1 Ex Vivo Porcine Corneal Wound Healing Following Exposure to Ophthalmic Ketorolac Solutions Mark McDermott, MD 1 ; Linda Villanueva, COT 2 ; Rhett M. Schiffman, MD, MS, MHSA 2 ; David A. Hollander, MD 2 1 The Kresge Eye Institute, Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI; 2 Allergan, Inc., Irvine, CA Study funded by Allergan, Inc. Dr. Mark McDermott has received research funding Allergan Inc. Ms. Linda Villanueva and Drs. Rhett M. Schiffman and David A. Hollander are employees of Allergan, Inc.

2Background The ketorolac family of topical nonsteroidals is used to treat pain and/or inflammation following cataract or corneal refractive surgery. 1-3 Ketorolac is potent inhibitor of prostaglandin production via suppression of both cyclooxygenase-1 and -2 (COX-1) and (COX-2) enzymes. 4 In the past, topical NSAIDS have been associated with corneal toxicity and delays in wound healing. 5 A novel formulation of preservative-free ketorolac 0.45% (Acuvail ® ; Allergan, Inc.; Irvine, CA) was recently introduced that also contains carboxymethylcellulose (CMC). 1 Previous formulations of ketorolac include a 0.5% aqueous solution preserved with 0.01% benzalkonium chloride (BAK) (Acular ® ; Allergan, Inc.; Irvine, CA ) 2 and a 0.4% aqueous solution preserved with 0.006% BAK (Acular LS ® ; Allergan, Inc.; Irvine, CA ). 3 (see next slide) We hypothesized that the addition of CMC and removal of BAK in the ketorolac 0.45% formulation would lead to improved corneal epithelial wound healing compared to prior ketorolac formulations. We tested this hypothesis using a porcine-derived, corneal organ culture model.

3 Ketorolac 0.45% Is the Only Preservative-Free Formulation That Contains CMC Brand Name Active ingredient Ketorolac 0.45% 1 ACUVAIL ® Ketorolac 0.5% 2 ACULAR ® Ketorolac 0.4% 3 ACULAR LS ® Inactive ingredientsCMC, Na citrateEDTA, octoxynol PreservativeNo preservativeBAK (0.01%)BAK (0.006%) pH CMC = carboxymethylcellulose; BAK = benzalkonium chloride; EDTA = ethylenediaminetetraacetic acid (sodium edetate).

4 Methods: Wound Healing Model 5 mm central epithelial wound made in freshly isolated porcine cornea with a trephine. After 24 hours, corneas were incubated for 10 minutes in either: –50 µL Minimum Essential Medium (MEM) (negative control) –Triton X-100 (positive control) –NSAID (ketorolac 0.45%, ketorolac 0.4%, or ketorolac 0.5%) Corneas rinsed twice with 3 mL PBS, and 2 mL fresh MEM was added. Corneas allowed to heal for another 24 hours prior to staining with Richardson's staining solutions. Remaining wound area was photographed and the intensity of staining was quantified using Adobe ® Photoshop ® and expressed as area in pixels. Original Wound

5 Results: Ketorolac 0.45% Improved Wound Healing Wound Healing Model (Porcine Eyes) Control MEM Ketorolac 0.45% Ketorolac 0.4% Ketorolac 0.5% Ketorolac 0.45%–treated corneas showed greater wound closure than the Triton X-100 positive control and prior ketorolac formulations (Acular ®, Acular LS ® ) (P <.05). Individual porcine corneas shown 48 hours after lesion Ketorolac 0.45%–treated corneas demonstrated superior wound closure than the MEM negative control (no drug) (P <.05).

6 Results: Ketorolac 0.45% Improved Wound Healing n = 3-5 per group a,b a b P <.05 versus all other treatment groups. Corneal wound area was significantly reduced in the ketorolac 0.45% group compared to eyes that received no drug (P <.05). Wound area was also significantly reduced in the ketorolac 0.45% group compared to previous formulations (P <.05).

7Discussion Our results demonstrate that ketorolac 0.45% led to more rapid wound healing than prior formulations of ketorolac in an animal model.  The addition of CMC to the active agent led to more rapid wound healing than tissue culture medium alone.  More rapid epithelial recovery was seen in ketorolac 0.45%- treated corneas than those treated with ketorolac 0.4%.  Additional studies are necessary to confirm that these results translate to more rapid wound healing in a clinical setting. The speed in epithelial recovery may stem from both the removal of BAK and the addition of CMC.  CMC has been shown to promote epithelial cell migration and re-epithelialization. 6

8Conclusions Ketorolac 0.45% exposure promoted corneal wound healing in an animal model. Differences in wound healing may be due to the presence of CMC and/or absence of BAK in ketorolac 0.45%.

9References 1.ACUVAIL ® [package insert]. Irvine, CA: Allergan, Inc.; ACULAR ® [package insert]. Irvine, CA: Allergan, Inc.; ACULAR LS ® [package insert]. Irvine, CA: Allergan, Inc.; 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): Tomas-Barberan S, Fagerholm P. Influence of topical treatment on epithelial wound healing and pain in the early postoperative period following photorefractive keratectomy. Acta Ophthalmol Scand. 1999;77(2): Garrett Q, Simmons PA, Xu S, et al. Carboxymethylcellulose binds to human corneal epithelial cells and is a modulator of corneal epithelial wound healing. Invest Ophthalmol Vis Sci. 2007;48(4):

10 Author Bio Dr. McDermott graduated from the University of Wisconsin Medical School with election to the medical honor society Alpha Omega Alpha. He completed a residency in Ophthalmology at the University of Wisconsin. After receiving a training grant from the National Institutes of Health; he completed a two year fellowship in Corneal Surgery at the Medical College of Wisconsin. Dr. McDermott is certified by the American Board of Ophthalmology. He is a fellow of the American Academy of Ophthalmology. For his efforts in education of practicing eye surgeons, Dr. McDermott has been awarded an Honor Award by the American Academy of Ophthalmology. He has published over 90 papers, book chapters, and abstracts covering all aspects of cataract and corneal surgery. Dr. McDermott is a member of the American Society of Cataract and Refractive Surgeons. He is a medical director for the Midwest Eye Bank and Transplantation Center. He holds a Masters in Business Administration from the University of Michigan Ann Arbor and is an elected member of the business honor society Beta Gamma Sigma. Dr. McDermott’s research interests include endothelial toxicology, endothelial response to phacoemulsification, and viscoelastics.