N-Acetylation of Etamicastat, a Reversible Dopamine-β-Hydroxylase Inhibitor

Abstract

Etamicastat [(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione hydrochloride] is a reversible dopamineb-hydroxylase inhibitor that decreases norepinephrine levels in sympathetically innervated tissues. After in vivo administration, N-acetylation of etamicastat was found to be a main metabolic pathway. The purpose of the current study was to characterize the N-acetylation of etamicastat by N-acetyltransferases (NAT1 and NAT2) and evaluate potential species differences in etamicastat N-acetylation using a sensitive and specific liquid chromatographymass spectrometry assay. Marked differences in etamicastat N-acetylation were observed among the laboratory species and humans. After oral administration, the rat, hamster, and human subjects presented the highest rates of etamicastat N-acetylation, whereas almost no acetylation was observed in the mouse, rabbit, minipig, and monkey and no acetylation was observed in the dog.In in vitro studies, rats and humans showed similar acetylation rates, whereas no acetylation was detected in the dog. Studies performed with human recombinant NAT1 4 and NAT2 4 enzymes revealed that both were able to conjugate etamicastat, although at different rates. NAT1 had lower affinity compared with NAT2 (K m , 124.8 6 9.031 mM and 17.14 6 3.577 mM, respectively). A significant correlation (r 2 = 0.65, P < 0.05) was observed in a comparison of etamicastat N-acetylation by human single-donor enzymes and sulfamethazine, a selective substrate to NAT2. No correlation was observed with p-aminosalicylic acid, a NAT1 selective substrate. In conclusion, these results suggest that NAT2 and, to a lesser extent, NAT1 contribute to etamicastat N-acetylation. Furthermore, the high interspecies and intraspecies differences in N-acetylation should be taken into consideration when evaluating the in vivo bioavailability of etamicastat.

Structural and Kinetic Characterization of a Novel N-acetylated Aliphatic Amine Metabolite of the PRMT Inhibitor, EPZ011652

“…The kinetic parameters measured for these two isoforms show at least an eightfold lower K m and a 63-fold higher V max with NAT2 than for NAT1, resulting in a 500-fold higher intrinsic clearance by NAT2 (110 mL/min/mg). This is in agreement with the recent report of N-acetylation of the aminoethyl group of etamicastat mediated by NAT2, with a lower affinity for NAT1 (Loureiro et al, 2013). Although NAT1 intrinsic clearance is much lower, it could still play a role in nonhepatic tissues, as NAT1 mRNA is uniformly expressed, whereas NAT2 in mostly expressed in the liver and gut (Husain et al, 2007a(Husain et al, , 2007b.…”

“…Because the capacity of NAT2 varies in humans, manifest as rapid, intermediate, and slow acetylator phenotypes (Hein, 2000), N-acetylation as the major elimination pathway can lead to variability in exposure in humans. This was the basis for the recent mechanistic work on the enzymology of etamicastat clearance, wherein high variability in etamicastat exposures in a rising, multiple dose study in healthy volunteers was dmd.aspetjournals.org associated with the polymorphic NAT2-mediated N-acetylation pathway (Nunes et al, 2010;Loureiro et al, 2013). The extent of systemic exposure to etamicastat in NAT2 slow acetylators was 1.5-6.7 times higher than in NAT2 fast acetylators, whereas exposure to etamicastat N-acetyl metabolite was 1.5-3.5 times higher in rapid NAT2 acetylators; thus, etamicastat pharmacokinetics were markedly affected by the NAT2 phenotype (Nunes et al, 2010).…”

Etamicastat, a new dopamine-ß-hydroxylase inhibitor, pharmacodynamics and metabolism in rat

“…However, BIA 5-998 was considered a minor metabolite in humans accounting for less than 1% of etamicastat related metabolites, thus providing additional evidence for inter-species differences in etamicastat metabolism. Intra and inter-species differences in etamicastat N-acetylation were previously observed (Loureiro et al, 2013a).…”

A Pharmacokinetic Modeling Approach to Predict the Contribution of Active Metabolites to Human Efficacious Dose

“…However, this does not rule out the potential for uptake-mediated clearance of MRK-1, and there are no data available on the in vivo clearance pathways for M1 and M2. The lack of conversion of M1 to M2 in dog hepatocytes was consistent with known N-acetylation species differences (Tibbitts, 2003;Gao et al, 2006;Loureiro et al, 2013). Further qualification of the PK model using rat data would therefore clearly have been desirable; however, the metabolism of M1 to M2 observed in rat blood, but not in dog or human blood, rendered such a validation questionable both in terms of feasibility and value.…”