On the action of methotrexate and 6-Mercaptopurine on M. avium subspecies paratuberculosis

Abstract

Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in proinflammatory cytokines. This has been presumed to indicate the “anti-inflammatory” mechanism of action of methotrexate and 6-MP. 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 hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP. The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in culture of two strains each of MAP (ATCC 19698 [bovine] & Dominic [human]) and M. avium (ATCC 28291 & 101) 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.) The positive control was clarithromycin (a macrolide with excellent coverage against MAP) the negative was the β lactam, ampicillin, which has no clinical role in the therapy of human mycobacterial diseases. With ATCC 19698 the positive control antibiotic, clarithromycin exhibits ≥ 86 %-ΔcGI at the lowest concentration evaluated (0.5 μg/ml). The negative control antibiotic ampicillin has a trivial (21%-ΔcGI) at 32 μg/ml. In contrast, 6-MP has an initial ≥ 43%-ΔcGI starting at 1μg/ml that is ≥ 84 % -ΔcGI at 4 μg/ml. Methotrexate has 40%-ΔcGI inhibition at 2 mg/ml and ≥75%-ΔcGI at 4μg/ml. With Dominic, methotrexate has ≥88%-ΔcGI by 4μg. In the other three isolates, although far more effective than ampicillin, 6-MP is consistently less effective than methotrexate in %-ΔcGI. We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different %-ΔcGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in proinflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. Antibiotic resistance, because of lack of appropriate poly antimycobacterial therapy may account for variation in response to antiMAP agents in IBD and be associated with clinical exacerbations such as toxic megacolon. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control or “placebo” groups.