3rd meeting of young researchers at UP 1 - IJUP - Universidade do ...

Scavenging activities of sulfasalazine and its metabolites,
sulfapyridine and 5-aminosalicylic acid, against reactive nitrogen
species
D. Couto, D. Ribeiro, D. Costa, A. Gomes, M. Freitas, E. Fernandes and J. L. F. C. Lima
1 Department of Physical-Chemistry, Faculty of Pharmacy, University of Porto, Portugal.
Sulfasalazine is a prodrug consisting on a molecule of 5-aminosalicylic acid (5-ASA)
and sulfapyridine (SP), linked by an azo bond, which has been shown to be useful in the
therapy of ulcerative colitis, as well as rheumatic diseases, such as rheumatoid arthritis
and ankylosing spondylitis. Nearly all the prodrug taken orally reaches the colon intact, where
it is converted in its two metabolites by colonic bacterial enzymes. SP is an antibacterial agent
and acts as a carrier to deliver 5-ASA to the colon. 5-ASA is the main active component of
sulfasalazine and acts as an anti-inflammatory agent. The mechanism of action of
sulfasalazine is not yet clearly understood, although the scavenging of pro-oxidant reactive
species may have an important role by preventing the oxidative stress status characteristic
of inflammatory processes. Thus, the present study was undertaken to evaluate the scavenging
activity for reactive nitrogen species (RNS), namely nitric oxide ( • NO) and peroxynitrite
(ONOO - ) by sulfasalazine and its metabolites. The ONOO - scavenging activity was measured
by monitoring the effect of the tested compounds on ONOO - -induced oxidation of nonfluorescent
dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123. In a parallel set of
experiments, the assays were performed in the presence of 25 mM NaHCO3 in order to
simulate the physiological CO2 concentrations. The • NO scavenging activity was measured by
monitoring the effect of the tested compounds on • NO-induced oxidation of non-fluorescent
4,5-diaminofluorescein (DAF-2) to fluorescent triazolofluorescein (DAF-2T).
The results showed that 5-ASA is a strong scavenger of • NO and ONOO - (with and without
bicarbonate), having achieved IC50s in the low micromolar range (1.06 ± 0.21, 2.88 ± 0.43, and
0.68 ± 0.09 µM, respectively). Sulfasalazine was also able to scavenge these RNS, although
with a much lower potency than 5-ASA (IC50s of 536 ± 89, 148 ± 16, and 90.6 ± 22.1 µM,
respectively). Sulfapyridine was unable to scavenge • NO in the tested concentrations but was
shown to scavenge ONOO - , with a higher strength when the assay was performed in the
presence of 25 mM of bicarbonate (IC50s of 257 ± 41, with bicarbonate and 1676 ± 35 µM,
without bicarbonate).
In conclusion, the RNS-scavenging effects of sulfasalazine and its metabolites shown in this
study may contribute to the anti-inflammatory effects mediated by sulfasalazine through the
prevention of the oxidative/nitrative/nitrosative damages caused by these species.
Acknowledgements:
Marisa Freitas acknowledges Fundação para a Ciência e Tecnologia (FCT) and Fundo Social
Europeu (FSE) her PhD grant (SFRH/BD/28502/2006).
Ana Gomes acknowledges FCT and FSE her post-doctoral grant (SFRH/BPD/63179/2009).
336 3 rd meeting of young researchers at UP