Pharmacokinetic properties of 2nd-generation fibroblast growth factor-1 mutants for therapeutic application

Abstract

Fibroblast growth factor-1 (FGF-1) is an angiogenic factor with therapeutic potential for the treatment of ischemic disease. FGF-1 has low intrinsic thermostability and is characteristically formulated with heparin as a stabilizing agent. Heparin, however, adds a number of undesirable properties that negatively impact safety and cost. Mutations that increase the thermostability of FGF-1 may obviate the need for heparin in formulation and may prove to be useful "2nd-generation" forms for therapeutic use. We report a pharmacokinetic (PK) study in rabbits of human FGF-1 in the presence and absence of heparin, as well as three mutant forms having differential effects upon thermostability, buried reactive thiols, and heparin affinity. The results support the hypothesis that heparan sulfate proteoglycan (HSPG) in the vasculature of liver, kidney and spleen serves as the principle peripheral compartment in the distribution kinetics. The addition of heparin to FGF-1 is shown to increase endocrine-like properties of distribution. Mutant forms of FGF-1 that enhance thermostability or eliminate buried reactive thiols demonstrate a shorter distribution half-life, a longer elimination half-life, and a longer mean residence time (MRT) in comparison to wild-type FGF-1. The results show how such mutations can produce useful 2nd-generation forms with tailored PK profiles for specific therapeutic application.

Figure 1. Relationship between WT FGF-1 and mutant proteins.

The FGF-1 structure (PDB accession 2AFG [62]) has three buried reactive thiols (Cys residues indicated) and heparin-binding functionality associated with surface loops 104–106 and 120–122 (indicated by blue color). Mutant M1 (based upon PDB accession 2HWM for Lys12Val/Cys117Val mutant [30]) includes stabilizing mutations Lys12Val and Pro134Val (indicated) combined with elimination of one buried thiol (Cys117Val). Mutant M2 (PDB accession 3FGM [31]) combines elimination of two buried thiols (Cys83Thr/Cys117Val) with two fully-buried stabilizing mutations (Leu44Phe/Phe132Trp; not shown) that offset the destabilizing effects of the Cys mutations (such that thermostability of M2 is equivalent to WT FGF-1). Mutant M3 (PDB accession 3O3Q, a Phe108Tyr form of M3 that promotes crystallization [63]) has enhanced thermostability and elimination of one buried thiol (Cys117Val) and is thus similar to M1; however, M3 also has loop deletions (blue turn regions in FGF-1) that effectively eliminate heparin-binding functionality.

Figure 2. PK profile and fitted two-compartment model for WT and mutant FGF-1 proteins (error bars for n = 3 data sets for each protein).

PBX IV bolus yielded no detectable endogenous FGF-1 for any time point (not shown). The inset on upper right shows a close-up of the 0–120 min time period.

Figure 3. Plasma triglyceride levels for pre-bleed (T = 0) control, 240, 480 and 1440 min samples.

PBX control is indicated in white, FGF-1 plus heparin is indicated in black, FGF-1 in the absence of heparin in gray, mutant M1 in red, mutant M2 in green, and mutant M3 in blue. Standard deviation for each protein measurement is indicated by vertical error bar. The normal range for triglyceride levels in NZW rabbits is indicated by the two horizontal dashed lines.

Figure 4. Plasma cholesterol levels for pre-bleed (T = 0) control, 240, 480 and 1440 min samples.

Standard deviation for each protein measurement is indicated by vertical error bar. The normal range for cholesterol levels in NZW rabbits is indicated by the two horizontal dashed lines.

Figure 5. Liver chemistry profile for the 1440 min time point plasma sample from PBX, FGF w/o heparin and M2 mutant protein.

The panel includes alanine transaminase (ALT), aspartate transaminase (AST), bilirubin, albumin and γ-glutamyltranspeptidase (GGT). The normal range of these components in the NZW rabbit , is indicated by dashed lines (note that the normal low range for GGT is 0.0 IU/L).

Figure 6. Plasma glucose levels for pre-bleed (T = 0) control and time points from 2 to 1440 min.

Standard deviation for each protein measurement are indicated by vertical error bar. The normal range for plasma glucose levels in NZW rabbits is indicated by the two horizontal dashed lines.

Figure 7. MRT values plotted for all proteins.

MRT is reduced for FGF-1+heparin, or for mutant M3 (which has a diminished heparin binding site), showing that heparin sequestration is a prime determinant of MRT.

Figure 8. Cp*t curves for all proteins.

Efficient distribution of FGF-1 from plasma to HSPG (via the heparin binding site) serves as a storage reservoir for latent redistribution of FGF-1 from HSPG into plasma, extending MRT.