Nafamostat is hydrolysed by human liver cytosolic long-chain acyl-CoA hydrolase

Abstract

Although the authors recently reported that nafamostat, a clinically used serine protease inhibitor, was mainly hydrolysed by carboxylesterase in human liver microsomes, the involvement of human liver cytosol has not been elucidated. The current study examined the in vitro metabolism of nafamostat with human liver cytosols. Kinetic analysis indicated that the V_max and K_m values in the liver cytosols were 9.82 nmol min⁻¹ mg⁻¹ protein and 197 µM for a liver sample HL-1, and 15.1 nmol min⁻¹ mg⁻¹ protein and 157 µM for HL-2, respectively. The V_max/K_m values in both cytosols were at least threefold higher than those in the corresponding microsomes. The liver cytosolic activity for nafamostat hydrolysis was inhibited by phenylmethylsulfonyl fluoride (PMSF) (43% inhibition at 100 µM), whereas diisopropyl fluorophosphate (DFP) and bis(p-nitrophenyl)phosphate (BNPP) failed to inhibit the activity. Furthermore, the hydrolytic activity was also reduced by palmitoyl-CoA (67% inhibition at 100 µM) but not by acetyl-CoA. Effects of PMSF, DFP and BNPP on cytosolic palmitoyl-CoA hydrolytic activity were comparable with those of the cytosolic nafamostat hydrolytic activity. In addition, the palmitoyl-CoA hydrolytic activity was competitively inhibited by nafamostat with the apparent K_i value of 164 µM for the liver cytosol from HL-2. These results suggest that an isoform of long-chain acyl-CoA hydrolase may be responsible for the nafamostat hydrolysis in human liver cytosol.

Key Words::

• Nafamostat
• hydrolysis
• human liver cytosol
• long-chain acyl-CoA hydrolase

Acknowledgements

This work was supported by the ‘Academic Frontier’ Project for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (2005–09), the Special Research Fund of Hokuriku University and the Torii Pharmaceutical Co., Ltd.