacokinetic properties of drugs. Multidrug resistance in cancer is often associated with overexpression of Pgp, which increases the efflux of chemotherapeutic agents from the cell. This, in turn, may prevent an effective treatment by reducing the effective intracellular concentrations of such agents. Consequently, identifying compounds that can either be transported out of the cell by Pgp (substrates) or impair Pgp function (inhibitors) is of great interest. Herein, using publically available data, we developed quantitative structure–activity relationship (QSAR) models of Pgp substrates and inhibitors. These models employed a variable-nearest neighbor (v-NN) method that calculated the structural similarity between molecules and hence possessed an applicability domain, that is, they used all nearest neighbors that met a minimum similarity constraint. The performance characteristics of these v-NN-based models were comparable or at times superior to those of other model constructs. The best v-NN models for identifying either Pgp substrates or inhibitors showed overall accuracies of >80% and κ values of >0.60 when tested on external data sets with candidate Pgp substrates and inhibitors. The v-NN prediction model with a well-defined applicability domain gave accurate and reliable results. The v-NN method is computationally efficient and requires no retraining of the prediction model when new assay information becomes available—an important feature when keeping QSAR models up-to-date and maintaining their performance at high levels.

297 - Fire and Brimstone: Turning the Gun on Cancer

Oxidative stress plays a role in the development of drug resistance in cancer cells. Cancer cells must constantly and rapidly adapt to changes in the tumor microenvironment, due to alterations in the availability of nutrients, such as glucose, oxygen and key transition metals (e.g., iron and copper). This nutrient flux is typically a consequence of rapid growth, poor vascularization and necrosis. It has been demonstrated that stress factors, such as hypoxia and glucose deprivation up-regulate master transcription factors, namely hypoxia inducible factor-1α (HIF-1α), which transcriptionally regulate the multi-drug resistance (MDR), transmembrane drug efflux transporter, P-glycoprotein (Pgp). Interestingly, in addition to the established role of plasma membrane Pgp in MDR, a new paradigm of intracellular resistance has emerged that is premised the ability of lysosomal Pgp to transport cytotoxic agents into this organelle. This mechanism is enabled by the topological inversion of Pgp via endocytosis resulting in the transporter actively pumping agents into the lysosome. In this way, classical Pgp substrates, such as doxorubicin (DOX), can be actively transported into this organelle. Within the lysosome, DOX becomes protonated upon acidification of the lysosomal lumen, causing its accumulation. This process efficiently traps DOX, preventing its cytotoxic interaction with nuclear DNA. These studies highlight a novel strategy by which redox-active and protonatable Pgp substrates can utilize lysosomal Pgp to gain access to this compartment, resulting in catastrophic lysosomal membrane permeabilization and cell death. Importantly, this mechanism is key to the activity of novel anti-cancer agent, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), that has entered multi-centre clinical trials for the treatment of a wide variety of advanced and resistant tumors (NCT02688101). Hence, a key MDR mechanism that utilizes Pgp (the gun) to sequester redox active drugs (“fire and brimstone”) within lysosomes can be turned on MDR cancer cells to destroy them from within.

Evaluation of the potential effect of Allium sativum, Momordica charantia, Eugenia jambolana, Ocimum sanctum, and Psidium guajava on intestinal p-glycoprotein in rats.

This study was evaluated synergistic effect of a polyherbal formulation (PHF) of Allium sativum L., Eugenia jambolana Lam., Momordica charantia L., Ocimum sanctum Linn., and Psidium guajava L. on p-glycoprotein (Pgp) of intestine. These five herbs were traditionally used for diabetes. These herbs are commonly present in Ayurvedic product as antidiabetics in India. PHF was prepared by five indigenous herbs. Different doses (50, 100 and 200 mg/kg/day) of were orally administered to Sprague-Dawley rats of different groups for multiple weeks except control groups. Alteration in Pgp expression was evaluated by real-time-polymerase chain reaction and western blotting while modulation in activity of Pgp was evaluated using rhodamine 123 (Rh123) as transport substrate by in-situ absorption and everted gut sac method. In PHF, pretreated group received 50, 100 and 200 mg/kg/day for 7 days, mRNA level decreased by 1.75, 2.45 and 2.37-fold, respectively, as compared to control. Similarly, when PHF at dose of 100 mg/kg/day was given consequently for 4 weeks, maximum decrease in Pgp expression level was observed only after 1 week and further increase in the treatment duration did not produce significant decrease compared to the 1st week treatment. Pgp mediated transport of Rh123 was significantly decreased with everted gut sac prepared from PHF pretreated rats (1 week) compared to those prepared from vehicle treated rats. We report that PHF pretreatment downregulated the expression of intestinal Pgp and this downregulated intestinal Pgp would result in decreased functional activity. In addition, this downregulated Pgp expression might affect the bioavailability of antidiabetic Pgp substrate drugs.

Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein

The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other’s interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis.

Alterations in P-Glycoprotein Expression and Function Between Macrophage Subsets.

Macrophages are an important cellular reservoir in HIV, and exist in two phenotypically dissimilar subsets, the pro-inflammatory M1 phenotype, and the anti-inflammatory M2 phenotype. The role of these two subsets is uncertain. We hypothesized that differences in drug efflux transporters exist between the subsets, which would result in altered intracellular drug concentrations between these cells. U937 monocytic cells were polarized to the M1 or M2 phenotype via treatment with interferon-gamma and LPS, or interleukins 4, 13, and LPS, respectively. PGP function was assessed with Hoechst 33342, and expression via western blotting. Intracellular lopinavir was assessed via LC-MS/MS. Data was confirmed with primary monocyte derived macrophages. We observed significant differences in intracellular concentrations of lopinavir, a PGP substrate, with higher concentrations in M1 cells. PGP function and expression was higher in the M2 macrophages. These results were confirmed with primary monocyte derived macrophages. This data shows that there are previously unreported differences in P-glycoprotein expression between macrophage subsets, and suggests that there may be differences for other transporters. These differences can play a role in intracellular drug concentrations in these cells, and may allow for low-level HIV replication.

Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors.

The blood–brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity.

Increasing nerve agent treatment efficacy by P-glycoprotein inhibition

One of the shortcomings of current treatment of nerve agent poisoning is that not all drugs effectively penetrate the blood-brain barrier (BBB), whereas most nerve agents easily do. P-glycoprotein (Pgp) efflux transporters at the BBB may contribute to this aspect. It was previously shown that Pgp inhibition by tariquidar enhanced the efficacy of nerve agent treatment when administered as a pretreatment.In the present study soman-induced seizures were also substantially prevented when the animals were intravenously treated with tariquidar post-poisoning, in addition to HI-6 and atropine. In these animals, approximately twice as much AChE activity was present in their brain as compared to control rats. The finding that tariquidar did not affect distribution of soman to the brain indicates that the potentiating effects were a result of interactions of Pgp inhibition with drug distribution. In line with this, atropine appeared to be a substrate for Pgp in in vitro studies in a MDR1/MDCK cell model. This indicates that tariquidar might induce brain region specific effects on atropine distribution, which could contribute to the therapeutic efficacy increase found.Furthermore, the therapeutic enhancement by tariquidar was compared to that of the less specific and less potent Pgp inhibitor cyclosporine A. This compound appeared to induce a protective effect similar to tariquidar.In conclusion, treatment with a Pgp inhibitor resulted in enhanced therapeutic efficacy of HI-6 and atropine in a soman-induced seizure model in the rat. The mechanism underlying these effects should be further investigated. To that end, the potentiating effect of nerve agent treatment should be addressed against a broader range of nerve agents, for oximes and atropine separately, and for those at lower doses. In particular when efficacy against more nerve agents is shown, a Pgp inhibitor such as tariquidar might be a valid addition to nerve agent antidotes.

Unraveling the Structural Basis of P‐glycoprotein Transport of Serotonin 5‐HT1B/1D Receptor Agonists: Eletriptan and Sumatriptan

P‐glycoprotein (Pgp) acts as the gatekeeper at the blood‐brain barrier by preventing the entry of toxic insults; however, drugs that are Pgp substrates cannot penetrate the blood‐brain barrier to treat central nervous system (CNS) disorders and diseases. Serotonin 5‐HT1B/1D receptor agonists are members of the triptan class of drugs that abort migraines during the onset of symptoms and are known Pgp substrates. While members of a drug class are structurally similar, eletriptan (Relpax®, ETT) and sumatriptan (Imitrex®, STT) have large differences in their Pgp efflux ratios. To understand the relationship between drug binding and efflux, ETT and STT were used to probe the structural basis of triptan transport. From ATPase activity assays, ETT stimulated Pgp ATP hydrolysis and was fit to the Michaelis‐Menten equation. In contrast, STT induced very little activation of the Pgp‐coupled hydrolysis. Protein fluorescence quenching was used to determine the binding affinities and the number of binding sites of ETT and STT. Each triptan drug was identified to have a single binding site. To test if these binding sites were overlapping, competition ATPase activity assays were performed. Conformational changes induced by each ETT and STT binding was determined by acrylamide quenching.Support or Funding InformationNIH ‐1021RR571390, AHA ‐1021RR571388, Pfizer, Inc., University of Georgia, Bioexpression and Fermentation Facility at UGA

Abstract P4-09-27: ABC transporter expression: Clues into chemoresistance of triple negative breast cancers

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive histological subtype with high rates of recurrence and metastatic disease. Intrinsic or acquired multidrug resistance facilitated by the over-expression of drug efflux pumps (ABC transporters: BCRP [ABCG2], MRP1 [ABCC1] and PGP [ABCB1]), may contribute to the aggressive nature of this disease. We examined the expression patterns of drug efflux pumps for insight on their potential role in chemoresistance of TNBC. Methods: 1393 TNBC patients molecularly profiled with a commercial assay (Caris Life Sciences) were evaluated retrospectively for expression of BCRP, MRP1 and PGP by immunohistochemistry. Antibodies used: PGP (C494), BCRP (6D171) and MRP1 (33A6). IHC threshold: positive = ≥1+ and ≥10%). This data set also included metachronous paired samples from 71 TNBC patients. JMP was used to ascertain distributional differences. Results: PGP and MRP1 positive expression rates were 8.4% (117/1393) compared to 78.2% (248/317), respectively, displaying an inverse association (p=0.0001). BCRP was over-expressed in 52% (90/173). Co-expression data for all three transporters was available for 153 patients. BCRP/MRP1 co-expression was most abundant, 39% (59/153), followed by MRP1/PGP at 4% (6/153) and BCRP/PGP at 2% (3/153). Furthermore, 12% (18/153) of TNBC exhibited positive expression for all three drug pumps (16/18 or 89% were from patients with metastatic disease) and 14% (22/153) exhibited negative expression for all three drug pumps (15/22 or 68% were from patients with metastatic disease). Interestingly, positive MRP1 status, but not BCRP or PGP, correlated with metastatic disease (p=0.0061). In the paired data analysis, PGP expression was absent in 77% (55/71) and retained lack of expression in subsequently profiled specimens, 3% (2/71) retained positive PGP status, 13% (9/71) lost expression of PGP and 7% (5/71) gained expression of PGP. 36/40 patients that lacked PGP expression and 8/9 patients that "lost" PGP expression, exhibited and retained expression of MRP1 in the initial and subsequently-profiled specimens. Conclusion: Biomarker expression patterns of drug efflux pumps may provide insight to the chemoresistance phenotype observed in TNBC. Expression of MRP1 is favored in TNBC and is correlated with metastatic disease status. Although PGP expression may be absent or lost during TNBC progression, MRP1 (and BCRP) expression are almost always retained. Utilization of chemotherapies that are substrates of PGP, but not MRP1, (based on expression status) may be worthy of future investigation. Citation Format: Feldman R, Abbott B, Reddy S, Gatalica Z, Castro M. ABC transporter expression: Clues into chemoresistance of triple negative breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-27.

Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter

Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in multidrug-resistant tumors where a stressful micro-environment exists.

METHODS OF IDENTIFICATION OF DRUGS AS P-GLYCOPROTEIN SUBSTRATES

The article presents an original method for determining belonging of drugs to the substrates of the transport protein P-glycoprotein (Pgp). This methodics is based on a comparison of the pharmacokinetic parameters of the test substrate before and after a course of administration of the inductor (rifampicin) and inhibitor (verapamil) of the transport protein. If the content of test substance in organism is reduced after Pgp inductor administration, and increased after the inhibitor administration, we conclude that this substance is Pgp substrate. For approbation of developed technique we examined by blind method the pharmacokinetics of fexofenadine - known Pgp substrate at a dose of 67,5 mg/kg, before and after the 14-days administration of rifampicin (40 mg/kg twice daily) and verapamil (a daily dose of 20 mg/kg three times a day). It was revealed that after a course administration of verapamil maximum concentration of fexofenadine, its area under the concentration-time curve and half-life were significantly increased, as well as the time of maximum concentration was decreased. In contrast, rifampicin course administration resulted in a decrease in the area under the curve concentration-time and an increase in total clearance of marker substrate of Pgp, wich proves the adequacy of methods.

The Interesting Case of Acyclovir Delivered Using Chitosan in Humans: Is it a Drug Issue or Formulation Issue?

Attempts to formulate acyclovir to improve its bioavailability and reduce the frequency of dosing from the present q4h have not materialized. It was thought that an approach using permeability enhancer such as chitosan may impart improved absorption profile to acyclovir; however, the recently published pharmacokinetic data suggested otherwise. The lack of promise of chitosan formulation was attributed to the muco-adhesive properties of chitosan to hold off acyclovir and preventing its transport across the gastrointestinal tract. However, the above hypothesis was refuted by another published human pharmacokinetic study of fexofenadine formulated with chitosan formulation - in this work it was unambiguously shown that chitosan helped in enhanced absorption of fexofenadine which is a well-known Pgp substrate. If one examines the pharmacokinetic disposition of acyclovir, it is clear that renal elimination is so rapid necessitating frequent dosing of acyclovir. In summary, the ability of chitosan based formulations to aid in the oral absorption of drugs may be drug dependent as enumerated by data obtained from acyclovir and fexofenadine. While chitosan favourably improved the pharmacokinetics of fexofenadine, acyclovir may not be ideal for chitosan type of formulation. The choice of the drug and the formulation type intended to deliver the drug need to be made in a diligent and pragmatic fashion.