Cytotoxicity of topical medications after cataract surgery for human corneal endothelial/epithelial cells, and conjuncti

1. Cytotoxicity of topical medications after cataract surgery for human corneal endothelial/epithelial cells, and conjunctival epithelial cells 1)Masahiko Ayaki, MD, 1)Shigeo Yaguchi, MD, PhD, 2)Atsuo Iwasawa, PhD, 3)Ryohei Koide, MD PhD. 1)Department of Ophthalmology and 2) Department of Clinical Pathology, Fujigaoka Hospital, Showa University School of Medicine, 3) Department of Ophthalmology, Showa University School of Medicine Authors have no financial interest.

2. Purpose and Methods • Purpose • Cataract surgeons sometimes encounter unexplained persistent corneal edema after surgery and drug toxicity may be one of the potential etiologies. The purpose of this study is to evaluate cytotoxicity of topical medications to corneal and conjunctival cells. • Cells • Human corneal endothelial cells (primary culture from eye bank eyes) • SIRC (human corneal epithelial cells, ATCC CCL-60,ATCC; American tissue and Cells Corporation) • Chang conjunctiva (human conjunctival epithelial cells, ATCC CCL-20.2, ATCC) • Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells after 48 hours exposure at 10, 100, and 1000-fold dilution. • Culture method (reference) • Masahiko Ayaki, Shigeo Yaguchi, Ryohei Koide, Atsuo IwasawaCytotoxicity of ophthalmic solutions with and without preservatives for human corneal endothelial cells, epithelial cells, and conjunctival epithelial cells. Exp Clin Ophthalmol, 2008：36（6）；553-559

3. Tested ophthalmic solutions • Antibiotics • gatifloxacin (GatifloR , Senjyu, Japan) • moxifloxacin (VegamoxR , Alcon) • levofloxacin (CravitR , Santen) • norfloxacin (NofloR, Banyu, Japan) • tosufloxacin (TosufloR, Nidek) • dibekacin (PanimycinR, Meiji, Japan) • cefmenoxime( BestronR, Kaken, Japan) • Anti-inflammatory; Non steroid • diclofenac (DiclodR, DiclostarR, Nitten, Japan, DiclostarRPF) • bromfenac (BronuckR , Senjyu) • pranoprofen (NiflanR, Senjyu) • Anti-inflammatory; Steroid • betamethasone (RinderonR, Shinogi, Japan, RinbetaR, Nitten, Japan, RinbetaRPF) • betamethason&fradiomycin (RinderonR A, Shionogi) • fluolomethorone (FlumethoronR, Santen) • Topical anesthetics • oxybuprocain (BenoxilR, Santen)

4. Preservatives in Tested Ophthalmic Solutions BAK=Benzalkonium Chloride Para= Parahydroxybenzoate EDTA=ethylene-diaminetetraacetic acid (edetic acid)

5. Results :Corneal Endothelia Antibiotics (10-fold dilution, 48 hours exposure) 140 120 100 80 60 40 20 0 Cell survival (%) Levofloxacin Moxifloxacin Gatifloxacin Norfloxacin Tosufloxacin Panimycin Bestron Anti-inflammatory (10-fold dilution, 48 hours exposure) 140 120 100 80 60 40 20 0 Cell survival (%) Oxybuprocain Diclofenac Bromfenac Pranoprofen Betamethason+Fradiomycin Betamethason Fluolomethorone 0.1% Fluolomethorone 0.02% Diclofenac(G) Diclofenac(G)-F Betamethason(G)-F

6. 140 120 100 80 60 40 20 0 Cell survival (%) Levofloxacin Moxifloxacin Gatifloxacin Norfloxacin Tosufloxacin Dibekacin Cefmenoxime Sulperin Results : Epithelia-Antibiotics Antibiotics (Corneal epithelia, 10-fold dilution, 48 hours exposure) Antibiotics (Conjunctival Epithelia, 10-fold dilution, 48 hours exposure) 140 120 100 80 60 40 20 0 Cell survival (%) Levofloxacin Moxifloxacin Gatifloxacin Norfloxacin Tosufloxacin Panimycin Bestron

7. Results : Epithelia-Antiinflammatory Antiinflamatory (Corneal epithelia, 10-fold dilution, 48 hours exposure) 140 120 100 80 60 40 20 0 Cell survival (%) Diclofenac Bromfenac Pranoprofen Betamethason+Fladiomycin Oxybuprocain Betamethason Diclofenac(G) Fluolomethorone 0.1% Fluolomethorone0.02% Diclofenac(G)-F Betamethason(G)-F Antiinflammatory (Conjunctival Epithelia , 10-fold dilution, 48 hours exposure) 140 120 100 80 60 40 20 0 Cell survival (%) Oxybuprocain Diclofenac Bromfenac Pranoprofen Betamethason+Fladiomycin Betamethason Fluolomethorone 0.1% Fluolomethorone 0.02% Diclofenac(G) Diclofenac(G)-F Betamethason(G)-F

8. Summary of Results • Most of tested solutions had corneal and conjunctival toxicity in 10-fold dilution (steroids < antibiotics < non steroidal anti-inflammatory medications). • It decreased (cell survival > 80%) after 1000-fold or more dilution and seemed to depend mostly on the components of ophthalmic solution such as benzalkonium chloride.

9. Cause of postoperative corneal edema (Liu, JCRS, 2001) • 1.Preexisting endothelial damage • (a) Fuchs‘ corneal endothelial dystrophy or advanced cornea guttata • (b) Posterior polymorphous corneal dystrophy • (c) Low endothelial cell count • 2.Surgical trauma • (a) Cavitational energy • (b) Direct touch by instruments or intraocular lens • (c) Turbulent flow of irrigation solution • (d) Lens or lens particle contact with the cornea • (e) Repeated anterior chamber collapse • (f) Descemet‘s detachment • (g) Corneal burn • 3.Use of unphysiologic or toxic intraocular fluids and drugs • (a) Hypoosmotic (under 200 mOsm) or hyperosmotic(over 400 mOsm) fluids • (b) Low or high pH value (under 6.8 or over 8.2) • (c) Lack of calcium in irrigating fluid • (d) Toxic concentrations of drugs ( antibiotics, local anesthetics, miotics) • (e) Detergents and sterilizing agents • (f) Preservatives • 4.Postoperative conditions • (a) Excessive inflammation • (b) High intraocular pressure • (c) Adherence of vitreous, iriis, or lens capsule • (d) Epithelial downgrowth

10. Conclusions • The postoperative topical medications had cytotoxicity and those preserved with benzalkonium showed higher toxicity than those without them. Considering actual concentration at corneal endothelium, they do not seem to cause endothelial damage. • Correspondence • Masahiko Ayaki MD, Showa University School of Medicine, Yokohama, Japan • ayaki@showaf.jp