Summary Although genome-wide hypomethylation is a hallmark of many cancers, roles for active DNA demethylation during tumorigenesis are unknown. Here, loss of the APC tumor suppressor gene causes upregulation of a DNA demethylase system and the concomitant hypomethylation of key intestinal cell fating genes. Notably, this hypomethylation maintained zebrafish intestinal cells in an undifferentiated state which was released upon knock down of demethylase components. Mechanistically, the demethylase genes are directly activated by Pou5f1 and Cebpβ, and indirectly repressed by retinoic acid, which antagonizes Pou5f1 and Cebpβ. Apc mutants lack retinoic acid, due to the transcriptional repression of retinol dehydrogenase l1 via a complex that includes Lef1, Groucho2, Ctbp1, Lsd1 and Corest. Our findings imply a model wherein APC controls intestinal cell fating through a switch in DNA methylation dynamics. Wildtype APC and retinoic acid downregulate demethylase components, thereby promoting DNA methylation of key genes and helping progenitors commit to differentiation.

N-acetylcysteine (NAC) is being investigated as an antioxidant for several conditions including traumatic brain injury, but the mechanism by which it crosses membrane barriers is unknown. We sought to understand how the transporter inhibitor, probenecid, affects NAC pharmacokinetics and to evaluate the interaction of NAC with transporters.Juvenile Sprague-Dawley rats were administered NAC alone or in combination with probenecid intraperitoneally. Plasma and brain samples were collected serially and NAC concentrations were measured. Transporter studies were conducted with human embryonic kidney-293 cells that overexpress organic anion transporter (OAT)1 or OAT3 and with human multi-drug resistance-associated protein (MRP)1 or MRP4 membrane vesicles.NAC area under the curve was increased in plasma (1.65-fold) and brain (2.41-fold) by probenecid. The apparent plasma clearance was decreased by 65%. Time- and concentration-dependent NAC uptake that was inhibitable by probenecid was observed with OAT1 and OAT3. No uptake of NAC was observed with MRP1 or MRP4.Our results indicate for the first time that NAC is substrate for OAT1 and OAT3 and that probenecid increases NAC plasma and brain exposure in vivo. These data provide insight regarding how NAC crosses biological barriers and suggest a promising therapeutic strategy to increase NAC exposure.

This proof-of-concept neuropharmacometabolomics assessment reveals alterations in known and previously unidentified metabolic pathways and supports therapeutic target engagement of the combination of probenecid and N-acetylcysteine treatment after severe traumatic brain injury in children.

Objective The objective of this study was to characterize the population pharmacokinetics of fentanyl and identify factors that contribute to exposure variability in critically ill pediatric patients. Methods We conducted a single-center, retrospective cohort study using electronic record data and remnant blood samples in the setting of a mixed medical/surgical intensive care unit (ICU) at a quaternary children's hospital. Children with a predicted ICU length of stay of at least 3 days and presence of an indwelling central venous or arterial line were included. Serum fentanyl measurements were performed for 278 unique remnant samples from 66 patients. Both one-and two-compartment models were evaluated to describe fentanyl disposition. Covariates were introduced into the model in a forward/backward, stepwise approach and included age, sex, race, weight, cytochrome P450 (CYP) 3A5 genotype, and the presence of CYP3A4 or CYP3A5 inducers or inhibitors. Simulations were performed using the successful model to depict the influence of inducers on fentanyl concentrations. Results A two-compartment base model best described the data. There was good agreement between observed and predicted concentrations in the final model. The typical fentanyl clearance for 70 kg (reference weight) and 20.1 kg (median weight) patients were 34.6 and 13.6 L/h, respectively. The magnitude of the unexplained random inter-individual variability was high for both clearance (60.7%) and apparent volume of the central compartment (V 1) (107.2%). Coadministration of the known CYP3A4/5 inducers fosphenytoin and/or phenobarbital was associated with significantly increased fentanyl clearance. Simulations demonstrate that the effect of inducer administration was most pronounced following discontinuation of a fentanyl infusion. Conclusions In this study we show the feasibility and utility of using electronic record data and remnant blood samples to successfully construct population pharmacokinetic models for a heterogeneous cohort of critically ill children. A clinically relevant effect of concomitant CYP3A4/5 inducers was identified. Scaling this population pharmacokinetic approach is necessary to craft precision approaches to fentanyl administration for critically ill children.