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

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.

scholarly journals Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5095-5103 ◽  
Author(s):  
G Hortelano ◽  
A Al-Hendy ◽  
FA Ofosu ◽  
PL Chang
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Ex Vivo ◽  
Cost Effective ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapy Strategy ◽  
Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

Adenovirus-mediated regulatable Expression of human Factor IX in vitro and in vivo

2003 ◽  
pp. 72-80
Author(s):  
M. A. Srour ◽  
H. Fechner ◽  
X. Wang ◽  
U. Siemetzki ◽  
T. Albert ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445 ◽  
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Abstract Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

Mesenchymal Stromal Cells Gene-Enhanced To Express Human Factor IX and Their In Vivo Use for Correction of Hemophilia B Phenotype in R333Q Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5136-5136
Author(s):  
Daniel L. Coutu ◽  
Jessica Cuerquis ◽  
May Griffith ◽  
Mark D. Blostein ◽  
Jacques Galipeau
Keyword(s):  
Gene Therapy ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Monogenic Disease ◽  
Human Factor Ix ◽  
B Mice

Abstract Hemophilia B is considered an appropriate disease target for gene therapy because it is a well characterized monogenic disease with a large therapeutic index. Despite promising preclinical and clinical trials in the last decade, safety and efficacy concerns associated with the in vivo administration of viral vectors still need to be addressed before gene therapy becomes part of the standard arsenal for clinicians. Our laboratory has developed a cell therapy approach using gene-enhanced autologous Mesenchymal Stromal Cells (MSCs) to deliver a therapeutic plasmatic protein which addresses these safety concerns. In this study, we tested whether MSCs engineered to express human Factor IX (hFIX) can be used to reverse the bleeding phenotype of R333Q hemophilia B mice developed by Stafford et al. We retrovirally engineered MSCs harvested from normal C57Bl/6 to express hFIX. A gene enhanced polyclonal population of MSCs was capable of producing carboxylated and fully active hFIX by in vitro clotting assays. By ELISA, the cells were shown to produce approximately 250ng of hFIX per million cells per 24h. Ten million of these cells were embedded in a collagen I gel matrix and implanted subcutaneously in R333Q hemophilia B mice (n=10). hFIX activity in mouse plasma (test and control groups) were followed weekly by aPTT assays. hFIX activity reached levels as high as 20% normal activity in some animals with an average +/− SEM of 11.2 +/− 2.1 (FIX activity in controls is <1%). The hFIX activity returned to baseline within 4 weeks. In conclusion, we demonstrate that gene-enhanced autologous MSCs can serve as an effective delivery of functional FIX for temporary correction of the hemophilia B phenotype. We hypothesize the presence of GFP co-expression by the implanted MSCs caused their immune rejection and we are currently testing this hypothesis.

scholarly journals Hemophilia B: Genetic Variants and Carrier Detection

1977 ◽  
Author(s):  
C.K. Kasper ◽  
B. Østerud ◽  
S.I. Rapaport
Keyword(s):  
Prothrombin Time ◽  
Genetic Variants ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Carrier Detection ◽  
Severe Hemophilia ◽  
Human Factor Ix ◽  
Normal Women

In 92 males with hemophilia B from 71 kindreds, we measured factor IX activity, prothrombin time using bovine thromboplastin (bovine tpln time), and factor IX antigen both by inhibitor neutralization using a human factor IX inhibitor and by electroimmunoassay using a precipitating rabbit anti-human-factor IX antiserum. Eighty patients with 3% or less factor IX activity could be divided into 4 groups:(1) 7 patients with greatly prolonged bovine tpln times and normal levels of factor IX antigen;(2) 17 patients with mildly prolonged bovine tpln times and factor IX antigen levels between about 25% and normal;(3) 8 patients with normal bovine tpln times and antigen levels between about 25% and normal; (4) 48 patients with normal bovine tpln times and no measureable antigen excess. Some of the latter group were also tested with a chicken anti-human-factor IX antiserum and no antigen was found. None of 12 patients with mild hemophilia B (factor IX activity of 4 to 22%) had a prolonged bovine tpln time although 4 patients had excess factor IX antigen over activity. Thus, about 1/3 of these 92 hemophilia B patients had evidence of an abnormal factor IX molecule. Factor IX activity was also measured in 48 normal women and in 51 definite carriers of severe hemophilia B. Probability curves were derived to estimate the chance of a woman being a carrier based upon her factor IX level and her degree of kinship to a definite carrier. The relation between factor IX activity and antigen was also delineated for normal women and for carriers. In kindreds in which affected males had excess antigen, some carriers could be distinguished from normal women on the basis of excess antigen over activity. In appropriate kindreds, prolonged bovine tpln times helped distinguish some carriers.

scholarly journals Recombinant human factor IX: replacement therapy, prophylaxis, and pharmacokinetics in canine hemophilia B

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2603-2610 ◽  
Author(s):  
KM Brinkhous ◽  
JL Sigman ◽  
MS Read ◽  
PF Stewart ◽  
KP McCarthy ◽  
...  
Keyword(s):  
Replacement Therapy ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Degree Of Saturation ◽  
Prophylactic Regimen ◽  
Human Factor Ix ◽  
In Vivo Testing ◽  
Recombinant Human Factor Ix

Recombinant human factor IX (rFIX) has been expressed in transduced cultured cell systems since 1985. Because there has been limited in vivo testing of rFIX in hemophilia B subjects, this study was undertaken using the severe hemophilia B canines of the Chapel Hill strain. Three groups of hemophilic dogs received either 50, 100, or 200 IU/kg of rFIX. As a control, a fourth group of hemophilic dogs received 50 IU/kg of a high purity, plasma-derived human FIX (pdFIX). The coagulant and hemostatic effects of rFIX and pdFIX were similar with all comparative dosing regimens. Based on activity data, the elimination half-life of rFIX was 18.9 +/- 2.3 hours and pdFIX was 17.9 +/- 2.1 hours. A prophylactic regimen administering rFIX daily resulted in a continuous therapeutic level of plasma FIX and was accompanied by a two-fold increase in recovery levels by day 5, compared to that observed with administration of a single bolus. The mechanisms of the high to complete recovery of FIX with the prophylactic regimen could depend not only on the degree of saturation of the vascular endothelial binding sites but also on the altered dynamics of the balance of FIX distribution between the intravascular and extravascular compartments. The pharmacokinetic (PK) parameters for rFIX and pdFIX were similar. However, the relative PK values for V1 and V5s of both products on day 5 differed greatly from day 1 and may reflect the changing equilibrium of FIX between compartments with elevated levels of plasma FIX. Neutralizing antihuman FIX antibodies resulting from human FIX antigen being administered to FIX deficient dogs were observed beginning at 14 days. The antigenicity of rFIX and pdFIX appeared to be comparable. Despite the very different procedures used for production of rFIX and pdFIX products, in vivo testing in hemophilia B dogs showed the functional behavior of these products is similar; they are highly effective for replacement therapy and for prophylaxis.

Neutrophil Elastase Cleavage of Human Factor IX Generates an Activated Factor IX-Like Product Devoid of Coagulant Function

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1287-1296 ◽  
Author(s):  
John A. Samis ◽  
Eunice Kam ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Active Site ◽  
Neutrophil Elastase ◽  
Human Factor ◽  
Sodium Dodecyl ◽  
Factor Ix ◽  
Human Neutrophil Elastase ◽  
Human Factor Ix ◽  
Sds Page

Abstract In preliminary studies, the generation of thrombin in vivo was found to induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F.IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost 69:1127, 1993 [abstr]). These changes were associated with evidence of increased elastase availability. To study the possibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F.IX by human neutrophil elastase (HNE) was performed in vitro. An activated partial thromboplastin time (aPTT) clotting assay demonstrated that, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that could not be distinguished from the respective heavy and light chain peptides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators. In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the active site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3) activate human factor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal sequencing indicated that HNE cleaved human F.IX at Thr140, Thr144, Ile164, Thr172, and Val181. The cleavages at Thr140/Thr144 and at Thr172/Val181 are both very close to the normal F.XIa -(Arg145) and β-(Arg180) cleavage sites, respectively. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of improper active site formation. These in vitro observations support the possibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as disseminated intravascular coagulation (DIC). © 1998 by The American Society of Hematology.

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