Sulfation of O-Demethyl Apixaban: Enzyme Identification and Species Comparison

Abstract

ABSTRACT:Apixaban, a potent and highly selective factor Xa inhibitor, is currently under development for treatment of arterial and venous thrombotic diseases. The O-demethyl apixaban sulfate is a major circulating metabolite in humans but circulates at lower concentrations relative to parent in animals. The aim of this study was to identify the sulfotransferases (SULTs) responsible for the sulfation reaction. Apixaban undergoes O-demethylation catalyzed by cytochrome P450 enzymes to O-demethyl apixaban, and then is conjugated by SULTs to form O-demethyl apixaban sulfate. Of the five human cDNA-expressed SULTs tested, SULT1A1 and SULT1A2 exhibited significant levels of catalytic activity for formation of O-demethyl apixaban sulfate, and SULT1A3, SULT1E1, and SULT2A1 showed much lower catalytic activities. In human liver S9, quercetin, a highly selective inhibitor of SULT1A1 and SULT1E1, inhibited O-demethyl apixaban sulfate formation by 99%; 2,6-dichloro-4-nitrophenol, another inhibitor of SULT1A1, also inhibited this reaction by >90%; estrone, a competitive inhibitor for SULT1E1, had no effect on this reaction. The comparable K m values for formation of O-demethyl apixaban sulfate were 41.4 M (human liver S9), 36.8 M (SULT1A1), and 70.8 M (SULT1A2). Because of the high level of expression of SULT1A1 in liver and its higher level of catalytic activity for formation of O-demethyl apixaban sulfate, SULT1A1 might play a major role in humans for formation of O-demethyl apixaban sulfate. O-Demethyl apixaban was also investigated in liver S9 of mice, rats, rabbits, dogs, monkeys, and humans. The results indicated that liver S9 samples from dogs, monkeys, and humans had higher activities for formation of O-demethyl apixaban sulfate than those of mice, rats, and rabbits.

Metabolism, pharmacokinetics and pharmacodynamics of the factor Xa inhibitor apixaban in rabbits

“…Three prominent metabolites M1, M2, and M14 were observed in the circulation of rabbits, of which M1 and M2 were inactive metabolites for inhibition of factor Xa activities [27]. M14 was a glucuronide conjugate and not likely to be an active metabolite for inhibition of factor Xa enzymes.…”

Comparative Metabolism of14C-Labeled Apixaban in Mice, Rats, Rabbits, Dogs, and Humans

“…This metabolite was found at much lower abundance relative to the parent in the plasma of mouse, rat, or dog. It is unclear what drives the difference in disposition and makes this metabolite a prominent circulating metabolite in humans, but not in animals as all species were capable of the demethylation and sulfate conjugation reactions based on the in vitro data (Wang et al, 2009). In addition, M1 was a prominent metabolite in rat bile.…”

“…In addition, M1 was a prominent metabolite in rat bile. Metabolites M1 and M2 were tested and would be inactive factor Xa inhibitors (Wang et al, 2009). Although M1 was disproportionately circulating in humans compared with animal species with the given doses, no additional safety testing was needed because it was a stable and inactive conjugation metabolite (Food and Drug Administration Guidance for Industry: Safety Testing of Drug Metabolites, February 2008, http:/www.fda.gov/Drugs/Guidances/).…”

In Vitro Assessment of Metabolic Drug-Drug Interaction Potential of Apixaban through Cytochrome P450 Phenotyping, Inhibition, and Induction Studies

“…The primary metabolic pathways of apixaban in humans included O-demethylation (M2) and hydroxylation (M4 and M7). M2 was further conjugated by sulfation to form a sulfate metabolite (M1) Wang et al, 2009). Other metabolites (M3, M5, and M6) previously identified as minor metabolites in animals and humans were also very minor metabolites formed in the in vitro incubations in this study and were not further evaluated.…”