Characterization of human insulin microcrystals and their absorption enhancement by protease inhibitors in rat lungs

Pulmonary route appears to be an attractive alternative as a non-invasive systemic delivery for peptide and protein drugs. An appropriate formulation, however, is important for increasing their bioavailability in lung. In this study, the human insulin microcrystals were produced. The particle size analysis and scanning electron microscopy (SEM) showed that the microcrystals were uniform and had a monodispersed size distribution (mean diameter = 0.95 μm) for pulmonary delivery. The physicochemical properties of the microcrystals developed were similar to those of the commercial crystalline powder in powder X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analyses. The percentage of high molecular weight proteins (%HMWP), the percentage of other insulin related compounds (%OIRC) and the percentage of A-21 desamido insulin (%D) of the microcrystals were very low. In addition, the cytotoxicity of microcrystals developed and protease inhibitors (aprotinin, bacitracin and soybean-trypsin inhibitor) was investigated, and the enhancement of insulin absorption in the presence of these protease inhibitors at various concentrations was studied. The cell viability of A549 was over 80% at various concentrations of aprotinin and soybean-trypsin inhibitor, except for bacitracin (below 60%). The percent of decrease in blood glucose (D%) was 42.68 ± 1.62% after intratracheal instillation of insulin microcrystals (5 U/kg). An enhancement of hypoglycemic effect with protease inhibitors was also found. Soybean-trypsin inhibitor (48.86 ± 3.24% at 10 mg/ml; 55.78 ± 0.71% at 5 mg/ml; 51.49 ± 5.27% at 1 mg/ml) and aprotinin (52.57 ± 8.78% at 10 mg/ml; 51.97 ± 1.98% at 5 mg/ml; 56.90 ± 3.42% at 1 mg/ml) were effective for absorption enhancement. These findings suggest that the use of insulin microcrystals and protease inhibitors would be useful to improve the hypoglycemic effect in pulmonary route.