Apixaban

Apixaban Biological Activity

Description:Apixaban is a highly selective, reversible inhibitor of Factor Xa with Ki of 0.08 nM and 0.17 nM in human and rabbit, respectively.

Target:IC50: 0.08 nM (Human Factor Xa), 0.17 nM (Rabbit Factor Xa)

In Vitro:Apixaban exhibits a high degree of potency, selectivity, and efficacy on Factor Xa with Ki of 0.08 nM and 0.17 nM for Human Factor Xa and Rabbit Factor Xa, respectively[1]. In vitro, Apixaban prolongs the clotting times of normal human plasma with the concentrations (EC2x) of 3.6 μM, 0.37 μM, 7.4 μM, and 0.4 μM, which are required respectively to double the prothrombin time (PT), modified prothrombin time (mPT), activated partial thromboplastin time (APTT) and HepTest. Besides, Apixaban shows the highest potency in human and rabbit plasma, but less potency in rat and dog plasma in both the PT and APTT assays.

In Vivo；Apixaban shows the excellent pharmacokinetics with very low clearance (Cl: 0.02 L/kg/h), and low volume of distribution (Vdss: 0.2 L/kg) in the dogs. Besides, Apixaban also exhibits a moderate half-life (T1/2: 5.8 hours) and good oral bioavailability (F: 58%)[1]. In the arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT) and electrically mediated carotid arterial thrombosis (ECAT) rabbit models, Apixaban produces dose-dependent antithrombotic effects with EC50 of 270 nM, 110 nM and 70 nM, respectively[2]. Apixaban significantly inhibits factor Xa activity with IC50 of 0.22 μM in rabbit ex vivo[3]. In chimpanzee, Apixaban also shows small volume of distribution (Vdss: 0.17 L/kg), low systemic clearance (Cl: 0.018 L/kg/h), and good oral bioavailability (F: 59%).

Kinase Assay:Purified FXa is obtained after activation with Russell’s viper venom followed by affinity chromatography. The resulting FXa is > 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 are determined using the chromogenic substrate S-2765, and are 36, 60, 240 and 70 μM, respectively. The substrate hydrolysis is 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 is determined in duplicate. The Ki values are 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.

Animal Admin:Briefly, male New Zealand White rabbits are anesthetized with  (50 mg/kg i.m.) and xylazine (10 mg/kg i.m.), and their femoral artery, jugular vein and femoral vein are catheterized. These anesthetics are supplemented as needed. Thrombosis is 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 is then disconnected and the silk thread covered with thrombus is weighed. As apixaban has an oral bioavailability of < 5% in rabbits (unpublished result), it is administered intravenously for in vivo studies. To achieve a stable plasma level with minimum experimental variability, apixaban, fondaparinux or vehicle is given by a continuous intravenous infusion 1 h prior to shunt placement. The infusion is continued throughout the experiment. Warfarin or vehicle is dosed orally once daily for 4 days. On the fourth day after the last oral dose of warfarin or vehicle, rabbits are anesthetized 1.5 h later, and the treatment effect is evaluated about 2 h postdose. Arterial blood samples for the determination of clotting times or plasma levels are collected 20 min after shunt placement. Plasma levels of apixaban are 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 are 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) is determined as described below. The apixaban group treatment consists of vehicle (10%N,N-dimethylacetamide; 30% 1,2-propanediol; 60% water) (n=4), and apixaban (mg/kg/h) at 0.03 (n=7), 0.1 (n=7), 0.3 (n=7), 1 (n=7), and 3 (n=3). The fondaparinux group treatment consists of vehicle (saline) (n=6), and fondaparinux (mg/kg/h1) 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 consists of vehicle (water) (n=6), and warfarin (mg/kg/day) at 0.1 (n=6), 0.3 (n=6), 1 (n=6), and 3 (n=6).

Density:1.4±0.1 g/cm3

Boiling Point:770.5±60.0 °C at 760 mmHg

Molecular Formula:C25H25N5O4

Molecular Weight:459.497

Flash Point:419.8±32.9 °C

Exact Mass:459.190643

PSA:110.76000

LogP:0.48

Vapour Pressure:0.0±2.6 mmHg at 25°C

Index of Refraction:1.705