Photoinduced Oxidation of Lipid Membranes in the Presence of the Nonsteroidal Anti-Inflammatory Drug Ketoprofen

Abstract

The damage of cell membranes induced by photosensitive drugs has attracted the significant attention of researchers in various fields of medicine. Ketoprofen (KP) is known to be the most photosensitive among the nonsteroidal anti-inflammatory drugs. The phototoxic side effects of KP and other non-steroidal anti-inflammatory drugs are associated with the action of free radicals, but there is insufficient information about the nature of these radicals. In the present study, free radicals formed upon KP irradiation within lipid membranes were studied using nuclear magnetic resonance (NMR) and chemically induced dynamic nuclear polarization (CIDNP) methods, as well as a molecular dynamics simulation. Our study confirmed the effective penetration of KP into the lipid bilayer and showed a significant effect of the nature of the medium on the photolysis mechanism. While, in a homogeneous solution, the main channel of KP photolysis is free radical-mediated monomolecular decomposition with formation of radical pairs of benzyl and CO₂H^● radicals, then, in the lipid membrane, the reaction route shifts towards the bimolecular reaction of KP photoreduction. In addition, the effect of the presence an electron donor (the amino acid tryptophan) on lipid oxidation has been studied. It was found that photoreaction of KP with tryptophan proceeds more efficiently than with lipid molecules.

Keywords: CIDNP; decarboxylation; free radicals; ketoprofen; lipid membranes; molecular dynamics; photosensitivity; phototoxicity; radical polymerization.

Figure 1

The structure of ketoprofen (KP, 2-(3-benzoylphenyl) propanoic acid, top), and tryptophan (bottom).

Figure 2

Structures of lipids DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DHPC (1,2-diheptanoyl-sn-glycero-3-phosphocholine).

Figure 3

1D NOESY spectra (blue and red lines) and ¹H NMR spectra (gray lines) of (a) 2 mM KP; (b) 2 mM KP + 4 mM Trp, KP protons; (c) 2 mM KP + 4 mM Trp, Trp protons in DHPC/DMPC bicelles at different pH values. Mixing time was 500 ms. Selective excitation of KP protons at 7.8 ppm (Figure 1, blue circles) and Trp protons at 7.2 ppm (Figure 1, red circle) was performed.

Figure 3

1D NOESY spectra (blue and red lines) and ¹H NMR spectra (gray lines) of (a) 2 mM KP; (b) 2 mM KP + 4 mM Trp, KP protons; (c) 2 mM KP + 4 mM Trp, Trp protons in DHPC/DMPC bicelles at different pH values. Mixing time was 500 ms. Selective excitation of KP protons at 7.8 ppm (Figure 1, blue circles) and Trp protons at 7.2 ppm (Figure 1, red circle) was performed.

Figure 4

Density profiles of the selected C (a) and O (b) atoms of ketoprofen in protonated and deprotonated states and atom selections (c). Vertical lines correspond to the centers of density profiles of DMPC N atoms.

Figure 5

Density profiles of the selected C and O atoms of ketoprofen (a) and tryptophan (b) in the protonated and deprotonated ketoprofen state. Vertical lines correspond to the centers of density profiles of DMPC N atoms.

Figure 6

Radial distribution function g(r) of the hydrogens of KP (blue circles) and CH₂-groups of DMPC (green circle) (a) in the absence of tryptophan; (b) in the presence of tryptophan.

Figure 7

¹H NMR and CIDNP spectra of KP in water solution and DHPC/DMPC bicelles at different pH of the media.

Scheme 1

Suggested reaction channels of KP photodegradation.

Figure 8

Absorption spectra of KP (0.1 mM) and Trp (0.2 mM) in bicelles at pH 7.4. Extinction coefficients are shown for wavelength of laser light, 308 nm.

Figure 9

CIDNP spectra detected during photolysis of a mixture of KP (2 mM) + tryptophan (4 mM) in phosphate buffer and DMPC/DHPC bicelles at various pH (aerated solution).

Figure 10

NMR spectra (fragments) of KP solutions in aerated PBS (pH 7.4) before (ini) and after photolysis (irr, 64 laser pulses). Concentrations of KP = 2 mM, Trp = 4 mM. The reaction products are marked with red rectangles.