Comparison of drug transporter gene expression and functionality in CACO-2 cells from 10 laboratories. Eur J Pharm Sci
Discovery DMPK and Bioanalytical Chemistry, AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden. European Journal of Pharmaceutical Sciences
(Impact Factor: 3.35).
09/2008; 35(5):383-96. DOI: 10.1016/j.ejps.2008.08.004
Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.
Available from: PubMed Central
- "We recognise that many factors can affect a specific measurement, e.g., the seeding density, age of the cells, pH and so on. An interlaboratory comparison (Hayeshi et al., 2008) indicated that while on occasion measurements could vary by more than an order of magnitude, overall the groupings were normally reasonably tight (say within a factor of 2–5). The question of P app values in Caco-2 cells has been brought into sharper focus by a recent article (Matsson et al., 2015a; Matsson et al., in press) that claimed unusually high rates for verapamil and propranolol, based on measurements in a specific earlier article (Avdeef et al., 2005) in which stirring had been performed at a massive rate (and one not used in any equivalent transporter kinetics measurements). "
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ABSTRACT: We bring together fifteen, nonredundant, tabulated collections (amounting to 696 separate measurements) of the apparent permeability (
) of Caco-2 cells to marketed drugs. While in some cases there are some significant interlaboratory disparities, most are quite minor. Most drugs are not especially permeable through Caco-2 cells, with the median
value being some 16 ⋅ 10
. This value is considerably lower than those (1,310 and 230 ⋅ 10
) recently used in some simulations that purported to show that
values were too great to be transporter-mediated only. While these values are outliers, all values, and especially the comparatively low values normally observed, are entirely consistent with transporter-only mediated uptake, with no need to invoke phospholipid bilayer diffusion. The apparent permeability of Caco-2 cells to marketed drugs is poorly correlated with either simple biophysical properties, the extent of molecular similarity to endogenous metabolites (endogenites), or any specific substructural properties. In particular, the octanol:water partition coefficient, log
, shows negligible correlation with Caco-2 permeability. The data are best explained on the basis that most drugs enter (and exit) Caco-2 cells via a multiplicity of transporters of comparatively weak specificity.
Available from: Tetsuya Terasaki
- "Transport capacities of membrane transporters are generally evaluated by means of transport experiments using specific substrates and/or inhibitors of target transporters. Among the many transporters expressed in Caco-2 cells and intestine  ,m a j o r drug transporters in Caco-2 cells, such as MDR1, MRP2, BCRP, and PEPT1, have often been analyzed  . However, other transporters have not been well analyzed, and indeed, specific substrates , inhibitors or effective siRNA are often not available. "
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ABSTRACT: The purpose of the present study was to evaluate and compare the absolute protein expression levels of 28 drug-related transporters in Caco-2 cell monolayers cultured for 2, 3, and 4 weeks. Plasma membrane fractions of Caco-2 cells cultured on Transwell inserts for 2, 3 and 4 weeks were prepared and digested with trypsin, and then simultaneous absolute quantification of 28 transporters and Na(+)/K(+)-ATPase was conducted using our established quantitative targeted absolute proteomic technique. Nine transporters and Na(+)/K(+)-ATPase were detected. MDR1, BCRP, PEPT1, OSTα and OSTβ were highly expressed (greater than 1 fmol/μg protein), while MRP2, MRP4, OATP2B1 and MCT1 were moderately expressed (0.328-0.871 fmol/μg protein). No significant difference was observed in the protein expression levels of these transporters or Na(+)/K(+)-ATPase among the 2-, 3- and 4-week cultures. The other 19 transporters, including MRP1, MRP3, OATP1A2, OATP3A1, OATP4A1, and OATP1B3, were not detected. This information about the rank order of transporter protein expression will be useful to predict what transporter(s) are likely or unlikely to influence the permeability of various compounds across monolayers of Caco-2 cells, which are widely used in drug development studies.
Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
Available from: Angela Cardinali
- "Although Caco-2 cells retain many morphological and functional properties of the enterocytes, such as the expression of specific transporters and polarity, the intestinal cell behavior in vivo might be highly different. In addition, permeability data obtained from the Caco-2 system can often vary between laboratories, even when standard protocols for transport studies are used (Englund et al., 2006; Hayeshi et al., 2008). Therefore, when not properly controlled, relative Food Research International 54 (2013) 132–138 Abbreviations: BHT, butylated hydroxytoluene; Isc, Short circuit current; MF, microfiltrated; OMWW, Olive Mill Waste Water; P app , apparent permeability coefficient; P app,tissue , the apparent permeability coefficient tissue; P app,total , apparent permeability coefficient total; PCO 2 , partial pressure of carbon dioxide; PO 2 , partial pressure of oxygen; "
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ABSTRACT: The intestinal absorption of verbascoside (VB), a phenylpropanoid glycoside, extracted and purified from olive mill waste water (OMWW), was investigated using viable and healthy human colonic tissues, mounted in an Ussing chamber. Additionally, VB absorption and transport through the intestinal mucosa were quantified using permeability coefficients. VB absorption was time-dependent and varied in relation to the specific colonic segments considered. In particular, major uptake/absorption (0.50 μg/cm 2) occurred between 5 and 15 min in the proximal tract of the colon, followed by descending colon (0.38 μg/cm 2) between 30 and 60 min, and sigmoid–rectum colon (0.34 μg/cm 2) at 60 min. Overall, VB was absorbed rapidly, with an average uptake of 0.29 μg VB per cm 2 , corresponding to a total accumulation efficiency of ~0.12%. Moreover, the presence of the VB in the basolateral side supported the hypothesis of its bioavailability in the extent of 0.1%. In addition, the permeability coefficient calculation has contributed in a deeper understanding of VB absorption and transport across the human intestinal barrier and could be utilized for other polyphenols present in food and in dietary supplements.
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