Nonclinical Safety Assessment of a Long-Acting Recombinant PEGylated Factor Eight (BAY 94-9027) With a 60 kDa PEG.

BAY 94-9027 (Jivi) is a site-specifically PEGylated human B-domain-deleted (BDD) recombinant factor VIII (FVIII), with a 60 kDa branched PEG molecule attached. The nonclinical safety of BAY 94-9027 was evaluated in a toxicology program that included 2 weeks intravenous (IV) toxicity studies in rats and rabbits, a juvenile toxicity study in rats as well as a 26-week chronic study in rats. Doses of 75, 750, or 2250 IU/kg given every other day for 2 weeks did not elicit any findings related to BAY 94-9027. Specifically, no thrombus formation or histological changes such as cellular vacuolation were seen. In the chronic toxicity study, 40, 400, and 1200 IU/kg of BAY 94-9027 given twice weekly did not induce adverse effects related to BAY 94-9027, and no tissue vacuolation was observed. There was no PEG detected in choroid plexus or other areas of the brain, cerebrospinal fluid or in spleen or kidneys. These results were supported by toxicity studies in rats and rabbits treated with PEG 60 kDa attached to the maleimide linker (PEG-60-Mal-Cys). No findings related to PEG-60-Mal-Cys were seen. These results demonstrate the safety of BAY 94-9027 for long-term use.

A new animal model of thrombophilia confirms that high plasma factor VIII levels are thrombogenic.

The thrombotic risk associated with elevated plasma levels of clotting factor VIII (FVIII) was investigated in a mouse model of thrombophilia. After the intravenous injection of recombinant human FVIII and/or of purified FVIII-free human von Willebrand factor (vWF), a controlled mild injury was inflicted on the carotid artery of FVB mice by irradiation with filtered green light in combination with intravenous injection of the dye rose bengal. Formation of a platelet-rich thrombus was continuously monitored for 40 min via transillumination and the thrombus size was measured via image analysis. Administration of recombinant human FVIII at 40 microg/kg led to initial FVIII plasma activities equivalent to 250% of normal human plasma FVIII activity and significantly enhanced thrombus size. Immunohistochemical staining illustrated the accumulation of FVIII within the thrombi. Human vWF, even at 10 mg/kg, had no effect on thrombus formation. The thrombotic tendency induced by FVIII was significantly inhibited by the administration of human vWF in a dose-dependent manner. Separate plasma measurements revealed that human FVIII has comparable affinities for human and murine vWF but that human vWF does not effectively bind murine platelets. The inhibition by human vWF of the thrombotic tendency induced by human FVIII could therefore be explained by a lack of accumulation of FVIII within the developing thrombus because of the reduced affinity of human vWF for murine platelets and the reduced occupancy of murine von Willebrand factor by human FVIII after injection of human vWF. These results show that vWF actively participates in FVIII accumulation in the arterial thrombus and provide experimental evidence for epidemiological findings that elevated plasma FVIII levels are associated with an increased thrombotic risk, also in arteries.

Assessment of potential toxic effects of treated Hemofil injection in rats and mice and on the systemic hemodynamics in dogs.

The toxic effects of treated Hemofil (T-AHF) injection were evaluated by acute and subchronic intravenous administration to rats, mice, and dogs. Acute iv administration of T-AHF to rats and mice at dosages of 1320 units (U) of Factor VIII/kg did not produce toxic signs. Both species were found to have an LD50 greater than 1320 U. Seven-day iv administration to rats of T-AHF at dosages of 20, 40, and 60 U/kg and 3-mo administration (3 times/wk for 13 wk) of T-AHF at dosages of 100 and 200 U/kg did not produce any signs of toxicity. There were no treatment-related effects on body weights, hematology, clinical chemistry, urinalysis, ocular tissues, or histopathology. Intravenous administration to dogs at 0.5, 1.0, and 5.0 ml/min . kg (28 U/ml, 100 U/kg at each rate) produced no significant adverse effects on mean arterial pressure, cardiac output, or heart rate. No adverse changes in pulmonary function, as reflected by arterial blood-gas profiles, were observed. It is concluded that animals tolerated well T-AHF administered at dosages and rates similar to or greater than dosages used clinically. The results obtained from these studies establish a reasonable margin of safety and support the acceptability of the T-AHF for clinical use.