重组人表皮生长因子生物粘附肠溶胶囊的研究
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摘要
重组人表皮生长因子（recombinant human epidermal growth factor, rhEGF）作为基因工程药品进入临床以来，在烧伤、创伤及各种慢性创面的治疗等方面取得较好的效果。随着研究的不断深入，应用范围逐步扩大，近年来，有人将其用于胃肠道损伤的修复治疗。临床上消化性溃疡，肠炎为一常见病和多发病，也是烧、创伤及各种严重感染等疾病的常见并发症，目前肠道损伤尚无理想的治疗药物，因此人们根据rhEGF具有促进粘膜上皮细胞增生，加快创面愈合的作用机理，进行了初步的治疗尝试，如应用内窥镜下局部注射或喷撒EGF治疗消化性溃疡，也有把rhEGF制成口服液用于胃溃疡治疗的报道，结果都取得了很好的治疗效果[31、35]。对于rhEGF用于局部治疗胃肠道损伤的疾病，显示了良好的应用前景。但应用内窥镜下局部注射或喷撒治疗，存在应用不便，病人难以接受的缺点，而把rhEGF制成口服液应用，则稳定性难以达到要求。为寻求用于肠道损伤修复的治疗药物及其方便实用的药物制剂，既能使药物通过胃液不被破坏，又能保证药物在通过肠道时缓缓释放出来，达到理想的治疗效果，我们根据rhEGF的理化性质和治疗特点，进行了rhEGF的生物粘附肠溶制剂的开发研究。
本研究采用动物小肠体内、体外粘附力测定模型，选择常用生物粘附剂制成颗粒，并对颗粒的粘附性能进行筛选与评价。结果证明，CP934P、CP940P、SCMC、HPC等生物粘附剂在动物小肠上均具有一定的粘附性，其中CP940P粘附性最强，但与CP934P无明显差异，HPC次之。其粘附性能与添加比例呈正相关。以CP940、HPC与乳糖的适当配比，可获得具有适宜粘附性能的生物粘附颗粒，随着上述粘附材料成分的添加比例的增加，其粘附性增强；以乳糖：CP：HPC（78～82：10～15：8～13）较为合适。能达到95%以上的滞留率。
本文参照《中国生物制品规程》分别建立了放射免疫分析法（RIA）法和细胞测活法测定rhEGF生物粘附性肠溶胶囊含量和生物活性，目前放射免疫分析法（RIA）法和细胞测活法是测定rhEGF的主要方法，二者在测定rhEGF羟基端的部分代谢产物时，其结果是一致的。
采用放射免疫分析法考察了rhEGF在人工胃肠液介质的稳定性，rhEGF在人工胃液（pH=2）和人工肠液（pH=6.8）中的含量随时间的延长而呈下降趋势，6h后人工胃液和人工肠液中rhEGF浓度分别下降44.44%和55.75%；但在酶的存在下6h后rhEGF浓度分别下降95.43%和97.64%。稳定性研究结果表明，rhEGF在人工胃、肠液中稳定性较好，尽管在
酶的存在下rhEGF分解速度可明显加快，但可满足胃肠损伤修复的治疗需要，另外生物粘附材料的保护和缓释作用还可减缓其失活速度。
本研究设计处方中主药rhEGF含量甚微，混匀工艺是关键。本文采用少量乳糖加水先和rhEGF混匀成糊状，然后冷冻干燥得到均匀的含药乳糖粉末，再进一步用于制备颗粒。并利用生物活性测定方法，考察了冷冻干燥制备工艺对rhEGF生物活性的影响，冷冻干燥使rhEGF活性稍有降低，但冷冻干燥法可使剂型中rhEGF的含量均匀性得到保证，为下一步制剂作好准备。因此rhEGF和乳糖混和后的冻干粉末可作为整个制剂工艺的中间品，以此时的rhEGF的含量作为下次投料的标准。
选择正交实验设计，对rhEGF生物粘附肠溶制剂处方组成及制备工艺进行了研究，以肠溶胶囊的释放度、粘附性、生物活性和含量均匀度作为考察指标，对处方工艺进行筛选。结果表明CP与HPC的比例对胶囊释药速率和粘附性有非常显著影响,混合方式对药物含量均匀度也有显著影响,以综合得分最高为标准，确定优化处方为4号处方（A2B1C2），即CP和HPC的比例为10：8，助流剂选1%二氧化硅，以4号处方制备的生物粘附肠溶胶囊粘附性适中，含药均匀度好，缓冲液中0.5h释药8.6%,1h释药12%，基本能满足实验设计和治疗的要求，
采用RIA法测定了家兔给药后血清EGF浓度的变化趋势，及组织中的分布情况，籍以考察rhEGF的肠道吸收和体内分布情况，为吸收的安全性评价和临床应用提供参考依据。结果证明，家兔口服rhEGF生物粘附肠溶胶囊前后，血清EGF浓度和组织中的分布无明显改变，说明肠腔对rhEGF吸收甚微，肠道局部使用rhEGF基本不会产生全身作用。
以制剂中rhEGF释放度、含量和生物活性为指标，连续配制三批rhEGF生物粘附肠溶胶囊样品，对其不同条件下的放置稳定性进了考察。结果表明，rhEGF生物粘附肠溶胶囊在4℃ 条件下放置6个月较为稳定，制剂活性仅下降了11%。而在25℃条件下贮存时活性虽有稍明显下降，但仍符合质量要求。
本文研究的rhEGF生物粘附肠溶胶囊，可作用小肠靶部位，延长药物在粘膜局部的作用时间、提高局部rhEGF浓度，促进损伤部位愈合，并对血清EGF浓度无明显影响。基本达到了设计要求，可作为新的用于肠道损伤治疗的药物制剂进一步开发。
Since recombinant human epidermal growth factor (rhEGF) released to clinical use as a genetic engineered product, some good effects have been achieved in treatment of burn, trauma and a variety of chronic surfaces of wound. With continuous study on the product, its clinical use has been gradually exended, including preliminary trials on repairing of lesions in the gastrointestinal tract in recent years with limitation only to local EGF injection or spry under the endoscopy for treatment of peptic ulcer. And some reports deal with administration of oral liquid of rhEGF for the same purpose. Lesions in the gastrointestinal tract have most commonly followed burn, trauma and a great deal of severe infections and there aren't satisfied approaches available to these lesions at present. In order to seek out some effective medications for repairing of lesions in the gastrointestinal tract, we conducted studies to develop an intestine-released capsule of biological adhesive rhEGF.
The present study deals with in vivo and in vitro animal experimental models of small intestines for measurement of conglutinative force of biological adhesives in the form of granules to screen and assess their adhesive property. The results proved that biological adhesives, such as CP934P, CP940P, SCMC and HPC, showed to some extent adhesion to the tested animal intestines. Of them CP940P as well as CP934P has the most adhesive force, while HPC takes second place. The adhesive property has a positive correlation to their additive proportion. CP940, HPC and lactose in an appropriate blending ratio could offer a biological adhesive granule with an applicable adhesion property. With increased additive proportion of the adhesive materials mentioned above, the adhesive force could rise accordingly. A satisfied mixture could be obtained with proportions of lactose:CP:HPC (78-82:10-15:8-13), having more than 95% retention rate.
We have established methods of radioimmunoassay (RIA) and alive-cell identification to measure its content and biological activity of the enteric capsule of biological adhesive rhEGF with reference to Guidance for Biological Products in China. At present both of RIA and alive-cell identification are still the leading
methods for measurement of rhEGF, both of the approaches having an identified result in terms of measuring the metabolites from the hydroxyl moiety of rhEGF.
In addition, RIA has been used to observe the stability of rhEGF under the condition of artificial peptic juices. The content of rhEGF in artificial gastric juice (pH=2) and intestinal juice (pH=6.8) was prone to decrease over the time, lowering 44.44% and 55.75% from their baselines 6 hours later, respectively. But the decrease in the content of rhEGF would reach 95.43% and 97.64% after 6 hours in the presence of peptic enzymes, respectively.rhEGF in the artificial peptic juices was relatively stable and could meet the need for repair of lesions in the alimentary canal, although the process of its breakdown could be greatly expedited in the presence of peptic enzymes. The breakdown process could be attenuated through modified release of the adhesive materials.
The key technique in this preparation may be the blending approach in that the prescription design in the present study includes very low amount of main active ingredient, rhEGF. Accounting for major part of excipients in the recipe, a proportion of lactose was first mixed with rhEGF for its dilution. The resultant mixture underwent lyophilized process, which was evaluated for its feasibility by biological assay for rhEGF. It was noted that the lyophilized process could keep a good uniformity in the content of rhEGF in the preparation and make ready for the next step, although the lyophilized process slightly attenuated its bioactivity. The lyophilized mixture of rhEGF and lactose could be taken as an intermediate product in whole the preparation process. The assayed value of rhEGF content was taken as a measure for the next step to decide the additive amount of rhEGF.
We screened pre
本研究采用动物小肠体内、体外粘附力测定模型，选择常用生物粘附剂制成颗粒，并对颗粒的粘附性能进行筛选与评价。结果证明，CP934P、CP940P、SCMC、HPC等生物粘附剂在动物小肠上均具有一定的粘附性，其中CP940P粘附性最强，但与CP934P无明显差异，HPC次之。其粘附性能与添加比例呈正相关。以CP940、HPC与乳糖的适当配比，可获得具有适宜粘附性能的生物粘附颗粒，随着上述粘附材料成分的添加比例的增加，其粘附性增强；以乳糖：CP：HPC（78～82：10～15：8～13）较为合适。能达到95%以上的滞留率。
本文参照《中国生物制品规程》分别建立了放射免疫分析法（RIA）法和细胞测活法测定rhEGF生物粘附性肠溶胶囊含量和生物活性，目前放射免疫分析法（RIA）法和细胞测活法是测定rhEGF的主要方法，二者在测定rhEGF羟基端的部分代谢产物时，其结果是一致的。
采用放射免疫分析法考察了rhEGF在人工胃肠液介质的稳定性，rhEGF在人工胃液（pH=2）和人工肠液（pH=6.8）中的含量随时间的延长而呈下降趋势，6h后人工胃液和人工肠液中rhEGF浓度分别下降44.44%和55.75%；但在酶的存在下6h后rhEGF浓度分别下降95.43%和97.64%。稳定性研究结果表明，rhEGF在人工胃、肠液中稳定性较好，尽管在
酶的存在下rhEGF分解速度可明显加快，但可满足胃肠损伤修复的治疗需要，另外生物粘附材料的保护和缓释作用还可减缓其失活速度。
本研究设计处方中主药rhEGF含量甚微，混匀工艺是关键。本文采用少量乳糖加水先和rhEGF混匀成糊状，然后冷冻干燥得到均匀的含药乳糖粉末，再进一步用于制备颗粒。并利用生物活性测定方法，考察了冷冻干燥制备工艺对rhEGF生物活性的影响，冷冻干燥使rhEGF活性稍有降低，但冷冻干燥法可使剂型中rhEGF的含量均匀性得到保证，为下一步制剂作好准备。因此rhEGF和乳糖混和后的冻干粉末可作为整个制剂工艺的中间品，以此时的rhEGF的含量作为下次投料的标准。
选择正交实验设计，对rhEGF生物粘附肠溶制剂处方组成及制备工艺进行了研究，以肠溶胶囊的释放度、粘附性、生物活性和含量均匀度作为考察指标，对处方工艺进行筛选。结果表明CP与HPC的比例对胶囊释药速率和粘附性有非常显著影响,混合方式对药物含量均匀度也有显著影响,以综合得分最高为标准，确定优化处方为4号处方（A2B1C2），即CP和HPC的比例为10：8，助流剂选1%二氧化硅，以4号处方制备的生物粘附肠溶胶囊粘附性适中，含药均匀度好，缓冲液中0.5h释药8.6%,1h释药12%，基本能满足实验设计和治疗的要求，
采用RIA法测定了家兔给药后血清EGF浓度的变化趋势，及组织中的分布情况，籍以考察rhEGF的肠道吸收和体内分布情况，为吸收的安全性评价和临床应用提供参考依据。结果证明，家兔口服rhEGF生物粘附肠溶胶囊前后，血清EGF浓度和组织中的分布无明显改变，说明肠腔对rhEGF吸收甚微，肠道局部使用rhEGF基本不会产生全身作用。
以制剂中rhEGF释放度、含量和生物活性为指标，连续配制三批rhEGF生物粘附肠溶胶囊样品，对其不同条件下的放置稳定性进了考察。结果表明，rhEGF生物粘附肠溶胶囊在4℃ 条件下放置6个月较为稳定，制剂活性仅下降了11%。而在25℃条件下贮存时活性虽有稍明显下降，但仍符合质量要求。
本文研究的rhEGF生物粘附肠溶胶囊，可作用小肠靶部位，延长药物在粘膜局部的作用时间、提高局部rhEGF浓度，促进损伤部位愈合，并对血清EGF浓度无明显影响。基本达到了设计要求，可作为新的用于肠道损伤治疗的药物制剂进一步开发。
Since recombinant human epidermal growth factor (rhEGF) released to clinical use as a genetic engineered product, some good effects have been achieved in treatment of burn, trauma and a variety of chronic surfaces of wound. With continuous study on the product, its clinical use has been gradually exended, including preliminary trials on repairing of lesions in the gastrointestinal tract in recent years with limitation only to local EGF injection or spry under the endoscopy for treatment of peptic ulcer. And some reports deal with administration of oral liquid of rhEGF for the same purpose. Lesions in the gastrointestinal tract have most commonly followed burn, trauma and a great deal of severe infections and there aren't satisfied approaches available to these lesions at present. In order to seek out some effective medications for repairing of lesions in the gastrointestinal tract, we conducted studies to develop an intestine-released capsule of biological adhesive rhEGF.
The present study deals with in vivo and in vitro animal experimental models of small intestines for measurement of conglutinative force of biological adhesives in the form of granules to screen and assess their adhesive property. The results proved that biological adhesives, such as CP934P, CP940P, SCMC and HPC, showed to some extent adhesion to the tested animal intestines. Of them CP940P as well as CP934P has the most adhesive force, while HPC takes second place. The adhesive property has a positive correlation to their additive proportion. CP940, HPC and lactose in an appropriate blending ratio could offer a biological adhesive granule with an applicable adhesion property. With increased additive proportion of the adhesive materials mentioned above, the adhesive force could rise accordingly. A satisfied mixture could be obtained with proportions of lactose:CP:HPC (78-82:10-15:8-13), having more than 95% retention rate.
We have established methods of radioimmunoassay (RIA) and alive-cell identification to measure its content and biological activity of the enteric capsule of biological adhesive rhEGF with reference to Guidance for Biological Products in China. At present both of RIA and alive-cell identification are still the leading
methods for measurement of rhEGF, both of the approaches having an identified result in terms of measuring the metabolites from the hydroxyl moiety of rhEGF.
In addition, RIA has been used to observe the stability of rhEGF under the condition of artificial peptic juices. The content of rhEGF in artificial gastric juice (pH=2) and intestinal juice (pH=6.8) was prone to decrease over the time, lowering 44.44% and 55.75% from their baselines 6 hours later, respectively. But the decrease in the content of rhEGF would reach 95.43% and 97.64% after 6 hours in the presence of peptic enzymes, respectively.rhEGF in the artificial peptic juices was relatively stable and could meet the need for repair of lesions in the alimentary canal, although the process of its breakdown could be greatly expedited in the presence of peptic enzymes. The breakdown process could be attenuated through modified release of the adhesive materials.
The key technique in this preparation may be the blending approach in that the prescription design in the present study includes very low amount of main active ingredient, rhEGF. Accounting for major part of excipients in the recipe, a proportion of lactose was first mixed with rhEGF for its dilution. The resultant mixture underwent lyophilized process, which was evaluated for its feasibility by biological assay for rhEGF. It was noted that the lyophilized process could keep a good uniformity in the content of rhEGF in the preparation and make ready for the next step, although the lyophilized process slightly attenuated its bioactivity. The lyophilized mixture of rhEGF and lactose could be taken as an intermediate product in whole the preparation process. The assayed value of rhEGF content was taken as a measure for the next step to decide the additive amount of rhEGF.
We screened pre
引文
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