Stabilization of somatropin by heparin.
Somatropin, human growth hormone (hGH), is an unstable protein, posing challenging problems for its formulation and long-term stability. Since hGH formed insoluble adducts with heparin our aim was to evaluate heparin as a stabilizing agent for the drug. These adducts were characterized by particle diameter, tertiary structure variations and release studies. Studies were also carried out to determine the stability of hGH in the presence and absence of heparin by an interfacial denaturation method and real-time stability studies by measuring hGH activity and particle diameter. Moreover, biological activity of hGH and hGH/UH (unfractionated heparin) adducts was identified by daily subcutaneous injections to hypophysectomized rats. There was a decrease in mean hydrodynamic particle diameter of hGH/UH adducts with increased pH (54.4 to 12.2 nm from pH 3 to pH 7) indicating that the adducts were either dissociating or dissolving at high pH. Furthermore, second-derivative spectroscopy indicated that complexation of hGH with heparin did not cause a major disruption in the tertiary structure of hGH but decreased the hydrophilic environment around the tyrosine residues. Release of hGH from hGH/UH adducts was pH and ionic strength dependent with the highest release at pH 8 (93%) and lowest release at pH 3 (0%) over the first hour. Interfacial denaturation methods indicated that vortex agitation over 120 s resulted in no change in the optical density of hGH/UH adducts compared with a substantial increase for hGH alone at pH 6.8. Real-time stability studies over 93 days demonstrated that hGH/UH adducts at both pH 3 and 7 with an excess of heparin produced the highest percent of active hGH remaining in the solution at 4 degrees C and 37 degrees C. The higher stability of hGH/UH adducts with excess heparin compared with the stoichiometric ratio was also confirmed by particle size measurements during storage. The biological activity of these adducts was comparable with hGH alone by weight-gain studies in hypophysectomized rats. The findings suggest the value of using hGH/heparin adducts to stabilize the protein.
Zamiri
Camellia
C
Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA.
Groves
Michael J
MJ
D000818:Animals; D004355:Drug Stability; D005260:Female; D006493:Heparin / Q000737:chemistry* / Q000494:pharmacology; D019382:Human Growth Hormone / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007016:Hypophysectomy; D010316:Particle Size; D011489:Protein Denaturation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013696:Temperature; D015430:Weight Gain / Q000187:drug effects*
D019382:Human Growth Hormone; D006493:Heparin