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Biomedical subjects

Ulla Hedner

Publications and source records attributed to Ulla Hedner.

At least 19 recordsLinked to original sources
PubMed2007

A variant of recombinant factor VIIa with enhanced procoagulant and antifibrinolytic activities in an in vitro model of hemophilia.

OBJECTIVE: Recombinant factor VIIa (rFVIIa, NovoSeven) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. A rFVIIa analog with mutations V158D/E296V/M298Q (NN1731) exhibits increased procoagulant activity in in vitro and in vivo models. The aim of this work was to define the effects of NN1731 toward factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability. METHODS AND RESULTS: In a cell-based in vitro model of hemophilia, rFVIIa and NN1731 similarly increased factor X activation on tissue factor-bearing cells; however, NN1731 exhibited 30-fold higher factor Xa generation on platelets than similar rFVIIa concentrations. NN1731-mediated thrombin generation depended on platelet activation, but NN1731 did not directly activate platelets. NN1731 produced 4- to 10-fold higher maximal thrombin generation rates than equal rFVIIa concentrations. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at 50-fold lower concentrations than were required of rFVIIa. In fibrinolytic conditions, both rFVIIa and NN1731 increased fibrin formation and stability; however, NN1731 was effective at 50-fold lower concentrations than were required of rFVIIa. CONCLUSIONS: By increasing factor Xa generation, NN1731 promotes the formation of thrombin and a stable clot to a greater degree than rFVIIa.

Antifibrinolytic Agents↗
PubMed2006

Cutaneous wound healing is impaired in hemophilia B.

We used a mouse model to test the hypothesis that the time course and histology of wound healing is altered in hemophilia B. Punch biopsies (3 mm) were placed in the skin of normal mice and mice with hemophilia. The size of the wounds was measured daily until the epidermal defect closed. All wounds closed in mice with hemophilia by 12 days, compared with 10 days in normal animals. Skin from the area of the wound was harvested at different time points and examined histologically. Hemophilic animals developed subcutaneous hematomas; normal animals did not. Macrophage infiltration was significantly delayed in hemophilia B. Unexpectedly, hemophilic mice developed twice as many blood vessels in the healing wounds as controls, and the increased vascularity persisted for at least 2 weeks. The deposition and persistence of ferric iron was also greater in hemophilic mice. We hypothesize that iron plays a role in promoting excess angiogenesis after wounding as it had been proposed to do in hemophilic arthropathy. We have demonstrated that impaired coagulation leads to delayed wound healing with abnormal histology. Our findings have significant implications for treatment of patients with hemophilia, and also highlight the importance of rapidly establishing hemostasis following trauma or surgery.

Animals↗
PubMed2006

Mechanism of action, development and clinical experience of recombinant FVIIa.

Recombinant FVIIa has been developed for treatment of bleedings in hemophilia patients with inhibitors, and has been found to induce hemostasis even during major surgery such as major orthopedic surgery. Recombinant FVIIa is being produced in BHK cell cultures and has been shown to be very similar to plasma-derived FVIIa. The use of rFVIIa in hemophilia treatment is a new concept of treatment and is based on the low affinity binding of FVIIa to the surface of thrombin activated platelets demonstrated in a cell-based in vitro model. By the administration of pharmacological doses of exogenous rFVIIa the thrombin generation on the platelet surface at the site of injury is enhanced independently of the presence of FVIII/FIX. As a result of the increased and rapid thrombin formation, a tight fibrin hemostatic plug is being formed. A tight fibrin structure has been found to be more resistant to fibrinolytic degradation thereby helping to maintain hemostasis. The general mechanism of action of pharmacological doses of rFVIIa shown to induce hemostasis not only in hemophilia, but also in patients with platelet defects, and with profuse bleedings triggered by extensive surgery or trauma, may very well be the capacity of generating a tight fibrin hemostatic plug through the increased thrombin generation. Such a fibrin plug will help to resist the overwhelming mostly local release of fibrinolytic activity triggered by the vast tissue damage occurring in extensive trauma. A release of fibrinlytic activity locally has also been demonstrated to occur in the gastrointestinal tract as well as during profuse postpartum bleedings. Pharmacological doses of rFVIIa have in fact, also been shown to induce hemostasis in such cases.

Factor VII↗
PubMed2006

Mechanism of action of recombinant activated factor VII: an update.

Bleeding episodes in patients with hemophilia and inhibitors must be managed using agents that are hemostatically active in the absence of factor VIII or IX. Activated prothrombin complex concentrates have long been used in this context. However, the search for safer and more effective agents has led to the development of recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark). This paper presents an update on the mechanism of action of rFVIIa, and describes how pharmacologic doses of this agent enhance thrombin production and thus contribute to the development of a stable, lysis-resistant fibrin plug at the site of vessel damage. This mechanism explains the reported efficacy of rFVIIa in a range of clinical situations characterized by impaired thrombin generation.

Blood Coagulation↗
PubMed2006

Mechanism of action of factor VIIa in the treatment of coagulopathies.

Recombinant factor VIIa (rFVIIa) has been developed for treatment of bleeding in patients with hemophilia who have inhibitors against factor VIII (FVIII) or FIX, and has been found to induce hemostasis during major orthopedic surgery. The use of rFVIIa treatment for hemophilia is a new concept and is based on the low-affinity binding of FVIIa to the surface of thrombin-activated platelets. Administration of pharmacologic doses of exogenous rFVIIa enhances thrombin generation on the platelet surface at the site of injury independently of the presence of FVIII or FIX. Pharmacologic doses of rFVIIa induce hemostasis not only in hemophilia patients, but also in patients with thrombocytopenia, functional platelet defects, and with profuse bleeding triggered by extensive surgery or trauma. The general mechanism of action of rFVIIa to induce hemostasis under these conditions may be its capacity to generate a tight fibrin hemostatic plug through increased thrombin generation. A tight fibrin plug will aid in resisting the overwhelming local release of fibrinolytic activity triggered by vast tissue damage occurring in extensive trauma. Local fibrinolytic activity also occurs in the gastrointestinal tract as well as during profuse postpartum bleeding. Pharmacologic doses of rFVIIa induce hemostasis in these cases also.

Blood Coagulation Disorders↗
PubMed2005

A study of the pharmacokinetics and safety of recombinant activated factor VII in healthy Caucasian and Japanese subjects.

In this randomized, placebo-controlled, double-blind, single-centre, dose escalation study, we report the first evaluation of the pharmacokinetics and safety of recombinant activated factor VII (rFVIIa) in healthy Caucasian and Japanese subjects. Thirty-two healthy subjects were stratified according to sex and ethnic group to receive single bolus intravenous injections of three different doses of rFVIIa (40, 80, 160 microg/kg rFVIIa) or placebo, each separated by a 7-day wash-out period. Blood samples were taken up to 24 h after dosing. The factor VII clotting activity appeared to be dose dependent, but independent of sex and ethnic group. Statistical analyses demonstrated no significant effect of dose, sex or ethnicity on the dose-normalized mean area under the plasma concentration-time curve AUC0-t, indicating dose proportionality. No serious adverse events or thromboembolic events were reported. Analyses of coagulation parameters did not suggest induction of systemic coagulation when dosing rFVIIa up to 160 microg/kg. In conclusion, the pharmacokinetics of rFVIIa in Caucasian and Japanese subjects are similar, and no safety issues were identified.

Adult↗
PubMed2005

High dose factor VIIa improves clot structure and stability in a model of haemophilia B.

Factor IX (FIX) deficiency results in haemophilia B and high dose recombinant activated factor VII (rFVIIa) can decrease bleeding. Previously, we showed that FIX deficiency results in a reduced rate and peak of thrombin generation. We have now used plasma and an in vitro coagulation model to examine the effect of these changes in thrombin generation on fibrin clot structure and stability. Low FIX delayed the clot formation onset and reduced the fibrin polymerisation rate. Clots formed without FIX were composed of thicker fibrin fibres than normal. rFVIIa shortened the clot formation onset time and improved the fibre structure of haemophilic clots. We also examined clot formation in the presence of a fibrinolytic challenge by including tissue plasminogen activator or plasmin in the reaction milieu. In these assays, normal FIX levels supported clot formation; however, clots did not form in the absence of FIX. rFVIIa partially restored haemophilic clot formation. These results were independent of the effects of the thrombin-activatable fibrinolysis inhibitor. Our data suggest that rFVIIa enhances haemostasis in haemophiliacs by increasing the thrombin generation rate to both promote formation of a structurally normal clot and improve clot formation and stability at sites with high endogenous fibrinolytic activities.

Blood Coagulation↗
PubMed2004

Dosing with recombinant factor viia based on current evidence.

Recombinant factor VIIa (rFVIIa; NovoSeven(R), Novo Nordisk, Bagsvaerd, Denmark) induces hemostasis in patients with severe hemophilia and inhibitors, and has been found to control hemorrhage associated with severe trauma and surgery in patients with basically normal hemostatic mechanisms from the start. By enhancing the generation of thrombin on activated platelets, rFVIIa facilitates the formation of a tight, stable fibrin plug that is resistant to premature lysis. Clinical efficacy has been achieved with doses of rFVIIa much lower than originally proposed by in vitro models. Based on early clinical experiences, a dosing schedule of 90 to 120 microg/kg every 2 hours for the first 24 hours was recommended for serious bleeds and surgical cover. This schedule has been shown to induce and maintain hemostasis in 83% to 95% of serious bleeding episodes, and in 90% to 100% of major surgical cases. However, "mega" doses of rFVIIa may demonstrate greater efficacy in the treatment of joint bleeds, as they are more likely to evoke a full thrombin burst. Interpatient variation in recovery rates, clearance rates, and the ability to generate thrombin on the activated platelet surface may influence the efficacy of rFVIIa. Optimal doses may thus vary not only between hemophilia patients, but also between patients treated for other bleeding disorders.

Blood Coagulation↗
PubMed2004

Effect of active site-inactivated factor VIIa on ischaemia/reperfusion injury in a porcine flap model.

In free flap surgery, restored blood flow following a lengthy ischaemic period may lead to necrosis as a result of ischaemia/reperfusion (IR) injury. This injury comprises both proinflammatory and prothrombotic events, where the tissue factor/factor VIIa complex probably has a key role. Active site-inactivated factor VIIa (FFR-rFVIIa) exerts an antithrombotic effect by binding to tissue factor without initiating coagulation. In this study we have evaluated the potential protective effects of FFR-rFVIIa in IR injury. Bilateral musculocutaneous latissimus dorsi flaps in 16 pigs were made ischaemic for eight hours, then given 1 mg/kg/flap of FFR-rFVIIa or vehicle intra-arterially, and reperfused for 10 hours. The viable:necrotic tissue ratio, and accumulation of radiolabelled leucocytes, fibrinogen, and platelets were measured. There was no effect on tissue survival, but radiolabelled components in viable tissue were increased, though not significantly so. We conclude that FFR-rFVIIa did not prevent IR injury, indicating that tissue factor-mediated coagulation is not an important determinant of IR injury in this setting.

Animals↗
PubMed2004

Recombinant factor VIIa increases the pressure at which rebleeding occurs in porcine uncontrolled aortic hemorrhage model.

In trauma patients, resuscitation to endpoints below normal blood pressure (BP) levels may reduce further blood loss due to the rebleeding often caused by more aggressive resuscitation. However, patients whose BP is maintained at lower levels for extended periods are at increased risk for organ failure. The purpose of this study was to determine whether recombinant activated factor VII (rFVIIa) raises the BP level at which rebleeding occurs in a prospective, randomized, blinded study using a porcine model of uncontrolled hemorrhage and resuscitation. Thirty anesthetized 40-kg pigs were assigned to three groups (n = 10/group): control, low-dose rFVIIa (180 microg/kg), or high-dose (720 microg/kg). Vehicle or drug was infused 5 min before creating a 2.0-mm infrarenal aortotomy. Ten minutes later, resuscitation with lactated Ringer's (LR) solution at 100 mL/min was begun. Hemorrhage and LR volumes and BP were recorded continuously. We found that pretreatment with rFVIIa increased the mean arterial pressure at which rebleeding occurred during resuscitation (45 +/- 3, 69 +/- 5, and 66 +/- 6 mmHg in the control, low-dose, and high-dose groups, respectively, P = 0.003). Rebleed hemorrhage volume was reduced with rFVIIa (39 +/- 9, 22 +/- 7, and 26 +/- 5 mL/kg for control, and low and high dose, respectively; P = 0.055). This is the first study to show that rFVIIa increases the BP at which rebleeding occurs during resuscitation in an injury to a major artery, suggesting the formation of a tight, stronger fibrin plug in the presence of high concentrations of rFVIIa.

Animals↗
PubMed2003

The effect of recombinant factor VIIa on noncoagulopathic pigs with grade V liver injuries.

BACKGROUND: Recombinant Factor VIIa (rFVIIa) has been used to decrease bleeding in a number of settings, including hemophilia, liver transplantation, intractable bleeding, and cirrhosis. It has also been shown to reduce bleeding in coagulopathic pigs with Grade V liver injuries when used as an adjunct to packing. This study was performed to determine if rFVIIa would reduce blood loss after a Grade V liver injury in noncoagulopathic pigs when used as sole therapy. STUDY DESIGN: Thirty normothermic animals were randomized to receive either 150 microg/kg of rFVIIa or normal saline intravenously. After laparotomy and splenectomy, a standardized Grade V liver injury was made with a liver clamp. Thirty seconds after injury, blinded therapy was given. Blood loss was measured 15 minutes after injury and the abdomen was closed. Animals were resuscitated to their baseline blood pressure and the study was continued for 2 hours. Serial coagulation parameters were obtained. Following the study period, blood loss was measured and an autopsy was performed. Grossly normal areas of lung were examined for evidence of intravascular thrombosis. RESULTS: Mean Factor VII:C levels increased 155-fold in the treatment group after infusion of rFVIIa. The mean prothrombin time in the treatment group decreased from 9.8 +/- 0.4 seconds to 7.3 +/- 0.2 seconds and remained significantly different from the control group throughout the study (p < 0.01). There were no differences in other coagulation parameters. Mean initial blood loss was 822 +/- 266 mL in the treatment group and 768 +/- 215 mL in the control group (p = 0.6). Rebleeding blood volume was 397 +/- 191 mL in the treatment group and 437 +/- 274 mL (p = 0.6) in the control group. Lung histology revealed no evidence of abnormal microvascular thrombosis. CONCLUSIONS: rFVIIa does not reduce blood loss after Grade V liver injury when it is used as sole therapy in warm noncoagulopathic pigs.

Animals↗
PubMed2003

Recombinant factor VIIa (NovoSeven) as a hemostatic agent.

Recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Denmark) induces hemostasis in life- and limb-threatening bleeds and in major surgery of hemophilia A and B patients, regardless of inhibitor titer. A total of more than 6,500 patients have been treated, and NovoSeven has been administered in more than 180,000 standard doses. Experience gained from these clinical situations suggests that NovoSeven should be administered as a 90- to 110-microg/kg bolus dose every second hour. Hemophilia patients with mild to moderate bleeding episodes require two to three doses to achieve complete hemostasis, whereas patients with severe bleeding episodes may require more doses. For major surgery and in cases of life-threatening bleeding, dosing every second hour for the first 24 hours may be required. Thereafter, the same dose, but with longer intervals between doses, is recommended. Recent in vitro experiments indicate that even higher doses of NovoSeven may be needed to achieve full thrombin generation in the absence of factor VIII (FVIII), factor IX (FIX), and factor XI (FXI).

Blood Coagulation↗
PubMed2003

rFVIIai in Acute Coronary Syndromes.

Following vessel wall injury, tissue factor (TF) is exposed and forms complexes with already activated factor VII (FVIIa) present in the circulating blood, thereby initiating the hemostatic process. After the first FXa is formed, the TF pathway inhibitor (TFPI) forms a complex with FXa, and a quaternary complex is formed, TF/FVIIa/ FXa/TFPI, which inhibits the first step of the hemostatic pathway. Recombinant activated FVII (rFVIIa) has been developed for use as a hemostatic agent (NovoNordisk A/S, Denmark). Active site-inactivated rFVIIa (rFVIIai) has also been prepared and was shown to have a faster association to and a slower dissociation from TF than rFVIIa, resulting in a lower calculated Kd of rFVIIai compared with rFVIIa. In various animal models rFVIIai has been demonstrated to prevent or diminish immediate thrombus formation at the site of vessel wall injury (athroplasty or other forms of mechanical injury) as well as the development of long-term intima thickening. The inflammatory response following endotoxin-induced sepsis was shown to decrease after administration of rFVIIai. Also, survival increased in the rFVIIai-treated animals in this study. In addition, ischemia-reperfusion injury was mitigated by rFVIIai. In a limited number of patients undergoing percutaneous transluminal coronary angioplasty (PTCA), rFVIIai was observed to allow PTCA to be performed at lower doses of heparin than what has been reported previously.

Angioplasty, Balloon, Coronary↗
PubMed2003

Potential role of recombinant factor VIIa as a hemostatic agent.

Recombinant factor VIIa (rFVIIa) has been shown to induce hemostasis in hemophilia patients with inhibitors against factor VIII or factor IX independent of factor VIII/factor IX. Factor VIIa binds to tissue factor (TF) exposed at the site of injury and generates, through factor X activation on the TF-bearing cells, enough thrombin to activate factors VIII, V, and XI, as well as platelets. The thrombin-activated platelets provided a perfect template for binding of activated factors VIII, IX, and V, further activation of factor X, and thrombin generation. Factor VIIa in high concentrations binds to thrombin-activated platelets and is capable of activating factor X, thereby generating thrombin independent of the presence of factor VIII or factor IX. Accordingly, rFVIIa has been shown to initiate hemostasis in severe hemophilila patients with inhibitors subjected to major surgery and suffering from serious limb- and life-threatening bleeding. Since rFVIIa enhances thrombin generation-thereby providing the formation of tight, stable fibrin hemostatic plugs resistance to premature lysis-it should be hemostatic in other situations characterized by impaired thrombin generation. A hemostatic effect has been reported in patients with various platelet disorders and factor XI deficiency. Further, a hemostatic effect of rFVIIa has been reported in patients subjected to trauma and extensive surgery who have developed profuse, excessive bleeding resulting in hemodilution and changes in coagulation patterns. rFVIIa was developed to treat bleeding in hemophilia patients with inhibitors against factor VIII or factor IX and has been shown to induce effective hemostasis in most such patients and also in life- and limb-threatening bleeding. It has also been used successfully to stop bleeding in patients who do not have hemophilia but who do have acquired antibodies against factor VIII (acquired hemophilia). rFVIIa initiates hemostasis by forming a complex with TF exposed as a result of vessel wall injury. Pharmacologic doses of rFVIIa can enhance thrombin generation on platelets that are already thrombin-activated, resulting in the formation of full thrombin burst. By enhancing thrombin generation, rFVIIa helps to form tight, stable, fibrin plugs resistant to premature fibrinolysis. This also maintains hemostasis in the absence of factor VIII or factor IX. Pharmacologic doses of rFVIIa may accordingly be of benefit in producing hemostasis in situations other than hemophilia characterized by profuse bleeding and impaired thrombin generation. There is now clinical experience indicating a hemostatic effect in patients with thrombocytopenia and functional platelet defects. rFVIIa has also been successfully used in acute trauma patients with profuse bleedings and in other bleeding situations.

Blood Platelet Disorders↗
PubMed2002

The effect of recombinant factor VIIa on coagulopathic pigs with grade V liver injuries.

BACKGROUND: Recombinant factor VIIa (rFVIIa) has been used to decrease bleeding in a number of settings including hemophilia, liver transplantation, intractable bleeding, and cirrhosis. Experience in the trauma setting is limited. This study was performed to determine whether rFVIIa would reduce bleeding after a grade V liver injury in hypothermic, dilutionally coagulopathic pigs when used as an adjunct to abdominal packing and to determine whether increasing the dose of the drug increased its hemostatic efficacy. METHODS: Thirty animals were randomized to receive 180 microg/kg of rFVIIa, 720 microg/kg of rFVIIa, or vehicle buffer control. After laparotomy and splenectomy, animals underwent a 60% blood volume isovolemic exchange transfusion with 5% human albumin. The animals' temperature was maintained at 33 degrees C and a standardized grade V liver injury was made with a liver clamp. Thirty seconds after injury, the abdomen was packed with laparotomy sponges, resuscitation was initiated, and blinded therapy was given. Animals were resuscitated to their baseline mean arterial pressure and the study was continued for 2 hours. Serial coagulation parameters were measured at the temperature they were drawn. After the study period, surviving animals were killed, posttreatment blood loss was measured, and an autopsy was performed. RESULTS: Ten animals were randomized to each group. After administration of study drug, factor VII clotting activity (FVII:C) was higher in the 720 microg/kg group than in the 180 microg/kg group (p < 0.01). FVII:C was higher in both treatment groups than in the control group (p < 0.01). The mean prothrombin time was shorter in the treatment groups than in the control group (p < 0.05). Mean arterial pressure was lower in the control group than in the treatment groups throughout the study (p < 0.01). Mean blood loss was less in the treatment groups than in the control group (p = 0.03). Mortality was not different between groups. There were no differences between the groups that received rFVIIa in any measured parameters except for FVII:C. Liver injuries were similar between groups and there was no evidence of microthrombosis on lung histology. CONCLUSION: rFVIIa reduces blood loss in hypothermic, dilutionally coagulopathic pigs with grade V injuries when used as an adjunct to packing. Increasing the dose does not enhance the hemostatic effect.

Animals↗
PubMed2002

General haemostatic agents--fact or fiction?

Haemophilia is the most serious bleeding model that nature has provided us with, indicating the importance of factor FVIII and FIX in haemostasis. According to current knowledge, haemostasis is initiated by the formation of a complex between tissue factor (TF), exposed as a result of a vessel wall injury, and activated FVII (FVIIa) that is normally present in circulating blood. The TF-FVIIa complexes convert FX into FXa on the TF-bearing cell. FXa then activates prothrombin into thrombin. This limited amount of thrombin activates FVIII, FV, FXI and platelets. Thrombin-activated platelets change shape, resulting in exposure of negatively-charged phospholipids, which form the perfect template for full thrombin generation involving FVIII and FIX. In patients with haemophilia FVIII or FIX is missing. These individuals generate only initial limited amounts of thrombin as its generation is dependent on the presence of FVIII and FIX. Full thrombin generation is necessary for complete activation of FXIII and thrombin activatable fibrinolytic inhibitor to occur. Furthermore, full thrombin generation is important for the fibrin structure of the haemostatic plug. In the case of impaired thrombin generation, fibrin plugs will be loose and highly permeable. Such fibrin plugs are easily dissolved by normal fibrinolytic activity and thus prevent full and maintained haemostasis from occurring. The addition of rFVIIa to FVIII- or FIX-deficient plasma has been shown to increase thrombin generation in a cell-based in vitro model. Furthermore, extra rFVIIa was found to normalise fibrin clot permeability in vitro and to tighten the fibrin structure as studied by three-dimensional confocal microscopy. These findings indicate that administration of rFVIIa is capable of compensating for the lack of FVIII and FIX. Accordingly, the administration of exogenous rFVIIa has been found to stop bleedings in haemophilia patients and, provided it is given in doses high enough, to allow major surgery to be performed in severe haemophiliacs with inhibitors. As rFVIIa enhances thrombin generation on already activated platelets, it has been suggested that rFVIIa may also help to improve haemostasis in other situations involving impaired thrombin generation, such as platelet disorders (thrombocytopenia and functional platelet defects). Preliminary clinical data appear to support this. Patients with profuse bleeding due to extensive surgery or trauma often develop a complex coagulation pattern which includes reduced plasma levels of fibrinogen, FVIII and FV, and decreased platelet counts. These patients may well have an impaired capacity to generate thrombin. Consequently, they may benefit from one or two doses of rFVIIa in order to assist in the generation of a thrombin peak sufficient to form a firm, stable fibrin haemostatic plug and thereby reduce bleeding. This would facilitate any mechanical repair necessary for full haemostasis. Preliminary results in a few patients may support such an effect for rFVIIa. As thrombin has such a crucial role in providing haemostasis, any agent that enhances the thrombin generation in situations with an impaired thrombin formation may be characterised as a 'general haemostatic agent'. Let us look forward to more 'facts' through the 'evidence-based route'.

Factor VII↗
PubMed2002

Evaluation of potential antigenicity of active-site-inhibited recombinant human FVIIa (FFR-rFVIIa) in an immune-tolerant rat model.

Recombinant human FVIIa (rFVIIa) was inactivated by coupling Phe-Phe-Arg-CK- (FFR) covalently to the active site of the enzyme. To test the chemically-modified human protein for potential antigenicity prior to clinical trial an immune-tolerant rat model was established. Intraperitoneal injection of the parent compound, human rFVIIa, within 30 h after birth, followed by repeated subcutaneous challenge with rFVIIa in Freunds incomplete adjuvant resulted in 79% non-responding rats at day 32. Monthly subcutaneous challenge showed that the induced tolerance was stable over the 3 months study period in 80% of the rats. The clinically relevant route, intravenous administration, was used for evaluating the potential antigenicity of FFR-rFVIIa. Repeated intravenous administration of different dosages of FFR-rFVIIa did not break tolerance, indicating that FFR-rFVIIa might not be antigenic, for a limited number of intravenous administrations in a clinical setting.

Amino Acid Chloromethyl Ketones↗