The hallmark of haemophilia is the joint morbidity resulting from haemarthrosis that accounts for the majority of the bleeds. The exact mechanisms underlying changes are not fully elucidated. Cytokines are speculated to be involved in the progression and in vitro studies have confirmed the presence of elevated levels of cytokines in synovial tissue and cartilage from patients with haemophilic synovitis. In this study, the presence of selected cytokines in synovial fluid from haemophilia A mice with experimentally induced haemarthroses treated with rFVIII, rFVIIa and an rFVIIa analogue were investigated. Ten cytokines previously shown to be involved in arthritic syndromes were evaluated. Interleukin (IL)-1 beta, IL-2, IL-4, IL-6, IL-10, IL-17, Tumor Necrosis Factor-alpha (TNF- alpha), keratinocyte-derived chemokine (KC), Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) and monocyte chemotactic protein-1 (MCP-1) were included. In this article, we demonstrate, for the first time, that bleeding in knee joints of haemophilia A mice resulted in correlated increased levels of the pro-inflammatory cytokines: IL-1 beta, IL-6, KC and the MCP-1 in synovial fluid. These results suggest an important role of MCP-1 in the recruitment of monocytes and furthermore that the inflamed synovium releases IL-1 beta, IL-6 and KC, which in turn might contribute to further progression of the inflammatory process.

Summary. Background: Hemophilia A is the most common serious bleeding disorder, and the hallmark of this disease is joint bleeding episodes. These result in hemophilic synovitis, an inflammatory and proliferative condition of the joint, which progresses into a chronic degenerative arthritis, hemophilic arthropathy. Methods: In this paper, we describe the effect of recombinant factor VIIa (rFVIIa), and an analogue NN1731 as well as rFVIII on needle-induced bleeding in hemophilia A mice. Conclusions: Here we show a reducing effect of rFVIIa and NN1731 on bleeding induced in hemophilic mice, and we show that preventive treatment with rFVIII normalizes bleeding.

N8 is a new recombinant factor VIII (rFVIII) compound produced and formulated without human- or animal-derived protein. The aims of the present studies were to evaluate the pharmacokinetics and pharmacodynamics properties of N8 and to compare with a commercially available rFVIII product (Advate(®)) in haemophilia A mice. The pharmacokinetics were evaluated after single i.v. administration of 80, 120 and 280 IU kg(-1) of N8 and Advate(®) and measurements of FVIII blood concentrations as a function of time. The efficacy and dose response curves of N8 and Advate(®) (1-200 IU kg(-1)) were evaluated in a tail bleeding model. Furthermore, the effects in a newly developed haemophilia knee joint haemarthrosis model were investigated. No significant differences were found in the pharmacokinetic parameters between N8 and Advate(®). The clearances were 11 ± 1 vs. 10 ± 2 mL h(-1) kg(-1) (P = 0.14) and the half-lives 7.2 ± 0.9 vs. 7.7 ± 1.4 h (P = 0.31) after administration of N8 and Advate(®) respectively. Dose-independent pharmacokinetics was shown, and comparable efficacy and potency were shown between N8 and Advate(®) in the tail bleeding model. Both compounds normalized the bleeding at the dose of 200 IU kg(-1), and for blood loss ED(50) values of 27 IU kg(-1) (N8) and 28 IU/kg (Advate(®)) were found (P = 0.97). In the haemarthrosis model, treatment with N8 and Advate(®) at 200 IU kg(-1) reduced the mean increase in the joint diameter significantly from 1.23 ± 0.19 to 0.32 ± 0.08 mm (P < 0.01) and 0.25 ± 0.08 mm (P < 0.001) respectively. Pharmacokinetics and pharmacodynamics of N8 and Advate(®) were comparable after i.v. administration to haemophilia A mice.

Animal models have contributed immensely to the understanding of and the improvement in treatment of haemophilia A and B. First, establishment of haemophilic dog colonies provided an invaluable opportunity to investigate the diseases and later, the advances in gene technologies resulting in small haemophilic animal models were a milestone in the preclinical research making it possible to address some of the many unanswered questions. This review provides an overview of animal models used in the study of haemophilia as well as a short overview of the contributions resulting from studies in these models.

Haemarthrosis is the most common clinical manifestation of haemophilia and is responsible for significant morbidity in haemophilic patients. The murine experimentally-induced knee bleeding model is an important model in haemophilia research but it is currently unknown if the use of analgesia in this model might impact on the inflammatory response. The aim was to investigate the inflammatory response after a needle induced knee bleed in haemophilia A mice treated with buprenorphine or saline. One hundred and sixty mice were randomized into two groups to blindly receive buprenorphine or saline. All the mice were anaesthetized and knee injury was induced by inserting a 30 G needle into the right knee joint. At t ¼ 6, 24, 48 and 72 h, 20 mice from each group were terminated and the following parameters were assessed: change in body weight and joint diameter, visual bleeding score (VBS), white blood counts, haematocrit, platelet concentrations, haemoglobin, plasma haptoglobin and plasma and synovial fluid levels of 23 cytokines. Twenty mice were terminated at t ¼ 0 receiving no injury or treatment to provide baseline measures. Twenty-one cytokines in plasma and 22 cytokines in synovial fluid, joint diameter change, VBS and blood parameters were not significantly altered by the administration of buprenorphine. Slight alterations of plasma haptoglobin at t ¼ 48 h, body weight, plasma and synovial eotaxin and plasma G-CSF were found in buprenorphine-treated mice. We demonstrated that buprenorphine does not overall impact on the inflammatory response, and the use of buprenorphine in the knee bleeding model in haemophilic mice should be continued.