Stability and interactions of recombinant human nerve growth factor in different biological matrices: In vitro and in vivo studies

The purpose of this investigation was to characterize the stability, activi ty, and interactions of recombinant human nerve growth factor (rhNGF) in va rious biological matrices in vitro and in vivo. rhNGF (10 mu g/ml) remained stable in human plasma for up to 4 days at 37 degrees C. There was a decre ase in the recovery of rhNGF after incubation at lower concentrations (20 n g/ml) and for longer time periods (3 and 5 days at 37 degrees C). Size excl usion HPLC analysis indicated that rhNGF forms high molecular weight (HMW) complexes after long incubation periods. We confirmed that alpha(2)-macrogl obulin (alpha(2)M) is the major plasma component that binds to rhNGF. Furth ermore, this interaction was considerably increased by treatment of plasma with primary amines such as CH3NH2. Changes in the pH environment did not a ffect the interaction of rhNGF with a2M. We also determined that the bindin g of rhNGF to CH3NH2-treated pure alpha(2)M or alpha(2)M present in human p lasma substantially diminished its immunoreactivity and bioactivity detecti on. The interaction of rhNGF with activated alpha(2)M was reversed and inhi bited by coincubation with dimethyl sulfoxide. Released rhNGF under these c onditions was fully bioactive. I-125-rhNGF also binds to alpha(2)M by formi ng similar I-125-rhNGF/HMW complexes in plasma after i.v. administration in rats and mice. Sixty minutes after dosing in rats, most of the labeled mat erial was in the form of a I-125-rhNGF/HMW complex. These studies have prov ided a better understanding of the nature of the interactions of rhNGF with plasma components as well as methods to enhance, reverse, and inhibit thes e interactions.