FORMULATION DESIGN OF ACIDIC FIBROBLAST GROWTH-FACTOR

The design of an aqueous formulation for acidic fibroblast growth fact or (aFGF) requires an understanding of the type of compounds that can either directly or indirectly stabilize the protein. To this end, spec trophotometric turbidity measurements were initially employed to scree n the ability of polyanionic ligands, less specific compounds, and var iations in solution conditions (temperature and pH) to stabilize aFGF against heat-induced aggregation. It was found that in addition to the well-known protection of aFGF by heparin, a surprisingly wide variety of polyanions (including small sulfated and phosphorylated compounds) also stabilizes aFGF. These polyanionic ligands are capable of raisin g the temperature at which the protein unfolds by 15-30-degrees-C. Man y commonly used excipients were also observed to stabilize aFGF in bot h the presence and the absence of heparin. High concentrations of some of these less specific agents are also able to increase the temperatu re of aFGF thermal unfolding by as much as 6-12-degrees-C as shown by circular dichroism and differential scanning calorimetry. Other compou nds were found which protect the chemically labile cysteine residues o f aFGF from oxidation. Aqueous formulations of aFGF were thus designed to contain both a polyanionic ligand that enhances structural integri ty by binding to the protein and chelating agents (e.g., EDTA) to prev ent metal ion-catalyzed oxidation of cysteine residues. While room-tem perature storage (30-degrees-C) leads to rapid inactivation of aFGF in physiological buffer alone, several of these aFGF formulations are st able in vitro for at least 3 months at 30-degrees-C. Three aFGF topica l formulations were examined in an impaired diabetic mouse model and w ere found to be equally capable of accelerating wound healing.