PAMPA Vs Caco-2 FAQs

Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. When cultured as a monolayer, Caco-2 cells differentiate to form tight junctions between cells to serve as a model of paracellular movement of compounds across the monolayer. In addition, Caco-2 cells express transporter proteins, efflux proteins, and Phase II conjugation enzymes to model a variety of transcellular pathways as well as metabolic transformation of test substances. In many respects, the Caco-2 cell monolayer mimics the human intestinal epithelium. One of the functional differences between normal cells and Caco-2 cells is the lack of expression of the cytochrome P450 isozymes and in particular, CYP3A4, which is normally expressed at high levels in the intestine. However, Caco-2 cells may be induced to express higher levels of CYP3A4 by treatment with vitamin D3. Caco-2 cell monolayers are usually cultured on semipermeable plastic supports that may be fitted into the wells of multi-well culture plates. Test compounds are then added to either the apical or basolateral sides of the monolayer. After incubation for various lengths of time, aliquots of the buffer in opposite chambers are removed for the determination of the concentration of test compounds and the computation of the rates of permeability for each compound (called the apparent permeability coefficients).

What are the differences between the PAMPA and the Caco-2 permeability assay?
Caco-2 permeability screen is considered to be more representative of human absorption in vivo than PAMPA (parallel artificial membrane permeability assay). PAMPA solely provides a measure of passive diffusion whereas the Caco-2 model provides better prediction of the human absorption for compounds which display active uptake or efflux or pass through the membrane via the paracellular route. The information from both assays used in conjunction can be valuable in identifying the root cause for poor absorption.

What is the relationship between Caco-2 permeability and human intestinal absorption?

The relationship between Caco-2 permeability (using pH7.4 HBSS buffers in both the apical and basolateral compartments) and human intestinal absorption is displayed in Figure 4. This correlation is typical of those observed in the literature for these two parameters4. It is important to note that this plot is influenced by the accuracy of the intestinal absorption data. The intestinal absorption values used in this plot are taken from Zhao et al. 20012. In this paper the human intestinal absorption has been extracted from various sources and varies considerably in quality. Several compounds are also known to exhibit dose-dependent absorption (shown by error bars on the graph).

What specific applications potentially benefit from PAMPA vs. Caco-2?
PAMPA allows high throughput screening of compounds more than that of Caco-2 model but in terms of results the Caco-2 model is more descriptive (These days Caco-2 is also used as high through screening model as 96-well plate and automation but the cost of experiment is higher). PAMPA is used only to measure passive permeability of the molecules while in Caco-2 cell monolayer passive as well as active transport can be studied.

What is limiting broader use of the PAMPA assay?
It can predict only permeability of passively transported compounds and may give false prediction of actively transported compounds.

 

0 Comments

Leave a reply

Your email address will not be published. Required fields are marked *

*

© 2018 | GVK Biosciences Private Limited, All Rights Reserved | Terms & Conditions

Log in with your credentials

Forgot your details?