In vitro evaluation of major in vivo drug metabolic pathways using primary human hepatocytes and HepaRG cells in suspension and in a dynamic three-dimensional bioreactor system

Number of tables: 2 Number of figures: 7 Word count for abstract: 246 (250 max) Word count for introduction: 607 (750 max) Number of words in the discussion: 1498 (1500 max) Number of references: 44 (60 max) Abbreviations: 2D, two-dimensional; 3D, three-dimensional; ACN, acetonitrile; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; Ct, threshold cycle; CYP, cytochrome P450; LC, liquid chromatography; MS, mass spectrometry; P450, cytochrome P450; RAM, radioactivity monitoring; UGT, UDP-glucuronosyltransferase Abstract Major human specific metabolites, not detected during in vivo and in vitro preclinical studies, may cause unexpected drug interactions and toxicity in human and delays in clinical programs. Thus, reliable preclinical tools for detection of major human metabolites are of high importance. The aim of this study was to compare major drug metabolic pathways in HepaRG cells, a human hepatoma cell line, to fresh human hepatocytes, cryopreserved human hepatocytes and human in vivo data. Further, the maintenance of P450 and UGT activities in a dynamic three-dimensional (3D) bioreactor were evaluated over time using HepaRG cells and human hepatocytes. 14 C-diclofenac and a candidate from our drug development program, 14 C-AZD6610, which are metabolized by cytochrome P450 (P450) and UDPglucuronosyltransferase (UGT) in vivo, were used as model substrates. The proportion of relevant biotransformation pathways of the investigated drug was clearly different in the various cell systems. The hydroxylation route was favoured in primary human hepatocytes, whereas the glucuronidation route was favoured in HepaRG cells. The human in vivo metabolite profile of AZD6610 was best represented by human hepatocytes, whereas all major diclofenac metabolites were detected in HepaRG cells. Moreover, the metabolite profiles in cryopreserved and fresh human hepatocytes were essentially the same. The liver bioreactor using both fresh human hepatocytes and HepaRG cells retained biotransformation capacity over one week. Thus, the incubation time can be increased from a few hours in suspension to several days in 3D cultures, which opens up for detection of metabolites from slowly metabolized drugs

HLA class I and II expression in oropharyngeal squamous cell carcinoma in relation to tumor HPV status and clinical outcome.

HPV-DNA positive (HPVDNA+) oropharyngeal squamous cell carcinoma (OSCC) has better clinical outcome than HPV-DNA negative (HPVDNA-) OSCC. Current treatment may be unnecessarily extensive for most HPV+ OSCC, but before de-escalation, additional markers are needed together with HPV status to better predict treatment response. Here the influence of HLA class I/HLA class II expression was explored. Pre-treatment biopsies, from 439/484 OSCC patients diagnosed 2000-2009 and treated curatively, were analyzed for HLA I and II expression, p16(INK4a) and HPV DNA. Absent/weak as compared to high HLA class I intensity correlated to a very favorable disease-free survival (DFS), disease-specific survival (DSS) and overall survival (OS) in HPVDNA+ OSCC, both in univariate and multivariate analysis, while HLA class II had no impact. Notably, HPVDNA+ OSCC with absent/weak HLA class I responded equally well when treated with induction-chemo-radiotherapy (CRT) or radiotherapy (RT) alone. In patients with HPVDNA- OSCC, high HLA class I/class II expression correlated in general to a better clinical outcome. p16(INK4a) overexpression correlated to a better clinical outcome in HPVDNA+ OSCC. Absence of HLA class I intensity in HPVDNA+ OSCC suggests a very high survival independent of treatment and could possibly be used clinically to select patients for randomized trials de-escalating therapy

Effect of timing of dosing in relation to food intake on the pharmacokinetics of esomeprazole

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