Preclinical assessment of a new recombinant ADAMTS-13 drug product (BAX930) for the treatment of thrombotic thrombocytopenic purpura.

UNLABELLED: Essentials ADAMTS-13-deficiency is a cause of thrombotic thrombocytopenic purpura (TTP). Preclinical safety of recombinant human ADAMTS-13 (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. Lämmle 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.

The biological efficacy profile of BAX 855, a PEGylated recombinant factor VIII molecule.

Prophylaxis prevents joint and other bleeding episodes in patients with haemophilia A. Development of new factor concentrates with longer circulating half-lives may encourage patients to start, continue or resume prophylaxis. The aim of this study was to compare the pharmacodynamic effect of a PEGylated full-length recombinant factor VIII (rFVIII) concentrate with that of an unmodified rFVIII concentrate with respect to the duration of prophylactic efficacy in a murine model of haemophilic joint bleeding. Mice were pretreated with BAX 855 or unmodified rFVIII at specified times before right knee puncture to induce haemarthrosis; left knee joints served as controls. Joint bleeding was evaluated using a combination of visual and histological assessments. Administration of a single dose of unmodified rFVIII before joint puncture prevented haemarthrosis in mice up to 24 h, whereas pretreatment with BAX 855 protected the joint from bleeding up to 48 h. This pharmacodynamic study showed prolonged efficacy of BAX 855 compared to ADVATE in a haemophilia A mouse joint bleeding model. This finding supports the possibility of using BAX 855 to increase FVIII trough levels and/or extend the dosing interval in patients with haemophilia A on prophylaxis, which may potentially improve prophylactic efficacy and long-term adherence.

BAX 855, a PEGylated rFVIII product with prolonged half-life. Development, functional and structural characterisation.

A longer acting recombinant FVIII is expected to serve patients' demand for a more convenient prophylactic therapy. We have developed BAX 855, a PEGylated form of Baxter's rFVIII product ADVATE™ based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses a novel PEG reagent. The production process was adjusted to yield a rFVIII conjugate with a low PEGylation degree of about 2 moles PEG per FVIII molecule. This optimised modification degree resulted in an improved PK profile for rFVIII without compromising its specific activity. PEGylation sites were identified by employing various HPLC- and MS-based methods. These studies not only indicated that about 60% of the PEG chains are localised to the B-domain, which is cleaved off upon physiological activation during the coagulation process, but also demonstrated an excellent lot to lot consistency with regard to PEGylation site distribution. Detailed biochemical characterization further showed that PEGylated FVIII retained all the physiological functions of the FVIII molecule with the exception of binding to the LRP clearance receptor which was reduced for BAX 855 compared to ADVATE™. This might provide an explanation for the prolonged circulation time of BAX 855 as reduced receptor binding might slow-down clearance. Preclinical studies showed improved pharmacokinetic behaviour and clinically relevant prolonged efficacy compared to ADVATE™ without any signs of toxicity or elevated immunogenicity. The comprehensive preclinical data package formed the basis for approval of the phase 1 clinical study by European authorities which started in 2011.

Biochemical, molecular and preclinical characterization of a double-virus-reduced human butyrylcholinesterase preparation designed for clinical use.

BACKGROUND AND OBJECTIVES: A human plasma-derived butyrylcholinesterase preparation manufactured on the industrial scale is described. MATERIAL AND METHODS: The human butyrylcholinesterase (hBChE) product was extensively investigated for its purity using immunological and electrophoretic methods and characterized by thorough glycoproteomic approaches. A comprehensive preclinical testing programme addressing safety and pharmacokinetic parameters supplemented the biochemical characterization. RESULTS: The high-purity hBChE preparation is tetrameric and has high specific activity and molecular integrity of the protein backbone. Acute toxicity studies and in vivo thrombogenicity studies provided evidence of a sufficient safety margin for use in humans. CONCLUSION: Extensive preclinical safety and pharmacokinetic testing confirmed that this hBChE preparation can be used for further efficacy testing as a bioscavenger for toxic organophosphate compounds in appropriate animal models and ultimately in humans.

Modulation of factor VIII-specific memory B cells.

The development of inhibitory antibodies against factor VIII (FVIII) is the major complication in patients with haemophilia A who are treated with FVIII products. Memory B cells play an essential role in maintaining established antibody responses. Upon re-exposure to the same antigen, they are rapidly re-stimulated to proliferate and differentiate into antibody-secreting plasma cells (ASC) that secrete high-affinity antibodies. It is, therefore, reasonable to believe that memory B cells have to be eradicated or inactivated for immune tolerance induction therapy to be successful in patients with haemophilia A and FVIII inhibitors. The aim of our studies was the development of strategies to prevent FVIII-specific memory B cells from becoming re-stimulated. We established a 6-day in vitro culture system that enabled us to study the regulation of FVIII-specific murine memory-B-cell re-stimulation. We tested the impact of the blockade of co-stimulatory interactions, of different concentrations of FVIII and of ligands for toll-like receptors (TLR). The blockade of B7-CD28 and CD40-CD40 ligand interactions prevented FVIII-specific murine memory B cells from becoming re-stimulated by FVIII in vitro and in vivo. Furthermore, high concentrations of FVIII blocked re-stimulation of FVIII-specific murine memory B cells. Triggering of TLR7 amplified re-stimulation by low concentrations of FVIII and prevented blockade by high concentrations of FVIII. We conclude that we defined modulators that either amplify or inhibit the re-stimulation of FVIII-specific murine memory B cells. Currently, we are investigating whether the same modulators operate in patients with haemophilia A and FVIII inhibitors.

Serum-derived immunoglobulins alter amyloid beta transport across a blood-brain barrier in vitro model.

Since passive immunization with serum-derived immunoglobulins (intravenous immunoglobulins) showed several positive effects in some patients with Alzheimer's disease (AD), intravenous immunoglobulins (IVIG) are discussed as a possible treatment option. IVIG, an antibody product derived from human plasma, contains natural antibodies against amyloid beta(Abeta) peptide. Until now it is not known, how IVIG interferes with pathogenesis in AD, but several proposed mechanisms are in discussion. Receptor types which are involved in transport processes at the BBB are LRP, RAGE and hFcRn. We were looking for an in vitro BBB model expressing these receptors and studied the alteration of transport of Abeta peptides across this model under the influence of immunoglobulins. Cell line ECV304 was found to be suitable for our experiments. We found evidence for involvement of an improved clearance of Abeta across the BBB as well as a decreased Abeta influx from blood to the brain probably following complex formation of immunoglobulins with free Abeta in the periphery. Furthermore, we were able to confirm the activity of IVIG preparations which acted the same way but showed slightly less efficacy in comparison to monoclonal anti-Abeta antibodies. Based on these results we suggest multiple mechanisms responsible for the efficacy of immunotherapy in Alzheimer's disease.

Optimization, refinement and reduction of murine in vivo experiments to assess therapeutic approaches for haemophilia A.

The tail cut bleeding model (CUT) is routinely used in factor VIII-deficient mice to assess pharmacodynamic effects of therapeutic strategies for haemophilia A. Results from this model are highly variable, many modifications to the model are reported and at times the animals' wellbeing may be compromised by recording survival as an endpoint. We therefore investigated if the ferric chloride carotid occlusion model (COM) used for thrombosis research can be applied to enhance data quality and animal welfare in haemophilia A research. Relative dose effects and relative dose variations were calculated for the CUT and COM. The requisite sample sizes were estimated and the importance of survival rates to assess rebleeds during recovery was evaluated by correlating initial blood loss to mortality. Relative dose effects increased with higher doses in both models. The COM was more sensitive at lower doses than the CUT, had up to 82% less variation across doses and clearly showed superior accuracy. Only 5% of the sample size required for the CUT would be needed to establish non-inferiority between a specific therapeutic dose in haemophilia A mice and healthy wild-type animals. A strong statistically significant correlation was found between initial blood loss and mortality within 24 h. Our findings clearly suggest that the COM is a valid tool for assessing haemophilia A treatment in vivo. The highly reproducible data means that significantly fewer animals are required and a more humane endpoint can be used by directly assessing clot stability instead of survival rate.

Development of a plasma- and albumin-free recombinant von Willebrand factor.

Baxter has developed a recombinant therapy for treating von Willebrand's disease. Recombinant VWF is co-expressed with the rFVIII in CHO cells used to produce the rFVIII product Advate. This rVWF is used as a drug component for a rVWF-rFVIII complex drug product. CHO cells produce partially processed and partially un-processed rVWF still containing the pro-peptide. In order to make a consistent preparation containing mature and processed rVWF only rVWF is exposed to recombinant furin to remove the pro-peptide. Recombinant VWF and furin are produced under serum- and protein-free conditions. It is highly purified by a series of chromatographic steps and formulated in a protein-free buffer and has a homogeneous multimer distribution. The specific activity is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. SDS agarose electrophoretic analysis shows the presence of ultra-high molecular weight multimers. The FVIII-binding capacity and affinity of rVWF to FVIII is comparable to VWF in plasma. Carbohydrate analysis shows an intact glycosylation pattern. Recombinant VWF binds to collagen and promotes platelet adhesion under shear stress. It stabilizes endogenous FVIII in VWF-deficient knock-out mice as seen by a secondary rise in murine FVIII.

Species-dependent variability of ADAMTS13-mediated proteolysis of human recombinant von Willebrand factor.

BACKGROUND: von Willebrand factor (VWF) is composed of a series of multimers, the sizes of which are regulated by the plasma metalloprotease ADAMTS13. OBJECTIVE: Proteolysis of human recombinant VWF (rVWF) by ADAMTS13 present in the plasma of different species typically used as preclinical animal models was investigated to evaluate the efficacy and safety of rVWF. METHODS: Degradation of rVWF was studied in vitro under moderate denaturing conditions and was monitored by multimer analysis, residual collagen binding, and immunoblot analysis. In vivo cleavage was determined by administration of rVWF to cynomolgus monkeys, rabbits and VWF-deficient mice and subsequent analysis of plasma samples by immunoblot. Plasma ADAMTS13 levels were determined with a synthetic human VWF peptide (FRETS-VWF73). RESULTS: From the animals tested, only rabbit plasma was as efficient as human plasma in proteolysing rVWF in vitro. Mouse plasma virtually failed to cleave rVWF. Administration of human rVWF resulted in ADAMTS13-specific cleavage products in rabbits and, to a lesser extent, in cynomolgus monkeys at various doses of rVWF. Virtually no cleavage occurred in mice. ADAMTS13 activity levels in rabbit and monkey plasma were similar to those in human plasma and were not significantly altered upon infusion of rVWF up to very high doses, indicating that rVWF did not lead to an exhaustion of endogenous ADAMTS13 in both species. CONCLUSIONS: The differences in susceptibility to cleavage of rVWF by different species need to be considered when interpreting the physiology of human rVWF from results of tests in animal models.

A guide to murine coagulation factor structure, function, assays, and genetic alterations.

Murine blood coagulation factors and function are quite similar to those of humans. Because of this similarity and the adaptability of mice to genetic manipulation, murine coagulation factors and inhibitors have been extensively studied. These studies have provided significant insights into human hemostasis. They have also provided useful experimental models for evaluation of the pathophysiology and treatment of thrombosis. This review contains recommendations for obtaining, processing and assaying mouse blood hemostatic components, and it summarizes the extensive literature on murine coagulation factor structure and function, assays and genetic alteration. It is intended to be a convenient reference source for investigators of hemostasis and thrombosis.

A new liquid, intravenous immunoglobulin product (IGIV 10%) highly purified by a state-of-the-art process.

BACKGROUND AND OBJECTIVES: The ultimate goal was to generate an industrial-scale process suitable to produce a high-yield, safe and stable immunoglobulin G (IgG) preparation for intravenous administration, which is ready to use for customer convenience. This new liquid 10% IgG preparation (IGIV 10%) was compared to Gammagard SD, a licenced lyophilized immunoglobulin in biochemical and preclinical testing. MATERIALS AND METHODS: The new process, which includes three dedicated virus clearance steps, is a streamlined combination of the currently applied and well-established manufacturing procedures. The biochemical characterization is done by standard methods focusing on purity, integrity and functionality of the preparation. Efficacy is demonstrated in vivo by mouse protection testing and in vitro by opsonization and protein A affinity chromatography. Pharmacokinetics in rats is evaluated after a single intravenous dose. The anaphylactoid potential is determined in rats and in guinea pigs, while thrombogenicity is assessed in a rabbit model. The influence of the products on vital functions is tested on dogs, while acute toxicity studies are carried out on mice and rats. RESULTS: The biochemical characterization data demonstrate the high purity of monomeric IgG in the product. The mouse protection test showed that the protective activity against systemic bacterial infections of IGIV 10% is at least as good as the reference Gammagard SD. This result is supported by the broad spectrum of antibodies in high titres against bacteria and viruses and the high functional integrity of the IgG molecule (> or = 90% functionally intact IgG) in IGIV 10%. The opsonic activity of all IGIV 10% lots is similar to the one of the reference Gammagard SD. In safety and thrombogenicity studies, no adverse effects of IGIV 10% were observed. Pharmacokinetic studies showed no statistically significant differences between the two products. In the acute toxicity animal studies, IGIV 10% compared favourably to the reference Gammagard SD. CONCLUSIONS: The new manufacturing process enables the production of a highly purified IgG preparation for intravenous administration. The product has an IgG subclass distribution similar to plasma and contains a broad spectrum of functionally intact antibodies. Preclinical studies demonstrate that the liquid IGIV 10% combines excellent qualities of efficacy, safety and tolerability.

Effects of alpha 1-acid glycoprotein on acute pancreatitis and acute lung injury in rats.

alpha 1-Acid glycoprotein (AAG), a highly negatively charged glycoprotein, well known for its capillary stabilizing effect, was tested in rat models of acute edematous pancreatitis, acute hemorrhagic-necrotizing pancreatitis, and acute respiratory distress syndrome (ARDS). In cerulein-elicited edematous pancreatitis AAG improved histological alterations at 200 mg/kg i.v. and plasma amylase activity at 1800 or 4200 mg/kg i.v. All other parameters (edema, plasma lipase) were not affected in a biologically relevant manner. In glycodeoxycholic acid-induced hemorrhagic-necrotizing pancreatitis AAG was without effect on parameters measured (plasma amylase, plasma lipase activity, histological scores) at 1800 or 4200 mg/kg i.v. At the extremely high dose of 1500 mg/kg i.v. plasma amylase and lipase levels were decreased. In lipopolysaccharide-mediated ARDS, AAG was tested at 50, 200 or 600 mg/kg i.v. AAG, but also the placebo formulation decreased the myeloperoxidase content in the bronchoalveolar lavage fluid. Histological alterations were improved by AAG, however, not by the placebo formulation. Lung water content was not significantly influenced by AAG, whereas Evans blue extravasation was significantly diminished by all three doses of AAG. It is concluded that the edematous pancreatitis is the first in vivo condition with increased extravascular fluid accumulation, in which AAG is not effective. Based on data presented here and literature data, there is evidence for a beneficial effect of AAG in acute lung injury.

[Preclinical investigation of alpha 1-acid glycoprotein (orosomucoid)]. / Präklinische Untersuchung von alpha 1-saurem Glykoprotein (Orosomucoid).

The molecular properties of alpha 1-acid glycoprotein are briefly discussed. This molecule has been shown in in vitro experiments to have both a stabilizing effect on vascular permeability and antiinflammatory properties. We were able to demonstrate these two effects in vivo in guinea pigs (skin, Evan's Blue extravasation) and in rats (paw, carrageenan induced inflammation). Further experiments were performed in rats relating to possible therapeutic indications for alpha 1-acid glycoprotein: (1) inhibitory effect on brain edema formation after experimental stroke, (2) therapeutic effect in the puromycin aminonucleoside-induced minimal change nephrosis, (3) improvement of vital parameters in hemorrhagic-hypovolemic shock, (4) increase in survival rate in septic peritonitis, and (5) promising effects in burn-induced remote lung injury. The high content of sialic acid and the high negative charge of alpha 1-acid glycoprotein are believed to be major contributors to its stabilizing effect on vascular permeability. The protein is bound to the glycocalyx of the endothelial cells (and presumably to structures of the glomerular basement membrane), thereby hindering the passage of other polyanionic molecules through the vascular wall. The antiinflammatory/immunomodulatory effect of alpha 1-acid glycoprotein appears mainly due to suppression of polymorphonuclear neutrophils. This action is dependent on the glycan part of the molecule, which is highly variable (microheterogeneity). It is obvious that there are differences between the different glycan forms as far as the antiinflammatory property of the protein is concerned. Together with data in the literature, the results presented here suggest a variety of potential indications for therapeutic use of alpha 1-acid glycoprotein in humans.

Effects of alpha 1-acid glycoprotein in different rodent models of shock.

It was the aim of the present study to investigate the effects of the acute phase protein alpha 1-acid glycoprotein in different models of shock. The human plasma preparation used was without effect on mortality in lipopolysaccharide-injected mice when administered in two different doses (1 or 0.33 g/kg i.v.) and according to different treatment schedules. The same preparation significantly increased survival rate (48 h) in rats with septic peritonitis. This effect was seen when alpha 1-acid glycoprotein (200 mg/kg i.v.) was given 15 min prior to and 24 h after cecal puncture. All other dose regimes tested were without significant effect on survival rate. A hemorrhagic/hypovolemic shock model (including a defined trauma) in rats resuscitated with 200 mg/kg alpha 1-acid glycoprotein resulted in significantly higher values of mean arterial blood pressure, cardiac output and stroke volume when compared to corresponding values obtained after resuscitation with Ringer's solution or 200 mg/kg albumin i.v. (free of alpha 1-acid glycoprotein; placebo formulation). Taking all other possible mechanisms of alpha 1-acid glycoprotein into consideration, the partially protective effects of the preparation are explained by enhancing the capillary barrier function and thereby maintaining perfusion of vital organs.

Studies on the effect of human lys-plasminogen in a rat model of global cerebral ischemia.

Human lys-plasminogen and the corresponding formulation buffer were tested in a rat model of global cerebral ischemia (clamping of both carotid arteries, withdrawal of 5 ml blood for 30 min). The two main parameters, tested in different experimental set-ups, were 1. brain edema (water content) 23.5 h after reperfusion and 2. assessment of neurological deficits 24, 48 and 72 h after reperfusion. In some groups of animals of the first set-up, brains were examined histologically for microvascular fibrin deposits. In a separate group of animals the fibrinolytic plasma activity of rats treated with 500 CU/kg lys-plasminogen was studied. Concerning brain water content lys-plasminogen completely antagonized the formation of brain edema when given with 500 caseolytic Units (CU)/kg i.v. with blood reperfusion and was still effective when given 30 min later. 200 CU/kg i.v. given with blood reperfusion as well as 500 CU/kg i.v. given 60 min after blood reperfusion proved ineffective. In none of the brains investigated microvascular fibrin deposits were found. In experiments with assessment of neurological deficits, animals treated with 500 CU/kg lys-plasminogen i.v. showed almost no disabilities (like sham operated animals) when compared to ischemic (positive) controls which were rather severely handicapped. The formulation buffer of lys-plasminogen, tested in an equivalent volume, was without any effect in both set-ups. No fibrinolytic activity was found in plasma samples of rats up to 240 min after treatment with 500 CU/kg lys-plasminogen i.v. It is concluded from these experiments that human lys-plasminogen has a protective effect in rats against the sequelae of global cerebral ischemia which is not related to the well-known fibrinolytic potential but might be a separate quality.

In vivo effect of alpha 1-acid glycoprotein on experimentally enhanced capillary permeability in guinea-pig skin.

Anesthetized guinea-pigs were intravenously injected with Evans blue. After intracutaneous injection of agonists (lys-plasminogen, histamine, platelet-activating factor, thrombin, bradykinin), the resulting wheals appeared blue in a dose-dependent manner, due to an enhanced capillary permeability, alpha 1-Acid glycoprotein, given i.v. in different doses (3.125-50 mg/kg) and at different times (30-180 min) before Evans blue administration, antagonized the effects of all agonists listed above. This was shown by a parallel shift of the agonist dose-response curves to the right. The effect was time-dependent (tmax: mainly 120 min) and dose-dependent. alpha 1-Acid glycoprotein antagonized the agonists in the following order: lys-plasminogen > histamine = platelet-activating factor > thrombin > bradykinin. As all agonist mentioned are suggested to play a major role in the shock-related increase in vascular permeability, a putatively beneficial role of alpha1-acid glycoprotein in shock is discussed.