The Cytotoxic Effects of Preserved and Preservative-Free Prostaglandin Analogs on Human Corneal and Conjunctival Epithelium In Vitro and the Distribution of Benzalkonium Chloride Homologs in Ocular Surface Tissues In Vivo

Abstract

Purpose: To investigate the cytotoxicity of benzalkonium chloride (BAC)-containing ophthalmic solutions of prostaglandin analogs (latanoprost, travoprost, bimatoprost, and preservative-free (PF) tafluprost), BAC mixture (BACmix) and BAC homologs with different alkyl chain lengths using human corneal epithelial (HCE) and conjunctival epithelial (IOBA-NHC) cell cultures. The distribution of BAC homologs in rabbit ocular surface tissues in vivo was examined.

Methods: The cells were exposed for one hour to prostaglandin analogs, BACmix and three homologs. Cytotoxicity was assessed with the WST-1 and lactate dehydrogenase (LDH) assays for cellular viability and cell membrane integrity. BAC 0.02% solution was instilled on the rabbit eye daily for 14 days and the concentrations of BAC homologs in external ocular tissues were determined.

Results: The order of decreasing cytotoxicity in the WST-1 test was latanoprost ≥ travoprost > bimatoprost ≥ PF tafluprost. IOBA-NHC cells were more sensitive than HCE cells. In HCE, only latanoprost diluted to 10% increased LDH leakage. In IOBA-NHC, LDH leakage was statistically significant with 3–10% travoprost and 10% latanoprost. The order of decreasing cytotoxicity of preservatives was C14 > C12 > BACmix > C16 in HCE and C12 > C14 > BACmix > C16 in IOBA-NHC. Following treatment with BAC 0.02% solution, the amounts of BAC-C12, -C14 and -C16 in rabbit cornea and conjunctiva, respectively were: 0.37 ± 0.08 and 2.64 ± 0.27 ng/mg; 0.42 ± 0.07 and 4.77 ± 0.43 ng/mg; 0.04 ± 0.01 and 0.54 ± 0.05 ng/mg.

Conclusions: The cytotoxic effects of latanoprost, travoprost, and bimatoprost were dependent on the BAC concentration in their formulations. BACmix was cytotoxic at the concentrations above those corresponding to 0.001% BAC in ophthalmic medications. PF tafluprost was the least toxic of the drugs tested. Within studied BAC homologs, those with longer alkyl chain and higher lipophility penetrated effectively into rabbit external ocular tissues.

Keywords::

• Prostaglandin analog
• Benzalkonium chloride
• Cytotoxicity
• Corneal epithelial cells
• Conjunctival epithelial cells
• Rabbit eye

ACKNOWLEDGEMENT

Mrs. Maija Koskela and Ms. Marja Mali are acknowledged for their technical assistance. Presented in part as E-poster at the 8^th International Symposium on Ocular Pharmacology and Therapeutics, Rome, Italy, December 2009.

Declaration of interest: The study was supported by the Finnish Association for Eye Research (Elsemay Björn Research Fund), the Eye and Tissue Bank Foundation, and the Competitive Research Funding of Pirkanmaa Hospital District Tampere University Hospital. Drs Huhtala and Uusitalo received a financial support from Santen Oy. Drs. Pellinen and Mäenpää and Mr. Lokkila are employees at Santen Oy.