The use of recombinant factor VIII in the management of Hemophilia

Recombinant factor VIII is currently in the late stages of clinical trials. The available studies indicate that the product is safe and well-tolerated, and appears to be free of virus diseases such as HIV and hepatitis infections. Based on these early studies, recombinant coagulation factors appear to have enonnous promise and potential for transfusion medicine. The synthesis of large quantities of safe material may lead to the development of techniques for daily administration of factor VIII aimed at the prevention of joint and soft tissue bleedings. There is also the promise of decreased costs, as techniques for the efficient synthesis of recombinant proteins are refined further.     

Intensive exposure to factor VIII is a risk factor for inhibitor development in mild hemophilia A

Summary.  Background : Inhibitors are rare in boys with mild hemophilia A (MHA; factor (F)VIII:C > 5%) but may arise following intense FVIII exposure, e.g. continuous infusion (CI). Objectives : To determine the impact of intense FVIII exposure in inhibitor formation in MHA at our institution and to compare this with previous reports. Patients and methods : We reviewed FVIII exposure and inhibitor development in boys (ages 0–18 years) with MHA followed at our institution from 1996 to 2001 and conducted a Medline search (1966–2002) on the experience of inhibitor development following intensive/CI exposure to FVIII. Results : We identified 54 boys with MHA. Twenty-nine (54%) had been exposed to FVIII. Seven had received FVIII by CI. Four developed inhibitors; three high titer (at ages 10 years, 16 years and 17 years) and one low titer (at 1 month old). All four had received a CI of recombinant (r) FVIII of at least 6 days within 6 weeks of developing inhibitors. Baseline FVIII levels fell to < 1% in all cases and the three with high-titer inhibitors developed severe bleeding. Immune tolerance therapy (ITT) was attempted in two boys and was successful in one. Our literature search identified 35 cases (only four children) with MHA developing inhibitors following intense FVIII exposure often in the context of surgery. Conclusions : The incidence of inhibitors in our MHA population was 7.4%. If expressed according to exposure the incidence was significantly higher: 14% (4/29) for any exposure to FVIII and 57% (4/7) for exposure by CI. A prospective study to address whether CI is associated with an increased incidence of inhibitor development in MHA is warranted.     

Prediction of factor VIII inhibitor development in the SIPPET cohort by mutational analysis and factor VIII antigen measurement

Essentials

• A residual factor VIII synthesis is likely to be protective towards inhibitor (INH) development.
• Mutation type‐inhibitor risk association was explored in 231 patients with severe hemophilia A.
• A 2‐fold increase in INH development for in silico null vs. non‐null mutations was found.
• A 3.5‐fold increase in INH risk for antigen negative vs. antigen positive mutations was found.

Summary

Background

The type of F8 mutation is the main predictor of inhibitor development in patients with severe hemophilia A. Mutations expected to allow residual synthesis of factor VIII are likely to play a protective role against alloantibody development by inducing immune tolerance. According to the expected full or partial impairment of FVIII synthesis, F8 variants are commonly classified as null and non‐null.

Objectives

To explore the mutation type–inhibitor risk association in a cohort of 231 patients with severe hemophilia A enrolled in the Survey of Inhibitors in Plasma‐Product Exposed Toddlers (SIPPET) randomized trial.

Methods

The genetic defects in these patients, consisting of inversions of intron 22 (n = 110) and intron 1 (n = 6), large deletions (n = 16), and nonsense (n = 38), frameshift (n = 28), missense (n = 19) and splicing (n = 14) variants, of which 34 have been previously unreported, were reclassified according to two additional criteria: the functional effects of missense and splicing alterations as predicted by multiple in silico analyses, and the levels of FVIII antigen in patient plasma.

Results

A two‐fold increase in inhibitor development for in silico null mutations as compared with in silico non‐null mutations (hazard ratio [HR] 2.08, 95% confidence interval [CI] 0.84–5.17) and a 3.5‐fold increase in inhibitor development for antigen‐negative mutations as compared with antigen‐positive mutations (HR 3.61, 95% CI 0.89–14.74] were found.

Conclusions

Our findings confirm an association between the synthesis of minute amounts of FVIII and inhibitor protection, and underline the importance of investigating the residual FVIII antigen levels associated with causative variants in order to understand their clinical relevance.

     

Clinical and immunologic effects of Staphylococcal protein A immunoadsorption therapy for inhibitor to factor VIII

A 74-year mild hemophiliac with an inhibitor to factor VIII underwent five 2-L plasma immunoadsorption procedures with a Staphylococcal protein A (Prosorba) column over 7 days. Initially, the inhibitor level increased from 56 to 154 BU/mL. Serum immunoglobulins, immune complexes, C3 and C4 fell progressively with each procedure: C3a was increased. Before therapy, the patient had increased cytotoxic T and activated T cells which decreased to the normal range with treatment. The proportion of T inducer cell and CD4:CD3 ratios increased progressively with each treatment. Natural killer cell activity remained unchanged. Unstimulated patient lymphocytes showed high proliferative activity, but after therapy there was markedly reduced response to mitogens. Both patient and normal lymphocytes showed marked increase in thymidine incorporation when incubated with adsorbed plasma (from column outlet), compared to incubation with unadsorbed plasma (column inlet). The patient's serum showed a progressive rise in this “mitogenicity” effect, maximal after the third procedure, then declining to pre-treatment levels after the fifth procedure. After the third immunoadsorption, inhibitor level was 70 BU/mL and he was given prednisone and cyclophosphamide; after the fifth procedure it was 32 BU/mL and 6 months later was undetectable. Although subsequent decrease in the inhibitor may have been due to the plasma immunoadsorption, it is important to note that this treatment may be paradoxically associated with a rise in antibody levels. In addition to antibody changes, Staph. protein A perfusion affected cellular immunity.     

Comparative thrombotic event incidence after infusion of recombinant factor VIIa versus factor VIII inhibitor bypass activity

Thrombosis is a rare but well-recognized potential complication of Factor VIII Inhibitor Bypass Activity (FEIBA) infusion. Recombinant factor VIIa (rFVIIa) is increasingly used as an alternative to FEIBA; however, the thrombotic safety profile of rFVIIa remains incompletely characterized. To determine the incidence rates of thrombotic adverse events (AEs) after infusion of rFVIIa and FEIBA. Data from the MedWatch pharmacovigilance program of the US Food and Drug Administration, as supplemented by published case reports, were used in conjunction with estimated numbers of infusions available from manufacturers to assess comparative incidence of thrombotic AEs in patients receiving rFVIIa or FEIBA in the period from April 1999 through June 2002. Reported thrombotic AEs were rare, with incidence rates of 24.6 per 10(5) infusions (CI, 19.1-31.2 per 10(5) infusions) for rFVIIa and 8.24 per 10(5) infusions (CI, 4.71-13.4 per 10(5) infusions) for FEIBA. Thrombotic AEs were significantly more frequent in rFVIIa than FEIBA recipients (incidence rate ratio, 2.98; CI, 1.71-5.52). The most commonly documented single type of thrombotic AE after rFVIIa infusion was cerebrovascular thrombosis, while myocardial infarction was the most frequent type in patients receiving FEIBA. Contrasting AE reporting patterns between rFVIIa and FEIBA may have contributed to the observed difference in thrombotic event incidence. Nevertheless, this comprehensive pharmacovigilance assessment does not support superior thrombotic safety of rFVIIa and suggests that thrombotic AE risk may be higher in rFVIIa than FEIBA recipients.     

Source and purity of factor VIII products as risk factors for inhibitor development in patients with hemophilia A

Summary. Background: Inhibitor development is influenced by several factors and the type of factor VIII (FVIII) products may play a role. Objectives: In order to explore such a role, we designed a cohort study whose novelty resides in the classification of products not only according to the source of FVIII (plasmatic, pd, or recombinant, r) but also to their degree of purity (expressed as specific activity). Patients/Methods: Treatment data up to inhibitor development or 150 exposure days were collected in 377 patients with hemophilia A. Results: Inhibitors developed in 111 patients (29%; 96 high‐responders, 25%). The cumulative incidence was progressively higher from patients treated with low/intermediate‐purity pdFVIII compared with those treated with high‐purity pd and rFVIII. The adjusted hazard ratio of inhibitor development was 4.9 with rFVIII and 2.0 with high‐purity pdFVIII (95% CI, 2.9–8.3 and 1.1–4.0), taking as reference low/intermediate‐purity pdFVIII. There was no difference in the frequency of inhibitor testing between treatment groups. Sensitivity analyses (in patients who never switched product type, previously untreated patients, those treated on‐demand and those with high‐risk F8 mutations) confirmed an increased inhibitor risk with rFVIII and high‐purity pdFVIII. Conclusions: This study shows that the degree of purity of FVIII products influences inhibitor development independently from other risk factors, and emphasizes that differences exist within pdFVIII products.     

Purified complex of factor VIII coagulant moiety and phospholipid: high factor VIII coagulant activity in factor VIII inhibitor plasma

A complex of factor VIII coagulant moiety and phospholipid was purified by means of immunoadsorbent chromatography of factor VIII concentrate and consecutive dissociation of the immobilized factor VIII complex by human placental phospholipid (Fibraccel). The complex of factor VIII coagulant moiety and phospholipid displayed factor VIII coagulant activity (VIII C) and factor VIII coagulant antigen (VIII C:Ag), but no factor VIII related antigen (VIII R:Ag). When incubated with factor VIII inhibitor plasma, the complex of factor VIII coagulant moiety and phospholipid exerted higher factor VIII coagulant activity than native factor VIII complex, and than purified factor VIII coagulant moiety alone. Our results prove that in activated prothrombin complex concentrates a complex of factor VIII coagulant moiety and phospholipid is the active mechanism exerting procoagulant activity in factor VIII inhibitor plasma.     

Rate of inhibitor development in previously untreated hemophilia A patients treated with plasma‐derived or recombinant factor VIII concentrates: a systematic review

Summary. Background: Different rates of inhibitor development after either plasma‐derived (pdFVIII) or recombinant (rFVIII) FVIII have been suggested. However, conflicting results are reported in the literature. Objectives: To systematically review the incidence rates of inhibitor development in previously untreated patients (PUPs) with hemophilia A treated with either pdFVIII or rFVIII and to explore the influence of both study and patient characteristics. Methods: Summary incidence rates (95% confidence interval) from all included studies for both pdFVIII and rFVIII results were recalculated and pooled. Sensitivity analysis was used to investigate the effect of study design, severity of disease and inhibitor characteristics. Meta‐regression and analysis‐of‐variance were used to investigate the effect of covariates (testing frequency, follow‐up duration and intensity of treatment). Results: Two thousand and ninety‐four patients (1167 treated with pdFVIII, 927 with rFVIII; median age, 9.6 months) from 24 studies were investigated and 420 patients were observed to develop inhibitors. Pooled incidence rate was 14.3% (10.4–19.4) for pdFVIII and 27.4% (23.6–31.5) for rFVIII; high responding inhibitor incidence rate was 9.3% (6.2–13.7) for pdFVIII and 17.4% (14.2–21.2) for rFVIII. In the multi‐way anova study design, study period, testing frequency and median follow‐up explained most of the variability, while the source of concentrate lost statistical significance. It was not possible to analyse the effect of intensity of treatment or trigger events such as surgery, and to completely exclude multiple reports of the same patient or changes of concentrate. Conclusions: These findings underscore the need for randomized controlled trials to address whether or not the risk of inhibitor in PUPs with hemophilia A differs between rFVIII and pdFVIII.     

Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors

Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable expression of hFVIII in platelets and correct hemophilia phenotype for long term. This approach has high potential to permanently treat FVIII deficiency with and without pre-existing anti-FVIII antibodies.     

Acquired factor VIII inhibitor in a patient with chronic myelogenous leukemia receiving interferon-alfa therapy

OBJECTIVE: To report a case of an acquired factor VIII inhibitor associated with the use of interferon-alfa. CASE SUMMARY: A 58-year-old white man with newly diagnosed chronic myelogenous leukemia (CML) was initially treated with hydroxyurea. Interferon-alfa therapy was started six weeks later in order to enhance the response, with gradual reduction and eventual discontinuation of hydroxyurea. Interferon-alfa was continued for one year. Following bone marrow aspiration at one year, the patient developed significant bleeding and bruising at the site of extraction. His hemoglobin decreased from 11.3 to 9.3 g/dL and his activated partial thromboplastin time was elevated at 72 seconds. The factor VIII concentration was 0.02 units/mL; factor VIII inhibitor concentration was 58 Bethesda units. A diagnosis of an acquired factor VIII inhibitor was made, and the patient was treated with activated factor VII concentrates and prednisone. Interferon-alfa was discontinued, and the inhibitor subsequently disappeared over the next six weeks. The patient did not have any further bleeding problems. DISCUSSION: Acquired factor VIII inhibitors other than in patients with hemophilia are rare. To date, there are no reported cases of factor VIII inhibitors associated with CML. Moreover, the temporal association with interferon-alfa administration suggests a causal relationship. There are only two previous case reports suggesting interferon-alfa as a cause of factor VIII inhibitors. CONCLUSIONS: Induction of factor VIII inhibitors is a serious potential complication of therapy with interferon-alfa. We suggest that a diagnosis of an acquired factor VIII inhibitor be considered in patients who experience unexplained bleeding with interferon-alfa therapy.     

The Canadian Hemophilia Registry as the basis for a national system for monitoring the use of factor concentrates

BACKGROUND: Canada's publicly funded blood system has recently introduced high-purity concentrates as the standard treatment for individuals with hemophilia. The added cost and the need to document patient outcomes have prompted the consideration of a national blood product monitoring system. STUDY DESIGN AND METHODS: This study investigates the suitability of the Canadian Hemophilia Registry (CHR) as the basis of such a monitoring system by assessing the degree to which it represents users of factor concentrates. RESULTS: Currently, there are 1978 individuals registered with the CHR, of whom 1594 (81%) have hemophilia A and 384 (19%) have hemophilia B. The total prevalence is 7.2 per 10(5) population, with the prevalence of severe cases being 2.3 per 10(5). This overall prevalence is similar to that seen in other countries with national registries. The CHR national prevalence also compares favorably with that in the province of Quebec, where registration of users of blood products is compulsory. The CHR figures indicate that the number of persons currently infected with human immunodeficiency virus, both alive and dead, is 652, which is similar to the number of applicants (658) to the federal government's assistance program. The registry is stable, and the number of persons with severe cases, other than young children, newly registered or lost to follow-up during the last 2 years is very small. CONCLUSION: The CHR includes the vast majority of factor concentrate users and is therefore ideal as the basis for a national monitoring system.     

Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A

BACKGROUND: Previous studies from our laboratory have demonstrated that lineage-targeted synthesis of factor VIII (FVIII) under the direction of the platelet-specific integrin alphaIIb gene promoter (2bF8) can correct the murine haemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model. OBJECTIVE: In this study, we assessed the efficacy of using a genetic therapy approach to correct haemophilia A in FVIII-deficient (FVIII(null)) mice by transplantation of bone marrow (BM) transduced with a lentivirus (LV)-based gene transfer cassette encoding 2bF8. RESULTS: Functional FVIII activity (FVIII:C) was detected in platelet lysates from treated mice and the levels were similar to 2bF8 heterozygous transgenic mice. Mice transplanted with 2bF8 LV-transduced BM survived tail clipping and we did not detected inhibitory or non-inhibitory FVIII antibodies over the period of this study (11 months). Furthermore, BM transferred from the primary transplant recipients into FVIII(null) secondary recipients demonstrated sustained platelet-FVIII expression leading to correction of the haemophilia A phenotype showing that gene transfer occurred within long-term repopulating haematopoietic stem cells. CONCLUSIONS: These results demonstrate that ectopic expression of FVIII in platelets by lentivirus-mediated bone marrow transduction/transplantation may be a promising strategy for gene therapy of haemophilia A in humans.