No changes in rabbit corneal esterase activities with dipivefrine hydrochloride instillation for 4 weeks.

Abstract

Dipivefrine hydrochloride (DPE), a prodrug of epinephrine, is hydrolyzed by corneal esterases [1] and especially by cholinesterase [13]. It is known that some types of esterase in liver are induced by both phenobarbital administration as well as the enzyme responsible for its degradation [10, 14, 15]. Since DPE is used in long-term glaucoma therapy, it is possible that its efficacy is decreased as a result of an alteration in esterase activity. In the present study we considered whether the instillation of DPE four times a day for 4 weeks in the rabbit cornea changes the activity of either cholinesterase (EC 3.1.1.8), acetylcholinesterase (EC 3.1.17), a mixture containing carboxylesterase (EC 3.1.1.1), acetylesterase (EC 3.1.1.6), and arylesterase (EC 3.1.1.2), or a nonspecific esterase. We also determined whether the rate of hydrolysis of DPE changed over this period. Male Japanese white rabbits weighing 2-3 kg were kept in an air-conditioned room and allowed free access to standard laboratory chow and water. Animal experiments conformed to the guidelines of the ARVO Resolution on the Use of Animals in Research. Rabbits were divided into two groups of equal weight. One group was instilled with one drop ofa 0.1% DPE solution four times a day in both eyes while the control group was instilled with its vehicle. After instillation for either 1, 2, 3, or 4 weeks, at each time the corneas of five rabbits were removed and the esterase activities determined as described below. The isolated cornea was rinsed with a physiological saline buffer and then homogenized in an ice-cold isotonic potassium chloride solution in a PotterElvehjem glass homogenizer. The homogenate was centrifuged at 10000 g and the supernatant used to assay for esterase activities. Cholinesterase and acetylcholinesterase were determined using butyrylthiocholine (BuSCh) and acetylthiocholine (ASCh) as specific substrates, respectively [2, 7]. The assay mixture contained either BuSCh or ASCh, corneal enzyme sample, 5,5'-dithiobis-(2-nitrobenzoic acid; 0.21 mM) and phosphate buffer (4 raM, pH 7.2). An aliquot of the homogenate was added to this mixture and incubated at 37°C for 10 min. The reaction was stopped by the addition of quinidine sulfate (0.55 raM), and the production of 5-thio-2-benzoic acid was measured in an absorption spectrophotometer (Shimadzu, UV-190) at 412 nm.