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A study on low molecular weight heparin conjugates for cancer chemoprevention and chemotherapy based on angiogenesis inhibition : 혈관 신생 억제 작용을 기반으로 한 화학적 암 예방 및 치료를 위한 헤파린 유도체의 개발에 관한 연구

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dc.contributor.advisor변영로-
dc.contributor.author김지영-
dc.date.accessioned2017-07-13T16:34:36Z-
dc.date.available2017-10-23T07:46:41Z-
dc.date.issued2014-08-
dc.identifier.other000000021766-
dc.identifier.urihttps://hdl.handle.net/10371/120082-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 약학과, 2014. 8. 변영로.-
dc.description.abstractThe important role of angiogenesis over the whole process of cancer development has been extensively studied. Conventionally, angiogenesis is defined as a new vessel formation from pre-existing vascular structures and triggered at tumor size of 1-2 mm3 for further growth. A numerous cytokines and growth factors are involved in this process, and thus they are good targets for cancer treatment. Consequently, a number of angiogenesis inhibitors with different mechanisms have been approved and widely used in the clinics. On the other hand, recently, increasing number of preclinical and clinical evidences also shows that angiogenic switch is already turned on in the premalignant stage including hyperplasia and dysplasia, which provides a rationale to target angiogenesis for cancer chemoprevention. Cancer chemoprevention, which is defined as a pharmacological intervention to impede, arrest, or reverse carcinogenesis at its earliest stage, is well-accepted as a promising strategy for cancer controlling strategy. By targeting angiogenesis, not only the transformation from premalignant lesions to malignant tissues, but the further progression of malignant tumors can also be properly prevented and inhibited. In addition, the development of inventive drug delivery systems targeting vascular structures might be required for the further improve the therapeutic efficacy of antiangiogenic drugs while reducing toxicity.

Low molecular weight heparin is a polydisperse and highly sulphated glycosaminoglycan with molecular weight about 5000 Daltons. It has been widely used as an anticoagulant drug in the clinics. However, due to electrostatic interactions with various growth factors and cytokines, its application into anticancer therapy also has been extensively studied. In this context, a series of LMWH-bile acid conjugates was synthesized as antiangiogenic drugs in the previous studies. Through the chemical modification of LMWH, while side effect such as hemorrhage was avoided, the therapeutic efficacy was enhanced. They demonstrated both significant anticancer effects by angiogenesis inhibition and pharmacokinetic properties via different administration routes.

In the first part of this research, we introduce a newly developed oral heparin derivative (LHTD4) for use as an inhibitor of angiogenesis and evaluate its antiangiogenic and preventive effects in an animal model of lung cancer. The antiangiogenic activities of LHTD4 were evaluated using tube formation and Matrigel assays. VEGF- and bFGF-induced tube formations were reduced by up to 77.2 and 67.3%, respectively, by LHTD4. Hemoglobin content was also significantly decreased by LHTD4 in the Matrigel plugs that were transplanted into mice. We also observed that the VEGF- and bFGF-mediated phosphorylation of the receptors VEGFR-2 and FGFR-1 was also inhibited by LHTD4. The in vivo anticancer effects of LHTD4 that developed following oral administration were also verified in a tumor xenograft model of human A549 lung cancer cells
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dc.description.abstractmost especially, tumor volume (60.2%). The expression of CD34 and Ki-67 in LHTD4-treated group was also affected. Finally, in our chemically induced lung carcinogenesis model, the number and area of each nodule were significantly reduced in the LHTD4-treated groups by 49.2% and 30.1%, respectively. In addition, the degree of angiogenesis in the lung tissue itself was affected in the treatment group. Taken together, these results suggest that LHTD4, which is an orally active heparin derivative, could be a promising candidate for the prevention of cancer by inhibiting angiogenesis.

Secondly, the combination effect of celecoxib and newly developed oral angiogenesis inhibitor, LHD4, on chemoprevention was evaluated to achieve a clinically rational regimen for cancer chemoprevention with improved efficacy and safety. The chemopreventive effects of celecoxib, LDH4, and the combination of celecoxib and LHD4 were evaluated in a murine colorectal carcinogenesis model. After 17 experimental weeks, mouse colon tissues were collected and examined in terms of polyp volume and degree of carcinogenesis, inflammation and angiogenesis. Mice in the celecoxib- or LHD4-treated groups bore total polyp volumes of 47.0 ± 9.7 and 120.1 ± 45.2 mm3, respectively, which represented decreases of 65.6% and 22.3% from the control (154.5 ± 33.5 mm3). However, the polyp volume in the combination group was 22.8 ± 9.3 mm3, a decrease of 85.2% from the control. In the comparison of carcinogenesis, the percentage of normal tissue (i.e., excluding proliferative tissue) was found to be 40.6% in the control, 51.7% in the celecoxib, 56.9% in the LHD4, and 81.7% in the combination group. In accordance with attenuated carcinogenesis, both inflammation and angiogenesis were also well controlled. Together, these results suggest that combinatory use of celecoxib and a newly developed oral heparin conjugate could be a promising regimen for chemoprevention by intervening both inflammation and angiogenesis.

Third, we have studied the COX-2 inhibition effect on antiangiogneic therapy. In present study, the induction of hypoxia and COX-2 overexpression were observed at tumor tissues that were treated with a multi-targeting angiogenesis inhibitor named as LHT7. In addition, while the recruitment of macrophage was also increased under angiogenesis inhibition, it was well-controlled by combination use of celecoxib and LHT7. On the other hand, the combination effect on tumor vasculature was also studied. The in vitro tube formation was inhibited by either LHT7 or celecoxib, but the inhibition effect was further enhanced by using them together. In addition, the in vivo tumor vessel formation and structure were also altered by treatment with LHT7, celecoxib, and combination use. However, even though the combination therapy was effective enough to inhibit tumor angiogenesis, it did not further enhance the inhibitory effect on tumor growth in terms of volume than single drug use. Moreover, even though this regimen could not significantly increase cellular apoptosis at tumor tissues, it retarded the tumor growth by affecting cell proliferation. Taken all, COX-2 inhibition might enhance the therapeutic effect of antiangiogenic drugs both by inhibiting the inflammatory reactions induced by hypoxia and by altering the vascular stabilization that is mediated by assembly with mural cells.

Finally, we have studied on the tumor vascular structure-targeted delivery of antiangiogenic drug. We found that the systemic administration of LHT7 in cationic nanolipoplex could substantially enhance the anticancer effects. Moreover, we found that co-delivery of LHT7 with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, in nanolipoplex could provide synergistic antitumor effect. LHT7/SAHA nanolipoplex was formulated by encapsulating SAHA inside cationic liposomes, followed by complexation of negatively charged LHT7 onto the cationic surfaces of SAHA-loaded liposomes (SAHA-L). The nanolipoplex form of LHT7 could alter its pharmacokinetics with 1.9-fold increased mean residence time compared to the free form of LHT7. LHT7/SAHA nanolipoplex showed highest antitumor efficacy in SCC-bearing mice, compared to LHT7, SAHA-L and sequential co-administration of LHT7 and SAHA-L. Consistent with the enhanced antitumor effect, the reduction of abnormal vessels in the tumor site was also the highest in the LHT7/SAHA nanolipoplex-treated group. These results suggested the potential of LHT7/SAHA nanolipoplex for enhanced tumor vasculature targeting, and the importance of nanolipoplex-mediated co-delivery with a histone deacetylase inhibitor for maximal anticancer effect.

In conclusion, a series of LMWH-bile acids conjugates could be promising cancer chemopreventive and therapeutic agents via angiogenesis inhibition in the future. In addition, the antiangiogenic potency might be further improved by utilizing a functionalized drug delivery system such as tumor vascular targeting carrier.
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dc.description.tableofcontentsAbstract i
Table of Contents vii
List of Tables xiv
List of Figures xv
Abbreviations xxiv

Chapter I. Introduction 1
1.1. Angiogenesis 2
1.1.1. Angiogenesis in physiological and pathological conditions 2
1.1.2. Angiogenesis and cancer 3
1.1.3. Anti-angiogenic drugs for cancer treatment 4
1.1.4. Limitations of antiangiogenic therapy 6
1.2. Cancer chemoprevention and angiogenesis 7
1.2.1. Cancer chemoprevention 7
1.2.2. Classifications and requirements of cancer chemoprevention 8
1.2.3. Combination chemoprevention 9
1.2.4. Clinical cancer chemoprevention by delay 10
1.2.5. Angiogenesis as a target for cancer chemoprevention 10
1.3. Low molecular weight heparin-bile acid conjugates as an angiogenesis inhibitor 11
1.3.1. Unfractionated heparin and low molecular weight heparin 11
1.3.2. LMWH and cancer 13
1.3.3. Heparin-bile acid conjugates as an angiogenesis inhibitor 14
1.3.4. Orally active heparin-bile acid conjugates 15
1.4. Drug delivery system for anti-angiogenic drugs 17
1.4.1. Nanoparticles as a drug delivery system 17
1.4.2. Pathological characteristics of tumor vascular structure and EPR effect 18
1.4.3. Vascular targeted delivery of anticancer drugs using nanoparticles 20
1.5. Research rationale 22

Chapter II. Chemoprevention effect of an orally active low-molecular-weight heparin conjugates on urethane-induced lung carcinogenesis via the inhibition of angiogenesis 35
2.1. Introduction 36
2.2. Materials and Methods 39
2.2.1. Materials 39
2.2.2. Synthesis and characterization of LHT7, LHTD4, and DCK 39
2.2.3. Tube formation assay 41
2.2.4. Matrigel plug assay 42
2.2.5. Western blot 43
2.2.6. Tumor growth inhibition test 44
2.2.7. Urethane-induced lung carcinogenesis model 45
2.2.8. Histological and serological evaluations 46
2.2.9. Statistical analysis 47
2.3. Results 47
2.3.1. Characterization of LHTD4 as an angiogenesis inhibitor 47
2.3.2. Antiangiogenic effects of LHTD4 on tumor growth were observed in the A549 human lung cancer xenograft model 49
2.3.3. LHTD4 demonstrates chemopreventive effects by decreasing nodule formation and the degree of angiogenesis in lung tissue 50
2.4. Discussion 51
2.5. Conclusions 56

Chapter III. Combinational chemoprevention effect of celecoxib and an oral anti-angiogenic LHD4 on colorectal carcinogenesis in mice 66
3.1. Introduction 67
3.2. Materials and Methods 70
3.2.1. Materials 70
3.2.2. Synthesis of LHD4 70
3.2.3. Animal treatment 71
3.2.4. Histopathological evaluation 72
3.2.5. Immunohistochemical analysis 74
3.2.6. Statistical analysis 75
3.3. Results 75
3.3.1. Clinical symptoms were not observed in mice that were co-treated with celecoxib and LHD4 75
3.3.2. The malignant progression of polyps was significantly attenuated by the combined use of celecoxib and LHD4 77
3.3.3. The degree of inflammation and angiogenesis was correlated with carcinogenesis and was regulated by co-treatment with celecoxib and LHD4 79
3.4. Discussion 80
3.5. Conclusions 86

ChapterIV. COX-2 inhibition effect on tumor growth under anti-angiogenic therapy using heparin conjugates 94
4.1. Introduction 95
4.2. Materials and Methods 98
4.2.1. Materials 98
4.2.2. Synthesis of LHT7 98
4.2.3. Tube formation assay 98
4.2.4. Tumor growth inhibition assay 99
4.2.5. Hypoxic probe staining 100
4.2.6. Vessel staining with lectin perfusion 101
4.2.7. Immunohistological evaluation 102
4.2.8. Statistical analysis 103
4.3. Results 103
4.3.1. Induction of hypoxia and COX-2 expression under anti-angiogenic therapy 103
4.3.2. Combination effect on macrophage recruitment on tumor tissue 104
4.3.3. The combination effect of celecoxib and LHT7 on vessel formation in vitro and vivo 105
4.3.4. The effect of combination use of celecoxib and LHT7 on tumor growth 106
4.4. Discussion 107
4.5. Conclusions 110

Chapter V. Tumor vasculature targeting following co-delivery of heparin-taurocholate conjugate and suberoylanilide hydroxamic acid using cationic nanolipoplex 118
5.1. Introduction 119
5.2. Materials and Methods 121
5.2.1. Materials 121
5.2.2. Synthesis of LHT7, FITC-LHT7, and Cy5.5-LHT7 122
5.2.3. Preparation of LHT7/SAHA nanolipoplex 123
5.2.4. Pharmacokinetic study 124
5.2.5. Inhibition effect of LHT7/SAHA nanolipoplex on tumor growth 125
5.2.6. Statistical analysis 127
5.3. Results 127
5.3.1. Pharmacokinetic parameters of LHT7 127
5.3.2. Inhibition effect of LHT7/SAHA nanolipoplex on tumor growth in SCC7-bearing mice 128
5.4. Discussion 129
5.5. Conclusions 133

Chapter VI. Conclusions 141


References 145
국문초록 159
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dc.formatapplication/pdf-
dc.format.extent4006884 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectangiogenesis-
dc.subjectheparin conjugate-
dc.subjectcancer chemoprevention-
dc.subjectcancer chemotherapy-
dc.subjectcyclooxygenase-2-
dc.subjectcombination therapy-
dc.subjectnanocomplex-
dc.subject.ddc615-
dc.titleA study on low molecular weight heparin conjugates for cancer chemoprevention and chemotherapy based on angiogenesis inhibition-
dc.title.alternative혈관 신생 억제 작용을 기반으로 한 화학적 암 예방 및 치료를 위한 헤파린 유도체의 개발에 관한 연구-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pages196-
dc.contributor.affiliation약학대학 약학과-
dc.date.awarded2014-08-
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