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P092. Co-administration of 5-aminosalicylic acid to 6-mercaptopurine reduces in vitro hepatotoxicity

M. Broekman, H.M. Roelofs, F. Hoentjen, M. Kerstholt, W.H. Peters, G.J. Wanten, D.J. de Jong, Radboud UMC, Gastroenterology & Hepatology, Nijmegen, Netherlands

Background

Thiopurines are widely used for the treatment of inflammatory bowel disease, but are associated with hepatotoxicity. 5-Aminosalicylic acid (5-ASA) preparations, can influence thiopurine metabolism by inhibiting thiopurine methyl transferase (TMPT) and subsequently reducing the formation of 6-methylmercaptopurine (6-MMP) metabolites. This is of particular interest as 6-MMP metabolites are associated with the development of hepatotoxicity, but it is unknown whether co-administration of 5-ASA with thiopurines reduces hepatotoxicity. Our aim is to clarify whether 5-ASA co-administration has the potential to decrease in vitro thiopurine-induced hepatotoxicity.

Methods

Three human hepatoma cell lines (Huh7, HepG2 and HepaRG) were cultured in William's E medium containing 2% dimethyl sulfoxide. Azathioprine (AZA), 6-mercaptopurine (6-MP) or tioguanine (TG) was added in a concentration range from 3.9 to 4000 µM. In separate co-administration experiments, a fixed non toxic dose of 200 µM 5-ASA was used in combination with 3.9 to 4000 µM of thiopurines. Water-soluble tetrazolium salt-1 (WST-1) cytotoxicity assays were conducted after 24, 48 and 72 hours of incubation. Culture medium plus examined drugs were refreshed every 24 hours. Cell survival curves and half maximal inhibitory concentrations (IC50) were obtained from three independent experiments conducted in triplicate.

Results

Addition of 5-ASA resulted in significant changes of the IC50 in 7 out of 27 (26%) experiments; in 5 out of 27 (19%) a decreased and in 2 out of 27 (7%) an increased cytotoxicity was seen. In HepaRG cells incubated with 6-MP, the IC50 increased from 679 (95% CI 502–920 µM) to 5704 µM (95% CI 2174–14962 µM) and from 412 µM (95% CI 321–530 µM) to 918 µM (753–1118 µM) for experiments with 48 and 72 hours incubation time, respectively. In HepaRG cells and HepG2 cells, IC50 was approximately 60 µM lower (more hepatotoxic) when incubated for 72 hours with the combination of AZA + 5-ASA in comparison to AZA alone. In Huh7 cells, the IC50 was 80 µM higher after 72 hours incubation with AZA + 5-ASA compared to AZA alone. Concentration based, TG was the most toxic thiopurine with an IC50 value of 18 µM after 72 hours incubation in HepaRG cells, compared to AZA (266 µM) and 6-MP (412 µM).

Conclusion

Addition of a fixed, non toxic dose of 5-ASA to 6-MP resulted in a decreased in vitro toxicity in hepatoma cells.