Background/Purpose: Methotrexate (MTX) is a first-line treatment drug used in rheumatoid arthritis (RA) patients. However, only 30-40% of patients tolerate the drug and achieve adequate therapeutic effects¹. Risk factors such as smoking, age, and disease severity are associated with MTX non-responsiveness in patients². Yet, these risk factors are limited in their ability to predict MTX non-responsiveness, and many of them are not modifiable. One hypothesis, pursued by our lab, is that the gut microbiome metabolizes MTX and interferes with its efficacy. This is significant because the gut microbiome can be both predictive and modifiable, and therefore can be leveraged to improve MTX efficacy and advance personalized medicine for patients with RA. In support of this hypothesis, we have previously shown that specific bacterial taxa and the abundance of bacterial genes are predictive of future MTX outcomes in RA patients³.Yet, it remains to be seen whether the gut microbial community can directly affect MTX pharmacology and which microbes are responsible for MTX metabolism. Here, we examined the causal impact of the microbiome on MTX pharmacokinetics and surveyed the spectrum of RA-associated gut microbes that metabolize MTX.

Methods: To test the contribution of microbial metabolism of in MTX pharmacology in vivo, we orally dosed germ-free (GF) mice and specific-pathogen free mice (SPF) (N=6 per colonization state) with MTX 50 mg/kg. Plasma was collected at 0, 15, 30, 60, 120, 240, and 480 minutes; the concentration of MTX and its metabolites were quantified by HPLC-MS-MS (Sciex 6500 QTRAP). To survey the spectrum of RA-associated gut microbes that metabolize MTX, we collected stool samples from treatment-naïve RA patients (N= 5) and isolated individual strains on rich media. Each isolate was incubated with MTX 50 ug/mL or vehicle control (i.e., DMSO) for 72 hours and bacterial growth was measured by spectrometry (i.e., OD600). MTX metabolism was assessed by HPLC by: (1) quantifying a decrease in MTX levels and (2) detection of novel peaks.

Results: Peak plasma concentrations of MTX were significantly higher in GF mice as compared to SPF mice. In contrast, plasma concentrations of MTX metabolites were negligible in GF mice, whereas SPF mice showed increasing levels of MTX metabolites over 4 hours. To determine which RA-associated microbes might be responsible for MTX metabolism, we harvested 120 bacterial isolates from 5 RA patients. Of the profiled 120 isolates, 30.83% of isolates (37/120) metabolized MTX.

Conclusion: The presence of a microbiome in mice results metabolism of orally-administered MTX in vivo leading to altered pharmacokinetics. Furthermore, a significant percentage of isolates harvested from RA patient microbial communities are capable of metabolizing MTX in vitro. These findings support the overarching hypothesis that the gut microbiome contributes to MTX efficacy in RA patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microbial-metabolism-of-methotrexate-contributes-to-its-pharmacokinetics-in-vivo/