Background/Purpose:  Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology that causes inflammation and irreversible damage in joints and other organs. Methotrexate (MTX) is the first-line therapy used in the treatment of RA. However, not all patients respond to MTX, and a tool to predict MTX responders would help clinicians identify the 50-60% of patients that require additional therapy. Because MTX affects evolutionarily conserved pathways that are present in both humans and bacteria, it is plausible that the microbiome may be affected by MTX or directly metabolizes the drug, which is a folic acid analogue. Additionally, gut bacteria have been shown in prior studies to metabolize many pharmacologic drugs. We hypothesize that the microbiome contributes to inter-individual variations in clinical outcome. Here, we focus on the response of bacteria to MTX and ask whether bacteria can metabolize MTX.

Methods: First, we tested the in vitro growth of a panel of 25 gut bacterial isolates in response to MTX. The minimal inhibitory concentration (MIC), or the concentration of MTX required to completely suppress bacterial growth, was identified for each isolate. Second, we used HPLC to ask whether gut bacteria metabolize methotrexate. In select cases, we also used UPLC-MS-MS to learn the identity of MTX metabolites.

Results:  Methotrexate inhibited the growth of 19 of the 25 isolates examined. Minimal inhibitory concentrations ranged from 2 ug/ml to >900 ug/ml in vitro. At the Phylum level, Bacteroidetes tended to be sensitive and Firmicutes tended to be resistant to antimicrobial effects of MTX (Fisher’s exact test, p=0.03). We next asked whether these species metabolize MTX, and found that 5 possessed this ability. At least two species metabolized MTX into polyglutamated methotrexate, which is a novel finding that has not been described previously in the literature. Interestingly, there was no ­­­­­association between bacteria that were resistant to MTX and those that metabolized it.

Conclusion: These findings suggest that methotrexate is an antibacterial in addition to being a chemotherapeutic agent and an immunosuppressant. Additionally, gut bacteria can metabolize MTX, perhaps even before this drug enters a patient’s bloodstream. The metabolite found in our study was polyglutamated MTX, which prior studies have shown to be associated with patient response. Our ongoing studies will examine the in vivo implications of these findings, but this supports the hypothesis that a patient’s response to methotrexate may be influenced by their gut microbiome. Thus, the microbiome may be an important factor in predicting patient response to MTX and perhaps other rheumatologic medications as well.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/methotrexate-is-an-antibacterial-drug-metabolized-by-human-gut-bacteria/