| Exendin-4 was originally isolated from the salivary secretions of the lizard Heloderma suspectum. It is investigated for its potential to address important unmet medical needs of many people with type 2 diabetes. Exendin-4 is a 39-amino acid peptide that exhibits potent anti-diabetic, or glucoregulatory activities, which are similar to those of mammalian hormone GLP-1, mediated by the pancreatic GLP-1 receptor. Glucoregulatory actions of Exendin-4 include enhancing glucose- dependent insulin secretion, suppressing glucose-dependent inappropriately high glucagon secretion, slowing gastric emptying and reducing food intake. Moreover, Exendin-4 is a very potent agent to promoteβ–cell proliferation and islet neogenesis from precursor cells both in vitro and in vivo. Though the effects of Exendin-4 treatment on glucose control are likely due to several actions that are similar to those of GLP-1, the activity of Exendin-4 is 5000-fold greater than that of GLP-1 in controlling hyperglycemia, which may results from its resistance to degradation by DPP-Ⅳwhile GLP-1 is degraded by DPP-Ⅳwith a half-life of about 2 min in mammals.Peptide drugs are susceptible to protease degradation, which makes this type of drugs lose their potency easily. The glucoregulatory actions of Exendin-4, combined with better pharmacokinetic profiles over GLP-1 result in much higher potency in vivo compared with native GLP-1. Although Exendin-4 is less susceptible to degradation by neutral endopeptidase compared with GLP-1, it still falls short of being a long-term stable acting agent. One of the most commonly taken measures to limit the peptide degradation is amino acid substitution in the sites subjected to proteolysis. This method results in a minimal change in its secondary structure or binding potency, and the results have shown that such modifications do not impair interactions of a drug with its receptor in vitro. However, the proteolysis characteristics have not been well known. Therefore, the aim of our work was to find sites that are relatively susceptible to degradation and find new Exendin-4 analogues that are long-term stable.In this study the stability of Exendin-4 in human plasma was evaluated in vitro. Exendin-4 was incubated in plasma at 37℃, extracted and subsequently analyzed using high performance liquid chromatography (HPLC). Exendin-4 was slowly broken down in plasma. Its t1/2 is 9.57 h. The degradation products were identified by mass-spectrum (MS). According to natural sequence of exedin-4, we deduced that the cleavage were between the Thr5 and Phe6 bond, Phe6 and Thr7 bond, and Thr7 and Ser8 bond of the N-terminus region of the peptide, and the results of ESI-TOF- MS proved that our primary conclusion was correct.After that we designed seven Exendin-4 analogues (Analogue A--Analogue G) because of the cleavage information. All of the peptides were synthesized and purified by high performance liquid chromatography (HPLC).Determined by ESI-TOF- MS, their molecular mass were right. We used the method that is same to Exendin-4`s to study the stability of these Analogues in human plasma ,and we used insulinoma(RINm5F) constructing a cell proliferation model, we compared the bioactivity of Exendin-4 and Analogues in activating insulinoma(RINm5F) proliferation through MTT. According to these research find two Analogues Analogue C and Analogue E they almost have the same bioactivity to Exendin-4 and their half-life rise up 100% and 200% individually compared to Exendin-4,we also known the relativity of some amino acids to the stability and bioactivity of Exendin-4 ,that is very important to our future work. |
PDF Full Text Request