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Metabolism and disposition of the DOT1L inhibitor, pinometostat (EPZ-5676), in rat, dog and human



The metabolism and disposition of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was investigated in rat and dog. Metabolite profiles were compared with those from adult patients in the first-in-man phase 1 study as well as the cross-species metabolism observed in vitro.


EPZ-5676 was administered to rat and dog as a 24-h IV infusion of [14C]-EPZ-5676 for determination of pharmacokinetics, mass balance, metabolite profiling and biodistribution by quantitative whole-body autoradiography (QWBA). Metabolite profiling and identification was performed by radiometric and LC–MS/MS analysis.


Fecal excretion was the major route of elimination, representing 79 and 81 % of the total dose in and rat and dog, respectively. QWBA in rats showed that the radioactivity was well distributed in the body, except for the central nervous system, and the majority of radioactivity was eliminated from most tissues by 168 h. Fecal recovery of dose-related material in bile duct-cannulated animals as well as higher radioactivity concentrations in the wall of the large intestine relative to liver implicated intestinal secretion as well as biliary elimination. EPZ-5676 underwent extensive oxidative metabolism with the major metabolic pathways being hydroxylation of the t-butyl group (EPZ007769) and N-dealkylation of the central nitrogen. Loss of adenine from parent EPZ-5676 (M7) was observed only in rat and dog feces, suggesting the involvement of gut microbiota. In rat and dog, steady-state plasma levels of total radioactivity and parent EPZ-5676 were attained rapidly and maintained through the infusion period before declining rapidly on cessation of dosing. Unchanged EPZ-5676 was the predominant circulating species in rat, dog and man.


The excretory and metabolic pathways for EPZ-5676 were very similar across species. Renal excretion of both parent EPZ-5676 and EPZ-5676-related material was low, and in preclinical species fecal excretion of parent EPZ-5676 and EPZ007769 accounted for the majority of drug-related elimination.

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Bile duct cannulated


Histone methyltransferase


High performance liquid chromatography




Lower limit of quantitation


Mass spectrometry


Start of infusion


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The authors thank Jessica Shoultz and Shelby Anderson at Q2 Solutions, and Steve Madden, Alda Pombo-Gentile and Sandra Dennis at Charles River Labs, for their technical and scientific support to these studies.

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Correspondence to Nigel J. Waters.

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NJW, SS, EJO, RMP, MPM and RC are all previous or current employees of Epizyme and hold stock in Epizyme.

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Waters, N.J., Smith, S.A., Olhava, E.J. et al. Metabolism and disposition of the DOT1L inhibitor, pinometostat (EPZ-5676), in rat, dog and human. Cancer Chemother Pharmacol 77, 43–62 (2016).

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  • MLL-r leukemia
  • Drug metabolism
  • Drug disposition
  • Histone methyltransferase inhibitor
  • DOT1L
  • Epigenetics