Optimized Pharmacodynamics
The pharmacodynamic effects of recombinant human G-CSF-ABD fusion protein were investigated in rats. One single injection of rhG-CSF-ABD fusion protein resulted in a dose dependent increase of blood granulocytes. Albumin association of G-CSF-ABD lead to a sustained elevated level of granulocytes compared to Neupogen® (rhG-CSF). The level did not return to baseline until 96 (low dose) and 120 (high dose) hours post-injection.
Prolonged pharmacodynamic effect. Rats were given a single s.c. injection of either equimolar amounts of rhG-CSF-ABD fusion protein or Neupogen® (rhG-CSF), a 10-fold lower amount of the rhG-CSF-ABD fusion protein, or vehicle control. The number of granulocytes was determined over time, using FACS analysis. Data are expressed as mean total granulocyte counts.
Fully Retained Activity
Neupogen® is a recombinantly produced human G-CSF used for treating neutropenia. The in vitro biological activity of a G-CSF-ABD fusion protein was determined using an NFS-60 cell proliferation assay. The G-CSF-ABD fusion protein shows same biological activity as Neupogen®, also when non-covalently attached to serum albumin.
Fully retained activity in vitro. G-CSF-ABD fusion protein (in a three times molar excess of HSA) and Neupogen® were titrated to NFS-60 cells. After three days of incubation the proliferation was assayed by addition of CCK8 reagent (Sigma) and absorbance readings at 450 nm.
Half-Life Extension The G-CSF-ABD fusion protein demonstrates improved pharmacokinetic properties compared to G-CSF alone. Association with albumin slows down the elimination and an effective concentration of the G-CSF-ABD fusion protein in the circulation is sustained for days.
10-fold increase in half-life. Rats were administered with a single i.v. injection of equimolar amounts of G-CSF-ABD fusion protein (0.4 mg/ rat; n=5) or G-CSF (Neupogen®) (0.3mg/rat; n=4) and their respective plasma concentrations over time were determined using a G-CSF specific ELISA.
Biodistribution The biodistribution of an ABD fusion protein and albumin were investigated in a dual labeling experiment performed in rat. This data indicate that the biodistribution profiles for the two molecules are similar. The ABD fusion protein benefits from the wide distribution, which is typical for albumin, and the half-life is extended as compared to the same non-fused molecule.
Similar biodistribution profiles of an ABD fusion protein and serum albumin from rat (RSA). 177Lu-labeled ABD fusion protein and 111In-labeled RSA were simultaneously administered to rat by intravenous injections. Selected organs were excised and the radioactivity was measured. Shown are the accessible interstitial volume corrected concentrations. Data are expressed as mean %IA/g (n=3).