Individual thrombin generation and spontaneous bleeding rate during personalized prophylaxis with Nuwiq® (human-cl rhFVIII) in previously treated patients with severe haemophilia A.

INTRODUCTION: For individuals with haemophilia A, prophylaxis with factor VIII (FVIII) is typically directed towards trough activity >1 IU/dL; however, some patients still experience spontaneous bleeding events (sBEs). AIM: Aims were to evaluate relationships of endogenous thrombin potential (ETP) and FVIII:C with occurrence of clinical bleeding. METHODS: GENA-21 was a prospective, open-label, phase IIIb study investigating the safety and efficacy of Nuwiq® (human-cl rhFVIII) in previously treated adults with severe haemophilia A. The study included a 72-hour pharmacokinetic (PK) evaluation phase and a 6-month personalized prophylaxis phase in which treatment was guided by PK parameters. This subanalysis assessed FVIII:C by one-stage assay and ETP by thrombin generation assay in blood samples. RESULTS: Baseline mean ETP was lower in the 7 patients who experienced sBEs during personalized prophylaxis versus 25 who did not (n = 32 with data from PK phase and prophylaxis phase; P = .0002). During personalized prophylaxis (n = 49), only patients with lower median trough ETP experienced sBEs (8/49 patients; ROC AUC = 0.9421; P < .0001); there was no significant relationship for FVIII:C in predicting sBEs (ROC AUC = 0.5838; P = .4750). Directly following infusion of human-cl rhFVIII, ETP was lower in patients who experienced sBEs versus those who did not (P = .0002), whereas FVIII:C did not differ significantly between these groups. CONCLUSIONS: In adults with severe haemophilia A and reduced thrombin generation, increased frequency of spontaneous bleeding was observed irrespective of trough FVIII levels. Thus, personalized prophylaxis should take into account variables other than FVIII:C. Large prospective trials are needed to verify ETP as a marker for spontaneous bleeding.

Postauthorization safety study of Clottafact® , a triply secured fibrinogen concentrate in acquired fibrinogen deficiency: a prospective observational study.

BACKGROUND AND OBJECTIVES: A postauthorization safety study was performed between 2009 and 2012 to describe the use of Clottafact® in acquired fibrinogen deficiency in real-life medical practice in France. MATERIALS AND METHODS: One hundred and fifty patients were planned for 28 days of prospective follow-up after infusion. The analysis of this observational study was descriptive and performed according to the type of treatment (curative or preventive) and the origin of the bleed. RESULTS: One hundred and fifty-six patients (16-87 years) were included in 13 centres and treated in five different medical bleeding situations: postpartum (59), other gynaecological/obstetrical (6), trauma (34), liver (13), cardiovascular (23) and other various bleeding situations (21). The mean follow-up time was 18·9 ± 12·3 days. Two patients presented adverse drug reactions: one a pulmonary embolism and the other a four-site venous thromboembolic episode. All were serious with a dubious causal relationship with the study treatment. Efficacy data were collected as a secondary objective. In 150 patients receiving curative treatment, 117 of 159 infusions (73·6%) were considered as successful by the investigators, 35 as moderate (22%) and seven as no response (4·4%). CONCLUSION: The Clottafact® safety profile observed during the study matched the known profile of fibrinogen during use.

First report on the safety and efficacy of an extended half-life glycoPEGylated recombinant FVIII for major surgery in severe haemophilia A.

BACKGROUND: N8-GP (turoctocog alfa pegol) is an extended half-life glycoPEGylated recombinant factor VIII (FVIII) product developed for the prevention and treatment of bleeds in haemophilia A patients. AIM: This is a planned interim analysis of pathfinder™3, an international, open-label, Phase 3 trial evaluating the efficacy and safety (including immunogenicity) of N8-GP administered before, during and after major surgery in severe haemophilia A patients aged ≥12 years. METHODS: Sixteen patients who underwent 18 major surgical procedures (including synovectomy, joint replacement and ankle arthrodesis) were included here. Postoperative assessments were conducted daily for days 1-6, and once for days 7-14. Primary endpoint was N8-GP haemostatic efficacy, assessed after completion of surgery using a four-point scale ('excellent', 'good', 'moderate', 'none'). RESULTS: Haemostasis was successful (rated 'excellent' or 'good') on completion of surgery in 17 (94.4%) procedures and rated as 'moderate' (5.6%) for one surgery in a patient with multiple comorbidities who needed an intraoperative N8-GP dose (20.7 IU kg-1 ). In the postoperative period, three bleeds occurred (one during days 1-6; two during days 7-14); all were successfully treated with N8-GP. Mean N8-GP consumption on day of surgery was 80.0 IU kg-1 ; patients received a mean of 1.7 doses (median: 2, range: 1-3). No safety concerns were identified. CONCLUSION: The data showed that N8-GP was effective and well tolerated for the prevention and treatment of bleeds during major surgery; such FVIII products with extended half-lives may modify current treatment schedules, enabling fewer infusions and earlier patient discharge.

Molecular cytogenetic characterization of five F8 complex rearrangements: utility for haemophilia A genetic counselling.

BACKGROUND: Genomic inversions are usually balanced, but unusual patterns have been described in haemophilia A (HA) patients for intron 22 (Inv22) and intron 1 (Inv1) inversions leading to the hypothesis of more complex rearrangements involving deletions or duplications. AIM: To characterize five abnormal patterns either in Southern blot and long-range PCR for Inv22 or in PCR for Inv1. MATERIALS AND METHODS: All patients were studied using cytogenetic microarray analysis (CMA). RESULTS: In all cases, CMA analysis found that each inversion was associated with complex Xq28 rearrangement. In three patients, CMA analysis showed large duplication ranging from 230 to 1302 kb and encompassing a various number of contiguous genes among which RAB39B. RAB39B duplication is a strong candidate gene for X-linked intellectual disability (XLID). Surprisingly, none of the severe HA patients with RAB39B duplication reported in this study or in the literature exhibited XLID. We hypothesise that F8 complex rearrangement down regulated RAB39B expression. In the two remaining patients, CMA analysis found Xq28 large deletion (from 285 to 522 kb). Moyamoya syndrome was strongly suspected in one of them who carried BRCC3 deletion. CONCLUSION: Because several F8 neighbouring genes are associated with other pathologies such as XLID and cardiovascular disease, all HA patients where complex Xq28 rearrangement was suspected should be referred to a geneticist for possible utility of a pangenomic study. Such investigation should be carefully considered in genetic counselling in female carriers to assess the risk of transmitting severe HA with a "contiguous gene syndrome".

Pharmacokinetics of a novel extended half-life glycoPEGylated factor IX, nonacog beta pegol (N9-GP) in previously treated patients with haemophilia B: results from two phase 3 clinical trials.

INTRODUCTION: Nonacog beta pegol (N9-GP) is a glycoPEGylated recombinant factor IX (FIX) with an extended half-life developed for routine prophylaxis and the prevention and treatment of bleeding episodes in patients with haemophilia B. AIM: The aim of this study was to evaluate the pharmacokinetics (PK) of N9-GP. METHODS: Data from 41 previously treated haemophilia B patients, enrolled globally (16 adolescents/adults and 25 children; FIX activity ≤0.02 IU mL-1 ) with no history of FIX inhibitors, were included. N9-GP was administered once-weekly as 10 IU kg-1 or 40 IU kg-1 in adolescents/adults and 40 IU kg-1 in children. Blood was sampled up to 168 h (1 week) post dose. Standard PK was estimated on the basis of plasma FIX activity vs. time (PK profiles) using non-compartmental methods. Furthermore, a population PK analysis and FIX activity predictions were performed. RESULTS: Incremental recoveries were 0.02 (IU mL-1 )/(IU kg-1 ) in both adolescents/adults and children. The extended half-life resulted in mean trough levels of 0.27 IU mL-1 for adolescents/adults and 0.17 IU mL-1 for children at steady-state after weekly dosing at 40 IU kg-1 . The population PK analysis confirmed a mono-exponential decay in FIX activity and allowed for predictions of FIX activity for adolescents/adults above 0.15 IU mL-1 at all times and 6.4 days week-1 in children. CONCLUSION: N9-GP has the potential to shift previously treated haemophilia B patients from a severe/moderate disease state into a mild- or non-haemophilic range for most of the dosing interval, which is expected to reduce the number of bleeding episodes.

Target plasma factor levels for personalized treatment in haemophilia: a Delphi consensus statement.

BACKGROUND: Prophylactic replacement with factor concentrate is the optimal treatment for persons with severe haemophilia to avoid or minimize bleeding. This ultimately prevents or reduces joint disease and improves life expectancy and quality of life towards values matching those in the normal population. However, uncertainty still exists around the optimal regimens to be prescribed for prophylaxis. An increasing number of treating physicians and patients are showing interest in patient-tailored approaches to prophylaxis, which aim to harmonize the prophylaxis regimen with the patients' bleeding phenotype, levels of physical activity and a variety of other variables. METHODS: A modified Delphi technique was adopted to generate consensus. The expert panel met in person to set the objectives, be trained on the Delphi technique and agree on the desired level of consensus. Three iterations were used to identify the targets, the scenarios and their combinations. RESULTS: Twenty-eight scenarios and eight target levels were identified and used to issue recommendations. The panel reached the desired level of consensus on positive or negative recommendations. Areas where consensus was not reached were identified and proposed as areas for future research. Prospective assessment of the validity of most of the proposed targets is recommended. CONCLUSIONS: We have generated, by expert consensus, target plasma levels of factor concentrate to be used to tailor treatment for persons with haemophilia.

Dosing regimens, FVIII levels and estimated haemostatic protection with special focus on rFVIIIFc.

AIM: To use Pharmacokinetic (PK) simulations to illustrate potential differences in clinical outcomes between prophylaxis with conventional recombinant factor VIII (rFVIII) and rFVIIIFc, an extended half-life rFVIII covalently fused to the Fc domain of human IgG1. METHODS: Population PK estimates from 180 (rFVIIIFc) and 46 (rFVIII) severe haemophilia A patients were used to simulate FVIII activity over time at various rFVIIIFc dosing regimens compared to rFVIII 30 IU kg(-1) three times weekly in a typical adult patient. RESULTS: rFVIII dosed 3x30 IU kg(-1) weekly gave trough levels of 2.7, 2.8 and 0.7 IU dL(-1) , and time spent below 1, 3 and 5 IU dL(-1) of 0.2/1.2/2.3 days week(-1) . rFVIIIFc 2 x 45 IU kg(-1) gave higher troughs (4.4 and 1.7 IU dL(-1) ) and shorter time spent below 1, 3 and 5 IU dL(-1) (0/0.6/1.3 days week(-1) ), with same total factor consumption. rFVIIIFc 2 x 30 IU kg(-1) gave similar troughs (3.0 and 1.2 IU kg(-1) ) and time spent below 1, 3 and 5 IU dL(-1) (0/1.0/2.1 days week(-1) ), despite total factor consumption being reduced by one-third. The same dose and interval of rFVIIIFc (3 x 30 IU kg(-1) ) gave substantially higher troughs (7.8, 8.5 and 3.3 IU dL(-1) ) and markedly shorter time spent below 1, 3 and 5 IU dL(-1) (0/0/0.4 days week(-1) ). CONCLUSION: The lower clearance of rFVIIIFc compared to conventional rFVIII gives rFVIIIFc the potential of improved bleed prevention and reduced injection frequency at similar factor consumption. Although additional clinical data are required to confirm the conclusions, the simulations clearly show the potential of rFVIIIFc of increased flexibility to tailor treatment to the individual patient, and to advance the standard of care in haemophilia.

Recombinant long-acting glycoPEGylated factor IX (nonacog beta pegol) in haemophilia B: assessment of target joints in multinational phase 3 clinical trials.

BACKGROUND: The paradigm(™) 2 and 4 phase 3 clinical trials investigated the safety and efficacy of nonacog beta pegol, a recombinant glycoPEGylated factor IX (FIX) with extended half-life, in previously treated haemophilia B patients. AIM: These post hoc analyses investigated the bleeding patterns in target joints. METHODS: Patients randomized to 40 or 10 IU kg(-1) once weekly prophylaxis who had at least one target joint were included. Baseline demographics and disease-specific data were collected. Bleeding patterns were assessed, and an International Society on Thrombosis and Haemostasis (ISTH) definition of target joints was used. RESULTS: A total of 67% and 8% of patients in the 40 and 10 IU kg(-1) arm, respectively, did not experience target joint bleeds during the paradigm(™) 2 trial. Twenty-four target joints were recorded in each prophylaxis arm at baseline. During the paradigm(™) 2 trial, no bleeds were reported in 17 (71%) and 7 (29%) target joints in the 40 and 10 IU kg(-1) arms respectively. All target joint bleeds in the 40 IU kg(-1) once weekly prophylaxis arm were controlled with a single injection of 40 IU kg(-1) nonacog beta pegol. By the latest ISTH definition, 90% and 58% of target joints in the 40 and 10 IU kg(-1) arms, respectively, were no longer considered target joints at the end of the paradigm(™) 2 trial. At the end of the paradigm(™) 4 extension trial, all target joints in the 40 IU kg(-1) arm were no longer considered target joints. CONCLUSION: Routine prophylaxis with 40 IU kg(-1) once weekly nonacog beta pegol has the potential for effective management of target joint bleeds in haemophilia B patients.

Characterization of five associations of F8 missense mutations containing FVIII B domain mutations.

INTRODUCTION: Haemophilia A (HA) is a bleeding disorder due to an absence or a reduced activity of coagulation factor VIII (FVIII) caused by mutations in F8 gene. Missense mutations represent approximately 45% of the reported molecular defects in HA. However, only few missense mutations in FVIII B domain have been described. AIM: The aim of this study was to characterize five genetic variations (three novels and two previously reported) localized in the FVIII B domain. In all cases, an additional missense variation located outside the FVIII B domain was found. We investigated each of these variations separately and in combination too for their contribution to HA phenotype. METHODS: F8 variants were transiently expressed in COS-1 cells. Media and cell lysates were collected after 72 h. Then, FVIII activity, secretion and thermostability were analysed and compared to FVIII wild-type. RESULTS: The 5 FVIII B domain variants showed normal FVIII: C (98.5-128.5%) and FVIII: Ag (97.7-154%). No synergistic effect was observed between the B domain variant and their associated mutations. In contrast, the variants located outside the B domain, p.V682L, p.S714L, p.V592D and p.C573F revealed significantly decrease of FVIII: C with values in the range 3.5-44.5% (p < 0.05). However, the p.G224R variant showed FVIII: C and FVIII: Ag values no significantly different from FVIII-WT. CONCLUSION: The FVIII B domain variants, p.D963N, p.S806T, p.G873D, p.H998Q and p.Q1225R may be considered as polymorphism or non-pathologic mutations. In five patients, clinical phenotype could be explained by the additional causative missense mutation. For the p.G224T variant further splicing studies are necessary to determine its pathogenicity.

The association of haemophilic arthropathy with Health-Related Quality of Life: a post hoc analysis.

BACKGROUND: The aim of replacement therapy in haemophilia is to improve Health-Related Quality of Life (HRQoL) by preventing bleeding and arthropathy. However, the association of arthropathy with HRQoL is unknown. AIM: To explore the association of haemophilic arthropathy with HRQoL. METHODS: A post hoc analysis on patients with severe/moderate haemophilia with SF36 questionnaire (SF36) and X-rays of ankles, knees and elbows made within 2.5-years. The SF36 scores of 'physical functioning' (SF36-PF, range 0-100, optimum 100) and Utility (SF6D-Utility, range 0-1, optimum 1) and radiological Pettersson scores (PS, range 0-78, optimum 0) were calculated. The association of PS with reduced SF6D-Utility and SF36-PF ( 21 points, the risk of reduced SF6D-Utility was stable (OR 4.16; 95% CI: 2.03-8.51) but SF36-PF continued to decrease: compared to lowest PS, OR for reduced SF36-PF was 5.69 (95% CI: 1.62-20.06) for PS 22-39 and 25.15 (95% CI: 6.53-96.81) for PS 40-78. CONCLUSION: Health-Related Quality of Life only showed a significant deterioration in patients with a Pettersson score of >21 points. This suggests that HRQoL is relatively insensitive to early joint changes.

Efficacy and safety of long-acting recombinant fusion protein linking factor IX with albumin in haemophilia B patients undergoing surgery.

INTRODUCTION: Recombinant factor IX fusion protein (rIX-FP) has been developed to improve the pharmacokinetic (PK) profile of factor IX (FIX), allowing maintenance of desired FIX activity between injections at extended intervals, ultimately optimizing haemophilia B treatment. AIM: To determine the efficacy and safety of rIX-FP in the perioperative setting. METHODS: Subjects were adult and paediatric patients with severe to moderately severe haemophilia B (FIX ≤ 2%) participating in three Phase III clinical trials and undergoing a surgical procedure. PK profiles were established prior to surgery for each patient. Haemostatic efficacy was assessed by the investigator for up to 72 h after surgery. Safety measurements during the study included adverse events and inhibitors to FIX. FIX activity was monitored during and after surgery to determine if repeat dosing was required. RESULTS: Twenty-one, both major and minor, surgeries were performed in 19 patients. Haemostatic efficacy was rated as excellent (n = 17) or good (n = 4) in all surgeries. A single preoperative dose maintained intraoperative haemostasis in 20 of 21 surgeries. Nine major orthopaedic surgeries were conducted in eight patients with a mean of 7 (range: 6-12) rIX-FP injections during surgery and the 14-day postoperative period. Median rIX-FP consumption for orthopaedic surgeries was 87 IU kg(-1) preoperatively and 375 IU kg(-1) overall. No subject developed inhibitors to FIX or antibodies to rIX-FP. CONCLUSION: Recombinant factor IX fusion protein was well tolerated and effectively maintained haemostasis during and after surgery. Stable FIX activity was achieved with a prolonged dosing interval and reduced consumption compared to conventional or currently available long-acting recombinant FIX.

Achievements, challenges and unmet needs for haemophilia patients with inhibitors: Report from a symposium in Paris, France on 20 November 2014.

Over the past 20 years, there have been many advances in haemophilia treatment that have allowed patients to take greater control of their disease. However, the development of factor VIII (FVIII) inhibitors is the greatest complication of the disease and a challenge in the treatment of haemophilia making management of bleeding episodes difficult and surgical procedures very challenging. A meeting to discuss the unmet needs of haemophilia patients with inhibitors was held in Paris on 20 November 2014. Topics discussed were genetic and non-genetic risk factors for the development of inhibitors, immunological aspects of inhibitor development, FVIII products and inhibitor development, generation and functional properties of engineered antigen-specific T regulatory cells, suppression of immune responses to FVIII, prophylaxis in haemophilia patients with inhibitors, epitope mapping of FVIII inhibitors, current controversies in immune tolerance induction therapy, surgery in haemophilia patients with inhibitors and future perspectives for the treatment of haemophilia patients with inhibitors. A summary of the key points discussed is presented in this paper.

Post-authorization safety study of Clottafact® , a triply secured fibrinogen concentrate in congenital afibrinogenemia. A prospective observational study.

BACKGROUND AND OBJECTIVES: A new fibrinogen concentrate Clottafact® was developed according to European guidelines on plasma-derived products. A post-authorization safety study was set up in 2009 as part of the risk management plan. This was a non-interventional, prospective, non-comparative, multicenter study of the use of fibrinogen concentrate for congenital afibrinogenemia in real-life medical practice in France. MATERIALS AND METHODS: The analysis was descriptive and performed on 3 subgroups: prophylaxis vs. on-demand treatment, age (<6, <12 and ≥12) and severity of the deficiency. RESULTS: Fourteen patients [1-78 years] were included in 7 centres and followed for 1 year. Twenty-one adverse drug reactions (ADRs) classically reported with fibrinogen (pallor, chills, cough, vomiting, headache, urticaria and erythematous rash) were reported in 5 of 14 patients. Two ADRs were serious: an anaphylactic shock and a subclavian venous thrombosis with a favourable outcome without sequelae. In the nine patients under prophylaxis, 365 of 367 infusions were considered as successful (99·5%) and 2 as failures. For the five patients treated on-demand, the efficacy was rated as excellent for 27 of 48 infusions and good for the 21 others. CONCLUSION: This study confirms that the benefit/risk balance for this fibrinogen concentrate is favourable.

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.

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-domain-deleted 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 full-length 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 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 Nuwiq(®) and plans to launch it in the USA and globally in 2015.

FEIBA in the treatment of acquired haemophilia A: results from the prospective multicentre French 'FEIBA dans l'hémophilie A acquise' (FEIBHAC) registry.

Factor VIII inhibitor bypass activity (FEIBA) is a recommended first-line bypassing agent for bleeding episodes in patients with acquired haemophilia A (AHA). Due to the low incidence of AHA, available clinical data on FEIBA treatment are limited. The study aim was to delineate practice patterns in FEIBA treatment of AHA patients, the haemostatic efficacy of FEIBA, including criteria for its assessment, and safety. A prospective registry was established of AHA patients receiving FEIBA for bleeding episodes or prophylaxis at the time of invasive procedures. Data were collected at 16 participating centres in France. Patients were followed up for 3 months. Haemostatic efficacy, FEIBA regimen and FEIBA-related adverse events were documented. Thirty-four patients averaging 81.8 years old with standard deviation (SD) 8.1 years were included in the study: 33 for acute bleeding and one for haematoma evacuation. The mean initial dose of FEIBA for acute bleeding was 75.4 U kg(-1) (SD, 7.7 U kg(-1) ), most often administered twice daily, and the median duration of FEIBA treatment was 4.0 days (interquartile range, 2.2-8.0 days). FEIBA was effective in managing 88.0% of bleeding episodes (95% confidence interval, 75.8-94.5%). No baseline variables influencing treatment response could be identified. The sensitivity and specificity of an objective haemostatic efficacy scale in predicting sequential investigator assessments of haemostatic efficacy were 45.3% and 84.1% respectively. Four patients experienced a total of six serious adverse events possibly related to FEIBA. In the first prospective study specifically focused on FEIBA treatment of patients with AHA, 88.0% of bleeding episodes were effectively managed.

In vivo efficacy of human recombinant factor IX produced by the human hepatoma cell line HuH-7.

INTRODUCTION: Post-translational modifications of the CHO-cell-derived-recombinant human factor IX (FIX) currently used for the treatment of hemophilia B (HB) are different from plasma derived FIX. Our previous studies described a rFIX (HIX) having better profile of post-translational modifications than rFIX produced by CHO cells. The aim of the study consisted to verify the improved post-translational modifications effect of HIX on in vivo recovery. MATERIALS AND METHODS: HIX has been produced in a bioreactor and then purified from supernatants. In vitro activation and activity were evaluated measured by thrombin generation tests (TGT) and compared to commercial molecules, Benefix(®) , Mononine(®) . The three molecules were then administrated (i.v.) to FIX-knockout mice and two minutes after injection, blood samples were collected and subjected to human FIX-specific-ELISA and TGT. RESULTS: The clotting function of HIX, activation courses of HIX by FXIa and FVIIa-TF complex appear normal as did activation of Benefix(®) , Mononine(®) and TG constants of each FIX were equivalent. After injection to HB mice, circulating HIX did not present any significant difference in term of antigen value with Benefix(®) . Intriguingly, TGT were clearly exhibiting a better velocity for HIX than Benefix(®) and Mononine(®) . These data suggested that HIX may improve in vivo coagulant efficacy in comparison with the two commercial FIX injected at the same dose. CONCLUSION: The study shows that HuH-7-derived-rFIX has better in vivo haemostatic activity in hemophilia B mice compared to the reference rFIX molecule despite similar in vivo recovery rates, suggesting that HuH-7 cells could represent an effective cellular system for production of rFIX.

Usefulness of an in vitro cellular expression model for haemophilia A carrier diagnosis: illustration with five novel mutations in the F8 gene in women with isolated factor VIII:C deficiency.

This study aims to determine the way to predict the haemophilia A (HA) carrier status and the potential severity in six females with low FVIII: C levels (<0.50 IU mL(-1) ), F8 gene variations and without family history of HA. Except p.Ser577Tyr, F8 gene variations that we reported have never been described (p.Leu107His, p.Pro521Leu, p.Val682Leu, p.Leu2032Pro, p.Ala315dup). Prediction of their potential causal impact was studied by two strategies: bioinformatics approaches and site-directed mutagenesis followed by FVIII cellular expression into COS-1 cell. FVIII clotting assay ( FVIII: C) and antigen ( FVIII: Ag) were assayed in vitro. In silico analysis showed the probably damaging effect of all substitutions and the full conservation of the residues across mammalian species, except for p.Leu2032Pro. The in vitro variant expression model showed abnormal intra and/or extracellular FVIII: C and FVIII: Ag levels for five mutations, which suggest their causality in HA and provide informations about the involved mechanism. We suspect a defect in synthesis and secretion for p.Leu107His, p.Ala315dup and p.Pro521Leu. The mutation p.Val682Leu only affects the FVIII function while p.Ser577Tyr alters function and synthesis. The variant p.Leu2032Pro is probably a polymorphism because no alteration of the FVIII protein expression was observed in vitro. In vitro results suggest that mutations p.Ser577Tyr and p.Ala315dup could led to a severe HA in men. This study demonstrates the ability of this in vitro cellular expression model to contribute to the diagnosis strategy for female suspected of being HA carrier, without HA family history and with a novel F8 gene variation and to provide new criteria for the genetic counselling.

The first recombinant FVIII produced in human cells--an update on its clinical development programme.

The development of inhibitors and the need for frequent venous access for FVIII injection are major challenges in current haemophilia treatment. Presently available recombinant FVIII (rFVIII) products produced in hamster cell lines are associated with inhibitor formation in up to 32% of previously untreated patients. The new human cell line-derived recombinant human FVIII (Human-cl rhFVIII) protein is the first native, unmodified truly human rFVIII product produced in a human cell line without additive animal proteins. The aim of using a human cell line for the production of rFVIII is the avoidance of non-human epitopes on rFVIII, thereby potentially reducing the rate of inhibitor development, avoiding allergic reactions and allowing personalized prophylaxis with the chance of fewer infusions. Studies to date show that prophylaxis with Human-cl rhFVIII prevents 96% of bleeding events in adults with severe haemophilia A when compared to on-demand treatment. Available pharmacokinetic data with a mean half-life of 17.1 h allow personalized prophylaxis with the chance of fewer infusions. Studies in previously treated children and adults indicate that Human-cl rhFVIII is efficacious and safe in the prevention and treatment of bleeding episodes and that none of the treated patients developed inhibitors or allergic reactions thus far.

Characterization of four novel molecular changes in the promoter region of the factor VIII gene.

Haemophilia A (HA) is an X-linked recessive bleeding disorder, caused by a wide variety of mutations in the factor VIII (F8) gene, leading to deficiency in the activity of coagulation FVIII. These mutations can affect all the F8 exons from the initiation codon to the termination codon, however, only few molecular changes in the promoter region of the F8 gene were reported so far. Here, we describe six nucleotide variations (c.-51G>A, c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A and c.-664G>A) detected in the F8 promoter and their correlation with clinical phenotype of the patients. Potential role of these mutations in HA was also assessed. Causality was demonstrated with transient transfection experiments using luciferase reporter gene plasmids and computational analysis. Two molecular changes (c.-51G>A and c.-664G>A) did not seem to affect the promoter function of the F8 gene whereas c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A mutations had an impact on the F8 promoter function and were responsible for HA. Furthermore, these mutations were associated with resistance to 1-deamino-8-D-argininevasopressin (desmopressin) therapy when they were causative. When molecular variation was detected in F8 promoter, we propose to use prediction software and to verify predictions by reporter gene analysis. If the mutation is causative, it will be probably associated with a lack of therapeutic response to desmopressin and this clinical implication should be considered by clinicians.

Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements.

Thirty per cent of patients with mild haemophilia A (MHA) present markedly different FVIII: C level when assayed by one-stage clotting and two-stage chromogenic assays. It is, therefore, a real clinical challenge to predict the individual bleeding risk of these patients. The aim of the present work was to study the relationship between the bleeding tendency of these patients with the results of a panel of phenotypic and genotypic tools. Thirty-six patients with MHA were included in this multicentre prospective clinical study. The severity of bleeding symptoms was evaluated using the ISTH/SSC score. FVIII:C levels were measured using an activated partial thromboplastin time-based one-stage FVIII assay (FVIII: C1) and three commercial chromogenic kits (FVIII:CR). FVIII antigen levels, thrombin generation measurement and FVIII gene mutation analysis were also performed. Our results showed that a one-stage FVIII: C assay cannot rule out the diagnosis of MHA, a combined use of FVIII:C1 with a FVIII:CR is suitable for detecting MHA. We observed that FVIII:CR results better reflected the clinical bleeding tendency of patients compared to FVIII:C1. We also observed a relationship between thrombin generation (TG) capacity and FVIII:CR of these patients. FVIII gene mutation analysis showed mutations previously reported in MHA patients with discrepant FVIII:C measurements, but with no predictive value of the individual bleeding phenotype of patients. Overall, we observed a relationship between chromogenic FVIII:C results, TG assay and bleeding tendency of patients with discrepant FVIII:C measurements, while FVIII:C1 was not well correlated with clinical bleeding phenotype in this particular population.