Modulatory Effects of 6-Carboxymethylthiopurine on Activated Murine Macrophages Marcio L. De Paula 1 , Fernanda G. Braga 1 , Elaine S. Coimbra 1 , Arturene M. L. Carmo 2 , Henrique C. Teixeira 1 , Adilson D. Da Silva 2 , Maria A. Souza 1 and Ana Paula Ferreira 1, * 1 Departamento de Parasitologia, Microbiologia e Imunologia, Instituto de CiÞncias Biológicas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil 2 Departamento de Quimica, Instituto de CiÞncias Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil *Corresponding author: Ana Paula Ferreira, ana.paula@ufjf.edu.br The immunological activity of macrophages against pathogens in hosts includes the phagocy- tosis and the production of nitric oxide. We report herein the investigation of the effect of 6-carbo- xymethylthiopurine on nitric oxide production by murine macrophages as well as its effect on the cell viability and proliferation after stimulus with Mycobacterium bovis bacille Calmette–Gue ´ rin, interferon-gamma or a combination of both. J774A.1 macrophages stimulated or not by bacille Calmette–Gue ´ rin (20 lg ⁄ mL), interferon-gamma or both, were cultured in the presence of 6-carbo- xymethylthiopurine (125, 250 and 500 lM). Nitric oxide production was measured by the Griess method and cell viability ⁄ proliferation by the diphenyltetrazolium assay [3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide]. We observed an increase of J774A.1 cell proliferation after stimulus with bacille Calmette–Gue ´ rin at 125, 250 and 500 lM (69.1, 124.0 and 89.7%, respectively) and with interferon-gamma at 125 and 250 lM (64.8% and 61.7%, respectively) (p < 0.05). In all cultures treated with 6-carbo- xymethylthiopurine, interferon-gamma-activated nitric oxide production by J774A.1 cells decreased as well as when subjected to interferon-gamma plus bacille Calmette–Gue ´ rin stimuli at 500 lM (p < 0.05). Altogether these data point to an anti- inflammatory effect of 6-carboxymethylthiopurine on stimulated macrophages. Key words: 6-carboxymethylthiopurine, immunological activity, inflammatory response, macrophage, nitric oxide Received 25 January 2008, revised 21 March 2008 and accepted for publication 1 April 2008 6-Mercaptopurine (6-MP) (Figure 1) is an orally administered, water- insoluble purine analog that is effective against acute lymphatic leukemia (1). Oral absorption of 6-MP, however, is quite erratic, with only 16–50% of the administered dose reaching the blood (2). With the goal of obtaining 6-MP derivatives more soluble for parenteral administration, various compounds were synthesized (3,4). 6-Mercaptopurine is a 6-thiopurine analog of naturally occurring purine bases: hypoxantine and guanine. It is a phase-specific drug, inhibiting cell division in the S-phase of the cell cycle and it is a prodrug requiring intracellular activation by thiopurine nucleotides to exert an antileukemic effect (5). 6-Mercaptopurine inhibits phos- phoribosylpyrophosphate amidotransferase by methylthioinosine nucleotides thus inhibiting de novo purine synthesis. Therefore, there is a purine deprivation, leading to an inhibition of DNA synthesis, decreased cell proliferation and antileukemic effect (6). 6-Mercaptopurine is an antimetabolite, which has been widely applied to treat childhood acute lymphoblastic leukemia, as well as being a major component of remission maintenance treatment (7,8). Although 6-MP and its prodrug azathioprine (AZA) have been used in clinical practice as immunosuppressive drugs for more than 20 years, their mechanism of action is little known. This is largely because of the fact that they are by themselves inactive and must be transformed intracellularly into active 6-thioguanine (6-TG) metabolites (9). 6-Thioguanine may be used to treat AZA and 6-MP- intolerant inflammatory bowel disease patients because 6-TG is considered to be an escape maintenance immunosuppressant (10). 6-Mercaptopurine and AZA act on T and B-lymphocytes in vitro, inhibit the expression of surface receptors and block mitogen-stimu- lated immunologic response. Low doses of 6-MP are applied to the treatment of ulcerative colitis, Crohn's disease and multiple sclero- sis (11–13). Different 6-alkylthiopurine and analogs were prepared and tested for the carcinostatic and leishmanial activity and have more proven inhibitory effects than the previously known 6-MP and have no toxic H N N N N SH Figure 1: Structure of 6-mercaptopurine. 563 Chem Biol Drug Des 2008; 71: 563–567 Research Article ª 2008 The Authors Journal compilation ª 2008 Blackwell Munksgaard doi: 10.1111/j.1747-0285.2008.00665.x