Free Radical Content of Drugs Used in Ophthalmology

To evaluate the presence of free radicals in various drugs used in ophthalmology. Twenty five samples of drugs that are used by intracameral or intravitreal route, eleven samples of drugs used as fortified eye drop, thirty samples of topical commercial preparations that are used as long term, and eight samples of autologous serum were analyzed for total free radical presence using a kit for total oxidant status. The highest free radical concentration was in Moxifloxacin (Vigamox) in intracameral group, clindamycin in intravitreal group, brinzolamide in antiglaucoma group. Free radicals are present in drugs used in ophthalmology. The surgeon should consider the risk of oxidative damage in the presence of diseased eye.


INTRODUCTION
The most important targets of free radicals are unit membranes, e.g.plasma, mitochondrial, rough endoplasmic reticulum, and nuclear membranes.Free radicals initiate toxic reactions at the plasma membrane and lead to cellular edema or necrosis (1)(2)(3).The unsaturated bonds of cholesterol and fatty acids in the membranes can readily react with free radicals and undergo peroxidation.This process can become autocatalytic after initiation and will yield lipid peroxides (2,3).Lipid peroxides have a sufficient lifetime, which means that they can migrate and damage other cellular components, including DNA, apart from the membranes (4).In a recent study, free radicals were detected in significant amount in agents used for intracameral injections after cataract surgery (5).Numbers of off label agents that are used in intravitreal and intracameral route are increasing continuously.Free radicals in these agents may cause undetectable damage to corneal endothelium, trabecular meshwork, and retina.
Ophthalmic eye drops contain preservative to prevent bacterial and fungal growth secondary to contamination from the patient.Common preservatives are benzalko-nium chloride, chlorobutanol, parahydroxbenzoates, and polysorbate.Drugs itself and preservative may act as free radicals and cause peroxidation of polyunsaturated lipids in cell membranes (6).Chronic use of ophthalmic eye drops such as antiglaucoma medications causes dry eye and failed trabeculectomy bleb (7).
Many off labeled fortified eye drops that are prepared from intravenous formulation are used for treatment of fungal and bacterial keratitis and endophthalmitis in which there is a diseased and impending perforated cornea.Fortified eye drops are used in very high concentration and in a frequent regimen such as hourly or half an hour.Frequent and long time use of fortified eye drops containing high level of free radicals may induce peroxidation of corneal epithelium and keratocyte cell membrane in the presence of diseased cornea with altered epithelium and Bowmans membrane.Thus, these drugs may induce corneal melting and perforation while eradicating the infecting organisms (8).Knowing the amount of free radicals in fortified eye drops will guide ophthalmologist to choose the least free radical containing agents.
In this study, we aimed to measure the amount of free radicals in agents used in intracameral and intravitreal injections, in fortified eye drops, in autologous serums, and in long term used topical commercial preparations.

MATERIALS AND METHODS
In our study, totally 74 samples of agents were analyzed for total oxidant and antioxidant capacity.0.2 mL of each samples were used for the analysis.

Autologous serum
Autologous serum from eight patients from different sex and age without any known disease were also analyzed (Table 4).

Total antioxidant status (TAS)
TAS levels were measured using commercially available kits (Rel Assay).The novel automated method is based on the bleaching of characteristic color of a more stable ABTS (2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]) radical cation by antioxidants.The commercial kit work colorimetric method is based on 660 nm absorbance.The results were expressed as mmol Trolox equivalent/L.

Total oxidant status (TOS)
TOS levels were measured using commercially available kits (Rel Assay).In this method, oxidants present in the sample oxidized the ferrous ion-o-dianisidine complex to ferric ion.The oxidation reaction was enhanced by glycerol molecules abundantly present in the reaction medium.The ferric ion produced a colored complex with xylenol orange in an acidic medium.The color intensity was related to the total amount of oxidant molecules present in the sample.The assay was calibrated with hydrogen peroxide and the results were expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmol H 2 O 2 equivalent/L).

DISCUSSION
The numbers of off-label drug used in intracameral and intravitreal injections are continuously increasing.
Intracameral agents include anaesthetic agents for iris anesthesia, antibiotics such as cefuroxime, moxifloxacin and vancomycin for endophthalmitis prophylaxis after cataract surgery, adrenalin for pupillary dilatation, carbachol for miosis, trypan blue for capsule staining, triamcinolone for identifying vitreous strands.Intravitreal agents that used in treatments are vancomycin and ceftazidim for bacterial endophthalmitis, voriconazole and amphotericin B for fungal endophthalmitis, clindamycin for toxoplasma retinitis, dexamethazone and triamcinolone for retinal and vitreous inflammation.Most of these drugs are prepared in the operation theatre and accuracy of concentration may not be obtained.
Corneal endothelium is very sensitive to any form of toxic exposure.Recently, studies have been published with regard to the oxidative damage of intracameral agents.Borazan et al. (9) documented the induction of apoptosis by intracameral preservative free lidocaine hydrochloride 2%, ropivacaine1%, and levobupivacaine 0.75% in rabbit corneal endothelium.Similarly, Demir NM et al. (10) showed immediate toxicity of levobupivacaine 0.5% on rabbit corneal endothelium and oxidative damage of lidocaine hydrochloride 2% on corneal and lens tissue.In another similar study, Ozlem YT et al (11) reported that cefuroxime made an oxidative damage on corneal tissue while vancomycin had no harmful effect.Intracameral injection of these antibiotics after removal of OVD which acts as free radical scavengers cause the corneal endothelium open to oxidative damage.Additionally, free radicals in intracameral agents may cause toxic anterior syndrome (12).In this study, we found that moxifloxacin from eye drop had the highest free radical content (Table 1).TAS levels were similar in this group.Non-toxic doses of many agents that are used in intravitreal injections were determined in various studies.(13).Clearance of an intravitreal drug takes longer time than intracameral drugs.Superior complexity of retinal anatomy is another factor that puts the retina to undetermined toxicities.In this study, among the intravitreal agents, clindamycin had the highest free radical content.TAS levels were similar in this group (Table 1).
Fortified eye drops contain high concentration of antibiotics or antifungal agents.It was shown that fortified antibiotic eyedrops had negative influence on corneal epithelial wound healing (14).Free radicals may have more damage on diseased corneal cells in the presence of keratitis.Additionally, poradic toxicity case reports secondary to fortified eye drops usage were reported (8,15,16) Fortified eye drop contains a higher amount of free radicals.Information about free radical content will help physician to choose the least free radical containing agents.In our study, we found that clindamycin had the highest free radical concentration (Table 2).TAS levels were similar in this group.The normal ocular tear film contains protective components such as lactoferrin, aldehyte dehydrogenase, superoxide dismutase, and glutathione which are suboptimal in eyes with ocular surface disease or tear film deficiency (17,18).Usage of multiple topical preparations may cause free radical related damage.Ocular surface disorders may develop secondary to preservative related oxidative injury in long term treated glaucoma patients (7).It was shown that, preservative free preparations have less cytotoxic effects than preserved preparations (19).The highest free content agents in the eye drop group were (Table 3); Brimonidine tartrate 0.15% (Alphagan P) and Travoprost 0.004% (Travatan) in anti-glaucoma drugs, Polyvidon (Oculotect) and N-acetyl-cystein 5% in drugs used for treatment of dry eye group, Sodium cromoglycate 2% (Allergocrom) in anti-allergic eye drop group, Ketorolac 0.4% (Acular LS) in anti-inflammatory group.TAS levels were similar in this group.Autologous serum eye drops are prepared from patients own blood and used for treatment of dry eye (20).In our study we found that TOS and TAS levels were similar for different aged patients (Table 4).
In conclusion, we have shown that free radicals are present in agents used in intravitreal and intracameral injections, in long term used commercial eye drops, in fortified eye drops, and in autologous serum eye drops.
The physician should use these drugs carefully by considering the amount of free radical content.More studies should be done to further investigate the free radical content of agents used in ophthalmology.

Table 1 .
Presence of free radicals (TOS) and antioxidant status (TAS) in agents used in intravitreal and intracameral injections.

Table 2 .
Presence of free radicals (TOS) and antioxidant status (TAS) in agents used in fortified eye drops.

Table 3 .
Presence of free radicals (TOS) and antioxidant status (TAS) in long term used commercial eye drops.