Bioactivity In Vitro And Pharmacokinetics In Vivo Of Hypoglycemic Protein E2HSA

Objective:This thesis was designed to study the bioactivity in vitro and pharmacokinetics in vivo of hypoglycemic protein E2HSA.Methods:The change of cAMP concentration in RINm-5F cells can reflect the vitro bioactivity of Exendin-4 and E2HSA.Meanwhile the cAMP levels can be measured by a cAMP immunoassay kit. A chemiluminescent enzyme immumoassay (CLEIA) for analyzing E2HSA and Exendin-4 in blood samples was established and validated. And this CLEIA was employed to study pharmacokinetics of E2HSA and Exendine-4. Furthermore, ammonium sulfate together with CLEIA were applied in studying metabolic conversion and active styles of E2HSA in rhesus monkey.Results and Conclusions:1. In response to the binding of GLP-1 molecule and its analogs (e.g. Exendin-4 and E2HSA), GLP-1R directly stimulates adenylate cyclase, leading to a rise of intracellular cAMP. So the change of cAMP concentration in RINm-5F cells can reflect the vitro bioactivity of Exendin-4, or E2HSA. E2HSA and Exendin-4 produced a dose-dependent stimulation of cAMP accumulation in RINm-5F cells. And the cAMP levels were measured with a cAMP immunoassay kit. Results showed that E2HSA with a linker sequence GGGGS had been proved to retain only 1% activity of Exendin-4 in vitro because of increased spatial blockade.2. A chemiluminescent enzyme immunoassay of speciality, sensitivity, precision and accuracy was established to detect the concentrations of E2HSA and Exendin-4. A good linearity was obtained over the serum concentration range 2-500 ng·mL^-1. The mean intra- and inter-precisions for E2HSA were 2.5%⁷.2% and 6.4%¹5.2%, respectively. The dilution integrity of the method was not found. The mean intra- and inter-precisions for Exendin-4 were 5.3 %-11.6% and 1.1 %-14.1 %, respectively.3. Following a single subcutaneouly administration with 0.3 mg·kg^-1 and 15 ug·kg^-1of E2HSA and Exendin-4 to rhesus monkey respectively, the blood samples were collected at different time points after dosing. All collected blood samples were centrifuged to obtain serum and the concentrations of E2HSA and Exendin-4 in serum were determined by CLEIA method described as above. Pharmacokinetic parameter calculations and pharmacokinetic modeling were carried out using the WinNonlin 5.2 Statistical software. It indicated that the exposure level of E2HSA in serum increased significantly. There was obviously prolongation of the t1/2 for E2HSA comparing with that of Exendin-4.4. Proteins with different molecular weight can be separated by ammonium sulfate precipitation according to their diverse solubility. Results showed that 60% saturated ammonium sulfate can completely precipitate the E2HSA in the sample, while Exendin-4 remained in the supernatant. Ammonium sulfate precipitation together with CLEIA can be used to detect the E2HSA metabolism features in vivo, which included the concentration of both E2HSA and its metabolite Exendin-4. The results revealed that following the administration of E2HSA to rhesus monkey, its metabolite Exendin-4 degraded gradually from the E2HSA till their concentrations reached a dynamic balance.