Biodistribution of the recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in rats

AbstractIntroductionThe recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) is undergoing clinical trials for prophylaxis and on-demand treatment of haemophilia B patients. The aim of this study was to investigate the pharmacokinetics, whole-body and knee joint distribution of rIX-FP following intravenous administration to rats, compared with a marketed, non-fused rFIX and recombinant human albumin.Material and Methods[3H]-rIX-FP, [3H]-rFIX or [3H]-albumin were administered to rats followed by quantitative whole-body autoradiography over 24 or 240hours, and the tissue distribution as well as elimination of radioactivity were measured.ResultsElimination of all radioactivity derived from the three proteins was shown to occur primarily via the urine. The tissue distribution of [3H]-rIX-FP and [3H]-rFIX (but not of [3H]-albumin) was comparable, both penetrating predominantly into bone, and well-perfused tissues, suggesting that the rIX moiety determines the distribution pattern of rIX-FP, while the albumin moity is responsible for the prolonged plasma and tissue retention. Detailed knee-joint analysis indicated rapid presence of [3H]-rIX-FP and [3H]-rFIX in synovial and mineralised bone tissue, mostly localised to the zone of calcified cartilage. Longest retention times were observed in the bone marrow and the endosteum of long bones. Intriguingly, [3H]-rIX-FP- and [3H]-albumin-derived radioactive signals were detectable up to 240hours, while [3H]-rFIX-derived radioactivity rapidly declined after 1hour post-dosing correlating to the extended plasma half-life of [3H]-rIX-FP.ConclusionThe prolonged plasma and tissue retention of rIX-FP achieved by albumin fusion may allow a reduction in dosing frequency leading to increased therapeutic compliance and convenience

In Vivo Methods for the Assessment of Topical Drug Bioavailability

This paper reviews some current methods for the in vivo assessment of local cutaneous bioavailability in humans after topical drug application. After an introduction discussing the importance of local drug bioavailability assessment and the limitations of model-based predictions, the focus turns to the relevance of experimental studies. The available techniques are then reviewed in detail, with particular emphasis on the tape stripping and microdialysis methodologies. Other less developed techniques, including the skin biopsy, suction blister, follicle removal and confocal Raman spectroscopy techniques are also described

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats

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

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain, a novel recombinant single-chain factor VIII

AbstractIntroductionrVIII-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 MethodsBinding 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 200–250IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice.ResultsrVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44pM 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.ConclusionsrVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models

Therapeutic treatment with rHA-Infestin-4 improves neurological performance after tMCAO in rats.

<p><b>(A)</b> Zea Longa score 24 h after transient middle cerebral artery occlusion (tMCAO) in rats. Rats were subjected to 90 min of tMCAO followed by reperfusion until 24 h. Rats were treated with 100 mg/kg rHA-Infestin-4 or saline (control) directly after start of reperfusion. Therapeutic rHA-Infestin-4 treatment significantly reduced the Zea Longa score compared to saline treated rats (n = 28-30/group). *p<0.05, Mann-Whitney test compared to saline treated animals. Horizontal solid lines indicate mean values (+SEM). <b>(B)</b> In addition, the Neuroscore was reduced in rHA-Infestin-4 treated rats compared to saline treated rats 24 h after tMCAO (n = 28-30/group). *p<0.05, Mann-Whitney test compared to saline treated animals. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals. <b>(C)</b> Functional performance on RotaRod and <b>(D)</b> grip strength in percent compared to the individual baseline of saline treated and rHA-Infestin-4 treated animals 24 h after tMCAO. rHA-Infestin-4 had no effect in these two assays compared to saline treated controls (n = 18-20/ group). p>0.05 between rHA-Infestin-4 treated and saline treated animals, Mann-Whitney test. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals.</p

Prophylactic rHA-Infestin-4 application is still active 24 h after tMCAO in rats.

<p>Prophylactic rHA-Infestin-4 application is still active 24 h after tMCAO in rats.</p