Absence of exaggerated pharmacology by recombinant ADAMTS13 in the rat and monkey.

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.

Evaluation of Factor VIII Polysialylation: Identification of a Longer-Acting Experimental Therapy in Mice and Monkeys.

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 full-length 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 VWF-independent 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 STATEMENT: Prolongation 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.

Coagulation phenotype of wild-type mice on different genetic backgrounds.

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.

Pharmacokinetics, disease-modifying activity, and safety of an experimental therapeutic targeting an immunological isoform of macrophage migration inhibitory factor, in rat glomerulonephritis.

New therapeutic agents are needed to overcome the toxicity and suboptimal efficacy observed in current treatment of glomerulonephritis (GN). BaxB01 is a fully human monoclonal antibody targeting a disease-related immunologically distinct isoform of Macrophage migration Inhibitory Factor (MIF), designated oxidized MIF (oxMIF) and locally expressed in inflammatory conditions. We report the pharmacokinetic profile of BaxB01, and its dose and exposure-related disease-modifying activity in experimentally induced rat GN. BaxB01 bound to rat oxMIF with high affinity and reduced rat macrophage migration in vitro. After intravenous administration in rats, BaxB01 demonstrated favorable pharmacokinetics, with a half-life of up to nine days. Disease modification was dose-related (≥ 10mg/kg) as demonstrated by significantly reduced proteinuria and diminished histopathological glomerular crescent formation. Importantly, a single dose was sufficient to establish an exposure-related, anti-inflammatory milieu via amelioration of glomerular cellular inflammation. Pharmacodynamic modeling corroborated these findings, consistently predicting plasma exposures that were effective in attenuating both anti-inflammatory activity and reducing loss of kidney function. This pharmacologic benefit on glomerular function and structure was sustained during established disease, while correlation analyses confirmed a link between the antibody's anti-inflammatory activity and reduced crescent formation in individual rats. Finally, safety assessment in rats showed that the experimental therapeutic was well tolerated without signs of systemic toxicity or negative impact on kidney function. These data define therapeutically relevant exposures correlated with mechanism-based activity in GN, while toxicological evaluation suggests a large therapeutic index and provides evidence for achieving safe and effective exposure to a MIF isoform-directed therapeutic in nephritis-associated disease.

Nonacog gamma, a novel recombinant factor IX with low factor IXa content for treatment and prophylaxis of bleeding episodes.

Nonacog gamma is a new recombinant factor IX to treat factor IX deficiency. It is indicated for control of bleeding episodes, perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia B. Nonacog gamma was first approved in the USA in June 2013 under the trade name RIXUBIS followed by market approvals in Australia and the EU in 2014, and marketing authorization decision is pending in Japan. Nonacog gamma is derived from a recombinant Chinese hamster ovary cell line using a state of the art biotechnological manufacturing process. Recombinant factor IX is produced by Baxter's protein-free fermentation technology, which was first developed for ADVATE. The product is purified and formulated in the absence of any human or animal-derived protein. Nonacog gamma was characterized both in comprehensive in vitro and in vivo non-clinical studies as well as in an extensive clinical trial program.