			Home About us Contact

Human Factor VIII (human + factor_viii)

Distribution by Scientific Domains

Life Sciences	44%
Medical Sciences	33%

Selected Abstracts

Characteristics of Rabbit Transgenic Mammary Gland Expressing Recombinant Human Factor VIII

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2009
P. Chrenek
Summary The objective of this research was to compare (i) the content of milk protein and recombinant human factor VIII (rhFVIII) in the milk of transgenic and non-transgenic rabbit females at three lactations and (ii) histological structure, ultrastructural morphology and occurrence of apoptosis in rabbit transgenic and non-transgenic mammary gland during third lactation and involution. Significant differences (t0.05) in milk protein content were found between transgenic and non-transgenic at all three lactations. The percentage of apoptotic cells was significantly higher (t0.01) in non-transgenic ones compared with transgenic mammary gland tissues (6.5% versus 2.4%) taken at the involution stage. Morphometrical analysis of histological preparations at the involution stage detected a significantly higher (t0.05) relative volume of lumen in transgenic animals compared with non-transgenic ones (60.00 versus 46.51%). Ultrastructural morphology of the transgenic mammary gland epithelium at the involution stage revealed an increased relative volume of protein globules (t0.05); at the lactation stage, a significantly higher volume of mitochondria (13.8%) compared with the non-transgenic (9.8%) ones was observed. These results, although revealing differences in some parameters of ultrastructure and histology, indicate no harmful effect of the mouse whey acid protein-hFVIII transgene expression on the state of mammary gland of transgenic rabbit females. [source]

Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation

ELECTROPHORESIS, Issue 16 2010
Gian Maria D'Amici
Abstract Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate®, Helixate NexGen® and Refacto®), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGen® showed the presence of impurities, such as Hsp70,kDa, haptoglobin and proapolipoprotein; Refacto® showed glutathione S -transferase and ,-lactamase, whereas Advate® apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants. [source]

Evaluation of pharmacokinetics, efficacy and safety of Immunate® solvent detergent in previously treated patients with severe haemophilia A

HAEMOPHILIA, Issue 1 2007
L. NEMES
Summary., Immunate® Solvent Detergent (S/D) is a plasma derived, purified, human factor VIII (FVIII) , von Willebrand factor (VWF) complex subjected to two virus inactivation/removal processes: S/D and vapor heat treatment. This prospective, multicentre, three-part clinical study evaluated the pharmacokinetics (in comparison to the predecessor product Immunate®), efficacy and safety of Immunate® S/D in 56 previously treated patients with severe haemophilia A. Subjects received Immunate® S/D on-demand, as a prophylactic regimen or both. The results of the pharmacokinetic population demonstrate that Immunate® and Immunate® S/D were equivalent with respect to the FVIII , and to the retrospectively VWF , parameters assessed. A total of 623 bleeding episodes were reported in 47/56 subjects. The duration of prophylaxis ranged from 0.1--5.2 months with a total of 175.6 months. The median number of bleeds per month in subjects on prophylaxis was 0 (range 0--10). Ninety-six percent of bleeding episodes were rated as having an excellent or good response. For most bleeding episodes (89%), subjects required only one infusion with a mean dose of 29.6 IU kg,1. No FVIII inhibitory antibodies were observed in any subject. No related serious adverse events were reported. Thus, the introduction of S/D treatment did not alter the PK characteristics and function of VWF and FVIII molecules of Immunate® S/D which is effective and safe for treatment of bleeding episodes, management of surgical procedures, and prophylaxis. [source]

Effect of metal cations on the conformation and inactivation of recombinant human factor VIII

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2004
Tiffany S. Derrick
Abstract Heavy metals have been implicated in the aggregation of proteins and the pathophysiology of several neurodegenerative diseases. Herein, we describe the interaction of recombinant human factor VIII (rhFVIII) with Al+3, Tb+3, Co+2, and Fe+3 using a combination of intrinsic fluorescence, circular dichroism, and high-resolution fourth-derivative absorbance analysis. rhFVIII in solution was titrated with the metal cations and the properties of the resulting complexes were examined. rhFVIII has a tendency to aggregate and inactivate slowly over time under physiological conditions, but this aggregation process is greatly accelerated in the presence of metals with Al+3 being the most efficient. This leads to a complete loss of activity of the protein. Al+3 -induced conformational changes in the protein were small but detectable with limited changes seen in secondary and tertiary structure. Because rhFVIII is a multidomain protein with subunits linked through divalent metal cations, the small intramolecular changes seen may be attributed to rearrangements of the subunits to an aggregation-competent conformer that is very similar to that of the native form. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:2549,2557, 2004 [source]

Establishment of embryonic stem cells secreting human factor VIII for cell-based treatment of hemophilia A

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2008
S. KASUDA
Summary.,Background:,Hemophilia A is an X-chromosome-linked recessive bleeding disorder resulting from an F8 gene abnormality. Although various gene therapies have been attempted with the aim of eliminating the need for factor VIII replacement therapy, obstacles to their clinical application remain. Objectives:,We evaluated whether embryonic stem (ES) cells with a tetracycline-inducible system could secrete human FVIII. Methods and results:,We found that embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted human FVIII after doxycycline induction. Moreover, use of a B-domain variant F8 cDNA (226aa/N6) dramatically enhanced FVIII secretion. Sorting based on green fluorescent protein (GFP),brachyury (Bry) and c-kit revealed that GFP,Bry+/c-kit+ cells during EB differentiation with serum contain an endoderm progenitor population. When GFP,Bry+/c-kit+ cells were cultured under the liver cell-promoting conditions, these cells secreted FVIII more efficiently than other populations tested. Conclusion:,Our findings suggest the potential for future development of an effective ES cell-based approach to treating hemophilia A. [source]

Comparative immunogenicity of recombinant B domain-deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2004
E. T. Parker
Summary., Hyate is a commercial plasma-derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI-1 is a recombinant B domain-deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI-1 or Hyate:C in a comparative immunogenicity trial. OBI-1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg,1. Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI-1 or Hyate:C at doses of 1 or 10 U kg,1, but were identified in 81% and 94% of mice given 100 U kg,1 of OBI-1 or Hyate:C, respectively. There was no significant difference between OBI-1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI-1-treated mice at 10 U kg,1 (P = 0.09). Total anti-FVIII antibodies to Hyate:C and OBI-1 were also measured by ELISA using immobilized purified plasma-derived porcine FVIII and OBI-1, respectively, as antigens. At the 10 and 100 U kg,1 doses, the mean anti-FVIII response was higher in Hyate:C-treated-mice than in OBI-1-treated mice (P = 0.02 and P = 0.004, respectively). The results using this model suggest that OBI-1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients. [source]

Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice

THE JOURNAL OF GENE MEDICINE, Issue 10 2004
Jiro Kikuchi
Abstract Background An Erratum has been published for this article in Journal of Gene Medicine 7(6), 2005, 836. Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). Methods CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied. Results We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 ± 0.1%) at MOI of 5 × 103 vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 ± 20.1 ng) from 106 CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5,10 × 105) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 ± 0.9 ng/mL, maximum 3.6 ± 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. Conclusions Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients. Copyright © 2004 John Wiley & Sons, Ltd. [source]