Bidirection permeability assay

Transporters P-gp assay (see Section 6.3.2.2) Other transporter assays Assessing transporter involvement in in vitro absorption experiments Predicting in vivo transporter-related habihties Assay ° Bidirectional permeability assays using Caco-2 or other cell lines ° 3-50 xM compound concentration Analysis ° PPT followed by LC-MS/MS... 

In the transport assays, the permeability of a compound in both absorption and secretion directions is measured using polarized epithelial cells that constitutively express high levels of P-gp (e.g. Caco-2) or have been transfected with the gene for a specific P-gp (e.g. MDR1-transfected MDCK or LLC-PK1 cells). Since P-gp is expressed on the apical membrane, ratios ofbasolateral-to-apical (B —> A) permeability versus apical-to-basolateral (A —> B) permeability greater than 1 may indicate an active efflux transport process. Bidirectional permeability measurements can also be performed in the presence of a specific P-gp inhibitor. Thus, apical-to-basolateral permeability increases and basolateral-to-apical permeability decreases such that... 

This buffer is used in both the unidirectional (permeability) and the bidirectional (Efflux, Section 4.3.2). In the permeability assay, NCEs are placed into the apical compartment of the Transwell. Duplicate samples are taken immediately after compound addition from the apical compartment (zero time) and then after 2h from both the apical and basolateral compartments for LC-MS/MS analysis. The integrity of the monolayer is confirmed by the measurement of the transepithelial electrical resistance (TEER), which must be above a certain limit to be used for transport experiments. In addition, with each experiment a transcellular and a paracellular marker are included for quality control. 

Absorption PAMPA (see Section 63.2.2) Permeability (Caco-2, MDCK cells, etc) (see Section 6.3.2.2) Bidirection permeability (see Section 6.3.2.2) Assessing in vitro membrane permeabUity Predicting in vivo absorption Assay ° Dual-chamber sandwich setup using either artificial or live cell membranes ° 3-50 rM compound concentration Analysis ° PPT foUowed by LC-MS/MS... 

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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