Glycopegylated Factor IX Enables Prolonged Efficacy After Subcutaneous Injection But Requires a Higher Than Expected Plasma Activity To Prevent Bleeding In Hemophilia B Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1104-1104
Author(s):  
Derek S Sim ◽  
Alan Brooks ◽  
Cornell Mallari ◽  
Yifan Xu ◽  
Rick I Feldman ◽  
...  
Keyword(s):  
Half Life ◽  
Subcutaneous Injection ◽  
Specific Activity ◽  
Periodate Oxidation ◽  
Factor Ix ◽  
Hemophilia B ◽  
Current Therapy ◽  
B Mice ◽  
Trough Levels ◽  
Plasma Activity

Abstract Background Reduced frequency of administration as well as subcutaneous (s.c.) injection would improve the treatment of Hemophilia B. Conjugation to polyethylene glycol (PEG) has been shown to increase the half-life of i.v. dosed Factor IX (FIX), but s.c. dosing of PEGylated FIX was not previously evaluated. Because s.c. dosing is limited by volume and bioavailability, we evaluated the combination of PEGylation with the increased specific activity variant R338A to reduce the amount of protein needed to provide therapeutic levels of FIX. Methods FIX-R338A was PEGylated on N-linked glycans in the activation peptide by periodate oxidation of sialic acid residues followed by conjugation to amino-oxy functionalized PEG. Pharmacokinetic (PK) profiles were determined in hemophilia B mice and cynomologous monkeys. Allometric scaling was used to predict dose regimens in humans. Prophylactic efficacy was determined in a Hemophilia B mouse tail bleeding model. Results 60kDaPEG-R338A had prolonged terminal half-life in mice (3-fold) and monkeys (5-fold) and the s.c. bioavailability was 44% and 35%, respectively. The volume of distribution was reduced 5-fold. To achieve a trough level of 3% FIX activity, s.c. dosing at weekly, bi-monthly and monthly intervals was predicted to require doses of 7, 25 and 220 IU/kg in patients. However, in a tail vein transection injury model, approximately 10-fold higher plasma FIX activity levels of PEGylated proteins were found to be needed to protect hemophilia B mice against bleeding than was required for i.v. dosed un-PEGylated recombinant FIX. This difference was observed for PEGylated wild-type and R338A proteins, dosed i.v or s.c. We hypothesize that this is related to the reduced distribution of PEGylated FIX to the extravascular compartment. Trough levels of 30% FIX activity were predicted to be achievable in humans after weekly and bi-monthly s.c. dosing at 70 and 260 IU/kg. Conclusions The PEGylation of FIX led to a significant improvement in both i.v. and s.c. PK. Unexpectedly, a 10-fold higher plasma activity was needed for PEGylated FIX to provide protection against bleeding in Hemophilia B mice, suggesting that trough levels of 10 to 30% of PEGylated FIX activity may be needed in patients to provide efficacy equivalent to current therapy of recombinant or plasma derived FIX. Nevertheless, 60kDaPEG-R338A has the potential to treat hemophilia B patients with once weekly or twice monthly subcutaneous injection. Disclosures: Sim: Bayer HealthCare: Employment. Brooks:Bayer HealthCare: Employment. Mallari:Bayer HealthCare: Employment. Xu:Bayer HealthCare: Employment. Feldman:Bayer HealthCare: Employment. Schneider:Bayer HealthCare: Employment. Patel:Bayer HealthCare: Employment. Blasko:Bayer HealthCare: Employment. Ho:Bayer HealthCare: Employment. Su:Bayer HealthCare: Employment. Liu:Bayer HealthCare: Employment. Laux:Bayer HealthCare: Employment. Murphy:Bayer HealthCare: Employment.

scholarly journals Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Blood ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2333-2341 ◽  
Author(s):  
Henrik Østergaard ◽  
Jais R. Bjelke ◽  
Lene Hansen ◽  
Lars Christian Petersen ◽  
Anette A. Pedersen ◽  
...  
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Duration Of Effect ◽  
B Mice ◽  
Bleeding Episodes ◽  
Activation Peptide ◽  
Extended Circulation

Abstract Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the Km for activation by FVIIa–tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood–based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Posttranslational modifications of recombinant myotube-synthesized human factor IX

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 130-138 ◽  
Author(s):  
Valder R. Arruda ◽  
James N. Hagstrom ◽  
Jeffrey Deitch ◽  
Terry Heiman-Patterson ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Posttranslational Modifications ◽  
Half Life ◽  
Specific Activity ◽  
Factor Ix ◽  
Serine Phosphorylation ◽  
Hemophilia B ◽  
Tyrosine Sulfation ◽  
Aav Vector ◽  
Carbohydrate Analysis

Abstract Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical γ-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and γ-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.

Factor IX variants improve gene therapy efficacy for hemophilia B

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2316-2323 ◽  
Author(s):  
Joerg Schuettrumpf ◽  
Roland W. Herzog ◽  
Alexander Schlachterman ◽  
Antje Kaufhold ◽  
Darrel W. Stafford ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Intramuscular Injection ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Promising Strategy ◽  
B Mice ◽  
Higher Doses ◽  
Vector Delivery

Abstract Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

Extravascular Administration of Factor IX: Potential for Replacement Therapy of Canine and Human Hemophilia B

1997 ◽  
Vol 77 (05) ◽  
pp. 0944-0948 ◽  
Author(s):  
Darla Liles ◽  
Charles N Landen ◽  
Dougald M Monroe ◽  
Celeste M Lindley ◽  
Marjorie s Read ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Vitamin K ◽  
Absorption Rate ◽  
Venous Access ◽  
Factor Ix ◽  
Hemophilia B ◽  
Current Therapy ◽  
Home Therapy ◽  
Routes Of Administration ◽  
Plasma Compartment

SummaryCurrent therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availibility of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (SC), intramuscular (IM), intra- peritoneal (IP) or intravenous (IV) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical IV dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the IV route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the IM injection in the canine resulted in a bioavailibility of 82.8%, while the SC injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with IV administration. These data show that significant levels of F.IX may be obtained via SC injection in canine and ‘ human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

scholarly journals Real World Factor Dispensation and Expenditures in us Patients with Hemophilia B: Standard Half-Life vs. Extended Half-Life Factor IX Replacement Products

2018 ◽  
Vol 21 ◽  
pp. S111
Author(s):  
A Chhabra ◽  
D Spurden ◽  
BJ Tortella ◽  
PF Fogarty ◽  
A Pleil ◽  
...  
Keyword(s):  
Real World ◽  
Half Life ◽  
Factor Ix ◽  
Hemophilia B ◽  
Life Factor

Prophylaxis for Hemophilia in the Era of Extended Half-Life Factor VIII/Factor IX Products

2016 ◽  
Vol 42 (05) ◽  
pp. 518-525 ◽  
Author(s):  
Erik Berntorp ◽  
Nadine Andersson
Keyword(s):  
Half Life ◽  
Safety Margin ◽  
Clinical Development ◽  
Factor Ix ◽  
Life Extension ◽  
Bleeding Events ◽  
Treatment And Prevention ◽  
Trough Levels ◽  
Life Factor

There are two main bioengineering approaches to extending the half-life of factor (F)VIII or FIX products used for hemophilia replacement therapy. These are fusion to Fc-immunoglobulin G (FVIII and FIX) or to albumin (FIX) or pegylation/glycopegylation (FVIII and FIX). Four FVIII and three FIX products are in clinical development or have recently been licensed in regions of the world. The reported half-life extension is approximately 1.5-fold for FVIII and 2.5-fold, or even longer, for FIX. Clinical trials have shown promising results with respect to extension of dose intervals and efficacy in the treatment and prevention of bleeding events. The role of these products in clinical practice has been discussed in terms of either improving convenience and adherence through prolongation of the interval between infusions or maintaining current intervals thereby increasing trough levels and the safety margin against bleeds. This review of extended half-life products addresses the possibilities and problems of their introduction in hemophilia treatment.

scholarly journals In silico evaluation of limited sampling strategies for individualized dosing of extended half-life factor IX concentrates in hemophilia B patients

2021 ◽  
Author(s):  
T. Preijers ◽  
M. W. F. van Spengler ◽  
K. Meijer ◽  
K. Fijnvandraat ◽  
K. Fischer ◽  
...  
Keyword(s):  
In Silico ◽  
Half Life ◽  
Factor Ix ◽  
Hemophilia B ◽  
Weekly Dose ◽  
Sampling Strategies ◽  
Time Profiles ◽  
Limited Sampling ◽  
Individualized Dosing ◽  
Life Factor

Abstract Purpose Hemophilia B is a bleeding disorder, caused by a factor IX (FIX) deficiency. Recently, FIX concentrates with extended half-life (EHL) have become available. Prophylactic dosing of EHL-FIX concentrates can be optimized by assessment of individual pharmacokinetic (PK) parameters. To determine these parameters, limited sampling strategies (LSSs) may be applied. The study aims to establish adequate LSSs for estimating individual PK parameters of EHL-FIX concentrates using in silico evaluation. Methods Monte Carlo simulations were performed to obtain FIX activity versus time profiles using published population PK models for N9-GP (Refixia), rFIXFc (Alprolix), and rIX-FP (Idelvion). Fourteen LSSs, containing three or four samples taken within 8 days after administration, were formulated. Bayesian analysis was applied to obtain estimates for clearance (CL), half-life (t1/2), time to 1% (Time1%), and calculated weekly dose (Dose1%). Bias and precision of these estimates were assessed to determine which LSS was adequate. Results For all PK parameters of N9-GP, rFIXFc and rIX-FP bias was generally acceptable (range: −5% to 5%). For N9-GP, precision of all parameters for all LSSs was acceptable (< 25%). For rFIXFc, precision was acceptable for CL and Time1%, except for t1/2 (range: 27.1% to 44.7%) and Dose1% (range: 12% to 29.4%). For rIX-FP, all LSSs showed acceptable bias and precision, except for Dose1% using LSS with the last sample taken on day 3 (LSS 6 and 10). Conclusion Best performing LSSs were LSS with samples taken at days 1, 5, 7, and 8 (N9-GP and rFIXFc) and at days 1, 4, 6, and 8 (rIX-FP), respectively.

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