As this is the principal clearance mechanism for free FVIII it is likely that this twofold increase represents a ceiling for our attempts to extend FVIII half-life by modifying this molecule. As detailed below,

this conclusion has largely been borne out by experience to date. We also know that the existing half-life of FVIII in normal individuals varies greatly, which reflects the corresponding wide variation in VWF half-life. A major determinant of this variation is ABO blood group which alters FVIII levels by varying EPZ-6438 cell line the clearance rate of VWF. Observed half-lives of infused FVIII do vary from 5–18 h and are shorter in those with blood group O than in those of non-O blood group, confirming the dominant effect of VWF clearance on FVIII survival [104, 105]. Most of the attempts to prolong FVIII half-life have proceeded by adding another molecule to the FVIII. These molecules include PEG, albumin and the Fc portion of the immunoglobulin chain. It may also be possible to prolong the half-life by altering the glycan structure of FVIII or by introducing amino acid changes in the molecule

that will alter its stability, binding to VWF, resistance to AZD0530 activated protein C or its interaction with the LRP clearance receptor. At present the only examples close to clinical use and with associated clinical trial data are those using PEG and the immunoglobulin Fc fraction. Polyethylene glycol is a synthetic polymer of ethylene oxide which can be made in various 上海皓元医药股份有限公司 branched or single chain structures to a wide variety of lengths. It can be coupled to proteins via amide linkages to lysine residues

but relying on this simple chemical coupling has been difficult to control. Multiple sites of PEG addition frequently result in loss of protein function and produce a heterogeneous population of molecules which are all PEGylated to different extents. Nonetheless, it has been successfully used to prolong the half-life of several biological and therapeutic molecules. One approach to regulating the attachment of PEG has been to use thiol coupling. In this system the PEG is modified by the addition of a maleimide residue to the molecule, and this is used to direct the PEG to free thiols on cysteine residues on the target molecule. In the case of FVIII this has been achieved by introducing additional cysteine residues on the surface of the molecule by mutagenesis. Mei et al. [106] studied a large number of FVIII molecules that had been modified in this way and were able to select those where the introduction of the cysteine molecule and its subsequent PEGylation did not significantly alter FVIII function. They were then able to show that the half-life of these PEGylated FVIII molecules was prolonged when injected into FVIII knockout mice.