Preparation, characterization, and in vivo evaluation of salmon calcitonin microspheres

Abstract

Purpose. This study was done to prepare, characterize, and evaluate salmon calcitonin (sCT) microspheres (ms) in vivo using a low molecular weight, hydrophilic 50:50 poly (D,L-lactide-coglycolide) polymer (PLGA). Methods. sCT ms were prepared by a dispersion/solvent extraction/evaporation process and characterized for drug content, particle size, surface morphology, and structural integrity of encapsulated peptide. Peptide stability and binding to the polymer was studied in 0.1 M phosphate buffer (PB), pH 7.4, and 0.1 M acetate buffer (AB), pH 4.0. Serum sCT levels were monitored for 2 weeks after subcutaneous injection of sCT ms to rats. Results. sCT ms were essentially free of discernible surface pores with a particle size distribution in the range of 16 to 89 mm and mean particle size of 51 and 53 mm for 2 batches. Fourier Transform Matrix-assisted Laser Desorption mass spectrometry of the extracted peptide showed that the encapsulation process did not alter its chemical structure. The peptide was substantially more stable in AB than in PB. Peptide binding to the polymer was dependent on pH and was markedly higher in PB than in AB. In vivo study proved that elevated serum sCT levels could be sustained for at least 10 days after administration of sCT ms to rats at a dose of 1.0 mg/kg. Conclusions. It was demonstrated that sCT could be incorporated into polymeric ms prepared from a low molecular weight, hydrophilic PLGA using a dispersion technique without altering molecular structure. A 2 -week formulation was prepared at a dose of 1.0 mg/kg.

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

“…At the end of 2 weeks, addition of free sCT at the 2 concentrations along with blank ms did not decrease the total amount of resorption as much as that with the addition of free sCT at 10 and 250 nM. Previous adsorption studies of sCT to blank ms had shown that as much as 20 µg of sCT was bound to 1 mg of blank ms at pH 7.4 (pH of the culture medium) [ 10]. Therefore, at concentrations of 10 nM (0.03 µg sCT/mL) and 250 nM (0.86 µg sCT/mL) almost all of the sCT should be adsorbed to 1 mg of blank ms (at the end of week 2) leaving none or very little available for binding to cells.…”

“…Furthermore, free sCT, either alone or along with blank ms, was added to the cultures only twice a week for 4 weeks. Previous studies at pH 7.4, 37°C had shown that almost 37% of sCT degraded within 24 hours and 100% by 3 days [10]. Consequently, the frequency of addition of free sCT was less than optimal and could have resulted in the lack of dose response.…”

“…In vitro release was calculated by subtracting the amount of intact sCT recovered at each time point from the initial amount of intact sCT present in the microspheres before culture. Previous studies had demonstrated that sCT that was encapsulated into PLGA ms remained intact, retaining its molecular structure [10]. sCT concentration in the culture medium (supernatant) under the conditions of our study was not measured since such a measurement was very difficult (and probably far less accurate) because of the presence of bovine serum proteins (the medium contained 15% fetal bovine serum).…”

“…Salmon calcitonin microspheres were prepared by a dispersion method followed by solvent extraction and evaporation [ 10]. Briefly, a solution of peptide in methanol (CH 3 OH) was combined with a solution of PLGA in methylene chloride (CH 2 Cl 2 ) and stirred until clear, then slowly injected into a 1-L reactor with baffles (Ace Glass Inc, Vineland, NJ) containing the continuous phase (CP, 0.35% [wt/vol] solution of PVA, pH 7.2) and stirred at 3500 rpm with a Silverson L4R homogenizer (Silverson Machines Ltd, Buckinghamshire, England).…”

“…The HPLC method used in this study was very powerful in detecting intact sCT and separating it from degradation peaks. This method has been used previously to evaluate the stability of intact sCT at different pH [10].…”