Pharmacokinetics and pharmacodynamics of thiopurines in an in vitro model of human hepatocytes: Insights from an innovative mass spectrometry assay

Marco Pelin, Elena Genova, Laura Fusco, Monzer Marisat, Ute Hofmann, Ute Hofmann, Diego Favretto, Marianna Lucafò, Andrea Taddio, Andrea Taddio, Stefano Martelossi, Alessandro Ventura, Alessandro Ventura, Gabriele Stocco, Matthias Schwab, Matthias Schwab, Matthias Schwab, Matthias Schwab, Giuliana Decorti, Giuliana Decorti

Research output: Contribution to journalArticlepeer-review


© 2017 Elsevier B.V. Aim To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity. Methods Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10 −4 M azathioprine and 1.1 × 10 −3 M mercaptopurine, concentrations corresponding to the IC 50 values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA). Results Azathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037). Conclusion Detailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability.
Original languageEnglish
Pages (from-to)189-195
Number of pages7
JournalChemico-Biological Interactions
Publication statusPublished - Sep 25 2017


  • Azathioprine
  • Hepatocytes
  • LC-MS/MS
  • Mercaptopurine
  • Thiopurines metabolites


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