Abstract

Pilocarpine has been widely used in ophthalmic preparations for the treatment of glaucoma and in oral preparations for the treatment of radiation-induced xerostomia and Sjögren syndrome. The major metabolic pathways of pilocarpine in human are hydrolysis and hydroxylation. It was found that CYP2A6 is responsible for the 3-hydroxylation, but the enzymes responsible for the hydrolysis have not been characterized. In this study, we attempted to identify esterases responsible for pilocarpine hydrolysis. Pilocarpine hydrolase activities in human liver microsomes and plasma were stimulated by the addition of CaCl₂, suggesting that the calcium-dependent esterase, paraoxonase (PON), was responsible for pilocarpine hydrolysis. To confirm this hypothesis, the pilocarpine hydrolase activity was measured using the recombinant human PONs (PON1, PON2, and PON3) established in this study, and the result was that only PON1 showed pilocarpine hydrolase activity. The effect of PON1 polymorphism (Q192R) on pilocarpine hydrolase activity was analyzed using recombinant human PON1 192Q and 192R and human plasma from 50 volunteers. The results showed that recombinant PON1 192R revealed significantly higher catalytic efficiency than PON1 192Q. In human plasma, the activity of the R/R genotype (117.0 ± 25.2 pmol · min⁻¹ · μl⁻¹, n = 23) was significantly higher than those of the Q/R and Q/Q genotypes (97.3 ± 21.0 pmol · min⁻¹ · μl⁻¹, n = 20 and 90.4 ± 26.2 pmol · min⁻¹ · μl⁻¹, n = 7, respectively). It is suggested that this polymorphism affects pilocarpine hydrolase activity. In this study, we found that human PON1 is the major enzyme for the catalytic efficiency of pilocarpine hydrolysis.