An in vitro method which assesses corneal epithelial toxicity due to antineoplastic, preservative and antimicrobial agents.

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.

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