Preparation Of Oral Butylphthalide Phosphatidylcholine Mixed Micelles And Its Application In Ischemic Stroke

Objective:N-butylphthalide is a kind of fat-soluble small molecular compound.As one of the innovative drugs independently developed in China,it has been used in the treatment of ischemic stroke and has a certain curative effect.It can inhibit ischemic stroke by inhibiting inflammatory reaction,increasing blood flow in ischemic area,inhibiting apoptosis and so on.Oral administration is a convenient,safe and most acceptable way of drug administration for patients.At present,the only n-butylphthalide oral product on the market is n-butylphthalide soft capsule produced by Shijiazhuang Pharmceutical Group.Because n-butylphthalide is an oily liquid,the preparation cost is relatively high,and the price is relatively expensive,on the other hand,the oral bioavailability of n-butylphthalide is relatively low,the oral dose is relatively high,and the treatment course of ischemic stroke is relatively long,which cause a great economic burden to the patients.Therefore,in view of the existing drug delivery problems,there is an urgent need for a new safe and efficient oral preparation of n-butylphthalide to improve its solubility and oral bioavailability,and to further develop its clinical application value in the treatment of ischemic stroke and solve the existing drug delivery problems.Nanotechnology has shown great advantages in oral drug delivery research,which can overcome the above difficulties to a certain extent.The phospholipid bile salt mixed micelles system is a kind of nano-drug biomimetic system which can be established by simulating the process of lipid absorption in vivo.It has high degree of biosafety and good physiological compatibility.Compared with single micelle,the stability of phosphatidylcholine bile salt mixed micelles is higher,the drug loading is larger,and the solubilization ability is stronger.Up to now,there is no research report on the application n-butylphthalide in mixed micelles system.The preparation of n-butylphthalein phospholipid bile salt mixed micelles（NBP-EPC-SC-MMs）is expected to improve the solubility and oral bioavailability of n-butylphthalide.In this study,egg yolk lecithin（EPC）and sodium cholate（SC）were used to prepare mixed micelles carrying n-butylphthalide,and NBP-EPC-SC-MMs were obtained.In addition,NBP-EPC-SC-MMs were studied in terms of appearance,cytology,pharmacokinetics,distribution and pharmacodynamics.Methods:（1）Preparation and characterization of NBP-EPC-SC-MMs.NBP-EPC-SC-MMs was prepared by thin film dispersion method and observed under transmission electron microscope（TEM）.Its particle size,surface zeta potential,drug loading and entrapment efficiency of NBP-EPC-SC-MMs were determined.The critical micelle concentration was determined by pyrene fluorescence probe method.The in vitro release of NBP-EPC-SC-MMs was studied by dynamic membrane dialysis method.The dilution stability and placement stability were also investigated.（2）Study on cytological of NBP-EPC-SC-MMs in vitro.The MTT method was used to investigate the cytotoxicity of n-butylphthalide original drug（NBP）and NBP-EPC-SC-MMs to Caco-2 cells.Cell uptake assay was used to investigate the time,temperature and energy dependence of NBP,and the NBP-EPC-SC-MMs uptake by Caco-2 cells was also investigated.（3）Study on pharmacokinetics of NBP-EPC-SC-MMs in vivo.After intragastric administration,the blood was taken from the orbit of rats at different time points to study the pharmacokinetics in vivo,and the change of blood concentration with time was investigated after oral administration.（4）Study on distribution of NBP-EPC-SC-MMs in vivo.After intragastric administration,the eyeballs of the mice were removed at different time points,and the mice were killed immediately after taking blood.Then the heart,liver,spleen,lung,kidney and brain were dissected to study the distribution of the drug in different organs of mice.（5）Study on pharmacodynamics of NBP-EPC-SC-MMs.The rat model of cerebral ischemia-reperfusion（I/R）was established by thread method,and the neurological deficit scores of rats in each group was scored.The volume of cerebral infarction was observed by TTC staining,the percentage of infarct volume was calculated.The morphological changes of brain tissue were observed by hematoxylin-eosin staining（HE）.The level of reactive oxygen species（ROS）in the brain tissue was detected by immunofluorescence.The activities of malondialdehyde（MDA）,superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）in brain homogenate were quantitatively measured to evaluate the pharmacodynamic effect of NBP-EPC-SC-MMs.Results:（1）NBP-EPC-SC-MMs was successfully prepared and the optimum prescription was obtained（NBP/SC/EPC mass ratio was 2:5:15）.The results of in vitro characterization showed that NBP-EPC-SC-MMs was the appearance of NBP-EPC-SC-MMs is well-dispersed liquid with opalescent light.TEM results showed that the mixed micelles were uniformly distributed round particles with uniform size and no adhesion.The particle size of NBP-EPC-SC-MMs was about 104.2 nm,zeta potential was about-32.3 m V,and the critical micelle concentration was 0.0002 mg/m L.The results of in vitro release showed that the release of NBP in simulated gastrointestinal fluid was slow,and the cumulative release rate was low,its release rate was only about7%in 12 hours,while the release of NBP-EPC-SC-MMs in simulated gastrointestinal fluid was faster,and the drug release reached more than 70%in 12 hours.The results of dilution stability test showed that there was no obvious change in the particle size of NBP-EPC-SC-MMs in a certain dilution range;the storage stability results showed that the particle size,potential and encapsulation efficiency of NBP-EPC-SC-MMs were almost unchanged when it was stored at 4℃for one week,indicating that the mixed micelles system was relatively stable.（2）Cytotoxicity experiments showed that solvent DMSO,blank micelles,original drug and NBP-EPC-SC-MMs had no obvious cytotoxicity to Caco-2 cells.Cell uptake assay showed that the uptake of NBP-EPC-SC-MMs by Caco-2 cells was 2.57 times higher than that of NBP,and there was significant difference between the two groups（p<0.01）.The uptake of NBP and NBP-EPC-SC-MMs by Caco-2 cells was time-dependent,independent of temperature and independent of energy.（3）Pharmacokinetic experiments showed that compared with oral administration of NBP,oral administration of NBP-EPC-SC-MMs showed good pharmacokinetic properties.The C_max and AUC_0-t in the preparation group were 4.44 and 4.27 times higher than those in the original drug group,and the maximum blood concentration and bioavailability of NBP-EPC-SC-MMs were significantly increased（p<0.01）.The in vivo clearance rates of NBP and NBP-EPC-SC-MMs were approximately 12.11 L/h/kg and 3.35 L/h/kg,respectively.Compared with NBP group,the clearance rate of NBP group was significantly lower than that of NBP group（p<0.05）,indicating that the clearance of NBP-EPC-SC-MMs in vivo was slower.（4）In vivo distribution experiments showed that after intragastric administration of NBP-EPC-SC-MMs to mice,the C_max and AUC_0-t of NBP-EPC-SC-MMs in blood,brain,heart,liver,spleen,and kidney increased significantly.The C_maxof the drug in blood,brain,heart,liver,spleen,and kidney were about 16.5,6.19,7.28,5.02,188.86,and 47.53 times of the original drug group,respectively,and AUC_0-twas 5.55,2.68,4.98,3.08,58.00 and 8.26 times of the original drug group,respectively.The above results showed that NBP-EPC-SC-MMs increased the oral bioavailability of n-butylphthalide and significantly improved the distribution of n-butylphthalide in various tissues in vivo.The results of brain targeting evaluation showed that the relative uptake rate（re）of NBP-EPC-SC-MMs in the brain was 3.48（re>1）,and the peak concentration ratio（Ce）was 9.04,indicating that NBP-EPC-SC-MMs increased the concentration of n-butylphthalide in brain tissue and had a certain brain targeting effect.（5）Pharmacodynamic experiments showed that the blank micelles had no obvious therapeutic effect on ischemia reperfusion rats.Compared with the original drug group,NBP-EPC-SC-MMs group reduced the neurological deficit scores of ischemia reperfusion rats to about 1.5 points,significantly improved the symptoms of neurological deficit.NBP-EPC-SC-MMs also reduced the cerebral infarction volume,reduced the necrosis and apoptosis of nerve cells,reduced the occurrence of oxidative stress and enhanced the activity of antioxidant enzymes of ischemia reperfusion rats,indicating that NBP-EPC-SC-MMs had a significant therapeutic effect on ischemic stroke.Conclusions:The NBP-EPC-SC-MMs prepared in this study can significantly improve the solubility and oral bioavailability of n-butylphthalide in vivo.The mixed micelles has a certain brain targeting and significant effect on ischemic stroke.It also has a broad application prospect in the clinical treatment of ischemic stroke.