臺灣博碩士論文加值系統

See English Abstract

Colorectal cancer (CRC) is a highly prevalent and the third common cause of cancer modalities in Taiwan in 2007. There is still no available cure for this malignant disease. To address this issue, we applied the multimodalities of molecular imaging to explore the diagnostic and therapeutic effectiveness of passively nanotargeted 111In-labelled PEGylated liposomal vinorelbine (VNB) in an animal model of human colorectal adenocarcinoma (HT-29) that stably expresses luciferase (luc) as a reporter. Prior to the preclinical therapeutic trial, the pharmacokinetics and biodistribution were estimated to determine the drug profile and targeting efficiency between 0.9 mol% and 6 mol% PEGylated 111In-liposomes. Gamma scintigraphy, whole-body autoradiography (WBAR), bioluminescence imaging (BLI) and 18F-FDG microPET were applied for intercomparison and to monitor the spatial and temporal distribution, and therapeutic response after drug administration. The survival in vivo was estimated and linked with the toxicological and histopathological analyses in order to determine the preclinical safety and feasibility of the nanomedicine.
Pharmacokinetic studies indicated that the terminal half-life (T1/2λz) and Cmax of 6 mol% PEG 111In-liposomes were similar to that of 0.9 mol% PEG 111In-liposomes. In the blood, the total body clearance (Cl) of 6 mol% PEG 111In-liposomes was about 1.7-fold lower and the area under the curve (AUC) was 1.7-fold higher than that of 0.9 mol% PEG 111In-liposomes. These results demonstrated that the effect of long-term circulation and localization of radioactivity in the plasma was achieved by 6 mol% PEGylated liposomes. The biodistribution of 6 mol% PEG 111In-liposomes showed significantly lower uptake in the liver, spleen, kidneys, small intestine and bone marrow, yet enhanced the uptake in the tumor than that of 0.9 mol% PEG 111In-liposomes. Prominent tumor uptake and the highest tumor/muscle ratios were found at 48 h post injection. Gamma scintigraphy at 48 h post injection also illustrated more distinct tumor uptake with 6 mol% PEG 111In-liposomes as compared to that of 0.9 mol% (p< 0.01). BLI and in vivo tumor growth tracing showed that growth in tumor volume could largely be inhibited by 6 mol% PEG 111In-liposomes.
In terms of therapeutic response of formulated 6 mol% PEG 111In-VNB-liposomes, the tumor growth and BLI monitoring showed that tumor volume could be completely inhibited by the combination therapy with 111In-VNB-liposomes and by chemotherapy with NanoX-VNB-liposomes (i.e. without Indium-111) (p< 0.01). The gamma scintigraphy and WBAR also confirmed the similar results as shown by biodistribution and the conspicuous inhibition of tumor growth with the combination therapy.
Nevertheless, on the basis of either the chemotherapy displayed a cure effect as good as that of combination therapy or both groups appeared minimal toxicity as shown by hematological analysis, the dosage design for the treatment was further modified in order to clarify the real therapeutic effectiveness of 6 mol% PEG 111In-VNB-liposomes via elevation of the radiation dosage and reduction in the concentration of chemotherapeutic agents. Selective tumor uptake was represented by cumulative deposition and the maximum accumulation was at 48 h post injection. The combination therapy exhibited an additive effect in terms of tumor growth suppression as tracked by caliper measurement, BLI and 18F-FDG microPET imaging. Furthermore, an improved survival rate and reduced tissue toxicity were closely correlated with the toxicological and histopathological results.
In summary, the results demonstrated that the use of 6 mol% PEG 111In-VNB-liposomes for passively targeted tumor therapy displayed an additive effect with combined therapy, not only by prolonging the circulation rate due to a reduction in the phagocytic effect of the RES, but also by enhancing tumor uptake. Thus, the preclinical study suggests that 6 mol% PEG 111In-VNB-liposomes have the potential to increase the therapeutic index and reduce the toxicity of the passively nanotargeted chemoradiotherapies.

ACKNOWLEDGEMENTS………………………………………………………4

ABSTRACT…………………………………………………………………6

1. INTRODUCTION
1.1. Colorectal Cancer (CRC) in Taiwan……………………8
1.2. Targeted Therapies……………………………………………………..……...8
1.3. Liposomes for Passive Targeting……………………..9
1.4. Vinorelbine (VNB)……………………………………….12
1.5. Indium-111 (111In)………………………………………13
1.6. Reporter Gene and Molecular Imaging……………….14

2. MATERIALS AND METHODS

PART 1. Diagnostic and Therapeutic Evaluation of 111In-Vinorelbine-Liposomes in a Human Colorectal Carcinoma HT-29/luc-Bearing Animal Model

2.1. Plasmid Construction and Transfection…………………18
2.2. Cell Culture…………………………………………………………..……18
2.3. In Vitro Tumor Growth Curve………………………………19
2.4. Tumor Xenografts and Experimental Design…………….19
2.5. Preparation of Liposomes………………………………….20
2.6. Radiolabeling of 111In-Oxine…………………………….20
2.7. Preparation of 111In-Liposomes………………………….20
2.8. Anti-Cancer Drug Encapsulation………………………….21
2.9. Pharmacokinetics and Biodistribution………………….21
2.10. Gamma Scintigraphy of HT-29/luc Tumor-Bearing Mice.22
2.11. Bioluminescence Imaging (BLI) of HT-29/luc Tumor-Bearing Mice…….........................................23
2.12. Whole-Body Autoradiography (WBAR)…….……………….23
2.13. Body Weight and Survival Assessment……………..……24
2.14. Biochemistry and Hematology Analyses…...……………24
2.15. Tissue Preparation for Histopathology…………………24
PART 2. Therapeutic Efficacy Evaluation of 111In-labeled Pegylated Liposomal Vinorelbine in Murine Colon Carcinoma with Multimodalities of Molecular Imaging

2.16. CT-26/tk-luc Tumor Cell Preparation……………………25
2.17. CT-26/tk-luc Tumor Xenografted Animal Model…………25
2.18. Preparation of PEGylated 111In-VNB-liposomes……….25
2.19. Biodistribution of 111In-VNB-liposomes in the CT-26/tk-luc Tumor-Bearing Mice…………………………………….26
2.20. Bioluminescence Imaging of CT-26/tk-luc Tumor-Bearing Mice………..…….........................................26
2.21. 18F-FDG MicroPET Imaging of CT-26/tk-luc Tumor-Bearing Mice……….…27
2.22. Statistical Analysis………………………………………………..…………….27
2.23. The Purpose of the Study……………………………………...……………….........27
2.24. The Experimental Design……………………………………………………….........29

3. RESULTS

PART 1. Diagnostic and Therapeutic Evaluation of 111In-Vinorelbine-Liposomes in a Human Colorectal Carcinoma HT-29/luc-Bearing Animal Model

3.1. In Vitro and in Vivo Growth Curve of HT-29/luc Tumor Cells………………........................................31
3.2. Pharmacokinetics of 0.9 mol% and 6 mol% PEG 111In-Liposomes in HT-29/luc Tumor-Bearing Mice……………………31
3.3. Biodistribution of 0.9 mol% and 6 mol% PEG 111In-Liposomes in HT-29/luc Tumor-Bearing Mice……………………34
3.4. Gamma Scintigraphy of HT-29/luc Tumor-Bearing Mice…37
3.5. Tumor Growth Inhibition of 0.9 mol% and 6 mol% PEG 111In-Liposomes in HT-29/luc Tumor-Bearing Mice……………37
3.6. Therapeutic Effectiveness of 111In-NanoX/VNB-Liposomes in HT-29/luc Tumor Bearing Mice……………………………………………….…………………..42
3.7. Whole Body Autoradiography (WBAR)……………………….48
3.8. Body Weights and Survivals Assessment………………….48
3.9. Toxicology Studies…………………………..………………48
3.10. Histopathology of Tumors and RES-Related Organs after Drug Treatment….........................................53
PART 2. Therapeutic Efficacy Evaluation of 111In-labeled Pegylated Liposomal Vinorelbine in Murine Colon Carcinoma with Multimodalities of Molecular Imaging

3.11. Biodistribution of 111In-VNB-liposomes in CT-26/tk-luc Tumor-Bearing Mice……….……………………………….55
3.12. Bioluminescence Imaging for Monitoring Therapeutic Response….…..……......................................58
3.13. 18F-FDG MicroPET Imaging for Monitoring the Therapeutic Response………62
3.14. Body Weights and Survivals Assessment……………....62
3.15. Toxicology Studies…………………………………….……68
3.16. Histopathological Examination after the Various Treatments………………….................................68

4. DISCUSSION………………………………………………….…….72

5. CONCLUSION…...………………………………………………….77

6. REFERENCE…..……………………………………………….....................78

APPENDIX………………………………………………………..…….84

PUBLICATION LIST……………………………………………………………….....89

1.Chen SP, Tsai ST, Jao SW, et al. Single nucleotide polymorphisms of the APC gene and colorectal cancer risk: a case-control study in Taiwan. BMC Cancer. 2006;6:83.
2.Kelly K, Alencar H, Funovics M, Mahmood U, Weissleder R. Detection of invasive colon cancer using a novel, targeted, library-derived fluorescent peptide. Cancer Res. 2004;64:6247-6251.
3.Gerber DE. Targeted therapies: a new generation of cancer treatments. Am Fam Physician. 2008;77:311-319.
4.Alekshun T, Garrett C. Targeted therapies in the treatment of colorectal cancers. Cancer Control. 2005;12:105-110.
5.Bege T, Lelong B, Viret F, et al. Bevacizumab-related surgical site complication despite primary tumor resection in colorectal cancer patients. Ann Surg Oncol. 2009;16:856-860.
6.Hegde SR, Sun W, Lynch JP. Systemic and targeted therapy for advanced colon cancer. Expert Rev Gastroenterol Hepatol. 2008;2:135-149.
7.Dechant M, Weisner W, Berger S, et al. Complement-dependent tumor cell lysis triggered by combinations of epidermal growth factor receptor antibodies. Cancer Res. 2008;68:4998-5003.
8.Goodin S. Progress in the development of monoclonal antibody therapies for colorectal cancer. Introduction. Am J Health Syst Pharm. 2008;65:S2.
9.Lin WL, Lin WC, Yang JY, et al. Fatal toxic epidermal necrolysis associated with cetuximab in a patient with colon cancer. J Clin Oncol. 2008;26:2779-2780.
10.Levy EM, Sycz G, Arriaga JM, et al. Cetuximab-mediated cellular cytotoxicity is inhibited by HLA-E membrane expression in colon cancer cells. Innate Immun. 2009;15:91-100.
11.Chow TH, Lin YY, Hwang JJ, et al. Diagnostic and therapeutic evaluation of 111In-vinorelbine-liposomes in a human colorectal carcinoma HT-29/luc-bearing animal model. Nucl Med Biol. 2008;35:623-634.
12. Lee WC, Hwang JJ, Tseng YL, et al. Therapeutic efficacy evaluation of 111In-VNB-liposome on human colorectal adenocarcinoma HT-29/luc mouse xenografts. Nucl Instrum Meth A. Oct 2006;569:497-504.
13.Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science. 2004;303:1818-1822.
14.Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov. 2005;4:145-160.
15.Messerer CL, Ramsay EC, Waterhouse D, et al. Liposomal irinotecan: formulation development and therapeutic assessment in murine xenograft models of colorectal cancer. Clin Cancer Res. 2004;10:6638-6649.
16.Bakker-Woudenberg IA, Lokerse AF, Roerdink FH. Antibacterial activity of liposome-entrapped ampicillin in vitro and in vivo in relation to the lipid composition. J Pharmacol Exp Ther. 1989;251:321-327.
17.Boerman OC, Storm G, Oyen WJ, et al. Sterically stabilized liposomes labeled with indium-111 to image focal infection. J Nucl Med. 1995;36:1639-1644.
18.Li SD, Huang L. Pharmacokinetics and biodistribution of nanoparticles. Mol Pharm. 2008;5:496-504.
19.Huang SK, Lee KD, Hong K, Friend DS, Papahadjopoulos D. Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice. Cancer Res. 1992;52:5135-5143.
20.Huang SK, Martin FJ, Jay G, Vogel J, Papahadjopoulos D, Friend DS. Extravasation and transcytosis of liposomes in Kaposi's sarcoma-like dermal lesions of transgenic mice bearing the HIV tat gene. Am J Pathol. 1993;143:10-14.
21.Northfelt DW, Dezube BJ, Thommes JA, et al. Pegylated-liposomal doxorubicin versus doxorubicin, bleomycin, and vincristine in the treatment of AIDS-related Kaposi's sarcoma: results of a randomized phase III clinical trial. J Clin Oncol. 1998;16:2445-2451.
22.Udhrain A, Skubitz KM, Northfelt DW. Pegylated liposomal doxorubicin in the treatment of AIDS-related Kaposi's sarcoma. Int J Nanomedicine. 2007;2:345-352.
23.Gordon AN, Fleagle JT, Guthrie D, Parkin DE, Gore ME, Lacave AJ. Recurrent epithelial ovarian carcinoma: a randomized phase III study of pegylated liposomal doxorubicin versus topotecan. J Clin Oncol. 2001;19:3312-3322.
24.Green AE, Rose PG. Pegylated liposomal doxorubicin in ovarian cancer. Int J Nanomedicine. 2006;1:229-239.
25.Harris L, Batist G, Belt R, et al. Liposome-encapsulated doxorubicin compared with conventional doxorubicin in a randomized multicenter trial as first-line therapy of metastatic breast carcinoma. Cancer. 2002;94:25-36.
26.Minisini AM, Andreetta C, Fasola G, Puglisi F. Pegylated liposomal doxorubicin in elderly patients with metastatic breast cancer. Expert Rev Anticancer Ther. 2008;8:331-342.
27.Katsumata N, Fujiwara Y, Kamura T, et al. Phase II clinical trial of pegylated liposomal doxorubicin (JNS002) in Japanese patients with mullerian carcinoma (epithelial ovarian carcinoma, primary carcinoma of fallopian tube, peritoneal carcinoma) having a therapeutic history of platinum-based chemotherapy: a Phase II Study of the Japanese Gynecologic Oncology Group. Jpn J Clin Oncol. 2008;38:777-785.
28.Heidenreich A, Sommer F, Ohlmann CH, et al. Prospective randomized Phase II trial of pegylated doxorubicin in the management of symptomatic hormone-refractory prostate carcinoma. Cancer. 2004;101:948-956.
29.Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated liposomal Doxorubicin: review of animal and human studies. Clin Pharmacokinet. 2003;42:419-436.
30.Papahadjopoulos D, Allen TM, Gabizon A, et al. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc Natl Acad Sci U S A. 1991;88:11460-11464.
31.Potier P. The synthesis of Navelbine prototype of a new series of vinblastine derivatives. Semin Oncol. 1989;16:2-4.
32.Bartsch R, Wenzel C, Altorjai G, et al. Results from an observational trial with oral vinorelbine and trastuzumab in advanced breast cancer. Breast Cancer Res Treat. 2007;102:375-381.
33.Ohe Y, Ohashi Y, Kubota K, et al. Randomized phase III study of cisplatin plus irinotecan versus carboplatin plus paclitaxel, cisplatin plus gemcitabine, and cisplatin plus vinorelbine for advanced non-small-cell lung cancer: Four-Arm Cooperative Study in Japan. Ann Oncol. 2007;18:317-323.
34.Uckun FM, Morar S, Qazi S. Vinorelbine-based salvage chemotherapy for therapy-refractory aggressive leukaemias. Br J Haematol. 2006;135:500-508.
35.Okouneva T, Hill BT, Wilson L, Jordan MA. The effects of vinflunine, vinorelbine, and vinblastine on centromere dynamics. Mol Cancer Ther. 2003;2:427-436.
36.Baweja M, Suman VJ, Fitch TR, et al. Phase II trial of oral vinorelbine for the treatment of metastatic breast cancer in patients > or = 65 years of age: an NCCTG study. Ann Oncol. 2006;17:623-629.
37.Howell RW, Narra VR, Sastry KS, Rao DV. On the equivalent dose for Auger electron emitters. Radiat Res. 1993;134:71-78.
38.Mariani G, Bodei L, Adelstein SJ, Kassis AI. Emerging roles for radiometabolic therapy of tumors based on auger electron emission. J Nucl Med. 2000;41:1519-1521.
39.Chen P, Cameron R, Wang J, Vallis KA, Reilly RM. Antitumor effects and normal tissue toxicity of 111In-labeled epidermal growth factor administered to athymic mice bearing epidermal growth factor receptor-positive human breast cancer xenografts. J Nucl Med. 2003;44:1469-1478.
40.Doubrovin M, Serganova I, Mayer-Kuckuk P, Ponomarev V, Blasberg RG. Multimodality in vivo molecular-genetic imaging. Bioconjug Chem. 2004;15:1376-1388.
41.Wu JC, Inubushi M, Sundaresan G, Schelbert HR, Gambhir SS. Optical imaging of cardiac reporter gene expression in living rats. Circulation. 2002;105:1631-1634.
42.Bhaumik S, Gambhir SS. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci U S A. 2002;99:377-382.
43.Tseng YL, Hong RL, Tao MH, Chang FH. Sterically stabilized anti-idiotype immunoliposomes improve the therapeutic efficacy of doxorubicin in a murine B-cell lymphoma model. Int J Cancer. 1999;80:723-730.
44.Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin. 2002;52:23-47.
45.Chen LC, Chang CH, Yu CY, et al. Biodistribution, pharmacokinetics and imaging of (188)Re-BMEDA-labeled pegylated liposomes after intraperitoneal injection in a C26 colon carcinoma ascites mouse model. Nucl Med Biol. 2007;34:415-423.
46.Dos Santos N, Allen C, Doppen AM, et al. Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding. Biochim Biophys Acta. 2007;1768:1367-1377.
47.Liu D, Mori A, Huang L. Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes. Biochim Biophys Acta. 1992;110 :95-101.
48.Torchilin VP, Lukyanov AN, Gao Z, Papahadjopoulos-Sternberg B. Immunomicelles: targeted pharmaceutical carriers for poorly soluble drugs. Proc Natl Acad Sci U S A. 2003;100:6039-6044.
49.Zhang JS, Liu F, Huang L. Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity. Adv Drug Deliv Rev. 2005;57:689-698.
50.Levchenko TS, Rammohan R, Lukyanov AN, Whiteman KR, Torchilin VP. Liposome clearance in mice: the effect of a separate and combined presence of surface charge and polymer coating. Int J Pharm. 2002;240:95-102.
51. Chow TH, Lin YY, Hwang JJ, et al. Improvement of Biodistribution and Therapeutic Index via Increase of Polyethylene Glycol on Drug-carrying Liposomes in an HT-29/luc Xenografted Mouse Model. Anticancer Res. 2009;29:2111-2120.
52.Massoud TF, Gambhir SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 2003;17:545-580.
53.Chang CH, Wang HE, Wu SY, et al. Comparative evaluation of FET and FDG for differentiating lung carcinoma from inflammation in mice. Anticancer Res. 2006;26:917-925.
54.Chang CH, Jan ML, Fan KH, et al. Longitudinal evaluation of tumor metastasis by an FDG-microPet/microCT dual-imaging modality in a lung carcinoma-bearing mouse model. Anticancer Res. 2006;26:159-166.
55.Torigian DA, Huang SS, Houseni M, Alavi A. Functional imaging of cancer with emphasis on molecular techniques. CA Cancer J Clin. 2007;57:206-224.
56.Weissleder R. Molecular imaging in cancer. Science. 2006;312:1168-1171.
57.El Hilali N, Rubio N, Martinez-Villacampa M, Blanco J. Combined noninvasive imaging and luminometric quantification of luciferase-labeled human prostate tumors and metastases. Lab Invest. 2002;82:1563-1571.
58.Thalmann GN, Anezinis PE, Chang SM, et al. Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res. 1994;54:2577-2581.
59.Contag CH, Spilman SD, Contag PR, et al. Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem Photobiol. 1997;66:523-531.
60.Contag CH, Jenkins D, Contag PR, Negrin RS. Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia. 2000;2:41-52.
61.Park HS, Chung JW, Jae HJ, et al. FDG-PET for evaluating the antitumor effect of intraarterial 3-bromopyruvate administration in a rabbit VX2 liver tumor model. Korean J Radiol. 2007;8:216-224.
62.Oya N, Nagata Y, Ishigaki T, et al. Evaluation of experimental liver tumors using fluorine-18-2-fluoro-2-deoxy-D-glucose PET. J Nucl Med. 1993;34:2124-2129.
63.Spaepen K, Stroobants S, Dupont P, et al. [(18)F]FDG PET monitoring of tumour response to chemotherapy: does [(18)F]FDG uptake correlate with the viable tumour cell fraction? Eur J Nucl Med Mol Imaging. 2003;30:682-688.
64.Juweid ME, Cheson BD. Positron-emission tomography and assessment of cancer therapy. N Engl J Med. 2006;354:496-507.
65.Oya N, Nagata Y, Tamaki N, et al. FDG-PET evaluation of therapeutic effects on VX2 liver tumor. J Nucl Med. 1996;37:296-302.