title: Structural and Kinetic Characterization of a Novel N-acetylated Aliphatic Amine Metabolite of the PRMT Inhibitor, EPZ011652.
abstract: Pharmacokinetic and metabolite identification studies were conducted to understand the clearance pathways of EPZ011652 [(2-aminoethyl)(methyl)({3-[4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl}methyl)amine], a potent protein arginine N-methyltransferase inhibitor. Metabolic clearance was the major pathway of EPZ011652 elimination in rats with structural elucidation of metabolites via liquid chromatography - mass spectrometry (LC-MS(n)) accurate mass measurement revealing the formation of a novel aliphatic N-acetylated metabolite (M1) located on the terminal nitrogen of the ethylene-diamine side chain. EPZ015564, a synthetic standard of the N-acetyl product, was prepared and was also generated by human and rat, but not dog hepatocytes. In rat hepatocytes, on incubation with EPZ011652, the concentration of EPZ015564 initially increased before decreasing with incubation time, suggesting that the metabolite is itself a substrate for other metabolizing enzymes, in agreement with the identification of metabolites M2, M3, and M4 in rat bile, all N-acetylated metabolites, undergoing sequential phase I (demethylation, oxidation) or phase II (sulfation) reactions. Reaction phenotyping with recombinant human N-acetyltransferase (NAT) isoforms revealed that both NAT1 and NAT2 are capable of acetylating EPZ011652, although with different catalytic efficiencies. Kinetic profiles of EPZ015564 formation followed classic Michaelis-Menten behavior with apparent Km values of >1000 μM for NAT1 and 165 ± 14.1 µM for NAT2. The in vitro intrinsic clearance for EPZ011652 by NAT2 (110 μL/min/mg) was 500-fold greater than by NAT1. In summary, we report the unusual N-acetylation of an aliphatic amine and discuss the implications for drug discovery and clinical development.
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Nathalie
authors.initials: N
authors.lastname: Rioux
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Lorna H
authors.initials: LH
authors.lastname: Mitchell
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Philip
authors.initials: P
authors.lastname: Tiller
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Katie
authors.initials: K
authors.lastname: Plant
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Joanne
authors.initials: J
authors.lastname: Shaw
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Kerry
authors.initials: K
authors.lastname: Frost
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Scott
authors.initials: S
authors.lastname: Ribich
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Mikel P
authors.initials: MP
authors.lastname: Moyer
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Robert A
authors.initials: RA
authors.lastname: Copeland
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.).
authors.forename: Richard
authors.initials: R
authors.lastname: Chesworth
authors.affiliation: Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.) nwaters@epizyme.com.
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Waters
mesh_terms: D000588:Amines / Q000378:metabolism*; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism; D001646:Bile / Q000378:metabolism; D001711:Biotransformation; D004285:Dogs; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005029:Ethylenediamines / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D053858:Metabolic Networks and Pathways; D011484:Protein-Arginine N-Methyltransferases / Q000037:antagonists & inhibitors*; D011720:Pyrazoles / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000588:Amines; C000604935:EPZ011652; D004791:Enzyme Inhibitors; D005029:Ethylenediamines; D007527:Isoenzymes; D011720:Pyrazoles; D011484:Protein-Arginine N-Methyltransferases; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; C478900:NAT2 protein, human