Preformulation studies of EFdA, a novel nucleoside reverse transcriptase inhibitor for HIV prevention (original) (raw)
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Journal of Pharmaceutical Sciences, 1998
0 The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody. Previous drug exposure of the cells showed that verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid (MKA) decreased the P-gp expression level as compared to the control. Permeation studies consolidated the theory that P-gp is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to basolateral could be measured. Incubation of the cell monolayer with MRK 16 reduced the secretion process to the apical side, but did not influence [ 3 H]mannitol flux. Caco-2 cells seem to be a suitable cell line model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.
Cell Culture Models for Drug Transport Studies
Principles and Applications, 2016
Page 1. 7 CELL CULTURE MODELS FOR DRUG TRANSPORT STUDIES D. NEDRA KARUNARATNE, PETER S. SILVERSTEIN, VEENA VASANDANI, AMBER M. YOUNG, ERIK RYTTING, BRADLEY YOPS, AND KENNETH ...
Journal of Pharmaceutical Sciences, 1990
Abatract 0 A human intestinal cell line, Cam-2, was used as a model to study the passive diffusion of drugs across intestinal epithelium. The cells formed continuous monolayers when grown on permeable filters of polycarbonate. After 10 days in culture, the monolayers had a transmembrane resistance of -260 ohmscm2 and a cell density of 0.9 x 10' cellslcm2. At this time the cells were impermeable to ["CJpolyethyleneglycol (MW 4000). These characteristics remained constant for 20 days (i.e., from day 10 to day 30). Six beta-blocking agents with a 2000-fold range of lipophilicity were studied for their transepithelial transport properties. The transport parameters were independent of drug concentration and transport direction. The apparent permeability coefficients ranged from 41.91 t 4.31 x lo-' cm/s for the most lipophilic drug, propranolol, to 0.203 2 0.004 x lO-'cm/s for the most hydrophilic drug, atenolol. The transport parameters were compared with those published for rat ileum. Drugs and Radiolabeled MarkereSH-Labeled and unlabeled atenolol (specific radioactivity 0.627 nCi/nmol), H216/44 [(S)-4-hydroxy-N-~2-((2-hydroxy-3-(4-~2-~2-~cyclopropylmethoxy)ethoxy)ethyl)phenoxy)-propyl)amino)ethyl)-l-piperidinecar~x~ide; specific radioactivity 16.7 pCi/nmoll, alprenolol (specific radioactivity 0.252 nCi/nmol), and metoprolol (specific radioactivity 0.475 pCi/nmol, as well as "C-labeled practolol (2.1 nCi/nmol) were generous iRs from Dr. KurtJtirgen Hoffman, AB HBssle, GBteborg, (MW 4000; specific radioactivity 15.0 Ci/mg) were obtained from New England Nuclear, Boston, MA. The radiolabeled compounds had a radiochemical purity of 96-99%, Propranolol and alprenolol were purchased from Sigma Chemical Company, St. Louis, MO. Octanol-water distribution coefficienta (0) and PK, values for the pblockers were kindly provided by Dr. KurtJtirgen Hoffman, AB Hlssle, GBteborg, Sweden, and have also been published elsewhere.1"14 Cells-Caco-2 cells, originating from a human colorectal carcinoma,lb were obtained from American Tissue Culture Collection, Rockville, MD. The cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum, 1% nonesaential amino acids, benzylpenicillin (100 U/mL), and streptomycin (10 pg/mL). Cells of passage number 85-95 were d. All tissue culture media were obtained from Gibco through Laboratorie Design AB, Lindingo, Sweden. The medium was changed every eecond day.
Journal of Pharmaceutical Sciences, 2012
The HT-29 cell line forms a confluent monolayer with tight junctions, but displays different phenotypes when cultured for 21 days in galactose-supplemented media (differentiated) versus glucose-supplemented media (dedifferentiated). This study is aimed at elucidating how media differences might affect selected drug transporter expression and peptide-based substrate transport toward reducing this variability. A vial of HT-29 cells was amplified and cultured over several passages in four different mediums (American Type Culture Collection recommended McCoy's 5A versus Dulbecco's modified Eagle's media containing glucose, galactose, or neither carbohydrate) with normal supplementation. Transporter mRNA expression was characterized at days 5 and 21 postseeding utilizing SABiosciences quantitative reversetranscriptase polymerase chain reaction (qRT-PCR) drug transporter arrays. Transport studies using [H]histidine, [ 3 H]glycylsarcosine, [ 3 H]valacyclovir, and [ 3 H]carnosine were performed to assess the functional effects of oligopeptide transporter expression changes in HT-29 cells grown in each media. qRT-PCR arrays illustrated variable, media-dependent transporter expression between both the initial and differentiated time points. Permeability studies illustrated considerable media-dependent differences in both paracellular and transcellular substrate fluxes. The results demonstrate that these cells exhibit differing monolayer characteristics and genotypic/phenotypic profile properties when cultured under different media. The results suggest a need for standardization of culture methodologies for reducing inter-and intralaboratory variability.
European Journal of Pharmaceutical Sciences, 2007
Intestinal absorption Transport medium Simulated intestinal fluid a b s t r a c t The purpose of this study was to identify simulated intestinal fluids (SIFs) containing nutrients compatible with the Caco-2 cell culture model and to examine the impact of the identified medium on the transport of a poorly aqueous soluble model compound, estradiol, and a substrate of efflux mechanisms, etoposide. Monolayer integrity was evaluated by transepithelial electrical resistance and cellular viability by release of lactate dehydrogenase to the apical compartment and cellular protein content. It was shown that the viability of Caco-2 cells was enhanced by use of the CO 2 independent nutritional medium, Leibovitz's L-15 compared to Hanks' balanced salt solution. SIF containing 5 mM sodium taurocholate and 1.25 mM phosphatidylcholine or lysophosphatidylcholine in Leibovitz's L-15 induced less release of lactate dehydrogenase than the traditional transport medium, HBSS.
Govaniadine Evaluation of Cytotoxicity and Permeability in Cell Culture
Revista Brasileira de Farmacognosia, 2020
Govaniadine (1), a tetrahydroprotoberberine-type alkaloid, is one of the main active constituent isolated from Corydalis govaniana Wall., Papaveraceae, with several biological activities: antinociceptive, anti-urease, and leishmanicidal. Some articles reporting tetrahydroprotoberberine and structurally related alkaloids cytotoxicity prompted us to evaluate the influence of compound 1 on cellular viability in two different cell lines: human hepatoma carcinoma (HepG2) and human embryonic kidney (HEK-293T) and its permeation across the human colon carcinoma cell line (Caco-2). Cellular viability reduction of compound 1 was observed between 30 and 100 μM (HepG2) and between 70 and 100 μM (HEK-293T). However, the effects were weaker than those in the positive controls (T-2 toxin and camptothecin). Prior to proceed the transport studies, a method for compound 1 quantification in Hank's Balanced Salt Solution medium was developed and validated by LC-MS/MS. The two calibration curves were linear over the concentration range of 6.3-200 nM and 0.2-10 μM. The apparent permeability coefficient for absorptive transport was 20.6 ± 3.9 × 10 −6 cm/s, indicating compound 1 crossed the cell monolayer by passive diffusion and it was not subjected to active efflux.
Evaluation of Drug Transport in MDCKII-Wild Type, MDCKII-MDR1, MDCKII-BCRP and Caco-2 Cell Lines
Current pharmaceutical biotechnology, 2017
Drug transporters function as gatekeepers and modulate drug access into body and various tissues. Thus, a thorough and precise understanding of transporter liability for compound uptake and efflux is critical during drug development. In the present study, we assessed the apparent permeability (Papp) and compared efflux ratio of various compounds in stably transfected Madin-Darby Canine Kidney (MDCKII) cells overexpressing human P-gp (MDCKII-MDR1), human BCRP (MDCKII-BCRP), wild-type (MDCKII-WT), and Caco-2 cell monolayers. We observed that quinidine, a substrate for MDR1 transporter, showed efflux ratio (Papp B-A/ Papp A-B) of 838 in MDCKII-MDR1 cells which plummeted to 14 in presence of verapamil, a known inhibitor of MDR1. With MDCKII-WT cells, Papp of quinidine dropped from 2 to 1, in the presence of verapamil. Caco-2 cells showed a diminutive decrease in efflux ratio of quinidine from 2.5 to 1.6 by verapamil. Prazosin and dantrolene were evaluated in MDCKII-BCRP cells and were f...
European Journal of Pharmaceutics and Biopharmaceutics, 2011
The aim of the present study was to investigate whether Calu-3 cell culture conditions influence drug and nutrient transport known to occur via carriers or transporters. Calu-3 cell layers, an in vitro model of the lung epithelium, were cultured using air interfaced culture (AIC) or liquid covered culture (LCC) on either polycarbonate or polyester as filter support material. We found that the development of the Calu-3 cell layer barrier function did not depend on the filter material but rather on the culture conditions as follows: (i) the apical uptake of Gly-Sar was significantly larger for cells grown in AIC compared to LCC, (ii) the TEER values for cells grown in LCC were approximately three times larger than for cells grown in AIC, (iii) the transepithelial transport in both AIC and LCC Calu-3 cells was polarized in the apical-basolateral direction of proline, glycine, a-methyl-D-glucoside, glipizide, taurocholic acid and estrone-3-sulfate, whereas inulin, mannitol and Gly-Sar showed no polarized transport. Etoposide showed polarized efflux (basolateral to apical transport) in AIC and LCC Calu-3 layers. These findings provide information about nutrient and drug transport in Calu-3 cells, and this may have implications for selecting culture conditions for transport studies in this in vitro model of the lung epithelium.