| Protamine Sulfate is an antagonist of heparin, it is recruited into the Pharmacopoeia of People’s Republic of China (version2010), and it is normally used to rescue heparin methysis on clinic. Protamine Sulfate is onest of action by injection; however, it is a kind of foreign proteins, and leads the allergy at times. Because of this risk, Protamine Sulfate is limited used to the suffers. The Low Molecular Weight Protamine (LMWP) is a kind of peptides, possesses similar function as protamine, and it can be prepared by using thermolysin. The LMWP remains the nature of protamine-the antagonist of heparin. Further more, it possesses the property of TAT, Polyarginine as a kind of Cell Penetrating Peptides (CPPs). When combine with the large molecule medicines, CPPs will promote the absorption of them.This thesis was researched on the assay, preparation&purification, in vivo and in vitro. This research established the LMWP assay and preparation methods, use the physical mixing to prepare LMWP-insulin complex, and confirmed the bioavailability. The results of this research provide a new thinking about the developing of oral insulin administration. The main conclusion as followed:1) Established the assay method of LMWP, compared the UPLC method with HPLC, chose the UPLC method for assay during the research followed;2) Established the preparation&purification method of LMWP, confirmed the product after the preparation&purification, and the product possessed high pureness;3) Used the Caco-2cell model to establish the uptake and transport research, proved the complex formed from physical conjugates of insulin possessed higher uptake and transport efficiency than insulin alone. The MTT test demonstrate LMWP do not show cell toxic below the highest concentration of this thesis research-500μg/ml.4) The rat in-situ loop absorption research demonstrate LMWP-insulin can be absorbed in the ileum. Compared with the insulin, the LMWP-insulin showed significant stronger hypoglycemia effect. |
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