Publications (20)68.09 Total impact
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Article: Ciprofloxacin is actively transported across bronchial lung epithelial using a Calu-3 air-interface cell model.
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ABSTRACT: Ciprofloxacin is a well-established broad-spectrum fluroquinolone antibiotic that penetrates well into the lung tissues; still the mechanisms of its transepithelial transport are unknown. Contributions of specific transporters including multidrug efflux transporters, organic cation transporters and organic anion transporting polypeptide transporters on the uptake of ciprofloxacin were investigated in vitro using an air interface bronchial epithelial model. Our results demonstrate that ciprofloxacin is subject to predominantly active influx and a slight efflux component.Antimicrobial Agents and Chemotherapy 03/2013; · 4.84 Impact Factor -
Article: Multiple dosing of simvastatin inhibits airway mucus production of epithelial cells: implications in the treatment of chronic obstructive airway pathologies.
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ABSTRACT: BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by mucus hyper-production. This pathology, together with other inflammatory contributions, leads to airway obstruction and breathing complications. Newer therapeutic approaches are of increased interest, including the use of HMG-CoA reductase inhibitors. Retrospective studies have shown that statins are effective in reducing patient mortality, and blood cytokines levels. These findings suggest statins may also provide a new therapeutic approach in COPD treatment. PURPOSE: The aim of the present work was to study the transport of SV across Calu-3 epithelial cells and to investigate its pharmacological action with respect to reduction in mucus production. METHODS: Calu-3 cells were grown under liquid covered culture (LCC) conditions for transport studies in order to demonstrate the ability of SV to transport across the monolayer. For mucus detection, cells were grown under air interface culture (AIC) conditions. Samples collected for microscope analysis, were stained with alcian blue; images of the stained cell surface were acquired and the mucus was quantified as the RGBB ratio. RESULTS: SV was transported through the cell monolayer and 'retained' inside the Calu-3 cells. Colour analysis of stained Calu-3 monolayers microscope-images, showed that chronic administration of SV for 14 days caused a significant inhibition in mucus production. CONCLUSION: These findings suggest that local delivery of SV directly to the lungs may provide a promising treatment and potential disease management approach of COPD, with significant effects on mucus reduction.European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 03/2013; · 3.15 Impact Factor -
Article: Salbutamol sulphate absorption across Calu-3 bronchial epithelia cell monolayer is inhibited in the presence of common anionic NSAIDs.
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ABSTRACT: Purpose: The aim of this study was to characterize the permeability kinetics of salbutamol sulphate, a commonly used β2-agonist in the treatment of asthma exacerbation, across Calu-3 respiratory epithelial cell monolayers in the presence of non-steroidal anti-inflammatory drugs (NSAIDs), as they have been implicated to be able to modulate organic cation transporters (OCT). Methods: Calu-3 cell monolayers were grown in a liquid covered culture (LCC) configuration on 0.33 cm(2) Transwell polyester cell culture supports. Monolayers, cultured between 11 and 14 days were evaluated for epithelial resistance, tight junction integrity and expression of OCT using Western blot analysis. The transport of salbutamol across the monolayer was studied as a function of concentration. Directional transport was investigated by assessing apical-basal (a-b) and basal- apical (b-a) directions. The influence of a non-specific OCT inhibitor (tetraethylammonium, TEA) and three NSAIDs (aspirin, ibuprofen and indomethacin) on the uptake of salbutamol was studied. Results: The flux of salbutamol sulphate increased with increasing concentration, before reaching a plateau suggesting the involvement of a transport mediated uptake mechanism. Western blot analysis detected the presence of OCT1-3 and N1 and N2 sub-types suggesting the presence of functioning transporters. The apparent permeability (P(app)) of 0.1 mM salbutamol across the epithelial monolayer displayed directional transport in the a-b direction which was inhibited by ˜70% in the presence of TEA, suggesting OCT mediated uptake. Likewise, the uptake of 0.1 mM salbutamol was decreased in the presence of all three NSAIDs supporting a mechanism whereby NSAIDs inhibit absorption of salbutamol across the bronchial epithelium via effects on the OCT transporters. Conclusion: This study demonstrates that NSAIDS influence the uptake kinetics of salbutamol in an in vitro Calu-3 cell system.Journal of Asthma 02/2013; · 1.52 Impact Factor -
Article: Breast Cancer-Derived Microparticles Display Tissue Selectivity in the Transfer of Resistance Proteins to Cells.
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ABSTRACT: Microparticles (MPs) play a vital role in cell communication by facilitating the horizontal transfer of cargo between cells. Recently, we described a novel "non-genetic" mechanism for the acquisition of multidrug resistance (MDR) in cancer cells by intercellular transfer of functional P-gp, via MPs. MDR is caused by the overexpression of the efflux transporters P-glycoprotein (P-gp) and Multidrug Resistance-Associated Protein 1 (MRP1). These transporters efflux anticancer drugs from resistant cancer cells and maintain sublethal intracellular drug concentrations. By conducting MP transfer experiments, we show that MPs derived from DX breast cancer cells selectively transfer P-gp to malignant MCF-7 breast cells only, in contrast to VLB100 leukaemic cell-derived MPs that transfer P-gp and MRP1 to both malignant and non-malignant cells. The observed transfer selectivity is not the result of membrane restrictions for intercellular exchange, limitations in MP binding to recipient cells or the differential expression of the cytoskeletal protein, Ezrin. CD44 (isoform 10) was found to be selectively present on the breast cancer-derived MPs and not on leukaemic MPs and may contribute to the observed selective transfer of P-gp to malignant breast cells observed. Using the MCF-7 murine tumour xenograft model we demonstrated the stable transfer of P-gp by MPs in vivo, which was found to localize to the tumour core as early as 24 hours post MP exposure and to remain stable for at least 2 weeks. These findings demonstrate a remarkable capacity by MPs to disseminate a stable resistant trait in the absence of any selective pressure.PLoS ONE 01/2013; 8(4):e61515. · 4.09 Impact Factor -
Article: Liposomal Nanoparticles Control the Uptake of Ciprofloxacin Across Respiratory Epithelia.
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ABSTRACT: PURPOSE: Liposomal ciprofloxacin nanoparticles were developed to overcome the rapid clearance of antibiotics from the lungs. The formulation was evaluated for its release profile using an air interface Calu-3 cell model and further characterised for aerosol performance and antimicrobial activity. METHODS: Liposomal and free ciprofloxacin formulations were nebulised directly onto Calu-3 bronchial epithelial cells placed in an in vitro twin-stage impinger (TSI) to assess the kinetics of release. The aerosol performance of both the liposomal and free ciprofloxacin formulation was characterised using the next generation impactor. Minimum inhibitory and bactericidal concentrations (MICs and MBCs) were determined and compared between formulations to evaluate the antibacterial activity. RESULTS: The liposomal formulation successfully controlled the release of ciprofloxacin in the cell model and showed enhanced antibacterial activity against Pseudomonas aeruginosa. In addition, the formulation displayed a respirable aerosol fraction of 70.5 ± 2.03% of the emitted dose. CONCLUSION: Results indicate that the in vitro TSI air interface Calu-3 model is capable of evaluating the fate of nebulised liposomal nanoparticle formulations and support the potential for inhaled liposomal ciprofloxacin to provide a promising treatment for respiratory infections.Pharmaceutical Research 07/2012; · 4.09 Impact Factor -
Article: Microparticle conferred microRNA profiles - implications in the transfer and dominance of cancer traits.
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ABSTRACT: BACKGROUND: Microparticles (MPs) are membrane vesicles which are released from normal and malignant cells following a process of budding and detachment from donor cells. MPs contain surface antigens, proteins and genetic material and serve as vectors of intercellular communication. MPs comprise the major source of systemic RNA including microRNA (miRNA), the aberrant expression of which appears to be associated with stage, progression and spread of many cancers. Our previous study showed that MPs carry both transcripts and miRNAs associated with the acquisition of multidrug resistance in cancer. RESULTS: Herein, we expand on our previous finding and demonstrate that MPs carry the transcripts of the membrane vesiculation machinery (floppase and scramblase) as well as nucleic acids encoding the enzymes essential for microRNA biogenesis (Drosha, Dicer and Argonaute). We also demonstrate using microarray miRNA profiling analysis, the selective packaging of miRNAs (miR-1228*, miR-1246, miR-1308, miR-149*, miR-455-3p, miR-638 and miR-923) within the MP cargo upon release from the donor cells. CONCLUSION: These miRNAs are present in both haematological and non-haematological cancer cells and are involved in pathways implicated in cancer pathogenesis, membrane vesiculation and cascades regulated by ABC transporters. Our recent findings reinforce our earlier reports that MP transfer 're-templates' recipient cells so as to reflect donor cell traits. We now demonstrate that this process is likely to occur via a process of selective packaging of nucleic acid species, including regulatory nucleic acids upon MP vesiculation. These findings have significant implications in understanding the cellular basis governing the intercellular acquisition and dominance of deleterious traits in cancers.Molecular Cancer 06/2012; 11(1):37. · 3.99 Impact Factor -
Article: Deposition, diffusion and transport mechanism of dry powder microparticulate salbutamol, at the respiratory epithelia.
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ABSTRACT: The deposition, dissolution and transport of salbutamol base (SB) and salbutamol sulfate (SS) inhalation powders were investigated using the Calu-3 air interface cell culture model and Franz diffusion cell. Drug uptake by cells was studied with respect to deposited dose, drug solubility and hydrophobicity. Furthermore, the role of active transport via organic cationic transporters (OCTs) was studied. SB and SS were processed to have similar diameters (3.09 ± 0.06 μm and 3.07 ± 0.03 μm, respectively) and were crystalline in nature. Analysis of drug wetting, dissolution and diffusion using a conventional in vitro Franz cell (incorporating a cell culture support Transwell polyester membrane) showed diffusion of SB to be slower than that of SS (98.57 ± 4.23 μg after 4 h for SB compared to 98.57 ± 4.01 μg after 15 min for SS). Such observations suggest dissolution to be the rate-limiting step. In comparison, the percentage transfer rate using the air interface Calu-3 cell model suggested SB transport to be significantly faster than SS transport (92.02 ± 4.47 μg of SB compared to 63.76 ± 8.84 μg of SS transported over 4 h), indicating that passive diffusion through the cell plays a role in transport. Furthermore, analysis of SB and SS transport, over a range of deposited doses, suggested the transport rate in the Franz diffusion cell to be limited by wetting of the particle and dissolution into the medium. However, for the cell monolayer, the cell membrane properties regulate the diffusion and transport rate. Analysis of the drug transport in the presence of triethylamine (TEA), a known inhibitor of OCTs, resulted in a significant decrease in drug transport, suggesting an active transport mechanism. The presence of OCTs in this cell line was further validated by Western blot analysis. Finally, the transport of SS from a commercial product (Ventolin Rotacaps) was studied and showed good agreement with the model SS system studied here.Molecular Pharmaceutics 05/2012; 9(6):1717-26. · 4.78 Impact Factor -
Article: Modification of disodium cromoglycate passage across lung epithelium in vitro via incorporation into polymeric microparticles.
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ABSTRACT: Two microparticle systems containing disodium cromoglycate (DSCG) alone or with polyvinyl alcohol (DSCG/PVA) were produced via spray drying and compared in terms of their physicochemical characteristics, aerosol performance and drug uptake across a pulmonary epithelial cell line (Calu-3), cultured under air interface conditions. The particle size distribution of DSCG and DSCG/PVA were similar, of spherical geometry, amorphous and suitable for inhalation purposes. Aerosolisation studies using a modified twin-stage impinger showed the DSCG/PVA to have greater aerosol performance than that of DSCG alone. Aerosol particles of DSCG and DSCG/PVA were deposited onto the surface of the Calu-3 air interface epithelium monolayer and the drug uptake from apical to basal directions measured over time. Drug uptake was measured across a range of doses to allow comparison of equivalent drug and powder mass deposition. Analysis of the data indicated that the percentage cumulative drug uptake was independent of the mass of powder deposited, but dependent on the formulation. Specifically, with the formulation containing DSCG, the diffusion rate was observed to change with respect to time (indicative of a concentration-dependent diffusion process), whilst DSCG/PVA showed a time-independent drug uptake (suggesting a zero-order depot release).The AAPS Journal 12/2011; 14(1):79-86. · 5.09 Impact Factor -
Article: Microparticle-associated nucleic acids mediate trait dominance in cancer.
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ABSTRACT: Drug resistance is a major cause of cancer treatment failure, with multidrug resistance (MDR) being the most serious, whereby cancer cells display cross-resistance to structurally and functionally unrelated drugs. MDR is caused by overexpression of the efflux transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). These transporters act to maintain sublethal intracellular drug concentrations within the cancer cell, making the population treatment unresponsive. Recently, we discovered a novel nongenetic basis to MDR whereby microparticles (MPs) transfer P-gp intercellularly from MDR donor cells to drug-sensitive recipient cells. MPs isolated from MDR leukemia and breast cancer cells were cocultured with their drug-sensitive counterparts. P-gp transfer was assessed by direct immunolabeling, and acquired transcripts and regulatory microRNAs by quantitative real-time PCR. We show that MDR MPs incorporate nucleic acids; MPs change recipient cells' transcriptional environment to reflect donor MDR phenotype, and distinct pathways exist among cancers of different origin that may be dependent on donor cells' ABCB1 overexpression. We demonstrate that this pathway exists for both hematological and nonhematological malignancies. By conferring MDR and "retemplating" the transcriptional landscape of recipient cells, MPs provide a novel pathway, having implications in the dissemination and acquisition of deleterious traits in clinical oncology.The FASEB Journal 09/2011; 26(1):420-9. · 5.71 Impact Factor -
Article: Epithelial profiling of antibiotic controlled release respiratory formulations.
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ABSTRACT: Release profiles of two ciprofloxacin hydrochloride formulations for the treatment of respiratory infection were evaluated using different in vitro methodologies and characterised for aerosol performance and toxicity. Spray-dried ciprofloxacin and ciprofloxacin spray-dried with polyvinyl alcohol as a controlled release (CR) agent at a 50:50 w/w ratio were formulated and physico-chemically characterised. Aerosol performances were assessed in vitro using a liquid impinger. Drug release was performed using a modified Franz cell and a validated air interface Calu-3-modified twin stage impinger (TSI). Ciprofloxacin toxicity was also established in vitro. Both formulations had a similar size distribution, while CR ciprofloxacin had superior aerosol performance and stability. The release profiles showed the CR formulation to have a higher transport rate compared to ciprofloxacin alone in the cell model. Contrary results were observed using the diffusion cell. Results suggest that the air interface cell model provides a more physiologically relevant model than the modified Franz cell. Toxicity analysis showed that the lung epithelial cells could tolerate a wide range of ciprofloxacin concentrations. This study establishes that the in vitro modified TSI air interface Calu-3 model is capable of evaluating the fate of inhaled powder formulations.Pharmaceutical Research 05/2011; 28(9):2327-38. · 4.09 Impact Factor -
Article: Chronic obstructive pulmonary disease: patho-physiology, current methods of treatment and the potential for simvastatin in disease management.
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ABSTRACT: INTRODUCTION: Chronic Obstructive Pulmonary Disease (COPD) is a severe disease that leads to a non-reversible obstruction of the small airways. The prevalence of this disease is rapidly increasing in developed countries, and in 2020 it has been predicted that this disease will reach the third cause of mortality worldwide. COPD patients do not respond well to current treatment modalities, such as bronchodilators and corticosteroids. AREAS COVERED: This review article focuses on the patho-physiology of COPD, explores current approaches to alleviate and treat the disease, and discusses the potential use of statins for treatment. Specifically, the mechanism of action and metabolism of simvastatin, the most known and studied molecule among the statin family, are critically reviewed. EXPERT OPINION: Various cellular pathways have been implicated in COPD, with alveolar macrophages emerging as pivotal inflammatory mediators in the COPD patho-physiology. Recently, emerging anti-cytokine therapies, such as PDE4 inhibitors and ACE inhibitors, have shown good anti-inflammatory properties that can be useful in COPD treatment. Recently, statins as a drug class have gained much interest with respect to COPD management, following studies which show simvastatin to exert effective anti-inflammatory effects, via inhibition of the mevalonic acid cascade in alveolar macrophages.Expert Opinion on Drug Delivery 05/2011; 8(9):1205-20. · 4.90 Impact Factor -
Conference Proceeding: Comparison of Albuterol Sulphate and Base Dry Powder Particulate Deposition Using the Calu-3 Lung Epithelial Model
Respiratory Drug Delivery; 05/2011 -
Article: Modulation of P-glycoprotein-mediated anticancer drug accumulation, cytotoxicity, and ATPase activity by flavonoid interactions.
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ABSTRACT: Flavonoids are components of plant foods and of many herbal medicines taken in combination with anticancer drugs. We have examined the potential of flavonoids to affect the accumulation and cytotoxicity of 3 cytotoxic drugs [vinblastine (VLB), daunorubicin (DNR), and colchicine (COL)] that are substrates for the ABC transporter, P-glycoprotein in a vinblastine-resistant T-cell leukemia, CEM/VBL(100), that overexpresses P-glycoprotein. The effects of the flavonoids on accumulation and cytotoxicity of these drugs were different depending on the P-gp substrate used. Most of the 30 flavonoids tested decreased DNR accumulation in the VBL-resistant, but not sensitive, leukemia cells. By contrast, flavonoids that inhibited DNR accumulation enhanced the accumulation of fluorescently labeled vinblastine. None of these flavonoids affected COL accumulation. The effects of the flavonoids on the cytotoxicities of these drugs paralleled their effects on accumulation; the same flavonoids decreased DNR cytotoxicity but increased VLB cytotoxicity and had no effect on COL. Verapamil reversed the accumulation deficit and cytotoxicity of all three P-gp substrates. These effects correlated with the effects of flavonoids on P-gp-ATPase activity. Flavonoids that decreased DNR accumulation stimulated DNR-activated P-gp ATPase, whereas flavonoids that increased fluorescently labeled VLB accumulation inhibited VBL-stimulated P-gp ATPase activity, thereby accounting for the decrease or increase in cancer drug accumulation in resistant cells. We conclude that flavonoids often ingested by cancer patients may have different effects on anticancer drugs and that these findings should be considered in designing future combination treatments for cancer patients.Nutrition and Cancer 03/2011; 63(3):435-43. · 2.78 Impact Factor -
Article: Characterization of PXR mediated P-glycoprotein regulation in intestinal LS174T cells.
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ABSTRACT: Intestinal P-glycoprotein (P-gp) is an important target in drug-drug interactions. Pregnane X receptor (PXR) mediates the induction of intestinal P-gp. The LS174T intestinal cell line has been used in several studies as an in vitro tool to identify the effect of PXR inducers on intestinal P-gp expression. In this study we aimed to further characterize this cell line by focusing on the time dependence of P-gp expression, localization and function in the presence of rifampicin, a P-gp inducer. P-gp protein expression was increased in a time and dose dependent manner following exposure of cells to rifampicin (5-50 μM). The induction of P-gp by rifampicin and its inhibition by ketoconazole (an inhibitor of PXR mediated P-gp induction) confirms the suitability of these cells for PXR induction studies. Confocal microscopy showed that P-gp translocated from intracellular compartments to plasma membrane over 7 days in LS174T cells. P-gp function, as established by rhodamine 123 (Rh123) intracellular accumulation, correlated with increasing P-gp expression and plasma membrane localization over this period. Our data demonstrates that LS174T cells provide a suitable in vitro model to test for the effect of PXR inducers/inhibitors on P-gp induction, localization and function over this culture period. This model also has application for the screening of drug candidates for effects on oral bioavailability via effects on the subcellular distribution and trafficking of P-gp.Pharmacological Research 11/2010; 62(5):426-31. · 4.44 Impact Factor -
Conference Proceeding: Characterization of P-gp Expression and Function vs. Time and Passage in the Calu-3 Air-Interface Model for Drug Delivery to the Lung
Respiratory Drug Delivery, Orlando, FL; 04/2010 -
Article: Time- and passage-dependent characteristics of a Calu-3 respiratory epithelial cell model.
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ABSTRACT: Although standard protocols for the study of drug delivery in the upper airways using the sub-bronchial epithelial cell line Calu-3 model, particularly that of the air-liquid interface configuration, are readily available, the model remains un-validated with respect to culture conditions, barrier integrity, mucous secretion, and transporter function. With respect to the latter, the significance of functional P-glycoprotein (P-gp) activity in Calu-3 cells has recently been questioned, despite previous reports demonstrating a significant contribution by the same transporter in limiting drug uptake across the pulmonary epithelium. Therefore, the aim of this study was the standardization of this model as a tool for drug discovery. Calu-3 cells were grown using air-interfaced condition (AIC) on polyester cell culture supports. Monolayers were evaluated for transepithelial electrical resistance (TEER), permeability to the paracellular marker fluorescein sodium (flu-Na), surface P-gp expression, and functionality. Mucous secretion was also identified by alcian blue staining. TEER and permeability values obtained for Calu-3 monolayers were shown to plateau between day 5 and day 21 in culture with values reaching 474 +/- 44 omega cm(2) and 2.33 +/- 0.36 x 10(-7) cm/s, respectively, irrespective of the passage number examined. 32.7 +/- 1.49% of Calu-3 cells cultured under these conditions detected positive for cell surface P-gp expression from day 7 onwards. Functional cell surface expression was established by rhodamine 123 drug extrusion assays. This study establishes a clear dependence on culture time and passage number for optimal barrier integrity, mucous secretion, and cell-surface P-gp expression and function in Calu-3 cells. Furthermore it provides initial guidelines for the optimization of this model for high throughput screening applications.Drug Development and Industrial Pharmacy 04/2010; 36(10):1207-14. · 1.49 Impact Factor -
Article: Dietary nucleotide supplements in infant formula modify the expression of P-glycoprotein in the intestinal epithelium in vitro.
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ABSTRACT: The effect of dietary nucleotides at concentrations found in supplemented infant formula on P-glycoprotein (P-gp) expression in colon cells was examined. We report that P-gp expression in colon cells was significantly decreased in a time- and concentration-dependent manner. When colon cells were co-cultured with lymphocytes, so as to mimic the involvement of gut-associated lymphoid tissue in normal gut pathophysiology, we observed a reversal of this effect with a demonstrated increase in P-gp expression. These findings have important implications on effects of nucleotide exposure on increasing drug bioavailability, reducing the capacity for xenobiotic efflux, and increasing the risk of inflammatory bowel disease in susceptible infants. Future studies are directed at defining both the mechanisms underlying these findings and effects of dietary nucleotide supplementation in vivo.International Journal for Vitamin and Nutrition Research 09/2009; 79(5-6):381-7. · 0.88 Impact Factor -
Article: Differential pharmacological regulation of drug efflux and pharmacoresistant schizophrenia.
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ABSTRACT: Pharmacoresistant schizophrenia is a significant impediment to the successful management of the disease. The expression and function of P-glycoprotein (P-gp) has recently been implicated in this phenomenon. P-gp is a multidrug efflux transporter that prevents drug substrates from crossing the blood-brain barrier (BBB). Although the direct interaction between individual antipsychotic agents and P-gp has been demonstrated, the effect of antipsychotic drug combinations used in disease management on P-gp transport function remains to be elucidated. This could have important clinical implications in some individuals as dosage adjustments based on plasma drug concentration changes may not always be appropriate if drug-drug interactions and the resulting changes in drug concentration in the brain are not considered. This paper introduces the potential impact that combination antipsychotic therapy may have on P-gp function at the BBB and discusses the consequences of this in the prevention and circumvention of unfavourable therapeutic response in schizophrenic disorders.BioEssays 03/2008; 30(2):183-8. · 4.95 Impact Factor -
Article: Effect of short-term morphine exposure on P-glycoprotein expression and activity in cancer cell lines.
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ABSTRACT: Multidrug resistance (MDR) is a common problem in various types of cancer. One important factor in the development of MDR is overexpression of P-glycoprotein, encoded by the MDR1 gene. Morphine is the opioid of choice for moderate to severe cancer pain, and is a substrate of P-glycoprotein. Recently, morphine has been shown to induce P-glycoprotein expression in the rat brain. Using Western blot analysis and cytotoxicity assays respectively, we have investigated the effects of short-term (72 h) morphine treatment on P-glycoprotein expression in a panel of human cancer cell lines, and its effects on cellular resistance to the known P-glycoprotein substrates, vinblastine and colchicine. The effect of morphine on P-glycoprotein expression and activity in the mouse fibroblast NIH-3T3 cell was assessed to establish whether morphine effects are species specific. Short-term exposure to morphine did not result in any significant differences in P-glycoprotein expression or activity in any cancer cell lines. Morphine pre-treatment resulted in a moderate but significant increase in sensitivity of NIH-3T3 cells to vinblastine, but not colchicine. This study suggests that morphine effects may be cell-type specific. Importantly, however, it appears that short-term morphine treatment does not affect the MDR phenotype of tumour cells.Oncology Reports 06/2004; 11(5):1091-5. · 1.84 Impact Factor -
Article: Dynamic and intracellular trafficking of P-glycoprotein-EGFP fusion protein: Implications in multidrug resistance in cancer.
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ABSTRACT: In our present study, a P-glycoprotein-EGFP (P-gp-EGFP) fusion plasmid was constructed and functionally expressed in HeLa cells to investigate the intracellular localization and trafficking of P-glycoprotein (P-gp). Using immunocytochemistry and fluorescent confocal microscopy techniques, colocalization studies showed that after transfection, P-gp-EGFP was progressively transported from the endoplasmic reticulum (ER) to the Golgi and finally to the plasma membrane within 12-48 hr. The degree of intracellular accumulation of daunorubicin was related to the particular localization of P-gp-EGFP. Significant daunorubicin accumulation occurred in transfected cells when P-gp-EGFP was localized predominantly within the ER, and accumulation remained high when P-gp-EGFP was mainly localized in the Golgi. However, there was little or no intracellular accumulation of daunorubicin when P-gp-EGFP was localized predominantly on the plasma membrane. Blocking the intracellular trafficking of P-gp-EGFP with brefeldin A (BFA) and monensin resulted in inhibition of traffic of P-gp-EGFP and retention of P-gp-EGFP intracellularly. Intracellular accumulation of daunorubicin also increased in the presence of BFA or monensin. Our study shows that P-gp-EGFP can be used to define the dynamics of P-gp traffic in a transient expression system, and demonstrates that localization of P-gp on the plasma membrane is associated with the highest level of resistance to daunorubicin accumulation in cells. Modulation of intracellular localization of P-gp with agents designed to selectively modify its traffic may provide a new strategy for overcoming multidrug resistance in cancer cells.International Journal of Cancer 04/2004; 109(2):174-81. · 5.44 Impact Factor
Top co-authors
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Institutions
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2013
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Woolcock Institute of Medical Research
Sydney, New South Wales, Australia
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2004–2012
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University of Sydney
- Faculty of Pharmacy
Sydney, New South Wales, Australia
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