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title page
Contents
List of Abbreviations 7
I. Introduction 11
1. Glucagon-like peptide-1 11
2. Nasal Delivery 17
3. PEGylation 19
4. MALDI-TOF mass spectrometry 25
5. Objective 28
II. Materials 29
1. Chemicals 29
2. Instruments 31
III. Methods 32
1. Preparation and purification of succinimidyl propionated-monomethoxy polyethylene glycol or Succinimidyl α-methyl butanoate-monomethoxy polyethyleneglycol- conjugated GLP-1 32
2. Separation and purification of Lys34-modified mono-PEG-GLP-1(이미지참조) 32
3. Characterization of GLP-1 and Lys34 mono-PEG-GLP-1 species(이미지참조) 34
3.1. MALDI-TOF mass spectrometry of GLP-1 and Lys34-mono PEG-GLP-1(이미지참조) 34
3.2. Endoproteinase Lys-c digestion 34
4. The stability of GLP-1 and Lys34-mono-PEGylated GLP-1(이미지참조) 35
4.1. (제목없음) 35
4.1.1. The stability in Dipeptidyl peptidase IV 35
4.1.2. HPLC analysis condition of GLP-1 and Lys34-mono PEG-GLP-1 in Dipeptidyl peptidase IV(이미지참조) 35
4.2. The stability of GLP-1 and Lys34-mono PEG-GLP-1 in rabbit nasal homogenates.(이미지참조) 36
4.2.1. Preparation of rabbit nasal homogenates 36
4.2.2. The stability in rabbit nasal homogenates 36
4.2.3. HPLC analysis condition of GLP-1 and Lys34-mono PEG-GLP-1 in rabbit nasal homogenates.(이미지참조) 37
5. Screening of insulinotropic action in vitro 37
5.1. (제목없음) 37
5.1.1. MIN6 cell culture 37
5.1.2. MIN6 cell insulin release test 38
5.2. Insulinotropic actions from isolated rat pancreatic islets. 38
6. Biological activity of of GLP-1 and Lys34-mono PEG-GLP-1 species in vivo(이미지참조) 39
6.1. db/db mice 39
6.2. Oral glucose tolerance test (OGTT) 39
IV. Results and Discussion 41
1. Preparation, separation, purification and characterization of Lys34-mono polyethylene glycol conjugated-GLP-1(이미지참조) 41
2. The stability of Native GLP-1 and Lys34-mono-PEG-GLP-1(이미지참조) 48
2.1. The stability of Native GLP-1 and Lys34-mono-PEG-GLP-1 in dipeptidyl peptidase IV(이미지참조) 48
2.2. The stability The stability of Native GLP-1 and Lys34-mono-PEG-GLP-1 in rabbit nasal homogenate(이미지참조) 48
3. Screening of insulinotropic action in vitro 55
3.1. The insulinotropic effect of Lys34-mono-PEG-GLP-1 species in MIN-6 cell(이미지참조) 55
3.2. The insulinotropic effect of Lys34-mono-PEG-GLP-1 species in isolated rat pancreatic islet(이미지참조) 55
4. Biological activity of mono-PEG-modified GLP-1 species in vivo 61
V. Conclusion 66
Reference 68
Abstract 72
국문 요약 74
Table 1. The fragment of Native GLP-1 and PEGylated GLP-1s were digested by endoproteinase Lys-C 47
Table 2. The degradation half-life of native GLP-1 and Lys34-mono-PEG-GLP-1 species in Dipeptidyl peptidase IV(이미지참조) 50
Table 3. The degradation half-life of native GLP-1 and Lys34-mono-PEGylated GLP-1 species in Rabbit nasal homogenates.(이미지참조) 53
Figure 1. Primary structure of glucagons-like peptide-1 (GLP-1) 14
Figure 2. Pancreatic and extra-pancreatic glucagon-like peptide-1 receptor agonist-dependent actions. CNS, central nervous system. (Baggio et al., 2004) 15
Figure 3. The ionic mechanism (KATP channel-dependent) of stimulus-secretion coupling in pancreatic β-cell (Patrick E. MacDonald 2002)(이미지참조) 16
Figure 4. Chemical structures of representative activated polyethylene glycol derivatives. 22
Figure 5. Schematic of a MALDI-TOF Mass Spectrometer 26
Figure 6. Heterogeneous nature of PEG molecule measured by MALDI-TOF mass spectrometry. The numbers on the peaks are the mass-to-charge ratio values of the corresponding peaks.... 27
Figure 7. Schematic diagram of PEGylation of GLP-1 with mPEG 33
Figure 8. Separation and purification chromatograms of Lys34-specific mono PEGylated GLP-1 species using RP-HPLC(이미지참조) 43
Figure 9. RP-HPLC chromatograms of Lys34-mono-PEG-GLP-1 species.(이미지참조) 44
Figure 10. MALDI-TOF Mass spectra of native GLP-1 (7-36) 45
Figure 11. MALDI-TOF mass spectra of Lys34-mono PEG-GLP-1 (with various PEG size)(m.w 1K, 2K, 5K, 10K) and digested PEG-GLP-1s by Lys-C(이미지참조) 46
Figure 12. RP-HPLC chromatograms of native GLP-1 and Lys34-modified PEG1K-GLP-1 in dipeptidyl peptidase IV(이미지참조) 51
Figure 13. The degradation kinetics of GLP-1 and Lys34-PEGylated GLP-1 species in dipeptidyl peptidase IV (n=3)(이미지참조) 52
Figure 14. The degradation kinetics of GLP-1 and Lys34-PEGylated GLP-1 species in Rabbit nasal homogenates (n=3).(이미지참조) 54
Figure 15. Insulinotropic actions of Lys34-mono-PEG-GLP-1 species in MIN6 cell.(n=4)(이미지참조) 58
Figure 16. Comparison of insulinotropic action tendency between MIN6 cell line test and Isolated rat islet test 59
Figure 17. Insulinotropic actions of Lys34-mono-PEG-GLP-1 species in isolated rat pancreatic islet (n=7)(이미지참조) 60
Figure 18. Glucose-Lowering effect of Lys34-PEG-GLP-1 species at 30 min after intranasal administration (n=6~11)(이미지참조) 62
Figure 19. Hypoglycemic Degree (%) of Lys34-PEG-GLP-1 species relative to control group ; The area under the curve (AUC) was calculated in concentration per time for 180 min.(이미지참조) 63
Figure 20. Glucose-Lowering effect of Lys34-PEG-GLP-1 species at 1hr min after intranasal administration (n=5~7)(이미지참조) 64
Figure 21. Hypoglycemic Degree (%) of Lys34-PEG-GLP-1 species relative to control group ; The area under the curve (AUC) was calculated in concentration per time for 180 min.(이미지참조) 65
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