Efflux Transport System Research Articles

Nonlinear Intestinal Absorption of Fluorescein Isothiocyanate Dextran 4, 000 Caused by Absorptive and Secretory Transporting System

The mechanism of the nonlinear concentration dependence of the intestinal absorption of fluorescein isothiocyanate dextran 4,000 (FD-4) was studied using in situ rat intestinal loops and the in vitro Ussing-type chamber method. The intestinal absorption rate constant of FD-4, as evaluated by the intestinal loop method, increased significantly in a nonlinear fashion as the FD-4 concentration increased up to 0.2 mM and tended to decrease at concentrations higher than 0.2 mM. The mucosal-to-serosal permeation of FD-4 across rat ileal sheets, as evaluated by the in vitro Ussing-type chamber method, also increased in a nonlinear fashion in the low concentration range (0.01 - 0.02 mM), before decreasing as the concentration increased further, whereas serosal-to-mucosal permeation decreased in a concentration-dependent manner. In addition, mucosal-to-serosal flux and serosal-to-mucosal flux were increased and reduced in the presence of the metabolic inhibitor 2, 4-dinitrophenol, respectively. These results suggest that FD-4 is predominantly secreted into the intestinal lumen by an efflux transport system.

Ranolazine-Tacrolimus Interaction

To report the case of a kidney allograft recipient on a stable regimen of tacrolimus who exhibited increased tacrolimus concentrations within 24 hours of initiating ranolazine. A 64-year-old kidney allograft recipient on a stable dose of tacrolimus (10 mg twice daily) was admitted for recent worsening of her chronic anginal pain. The patient was initiated on ranolazine 500 mg twice daily on hospital day 2. Tacrolimus concentrations rose from 7.0-10.1 ng/mL preadmission to 17.8 ng/mL within 24 hours of ranolazine initiation. Ranolazine therapy was continued due to the patient's beneficial response; therefore, the tacrolimus dose was eventually decreased by 70% to 3 mg twice daily to maintain steady-state trough concentrations between 6.6 and 7.9 ng/mL with ranolazine therapy. Ranolazine dechallenge on a subsequent admission produced subtherapeutic tacrolimus concentrations requiring dosage increases. Ranolazine, an antianginal agent, is both a substrate and a weak inhibitor of CYP3A as well as a substrate and moderate inhibitor of the P-glycoprotein (P-GP) efflux transport system. Tacrolimus, an immunosuppressant, is also a substrate of CYP3A and P-GP. Through possible inhibition of both P-GP- and CYP3A-mediated first-pass metabolism and CYP3A systemic metabolism, ranolazine may have significantly increased serum concentrations of tacrolimus necessitating an eventual 70% decrease in the tacrolimus dose. Based on the Horn Drug Interaction Probability Scale, this interaction is possible. We suggest that the eventual 70% decrease in tacrolimus dose after ranolazine initiation may indicate that ranolazine decreases the metabolism and clearance of tacrolimus, causing an elevation in tacrolimus concentrations and the potential for tacrolimus toxicity. Clinicians should be aware of this possible interaction when initiating ranolazine in patients on tacrolimus.

Involvement of influx and efflux transport systems in gastrointestinal absorption of celiprolol

Gastrointestinal absorption of several beta-blockers is inhibited by citrus juices, although molecular mechanism(s) lying on their small intestinal absorption has not yet been identified. Here, we attempted to demonstrate involvement of both influx and efflux transporters in vivo in gastrointestinal absorption of celiprolol in mice. Plasma concentration of celiprolol (3 mg/kg) after oral administration was mostly under the limit of quantification in wild mice, whereas that in mdr1a/b knockout (mdr1a/b(-/-)) mice was much more obvious, indicating P-glycoprotein-mediated efflux. Then, the oral absorption of celiprolol in mdr1a/b(-/-) mice was further examined to investigate influx transport mechanism with avoiding effect of P-glycoprotein. Coadministration of bromosulfophthalein (BSP), an inhibitor of various influx transporters including organic anion transporting polypeptide (OATP) reduced plasma celiprolol concentration. Inhibition by BSP of celiprolol uptake from apical membranes was confirmed in Ussing-type chamber of small intestinal tissues. Uptake of celiprolol by human small intestinal transporter OATP-A/1A2 was also confirmed in Xenopus Laevis oocytes. Interestingly, OATP-A/1A2 accepts various beta-blockers including acebutolol, atenolol and sotalol, oral absorption of which is inhibited by coadministration of citrus juice or telithromycin in human. Taken together, these findings have suggested fundamental role of influx transport system(s) in oral absorption of celiprolol.

Rho signaling is involved in HIV‐1‐mediated upregulation of P‐glycoprotein expression in human brain endothelial cells

Limited drug penetration into the brain is an obstacle in treatment of human immunodeficiency virus type‐1 (HIV)‐associated CNS diseases. Specific drugs used in HAART, particularly HIV‐1 protease inhibitors, are substrates for the efflux transport systems, such as P‐glycoprotein (P‐gp). P‐gp is a 170 kDa ATP‐dependent integral membrane protein and belongs to the family of ATP‐binding cassette (ABC) drug efflux transporters. We hypothesize that overexpression of P‐gp at the blood‐brain barrier level may contribute to enhanced removal of antiretroviral drugs from the brain and lead to the development of resistance to HAART. Treatment with HIV Tat protein markedly elevated GTP‐RhoA levels in human brain endothelial cells as assessed by the pull‐down assay, while the total level of RhoA was not changed. Moreover, Tat increased phosphorylation of Rho associated kinase (ROCK). Inhibition of the Rho signaling cascade by C3 exoenzyme or by Y27632 blocked Tat‐induced P‐gp overexpression. The present data indicate the critical role of the Rho signaling in upregulation of P‐gp in the brain in the context of HIV‐associated CNS diseases. It appears that the Rho cascade may be a potential target to prevent the development of HAART resistance in HIV‐infected patients. Supported by the NIH grants: MH 63022, MH 072567, NS 39254, and P42 ES 07380.

Future prospects of nanoparticles on brain targeted drug delivery

To the editor Human brain is restricted and separated from circulatory network by a highly efficient blood brain barrier. This is constituted by relatively impermeable endothelial cells with tight junctions, enzymatic activity and active efflux transport systems. Physiologically, blood-brain barrier is designed in such a manner that it can only permit the transport of molecules essential for functional activity of brain. It efficiently prevents flow of water-soluble molecules from blood circulation into central nervous system, and can also decrease concentration of lipid-soluble molecules by the action of enzymes or efflux pumps [1]. Presently, drug delivery to central nervous system is a major menace as multiple cerebral diseases like Alzheimer’s, brain tumors, prion diseases are cropping up. The blood brain barrier (BBB) represents an insurmountable obstacle for a large number of drugs including antibiotics, antineoplastic agents and a variety of central nervous system (CNS)-active drugs like neuropeptides [1, 2]. This creates a considerable threat for the therapy of cerebral diseases. Currently, nanoparticles are utilized as a drug delivery vehicle. The use of nanoparticles to deliver drugs to the brain by infiltrating blood brain barrier (BBB) may provide a significant strategy to break this impasse. The primary advantage of nanoparticle carrier technology is that it can cross blood brain barrier entrapping the original characteristics of the therapeutic drug molecule. Furthermore, this system may reduce drug leaching in the brain and decrease peripheral toxicity [3]. Drugs that have successfully been transported into the brain using nanoparticle carrier include the hexapeptide dalargin, the dipeptide kytorphin, loperamide, tubocurarine, the NMDA receptor antagonist MRZ 2/ 576, doxorubicin, etc. The nanoparticles may be special vehicle for the treatment of the brain tumors [2] especially for primary and metastatic brain tumors [4]. Currently, nanoparticle iron chelators have been used to treat Alzheimer disease and other neurologic disorders associated with trace metal imbalance [5]. It has been reported that PEGylated polymeric nanoparticles are efficient drug carrier for the delivery of active therapeutic molecules in prion diseases [6]. Strategies for nanoparticle targeting to the brain rely on the presence of its interaction with specific receptor-mediated transport systems in the BBB. For example, polysorbate 80/LDL, transferrin receptor binding antibody (such as OX26), lactoferrin, cellpenetrating peptides and melanotransferrin have been shown its efficacy of delivering a self non-transportable drug into the brain via chimeric construct that can undergo receptor-mediated transcytosis [7–11]. It has been reported that poly (butylcyanoacrylate) nanoparticles is able to deliver hexapeptide dalargin, doxorubicin and other agents into the brain which C. Chakraborty Department of Biotechnology, College of Engineering and Technology, IILM Academy of Higher Learning, Knowledge Park-II, Greater Noida, India

Food–drug interaction of (−)-epigallocatechin-3-gallate on the pharmacokinetics of irinotecan and the metabolite SN-38

The aim of the present study was to investigate the effect of (−)-epigallocatechin-3-gallate (EGCG) on the pharmacokinetics of irinotecan (CPT-11) and its metabolite SN-38. EGCG was potentially used to modulate the ATPase activity of P-glycoprotein (P-gp). Experimental Sprague–Dawley rats were treated with EGCG (20 mg/kg, i.v.) 10 min before CPT-11 (10 mg/kg, i.v.) administration, whereas the control group received CPT-11 (10 mg/kg, i.v.) only. The biological samples were prepared by the protein precipitation and detected by HPLC-fluorescence detection which provided a good separation of CPT-11 and SN-38 within 10 min. The pharmacokinetic data indicate that the area under the plasma concentration–time curves (AUC) of CPT-11 and SN-38 were increased by 57.7 and 18.3%, and AUC in bile were decreased by 15.8 and 46.8%, respectively, for the group pretreated with EGCG. The blood to bile distribution ratio (AUC bile/AUC blood) was significantly reduced after group coadministration of EGCG, it can be seen that the bile efflux transport system of CPT-11 and SN-38 may be markedly reduced by the treatment of EGCG which plays the role of P-gp inhibitor. In conclusion, EGCG was found to inhibit the transport of CPT-11 and SN-38 into the biliary elimination and their half-lives in plasma could be substantially prolonged. Based on the food–drug interaction, persons taking daily nutritional supplements should be warned of this interaction possibility.

Regulation of Choline Transport by Oxidative Stress at the Blood-Brain Barrier In Vitro Model

In the present study, we examined how the transport of choline is regulated at the blood-brain barrier (BBB) under the central nervous system (CNS) cellular damages by oxidative stress using a conditionally immortalized rat brain capillary endothelial cells (TR-BBB), in vitro the BBB model. It was also tested whether the choline uptake is influenced by membrane potential, extracellular pH, protonophore (FCCP) and amiloride in TR-BBB cells. In result, uptake was inhibited by FCCP and dependent on extracellular pH. The treatment of TR-BBB cells with 20 ng/mL tumor necrosis , 10 ng/mL lipopolysaccharide (LPS), 100 diethyl maleate (DEM) and 100 glutamate resulted in 3.0-fold, 2.6-fold, 1.8-fold and 2.0-fold increases of uptake at the respective peak time, respectively. In contrast, hydrogen peroxide and raffinose did not show any significant effects on choline uptake. In addition, choline efflux was significantly inhibited by , LPS and DEM producing cell damage states. In conclusion, the influx and efflux transport system for choline existed in TR-BBB cell line and this process was affected by several oxidative stress inducing agents.

Assessment of Intestinal Absorption of Vitexin-2″-O-Rhamnoside in Hawthorn Leaves Flavonoids in Rat Using In Situ and In Vitro Absorption Models

The purpose of this study was to investigate the absorption mechanism of vitexin-2″-O-rhamnoside, the index component in hawthorn leaves flavonoids (HLF) in the rat intestine, using two different absorption models, the in situ single-pass intestinal perfusion and the in vitro everted gut sac model. The effective permeability coefficients (Peff) in the in situ single-pass intestinal perfusion experiments and the apparent permeability coefficients (Papp) in the in vitro everted gut sac experiments were calculated. Furthermore, the influences of the P-glycoprotein inhibitors, verapamil and digoxin, on the intestinal absorption of vitexin-2″-O- rhamnoside in HLF were studied using the above-mentioned two models. Results showed that there were no significant differences in the absorption of vitexin-2″-O-rhamnoside in HLF in four segments of the rat intestine, duodenum, jejunum, ileum, and colon, and at different concentrations of HLF ranging from 0.05 mg/ml to 0.5 mg/ml (P > 0.05). The Peff values for vitexin-2″-O-rhamnoside in the rat jejunal perfusion at the concentration of 0.05, 0.1, 0.25, and 0.5 mg/ml were (2.48 ± 0.33) × 10−5; (2.23 ± 0.67) × 10−5; (2.18 ± 0.48) × 10−5; and (2.25 ± 0.17) × 10−5 cm/s, respectively. But there was significant difference between absence and presence of verapamil or digoxin (P < 0.05). Peff and Papp values of vitexin-2″-O-rhamnoside in HLF were increased in the presence of verapamil or digoxin. In conclusion, vitexin-2″-O-rhamnoside can be classified into high permeability class drug according to the biopharmaceutical classification system. Passive diffusion dominates the absorptive transport behavior of vitexin-2″-O-rhamnoside in HLF. The absorption and secretion are mediated by the efflux transport system, P-gp. The absorption of vitexin-2″-O-rhamnoside in HLF can be enhanced administered together with P-gp inhibitors.

Approach to enhancing the distribution of glycyrrhizin in the liver: intravenous infusion studies in a rat model of bile duct ligation

We adopted a pharmaceutical approach to enhancing the distribution of glycyrrhizin (GZ) in the liver based on continuous administration of GZ for chronic hepatitis therapy. The present study characterizes the changes in GZ concentration in serum, bile, and liver under steady-state conditions after drip GZ infusion (0.5-2.0 mg/ml/h) in rats with bile duct fistulas. Eight hours after infusion, approximately 90% of GZ was excreted into bile as an intact compound regardless of the GZ dose tested. We observed a non-linear relationship between steady-state serum GZ concentrations and GZ dose (i.e. serum GZ concentrations abruptly increased with increasing GZ dose). GZ concentrations in the liver 8 h after infusion paralleled those in serum under steady-state conditions, but did not parallel those in bile. These results led us to conclude that under our experimental conditions transport activity responsible for the excretion of GZ to bile was in a rate-limiting step compared to that responsible for the uptake of GZ by hepatocytes. We therefore predicted that GZ concentration in the liver will increase with obstructing an efflux transport system such as canalicular multi-specific organic anion transporters. Probenecid among inhibitors or substrates for efflux transporters significantly enhanced the liver GZ concentration in the infusion studies. In conclusion, the concomitant administration of probenecid would be useful for liver protection therapy under continuous injection of the limited amount of GZ.

Magnesium transport in hypertension

Epidemiological, clinical and experimental evidence indicates an inverse association between Mg 2+ levels (serum and tissue) and blood pressure. Magnesium may influence blood pressure by modulating vascular tone and structure through its effects on numerous biochemical reactions that control vascular contraction/dilation, growth/apoptosis, differentiation and inflammation. Magnesium acts as a calcium channel antagonist, it stimulates production of vasodilator prostacyclins and nitric oxide and it alters vascular responses to vasoactive agonists. Mammalian cells regulate Mg 2+ concentration through specialized influx and efflux transport systems that have only recently been characterized. Magnesium efflux occurs via Na 2+-dependent and Na 2+-independent pathways. Mg 2+ influx is controlled by recently cloned transporters including Mrs2p, SLC41A1, SLC41A1, ACDP2, MagT1, TRPM6 and TRPM7. Alterations in some of these systems may contribute to hypomagnesemia and intracellular Mg 2+ deficiency in hypertension. In particular increased Mg 2+ efflux through altered regulation of the vascular Na +/Mg 2+ exchanger and decreased Mg 2+ influx due to defective vascular and renal TRPM6/7 expression/activity may be important. This review discusses the role of Mg 2+ in vascular biology and implications in hypertension and focuses on the putative transport systems that control vascular magnesium homeostasis. Much research is still needed to clarify the exact mechanisms of Mg 2+ regulation in the cardiovascular system and the implications of aberrant transcellular Mg 2+ transport in the pathogenesis of cardiovascular disease.

Nesfatin-1 crosses the blood–brain barrier without saturation

Nesfatin-1 is an 82 amino acid peptide that suppresses food intake after intracerebroventricular injection. Nesfatin-1 and its precursor NUCB2 were identified by subtraction cloning in cell lines of both neuronal and adipocytic origin. This provides a strong basis for studies to determine how peripherally derived nesfatin-1 permeates the blood–brain barrier (BBB) to participate in its CNS actions and whether pharmacological delivery by the peripheral route is feasible. In this study, nesfatin-1 remained stable in blood at least 20 min after intravenous injection and permeated the BBB by a non-saturable mechanism. The influx rate of nesfatin-1 after intravenous delivery was 0.27 ± 0.11 μl/g-min, and 0.3% of nesfatin-1 reached brain parenchyma 10 min after injection. The lack of saturation of influx was shown by use of excess unlabeled nesfatin-1 in multiple-time regression analysis, capillary depletion, and in situ brain perfusion. After intracerebroventricular injection, nesfatin-1 had a half-time disappearance of 23.8 min, which was not significantly different from that of albumin. This indicates that nesfatin-1 exited the brain by bulk absorption of cerebrospinal fluid without a specific efflux transport system. We conclude that the permeation of nesfatin-1 is a non-saturable process in either the blood-to-brain or brain-to-blood direction. Thus, the limited penetration under physiological conditions does not limit the pharmacological delivery of this satiety peptide as a potential therapeutic agent.

Brain endothelium serotonin (5-HT) transporter contributes 5-HT homeostasis within the brain

Serotonergic neurons have auto-regulation system, i.e. auto-receptor (5-HT1A receptor) and re-uptake mechanism (5-HT transporter). Recent in vitro studies (Brust et al. 2000; Wakayama et al. 2002) have revealed that 5-HT transporter is present not only in the synaptic terminals of 5-HT neurons but also in the brain endothelial cells, constituting blood–brain barrier (BBB). However, the functional role of 5-HT transporter in BBB is unclear. We hypothesize that 5-HT transporter in BBB would act as an efflux transport system for augmented brain 5-HT.

P-glycoprotein mediates brain-to-blood efflux transport of buprenorphine across the blood–brain barrier

The involvement of P-glycoprotein (P-gp) in buprenorphine (BNP) transport at the blood–brain barrier (BBB) in rats was investigated in vivo by means of both the brain uptake index technique and the brain efflux index technique. P-gp inhibitors, such as cyclosporin A, quinidine and verapamil, enhanced the apparent brain uptake of [3H]BNP by 1.5-fold. The increment of the BNP uptake by the brain suggests the involvement of a P-gp efflux mechanism of BNP transport at the BBB. [3H]BNP was eliminated with an apparent elimination half-life of 27.5 min after microinjection into the parietal cortex area 2 regions of the rat brain. The apparent efflux clearance of [3H]BNP across the BBB was 0.154 ml/min/g brain, which was calculated from the elimination rate constant (2.52 × 10− 2 min− 1) and the distribution volume in the brain (6.11 ml/g brain). The efflux transport of [3H]BNP was inhibited by range from 32 to 64% in the presence of P-gp inhibitors. The present results suggest that BNP is transported from the brain across the BBB via a P-gp-mediated efflux transport system, at least in part.

Decreased blood–brain barrier permeability to fluorescein in streptozotocin-treated rats

Investigations of the blood–brain barrier (BBB) in diabetes have yielded contradictory results. It is possible that diabetes differentially affects paracellular and transcellular permeabilities via modulation of tight junction and transport proteins, respectively. Fluorescein (FL), a marker for paracellular permeability, is a substrate for the transport proteins organic anion transporter (OAT)-3 and multidrug resistance protein (MRP)-2 at the BBB. Furthermore, MRP-2-mediated efflux of FL can be upregulated by glucose. In this study, streptozotocin-induced diabetes led to decreased brain distribution of FL measured by in situ brain perfusion, consistent with activation of an efflux transport system for FL at the BBB. This change was paralleled by increased protein expression of MRP-2, but not OAT-3, in cerebral microvessels. These data indicate that diabetes may lead to changes in efflux transporters at the BBB and have implications for delivery of therapeutics to the central nervous system.

The mef (E)-Carrying Genetic Element (mega) of Streptococcus pneumoniae : Insertion Sites and Association with Other Genetic Elements

The structure of the macrolide efflux genetic assembly (mega) element, its genomic locations, and its association with other resistance determinants and genetic elements were investigated in 16 Streptococcus pneumoniae isolates carrying mef(E), of which 1 isolate also carried tet(M) and 4 isolates also carried tet(M) and erm(B). All isolates carried a mega element of similar size and structure that included the operon mef(E)-msr(D) encoding the efflux transport system. Among tetracycline-susceptible isolates, six different integration sites were identified, five of which were recognized inside open reading frames present in the R6 genome. In the five isolates also carrying tet(M), mega was inserted in different genetic contexts. In one isolate, it was part of previously described Tn916-like element Tn2009. In another isolate, mega was inserted in a transposon similar to Tn2009 that also included an erm(B) element. This new composite transposon was designated Tn2010. Neither Tn2009 nor Tn2010 could be transferred by conjugation to pneumococcal or enterococcal recipients. In the three isolates in which mega was not physically linked with tet(M), this gene was associated with erm(B) in transposon Tn3872, a Tn916-like element. Homologies between the chromosomal insertions of these composite transposons and sequences of multidrug-resistant pneumococcal genomes in the databases indicate the presence of preferential sites for the integration of composite Tn916-like elements carrying multiple resistance determinants in S. pneumoniae.

HIV-TAT Protein Upregulates Expression of Multidrug Resistance Protein 1 in the Blood–Brain Barrier

Central nervous system (CNS) complications of human immunodeficiency virus (HIV) infection remain a serious health risk in HIV/acquired immunodeficiency syndrome despite significant advances in highly active antiretroviral therapy (HAART). Specific drugs used for HAART are substrates for the efflux transport systems, such as the multidrug resistance-associated proteins (MRPs), which are present on brain microvascular endothelial cells (BMEC) and astrocytes, that is, the main cell types that form the blood-brain barrier (BBB). Thus, drugs employed in HAART are actively removed from the CNS and do not efficiently inhibit HIV replication in the brain. To study the potential mechanisms of this process, the aim of the present research was to address the hypothesis that HIV Tat protein can contribute to upregulation of MRP expression at the BBB level. Tat is a protein produced and released by HIV-infected cells, which may play an important role in brain vascular pathology in the course of HIV infection. Among the family of MRPs, exposure to Tat specifically induced MRP1 messenger ribonucleic acid and protein expression both in BMEC and astrocytes. These alterations were accompanied by enhanced MRP1-mediated efflux functions. Furthermore, activation of the mitogen-activated protein kinase signaling cascade was identified as the mechanism involved in Tat-mediated overexpression of MRP1. These results indicate that Tat exposure can lead to alterations of the BBB functions and decrease HAART efficacy in the CNS through overexpression of drug efflux transporters.

ATP-Binding Cassette Transporter G2 Mediates the Efflux of Phototoxins on the Luminal Membrane of Retinal Capillary Endothelial Cells

The purpose of this study was to clarify the localization and function of the ATP-binding cassette transporter G2 (ABCG2; BCRP/MXR/ABCP) in retinal capillary endothelial cells, which form the inner blood-retinal barrier, as an efflux transport system. The expression was determined by reverse transcriptase polymerase chain reaction and Western blotting. The localization was identified by immunostaining. The transport function of ABCG2 was measured by flow cytometry. Western blotting indicated that ABCG2 was expressed as a glycosylated disulfide-linked complex in the mouse retina and in peripheral tissues, including liver, kidney, and small intestine. Double immunolabeling of ABCG2 and glucose transporter 1 suggested that ABCG2 was localized on the luminal membrane of mouse retinal capillary endothelial cells. ABCG2 mRNA and protein were found to be expressed in a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB, and rat retina. Treatment with Ko143, an ABCG2 inhibitor, restored the accumulation of pheophorbide a and protoporphyrin IX in TR-iBRB cells. ABCG2 is expressed on the luminal membrane of retinal capillary endothelial cells, where ABCG2 acts as the efflux transporter for photosensitive toxins such as pheophorbide a and protoporphyrin IX. ABCG2 could play an important role at the inner blood-retinal barrier in restricting the distribution of phototoxins and xenobiotics in retinal tissue.

Bacteroides fragilis BmeABC efflux systems additively confer intrinsic antimicrobial resistance

To determine the prevalence of expression and function(s) of Bacteroides fragilis RND family efflux transport systems (bmeABC1-16). The mRNA transcripts of bmeB efflux pump genes were detected in a wild-type strain ADB77 by RT-PCR and expression in different strains was quantified by comparative quantitative real-time RT-PCR. In order to determine independent or additive functions, BmeB 1, 3, 12 and 15 (the first efflux pumps identified) were deleted as singles, doubles, triples or quadruples by the double cross-over technique with pADB242 and antimicrobial susceptibility was assayed by the spiral gradient endpoint technique. All efflux pumps except bmeB9 were expressed in the wild-type parental strain. Susceptibility to beta-lactams, fluoroquinolones, ethidium bromide, SDS and triclosan was increased in ADB77DeltabmeB3 (up to 3-fold) and ADB77DeltabmeB1DeltabmeB3DeltabmeB12 (up to 5-fold). Expression of bmeB9 was increased and that of bmeB11 repressed in the latter deletant. A quadruple deletant (ADB77DeltabmeB1DeltabmeB3DeltabmeB12DeltabmeB15) had similar changes as well as a 2-fold increase in expression of bmeB16 and norfloxacin resistance. Expression of bmeB3 was increased in two triple deletants ADB77DeltabmeB1DeltabmeB12DeltabmeB15-type I (2-fold) and ADB77DeltabmeB1DeltabmeB12DeltabmeB15-type II (5.8-fold). Antimicrobial MICs were also increased in the latter deletant; ampicillin (2.6-fold), cefoperazone (3.4-fold), cefoxitin (1.8-fold), tetracycline (36.4-fold), SDS (1.7-fold) and triclosan (2-fold). These data demonstrate that constitutive bmeB expression is prevalent in B. fragilis. At least seven BmeB efflux pumps are functional in transporting antimicrobials and have overlapping substrate profiles, and at least four confer intrinsic resistance.

Dose dependency in the oral bioavailability of an organic cation model, tributylmethyl ammonium (TBuMA), in rats: Association with the saturation of efflux by the P-gp system on the apical membrane of the intestinal epithelium

The oral bioavailability of tributylmethyl ammonium (TBuMA), an organic cation (OC), exhibited a dose-dependency (i.e., 17, 27, and 35% at doses of 0.4, 4, or 12 micromol/kg, respectively) in the rat. Relevant mechanisms were investigated in the present study by estimating the mucosal to serosal (m-s) and serosal to mucosal (s-m) transport of TBuMA across the rat ileum in an Ussing chamber experiment. The m-s permeability rapidly increased with TBuMA concentration in the mucosal side, and then becoming constant at high TBuMA concentrations. Various studies, including temperature- and potential-dependency and inhibition experiments, revealed that carrier-mediated transport mechanisms (most likely OCT1, OCT3, and P-gp) are involved in the s-m transport of TBuMA, and the saturation of the transport at higher concentrations is responsible for the concentration-dependency in the m-s permeability or dose-dependency of the bioavailability of TBuMA. A nonlinear regression of the m-s transport, based on the assumption of a mixed process of linear diffusion and saturable efflux, exhibited a clearance (CLlinear) of 0.343 microL/min/cm2 for the passive diffusion, and an apparent Km of 241 microM for the saturable process. The Km value is consistent with the concentration range in the intestine which is expected to be achieved after the oral dosing of TBuMA at a dose of 0.4 micromol/kg (i.e., 68 approximately 185 microM). Interestingly, the m-s transport of TBuMA was increased by the presence of P-gp substrates or inhibitors in the mucosal side, but not by the mucosal presence of OCT substrates or inhibitors, suggesting that only efflux transport systems on the apical membrane (e.g., P-gp), but not those on the serosal membrane (e.g., OCT1 and OCT3), of the intestinal epithelial cells, are involved in the dose-dependency or concentration dependency. A similar relationship seems likely for drugs that are substrates of efflux transporters on the apical membrane of the intestinal epithelium.

Efflux transport systems at the blood–brain barrier and blood CSF barrier

Drugs acting in the central nervous system (CNS) have to cross the blood-brain barrier (BBB) and/or blood–cerebrospinal fluid barrier (BCSFB). These two barriers are formed by brain capillary endothelial cells and choroid plexus epithelial cells. P-glycoprotein (P-gp) was initially identified as an efflux transporter at the BBB. It extrudes its substrate drugs into the blood, and acts as one of barrier functions against invasion of xenobiotics into the central nervous system. Thus, recognition by P-gp as a substrate is a major disadvantage for drugs used to treat CNS disease. In addition to P-gp, accumulating evidence has revealed the presence of efflux transport systems for anionic drugs. The transporters involved in the efflux transport of organic anions from the CNS are being identified in the BBB and BCSFB, and include the members of organic anion transporting polypeptides (Oatp1a4 and Oatp1a5) and organic anion transporter (Oat3) which are involved in the efflux of amphipathic and hydrophilic organic anions, respectively. The transporters located on the membranes facing the blood still remain to be identified although several candidate primary active transporters, such as MRP and BCRP, have already been proposed. Further clarification of influx and efflux transporters in the barriers will enable us to deliver drugs efficiently into the brain and to understand the mechanisms of drug–drug interactions and interindividual differences in the therapeutic CNS effects and/or adverse effects caused by drugs. The present manuscript summarizes the recent advance in the characterization of efflux transport systems at the BBB and BCSFB from our laboratory and others.