Effect of gelation on the chemical stability and conformation of leuprolide.

PURPOSE

The purpose of this study was to characterize the conformation, aggregation, and stability of leuprolide on gelation.

METHODS

Infrared spectra (FTIR) of leuprolide solutions and gels were collected in water, propylene glycol (PG), dimethyl sulfoxide (DMSO), and trifluoroethanol (TFE). Leuprolide solution and gel stability data were obtained by SEC and RP-HPLC.

RESULTS

Leuprolide was induced to gel with increasing peptide concentration, introduction of salts, and gentle agitation. Leuprolide dissolved in water (400 mg/ml) demonstrated FTIR spectra consisting of two major bands of equal intensity at 1615 cm(-1) and 1630 cm(-1), similar to inter- and intra-molecular beta-sheet structure in proteins. When samples were gently agitated for 24 hours at 25 degrees C, the formulation was observed to change from a viscous liquid to an opaque gel with a concomitant shift in infrared spectra from the equal intensity bands to mostly 1630 cm(-1), indicating a shift to a preferred beta-sheet structure. Incubation of leuprolide with 20-200 mM salts at 25 degrees C and 37 degrees C also produced gels ranging from clear to cloudy and stringy white precipitates. The gel and precipitate were marked by a shift of the predominant beta-sheet band to 1630 cm(-1) and 1615 cm(-1), respectively. Leuprolide was also observed to gel and/or precipitate in mixtures of water, PG or TFE, but not in DMSO.

CONCLUSIONS

Birefringence was noted in many of the firmer gels. Both solutions and gels demonstrated minimal dimer or trimer formation, with no larger order aggregates detected. The chemical stability profile of gelled leuprolide was similar to that of the non-gelled water formulation by RP-HPLC.