Clotting Factor VIII Overexpression Shows Signs of ER Stress but Does Not Cause Toxicity upon Gene Transfer

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3238-3238
Author(s):  
Irene Zolotukhin ◽  
Brett Palaschak ◽  
David M Markusic ◽  
Roland Herzog
Keyword(s):  
Stress Response ◽  
Gene Transfer ◽  
Factor Viii ◽  
Er Stress ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Clotting Factor ◽  
Potent Inducer ◽  
Cost Effective Treatment ◽  
Human Factor Ix

Abstract Hemophilia A is the X-linked bleeding disorder resulting from the loss of functional clotting factor VIII (FVIII). Hemophilia A patients with severe disease (<1% residual FVIII activity) experience spontaneous bleeds into the joints and closed spaces with severe morbidity. Restoration of hemostasis is managed by repeated infusions (2-3 times per week) of plasma derived or recombinant FVIII protein. While a standard treatment is available for patients, life-long infusions of FVIII protein is very expensive, has a negative impact on the patient's quality of life, and FVIII protein products are not available worldwide. Hence, there is a need to develop a more robust and cost effective treatment for hemophilia A patients. Liver-directed gene therapy using adeno-associated virus (AAV) represents a promising approach to treat hemophilia A. However, previous studies have shown that overexpression of human FVIII protein in the context of hydrodynamic delivery of plasmid vectors induces ER stress mediated through the unfolded protein response (UPR). Because human FVIII protein is inefficiently secreted into circulation, high AAV vector doses will be required to obtain therapeutic expression levels. Therefore, we sought to determine if AAV-FVIII gene delivery also triggers cellular UPR in hepatocytes in vivo. To this end, we selected to use a codon-optimized FVIII cDNA, which has been shown by our lab to significantly increase FVIII protein expression, and a high vector dose of AAV8-ApoE-hAAT-cohF8, containing a hepatocyte-specific enhancer/promoter combination. We evaluated this vector at doses of 1 x 1011 vg (4x1012 vg/kg) and 1 x 1012 vg (4x1013 vg/kg) in hemophilia A mice on a 129/BL6 background. Intravenous administration of the highest vector dose completely restored hemostasis, which was sustained and achieved super-physiological levels in some animals. Importantly, none of these mice developed inhibitors against FVIII. Next, we administered the vector at the same 2 doses to C67BL/6 mice, which show higher hepatic transduction efficiency than other strains. Experimental controls consisted of mice, with no vector or 1x1012 vg of AAV8-ApoE-hAAT-F9, expressing human factor IX (FIX) protein. Injection of tunicamycin, a potent inducer of the ER stress response, served as a positive control for all assays. Vector-treated mice were studied 2 and 4 weeks after gene transfer (n=3-4 per group). First, we evaluated the status of key molecular chaperones, known to be the mediators of the UPR: Bip, p-PERK, and p-eIF2a. Western blotting performed on the liver lysates indicated modest up-regulation of all three markers compared to normal control, but that effect was neither dose nor gene dependent. In addition, we tested the splicing of Xbp1 mRNA by PCR assay and observed low level of the 26 bp spliced fragment, indicative of the UPR, at the high vector dose. Immunohistochemistry on liver sections from each of our experimental groups including H&E staining, Tunnel staining for apoptotic cells, and reactive oxygen species staining. None of the stains yielded evidence for liver damage even in the 1x1012 AAV8-cohF8 treated mice compared to untreated controls. There was also no elevation of liver enzyme levels in plasma samples. Analysis of plasma from vector injected mice showed systemic levels of human FVIII and FIX proteins at ~30 ng/ml and ~6300 ng/ml, respectively (these ELISA-based measurements likely underestimate FVIII levels due to interference by von Willebrand factor). Overall our results suggest that over-expression of coagulation factors in hepatocytes from AAV vectors causes a mild cell stress response that is not strong enough to cause liver toxicity, is not specific for FVIII, and does impact expression or immunogenicity. Disclosures No relevant conflicts of interest to declare.

Piggybac Mediated Gene Transfer To Correct Hemophilia A

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2900-2900
Author(s):  
Janice M Staber ◽  
Molly Pollpeter ◽  
Angela Arensdorf ◽  
Patrick L Sinn ◽  
Thomas D Rutkowski ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Viral Vector ◽  
Conflicts Of Interest ◽  
Vector System ◽  
Expression Vectors ◽  
Dna Repeats ◽  
Tail Clip

Abstract Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder, affecting about 1 out of 5,000 males; those affected suffer disabling joint and muscle hemorrhages. Hemophilia A is an attractive gene therapy candidate, because even small increases in FVIII levels (5-10%) will alter the phenotype. Non-viral vector systems are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize that a PB transposon vector carrying a codon-optimized human FVIII cDNA along with a hyperactive transposase (iPB7) will confer persistent gene expression and correction of the hemophilia A bleeding phenotype. We engineered PB transposon to carry a codon-optimized human FVIII B-domain deleted cDNA (coFVIII-BDD). We evaluated the in vivo gene transfer efficiency in hemophilia A mice by hydrodynamic tail-vein injection using PB coFVIII-BDD driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Factor VIII null mice received 25 micrograms each of the PB coFVIII-BDD transposon and iPB7 to determine long term expression and phenotypic correction. FVIII activity and antigen levels were measured prior to injection and then every 4 weeks for 24 weeks. Results revealed therapeutic levels (50-225%) of factor VIII activity and antigen post gene transfer with stable expression for 24 weeks in most mice. A goal of gene transfer based therapies is to develop the most efficacious expression vectors with the least toxicity. To assess endoplasmic reticulum stress in the livers of treated and untreated mice, we evaluated BiP, CHOP, and EDEM levels via q-PCR. All experimental mice, null mice, and transposon treated mice without the coFVIII-BDD cassette revealed no evidence of cell stress. These data indicate codon-optimized FVIII and the piggyBac transposon vector system may provide a safe long term gene transfer strategy. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. More than 75% of mice receiving PB coFVIII-BDD transposon and iPB7 demonstrated functional correction via tail clip. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression and phenotypic correction. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ultrasound Combined with Microbubbles Mediated Factor VIII Plasmid Gene Therapy in Hemophilia a Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2039-2039
Author(s):  
Shuxian Song ◽  
James Harrang ◽  
Bryn Smith ◽  
Carol H. Miao
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Liver Perfusion ◽  
Transfer Efficiency ◽  
Clotting Factor ◽  
Gene Transfer Efficiency ◽  
Fviii Activity

Abstract Hemophilia A is a genetic bleeding disorder resulted from a deficiency of blood clotting factor VIII. In order to develop the efficient approach to gene therapy for hemophilia A, we previously explored reporter gene transfer mediated by ultrasound (US) combined with microbubbles (MBs). It was demonstrated that US/MB can significantly enhance gene transfer efficiency and serve as an efficient non-viral physical delivery strategy. In this study, we further delivered a therapeutic FVIII plasmid into the livers of hemophilia A (HA) mice. In consideration of FVIII synthesis from multiple tissues/cell lines, we first explored the distribution of gene expression using a pGL4.13 [luc2/SV40] luciferase plasmid driven by a ubiquitous promoter. One day following gene transfer, hepatocytes and endothelia cells were isolated from treated lobes by liver perfusion and centrifuge method. Evaluation of luciferase levels in two cell populations indicated that luciferase predominantly expressed in hepatocytes (5.35´104 RLU/107 cells vs. 1.46´103 RLU/107 cells in endothelia cells). Furthermore, gene transfer of pGFP (driven by a ubiquitous CMV promoter) mediated by US/MB also showed fluorescence distribution mostly in hepatocytes. These results indicate that hepatocyte is the predominant site of gene expression following US/MB mediated gene transfer into the liver. Based on these results, a hepatocyte-specific human FVIII plasmid (pBS-HCRHP-hFVIII/N6A) was used for US/MB mediated gene transfer in HA mice. In the short-term experiment, FVIII activity levels of treated HA mice ranged from 4-40% of normal FVIII activity. To follow FVIII expression for longer term, HA mice were pretreated with IL-2/IL-2 mAb (JES6-1) complexes on day −5, −4, and −3 to prevent immune response. In addition, the mice were infused with normal mouse plasma and human FVIII protein prior to gene transfer to maintain hemostasis. Subsequently, FVIII plasmids and 5 Vol% NUVOX MBs were injected into the mouse liver under simultaneous US exposure (1.1MHz transducer H158A driven by a pulse generator and high-power radio frequency amplifier capable of generating up to 1000W). Blood and liver samples were collected at serial time points after treatment to determine FVIII activity in plasma and liver damage. Following gene transfer, 10-30% of FVIII activity was achieved on day 4 and persisted in the average level of 20% by day 28. In a separate long-term follow-up experiment (n=3), 2 of 3 mice still maintained 10-30% activity after 120 days. Both transaminase levels (alanine aminotransferase and aspartate aminotransferase) and histological examination showed that the procedure of plasmid/MBs portal-vein injection and pulse-train acoustic exposure produced transiently localized liver damages however the damages were repaired and the liver recovered rapidly. Phenotypic correction of HA mice was further examined by tail clip assay. Blood loss of US/MB treated mice was significantly reduced compared with naive HA mice. Furthermore, a novel plasmid encoding a B domain-deleted FVIII variant containing mutations of 10 amino acids in the A1 domain (BDDFVIII-X10, a kind gift from Weidong Xiao) was constructed. Preliminary results from ongoing study showed that the gene transfer efficiency could be further improved with better plasmid and more efficient immune modulation. Together all the results indicate that US/MB mediated gene transfer is highly promising for efficient and safe gene therapy of hemophilia A. Disclosures No relevant conflicts of interest to declare.

Stable High Level Coagulation Factor VIII Expression In Vivo Following Gene Transfer Using a Novel Expression Cassette Encoding a More Potent FVIII Variant

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 250-250 ◽  
Author(s):  
Jenny McIntosh ◽  
Peter J Lenting ◽  
Edward Tuddenham ◽  
Motunrayo Sotannde ◽  
Simon Waddington ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Blot Analysis ◽  
Hemophilia A ◽  
Expression Cassette ◽  
Clotting Factor ◽  
High Dose ◽  
Dose Cohort ◽  
Tail Vein ◽  
Transfer Strategies

Abstract Abstract 250 Hemophilia A (HA, or clotting factor VIII deficiency), the commonest inherited bleeding disorder, is a good model for early proof of concept gene therapy studies. This is primarily because its clinical manifestations are entirely attributable to the lack of a single gene product (FVIII) that circulates in minute amounts (200ng/ml) in the plasma. Furthermore, a modest increase in the level of FVIII (>1% of normal) can ameliorate the severe phenotype. Several different gene transfer strategies for FVIII replacement have been evaluated but these have been hampered by the fact that hFVIII protein expression is highly inefficient. In addition the relatively large size of the hFVIII cDNA, (≂f7.0 kb) far exceeds the normal packaging capacity of adeno-associated viral vectors (AAV), which are currently the vectors of choice for the correction of genetic disorders such as hemophilia A. We have begun to address some of these limitations through the development of a 5.7kb AAV expression cassette (rAAV-HLP-codop-hFVIII-N6) which consists of a novel more potent hFVIII (codop-hFVIII-N6) which contains a short 226 amino-acid B-domain spacer, rich in asparagine-linked oligosaccharides, which is currently the most efficiently expressed hFVIII variant. This variant is under the control of a small ≂f200bp liver specific promoter. This expression cassette can be efficiently packaged into a single AAV vector, without significantly compromising vector yields. Transient transfection of this rAAV expression cassette into the HuH7 liver cell-line resulted in hFVIII expression that was between 4 and 8 (0.05±0.02IU/ml/24h) fold higher than that achieved with the B domain deleted (BDD-hFVIII) and N6-hFVIII variants respectively. Tail vein injection of serotype 5 or 8 pseudotyped vector rAAV-HLP-codop-hFVIII-N6 in C57Bl/6 mice resulted in detectable hFVIII within two weeks of gene transfer, reaching steady state levels of 23±6 IU/ml and 54±12 IU/ml respectively by 10 weeks. This level of expression is at least 400-fold greater than required for therapeutic efficacy (0.05IU/ml) and at least 10 fold higher than achieved in mice transduced with a comparable dose of rAAV encoding either the BDD or N6 variant of hFVIII. Southern blot analysis of DNA extracted from the liver of rAAV-HLP-codop-hFVIII-N6 transduced mice revealed head-to-tail and head-to-head concatemer fragments of ≂f5kb and ≂f10kb respectively in the expected ratio of 3:1. Western blot analysis showed that the rAAV-HLP-codop-hFVIII-N6 cassette mediated the synthesis and secretion of a single chain 210kd protein. To confirm correction of the bleeding phenotype, either 4×1011 (low-dose cohort, n=3) or 4×1012 (high-dose cohort, n=3) rAAV5-HLP-codop-hFVIII-N6 vector genomes were injected into the tail vein of haemophilia A knockout mice. Peak hFVIII levels, as determined by a one-stage clotting assay, were 137±27% and 374±18% of normal levels in the low and high-dose cohorts of F8-/- mice respectively. These levels were sufficient to arrest bleeding in a modified tail clip assay. Anti-hFVIII antibodies were not detected in the rAAV treated HA mice at any stage. Therefore, the higher potency of our novel codop-hFVIII-N6 construct and the ability to package this FVIII variant within AAV virions has substantially improved the prospects of effective gene transfer for Hemophilia A. Disclosures: No relevant conflicts of interest to declare.

Increased Prevalence of Inhibitors in Mexican-Hispanic Patients with Severe Hemophilia A Enrolled in the Universal Data Collection Project.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3488-3488 ◽  
Author(s):  
Shannon Carpenter ◽  
J. Michael Soucie ◽  
Sophia Sterner ◽  
Rodney J Presley
Keyword(s):  
African Americans ◽  
Data Collection ◽  
Factor Viii ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Treatment Center ◽  
Hispanic Population ◽  
Severe Hemophilia ◽  
Inhibitor Development ◽  
Insurance Type

Abstract Abstract 3488 Poster Board III-425 Neutralizing inhibitor formation occurs in up to 20-30% of patients with severe factor VIII deficiency, leading to significantly increased morbidity in affected individuals. It has been well-established that patients of African descent have a higher prevalence of inhibitor development. [Oldenburg, J et al. Semin Hematol, 2004] The Hispanic population also has been assumed to have an increase in inhibitor development when compared with Caucasians. The study presented here is the first to definitively demonstrate an increased prevalence of inhibitors in the Hispanic population. We compared inhibitor prevalence among various racial and ethnic groups in a cross-sectional analysis of 6198 males with severe hemophilia A that participated in the Universal Data Collection project sponsored by the Centers for Disease Control and Prevention. We used logistic regression analysis to control for potential confounding variables including age, insurance type (as a proxy for access to care and socio-economic status), age at first bleed, age at diagnosis and use of prophylaxis. The included table shows those variables that were determined to be independently predictive of inhibitors. We assigned Mexican derivation to participants who labeled themselves as Hispanic and who were born either in Mexico, in states bordering Mexico or in states with large Mexican populations as established by Census data. The prevalence of high titer inhibitors in the Mexican-Hispanic population was 26.3% compared to 16.4% for Caucasian patients [OR 1.5, 95% CI 1.1, 1.9], and 26.8% for African-Americans. The underlying cause of increased inhibitor prevalence in these populations is still unknown, though a recent study in African-Americans demonstrated wild-type factors unique from commercially available product. [Viel KR, et al. Inhibitor of Factor VIII in Black Patients with Hemophilia. N Engl J Med, 2009] Further investigation of this phenomenon in the Mexican-Hispanic population, as well as the potential impact of differing immune responses, is warranted. Multivariate analysis of ethnicity and other variables found to be independently predictive of a prevalent inhibitor Characteristic Odds Ratio 95% CI Race/Ethnicity African-American 1.5 1.2 - 1.9 Mexican Hispanic 1.5 1.1 - 1.9 Hispanic 1.2 0.9 - 1.7 Other 1.2 0.9 - 1.6 White Ref Age* (years) <2 4.2 3.0 - 5.9 2-5 6.4 5.1 - 8.0 6-10 2.8 2.2 - 3.5 11-18 1.7 1.4 – 2.1 >18 Ref Insurance type Medicare 1.8 1.4 - 2.3 Medicaid 1.3 1.1 - 1.5 State program 1.1 0.6 - 1.9 TRICARE 1.0 0.4 - 2.1 Other 0.8 0.6 - 1.2 Uninsured 1.6 1.0 - 2.4 Commercial Ref Prophylaxis Yes 0.6 0.5 - 0.7 No Ref * Age with inhibitor or last UDC visit if no inhibitor The authors wish to acknowledge the contributions of the Hemophilia Treatment Center Network Investigators in the completion of this study. Disclosures: No relevant conflicts of interest to declare.

Inhibitor Development In Patients with Mild and Moderate Hemophilia A: Results From a Single Centre.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1407-1407 ◽  
Author(s):  
Yohann Repesse ◽  
Philippe Gautier ◽  
Annie Borel-Derlon
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Immunosuppressive Treatment ◽  
Gene Promoter ◽  
Missense Mutations ◽  
Bleeding Events ◽  
Inhibitor Development ◽  
Treatment Centre

Abstract Abstract 1407 The development of factor VIII (FVIII) inhibitors is usually considered uncommon among patients with mild and moderate hemophilia A (HA) and less frequent than in patients with severe HA. We report here the prevalence of FVIII inhibitors and their caracteristics in 167 patients with mild and moderate HA followed in Caen Hemophilia Treatment Centre (Table). FVIII molecular defects were identified by direct sequencing in 167 patients including 30 and 137 with mild and moderate HA, respectively. Following FVIII concentrates infusions, FVIII inhibitors occured in 7.8% of patients (13/167). Fifteen percent (2/13) were low-responding inhibitors. The risk of inhibitor development appeared to be associated with high-risk FVIII genotypes clustered in the A2 and C2 domains, especially p.Arg2150His (50%) and p.Arg593Cys mutations. Interestingly, we described inhibitor development associated with novel missense-mutations (p.Tyr1786Ser, p.Asp115Tyr and -219C>T substitutions in FVIII gene promoter). In addition, high regimen infusion of FVIII concentrates appeared as risk factor for FVIII inhibitors development. Indeed, 60% (8/13) developped FVIII inhibitors following massive infusion of FVIII concentrates associated with FVIII:C levels above 1.2 UI/dL. Inhibitors in mild HA usually cross-react with endogenous factor VIII reducing the circulating basal FVIII:C level and are associated with more bleeding events. Similarly, we observed the evolution of bleeding patterns in our cohort to severe phenotypes. Bleedings were treated with FVIII concentrates and bypassing therapies (activated FVII and activated-prothrombin complex). About 25% (3/13) of these inhibitors disappeared spontaneously. Induction of Immune Tolerance (ITI) protocoles with high doses of FVIII were initiated for 7 high-responding patients with a success rate of 85 % (6/7). However, inhibitors persisted long-term and remained troublesome in 1 of these patients despite of ITI protocole. For two patients, immunosuppressive treatment with corticosteroids was started. Inhibitors disappeared and the levels of FVIII:C became detectable within 6 months. Development of FVIII inhibitors, their disappearance and the efficacy of ITI regimen seem to be different from our experience between patients with mild or moderate HA and severe HA. Disclosures: No relevant conflicts of interest to declare.

An Increase in Factor VIII Levels with Increasing Age in a Subgroup of Individuals with Mild Hemophilia A

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2278-2278 ◽  
Author(s):  
Lauren J Lee ◽  
John K. Wu ◽  
Man-Chiu Poon ◽  
Shannon Jackson
Keyword(s):  
Hepatitis C ◽  
Factor Viii ◽  
Medical Records ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Bleeding Disorder ◽  
Physiological Age ◽  
Management Options ◽  
Southern Alberta ◽  
The Mean

Abstract Abstract 2278 Background: Hemophilia A is an X-linked hereditary disorder resulting in absent or reduced levels of functional factor VIII (FVIII:C). The baseline FVIII:C levels are important in the determination of treatment and management options in individuals with mild hemophilia who by definition exhibit FVIII:C levels between 6–40 U/dL. In individuals without hemophilia, FVIII:C increases with physiological age and this trend has not been well characterized in mild hemophilia. This is important to consider in mild hemophilia because treatment with factor concentrate or DDAVP may paradoxically increase the risk of venous or arterial thrombosis in older patients. Objectives: To describe the changes in baseline FVIII:C levels with time in a cohort of pediatric and adult subjects with mild hemophilia registered in the British Columbia Provincial and Southern Alberta Bleeding Disorder programs. Methods: All medical records for subjects with FVIII:C levels 6–40 U/dL registered with the BC Provincial and Southern Alberta Bleeding Disorder programs were reviewed for eligibility. Male subjects with a minimum of 2 FVIII:C level measurements at least 5 years apart were included in the analysis. Retrospective data was extracted from database/medical records, including age, gender, blood group, FVIII mutation, historical FVIII:C levels, historical DDAVP response (>3 fold and/or >50 U/dL), and co-infection (Hepatitis C, B and HIV) status. Linear mixed effects regression models were used to examine time trend of FVIII:C levels and subjects were stratified into 3 groups based on baseline FVIII:C (FVIII:C<15 U/dL, 15–24 U/dL, and ≥25 U/dL). Results: 198 records were reviewed and 116 subjects excluded from the analysis (chart unavailable, n=28; inadequate FVIII:C data, n=79; female, n=9) leaving 82 subjects who met eligibility criteria. The mean age at first FVIII:C measurement was 24.0 years (SD 19.6; Range 0.0–77.8). The mean follow-up time was 16.5 years (SD 19.6; Range 4.8–44.8). There was no observable trend of FVIII:C with time in the primary analysis of the whole cohort (p=0.667). However, for subjects with baseline FVIII:C<15 U/dL, who were ≥25 years of age (n=31), an increase in baseline FVIII:C levels of 0.11 percent per year (p<0.05) was observed. Subjects with baseline FVIII:C<15 U/dL (n=40) also showed less FVIII:C variability between measurements than subjects with FVIII:C≥15 U/dL (n=42). Subjects with baseline FVIII:C≥25 U/dL (n=18) demonstrated a decreasing trend of −0.15 percent per year (p<0.05), however also demonstrated the greatest variability between FVIII:C measurements. Co-infections were present in 24% (n=20) of subjects and included Hepatitis C (n=18), Hepatitis B (n=1), HIV (n=1). 73 subjects had known DDAVP response; >3-fold but ≤50 U/dL (n=11), ≤3-fold but >50 U/dL (n=15), >3-fold & >50 U/dL (n=23), ≤3-fold & ≤50 U/dL (n=24). Conclusion: This study suggests that there is an increase in FVIII:C with time in the subgroup of individuals with mild hemophilia ≥25 years of age and baseline FVIII:C levels <15 U/dL. Further long-term data particularly in the older cohort of subjects is needed to confirm this preliminary finding and further characterize this trend in subgroups with different baseline FVIII:C. Disclosures: No relevant conflicts of interest to declare.

Pharmacokinetics of Continuous Infusion Therapy of Factor VIII Concentrates in Hemophilia A Patients with Inhibitors,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3320-3320
Author(s):  
Katsumi Nishiya ◽  
Ichiro Tanaka ◽  
Keiji Nogami ◽  
Kenichi Ogiwara ◽  
Koji Yada ◽  
...  
Keyword(s):  
Factor Viii ◽  
Continuous Infusion ◽  
Infusion Rate ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
High Titer ◽  
Infusion Therapy ◽  
Target Level ◽  
Ci Therapy ◽  
The Difference

Abstract Abstract 3320 Continuous infusion (CI) of factor VIII (FVIII) concentrates is aimed at maintaining a steady hemostatic level of FVIII activity (FVIII:C) in hemophilia A patients during various surgeries. However, there are few reports that mentioned the difference of pharmacokinetics of CI therapy in hemophilia A patients with inhibitors. We investigated the relationship between the FVIII:C levels and the rate of CI, and the difference of clearance (CL) and volume of distribution (Vd) of FVIII in hemophilia A patients with/without inhibitors. 8 severe hemophilia A patients without inhibitors (arthroscopic synovectomy; 4 cases, total knee arthroplasty; 2 cases, total nephrectomy; 1 case, partial hepatectomy; 1 case), 3 patients with low-titer (2.0–2.9 BU) inhibitors and 3 patients with high-titer (6.0–9.0 BU) inhibitors (insertion or removal of a central venous access device), were enrolled in this study between 2005 and 2010. According to the Japanese guideline for hemophilia treatment, we should do CI therapy to keep target level 80–100% for 5–10 days for joint surgery and other major surgeries. An initial bolus infusion (BI) of FVIII concentrates was administered to achieve this level prior to CI. In addition, we have to neutralize the inhibitors by FVIII concentrates in case of the patients with inhibitors. FVIII:C was measured using one-stage clotting assays and FVIII inhibitor assays were performed using the Bethesda method. All therapy was conducted after obtaining fully informed consent. The median FVIII:C level after BI was 120.2% (range: 90–150) in the patients without inhibitors, 72.0% (range: 68–160) with low-titer inhibitors, and 20.0% (range: 9.4–30) with high-titer inhibitors, respectively. The target level of FVIII:C was adjusted to approximately 100%. The initial infusion rate was 3.7 U/kg/hr (range: 2.2–5.0), 8.3 U/kg/hr (range: 8.0–8.5) and 18.5 U/kg/hr (range: 15–22), respectively. After adjustment for the target level, the final infusion rate decreased to 2.6 U/kg/hr (range: 1.5–5.4), 4.7 U/kg/hr (range: 3.0–5.6) and 8.0 U/kg/hr (range: 7.0–9.0), respectively. CL was 2.3 ml/hr/kg (range: 1.5–3.9), 4.0 ml/hr/kg (range: 2.3–5.1) and 9.3 ml/hr/kg (range: 9.0–9.6), respectively. Vd was 0.04 L/kg (range: 0.031–0.047), 0.18 L/kg (range: 0.12–0.29) and 1.54 L/kg (range: 0.95–2.43), respectively. No unexpected safety concerns associated with CI, such as thrombosis, was identified during the study. On CI therapy, we could keep target level of the patients without inhibitors and with low-titer inhibitors easier than those with high-titer inhibitors. One of the reason is that CL and Vd in patients with inhibitors are higher than those in patients without inhibitors. CI with appropriate monitoring of FVIII:C level and concerning CL and Vd forms a safe method for perioperative care in hemophilia A patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

Source and Purity of Factor VIII Products As Risk Factors for Inhibitor Development In Previously Untreated Patients with Severe Hemophilia A

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 25-25
Author(s):  
Maria Elisa Mancuso ◽  
Pier Mannuccio Mannucci ◽  
Angiola Rocino ◽  
Isabella Garagiola ◽  
Annarita Tagliaferri ◽  
...  
Keyword(s):  
Risk Factors ◽  
Factor Viii ◽  
High Purity ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Sensitivity Analyses ◽  
Severe Hemophilia ◽  
Inhibitor Development ◽  
Treatment Data

Abstract Abstract 25 Background: Inhibitor development is influenced by several genetic and environmental factors and the type of factor VIII (FVIII) products may play a role. Methods: We designed a cohort study whose novelty resides in the classification of products not only according to the plasmatic (pdFVIII) or recombinant (rFVIII) source of FVIII but also to the degree of purity expressed as FVIII specific activity per mg of protein. The role of FVIII product as risk factor for inhibitor development was evaluated in a multivariate model adjusting for potential confounders (i.e. age at first FVIII exposure, intensive treatment and prophylaxis). Cumulative incidences of all and high-responding inhibitors were calculated for the whole cohort of 721 patients with severe and moderate hemophilia A followed-up in 3 Italian Hemophilia Centers. Detailed treatment data from the first FVIII infusion up to inhibitor development or 150 exposure days were available for 377 patients and in this group of patients risk factors for inhibitor development including the type of FVIII product and its degree of purity (i.e. low/intermediate-, high-purity pdFVIII and rFVIII) were analysed. Results: The overall cumulative incidence of inhibitors was 22% (n=160; 130 high-responders, 18%) and it was lower in patients first treated with pdFVIII (107/586, 18%) than in those treated with rFVIII (53/135, 39%). Similar results were obtained by evaluating only high-responding inhibitors and patients with severe hemophilia. 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 in patients first treated with rFVIII or high-purity pdFVIII than in those treated with low/intermediate-purity pdFVIII. In fact, in all the aforementioned subgroups by multivariate analysis the risk of inhibitor development was invariably 3- to 6-fold higher in patients first treated with rFVIII than in those first treated with pdFVIII, and similar results were obtained for both all inhibitors and high-responding inhibitors. 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 also within pdFVIII products. Disclosures: No relevant conflicts of interest to declare.

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