1. “An Anterior Chamber Toxicity Study Evaluating Besivance, AzaSite, ciprofloxacin and BSS” Authors: Peter J. Ness, Nick Mamalis, Liliana Werner, Surekha Maddula, Don K. Davis, Eric D. Donnenfeld, Randall J. Olson From the: John A. Moran Eye Center, University of Utah - The authors have no financial or proprietary interest in any product mentioned in this poster; Randall J. Olson is a consultant for Allergan, Inc. - This study is supported by unrestricted grants from Allergan, Inc. and Research to Prevent Blindness, Inc.

2. Background Postoperative endophthalmitis is an uncommon but devastating complication of cataract surgery. Postoperative endophthalmitis prophylaxis 1 –Widely used around the world –All antibiotics are used “off-label” in this setting in the US –Has been shown to decrease risk of endophthalmitis Sutureless clear corneal cataract surgery 2,3 –Decreases astigmatism –Not all wounds are as well sealed as we wish –Leaky wounds allow the tear film to enter the anterior chamber –The tear film entering the eye contains antibiotics and other medications being administered

3. Background Besivance ® * (besifloxacin) and AzaSite ® ** (azithromycin) –The first drugs using the DuraSite ® *** bio-adhesive vehicle –Approved in the US to treat bacterial conjunctivitis DuraSite benefits: –Prolonged administration of the medication on the ocular surface as the antibiotic-embedded polymer is slowly broken down –Less frequent dosing is required for equivalent efficacy 4 –Better patient compliance Why not use these helpful antibiotics for postoperative endophthalmitis prophylaxis? * Bausch & Lomb, Rochester, NY, USA ** Inspire Pharmaceuticals, Inc., Durham, NC, USA *** InSite Vision Inc., Alameda, CA, USA

4. Objective No studies, to date, have investigated the effects of DuraSite-based medications in the anterior chamber. Our aim in this study was to evaluate the possible toxicity of DuraSite-based medications, delivered as a large bolus, into the anterior chamber of rabbit eyes, simulating an extremely leaky clear corneal wound.

5. Methods/Materials Subjects: 20 New Zealand White Rabbits Study groups: Besivance 0.6%, AzaSite 1.0%, ciprofloxacin 0.3% and balanced salt solution (BSS) (10 eyes randomized into each group) Surgical technique: sterile aspiration of 0.1 mL of aqueous from the anterior chamber using a 30 g needle, then injection of 0.1 mL of the study material through the same needle Postoperative examinations: slit-lamp exams (by a masked physician) at 24 and 48 hours after injection, focusing on inflammatory signs Sacrifice: each rabbit was humanely euthanized at 48 hours post-injection and all eyes enucleated Data analysis: –Corneal vital staining: 2 eyes randomized from each group –Histopathology: remaining 8 eyes from each group –Analysis focused on damage to the corneal endothelial cell layer and other signs of anterior segment damage Outcome measures: clinical and pathologic signs of toxicity * Falcon Pharmaceuticals, Fort Worth, TX, USA

6. Results Clinical Slit-Lamp Exam (DuraSite-based groups) –Severe, diffuse corneal edema (20 of 20 eyes) –Corneal ectasia and bullous keratopathy (20 of 20 eyes) –Profound conjunctival injection –Moderate limbal vascularity –Generally increased globe size –No statistically significant difference between Besivance and AzaSite examination scores Figure 1. Diffuse corneal edema after injection of Besivance Figure 2. Ruptured bullae and corneal edema after injection of Besivance Figure 3. Corneal ectasia and bullous keratopathy after injection of AzaSite

7. Results Clinical Slit-Lamp Exam (Non-DuraSite-based groups) –No corneal opacity (19 of 20 eyes) –Mild conjunctival injection (12 of 20 eyes) –Mild limbal vascularity (16 of 20 eyes) –Mild conjunctival injection & discharge with moderate diffuse corneal opacification and limbal vascularity (1 eye injected with ciprofloxacin [Figure 6]) –No statistically significant difference between ciprofloxacin and BSS examination scores Figure 4. Clear cornea and mild conjunctival injection after injection of ciprofloxacin Figure 5. Clear cornea with no signs of inflammation after injection of ciprofloxacin Figure 6. Diffuse moderate corneal edema after injection of ciprofloxacin

8. Results Corneal vital staining DuraSite-based eyes revealed: –Severe alteration of endothelial cell size and shape indicative of damage Non-DuraSite-based eyes showed: –Mild intracellular edema –Iintact hexagonal shape of endothelial cells Figure 7. Corneal vital staining with morphologically damaged endothelial cells after injection of AzaSite Figure 8. Corneal vital staining with mild intracellular edema after injection of BSS BesivanceAzaSiteCiprofloxacinBSS Globe volume (standard deviation) [cm 3 ] 3.05 (0.17)3.16 (0.52)2.46 (0.13)2.56 (0.17) Table. Globe volume by gross measurements after enucleation

9. Results Figure 9. Histopathologic slide showing fibrin, amorphous material in iridocorneal angle and acute inflammatory cells after injection of Besivance, H&E, 100x Figure 10. Histopathologic slide showing a large epithelial bulla, corneal edema and fibrin in the anterior chamber after injection of Besivance, H&E, 40x Histopathology DuraSite-based eyes showed (to varying degrees in each eye): –Bullous keratopathy –Corneal stromal thinning –Anterior chamber fibrin –Extensive endothelial cell attenuation –Peripheral anterior synechia (in some eyes) –Amorphous eosinophilic material within the iridocorneal angle and trabecular meshwork Non-DuraSite-based eyes showed: –No signs of inflammation or anterior segment damage

10. Discussion Although the literature has clearly shown benefits of Besivance and AzaSite, their safety in the setting of sutureless clear corneal wounds (i.e. post cataract surgery) has not been investigated. DuraSite medications seem to cause glaucomatous and toxic damage in the anterior chamber when injected intracamerally as a large bolus. The difference in effect between DuraSite-based and non- DuraSite-based medications was statistically significant.

11. Discussion/Conclusions Each medication is composed of various components: antibiotic, benzalkonium chloride (BAK) preservative, vehicle and other inactive ingredients. BAK is contained in all 3 medications at differing concentrations (Besivance: 0.01%, AzaSite: 0.003%, ciprofloxacin: 0.006%) –These concentrations are within the range previously reported to possibly cause endothelial toxicity in rabbits 5 ; therefore, it is logical to conclude that BAK caused the toxic reaction –If BAK had caused this toxicity, we would expect some dose-response relationship, BUT instead there was a poor correlation between BAK concentration and toxicity (e.g. Besivance and AzaSite appeared equally toxic); therefore, it is unlikely to have caused the noted toxicity The vehicle (DuraSite) alone was not used as a control due to commercial unavailability, so the authors used a variety of DuraSite and non-DuraSite medications (all commonly used in ophthalmic practice) for comparison We deduce that the DuraSite component of Besivance and AzaSite caused the toxicity and glaucomatous damage We recommend: –Further study of these medications at lower volumes in the anterior chamber –Until the safety is better established, surgeons should consider placing a suture over a clear corneal wound if DuraSite-based medications may be used

12. References 1.O'Brien TP, Arshinoff SA, Mah FS. Perspectives on antibiotics for postoperative endophthalmitis prophylaxis: potential role of moxifloxacin. J Cataract Refract Surg 2007;33:1790-1800. 2.Herretes S, Stark WJ, Pirouzmanesh A, Reyes JM, McDonnell PJ, Behrens A. Inflow of ocular surface fluid into the anterior chamber after phacoemulsification through sutureless corneal cataract wounds. Am J Ophthalmol 2005;140:737-740. 3.Taban M, Sarayba MA, Ignacio TS, Behrens A, McDonnell PJ. Ingress of India ink into the anterior chamber through sutureless clear corneal cataract wounds. Arch Ophthalmol 2005;123:643-648. 4.McDonald MB, Protzko EE, Brunner LS, et al. Efficacy and safety of besifloxacin ophthalmic suspension 0.6% compared with moxifloxacin ophthalmic solution 0.5% for treating bacterial conjunctivitis. Ophthalmology 2009;116:1615-1623 e1. 5.Green K, Hull D, Vaughn E, Malizia A, Bowman K. Rabbit endothelial response to toxic preservatives. Arch Ophthalmol 1977;95:2218-2221.