【作者】 谢斌；

【导师】 方建国；

【作者基本信息】 华中科技大学 ， 中西医结合药理学， 2011， 博士

【摘要】 处方工艺的研究是口服缓释制剂开发的物质基础和关键。体外释放度考查是口服缓释制剂开发的重点内容,也是处方工艺筛选和评价的重要指标。在缓释制剂的其他基本技术指标符合药典制剂通则要求的前提下,达到预期的体外释放行为是临床前处方工艺筛选的目标。但是,体外释放特性有时会与体内药物释放行为不一致的情况,因此,处方工艺的筛选研究必须经过动物药代动力学研究,甚至与临床试验相结合起来,根据动物药代动力学研究和临床试验结果进行处方和工艺的调整。临床研究前确定的处方工艺往往只是一个阶段性的研究结果,体内试验结果才是处方工艺验证的最终指标。体外释放度是口服缓释制剂处方工艺筛选的重要指标,也是有效控制产品质量的重要指标。建立的释放度检查方法,如果能够结合体内试验研究结果,建立体内外相关性,则体外释放度检查可以在一定程度上预测产品的体内行为。因此,在处方工艺探讨和研究中,通过必要的动物药代动力学试验初步考察制剂的体内药物释放行为可以在较大程度上化解体内外不相关的技术风险。动物药代动力学研究对于处方工艺的选择具有重要意义,其目的在于验证研究的药物制剂进入机体后是否存在突释的可能性,以及药物在体内外释放行为是否一致,即药物在体内与体外的相关性。目的：建立一种制剂处方筛选和评价的方法。为降低药物在体内试验失败的风险,提高处方和工艺的把握度,在进行缓释制剂的处方筛选中,我们把大鼠的体内外相关性作为口服缓释制剂处方筛选过程中的关键性筛选指标。这样一方面有利于选出具有恰当体内释放速度的处方,同时,也为Beagle犬体内试验和人体临床试验奠定分析方法学的基础。本实验以布洛芬缓释胶囊和尼美舒利缓释胶囊为例,以大鼠为实验动物模型,进行大鼠药代动力学对比研究,为筛选最佳的口服缓释制剂的处方和工艺奠定方法学的基础。方法：1、建立口服缓释制剂的大鼠体内高效液相色谱测定方法(方法专属性研究、标准曲线及线性回归方程建立、精密度与准确度试验、绝对回收率试验、稳定性试验)。2、在预实验的基础上,分别口服给予实验动物自制口服缓释制剂和参比制剂(相同活性成分、规格和剂型的口服缓释制剂),在不同时间点采集血样,选择合适的分析仪器,对血样进行分析。3、运用DAS软件,对数据进行统计分析,分析体内和体外的相关性。如体内外不相关,则根据数据统计分析,提出处方修改意见。结果：通过自制的布洛芬缓释胶囊和尼美舒利缓释胶囊与已上市口服缓释制剂的大鼠药代动力学对比研究,探讨自制和已上市口服缓释制剂的体外释放特性一致不代表其体内释放特性一致,从而得出以大鼠为模型筛选口服缓释制剂处方和工艺的价值。结论：本文建立的大鼠割尾采血的药动学模型筛选和评价缓释制剂的处方的方法,在国内外罕有报道。该方法对于处方和工艺的选择具有重要意义,很大程度上化解了Beagle犬药动学评价和人体临床研究的风险。我们建立这种方法的目的不是为了取代缓释制剂的Beagle犬药动学评价和人体的临床研究,而是为缓释制剂Beagle犬药动学研究和临床研究提供有价值的参考,这在方法学上是一种创新。值得一提的是,以大鼠为动物模型对不同生产厂家生产的相同活性成分、相同剂型和规格的产品的质量评价也具有一定的借鉴意义。目前,肥胖已经危害人类健康的重要问题,肥胖症作为一种全身性代谢性疾病,日益受到人们的重视。肥胖与心血管疾病、Ⅱ型糖尿病和代谢性疾病有非常紧切的关系,是此类疾病的高诱发因素。最新的科学研究表明,前脂肪细胞的增殖、分化与肥胖密不可分,脂肪细胞快速增生导致细胞数目增多可能是引起肥胖的机制之一。因此,抑制前脂肪细胞的增殖、分化,以及促进前脂肪细胞的凋亡将对抑制肥胖的产生具有重要的作用。荷叶(Nelumbinis Folium)为睡莲科植物Nelumbo nucifera Gaertn的干燥叶。苦,平。归肝、脾、胃经。始载于唐朝孟诜《食疗本草》,历版《中国药典》亦均有收载,具有清热解暑,升发清阳,凉血止血之功效。作为一味传统中药,荷叶在减肥降脂方面的保健和治疗效果突出。荷叶碱为荷叶提取物的主要成分,治疗肥胖的作用机理尚不清楚。本实验以3T3-L1前脂肪细胞为靶细胞,研究了荷叶碱潜在的减肥作用。目的：本研究的目的是以大鼠3T3-L1前脂肪细胞为靶细胞,考察荷叶碱对其增殖、分化和凋亡的影响。方法：我们研究了荷叶碱对3T3-L1前脂肪细胞的影响,以MTT法分析了荷叶碱对3T3-L1前脂肪细胞的增殖的影响,以油红O染色法分析了荷叶碱对3T3-L1前脂肪细胞分化的影响,以AnnexinⅤ-FITC/PI双染法和流式细胞术法分析了荷叶碱对3T3-L1前脂肪细胞凋亡的影响。结果：我们研究了荷叶碱对3T3-L1前脂肪细胞的影响。MTT法检测荷叶碱对3T3-L1前脂肪细胞增殖的影响,发现荷叶碱能够抑制3T3-L1前脂肪细胞的增殖,并且荷叶碱对3T3-L1前脂肪细胞具有时间和浓度依赖性。随荷叶碱浓度的增加及荷叶碱作用时间的延长,对3T3-L1前脂肪细胞增殖的抑制率也增加。油红O染色法检测荷叶碱对3T3-L1前脂肪细胞分化的影响,发现荷叶碱能够抑制3T3-L1前脂肪细胞的分化,并且荷叶碱对3T3-L1前脂肪细胞的抑制作用具有时间和浓度依赖性。随荷叶碱浓度的增加及荷叶碱作用时间的延长,对3T3-L1前脂肪细胞分化的抑制率也增加。AnnexinV-FITC/PI双染法检测荷叶碱对3T3-L1前脂肪细胞凋亡的影响中,考察荷叶碱作用于3T3-L1前脂肪细胞不同时间点细胞的凋亡情况,发现荷叶碱能够促进3T3-L1前脂肪细胞的凋亡,并且具有浓度依赖性,随浓度的升高和作用时间的延长,3T3-L1前脂肪细胞的凋亡率越高。结论：我们研究了荷叶碱对3T3-L1前脂肪细胞增殖、分化和凋亡作用,发现荷叶碱在抑制3T3-L1前脂肪增殖和分化,促进3T3-L1前脂肪细胞的凋亡,在一定的浓度范围内,具有浓度依赖性,随浓度的升高其抑制率和凋亡率越高。更多还原

【Abstract】 Process of prescriptions is not only the material basis of oral sustained-release formulations, but also it is pivitol points. In vitro accumulation release rate test is the focus of research and development of oral sustained-release formulations, it is also is an important indicator of prescription. Under the premise of the sustained release formulation’s basic technical indicators in line with pharmacopoeia general requirements, to achieve the desired accumulated release rate of formulation and process in vitro is a pre-clinical screening goal, but sometimes accumulated release rate in vitro and drug release behavior in vivo is not consistency. Therefore, screening studies of formulation and process must be combined with animesulideal pharmacokinetic studies, even with clinical trials. Prescription and process should be adjusted according to the results of animal pharmacokinetic studies and clinical trials.Prescription and process which is determined at pre-clinical phase is often the findings of some phase. Verifying the formulation and process through experimental results in vivo is the ultimate target. Accumulated release rate in vitro of oral sustained release formulation is not only an important indicator of prescription and process screening, but also it is an effective and important indicator of product quality control. If the correlation in vivo and in vivo has been established through accumulation release rate test, the results of the accumulated release rate in vitro can predict release behavior in vivo to a certain extent. Therefore, evaluating prescription and process through the necessary preliminary pharmacokinetic study in vivo can decrease technical risks of sustained release formulations to a greater extent.Animal pharmacokinetic studies for the choice of formulation and process is important, its main purpose is to verify whether the agent is of the possibility of burst release or not and evaluate whether release behavior in vivo and in vitro are consistent or not. That is to say, release behavior of drug in vitro is correlation to release behavior of drug in vivo or not.ObjectiveTo establish a method of formulation prescription screening and evaluation. In order to reduce the risk of failure in vitro and to improve the degree of success prescription, we regard the correlation of test in vitro and in vivo as a pivitol indicator of prescription screening. On the one hand, it is useful to screen successful prescription and process, on the other hand, it will be a methological basis of the Beagle dogs for testing and human clinical trials. In this experiment, we take ibuprofen and nimesulide release capsules for examples, we have developed the comparison studies of pharmacokinetics in rat in order to screen optimized prescriptions and process.MethodsFirstly, to establish high performance liquid chromatographic method of oral sustained-release formulations in rats (method specificity, the standard curve and linear regression equation, precision and accuracy test, absolute recovery test, stability test).Secondly, on the basis of preliminary experiments, animesulideals were given oral homemade oral sustained-release formulations and reference preparation (the same active ingredients, specifications and formulations of oral sustained-release formulations). Blood samples of rats were collected at different time and analysized by the appropriate analytical instruments.Thirdly, the relevance of oral homemade oral sustained-release formulations and reference preparation in vivo and in vitro is analysized by the use of DAS software. If the relevance of oral homemade oral sustained-release formulations and reference preparation in vivo and in vitro is not good, prescription revisions will be given to the researchers. ResultsTake ibuprofen sustained release capsules and nimesulide sustained release capsules for example, comparative pharmacokinetics study of oral self-made preparation and reference preparation have been used to verify that consistency of the results in vitro can’t stand for consistency of the results in vivo. Then, we conclude that it is very valuable to screen pharmaceutical prescription and process by the rat model.ConclusionWe established the pharmacokinetic method by cutting rat tail of screening and evaluating sustained release formulation prescription and process which is rarely reported at home and abroad. The method is valuable for selection of formulation prescription and process. It defused the risk of fail Beagle dog and human experiments. Our goal is not to replace the pharmacokinetic study in Beagle dogs and clinical study in patients, but to provide an innovative methodology. It is worth mentioning that it is valuable to evaluate quality of different products which is characteristics of the same active ingredients, formulations and specifications. Currently, obesity is a major hazard to human health problems. As a kind of systemic metabolic diseases, obesity has been paid increasing attention. Obesity has been considered to be a risk factor associated with the genesis or development of various diseases, including cardiovascular disease, type 2 diabetes mellitus and metabolic syndrome, which resulting in an increasing morbidity and mortality. Recent reports have proposed that the preadipocytes play a key role by differentiating into mature adipocytes and increasing fat mass. Obesity is characterized by the accumulation of adipose tissue, which expands due to an increase in adipocyte size and number. Therefore, inhibition of adipogenesis from preadipocytes may regulate the amount of adipose tissue.Nelumbinis Folium, the dried leaf of Nelumbo nucifera Gaertn, is bitter, slightly sweet and neutral. Nelumbinis Folium can be used to relieve summer-heat, to invigorate the spleen function of the spleen and arrest bleeding by reducing heat in blood. It was first recorded in the book of Herbal in Tang Dynasty, the every version of Chinese Pharmacopoeia also has close set. As a traditional chinese medicine, the leaf of Nelumbo nucifera Gaertn is prominent in the diet’s health and treatment of lipid-lowering effect. Studies on antiobesity and lipid-lowering effect of the leaf of Nelumbo nucifera Gaertn have become the focus of attention of scholars in the world. Nuciferine is the main component of the the leaf of Nelumbo nucifera Gaertn extract. However, the literature regarding the effect of alkaloids in the leaf of Nelumbo nucifera for treatment of obesity still remains unclear. In the present study, we investigated the antiobesity potential of Nelumbo nucifera alkaloid (NNA) using 3T3-L1 preadipocytes.ObjectiveThe objective of this study was focused on the induction of apoptosis and the inhibition of preadipocyte proliferation and differentiation by NNA in 3T3-L1 preadipocytes.MethodsWe investigated the effects of Nuciferine on 3T3-L1 preadipocytes in this study. We determined the proliferation of 3T3-L1 preadipocytes by MTT assays and the differentiation of 3T3-L1 preadipocytes by oil red staining, and measured the apoptosis by AnnexinⅤ-FITC/PI staining and Flow Cytometry (FCM).ResultsIn this study we investigated the effects of nuciferine on 3T3-L1 preadipocytes. Results showed that nuciferine significantly inhibited the proliferation and differentiation of 3T3-L1 preadipocytes and its inhibition of proliferation and differentiation in a concentration-dependent and time-dependent manner. At the same time nuciferine also promoted the apoptosis of 3T3-L1 preadipocytes, this was also a concentration-dependent and time-dependent manner.ConclusionWe evaluated the proliferation and differentiation inhibition, apoptosis promotion on 3T3-L1 preadipocytes of nuciferine, and prevention of weight increase in vivo. This is vital important for the further research of nuciferine in vivo and vitro.更多还原