On the Action of 5-Amino-Salicylic Acid and Sulfapyridine on M. avium including Subspecies paratuberculosis

PLOS ONE, Jun 2007

Background Introduced in 1942, sulfasalazine (a conjugate of 5-aminosalicylic acid (5-ASA) and sulfapyridine) is the most prescribed medication used to treat “inflammatory” bowel disease (IBD.) Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We have shown that two other agents used in the therapy of IBD (methotrexate and 6-MP) profoundly inhibit MAP growth. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. We herein hypothesize that the mechanism of action of 5-ASA and/or sulfapyridine may also simply be to inhibit MAP growth. Methodology The effect on MAP growth kinetics by sulfasalazine and its components were evaluated in bacterial culture of two strains each of MAP and M. avium, using a radiometric (14CO2 BACTEC®) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (%−ΔcGI). Principal Findings There are disparate responses to 5-ASA and sulfapyridine in the two subspecies. Against MAP, 5-ASA is inhibitory in a dose-dependent manner (MAP ATCC 19698 46%−ΔcGI at 64 µg/ml), whereas sulfapyridine has virtually no effect. In contrast, against M. avium ATCC 25291, 5-ASA has no effect, whereas sulfapyridine (88%−ΔcGI at 4 µg/ml) is as effective as methotrexate, our positive control (88%−ΔcGI at 4 µg/ml). Conclusions 5-ASA inhibits MAP growth in culture. We posit that, unknowingly, the medical profession has been treating MAP infections since sulfasalazine's introduction in 1942. These observations may explain, in part, why MAP has not previously been identified as a human pathogen. We conclude that henceforth in clinical trials evaluating antiMAP agents in IBD, if considered ethical, the use of 5-ASA (as well as methotrexate and 6-MP) should be excluded from control groups.

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On the Action of 5-Amino-Salicylic Acid and Sulfapyridine on M. avium including Subspecies paratuberculosis

Brown ST (2007) On the Action of 5-Amino-Salicylic Acid and Sulfapyridine on M. avium including Subspecies paratuberculosis. PLoS ONE 2(6): e516. doi:10.1371/journal.pone.0000516 On the Action of 5-Amino-Salicylic Acid and Sulfapyridine on M. avium including Subspecies paratuberculosis Robert J. Greenstein 0 1 Liya Su 0 1 Azra Shahidi 0 1 Sheldon T. Brown 0 1 0 Academic Editor: Naomi Ward, The Institute for Genomic Research , United States of America 1 1 Department of Surgery, VA Medical Center, Bronx, New York, United States of America, 2 Laboratory of Molecular Surgical Research, VA Medical Center, Bronx, New York, United States of America , 3 Microbiology , VA Medical Center, Bronx, New York, United States of America , 4 Infectious Diseases , VA Medical Center , Bronx, New York , United States of America Background. Introduced in 1942, sulfasalazine (a conjugate of 5-aminosalicylic acid (5-ASA) and sulfapyridine) is the most prescribed medication used to treat ''inflammatory'' bowel disease (IBD.) Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We have shown that two other agents used in the therapy of IBD (methotrexate and 6-MP) profoundly inhibit MAP growth. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. We herein hypothesize that the mechanism of action of 5-ASA and/or sulfapyridine may also simply be to inhibit MAP growth. Methodology. The effect on MAP growth kinetics by sulfasalazine and its components were evaluated in bacterial culture of two strains each of MAP and M. avium, using a radiometric (14CO2 BACTECH) detection system that quantifies mycobacterial growth as arbitrary ''growth index units'' (GI). Efficacy data are presented as ''percent decrease in cumulative GI'' (%2DcGI). Principal Findings. There are disparate responses to 5-ASA and sulfapyridine in the two subspecies. Against MAP, 5-ASA is inhibitory in a dose-dependent manner (MAP ATCC 19698 46%2DcGI at 64 mg/ml), whereas sulfapyridine has virtually no effect. In contrast, against M. avium ATCC 25291, 5-ASA has no effect, whereas sulfapyridine (88%2DcGI at 4 mg/ml) is as effective as methotrexate, our positive control (88%2DcGI at 4 mg/ml). Conclusions. 5-ASA inhibits MAP growth in culture. We posit that, unknowingly, the medical profession has been treating MAP infections since sulfasalazine's introduction in 1942. These observations may explain, in part, why MAP has not previously been identified as a human pathogen. We conclude that henceforth in clinical trials evaluating antiMAP agents in IBD, if considered ethical, the use of 5-ASA (as well as methotrexate and 6-MP) should be excluded from control groups. - INTRODUCTION In 1942 sulfasalazine (Salazopyrin) was introduced into clinical practice for ulcerative colitis. [1] Sulfasalazine has become, because of empirically observed clinical efficacy, the most common medicine used to treat Inflammatory Bowel Disease (IBD) [2] with greatest efficacy in ulcerative colitis. [35] Sulfasalazine is a conjugate of sulfapyridine and 5- aminosalicylic acid (5-ASA.) It is cleaved into its two component molecules following ingestion. [2] The sulfapyridine moiety [(2-(p aminobenzenesulphonamido) pyridine] is an acknowledged antibiotic. [6,7] However, prevailing medical dogma concludes that it is unlikely that (sulfasalazines) antibacterial activity accounts for its clinical efficacy. [2] In 1977, a two-week study on ulcerative proctitis, compared 5-ASA to sulfapyridine. Because of a decrease in inflammation in the 5-ASA group, the authors concluded, that in the therapy of ulcerative colitis the active moiety of sulfasalazine was 5-ASA. [8] As a consequence therapy with 5-ASA is called anti-inflammatory although the mechanism of action of 5-ASA in IBD is uncertain. [2] Although controversial, there are increasingly compelling data that all [9,10] or some of IBD may be caused by a single infectious agent Mycobacterium avium subspecies paratuberculosis (MAP.) [913] (& See [14] for review.) We have shown that two agents, methotrexate and 6-mercaptopurine (6-MP) [10], presumed to have immunomodulatory actions in IBD, [15,16] are potent antiMAP antibiotics. We suggested that the decreases in proinflammatory cytokines in IBD immunomodulatory therapy might simply reflect a normal, secondary, physiological response, as the instigating MAP infection was effectively treated. We concluded that henceforth methotrexate and 6-MP should be excluded from the placebo group when evaluating antiMAP therapies in IBD. [10] In this study we test the hypothesis that the anti-inflammatory action of 5-ASA and/or sulfapyridine could simply be due to one or both of sulfasalazines components acting as an antiMAP antibiotic(s.) If correct the anti-inflammatory effect would simply represent a normal, secondary, physiological response as the causative MAP in (...truncated)


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Robert J. Greenstein, Liya Su, Azra Shahidi, Sheldon T. Brown. On the Action of 5-Amino-Salicylic Acid and Sulfapyridine on M. avium including Subspecies paratuberculosis, PLOS ONE, 2007, Volume 2, Issue 6, DOI: 10.1371/journal.pone.0000516