To cite this article: Wong PC, Crain EJ, Xin B, Wexler RR, Lam PYS, Pinto DJ, Luettgen JM, Knabb RM. Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies. J Thromb Haemost 2008; 6: 820-9.Summary. 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 2· (lM, concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 lM did not alter human and rabbit platelet aggregation to ADP, cthrombin, 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 3· (dose that increased BT by 3-fold) were 0.27 ± 0.03, 0.11 ± 0.03, 0.07 ± 0.02 and > 3 mg kg warfarin, respectively. Conclusions: In summary, apixaban was effective in the prevention of experimental thrombosis at doses that preserve hemostasis in rabbits.

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.

The present study describes a new analytical approach for the detection and characterization of GSH-trapped reactive metabolites using multiple reaction monitoring (MRM) as the survey scan to trigger the acquisition of enhanced product ion (EPI) spectra on a triple quadrupole linear ion mass spectrometer. The MRM scan step was carried out following up to 114 MRM transitions from the protonated molecules of potential GSH adducts to their product ions derived from a neutral loss of 129 or 307 Da. MRM transition protocols were constructed on the basis of common bioactivation reactions predicted to occur in human liver microsomes (HLM). The effectiveness and reliability of the approach were evaluated using acetaminophen, diclofenac, and carbamazepine as model compounds. The total ion chromatograms of the MRM for the HLM incubations with these compounds and GSH clearly displayed a number of GSH adducts, including acetaminophen-GSH adducts and carbamazepine-GSH adducts that were not previously observed in HLM incubations. In addition, clomipramine and mefenamic acid that have the frame structures susceptible to P450-mediated bioactivation were investigated. As a result, the MRM-EPI analysis revealed multiple GSH adducts of clomipramine and mefenamic acid in HLM incubations possibly mediated by epoxide and/or quinone imine intermediates. Compared with the neutral loss (NL) and precursor ion (PI) scanning analysis, the MRM-based approach provided superior sensitivity and selectivity for GSH adducts. It also enabled the sensitive acquisition of EPI spectra with rich fragmentation in the same LC/MS run, which were useful for the rapid structure elucidation of GSH adducts and the elimination of false positives. The MRM-EPI experiment can be employed for high throughput screening of reactive metabolites and should be especially applicable to compounds of the same chemotype. Also, it can be applied in conjunction with the PI or NL scan as a comprehensive method for the analysis of reactive metabolites in a drug discovery setting.

To cite this article: Wong PC, Crain EJ, Watson CA, Xin B. Favorable therapeutic index of the direct factor Xa inhibitors, apixaban and rivaroxaban, compared with the thrombin inhibitor dabigatran in rabbits. J Thromb Haemost 2009; 7: 1313-20.Summary. Background: Apixaban is an oral, direct factor Xa (FXa) inhibitor in late-stage clinical development. This study assessed effects of the direct FXa inhibitors, apixaban and rivaroxaban, vs. the direct thrombin inhibitor, dabigatran, on venous thrombosis (VT), bleeding time (BT) and clotting times in rabbits. Methods: We induced the formation of non-occlusive thrombus in VT models by placing threads in the vena cava, and induced bleeding by the incision of cuticles in anesthetized rabbits. Apixaban, rivaroxaban and dabigatran were infused IV to achieve a stable plasma level. Clotting times, including the activated partial thromboplastin time (aPTT), prothrombin time (PT), modified PT (mPT) and thrombin time (TT), were measured. Results: Apixaban, rivaroxaban and dabigatran exhibited dose-related efficacy in preventing VT with EC 50 of 65, 33 and 194 nM, respectively. At doses for 80% reduction of control thrombus, apixaban, rivaroxaban and dabigatran prolonged BT by 1.13 ± 0.02-, 1.9 ± 0.1-* and 4.4 ± 0.4-fold*, respectively (*P < 0.05, vs. apixaban). In the treatment model, these inhibitors equally prevented growth of a preformed thrombus. Antithrombotic doses of apixaban and rivaroxaban prolonged aPTT and PT by <3-fold with no effect on TT. Dabigatran was ‡50-fold more potent in prolonging TT than aPTT and PT. Of the clotting assays studied, apixaban, rivaroxaban and dabigatran responded the best to mPT. Conclusion: Comparable antithrombotic efficacy was observed between apixaban, rivaroxaban and dabigatran in the prevention and treatment of VT in rabbits. Apixaban and rivaroxaban exhibited lower BT compared with dabigatran at equivalent antithrombotic doses. The clinical significance of these findings remains to be determined.