The inhibitory effect of glutathione on buccal enzymatic degradation of therapeutic peptides (leu-enkephalin, luteinizing hormone-releasing hormone and pituitary adenylate cyclase activating peptide)

The purpose of this study was to evaluate the potential of glutathione (GSH) as an auxiliary agent for buccal drug delivery systems to protect therapeutic peptides from enzymatic degradation. Stability of the model peptide drugs leu-enkephalin (Leu-Enke), luteinizing hormone-releasing hormone (LHRH) and pituitary adenylate cyclase activating peptide (PACAP) in the absence and presence of GSH on degradation by buccal enzymes was investigated. Inhibition studies were performed with isolated aminopeptidase N representing the most abundant peptidase on the buccal mucosa and on excised intact buccal mucosa from pigs. Enzymatic degradation of peptides was quantified by HPLC. The absorption of GSH across freshly excised porcine buccal mucosa was studied using Ussing type diffusion chambers. The proteolytic activity of isolated aminopeptidase N was significantly inhibited by the addition of 2% (m/v) GSH. Without the addition of enzyme inhibitors Leu-Enke was completely degraded by aminopeptidase N, whereas 34.8 ± 27.2% of LHRH and 74.2 ± 12.0% of PACAP remained intact. In contrast, by the addition of 2% (m/v) GSH 33.2 ± 2.2% of Leu-Enke, 88.6 ± 12.8% of LHRH and 97.0 ± 6.2% of PACAP remained stable after 5 h of incubation. Additionally, it was shown that only 0.2 ± 0.0% of GSH was able to permeate the buccal mucosa within 5 h, demonstrating that GSH remains concentrated at the site of drug absorption. After incubation for 5 h on the buccal mucosa 0.8 ± 0.2% of Leu-Enke, 73.2 ± 6.6% of LHRH and 82.9 ± 8.8% of PACAP remained stable. In contrast, in the presence of 2% (m/v) GSH 103.1 ± 12.7% of Leu-Enke, 98.5% ± 5.4% of LHRH and 97.2 ± 4.2% of PACAP were stable. According to these results GSH seems to represent a promising inhibitor for the buccal administration of peptide drugs.