N-acetyltransferase 2 genotype-dependent N-acetylation of hydralazine in human hepatocytes

Abstract

Hydralazine is used in the treatment of essential hypertension and is under investigation for epigenetic therapy in the treatment of neoplastic and renal diseases. N-acetyltransferase (NAT) 2 exhibits a common genetic polymorphism in human populations. After recombinant expression in yeast, human NAT2 exhibited an apparent Lineweaver-Burk constant (Km) value (20.1 ± 8.8 μM) for hydralazine over 20-fold lower than the apparent Km value (456 ± 57 μM) for recombinant human NAT1 (P = 0.0016). The apparent Vmax value for recombinant human NAT1 (72.2 ± 17.9 nmol acetylated/min/mg protein) was significantly (P = 0.0245) lower than recombinant human NAT2 (153 ± 15 nmol acetylated/min/mg protein), reflecting 50-fold higher clearance for recombinant human NAT2. Hydralazine NAT activities exhibited a robust acetylator gene dose response in cryopreserved human hepatocytes both in vitro and in situ. Hydralazine NAT activities in vitro differed significantly with respect to NAT2 genotype at 1000 (P = 0.0319), 100 (P = 0.002), and 10 μM hydralazine (P = 0.0029). Hydralazine NAT activities differed significantly (P < 0.001) among slow acetylator hepatocytes, (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A). The in situ hydralazine N-acetylation rates differed significantly with respect to NAT2 genotype after incubation with 10 (P = 0.002) or 100 μM (P = 0.0015) hydralazine and were higher after incubation with 100 μM (10-fold) than with 10 μM (4.5-fold) hydralazine. Our results clearly document NAT2 genotype–dependent N-acetylation of hydralazine in human hepatocytes, suggesting that hydralazine efficacy and safety could be improved by NAT2 genotype–dependent dosing strategies.