Preparation,in Vitro And In Vivo Evaluation Of Sinomenine Hydrochloride Transfersomes

Objective To optimize the formulation of sinomenine hydrochloride transfersomes（SHTs）and to clarify its characteristics by vehicle characterization,in vitro percutaneous permeation and skin deposition,in vivo pharmacokinetics and pharmacodynamics.To verify their therapeutic effects on rheumatoid arthritis in rats.Methods SHTs were prepared by ethanol injection method.After Single factor experiments on the amount of cholesterol and sodium deoxycholate,pH and relative ionic concentration of phosphate buffered saline,the formulation was optimized by an orthogonal test,which was based on the elasticity of TFSs.Morphology of SHTs was observed by transmission electron microscope,mean particle size,polydispersity index（PDI）and Zeta potential were determined by laser scattering particle size analyzer.Elasticity was measured by constant pressure extrusion method and entrapment efficiency was measured by HPLC combined with centrifugation ultrafiltration.The skin permeation and deposition test of SHTs and SHLs were carried out by a Franz diffusion cell with excised abdominal skin of male Wistar rats.The effects of two different vesicles on percutaneous permeation of SH were investigated.Male Wistar rats were selected as experimental animals for the investigation of skin and blood pharmacokinetic properties of SHTs after transdermal administration on the abdominal skin,and SHLs was used as reference.Synchronous microdialysis technique was applied in pharmacokinetics study and UPLC-ESI-MS/MS method was for the determination of drug in microdialysis samples.The model of rheumatoid arthritis was established by subcutaneous injection of type Ⅱ collagen into Wistar rats’tail.The therapeutic effects of the SHTs on rheumatoid arthritis in rats were evaluated based on ankle joint score,swelling degree,content of TNF-αand IL-1βin serum as well as histological changes including inflammatory cell infiltration,pannus formation,cartilage destruction,and bone erosion.Results SHTs were prepared by ethanol injection and optimized by Single factor and orthogonal test.The optimized formulation was as follows:egg phospholipid 300 mg,cholesterol 30 mg,Sinomenine hydrochloride 100 mg,sodium deoxycholate 60 mg,vitamin E 5 mg,phosphate buffered saline（pH 8.0）23 mL,and absolute ethyl alcohol2 mL.The optimized SHTs were round to roundish vesicles,had an average size of（83.31±0.08）nm,Zeta potential of（-32.57±3.27）mV,deformability index of 38.69±1.66 and entrapment efficiency of（39.82±0.97）%.The in vitro skin permeation experiment showed that SHTs had an accumulated skin penetration amount of（499.57±122.64）μg·cm^-2,permeation percentage of（87.73±21.54）%,steady-state permeation rate of（27.61±8.50）μg·cm^-2·h^-1,which was approximately 1.7,1.7,2 times as much as that of SHLs,respectively.T_lagof SHTs was（0.75±0.41）h,shorter than that of SHLs.It indicated that SHTs could quickly penetrate into the skin.Combined with in vitro skin permeation experiment,the in vitro skin deposition experiment showed that the deposition amount of SHTs was（71.70±44.73）μg·cm^-2 in the whole skin and（20.19±19.86）μg·cm^-2 in the stratum corneum at 36 h,which was less than that of SHLs.The percentage of drug deposition of SHTs was much higher than that of SHLs,indicating that SHTs could penetrate into deeper skin.The skin pharmacokinetic data showed that the C_maxand AUC_0-tof SHTs was 7.4,7.7 times as much as that of SHLs,and the MRT_0-twas shorter.The blood pharmacokinetic data revealed that the C_maxand AUC_0-tof SHTs was 2.6,2.9 times as much as that of SHLs,indicating that SHTs could penetrate through the stratum corneum barrier of the skin much easier into the dermis than SHLs and had better transdermal permeability.The results of pharmacodynamics test revealed that the preparation could significantly reduce joint swelling caused by rheumatoid arthritis（P<0.01）and lower TNF-αand IL-1βcontent in serum（P<0.01）,effectively alleviate inflammatory response and improve histological changes of ankle joint.Conclusion The ethanol injection method is feasible for the preparation of SHTs and their quality can be controlled.The optimized SHTs have a good permeability and are effective for treatment of rheumatoid arthritis in rats.