On the action of 5-amino-salicylic acid and sulfapyridine on M. avium including subspecies paratuberculosis

Abstract

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 ((14)CO(2) BACTEC(R)) detection system that quantifies mycobacterial growth as arbitrary "growth index units" (GI). Efficacy data are presented as "percent decrease in cumulative GI" (%-DeltacGI).

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%-DeltacGI at 64 microg/ml), whereas sulfapyridine has virtually no effect. In contrast, against M. avium ATCC 25291, 5-ASA has no effect, whereas sulfapyridine (88%-DeltacGI at 4 microg/ml) is as effective as methotrexate, our positive control (88%-DeltacGI at 4 microg/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.

Figure 1. Cumulative GI data when 2.7×10⁴ CFU/vial of MAP ATCC 19698 was inoculated into each Bactec vial.

Each drug dilution was studied in duplicate. Error bars are SD. The positive control is methotrexate, with maximal inhibition by 8 mg/ml. Neither the negative control sulfasalazine, nor sulfapyridine, have any inhibition. Both and 5-ASA alone and in combination with sulfapyridine have dose dependent inhibition.

Figure 2. A composite graph of the two MAP and two M. avium bacterial strains studied.

Each dose was studied in singlicate. For MAP ATCC 19698, 6×10⁴ CFU's were inoculated/vial. Sulfasalazine is the negative control for both MAP and M. avium strains. In all strains there is dose dependent increase in cGI. There is no inhibition by sulfapyridine alone with either MAP strain. 5-ASA has dose dependent inhibition on both MAP strains. Note the subtle synergy up to 16 µg/ml for both MAP strains for the 5-ASA+sulfapyridine group compared to 5-ASA alone. The positive control in the Dominic study was clarithromycin which, because it was diluted in methanol, is off scale. Accordingly, the clarithromycin data are not presented.