| Endometriosis is a gynecological disease that the endometrium which has growthfunction in places outside the uterus covered surface growth and reproduction, which canlead to symptoms of dysmenorrhea, irregular menstruation, infertility and othergynecological. The gonadotropin-releasing hormone agonist (GnRH) was chosen as thepreferred treatment substance. Alarelin frozen-powder injection used on the domesticmarket for this disease has shown good effects, while the formulation administered150μgper day during a course of treatment for3to6months. This treatment program requiresfrequent injections that decrease the compliance of patients. The aim of this study is todevelop the sustainedly released formulations of alarelin in order to solve the deficiency ofalarelin powder injectionAt first, a HPLC method was established for the determination of alarelin. Thestability of alarelin in various temperatures and pH was studied. The optimized storagecondition for alarelin solution was4℃and pH5.6.Secondly, the alarelin loaded microparticles (AR-MPs) were prepared with W1/O/W2double emulsion-solvent evaporation method. The preparation process was optimizedusing the encapsulation efficiency and particle size as evaluation indicators.Theinvestigated factors include the viscosity of internal aqueous phase, PLGAconcentration, the temperature and the ultrasonic power using in the preparation of W1/Oemulsion, PVA concentration in external aqueous phase, the volume of external aqueousphase, the stirring speed, the osmolality of external aqueous phase, alarelin concentrationand so on. Then, an orthogonal test was utilized to investigate the effects of themainvariables.. The optimized factors were the power of200w, PLGA concentration of40%(w/v), the temperature of4℃andthe PVA concentration of3%. The microparticleswere of particle size as75μm, EE%as97.31.5%andthe initial release as6.51.5%.The optimized AR-MPs could sustainly release in vitro for35days. The residual organicsolvents of AR-MPs were qualified.Thirdly,60Co irradiation was chosen as sterilization method for AR-MPs. Theinfluences of different dosages of irradiation on AR-MPs were investigated, such asmorphological characterization, alarelin content in AR-MPs, in vitro release tendency and so on. The results showed no significant changes in the morphology of AR-MPs and in thein vitro release tendency before and after the AR-MPs treated with different dosages of60Co radiation. Alarelin in the microparticles reduced with5%and the initial burst releasealso decreased after being irradiated under25kGy. Tg values of AR-MPs treated with25kGy irradiation were significantly different (P<0.05). The molecule polydispersity ofcarrier decreased with irradiation dosage increased. AR-MPs were most stable under4℃/35%RH. We draw a conclusion that the the60Co radiation sterilization method can beapplied for AR-MPs. The morphology and related process parameters of the AR-MPsdidn’t significantly change after being treated with different dosages of irradiation.Fourthly, pharmacokinetics of AR-MPs with and without60Co irradiation in beagleswere investigated and compared with alarelin powder. The relative parameters includehalf-life (t1/2), the maximum plasma concentration (Cmax), the peak time (tmax), meanresidence time (MRT) and the bioavailability were investigated. The half-life of pre-proAR-MPs was65.87times and68.74times when compared with a single dose of alrelin.The MRT of pre-pro AR-MPs were65.87times and68.74times higher than that of alrelin.Compared to alarelin powder, the relative bioavailability of AR-MPs with and withoutbeing irradiatedr was260.4752.71%and263.8258.68%respectively. Microparticlesprolonged the release time of alarelin and maintained therapeutic alarelin concentrationsfor at least35days. The behaviors of alarelin released in vitro and in vivo have goodcorrelation.In summary, this paper draws up the AR-MPs used the W1/O/W2double emulsionsolvent evaporation method and optimizes the preparation process according to theencapsulation efficiency and initial burst release. As the result, a microspherepreparationmethod with stable quality and good reproducibility was acquired. TheAR-MPs showed sustained release behaviors in5weeks. The60Co irradiation was verifiedto be the final sterilization of miccrospheres for the late clinical research. Thepharmacokinetic study of the AR-MPs in beagle dogs proved the microparticles have thesustainedly released characteristics in vivo. It also offered a great potential for the AR-MPsto be used in clinic. |
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