Caco-2 drug transport assays

The apparent permeability (Papp) in units of centimeters per second, can be calculated for Caco-2 drug transport assays using the following equation ... 

In conclusion, there are several drawbacks to the use of Caco-2 cells in studies of active drug transport. Despite these drawbacks, we note that a recent comprehensive study comparing various P-glycoprotein drug efflux assays in drug discovery came to the conclusion that the Caco-2 transport assay is the method of choice, since it displays a biased responsiveness towards compounds with low or moderate permeability - in other words, towards compounds whose intestinal permeability is most likely to be significantly affected by drug efflux mechanisms [101]. 

We then used this Caco-2 cell assay to categorize representative fluoroquinolone drug substance permeability [50], The drugs demonstrated some concentration-dependent permeability indicative of active drug transport. Based upon comparison to labetalol, ciprofloxacin was classified as a LP drug, whereas levofloxacin, lomefloxacin, and ofloxacin were classified as HP drugs, which matched their human in vivo bioavailabilities. All four fluoroquinolone drugs were subject to efflux transport (ciprofloxacin > lomefloxacin > rhodamine 123 > levofloxacin > ofloxacin). 

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. 

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

Here, we briefly describe the automated Caco-2 assay used at the research site in AstraZeneca R D Molndal. The solubility of the test compounds is measured (or theoretically predicted) before they are run in the Caco-2 assay. In order to be able to make correct determinations of the permeability coefficient, the substance must be dissolved when added to cell monolayer in the transport experiment. Compounds with insufficient solubility are therefore not tested. We generally apply a test concentration of 10 pM, but in specific projects or under certain circumstances a concentration of only 1 pM is applied. The test compounds are first prepared in DM SO solution (1 mM) on a parent plate and are then diluted in transport buffer to give a final drug concentration of 10 pM (solution containing 1% DMSO) when added to the cell monolayers. 

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. 

Preliminary pharmacokinetic behavior can be tested through a number of whole cell assays. Most commercially successful drugs are administered orally, meaning the drug must be able to enter the bloodstream by crossing membranes in the intestines. The most common membrane permeability assay is performed by monitoring the absorption and secretion of a compound by colon carcinoma cells (Caco-2). Diffusion across Caco-2 cell membranes is considered to be a valid model for molecular transport in the small intestines.16... 

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