A Novel Glucagon-like Peptide-1 Analog And Its Long-acting Technology

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that is released from intestinal L-cells into the circulation in response to orally ingested nutrients. The multifaceted physiological actions of GLP-1 include stimulation of insulin gene expression, stimulation of insulin secretion, tropic effects on beta-cells, inhibition of glucagon secretion, promotion of satiety, inhibition of food intake, and reduction of gastric emptying. all of which contribute to normalizing elevated glucose levels and make GLP-1 attractive as a candidate for the treatment of type 2 diabetes mellitus. However, its rapid degradation by dipeptidyl-peptidase IV (DPP-IV) and its renal clearance have limited the use of the native peptide in clinical settings. Consequently. multiple efforts that focus on the design of novel DPP-IV-resistant GLP-1 analogs or sustained-release formulation of GLP-1 are being pursued.In this study, by exploiting the affinity of cellulose binding domain (CBD) binding for cellulose, we creatively activated and connected biomatrices of micron-sized MCMS with SA. In addition, using biotinylated Oligo(dT) a one-step isolation method of mRNA from cells or tissues was developed and achieved. On this basis we successfully isolated mRNA from rat islet tissue and the gene encoding GLP-1R was amplified. The recombinant plasmid pcDNA3.1(+)/GLP-1R was conducted and transfected into a CHO/EGFP cell line. After resistance screening, flow cytometry sorting and functional analysis, the resulting stable recombinant CHO/EGFP/GLP-1R cell line was developed. This functional reporter gene assay system can be used to identify and for the quantitative analysis of in vitro activity of GLP-1R agonists. The results laid the approach foundation for the research and development on the long-acting GLP-1 analogs.Then we reported a novel GLP-1 analog designated KGLP-1, which was designed with an extra Lys in front of the N-terminal His of the native GLP-1. By using the above constructed CHO/EGFP/GLP-1R cells and DPP-IV extract, we determined its plasma stability and biological activity in vitro. The results suggested that KGLP-1 activated GLP-1R in a dose-dependent manner. The EC50 for KGLP-1 was 2.44 nmol/L, which was higher than that of native GLP-1 (0.83 nmol/L). But KGLP-1 revealed a marked enzymatic resistance to DPP-IV. After 12 h incubation with the DPP-IV extract at 37℃,80% of active KGLP-1 still remained. Further animal experiments confirmed that KGLP-1 reduced the blood glucose levels after intraperitoneal administration to KM mice in a dose-dependent manner. At the same moderate dose, the glucose-lowering ability of KGLP-1 was comparable with native GLP-1. Although native GLP-1 immediately showed significant glucose-lowering and insulin-secreting effects after administration in normal or diabetic model mice, the effect disappeared 1h after administration. Compared with native GLP-1, the significant blood glucose-lowering and insulinotropic activity of KGLP-1 lasted for at least 2 h after administration, demonstrating that KGLP-1 had a longer biological action.To delay the rapid renal clearance, we prepared a fusion protein of KGLP-1 with HSA. And the therapeutic potential of KGLP-1/HSA, with respect to its biological activity and metabolic stability in vitro and its in vivo antihyperglycemic and insulinotropic effects were investigated. It was found in vitro study that KGLP-1/HSA activated GLP-1R in a dose-dependent manner that paralleled GLP-1 and KGLP-1. The EC 50 value for KGLP-1/HSA was calculated to be 314 nmol/L, while KGLP-1 and native GLP-1, exhibited much smaller EC50 values. But KGLP-1/HSA revealed significant enzymatic resistance, and it still stimulated EGFP expression at 84.0% activity after incubation for 12 h. The in vivo test confirmed that although KGLP-1/HSA did not show marked antihyperglycemic and insulinotropic effects until 1 h after administration, the glucose-lowering and insulin-releasing effect of KGLP-1/HSA was still evident up to 8 h after administration. In contrast, although the blood glucose and insulin responses for both GLP-1 and KGLP-1 showed significant glucose-lowering and insulin-releasing effects immediately after administration, the effects of GLP-1 or KGLP-1 at a dose of 0.1μmol/kg disappeared 1-2 h after administration. Overall, The fusion protein KGLP-1/HSA maintained the glucose-lowering and insulinotropic action of native GLP-1, together with increased resistance to DPP-IV mediated inactivation and prolonged duration of action. Meanwhile, we made active exploration on injectable sustained-release formulation of KGLP-1. Firstly, using W/O/W emulsion method, we prepared KGLP-1-loaded PLA MS and PLGA MS, and examined the type of polymer, polymer average molecular weight on morphology, average particle size, encapsulation efficiency and release characteristics in vitro of MS. The results showed that their encapsulation efficiencies were all below 40%, which would cause big losses of drugs. Then KGLP-1 entrapped in long-acting injectable PLGA MS were prepared using the S/O/O solvent extraction method. The surface morphology of MS was examined by SEM and micrographs showed that the MS had a regular spherical shape with a relatively smooth surface and a homogeneous size. The particle diameter was 33.5μm, and the encapsulation efficiency was 85.5%. The MS released the entrapped peptide with a release rate of approximately 15.3% peptide during the first 6 h, followed by the release of 54.2% of the peptide over 2 weeks. Animal studies using alloxan-induced diabetic rats have been performed and the results suggested that the continuous release of KGLP-1 from the subcutaneously injected KGLP-1-loaded PLGA MS had sustained glucose-lowering and insulinotropic effects. The KGLP-1 MS formulation achieved controlled release in vivo for 10 days and exhibited sustained long-term pharmacological efficacy to maintain stable blood glucose level in a diabetic model.In summary, the thesis made use of self-developed SA-CBD-MCMS, isolated the mRNA of rat islet tissue and cloned the gene coding rat GLP-1R. Then the functional cell assay system CHO/EGFP/GLP-1R containing EGFP reporter gene was successfully developed for the analysis of in vitro activity of GLP-1R agonists. Using this cell line, the plasma stability and biological activity in vitro of a novel GLP-1 analogue (KGLP-1) and its fusion protein with HSA (KGLP-1/HSA) were examined. In combination with tests in KM mice and alloxan-induced diabetic animals, we confirmed that KGLP-1 is an effective GLP-1 analog with marked resistance to DPP-IV degradation and retained bioactivity that is normally associated with native GLP-1 both in vitro and in vivo. And the fusion protein of KGLP-1 with HSA maintained the glucose-lowering and insulinotropic action of native GLP-1, together with increased resistance to DPP-Ⅳmediated inactivation and prolonged duration of action. Furthermore, KGLP-1-loaded PLGA MS were prepared using the S/O/O solvent extraction method and, after a single injection in diabetic rats, the effects of KGLP-1 on glucose control were sustained for 10 days. It laid the well foundation for the development of long-acting GLP-1 drugs and their sustained-release formulations for the treatment of 2DM.