發(fā)布時間：2018-01-06 05:29

  本文關鍵詞：二烯丙基三硫醚肺損傷保護機制及其固體制劑的初步研究　出處：《山東大學》2016年博士論文　論文類型：學位論文

  更多相關文章： 二烯丙基三硫 肺損傷 抗氧化 抗炎 腸溶微囊

【摘要】：肺部損傷主要由過度炎癥反應和氧化應激等發(fā)病機制所致病的,因此,炎癥消除和去除氧化應激在肺部損傷的治療中起著關鍵作用。臨床治療肺部損傷常用藥物為抗生素、激素類和抗氧化劑等。所使用的抗生素類藥物如慶大霉素、丁胺卡那霉素、四環(huán)素等具有肝、腎損傷等毒副作用；另外,抗生素類藥物長期用藥容易產(chǎn)生耐藥性。激素類藥物能有效地改善肺泡毛細血管膜的通透性,減少毛細血管滲出,抑制炎癥反應,阻止過度損傷。但是,激素類的副作用也是臨床醫(yī)生所敬畏的。世界著名制藥公司默克公司公布的一項臨床研究結果：激素類藥物對少年兒童的身高增長有明顯抑制�？寡趸瘎┌磥碓纯煞譃樘烊豢寡趸瘎�(如超氧化物歧化酶、谷胱甘肽過氧化物酶、過氧化氫酶以及還原型谷胱甘肽、維生素C和維生素K等)和人工合成或化學修飾的抗氧化劑,如叔丁基對甲氧酚(BHA)、二叔丁基對甲酚(BHT)、乙氧奎(EQ)、MnPAM和MnBAM等。抗氧劑的未知副反應,也是目前食品藥品行業(yè)關注的熱點。臨床上關于肺損傷的治療多是單一藥物治療為主,少見多種通路共同治療的方案。前期大量文獻調(diào)研發(fā)現(xiàn),DATS在抗炎和抗氧化方面均具有明顯的治療效果,但目前并未見關于該化合物在肺部保護方面的詳細深入報道。因此,本課題致力于驗證和探索DATS肺保護方面的機制。本課題的內(nèi)容包括四個方面。第一,DATS的藥理活性評價,主要考察DATS對于肺損傷的治療作用和作用機制；第二,DATS的基本性質(zhì)評價以及體外檢測方法學建立；第三,DATS的固化工藝和腸溶制劑進行初步研究,通過不同固體化工藝制備DATS三種固體劑型,進一步制備腸溶制劑并對其評價；第四,DATS制劑生物藥劑學的初步研究,對主要代謝產(chǎn)物進行了合成和鑒定,并對制劑的的代謝產(chǎn)物的藥動學過程進行了研究。首先,建立體外研究模型,從體外系統(tǒng)研究DATS對肺損傷保護以及治療的研究機制。A549細胞系是人體肺腺癌上皮細胞系,是正常肺上皮細胞的癌變細胞系,具有正常肺上皮細胞的特征。本章選擇A549細胞系為肺部上皮組織的體外研究模型,測定在萘和DATS處理的情況下細胞分泌物、細胞蛋白及細胞核RNA的量的變化,評價DATS對上皮細胞的保護作用。數(shù)據(jù)表明,DATS能夠顯著抑制萘誘導的細胞毒性,同時抑制了促炎性細胞因子(TNF-α,IL-6和IL-8)的產(chǎn)生,表現(xiàn)出較高的抗炎活性。DATS也能抑制萘引起的SOD的活性降低,抗氧化活性劑極高,這一結果和體外抗氧化活性實驗的結果一致。Western blot和免疫熒光結果表明DATS的抗炎和抗氧化作用是由核轉錄因子NF-κB的活化調(diào)節(jié)來實現(xiàn)的,而NF-κB p65的核轉位通過減少IκBα的降解來達到。RT-PCR結果顯示,萘處理組的IL-8基因高水平表達,而DATS處理對這一高水平表達有明顯抑制。為了進一步研究DATS對萘引起的炎癥和氧化損傷的保護機制,課題組一次性給一定劑量的萘(100mg/kg)建立昆明小鼠炎癥和氧化損傷模型,通過測定血清和組織相關生化指標以及組織病理學改變,評價DATS抗損傷的效果。進一步通過免疫組化學,從分子生物角度探討該保護機制的通路。DATS處理明顯抑制了MDA的產(chǎn)生,伴隨著血清ALT和AST的活性變化,這一結果指示DATS能夠顯著減少體內(nèi)的脂質(zhì)過氧化和細胞的損傷,從而保護肝組織免受萘引起的氧化損傷。這些數(shù)據(jù)表明,DATS顯示抗氧化作用,這可能與體內(nèi)抗氧化酶活性變化有關(LDH,SOD和MPO)。這項研究還表明,大蒜素可通過抑制肺組織炎癥細胞浸潤抑制血清TNF-α口IL-8的生產(chǎn)。這種效應似乎是通過抑制中性粒細胞浸潤,降低肺組織中炎癥因子的過分表達。結果表明,DATS有保護組織免受氧化劑和炎癥損傷的作用。體內(nèi)研究結果和體外結果相一致,共同證明了DATS的抗氧化及抗炎作用。DATS固體制劑的開發(fā)是本課題關注的另一方面,在處方開發(fā)前,本論文對DATS建立了體外檢測方法學。本實驗所選用的高效液相色譜法測定DATS的含量,專屬性好,精密度符合要求,靈敏度和回收率高,操作方法簡單,可適用于DATS的體外定性和定量分析。線性關系考察結果表明,DATS在5-200μg/mL范圍內(nèi)濃度與色譜峰面積的線性關系良好。測定了藥物在水中的溶解度,并考察了增溶劑的加入對其水中溶解度的改善效果。通過將藥物純化,考察了純化后藥物的穩(wěn)定性,并在此基礎上,評價了溫度及抗氧化劑對DATS純度的影響。結果可見,抗氧化劑均對DATS長期存放的穩(wěn)定性有一定增益,同時純化后的DATS在低溫或者冷凍環(huán)境下能夠較長時間保持高純度(90 d)。抗氧化劑和增溶劑的研究為后期處方的開發(fā)提供了數(shù)據(jù)支持。前期調(diào)研發(fā)現(xiàn),DATS在固體制劑研究方面仍處于初步探索階段。在前期多種手段嘗試后,選擇液固載藥系統(tǒng)、包合物和固體分散體三種揮發(fā)油類藥物開發(fā)常用的常規(guī)劑型進行基礎研究,制備出載藥的固體顆粒,為后期更穩(wěn)定和性質(zhì)更優(yōu)良的處方的發(fā)現(xiàn),提供基礎。通過本章實驗篩選發(fā)現(xiàn),以上常規(guī)劑型能夠實現(xiàn)液體形式藥物的固體化,但在穩(wěn)定性方面仍有待改善。同時,在藥物刺激性味道的掩蓋方面仍存在較大問題。OSCs類化合物具有刺激性辛辣氣味,直接口服對胃腸道有強烈刺激性。通過實驗驗證,DATS具有強烈胃刺激性,對胃粘膜有嚴重損傷。因此開發(fā)腸溶性固體制劑,可以增強病人適應性,同時也能降低藥物自身的毒副作用。本文選擇適于工業(yè)大生產(chǎn)的和揮發(fā)油類化合物的固體腸溶制劑微囊進行開發(fā)。首先,通過效應面優(yōu)化法對微囊處方進行優(yōu)化；其次通過SEM對微囊外觀進行評價,同時結合多種固體制劑評價手段,比如粒徑、流動性和體外溶等對這一制劑進行綜合評價,為這一劑型的后期開發(fā)提供大量數(shù)據(jù)支持；最后,在最優(yōu)處方的基礎上,結合前期篩選的藥物固化手段,先將藥物固化后再進行微囊的制備,同樣的分析和評價手段,制備出性能更優(yōu)越的微囊制劑。該制劑,具有較高的載藥量,同時也在一定程度上增加了制劑的穩(wěn)定性。結合課題組多項研究基礎,多次嘗試發(fā)現(xiàn)藥物在體內(nèi)的基本代謝特點,選擇其中兩個具有代表性的代謝產(chǎn)物進行監(jiān)測,發(fā)現(xiàn)DATS在體內(nèi)主要以被氧化為主,含量比較高的代謝產(chǎn)物為AMSO和AMSO2。以GC-MS為檢測手段,分別以全掃描和選擇離子掃描建立這兩種化合物的體內(nèi)分析方法并對其方法學進行驗證。以SD大鼠為實驗動物模型,通過口服不同制劑形式,對藥物的體內(nèi)代謝動力學行為進行評價。這兩種代謝產(chǎn)物均表現(xiàn)出先增高后降低的趨勢。注射劑組,二者均在8h達到最高值,相對于注射劑組,固體制劑組達峰時間延遲至12h,延長了藥物在體內(nèi)的循環(huán)時間。本論文從體內(nèi)外兩個角度,從分子水平到基因水平,深入探討了DATS對肺損傷的保護機制。實驗結果證明了,藥物同時從抗炎和抗氧化兩個角度對機體產(chǎn)生保護作用,為該化合物在臨床上的應用提供藥理研究支持。另外,從多角度展開對該液體藥物的固體制劑劑型的開發(fā)。通過聯(lián)用液固系統(tǒng)和微囊制備技術,制備出同時具有較高載藥量和較高穩(wěn)定性的固體制劑形式,為擴大該化合物的工業(yè)開發(fā),提供了大量的數(shù)據(jù)支持。本論文合成DATS體外的主要代謝產(chǎn)物,并建立了該化合物全掃描和選擇性單離子掃描的GC-MS檢測方法,對制劑的體內(nèi)行為進行了評價。為該化合物的全面代謝評價提供前期的數(shù)據(jù)支持。但是,由于該化合物在體內(nèi)代謝產(chǎn)物種類較多,在攻讀學位期間未完成所有代謝產(chǎn)物的定性和定量分析,該工作仍需要課題組其他人員的繼續(xù)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.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R563
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本文編號：1386502