| Stroke,also known as apoplexy,is one of the most prevalent diseases to endanger human's lives and listed as the most serious disease among four major stubborn diseases:apoplexy,tuberculosis,ascites and phrenodynia by herbalist doctors,with characteristic of high morbidity,disability,mortality and recurrence rate.The Xingnaojing(XNJ)injection,based on the famous traditional Chinese medicine prescription "AnGongNiuHuangWan",is composed of musk,gardenia,borneol and curcumae,which can eliminating heat and purging fire,cooling blood detoxification,refreshing and resuscitating,it is an effective clinical drug used to treat acute stroke.However,due to its inconvenience to use and clinical adverse events,slow action and low bioavailability of the related oral preparations;it is high time to develop a new dosage form and administration route to protect the compliance and security of patient medication,which also show rapid action and good brain targeting efficiency.Intranasal administration is an effective way to treat encephalopathy,with certain brain targeting,and has been proved by traditional medicine theory and modern medical studies.In the previous research in our lab,nasal administration of XNJ had significant brain targeting compared with injection administration,and the preparation technology of XNJ microemulsion(XM)had been accomplished.While considering the drug and surfactant have certain irritating effect on nasal mocosa,XM partly modified by methoxy poly(ethylene glycol)-poly(lactide)(mPEG-PLA)(XMM)was prepared in our previous research,and its mucosa irritating effect was reduced to a certain extent.This subject intends to investigate the pharmacokinetic characteristics from nose to brain and the main way of delivery from aspects of the in vivo pharmacokinetics and tissue distribution,and to evaluate the possibility of mPEG2000-PLA to improve long circulation and brain-targeting of microemulsion.The main works were as follows:1 The study on in vitro release of effective components of XM and XMMThe determination method for bomeol and muscone by GC method was firstly established.The method of dialysis was applied to compare the release characteristics of borneol and geniposide in two kinds of microemulsion.The results indicated that the release of bortneol in two kinds of microemulsions was close to the Higuchi model within 12 h and the release of geniposide was close to the Weibell model,and the accumulative release rate of borneol in XMM was significantly higher than that of XM,while the release of geniposide in two kinds of microemulsions presented no difference.While during the experiment,the muscone release was not discovered.Possibly due to the strong fat soluble and dissolution in the inner phase of microemulsion,muscone was difficult to release.The release results can not only reflect the stability of microemulsion,also has significance for the release rule of lipophilic drugs encapsulated in microemulsion and the existence form of microemulsion entering the body.2 The study of pharmacokinetics of borneol in plasma and brain samples by different routes of administration of XM and XMMWe firstly established the analysis method of borneol in mice by GC-FID.This study used GC-FID method to determinethe concentration of borneol in biological samples,and liquid-liquid extraction method was selected in processing plasma and brain samples,which needs small amount of biological samples and shows simple and fast in the pretreatment.Compared with the blank biological samples,the separation of borneol and octodecane in biological samples was good,without interference from the metabolites and endogenous substances.After verification of methodology,linearity,precision,recovery rate and stability all met the requirements.This method can satisfy the requirements in studying pharmacokinetics of borneol in plasma and brain samples in different ways of administration.In this study,we used ICR male mice as model animal and they were intranasally administrated by 4 ?L per animal or intravenously administrated by 0.2 mL per animal(i.e.,8 mg/kg borneol).The eyeballs were removed to get blood and then the brain was separated and homogenated.We used GC as analysis method and explore pharmacokinetics of borneol in plasma and brain samples by intranasal and intravenous administrations of XM and XMM.Kinetica pharmacokinetic software was applied to calculate the pharmacokinetic parameters in non-compartmental model.The plasma pharmacokinetics results showed that:The Cmax values of every group were 5.83±1.32 ?g· mL-1(I),7.28±1.84 ?g· mL-1(?I),9.02±3.62 ?g· mL-1(?I),9.68±2.14 ?g· mL-1(?)and the AUC values were 123.33±35.76 ?g· mL-1·min(?),113.02±43.79 ?g· mL-1·min(?),154.89±56.07 ?g· mL-1·min(?)and 236.62±36.11 ?g· mL-1·min(?).After nasal administration,XM and XMM could be quickly absorbed into the blood(Tmax<2 min),and both AUC values were greater than that of intravenous administration.The absolute bioavailability of F were 125.59%and 209.36%respectively,which were higher than that of nasal drop group(57.06%)in the previous study showing the higher level entering into blood of microemulsion.The brain pharmacokinetics results showed that:The Cmax values of every group were 11.73±0.91 ?g·g-1(?),10.00±0.79 ?g·g-1(?),2.82±0.58 ?g·g-1(?),3.63±0.49 ?g·g-1(?V)and the AUC values were 121.38±31.84 ?g·g-1·min(?),87.50±51.25 ?g·g-1·min(?),163.36±6.61 ?g·g-1·min(?)and 173.32±98.30 ?g·g-1·min(?).After intravenous administration of XM and XMM,the brain Cmax values were higher than that of blood samples and higher than that of brain samples administrated intranasally,which indicated that the microemulsion administrated intravenously distributed into tissues rapidly;and the blood Cmax value of XMM was higher than that of XM while brain Cmax value was lower than that of XM after intravenous administration,exhibiting that the introduction of mPEG segment reduced tissue binding with microemulsion and decreased the phagocytosis and capture on microemulsion oil phase by tissues,conducive to the realization of long circulating of drugs in oil phase.Two microemulsions could quickly enter into the brain after intranasal administration and AUC was greater than intravenous administration,Re values were more than 100%(XM 134.59%and XMM 198.09%).Comparing to the results of XNJ nasal drops(Re value(81.42%),Tmax(6.8 min)),the microemulsion after intranasal administration came into the brain much faster and of a higher degree,which presented the significant advantage of the brain targeting;the MRT of intranasal administration was significantly higher than intravenous administration which showed that the metabolism of microemulsion in brain was slow.The DTI value of XM was higher,but the levels of entering into blood and brain of XMM after intranasal administration(F and Re)were greater than XM.3 The study of pharmacokinetics of geniposide in plasma and brain samples by different routes of administration of XM and XMMIn this study,we used ICR male mice as model animal and they were intranasally administrated by 4 ?L per animal or intravenously administrated by 0.2 mL per animal(i.e.,4.12 mg/kg geniposide).The eyeballs were removed to get blood and then the brain was separated and homogenated.We used HPLC and UPLC-MS/MS as analysis methods,and explored pharmacokinetics of geniposide in plasma and brain samples by intranasal and intravenous administrations of XM and XMM.Kinetica pharmacokinetic software was applied to calculate the pharmacokinetic parameters in non-compartmental model.The plasma pharmacokinetics results showed that:The Cmax values of every group were 4.05±0.61 ?g·mL-1(?),3.26±0.46?g·mL-1(?),1.31 ±0.14?g·mL-1(?),1.57±0.81 ?g·mL-1(?)and the AUC values were 45.72±2.96 ?g·mL-1·min(?),29.72±2.42 ?g·mL-1·min(?),24.994±3.96 ?g·mL-1·min(?)and 30.79±3.13 ?g·mL-1·min(?).After intravenous administration,the AUC and Cmax values of XMM were lower than that of XM,indicating that the trend and degree of distribution in tissues of geniposide in XMM after intravenous injection were stronger than XM.After intranasal administration,the AUC value of XMM was significantly larger than XM(P<0.05),showing that geniposide of XMM had greater degree into blood due to its much less nasal irritation.The F value of XMM was significantly higher than that of XM,suggesting that XMM was propitious to improve bioavailability of geniposide intranasally.The brain pharmacokinetics results showed that:The Cmax values of every group were 136.75±43.56 ng.g-1(?),133.60±48.48 ng·g-1(?),96.47± 13.09 ng·g-1(?),114.89±23.41 ng·g-1(?)and the AUC values were 3654.70±784.18 ng·g-1·min(I),3411.22±366.13 ng.g-1··min(?),3278.19±181.68ng.g-1·min(?),6424.51±1071.89 ng·g-1·min(IV).After intravenous administration,the Cmax values and AUC values of two microemulsions had no significant difference,but the Te value(11.48%)of XMM was higher than that of XM(7.99%),which verified the conclusion of pharmacokinetics that the water-soluble components of XMM after intravenous administration was more conducive into organizations(brain).After intranasal administration of two microemulsions,brain AUC value of geniposide in XMM was significantly higher than that of XM(P<0.01)and the Re value was also higher than that of XM(188.33%and 89.70%),and XMM had significant advantages in brain-targeting.And comparing to the DTI value(1.29)of geniposide in XNJ nasal drops after intranasal administration,XM(1.64)and XMM(1.82)were higher,which indicated that microemulsion could improve the brain targeting of drugs through nasal cavity and XMM was better than XM.4 Preliminary studies on characteristics of drug delivery to brain and tissue distribution of XM and XMMThe near infrared dye DiR which shows strong fat soluble was selected to mark XM and XMM and prepared XM carrying DiR(D-XM)and XMM carrying DiR(D-XMM),and their pharmaceutical properties were investigated.The results showed that the pharmaceutical properties of D-XM and D-XMM were similar to that of the two microemulsions without DiR and D-XM and D-XMM showed stable property;DiR with high fat solubility,was encapsulated in the oil phase in microemulsion,and exhibited no leakage in 12 h.We used CD-1 nude mice as model animal and divided into D-XD nasal administration group(?),D-XM nasal administration group(?),D-XMM nasal administration group(?),D-XM intravenous administration group(IV),D-XMM intravenous administration group(?),DiR solution intravenous administration group(VI),and groups(?)(?)(?)were administrated by 4 ?L per animal(DiR 0.20 mg/kg),(?)(?)(?)were administrated by 0.2 mL per animal(DiR 2 mg/kg).Experimental animals were taken pictures for fluorescence at different time points after administration by the small animal imaging instrument.After taking pictures,the brain and other organs of mice were removed which were then taken images for fluorescence to compare the delivery and distribution characteristics of drugs in two microemulsions.The imaging results showed that microemulsion was rapidly distributed in tissues after intravenous administration and due to the introduction of mPEG segment of XMM,the combination between tissue and microemulsion was reduced and the phagocytosis and capture on microemulsion oil phase by tissues was decreased compared to XM,which was consistent with the conclusion drawn by borneol and geniposide pharmacokinetics.After intranasal administration,microemulsion mainly entered in the brain and had significant brain-targeting,which was notably higher than intravenous administration.Compared to XM,XMM still had a lot of drugs in the brain after intranasal administration for 4 h,suggesting that the oil phase of XMM was absorbed slowly into the brain after administration and had the greater degree entering in brain,which may be related to its smaller nasal irritation.In addition,the distribution in the brain of XM and XMM were same that was mainly distributed in the olfactory bulb,and the concentraton was reduced gradually from the olfactory bulb to the inner brain,so we inferred that XM and XMM transported to the olfactory bulb through olfactory nerve pathway.From the results above,we can found that after i.n.administration XMM could improve the brain-targeting of fat-soluble and water-soluble drugs,and after i.v.administration,XMM increased the binding with tissues of water-soluble components and reduced the combination of fat-soluble components with tissues.In addition,the application of mPEG-PLA block copolymer in the microemulsion has been reported rarely at home and abroad,therefore our study has a certain guiding significance to the application of mPEG-PLA in microemulsion and its effect on the microemulsion drug. |
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