Intestinal drug absorption is one of the factors that determine the success of an oral drug product along with its efficacy, pharmacokinetics, and toxicity. Therefore, models for predicting intestinal drug absorption are becoming more important in early drug development to accelerate identification of promising or troublesome compounds. The use of cell cultures provides a method to predict drug permeability by utilizing cell monolayers in a two-chamber diffusion system to simulate the passage of drugs from the intestinal lumen into the blood. The cell model is simple and easy to use and avoids the usage of animal models for pharmacological and toxicological studies, so it is cost-effective and can produce reliable and reproducible results for understanding and evaluating the permeability characteristics of the potential lead drug candidates.
Many cell monolayer models have been developed to mimic human intestinal epithelium and are gaining in popularity. These models use immortalized cells that grow rapidly into confluent monolayers and undergo spontaneous differentiation. Therefore, these cell monolayer models provide an ideal system for the study of intestinal drug absorption. Several cell lines are in use for modelling human intestinal absorption, including Madin Darby canine kidney (MDCK), TC-7, HT29-MTX, 2/4/A1, and the most popular, Caco-2 cells.
Caco-2 cell represent a reference model for predicting drug permeability, and are routinely used to study transepithelial drug transport for the passive transcellular route, paracellular route, carrier-mediated route and transcytosis. Caco-2 cell lines, derived from human colorectal carcinoma, are cultivated on semipermeable filters for 21-23 days. After differentiation, the cells form a polarized monolayer with brush border, microvilli and tight junctions on the apical and basolateral sides, and express P-gp and several relevant efflux transporters and enzymes.
The Madin-Darby canine kidney (MDCK) is considered as an alternative to Caco- 2 cell for permeability studies. MDCK cells exhibit a shorter culture time (3-5 days) and lower transepithelial electrical resistance (TEER) values compared with Caco-2 cells (MDCK values are much closer to the in vivo TEER of the small intestine). However, these cells are derived from canine kidney and therefore the expression levels and metabolic activity of some transporters are quite different as compared to Caco-2 cells.
TC7, a subclone of Caco-2 cell, is also used for permeability screening. The TC7 shows morphological characteristics of brush border membrane, microvilli and tight junctions similar to Caco-2 monolayer. Therefore, the TC7 model offers an alternative to Caco-2 to evaluate the intestinal permeability of test compounds. In addition, TC7 has an advantage over Caco-2 by expressing high levels of CYP3A4 enzymes well represented in the intestine. However, TC7 lacks transport proteins, so its application is biased towards drug metabolized by CYP3A4.
The human adenocarcinoma HT29-MTX model is used to study the role of intestinal mucus on drug absorption across the intestinal barrier. HT29-MTX is conditioned to acquire the morphological and mucin producing features of goblet cells by culturing parental HT29 in a medium containing methotrexate (MTX) for 6 months. Unlike Caco-2, HT29-MTX develops sparse microvilli on the apical side and reaches confluence 3 days later than the former. However, the expression of goblet cells in HT29-MTX increases absorption of lipophilic compounds compared to Caco-2 monolayer.
2/4/A1 originates from the intestine of fetal rat and is believed to mimic intestinal passive paracellular permeability in humans better than Caco-2 monolayer. This immortalized cell is reported to differentiate into a monolayer with tight junctions, brush-border membrane enzymes and transporter proteins. Unlike Caco-2, the tight junctions expressed in 2/4/A1 are loose and better for studying compounds absorbed in the human intestine through the paracellular route.
IEC-18 cell line, also of rat origin, derived from native ileal crypts and is a valuable model for studying the permeability and paracellular transport across intestinal epithelium. These cells are also used to study the effects of enzymes and receptors on the permeability of drugs. Nevertheless, because they are less well differentiated than Caco-2 cells, some of the carrier-mediated transport is absent.
LLC-PK1 cells derived from pig kidney epithelium have also been employed as an alternative model to Caco-2 cells to assess the permeability of test compounds. Studies have reported the utility of LLC-PK1 in characterizing the passive (transcellular and paracellular) absorption of test compounds
T84 is a cell line derived from human colon carcinoma, which spontaneously differentiates to form polarized monolayer with well-formed tight junctions. T84 cells are similar to adult colonic crypt cells in morphology, tight junctions, and ion transport characteristics. Unlike Caco-2 cells, T84 cells are less prone to differentiate into sublines with altered characteristics.
Table 1. Comparison of cell-based models for intestinal permeability studies.
| Cell lines | Origin | Characteristics | Advantages | Limitations |
|---|
| Caco-2 | Human colon adenocarcinoma | - Polarized monolayers with tight junction, brush border and apical microvilli
- Expression of some relevant efflux transporters
| - Well-developed and characterized
- Easy to maintain
- Good reproducibility, robustness and functional property of human intestinal cells
| - Absence of mucous secreting goblet cells
- Expression of influx transporters is variable (differs laboratory-to-laboratory)
- Not suitable for paracellular transport
- 21-days culture period
|
| MDCK | Dog kidney | - Polarized cells with low intrinsic expression of ABC transporters
| - 3-5 days culture period
- Ideal for transfections
| - Under-expression of major efflux transporters
- Not for active and efflux studies
- Not for mechanistic studies
|
| TC7 | Caco-2 subclone | - Low expression of P-gp
- Stable expression of CYP3A4
| - Higher levels of CYP3A4 and 3A5
- Useful to evaluate metabolic effects during transport
- Greater homogeneity ensuring more consistent results with lower variability
| |
| HT29-MTX | Human colon carcinoma | - Multilayer of undifferentiated cells
- Lack tight junction & functional polarity
| - Contain mucus-producing goblet cells
| - Not suitable for carrier-mediated transport
- Unstable mucus layer
|
| 2/4/A1 | Fetal rat intestine | - Polarized monolayers with tight junction, brush border membrane enzymes and transporter proteins
| - Temperature-sensitive
- Ideal for paracellularly absorbed compounds (leakier pores)
- Good for medium to low throughput screening
| - Lack enzyme systems, transporters fond in Caco-2 and small intestine
- Less pharmaceutical use
|
| IEC-18 | Rat intestine | - Poor cell differentiation
- Leakier paracellular pathway
| - Better model for epithelial permeability study of hydrophilic molecules than Caco-2
- Provides a size-selective barrier for paracellularly transported compounds
| - Not for cell mediated transport studies
|
| LLC-PK1 | Pig kidney | - Polarized cells with low intrinsic transporter expression
| - Characterization of passive transport
- Ideal for transfections
| - Not for active and efflux studies
- Not for mechanistic studies
|
| T84 | Human colon cancer | - Not well differentiated cells
| - Resemble colonic crypt cell phenotype
| - Not adequate for drug studies and carrier mediated process
|
Although all of these cell models show good or moderate correlations with passively absorbed drug permeability in humans, correlations with active transport are variable and mainly low. However, even if in vivo correlation is slow, they are interesting models for determining drug transport mechanism, and extensive studies are needed to identify the relevant carriers and active transport mechanisms.
References
- Pereira, C. et al.; Cell-based in vitro models for intestinal permeability studies. Concepts and Models for Drug Permeability Studies, 2016, 57-81.
- Balimane, P. V. et al.; Cell culture-based models for intestinal permeability: A critique. Drug Discovery Today, 2005,10(5): 335-343.
- Youhanna, S. et al.; The Past, Present and Future of Intestinal In Vitro Cell Systems for Drug Absorption Studies. Journal of Pharmaceutical Sciences, 2020.
- Van Breemen, R. B. et al.; Caco-2 cell permeability assays to measure drug absorption. Expert Opin Drug Metab Toxicol. 2005, 1(2): 175-185.
- Awortwe C. et al.; Application of Caco-2 cell line in herb-drug interaction studies: current approaches and challenges. J Pharm Pharm Sci. 2014, 17(1): 1-19.
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