Solubility and intestinal permeability are the most important factors determining oral absorption of drugs. Caco-2 (human colon adenocarcinoma cells) assay is the model most widely used in drug discovery for evaluation of compound permeability properties. When grown to confluence and allowed to differentiate the cells form a monolayer resembling luminal epithelium of human intestine by structure and properties. Unlike the PAMPA assay used for evaluation of passive transfer, Caco-2 cells have a variety of active transporters, which are relevant to the absorption process in the gastro-intestinal tract. For this reason, this assay has a good correlation to in vivo studies of absorption.
For Caco-2 permeability assay, cells are grown on semi permeable supports inside inserts in multi well plates. The system is composed in the way that semi permeable support separates apical and basal compartments. Caco-2 monolayer is asymmetrical, it’s apical part is different from basal part. Tested compounds are added to either apical or basal compartment and the only way to get to the opposite compartment is through Caco-2 monolayer. Permeability of test compounds can be measured in either direction (apical to basal or basal to apical), typically the former is assessed. After certain incubation time, the initial solution of tested compounds and samples from appropriate compartments are taken and analyzed by LC-MS/MS. Based on compound concentrations measured, apparent permeability coefficient (Papp), reflecting the ability of a compound to penetrate cell monolayer, is calculated.
Caco-2 (human colorectal adenocarcinoma) was obtained from ATCC (cat. # HTB-37) and cultivated according to the supplier’s recommendations. All steps of the Caco-2 permeability assay are done according to BD Cat. No. 354802 BIOCOAT® HTS Caco-2 Assay System manual. Incubation time for tested compounds is 1-2 hrs. The assay is typically run in duplicates.
A minimal weighted amount of dry compound, 1 mg, or 50 µL of 10mM stock DMSO solution, is required for this assay.
Caco-2 Method Validation data can be accessed here.