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研究生:趙志浩
研究生(外文):Chao, Chih-hao
論文名稱:以動物模式探討奈米化Clarithromycin之特性及其對急性肺損傷療效研究
論文名稱(外文):Characterize Nano-Clarithromycin and evaluate the therapeutic effect on acute lung injury
指導教授:楊重熙吳杰亮吳杰亮引用關係
指導教授(外文):Yang, Chung-ShiWu, Chieh-Liang
口試委員:吳立真
口試委員(外文):Wu, Li-chen
口試日期:2011-07-25
學位類別:碩士
校院名稱:國立暨南國際大學
系所名稱:生物醫學科技研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:55
中文關鍵詞:急性肺損傷奈米巨環抗生素
外文關鍵詞:Acute lung injurynanoparticleClarithromycinPLGA
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簡介:
急性呼吸窘迫症(Acute respiratory distress syndrome, ARDS)是加護病房中常見的急性呼吸衰竭症候群,病人的肺部呈現兩側廣泛性浸潤及嚴重低血氧(動脈血氧濃度/吸氧濃度<200mmHg,PaO2/FiO2 < 200mmHg)之現象。病人如果有上述相同症狀,而PaO2/FiO2<300mmHg者,稱之為急性肺損傷(Acute lung injury, ALI)。ALI/ARDS是一種強烈的肺部發炎反應,除了各種發炎介質的釋放外,還有大量嗜中性白血球進入肺組織,破壞肺泡與微血管的結構。ALI/ARDS治療過程消耗相當多的醫療資源,但死亡率仍高達30~40%。目前除了支持性的治療外,仍然沒有直接有效的治療藥物。
Clarithromycin是一種巨環抗生素,透過與50 S ribosome作用抑制細菌蛋白合成。這藥物也可以透過NF-B之抑制,降低LPS引起的發炎反應。Clarithromycin是否可以減低急性肺損傷時肺部傷害的程度仍有待研究。
奈米化的藥物可以透過靜脈注射、口服、肺部、或皮膚給予。可以增加藥物的生物可用率、藥物溶解度、血中停留時間和活性,因此理論上可以減少使用的藥物劑量。我們已初步研製成將Clarithromycin作成奈米的藥物型態。我們將利用這奈米化的Clarithromycin進一步探討其對肺部發炎反應是否具有療效。
方法:
首先我們將探討奈米化Clarithromycin的藥物包覆率、奈米微粒的大小、形狀及表面電荷。注入大鼠體內後在血液中的濃度。然後我們從大鼠氣管內噴入脂多糖 (lipopolysaccharide)誘發急性肺損傷,並同時給予腹膜或靜脈藥物注射,在16小時後我們將老鼠犠牲,並進行肺泡灌洗,我們測量灌洗液中的全細胞數目,蛋白質濃度及細胞激素濃度,此外也會測量肺組織內的髓過氧化物酶 (myeloperoxidase) 以及計算肺部組織切片中的肺損傷分數 (Lung injury score)。
結果:
製作出來的奈米化clarithromycin的粒子大小為25.2 (以PEG2000-PE合成) 和32.5奈米 (以PEG2000-PE-PLGA合成)。腹膜內clarithromycin的注射可以有效地緩解肺部的發炎,但奈米化的clarithromycin並無法抑制肺部發炎,靜脈注射clarithromycin也同樣沒有療效。單獨給予沒有包含clarithromycin藥物的奈米分子 (PEG2000-PE配方)會導致沒有接受脂多糖處理的老鼠產生肺部損傷,而在已接受氣管內脂多糖處理的老鼠,給予奈米分子並不會再加重肺部的發炎。
結論:
clarithromycin腹腔內注射可以有效抑制肺部發炎反應,但奈米化clarithromycin沒有療效,合成奈米分子所使用的成份可能會引起肺部傷害。

Introduction: Acute lung injury (ALI) and its most severe manifestation, the acute respiratory distress syndrome (ARDS), still had high mortality rate. Till now, there is no specific pharmacologic therapy for ALI/ARDS. Clarithromycin inhibits LPS-induced inflammatory response through NF-κB pathway. It also can suppress inflammation caused by sepsis and pneumonia. Nanoparticles had been shown to enhance drug targeting, prolong serum half life, improve drug bioactivity and stability, and reduce effective dosage. We synthesized two formulations of nano-clarithromycin (PEG2000-PE and PEG2000-PE-PLGA) and tested their effect on LPS-induced ALI.
Method: Rats received intra-tracheal instillation of LPS, followed by intraperitoneal or intravenous drugs administration immediately. Rats were sacrificed 16 hours later and bronchoalveolar larvage and tissue dissection were done. Total cell counts, total protein concentrations and cytokine levels in BALF, and MPO levels in lung parenchyma were measured. The histological severity of lung injury was scored.
Results: The diameter of nano-clarithromycin containing PEG2000-PE and PEG2000-PE-PLGA was 25.2 nm and 32.5 nm respectively by particle size analyzer. Nano-clarithromycin synthesized by PEG2000-PE and PLGA showed favorable pharmacokinetic parameters. Intra-peritoneal free clarithromycin effectively reduced LPS – induced ALI. But both formulations of nano-clarithromycin failed to ameliorate acute lung injury. Intravenous free clarithromycin had no therapeutic effect, either. Empty PEG2000-PE nanoparticles without clarithromycin entrapment resulted in lung injury in rats without intra-tracheal LPS instillation, and this effect obscured in rats with LPS-induced lung injury.
Conclusion: Intra-peritoneal administration of clarithromycin protected LPS-induced ALI but nano-formulated clarithromycin synthesized by PE-PEG2000 or PE-PEG2000-PLGA did not. Intravenous empty naroparticles might lead to lung injury in healthy rats.

致謝…………………………………………………………………………………….I
Chinese abstract……………………………………………………………………….II
English abstract………………………………………………………………………IV
Table of contents………………………………………………………………..……VI
List of tables……………………………………………………………………...….IX
List of figures………………………………………………………………...…….....X
Chapter 1 Introduction…………………………………………………………………1
1-1 Acute lung injury/acute respiratory distress syndrome……………………....1
1-2 The etiology of ALI/ARDS…………………………………………...……...1
1-3 The pathogenesis of ALI/ARDS…………………………………………..…2
1-3-1 Neutrophils……………………………………………………….…2
1-3-2 Cytokines……………………………………………………………2
1-3-3 Other cells and mediators…………………………………………...3
1-4 Clarithromycin…………………………………………………………….....3
1-4-1 The mechanism of clarithromycin action…………………………...3
1-4-2 Anti-inflammatory effect of clarithromycin in chronic lung disease.4
1-4-3 Anti-inflammatory effect of clarithromycin in acute infection/inflammation…………………………………………...…4
1-5 Nanoparticles………………………………………………………………...5
1-5-1 Characteristic of nanoparticles……………………………………...5
1-5-2 Poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles………....5
1-5-3 Polyethylene glycol /phosphatidylethanolamine (PEG2000-PE) nanoparticles………………………………………………………...6
1-5-4 Nano-formulated clarithromycin (nano-clarithromycin)……………6
1-6 Motivations…………………………………………………………………..6
Chapter 2 Materials and Methods
2-1 Reagents……………………………………………………………………...8
2-2 Equipments………………………………………………………………..….8
2-3 Methods……………………………………………………………………....9
2-3-1 Study designs…………………………………………………………9
2-3-2 Animals and induction of acute lung injury........................................10
2-3-3 Clarithromycin preparation.................................................................11
2-3-4 Formation of Nano-clarithromycin.....................................................11
2-3-5 Determination of encapsulation efficiency and loading content of Clarithromycin....................................................................................12
2-3-6 HPLC analysis....................................................................................12
2-3-7 Transmission electron microscopy......................................................13
2-3-8 Particle size analyses..........................................................................13
2-3-9 In vitro drug release analysis..............................................................13
2-3-10 Severity of lung injury......................................................................13
2-3-11 Myeloperoxidase (MPO) assay.........................................................14
2-3-12 Collection of BALF..........................................................................14
2-3-13 BALF analyses….............................................................................15
Chapter 3 Results..........................................................................................................16
3-1 Size and morphologic characterization of the nano-clarithromycin..............16
3-2 In vitro drug release analysis..........................................................................17
3-3 Pharmacokinetic study...................................................................................18
3-4 Intra-peritoneal free-clarithromycin inhibited acute lung injury induced by intra-tracheal LPS instillation.........................................................................21
3-5 Intravenous 1 % phosphoric acid/saline had no significant injurious effect in the lungs in rats with or without LPS-induced lung injury............................25
3-6 Intravenous free clarithromycin had no therapeutic effect on LPS-induced acute lung injury.............................................................................................30
3-7 Intravenous nano-clarithromycin had no therapeutic effect in a dosage equivalent to 20 mg/kg clarithromycin..........................................................34
3-8 Intravenous PEG2000-PE nanoparticles would induce lung injury in healthy rats..................................................................................................................39
3-9 The injurious effect of PEG2000-PE nanoparticles was not significant after LPS-induced lung injury.................................................................................43
Chapter 4 Discussion....................................................................................................47
Chapter 5 Future works................................................................................................51
Chapter 6 References....................................................................................................52

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