1. 1 A Comparison of Ocular Concentrations and Anti-inflammatory Activity of Ketorolac 0.45% and Bromfenac 0.09% in a Rabbit Model of Intraocular Inflammation L. David Waterbury, PhD 1 ; Linda Villanueva, COT 2 ; Milan Patel, BA 2 ; Lisa Borbridge, MSc 2 ; Rhett M. Schiffman, MD, MS, MHSA 2 ; David A. Hollander, MD 2 1 Raven Biosolutions LLC, San Carlos, CA; 2 Allergan, Inc., Irvine, CA Study funded by Allergan, Inc. Dr. L. David Waterbury has received research funding and travel expense reimbursement from Allergan Inc. Mr. Milan Patel, Ms. Lisa Borbridge, Ms. Linda Villanueva and Drs. Rhett M. Schiffman and David A. Hollander are employees of Allergan, Inc.

2. 2 Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used perioperatively for the prevention and treatment of ocular inflammation and pain following cataract surgery. 1  Ketorolac, a COX-1 and COX-2 inhibitor, is the most extensively investigated NSAID. 2-4 A novel formulation of preservative-free ketorolac containing carboxymethylcellulose (CMC) was recently introduced (ketorolac 0.45%, Acuvail ® ; Allergan, Inc.; Irvine, CA). 5  Ophthalmic bromfenac solution 0.09% (Xibrom ® ; ISTA Pharmaceuticals; Irvine, CA) is another NSAID that selectively inhibits COX-2. 4,6 Ocular inflammation can be modeled in animals following intravenous injections of the endotoxin lipopolysaccharide (LPS). 4,7,8 In this model, the efficacy of NSAIDs in suppressing ocular inflammation can be compared throughout the 12-hour dosing cycle by assessing aqueous and iris-ciliary body concentrations as well as inhibition of inflammation. This study was designed to compare ocular penetration and efficacy of ketorolac 0.45% and bromfenac 0.09% in a rabbit model of LPS-induced ocular inflammation.

3. 3 Methods: LPS Rabbit Model of Inflammation Peak group (see diagram)  One hour after initial dosing, rabbits received intravenous injections of LPS (10 µg/kg) and fluorescein isothiocyanate-dextran (FITC-dextran; MW ~ 70,000, 30 mg/kg).  Two hours after initial dosing (1 hour after LPS), aqueous and iris-ciliary body samples were collected for analysis. 1 Peak Trough 2 LPS and FITC-dextran injections Sample withdrawal Time (hours) LPS and FITC-dextran injections Sample withdrawal 1011 Study product 0 Peak and trough concentrations of ketorolac and bromfenac were plotted against previously described COX-1 and COX-2 inhibition curves. 4 Trough group (see diagram)  Ten hours after initial dosing, rabbits received intravenous injections of LPS (10 µg/kg) and FITC-dextran; MW ~ 70,000, 30 mg/kg.  Eleven hours after initial dosing (1 hour after LPS), aqueous and iris-ciliary body samples were collected for analysis. At hour 0, ketorolac 0.45%, bromfenac 0.09%, or artificial tears were instilled every 20 minutes for 3 doses to 42 New Zealand white rabbits which were previously randomized into a peak or trough group.. FITC-dextran (fluorophotometry) concentrations, PGE 2 concentrations (immunoassay), and study drug concentrations were measured.

4. 4 a a a Peak ConcentrationsTrough Concentrations Ketorolac 0.45% (n = 6) Bromfenac 0.09% (n = 6) Ketorolac 0.45% (n = 6) Bromfenac 0.09% (n = 6) Results: Higher Ocular Concentrations With Ketorolac 0.45% At peak, the ketorolac aqueous concentrations were 7.8-fold higher than bromfenac aqueous concentrations (737.8 ± 64.7 ng/mL vs 94.2 ± 13.8 ng/mL, respectively; P <.0001) and the ketorolac iris-ciliary body concentrations were 12.1 fold higher than bromfenac iris-ciliary body concentrations (556.0 ± 36.0 ng/mL vs 45.9 ± 8.4 ng/mL, respectively; P <.0001). At trough, the ketorolac aqueous concentrations were 7.7-fold higher than bromfenac aqueous concentrations (127.0 ± 18.9 ng/mL vs 16.5 ± 2.7 ng/mL, respectively; P <.001) and the iris-ciliary body concentrations for ketorolac were 7.2 fold higher than for bromfenac (59.9 ± 6.5 ng/mL vs 8.3 ± 1.3 ng/mL; P =.0003). NSAID Concentration (ng/mL) NSAID Concentration ng/mL a P <.001 a P <.0001 a

5. 5 a b a P <.01 and b P <.05 compared to LPS + artificial tears. c Same for peak and trough, shown twice for simplicity. a b Aqueous PGE 2 Concentration (pg/mL) LPS + artificial tear (n = 6) No LPS (n = 6) C LPS + bromfenac 0.09% (n = 6) LPS + ketorolac 0.45% (n = 6) Results: NSAIDs Reduce PGE 2 Concentrations at Peak and Trough LPS significantly increased PGE 2 concentrations in the aqueous of artificial tear–treated animals compared to those that did not receive LPS (8430 ± 1055 pg/mL vs 2115 ± 259 pg/mL, P <.001 ). Ketorolac and bromfenac significantly inhibited LPS-induced aqueous PGE 2 elevation at both peak (1681 ± 224 pg/mL and 1952 ± 313 pg/mL vs 8430 ± 1055 pg/mL, respectively; P <.01) and trough (2250 ± 317 pg/mL and 2323 ± 308 pg/mL vs 8810 ± 2201 pg/mL, respectively; P <.05).

6. 6 a a P <.001 and b P <.01 compared to LPS. c P <.05 compared to LPS + bromfenac 0.09%. d Same for peak and trough. Shown twice for simplicity. a b,c LPS + artificial tear (n = 6) No LPS (n = 6) d LPS + bromfenac 0.09% (n = 6) LPS + ketorolac 0.45% (n = 6) Aqueous FITC-Dextran Concentration (μg/mL) LPS significantly increased FITC-dextran concentrations in the aqueous of artificial tear-treated animals compared to those that did not receive LPS (16.05 ± 2.57 μg/mL vs 0.06 ± 0.04 μg/mL, P <.001). Treatment with ketorolac or bromfenac significantly reduced LPS-induced aqueous FITC-dextran elevation at peak (0.26 ± 0.15 μg/mL and 1.24 ± 0.60 μg/mL vs 16.05 ± 2.57 μg/mL, respectively; P <.001). However, only ketorolac not bromfenac significantly reduced LPS-induced aqueous FITC-dextran elevation at trough (4.70 ± 0.75 μg/mL vs 15.37 ± 2.45 μg/mL; P <.01). Aqueous FITC-dextran concentration after trough dosing of ketorolac 0.45% was significantly less than that of bromfenac 0.09% (4.70 ± 0.75 μg/mL vs 10.1 ± 1.61 μg/mL; P =.0276). Results: Ketorolac 0.45% Reduces FITC- Dextran Concentrations at Peak and Trough

7. 7 COX-1 Activity (%) -11-10-9-8-7 -6-5 Ketorolac 0.45% Bromfenac 0.09% -4 NSAID Concentration (Log M) Aqueous peak Aqueous trough Iris-ciliary body peak Iris-ciliary body trough Ketorolac mean peak and trough concentrations in the aqueous and iris-ciliary body exceeded its IC 50 value for previously determined COX-1 (7.5 ng/mL; 2 x 10 -8 M). 4 However, with the exception of peak aqueous concentration, bromfenac mean peak and trough concentrations in aqueous and iris-ciliary body did not exceed its IC 50 value for previously determined COX-1 (70 ng/mL; 21 x 10 -8 M). 4 Results: Ketorolac 0.45% Achieved Concentrations That Exceeded Its IC 50 Values for COX-1 Mean peak concentrations (ng/mL) Mean trough concentrations (ng/mL) Ketorolac 0.45% Aqueous737.8 127.0 Iris-ciliary body556.259.9 Bromfenac 0.09% Aqueous94.216.5 Iris-ciliary body45.98.3 See Slide 4 COX-1 Activity (%)

8. 8 -11-10-9-8-7-6-5-4 NSAID Concentration (Log M) COX-2 Activity (%) Ketorolac 0.45% Bromfenac 0.09% Aqueous peak Aqueous trough Iris-ciliary body peak Iris-ciliary body trough Ketorolac mean peak and trough concentrations in the aqueous and iris-ciliary body exceeded its IC 50 value for previously determined COX-2 (45 ng/mL; 12 x 10 -8 M). 4 The mean peak and trough concentrations of bromfenac in aqueous and iris-ciliary body also exceeded its IC 50 value for previously determined COX-2 (2.2 ng/mL; 0.66 x 10 -8 M). 4 Results: Ketorolac 0.45% Achieved Concentrations That Exceeded Its IC 50 Values for COX-2 Mean peak concentrations (ng/mL) Mean trough concentrations (ng/mL) Ketorolac 0.45% Aqueous737.8 127.0 Iris-ciliary body556.259.9 Bromfenac 0.09% Aqueous94.216.5 Iris-ciliary body45.98.3 See Slide 4

9. 9 Discussion Controlling inflammation following cataract surgery is important for achieving optimal surgical outcomes. Our study demonstrates that ketorolac achieved at least 7-fold higher concentrations in the aqueous and iris-ciliary body than did bromfenac at both peak and trough. Ocular inflammation induced by LPS resulted in an overt inflammatory response as evidenced by a 4-fold and > 267-fold increase in aqueous PGE 2 and FITC-dextran levels, respectively.  The nonselective COX inhibitor ketorolac 0.45% significantly inhibited LPS-induced elevation of both PGE 2 and FITC-dextran concentrations at both peak and trough.  The COX-2 selective inhibitor bromfenac 0.09% also significantly inhibited LPS-induced aqueous PGE 2 elevation at peak and trough, but it did not inhibit FITC-dextran at trough. Failure of bromfenac 0.09% to inhibit FITC-dextran leakage at trough may stem, at least in part, from its insufficient ability to inhibit COX-1 in the iris-ciliary body, the site of FITC-dextran leakage from blood vessels. Ketorolac 0.45% provides more sustained antiinflammatory activity throughout its dosing cycle than does bromfenac 0.09% by achieving higher ocular concentrations, which exceed its IC 50 value for both COX-1 and COX-2. Our findings suggest that inhibition of both COX-1 and COX-2 is necessary to alleviate prostaglandin-mediated inflammation.

10. 10 Conclusions Ketorolac 0.45% achieved aqueous and iris-ciliary body concentrations that exceeded its IC 50 values for both COX-1 and COX-2 and were 7 to 12-fold higher than those achieved by bromfenac 0.09% at both peak and trough. At peak, ketorolac 0.45% and bromfenac 0.09% inhibited LPS-induced anterior chamber inflammation. At trough, only ketorolac 0.45%, not bromfenac 0.09%, significantly prevented FITC-dextran leakage.

11. 11 References 1.O'Brien TP. Emerging guidelines for use of NSAID therapy to optimize cataract surgery patient care. Curr Med Res Opin. 2005;21(7):1131-1137. 2.Buckley MM, Brogden RN. Ketorolac. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential. Drugs.1990;39(1):86-109. 3.Sinha VR, Kumar RV, Singh G. Ketorolac tromethamine formulations: an overview. Expert Opin Drug Deliv. 2009;6(9):961-975. 4.Waterbury LD, Silliman D, Jolas T. Comparison of cyclooxygenase inhibitory activity and ocular anti- inflammatory effects of ketorolac tromethamine and bromfenac sodium. Curr Med Res Opin. 2006;22(6):1133- 1140. 5.Acuvail ® [package insert]. Irvine, CA: Allergan, Inc.; 2009. 6.Xibrom ® [package insert]. Irvine, CA: ISTA Pharmaceuticals, Inc.; 2006. 7.Waterbury LD, Flach AJ. Efficacy of low concentrations of ketorolac tromethamine in animal models of ocular inflammation. J Ocul Pharmacol Ther. 2004;20(4):345-352 8.Waterbury LD, Flach AJ. Comparison of ketorolac tromethamine, diclofenac sodium, and loteprednol etabonate in an animal model of ocular inflammation. J Ocul Pharmacol Ther. 2006;22(3):155-159.

12. 12 Author Bio and Photo L. David Waterbury, PhD  Director, Raven Biosolutions LLC, San Carlos, CA L. David Waterbury, PhD, received his bachelor’s degree from the University of Michigan and his doctorate from the University of Vermont. He completed his postdoctoral training at Baylor College of Medicine in Houston, and went on to accept a position of assistant professor of pharmacology at Wake Forest University. Dr. Waterbury was employed for several years at Syntex Research in Palo Alto, CA, which later became part of Roche Bioscience. His scientific interests include ocular inflammation, corneal and lens research, and systemic inflammatory diseases. Dr. Waterbury conducts studies in ocular pharmacology, and has authored more than 75 papers and patents. He lives in San Carlos, CA.