RESUMEN
In childhood, developmental changes and environmental interactions highly affect orally dosed drug disposition across the age range. To optimize dosing regimens and ensure safe use of drugs in pediatric patients, understanding this age-dependent biology is necessary. In this proof-of-concept study, we aimed to culture age-specific enteroids from infant tissue which represent its original donor material, specifically for drug transport and metabolism. Enteroid lines from fresh infant tissues (n = 8, age range: 0.3-45 postnatal weeks) and adult tissues (n = 3) were established and expanded to 3D self-organizing enteroids. The gene expression of drug transporters P-gp (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), and PEPT1 (SLC15A1) and drug metabolizing enzymes CYP3A4, CYP2C18, and UGT1A1 was determined with RT-qPCR in fresh tissue and its derivative differentiated enteroids. Expression levels of P-gp, BCRP, MRP2, and CYP3A4 were similar between tissues and enteroids. PEPT1 and CYP2C18 expression was lower in enteroids compared to that in the tissue. The expression of UGT1A1 in the tissue was lower than that in enteroids. The gene expression did not change with the enteroid passage number for all genes studied. Similar maturational patterns in tissues and enteroids were visually observed for P-gp, PEPT1, MRP2, CYP3A4, CYP2C18, and VIL1. In this explorative study, interpatient variability was high, likely due to the diverse patient characteristics of the sampled population (e.g., disease, age, and treatment). To summarize, maturational patterns of clinically relevant ADME genes in tissue were maintained in enteroids. These findings are an important step toward the potential use of pediatric enteroids in pediatric drug development, which in the future may lead to improved pediatric safety predictions during drug development. We reason that such an approach can contribute to a potential age-specific platform to study and predict drug exposure and intestinal safety in pediatrics.
RESUMEN
Background: The gut and its microbiome have a major impact on many aspects of health and are therefore also an attractive target for drug- or food-based therapies. Here, we report on the added value of combining a microbiome screening model, the i-screen, with fresh intestinal tissue explants in a microfluidic gut-on-a-chip model, the Intestinal Explant Barrier Chip (IEBC). Methods: Adult human gut microbiome (fecal pool of 6 healthy donors) was cultured anaerobically in the i-screen platform for 24 h, without and with exposure to 4 mg/mL inulin. The i-screen cell-free culture supernatant was subsequently applied to the luminal side of adult human colon tissue explants (n = 3 donors), fixed in the IEBC, for 24 h and effects were evaluated. Results: The supplementation of the media with inulin promoted the growth of Anaerostipes, Bifidobacterium, Blautia, and Collinsella in the in vitro i-screen, and triggered an elevated production of butyrate by the microbiota. Human colon tissue exposed to inulin-treated i-screen cell-free culture supernatant or control i-screen cell-free culture supernatant with added short-chain fatty acids (SCFAs) showed improved tissue barrier integrity measured by a 28.2%-34.2% reduction in FITC-dextran 4000 (FD4) leakage and 1.3 times lower transport of antipyrine. Furthermore, the release of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α was reduced under these circumstances. Gene expression profiles confirmed these findings, but showed more profound effects for inulin-treated supernatant compared to SCFA-supplemented supernatant. Conclusion: The combination of i-screen and IEBC facilitates the study of complex intestinal processes such as host-microbial metabolite interaction and gut health.
RESUMEN
After oral administration, the intestine is the first site of drug absorption, making it a key determinant of the bioavailability of a drug, and hence drug efficacy and safety. Existing non-clinical models of the intestinal barrier in vitro often fail to mimic the barrier and absorption of the human intestine. We explore if human enteroid monolayers are a suitable tool for intestinal absorption studies compared to primary tissue (Ussing chamber) and Caco-2 cells. Bidirectional drug transport was determined in enteroid monolayers, fresh tissue (Ussing chamber methodology) and Caco-2 cells. Apparent permeability (Papp) and efflux ratios for enalaprilat (paracellular), propranolol (transcellular), talinolol (P-glycoprotein (P-gp)) and rosuvastatin (Breast cancer resistance protein (BCRP)) were determined and compared between all three methodologies and across intestinal regions. Bulk RNA sequencing was performed to compare gene expression between enteroid monolayers and primary tissue. All three models showed functional efflux transport by P-gp and BCRP with higher basolateral to apical (B-to-A) transport compared to apical-to-basolateral (A-to-B). B-to-A Papp values were similar for talinolol and rosuvastatin in tissue and enteroids. Paracellular transport of enalaprilat was lower and transcellular transport of propranolol was higher in enteroids compared to tissue. Enteroids appeared show more region- specific gene expression compared to tissue. Fresh tissue and enteroid monolayers both show active efflux by P-gp and BCRP in jejunum and ileum. Hence, the use of enteroid monolayers represents a promising and versatile experimental platform to complement current in vitro models.
RESUMEN
Drug metabolism in the intestinal wall affects bioavailability of orally administered drugs and is influenced by age. Hence, it is important to fully understand the drug metabolizing capacity of the gut to predict systemic exposure. The aim of this study was to investigate the potential of enteroids as a tool to study CYP3A4/5 -mediated metabolism in both children and adults. Bioconversion of midazolam, a CYP3A4/5 model substrate, was studied using enteroid monolayers as well as tissue explants in the Ussing chamber, both derived from pediatric [median (range age): 54 weeks (2 days - 13 years), n = 21] and adult (n = 5) tissue. Caco-2 cellular monolayers were employed as controls. In addition, mRNA expression of CYP3A4 was determined in enteroid monolayers (n = 11), tissue (n = 23) and Caco-2 using RT-qPCR. Midazolam metabolism was successfully detected in all enteroid monolayers, as well as in all tissue explants studied in the Ussing chamber, whereas Caco-2 showed no significant metabolite formation. The extracted fraction of midazolam was similar between enteroid monolayers and tissue. The fraction of midazolam extracted increased with age in enteroid monolayers derived from 0 to 70 week old donors. No statistically significant correlation was observed in tissue likely due to high variability observed and the smaller donor numbers included in the study. At the level of gene expression, CYP3A4 increased with age in tissues (n = 32), while this was not reflected in enteroid monolayers (n = 16). Notably, asymmetric metabolite formation was observed in enteroids and tissue, with higher metabolite formation on the luminal side of the barrier. In summary, we demonstrated that enteroids can be used to measure CYP3A4/5 midazolam metabolism, which we show is similar as observed in fresh isolated tissue. This was the case both in children and adults, indicating the potential of enteroids to predict intestinal metabolism. This study provides promising data to further develop enteroids to study drug metabolism in vitro and potentially predict oral absorption for special populations as an alternative to using fresh tissue.