Safety, pharmacokinetics and efficacy of factor VIIa formulated with PEGylated liposomes in haemophilia A patients with inhibitors to factor VIII--an open label, exploratory, cross-over, phase I/II study.

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.

Enhancement of factor VIIa haemostatic efficacy by formulation with PEGylated liposomes.

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.

A modified chromogenic assay for the measurement of very low levels of factor VIII activity (FVIII:C).

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.

Identification of a region in glycoprotein IIIa involved in subunit association with glycoprotein IIb: further lessons from Iraqi-Jewish Glanzmann thrombasthenia.

The most frequent mutation causing Glanzmann thrombasthenia in Iraqi-Jews (IJ-1) is an 11-bp deletion in exon 13 of the glycoprotein (GP) IIIa gene. This deletion predicts a frameshift that results in the elimination of the C406-C655 disulfide bond and a premature termination codon shortly before the transmembrane domain. To determine the contribution of each of these alterations to the thrombasthenic phenotype, Chinese hamster ovary or baby hamster kidney cells were cotransfected with normal GPIIb complementary DNA (cDNA) and the following GPIIIa cDNAs: normal, cDNA bearing IJ-1 mutation, 2011T>A mutated cDNA predicting C655S (single-letter amino acid codes) substitution, and 2019A>T mutated cDNA predicting Stop657. Elimination of the C406-C655 disulfide bond by C655S substitution did not affect GPIIb/IIIa surface expression or binding of the transfected cells to immobilized fibrinogen, whereas elimination of the transmembrane and cytoplasmic domains in IJ-1 and Stop657 mutants prevented both surface expression and binding of the transfected cells to immobilized fibrinogen. Immunohistochemical staining and immunoprecipitation demonstrated that the elimination of amino acids 657-762 in IJ-1 and Stop657 prevented intracellular GPIIb/IIIa complex formation, and differential immunofluorescence staining of GPIIIa and cellular organelles suggested that the truncated uncomplexed GPIIIa protein was retained in the endoplasmic reticulum. Because the use of GPIIIa Stop693 and normal GPIIb cDNAs yielded GPIIb/IIIa complex formation, though with lower efficiency, it is suggested that amino acids 657-692 of GPIIIa are essential for the intracellular association of GPIIb and GPIIIa. (Blood. 2001;98:1063-1069)

Glanzmann thrombasthenia in two Iraqi-Jewish siblings is caused by a novel splice junction mutation in the glycoprotein IIb.

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.

Glanzmann thrombasthenia caused by an 11.2-kb deletion in the glycoprotein IIIa (beta3) is a second mutation in Iraqi Jews that stemmed from a distinct founder.

Glanzmann thrombasthenia (GT) is a rare bleeding disorder resulting from mutations in either glycoprotein (GP) IIb or GPIIIa genes. The disease is relatively frequent in highly inbred populations such as Iraqi Jews. The molecular basis of GT in 6 unrelated Iraqi-Jewish patients was previously identified as an 11-bp deletion in exon 12 of the GPIIIa gene. We now describe a second mutation found in 3 unrelated Iraqi-Jewish families that consists of an 11.2-kb deletion between an Alu repeat in intron 9 and exon 13 of the GPIIIa gene. The mutant DNA is transcribed into mRNA in which exons 10 through 13 are absent. Splicing of exon 9 directly to exon 14 leads to a shift in the reading frame resulting in a stop codon. The predicted protein is truncated in the middle of the third cysteine-rich domain before the transmembrane domain. Simple DNA-based methods were devised for identification of both mutations in Iraqi Jews for the purpose of carrier detection and prenatal diagnosis enabling prevention of GT. A survey of the general Iraqi-Jewish population for the first 11-bp deletion and the second 11.2-kb deletion disclosed that the allele frequency of the first mutation was 0.0043, whereas none of 700 individuals examined bore the second mutation (allele frequency <0.0007). Among 40 GT patients of Iraqi-Jewish origin 31 were homozygous for the first mutation, 4 were compound heterozygotes for the first and second mutations, and 2 were homozygous for the second mutation. Haplotype analyses using 4 polymorphic markers in the GPIIIa gene showed that each mutation originated in a distinct founder.

One of the two common mutations causing factor XI deficiency in Ashkenazi Jews (type II) is also prevalent in Iraqi Jews, who represent the ancient gene pool of Jews.

In recent years four mutations causing factor XI deficiency have been identified in Jews of Ashkenazi (European) origin. Two of them, type II (a nonsense mutation) and type III (a missense mutation), were found to prevail among 125 unrelated Ashkenazi Jews with severe factor XI deficiency. A finding of type II mutation in four unrelated Iraqi-Jewish families raised the possibility that this mutation is also common in Iraqi Jews, who represent the ancient gene pool of the Jews. A molecular-based analysis performed in 1,040 consecutively hospitalized patients disclosed the following results: Among 531 Ashkenazi-Jewish patients, the type II allele frequency was 0.0217 and among 509 Iraqi-Jewish patients, 0.0167 (P = .50). The type III allele frequency in the Ashkenazi-Jewish patients was 0.0254, whereas none of 502 Iraqi-Jewish patients examined had this mutation. These data suggest that the type II mutation was present in Jews already 2.5 millenia ago. The data also indicate that the estimated risk for severe factor XI deficiency in Ashkenazi Jews (due to either genotype) is 0.22% and in Iraqi Jews, 0.03%, and that the estimated risk of heterozygosity in Ashkenazi Jews is 9.0% and in Iraqi Jews, 3.3%. As patients with severe factor XI deficiency are prone to bleeding after injury and patients with partial deficiency may have similar bleeding complications when an additional hemostatic derangement is present, the observed high frequencies should be borne in mind when surgery is planned for individuals belonging to these populations.