Enteroids to Study Pediatric Intestinal Drug Transport

Abstract

Intestinal maturational changes after birth affect the pharmacokinetics (PK) of drugs, having major implications for drug safety and efficacy. However, little is known about ontogeny-related PK patterns in the intestine. To explore the accuracy of human enteroid monolayers for studying drug transport in the pediatric intestine, we compared the drug transporter functionality and expression in enteroid monolayers and tissue from pediatrics and adults. Enteroid monolayers were cultured of 14 pediatric [median (range) age: 44 weeks (2 days-13 years)] and 5 adult donors, in which bidirectional drug transport experiments were performed. In parallel, we performed similar experiments with tissue explants in Ussing chamber using 11 pediatric [median (range) age: 54 weeks (15 weeks-10 years)] and 6 adult tissues. Enalaprilat, propranolol, talinolol, and rosuvastatin were used to test paracellular, transcellular, and transporter-mediated efflux by P-gp and breast cancer resistance protein (BCRP), respectively. In addition, we compared the expression patterns of ADME-related genes in pediatric and adult enteroid monolayers with tissues using RNA sequencing. Efflux transport by P-gp and BCRP was comparable between the enteroids and tissue. Efflux ratios (ERs) of talinolol and rosuvastatin by P-gp and BCRP, respectively, were higher in enteroid monolayers compared to Ussing chamber, likely caused by experimental differences in model setup and cellular layers present. Explorative statistics on the correlation with age showed trends of increasing ER with age for P-gp in enteroid monolayers; however, it was not significant. In the Ussing chamber setup, lower enalaprilat and propranolol transport was observed with age. Importantly, the RNA sequencing pathway analysis revealed that age-related variation in drug metabolism between neonates and adults was present in both enteroids and intestinal tissue. Age-related differences between 0 and 6 months old and adults were observed in tissue as well as in enteroid monolayers, although to a lesser extent. This study provides the first data for the further development of pediatric enteroids as an in vitro model to study age-related variation in drug transport. Overall, drug transport in enteroids was in line with data obtained from ex vivo tissue (using chamber) experiments. Additionally, pathway analysis showed similar PK-related differences between neonates and adults in both tissue and enteroid monolayers. Given the challenge to elucidate the effect of developmental changes in the pediatric age range in human tissue, intestinal enteroids derived from pediatric patients could provide a versatile experimental platform to study pediatric phenotypes.

Keywords: Ussing chamber; drug transporters; enteroids; intestinal organoids; intestine; pediatrics; pharmacokinetics.

Figure 1

Apparent permeability (P_app) and ER determined in enteroid monolayers. A-to-B: apical to basolateral transport. B-to-A: basolateral to apical transport. Succeeded experiments per substrate are described by n = 14 pediatric and 5 adult donor succeeded experiments. (A) P_app enalaprilat (paracellular transport). (B) P_app propranolol (transcellular transport). (C) ER enalaprilat with age. (D) ER propranolol with age. (E) P_app talinolol (P-gp transport). (F) P_app rosuvastatin (BCRP transport). (G) ER talinolol with age. (H) ER rosuvastatin with age. Bidirectional drug transport in human intestinal tissues.

Figure 2

Apparent permeability (P_app) and ER determined in tissue with the Ussing chamber. A-to-B: apical to basolateral transport. B-to-A: basolateral to apical transport. Succeeded experiments per substrate are described by n = A-to-B/B-to-A. (A) P_app enalaprilat (paracellular transport) n = 9/10 pediatrics, n = 4/5 adults. (B) P_app propranolol (transcellular transport) n = 10 pediatrics, n = 5 adults. (C) ER enalaprilat with age, n = 8 pediatric, n = 4 adults. (D) ER propranolol with age, n = 9 pediatric, n = 4 adults. (E) P_app talinolol (P-gp transport) n = 9/10 pediatrics, n = 4/6 adults. (F) P_app rosuvastatin (BCRP transport) n = 10/10 pediatrics, n = 4/6 adults. (G) ER talinolol with age, n = 8 pediatric, n = 4 adults. (H) ER rosuvastatin with age, n = 9 pediatric, n = 4 adults.

Figure 3

PCA of 0–6 month old donors and adults. (A) PCA of enteroid monolayers, red open squares: 0–6 month old enteroids n = 4, blue open circles: adult enteroids n = 5. (B) PCA of tissue, red filled squares: 0–6 month old tissue n = 5, blue filled circles: adult tissue donors n = 7.

Figure 4

PCA of 0–15 month old donors and adults. (A) PCA of enteroid monolayers, red open squares: 0–15 month old enteroids n = 11, blue open circles: adult enteroids n = 5. (B) PCA of tissue, red filled squares: 0–15 month old tissue n = 22, blue filled circles: adult tissue donors n = 7.

Figure 5

Venn diagram showing more DEGs in tissue compared to enteroid monolayers in neonates (n_tissue = 5, n_enteroids = 4) versus adults (n_tissue = 7, n_enteroids = 5).

Figure 6

Gene expression of ABCB1 (P-gp) and ABCG2 (BCRP) in tissue n = 34 and in enteroid monolayers n = 18 across the age range. Black dots are donors with tissue and enteroid measurement. Gray dots miss one of the two models. (A) ABCB1 in tissue, (B) ABCB1 in enteroid monolayers, (C) correlation of ABCB1 expression between tissue and enteroid monolayers, (D) ABCG2 in tissue, (E) ABCG2 in enteroid monolayers, (F) correlation of ABCG2 expression between tissue and enteroid monolayers, (G) EPCAM in tissue, (H) EPCAM in enteroid monolayers, (I) correlation of EPCAM expression between tissue and enteroid monolayers, (J) VIL1 in tissue, (K) VIL1 in enteroid monolayers, and (L) correlation of VIL1 between tissue and enteroid monolayers.

Figure 7

Top 20 pathways from WikiPathways (EnrichR) that were significantly enriched between 0 and 6 month old and adult enteroid monolayers (A) and tissue (B).