3-(6-isobutyl-9-methoxy-1-4-dioxo-1-2-3-4-6-7-12-12a-octahydropyrazino(1--2--1-6)pyrido(3-4-b)indol-3-yl)propionic-acid-tert-butyl-ester and verlukast

Demethoxycurcumin (DMC) is a safe and natural food-coloring additive, as well as an agent with several therapeutic properties. However, extensive glucuronidation in vivo has resulted in its poor bioavailability. In this study, we aimed to investigate the formation of DMC-O-glucuronides by uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) and its transport by breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in HeLa cells stably transfected with UGT1A1 (named HeLa1A1 cells). The chemical inhibitors Ko143 (a selective BCRP inhibitor) and MK571 (a pan-MRP inhibitor) both induced an obvious decrease in the excretion rate of DMC-O-glucuronides and a significant increase in intracellular DMC-O-glucuronide concentrations. Furthermore, BCRP knock-down resulted in a marked reduction in the level of excreted DMC-O-glucuronides (maximal 55.6%), whereas MRP1 and MRP4 silencing significantly decreased the levels of excreted DMC-O-glucuronides (a maximum of 42.9% for MRP1 and a maximum of 29.9% for MRP3), respectively. In contrast, neither the levels of excreted DMC-O-glucuronides nor the accumulation of DMC-O-glucuronides were significantly altered in the MRP4 knock-down HeLa cells. The BCRP, MRP1 and MRP3 transporters were identified as the most important contributors to the excretion of DMC-O-glucuronides. These results may significantly contribute to improving our understanding of mechanisms underlying the cellular disposition of DMC via UGT-mediated metabolism.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; Biological Availability; Diarylheptanoids; Diketopiperazines; Food Coloring Agents; Gene Silencing; Glucuronides; Glucuronosyltransferase; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Propionates; Protein Transport; Quinolines; Transfection

Active efflux transport of glucuronides out of cells is a critical process in elimination of drugs and food-derived compounds. Wushanicaritin, a natural polyphenol from Epimedium species, has shown many biological activities. However, the transporters responsible for excretion of wushanicaritin glucuronides still remain undefined. Herein, chemical inhibitors (Ko143, MK571, dipyridamole and leukotriene C4) and single stable knocked-down efflux transporters (BCRP, MRP1, MRP3 and MRP4) were used to determine the contributions of efflux transporters to glucuronide efflux and cellular glucuronidation in UGT1A1-overexpressing HeLa cells (HeLa1A1). Knock-down of transporters was performed by stable transfection of short hairpin RNA (shRNA) using lentiviral vectors. The HeLa1A1 cell lysate catalyzed wushanicaritin glucuronidation, generating wushanicaritin-3-O-glucuronide and wushanicaritin-7-O-glucuronide. Ko143 (a dual inhibitor of BCRP, 5-20 μM) caused a marked decrease in excretion rate (maximal 53.4%) and increase of intracellular glucuronides (maximal 86.0%), while MK-571 (an inhibitor of MRPs, 5-20 μM) resulted in a significant reduction in excretion rate (maximal 64.6%) and rise of intracellular glucuronides (maximal 98.0%). By contrast, dipyridamole and leukotriene C4 showed no inhibitory effects on glucuronide excretion. Furthermore, shRNA-mediated silencing of a target transporter led to a marked reduction in the excretion rate of wushanicaritin glucuronides (maximal 33.8% for BCRP; 25.9% for MRP1; 26.7% for MRP3; 39.3% for MRP4). Transporter silencing also led to substantial decreases in efflux clearance (maximal 61.5% for BCRP; 48.7% for MRP1; 35.1% for MRP3; 63.1% for MRP4). In conclusion, chemical inhibition and gene silencing results suggested that BCRP, MRP1, MRP3 and MRP4 were significant contributors to excretion of wushanicaritin glucuronides.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; Biological Transport; Diketopiperazines; Dipyridamole; Drugs, Chinese Herbal; Epimedium; Flavonoids; Gene Knockdown Techniques; Gene Silencing; Glucuronides; Glucuronosyltransferase; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Propionates; Quinolines; RNA, Small Interfering

Neobavaisoflavone is one of flavonoids of traditional Chinese medicine Psoralea corylifolial. It has numerous biological properties such as antibacterial, anti-inflammatory, anti-cancer, and anti-osteoporosis effects. This paper aimed to investigate the absorption mechanism of neobavaisoflavone in Caco-2 cell monolayer model. The analyte and osalmide were separated on Thermo Syncronis C18 column with methanol-0.1% formic acid solution (90∶10) as the mobile phase, at a flow rate of 0.2 mL•min⁻¹. The concentration of neobavaisoflavone was determined in eletrospray ionization(ESI) positive ion mode with osalmide as an the internal standard. The effects of time, concentration, P-gp inhibitor verapamil, MRP-2 inhibitor MK-571 and BCRP inhibitor Ko143 on the absorption of neobavaisoflavone were investigated. According to the results, neobavaisoflavone showed a good linearity within the concentration of 10-2 000 μg•L⁻¹, and the results of its specificity, matrix effect, extraction recovery, precision, accuracy and stability all met the requirements. In the Caco-2 cell monolayer model, the transport volume of neobavaisoflavone was correlated positively with the time and concentration. The ER values of 15, 30, 50 μmol•L⁻¹ neobavaisoflavone were 1.64, 1.94,0.99, respectively. As compared with the control group, all of verapamil hyduochloride, MK-571 and Ko143 could promote the transportation of neobavaisoflavone, and the effect was more obvious in verapamil hyduochloride and Ko143. The absorption of neobavaisoflavone may be mainly of active transport in Caco-2 cell monolayer model, and also involve passive transport. Excretion mechanism of intestinal transport protein may be also involved.

Topics: Biological Transport; Caco-2 Cells; Diketopiperazines; Heterocyclic Compounds, 4 or More Rings; Humans; Intestinal Absorption; Isoflavones; Multidrug Resistance-Associated Protein 2; Propionates; Quinolines

Bazedoxifene, a novel selective estrogen receptor modulator, has complex pharmacokinetics with rapid absorption, high metabolic clearance, low oral bioavailability (6.25 %) and a slow elimination phase. Our hypothesis is that drug uptake and efflux transporters may play an important role in its disposition. To adequately cover all aspects of bazedoxifene transport, several approaches were undertaken: PAMPA assay, ATPase assay, membrane inside-out vesicles and Caco-2 and CHO cell lines. The results obtained from PAMPA experiments showed moderate passive permeability of bazedoxifene (P app ≈ 2 × 10(-6)cm/s), suggesting the existence of an active transport during the rapid absorption phase. The Caco-2 transport assay showed large and significant changes in the measured efflux ratios of bazedoxifene when selective transporter inhibitors were applied: verapamil (a Pgp inhibitor), MK571 (an MRP inhibitor), Ko143 (a BCRP inhibitor) and DIDS (an OATP inhibitor). Additionally, membrane preparation experiments demonstrated the interaction of bazedoxifene with P-gp, MRP2 and BCRP. CHO experiments did not show any interactions of bazedoxifene with OATP1B1 or OATP1B3; therefore, bazedoxifene may be a substrate of other OATP isoform(s). The comprehensive in vitro study indicates a strong involvement of Pgp, MRP, BCRP and OATP in bazedoxifene disposition.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; Biological Transport, Active; Caco-2 Cells; Cell Line; Cell Line, Tumor; CHO Cells; Cricetulus; Diketopiperazines; Heterocyclic Compounds, 4 or More Rings; Humans; Indoles; Liver-Specific Organic Anion Transporter 1; Membrane Transport Proteins; Multidrug Resistance-Associated Proteins; Organic Anion Transporters, Sodium-Independent; Permeability; Propionates; Quinolines; Verapamil

The purpose of this study was to determine whether quercetin decreases the uptake of aristolochic acid I (AAI) from the apical membranes of Caco-2 cells via H(+) -linked MCTs at neutral pH as well as to confirm the secretion of AAI through the Caco-2 cell monolayers via ABC transporters.. Caco-2 cells cultured on the dishes or permeable membranes were incubated with AAI in the absence or presence of quercetin or transporter inhibitors.. Coincubation with quercetin decreased the uptake of AAI by Caco-2 cells cultured on the dishes at pH 7.4, and a similar decrease in AAI uptake was found when the cells were coincubated with acetic acid or benzoic acid. In contrast, the basolateral-to-apical transport of AAI was higher than the apical-to-basolateral transport of AAI at pH 7.4, and the former transport was decreased by quercetin and the BCRP inhibitors of Ko-143 and mitoxantrone, but not by P-gp or MRP2 inhibitors.. AAI appears to be secreted from the apical membranes of Caco-2 cells via BCRP at neutral pH, although a small amount of AAI is taken up from the apical membranes via H(+) -linked MCTs, and quercetin may decrease both the BCRP-mediated efflux and the MCT-mediated influx of AAI.

Topics: Acetic Acid; Aristolochic Acids; ATP-Binding Cassette Transporters; Benzoic Acid; Biological Transport; Caco-2 Cells; Cells, Cultured; Cyclosporine; Diketopiperazines; Heterocyclic Compounds, 4 or More Rings; Humans; Hydrogen-Ion Concentration; Indomethacin; Intestinal Absorption; Mitoxantrone; Pravastatin; Propionates; Quercetin; Quinidine; Quinolines

Efflux transport is a critical determinant to the pharmacokinetics of sulfate conjugates. Here we aimed to establish SULT1A3 stably transfected HEK293 cells, and to determine the contributions of BCRP and MRP transporters to excretion of chrysin and apigenin sulfates. The cDNA of SULT1A3 was stably introduced into HEK293 cells using a lentiviral vector, generating a sulfonation active cell line (i.e., SULT293 cells). Identification of sulfate transporters was achieved through chemical inhibition (using chemical inhibitors) and biological inhibition (using short-hairpin RNAs (shRNAs)) methods. Sulfated metabolites were rapidly generated and excreted upon incubation of SULT293 cells with chrysin and apigenin. Ko143 (a selective BCRP inhibitor) did not show inhibitory effects on sulfate disposition, whereas the pan-MRP inhibitor MK-571 caused significant reductions (38.5-64.3%, p<0.001) in sulfate excretion and marked elevations (160-243%, p<0.05) in sulfate accumulation. Further, two efflux transporters (BCRP and MRP4) expressed in the cells were knocked-down by shRNA-mediated silencing. Neither sulfate excretion nor sulfate accumulation was altered in BCRP knocked-down cells as compared to scramble cells. By contrast, MRP4 knock-down led to moderate decreases (17.1-20.6%, p<0.05) in sulfate excretion and increases (125-135%, p<0.05) in sulfate accumulation. In conclusion, MRP4 was identified as an exporter for chrysin and apigenin sulfates. The SULT1A3 modified HEK293 cells were an appropriate tool to study SULT1A3-mediated sulfonation and to characterize BCRP/MRP4-mediated sulfate transport.

Topics: Adenosine; Apigenin; Arylsulfotransferase; Diketopiperazines; Dose-Response Relationship, Drug; Flavonoids; Gene Expression Regulation; Gene Knockdown Techniques; HEK293 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Sulfates

Excretion of sulfate metabolites is an essential process in disposition of raloxifene via the sulfonation pathway. However, the transporters responsible for excretion of raloxifene sulfates remain undefined. Here, sulfonation of raloxifene and excretion of its sulfate metabolites were investigated using SULT1A3-overexpressing HEK293 cells (or SULT293 cells) with significant expression of BCRP and MRP4. SULT293 cell lysate catalyzed the sulfonation of raloxifene at both 6-OH and 4'-OH groups, generating raloxifene-6-sulfate (R-6-S) and raloxifene-4'-sulfate (R-4'-S), respectively. Sulfate formation followed the Michaelis-Menten kinetics (Km = 0.49 μM and Vmax = 5.79 pmol/min/mg for R-6-S; Km = 0.33 μM and Vmax = 1.25 pmol/min/mg for R-4'-S). As expected, the recombinant SULT1A3 enzyme showed a high similarity in raloxifene sulfonation profiles with the lysate preparation. Ko143, a selective inhibitor of BCRP, significantly decreased the excretion rates of raloxifene sulfates (maximal 66.1%) while increasing the intracellular sulfates (maximal 282%). As a result, the apparent efflux clearance (CLef,app, representing the efflux efficiency of raloxifene sulfates) was substantially reduced (maximal 85.6%). Likewise, the pan-MRP inhibitor MK-571 significantly deceased the excretion rates (maximal 69.6%) and CLef,app values (maximal 96.0%) of raloxifene sulfates while increasing the intracellular sulfates (maximal 667%). Further, the short-hairpin RNA (shRNA) targeting BCRP significantly reduced (maximal 35.0%) sulfate excretion. Use of BCRP shRNA also caused significant decreases (maximal 52.5%) in the CLef,app values. Silencing of MRP4 by shRNA led to a substantial alteration in sulfate disposition (i.e., 28.6-37.8% reductions in sulfate excretion, 30.5-59.3% elevations in intracellular sulfates, and 44.8-47.7% deceases in CLef,app values). In conclusion, two sulfate metabolites R-6-S and R-4'-S were generated from raloxifene in SULT293 cells. Cellular excretion of the raloxifene sulfates was mainly mediated by BCRP and MRP4.

Topics: Arylsulfotransferase; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Diketopiperazines; Enzyme Induction; HEK293 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Kinetics; Metabolic Detoxication, Phase II; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Propionates; Quinolines; Raloxifene Hydrochloride; RNA Interference; Selective Estrogen Receptor Modulators; Sulfates; Transfection

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4(-/-) and Mrp4(+/+) mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4(-/-) and Mrp4(+/+) mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4(+/+) or Mrp4(-/-) mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.

Topics: Adenosine; Allergens; Animals; Asthma; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Bronchoalveolar Lavage Fluid; Cyclic AMP; Cytokines; Diketopiperazines; Eosinophils; Heterocyclic Compounds, 4 or More Rings; Lipopolysaccharides; Lung; Mice; Multidrug Resistance-Associated Proteins; Neutrophils; Ovalbumin; Phosphodiesterase 4 Inhibitors; Propionates; Pulmonary Disease, Chronic Obstructive; Quinolines; Rolipram; Smoking; Th2 Cells; Time Factors

Aconitum alkaloids including aconitine (AC), mesaconitine (MA), hypaconitine (HA), are highly toxic. Their hydrolysates, such as benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine, and mesaconine, are considerably less toxic. Efflux transporters, including P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein isoform 2 (MRP2), act as a first line of defence and play key roles in toxicity prevention. The aim of the present study was to determine the role of efflux transporters in the transport of Aconitum alkaloids using cultured Caco-2, MDR1-MDCKII and BCRP-MDCKII cells. Bidirectional transport assays of the Aconitum alkaloids were performed with or without P-gp (cyclosporine A and verapamil), BCRP (Ko143) and MRP2 (MK571) inhibitors. The efflux ratios (Er) of AC, MA, and HA in Caco-2 cells were 34.6±4.2, 29.7±2.1, and 15.6±2.1, respectively; those of BAC, BMA, and BHA were approximately 4, and those of aconine and mesaconine were equal to 1. The Er values of AC, MA, and HA in MDR1-MDCKII and BCRP-MDCKII cells were significantly higher than those in parental MDCKII cells. Taken together the results of Er values and intracellular amounts in the presence of inhibitors, P-gp and BCRP were involved in the transport of AC, MA and HA; and MRP2 might transport AC, MA, HA, BAC, BMA and BHA.

Topics: Aconitine; Adenosine; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Caco-2 Cells; Cyclosporine; Diketopiperazines; Dogs; Heterocyclic Compounds, 4 or More Rings; Humans; Intestinal Absorption; Madin Darby Canine Kidney Cells; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Propionates; Quinolines; Transfection; Verapamil

The purpose of the present study was to investigate the role of efflux transporters on the intestinal absorption of amtolmetin guacyl (MED-15).. The effects of P-glycoprotein (P-gp), multiple resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on intestinal absorption amount of MED-5 (tolmetin-glycine amide derivative), the metabolite formed from MED-15 in the intestinal epithelial cells were studied in the in vitro everted gut sac experiments. Moreover, the in situ single-pass intestine perfusion was adopted to clarify the role of efflux transporters in excreting MED-5 in knockout mice. The plasma concentration of MED-5 and tolmetin, the metabolite formed from MED-5 was determined in Bcrp1 knockout mice and wild-type mice.. BCRP inhibitor Ko143 (50 μM and 100 μM) significantly increased the intestinal absorption amount in jejunum, ileum and colon (p<0.05). However, no effect was observed in the presence of P-gp inhibitor verapamil and MRP2 inhibitor MK571 in each intestinal segment. Furthermore, the plasma concentration MED-5 and tolmetin, metabolites of MED-15, increased 2-fold and 4-fold, respectively, in Bcrp1 knockout mice compared with wild-type mice after the single-pass perfusion of small intestine with MED-15.. It may be concluded that BCRP plays an important role in the intestinal efflux of MED-5 and limits the bioavailability after oral administration of MED-15.

Topics: Adenosine; Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Availability; Diketopiperazines; Glycine; Heterocyclic Compounds, 4 or More Rings; Intestinal Absorption; Intestinal Mucosa; Leukotriene Antagonists; Male; Mice; Mice, Knockout; Propionates; Pyrroles; Quinolines; Rats; Rats, Sprague-Dawley; Tolmetin; Vasodilator Agents; Verapamil

Forsythiaside was characterized by low intestinal absorption by in situ rat experiment and Caco-2 cells. The mechanisms behind this low absorption had not yet been elucidated. The purpose of this study was to investigate the role of efflux transporters in the intestinal absorption of forsythiaside as a potential mechanism for its low small-intestinal absorption following oral administration. Polarized MDCKII cell lines stably transfected with human or murine complementary DNA encoding for various efflux transporters (P-gp/MDR1, MRP2 and Bcrp1) were used to study transepithelial transport of forsythiaside and compare results with the MDCKII-Wild type cells. The transportation inhibitors GF120918, MK571 and Ko143 were used to investigate the transport mechanism. The active transport of forsythiaside was found in MDCKII-WT cells. The MDCKII-MRP2 and MDCKII-Bcrp1 cells significantly increased forsythiaside efflux ratio compared with the parental cells due to the apically directed transport by MRP2 and Bcrp1, respectively. The efflux ratios in MRP2 and Bcrp1 transfected cell lines were greatly decreased in the presence of MK-571 and Ko143, respectively, which indicated that forsythiaside efflux by MRP2 and Bcrp1 were significantly inhibited by their selective inhibitors. MDCKII-MDR1 cells did not exhibit a significant reduction in the forsythiaside efflux compared with the parental cells, indicating that it was not a good substrate for MDR1. And the results were then validated by the in situ experiment. This study presents direct evidence that forsythiaside is effluxed by both MRP2 and Bcrp1, which may contribute to its poor oral bioavailability.

Topics: Adenosine; Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cells, Cultured; Diketopiperazines; Dogs; Drugs, Chinese Herbal; Glycosides; Heterocyclic Compounds, 4 or More Rings; Humans; Intestinal Absorption; Mice; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Propionates; Quinolines; Rats; Rats, Wistar

Efflux transporters expressed in the apical membrane of intestinal enterocytes have been implicated in drug oral absorption. The current study presents a strategy and tools to quantitatively predict the impact of efflux on oral absorption for new chemical entities (NCEs) in early drug discovery. Sixty-three marketed drugs with human absorption data were evaluated in the Caco-2 bidirectional permeability assay and subjected to specific transporter inhibition. A four-zone graphical model was developed from apparent permeability and efflux ratios to quickly identify compounds whose efflux activity may distinctly influence human absorption. NCEs in "zone 4" will probably have efflux as a barrier for oral absorption and further mechanistic studies are required. To interpret mechanistic results, we introduced a new quantitative substrate classification parameter, transporter substrate index (TSI). TSI allowed more flexibility and considered both in vitro and in vivo outcomes. Its application ranged from addressing the challenge of overlapping substrate specificity to projecting the role of transporter(s) on exposure or potential drug-drug interaction risk. The potential impact of efflux transporters associated with physicochemical properties on drug absorption is discussed in the context of TSI and also the previously reported absorption quotient. In this way, the chemistry strategy may be differentially focused on passive permeability or efflux activity or both.

Topics: Adenosine; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Sub-Family B Member 4; ATP-Binding Cassette Transporters; Biological Transport; Caco-2 Cells; Chromatography, Liquid; Dibenzocycloheptenes; Diketopiperazines; Drug Discovery; Heterocyclic Compounds, 4 or More Rings; Humans; Intestinal Absorption; Mass Spectrometry; Models, Biological; Neoplasm Proteins; Pharmaceutical Preparations; Predictive Value of Tests; Propionates; Quinolines; Substrate Specificity

The purpose of the study is to screen the interactions of fourth generation fluoroquinolone-gatifloxacin with efflux pumps, i.e., P-gp, MRP2 and BCRP. Mechanism of gatifloxacin interaction with efflux transporters may explain its acquired resistance. Such clarification may lead to the development of strategies to overcome efflux and enhance its bioavailability at target site. This process will aid in the reduction of dose volume, further eliminating the chances of systemic toxicity from topical gatifloxacin eye drops. MDCK cell lines transfected with the targeted efflux transporters were used for this study. [(14)C] Erythromycin was selected as a model substrate for P-gp and MRP2 whereas Hoechst 33342 was employed as a substrate for BCRP. Uptake and transport studies of these substrates were performed in the presence of gatifloxacin to delineate its interaction with efflux transporters. Further the efflux ratio in the presence of gatifloxacin was calculated from bidirectional transport studies. The concentration of [(14)C] erythromycin and Hoechst 33342 was measured using scintillation counter and fluorescence plate reader, respectively. A concentration dependent inhibition effect in the presence of gatifloxacin was revealed on [(14)C] erythromycin uptake. The efflux ratio (BL-AP/AP-BL) of substrates was found to approach unity at higher gatifloxacin concentrations. Increased concentration of gatifloxacin did not elevate uptake of Hoechst 33342. All these studies were validated with known inhibitors as positive control. Uptake and transport studies support the hypothesis that gatifloxacin is a substrate for P-gp, MRP2 but not for BCRP. Possible interactions of gatifloxacin with P-gp and MRP2 may be a possible mechanism for acquired resistance of gatifloxacin. This information can be further extended to design prodrugs or formulations in order to prevent development of acquired resistance and improve therapeutic efficacy with its reduction in side effects.

Topics: Adenosine; Adenosine Triphosphate; Animals; Anti-Bacterial Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Binding, Competitive; Biological Transport; Cell Line; Cell Polarity; Diketopiperazines; Dogs; Dose-Response Relationship, Drug; Drug Resistance, Bacterial; Erythromycin; Fluoroquinolones; Gatifloxacin; Heterocyclic Compounds, 4 or More Rings; Humans; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Propionates; Quinidine; Quinolines; Transfection

To study the inhibition patterns of the three major human ABC transporters P-gp (ABCB1), BCRP (ABCG2) and MRP2 (ABCC2), using a dataset of 122 structurally diverse drugs.. Inhibition was investigated in cellular and vesicular systems over-expressing single transporters. Computational models discriminating either single or general inhibitors from non-inhibitors were developed using multivariate statistics.. Specific (n = 23) and overlapping (n = 19) inhibitors of the three ABC transporters were identified. GF120918 and Ko143 were verified to specifically inhibit P-gp/BCRP and BCRP in defined concentration intervals, whereas the MRP inhibitor MK571 was revealed to inhibit all three transporters within one log unit of concentration. Virtual docking experiments showed that MK571 binds to the ATP catalytic site, which could contribute to its multi-specific inhibition profile. A computational model predicting general ABC inhibition correctly classified 80% of both ABC transporter inhibitors and non-inhibitors in an external test set.. The inhibitor specificities of P-gp, BCRP and MRP2 were shown to be highly overlapping. General ABC inhibitors were more lipophilic and aromatic than specific inhibitors and non-inhibitors. The identified specific inhibitors can be used to delineate transport processes in complex experimental systems, whereas the multi-specific inhibitors are useful in primary ABC transporter screening in drug discovery settings.

Topics: Acridines; Adenosine; Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Computer Simulation; Diketopiperazines; Heterocyclic Compounds, 4 or More Rings; Humans; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Propionates; Quinolines; Tetrahydroisoquinolines