Recombinant Lactococcus lactis: A novel tool for oral delivery of glucagon like peptide-1 for treatment of type-2 diabetes.

摘要

Glucagon like peptide-1 (GLP-1) is a promising therapeutic agent for the treatment of type-2 diabetes mellitus (T2DM). However, its short in vivo half life (2-5 min) demands for frequent injections or infusions that limit the patient's compliance. As for almost other protein/peptide drugs, the oral delivery of GLP-1 has been being a great challenge because of the extensive degradation and limited absorption. The objective of this project was to develop a genetically modified normal flora for sustained oral delivery of GLP-1. To attain the objective, GLP-1 (7-36) c-DNA was designed and synthesized and fused to the two restriction sites SalI and BamHI in the expression cassette of pUB1000 downstream to usp45 secretion signal peptide to get the recombinant plasmid pUBGLP-1. Finally, pUBGLP-1 was transformed into the competent L. lactis cells by electroporation. The transformed L. lactis (LL-pUBGLP-1) was characterized by ELISA, PCR and restriction enzyme digestion.;The bioactivity of the r-GLP-1 secreted by LL-pUBGLP-1 was investigated by its insulinotropic activity on HIT-T15 cells. Transport of r-GLP-1 across MDCK cell monolayer when delivered by LL-pUBGLP-1 was studied. The therapeutic effect of the LL-pUBGLP-1 after oral administration was investigated in ZDF rats as well.;The results show that that 104 cfu/mL of the LL-pUBGLP-1 was able to secrete 60 pM of r-GLP-1 in 12-hrs in M17. LL-pUBGLP-1 stimulated the insulin secretion by the HIT-T15 cells, which was 2.9 times (p 0.05) of that by the blank M17 with or without the untransformed L. lactis (LL-pUB1000). The insulinotropic activity of the r-GLP-1 by the LL-pUBGLP-1 was glucose-dependent, a characteristic of the native GLP-1. The transport of r-GLP-1 by LL-pUBGLP-1 across the MDCK monolayer was almost 8 times greater than that by the free solution form (p 0.01) with or without the untransformed L. lactis, demonstrating a great advantage of this living delivery system.;Oral administration of 109 cfu of LL-pUBGLP-1 in ZDF rats showed a significant decrease (10-20%, p 0.05) in the blood glucose levels from the baseline during 2-11-hrs post-dosing, and a significant increase of insulin secretion. In contrast, the oral administration of 109 LL-pUB1000 with 1000 nanomoles/kg of GLP-1 (control group) caused an increase of 20-30% in the blood glucose levels from the baseline throughout the course of the study and no change of insulin level. The insulin AUC (0.11 hr) of the LL-pUBGLP-1 group was 2.5 times greater than that of the control group (p 0.01). In conclusion, the present study demonstrates that the LL-pUBGLP-1 can be used as efficient and sustained oral delivery system for GLP-1 for the treatment of type-2 diabetes. Furthermore, gene transformed normal flora can be utilized as a feasible approach for non-invasive delivery for protein/peptide drugs.