FRONTIER1: a partially randomized phase 2 study assessing the safety, pharmacokinetics, and pharmacodynamics of Mim8, a factor VIIIa mimetic.

BACKGROUND: Mim8 (denecimig) is a factor VIII (FVIII) mimetic bispecific antibody in development for the treatment of hemophilia. Data from the phase 1 part of FRONTIER1 (EudraCT: 2019-000465-20, NCT04204408, and NN7769-4513) suggested that Mim8 was well tolerated in healthy participants and exhibited pharmacokinetic (PK) properties consistent with dose proportionality. OBJECTIVES: The partially randomized, phase 2, multiple ascending dose (MAD) part of FRONTIER1 aimed to evaluate the safety, PK, pharmacodynamics (PD), and exploratory efficacy of Mim8 in participants with hemophilia A with or without FVIII inhibitors. METHODS: The MAD part of FRONTIER1 consisted of 42 participants, assigned to 5 cohorts, with participants in cohorts 3 and 4 randomized 1:1 to dosing weekly or every 4 weeks, respectively. Four of the 42 participants (9.5%) had FVIII inhibitors prior to study enrolment. The primary endpoint was treatment-emergent adverse events (TEAEs). PK and PD were evaluated by Mim8 plasma concentration and thrombin generation, respectively. Exploratory efficacy was assessed via the number of treated bleeds. Safety and PD parameters were also evaluated from an exploratory cohort treated with emicizumab. RESULTS: Mim8 was well tolerated, with 1 serious TEAE (anxiety-related chest pain) deemed unrelated to Mim8. There was no dose dependency on the number, causality, type, or severity of TEAEs. PK/PD properties supported weekly to monthly dosing approaches, and few participants experienced treated bleeds beyond the lowest dose cohort (1 in cohorts 2 and 3, and 3 in cohort 5). CONCLUSION: These data support the continued clinical development of Mim8, and FRONTIER1 has proceeded onto an extension phase.

Recombinant B domain deleted porcine factor VIII for the treatment of bleeding episodes in adults with acquired hemophilia A.

Hemophilia A is an inherited deficiency of clotting factor VIII (FVIII) often complicated by inhibitor development (CHAWI) in which neutralizing antibodies block the therapeutic benefit of replacement therapy. Inhibitors to FVIII can also be seen in an auto-immune disease known as acquired hemophilia A (AHA). 'Bypassing' therapies have been shown to provide hemostasis but dosing must be done empirically because current assays cannot measure objective markers of treatment efficacy and safety. A recombinant porcine sequence factor VIII (r-pFVIII) has been developed for the management of AHA. Preclinical, Phase I and Phase II clinical research studies in CHAWI subjects showed therapeutic potential and safety of this agent. A Phase II/III study in AHA with serious bleeding episodes shows a positive response in all subjects after administration. Based on current preclinical and clinical trial data, r-pFVIII should become the first line of treatment in the management of hemorrhage in patients with AHA.

Reversal of rivaroxaban-induced alterations on hemostasis by different coagulation factor concentrates ­ in vitro studies with steady and circulating human blood.

BACKGROUND: Despite the good safety of rivaroxaban, there is limited information on strategies for urgent reversal of its antihemostatic effects. METHODS AND RESULTS: Alterations of hemostasis induced by rivaroxaban (230 ng/ml) were assessed by using several tests applied to steady and circulating human blood. Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were measured. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with circulating blood. The potential reversal of prothrombin complex concentrates (PCCs; 50 IU/kg), activated PCCs (aPCCs; 75 IU/kg), or recombinant factor VIIa (rFVIIa; 270 µg/kg) was evaluated. Impairment of TG parameters induced by rivaroxaban were corrected by the different concentrates (aPCC≥PCC>rFVIIa). Prolonged clotting times and reduced clot firmness caused by rivaroxaban on TEM tests were improved by different concentrates (rFVIIa≥aPCC>PCC). Rivaroxaban significantly reduced platelets and fibrin interactions with damaged vascular surfaces in perfusion studies. While alterations of platelet interactions were favourably counteracted by rFVIIa or aPCCs, reductions in fibrin formation were only partially restored by the different factor concentrates (rFVIIa>aPCC≥PCC). CONCLUSIONS: Rivaroxaban-induced alterations on coagulation parameters measured through assays performed under static conditions were easily reversed by the different concentrates. Studies under flow conditions revealed that these concentrates normalized the action of rivaroxaban on platelets, and significantly improved fibrin formation; although in the later case, levels were not restored to the pre-treatment value.

Erythrocyte-derived microparticles supporting activated protein C-mediated regulation of blood coagulation.

Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte-derived microparticle surface is suitable for the anticoagulant reactions of the protein C system, which may be important to balance the initiation and propagation of coagulation in vivo.

Cyclic peptide analogs of 558-565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants.

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII-Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558-565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa-Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.

A soluble tissue factor-annexin V chimeric protein has both procoagulant and anticoagulant properties.

Tissue factor (TF) initiates blood coagulation, but its expression in the vascular space requires a finite period of time. We hypothesized that targeting exogenous tissue factor to sites of vascular injury could lead to accelerated hemostasis. Since phosphatidylserine (PS) is exposed on activated cells at sites of vascular injury, we cloned the cDNA for a chimeric protein consisting of the extracellular domain of TF (called soluble TF or sTF) and annexin V, a human PS-binding protein. Both the sTF and annexin V domains had ligand-binding activities consistent with their native counterparts, and the chimera accelerated factor X activation by factor VIIa. The chimera exhibited biphasic effects upon blood coagulation. At low concentrations it accelerated blood coagulation, while at higher concentrations it acted as an anticoagulant. The chimera accelerated coagulation in the presence of either unfractionated or low-molecular-weight heparins more potently than factor VIIa and shortened the bleeding time of mice treated with enoxaparin. The sTF-annexin V chimera is a targeted procoagulant protein that may be useful in accelerating thrombin generation where PS is exposed to the vasculature, such as may occur at sites of vascular injury or within the vasculature of tumors.

Kinetics of human factor VII activation.

In this study the activation of human factor VII by a variety of potential activators in the presence and absence of mixed phospholipid vesicles [25% phosphatidylserine (PS), 75% phosphatidylcholine (PC)] is evaluated. At the plasma concentration of factor VII, 10 nM, the activation rate of the zymogen by 0.05 nM factor Xa is anionic phospholipid (PCPS) dependent and achieves a maximum value of 18 pM/s at 5-20 microM PCPS; further increases in the levels of PCPS decrease the activation rate of factor VII. The maximum activation rate of factor VII (10 nM) by the factor VIIa-tissue factor complex (0.1 nM), 0.76 pM/s, is achieved at 200 microM PCPS. No detectable activation of 10 nM factor VII is observed under similar conditions when either thrombin (0.1 nM) or factor IXa (0.1 nM) is used as an activator. Factor VIIa (10 nM) and factor XIa (1 nM) are not observed to activate factor VII at detectable rates. The observed Michaelis-Menten constants (KM) for factor VII activation in the presence of PCPS at optimal concentrations vary from 1.2 microM for factor Xa to 3.2 microM for the factor VIIa-tissue factor complex. The highest catalytic constant (kcat) value (15.2 s-1) is observed for factor Xa-PCPS. The factor VIIa-tissue factor complex, factor IXa, and thrombin kcat values are 1.4, 0.32, and 0.061 s-1, respectively. Tissue factor does not increase the factor VII activation rate by factor Xa, factor IXa, or thrombin. Factor VIIIa in the presence of PCPS has no effect on factor VII activation by factor IXa. In contrast, factor Va decreases the factor VII activation rate by factor Xa, reaching saturation at concentrations consistent with complete prothrombinase complex formation. The formed prothrombinase complex activates factor VII at approximately 30% the rate of factor Xa bound to phospholipids. These data allow us to conclude that the predominant physiological factor VII activator is, most likely, membrane-bound factor Xa.

The role of human factor X activation peptide in activation of factor X by factor IXa.

We studied the interaction of factor X activation peptide (XAP) with factor IXa and factor Xa and the effect of XAP on factor IXa-catalyzed activation of factor X. XAP associated with factor Xa in the presence of 5 mM Ca2+ was dissociated from factor Xa by gel chromatography using Ultrogel AcA54 in 5 mM EDTA, or in 8 M urea-0.1% SDS. An exogenous isolated XAP inhibited the factor IXa-catalyzed factor X activation both in the presence and absence of factor VIIIa. 4-Amidinophenylmethylsulfonyl (aPMS)-factor Xa independent of XAP also inhibited the factor X activation more effectively than XAP alone in the presence of factor VIIIa. However, aPMS-factor Xa independent of XAP hardly inhibited the factor X activation in the absence of factor VIIIa. The binding of 125I-labeled factor X to the aPMS-factor IXa fixed to a microwell plate was inhibited by unlabeled factor X or XAP, but not by aPMS-factor Xa with or without XAP. Factor IXa directly bound to XAP and aPMS-factor Xa with XAP, but did not bind to aPMS-factor Xa without XAP. These findings suggest that the region of XAP in factor X directly interacts with factor IXa, and factor Xa region other than XAP interacts with factor VIIIa. Desialation or deletion of N-linked carbohydrates of XAP reduced the inhibitory activity of XAP for the factor X activation by factor IXa to approximately 50% of that of the intact XAP. This suggests that the sialic acids in the carbohydrate chains of the XAP region partly contribute to the interaction with factor IXa during its activation.

Mechanism-based isocoumarin inhibitors for blood coagulation serine proteases. Effect of the 7-substituent in 7-amino-4-chloro-3-(isothioureidoalkoxy)isocoumarins on inhibitory and anticoagulant potency.

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine. With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.