Efflux Transporter Activity Research Articles

Silicon efflux transporters isolated from two pumpkin cultivars contrasting in Si uptake

The accumulation of silicon (Si) differs greatly with plant species and cultivars due to different ability of the roots to take up Si. In Si accumulating plants such as rice, barley and maize, Si uptake is mediated by the influx (Lsi1) and efflux (Lsi2) transporters. Here we report isolation and functional analysis of two Si efflux transporters (CmLsi2-1 and CmLsi2-2) from two pumpkin (Cucurbita moschata Duch.) cultivars contrasting in Si uptake. These cultivars are used for rootstocks of bloom and bloomless cucumber, respectively. Different from mutations in the Si influx transporter CmLsi1, there was no difference in the sequence of either CmLsi2 between two cultivars. Both CmLsi2-1 and CmLsi2-2 showed an efflux transport activity for Si and they were expressed in both the roots and shoots. These results confirm our previous finding that mutation in CmLsi1, but not in CmLsi2-1 and CmLsi2-2 are responsible for bloomless phenotype resulting from low Si uptake.

Pharmacokinetic characterization of decursinol derived from Angelica gigas Nakai in rats

Decursinol is a major coumarin derived from the roots of Angelica gigas and has various pharmacological effects against inflammation, angiogenesis, nociceptive pain and Alzheimer’s disease. In vitro and in vivo studies were conducted to characterize the metabolism and pharmacokinetics of decursinol.Decursinol exhibited high stability to oxidative and glucuronic metabolism in human and rat liver microsomes.In Caco-2 cell monolayers, decursinol showed high permeability (>14 × 10−6 cm/s) at all tested concentrations in the absorptive direction, which saturated at 100 μM. Secretion increased in a concentration-dependent manner, with an efflux ratio of more than 2 at 50 μM, indicating the participation of an active efflux transporter such as P-glycoprotein, multidrug resistance protein 2 or breast cancer resistance protein.The fraction of decursinol not bound to plasma proteins was 25–26% in the rat and 9–18% in humans. In human plasma, but not rat plasma, the percentage of unbound decursinol was concentration dependent.Following intravenous administration in rats, non-linear elimination of decursinol was observed with Km and Vmax values of 2.1 μg/mL and 2.5 mg·h−1·kg−1, respectively. Following oral administration, decursinol exhibited high oral bioavailability (>45%) and rapid absorption (Tmax, 0.4–0.9 h) over the dose range studied. In addition, dose-dependent absorption and elimination were observed at 20 mg/kg.

Delivery of molecularly targeted therapy to malignant glioma, a disease of the whole brain

Glioblastoma multiforme, because of its invasive nature, can be considered a disease of the entire brain. Despite recent advances in surgery, radiotherapy and chemotherapy, current treatment regimens have only a marginal impact on patient survival. A crucial challenge is to deliver drugs effectively to invasive glioma cells residing in a sanctuary within the central nervous system. The blood-brain barrier (BBB) restricts the delivery of many small and large molecules into the brain. Drug delivery to the brain is further restricted by active efflux transporters present at the BBB. Current clinical assessment of drug delivery and hence efficacy is based on the measured drug levels in the bulk tumour mass that is usually removed by surgery. Mounting evidence suggests that the inevitable relapse and lethality of glioblastoma multiforme is due to a failure to effectively treat invasive glioma cells. These invasive cells hide in areas of the brain that are shielded by an intact BBB, where they continue to grow and give rise to the recurrent tumour. Effective delivery of chemotherapeutics to the invasive glioma cells is therefore critical, and long-term efficacy will depend on the ability of a molecularly targeted agent to penetrate an intact and functional BBB throughout the entire brain. This review highlights the various aspects of the BBB, and also the brain-tumour-cell barrier (a barrier due to expression of efflux transporters in tumour cells), that together can significantly influence drug response. It then discusses the challenge of glioma as a disease of the whole brain, which lends emphasis to the need to deliver drugs effectively across the BBB to reach both the central tumour and the invasive glioma cells.

A Critical Analysis of the Interplay between Cytochrome P450 3A and P-Glycoprotein: Recent Insights from Knockout and Transgenic Mice

CYP3A is one of the most important drug-metabolizing enzymes, determining the first-pass metabolism, oral bioavailability, and elimination of many drugs. It is also an important determinant of variable drug exposure and is involved in many drug-drug interactions. Recent studies with CYP3A knockout and transgenic mice have yielded a number of key insights that are important to consider during drug discovery and development. For instance, studies with tissue-specific CYP3A-transgenic mice have highlighted the importance of intestinal CYP3A-dependent metabolism. They also revealed that intestinal CYP3A plays an important role in the regulation of various drug-handling systems in the liver. Intestinal CYP3A activity can thus have far-reaching pharmacological effects. Besides CYP3A, the active drug efflux transporter P-glycoprotein also has a strong effect on the pharmacokinetics of numerous drugs. CYP3A and P-glycoprotein have an extensive overlap in their substrate spectrum. It has been hypothesized that for many drugs, the combined activity of CYP3A and P-glycoprotein makes for efficient intestinal first-pass metabolism of orally administered drugs as a result of a potentially synergistic collaboration. However, there is only limited in vitro and in vivo evidence for this hypothesis. There has also been some confusion in the field about what synergy actually means in this case. Our recent studies with Cyp3a/P-glycoprotein combination knockout mice have provided further insights into the CYP3A-P-glycoprotein interplay. We here present our view of the status of the synergy hypothesis and an attempt to clarify the existing confusion about synergy. We hope that this will facilitate further critical testing of the hypothesis and improve communication among researchers. Above all, the recent findings and insights into the interplay between CYP3A and P-glycoprotein may have implications for improving oral drug bioavailability and reducing adverse side effects.

Transport of silicon from roots to panicles in plants

Silicon (Si) is the most abundant minerals in soil and exerts beneficial effects on plant growth by alleviating various stresses. The transport of Si from soil to the panicles is mediated by different transporters. Lsi1, belonging to a NIP group of the aquaporin family, is responsible for the uptake of Si from soil into the root cells in both dicots and monocots although its expression patterns and cellular localization differ with plant species. The subsequent transport of Si out of the root cells towards the stele is medicated by an active efflux transporter, Lsi2. Lsi1 and Lsi2 are polarly localized at the distal and proximal sides, respectively, of both exodermis and endodermis in rice root. Silicon in the xylem sap is presented in the form of monosilicic acid and is unloaded by Lsi6, a homolog of Lsi1 in rice. Lsi6 is also involved in the inter-vascular transfer of Si at the node, which is necessary for preferential Si distribution to the panicles.

Transwell-Grown HepG2 Cell Monolayers as In Vitro Permeability Model to Study Drug–Drug or Drug–Food Interactions

HepG2 cell monolayers, formed during cell growth on collagen-coated Transwell® (Corning® Inc., Corning, NY, USA) inserts, can be used for the evaluation of interactions between food supplements and drugs that are substrates for P-glycoprotein (Pgp) and/or multidrug resistance-associated protein 2 (MRP-2). Samples obtained during such permeability studies were relatively free of intracellular proteins or cell debris compared to usually performed uptake experiments with HepG2 cells; therefore no special preparation protocol prior to the analysis was needed. In the presence of aged garlic extract the activities of hepatic efflux transporters (Pgp, MRP-2) changed, which was observed as significant permeability changes of tested human immunodeficiency virus (HIV) protease inhibitors. Darunavir efflux significantly increased, whereas that of saquinavir significantly decreased. Because of the observed in vitro interactions between aged garlic extract and HIV protease inhibitors (darunavir, saquinavir), any alterations of in vivo liver transport in the presence of garlic phytochemicals could also significantly influence darunavir/saquinavir hepatocyte intracellular concentrations and hence their bioavailabilities. Therefore this aspect needs further in vivo animal evaluation.

Kaempferide Targets Side Population, the Putative Cancer Stem Cell, In Myeloma and Induced Apoptosis In Dose-Dependant Manner

AbstractAbstract 5029Multiple myeloma (MM), cancer of the plasma cells, remains incurable despite advancement in therapeutic regiments. Studies showed that the ‘side population (SP)‘, a subpopulation enriched with CSC in various cancers, has higher drug transporter activity than the bulk tumor; suggesting the reason why these cells survived despite post-chemotherapy. The increasing evidence of cancer stem cells (CSC) suggests that anti-cancer drugs targeting this subpopulation may lead to eradication of the root of cancer recurrence. In this study, we explored the capability of kaempferide (KFD), a flavanoid in propolis (bee glue) that can reverse drug transporter activity, to combat SP cells in myeloma. KFD was one of the compounds in Brazilian propolis that reduced SP percentage in myeloma cell lines. We report for the first time that KFD is able to induce apoptosis in the SP cells in myeloma. The ability of KFD to inhibit growth of unfractionated KMS-11 cells was first investigated. Parthenolide (PAR), a natural product shown to inhibit growth of putative CSC in acute and chronic myelogenous leukemia was included as control. Unfractionated cells were seeded at 20000 cells per well in a 96 well plate. After 24h of incubation with various concentration of KFD, PAR, and DMSO (vehicle control), MTS solution was added and absorbance at 490 nm was determined. The IC50 of KFD and PAR in KMS-11 cells was 26 μM and 5 μM with 95% confidence intervals between 17.7 to 33.3 μM and 4.0 to 5.8 μM respectively. This is shown in the dose-response curve in figure 1A. No significant growth inhibition was observed in DMSO (0.1% to 0.5 % v/v) treated cells. We then examined if the KFD causes apoptosis in the sorted-SP cells. Sorted-SP cells were treated with 26 μM KFD, 5 μM PAR, or 0.2% v/v DMSO as mentioned above. After 1, 3, and 6 hour of incubation, cells were harvested and stained with Annexin V and propidium iodide and then analyzed using FACS Calibur. Result showed that percentage of Annexin V+ apoptotic cells in KFD-treated cells increased in a time series manner (1, 3, 6 h) (figure 1B). Because KFD was reported to be capable to reverse the activity of drug efflux transporter, further studies to investigate the synergistic effect of KFD with conventional drugs to treat myeloma will be carried out. Alternative medicines employing natural products have become increasingly sought after when conventional drugs cause immense side effects. Component in propolis that was reported to have anti-cancer properties, has low toxicity at high concentration, and spare normal cells, appeals to be a potential compound to combat myeloma. Exploitation of KFD that has the dual effect to induce apoptosis in putative CSC in myeloma and reverse drug transporter activity offers great opportunity in cancer drug development and future clinical trials in patients with myeloma.ABAnnexin V-FITC (FL-1)Propidium iodied (FL-3) [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Inhibition of P-Glycoprotein Pumps by PEO–PPO Amphiphiles: Branched Versus Linear Derivatives

Inhibition of the activity of efflux transporters may relevantly improve the chemotherapy of cancer and infectious diseases. To explore the ability of poloxamines (Tetronic(®), X-shaped structure with a central ethylendiamine group and four branches of poly[ethylene oxide]-poly[propylene oxide] [PEO-PPO]) to inhibit the activity of P-glycoprotein (P-gp) on Caco-2 cell monolayers and to elucidate the incidence of the molecular architecture of PEO-PPO block copolymers on the intracellular accumulation of a relevant substrate, doxorubicin, by comparison with poloxamers (Pluronic(®), linear triblock copolymers), well-known inhibitors of this efflux transporter. Both pristine and N-methylated poloxamines displaying a wide range of molecular weights and EO/PO ratios were tested regarding cytocompatibility and accumulation of doxorubicin in Caco-2 monolayers. Verapamil was used as a control. The most active anti-P-gp poloxamines (which enhanced two- to three-fold doxorubicin accumulation compared with verapamil) resulted to be pristine medium-to-high hydrophobic T304, T904, T1301, T901 and T150R1. A notable dependence of the anti-P-gp activity on the copolymer concentration was found. A joint diagram of the inhibitory activity of poloxamers and poloxamines as a function of the effective length of the PPO block is proposed. The anti-P-gp activity is maxima for block copolymers possessing a low-to-medium hydrophilic-lipophilic balance and an 'effective number' of PO units ranging from 30 to 50.

Evaluation of the endothelin receptor antagonists ambrisentan, darusentan, bosentan, and sitaxsentan as substrates and inhibitors of hepatobiliary transporters in sandwich-cultured human hepatocytesThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

To evaluate potential mechanisms of clinical hepatotoxicity, 4 endothelin receptor antagonists (ERAs) were examined for substrate activity and inhibition of hepatic uptake and efflux transporters in sandwich-cultured human hepatocytes. The 4 transporters studied were sodium-dependent taurocholate cotransporter (NTCP), organic anion transporter (OATP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2). ERA transporter inhibition was examined using the substrates taurocholate (for NTCP and BSEP), [(3)H]estradiol-17beta-D-glucuronide (for OATP), and [2-D-penicillamine, 5-D-penicillamine]enkephalin (for MRP2). ERA substrate activity was evaluated using probe inhibitors ritonavir (OATP and BSEP), bromosulfalein (OATP), erythromycin (P-glycoprotein), probenecid (MRP2 and OATP), and cyclosporin (NTCP). ERAs were tested at 2, 20, and 100 micromol*L-1 for inhibition and at 2 micromol*L-1 as substrates. OATP, NTCP, or BSEP transport activity was not reduced by ambrisentan or darusentan. Bosentan and sitaxsentan attenuated NTCP transport at higher concentrations. Only sitaxsentan decreased OATP transport (52%), and only bosentan reduced BSEP transport (78%). MRP2 transport activity was unaltered. OATP inhibitors decreased influx of all ERAs. Darusentan influx was least affected (84%-100% of control), whereas bosentan was most affected (32%-58% of control). NTCP did not contribute to influx of ERAs. Only bosentan and darusentan were shown as substrates for both BSEP and P-glycoprotein efflux. All ERAs tested were substrates for at least one hepatic transporter. Bosentan and sitaxsentan, but not ambrisentan and darusentan, inhibited human hepatic transporters, which provides a potential mechanism for the increased hepatotoxicity observed for these agents in the clinical setting.

Lack of brain‐to‐blood efflux transport activity of low‐density lipoprotein receptor‐related protein‐1 (LRP‐1) for amyloid‐β peptide(1–40) in mouse: involvement of an LRP‐1‐independent pathway

The contribution of low-density lipoprotein receptor-related protein-1 (LRP-1) to the brain-to-blood amyloid-beta peptide (A beta) efflux transport across the blood-brain barrier (BBB) remains controversial. The purpose of the present study was to clarify whether or not LRP-1 plays a role in efflux transport of A beta at the BBB. After microinjection of [(125)I]activated alpha2-macroglobulin (alpha 2M), a typical LRP-1 ligand, into mouse secondary somatosensory cortex region under ketamine-xylazine anesthesia, residual radioactivity was not significantly decreased up to 90 min, whereas after microinjection of [(125)I]human A beta(1-40) [hA beta(1-40)], the residual radioactivity decreased time-dependently. Co-administration of receptor-associated protein, an inhibitor of LRP-1, did not influence [(125)I]hA beta(1-40) elimination from mouse brain, suggesting that members of the LDL receptor gene family, including LRP-1, do not contribute to hA beta(1-40) elimination from mouse brain across the BBB. There was no significant difference between the uptakes of [(125)I]activated alpha 2M and [(14)C]inulin by mouse brain slices, suggesting that activated alpha 2M was not significantly bound to and/or taken up by parenchymal cells. In conclusion, our results show that LRP-1 does not play a significant role in the brain-to-blood efflux transport of A beta(1-40) at the mouse BBB, but an unidentified transporter(s) appears to be involved.

Abstract 1527: Pharmacogenomic identification of compounds selectively killing multidrug-resistant cancer cells with high glutathione metabolism

Abstract The ability of cancer cells to become simultaneously resistant to different drugs - a trait known as multidrug resistance (MDR) - remains a significant impediment to successful cancer therapy. Cancer cells develop MDR via several possible mechanisms, including (1) extrusion of drugs by increasing activity of efflux transporters, (2) reduction of drug uptake and (3) activation of detoxifying pathways such as those mediated by glutathione (GSH). SLC7A11 encodes a subunit of a transporter, designated xc−, which is responsible for cellular uptake of the amino acid cystine in exchange for intracellular glutamate. Once inside the cell, cystine is rapidly reduced to cysteine, a rate limiting precursor for biosynthesis of GSH. Using a pharmacogenomic approach, we have previously demonstrated that increased expression of SLC7A11 confers GSH-mediated resistance of tumor cells to anticancer drugs, since its expression level was negatively correlated with drug potency across the National Cancer Institute's 60 cancer cell lines (NCI-60). The number of significant SLC7A11-drug correlations was much greater than those of known resistance genes such as ABCB1 and GSTs. To further investigate the role of SLC7A11 on chemosensitivity, we analyzed the correlation between SLC7A11 expression and activity of 1,400 chemical compounds on the NCI-60 and identified 39 compounds with positive SLC7A11 correlations. Interestingly, many of these compounds are analogs of the anticancer drug ellipticinium. All the eight ellipticinium analogs among the 1,400 compounds showed significant positive correlations with SLC7A11, while all the ellipticine analogs had no such correlation. Since the positive gene-drug correlation suggests that cell lines with higher SLC7A11 expression and higher GSH metabolism showed higher sensitivity to ellipticiniums, these compounds could exploit, rather than suppress the GSH pathways to increase tumor chemosensitivity. We examined the effect of the xc− transporter inhibitor sulfasalazine on SLC7A11 over-expressing and drug resistant glioma cell lines SNB-19 and SF-295. Sulfasalazine treatment (0.15 mM) significantly reduced the cytotoxicity of ellipticiniums but not that of ellipticines, consistent with the results obtained by the pharmacogenomic analysis. In view of the above exciting results, further investigations are being conducted to validate the interaction between the xc− transporter and ellipticinium analogs and to study the mechanism of the selective cytotoxicity. It may be feasible to take advantage of high level of SLC7A11 (or other genes in the GSH regulatory pathway) in some tumors for targeted cancer therapy. Taken together, the discovery of the MDR-selective compounds among the ellipticinium analogs demonstrated the robustness of the developing field of MDR-targeting therapy as a new strategy for overcoming GSH-mediated MDR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1527.

Abstract 3578: New Angiochem-modified doxorubicin with increased brain penetration and efficacy against brain tumors

Abstract The blood-brain barrier (BBB) is mainly formed by brain capillary endothelial cells which are closely sealed by tight junctions and express high levels of active efflux transport proteins, including P-glycoprotein (Pgp). As a result, the overwhelming majority of small molecules, proteins and peptides do not cross the BBB. Angiochem's engineered peptide compounds (EPiC) provides a non-invasive and flexible platform for small and large molecules to treat brain diseases. Based on these properties, we have created a portfolio of new drug entities composed of siRNA, peptides and mAbs, the most advanced of which is ANG1005 formed by chemical conjugation of our peptide to three molecules of paclitaxel. ANG1005 demonstrated safety and efficacy in two phase ½ clinical trials for the treatment of primary and secondary brain tumors in humans. In the present study, we have investigated the brain uptake of a new chemical entity formed by conjugation of the peptide Angiopep-2 (An2) with 3 molecules of the anti-cancer drug doxorubicin. Despite the clinical effectiveness of doxorubicin in the treatment of many malignant tumors, clinical trials involving systemic administration have demonstrated very limited efficacy in the treatment of gliomas. This limited efficacy can be explained by the poor penetration of the drug thru the BBB and by the effect of the multidrug resistance pump, P-gp. Here, we show by mice in-situ brain perfusion that the ANG-doxorubicin (ANG1007) is transported very efficiently across the BBB. The transport rate is higher than that of unconjugated doxorubicin by at least 10-fold. In vitro, ANG1007 inhibits cancer cell proliferation, with highly cytotoxic activities against various tumor cell lines. In addition, brain perfusion studies performed with P-gp knock-out mice showed that ANG1007 bypasses the drug efflux pump P-gp at the BBB. Furthermore, ANG1007 inhibits growth of glioblastoma s.c. tumors and increases survival of mice implanted with brain tumors. In conclusion, these data confirm that conjugation of chemotherapeutic drugs such as doxorubicin to the peptide Angiopep-2 significantly enhances their entry into the brain and further validate the use of Angiochem's technology for new brain tumor therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3578.

Transporters in the Brain Endothelial Barrier

The blood-brain barrier (BBB) not only impedes the influx of intravascular substances from blood to brain, but also promotes transport of substances from blood to brain or from brain to blood through several transport systems such as carrier-mediated transport, active efflux transport, and receptor-mediated transport systems. The multidrug resistance transporter P-glycoprotein (P-gp) is an ATP-dependent efflux pump and contributes to efflux of undesirable substances such as amyloid-beta:(Abeta) proteins from the brain into the blood as well as many drugs such as anti-cancer drugs. The inhibition of P-gp has favorable and unfavorable effects on living bodies. P-gp deficiency at the BBB induces the increase of Abeta:deposition in the brain of an Alzheimer disease mouse model. It is also known that the Abeta:deposition is inversely correlated with P-gp expression in the brains of elderly non-demented humans. However, the transient inhibition of P-gp by antidepressants enables medicines such as anti-cancer drugs to enter the brain. Concerning Abeta:clearance in the brain, the low-density lipoprotein receptor-related protein 1 (LRP1) is a major efflux transporter for Abeta, while the receptor for advanced glycation end products (RAGE) is a major influx transporter for Abeta:across the BBB. Dysfunction of the BBB with efflux and influx transporters may contribute to the pathogenesis of some degenerative neuronal disorders. This review will focus on several transporters and discuss how medicines pass the BBB to reach the brain parenchyma.

Transport of Alzheimer Disease Amyloid-β-Binding d-Amino Acid Peptides across an In Vitro Blood–Brain Barrier Model

Previously, two D-enantiomeric amino acid peptides, D1 and D3, which specifically bind to the amyloid-beta peptide Abeta(1-42), were identified by phage display selection. To assess the diagnostic and therapeutic potentials of D1 and D3 for the diagnosis and treatment of Alzheimer disease, the blood-brain barrier transport of these D-peptides was quantitatively evaluated in vitro. Our results showed that the apical-to-basolateral transport of D3 was more efficient than that of D1. An active efflux transport mechanism seems to oppose the transport of D1, whereas D3 is likely to be transported through the blood-brain barrier via an adsorptive-mediated transcytosis mechanism.

MDR1 C2005T polymorphism changes substrate specificity

The current study is to determine the alterations of efflux transport activity to Rh123 and sensitivity to anticancer agents mediated by a MDR1 C2005T polymorphism. Expressions of mRNA and protein of MDR1 were measured by real-time PCR and immunoblotting, respectively, and localization of P-glycoprotein (P-gp) by confocal microscopy. Cell cytotoxicity and efflux transport activity were determined by MTT and Rh123 transepithelial permeability assay, respectively. MDR1 C2005T polymorphism did not affect the expression level of the MDR1 mRNA and protein and had no effect on the trafficking of P-gp to plasma membrane. A cytotoxicity study showed that MDR1wt and MDR1 (2005T) cells exhibited similar resistance, as measured by IC(50) values, to vinblastine (30.3 +/- 2.5 vs. 32.5 +/- 1.7 nM) and vincristine (104.1 +/- 1.9 vs. 110.3 +/- 3.5 nM). However, MDR1 (2005T) cells were less resistant to paclitaxel (28.2 +/- 2.1 vs. 91.8 +/- 3.5 nM; P < 0.05) and etoposide (119.7 +/- 6.5 vs. 546.8 +/- 9.5 nM; P < 0.05). The apparent transepithelial permeability ratios of Rh123 in MDR1 (wt) and MDR1 (2005T) cells were 2.12 +/- 0.46 and 3.64 +/- 0.78 (P < 0.05), respectively. The MDR1 C2005T polymorphism alters the transepithelial permeability of a fluorescent substrate and sensitivity to select cytotoxic agents, which may influence drug disposition and the therapeutic efficacy of some P-gp substrates.

N-desmethyl-Loperamide Is Selective for P-Glycoprotein among Three ATP-Binding Cassette Transporters at the Blood-Brain Barrier

[(11)C]N-desmethyl-Loperamide ([(11)C]dLop) is used in positron emission tomography (PET) to measure the in vivo activity of efflux transporters that block the passage of drugs across the blood-brain barrier. The three most prevalent ATP-binding cassette efflux transporters at the blood-brain barrier are P-glycoprotein (P-gp), multidrug resistance protein 1 (Mrp1), and breast cancer resistance protein (BCRP). We sought to measure the selectivity of dLop among these three transporters. The selectivity of dLop at low concentrations (< or =1 nM) was measured both as the accumulation of [(3)H]dLop in human cells that overexpress each transporter and as the uptake of [(11)C]dLop in brains of mice that lack genes encoding P-gp, Mrp1, or BCRP. The selectivity of dLop at high concentrations (> or =20 microM) was measured as the inhibition of uptake of a fluorescent substrate and the change in cytotoxicity of drugs effluxed at each transporter. Accumulation of [(3)H]dLop was lowest in cells overexpressing P-gp, and the uptake of [(11)C]dLop was highest in brains of mice lacking P-gp. At high concentrations, dLop selectively inhibited P-gp function and also decreased the resistance of only the P-gp-expressing cells to cytotoxic agents. dLop is selective for P-gp among these three transporters, but its activity is dependent on concentration. At low concentrations (< or =1 nM), dLop acts only as a substrate; at high concentrations (> or =20 microM), it acts as both a substrate and an inhibitor (i.e., a competitive substrate). Because low concentrations of radiotracer are used for PET imaging, [(11)C]dLop acts selectively and only as a substrate for P-gp.

Intestinal efflux transport kinetics of green tea catechins in Caco-2 monolayer model

The bioavailability of green tea catechins (GTCs), including epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC) is low in both animals and humans. The contribution of intestinal efflux to this low bioavailability has been suggested by previous studies. The objective of the present study was to investigate the kinetics of efflux transport of the four major GTCs in Caco-2 cell lines, to provide comparison on the efflux transport between each GTC. The basal-to-apical transport of each GTC at concentrations ranging from 15 to 265 microM was examined using the Caco-2 cell monolayer model. Transported amount of GTC was measured by high-performance liquid chromatography with electrochemical detection. Kinetic parameters, V(max), K(m) and V(max)/K(m) were determined and compared among the four studied GTCs. The extent of basal-to-apical transport was, in descending order, EC > EGC > ECG approximately EGCG. Kinetic studies indicated that active and saturable efflux transport of EC took place in Caco-2 cells, with a K(m) of 131 microM, a V(max) of 0.0249 nmol min cm(-2) and an intrinsic clearance (V(max)/K(m)) of 0.19 microL min cm(-2). No saturation could be observed for the efflux transport of EGC, ECG and EGCG even at concentrations up to about 200 microM, which may be due to their low affinity towards the transporters at the concentration range studied. In conclusion, the extent of efflux transport of GTCs in Caco-2 cells was, in descending order, EC > EGC > ECG approximately EGCG, which may reflect the order of elimination occurring in the intestine. The kinetic studies showed the importance of efflux transporters in basal-to-apical transport of EC and suggests their role in the limited oral bioavailability of EC.

Active‐Site Concentrations of Chemicals – Are They a Better Predictor of Effect than Plasma/Organ/Tissue Concentrations?

Active-site concentrations can be defined as the concentrations of unbound, pharmacologically active substances at the site of action. In contrast, the total concentrations of the drug in plasma/organ/tissue also include the protein- or tissue-bound molecules that are pharmacologically inactive. Plasma and whole tissue concentrations are used as predictors of effects and side effects because of their ease of sampling, while the concentrations of unbound drug in tissue are more difficult to measure. However, with the introduction of microdialysis and subsequently developed techniques, it has become possible to test the free drug hypothesis. The brain is an interesting organ in this regard because of the presence of the blood-brain barrier with its tight junctions and active efflux and influx transporters. We have proposed that research into brain drug delivery be divided into three main areas: the rate of delivery (PS, CL(in)), the extent of delivery (K(p,uu)) and the non-specific affinity of the drug to brain tissue, described by the volume of distribution of unbound drug in the brain (V(u,brain)). In this way, the concentration of unbound drug at the target site can be estimated from the total brain concentration and the plasma concentration after measuring the fraction of unbound drug. Results so far fully support the theory that active site concentrations are the best predictors when active transport is present. However, there is an urgent need to collect more relevant data for predicting active site concentrations in tissues with active transporters in their plasma membranes.

POLICY PAPER - COST Exploratory Workshop on Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU

POLICY PAPER - COST Exploratory Workshop on Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU

The Influence of Aged Garlic Extract on the Uptake of Saquinavir and Darunavir into HepG2 Cells and Rat Liver Slices

Bioavailability and therapeutic outcome of treatment with HIV-protease inhibitors depends on intestinal and hepatic transporter-enzyme interplay. Liver transport of HIV protease inhibitors (saquinavir, darunavir) was assessed in the presence of aged garlic extract, because the HIV-infected often consume garlic supplements together with prescribed therapy. The in vitro uptake of both drugs into HepG2 cells and precision cut rat liver slices significantly increased in the presence of Pgp and MRP-2 inhibitor ritonavir. The incubation medium containing aged garlic extract caused significant inhibition of saquinavir efflux from HepG2 cells and precision cut liver slices, while the activity of darunavir efflux transporters in both liver models significantly increased. Due to opposite in vitro interactions observed between aged garlic extract and HIV protease inhibitors, darunavir and saquinavir most probably bind to different binding sites on one or both efflux transporters. Based on this study, coadministration of investigated compounds with garlic supplements could result in significant in vivo modification of hepatic transport-enzyme interplay, possibly leading to further bioavailability change.