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Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium–bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance–associated protein (MRP) 2, MRP3, organic anion–transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0–2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location–specific, age-related changes. SIGNIFICANCE STATEMENT This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry–based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.

Although intestinal metabolism plays an important role in drug disposition, early predictions of human outcomes are challenging, in part because of limitations of available in vitro models. To address this, we have evaluated three in vitro models of human intestine (microsomes, permeabilized enterocytes, and cryopreserved intestinal mucosal epithelium) as tools to assess intestinal metabolism and estimate the fraction escaping gut metabolism (fg) in drug discovery. The models were tested with a chemically diverse set of 32 compounds, including substrates for oxidoreductive, hydrolytic, and conjugative enzymes. Liquid chromatography–high-resolution mass spectrometry was used to quantify substrate disappearance [intrinsic clearance (CLint)] and qualify metabolite formation (quantitative-qualitative bioanalysis). Fraction unbound in the incubation (fu,inc) was determined by rapid equilibrium dialysis. Measured in vitro results (CLint and fu,inc) were supplemented with literature data [passive Caco-2 apical to basolateral permeability, enterocyte blood flow, and intestinal surface area (A)] and combined using a midazolam-calibrated Qgut model to predict human fg values. All three models showed reliable CYP and UDP-glucuronosyltransferase activities, but enterocytes and mucosa may offer advantages for low-clearance compounds and alternative pathways (e.g., sulfation, hydrolases, and flavin-containing monooxigenases). Early predictions of human fg values were acceptable for the high-fg compounds (arbitrarily fg > 0.7). However, predictions of low- and moderate-fg values (arbitrarily fg Significance Statement We found that cellular models of the human gut (permeabilized enterocytes and cryopreserved intestinal mucosa) offer an alternative to and potential advantage over intestinal microsomes in studies of drug metabolism, particularly for low-clearance compounds and alternative pathways (e.g., sulfation, hydrolases, and flavin-containing monooxigenases). The predictivity of human fraction escaping gut metabolism for common CYP and UDP-glucuronosyltransferase substrates based on the Qgut model is still limited, however, and appropriate further evaluation is recommended.

After graduating with an Oceanography degree from Swansea University, Lloyd has spent over 20 years in the field of bioanalysis and metabolite profiling. He started his career in large pharma at Wyeth UK, where he was involved in setting up the first GC and LC–MS/MS systems for both QC and early DMPK assays, employing EI/CI, thermospray, and the then new electrospray ionization techniques. Lloyd then joined Celltech, now UCB, where he is primarily tasked with metabolite profiling by LC–MS and NMR to support both early research projects and late-stage clinical studies. The application of liquid chromatography high-resolution mass spectrometry for simultaneous quantitative and qualitative (quan/qual) analysis has gained momentum across a range of different scientific arenas in recent years. The ability to acquire high quality quantitative data, whilst also capturing qualitative data for either parallel or retrospective analysis, is a powerful resource, especially in view of ever-reducing cycle times, laboratory space and budgets. The quan/qual approach employing a Q-Exactive™ Orbitrap high-resolution mass spectrometer has been successfully introduced into UCB's research DMPK department. This article describes our experiences in introducing quan/qual, issues that we discovered in establishing this new working paradigm, the evolution of the strategy and its future potential.

Phosphoinositide 3-kinases (PI3K) are key signaling enzymes regulating cellular survival, development, and function. Expression of the PI3K δ isoform is largely restricted to leukocytes and it plays a key role in immune cell development and function. Seletalisib is a novel small-molecule inhibitor of PI3K δ that was evaluated in biochemical assays, cellular assays of adaptive and innate immunity, and an in vivo rat model of inflammation. Our findings show that seletalisib is a potent, ATP-competitive, and selective PI3K δ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N -formyl peptide–stimulated but not phorbol myristate acetate–stimulated superoxide release from human neutrophils, consistent with a PI3K δ -specific activity. No indications of cytotoxicity were observed in peripheral blood mononuclear cells (PBMCs) or other cell types treated with seletalisib. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. Seletalisib showed dose-dependent inhibition in an in vivo rat model of anti-CD3-antibody-induced interleukin 2 release. Collectively, these data characterize seletalisib as a selective PI3K δ inhibitor and potential therapeutic candidate for the treatment of B-cell malignancies and autoimmune diseases driven by dysregulated proinflammatory cytokine secretion.