Efflux Transporter Activity Research Articles

Overview of P-glycoprotein inhibitors: a rational outlook

P-glycoprotein (P-gp), a transmembrane permeability glycoprotein, is a member of ATP binding cassette (ABC) super family that functions specifically as a carrier mediated primary active efflux transporter. It is widely distributed throughout the body and has a diverse range of substrates. Several vital therapeutic agents are substrates to P-gp and their bioavailability is lowered or a resistance is induced because of the protein efflux. Hence P-gp inhibitors were explored for overcoming multidrug resistance and poor bioavailability problems of the therapeutic P-gp substrates. The sensitivity of drug moieties to P-gp and vice versa can be established by various experimental models in silico, in vitro and in vivo. Ever since the discovery of P-gp, the research plethora identified several chemical structures as P-gp inhibitors. The aim of this review was to emphasize on the discovery and development of newer, inert, non-toxic, and more efficient, specifically targeting P-gp inhibitors, like those among the natural herb extracts, pharmaceutical excipients and formulations, and other rational drug moieties. The applications of cellular and molecular biology knowledge, in silico designed structural databases, molecular modeling studies and quantitative structure-activity relationship (QSAR) analyses in the development of novel rational P-gp inhibitors have also been mentioned.

Campylobacter jejuni type VI secretion system: roles in adaptation to deoxycholic acid, host cell adherence, invasion, and in vivo colonization.

The recently identified type VI secretion system (T6SS) of proteobacteria has been shown to promote pathogenicity, competitive advantage over competing microorganisms, and adaptation to environmental perturbation. By detailed phenotypic characterization of loss-of-function mutants, in silico, in vitro and in vivo analyses, we provide evidence that the enteric pathogen, Campylobacter jejuni, possesses a functional T6SS and that the secretion system exerts pleiotropic effects on two crucial processes – survival in a bile salt, deoxycholic acid (DCA), and host cell adherence and invasion. The expression of T6SS during initial exposure to the upper range of physiological levels of DCA (0.075%–0.2%) was detrimental to C. jejuni proliferation, whereas down-regulation or inactivation of T6SS enabled C. jejuni to resist this effect. The C. jejuni multidrug efflux transporter gene, cmeA, was significantly up-regulated during the initial exposure to DCA in the wild type C. jejuni relative to the T6SS-deficient strains, suggesting that inhibition of proliferation is the consequence of T6SS-mediated DCA influx. A sequential modulation of the efflux transporter activity and the T6SS represents, in part, an adaptive mechanism for C. jejuni to overcome this inhibitory effect, thereby ensuring its survival. C. jejuni T6SS plays important roles in host cell adhesion and invasion as T6SS inactivation resulted in a reduction of adherence to and invasion of in vitro cell lines, while over-expression of a hemolysin co-regulated protein, which encodes a secreted T6SS component, greatly enhanced these processes. When inoculated into B6.129P2-IL-10tm1Cgn mice, the T6SS-deficient C. jejuni strains did not effectively establish persistent colonization, indicating that T6SS contributes to colonization in vivo. Taken together, our data demonstrate the importance of bacterial T6SS in host cell adhesion, invasion, colonization and, for the first time to our knowledge, adaptation to DCA, providing new insights into the role of T6SS in C. jejuni pathogenesis.

Effects of aripiprazole and its active metabolite dehydroaripiprazole on the activities of drug efflux transporters expressed both in the intestine and at the blood–brain barrier

The inhibition potencies of aripiprazole and its active metabolite, dehydroaripiprazole, on the activities of human multidrug resistance protein 1 (MDR1/ABCB1; P-glycoprotein), breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4), that are drug efflux transporters expressed both in the intestine and at the blood-brain barrier (BBB), were investigated. Aripiprazole and dehydroapripiprazole showed relatively strong inhibitory effects on human MDR1 with IC(50) values of 1.2 and 1.3 µm in human MDR1-transfected Mardin-Darby canine kidney (MDCKII-MDR1) cells, respectively. The inhibition potencies of other atypical antipsychotics (risperidone, paliperidone, olanzapine and ziprasidone) for human MDR1 were also evaluated using the same in vitro experimental system and IC(50) values were more than 10-fold higher than those of the two compounds. Aripiprazole and dehydroaripiprazole also had inhibition potencies against human BCRP with IC(50) values of 3.5 and 0.52 µm, respectively. The ratios of steady-state unbound concentrations of aripiprazole and dehydroaripiprazole to their IC(50) values against human MDR1 and BCRP activities were less than 0.1, whereas the theoretically maximum gastrointestinal concentration of aripiprazole ([I](2) ) to its IC(50) values was much higher than the cut-off value of 10, proposed by the International Transporter Consortium (ITC) and the Food and Drug Administration (FDA). In contrast, aripiprazole and dehydroaripiprazole showed almost no inhibitory effect against the activity of human MRP4. These findings indicate that aripiprazole is unlikely to cause drug-drug interactions (DDIs) at the BBB when co-administered with substrate drugs of these drug transporters investigated. However, interactions at the intestinal absorption process may be of concern.

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Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells

The blood-brain barrier (BBB) plays an important role in brain health and is often compromised in disease. Moreover, as a result of its significant barrier properties, this endothelial interface restricts neurotherapeutic uptake. Thus, a renewable source of human BBB endothelium could prove enabling for brain research and pharmaceutical development. Herein, we demonstrate that endothelial cells generated from human pluripotent stem cells (hPSCs) can be specified to possess many BBB attributes, including well-organized tight junctions, expression of nutrient transporters, and polarized efflux transporter activity. Importantly, hPSC-derived BBB endothelial cells respond to astrocytic cues yielding impressive barrier properties as measured by transendothelial electrical resistance (1450±140 Ωxcm2) and molecular permeability that correlates well with in vivo brain uptake. In addition, specification of hPSC-derived BBB endothelial cells occurs in concert with neural cell co-differentiation via Wnt/β-catenin signaling, consistent with previous transgenic studies. This study represents the first example of organ-specific endothelial differentiation from hPSCs.

Estrone‐3‐Sulfate is a substrate to verify functionality of uptake transporters in primary hepatocytes

BackgroundPrimary mammalian hepatocytes are used for several in vitro applications like testing of drug metabolism, toxicity and transporter assays. However, little is known about species specific differences or similarities in the activity of uptake and efflux transporter. Therefore, we started a species specific characterization and comparison of uptake transporters functionalities in relevant species used for drug testing. Here, we report the uptake of estrone‐3‐sulfate (E3S) in primary hepatocyte cultures of human, rat and monkey.MethodsHepatocytes were incubated in serum free media. Two ‐ four days after cell isolation a time and concentration dependent uptake of [3H]‐E3S was measured using liquid scintillation counting. Rifampicin was added in various concentrations to measure its effects on E3S uptake.ResultsAll hepatocytes showed at 37 °C a time‐dependent and saturable increase in E3S uptake compared to the uptake at 4 °C. E3S revealed a high affinity to human (Km = 12.9 ± 10.1 μmol/l; Vmax = 84.2 ± 30.3 pmol/mg × min) and monkey (Km = 8.2 ± 2.3 μmol/l; Vmax = 31.3 ± 3.6 pmol/mg × min) but not to rat hepatocytes. Rifampicin clearly inhibited the E3S uptake in monkey hepatocytes while rat hepatocytes were not influenced.ConclusionOur data suggest that [3H]‐E3S is a suitable substrate to verify the functionality of uptake transporters in primary hepatocytes of humanoids but not of rats.

Blood–Brain Barrier Permeation and Efflux Exclusion of Anticholinergics Used in the Treatment of Overactive Bladder

Overactive bladder (OAB) is a common condition, particularly in the elderly. Anticholinergic agents are the mainstay of pharmacological treatment of OAB; however, many anticholinergics can cross the blood-brain barrier (BBB) and may cause central nervous system (CNS) effects, including cognitive deficits, which can be especially detrimental in older patients. Many anticholinergics have the potential to cause adverse CNS effects due to muscarinic (M(1)) receptor binding in the brain. Of note, permeability of the BBB increases with age and can also be affected by trauma, stress, and some diseases and medications. Passive crossing of a molecule across the BBB into the brain is dependent upon its physicochemical properties. Molecular characteristics that hinder passive BBB penetration include a large molecular size, positive or negative ionic charge at physiological pH, and a hydrophilic structure. Active transport across the BBB is dependent upon protein-mediated transporter systems, such as that of permeability-glycoprotein (P-gp), which occurs only for P-gp substrates, such as trospium chloride, darifenacin and fesoterodine. Reliance on active transport can be problematic since genetic polymorphisms of P-gp exist, and many commonly used drugs and even some foods are P-gp inhibitors or are substrates themselves and, due to competition, can reduce the amount of the drug that is actively transported out of the CNS. Therefore, for drugs that are preferred not to cross into the CNS, such as potent anticholinergics intended for the bladder, it is optimal to have minimal passive crossing of the BBB, although it may also be beneficial for the drug to be a substrate for an active efflux transport system. Anticholinergics demonstrate different propensities to cross the BBB. Darifenacin, fesoterodine and trospium chloride are substrates for P-gp and, therefore, are actively transported away from the brain. In addition, trospium chloride has not been detected in cerebrospinal fluid assays and does not appear to have significant CNS penetration. This article reviews the properties of anticholinergics that affect BBB penetration and active transport out of the CNS, discusses issues of increased BBB permeability in patients with OAB, and examines the clinical implications of BBB penetration on adverse events associated with anticholinergics.

Zidovudine and Ursodeoxycholic Acid Conjugation: Design of a New Prodrug Potentially Able To Bypass the Active Efflux Transport Systems of the Central Nervous System

We have synthesized a new prodrug obtained by the 5'-ester conjugation of zidovudine (AZT), an antiviral agent substrate of active efflux transport systems (AET), with ursodeoxycholic acid (UDCA), a bile acid able to permeate into the central nervous system (CNS). We have demonstrated, by HPLC analysis, that UDCA-AZT is quickly hydrolyzed in rat plasma and whole blood (half-life <10 s). The same compound was hydrolyzed with slower rates in human plasma (half-life =7.53 ± 0.44 h) and whole blood (half-life =3.71 ± 0.16 h), allowing to control the AZT release. UDCA-AZT appeared hydrolyzed also in rat brain (half-life = 7.24 ± 0.45 min) and liver homogenates (half-life = 2.70 ± 0.14 min). In the aim to study the permeation properties of the UDCA-AZT across physiological barriers, we have used an established human retinal pigment epithelium (HRPE) cell line to obtain a polarized cell monolayer showing epithelial features. The bidirectional permeation of 30 μM AZT across this monolayer was regulated by apparent permeability coefficients (P(E)) higher from the apical to basolateral compartments (P(E) = 209 ± 4 × 10⁻⁵ cm/min) than in the opposite way (P(E) = 133 ± 8 × 10⁻⁵ cm/min), in conformity with the in vivo behavior of AZT, actively effluxed from the CNS. The influx (P(E) = 39.1 ± 1.2 × 10⁻⁵ cm/min) and efflux (P(E) = 31.3 ± 3.6 × 10⁻⁵ cm/min) permeability coefficients of 30 μM UDCA-AZT were instead the same, suggesting the ability of the prodrug to avoid the AET systems and, potentially, to allow its accumulation in the CNS. The relatively low P(E) values of UDCA-AZT were associated with a partial hydrolysis during its permeation across the cell monolayer.

In Silico Modeling for the Nonlinear Absorption Kinetics of UK-343,664: A P-gp and CYP3A4 Substrate

The aim of this work was to extrapolate in vitro and preclinical animal data to simulate the pharmacokinetic parameters of UK-343,664, a P-glycoprotein (P-gp) and CYP3A4 substrate, in human. In addition, we aimed to develop a simulation model to demonstrate the involvement and the controversial complex interaction of intestinal P-gp and CYP3A4 in its nonlinear absorption, first-pass extraction, and pharmacokinetics using the advanced compartmental absorption and transit (ACAT) model. Finally, we aimed to compare the results predicted from the model to the reported findings in human clinical studies. In situ perfusion, allometric scaling, PBPK Rodger mechanistic approach, in vitro metabolism, and fitting to in vivo data were used to mechanistically explain the absorption, distribution and metabolism, respectively. GastroPlus was used to build the integrated simulation model in human for UK-343,664 to mechanistically explain the observed clinical data at 30, 100, 200, 400, and 800 mg oral doses. The measured in vitro value for CYP3A4 K(m) (465 μM) in rCYPs was converted to units of μg/mL, corrected for assumed microsomal binding (17.8%) and applied to all metabolic processes. The measured in vitro values of V(max) for CYP3A4 (38.9 pmol/min/pmol), 2C8, 2C9, 2C19, and 2D6 were used along with the in vitro CYP3A4 K(m) to simulate liver first pass extraction and systemic clearance. The measured in vitro values of V(max) for CYP3A4 and 2D6 were used along with the in vitro CYP3A4 K(m) to simulate gut first pass extraction. V(max) and K(m) values for P-gp were obtained by fitting to in vivo data and used to simulate gut efflux transport activity. Investigation of the interaction mechanism of P-gp and CYP3A4 in the intestine was achieved by comparing the influence of a virtual knockout of P-gp or gut metabolism on the fraction absorbed, fraction reaching the portal vein, and fraction metabolized in the gut. Comparison between simulation and in vivo results showed that the in silico simulation provided a mechanistic explanation of the observed nonlinear absorption kinetics of UK-343,664 in human following its administration in the range of 30-800 mg as oral solutions. The simulation results of the pharmacokinetic parameters, AUC and C(max), by GastroPlus were comparable with those observed in vivo. This simulation model is one possible mechanistic explanation of the observed in vivo data and suggests that the nonlinear dose dependence could be attributed to saturation of both the efflux transport by P-gp and the intestinal metabolism. However, the concentration ranges for either protein saturation did not overlap and resulted in much greater than dose proportional increases in AUC. At low doses, producing intraenterocyte concentrations below the fitted value of K(m) for P-gp, the influence of P-gp appears to be protective and results in a lower fraction of gut 3A4 metabolism. At higher doses, as P-gp becomes saturated the fraction of gut 3A4 extraction increases, and eventually at the highest doses, where 3A4 becomes saturated, the fraction of gut 3A4 extraction again decreases. Such a complex interpretation of this in vitro-in vivo extrapolation (IVIVE) is another example of the value and insight obtained by physiologically based absorption simulation.

ES2-2: Mouse Models of Basal-Like Breast Cancer.

Abstract Mouse models of human cancer provide powerful in vivo tools to study the mechanisms underlying drug response and acquired resistance. Once these processes are understood in sufficient detail it may be possible to design (combination) therapies that not only cause complete remissions but also eliminate remnant cells that might elicit recurrent disease. Women carrying germline mutations in BRCA1 are strongly predisposed to developing basal-like breast cancers, which frequently contain TP53 mutations. To study the role of BRCA1 loss-of-function in development of basal-like breast cancer, we and others have established human tumor xenograft models for basal-like-breast cancer as well as genetically engineered mouse models (GEMMs) for BRCA1-mutated basal-like breast cancer based on tissue-specific deletion of p53 and Brca1 [1]. The mammary tumors that arise in these mouse models show strong similarity to basal-like breast cancer with respect to expression of basal cell markers, high tumor grade, and lack of expression of hormone receptors and HER2 [2]. In addition, the BRCA1-mutated basal-like tumors in our mouse models are characterized by a high degree of genomic instability and hypersensitivity to DNA-damaging agents due to loss of homology-directed double-strand break (DSB) repair [3]. We have successfully used the xenograft models and GEMMs of BRCA1-deficient basal-like breast cancer for preclinical evaluation of therapy response and elucidation of mechanisms of acquired drug resistance. BRCA-deficient mammary tumors are highly sensitive to PARP inhibitors and platinum drugs, but none of these drugs is capable of causing tumor eradication and all tumors grow back after drug treatment. Using functional genetic screens and mouse genetics, we found that therapy response and resistance is affected by several factors, including drug efflux transporter activity [3,4], type of BRCA1 founder mutation and 53BP1 status [5]. The lack of tumor eradication prompted us to look for additional drugs targeting BRCA-deficient tumors. Using a cell-based screening approach, we found that bifunctional alkylators such as nimustine may cause durable complete remission of BRCA-deficient mouse mammary tumors [6], suggesting that BRCA-mutated hereditary breast cancers and BRCA-like sporadic tumors may be eradicated by dose-intensive treatment with bifunctional alkylators. In support of this notion, patients with breast cancers that display a BRCA1-like profile of genomic aberrations show a high complete remission rate and long progression-free survival after treatment with high-dose alkylating chemotherapy [7].

Drug Transporters in Imatinib Resistance: A New Therapeutic Targeted Approach In CML

Abstract 5010Chronic myeloid leukaemia (CML) is a mieloproliferative disorder characterized by the presence of the BCR-ABL gene fusion, which encodes an oncoprotein with a deregulated tyrosine kinase activity. This oncoprotein became the main therapeutic target of the disease. In fact, the first-line treatment is Imatinib, a specific tyrosine inhibitor that blocks BCR-ABL tyrosine kinase activity. Although, the good results with Imatinib, the development of drug resistance is a reality and can be mediated by different pathways, besides the BCR-ABL mutations, like the unbalance between influx and efflux drug transporters expression. Therefore, the knowledge of these changes would enable the use of new therapeutics approaches in CML.The aims of this study are to evaluate the role of drug influx and efflux transporters in Imatinib resistance and the potential therapeutic of Reversin 205 in overcoming the resistance to Imatinib. For this purpose, we used a CML cell line sensitive to Imatinib, the K562 cells, and generated two sub-cell lines resistant to Imatinib, the K562-RC and K562-RD cells. To obtain these resistant cells, K562 cells were exposed to the drug following two strategies: continuous exposure of increasing concentrations of Imatinib (K562-RC cells) and discontinuous exposure, with interchanged mediums with and without Imatinib (K562-RD cells). Drug influx and efflux transporters expression levels, namely OCT1, OCTN2, PgP and BCRP, were evaluated by flow cytometry (FC) using monoclonal antibodies labeled with fluorescent probes. Functional activity of PgP was assessed by a kinetic study using the radiosensitizer 99mTc Sestamibi. To evaluate the effect of Imatinib and Reversin 205, a P-gP inhibitor, on cell viability, all cell lines were treated in the absence and presence of different drug’s concentrations and analyzed by the resazurin assay. Cell death was analyzed by optical microscopy (May-Grünwald staining) and by FC using the Annexin V and Propidium Iodide double staining and caspases expression levels.Our results show that the half maximal inhibitory concentration (IC50) of Imatinib in K562 sensitive cells is 75nM, whereas in K562-RC cells this value is increased eight times (to 605nM) and in K562-RD cells is around eighteen times higher (1389nM). On the other hand, we observed a decrease in OCT1 and OCT2 expression levels and an increase in PgP and BCRP expression in resistant cells compared with the sensitive cell line, which may contribute to the resistance to Imatinib. Besides the alterations in proteins expression levels we found, in resistant cell lines, a lower uptake profile of the radiosensitizer, demonstrating the functional activity of the MDR protein efflux transporter, Pgp. Concordantly with these results, we observed that when resistant cell lines were exposed to the combination of Reversin 205 (5μM), plus increasing concentrations of Imatinib, the IC50 decreased to 170nM in K562 RC and to 367nM in K562 RD. These results suggest that Reversin 205 may circumvent resistance to imatinib in CML cells. On the other hand, at higher doses, this compound revealed a cytotoxic effect in sensitive and resistant cells, and induces cell death by apoptosis in a time- and dose- dependent manner. The IC50 at 48 hours of exposure was of 12μM to K562, 18,9 μM to K562-RD and 18,7 μM to K562-RC cells.In conclusion, our results suggest that discontinuous drug exposure induces higher resistance levels and drug's transporters are involved in the acquisition of resistance to Imatinib. The use of Reversin 205 or other drug efflux transporters inhibitors might circumvent this resistance and could be used as a new potential therapeutic targeted approach in CML. Disclosures:No relevant conflicts of interest to declare.

Predicting Phenolic Acid Absorption in Caco-2 Cells: A Theoretical Permeability Model and Mechanistic Study

There is a considerable need to rationalize the membrane permeability and mechanism of transport for potential nutraceuticals. The aim of this investigation was to develop a theoretical permeability equation, based on a reported descriptive absorption model, enabling calculation of the transcellular component of absorption across Caco-2 monolayers. Published data for Caco-2 permeability of 30 drugs transported by the transcellular route were correlated with the descriptors 1-octanol/water distribution coefficient (log D, pH 7.4) and size, based on molecular mass. Nonlinear regression analysis was used to derive a set of model parameters a', β', and b' with an integrated molecular mass function. The new theoretical transcellular permeability (TTP) model obtained a good fit of the published data (R² = 0.93) and predicted reasonably well (R² = 0.86) the experimental apparent permeability coefficient (P(app)) for nine non-training set compounds reportedly transported by the transcellular route. For the first time, the TTP model was used to predict the absorption characteristics of six phenolic acids, and this original investigation was supported by in vitro Caco-2 cell mechanistic studies, which suggested that deviation of the P(app) value from the predicted transcellular permeability (P(app)(trans)) may be attributed to involvement of active uptake, efflux transporters, or paracellular flux.

Prodrugs and Endogenous Transporters: Are They Suitable Tools for Drug Targeting into the Central Nervous System?

Hydrophilic drugs, or neuroactive agents characterized by high molecular weight, do not have the physico-chemical properties required for passive diffusion across the blood brain barrier (BBB). The prodrug approach by lipidization of hydrophilic drugs generally allows to sensibly increase their permeability across BBB, even if this phenomenon is often not associated to an effective entry into the brain of the lipidized drugs. It has been understood that active efflux transporters (AET) can have a very important role in extruding from the brain not only prodrugs obtained by lipidization processes, but also lipophilic drugs. On the other hand, it has been also demonstrated that carrier mediated transporters (CMT), able to transfer essential nutrients and hormones from the bloodstream to the CNS, can be employed for the brain targeting of appropriated designed prodrugs. This approach consists on the chemical modification of a drug into a "pseudonutrient" or, differently, on drug conjugation to essential nutrients transported by CMT systems. This review focuses the molecular aspects that regulate the activity of the CMT and AET systems for the transport of their substrates, taking into account the in vitro and in vivo studies related to these transporters. The studies are described and summarized in the aim to evaluate the molecular keys for the design of prodrugs efficacious in the brain targeting. Among these, the molecular Trojan horses systems are briefly illustrated as carriers for the transport in the brain of large molecular weight neuroactive agents.

Influence of time to achieve substrate distribution equilibrium between brain tissue and blood on quantitation of the blood–brain barrier P-glycoprotein effect

Active efflux transport processes at the blood–brain barrier (BBB), such as P-glycoprotein (P-gp)-mediated efflux, can limit brain uptake of therapeutics. Accurate determination of the consequent impact on brain uptake is assumed to require sampling post-attainment of brain-to-blood distribution equilibrium. Because this approach is not always feasible, understanding the relationship between apparent degree of efflux (e.g., calculated BBB P-gp effect) and the fraction of time remaining until distribution equilibrium is achieved (FTDE) would be advantageous. This study employed simulation strategies to explore this relationship in the simplest relevant system (absence of protein binding, saturable uptake, or metabolism at the BBB). Concentration-time profiles were simulated with a 4-compartment system (blood, peripheral tissues, BBB endothelium and brain parenchyma). A unidirectional endothelium-to-blood rate constant, PSe, represented P-gp-mediated efflux. A parameter space was selected to simulate an 18-fold P-gp effect, (Kp,brain at distribution equilibrium in the absence [Kp,brain=82] vs. presence [Kp,brain=4.5] of P-gp-mediated flux), as observed for paclitaxel in P-gp-deficient vs. P-gp-competent mice. Hypothetical compounds with different P-gp effects, peripheral compartment distribution kinetics, or times to achieve distribution equilibrium were simulated by perturbing the values of relevant model parameters. P-gp effects calculated prior to attainment of distribution equilibrium may be substantially erroneous. However, reasonably accurate estimates can be obtained relatively early in the net distributional phase (under 20% error at FTDE>0.36 or 0.11 for bolus or infusion administration, respectively). Potential errors associated with non-equilibrium calculations are dependent on both P-gp-mediated and P-gp-independent components of flux across the BBB.

PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters In Vivo

Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer's and Parkinson's disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications.

Abstract C69: Ontogenetic switches in activity and location of multidrug efflux transporters during embryonic development

Abstract Multidrug resistance, mediated by ATP binding cassette (ABC) transporters, is a major obstacle to the treatment of metastatic cancer. This phenomenon has eluded treatment by direct pharmacological inhibition of the multidrug transporters responsible for resistance. An alternative strategy could be to control drug resistance by targeting the cellular regulation of insertion and retrieval of transporters from the plasma membrane. However, the pleiomorphic nature of cancer cells and tumors complicates the identification of core machinery involved in regulation of transporter dynamics. Embryos have natural, rapid switches in multidrug efflux activity, coincident with the normal reorganization of cell surfaces at fertilization and at epithelial to mesenchymal transitions. We are focusing on two major changes in surface organization and transport activity occurring during the first 24 hours of sea urchin development. As basal deuterostomes, the branch of evolution leading to vertebrates, sea urchins provide a tractable system in which core machinery regulating multidrug efflux transporters can be manipulated and characterized. The first occurs at fertilization and involves the activation of transport by shortrange, actin-dependent translocation of ABCB1a (aka P-gp, MDR1). Super-resolution fluorescence microscopy reveals that transporters move to the to the distal tips of microvilli, possibly to overcome diffusive limits in the boundary layer between microvilli. The second switch in activity is a selective downregulation of multidrug efflux transport that occurs in germline progenitors of these animals and results in long-term reduction of transport. It is mediated by endocytosis of the proteins in those cells coincident with changes in apicobasal cell polarity. We are currently investigating the sequence of events that leads to pump retrieval in these cells. The implications of this work are new insights into the molecular switches that govern acquisition and loss of transporter-mediated drug resistance during differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C69.

Inhibition of breast cancer resistance protein at the murine blood-brain barrier by Ko143 studied with [11C]tariquidar and PET

Background The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are expressed in the blood-brain barrier (BBB), where they impede brain uptake of their substrates by active efflux transport. BCRP has recently been shown to be the quantitatively most important ABC transporter at the human BBB. Inhibition of BCRP by inhibitors such as the fumitremorgin C derivative Ko143 [1] may be an interesting strategy to improve brain uptake of BCRP substrates. The aim of this study was to assess the dose-response relationship of Ko143 for inhibition of Bcrp1 at the murine BBB using small-animal positron emission tomography (PET) together with the dual P-gp/BCRP substrate radiotracer [C]tariquidar.

11C]Elacridar as a novel P-glycoprotein PET tracer, assessment of whole-body distribution and radiation dosimetry in humans

Background The ATP-binding cassette transporter P-glycoprotein (P-gp) is expressed at the blood-brain barrier (BBB) where it protects the brain from toxic substances and xenobiotics by active efflux transport. The C-labelled third-generation P-gp inhibitor [C]elacridar was developed as a positron emission tomography (PET) tracer for the in vivo quantification of P-gp expression levels in different organs. The aim of this study was to provide human dosimetry estimates for [C]elacridar based on whole-body PET.

Differential effect of P-gp and MRP2 on cellular translocation of gemifloxacin

Fluoroquinolones are broad spectrum antibiotics widely indicated in the treatment of both human and animal diseases. The primary objective of this study was to assess short and long term affinities of gemifloxacin towards efflux transporters (P-gp, MRP2) and nuclear hormone receptor (PXR). Uptake and dose dependent inhibition studies were performed with [ 14C] erythromycin (0.25 μCi/ml) on MDCKII-MDR1 and MDCKII-MRP2 cells. Cellular accumulation of calcein-AM was further determined to confirm the affinity of gemifloxacin towards P-gp and MRP2. Transport studies were conducted to determine bi-directional permeability and to assess efflux ratio of gemifloxacin. LS-180 cells were treated with three different concentrations of gemifloxacin for 72 h and real-time PCR analysis was performed to study the quantitative gene expression levels of PXR, MDR1 and MRP2. Further, [ 14C] erythromycin uptake was also performed on LS-180 treated cells to better delineate the functional activity of efflux transporters. Results from our study suggest that gemifloxacin may be a substrate of both the efflux transporters studied. This compound inhibited both P-gp and MRP2 mediated efflux of [ 14C] erythromycin in a dose dependent manner with IC 50 values of 123 ± 2 μM and 16 ± 2 μM, respectively. The efflux ratio of [ 14C] erythromycin lowered from 3.56 to 1.63 on MDCKII-MDR1 cells and 4.93 to 1.26 on MDCKII-MRP2 cells. This significant reduction in efflux ratio further confirmed the substrate specificity of gemifloxacin towards P-gp and MRP2. Long term exposure significantly induced the expression of PXR (18 fold), MDR1 (6 fold) and MRP2 (6 fold). A decrease (20%) in [ 14C] erythromycin uptake further confirmed the elevated functional activity of P-gp and MRP2. In conclusion, our studies demonstrated that gemifloxacin is effluxed by both P-gp and MRP2. Long term exposure induced their gene expression and functional activity. This substrate specificity of gemifloxacin towards these efflux transporters may be one of the major factors accounting for low oral bioavailability (71%). Better understanding of these mechanistic interactions may aid in the development of newer strategies to achieve adequate therapeutic levels and higher bioavailability.

Induction of expression and functional activity of P-glycoprotein efflux transporter by bioactive plant natural products

The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug’s therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived γ-tocotrienol, the extra-virgin olive oil-derived secoiridoid oleocanthal, and the triterpene acid asiatic acid derived from Melaleuca ericifolia and abundant in several other common plant dietary supplements. Treatment with 25μM of cembratriene, oleocanthal, γ-tocotrienol, or asiatic acid showed 2.3–3.0-fold increase in P-gp expression as demonstrated by Western blotting. These results were consistent with those obtained by quantitative analysis of fluorescent micrographs for P-gp. Accumulation studies demonstrated 31–38% decrease in rhodamine 123 intracellular levels when LS-180 cells were treated with the investigated compounds as a result of P-gp induction. Bioactive natural products can up-regulate the P-gp expression and functionality, which may induce herb/food-drug interactions when concomitantly used with P-gp substrate drugs.