title: Hepatocyte spheroids as a competent in vitro system for drug biotransformation studies: nevirapine as a bioactivation case study.
abstract: The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12 days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with N-acetyl-L-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Lisbon, Portugal.|Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.
authors.forename: Pedro F
authors.initials: PF
authors.lastname: Pinheiro
authors.affiliation: Centro de Estudos de Doenças Crónicas, CEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Campo dos Mártires da Pátria, 130, 1169-056, Lisbon, Portugal.
authors.forename: Sofia A
authors.initials: SA
authors.lastname: Pereira
authors.affiliation: Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.
authors.forename: Shrika G
authors.initials: SG
authors.lastname: Harjivan
authors.affiliation: Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.
authors.forename: Inês L
authors.initials: IL
authors.lastname: Martins
authors.affiliation: Centro de Estudos de Doenças Crónicas, CEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Campo dos Mártires da Pátria, 130, 1169-056, Lisbon, Portugal.
authors.forename: Aline T
authors.initials: AT
authors.lastname: Marinho
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Lisbon, Portugal.
authors.forename: Madalena
authors.initials: M
authors.lastname: Cipriano
authors.affiliation: Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.
authors.forename: Cristina C
authors.initials: CC
authors.lastname: Jacob
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Lisbon, Portugal.
authors.forename: Nuno G
authors.initials: NG
authors.lastname: Oliveira
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Lisbon, Portugal.
authors.forename: Matilde F
authors.initials: MF
authors.lastname: Castro
authors.affiliation: Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.
authors.forename: M Matilde
authors.initials: MM
authors.lastname: Marques
authors.affiliation: Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal. alexandra.antunes@tecnico.ulisboa.pt.
authors.forename: Alexandra M M
authors.initials: AMM
authors.lastname: Antunes
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003, Lisbon, Portugal. jmiranda@ff.ulisboa.pt.
authors.forename: Joana P
authors.initials: JP
authors.lastname: Miranda
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D015239:Arylsulfotransferase / Q000378:metabolism; D001711:Biotransformation; D018929:Cell Culture Techniques / Q000379:methods; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008658:Inactivation, Metabolic; D019829:Nevirapine / Q000493:pharmacokinetics*; D051381:Rats; D015203:Reproducibility of Results; D018874:Spheroids, Cellular / Q000187:drug effects*
chemical_list: D019829:Nevirapine; D015239:Arylsulfotransferase; C573795:Sult1a1 protein, rat; D000111:Acetylcysteine
keywords: 3D culture; Bioactivation; Covalent protein adducts; Hepatocytes; SULT1A1 modulation