The mechanism of action of the opthalmic preservative Purite™ and a comparison to other preservatives
The use of preservatives in ophthalmic pharmaceuticals is mandatory to inhibit the growth of micro-organisms whilst on the shelf and during use. Traditional preservatives used in this application, over the long-term, are reported to cause serious patient reactions limiting their use. The novel oxy-chloro compound Purite™; is a newer generation ocular preservative that overcomes some of the patient related problems. Although the major constituent of Purite™; is sodium chlorite, very little is known about the exact mechanism for the activity of chlorite solutions as preservatives and the reasons for the differential toxicity between micro-organisms and ocular tissue. Electrospray mass spectrometry and NMR were used to investigate the types of reactions Purite™; was capable of undergoing in the presence of biological molecules in vitro. When added to preparations of phospholipids, at concentrations shown to be toxic to microbial cells, Purite™;was found to be a very mild oxidant of phospholipid vesicles generating very low levels of lipid hydroperoxides and no lipid chlorohydrins. However, compared to other oxidants, Purite™; was found to rapidly oxidise the reduced form of glutathione in vitro. Experiments on mammalian and microbial cells showed that the resistance could not be correlated to native membrane phospholipid profiles. Also, there was no membrane phospholipid oxidation observed in organisms treated with toxic doses of Purite™;. The differential resistance of test organisms to Purite™; related to their native levels of the antioxidant glutathione. Further research showed close correlation between cellular resistance and the ability of cells to maintain their cytosolic glutathione with increasing Purite™; doses. Anti-oxidant inhibitors were used to deplete specific resistance pathways in each test organism. The loss of resistance to Purite™; in specific organisms was found to correlate well not only to glutathione depletion but also catalase depletion. This study shows that Purite™; preferentially oxidises cellular glutathione to induce oxidative stress and resistance depends upon the anti-oxidant repertoire of each organism.