Caco-2 Permeability Assay

Creative Bioarray is offering Caco-2 permeability assay to help determine the absorption and the bioavailability of drug candidates, facilitating the lead optimization process in drug discovery.

Caco-2 Permeability Assay Introduction

  • The Intestinal barrier controls drug absorption

Oral delivery is the most convenient form of administration of pharmaceutical agents in patient compliance. However, intestinal absorption is a formidable barrier that restricts the oral bioavailability of many potential new drugs.

In the human intestine, absorption is controlled by a barrier composed of a single layer of polarized epithelial cells arranged on the intestinal wall. Among the factors influencing intestinal absorption of drugs, their solubility, intestinal permeability, and dissolution rate are the most influential.

  • Why Caco-2 cells?

Caco-2 cell is a human colon epithelial cancer cell line. Under specific culture conditions, the Caco-2 cells become differentiated and polarized with intercellular tight junctions, a well-differentiated brush border, and typical small-intestinal nutrient transporters, resembling the enterocytes lining the small intestine, making it ideal for intestinal absorption simulations.

Caco-2 permeability assay is one of the recommended in vitro methods in the United States FDA guidelines to evaluate drug permeability. It has become the gold standard method to evaluate both the passive and active transport and the absorption of orally administered drugs.

Brief protocol

  • Caco-2 cells culture stage

Caco-2 cells are cultured on semi-permeable polycarbonate surfaces on inserts that fit into an assay chamber, establishing apical and basolateral chambers. These two chambers are connected only through the monolayer of cells and their semi-permeable substrate (Figure 1). The apical and basolateral chambers represent gastrointestinal tract's luminal and blood/mesenteric lymph sides. Typically, Caco-2 cells are cultured for ~ 21 days to reach confluence and differentiate into enterocytes exhibiting transporter proteins and tight junctions.

Caco-2 Permeability AssayFigure 1. Caco-2 intestinal epithelial cell assay for intestinal absorption(van Breemen & Li, 2005).

  • Evaluating the integrity of the monolayers

Parameters such as transepithelial resistance (TEER) and Lucifer Yellow (LY) can be used to verify whether cell integrity meets the criteria. The specifications after 21 days of differentiation are:

MeasurementValue
TEER450Ω·cm2
LY<1.0%
  • Test compound permeability assay

Compounds were diluted at a concentration of 10 µM and applied to the apical or basolateral side of the cell monolayer. The apparent permeability and efflux ratio of the test compounds was determined in duplicate. Compounds were quantified by LC-MS/MS analysis based on the peak area.

Some compounds can be used as control tests. The main recommendation is to validate the assay using a high permeable molecule, such as Propanolol, and a low permeability compound, such as Atenolol, in triplicates for each group.

Case study: Papp and Efflux Ratio Determination in Caco-2 Cells
Compound IDMean Papp (10-6 cm/s)Efflux RatioMean Recovery %Rank
AP to BLBL to APAP to BLBL to APPapp
Atenolol0.700.741.0690.8196.87Low
Propranolol24.8315.500.6277.9094.27High
Digoxin0.2616.5863.5194.0496.07Low
Sample#10.843.684.3685.97110.98Low

The transport rate for a particular concentration of a test compound is typically expressed as the apparent permeability coefficient (Papp), which is calculated using the following equation:

Caco-2 Permeability Assay

Efflux ratio = Papp [BL>AP] / Papp [AP>BL]; %Recovery = 100 x [(Vr x Cr) + (Vd x Cd)] / (Vd x C0).

where:

dQ/dt: the permeability rate.

A: the surface area of the cell monolayer.

C0: the initial concentration in the donor compartment.

Vr: the solution volume in the receiver chamber.

Vd: the volume in the donor chambers.

Cd and Cr: the final concentrations of transport compound in donor and receiver chambers, respectively

Binning Criteria:

Papp ≤0.500 (x10-6 cm/s)Low permeability
0.500 < Papp < 2.50 (x10-6 cm/s)Moderate permeability
Papp ≥2.50 (x10-6 cm/s)High permeability

Quotation and Ordering

If you have any special needs or questions regarding our services, please feel free to contact us. We look forward to cooperating with you in the future.

Reference

  1. van Breemen, R. B.; Li, Y. Caco-2 cell permeability assays to measure drug absorption. Expert Opinion on Drug Metabolism & Toxicology, (2005), 1(2), 175-185.

For research use only. Not for any other purpose.