99mTc과 188Re을 이용한 암 표적 영상진단 및 치료용 방사성표지화합물 개발 = Development of Radiolabeled Compounds for Targeted Cancer Diagnosis and Therapy Using 99mTc and 188Re

다국어 초록 (Multilingual Abstract)

The development of improved modality for cancer treatment is likely to become an increasingly important issue of public health care. One of the promising approaches for cancer therapy is the use of inhibitors against proteins that are over-expressed in cancer, known as targeted therapy. In particular, among various treatment modalities, a radiopharmaceutical is regarded as a promising means for effective treatment of a number of widely disseminated diseases. As prepared by conjugating the radionuclide to the biomolecules which have specificity for in vivo targeting of the diseases, radiopharmaceuticals can effectively deliver therapeutic doses of ionizing radiation with a high selectivity to the cancerous sites, even though their location in the body is unknown.
In order to develop the radiopharmaceuticals for imaging and therapy using 99mTc and 188Re, one chelator conjugated cyclized somatostatin analogue for somatostatin receptor targeting and two chelator conjugated cyclized α-melanocyte-stimulating hormone (α-MSH) analogues for melanocortin-1 receptor (MC1-R) targeting were newly prepared. The amino acid sequences of somatostatin analogue was Mercaptoacetyl-Gly-Cys-Gly-Dap-D-Phe-Trp-Lys- Val-Asp-Thr (Cyclized Dap-Asp, N2S2-somatostatin) in which a novel bifunctional chelating agent of diaminodithiolate (N2S2), Mercaptoacetyl-Gly- Cys, was introduced for the efficient radiolabeling with 99mTc and 188Re. In addition, the amino acid sequences of α-MSH analogues were Mercaptoacetyl- Gly-Cys-Gly-Dap-Glu-His-D-Phe-Arg-Trp-Asp-Lys-Pro-Val-NH2 (Cyclized Dap- Asp, N2S2-α-MSH) and Mercaptoacetyl-Lys(Glucose)-Cys-Gly-Dap-Glu-His- D-Phe-Arg-Trp-Asp-Lys-Pro-Val-NH2 (Cyclized Dap-Asp, N2S2-glucose-α-MSH) in which Mercaptoacetyl-(Gly or Lys)-Cys was introduced as a bifunctional chelating agent for radiolabeling with 99mTc and 188Re. Those peptides were prepared by using a conventional method of solid phase peptide synthesis. Cyclization of peptide sequences was accomplished by the formation of lactam between the β-carboxylic acid of aspartic acid (Asp) and β-amine of diaminopropionic aicd (Dap) to increase the stability and the biological activity in vivo. The amide coupling between side chain of Dap and that of Asp was the first attempt to prepare the cyclized peptide for the development of radiopharmaceuticals.
The radiolabeling of peptide with 99mTc and 188Re was performed by using a ligand exchange partner, a glucoheptonate (GH) kit, which was originally used as a clinical pharmaceutical. The radio-peptide was prepared in a high yield (>98%) at a low concentration of a peptide (1 nmol) and showed a high radiochemical stability (>98%) in serum at 37℃ for 72 h.
Although biodistribution study of 188Re-N2S2-somatostatin demonstrated a low tumor uptake in Calu-6 bearing mice, most of the injected radioactivity was excreted at a fast rate through the hepatobiliary route within 24 h post-injection without non-specific binding to the main organs. It is observed that the Calu-6 cell line is not a proper cell for the evaluation of its potential as a pharmaceutical to target the somatostatin receptor.
Biodistribution studies of 188Re-N2S2-α-MSH using B16/F1-bearing mice demonstrated a high tumor uptake and extended tumor retention. Tumor uptake values and were 8.42±1.48 at 2 h and 1.66±0.38 %ID/g at 24 h, respectively. Since the clearance kinetics was rapid, most of the injected radioactivity was detected in large intestine in 2 h post-injection. The tumor-to-blood ratios were 7.0 and 5.4 at 2 and 24 h post-injection, respectively. The treatment effects of 188Re-N2S2-α-MSH on tumor growth rate in the therapy study revealed that the single-dose administration of 188Re-N2S2-α-MSH (250 μCi) caused substantial tumor growth inhibition compared with the untreated tumor control group. The average tumor volume was reduced to about 38% in comparison with that of mice in placebo control group at 14 day after inoculation. The me야an survival time for the treated tumor group was prolonged to 24 day, whereas the untreated group exhibited a median survival time of 14 day.
Furthermore, biodistribution studies of 188Re-N2S2-glucose-α-MSH using B16/F1 (melanoma) bearing mice demonstrated a high tumor uptake and extended tumor retention of 2.82±0.11 at 2 h and 1.91±0.31 %ID/g at 24 h post-injection, respectively. However, the main route of overall excretion was the urinary track which was attributed to the decrease of lipophilicity. The introduction of glucose derivative to the peptide increased the excretion rate of unbounded radiopeptide to its receptor in vivo and sustained its binding to a receptor expressed on the tumor.
Since the prepared peptide of α-MSH analogues showed their potential to target the MC1-R positive tumor, those can be applicable as target-medicated radiopharmaceuticals for both diagnosis and therapy of melanoma. In particular, the 188Re-N2S2-α-MSH is a potential candidate for the treatment of melanoma.
The established methods for peptide synthesis, radiolabeling and in vitro/in vivo evaluation can be applied to develop a new radiopharmaceutical for the targeted imaging and therapy of a cancerous tumor. Furthermore, the prepared radiopeptides, N2S2-somatostatin, N2S2-α-MSH and N2S2-glucose-α-MSH can be applicable to tumor imaging and therapy for their receptor positive tumors.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• 1. Cancer 1
• 2. Targeted Therapy of Cancer 5
• 3. Research Purposes 7
• Chapter 2. Theoretical Background 10
• 1. Radiopharmaceuticals 10
• 2. Technetium and Rhenium in Radiopharmaceuticals 12
• 3. Development of Target Specific Radiopharmaceuticals 17
• 4. Peptide Receptors in Oncology 18
• 5. Design of Peptide-based Radiopharmaceuticals 23
• 6. Somatostatin Receptor Targeting 29
• 7. Melanocortin-1 Receptor(MC1-R) 35
• Chapter 3. Approaches and Experiments 43
• 1. Approaches 43
• 2. Experiments 50
• Chapter 4. Results and Discussion 60
• 1. Synthesis of Peptide 60
• 2. Radiolabeling of Peptides with 99mTc and 188Re 78
• 3. Biodistribution Studies 96
• 4. Radiolnuclide Therapy of 188Re-N2S2-α-MSH 102
• Chapter 5. Conclusions 105
• References 109
• 국문 요약 118