Permeability Assays

Rugged Data and Expert Interpretation from Industry Leaders

Permeability across a monolayer of polarized epithelial cells is a fundamental property of a compound that can be measured through our ExpressPlus™ assays.

We do more assays with Caco-2 cell monolayers than anyone in the world.
No one is more knowledgeable than Absorption Systems when it comes to Caco-2 cells. A combination of experience and rigorous cell biology QC processes makes it possible for us to interpret your compound’s test results. 

Permeability Assays

Express Unidirectional Permeability Through Caco-2 Monolayers
Express Bidirectional Permeability Through Caco-2 Monolayers
Express MDR1-MDCK for BBB Penetration Potential
Express P-gp Substrate Assessment Using MDR1-MDCK Cell Monolayers
Express P-gp Inhibitor Assessment Across Cell Monolayer
Express BCRP Inhibitor Assessment
Express PepT1 Substrate Assessment in Caco-2 Cell Monolayers
Express P-gp Substrate Assessment in CellPort CPT-B1 BCRP Knockdown Cells
Express BCRP Substrate Assessment in CPT-B1 BCRP Knockdown Cells
Express BCRP Substrate in CPT-B1
Express P-gp Substrate in CPT-P1

Different cell lines are used as in vitro models for different purposes.
Caco-2 cell monolayers predict not only intestinal absorption but also drug-drug interactions involving transporters. Likewise, MDR1-MDCK cell monolayers are used to predict drug-drug interactions involving the efflux transporter P-glycoprotein as well as blood-brain barrier (BBB) penetration.

"We know when the test worked and what the results mean vs. when the test didn’t work because the cells didn’t tolerate the compound."

Other ExpressPlus assays utilizing the same test format identify substrates and inhibitors of transporters. Taken together, these studies help identify compounds with drug-like properties. The fixed protocols and rapid turnaround time make them ideal for screening large numbers of compounds early in drug discovery. At a later stage, when the same tests are run as custom assays, including test conditions optimized for a given compound, additional bioanalytical rigor, etc., you can count on getting the same results. That’s because the Absorption Systems philosophy is, wherever possible, to use the same rigorously validated test systems throughout the drug development continuum.

Bidirectional assay format

Background on Permeability Testing Tools and Methods
Caco-2 cells have become both an industry and academic standard assay system for the study of active and passive intestinal transport. Most importantly, they are of human origin.

Redefining the Threshold for Permeability Classification
The permeability threshold defined by Absorption Systems’ High Permeability Internal Standard (HPIS) is the lowest available.

Caco-2 Application Pioneered by Ismael Hidalgo
Caco-2 is a human cell line that accurately predicts human intestinal absorption. This application of Caco-2 cell monolayers was pioneered in the late 1980s by Absorption Systems co-founder and Chief Scientific Officer Ismael Hidalgo, Ph.D.

CellPort Transporters
Transporters can have an enormous influence on the pharmacokinetics of drugs that are substrates, either limiting their intestinal absorption, mediating their excretion via bile or urine, or determining how much gets into the brain, cancer cells, or placenta.

In Situ Organ Perfusion
These unique brain, liver, and intestinal models provide the flexibility that you require when narrowing down your compound choice with a higher tolerability than similar in vitro test systems.

Simplified with Exemplary Service
Absorption Systems provides a clear advantage with results you can trust. Research managers are dedicated to your project, acting as your personal Data Concierge™ — delivering projects on time and on budget.

Permeability Lead Optimization Tabbed Module

Bidirectional Monolayer Format
For permeability assays we use the bidirectional monolayer transport format, a confluent monolayer of polarized epithelial cells established on a semi-permeable filter separating the two chambers of a Transwell® apparatus. Caco-2 cells form a polarized monolayer in about 21 days; MDR1-MDCK cells take about 7 days. The rate of transport of a test compound across the monolayer is measured in both directions, by adding the compound to the apical chamber and sampling from the basolateral chamber (apical-to-basolateral or A-to-B permeability) or by doing the opposite (for B-to-A permeability).

EA201 Express Unidirectional Permeability Through Caco-2 Monolayers
EA202
Express Bidirectional Permeability Through Caco-2 Monolayers
EA203
Express MDR1-MDCK for BBB Penetration Potential
EA204
Express P-gp Substrate Assessment Determined Using MDR1-MDCK Cell Monolayers
EA222 Express P-gp Inhibitor Assessment Across Cell Monolayers
EA223 Express BCRP Inhibitor Assessment
EA224 Express PepT1 Substrate Assessment in Caco-2 Cell Monolayers
EA250 Express P-gp Substrate Assessment in CellPort CPT-B1 BCRP Knockdown Cells
EA251 Express BCRP Substrate Assessment in CPT-B1 BCRP Knockdown Cells
EA251 Express BCRP Substrate in CPT-B1
EA252 Express P-gp Substrate in CPT-P1

>> View all assays

EA701 Express Plasma Protein Binding (human plasma) Assay Data Sheet
EA702
Express Plasma Protein Binding (rat plasma) Assay Data Sheet
EA703 PPB – Ultracentrifugation
EA704 PPB – Equilibrium Dialysis
EA705 PPB – Ultrafiltration
EA706 Red Blood Partitioning
EA707
Express Fraction Unbound (rat or mouse brain)
EA708
Express Plasma Protein Binding (mouse plasma) Assay Data Sheet
EA709
Express Plasma Protein Binding (primate plasma) Assay Data Sheet
EA710
Express Plasma Protein Binding (dog plasma) Assay Data Sheet
EA713
Express Blood to Plasma Partitioning in Mice Rats and Humans
EA813
Express ABT Exposure in Rats
EA982
Express Formulation Assessment

>> View all assays

Artursson P, et al., Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv Drug Deliv Rev. 2001 Mar 1; 46(1-3):27-43.

Avdeef A, et al., Physicochemical profiling in drug research: a brief survey of the state-of-the-art of experimental techniques. Cell Mol Life Sci. 2002 Oct; 59(10):1681-9.

Balani SK, et al., Strategy of Utilizing In Vitro and In Vivo ADME Tools for Lead Optimization and Drug Candidate Selection.  Curr Top Med Chem. 2005;5(11):1033-8.

Bhardwaj RK, et al., Delineation of human peptide transporter (hPepT1)-mediated uptake and transport of substrates with. J Pharm Exp Ther. 2005 Sept; 314(3):1093-1100.

Bohets H, et al., Strategies for absorption screening in drug discovery and development. Curr Top Med Chem. 2001 Nov; 1(5):367-83.

Borchardt RT, et al., Are MDCK cells transfected with the human MRP2 gene a good model of the human intestinal mucosa?. Pharm Res. 2002 Jun; 19(6):773-9.

Chen C, et al., P-glycoprotein limits the brain penetration of nonsedating but not sedating H1-antagonists. Drug Metab Dispos. 2003 Mar; 31(3):312-8.

Clark DE, In silico prediction of blood–brain barrier permeation. DDT Vol. 8, No. 20 October 2003.

Cohen LH, et al., In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates.   Drug Metab Dispos. 2003 Aug;31(8):1005-15.

de Vrueh, RLA, et al., Transport of L-Valine-Acyclovir via the oligopeptide transporter in the human intestinal cell line, Caco-2. J Pharm Exp Ther. 1998 Sept; 286(3):1166-70.

Ee, PLR, et al., Modulation of breast cancer resistance protein (BCRP/ABCG2) gene expression using RNA interference. Mol Cancer Ther. 2004 Dec; 13(12):1577-83.

Fabre G, et al., Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line. Pharm Res. 1998 May; 15(5):726-33.

Gotoh Y, et al., The advantages of the Ussing chamber in drug absorption studies. J Biomol Screen. 2005 Aug; 10(5):517-23.

Hidalgo IJ, et al., Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterol. 1989; 96:736-49.

Hunter J, et al., Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators. J Biol Chem. 1993 Jul 15; 268(20):14991-7.

Korinth G, et al., Is the permeability coefficient Kp a reliable tool in percutaneous absorption studies?. Arch Toxicol. 2005 Mar; 79(3):155-9. Epub 2004 Dec 31

Lin JH, et al., Role of pharmacokinetics and metabolism in drug discovery and development. Pharmacol Rev. 1997 Dec; 49(4):403-49.

Polli JW, et al., Rational Use of in Vitro P-glycoprotein Assays in Drug Discovery. J Pharmacol Exp Ther. 2001 Nov; 299(2):620-8.

Pritchard JF, et al., Making better drugs: Decision gates in non-clinical drug development. Nat Rev Drug Discov. 2003 Jul; 2(7):542-53.

Rautio, J, et al., In Vitro P-glycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: A recommendation for probe substrates. Drug Metab Disp. 2006; 34 (5): 786-92.

Sambuy Y, et al., The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol. 2005 Jan; 21(1):1-26.

Summerfield SG, et al., Central nervous system drug disposition: the relationship between in situ brain permeability and brain free fraction. J Pharmacol Exp Ther. 2007 Jul; 322(1):205-13.

Tanaka H, et al., Drug-protein binding and blood-brain barrier permeability. J Pharmacol Exp Ther. 1999 Mar; 288(3):912-8

Taub ME, et al., Functional assessment of multiple Pglycoprotein (P-gp) probe substrates: influence of cell line and modulator concentration on P-gp activity. Drug Metab Dispos. 2005 Nov; 33(11):1679-87. Epub 2005 Aug 10.

Wang Q, et al., Evaluation of the MDR-MDCK cell line as a permeability screen for the blood-brain barrier. Int J Pharm. 2005 Jan 20; 288(2):349-59.

Zhou R, et al., pH-dependent dissolution in vitro and absorption in vivo of weakly basic drugs: development of a canine model. Pharm Res. 2005 Feb; 22(2):188-92.

In Vitro and In Vivo Rabbit Models of Ocular Drug Absorption (Poster #T2086, AAPS 2010)

Evaluation of Corneal Orbs Created from Human Stem Cells as an In Vitro Model for Studying Ocular Drug Absorption (Poster #T2085, AAPS 2010)

In Vitro Corneal Model for Prediction of In Vivo Ocular Absorption Following Topical Administration (Poster, AAPS 2009)

Utility of the P-gp Knockout Mouse In Situ Brain Perfusion to Determine the Brain Penetration Potential of P-gp Substrate Drugs (Poster, AAPS 2007)

>> Resource Library

When would I use one of your excised tissue permeability models?
To ask mechanistic questions, e.g.:
Is my prodrug converted to the active moiety during absorption?
Is the poor oral bioavailability of my drug due to enzymatic degradation during absorption?
Is the absorption of my drug limited to a specific region of the gut?

Why do you use both Caco-2 and MDR1-MDCK cell monolayers?
We use them in a complementary fashion, particularly for P-gp interactions.

For P-gp interactions, MDR1-MDCK cells offer the advantage of over-expressing only the transporter of interest; however, we have seen both false positives and false negatives with this model.

Caco-2 cells express multiple efflux transporters, which can make it difficult to identify which transporter is involved.

Caco-2 cell monolayers predict intestinal absorption, whereas MDR1-MDCK cell monolayers are better for prediction of BBB penetration.

>> Learn more about ExpressPlus

We are the drug absorption experts
No one does more Caco-2 cell assays than Absorption Systems.

Multiple uses for the same test system

Multiple test systems for different purposes

The same well-validated in vitro test systems from screening through NDA

 

What are the advantages and uses of the Caco-2 monolayer model?
Caco-2 is a human cell line.

The correlation between in vitro Caco-2 permeability and in vivo human absorption of orally administered drugs is well-established.

CYP-mediated drug metabolism is essentially nil, which simplifies the bioanalysis and interpretation of many permeability experiments.

We have validated the Caco-2 model for both permeability and transporter interactions, which can help you avoid having to do certain clinical drug-drug interaction studies.

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