| A recombinant expression plasmid consisting of the whole sequence of Nattokinase had been constructed according to the special construction features and physic-chemical property of Nattokinase by our laboratory. The NK can be expressed effectively through Bacillus subtilis which was regarded as expression vector.The oral biological availability of NK is very low because of its biomacromolecule property. As a kind of macromolecular drugs, NK is characteristic of a low bioavailability through direct oral administration. According to the related reports about NK’s zymological properties, it keeps stable in pH6～12, below50℃. It also has ability of antitrypsin and antipesin. But it is sensitive to acid environment. Therefore, in order to improve NK’s bioavailability by mouth, the most important things are to prevent the drugs from degradation in gastric acid environment and to promote the absorption by intestinal.In our experiment, we used the environmental-friendly supercritical CO2fluid to replace the traditional organic solvents in this tentative, prepared enteric-coated microspheres of NK, studied the response parameters and investigated the drugs’ activity and release behavior in simulated gastral and intestinal environment, and finally finished the initial study of the pharmacodynamics experiment and bioavailability research.The main ideas of our research are:1. Fermentation at shake flask and the purification and save of r-NK.2. Prove the artwork of supercritical CO2fluid, and prepare r-NK enteric-coated microspheres. 3. Preparation and purification the r-NK polyclonal antibody from rabbit.4. Finished the pharmacodynamics experiment and bioavailability research of r-NK enteric-coated microspheres.the NK-loaded microspheres subsequently are coated by Eudragit L100-55. This material does not dissolve in acid medium, but only dissolve in the media of which pH over5.5. So it can help the microspheres to resist acid environment, and rapidly dissolve in the neutral or slightly alkaline environment. Solvent emulsion evaporation (SEE) and supercritical fluid precipitation (SFP) are two techniques used for controlled release preparations. It is reported that many controlled release microparticles have been prepared by supercritical fluid. But traditional coating process requires a variety of chemical solvents and a large number of organic solvents which will inevitablely result in serious air and water pollution.The coated process was developed and carried out at the optimum conditions, with the ratio between the coating material and the pill1:2, the pressure20MPa, the temperature35℃, both plasticizer triethyl citrate and auxiliary solvent ethanol10%(v/m), talcum powder as antisticking agent.The in vitro test showed that the drug was released only9.7%in the simulated gastric fluid (pH1.2) and the relative activity was remained nearly90%in2h. The drug cumulative release percentage reached75%when it was exposed to the simulated intestinal fluid in2h. The r-NK was not released in the simulated gastric fluid, which suggested that the r-NK enteric-coated microspheres protect drug from being degraded by gastric acid. However, the drug was released quickly and completely from coated microspheres in the simulated intestinal fluid, which would contribute to the absorption of r-NK.The pharmacodynamics experiment and pharmacokinetics research of NK enteric-coated microspheres were carried out in rabbits. In the research of pharmacodynamics, the FIB and FDP values in administration group are changed signally compared to the control group. We predicate that r-NK have been absorbed and the changes of FIB and FDP values result from r-NK’s fibrinolytic activity. Through the followed research of Bioavailability with r-NK enteric-coated microspheres, we further testified that r-NK can be absorbed into blood system by intestinal mucous membrane soundly. |
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