Involvement of Human Blood Arylesterases and Liver Microsomal Carboxylesterases in Nafamostat Hydrolysis

Summary:

Metabolism of nafamostat, a clinically used serine protease inhibitor, was investigated with human blood and liver enzyme sources. All the enzyme sources examined (whole blood, erythrocytes, plasma and liver microsomes) showed nafamostat hydrolytic activity. V_max and K_m values for the nafamostat hydrolysis in erythrocytes were 278 nmol/min/mL blood fraction and 628 μM; those in plasma were 160 nmol/min/mL blood fraction and 8890 μM, respectively. Human liver microsomes exhibited a V_max value of 26.9 nmol/min/mg protein and a K_m value of 1790 μM. Hydrolytic activity of the erythrocytes and plasma was inhibited by 5, 5′-dithiobis(2-nitrobenzoic acid), an arylesterase inhibitor, in a concentration-dependent manner. In contrast, little or no suppression of these activities was seen with phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), bis(p-nitrophenyl)phosphate (BNPP), BW284C51 and ethopropazine. The liver microsomal activity was markedly inhibited by PMSF, DFP and BNPP, indicating that carboxylesterase was involved in the nafamostat hydrolysis. Human carboxylesterase 2 expressed in COS-1 cells was capable of hydrolyzing nafamostat at 10 and 100 μM, whereas recombinant carboxylesterase 1 showed significant activity only at a higher substrate concentration (100 μM). The nafamostat hydrolysis in 18 human liver microsomes correlated with aspirin hydrolytic activity specific for carboxylesterase 2 (r = 0.815, p < 0.01) but not with imidapril hydrolysis catalyzed by carboxylesterase 1 (r = 0.156, p = 0.54). These results suggest that human arylesterases and carboxylesterase 2 may be predominantly responsible for the metabolism of nafamostat in the blood and liver, respectively.