3. Photoprotective effect of NSAIDs toward amino acids and peptides oxidation

In order to evaluate an eventual antioxidant/protective effect of NSAIDs towards biologically relevant substrates, amino acids (AA) and peptides may be employed as typical oxidizable targets in a proteinaceous medium.

Tryptophan (Trp) and tyrosine (Tyr) are AAs that can be affected by photo-damages through photodynamic activity [23, 24]. They are known quenchers of <sup>3</sup> Rf� with <sup>3</sup> k<sup>q</sup> of 2:5 · 109 M<sup>−</sup><sup>1</sup> s−1 and 1:0 · 109 M<sup>−</sup><sup>1</sup> s<sup>−</sup>1, respectively [13]. In order to evaluate the eventual protective effect of NSAIDs against photooxidation, Rf-photosensitized experiments were performed using each of these AA and the oxicam PIR. For comparative purposes, the trials were also performed replacing Rf by RB which ensures that the prevalent oxidation process is due to O2ð 1 ΔgÞ. As a measure of the global photooxidative process, the rates of oxygen consumption were determined in each trial monitoring up to 10% conversion of the substrate under study.

PIR and Trp, as isolated substrates, are efficient O2ð 1 ΔgÞ chemical scavengers. Their k<sup>r</sup> values are virtually identical, while the kr=k<sup>t</sup> relationship presents also very similar values. For the interaction Trp-O2ð 1 <sup>Δ</sup>g<sup>Þ</sup> it has been reported the rate constant values <sup>k</sup><sup>t</sup> <sup>¼</sup> <sup>7</sup>:<sup>2</sup> · 107 M<sup>−</sup><sup>1</sup> s<sup>−</sup>1. and <sup>k</sup><sup>t</sup> <sup>¼</sup> <sup>4</sup>:<sup>7</sup> · 107 M<sup>−</sup><sup>1</sup> s<sup>−</sup>1.[13, 25]. Using RB as the sensitizer, the rates of oxygen uptake for the mixture PIR + Trp were approximately equal to the rates of PIR and Trp individually considered, which may be due to the fact that they react through a pure O2ð 1 ΔgÞ-mediated process (Figure 7). Employing Rf as the photosensitizer, the mixture PIR + Trp presented a rate of oxygen consumption significantly lower than the addition of the respective rates for each substrate. A possible explanation is that both compounds present a high <sup>3</sup> kqvalue, so the simultaneous action of them may decrease the O2ð 1 ΔgÞ concentration leading to the lower rate observed with the presence of the mixture.

In neutral pH, Tyr is present in a very low reactive form. The interaction Tyr with O2ð 1 ΔgÞ mostly operates by physical deactivation of the ROS with a reported rate constant value

Figure 7. Bar diagrams for the relative rates of oxygen consumption upon RB (A560 = 0.4) photosensitization in pH 7 buffered aqueous solution by: 0.5 mM PIR; 0.5 mM Trp; 0.5 mM PIR plus 0.5 mM Trp. And upon Rf (A445 = 0.5) photosensitization in pH 7 buffered aqueous solution by: 0.5 mM PIR; 0.5 mM Trp; 0.5 mM PIR plus 0.5 mM Trp. Reprinted from Ferrari et al. [11], © (2015), with permission from Elsevier B.V.

<sup>k</sup><sup>t</sup> <sup>¼</sup> <sup>1</sup>:<sup>5</sup> · 107 M<sup>−</sup><sup>1</sup> s<sup>−</sup><sup>1</sup> [22, 26]. The very low kr=k<sup>t</sup> may be due to the clear decrease in the rate of oxygen uptake by the mixture PIR + Tyr as compared to the one for the isolated PIR with RB as the photosensitizer. (Figure 8) With Rf as a sensitizer, the corresponding rates for PIR alone and the one for the mixture are practically equal.

A relevant result was that PIR in the presence of Rf showed an interesting degree of protection against Trp or Tyr oxidation by the in situ-photogenerated ROS. This fact has been revealed by

Figure 8. Bar diagrams for the relative rates of oxygen consumption upon RB (A560 = 0.4) photosensitization in pH 7 buffered aqueous solution by: 0.5 mM PIR; 0.5 mM Tyr; 0.5 mM PIR plus 0.5 mM Tyr. And upon Rf (A445 = 0.5) photosensitization in pH 7 buffered aqueous solution by: 0.5 mM PIR; 0.5 mM Tyr; 0.5 mM PIR plus 0.5 mM Tyr. Reprinted from Ferrari et al. [11], © (2015), with permission from Elsevier B.V.

the lower rates of oxygen consumption of the mixture oxicam-AA as compared to the ones for the individual substrates.

The dipeptide Trp-Tyr in a 0.5 mM aqueous solution was employed as a biologically relevant model compound, with RB or Rf as photosensitizers and IMT or DFN as potential photoprotective substrates. The O2ð 1 ΔgÞ- mediated process of Trp-Tyr could be studied using RB alone. Its rate constant value <sup>k</sup><sup>r</sup> <sup>¼</sup> <sup>5</sup>:<sup>9</sup> · 107 M<sup>−</sup><sup>1</sup> s<sup>−</sup><sup>1</sup> had already been reported [24]. The comparison of the relative rates of oxygen consumption in the presence and in the absence of 0.5 mM IMT showed that the value for the mixture Trp-Tyr + IMT was close to the simple addition of the respective individual rates (Figure 9).

<sup>k</sup><sup>t</sup> <sup>¼</sup> <sup>1</sup>:<sup>5</sup> · 107

M<sup>−</sup><sup>1</sup>

254 Pain Relief - From Analgesics to Alternative Therapies

and the one for the mixture are practically equal.

Reprinted from Ferrari et al. [11], © (2015), with permission from Elsevier B.V.

s<sup>−</sup><sup>1</sup> [22, 26]. The very low kr=k<sup>t</sup> may be due to the clear decrease in the rate of

oxygen uptake by the mixture PIR + Tyr as compared to the one for the isolated PIR with RB as the photosensitizer. (Figure 8) With Rf as a sensitizer, the corresponding rates for PIR alone

Figure 7. Bar diagrams for the relative rates of oxygen consumption upon RB (A560 = 0.4) photosensitization in pH 7 buffered aqueous solution by: 0.5 mM PIR; 0.5 mM Trp; 0.5 mM PIR plus 0.5 mM Trp. And upon Rf (A445 = 0.5) photosensitization in pH 7 buffered aqueous solution by: 0.5 mM PIR; 0.5 mM Trp; 0.5 mM PIR plus 0.5 mM Trp.

A relevant result was that PIR in the presence of Rf showed an interesting degree of protection against Trp or Tyr oxidation by the in situ-photogenerated ROS. This fact has been revealed by

Figure 8. Bar diagrams for the relative rates of oxygen consumption upon RB (A560 = 0.4) photosensitization in pH 7 buffered aqueous solution by: 0.5 mM PIR; 0.5 mM Tyr; 0.5 mM PIR plus 0.5 mM Tyr. And upon Rf (A445 = 0.5) photosensitization in pH 7 buffered aqueous solution by: 0.5 mM PIR; 0.5 mM Tyr; 0.5 mM PIR plus 0.5 mM Tyr.

Reprinted from Ferrari et al. [11], © (2015), with permission from Elsevier B.V.

Figure 9. Bar diagram for the relative rates of oxygen consumption upon RB (A560 = 0.4) photosensitization in pH 7 buffered aqueous solution of: 0.5 mM Trp-Tyr; 0.5 mM IMT; 0.5 mM DFN; 0.5 mM Trp-Tyr plus 0.5 IMT; 0.5 mM Trp-Tyr plus 0.5 DFN. Reprinted from Purpora et al. [10], © (2013), with permission from The American Society of Photobiology, a Wiley Company, John Wiley & Sons, Inc.

Figure 10. Bar diagram for the relative rates of oxygen consumption upon Rf (A445 = 0.5) photosensitization in pH 7 buffered aqueous solution of: 0.5 mM Trp-Tyr; 0.5 mM IMT; 0.5 mM DFN; 0.5 mM Trp-Tyr plus 0.5 IMT; 0.5 mM Trp-Tyr plus 0.5 DFN. Reprinted from Purpora et al. [10], © (2013), with permission from The American Society of Photobiology, a Wiley Company, John Wiley & Sons, Inc.

Meanwhile, the rate for the mixture Trp-Tyr + DFN decreased more than 50% of the one for the isolated dipeptide. Upon Rf-sensitization, similar results were obtained for DFN and IMT (Figure 10). This fact suggested that the photoxidation occurs mainly by reaction with the Rf-photogenerated O2ð 1 ΔgÞ.
