Journal of Analytical & Bioanalytical Techniques
Open Access

Abstract

Objective : Th is thesis was designed to study the bioactivity in vitro and pharmacokinetics in vivo of hypoglycemic protein E2HSA Methods : Th e change of cAMP concentration in RINm-5F cells can refl ect the vitro bioactivity of Exendin-4 and E2HSA.Meanwhile the cAMP levels be measured by a cAMP immunoassay kit. A chemiluminescent enzyme immunoassay (CLEIA) for analyzing E2HSA and Exendin-4 in blood samples was established and validated. CLEIA was employed to study pharmacokinetics of E2HSA and Exendine-4. Furthermore, ammonium sulfate and CLEIA were applied in studying metabolic conversion and active styles of E2HSA in rhesus monkey. Results and Conclusions: In response to the GLP-1 molecule and its analogs (e.g. Exendin-4 and E2HSA) binding, GLP-1R directly stimulates adenyl cyclase, leading to a rise of intracellular cAMP. Th e change of cAMP concentration in RINm-5F cells refl ected the bioactivity of Exendin-4 and E2HSA. Th e cAMP levels were measured using a cAMP immunoassay kit. E2HSA and Exendin-4 exhibited a dose-dependent stimulation of cAMP accumulation in RINm-5F cells. E2HSA proved to retain only 1% activity of Exendin-4 in vitro . Following a single subcutaneous administration with a dosage 0.3 mg·kg -1 and 15 μg·kg -1 of E2HSA and Exendin-4 to rhesus monkey, the blood samples were collected at diff erent time points. All collected blood samples were centrifuged to obtain serum and the concentration of E2HSA and Exendin-4 in serum were determined by CLEIA method. Pharmacokinetic parameter calculations and pharmacokinetic modeling were carried out using the WinNonlin 5.2 Statistical soft ware. It indicated that the exposure level of E2HSA in serum was increased signifi cantly. Th ere was obviously prolongation of the t 1/2 for E2HSA comparing with Exendin-4. Proteins with diff erent molecular weight can be separated by ammonium sulfate precipitation according to their solubility. Results showed that 60% saturated ammonium sulfate can completely precipitate the E2HSA in the serum, while Exendin-4 remained in the supernatant. Ammonium sulfate precipitation 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.

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