High-performance cation electrokinetic concentrator based on a γ-CD/QCS/PVA composite and microchip for evaluating the activity of P-glycoprotein without any interference from serum albumin.

The development of cation electrokinetic concentrators (CECs) has been hindered by the lack of commercial anion-exchange membranes (AEMs). This paper introduces a γ-cyclodextrin-modified quaternized chitosan/polyvinyl alcohol (γ-CD/QCS/PVA) composite as an AEM, which is combined with a microchip to fabricate a CEC. Remarkably, the CEC only concentrates cationic species, thereby overcoming the interference of the highly abundant, negatively charged serum albumin in the blood sample. P-Glycoprotein (P-gp) is recognized as an efflux transporter protein that influences the pharmacokinetics (PK) of various compounds. The CEC was used to evaluate the activity of P-gp by detecting the positively charged rhodamine 123 (Rho123 is a classical substrate of P-gp) with no interference from serum albumin in the serum sample. Using the CEC, the enrichment factor (EF) of Rho123 exceeded 105-fold under DC voltage application. The minimal sample consumption of the CEC (<10 µL) enables reduction of animal sacrifice in animal experiments. Here, the CEC has been applied to evaluate the transport activity of P-gp in in vitro and in vivo experiments by detecting Rho123 in the presence of P-gp inhibitors or agonists. The results are in good agreement with those reported in previous reports. Therefore, the CEC presents a promising application potential, owing to its simple fabrication process, high sensitivity, minimal sample consumption, lack of interference from serum albumin and low cost.

Drug Transcellular Transport Assay Using a High Porosity Honeycomb Film.

In vitro transport studies across cells grown on culture inserts are widely used for evaluating pharmacokinetic characteristics such as intestinal membrane permeability. However, measurements of the apparent permeability coefficient of highly lipophilic compounds are often limited by transport across the membrane filters, not by transport across the cultured cells. To overcome this concern, we have investigated the utility of a high-porosity membrane honeycomb film (HCF) for transcellular transport studies. Using the HCF inserts, the apparent permeability coefficient (Papp) of the drugs tested in LLC-PK1 and Caco-2 cells tended to increase with an increase in lipophilicity, reaching a maximum Papp value at Log D higher than 2. In contrast, using the commercially available Track-Etched membrane (TEM) inserts, a maximum value was observed at Log D higher than 1. The basolateral to apical transport permeability Papp(BL→AP) of rhodamine 123 across LLC-PK1 cells that express P-glycoprotein (P-gp) cultured on HCF inserts and TEM inserts was 2.33 and 2.39 times higher than the reverse directional Papp(AP→BL) permeability, respectively. The efflux ratio (Papp(B-A)/Papp(A-B)) of rhodamine 123 in LLC-PK1 expressing P-gp cells using HCF inserts was comparable to that obtained using TEM inserts, whereas the transported amount in both directions was significantly higher when using the HCF inserts. Accordingly, due to the higher permeability and high porosity of HCF membranes, it is expected that transcellular transport of high lipophilic as well as hydrophilic compounds and substrate recognition of transporters can be evaluated more accurately by using HCF inserts.

Transfer of rhodamine-123 into the brain and cerebrospinal fluid of fetal, neonatal and adult rats.

BACKGROUND: Adenosine triphosphate binding cassette transporters such as P-glycoprotein (PGP) play an important role in drug pharmacokinetics by actively effluxing their substrates at barrier interfaces, including the blood-brain, blood-cerebrospinal fluid (CSF) and placental barriers. For a molecule to access the brain during fetal stages it must bypass efflux transporters at both the placental barrier and brain barriers themselves. Following birth, placental protection is no longer present and brain barriers remain the major line of defense. Understanding developmental differences that exist in the transfer of PGP substrates into the brain is important for ensuring that medication regimes are safe and appropriate for all patients. METHODS: In the present study PGP substrate rhodamine-123 (R123) was injected intraperitoneally into E19 dams, postnatal (P4, P14) and adult rats. Naturally fluorescent properties of R123 were utilized to measure its concentration in blood-plasma, CSF and brain by spectrofluorimetry (Clariostar). Statistical differences in R123 transfer (concentration ratios between tissue and plasma ratios) were determined using Kruskal-Wallis tests with Dunn's corrections. RESULTS: Following maternal injection the transfer of R123 across the E19 placenta from maternal blood to fetal blood was around 20 %. Of the R123 that reached fetal circulation 43 % transferred into brain and 38 % into CSF. The transfer of R123 from blood to brain and CSF was lower in postnatal pups and decreased with age (brain: 43 % at P4, 22 % at P14 and 9 % in adults; CSF: 8 % at P4, 8 % at P14 and 1 % in adults). Transfer from maternal blood across placental and brain barriers into fetal brain was approximately 9 %, similar to the transfer across adult blood-brain barriers (also 9 %). Following birth when placental protection was no longer present, transfer of R123 from blood into the newborn brain was significantly higher than into adult brain (3 fold, p < 0.05). CONCLUSIONS: Administration of a PGP substrate to infant rats resulted in a higher transfer into the brain than equivalent doses at later stages of life or equivalent maternal doses during gestation. Toxicological testing of PGP substrate drugs should consider the possibility of these patient specific differences in safety analysis.

Influene of Pharmaceutical Excipients on the Membrane Transport of a P-glycoprotein Substrate in the Rat Small Intestine.

BACKGROUND AND OBJECTIVES: Generic drugs are generally used worldwide because of affordability compared to brand-name drugs. One of the main differences between brand-name and generic drugs is pharmaceutical excipients. We previously reported the effects of pharmaceutical excipients on the membrane permeation of drugs via the paracellular and transcellular routes, which are passive transport routes. P-glycoprotein (P-gp) is a typical ATP-binding cassette transporter and is mostly responsible for drug-drug interactions involving transporters. In the present study, rhodamine 123 (Rho123) was selected as the P-gp substrate, and the effects of pharmaceutical excipients on its membrane transport in the rat jejunum and ileum were examined. METHODS: Twenty major pharmaceutical excipients widely used in the pharmaceutical industry were selected. The in vitro diffusion chamber method using the rat jejunum and ileum was employed to investigate the effects of pharmaceutical excipients on the membrane permeation of Rho123. RESULTS: The results obtained showed that the membrane permeability of Rho123 significantly (P < 0.05) changed under certain dosage conditions of pharmaceutical excipients such as sodium carboxymethyl starch, pullulan, glyceryl monostearate and so on. Furthermore, the effects of pharmaceutical excipients were site specific in the small intestine. CONCLUSION: The present results demonstrated that some pharmaceutical excipients altered the membrane permeability of Rho123 in the rat small intestine.

Effects of MDR1 1236C > T-2677G > T-3435C > T polymorphisms on the intracellular accumulation of tacrolimus, cyclosporine A, sirolimus and everolimus.

1. Overexpression of P-glycoprotein (P-gp, encoded by MDR1) mediates resistance to multiple immunosuppressors. Several common MDR1 variants (1236C > T, 2677G > T, 3435C > T) impact the efflux activity of P-gp-mediated substrates. We assessed the effect of these polymorphisms on the sensitivity, intracellular accumulation, and efflux of tacrolimus, cyclosporine A, sirolimus and everolimus in transfected LLC-PK1 cells. 2. LLC-PK1 cell lines were transfected with empty vector (pcDNA3.1) and recombinant MDR1T-T-T, MDR1C-T-T, MDR1C-G-T and MDR1C-G-C vectors, respectively and further screened in the presence of puromycin. The IC50 values, intracellular accumulation, and apparent permeability ratios of tacrolimus, cyclosporine A, sirolimus and everolimus were evaluated. 3. MDR1 overexpression increased the resistance of LLC-PK1 cells to tacrolimus, cyclosporine A, sirolimus and everolimus. The resistance of cells expressing MDR1C-G-C wild-type haplotypes to tacrolimus were increased compared to MDR1T-T-T, MDR1C-T-T, MDR1C-G-T variant haplotypes. The efflux ability of P-gp-mediated tacrolimus in cells transfected with MDR1C-G-C was higher than cells overexpressing MDR1T-T-T, MDR1C-T-T, MDR1C-G-T variant haplotypes. In addition, the resistance of cells expressing MDR1C-G-C wild-type haplotypes to sirolimus were increased compared to MDR1C-T-T, MDR1C-G-T variant haplotypes. The efflux ability of P-gp-mediated sirolimus in cells overexpressing MDR1C-G-C was higher than cells transfected with MDR1C-T-T, MDR1C-G-T variant haplotypes. 4. These findings indicate that wild-type MDR1 exports tacrolimus and sirolimus more efficiency than the MDR1T-T-T, MDR1C-T-T, MDR1C-G-T variant protein. This observation indicates that 1236C > T, 2677G > T, 3435C > T variant haplotypes drastically decrease the efflux ability of P-gp-mediated tacrolimus and sirolimus in a substrate-specific manner.

A multi-chamber microfluidic intestinal barrier model using Caco-2 cells for drug transport studies.

This paper presents the design and fabrication of a multi-layer and multi-chamber microchip system using thiol-ene 'click chemistry' aimed for drug transport studies across tissue barrier models. The fabrication process enables rapid prototyping of multi-layer microfluidic chips using different thiol-ene polymer mixtures, where porous Teflon membranes for cell monolayer growth were incorporated by masked sandwiching thiol-ene-based fluid layers. Electrodes for trans-epithelial electrical resistance (TEER) measurements were incorporated using low-melting soldering wires in combination with platinum wires, enabling parallel real-time monitoring of barrier integrity for the eight chambers. Additionally, the translucent porous Teflon membrane enabled optical monitoring of cell monolayers. The device was developed and tested with the Caco-2 intestinal model, and compared to the conventional Transwell system. Cell monolayer differentiation was assessed via in situ immunocytochemistry of tight junction and mucus proteins, P-glycoprotein 1 (P-gp) mediated efflux of Rhodamine 123, and brush border aminopeptidase activity. Monolayer tightness and relevance for drug delivery research was evaluated through permeability studies of mannitol, dextran and insulin, alone or in combination with the absorption enhancer tetradecylmaltoside (TDM). The thiol-ene-based microchip material and electrodes were highly compatible with cell growth. In fact, Caco-2 cells cultured in the device displayed differentiation, mucus production, directional transport and aminopeptidase activity within 9-10 days of cell culture, indicating robust barrier formation at a faster rate than in conventional Transwell models. The cell monolayer displayed high TEER and tightness towards hydrophilic compounds, whereas co-administration of an absorption enhancer elicited TEER-decrease and increased permeability similar to the Transwell cultures. The presented cell barrier microdevice constitutes a relevant tissue barrier model, enabling transport studies of drugs and chemicals under real-time optical and functional monitoring in eight parallel chambers, thereby increasing the throughput compared to previously reported microdevices.

Excipient-drug pharmacokinetic interactions: Effect of disintegrants on efflux across excised pig intestinal tissues.

Pharmaceutical excipients were designed originally to be pharmacologically inert. However, certain excipients were found to have altering effects on drug pharmacodynamics and/or pharmacokinetics. Pharmacokinetic interactions may be caused by modulation of efflux transporter proteins, intercellular tight junctions and/or metabolic enzyme amongst others. In this study, five disintegrants from different chemical classes were evaluated for P-glycoprotein (P-gp) related inhibition and tight junction modulation effects. Bi-directional transport studies of the model compound, Rhodamine 123 (R123) were conducted in the absence (control group) and presence (experimental groups) of four concentrations of each selected disintegrant across excised pig jejunum tissue. The results showed that some of the selected disintegrants (e.g. Ac-di-sol® and Kollidon® CL-M) increased R123 absorptive transport due to inhibition of P-gp related efflux, while another disintegrant (e.g. sodium alginate) changed R123 transport due to inhibition of P-gp in conjunction with a transient opening of the tight junctions in a concentration dependent way. It may be concluded that the co-application of some disintegrants to the intestinal epithelium may lead to pharmacokinetic interactions with drugs that are susceptible to P-gp related efflux. However, the clinical significance of these in vitro permeation findings should be confirmed by means of in vivo studies.

Inhibitory Effects of Benzaldehyde, Vanillin, Muscone and Borneol on P-Glycoprotein in Caco-2 Cells and Everted Gut Sac.

AIMS: In clinical practice, herbal medicines have played an important role in the modulation of drug transporters through the combination of conventional prescription drugs, which necessitates the elucidation of herb-drug interactions. The present study was designed to investigate the inhibitory effects and mechanisms of benzaldehyde, vanillin, muscone, and borneol on P-glycoprotein (P-gp). METHODS: The effects of the 4 compounds on the intracellular accumulation of rhodamine-123 (Rho-123) in vinblastine-treated Caco-2 (VB-Caco-2) cells were studied by monitoring fluorescence intensity through a flow cytometry assay, and the effects of these compounds on Rho-123 transport through VB-Caco-2 monolayers and Rho-123 intestinal absorption in the rat everted gut sac were investigated by high-performance liquid chromatography. Moreover, P-gp expression in VB-Caco-2 cells was assessed using flow cytometry and Western blot analysis, and the relative ABCB1 mRNA level was determined by Real-time RT-PCR. KEY FINDINGS: The results showed that benzaldehyde, vanillin, muscone, and borneol significantly increased Rho-123 uptake in VB-Caco-2 cells, increased the absorption rate and apparent permeability coefficient of Rho-123 in rat jejunum and ileum, and decreased the efflux ratio of Rho-123 from 6.52 to less than 2 during transport across VB-Caco-2 cell monolayers. In addition, these compounds reduced the protein and ABCB1 mRNA levels of P-gp in VB-Caco-2 cells. CONCLUSIONS: These data indicate that benzaldehyde, vanillin, muscone and borneol could effectively reverse multidrug resistance via inhibiting the P-gp function and expression pathway. The data provide fodder for further investigation into the interaction between the 4 compounds and other drugs transported by P-gp.

Drug Clearance from Cerebrospinal Fluid Mediated by Organic Anion Transporters 1 (Slc22a6) and 3 (Slc22a8) at Arachnoid Membrane of Rats.

Although arachnoid mater epithelial cells form the blood-arachnoid barrier (BAB), acting as a blood-CSF interface, it has been generally considered that the BAB is impermeable to water-soluble substances and plays a largely passive role. Here, we aimed to clarify the function of transporters at the BAB in regulating CSF clearance of water-soluble organic anion drugs based on quantitative targeted absolute proteomics (QTAP) and in vivo analyses. Protein expression levels of 61 molecules, including 19 ATP-binding-cassette (ABC) transporters and 32 solute-carrier (SLC) transporters, were measured in plasma membrane fraction of rat leptomeninges using QTAP. Thirty-three proteins were detected; others were under the quantification limits. Expression levels of multidrug resistance protein 1 (Mdr1a/P-gp/Abcb1a) and breast cancer resistance protein (Bcrp/Abcg2) were 16.6 and 3.27 fmol/µg protein (51.9- and 9.82-fold greater than in choroid plexus, respectively). Among those organic anion transporters detected only at leptomeninges, not choroid plexus, organic anion transporter 1 (oat1/Slc22a6) showed the greatest expression (2.73 fmol/µg protein). On the other hand, the protein expression level of oat3 at leptomeninges was 6.65 fmol/µg protein, and the difference from choroid plexus was within two-fold. To investigate oat1's role, we injected para-aminohippuric acid (PAH) with or without oat1 inhibitors into cisterna magna (to minimize the contribution of choroid plexus function) of rats. A bulk flow marker, FITC-inulin, was not taken up from CSF up to 15 min, whereas uptake clearance of PAH was 26.5 µL/min. PAH uptake was completely blocked by 3 mM cephalothin (inhibits both oat1 and oat3), while 17% of PAH uptake was inhibited by 0.2 mM cephalothin (selectively inhibits oat3). These results indicate that oat1 and oat3 at the BAB provide a distinct clearance pathway of organic anion drugs from CSF independently of choroid plexus.

Pharmacokinetic interaction of green tea beverage containing cyclodextrins and high concentration catechins with P-glycoprotein substrates in LLC-GA5-COL150 cells in vitro and in the small intestine of rats in vivo.

OBJECTIVES: Possible interaction of green tea beverage (GT) containing cyclodextrins and high concentration catechins, a drinking water, with P-glycoprotein (P-gp) substrates was examined in vitro and in vivo. METHODS: Effects of GT on the uptake of rhodamine 123 by LLC-GA5-COL150 cells and intestinal efflux of rhodamine 123 from blood, intestinal absorption of quinidine from ileum loop and oral absorption of digoxin were examined in rats. Effects of GT and GT components on digoxin solubility were also examined. KEY FINDINGS: Green tea increased the uptake of rhodamine 123 by LLC-GA5-COL150 cells, suppressed the intestinal efflux of rhodamine 123 from blood and increased the absorption of quinidine in the ileum of rats. Also, GT increased the solubility of digoxin, and ingestion of GT significantly increased the oral absorption of digoxin given at a high dose in rats. CONCLUSIONS: Green tea suppressed the P-gp-mediated efflux transport of hydrophilic compounds and increased the solubility of lipophilic compounds. Thus, GT may cause interaction with various P-gp substrates, due to the combined effects of catechins and cyclodextrins. Especially, cyclodextrin alone can cause interaction with various low-solubility compounds in vivo. In taking low-solubility drugs including low-solubility P-gp substrates, cyclodextrin-containing foods and beverages such as GT should be avoided.

CAN a P-gp modulator assist in the control of methotrexate concentrations in the rat brain? -inhibitory effects of rhodamine 123, a specific substrate for P-gp, on methotrexate excretion from the rat brain and its optimal route of administration.

Although methotrexate (MTX) is mainly transported by reduced folate carrier, P-gp and MRP1 may also be involved in its transport. In our previous study, a potent P-gp and MRP1 modulator, Cyclosporine A, potentiated MTX concentration in rat brain. Since it is important for MTX therapy for brain tumor to clarify which transporter is dominant, we herein determined whether the specific P-gp substrate, rhodamine123 (Rho123), potentiates the transport and retention of MTX in the brain. Rho123 was injected intravenously or intrathecally into rats immediately after injection of MTX. 6 or 12 hr after the MTX injection, brains were isolated just after the sampling of cerebrospinal fluid (CSF). Blood was also collected intermittently. MTX concentrations were determined in plasma, CSF and the brain using high-performance liquid chromatography with UV detection. When MTX was intravenously injected, Rho123 didn't affect MTX concentrations in the brain. However, Rho123 resulted in significantly higher MTX concentrations in the brain at 12 hr after injection when MTX was intrathecally injected. It is suggested that Rho123 inhibits the excretion of MTX from the brain, but does not potentiate its distribution from the blood into the brain. This reveals that P-gp can be one of the major transporters of MTX in rat brain. Therefore, treatments with P-gp modulators may contribute to intrathecal MTX therapy for brain tumor. Since plasma concentration-time curves of MTX were not affected by Rho123, treatments with P-gp modulators may not potentiate the adverse effects of MTX.

A Novel Model of P-Glycoprotein Inhibitor Screening Using Human Small Intestinal Organoids.

P-glycoprotein (P-gp), an important efflux transporter in intestine, regulates the bioavailability of orally taken drugs. To develop an in vitro model that preferably mimics the physiological microenvironment of human intestine, we employed the three-dimensionally (3D) cultured organoids from human normal small intestinal epithelium. It was observed that the intestinal crypts could efficiently form cystic organoid structure with the extension of culture time. Furthermore, the physiological expression of ABCB1 was detected at both mRNA and protein levels in cultured organoids. Rhodamine 123 (Rh123), a typical substrate of P-gp, was actively transported across 3D organoids and accumulated in the luminal space. This transport process was also inhibited by verapamil and mitotane. In summary, the above-mentioned model based on human small intestinal 3D organoids is suitable to imitate the small intestinal epithelium and could be used as a novel in vitro model especially for P-gp inhibitor screening.

Effect of Curcuma comosa extracts on the functions of peptide transporter and P-glycoprotein in intestinal epithelial cells.

Curcuma comosa has been widely used as a herbal medicine in Thailand; however, it remains unclear whether C. comosa influences the absorption of drugs that are substrates for the transporters in the small intestine. In this study, we investigated the effect of C. comosa extracts on the functioning of peptide transporter 1 (PEPT1), an influx transporter, and P-glycoprotein (P-gp), an efflux transporter, in Caco-2 cells and rat intestine. In Caco-2 cells, the ethanolic extract of C. comosa (CCE) lowered the uptake of glycylsarcosine (Gly-Sar), a PEPT1 substrate, while it enhanced the uptake of rhodamine 123 (Rho123), a P-gp substrate, in a concentrationdependent manner. In addition, CCE inhibited apical-to-basal transport of Gly-Sar and basal-to-apical transport of Rho123. Furthermore, the absorption of cephalexin, another PEPT1 substrate, and the exsorption of Rho123 across the rat intestine were inhibited by CCE. Conversely, CCW, the hot water extract of C. comosa, suppresses the function of PEPT1 but not of P-gp in Caco-2 cells. These results suggest that C. comosa used as a herbal medicine in Thailand may affect the intestinal absorption of certain drugs.

Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin.

P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp.

In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs).

BACKGROUND: Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection. METHODS: To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-PAM is a substrate for common brain efflux pumps, experiments were performed in the MDCKII-MDR1 cell line, transfected to overexpress the P-gp efflux pump, and the MDCKII-FLuc-ABCG2 cell line, transfected to overexpress the BCRP efflux pump. To determine how transcellular transport influences enzyme reactivation, we developed a modified transwell assay where the inhibited acetylcholinesterase enzyme, substrate, and reporter are introduced into the basolateral chamber. Enzymatic activity was inhibited using paraoxon and parathion. RESULTS: The permeability of 2-PAM is about 2 × 10(-6) cm s(-1) in MDCK cells and about 1 × 10(-6) cm s(-1) in BC1-hBMECs. Permeability is not influenced by pre-treatment with atropine. In addition, 2-PAM is not a substrate for the P-gp or BCRP efflux pumps. CONCLUSIONS: The low permeability explains poor brain penetration of 2-PAM and therefore the slow enzyme reactivation. This elucidates one of the reasons for the necessity of sustained intravascular (IV) infusion in response to organophosphate poisoning.

Response of heterogeneous cancer cells on targeted nanoparticles.

Heterogenous cancer cells possess cancer multidrug resistance (MDR) due to their relative quiescence and ABC-transporter expression. Heterogenous cancer cells can be detected by an Rh123 exclusion assay for identifying Rh123low population. In the present study, we fabricated targeted nanoparticles entrapped with Rh123 (Rh123 NPs) to investigate the effect of these targeted nanoparticles on an Rh123low population. The Rh123low population stained by Rh123 NPs exhibited similar heterogeneity to that stained by Rh123. In addition, the ABC-transporters did not contribute to the uptake of Rh123 or Rh123 NPs. Interestingly, ABC-transporters in the Rh123low population stained by Rh123 were possibly responsible for Rh123 efflux, while Rh123 NPs were not susceptible to ABC-transporters in the Rh123low population. It is plausible that the synergistic effect of NPs caused a targeted and endocytic effect which promoted the cellular uptake of Rh123 NPs, and the targeted effect played a more important role.

Gibberellin derivative GA-13315 sensitizes multidrug-resistant cancer cells by antagonizing ABCB1 while agonizes ABCC1.

PURPOSE: GA-13315 is a gibberellin derivative that reveals antitumor and antineoplastic effects both in vitro and in vivo. In the present study, the chemosensitizing effects of GA-13315 in multidrug-resistant cell lines were examined and the underlying mechanisms were investigated. METHODS: Cytotoxicity and chemosensitizing effects of GA-13315 were determined by MTT assay. Function of ABC transporter was analyzed by measuring intracellular drug accumulation of doxorubicin and rhodamine 123 and by determining the ATPase activity of ABC transporter. Expression levels of apoptosis regulators were analyzed using real-time quantitative PCR and Western blot. RESULTS: GA-13315 selectively killed MCF-7/adr cells that overexpress P-glycoprotein (ABCB1) over the parent MCF-7 cells. In combination with conventional chemotherapeutic agents, GA-13315 at sub-toxic concentrations reversed the multidrug resistance mediated by ABCB1 but exacerbated the resistance conferred by multidrug resistance-associated protein 1 (ABCC1). GA-13315 increased intracellular accumulation of doxorubicin and rhodamine 123 in MCF-7/adr cells and in ABCB1-transfected HEK293 cells but facilitated drug flush-out from cells that overexpress ABCC1. GA-13315 inhibited the ATPase activity of ABCB1 while stimulated that of ABCC1. Moreover, the downregulated expression of Bax in MCF-7/adr cells was restored by GA-13315 markedly. CONCLUSION: These data suggest that GA-13315 sensitizes multidrug-resistant cells at least partially by impeding the efflux function of ABCB1. The upregulation of Bax by GA-13315 may also contribute to the sensitizing action. The opposite effects of GA-13315 on different ATP-binding cassette transporters and their implications in overcoming drug resistance require further investigation.

Organic cation rhodamines for screening organic cation transporters in early stages of drug development.

The aim of this study was to investigate the suitability of rhodamine-123, rhodamine-6G and rhodamine B as non-radioactive probes for characterizing organic cation transporters in respiratory cells. Fluorescent characteristics of the compounds were validated under standard in vitro drug transport conditions (buffers, pH, and light). Uptake/transport kinetics and intracellular accumulation of the compounds were investigated. Uptake/transport mechanisms were investigated by comparing the effect of pH, temperature, concentration, polarity, OCTs/OCTNs inhibitors/substrates, and metabolic inhibitors on the cationic dyes uptake in Calu-3 cells. Fluorescence stability and intensity of the compounds were altered by buffer composition, light, and pH. Uptake of the dyes was concentration-, temperature- and pH-dependent. OCTs/OCTNs inhibitors significantly reduced intracellular accumulation of the compounds. Whereas rhodamine-B uptake was sodium-dependent, pH had no effect on rhodamine-123 and rhodamine-6G uptake. Transport of the dyes across the cells was polarized: (AP→BL>BL→AP transport) and saturable: {Vmax=14.08±2.074, Km=1821±380.4 (rhodamine-B); Vmax=6.555±0.4106, Km=1353±130.4 (rhodamine-123) and Vmax=0.3056±0.01402, Km=702.9±60.97 (rhodamine-6G)}. The dyes were co-localized with MitoTracker®, the mitochondrial marker. Cationic rhodamines, especially rhodamine-B and rhodamine- 6G can be used as organic cation transporter substrates in respiratory cells. During such studies, buffer selection, pH and light exposure should be taken into consideration.

The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation.

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10(-6) cm/sec, followed by amodiaquine around 20 x 10(-6) cm/sec; both mefloquine and artesunate were around 10 x 10(-6) cm/sec. Methylene blue was between 2 and 6 x 10(-6) cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.

Psoralen reverses the P-glycoprotein-mediated multidrug resistance in human breast cancer MCF-7/ADR cells.

The resistance of cancer to chemotherapeutic agents is a major obstacle during chemotherapy. Clinical multidrug resistance (MDR) is commonly mediated by membrane drug efflux pumps, including ATP­binding cassette subfamily B member 1, also termed P-glycoprotein (P-gp). P-gp is a membrane transporter encoded by the MDR1 gene. The current study aimed to investigate the impact of psoralen on the expression and function of P­gp. The 10% inhibitory concentration (IC10) of psoralen, and its capacity to reduce MDR in adriamycin (ADR)­resistant MCF­7/ADR cells were determined using MTT assay. The ability of psoralen to modulate the transport activity of P­gp in MCF­7/ADR cells was evaluated by measuring the accumulation and efflux of rhodamine 123 (Rh 123) and adriamycin with flow cytometry. The present study evaluated the mRNA level of MDR1 in MCF­7 and MCF­7/ADR cells treated with psoralen using reverse transcription-quantitative polymerase chain reaction. The protein expression level of P­gp was examined by western blot analysis. The current study demonstrated that the IC10 of psoralen in MCF­7/ADR cells was 8 µg/ml. At 8 µg/ml, psoralen reduced MDR and the sensitivity of the MCF­7/ADR cells to ADR compared with untreated cells. Additionally, psoralen significantly increased the intracellular accumulation of ADR and Rh 123. However, the IC10 of psoralen did not affect the protein expression levels of P­gp or mRNA levels of MDR1 (P>0.05). Psoralen reduces MDR by inhibiting the efflux function of P­gp, which may be important for increasing the efficiency of chemotherapy and improving the clinical protocols aiming to reverse P-gp-mediated MDR.