title: Investigations into the liver effects of ximelagatran using high content screening of primary human hepatocyte cultures.
abstract: BACKGROUND
  Ximelagatran, the first oral agent in the new class of direct thrombin inhibitors, was withdrawn from the market due to increased rates of liver enzyme elevations in long-term treatments. Despite intensive pre clinical investigations the cellular mechanisms behind the observed hepatic effects remain unknown.
  
  
  OBJECTIVE
  The aim of this study was to assess drug-induced cytotoxicity in primary human hepatocyte cultures by ximelagatran and other reference pharmaceutical agents with known in vivo hepatotoxic profiles.
  
  
  METHODS
  Drugs cause liver injury by many distinct mechanisms that result in abnormal cellular functioning and different patterns of injury. To address many potential toxic mechanisms in a human-relevant model, freshly isolated human hepatocytes were used in automated imaging assays. Ximelagatran was used as a test compound to study biochemical and morphological changes in human hepatocytes. In addition, 11 control, reference and comparator compounds with known liver-toxic potential in humans were used. The response to these compounds was assessed across five different hepatocyte donor preparations.
  
  
  RESULTS
  Cytotoxicity induced by a number of compounds was quantitatively monitored using an automated imaging technique. A variety of morphological changes in hepatocyte cytoskeleton and mitochondrial function could be identified at sublethal doses of test compounds. Doses of ximelagatran up to 500 microM did not cause a cytotoxic response in the majority of preparations and no subcytotoxic response was observed at doses below 125 microM.
  
  
  CONCLUSIONS
  The experiments described here demonstrate that primary human hepatocytes may be used in a medium-throughput format for screening using imaging-based assays for the identification of cellular responses. Overall, it is concluded that ximelagatran did not cause a significant decrease in cell viability when incubated for 24 h at considerably higher concentrations than are found in plasma following therapeutic dosing.
authors.affiliation: Advanced Science and Technology Laboratory, AstraZeneca R&D Charnwood, Loughborough, UK.
authors.forename: Edward K
authors.initials: EK
authors.lastname: Ainscow
authors.forename: James E
authors.initials: JE
authors.lastname: Pilling
authors.forename: Nick M
authors.initials: NM
authors.lastname: Brown
authors.forename: Alexandra T
authors.initials: AT
authors.lastname: Orme
authors.forename: Michael
authors.initials: M
authors.lastname: Sullivan
authors.forename: Andy C
authors.initials: AC
authors.lastname: Hargreaves
authors.forename: Emma-Louise
authors.initials: EL
authors.lastname: Cooke
authors.forename: Elaine
authors.initials: E
authors.lastname: Sullivan
authors.forename: Stefan
authors.initials: S
authors.lastname: Carlsson
authors.forename: Tommy B
authors.initials: TB
authors.lastname: Andersson
mesh_terms: D000925:Anticoagulants / Q000008:administration & dosage / Q000009:adverse effects*; D001384:Azetidines / Q000008:administration & dosage / Q000009:adverse effects*; D001596:Benzylamines / Q000008:administration & dosage / Q000009:adverse effects*; D002470:Cell Survival / Q000187:drug effects; D003599:Cytoskeleton / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008930:Mitochondria, Liver / Q000187:drug effects
chemical_list: D000925:Anticoagulants; D001384:Azetidines; D001596:Benzylamines; C426686:ximelagatran