Preclinical toxicity biomarkers for combination treatment with clotting factors rFXIII and rFVIIa.

Combination treatment with the clotting factors recombinant activated factor VII (rFVIIa), serine protease, and recombinant factor XIII (rFXIII), protransglutaminase, is being explored for haemostatic therapy. We performed a single-dose toxicology study in the cynomolgus monkey, with four dose groups receiving 0.1 + 0.34 mg kg(-1) (group 1), 0.33 + 1.12 mg kg(-1) (group 2), 1.67 + 5.60 mg kg(-1) (group 3) and 5.00 + 16.80 mg kg(-1) (group 4) of a rFVIIa + rFXIII combination. In the three lower dose groups, no clinical, histopathological or blood chemistry changes were observed. In group 4, the animals died at 4 h post-dosing, with histopathology revealing a systemic coagulopathy resembling, but distinct from, disseminated intravascular coagulation. Due to the absence of toxicity warning signs, toxicity biomarkers were identified by a Western blot-based screening of approximately 20 plasma proteins known to be involved in the clotting cascade. Three of the examined proteins were specifically affected by rFVIIa + rFXIII treatment. Fibronectin and fibrinogen exhibited dose-dependent reductions from less than 10% reduction (group 2) to more than 90% reduction (group 4). These reductions were reversible, and specific. For vitronectin, a dose-dependent conversion to the 65-kDa form was found to occur in groups 3 and 4. Thus, fibrinogen, fibronectin and vitronectin represent the first biomarkers for clotting factor toxicity.

Preclinical safety and pharmacokinetics of recombinant human factor XIII.

Factor XIII (FXIII) is a thrombin-activated protransglutaminase responsible for fibrin clot stabilization and longevity. Deficiency in FXIII is associated with diffuse bleeding and wound-healing disorders in humans. This report summarizes results from several studies conducted in adult cynomolgus monkeys (M. fascicularis) to evaluate the safety and pharmacokinetics of recombinant human factor XIII A(2) dimer (rFXIII). Intravenous slow bolus injection of rFXIII resulted in the expected formation of the heterotetramer rA(2)cnB(2), prolonged circulating half-life (5-7 days), and increased plasma transglutaminase activity. Recombinant FXIII was well tolerated as a single dose up to 20 mg/kg rFXIII (2840 U/kg), as repeated daily doses up to 6 mg/kg (852 U/kg) for 14 days, and as 3 repeated doses of 8 mg/kg (1136 U/kg) separated by 14 days. Overt toxicity occurred after a single intravenous injection of = 22.5 mg/kg rFXIII (3150 U/kg), or with 2 doses of = 12.5 mg/kg (1775 U/kg) administered within 72 hours. The rFXIII-mediated toxicity was expressed as an acute systemic occlusive coagulopathy. Evaluation of plasma samples from dosed animals demonstrated formation of cross-linked fibrin/fibrinogen oligomers and higher-order protein aggregates, which are hypothesized to be responsible for the observed vessel occlusion and associated embolic sequelae. These results demonstrate that rFXIII-mediated toxicity results from exaggerated pharmacological activity of the molecule at supraphysiological concentrations. The absence of observed toxicological effect with repeated intravenous doses up to 8 mg/kg (1136 U/kg) was used to support an initial clinical dose range of 0.014 to 0.35 mg/kg (2-50 U/kg).

Mediastinal fibrin glue: hemostatic effect and tissue response in calves.

There is continued controversy regarding the effectiveness and potential adverse effects of fibrin glue. Thus, we chose to evaluate it in a model of experimental calf aortic valve replacement that has been previously well established. Concentrated fibrinogen and topical thrombin were sprayed to form a thin layer of fibrin glue over the mediastinal tissues of 20 consecutive calves undergoing aortic valve replacement. Chest tube outputs of these animals were compared with those of the preceding 20 consecutive calves undergoing aortic valve replacement without fibrin glue. All procedures were performed by the same surgeon, and no other technical changes were made between the two series. Total postoperative chest tube output (mean +/- standard error) was 553 +/- 50 mL for the calves treated with fibrin glue and 1,155 +/- 103 mL for the control calves (p less than 0.001). On histological examination of mediastinal tissues from 5 treated calves killed 6 weeks after operation, there was no evidence of inflammation, fibrosis, or residual fibrin. To our knowledge, this is the first controlled laboratory study to show that fibrin glue spray is an effective hemostatic agent and that it produces no long-term tissue reaction.

Autologous fibrin tissue adhesive for cerebrospinal fluid leaks: a controlled study of neurotoxicity.

Postoperative cerebrospinal fluid (CSF) leakage continues to be one of the most common and potentially serious complications following translabyrinthine surgery despite numerous strategies aimed at its prevention. Fibrinogen-based tissue adhesives may be helpful in decreasing this complication rate. Although commercial glues have been used widely in Europe (especially for dural repairs), they are not approved for use in the United States. Recent investigation has provided a relatively simple technique for producing a comparable autologous glue that obviates the risks of the commercial product. Since this glue will bind fascia, fat, and dura in the watertight fashion, it is potentially ideal for preventing CSF leaks. Experimental studies in rabbits reveal that autologous tissue adhesive can be used safely around intact nerves, suggesting it can be used safely to supplement fat, fascia, or muscle plugs for closing translabyrinthine defects. Clinical trials to test the efficacy of tissue adhesives in this application are currently under way.

Autologous fibrin tissue adhesive biodegration and systemic effects.

A series of experiments was conducted to investigate the rate of Autologous Fibrin Tissue Adhesive (AFTA) degradation by the fibrinolysis inhibitor, epsilon amino caproic acid (EACA). The duration of AFTA clots in vitro, subcutaneous, and in the middle ear was prolonged for a time interval that was proportional to the concentration of EACA in Component II of the adhesive. No toxic reactions were observed in the middle or inner ear. Systemic pathology (thrombosis or emboli) could not be related to the presence of EACA applied in the middle ear or directly into the blood stream at concentrations (mg/kg body weight) up to 1,500 times that expected to occur during surgery on humans.

Effect of human fibrin adhesive on the ear. An electrophysiological study of auditory function in the guinea pig correlated to light and electron microscopy of middle ear mucosa and inner ear structures.

The effect of human fibrin adhesive applied to the middle ear has been studied in guinea pig. Auditory function was measured using acoustically evoked brainstem responses. Middle and inner ear structures were studied with light, transmission and scanning electron microscopy. A transitory conductive hearing loss was observed, but after 8 weeks the auditory function appeared normal. Microscopy of the middle and inner ear failed to show any tissue damage.