Background We have recently reported on a recombinant von Willebrand factor (VWF) D ' D3 albumin fusion protein (rD ' D3-FP) developed to extend the half-life of coagulation factor VIII (FVIII) for the treatment of hemophilia A. Based on predictive modelling presented in this study, we hypothesized that modifying rD ' D3-FP to improve FVIII interaction would reduce exchange with endogenous VWF and provide additional FVIII half-life benefit. Objectives The aim of this study was to identify novel rD ' D3-FP variants with enhanced therapeutic efficacy in extending FVIII half-life. Methods Through both directed mutagenesis and random mutagenesis using a novel mammalian display platform, we identified novel rD ' D3-FP variants with increased affinity for FVIII (rVIII-SingleChain) under both neutral and acidic conditions and assessed their ability to extend FVIII half-life in vitro and in vivo. Results In rat preclinical studies, rD ' D3-FP variants with increased affinity for FVIII displayed enhanced potency, with reduced dose levels required to achieve equivalent rVIII-SingleChain half-life extension. In cell-based imaging studies in vitro, we also demonstrated reduced dissociation of rVIII-SingleChain from the rD ' D3-FP variants within acidic endosomes and more efficient co-recycling of the rD ' D3-FP/rVIII-SingleChain complex via the FcRn recycling system. Conclusions In summary, at potential clinical doses, the rD ' D3-FP variants provide marked benefits with respect to dose levels and half-life extension of co-administered FVIII, supporting their development for use in the treatment of hemophilia A.

Hemophilia A and hemophilia B are X-linked inherited bleeding disorders caused by a deficiency of coagulation factor VIII and IX, respectively. Standard of care is prophylactic factor replacement therapy; however, the development of neutralizing antibodies against these factors represents serious complications underlining the need for alternative treatment approaches. Human coagulation factor X has a central role within the blood coagulation system making it an attractive target for the development of alternative treatment strategies for patients with hemophilia. This study focuses on a modified variant of the human coagulation factor X with enhanced hemostatic bypass activity due to insertion of a factor IX derived activation sequence. This molecule design leads to the direct activation of the modified factor X protein by factor XIa allowing it to bypass the need for coagulation factor VIIIa/factor IXa. The modified variant was able to correct in-vitro activated partial prothrombin time of human and murine factor VIII/factor IX deficient plasma. Furthermore, reduced blood loss in factor VIII knock-out mice was observed after intravenous application of the modified factor X variant. In conclusion, these data suggest that the factor X variant described here could potentially serve as a bypassing agent independent of the inhibitor status of hemophilia patients. However, more research is needed to further investigate the potential of this molecule.

A novel mechanism for extending the circulatory half-life of coagulation factor VIII (FVIII) has been established and evaluated preclinically. The FVIII binding domain of von Willebrand factor (D'D3) fused to human albumin (rD'D3-FP) dose dependently improved pharmacokinetics parameters of coadministered FVIII in all animal species tested, from mouse to cynomolgus monkey, after IV injection. At higher doses, the half-life of recombinant FVIII (rVIII-SingleChain) was calculated to be increased 2.6-fold to fivefold compared with rVIII-SingleChain administered alone in rats, rabbits, and cynomolgus monkeys, and it was increased 3.1-fold to 9.1-fold in mice. Sustained pharmacodynamics effects were observed (ie, activated partial thromboplastin time and thrombin generation measured ex vivo). No increased risk of thrombosis was observed with coadministration of rVIII-SingleChain and rD'D3-FP compared with rVIII-SingleChain alone. At concentrations beyond the anticipated therapeutic range, rD'D3-FP reduced the hemostatic efficacy of coadministered rVIII-SingleChain. This finding might be due to scavenging of activated FVIII by the excessive amount of rD'D3-FP which, in turn, might result in a reduced probability of the formation of the tenase complex. This observation underlines the importance of a fine-tuned balance between FVIII and its binding partner, von Willebrand factor, for hemostasis in general.

Scientific Research Question: A recombinant VWF D'D3 albumin fusion protein (rD'D3-FP) has been developed to extend the half-life of co-administered coagulation factor VIII (FVIII), for the treatment of hemophilia A. Pharmacokinetic (PK) studies in rats, rabbits, cynomolgus monkeys and FVIII knockout mice have demonstrated a significant improvement in rVIII-SingleChain PK parameters upon co-administration with rD'D3-FP. Interestingly the half-life of rVIII-SingleChain, although increased, remained less than that of the co-administered rD'D3-FP. It is hypothesized that FVIII exchange between rD'D3-FP and endogenous VWF in circulation combined with a reduced recycling efficiency for FVIII contribute to this shorter half-life. To overcome these issues, novel rD'D3-FP variants with reduced dissociation for FVIII under both neutral and acidic conditions have been generated. This study aimed to investigate if rD'D3-FP variants with enhanced FVIII binding are more effective at improving the PK of co-administered FVIII than wild-type rD'D3-FP.

BACKGROUND AND PURPOSE:Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach.METHODS:For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation.RESULTS:Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis.CONCLUSIONS:With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.

Haemostasis including blood coagulation is initiated upon vessel wall injury and indispensable to limit excessive blood loss. However, unregulated pathological coagulation may lead to vessel occlusion, causing thrombotic disorders, most notably myocardial infarction and stroke. Furthermore, blood exposure to foreign surfaces activates the intrinsic pathway of coagulation. Hence, various clinical scenarios, such as extracorporeal membrane oxygenation, require robust anticoagulation consequently leading to an increased bleeding risk. This study aimed to further assess the antithrombotic efficacy of the activated factor XII (FXIIa) inhibitor, rHA-Infestin-4, in several thrombosis models. In mice, rHA-Infestin-4 decreased occlusion rates in the mechanically-induced arterial (Folt's) and the FeCl3 -induced venous thrombosis model. rHA-Infestin-4 also protected from FeCl3 -induced arterial thrombosis and from stasis-prompted venous thrombosis in rabbits. Furthermore, rHA-Infestin-4 prevented occlusion in the arterio-venous shunt model in mice and rabbits where thrombosis was induced via a foreign surface. In contrast to heparin, the haemostatic capacity in rabbits was unaffected by rHA-Infestin-4. Using rodent and non-rodent species, our data demonstrate that the FXIIa inhibitor rHA-Infestin-4 decreased arterial, venous and foreign surface-induced thrombosis without affecting physiological haemostasis. Hence, we provide further evidence that targeting FXIIa represents a potent yet safe antithrombotic treatment approach, especially in foreign surface-triggered thrombosis.

Ammonia is a well-known natural refrigerant which has been widely used in refrigeration technology due to its favorable thermodynamic properties. In this paper, an absorption-resorption refrigeration system is studied. The main advantages of an absorption-resorption system compared to commonly used absorption refrigeration systems are the lower pressure levels and the use of lower temperatures at the desorber and the resorber. Thus, resorption technology opens up new possibilities for using cheaper assembly materials, for example polymeric heat exchangers, due to the lower working pressures.In this work, the modeling of an absorption-resorption refrigeration system using Engineering Equation Solver (EES) is used to analyze the performance of the cycle working at moderated pressure levels. The study was divided in two parts: First, taking into account only the internal variables of the cycle, the influence of the pressure and temperatures on the cycle performance (/composition of the solution circuits, COP and cooling capacity) has been studied. Secondly, it has been carried out a preliminary comparison between the calculated and experimental results obtained in an ammonia/water absorption-resorption refrigeration chiller, which was set up at TU Dresden by Makatec GmbH. For this second task, a model has been developed that takes the external streams into account.

Cost efficient power generation from low temperature heat sources requires an optimal usage of the available heat. In addition to the ORC (Organic Rankine Cycles), cycles with ammonia and water as working fluid show promising results regarding efficiency. Due to their non-isothermal phase change, mixtures can adapt well to a liquid heat source temperature profile and reduce the exergetic losses. In this analysis thermodynamic calculations on the layouts of two existing ammonia-water cycles are compared: a geothermal power plant based on a Siemens' patent and a modified lab plant based on a patent invented by Kalina (KCS-34). The difference between the two cycles is the position of the internal heat recovery. Cycle simulations were carried out at defined boundary conditions in order to identify optimal operation parameters. For the selected heat source of 393.15 K (hot water) the ammonia mass fraction between 80% and 90% results in the best performance in both configurations. In general, the layout of Siemens achieves a slightly better efficiency compared to the KCS-34. Compared to an ORC using R245fa as working fluid, the exergetic efficiency can be increased by the ammonia/water based cycles by approximately 25%. (C) 2015 Elsevier Ltd. All rights reserved.

Introduction: Current state of the art treatment of severe Hemophilia A is prophylactic or on-demand replacement of the deficient factor VIII (FVIII) using plasma derived or recombinant factors [Keeling 2008; Srivastava 2012; Wong 2011]. The compliance to existing therapies is hampered by the short half-life of available FVIII products necessitating multiple intravenous injections per week for adequate prophylaxis [Bjorkman 2009; Sheridan 2011]. Thus, FVIII products with improved pharmacokinetic profiles are currently under development or have recently been made available [Oldenburg 2014]. FVIII's pharmacokinetic (PK) properties are believed to be strongly dictated by its binding to von Willebrand Factor (VWF) [Lenting 2007; Dasgupta 2007; Saenko 2006]. The aim of this study was to compare the binding affinity of different recombinant FVIII (rFVIII) products to plasma derived VWF (pd VWF), and characterize their PK properties in different animal species.

INTRODUCTION:rVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII. MATERIALS AND METHODS:Binding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate(®)) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl3-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200-250 IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice. RESULTS:rVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44 pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl3-induced arterial occlusion model. CONCLUSIONS:rVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.

Summary The prophylactic treatment of haemophilia B and the management of haemophilia A or B with inhibitors demand frequent administrations of coagulation factors due to the suboptimal half-lives of the products commercially available and currently in use, e.g. recombinant factor IX (rFIX) and recombinant factor VIIa (rFVIIa), respectively. The extension of the half-lives of rFIX and rFVIIa could allow for longer intervals between infusions and could thereby improve adherence and clinical outcomes and may improve quality of life. Albumin fusion is one of a number of different techniques currently being examined to prolong the half-life of rFIX and rFVIIa. Results from a phase I clinical trial demonstrated that the recombinant fusion protein linking FIX to albumin (rIX-FP) has a five-times longer half-life than rFIX, and preclinical studies with the recombinant fusion protein linking FVIIa to albumin (rVIIa-FP) suggest that rVIIa-FP possesses a significantly extended half-life versus rFVIIa. In this review, we describe albumin fusion technology and examine the recent progress in the development of rIX-FP and rVIIa-FP.

INTRODUCTION:The preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII, was assessed in a series of animal studies. MATERIALS AND METHODS:In the tail-clip bleeding model, hemophilia A mice were injected with escalating doses (1-150 IU/kg) of rVIII-SingleChain, B-domain deleted (BDD) rFVIII (ReFacto AF(®)), or full-length rFVIII products (Advate(®), Helixate(®)). Total blood loss and the percentage of animals in which hemostasis occurred were assessed in this observer-blinded, randomized study. In a second non-randomized study in hemophilia A mice, thromboelastographic analysis, thrombin generation, and activated partial thromboplastin time assays were performed. General safety and toxicity were assessed in three animal species, including determination of the prothrombotic potential of rVIII-SingleChain in a rabbit venous thrombosis model. RESULTS:Under acute bleeding conditions, the effect of rVIII-SingleChain on total blood loss and hemostasis was indistinguishable from BDD and full-length rFVIII. rVIII-SingleChain and full-length rFVIII (both 20 IU/kg) corrected thromboelastographic parameters, activated partial thromboplastin time, and thrombin generation to a similar degree in hemophilia A mice. In a thrombosis model, the effect of rVIII-SingleChain on thrombus incidence was non-significant and comparable to BDD rFVIII at doses up to 500 IU/kg. Treatment with rVIII-SingleChain did not cause anaphylactic reaction or local intolerance in safety and toxicity studies, and demonstrated an excellent overall safety profile. CONCLUSIONS:rVIII-SingleChain showed convincing hemostatic efficacy and excellent tolerability in animal studies, warranting continued investigation in human Phase I/III trials (AFFINITY).

BackgroundRecombinant factor VIIa (rFVIIa) is approved for use in controlling bleeding episodes in people with hemophilia who have developed inhibitors to replacement therapy. Due to its short half-life (t(1/2)), frequent injections are required, limiting its use as a prophylactic treatment. A novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) has been developed to extend the t(1/2) of rFVIIa.ObjectivesThe aim of our studies was to investigate the pharmacokinetic/pharmacodynamic characteristics of rVIIa-FP in preclinical animal species.MethodsPharmacokinetic (PK) parameters were derived after single intravenous dosing in hemophilia A mice, rats, rabbits and monkeys. PK analysis was based on human FVII plasma levels determined by measuring FVII antigen levels by ELISA in mice and rats, and FVIIa activity using STACLOT((R)) VIIa-rTF in rabbits and monkeys. Induction of thrombin generation was investigated in mice, while hemostatic activity was assessed by thrombus formation in rabbits.ResultsCompared with rFVIIa, rVIIa-FP displayed a prolonged t(1/2), enhanced in vivo recovery and reduced clearance in all species investigated. In mice, 16h after treatment with rVIIa-FP, thrombin levels were quantifiable, indicating prolonged efficacy, whereas values had approached baseline at this time after treatment with rFVIIa. After 12h, hemostatic efficacy was negligible in rFVIIa-treated rabbits, but sustained in animals receiving rVIIa-FP.ConclusionsThese studies indicate that the longer t(1/2) of rVIIa-FP compared with rFVIIa translates into extended activity. These findings suggest that rVIIa-FP has the potential to be administered less frequently than rFVIIa-containing concentrates in clinical use.