吲哚美辛生物粘附凝胶小球的研究
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摘要
目的：吲哚美辛是一种高效的非甾体抗炎药之一，临床上主要应用于风湿性及类风湿性关节炎的治疗。但从将近几十年的临床应用来看，吲哚美辛具有较大的胃肠道副反应，这也成为了限制吲哚美辛应用前景的客观因素所在。基于该点考虑，本研究将采用壳聚糖、海藻酸钠、氯化钙、卡波普等为辅料制备吲哚美辛生物粘附凝胶小球。由于粘附材料的使用，凝胶小球与肠道粘膜发生粘附作用，延长了药物在吸收部位的停留时间，达到了长效及肠道释药的目的。
方法：在预实验基础上初步确定吲哚美辛生物粘附凝胶小球的制备处方，然后通过四因素三水平正交实验对各种辅料用量进行筛选，同时采用扫描电镜、差示热分析等手段考察吲哚美辛生物粘附凝胶小球的外观形态并进行药物释放机理以及凝胶小球成型机理的研究。通过大鼠实验验证吲哚美辛生物粘附凝胶小球的体内外粘附性能及其药代动力学特征，最后进行体内外相关性研究及吲哚美辛生物粘附凝胶小球的质量检查和稳定性实验。
凝胶小球的制备：通过预实验初步确定具有良好粘附性能及药物释放性能的处方。在对粘附凝胶小球制备过程中的各种影响药物释放的条件进行单因素考察的基础上，通过四因素三水平正交实验L9(34)，pH6.8磷酸盐缓冲液为释放介质进行溶出实验。选用Higuchi缓释模型拟合参数r以及粘附滞留率为指标对各个处方进行筛选、评分，并对评分结果进
行极差分析，最后确定吲哚美辛生物粘附凝胶小球的最佳制备处方工艺。
形态考察：利用扫描电镜观察吲哚美辛生物粘附凝胶小球在干燥状态下的外观以及断面形态，同时将进行溶出实验4h后的凝胶小球留样，观察外观及断面形态。比较凝胶小球在溶出前后的形态变化，进行药物释放机理的探讨。
含量测定方法：选用简单快速的紫外分光光度法测定吲哚美辛生物粘附凝胶小球中的药物含量。
释放度考察：根据中国药典(2000年版)附录XD溶出度第二法桨法，在规定时间点取样并于波长320nm处测定吸光度A值，计算累积释放百分率。
稳定性考察：将最佳处方的吲哚美辛生物粘附凝胶小球进行稳定性影响因素实验以及长期实验。通过实验证明粘附小球对湿、热的稳定性。
粘附性实验：采用大鼠离体小肠作为生物组织，将凝胶小球进行离体粘附实验，考察凝胶小球的体外粘附性能，同时进行大鼠在体粘附实验。
病理学研究：使用病理学手段观察大鼠小肠粘膜在凝胶小球粘附前与粘附后的绒毛结构变化，以评价凝胶小球的性能。
小球成型机理研究：通过差示热分析手段对吲哚美辛生物粘附凝胶小球进行成型机理探讨。即通过比较各种辅料及主药在成型前后的差示热分析谱图变化说明在小球制备过程当中有可能发生的各种反应。
生物利用度实验：(1)血浆中的吲哚美辛浓度测定选择内标法，内标为对羟基苯甲酸丁酯。色谱条件为：色谱柱：
DIKMA?C18柱(4.6mm×250mm,5μm)；流动相：甲醇-水-磷酸(75:25:0.025)；流速：1.0mL·min-1；检测波长：256nm；温度：室温。
(2)将大鼠作为模型动物，采用随机分组的方法将大鼠分为两组，分别口服吲哚美辛生物粘附凝胶小球与自制对照吲哚美辛混悬液。在不同时间点分别取样，使用高效液相色谱仪测定血浆中吲哚美辛的浓度，血药浓度数据采用缓释药物动力学程序处理。
结果：通过单因素考察以及正交实验筛选出了最佳处方，根据最佳处方制备的吲哚美辛生物粘附凝胶小球外观圆整，呈类白色，流动性良好。从正交结果的极差分析得知，在考察的各种辅料用量中，壳聚糖的用量对凝胶小球中药物的释放影响最大。
形态考察：通过光学显微镜观察到的小球粒径主要分布在750~850μm范围内。使用扫描电镜可观察到小球表面有少许裂缝。将凝胶小球在pH6.8磷酸盐缓冲液中进行溶出实验4h后，小球外膜破裂，有吲哚美辛晶体暴露在释放介质中。从断面结构可明显观察到各种辅料已形成水凝胶，且基质随着时间延长逐渐溶解，吲哚美辛结晶完全暴露在溶出介质中。
含量测定：吲哚美辛在浓度1.48~29.5μg·mL-1范围内线性关系良好，标准曲线方程为：
C=52.03A-0.01948，r=0.9999。
根据选用测定方法测定吲哚美辛生物粘附凝胶小球含量，所得药物含量平均值为39.9%±1.3%(n=6)。
释放度实验：最佳处方制备的凝胶小球在1h时累积释
放百分率为29.47%，4h时为55.70%，8h的累积释放百分率为90.00%。分别使用Higuchi模型、零级模型和一级模型对溶出曲线进行拟合，相关系数为：Higuchi模型r=0.9995,零级模型r=0.9890,一级模型r=0.9209。
稳定性考察：稳定性影响因素实验中，高湿实验结果：凝胶小球在相对湿度为92%环境下放置5天后吸湿增重﹥5%，且小球外观呈黄色。放入相对湿度75%环境第5天时凝胶小球吸湿 <5%，外观无变化。高温放置5天和10天后取样，外观无变化，释放度实验数据没有显著性差别。稳定性长期实验的每个月末取样做释放度实验，结果无明显变化。
粘附性实验：大鼠离体实验中，凝胶小球的平均滞留率在90%左右；大鼠在体粘附实验中, 2.5h后凝胶小球大部分已经排空到小肠，实验数据与各项实验结果表明凝胶小球在体外与体内均有较好的粘附性能。
病理学实验：结果显示小肠绒毛浅层靠近腔面近1/4处有轻微损伤,?
Objective: Indomethacin is one of the effective no-steroid antiinflammatories. It is used to treat rheumatism and atrophic arthritis in clinic. However, the clinical applications of Indomethacin in recent years shows that it can cause serious side-effects on gastrointestinal tract ,which limits its application. In order to eliminite bad effect of Indomethacin, Chitosan, Sodium Alginic Acid, Calcium chloride and Carbopol were adopted as additives to make bioadhesive gel beads.It had long effects on intestinal tract since it could adhere to mucosa.
Method: Through the pre-experiment, the preliminary formula of gel beads were defined and the orthogonal experiment was adopted to select the ratio of additives. Meanwhile, scanning electron microscope and differential scanning calorimetry were used to study the appearance of gel beads , drug release and the gel beads formation mechanism. Rats were employed to verify bioadhesive behavior and pharmacokinetics of gel beads. At last,the correlation between in vitro and in vivo, quality determination and stability of gel beads were also studied.
Preparation of gel beads: through pre-experiment the fomula were defined that possed the behavior of good adhesiveness and drug release. Based on the analysis of the
factors which influenced the drug release behavior in phosphate buffer pH6.8, the orthogonal experiment design L9(34) was used to select the optimal fomula. In the course of that, the parameters of sustained-release model and adhesive retention rate were used to score every prescription.
The scanning electron photomicrographs of surface and interior were taken before and after dissolution testing with paddle method for four hours.
Through analysing the changes of gel beads before and after dissolution testing, the release mechanism of drug was studied. The content of Indomethacin in bioadhesive gel beads was determined by UV spectrophotography.
Paddle method was employed in the experiment of dissolution and the concentration of Indomethacin was determined by UV spectrophotometer at 320nm so as to calculate accumulative release percentage.
The chemical and physical stability of the optimal formula was investigated, including stress testing(high humidity testing, high temperature testing) and long-term testing.
The small intestines of rats were used as organism tissue to test adhesive behavior of gel beads, including in vitro and in vivo.
The studies of Patholog: the structure changes of rats intestinal villi after bioadhesion were observed by means of pathology methods in order to evaluate the property of gel beads.
The formation mechanism of bioadhesive gel beads of Indomethacin was studied by means of differential scanning calorimetry. The informations taken from the diagrams showed that additives had reacted with each other.
Bioavailability(1)High-performance Liquid Chromatogra- ph with Ultraviolet detector was adopted to determine the content of Indomethacin in plasma in which butyl ester of p-hydroxybenzoic acid was used as internal standard. The Liquid Chromatograph was equipped with a 256nm detector and a DIKMA? C18 column (4.6mm×250mm, 5 μm).Mobile phase was a suitable mixture of methylalcohol, water and phosphoric acid (75:25:0.025). The flow rate was about 1.0mL per minute and temperature was 25℃.(2)gel beads of Indomethacin and contrast drug were given to rats which had been divided into two groups in random. Sampling on suitable time and using HPLC to determine the concentration of Indomethacin in plasma. The pharmacokinetics parameters were fitted and calculated by sustain-release program.
Result: The optimal formula was defined through the orthogonal experiment. The bioadhesive gel beads made from the optimal formula were white and smooth, with good fluidity, etc. The analysis of orthogonal results showed that the concentration of Chitosan had much more influence on drug release than any others. Crack on the surfaces of gel beads was observed after dissolution experiment in phosphate buffer pH6.8 for four hours, crystal of drug
方法：在预实验基础上初步确定吲哚美辛生物粘附凝胶小球的制备处方，然后通过四因素三水平正交实验对各种辅料用量进行筛选，同时采用扫描电镜、差示热分析等手段考察吲哚美辛生物粘附凝胶小球的外观形态并进行药物释放机理以及凝胶小球成型机理的研究。通过大鼠实验验证吲哚美辛生物粘附凝胶小球的体内外粘附性能及其药代动力学特征，最后进行体内外相关性研究及吲哚美辛生物粘附凝胶小球的质量检查和稳定性实验。
凝胶小球的制备：通过预实验初步确定具有良好粘附性能及药物释放性能的处方。在对粘附凝胶小球制备过程中的各种影响药物释放的条件进行单因素考察的基础上，通过四因素三水平正交实验L9(34)，pH6.8磷酸盐缓冲液为释放介质进行溶出实验。选用Higuchi缓释模型拟合参数r以及粘附滞留率为指标对各个处方进行筛选、评分，并对评分结果进
行极差分析，最后确定吲哚美辛生物粘附凝胶小球的最佳制备处方工艺。
形态考察：利用扫描电镜观察吲哚美辛生物粘附凝胶小球在干燥状态下的外观以及断面形态，同时将进行溶出实验4h后的凝胶小球留样，观察外观及断面形态。比较凝胶小球在溶出前后的形态变化，进行药物释放机理的探讨。
含量测定方法：选用简单快速的紫外分光光度法测定吲哚美辛生物粘附凝胶小球中的药物含量。
释放度考察：根据中国药典(2000年版)附录XD溶出度第二法桨法，在规定时间点取样并于波长320nm处测定吸光度A值，计算累积释放百分率。
稳定性考察：将最佳处方的吲哚美辛生物粘附凝胶小球进行稳定性影响因素实验以及长期实验。通过实验证明粘附小球对湿、热的稳定性。
粘附性实验：采用大鼠离体小肠作为生物组织，将凝胶小球进行离体粘附实验，考察凝胶小球的体外粘附性能，同时进行大鼠在体粘附实验。
病理学研究：使用病理学手段观察大鼠小肠粘膜在凝胶小球粘附前与粘附后的绒毛结构变化，以评价凝胶小球的性能。
小球成型机理研究：通过差示热分析手段对吲哚美辛生物粘附凝胶小球进行成型机理探讨。即通过比较各种辅料及主药在成型前后的差示热分析谱图变化说明在小球制备过程当中有可能发生的各种反应。
生物利用度实验：(1)血浆中的吲哚美辛浓度测定选择内标法，内标为对羟基苯甲酸丁酯。色谱条件为：色谱柱：
DIKMA?C18柱(4.6mm×250mm,5μm)；流动相：甲醇-水-磷酸(75:25:0.025)；流速：1.0mL·min-1；检测波长：256nm；温度：室温。
(2)将大鼠作为模型动物，采用随机分组的方法将大鼠分为两组，分别口服吲哚美辛生物粘附凝胶小球与自制对照吲哚美辛混悬液。在不同时间点分别取样，使用高效液相色谱仪测定血浆中吲哚美辛的浓度，血药浓度数据采用缓释药物动力学程序处理。
结果：通过单因素考察以及正交实验筛选出了最佳处方，根据最佳处方制备的吲哚美辛生物粘附凝胶小球外观圆整，呈类白色，流动性良好。从正交结果的极差分析得知，在考察的各种辅料用量中，壳聚糖的用量对凝胶小球中药物的释放影响最大。
形态考察：通过光学显微镜观察到的小球粒径主要分布在750~850μm范围内。使用扫描电镜可观察到小球表面有少许裂缝。将凝胶小球在pH6.8磷酸盐缓冲液中进行溶出实验4h后，小球外膜破裂，有吲哚美辛晶体暴露在释放介质中。从断面结构可明显观察到各种辅料已形成水凝胶，且基质随着时间延长逐渐溶解，吲哚美辛结晶完全暴露在溶出介质中。
含量测定：吲哚美辛在浓度1.48~29.5μg·mL-1范围内线性关系良好，标准曲线方程为：
C=52.03A-0.01948，r=0.9999。
根据选用测定方法测定吲哚美辛生物粘附凝胶小球含量，所得药物含量平均值为39.9%±1.3%(n=6)。
释放度实验：最佳处方制备的凝胶小球在1h时累积释
放百分率为29.47%，4h时为55.70%，8h的累积释放百分率为90.00%。分别使用Higuchi模型、零级模型和一级模型对溶出曲线进行拟合，相关系数为：Higuchi模型r=0.9995,零级模型r=0.9890,一级模型r=0.9209。
稳定性考察：稳定性影响因素实验中，高湿实验结果：凝胶小球在相对湿度为92%环境下放置5天后吸湿增重﹥5%，且小球外观呈黄色。放入相对湿度75%环境第5天时凝胶小球吸湿 <5%，外观无变化。高温放置5天和10天后取样，外观无变化，释放度实验数据没有显著性差别。稳定性长期实验的每个月末取样做释放度实验，结果无明显变化。
粘附性实验：大鼠离体实验中，凝胶小球的平均滞留率在90%左右；大鼠在体粘附实验中, 2.5h后凝胶小球大部分已经排空到小肠，实验数据与各项实验结果表明凝胶小球在体外与体内均有较好的粘附性能。
病理学实验：结果显示小肠绒毛浅层靠近腔面近1/4处有轻微损伤,?
Objective: Indomethacin is one of the effective no-steroid antiinflammatories. It is used to treat rheumatism and atrophic arthritis in clinic. However, the clinical applications of Indomethacin in recent years shows that it can cause serious side-effects on gastrointestinal tract ,which limits its application. In order to eliminite bad effect of Indomethacin, Chitosan, Sodium Alginic Acid, Calcium chloride and Carbopol were adopted as additives to make bioadhesive gel beads.It had long effects on intestinal tract since it could adhere to mucosa.
Method: Through the pre-experiment, the preliminary formula of gel beads were defined and the orthogonal experiment was adopted to select the ratio of additives. Meanwhile, scanning electron microscope and differential scanning calorimetry were used to study the appearance of gel beads , drug release and the gel beads formation mechanism. Rats were employed to verify bioadhesive behavior and pharmacokinetics of gel beads. At last,the correlation between in vitro and in vivo, quality determination and stability of gel beads were also studied.
Preparation of gel beads: through pre-experiment the fomula were defined that possed the behavior of good adhesiveness and drug release. Based on the analysis of the
factors which influenced the drug release behavior in phosphate buffer pH6.8, the orthogonal experiment design L9(34) was used to select the optimal fomula. In the course of that, the parameters of sustained-release model and adhesive retention rate were used to score every prescription.
The scanning electron photomicrographs of surface and interior were taken before and after dissolution testing with paddle method for four hours.
Through analysing the changes of gel beads before and after dissolution testing, the release mechanism of drug was studied. The content of Indomethacin in bioadhesive gel beads was determined by UV spectrophotography.
Paddle method was employed in the experiment of dissolution and the concentration of Indomethacin was determined by UV spectrophotometer at 320nm so as to calculate accumulative release percentage.
The chemical and physical stability of the optimal formula was investigated, including stress testing(high humidity testing, high temperature testing) and long-term testing.
The small intestines of rats were used as organism tissue to test adhesive behavior of gel beads, including in vitro and in vivo.
The studies of Patholog: the structure changes of rats intestinal villi after bioadhesion were observed by means of pathology methods in order to evaluate the property of gel beads.
The formation mechanism of bioadhesive gel beads of Indomethacin was studied by means of differential scanning calorimetry. The informations taken from the diagrams showed that additives had reacted with each other.
Bioavailability(1)High-performance Liquid Chromatogra- ph with Ultraviolet detector was adopted to determine the content of Indomethacin in plasma in which butyl ester of p-hydroxybenzoic acid was used as internal standard. The Liquid Chromatograph was equipped with a 256nm detector and a DIKMA? C18 column (4.6mm×250mm, 5 μm).Mobile phase was a suitable mixture of methylalcohol, water and phosphoric acid (75:25:0.025). The flow rate was about 1.0mL per minute and temperature was 25℃.(2)gel beads of Indomethacin and contrast drug were given to rats which had been divided into two groups in random. Sampling on suitable time and using HPLC to determine the concentration of Indomethacin in plasma. The pharmacokinetics parameters were fitted and calculated by sustain-release program.
Result: The optimal formula was defined through the orthogonal experiment. The bioadhesive gel beads made from the optimal formula were white and smooth, with good fluidity, etc. The analysis of orthogonal results showed that the concentration of Chitosan had much more influence on drug release than any others. Crack on the surfaces of gel beads was observed after dissolution experiment in phosphate buffer pH6.8 for four hours, crystal of drug
引文
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