Haemophilia (2015), 21 (Suppl. 1), 1–12
DOI: 10.1111/hae.12582
REVIEW ARTICLE
Spotlight on the human factor: building a foundation for the future of haemophilia A management Report from a symposium on human recombinant FVIII at the World Federation of Hemophilia World Congress, Melbourne, Australia on 12 May 2014 C. KESSLER,* J. OLDENBURG,† C. ESCURIOLA ETTINGSHAUSEN,‡ A. TIEDE,§ K. KHAIR,¶ R I E R * * and R . K L A M R O T H † † C . N EG *Division of Hematology and Oncology, The Vincent Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA; †The Institute of Experimental Haematology and Transfusion Medicine and the Haemophilia Centre at the University Clinic, Bonn, Germany; ‡Haemophilia Centre Rhein-Main HZRM, M€ orfelden-Walldorf, Germany; §Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; ¶Great Ormond Street Hospital for Children NHS Trust, London, UK; **Hematology Division, Director Hemophilia Comprehensive Care Center, H^ opital Edouard Herriot Pavillon E, Universite Lyon, Lyon, France; and ††The Haemophilia Treatment Centre, Vivantes Klinikum im Friedrichshain, Berlin, Germany
Summary. Inhibitor development is the most serious and challenging complication in the treatment of severe haemophilia A. Up to 38% of such patients develop inhibitors with current recombinant factor VIII (rFVIII) products produced in hamster cell lines. Human-cl rhFVIII is a new generation fully sulfated B-domaindeleted FVIII coagulant glycoprotein, which is generated from a human cell line. Thus, there are no non-human epitopes which would be potentially immunogenic. This molecule has significantly higher VWF-binding affinity compared with existing fulllength rFVIII produced in hamster cell lines. The development aim of Human-cl rhFVIII is to address the challenges of FVIII inhibitors and frequent infusions during prophylaxis. Human-cl rhFVIII’s mean half-life is very comparable to some of the newer products which involve modification of the FVIII molecule to extend the circulating half-life. There are promising data concerning the use of a personalized prophylaxis
Correspondence: Dr Craig M. Kessler, Division of Hematology and Oncology, The Vincent Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA. Tel.: +1 202 444 8676; fax: +1 202 444 1229; e-mail: [email protected] and Dr Johannes Oldenburg, The Institute of Experimental Haematology and Transfusion Medicine and the Haemophilia Centre at the University Clinic, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Tel.: +49 (0) 228 287 15175; fax: +49 (0) 228 287 14783; e-mail: [email protected] © 2014 John Wiley & Sons Ltd
regimen with Human-cl rhFVIII. Preliminary data indicate a median dosing interval of 3.5 days with 66.7% of the patients on a twice per week or fewer infusions schedule combined with a low bleeding rate and no increased FVIII consumption when compared to standard prophylaxis. No product-specific laboratory assay is required to monitor the coagulation activity for Human-cl rhFVIII. The results of registration clinical trials with Human-cl rhFVIII as well as the ongoing studies in previously untreated patients (NuProtect) and personalized prophylaxis study in previously treated patients (NuPreviq), will be discussed. The manufacturer has received marketing authorization for Human-cl rhFVIII in Europe and Canada under the name Nuwiqâ and plans to launch it in the USA and globally in 2015. Keywords: assays, clinical trials, haemophilia A, Human-cl rhFVIII, inhibitors, personalized prophylaxis
Introduction (Craig Kessler, United States) For the last 20 years, haemophilia A patients have been treated with recombinant factor VIII (rFVIII) concentrates which have all been produced in hamster derived cell lines. We now have on the horizon an opportunity to use a recombinant B-domain-deleted FVIII concentrate that has been produced in human embryonic kidney cells. This represents a major advance in the manufacture of genetically engineered clotting factor replacement products. Such a product might be useful in the armamentarium of treatment for patients with 1
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haemophilia A as it has the potential to address some of the unmet needs in the care of these patients. The major unmet need is the development of replacement products which have lower potential to induce the development of alloantibody inhibitors against FVIII in previously untreated patients (PUPs) [1]. Furthermore, the development of alloantibody inhibitors throughout the lifetime in previously treated patients (PTPs) with haemophilia is becoming much more apparent, as a result of an increased number of surgeries being performed in patients with mild and moderate haemophilia, who concurrently receive large amounts of replacement therapy, perhaps within the context of ‘triggers’ of inflammation and stress to the immune system. There is also a rising incidence of inhibitors in older patients with severe haemophilia A [1]. The benefits of prophylaxis strategies, particularly started early, in patients with severe haemophilia have been well demonstrated [2,3]. Successful prophylaxis regimens will enhance the quality of our patients’ lives and will allow them to be able to lead much more normal physically active lives. However, a ‘one-sizefits-all’ strategy for prophylaxis may pose an unnecessary bleeding risk or theoretically a hypercoagulable thrombotic risk. Differences in intra- and inter-individual FVIII half-lives suggest that tailored dosing approaches would be desirable to ensure a high quality of life and can be adjusted to meet the aspirational activity goals of the individual. There remain some concerns with the future and recently USA FDA-approved treatments for haemophilia A, including the incidence of inhibitor formation, which the new generation of manipulated and posttranslationally modified rFVIII molecules may potentially induce. Furthermore, physicians will need to learn how to monitor their in vivo haemostatic activities since some of the new products appear to require product-specific laboratory assay systems. These points were discussed in more detail at a symposium on human recombinant FVIII at the World Federation of Hemophilia World Congress, Melbourne, Australia on 12 May 2014 and a report of the meeting is presented below.
Inhibitors in haemophilia: perspectives and challenges (Carmen Escuriola Ettingshausen, Germany) Inhibitor development is the most serious and very challenging complication in the treatment of patients with haemophilia [1,4]. Around 35% of severe haemophilia A patients and around 5% of severe haemophilia B patients develop inhibitors [5,6]. Characteristically, this complication occurs during the initial phase of FVIII exposure, meaning that it usually occurs in very small children during the first 20 expoHaemophilia (2015), 21 (Suppl. 1), 1--12
sure days (EDs). It is associated with a reduction of efficacy of haemostatic therapy with FVIII and increased morbidity, particularly in patients with high responding inhibitors (>5 BU). In addition to the fact that these patients quickly become disabled, there are high treatment costs to control the bleeding episodes.
Risk factors for the development of inhibitors There are genetic endogenous host risk factors that support the development of inhibitors, i.e. there are different underlying gene defects for haemophilia that are associated with a different risk of inhibitor development. While the underlying F8 or F9 gene mutation is a significant predictor of inhibitor development, there are also less strong risk factors, including replacement product immunogenicity, the human leucocyte antigen (HLA) type, and the ethnic origin of the patient [7]. Exogenous/environmental risk factors, which predispose to the development of alloantibody inhibitors, are also very challenging and include age at first exposure to FVIII, the therapeutic regimen employed (prophylaxis or on-demand treatment – patients treated prophylactically have a lower inhibitor incidence than those treated on-demand) and the intensity of treatment. From the CANAL [8] and RODIN [5] studies it was shown that surgical procedures as well as peak treatment levels are extremely important and put the patient at risk of developing an inhibitor. The type of FVIII concentrate used has also been a topic of great debate [9]. The large RODIN study seemed to indicate that there was no difference concerning inhibitor development between plasma-derived (pd) and rFVIII concentrates although the results of a prospective randomized study, the SIPPET study, which investigates inhibitor development with the use of pdFVIII or rFVIII in PUPs have not yet been published [10]. With regard to the differences that might account for the reason that pdFVIII products could be associated with a lower inhibitor incidence, pd products contain a considerable amount of von Willebrand factor (VWF). VWF may mask B-cell epitopes on the light chain of FVIII [11]. VWF prevents internalization of FVIII by bone marrow dendritic cells and hinders antigen recognition of FVIII [12]. rFVIII-treated and pdFVIII-treated mice have different subsets of CD4+ T–cells [13], which suggests that these mice might produce different amounts of anti-FVIII antibodies. In pdFVIII-treated mice, the amount of antibodies produced might be lower. To compare pdFVIII and rFVIII concentrates, the impact of the posttranslational modifications (PTMs) of these FVIII molecules should be considered. PTMs are the biochemical modifications of a protein after its translation from DNA to amino acid sequence. All human plasma proteins undergo PTMs. Different © 2014 John Wiley & Sons Ltd
HUMAN-CL rhFVIII FOR HAEMOPHILIA A
expression systems (e.g. bacterial, yeast, fungal, plant, insect, hamster and human) produce different PTMs for the same protein sequence. For rFVIII, the PTMs such as glycosylation and sulfation of Tyr1680 have been shown to be vital for functionality and VWF-binding affinity. In the absence of tyrosine sulfation at Y1680 in rFVIII, the affinity for VWF is reduced five-fold [14,15]. Octanateâ is a human pdFVIII naturally stabilized with VWF and is a fully sulfated protein. The ratio of FVIII to VWF in Octanateâ is in the order of 0.4 with a mean specific activity of >100 IU FVIII:C/mg total protein; albumin is not added as a stabilizer. The product undergoes double virus inactivation by solvent/detergent and terminal dry-heat treatment to inactivate both enveloped and non-enveloped viruses. Octanate has been on the market for 16 years and, therefore, there is a large clinical experience. The ongoing Good Clinical Practice (GCP)–PUP study with Octanate has recruited 51 predominantly severe haemophiliacs. With regard to genetic risk factors, a total of 70.6% of the patients had high-risk mutations, most commonly intron 22 mutations (51%), whereas a minority had low-risk mutations such as missense (18%) or splice site mutations (4%) [16]. Patients were first exposed to Octanate at the age of around 1 year. There was a family history of haemophilia A and inhibitors in three patients (6%) [16]. Of the 51 patients, 14 were treated on-demand for ≥50 EDs, 15 were treated on-demand for ≥20 EDs before being switched to prophylactic administration, 20 were treated exclusively prophylactically (i.e. during the high-risk phase) and two patients were treated on-demand for 5 BU); however, in two cases, these inhibitors were transient and disappeared under regular treatment with FVIII. All inhibitors developed in patients treated on an on-demand basis; no inhibitor developed in patients undergoing prophylaxis. The overall inhibitor rate at the current time, including the two transient inhibitors, is 9.8% (five of 51 patients) and the incidence of clinically relevant inhibitors is 5.9% (three of 51 patients). In an ongoing international ITI study (Observational Immune Tolerance Induction, ObsITI), Octanate provided an ITI success rate of >70% in patients with poor prognosis factors [17]. No cases of haemolysis and no accumulation of VWF during high-dose ITI have been reported [18].
Human-cl rhFVIII Glycosylation alters the structural and functional properties of a protein and is responsible for physicochemical properties, immunological properties, receptor binding/affinity and intracellular sorting. A recent study by Kannicht et al. to characterize PTMs of Human-cl rhFVIII (simoctocog alfa) showed the product to be glycosylated with complex-, hybrid- and high mannose-type glycosylation present at the same sites as in human pdFVIII [15]. In addition, as a consequence of the expression in a human cell line, it is devoid of the non-human antigenic carbohydrate epitopes, in particular Gala1-3Galb1-GlcNAc-R (a-Gal) and N-glycolylneuraminic acid (Neu5Gc), Fig. 1 [15,19–21]. These residues are typically found in rFVIII molecules produced in rodent cells, such as Chinese hamster ovarian (CHO) cells and baby hamster kidney (BHK) cells, Table 1 [15].
Fig. 1. N-glycosylation: carbohydrate epitopes on rFVIII molecules produced in human embryonic kidney (HEK), CHO and BHK cell lines [15,19–21].
© 2014 John Wiley & Sons Ltd
Haemophilia (2015), 21 (Suppl. 1), 1--12
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Table 1. Terminal structures of N-glycans of human serum glycoproteins in comparison with plasma-derived and recombinant FVIII [15]. Core structure
Terminal structure
GlcNAc2-Man3 core Fuc
High mannose GlcNAc-Gal type 1 or 2 a-2-3/a-2,6 linked sialyl type 1 or 2 Antennae fucosylation, e.g. LeX or LeA or LDN(F) Blood group ABO Neu5Gc Gala1-3Gal
Human plasma proteins
Human pdFVIII (e.g. Octanateâ)
+ + +/+ +
+ + +/+ +
+
+
Human-cl rhFVIII + + +/+ +
CHO or BHK-derived rFVIII + + +/ +
+ (CHO) + (BHK)
Fig. 2. Extracted ion chromatograms showing the sulfated (major peak) and non-sulfated (minor or absent peak) Y1680-containing peptides [15].
Human-cl rhFVIII is a fully sulfated human protein (Fig. 2 [15]). It has significantly higher VWF-binding affinity compared with existing full-length rFVIII produced in hamster cell lines. In a study extracted ion chromatograms showed the sulfated and non-sulfated (minor or absent peak) Tyr1680-containing peptides. For Octanate and Human-cl rhFVIII, there was only a sulfated peak, whereas for full-length rFVIII (CHO), full-length rFVIII (BHK) and B-domain-deleted rFVIII (CHO), there was both a sulfated peak and a minor non-sulfated peak [15] in rFVIII molecules expressed by rodent cells. With regard to the importance of the binding activity to VWF, two other experiments have been performed comparing the human cell line rFVIII with FVIII concentrate produced by rodent cells: these were the SPR-based affinity measurements and binding to VWF–Sepharose to determine free rFVIII. In the first experiment, the dissociation constant was lower in the Human-cl rhFVIII (193 36 pM) compared to the rodent cell line derived FVIII (full-length rFVIII [CHO] 320 39, full-length rFVIII [BHK] 342 35, B-domain-derived rFVIII [CHO] 230 31), meaning that there is a higher binding affinity for VWF [22]. The second experiment showed a significantly higher amount of unbound FVIII for products expressed by the rodent cell lines compared to Human-cl rhFVIII [22]. Thus, both experiments showed a significantly Haemophilia (2015), 21 (Suppl. 1), 1--12
higher VWF-binding affinity for Human-cl rhFVIII than the comparator rFVIII proteins that were tested. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed
Fig. 3. Human-cl rhFVIII (BDD rFVIII): comparable FVIII activities in one-stage* (OS) and chromogenic** (CS) release assays. (*BCSâXP using Dade FVIII-deficient plasma and Actinâ; **Coatest SPâ FVIII kit) [22].
© 2014 John Wiley & Sons Ltd
HUMAN-CL rhFVIII FOR HAEMOPHILIA A
to lower the intrinsic immunogenicity of Human-cl rhFVIII. It is also extremely important that if these agents are to be used in the clinical setting, they should be suitable for all FVIII determination assays. A study was conducted with 17 batches of Human-cl rhFVIII which were tested using the one-stage assay as well as the chromogenic assay. The results were quite comparable (Fig. 3 [22]) so it can be concluded that both types of FVIII activity tests can be used for Human-cl rhFVIII.
The new Human-cl rhFVIII: overview of clinical trial experience in children and adults with haemophilia A (Andreas Tiede, Germany) Switching the cell line and having a slightly different B-domain-deleted FVIII molecule led to the development of a new drug with the generic name simoctocog alfa (Human-cl rhFVIII). The vision with this new product was to try and reduce inhibitor rates with a rFVIII concentrate that in terms of glycosylation might be more similar to the natural human FVIII. Three pivotal studies with Human-cl rhFVIII in PTPs have reported results: GENA–01 (ondemand) in adults and adolescents looking at pharmacokinetics, efficacy, safety and immunogenicity, GENA–08 (prophylaxis) in adults and adolescents looking at efficacy, safety and immunogenicity, and GENA–03 (prophylaxis) in children looking at pharmacokinetics, efficacy, safety and immunogenicity (Table 2).
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Looking first at the efficacy of on-demand treatment in GENA–01, in 94.4% of cases treatment of bleeding episodes (986 bleeding episodes in total) was rated excellent or good. A total of 96.7% of all bleeds were managed with one (90.3%) or two (6.4%) infusions. The median dose per infusion was 30.0 IU kg 1 and the median dose per bleeding episode was 30.9 IU kg 1. Results for the prophylactic study (GENA–08) showed that there was no major or life-threatening bleeding and 50% of patients did not experience any bleeding episodes. In 34.4%, there was one bleeding episode, which in most cases was minor and in 15.6% of cases there were five or more bleeding episodes. The mean annualized bleeding rate (ABR) was 2.28 3.73 bleeds per year, Fig. 4 [19]. The GENA–01 and GENA–08 studies were two very similar cohorts with an identical way of collecting the data. This shows that prophylaxis also prevents bleeds in adults: there was a 96% reduction in mean ABR with prophylaxis versus on-demand treatment. Increasingly, there are more and more studies showing that prophylaxis in adults can prevent bleeds.
Paediatric study In the paediatric study (GENA–03), the mean age was 6.1 2.97 years, all children were white and in 53 of the 59 patients prophylaxis had been given prior to the study. The main efficacy outcome was breakthrough bleeds during prophylactic treatment. In all patients pooled together, the number of breakthrough
Table 2. Clinical trials with Human-cl rhFVIII that have reported results. Adults and adolescents GENA–01 (on-demand) Pharmacokinetics Efficacy, safety and immunogenicity Treatment
✔ ✔
Development phase Number of centres Number of countries
II 9 3 (USA, Germany, Bulgaria)
30–40 IU kg 1 every other day/treatment of breakthrough bleeds for ≥6 months and ≥50 EDs III 11 4 (Austria, Bulgaria, Germany, UK)
Number of PTPs Coordinating investigator
22 Marilyn J. Manco-Johnson, USA
32 Johannes Oldenburg, Germany
≥6 months and ≥50 EDs
Children 2–12 years GENA–03 (prophylaxis)
GENA–08 (prophylaxis) ✔
✔ ✔ 30–40 IU kg 1 every other day or three times per week for ≥6 months and ≥50 EDs III 15 7 (UK, Poland, France, Russia, Turkey, Romania, Czech Republic) 59 Raina J. Liesner, UK
Fig. 4. Mean annualized bleeding rate per regimen with Human-cl rhFVIII: on-demand versus prophylaxis. Adult patients on prophylactic regimens experienced significantly fewer bleeding episodes than those using on-demand treatment [19].
© 2014 John Wiley & Sons Ltd
Haemophilia (2015), 21 (Suppl. 1), 1--12
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Fig. 5. Mean annualized bleeding rate (ABR) in children undergoing prophylaxis (GENA–03) [19].
bleeds per year was 4.1, this equates to 2.6 bleeds in the younger children (2–5 years) and 5.6 bleeds in the older children (6–12 years), Fig. 5 [19]. A significant number of the bleeds, especially in older children, were traumatic. There were no major or life-threatening bleeds. Twenty children did not experience any bleeds. The overall mean ABR of spontaneous breakthrough bleeds was 1.5. In terms of tolerability in the 135 PTPs (adults and children), no inhibitors were reported in these studies. In addition, there were no drug-related serious or severe adverse reactions.
The NuProtect study: can inhibitor incidence in PUPs be reduced? (Kate Khair, United Kingdom) The development of inhibitors in children with haemophilia A presents a major clinical problem in terms of both the success and cost of treatment. Inhibitor incidence with currently marketed rFVIII products produced in hamster cells is up to 38% [8]. The reduction of the overall immunological challenge is one of the unmet needs of the global haemophilia community. GCP–PUP studies should be performed for all novel rFVIII products to investigate efficacy and safety in this specific patient population [23]. The PUP study should start prior to marketing authorization and PUPs are excluded from the licensed indication of new products until data from 50 PUPs investigated for efficacy and safety are available and have been examined by the authorities. The Great Ormond Street Hospital for Children is a tertiary referral centre for children and the lead paediatric centre for North London. Guidelines in the UK mean that only rFVIII products are used for newborn babies. In this centre, there are about 10 new severe PUPs per year on average with an inhibitor rate of about 60%, a high number, which may reflect the presence of Sub-Saharan African patients in addition to those of Caucasian ethnicity. There is a neonatal treatment centre and children are Haemophilia (2015), 21 (Suppl. 1), 1--12
referred with haemophilia who may have had an intracranial haemorrhage so babies can be exposed to rFVIII as neonates. From the current studies of the licensed products inhibitor rates are between 28% and 38% with different marketed rFVIII products produced in hamster cell lines [5]. Based on the absence of non-human immunogenic epitopes, as seen in other rFVIII concentrates from hamster cell lines, it is hypothesized that Human-cl rhFVIII may be less immunogenic. This has been tested in NuProtect, a prospective, multicentre, multinational, open-label, non-controlled phase III study in 100 PUPs with severe haemophilia (20 IU FVIII kg 1 body weight with the first bleeding episode. For on-demand treatment, the recommended dose is 20–80 IU FVIII kg 1 body weight depending on the severity of bleeding. There will be a number of optional investigations aiming to identify predictive markers of inhibitor development to accurately identify individuals at risk of inhibitor formation. The optional substudies will look at immunogenotyping (HLA typing, immune response gene profiling, FVIII ethnic haplotype determination) to find out if there is anything that could predict development of FVIII inhibitors in PUPs. RNA expression profiling will also be carried out to look at immune response genes to see if there are © 2014 John Wiley & Sons Ltd
HUMAN-CL rhFVIII FOR HAEMOPHILIA A
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Potential role of the thrombin generation assay (TGA) in personalized prophylaxis (Claude Negrier, France)
Fig. 6. Inhibitor development in PUPs: patients are at highest risk during the first 20 exposure days [8].
any genes that predict the development of inhibitors in children. There is also an optional ex vivo immunogenicity study where blood is taken before the first ED to culture peripheral blood monocytes to examine natural T-cell responses in these children and to assess if that is in any way predictive of inhibitor development. If children do develop an inhibitor then epitope mapping can be carried out to investigate the antibody response/specificity against FVIII. It is known that inhibitor development in PUPs will occur fairly early on, usually within the first 20–50 EDs (Fig. 6 [8]), which is why these children are being screened so intensively. The current enrolment is shown in Fig. 7 – 33 patients have so far been screened, of whom 24 have been treated. The inhibitor rate with the new-generation, artificially enlarged rFVIII products remains unknown. GCP–PUP studies should be performed for all novel rFVIII products to investigate efficacy and safety in this specific patient population.
The thrombin generation assay (TGA) has been the focus of investigations to try and obtain a more personalized prophylaxis. In 2004, Mann et al. took blood samples from 13 healthy controls and measured the production of thrombin every 3 months by these individuals [24]. Results showed that there was very little variability for the same individual in thrombin–antithrombin (TAT) complex levels over a year. However, between subjects there were differences in the TAT levels even in these normal individuals (Fig. 8 [24]). Taking into account the lowest and highest values of normal range of the procoagulant and anticoagulant molecules, respectively, a program can be run to measure thrombin formation. A ‘low healthy’ population group for the production of thrombin can be identified. Then, the opposite is done, the control highest values of the procoagulant molecules and the lowest values of the anticoagulant molecules are taken and the software is run again, resulting in a curve for the ‘high healthy’ population (Fig. 9 [24]).
Fig. 8. Individual thrombin production capacity as assessed by thrombin– antithrombin III (TAT) complex formation for 13 control individuals is shown as a bar graph of the mean standard deviation for 6 months. Subjects 1–13 are labelled below the appropriate bar [24].
Fig. 7. Patient enrolment in NuProtect (status as at May 10, 2014).
© 2014 John Wiley & Sons Ltd
Haemophilia (2015), 21 (Suppl. 1), 1--12
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(B)
Fig. 9. Hypothetical thrombin generation curves (5 pM TF) as functions of FVIII concentration for hypothetical individuals whose clotting factors were at the high or low ends of the healthy population [24].
From the y-axis for the ‘high healthy’ population, it can be seen that this population produces much more thrombin, about twice that of the ‘low healthy’ population. But both these populations are healthy, reflecting the fact that there is variability among individuals in their capacity to produce thrombin. In the haemophilia population, there is a failure of platelet-surface factor X activation, leading to a decrease in platelet-surface thrombin generation and ineffective clot formation [25]. The initiation phase proceeds normally but the propagation phase is absent or significantly decreased due to the deficiency of FVIII or FIX so factor Xa cannot be generated on the platelet surface. With new instruments developed by several manufacturers, there is now the capacity to measure thrombin generation as a function of time. Coagulation is triggered either through the extrinsic or intrinsic pathways and production of thrombin increased up to a maximum (peak height) and then coagulation in the test tube is shut down by the regulators. The individual’s capacity to produce thrombin is measured as a function of time. Different parameters, e.g. the area under the curve (endogenous thrombin potential) and time to tail can be deduced from this type of graph, Fig. 10 [26]. The advantages of prophylaxis are decreased to absent bleeding, minimal to reduced joint damage, greater activities/sports participation, normalization of family and social life, and minimization of school/ work absences. We looked at whether the TGA could be used in the inhibitor population because in this population there is also variability in phenotype: some patients bleed more than others despite the fact that Haemophilia (2015), 21 (Suppl. 1), 1--12
FVIII:C is 5%); D) normal control [29].
In non-inhibitor patients, thrombin generation was demonstrated to be associated to some extent with the clotting activity of the missing factor in the plasma [29] (Fig. 11). Blood samples were taken and thrombin generation was measured in severe, moderate and mild haemophilia populations as well as in normal control subjects. Although there was some overlap between the different categories, if a patient has severe haemophilia with an ETP of 394 186 the concept of prophylaxis is to change him to a category of a patient with moderate or mild haemophilia (Table 3 [29]). For individuals with the same clotting activity in terms of FVIII:C, not all patients have the same capacity to produce thrombin, reflecting differences in the proand anticoagulant molecules. For patients with severe haemophilia 150 EDs) with severe haemophilia A (
DOI: 10.1111/hae.12582
REVIEW ARTICLE
Spotlight on the human factor: building a foundation for the future of haemophilia A management Report from a symposium on human recombinant FVIII at the World Federation of Hemophilia World Congress, Melbourne, Australia on 12 May 2014 C. KESSLER,* J. OLDENBURG,† C. ESCURIOLA ETTINGSHAUSEN,‡ A. TIEDE,§ K. KHAIR,¶ R I E R * * and R . K L A M R O T H † † C . N EG *Division of Hematology and Oncology, The Vincent Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA; †The Institute of Experimental Haematology and Transfusion Medicine and the Haemophilia Centre at the University Clinic, Bonn, Germany; ‡Haemophilia Centre Rhein-Main HZRM, M€ orfelden-Walldorf, Germany; §Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; ¶Great Ormond Street Hospital for Children NHS Trust, London, UK; **Hematology Division, Director Hemophilia Comprehensive Care Center, H^ opital Edouard Herriot Pavillon E, Universite Lyon, Lyon, France; and ††The Haemophilia Treatment Centre, Vivantes Klinikum im Friedrichshain, Berlin, Germany
Summary. Inhibitor development is the most serious and challenging complication in the treatment of severe haemophilia A. Up to 38% of such patients develop inhibitors with current recombinant factor VIII (rFVIII) products produced in hamster cell lines. Human-cl rhFVIII is a new generation fully sulfated B-domaindeleted FVIII coagulant glycoprotein, which is generated from a human cell line. Thus, there are no non-human epitopes which would be potentially immunogenic. This molecule has significantly higher VWF-binding affinity compared with existing fulllength rFVIII produced in hamster cell lines. The development aim of Human-cl rhFVIII is to address the challenges of FVIII inhibitors and frequent infusions during prophylaxis. Human-cl rhFVIII’s mean half-life is very comparable to some of the newer products which involve modification of the FVIII molecule to extend the circulating half-life. There are promising data concerning the use of a personalized prophylaxis
Correspondence: Dr Craig M. Kessler, Division of Hematology and Oncology, The Vincent Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA. Tel.: +1 202 444 8676; fax: +1 202 444 1229; e-mail: [email protected] and Dr Johannes Oldenburg, The Institute of Experimental Haematology and Transfusion Medicine and the Haemophilia Centre at the University Clinic, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Tel.: +49 (0) 228 287 15175; fax: +49 (0) 228 287 14783; e-mail: [email protected] © 2014 John Wiley & Sons Ltd
regimen with Human-cl rhFVIII. Preliminary data indicate a median dosing interval of 3.5 days with 66.7% of the patients on a twice per week or fewer infusions schedule combined with a low bleeding rate and no increased FVIII consumption when compared to standard prophylaxis. No product-specific laboratory assay is required to monitor the coagulation activity for Human-cl rhFVIII. The results of registration clinical trials with Human-cl rhFVIII as well as the ongoing studies in previously untreated patients (NuProtect) and personalized prophylaxis study in previously treated patients (NuPreviq), will be discussed. The manufacturer has received marketing authorization for Human-cl rhFVIII in Europe and Canada under the name Nuwiqâ and plans to launch it in the USA and globally in 2015. Keywords: assays, clinical trials, haemophilia A, Human-cl rhFVIII, inhibitors, personalized prophylaxis
Introduction (Craig Kessler, United States) For the last 20 years, haemophilia A patients have been treated with recombinant factor VIII (rFVIII) concentrates which have all been produced in hamster derived cell lines. We now have on the horizon an opportunity to use a recombinant B-domain-deleted FVIII concentrate that has been produced in human embryonic kidney cells. This represents a major advance in the manufacture of genetically engineered clotting factor replacement products. Such a product might be useful in the armamentarium of treatment for patients with 1
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haemophilia A as it has the potential to address some of the unmet needs in the care of these patients. The major unmet need is the development of replacement products which have lower potential to induce the development of alloantibody inhibitors against FVIII in previously untreated patients (PUPs) [1]. Furthermore, the development of alloantibody inhibitors throughout the lifetime in previously treated patients (PTPs) with haemophilia is becoming much more apparent, as a result of an increased number of surgeries being performed in patients with mild and moderate haemophilia, who concurrently receive large amounts of replacement therapy, perhaps within the context of ‘triggers’ of inflammation and stress to the immune system. There is also a rising incidence of inhibitors in older patients with severe haemophilia A [1]. The benefits of prophylaxis strategies, particularly started early, in patients with severe haemophilia have been well demonstrated [2,3]. Successful prophylaxis regimens will enhance the quality of our patients’ lives and will allow them to be able to lead much more normal physically active lives. However, a ‘one-sizefits-all’ strategy for prophylaxis may pose an unnecessary bleeding risk or theoretically a hypercoagulable thrombotic risk. Differences in intra- and inter-individual FVIII half-lives suggest that tailored dosing approaches would be desirable to ensure a high quality of life and can be adjusted to meet the aspirational activity goals of the individual. There remain some concerns with the future and recently USA FDA-approved treatments for haemophilia A, including the incidence of inhibitor formation, which the new generation of manipulated and posttranslationally modified rFVIII molecules may potentially induce. Furthermore, physicians will need to learn how to monitor their in vivo haemostatic activities since some of the new products appear to require product-specific laboratory assay systems. These points were discussed in more detail at a symposium on human recombinant FVIII at the World Federation of Hemophilia World Congress, Melbourne, Australia on 12 May 2014 and a report of the meeting is presented below.
Inhibitors in haemophilia: perspectives and challenges (Carmen Escuriola Ettingshausen, Germany) Inhibitor development is the most serious and very challenging complication in the treatment of patients with haemophilia [1,4]. Around 35% of severe haemophilia A patients and around 5% of severe haemophilia B patients develop inhibitors [5,6]. Characteristically, this complication occurs during the initial phase of FVIII exposure, meaning that it usually occurs in very small children during the first 20 expoHaemophilia (2015), 21 (Suppl. 1), 1--12
sure days (EDs). It is associated with a reduction of efficacy of haemostatic therapy with FVIII and increased morbidity, particularly in patients with high responding inhibitors (>5 BU). In addition to the fact that these patients quickly become disabled, there are high treatment costs to control the bleeding episodes.
Risk factors for the development of inhibitors There are genetic endogenous host risk factors that support the development of inhibitors, i.e. there are different underlying gene defects for haemophilia that are associated with a different risk of inhibitor development. While the underlying F8 or F9 gene mutation is a significant predictor of inhibitor development, there are also less strong risk factors, including replacement product immunogenicity, the human leucocyte antigen (HLA) type, and the ethnic origin of the patient [7]. Exogenous/environmental risk factors, which predispose to the development of alloantibody inhibitors, are also very challenging and include age at first exposure to FVIII, the therapeutic regimen employed (prophylaxis or on-demand treatment – patients treated prophylactically have a lower inhibitor incidence than those treated on-demand) and the intensity of treatment. From the CANAL [8] and RODIN [5] studies it was shown that surgical procedures as well as peak treatment levels are extremely important and put the patient at risk of developing an inhibitor. The type of FVIII concentrate used has also been a topic of great debate [9]. The large RODIN study seemed to indicate that there was no difference concerning inhibitor development between plasma-derived (pd) and rFVIII concentrates although the results of a prospective randomized study, the SIPPET study, which investigates inhibitor development with the use of pdFVIII or rFVIII in PUPs have not yet been published [10]. With regard to the differences that might account for the reason that pdFVIII products could be associated with a lower inhibitor incidence, pd products contain a considerable amount of von Willebrand factor (VWF). VWF may mask B-cell epitopes on the light chain of FVIII [11]. VWF prevents internalization of FVIII by bone marrow dendritic cells and hinders antigen recognition of FVIII [12]. rFVIII-treated and pdFVIII-treated mice have different subsets of CD4+ T–cells [13], which suggests that these mice might produce different amounts of anti-FVIII antibodies. In pdFVIII-treated mice, the amount of antibodies produced might be lower. To compare pdFVIII and rFVIII concentrates, the impact of the posttranslational modifications (PTMs) of these FVIII molecules should be considered. PTMs are the biochemical modifications of a protein after its translation from DNA to amino acid sequence. All human plasma proteins undergo PTMs. Different © 2014 John Wiley & Sons Ltd
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expression systems (e.g. bacterial, yeast, fungal, plant, insect, hamster and human) produce different PTMs for the same protein sequence. For rFVIII, the PTMs such as glycosylation and sulfation of Tyr1680 have been shown to be vital for functionality and VWF-binding affinity. In the absence of tyrosine sulfation at Y1680 in rFVIII, the affinity for VWF is reduced five-fold [14,15]. Octanateâ is a human pdFVIII naturally stabilized with VWF and is a fully sulfated protein. The ratio of FVIII to VWF in Octanateâ is in the order of 0.4 with a mean specific activity of >100 IU FVIII:C/mg total protein; albumin is not added as a stabilizer. The product undergoes double virus inactivation by solvent/detergent and terminal dry-heat treatment to inactivate both enveloped and non-enveloped viruses. Octanate has been on the market for 16 years and, therefore, there is a large clinical experience. The ongoing Good Clinical Practice (GCP)–PUP study with Octanate has recruited 51 predominantly severe haemophiliacs. With regard to genetic risk factors, a total of 70.6% of the patients had high-risk mutations, most commonly intron 22 mutations (51%), whereas a minority had low-risk mutations such as missense (18%) or splice site mutations (4%) [16]. Patients were first exposed to Octanate at the age of around 1 year. There was a family history of haemophilia A and inhibitors in three patients (6%) [16]. Of the 51 patients, 14 were treated on-demand for ≥50 EDs, 15 were treated on-demand for ≥20 EDs before being switched to prophylactic administration, 20 were treated exclusively prophylactically (i.e. during the high-risk phase) and two patients were treated on-demand for 5 BU); however, in two cases, these inhibitors were transient and disappeared under regular treatment with FVIII. All inhibitors developed in patients treated on an on-demand basis; no inhibitor developed in patients undergoing prophylaxis. The overall inhibitor rate at the current time, including the two transient inhibitors, is 9.8% (five of 51 patients) and the incidence of clinically relevant inhibitors is 5.9% (three of 51 patients). In an ongoing international ITI study (Observational Immune Tolerance Induction, ObsITI), Octanate provided an ITI success rate of >70% in patients with poor prognosis factors [17]. No cases of haemolysis and no accumulation of VWF during high-dose ITI have been reported [18].
Human-cl rhFVIII Glycosylation alters the structural and functional properties of a protein and is responsible for physicochemical properties, immunological properties, receptor binding/affinity and intracellular sorting. A recent study by Kannicht et al. to characterize PTMs of Human-cl rhFVIII (simoctocog alfa) showed the product to be glycosylated with complex-, hybrid- and high mannose-type glycosylation present at the same sites as in human pdFVIII [15]. In addition, as a consequence of the expression in a human cell line, it is devoid of the non-human antigenic carbohydrate epitopes, in particular Gala1-3Galb1-GlcNAc-R (a-Gal) and N-glycolylneuraminic acid (Neu5Gc), Fig. 1 [15,19–21]. These residues are typically found in rFVIII molecules produced in rodent cells, such as Chinese hamster ovarian (CHO) cells and baby hamster kidney (BHK) cells, Table 1 [15].
Fig. 1. N-glycosylation: carbohydrate epitopes on rFVIII molecules produced in human embryonic kidney (HEK), CHO and BHK cell lines [15,19–21].
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Table 1. Terminal structures of N-glycans of human serum glycoproteins in comparison with plasma-derived and recombinant FVIII [15]. Core structure
Terminal structure
GlcNAc2-Man3 core Fuc
High mannose GlcNAc-Gal type 1 or 2 a-2-3/a-2,6 linked sialyl type 1 or 2 Antennae fucosylation, e.g. LeX or LeA or LDN(F) Blood group ABO Neu5Gc Gala1-3Gal
Human plasma proteins
Human pdFVIII (e.g. Octanateâ)
+ + +/+ +
+ + +/+ +
+
+
Human-cl rhFVIII + + +/+ +
CHO or BHK-derived rFVIII + + +/ +
+ (CHO) + (BHK)
Fig. 2. Extracted ion chromatograms showing the sulfated (major peak) and non-sulfated (minor or absent peak) Y1680-containing peptides [15].
Human-cl rhFVIII is a fully sulfated human protein (Fig. 2 [15]). It has significantly higher VWF-binding affinity compared with existing full-length rFVIII produced in hamster cell lines. In a study extracted ion chromatograms showed the sulfated and non-sulfated (minor or absent peak) Tyr1680-containing peptides. For Octanate and Human-cl rhFVIII, there was only a sulfated peak, whereas for full-length rFVIII (CHO), full-length rFVIII (BHK) and B-domain-deleted rFVIII (CHO), there was both a sulfated peak and a minor non-sulfated peak [15] in rFVIII molecules expressed by rodent cells. With regard to the importance of the binding activity to VWF, two other experiments have been performed comparing the human cell line rFVIII with FVIII concentrate produced by rodent cells: these were the SPR-based affinity measurements and binding to VWF–Sepharose to determine free rFVIII. In the first experiment, the dissociation constant was lower in the Human-cl rhFVIII (193 36 pM) compared to the rodent cell line derived FVIII (full-length rFVIII [CHO] 320 39, full-length rFVIII [BHK] 342 35, B-domain-derived rFVIII [CHO] 230 31), meaning that there is a higher binding affinity for VWF [22]. The second experiment showed a significantly higher amount of unbound FVIII for products expressed by the rodent cell lines compared to Human-cl rhFVIII [22]. Thus, both experiments showed a significantly Haemophilia (2015), 21 (Suppl. 1), 1--12
higher VWF-binding affinity for Human-cl rhFVIII than the comparator rFVIII proteins that were tested. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed
Fig. 3. Human-cl rhFVIII (BDD rFVIII): comparable FVIII activities in one-stage* (OS) and chromogenic** (CS) release assays. (*BCSâXP using Dade FVIII-deficient plasma and Actinâ; **Coatest SPâ FVIII kit) [22].
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to lower the intrinsic immunogenicity of Human-cl rhFVIII. It is also extremely important that if these agents are to be used in the clinical setting, they should be suitable for all FVIII determination assays. A study was conducted with 17 batches of Human-cl rhFVIII which were tested using the one-stage assay as well as the chromogenic assay. The results were quite comparable (Fig. 3 [22]) so it can be concluded that both types of FVIII activity tests can be used for Human-cl rhFVIII.
The new Human-cl rhFVIII: overview of clinical trial experience in children and adults with haemophilia A (Andreas Tiede, Germany) Switching the cell line and having a slightly different B-domain-deleted FVIII molecule led to the development of a new drug with the generic name simoctocog alfa (Human-cl rhFVIII). The vision with this new product was to try and reduce inhibitor rates with a rFVIII concentrate that in terms of glycosylation might be more similar to the natural human FVIII. Three pivotal studies with Human-cl rhFVIII in PTPs have reported results: GENA–01 (ondemand) in adults and adolescents looking at pharmacokinetics, efficacy, safety and immunogenicity, GENA–08 (prophylaxis) in adults and adolescents looking at efficacy, safety and immunogenicity, and GENA–03 (prophylaxis) in children looking at pharmacokinetics, efficacy, safety and immunogenicity (Table 2).
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Looking first at the efficacy of on-demand treatment in GENA–01, in 94.4% of cases treatment of bleeding episodes (986 bleeding episodes in total) was rated excellent or good. A total of 96.7% of all bleeds were managed with one (90.3%) or two (6.4%) infusions. The median dose per infusion was 30.0 IU kg 1 and the median dose per bleeding episode was 30.9 IU kg 1. Results for the prophylactic study (GENA–08) showed that there was no major or life-threatening bleeding and 50% of patients did not experience any bleeding episodes. In 34.4%, there was one bleeding episode, which in most cases was minor and in 15.6% of cases there were five or more bleeding episodes. The mean annualized bleeding rate (ABR) was 2.28 3.73 bleeds per year, Fig. 4 [19]. The GENA–01 and GENA–08 studies were two very similar cohorts with an identical way of collecting the data. This shows that prophylaxis also prevents bleeds in adults: there was a 96% reduction in mean ABR with prophylaxis versus on-demand treatment. Increasingly, there are more and more studies showing that prophylaxis in adults can prevent bleeds.
Paediatric study In the paediatric study (GENA–03), the mean age was 6.1 2.97 years, all children were white and in 53 of the 59 patients prophylaxis had been given prior to the study. The main efficacy outcome was breakthrough bleeds during prophylactic treatment. In all patients pooled together, the number of breakthrough
Table 2. Clinical trials with Human-cl rhFVIII that have reported results. Adults and adolescents GENA–01 (on-demand) Pharmacokinetics Efficacy, safety and immunogenicity Treatment
✔ ✔
Development phase Number of centres Number of countries
II 9 3 (USA, Germany, Bulgaria)
30–40 IU kg 1 every other day/treatment of breakthrough bleeds for ≥6 months and ≥50 EDs III 11 4 (Austria, Bulgaria, Germany, UK)
Number of PTPs Coordinating investigator
22 Marilyn J. Manco-Johnson, USA
32 Johannes Oldenburg, Germany
≥6 months and ≥50 EDs
Children 2–12 years GENA–03 (prophylaxis)
GENA–08 (prophylaxis) ✔
✔ ✔ 30–40 IU kg 1 every other day or three times per week for ≥6 months and ≥50 EDs III 15 7 (UK, Poland, France, Russia, Turkey, Romania, Czech Republic) 59 Raina J. Liesner, UK
Fig. 4. Mean annualized bleeding rate per regimen with Human-cl rhFVIII: on-demand versus prophylaxis. Adult patients on prophylactic regimens experienced significantly fewer bleeding episodes than those using on-demand treatment [19].
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Fig. 5. Mean annualized bleeding rate (ABR) in children undergoing prophylaxis (GENA–03) [19].
bleeds per year was 4.1, this equates to 2.6 bleeds in the younger children (2–5 years) and 5.6 bleeds in the older children (6–12 years), Fig. 5 [19]. A significant number of the bleeds, especially in older children, were traumatic. There were no major or life-threatening bleeds. Twenty children did not experience any bleeds. The overall mean ABR of spontaneous breakthrough bleeds was 1.5. In terms of tolerability in the 135 PTPs (adults and children), no inhibitors were reported in these studies. In addition, there were no drug-related serious or severe adverse reactions.
The NuProtect study: can inhibitor incidence in PUPs be reduced? (Kate Khair, United Kingdom) The development of inhibitors in children with haemophilia A presents a major clinical problem in terms of both the success and cost of treatment. Inhibitor incidence with currently marketed rFVIII products produced in hamster cells is up to 38% [8]. The reduction of the overall immunological challenge is one of the unmet needs of the global haemophilia community. GCP–PUP studies should be performed for all novel rFVIII products to investigate efficacy and safety in this specific patient population [23]. The PUP study should start prior to marketing authorization and PUPs are excluded from the licensed indication of new products until data from 50 PUPs investigated for efficacy and safety are available and have been examined by the authorities. The Great Ormond Street Hospital for Children is a tertiary referral centre for children and the lead paediatric centre for North London. Guidelines in the UK mean that only rFVIII products are used for newborn babies. In this centre, there are about 10 new severe PUPs per year on average with an inhibitor rate of about 60%, a high number, which may reflect the presence of Sub-Saharan African patients in addition to those of Caucasian ethnicity. There is a neonatal treatment centre and children are Haemophilia (2015), 21 (Suppl. 1), 1--12
referred with haemophilia who may have had an intracranial haemorrhage so babies can be exposed to rFVIII as neonates. From the current studies of the licensed products inhibitor rates are between 28% and 38% with different marketed rFVIII products produced in hamster cell lines [5]. Based on the absence of non-human immunogenic epitopes, as seen in other rFVIII concentrates from hamster cell lines, it is hypothesized that Human-cl rhFVIII may be less immunogenic. This has been tested in NuProtect, a prospective, multicentre, multinational, open-label, non-controlled phase III study in 100 PUPs with severe haemophilia (20 IU FVIII kg 1 body weight with the first bleeding episode. For on-demand treatment, the recommended dose is 20–80 IU FVIII kg 1 body weight depending on the severity of bleeding. There will be a number of optional investigations aiming to identify predictive markers of inhibitor development to accurately identify individuals at risk of inhibitor formation. The optional substudies will look at immunogenotyping (HLA typing, immune response gene profiling, FVIII ethnic haplotype determination) to find out if there is anything that could predict development of FVIII inhibitors in PUPs. RNA expression profiling will also be carried out to look at immune response genes to see if there are © 2014 John Wiley & Sons Ltd
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Potential role of the thrombin generation assay (TGA) in personalized prophylaxis (Claude Negrier, France)
Fig. 6. Inhibitor development in PUPs: patients are at highest risk during the first 20 exposure days [8].
any genes that predict the development of inhibitors in children. There is also an optional ex vivo immunogenicity study where blood is taken before the first ED to culture peripheral blood monocytes to examine natural T-cell responses in these children and to assess if that is in any way predictive of inhibitor development. If children do develop an inhibitor then epitope mapping can be carried out to investigate the antibody response/specificity against FVIII. It is known that inhibitor development in PUPs will occur fairly early on, usually within the first 20–50 EDs (Fig. 6 [8]), which is why these children are being screened so intensively. The current enrolment is shown in Fig. 7 – 33 patients have so far been screened, of whom 24 have been treated. The inhibitor rate with the new-generation, artificially enlarged rFVIII products remains unknown. GCP–PUP studies should be performed for all novel rFVIII products to investigate efficacy and safety in this specific patient population.
The thrombin generation assay (TGA) has been the focus of investigations to try and obtain a more personalized prophylaxis. In 2004, Mann et al. took blood samples from 13 healthy controls and measured the production of thrombin every 3 months by these individuals [24]. Results showed that there was very little variability for the same individual in thrombin–antithrombin (TAT) complex levels over a year. However, between subjects there were differences in the TAT levels even in these normal individuals (Fig. 8 [24]). Taking into account the lowest and highest values of normal range of the procoagulant and anticoagulant molecules, respectively, a program can be run to measure thrombin formation. A ‘low healthy’ population group for the production of thrombin can be identified. Then, the opposite is done, the control highest values of the procoagulant molecules and the lowest values of the anticoagulant molecules are taken and the software is run again, resulting in a curve for the ‘high healthy’ population (Fig. 9 [24]).
Fig. 8. Individual thrombin production capacity as assessed by thrombin– antithrombin III (TAT) complex formation for 13 control individuals is shown as a bar graph of the mean standard deviation for 6 months. Subjects 1–13 are labelled below the appropriate bar [24].
Fig. 7. Patient enrolment in NuProtect (status as at May 10, 2014).
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(B)
Fig. 9. Hypothetical thrombin generation curves (5 pM TF) as functions of FVIII concentration for hypothetical individuals whose clotting factors were at the high or low ends of the healthy population [24].
From the y-axis for the ‘high healthy’ population, it can be seen that this population produces much more thrombin, about twice that of the ‘low healthy’ population. But both these populations are healthy, reflecting the fact that there is variability among individuals in their capacity to produce thrombin. In the haemophilia population, there is a failure of platelet-surface factor X activation, leading to a decrease in platelet-surface thrombin generation and ineffective clot formation [25]. The initiation phase proceeds normally but the propagation phase is absent or significantly decreased due to the deficiency of FVIII or FIX so factor Xa cannot be generated on the platelet surface. With new instruments developed by several manufacturers, there is now the capacity to measure thrombin generation as a function of time. Coagulation is triggered either through the extrinsic or intrinsic pathways and production of thrombin increased up to a maximum (peak height) and then coagulation in the test tube is shut down by the regulators. The individual’s capacity to produce thrombin is measured as a function of time. Different parameters, e.g. the area under the curve (endogenous thrombin potential) and time to tail can be deduced from this type of graph, Fig. 10 [26]. The advantages of prophylaxis are decreased to absent bleeding, minimal to reduced joint damage, greater activities/sports participation, normalization of family and social life, and minimization of school/ work absences. We looked at whether the TGA could be used in the inhibitor population because in this population there is also variability in phenotype: some patients bleed more than others despite the fact that Haemophilia (2015), 21 (Suppl. 1), 1--12
FVIII:C is 5%); D) normal control [29].
In non-inhibitor patients, thrombin generation was demonstrated to be associated to some extent with the clotting activity of the missing factor in the plasma [29] (Fig. 11). Blood samples were taken and thrombin generation was measured in severe, moderate and mild haemophilia populations as well as in normal control subjects. Although there was some overlap between the different categories, if a patient has severe haemophilia with an ETP of 394 186 the concept of prophylaxis is to change him to a category of a patient with moderate or mild haemophilia (Table 3 [29]). For individuals with the same clotting activity in terms of FVIII:C, not all patients have the same capacity to produce thrombin, reflecting differences in the proand anticoagulant molecules. For patients with severe haemophilia 150 EDs) with severe haemophilia A (
