Identification and determination of GnRH antagonist gelling at injection site.

The purpose of this study was to first observe whether orntide, a GnRH antagonist, gels at the injection site and if so, to develop and validate an extraction method to quantitate the peptide amount as well as assess chemical stability in the gel. After subcutaneous injection of a large dose of orntide acetate solution, a white gel and local traumatized effect were observed at the injection site. Orntide remaining at the injection site was recovered by tissue excision, homogenization and tissue protein precipitation with perchloric acid and quantified by high performance liquid chromatography (HPLC) following separation on a C18 column. The standard curve was linear in the detection range and there was no interference from either blank tissue or excipients of the orntide formulation. The recovery from spiked tissue or that immediately following injection was in the range of 90-110%. MALDI-FT mass spectrometry (MS) of the peak fraction indicated that the orntide recovered from the injection site was in the intact form. The results showed that orntide solution, when injected at a large dose, formed a gel at the injection site. The gel delayed the release from the injection site and caused discernible tissue reaction.

A study of the antiresorptive activity of salmon calcitonin microspheres using cultured osteoclastic cells.

The purpose of this study was to evaluate salmon calcitonin (sCT) microspheres in vitro for their antiresorptive activity using cultured osteoclastic cells. The antiresorptive activity of sCT-loaded microspheres, prepared from a low molecular weight hydrophilic poly (lactide-co-glycolide) polymer (PLGA), was studied using bone marrow culture cells harvested from juvenile rats and cultured on slices of devitalized bone for up to 4 weeks. The resorptive activity of osteoclastic cells was quantified in terms of number and type of resorption pits and total area of resorption. Microspheres containing 5.1% sCT released 70% peptide in 2 weeks and 88% in 4 weeks. All sCT treatments inhibited total resorptive activity. A dose-dependent decrease in resorption was observed with sCT microspheres at 2 weeks. The high dose (10 mg of microspheres) produced a 99.5% decrease in resorption at 3 weeks, while the low dose (1 mg) produced an 80% reduction. Exposure of cultures to soluble sCT and sCT-loaded microspheres caused a decrease in the number of large pits, which were the predominant type formed in control cultures. Thus, this system could serve as an in vitro method to evaluate the antiresorptive effect of PLGA-sCT microspheres.