Abstract
Abstract: :
Purpose: It has been reported that dry-eye patients have reduced levels of mucin, a secretion found in tear fluid that contributes to the stability of the tear. Various techniques are typically used to measure mucin levels, including methods that detect the components of the constituent sugar of mucin and ELISA methods for measuring anti-mucin antibodies. We previously reported results from a study that used the alcian blue dying method based on the fact that alcian blue dye forms a complex with glycosaminoglycan and that OPC-12759 ophthalmic suspensions increase the mucin levels on the ocular surface. In this report, we investigate the mucin increasing action of an OPC-12759 ophthalmic suspension using a combination of gel filtration and enzyme-linked lectin assay (ELLA) methods to measure mucin levels. Methods: A 1% OPC-12759 ophthalmic suspension was applied 6 times a day for a period of 14 days to rabbits with reduced ocular mucin levels as a result of pre-treatments with N-acetylcysteine (NAC). Following the final administration of the test substances, tissues were collected, samples from the extracted rabbit corneas, conjunctivae, and tear fluid were filtered, and the filtrate from the mucin peaks in the vicinity of the void volumes were collected. Mucin levels were measured using an ELLA method that utilizes the lectin wheat germ agglutinin (WGA). Results: Corneal and conjunctival mucin levels were significantly reduced in NAC-treated eyes when compared with the normal eyes. In the cornea of the NAC-treated eyes, the average mucin level in the group treated with a 1% OPC-12759 ophthalmic suspension was 603.3397.16 ng, while the level in the vehicle treated group was 1460.67199.05 ng. OPC-12759 also increased conjunctival mucin levels by a factor of about 1.8 when compared with the vehicle treated group. In contrast, we found no significant differences between the levels of tear fluid mucin in the normal eye and in the NAC treated eye. Conclusion: We consider the methods used in this experiment to measure mucins to be of high specificity because the mucins we measured using gel filtration were of masses greater than 667kDa and because of the lectin WGA used in the ELLA method is able to detect N-acetylglucosamine and sialic acid, major components of the constituent sugars of mucin. With these methods we were able to confirm increased levels of mucin on the cornea and conjunctiva after treatment with the OPC-12759 ophthalmic suspension. We therefore consider that the OPC-12759 ophthalmic suspension has the potential to become a new type of therapeutic agent with a mucin increasing action suitable for treating dry eye.
Keywords: 390 drug toxicity/drug effects • 375 cornea: surface mucins • 376 cornea: tears/tear film/dry eye