本文選題：利福平(RFP) + 聚乳酸-羥基乙酸共聚物(PLGA)　； 參考：《中南大學(xué)》2013年博士論文

【摘要】：目的：①擬通過(guò)O/w乳化溶劑揮發(fā)法制備RFP-PLGA微球,以形態(tài)、粒徑分布、包封率和載藥量為指標(biāo)考察各處方因素的影響,并篩選出最優(yōu)工藝；②擬采用前期優(yōu)化處方制備緩釋微球,與骨支架結(jié)合,并觀(guān)察微球的骨填充情況及體外釋放行為；③采用新西蘭大白兔作為動(dòng)物模型,對(duì)RFP-PLGA骨緩釋復(fù)合體中RFP在體內(nèi)的分布及緩釋行為進(jìn)行了相關(guān)研究,同時(shí)考察了該復(fù)合物在體內(nèi)的相容性。為探討術(shù)后脊柱結(jié)核患者抗結(jié)核治療,減少其副作用提供實(shí)驗(yàn)依據(jù)。 方法：①通過(guò)乳化-溶劑揮發(fā)法制備緩釋微球,以粒徑、載藥量、包封率為考察指標(biāo)對(duì)處方中的不同因素如PLGA濃度,表面活性劑的種類(lèi)及濃度,油水比,藥載比進(jìn)行了單因素考察。并選取重要的影響因素進(jìn)行正交實(shí)驗(yàn)對(duì)處方進(jìn)一步優(yōu)化,得出各影響因素對(duì)制備過(guò)程影響強(qiáng)度,并篩選出最優(yōu)處方；②采用超聲振蕩法將RFP-PLGA緩釋微球灌入異體骨內(nèi),再澆蓋一層殼聚糖溶液,真空干燥噴金后,用電子掃描顯微鏡和骨密度儀分別觀(guān)察RFP-PLGA緩釋復(fù)合體的填充狀態(tài)、外觀(guān)表面形態(tài)及灌注前后骨密度的變化；體外釋放實(shí)驗(yàn)分成三組：A組RFP晶體、B組RFP緩釋微球及C組RFP-PLGA骨緩釋復(fù)合體,并采用透析袋法進(jìn)行體外釋放研究；③體內(nèi)藥動(dòng)學(xué)研究分成A組RFP口服給藥組、B組RFP-PLGA骨緩釋復(fù)合體組、C組RFP晶體-骨復(fù)合體組,后兩組植入兔腰椎骨性缺損處,并通過(guò)大體觀(guān)察、影像學(xué)觀(guān)察、骨密度觀(guān)察、組織分布結(jié)果、生化指標(biāo)檢測(cè)、組織形態(tài)學(xué)觀(guān)察六個(gè)方面進(jìn)行對(duì)比。采用HPLC-UV法測(cè)定血漿及各組織中的藥物濃度。采用SPSS17.0軟件包進(jìn)行分析,結(jié)果以均數(shù)±標(biāo)準(zhǔn)差即x±S表示,采用多種統(tǒng)計(jì)學(xué)分析方法,如t檢驗(yàn)、方差分析及組間比較的SNK-q檢驗(yàn)、LSD-t檢驗(yàn)等,檢驗(yàn)水準(zhǔn)a=0.05,P值0.05為差異有顯著性意義。 結(jié)果：①通過(guò)單因素考察得知對(duì)考察指標(biāo)具有較大影響的處方因素,在此基礎(chǔ)上對(duì)處方進(jìn)行了優(yōu)化,結(jié)果中各處方因素的影響強(qiáng)度如下：藥載比PVA濃度PLGA濃度油水比,并確定較優(yōu)工藝為：PLGA濃度15mg·ml-1、藥載比1：2、PVA濃度2%、油水比1：5；平行制得3批微球粒徑為12.23±0.98μ m；載藥量、包封產(chǎn)率分別為(19.63±0.62)%、(59.49±2.62)%,微球冷凍干燥后,再分散性及外觀(guān)良好； ②體外釋放顯示：A組RFP晶體在24h內(nèi)釋放基本完全,B組RFP緩釋微球組及C組RFP.PLGA骨緩釋復(fù)合體組釋藥平緩,其中RFP緩釋微球組31d累計(jì)釋藥率達(dá)到近95%,釋放進(jìn)行動(dòng)力學(xué)方程擬合,顯示符合零級(jí)動(dòng)力學(xué)方程F=0.168*t;RFP-PLGA骨緩釋復(fù)合體組約31d累計(jì)釋藥率達(dá)到近92.31%,未見(jiàn)無(wú)明顯的突釋,進(jìn)行動(dòng)力學(xué)方程擬合,顯示符合零級(jí)動(dòng)力學(xué)方程F=0.15*t；骨密度檢測(cè)：三組灌注前后及B、C組骨密度無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05)； ③體內(nèi)實(shí)驗(yàn)表明：A組即口服給藥組雖然在局部腰椎體組織中能維持較高的有效濃度,但其在肝、脾、心、肺等臟器中也存在較高的濃度;C組即RFP晶體-骨復(fù)合體植入組,盡管在維持靶組織有效濃度的同時(shí)減小藥物在其他臟器中的分布,但維持時(shí)間甚短,15天后已低于最低檢測(cè)濃度；相對(duì)而言,B組RFP-PLGA骨緩釋復(fù)合體植入新西蘭大兔給藥后,3天時(shí)RFP濃度為11±0.7μg/ml-1,在7天時(shí)RFP濃度為13.3±0.4μg/ml-1,在15天時(shí)RFP濃度為8.5±1.6μg/ml-1,在30天時(shí)RFP濃度為1.6±0.4μg/ml-1,在40天時(shí)RFP濃度為0.45±0.21μg/ml-1,均明顯高于有效抑菌濃度,在體內(nèi)組織中緩慢下降,不僅其他臟器中的藥物濃度低,而且40天時(shí)腰椎椎體RFP濃度仍可維持有效抑菌濃度,相對(duì)于C組在局部腰椎椎體組織中RFP的有效濃度持續(xù)時(shí)間較長(zhǎng)；骨密度檢測(cè)：a.術(shù)后第7天：三組統(tǒng)計(jì)學(xué)比較分析：P0.05,無(wú)明顯統(tǒng)計(jì)學(xué)意義；b.術(shù)后第30天：A組與B、C組比較：P0.05,有明顯統(tǒng)計(jì)學(xué)意義；B組與C組比較：P0.05,無(wú)明顯統(tǒng)計(jì)學(xué)意義；c.術(shù)后第7天與第30天不同組別比較：A7組與A30組比較：P0.05,無(wú)明顯統(tǒng)計(jì)學(xué)意義,B7組與B30組比較：P0.05,有明顯統(tǒng)計(jì)學(xué)意義,C7組與C30組比較：P0.05,有明顯統(tǒng)計(jì)學(xué)意義；肝毒性檢測(cè)：三組給藥前后比較未見(jiàn)明顯統(tǒng)計(jì)學(xué)差異；RFP-PLGA骨緩釋復(fù)合體行內(nèi)植入腰椎骨性組織后,腰椎和腰大肌組織均未見(jiàn)明顯炎癥反應(yīng),亦未見(jiàn)相關(guān)正常組織的破壞或形態(tài)異常。 結(jié)論： ①1、乳化-溶劑揮發(fā)法中諸多因素對(duì)微球成形和質(zhì)量具有明顯影響；2、篩選出最優(yōu)處方即PLGA濃度15mg·mL-1、PVA濃度2%、藥載比1：2及油水比1：5。 ②RFP微球形態(tài)良好,粒徑分布均勻,超聲波振蕩的方法可以與多孔隙的骨制備成RFP-PLGA骨緩釋復(fù)合體,其填充效果好,三組灌注前后骨密度無(wú)明顯統(tǒng)計(jì)學(xué)差異,具有良好的體外緩釋釋放性能,檢測(cè)各時(shí)間點(diǎn)的濃度均在有效抑菌濃度以上。 ③RFP-PLGA骨緩釋復(fù)合體體內(nèi)椎旁植入后,局部組織內(nèi)RFP抗結(jié)核有效濃度可維持在持續(xù)到約40天以上,無(wú)明顯心、肝、肺及脾等組織蓄積作用及毒性,骨密度無(wú)明顯影響,組織相容性良好。
[Abstract]:Objective: (1) to prepare RFP-PLGA microspheres by O/w emulsification solvent evaporation method, the effects of various prescription factors were investigated by morphology, particle size distribution, encapsulation efficiency and drug loading, and the optimal process was screened. (3) using New Zealand white rabbits as animal models, the distribution and release behavior of RFP in the RFP-PLGA bone sustained release complex were studied, and the compatibility of the compound in the body was investigated, and the experimental basis was provided to explore the anti tuberculosis treatment and reduce the side effects of postoperative spinal tuberculosis.
Methods: (1) the sustained release microspheres were prepared by emulsification solvent evaporation method. The particle size, drug loading and encapsulation rate were used to investigate the different factors in the prescription, such as the concentration of PLGA, the species and concentration of surfactant, the ratio of oil to water and the ratio of drug load. The effect of various factors on the strength of the preparation process was obtained and the optimal prescription was screened. Secondly, the RFP-PLGA microspheres were injected into the allogenic bone by ultrasonic oscillation, and then a chitosan solution was poured and covered. After the vacuum drying, the filling state of the RFP-PLGA sustained release complex was observed by the electron scanning microscope and the bone densitometer, and the appearance surface shape was observed. The changes of bone density before and after perfusion were divided into three groups: group A RFP, B group RFP sustained-release microspheres and C group RFP-PLGA bone slow release complex, and the release study was carried out by dialysis bag method, and the pharmacokinetic study in vivo was divided into A group RFP oral administration group, B group RFP-PLGA bone slow release complex group, C group RFP crystal bone compound The body group and the latter two groups were implanted in the bone defect of the lumbar spine, and compared the six aspects of the gross observation, the imaging observation, the bone density observation, the tissue distribution, the biochemical index detection and the histomorphology observation. The concentration of the drugs in the plasma and the tissues was measured by the HPLC-UV method. The results were analyzed with the SPSS17.0 software package. The standard deviation is x + S, using a variety of statistical analysis methods, such as t test, variance analysis and SNK-q test of group comparison, LSD-t test, test level a=0.05, P value 0.05 is significant difference.
Results: (1) on the basis of single factor investigation, the prescription factors which had great influence on the index were optimized. On this basis, the prescription was optimized. The influence intensity of each prescription factor was as follows: the ratio of PVA concentration PLGA concentration to oil and water ratio was as follows: PLGA concentration 15mg. Ml-1, 1:2, PVA concentration 2%, and oil and water ratio 1 5: the diameter of the 3 batch microspheres was 12.23 + 0.98 mu m, the drug loading, the encapsulation yield was (19.63 + 0.62)%, (59.49 + 2.62)% respectively, and the microspheres were redispersible and the appearance was good after the microspheres were frozen and dried.
In vitro release, the release of RFP crystals in group A was basically complete in 24h. The release of drug release in group B RFP sustained-release microsphere group and C group RFP.PLGA bone slow release complex was slow, and the cumulative release rate of 31d in RFP sustained-release microspheres reached nearly 95%, and the release was fitted by kinetic equation, showing F=0.168*t of zero order kinetic equation and RFP-PLGA bone sustained release complex group. The cumulative release rate of about 31d reached nearly 92.31%, and no obvious sudden release was found. The kinetic equation was fitted to fit the zero order kinetic equation F=0.15*t. Bone mineral density was detected in the three groups before and after perfusion and B, and there was no significant difference in bone mineral density in group C (P0.05).
(3) in vivo experiments showed that, although the A group, the oral administration group, can maintain a high effective concentration in the local lumbar vertebra tissue, it also has a high concentration in the liver, spleen, heart, lung and other viscera. Group C, RFP crystal bone complex implantation group, although it can reduce the distribution of drug in other organs while maintaining the effective concentration of the target tissue. The maintenance time was very short, 15 days later, it was lower than the minimum detection concentration. Relative, the concentration of RFP was 11 + 0.7 mu g/ml-1 at 3 days and the concentration of RFP was 13.3 + 0.4 g/ml-1 at 7 days, and at 15 days, the concentration of RFP was 8.5 + 1.6 mu g/ml-1, and the concentration of RFP was 1.6 + 0.4 g/ml-1 and RF on 30 days, RF, at the time of 15 days. The concentration of P was 0.45 + 0.21 mu g/ml-1, which was obviously higher than the effective inhibitory concentration and decreased slowly in the body tissues, not only the drug concentration in other organs was low, but also the effective inhibitory concentration of the lumbar vertebra RFP concentration was still maintained at 40 days. Compared with the group C in the local lumbar vertebra tissue, the effective concentration of RFP was longer and the bone density was detected. Seventh days after A.: three groups of statistical analysis: P0.05, no significant statistical significance; thirtieth days after B.: group A and B, C group: P0.05, there was significant statistical significance; B group and C group: P0.05, no significant statistical significance; C. postoperative seventh days and thirtieth days to compare: A7 group and the group comparison: there is no obvious unification Study significance, group B7 and group B30 compared with group P0.05, there was significant statistical significance, C7 group compared with the C30 group: P0.05, there was significant statistical significance; hepatotoxicity test: three groups before and after the drug delivery was not significantly different; RFP-PLGA bone slow release complex after the implantation of lumbar vertebra bone tissue, lumbar and the lumbar major muscle tissue are not obvious inflammation There was no damage or abnormal morphology of normal tissues.
Conclusion:
(1) 1, many factors in the emulsification solvent evaporation method have obvious influence on the formation and quality of microspheres. 2, the optimum prescription is PLGA concentration 15mg mL-1, PVA concentration 2%, drug load ratio 1:2 and oil and water ratio 1:5.
The morphology of RFP microspheres was good and the particle size distribution was uniform. The method of ultrasonic oscillation could be prepared with the porous bone to prepare the sustained release complex of RFP-PLGA bone, and its filling effect was good. There was no significant difference in bone density between the three groups before and after perfusion. It had good release performance in vitro, and the concentration of each time point was above the effective bacteriostasis concentration.
(3) after the paravertebral implantation in the RFP-PLGA bone sustained release complex, the effective concentration of RFP in the local tissue was maintained for more than 40 days, without obvious heart, liver, lung and spleen and other tissue accumulation and toxicity, bone density had no obvious influence, and the histocompatibility was good.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R318.08

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