title: The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes.
abstract: Recombinant activated factor VII (rFVIIa; NovoSeven®) is a heterogeneously glycosylated serine protease used for treatment of haemophiliacs with inhibitors. The drug substance contains a subpopulation consisting of ~20% of rFVIIa molecules which are unsialylated and consists of carbohydrate moieties with terminally exposed galactose and N-acetyl-D-galactosamine (GalNAc). Recently, data from an in situ perfused liver model showed that a subpopulation of rFVIIa, appearing to be unsialylated rFVIIa, was cleared by the liver, thus suggesting a carbohydrate-moiety mediated mechanism. The parenchymal cells of the liver, hepatocytes, are known to abundantly express functional carbohydrate-specific receptors and in this study we therefore used primary rat hepatocytes to study binding and intracellular fate of rFVIIa at a cellular level. Immunofluorescence microscopy showed that rFVIIa was distributed into distinct intracellular vesicles and electron microscopic autoradiography revealed that radioiodinated rFVIIa distributed only into cytoplasmic free vesicles resembling endosomes and lysosomes. These findings suggest that endocytosis of rFVIIa in hepatocytes could be partly mediated via initial membrane binding to a receptor. Quantitative binding studies showed that the presence of excess unlabelled asialo-orosomucoid, asialo-rFVIIa and GalNAc significantly decreased binding of 125I-rFVIIa. An antibody which specifically binds to the carbohydrate recognition domain of the asialoglycoprotein receptor (ASGPR) significantly decreased binding of asialo-rFVIIa by ~36% and rFVIIa by ~19%. Together our data showed that a receptor-mediated mechanism involving the ASGPR is able to bind a subpopulation of unsialylated rFVIIa, while a hepatic mechanism for binding and clearing sialylated rFVIIa is still unknown.
authors.affiliation: Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark. tojo@novonordisk.com
authors.forename: Torben
authors.initials: T
authors.lastname: Seested
authors.forename: Hanne M
authors.initials: HM
authors.lastname: Nielsen
authors.forename: Erik I
authors.initials: EI
authors.lastname: Christensen
authors.forename: Rupa S
authors.initials: RS
authors.lastname: Appa
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D037263:Asialoglycoprotein Receptor / Q000276:immunology / Q000378:metabolism*; D001345:Autoradiography; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D004705:Endocytosis; D015942:Factor VIIa / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008297:Male; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000906:Antibodies; D037263:Asialoglycoprotein Receptor; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; C103587:recombinant FVIIa; D015942:Factor VIIa