Related Experiment Video
Updated: Aug 18, 2026
Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation
Published on: May 2, 2020
An in vitro method which assesses corneal epithelial toxicity due to antineoplastic, preservative and antimicrobial
H M Lazarus1, P S Imperia, R E Botti
1Department of Medicine, University Hospitals of Cleveland, Case Western Reserve University, Ohio 44106.
Abstract:
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.
Insights
This study developed an in vitro model to assess drug toxicity on corneal cells, finding that deoxycytidine and prednisolone phosphate reduced keratitis in leukemia patients. The model also identified varying toxicity levels among ophthalmic preservatives and antiviral agents.
Area of Science:
- Ophthalmology
- Toxicology
- Pharmacology
Background:
- Corneal epithelial toxicity is a significant concern with topical ophthalmic agents.
- Existing methods for assessing cytotoxicity may not fully capture in vivo responses.
- Developing reliable in vitro models is crucial for predicting ocular drug safety.
Purpose of the Study:
- To establish and validate an in vitro model for evaluating the cytotoxicity of pharmacologic agents on corneal epithelium.
- To assess the protective effects of deoxycytidine against chemotherapy-induced ocular toxicity.
- To compare the toxicity of common ophthalmic preservatives and antiviral agents.
Main Methods:
- Primary rabbit corneal epithelial cell cultures were utilized for in vitro cytotoxicity assays.
- 3H-thymidine incorporation was measured to quantify cellular proliferation and drug effects.
- Dose-response curves were generated for various chemotherapeutic agents, preservatives, and antivirals.
- An in vivo study in leukemia patients assessed the efficacy of topical deoxycytidine and prednisolone phosphate in preventing keratitis.
Main Results:
- Chemotherapeutic agents like cytosine arabinoside, methotrexate, and 5-fluorouracil showed significant cytotoxicity at clinical concentrations.
- Topical deoxycytidine and prednisolone phosphate effectively reduced keratitis symptoms in patients receiving high-dose cytosine arabinoside.
- Ophthalmic preservatives varied in toxicity: benzalkonium chloride was most toxic, followed by thimerosal, with chlorobutanol being least toxic.
- Among antiviral agents, (E)-5(2-bromovinyl)-2'-deoxyuridine (BVDU) was non-toxic, while trifluridine, ethyldeoxuridine, and idoxuridine showed dose-dependent toxicity.
- Aminoglycoside antibiotics demonstrated differential toxicity, with tobramycin and amikacin being less toxic than gentamicin and neomycin.
Conclusions:
- The developed in vitro model accurately predicts corneal epithelial cytotoxicity of various ophthalmic agents.
- The findings support the use of deoxycytidine and prednisolone phosphate for preventing chemotherapy-induced keratitis.
- The study provides valuable data on the relative toxicity of common ophthalmic preservatives and antiviral drugs.
- This in vitro model serves as a useful tool for the toxicologic assessment of novel topical ophthalmic agents.
