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

unctival 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.

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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 Iwasawa 　 Cytotoxicity 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

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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)

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Preservatives in Tested Ophthalmic Solutions

Trade Name ActiveComponent Preservative

Levoquin, Cravit

Levofloxacine No

Vegamox Moxifloxacin No

Zymer, Gatiflo Gatifloxacin No

Noflo Norfloxacin No

Tosflo Tosufloxacin No

Panimycin Dibekacin BAK

Bestron Cefmenoxime Methyl Para, Propyl Para, EDTA, Boric acid

Trade Name ActiveComponent Preservative

Rinderon Betamethason Methyl Para, Propyl Para

Rinderon A Betamethason, Fradiomycin

Methyl Para, Propyl Para

Rinbeta Betamethason

(Generic)

Boric acid, EDTA

RinbetaPF Betamethason (Generic with filter in the tip)

Removed by filtration

Flumethoron 0.1% Fluolomethoron 0.1% BAK, Polysorbate 80, EDTA

Flumethoron 0.02% Fluolomethoron 0.02% BAK, Polysorbate 80, EDTA

Diclod (Voltaren) Diclofenac Chlorobutanol, Polysorbate 80 Boric Acid

Diclostar Diclofenac (Generic) Boric Acid, EDTA

Diclostar PF Diclofenac (Generic with filter in the tip)

Removed by filtration

Bronuck (Zybrom) Bromfenac BAK, Polysorbate 80

Niflan Pranoprofen BAK, Polysorbate 80, Boric Acid, EDTA

Benoxil Oxybuprocain BAK, EDTA

BAK=Benzalkonium Chloride

Para= Parahydroxybenzoate

EDTA=ethylene-diaminetetraacetic acid (edetic acid)

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Results Results :Corneal Endothelia:Corneal EndotheliaC

ell s

urv

ival

(%

)

Anti-inflammatory (10-fold dilution, 48 hours exposure)140

120

100

80

60

40

20

0

Betamethason

Betamethason(G)-F

Fluolomethorone

0.1%Fluolomethorone

0.02% Diclofenac(G)

Diclofenac(G)-F

Pranoprofen

Oxybuprocain

Bromfenac

Diclofenac

Betamethason+

Fradiomycin

Antibiotics (10-fold dilution, 48 hours exposure)C

ell s

urv

ival

(%

)140

120

100

80

60

40

20

0Levofloxacin Moxifloxacin Gatifloxacin Norfloxacin Tosufloxacin Panimycin Bestron

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Results Results : : EpitheliaEpithelia--AntibioticsAntibioticsAntibiotics (Corneal epithelia, 10-fold dilution, 48 hours exposure)

Cel

l su

rviv

al

(%)

140

120

100

80

60

40

20

0Levofloxacin Moxifloxacin Gatifloxacin Norfloxacin Tosufloxacin Dibekacin Cefmenoxime Sulperin

Cel

l su

rviv

al

(%)

Antibiotics (Conjunctival Epithelia, 10-fold dilution, 48 hours exposure)140

120

100

80

60

40

20

0Levofloxacin Moxifloxacin Gatifloxacin Norfloxacin Tosufloxacin Panimycin Bestron

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Results Results : : EpitheliaEpithelia--AntiinflammatoryAntiinflammatoryC

ell s

urv

ival

(%

)140

120

100

80

60

40

20

0

Betamethason

Betamethason(G)-F

Fluolomethorone

0.1%Fluolomethorone 　

0.02% Diclofenac(G)

Diclofenac(G)-F

Pranoprofen

Oxybuprocain

Bromfenac

Diclofenac

Betamethason+

Fladiomycin

Cel

l su

rviv

al

(%)

140

120

100

80

60

40

20

0

Betamethason

Betamethason(G)-F

Fluolomethorone

0.1% Fluolomethorone

0.02% Diclofenac(G)

Diclofenac(G)-F

Pranoprofen

Oxybuprocain

Bromfenac

Diclofenac

Betamethason

+Fladiomycin

Antiinflamatory (Corneal epithelia, 10-fold dilution, 48 hours exposure)

Antiinflammatory (Conjunctival Epithelia , 10-fold dilution, 48 hours exposure)

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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.

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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

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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 Medicin

e, Yokohama, Japan• ayaki@showaf.jp