802
Newer Hemostatic Agents Massimo Franchini, MD1
Emmanuel J. Favaloro, PhD FFSc (RCPA)2
1 Department of Hematology and Transfusion Medicine, Azienda
Ospedaliera Carlo Poma, Mantova, Italy 2 Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Pathology West, Westmead Hospital, Westmead, New South Wales, Australia 3 Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy
Giuseppe Lippi, MD3
Address for correspondence Massimo Franchini, MD, Department of Hematology and Transfusion Medicine, Azienda Ospedaliera Carlo Poma, Mantova, Italy (e-mail: [email protected]).
Abstract
Keywords
► clotting factor concentrates ► new hemostatic products ► extended half-life ► hemophilia ► inhibitors
The mainstay of treatment of inherited coagulation disorders is based on the infusion of the deficient clotting factor, when available. Significant advances have been made over the past two decades in the production and availability of factor replacement products. In spite of such progression, several issue are still unsolved, the most important being the need for frequent factor concentrate infusions and the development of inhibitory alloantibodies. To overcome these important limitations, several newer hemostatic agents with an extended half-life are at an advanced stage of clinical development. After a brief overview of hemostasis, this narrative review summarizes the current knowledge on the most promising novel products for hemostasis. The current status of gene therapy for hemophilia, the only therapeutic option to definitively cure this inherited bleeding disorder, is also concisely discussed.
The availability of highly purified plasma-derived and recombinant clotting factor concentrates over the last two decades has dramatically improved the quality of life and the life expectancy of severely affected hemophilia A and B patients, as it has greatly facilitated the widespread adoption of home replacement therapy with the broad implementation of prophylactic treatment regimens.1–6 Indeed, regular prophylaxis has proved to be an extraordinarily effective therapeutic defense able to remarkably reduce, compared with on-demand therapy, spontaneous and life-threatening bleeds and to protect hemophiliacs from the development and progression of arthropathy.7 However, several issues still remain incompletely resolved in the management of hemophilia. First, the short median half-life of factor VIII (FVIII) and factor IX (FIX) limits the ongoing efficacy of replacement therapy, requiring frequent infusions, especially when prophylactic regimens are adopted in pediatric and young adult hemophiliacs, making adherence to treatment difficult for these patients.5 Another major clinical challenge is the management of inhibitors, which develop in many patients and render hemostatic control extremely difficult and complicated, necessitating the use of bypassing agents (i.e., recombinant activated factor VII
published online April 20, 2015
Issue Theme Tissue Factor in Arterial and Venous Thrombosis: From Pathophysiology to Clinical Implications; Guest Editors: Vincenzo Toschi, MD, and Massimo Franchini, MD.
[rFVIIa] and activated prothrombin complex concentrates [APCC]) which remain, however, less effective than standard replacement therapy.8–11 Thus, major research in this field has focused on development of coagulation factor concentrates that are less immunogenic and with a prolonged half-life, aiming by less frequent infusions, at decreasing bleeding rate and reducing the burden of treatment. In this narrative review, we summarize the new technologies and the current development status of the novel products for hemostasis, focusing in particular on those with half-life extension for treatment of hemophilia. A brief overview of the current status of gene therapy for hemophilia is also presented.
Overview of Hemostasis Hemostasis represents the mechanism whereby the body maintains circulatory blood flow. It can be represented by Virchow triad (►Fig. 1A) or, more commonly, as a balance of procoagulant and anticoagulant mechanisms or pathways, inclusive of fibrinolysis.12,13 In brief, damage to the
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0034-1544004. ISSN 0094-6176.
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Semin Thromb Hemost 2015;41:802–808.
Fig. 1 (A) Virchow triad. (B) Secondary hemostasis. (C) The fibrinolytic system.
vasculature will initiate two major integrated “procoagulant” pathways, termed primary and secondary hemostasis, which will lead to eventual plug formation to prevent excessive hemorrhage. The primary pathway involves activation of platelets, the recruitment of von Willebrand factor (VWF) to promote platelet attachment to the site of injury, and the engagement of secondary hemostasis (►Fig. 1B) through various procoagulant proteins including fibrinogen, and factors (F) V, VIII, and IX. Secondary hemostasis is also initiated directly by damage to the vasculature, primarily via the tissue factor (TF) pathway involving FVII, but also via
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the contact pathway, and as amplified by the primary pathway (►Fig. 1B). The process of secondary hemostasis essentially represents the progression of blood coagulation, involving a series of coordinated and calcium-dependent conversions of proenzymes to the respective serine proteases, culminating in the conversion (activation) of prothrombin (FII) to thrombin (FIIa). Moreover, the interaction of these interconnected pathways with cellular components is critical for hemostasis in vivo.14 The so-called extrinsic (TF) pathway is initiated at the site of injury in response to the release of TF (factor III) (►Fig. 1B). TF is a cofactor in the activated FVII (FVIIa)catalyzed activation of FX, which cleaves FX to FXa. According to the amplification model, each FVIIa-TF complex can produce many FXa molecules, which subsequently produce an even greater amount of thrombin. The so-called intrinsic (contact) pathway (►Fig. 1B), involving the clotting factors VIII, IX, X, XI, and XII, is less significant for in vivo hemostasis under normal physiologic conditions. Nevertheless, the major hemophilias are represented by deficiencies of FVIII (hemophilia A), FIX (hemophilia B), and, to a lesser extent, FXI (hemophilia C). Initiation of the intrinsic pathway theoretically occurs when prekallikrein, high-molecular-weight kininogen, FXI, and FXII are exposed to a negatively charged surface (“the contact phase”). The assemblage of contact phase components results in conversion of prekallikrein to kallikrein, which in turn activates FXII to FXIIa. FXIIa then activates FXI to FXIa, which in turn activates FIX to FIXa. The subsequent activation of FXa requires assemblage of the tenase complex (Ca2þ and factors VIIIa, IXa, and X) on the surface of activated platelets. In this process, FVIIIa acts as a cofactor for factors IXa and X. The final protease generated is thrombin (FIIa), which converts fibrinogen into an insoluble fibrin gel, which is stabilized by covalent cross-linking catalyzed by FXIIIa. Several lines of evidence now attest that the physiological role of FXII in activating the intrinsic pathways is probably meaningless in vivo, because FXII deficiency (even at homozygous state) does not significantly impair the hemostatic response,15 so that the effective function of this factor in the initiation phase of blood coagulation remains largely controversial. A more genuine in vivo role of the intrinsic pathway in blood coagulation seems hence attributable to the amplification of the coagulation cascade, which results from the activation of factors V, VIII, and possibly XI by thrombin, as well as by direct activation of FVII. It is important to recognize that that the dichotomy between intrinsic and extrinsic pathways as assessed by in vitro coagulation assays does not accurately reflect the hemostasis activation in vivo. In an in vitro cell-based model, adding increasing amounts of FVIIa to activated platelets produces increase in generation of FXa independently of the presence of TF on the platelet surface.16 Therefore, FVIIa at pharmacologic doses, being much higher than those generated during normal hemostasis (i.e., 10–100 times higher), provides an effective bypassing agent, which can directly activate FX and lead to an additional burst of thrombin in a broad spectrum of congenital and acquired bleeding conditions, including Seminars in Thrombosis & Hemostasis
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hemophilia A and B, as well as in the presence of factor inhibitors (►Fig. 1). Secondary blood coagulation is under the influence of several negative regulators at different steps, including tissue factor pathway inhibitor (TFPI), antithrombin (AT), and the components of the protein C system (protein C, protein S, and thrombomodulin). The protein C system and AT are both effective in regulating the propagation phase of thrombin, whereas TFPI is primarily effective in regulating the initiation phase of the reaction, by inhibiting the FVIIa-TF complex.
Hemostatic Agents with Extended Half-Life The half-life of human FVIII is 10 to 12 hours, for FIX is 18 to 34 hours, and the half-life of FVIIa is approximately 2.5 hours, being the shortest among all the coagulation factors. Several technologies are being applied to prolong the half-life of FVIIa, FVIII, and FIX, including PEGylation, glycoPEGylation, Fc-fusion, and recombinant albumin fusion17,18 (►Table 1).
New FVIII Products A B-domain truncated modified O-glycoPEGylated recombinant FVIII (rFVIII) is in development (N8-GP, Novo Nordisk, Bagsvaerd, Denmark), and is scheduled for commercialization in 2015. N8-GP has a 40 kDa PEG attached to a B-domain truncated molecule of rFVIII (turoctocog alfa). Upon activation by thrombin, the B-domain containing PEG is released, leaving active rFVIII.19 Preclinical studies evaluating the pharmacokinetic (PK) and pharmacodynamic (PD) properties of N8-GP in dogs with congenital hemophilia showed a halflife of 22 hours and extended PD activity.20 Similarly, a clinical dose-escalation study in 26 previously treated patients with hemophilia A showed a linear-dose PK profile, a mean halflife of 19 hours, and a low frequency of adverse events without inhibitors.21 A phase III trial in adults is underway
(ClinicalTrials.gov identifier: NCT01480180), as is a pediatric study (ClinicalTrials.gov identifier: NCT01731600), while a trial evaluating efficacy during surgery is currently recruiting (ClinicalTrials.gov identifier: NCT01489111). Another B-domain–deleted PEGylated rFVIII in development is BAY 94–9027 (Bayer Healthcare AG, Leverkusen, Germany), which contains a single, large-branched PEG molecule conjugated to a specific amino acid on rFVIII.22 In a phase I clinical study in previously treated patients with hemophilia A, BAY 94–9027 demonstrated equivalent recovery and improved PK profile versus rFVIII formulated with sucrose (rFVIII-FS) (19 vs. 13 hours), with no immunogenicity.23 A phase III trial (ClinicalTrials.gov Identifier: NCT01775618) is ongoing, whereas another (ClinicalTrials.gov Identifier: NCT01580293) is recruiting. Baxter (Baxter Innovations GmbH, Vienna, Austria) is developing a PEGylated version of its full-length rFVIII ADVATE (BAX 855) with extended half-life. This technique provides two moles (20 kDa) of PEG per rFVIII molecule in a non–site-specific manner, which improves the PK profile without compromising specific activity.24 Preclinical studies in various animal models have demonstrated a twofold prolongation of terminal half-life.24 A phase II/III clinical trial of BAX 855 (ClinicalTrials.gov Identifier: NCT01736475) in previously treated patients with severe hemophilia A is ongoing, with a further trial currently recruiting patients undergoing surgical procedures (ClinicalTrials.gov Identifier: NCT01913405). BAX 855 is expected to reach the market toward the end of 2016. Biogen Idec (Weston, MA) is developing a rFVIII fusion protein (rFVIII-Fc), which is a recombinant fusion of a B-domain deleted FVIII molecule and the dimeric constant region (Fc) of IgG1.25,26 The first human single-dosing study in 16 previously treated patients showed that the elimination of rFVIIIFc was prolonged by 1.5- to 1.7-fold compared with a
Table 1 Products with extended half-life for hemophilia treatment in clinical development Product
Company
Technology
Terminal half-life
Stage of development
Novo Nordisk
Site-specific glycoPEGylation
22 h
Phase III ongoing
FVIII N8-GP BAY 94–9027
Bayer Healthcare
Site-specific glycoPEGylation
19 h
Phase III ongoing
BAX855
Baxter Innovations
Non–site-specific glycoPEGylation
2-fold (preclinical studies)
Phase III ongoing
rFVIII-Fc (ELOCTATE)
Biogen Idec
Fusion to IgG1-Fc
19 h
Marketed in the USA
N9-GP
Novo Nordisk
Site-specific glycoPEGylation
110 h
Phase III ongoing
rIX-Fc (ALPROLIX)
Biogen Idec
Fusion to IgG1-Fc
82 h
Marketed in the USA
rFIX-FP
CSL Behring
Fusion protein with albumin
92 h
Phase III ongoing
CSL Behring
Fusion protein with albumin
8.5 h
Phase II/III studies are expected to commence in late 2014
FIX
FVIIa rFVIIa-FP
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native rFVIII.27 Similarly, the 52-week, open-label, phase III (A-LONG) study conducted in 165 previously treated patients with hemophilia A showed that the terminal half-life of rFVIII-Fc was 19 hours versus 12.4 for rFVIII.28 rFVIII-Fc resulted in an annualized bleeding rate (ABR) of 1.6 in those receiving prophylaxis with rFVIII-Fc every 3 to 5 days (median weekly dose 77.7 IU/kg), 3.6 with weekly prophylaxis (fixed weekly dose of 65 IU/kg), and 33.6 for episodic treatment (10–50 IU/kg).28 In this study, 87.3% of all bleeding episodes were resolved with one and 97.8% with two or fewer injections, and 45.3% of patients experienced no bleeding episodes during the study.28 Recent updates of these studies confirmed the efficacy and safety of rFVIII-Fc in both prophylaxis and on-demand regimens.29,30 A trial in pediatric patients is ongoing (ClinicalTrials.gov Identifier: NCT01458106). rFVIIIFc was launched in the United States in September 2014 under the brand name of ELOCTATE (Biogen Idec, Weston, MA).
New FIX Products In a preclinical study, the glycoPEGylated rFIX nonacog β pegol (N9-GP, Novo Nordisk) exhibited a greater than twofold increase in in vivo recovery and a markedly prolonged half-life in minipigs and hemophilia B dogs compared with rFIX.31 The first human dose trial (ClinicalTrials.gov identifier: NCT00956345) assessed three doses of N9-GP in 16 patients with hemophilia B and demonstrated an extended half-life (mean 93 h), which was five times longer than in hemophilia B patients previously treated with conventional FIX products.32 Population PK modeling based on these results has suggested that weekly prophylaxis may be possible with N9-GP.33 Results of a phase III clinical trial were presented recently, assessing prophylaxis and on-demand treatment in 74 patients with hemophilia B, with no patients developing inhibitors. The mean half-life was 110 hours and median ABR with prophylactic therapy of 40 U/kg and 10 U/kg per week were 1.0 and 2.9 episodes per year, respectively, and 15.9 for on-demand treatment, with 99% of bleeds resolved following a single injection.34 An extension trial (ClinicalTrials. gov identifier: NCT01395810) and a pediatric trial (ClinicalTrials.gov identifier: NCT01467427) are ongoing, and a trial in surgical patients has recently finished (ClinicalTrials.gov identifier: NCT01386528). The fusion of the Fc-portion of immunoglobulin G to a single molecule of rFIX (rFIX-Fc) (Biogen Idec) has also been explored to increase its circulation time.35 In animal models of hemophilia B, rFIX-Fc exhibited an extended half-life of up to 48 hours, as compared with the standard rFIX half-life of approximately 18 hours.36 A phase I/II clinical study (ClinicalTrials.gov identifier: NCT00716716) reported that rFIX-Fc was welltolerated, with a threefold longer half-life than native rFIX.37 An open-label multicenter phase III study (B-LONG Study; ClinicalTrials.gov identifier: NCT01027364), enrolling 123 patients and aiming at evaluating safety, PK, and efficacy of rFIX-Fc in previously treated patients with severe and moderately severe hemophilia B, was recently completed.38 The mean half-life of rFIX-Fc was 82.1 hours compared with 33.8 hours for rFIX, with a mean ABR in the weekly prophylaxis arm
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(starting dose 50 IU/kg) of 2.95 versus 1.38 in the individualized prophylaxis arm (starting with doses of 100 IU/kg every 10 days) and 17.69 in the on-demand arm. The overall median dosing individualized-interval prophylaxis was 12.5 days, whereas it was 13.8 days during the last 6 months of the study. No inhibitors were detected throughout the study period, where 93.5% of bleeding episodes resolved with a single rFIX-Fc infusion.38 An extension trial is enrolling (ClinicalTrials.gov identifier: NCT01425723), and a pediatric trial is currently recruiting participants (ClinicalTrials.gov identifier: NCT01440946). rFIX-Fc was approved in March 2014 by the Food and Drug Administration (FDA) and is now marketed in the United States under the brand name of ALPROLIX (Biogen Idec, Weston, MA). Fusion of rFIX to recombinant albumin (rFIX-FP, CSL Behring LLC, King of Prussia, PA) has achieved a fivefold lengthening in half-life compared with licensed rFIX in preclinical studies.39 A phase I dose-escalation study conducted in 25 patients with severe hemophilia B demonstrated a fivefold increase in half-life, a 44% increase in recovery, a sevenfold reduction in clearance compared with rFIX, and a linear dose response with no allergic reactions and no development of inhibitors.40 Preliminary results from the open-label PROLONG9-FP phase I/II trial, which included both an on-demand and a weekly prophylaxis treatment arms, indicated an excellent safety profile based on more than 600 exposure days, with no serious adverse events.41 After 11 months of treatment, the 13 patients on prophylaxis had a mean ABR of 1.26, and those switching from pretrial on-demand therapy to prophylaxis (n ¼ 3) had a greater than 80% reduction in bleeding episodes.42 Furthermore, the efficacy of rFIX-FP was confirmed as all bleeding events were treated successfully with two or fewer injections and approximately 90% successfully treated with a single injection.42 A phase II/III trial (ClinicalTrials.gov identifier: NCT01496274) is ongoing, whereas two other trials (an extension trial [ClinicalTrials.gov identifier: NCT02053792] and a pediatric trial [ClinicalTrials.gov identifier: NCT01662531]) are currently recruiting participants.
New FVII Products Though a long-acting rFVIIa-Fc (Biogen Idec) is still at a preclinical stage,43 rFVII-FP (CSL Behring), where rFVIIa and recombinant albumin are fused, is at a more advanced phase of development. PK studies in rodents indicated that the half-life of rFVIIa-FP was 5.8-fold longer and recovery was improved by 1.4-fold compared with rFVIIa.44 Results from the phase I double-blind study were reported recently, showing that rFVIIa-FP was well tolerated after single-dose administration to 40 healthy individuals, with no incidences of inhibitor or antidrug antibody development.45 In humans, the half-life was increased to 8.5 hours with a dose of 1000 µg/kg, and the clearance was reduced by three- to fourfold.45 A phase II/III trial is expected to commence in late 2014. Finally, it is important to highlight that for each new hemostatic product that successfully reaches the market, several products under development are stopped after clinical trial “failures.” For instance, Bayer stopped PEGylated Seminars in Thrombosis & Hemostasis
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liposome associated FVIII Bay 79–4980 program after clinical data showing lower efficacy than standard prophylaxis regimen with Kogenate46 and a bioengineered rFVIIa (BAY86–6150) with a fivefold increased half-life extension was ceased because of an increased immunogenicity.47 Similarly, Novo Nordisk stopped their clinical trial program of the glycoPEGylated rFVIIa (N7-GP) because of lack of efficacy in a phase II/III clinical trial48 and of a rFVIIa analogue with a higher activity (vatreptacog alfa) because of a high incidence of antidrug antibody development.49
Other Investigational Hemostatic Agents As previously described, TFPI is a major regulator of clotting initiation by direct inhibition of the TF-FVIIa complex, and should therefore be regarded as a promising target for both pro- and anticoagulation therapy. A high-affinity, specific TFPI antagonist designed to improve hemostasis was recently developed by Baxter, under the name of BAX499 (previously known as ARC19499).50 Early in vitro studies have shown that this novel compound is effective for improving the clotting times of samples from hemophilic patients in a concentration-dependent fashion, thus producing clotting profiles that were similar to those of healthy controls.51 However, the BAX499 clinical program appears to have been ceased after an increased number of bleeding episodes during a phase I/II clinical trial (ClinicalTrials.gov identifier: NCT01191372).52 OBI-1 (Baxter International Inc.) is a bioengineered, highly purified form of porcine rFVIII, which retains the structural and procoagulant properties of porcine plasma–derived FVIII, but it can be easily manufactured and also exhibits a lower risk of toxicity and infection.53 In a first phase I study, a single dose of OBI-1 was shown to have higher bioavailability than porcine FVIII in patients with hemophilia A without inhibitors, and was also well tolerated.54 Preliminary data of an ongoing phase ⅔ study are available (ClinicalTrials.gov identifier: NCT01178294). Briefly, 18 patients presenting with a serious bleed were treated with an initial dose of OBI-1 (200 U/kg), followed by additional doses based on personal profiles, including clinical evaluations and target factor VIII activity levels. All patients responded positively to treatment in the first 24 hours (14 effective and 4 partially effective) and no serious adverse events were recorded. Nonserious mild adverse events were described in two (11.1%) patients, who also developed antiporcine inhibitors to OBI-1.
Gene Therapy: Where Do We Stand, Now? Gene therapy is conventionally known as delivering therapeutic DNA (more or less as a drug) into patient cells to treat a certain disease. This approach is particularly appealing for hemophiliacs, because it would help patients synthesize the deficient factor, thus alleviating or reducing the need of infusions and achieving access to care on a broader scale, especially for those living in developing countries.55 Some gene therapy research trials have been performed in humans, with mixed results56 and this is also attributable to the Seminars in Thrombosis & Hemostasis
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fact that the various viral vector systems possess specific properties that characterize the degree of gene delivery to target tissues and their specific function in vivo. A recent study based on a single-dose infusion of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human FIX transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein of six patients with severe hemophilia B has shown promising results.57 AAV-mediated expression generated plasma FIX levels comprised between 2 and 11% in all participants. Four out of the six patients suspended prophylaxis and remained free of spontaneous hemorrhage, whereas the interval between prophylactic injections was significantly increased in the remaining two patients. A phase I/II study, based on delivering AAV8-hFIX19 vector (Spark Therapeutics, LLC Philadelphia, PA) to liver cells is currently recruiting (ClinicalTrials.gov identifier: NCT01620801), but the primary completion date is expected late in 2019. Another attractive strategy has recently been attempted, which is mainly based on a lentiviral vector encoding the integrin αIIb (ITGA2B) gene promoter, which enables megakaryocyte-specific expression of a hybrid FVIII molecule fused with the VWF propeptide-D2. A recent study showed that this approach may be effective to induce storage and release of FVIII from activated platelets in animals.58 Nevertheless, several hurdles still remain before gene therapy for hemophilia can make the transition to clinical practice. These mostly involve the elimination of contaminating empty vector capsids, the development of FVIII and FIX modified transgenes to improve the function of the transgene product, as well as the need for developing pharmacological therapies that reduce interference from naturally occurring antibodies against vectors.59 Because of the larger size of FVIII gene compared with the FIX gene, it can be predicted that the route toward the development of an effective gene therapy for hemophilia A will be much more challenging than that for hemophilia B.
Conclusion and Future Prospects The analysis of the literature data documents that several novel products for hemostasis are actually at an advanced stage of development and will reach (or have already reached) the US and European market in the near future. Although their clinical safety, efficacy, and potential superiority to traditional products need to be validated by post-marketing (real-world experiences), several other open issues remain. In particular, one can envisage additional complexity for future laboratory practice that will include the laboratory monitoring of a broad range of possible therapies, including the longacting hemostatic products. Indeed, the licensing of novel products will require product-specific assays, which for most will likely be chromogenic assays. One-stage assays, which are still widely used in hemostasis laboratories in the United States and Europe, may not adequately reflect the biological activity of the new proteins. To overcome this problem, compatibility of some products have been assessed several assays including current procedures,60,61 while for other
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products alternative strategies are being assessed, such as product-specific reference standards and conversion factors. Irrespective of the approaches finally recommended, it will be challenging for many laboratories including hemophilia treatment centers to introduce such new assays and procedures for the routine and ongoing clinical management of hemophilia patients.
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glycoPEGylated recombinant factor VIII in patients with hemophilia A. J Thromb Haemost 2013;11(4):670–678 Mei B, Pan C, Jiang H, et al. Rational design of a fully active, longacting PEGylated factor VIII for hemophilia A treatment. Blood 2010;116(2):270–279 Coyle TE, Reding MT, Lin JC, Michaels LA, Shah A, Powell J. Phase I study of BAY 94-9027, a PEGylated B-domain-deleted recombinant factor VIII with an extended half-life, in subjects with hemophilia A. J Thromb Haemost 2014;12(4):488–496 Turecek PL, Bossard MJ, Graninger M, et al. BAX 855, a PEGylated rFVIII product with prolonged half-life. Development, functional and structural characterisation. Hamostaseologie 2012;32 (Suppl 1):S29–S38 Dumont JA, Liu T, Low SC, et al. Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs. Blood 2012;119(13):3024–3030 Peters RT, Toby G, Lu Q, et al. Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein. J Thromb Haemost 2013;11(1):132–141 Powell JS, Josephson NC, Quon D, et al. Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients. Blood 2012;119(13):3031–3037 Mahlangu J, Powell JS, Ragni MV, et al; A-LONG Investigators. Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A. Blood 2014;123(3):317–325 Shapiro A, Chowdary P, Quon D, et al. Recombinant factor VIII Fc fusion protein as episodic treatment for bleeding: analysis from the A-LONG Study. [Abstract] Haemophilia 2014;20(Suppl 3):16 Kulkarni R, Ragni MV, Baker RI, et al. Analysis of clinical target joint bleeding on prophylactic regimens with recombinant factor VIII Fc fusion protein in patients with severe hemophilia A. [Abstract] Haemophilia 2014;20(Suppl 3):94 Østergaard H, Bjelke JR, Hansen L, et al. Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide. Blood 2011;118(8): 2333–2341 Negrier C, Knobe K, Tiede A, Giangrande P, Møss J. Enhanced pharmacokinetic properties of a glycoPEGylated recombinant factor IX: a first human dose trial in patients with hemophilia B. Blood 2011;118(10):2695–2701 Collins PW, Møss J, Knobe K, Groth A, Colberg T, Watson E. Population pharmacokinetic modeling for dose setting of nonacog beta pegol (N9-GP), a glycoPEGylated recombinant factor IX. J Thromb Haemost 2012;10(11):2305–2312 Collins PW, Colberg T, Young G, et al. Safety, efficacy and pharmacokinetics of nonacog beta pegol (N9-GP) for prophylaxis and treatment of bleeding episodes in patients with haemophilia B. J Thromb Haemost 2013;11(Suppl 2):19 Valentino LA. Recombinant FIXFc: a novel therapy for the royal disease? Expert Opin Biol Ther 2011;11(10):1361–1368 Peters RT, Low SC, Kamphaus GD, et al. Prolonged activity of factor IX as a monomeric Fc fusion protein. Blood 2010;115(10): 2057–2064 Shapiro AD, Ragni MV, Valentino LA, et al. Recombinant factor IXFc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients. Blood 2012;119(3):666–672 Powell JS, Pasi KJ, Ragni MV, et al; B-LONG Investigators. Phase 3 study of recombinant factor IX Fc fusion protein in hemophilia B. N Engl J Med 2013;369(24):2313–2323 Metzner HJ, Weimer T, Kronthaler U, Lang W, Schulte S. Genetic fusion to albumin improves the pharmacokinetic properties of factor IX. Thromb Haemost 2009;102(4):634–644 Santagostino E, Negrier C, Klamroth R, et al. Safety and pharmacokinetics of a novel recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in hemophilia B patients. Blood 2012;120(12):2405–2411
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safety, efficacy and PK study of a recombinant coagulation factor IX albumin fusion protein (rIX-FP) in subjects with hemophilia B. Thromb Res 2013;131(Suppl 2):S11–S14 Martinowitz U, Lubetsky A, Santagostino E, et al. Efficacy, PK and safety results of a Phase I/II clinical study of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in previously treated patients with hemophilia B. J Thromb Haemost 2013;11(Suppl 2):240–241 Salas J, Liu T, Kistanova E, et al. Enhanced pharmacokinetics of factor VIIA as a monomeric FC fusion. J Thromb Haemost 2011;9 (Suppl 2):268 Weimer T, Wormsbächer W, Kronthaler U, Lang W, Liebing U, Schulte S. Prolonged in-vivo half-life of factor VIIa by fusion to albumin. Thromb Haemost 2008;99(4):659–667 Golor G, Bensen-Kennedy D, Haffner S, et al. Safety and pharmacokinetics of a recombinant fusion protein linking coagulation factor VIIa with albumin in healthy volunteers. J Thromb Haemost 2013;11(11):1977–1985 Powell J, Martinowitz U, Windyga J, et al; LipLong Study Investigators. Efficacy and safety of prophylaxis with once-weekly BAY 79-4980 compared with thrice-weekly rFVIII-FS in haemophilia A patients. A randomised, active-controlled, double-blind study. Thromb Haemost 2012;108(5):913–922 Mahlangu JN, Coetzee MJ, Laffan M, et al. Phase I, randomized, double-blind, placebo-controlled, single-dose escalation study of the recombinant factor VIIa variant BAY 86-6150 in hemophilia. J Thromb Haemost 2012;10(5):773–780 Møss J, Rosholm A, Laurén A. Safety and pharmacokinetics of a glycoPEGylated recombinant activated factor VII derivative: a randomized first human dose trial in healthy subjects. J Thromb Haemost 2011;9(7):1368–1374 Mahlangu J, Karim FA, Gorska-Kosicka M, et al. Anti-drug antibody formation in hemophilia patients with inhibitors after receiving recombinant activated FVII analogue (vatreptacog alfa). Haemophilia 2013;19(Suppl 2):26 Parunov LA, Fadeeva OA, Balandina AN, et al. Improvement of spatial fibrin formation by the anti-TFPI aptamer BAX499:
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changing clot size by targeting extrinsic pathway initiation. J Thromb Haemost 2011;9(9):1825–1834 Gorczyca ME, Nair SC, Jilma B, et al. Inhibition of tissue factor pathway inhibitor by the aptamer BAX499 improves clotting of hemophilic blood and plasma. J Thromb Haemost 2012;10(8): 1581–1590 Dockal M, Pachlinger R, Hartmann R, et al. Biological Explanation of Clinically Observed Elevation of TFPI Plasma Levels After Treatment with TFPI-Antagonistic Aptamer BAX 499 Blood (ASH Annual Meeting Abstracts), November 2012;120:1104 Toschi V. OBI-1, porcine recombinant Factor VIII for the potential treatment of patients with congenital hemophilia A and alloantibodies against human Factor VIII. Curr Opin Mol Ther 2010;12(5): 617–625 Kempton CL, Abshire TC, Deveras RA, et al. Pharmacokinetics and safety of OBI-1, a recombinant B domain-deleted porcine factor VIII, in subjects with haemophilia A. Haemophilia 2012;18(5):798–804 Skinner MW. Gene therapy for hemophilia: addressing the coming challenges of affordability and accessibility. Mol Ther 2013;21(1): 1–2 Lippi G, Franchini M, Saenko EL. Gene therapy for hemophilia A. Friend or foe? Blood Coagul Fibrinolysis 2009;20(6):395–399 Nathwani AC, Tuddenham EG, Rangarajan S, et al. Adenovirusassociated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 2011;365(25):2357–2365 Du LM, Nurden P, Nurden AT, et al. Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A. Nat Commun 2013;4:2773 Monahan PE, Gui T. Gene therapy for hemophilia: advancing beyond the first clinical success. Curr Opin Hematol 2013;20(5): 410–416 Viuff D, Barrowcliffe T, Saugstrup T, Ezban M, Lillicrap D. International comparative field study of N8 evaluating factor VIII assay performance. Haemophilia 2011;17(4):695–702 Sommer JM, Moore N, McGuffie-Valentine B, et al. Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories. Haemophilia 2014;20(2):294–300
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Newer Hemostatic Agents Massimo Franchini, MD1
Emmanuel J. Favaloro, PhD FFSc (RCPA)2
1 Department of Hematology and Transfusion Medicine, Azienda
Ospedaliera Carlo Poma, Mantova, Italy 2 Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Pathology West, Westmead Hospital, Westmead, New South Wales, Australia 3 Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy
Giuseppe Lippi, MD3
Address for correspondence Massimo Franchini, MD, Department of Hematology and Transfusion Medicine, Azienda Ospedaliera Carlo Poma, Mantova, Italy (e-mail: [email protected]).
Abstract
Keywords
► clotting factor concentrates ► new hemostatic products ► extended half-life ► hemophilia ► inhibitors
The mainstay of treatment of inherited coagulation disorders is based on the infusion of the deficient clotting factor, when available. Significant advances have been made over the past two decades in the production and availability of factor replacement products. In spite of such progression, several issue are still unsolved, the most important being the need for frequent factor concentrate infusions and the development of inhibitory alloantibodies. To overcome these important limitations, several newer hemostatic agents with an extended half-life are at an advanced stage of clinical development. After a brief overview of hemostasis, this narrative review summarizes the current knowledge on the most promising novel products for hemostasis. The current status of gene therapy for hemophilia, the only therapeutic option to definitively cure this inherited bleeding disorder, is also concisely discussed.
The availability of highly purified plasma-derived and recombinant clotting factor concentrates over the last two decades has dramatically improved the quality of life and the life expectancy of severely affected hemophilia A and B patients, as it has greatly facilitated the widespread adoption of home replacement therapy with the broad implementation of prophylactic treatment regimens.1–6 Indeed, regular prophylaxis has proved to be an extraordinarily effective therapeutic defense able to remarkably reduce, compared with on-demand therapy, spontaneous and life-threatening bleeds and to protect hemophiliacs from the development and progression of arthropathy.7 However, several issues still remain incompletely resolved in the management of hemophilia. First, the short median half-life of factor VIII (FVIII) and factor IX (FIX) limits the ongoing efficacy of replacement therapy, requiring frequent infusions, especially when prophylactic regimens are adopted in pediatric and young adult hemophiliacs, making adherence to treatment difficult for these patients.5 Another major clinical challenge is the management of inhibitors, which develop in many patients and render hemostatic control extremely difficult and complicated, necessitating the use of bypassing agents (i.e., recombinant activated factor VII
published online April 20, 2015
Issue Theme Tissue Factor in Arterial and Venous Thrombosis: From Pathophysiology to Clinical Implications; Guest Editors: Vincenzo Toschi, MD, and Massimo Franchini, MD.
[rFVIIa] and activated prothrombin complex concentrates [APCC]) which remain, however, less effective than standard replacement therapy.8–11 Thus, major research in this field has focused on development of coagulation factor concentrates that are less immunogenic and with a prolonged half-life, aiming by less frequent infusions, at decreasing bleeding rate and reducing the burden of treatment. In this narrative review, we summarize the new technologies and the current development status of the novel products for hemostasis, focusing in particular on those with half-life extension for treatment of hemophilia. A brief overview of the current status of gene therapy for hemophilia is also presented.
Overview of Hemostasis Hemostasis represents the mechanism whereby the body maintains circulatory blood flow. It can be represented by Virchow triad (►Fig. 1A) or, more commonly, as a balance of procoagulant and anticoagulant mechanisms or pathways, inclusive of fibrinolysis.12,13 In brief, damage to the
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0034-1544004. ISSN 0094-6176.
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Fig. 1 (A) Virchow triad. (B) Secondary hemostasis. (C) The fibrinolytic system.
vasculature will initiate two major integrated “procoagulant” pathways, termed primary and secondary hemostasis, which will lead to eventual plug formation to prevent excessive hemorrhage. The primary pathway involves activation of platelets, the recruitment of von Willebrand factor (VWF) to promote platelet attachment to the site of injury, and the engagement of secondary hemostasis (►Fig. 1B) through various procoagulant proteins including fibrinogen, and factors (F) V, VIII, and IX. Secondary hemostasis is also initiated directly by damage to the vasculature, primarily via the tissue factor (TF) pathway involving FVII, but also via
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the contact pathway, and as amplified by the primary pathway (►Fig. 1B). The process of secondary hemostasis essentially represents the progression of blood coagulation, involving a series of coordinated and calcium-dependent conversions of proenzymes to the respective serine proteases, culminating in the conversion (activation) of prothrombin (FII) to thrombin (FIIa). Moreover, the interaction of these interconnected pathways with cellular components is critical for hemostasis in vivo.14 The so-called extrinsic (TF) pathway is initiated at the site of injury in response to the release of TF (factor III) (►Fig. 1B). TF is a cofactor in the activated FVII (FVIIa)catalyzed activation of FX, which cleaves FX to FXa. According to the amplification model, each FVIIa-TF complex can produce many FXa molecules, which subsequently produce an even greater amount of thrombin. The so-called intrinsic (contact) pathway (►Fig. 1B), involving the clotting factors VIII, IX, X, XI, and XII, is less significant for in vivo hemostasis under normal physiologic conditions. Nevertheless, the major hemophilias are represented by deficiencies of FVIII (hemophilia A), FIX (hemophilia B), and, to a lesser extent, FXI (hemophilia C). Initiation of the intrinsic pathway theoretically occurs when prekallikrein, high-molecular-weight kininogen, FXI, and FXII are exposed to a negatively charged surface (“the contact phase”). The assemblage of contact phase components results in conversion of prekallikrein to kallikrein, which in turn activates FXII to FXIIa. FXIIa then activates FXI to FXIa, which in turn activates FIX to FIXa. The subsequent activation of FXa requires assemblage of the tenase complex (Ca2þ and factors VIIIa, IXa, and X) on the surface of activated platelets. In this process, FVIIIa acts as a cofactor for factors IXa and X. The final protease generated is thrombin (FIIa), which converts fibrinogen into an insoluble fibrin gel, which is stabilized by covalent cross-linking catalyzed by FXIIIa. Several lines of evidence now attest that the physiological role of FXII in activating the intrinsic pathways is probably meaningless in vivo, because FXII deficiency (even at homozygous state) does not significantly impair the hemostatic response,15 so that the effective function of this factor in the initiation phase of blood coagulation remains largely controversial. A more genuine in vivo role of the intrinsic pathway in blood coagulation seems hence attributable to the amplification of the coagulation cascade, which results from the activation of factors V, VIII, and possibly XI by thrombin, as well as by direct activation of FVII. It is important to recognize that that the dichotomy between intrinsic and extrinsic pathways as assessed by in vitro coagulation assays does not accurately reflect the hemostasis activation in vivo. In an in vitro cell-based model, adding increasing amounts of FVIIa to activated platelets produces increase in generation of FXa independently of the presence of TF on the platelet surface.16 Therefore, FVIIa at pharmacologic doses, being much higher than those generated during normal hemostasis (i.e., 10–100 times higher), provides an effective bypassing agent, which can directly activate FX and lead to an additional burst of thrombin in a broad spectrum of congenital and acquired bleeding conditions, including Seminars in Thrombosis & Hemostasis
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hemophilia A and B, as well as in the presence of factor inhibitors (►Fig. 1). Secondary blood coagulation is under the influence of several negative regulators at different steps, including tissue factor pathway inhibitor (TFPI), antithrombin (AT), and the components of the protein C system (protein C, protein S, and thrombomodulin). The protein C system and AT are both effective in regulating the propagation phase of thrombin, whereas TFPI is primarily effective in regulating the initiation phase of the reaction, by inhibiting the FVIIa-TF complex.
Hemostatic Agents with Extended Half-Life The half-life of human FVIII is 10 to 12 hours, for FIX is 18 to 34 hours, and the half-life of FVIIa is approximately 2.5 hours, being the shortest among all the coagulation factors. Several technologies are being applied to prolong the half-life of FVIIa, FVIII, and FIX, including PEGylation, glycoPEGylation, Fc-fusion, and recombinant albumin fusion17,18 (►Table 1).
New FVIII Products A B-domain truncated modified O-glycoPEGylated recombinant FVIII (rFVIII) is in development (N8-GP, Novo Nordisk, Bagsvaerd, Denmark), and is scheduled for commercialization in 2015. N8-GP has a 40 kDa PEG attached to a B-domain truncated molecule of rFVIII (turoctocog alfa). Upon activation by thrombin, the B-domain containing PEG is released, leaving active rFVIII.19 Preclinical studies evaluating the pharmacokinetic (PK) and pharmacodynamic (PD) properties of N8-GP in dogs with congenital hemophilia showed a halflife of 22 hours and extended PD activity.20 Similarly, a clinical dose-escalation study in 26 previously treated patients with hemophilia A showed a linear-dose PK profile, a mean halflife of 19 hours, and a low frequency of adverse events without inhibitors.21 A phase III trial in adults is underway
(ClinicalTrials.gov identifier: NCT01480180), as is a pediatric study (ClinicalTrials.gov identifier: NCT01731600), while a trial evaluating efficacy during surgery is currently recruiting (ClinicalTrials.gov identifier: NCT01489111). Another B-domain–deleted PEGylated rFVIII in development is BAY 94–9027 (Bayer Healthcare AG, Leverkusen, Germany), which contains a single, large-branched PEG molecule conjugated to a specific amino acid on rFVIII.22 In a phase I clinical study in previously treated patients with hemophilia A, BAY 94–9027 demonstrated equivalent recovery and improved PK profile versus rFVIII formulated with sucrose (rFVIII-FS) (19 vs. 13 hours), with no immunogenicity.23 A phase III trial (ClinicalTrials.gov Identifier: NCT01775618) is ongoing, whereas another (ClinicalTrials.gov Identifier: NCT01580293) is recruiting. Baxter (Baxter Innovations GmbH, Vienna, Austria) is developing a PEGylated version of its full-length rFVIII ADVATE (BAX 855) with extended half-life. This technique provides two moles (20 kDa) of PEG per rFVIII molecule in a non–site-specific manner, which improves the PK profile without compromising specific activity.24 Preclinical studies in various animal models have demonstrated a twofold prolongation of terminal half-life.24 A phase II/III clinical trial of BAX 855 (ClinicalTrials.gov Identifier: NCT01736475) in previously treated patients with severe hemophilia A is ongoing, with a further trial currently recruiting patients undergoing surgical procedures (ClinicalTrials.gov Identifier: NCT01913405). BAX 855 is expected to reach the market toward the end of 2016. Biogen Idec (Weston, MA) is developing a rFVIII fusion protein (rFVIII-Fc), which is a recombinant fusion of a B-domain deleted FVIII molecule and the dimeric constant region (Fc) of IgG1.25,26 The first human single-dosing study in 16 previously treated patients showed that the elimination of rFVIIIFc was prolonged by 1.5- to 1.7-fold compared with a
Table 1 Products with extended half-life for hemophilia treatment in clinical development Product
Company
Technology
Terminal half-life
Stage of development
Novo Nordisk
Site-specific glycoPEGylation
22 h
Phase III ongoing
FVIII N8-GP BAY 94–9027
Bayer Healthcare
Site-specific glycoPEGylation
19 h
Phase III ongoing
BAX855
Baxter Innovations
Non–site-specific glycoPEGylation
2-fold (preclinical studies)
Phase III ongoing
rFVIII-Fc (ELOCTATE)
Biogen Idec
Fusion to IgG1-Fc
19 h
Marketed in the USA
N9-GP
Novo Nordisk
Site-specific glycoPEGylation
110 h
Phase III ongoing
rIX-Fc (ALPROLIX)
Biogen Idec
Fusion to IgG1-Fc
82 h
Marketed in the USA
rFIX-FP
CSL Behring
Fusion protein with albumin
92 h
Phase III ongoing
CSL Behring
Fusion protein with albumin
8.5 h
Phase II/III studies are expected to commence in late 2014
FIX
FVIIa rFVIIa-FP
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native rFVIII.27 Similarly, the 52-week, open-label, phase III (A-LONG) study conducted in 165 previously treated patients with hemophilia A showed that the terminal half-life of rFVIII-Fc was 19 hours versus 12.4 for rFVIII.28 rFVIII-Fc resulted in an annualized bleeding rate (ABR) of 1.6 in those receiving prophylaxis with rFVIII-Fc every 3 to 5 days (median weekly dose 77.7 IU/kg), 3.6 with weekly prophylaxis (fixed weekly dose of 65 IU/kg), and 33.6 for episodic treatment (10–50 IU/kg).28 In this study, 87.3% of all bleeding episodes were resolved with one and 97.8% with two or fewer injections, and 45.3% of patients experienced no bleeding episodes during the study.28 Recent updates of these studies confirmed the efficacy and safety of rFVIII-Fc in both prophylaxis and on-demand regimens.29,30 A trial in pediatric patients is ongoing (ClinicalTrials.gov Identifier: NCT01458106). rFVIIIFc was launched in the United States in September 2014 under the brand name of ELOCTATE (Biogen Idec, Weston, MA).
New FIX Products In a preclinical study, the glycoPEGylated rFIX nonacog β pegol (N9-GP, Novo Nordisk) exhibited a greater than twofold increase in in vivo recovery and a markedly prolonged half-life in minipigs and hemophilia B dogs compared with rFIX.31 The first human dose trial (ClinicalTrials.gov identifier: NCT00956345) assessed three doses of N9-GP in 16 patients with hemophilia B and demonstrated an extended half-life (mean 93 h), which was five times longer than in hemophilia B patients previously treated with conventional FIX products.32 Population PK modeling based on these results has suggested that weekly prophylaxis may be possible with N9-GP.33 Results of a phase III clinical trial were presented recently, assessing prophylaxis and on-demand treatment in 74 patients with hemophilia B, with no patients developing inhibitors. The mean half-life was 110 hours and median ABR with prophylactic therapy of 40 U/kg and 10 U/kg per week were 1.0 and 2.9 episodes per year, respectively, and 15.9 for on-demand treatment, with 99% of bleeds resolved following a single injection.34 An extension trial (ClinicalTrials. gov identifier: NCT01395810) and a pediatric trial (ClinicalTrials.gov identifier: NCT01467427) are ongoing, and a trial in surgical patients has recently finished (ClinicalTrials.gov identifier: NCT01386528). The fusion of the Fc-portion of immunoglobulin G to a single molecule of rFIX (rFIX-Fc) (Biogen Idec) has also been explored to increase its circulation time.35 In animal models of hemophilia B, rFIX-Fc exhibited an extended half-life of up to 48 hours, as compared with the standard rFIX half-life of approximately 18 hours.36 A phase I/II clinical study (ClinicalTrials.gov identifier: NCT00716716) reported that rFIX-Fc was welltolerated, with a threefold longer half-life than native rFIX.37 An open-label multicenter phase III study (B-LONG Study; ClinicalTrials.gov identifier: NCT01027364), enrolling 123 patients and aiming at evaluating safety, PK, and efficacy of rFIX-Fc in previously treated patients with severe and moderately severe hemophilia B, was recently completed.38 The mean half-life of rFIX-Fc was 82.1 hours compared with 33.8 hours for rFIX, with a mean ABR in the weekly prophylaxis arm
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(starting dose 50 IU/kg) of 2.95 versus 1.38 in the individualized prophylaxis arm (starting with doses of 100 IU/kg every 10 days) and 17.69 in the on-demand arm. The overall median dosing individualized-interval prophylaxis was 12.5 days, whereas it was 13.8 days during the last 6 months of the study. No inhibitors were detected throughout the study period, where 93.5% of bleeding episodes resolved with a single rFIX-Fc infusion.38 An extension trial is enrolling (ClinicalTrials.gov identifier: NCT01425723), and a pediatric trial is currently recruiting participants (ClinicalTrials.gov identifier: NCT01440946). rFIX-Fc was approved in March 2014 by the Food and Drug Administration (FDA) and is now marketed in the United States under the brand name of ALPROLIX (Biogen Idec, Weston, MA). Fusion of rFIX to recombinant albumin (rFIX-FP, CSL Behring LLC, King of Prussia, PA) has achieved a fivefold lengthening in half-life compared with licensed rFIX in preclinical studies.39 A phase I dose-escalation study conducted in 25 patients with severe hemophilia B demonstrated a fivefold increase in half-life, a 44% increase in recovery, a sevenfold reduction in clearance compared with rFIX, and a linear dose response with no allergic reactions and no development of inhibitors.40 Preliminary results from the open-label PROLONG9-FP phase I/II trial, which included both an on-demand and a weekly prophylaxis treatment arms, indicated an excellent safety profile based on more than 600 exposure days, with no serious adverse events.41 After 11 months of treatment, the 13 patients on prophylaxis had a mean ABR of 1.26, and those switching from pretrial on-demand therapy to prophylaxis (n ¼ 3) had a greater than 80% reduction in bleeding episodes.42 Furthermore, the efficacy of rFIX-FP was confirmed as all bleeding events were treated successfully with two or fewer injections and approximately 90% successfully treated with a single injection.42 A phase II/III trial (ClinicalTrials.gov identifier: NCT01496274) is ongoing, whereas two other trials (an extension trial [ClinicalTrials.gov identifier: NCT02053792] and a pediatric trial [ClinicalTrials.gov identifier: NCT01662531]) are currently recruiting participants.
New FVII Products Though a long-acting rFVIIa-Fc (Biogen Idec) is still at a preclinical stage,43 rFVII-FP (CSL Behring), where rFVIIa and recombinant albumin are fused, is at a more advanced phase of development. PK studies in rodents indicated that the half-life of rFVIIa-FP was 5.8-fold longer and recovery was improved by 1.4-fold compared with rFVIIa.44 Results from the phase I double-blind study were reported recently, showing that rFVIIa-FP was well tolerated after single-dose administration to 40 healthy individuals, with no incidences of inhibitor or antidrug antibody development.45 In humans, the half-life was increased to 8.5 hours with a dose of 1000 µg/kg, and the clearance was reduced by three- to fourfold.45 A phase II/III trial is expected to commence in late 2014. Finally, it is important to highlight that for each new hemostatic product that successfully reaches the market, several products under development are stopped after clinical trial “failures.” For instance, Bayer stopped PEGylated Seminars in Thrombosis & Hemostasis
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liposome associated FVIII Bay 79–4980 program after clinical data showing lower efficacy than standard prophylaxis regimen with Kogenate46 and a bioengineered rFVIIa (BAY86–6150) with a fivefold increased half-life extension was ceased because of an increased immunogenicity.47 Similarly, Novo Nordisk stopped their clinical trial program of the glycoPEGylated rFVIIa (N7-GP) because of lack of efficacy in a phase II/III clinical trial48 and of a rFVIIa analogue with a higher activity (vatreptacog alfa) because of a high incidence of antidrug antibody development.49
Other Investigational Hemostatic Agents As previously described, TFPI is a major regulator of clotting initiation by direct inhibition of the TF-FVIIa complex, and should therefore be regarded as a promising target for both pro- and anticoagulation therapy. A high-affinity, specific TFPI antagonist designed to improve hemostasis was recently developed by Baxter, under the name of BAX499 (previously known as ARC19499).50 Early in vitro studies have shown that this novel compound is effective for improving the clotting times of samples from hemophilic patients in a concentration-dependent fashion, thus producing clotting profiles that were similar to those of healthy controls.51 However, the BAX499 clinical program appears to have been ceased after an increased number of bleeding episodes during a phase I/II clinical trial (ClinicalTrials.gov identifier: NCT01191372).52 OBI-1 (Baxter International Inc.) is a bioengineered, highly purified form of porcine rFVIII, which retains the structural and procoagulant properties of porcine plasma–derived FVIII, but it can be easily manufactured and also exhibits a lower risk of toxicity and infection.53 In a first phase I study, a single dose of OBI-1 was shown to have higher bioavailability than porcine FVIII in patients with hemophilia A without inhibitors, and was also well tolerated.54 Preliminary data of an ongoing phase ⅔ study are available (ClinicalTrials.gov identifier: NCT01178294). Briefly, 18 patients presenting with a serious bleed were treated with an initial dose of OBI-1 (200 U/kg), followed by additional doses based on personal profiles, including clinical evaluations and target factor VIII activity levels. All patients responded positively to treatment in the first 24 hours (14 effective and 4 partially effective) and no serious adverse events were recorded. Nonserious mild adverse events were described in two (11.1%) patients, who also developed antiporcine inhibitors to OBI-1.
Gene Therapy: Where Do We Stand, Now? Gene therapy is conventionally known as delivering therapeutic DNA (more or less as a drug) into patient cells to treat a certain disease. This approach is particularly appealing for hemophiliacs, because it would help patients synthesize the deficient factor, thus alleviating or reducing the need of infusions and achieving access to care on a broader scale, especially for those living in developing countries.55 Some gene therapy research trials have been performed in humans, with mixed results56 and this is also attributable to the Seminars in Thrombosis & Hemostasis
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fact that the various viral vector systems possess specific properties that characterize the degree of gene delivery to target tissues and their specific function in vivo. A recent study based on a single-dose infusion of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human FIX transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein of six patients with severe hemophilia B has shown promising results.57 AAV-mediated expression generated plasma FIX levels comprised between 2 and 11% in all participants. Four out of the six patients suspended prophylaxis and remained free of spontaneous hemorrhage, whereas the interval between prophylactic injections was significantly increased in the remaining two patients. A phase I/II study, based on delivering AAV8-hFIX19 vector (Spark Therapeutics, LLC Philadelphia, PA) to liver cells is currently recruiting (ClinicalTrials.gov identifier: NCT01620801), but the primary completion date is expected late in 2019. Another attractive strategy has recently been attempted, which is mainly based on a lentiviral vector encoding the integrin αIIb (ITGA2B) gene promoter, which enables megakaryocyte-specific expression of a hybrid FVIII molecule fused with the VWF propeptide-D2. A recent study showed that this approach may be effective to induce storage and release of FVIII from activated platelets in animals.58 Nevertheless, several hurdles still remain before gene therapy for hemophilia can make the transition to clinical practice. These mostly involve the elimination of contaminating empty vector capsids, the development of FVIII and FIX modified transgenes to improve the function of the transgene product, as well as the need for developing pharmacological therapies that reduce interference from naturally occurring antibodies against vectors.59 Because of the larger size of FVIII gene compared with the FIX gene, it can be predicted that the route toward the development of an effective gene therapy for hemophilia A will be much more challenging than that for hemophilia B.
Conclusion and Future Prospects The analysis of the literature data documents that several novel products for hemostasis are actually at an advanced stage of development and will reach (or have already reached) the US and European market in the near future. Although their clinical safety, efficacy, and potential superiority to traditional products need to be validated by post-marketing (real-world experiences), several other open issues remain. In particular, one can envisage additional complexity for future laboratory practice that will include the laboratory monitoring of a broad range of possible therapies, including the longacting hemostatic products. Indeed, the licensing of novel products will require product-specific assays, which for most will likely be chromogenic assays. One-stage assays, which are still widely used in hemostasis laboratories in the United States and Europe, may not adequately reflect the biological activity of the new proteins. To overcome this problem, compatibility of some products have been assessed several assays including current procedures,60,61 while for other
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products alternative strategies are being assessed, such as product-specific reference standards and conversion factors. Irrespective of the approaches finally recommended, it will be challenging for many laboratories including hemophilia treatment centers to introduce such new assays and procedures for the routine and ongoing clinical management of hemophilia patients.
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