alpha-chymotrypsin and ethylenediamine

Atom transfer radical polymerization (ATRP) has been successfully employed to obtain a new derivative of hyaluronic acid (HA) able to change its solubility as a function of external pH and then to be potentially useful for intestinal release of bioactive molecules, included enzymes and proteins. In particular, a macroinitiator has been prepared by linking 2-bromo-2-methypropionic acid (BMP) to the amino groups of ethylenediamino derivative of tetrabutyl ammonium salt of HA (HA-TBA-EDA). This macroinititor, named HA-TBA-EDA-BMP has been used for the ATRP of sodium methacrylate (MANa) using a complex of Cu(I) and 2,2'-bipyridyl (Byp) as a catalyst. The resulting copolymer, named HA-EDA-BMP-MANa, has been characterized by (1)H NMR and size exclusion chromatography (SEC) analyses. A turbidimetric analysis has showed its pH sensitive behavior, being insoluble in simulated gastric fluid but soluble when pH increases more than 2.5. To confirm the ability of HA-EDA-BMP-MANa in protecting peptides or proteins from denaturation in acidic medium, α-chymotrypsin has been chosen as a model of protein molecule and its activity has been evaluated after entrapment into HA-EDA-BMP-MANa chains and treatment under simulated gastric conditions. Finally, cell compatibility has been evaluated by performing a MTS assay on murine dermal fibroblasts cultured with HA-EDA-BMP-MANa solutions.

Topics: Administration, Oral; Animals; Cells, Cultured; Chromatography, Gel; Chymotrypsin; Ethylenediamines; Fibroblasts; Hyaluronic Acid; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Nephelometry and Turbidimetry; Polymerization; Quaternary Ammonium Compounds; Rats; Rats, Wistar

Theophylline-polyrotaxane conjugates were synthesized by coupling theophylline with alpha-cyclodextrins (alpha-CDs) in the polyrotaxane. The polyrotaxane is a molecular assembly in which many alpha-CDs are threaded onto a poly(ethylene glycol) (PEG) chain capped with L-phenylalanine (L-Phe). Theophylline-7-acetic acid was activated by coupling with 4-nitrophenol, and then ethylenediamine was allowed to react with the active ester in order to obtain N-aminoethyl-theophylline-7-acetoamide. This derivative was coupled with a 4-nitrophenyl chloroformate-activated polyrotaxane to obtain the theophylline-polyrotaxane conjugates. The conjugates formed a specific association under physiological conditions, depending upon interactions between the theophylline molecules and/or the terminal l-Phe moiety in the conjugates. In vitro degradation of the conjugates revealed that theophylline-immobilized alpha-CDs were completely released by hydrolysis of the terminal peptide linkage in the polyrotaxane. This result indicates that the association of the conjugates does not induce the steric hindrance but rather enhances the accessibility of enzymes to the terminal peptide linkages. It is suggested that our designed drug-polyrotaxane conjugates can release the drugs via the dissociation of the supramolecular structure without steric hindrance of enzymatic accessibility to the terminal peptide linkages.

Topics: alpha-Cyclodextrins; Bronchodilator Agents; Chemistry, Pharmaceutical; Chymotrypsin; Cyclodextrins; Delayed-Action Preparations; Drug Carriers; Ethylenediamines; Nitrophenols; Papain; Phenylalanine; Polyethylene Glycols; Polymers; Solubility; Theophylline