Oxidation of Mesalamine under Phenoloxidase- or Peroxidase-like Enzyme Catalysis

Abstract

Mesalamine, also called 5-ASA (5-aminosalicylic acid), is a largely used anti-inflammatory agent and is a main choice to treat Ulcerative Colitis. This report is aimed to investigate enzymatic processes involved in the oxidation of mesalamine to better understand some of its side-effects. Oxidation with oxygen (catalyzed by ceruloplasmin) or with hydrogen peroxide (catalyzed by peroxidase or hemoglobin) showed that these oxidases, despite their different mechanisms of oxidation, could recognize mesalamine as a substrate and trigger its oxidation to a corresponding quinone-imine. These enzymes were chosen because they may recognize hydroquinone (a p-diphenol) as substrate and oxidize it to p-benzoquinone and that mesalamine, as a p-aminophenol, presents some similarities with hydroquinone. The UV-Vis kinetics, FTIR and ¹H NMR supported the hypothesis of oxidizing mesalamine. Furthermore, mass spectrometry suggested the quinone-imine as reaction product. Without enzymes, the oxidation process was very slow (days and weeks), but it was markedly accelerated with the oxidases, particularly with peroxidase. Cyclic voltammetry supported the hypothesis of the oxidative process and allowed a ranking of susceptibility to oxidizing mesalamine in comparison with other oxidizable drug molecules with related structures. The susceptibility to oxidation was higher for mesalamine, in comparison with Tylenol (acetaminophen) and with aspirin (salicylic acid).

Keywords: 5-ASA (5-aminosalicylic acid); acetaminophen; ceruloplasmin; hemoglobin; hydrogen peroxide; hydroquinone; inflammatory bowel diseases; laccase; mesalamine; peroxidase.

Figure 1

UV–Vis (200–600 nm) spectra of MS recorded at various times of (a) MS (0.55 mM) alone at 24 h, 2, and 6 weeks and (b) MS (0.55 mM) in presence of H₂O₂ (0.05%) up to 24 h in PBS (50 mM). The spectra are representative of at least n = 3 scans for each experimental condition (all run at room temperature).

Figure 2

UV–Vis (200–600 nm) of MS (0.55 mM) and its oxidation product in PBS (50 mM) at room temperature in presence of (a) Laccase (0.030 U); (b) Ceruloplasmin (0.030 U) prepared to present the same number of enzyme units as laccase. Spectra are representative of at least n = 3 scans for each experimental condition.

Figure 3

The UV–Vis spectra (200–600 nm) of MS (0.55 mM in PBS 50 mM) recorded at various times at room temperature with H₂O₂ 0.05% in the presence of (a) Horseradish peroxidase (1.20 mU), or (b) Hemoglobin (1.20 mU), in 50 mM PBS. Spectra are representative of at least n = 3 scans for each experimental condition.

Figure 4

Anodic cyclic voltammetry scans of hydroquinone: 0.62 V (vs. Ag/AgCl) (a) of 4-aminophenol: 0.55 V (vs. Ag/AgCl) (b), of mesalamine: 0.58 V (vs. Ag/AgCl) (c), of acetaminophen: 0.75 V (vs. Ag/AgCl) (d), and of salicylic acid: 1.1 V (vs. Ag/AgCl) (e), in aqueous solution. Molecular models schematize the possible oxidation pathways.

Figure 5

Canonical chemical structures and appearance of (a) mesalamine powder before oxidation processing and (b) of lyophilized mesalamine after non catalytical, spontaneous oxidation in aqueous solution for 6 weeks at room temperature.

Figure 6

Fourier-transform infrared (FTIR) spectra of (a) mesalamine and of (b) oxidized mesalamine.

Figure 7

¹H NMR of (a) MS and of (b) oxidized MS form (b), (300 MHz, DMSO).

Figure 8

High resolution mass spectrometry (HRMS) corresponding to the oxidized mesalamine.

Figure 9

Suggested oxidation mechanism of MS in the human body. [O] stands for both hydrogen peroxide and oxygen species.