Establishment of an in vitro method to measure the activity of human amylin analogue pramlintide

The method for measuring the activity of human amylin analogue pramlintide in vitro was established，which could provide a reliable cell model and test method for the quality control of pramlintide and its preparations.The L6 cell line was cultured and differentiated into mature skeletal muscle cell with obvious myotubes-like structure.The effect of pramlintide on glucose uptake of differentiated L6 cell cultured in high glucose medium was detected by the method of glucose oxidase-peroxidase (GOD-POD).The results showed that,within the concentration range of 1×10^-9-1×10^-5 mol/L,pramlintide could significantly increase the glucose uptake of differentiated L6 cells,and could reach the maximum at 16 hour.A good linear relationship between the negative logarithm of the concentrations of pramlitide and the incremental amount of glucose uptake (y=-4.750x+59.544，R²=0.991 9) was observed.The activity of commercially available drug pramlintide acetate injection was detected by the established method.The incremental glucose uptake (%) of differentiated L6 cells after incubation in 1×10^-6,1×10^-7 and 1×10^-8 mol/L pramlintide injection for 16 hours were (61.89±9.54)%,(43.68±10.06)% and (33.30±13.03)%,respectively (P <0.01);Combined administration of pramlintide injection and insulin could further promote the glucose uptake of differentiated L6 cells compared to the insulin group (P<0.01),and it was consistent with the clinical results of pramlintide injection.Through this study,we provide a stable and reliable cell model for the activity detection of pramlintide,which can replace the existing models.