Antithrombin lowering in hemophilia: a closer look at fitusiran

Thrombin is a key enzyme in the maintenance of normal hemostatic function and is the central product of an interconnected set of simultaneously occurring cellular and proteolytic events. Antithrombin (AT) is a natural anticoagulant that downregulates different components of the clotting process, particularly thrombin generation. In good health, well-regulated hemostasis is the result of a balance between procoagulant and anticoagulant elements. Cumulative understanding of the regulation of thrombin generation and its central role in hemostasis and bleeding disorders has led to the clinical development of therapeutic strategies that aim to rebalance hemostasis in individuals with hemophilia and other coagulation factor deficiencies to improve bleeding phenotype. The aim of this review is to discuss the rationale for AT lowering in individuals with hemophilia, with a focus on fitusiran, its mechanism of action, and its potential as a prophylactic therapy for individuals with hemophilia A or B, with or without inhibitors. Fitusiran is an investigational small, interfering RNA therapeutic that targets and lowers AT. It is currently in phase III clinical trials and results have shown its potential to increase thrombin generation, leading to enhanced hemostasis and improved quality of life while reducing the overall treatment burden.


| I N T R O D U C T I O N
The hemostatic system is designed to respond to vascular injury, aiming to reduce blood loss and maintain hemostasis and the integrity of blood circulation, thereby preventing life-threatening hemorrhage [1][2][3]. In 2001, Hoffman and Monroe proposed the thrombin-centric, cell-based model of coagulation in which coagulation takes place on different cell surfaces in 3 phases (Figure 1). This cell-based model describes how coagulation is prevented from spreading throughout the vascular system and is confined to the site of injury. Additionally, the cell-based model allows a more detailed understanding of how hemostasis occurs in vivo compared with the canonical coagulation cascade model and can be used to help explain the pathophysiological mechanisms of certain coagulation disorders including hemophilia [4].
Despite differences in the detailed description of how coagulation functions, all models have in common that they culminate in the formation of thrombin as the key enzyme necessary for the formation of a fibrin network and stable clot [5][6][7].
The regulation of thrombin formation is the result of a balance between procoagulant and anticoagulant proteins, and the absence of key proteins in this process may result in thrombotic or hemorrhagic complications [2]. For example, in individuals with hemophilia A or B, deficiency or dysfunction of factor (F)VIII (FVIII) or factor FIX, respectively, results in insufficient thrombin generation resulting in impaired hemostasis and uncontrolled or excessive bleeding, whereas antithrombin (AT) deficiency alone otherwise leads to thrombosis ( Figure 2) [2].
To restore hemostasis in individuals with hemophilia the standard of care is prophylaxis, which aims to achieve no spontaneous bleeding [8]. Available treatment strategies rely on either replacing or mimicking the missing factors [9]. The major complication of clotting factor concentrate (CFC) therapy is the development of inhibitors, or neutralizing antibodies to replacement factor, with 30% of individuals with severe hemophilia A [10] and 10% of those with severe hemophilia B developing inhibitors [11].

Essentials
• Hemostasis is achieved when sufficient thrombin is generated to form a stable fibrin clot.
• Fitusiran is a nonfactor therapy that lowers antithrombin (AT) to enhance thrombin generation.
• Fitusiran dosing now targets AT levels of 15% to 35% aiming to enhance its benefit-risk profile.
• Risk of thrombosis is greater with AT levels <10%, even in individuals with hemophilia. In individuals with hemophilia and high-titer inhibitors, bypassing agents (BPAs) such as activated prothrombin complex concentrate and activated recombinant FVII (rFVIIa) can be used to treat acute bleeding events and as prophylaxis [12]. However, the use of BPAs has several limitations, including a short half-life, which means that they have limited efficacy as prophylactic agents used for the prevention of bleeding. They also require frequent infusions, increase the risk of thrombosis, and come at a high cost, which restricts access [13][14][15][16][17].
Nonfactor therapies are currently being used or investigated for prophylaxis in individuals with hemophilia and currently fall under 2 categories: therapies that mimic FVIIIa (substitution agents, ie, humanized anti-FIXa/FX bispecific antibodies such as emicizumab or investigational Mim8) and therapies that interfere with anticoagulant pathways (rebalancing agents; ie, AT-lowering, small interfering RNA therapy, such as fitusiran, anti-tissue factor pathway inhibitor antibodies such as concizumab or marstacimab, or therapies that target APC-dependent pathways) [18][19][20]. All of these therapies can be administered subcutaneously, most have long half-lives and necessitate, in general, infrequent administration, which may reduce treatment burden and increase the ability to deliver prophylaxis [19,21].
While these treatments are promising for prophylaxis, other hemostatic agents (CFC and BPAs) continue to be required for breakthrough bleeding or surgery [21].
The purpose of this review is to raise awareness of the rationale for the AT-lowering approach as a treatment strategy for people with hemophilia focusing on fitusiran, its mechanism of action, and its potential use as prophylactic treatment for individuals with hemophilia A or B, with or without inhibitors.

| Hemophilia and AT deficiency
Hemophilia bleeding phenotype is defined by the clinical severity of bleeding as a balance of all hemostatic parameters, as well as the levels of the deficient or dysfunctional factors [22]. The severity of bleeding phenotype for individuals with hemophilia appears to correlate with thrombin generation, which is measured by global hemostatic assays [23][24][25][26]. Part of this variation in thrombin generation and clinical phenotype can be explained by the variation in levels of anticoagulant proteins among individuals [27].
AT is the key anticoagulant enzyme of the hemostatic system ( Figure 2). Heparin-activated AT inhibits coagulation by neutralizing thrombin and FXa and to a lesser extent FIXa, FXIa, FXIIa, and other procoagulants [2,14,15]. Given its capacity to neutralize multiple targets, AT not only interferes with the generation of thrombin but also efficiently inhibits thrombin once generated [2]. Evidence suggests there is a modulation of bleeding tendency by factors in the anticoagulant and fibrinolytic systems [16].
-3 of 13 AT deficiency was first described in 1965 [17]. and is associated with an increased risk for venous thrombosis as a result of reduced regulation of procoagulant proteins [28]. The coinheritance of prothrombotic traits in those with hemophilia, such as AT deficiency, has been found to be associated with a milder bleeding phenotype and an increased event-free bleeding survival rate [16,29]. Thus, reduced AT levels are hypothesized to improve thrombin generation and promote hemostasis, resulting in a potentially milder bleeding phenotype in individuals with hemophilia [16,29]. Antithrombin was therefore deemed an attractive, thrombin-target therapeutic strategy to explore for enhancing hemostasis in people with hemophilia [30].

| Unique mechanism of action of fitusiran
In 1998, Fire and Mello published their landmark article, providing the first demonstration that RNA interference (RNAi) is triggered by double-stranded RNA and could repress the expression of a single gene [31]. These findings paved the way for the development of treatments, such as fitusiran, which is a subcutaneously administered, small, interfering RNA therapeutic agent that harnesses natural cellular RNAi mechanisms to cleave and degrade AT mRNA and reduce AT levels [32].
Small interfering RNA therapeutics are the most commonly used RNAi tools that harness the natural RNAi process. They consist of a synthetic RNA duplex designed to specifically target a particular mRNA for degradation. This prevents translation of the specific target mRNA, thus inhibiting protein synthesis [33].
AT, a natural anticoagulant with normal levels in the range of 80 to 120 IU/dL, is synthesized in the liver. Fitusiran is targeted to the liver by conjugation to N-acetylgalactosamine, a ligand for the asialoglycoprotein receptor located on hepatocytes [32][33][34][35]. Fitusiran utilizes enhanced stabilization chemistry-N-acetylgalactosamine conjugate technology, which enables subcutaneous dosing, with increased potency and durability [34,36,37]. Fitusiran is not thought to be suitable for pediatric populations aged <1 year, as AT levels increase with age and do not reach adult values until about 6 months of age [38].
Pharmacokinetic analysis in clinical studies has shown that fitusiran has a short half-life in plasma (3-5 hours); however, lower AT levels persist for several months after discontinuation of fitusiran at all dose regimens tested, with a mean rate of AT recovery of 10% to 15% per month accompanied by a decrease in thrombin generation and an increase in bleeding events [32,39]. The median percent AT was found to increase to >60% after a 5-month period compared with the last measurement before dosing interruption [40].
The target pharmacodynamic effect of AT lowering has been shown to occur between 15 and 28 days after the administration of the first dose of fitusiran. After this onset period, it has been found that while there is interindividual variation in AT levels, there is minimal intraindividual variation in AT lowering, which potentially allows for more constant hemostatic protection among variable doses [32,39].
It has been proposed that reversal agents for nonfactor therapies may be useful, as the risk of thrombosis may potentially be increased when combining nonfactor therapies with other hemostatic agents to manage breakthrough bleeds. Currently, fitusiran is the only nonfactor therapy that has a specific reversal agent available in the form of recombinant or plasma-derived AT concentrates [41]. Evidence also demonstrates that decreased AT levels in plasma do not affect standard coagulation laboratory assays. This is important as it means that hemostasis and factor levels can easily be monitored in patients receiving fitusiran who may require CFC or BPAs to treat breakthrough bleeds and during some surgeries [42].

| Preclinical proof of concept for fitusiran
Preclinical studies in FVIII-deficient mice with heterozygous AT deficiency showed that moderately reduced AT levels increased thrombin generation and decreased bleeding after tail clipping, suggesting that bleeding phenotypes can be modulated by the balance between procoagulant and anticoagulant proteins [43]. In addition to this, a second study demonstrated that when administered subcutaneously, fitusiran showed potent, dose-dependent, and durable reductions in AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates with anti-FVIII inhibitors, resulting in improved thrombin generation [44]. Seghal et al. [44]    To note, as of December 2020, protocol amendments were made affecting all ongoing studies in the fitusiran clinical development program to mitigate the risk of thrombosis with fitusiran. These changes are discussed in detail in section 2.6. The efficacy data reported here relates to data published prior to the protocol amendments, which affected the dose and regimen of fitusiran.

| Reported efficacy and potential use of fitusiran prophylaxis in people with hemophilia
Fitusiran aims to rebalance hemostasis through AT lowering in individuals with hemophilia, irrespective of inhibitor status, leading to a sustained increase in thrombin generation and improved stable clot formation ( Figure 2).
In part C of the phase I fitusiran clinical trial, once-monthly s.c. administration of fitusiran demonstrated dose-dependent mean maximum lowering in AT levels by 70% to 89% from baseline and increased thrombin generation in participants with hemophilia A or B without inhibitors ( Figure 4A) [32]. A post hoc exploratory analysis determined that monthly fitusiran dosing resulted in fewer bleeding episodes per month following treatment with fitusiran than before treatment [32]. Consistent with these results, in part D of the phase I trial, participants with hemophilia A or B with inhibitors, who received once-monthly administration of subcutaneous doses of fitusiran at 50 mg and 80 mg and were followed up for 112 days, experienced AT reductions from baseline of 82.0% and 87.4%, respectively, at nadir ( Figure 4B). The reduction in AT activity was associated with increased thrombin generation [39]. Additionally, 64.7% of participants had no bleeds during the observation period (4 weeks after the first dose to 8 weeks after the last dose; mean,

days) with mean changes from baseline in the Haemophilia
Quality of Life Questionnaire for Adults total and physical health domain scores, suggesting that there was a clinically meaningful improvement in quality of life compared with published thresholds; reductions of 10-and 7-units in the "Physical health" and "Total score" domains, respectively [39,54].
As of March 10, 2020, an interim analysis of the phase II openlabel extension study showed that once-monthly fitusiran dosing achieved sustained AT lowering of 80%, resulting in peak thrombin levels approaching the lower range observed in healthy volunteers [55]. Exploratory post hoc analysis of bleeding events (follow-up of up to 4.7 years; median, 2.6 years) revealed a lower rate of bleeding episodes than before study enrollment. The median annualized bleeding rate (ABR) and the median annualized spontaneous bleeding rate were 0.84 and 0.34, respectively, for all participants (with or without inhibitors) during the observation period (the period following the 28-day onset period for fitusiran during which AT levels are expected to be within the target range) [55].  For situations requiring higher doses, more frequent administration, or multiple repeated doses, discussion with study medical monitor and clinical advicer is recommended, and AT replacement should be considered.
Antifibrinolytics should not be used in combination with factor or BPA while on fitusiran.
APCC, activated prothrombin complex concentrate; AT, antithrombin; BPA, bypassing agent; IU, international unit; rFVIIa, recombinant activated factor VII. 50.6% of participants in the fitusiran arm with zero treated bleeding events [57]. A statistically significant improvement in physical health domain score with fitusiran vs. the on-demand arm was also observed in both studies indicating a meaningful improvement in health-related quality of life [56,57]. In addition, recent analysis from the ATLAS-PPX study has revealed that once-monthly administration of 80 mg fitu-

| Safety of fitusiran
In the phase I trial, 76% of the 25 participants with hemophilia who received fitusiran reported an adverse event (AE), with most of the events being mild to moderate in severity [32]. In part D of the phase I trial, no participants experienced serious drug-related AEs [39,59].
The most common drug-related AEs in the phase I study were injection-site pain and injection-site erythema. No AEs led to treatment discontinuation [32,39].
In phase I/II studies, a monthly fixed dose of fitusiran administered subcutaneously was evaluated in 25 participants with hemophilia A or B, with or without inhibitors. Following the release of phase II interim data, the study was placed on clinical hold on September 1, 2017, due to a fatal event of cerebral venous sinus thrombosis initially misdiagnosed as subarachnoid hemorrhage and accordingly treated with recommended doses of CFC per protocol [30,40]. Thrombotic risk mitigation strategies were implemented in November 2017, including revised breakthrough bleeding guidance informed by in silico modeling ( Table 2), education of investigators and participants, and evaluation of symptoms suggestive of thrombosis [60]. Use of activated prothrombin complex concentrate and rFVIIa continued to be permitted for bleed treatment in participants receiving fitusiran but with a reduction in dose and frequency of infusions of hemostatic agents and avoidance of concomitant use of antifibrinolytics [30,60]. Following this, the study was restarted in The revised dose and regimen aimed at mitigating the thrombosis risk by modification of the fitusiran dose, dosing regimen, and target AT levels ( Figure 5) [62]. The amended target AT levels were 15% to 35% rather than the previous goal of <10%. These changes are based on  For all adult and adolescent patients exposed to at least 1 dose of fitusiran, the total patient-years for each of the 3 AT categories was calculated: <10%, 10% to 20%, and >20%. The patients with vascular thrombotic events were then included in the AT category representative of their level for the greatest amount of time during fitusiran exposure and an incident rate per 100 patient-years was derived.
c Adverse event data as of October 20, 2020. YOUNG ET AL.

| C O N C L U S I O N S
Fitusiran is an investigational small interfering RNA therapeutic for subcutaneous prophylaxis in individuals with hemophilia A or B, irrespective of their inhibitor status, that has the potential to be transformative in hemophilia management through rebalancing of thrombin generation resulting in a milder bleeding phenotype, impacting the quality of life and reducing overall treatment burden.
Ongoing clinical studies will provide further evidence on the efficacy and safety of fitusiran and its impact on patient-reported outcomes. It has been shown in preclinical in vitro and in silico studies that thrombin generation improves when fitusiran is added to plasma taken from patients with severe deficiency of FV, FVII, or FX [30]. Owing to its mechanism of action and thrombin-targeted approach, fitusiran therefore may be of use in other rare bleeding disorders that arise from insufficient thrombin generation, but further clinical studies are F I G U R E 5 Fitusiran revised dose and dose regimen, targeting an antithrombin range from 15% to 35% [62]. The revised dose and dose regimen was introduced as of December 2020. BPA, bypassing agent; CFC, clotting factor concentrate; Q2M, every other month; SS, steady state. Source: Figure adapted with permission from Pipe et al. [62]. a Participants are eligible for dose escalation if >4 doses of fitusiran have been administered at the current dose level, and they experienced >2 predose antithrombin (AT) activity levels >35% (as per central laboratory) after their second dose at the current dose, and fitusiran administration and AT activity assessments occurred as per schedule at the current dose level. b Participants previously escalated to a dose of 20 mg every month (QM), 50 mg QM or 80 mg QM due to AT >35% who experience >1 AT activity level <15% within a 12 month period must either permanently discontinue fitusiran prophylaxis, or in consultation with the Study Medical Manager may have the option to be de-escalated to their prior dose level. c Start of dosing after de-escalation from higher dose to occur only after centrally measured AT levels ≥22%. Participants receiving fitusiran at a dose of 20 mg every other month who experience ≥1 AT activity level <15% (as per central laboratory) within a 12-month period must permanently discontinue fitusiran treatment. d Participants with QM dosing bleeding episodes during the first 8 weeks at the current dose level or every other month dosing bleeding episodes during the first 12 weeks at the current dose level will not be considered for this judgment.
needed to confirm this hypothesis [30,44,64]. Overall, the evidence from clinical studies of fitusiran suggests that the benefits of the drug outweigh the risks [55][56][57]. and that fitusiran has the potential to change future clinical practice in hemophilia.

RELATIONSHIP DISCLOSURE
G.Y. has received contracts for Sanofi clinical trials, consulting fees, and support from Sanofi for travel to attend meetings. P.J.L. has received grants or contracts from Sanofi and has given lectures for Sanofi. S.E.C. has received grants or contracts from Sanofi and has participated on an advisory board for Sanofi. B.N. has received contracts for Sanofi clinical trials. A.S. has received grants or contracts from Sanofi and has participated in advisory boards for Sanofi.

ACKNOWLEDGMENTS
The authors would like to thank the patients, investigators, and study staff who participated in the fitusiran studies. Viridiana Cano from Sanofi contributed to the manuscript concept and checked the accuracy of the data included. Medical writing/editorial support was provided by Samuel Thomas, MSc, BSc, and Niki Panagiotaki, PhD, from Lucid Group Communications Ltd.

Medical writing support was funded by Sanofi in accordance with
Good Publication Practice (GPP3) guidelines.

AUTHOR CONTRIBUTIONS
All authors contributed to the organization of the work and contributed to the writing, reviewing, and revising of the manuscript critically for accuracy and completeness.