Establishment and Characterization of Novel Human Intestinal In Vitro Models for Absorption and First-Pass Metabolism Studies

Abstract

Commonly used intestinal in vitro models are limited in their potential to predict oral drug absorption. They either lack the capability to form a tight cellular monolayer mimicking the intestinal epithelial barrier or the expression of cytochrome P450 3A4 (CYP3A4). The aim of this study was to establish a platform of colorectal cancer patient-derived cell lines for evaluation of human intestinal drug absorption and metabolism. We characterized ten 2D cell lines out of our collection with confluent outgrowth and long-lasting barrier forming potential as well as suitability for high throughput applications with special emphasis on expression and inducibility of CYP3A4. By assessment of the transepithelial electrical resistance (TEER) the cells barrier function capacity can be quantified. Very high TEER levels were detected for HROC60. A high basal CYP3A4 expression and function was found for HROC32. Eight cell lines showed higher CYP3A4 induction by stimulation via the vitamin D receptor compared to Caco-2 cells (5.1- to 16.8-fold change). Stimulation of the pregnane X receptor led to higher CYP3A4 induction in two cell lines. In sum, we identified the two cell lines HROC183 T0 M2 and HROC217 T1 M2 as useful tools for in vitro drug absorption studies. Due to their high TEER values and inducibility by drug receptor ligands, they may be superior to Caco-2 cells to analyze oral drug absorption and intestinal drug–drug interactions. Significance statement: Selecting appropriate candidates is important in preclinical drug development. Therefore, cell models to predict absorption from the human intestine are of the utmost importance. This study revealed that the human cell lines HROC183 T0 M2 and HROC217 T1 M2 may be better suited models and possess higher predictive power of pregnane X receptor- and vitamin D-mediated drug metabolism than Caco-2 cells. Consequently, they represent useful tools for predicting intestinal absorption and simultaneously enable assessment of membrane permeability and first-pass metabolism.

Bioengineered human colon organoids within vivo-like complexity and function

“…Even though these cells are of colonic origin and share some features with small intestinal enterocytes, their transformed character and genomic instability raise doubts about their use for basic and translational research. Moreover, Caco-2 cells exhibit aberrant expression of carboxylesterase 1 (CES1) isoenzyme instead of CES2, though levels of CES1 are extremely low in the native human intestine 33 . More importantly, for drug development, cytochrome P450 CYP3A4 is barely expressed in Caco-2 cells, restricting analyses involving CYP3A4-mediated drug metabolism.…”

“…More importantly, for drug development, cytochrome P450 CYP3A4 is barely expressed in Caco-2 cells, restricting analyses involving CYP3A4mediated drug metabolism. Genome editing is often used to induce the expression of functional CYP3A4 and CES2 in Caco-2 cells [33][34][35] . In comparison with this work, qPCR revealed significantly higher CYP3A4 and CES2 expression and the absence of liver-specific CES1 expression in human mini-colons and organoids (Figure S2D).…”

Novel In Vitro Models for Cell Differentiation and Drug Transport Studies of the Human Intestine

“…The presented cell panel consists of eight lines derived from human colon cancer; further, two lines were from human rectal cancer. These lines were used with low passage numbers but are immortal and have been tested before for application in standard in vitro assays and for fulfilling basic requirements for intestinal in vitro models [12].…”

“…Caco-2 cells were purchased from CLS (Eppelheim, Germany). Cell culture was performed as previously described [12].…”

“…In a previous study, we described a panel of ten 2D colorectal cancer cell lines with longlasting barrier integrity due to their basal activity and the induction potential of Cytochrome P450 (CYP) 3A4. Of those lines, two candidates with higher CYP3A4 induction potentials than Caco-2 cells were identified [12]. The aim of the present study was to deepen this analysis by assessing intestinal cell differentiation and DT regulation at the transcriptional and protein levels.…”