Factor Xa (Ki = 0.08 nM and 0.17 nM for human and rabbit Factor Xa)

At concentrations (EC2x) of 3.6 μM, 0.37 μM, 7.4 μM, and 0.4 μM, respectively, apixaban (BMS-562247-01) prolongs the clotting times of normal human plasma, which is needed to double the prothrombin time (PT), modified prothrombin time (mPT), activated partial thromboplastin time (APTT), and HepTest. Furthermore, in both the PT and APTT tests, Apixaban exhibits the highest potency in human and rabbit plasma but less potency in rat and dog plasma[2].

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Selectivity and Liability Profiling. [1]
Compound 40/Apixaban shows a high degree of selectivity versus other proteases (see supplemental section), even compared to compounds 47a and 5.15a Additionally, the compound shows weak activity against various P450 isozymes (IC50 > 25μM) and weak activity against the hERG potassium channel (IC50 > 25 μM, patch clamp assay).26a-e The solubility of compound 40 was shown to be approximately 40−50 μg/mL.27 In the human liver microsome assay, compound 40 was very stable with a T1/2 of >100 min (the HLM T1/2 of 47 was not measured). The Caco-2 permeability values for compounds 40 (Papp = 0.9 × 10-6 cm s-1) and 47 (Papp = 2.5 × 10-6 cm s-1) were moderate to high.

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In vitro, Apixabanis potent and selective, with a K(i) of 0.08 nm for human FXa. It exhibited species difference in FXa inhibition [FXa K(i) (nm): 0.16, rabbit; 1.3, rat; 1.7, dog] and anticoagulation [EC(2x) (microm, concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 microm did not alter human and rabbit platelet aggregation to ADP, gamma-thrombin, and collagen. [2]

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Enzyme assays [2]
The Lineweaver–Burk plot of inhibition of human FXa by Apixaban indicates that apixaban is a competitive inhibitor vs. the chromogenic peptide substrate S‐2765, with a Ki of 0.08 nm (Fig. 1). Apixaban also inhibited FXa from rats, dogs, and rabbits (Table 1). In terms of FXa Ki at 25 °C, apixaban has similar potency in inhibiting human and rabbit FXa, but is 10–20 times less potent against rat and dog FXa (Table 1).

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Clotting assays [2]
As expected for an inhibitor of FXa, addition of Apixaban to normal human plasma prolonged clotting times, including APTT, PT, mPT, and HepTest. Among the three clotting time assays, it appears that the mPT and HepTest are 10–20 times more sensitive than APTT and PT in monitoring the in vitro anticoagulant effect of apixaban in human plasma (Table 1). In both the PT and APTT assays, apixaban had the highest potency in human and rabbit plasma, but was less potent in rat and dog plasma (Table 1).

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Platelet aggregation [2]
In vitro platelet aggregation responses to ADP, γ‐thrombin and collagen averaged 47 ± 5%, 53 ± 4% and 51 ± 5%, respectively in human PRP, and 50 ± 5%, 56 ± 5% and 60 ± 1%, respectively, in rabbit PRP. These platelet responses were not significantly changed by Apixaban at 1, 3 and 10 μm, but were almost completely inhibited by the GPIIb/IIIa antagonist DMP802 at 3 μm (data not shown).

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Pharmacodynamic effects of Apixaban in rabbit and human plasma in vitro [3]
As plasma Apixaban concentrations increased, in vitro FXa activity decreased (Fig. 1); concentration–response curves were similar in rabbits and humans. The IC50 values determined from the in vitro plasma samples spiked with various concentrations of apixaban are summarized in Table 1. IC50 values for rabbit and human plasma were equivalent: 0.25 μM (±0.01). The plasma concentrations required to double the PT and mPT were previously reported for human and rabbit plasma.

In dogs, Apixaban (BMS-562247-01) has good pharmacokinetics with a very low volume of distribution (Vdss: 0.2 L/kg) and a very low clearance (Cl: 0.02 L/kg/h). Additionally, Apixaban has a decent oral bioavailability (F: 58%) and a reasonable half-life (T1/2: 5.8 hours)[1]. Apixaban exhibits dose-dependent antithrombotic effects in rabbit models of arteriovenous shunt thrombosis (AVST), venous thrombosis (VT), and electronically mediated carotid arterial thrombosis (ECAT) with EC50 values of 270 nM, 110 nM, and 70 nM, respectively[2]. In rabbits ex vivo, apixaban dramatically reduces factor Xa activity with an IC50 of 0.22 μM[3]. Apixaban also exhibits low systemic clearance (Cl: 0.018 L/kg/h), a modest volume of distribution (Vdss: 0.17 L/kg), and good oral bioavailability (F: 59%) in chimpanzees[4].

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Dog Pharmacokinetics and Rabbit Antithrombotic Efficacy. [1]
As a result of the excellent in vitro potency and selectivity of compounds 40/Apixaban and 47, the pharmacokinetic profiles of both compounds were studied in dogs using a cassette dosing paradigm (“N-in-one” study, Table 6).7a,34a,b The acetylated N-methyl analogue 47 was orally bioavailable; but showed high clearance (Cl = 2.8 L kg-1 h-1), moderate volume of distribution (Vdss = 1.7 L kg-1), and unacceptable half life. The dog pharmacokinetics for compound 40 was outstanding with very low clearance (Cl = 0.02 L kg-1 h-1), and low volume of distribution (Vdss = 0.2 L kg-1). These values were significantly lower than those observed with compounds 4 and 5. FXa being a vascular target, the pharmacokinetic profile for compound 40 was viewed as highly desirable and less likely to have nontarget-related adverse effects. Importantly, compound 40 had a moderate half-life (T1/2 = 5.8 h) and good oral bioavailability (F = 58%). The human serum protein binding as measured by equilibrium dialysis for 40 was 87%.25 In the rabbit AVShunt thrombosis model (Figure 4), compound 40 inhibited thrombus formation in a dose-dependent manner with an IC50 value of 329 nM.15d,e This is comparable to the antithrombotic potency obtained for compound 4 (IC50 = 340 nM) in the same model.

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AVST model [2]
Mean thrombus weights in the different vehicle‐treated AVST rabbits were similar, and ranged from 290 ± 11 to 327 ± 15 mg (n = 6 per group). As shown in Fig. 2, Apixaban, fondaparinux and warfarin were efficacious in the AVST rabbits and produced dose‐dependent antithrombotic effects; their ED50 values are reported in Table 2. At their top doses studied in this model, apixaban at 3 mg kg−1 h−1 i.v., fondaparinux at 1 mg kg−1 h−1 i.v. and warfarin at 3 mg kg−1 day−1 p. o. reduced thrombus weight by 98%, 86% and 77%, respectively, relative to their corresponding vehicle group. We observed that apixaban at 0.03, 0.1, 0.3, 1 and 3 mg kg−1 h−1 i.v. produced linear dose‐proportional increases in plasma levels of 34 ± 2, 121 ± 9, 490 ± 104, 1155 ± 153 and 3705 ± 525 nm, respectively (n = 3–7 per group). The EC50 for apixaban was estimated to be 357 ± 90 nm.

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VT model [2]
Mean thrombus weights in the different vehicle‐treated VT rabbits were similar, and ranged from 64 ± 2 to 79 ± 7 mg (n = 6 per group). In this model, Apixaban, fondaparinux and warfarin produced dose‐dependent antithrombotic effects (Fig. 2); their ED50 values are given in Table 2. At their top doses studied in this model, apixaban at 1 mg kg−1 h−1, fondaparinux at 0.3 mg kg−1 h−1 and warfarin at 3 mg kg−1 day−1 reduced thrombus formation by 83%, 74% and 84%, respectively, relative to their corresponding vehicle group. Arterial thrombosis model Figure 3 (top panel) shows the effects of vehicle and apixaban on carotid blood flow after electrical stimulation. Basal carotid blood flow in the vehicle‐treated animals averaged 21 ± 4 mL min−1. After the initiation of thrombosis, blood flow was gradually decreased, and the artery was totally occluded in about 35 min in vehicle‐treated animals. Apixaban at 0.01–1 mg kg−1 h−1 i.v. produced a dose‐dependent increase in duration of the patency of the injured artery. At 0.03–1 mg kg−1 h−1 i.v., there was no occlusion in any of the animals up to 90 min.

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Ex vivo coagulation markers [2]
Figure 4 shows the summary of the ex vivo APTT and PT responses to Apixaban, fondaparinux and warfarin obtained from the AVST, VT and ECAT studies. Apixaban significantly prolonged ex vivo APTT at 3 mg kg−1 h−1, and PT at doses of 0.3 mg kg−1 h−1 and higher (Fig. 4). Fondaparinux at the doses studied did not have significant effects on ex vivo APTT and PT. Warfarin significantly prolonged ex vivo APTT at doses of 0.3 mg kg−1 day−1 and higher, and PT at doses of 0.1 mg kg−1 day−1 and higher (Fig. 4).

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Cuticle bleeding time model [2]
Mean cuticle BT in the vehicle‐treated groups of Apixaban, fondaparinux and warfarin were 172 ± 2, 181 ± 7 and 183 ± 7 s, respectively (n = 6 per group). Warfarin at 0.1, 0.3, 1 and 3 mg kg−1 day−1 p.o. produced dose‐dependent increases in BT (228 ± 14, 371 ± 24, 929 ± 70 and 1129 ± 43 s, respectively), with an ED3× of 0.70 mg kg−1 day−1 (Table 2). As compared to vehicle, warfarin at 1 and 3 mg kg−1 day−1 p.o. increased BT significantly by 5.1‐fold and 6.2‐fold, respectively (P < 0.05). In contrast, fondaparinux and apixaban at antithrombotic doses increased BT slightly (fondaparinux, 166 ± 4, 210 ± 12 and 213 ± 11 s at 0.3, 1 and 3 mg kg−1 h−1 i.v., respectively; apixaban, 191 ± 8 and 228 ± 14 s at 1 and 3 mg kg−1 h−1 i.v., respectively). At 3 mg kg−1 h−1 i.v., fondaparinux and apixaban increased BT by 1.3‐fold and 1.2‐fold relative to their vehicle, respectively with ED3× of > 3 mg kg−1 h−1 for both compounds (Table 2).

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Apixaban has similar affinity for human and rabbit factor Xa (FXa). Rabbits are commonly used in development of thrombosis disease models; however, unlike in other species, Apixaban demonstrated poor oral bioavailability (F = 3%) and a high clearance rate (2.55 l/h/kg) in rabbits. Oxidative metabolism of [14C] apixaban by liver microsomes was approximately 20 times faster in rabbits than in rats or humans. Following an intravenous (IV) dose of 5 mg/kg, circulating levels of [14C] apixaban decreased from the earliest sampling time (5 min) to undetectable at 4 h. After an oral dose of 30 mg/kg, levels of [14C] apixaban were only detected at 1 and 4 h. Radioactivity profiling showed that apixaban was a significant component in plasma only after IV administration; O-demethyl apixaban (M2), O-demethyl apixaban glucuronide (M14) and O-demethyl apixaban sulfate (M1) were prominent metabolites after both IV and oral administration. Studies of apixaban in rabbits showed a good correlation between apixaban concentrations and inhibition of FXa activity, prolongation of prothrombin time and modified prothrombin time, with no lag time between these ex vivo pharmacodynamic markers and plasma drug levels. The apixaban concentration required for 50% inhibition (IC50) of FXa activity ex vivo (0.22 +/- 0.02 microM) agreed with the IC50 from in vitro experiments in rabbit and human plasma. In summary, apixaban shows similar affinity for human and rabbit FXa. It produces a rapid onset of action, predictable concentration-dependent pharmacodynamic responses, and, unlike rats or humans, a rapid hepatic metabolism in rabbits.[3]

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Apixaban is a potent, highly selective, reversible, oral, direct factor Xa (fXa) inhibitor in development for thrombosis prevention and treatment. The preclinical pharmacokinetic (PK) attributes of Apixaban feature small volume of distribution (Vd), low systemic clearance (CL), and good oral bioavailability. Apixaban is well absorbed in rat, dog, and chimpanzee, with absolute oral bioavailability of approximately 50% or greater. The steady-state Vd of apixaban is approximately 0.5, 0.2, and 0.17 l/kg in rats, dogs, and chimpanzees, while CL is approximately 0.9, 0.04, and 0.018 l/h/kg, respectively. In vitro metabolic clearance of apixaban is also low. Renal clearance comprises approximately 10-30% of systemic clearance in rat, dog, and chimpanzee. Anti-fXa activity, prothrombin time (PT), and HEPTEST(®) clotting time (HCT) prolongation correlated well with plasma apixaban concentration in rat, dog and chimpanzee. There was no lag time between apixaban plasma concentration and the pharmacodynamic (PD) markers, suggesting a rapid onset of action of apixaban. The PK/PD analyses were performed using an inhibitory E (max) model for anti-fXa assay and a linear model for PT and HCT assays. The IC(50) values for anti-fXa activity were 0.73 ± 0.03 and 1.5 ± 0.15 μM for rat and dog, respectively. The apparent K ( i ) values for PT were approximately 1.7, 6.6, and 4.8 μM for rat, dog and chimpanzee, respectively. The apparent K ( i ) for HCT was approximately 1.3 μM for dog. Apixaban exhibits desirable PK and PD properties for clinical development with good oral bioavailability, small Vd, low CL, and direct, predictable, concentration-dependent PD responses [4].

Enzyme Affinity Assays. [1]
All enzyme Ki values were obtained from purified human enzymes. All fXa assays were run in microtiter plates using a total volume of 250 μL in 0.1 M sodium phosphate buffer containing 0.2 M NaCl and 0.5% polyethylene glycol 6000 at pH 7.0. The compounds were run at 10, 3.16, 1.0, 0.316, 0.1, 0.0316, 0.01, and 0.003 16 μM. Plates were read for 30 min at 405 nm. Rates were determined in the presence of the controls (no inhibitor) and for the inhibitors. Percent enzyme activity was determined from these rates and used in the following formula to determine Ki:
where S is the substrate concentration and ACT is the fraction of percent enzyme activity for inhibitor rates. All compounds were tested in duplicate studies and were compared with the same internal standards. The intraassay and interassay variabilities are 5% and 20%, respectively. These assays are described in detail in refs 28 and 29. All of the enzyme assays were conducted in pH 7.4 buffer at room temperature. All enzymes were purified from human tissues and were obtained from commercially available sources. Individual enzyme and substrate Km were determined in separate experiments and were close to values established in the literature. Steady-state inhibition of enzyme activity was determined by incubating a range of inhibitor concentrations (1 nM to 50 μM, in duplicate) with fixed enzyme (0.1−100 nM) and peptide substrate (200−1000 μM) concentration for up to 30 min. The Ki was calculated, assuming competitive inhibition and one-site binding, either from the IC50 or from the extent of inhibition at each inhibitor concentration.

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In Vitro Coagulation Assays (PT/APTT). [1]
Standard clotting assays were performed in a temperature-controlled automated coagulation device. Blood was obtained from healthy volunteers by venipuncture and anticoagulated with 1/10 volume of 0.11 M buffered sodium citrate. Plasma was obtained after centrifugation at 2000g for 10 min and kept on ice prior to use. An initial stock solution of the inhibitor at 10 mM was prepared in DMSO. Subsequent dilutions were done in plasma. Plasma solutions containing inhibitor were kept on ice prior to assay. Clotting time was determined on control plasma and plasma containing five to seven different concentrations of inhibitor. Determinations at each plasma concentration were done in duplicate. The clotting time at each concentration was compared with the control clotting time for each pooled plasma. The prothrombin time test was performed using Dade Thromboplastin C Plus according to the reagent instructions. Plasma (50 μL) was warmed to 37 °C for 3 min before adding Dade Thromboplastin C Plus (100 μL). The activated partial thromboplastin time (aPTT) was performed using AlexinTM according to the reagent instructions. Plasma (50 μL) was warmed to 37 °C for 1 min before adding aPTT reagent (50 μL). Three minutes later calcium chloride (50 μL) was added.

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Determination of FXa activity [3]
FXa activity was measured using a commercially available Factor X Kit containing assay buffer, Russell’s viper venom, calcium chloride and FXa chromogenic substrate. All reagents were prepared and used according to the package insert instructions. The buffer was 50 mM Tris buffer (pH 7.8) containing 20 mg/l polybrene (hexadimethrine bromide). For ex vivo samples, rabbit plasma at each time point was diluted 21-fold (10 μl + 200 μl buffer). For in vitro samples, pooled normal human or rabbit plasma was spiked to achieve final concentrations of Apixaban from 0.1 to 12.8 μM, then diluted 21-fold with assay buffer. Additional plasma dilutions, without inhibitor, were prepared to confirm that FXa activity was directly proportional to the amount of plasma added. In a 96-well microtiter plate (Costar 3474; Corning Inc., Lowell, MA), an aliquot of 50 μl diluted plasma was added and incubated at 37°C for 10 min inside the plate reader. An aliquot of 50 μl factor X substrate was then added and incubated at 37°C for 4 min inside the plate reader. Reactions were initiated by adding 50 μl Russell’s viper venom/calcium, which was pre-warmed to 37°C in a water bath. Hydrolysis of the substrate resulted in the release of para-nitroaniline, which was monitored spectrophotometrically by measuring the increase in absorbance at 405 nm every 15 s for up to 20 min. The rate of absorbance change, expressed as mOD/min (1000 times change in optical density per minute), is proportional to enzyme activity. Initial rates of substrate hydrolysis were used for analysis.

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Determination of anticoagulant activity[3]
Prothrombin time (PT) assays were performed according to manufacturer’s directions using an automated coagulation analyzer. The traditional PT assay measures the overall coagulation cascade reactions triggered through the extrinsic pathway. For this assay, an aliquot of 50 μl plasma was incubated at 37°C for 2 min, followed by addition of 100 μl PT reagent. The modified PT (mPT) assay was performed by diluting 1 ml Thromboplastin C Plus with 1.25 ml of 100 mM calcium chloride and using this diluted reagent in place of the normal PT reagent. Clotting time was recorded by the automated analyzer. Clotting times greater than 120 s were set equal to 120 s.

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Determination of protein binding[3]
Equilibrium dialysis was used to determine the protein binding of Apixaban in rabbit and human serum. All sera were thawed at room temperature and centrifuged at 2000×g for 10 min to remove residual clotted proteins. Dialysis membranes were preconditioned with water and, subsequently, with 0.133 M potassium phosphate buffer (pH 7.4). Serum containing 1, 3 or 10 μM apixaban (0.75 ml) was added to one side of the cell and an equal volume of buffer (0.133 M potassium phosphate buffer, pH 7.4) was added to the opposite side. Equilibrium was achieved by rotating the cells at 3–5 rpm at 37°C for 3 h. After incubation, aliquots of the serum and the buffer sides were collected separately and mixed with an equal volume of the opposite matrix.

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Enzyme assays [2]
Using established protein purification procedures, FX was isolated from citrated plasma obtained from healthy dogs, rats, and rabbits. Purified FXa was obtained after activation with Russell’s viper venom followed by affinity chromatography. The resulting FXa was > 95% pure as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis. The substrate affinity values for FXa, expressed as the Michaelis–Menten–Henri constant (Km), for human, rabbit, rat and dog FXa were determined using the chromogenic substrate S‐2765, and were 36, 60, 240 and 70 μm, respectively. The substrate hydrolysis was monitored by measuring absorbance at 405 nm at 25 °C for up to 30 min using a SpectraMax 384 Plus plate reader and SoftMax. FXa activity for each substrate and inhibitor concentration pair was determined in duplicate. The Ki values were calculated by non‐linear least‐squares fitting of the steady‐state substrate hydrolysis rates to the equation for competitive inhibition (Equation 1) using GRAFIT, where v equals reactions velocity in OD min−1, Vmax equals maxiumum reaction velocity, S equals substrate concentration, and I equals inhibitor concentration.

Clotting assays [2]
Blood samples were collected in tubes containing 1/10 volume of 3.2% sodium citrate, and platelet‐poor plasma was obtained after centrifuging at > 2000 × g for 10 min. Clotting times were measured with an automated coagulation analyzer. PT, APTT, and HepTest reagents were reconstituted and assays were performed according to the manufacturer’s instructions. The modified PT (mPT) assay was performed by diluting 1 mL of thromboplastin C Plus with 1.25 mL of 100 mm calcium chloride and using this diluted reagent in place of the normal PT reagent. For PT and mPT, plasma (50 μL) was warmed to 37 °C for 3 min before adding PT reagent (100 μL). For APTT, plasma (50 μL) was warmed to 37 °C for 1 min before adding APTT reagent (50 μL). After two more minutes, 25 mm calcium chloride (50 μL) was added. For HepTest, plasma (50 μL) was warmed to 37 °C for 2 min before bovine FXa (50 μL) was added. After two more minutes, HepTest ReCal mix (50 μL) was added. Determinations were performed in duplicate and expressed as a mean ratio of treated vs. baseline control. The concentrations required to prolong clotting time by 2‐fold (EC2×) were expressed as total plasma concentrations, not final assay concentrations after addition of clotting assay reagents. For in vitro studies, Apixaban was serially diluted into citrated plasma obtained from healthy dogs, rats, and rabbits, beginning with a 10 mm dimethylsulfoxide stock solution.

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Platelet aggregation assays [2]
Platelet aggregation was measured in citrated human and rabbit platelet‐rich plasma (PRP) in vitro with a platelet aggregometer. PRP was obtained from citrated blood after centrifuging at 250 × g for 6 min. Citrated PRP (250 μL) was mixed with 20 μL of vehicle, DMP802 at 3 μm or Apixaban at 1–10 μm, and incubated for 3 min at 37 °C. DMP802, a glycoprotein (GP)IIb/IIIa receptor antagonist, was included as a positive control (IC50 = 29 nm against human platelet aggregation response to 10 μm ADP). Peak platelet aggregation was determined after the addition of 20 μL of the agonist (ADP at 10 μm, γ‐thrombin at 35 nm, and collagen at 10 μg mL−1, final concentration).

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Determination of Apixaban concentrations and measures of radioactivity[3]
The concentrations of apixaban in plasma and other biological matrices from studies with non-radiolabeled apixaban were determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Levels of radioactivity in the plasma, urine and feces samples were determined by liquid scintillation counting (LSC). A Model A0387 sample oxidizer was used to combust samples. In order to quantify the radioactivity, the resulting 14CO2 was trapped with Carbo-Sorb E and mixed with Permafluor E+ scintillation fluid.

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Metabolite profiles, quantitation and identification[3]
Sample analysis by HPLC was performed on a Shimadzu LC-10AT system equipped with a photodiode array ultraviolet detector and an Ace 3, C18 (3 μm), 150 × 4.6 mm column. The mobile phase flow rate was 0.7 ml/min. The retention times of Apixaban and M2 were confirmed by their ultraviolet spectra using the Shimadzu diode-array detector. For quantitation of radioactivity, the HPLC effluent was collected in 0.26-min intervals using a Gilson Model 204 fraction collector. The plates were dried in an Automatic Environmental Speed Vac and counted for radioactivity for 10 min using a Packard TopCount NXT microplate scintillation and luminescence counter. Radiochromatograms were reconstructed from the TopCount data using Microsoft® Excel software.

Urine and the extracts of pooled plasma and fecal samples were analyzed by LC–MS/MS using a Finnigan LTQ ion trap mass spectrometer and Agilent HPLC. LC–MS analysis was performed with an ESI probe in the positive ion mode. The HPLC eluate was split such that 25% of the eluate was directed to the mass spectrometer. Flow was diverted from 0 to 5 min. The eluate flow was directed to the mass spectrometer from 5 min until the end of the HPLC run. The capillary temperature for analysis was set to 230°C. The nitrogen gas flow rate, spray current and voltages were adjusted to give the maximum sensitivity for Apixaban.

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In vitro incubations[3]
A total of 10 μM of [14C] Apixaban (the maximum concentration [Cmax] after IV administration) was incubated with pooled liver microsomes from rabbit, rat and human subjects (1 mg/ml protein concentration, BD Biosciences, Woburn, MA) in 100 mM phosphate buffer (pH 7.4) containing 1 mM of nicotinamide adenine dinucleotide phosphate for 60 min at 37°C. After protein precipitating with acetonitrile, the samples were analyzed by HPLC, fraction collection, radioactivity measurement and LC–MS analysis.

Dog “N-in-One” Pharmacokinetic Study. [1]
Compounds were dissolved in N,N-dimethylacetamide (DMAC) to a concentration of 20 mg/mL. Compounds were combined in a final dosing solution containing 0.2 mg/mL of each compound (e.g. Apixaban) in 10:10:10:70 % v/v DMAC/ethanol/propylene glycol/water. Beagle dogs were administered 2.5 mL kg-1 h-1 for 1 h by intravenous infusion or 1 mL kg-1 by oral gavage. At timed intervals, blood samples were drawn into 1/10 volume of 3.2% sodium citrate and placed on ice. Plasma was obtained after centrifuging blood at 2000g for 10 min at 4 C. Urine was collected up to 24 h after dosing. Plasma and urine were frozen on dry ice and stored at −70 °C for later analysis. Samples from the pharmacokinetic studies were analyzed with LC−MS/MS methods. High-throughput technologies such as the turbulent-flow column-switching technique and direct plasma sample injection were applied to some studies. In general, the analytical methods were specific and sensitive with a quantification level of 1 nM. The intraday variability was less than 30%. Average run time was about 6 min for each sample. AVST model [1]
The rabbit AVST model, described by Wong et al, was used in this study. Briefly, male New Zealand White rabbits were anesthetized with ketamine (50 mg kg−1 i.m.) and xylazine (10 mg kg−1 i.m.), and their femoral artery, jugular vein and femoral vein were catheterized. These anesthetics were supplemented as needed. Thrombosis was induced by an arteriovenous (AV)‐shunt device containing a silk thread. Blood flowed from the femoral artery via the AV shunt into the opposite femoral vein for 40 min. The shunt was then disconnected and the silk thread covered with thrombus was weighed.

As Apixaban has an oral bioavailability of < 5% in rabbits (unpublished result), it was administered intravenously for in vivo studies. To achieve a stable plasma level with minimum experimental variability, apixaban, fondaparinux or vehicle was given by a continuous intravenous infusion 1 h prior to shunt placement. The infusion was continued throughout the experiment. Warfarin or vehicle was dosed orally once daily for 4 days. On the fourth day after the last oral dose of warfarin or vehicle, rabbits were anesthetized 1.5 h later, and the treatment effect was evaluated about 2 h postdose. Arterial blood samples for the determination of clotting times or plasma levels were collected 20 min after shunt placement. Plasma levels of apixaban were measured by a specific and sensitive liquid chromatographic mass spectrometry method (LC/MS/MS). In rabbits treated with apixaban, fondaparinux or warfarin, the antithrombotic effects of these agents were expressed as percentage inhibition of thrombus formation based on the treated vs. the corresponding mean vehicle. The ED50 value (dose that produced 50% inhibition of thrombus formation) was determined as described below.

The Apixaban group treatment consisted of vehicle (10%N,N‐dimethylacetamide; 30% 1,2‐propanediol; 60% water) (n = 4), and apixaban (mg kg−1 h−1) at 0.03 (n = 7), 0.1 (n = 7), 0.3 (n = 7), 1 (n = 7), and 3 (n = 3). The fondaparinux group treatment consisted of vehicle (saline) (n = 6), and fondaparinux (mg kg−1 h−1) at 0.01 (n = 5), 0.03 (n = 5), 0.1 (n = 5), 0.3 (n = 5), and 1 (n = 5). The warfarin group treatment consisted of vehicle (water) (n = 6), and warfarin (mg kg−1 day−1) at 0.1 (n = 6), 0.3 (n = 6), 1 (n = 6), and 3 (n = 6).

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VT model [1]
The rabbit VT model, described by Hollenbach et al, was used in this study with modifications. Briefly, rabbits were anesthetized as above. The left femoral vein was catheterized, using 11‐cm IntraMedic polyethylene tubing. A prosthetic device was passed through the PE‐200 tubing into the abdominal vena cava. The prosthetic device consisted of a single strand of #10 awg braided copper wire (14 cm) terminated with eight pieces of 4‐0 silk threads 3 cm in length. The silk threads were positioned in the abdominal vena cava by advancing the copper guide wire. Thrombi were formed on the silk threads in a time‐dependent fashion.

Apixaban and fondaparinux were given intravenously as described above 1 h prior to the placement of the prosthetic VT device. Warfarin or its vehicle was dosed orally once daily for 4 days in rabbits as described above, and the prosthetic VT device was placed 2 h after the last oral dose. Ninety minutes after the placement of the prosthetic device, the abdominal vena cava was isolated through a midline abdominal incision. The vena cava was ligated just above and below the prosthetic device with 2‐0 silk. The vena cava segment between the ligations was excised, and the threads with associated thrombus were removed, blotted twice on paper, and weighed. The weight of thrombus formed on the threads was calculated by subtracting the average weight of eight pieces of 4‐0 silk threads 3 cm in length. Clotting times of apixaban, fondaparinux and warfarin in plasma samples collected during VT were measured as above.

In the VT study, the Apixaban group treatment consisted of vehicle (10%N,N‐dimethylacetamide; 90% of 5% dextrose) (n = 6), and apixaban (mg kg−1 h−1) at 0.03 (n = 6), 0.1 (n = 6), 0.3 (n = 6), and 1 (n = 6). The fondaparinux group treatment consisted of vehicle (saline) (n = 6), and fondaparinux (mg kg−1 h−1) at 0.01 (n = 6), 0.03 (n = 6), 0.1 (n = 6), and 0.3 (n = 6). The warfarin group treatment consisted of vehicle (water) (n = 6), and warfarin (mg kg−1 day−1) at 0.1 (n = 5), 0.3 (n = 5), 1 (n = 5), and 3 (n = 6). The ED50 value (dose that produced 50% inhibition of mean vehicle thrombus weight) was determined as described below.

Arterial thrombosis model The rabbit ECAT model, described by Wong et al, was used in this study. Briefly, male New Zealand White rabbits were anesthetized as above. An electromagnetic flow probe was placed on a segment of an isolated carotid artery to monitor blood flow. Thrombosis was induced by electrical stimulation of the carotid artery for 3 min at 4 mA, using an external stainless‐steel bipolar electrode. Carotid blood flow was measured continuously over a 90‐min period to monitor thrombosis‐induced occlusion. Integrated carotid blood flow over 90 min was measured by the area under the flow–time curve, calculated using the trapezoidal rule, and expressed as percentage of total control carotid blood flow, which would result if control blood flow had been maintained continuously for 90 min. The administration of Apixaban and fondaparinux was initiated intravenously as described above 1 h prior to the artery injury. Warfarin or its vehicle was dosed orally once daily for 4 days in rabbits, and thrombosis was initiated 2 h after the last oral dose. Clotting times of apixaban, fondaparinux and warfarin, and concentrations of apixaban in plasma samples, taken during electrically induced arterial thrombosis, were measured as above. In addition, we also measured ex vivo anti‐FXa and antithrombin activities.

In this study, the Apixaban group treatment consisted of vehicle (10%N,N‐dimethylacetamide; 90% of 5% dextrose), and Apixaban (mg kg−1 h−1) at 0.01, 0.03, 0.1, 0.3, and 1 (n = 6 per group). The fondaparinux group treatment consisted of vehicle (saline), and fondaparinux (mg kg−1 h−1) at 0.1, 0.3, 1, and 3 (n = 6 per group). The warfarin group treatment consisted of vehicle (water) (n = 6), and warfarin (mg kg−1 day−1) at 0.03, 0.1, 0.3, 1, and 3 (n = 6 per group). The ED50 (dose that increased carotid blood flow to 50% of the control) of compounds and the EC50 (plasma concentration that increased carotid blood flow to 50% of the control) of apixaban were estimated as described below.

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Cuticle bleeding model [1]
The rabbit cuticle BT model was used in this study. Briefly, rabbits were anesthetized as described above. A standard cut was made at the apex of the cuticle with a razor blade. Blood was allowed to flow freely by keeping the bleeding site in contact with 37 °C lactated Ringer’s solution. BT was defined as the time after transection when bleeding ceased. It was measured by averaging the bleeding time of three nail cuticles. The maximum bleeding recorded was 20 min. Apixaban, fondaparinux and warfarin were administered as described above. In rabbits treated with anticoagulants, the BT effect was expressed as a ratio of treated vs. the mean vehicle value. The ED3× (dose that increased BT 3‐fold) values of compounds were estimated as described below.

The Apixaban group treatment consisted of vehicle (10%N,N‐dimethylacetamide; 30% 1,2‐propanediol; 60% water) (n = 6), and apixaban (mg kg−1 h−1) at 1 (n = 6) and 3 (n = 6). The fondaparinux group treatment consisted of vehicle (saline) (n = 6), and fondaparinux (mg kg−1 h−1) at 0.3 (n = 6), 1 (n = 6), and 3 (n = 6). The warfarin group treatment consisted of vehicle (water) (n = 6), and warfarin (mg kg−1) at 0.1 (n = 5), 0.3 (n = 5), 1 (n = 5), and 3 (n = 5).

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Apixaban was administered intravenously over 10 min to three male rabbits at 2.5 mg/kg (2.5 mg/ml, 1 ml/kg), and orally to three rabbits at 10 mg/kg (2.5 mg/ml, 4 ml/kg). The dose was formulated in 35% hydroxypropyl β-cyclodextrin (HPBCD) in 10 mM phosphate buffer (pH 7.0) as a solution. Blood samples were collected into one-tenth volume of 3.8% sodium citrate at the following time intervals: pre-dose and then 10 (IV only), 15, 30 and 45 min and 1, 2, 4, 6 and 8 h relative to the start of the infusion or post oral dosing. [3]
Metabolism studies were conducted in two groups each comprising three female rabbits: Group 1 received a single IV bolus dose of [14C] Apixaban 5 mg/kg (26.5 μCi/kg) in cyclodextrin buffer; Group 2 received a single oral gavage dose of [14C] apixaban 30 mg/kg (45 μCi/kg) in 0.5% (v/v) Tween 80 in Labrafil®. The actual amount of dosing solution administered to each rabbit was determined by weighing the dosing syringe before and after dose administration. For both groups, urine and feces samples were collected at 12-h intervals (after dosing) over a 48-h period. Blood samples (4 ml) were collected into tubes containing potassium ethylenediaminetetraacetic acid (EDTA) at pre-dose and at the following time intervals after dosing: 0.083, 0.25, 0.5, 1, 4, 12, 24 and 48 h; blood samples were placed on ice immediately after collection and each centrifuged within 30 min of collection to harvest the plasma. Duplicate gravimetric aliquots (approximately 50 mg) of each plasma sample were analyzed for all-trans retinoic acid (or tretinoin) at the test site. All samples were stored at −70°C. [3]

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Dssolved in 10% N,N-dimethylacetamide; 30% 1,2-propanediol; 60% water; ≤3 mg/kg/h; i.v. injection

Arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT) and electrically mediated carotid arterial thrombosis (ECAT) rabbit models

Absorption, Distribution and Excretion
Apixaban is approximately 50% bioavailable though other studies report 43-46% oral bioavailability.
56% of an orally administered dose is recovered in the feces and 24.5-28.8% of the dose is recovered in the urine. 83-88% of the dose recovered in the urine was the unchanged parent compound.
Approximately 21L.
3.3L/h though other studies report 4876mL/h.
Distribution in pregnant rats/fetuses: Cmax in amnion was high. Significant concentrations were found in placenta and fetal blood, kidney and liver. Toxicokinetic data collected in the reproductive and developmental toxicity studies in rats, mice and rabbits showed that generally fetal plasma concentrations of apixaban were lower than those in the dams.
Two single dose radiolabel distribution studies were provided in rats. The data show a wide distribution, with the highest values in excretory organs (liver, kidney, urinary bladder (and contents), bile) and intestinal tract (and contents). After a dose of 20 mg/kg in male Long-Evans rats also relatively high Cmax and AUC were found in adrenals, lungs, thyroid gland, but after a dose of 5 mg/kg in Sprague Dawley rats (both sexes) these organs showed Cmax similar to most other organs and tissues. There was no qualitative difference in distribution between male and female rats, but the female rats showed higher Cmax values in the intestinal tract.
Protein binding differs between the species. The unbound fraction at concentrations of 1-10 uM is about 13% in human vs about 4% in rats and 8% in dogs. At the tested concentrations there was no effect of concentration or gender. In mice protein binding is much lower, with 44-6 % unbound, dependent on the tested concentration (range 100-2000 ng apixaban/mL).
Plasma to blood ratios of about one in dog and human blood indicate uniform distribution between plasma and red blood cells and thus no specific distribution to red blood cells.
For more Absorption, Distribution and Excretion (Complete) data for Apixaban (12 total), please visit the HSDB record page.

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Metabolism / Metabolites
50% of the orally administered dose is excreted as the unchanged parent compound, however 25% of the dose is excreted as O-demethyl apixaban sulfate. All apixaban metabolites account for approximately 32% of the excreted dose though the structure of all metabolites are not well defined. Apixaban is mainly metabolized by cytochrome p450(CYP)3A4 and to a lesser extent by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2J2.
Approximately 25% of an orally administered apixaban dose is recovered in urine and feces as metabolites. Apixaban is metabolized mainly via CYP3A4 with minor contributions from CYP1A2, 2C8, 2C9, 2C19, and 2J2. O-demethylation and hydroxylation at the 3-oxopiperidinyl moiety are the major sites of biotransformation. Unchanged apixaban is the major drug-related component in human plasma; there are no active circulating metabolites.
Apixaban is mainly metabolized by CYP3A4/5 with conjugation via SULT1A1, but several other CYP and SULT isozymes are also involved. No apixaban metabolites were found to have pharmacological activity and there were no unique human metabolites.
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 hr 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.
The metabolism and disposition of (14)C-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 (14)C-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.
... 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 microM (human liver S9), 36.8 microM (SULT1A1), and 70.8 microM (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.

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Biological Half-Life
12.7±8.55h.
Apixaban has ... an apparent half-life of approximately 12 hours following oral administration.
In a comparative study elimination half-life in rats (2-3 hrs) was shorter than in dogs (5-6 hrs) and chimpanzees (5-7 hrs). Distribution volume is relatively low in rats (0.31 L/kg), dogs (0.30 L/kg) and chimpanzees (0.17 L/kg).
... After a single oral administration ... the elimination half life of radioactivity in blood was 1.7 to 4.2 hr.

Toxicity Summary
IDENTIFICATION AND USE: Apixaban white to pale-yellow powder formulated into a film-coated tablet. Apixaban is indicated in patients to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. It is also indicated for the prophylaxis of deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE), in patients who have undergone hip or knee replacement surgery. It is also indicated for the treatment of PE and for the treatment of DVT. Finally, apixaban is indicated it patients to reduce the risk of recurrent DVT and PE following initial therapy. HUMAN EXPOSURE AND TOXICITY: Premature discontinuation of any oral anticoagulant, including apixaban, in the absence of adequate alternative anticoagulation increases the risk of thrombotic events. An increased rate of stroke was observed during the transition from apixaban to warfarin in clinical trials in atrial fibrillation patients. Apixaban increases the risk of hemorrhage and can cause serious, potentially fatal, bleeding. The drug should be discontinued if active pathological hemorrhage occurs. Epidural or spinal hematomas may occur in patients treated with apixaban who are receiving neuraxial anesthesia or undergoing spinal puncture. These hematomas may result in long-term or permanent paralysis. Factors that can increase the risk of developing epidural or spinal hematomas in these patients include: use of indwelling epidural catheters concomitant use of other drugs that affect hemostasis, such as nonsteroidal anti-inflammatory drugs (NSAIDs), platelet inhibitors, other anticoagulants a history of traumatic or repeated epidural or spinal punctures a history of spinal deformity or spinal surgery. Apixaban should not be used in people with mechanical heart valves. ANIMAL STUDIES: Single dose oral studies in mice (up to 4000 mg/kg), rats (up to 4510 mg/kg), dogs (up to 1500 mg/kg) and cynomolgus monkeys (up to 300 mg/kg) revealed no other drug-related effects than those related to the pharmacodynamic action of apixaban. In particular some monkeys died due to excessive bleeding after blood sampling. Apixaban was not carcinogenic when administered to mice and rats for up to 2 years. The systemic exposures (AUCs) of unbound apixaban in male and female mice at the highest doses tested (1500 and 3000 mg/kg/day) were 9 and 20 times, respectively, the human exposure of unbound drug at the MRHD of 10 mg/day. Systemic exposures of unbound apixaban in male and female rats at the highest dose tested (600 mg/kg/day) were 2 and 4 times, respectively, the human exposure. Apixaban had no effect on fertility in male or female rats when given at doses up to 600 mg/kg/day, a dose resulting in exposure levels that are 3 and 4 times, respectively, the human exposure. Apixaban administered to female rats at doses up to 1000 mg/kg/day from implantation through the end of lactation produced no adverse findings in male offspring (F1 generation) at doses up to 1000 mg/kg/day, a dose resulting in exposure that is 5 times the human exposure. Adverse effects in the F1-generation female offspring were limited to decreased mating and fertility indices at 1000 mg/kg/day. Apixaban was neither mutagenic in the bacterial reverse mutation (Ames) assay, nor clastogenic in Chinese hamster ovary cells in vitro, in a 1-month in vivo/in vitro cytogenetics study in rat peripheral blood lymphocytes, or in a rat micronucleus study in vivo.

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Hepatotoxicity
Apixaban is associated with serum aminotransferase elevations greater than 3 times the upper limit of normal in 1% to 2% of treated patients. This rate is similar or lower than rates with warfarin or comparator arms. In premarketing studies, no instances of clinically apparent liver injury were reported, but subsequent to its approval and more wide scale use, several reports of mild but clinically apparent liver injury have been published. The liver injury arose within days of starting apixaban and the pattern of liver enzyme elevations was hepatocellular. Immunoallergic and autoimmune features were not present. In most cases, recovery was rapid once apixaban was stopped. In one analysis of a national health care database, the incidence of hospitalization for acute liver injury after initiation of apixaban therapy was 1 per 2,200 patients treated, a rate similar to that of rivaroxaban.
Likelihood score: B (possible rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Information from four mothers indicates that apixaban levels in milk are rather high. An alternate drug is preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
92-94%.

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Interactions
Current evidence suggests that addition of apixaban to standard antiplatelet therapy (e.g., aspirin, clopidogrel) does not substantially reduce the rate of recurrent ischemic events in patients with acute coronary syndrome (ACS) and may increase the risk of major, sometimes fatal, bleeding. /NOT included in US product label/
In a study in healthy individuals, concomitant administration of apixaban and enoxaparin had no effect on the pharmacokinetics of apixaban, but had an additive effect on anti-factor Xa activity. The increased pharmacodynamic effect was considered to be modest. When administration of apixaban and enoxaparin was separated by 6 hours in this study, the additive effect on anti-factor Xa activity was attenuated.
In the principal efficacy study of apixaban, bleeding risk was increased in patients who received concomitant apixaban and aspirin therapy. Additionally, a placebo-controlled study of patients with acute coronary syndromes was terminated early after an increased risk of bleeding was observed in patients receiving apixaban in combination with aspirin and clopidogrel. In drug interaction studies in healthy individuals, apixaban did not substantially alter the pharmacokinetics of aspirin, and a pharmacodynamic interaction was not observed with such concomitant therapy.
Concomitant use of apixaban and drugs that affect hemostasis (e.g., aspirin or other antiplatelet drugs, heparin or other anticoagulants, fibrinolytics, selective serotonin reuptake inhibitors (SSRIs), serotonin norepinephrine reuptake inhibitors (SNRIs), nonsteroidal anti-inflammatory agents (NSAIAs)) increases the risk of bleeding.
For more Interactions (Complete) data for Apixaban (11 total), please visit the HSDB record page.

[1]. Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa. J Med Chem. 2007 Nov 1;50(22):5339-56.

[2]. Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies. J Thromb Haemost. 2008 May;6(5):820-9.

[3]. Metabolism, pharmacokinetics and pharmacodynamics of the factor Xa inhibitor apixaban in rabbits. J Thromb Thrombolysis. 2010 Jan;29(1):70-80.

[4]. Preclinical pharmacokinetics and pharmacodynamics of apixaban, a potent and selective factor Xa inhibitor. Eur J Drug Metab Pharmacokinet. 2011 Sep;36(3):129-39.

Therapeutic Uses
Eliquis (apixaban) is indicated to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. /Included in US product label/
Eliquis is indicated for the prophylaxis of deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE), in patients who have undergone hip or knee replacement surgery. /Included in US product label/
Eliquis is indicated for the treatment of pulmonary embolism (PE). /Included in US product label/
Eliquis is indicated for the treatment of deep vein thrombosis (DVT). /Included in US product label/
For more Therapeutic Uses (Complete) data for Apixaban (7 total), please visit the HSDB record page.

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Drug Warnings
/BOXED WARNING/ WARNING: PREMATURE DISCONTINUATION OF ELIQUIS INCREASES THE RISK OF THROMBOTIC EVENTS. Premature discontinuation of any oral anticoagulant, including Eliquis, increases the risk of thrombotic events. If anticoagulation with Eliquis is discontinued for a reason other than pathological bleeding or completion of a course of therapy, consider coverage with another anticoagulant.
/BOXED WARNING/ WARNING: SPINAL/EPIDURAL HEMATOMA. Epidural or spinal hematomas may occur in patients treated with Eliquis who are receiving neuraxial anesthesia or undergoing spinal puncture. These hematomas may result in long-term or permanent paralysis. Consider these risks when scheduling patients for spinal procedures. Factors that can increase the risk of developing epidural or spinal hematomas in these patients include: use of indwelling epidural catheters concomitant use of other drugs that affect hemostasis, such as nonsteroidal anti-inflammatory drugs (NSAIDs), platelet inhibitors, other anticoagulants a history of traumatic or repeated epidural or spinal punctures a history of spinal deformity or spinal surgery optimal timing between the administration of Eliquis and neuraxial procedures is not known. Monitor patients frequently for signs and symptoms of neurological impairment. If neurological compromise is noted, urgent treatment is necessary. Consider the benefits and risks before neuraxial intervention in patients anticoagulated or to be anticoagulated.
FDA Pregnancy Risk Category: B /NO EVIDENCE OF RISK IN HUMANS. Adequate, well controlled studies in pregnant women have not shown increased risk of fetal abnormalities despite adverse findings in animals, or, in the absence of adequate human studies, animal studies show no fetal risk. The chance of fetal harm is remote but remains a possibility./
It is unknown whether apixaban or its metabolites are excreted in human milk. Rats excrete apixaban in milk (12% of the maternal dose). Women should be instructed either to discontinue breastfeeding or to discontinue Eliquis therapy, taking into account the importance of the drug to the mother.
For more Drug Warnings (Complete) data for Apixaban (17 total), please visit the HSDB record page.

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Pharmacodynamics
Apixaban selectively inhibits factor Xa in its free and bound forms, independant of antithrombin III. Apixaban also inhibits prothrominase. These effects prevent the formation of a thrombus.

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Apixaban is a pyrazolopyridine that is 7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide substituted at position 1 by a 4-methoxyphenyl group and at position 6 by a 4-(2-oxopiperidin-1-yl)phenyl group. It is used for the prevention and treatment of thromboembolic diseases. It has a role as an anticoagulant and an EC 3.4.21.6 (coagulation factor Xa) inhibitor. It is a pyrazolopyridine, a member of piperidones, a lactam and an aromatic ether.
Apixaban is an oral, direct, and highly selective factor Xa (FXa) inhibitor of both free and bound FXa, as well as prothrombinase, independent of antithrombin III for the prevention and treatment of thromboembolic diseases. It is marketed under the name Eliquis. Apixaban was approved by the FDA on December 28, 2012.

Apixaban is a Factor Xa Inhibitor. The mechanism of action of apixaban is as a Factor Xa Inhibitor.
Apixaban is an oral anticoagulant and direct inhibitor of factor Xa which is used to decrease the risk of venous thromboses, systemic embolization and stroke in patients with atrial fibrillation, and lower the risk of deep vein thrombosis and pulmonary embolus after knee or hip replacement surgery. Apixaban has been linked to a low rate of serum aminotransferase elevations during therapy and to rare instances of clinically apparent liver injury.
Apixaban is an orally active inhibitor of coagulation factor Xa with anticoagulant activity. Apixaban directly inhibits factor Xa, thereby interfering with the conversion of prothrombin to thrombin and preventing formation of cross-linked fibrin clots.
APIXABAN is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2011 and has 8 approved and 20 investigational indications. This drug has a black box warning from the FDA.

Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P1 moieties that resulted in the identification of the p-methoxyphenyl P1, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P4 ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4.[1]

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Background: Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. Objective: We evaluated the in vitro properties of apixaban and its in vivo activities in rabbit models of thrombosis and hemostasis. Methods: Studies were conducted in arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), electrically mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. Results: In vitro, apixaban is potent and selective, with a K(i) of 0.08 nm for human FXa. It exhibited species difference in FXa inhibition [FXa K(i) (nm): 0.16, rabbit; 1.3, rat; 1.7, dog] and anticoagulation [EC(2x) (microm, concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 microm did not alter human and rabbit platelet aggregation to ADP, gamma-thrombin, and collagen. In vivo, the values for antithrombotic ED(50) (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT and the values for BT ED(3x) (dose that increased BT by 3-fold) were 0.27 +/- 0.03, 0.11 +/- 0.03, 0.07 +/- 0.02 and > 3 mg kg(-1) h(-1) i.v. for apixaban, 0.05 +/- 0.01, 0.05 +/- 0.01, 0.27 +/- 0.08 and > 3 mg kg(-1) h(-1) i.v. for the indirect FXa inhibitor fondaparinux, and 0.53 +/- 0.04, 0.27 +/- 0.01, 0.08 +/- 0.01 and 0.70 +/- 0.07 mg kg(-1) day(-1) p.o. for the oral anticoagulant warfarin, respectively.[2]

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In summary, apixaban was as effective as the current anticoagulant standard of care in the prevention of thrombosis at doses that preserve hemostasis in rabbits. HepTest, mPT and chromogenic anti‐FXa assays were potential markers to monitor the anticoagulant and plasma levels of apixaban. Because of the favorable preclinical profile of apixaban, this compound was selected for clinical development. Similar to its favorable preclinical findings, initial phase II studies with apixaban have demonstrated its efficacy and safety in the prevention and treatment of venous thromboembolism. Other potential indications for apixaban in the treatment and prevention of various life‐threatening thromboembolic events are currently being elucidated in the clinic.[2]

C25H25N5O4

459.5

459.19

C, 65.35; H, 5.48; N, 15.24; O, 13.93

503612-47-3

Apixaban-13C,d3;1261393-15-0;Apixaban-d3;1131996-12-7

10182969

Off-white to yellow solid powder

1.4±0.1 g/cm3

770.5±60.0 °C at 760 mmHg

419.8±32.9 °C

0.0±2.6 mmHg at 25°C

1.705

0.48

1

5

5

34

777

0

COC1=CC=C(C=C1)N2C3=C(CCN(C3=O)C4=CC=C(C=C4)N5CCCCC5=O)C(=N2)C(=O)N

QNZCBYKSOIHPEH-UHFFFAOYSA-N

InChI=1S/C25H25N5O4/c1-34-19-11-9-18(10-12-19)30-23-20(22(27-30)24(26)32)13-15-29(25(23)33)17-7-5-16(6-8-17)28-14-3-2-4-21(28)31/h5-12H,2-4,13-15H2,1H3,(H2,26,32)

1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5-dihydropyrazolo[5,4-c]pyridine-3-carboxamide

BMS56224701; BMS 56224701; Apixaban; 503612-47-3; Eliquis; BMS-562,247-01; BMS-562,247; BMS 562,247-01; 1-(4-Methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide; apixabanum; BMS 562247-01; Apixaban, BMS-56224701; brand name: Eliquis

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.44 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.44 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)