| With the increasingly prominent problem of the aging of the population,the morbidity of Alzheimer's disease(AD)increases myltiplyingly with age increasing,therefore,AD prevention and treatment of drug research and development has become the focus of attention. In recent years,survey study found that the incidence of AD is related with estrogen.For the time being,A 17-βestradiol-based estrogen replacement therapy(ERT)has been used in clinical,the most commonly method is oral,vaginal and percutaneous replacement therapy. However,three methods have some limitations.This study aimed at exploring a suitable route of administration of estradiol and reasonable dosage form,to increase the concentration of drug reaching the target of the brain,reduce dose administration,thereby reducing E2 systemic absorption and adverse reactions.Intranasal administration is one of the drug delivery system being given more research in recent years.It has the advantage of absorption rapidly,no first pass effect,the administration convenience,and good compliance.In addition,it's also the route for brain-targeting.These characteristics are useful for the clinical application of estradiol.After determining the solubility at different pH conditions and the oil-water partition coefficient of E2,the result showed that E2 is water insoluble,and less affected by pH,logP is 3.14-certain lipophilic property,which foretells good nasal absorption.An approach for the preparation of E2 nasal chitosan nanoparticles is based on an ionic gelation process.E2,a small molecule and fat-soluble drugs,to be made into chitosan nanoparticles,and reach a certain concentration,must be solubilized in water.First,we prepared inclusion complex with Randomly methylatedβ-cyclodextrin(RAMEB),and then made use of cross-linking reaction between chitosan and TPP to prepare nanoparticles.Taking the particle size and zeta potential as indicator,we inspected the concentration of CS and TPP, the mass ratio of CS and TPP,dropping speed,mixing speed,temperature,as well as the addition of drugs,and finally determined the formula and preparation process:chitosan was dissolved in 1%(v/v)acetic acid solution to get the concentration at 2.0 mg·mL-1,and then shifted precisely 5.0 mL,the estradiol inclusion complex(E2 2.0 mg·ml-1)was added into it,2,0 mL TPP(1.0 mg·mL-1)solution was added slowly with syringe,at 2.0 mL·h-1dropping speed under the magnetic stirring,and lasted for 30 min.Ajusting pH value to 5.0,and we got chitosan nanoparticles,of which the particle size was about 270 nm,ξ-potential was over +25 mV,and the encapsulation efficiency of E2 in chitosan nanoparticles suspension was 65% around,loading capacity reached 1.92 mg·mL-1.A two stage pressure homogenizer was used to prepare E2 submicron emulsion.Oil phase is determined,the ratio of the emulsifiers and the dosage of oleate were optimized.The stirring intensity and time,homogenization temperature,pressure,times and the way of addition of lecithin were studied,and the effect of pH was also detected,taking appearance, mean diameter,zeta potential,stability parameter,content and encapsulation efficiency as index.According to those results,the final formulation and preparation were determined: 2.5%glycerol,0.5%F68were heated and dissolved into distilled water.The oil phase was produced by dispersing 0.2%E2 in a mixture of MCT and soybean oil(1:1,20%)at 80℃,with 2.0%lethincin,0.06%oleate,till lethincin was dissolved.A primary emulsion was achieved by mixing the oil and aqueous phases forl5 rain using a high-shear mixer 14,000rpm.After adjusting the pH to about 5.5,final submicron emulsion was obtained by passing the coarse emulsion through a high pressure homogenizer.The homogenization condition was 40℃,90 MPa and 8 cycles.Submicron emulsion in the samples were bubbled with nitrogen gas and then sterilized on a water bath at 100 for 30 min℃with stirring.The particle size of E2 submicron emulsion was 220nm in mean diameter,-29.0 mV inξ-potential.The E2 loading capacity was 1.89 mg·mL-1,and the entrapment efficiency was above 85%.With the application of in situ rat nasal perfusion method,we inspected the nasal absorption kinetics of the E2 CS-NP and SE with different concentrations.The results showed that,they were well absorbed into nasal mucosa,and absorption rate constant got a rising trend with the increase of concentration,and what's more,the absorption of E2 SE was better than CS-NP.HPLC fluorometric method was performed to detect the concentration of E2 in rats' plasma and CSF,which was simple and precise.The pharmacokinetic study of E2 CS-NP and E2 SE after intravenous administration and nasal administration in rats was performed, compared with E2 inclusion complex.The pharmacokinetic parameters were studied according to non- compartment model.The results showed that,after intravenous administration of E2 inclusion complex,E2-CS-NP and E2-SE,Cmaxreached(170.08±64.67),(151.4±28.2)and(123.36±24.51)ng·mL-1respectively,the AUC value were(7844.60 ±2783.13),(9183.88±2631.10)and(11544.94±4003.41)ng·min·mL-1.As intranasal administration was concerned,three preparations exhibited an absorption process in concentration-time curve,and Cmaxwere(27.75±10.12),(32.69±10.11)and(34.17±8.82) ng·mL-1respectively at 20,28 and 26 min.The AUC value were(4117.244±602.50),(6057.11±2385.05)and(6404.59±2154.96)ng-min-mL-1.Apparently,the plasma concentration after i.n.administration was far lower than that after i.v.administration for the three preparations.The study on the brain-targeting of the three preparations was also performed. Microdialysis technique was used to determine the E2 concentration in CSF after intranasal administration,comparing with intravenous administration.Probe recovery in vitro was 61.64%by no net flux method,on the basis of it,in vivo recovery was 41.7%determined by retrodialysis method,which was lower than in vitro recovery.The results showed that,for E2 inclusion complex,E2-CS-NP,and E2-SE,Tmaxof E2 in CSF by i.n.administration was earlier than that by i.v.administration,and Cmaxof E2 in CSF by i.n.administration was higher that by i.v.administration,which exhibited E2 could access directly to the brain by olfactory pathway preliminarily.In order to more clearly understand the direct transfer of E2 between nose and brain after i.n.administration,drug brain targeting index(DTI)and percentage of drug direct transshipment to brain(DTP)were introduced,the results showed that DTI values were over 1 for three preparations,2.61,3.15 and 3.80 respectively,which noted a certain brain-targeting property for each preparation,and the order was:E2 inclusion complex＜E2-CS-NP＜E2-SE.The DTP values were 61.91%,68.43%and 73.63%.All of these data supported strongly that there was direct access from nose to brain for E2,getting into brain through olfactory pathway.
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