二烯丙基三硫醚肺损伤保护机制及其固体制剂的初步研究

发布时间：2018-01-06 05:29

  本文关键词：二烯丙基三硫醚肺损伤保护机制及其固体制剂的初步研究　出处：《山东大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 二烯丙基三硫 肺损伤 抗氧化 抗炎 肠溶微囊

【摘要】：肺部损伤主要由过度炎症反应和氧化应激等发病机制所致病的,因此,炎症消除和去除氧化应激在肺部损伤的治疗中起着关键作用。临床治疗肺部损伤常用药物为抗生素、激素类和抗氧化剂等。所使用的抗生素类药物如庆大霉素、丁胺卡那霉素、四环素等具有肝、肾损伤等毒副作用；另外,抗生素类药物长期用药容易产生耐药性。激素类药物能有效地改善肺泡毛细血管膜的通透性,减少毛细血管渗出,抑制炎症反应,阻止过度损伤。但是,激素类的副作用也是临床医生所敬畏的。世界著名制药公司默克公司公布的一项临床研究结果：激素类药物对少年儿童的身高增长有明显抑制。抗氧化剂按来源可分为天然抗氧化剂(如超氧化物歧化酶、谷胱甘肽过氧化物酶、过氧化氢酶以及还原型谷胱甘肽、维生素C和维生素K等)和人工合成或化学修饰的抗氧化剂,如叔丁基对甲氧酚(BHA)、二叔丁基对甲酚(BHT)、乙氧奎(EQ)、MnPAM和MnBAM等。抗氧剂的未知副反应,也是目前食品药品行业关注的热点。临床上关于肺损伤的治疗多是单一药物治疗为主,少见多种通路共同治疗的方案。前期大量文献调研发现,DATS在抗炎和抗氧化方面均具有明显的治疗效果,但目前并未见关于该化合物在肺部保护方面的详细深入报道。因此,本课题致力于验证和探索DATS肺保护方面的机制。本课题的内容包括四个方面。第一,DATS的药理活性评价,主要考察DATS对于肺损伤的治疗作用和作用机制；第二,DATS的基本性质评价以及体外检测方法学建立；第三,DATS的固化工艺和肠溶制剂进行初步研究,通过不同固体化工艺制备DATS三种固体剂型,进一步制备肠溶制剂并对其评价；第四,DATS制剂生物药剂学的初步研究,对主要代谢产物进行了合成和鉴定,并对制剂的的代谢产物的药动学过程进行了研究。首先,建立体外研究模型,从体外系统研究DATS对肺损伤保护以及治疗的研究机制。A549细胞系是人体肺腺癌上皮细胞系,是正常肺上皮细胞的癌变细胞系,具有正常肺上皮细胞的特征。本章选择A549细胞系为肺部上皮组织的体外研究模型,测定在萘和DATS处理的情况下细胞分泌物、细胞蛋白及细胞核RNA的量的变化,评价DATS对上皮细胞的保护作用。数据表明,DATS能够显著抑制萘诱导的细胞毒性,同时抑制了促炎性细胞因子(TNF-α,IL-6和IL-8)的产生,表现出较高的抗炎活性。DATS也能抑制萘引起的SOD的活性降低,抗氧化活性剂极高,这一结果和体外抗氧化活性实验的结果一致。Western blot和免疫荧光结果表明DATS的抗炎和抗氧化作用是由核转录因子NF-κB的活化调节来实现的,而NF-κB p65的核转位通过减少IκBα的降解来达到。RT-PCR结果显示,萘处理组的IL-8基因高水平表达,而DATS处理对这一高水平表达有明显抑制。为了进一步研究DATS对萘引起的炎症和氧化损伤的保护机制,课题组一次性给一定剂量的萘(100mg/kg)建立昆明小鼠炎症和氧化损伤模型,通过测定血清和组织相关生化指标以及组织病理学改变,评价DATS抗损伤的效果。进一步通过免疫组化学,从分子生物角度探讨该保护机制的通路。DATS处理明显抑制了MDA的产生,伴随着血清ALT和AST的活性变化,这一结果指示DATS能够显著减少体内的脂质过氧化和细胞的损伤,从而保护肝组织免受萘引起的氧化损伤。这些数据表明,DATS显示抗氧化作用,这可能与体内抗氧化酶活性变化有关(LDH,SOD和MPO)。这项研究还表明,大蒜素可通过抑制肺组织炎症细胞浸润抑制血清TNF-α口IL-8的生产。这种效应似乎是通过抑制中性粒细胞浸润,降低肺组织中炎症因子的过分表达。结果表明,DATS有保护组织免受氧化剂和炎症损伤的作用。体内研究结果和体外结果相一致,共同证明了DATS的抗氧化及抗炎作用。DATS固体制剂的开发是本课题关注的另一方面,在处方开发前,本论文对DATS建立了体外检测方法学。本实验所选用的高效液相色谱法测定DATS的含量,专属性好,精密度符合要求,灵敏度和回收率高,操作方法简单,可适用于DATS的体外定性和定量分析。线性关系考察结果表明,DATS在5-200μg/mL范围内浓度与色谱峰面积的线性关系良好。测定了药物在水中的溶解度,并考察了增溶剂的加入对其水中溶解度的改善效果。通过将药物纯化,考察了纯化后药物的稳定性,并在此基础上,评价了温度及抗氧化剂对DATS纯度的影响。结果可见,抗氧化剂均对DATS长期存放的稳定性有一定增益,同时纯化后的DATS在低温或者冷冻环境下能够较长时间保持高纯度(90 d)。抗氧化剂和增溶剂的研究为后期处方的开发提供了数据支持。前期调研发现,DATS在固体制剂研究方面仍处于初步探索阶段。在前期多种手段尝试后,选择液固载药系统、包合物和固体分散体三种挥发油类药物开发常用的常规剂型进行基础研究,制备出载药的固体颗粒,为后期更稳定和性质更优良的处方的发现,提供基础。通过本章实验筛选发现,以上常规剂型能够实现液体形式药物的固体化,但在稳定性方面仍有待改善。同时,在药物刺激性味道的掩盖方面仍存在较大问题。OSCs类化合物具有刺激性辛辣气味,直接口服对胃肠道有强烈刺激性。通过实验验证,DATS具有强烈胃刺激性,对胃粘膜有严重损伤。因此开发肠溶性固体制剂,可以增强病人适应性,同时也能降低药物自身的毒副作用。本文选择适于工业大生产的和挥发油类化合物的固体肠溶制剂微囊进行开发。首先,通过效应面优化法对微囊处方进行优化；其次通过SEM对微囊外观进行评价,同时结合多种固体制剂评价手段,比如粒径、流动性和体外溶等对这一制剂进行综合评价,为这一剂型的后期开发提供大量数据支持；最后,在最优处方的基础上,结合前期筛选的药物固化手段,先将药物固化后再进行微囊的制备,同样的分析和评价手段,制备出性能更优越的微囊制剂。该制剂,具有较高的载药量,同时也在一定程度上增加了制剂的稳定性。结合课题组多项研究基础,多次尝试发现药物在体内的基本代谢特点,选择其中两个具有代表性的代谢产物进行监测,发现DATS在体内主要以被氧化为主,含量比较高的代谢产物为AMSO和AMSO2。以GC-MS为检测手段,分别以全扫描和选择离子扫描建立这两种化合物的体内分析方法并对其方法学进行验证。以SD大鼠为实验动物模型,通过口服不同制剂形式,对药物的体内代谢动力学行为进行评价。这两种代谢产物均表现出先增高后降低的趋势。注射剂组,二者均在8h达到最高值,相对于注射剂组,固体制剂组达峰时间延迟至12h,延长了药物在体内的循环时间。本论文从体内外两个角度,从分子水平到基因水平,深入探讨了DATS对肺损伤的保护机制。实验结果证明了,药物同时从抗炎和抗氧化两个角度对机体产生保护作用,为该化合物在临床上的应用提供药理研究支持。另外,从多角度展开对该液体药物的固体制剂剂型的开发。通过联用液固系统和微囊制备技术,制备出同时具有较高载药量和较高稳定性的固体制剂形式,为扩大该化合物的工业开发,提供了大量的数据支持。本论文合成DATS体外的主要代谢产物,并建立了该化合物全扫描和选择性单离子扫描的GC-MS检测方法,对制剂的体内行为进行了评价。为该化合物的全面代谢评价提供前期的数据支持。但是,由于该化合物在体内代谢产物种类较多,在攻读学位期间未完成所有代谢产物的定性和定量分析,该工作仍需要课题组其他人员的继续努力。
[Abstract]:The pathogenesis of lung injury mainly by excessive inflammation and oxidative stress in the pathogenesis, therefore, eliminate inflammation and remove oxidative stress in the treatment of lung injury plays a key role in the treatment of lung injury. The clinical commonly used drugs for antibiotics, hormones and antioxidants. The use of antibiotic drugs such as gentamicin, amikacin, kanamycin Su, tetracycline with liver toxicity, renal injury; in addition, long-term use of antibiotics resistance. The permeability of hormone drugs can effectively improve the alveolar capillary membrane, reduce capillary permeability, inhibit inflammation, prevent excessive damage. However, side effects of hormones is the fear of a clinician. Study in the world famous pharmaceutical company Merck Co announced: hormone drugs have obvious inhibition on children's height growth. Antioxidant Agents can be classified according to source of natural antioxidants (such as superoxide dismutase, glutathione peroxidase, catalase and glutathione, vitamin C and vitamin K) and synthetic or chemical modification of antioxidants, such as Uncle Ding Ji p-methoxyphenol (BHA), di Ding Ji paracresol (BHT), ethylene oxide Quinn (EQ), MnPAM and MnBAM. The unknown side effects of antioxidants, which is currently the hot food and drug industry attention. Clinical on the treatment of lung injury is a single drug therapy, a variety of rare pathways treatmentscheme. Found a large number of literature research, DATS has obvious therapeutic effect on anti-inflammatory and antioxidant but at present, and no on the compound in lung protection with in-depth coverage. Therefore, this thesis focuses on the mechanism of development and validation of DATS lung protection. This topic includes four aspects . first, the pharmacological activity of DATS, mainly on the DATS for the treatment effect and the mechanism of lung injury; second, basic properties and in vitro evaluation of DATS detection methods established; third, a preliminary study on the curing process and enteric preparation of DATS by different solid preparation, three solid dosage forms of DATS process. The preparation of enteric preparation and its evaluation; fourth, preliminary study on Preparation of biological pharmacy DATS, the main metabolites were synthesized and identified, and the preparation of the metabolites of pharmacokinetics were studied. First, establish the research model in vitro, from in vitro system research on DATS mechanism of lung protection damage and treatment of.A549 cell line of human lung adenocarcinoma epithelial cell line, cancer cells of normal lung epithelial cells, with characteristics of normal lung epithelial cells. This chapter A549 cell lines of lung In vitro model of epithelial tissue, cell secretion in determination of naphthalene and DATS processing, the changes of cell protein and nuclear RNA content, protective effect evaluation of DATS on epithelial cells. Data showed that DATS could significantly inhibit the cytotoxicity induced by naphthalene, and inhibition of proinflammatory cytokines (TNF- alpha IL-6, and IL-8) have decreased the anti-inflammatory activity of.DATS was higher and also inhibited the activity of naphthalene induced SOD, antioxidant activity of this agent is extremely high, and the antioxidant activity in vitro experimental results consistent with.Western blot and immunofluorescence results showed that the anti-inflammatory and antioxidant effects of DATS by activation of nuclear transcription factor kappa NF- the regulation of B to achieve, and the NF- kappa B nuclear translocation of p65 by reducing the degradation of I kappa B alpha to achieve the results of.RT-PCR showed that the expression of naphthalene groups of IL-8 high level gene, and DATS treatment on the high level of expression Significantly decreased. In order to protect the further study of the mechanism of DATS on inflammation and oxidative damage caused by naphthalene, group disposable to a certain dose of naphthalene (100mg/kg) of inflammation and oxidative damage model of Kunming mice by determination of serum and tissue biochemical indexes and pathological changes, evaluation of DATS anti injury effect. Through further immune group of chemical, explore the protection mechanism of the.DATS pathway inhibited the production of MDA from molecular biology angle, accompanied by changes in the activity of serum ALT and AST, this result indicates that DATS can significantly reduce the lipid peroxidation and cell damage, thereby protecting the liver from oxidative damage caused by naphthalene. These data show that DATS showed antioxidant effect, which may be related to in vivo activity of antioxidant enzymes (LDH, SOD and the MPO). This study also showed that allicin can inhibit lung tissue The infiltration of inflammatory cells inhibited serum TNF- IL-8 alpha production. This effect appears to be mediated by inhibition of neutrophil infiltration, reduce the excessive expression of inflammatory cytokines in lung tissue. The results show that DATS can protect tissues from oxidative damage and inflammatory agent role. The research results and in vivo results are consistent, that the development of antioxidant and anti-inflammatory effects of.DATS solid preparation of DATS is another aspect of the topic of concern, in the prescription before development, the establishment of DATS detection method in vitro. The high performance liquid chromatography method for the determination of DATS content, good specificity, sensitivity and precision meet the requirements, high recovery rate, simple operation method, qualitative and quantitative analysis can be applied to DATS in vitro. The linear relationship between the investigation shows that the linear relationship between the concentration of DATS in the 5-200 g/mL range and the peak areas. The determination of good medicine Solubility in water, and the effects of solvent addition on its solubility in water. The effect of drug purification, investigated the stability after purification of drugs, and on this basis, to evaluate the effects of temperature and antioxidants on the purity of DATS. Results showed that some antioxidants gain stability of DATS in long-term storage. At the same time, the purified DATS can maintain high purity or a longer period of time in the low temperature freezing environment (90 d). Study on the antioxidant and the solutizer provides data support for the development of late prescription. Preliminary investigation found that DATS studies in solid preparation is still at the preliminary stage. In the early try various means, selection of liquid solid the drug delivery system, inclusion complexes and solid dispersions of three kinds of conventional forms of volatile oil used for drug development research, preparation of drug loaded solid particles, is more stable and late Provide the basic properties of prescription more excellent discovery. This chapter through screening experiments found that more conventional forms can achieve solid liquid form of drugs, but in terms of stability remains to be improved. At the same time, with a pungent odor in drug stimulating taste cover there are still major problems of.OSCs compounds, direct oral administration strong stimulation of the gastrointestinal tract. Through the experiment, DATS has a strong stomach irritation, severe injury of gastric mucosa. Therefore the development of enteric solid preparation, can enhance the adaptability of patients, but also can reduce the drug toxicity. The solid enteric preparation microcapsules is suitable for industrial production and the volatile oil compounds development. First, the microcapsules were optimized by response surface methodology; followed by SEM of microcapsules was evaluated, combined with a variety of solid preparations The evaluation means, such as particle size, mobility and in vitro dissolution of a comprehensive evaluation of the preparation, provide a lot of data to support for the later development of the dosage form; finally, based on the optimal prescription, combined with drug curing means early screening, the first drug after solidification in microcapsule preparation, the same the analysis and evaluation methods, preparation of microcapsule superior performance. The preparation has high drug loading, but also increase the stability of the preparation to a certain extent. Based on the basis of a number of studies, attempts to find the basic metabolic characteristics of drugs in the body, by monitoring selected the two representative the metabolites of DATS in vivo, found mainly in the oxidation of main metabolites with high content of AMSO and AMSO2. by means of GC-MS, respectively, with full scan and selected ion scan to establish the two. In vivo analysis method and to verify its methodology. Using SD rats as experimental animal model, through the different oral dosage form to evaluate the dynamic behavior of drug metabolism in vivo. The two metabolites showed increased first and then decreased. The injection group, two were the highest in 8h compared with injection group, solid preparation, peak time delay to 12h, prolong the circulation time of the drug in vivo. The in vitro and in vivo in two aspects, from the molecular level to gene level, in-depth study of the protective mechanism of DATS on lung injury. The experimental results show that, from the perspective of two anti-inflammatory drugs at the same time and antioxidant on the body to produce protective effect, to provide support for the application of pharmacological study of the compound in the clinic. In addition, the development of solid dosage forms of liquid medicine from many aspects. Through the combination of liquid solid and micro system Capsule preparation technology, prepared with solid dosage forms with high loading and high stability, to expand the industrial development of the compound, with a large amount of data support. The main metabolites of DATS were synthesized in vitro, and established the GC-MS detection method of the compound scan and selective ion scan the in vivo behavior of preparation was evaluated. The evaluation provides data support for the overall metabolism of the compound. However, due to the in vivo metabolites of many types, qualitative and quantitative analysis of all metabolites was not completed during his degree, the work still needs our staff to continue our efforts.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R563
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本文编号：1386502