Intravascular inhibition of factor VIIa and the analogue NN1731 by antithrombin

Abstract

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.

Fig 1

FVIIa clot activity and clot formation during in vivo elimination of NN1731 or rFVIIa in haemophilia A dogs. A bolus of 0·28 mg/kg NN1731 (squares) or rFVIIa (circles) was administered i.v. to haemophilia A dogs. (A) FVIIa clotting activities (closed symbols) measured according to standard procedures using a rFVIIa calibrator for analysis of both NN1731 and rFVIIa samples. (B) Clot propagation (α-angle) (open symbols) measured by thrombelastographic profiling. Data are means ± SD (n = 2).

Fig 2

Pharmacokinetics of in vivo elimination of NN1731 and rFVIIa. Dogs received 0·27 mg/kg bw NN1731 (A) or rFVIIa (B) in absence (full lines) and presence of 200 iu/kg of un-fractionated heparin pre-dosed i.v. 10 min before treatment with NN1731 or rFVIIa (dotted lines). Plasma was sampled at various time points and FVIIa antigen (ag) concentration, FVIIaAT antigen concentration and FVIIa clot activity were measured as indicated. Note that the activity of NN1731 was calibrated against a rFVIIa standard and given as rFVIIa equivalents in ng/ml. Data are means ± SD (n = 2–4).

Fig 3

NN1731/rFVIIa-AT complex formation during in vivo elimination of NN1731 and rFVIIa. NN1731 or rFVIIa (0·27 mg/kg) was administered i.v. to dogs, and plasma was sampled at various time points as indicated. Plasma was subjected to non-reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis and FVIIa Western Blot analysis. Arrows indicate free NN1731/rFVIIa and complexes with AT with molecular masses of c. 50 and 100 kDa, respectively.

Fig 4

Time courses of FVIIa clot activity, FVIIa antigen and FVIIa-AT antigen concentrations in canine plasma spiked with NN1731 or rFVIIa in the absence and presence of heparin. Canine plasma stabilized with TAP and hirudin was spiked with 3 µg/ml NN1731 (lower panels) or 3 µg/ml rFVIIa (upper panels) at room temperature and incubated for various times in the absence (left panel) and presence of 2 u/ml heparin (right panels). FVIIa antigen concentration (closed circles), FVIIa clot activity (open circles) and FVIIa-AT antigen concentration (open squares) were measured.