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Ultralarge multimers (ULM) of VWF are considered to be the most active with respect to binding to platelets and to subendothelial structures and therefore are of critical importance for the function of VWF in stabilizing the primary hemostatic plug. In contrast to plasma-derived FVIII-VWF concentrates, human rVWF obtained from mammalian cell culture retains the full-spectrum of intact multimers, including ULM, as physiologically formed in the Golgi apparatus and stored in platelet α-granules and endothelial cell Weibel-Palade bodies. In the course of physico and biochemical, functional and animal studies, rVWF exhibited superiority in structure and function compared to pdVWF. These effects seemed to correlate with the multimer size and therefore might be attributed to the presence of ULM in rVWF preparations. The pharmacokinetic (PK), safety and efficacy characteristics seen in preclinical studies were further demonstrated in clinical trials.Den hochmolekularen Multimeren des VWF wird die höchste Aktivität zugeschrieben, an Thrombozyten und subendotheliale Strukturen zu binden und sind daher von kritischer Relevanz für die hämostatischen Funktionen von VWF bei der Stabilisierung des Thrombus. Im Gegensatz zu allen FVIII/VWF-haltigen Konzentraten, die aus Plasma gewonnen werden, behält humaner rVWF, der aus Säugetierzellkulturen gewonnen wird, ein intaktes Multimerspektrum, welches physiologisch im Golgi Apparat gebildet und in den α-Granula der Thrombozyten und in Weibel-Palade-Körperchen der Endothelzellen gespeichert wird. Im Rahmen physiko-und biochemischer, funktioneller und tierexperimenteller Studien wurde festgestellt, dass rVWF bessere Eigenschaften in Struktur und Funktion als plasmatischer VWF besitzt. Diese Eigenschaften korrelieren möglicherweise mit der Größe der Multimere und könnten daher durch die hochmolekularen Multimere in rVWF hervorgerufen werden. In den klinischen Studien, die mit rVWF im Vergleich zu einem plasmatischen FVIII-VWF-Konzentrat durchgeführt wurden, konnten die für rVWF anderen pharmakologischen und pharmakokinetischen Eigenschaften als für pdVWF bestätigt werden.

Hemophilia A is a rare genetic bleeding disorder caused by missing or defective factor VIII (FVIII), a crucial factor in blood coagulation. Baxalta Innovations / Shire is currently developing SHP 826 (BAX 826), as the next generation EHL rFVIII replacement for the treatment of hemophilia A. SHP 826 is the first EHL rFVIII to utilize novel technology in which polysialic acid (PSA) is conjugated to Baxalta's licensed rFVIII product (Advate) to extend the circulation half-life of FVIII. The potential toxicity of SHP 826 was evaluated in Sprague Dawley rats and cynomolgus monkeys. Acute toxicity was assessed in a dose-escalation study in monkeys that were intravenously administered 350 and 1800 IU rFVIII/kg SHP 826. Repeat dose toxicity was assessed in rats at doses of 80, 350, and 800 IU rFVIII/kg, in monkeys at doses of 800 IU rFVIII/kg every 5 days for 4 weeks, and in monkeys at doses of 80, 350, or 600 IU rFVIII/kg every five days for 31 days. Cardiovascular and respiratory safety was assessed in one of the repeat dose toxicity studies in monkeys. The thrombogenic potential of SHP 826 was evaluated after a single intravenous administration in rabbits (900 rFVIII/kg BW) using a stasis model developed by Wessler et al. (1959). The licensed rFVIII product was used as a comparator in the acute toxicity study and in the Wessler test. Parameters evaluated included local tolerance at the injection site, body weight, clinical pathology, blood gas analysis, analysis of neutralizing and binding antibodies in plasma, organ weight, necropsy observation, and histopathological evaluation. In addition, seminology (epididymal and testicular sperm) was assessed in rats, and urinalysis, ophthalmic and telemetric cardiovascular examination and respiratory rate measurement was performed in monkeys. SHP 826 showed a favorable safety/toxicity profile in both species used, with no abnormalities directly caused by test item administration noted in vivo or during clinical or histopathological examination. The NOAEL was set to the highest dose tested in each study (i.e. 800 IU rFVIII/kg BW in rats and 600 IU rFVIII/ kg BW in monkeys). Toxicokinetic evaluation indicated dose proportional PK. Exposure to FVIII decreased after repeated treatments due to appearance of anti-FVIII antibodies in both species. Development of anti-FVIII antibodies is an expected immune response after repeated application of heterologous proteins, and is not predictive of any potential for triggering development of anti-FVIII antibodies in humans. Safety pharmacology studies with SHP 826 revealed no evidence of thrombogenic potential in rabbits. SHP 826 administered intravenously was well tolerated in conscious telemetered cynomolgous monkeys (part of the repeat dose toxicity study) and did not cause any adverse clinical, cardiovascular, or respiratory effects. The results of these safety studies indicate sufficient safety margins to support the anticipated maximum clinical dose, and therefore entry into clinical development. Disclosures Ehrlich: Shire: Employment. Leidenmuehler:Shire: Employment. Dietrich:Shire: Employment. Hoebarth:Shire: Employment. Ruthsatz:Shire: Employment. Turecek:Shire: Employment. Wolfsegger:Shire: Employment, Equity Ownership. Weber:Shire: Employment. Gritsch:Shire: Employment. Hoellriegl:Shire: Employment. Turecek:Shire: Employment.

To support the BAX 604 (FEIBA STAR) clinical study assessing different infusion rates and low volume reconstitution, the following nonclinical program was initiated: Two studies were performed in normal rats and FVIII-inhibited rabbits to evaluate the acute toxicity of FEIBA using different infusion rates. Three further studies were conducted using FEIBA at a reduced reconstitution volume (50% of the currently licensed amount of water for injection, internally identified as BAX 604) to evaluate efficacy, thrombogenicity, and local tolerance in normal and FVIII-inhibited rabbits. The two acute toxicity studies assessed the effect of administering 100 U/kg FEIBA twice 6 hours apart at infusion rates ranging from 2 to 100 U/kg/min. In rats, an exaggerated pharmacological effect was observed with all rates, the appearance and severity of which did not correlate with infusion rate. In FVIII-inhibited rabbits, the incidence and/or severity of thrombi also did not increase with infusion rate. These results revealed no correlation between an increasing infusion rate and increasing thrombogenicity of FEIBA. The effect of a reduced reconstitution volume on efficacy and thrombogenicity was assessed in FVIII-inhibited and normal rabbits. Efficacy was tested in a FVIII-inhibitor nail clip model. Each rabbit was treated intravenously with heat-inactivated FVIII-inhibitor goat plasma 45 minutes prior to nail cut. The nail on digit II of the right hind limb was cut and blood was collected. Rabbits were treated intravenously with BAX 604 or FEIBA at doses of 10, 50, or 100 U/kg BW and blood was collected for another 30 minutes. Endpoints included blood loss before and after treatment. No spontaneous deaths or signs of acute toxicity were observed. Treatment with BAX 604 or FEIBA resulted in a dose-related lower relative blood loss (71%, 2%, and 4% for BAX 604; 104%, 3%, and 3% for FEIBA) that was statistically significant (at the 5% level) for doses of 50 or 100 U/kg. The thrombogenic potential of BAX 604 vs. FEIBA was evaluated after a single intravenous administration of 4 U/kg BW, which results in an infusion rate of 16 U/kg/min. Thrombogenicity was determined semi-quantitatively, scoring thrombus formation in the isolated jugular vein on a scale of 0 to 4. The thrombus scores obtained - 1 (n=5), 0.5 (n=1), and 0 (n=2) for BAX 604, and 2 (n=4), 1 (n=2), 0.5 (n=1) and 0 (n=1) for FEIBA - demonstrated that BAX 604 is no more thrombogenic than FEIBA in the rabbit stasis model using an infusion rate of 16 U/kg/min (1.6-fold maximum infusion rate planned for the clinical trial). Based on these results, BAX 604 is considered to be as safe as FEIBA with regard to thrombogenicity. The local tolerance of BAX 604 and FEIBA was evaluated after intravenous, intra-arterial (2 mL/animal), or paravenous injection (0.5 mL/animal) into rabbits' ears. BAX 604 was well tolerated after intravenous and intra-arterial injection. Following a single paravenous injection, mild local irritation was observed for both products. This effect was slightly more pronounced for BAX 604 than for FEIBA, indicating that misapplication of either product should be avoided. The results of these nonclinical studies indicate that the efficacy and safety profiles of FEIBA and BAX 604 are comparable. Infusion rates of up to 100 U/kg/min (10-fold the planned clinical infusion rate) of the low reconstitution product did not reveal any concerns with regard to adverse clinical signs. As the infusion is based on U/kg/min (and not ml/kg/min), these studies also support using the low reconstitution volume. In conclusion, neither a faster infusion rate nor lower reconstitution volume of FEIBA are predicted to pose additional risks to human subjects when tested under the conditions of a clinical study. Disclosures Hoebarth: Shire: Employment. Kopic:Shire: Employment. Wolfsegger:Shire: Employment, Equity Ownership. Bauer:Shire: Employment. Turecek:Shire: Employment. Leidenmuehler:Shire: Employment. Benamara:Shire: Employment. Hoellriegl:Shire: Employment. Turecek:Shire: Employment.