【作者】 钟海军；

【导师】 邓英杰；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2005， 博士

【摘要】 灯盏花素是从菊科飞蓬属植物灯盏花[Erigeron breviscapus （Vant．） Hand-Mazz]中提取分离的黄酮类有效成份，是以灯盏乙素（scutellarin）为主，含少量甲素（apigenin-7-0-glucuronide）及其它黄酮类成分的混合物。灯盏花素具有扩张血管、增加脑血流量和心脏冠脉流量、降低血液粘度、抑制血小板聚集、改善微循环等作用。其现有制剂，如片剂、注射剂等，临床上主要用于治疗脑血栓、脑梗塞、中风后瘫痪、冠心病、心绞痛等疾病，疗效确切。近几年来，灯盏花素在我国已成为药物研发的一个热点，从2001年起，已经公开的有关灯盏花素的中国专利超过40个，其中一半以上针对灯盏花素的新剂型创新。灯盏花素口服给药生物利用度低，注射或口服给药后在体内均迅速消除。本论文的主要研究目的，是通过脂质体新剂型的研究提高灯盏花素口服生物利用度，延长其注射给药的体内滞留时间。为此，在处方前研究的基础上，设计了灯盏花素几种不同类型的脂质体，包括灯盏花素的普通脂质体（CLB）、壳聚糖N-三甲基化衍生物（TMC）包衣脂质体（TLB）和多囊脂质体（MLB）。首先，进行了灯盏花素的处方前研究工作，测定了灯盏花素电离常数pKa、真实油水分配系数、在不同溶剂以及不同pH水溶液中的溶解度，同时研究了灯盏花素在水溶液中的稳定性。结果表明，该药物是一个弱酸，pKa为3.29，在水中的溶解度受pH的影响很大，真实正辛醇／水分配系数为0.27，在各种有机溶剂中的溶解度较小。在25℃时，灯盏花素在pH 2～5水溶液中较稳定，在37℃时，pH 3～5较稳定，在水中存在专属性酸、碱催化降解机制。EDTA-2Na和NaHSO₃可以明显提高灯盏花素在水中的稳定性，两者合用比单用其中一种效果更好。在乙醇注入法的基础上，采用冷冻干燥-水化重建法制备了用于肌注给药的CLB，采用喷雾干燥-水化重建法制备了用于口服给药的CLB。通过单因素和正交试验，较为细致地考察了各种因素对乙醇注入法制备CLB包封率的影响，结果表明，磷脂浓度、水相介质种类及离子强度对CLB包封率影响较大，而其它因素影响较小，最后得到了包封率高的CLB，并对其性质进行了研究。该脂质体体外药物释放速度较快，室温放置稳定性需要进一步提高。冻干与喷干对脂质体有关性质影响的研究结果表明，采用冻干保护剂后加的方式制备，冻干重建的脂质体包封率高；加入冻干保护剂冻干的脂质体，重建后粒径稍微增大，药物在体外释放也较快。采用支持剂后加、喷前除去乙醇的方法制备，喷干-水化重建的脂质体包封率高；当支持剂用量适当时，喷干对脂质体粒径影响不明显；喷干过程对磷脂和药物的稳定性无显著影响。建立了稳定、快速、准确的灯盏花素血浆样品分析方法用于灯盏花素脂质体的药动学评价。通过冷冻贮存前在血浆样品中和在进样分析时用于溶解已处理好的样品的溶剂中加入NaHSO₃和EDTA，提高了样品在贮存和分析放置过程中的稳定性。CLB大鼠口服药物动力学研究表明，CLB和灯盏花素溶液（BS）药物的平均绝对生物利用F（％）分别为14.96％、4.77％，CLB的C_max和AUC（0→t）较BS分别提高2.27、2.13倍，统计分析有显著性差异（P＜0.001）。大鼠肌肉注射给药的药动学研究结果显示，CLB的药物平均滞留时间(MRT_（0→t）)，较BS延长了2.29倍，统计分析有显著性差异（P＜0.001）。灯盏花素脂质体中，很可能存在药物与磷脂分子之间的相互作用。为了证明这种推测并阐明其机理，并且对研究过程所出现的一些现象进行解释，采用脂质体／水分配系数、顺磁共振（EPR）、热分析（DSC）、红外光谱（IR）等手段，对灯盏花素脂质体中药物与磷脂的相互作用进行了研究。结果表明，在脂质体中灯盏花素与磷脂膜的作用力中疏水力的作用较小，而其它力（如氢键、静电等）占很大比例。在脂质体的脂质双层中，灯盏花素主要处于靠近磷脂极性头基的位置，而没有进入脂质双层的碳氢双链区域。灯盏花素分子中的羧基阴离子与磷脂分子胆碱部分中带正电性的季胺部位可能有离子间静电相互作用，灯盏花素的酚羟基与磷脂的P-O^-基之间形成了氢键。通过以上研究对前面的一些实验结果进行了解释。为进一步提高灯盏花素的生物利用度，合成了具有一定吸收促进效应，又有一定生物粘附性的N-三甲基壳聚糖盐酸盐（TMC），并用其对灯盏花素脂质体包衣，以进一步探索提高口服难以吸收的小分子药物生物利用度的方法和手段。研究结果表明，所得合成产物TMC的三甲基取代度为36.8％，TMC在中性或偏碱性介质中的溶解能力较壳聚糖明显提高。随着TMC浓度增加，TLB粒径增大。提示在脂质体表面TMC包衣层的形成。在4℃冰箱6个月的放置期间，不同浓度的TMC包衣所得的TLB均较为稳定。而又以0.5％（w／v）TMC所得的TLB物理稳定性最好。TMC包衣后，脂质体的药物包封率与包衣前相比没有明显变化。采用透射电子显微镜观察结果表明，0.5％TMC所得的TLB没有聚集，形态保持球形。TLB的药物释放总体比CLB慢，而且释放更彻底。在用不同浓度TMC包衣所得的TLB中，0.5％TMC药物释放最慢。TLB显示一定的生物粘附性。TLB提高了灯盏花素的口服生物利用度，TLB大鼠灌胃给药后药物的绝对生物利用度F（％）为21.48％，是BS的4.50倍，CLB的1.44倍。CLB肌注后药物平均滞留时间（MRT）虽然较BS延长了2.29倍，但也只有6～7小时，从给药频率看，至少还需要一天一次，比较频繁。为此，在选择合适制备工艺的基础上，通过单因素试验和正交试验筛选优化处方，制备了MLB并对其进行体内外评价，以研究制备效果更好的灯盏花素可注射缓控释新剂型，减少给药次数，方便患者。MLB的制备与性质研究结果表明，磷脂、胆固醇和三甘油酯等的用量在一定范围内对药物包封率影响较大，而脂药比影响较小。最优处方制备的MLB体外释放较慢，持续时间达6 d。大鼠药动学研究结果表明，MLB药物平均滞留时间（MRT）达到32.98 h，是BS的16.6倍，CLB的5.04倍，体内药物缓释的持续时间能够达到4～5 d。MLB的体内外相关性较好（r=0.9834），为选用人血浆作为释放介质的合理性提供了一个佐证。病理切片研究表明，MLB和CLB的生物相容性均良好。更多还原

【Abstract】 Breviscapine, a well-known bioactive flavonoid ingredient extracted from Erigeron breviscapus （Vant.） Hand.-Mazz., is mainly composed of scutellarin. Recent studies have shown that scutellarin possesses potent pharmacological effects in reducing blood viscosity, dilating blood vessel, increasing cerebral blood flow, inhibiting platelet aggregation, improving microcirculation, etc. The preparations of breviscapine, such as injections, tablets etc., are extensively used in clinic to treat ischemic cerebrovascular and cardiovascular diseases in China, such as cerebral infarction, apoplexy, coronary heart disease and angina pectoris, etc. Due to the prominent efficacy of breviscapine in the clinical treatment of these diseases, the research of breviscapine has become a hot topic in china in recent years. More than forty Chinese patents on breviscapine have been published since 2001. Over half of the patents focus on the renovation of the dosage form of breviscapine.It has been reported that the oral bioavailability of breviscapine is low and the residence time of breviscapine in the circulation is short. Therefore, the purposes of the present study were to increase its bioavailability after oral administration and to prolong its duration in the circulation after im administration. Several kinds of liposomes, including conventional liposomes, N-trimethyl chitosan chloride （TMC） coated liposomes, multivisicular liposomes, were used as the means of fulfilling the two purposes.Before the design of the liposomal formulations for breviscapine, the ionization constant （pKa）, the true n-octanol/water partition coefficient, the solubilities of scutellarin in various organic solvent and at various pH aqueous media were determined. Meanwhile, the stability of scutellarin in aqueous media was investigated. The results indicated those as follows: Scutellarin is a weak acid, its pKa was 3.29, and its solubility was markedly affected by the pH of the environment. Its true n-octanol/water partition coefficient was 0.27. The solubilities of scutellarin in various organic solvents were low. Scutellarin was more stable at pH 2-5 at 25℃and at pH 3-5 at 37℃than at the other pH aqueous media at the same temperature. The mechanism of degradation of scutellarin can be explained by specific acid/base catalysis. The addition of EDTA-2Na or/and NaHSO₃ can improve the stability of scutellarin at aqueous media, especially the addition of both EDTA-2Na and NaHSO₃.The conventional liposomes containing breviscapine （CLB） were prepared by ethanol injection method. Based on this, the liposomes for oral and intramuscular injection administration were prepared by spray-drying-reconstitution, and lyophilization-reconstitution, respectively. Single factor experiments and orthogonal experimental design were used to optimize the formulation and investigate the effects of various factors on the encapsulation efficiency （EE） of CLB. The results indicated that the concentration of phosphatidylcholine （PC）, different medium and its ionic strength had larger effect on EE than the other factors. The high EE above 80% could be obtained in the optimized formulation. Then its characteristics were investigated. The results indicated that the drug release rate from the liposomes in vitro was rapidly, and that the store stability of the liposomes at room temperature was not quite good. The effects of lyophilization and spray-drying on the characteristics of liposomes were studied. As for lyophilization-reconstitution liposomes, the higher EE could be obtained by the addition of sugars to the prepared liposomes by ethanol injection method. The particle size after reconstitution became a little larger, and the drug release in vitro from the reconstitution liposomes became faster. As for spray-drying-reconstitution liposomes, the higher EE could be obtained when the sugars were added to the prepared liposomes by ethanol injection method and the ethanol was removed before spray-drying. At appropriate amount of sugar, spray-drying had no significant effect on the particle size. No significant effects on the stability of both PC and drug were found during spray-drying.To study the pharmacokinetics behaviour of liposomal breviseapine, a stable, rapid, and accurate method was developed for the determination of scutellarin in rat plasma. By the addition of both NaHSO₃ and EDTA, the sample stability in both store and analysis process was improved to be suitable for the determination of scutellarin in the pharmacokinetic studies. The analytic time was shortened using scoparone （6, 7-dimethoxyeoumarin） as the internal standard. The results obtained from oral pharmacokinetics study of CLB indicated that the oral absolute bioavailabilities of scutellarin for CLB and breviscapine solution （BS） were 14.96 and 4.77%, respectively. The mean C_max and AUC_（0→t） of CLB were 3.3 and 3.1-fold higher than those of BS, respectively. The two parameters between CLB and BS showed a highly significant difference （P＜0.001）. The results obtained from pharmacokinetics study of CLB via im administration indicated that the mean MRT_0→t of CLB had a 3.29-fold increase compared with that of BS, so the MRT_（0→t） of CLB were significantly prolonged （P＜0.001）.In the study process, we felt that the molecular interaction between breviseapine and PC might exist. To confirm and then illuminate that, and to give some explains for some results obtained in earlier studies, the molecular interaction between breviscapine and PC was investigated by liposome/water partition coefficient, electron paramagnetic resonance （EPR）, DSC, and IR. The results indicated those as follows: The other interaction besides hydrophobic interaction, such as ionic interaction, hydrogen bond etc, play a main role in the molecular interaction between breviscapine and PC membrane. Breviscapine was mainly located at the polar phospho-diester groups, closed to the surface of the lipid bilayers, and did not enter the hydrocarbon chain region, especially the end of the hydrophobic chain. The anion of carboxy group in scutellarin molecule and cation of quaternary amine in PC molecule might exist electrostatic interaction. Hydrogen-bond might be formed between phenolic hydroxyl group in scutellarin molecule and P-O^- group in PC molecule. All of these could be used to explain some of the earlier results partly.The oral bioavailability of breviscapine was improved using conventional liposomal formulation. However, it was still only 14.96%. To increase it further, TMC, a polymeric absorption enhancer with mucoadhesive property, was synthesized and used to prepare TMC-coated liposomes. Then the characteristics of TMC-coated liposomes containing breviscapine （TLB） were evaluated in vitro and in vivo. The results indicated those as follows: The degree of quatemization of TMC was 36.8%. The solubilities of TMC at pH 7.4 aqueous media were significantly increased compared with those of ehitosan. The particle size of TLB was increased with the increasing concentration of TMC solution used in the coating, suggesting the formation of coating layer on the surface of the liposomes. All of TLBs coated with TMCs of different concentration were stable during the six-month storage at 4℃, especially with 0.5% （w/v） TMC. The TMCs coating did not change EE significantly. The liposomes kept sphericai and no significant aggregation was observed by transmission electron microscopy after coated with 0.5% （v/w） TMC. TMCs coating made the drug release from liposome became a little slower, especially 0.5% TMC coating, however, the maximum of accumulative release became larger. TLB showed mucoadhesive property. The absolute oral bioavailability was 21.48%, which was 4.50-fold and 1.44-fold increase as compared with BS and CLB, respectively; so oral absorption of breviscapine was further improved by the TMC-coated liposome formulation.Although the mean MRT_0→t of CLB had a 3.29-fold increase compared with that of BS, it was only 6～7h. The frequency of injection administration needs to be at least once a day if CLB is used to clinic. This motivated us to design a better formulation for breviscapine in order to provide a longer sustained-delivery duration, reduce the frequency of injection administration and therefore afford patient compliance. Multivesicular liposome, a unique lipid-based depot-delivery system, was utilized as drug delivery vehicles for the purpose. Single factor experiments and orthogonal experimental design were used to optimize the formulation based on the selection of feasible preparation technology. Then the characteristics of multivesicular liposome containing breviscapine （MLB） were evaluated in vitro and in vivo. The results indicated those as follows: Within a certain scope, the amounts of PC, cholesterol, and triglycerides used in the experiment have larger effect on EE than the other factors, such as the ratio of drug/PC. The drug durations both in vitro and in vivo were significantly prolonged for MLB, and that the drug release in vitro and the absorption in vivo showed a good linear correlation （R=0.9834）, which provided an evidence for the suitability to select human plasma as the medium of drug release from MLB in vitro. Drug release from MLB （triolein/tricaprylin 10/0） in vitro extended a long period of 5～6 days. The MRT_0→t obtained from the pharmacokinetics study of MLB after im administration was about 16.6-fold and 5.04-fold longer than those of BS and CLB, respectively. A duration in vivo for a period of 4～5 day was fulfilled for MLB. The results from light micrographs of pathological section of rat thigh muscle indicated that both MLB and CLB were well biocompatible.更多还原