Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation.

BACKGROUND: We previously reported that a humanized anti-factor IXa/X bispecific antibody, hBS23, mimics the function of FVIII even in the presence of FVIII inhibitors, and has preventive hemostatic activity against bleeding in an animal model of acquired hemophilia A. After further molecular engineering of hBS23, we recently identified an improved humanized bispecific antibody, ACE910, for clinical investigation. OBJECTIVES: To elucidate the in vivo hemostatic potency of ACE910 by examining its effect against ongoing bleeds, and to determine its pharmacokinetic parameters for discussion of its potency for prophylactic use. METHODS: A non-human primate model of acquired hemophilia A was established by injecting anti-primate FVIII neutralizing antibody. When bleeds emerged following an artificial bleed-inducing procedure, either ACE910 or recombinant porcine FVIII (rpoFVIII) was intravenously administered. rpoFVIII was additionally administered twice daily on the following 2 days. Bleeding symptoms were monitored for 3 days. A pharmacokinetic study and multiple-dosing simulations of ACE910 were also performed. RESULTS: A single bolus of 1 or 3 mg kg-1 ACE910 showed hemostatic activity comparable to that of 10 U kg-1 (twice daily) rpoFVIII against ongoing bleeds. The determined ACE910 pharmacokinetic parameters included a long half-life (3 weeks) and high subcutaneous bioavailability (nearly 100%). The simulation results based on pharmacokinetic parameters indicated that the above hemostatic level could be maintained with once-weekly subcutaneous administration of ACE910, suggesting the possibility of more effective prophylaxis. CONCLUSIONS: ACE910 may offer an alternative on-demand treatment option for patients with hemophilia A, as well as user-friendly and aggressive routine supplementation.

Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation.

BACKGROUND: We previously reported that a humanized anti-factor IXa/X bispecific antibody, hBS23, mimics the function of FVIII even in the presence of FVIII inhibitors, and has preventive hemostatic activity against bleeding in an animal model of acquired hemophilia A. After further molecular engineering of hBS23, we recently identified an improved humanized bispecific antibody, ACE910, for clinical investigation. OBJECTIVES: To elucidate the in vivo hemostatic potency of ACE910 by examining its effect against ongoing bleeds, and to determine its pharmacokinetic parameters for discussion of its potency for prophylactic use. METHODS: A nonhuman primate model of acquired hemophilia A was established by injecting anti-primate FVIII neutralizing antibody. When bleeds emerged following an artificial bleed-inducing procedure, either ACE910 or recombinant porcine FVIII (rpoFVIII) was intravenously administered. rpoFVIII was additionally administered twice daily on the following 2 days. Bleeding symptoms were monitored for 3 days. A pharmacokinetic study and multiple-dosing simulations of ACE910 were also performed. RESULTS: A single bolus of 1 or 3 mg kg⁻¹ ACE910 showed hemostatic activity comparable to that of 10 U kg⁻¹ (twice daily) rpoFVIII against ongoing bleeds. The determined ACE910 pharmacokinetic parameters included a long half-life (3 weeks) and high subcutaneous bioavailability (nearly 100%). The simulation results based on pharmacokinetic parameters indicated that the above hemostatic level could be maintained with once-weekly subcutaneous administration of ACE910, suggesting the possibility of more effective prophylaxis. CONCLUSIONS: ACE910 may offer an alternative on-demand treatment option for patients with hemophilia A, as well as user-friendly and aggressive routine supplementation.

Factor XI contributes to thrombus propagation on injured neointima of the rabbit iliac artery.

BACKGROUND: Thrombus formation through the activation of tissue factor (TF) and factor (F) XI is a critical event in the onset of cardiovascular disease. TF expressed in atherosclerotic plaques and circulating blood is an important determinant of thrombogenicity that contributes to fibrin-rich thrombus formation after plaque disruption. However, the contribution of FXI to thrombus formation on disrupted plaques remains unclear. METHODS: A mouse monoclonal antibody against FXI and activated FXI (FXIa) (XI-5108) was generated by immunization with activated human FXI. Prothrombin time (PT), activated partial thromboplastin time (APTT), bleeding time, and ex vivo platelet aggregation in rabbits were measured before and after an intravenous bolus injection of XI-5108. We investigated the role of FXI upon arterial thrombus growth in the rabbit iliac artery in the presence of repeated balloon injury. RESULTS: The XI-5108 antibody reacted to the light chain of human and rabbit FXI/FXIa, and inhibited FXIa-initiated FXa and FXIa generation. Fibrin-rich thrombi developed on the injured neointima that was obviously immunopositive for glycoprotein IIb-IIIa, fibrin, TF, and FXI. Intravenous administration of XI-5108 (3.0 mg kg(-1)) remarkably reduced thrombus growth, and the APTT was significantly prolonged. However, PT, bleeding time and platelet aggregation were not affected. CONCLUSIONS: These results indicate that plasma FXI plays a potent role in thrombus growth on the injured neointima. Inhibition of plasma FXI activity might help to reduce thrombus growth on ruptured plaques without prolonging bleeding time.

Contribution of Na+/Ca2+ exchanger to glucose-induced [Ca2+]i increase in rat pancreatic islets.

The present study was carried out to elucidate the role of the reverse mode of the Na+/Ca2+ exchanger in an increase in intracellular Ca2+ concentration ([Ca2+]i) induced by a stimulatory concentration of glucose in rat pancreatic islets. The effects of KB-R7943, a selective inhibitor of reverse Na+/Ca2+ exchanger, on Na+o removal-induced [Ca2+]i changes were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. Na+o removal induced a rapid increase in [Ca2+]i under 100 or 5 mM K+ conditions, respectively. The increases in [Ca2+]i induced by Na+o removal were inhibited by KB-R7943. The net amount of the [Ca2+]i increases during Na+o removal (Delta[Ca2+]i), obtained by subtracting the KB-R7943-independent Delta[Ca2+]i in the presence of KB-R7943 from Delta[Ca2+]i in the absence of KB-R7943, was significantly increased when extracellular K+ was raised. Increasing the external glucose concentration from 3 to 20 mM caused a biphasic increase in [Ca2+]i, which exhibited a transient increase (first phase) followed by a sustained increase (second phase) in [Ca2+]i. KB-R7943 (10 microM) partially inhibited the second phase of the [Ca2+]i increase rather than the first phase. These results suggest that the increase in [Ca2+]i induced by Na+o removal may be enhanced when plasma membrane is depolarized, and consequently, Ca2+ influx through the reverse Na+/Ca2+ exchanger may partially contribute to the glucose-induced [Ca2+]i dynamics in rat pancreatic islet cells.

Contribution of Na+/Ca2+ exchanger in maintaining [Ca2+]c at a stable state in rat pancreatic islets.

The effects of lowering extracellular Na+ concentration [Na+]o, on cytosolic Ca2+ concentration, [Ca2+]c were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. The total replacement of extracellular Na+ (Na+o) by equimolar N-methyl-D-(--)-glucamine caused a rapid rise in [Ca2+]c, and partial replacement of Na+o resulted in correlative rises in [Ca2+]c in accordance with the magnitude of reduced [Na+]o. The rise in [Ca2+]c induced by Na+o removal was strongly inhibited in the Ca2+o-deficient environment or by Ni2+. The [Ca2+]c rise, however, remained almost unchanged in the presence of nifedipine or SK&F 96365, and was enhanced by the addition of ouabain. The electrochemical gradients for Ca2+ (delta mu Ca2+) and Na+ (delta mu Na+) were calculated to be 39.08 and 12.8 kJ/mol, respectively, in this study, indicating a stoichiometry of 3Na+: 1 Ca2+. These results indicate that, in rat pancreatic islets, the rise in [Ca2+]c induced by lowering [Na+]o is mainly due to Ca2+ entry medicated by the Na+/Ca2+ exchanger operating with the stoichiometry of 3Na+:1 Ca2+, and that the Na+/Ca2+ exchanger plays an important role in maintaining stable-state [Ca2+]c.

Serial imaging of bilateral striatal necrosis associated with acidaemia in adults.

Bilateral striatal necrosis in acute encephalopathy has been reported in a small number of adults with methanol or cyanide intoxication, hypoxic encephalopathy or haemolytic-uraemic syndrome. Acute encephalopathy with bilateral striatal necrosis has been reported in infants and children. However, the pathogenesis of the necrosis remains unclear. This is the first report of serial imaging from the very early to chronic stage in two acute encephalopathic adults with bilateral striatal necrosis. A clinicoradiological study is presented for clarification of the pathological process and pathogenesis. Striatal lesions were not detected in the very early stages, but only thereafter. Serial studies suggested that the lesions were caused by delayed neuronal death. These patients had severe lactic acidosis, near the limit for survival. There have been few reports of adults with acute encephalopathy and bilateral striatal necrosis in whom arterial pH was described; all these exhibited marked acidosis. The common pathophysiological condition among these encephalopathies with bilateral striatal necrosis could be lactic acidosis elicited by impairment of ATP generation through the Krebs cycle. The striatum might represent one of the target areas of Krebs-cycle blockade.

Basic studies of radiopaque resin monomer (III). Physical properties of trial composite resins.

A series of studies has been conducted on the synthesis of radiopaque monomers and the development of a composite resin having these monomers. Using octachlorocyclotetraphosphazene, P4N4Cl8 (4PNC), three kinds of radiopaque cyclophosphazene monomers, 4PN(Br3Ph)1-3-(EMA)7-5, were synthesized by reacting 1-3 mols of tribromophenol (Br3Ph) and 7-5 mols of 2-hydroxyethyl methacrylate (HEMA). As the monomer for an organic composite filler, 70% (wt) synthesized monomer was used with silica (OX-50) treated with silane mixed at 30% and ground after heat-polymerization and then run through a 325-mesh sieve after polymerization. As a base monomer, 25% urethane monomer (U-2TH) was mixed in 25% synthesized monomer and photosensitizer was added. The composite resin was prepared by mixing 50% organic composite filler with 50% base monomer, and polymerized with a Dentacolor XS (Kulzer) visible light-curing apparatus by irradiation for 90 s on each side, 180 s in total. Mechanical properties did not vary with the increase in the number of tribromophenol replacements. Compressive yield strength was more than 120 MPa in all cases. Transverse strength was 60-75 MPa and hardness was HK 26-30. However, the aluminum equivalent increased with the increase in the number of tribromophenol replacements. In the case of 4PN-(Br3Ph)3, the value was 8.8 mm, whereas in the case of commercial composite resin, it was 0.4-10.4 mm.

Synthesis of radiopaque cyclophosphazene monomers, properties of bulk polymers and their application to composite resin.

A series of studies was conducted on the synthesis of polyfunctional cyclophosphazene monomers having radiopacity and a polymerization group in the same molecule, and their properties and applicability to composite resin were examined. Using octachlorocyclotetraphosphazene, P4N4Cl8 (4PNC), monomers were synthesized by replacing the 1-4 of chlorine (Cl) with p-bromophenol (BrC6H4OH, BrPh), and replacing the residual number of Cl, 7-4, with 2-hydroxyethyl methacrylate [CH2:C(CH3)COOCH2CH2OH](HEMA), so as to obtain four kinds of transparent monomer having radiopacity and a polymerization group in the same molecule. We then analyzed these monomers and examined their physical properties after bulk-polymerization. Next, we prepared an organic composite filler using 4PN-(BrPh)3-(EMA)5 monomer, which showed comparatively good radiopacity, to produce a new experimental radiopaque composite resin. Although radiopacity improved in accordance with the increase in the number of BrPh molecules replaced, the mechanical properties of the polymer became poorer. Similarly it was proved that the radiopacity of composite resin made with 4PN-(BrPh)3-(EMA)5 monomer was equivalent or even superior, compared with the radiopacity of the front tooth. Consequently, it was shown that these synthesized monomers can be applied to visible light-cured radiopaque composite resin.

[The development of visible light-cured composite resin. Kinds of fillers to be mixed with cyclophosphazene system monomers and its amount and its physical properties].

A visible light-cured composite resin was developed. Cyclophosphazene monomer, 4 PN-(TF)1-(EMA)7 was prepared as a monomer. The ratio of brush abrasion, mechanical properties, water sorption, thermal expansion coefficient and the surface of abrasion were examined after mixing with fillers of different particle size (R-972, OX-50 and VL-30). The ratio of brush abrasion showed a tendency to be small when more than 50 wt% of VL-30 with a large particle size was mixed with 4 PN-(TF)1-(EMA)7 monomer. However its abrasion surface was rough compared with that of the microparticle filler. When the microparticle filler (50 wt% of OX-50) was mixed with the monomer, its mechanical properties were good for the mixture with 50 wt% OX-50. In that case, the ratio of brush abrasion was 0.268, compressive and transverse strength, 124.3 and 86.3 MPa respectively, hardness, 43.2 Hk, water absorption 14.2 micrograms/mm3 and thermal expansion coefficient, 47.4 x 10(-6)/degrees C.