Investigating clearance mechanisms for recombinant activated factor VII in a perfused liver model

Rupa S. Appa; Charlotte Theill; Lene Hansen; Judi Møss; Carsten Behrens; Else Marie Nicolaisen; Niels K. Klausen; Michael S. Christensen

doi:10.1160/TH09-10-0723

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2010; 104(02): 243-251
DOI: 10.1160/TH09-10-0723

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Investigating clearance mechanisms for recombinant activated factor VII in a perfused liver model

Authors

Rupa S. Appa

¹Exploratory ADME, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark
Charlotte Theill

¹Exploratory ADME, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark
Lene Hansen

¹Exploratory ADME, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark
Judi Møss

²M&S Hematology and Clinical Pharmacology, Novo Nordisk A/S, Bagsværd, Denmark
Carsten Behrens

³Biopharm Chemistry, Novo Nordisk A/S, Måløv, Denmark
Else Marie Nicolaisen

⁴NovoSeven® Research, Novo Nordisk A/S, Måløv, Denmark
Niels K. Klausen

⁵Protein Chemistry, Biopharm, CMC Analysis and Formulation, Novo Nordisk A/S, Gentofte, Denmark
Michael S. Christensen

⁶DMPK and Bioanalysis Preclinical Development, Novo Nordisk A/S, Måløv, Denmark

Abstract

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Summary

Clearance mechanisms for recombinant activated human FVII (rFVIIa; NovoSeven®), a heterogeneously glycosylated protein, have yet to be fully elucidated, but may involve the liver. The effects of the γ-carboxy glutamic acid (Gla) domain and the sialic acid content of the protein on rFVIIa clearance were investigated following intravenous administration of rFVIIa lacking the Gla domain, des(1–44) rFVIIa and asialo-rFVIIa in pharmacokinetic (PK) studies and perfused rat livers. PK parameters for both rFVIIa and des(1–44) rFVIIa had similar biphasic clearance profiles, as well as half-lives ([t½]=80 and 88 minutes, respectively), while asialo-rFVIIa was cleared quickly (t½=21 minutes) with a linear clearance profile. Perfused liver studies with all proteins (10 nM) mirrored the trends in profiles observed in the PK study. rFVIIa and des(1–44) rFVIIa were cleared to a similar extent, 41% and 35%, respectively, after 1 h, whereas plasma-derived FVII from humans (which has a higher sialylation content than rFVIIa) was cleared to a lesser extent (21%). Asialo-rFVIIa, on the other hand, was almost totally cleared and when an excess of asialo-orosomucoid was added to the perfusate, its clearance was significantly reduced (by 34%) and also for rFVIIa, albeit to a lesser extent (by 14%). Together these data suggest that carbohydrate receptor(s) (e.g. the asialoglycoprotein receptor, ASGPR) play a role in asialo-rFVIIa and rFVIIa clearance. In vivo and liver clearance data correlated well showing similar trends and indi-cated that rFVIIa clearance is not affected by the Gla domain, but rather by a subpopulation of N-glycosylated structures on rFVIIa.

Keywords

Recombinant activated factor VII (rFVIIa) - in situ rat liver perfusion - asialoglycoprotein receptor (ASGPR) - asialo-rFVIIa - asialo-orosomu-coid

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References

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