As a result of this investigation Bayer chose a recombinant B-domain-deleted FVIII molecule with a single site modification to which a 60 kDa PEG molecule was attached for further development. A Phase 1 study in 14 patients showed that this molecule had an extended half-life of 19 h compared to 13 h in controls . This therefore represented an approximately 1.5-fold increase in half-life and was achieved without any adverse events or inhibitor development. Notwithstanding the limitations
described above, Baxter have pursued a chemical modification method to modify FVIII. Careful control of the reaction DNA Synthesis inhibitor conditions resulted in a full length FVIII molecule PEGylated in a 2:1 molar ratio using a 20 kDa PEG molecule. Further analysis showed that 60% of the PEG was attached to the B domain, which may be advantageous as this will be removed during FVIII activation. A Phase 1 study in 10 patients showed a half-life extension of approximately Selleckchem OTX015 1.5-fold, again with no significant adverse events . A third approach to PEG modification of FVIII has
been followed by Novo Nordisk. They noted that their B-domain-deleted FVIII molecule retained a single O-linked glycan in the residual B domain. Following desialylation of the FVIII, a specific transferase was used to transfer a sialic acid-modified PEG molecule onto the remaining O-linked glycan chain, following which the remaining N-linked glycans were resialylated. The FVIII molecule therefore contained a single 40 kDa PEG addition which resulted in a half-life extension of approximately 1.6-fold in a Phase 1 study [109, 110]. Thus, all three PEG modification strategies have received similar modest prolongations of FVIII half-life. The addition of the immunoglobulin Fc fragment to molecules results in their attachment to the neonatal Fc receptor after
cellular uptake and protects them from breakdown in endosomes, eventually resulting in their return to circulation. This technique has been successfully used to prolong the half-life of other therapeutic molecules and Powell et al. Acetophenone reported on the effect of modifying FVIII in this way in a Phase 1 study of patients with haemophilia A. In 16 previously treated patients (PTPs), the half-life of FVIII was prolonged from 11 h to 18.8 h, representing a mean 1.7-fold increase in half-life. There were no adverse events and no antibody production, but again the prolongation is relatively modest . Overall, it is clear from the studies reported so far that none of the modified molecules have been able to exceed the twofold extension in half-life produced by LRP knockout. It is worth noting that this modest prolongation of half-life is in contrast to the threefold or greater increase in half-life achieved using similar techniques to modify factor IX .