ABSTRACT
Turoctocog alfa (NovoEight®) is a new recombinant factor VIII (rFVIII) with a truncated B domain and a high degree of tyrosine sulphation, similar to plasma-derived FVIII products. The manufacturing process includes double nanofiltration with a 20-nm pore size and immunoaffinity chromatography with monoclonal F25 anti-FVIII antibodies. Treatment with turoctocog alfa can be monitored with both one-stage and chromogenic substrate assays without a product-specific laboratory standard. In total, 213 previously-treated patients with severe haemophilia A participated in the pivotal part of the clinical trial programme guardianTM. The median annualised bleeding rate during turoctocog alfa prophylaxis was 3.7 and 3.0 in adolescents/adults and children, respectively, with marked differences between participating countries. The success rate for the treatment of breakthrough bleeds was 85% (adults/adolescents) and 94% (children). A total of 41 surgical procedures (15 major, 26 minor) were performed in 33 patients, with a successful haemostatic response reported in all cases. No patient developed confirmed inhibitors in any of the trials.
Subject(s)
Drug Compounding/methods , Factor VIII/administration & dosage , Factor VIII/chemical synthesis , Hemophilia A/drug therapy , Hemophilia A/prevention & control , Protein Engineering/methods , Adolescent , Child , Child, Preschool , Dose-Response Relationship, Drug , Evidence-Based Medicine , Factor VIII/genetics , Female , Hemophilia A/diagnosis , Hemostatics/administration & dosage , Hemostatics/chemical synthesis , Humans , Infant , Infant, Newborn , Male , Treatment OutcomeABSTRACT
Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser558-Gln565 region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa-FIXa interaction. Based on this, a series of linear peptides, analogs of the 558-565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.
Subject(s)
Anticoagulants/chemical synthesis , Anticoagulants/metabolism , Factor VIII/chemical synthesis , Factor VIII/metabolism , Peptides/chemical synthesis , Peptides/metabolism , Solid-Phase Synthesis Techniques/methods , Amino Acid Motifs , Amino Acid Sequence , Anticoagulants/chemistry , Drug Design , Factor IXa/metabolism , Factor VIII/chemistry , Factor VIII/genetics , Factor X/metabolism , Humans , Molecular Sequence Data , Peptides/chemistry , Peptides/genetics , Protein BindingABSTRACT
Treatment with solvent/detergent is a widely used method for ensuring the virus safety of plasma products. In the present study, virus inactivation by a novel solvent/detergent combination, i.e. TnBP (tri-n-butyl phosphate) and polysorbate 20 during the manufacture of the factor VIII/VWF concentrate Optivate has been investigated. The inactivation of most enveloped viruses was rapid, i.e. > 5 log in 2 min, although the inactivation of vaccinia virus was slower, i.e. 4 log in 1h. Virus inactivation was effective over a wide range of conditions, i.e. solvent/detergent concentration, protein concentration and temperature, irrespective of whether tested individually or in combination. This confirms the effectiveness and robustness of this alternative version of the solvent/detergent procedure, and allows appropriate control limits to be set for this manufacturing step. Polysorbate 20 provides an alternative to the non-ionic detergents currently in use with the solvent/detergent procedure.
Subject(s)
Detergents/pharmacology , Factor VIII/standards , Polysorbates/pharmacology , Virus Inactivation , von Willebrand Factor/standards , Animals , Cell Survival/drug effects , Cells, Cultured , Chemical Precipitation , Chlorocebus aethiops , Drug Combinations , Drug Contamination/prevention & control , Factor VIII/chemical synthesis , Factor VIII/chemistry , Organophosphates/pharmacology , Solvents/pharmacology , Temperature , Vero Cells , Virus Diseases/blood , Virus Diseases/pathology , Virus Diseases/prevention & control , von Willebrand Factor/chemical synthesis , von Willebrand Factor/chemistryABSTRACT
Peptides that mimic protein epitopes are interesting drug candidates. However, the design of effective peptidic drugs is difficult for several reasons, such as the fast degradation of peptides, their high flexibility, and thus high entropy loss on binding to the target. We therefore propose an in silico method for the automated design of peptides that are optimal with respect to several objectives. We present a Pareto-based multiobjective evolutionary algorithm for in silico peptide design. Using a simple molecular model, we apply the method to the design of peptides that (a) mimic antibody epitopes of the proteins thrombin and blood coagulation factor VIII, respectively, that (b) are short, and (c) are conformationally stable.
Subject(s)
Computational Biology , Directed Molecular Evolution/methods , Epitopes/genetics , Peptide Fragments/genetics , Algorithms , Epitopes/chemistry , Factor VIII/chemical synthesis , Factor VIII/genetics , Humans , Mutation , Peptide Fragments/chemical synthesis , Protein Folding , Thrombin/chemical synthesis , Thrombin/geneticsABSTRACT
The monoclonal anti-factor VIII (FVIII) antibody C4 has previously been reported to inhibit the binding of purified FVIII to immobilized von Willebrand factor (vWF). The binding area of C4 was identified to be within fifteen amino acid residues (1670-1684) based on the ability of a synthetic FVIII peptide consisting of amino acid residues 1670-1684 to completely inhibit the binding of C4 to FVIII. We now report the further localization of the binding region of C4 to within eight amino acid residues (1677-1684) of FVIII light chain. Nine new overlapping FVIII peptides were synthesized based on the amino acid sequence of the acidic region of FVIII light chain and tested, along with seven previously tested peptides, for the ability to inhibit C4 binding to FVIII in an ELISA assay. Three synthetic FVIII peptides 1670-1684, 1675-1690, and 1677-1684 demonstrated dose dependent inhibition of C4 binding to FVIII. The three reactive peptides contain residues 1677-1684 in common. Since C4 can completely inhibit the binding of FVIII to vWF, this report further localizes an eight amino acid residue region of FVIII which may be important in the mediation of vWF binding.
Subject(s)
Factor VIII/metabolism , Peptide Fragments/metabolism , von Willebrand Factor/metabolism , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Binding Sites, Antibody , Binding, Competitive , Enzyme-Linked Immunosorbent Assay , Factor VIII/chemical synthesis , Factor VIII/immunology , Humans , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Protein Binding/immunologyABSTRACT
The biotechnology industry is thriving, and many predicted accomplishments have actually occurred during the last decade. Cloning and expression of genetic information is now simple and routine. Initial commercial products have been realized, but there is much yet to be accomplished in evaluating the clinical significance of many other gene products made available by biotechnology resources. During the next decade, human health care and the pharmaceutical industry should be affected substantially by first- and second-generation recombinant DNA products. Recombinant vaccines, blood coagulation factors, and known biological modulators produced by rDNA technologies should be widely used. Further opportunities will be realized with increasing discoveries of new bioactive molecules and identification of NANB hepatitis and AIDS infectious agents. Full exploitation of health care products will depend on innovative new delivery systems or the ability to reconstruct mammalian and plant genes, providing for in-situ delivery of the necessary gene products.
