Inhibition of Tissue Factor Pathway Inhibitor (TFPI) as a Treatment for Haemophilia: Rationale with Focus on Concizumab

Inhibition of Tissue Factor Pathway Inhibitor (TFPI) as a Treatment for Haemophilia: Rationale... Drugs https://doi.org/10.1007/s40265-018-0922-6 LEADING ARTICLE Inhibition of Tissue Factor Pathway Inhibitor (TFPI) as a Treatment for Haemophilia: Rationale with Focus on Concizumab Pratima Chowdary The Author(s) 2018, corrected publication June/2018 Abstract Replacement therapy with missing factor Pharmacokinetic parameters were influenced by binding to (F) VIII or IX in haemophilia patients for bleed manage- the target (TFPI), demonstrating target mediated drug ment and preventative treatment or prophylaxis is standard disposition. A trend towards decreasing bleeding tendency of care. Restoration of thrombin generation through novel was observed and this preventative effect is being studied mechanisms has become the focus of innovation to over- in Phase 2 studies with additional data gathered to improve come limitations imposed by protein replacement therapy. our understanding of the therapeutic window and potential Tissue factor pathway inhibitor (TFPI) is a multivalent for thrombosis. Kunitz-type serine protease inhibitor that regulates tissue factor (TF)-induced coagulation through a FXa-dependent feedback inhibition of the TF.FVIIa complex in plasma and Key Points for Decision Makers on endothelial surfaces. Concizumab is a monoclonal, humanised antibody, specific for the second Kunitz domain Restoration of thrombin generation is increasingly of TFPI that binds and inhibits FXa, abolishing the inhi- considered as a therapeutic intervention to overcome bitory effect of TFPI. Concizumab restored thrombin the limitations of protein replacement therapy. generation in FVIII and FIX deficient plasmas and Anti-TFPI monoclonal antibodies restore thrombin decreased blood loss in a rabbit haemophilia model. Phase generation by abolishing the inhibitory effect of 1 single and multiple dose escalation studies in haemo- TFPI on the initiation of coagulation. philia patients demonstrated a dose dependent decrease in TFPI levels and a pro-coagulant effect with increasing A dose-dependent pro-coagulant effect has been d-dimers and prothrombin fragment 1 ? 2. A dose noted in Phase 1 clinical studies with anti-TFPI dependent increase in peak thrombin and endogenous antibodies with potentially a decrease in bleeding thrombin potential was observed with values in the normal tendency, which requires confirmation in larger range when plasma TFPI levels were nearly undetectable. studies over a longer duration. A few haemophilia patients in the highest dose cohorts with complete inhibition of plasma TFPI showed a decreased fibrinogen concentration with normal levels of 1 Introduction anti-thrombin and platelets and no evidence of thrombosis. 1.1 Haemophilia The original version of this article was revised: Due to Figure 1 caption update. Haemophilia A and B are inherited bleeding disorders & Pratima Chowdary characterised by a deficiency or absence of factor (F) VIII p.chowdary@ucl.ac.uk and FIX, respectively. The incidence of haemophilia A is 1 1 in 5000 male live births, and that of haemophilia B is 1 in Katharine Dormandy Haemophilia and Thrombosis Centre, 30,000 [1]. The FVIII and FIX Subcommittee of the Royal Free Hospital, Pond Street, London NW3 2 QG, UK P. Chowdary International Society of Thrombosis and Haemostasis has treatment administration to be modified to their personal recommended the use of plasma levels for classifying the circumstances, to improve treatment outcomes, potentially severity of haemophilia. Three patient groups are recog- increasing treatment burden [11–13]. nised based on their plasma levels: severe haemophilia Current management of bleeding in patients with inhi- (FVIII or FIX \ 1 IU/dL), moderate haemophilia (FVIII or bitors includes treatment with bypassing agents, either FIX between 1 and 5 IU/dL) and mild haemophilia (FVIII recombinant activated factor VIIa (rFVIIa) or activated or FIX between 6 IU/dL and 40 IU/dL) [2]. The classifi- prothrombin complex concentrate (APCC) [10, 14]. The cation usually predicts the bleeding phenotype and patients mechanisms underpinning their haemostatic efficacy are with a severe disorder present with recurrent spontaneous based on restoration of thrombin generation through path- and trauma-related bleeding [2]. In an untreated state, ways that potentially contribute a minimal amount of recurrent and spontaneous bleeding into joints and muscles thrombin under normal conditions [15–17]. Besides bleed results in disability, with bleeding into vital organs or from management, they are used for secondary and tertiary a mucosal surface being the most common cause of death prophylaxis, but they are less effective when compared to [3]. FVIII and FIX prophylaxis in non-inhibitor patients and have been associated with occasional episodes of throm- 1.2 Modern Haemophilia Treatment—Principles bosis [18–21]. and Limitations 1.3 Haemophilia Treatment—New Approaches Modern haemophilia treatment includes replacement ther- apy with missing FVIII or FIX with correction of bleeding Restoration of thrombin generation through novel mecha- tendency and a near normal life span [1, 4]. Besides nisms has become the focus of innovation in the last decade management of bleeds with replacement therapy, regular to overcome some of the limitations imposed by protein intravenous infusion either by parents or patients on aver- replacement therapy. Two broad approaches that restore age between two-to-four times per week improves the thrombin generation are in clinical trials. The first approach bleeding phenotype. This practice of preventive treatment includes a bi-specific antibody, which acts as a FVIIIa or prophylaxis has been the cornerstone of haemophilia mimetic bridging FIXa and FX with the generation of FXa. treatment for the last five decades. Prophylaxis, by The FXa generation is regulated by substrate availability increasing the baseline level to 1% or greater aims to rather than through inhibition of the bi-specific antibody convert a severe bleeding tendency to a moderate pheno- [22, 23]. The second approach results in reduced function type, thereby decreasing the number of spontaneous bleeds of natural inhibitors. This is achieved either through a [5]. decrease in activity of anti-thrombin (AT) or tissue factor Different types of prophylactic regimens are recognised pathway inhibitor (TFPI). AT is the principle serine pro- based on the timing of prophylaxis initiation. In primary tease inhibitor regulating the common, initiation and prophylaxis, it is commenced before or after the first joint amplification pathways [24], whereas TFPI is a dominant bleed, but before the second joint bleed. In secondary inhibitor of the initiation pathway [25, 26]. prophylaxis it is initiated after two or more joint bleeds but before the onset of joint disease, and tertiary prophylaxis is started after the onset of joint disease [6–8]. Primary pro- 2 Tissue Factor Pathway Inhibitor—Structure phylaxis or early secondary prophylaxis results in near and Distribution normal joint health and normal life span. Limitations of current treatment include: implementa- 2.1 Structure tion of prescribed prophylactic regimens [9], development of inhibitory antibodies that makes treatment ineffective TFPI is a multivalent Kunitz-type serine protease inhibitor [10], requirement for regular intravenous infusions, diffi- that regulates TF-induced coagulation [27] through an culties with venous access, patient compliance, cost of FXa-dependent feedback inhibition of the FVIIa. TF drugs, progression of joint disease, risk of intracranial complex, which initiates coagulation [27, 28]. TFPI was bleeding, and modest treatment goals, all of which impact cloned in 1988 and the amino acid sequence shows a on quality of life. Further, regular prophylaxis results in negatively charged amino acid terminus, three tandem oscillation of factor levels and coagulation potential, and Kunitz-type inhibitory domains (K1, K2, and K3), and a while this achieves a minimum trough level of 1% or positively charged carboxyl terminus [29]. The historical greater with a decrease in the number of spontaneous discovery, its role in coagulation regulation and disorders bleeds to single figures and potentially to zero, it does not of coagulation have been extensively reviewed [30–32]. prevent traumatic bleeds. Thus, individual patients require Anti-TFPI Monoclonal Antibodies for Haemophilia Treatment Multiple isoforms of TFPI have been described sec- temporally, and localisation is crucial for limiting blood ondary to alternative mRNA splicing events, and the two loss without compromising blood flow through excessive important isoforms include TFPI alpha (TFPIa) and TFPI clot formation [41, 42]. Inadequate thrombin generation beta (TFPIb)[31]. TFPIa is a 276-residue glycoprotein secondary to inherited or acquired deficiency of coagula- with an acidic amino terminus followed by three Kunitz tion factors results in a bleeding diathesis [43]. Replace- domains and a basic carboxy terminus. In TFPIb, the car- ment therapy or bypass agents restore thrombin generation boxy terminus contains a glycosylphosphatidylinositol and clot formation and improve the bleeding tendency (GPI) anchor replacing the K3 domain [30]. The K1 and [10, 44]. K2 domains bind and inhibit FVIIa and FXa, respectively. Following tissue injury, the extrinsic pathway or initia- The K3 domain has no known inhibitory function [30, 31]. tion pathway forms the extrinsic tenase complex (TF.FVIIa) and provides the initial FXa for the pro- 2.2 Distribution thrombinase complex. The intrinsic pathway or amplifica- tion pathway becomes the source of additional FXa fol- Endothelial cells and megakaryocytes are the main cells lowing inhibition of extrinsic tenase complex by TFPI producing TFPI [32, 33]. TFPIb is the predominant TFPI [27, 45]. Pro-thrombinase complex comprimised of FXa isoform expressed on endothelium where its association is bound to activated cellular surfaces in the presence of its maintained via a GPI anchor [34, 35]. TFPIa is secreted by co-factor FVa, converts prothrombin to thrombin (common human endothelial cells and is present in plasma. TFPI pathway). Thrombin mediates platelet activation and fibrin levels are increased two- to four-fold following heparin deposition enabling blood clot formation [41, 42]. infusion where TFPIa is the main isoform [30]. This hep- The inhibitory activity of TFPI is initiated by the arin-releasable pool may be bound to cell-surface gly- binding of the K2 domain to FXa resulting in its inhibition cosaminoglycans through its basic C-terminal region, and the formation of a binary TFPI.FXa complex. This is although the exact mechanism is not well described [32]. closely followed by binding of the K1 domain to FVIIa in TFPIa is also produced by megakaryocytes and stored the TF.FVIIa binary complex to form a quaternary complex within quiescent platelets. Platelet TFPI is available for that inhibits further activation of FX. The rate-limiting step release following platelet activation when it can exhibit its in these sequential reactions is the inhibition of FXa and inhibitory activity [36]. At the site of vascular injury, local not the inhibition of TF.FVIIa [28, 45]. Protein S binds the TFPI concentrations appear to increase through the release K3 domain of TFPIa, localising it to membrane surfaces of TFPI from accumulating platelets within the thrombus and enhancing the inhibition of FXa by the K2 domain. and this release is facilitated by dual activation with col- This is particularly relevant at physiological concentrations lagen and thrombin [35]. of TFPI [46–48]. Further, TFPIa is also able to inhibit FXa- The mean plasma circulating TFPI concentration in activated FVa and platelet FVa formed in the early stages normal individuals is 1.6–2.5 nM or * 70 ng/mL [30, 37]. of clot initiation resulting in inhibition of the pro-throm- In plasma, 80% of TFPI is predominantly bound to low- binase complex at physiological levels. This interaction is density lipoproteins (LDL), and is carboxy-terminal trun- mediated through a high-affinity exosite interaction cated, with levels of the former having an impact on the between the basic region of TFPIa and the FV acidic TFPI levels [30]. The remaining 20% of TFPI circulates in region [49]. the free form defined by the presence of the K3 domain. It In summary, TFPI inhibits the activity of two major consists of either FL-TFPIa contributing most to the anti- protease–co-factor complexes: extrinsic tenase complex coagulant activity or carboxy-terminal truncated TFPI [30]. generating FXa and early pro-thrombinase complex that The platelet TFPI pool exclusively consists of FL-TFPIa, includes FXa activated FVa [31]. Attenuation of this the amount equal to the circulating active full length TFPI. inhibition results in restoration of thrombin generation. The At baseline, the plasma TFPI pool is around 3% of the total relative contribution of the two pathways in this restoration vascular TFPI pool. Readily available mature TFPI appears is not known. to consist of 95% GPI-anchored TFPIb [38]. The potential for using TFPI inhibition to manage hae- mophilia A and B was conceived as early as 1991. In an in vitro laboratory study of haemophilic plasma, a pro- 3 Rationale for Inhibition of TFPI coagulant effect was demonstrated by shortening of the dilute thromboplastin time following the addition of anti- The blood haemostatic response initiated following dam- TFPI antibody [50]. A subsequent study tested the clinical age to the vasculature results in thrombin generation, pla- effect of a polyclonal anti-TFPI IgG antibody in rabbits telet activation and aggregation, enabling clot formation with anti-FVIII antibody-induced haemophilia. The [39, 40]. This response is localised both spatially and bleeding time shortened significantly from 26 to 11 min P. Chowdary but did not normalise (normal mean bleeding time in non- medicinal product. The studies include explorer 1, a Phase 1 haemophilia rabbits: 5 min). Further, correction of coagu- single-dose escalation study [25]; explorer 3, a Phase 1 lation tests was seen at doses of anti-TFPI antibody that multiple dose study [53]; explorer 4, a Phase 2 proof of were lower than doses required to correct bleeding ten- concept multiple-dose study in inhibitor patients (Clini- dency [51]. Additionally, in a mouse model of calTrials.gov Identifier: NCT03196284); and explorer 5, a haemophilia, an anti-TFPI polyclonal antibody decreased Phase 2 proof of concept study in non-inhibitor patients blood loss in a tail clip assay. Blood loss continued to (ClinicalTrials.gov Identifier: NCT03196297). BAY- decrease beyond complete inhibition of plasma and 1093884 from Bayer is a monoclonal antibody against both endothelial TFPI pools, suggesting a putative role for Kunitz-1 and Kunitz-2, presently recruiting to Phase 1 sin- inhibition of platelet TFPI released at the site of injury gle- and multiple-dose escalation studies (ClinicalTrials.gov [52]. Identifier: NCT02571569) [54, 55]. Similarly, PF-06741086 from Pfizer is a monoclonal antibody against the Kunitz-2 domain recruiting to multiple-dose studies in non-inhibitor 4 Anti-TFPI Antibodies in Clinical Trials patients (ClinicalTrials.gov Identifier: NCT02974855) [56]. Three monoclonal antibodies against TFPI are currently in various phases of clinical trials and are illustrated in Fig. 1. 5 Concizumab (mAb 2021) Concizumab (mAb-2021) from Novo Nordisk is a mono- clonal, humanised IgG4 antibody specific for the K2 domain Following the initial characterisation of TFPI, site-directed TM of TFPI [26]. The explorer studies are a series of clinical mutagenesis confirmed that the K2 domain was required trials conducted with concizumab as the investigational for efficient binding and inhibition of FXa, and both the K1 Fig. 1 TFPI mechanism of action and inhibition by anti-TFPI b Inhibition of FXa and FVIIa by TFPI. c Binding of the different antibodies. a Tissue factor (TF) based initiation of coagulation and Kunitz (K) domains by the various anti-TFPI antibodies generation of FXa by the extrinsic tenase complex (FVIIa.TF.FX). Anti-TFPI Monoclonal Antibodies for Haemophilia Treatment and K2 were required for inhibition of FVIIa.TF activity. K2 domain of human TFPI defining the antibody-binding Alterations of the active-site residues of the K3 domain had epitope are all conserved in rabbit TFPI. Rabbits were no significant effect on either function. The production and induced with haemophilia with an anti-factor VIII anti- characterisation of concizumab were detailed by Hilden body. Administration of concizumab significantly reduced et al., and a summary is provided below [26]. cuticle bleeding in haemophilia rabbits when anti-TFPI antibody was administered 30 min prior to induction of 5.1 Isolation and Characterisation bleeding and blood loss was also reduced when adminis- tered within 5 min of onset of bleeding. No effect of In brief, human recombinant TFPI was isolated from a concizumab was observed when administered 15 or 30 min baby hamster kidney cell line. Mice were subsequently after induction of bleeding [57]. A dose-dependent effect immunised with full-length TFPI and hybridomas were was noted in the amount of blood loss and duration of generated from mice splenic cells with aid of myeloma effect. Efficacy was also demonstrable after subcutaneous cells and supernatant screened for anti-TFPI antibodies. administration. The anti-TFPI antibodies were subsequently screened for their reactivity against human TFPI. Antibodies of interest were selected based on their ability to inhibit FXa and form 6 Concizumab Clinical Studies a functional quaternary complex. Subsequently, the anti- body was humanised and cloned into a human IgG4 format. 6.1 Phase 1 Clinical Study X-ray crystallography of concizumab demonstrated an extensive overlap between the binding epitope of con- The first in human Phase 1 study (explorer 1) was con- cizumab and predicted FXa contact region of the K2 ducted in patients with severe haemophilia A and B and domain, providing a structural basis for the inhibition. healthy volunteers. The study design and outcomes are Concizumab also demonstrated high affinity binding to described in brief below [25]. both soluble (K2 domain and FL-TFPI) and cell surface bound (human umbilical vein endothelial cells) TFPI. 6.2 Study Design 5.2 Ex Vivo Efficacy The study was a multicentre, randomised, double-blind, placebo-controlled, single-dose, dose-escalation trial. In The attenuation and abrogation of TFPI activity by con- each dose cohort, trial participants were randomised 3:1 to cizumab has been demonstrated by the lack of TFPI inhi- receive a single dose of concizumab (n = 3) or placebo bition of FXa generation in purified systems and cell-based (n = 1). Following drug administration, patients were seen assays. This effect was also demonstrated in a modified regularly, and samples were collected for pharmacokinetic prothrombin assay, in which tissue factor (innovin or (PK), pharmacodynamics (PD), and safety assessments. thromboplastin) concentration was adjusted to give a clot Initial dosing was performed in healthy volunteers and time of * 200 s with normal plasma. A dose-dependent when pre-defined criteria were reached, the drug was decrease in clotting was observed following the addition of administered to haemophilia patients, thus restricting the concizumab. Concizumab restored thrombin generation number of haemophilia patients required to conduct of the and this was demonstrated in FVIII immune depleted study. The switching criteria were consistent elevation of plasma supplemented with platelets at 150 9 10 /L. d-dimers and/or prothrombin fragment 1 ? 2 above the Thrombin generation was activated with low doses of normal reference range for 24 h or more in two or more innovin (0.12 pM TF) and dose-dependent changes were healthy volunteers, and/or when a maximum intravenous observed with increased peak, increased area under curve, (IV) dose of 250 lg/kg and a maximum subcutaneous (SC) and shorter lag times. In whole blood samples from healthy dose of 1000 lg/kg was administered to healthy volun- volunteers rendered haemophilic with sheep anti-FVIII teers. Randomised haemophilia patients were in either the IgG, a dose-dependent decrease in clotting time and max- IV cohorts (single doses included: 250 , 1000, 3000 and imum thrombus formation to within normal ranges was 9000 lg/kg) or in SC cohorts (doses included 1000 and observed when tested by thromboelastography when spiked 3000 lg/kg). with concizumab. 6.3 Endpoints 5.3 In Vivo Efficacy The primary endpoint was safety and secondary endpoints The in vivo efficacy of concizumab has been demonstrated included PK and PD parameters. Table 1 describes the in a rabbit cuticle bleeding model as the amino acids in the various parameters [25]. P. Chowdary Table 1 Study endpoints a a Safety endpoints Pharmacokinetics (PK) Pharmacodynamics (PD) Adverse events over 43 days PK endpoints—area under curve (AUC), D-dimer and prothrombin fragment maximum plasma concentration (C ), 1 ? 2 concentrations max Clinical assessments including ECG clearance (CL), half-life (t ), mean absorption Free TFPI in plasma by ELISA (i.e. Local injection-site reactions time, volume of distribution at steady state, TFPI not bound to concizumab, Lab assessments—routine and for evidence of bioavailability, and time to maximum Asserachrom TFPI) disseminated intravascular coagulation (PT, concentration Residual TFPI functionality— aPTT, fibrinogen concentration, platelet count, Plasma concizumab concentrations (ELISA assay detection of FXa generation with protein C, S & AT activity) for both free concizumab & concizumab chromogenic assay, S2222; Anti-concizumab antibodies by ELISA technique complexed with TFPI) Chromogenix) aPPT activated partial thromboplastin time, ECG electrocardiogram, TFTI tissue factor pathway inhibitor, PT prothrombin time Blood samples for PK and PD analysis were collected at baseline, at 0 (i.e. completion of injection), 5, 15, and 30 min; 1, 4, 8, 12, 24, 36, and 48 h; and 3, 4, 5, 6, 7, 10, 14, 21, and 43 days post-dosing. Blood samples from 5 and 15 min were omitted when the drug was administered subcutaneously 6.3.1 Results—Safety 6.3.3 Results—Pharmacodynamics Fifty-two subjects (28 healthy male volunteers and 24 Two assays were used to assess the impact of concizumab. haemophilia patients: 21 with haemophilia A and 3 with The first was a quantitative assay to determine the total haemophilia B) were enrolled and randomised to treatment amount of TFPI by ELISA in the plasma that does not bind or placebo. During the trial, there were no reports of seri- to the drug. A dose-dependent decrease in concizumab ous adverse events (SAEs). Five of the other reported AEs free-total TFPI concentration was observed. The levels were possibly or probably treatment related, including two were low for up to 2 weeks in the highest dose cohort. The in the placebo group and three in concizumab group. The second assay was a functional assay that measured FXa latter included a single episode of a short segment of generation as a function of TFPI. The residual functional superficial thrombophlebitis in a healthy volunteer in the TFPI levels decreased with the total TFPI levels. A dose- 1000 lg/kg, SC cohort. The patient presented with skin dependent pro-coagulant response as assessed by d-dimer tenderness 5 days post-drug administration and an ultra- and prothrombin fragment 1 ? 2 levels was seen. The sound confirmed a short segment of phlebitis. Injection site steepest increase was observed in healthy volunteers fol- reactions were seen in a few patients. lowing IV administration. Importantly, haemophilia In the haemophilia patients randomised to the higher SC patients showed similar d-dimer response compared with dose cohorts (1000 or 3000 lg/kg), transient elevation of healthy volunteers when they received an approximately troponin T was noted in 3 patients with no significant ECG 36-fold higher dose of concizumab. abnormalities. Similarly, a couple of haemophilia patients randomised to the higher IV dose cohorts (1000 or 6.4 Thrombin Generation and Concizumab 3000 lg/kg) halved their fibrinogen concentration with no concurrent decrease in protein C, protein S, AT, or platelet Ex vivo spiking of samples from patients with haemophilia count. A and B with concizumab displayed a dose-dependent increase in peak thrombin and endogenous thrombin 6.3.2 Results—Pharmacokinetics potential (ETP) with shortening of the lag time. The influence on peak thrombin was more marked when com- Concizumab was detected in plasma up to 43 days after pared with ETP. A similar effect was seen in samples taken dosing. There were no differences in the PK profiles from healthy volunteers dosed with concizumab [58]. between healthy volunteers and haemophilia subjects. PK profiles were consistent with target-mediated drug dispo- 6.5 Clinical Study—Multiple Dose sition (TMDD) in which binding of concizumab to TFPI influenced drug distribution, elimination, and plasma The findings in explorer 1 study, were replicated in concentration. explorer 3, which was a placebo-controlled, multiple-dose, dose-escalation study where concizumab was administered SC [53]. A dose-dependent decrease in concizumab free- total TFPI and procoagulant effect were noted. Further, a Anti-TFPI Monoclonal Antibodies for Haemophilia Treatment dose-dependent increase in peak thrombin and ETP was can decrease with increasing dose [61, 62]. In this context, seen, and in the higher dose cohort, thrombin generation it is important to identify the drug- and target-specific parameters were within normal range. parameters that influence exposure–response relationships, and patient-specific characteristics that account for inter- 6.6 Clinical Study—Phase 2 Proof of Concept subject PD variability [61]. Both explorer 1 and explorer 3 demonstrated a decreasing 7.2 TMDD and Concizumab bleeding tendency with higher concizumab doses, but the trials were not designed to demonstrate proof of efficacy To understand the impact of TMDD on the PK profile of [25]. explorer 4 and 5 will explore the ability of con- concizumab, several experiments were undertaken on cizumab to decrease bleeding tendency in patients with Cynomolgus monkeys [62]. A classical TMDD model was severe haemophilia A and B with and without inhibitors, used for analysing PK, which included compartments for respectively. Due to the significant impact of TMDD, both standard drug disposition and target binding. Fol- concizumab will be administered daily, and to decrease lowing subcutaneous administration, bioavailability of treatment burden, it will be administered SC [53]. The concizumab was estimated at 93%, absorption half-life was starting dose in the trials is 0.15 mg/kg (150 lg/kg) with estimated at 72 h and terminal half-life was dependent on dose escalation to a maximum of 0.25 mg/kg (250 lg/kg). plasma concentration. At low levels of concizumab, non- The bleed frequency will be observed for a period of linear clearance was the most important elimination route, 24 weeks in the first instance, followed by a 12-month secondary to TFPI binding. Maximum elimination rate extension phase. Patients will continue to administer FVIII (V ) was estimated at 11 lg/kg/h. When levels reached max or rFVIIa for management of bleeds. In addition to over 100 lg/mL, conventional linear clearance dominated demonstrating efficacy of concizumab, the trials could and was estimated at 0.14 mL/h/kg [62]. potentially shed light on the level of TFPI inhibition TMDD was confirmed in the Phase 1 studies and, at low required for decreasing the bleeding tendency. concizumab concentrations, binding to TFPI was the dominant influence resulting in faster nonlinear clearance; whereas at higher levels, a slower more linear clearance 7 Target-Mediated Drug Disposition (TMDD) was noted [25]. 7.1 TMDD—Definition and Description 7.3 Challenges of Pro-Coagulant Therapies The PK profile of small drug molecules is typically linear, A major challenge for the widespread use of pro-coagulant with dose increases resulting in a proportional increase in therapies is the lack of data around the overlap between the plasma concentration with binding to receptors having a therapeutic window, where bleed prevention is achieved, minimal effect. Non-linear PK is also common, where and the toxicity window where there is a potential there is a lack of proportional increase in plasma concen- increased risk of thrombosis. Under baseline conditions of tration with increases in drug dose. This non-proportional regular prophylaxis, such overlap is unlikely. Further, the response impacts on the relationship between drug con- interactions between replacement therapy and bypass centration and desired pharmacological effect and unde- agents administered for bleed management, and the novel sired toxicity [59]. The term TMDD has been used to pro-coagulant therapies need to be investigated urgently. In describe non-linear PK, where a significant proportion of this context, transient hypercoagulability is to be expected, the drug is bound with high affinity to the pharmacological but there are limited data on the magnitude, duration, and target, relative to the dose administered [60]. clinical impact of this hypercoagulability. Further, there The PK consequences of TMDD are pronounced when has been a tendency towards a one-size-fits-all approach binding to the target contributes to significant elimination when evidence from thrombin generation studies shows a of the drug. Further, if the binding is saturable it results in wide range of values both in the normal population and different concentration–time profiles for different individ- patients with haemophilia A and B. ual doses [61]. With saturable target binding, at low drug Further, the in vivo mechanisms that underpin clot for- concentration, administration of increasing doses is asso- mation and thrombin generation need to be elucidated. ciated with an apparent decreasing steady-state volume of Elevated d-dimers represent a pro-coagulant state, but distribution until the target is saturated. Following satura- borderline elevations could also potentially represent tion of the target binding, a limiting value is reached with a exaggerated basal coagulation with no impact on either long terminal elimination half-life. Therefore, when target bleeding or thrombotic tendency [63]. binding contributes significantly to clearance, clearance P. Chowdary Society on Thrombosis and Haemostasis. Thromb Haemost. Concizumab clinical trials have shown that the doses 2001;85(3):560. selected for the Phase 2 studies inhibit most of the plasma 3. Larsson SA. Life expectancy of Swedish haemophiliacs, pool and a proportion of the intravascular pool, and one 1831-1980. Br J Haematol. 1985;59(4):593–602. could speculate that this might provide protection against 4. Peyvandi F, Garagiola I, Young G. The past and future of hae- mophilia: diagnosis, treatments, and its complications. 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Varadi K, Tangada S, Loeschberger M, Montsch P, Schrenk G, Open Access This article is distributed under the terms of the Ewenstein B, Turecek PL. Pro- and anticoagulant factors facili- Creative Commons Attribution-NonCommercial 4.0 International tate thrombin generation and balance the haemostatic response to License (http://creativecommons.org/licenses/by-nc/4.0/), which per- FEIBA((R)) in prophylactic therapy. Haemophilia. mits any noncommercial use, distribution, and reproduction in any 2016;22(4):615–24. medium, provided you give appropriate credit to the original 18. Leissinger C, Gringeri A, Antmen B, et al. Anti-inhibitor coag- author(s) and the source, provide a link to the Creative Commons ulant complex prophylaxis in haemophilia with inhibitors. N Engl license, and indicate if changes were made. J Med. 2011;365(18):1684–92. 19. Leissinger CA, Becton DL, Ewing NP, Valentino LA. 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Cell-derived microparticles circulate in healthy humans 61. Mager DE. Target-mediated drug disposition and dynamics. and support low grade thrombin generation. Thromb Haemost. Biochem Pharmacol. 2006;72(1):1–10. 2001;85(4):639–46. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Drugs Springer Journals

Inhibition of Tissue Factor Pathway Inhibitor (TFPI) as a Treatment for Haemophilia: Rationale with Focus on Concizumab

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Drugs https://doi.org/10.1007/s40265-018-0922-6 LEADING ARTICLE Inhibition of Tissue Factor Pathway Inhibitor (TFPI) as a Treatment for Haemophilia: Rationale with Focus on Concizumab Pratima Chowdary The Author(s) 2018, corrected publication June/2018 Abstract Replacement therapy with missing factor Pharmacokinetic parameters were influenced by binding to (F) VIII or IX in haemophilia patients for bleed manage- the target (TFPI), demonstrating target mediated drug ment and preventative treatment or prophylaxis is standard disposition. A trend towards decreasing bleeding tendency of care. Restoration of thrombin generation through novel was observed and this preventative effect is being studied mechanisms has become the focus of innovation to over- in Phase 2 studies with additional data gathered to improve come limitations imposed by protein replacement therapy. our understanding of the therapeutic window and potential Tissue factor pathway inhibitor (TFPI) is a multivalent for thrombosis. Kunitz-type serine protease inhibitor that regulates tissue factor (TF)-induced coagulation through a FXa-dependent feedback inhibition of the TF.FVIIa complex in plasma and Key Points for Decision Makers on endothelial surfaces. Concizumab is a monoclonal, humanised antibody, specific for the second Kunitz domain Restoration of thrombin generation is increasingly of TFPI that binds and inhibits FXa, abolishing the inhi- considered as a therapeutic intervention to overcome bitory effect of TFPI. Concizumab restored thrombin the limitations of protein replacement therapy. generation in FVIII and FIX deficient plasmas and Anti-TFPI monoclonal antibodies restore thrombin decreased blood loss in a rabbit haemophilia model. Phase generation by abolishing the inhibitory effect of 1 single and multiple dose escalation studies in haemo- TFPI on the initiation of coagulation. philia patients demonstrated a dose dependent decrease in TFPI levels and a pro-coagulant effect with increasing A dose-dependent pro-coagulant effect has been d-dimers and prothrombin fragment 1 ? 2. A dose noted in Phase 1 clinical studies with anti-TFPI dependent increase in peak thrombin and endogenous antibodies with potentially a decrease in bleeding thrombin potential was observed with values in the normal tendency, which requires confirmation in larger range when plasma TFPI levels were nearly undetectable. studies over a longer duration. A few haemophilia patients in the highest dose cohorts with complete inhibition of plasma TFPI showed a decreased fibrinogen concentration with normal levels of 1 Introduction anti-thrombin and platelets and no evidence of thrombosis. 1.1 Haemophilia The original version of this article was revised: Due to Figure 1 caption update. Haemophilia A and B are inherited bleeding disorders & Pratima Chowdary characterised by a deficiency or absence of factor (F) VIII p.chowdary@ucl.ac.uk and FIX, respectively. The incidence of haemophilia A is 1 1 in 5000 male live births, and that of haemophilia B is 1 in Katharine Dormandy Haemophilia and Thrombosis Centre, 30,000 [1]. The FVIII and FIX Subcommittee of the Royal Free Hospital, Pond Street, London NW3 2 QG, UK P. Chowdary International Society of Thrombosis and Haemostasis has treatment administration to be modified to their personal recommended the use of plasma levels for classifying the circumstances, to improve treatment outcomes, potentially severity of haemophilia. Three patient groups are recog- increasing treatment burden [11–13]. nised based on their plasma levels: severe haemophilia Current management of bleeding in patients with inhi- (FVIII or FIX \ 1 IU/dL), moderate haemophilia (FVIII or bitors includes treatment with bypassing agents, either FIX between 1 and 5 IU/dL) and mild haemophilia (FVIII recombinant activated factor VIIa (rFVIIa) or activated or FIX between 6 IU/dL and 40 IU/dL) [2]. The classifi- prothrombin complex concentrate (APCC) [10, 14]. The cation usually predicts the bleeding phenotype and patients mechanisms underpinning their haemostatic efficacy are with a severe disorder present with recurrent spontaneous based on restoration of thrombin generation through path- and trauma-related bleeding [2]. In an untreated state, ways that potentially contribute a minimal amount of recurrent and spontaneous bleeding into joints and muscles thrombin under normal conditions [15–17]. Besides bleed results in disability, with bleeding into vital organs or from management, they are used for secondary and tertiary a mucosal surface being the most common cause of death prophylaxis, but they are less effective when compared to [3]. FVIII and FIX prophylaxis in non-inhibitor patients and have been associated with occasional episodes of throm- 1.2 Modern Haemophilia Treatment—Principles bosis [18–21]. and Limitations 1.3 Haemophilia Treatment—New Approaches Modern haemophilia treatment includes replacement ther- apy with missing FVIII or FIX with correction of bleeding Restoration of thrombin generation through novel mecha- tendency and a near normal life span [1, 4]. Besides nisms has become the focus of innovation in the last decade management of bleeds with replacement therapy, regular to overcome some of the limitations imposed by protein intravenous infusion either by parents or patients on aver- replacement therapy. Two broad approaches that restore age between two-to-four times per week improves the thrombin generation are in clinical trials. The first approach bleeding phenotype. This practice of preventive treatment includes a bi-specific antibody, which acts as a FVIIIa or prophylaxis has been the cornerstone of haemophilia mimetic bridging FIXa and FX with the generation of FXa. treatment for the last five decades. Prophylaxis, by The FXa generation is regulated by substrate availability increasing the baseline level to 1% or greater aims to rather than through inhibition of the bi-specific antibody convert a severe bleeding tendency to a moderate pheno- [22, 23]. The second approach results in reduced function type, thereby decreasing the number of spontaneous bleeds of natural inhibitors. This is achieved either through a [5]. decrease in activity of anti-thrombin (AT) or tissue factor Different types of prophylactic regimens are recognised pathway inhibitor (TFPI). AT is the principle serine pro- based on the timing of prophylaxis initiation. In primary tease inhibitor regulating the common, initiation and prophylaxis, it is commenced before or after the first joint amplification pathways [24], whereas TFPI is a dominant bleed, but before the second joint bleed. In secondary inhibitor of the initiation pathway [25, 26]. prophylaxis it is initiated after two or more joint bleeds but before the onset of joint disease, and tertiary prophylaxis is started after the onset of joint disease [6–8]. Primary pro- 2 Tissue Factor Pathway Inhibitor—Structure phylaxis or early secondary prophylaxis results in near and Distribution normal joint health and normal life span. Limitations of current treatment include: implementa- 2.1 Structure tion of prescribed prophylactic regimens [9], development of inhibitory antibodies that makes treatment ineffective TFPI is a multivalent Kunitz-type serine protease inhibitor [10], requirement for regular intravenous infusions, diffi- that regulates TF-induced coagulation [27] through an culties with venous access, patient compliance, cost of FXa-dependent feedback inhibition of the FVIIa. TF drugs, progression of joint disease, risk of intracranial complex, which initiates coagulation [27, 28]. TFPI was bleeding, and modest treatment goals, all of which impact cloned in 1988 and the amino acid sequence shows a on quality of life. Further, regular prophylaxis results in negatively charged amino acid terminus, three tandem oscillation of factor levels and coagulation potential, and Kunitz-type inhibitory domains (K1, K2, and K3), and a while this achieves a minimum trough level of 1% or positively charged carboxyl terminus [29]. The historical greater with a decrease in the number of spontaneous discovery, its role in coagulation regulation and disorders bleeds to single figures and potentially to zero, it does not of coagulation have been extensively reviewed [30–32]. prevent traumatic bleeds. Thus, individual patients require Anti-TFPI Monoclonal Antibodies for Haemophilia Treatment Multiple isoforms of TFPI have been described sec- temporally, and localisation is crucial for limiting blood ondary to alternative mRNA splicing events, and the two loss without compromising blood flow through excessive important isoforms include TFPI alpha (TFPIa) and TFPI clot formation [41, 42]. Inadequate thrombin generation beta (TFPIb)[31]. TFPIa is a 276-residue glycoprotein secondary to inherited or acquired deficiency of coagula- with an acidic amino terminus followed by three Kunitz tion factors results in a bleeding diathesis [43]. Replace- domains and a basic carboxy terminus. In TFPIb, the car- ment therapy or bypass agents restore thrombin generation boxy terminus contains a glycosylphosphatidylinositol and clot formation and improve the bleeding tendency (GPI) anchor replacing the K3 domain [30]. The K1 and [10, 44]. K2 domains bind and inhibit FVIIa and FXa, respectively. Following tissue injury, the extrinsic pathway or initia- The K3 domain has no known inhibitory function [30, 31]. tion pathway forms the extrinsic tenase complex (TF.FVIIa) and provides the initial FXa for the pro- 2.2 Distribution thrombinase complex. The intrinsic pathway or amplifica- tion pathway becomes the source of additional FXa fol- Endothelial cells and megakaryocytes are the main cells lowing inhibition of extrinsic tenase complex by TFPI producing TFPI [32, 33]. TFPIb is the predominant TFPI [27, 45]. Pro-thrombinase complex comprimised of FXa isoform expressed on endothelium where its association is bound to activated cellular surfaces in the presence of its maintained via a GPI anchor [34, 35]. TFPIa is secreted by co-factor FVa, converts prothrombin to thrombin (common human endothelial cells and is present in plasma. TFPI pathway). Thrombin mediates platelet activation and fibrin levels are increased two- to four-fold following heparin deposition enabling blood clot formation [41, 42]. infusion where TFPIa is the main isoform [30]. This hep- The inhibitory activity of TFPI is initiated by the arin-releasable pool may be bound to cell-surface gly- binding of the K2 domain to FXa resulting in its inhibition cosaminoglycans through its basic C-terminal region, and the formation of a binary TFPI.FXa complex. This is although the exact mechanism is not well described [32]. closely followed by binding of the K1 domain to FVIIa in TFPIa is also produced by megakaryocytes and stored the TF.FVIIa binary complex to form a quaternary complex within quiescent platelets. Platelet TFPI is available for that inhibits further activation of FX. The rate-limiting step release following platelet activation when it can exhibit its in these sequential reactions is the inhibition of FXa and inhibitory activity [36]. At the site of vascular injury, local not the inhibition of TF.FVIIa [28, 45]. Protein S binds the TFPI concentrations appear to increase through the release K3 domain of TFPIa, localising it to membrane surfaces of TFPI from accumulating platelets within the thrombus and enhancing the inhibition of FXa by the K2 domain. and this release is facilitated by dual activation with col- This is particularly relevant at physiological concentrations lagen and thrombin [35]. of TFPI [46–48]. Further, TFPIa is also able to inhibit FXa- The mean plasma circulating TFPI concentration in activated FVa and platelet FVa formed in the early stages normal individuals is 1.6–2.5 nM or * 70 ng/mL [30, 37]. of clot initiation resulting in inhibition of the pro-throm- In plasma, 80% of TFPI is predominantly bound to low- binase complex at physiological levels. This interaction is density lipoproteins (LDL), and is carboxy-terminal trun- mediated through a high-affinity exosite interaction cated, with levels of the former having an impact on the between the basic region of TFPIa and the FV acidic TFPI levels [30]. The remaining 20% of TFPI circulates in region [49]. the free form defined by the presence of the K3 domain. It In summary, TFPI inhibits the activity of two major consists of either FL-TFPIa contributing most to the anti- protease–co-factor complexes: extrinsic tenase complex coagulant activity or carboxy-terminal truncated TFPI [30]. generating FXa and early pro-thrombinase complex that The platelet TFPI pool exclusively consists of FL-TFPIa, includes FXa activated FVa [31]. Attenuation of this the amount equal to the circulating active full length TFPI. inhibition results in restoration of thrombin generation. The At baseline, the plasma TFPI pool is around 3% of the total relative contribution of the two pathways in this restoration vascular TFPI pool. Readily available mature TFPI appears is not known. to consist of 95% GPI-anchored TFPIb [38]. The potential for using TFPI inhibition to manage hae- mophilia A and B was conceived as early as 1991. In an in vitro laboratory study of haemophilic plasma, a pro- 3 Rationale for Inhibition of TFPI coagulant effect was demonstrated by shortening of the dilute thromboplastin time following the addition of anti- The blood haemostatic response initiated following dam- TFPI antibody [50]. A subsequent study tested the clinical age to the vasculature results in thrombin generation, pla- effect of a polyclonal anti-TFPI IgG antibody in rabbits telet activation and aggregation, enabling clot formation with anti-FVIII antibody-induced haemophilia. The [39, 40]. This response is localised both spatially and bleeding time shortened significantly from 26 to 11 min P. Chowdary but did not normalise (normal mean bleeding time in non- medicinal product. The studies include explorer 1, a Phase 1 haemophilia rabbits: 5 min). Further, correction of coagu- single-dose escalation study [25]; explorer 3, a Phase 1 lation tests was seen at doses of anti-TFPI antibody that multiple dose study [53]; explorer 4, a Phase 2 proof of were lower than doses required to correct bleeding ten- concept multiple-dose study in inhibitor patients (Clini- dency [51]. Additionally, in a mouse model of calTrials.gov Identifier: NCT03196284); and explorer 5, a haemophilia, an anti-TFPI polyclonal antibody decreased Phase 2 proof of concept study in non-inhibitor patients blood loss in a tail clip assay. Blood loss continued to (ClinicalTrials.gov Identifier: NCT03196297). BAY- decrease beyond complete inhibition of plasma and 1093884 from Bayer is a monoclonal antibody against both endothelial TFPI pools, suggesting a putative role for Kunitz-1 and Kunitz-2, presently recruiting to Phase 1 sin- inhibition of platelet TFPI released at the site of injury gle- and multiple-dose escalation studies (ClinicalTrials.gov [52]. Identifier: NCT02571569) [54, 55]. Similarly, PF-06741086 from Pfizer is a monoclonal antibody against the Kunitz-2 domain recruiting to multiple-dose studies in non-inhibitor 4 Anti-TFPI Antibodies in Clinical Trials patients (ClinicalTrials.gov Identifier: NCT02974855) [56]. Three monoclonal antibodies against TFPI are currently in various phases of clinical trials and are illustrated in Fig. 1. 5 Concizumab (mAb 2021) Concizumab (mAb-2021) from Novo Nordisk is a mono- clonal, humanised IgG4 antibody specific for the K2 domain Following the initial characterisation of TFPI, site-directed TM of TFPI [26]. The explorer studies are a series of clinical mutagenesis confirmed that the K2 domain was required trials conducted with concizumab as the investigational for efficient binding and inhibition of FXa, and both the K1 Fig. 1 TFPI mechanism of action and inhibition by anti-TFPI b Inhibition of FXa and FVIIa by TFPI. c Binding of the different antibodies. a Tissue factor (TF) based initiation of coagulation and Kunitz (K) domains by the various anti-TFPI antibodies generation of FXa by the extrinsic tenase complex (FVIIa.TF.FX). Anti-TFPI Monoclonal Antibodies for Haemophilia Treatment and K2 were required for inhibition of FVIIa.TF activity. K2 domain of human TFPI defining the antibody-binding Alterations of the active-site residues of the K3 domain had epitope are all conserved in rabbit TFPI. Rabbits were no significant effect on either function. The production and induced with haemophilia with an anti-factor VIII anti- characterisation of concizumab were detailed by Hilden body. Administration of concizumab significantly reduced et al., and a summary is provided below [26]. cuticle bleeding in haemophilia rabbits when anti-TFPI antibody was administered 30 min prior to induction of 5.1 Isolation and Characterisation bleeding and blood loss was also reduced when adminis- tered within 5 min of onset of bleeding. No effect of In brief, human recombinant TFPI was isolated from a concizumab was observed when administered 15 or 30 min baby hamster kidney cell line. Mice were subsequently after induction of bleeding [57]. A dose-dependent effect immunised with full-length TFPI and hybridomas were was noted in the amount of blood loss and duration of generated from mice splenic cells with aid of myeloma effect. Efficacy was also demonstrable after subcutaneous cells and supernatant screened for anti-TFPI antibodies. administration. The anti-TFPI antibodies were subsequently screened for their reactivity against human TFPI. Antibodies of interest were selected based on their ability to inhibit FXa and form 6 Concizumab Clinical Studies a functional quaternary complex. Subsequently, the anti- body was humanised and cloned into a human IgG4 format. 6.1 Phase 1 Clinical Study X-ray crystallography of concizumab demonstrated an extensive overlap between the binding epitope of con- The first in human Phase 1 study (explorer 1) was con- cizumab and predicted FXa contact region of the K2 ducted in patients with severe haemophilia A and B and domain, providing a structural basis for the inhibition. healthy volunteers. The study design and outcomes are Concizumab also demonstrated high affinity binding to described in brief below [25]. both soluble (K2 domain and FL-TFPI) and cell surface bound (human umbilical vein endothelial cells) TFPI. 6.2 Study Design 5.2 Ex Vivo Efficacy The study was a multicentre, randomised, double-blind, placebo-controlled, single-dose, dose-escalation trial. In The attenuation and abrogation of TFPI activity by con- each dose cohort, trial participants were randomised 3:1 to cizumab has been demonstrated by the lack of TFPI inhi- receive a single dose of concizumab (n = 3) or placebo bition of FXa generation in purified systems and cell-based (n = 1). Following drug administration, patients were seen assays. This effect was also demonstrated in a modified regularly, and samples were collected for pharmacokinetic prothrombin assay, in which tissue factor (innovin or (PK), pharmacodynamics (PD), and safety assessments. thromboplastin) concentration was adjusted to give a clot Initial dosing was performed in healthy volunteers and time of * 200 s with normal plasma. A dose-dependent when pre-defined criteria were reached, the drug was decrease in clotting was observed following the addition of administered to haemophilia patients, thus restricting the concizumab. Concizumab restored thrombin generation number of haemophilia patients required to conduct of the and this was demonstrated in FVIII immune depleted study. The switching criteria were consistent elevation of plasma supplemented with platelets at 150 9 10 /L. d-dimers and/or prothrombin fragment 1 ? 2 above the Thrombin generation was activated with low doses of normal reference range for 24 h or more in two or more innovin (0.12 pM TF) and dose-dependent changes were healthy volunteers, and/or when a maximum intravenous observed with increased peak, increased area under curve, (IV) dose of 250 lg/kg and a maximum subcutaneous (SC) and shorter lag times. In whole blood samples from healthy dose of 1000 lg/kg was administered to healthy volun- volunteers rendered haemophilic with sheep anti-FVIII teers. Randomised haemophilia patients were in either the IgG, a dose-dependent decrease in clotting time and max- IV cohorts (single doses included: 250 , 1000, 3000 and imum thrombus formation to within normal ranges was 9000 lg/kg) or in SC cohorts (doses included 1000 and observed when tested by thromboelastography when spiked 3000 lg/kg). with concizumab. 6.3 Endpoints 5.3 In Vivo Efficacy The primary endpoint was safety and secondary endpoints The in vivo efficacy of concizumab has been demonstrated included PK and PD parameters. Table 1 describes the in a rabbit cuticle bleeding model as the amino acids in the various parameters [25]. P. Chowdary Table 1 Study endpoints a a Safety endpoints Pharmacokinetics (PK) Pharmacodynamics (PD) Adverse events over 43 days PK endpoints—area under curve (AUC), D-dimer and prothrombin fragment maximum plasma concentration (C ), 1 ? 2 concentrations max Clinical assessments including ECG clearance (CL), half-life (t ), mean absorption Free TFPI in plasma by ELISA (i.e. Local injection-site reactions time, volume of distribution at steady state, TFPI not bound to concizumab, Lab assessments—routine and for evidence of bioavailability, and time to maximum Asserachrom TFPI) disseminated intravascular coagulation (PT, concentration Residual TFPI functionality— aPTT, fibrinogen concentration, platelet count, Plasma concizumab concentrations (ELISA assay detection of FXa generation with protein C, S & AT activity) for both free concizumab & concizumab chromogenic assay, S2222; Anti-concizumab antibodies by ELISA technique complexed with TFPI) Chromogenix) aPPT activated partial thromboplastin time, ECG electrocardiogram, TFTI tissue factor pathway inhibitor, PT prothrombin time Blood samples for PK and PD analysis were collected at baseline, at 0 (i.e. completion of injection), 5, 15, and 30 min; 1, 4, 8, 12, 24, 36, and 48 h; and 3, 4, 5, 6, 7, 10, 14, 21, and 43 days post-dosing. Blood samples from 5 and 15 min were omitted when the drug was administered subcutaneously 6.3.1 Results—Safety 6.3.3 Results—Pharmacodynamics Fifty-two subjects (28 healthy male volunteers and 24 Two assays were used to assess the impact of concizumab. haemophilia patients: 21 with haemophilia A and 3 with The first was a quantitative assay to determine the total haemophilia B) were enrolled and randomised to treatment amount of TFPI by ELISA in the plasma that does not bind or placebo. During the trial, there were no reports of seri- to the drug. A dose-dependent decrease in concizumab ous adverse events (SAEs). Five of the other reported AEs free-total TFPI concentration was observed. The levels were possibly or probably treatment related, including two were low for up to 2 weeks in the highest dose cohort. The in the placebo group and three in concizumab group. The second assay was a functional assay that measured FXa latter included a single episode of a short segment of generation as a function of TFPI. The residual functional superficial thrombophlebitis in a healthy volunteer in the TFPI levels decreased with the total TFPI levels. A dose- 1000 lg/kg, SC cohort. The patient presented with skin dependent pro-coagulant response as assessed by d-dimer tenderness 5 days post-drug administration and an ultra- and prothrombin fragment 1 ? 2 levels was seen. The sound confirmed a short segment of phlebitis. Injection site steepest increase was observed in healthy volunteers fol- reactions were seen in a few patients. lowing IV administration. Importantly, haemophilia In the haemophilia patients randomised to the higher SC patients showed similar d-dimer response compared with dose cohorts (1000 or 3000 lg/kg), transient elevation of healthy volunteers when they received an approximately troponin T was noted in 3 patients with no significant ECG 36-fold higher dose of concizumab. abnormalities. Similarly, a couple of haemophilia patients randomised to the higher IV dose cohorts (1000 or 6.4 Thrombin Generation and Concizumab 3000 lg/kg) halved their fibrinogen concentration with no concurrent decrease in protein C, protein S, AT, or platelet Ex vivo spiking of samples from patients with haemophilia count. A and B with concizumab displayed a dose-dependent increase in peak thrombin and endogenous thrombin 6.3.2 Results—Pharmacokinetics potential (ETP) with shortening of the lag time. The influence on peak thrombin was more marked when com- Concizumab was detected in plasma up to 43 days after pared with ETP. A similar effect was seen in samples taken dosing. There were no differences in the PK profiles from healthy volunteers dosed with concizumab [58]. between healthy volunteers and haemophilia subjects. PK profiles were consistent with target-mediated drug dispo- 6.5 Clinical Study—Multiple Dose sition (TMDD) in which binding of concizumab to TFPI influenced drug distribution, elimination, and plasma The findings in explorer 1 study, were replicated in concentration. explorer 3, which was a placebo-controlled, multiple-dose, dose-escalation study where concizumab was administered SC [53]. A dose-dependent decrease in concizumab free- total TFPI and procoagulant effect were noted. Further, a Anti-TFPI Monoclonal Antibodies for Haemophilia Treatment dose-dependent increase in peak thrombin and ETP was can decrease with increasing dose [61, 62]. In this context, seen, and in the higher dose cohort, thrombin generation it is important to identify the drug- and target-specific parameters were within normal range. parameters that influence exposure–response relationships, and patient-specific characteristics that account for inter- 6.6 Clinical Study—Phase 2 Proof of Concept subject PD variability [61]. Both explorer 1 and explorer 3 demonstrated a decreasing 7.2 TMDD and Concizumab bleeding tendency with higher concizumab doses, but the trials were not designed to demonstrate proof of efficacy To understand the impact of TMDD on the PK profile of [25]. explorer 4 and 5 will explore the ability of con- concizumab, several experiments were undertaken on cizumab to decrease bleeding tendency in patients with Cynomolgus monkeys [62]. A classical TMDD model was severe haemophilia A and B with and without inhibitors, used for analysing PK, which included compartments for respectively. Due to the significant impact of TMDD, both standard drug disposition and target binding. Fol- concizumab will be administered daily, and to decrease lowing subcutaneous administration, bioavailability of treatment burden, it will be administered SC [53]. The concizumab was estimated at 93%, absorption half-life was starting dose in the trials is 0.15 mg/kg (150 lg/kg) with estimated at 72 h and terminal half-life was dependent on dose escalation to a maximum of 0.25 mg/kg (250 lg/kg). plasma concentration. At low levels of concizumab, non- The bleed frequency will be observed for a period of linear clearance was the most important elimination route, 24 weeks in the first instance, followed by a 12-month secondary to TFPI binding. Maximum elimination rate extension phase. Patients will continue to administer FVIII (V ) was estimated at 11 lg/kg/h. When levels reached max or rFVIIa for management of bleeds. In addition to over 100 lg/mL, conventional linear clearance dominated demonstrating efficacy of concizumab, the trials could and was estimated at 0.14 mL/h/kg [62]. potentially shed light on the level of TFPI inhibition TMDD was confirmed in the Phase 1 studies and, at low required for decreasing the bleeding tendency. concizumab concentrations, binding to TFPI was the dominant influence resulting in faster nonlinear clearance; whereas at higher levels, a slower more linear clearance 7 Target-Mediated Drug Disposition (TMDD) was noted [25]. 7.1 TMDD—Definition and Description 7.3 Challenges of Pro-Coagulant Therapies The PK profile of small drug molecules is typically linear, A major challenge for the widespread use of pro-coagulant with dose increases resulting in a proportional increase in therapies is the lack of data around the overlap between the plasma concentration with binding to receptors having a therapeutic window, where bleed prevention is achieved, minimal effect. Non-linear PK is also common, where and the toxicity window where there is a potential there is a lack of proportional increase in plasma concen- increased risk of thrombosis. Under baseline conditions of tration with increases in drug dose. This non-proportional regular prophylaxis, such overlap is unlikely. Further, the response impacts on the relationship between drug con- interactions between replacement therapy and bypass centration and desired pharmacological effect and unde- agents administered for bleed management, and the novel sired toxicity [59]. The term TMDD has been used to pro-coagulant therapies need to be investigated urgently. In describe non-linear PK, where a significant proportion of this context, transient hypercoagulability is to be expected, the drug is bound with high affinity to the pharmacological but there are limited data on the magnitude, duration, and target, relative to the dose administered [60]. clinical impact of this hypercoagulability. Further, there The PK consequences of TMDD are pronounced when has been a tendency towards a one-size-fits-all approach binding to the target contributes to significant elimination when evidence from thrombin generation studies shows a of the drug. Further, if the binding is saturable it results in wide range of values both in the normal population and different concentration–time profiles for different individ- patients with haemophilia A and B. ual doses [61]. With saturable target binding, at low drug Further, the in vivo mechanisms that underpin clot for- concentration, administration of increasing doses is asso- mation and thrombin generation need to be elucidated. ciated with an apparent decreasing steady-state volume of Elevated d-dimers represent a pro-coagulant state, but distribution until the target is saturated. Following satura- borderline elevations could also potentially represent tion of the target binding, a limiting value is reached with a exaggerated basal coagulation with no impact on either long terminal elimination half-life. Therefore, when target bleeding or thrombotic tendency [63]. binding contributes significantly to clearance, clearance P. Chowdary Society on Thrombosis and Haemostasis. Thromb Haemost. Concizumab clinical trials have shown that the doses 2001;85(3):560. selected for the Phase 2 studies inhibit most of the plasma 3. Larsson SA. Life expectancy of Swedish haemophiliacs, pool and a proportion of the intravascular pool, and one 1831-1980. Br J Haematol. 1985;59(4):593–602. could speculate that this might provide protection against 4. Peyvandi F, Garagiola I, Young G. The past and future of hae- mophilia: diagnosis, treatments, and its complications. 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DrugsSpringer Journals

Published: May 29, 2018

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