Phase 1/2 Trial of Subcutaneously Administered Factor IX Variant CB2679d/ISU304: Pharmacokinetics and Activity

Abstract Marketed factor IX (FIX) products require intravenous (IV) administration to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous (SQ) administration is a preferred route of administration but has been limited by low bioavailability and potency of the marketed FIX products. CB2679d/ISU304 with enhanced biological properties was developed using a rational protein design approach and has resistance to inhibition by ATIII, increased affinity for FVIIIa, increased catalytic activity, which result in 22-fold enhanced potency in vitro (clotting activity) and in vivo (the tail clip model) and 8-fold increased duration of aPTT activity in vivo compared with recombinant wild-type FIX dosed at the same mass and may allow SQ administration to provide prophylaxis. Methods: The trial design is provided below. IV pharmacokinetics (PK) (antigen and activity) was sampled at predose, 0, 0.25, 0.5, 1, 3, 6, 9, 24, 48 and 72 hours. SQ PK was sampled at predose, 1, 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours. Cohort 5 has PK sampled before each injection, 6 hours after first and 6th injection and 24 hours after 6th daily injection. Hematology, chemistry and coagulation was measured at Seoul Clinical Laboratories (Yongin-si, South Korea). FIX antigen and FIX activity, anti-drug antibody to BeneFIX and ISU304 and neutralizing antibody were measured at Haematologic Technologies Inc (Essex Junction, VT). A safety follow-up was done 2 weeks after last visit. FIX antigen was measured using VisuLizeTM Factor IX Antigen KitAG (Affinity Biologicals, Inc, Ancaster, ON, Canada) and FIX activity was measured using a one-stage clotting assay using ACL TOP 700 and Instrumentation Laboratories (Bedford, MA) reagents. The calculation of AUC was based on the trapezoidal rule. To calculate the additional area for AUC0-inf, the log-linear regression line for the last three time points was fit and extrapolated to the x-intercept. The calculation of half-life was based on the use of Demitasse 2000 (version 1.1.3, M. Lee, 2000) which uses an iterative piecewise fitting algorithm based on a robust (M-regression) log-linear model (Lee, 1990, 1997). All activity data were adjusted for baseline before analysis, assuming exponential falloff after IV administration and a half-life of 20 hours. Bioavailability was calculated from the AUC0-t for the IV and SQ data using FIX activity data. Subject safety was reviewed by an external Data Safety Monitoring Board and also an internal Data Monitoring Committee. Results: PK and activity of ISU304 demonstrate the 22-fold greater potency over BeneFIX and longer mean residence time (Cohort 1 figure, Table 1). Bioavailability of 18.2-23.6%, SQ beta half-life 66-103 hours and Tmax 6-24 hours (Cohort 2 figure, Table 2) One subject reported transient fever and a mild SQ injection site reaction. Conclusion: Interim study results support the aim of achieving normal or high mild hemophilia FIX levels in individuals with hemophilia B with repeated SQ dosing. Complete PK and activity and steady-state levels after 6 daily doses will be reported from this phase 1/2 subcutaneous dosing study. Figure Figure. Disclosures Levy: Catalyst Biosciences: Employment, Equity Ownership. Lee: Catalyst Biosciences: Consultancy. Hong: ISU Abxis: Employment. Siegel: Catalyst Biosciences: Consultancy. Park: ISU Abxis: Employment.

Download Full-text

Effect Of Combined Heparin- and Antithrombin-Binding Exosite Mutations On Human Factor IX(a) Coagulant Activity, Thrombin Generation, and Protease Half-Life In Human Plasma

Blood ◽

10.1182/blood.v122.21.2333.2333 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2333-2333

Author(s):

Pamela R. Westmark ◽

Pansakorn Tanratana ◽

John P. Sheehan

Keyword(s):

Human Plasma ◽

Half Life ◽

Research Funding ◽

Thrombin Generation ◽

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Coagulant Activity ◽

Plasma Half Life

Abstract Introduction Hemophilia B is an X-linked genetic disorder characterized by defective factor IX activity. Recombinant factor IX (rFIX) is employed as protein replacement for the treatment and prophylaxis of bleeding episodes. Antithrombin is the primary plasma inhibitor of activated factor IX (FIXa), and inhibition is enhanced by heparin/heparan sulfate. We hypothesize that selective disruption of protease interactions with heparin and antithrombin via mutations in the respective heparin- and antithrombin-binding exosites may enhance rFIX(a) efficacy by prolonging protease half-life in vivo. Aim To assess the effect of mutations in the FIX(a) heparin- and antithrombin-binding exosites on traditional coagulant activity, thrombin generation, and protease half-life in human plasma. Methods Human FIX cDNA constructs with alanine substitutions (chymotrypsinogen numbering) in the heparin exosite (K126A, K132A, K126A/K132A), antithrombin exosite (R150A), or both (K126A/R150A, K132A/R150A, K126A/K132A/R150A) were expressed in HEK293 cell lines. Recombinant zymogens were purified from conditioned media, and a portion activated to protease with human factor XIa. Zymogen and protease forms were characterized in APTT-based clotting assays, and tissue factor (TF) and FIXa-initiated thrombin generation (TG) assays in pooled human FIX-deficient plasma, respectively. Comparisons were made with human plasma-derived factor IX (pFIX) and recombinant FIX wild type (WT). Protease half-life in pooled, citrated human plasma was determined using a novel assay that detects FIXa activity by TG response. Results Zymogen coagulant activities (% WT ± S.E) were: pFIX 105.2 ± 2.8, WT 100 ± 7.1, K132A/R150A 75.8 ± 3.4, K126A 63.3 ± 2.3, R150A 62.4 ± 4.0, K132A 30.9 ± 1.0, K126A/R150A 27.0 ± 2.1, K126A/K132A 20.6 ± 9.2, and K126A/K132A/R150A 7.3 ± 3.8. Similarly, protease coagulant activities were: WT 100 ± 6.1, pFIXa 98.4 ± 11.4, K132A 91.4 ± 1.6, K132A/R150A 84.9 ± 2.8, R150A 77.1 ± 5.8, K126A 39.5 ± 2.4, K126A/R150A 25.3 ± 2.8, K126A/K132A/R150A 10.9 ± 0.6, and K126A/K132A 9.3 ± 0.6. In contrast to their relative coagulant activities, FIX K126A (1.9-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased peak thrombin concentrations during TF-triggered TG; pFIX, FIX K132A and K126A/R150A were similar to WT; and FIX K126A/K132A/R150A (0.6-fold) and K126A/K132A (0.2-fold) demonstrated marked reductions in peak thrombin relative to WT. In the FIXa-initiated TG assay, FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased peak thrombin concentrations; pFIXa, FIXa K132A, R150A, and K126A (0.8-1.0 fold) were similar to WT; while FIXa K126A/K132A and K126A/K132A/R150A demonstrated markedly reduced (0.2-0.3 fold) and delayed peak thrombin concentrations. In pooled, citrated FIX-deficient plasma, FIXa WT (40.9 ± 1.4 min) and K126A/K132A (37.2 ± 0.7 min) demonstrated similar half-lives, while FIXa R150A, K126A/R150A, and K132A/R150A all had half-lives > 2 hr. Conclusions Single exosite mutations resulted in mild to moderate reductions in coagulant activity, while the double mutation in the heparin exosite (K126A/K132A) markedly reduced activity, likely due to a synergistic effect on cofactor binding. Traditional coagulant activity did not accurately represent the ability of the mutant proteins to support thrombin generation. Despite variable reductions in coagulant activity, FIX K126A, K132A, R150A, K126A/R150A and K132A/R150A supported levels of plasma thrombin generation that were equal to or greater than FIX WT. The plasma half-life of FIXa WT activity was remarkably lengthy, and while mutations in the heparin exosite had negligible effects, R150A in the antithrombin exosite substantially increased protease half-life, consistent with a primary role for antithrombin in the plasma inhibition of FIXa. Thus, single exosite mutations did not significantly disrupt the procoagulant function of human FIX(a), and combined exosite mutations (K126A/R150A and K132A/R150A) maintain or enhance plasma thrombin generation while disrupting exosite-mediated regulatory mechanisms. The combination of intact procoagulant function with disruption of antithrombin- and heparin-mediated regulation of FIX(a) will potentially enhance in vivo recovery, prolong plasma half-life, and enhance the efficacy of hemophilia B replacement therapy. Disclosures: Sheehan: Novo Nordisk Access to Insight Basic Research Grant: Research Funding; Bayer Hemophilia Awards Program: Research Funding; Diagnostica Stago: reagents, reagents Other.

Download Full-text

Purified factor IX using monoclonal immunoaffinity technique: clinical trials in hemophilia B and comparison to prothrombin complex concentrates

Blood ◽

10.1182/blood.v79.3.568.568 ◽

1992 ◽

Vol 79 (3) ◽

pp. 568-575 ◽

Cited By ~ 55

Author(s):

HC Kim ◽

CW McMillan ◽

GC White ◽

GE Bergman ◽

MW Horton ◽

...

Keyword(s):

Monoclonal Antibody ◽

Vitamin K ◽

Half Life ◽

Viral Infections ◽

Factor Ix ◽

Hemophilia B ◽

Terminal Phase ◽

Factor Ix Deficiency ◽

In Vivo Recovery

Abstract Replacement therapy for hemophilia B (factor IX deficiency) using prothrombin complex concentrate (PCC) has been associated with serious complications of thromboembolic events and transmission of viral infections. Monoclonal antibody-purified factor IX (Mononine) provides a highly purified factor IX concentrate, while eliminating other vitamin K-dependent factors (II, VII, and X). Mononine was evaluated for in vivo recovery, half-life, and for its safety and efficacy in 10 patients with hemophilia B. The in vivo recovery of factor IX with Mononine was a 0.67 +/- 0.14 U/dL (mean +/- SD) increase per 1U/kg of infused factor IX, and the biologic half-life (t1/2), determined using the terminal phase of elimination, was 22.6 +/- 8.1 hours. Comparison of in vivo recovery of other vitamin K-dependent factors following a single infusion of either Mononine or PCC showed that, whereas Mononine infusion caused no changes in other vitamin K-dependent factors or in prothrombin activation fragment (F1+2), PCC infusion was associated with significant increases of factors II (2.7 U/dL per 1 U/dL of IX increase) and X (2.2 U/dL for 1 U/dL for 1 U/dL of IX). Patients who used Mononine as their sole therapeutic material during the 12-month period showed an excellent response in hemostasis for their bleeding episodes. Their experience with long-term use of Mononine was at least equivalent to their previous experience with PCC in the frequency and amount of factor usage. No patients developed antibody against mouse IgG or an increase in IX inhibitor during the 12-month period. These results indicate that monoclonal antibody-purified factor IX concentrate provides hemostatically effective factor IX replacement while avoiding extraneous thrombogenic substances.

Download Full-text

A Pharmacokinetic Study of the Plasma-Derived Factor IX AlphaNine® and Its Comparison with the Recombinant Factor IX BeneFIX®, in Previously Treated Patients with Severe Hereditary Hemophilia B.

Blood ◽

10.1182/blood.v110.11.3955.3955 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3955-3955

Author(s):

Vicente R. Cortina ◽

T. Lissichkov ◽

K. Zavilska ◽

M. Matysiak ◽

L. Gercheva ◽

...

Keyword(s):

Half Life ◽

Factor Ix ◽

Hemophilia B ◽

Severe Haemophilia ◽

Open Label ◽

Recombinant Factor Ix ◽

Previously Treated Patients ◽

Previously Treated ◽

In Vivo Recovery

Abstract Objectives The objective of the present study was two fold: first, to determine the pharmacokinetic (PK) profile of the plasma-derived FIX concentrate AlphaNine® in patients with congenital severe haemophilia B (FIX:C 2%). To do this, two PK studies were carried out one six months after the first. The second objective was a comparison of the Alphanine® PK profile with the recombinant Factor IX, BeneFIX®. Patients and methods The first study was a prospective, five-center, open-label, comparative, PK study carried out in 25 severe hemophilia B patients who received 2 single doses of 65–75 IU/kg of AlphaNine® within 6 months (t=0 and t=6). The following parameters were assessed: in vivo recovery, half-life, AUC, mean residence time and clearance. As an extension of the study, a single dose of 65–75 IU/kg of BeneFIX® was administered in 9 out of 25 patients, after a wash-out period of 7–15 days. Results Table 1 summarizes the results obtained when comparing AlphaNine® within a period of time of 6 months (PK1 vs PK2) in 25 patients. Table 2 shows the results obtained when comparing the in vivo recovery of AlphaNine ® vs BeneFIX ® in the 9 patients studied. Conclusions These results confirm that AlphaNine® PK has similar profile as other plasma derived FIX products presently available to treat Hemophilia B patients. In addition, our results show that the recombinant FIX studied, BeneFIX® has a reduced in vivo recovery when is compared to AlphaNine®. Table 1 Parameter AlphaNine® (PK1) t=0 m AlphaNine® (PK2) t=6 m Results are expressed as Mean (SD) In vivo recovery (IU/dl:IU/kg) 1.0 (0.2) 1.2 (0.4) Half-life (h) 34.5 (6.2) 33.7 (5.4) Clearance (ml/min) 0.07 (0.01) 0.07 (0.01) AUC0-inf (IUxh/dl) 1602 (312) 1644 (360) MRT0-inf (h) 35.8 (5.4) 34.6 (5.2) Table 2 Parameter AlphaNine® (PK2) BeneFIX® Results are expressed as Mean (SD); * p<0.05 for the comparison of the in vivo recovery for the BeneFIX® group with the AlphaNine® PK2 In vivo recovery (IU/dl:IU/kg) 1.3 (0.5) 0.8 (0.2)*

Download Full-text

IB1001, an Investigational Recombinant Factor IX, Pharmacokinetics in Pediatric Patients with Hemophilia B.

Blood ◽

10.1182/blood.v120.21.2225.2225 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2225-2225

Author(s):

Edward D. Gomperts ◽

Shashikant Apte ◽

Utpal Chaudhuri ◽

Joseph M John ◽

Vijay Ramanan ◽

...

Keyword(s):

Half Life ◽

Research Funding ◽

Pediatric Patients ◽

Robust Regression ◽

Factor Ix ◽

Hemophilia B ◽

Maximum Plasma ◽

Recombinant Factor Ix ◽

In Vivo Recovery

Abstract Abstract 2225 Introduction IB1001 is a recombinant factor IX product being investigated for the treatment and prevention of bleeding in individuals with hemophilia B. Pharmacokinetics (PK) in adults (>12 years) demonstrated that IB1001 had results similar to the currently available recombinant FIX with respect to parameters such as terminal phase half-life and incremental recovery. We report the interim findings from a PK assessment in children <12 years, with severe hemophilia B (FIX <2%), >50 prior exposure days to FIX, and no history of or currently detectable inhibitor to FIX. Methods Non-randomized, open-label PK study with patients receiving 75±5 IU/kg of IB1001 following a washout period of ≥4 days from a previous FIX infusion. Factor IX levels were determined pre-infusion and at 15–30 minutes, 4–6, 24–26, and 68–72 hours post-infusion. Additional samples could be drawn at 1–3 and 10–14 hours. Calculated PK parameters were: half-life (β-phase t1/2, determined using a robust regression approach [Lee ML et al. XVIth ISTH Congress, Florence, Italy, 1997]) but generally assuming a single compartmental model because of the small number of points, maximum plasma concentration (Cmax), in vivo recovery (IVR) and AUC(0-∞) (determined by the trapezoidal rule). In addition, the AUC(0-t) and mean residence time (MRT) were calculated. Results When compared to the findings previously reported with IB1001 in adult (≥12 years of age) subjects (Martinowitz U et al. Haemophilia, 18, 2012), the results in pediatric patients demonstrate a more rapid metabolism of factor IX as is indicated by the shorter terminal half-life (mean±SD of 19.3±7.8 h versus 29.6±18.2 h in adults) and the smaller AUC0-∞ (mean±SD of 1059±264 versus 1668±598 in adults). In addition, the in vivo recovery was lower (mean±SD of 0.69±0.21) versus that seen in adults (mean±SD of 0.98±0.22). These results are similar to those reported by Berntorp et al (Haemophilia, 7, 2001) with nonacog alfa. Conclusions The pharmacokinetics of IB1001 has previously been shown to be non-inferior to nonacog alfa, another recombinant factor IX, in hemophilia B individuals >12 years of age. The current study is intended to provide information on children <12 and, particularly, <6 years of age. IB1001 is metabolized faster and has a lower recovery than the comparable findings in patients >12 years of age. Although the study is ongoing, these may represent important implications for the potential use of IB1001 in pediatric patients. Disclosures: Gomperts: Inspiration Biopharmaceuticals Inc: Consultancy. Apte:Inspiration Biopharmacauticals Inc: Research Funding. Chaudhuri:Inspiration Biopharmaceuticals Inc: Research Funding. John:Inspiration Biopharmaceuticals Inc: Research Funding. Ramanan:Inspiration Biopharmaceuticals Inc: Research Funding. Liesner:Inspiration Biopharmaceuticals Inc: Research Funding. Shapiro:Inspiration Biopharmaceuticals Inc: Honoraria, Research Funding. Mills:Inspiration Biopharmaceuticals Inc: Employment. Lee:Inspiration Biopharmaceuticals Inc: Employment.

Download Full-text

In vivo recovery and half-life time of a steam-treated factor IX concentrate in hemophilia B patients

Blut Zeitschrift für die gesamte Blutforschung ◽

10.1007/bf00320754 ◽

1988 ◽

Vol 57 (6) ◽

pp. 341-345 ◽

Cited By ~ 11

Author(s):

M. K�hler ◽

E. Seifried ◽

P. Hellstern ◽

G. Pindur ◽

C. Miyashita ◽

...

Keyword(s):

Half Life ◽

Life Time ◽

Factor Ix ◽

Hemophilia B ◽

In Vivo Recovery

Download Full-text

Pharmacokinetics of Human Factor IX in a dog with Severe Hemophilia B.

10.1055/s-0039-1682760 ◽

1977 ◽

Author(s):

P.A. Gentry ◽

A.R. Thompson ◽

A.W. Forrey

Keyword(s):

Human Factor ◽

Numerical Procedure ◽

Factor Ix ◽

Hemophilia B ◽

High Yield ◽

Severe Hemophilia ◽

Activity Data ◽

Human Factor Ix

In preparing a factor IX concentrate with a high yield and low hepatitis and thromboembolic risks, we have tested this material for survival in an in vivo system, the hemophiliac dog. By following the disappearance of radiolabeled, isolated factor IX in addition to the classic clotting assays, data on protein survival and more accurate kinetic parameters were obtained.Crude factor IX concentrate was prepared by batchwise adsorption-elution with DEAE-Sephadex using cryoprecipitate-poor human plasma. Isolated human factor IX was radiolabeled with 125I by chloramine-T without in vitro loss of clotting activity (Thompson, J Clin Invest, in press, 1977). A preparation containing both crude and isolated factor IX was then subjected to filtration (0.22 μm) and lyophilization; clotting and radioactivity were not altered by these steps.Following infusion of the combined preparation into a dog with severe hemophilia B (0% baseline factor IX) 10 post infusion samples were taken over 96 h for determination of radioactivity and factor IX clotting activity. These data were then analyzed by fitting to a two exponential expression using a Marquart non-linear least squares numerical procedure for a two compartment open model. The central volume was 14.5% of the animal’s body weight; the total volume of distribution was 28% with a t 1/2 distribution of 114 min. The t 1/2 elimination was 20 h; the slower phase of elimination (β, or that affected by redistribution) had a t 1/2 of 40 h. Factor IX clotting activity from the crude concentrate closely paralleled radioactivity from the isolated factor IX throughout the 96 h; t 1/2 β was slightly longer from the clotting activity data.

Download Full-text

Purified factor IX using monoclonal immunoaffinity technique: clinical trials in hemophilia B and comparison to prothrombin complex concentrates

Blood ◽

10.1182/blood.v79.3.568.bloodjournal793568 ◽

1992 ◽

Vol 79 (3) ◽

pp. 568-575

Author(s):

HC Kim ◽

CW McMillan ◽

GC White ◽

GE Bergman ◽

MW Horton ◽

...

Keyword(s):

Monoclonal Antibody ◽

Vitamin K ◽

Half Life ◽

Viral Infections ◽

Factor Ix ◽

Hemophilia B ◽

Terminal Phase ◽

Factor Ix Deficiency ◽

In Vivo Recovery

Replacement therapy for hemophilia B (factor IX deficiency) using prothrombin complex concentrate (PCC) has been associated with serious complications of thromboembolic events and transmission of viral infections. Monoclonal antibody-purified factor IX (Mononine) provides a highly purified factor IX concentrate, while eliminating other vitamin K-dependent factors (II, VII, and X). Mononine was evaluated for in vivo recovery, half-life, and for its safety and efficacy in 10 patients with hemophilia B. The in vivo recovery of factor IX with Mononine was a 0.67 +/- 0.14 U/dL (mean +/- SD) increase per 1U/kg of infused factor IX, and the biologic half-life (t1/2), determined using the terminal phase of elimination, was 22.6 +/- 8.1 hours. Comparison of in vivo recovery of other vitamin K-dependent factors following a single infusion of either Mononine or PCC showed that, whereas Mononine infusion caused no changes in other vitamin K-dependent factors or in prothrombin activation fragment (F1+2), PCC infusion was associated with significant increases of factors II (2.7 U/dL per 1 U/dL of IX increase) and X (2.2 U/dL for 1 U/dL for 1 U/dL of IX). Patients who used Mononine as their sole therapeutic material during the 12-month period showed an excellent response in hemostasis for their bleeding episodes. Their experience with long-term use of Mononine was at least equivalent to their previous experience with PCC in the frequency and amount of factor usage. No patients developed antibody against mouse IgG or an increase in IX inhibitor during the 12-month period. These results indicate that monoclonal antibody-purified factor IX concentrate provides hemostatically effective factor IX replacement while avoiding extraneous thrombogenic substances.

Download Full-text

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

Blood ◽

10.1182/blood-2011-02-336172 ◽

2011 ◽

Vol 118 (8) ◽

pp. 2333-2341 ◽

Cited By ~ 95

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.

Download Full-text

Pharmacokinetics of Subcutaneously Administered CB2679d/ISU304 in Wild-Type and Hemophilia B Mice

Blood ◽

10.1182/blood.v128.22.1389.1389 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1389-1389 ◽

Cited By ~ 1

Author(s):

Seung-Beom Hong ◽

Howard Levy ◽

Jae Yong Jung ◽

Minkyung Park ◽

A Rim Seo ◽

...

Keyword(s):

Specific Activity ◽

Phase 1 ◽

Sandwich Elisa ◽

High Specific Activity ◽

Biological Properties ◽

Factor Ix ◽

Hemophilia B ◽

Wild Type

Abstract The rapid clearance of factor IX (FIX) necessitates frequent intravenous IV administrations to achieve effective prophylaxis for patients with hemophilia B (HB). Subcutaneous (SC) administration would be a preferred route of administration but has been limited by low bioavailability and potency of the marketed FIX products. CB2679d/ISU304 with enhanced biological properties was developed using a rational protein design approach and has resistance to inhibition by ATIII, increased affinity for FVIIIa, increased catalytic activity and resultant 20-fold enhanced potency in vitro (clotting activity) and in vivo (the tail clip model) and 8-fold increased duration of aPTT activity in vivo compared to recombinant wild-type FIX dosed at the same mass. ISU304 (4622 IU/mg) was injected into HB mice SC with a single dose ISU304 at 0.02, 0.05 or 0.15 mg/kg and sampled at 4, 6, 8, 24 hours. Groups of wild-type mice received ISU304 0.02, 0.05 or 0.15 mg/kg SC and BeneFIX (273 IU/mg) 0.15 mg/kg SC, and sampled at 0.25, 1, 4, 8, 24, 48, 72, and 96 hours. Daily SC injection in HB mice of ISU304 at 0.05 mg/kg was sampled at 24, 48, 72 and 96 hours. FIX antigen was measured using a sandwich ELISA and FIX activity was measured using a one-stage clotting assay on Stago Compact. Pharmacokinetics of FIX was performed using PKSolver. There was a dose-dependent increase of plasma FIX antigen with SC ISU304. Mass-based pharmacokinetic profiles of ISU304 (t1/2, 18 hours; Tmax,8 hours, Bioavailability, 19-22%) were similar to those of BeneFIX (t1/2, 20 hours; Tmax, 8 hours, Bioavailability, 16%). Due to ISU304 high specific activity, SC dose of ISU304 yields much higher FIX activities in mouse plasma compared with the same mass dose of BeneFIX. Daily SC dosing of ISU304 230 IU/kg reached steady-state plateau FIX 8% activity after three injections. The bioavailability and increased potency of CB2679d/ISU304 facilitates the initiation of the Phase 1 subcutaneous dosing study in individuals with hemophilia B. Figure 1 Figure 1. Table 1 Table 1. Figure 2 Figure 2. Disclosures Hong: ISU Abxis: Employment. Levy:Catalyst Biosciences: Employment. Jung:ISU Abxis: Employment. Park:ISU Abxis: Employment. Seo:ISU Abxis: Employment. Seo:ISU Abxis: Employment. Madison:Catalyst Biosciences: Employment, Equity Ownership, Patents & Royalties.

Download Full-text

Posttranslational modifications of recombinant myotube-synthesized human factor IX

Blood ◽

10.1182/blood.v97.1.130 ◽

2001 ◽

Vol 97 (1) ◽

pp. 130-138 ◽

Cited By ~ 83

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.

Download Full-text