Studies On The Preparation, Physicochemical Properties And Bioactivity Of Ins-Ltmc

Insulin has been applied for about seventy years in clinic as the choice drug for diabetes. As a polypeptide drug, which can be hydrolyzed easily by protease in gastrointestinal tract, insulin is invalid by oral administration. For many years, insulin has always been administrated by injection in clinic. However, injecting insulin frequently can lead to many adverse effects, such as allergy, tissue fibrosis, etc. and it can also bring psychologic and economic burdens, besides burden of body. So the studies of non-injection insulin have always been the common issue at home and abroad.In our topic, we chose LTMC to modify insulin by the electrostatic interaction and prepared non-injection insulin. The conditions of modification were mild, the technology was easy, the yield was high and the bioactivity of modified insulin was not influenced. As a modifier, LTMC can protect insulin and enhance it' s absorbance. In addition, we studied and confirmed that Ins-LTMC has significant effects of dropping blood sugar by sublingual and oral administration. So the modified insulin can be applied to the studies of non-injection administration and it' s preparation.The main contents, results and conclusions were derived from theexperiments as following.1. The preparation and the physicochemical properties of LTMCOn the basis of protophase studies, we improved some technology of preparation of LTMC. We prepared LTMC from CTS and studied the physicochemical of LTMC. The viscosity-average molecular weight of LTMC was about 7KDa, the quaternary ammonium degree was 46. 64%, the molecular weight distribution was uniform, the purity was better and we determined the quaternary ammonium groups and it' s feature according to UV and IR.2. The studies on preparation of Ins-LTMCAccording to mass rate, we chose the usage of Ins and LTMC, optimum pH and. In additions, we studied the influence of temperature, time and the volume of solution with orthogonal experimental design on the preparation technology of Ins-LTMC. As a result, in the pH6.5 solution, after standing 8h in 4°C, the binding of Ins and LTMC was more complete when the usage of LTMC is 1~1. 25 times of Ins.3. The physicochemical properties of Ins-LTMCWe studied the physicochemical properties of Ins-LTMC with physical and chemical methods. Ins-LTMC was white or off-white powder, was dissolved easily in the mineral acid and alkaline water. The content of LTMC was about 53% and insulin was about 47% in Ins-LTMC. The pi is 6.29, the distribution of molecular weight was uniform and the purity was good. The structure and bioactivity of insulin,remained better. The modified insulin had stable physicochemical properties. Except for the change of pi, the structure and properties had hardly any changes.4. The biological value of Ins-LTMCWe determined the biologic value of Ins-LTMC according to the value determination of insulin, calculated the value and experimental error with graded response parallel line assay and double cross design of biological standardization statistical method consulted of Chinapharmacopoeia 2000. The biologic value of Ins-LTMC was 12. 386U/mg, the confidence level was 8.54%, which was consistent with pharmacopoeia. The value that we determined in our studies is corresponded with the estimated value. The result shows that the modification had least influence on bioactivity of insulin.5. The dropping blood glucose effects of Ins-LTMC after sublingual administrationWe studied the dropping blood sugar effects of different modified insulin by sublingual administration and calculated the relative bioavailability of Ins-LTMC. Although each sample group had different dropping blood sugar effect, they all had highly significant difference (K0.01) compared to blank control group and insulin group.Ins-LTMC(M=7KDa, QD%=46. 6%) group had no significant difference (P>0. 05)in dropping blood sugar effects compared to insulin injected subcutaneously. The maximum effect appeared about 60min after administration, the blood sugar value dropped 59.83%, the relative bioavailability was 52.53%. So the experiment confirmed Ins-LTMC had better dropping blood sugar effects by sublingual administration. It could be administrated by non-injection and be more fit to control the diabetic postprandial blood sugar.6. The dropping blood sugar effects of Ins-LTMC by oral administration and the influence of LTMC on the tryptic bioactivityWe studied the dropping blood sugar effects of Ins-LTMC by oral administration and compared with the insulin control group. Results showed that they had highly significant difference in dropping blood sugar effect. The blood sugar value dropped 21. 03% ±6. 57% after 120min. It showed that the stability and absorbability of modified insulin enhanced significantly in gastrointestinal tract. In addition, we studied the influence of different concentration of LTMC on tryptic activity bydetermining the tryptic value in vitro. Results showed that LTMC had no influence on tryptic activity. So we concluded that LTMC protected insulin by decreasing the touch of insulin and trypsin.Our studies are not reported now at home and abroad. It is the first time that we modified insulin with LTMC. The method is feasible, yield is higher and the compound has better dropping blood sugar effects. It is the first time that we determined insulin and Ins-LTMC with HPLC at home and set up the qualitation and qualitative methods of insulin. So our studies provided theory basis, technology and detection methods for non-injection insulin and it' s preparation. So it has scientific theory purpose and better application perspective.