Karin Knobe

Enhanced pharmacokinetic properties of a glycoPEGylated recombinant factor IX: a first human dose trial in patients with hemophilia B

Replacement therapy with factor IX (FIX) concentrates is the recommended treatment for patients with hemophilia B, an X-linked bleeding disorder occurring in 1:25,000 male births. N9-GP is a recombinant FIX molecule with a prolonged half-life which is obtained by site-directed glycoPEGylation where a 40-kDa polyethylene glycol molecule is attached to the activation peptide of FIX. This first human dose trial in patients with hemophilia B investigated the safety and pharmacokinetic properties of a single IV dose of N9-GP. Sixteen previously treated patients received one dose of their previous FIX product followed by one dose of N9-GP at the same dose level (25, 50, or 100 U/kg). None of the patients developed inhibitors. One patient developed transient hypersensitivity symptoms during administration of N9-GP and was excluded from pharmacokinetic analyses. In the remaining 15 patients, N9-GP was well-tolerated. The half-life was 93 hours, which was 5 times higher than the patients previous product. The incremental recovery of N9-GP was 94% and 20% higher compared with recombinant and plasma-derived products, respectively. These results indicate that N9-GP has the potential to reduce dosing frequency while providing effective treatment of bleeding episodes with a single dose. The trial was registered at www.clinicaltrials.gov as NCT00956345.

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Joint protection in haemophilia

Summary.  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.

Recombinant long-acting glycoPEGylated factor IX in hemophilia B: a multinational randomized phase 3 trial

This multinational, randomized, single-blind trial investigated the safety and efficacy of nonacog beta pegol, a recombinant glycoPEGylated factor IX (FIX) with extended half-life, in 74 previously treated patients with hemophilia B (FIX activity ≤2 IU/dL). Patients received prophylaxis for 52 weeks, randomized to either 10 IU/kg or 40 IU/kg once weekly or to on-demand treatment of 28 weeks. No patients developed inhibitors, and no safety concerns were identified. Three hundred forty-five bleeding episodes were treated, with an estimated success rate of 92.2%. The median annualized bleeding rates (ABRs) were 1.04 in the 40 IU/kg prophylaxis group, 2.93 in the 10 IU/kg prophylaxis group, and 15.58 in the on-demand treatment group. In the 40 IU/kg group, 10 (66.7%) of 15 patients experienced no bleeding episodes into target joints compared with 1 (7.7%) of 13 patients in the 10 IU/kg group. Health-related quality of life (HR-QoL) assessed with the EuroQoL-5 Dimensions visual analog scale score improved from a median of 75 to 90 in the 40 IU/kg prophylaxis group. Nonacog beta pegol was well tolerated and efficacious for the treatment of bleeding episodes and was associated with low ABRs in patients receiving prophylaxis. Once-weekly prophylaxis with 40 IU/kg resolved target joint bleeds in 66.7% of the affected patients and improved HR-QoL. This trial was registered at www.clinicaltrials.gov as #NCT01333111.

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

Summary.  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.

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.

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.

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 kcat 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 Å from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 Å 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. Proteins 1999;35: 218–234.

Haemophilia and joint disease: pathophysiology, evaluation and management

In patients with haemophilia, regular replacement therapy with clotting factor concentrates (prophylaxis) is effective in preventing recurrent bleeding episodes into joints and muscles. However, despite this success, intra-articular and intramuscular bleeding is still a major clinical manifestation of the disease. Bleeding most commonly occurs in the knees, elbows, and ankles, and is often evident from early childhood. The pathogenesis of haemophilic arthropathy is multifactorial, with changes occurring in the synovium, bone, cartilage, and blood vessels. Recurrent joint bleeding causes synovial proliferation and inflammation (haemophilic synovitis) that contribute to end-stage degeneration (haemophilic arthropathy); with pain and limitation of motion severely affecting patients’ quality of life. If joint bleeding is not treated adequately, it tends to recur, resulting in a vicious cycle that must be broken to prevent the development of chronic synovitis and degenerative arthritis. Effective prevention and management of haemophilic arthropathy includes the use of early, aggressive prophylaxis with factor replacement therapies, as well as elective procedures, including restorative physical therapy, analgesia, aspiration, synovectomy, and orthopaedic surgery. Optimal treatment of patients with haemophilia requires a multidisciplinary team comprising a haematologist, physiotherapist, orthopaedic practitioner, rehabilitation physician, occupational therapist, psychologist, social workers, and nurses.

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

Summary.  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.