The experiment was to investigate interaction of candidate drug compounds: Verapamil and two benzodiazepine derivatives: CC175 (Trizolophenylalaine) and CC181 (Trizolovaline) with PGP. A fluorescent substrate transported by PGP acts as a marker for active transport function by the measurement of retained fluorescence per cell. In this case, Rhodamine123 (R123) is used as a fluorescent marker._x000D_
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Cell culture is needed to grow Caco-2 cells in a controlled and sterile environment. When 80% confluence has been reached, R123 efflux assay can be performed. Cells are incubated with R123 and the drug chosen for each run. Two additional culture dishes are needed. A drug free control and also one incubated with Verapamil to inhibit PGP growth as a positive control. Interactions of the benzodiazepine derivatives with PGP can be measured and compared to the controls. Protein assay and fluorimetric analysis is performed on each sample to evaluate the interaction and the level of inhibition in any of the candidate drugs. The level of R123 retained in each lysate sample can be corrected (normalised) to represent fluorescence responses for each particular drug used. As R123 is affected by other substrates/inhibitors the data collected can be evaluated to explain the interaction._x000D_
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The aim of this study was to evaluate the interaction of some candidate drug compounds with P-glycoprotein (potential to modify absorption or distribution). Caco-2 cell monolayers are used as a model of in vivo permeability to many drugs including, Verapamil and benzodiazepine derivatives using rhodamine 123 as a marker._x000D_
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The objective was to establish and grow up a caco-2 cell line and to investigate the interaction of therapeutic drugs,Verapamil and benzodiazepines with P-gp to observe whether they were absorbed in the gut. The objective was also to generate expression of PGP in cells and to perform a R123 efflux assay which included protein assay and fluorimetric analysis._x000D_
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The cell media used for the cells was Dulbecco’s Modified Eagles medium high glusose (DMEM). In 500ml of DMEM, 5.5ml of (4mM) glutamine was added together with 50ml of 10 % fetal bovine serum and 2.5ml of 50IU/ml penicillin and 50µg/ml streptomycin to prevent the growth of any bacteria. To check if the media is safe to use on the cells, some of it was poured into a universal tube which was placed into an incubator. After 3 days, the antibiotic was used up, and the media was checked if there was anything growing in it. The media was clear but if there was something growing it needed to be thrown away._x000D_
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The cells media was changed every two days in the microwflow class ΙΙ safety cabinet. The used media was poured out and the flask was washed with HBSS and 20ml of media was poured in. The flask was placed back into the sterile incubator, slightly opening the lid to allow oxygen to go in._x000D_
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The cells were obtained from Manchester and grown in flasks. The cells were split in order to grow them in a new flask. This technique was carried out in a clean microwflow class ΙΙ safety cabinet which was disinfected with ethanol before use. Splitting the cells involved washing the cells from the previous flask twice with Hanks Balanced Salt solution (HBSS) which was formulated without magnesium or calcium. The salt solution helped loosen the cells. Diluted 1.5ml trysin (1:1 with HBSS) was added to cells to loosen the cells that are stuck at the bottom of the flask. The trypsined cells were placed in an incubator for approximately 10- 15 minutes or until the cells fell of which could be seen by looking under the microscope. It was important to make sure all the cells fell off in order to capture all the properties. The cells that fell off first from the trypsin could have phenotypic qualities that are vulnerable to trypsin. 5ml of media was poured into a tube and then into the flask to neutralise the trypsin. Pour the mixture of cells and media from the flak into a second tube. This step was repeated again to clear out the flask. 1.5ml was pipette out of the tube with the cells and into a new flask. To make it up to 20ml media was poured into the flask. The flask was placed into the sterile incubator after the flak was dated the passage number was noted._x000D_
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Sometimes when seeding a new flask, wells were also seeded to measure the cells’ viability over a number of days. From the method above (2.2 Splitting the cells/Seeding a flask), after seeding the new flask, 20µl of the remaining cells that were in the universal tube were pipetted out and 20µl of tripam blue was added to them. Tripam blue stained the dead cells to enable the counting of the viable cells using a haemocytometer. It was then worked out in microlitres how much cells needed to put pipetted in each well. The wells were then placed in the incubator after adding media. The media was changed every two days._x000D_
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The cells from the wells were used to determine cell number and viability. Three wells were used each time. The media was pipetted out of the wells. 100µl of HBSS was pipette into the wells. The cells were scraped and pipette into epindorf tubes and mixed using a pipette. 20µl was pipette into another epindorf tube together with 20µl of tripam blue. A haemocytometer was used to determine the cell number from each epindorf. Cell viability was determined by dividing the total number of live cells by the total number of cells in each well multiplied by 100. This determined the cell viability as a percentange._x000D_
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Rhodamine 123. -Provided by Sigma._x000D_
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Verapamil. – Provided by Sigma._x000D_
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Benzodiazepines – Benzodiazepines CC175 (Trizolophenylalaine) and CC181(Trizolovaline) were provided by the University of Huddersfield._x000D_
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STRUCTURES OF BENZO-DERIVATIVES (provided by Dr Karl Hemming, University of Huddersfield)_x000D_
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A lysing solution was used in the Rhodamine 123 efflux assay. The solution consisted of 0.5% deoxycholate, 1% Triton X-100,1mM phenyl methyl sulfonyl and KH2PO4 at pH7.4_x000D_
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Petri dishes were each with HBSS (1ml). Rhodamine (7µl) was pipetted into the media solution(7ml) based on a 1:1000 dilution and Verapamil (3µl) was pipetted into media(3.5ml). CC175(3µl) and CC181(3µl) were each pipetted in media solution(3ml) respectively. The Rhodamine, Verapamil and CC175 and CC181 solutions were pipetted into the culture dishes as shown on the table1:_x000D_
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Petri Dishes_x000D_
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Solution added_x000D_
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1_x000D_
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ImL of Rhodamine (control)_x000D_
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2_x000D_
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1mL of Rhodamine (control)_x000D_
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3_x000D_
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1mL of Rhodamine (control)_x000D_
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4_x000D_
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1mL of Rhodamine + 1mL of Verapamil_x000D_
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5_x000D_
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1mL of Rhodamine + 1mL of Verapamil_x000D_
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6_x000D_
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1mL of Rhodamine + 1mL of Verapamil_x000D_
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7_x000D_
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1mL of Rhodamine + 1mL of CC175_x000D_
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8_x000D_
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1mL of Rhodamine + 1mL of CC175_x000D_
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9_x000D_
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1mL of Rhodamine + 1mL of CC175_x000D_
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10_x000D_
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1ml of Rhodamine + 1mL of CC181_x000D_
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11_x000D_
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1mL of Rhodamine + 1mL of CC181_x000D_
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12_x000D_
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1mL of Rhodamine + 1mL of CC181_x000D_
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The petri dishes were incubated for 90 minutes at 370. Following incubation the petri dishes were quickly washed 5 times with ice cold HBSS and lysed in a 1.5mL solutionof the lysing solution at 370 for 10 minutes. The cells were then shaken for 20 minutes and scraped off the surface of the petri dishes to ensure complete lysis of the cells. A sample of the lysate (0.5ml) was taken out for the lowry protein assay, to assess the protein content, and the remainder was centrifuged at 13,000 rmp for 10 minutes. The supernatant was used to fluorimetrically measure the level of R123 in the cells at 492nm and 535nm._x000D_
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The protein in the cells was determined using the lowry protein assay which involved using these reagents;_x000D_
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Solution A: 2% Na2CO3 in 0.1M-NaOH._x000D_
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Solution B: 0.5% CuSO4·5H2O in 1% sodium or potassium tartate._x000D_
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Solution C: 50mL of solution A was mixed with 1mL of solution B and this was renewed each day we did the assay._x000D_
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Solution D: Commercial Folin-Ciocalteu reagent was diluted 2-fold with water to make it 1M in acid._x000D_
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A protein standard curve was prepared in duplicate containing 0, 20, 40, 80, 100 and 120µg of bovine serum albumin serum (4mg/mL). This equated to 0, 5, 10, 20, 30 and 40µl of BSA solution made up to 100µL with distilled water. Glass test tubes were used to prepare this standard solution._x000D_
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2µL and 10µL of the protein samples were pipetted into glass test tubes and made up to 100µL with distilled water. 1mL of Solution C was added to each tube, vortex mixed and left for 5 minutes. 0.1mL of Solution D was added to each tube, vortex mixed and left to stand for 30 minutes. 1mL o distilled water was added to each tube and the absorbance read at 750nm for each tube.