Screening Of Orally Deliverable Glucagon-like Peptide-1 Analogs And Their Applications

Diabetes mellitus is a major contributor to the global disease burden and is currently experiencing a dramatic rise in prevalence. In 1985 an estimated 30 million people worldwide had diabetes, and in 2000, the figure had risen to over 150 million. This figure is expected to rise to almost 350 million by 2025.Type 2 diabetes will account for most of the projected increase, which reflects not only the demographics of an aging population, but also increasing numbers of overweight and obese people. Randomized, controlled trials have provided compelling evidence that achieving strict glycaemic control can reduce the long-term complications of diabetes. Despite this knowledge, achievement of glycaemic control in clinical practice is far from satisfactory, many patients failing to achieve long-term glycaemic control even when receiving optimal treatment with current anti-diabetes medications. Several reasons may account for this failure, including difficulty in implementing and maintaining adequate lifestyle modifications, poor compliance with therapy, limited efficacy and/or side effects of current antidiabetes therapy, and the underlying disease pathophysiology.There is a need for the continued development of new treatments in an attempt to gain greater efficacy and an improved tolerability profile. Glucagon-like peptide-(GLP-1) attracts a lot interests for scientists. It exists in two forms, a 30- and a 31-amino acid peptide, and is secreted by the L-cells of the large bowel and the last section of the small bowel. In theory, there are several attractive features of GLP-1 that would make it a particularly effective treatment for diabetes. The fact that GLP-1 induces both secretion and production of insulin, and that its activities are mainly glucose dependent, indicates that GLP-1 may have unique advantages over sulfonylurea drugs in the treatment of diabetes. Additionally, GLP-1 lowers glucagon concentrations, slows gastric emptying, reduces food intake, and may enhance insulin sensitivity and stimulateβ-cell neogenesis. Therefore, in many aspects, GLP-1 opposes the diabetic phenotype. Recently, a GLP-1 analog called Byetta has been approved in European and American.Unfortunately, in vivo half-life time of GLP-1 is only 2-3 minutes, which limits its application. It's quickly degraded when released in serum by so named dipeptidyl peptidaseâ…£(DPPâ…£) at the second amino acid residue Ala. In this thesis, Ala was substituted by Ser; meanwhile, Lys²⁶ and Lys³⁴ were substituted by Gln and Asp, respectively, made the GLP-1 analog named DTrGLP-1 resistant to both DPPâ…£and trypsin. The gene was cloned into E. coli vector pET-32a(+) and genetic strain BL21(DE3)/pET-32a-DTrGLP-1 was achieved. Expression and purification conditions were optimized and fusion protein was achieved. A dose of 36nmol·L^-1·kg^-1 was fused in mice via intragastric administration, and the serum glucose level of GLP-1 analog fused group was significantly decreased compared with the saline group until 90min after administration.To improve half-life time of GLP-1 analog, further experimsnts were performed. Arg³⁶ was substituted with Ala, Gly,Leu, Asp, Glu, Try, Gln or His, respectively and were named A³⁶-DTrGLP-1,G³⁶-DTrGLP-1,L³⁶-DTrGLP-1,D³⁶-DTrGLP-1, E³⁶-DTrGLP-1,T³⁶-DTrGLP-1,Q³⁶-DTrGLP-1 or H³⁶-DTrGLP-1.Constructions were achieved and mutant peptides were expressed in E. coli by auto-induction methods and were purified with affinity chromatography. Activities of trypsin digested GLP-1 analog fusion proteins mixture were performed on MIN6 cells, result showed that all analogs stimulated insulin secretion with slightly different levels. Oral administration of fusion GLP-1 analogs in mice showed T³⁶-DTrGLP-1, D³⁶-DTrGLP-1,G³⁶-DTrGLP-1 and A³⁶-DTrGLP-1 had significantly lowered serum glucose levels, whilst anthers didn't. Then lactic acid bacterium expression system and plant expression system were considered as better expression systems as they can be taken directly without any purification.NICE system was chosen as lactic acid bacterium expression system and codon optimized DTrGLP-1 gene was cloned into pNZ8149 and then transformed into NZ3900.With induction of nisin, DTrGLP-1 peptide was detected by SDS-PAGE and Western blot. Oral administration of whole NZ3900/pNZ8149-LAB-DTrGLP-1 reduced mice weight gain compared with NZ3900 group after 3 weeks, showing that DTrGLP-1 in L.lactis has bioactivities, though lowering serum glucose level effect wasn't detected. Further more, GLP-1 analogs were cloned into PVX/GFP viral expression system and were expressed in tomato, which has commercial advantages. PVX-X³⁶-DTrGLP-1-GFP (X represents A, G,D or T) were obtained and were transcript in vitro. Infectious recombinant viral RNAs were inoculated onto N. benthemiana.10 days posting inoculation, GLP-1 analog-GFP fusion proteins were observed and detected by western blot. Viral particles containing GLP-1 analog genes were inoculated onto cherry tomato or AC tomato and expression of GLP-1 analogs were detected by RT-PCR 2 weeks after inoculation. Tomato fruit was grand and administrated with a dose of 0.2mg/kg to mice. Results showed that serum glucose level of mice given T³⁶-DTrGLP-1-GFP and D³⁶-DTrGLP-1-GFP was significantly decreased lasting at least 60 minutes (210 minutes for G³⁶-DTrGLP-1-GFP group).In conclusion, oral deliverable long-term GLP-1 analogs were successfully achieved by PCR site-direct mutation. Lactic acid bacterium and tomatoes which expressed GLP-1 analog fusion proteins had GLP-1 bioactivities, which may probably be made commercial available.