Abstract
Purpose: :
To determine the relative cytotoxic effects of latanoprost free-acid, without the commercially used preservative benzalkonium chloride (BAC), in an in vitro assay of human corneal keratocytes and fibroblasts.
Methods: :
Two groups of cells were tested; primary keratocytes isolated from human donor corneas in serum-free medium (phenotype confirmed by positive immunostaining for ALDH3A1 and negative staining for Thy1) and fibroblasts (passage 4-6) cultured in 10% FBS. Cells were treated for 24 hours with 0.01 - 0.1% latanoprost free-acid (LFA) plus methyl acetate (MA). Stock 1% LFA dissolved in MA was diluted with serum-free culture producing a ratio of LFA to MA in all treatments of 1:100. A parallel experiment was performed with equivalent concentration of MA without the LFA. BAC (0.02%) was used as a positive control. At the end of 24 hours, cytotoxicity was assayed using the WST-1 reagent.
Results: :
Both LFA+MA and MA alone produced a dose-dependent cytotoxic effect with no statistical difference between the two. Notable cytotoxicity (60% viability or less) began to be observed at 0.04% LFA/4% MA. In both LFA+MA and MA alone the keratocytes showed a higher percentage (8-14%) of cell viability than fibroblasts at the highest treatment concentration tested (p=0.0613, 0.0024 respectively). The 0.01% LFA+1% MA concentration showed minimal (approx. 10%) loss of cell viability as compared to the untreated cells. Morphology of post-treated cells was indicative of a different mechanism of cell death comparing BAC to MA.
Conclusions: :
The cytotoxicity of LFA up to the point at which the MA solvent began to show toxicity was tested. Results show that MA is much less toxic to cells than previous reports of BAC and helps to substantiate that toxic effects noted from latanoprost/BAC medications are due to the BAC rather than the latanoprost. It is apparent that LFA has minimal cytoxic effect at, and below 0.01%, twice the amount contained in the topically applied drug Xalatan, and is presumably several times greater than the concentration stromal cells would be exposed to during daily topical application. It was interesting to note that keratocytes showed greater resiliency against the higher concentrations than fibroblasts.
Keywords: cornea: stroma and keratocytes • drug toxicity/drug effects • cell survival