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, activity, and interactions of recombinant human nerve growth factor (rhNGF) in various biological matrices in vitro and in vivo. rhNGF (10 microg/ml) remained stable in human plasma for up to 4 days at 37 degrees C. There was a decrease in the recovery of rhNGF after incubation at lower concentrations (20 ng/ml) and for longer time periods (3 and 5 days at 37 degrees C). Size exclusion HPLC analysis indicated that rhNGF forms high molecular weight (HMW) complexes after long incubation periods. We confirmed that alpha(2)-macroglobulin (alpha(2)M) is the major plasma component that binds to rhNGF. Furthermore, this interaction was considerably increased by treatment of plasma with primary amines such as CH(3)NH(2). Changes in the pH environment did not affect the interaction of rhNGF with alpha(2)M. We also determined that the binding of rhNGF to CH(3)NH(2)-treated pure alpha(2)M or alpha(2)M present in human plasma substantially diminished its immunoreactivity and bioactivity detection. The interaction of rhNGF with activated alpha(2)M was reversed and inhibited by coincubation with dimethyl sulfoxide. Released rhNGF under these conditions was fully bioactive. (125)I-rhNGF also binds to alpha(2)M by forming similar (125)I-rhNGF/HMW complexes in plasma after i.v. administration in rats and mice. Sixty minutes after dosing in rats, most of the labeled material was in the form of a (125)I-rhNGF/HMW complex. These studies have provided a better understanding of the nature of the interactions of rhNGF with plasma components as well as methods to enhance, reverse, and inhibit these interactions.
Nguyen
C B
CB
Department of Pharmacokinetics-Metabolism, Genentech, Inc., South San Francisco, California 94080, USA.
Szönyi
E
E
Sadick
M D
MD
Hotaling
T E
TE
Mendoza-Ramirez
J L
JL
Escandón
E
E
D000818:Animals; D001798:Blood Proteins / Q000187:drug effects / Q000378:metabolism; D002850:Chromatography, Gel; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D051379:Mice; D009414:Nerve Growth Factors / Q000097:blood / Q000378:metabolism* / Q000494:pharmacology; D016716:PC12 Cells; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000097:blood / Q000378:metabolism / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000378:metabolism
D001798:Blood Proteins; D007457:Iodine Radioisotopes; D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D000511:alpha-Macroglobulins; D004121:Dimethyl Sulfoxide