PEG Modified rVWF Prolongs the Survival of Native rFVIII in Hemophilia A Knock-Out Mice.

Mature CHO cell derived and highly purified rVWF was chemically derivatized by binding of 20 kDa polyethylene glycol (PEG) succinimidyl glutarate, to the primary amino groups in rVWF with covalent bond formation, primarily with lysine residues. PEG conjugates of rVWF were further purified and pharmaceutically formulated for application into hemophilia A knock-out mice. Groups of 6 mice each were treated with respective test and control articles to measure pharmacokinetics. Mice were treated with chemically modified rVWF in combination with recombinant human FVIII (rFVIII, Advate). For control purposes, mice were treated with unmodified rVWF plus rFVIII. Citrated plasma was prepared from their blood and measured for FVIII activity with a chromogenic FVIII assay.

Mice received 300 IU/kg rFVIII together with 3.6 mg/kg PEG-rVWF or 1.6 mg/kg unmodified rVWF. FVIII levels were measured at time points 5 minutes and 3, 9, 24 and 32 hours after injection. FVIII levels found 5 minutes after injection were set as 100%. At all time points FVIII levels were substantially higher in the groups co-administered with PEG-rVWF. Mice co-administered with PEG-rVWF showed a FVIII level of 3% at 24 hours which was maintained at 2% after 32 hours, while in the controls treated with rFVIII and unmodified rVWF, FVIII levels were close to the limit of detection at 24 hours (~ 0,6%) and had completely disappeared at 32 hours. Assuming a one compartmental model, a mean half life of 4.4 hrs of rFVIII in the presence of PEG-rVWF and 1.2 hrs in the presence of unmodified rVWF was calculated (Scientist program, MicroMath, Saint Louis, MO, USA). The FVIII AUC was increased by a factor of 2.5 for the group treated with PEG-rVWF (MS EXCEL, trapezoidal model). Our study demonstrates that chemical modification by PEGylation of rVWF can be used to improve the survival of co-administered native rFVIII in the circulation of hemophilic mice.