Improving the pharmacodynamics of protein drugs has the potential to improve the care and the qua... more Improving the pharmacodynamics of protein drugs has the potential to improve the care and the quality of life of patients suffering from a variety of diseases. Four approaches to improve protein drugs are described: PEGylation, amino acid substitution, fusion to carrier proteins and encapsulation. A new platform technology based on the binding of proteins/peptides to the outer surface of PEGylated liposomes (PEGLip) is then presented. Binding of proteins to PEGLip is non-covalent, highly specific and dependent on an amino acid consensus sequence within the proteins. Association of proteins with PEGLip results in substantial enhancement of the pharmacodynamic properties of proteins following administration. This has been demonstrated in preclinical studies and clinical trials with coagulation factors VIII and VIIa. It has also been demonstrated in preclinical studies with granulocyte colony-stimulating factor. A mechanism is presented that explains the improvements in hemostatic efficacy of PEGLip-formulated coagulation factors VIII and VIIa. The reader will gain an understanding of the advantages and disadvantages of each of the approaches discussed. PEGLip formulation is an important new approach to improve the pharmacodynamics of protein drugs. This approach may be applied to further therapeutic proteins in the future.
Summary. A precise and sensitive chromogenic assay for the measurement of very low levels of fac... more Summary. A precise and sensitive chromogenic assay for the measurement of very low levels of factor VIII (FVIII) in plasma has been developed. The assay is based on modifications of a commercially available chromogenic assay. The modifications include reduction of sample final dilution factor and prolongation of the development period. The modified assay allows accurate and precise measurement of FVIII in the range of 0.001–0.02 IU mL−1. The detection limit is 0.0005 IU mL−1 and the quantitation limit is 0.0015 IU mL−1. This assay can be used in research and to study the clinical efficacy of low circulating levels of FVIII in haemophilia A patients.
Summary. Recombinant activated factor VII (rFVIIa) is an effective treatment of the haemophilia ... more Summary. Recombinant activated factor VII (rFVIIa) is an effective treatment of the haemophilia patient with inhibitors and acquired haemophilia. However, on account of its relatively short half-life (HL), achieving therapeutic efficacy with FVIIa requires repeated injections. The development of a long-acting FVIIa product would therefore be beneficial. The formulation of factor VIII with PEGylated liposomes (PEGLip) was previously shown to extend the bleeding-free period in haemophilia patients. We report here an enhancement of haemostatic efficacy by similarly formulating FVIIa with PEGLip. Surface plasmon resonance analysis indicated that FVIIa binds non-covalently but with high affinity and specificity to PEGLip. A one-stage clotting assay demonstrated that formulation of FVIIa with PEGLip does not affect FVIIa activity and stability. A pharmacokinetic study in rats demonstrated that PEGLip formulation of FVIIa extends circulation time and results in higher FVIIa levels several hours after injection. Thromboelastography experiments indicated that PEGLip-FVIIa induces faster clot formation and higher clot stability than standard formulated FVIIa. These results suggest that formulation of FVIIa with PEGLip may lead to a safe and effective long-acting FVIIa that improves the care of haemophilic patients with inhibitors and acquired haemophilia.
An A-->G transition in the acceptor splice site at the intron 19/exon 20 junction of the g... more An A-->G transition in the acceptor splice site at the intron 19/exon 20 junction of the glycoprotein IIb gene was defined as a novel mutation causing Glanzmann thrombasthenia in two Iraqi-Jewish siblings. This mutant DNA was transcribed into four distinct species of mRNA, one of which resulted in a premature termination codon and the other three predicting deletions of 50, 61 or 72 amino acids, respectively.
Summary. Recombinant activated factor VIIa (FVIIa) is a bypassing agent used to treat bleeding e... more Summary. Recombinant activated factor VIIa (FVIIa) is a bypassing agent used to treat bleeding episodes in haemophilia patients with inhibitors to factor VIII (FVIII) and factor IX. The pharmacological effect of FVIIa is short-lived and therefore with the recommended dose of 90 μg kg−1, a bleeding episode is treated with multiple injections. A long-acting form of FVIIa that can ensure adequate haemostasis with a single infusion, without increasing the thrombotic risk, would therefore be beneficial. PEGylated liposomes (PEGLip) have been shown to bind FVIIa and to improve haemostatic efficacy in preclinical experiments. In the present phase I/II clinical trial, we assessed the safety and efficacy of PEGLip-formulated FVIIa in severe haemophilia A patients (FVIII ≤ 1%) with inhibitors to FVIII. Each patient received one prophylactic infusion of standard FVIIa and one prophylactic infusion of PEGLip-formulated FVIIa. The order of the infusions was randomized and the two infusions were separated by a ten-day washout period. Efficacy assessed by thromboelastography revealed that PEGLip-FVIIa induced significantly shorter clotting times and produced higher clot firmnesses than standard FVIIa. Thrombin generation assays showed that PEGLip-FVIIa induced faster thrombin generation and higher peak levels of thrombin than standard FVIIa. These effects lasted up to 5 h postinfusion. Measurements of D-dimer, prothrombin fragment 1 + 2 and fibrinogen showed no significant differences between the PEGLip-FVIIa and standard FVIIa treatments. PEGLip-FVIIa therefore showed improved haemostatic efficacy without increased risk of thrombosis and may be further developed for the treatment for bleeding episodes in haemophilia patients with inhibitors.
Improving the pharmacodynamics of protein drugs has the potential to improve the care and the qua... more Improving the pharmacodynamics of protein drugs has the potential to improve the care and the quality of life of patients suffering from a variety of diseases. Four approaches to improve protein drugs are described: PEGylation, amino acid substitution, fusion to carrier proteins and encapsulation. A new platform technology based on the binding of proteins/peptides to the outer surface of PEGylated liposomes (PEGLip) is then presented. Binding of proteins to PEGLip is non-covalent, highly specific and dependent on an amino acid consensus sequence within the proteins. Association of proteins with PEGLip results in substantial enhancement of the pharmacodynamic properties of proteins following administration. This has been demonstrated in preclinical studies and clinical trials with coagulation factors VIII and VIIa. It has also been demonstrated in preclinical studies with granulocyte colony-stimulating factor. A mechanism is presented that explains the improvements in hemostatic efficacy of PEGLip-formulated coagulation factors VIII and VIIa. The reader will gain an understanding of the advantages and disadvantages of each of the approaches discussed. PEGLip formulation is an important new approach to improve the pharmacodynamics of protein drugs. This approach may be applied to further therapeutic proteins in the future.
Summary. A precise and sensitive chromogenic assay for the measurement of very low levels of fac... more Summary. A precise and sensitive chromogenic assay for the measurement of very low levels of factor VIII (FVIII) in plasma has been developed. The assay is based on modifications of a commercially available chromogenic assay. The modifications include reduction of sample final dilution factor and prolongation of the development period. The modified assay allows accurate and precise measurement of FVIII in the range of 0.001–0.02 IU mL−1. The detection limit is 0.0005 IU mL−1 and the quantitation limit is 0.0015 IU mL−1. This assay can be used in research and to study the clinical efficacy of low circulating levels of FVIII in haemophilia A patients.
Summary. Recombinant activated factor VII (rFVIIa) is an effective treatment of the haemophilia ... more Summary. Recombinant activated factor VII (rFVIIa) is an effective treatment of the haemophilia patient with inhibitors and acquired haemophilia. However, on account of its relatively short half-life (HL), achieving therapeutic efficacy with FVIIa requires repeated injections. The development of a long-acting FVIIa product would therefore be beneficial. The formulation of factor VIII with PEGylated liposomes (PEGLip) was previously shown to extend the bleeding-free period in haemophilia patients. We report here an enhancement of haemostatic efficacy by similarly formulating FVIIa with PEGLip. Surface plasmon resonance analysis indicated that FVIIa binds non-covalently but with high affinity and specificity to PEGLip. A one-stage clotting assay demonstrated that formulation of FVIIa with PEGLip does not affect FVIIa activity and stability. A pharmacokinetic study in rats demonstrated that PEGLip formulation of FVIIa extends circulation time and results in higher FVIIa levels several hours after injection. Thromboelastography experiments indicated that PEGLip-FVIIa induces faster clot formation and higher clot stability than standard formulated FVIIa. These results suggest that formulation of FVIIa with PEGLip may lead to a safe and effective long-acting FVIIa that improves the care of haemophilic patients with inhibitors and acquired haemophilia.
An A-->G transition in the acceptor splice site at the intron 19/exon 20 junction of the g... more An A-->G transition in the acceptor splice site at the intron 19/exon 20 junction of the glycoprotein IIb gene was defined as a novel mutation causing Glanzmann thrombasthenia in two Iraqi-Jewish siblings. This mutant DNA was transcribed into four distinct species of mRNA, one of which resulted in a premature termination codon and the other three predicting deletions of 50, 61 or 72 amino acids, respectively.
Summary. Recombinant activated factor VIIa (FVIIa) is a bypassing agent used to treat bleeding e... more Summary. Recombinant activated factor VIIa (FVIIa) is a bypassing agent used to treat bleeding episodes in haemophilia patients with inhibitors to factor VIII (FVIII) and factor IX. The pharmacological effect of FVIIa is short-lived and therefore with the recommended dose of 90 μg kg−1, a bleeding episode is treated with multiple injections. A long-acting form of FVIIa that can ensure adequate haemostasis with a single infusion, without increasing the thrombotic risk, would therefore be beneficial. PEGylated liposomes (PEGLip) have been shown to bind FVIIa and to improve haemostatic efficacy in preclinical experiments. In the present phase I/II clinical trial, we assessed the safety and efficacy of PEGLip-formulated FVIIa in severe haemophilia A patients (FVIII ≤ 1%) with inhibitors to FVIII. Each patient received one prophylactic infusion of standard FVIIa and one prophylactic infusion of PEGLip-formulated FVIIa. The order of the infusions was randomized and the two infusions were separated by a ten-day washout period. Efficacy assessed by thromboelastography revealed that PEGLip-FVIIa induced significantly shorter clotting times and produced higher clot firmnesses than standard FVIIa. Thrombin generation assays showed that PEGLip-FVIIa induced faster thrombin generation and higher peak levels of thrombin than standard FVIIa. These effects lasted up to 5 h postinfusion. Measurements of D-dimer, prothrombin fragment 1 + 2 and fibrinogen showed no significant differences between the PEGLip-FVIIa and standard FVIIa treatments. PEGLip-FVIIa therefore showed improved haemostatic efficacy without increased risk of thrombosis and may be further developed for the treatment for bleeding episodes in haemophilia patients with inhibitors.
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Papers by Rivki Yatuv