title: Quantification of darunavir and etravirine in human peripheral blood mononuclear cells using high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), clinical application in a cohort of 110 HIV-1 infected patients and evidence of a potential drug-drug interaction.
abstract: OBJECTIVE
  To describe the validation of a sensitive high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method allowing the simultaneous quantification of darunavir (DRV) and etravirine (ETR) in peripheral blood mononuclear cells (PBMCs) and its application in a cohort of HIV-1 infected patients.
  
  
  METHODS
  Blood samples were obtained from 110 patients. PMBCs were isolated using density gradient centrifugation. Drug extraction from PBMCs was performed with a 60:40 methanol-water (MeOH-H2O) solution containing deuterated IS (DRV-d9 and ETR-d8). The chromatographic separation was performed on a RP18 XBridge™ column.
  
  
  RESULTS
  The geometric mean (GM) of cell associated concentration ([DRV]CC) and plasmatic concentration ([DRV]plasma) were 360.5ng/mL (CI95%:294.5-441.2) and 1733ng/mL (CI95%:1486-2021), respectively. A geometric mean intracellular (IC)/plasma ratio (GMR) of 0.21 (CI95%:0.18-0.24) was calculated. Adjusted for dose/body surface area and post-intake time, a statistically significant correlation was observed between [DRV]Plasma and the eGFR (p=0.002) and between [DRV]Plasma and the concomitant use of ETR (p=0.038). For the 10 patients receiving ETR in addition to DRV, the GM of [ETR]Plasma (available for 8 out of 10 patients) and [ETR]CC were 492.3ng/mL and 2951ng/mL respectively. The GMR of ETR was 7.6 (CI95%: 3.61-13.83).
  
  
  CONCLUSIONS
  A handy and sensitive high performance LC-MS/MS method allowing the simultaneous quantification of DRV and ETR in PBMCs has been described and successfully applied in the largest cohort of DRV-treated patients reported to date. ETR accumulates more efficiently in PBMCs compared to DRV. We have also highlighted a possible impact of ETR on DRV plasma concentrations requiring further investigations.
authors.affiliation: AIDS Reference Centre, Department of Internal Medicine, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium; Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium. Electronic address: leila.belkhir@uclouvain.be.
authors.forename: Leïla
authors.initials: L
authors.lastname: Belkhir
authors.affiliation: Department of Clinical Chemistry, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Morgane
authors.initials: M
authors.lastname: De Laveleye
authors.affiliation: AIDS Reference Centre, Department of Internal Medicine, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Bernard
authors.initials: B
authors.lastname: Vandercam
authors.affiliation: AIDS Reference Centre, Department of Internal Medicine, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Francis
authors.initials: F
authors.lastname: Zech
authors.affiliation: Department of Clinical Chemistry, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Kevin-Alexandre
authors.initials: KA
authors.lastname: Delongie
authors.affiliation: Department of Clinical Chemistry, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Capron
authors.affiliation: AIDS Reference Centre, Department of Internal Medicine, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Jean
authors.initials: J
authors.lastname: Yombi
authors.affiliation: AIDS Reference Centre, Department of Internal Medicine, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Anne
authors.initials: A
authors.lastname: Vincent
authors.affiliation: Integrated PharmacoMetrics, PharmacoGenomics and PharmacoKinetics, Louvain Drug Research Institute, Université Catholique de Louvain, Avenue Hippocrate 10, 1200Brussels, Belgium.
authors.forename: Laure
authors.initials: L
authors.lastname: Elens
authors.affiliation: Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium; Department of Clinical Chemistry, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: Vincent
authors.initials: V
authors.lastname: Haufroid
mesh_terms: D015415:Biomarkers / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015331:Cohort Studies; D000069454:Darunavir / Q000097:blood* / Q000627:therapeutic use; D004347:Drug Interactions; D016903:Drug Monitoring / Q000379:methods; D005260:Female; D005500:Follow-Up Studies; D006678:HIV / Q000187:drug effects; D015658:HIV Infections / Q000097:blood* / Q000188:drug therapy; D017320:HIV Protease Inhibitors / Q000097:blood / Q000627:therapeutic use; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D009570:Nitriles; D011379:Prognosis; D011724:Pyridazines / Q000097:blood* / Q000627:therapeutic use; D011743:Pyrimidines; D018894:Reverse Transcriptase Inhibitors / Q000097:blood / Q000627:therapeutic use; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015415:Biomarkers; D017320:HIV Protease Inhibitors; D009570:Nitriles; D011724:Pyridazines; D011743:Pyrimidines; D018894:Reverse Transcriptase Inhibitors; C451734:etravirine; D000069454:Darunavir
keywords: Anti-retroviral drugs; Darunavir; Drug interaction; Etravirine; HIV-1; Intracellular; LC–MS/MS; PBMCs