1. Anti-Inflammatory Activity of Topical Azithromycin on Corneal Innate Immune Responses Zahra Sadrai MD, Amir Reza Hajrasouliha MD, Sunil Chauhan PhD, Daniel Saban PhD, Reza Dana MD, MPH, MSc. Schepens Eye Research Institute, Massachusetts Eye & Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA Financial Disclosure: The authors have no financial interest in the product mentioned in this poster. Funding Sources: Inspire Pharmaceuticals provided research support for this study.

2. Corneal inflammation is a critical facet of many ocular pathologies, including corneal angiogenesis and corneal allograft rejection and represents a leading cause of blindness worldwide (1). BM-derived antigen presenting cells (APCs) in the cornea and ocular surface comprise diverse subsets of CD45+ cells including macrophages (CD11b+) that normally reside in the stroma, and CD11c+ dendritic cells in the epithelium (2, 3). Innate immunity, the major mechanism for acute inflammatory response, involves cellular trafficking into the cornea in response to traumatic, noxious, or microbial stimuli (2, 4-6). Cytokines, the molecular components of innate immune responses, coordinate leukocyte migration in immunity and inflammation (3). Macrolides are broad-spectrum antibiotics that are widely used to treat bacterial infections (7). There is also in vitro evidence that macrolides have anti-inflammatory and immunomodulatory activities. Studies have demonstrated that macrolides suppress the activation of NF-kB and the release of pro-inflammatory cytokines in vivo (8, 9). We hypothesized here that Azithromycin (AZM), a broad-spectrum macrolide antibiotic recently approved for ocular infection, has potential immunomodulatory effects on corneal inflammation. INTRODUCTION Anti-Inflammatory Activity of Topical Azithromycin on Corneal Innate Immune Responses

3. PURPOSE Anti-Inflammatory Activity of Topical Azithromycin on Corneal Innate Immune Responses To investigate the potential anti-inflammatory effects of AZM on corneal innate immune responses.

4. Cornea of 6-8 week old BALB/c mice underwent thermal cautery to induce inflammation and leukocyte influx. Fifteen corneas were randomly divided into three groups treated topically either with AZM ophthalmic solution 1% (AzaSite®; Inspire Pharmaceuticals, Inc, NC, USA), the relevant vehicle (DuraSite®; Inspire Pharmaceuticals, Inc, NC, USA), or prednisolone acetate 1% twice per day. Corneas were harvested at various time-points to characterize the inflammatory infiltrate via FACS analysis, and to quantitate expression of cytokines via real time PCR. All experiments were approved by the IACUC of the Schepens Eye Research Institute and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Naïve Cauterized Cornea Vehicle (Bid) Cauterized Cornea AZM (Bid) Real Time PCR IL-10, IL-6 Flow cytometry CD45, CD11c Days 1, 3, 7, 10, 14 METHODS Cauterized Cornea Prednisolone (Bid) Anti-Inflammatory Activity of Topical Azithromycin on Corneal Innate Immune Responses

5. Cell numbers per cornea RESULTS A.B. * * Amelioration of Corneal Inflammation by Topical Azithromycin (AZM) Figure 1. Relative to the vehicle control, the AZM-treated group showed a significant reduction in total infiltration of CD45+ (pan-leukocyte marker) cells at day 1 (30%) and day 7 (39%), which was similarly observed in the prednisolone-treated group (A). Dendritic cells (CD11c+) demonstrated a reduced infiltration at day 7 in the AZM-treated (35% reduction) and prednisolone-treated (40% reduction) groups. Anti-Inflammatory Activity of Topical Azithromycin on Corneal Innate Immune Responses

6. Figure 2. Corneas treated with AZM increased the expression level of IL-10 in all time-points (A). The relative expression of IL-6 was variable at different time-points (B). Anti-Inflammatory Activity of Topical Azithromycin on Corneal Innate Immune Responses IL-10IL-6 Fold increase compared to naive

7. Anti-Inflammatory Activity of Topical Azithromycin on Corneal Innate Immune Responses CONCLUSIONS Following an inflammatory insult, topical AZM reduced infiltration of leukocytes considerably to levels comparable to the prednisolone-treated group. This was further supported by an associated increase in anti- inflammatory cytokine IL-10. Thus, these data suggest that topical AZM holds endogenous anti-inflammatory properties.

8. 1.Streilein JW. Immunology and immunopathology of corneal transplantation. Chem Immunol 1999:73:186-206. 2. Dana MR. Corneal antigen-presenting cells: diversity, plasticity, and disguise. The Cogan lecture. Invest Ophthalmol Vis Sci. 2004;45:722-727. 3. Delves PJ, Roitt IM. The immune system. First of two parts. N Engl J Med. 2000 6;343(1):37-49. 4. Hamrah P, Huq SO, Liu Y, Zhang Q, Dana MR. Corneal immunity is mediated by heterogeneous population of antigenpresenting cells. J Leukoc Biol 2003;74:172-178. 5.Hamrah P, Zhang Q, Liu Y, Dana MR. Novel Characterization of MHC Class II–Negative Population of Resident Corneal Langerhans Cell–Type Dendritic Cells. Invest Ophthalmol Vis Sci. 2003;43:639-646. 6.Hamrah P, Zhang Q, Liu Y, Dana MR. The Corneal Stroma Is Endowed with a Significant Number of Resident Dendritic Cells. Invest Ophthalmol Vis Sci. 2003;44:581-589. 7.G.G. Zhanel, M. Dueck, D.J. Hoban, L.M. Vercaigne, J.M. Embil, A.S. Gin and J.A. Karlowsky, Review of macrolides and ketolides: focus on respiratory tract infections, Drugs 61 2001; 61:443–498. 8.O. Culic, V. Erakovic and M.J. Parnham, Anti-inflammatory effects of macrolide antibiotics, Eur. J. Pharmacol. 2001; 429: 209–229. 9.M.T. Labro, Cellular and molecular effects of macrolides on leukocyte function, Curr. Pharm. 2004: 3067–3080. References Contact Info reza.dana@schepens.harvard.edu z.sadrai@schepens.harvard.edu Anti-Inflammatory Activity of Topical Azithromycin on Corneal Innate Immune Responses