Preparation And Properties Of Betamethasone Phospholipid Complex Submicron Emulsion

Objective: Betamethasone is an isomer of glucocorticoid dexamethasone.The currently marketed betamethasone injections are betamethasone sodium phosphate injection and compound betamethasone injection.The former is is usually administered in a low dose,multiple injection in clinical use.The latter is a suspension that requires deep intramuscular injection of the buttocks,which requires higher requirements for medical staff.In addition,the suspension has higher requirements for particle size uniformity and stability.The process of aseptic production is quite difficult,and the incidence of adverse drug reactions(ADR)in clinical practice is high.In view of the shortcomings of betamethasone,this study intends to develop betamethasone phospholipid complex submicron emulsion by combining phospholipid complex technology and drug-loaded lipid emulsion technology.Methods:1.The pre-formulation study of BSE.The determination method of BMS content was established and verified by methodology,includeing specificity,linearity,recovery,repeatability,stability,LOD and LOQ.The solubility of BSM in different solvents was investigated as well.2.The preparation technology and the characterizations of BPC.The complexation rate(P value)was used as the evaluation standard,and the calculation method was verified by methodology.The effects of reaction solvent,drug-lipid ratio,drug concentration,reaction time and reaction temperature on the formation of phospholipid complex were investigated by a single factor.Box-Behken response surface method was used to optimize the prescription design.The structure was characterized by UV,IR and DSC.The oil-water distribution was investigated.3.The preparation and quality evaluation of BSE.The evaluation system of the submicron emulsion was established.The single factor experiment focused on the effects of oil-water ratio,emulsifier dosage,ultrasonic intensity and time,homogenization pressure and cycle times,and stabilizer type and dosage on the formation of the submicron emulsion.Plackett-Burman experimental design was used to identify the factors that had significant influence on the quality of the submicron emulsion.The prescription process was further optimized by Box-Behnken response surface methodology.At last the sterilization conditions,p H environment and osmotic pressure of the submicron emulsion were supplemented and screened.4.The stability study of BSE.Three batches of betamethasone submicron emulsion were continuously produced for the stability investigation,and the content,p H,particle size,ζ-potential,drug loading,encapsulation efficiency,centrifugal parameters,osmotic pressure,electron microscope scanning,and residual solvents were examined.The quality changes of samples under high temperature,light,acceleration,freeze-thaw were investigated.The experimental model of release study was established in vitro and the release curve was drawn.5.Preliminary safety evaluation of BSE.Hemolysis,vascular stimulation,muscle stimulation and cytotoxicity were examined.Results:1.Pre-formulation study of BSE.The determination method of betamethasone content has good specificity.The peaks of main drug,excipients and solvent do not interfere with each other.The linear relationship is good in the concentration range of 10-400 μg/m L.The recovery rates of low,medium and high concentrations of preparations are accurate,repeatability,and the stability of solution at room temperature meets the determination requirements.BSE is soluble in methanol,slightly soluble in ethanol,insoluble in dichloromethane,chloroform and ethyl acetate.2.Preparation and characterizations of BPC.The prescription process with the complexation rate higher than 99 % was found as follows: the drug-lipid ratio was 1:2.5,the reaction concentration was 8 mg/ml,the reaction solvent was dichloromethane,the reaction time was 1 h,and the reaction temperature was 40℃.The structure was characterized by UV,IR and DSC,demonstrating that betamethasone interacted with some groups of soybean phospholipids,but no new compound was formed.Oil-water distribution test showed that the lipophilicity was significantly improved and the formation of phospholipid complex was indirectly confirmed as well.3.Preparation and the quality evaluation of BSE.The comprehensive evaluation system of submicroemulsion process was successfully established.The effects of various factors on the preparation of BSE were investigated by single factor experiment.Plackett-Burman experimental design was used to determine the four factors that had significant influence on the quality of the submicron emulsion from large to small: ultrasonic temperature>oil volume> soybean phospholipid dosage > poloxamer 188 dosage.Box-Behnken response surface method was used to determine the prescription process with the highest comprehensive score as follows: ultrasonic temperature 55℃,oil-water ratio 22:78,soybean phospholipid 1.5% and poloxamer 0.75 %.4.The stability study of BSE.Three batches of samples were continuously produced,p H 6.54 ± 0.03,ζ-potential ﹣20.0±0.2,particle size 159±3 nm,PDI 0.257±0.06,drug loading 99.9±0.4%,encapsulation efficiency 98.0±0.5%,centrifugal parameters 0.106±0.02,osmotic pressure 300.0±2.0 m Osm/L.Microscopic observation showed that the droplet structure was complete and the edge was clear.Dichloromethane was not detected by residual solvent examination.The results of influencing factor experiments showed that the betamethasone submicron emulsion was unstable to high temperature and freeze-thaw test while stable to light at low temperature,suggesting that freezing and high temperature storage should be avoided.The in vitro release curve satisfies the first order equation.5.Preliminary safety evaluation of BSE.BSE did not cause hemolysis and agglutination reaction.BSE has no obvious vascular irritation and cytotoxicity,but have potential muscle irritation.Conclusion: In this study,the formulation and process of betamethasone submicron emulsion with phospholipid as the carrier were developed and optimized,and the BPC and BSE were characterized.The quality standard of BSE was preliminarily established,and the stability study and safety evaluation were carried out,which laid a solid foundation for the further development of the project.