Comparative burden of arthropathy in mild haemophilia: a register-based study in Sweden.

INTRODUCTION: Mild haemophilia is a congenital bleeding disorder affecting males. The burden of arthropathy in mild haemophilia has not been comprehensively described. AIM: The aim of this study was to compare the incidence, age at diagnosis and surgery for arthropathy and related hospitalizations between people with mild haemophilia and the general population in Sweden. METHODS: This was a register-based cohort study. Eligible participants were those with mild haemophilia born between 1941 and 2008 and a randomly selected, birthdate and sex-matched comparison group from the general population. Follow-up was from birth (or earliest 1984) until death, emigration or end of the study in 2008. Data on arthropathy were obtained from a national patient register. Negative binomial and competing risk regression and Kaplan-Meier estimate curves were used in the analysis. RESULTS: Overall, 315 people with haemophilia and 1529 people in the comparison group were included. Participants with haemophilia born between 1984 and 2008 had a ninefold (95% CI: 3.3-27.2) and 16-fold (95% CI: 6.7-36.5) increased incidence of arthropathy-related hospital admission and arthropathy diagnosis respectively. None in this cohort underwent surgery. Among participants with haemophilia born prior to 1984, the rates of arthropathy diagnosis and surgery of the index joints (knee, elbow, ankle) were increased twofold (95% CI: 1.0-3.2) and fivefold (95% CI: 1.7-17.8) respectively. CONCLUSION: Our data suggested a higher burden of arthropathy among individuals with mild haemophilia compared to the general population. Further research should investigate the need for targeted joint screening programmes among individuals with mild haemophilia.

Changes in the amino acid sequence of the recombinant human factor VIIa analog, vatreptacog alfa, are associated with clinical immunogenicity.

BACKGROUND: Vatreptacog alfa, a recombinant human factor VIIa (rFVIIa) analog developed to improve the treatment of bleeds in hemophilia patients with inhibitors, differs from native FVIIa by three amino acid substitutions. In a randomized, double-blind, crossover, confirmatory phase III trial (adept(™) 2), 8/72 (11%) hemophilia A or B patients with inhibitors treated for acute bleeds developed anti-drug antibodies (ADAs) to vatreptacog alfa. OBJECTIVES: To characterize the formation of anti-vatreptacog alfa ADAs in hemophilia patients with inhibitors. METHODS/PATIENTS: This was a post hoc analysis of adept(™) 2. Immunoglobulin isotype determination, specificity analysis of rFVIIa cross-reactive antibodies, epitope mapping of rFVIIa single mutant analogs and pharmacokinetic (PK) profiling were performed to characterize the ADAs. RESULTS: Immunoglobulin isotyping indicated that the ADAs were of the immunoglobulin G subtype. In epitope mapping, none of the rFVIIa single mutant analogs (V158D, E296V or M298Q) contained the complete antibody epitope, confirming that the antibodies were specific for vatreptacog alfa. In two patients, for whom PK profiling was performed both before and after the development of ADAs, vatreptacog alfa showed a prolonged elimination phase following ADA development. During the follow-up evaluation, the rFVIIa cross-reactivity disappeared after the last vatreptacog alfa exposure, despite continued exposure to rFVIIa as part of standard care. CONCLUSIONS: Results from the vatreptacog alfa phase III trial demonstrate that the specific changes made, albeit relatively small, to the FVIIa molecule alter its clinical immunogenicity.

Enhancing the pharmacokinetic properties of recombinant factor VIII: first-in-human trial of glycoPEGylated recombinant factor VIII in patients with hemophilia A.

BACKGROUND: N8-GP is a recombinant factor VIII (FVIII) with a site-directed glycoPEGylation for the purpose of half-life prolongation. OBJECTIVES: To evaluate the safety and pharmacokinetic profiles of N8-GP in comparison with those of the patients' previous FVIII products. PATIENTS/METHODS: This dose-escalation trial included previously treated patients with severe hemophilia A who received one of three dose levels (25, 50 or 75 U kg(-1) ) of N8-GP and FVIII product. Each dose escalation was preceded by safety and pharmacokinetic assessment. The trial was registered at www.clinicaltrials.gov (NCT01205724). RESULTS: Twenty-six patients each received one dose of their previous FVIII product followed by the same, single dose of N8-GP. N8-GP, at any tested dose, was well tolerated, with a low frequency of adverse events. No new inhibitors against FVIII or N8-GP and no binding antibodies against N8-GP developed during the trial. The pharmacokinetics of N8-GP were dose-linear. The incremental recovery of N8-GP was 0.025 [(U mL(-1) )/(U kg(-1) )]. The clearance was 1.79 mL(-1)  h(-1)  kg(-1) . The estimated time from dosing of 50 U kg(-1) N8-GP to a plasma activity of 1% was 6.5 days (range: 3.6-7.9 days). The mean terminal half-life of N8-GP was 19.0 h (range: 11.6-27.3 h), 1.6-fold longer than that of the patients' previous products. CONCLUSIONS: A single dose of up to 75 U kg(-1) N8-GP was well tolerated in patients with hemophilia A, with no safety concerns. N8-GP had a prolonged half-life, and FVIII:C activity remained at > 1% for longer than the patient's previous product. These results indicate that N8-GP has the potential to reduce dosing frequency during prophylaxis.

Population pharmacokinetic modeling for dose setting of nonacog beta pegol (N9-GP), a glycoPEGylated recombinant factor IX.

BACKGROUND: nonacog beta pegol (N9-GP) is a glycoPEGylated recombinant factor IX (rFIX) molecule with a prolonged half-life. OBJECTIVES: To provide information on potential dose regimens for N9-GP for phase 3 pivotal and surgery trials. METHODS: A population pharmacokinetic model was developed from single-dose data derived from the first human-dose trial with N9-GP in hemophilia B patients, and was used to extrapolate to steady-state conditions for different N9-GP dose regimens for prophylaxis. The model was also used to compare prophylaxis using N9-GP with standard prophylactic regimens using rFIX or plasma-derived (pd) FIX (40 IU kg(-1) every third day). Plasma activity following dosing with N9-GP, rFIX and pdFIX for surgery and on-demand treatment of bleeds was also simulated. RESULTS: A linear two-compartmental model best described the pharmacokinetic profiles of N9-GP, rFIX and pdFIX. A prophylactic regimen of 10 U kg(-1) N9-GP once weekly predicted mean peak and trough levels of 18 and 4.2 U dL(-1) , while 40 U kg(-1) once weekly predicted values of 72 and 17 U dL(-1) , respectively. Standard prophylactic regimens with rFIX and pdFIX predicted mean peak and trough levels of 34 and 3.9 IU dL(-1) for rFIX, and mean values of 43 and 2.1 IU dL(-1) for pdFIX. Additional simulations predicted significantly reduced dosing frequency and factor concentrate consumption for N9-GP vs. rFIX and pdFIX for surgery and the treatment of bleeds. CONCLUSIONS: N9-GP may allow prophylaxis, surgical dosing regimens and on-demand treatment of bleeding episodes with less frequent injections and lower factor concentrate consumption; this possibility is being investigated in prospective clinical trials.

Investigation of disease-associated factors in haemophilia A patients without detectable mutations.

To investigate disease causing mechanism in haemophilia A patients without detectable mutation. Screening for F8 mutations in 307 haemophilia A patients using: re-sequencing and inversion PCR, reverse transcription (RT-PCR) of mRNA, MLPA analysis, haplotyping using SNP and microsatellite markers. No F8 mutations were detected in 9 of the 307 patients (2.9%) using re-sequencing and inversion PCR. MLPA analysis detected duplication in exon 6 in one patient and RT-PCR showed no products for different regions of mRNA in four other patients, indicating failed transcription. No obvious associations were observed between the phenotypes of the nine patients, their F8 haplotypes and the putative mutations detected. The mutation-positive patients carrying the same haplotypes as the mutation-negative patients show a multitude of different mutations, emphasizing the lack of associations at the haplotype level. VWF mutation screening and factor V measurements ruled out type 2N VWD and combined factor V and VIII deficiency respectively. To further investigate a possible role for FVIII interacting factors the haplotypes/diplotypes of F2, F9, F10 and VWF were compared. The nine patients had no specific haplotype/diplotype combination in common that can explain disease. Duplications and faulty transcription contribute to the mutational spectrum of haemophilia A patients where conventional mutation screening fail to identify mutations.

Joint protection in haemophilia.

Haemarthroses (intra-articular haemorrhages) are a frequent finding typically observed in patients with haemophilia. Diagnosis and treatment of these bleeding episodes must be delivered as early as possible. Additionally, treatment should ideally be administered intensively (enhanced on-demand treatment) until the resolution of symptoms. Joint aspiration plays an important role in acute and profuse haemarthroses as the presence of blood in the joint leads to chondrocyte apoptosis and chronic synovitis, which will eventually result in joint degeneration (haemophilic arthropathy). Ultrasonography (US) is an appropriate diagnostic technique to assess the evolution of acute haemarthrosis in haemophilia, although magnetic resonance imaging remains the gold standard as far as imaging techniques are concerned. Some patients experience subclinical haemarthroses, which eventually tend to result in some degree of arthropathy, especially in the ankles. Nowadays, the most effective way of protecting these patients is primary prophylaxis, which in practice changes severe haemophilia into moderate haemophilia, preventing or at least minimizing the occurrence of haemarthrosis. If primary prophylaxis is, for whatever reason not an option, secondary prophylaxis and enhanced on demand treatment should be considered. Two alternatives are available for inhibitor patients: (i) control of haemostasis using by-passing agents (rFVIIa or aPCCs) either as enhanced on demand treatment or secondary prophylaxis, as appropriate, following the same basic principles used for non-inhibitor patients and (ii) immune tolerance induction (ITI) to eradicate the inhibitor.

Why does the mutation G17736A/Val107Val (silent) in the F9 gene cause mild haemophilia B in five Swedish families?

The mutation G17736A/Val107Val (silent) was found in five of a total of 86 families with haemophilia B in Sweden. It is unlikely that five families with analogous clinical expression will have the same polymorphism, which is not found in other patients or normal subjects, or that they will be the only families in the population without any other causative mutation. All affected individuals in the five families were found to have factor IX (F9) coagulation activity 15-20 U dL(-1), corresponding F9 protein levels and the same clinical history of mild haemophilia. Lymphocyte mRNA was extracted from one of the haemophiliacs and from a healthy male. RT-PCR of the mRNA and subsequent PCR amplification produced cDNA fragments of the same length from the patient and the normal subject, indicating no exon skipping or retention of introns. Sequencing of cDNA from codon 68 in exon D to codon 180 in exon F revealed that the patient had the G17736A mutation but no other abnormalities. We conclude that G17736A/Val107Val causes mild haemophilia B. Although, exon skipping and retention of introns can be excluded as pathophysiological mechanisms, it is plausible that the studied mutation has more subtle effects on a splicing site or interferes with a splicing enhancer site. Also, changes to synonymous codons may reduce the translation rate and thereby alter F9 protein folding in vivo, which would explain the phenotype. Confirmation of these assumptions requires methods that are more sensitive than those available today, and our discussion illustrates the existing obstacles.

Functional analysis of the EGF-like domain mutations Pro55Ser and Pro55Leu, which cause mild hemophilia B.

We studied the functional role of two mutations, Pro55Ser and Pro55Leu, located in the N-terminal Epidermal Growth Factor-like domain (EGF1) of coagulation factor (F) IX. Both mutations cause mild hemophilia B with habitual FIX coagulant activities of 10-12% and FIX antigen levels of 50%. We found that activation by FVIIa/TF and FXIa was normal for FIXPro55Ser, but resulted in proteolysis of FIXPro55Leu at Arg318-Ser319 with a concomitant loss of amidolytic activity, suggesting intramolecular communication between EGF1 and the serine protease domain in FIX. This was further supported by experiments using an anti-EGF1 monoclonal antibody. Activation of FX by FIXaPro55Ser was impaired in both the presence and the absence of phospholipid or FVIIIa, indicating that Pro55 is not directly involved in binding to FVIIIa. We also studied the effect of the two Pro55 mutations on Ca2+ affinity and found only small changes. Thus, the Pro55Ser mutation causes hemophilia primarily through to an impaired ability to activate FX whereas at least in vitro the Pro55Leu defect interferes with the activation of FIX.

Inhibitors in the Swedish population with severe haemophilia A and B: a 20-year survey.

AIM: To survey the entire population (n = 116) afflicted with severe haemophilia A or B born in Sweden over a 20-y period (1980-1999), and to examine the epidemiological, genetic and clinical aspects of development of inhibitors to factors VIII and IX (FVIII/FIX). METHODS: One hundred of the subjects had haemophilia A and 16 had haemophilia B. All of these subjects had received prophylactic treatment and had a check-up of inhibitor status at least twice a year. Sixty-one were born between 1980 and 1989 and 55 between 1990 and 1999. RESULTS: Nineteen percent (19/100) of those with haemophilia A and 37% (6/16) with haemophilia B developed inhibitors at 12-18 mo of age, after exposure to FVIII/FIX concentrates for an average of 14 d in the case of haemophilia A and 16 d in haemophilia B. All patients with inhibitors carried mutations that impaired protein synthesis. The high incidence of FIX inhibitors may have been due to the large number of complete deletions (13%) in the Swedish haemophilia B population. Patients with haemophilia A showed no significant increase (p = 0.65) in incidence of inhibitors (n = 10/48, total incidence 21%) in the 1990s, when they were treated mainly with recombinant products, as compared to the 1980s (n = 9/52, 17%), when they received intermediate/high-purity plasma-derived concentrates. CONCLUSION: Our population-based study verifies that genotype has a general impact on the incidence of FVIII/FIX inhibitors, and that recombinant FIII/FIX concentrates are not a predisposing factor for inhibitor development.

Breastfeeding does not influence the development of inhibitors in haemophilia.

Our aim was to test the hypothesis that breastfeeding may reduce development of inhibitors in male infants with haemophilia by inducing an oral immune tolerance to factor VIII. To achieve that goal, we performed a structured epidemiological survey comprising all males born with severe haemophilia A (in all 100 patients, 19 with inhibitors) or haemophilia B (in all 16 patients, six with inhibitors) in Sweden in 1980-99. Our results show no protective effect of breastfeeding.

An anti-EGF monoclonal antibody that detects intramolecular communication in factor IX.

Coagulation factor IX contains a gamma-carboxyglutamic acid (Gla) module, two epidermal growth factor-like (EGF) modules, and a serine protease region. We have characterized a mouse monoclonal antibody that binds the N-terminal EGF-like module of human factor IX with high affinity. Studies of recombinant factor IX mutants indicated that the epitope is located in the C-terminal end of the EGF-like module, which is consistent with the binding being non-Ca(2+)-dependent. The antibody bound factor IXa (K(D) = 7.6 x 10(-10) M) with about 10-fold higher affinity than factor IX (K(D) = 6.2 x 10(-9) M). Binding of the antibody to factor IXa did not affect the amidolytic activity of the protein, nor was binding affected by active site inhibition of factor IXa. These results are consistent with long-range interactions between the serine protease region and the N-terminal EGF-like module in factor IX.

Factor VIII inhibitors in two families with mild haemophilia A: structural analysis of the mutations.

The development of inhibitory antibodies against coagulation factor VIII (FVIII) in patients with mild haemophilia A is uncommon. We describe here two families in which three or two members have developed inhibitors, suggesting a familial predisposition. The mutations found, in the A2 (Arg593Cys) and C1 domains (Tyr2105Cys), have been reported to give rise to inhibitor development in single individuals in addition to the family cluster we describe, strongly suggesting that these amino acid substitutions give rise to a more immunogenic protein. The analysis of structural models of activated factor VIII revealed that Arg593 is solvent-exposed and involved in a network of electrostatic interactions while Tyr2105 is partially buried and has hydrophobic interactions essentially with Ile2144. All these residues are strictly conserved in the FVIII amino acid sequence from man, pig and mouse, suggesting, at least, that they have structural roles. We propose that the two mutations in these families could cause mild haemophilia A because they induce local conformational changes (and possible secretion or intermolecular interaction problems, e.g., with von Willebrand factor) compatible with immunogenicity and production of inhibitors against the infused wild-type FVIII.

Haemophilia B carrier detection by factor IX:C analysis; no impact of the type of mutation or severity of disorder.

Haemophilia B, an X-linked recessive bleeding disorder characterized by lack or deficiency of factor IX, has been shown to be caused by any of a variety of DNA abnormalities (partial or total deletions, nonsense or missense mutations). Since in most countries carrier detection is based on factor IX coagulant activity (FIX:C) assay, this study was designed to determine whether carriers' FIX:C values are dependent on the severity of haemophilia (mild, moderate or severe) or on the genetic anomaly in the family. FIX:C concentrations were studied in 28 obligate carriers, 39 women known to carry the mutation and 33 verified noncarriers subgrouped by severity of disorder or genetic anomaly. No significant subgroup differences in FIX:C values were found, thus suggesting the level of FIX:C concentrations in carriers to be unaffected by the severity of haemophilia, or by its expression (i.e. deficient or dysfunctional factor IX). The specificity and sensitivity of FIX:C analysis for the purpose of carrier diagnosis was judged by receiver operating characteristic curve analysis, where an FIX:C cut-off level of 75 IU dL-1 was found to be optimal (sensitivity 93% and specificity 88%).

Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling.

Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM), a membrane receptor present on the surface of endothelial cells. The understanding of this complex biological system is in part limited due to the lack of integration of experimental and structural data. In the work presented here, we analyze the PC-T-TM pathway in the context of both types of information. First, structural analysis of the serine protease domain of PC suggests that a positively charged cluster of amino acids could be involved in the activation process. To investigate the importance of these basic amino acids, two recombinant PC mutants were constructed using computer-guided site-directed mutagenesis. The double mutant had the K62[217]N/K63[218]D substitution and in the single mutant, K86[241] was changed to S. Both mutants were activated by free thrombin at rates equivalent to that of wild-type PC (wt-PC) and they demonstrated similar calcium-dependent inhibition of their activation. The K86[241]S mutant and wt-PC were activated by thrombin bound to soluble TM at a similar rate. In contrast, the K62[217]N/ K63[218]D mutant was activated by the T-TM complex at a 10-fold lower catalytic efficiency due to a lowering in k(cat) and increase in Km. Molecular models for PC and thrombin bound to a segment of TM were developed. The experimental results and the modeling data both indicate that electrostatic interactions are of crucial importance to orient PC onto the T-TM complex. A key electropositive region centered around loops 37[191] and 60[214] of PC is defined. PC loop 37[191] is located 7-8 A from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 A away from TM EGF4. Both loops are far from thrombin. A key function of TM could be to create an additional binding site for PC. The Gla domain of PC points toward the membrane and away from thrombin or the EGF modules of TM during the activation process.

Distribution of preproatrial natriuretic peptide mRNA in rat brain detected by in situ hybridization of DNA oligonucleotides: enrichment in hypothalamic and limbic regions.

The expression and distribution of mRNA encoding preproatrial natriuretic peptide (ppANP) in rat brain has been investigated by in situ hybridization of two 35S-labeled synthetic DNA oligonucleotides, based on a cDNA clone sequence that encodes rat ppANP. The highest relative concentrations of ppANP mRNA were detected in the medial preoptic hypothalamic nucleus ("anteroventral/third ventricle region") and the medial habenula. Moderate concentrations of ppANP mRNA were observed in the CA1 pyramidal cells of the hippocampus, the endopiriform nucleus, the arcuate nucleus, the zona incerta, and cells of the pontine tegmental and peduculopontine nuclei. Several of these regions, including the habenula and the hypothalamic areas, have previously been reported to contain atrial natriuretic peptide (ANP)-like immunoreactivity, but the expression of ppANP mRNA in CA1 pyramidal cells suggests the occurrence of differential translation of ppANP mRNA into protein product in different brain regions, or the existence of different immunological forms of the peptide. The abundance of ppANP mRNA in brain was relatively low in comparison with that previously reported for many other mRNA species encoding other brain neuropeptides. These results demonstrate that ANP gene expression occurs in discrete neuronal populations of the CNS and that studies of the regulation of this expression should now be possible using quantitative in situ hybridization.

Preprogastrin-releasing peptide messenger ribonucleic acid: neuroanatomical localization in rat brain by in situ hybridization with synthetic oligodeoxynucleotide probes.

Gastrin-releasing peptide (GRP) is a 27 amino acid peptide that is present in both the central and peripheral nervous systems and that shares immunological and functional properties with the amphibian peptide, bombesin. GRP has multiple putative biological functions including effects on feeding behaviour and carbohydrate metabolism, body temperature, and effects on hormone release, but little is known about the regulation of GRP gene expression in the brain. This study examined the distribution of neurones expressing preproGRP mRNA in rat brain by in situ hybridization of [35S]-labelled DNA oligonucleotides. PreproGRP mRNA was detected in several regions of brain, with highest concentrations in the parvocellular paraventricular and suprachiasmatic nuclei of the hypothalamus, the lateral and basolateral nuclei of the amygdala, the amygdaloid-hippocampal area and the ventral part of the granule cell layer of the dentate gyrus. Moderate levels were seen in layers II and III of the cingulate and retrosplenial cortex, the medial and mediobasal nuclei of the amygdala, the anteroventral thalamic nucleus; medial geniculate nucleus and the parabrachial nucleus. These findings are largely consistent with the cellular localization of GRP-like immunoreactivity in rat brain and recent studies of preproGRP mRNA localization using cRNA probes. The distribution of preproGRP mRNA observed further suggests the involvement of GRP in the central regulation of several functions including regulation of hypothalamic/pituitary hormone release.