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P048. In vitro pancreas toxicity by azathioprine but not 6-mercaptopurine

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

Background

Among drugs often used in the treatment of inflammatory bowel disease (IBD), thiopurines and 5-aminosalicylic acid (5-ASA) can cause pancreatitis. The underlying mechanism remains largely unclear, but may include an immune mediated drug reaction. Knowing that azathioprine (AZA) can stimulate pancreas secretion, we postulate that, like in the cerulein model of acute pancreatitis, this might cause acinar cell damage and consequently to autodigestion of the pancreas by its proteases. To bolster this hypothesis, we evaluated in vitro cytotoxic effects of thiopurines and 5-ASA, tested as single drugs and as combination treatment, on three pancreatic cell lines.

Methods

The human pancreatic cell lines PANC-1 (epithelioid carcinoma), AsPC-1 and Capan-1 (both adenocarcinoma) were cultured in Dulbecco's modified Eagle's medium (DMEM) (PANC-1) or RPMI medium (AsPC-1 and Capan-1). After seeding in 96-wells plates (1.48×105 cells/cm2), cells were incubated with the single drugs AZA, 6-mercaptopurine (6-MP), tioguanine (TG) or 5-ASA in the concentration range of 3.9–4000 µM. Every 24 hours culture medium and drugs were refreshed. After 24, 48 or 72 hours cytotoxicity was established by performing the water-soluble tetrazolium salt-1 (WST-1) assay. Cell survival curves were obtained and half maximal inhibitory concentrations (IC50) were calculated. Next, combination experiments were conducted, with various concentrations AZA, 6-MP or TG (3.9–4000 µM) in combination with a fixed non toxic concentration of 5-ASA (200 µM). Three independent experiments were conducted in triplicate.

Results

IC50 values of AZA were between 300 and 530 µM after 72 hours of incubation in the three cell lines. Incubation with TG resulted in IC50 values, ranging from 360 to 2508 µM, depending on the cell line used. Incubations with 6-MP or 5-ASA did not result in decreased cell survival. Combinations of thiopurines with 5-ASA resulted in increased toxicity of AZA and TG in AsPC-1 cells, and decreased toxicity of AZA in Capan-1 cells. TMPT genotyping did not reveal polymorphisms associated with decreased TPMT activity in the three cell lines.

Conclusion

AZA, and to a much lesser extent TG, but not 6-MP and 5-ASA, exerted a cytotoxic effect on the tested pancreatic cell lines in the present study. The difference in toxicity between AZA and 6-MP could be explained by differences in their metabolism. A large comparative study between AZA and 6-MP would be necessary to verify if there is also an in vivo difference.