Feasibility Study Of Long-acting Sustained-release Microspheres Loading Factor Ⅷ

Hemophilia is a coagulation disorder acquired genetically.The patient lacks of coagulation factor,and therefore are blooding traumatically or even with no obvious trauma.Hemophilia A is also called factor Ⅷ deficiency disease,which is a coagulation disorder genetic disease caused by factor Ⅷ encoding gene mutation leading to the function defect of coagulation factor,and the most sophisticated and effective treatment is to supply factor Ⅷ for life.If the long-acting preparation could be achieved,it will substantially increase patient compliance.Achieving extended activity of protein drugs after a single injection remains to be a thorny challenge.There are only few products may reach weekly or biweekly activity post injection due to chemical or biological modifications.This structure modification has never been successed for a large protein like factor Ⅷ whose molecular weight is over 500 K.There has yet to be a single product of sustained-release dosage forms for native proteins despite 41 years of research efforts.The laboratory where this work was completed developed a series of technologies for formulating sustained-release microspheres for fragile protein medicines,comprising protein pre-formulation through aqueous-aqueous emulsion or freezing induced aqueous phase separation,SPG membrane-added sedmentation,and phase-guided partition into porous microspheres.These methods were used successfully in producing sustained-release microspheres of relative small proteins,such as erythropoietin and human growth hormone,which have entered the stage of product development.However,the molecular weight of factor Ⅷ is far larger than the cytokine proteins,and thus more difficult to maintain the native conformuation during the process of microencapsulation.To examine the feasibility of factor Ⅷ for microencapsulation formulation,we utilized two of our microencapsulation strategies.1)Pre-formulating the protein into dextran particles through freezing induced aqueous phase saparation prior to SPG membrane-added sedmentation.2)Phase-guided partion of the protein into pre-maded porous microspheres,followed by pore-sealing in partially swollen state.The results of scanning electron microscope,laser particle size analysis,in vitro release test,and ELISA for quantitative analysis confirmed the feasibility of the protein for being formulated in sustained-release microspheres.The average particle diameters of the factor Ⅷ microspheres ranged around 39.1±8.1μm,and the encapsulation efficiency approached 90%.Annealing treatment in an aqueous solution of PEG20000 resulted in sealing of the pores of the PLGA microspheres at 37°C,a temperature substantially lower than the polymer’s Tg.The in vitro release test indicated that the microspheres formed via the phase-guided partition method released factor Ⅷ rapidly,and the rate was relavent to the extent pore sealing of the microspheres.Those made by SPG-added sedimentation released factor Ⅷ very slowly,so that the protein concentration was below the detection limit.While two release kinatics of severe burst and retardation were observed,the two extreme case indicates that adjusting the release rate of factor Ⅷ within a reasonable range is feasible.Taking our previous study for activity preservation of the protein into account,we may conclude that optimizing the pore sealing process of microspheres of phase-guided partition will lead to satisfied factor Ⅷ microspheres.