本文選題：超聲造影劑 + 微泡��； 參考：《南方醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究背景 超聲造影劑(Ultrasound Contrast Agents, UCAs)是一類能夠顯著增強(qiáng)醫(yī)學(xué)超聲檢測(cè)信號(hào)的診斷藥物,是一種含有高濃度微氣泡的制劑。包裹氣體的微泡制劑具有強(qiáng)烈的超聲波散射性能,經(jīng)靜脈注射到達(dá)體內(nèi)各器官微循環(huán)后,可使超聲回波信號(hào)顯著增強(qiáng),組織、器官圖像質(zhì)量顯著改善,從而大大提高超聲診斷效果。UCAs的出現(xiàn)顯著提高了超聲診斷的敏感性與特異性,進(jìn)一步拓展了超聲診斷領(lǐng)域,使超聲診斷由“平掃時(shí)代”進(jìn)入“增強(qiáng)時(shí)代”,被譽(yù)為超聲診斷領(lǐng)域的第三次革命。更值得注意的是,對(duì)于某些診斷而言,它們將是幫助超聲有效競(jìng)爭MRI、CT的關(guān)鍵技術(shù)之一。 新型UCAs的制備與應(yīng)用研究是近年來超聲醫(yī)學(xué)研究的熱點(diǎn)與前沿。UCAs的發(fā)展經(jīng)歷了從第1代包裹空氣的微泡制劑到第2代包裹氟碳類惰性氣體的微泡制劑,造影劑在穩(wěn)定性和有效性方面均取得了突破性的進(jìn)展。目前,UCAs研制的重要發(fā)展方向是成膜材料的改進(jìn),目的是使造影劑使用更安全、造影持續(xù)時(shí)間更長。從UCAs研制的歷史看,九十年代初期研制的造影劑多采用人血白蛋白作為成膜材料,九十年代后期開始向脂質(zhì)材料或多聚體材料方向發(fā)展。原因在于,從安全方面考慮,人血白蛋白作為血液制品存在傳播血液傳播性疾病的可能,安全性不如脂膜造影劑；從造影效果看,脂膜造影劑由大分子脂質(zhì)構(gòu)成的脂膜比變性白蛋白形成的膜更穩(wěn)定、更有彈性,造影效果更好,持續(xù)時(shí)間更長,更能滿足臨床的需要。因此,脂質(zhì)微泡的研發(fā)是目前UCAs發(fā)展的主流。 UCAs市場(chǎng)是一個(gè)新興而蓬勃發(fā)展的市場(chǎng),國外已有的UCAs價(jià)格十分昂貴,因此,在國內(nèi)研發(fā)UCAs具有重要價(jià)值。歐美國家已經(jīng)先后開發(fā)出新一代的脂質(zhì)UCAs,如SonoVue、Definity等,目前UCAs市場(chǎng)均被歐美國家所壟斷。國內(nèi)UCAs的研究以我科為先導(dǎo),先后開發(fā)出“東冠注射液”、“全氟顯”等以白蛋白為外殼的UCAs,其聲學(xué)活性在許多方面均優(yōu)于國外同類產(chǎn)品。由于與人血白蛋白UCAs相比,脂質(zhì)UCAs存在著明顯的優(yōu)勢(shì),所以后續(xù)開發(fā)上市的造影劑都以脂質(zhì)UCAs為主。因此,本課題組正致力進(jìn)行新型脂質(zhì)微泡UCAs的研究開發(fā)工作,以填補(bǔ)國內(nèi)脂質(zhì)UCAs市場(chǎng)的空白。 目的 1對(duì)脂質(zhì)微泡UCAs的處方、制備工藝進(jìn)行改進(jìn)、優(yōu)化,以制備出高濃度、粒徑適宜、穩(wěn)定有效的脂質(zhì)微泡制劑,為脂質(zhì)UCAs勺新藥研發(fā)提供試驗(yàn)依據(jù)。 2對(duì)脂質(zhì)微泡UCAs的理化性質(zhì)進(jìn)行評(píng)價(jià),包括其外觀形態(tài)、濃度、粒徑、分布、內(nèi)含氣體全氟丙烷含量測(cè)定、耐壓穩(wěn)定性、以及液體制劑貯存穩(wěn)定性,以初步探索脂質(zhì)UCAs的質(zhì)量標(biāo)準(zhǔn)。 3進(jìn)一步探討脂質(zhì)微泡UCAs的動(dòng)物藥效學(xué)特性,對(duì)其在新西蘭大白兔肝和腎的顯影效果進(jìn)行評(píng)價(jià)。 方法 1工藝參數(shù)及處方優(yōu)化研究 1.1工藝優(yōu)化 采用高速剪切法制備脂質(zhì)微泡UCAs,單因素試驗(yàn)分析脂質(zhì)微泡的制備工藝參數(shù),包括剪切速度、剪切時(shí)全氟丙烷氣體通氣時(shí)間、剪切時(shí)間。每個(gè)因素分別設(shè)不同水平,剪切速度(4檔、5檔、6檔)、剪切時(shí)全氟丙烷氣體通氣時(shí)間(15s、30s、45s、60s)、剪切時(shí)間(90s、110s、120s、130s、150s),以微泡的濃度、平均粒徑為指標(biāo),通過方差分析優(yōu)選出最佳工藝參數(shù)。 1.2處方優(yōu)化 采用優(yōu)選的剪切工藝參數(shù),以混合磷脂為成膜材料,全氟丙烷氣體為內(nèi)核材料制備脂質(zhì)微泡。磷脂總濃度、DPPE-PEG5000的比例、DPPA的比例以及NaCl濃度是影響脂質(zhì)微泡濃度及粒徑的重要因素,因此,以磷脂總濃度(A)、DPPE-PEG5000匕例(B)、DPPA比例(C)、NaCl用量(D)作為自變量,以脂質(zhì)微泡的濃度作為因變量,進(jìn)行正交設(shè)計(jì)L9(34),篩選出脂質(zhì)微泡UCAs的最優(yōu)配方,為脂質(zhì)UCAs的新藥研發(fā)提供試驗(yàn)依據(jù)。 2脂質(zhì)微泡UCAs的理化特性評(píng)價(jià) 2.1微泡外觀形態(tài)觀察 取脂質(zhì)微泡混懸液滴加于載玻片上,蓋上蓋玻片,于光學(xué)顯微鏡下觀察其外觀形態(tài)。 2.2庫爾特計(jì)數(shù)器檢測(cè) 采用50μm小孔管,0.9%NaCl電解液,分析體積500μl,粒徑測(cè)量范圍1.1μm～30μm。取微泡混懸液20μl,用生理鹽水稀釋5000倍,庫爾特計(jì)數(shù)器測(cè)定微泡的濃度、平均粒徑及其粒徑分布。 2.3全氟丙烷氣體含量測(cè)定 氣相色譜-質(zhì)譜聯(lián)用儀測(cè)定制劑中全氟丙烷氣體的含量。氣相色譜條件：色譜柱為DB-5ms(30m×250μm×0.25μm),柱溫35℃,載氣為氦氣,柱流速為0.8ml/min。質(zhì)譜條件：EI電離源,倍增電壓1900ev,選擇離子：m/z(質(zhì)荷比)69、169。樣品處理：分別精密吸取2ml微泡溶液,注入裝有500ml高純氮?dú)獾牟蓺獯鼉?nèi),于超聲破碎儀上水浴超聲10min使微泡完全破裂釋放出OFP氣體。吸取50μl進(jìn)行氣相色譜-質(zhì)譜檢測(cè),外標(biāo)法算出氣體含量。 2.4耐壓穩(wěn)定性 用5ml注射器抽取4ml脂質(zhì)微泡混懸液,通過三通管連接eagle4000監(jiān)護(hù)儀,分別持續(xù)施加150mmHg、300mmHg壓力1min、3min、5min、10min,再用庫爾特計(jì)數(shù)器測(cè)量其濃度、粒徑及分布。 2.5液體制劑貯存穩(wěn)定性 制備脂質(zhì)微泡6批,于4℃±2℃冰箱保存,分別于第0月,1月、2月、3月、6月取樣品用庫爾特計(jì)數(shù)器分析其濃度、平均粒徑及分布,以研究液體微泡的貯存穩(wěn)定性。 3藥效學(xué)評(píng)價(jià) 3.1給藥劑量選擇 取新西蘭大白兔3只,分別耳緣靜脈給藥0.005、0.01、0.02、0.06、0.1ml/kg,分別進(jìn)行腎臟、肝臟超聲造影,隨機(jī)安排各劑量組的給藥次序,每個(gè)劑量組重復(fù)給藥測(cè)定5次,記錄圖像數(shù)據(jù)至造影效果出現(xiàn)明顯減退為止。采用儀器自帶的分析軟件Q-lab對(duì)圖像進(jìn)行分析。 3.2造影效果評(píng)價(jià) 取新西蘭大白兔3只,脂質(zhì)微泡UCAs給藥劑量為0.01ml/kg,分別進(jìn)行腎臟、肝臟超聲造影,每個(gè)臟器檢查重復(fù)3次,記錄圖像數(shù)據(jù)至造影圖像廓清為止。采用儀器自帶的分析軟件Q-lab對(duì)圖像進(jìn)行分析。 結(jié)果 1按單因素試驗(yàn)考察脂質(zhì)微泡制備工藝相關(guān)參數(shù),得出全氟丙烷脂質(zhì)微泡最佳的制備工藝參數(shù)為：剪切檔數(shù)6檔,通氣時(shí)間30s,剪切時(shí)間2min。 2按正交設(shè)計(jì)方法進(jìn)行制劑處方優(yōu)化實(shí)驗(yàn),得出全氟丙烷脂質(zhì)微泡最佳制備處方：磷脂總濃度為1×10-3mol/L, DPPE-PEG5000比例為8%,DPPA比例為10%,NaCl用量為4.5mg/ml。 3按最優(yōu)工藝制備的脂質(zhì)微泡混懸液為白色乳狀液,光學(xué)顯微鏡下可見微泡呈透亮球形,分布均勻,彼此無聚集現(xiàn)象。微泡濃度為(2.99±0.19)×109個(gè)/ml,平均粒徑為(2.46±0.05)μm,97%微泡粒徑小于7μm,OFP含量為(387.81±35.28)1μg/ml(每毫升脂質(zhì)微泡OFP含量)。 4微泡的耐壓穩(wěn)定性良好,耐受150mmHg壓力后,各時(shí)間組濃度并無顯著性差異(P=0.051)；耐受300mmHg壓力后在5min和10min時(shí)與對(duì)照組(0min)相比濃度有顯著性差異(P0.001),但濃度仍大于2×109個(gè)/ml,仍能滿足超聲顯影要求。 5對(duì)脂質(zhì)微泡混懸液進(jìn)行6個(gè)月的穩(wěn)定性考察,貯存于4±2℃冰箱中,在3個(gè)月內(nèi)微泡濃度、平均粒徑?jīng)]有顯著性變化(P均0.05)。第6個(gè)月時(shí),微泡的濃度和粒徑都有所下降,與剛制備時(shí)(0月)相比差異顯著(P0.001),但濃度仍大于2×109個(gè)/ml,平均粒徑也仍然滿足最佳粒徑要求。 6藥效學(xué)實(shí)驗(yàn)表明：脂質(zhì)微泡UCAs最佳給藥劑量為0.01ml/kg。脂質(zhì)微泡給藥劑量為0.01ml/kg時(shí),所有實(shí)驗(yàn)兔均獲得滿意的腎臟、肝臟聲學(xué)圖像,造影劑填充均勻,與周圍組織分界清晰。脂質(zhì)微泡腎臟造影時(shí),其峰值減半時(shí)間為603±47s,廓清時(shí)間為726±6s；肝臟造影時(shí)其峰值減半時(shí)間為388±97s,廓清時(shí)間為718±89s,可以滿足臨床應(yīng)用要求。 結(jié)論 1用混合磷脂和全氟丙烷氣體作基本原料,經(jīng)高速剪切分散處理水合磷脂可制備出直徑小于7μm、濃度大于2.0×109個(gè)/ml、穩(wěn)定性較好的全氟丙烷脂質(zhì)微泡。 2使用庫爾特顆粒分析儀測(cè)定脂質(zhì)微泡的濃度、粒徑及分布,方法簡便可靠,是對(duì)脂質(zhì)微泡進(jìn)行質(zhì)量控制的主要手段。 3脂質(zhì)微泡中全氟丙烷氣體是其超聲顯影的主要成分,其含量是制劑質(zhì)控的關(guān)鍵指標(biāo)。氣相色譜—質(zhì)譜聯(lián)用法測(cè)定全氟丙烷氣體含量,具有簡便、快速、靈敏、準(zhǔn)確、精密度高等優(yōu)點(diǎn),可作為全氟丙烷類UCAs的有效質(zhì)控手段。 4低溫保存有利于脂質(zhì)微泡制劑的穩(wěn)定。 5制備的脂質(zhì)微泡在體外具有良好的耐壓能力,提示其在人體血管內(nèi)應(yīng)用后將有良好的穩(wěn)定性,可以達(dá)到較好的造影效果。 6制備的脂質(zhì)微泡對(duì)新西蘭大白兔腎臟、肝臟超聲造影圖像清晰,顯影時(shí)間長,提示其在動(dòng)物應(yīng)用后具有良好的顯影效果。
[Abstract]:Background of the study

Ultrasound Contrast Agents ( UCAs ) is a kind of diagnostic medicine capable of significantly enhancing the medical ultrasonic detection signal . It is a kind of preparation with high concentration of micro - bubbles .

The research on the preparation and application of new UCAs is the hot spot and the leading edge of the research of ultrasonic medicine in recent years . The development of UCAs has experienced breakthrough in the stability and effectiveness . At present , the important development direction of UCAs is the improvement of film - forming material .
The results showed that the lipid membrane composed of macromolecular lipids was more stable , more elastic , better in contrast , longer in duration and more responsive to clinical needs .

UCAs market is a new and booming market , UCAs is very expensive in foreign countries . As a result , UCAs has been developed by European and American countries .

Purpose

1 . The formulation and preparation technology of lipid vesicles UCAs were improved and optimized to prepare lipid vesicles with high concentration and proper particle size , which provided experimental basis for the research and development of lipid UCAs spoon .

2 The physical and chemical properties of UCAs were evaluated , including its appearance , concentration , particle size , distribution , content determination of perfluoropropane content , stability of pressure resistance , and storage stability of liquid preparation , so as to explore the quality standard of UCAs .

3 . To further study the pharmacodynamic properties of lipid vesicles UCAs , and to evaluate the development of liver and kidney of rabbits in New Zealand .

method

1 Process Parameter and Prescription Optimization Study

1.1 Process Optimization

The preparation process parameters of lipid microvesicles were prepared by high - speed shear method . The parameters of the preparation of lipid vesicles were analyzed by single factor test , including shear rate , shear rate ( 4 , 5 , 6 ) , perfluoropropane gas aeration time ( 15 s , 30s , 45s , 60s ) , shear time ( 90s , 110s , 120s , 130s , 150s ) , and the best process parameters were determined by variance analysis .

1.2 Formulation Optimization

The perfluoropropane gas in 3 lipid vesicles is the main component of its ultrasonic development . The content of perfluoropropane gas is the key indicator of quality control of the preparation . The content of perfluoropropane gas is determined by GC - MS . It is simple , rapid , sensitive , accurate and high in precision . It can be used as an effective control means of perfluoropropane UCAs .

Evaluation of physical and chemical properties of lipid vesicles UCAs

2.1 Observation of Appearance of Microbubbles

The lipid microbubble suspension is applied to the slide glass , the cover glass is covered on the cover glass , and the appearance of the glass slide is observed under an optical microscope .

2.2 Coulter counter detection

The concentration of microvesicles , mean particle size and particle size distribution of microvesicles were determined by using a 50.mu . m orifice tube , 0.9 % NaCl electrolyte , an analysis volume of 500 渭l and a particle size measuring range of 1.1 渭m ~ 30 渭m . The microvesicles were diluted 5000 times with physiological saline . The concentration , mean particle size and particle size distribution of the microvesicles were determined by Coulter counter .

2.3 Determination of perfluoropropane gas content

Gas chromatography - mass spectrometry ( GC - MS ) was used to determine the content of perfluoropropane gas in the preparation . GC conditions : The column was DB - 5ms ( 30 m 脳 250 渭m 脳 0.25 渭m ) , column temperature was 35 鈩，

本文編號(hào)：2073955