【作者】 赵领；

【导师】 余瑜；

【作者基本信息】 重庆医科大学 ， 生物制药与生物医用材料， 2009， 博士

【摘要】 背景在我国,肺癌、肺结核等肺部疾病发病率较高且呈快速递增发展趋势。为了治疗这些难治性疾病,我国已投入大量的人力物力,并在药物治疗领域取得了一系列重要进展,但现有治疗药物体内分布不具目标选择性,且用药量大、周期长,产生严重毒副反应与耐药性。对此,理想的治疗策略是开发肺靶向给药系统。在肺靶向给药系统中,脂质体作为一种毒性小,生物相容性好的载体,因具有缓释、增加肿瘤靶向、提高药物稳定性以及改善药物动力学特性等优点而倍受关注,但其研究水平、生产技术及产业化能力有待不断提高。为此,突破脂质体载体给药系统研究与产业化的关键技术,重点推进抗肺癌药、抗肺结核药等肺靶向脂质体载体给药系统研究非常必要。目的本文以针对非小细胞肺癌（NSCLC）的靶向给药系统及其生产急需解决的实际问题为切入点。选用多烯紫杉醇（DTX）为模型药物研制DTX-LP,望能通过脂质体给药系统将DTX传递到肺部,真正达到提高疗效,降低毒副作用的目的,并切实解决脂质体生产瓶颈及肺部靶向性两大关键科学问题。方法1.首次将固体分散技术与泡腾水化技术组合制备DTX-LP,即先采用固体分散技术制备DTX前体脂质体,再通过泡腾水化制备DTX-LP。2.采用光学显微镜及透射电镜法,观察DTX-LP的形态。3.采用马尔文激光粒度测定仪,测定DTX-LP的粒径和表面电荷。4.建立测定DTX-LP制剂的HPLC分析方法,并进行方法学确证。5.采用透析法,测定DTX-LP的包封率;考察DTX-LP的体外释药特性。6.采用单因素法,以脂质体粒径、形态为指标,考察筛选显著影响DTX-LP制备的主要因素。7.采用正交设计法,以脂质体粒径分布、包封率、稳定性为指标,优化并确定DTX-LP的制备工艺。8.建立体内生物样品中DTX含量测定的HPLC分析方法,并进行方法学确证。9.基于正交优化的处方及工艺,制备不同电荷、不同粒径的DTX-LP并测定其在兔体内1.5h的分布,进一步探讨其肺靶向作用并筛选确定其最佳的处方及工艺。10.采用药代动力学方法,考察DTX-LP的药代动力学特征。11.采用5个靶向参数为指标,评价DTX-LP的肺靶向性。12.采用单次给药和连续多次方法,考察DTX-LP的毒性作用。13.建立荷A549肺癌的裸鼠模型,考察DTX-LP的抗肿瘤活性。结果一、药学部分1.处方及工艺的研究:经单因素试验发现,影响脂质体的粒径分布、稳定性及包封率的主要因素有DTX和吐温-80的用量、溶液液面温度、枸橼酸与NaHCO₃比例。经正交试验确定的工艺所制备的DTX前体脂质体为粉末和（或）颗粒状。所得DTX-LP形态圆整、分散性好,表面电荷为-23.4mv,平均粒径为0.90gm,包封率为90.5%。2.最佳处方及工艺的确定:在前面研究的基础上,制备不同粒径和表面电荷的DTX-LP并测定其体内组织分布,以肺靶向性为指标确定的最佳处方及工艺:即称取处方量的DTX、HSPC、胆固醇、吐温-80、枸橼酸,溶于适量热乙醇中,趁热过滤,温度维持在55℃,搅拌下加入NaHCO₃,除去溶剂得到粉末和（或）颗粒状的DTX前体脂质体。用时称取DTX前体脂质体1g,加10mL 5%NaHCO₃溶液泡腾水化得DTX-LP。3.DTX-LP质量研究:采用最佳处方及工艺制备的DTX前体脂质体颗粒流动性好;与5%NaHCO₃溶液15min内泡腾水化完全。DTX-LP混悬液的pH值为5.5±0.3,脂质体形态圆整,粒径为0.95±0.12μm,电荷为-23±0.21mv;包封率为90.57±0.32%;体外显示缓慢释药;在密封、遮光和2～8℃干燥条件下保存至少1年质量稳定。二、药理毒理学部分1.体内生物样品的分析方法:建立了体内生物样品的HPLC测定方法,该方法专属性强,干扰峰少,且不影响主药测定;回收率为80.9%～113.4%,日内、日间精密度RSD均小于10%,萃取回收率均大于70%,满足生物样品分析要求。2.肺靶向作用研究:经不同粒径和表面电荷的DTX-LP兔体内组织分布研究。其结果显示,在粒径大致相同,带负电荷DTX-LP的肺部药物浓度显著高于带正电荷脂质体和注射液,且粒径在1μm左右和负电荷大于20mv的DTX-LP具有最高的肺组织分布。3.体内组织分布及肺靶向性评价:经兔体内组织分布试验结果表明,本文研制的DTX-LP在各时间点肺组织中DTX浓度均显著高于DTX注射液。从靶向参数角度分析,DTX-LP的肺部相对摄取率（Re）为28.91,显著高于其他组织和血液,表明DTX-LP显著增加了肺的靶向性。DTX-LP的肺相对其他组织或血液的靶向效率（Te）值均远远大于1,其中相对肝、脾和肾分别为3.16、23.00和27.83;且（Te）liposomes/（Te）injection的比值显著增加,由此表明,DTX-LP显著增加了肺部的选择性。DTX-LP与DTX注射液在肺部药物峰浓度比（Ce）为74.28,而肝、脾和肾分别为2.93、1.09和1.41,由此表明了DTX-LP完全改变了DTX在兔体内的分布,主要被浓集在肺部。DTX-LP肺组织的总靶向系数（TCe）为63.41,远高于其它器官组织,比DTX注射液提高了6倍。由重量-总靶向系数（TQe）值可知,DTX注射液中DTX在肺分布仅为10.445%,而脂质体制剂中有高达52.746%的DTX富集于肺,这将有助于肺部肿瘤的治疗,有一定的临床应用价值。4.体内药动学研究:DTX-LP和DTX注射液静脉给药后,在血液中,两制剂的药动学均符合三室模型,AUC_（0-t）分别为0.893和2.130mg/L^*h,DTX注射液的分布半衰期t_1/2α和消除半衰期t_1/2β,分别是0.122和0.207h,而DTX-LP的t_1/2α显著缩短（0.023h）,表明脂质体能快速从体循环中分布到靶器官,t_1/2β显著延长（2.186h）,有助于发挥DTX的治疗作用。DTX-LP和DTX注射液在肺部靶器官中药物经时变化规律经拟合分别为二室模型和三室模型,AUC_（0-t）分别为2428.65和84.00mg/L^*h,t_1/2α分别为1.414和0.081h,t_1/2β分别为8.808和3.481h。由此可见,DTX-LP在血液及肺靶器官中药动学行为有显著性差异,本文中的DTX-LP在血液中的药动学参数已不能反映靶器官—肺的药动学行为。5.毒性作用的研究:从生存情况方面,与DTX注射液组比较,DTX-LP组动物的平均生存时间提高近一倍;病理切片分析结果可知,DTX注射液组发生了严重的病理改变,而DTX-LP组几乎未见异常变化,由此表明了DTX-LP显著降低了DTX的全身毒副作用。6.药效学研究:经实验结果表明,DTX注射液（12mg/kg）的抑瘤率为75.72%;DTX-LP三个剂量组（3mg/kg、6mg/kg和12mg/kg）的抑瘤率分别为61.48%、94.40%和95.40%。结论1.首次采用固体分散技术与泡腾技术组合成功制备DTX-LP。该组合技术克服了现有脂质体生产批量小和质量不稳定等瓶颈问题,具有良好的工业化生产前景。2.首次提出带负电荷DTX-LP（1μm左右）具有高度肺靶向的新观点。3.本文所研制的DTX-LP是一种有效的肺靶向给药载体,改变了DTX的药代动力学行为,能达到减少DTX的用药剂量,提高抗肿瘤作用,降低毒副作用的目的,具有广阔的临床应用前景。4.本文关于DTX-LP的研究在国内外尚未见报道,具有独立自主知识产权,该研究成果将有助于推动前体脂质体制剂产品的开发,为肺癌、肺结核等肺部疾病的治疗提供更有效的给药系统。更多还原

【Abstract】 BackgroundThe high incidence of lung diseases such as lung cancer and tuberculosis is increasing in china. Our country has launched a lot of manpower and material resource into treatment of these intractable diseases. Though there are a series of important progression in the field of medicine therapy, the medicine results in high dosage, long period, serious side effects and drug tolerance because of the lack of target distribution in vivo. Lung-targeting delivery system is the ideal treatment strategies for lung diseases. Among the lung-targeting drug carrier, Liposomes, as a little toxic and well biocompatible carrier, more and more attention is being paid to them because they have many advantages such as sustained release drug, increasing tumor targeting, increasing pharmaceutical stability and improving Pharmacokinetics. But its research level, manufacture technique and industrialization need further improvement. Therefore, the key technology of liposome carrier delivery system research and industrialization are broken through, lung-targeting delivery liposome carrier system of lung cancer and tuberculosis drug and so on is developed, which are very necessary.ObjectiveThis study takes lung-targeting delivery system for treatment of non-small cell lung cancer and actual problem solved urgently in production as cut in point. Docetaxel （DTX） was chosen as model drug to prepare DTX-LP, and carried to lung through lung-targeting liposome carrier delivery system to improve therapy index and reduce side effects. Furthermore, the two key science problems of the bottleneck problem of production of liposome preparation and lung-targeting effect are effectively solved.Method1. A combination of solid dispersion and effervescent technology was used to prepare DTX-LP for the first time. Concretely speaking, first, the solid dispersion technology was used to prepare DTX proliposomes. Second, DTX-LP were obtained from DTX proliposomes by effervescent technology2. The morphology of liposome was observed by the light microscopy and transmission microscopy.3. The particle size and zeta-potential were determined by Malvern ZEN3600.4. HPLC analysis method was established and validated for analysis of DTX-LP preparation.5. Entrapment efficiency and the vitro release property of DTX-LP were studied by dialysis method.6. The significant influence factors on DTX-LP were studied using single factor method with particle size and morphology of liposome as index.7. The formulation and preparation of DTX-LP was optimized and initial determined using orthogonal design with particle size distribution, entrapment efficiency and stability as marker.8. HPLC analysis method was established and validated for determination of DTX in biosamples.9. On the basis of the initial optimized formulation and preparation, DTX-LP with various surface charges and particle size were prepared, and then the distribution of DTX was determined after intravenous administration of these liposomal DTX at 1.5h in rabbits to study lung-targeting effect and determined the best formulation and preparation.10. The vivo distribution and Pharmacokinetics of liposomal DTX were studied using pharmacokinetic method.11. Lung-targeting effect of liposomal DTX was evaluated with five targeting parameters as index.12. The toxity after intravenous administration of single-dose and series multiple dose of liposomal and injectable DTX was studied in rabbits.13. The transplantation tumor model of nude mice bearing A549 lung cancer was established to study inhibition effect of liposomal DTX on tumorResult一、Pharmacy study1. Formulation and preparation study: According to single factor, citric acid and sodium bicarbonate, surface temperature of solution on particle distribution, stability and entrapment efficiency was much significant. The initial optimized formulation and preparation was obtained using orthogonal design. The resulted DTX proliposomes were powder and （or） granules. The resulted DTX-LP were round in shape and dispersed well. Zeta-potential, particle size range and entrapment efficiency were about -23.4 mv, 0.90μm and 90.5%, respectively.2. Determination of the best formulation and preparation: On the basis of initial study, the best formulation and preparation was further determined with lung-targeting effect as marker by determination of vivo distribution of DTX-LP with various particle size and surface charges. The certain optimized formulation and preparation as follows: DTX, HSPC, cholesterol, tween-80 and citric acid were dissolved in ethanol with ultrasound instrument at 45℃. Then the solution was filtered through a 0.22μm hydrophobic membrane and placed in a round bottom flask. Under a constant stirring at 55℃, Sodium bicarbonate was added to evaporate organic to obtain solid granules （DTX proliposomes）. Appropriate DTX proliposomes （1g） were hydrated under the effervescence produced by introduction of 5% sodium bicarbonate solution to obtain DTX-LP.3. DTX-LP quality study: The study of quality of samples prepared by the best formulation and preparation showed that the DTX proliposomes had good flow property and were completely hydrated with 5% sodium bicarbonate solution for 15min. The pH value of the resulted DTX-LP ranged from 5.2 to 5.8. The liposomes were round and dispersed well. The particle size, zeta-potential and entrapment efficiency were 0.95±0.12μm, -23±0.21mv and 90.57±0.32%. The in vitro release study showed sustained release behavior. The sample was stable for at least one year at 2-8℃in sealed containers in a dark place.二、Pharmacology and toxicity studies1. Analysis of vivo biosamples: The selectivity of the HPLC conditions is good, sample preparation method is appropriate, no significant interference peaks was observed. The recoveries were 80.9-113.4% and the precision values （expressed as RSD） of intra-and inter were below 10% for these biosamples. The extraction recoveries were more than 70%. The HPLC method could meet biosamples analysis.2. Lung-targeting effect study: The effect of liposomal DTX with various mean diameter and zeta-potential on biodistribution in vivo showed that drug concentration in lung for negatively charged DTX-LP of about same particle size was much higher than that of possively charged DTX-LP. Furthermore, DTX-LP of about 1μm diameter with negative surface charge of greater than 20mv showed the highest tissue distribution of DTX in lung.3. Tissue distribution and lung-targeting evaluation: The result of distribution in rabbits showed that drug concentration of DTX-LP in lung was markedly higher than that of DTX injection at different time points.As far as targeting parameters are concerned, the Re value of lung in the case of the liposomal DTX was 28.91, which was much higher than that of other tissues and blood. It showed that the liposome carrier greatly increase lung-targeting effect. When the Te and the ratio of Te_{（liposomes）} to Te_{（injection）} of lung were 1.00, compared with liver, spleen, kidney, the Te value of lung increased by a factor of 4.96, 8.51, 18.37, respectively, and the ratio of Te increased by a factor of 3.16, 23.0, 27.83, respectively. It showed that the liposome carrier markedly enhance selectivity to lung. The value of Ce in lung increased by a factor of 74.28 compared with DTX injection in the case of DTX-LP, however, the Ce value in liver, spleen and kidney only increased by the factors of 2.93, 1.09 and 1.41, respectively. Therefore, it was known that the liposome carrier changed completely DTX biodistribution in vivo in rabbits; furthermore, the drug was greatly concentrated in lung. The TCe value in lung was 63.41 in the case of liposomal DTX, which was higher than that of other tissues. The distribution ratio of DTX in lung was 6-folds greater than that of injectable DTX in rabbits. It can be seen from TQe value that only 10.445% of total DTX content was distributed in lung for DIX injection, however, 52.746% of total DTX content was concentrated in lung for liposomal DTX. It was helpful to treat lung cancer and had clinical application.4. Pharmacokinetic study: In blood, after intravenous administration of liposomal and injectable DTX in rabbit, three-compartment model gave the best fit to the plasma drug concentration time curves. AUC_（0-t） was 0.893and 2.130mg/L^*h, respectively. The t_1/2α and t_1/2β value of injectable DTX were 0.122 and 0.207h, respectively. However, the parameter of t_1/2α （0.023h） of liposomal DTX was shortened, which showed that the DTX-LP could rapidly distribute to targeting organ-lung, and the parameter of t_1/2β （2.186h） was longer, which showed that DTX-LP could prolong drug action. In lung, the kinetic parameter of liposomal and injectable DTX showed that drug concentration-time curve fitted to a two-compartment model and three-compartment model,respectively. AUC_（0-t） was 2428.65 and 84.00mg/L^*h,respectively.The t_1/2α value were 1.414 and 0.081h, and t_1/2β value were 8.808 and 3.481h, respectively.It can be seen from these results that significant difference was observed in kinetic parameter of liposomal DTX in lungs and in plasma. For lung-targeting DTX-LP, the pharmacokinetic parameter in blood could not reflect pharmacokinetic behavior in target organ-lung.5. Toxicity study: survive time of DTX-LP group was about one time higher than that of DTX injection group. On the other hand, only DTX injection group, significant change in pathological biopsy of various tissues was observed. The result showed that DTX-LP could reduce toxicity of chemotherapeutic drug.6. Pharmacodynamic study: The result of pharmacodynamics showed that the inhibition rate to A549 lung cancer of injectable DTX （12 mg/kg） and liposomal DTX （3, 6, 12 mg/kg） were 75.72%, 61.48%, 94.40% and 95.40%, respectively.Conclusion1. In this study, a combination of solid dispersion and effervescent technology was used to prepare DTX-LP successfully for the first time. This technology overcame bottleneck problems of liposome such as small product batch and quality instability and so on, which showed good foreground of industrialization.2. The new viewpoint of negatively charged liposome of about 1μm diameter showed the greatest lung-targeting effect was reported for the first time3. DTX-LP prepared in this study was efficient lung-targeting delivery carrier and could completely changed pharmacokinetic behavior of DTX to decrease dosage and improve therapeutic index, and reduce side effects, which are important for clinical application.4. The study is totally new without any report at home and abroad up to now and has our own independent intellectual property. The research will be helpful to promote development of proliposome preparation products and provide a more efficient drug delivery system for treatment of lung cancer, tuberculosis and other lung disease.更多还原