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T. Abache et al.
bleeding continues to occur. Second, it is based on the use of a zymogen thereby being a unique strategy respecting the coagulation/anticoagulation balance. Actiten liberates a wild-type FXa, a natural target of the anticoagulation system for which antithrombin could easily be used as an antidote, if needed. Third, as the duration of the treatment is based on the half-life of FX, this strategy can offer a four-fold longer persistence of treatment efficacy than that using FVIII. Lastly, actiten was demonstrated to be active in several coagulation defects, offering a supplementary therapeutic option for rare coagulation defects such as hemophilia B with inhibitors (Figure 6E).
In order to generate actiten, the cleavage site liberating the activation peptide was targeted to change the recogni- tion of FX. The addition of the 10-amino acid peptide offers the possibility of two cleavage sites during actiten activation. As observed by mass spectroscopy following RVV-X cleavage, only the activation peptide containing the complete added peptide was detected, suggesting a preferential cleavage between LDRP and IVV from the heavy chain. Moreover, there was no loss in the activity of the FXa liberated from actiten with regard to pdFXa. These data confirm that the active species liberated corre- sponds to a wild-type FXa.
The modification of activation was demonstrated since the presence of thrombin allowed the generation of FXa from actiten but not pdFX. Importantly, the recognition of the molecule by the FVIIa/TF complex was minimally affected and still allowed efficient participation of actiten in the initiation of the coagulation leading to the genera- tion of primary traces of FXa and thrombin. The ability of the molecule to correct FX-deficient plasma confirmed that the activation of actiten by FVIIa/TF was sufficient to replace the endogenous FX. In contrast, the important loss of activation by the FVIIIa/FIXa complex is less crucial for the function of actiten in hemophilia since the molecule is designed to substitute for their absence.
In vitro, actiten showed a velocity, a peak height and an endogenous thrombin potential sufficient to correct factor- deficient plasma samples. However, an increased lag time to clotting was observed in all factor-deficient samples of plasmas. This delay likely results from: (i) the moderate loss of FVIIa/TF recognition to activate the molecule; (ii) activation by thrombin that might not be as efficient as natural initiation of coagulation; and/or (iii) the time required to generate the first traces of thrombin to amplify the coagulation.22 Nevertheless, this delay did not translate in vivo into a lack of efficiency since the presence of actiten allowed clotting in antibody-treated rabbits with the same kinetics as in wild-type animals. Factor V contained within platelets actively participates in the generation of thrombin through the prothrombinase complex.23-25 Thus, in vivo, there may be some elements favoring clotting, such as fac- tor V and platelets, that are lacking from the in vitro assays. The presence of such elements might reinforce the clotting efficiency of actiten and eliminate differences in lag time, compared to that in normal plasma, observed in vitro.
To evaluate actiten in vivo, a rabbit model was estab- lished since human FXa is equally efficient in rabbit and human plasma.26 The classic hemophilia A mouse model would have been preferred to evaluate the efficacy of actiten, but the compound is less efficient in mouse plas- ma, rendering this model poorly predictive (data not shown). Despite the fact that the pair of monoclonal anti- bodies severely impaired rabbit coagulation in vitro, a lim- itation of the model is that it was not possible to ascertain full FVIII inhibition in vivo.
It has been reported that the half-life of FXa in blood is extremely short (<1.5 min).27 In our in vivo model, the rab- bit nail cuticle was cut 35 min after infusion of the mole- cule. The occurrence of normal clotting at this time point demonstrated that the molecule was kept under a zymo- gen form in the circulation and that it was mobilized when needed.
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Table 1. Thrombin generation parameters of normal and factor-deficient plasma samples, containing inhibitors or not, supplemented with actiten.
TG Parameter
Normal FVIII-def FVIII-def Hem. A Hem. A Hem. A Hem. A FIX-def FIX-def FIX-def FIX-def FX-def FX-def FXI-def FXI-def plasma plasma plasma TF/PL TF/PL + inhibit. + inhibit. plasma plasma plasma + plasma + plasma plasma plasma plasma TF/PL TF/PL TF/PL (n=3) (n=5) TF/PL TF/PL TF/PL TF/PL inhibit. inhibit. TF/PL TF/PL TF/PL TF/PL
(n=13) (n=5) (n=5) (n=3) (n=3) (n=2) (n=4) TF/PL TF/PL (n=3) (n=6) (n=2) (n=4) (n=2) (n=4)
+ buffer + buffer + actiten + buffer + actiten + buffer + actiten + buffer + actiten + buffer + actiten + buffer + actiten + buffer + actiten
Lagtime 7.2 8.7 10.2 5.3 7.7 7.1 9.2 8.2 16.2 8.6 12.8 31.3 12.1 10.7 18.6 (min) ±1.7 ±1.4 ±1.1 ±2.2 ±1.6 ±0.8 ±0.9 ±4.8 ±6.1 ±4.1 ±3.1 ±6.3 ±1.8 ±3.3 ±8.7
ETP 1510 543 1819 213 812 199 689 155 1436 186 1394 0 1010
430 1285
(nM.min IIa) ±216 ±77
±202 ±22 ±243 ±3.0 ±24 ±56 ±101 ±263 ±87 ±0 ±73 224 11.9 81.3 9.4 76 5.3 168 9.5 157 0,3 119
±29 ±119
Peak 195 19.6 (nM IIa) ±87 ±4.0
±59 ±2.3 ±31.4 ±0.3
19.0 146 ±11 ±2.7 ±34 ±10.2 ±30 ±0.1 ±34 ±3.5 ±52
ttPeak 12.1 25.7
15.5 17.0 15.6 22.9
16.8 28.2 23.1 35 19.5 42.6 17.9 21.7 25.6
(min) ±2.6 ±2.1
±2.7 ±7.4 ±1.2 ±0.8
±0.8 ±12.3 ±6.6 ±26 ±3.0 ±4.0 ±1.4 ±4.2 ±10.3
StartTail 32.9 67.8 (min) ±6.1 ±6.8
38.8 48 35.5 ±6.6 ±11 ±2.3
53,8 33.0 63.5 40.9 28 39.0 18.7 37.6 57 44.6 ±1.2 ±2.2 ±11 ±6.9 ±39 ±4.4 ±32.3 ±4.1 ±19.8 ±12.2
Velocity 40.4 1.1 48.3 1.2 10.7
0.6 10.1 0.3 25.2 0.8 25.2 0.1 21.8 1.7 23.3
(nM IIa/min) ±25.4 ±0.25 ±22.8 ±0.6 ±5.3
±0.1 ±2.5 ±0.2 ±8.7 ±1.0 ±8.7 ±0.01 ±9.7 ±0.2 ±13.4
Thrombin generation assays were performed in normal lyophilized pooled plasma (normal plasma), lyophilized pooled factor VIII-deficient plasma (FVIII-def.), lyophilized hemophilia A plasma (Hem. A), lyophilized hemophilia A plasma with 292 BU/mL inhibitors (Hem. A + inhibit.), lyophilized pooled factor IX-deficient plasma (FIX-def. plasma), lyophilized pooled FIX-deficient plasma spiked with 17 BU/mL inhibitors from a polyclonal anti-FIX antibody (FIX-def.plasma + inhibit.),lyophilized pooled factor X-deficient plasma (FX-def.plasma) and lyophilized pooled FXI-deficient plasma (FXI-def.plasma).The thrombin generation was initiated by 0.5 pM tissue factor and 4 μM phospholipids (TF/PL) in the presence of actiten (20 μg/mL) or buffer.N:number of duplicates performed.