, 1999, Förster et al., 2005 and Cohen-Kashi Malina et al., 2009). Indeed, some are used commercially ( Culot et al., 2008 and Vandenhaute et al., 2012). A key question is the degree to which permeability data from an in vitro model reflect in vivo BBB permeability, i.e., the quality of in vitro–in vivo correlation (IVIVC). But Selleckchem LY2109761 often overlooked are the influence of the aqueous boundary layer (ABL) and variable/low-TEER
on in vitro permeability measurement. The ABL, also referred to as the unstirred water layer (UWL), is a region of poorly-stirred solution adjacent to the cell layer of interest (Korjamo et al., 2008). In vivo, the cerebral capillary network has an irregular highly branched course and a high velocity of red blood cells in the circulation ( Hudetz, 1997); even in capillaries with low or no red blood cell traffic, plasma flow has the same stirring effect ( Villringer et al., 1994). Therefore, the ABL in vivo is minimal. However, in both epithelia and endothelia in vitro, a significant ABL is present adjacent to the cell membrane as a result of inefficient stirring during
the experiment ( Barry and Diamond, 1984, Youdim et al., 2003 and Korjamo et al., 2008) ( Fig. 1). Permeation through the ABL is by passive diffusion. Hence, the ABL is a rate-limiting step for permeation of lipophilic compounds resulting in reduction of the apparent permeability ( Hidalgo et al., 1991, Karlsson and Artursson, 1991, Ruell et al., 2003, Avdeef et al., 2004, Katneni et al., 2008 and Velický et al., 2010), leading Trametinib to reduced dynamic range and lower resolution in rank-ordering compound permeation. The ABL can also be a source of bias in determining the Michaelis–Menten transport kinetic Km because of the concentration gradient created within the ABL ( Wilson and Dietschy, 1974 and Balakrishnan et al., 2007) ( Fig. 1). The ABL can also mask inhibition of specific carrier-mediated transport based on similar apparent permeability nearly measured for transporter substrate in
the absence and presence of inhibitors ( Naruhashi et al., 2003). If the ABL effect is ignored, the permeability measured in vitro will not reflect the true permeability in vivo. Currently there is no quantitative correction for ABL used routinely for in vitro BBB permeability data. An early study on the effect of ABL on in vitro BBB permeability by Ng et al. (1993) prompted awareness of the problem. Since then, most researchers have used stirring during permeability experiments to minimize the ABL effect. However, full ABL correction from analysis of in vitro permeability data is rarely used. The most common method to correct for ABL in in vitro BBB permeability data analysis is subtraction of the permeability of compounds through blank filter inserts, Pfilter (without cells) from apparent endothelial cell permeability, Papp, to obtain permeability through the cell monolayers, Pe (e.g.