We developed an in vitro model for studying the cytotoxicity of pharmacologic agents on corneal epithelium employing 3H-thymidine incorporation. Primary rabbit corneal epithelial cell cultures were established, and the cells plated prior to each experiment. 3H-thymidine incorporation was measured after the addition of
drug or vehicle to these confluent cells, and dose-response curves were generated. Marked inhibition of 3H-thymidine incorporation was reached at chemotherapeutic concentrations achieved clinically for
cytosine arabinoside (10(-7) M),
methotrexate (10(-3) M), and
5-fluorouracil (10(-6) M).
A 10(-4) M concentration of 2-deoxycytidine, a naturally occurring competitive inhibitor of
cytosine arabinoside, protected cells up to a concentration of 10(-5) M. We utilized these data to undertake an in vivo prophylaxis study in 13
leukemia patients receiving high-dose iv
cytosine arabinoside. Topical
deoxycytidine 10(-4) M and 1%
prednisolone phosphate, given 12 hours prior to the start of antileukemic
therapy, were effective in reducing symptoms and signs of
keratitis; both were better than historical placebo-treated eyes. Ophthalmic preservatives were studied in vitro at concentrations used clinically:
benzalkonium chloride (BAC) (0.004-0.02%) was the most toxic,
thimerosal (TMS) (0.001-0.004%) intermediate, and
chlorobutanol (CHB) (0.2-0.5%) the least toxic.
Antiviral agents (final concentration) included:
trifluridine (TFT) (1.0%), ethyldeoxuridine (EDU) (2.0%), and
idoxuridine (IDU) (0.1%). Dose but not time-dependent concentrations of these 3 agents were noted to cause toxicity; however, (E)-5(2-bromovinyl)-2'-deoxyuridine (BVDU) (0.1%) was non-toxic. Similarly,
tobramycin and
amikacin were significantly less toxic than
gentamicin and
neomycin in this system. These in vitro cytotoxicity data correlate well with previous in vivo and pre-clinical corneal epithelial toxicity studies. Our model may be useful in the toxicologic study of future topical ophthalmic agents.