Macitentan is a dual endothelin receptor antagonist under phase 3 investigation in pulmonary arterial hypertension. We investigated the effect of cyclosporine (Cs) and rifampin on the pharmacokinetics of macitentan and its metabolites ACT-132577 and ACT-373898 in healthy male subjects. In addition, in vitro studies were performed to investigate interactions between macitentan and its active metabolite ACT-132577 with human organic anion-transporting polypeptides (OATPs). The clinical study (AC-055-111) was conducted as a two-part, one-sequence, crossover study. Ten subjects in each part received multiple-dose macitentan followed by multiple-dose co-administration of Cs (part A) or rifampin (part B). In the presence of Cs, steady-state area under the plasma concentration–time profiles during a dose interval (AUCτ) for macitentan and ACT-373898 increased 10% and 7%, respectively, and decreased 3% for ACT-132577. Steady-state AUCτ of macitentan and ACT-373898 in the presence of rifampin decreased 79% and 64%, respectively. For ACT-132577, no relevant difference in AUCτ between the two treatments was observed. Macitentan co-administered with Cs or rifampin was well tolerated. The complementary in vitro studies demonstrated no marked differences in uptake rates of macitentan and ACT-132577 between the wild-type and OATP over-expressing cells over the concentration range tested. Concomitant treatment with Cs did not have any clinically relevant effect on the exposure to macitentan or its metabolites, at steady-state. Concomitant treatment with rifampin reduced significantly the exposure to macitentan and its metabolite ACT-373898 at steady-state but did not affect the exposure to the active metabolite ACT-132577 to a clinically relevant extent.

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The rotational thromboelastography (ROTEG) assay system allows the real-time analysis of clot formation (fibrin formation) in a whole-blood assay format. The ROTEG system provides significant advantages over the current plasma-based assay systems as it includes the important interactions between cellular and plasmatic coagulation factors. We have employed the ROTEG system to characterize clot formation dynamics in factor VIII- deficient mouse whole blood and examined the ability of recombinant FVIII (rFVIII) supplementation to restore the normal phenotype. The ability to generate a clear dose-response relationship by adding rFVIII to FVIII-deficient murine whole blood (FVIII-/-) demonstrates the feasibility of this approach. A dose-response from 1 U to 0.00001 U mL(-1) demonstrates the enhanced sensitivity of the ROTEG system. Further characterization of this experimental approach may provide a potential tool for comparing the activity of FVIII concentrates and/or evaluating FVIII mutants.

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Current treatment of patients with hemophilia A often requires the frequent infusion of Factor VIII (FVIII) due to its short circulating half-life. A longer-acting FVIII molecule could profoundly impact patients’ lives by extending bleeding protection with a reduced frequency of infusions. Several strategies to prolong plasma concentrations of FVIII have been attempted. In particular, targeting domains on FVIII that bind to LRP, the putative clearance receptor, has been a popular strategy. We have investigated the use of site-directed pegylation of B-domain deleted (BDD) FVIII to evaluate the utility of PEG as a method to decrease FVIII clearance through steric hindrance of LRP binding, or other unknown clearance mechanisms, while minimizing decreases in vWF binding and in vivo activity. The evaluation of novel constructs required the development of in vivo pharmacokinetic models and a FVIII-dependent bleed model. We describe the development of an acute bleed model following uniform tail transection in the hemophilia A mouse that is FVIII dependent and allows the evaluation of the acute pharmacologic effects of FVIII or variants in vivo. Pharmacokinetic analysis of recombinant FVIII (rFVIII) and its variants was performed in rabbits over 32-hours and rFVIII or variants were measured using a modified Coatest® to differentiate endogenous rabbit FVIII from the administered human FVIII. For efficacy evaluations, hemophilia A mice were anesthetized with isoflurane and their pre-warmed tail was cut by a scalpel and placed into a new tube of warmed saline (37–40°C). Blood was collected over 40 minutes and blood loss was measured gravimetrically. Three modes of treatment were evaluated: prevention of bleeding (drug was administered 5 minutes before injury), treatment of an acute bleeding event (drug was administered 5 minutes after injury), and a delayed injury model (tail cut occurred at 20 or 24 hours after the drug administration). Over the course of 40 minutes control (C57BL6) mice demonstrated negligible bleeding (approximately 41 ± 8 μL) compared to 919 ± 26 μL in hemophilia A mice. A dose response curve was constructed for doses ranging from 0.1 to 5.0 IU of human rFVIII per mouse. Hemophilia A mice treated with 200 IU/kg of human rFVIII (5 IU/mouse) lost a similar volume of blood as control mice. The protective effect was rFVIII dose dependent over a range of 4–200 IU/kg (0.1–5 IU/mouse). In contrast, more rFVIII was required to stop an acute bleeding event when administered after the injury. In the delayed injury model, mice injured 24 hours after drug administration had a significantly larger mean blood volume loss compared to mice injured 20 hours post drug administration. Pegylated rFVIII constructs with longer half-lives also had increased activity over time compared to non-pegylated rFVIII in this mouse model. These results describe a superior hemophilia A tail bleed model that demonstrates FVIII-dependent bleeding reduction in response to acute hemorrhage over a 40 minute time course. This is the first demonstration of a hemophilia A mouse model in which all untreated animals uniformly bleed and all control animals demonstrate negligible bleeding. This model was used to evaluate the in vivo hemostatic efficacy of new rFVIII molecules that were designed to have superior pharmacologic and/or pharmacokinetic properties compared to rFVIII.

To cite this article: Landskroner KA, Olson NC, Jesmok GJ. Enhanced factor VIII activity measurements using ROTEG and factor VIII-/-mice whole blood. J Thromb Haemost 2004; 2: 2274-5.To quantify the rate of fibrin formation after recombinant FVIII (rFVIII) administration to FVIII-deficient mouse whole blood we have employed the rotational thromboelestography (ROTEG, Pentapharm) assay system. This system demonstrated greater sensitivity than traditional assay systems and allows the analysis of clot (fibrin) formation in real time. This system uses FVIII-deficient mouse whole blood and provides a more relevant fibrin formation assay system than plasma-based systems. In contrast to ROTEG, current assays used to measure FVIIIa-dependent fibrin clot formation, such as the activated partial thromboplastin time (APTT), are less sensitive. Reasons for the decreased sensitivity are due, in part, to the absence of platelets that are necessary for optimal tenase and prothrombinase assembly. While there has always been interest in measuring low FVIIIa activity, i.e. < 0.001 U mL , the technology and methodology were not available. Thromboelastography (TEG) systems have been available for decades; however, recent ROTEG technology has improved ease of use and reproducibility [1,2]. In order to evaluate the detection sensitivity of ROTEG at low levels of FVIII we examined the thromboelastographic profile of FVIII-/-mice [3] whole blood supplemented with rFVIII over a 100 000-fold concentration range. The ability to generate a clear doseresponse relationship between FVIII levels of 0.00001 U mL )1 (0.001%) to 1 U mL )1 (100%) and the rate of fibrin formation demonstrates the enhanced sensitivity of the approach. Briefly, blood from mice was drawn from the descending vena cava under anesthesia and immediately anticoagulated with 3.8% citrate in a 1 : 9 dilution (citrate : blood). For comparison, blood from C57BL/6 mice (FVIII+/+, background strain) was used as control. Blood samples were pooled from three to five animals for analysis. Within 8 h of sampling the blood was assayed with the ROTEG system using the NATEG protocol provided by the manufacturer [1,2]. Coagulation was allowed to proceed without the exogenous addition of tissue factor. Recombinant FVIII (Refacto, Wyeth) at each dilution was added in a total volume of 3.3 lL to 330 lL of whole blood. The coagulation reaction was started by the addition of 20 lL of 200 mM CaCl 2 . Using this protocol the following measurements were made: clotting time (CT), the time delay from start of sample run to point where trace is 1 mm wide; clot formation time (CFT), the time between the 1 mm wide point to the 20 mm wide point; and alpha angle, which is an indirect measurement of the rate of clot formation [1,2]. The tests were halted after the alpha angle was calculated by the software program, or 2 h, whichever happened first. Experiments consisted of drawing blood from three to five animals, pooling the blood, and adding dilutions of rFVIII. Each experiment was repeated three to f...