Study Of Lung Targeted Tetrandrine Polylactic Acid Microspheres

Tetrandrine (TET) is a kind of extract from the root of Stephanh tetrandra S. Moore, a kind of benzyl bases quinoline alkaloid. TET has the pharmacological effects of antihypertension, anti-myocardial Ischemia, improving reperfusion injury, anti- Ca~2+ channel, anti-inflammation, immunosuppression, anti-silicosis, antitumour, anti-diabetes and anti-free radical damage. TET have been already applied clinically to treat hypertension, angina pectoris, silicosis, portal hypertension with liver cirrhosis, tec.TET tablet and its injection have already been produced at China. TET is difficult to dissolve in water, therefore, it is poorly absorbed by oral administration, which causes low bioavailability, and unstable therapeutic effect. The TET therapeutic effect is much influenced because TET ordinaty injection is distributed widely in the body without targeting effect and high concentration in diseased organ or tissue.The research of microspheres has made very great progress in recent years. It shows controlled drug release effect with the result of achieving prolonged or sustained release, increasing drug targeting, reduing side effect, improving therapeutic effect.In order to solve the clinical problem of TET mentioned above, this thesis explores several respects of TET microspheres. It is expected to have therapeutic effect of targeting to those specific organ disease such as hepatic fibrosis, lung cancer, silicosis. It is greatly meaningful that targeted therapeutic effect will improve drug therapy, reduce side effect, improve drug safety and efficacy and its reliability.Sustained release of lung targeted tetrandrine polylactic acid microspheres has been relevantly reported at home or abroad so far. Polylactic acid (PLA) is a biodegradable polymer which has been extensively used in sustained drug release system. TET microspheres are produced by PLA with molecular weight of 10kD, which shows good lung targeted effect.This project includes the study of tetrandrine microspheres preparation, content determination, stability of tetrandrine microspheres. We establish a method for determining the plasma concentration of tetrandrine in rabbit, we also research the pharmacokinetics of Tetrandrine polylactic acid microspheres in in rabbit and its distribution in vivo in mouse. The acute toxicity and pharmacodynamics of TET microspheres are studied tooOptimization of preparation of etrandrine microspheresWhen exploring the preparation method of microspheres, adopting the continuous liquid volatilization method, the microsphere's adhesion may cause very serious crystallized phenomenon, which will influence microsphere's quality. In order to improve this phenomenon , we turn to adopt liquid volatilization/extraction method instead. PLA has good solubility in dichloromethane, therefore tetrandrine is dissolved in dichloromethane as organic phase, gelatin is dissolved in water as water phase, then TET-PLA microspheres are prepared.Uniform design is adopted to optimize the preparation of TET-PLA microspheres. When exploring microsphere preparation method, the factors influencing TET-PLAmicrospheres are: PLA concentration (A), TET concentration (B), gelatin solution concentration (C) , the speed of mixing (D) at the time of emulsification. Thus we select the above four factors dividing to 12 levels , select form of Uo (13n ), and uniform design, with encapsulated efficiency, drug content, particle diameter and its shape as index, constitute U)2 form, optimizing the preparation of microspheres. The equation adopted by multivariate progressive regression analysis is:Y=35.306-0.676A"9.248*10-3B+1.819C-2.261D, R=0.902, F=7.668,The calculated results is drawn :Ymax=27.1453 ±3.2515, The range of prediction of namely Y is between 23.8918-30.3698. Combine the actual conditions, choose A =4,B =5,C =2,D =900, in order to optimize the condition.But, consider actual experiment, PLA , and Tetrandrine to can is it investigate minimum of range to adopt. Because ,if PLA adopts the minimum, then PLA concentration and viscidity are not big enough, the microsphere presents more and analyses brilliantly with the adhesion. And TET is too small, the drug amount is too small in year. Combine actual conditions copies of experiment adopt size three terms, experiment of group as terms of optimizing directly.Preparing three batches of microspheres according to the optimizing condition, in the total amount of 500 microspheres, the 7-15um range of a foot-path is 80.3% , the drug amount in microspheres is 46.95%, the seal rate is 61. 71%, and have better shapes . the identification should prepare the craft and have feasibility .Determination of drug content in microsphere.TET can become salt with hydrochloric acid and dissolve, but carrier material PLA does not dissolve in hydrochloric acid, so PLA can be removed through strain ,there are no absorb interference to the determination of drug.regard hydrochloric acid solution as and determine medium and blank solution, divide all luminosity law and determine the content with purple outside, determinewavelength 279. 5nm, determines the method simple and easy to do. Study research after carrying on the method, the method is verify to be reliable and accurate.Study on the Stability of Tetrandrine Microsphere.Adopt with higher speed test and room temperature test, investigate the indexes, such as properties of appearance, amount of drug loaded , seal rate, seepage rate, microbial stability,etc. Results: Through the test of six months,properties of appearance, amount of drug loaded , seal rate, seepage rate, microbial stability have not obviously change, but after testing for 6 months with higher temperature,the seal rate is reduced,and the seepage rate mcreasing.Conclusions: Tetrandrine microsphere is steady under the room temperature condition,but is unstable to hot, and ought to keep in conformity with low-temperature.Determination on blood drug level of Tetrandrine in in rabbit.A method that determinate the blood drug level of Tetrandrine in in rabbit byu HPLC determines is establishd The terms of the chromatogram are HPHypersilODS-C18 posts (5um, 4.6mmX 150mm); Flow phase is methyl alcohol -water (45:55 ); Velocity of flow is 0.8ml/min , Measure wavelength is 210nm; inject volume is lOul. With this condition, Tetrandrine satisfied separated with reference substance ,with short reserve time - 4.67and5.83, the endogenous materials are noiseless to the two of the plasma.Treatment of Plasma sample: Prepare 0.5ml plasma samples accurately, filled in the leadership of 10ml scale, add reference substance 15ul accurately, regulate PH to 10 with NaOH solution, puts into Nitrile 0.5ml, chloroform 5ml, mixing well, Swirl mixes 2min, at odds with the community or the leadership 5min (3000r/min ). Move to evaporate ware chloroform, surplus water looks in addition chloroform 4ml, extracted and at odds with the community or the leadership with the law.Amalgamate and extract the liquid twice, the water bath is waved and done, the residue holds lmldefinitely with the methyl alcohol, over 0.45 pm capillary strain membrane.The dissolvent of Tetrandrine in rabbit plasma is strengthen , draw with the chloroform after alkalizing, it is relatively ideal to add the precision of rate of recovery of the style.Determine blood drug level of Tetrandrine on 10 point arrange of 5-180min.Pharmacokinetics research of Tetrandrine microsphere in rabbit.6 rabbits, weight 2.5kg, dosage is 8mg.kg"!, an arterial carotis intubate after the fifth barbital sodium (80mg.kg-l ) injects and anaesthetizes in the abdominal cavity, ear reason intravenous injection Tetrandrine microsphere, in order to prolong moustache ask for second mark while acting as usually, determine tetrandrine plainly in rabbit's concentration of blood drug with HPLC law. Not presenting the unusual phenomenon in the course of experiment, the plasma has not had haemolysis . Use and dialyse bag, release degree by dividing all luminosity law and determining the body purple of two stepses of derivatives.Move calculation procedure of studying of 3P87 the drugs adopting blood drug level of Tetrandrine, draft compartment the models. Adopt Wagner-Nelsion method to study the external dependence in the body.Examine blood drug level of Tetrandrine, map with logarithm and time ,become a curve. So, pharmacokinetics model in rabbit is two room room model, calculate the parameter of pharmacokinetics.Result of study of internal and external dependence:The return equation is: Y =2.3692X-40.173,R =0.9960, is greater than the critical value , indicate that it is remarkable to return to the equation, dependence is good. Making it known and releasing outside the body has good dependence with the blood drug level.Distribution of Tetrandrine microsphere in mouse.Fetch 80 mouse of NIH , weight 18g-22g , divided into 20 groups , 4 everygroups at random. Intravenous inject TET microsphere of mouse's tail (dosage is lmg / lOg), and contrast with TET injection (dosage is 0.469mg/10g) .The result show ,in the same dosage ,The concentration of TET microsphere in mouse lung is high than that of TET injection, in time 15min to 12h, TET concentration is more than 1 time of water needle; The concentration of TET microsphere in mouse lung is 2 times of that in the liver,on the time of 0.08,0.15 ,0.5 , 1 ,2, 5h .among them, on the time of 0.15 , 0.5and lh, the concentration TET in lung is more than 4 times of that in the liver; According to distributing the percentage, TET is higher than otheT internal organs group notably too in the distribution percentage of the lung. Therefore can think tentatively , Tetrandrine microspherethat this craft makes has obvious lung target tropism.From the concentration of TET in mouse internal organs on different time mouse, we can find , Tetrandrine microsphere reduce relatively slowly in lung. We can maintain certain concentration in drug within longer time (12h ), maintains 13.274 mg/ml In 72h. Obviously higher than other organizations and plasma groups .Therefore .Tetrandrine microspherehas clearing up slowly in the lung.Study on Acute toxicity of Tetrandrine microsphere.Study on Acute toxicity with mouse, tetrandrine plain little acute toxicity of ball.determined the LD50 of Tetrandrine microsphere.the toxic response of Tetrandrine microsphereacute is similar to that of tetrandrine. we see ,mouse activity reducing , piloerection , having difficulty in breathing , not taking food after using drug, and death.Checking the corpse, there is extravasated blood in the lung, the intestines congestion is expanded, have not seen that obviously change with other viscera.Tetrandrine microsphere group situation, extravasated blood is more serious than that of Tetrandrine, this may relate to the fact that the drug in the lung is higher.LD5o of Tetrandrine and Tetrandrine microsphere is 46.43 + 19.35 mg ? kg'1^ 96.43± 19.35 mg'kg"1.Pharmacodynamics research, carried on the ecffect of anti- liver fibrosis and the experimental cerebral infarction.Research on The Anti-liver Fibrosis Ecffect of Tetrandrine Microsphere.40 big mice of male ,SD, weight 180-220g, divided into 4 groups at random: Contrast group , liver fibre group , Tetrandrine group , and Tetrandrine Microsphere group, 10 every groups. Besides contrast group, every group inject tetrachloride carbon. The fibre hierarchical data processing of liver adopts the order and examines; The others adopt analysis of variance .The result shows , 4 groups of serum AST and ALT activation , HA content and liver fibre degree are through comparing.Compared with liver fibre Group,, AST and ALT activation and HA content drop obviously (p <0.01 or p <0.05) in the Tetrandrine group , and the Tetrandrine Microsphere group; Compared with contrast group, AST and ALT activation increase notably(p <0.05), Indicate that Tetrandrine can protect liver cell , prevent it to changing bad, reduce liver organize inflammation react.HA is a sensitive index that can indicates liver endothelial cell function and the liver fibre degree, it is lower than the model group notably , so TET not ongly protects the liver cell ,but also obviously suppressed the synthetic of cell's outer room quality in the liver, thus resist fibre functionUnder the microscope, degree of liver fibre of contrast group , liver fibre group, Tetrandrine group and Tetrandrine Microsphere group is 0, 2.8,1.3,1.5 grades; Compare with the liver fibre group, degree of liver fibre reducing notably (p <0.01) in Tetrandrine group, and Tetrandrine Microsphere group.Influence of TET Microspbere to experimental cerebral infarction.Fetch 44 big normal mice , divided into 4 groups , 9 every groups at random: (1) shamoperation, (2) ischemic, (3) ischemic + tetrandrine,(4) ischemic + tetrandrine microsphere. The above is used in the determination of the volume of brain block . Break end and fetch the brain organization, and examine Ca2 + , Na + , K concentration in 24h after the skill.The result show, in brain district of blood lack ,Ca2 + concentration is obviously higher than that of contrast group, organize Ca2 + usually with Tetrandrine rise obvious, obviously lower than the group that lacking blood. This prove ,Tetrandrine can resist Ca2 + of brain lacking blood, reduce person who block up Na + content lighten the brain hydroncus while being short of money.By the result, tetrandrine has usually failed to totally stop lacking the blood venereal disease and pay attention to the process changed, but take the neuron to have certain protective action to the edge, it is one of the reasons to reduce of blocking up area, and Tetrandrine to through being short of money to resist Ca2 + indraft, reduce blood platelet build up, maintain blood vessel stability, blood not to circulate smooth a lot of link, have obtained and blocked the good result treated to the experiment big mouse MCAO office cooking stove brain.