Increased in–vitro Toxicity of Unpreserved Compared With Preserved HP–Guar

Abstract

Purpose: : The use of preservatives usually increases the toxicity of pharmaceutical tear substitutes such as hydroxypropylmethylcellulose (HPMC). HP–Guar has been introduced recently as a new artificial tear substitute. To reduce toxicity it is preserved with polyquad (0.001%), which is considered to be not toxic. We therefore examined the effect of preserved (cetrimide 0.01%) and unpreserved HPMC and HP–guar in dose– and time–response experiments in a human corneal epithelial cell culture model.

Methods: : The artificial tears were diluted in culture medium to the concentrations of 100, 50, 25, 12.5, 6.25 and 3.125%. SV–40 immortalised human conjunctival and corneal epithelial cells were cultured in 96–well plates at 37°C, 5% CO_2.. At 30% confluency the cell cultures were exposed to the test solutions and proliferation quantified by means of a luminescence–based ATP–assay. Cytotoxicity was assessed in cell cultures at 100% confluency incubated with the test solutions for 24 and 48 hours and was visualised with a double fluorescent viability staining (Calcein AM/Ethidium–homodimerD–1). In addition cell migration was assessed in a colony dispersion assay. All experiments were performed in triplicates and repeated at least once. The significance of differences in cell proliferation was determined with an unpaired two–sided t test.

Results: : HPMC with preservative was severely cytotoxic at all concentrations tested. Unpreserved HPMC however showed an inhibition of proliferation only at 100% and no toxicity in the fluorescent viability or colony dispersion assay. HP–Guar with and without preservative were found to be more toxic than unpreserved HPMC, but the unpreserved solution was found to reduce cellular ATP levels and to permeabilise cell membranes significantly more than the preserved solution.

Conclusions: : As expected unpreserved HPMC was found to be significantly less cytotoxic than the preserved solution. Paradoxcially HP–Guar without polyquad was found to be more toxic than the unpreserved solution. Possible explanations and limitations of the test model are discussed.

Keywords: cornea: epithelium • cornea: tears/tear film/dry eye • cell survival