Establishment Of Verification Method About GLP-1 Similar Drugs Activity In Vitro And Screening Of Natural Active Products

Glucagon-like Peptide-1 is mainly secreted by epidermal L cell at small intestine,which can be used for the treatment of type 2 diabetes on clinic.At present,the assay methods of activity in vitro for this kind of drugs are including luminescent bead interactions to detect cAMP’s quantity and Promega kit to test residual ATP’s quantity.The determination results of these methods are greatly influenced by the environmental factors,and other disadvantages such as short half-life,high cost and poor stability appeared in application.Therefore it needs to develop a new verification method in vitro for GLP-1 similar drugs with features as easy operation,high sensitivity and stability.A stable GFP labeled cell lines built previously was used as a cell model,with a high content screening equipment,a high specificity,rapid and sensitive verification method in vitro was established in this study.The linear regression,accuracy,precision(routine)and specificity of this new method were investigated intensively.The linear regression of this new method within the scope of the standard±50%showed excellent performance with the R2≥0.98;the accuracy is also very high,at 50%,80%,100%,120%,150%,the biases of five levels between the measured value with the theoretical value is less than 8%and the precision is satisfactory,the determination results of RSD is less than 10%,the accuracy and precision were significantly superior to the standard of 20%in 2015 edition of "Chinese Pharmacopoeia".This method also showed a very good specificity,this model reacted only with the GLP-1 similar drugs and showed dose-response relationship among six kinds of drugs related to regulate insulin secretion such as Glucagon,Liraglutide,Growth hormone,Essex et al.Results also showed that the data of the new method can keep stable for nearly 100 h,which was better than the method of Promega kit that is routinely used domestic and only keep stable no more than 1h.The newly constructed cell line can be used continuously more than 47 generations,and cell line used in Promega kit only can be used to 33 generations.For a 96-well plate,each test only costs RMB 200,which is significantly lower than the cost of RMB 1,000 for Promega method.Totally,12 batches of various GLP-1 similar drugs in bulk or injection formula from 3 different manufacturers were tested by this method,and the results are validated,which confirmed the reliability of this method.Quantitive assay of cAMP generated in downstream signal molecule of the constructed cell line were performed and the results showed that although the C end of recombinant receptors was labeled with GFP,but it did not interfere with the information transfer of downstream signal.Finally,a primarily platform to screening GLP-1 similar natural active ingredients from Chinese herbal medicine with our constructed cell line was established,and almost 100 kinds of natural products including Notoginseng,Radix astragali,Rhizoma coptidis and Gardenia were screened under this platform.In conclusion,a novel verification method for biological activity in vitro of GLP-1 similar drugs was established successfully in this paper,the method presents some advantages about strong specificity,high stability,simple operation and low cost,which is suitable for testing in vitro biological activity of all GLP-1 similar drugs.And the cell screening method of natural active products similar to GLP-1 was initially established as well,the preliminary results show that the Notoginseng and Gastrodia Elata may contain the active ingredients of agonist,which provided the practical foundation for the future development of oral small molecule drugs for the treatment of type 2 diabetes.