Sang June Kim 1 , Ha Bum Lee 1 , Hungwong Tchah 2 Hye Young Park 1

1. Effect of intracameral injection of bevacizumab on the rabbit corneal endothelium 1.Department of Ophthalmology College of Hallym University Hallym Medical Center Seoul, South Korea 2.Department of Ophthalmology University of Ulsan College of Medicine ASAN Medical Center Seoul, South Korea Sang June Kim1, Ha Bum Lee1, Hungwong Tchah2 Hye Young Park1

2. Avery RL, 2006; Iturralde D et al, 2006; Rosenfeld PJ et al, 2005; Spaide RF & Fisher YL, 2006; Spaide RF et al, 2006 T therapeutic benefits of intravitreal bevacizumab injection for various Intraocular neovascular diseases Grisanti S et al, 2006 intracameral bevacizumab injection resulted in encouraging preliminary outcomes in neovascular glaucoma Introduction • Bevacizumab(Avastin®, Genentech, San Francisco, California, USA) recombinant humanized monoclonal Ig G1 antibody directed against the vascular endothelial growth factor (VEGF) • Systemic use first anti-angiogenic agent approved by the FDA for intravenous use against metastatic colorectal cancer • Ophthalmic use

3. Purpose • Background While studies on retinal cells provided information regarding safe and effective doses of bevacizumab for intravitreal injection (Feiner L et al , 2006;Inan UU et al, 2007;Luthra S et al, 2006;Spitzer MS et al, 2006) , similar studies on corneal endothelial cells have not been undertaken. Such information would assist in determining safe and effective concent rations of intracameral bevacizumab injections.  • Purpose The present study examined the effect of intracameral bevacizumab injection on corneal endothelial cell morphology and viability in a rabbit model.

4. Materials and Methods • Materials Animals : 20 eyes of 10 New Zealand White rabbits (1.3∼1.7 kg) * The animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences, Asan Medical Center. The committee abides by the Institute of Laboratory Animal Resources (ILAR) guide. Measurement Specular microscopy(Topcon SP2000P) : mean value from 3 consecutive measurement Pachymetry(pachymetry; DGH-550 Pachette 2) : mean value from 5 consecutive measurement Slit lamp examination (Kowa, SL-15, portable slit lamp)

5. Materials and Methods • Procedures and Examinations Anesthesia: intramuscular injection of ketamine hydrochloride and Xylazine Grouping into 4 subgroups * Avastin® , Genentech, San Francisco, California, USA BSS® , Santen, Osaka, Japan Post injection examination schedule 2 hours, 1 week, 4 weeks later after injection Measurements specular microscope, pachymeter and slit-lamp examination

6. Materials and Methods • Tissue Preparation and Examinations Corneal buttoning 4 weeks after injection, 2 cornea button was obtained from each subgroup Stain methods alizarin red (Sigma) and trypan blue (TCI) according to Taylor and Hunt’s method (Spence DJ & Peyman GA, 1976; Taylor MJ & Hunt CJ, 1981). Scanning Electron Microscopy(SEM) method of Wang and Hu (Wang I-J & Hu F-R, 1997). • Statistical Analysis Wilcoxon signed rank test : compare data among groups Mann-Whitney U-tests : compare between each group * Differences were considered statistically significant when P < 0.05.

7. For each individual subgroup, there was no significant change in any parameter during the study. In addition, there was no statistical difference in these parameter upon comparison between groups (See table 1, 2, 3) Results Slit-lamp microscopic examinations revealed no apparent abnormalities in the corneas, anterior chambers or lenses over the duration of the study • Portable slit lamp examination (Kowa, Sl-15, portable slit lamp) . • Specular Microscope (Topcon SP2000P) Table 1. Specular microscopic result - cell density (CD) (cells/mm2) of each group before-and after injection (mean±SE)

8. Results Table 2. Specular microscopic result - coefficient of variation (CV) of each group before-and after injection (mean±SE) Table 3. Specular microscopic result – average cell area (AVG) (unit/mm2) of each group before-and after injection (mean±SE)

9. The corneal thickness values for the 4 subgroups are shown in Table 4. These pachymetric data showed there were no significant differences between the subgroups. Results • Pachymeter (pachymetry; DGH-550 Pachette 2) Table 4. Corneal thickness(㎛) of each group before-and injection (mean±SE)

10. Histology ( Light Microscopic Image, magnification X 200) Histology ( Scanning Electron Micrographs of corneal endothelial cells. ) B - 1 0.1mL BSS injection B A D C Untreated normal cornea 0.05mL (1.25mg) bevacizumab injection 0.05mL BSS injection C 0.1mL BSS injection bevacizumab injection 0.05ml(1.25mg) 0.1ml BSS injection Giantcell (black arrow) Giant cell (black arrow) 0.05mL BSS injection 0.1mL (2.5mg) bevacizumab injection ( ) Giant cell balck arrow Giant cell (black arrow) 0.05ml BSS injection 0.1ml(2.5mg) bevacizumab injection (black arrow) ( Giant cell (black arrow) Results Click here, you can see LM 5 images Click here, you can see 4 SEM images While giant cells were observed in all subgourps, but cell borders and microvilli were well preserved. There was no evidence of abnormal endothelial cell formation such as blebbing or cellular border disintegration

11. 1 2 3 (Grisanti S et al, 2006) Intracameral bevacizumab injection significantly inhibit iris neovascularization. (Aggio FB et al, 2007) Intravitreal injection is Also effective against iris rubeosis, but endoph- thalmitis and RPE tears risk increased. So, Intracameral injection is superior method, if it is safe in terms of corneal endothelium. Conclusion • Angiogenesis associated wound healing associated many ocular pathogenesis Ex) NeoVascular Glaucoma , Diabetic Retinopathy Corneal Neovascularization • Use of Bevacizumab on NVG 1. Intracameral injection 2. Intravitreal injection VS

12. Conclusion Click here !! Yoeruek E et al(2007) 5.0mg/ml bevacizumab did not affect cultured human corneal endothelial cell viability • Previous experimental study about corneal toxicity of Bevacizumab • Do you want our conclusion? Click here again !! Bevacizumab (up to 2.5 mg/0.1 mL) had no effect on endothelial cell viability or morphology in the rabbit cornea. Further studies are required to examine the effect of repeated bevacizumab intracameral injections on corneal endothelium such as would be used in clinical applications.