A novelin vitro release technique for peptide-containing biodegradable microspheres

Abstract

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.

IVIVC from Long Acting Olanzapine Microspheres

“…Figure 1 shows the in vitro release from Olanzapine microspheres, measured using the modified dialysis method [26]. An initial burst of almost 10% (day 1) was observed from Formulations A and B after which drug release from these batches was very similar through day 30.…”

“…In vitro release ( n = 3) was performed using a modified dialysis method [26]. Briefly, Olanzapine microspheres were accurately weighed and placed in a 7 mL dialysis tube (Tube-O-Dilalyzer, MWCO 300,000 Da) filled with 5 mL 0.5 M PBS (phosphate buffered saline), pH 7.4, containing 0.05% Tween 80 and 0.1% sodium azide (inner media), which in turn was placed in a 50 mL tube containing 40 mL of the same release medium (outer media).…”

“…As drug is released from the dosage form and into the inner media, it diffuses through the dialysis membrane into the outer sink. This scenario mimics in vivo conditions where the long acting injectable is immobilized upon subcutaneous or intramuscular administration and surrounded by a stagnant layer leading to slow drug diffusion due to non-maintenance of sink conditions [25, 26]. Several adaptations of the dialysis based techniques (e.g., dialysis bags) have been evaluated, including the modified dialysis method that was successfully used to study drug release from large molecules like peptides [27].…”

Engineering cells with intracellular agent–loaded microparticles to control cell phenotype

“…Release of small molecules from microparticles in vitro can be determined by using dialysis as previously described 33,34 . Although release kinetics of drug from particles internalized within cells is influenced by the intracellular environment (e.g., the presence of enzymes or altered pH), the simplified dialysis system is an important tool that can provide insight into the release kinetics and it should highlight relevant trends and pitfalls including excessive burst release and incomplete release.…”

In-vitro dissolution methods for controlled release parenterals and their applicability to drug-eluting stent testing

“…Nevertheless, there are very few reports on such test systems for non‐oral dosage forms. IVIVC at different correlation levels has been established for some parenteral products [95–101] . However, most IVIVCs have been achieved without employing physiologically relevant testing conditions in the in‐vitro system [23] .…”