Discovery of betrixaban (PRT054021), N-(5-chloropyridin-2-yl)-2-(4-(N,N-dimethylcarbamimidoyl)benzamido)-5-methoxybenzamide, a highly potent, selective, and orally efficacious factor Xa inhibitor.

Systematic SAR studies of in vitro factor Xa inhibitory activity around compound 1 were performed by modifying each of the three phenyl rings. A class of highly potent, selective, efficacious and orally bioavailable direct factor Xa inhibitors was discovered. These compounds were screened in hERG binding assays to examine the effects of substitution groups on the hERG channel affinity. From the leading compounds, betrixaban (compound 11, PRT054021) has been selected as the clinical candidate for development.

1-(2-Naphthyl)-1H-pyrazole-5-carboxylamides as potent factor Xa inhibitors. Part 2: A survey of P4 motifs.

A variety of P4 motifs have been examined to increase the binding affinity and in vitro anticoagulant potency of our biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamide-based fXa inhibitors. Highly potent 2-naphthyl-P1 fXa inhibitors (K(i)< or =2 nM) with improved in vitro anticoagulant activity (2xTG< or =1 microM) and respectable pharmacokinetic properties have been discovered.

1-(2-Naphthyl)-1H-pyrazole-5-carboxylamides as potent factor Xa inhibitors. Part 3: Design, synthesis and SAR of orally bioavailable benzamidine-P4 inhibitors.

Using N,N-dialkylated benzamidines as the novel P4 motifs, we have designed and synthesized a class of 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides as highly potent and selective fXa inhibitors with significantly improved hydrophilicity and in vitro anticoagulant activity. These benzamidine-P4 fXa inhibitors have displayed excellent oral bioavailability and long half-life.

Risk of early surgery for Crohn's disease: implications for early treatment strategies.

OBJECTIVES: In this study we aimed to define the rate of early surgery for Crohn's disease and to identify risk factors associated with early surgery as a basis for subsequent studies of early intervention in Crohn's disease. METHODS: We assembled a retrospective cohort of patients with Crohn's disease diagnosed between 1991 and 1997 and followed for at least 3 yr, who were identified in 16 community and referral-based practices in New England. Chart review was performed for each patient. Details of baseline demographic and disease features were recorded. Surgical history including date of surgery, indication, and procedure were also noted. Risk factors for early surgery (defined as major surgery for Crohn's disease within 3 yr of diagnosis, exclusive of major surgery at time of diagnosis) were identified by univariate analysis. Multiple logistic regression was used to identify independent risk factors. RESULTS: Of 345 eligible patients, 69 (20.1%) required surgery within 3 yr of diagnosis, excluding the 14 patients (4.1%) who had major surgery at the time of diagnosis. Overall, the interval between diagnosis and surgery was short; one half of all patients who required surgery underwent operation within 6 months of diagnosis. Risk factors identified by univariate analysis as significantly associated with early surgery included the following: smoking; disease of small bowel without colonic involvement; nausea and vomiting or abdominal pain on presentation; neutrophil count; and steroid use in the first 6 months. Disease localized to the colon only, blood in the stool, use of 5-aminosalicylate, and lymphocyte count were inversely associated with risk of early surgery. Logistic regression confirmed independent associations with smoking as a positive risk factor and involvement of colon without small bowel as a negative risk factor for early surgery. CONCLUSIONS: The rate of surgery is high in the first 3 yr after diagnosis of Crohn's disease, particularly in the first 6 months. These results suggest that improved risk stratification and potent therapies with rapid onset of action are needed to modify the natural history of Crohn's disease.

Inhibition of purified factor Xa amidolytic activity may not be predictive of inhibition of in vivo thrombosis: implications for identification of therapeutically active inhibitors.

OBJECTIVE: In this study we test the hypothesis that blood/plasma-based prothrombinase assays, rather than inhibition of purified factor Xa (fXa), are predictive of in vivo antithrombotic activity. METHODS AND RESULTS: Six fXa inhibitors with equivalent nanomolar Ki were studied in thrombin generation assays using human plasma/blood and endogenous macromolecular substrate. In all assays, benzamidine inhibitors were more potent (100 to 800 nmol/L) than the aminoisoquinolines (5 to 58 micromol/L) or neutral inhibitors (3 to 10 micromol/L). A similar rank order of compound inhibition was also seen in purified prothrombinase assays as well as in a rabbit model of deep vein thrombosis. CONCLUSIONS: Assays using prothrombinase with protein substrates are better predictors of in vivo efficacy than fXa Ki using amidolytic substrates.

Differences between human and rabbit coagulation factor X-implications for in vivo models of thrombosis.

The activation of factor X (fX) to factor Xa (fXa) marks the penultimate step in the coagulation cascade and modulating fXa activity may be effective for antithrombotic therapy. Even though fXa inhibitors are screened using in vitro inhibition of human fXa (HfXa) while subsequent evaluation uses in vivo rabbit models, there is limited knowledge of species differences between the coagulation proteins. When comparing amino acid sequences for the human (HfX) and rabbit (RafX) protein, differences are found in the activation peptide and active site regions. In order to study the relative functional characteristics of HfX and RafX, we asked (1) whether fX from the two species is immunologically related, (2) whether the two proteins are activated to fXa in a similar manner, (3) whether HfXa and rabbit factor Xa (RafXa) have similar catalytic activities toward tripeptide substrates. To answer (1), we expressed RafX-glutathione S-transferase (RafX-GST) fusion protein in bacteria and purified the protein for use as an antigen. The resulting monoclonal antibodies were suitable for affinity purification of plasma RafX and for effective anticoagulation in rabbit plasma clotting assays. We found two antibodies (mAb 214 and mAb 290) that anticoagulated rabbit plasma in a dose responsive manner but did not cross-react with human plasma. At a concentration of 500 nM, mAb 214 attained a two-fold extension of rabbit plasma activated partial thromboplastin time (aPTT). To answer (2), we purified plasma RafX and compared the activation of HfX and RafX with Russell's viper venom (RVV-X). Under equivalent reaction conditions, conversion was 30% slower for the rabbit protein. To answer (3), amidolytic activity of HfXa and RafXa were assayed by cleavage of three para-nitroanilide (pNA) substrates (S2222 [Bz-Ile-Glu(gamma-OR)-Gly-Arg-pNA.HCl], S2765 [Z-D-Arg-Gly-Arg-pNA.HCl] and Spectrozyme Xa [MeO-CO-D-CHG-Gly-Arg-pNA.AcOH]). Michaelis constants (K(m)) for the rabbit protein were 187, 72 and 69 microM, respectively, and for the human analog, 255, 63 and 135 microM, respectively. Comparing the extent of substrate turnover (V(max)) for HfXa and RafXa, the latter was shown to cleave all three substrates at a reduced rate. Based on these observations, it can be speculated that the relative antithrombotic potency of active site directed fXa inhibitors might be different between the two species. Predicted human therapeutic doses derived from in vivo results in rabbit models should therefore take species variation into consideration.