Efflux Experiments Using CACO-2 Cells

MDR1 and members of the MRP family have substantial substrate overlaps (Borst et al. 2000). 

BCRP is an ABC half-transporter and is expressed at very high levels in the intestine, liver, kidney and blood-brain barrier. However, the identified list of substrates and/or inhibitors is limited yet Mitox-antrone, topotecan, doxorubicin, flavopiridol, SN-38, Lysotracker green, BBR 3390, BODIPY-Prazosin, Rhodamin 123. As inhibitors GF 120918 and fu-mitremorgin C were identified (Doyle et al. 1998 Litman et al. 2000). 

In the small intestine expression data for MDR1, ABCG2, MRP s and LRP have been detected (Kool 

Many laboratories use CACO-2 cells as a standard method for assessment of efflux. Since the standard CACO-2 cell assay is very well established, easy to use, reproducible and reliable the corresponding efflux assay can give valuable and helpful data for project support in a screening approach. A prerequisite for the interpretation of efflux data is a characterization of efflux transporters present in the system used and a set of standard efflux markers checked regularly (like digoxin for MDR1). 

As experimental buffer for washing, permeability and efflux assay HBSS, pH 7.4 is proposed. 

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II.D.5 Efflux Inhibition Experiments Using CACO-2 Cells... 

The same quinolone CNV97100 was used to perform bidirectional experiments in Caco-2 cells to obtain the apical to basal Pai, and basal to apical Pya permeabilities at different initial concentrations. The relationship between previously obtained in situ rat permeability values and in vitro Caco-2 of CNV97100 was examined. In each system the in vitro and in situ paracellular permeability of CNV97100 was considered to be negligible. The systems differed in effective surface area (Sj) and in efflux transporter expression level. Although the Caco-2 expression level of P-gp is similar to the expression levels in rat ileum, the model allows for system differences (EJj. 

Intestinal excretion is an important mechanism in the elimination of flavonoid conjugates [82-84]. This is likely mediated by the multidrug resistance-associated protein (MRP) pumps. MRPs actively export conjugated metabolites out of the small intestine back into the intestinal lumen and so prevent or reduce systemic circulation [85]. An experiment using cultured Caco-2 cells showed that two metabolites of epicatechin were excreted on the apical side of the cells. Their elimination has been attributed to MRP-2, as efflux was significantly reduced by a competitive MRP-2 inhibitor [86]. Conversely, intestinal perfusion experiments with catechin indicate that catechin metabolites are not substrates for these transport proteins [49]. 

Modifications include all details described in the method above (time of permeability experiment, concentration ranges, shaking, use of standards, quality criteria and acceptance parameters). Since in CACO-2 cells efflux for standard compounds varies due to heterogenicity and batch variations several teams used... 

In addition to the unidirectional mode described above, Caco-2 assays can also be performed in the bidirectional mode. A bidirectional assay essentially consists of two independent unidirectional assays, with compounds added to the apical side in one experiment, and to the basolateral side in the other. Unidirectional Caco-2 assays yield permeability values only in A > B direction, whereas bidirectional assays yield values in both A > B and B > A directions. Since many active transporter proteins are expressed in the Caco-2 cell line, the ratios of Pa>b to Pb>a in bidirectional assays can be used to assess transporter involvement in drug permeability. If Pa>b Pb>a, then the compound is likely to be actively transported for uptake if Pb>a/Pa>b > 3, then the compound is likely a substrate for efflux transporters. Additional assays using the same bidirectional format can be used to further identify whether the test compounds are substrates or inhibitors of the transporters, therefore providing liabihty assessments of transporter-related DDI potential as either victims (substrates) or perpetrators (inhibitors) [77]. 

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