EFFECT OF PROTEASE INHIBITORS ON DEGRADATION OF RECOMBINANT HUMAN EPIDERMAL GROWTH-FACTOR IN SKIN TISSUE

Recombinant human epidermal growth factor (rhEGF), a polypeptide of 53 amino acid residues, is subject to degradation by numerous enzymes, e specially proteases, when it is applied on the skin for the treatment of open wound. Amastatin, aprotinin, bestatin, EDTA, EGTA, gabexate, g entamicin, leupeptin, and TPCK were investigated for the possible prot ease inhibitors, which may use to protect rhEGF from degradation by th e enzymes in the skin. Skin homogenates containing protease inhibitors and rhEGF were incubated at 37 degrees C for 30 minutes. After the re action was stopped with trifluoroacetic acid, the amount of rhEGF rema ining in the sample was determined with an HPLC method. The percentage s of rhEGF degraded, at the skin/PBS ratio of 0.25, in the mouse, rat, and human skin homogenate were 85%, 70%, and 46%, respectively. The d egree of degradation of rhEGF in the cytosolic fraction was higher tha n that in the membrane fraction and these enzyme reactions were comple ted in 30 minutes. Bestatin, EGTA, and TPCK showed significant inhibit ory effects on the degradation of rhEGF in the two fractions (p<0.05), while the other protease inhibitors had no significant inhibitory eff ects or, even resulted in deleterious effects. Therefore, the formulat ion containing one or several inhibitors among these effective inhibit ors would be a promising topical preparation of rhEGF for the treatmen t of open wound.