Predict intestinal permeability and oral absorption of compounds

• The Caco-2 monolayer is widely used across the pharmaceutical industry as an in vitro model of the human small intestinal mucosa to predict the absorption of orally administered drugs.¹⁻⁴

• The Caco-2 cell model mimics processes such as transcellular transport, paracellular transport, and some aspects of efflux and active transport.

Readout: P_app (A→B),  P_app (B→A),  Efflux ratio, % Recovery

Controls: with and without Pgp inhibitor; Atenolol, Propanolol, Taxel-1

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.

Data Analysis:

The parameters P_app (apparent permeability) and efflux ratio are calculated as follows:

P_app = (dQ/dt) × (1/C₀) × (1/A)

Efflux ratio = P_app [B → A] / P_app [A → B]

where dQ/dt is the permeability rate, C₀ is the initial concentration in the donor compartment, and A is the surface area of the cell monolayer (0.33cm²). The P_app value is a rate measured in cm/s.

Calculated P_app is ranked as low (<1×10^-6 cm/s), moderate (1-10×10^-6 cm/s), and high (>10×10^-6 cm/s).

Comparing the efflux ratios generated in the presence and absence of a Pgp inhibitor identifies whether the test article is a Pgp substrate. A compound is considered to be a Pgp substrate when the efflux ratio in the absence of inhibitor is >1.99 and is significantly reduced (≤ 1) in the presence of an inhibitor.

Recovery is calculated as follows:

%Recovery = (Total compound mass in donor and receiver compartments at the end of the incubation / Initial compound mass in the donor compartment) x 100.

Abbreviations:

DMEM                   Dulbecco’s Modified Eagle Medium

DMSO                    Dimethylsulfoxide

HBSS                      Hank’s Buffered Salt Solution

HEPES                    4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC                      High-performance liquid chromatography

Pgp                          P-glycoprotein 1, also known as multidrug resistance protein 1 (MDR1)

LC                            Liquid chromatography

MS                           Mass spectrometry

TEER                      Transepithelial Resistance (in Ohm/cm²)

Literature:

• Artursson, P.; Karlsson, J. “Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells“; Biochem. Biophys. Res. Commun. 175, 880, (1991).

• Bohets, H.; Annaert, P.; Van Beijsterveldt, L.; Anciaux, K., Verboven, P.; Meuldermans, W., Lavrijsen, K. “Strategies for absorption screening in drug discovery and development”; 1, 367, (2001).

• Artursson, P.; Palm, K.; Luthman, K. “Caco-2 monolayers in experimental and theoretical predictions of drug transport”; Adv. Drug Del. Rev. 64, 280, (2012).

• Shah, P.; Jogani, V.; Bagchi, T.; Misra, A. “Role of Caco-2 cell monolayers in prediction of intestinal drug absorption“; Biotechnol. Progr. 22, 186, (2006).

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