RESUMEN
Inherited antithrombin (AT) deficiency is one of the most clinically significant forms of congenital thrombophilia and follows an autosomal dominant mode of inheritance. We analyzed SERPINC1 in a patient who developed deep-vein thrombosis and low AT activity during pregnancy, and identified a novel missense mutation c.259A>G (p.Asn87Asp; N87D). Surprisingly, analysis of the parents' DNA showed that they did not possess this mutant, and thus, it may have been due to a de novo mutation. We also expressed this mutant AT protein in COS-1 cells and compared its intracellular localization and intracellular and extracellular antigen levels with that of wild-type AT. The expression experiment did not reveal a significant difference in the antigen levels of the mutant and wild-type AT in the cell lysate, but the mutant AT antigen level was markedly lower than that of its wild-type counterpart in the COS-1 cell supernatant. Immunofluorescence did not indicate any difference between the mutant and wild-type AT in terms of cytoplasmic localization of fluorescence signals. Our findings suggest that the patient's AT deficiency may have been caused by impaired extracellular secretion of mutant AT protein p.Asn87Asp.
Asunto(s)
Antígenos/metabolismo , Deficiencia de Antitrombina III/genética , Antitrombina III/genética , Proteínas Antitrombina/genética , Proteínas Antitrombina/fisiología , Mutación Missense , Complicaciones del Embarazo/genética , Adulto , Animales , Proteínas Antitrombina/inmunología , Proteínas Antitrombina/metabolismo , Células COS , Chlorocebus aethiops , Femenino , Humanos , Embarazo , Trombofilia/genética , Trombosis de la VenaRESUMEN
AIM: NN1731 is a recombinant activated factor VII (rFVIIa) analogue with enhanced activity. The objective of the present study was to evaluate the clearance mechanisms of rFVIIa and NN1731 after intravenous administration to Beagle dogs. METHODS: The study was performed in Beagle dogs administered with a single dose of 5.4 nmol/kg rFVIIa or NN1731 intravenously. Plasma samples collected up to 12-h post-administration were analysed using three different assays to determine FVIIa clot activity (FVIIa:C), total FVIIa antigen, and levels of FVIIa-antithrombin (AT) complexes. Pharmacokinetic parameters were determined by use of standard non-compartmental and non-linear mixed effects methods. RESULTS: For both compounds, complex formation with AT accounted for the observed difference between the activity and the antigen curves and constituted 60-70% of the total clearance. The clearance of rFVIIa and NN1731 was estimated to be 73 and 214 mL/h/kg, respectively, accordingly, AT complex formation occurred around three times faster for NN1731. The difference in activity observed in the initial phase, resulting in distribution half-lives of 0.71 and 0.22 h for rFVIIa and NN1731, was mainly caused by the 3-fold difference in clearance. The terminal half-life of rFVIIa and NN1731 was estimated to be 2.1 and 2.5 h, respectively. The non-compartmental analysis resulted in almost identical parameters. CONCLUSION: The present study demonstrates that the difference between the activity and the antigen profiles of rFVIIa and NN1731 in Beagle dogs is the result of complex formation with AT which constitutes a major pathway for the clearance of rFVIIa activity.
Asunto(s)
Factor VII/farmacocinética , Factor VIIa/farmacocinética , Modelos Biológicos , Animales , Proteínas Antitrombina/fisiología , Coagulación Sanguínea/efectos de los fármacos , Interpretación Estadística de Datos , Perros , Factor VII/administración & dosificación , Factor VII/farmacología , Factor VIIa/administración & dosificación , Factor VIIa/farmacología , Semivida , Inyecciones Intravenosas , Tasa de Depuración Metabólica , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologíaRESUMEN
NN1731 is a recombinant activated factor VII (rFVIIa) analogue with increased intrinsic activity. This also applies to its reactivity towards antithrombin (AT), the role of which was investigated in a pharmacokinetic (PK) study. NN1731 or rFVIIa was administered to normal and haemophilia A dogs and elimination was measured by FVIIa clot activity, FVIIa- and FVIIa-AT antigen. In vitro AT complex formation was studied in canine plasma spiked with NN1731 or rFVIIa. Based on FVIIa antigen concentrations, PK profiles in normal and haemophilia A dogs were similar for NN1731 and rFVIIa with antigen half lives, t(½) ≈1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t(½) ≈0·5 h) pro-coagulant response, whereas rFVIIa induced a lower, longer lasting (t(½) ≈1·1 h) response. Western Blot and FVIIa-AT antigen analysis demonstrated in vivo AT complex formation that accounted for these divergences. AT complex formation with FVIIa or NN1731 in vitro in canine plasma was considerably slower than the in vivo reaction. The results suggest that in vivo inhibition by AT contributes significantly to define drug duration in haemophilia treatment with rFVIIa and in particular with the NN1731 analogue.
