BACKGROUND: Oral thiopurines are effective and widely used in treatment of inflammatory bowel disease (IBD) in humans, although their use is limited due the development of adverse events. Here, we examine the efficacy and toxicity of oral treatment with 6-tioguanine (6-TG) and azathioprine (AZA) in a murine model of IBD. METHODS: We induced acute or chronic colitis in BALB/c mice by one or four cycles of 3% dextran sulphate sodium (DSS), respectively. Mice were treated by daily gavages of various dosages of 6-tioguanine, azathioprine, or by phosphate buffered saline (PBS) starting the first day of DSS or after two cycles of DSS, respectively. We monitored the efficacy and toxicity by measuring the weight change and serum alanine aminotransferase (ALT) activity and by disease severity and histology, at the end of the experiment. Moreover, we measured cytokine production after colon fragment cultivation by enzyme-linked immunoabsorbent assay and numbers of apoptotic cells in the spleen by flow cytometry. RESULTS: 6-TG is effective in the treatment of acute DSS-induced colitis in a dose-dependent manner and 40 μg of 6-TG is significantly more effective in the treatment of acute colitis than both AZA and PBS. This effect is accompanied by decrease of IL-6 and IFN-γ production in colon. We did not observe histological abnormalities in liver samples from control (PBS) or 6-TG treated mice. However, liver samples from most mice treated with AZA showed mild, yet distinct signs of hepatotoxicity. In chronic colitis, all thiopurine derivatives improved colitis, 20 μg of 6-TG per dose was superior. High doses of 6-TG led to significant weight loss at the end of the therapy, but none of the thiopurine derivatives increased levels of serum ALT. Both thiopurine derivatives reduced the proportion of apoptotic T helper cells, but a high production of both IL-6 and TGF-β was observed only in colon of AZA-treated mice. CONCLUSIONS: Use of 6-TG in the treatment of experimental colitis in mice appears superior to AZA administration and placebo. In contrast to 6-TG, the use of AZA resulted in histological liver abnormalities.

Department of Immunology and Gnotobiology Institute of Microbiology Academy of Sciences of Czech Republic Videnska 1083 14220 Prague 4 Czech Republic

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Candy S, Wright J, Gerber M, Adams G, Gerig M, Goodman R. A controlled double blind study of azathioprine in the management of Crohn's disease. Gut. 1995;37:674–678. doi: 10.1136/gut.37.5.674. PubMed DOI PMC

Fraser AG, Orchard TR, Jewell DP. The efficacy of azathioprine for the treatment of inflammatory bowel disease: a 30 year review. Gut. 2002;50:485–489. doi: 10.1136/gut.50.4.485. PubMed DOI PMC

Present DH, Korelitz BI, Wisch N, Glass JL, Sachar DB, Pasternack BS. Treatment of Crohn's disease with 6-mercaptopurine. A long-term, randomized, double-blind study. N Engl J Med. 1980;302:981–987. doi: 10.1056/NEJM198005013021801. PubMed DOI

Tiede I, Fritz G, Strand S, Poppe D, Dvorsky R, Strand D, Lehr HA, Wirtz S, Becker C, Atreya R. et al.CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes. J Clin Invest. 2003;111:1133–1145. PubMed PMC

Thomas CW, Myhre GM, Tschumper R, Sreekumar R, Jelinek D, McKean DJ, Lipsky JJ, Sandborn WJ, Egan LJ. Selective inhibition of inflammatory gene expression in activated T lymphocytes: a mechanism of immune suppression by thiopurines. J Pharmacol Exp Ther. 2005;312:537–545. PubMed

de Jong DJ, Derijks LJ, Naber AH, Hooymans PM, Mulder CJ. Safety of thiopurines in the treatment of inflammatory bowel disease. Scand J Gastroenterol Suppl. 2003. pp. 69–72. PubMed

Jharap B, Seinen ML, de Boer NK, van Ginkel JR, Linskens RK, Kneppelhout JC, Mulder CJ, van Bodegraven AA. Thiopurine therapy in inflammatory bowel disease patients: Analyses of two 8-year intercept cohorts. Inflamm Bowel Dis. 2010;19:1541–1549. PubMed

Pearson DC, May GR, Fick GH, Sutherland LR. Azathioprine and 6-mercaptopurine in Crohn disease. A meta-analysis. Ann Intern Med. 1995;123:132–142. PubMed

de Boer NK, van Bodegraven AA, Jharap B, de Graaf P, Mulder CJ. Drug Insight: pharmacology and toxicity of thiopurine therapy in patients with IBD. Nat Clin Pract Gastroenterol Hepatol. 2007;4:686–694. PubMed

Herrlinger KR, Deibert P, Schwab M, Kreisel W, Fischer C, Fellermann K, Stange EF. Remission maintenance by tioguanine in chronic active Crohn's disease. Aliment Pharmacol Ther. 2003;17:1459–1464. doi: 10.1046/j.1365-2036.2003.01590.x. PubMed DOI

Dubinsky MC, Vasiliauskas EA, Singh H, Abreu MT, Papadakis KA, Tran T, Martin P, Vierling JM, Geller SA, Targan SR, Poordad FF. 6-thioguanine can cause serious liver injury in inflammatory bowel disease patients. Gastroenterology. 2003;125:298–303. doi: 10.1016/S0016-5085(03)00938-7. PubMed DOI

Geller SA, Dubinsky MC, Poordad FF, Vasiliauskas EA, Cohen AH, Abreu MT, Tran T, Martin P, Vierling JM, Targan SR. Early hepatic nodular hyperplasia and submicroscopic fibrosis associated with 6-thioguanine therapy in inflammatory bowel disease. Am J Surg Pathol. 2004;28:1204–1211. doi: 10.1097/01.pas.0000128665.12063.97. PubMed DOI

Seiderer J, Zech CJ, Reinisch W, Lukas M, Diebold J, Wrba F, Teml A, Chalupna P, Stritesky J, Schoenberg SO. et al.A multicenter assessment of liver toxicity by MRI and biopsy in IBD patients on 6-thioguanine. J Hepatol. 2005;43:303–309. doi: 10.1016/j.jhep.2005.02.051. PubMed DOI

de Boer NK, Derijks LJ, Gilissen LP, Hommes DW, Engels LG, de-Boer SY, den Hartog G, Hooymans PM, Makelburg AB, Westerveld BD. et al.On tolerability and safety of a maintenance treatment with 6-thioguanine in azathioprine or 6-mercaptopurine intolerant IBD patients. World J Gastroenterol. 2005;11:5540–5544. PubMed PMC

Ansari A, Elliott T, Fong F, Arenas-Hernandez M, Rottenberg G, Portmann B, Lucas S, Marinaki A, Sanderson J. Further experience with the use of 6-thioguanine in patients with Crohn's disease. Inflamm Bowel Dis. 2008;14:1399–1405. doi: 10.1002/ibd.20492. PubMed DOI

Derijks LJ, Gilissen LP, de Boer NK, Mulder CJ. 6-Thioguanine-related hepatotoxicity in patients with inflammatory bowel disease: dose or level dependent? J Hepatol. 2006;44:821–822. doi: 10.1016/j.jhep.2005.11.049. PubMed DOI

Gilissen LP, Derijks LJ, Driessen A, Bos LP, Hooymans PM, Stockbrugger RW, Engels LG. Toxicity of 6-thioguanine: no hepatotoxicity in a series of IBD patients treated with long-term, low dose 6-thioguanine. Some evidence for dose or metabolite level dependent effects? Dig Liver Dis. 2007;39:156–159. doi: 10.1016/j.dld.2006.10.007. PubMed DOI

Qasim A, McDonald S, Sebastian S, McLoughlin R, Buckley M, O'Connor H, O'Morain C. Efficacy and safety of 6-thioguanine in the management of inflammatory bowel disease. Scand J Gastroenterol. 2007;42:194–199. doi: 10.1080/00365520600825166. PubMed DOI

De Boer NK, Tuynman H, Bloemena E, Westerga J, Van Der Peet DL, Mulder CJ, Cuesta MA, Meuwissen SG, Van Nieuwkerk CM, Van Bodegraven AA. Histopathology of liver biopsies from a thiopurine-naive inflammatory bowel disease cohort: prevalence of nodular regenerative hyperplasia. Scand J Gastroenterol. 2008;43:604–608. doi: 10.1080/00365520701800266. PubMed DOI

Derijks LJ, Gilissen LP, Engels LG, Bos LP, Bus PJ, Lohman JJ, Curvers WL, Van Deventer SJ, Hommes DW, Hooymans PM. Pharmacokinetics of 6-mercaptopurine in patients with inflammatory bowel disease: implications for therapy. Ther Drug Monit. 2004;26:311–318. doi: 10.1097/00007691-200406000-00016. PubMed DOI

Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest. 1993;69:238–249. PubMed

Kverka M, Zakostelska Z, Klimesova K, Sokol D, Hudcovic T, Hrncir T, Rossmann P, Mrazek J, Kopecny J, Verdu EF, Tlaskalova-Hogenova H. Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition. Clin Exp Immunol. 2011;163:250–259. doi: 10.1111/j.1365-2249.2010.04286.x. PubMed DOI PMC

Dieleman LA, Palmen MJ, Akol H, Bloemena E, Pena AS, Meuwissen SG, Van Rees EP. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 1998;114:385–391. doi: 10.1046/j.1365-2249.1998.00728.x. PubMed DOI PMC

Petit E, Langouet S, Akhdar H, Nicolas-Nicolaz C, Guillouzo A, Morel F. Differential toxic effects of azathioprine, 6-mercaptopurine and 6-thioguanine on human hepatocytes. Toxicol In Vitro. 2008;22:632–642. doi: 10.1016/j.tiv.2007.12.004. PubMed DOI

Colombel JF, Ferrari N, Debuysere H, Marteau P, Gendre JP, Bonaz B, Soule JC, Modigliani R, Touze Y, Catala P. et al.Genotypic analysis of thiopurine S-methyltransferase in patients with Crohn's disease and severe myelosuppression during azathioprine therapy. Gastroenterology. 2000;118:1025–1030. doi: 10.1016/S0016-5085(00)70354-4. PubMed DOI

Lennard L, Gibson BE, Nicole T, Lilleyman JS. Congenital thiopurine methyltransferase deficiency and 6-mercaptopurine toxicity during treatment for acute lymphoblastic leukaemia. Arch Dis Child. 1993;69:577–579. doi: 10.1136/adc.69.5.577. PubMed DOI PMC

Dubinsky MC, Yang H, Hassard PV, Seidman EG, Kam LY, Abreu MT, Targan SR, Vasiliauskas EA. 6-MP metabolite profiles provide a biochemical explanation for 6-MP resistance in patients with inflammatory bowel disease. Gastroenterology. 2002;122:904–915. doi: 10.1053/gast.2002.32420. PubMed DOI

Gisbert JP, Luna M, Gonzalez-Lama Y, Pousa ID, Velasco M, Moreno-Otero R, Mate J. Liver injury in inflammatory bowel disease: long-term follow-up study of 786 patients. Inflamm Bowel Dis. 2007;13:1106–1114. doi: 10.1002/ibd.20160. PubMed DOI

Vernier-Massouille G, Cosnes J, Lemann M, Marteau P, Reinisch W, Laharie D, Cadiot G, Bouhnik Y, De Vos M, Boureille A. et al.Nodular regenerative hyperplasia in patients with inflammatory bowel disease treated with azathioprine. Gut. 2007;56:1404–1409. doi: 10.1136/gut.2006.114363. PubMed DOI PMC

Holtmann M, Schreiner O, Kohler H, Denzer U, Neurath M, Galle PR, Hohler T. Veno-occlusive disease (VOD) in Crohn's disease (CD) treated with azathioprine. Dig Dis Sci. 2003;48:1503–1505. doi: 10.1023/A:1024755521423. PubMed DOI

Russmann S, Kaye JA, Jick SS, Jick H. Risk of cholestatic liver disease associated with flucloxacillin and flucloxacillin prescribing habits in the UK: cohort study using data from the UK General Practice Research Database. Br J Clin Pharmacol. 2005;60:76–82. doi: 10.1111/j.1365-2125.2005.02370.x. PubMed DOI PMC

de Boer NK, Zondervan PE, Gilissen LP, den Hartog G, Westerveld BD, Derijks LJ, Bloemena E, Engels LG, van Bodegraven AA, Mulder CJ. Absence of nodular regenerative hyperplasia after low-dose 6-thioguanine maintenance therapy in inflammatory bowel disease patients. Dig Liver Dis. 2008;40:108–113. doi: 10.1016/j.dld.2007.10.013. PubMed DOI

Wanless IR. Micronodular transformation (nodular regenerative hyperplasia) of the liver: a report of 64 cases among 2,500 autopsies and a new classification of benign hepatocellular nodules. Hepatology. 1990;11:787–797. doi: 10.1002/hep.1840110512. PubMed DOI

Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine UI, Liotta LA, Falanga V, Kehrl JH. et al.Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA. 1986;83:4167–4171. doi: 10.1073/pnas.83.12.4167. PubMed DOI PMC

Lawrance IC, Maxwell L, Doe W. Inflammation location, but not type, determines the increase in TGF-beta1 and IGF-1 expression and collagen deposition in IBD intestine. Inflamm Bowel Dis. 2001;7:16–26. PubMed

Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. doi: 10.1038/nature04753. PubMed DOI

Mangan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, Hatton RD, Wahl SM, Schoeb TR, Weaver CT. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature. 2006;441:231–234. doi: 10.1038/nature04754. PubMed DOI

Deibert P, Dilger K, Fischer C, Hofmann U, Nauck S, Stoelben S, Kreisel W. High variation of tioguanine absorption in patients with chronic active Crohn's disease. Aliment Pharmacol Ther. 2003;18:183–189. doi: 10.1046/j.1365-2036.2003.01652.x. PubMed DOI

Dubinsky MC, Hassard PV, Seidman EG, Kam LY, Abreu MT, Targan SR, Vasiliauskas EA. An open-label pilot study using thioguanine as a therapeutic alternative in Crohn's disease patients resistant to 6-mercaptopurine therapy. Inflamm Bowel Dis. 2001;7:181–189. doi: 10.1097/00054725-200108000-00001. PubMed DOI