【摘要】：肺癌是導(dǎo)致人類疾病的主要癌癥之一,嚴(yán)重威脅人類健康和生命,到2025年,中國將成為世界第一肺癌大國。肺癌有非小細(xì)胞肺癌(NSCLC)和小細(xì)胞肺癌兩大類,而肺癌中大約有85%為非小細(xì)胞癌。肺癌的治療方式和藥物選擇正日趨多樣化。其中具有高效、低毒、且具有一定靶向性的藥物,越來越成為研究熱點(diǎn)。 蓓薩羅丁(Bexarotene)為新型維甲酸類似物,其口服軟膠囊和外用凝膠劑獲FDA批準(zhǔn)于2000年在美國上市,用于治療皮膚T-細(xì)胞淋巴瘤。目前,國外正在開展蓓薩羅丁三期臨床試驗(yàn),主要用于治療非小細(xì)胞肺癌、乳腺癌及牛皮癬�；衔镙硭_羅丁對肺癌有顯著的治療效果,但是其屬于生物藥劑學(xué)分類系統(tǒng)(BCS) Class Ⅱ類化合物,溶解性差,生物利用度低,以及給藥靶向性不好等諸多因素嚴(yán)重的限制了其臨床的應(yīng)用。本課題主要以藥物蓓薩羅丁為研究重點(diǎn),以期解決上述難題,并為蓓薩羅丁開發(fā)為治療肺癌新的劑型提供基礎(chǔ)。 本課題研究以蓓薩羅丁為模型藥物,采用沉淀法與高壓均質(zhì)法結(jié)合(Precipitation-Microfluidisation)技術(shù)將蓓薩羅丁制備成納米制劑,構(gòu)建了藥物納米混懸劑給藥系統(tǒng),建立了蓓薩羅丁藥物體外以及其生物樣品的分析方法,考察其制劑學(xué)特征、大鼠體內(nèi)藥物動(dòng)力學(xué)以及小鼠體內(nèi)組織分布特征。并對納米制劑的體內(nèi)外抗腫瘤活性進(jìn)行考察,為肺癌靶向制劑的開發(fā)提供理論和實(shí)驗(yàn)依據(jù)。本研究的主要內(nèi)容包括： 1、蓓薩羅丁納米混懸劑處方前研究 根據(jù)處方前研究,利用紫外分光光度法,對蓓薩羅丁樣品進(jìn)行紫外掃描,確定了蓓薩羅丁藥物最適宜的檢測波長,建立了藥物的高效液相測定方法,方法學(xué)考察結(jié)果表明,蓓薩羅丁在濃度范圍0.05～40.0μg·mL-1線性關(guān)系良好含量測定方法的日內(nèi)、間精密度、穩(wěn)定性、重現(xiàn)性以及回收率均符合方法學(xué)考察的要求。 2、蓓薩羅丁納米混懸劑的工藝研究與處方優(yōu)化 采用正交設(shè)計(jì)法蓓薩羅丁納米混懸劑進(jìn)行處方優(yōu)化篩選,以輔料的用量為考察因素,以不同穩(wěn)定劑對納米混懸劑Zeta-電位的影響作為篩選指標(biāo)。實(shí)驗(yàn)結(jié)果顯示,各因素對Zeta-電位的影響大小順序依次為：卵磷脂Pluronic F68＞PVPK30。制備得到納米混懸劑屬于熱力學(xué)不穩(wěn)定系統(tǒng),為提高納米混懸劑的穩(wěn)定性,采用冷凍干燥法將其制備成了凍干粉。在凍干過程中,為保證蓓薩羅丁納米混懸劑良好的分散性和物理化學(xué)穩(wěn)定性,對其凍干工藝進(jìn)行考察。通過對蓓薩羅丁納米混懸劑共晶點(diǎn)的測定和凍干保護(hù)劑的篩選,確定冷凍干燥工藝為：采用沉淀法進(jìn)行預(yù)處理,同時(shí)結(jié)合高壓均質(zhì)技術(shù)制備蓓薩羅丁納米混懸劑,加入5%甘露醇作為凍干保護(hù)劑,置冷凍干燥機(jī)中,凍干24h,得到納米混懸劑凍干粉(NC-Bexarotene)。 3、蓓薩羅丁納米混懸劑制劑學(xué)性質(zhì)的研究 經(jīng)過凍干工藝制備得到蓓薩羅丁納米混懸劑之后,對其進(jìn)行制劑學(xué)性質(zhì)的考察。采用DelsaTM Nano C Particle Analyzer測得NC-Bexarotene的粒徑及Zeta-電位；對蓓薩羅納米混懸劑進(jìn)行掃描電子顯微鏡(SEM)觀察,從納米混懸劑NC-Bexarotene放大倍數(shù)為5,000和10,000的圖像中可以看出,納米混懸劑粒度分布較均勻,且外觀呈不規(guī)則的顆粒狀；透射電鏡(TEM)觀察結(jié)果表明蓓薩羅丁納米混懸劑基本上呈完整球形外觀,且粒度分布均一；從NC-Bexarotene原子力顯微鏡(AFM)分析的3D表面形態(tài)圖像中可以看出,納米制劑在水中復(fù)溶后并沒有出現(xiàn)聚集的現(xiàn)象,說明制劑凍干之后是穩(wěn)定的。 將藥物與輔料處方比例的物理混合物,NC-Bexarotene樣品和三個(gè)月后的NC-Bexarotene進(jìn)行差示掃描量熱分析(DSC), X-射線衍射(?)XRPD)測定和共聚焦顯微拉曼光譜檢測。DSC曲線中可以看到各樣品中的蓓薩羅丁有相同的吸熱峰；蓓薩羅丁的特征峰均可以在X-射線衍射(XRPD)的衍射圖譜中找到；在拉曼譜圖中,我們可以看到1607cm-1的峰顯示的是蓓薩羅丁的C=C。根據(jù)DSC,XRPD和拉曼譜圖,可以充分的表明在以沉淀法-高壓均質(zhì)法制備制劑以及冷凍干燥的過程中蓓薩羅丁的晶型并沒有發(fā)生改變,體現(xiàn)出了良好的物理化學(xué)穩(wěn)定性。 用恒溫電磁攪拌法對NC-Bexarotene的飽和溶解度進(jìn)行考察,實(shí)驗(yàn)結(jié)果表明其PBS (pH=7.4)中溶解度顯著提高。體外溶出采用槳法進(jìn)行測定,結(jié)果表明與原料藥相比,納米制劑很大程度的改善了蓓薩羅丁的溶出速率。實(shí)驗(yàn)中為了對照納米制劑和已上市軟膠囊藥物(Soft Gelatin Capsule, SGC)的體外釋放情況,采用透析的方法進(jìn)行比較,納米制劑和已上市軟膠囊藥物的體外釋放實(shí)驗(yàn)結(jié)果表明,納米制劑的釋放速度比SGC有明顯的優(yōu)越性。NS-Bexarotene和NC-Bexarotene的初步穩(wěn)定考察以外觀,粒徑以及分布為指標(biāo),結(jié)果證明了將NS-Bexarotene制備成NC-Bexarotene顯著的增加了制劑的穩(wěn)定性。 4、蓓薩羅丁納米混懸劑在大鼠體內(nèi)的藥代動(dòng)力學(xué)研究 本文建立了蓓薩羅丁生物樣品的HPLC檢測方法,考察了NC-Bexarotene分別經(jīng)口服給藥和靜脈注射給藥后在大鼠體內(nèi)的藥物動(dòng)力學(xué)過程,探討了納米制劑在改善藥物動(dòng)力學(xué)特征方面所起到的作用。 分別研究了經(jīng)灌胃給藥以及靜脈內(nèi)給藥后藥物在大鼠體內(nèi)的藥代動(dòng)力學(xué)行為,根據(jù)口服藥動(dòng)學(xué)參數(shù)具體分析,蓓薩羅丁原料藥對照組和納米混懸劑NC-Bexarotene組口服給藥均符合二室模型。根據(jù)藥動(dòng)學(xué)參數(shù)分析,NC-Bexarotene制劑組的半衰期T1/2明顯長于溶液組；而NC-Bexarotene混懸劑組AUC0-∞為接近溶液組的2倍,納米釋藥系統(tǒng)維持相對持久且平穩(wěn)的藥-時(shí)曲線,主要由于其具有良好的生物黏附性的特點(diǎn),延長了藥物在胃腸道的滯留時(shí)間,很大程度提高了藥物生物利用度。而經(jīng)靜脈注射后,藥物能夠迅速進(jìn)入到血液循環(huán),NC-Bexarotene以注射用水為溶劑,避免了有機(jī)溶劑對機(jī)體的刺激和損害,比起蓓薩羅丁溶液組有更好的順應(yīng)性。 5、納米混懸劑在小鼠體內(nèi)組織分布的研究 為了考察納米制劑對藥物體內(nèi)分布特征的改變,該實(shí)驗(yàn)研究了NC-蓓薩羅丁制劑在小鼠體內(nèi)組織分布的特征。對口服后藥物在血漿的動(dòng)力學(xué)過程進(jìn)行分析,與Bexarotene-Sol相比,NC-Bexarotene明顯的降低了藥物的初始濃度,并且使藥-時(shí)曲線更加平穩(wěn)且持久。MRT從3.82h增加到了16.58h, AUC從17.02h·μg·g-1增加至103.2h·μg·g-1;而注射組的MRT變化卻不大,但AUC由12.68h·μg·g-1提高到了18.65h·μg·g-1。與Bexarotene-Sol相比,不論通過口服給藥還是經(jīng)靜脈注射給藥,NC-Bexarotene均可降低心臟以及腎臟中最高藥物濃度,可以避免過高的藥物濃度對心臟以及腎臟機(jī)能產(chǎn)生不良影響。由于NC-Bexarotene明顯延長了藥物在體內(nèi)的滯留時(shí)間,Bexarotene在心臟中的MRT也由1.96h增加到3.15h(口服),1.73h增加到2.90h(注射),但在心臟中的分布百分率與溶液相比卻大大降低。根據(jù)肝臟組織分布結(jié)果,口服過程中,NC-Bexarotene在肝臟中的MRT雖也有所延長,但藥物AUC變化并不明顯,而且分布百分比也并不突出；注射組中,MRT略有延長,AUC相對于Bex-Bexarotene有一定的提高,分布百分率也增加�？诜约白⑸銷C-Bexarotene之后,藥物在脾中的分布均較溶液組有明顯的提高,藥物在脾中的AUC、AUQ值明顯增大,MRT值也明顯的延長,rCe為1.50(口服組)和1.25(注射組)均較肝臟的靶向性明顯,但從藥物的分布百分比圖中可看出腎臟中藥物百分比明顯低于其他臟器。在肺組織分布中,藥物在肺中的分布情況幾乎與脾中的分布情況相似。不論是AUC、AUQ還是MRT均得到增加,口服中AUC由48.25增加到79.93,MRT由1.96延長到3.80；注射組中,AUC由32.85增加到54.94,MRT由2.69延長至6.34。rCe為1.7(肺脾肝)。而且根據(jù)相對分布百分比率圖比較,相比于溶液組有明顯的提高。與溶液組相比,NC-Bexarotene幾乎在各時(shí)間點(diǎn)的腎臟濃度均有明顯的降低,因此將蓓薩羅丁制備成NC-Bexarotene后有利于降低Bexarotene可能帶來的腎臟毒性。 6、蓓薩羅丁納米混懸劑對人肺腺癌細(xì)胞A549及其裸鼠移植瘤的藥效學(xué)考察 從細(xì)胞學(xué)實(shí)驗(yàn)和在體裸鼠移植瘤實(shí)驗(yàn)結(jié)果表明,蓓薩羅丁和蓓薩羅丁納米混懸劑可抑制人體肺腺癌上皮A549細(xì)胞系增殖,蓓薩羅丁納米混懸劑作用相對較強(qiáng)；蓓薩羅丁納米混懸劑顯著誘導(dǎo)人肺腺癌上皮A549細(xì)胞的凋亡,細(xì)胞周期的G1期阻滯；通過細(xì)胞滲漏乳酸脫氫酶(LDH)含量測定證實(shí)蓓薩羅丁和蓓薩羅丁納米混懸劑對人體肺腺癌上皮A549細(xì)胞沒有明顯的毒性作用；在給藥期間,荷瘤模型組裸鼠的體重下降更明顯,蓓薩羅丁納米混懸劑組裸鼠死亡率最低；在給藥期間,蓓薩羅丁和蓓薩羅丁納米混懸劑可以抑制裸鼠移植瘤瘤體積的增長,但與荷瘤模型組比較作用不顯著；給藥第13天和第17天之后,蓓薩羅丁納米混懸劑組可明顯抑制裸鼠移植瘤相對體積RTV；解剖后離體的裸鼠移植瘤體積和重量,蓓薩羅丁納米混懸劑組最低,但各組間無顯著性差異。從在體實(shí)驗(yàn)結(jié)果看,要達(dá)到更有效的治療效果,還需要延長給藥時(shí)間,但可能由于人肺腺癌上皮A549細(xì)胞的侵襲性比較強(qiáng),給藥后期裸鼠死亡的比例加大,裸鼠的體重偏低,特別是荷瘤模型組的裸鼠。相對蓓薩羅丁,蓓薩羅丁納米混懸劑有更好的抑制人體肺腺癌上皮A549細(xì)胞增殖、誘導(dǎo)凋亡、引起細(xì)胞周期阻滯的作用,荷瘤鼠死亡率低,無明顯的毒副作用。 本課題首次構(gòu)建了蓓薩羅丁納米混懸劑釋藥系統(tǒng),首次對蓓薩羅丁納米混懸劑的口服以及注射制劑在動(dòng)物體內(nèi)的藥物動(dòng)力學(xué),組織分布特征進(jìn)行了評價(jià),并首次對蓓薩羅丁納米混懸劑對人肺腺癌細(xì)胞A549及其裸鼠移植瘤的藥效學(xué)進(jìn)行考察,本文豐富了納米釋藥系統(tǒng)被動(dòng)靶向的研究內(nèi)容,對蓓薩羅丁口服納米制劑以及注射制劑的開發(fā)和應(yīng)用提供了一定實(shí)驗(yàn)參考,對抗癌藥物蓓薩羅丁的臨床開發(fā)有著重要的意義。
[Abstract]:Lung cancer is one of the main cancers causing human diseases, which seriously threatens human health and life. By 2025, China will become the world's largest lung cancer country. Lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer, and about 85% of lung cancer is non-small cell cancer. Drugs with high efficiency, low toxicity and certain targeting properties have increasingly become a research hotspot.
Bexarotene is a new retinoic acid analogue. Its oral soft capsules and topical gels were approved by FDA to be marketed in the United States in 2000 for the treatment of cutaneous T-cell lymphoma. It has a significant therapeutic effect on lung cancer, but it belongs to Class II compounds of Biopharmaceutics Classification System (BCS). Its poor solubility, low bioavailability, and poor drug targeting seriously limit its clinical application. Beza Rodin provides a basis for the development of new dosage forms for lung cancer.
In this study, Besarodine was used as a model drug to prepare nanoparticles by precipitation and high pressure homogenization (Precipitation-Microfluidisation) technology. The drug nanosuspension delivery system was constructed, and the analytical method of Besarodine in vitro and its biological samples was established. Pharmacokinetics in vivo and tissue distribution in mice were studied. The antitumor activity of nanoparticles in vitro and in vivo was investigated to provide theoretical and experimental basis for the development of lung cancer targeting agents.
1, pre formulation study of Bei sardin nanosulfan.
According to the pre-prescription study, the Besarodine samples were scanned by ultraviolet spectrophotometry, and the optimum detection wavelength was determined. A high performance liquid chromatographic method for the determination of Besarodine was established. The results of methodological investigation showed that Besarodine had a good linear relationship in the concentration range of 0.05-40.0 ug.mL-1. Intraday density, stability, reproducibility and recovery were in line with methodological requirements.
2, the technology research and Prescription Optimization of Bei sardin nano suspensions.
The orthogonal design method was used to optimize the formulation of Bexarodin nanosuspension. The influence of different stabilizers on Zeta-potential of the nanosuspension was taken as the screening index. The results showed that the order of influence of various factors on Zeta-potential was lecithin Pluronic F68 > PVPK30. In order to improve the stability of nano-suspension, freeze-drying method was used to prepare freeze-dried powder. In order to ensure good dispersion and physicochemical stability of nano-suspension, the freeze-drying process was investigated. The determination of eutectic point and the selection of freeze-drying protectant were carried out. The freeze-drying process was determined as follows: pretreatment by precipitation method, preparation of Bexarotine nanosuspension by high-pressure homogenization technology, adding 5% mannitol as freeze-drying protectant, freeze-drying machine, and freeze-drying for 24 hours to obtain NC-Bexarotene nanosuspension powder.
3, the study of the properties of Bei sardin nano suspensions.
Bexarotine nanosuspension was prepared by freeze-drying process and its pharmacological properties were investigated. The particle size and Zeta-potential of NC-Bexarotene were measured by DelsaTM Nano-C Particle Analyzer, and the magnification of NC-Bexarotene nanosuspension was observed by scanning electron microscopy (SEM). The results of transmission electron microscopy (TEM) showed that the particle size distribution of the nanosuspension was uniform and the appearance was irregular. The results of TEM showed that the nanosuspension was basically spherical and uniform in size distribution. The surface morphology of the nanosuspension was analyzed by NC-Bexarotene atomic force microscopy (AFM). It can be seen that there is no aggregation of nanoparticles in water after re-dissolution, indicating that the preparation is stable after lyophilization.
Differential scanning calorimetry (DSC), X-ray diffraction (?) XRPD and confocal microscopic Raman spectroscopy were used to determine the ratio of drug to excipient, NC-Bexarotene samples and NC-Bexarotene samples three months later. The peaks can be found in the X-ray diffraction (XRPD) spectra. In the Raman spectra, we can see that the peak of 1607 cm-1 shows C=C of Besarodine. According to DSC, XRPD and Raman spectra, it can be fully shown that the crystalline form of Besarodine prepared by precipitation-high pressure homogenization method and freeze-drying process is not found. The change shows good physical and chemical stability.
The saturated solubility of NC-Bexarotene was investigated by electromagnetic stirring at constant temperature. The results showed that the solubility of NC-Bexarotene in PBS (pH=7.4) was significantly improved. The dissolution of NC-Bexarotene in vitro was determined by paddle method. The results showed that the dissolution rate of NC-Bexarotene was greatly improved by nano-preparation compared with the raw material. In vitro release of marketed soft gelatin capsule (SGC) was compared by dialysis. The results of in vitro release experiments of nano-preparations and marketed soft capsules showed that the release rate of nano-preparations was obviously superior to that of SGC. The results showed that the preparation of NC-Bexarotene from NS-Bexarotene significantly increased the stability of the preparation.
4, pharmacokinetics of Beza Rodin nano suspensions in rats
A method for the determination of Bexarotine in biological samples by HPLC was developed. The pharmacokinetic process of NC-Bexarotene in rats after oral administration and intravenous administration was investigated. The role of nanoparticles in improving the pharmacokinetic characteristics was discussed.
The pharmacokinetic behavior of the drug in rats after intragastric administration and intravenous administration was studied. According to the specific analysis of oral pharmacokinetic parameters, the two compartment model was established in the control group of Bexarotine and the NC-Bexarotene group. Phase T1/2 was significantly longer than that of the solution group, while AUC0-uuuuuuuuuuuuuuuuuuuuuuuuuuu After intravenous injection, the drug can quickly enter the blood circulation, NC-Bexarotene injection of water as a solvent, to avoid organic solvent stimulation and damage to the body, compared with the Bexarotine solution group has better compliance.
5, the tissue distribution of nanossuspensions in mice.
To investigate the effect of nanoparticles on the distribution of NC-Bexarotine in mice, the tissue distribution of NC-Bexarotine in mice was studied. MRT increased from 3.82 hours to 16.58 hours, AUC increased from 17.02 hours to 103.2 hours. MRT did not change much in the injection group, but AUC increased from 12.68 hours to 18.65 hours. Because NC-Bexarotene significantly prolonged the drug retention time in the body, the MRT of Bexarotene in the heart increased from 1.96 h to 3.15 h (oral) and from 1.73 h to 2.90 h (injection), but the percentage of distribution in the heart increased from 1.96 h to 2.90 h (injection). According to the results of liver tissue distribution, the MRT of NC-Bexarotene in the liver was prolonged, but the change of AUC was not obvious and the percentage of distribution was not prominent. In the injection group, the MRT was slightly prolonged, the percentage of distribution of AUC was increased compared with that of Bex-Bexarotene. After taking NC-Bexarotene and injecting NC-Bexarotene, the distribution of the drug in spleen was significantly higher than that of the solution group, the AUC and AUQ values in spleen were significantly increased, and the MRT values were significantly prolonged. Both rCe 1.50 (oral group) and 1.25 (injection group) were more targeted to the liver, but the percentage distribution of the drug in kidney could be seen from the drug percentage map. The distribution of drugs in lung tissue was similar to that in spleen. Both AUC, AUQ and MRT increased, AUC increased from 48.25 to 79.93, MRT from 1.96 to 3.80 in oral administration, AUC increased from 32.85 to 54.94 in injection group, and MRT from 2.69 to 6.34.rCe to 1.7 (lung, spleen and liver). Compared with the solution group, the concentration of NC-Bexarotene in the kidney decreased significantly at almost every time point. Therefore, the preparation of Bexarotene into NC-Bexarotene can reduce the possible renal toxicity of Bexarotene.
6, the pharmacodynamics of Bei sardin nano suspension on human lung adenocarcinoma cell A549 and nude mice transplanted tumor.
Cytological and xenograft tumors in nude mice showed that besarotine and besarotine nanosuspensions could inhibit the proliferation of human lung adenocarcinoma epithelial A549 cell line, and the effect of besarotine nanosuspension was relatively strong. The results showed that Besarodine and Besarodine nanosuspensions had no obvious toxic effect on human lung adenocarcinoma epithelial A549 cells. During the administration period, the weight of nude mice in tumor-bearing model group decreased more significantly, and the mortality of nude mice in Besarodine nanosuspension group was the lowest. Besarodine and Besarodine nanosuspensions inhibited the growth of tumor volume in nude mice, but had no significant effect compared with the tumor-bearing model group; Besarodine nanosuspension group significantly inhibited the relative volume of tumor RTV in nude mice after 13 and 17 days of administration; Anatomically isolated tumor volume and weight in nude mice Salodine nanosuspension group was the lowest, but there was no significant difference between the groups. From the in vivo experimental results, to achieve more effective treatment, we need to extend the administration time, but it may be due to the strong invasiveness of human lung adenocarcinoma epithelial A549 cells, increase the proportion of death in nude mice, nude mice with low body weight, especially tumor-bearing model. Compared with Besarodine, Besarodine nanosuspension could inhibit the proliferation of human lung adenocarcinoma epithelial A549 cells, induce apoptosis, and cause cell cycle arrest. The mortality of tumor-bearing mice was low without obvious side effects.
The release system of Besarodine nanosuspension was constructed for the first time. The pharmacokinetics and tissue distribution of Besarodine nanosuspension were evaluated in vivo for the first time. The pharmacodynamics of Besarodine nanosuspension on human lung adenocarcinoma cell line A549 and its transplanted tumor in nude mice were studied for the first time. This study enriched the research contents of passive targeting of nanodrug delivery system, oral nanopreparations and injection preparation of Besarodine.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R734.2;R943

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