FVIIa Inhibitor Research Articles

Screening of a Library for Factor VIIa Inhibitors

Background:As an alternative to the anticoagulant’s strategy using direct or indirect anti-Xa drugs, considering other targets upstream in the coagulation cascade such as anti-Factor VIIa could represent an effective and safer strategy in coagulation and pathological angiogenesis.Objective:The objective of the study was to assess a high technology methodology composed of virtual screening, anticoagulant, and anti-angiogenesis assays to identify potent small-molecule FVIIa inhibitors.Methods:Chemical databanks were screened to select molecules bearing functional groups that could fit into the active site of FVIIa, which were then tested. Ligands assigned with the lowest scores were retained and then biologically assessed.Results:From the 500 molecules considered, 8 chemical structures revealed to be effective compounds in vitro and to inhibit angiogenesis in the chick chorioallantoic membrane (CAM) model.Conclusion:New potent small-molecule FVIIa inhibitors have been identified; further biochemical and chemical developments would be investigated directly from the selected scaffolds.

A Phase 2 Study of PCI-27483, a Factor VIIa Inhibitor in Combination with Gemcitabine for Advanced Pancreatic Cancer

Objectives: Tissue factor overexpression is associated with tumor progression, venous thromboembolism, and worsened survival in patients with cancer. Tissue factor and activated factor VII (FVIIa) complex may contribute to tumor invasiveness by promoting cell migration and angiogenesis. The study objective was to evaluate safety, pharmacokinetics, and efficacy of PCI-27483, a selective FVIIa inhibitor. Methods: This was an open-label, multicenter phase 2 trial of patients with advanced pancreatic cancer. Part A of the study was an intrapatient dose escalation lead-in portion in patients concurrently receiving gemcitabine, and in part B, patients were randomized 1: 1 to the recommended phase 2 dose combination PCI-27483–gemcitabine versus gemcitabine alone. Results: Target international normalized ratio (between 2.0–3.0) was achieved following PCI-27483 treatment. Overall safety of PCI-27483–gemcitabine (n = 26) was similar to gemcitabine alone (n = 16), with a higher incidence of mostly low-grade bleeding events (65% vs. 19%). Progression-free survival (PFS) and overall survival (OS) were not significantly different between patients treated with PCI-27483–gemcitabine (PFS: 3.7 months, OS: 5.7 months) and those treated with gemcitabine alone (PFS: 1.9 months, OS: 5.6 months). Conclusions: Targeted inhibition of the coagulation cascade was achieved by administering PCI-27483. PCI-27483–gemcitabine was well tolerated, but superiority to single agent gemcitabine was not demonstrated.

Mechanism of Tissue Factor (TF) Enhancing Factor (F)VIII Activity on FXa Generation in Initial Phase of Coagulation and Interaction Between TF and FVIII C2 Domain

In the cell-based coagulation model, factor (F)VIIa complex with tissue factor (TF) initiates the blood coagulation by generating FXa as the extrinsic tenase complex and activates FIX which composes the intrinsic tenase complex. We demonstrated that FVIIa/TF directly activated FVIII in an early coagulation phase (Soeda, JTH, 2010), and TF enhanced the intrinsic tenase activity via possible interaction with FVIIIa (Ogiwara, ASH, 2010). In this study, we clarified the enhancing mechanism of intrinsic tenase activity in the TF-related up-regulation of FVIII, and identified the TF-interactive region on FVIII.To explore the enhancing mechanism of TF for FVIII regulation, we performed the FXa generation assay with various amounts of FVIII or thrombin-mediated FVIIIa, constant FIXa (1 nM), FX (300 nM) and phospholipid vesicles (PL; 20 µM) in the presence of recombinant lipidated TF (rTF, Innovin®). The Km value for FVIII in the presence of rTF was ~2.4-fold lower than that its absence (Km; 5.6±0.5, 13.3±3.7 nM, respectively, p<0.05). Similarly, the Km for FVIIIa in the presence of TF was ~1.5-fold lower than that in its absence (Km; 6.3±0.6 nM, 9.7±1.6 nM, respectively, p<0.05), supporting that the presence of TF could promote the FXa-catalyzed activation of FVIII and FVIIIa-dependent generation of intrinsic FXa. To further evaluate the effect of TF on FVIII-dependent FXa generation, the FXa generation assay with FVIIa/TF-activated FVIIIa (FVIIIa-VIIa/TF) was also performed. The initial velocity on FXa generation with FVIIIa-VIIa/TF was 22.6 nM/min. However, the initial velocity on FXa generation with FVIIIa-VIIa/TF by addition of FVIIa-inhibitor (E-76), not to generate FVIIa/TF-dependent FXa, was 3.4 nM/min, and that with FVIII alone was 0.05 nM/min. In addition, the initial velocity with FVIIa/TF alone was 10.4 nM/min. These findings supported that the TF increased FXa generation greater than the additive effect of FVIII-dependent and FVIIa/TF-dependent FXa generation in early initiation phase of coagulation prior to thrombin generation. Since tissue factor pathway inhibitor (TFPI) is present in physiological circulating whole blood, a similar experiment on FXa generation assay was repeated under the presence of TFPI, estimated to be present at 0.5 nM in the pre-coagulant state or at 15 nM in the coagulant state in circulating blood. The initial velocity on FXa generation with FVIIIa-VIIa/TF was reduced by the presence of 0.5 or 15 nM TFPI (17.9 and 12.6 nM/min, respectively). By contrast, the initial velocity on FVIIIa-VIIa/TF-dependent FXa generation with addition of FVIIa inhibitor was little reduced by 0.5 nM TFPI, whilst was reduced by 15 nM TFPI (3.5 and 2.5 nM/min, respectively). These findings supported that the TFPI possibly didn’t inhibit TF on the enhanced intrinsic tenase on association with FVIII in the pre-coagulant state.We further reported that TF enabled FXa to activate FVIII, irrespective of von Willebrand factor (VWF), and the direct association of rTF and non-lipidated TF with FVIII (Furukawa, ISTH, 2015). Since TF is transmembrane protein, however, we performed a surface plasmon resonance (SPR)-based assay (BIAcore®) and solid phase-based ELISA to identify the interactive region(s) on FVIII to recombinant soluble TF (sTF; Altor BioScience), a portion of TF outside of the PL membrane. An SPR-based assay revealed the direct binding of intact FVIII, LCh (a3-A3C1C2, A3C1C2) subunit, C2 domain to immobilized sTF (Kd; 2.3±0.6, 5.8±1.0, 10.5±3.5, 11.8±0.5 nM, respectively). The intact HCh, A1 or A2 domain to sTF failed to bind, however. A non-equilibrium ELISA also revealed that sTF bound to immobilized C2 domain with moderate affinity (Kdapp; 16.9±2.2 nM), and the interaction was dependent on ionic strength and Ca2+. In addition, the presence of VWF significantly competitively inhibited the C2 and sTF binding by ~90% (IC50; 5.7 µg/ml) at the maximal concentration employed, suggesting that the C2 domain-TF interaction could activate FVIII by FXa even in the presence of VWF.We concluded that it might be possible that TF enhanced the FVIII-mediated FXa generation by not only FVIIa but also FXa, additionally this enhancing mechanism might not be suppressed by TFPI in the initiation phase of coagulation. Furthermore, TF might function to FVIII activation, irrespective of presence of VWF, by the binding to C2 domain through the competition with VWF. DisclosuresNogami:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Shima:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Design and Synthesis of Novel Meta-Linked Phenylglycine Macrocyclic FVIIa Inhibitors.

Two novel series of meta-linked phenylglycine-based macrocyclic FVIIa inhibitors have been designed to improve the rodent metabolic stability and PK observed with the precursor para-linked phenylglycine macrocycles. Through iterative structure-based design and optimization, the TF/FVIIa Ki was improved to subnanomolar levels with good clotting activity, metabolic stability, and permeability.

Atropisomer Control in Macrocyclic Factor VIIa Inhibitors.

Incorporation of a methyl group onto a macrocyclic FVIIa inhibitor improves potency 10-fold but is accompanied by atropisomerism due to restricted bond rotation in the macrocyclic structure, as demonstrated by NMR studies. We designed a conformational constraint favoring the desired atropisomer in which this methyl group interacts with the S2 pocket of FVIIa. A macrocyclic inhibitor incorporating this constraint was prepared and demonstrated by NMR to reside predominantly in the desired conformation. This modification improved potency 180-fold relative to the unsubstituted, racemic macrocycle and improved selectivity. An X-ray crystal structure of a closely related analogue in the FVIIa active site was obtained and matches the NMR and modeled conformations, confirming that this conformational constraint does indeed direct the methyl group into the S2 pocket as designed. The resulting rationally designed, conformationally stable template enables further optimization of these macrocyclic inhibitors.

Structure-Based Design of Macrocyclic Coagulation Factor VIIa Inhibitors.

On the basis of a crystal structure of a phenylpyrrolidine lead and subsequent molecular modeling results, we designed and synthesized a novel series of macrocyclic FVIIa inhibitors. The optimal 16-membered macrocycle was 60-fold more potent than an acyclic analog. Further potency optimization by incorporation of P1' alkyl sulfone and P2 methyl groups provided a macrocycle with TF/FVIIa Ki = 1.6 nM, excellent selectivity against a panel of seven serine proteases, and FVII-deficient prothrombin time EC2x = 1.2 μM. Discovery of this potent, selective macrocyclic scaffold opens new possibilities for the development of orally bioavailable FVIIa inhibitors.

Design and synthesis of potent, selective phenylimidazole-based FVIIa inhibitors.

Heterocyclic amide isosteres were incorporated into a phenylglycine-based tissue factor/factor VIIa (TF-FVIIa) inhibitor chemotype, providing potent inhibitors. An X-ray co-crystal structure of phenylimidazole 19 suggested that an imidazole nitrogen atom effectively mimics an amide carbonyl, while the phenyl ring forms key hydrophobic interactions with the S1' pocket. Exploration of phenylimidazole substitution led to the discovery of potent, selective and efficacious inhibitors of TF-FVIIa.

Activated prothrombin complex concentrate (APCC)-mediated activation of factor (F)VIII in mixtures of FVIII and APCC enhances hemostatic effectiveness

Activated prothrombin complex concentrates (APCCs), utilized in bypassing therapy for hemophiliacs with inhibitor, contain factors (Fs) VII, FII, FIX and FX, and their active forms. A recent report has demonstrated that mixtures of APCC and FVIII potentiated thrombin generation, in vitro, in plasma from patients with severe hemophilia A, but the mechanism(s) involved remains unknown. APCC (0.05UmL(-1) ) increased FVIII activity ~4-fold within 1min in one-stage clotting assays, followed by a return to initial levels within 10min. This reaction was dependent on the presence of tissue factor and phospholipid. Thrombin generation produced from APCC was ~3.5-fold greater in the presence of FVIII than that in its absence. SDS-PAGE analysis revealed that APCC sequentially proteolyzed the heavy chain of FVIII at Arg(372) and Arg(740) , followed by cleavage at Arg(336) . Proteolysis was prevented by FVIIa inhibitor, but not by hirudin, supporting the concept that APCC itself possessed the potential to activate FVIII in early coagulation phases, and that FVIIa in APCC contributed mainly to this reaction. APCC-mediated FVIII activation was unaffected by the addition of anti-FVIII inhibitor antibodies, irrespective of epitope specificity. Anti-C2 type 1 inhibitors, however, diminished the inactivation phase of the APCC reaction by inhibiting cleavage at Arg(336) . Small amounts of APCC, relative to the standard concentration used for clinical purposes, could activate FVIII directly, even in the presence of anti-FVIII antibodies. Combination therapy based on mixtures of APCC and FVIII could have significant beneficial implications for the treatment of hemophilia A patients with inhibitors.

Direct inhibition of factor VIIa by TFPI and TFPI constructs

Tissue factor pathway inhibitor (TFPI) is a multi-Kunitz domain protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. To investigate the role of the three Kunitz domains (KDs) of TFPI in FVIIa inhibition using full-length TFPI (TFPIfl ) and truncated TFPI constructs. Inhibition of FVIIa with/without relipidated tissue factor (TF) or soluble TF (sTF) by TFPIfl /TFPI constructs was quantified with a FVIIa-specific chromogenic substrate. TFPIfl inhibited TF-FVIIa via a monophasic reaction, which is rather slow at low TFPIfl concentrations (t½ ≈5min at 2nm TFPI) and has a Ki of 4.6nm. In the presence of sTF and without TF, TFPIfl was a poor FVIIa inhibitor, with Ki values of 122nm and 1118nm, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPIfl . TFPI constructs without the KD3-c-terminus (TFPI1-150 and KD1-KD2) were 7-10-fold less effective than TFPIfl in inhibiting TF-FVIIa and sTF-FVIIa, indicating that the KD3-C-terminus significantly contributes to direct inhibition of FVIIa by TFPI. Compared with KD1-KD2, KD1 was a poor TF-FVIIa inhibitor (Ki =434nm), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF-FVIIa inhibition by TFPIfl (Ki =0.7nm). In the presence of FXa, a tight quaternary TF-FVIIa-TFPI-FXa complex is formed with TFPIfl , TFPI1-150 and KD1-KD2, with Ki values of <0.15nm, 0.5nm and 0.8nm, respectively, indicating the KD3-C-terminus is not a prerequisite for quaternary complex formation. Phospholipids and the Gla-domain of FXa are required for quaternary complex formation.

Discovery and gram-scale synthesis of BMS-593214, a potent, selective FVIIa inhibitor

A 6-amidinotetrahydroquinoline screening hit was driven to a structurally novel, potent, and selective FVIIa inhibitor through a combination of library synthesis and rational design. An efficient gram-scale synthesis of the active enantiomer BMS-593214 was developed, which required significant optimization of the key Povarov annulation. Importantly, BMS-593214 showed antithrombotic efficacy in a rabbit arterial thrombosis model. A crystal structure of BMS-593214 bound to FVIIa highlights key contacts with Asp 189, Lys 192, and the S2 pocket.

Design and synthesis of bicyclic pyrazinone and pyrimidinone amides as potent TF–FVIIa inhibitors

Bicyclic pyrazinone and pyrimidinone amides were designed and synthesized as potent TF–FVIIa inhibitors. SAR demonstrated that the S2 and S3 pockets of FVIIa prefer to bind small, lipophilic groups. An X-ray crystal structure of optimized compound 9b bound in the active site of FVIIa showed that the bicyclic scaffold provides 5 hydrogen bonding interactions in addition to projecting groups for interactions within the S1, S2 and S3 pockets. Compound 9b showed excellent FVIIa potency, good selectivity against FIXa, Xa, XIa and chymotrypsin, and good clotting activity.

Tissue Factor–Activated Coagulation Cascade in the Tumor Microenvironment Is Critical for Tumor Progression and an Effective Target for Therapy

Tissue factor (TF), a rate-limiting enzyme cofactor in activating coagulation, is highly expressed in a wide spectrum of human tumor and tumor stromal cells. Using TF-deficient cancer cells and a conditional TF-knockout mouse model, we show that TF expressed by cancer cells, but not by the host stromal cells, plays a critical role in tumor growth. In the tumor microenvironment, serum coagulation factors are readily extravasated and therefore lead to continuous TF-mediated activation of coagulation proteases. To target this highly specific cascade of serine proteases, we used both a TF:VIIa inhibitor and doxorubicin-based prodrugs that are selectively activated by TF:FVIIa, FXa, and thrombin. Treatment with the TF:FVIIa inhibitor led to growth retardation in breast tumor models. In contrast, treatment with the prodrug eliminated primary tumor cells and lung metastases without apparent toxicity. Our findings offer preclinical proof of principle that targeting the coagulation cascade that is activated in the tumor microenvironment can be a highly effective approach for cancer therapy.

Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-D-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K(i)=8μM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.

BMS-593214, an active site-directed factor VIIa inhibitor: Enzyme kinetics, antithrombotic and antihaemostatic studies

Factor (F) VIIa in association with tissue factor (TF) is the primary in vivo initiator of blood coagulation and activates FX and FIX to generate thrombin, which plays a key role in the pathogenesis of thrombosis. We evaluated the enzyme kinetics, antithrombotic and antihaemostatic properties of BMS-593214, an active-site, direct FVIIa inhibitor. Studies were conducted in enzymatic assays, and in anesthetised rabbit models of electrically-induced carotid arterial thrombosis (AT), thread-induced vena cava venous thrombosis (VT) and cuticle bleeding time (BT). Antithrombotic efficacy of BMS-593214 given intravenously was evaluated for both the prevention and treatment of AT and VT. BMS-593214 displayed direct, competitive inhibition of human FVIIa in the hydrolysis of a tripeptide substrate with Ki of 5 nM. However, it acted as a noncompetitive inhibitor of the activation of the physiological substrate FX by TF/VIIa with Ki of 9.3 nM. BMS-593214 showed selectivity for FVIIa and exhibited species differences in TF-FVIIa-dependent anticoagulation with similar potency in human and rabbit plasma. BMS-593214 was efficacious in the prevention and treatment models of AT and VT with ED50 values of 1.1 to 3.1 mg/kg. Furthermore, BMS-593214 exhibited a wide therapeutic window with respect to BT. These results suggest that inhibition of FVIIa with small-molecule active-site inhibitors represents a promising antithrombotic approach for the development of new therapies for the prevention and treatment of AT and VT.

N,N′-Dihydroxyamidines: A New Prodrug Principle To Improve the Oral Bioavailability of Amidines

N, N'-dihydroxybenzamdine represents a model compound for a new prodrug principle to improve the oral bioavailability of drugs containing amidine functions. The activation of the prodrug could be demonstrated in vitro by porcine and human subcellular enzyme fractions, the mitochondrial benzamidoxime reducing system, and porcine hepatocytes. In vivo, the bioavailability of benzamidine after oral application of N, N'-dihydroxybenzamidine was about 91% and exceeded that of benzamidine after oral application of benzamidoxime, being about 74% (Liu, L.; Ling, Y.; Havel, C.; Bashnick, L.; Young, W.; Rai, R.; Vijaykumar, D.; Riggs, J. R.; Ton, T.; Shaghafi, M.; Graupe, D.; Mordenti, J.; Sukbuntherng, J. Species comparison of in vitro and in vivo conversion of five N-hydroxyamidine prodrugs of fVIIA inhibitors to their corresponding active amidines. Presented at the 13th North America ISSX Meeting, Maui, HI, 2005).

Structure of human factor VIIa/tissue factor in complex with a peptide-mimetic inhibitor: high selectivity against thrombin by introducing two charged groups in P2 and P4.

The crystal structure of human factor VIIa/soluble tissue factor (FVIIa/sTF) in complex with a highly selective peptide-mimetic FVIIa inhibitor which shows 1670-fold selectivity against thrombin inhibition has been solved at 2.6 A resolution. The inhibitor is bound to FVIIa/sTF at the S1, S2 and S3 sites and at the additional S1 subsite. Two charged groups, the amidino group in P2 and the carboxylate group in P4, form ionic interactions with Asp60 and Lys192 of FVIIa, respectively. Structural comparisons between factor VIIa and thrombin show that thrombin has oppositely charged residues, Lys60F and Glu192, in the S2 site and the S1 subsites, respectively. These data suggest that the utilization of the differences of charge distribution in the S2 site and the S1 subsites between FVIIa and thrombin is critical for achieving high selectivity against thrombin inhibition. These results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.

Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents

Cardiovascular and cerebrovascular diseases continue to be the leading cause of death throughout the world. Over the past two decades, great advances have been made in the pharmacological treatment and prevention of thrombotic disorders (e.g., tissue plasminogen activators, platelet GPIIb/IIIa antagonists, ADP receptor antagonists such as clopidogrel, low-molecular weight heparins, and direct thrombin inhibitors). New research is leading to the next generation of antithrombotic compounds such as direct coagulation FVIIa inhibitors, tissue factor pathway inhibitors, gene therapy, and orally active direct thrombin inhibitors and coagulation Factor Xa (FXa) inhibitors. Animal models of thrombosis have played a crucial role in discovering and validiting novel drug targets, selecting new agents for clinical evaluation, and providing dosing and safety information for clinical trials. In addition, these models have provided valuable information regarding the mechanisms of these new agents and the interactions between antithrombotic agents that work by different mechanisms. This review briefly presents the pivitol preclinical studies that led to the development of drugs that have proven to be effective clinicallly. The role that animal models of thrombosis are playing in the discovery and development of novel antithrombotic agents is also described, with specific emphasis on FXa inhibitors. The major issues regarding the use of animal models of thrombosis, such as the use of positive controls, appropriate pharmacodynamic markers of activity, safety evaluation, species-specificity, and pharmacokinetics, are highlighted. Finally, the use of genetic models in thrombosis/hemostasis research and pharmacology is presented using gene-therapy for hemophilia as an example of how animal models have aided in the development of these therapies that are now being evaluated clinically. In summary, animal models have contributed greatly to the discovery of currently available antithrombotic agents and will play a primary role in the discovery and characterization of the novel antithrombotic agents that will provide safe and effective pharmacological treatment for life-threatening thrombotic diseases.