Tetsumi Irie

Protection Afforded by Maltosyl-β-cyclodextrin Against α-Chymotrypsin-Catalyzed Hydrolysis of a Luteinizing Hormone-Releasing Hormone Agonist, Buserelin Acetate

AbstractPurpose. The present study addresses how maltosyl-β-cyclodextrin (G2-β-CyD) impacts upon the α-chymotrypsin-catalyzed hydrolysis of buserelin acetate, an agonist of luteinizing hormone-releasing hormone with emphasis upon the direct effect of G2-β-CyD on the activity of the protease.nMethods. Kinetic and solubility studies were performed in isotonic phosphate buffer (pH 7.4) at 25°C and 37°C. The interaction of α-chymotrypsin with G2-β-CyD in the buffer solution was examined by differential scanning calorimetry.nResults. G2-β-CyD decelerated the α-chymotrypsin-catalyzed hydrolysis of buserelin acetate to give the 1−3 tripeptide and the 4−9 hexapeptide fragments. This deceleration can be explained solely by a nonproductive encounter between a complex of the substrate with G2-β-CyD and the protease at relatively low CyD concentrations, while the direct inhibitory effect of G2-β-CyD on the proteolytic activity made a considerable contribution to the overall deceleration of the hydrolysis at higher CyD concentrations. Calorimetric studies indicate the presence of intermediate states in the thermal unfolding of α-chymotrypsin, simultaneously accompanied by the autolysis. By contrast, a two-state thermal unfolding of α-chymotrypsin was observed in the presence of G2-β-CyD, suggesting reduced proteolytic activity upon binding to G2-β-CyD.nConclusions. These results suggest that G2-β-CyD at higher concentrations inhibits the proteolytic action of α-chymotrypsin through direct interaction with the protease, as well as through the formation of a non-productive complex with the substrate.

Controlled release of the LHRH agonist buserelin acetate from injectable suspensions containing triacetylated cyclodextrins in an oil vehicle

Abstract Heptakis (2,3,6-tri- O -acetyl)-β-cyclodextrin) ( TA ue5f8 β ue5f8 CyD ) and octakis (2,3,6-tri- O -acetyl- γ - cyclodextrin) (TAue5f8βue5f8CyD) were prepared for use as hydrophobic carriers of buserelin acetate (BLA), an agonist of luteinizing hormone-releasing hormone. The results from this study suggest that the in vitro release of BLA from the peanut oil suspension into the aqueous phase was retarded by complexation with TAue5f8CyDs. A single subcutaneous injection of the oily suspension of BLA containing TAue5f8βue5f8CyD and TAue5f8γue5f8CyD in rats led to retardation of plasma levels of BLA containing in 25- and 39-fold longer mean residence times, respectively, than that of BLA alone. Simultaneously with the suppression of plasma testosterone to castrate level, the pharmacological effectiveness of BLA continued for 1–2 weeks and significant weight reduction of genital organs was observed due to the antigonadal effect. Since TAue5f8βue5f8CyD and TAue5f8γue5f8CyD were degraded enzymatically in rat skin homogenates, both TAue5f8CyDs can be useful as bioabsorbable sustained-release carriers for hydrophilic peptides following the subcutaneous injection of an oily suspension.

Combined use of 2-hydroxypropyl-β-cyclodextrin and a lipophilic absorption enhancer in nasal delivery of the LHRH agonist, buserelin acetate, in rats

Abstract The potential use of 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) as a biocompatible solubilizer for the lipophilic absorption enhancer, 1-[2-(decylthio)ethyl] azacyclopentane-2-one (HPE-101), was assessed in the nasal absorption of an LHRH agonist, buserelin acetate (BLA), in rats. HP-β-CyD increased the solubility of HPE-101 in water through the formation of an inclusion complex, and hence facilitated the transfer of HPE-101 into the nasal mucosa. The nasal administration of the combined system of HPE-101 and HP-β-CyD led to a significant increase in plasma BLA levels in the rats. This enhancement effect was ascribable to the lowering of both enzymatic and permeation barriers to the peptide in the nasal mucosa. The release of membrane proteins and scanning electron microscopic observations indicated that local mucosal damage due to the coadministration of HPE-101 and HP-β-CyD may not be a serious obstacle to their safe use. The results obtained here suggest that HP-β-CyD potentiates the action of the lipophilic absorption enhancers at the appropriate combination ratio without causing severe local irritation, and that this combination may be useful for designing an aqueous nasal formulation of BLA.

Application of a β-cyclodextrin sulfate-immobilized precolumn to selective on-line enrichment and separation of heparin-binding proteins by column-switching high-performance liquid chromatography

Abstract A column-switching high-performance liquid chromatography (HPLC) system which consisted of a β-cyclodextrin (β-CD) sulfate-immobilized hydrophilic vinyl–polymer gel precolumn and a reversed-phase analytical column was developed for the selective on-line enrichment and separation of heparin-binding proteins. Of 15 proteins investigated, 10 proteins having heparin-binding activity were retained on the β-CD sulfate precolumn almost quantitatively, in contrast 5 proteins having no heparin-binding activity were not retained. Calibration graphs for basic fibroblast growth factor constructed at various sample volumes were nearly identical, indicating that the protein could be enriched by this system. The system was successfully used for the selective separation of lysozyme in egg white. The β-CD sulfate-immobilized precolumn showed no loss of analytical performance over 2 years during which about 400 samples were analysed.