A novel in vitro release technique for peptide containing biodegradable microspheres

The purpose of this study was to develop and evaluate a dialysis in vitro release technique for peptide-containing poly(d, l-lactide-co-glycolide) (PLGA) microspheres (ms) that would correlate with in vivo data. Using a luteinizing hormone- releasing hormone analogue (LHRH), Orntide acetate, solubility and stability were determined in 0.1 M phosphate buffer (PB), pH 7.4, and in 0.1 M acetate buffer (AB), pH 4.0, with high-performance liquid chromotography (HPLC), and peptide permeability through a dialysis membrane (molecular weight cut-off 300,000) was determined. Orntide ms were prepared by a dispersion/solvent extraction/evaporation method and characterized for drug content (HPLC), particle size distribution (laser diffraction method), and surface morphology (scanning electron microscopy). In vitro release was studied in PB using a conventional extraction method and with a new dialysis method in AB. Gravimetric analyses of polymer mass loss and matrix hydration, and peptide adsorption to blank PLGA ms (50:50, M(w) 28 022) were carried out in PB and AB upon incubation at 37 degrees C. Serum Orntide and testosterone levels in rats after administration of Orntide ms were determined by radioimmunoassay. Orntide acetate solubility was influenced by pH; approximately 2.3 mg/mL dissolved in PB and > 18 mg/mL in AB. Stability was pH- and temperature-dependent. The peptide was very stable at pH 4.0, 4 degrees C, but degraded rapidly at pH 7.4, 37 degrees C. Peptide permeability through the dialysis membrane was accelerated by agitation and >95% equilibrium was reached within 48 hours. The overall release rate was higher with the dialysis method. Mass loss of the Orntide ms was faster in AB (50% loss in 3 weeks; 95% in 35 days) than in PB (65% in 35 days). In contrast, hydration after 35 days was 4-fold higher in PB. The nonspecific adsorption to blank ms was greater in PB (128 microg Orntide/10 mg PLGA) compared with AB (< 5 microg Orntide/ 10 mg PLGA). Administration of 30-day Orntide PLGA ms to rats resulted in an initial serum Orntide level of 21 ng/mL after 6 hours and a Cmax of 87 ng/mL after 6 days. Testosterone levels were suppressed immediately after ms administration (3 mg Orntide /Kg) from 5.2 ng/mL to 0.3 ng/mL (after 24 hours) and remained suppressed for 38 days. Orntide acetate solubility and degradation kinetics were markedly influenced by pH of the buffer systems and mass loss; matrix hydration, as well as the nonspecific adsorption to blank ms, was pH-dependent. The in vitro release profile obtained with the dialysis method in AB correlated well with the in vivo data, thereby providing a more reliable prediction of in vivo performance.