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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.

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.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111 In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model ( CKP ). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.