ABSTRACT:The metabolism and disposition of [ 14 C]apixaban, an orally bioavailable, highly selective, and direct acting/reversible factor Xa inhibitor, was investigated in 10 healthy male subjects without (group 1, n ‫؍‬ 6) and with bile collection (group 2, n ‫؍‬ 4) after a single 20-mg oral dose. Urine, blood, and feces samples were collected from all subjects. Bile samples were also collected for 3 to 8 h after dosing from group 2 subjects. There were no serious adverse events or discontinuations due to adverse effects. In plasma, apixaban was the major circulating component and O-demethyl apixaban sulfate, a stable and water-soluble metabolite, was the significant metabolite. The exposure of apixaban (C max and area under the plasma concentration versus time curve) in subjects with bile collection was generally similar to that in subjects without bile collection. The administered dose was recovered in feces (group 1, 56.0%; group 2, 46.7%) and urine (group 1, 24.5%; group 2, 28.8%), with the parent drug representing approximately half of the recovered dose. Biliary excretion represented a minor elimination pathway (2.44% of the administered dose) from group 2 subjects within the limited collection period. Metabolic pathways identified for apixaban included O-demethylation, hydroxylation, and sulfation of hydroxylated O-demethyl apixaban. Thus, apixaban is an orally bioavailable inhibitor of factor Xa with elimination pathways that include metabolism and renal excretion.Thromboembolic events, including acute myocardial infarction, unstable angina, deep vein thrombosis, pulmonary embolism, and ischemic stroke continue to be the leading cause of morbidity and mortality in the United States and other Western countries (Heit et al., 2005;Rosamond et al., 2007). Current therapies for the treatment and prevention of thromboembolic events, such as vitamin K antagonists (e.g., warfarin), heparin, and low-molecular-weight heparin (e.g., enoxaparin), are suboptimal (O'Donnell and Weitz, 2004;Wittkowsky, 2004;Campbell, 2006). However, the requirement for intravenous or subcutaneous injection and/or the need for careful monitoring because of the risk of excessive bleeding or unpredictable/inconsistent pharmacokinetics (PK) can complicate administration and present barriers to the use of these agents (O'Brien and Caro, 2002;Wittkowsky, 2004;Campbell, 2006). Therefore, new, orally active anticoagulants with predictable pharmacokinetic profiles that can be administered with a reduced need for monitoring are needed.Factor Xa is a key serine protease in the coagulation cascade and is a promising target enzyme for new therapeutic agents for the treatment and prevention of arterial and venous thrombosis (Kaiser, 2002;Samama, 2002;Walenga et al., 2003). In particular, factor Xa plays a critical role in blood coagulation, serving as the juncture between the extrinsic (tissue factor initiated) and intrinsic (surface activation and amplification) systems (Mann et al., 2003). Factor Xa forms the prothrombinase complex with phospholi...

ABSTRACT:Apixaban is an oral, direct, and highly selective factor Xa inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. The metabolic drug-drug interaction potential of apixaban was evaluated in vitro. The compound did not show cytochrome P450 inhibition (IC 50 values >20 M) in incubations of human liver microsomes with the probe substrates of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4/5. Apixaban did not show any effect at concentrations up to 20 M on enzyme activities or mRNA levels of selected P450 enzymes (CYP1A2, 2B6, and 3A4/5) that are sensitive to induction in incubations with primary human hepatocytes. Apixaban showed a slow metabolic turnover in incubations of human liver microsomes with formation of O-demethylation (M2) and hydroxylation products (M4 and M7) as prominent in vitro metabolites. Experiments with human cDNA-expressed P450 enzymes and P450 chemical inhibitors and correlation with P450 activities in individual human liver microsomes demonstrated that the oxidative metabolism of apixaban for formation of all metabolites was predominantly catalyzed by CYP3A4/5 with a minor contribution of CYP1A2 and CYP2J2 for formation of M2. The contribution of CYP2C8, 2C9, and 2C19 to metabolism of apixaban was less significant. In addition, a human absorption, distribution, metabolism, and excretion study showed that more than half of the dose was excreted as unchanged parent (f m CYP <0.5), thus significantly reducing the overall metabolic drug-drug interaction potential of apixaban. Together with a low clinical efficacious concentration and multiple clearance pathways, these results demonstrate that the metabolic drug-drug interaction potential between apixaban and coadministered drugs is low.Apixaban (Fig. 1), a novel and highly selective inhibitor of factor Xa (Luettgen et al., 2006;Pinto et al., 2007;Wong et al., 2008), is under development for the treatment and prevention of thromboembolic disorders, prevention of stroke in patients with atrial fibrillation, and secondary prevention in patients with acute coronary syndromes (APPRAISE Steering Committee and Investigators, 2009). In addition to demonstrating high oral availability (Frost et al., 2007), clinical studies of apixiban have shown prevention of venous thromboembolic events in patients after knee replacement surgery (Lassen et al., 2007). Apixaban is efficacious and well tolerated in the treatment of patients with acute symptomatic deep vein thrombosis (Buller et al., 2008). After oral administration, apixaban was slowly metabolized and thus was mostly unchanged in the circulation although apixaban was metabolized by multiple pathways in animals and humans (Zhang et al., , 2010. 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 als...

The in vitro and in vivo disposition of DPC 423 was investigated in mice, rats, dogs and humans and the metabolites characterized by LC/MS, LC/NMR and high field-NMR. The rodents produced several metabolites that included an aldehyde (M1), a carboxylic acid (M2), a benzyl alcohol (M3), glutamate conjugates (M4 and M5), an acyl glucuronide (M6) and its isomers; a carbamyl glucuronide (M7); a phenol (M8) and its glucuronide conjugate (M9), two glutathione adducts (M10 and M11), a sulfamate conjugate (M12), isomers of an oxime metabolite (M13), and an amide (M14). Humans and dogs produced less complex metabolite profiles than rats. While unchanged DPC 423 was the major component in plasma and urine samples, differences in the metabolic disposition of this compound among species were noted. M1 is believed to be rapidly oxidized to the carboxylic acid (M2), which forms the potentially reactive acyl glucuronide (M6). The formation of novel glutamate conjugates (M4 and M5) and their role in depleting endogenous glutathione have been described previously. The carbamyl glucuronide M7, found as the major metabolite in rats and in other species, was considered nonreactive and was easily hydrolyzed to the parent compound in the presence of beta-glucuronidase. The identification of GSH adducts M10 and M11 led us to postulate the existence of at least two reactive intermediates responsible for their formation, an epoxide and possibly a nitrile oxide, respectively. Although the formation of GSH adducts such as M10 from epoxides has been described before, there are no reports to date describing the existence of a GSH adduct (M11) of an oxime. The formation of a sulfamate conjugate (M12) formed by direct coupling of sulfate to the nitrogen of benzylamine is described. A mechanism is proposed for the formation of the oxime (M13) that involves sequential oxidation of the benzylamine to the corresponding hydroxylamine and nitroso intermediate. The rearrangement of the nitroso intermediate is believed to produce the oxime (M13). In vitro studies suggested that both the oxime (M13) and the aldehyde (M1) were precursors to the carboxylic acid (M2). This is the first demonstration of carboxylic acid formation via an oxime intermediate produced from an amine. The stability of DPC423 in plasma obtained from several species was studied. Significant species differences in the plasma stability of DPC 423 were observed. The formation of the aldehyde metabolite (M1) was found to be catalyzed by a semicarbazide-sensitive monoamine oxidase (SSAO) found in plasma of rabbits, dogs, and rhesus monkeys. Rat, chimpanzee, and human plasma did not form M1.

The in vivo and in vitro disposition of DPC 423, a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa, has recently been described. Several metabolites, some of which were considered potentially reactive, were identified in rats. A novel GSH adduct, the structure of which was not determined conclusively, was isolated from bile of rats dosed with DPC 423. Herein, we describe the complete structural elucidation of this unique GSH conjugate employing LC/MS and high-field NMR. Similar GSH adducts of DPC 602, [13CD2]DPC 602, and SX 737, all structural analogues of DPC 423, were isolated, characterized spectroscopically, and shown to have identical mass fragmentation pathways. The structures of these conjugates were initially suspected to be either an amide with N-S bond or a nitrogen-oxygen juxtaposed amide with a C-S bond. Studies conducted with [13CD2]DPC 602 indicated an aldoxime structure. The concluding evidence came from HMBC NMR spectrum of the conjugate, which showed strong correlation of the cysteine methylene protons with the imino carbon. Further spectroscopic studies with chemically prepared GSH adduct from benzaldehyde oxime confirmed this pattern of correlation. In vivo and in vitro studies with the synthetic oxime intermediate from DPC 423 showed an adduct identical to the one isolated from the bile of rats dosed with DPC 423. This supported the intermediacy of an aldoxime as a precursor to the GSH adducts. It is postulated that the benzylamine moiety of DPC 423 (and its analogues) is oxidized to a hydroxylamine, which is subsequently converted to a nitroso intermediate. Subsequent rearrangement of the nitroso leads to an aldoxime which in turn is metabolized by P450 to a reactive intermediate. The formation of oxime from DPC 423 (and its analogues) was found to be mediated by rat CYP 3A1/2, which were also responsible for converting the oxime to the GSH trappable reactive intermediate. It is postulated that the aldoxime produces a radical or a nitrile oxide intermediate that reacts with GSH and hence produces this unusual GSH adduct. On the basis of synthetic analogy, it is more likely that the nitrile oxide resulting from two-electron oxidation of the aldoxime is the reactive intermediate. Intramolecular kinetic isotope effects were studied with [13CD2]DPC 602 to assess the importance of the metabolic cleavage of the aminomethyl carbon-hydrogen bond in forming this GSH adduct. The lack of isotope effect in forming the aldoxime from [13CD2]DPC 602 suggests its formation does not occur through the imine intermediate. Instead the data supports the postulated mechanism of hydroxylamine and nitroso intermediates as precursors to the aldoxime. However, the formation of the GSH adduct from [13CD2]DPC 602 did show a significant intramolecular kinetic isotope effect (kH/kD = 2.3) since a carbon-deuterium bond had to be broken on the aldoxime prior to the formation of the adduct. A stable nitrile oxide derived from DPC 602 was postulated as the reactive intermediate respo...