Permeability screening assays

Compared to in vivo studies, the Caco-2 model substantially increases the speed at which absorption potential can be estimated and reduces the amount of drug substance needed. However, manually performed assays are still too slow and labor intensive compared to biological high-throughput screening assays. Caco-2 cells take about 3 weeks to form monolayers of fully differentiated cells. At this point, Caco-2 monolayers are used to evaluate absorption potential under a variety of permeability protocols. In order to further expedite the process of absorption potential assessment, efforts have been made to increase the throughput of Caco-2 transport experiments. 

Another in vitro method for permeability screening was parallel artificial membrane permeation assay (PAMPA) initially reported by Kansy. In a PAMPA permeability screen, the Caco-2 cell mono-layer membrane is replaced by an artificially generated membrane. Versions of different artificial membranes that lack active transporter systems and pores have been developed to mimic the in vivo transcellular intestinal epithelial cell barrier. Therefore, the PAMPA screen only measures the intrinsic... 

The majority of permeability screening is based on cellular assays mimicking intestinal adsorptive cells CACO-2 cells (human colon carcinoma), and the CACO-2 clone TC7, HT-29, T84, IL-6 or MDCK cells (Madine Darby Canine Kidney). In this widely used and accepted cellular assays compounds are classified into 3 classes of permeability high, medium, low. Moreover, the absorption potential of compounds within a chemical series can be compared concerning their apparent permeability coefficients (Artursson 1991 Hillgren et al. 1995 Artursson and Borchardt 1997). 

Solubility measurements are made to determine an intrinsic property, which influences the absorption potential of a compound [3]. Even though solubility itself does not directly dictate the absorption of a drug, it is important to consider solubility in relation to permeability and potency. In addition, in medicinal chemistry projects, there are other issues to consider that may also be affected by poor solubility, particularly insolubility under screening assay conditions. 

Our experience with trying to build computational models based on experimental permeability screening in Caco-2 cell culture illustrates the problem introduced by multiple mechanisms. We found that deviation from a single mechanism could arise either in the assay per se or could arise from the compounds that were screened in the assay. One aspect of the multiple mechanism problem is the presence of active multiple biological transport mechanisms for both enhancing and reducing absorption in cell culture assays. This issue is well documented and is outside the scope of this chapter. 

Poor aqueous solubility, a compound related factor rather than an assay related factor, has a major effect by introducing noise into permeability screening and hence has an effect on making computational model building very difficult. It must be stressed that the compound solubility factor virtually never appears as an explicit consideration in the published permeability literature. Compound sets are published that are used to validate in vitro cell based absorption assays. Validation usually means obtaining an acceptable correlation between human fraction absorbed data and in vitro permeability data. The absorption data always include the experimentally very well controlled but compound number limited human fraction absorbed data that are used to define absorption ranges in the FDA bioavailability waiver guidelines. This limited compound set is then supplemented with additional compounds chosen from published human absorption literature. In our own work we have been able to accumulate... 

Figure 16.2 sets the stage for the types of solubility among currently synthesized compounds that are likely to be submitted to a permeability screen like a Caco-2 assay. In this type of assay a variety of biological transporters are present that mediate both absorption and efflux. The movement of a compound through the Caco-2 polarized cell layer through the action of... 

Blattner, J. R. He, L. Lemasters, J. J. Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader. Anal. Biochem. 2001,295, 220-226. 

Kansy, M., Senner, F., Gubemator, K. Physicochemical high throughput screening parallel artificial membrane permeability assay in the description of passive absorption processes. J. Med. Chem. 1998, 41, 1007-1010. 

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