Efficacy And Safety Evaluation Of Non-injection Third-generation Human Insulin

Research Background: Diabetes mellitus is a common and frequently occurring disease,which is caused by the relative or absolute deficiency of insulin secretion,and is characterized by elevated blood glucose.Insulin is the only hormone in the body that can reduce blood sugar and promote the synthesis of glycogen,protein and fat.Exogenous insulin is the main treatment of diabetes.Insulin injection has the advantages of quick effect and obvious effect.However,repeated injection will exert a lot of pressure and pain on patients,such as bleeding,pain and fatty degeneration at the injection site.In addition,insulin injection may cause hyperinsulinemia,resulting in hypoglycemia,coma and even shock.Buccal mucosa and sublingual mucosa in oral cavity are not keratinized,which contain low concentration of proteolytic enzyme and abundant blood vessels.Drugs can reach the whole body through submucosal blood vessels,which provides a potential non injection route for insulin and other macromolecular drugs,and has the characteristics of convenient administration.At the early stage of our research group,we designed the third-generation insulin p Insulin which can be administered through sublingual mucosa.p Insulin can penetrate the skin or mucous membrane efficiently under the guidance of transmembrane peptide,and contain the site of enzyme cleavage.After entering the tissue,C peptide can be cut off under the effect of enzyme cleavage,and processed into mature insulin,so as to play its hypoglycemic role.Research Objective: To prepare and optimize the preparation conditions of p Insulin,study its hypoglycemic effect,evaluate its safety,and provide theoretical basis for the further development and application of pInsulin insulin.Research Methods:In this study,the protein of p Insulin was expressed by E.coli prokaryotic expression system,purified by Ni column affinity chromatography,refold by pulse dilution.p Insulin lyophilized powder was prepared by using safe and non-toxic mannitol as auxiliary material,which is convenient for long-term stable storage.Then,the biological titer of p Insulin was determined by the mouse blood glucose method,which provided a clear dose of p Insulin sample for the follow-up experiment.We established a diabetic mouse model by intraperitoneal injection of STZ,and evaluated the feasibility and hypoglycemic effect of insulin p Insulin through sublingual mucosa by pharmacodynamic experiment in vivo.Finally,the safety of p Insulin was evaluated by acute toxicity test and subchronic toxicity test(90 days).Results: 1.p Insulin sample preparationThe protein of p Insulin was successfully expressed by E.coli prokaryotic expression system,purified by Ni column affinity chromatography,refold by pulse dilution.After renaturation,20-40 mg p Insulin can be obtained per liter of fermentation liquid.With mannitol as auxiliary material,p Insulin freeze-dried powder samples were successfully prepared.The biological titer of p Insulin was 13.15 IU/mg by mouse blood glucose method.2.Pharmacodynamic experiment of p InsulinThe diabetic model mice were successfully constructed by STZ.After intraperitoneal injection of p Insulin at a dose of 4.5 IU /Kg,the blood glucose of diabetic model mice decreased significantly.After 1 hour of administration,the blood glucose decreased to the normal range.The AAC(Area above the curve)of the thirdgeneration recombinant human insulin(Humalog R25)was 2821.4 ± 114.8 by intraperitoneal injection of 4.5 IU/Kg,and the AAC of the intraperitoneal injection of p Insulin(4.5 U / Kg)was 2761.2 ± 111.8.There was no statistical difference between the two.It showed that the intraperitoneal injection of p Insulin and the intraperitoneal injection of Humalog R25 had similar hypoglycemic effect.The blood glucose of diabetic model mice can be significantly reduced by injecting p Insulin through sublingual mucosa at 150 IU/Kg body weight.After 2 hours of administration,the blood glucose of diabetic model mice can be reduced to 1 / 3 of that before administration.However,the hypoglycemic effect of Humalog R25(150 IU/Kg)was not significant.The AAC of UBR 25 was 299 ± 91.11,and the relative bioavailability of Humalog R25 was 0.32%.The AAC of p Insulin was 2282.5 ± 181.13,and the relative bioavailability was 2.43%,which was 7.6 times of that of Humalog R25.The results showed that p Insulin was superior to Humalog R25 in sublingual administration.The results of pharmacodynamic stability test showed that compared with the control group(p Insulin solution prepared on the day of the experiment),p Insulin stored at 4 ? was more stable,and the hypoglycemic effect was not significantly reduced,while p Insulin stored at 25 ? and 37 ? was significantly reduced.It is suggested that p Insulin is easily inactivated at room temperature or higher and should be stored at 4 ?.3.Acute toxicity test of pInsulinThe maximum dose method was used to evaluate the acute toxicity of p Insulin.After 15 minutes of subcutaneous injection of p Insulin at 131.5 IU/Kg body weight for each animal in the experimental group,the animals in the experimental group began to suffer from mental depression and decreased activity.With the increase of time,some animals gradually developed into quadriplegia,muscle spasm,drowsiness,hypothermia and other symptoms.The animals began to die about 2 hours after administration,and all the dead animals appeared within 8 hours after administration.Among them,2 females died,accounting for 20% of the total number of females,3 males died,accounting for 30% of the total number of males,and the total mortality rate was 25%.The results showed that the LD50 of p Insulin was more than 131.5 IU/Kg body weight,which was 41 times of the clinical equivalent dose.No obvious abnormality was observed in the heart,liver,spleen,lung,kidney,brain and other organs of the dead animals.The surviving animals were killed two weeks after administration.The rats were in good mental condition,with bright hair and normal activity during feed.No obvious abnormality was observed in heart,liver,spleen,lung,kidney,brain and other organs,and no significant difference was found in organ coefficient of each organ.4.Subchronic toxicity experiment of p InsulinDuring 90 days of administration,the weight of rats in each group continued to increase,and there was no significant statistical difference between the experimental group and the solvent control group.The rats were in good mental condition and normal activity before death.After gross dissection,the pleura and peritoneum of the rats were smooth without obvious effusion and adhesion.Some animals have lung tissue degeneration,occasionally nodule formation,and some animals have slight redness and swelling of spleen.In the high dose group,the liver of a small part of the animals was inflamed.There was no significant dose-response relationship in the incidence of each group.Other organs such as heart,kidney and brain were normal in shape,color and position.The organ coefficient of each organ was compared with that of the solvent control group according to sex,and it was found that the organ coefficient of liver in the high dose group was significantly higher.Further HE staining showed that most of the tissues were normal.In a small number of animals,the alveolar epithelium atrophied in varying degrees,the alveolar septum widened,red blood cells oozed,chronic inflammatory cells infiltrated,local pulmonary fibrosis and other changes were found.There was no significant dose-response relationship between the incidence of lesions.A small part of spleen tissue had hyperemia and hemosiderin deposition,and the incidence of lesions had no dose-response relationship.In the high dose group,there was cellular edema in part of the liver,but the structure of the liver lobule was complete and the hepatic cord was clear.In the high dose group,the local blood vessels were dilated and congested,but no obvious myocardial cell degeneration and necrosis and inflammatory cell infiltration were found.Compared with the solvent control group,the biochemical indexes of each experimental group had no significant difference except ALT / AST of the high dose group of male rats and urea of the middle dose group of female rats.Conclusion 1.The p Insulin fusion protein was successfully prepared and its biological titer was 13.15 IU / mg,the confidence limit rate was 19.36%.2.p Insulin has good hypoglycemic effect when administered by intraperitoneal injection / sublingual mucosa.3.The median lethal dose of p Insulin is more than 131.5 IU / kg,which is 41 times of the clinical equivalent dose.4.Subchronic toxicity test showed that the no observed adverse effect level(NOAEL)of p Insulin was 6.4 IU / kg,twice the clinical equivalent dose.