Preparation Of Oleanolic Acid Nanosuspension By Two Methods And Pharmacokinetic Study

As a kind of pentacyclic triterpenoid compound,Oleanolic acid（OLA）has many pharmacological effects such as liver protection,anti-inflammatory,anti-cancer,etc.It can be used to treat viral hepatitis and inhibit the development of liver cancer.The clinical applications of OLA are limited by low oral bioavailability because it belongs to BCSⅣdrug.Nanosuspension（Nanosuspension,NS）can reduce poorly soluble drugs to nanometer size through a variety of preparation methods,and improve the bioavailability in vivo after oral administration to varying degrees.There are many methods for preparing nanosuspensions,and different methods have different effects on dissolution and bioavailability.This study used high-pressure homogenization and grinding to prepare oleanolic acid nanosuspension（OLA-NS）.The particle size and polydispersity（PDI）were used as the evaluation index to screen the best prescription composition of OLA-NS and optimize the process of preparation and freeze-dried.OLA-NS were characterized through various methods and determined dissolution rate of OLA and OLA-NS in different dissolution media.The stability of the two OLA-NS were investigated.Rats were intragastrical administered OLA drug and OLA-NS to compare the differences in pharmacokinetic parameters between groups,explore the influence of preparation methods on the in vivo behavior of OLA,and provide the reference for the selection of preparation methods for OLA oral nano-formulations.It provided a reference for the selection of preparation methods in OLA oral nano-formulations.Objective:Methods for the determination of OLA content,cumulative dissolution and concentration in rat plasma were established.Two kinds of OLA-NS were prepared by high-pressure homogenization method and grinding method,and the optimal prescription and technology were screened out.The physical characterization and in vitro dissolution determination of the OLA-NS were carried out.The influencing factors of OLA-NS stability were investigated.The pharmacokinetic studies of OLA and OLA-NS prepared by different methods was conducted.Methods:1.The determination method of OLA content and dissolution by HPLC were established,and the methodological investigations were conducted.2.OLA-NS was prepared by grinding method and high-pressure homogenization method respectively.And the best prescription and preparation process of OLA-NS were determined which taking particle size and PDI as indicators.The type and proportion of freeze-dried protective agent were determined by comparing the changes in particle size and appearance of OLA-NS freeze-dried powder.3.OLA-NS were characterized by TEM,DSC,XRD,FT-IR.The cumulative dissolution rate of OLA and OLA-NS in different dissolution media,and the influence factors of OLA-NS freeze-dried powder were investigated.4.The UPLC-MS/MS method for determining the concentration of OLA in rat plasma was established.18 Wistar rats were randomly divided into 3 groups,OLA and OLA-NS prepared by two different methods were given by gavage respectively.The concentration of OLA was determine with the glycyrrhetinic acid as the internal standard.The relevant parameters were calculated and compared the differences between groups.Result:1.The HPLC method was used to determine the content of OLA.The method had a good specificity and linear range was 8.517～102.200μg/m L.The standard curve equation was Y=5.1432X+3.5346（r=0.9992）.The recovery rate was between 97.21～102.51%,and the RSD were less than 2%,which met the requirements.The HPLC method was established to determine the dissolution rate of OLA which had a good specificity.The standard curve equation was Y=5.1716X+0.6794（r=0.9997）which was used to determine the dissolution rate,and the linear range was2.044～40.88μg/m L,the recovery rate was 100.93～103.32%,the RSD of precision,repeatability,recovery rate and stability were less than 2%,which met the requirements.2.The types and proportions of stabilizers were screened with the particle size and PDI of OLA-NS as evaluation indicators.The best formulation composition was OLA:PLX-407:SDS=100:100:1.In order to prepare OLA-NS with the same particle size and smaller PDI by the two methods,grinding method investigated the grinding speed and time,and high-pressure homogenization method investigated the influence of homogenization pressure and time.The preparation process was optimized,and the preparation process of the high-pressure homogenization method was finally determined as:the mass concentration of OLA was 3%,200 bar,500 bar,800 bar,and 1000 bar were respectively homogenized for 4 min;the preparation process parameters of the grinding method were:the mass concentration of OLA was 3%,the volume ratio of initial suspension and 0.4～0.6 mm zirconia beads was 1:1,forward rotation(450 r·min^-1)3 min-stop 2 min-reverse rotation(450 r·min^-1)3 min-stop 2 min,grinding time was 120 min.The types and mass concentration of protective agents with ideal freeze-drying effects were screened by comparing the appearance and reconstitution of OLA-NS freeze-dried powder,measuring the particle size and PDI.The results showed that the particle size of the two OLA-NS freeze-dried powders were similar and small,about 350 nm if2%maltose+3%mannitol was used as protective agent.3.OLA,stabilizer,freeze-dried protective agent and two OLA-NS were characterized.TEM results showed that two OLA-NS had the same morphology,and they were all rod-shaped crystals with a size of about300 nm;DSC spectra showed that the spectra of the two OLA-NS were the same.There were the characteristic melting point peak of OLA,stabilizer PLX-407,freeze-dried protectant maltose and mannitol.But it was difficult to observe SDS,which might be due to the low concentration of SDS.It indicated that the crystal form of OLA was not been changed by grinding method and high-pressure homogenization method which the characteristic melting point peak of OLA in the freeze-dried powder was existed.XRD results showed that two OLA-NS freeze-dried powders had the characteristic diffraction peaks of OLA,stabilizer,and freeze-dried protective agent.It indicated that the crystal form of OLA was not been changed after being made into NS.The FT-IR spectrum showed the two OLA-NS freeze-dried powders were basically same,which including C=O and-OH characteristic peaks at 1691.66 cm^-1and 3548.97 cm^-1of OLA.It indicated that there was no interaction.The above results showed that OLA still existed as crystalline form in the two OLA-NS,and the high-pressure homogenization method and grinding method did not change the crystal structure of OLA.The in vitro dissolution test results showed that OLA-NS-Y freeze-dried powder dissolved faster at 0-15 min and the cumulative dissolution rate of OLA-NS-Y freeze-dried powder was 2 to 3 times that of OLA-NS-G in the dissolution medium of pure water.With 0.5%SDS aqueous solution as the dissolution medium,the dissolution rate of OLA-NS-G freeze-dried powder was faster at 0-30 min,the cumulative dissolution rate of OLA-NS-Y freeze-dried powder was slightly higher after 30 min,and the cumulative dissolution rate reached about 80%at 120 min.When Fa SSIF-V2 was used as the dissolution medium,OLA-NS-G lyophilized powder dissolved faster in 0-10 min and stabilized after 15 min.The cumulative dissolution rate at 120 min reached 11.52%,which was about1/2 of OLA-NS-Y freeze-dried powder.The OLA in pure water and Fa SSIF-V2 were almost insoluble.OLA-NS could significantly improve the in vitro dissolution of OLA,and the dissolution behavior of OLA-NS-Y freeze-dried powder in related biological media was better than that of freeze-dried powder prepared by high-pressure homogenization.The experimental study of influencing factors found that OLA-NS freeze-dried powder slightly agglomerated under high temperature and high humidity conditions.The dissolution rate of OLA-NS-Y freeze-dried powder changed greatly under high temperature conditions.The OLA-NS-G freeze-dried powder had strong hygroscopicity.The particle size of the two freeze-dried powders and the PDI of the OLA-NS-Y freeze-dried powder were easily affected by humidity.The appearance,content,dissolution,particle size and PDI of two OLA-NS freeze-dried powder were almost unchanged under strong light conditions.Therefore,OLA-NS freeze-dried powder should pay attention to avoiding high temperature and humidity during storage,choosing a low temperature,dry storage environment.4.With glycyrrhetinic acid as the internal standard,the UPLC-MS/MS method was established to determine the concentration of OLA in rat plasma.The standard curve equation was Y=1225.98310X+5597.33052（r=0.99980）.The concentration range was4.088～1635.200 ng/m L.and the lowest limit of quantification was 4.088ng/m L;the accuracy of low（10.220 ng/m L）,medium（204.400 ng/m L）,high（1226.400 ng/m L）concentration QC samples were between 0.89%and 7.33%;the matrix effect of QC samples and glycyrrhetinic acid were91.30%to 101.42%,and the extraction recovery rate were between93.89%and 96.75%;the precision and the stability RSD of QC samples were less than 8%,and the methodological inspections met the requirements.Pharmacokinetic studies had shown that making OLA into NS could significantly shorten the peak time and increase the peak concentration.OLA-NS-G and OLA-NS-Y plasma concentrations were higher than OLA of varying degrees.The relative bioavailability of APIs respectively were 338.43%and 473.01%;statistical analysis found that there were significant differences in multiple parameters between the OLA-NS group and the OLA group（P<0.05）.Conclusion:The analytical methods of HPLC and UPLC-MS/MS could determinate OLA in samples.OLA-NS could significantly improve the dissolution and bioavailability of OLA,which prepared by high-pressure homogenization method and grinding method,and OLA-NS-Y showed greater advantages.