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ACR 2017 | Daily Highlights
Methotrexate Is an Antibacterial Drug Metabolized By Human Gut Bacteria
Authors: Renuka R. Nayak1, Kye Stapleton-Gray2, Colleen O'Loughlin3, Michael Fischbach4 and Peter J. Turnbaugh5, 1Department of Medicine, Division of Rheumatology, Rosalind Russell / Ephraim P. Engleman Rheumatology Research Center, San Francisco, CA, 2Carnegie Mellon University, Pittsburgh, PA, 3University of California, San Francisco, San Francisco, CA, 4Department of Bioengineering and Therapeutic Sciences,, University of California, San Francisco, San Francisco, CA, 5Microbiology and Immunology, University of California, San Francisco, San Francisco, CA
Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology causing inflammation and irreversible damage in joints and other organs. Methotrexate (MTX) is first-line therapy used in the treatment of RA. However, not all patients respond to MTX — about 50-60% of patients require additional therapy. Because MTX is a folic acid analogue that may affect evolutionarily conserved pathways found in bacteria, we hypothesized that the gut microbiome is altered by MTX and that gut bacteria metabolize the drug. Since gut bacteria have been shown previously to metabolize many pharmacologic drugs, we also sought to investigate the impact of the microbiome on inter-individual variations in MTX response. Here, we focus on the response of bacteria to MTX and ask whether bacteria can metabolize MTX.
We tested the in vitro growth of 40 gut bacterial isolates in response to MTX. The minimal inhibitory concentration (MIC), or the concentration of MTX required to suppress bacterial growth > 90%, was identified for each isolate. We asked if these in vitro findings were recapitulated in vivo by colonizing germ-free mice with human gut bacteria and treating with daily oral MTX at high (50 mg/kg) and low (1 mg/kg) doses. Next, we asked if bacteria metabolized MTX by examining either pure bacterial cultures in vitro or human stool sample ex vivo. Samples were incubated with MTX and metabolism was measured using HPLC. In select cases, we also used UPLC-MS-MS to learn the identity of MTX metabolites.
MTX inhibited the growth of 33 of the 40 isolates examined. MICs 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 the antimicrobial effects of MTX (Wilcoxon rank sum, p=0.005). In vivo studies showed that high-dose MTX altered the humanized gut microbiome of mice compared to those that were saline-treated (ANOSIM, p=0.001). The relative abundance of Bacteroidetes decreased while Firmicutes increased, recapitulating what was seen in vitro. Low-dose MTX also produced changes to the microbiome, but this effect was subtler. We next asked whether gut bacteria metabolize MTX, and found that 8 possessed this ability in vitro. At least two species metabolized MTX into polyglutamated MTX, which is a novel finding that has not been described previously in the literature. In ex vivo studies, human fecal slurries incubated with MTX produced known as well as novel MTX metabolites.
We conclude that MTX is an antibacterial drug. Furthermore, we find that gut bacteria metabolize MTX. One metabolite found in our study was polyglutamated MTX, which prior studies have shown to be associated with patient response. Our ongoing and future studies will examine the in vivo implications of these findings in mice and examine whether bacterial metabolism of MTX is associated with clinical response in patients. Our findings support the hypothesis that a patient’s response to MTX 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.
R. R. Nayak, None; K. Stapleton-Gray, None; C. O'Loughlin, None; M. Fischbach, None; P. J. Turnbaugh, None.
“Antique” therapeutic agents hide many surprises. As a representative of those pertaining to methotrexate, this study combines in vitro inhibition of gut bacteria growth by methotrexate, the effect of methotrexate gut microbes of mice colonized with human gut bacteria and the metabolism of methotrexate by human gut bacteria and the identification of metabolites.
Bacteroidetes tended to be sensitive and Firmicutes tended to be resistant to the antimicrobial effects of MTX (Wilcoxon rank sum, p=0.005). In vivo high-dose MTX decreased bacteroidetes and increased firmicutes in the humanized gut microbiome of mice compared to saline-treated (ANOSIM, p=0.001). MTX was converted into polyglutamylated MTX, the form in which methotrexate is retained in cells.
These results suggest that methotrexate response may be linked to the gut microbiome and that methotrexate may modulate the composition of the microbiome and thereby mediate some therapeutic effect.
Other abstracts address the mechanism of methotrexate in reducing anti-drug antibodies in conjunction with BAFF (1463 and 1823) and its synergy with the even more antique colchicine (1445).
Prof. Dr. Paul Hasler