Study On Trehalose As A Pharmaceutical Excipient For Injection And The Mechanism Of Its Stabilizing Effect On Bioactive Molecules

Pharmaceutical excipients are important part of pharmaceutical preparations,not only make drugs exist in different dosage forms,but also improve the therapeutic efficacy of drugs,reduce the adverse reaction to a certain extent.However,the status of excipients especially for injection in China is still fall behind,which become one of the key elements for blocking the development of new preparation.On the basis of clinical trial research from marine biotoxins,with consulting a large number of literature at home and abroad,for filling the blank of the excipients for injection in the domestic,this paper conduct the research on trehalose as a pharmaceutical excipient and the mechanism of its stabilizing effect on bioactive moleculesTrehalose is a non-reducing sugar with clear chemical structure,formed by the combination of two glucose molecules through the hemiacetal hydroxyl,with a 1,1-glycosidic bond.Trehalose widely exists in plants,insects,microorganisms and other organisms,it can stabilize cell membrane and protein structure,maintain its function,and has broad application prospects in the field of biology and medicine.In this study.we used food grade trehalose as raw material,eonstructed the purification process in the needs of pharmaceutical excipient for injection,obtained high-purity trehalose sample as pharmaceutical excipient for injection,with the purity above 99%,bacterial endotoxin under 0.1 EU/mg,and sterility test passedAn analytical method with high sensitivity detection for trehalose had been established based on ion chromatography.The limit of quantification was 10 ?g/L,the linear range was 62.5?10000 g/L(r=0.9999),the precision was RSD=0.42%,the recovery rate was 98.09?100.0%.The detection method can meet the requirements of pharmaceutical excipients detectionAn ion chromatography method were established for detecting trehalose in rat plasma The limit of quantification was 20 mg/L,the linear range was 0.2?10mg/L.The relative standard deviations(RSD)of within-run and between-run precision at low,medium and high concentrations were between 0.96?8.33%,and the accuracy was within the range of 80.09?114.99%.The study of trehalose metabolism in rat after a single intramuscular inj ection had been finish.The overall safety of trehalose used as pharmaceutical excipients had been system studied and evaluated.The results showed that the trehalose had non-acute oral toxicity in SD rats,LD50 in KM mice by intravenous inJ ection was above 5 g/kg;it had no irritation to rabbit skin and eyes,the sensitized strength tor guinea pig skin was weak sensitizer;90 d intramuscular administration triail in SD rats showed that trehalose had no significant toxicity,its toxic effect dose was above 200 mg/kg.In accordance with the requirements of national standard sample preparation,the key technology problem during the development process was researched.Monocrystal of trehalose were obtained through the process of natural crystallization from saturated solution;NMR,X-ray diffraction methods were used to identify the structure of trehalose;established standard sample detection nethod were used to evaluated the homogeneity and stability;the study on fixed value and measurement uncertainty was completed in collaboration with 8 laboratories with analytical qualifications in the domestic.The results showed that the prepared standard sample had good homogeneity within 95%conficlence interval;samples can be stable under 40? more than 24 months;The fixed value of purity was 99.72%,and the relative expansion uncertainty was 0.26%(k=1.96).Trehalose had a great effect in stabilizing bioactive molecules.Heat inactivation results showed that?trehalose had stronger stability effects on superoxide dismutase(SOD)and Scygonadin2 protein(Scy2),SOD freeze-dried powder setted under 90?for 10 d,the residual enzyme activity still remained above 90%;Scy2 freeze dried-powder setted under 25 C for 10 d.the MIC was kept at 25 ?mol/L.The stabilizing mechanism of trehalose for bioactive molecules was studied by various modern analytical techniques.SPR results showed that,compared to other disaccharides or monoaccharides,trehalose had the similar dynamic process for combining with SOD or Scy2,indicated that the binding force between the protein and trehalose had no direct relationship with the effect of thermal stability from trehalose;DSC and bioactive testing data showed that the thermo stability effect of lactose in amorphous state was weaker than mannitol even sucrose in crystalline state,indicated that crystalline state was not necessary for maintaining the bioactivity of protein;Low-field NMR data showed that freeze-dried trehalose does not contain free water which greatly destruct the stability of bioactive molecules,indicating that the strong binding ability to water,may be the key to the exert the stabilizing effect.In summary,this paper researched the preparation,detection,safety and standard sample development of trehalose as pharmaceutical excipients for injection,discussed the stability mechanism of trehalose on biological molecules.The research results provide solid technical support and important scientific basis tor the application of trehalose as pharmaceutical injection excipient,which has potential significant application value.