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研究生:蘇芳儀
研究生(外文):Su, Fang-Yi
論文名稱:可抑制酵素活性及促進藥物吸收之功能性奈米微粒做為胰島素口服釋放載體之評估
論文名稱(外文):Protease Inhibition and Absorption Enhancement by Functional Nanoparticles for Effective Oral Insulin Delivery
指導教授:宋信文
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
論文頁數:39
中文關鍵詞:口服胰島素金屬離子螯合劑分子模擬抑制蛋白酶活性adherens junction
外文關鍵詞:oral protein deliverycomplexing agentmolecular dynamic simulationproteolytic inhibitionadherens junction
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金屬離子螯合劑,例如:diethylene triamine pentaacetic acid (DTPA),可藉由螯合二價金屬離子,達到打開細胞間緊密連接蛋白(tight junction)及抑制酵素活性的功效。本研究以DTPA修飾聚麩胺酸[poly(γ-glutamic acid), γ-PGA],與幾丁聚醣(chitosan, CS)形成離子鍵結型奈米微粒,作為胰島素的口服釋放載體。由於CS具有黏膜吸附性,可以使DTPA集結在小腸黏膜,於釋放藥物的局部發揮抑制酵素活性及促進paracellular pathway通透性的功能。由體外(In vitro)實驗結果證實,所合成出的γPGA-DTPA確實可表現出促進細胞間滲透性及抑制酵素活性的效力;γPGA-DTPA可和CS形成具有pH值敏感性的奈米微粒,其粒徑大小會隨pH值增加而逐漸膨脹,且在pH 7.0以上的環境中崩解。此外,本研究以雷射掃描共軛焦顯微鏡及單光子放射斷層掃描(SPECT)觀察CS/γPGA-DTPA奈米微粒在生物體內的分布情形。由實驗結果得知,CS/γPGA-DTPA奈米微粒可促進胰島素在整個小腸區段中被吸收,且可在實驗鼠的腎臟及膀胱中清楚地觀察到被吸收的胰島素。此外,藥效學及藥物動力學實驗結果直接地證實:藉由此功能性奈米微粒所投遞的胰島素,確實可降低血糖值並延長低血糖持續的時間(與皮下注射胰島素溶液相比);在投藥後四小時,胰島素可在實驗鼠體內達到最高的濃度,且藥物生體可用率(bioavailability)大約為20%。綜合本研究的實驗結果,此CS/γPGA-DTPA功能性奈米微粒相當具有潛力做為胰島素的口服釋放載體。
Complexing agents such as diethylene triamine pentaacetic acid (DTPA) are known to disrupt intestinal tight junctions and inhibit intestinal proteases by chelating divalent metal ions. This study attempts to incorporate these benefits of DTPA in functional nanoparticles (NPs) for oral insulin delivery. To maintain the complexing agent concentrated on the intestinal mucosal surface, where the paracellular permeation enhancement and enzyme inhibition are required, DTPA was covalently conjugated on poly(γ-glutamic acid) (γPGA). The functional NPs were prepared by mixing cationic chitosan (CS) with anionic γPGA-DTPA conjugate. The γPGA-DTPA conjugate inhibited the intestinal proteases substantially, and produced a transient and reversible enhancement of paracellular permeability. The prepared NPs were pH-responsive; with an increasing pH, CS/γPGA-DTPA NPs swelled gradually and disintegrated at a pH value above 7.0. Additionally, the biodistribution of insulin orally delivered by CS/γPGA-DTPA NPs in rats was examined by confocal microscopy and scintigraphy. Experimental results indicate that CS/γPGA-DTPA NPs can promote the insulin absorption throughout the entire small intestine; the absorbed insulin was clearly identified in the kidney and bladder. In addition to producing a prolonged reduction in blood glucose levels, the oral intake of the enteric-coated capsule containing CS/γPGA-DTPA NPs showed a maximum insulin concentration at 4 h after treatment. The relative oral bioavailability of insulin was approximately 20%. Results of this study demonstrate the potential role for the proposed formulation in delivering therapeutic proteins by oral route.
致 謝..I
摘 要..II
Abstract..III
Table of Contents..IV
List of Figures..VI
List of Tables..VIII
Chapter 1. Introduction..1
1.1 Insulin delivery..1
1.2 Strategies for oral insulin delivery..1
1.3 Nanoparticles composed of Chitosan and poly(γ-glutamic acid)..2
1.4 Inhibition of peptidases by complexing agents..5
1.5 Paracellular permeation enhancement by complexing agents..5
1.6 Objectives and specific aims of this study..5
Chapter 2. Materials and Methods..8
2.1 Synthesis and characterization of γPGA-DTPA..8
2.1.1 Synthesis of γPGA-hexanediamine..9
2.1.2 Synthesis of γPGA-DTPA..9
2.1.3 Degree of substitution of DTPA in the synthesized conjugate..9
2.1.4 Divalent ion binding assay..9
2.2 Inhibition of enzymatic degradation..10
2.3 Preparation and characterization of CS/γPGA-DTPA NPs..10
2.4 Enhancement of paracellular permeability..11
2.5 Animal study..12
2.5.1 Mucoadhesion and absorption enhancement..12
2.5.2 Biodistribution study..13
2.5.3 PD and PK profiles..13
Chapter 3. Results and Discussion..15
3.1 Characteristics of the synthesized γPGA-DTPA..15
3.2 Inhibition of enzymatic degradation..16
3.3 Characteristics of CS/γPGA-DTPA NPs..18
3.4 Enhancement of paracellular permeability..19
3.5 Animal study..20
3.5.1 Mucoadhesion and absorption enhancement..20
3.5.2 Biodistribution study..22
3.5.3 PD and PK profiles..23
Chapter 4. Conclusions..26
References..27
List of Publications..30
[1] C. Binder, T. Lauritzen, O. Faber, S. Pramming, Insulin Pharmacokinetics, Diabetes Care, 7 (1984) 188-199.
[2] S. Khafagy el, M. Morishita, Y. Onuki, K. Takayama, Current challenges in non-invasive insulin delivery systems: a comparative review, Adv Drug Deliv Rev, 59 (2007) 1521-1546.
[3] D.R. Owens, B. Zinman, G. Bolli, Alternative routes of insulin delivery, Diabet Med, 20 (2003) 886-898.
[4] G.P. Carino, E. Mathiowitz, Oral insulin delivery, Adv Drug Deliver Rev, 35 (1999) 249-257.
[5] M. Mesiha, F. Plakogiannis, S. Vejosoth, Enhanced Oral Absorption of Insulin from Desolvated Fatty-Acid Sodium Glycocholate Emulsions, Int J Pharm, 111 (1994) 213-216.
[6] M.K. Marschutz, A. Bernkop-Schnurch, Oral peptide drug delivery: polymer-inhibitor conjugates protecting insulin from enzymatic degradation in vitro, Biomaterials, 21 (2000) 1499-1507.
[7] D.R. Owens, B. Zinman, G. Bolli, Alternative routes of insulin delivery, Diabetic Med, 20 (2003) 886-898.
[8] Y.H. Lin, C.T. Chen, H.F. Liang, A.R. Kulkarni, P.W. Lee, C.H. Chen, H.W. Sung, Novel nanoparticles for oral insulin delivery via the paracellular pathway, Nanotechnology, 18 (2007).
[9] K. Sonaje, Y.H. Lin, J.H. Juang, S.P. Wey, C.T. Chen, H.W. Sung, In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery, Biomaterials, 30 (2009) 2329-2339.
[10] K. Sonaje, Y.J. Chen, H.L. Chen, S.P. Wey, J.H. Juang, H.N. Nguyen, C.W. Hsu, K.J. Lin, H.W. Sung, Enteric-coated capsules filled with freeze-dried chitosan/poly(gamma-glutamic acid) nanoparticles for oral insulin delivery, Biomaterials, 31 (2010) 3384-3394.
[11] L. Illum, Chitosan and its use as a pharmaceutical excipient, Pharmaceut Res, 15 (1998) 1326-1331.
[12] A. Bernkop-Schnurch, The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins, J Control Release, 52 (1998) 1-16.
[13] A.B.J. Noach, Y. Kurosaki, M.C.M. Blomroosemalen, A.G. Deboer, D.D. Breimer, Cell-Polarity Dependent Effect of Chelation on the Paracellular Permeability of Confluent Caco-2 Cell Monolayers, Int J Pharm, 90 (1993) 229-237.
[14] X.D. Zhao, L.Z. Song, Z.H. Zhang, R. Wang, J. Fu, Adsorption investigation of MA-DTPA chelating resin for Ni(II) and Cu(II) using experimental and DFT methods, J Mol Struct, 986 (2011) 68-74.
[15] M. Tomita, M. Hayashi, S. Awazu, Absorption-enhancing mechanism of EDTA, caprate, and decanoylcarnitine in Caco-2 cells, J Pharm Sci, 85 (1996) 608-611.
[16] S. Citi, Protein-Kinase Inhibitors Prevent Junction Dissociation Induced by Low Extracellular Calcium in Mdck Epithelial-Cells, J Cell Biol, 117 (1992) 169-178.
[17] X. Guo, J.N. Rao, L. Liu, T.T. Zou, D.J. Turner, B.L. Bass, J.Y. Wang, Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines, Am J Physiol-Cell Ph, 285 (2003) C1174-C1187.
[18] M. Thanou, J.C. Verhoef, H.E. Junginger, Oral drug absorption enhancement by chitosan and its derivatives, Adv Drug Deliver Rev, 52 (2001) 117-126.
[19] M. Sakai, T. Imai, H. Ohtake, H. Azuma, M. Otagiri, Effects of absorption enhancers on cytoskeletal actin filaments in Caco-2 cell monolayers, Life Sci, 63 (1998) 45-54.
[20] X.X. Wen, E.F. Jackson, R.E. Price, E.E. Kim, Q.P. Wu, S. Wallace, C. Charnsangavej, J.G. Gelovani, C. Li, Synthesis and characterization of poly(L-glutamic acid) gadolinium chelate: A new biodegradable MRI contrast agent, Bioconjugate Chem, 15 (2004) 1408-1415.
[21] A. BernkopSchnurch, C. Paikl, C. Valenta, Novel bioadhesive chitosan-EDTA conjugate protects leucine enkephalin from degradation by aminopeptidase N, Pharmaceut Res, 14 (1997) 917-922.
[22] I. Legen, A. Kristl, Factors affecting the microclimate pH of the rat jejunum in ringer bicarbonate buffer, Biol Pharm Bull, 26 (2003) 886-889.
[23] M.C. Chen, K. Sonaje, K.J. Chen, H.W. Sung, A review of the prospects for polymeric nanoparticle platforms in oral insulin delivery, Biomaterials, 32 (2011) 9826-9838.
[24] M. Sameti, G. Bohr, M.N.V.R. Kumar, C. Kneuer, U. Bakowsky, M. Nacken, H. Schmidt, C.M. Lehr, Stabilisation by freeze-drying of cationically modified silica nanoparticles for gene delivery, Int J Pharm, 266 (2003) 51-60.
[25] M.T. Nelson, W. Humphrey, A. Gursoy, A. Dalke, L.V. Kale, R.D. Skeel, K. Schulten, NAMD: A parallel, object oriented molecular dynamics program, Int J Supercomput Ap, 10 (1996) 251-268.
[26] D.F. Evans, G. Pye, R. Bramley, A.G. Clark, T.J. Dyson, J.D. Hardcastle, Measurement of Gastrointestinal Ph Profiles in Normal Ambulant Human-Subjects, Gut, 29 (1988) 1035-1041.
[27] S. Yamashita, T. Takashima, M. Kataoka, H. Oh, S. Sakuma, M. Takahashi, N. Suzuki, E. Hayashinaka, Y. Wada, Y.L. Cui, Y. Watanabe, PET Imaging of the Gastrointestinal Absorption of Orally Administered Drugs in Conscious and Anesthetized Rats, J Nucl Med, 52 (2011) 249-256.
[28] M.C. Chen, H.S. Wong, K.J. Lin, H.L. Chen, S.P. Wey, K. Sonaje, Y.H. Lin, C.Y. Chu, H.W. Sung, The characteristics, biodistribution and bioavailability of a chitosan-based nanoparticulate system for the oral delivery of heparin, Biomaterials, 30 (2009) 6629-6637.
[29] H.N. Nguyen, S.P. Wey, J.H. Juang, K. Sonaje, Y.C. Ho, E.Y. Chuang, C.W. Hsu, T.C. Yen, K.J. Lin, H.W. Sung, The glucose-lowering potential of exendin-4 orally delivered via a pH-sensitive nanoparticle vehicle and effects on subsequent insulin secretion in vivo, Biomaterials, 32 (2011) 2673-2682.
[30] L.L. Chang, J.P.F. Bai, Evidence for the existence of insulin-degrading enzyme on the brush-border membranes of rat enterocytes, Pharmaceut Res, 13 (1996) 801-803.
[31] C. Damge, P. Maincent, N. Ubrich, Oral delivery of insulin associated to polymeric nanoparticles in diabetic rats, J Control Release, 117 (2007) 163-170.
[32] B.A. Teply, R. Tong, S.Y. Jeong, G. Luther, I. Sherifi, C.H. Yim, A. Khademhosseini, O.C. Farokhzad, R.S. Langer, J. Cheng, The use of charge-coupled polymeric microparticles and micromagnets for modulating the bioavailability of orally delivered macromolecules, Biomaterials, 29 (2008) 1216-1223.
[33] B.S. Inbaraj, J.S. Wang, J.F. Lu, F.Y. Siao, B.H. Chen, Adsorption of toxic mercury(II) by an extracellular biopolymer poly(gamma-glutamic acid), Bioresour Technol, 100 (2009) 200-207.
[34] V. Darras, M. Nelea, F.M. Winnik, M.D. Buschmann, Chitosan modified with gadolinium diethylenetriaminepentaacetic acid for magnetic resonance imaging of DNA/chitosan nanoparticles, Carbohyd Polym, 80 (2010) 1137-1146.
[35] A. Bernkop-Schnurch, J. Freudl, Comparative in vitro study of different chitosan-complexing agent conjugates, Pharmazie, 54 (1999) 369-371.
[36] M.K. Marschutz, P. Caliceti, A. Bernkop-Schnurch, Design and in vivo evaluation of an oral delivery system for insulin, Pharmaceut Res, 17 (2000) 1468-1474.
[37] A. Bernkop-Schnurch, Chitosan and its derivatives: potential excipients for peroral peptide delivery systems, Int J Pharm, 194 (2000) 1-13.
[38] Y. Aoki, M. Morishita, K. Asai, B. Akikusa, S. Hosoda, K. Takayama, Region-dependent role of the mucous/glycocalyx layers in insulin permeation across rat small intestinal membrane, Pharmaceut Res, 22 (2005) 1854-1862.
[39] K. Sonaje, K.J. Lin, S.P. Wey, C.K. Lin, T.H. Yeh, H.N. Nguyen, C.W. Hsua, T.C. Yen, J.H. Juang, H.W. Sung, Biodistribution, pharmacodynamics and pharmacokinetics of insulin analogues in a rat model: Oral delivery using pH-Responsive nanoparticles vs. subcutaneous injection, Biomaterials, 31 (2010) 6849-6858.
[40] C.M. Lehr, Bioadhesion Technologies for the Delivery of Peptide and Protein Drugs to the Gastrointestinal-Tract, Crit Rev Ther Drug, 11 (1994) 119-160.
[41] I.A. Sogias, A.C. Williams, V.V. Khutoryanskiy, Why is chitosan mucoadhesive?, Biomacromolecules, 9 (2008) 1837-1842.
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