【作者】 游剑；

【导师】 崔福德；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2005， 博士

【摘要】 目前在药物剂型设计中，液态药物以及液态载药系统的固体化（应用于口服给药）制剂的研究越来越受到药学工作者的关注。这个研究领域的关键是如何提高液态药物或者油性药物在载体中的含药量、提高药物的物理化学稳定性和口服生物利用度。本论文以具有较强的抗病毒和抗肿瘤作用的莪术油为模型药物，运用乳化溶剂扩散方法制备具有自乳化功能的微球。莪术油在载体中呈高度分散实现油性药物的固体化，在水性介质里能自发成乳，显著增强其溶出，以达到提高药物口服生物利用度的目的。本论文首先分离纯化莪术油中有效成分，制备了莪术油及其制剂的质量控制用对照品；通过对莪术油中三种指标性成份吸收机理的研究，为剂型设计提供指导和依据；通过大鼠和家兔静脉给药研究了莪术油中吉玛酮药物动力学，了解莪术油体内药动学行为；以提高莪术油物理化学稳定性为目，筛选出莪术油稳定剂（油性基质）；制备两种莪术油微球制剂，即普通型和缓释型（分别应用于抗病毒和抗肿瘤），并对制备、质量和稳定性进行了评价；为揭示莪术油微球自乳化特点而进行了一系列研究；最后评价了两种微球制剂的口服相对生物利用度。经柱层析，从莪术油中分离纯化出吉玛酮和莪术二酮，为莪术油及其制剂质量控制提供对照品。经过TLC和配备有二极管阵列检测器的HPLC分析，两种晶体纯度均大于95％；两种晶体分别经IR、MS、¹⁴C-NMR和¹H-NMR鉴定，与文献报道一致。通过大鼠单向灌流模型同时考察了吉玛酮、莪术醇和莪术二酮在四个肠段（十二指肠、空肠、回肠和盲肠）的吸收情况，结果表明灌流液中莪术油质量浓度在0.4、0.8和1.2mg·mL^-1时，莪术醇、莪术二酮、吉玛酮表观吸收系数均较低，且无显著性差异，药物浓度对吸收无抑制作用，表明三成分肠黏膜的转运均为被动扩散过程，各肠段对三成分表观吸收系数也无显著性差异，低的表观吸收系数说明莪术油肠道吸收一般。静脉给予大鼠莪术油乳剂，以莪术油中吉玛酮为指标性成分，研究了莪术油大鼠体内药物动力学；以提高检测灵敏度和方法的可靠性为主要目的，在所建立的大鼠体内分析方法基础上，进一步改善体内分析方法，并研究了莪术油中吉玛酮家兔体内药物动力学。，吉玛酮在大鼠和家兔的药动学行为均符合双隔室模型。对于大鼠，一个吉玛酮剂量水平(8.9 mg·kg^-1)T_1／2α、T_1／2β分别为9.89、41.53 min；对于家兔，三个吉玛酮剂量水平(3.2、6.4和9.6mg·kg^-1)T_1／2α分别为0.159h、0.153h和0.148h，T_1／2β分别为6.660h、9.502h和8.880h并显示在吉玛酮3.2-9.6 mg·kg^-1剂量范围内呈现线性药动学行为。吉玛酮在大鼠及家兔体内均呈现快速分布，家兔在给药后初期表现为快速消除（与大鼠体内研究现象一致），后期表现为缓慢下降，可能是由于肝肠循环所致，但需进一步研究证实。以莪术油及莪术油中三个指标性成分（吉玛酮、莪术醇和莪术二酮）的含量为指标，筛选利于莪术油稳定的油性基质。比较油性基质和莪术油的物理混合物在60℃环境下莪术油的稳定性；制备含不同油性基质的莪术油微球制剂，考察了不同处方微球在高温60℃（包括开放和密闭）、高湿RH75％和光照（4500LX）条件下莪术油稳定性。结果表明，以蓖麻油为油性基质在所考察的几种基质中，具有最好的稳定微球中莪术油的作用。通过处方考察筛选，以肠溶材料醋酸羟丙基甲基纤维素琥珀酸酯（HAS-LG）为成球骨架材料、以Aerosil200为吸附剂和以Talc为分散支持剂，运用乳化溶剂扩散法成功地制备了莪术油普通微球；采用阻滞性高分子HAS-HG以及Aerosil200和Talc成功地制备了莪术油缓释微球。两种微球含油率均较高（～50％），且微球中莪术油以及三指标成分包封收率均在90％以上。扫描电镜结果表明微球外观圆整，质地均一致密。释放度试验表明：对于普通微球，在模拟胃液液中(含1.2％聚三梨酯-80的0.1mol·L^-1盐酸溶液)2小时药物累计释放20％左右，在接着的模拟小肠液（含1.2％聚三梨酯-80的pH5.8磷酸盐缓冲溶液）中释放迅速，1小时后能达到全部释放，不同处方量微球中高分子、吸附剂和分散支持剂对微球在模拟胃液中的释放有较大影响，而对在模拟小肠液中的释放影响不显著；对于缓释微球，在含1.2％聚三梨酯-80蒸馏水介质中12h持续释药，具有明显的缓释特征，微球释药行为主要受高分子与吸附剂比例控制。以微球中莪术油及三指标成分含量为指标，进行稳定性研究，结果表明市售包装下普通和缓释微球在冷藏及常温下放置6个月稳定，加速试验较不稳定。微球中的莪术油通过Aerosil粒子和高分子聚合物HAS稳定达到自发成乳的目的，为了了解Aerosil粒子和高分子聚合物稳定乳剂的特点，从而揭示莪术油微球自乳化的特点，并对自乳化莪术油微球的处方设计提供理论依据，我们研究了不同类型的高分子聚合物和Aerosil微粒联合运用稳定莪术油乳剂的特点，以及不同高分子聚合物／Aerosil比例与乳剂质量的关系。为此对采用超声乳匀法制备的含有不同高分子聚合物和Aerosil类型以及他们之间不同比例的莪术油乳剂进行了研究并与微球自乳化所形成的乳剂进行了比较。结果显示，对于超声乳匀法所制备的乳剂，不同类型的高分子聚合物和Aerosil对乳剂稳定性有较大影响，不同Aerosil／高分子聚合物比例同样显著影响着乳剂的稳定性和乳滴粒径，且随比例的增加乳剂的稳定性和粒径将显著增大。对于含不同Aerosil／高分子聚合物比例微球自乳化所形成的乳剂，同样存在着上述现象。一个最佳的Aerosil／高分子聚合物比例范围被发现是在这种情况下：溶液中Aerosil粒子能完全被高分子聚合物所吸附辅盖，同时溶液中无剩余的高分子聚合物存在。这一理想比例范围可以通过Aerosil对高分子聚合物的吸附等温线来进行预测，这将对莪术油自乳化微球处方设计与筛选提供有力的指导。通过家兔分别口服给予通常意义上的莪术油自乳化系统、具有自乳化能力的普通和缓释莪术油微球，研究了普通和缓释微球的口服相对生物利用度。结果表明，两种微球口服后较参比制剂具有更大的生物利用度，普通和缓释微球相对生物利用度分别为157.7％和135.6％，缓释微球与前两者制剂比较具有明显的缓释特征。三制剂口服后体内血药浓度达峰时间与制剂体外介质中自乳化快慢相一致。制剂自乳化后所形成乳滴的粒径与相对生物利用度有着密切的关系，即乳滴粒径小，药物分散度高，致使口服相对生物利用度提高。本论文首次研究提供了莪术油中三个指标性成分（吉玛酮、莪术醇和莪术二酮）在大鼠不同肠段吸收数据。首次研究了莪术油中吉玛酮大鼠及家兔体内药物动力学行为。研究了一种有效的制各液体油性药物固体化制剂的方法，首次较深入地揭示了高分子聚合物和固体微粒联合运用稳定乳剂的特点。一种新颖的液态药物固体化制剂在这里被设计，与以往研究相比制剂具有如下特点：1．具有高含油率且稳定，含油率达50％（w／w）；2．制剂中药物高度分散，有利于口服生物利用度的提高；3．制剂中药物具有自乳化功能，能提高微球中莪术油的释放和溶出，而这种药物被乳化和形成稳定乳剂的机理显著不同于通常意义下的自乳化制剂，充实了自乳化药物传递系统的领域。更多还原

【Abstract】 Solidification of liquid systems has been a challenge that has attracted wide attention at present. Various attempts have been reported in literature to transform liquids into solids. The main goals in this study field are to obtain the solid dose form with high content of liquid drug and to improve the stability and bioavailability of the drug. In this study, the microspheres with self-emulsifying ability containing zedoary turmeric oil （ZTO）, which is as a poorly water-soluble model drug with pharmacological action including suppression of tumors and anti-bacterial, were prepared by the quasi-emulsion solvent diffusion method. To evaluate the drug content in microspheres, two pure components were separated from ZTO as the standards. Then, the absorption of three indexical components （curdione, germacrone, curcumol） found in ZTO in rat intestine was investigated to direct the formulation design. And, pharmacokinetic of ZTO was studied in rats and rabbits after intravenous administration of ZTO emulsion. In pre-formulation studies, the oily excipients were selected to improve the stability of ZTO in microspheres. Then, two kinds of ZTO microspheres （conventional ZTO microspheres and sustained-release ZTO microspheres） were prepared and evaluated with the appearance, drug stability and release. The efficiency of self-emulsification of microspheres was also investigated. Finally, the bioavailabilities of two types of microspheres were evaluated in rabbits after oral administration.Two components were separated from ZTO by the column chromatography. The both contents were evaluated to be > 95% by the methods of TLC and HPLC. The chemical structures of two components were confirmed to be germacrone and curdione, respectively, by the methods of IR, MS, ¹⁴C-NMR and ¹H-NMR.To study the absorption of ZTO in rat intestine, In situ single pass perfusion model was used and the concentrations of three components in perfusate were determined by HPLC in combination with diode array detection. It was shown that the P_apps of curcumol, curdione and germacrone were all low and had no significant difference （P>0.05） at zedoary oil concentration of 0.4, 0.8 and 1.2 mg-mL^-1 in transmucosal fluid or in four different regions of intestine of rat [duodenum, jejunum, ileum, colon ]. The absorption rates of germacrone and curdione were faster than curcumol’s in this study. It was concluded that ZTO concentration in transmucosal fluid had no significant effect on the P_apps within the scope of 0.4-1.2 mg·mL^-1. The absorption of curcumol, curdione and germacrone showed the passive diffusion process, and didn’t contain a special absorption window. An economical HPLC method to determine the amount of germacrone in plasma was presented to investigate the pharmacokinetic of ZTO in rats. To obtain sufficient sensitivity, the method had been improved for the ZTO pharmacokinetics study in rabbits. It was found that the plasma germacrone concentration-time datas fit to a two-compartment intravenous model in rats and rabbits after intravenous administration of ZTO microcmulsion. The phannacokinetic parameters of T_1/2α and T_1/2β were 9.890 and 41.53 min, respectively, after administration of 8.9 mg·kg^-1 of germacrone in rats. T_1/2α were 0.159, 0.153 and 0.148h, respectively, and T_1/2β were 6.66, 9.502 and 8.88h, respectively, after administration of three germacrone dose levels of 3.2, 6.4 and 9.6mg.kg（-1） in rabbits. Germacrone exhibited linear phannacokinetics after intravenous administration to rabbits over the dose range of 3.2-9.6 mg·kg^-1. Rapid in vivo distribution of germacrone was shown from the concentration-time profiles in rats and rabbits, while slow in vivo elimination of germacrone were shown in rabbits, which can be the reason that germacrone has undergone the circulation between liver and intestine.To improve the stability of ZTO in microsphcres, some oily excipients as ZTO stabilizer were added into the formulations. The stability studies were carded out at the opening environment of 60℃, airtight environment of 60℃, opening environment of RH 75％and opening environment of illumination （4500LX）, in which the ZTO stability was evaluated by determining the contents of ZTO and three indexical components in microspheres, such as germacrone, curdione and curcumol. It was shown that castor oil is the most helpful to the ZTO stability in selected oily excipients.Two kinds of ZTO microsphcrcs with oil （containing oil drug and excipicnt） content of～50％, such as conventional and sustained-release microspheres, had been prepared by the emulsion-solvent-diffusion method. The enterosoluble polymer, such as hydroxypropyl methylcellulose acetate succinate （HPMCAS-LG）, was used in the formulation of conventional microsphcrcs, while HPMCAS-HG as a retarding agent was employed in the formulation of sustained-release microspheres. Aerosi1200 and Talc were used as adsorbent and dispersing agent, respectively, in both formulations. Satisfying incorporation efficiency over 90％was obtained for ZTO and three indexical components in conventional and sustained-release microspheres. The appearance, drug release and stability of both kinds of resultant microspheres were investigated. It was shown that microspheres were spherical and exhibited uniform and compact internal structure by the observation of SEM. The ZTO release rate was restrained in the pH1.2 HCL solution （containing 1.2％, W/V, tween-80 to meet the sink condition） and, subsequently, rapid release was shown in the pH5.8 phosphate buffer （containing 1.2％, W/V, tween-80） for the conventional microspheres. The amounts of HPMCAS-LG, Aerosi1200 and Talc in the formulation had significant effect on ZTO release rate in the pH1.2 HCL solution （containing 1.2％, W/V, tween-80）, while did not show the obvious different to release rate in pH 5.8 phosphate buffer （containing 1.2％, W/V, tween-80）. The ZTO release rate from the sustained-release microspheres was mainly controlled by the ratio of HPMCAS to Aerosi1200 when microspheres were put into the medium of distilled water containing 1.2％（W/V） tween-80. Stability studies of two kinds of microspheres, which were packed under simulating them at counter, were carded out. It was shown that above microspheres were stable under the normal temperature （25℃, RH 60％） and cool environment （4-8℃）, while had not sufficient stability under accelerating environment （40℃, RH 75％）.To investigate the character of microsphere self-emulsification, we dealt with a system consisting of Aerosil, HPMCAS, ZTO and pH6.8 phosphate buffer. A series of emulsions was prepared by ultrasonic-emulsifying method and evaluated with the stability and droplet size in order to investigate the effect of different types of Aerosil and polymer as well as the ratio of them to stabilize ZTO emulsion. In addition, the ZTO microspheres containing different ratios of Aerosil to polymer were prepared. The stability and droplet size of resultant emulsion produced from self-emulsification of microspheres were compared with them of emulsions prepared by ultrasonic-emulsifying method. It was shown that emulsion stability was defined strongly by the type of Aerosil and polymer as well as the ratio of these excipients. Droplet size and stability of emulsions, which were produced by ultrasonic-emulsifying method and microsphere self-emulsification, increased on the increase of Aerosil/polymer ratio, while the smaller size of droplets and stabler emulsion were obtained by microsphere self-emulsification comparing with them of emulsions prepared by ultrasonic-emulsifying method when at the same Aerosil/polymer ratio. An ideal range of Aerosil/HPMCAS ratio seems to be given in the case of almost complete coverage of the Aerosil particles without having a substantial amount of unbonded polymer in the bulk solution. The best ratio of Aerosil to HPMCAS-LG or -HG was predictable from the Langumuir-fit of the adsorption isotherms, which is very useful for the design of our microsphere formulation.The bioavailability studies were carded out in rabbits after oral administration of conventional ZTO microspheres, sustained-release ZTO microspheres and conventional self-emulsifying ZTO formulation （conventional SES）. The relative bioavailabilities of conventional and sustained-release ZTO microspheres were greatly improved over the conventional SES, which were 157.7％and 135.6％, respectively. It was evident that the sustained-release ZTO microspheres achieved a more prolonged absorption profile compared with other two preparations. More rapid rate of self-emulsification resulted in shorter time of getting maximum germacrone concentration after oral administrations of above three ZTO preparations. Decrease of droplet size of resultant emulsions produced from self-emulsifying preparations significantly enhanced the in vivo absorption of ZTO and thus improved the bioavailability of oral administration.Some investigations, which have not reported at precent, have been carried out in this work, such as the study of absorption of ZTO in intestine, pharmacokinetics of ZTO in rats and rabbits and the study of stabilizing ZTO emulsions by a united use of Aerosil and polymer. A novel formulation of oily drug, ZTO microspheres with self-emulifying ability, has been designed here. The microspheres, as a solid dose form of liquid drug, have three advantages, such as high content of oily drug in microspheres, greatly dispersed degree of drug and self-emulsifying ability of microspheres, which were significantly different from the conventional self-emulsifying formulation.更多还原