Understand the suitability of your compound for oral dosing by using out Caco-2 permeability assay. The Caco-2 assay is widely used across pharmaceutical industry as an in vitro model of the human small intestinal mucosa to predict the absorption of orally administered drugs. The Caco-2 cell model mimics processes such as transcellular transport, paracellular transport, and some aspects of efflux and active transport. So, Caco-2 assay is used to predict human intestinal permeability and investigate drug efflux.
Monolayers based on Caco-2 cells (a human colon carcinoma cell line) express a wide range of transporter proteins on its cell membranes similar to those of intestinal endothelium cells (Siissalo S et al. 2007, Calcagno AM et al. 2006), thus this cell line is ideal for intestinal absorption simulations. In fact, in the last decade, the utilization of Caco-2 cells has become an industry standard for the investigation of intestinal absorption, permeability and drug-drug interactions (DDIs) (Oh DM et al. 2002.).
Caco-2 permeability Assay is a part of our portfolio of in vitro ADME screening services. GVK BIO delivers consistent, high quality Caco-2 Assay data with cost-efficiency that comes from a highly automated approach.
Caco-2 permeability assay to investigate intestinal permeability
- GVK BIO’s Caco-2 Permeability assay uses an established method for predicting the in vivo absorption of drugs across the gut wall by measuring the rate of transport of a compound across the Caco-2 cell line.
- The Caco-2 cell line is derived from a human colon carcinoma. The cells have characteristics that resemble intestinal epithelial cells such as the formation of a polarised monolayer, well-defined brush border on the apical surface and intercellular junctions.
- Assessing transport in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer enables an efflux ratio to be determined which provides an indicator as to whether a compound undergoes active efflux.
- The P-glycoprotein (P-gp) inhibitor, verapamil, can be included to identify whether active transport is mediated by P-gp.
Caco-2 Permeability Studies available:
- Passive permeability in Caco-2
- MDR1 substrate assesment in Caco-2
- BCRP substrate assessment in Caco-2
- Inhibitor assessment in Caco-2: Drug-Drug Interaction studies: MDR1 and BCRP
- Caco-2 studies can be performed in different setups. SOLVO offers a number of standard setups, which differ in terms of the derivable information and cost. These standard setups serve as starting points to define the optimal final study parameters. All setups contain a standard number of controls (e.g. low permeability, high permeability, transporter function)
1. Basic – Studies at one concentration
2. Extended – Studies at several concentrations, more controls
3. FDA Guidelines – Studies according to FDA draft guidance (2006)
These setups can be customized to suit your needs.
Caco-2 Permeability Assay Description:
The Caco-2 cells are cultured to confluency, trypsinized and seeded onto a filter transwell insert at a density of ~32,000 cells/well in DMEM cell culture medium. Cells are grown in a humidified atmosphere of 5% CO2 at 37 °C. Following an overnight attachment period (24 h after seeding), the cell medium is replaced with fresh medium in both the apical and basolateral com-partments every other day. The cell monolayers are used for transport studies 21 days post seeding after measuring the TEER values (>600 Ohms/cm2). The apical sides and basolateral sides are washed consecutively with HBSS 2.5% (v/v), HEPES (pH 7.4) or HBSS 2.5% (v/v), HEPES 10% (v/v), and Fetal Bovine Serum (pH 7.4) at 37 °C in an incubator under an atmosphere of 5% CO2.
Donor working solution is prepared by dilution of DMSO stock of test article or positive control with transport media to 10 µM.
For A → B directional transport, the donor working solution (with test article or positive control, with or without Pgp inhibitor) is added to the apical (A) compartment and the transport media as receiver working solution is added to the basolateral (B) compartment. For B → A directional transport, the donor working solution (with positive control or test article, with or without Pgp inhibitor) is added to the basolateral (B) compartment and transport media as receiver working solution is added to the apical (A) compartment.
The cells are incubated in a humidified atmosphere of 5% CO2 at 37 °C for 90 minutes.
At the end of the incubation, samples are taken from both donor and receiver compartments and transferred into 96-well assay plates containing internal standard solution (IS) in each well. After centrifugation, the supernatant solutions are transferred to clean 96 well plates and analyzed by LC-MS/MS. The MS detection is performed using a Sciex API 4000 instrument. Each compound is analyzed by reversed phase HPLC.
Caco-2 Permeability Assay Service details
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 Caco-2 assay is typically run in duplicates.
Caco-2 Assay Sample Requirement/Submission
A minimal weighted amount of dry compound, 1 mg, or 50 µL of 10mM stock DMSO solution, is required for Caco-2 assay.
Visit our DMPK studies page for a more comprehensive list of assays offered.