Caco-2 monolayer, a model for human drug intestinal permeability, is of great interest. Kinetics of intestinal drug absorption, permeation enhancement, chemical moiety structure-permeability relationships, dissolution testing, in-vitro/in-vivo correlation, bioequivalence, and the development of novel polymeric materials are closely associated with the concept of Caco-2. Literature reveals only limited information particularly on the correlation between oral drug absorption in humans and the apparent drug permeability coefficients obtained from the Caco-2 monolayer model.
It is unacceptable for a drug candidate to demonstrate low oral absorption particularly during clinical trials because by the time it reaches patient investigation there is high expenditure involved and membrane-based drug assays could be used as early screens for their oral absorption potential in drug discovery and development stages. Various in-vitro methods are listed in United States FDA guidelines, acceptable to evaluate the permeability of a drug substance, includes monolayer of suitable epithelial cells and one such epithelial cell line that has been widely used as a model system of intestinal permeability is the Caco-2 cell line. Since most drugs are known to absorb via intestines without using cellular pumps, passive permeability models came in the limelight.
In 1990s membrane-based drug assays led to the passage of drugs through the intestinal mucosa and an important Caco-2 assay emerged in the pharmaceutical research (Hilgars, A.R., Conradi. R.A., Burton, P.S.; Pharm. Res. 7, 902-910, 1990 and Karlsson, J., Arturrson, P.; Int. J. Pharmaceutics 71, 55-64, 1991). In a typical Caco-2 experiment, a monolayer of cells is grown on a filter separating two stacked micro well plates. The permeability of drugs through the cells is determined after the introduction of a drug on one side of the filter. The entire process is automated, and when used in conjunction with chromatography and/or mass spectroscopy detection, it enables any drug's permeability to be determined. The method requires careful sample analysis to calculate permeability correctly.
Limitations of Caco-2 experiments are 21-days for preparing a stable mono layer, stringent storage conditions; however, tight-junction formation prior to use is the better choice. The villus in the small intestine contains more than one cell type, the Caco-2 cell line does not produce the mucus as observed in the small intestine and no P-450 metabolizing enzyme activity has been found in the Caco-2 cell line. Test compound solubility may pose a problem in Caco-2 assays because of the assay conditions. Finally, Caco-2 cells also contain endogenous transporter and efflux systems, the later of which works against the permeability process and can complicate data interpretation for some drugs.
Caco-2 cell model has the advantages of simplicity and reproducibility. Values of Lucifer Yellow Rejection (LYR) and Trans Epithelial Electrical Resistance (TEERS in Ohms*cm2) achieved with a 21 day culture of Caco-2 cells on the MultiScreen Caco-2 plate is of importance. Recently, use of the 96-well system increases screening throughput 4 times over current 24-well systems with no compromise to cell growth or sensitivity for analysis. Each well and basolateral access hole is aligned to complement use with automated probes.
US FDA recognizes Caco-2 to measure permeability as part of the bioequivalence waiver process. Based on the Biopharmaceutics Classification Systems, pharmaceutical companies can request a waiver from bioequivalence studies. A drug substance with respect to solubility and permeability can be classified in 4 categories. Category-I: high solubility and high permeability, II: low solubility and high permeability, III: high solubility and low permeability, and IV: low solubility and low permeability. A biowaiver can be requested only for an immediate release dosage form of category-I drug substance.
-- The author is a Certified Bioinformatics Specialist, Cert. in SAS Programming and associated with Pharmaceutical Research and Clinical Trials, PRACT Advisory Service, Alexandria, Virginia (USA)