Effect of salts on the structure of a potent analog of growth hormone releasing hormone as determined by optical spectroscopy.

Optical spectroscopic methods (circular dichroism, analytical ultracentrifugation, and static light scattering) were employed to study the solution behavior of an N-terminal-acylated 76-residue analog of growth hormone releasing hormone (GHRH). The GHRH analog had a 30% helical configuration in aqueous acidic solution, unlike other GHRH analogs that had a random coil configuration in aqueous solutions, and self-associated. High concentrations (7 M) of urea were required to obtain monomeric peptide, but such urea concentrations unfolded the peptide. Therefore, the folding and self-association were related events. The self-association and helix content were increased by the addition of various cations (e.g., Na+, Ca2+). In 7 M urea, when these cations were added, the peptide started to refold toward its aqueous conformation in a pH dependent manner; at low pH (2.5-3.5) the peptide folded to approximately 50% of the native configuration. At neutral pH (> 6) only small changes were seen when salts were added. When CaCl2 was added to a solution containing 7 M urea at pH 2.5, the self-association of the peptide increased with the concentration of CaCl2. Therefore, the salt dependent self-association observed in aqueous solvents was present in 7 M urea. Residues 45-76 in this GHRH analog, which are not present outside the hypothalamus, are probably needed to interact with a folding chaperone in vivo and provide stability for successful membrane transport and maintenance of biological activity.

Effect of impurities on estradiol crystallization in a sustained-release implant.

During the development of a silicone rubber implant for the delivery of estradiol 17-beta some batches of implants made from a certain lot of commercial estradiol inexplicably developed surface crystals of estradiol after several days of storage. An impurity profile was obtained for 28 lots of estradiol by a newly developed HPLC method. One or more impurities may have had a role in the spontaneous crystal growth on the surface of the implants, because the one lot of estradiol that initially had surface crystals on aging produced acceptable implants after multiple recrystallizations. Attempts to isolate suspected impurities for characterization were unsuccessful. During the manufacture of the implants, temperatures sufficient to melt the estradiol (mp, 173-179 degrees C) were used. It was expected that, upon implant cooling, melted impure estradiol would form a thermodynamically more active (i.e., noncrystalline) physical form. This metastable form could have migrated to the implant surface, where ambient conditions favored crystallization. Because melted estradiol of a higher purity tended to crystallize more readily, it was less likely to form a glass upon cooling. The phenomenon of surface crystallization was limited to one lot of estradiol with the highest level of impurities. Data from differential scanning calorimetry studies supported this conclusion.