Experiment Study Of A Fusion Protein Of Peptide Stimulating Insulin Secretion And Human Serum Albumin On Primary Pharmacodynamics And Pharmacokinetics

PartⅠThe Pharmacodynamics of a Fusion Protein of Peptide Stimulating Insulin Secretion and Human Serum Albumin on C57BL/6J MiceObjective: To Study the effect of a Fusion Protein (Betalog) of Peptide stimulating insulin secretion and Human Serum Albumin to Glucose tolerance and plasma insulin of C57BL/6J Mice.Methods:①32 C57BL/6J mice were used to study acute glucose-lowering effect of Betalog.They were divided into 4 groups.Control group (Human Serum Albumin) or experimental groups (Betalog)were administered by IP injection. Injected dosage of Betalog were 50μg·kg-1, 150μg·kg-1, 450μg·kg-1, respectively. The blood samples were taken from the tail vein at before drug administration and 0,10,20,30,60,90,120min after glucose of dose 1.5mg·g-1 administration and blood glucose level was detected.②40 C57BL/6J mice were used to study long-lasting glucose-lowering effect. They were also divided into 4 groups. Control group (HSA) or experimental groups were administered by IP injection .Test was defferentiated by time of injection.③12 C57BL/6J Mice were used to study insulin-releasing effect. They were divided into Control(HSA) or experimental group. For plasma insulin determination, blood samples (100μL for each) was taken from the tail vein during the 20min and 60min time period after glucose administration.Results: In the study of acute study acute glucose-lowering effect, the difference of blood glucose between HSA group and Betalog group(middle dose) in every time point was significant. Blood glucose of Betalog group was lower than HSA group. The difference between the 3 Betalog groups (high dose, middle dose, low dose) was significant in the time point of 20 min and 30 min, and was no significant in the other time points. In the study of long-lasting glucose-lowering effect, the difference between every group was significant in the time points of 10min, 20 min, 30 min, 60 min, 90 min, 120 min. In the time points of 20 min and 30 min, the differences between HSA group and 1h group, 24h group, 48h group were significant. In the time point of 60 min, the differences between HSA group and 1h group were significant. In the time point of 90min, the differences between HSA group and 1h group, 48h group were significant, and no significant between HSA group and 24h group. In the study of insulin-releasing effect, the differences between HSA group and Betalog groups ( middle dose) were significant in every time point.Conclusion: Betalog can effectively control blood glucose level after the injection of glucose in C57BL/6J mice, the effect continued to 48h after dministration and was different with dose. Betalog can significantly increase insulin levels.PartⅡThe Pharmacodynamics of a Fusion Protein of Peptide Stimulating Insulin Secretion and Human Serum Albumin on GK ratsObjective: To Study the effect of Betalog to Glucose tolerance and plasma insulin of GK rat.Methods: 12 male GK rats were used to study glucose-lowering effect and plasma insulin of Betalog.They were divided into 2 groups.Control (HSA of 1mg?kg-1) or test (Betalog of 1mg?kg-1) were administered by IP injection. Blood samples were taken from the tail vein at 0.5h,2h,4h,8h,12h,24h,48h after glucose of 1.5mg/g administration. Plasma insulin determination was carried out at 2h,8h,48h after glucose administration. Results: The difference of blood glucose level between HSA group and Betalog group were significant in the time points of 8h, 12h, 24h, 48h, and no significant in the time points of 0.5h, 2h, 4h. The difference of blood insulin level between the two groups was significant in the time points of 8h and 48h, and no significant in the time point of 2h. Blood insulin level of Betalog group was higher than HSA group in the time points of 8h and 48h .Conclusion: Blood glucose level can be controlled and secretion of insulin can be promoted by Betalog within 48h after administration in GK rats. PartⅢPharmacokinetics,Biodistribution and Excretion of a Fusion Protein of Peptide stimulating insulin secretion and Human Serum AlbuminObjective: To investigate the preclinical pharmacokinetics, bioditribution and excretion of Betalog, to further evaluate the possibility for its clinical application.Methods:①The pharmacokinetics, bioditribution and excretion of Betalog were investigated with radiolabels combined with Trichloroacetic acid (TCA) precipitation .②Betalog were labeled with 125I by Chloramine-T (Ch-T) method. TCA precipitation method were used to determine their radiochemical purities and separate the fusion protein from its metabolite, and radioactivities of all samples were counted by a dual-window gamma counter.③SD rats were used in the trial. A group of SD rats were injected with 125I-Betalog of the dose 150μg·kg-1, 300μg·kg-1, 600μg·kg-1 respectively. After the injection, serial blood samples were withdrawn and radioactivities were determinated. The blood clearance rates of 125I-Betalog were illustrated as its concentration over time. The main parameters of pharmacokinetics were calculated by DAS2.0 software.④The biodistribution of 125I-Betalog in rats were determinated by detecting the radioactivity of their organs.⑤The urine and feces and bile of administrated rats were collected during a certain interval and radioactivities of aliquot urine , feces and bile were counted. The cumulated excretion fractions of rats were calculated.Results:①The radiochemical purities of 125I-Betalog were more than 90%. There is no change of its character after labeled with iodine-125.②The accuracy is 96%~112%. In the trial, radioactivity is linear to 125I-Betalog content (r>0.9979). The TCA fraction of 125I-Betalog is more than 90%, and that of 125I is only 5.10%±0.58%.③The concentration of 125I-Betalog in rats declined as a one-component model in three kinds of doses. The half-lives of 125I-Betalog in rats were 15.2h～16.7h(T1/2).The area under curve (AUC) is linear related with the injected doses.Most of 125I labeled fusion protein in murine's plasma existed in its intact form Betalog after 24h.④Thyroid, kidney and lung were the main retention organs in rats at the beginning of metabolism. There was only less 125I-Betalog in murine tissues except thyroid and kidney after 72h post-injection. It is important that murine ovary was also a major organ for deposition of Betalog, which indicated that its reproduction toxicity should be considered. 125I-Betalog cannot penetrate through blood brain barrier (BBB) of rat .⑤The kidneys were the major organ of elimination of Betalog. At 144h post-injection, 95% Betalog were eliminated through kidneys in rats. Most of 125I- Betalog in their urine existed in its metabolite. Only about 11.67% and 1.5% Betalog was eliminated through their bile and feces respectively.Conclusion: The concentration of 125I-Betalog in rats decreased as a one-component model. The main retention organs were thyroid, kidney and lung. Kidney is the major excretion organ of 125I-Betalog. Betalog had shown favorable pharmacokinetic properties. These investgation of preclinical pharmacokinetics is benefit to its advanced research and development.