Essentials• ADAMTS-13-deficiency is a cause of thrombotic thrombocytopenic purpura (TTP).• Preclinical safety of recombinant human (BAX930) was shown in animal models.• Preclinical efficacy of BAX930 was shown in a mouse model of TTP.• BAX930 showed advantageous efficacy over fresh frozen plasma, the current standard of care.Click to hear Dr Cataland and Prof. L€ ammle present a seminar on Thrombotic Thrombocytopenic Purpura (TTP): new Insights in Pathogenesis and Treatment Modalities.Summary. Background: Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder characterized by microthrombosis in small blood vessels of the body, resulting in a low platelet count. Baxalta has developed a new recombinant ADAMTS-13 (rADAMTS-13) product (BAX930) for on-demand and prophylactic treatment of patients with hereditary TTP (hTTP). Objectives: To evaluate the pharmacokinetics, efficacy and safety of BAX930 in different species, by use of an extensive preclinical program. Methods: The prophylactic and therapeutic efficacies of BAX930 were tested in a previously established TTP mouse model. Pharmacokinetics were evaluated after single intravenous bolus injection in mice and rats, and after repeated dosing in cynomolgus monkeys. Toxicity was assessed in rats and monkeys, safety pharmacology in monkeys, and local tolerance in rabbits. Results: BAX930 was shown to be efficacious, as demonstrated by a stabilized platelet count in ADAMTS-13 knockout mice that were thrombocytopenic when treated. Prophylactic efficacy was dose-dependent and comparable with that achieved by treatment with fresh frozen plasma, the mainstay of hTTP treatment. Therapeutic efficacy was treatment interval-dependent. Safety pharmacology evaluation did not show any deleterious effects of BAX930 on cardiovascular and respiratory functions in monkeys. The compound's pharmacokinetics were similar and dose-proportional in mice, rats, and monkeys. BAX930 was well tolerated in rats, monkeys, and rabbits, even at the highest doses tested. Conclusions: These results demonstrate that BAX930 has a favorable preclinical profile, and support the clinical development of rADAMTS-13 for the treatment of hTTP.

Genetically engineered mouse models are used to investigate beneficial treatment in haemophilia by comparison with wild-type mice. It has been recognized that wild-type and haemophilic mice of different genetic backgrounds show different bleeding phenotypes. We assessed ex-vivo coagulation parameters in nine wild-type substrains of 129S1/Sv, BALB/c and C57BL/6 mice applying thromboelastography (TEG), activated partial thromboplastin time (aPTT), prothrombin time (PT) and fibrinogen levels. The comprehensive ex-vivo data are discussed in view of results from a tail-tip bleeding assay. Time to first clot formation (R-time) showed higher within-substrain (CV range: 28–54%) and higher between-substrain (median range: 25.53–42.60 min) variation for BALB/c than for C57BL/6 mice (CV range: 14–31%; median range: 22.45–24.93 min). Median R-time for 129S1/Sv mice was 30.42 min (CV: 33%). No distinct strain differences were observed for maximum amplitude (MA), aPTT, or PT, but males generally showed higher MA and shorter aPTT than females. Males of all substrains had higher fibrinogen levels than females. The heightened in-vivo variability (CV range: 81–171%; median range: 36.00–469.50 mg) in the tail-tip bleeding assay and increased blood loss in wild-type C57BL/6 male mice was not reflected in ex-vivo coagulation parameters. In general, ex-vivo coagulation results appeared consistent within substrains, but showed substrain and sex differences of variable magnitudes. We conclude that alignment of the mouse substrain genetic background to the experimental model is critical to reduce data variability and animal numbers.

Extended half-life (EHL) factor therapies are needed to reduce the burden of prophylaxis and improve treatment adherence in patients with hemophilia. BAX 826 is a novel polysialylated fulllength recombinant factor VIII [polysialyic acid (PSA) rFVIII] with improved pharmacokinetics (PK), prolonged pharmacology, and maintained safety attributes to enable longer-acting rFVIII therapy. In factor VIII (FVIII)-deficient hemophilic mice, PSArFVIII showed a substantially higher mean residence time (.2-fold) and exposure (.3-fold), and prolonged efficacy in tail-bleeding experiments (48 vs. 30 hours) compared with unmodified recombinant FVIII (rFVIII), as well as a potentially favorable immunogenicity profile. Reduced binding to a scavenger receptor (low-density lipoprotein receptor-related protein 1) and von Willebrand factor (VWF) as well as a largely VWFindependent circulation time in mice provide a rationale for prolonged BAX 826 activity. The significantly improved PK profile versus rFVIII was confirmed in cynomolgus monkeys [mean residence time: 23.4 vs. 10.1 hours; exposure (area under the curve from time 0 to infinity): 206 vs. 48.2 IU/ml×h] and is in line with results from rodent studies. Finally, safety and toxicity evaluations did not indicate increased thrombogenic potential, and repeated administration of BAX 826 to monkeys and rats was well tolerated. The favorable profile and mechanism of this novel experimental therapeutic demonstrated all of the requirements for an EHL-rFVIII candidate, and thus BAX 826 was entered into clinical assessment for the treatment of hemophilia A. SIGNIFICANCE STATEMENTProlongation of FVIII half-life aims to reduce the burden of prophylaxis and improve treatment outcomes in patients with hemophilia. This study shows that polysialylation of PSArFVIII resulted in prolongations of rFVIII circulation time and procoagulant activity, together with a favorable nonclinical safety profile of the experimental therapeutic.This study was funded by Baxalta Innovations GmbH, a Member of the Takeda Group of Companies.

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Insufficiency of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif repeats-13) is the cause of thrombotic thrombocytopenic purpura (TTP) and contributes in microangiopathy in sickle cell disease (SCD). Recombinant ADAMTS13 effectively cleaves prothrombotic ultra-large von Willebrand factor (VWF) multimers. It is being tested as replacement therapy for TTP, and at supra-physiologic concentrations, for moderating vaso-occlusive crisis in SCD. Deficiencies of VWF, or concomitant treatment with antithrombotic drugs, could pose risks for increased bleeds in these patient populations. The purpose of the experiments was to evaluate the potential of exaggerated pharmacology and temporary bleeding risks associated with rADAMTS13 administration. We utilized safety studies in monkey and tested the effects of administering maximum-feasible doses of rADAMTS13 on nonclinical safety and spontaneous or aggressive bleeds in the rat model. Evaluation of pharmacokinetics, toxicity profiles, and challenge in a tail-tip bleeding model show that treatment with rADAMTS13 did not increase bleeding tendency, either alone, or in combination with enoxaparin or acetylsalicylic-acid. These novel findings demonstrate absence of rADAMTS13 exaggerated pharmacology without spontaneous or aggravated bleeds even at supra-physiologic (>100-fold) plasma concentrations.