| Background and objectives：In1975，Swedish scientists G.Boman induced Hyatophora Ceropia with Enterobactercloacae and Escherichia coli and got one substance separated from its hemolymph.The lateranalysis showed that this substance, named Cecropins,which is a polypeptide withantimicrobial activity.This is the first kind of antimicrobial peptides found in the world.Atpresent,there are many kinds of antimicrobial peptides have been found.These peptides areencoded by genes,and exist in different species and different tissues.The number of Humanantimicrobial peptides is less than others.The defensins family，cathelicidins family，histatinsand hGlyrichi family had been proved currently.Defensins is a kind of cationic peptides,which have been found in the process ofmammalian and insect and other’s defense and resistance to microbial pathogens invasion andinfection. According to the connection position of the cysteine residues and disulfide bond,thehuman defensins can be divided into α and β defensin. There are6kinds of α defense,4kindsof myeloid-derived defenses are secreted by neutrophils,2kinds of intestinal defensins comefrom Paneth cells and small intestinal epithelial cells. In the effective doses range,thedefensins couldn’t damage the human normal cells of the body, nor induce drug resistancewithin a effective concentration for The defensins has identification of pathogenicmicroorganisms and normal cells. therefore, the defensin is expected to become a newantibiotics. Human alpha defensin5(HD5) is mainly expressed by intestinal epithelial cellsand genital mucosal epithelial cells with prominent secretory expression, antibacterial and antiHIV, HPV and HSV-2virus, which is one of the most prominent human alpha defensin withantimicrobial spectrum and antimicrobial activity, has great development and applicationvalue. Pharmaceutical excipients plays a key role in medicine preparation, with the name of allsubstances without active ingredients,which is the basic materials and important part inpharmaceutical manufacturing. Good excipients can not only make the drug develop toneeded pharmaceutical dosage form and improve the use of drug effectiveness, but alsoreduce its side effect.at present, there are40classes of pharmaceutical excipients withthousands kinds, including Preservatives, excipient and stabilizer, antioxidant, solvent and soon.for the reason, to choose a right excipient which was less side effect and with known safedose is the basic guarantee for dug safety. In order to keep the biological activity of peptideand medicinal proteins in the process of preparation and stockpile,some pharmaceuticalexcipients are added such as alcohol(Mannitol, glycerin, sorbitol), saccharides(Lactose,sucrose and glucose), amino acids(glycine).As we know,the biological activity of most defensins are affected by externalcircumstances, so in order to find the excipients for polypeptide HD5,we made the medcineinto external spray to test in vitro antibacterial activity and expedite the drug withantibacterial rates as the response values, and chose the best excipient group according to theeffect of excipients on the biological activity and stability of HD5. meanwhile,through theanimal experiment,we analyzed the preventive effect of HD5peptides with excipients onwound infection,which lay the foundation for HD5peptides gene engineering to producepolypeptide medicine.Methods：1. According to the literature,Possible excipients were screened for HD5on the basis ofthe physicochemical properties of HD5and spray formulation requirements.And settingexcipients concentration through the relevant literature and the relevant requirements of"Chinese Pharmacopoeia" and "Chinese medicinal materials".2. Confect these excipients to corresponding concentration range One by one. Theinfluence of excipients on the antibacterial activity of HD5polypeptide was analyzed throughthe data of in vitro antibacterial test. Compare their inhibitory rate to screen excipients typeand concentration in the range of approximately.3. A variety of optional excipients with corresponding concentration combined with HD5to mixed liquid in various combinations. And set the corresponding only excipientscombination as control group. Analyzed the influence of excipients combination on the antibacterial activity of HD5polypeptide through the data of in vitro antibacterial test. thebest excipients combination was determined for HD5according the finding,and then be put in37℃for1~6months. Antibacterial activity in vitro of HD5was been Analyzed again.5. The animal (mouse) wound Pseudomonas aeruginosa infection model was established.Observe the effect of HD5polypeptide biological products on prevention and treatment ofanimal wound infection.and provide a reliable experimental basis for the application of HD5polypeptide.Results：1. Obtained the available spare accessories of HD5and the approximate concentrationrange. That is to say, there is no obvious influence on HD5antibacterial activity whenseparately adding glycerin whose concentration were less than5%, sucrose whoseconcentration were less than12%, Twain80whose concentration were less than0.2%,HP-β-CD whose concentration were less than5%, or Methyl Paraben sodium whoseconcentration were less than0.05%.2. Obtained the optimum combination of accessories of HD5, namely5%Glycerol+2.5%HP-β-CD. The corresponding experiments has proved it will not affect the antibacterialactivity of HD5. And5%Glycerol+0.5%Twain-80+2.5%HP-beta-CD and5%Glycerol+2.5%HP-β-CD as HD5accessories, will not affect the antibacterial activity ofHD5, and can play a stabilizing role3. Successfully established mice model with back skin pseudomonas aeruginosainfection, and confirmed that5%Glycerol+2.5%HP-β-CD+HD5can effectively controlthe wound infection caused by pseudomonas aeruginosa. But HD5would not induce drugresistance within the effective dose, and this characteristic makes HD5has a very gooddevelopment prospects.4. Preliminary assessed pseudomonas aeruginosa infection in animals by measuring theamount of bacteria under callus, and provided the basis for accurate judgement of infectioncontrol.Conclusions：1. The study has preliminarily tested several commonly used pharmaceutical excipientsof HD5and their concentration range through antibacterial experiment, and the optimaladjuvant of HD5has been obtained. 2. The study has established a method for HD5polypeptide drugs formula selection, andthe relevant data could provide some reference for HD5aerosol formula.3. Back skin pseudomonas aeruginosa infection in mice model was established, and theinfection control situation was preliminary judged according to the amount of bacteria undercallus. |
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