Robert M. Knabb

Comparative Metabolism of 14C-Labeled Apixaban in Mice, Rats, Rabbits, Dogs, and Humans

The metabolism and disposition of [14C]apixaban, a potent, reversible, and direct inhibitor of coagulation factor Xa, were investigated in mice, rats, rabbits, dogs, and humans after a single oral administration and in incubations with hepatocytes. In plasma, the parent compound was the major circulating component in mice, rats, dogs, and humans. O-Demethyl apixaban sulfate (M1) represented approximately 25% of the parent area under the time curve in human plasma. This sulfate metabolite was present, but in lower amounts relative to the parent, in plasma from mice, rats, and dogs. Rabbits showed a plasma metabolite profile distinct from that of other species with apixaban as a minor component and M2 (O-demethyl apixaban) and M14 (O-demethyl apixaban glucuronide) as prominent components. The fecal route was a major elimination pathway, accounting for >54% of the dose in animals and >46% in humans. The urinary route accounted for <15% of the dose in animals and 25 to 28% in humans. Apixaban was the major component in feces of every species and in urine of all species except rabbit. M1 and M2 were common prominent metabolites in urine and feces of all species as well as in bile of rats and humans. In vivo metabolite profiles showed quantitative differences between species and from in vitro metabolite profiles, but all human metabolites were found in animal species. After intravenous administration of [14C]apixaban to bile duct-cannulated rats, the significant portion (approximately 22%) of the dose was recovered as parent drug in the feces, suggesting direct excretion of the drug from gastrointestinal tracts of rats. Overall, apixaban was effectively eliminated via multiple elimination pathways in animals and humans, including oxidative metabolism, and direct renal and intestinal excretion.

Isoxazolines and isoxazoles as Factor Xa inhibitors

3,4,5-Trisubstituted isoxazolines (2) and isoxazoles (3) were prepared and evaluated for their in vitro and in vivo antithrombotic efficacy. They were compared to 3,5,5-trisubstituted isoxazolines (1) for Factor Xa selectivity and potency. They were also compared in an arterio-venous (A-V) shunt model of thrombosis.

Nonpeptide Factor Xa Inhibitors III: Effects of DPC423, an Orally-Active Pyrazole Antithrombotic Agent, on Arterial Thrombosis in Rabbits

DPC423 [1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2′-(methylsulfonyl)[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide] is a synthetic, competitive, and selective inhibitor of coagulation factor Xa (fXa) (K i: 0.15 nM in humans, 0.3 nM in rabbit). The objective of this study was to compare effects of DPC423, enoxaparin (low-molecular-weight heparin), and argatroban (thrombin inhibitor) on arterial thrombosis and hemostasis in rabbit models of electrically induced carotid artery thrombosis and cuticle bleeding, respectively. Compounds were infused i.v. continuously from 60 min before artery injury or cuticle transection to the end of experiment. Carotid blood flow was used as a marker of antithrombotic effect. Antithrombotic ED50 values were 0.4 mg/kg/h for enoxaparin (n = 6), 0.13 mg/kg/h for argatroban (n = 6), and 0.6 mg/kg/h for DPC423 (n = 12). DPC423 at the maximum antithrombotic dose increased activated partial thromboplastin time and prothrombin time (n = 6) by 1.8 ± 0.07- and 1.8 ± 0.13-fold, respectively, without changes in thrombin time and ex vivo thrombin activity. The antithrombotic effect of DPC423 was significantly correlated with its ex vivo anti-fXa activity (r = 0.86). DPC423 at 1, 3, and 10 mg/kg p.o. increased carotid blood flow (percent control) at 45 min to 10 ± 4, 24 ± 6, and 74 ± 7, respectively (n= 6/group). Cuticle bleeding times (percent change over control) determined at the maximum antithrombotic dose were 88 ± 12 for argatroban, 69 ± 13 for heparin, 4 ± 3 for enoxaparin, 5 ± 4 for DPC423, and −3 ± 2 for the vehicle (n = 5–6/group), suggesting dissociation of antithrombotic and bleeding time effects for DPC423 and enoxaparin. The combination of aspirin and DPC423 at ineffective antithrombotic doses produced significant antithrombotic effect. Therefore, these results suggest that DPC423 is a clinically useful oral anticoagulant for the prevention of arterial thrombosis.

Rational design of boropeptide thrombin inhibitors: β,β-dialkyl-phenethylglycine P2 analogs of DuP 714 with greater selectivity over complement factor I and an improved safety profile

The potent boropeptide thrombin inhibitor DuP 714 caused side effects in laboratory animals that appear to be related to its ability to inhibit complement factor I, thereby activating the complement cascade. Using X-ray crystal structure information, we have designed compounds that have greater selectivity for thrombin over factor I and that have reduced tendency to produce these side effects.

Sulfation of O-demethyl apixaban: Enzyme identification and species comparison

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 Km values for formation of O-demethyl apixaban sulfate were 41.4 μM (human liver S9), 36.8 μM (SULT1A1), and 70.8 μM (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.

Synthesis and SAR of benzamidine factor Xa inhibitors containing a vicinally-substituted heterocyclic core.

The selective inhibition of coagulation factor Xa has emerged as an attractive strategy for the discovery of novel antithrombotic agents. Here we describe highly potent benzamidine factor Xa inhibitors based on a vicinally-substituted heterocyclic core.

Pyrazoles, 1,2,4-triazoles, and tetrazoles as surrogates for cis-amide bonds in boronate ester thrombin inhibitors.

Substituted pyrazoles, 1,2,4-triazoles, and tetrazoles are good surrogates for cis-amide bonds in a series of boronate ester thrombin inhibitors.

DESIGN AND SYNTHESIS OF RING-CONSTRAINED BOROPEPTIDE THROMBIN INHIBITORS

Abstract Ring-constrained boropeptide thrombin inhibitors were designed using information from the X-ray crystal structure of 1 (3-Phenylpropionyl-Pro-boroLys-OH ·HCl) bound to thrombin. The constraints utilized cyclohexane and pyrrolidine rings to preorganize an aromatic ring in an orientation allowing optimum edge-to-face interaction with the tryptophan 215 side chain located in the S3 specificity pocket of thrombin.

Anticoagulant activity of a peptide boronic acid thrombin inhibitor by various routes of administration in rats

The peptide boronic acid analog Ac-(D)Phe-Pro-boroArg-OH (I) is a potent and selective inhibitor of thrombin. The objective of this study was to determine whether I is active orally or when administered by alternative transmucosal routes. The measured effect was the time for clotting of plasma after initiation with thrombin. With this assay there was a narrow window from no measurable effect to the maximal effect, a clotting time greater than 300 seconds. Intravenous I at a 0.15 mg/kg dose in rats, a nasal 0.45 mg/kg dose, and 3 mg/kg doses administered orally, colonically, or rectally all produced maximal effects. Therefore, although bioavailability cannot be estimated, it is demonstrated that this peptide analog was absorbed by each of these routes.

Reductive Isoxazole Ring Opening of the Anticoagulant Razaxaban Is the Major Metabolic Clearance Pathway in Rats and Dogs

Razaxaban is a selective, potent, and orally bioavailable inhibitor of coagulation factor Xa. The molecule contains a 1,2-benzisoxazole structure. After oral administration of [14C]razaxaban to intact and bile duct-cannulated rats (300 mg/kg) and dogs (20 mg/kg), metabolism followed by biliary excretion was the major elimination pathway in both species, accounting for 34 to 44% of the dose, whereas urinary excretion accounted for 3 to 13% of the dose. Chromatographic separation of radioactivity in urine, bile, and feces of rats and dogs showed that razaxaban was extensively metabolized in both species. Metabolites were identified on the basis of liquid chromatography/tandem mass spectrometry and comparison with synthetic standards. Among the 12 metabolites identified, formation of an isoxazole-ring opened benzamidine metabolite (M1) represented a major metabolic pathway of razaxaban in rats and dogs. However, razaxaban was the major circulating drug-related component (>70%) in both species, and M1, M4, and M7 were minor circulating components. In addition to the in vivo observations, M1 was formed as the primary metabolite in rat and dog hepatocytes and in the rat liver cytosolic fraction. The formation of M1 in the rat liver fraction required the presence of NADH. Theses results suggest that isoxazole ring reduction, forming a stable benzamidine metabolite (M1), represents the primary metabolic pathway of razaxaban in vivo and in vitro. The reduction reaction was catalyzed by NADH-dependent reductase(s) in the liver and possibly by intestinal microflora on the basis of the recovery of M1 in feces of bile duct-cannulated rats.