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[Preprint]. 2024 Dec 2:2024.11.26.625478.
doi: 10.1101/2024.11.26.625478.

Metabolomic Response to Non-Steroidal Anti-Inflammatory Drugs

Soumita Ghosh  1 , Nick Lahens  1 , Kayla Barekat  1 , Soon-Yew Tang  1 , Katherine N Theken  2 , Emanuela Ricciotti  3 , Arjun Sengupta  3 , Robin Joshi  1 , Frederic D Bushman  4 , Aalim Weljie  3 , Tilo Grosser  1   5 , Garret A FitzGerald  1   6
Affiliations

Metabolomic Response to Non-Steroidal Anti-Inflammatory Drugs

Soumita Ghosh et al. bioRxiv. .

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) are popular choices for the mitigation of pain and inflammation; however, they are accompanied by side effects in the gastrointestinal and cardiovascular systems. We compared the effects of naproxen, a traditional NSAID, and celecoxib, a cyclooxygenase -2 (Cox-2) inhibitor, in humans. Our findings showed a decrease in tryptophan and kynurenine levels in plasma of volunteers treated with naproxen. We further validated this result in mice. Additionally, we find that the depression of tryptophan was independent of both Cox-1 and Cox-2 inhibition, but rather was due to the displacement of bound tryptophan by naproxen. Supplementation of tryptophan in naproxen-treated mice rescued fecal blood loss and inflammatory gene expression driven by IL-1β in the heart.

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Figures

Fig.1:
Fig.1:. Metabolic profiles in volunteers in Naproxen alters wrt to Celecoxib and Placebo.
A) The scheme for the clinical trial experiment for the human volunteers. B) Cox-1 inhibition ex vivo (Adapted from ref 8) and C) Cox-2 inhibition ex vivo by treatments (Adapted from ref 8) in a mixed effect analysis. D) OPLS-DA scores plot of plasma metabolites from Placebo, Celecoxib and Naproxen at T0. E) OPLS-DA scores plot of plasma metabolites from Placebo, Celecoxib and Naproxen at T4. F) Pathway analysis of the metabolites in global metabolomics platform. G) Univariate analysis of Tryptophan in Placebo, Celecoxib and Naproxen using two-way ANOVA. H) Univariate analysis of Kynurenine in Placebo, Celecoxib, and Naproxen using two-way ANOVA. I) The metabolic pathway of tryptophan metabolism. J-L) ROC plot of tryptophan in Placebo vs. Naproxen, Celecoxib vs. Naproxen, and Placebo vs. Celecoxib. M-O) ROC plot of Kynurenine in Placebo vs. Naproxen, Celecoxib vs. Naproxen and Placebo vs. Celecoxib. O). Black, blue, and green dots refer to control celecoxib and naproxen treatments. * designates statistical significance, *p ≤ 0.05, *** p≤ 0.001, **** p≤ 0.001.
Fig.2:
Fig.2:. Plasma metabolic profiles of mice are altered by Naproxen diet.
A) The scheme of the mouse experiment is to investigate the metabolic effects of Naproxen vs. the control diet. B) Targeted analysis of plasma tryptophan levels C) Plasma kynurenine levels. D) Plasma kynurenic acid. E) Plasma xanthurenic acid. F) Plasma anthranilic acid in mice by LC/MS/MS in a two-way ANOVA. G) ROC plot of Tryptophan in Control vs. Naproxen. H) ROC plot of Kynurenine in Control vs. Naproxen. I) Plasma tryptophan levels in Control mice and in Cox-DKO in control chow and Naproxen diet. J) Comparison of change in the tryptophan levels in control mice and Cox-DKO mice in Naproxen diet with respect to Control chow in a two-way ANOVA. K) Spearman correlation plot of human urinary prostaglandin metabolites and the plasma metabolite levels. L) Plasma Tryptophan levels of Control mice in Naproxen and R-2-Phenylpropionic acid diet (PPA) and “Cox-DKO” mice in PPA and control chow diet using One-Way-ANOVA. M) Plasma Kynurenine levels in Control mice in Naproxen and PPA diet and “Cox-DKO” mice in PPA and control chow diet using One-Way-ANOVA. The green, blue, purple and black circles denote the control mice in the Naproxen diet, control mice in the PPA diet, “Cox-DKO” mice in the PPA diet, and “Cox-DKO” mice in the control chow diet, respectively. All the data are expressed in mean ± SD * Designates statistical significance, **** p≤ 0.001.
Fig.3:
Fig.3:. Naproxen displaces tryptophan from albumin binding.
A) % bound tryptophan to albumin in plasma samples of volunteers in three different treatments: Placebo, Celecoxib and Naproxen analyzed in One-Way-ANOVA. B) Free tryptophan levels in Placebo, Celecoxib, and Naproxen in the buffer chamber of the RED plate analyzed by One-Way-ANOVA. C) Spearman correlation between plasma Naproxen and Tryptophan in the human volunteers. D) %Bound tryptophan concentration in mouse plasma treated with increasing doses of Naproxen. The data is expressed as mean ± sd. * Designates statistical significance, ** p≤ 0.01.
Fig.4:
Fig.4:. Naproxen alters microbiome and microbiome derived metabolites.
A) Proportions of Coprococcus and Ruminococcaceae in human stool after Naproxen treatment. B) Urinary Indole 3 Acetic acid levels in Naproxen and Placebo treatment in human volunteers analyzed by paired Wilcox test. C) Urinary Indole 3 Acetic acid levels in Naproxen and Placebo treatment in mice by unpaired Welch test. The black and the green circles represent Placebo and Naproxen, respectively. The data is expressed as mean ± SD. * Designates statistical significance, * p≤ 0.05.
Fig.5:
Fig.5:. Tryptophan Supplementation experiment in mice during Naproxen treatment ameliorate inflammation.
A) Scheme of Tryptophan supplementation experiment in mice. B) Plasma Tryptophan levels in three different groups (Control, Naproxen, Naproxen+ Tryptophan). C) Heatmap showing “rescue genes” in the heart after the supplementation of tryptophan with Naproxen. D) Canonical Pathway analysis for the rescue genes in Ingenuity Pathway Analysis (IPA). E) Upstream regulators of the rescue genes in IPA. F) IL-1β levels in the mouse heart after tryptophan supplementation in Naproxen treatment analyzed by One-Way-ANOVA. G) NLRP3 levels in mouse heart following tryptophan supplementation in Naproxen treatment One-Way-ANOVA. H) Target genes of IL-1β that are rescued by tryptophan in Naproxen treatment. Pathway analysis of the target genes. I)Venn diagram showing the target genes of IL-1β that are part of possible cardiac phenotypes in mice in Naproxen treatment in IPA analysis. Black circles, green triangles, and blue diamonds refer to control, naproxen, and naproxen+tryptophan groups. The data is expressed as mean ± sd. * designates statistical significance, * p≤ 0.05, ** p≤ 0.01 respectively.
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