ABC drug transporter, MDR1, is a drug flippase that moves a variety of hydrophobic molecules from the inner to the outer leaflet of the plasma membrane. We have previously reported that MDR1 can function as a glycolipid flippase, being one of the mechanisms responsible for the translocation of glucosylceramide into the Golgi for neutral, but not acidic, glycosphingolipids (GSLs) synthesis. The interplay between GSLs and MDR1 could provide a whole new spectrum of innovative therapeutic options. We found that cell surface MDR1 partially co-localized with globotriaosyl ceramide (Gb3) in MDR1 transfected cells. Inhibition of GSL biosynthesis results in the loss of drug resistance and of cell surface MDR1. We speculated that an association of MDR1 and cell surface GSLs, in particular Gb3, may be functional at the cell surface, as MDR1 partitions into plasma membrane lipid rafts regulating MDR1 function. We therefore tested adamantyl Gb3 (adaGb3), a water soluble analog of Gb3, on MDR1 functions. AdaGb3 was able to inhibit MDR1-mediated rhodamine 123 drug efflux from MDR1 expressing cells, like cyclosporin A (CsA), a classical MDR1 inhibitor. AdaGb3 was also able to reverse vinblastine drug resistance in cell culture, whereas adamantyl galactosylceramide had no effect on drug resistance. The strong MDR1 reversal effects of adaGb3, as well as its favourable in vivo features make it a possible choice for inhibition of MDR1 to increase bioavailability of drugs across the intestinal epithelium (De Rosa et al., 2008). Thus, specific GSL analogs provide a new approach to MDR reversal. We have previously shown that MDR1 inhibitor CsA depletes Fabry cell lines of Gb3, the characteristic GSL accumulated in this disease, by preventing its de novo synthesis, and can also deplete Gaucher lymphoid cell lines of accumulated GlcCer (Mattocks et al., 2006). Liver and heart sections of Fabry mice treated with third generation MDR1 inhibitors showed significantly less Gb3 than liver and heart sections of untreated Fabry mice. Thus, MDR1 inhibition offers a potential alternative therapeutic approach not only for Fabry disease given the extraordinary cost of conventional enzyme replacement therapy, but also for other neutral GSL storage diseases, such as Gaucher disease.

Resistance to anticancer drugs is a serious obstacle to cancer chemotherapy. A common form of multidrug resistance (MDR) is caused by the overexpression of transmembrane transporter proteins P-glycoprotein and MRP1, encoded by MDR1 and MRP1 genes, respectively. These proteins lead to reduced intracellular drug concentration and decreased cytotoxicity by means of their ability to pump the drugs out of the cells. Breast cancer tumor resistance is mainly associated with overexpression of P-gp/MDR1. Although some chemical MDR modulators aim to overcome MDR by impairing the function of P-gp, they exhibit severe toxicities limiting their clinical relevance. Consequently, selective blocking of the expression of P-gp/MDR1 specific mRNA through RNA interference strategy may be an efficient tool to reverse MDR phenotype and increase the success of chemotherapy. Aim of this study was re-sensitizing doxorubicin resistant breast cancer cells to anticancer agent doxorubicin by selective downregulation of P-gp/MDR1 mRNA. The effect of the selected MDR1 siRNA and MRP1 expression after MDR1 silencing was determined by qPCR analysis. XTT cell proliferation assay was performed to v determine the effect of MDR1 silencing on doxorubicin sensitivity.Intracellular drug accumulation and localization was investigated by confocal laser scanning microscopy after treatment with MDR1 siRNA or other MDR modulators / verapamil or promethazine. The role of P-gp in migration characteristics of resistant cells was evaluated by wound healing assay. The results demonstrated that approximately 90% gene silencing occurred by the selected siRNA targeting MDR1 mRNA. However the level of MRP1 mRNA did not change after MDR1 downregulation. Introduction of siRNA resulted in about 70% re-sensitization to doxorubicin. Silencing of P-gp encoding MDR1 gene resulted in almost complete restoration of the intracellular doxorubicin accumulation and re-localization of the drug to the nuclei. Despite the considerably high concentration of the modulators, verapamil and promethazine were not as effective as siRNA for reversal of the drug efflux. According to wound healing assay, MDR1 silencing did not have any effect on migration characteristics of resistant cells, that is, P-gp expression does not seem to affect the motility of the cells. Selected siRNA duplex was shown to effectively inhibit MDR1 gene expression, restore doxorubicin accumulation and localization, and enhance chemo-sensitivity of resistant cells, which makes it a suitable future candidate for therapeutic applications.

P-glycoprotein (Pgp), which leads to multidrug resistance in tumour cells, is an ATP-dependent secretory drug efflux pump. In the intestine, as well as at specific other epithelial and endothelial sites, P-glycoprotein expression is localised to the apical membrane, consistent with secretory detoxifying and absorption limitation functions. The primary function of Pgp is to clear the membrane lipid bilayer of lipophilic drugs. Results from in vitro studies with human Caco-2 cells provide direct evidence for Pgp limiting drug absorption. Limitation has non-linear dependence of absorption on substrate (eg. vinblastine) concentration, increased absorption upon saturation of secretion and increased absorption upon inhibition of Pgp function, with modulators such as verapamil. The aim of this study was to investigate the effect of a known Pgp inhibitor (verapamil) and grapefruit juice components (naringenin, quercetin and bergamottin) on the transport of Rhodamine 123 across rat jejunum and to compare these results with those obtained in similar studies done in Caco-2 cells and in rat intestine (monodirectional). Verapamil, naringenin (442 µM, 662 µM and 884 µM), quercetin (73 µM, 183 µM and 292 µM) and bergamottin (12 µM, 30 µM and 48 µM) were evaluated as modulators of rhodamine 123 transport across rat jejunum using Sweetana-Grass diffusion cells. This study was done bidirectionally, with three cells measuring transport in the apical to basolateral direction (AP / BL) and three cells measuring transport in the basolateral to apical direction (BL / AP). The rate of transport was expressed as the apparent permeability coefficient (Papp) and the extent of active transport was expressed by calculating the ratio of BL/AP to AP/BL. The BL-AP/AP-BL ratio calculated for Rhodamine 123 with no modulators added was 2.31. The known modulator verapamil decreased the BL-AP/AP-BL ratio to 1.52. This was statistically significant and inhibition of active transport was clearly demonstrated. All modulators inhibited active transport. Only naringenin 884 µM, quercetin 183 µM and bergamottin 30 µM did not show a statistically significant decrease in the BL-AP/AP-BL ratio. All three components of grapefruit juice showed inhibition of active transport and should have an effect on the bioavailability of the substrates of Pgp and other active transporters. The results obtained in this study are similar to the results found in Caco-2 cells, which suggests that Sweetana-Grass diffusion method can be used for diffusion studies. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Many orally administered drugs must overcome several barriers before reaching their target site. The first major obstacle to cross is the intestinal epithelium. Although lipophilic compounds may readily diffuse across the apical plasma membrane, their subsequent passage across the basolateral membrane and into blood is by no means guaranteed. Efflux proteins located at the apical membrane, which include P-glycoprotein (P-gp, MDR1) and Multidrug Resistance-associated Protein (MRP2), may drive compounds from inside the cell back into the intestinal lumen, preventing their absorption into the blood. Intestinal P-gp is localised to the villus tip enterocytes, i.e. the main site of absorption for orally administered compounds and in close proximity to the lumen. P-gp is therefore ideally positioned to limit the absorption of compounds by driving efflux back into the lumen. Drugs may also be modified by intracellular phase I and phase II metabolizing enzymes. This process may not only render the drug ineffective, but it may also produce metabolites that are themselves substrates for P-gp and/or MRP2. Drugs that reach the blood are then passed to the liver, where they are subjected to further metabolism and biliary excretion, often by a similar system of ATP binding cassette (ABC) transporters and enzymes to that present in the intestine. Thus a synergistic relationship exists between intestinal drug metabolizing enzymes and apical efflux transporters, a partnership that proves to be a critical determinant of oral bioavailability. Aim: The aim of this study was to investigate the effect of selected methoxy flavonoids (3-methoxyflavone, 5-methoxyflavone, 6-methoxyflavone and 7- methoxyflavone) on the mean ratio of Rhodamine123 (Rho 123) transport across rat intestine (jejunum) and to investigate structure activity relationships (SAR) of the selected flavonoids with reference to inhibition of P-gp. Methods: 3-Methoxyflavone, 5- methoxyflavone, 6-methoxyflavone and 7-methoxyflavone were evaluated at a concentration of 10μM and 20μM as modulators of Rho 123 transport across rat jejunum. The Sweetana-Grass diffusion cells were used to determine the transport of Rho 123. Each modulator was studied bidirectionally with two cells measuring transport in the apical to basolateral direction (AP/BL) and two cells measuring transport in the basolateral to apical direction (BUAP). The rate of transport was expressed as the apparent permeability coefficient (Papp)and the extent of active transport was expressed by calculating the ratio of BUAP to AP/BL. Each modulators Papp ratio was then compared with that of the control. Results: 3-Methoxyflavone decreased the Papp ratio from 3.34 (control) to 1.66 (10μM) and 1.33 (20μM) and showed statistical significant differences. 7-Methoxyflavone decreased the Papp ratio to 1.94 (10μM) and 1.55 (20μM) but only showed a statistical significant difference at 10μM. 5- Methoxyflavone decreased the Papp ratio to 2.41 (10μM) and 1.71 (20μM) and 6- methoxyflavone decreased the Papp to 3.03 (10μM) and 2.49 (20μM). Both 5- and 6- methoxyflavone showed no statistical significant differences from the control. The structure activity relationships with reference to P-gp inhibition clearly indicated that the C3 and C7 positioning of the methoxy-group on the A ring played a major role in the inhibition of Rho 123 transport. Conclusion: All the selected modulators showed inhibition of Rho 123 transport across the jejunum. This should affect the bioavailability of the substrates of P-gp and other active transporters. In summary, this study describe the inhibitory interaction of selected flavonoids with P-gp. Structure activity relationships were identified describing the inhibitory potency of the flavonoids based on methoxy groups positioning. The inhibitory potency results were 3-methoxyflavone > 7- methoxyflavone > 5-methoxyflavone> 6-methoxyflavone / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Background: Multidrug resistance (MDR) is resistance of cancer cells to multiple classes of chemotherapeutic drugs that can be structurally unrelated. MDR involves altered membrane transport that results in a lower cell concentration of cytotoxic drugs which plays an important role during cancer treatment. P-glycoprotein (Pgp) is localised at the apical surface of epithelial cell in the intestine and it functions as a biological barrier by extruding toxic substances and xenobiotics out of cells (Lin, 2003:54). The ATP-binding-cassette superfamily is a rapidly growing group of membrane transport proteins and are involved in diverse physiological processes which include antigen presentation, drug efflux from cancer cells, bacterial nutrient uptake and cystic fibrosis (Germann, 1996:928; Kerr, 2002:47). A number of drugs have been identified which are able to reverse the effects of Pgp, multidrug resistance protein (MRPI) and their associated proteins on multidrug resistance. The first MDR modulators discovered and studied during clinical trials were associated with definite pharmacological actions, but the doses required to overcome MDR were associated with the occurrence of unacceptable side effects. As a consequence, more attention has been given to the development of modulators with proper potency, selectivity and pharmacokinetic characteristics that it can be used at a lower dose. Several novel MDR reversing agents (also known as chemosensitisers) are currently undergoing clinical evaluation for the treatment of resistant tumours (Teodori et al., 2002:385). Aim: The aim of this study was to investigate the effect of selected flavonoids (morin, galangin, kaempferol and quercetin) at two different concentrations (10 μM and 20 μM) on the transport of a known Pgp substrate, Rhodamine 123 (Rho 123) across rat intestine (jejunum) and to investigate structure activity relationships (SAR) of the selected flavonoids with reference to the inhibition of Pgp. Methods: Morin, galangin, kaempferol and quercetin were evaluated as potential modulators of Rho 123 transport, each at a concentration of 10 μM and 20 μM across rat jejunum using Sweetana-Grass diffusion cells. This study was done bidirectionally, with two cells measuring transport in the apical to basolateral direction (AP-BL) and two cells measuring transport in the basolateral to apical direction (BL-AP). The rate of transport was expressed as the apparent permeability coefficient (Pap,) and the extent of active transport was expressed by calculating the ratio of BL-AP to AP-BL. Results: The BL-AP to AP-BL ratio calculated for Rho 123 with no modulators added was 3.29. Morin decreased the BL-AP to AP-BL ratio to 1.88 at a concentration of 10 μM and to 1.49 at a concentration of 20 μM. Galangin decreased the BL-AP to AP-BL ratio to 1.60 at a concentration of 20 μM. These two flavonoids showed statistically significant results and inhibition of active transport were clearly demonstrated. However, the other flavonoids inhibited active transport of Rho 123 but according to statistical analysis, the results were not significantly different. The two different concentrations (10 μM and 20 μM) indicated that galangin, kaempferol and quercetin showed practically significant differences according to the effect sizes. Morin, however, did not show any practically significant differences at the different concentrations. Regarding .the SAR, it was shown by Boumendjel and co-workers (2002:512) that the presence of a 5-hydroxyl group and a 3-hydroxyl group as well as the C2-C3 double bond are required for high potency binding to the nucleotide binding domain (NBD) of Pgp. All the flavonoids tested had the above-mentioned characteristics. Conclusion: All the selected flavonoids showed inhibition of active transport of Rho 123 and should have an effect on the bioavailability of the substrates of Pgp and other active transporters. This study described the inhibitory interaction of selected flavonoids on Pgp activity. Practical significant differences between the same modulator at different concentrations were also observed. Structure activity relationships were identified describing the inhibitory potency of the flavonoids based on hydroxyl group positioning / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

P-glycoprotein (Pgp, ABCB1) is a polyspecific efflux transporter implicated in multidrug resistance in human cancers. In this study, tetramethylrhodamine-5-carbonyl azide (AzTMR) was covalently crosslinked to the Pgp drug binding pocket with a stoichiometry of 1. The Pgp-AzTMR adduct was functionally equivalent to unlabelled Pgp and retained its ability to transport Hoechst 33342. The binding site of AzTMR in Pgp was nonpolar, with a similar environment to that of propanol. Pgp-AzTMR could bind a second drug molecule, with a higher affinity for H-site drugs and lower affinity for other R-site drugs. Unlabelled Pgp interacted with dimeric versions of known Pgp modulators, binding them with higher affinity than the monomer. These compounds were also found to either stimulate or inhibit Pgp ATPase activity depending on the concentration. Pgp-AzTMR was able to bind dimeric drugs, indicating that 3 substrate moieties can fit into the binding pocket. / The Canadian Cancer Society

No description available.

INTRODUCTION: The blood brain barrier (BBB), formed by the brain capillary endothelial cells separating the blood from the brain. Furthermore, the brain endothelial cells also express numerous transporter systems which help regulate and maintain the brain microenvironment. The protective function of the BBB and their transporter systems under pathological disease states, including brain tumor, can be an obstacle for the entry of therapeutic agents to the brain. OBJECTIVES: The current study set out to characterize brain tumor-induced alterations of the BBB of a mouse brain tumor model. Studies were performed to address changes in BBB permeability to P-gp dependent solutes using Rhodamine (R800). Furthermore, the use of lysophosphatidic acid (LPA) to modulate BBB permeability was also examined in healthy mice and tumor-bearing mice. METHODS: Tumors were induced by injecting Lewis Lung carcinoma (3LL) cells into the right hemisphere of female Balb/c mice. Changes in BBB permeability were assessed at various stages of tumor development, using both gadolinium contrast-enhanced agent (Gad) and 3H-mannitol. Functional activity of P-gp in the BBB was examined in adult mice following i.v. injection of R800 in the presence and absence of GF120918 (a P-gp inhibitor). Alterations in BBB permeability were characterized in healthy and tumor-bearing mice using a small (Gad) and large (IRdye800cw PEG) vascular permeability agent as well as R800 (changes in P-gp mediated permeability). RESULTS: Median mouse survival following 3LL injection was 17 days. The BBB was largely intact during tumor development with disruptions observed at the later stages of tumor development as indicated by Gad permeability. By inhibiting the function of P-gp with GF120918, the distribution of R800 in the brain increased by 4-fold. The enhancement effect of LPA on BBB permeability occurs within 3-6 minutes of injection with the barrier being restored back to its normal function within 20 minutes. Furthermore, an increased in brain penetration of IRdye800ce PEG and R800 were observed following LPA injection in both healthy and tumo-bearing mice. CONCLUSION: These studies provide the initial proof of concept for the use of BBB modulators including LPA and GF120918 to enhance drug delivery to the brain and the tumor sites.

Cystic fibrosis is caused primarily by deletion of Phe508. An exciting discovery was that CFTR’s sister protein, the P-glycoprotein (P-gp) containing the equivalent mutation (ΔY490), could be repaired by a drug-rescue approach. Drug substrates showed specificity, and their mechanism involves direct binding to the transmembrane domains (TMDs) since arginine suppressor mutations were identified in TMDs that mimicked drug-rescue to promote maturation. We tested the possibility of rescuing CFTR processing mutants with a drug-rescue approach. 1) Arginine mutagenesis was performed on TM6, 8, and 12. 2) Correctors were tested for specificity. 3) Truncation mutants were used to map the VX-809 rescue site. Correctors 5a, 5c, and VX-809 were specific for CFTR. VX-809 appeared to specifically rescue CFTR by stabilizing TMD1. Therefore, the TMDs are potential targets to rescue CFTR. Rescue of P-gp and CFTR appeared to occur by different mechanisms since no arginine suppressor mutations were identified in CFTR.

Cystic fibrosis is caused primarily by deletion of Phe508. An exciting discovery was that CFTR’s sister protein, the P-glycoprotein (P-gp) containing the equivalent mutation (ΔY490), could be repaired by a drug-rescue approach. Drug substrates showed specificity, and their mechanism involves direct binding to the transmembrane domains (TMDs) since arginine suppressor mutations were identified in TMDs that mimicked drug-rescue to promote maturation. We tested the possibility of rescuing CFTR processing mutants with a drug-rescue approach. 1) Arginine mutagenesis was performed on TM6, 8, and 12. 2) Correctors were tested for specificity. 3) Truncation mutants were used to map the VX-809 rescue site. Correctors 5a, 5c, and VX-809 were specific for CFTR. VX-809 appeared to specifically rescue CFTR by stabilizing TMD1. Therefore, the TMDs are potential targets to rescue CFTR. Rescue of P-gp and CFTR appeared to occur by different mechanisms since no arginine suppressor mutations were identified in CFTR.