Preliminary Pharmacological Study Of Recombinant Human Glucagon-like Peptide-2? And Its Protective Effect On Intestinal Injury

Glucagon-like peptide-2(GLP-2)is a proglucagon-derived peptide hormons liberated from proglucagon in enteroendocrine L cells in intestines and encoded by Proglucagon(PG)gene.It has been reported that GLP-2 has the function of enhancing intestinal barrier function,increasing blood flow and crypt-villus height,promoting the survival of crypt cell and has obvious therapeutic effect on short bowel syndrome patients.However,the therapeutic effect and mechanism of GLP-2 on intestinal lesions is largely unknown.Currently,the GLP-2 and its analogs used in clinical research are usually synthesized by chemical methods.There are still some problems,such as complex preparation process,long cycle time,high cost and short half-life.All these factors have greatly limited the application of GLP-2 in clinical research.Therefore,it is valuable to develop an effective GLP-2 with longer half-life and further study their therapeutic effects and mechanism in intestinal injury diseases.Based on the optimization of the GLP-2 structure,our group has found a new GLP-2 dimeric analog which was designed and prepared using genetic engineering technology.It was named GLP-2(2).The gene engineering expression product has obtained China National invention patent(CN103159848A).We conducted a series of studies on the biological functions of GLP-2(2).The results of the experiments showed that:(1)CCK-8 experiments showed that GLP-2(2)had similar pro-proliferative effects to chemically synthesized GLP-2 monomers.(2)Animal experiments and HE staining revealed that both GLP-2(2)and GLP-2 can promote the intestinal weight,length and villus height in normal mice.(3)Injection of BrdU and its specific antibody labeling showed that both GLP-2(2)and GLP-2 could significantly promote the proliferation of intestinal cells.The therapeutic effect of GLP-2(2)was better than GLP-2.The above results suggest that recombinant GLP-2(2)not only has intestinal protective effect,but also has better effect than chemically synthesized GLP-2 monomer.Furthermore,does GLP-2(2)have better repair effect than monomer for the pathological state of intestinal injury? What is the mechanism? Then,in order to further investigate the therapeutic effects of GLP-2(2)on intestinal lesions,we will conduct the following studies:First of all,we intended to establish the seed bank of engineering bacteria of GLP-2(2),and evaluated the genetic stability of the strains.Then,based on the completed laboratory studies,the process was further scaled up to establish pilot production process.At the same time,this project plans to establish the quality methods of GLP-2(2)to ensure the stability,effectiveness,and batch consistency of the samples.Secondly,the effect of GLP-2(2)on inflammatory bowel disease(IBD)and radiation enteropathy was systematically studied through the construction of IBD models and radiation enteropathy models in vivo and in vitro.Finally,we would like to reveal the mechanism of GLP-2(2)treatment for repair of injury by the study of pharmacokinetics and signal pathway.Through the above experimental,we obtained the following results:(1)The bacteria seed banks of GLP-2(2)were established,and the production strains was completely examined.The pilot fermentation process of GLP-2(2)was established,the fermentation scale was sacled up to 30 L,and the amount of bacteria that can be obtained was always greater than 30 grams per liter.The pilot purification process of GLP-2(2)was established,the raw material of GLP-2(2)with a putity greater than 95% could be obtained for 2.48±0.13 grams per batch.Furthermore,we preliminarily established a GLP-2(2)quality testing methods,for example,the biological activity of GLP-2(2)was assayed by CCK-8,the purity of GLP-2(2)was tested by HPLC,etc.,the results showed that all the items met the requriements.(2)IBD cell model and animal model showed that both GLP-2 and GLP-2(2)can significantly promote the secretion of inflammatory cytokines in CCD-18 Co cells induced by LPS and reduce the apoptosis of CCD-18 Co cells.Meanwhile,GLP-2 and GLP-2(2)can reduce the weight loss of mice and delay the pathogenesis of IBD,reduce intestinal structural damage,downregulate the inflammatory response caused by DSS,promote the survival of intestinal crypt cells,reduce the inflammation and induced intestinal cells apoptosis,and GLP-2(2)is superior to GLP-2 monomer.(3)The resules of radiation-induced intestinal disease cell model and animal model showed that GLP-2(2)can significantly reduce the radiation-induced apoptosis,necrosis and G0/G1 phase cell cycle arrest of CCD-18 Co cells.Both GLP-2 and GLP-2(2)can prolong the survival time of mice,reduce the expression of inflammatory cytokines and reduce intestinal damage.However,GLP-2(2)is more effective than GLP-2 monomer.(4)The results of preliminary pharmacokinetics and cAMP analysis showed that GLP-2(2)has longer half-life,stability and receptor activation in vitro and in vivo.(5)Molecularly,GLP-2(2)can participate in the repair of intestinal injury through multiple cell survival and apoptosis signaling pathways.Taken together,we established the seed banks of GLP-2(2),the pilot fermentation and purification process,and the quality teting standard.We confirmed that GLP-2(2)has a significant therapeutic effect in IBD and radioactive enteropathy.The therapeutic effect of GLP-2(2)is better than GLP-2 monomer.Pharmacokinetic results showed that GLP-2(2)has a longer half-life and stability in vivo and in vitro.Molecularly,we further demonstrated that GLP-2(2)rapidly activates divergent intracellular signaling pathways involved in cell survival and apoptosis.However,the detailed mechanism of GLP-2(2)activation pathway higher than GLP-2 monomer needs further study.Our data revealed a potential novel and safe peptide drug for limiting the adverse effect of radiotherapy on the gastrointestinal system.