Caco-2 Method Validation

Unidirectional (A-B) Caco-2 Assay

Figure 1.  Highly and poorly permeable compounds in Caco-2 unidirectional assay (A-B)
(according to the experimental data obtained in Bienta laboratory in 2017).

  • The data for atenolol, propranolol and quinidine are mean of seven independent experiments assayed in triplicates;
  • The data for all another tested compounds are mean of two independent experiments assayed in triplicates.

 According to US FDA “Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, Guidance for Industry” (Dec, 2017):

“METHOD SUITABILITY: One of the critical steps in using in vivo or in situ perfusion, or in vitro permeability methods for permeability classification is to demonstrate the suitability of the method. To demonstrate suitability of a permeability method intended for BCS-based permeability determination, a rank-order relationship between experimental permeability values and the extent of drug absorption data in human subjects should be established using a sufficient number of model drugs. (…) for in vitro (…) cell monolayer methods, twenty model drugs are recommended. This relationship should allow accurate differentiation between drug substances of low and high intestinal permeability attributes.
To demonstrate the suitability of a method, model drugs should represent a range of zero, low (e.g., < 50 percent), moderate (e.g., 50 – 84 percent), and high (≥ 85 percent) absorption. Sponsors/applicants may select compounds from the list of drugs and/or chemicals provided in Attachment A, or they may select other drugs for which there is information available on mechanism of absorption and reliable estimates of the extent of drug absorption in humans.”

According to FDA recommendations for in vitro permeability assessment using cell cultures, twenty model drugs with well established properties were used for Caco-2 method validation in Bienta laboratory. Apparent permeability (Papp) values obtained for these compounds (with error bars representing standard deviation) are shown on the two bar graphs above. According to these data, propranolol and theophylline belong to highly permeable compounds, whereas sulpiride, acyclovir and famotidine are poorly permeable compounds in the Caco-2 test system setting. This is in good correlation with systemic absorption data for these drugs represented in Attachment A to the FDA guidance. It is generally accepted that Papp values < 1 х 10-6 cm/s indicate poor permeability (which, in most cases, corresponds to low intestinal absorption), and Papp > 10 х 10-6 cm/s indicate high permeability (and high intestinal absorption). For moderate permeability range, 10 х 10-6 cm/s < Papp < 10 х 10-6 cm/s, correlation between Caco-2 permeability and intestinal absorption is poor. Model compounds with moderate permeability are basically used in order to mark the borderline between the high and low permeability compounds.


Bidirectional (B-A)/(A-B) Caco-2 Assay

Fig.2. Efflux ratio (B-A)/(A-B) for 4 reference compounds in bidirectional Caco-2 assay.

  • Screening compounds in both the A to B and B to A direction provides a ratio of B-A/A-B (efflux ratio). When a compound has an efflux ratio of greater than 2, it suggests that the compound may be subject to active efflux. The data for compounds are mean of two independent experiments assayed in triplicates.

According to US FDA “Waiver of In Vivo Bioavailability…”, in vitro methods, such as those using cultured monolayers of human epithelial cells, are considered appropriate for passively transported drugs only.  For proper permeability estimation of the drug candidates, efflux ratio should be evaluated for tested compounds, as follows:

“The observed low permeability of some drug substances in humans could be caused by efflux of drugs via membrane efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance  protein (BCRP) and/or multidrug resistance associated protein 2 (MRP2). When the efflux transporters are absent in these models, or their degree of expression is low compared to that in  humans, there may be a greater likelihood of misclassification of permeability class for a drug subject to efflux compared to a drug transported passively. Expression of known transporters in selected study systems should be characterized. Functional expression of efflux systems (e.g., Pgp, BCRP, MRP2) can be demonstrated with techniques such as bidirectional transport studies,  demonstrating a higher rate of transport in the basolateral-to-apical direction as compared to apical-to-basolateral direction (efflux ratio >2), 9,10 using selected model drugs or chemicals  at  concentrations that do not saturate the efflux system (e.g., digoxin, vinblastine, rhodamine 123, methotrexate). The use of animal or in vitro permeability test methods is recommended only for  drug substances that are transported by passive mechanisms (efflux ratio of the test drug should be < 2).”

Typical results of the efflux ratio (B-A)/(A-B) evaluation for 4 reference compounds in bidirectional Caco-2 assay are shown on the Figure 2. These experimental data obtained at Bienta  show good correlation with the literature data for efflux substrates.