Cell-based permeability assay

Low permeability can itself be the cause of apparent discrepancies between biochemical and cell-based assays and may or may not have physiological relevance. Independent of the solubility limitation mentioned above, the selection of an appropriate loading concentration in cell-based permeability assays impacts on the assay outcome and depends on what information one wants to extract from the measurement loading at high concentration (i.e., 100 pM) will essentially cancel the effect of active transports unless passive diffusion is low. When high loading concentrations are used, poor recovery and bioanalytics are usually not an issue. 

In contrast to cell-based assays, noncell-based permeability assays using artificial membranes supported on filters are fast, flexible, cheap, and fully automatable. They are therefore ideally placed for use in high throughput. There have been many variations of this assay in terms of the fine details of the experiment and these will be discussed in due course however, the basic principles remain the same, based on a 96-well microtiter plate format. 

Poor aqueous solubility, a compound-related factor rather than an assay-related factor, has a major effect by introducing noise into absorption screening and thus 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... 

Many organizations use colon adenocarcinoma (Caco-2) for detailed study of permeability however, this method can be resource intensive. Parallel artificial-membrane permeability (PAMPA) [19] has proven to be a reliable predictor of passive transcellular permeability for intestinal absorption prediction. It is also useful to interpret results of cell-based discovery assays, in which cell-membrane permeability is limiting. Finally, pTf provides insight into the pH dependence of solubility and permeability. It can be measured [20] or calculated to get an understanding of the regions of the intestine in which the compound will be best absorbed, as well as to anticipate the effect of pH on solubility and pemieability. Permeability at the blood-brain barrier (BBB) also can be rapidly profiled [21]. 

Figure 15.5 shows a hypothetical model PLS model for a set of compounds. The analysis shows that the cell-based lunclional assays (isopropionic acid) (IPA)-rat, IPA-human, and bioassay) do not correlate well with the receptorbinding assay. On the other hand, the cell-based assays are correlated well with the pharmaceutical properties, such as solubility and permeability. The model shows that increased MW and hydrogen-bonding capacity will improve potency. That, however, will decrease solubility and permeability and make them less druglike. So, for this series of compounds, it is important to find a balance between potency and druglike properties. 

PAMPA is typically used to make a prediction of the passive, transcellular absorption of a compound. Compounds which may be absorbed by a paracellular mechanism or may be substrates for active transport (uptake or efflux) are usually better assessed in a cell based system. A combination of assays can be applied to gain a greater understanding of the permeability and transport properties of a compound. 

There are several approaches to estimating absorption using in vitro methods, notably Caco-2 and MDCK cell-based methods or using methods that assess passive permeability, for example the parallel artificial membrane permeation assay (PAMPA) method. These are reviewed elsewhere in this book. The assays are very useful, and usually have an important role in the screening cascades for drug discovery projects. However, as discussed below, the cell-based assays are not without their drawbacks, and it is often appropriate to use ex vivo and/or in vivo absorption assays. 

The Caco-2 permeability assay is usually performed in a Transwell device (Figure 18.1). The Transwell contains two compartments a donor and a receiver compartment. The apical donor compartment contains a porous membrane that supports the growth of the Caco-2 monolayer. Caco-2 cells are seeded on the porous membrane. Upon confluency of the cell culture, the compound is added into the donor compartment at a concentration range from one to several hundred micromolar. Samples are collected from the receiver compartment for up to 2 h, then LC-UV or LC-MS methods are used to quantify compound in each sample. The permeability coefficient of the compound is calculated based on the following equation ... 

Secondary assays depend on the project. Where the primary screen was a cell-based assay, the secondary assay may be a radioligand competition binding assay. In other cases, such as where the primary screen was a biochemical assay, the secondary assay may be a cellular assay, and may be functional or mechanistic. One of the issues that may arise at this stage is that compounds with reasonable activity in the primary assay may not show activity in the secondary assay. There can be a number of reasons for this, including insufficient potency, inability of the compound to get into cells, or a higher intracellular concentration of the natural ligand (e.g., ATP) if the inhibitor is a competitive inhibitor. It is often necessary at this stage to prepare additional compounds in the series to get compounds of sufficient potency and/or permeability so that cellular activity can be demonstrated. 

Kinetic solubility This pragmatic approach starts with a concentrated compound solution in pure DM SO further diluted in a buffer medium. The amount of compound in solution is measured after a few minutes incubation either by recording its UV absorbance (with or without a chromatographic step) or precipitate formation using an optical method (turbidimetry, nephelometry or flow cytometry). This approach mimics the typical path of the compound in biochemical, cellular assays or in vivo animal models. Kinetic solubility usually serves as a quality filter prior to cell based assays (see paragraphs on solubility, permeability and cellular assays). 

Significant interlaboratory differences in permeability measurements are observed with cell-based assays. It is important to standardize culture conditions and characterize a cell line within one s own laboratory. Permeability differences can be attributed to a number of factors, for example, heterogenecity of cell line, passage number, culture conditions, characteristics of the filter membrane, age of mono-layers and level of differentiation and experimental methodology used. Active... 

One of the main in vitro permeability assays used in the pharmaceutical industry has been for many years the Caco-2 monolayer. Therefore, most of the in silica models developed to predict permeability were based on Caco-2 data. Hou and Johnson produced a couple of reviews that comprehensibly summarizes the recent efforts using Caco-2 permeability data [92, 94]. All those models are designed to predict the influx or apparent permeability of drugs in the same direction as intestinal absorption occurs, that is, from the apical to the basal side of the cell line, regardless of the extent of active transport involved in the permeation process. 

Volpe Donna, A. (2008) Variability in Caco-2 and MDCK cell-based intestinal permeability assays. Journal of Pharmaceutical Sciences, 97, 712-725. 

In contrast to enzyme assays, cell-based assays present the target in a more physiological milieu. With enzyme assays, it may be difficult to purify and express active kinases and phosphatases in their full-length forms and they may require the use of fusion proteins with kinase activity domains. Cell-based technologies, on the other hand, present the opportunity to express the targets with regulatory domains included. Furthermore, cell-based assays usually detect only cell-permeable inhibitors and have the potential to identify more unusual mechanisms, as described earlier. 

In the assay described here cells are permeablized using a detergent-based lysis method. Care must be taken to inhibit the membrane-bound enzyme, y glutamyl-transferase (y-GT), which metabolizes glutathione released from the cell (12). This is achieved by the use of an acidified lysis buffer (8). The lysate is then assayed for total protein and glutathione content. 

Permeability is a kinetic process, and is quoted as a rate. In cell-based assays such as Caco-2, a number of time points are generally taken from both the donor (apical) wells and the receiver (basolateral) wells. Because of this, retention on the membranes is not determined and the permeability values quoted are apparent, Pa. The main benefit of PAM PA type assays is their usefulness as high-throughput tools and therefore taking time point measurements will create a bottleneck. Measurements of the donor and receiver wells are made at the end of the incubation period and referenced back to the starting concentration of the solute in the donor wells. 

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