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Novel radiolabeled peptides to improve breast and prostate cancer diagnosis by PET Pourghiasian, Maral 2015

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NOVEL RADIOLABELED PEPTIDES TO IMPROVE BREAST AND PROSTATE CANCER DIAGNOSIS BY PET  by  Maral Pourghiasian  B.Sc. of Radiology, Tehran University of Medical Sciences, 2005  M.Sc. of Oncology, Vrije Universiteit Amsterdam, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Doctor of Philosophy  in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Interdisciplinary Oncology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   April 2015  © Maral Pourghiasian, 2015 ii  Abstract   In the past years, peptide based radiopharmaceuticals have turned into favorable molecular imaging agents for specific targeting of cancer. This is mainly because many tumors happen to overexpress certain regulatory peptide receptors.  For instance, the gastrin releasing peptide (GRP) receptors are overexpressed in prostate cancer–the most common malignancy among men–and somatostatin 2a (SST2a), and neuropeptide Y1 (NPY1) receptors are overexpressed in breast cancer–the most common cancer among women. There are disadvantages to most existing imaging techniques used for early detection of prostate and breast cancer. Thus, the objective of the work presented in this thesis was to develop a novel and specific diagnostic approach using radiolabeled peptides for PET imaging to localize lesions of breast and prostate cancers. Towards this end, different derivatives of GRP, SST2a, and NPY1 peptides were synthesized and their binding affinity was confirmed in vitro. The promising candidates were radiolabeled with 18F or 68Ga–the ideal radioisotopes in PET applications. Two different 18F labeling methods (click chemistry and trifluoroborate exchange reaction) were conducted. Finally, the biological evaluation of radiopharmaceuticals was performed in vivo by using animal models of prostate and breast cancer.  In the click chemistry approach, introducing PEG spacers to GRP derivatives improved the in vitro properties and the pharmacokinetics in the prostate tumor model, leading to better tumor visualization by PET imaging. The trifluoroborate exchange reaction proved to be a superior technique–by both radiochemistry and biological criteria–in GRP labeling, resulting in an excellent tumor uptake with 18F-AmBF3-MJ9. The same approach was also successful for iii  targeting SST2a receptors in mice bearing rat pancreatic tumor cells. The data achieved with this labeling method suggest the potential application of these radiopharmaceuticals for diagnosis in cancer patients.  A high tumor to background ratio was achieved in the Zr-75-1 tumor model with 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3. Hence, this cell line is a promising breast cancer model for SST2a imaging. The NPY1R compound 18F-ALK-BVD15 was not metabolically stable and showed low receptor-mediated tumor uptake in NPY1R-positive tumors. Different strategies will need to be explored in order to modify and improve the stability of the peptide.  iv  Preface  Chapters 2 and 6 are based on work conducted at BC Cancer Agency Research Centre with the collaboration of TRIUMF and Université de Sherbrook. Peptides used in these chapters were synthesized in Dr. Brigitte Guérin's lab in Sherbrook. Radiolabeling was done by Dr. James A. Inkster at TRIUMF and the BC Cancer Agency. I performed all the biological experiments including in vitro, in vivo and ex vivo and data analysis except for Figure 2.3. A version of chapter 2 will be submitted under:  “F-18 labeled PEGylated bombesin derivatives for improved gastrin releasing peptide receptor imaging in prostate cancer by positron emission tomography”, Maral Pourghiasian, James Inkster, Kuo-Shyan Lin, Brigitte Guerin, Samia Ait-Mohand, Navjit Hundal, Thomas J. Ruth, François Bénard.   The work in Chapter 3 and 4 is done with the collaboration of department of Chemistry at UBC. The chemistry and radiochemistry was conducted by Dr. Zhibo Liu at UBC and the BC Cancer Agency. The biological work and data analysis was done by me.  A version of chapter 3 is in press in the journal of Bioorganic and Medicinal Chemistry, which I contributed in performing the biological experiments and writing the manuscript with the editorial help of Drs. François Bénard and Kuo-Shyan Lin:    “18F-AmBF3-MJ9: a novel radiofluorinated bombesin derivative for prostate cancer imaging”, Maral Pourghiasian, Zhibo Liu, Jinhe Pan, Zhengxing Zhang, Nadine Colpo, Kuo-Shyan Lin, David M. Perrin, François Bénard. v  A version of chapter 4 has been published which I am a co-first author and performed and wrote the biological work with the editorial help of Dr. François Bénard: Liu Z, Pourghiasian M, Benard F, Pan J, Lin KS, Perrin DM: Preclinical Evaluation of a High-Affinity 18F-Trifluoroborate Octreotate Derivative for Somatostatin Receptor Imaging. Journal of nuclear medicine: official publication, Society of Nuclear Medicine 2014, 55(9):1499-1505.  Chapter 5 was conducted at the BC Cancer Agency Research Centre. The chemistry and radiochemistry was done by Drs. Jinhe Pan and Zhengxing Zhang. All the biological experiments and data analysis were done by me except for Figures 5.7-5.10. A version of chapter 5 will be submitted under: “Comparison of radiolabeled somatostatin receptor agonist and antagonists in a mouse model of human breast cancer”, Maral Pourghiasian, Zhengxing Zhang, Navjit Hundal, Guillaume V Amouroux, Silvia Jenny, Kuo-Shyan Lin, François Bénard. All the animal studies performed in this thesis were under animal protocol A13-0032, “Radiolabeled peptides to improve cancer diagnosis by Positron emission tomography and Single-photon emission tomography” that was approved by the Institutional Animal Care Committee of the University of British Columbia and was performed in compliance with the Canadian Council on Animal Care Guidelines. vi  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents .......................................................................................................................... vi List of Tables ............................................................................................................................... xiii List of Figures .............................................................................................................................. xv List of Schemes ............................................................................................................................ xx List of Abbreviations and Symbols ........................................................................................... xxi Acknowledgements .................................................................................................................... xxv Dedication ................................................................................................................................. xxvii Chapter 1: Introduction ................................................................................................................ 1 1.1 Molecular imaging in nuclear medicine .......................................................................... 1 1.2 Positron emission tomography (PET) ............................................................................. 2 1.3 Management of low metabolic rate cancers in nuclear medicine .................................... 6 1.3.1 Prostate cancer ............................................................................................................. 6 1.3.1.1 Common procedures in nuclear medicine for detection of prostate cancer ........ 7 1.3.1.1.1 PSMA targeting agents .................................................................................. 7 1.3.1.1.2 Non-prostate specific agents .......................................................................... 8 1.3.1.1.3 Androgen receptor imaging agents ................................................................ 8 1.3.1.1.4 Protein synthesis imaging agents ................................................................... 9 1.3.1.1.5 Bone imaging agents ...................................................................................... 9 1.3.2 Breast cancer.............................................................................................................. 11 vii  1.3.2.1 Common procedures in nuclear medicine for detection of breast cancer.......... 12 1.3.2.1.1 SPECT imaging ........................................................................................... 12 1.3.2.1.2 PET imaging ................................................................................................ 13 1.4 Regulatory peptides and peptide receptors .................................................................... 13 1.5 Peptide-based radiopharmaceuticals for cancer imaging .............................................. 18 1.5.1 Development of peptide-based radiopharmaceuticals ............................................... 19 1.6 Hypothesis and objectives ............................................................................................. 22 1.6.1 Specific aim 1 ............................................................................................................ 22 1.6.2 Specific aim 2 ............................................................................................................ 22 1.6.3 Specific aim 3 ............................................................................................................ 23 Chapter 2: GRP receptors, click chemistry approach ............................................................. 24 2.1 Background .................................................................................................................... 24 2.2 Materials and methods ................................................................................................... 27 2.2.1 Cell culture ................................................................................................................ 27 2.2.2 Peptide synthesis and radiosynthesis of bombesin derivatives ................................. 27 2.2.3 LogD7.4 measurement................................................................................................. 30 2.2.4 Metabolic stability study ........................................................................................... 30 2.2.5 Immunohistochemistry .............................................................................................. 30 2.2.6 In vitro receptor binding assays ................................................................................. 31 2.2.7 Biodistribution studies in tumor-bearing mice .......................................................... 31 2.2.8 PET/CT imaging and kinetic analysis ....................................................................... 32 2.2.9 Statistical analysis ..................................................................................................... 33 2.3 Results ........................................................................................................................... 33 viii  2.3.1 Chemistry and radiochemistry ................................................................................... 33 2.3.2 LogD7.4 measurements ............................................................................................... 34 2.3.3 In vitro binding assays ............................................................................................... 34 2.3.4 Immunohistochemistry .............................................................................................. 35 2.3.5 Metabolic stability in mouse plasma ......................................................................... 36 2.3.6 Biodistribution studies in tumor bearing mice .......................................................... 38 2.3.7 PET/CT imaging and kinetic analysis ....................................................................... 43 2.4 Discussion ...................................................................................................................... 47 2.5 Conclusion ..................................................................................................................... 49 Chapter 3: GRP receptors, trifluoroborate isotope exchange reaction approach ................ 50 3.1 Background .................................................................................................................... 50 3.2 Materials and methods ................................................................................................... 51 3.2.1 HPLC analysis ........................................................................................................... 52 3.2.2 Synthesis of AmBF3-MJ9 .......................................................................................... 53 3.2.2.1 Synthesis of N-propargyl-N,N-dimethyl-ammoniomethyl boronylpinacolate .. 53 3.2.2.2 Synthesis of azidoacetyl-MJ9 ............................................................................ 53 3.2.2.3 Synthesis of AmBF3-MJ9 .................................................................................. 54 3.2.3 In vitro receptor binding assays ................................................................................. 54 3.2.4 Radiolabeling ............................................................................................................. 55 3.2.5 Metabolic stability study in mouse plasma................................................................ 56 3.2.6 Internalization studies ................................................................................................ 56 3.2.7 Biodistribution studies in tumor-bearing mice .......................................................... 57 3.2.8 PET/CT imaging ........................................................................................................ 58 ix  3.3 Results ........................................................................................................................... 58 3.3.1 Synthesis of AmBF3-MJ9 .......................................................................................... 58 3.3.2 In vitro binding assay ................................................................................................ 59 3.3.3 Radiochemistry .......................................................................................................... 59 3.3.4 Metabolic stability in mouse plasma ......................................................................... 61 3.3.5 Internalization studies ................................................................................................ 62 3.3.6 Biodistribution and imaging studies .......................................................................... 63 3.4 Discussion ...................................................................................................................... 67 3.5 Conclusion ..................................................................................................................... 70 Chapter 4: Somatostatin receptors, trifluoroborate isotope exchange reaction approach .. 71 4.1 Background .................................................................................................................... 71 4.2 Materials and methods ................................................................................................... 74 4.2.1 HPLC Analysis .......................................................................................................... 74 4.2.2 Peptide synthesis and 18F labeling ............................................................................. 74 4.2.3 Synthesis of N-propargyl-N, N-dimethyl-ammoniomethyl-boronylpinacolate ......... 75 4.2.4 Synthesis of AmBF3-TATE ....................................................................................... 75 4.2.5 In vitro binding assays ............................................................................................... 77 4.2.6 Metabolic stability in mouse plasma ......................................................................... 77 4.2.7 Radiolabeling ............................................................................................................. 78 4.2.8 Internalization studies ................................................................................................ 78 4.2.9 Animal models and biodistribution studies ............................................................... 79 4.2.10 PET/CT imaging ........................................................................................................ 79 4.3 Results ........................................................................................................................... 80 x  4.3.1 In vitro binding assay ................................................................................................ 80 4.3.2 Radiosynthesis ........................................................................................................... 81 4.3.3 Metabolic stability in mouse plasma ......................................................................... 82 4.3.4 Internalization studies ................................................................................................ 83 4.3.5 Biodistribution studies ............................................................................................... 84 4.3.6 PET/CT imaging ........................................................................................................ 86 4.3.7 Kinetic analysis ......................................................................................................... 88 4.4 Discussion ...................................................................................................................... 89 4.5 Conclusion ..................................................................................................................... 91 Chapter 5: Evaluation of the potential of positron-emitting somatostatin analogs to detect breast cancer ..................................................................................................................... 93 5.1 Background .................................................................................................................... 93 5.2 Materials and methods ................................................................................................... 94 5.2.1 Peptide synthesis ....................................................................................................... 95 5.2.1.1 NOTA-BASS ..................................................................................................... 95 5.2.1.2 NatGa-NOTA-BASS ........................................................................................... 96 5.2.1.3 DOTA-TATE .................................................................................................... 97 5.2.1.4 NatGa-DOTA-TATE ........................................................................................... 98 5.2.1.5 NODAGA-LM3 ................................................................................................ 98 5.2.1.6 NatGa-NODAGA-LM3 ....................................................................................... 99 5.2.2 Cell culture and membrane isolation ......................................................................... 99 5.2.3 In vitro binding assays ............................................................................................. 100 5.2.4 Radiolabeling ........................................................................................................... 101 xi  5.2.4.1 68Ga-DOTA-TATE .......................................................................................... 101 5.2.4.2 68Ga-NODAGA-LM3 ...................................................................................... 101 5.2.5 Flow cytometry ........................................................................................................ 102 5.2.6 Internalization studies .............................................................................................. 102 5.2.7 Animal models and biodistribution studies ............................................................. 103 5.2.8 PET/CT imaging ...................................................................................................... 104 5.3 Results ......................................................................................................................... 105 5.3.1 Peptide synthesis ..................................................................................................... 105 5.3.2 Radiolabeling ........................................................................................................... 105 5.3.3 In vitro binding assays ............................................................................................. 106 5.3.4 Internalization studies .............................................................................................. 111 5.3.5 Flow cytometry ........................................................................................................ 112 5.3.6 Biodistribution studies ............................................................................................. 115 5.3.7 PET/CT imaging and kinetic analysis ..................................................................... 119 5.4 Discussion .................................................................................................................... 125 5.5 Conclusion ................................................................................................................... 127 Chapter 6: Neuropeptide Y1 receptors ................................................................................... 129 6.1 Background .................................................................................................................. 129 6.2 Materials and methods ................................................................................................. 132 6.2.1 Cell culture .............................................................................................................. 132 6.2.2 Peptide synthesis and radiosynthesis of NPY1 derivative....................................... 132 6.2.3 Immunohistochemistry ............................................................................................ 133 6.2.4 In vitro receptor binding assays ............................................................................... 134 xii  6.2.5 Biodistribution studies in tumor-bearing mice ........................................................ 135 6.2.6 PET/CT imaging and kinetic analysis ..................................................................... 136 6.2.7 Metabolic stability study in mouse plasma.............................................................. 136 6.2.8 Liquid chromatography tandem ms analysis ........................................................... 137 6.2.9 Statistical analysis ................................................................................................... 138 6.3 Results ......................................................................................................................... 138 6.3.1 Chemistry and radiochemistry ................................................................................. 138 6.3.2 In vitro binding assays ............................................................................................. 138 6.3.3 Biodistribution studies ............................................................................................. 140 6.3.4 PET/CT imaging ...................................................................................................... 143 6.3.5 Immunohistochemistry ............................................................................................ 144 6.3.6 Metabolic stability in mouse plasma ....................................................................... 144 6.3.7 Mass spectrometry analysis ..................................................................................... 146 6.4 Discussion .................................................................................................................... 147 6.5 Conclusion ................................................................................................................... 150 Chapter 7: Summary and conclusion ...................................................................................... 152 Bibliography ............................................................................................................................... 158   xiii  List of Tables  Table 1.1 The characteristics of the most common radionuclides used in PET.  ....................... 5 Table 1.2 Age-specific female breast cancer incidence and mortality rates in the US in 2013 ) .................................................................................................................................................. 11 Table 1.3 Peptide receptor expression patterns.  Common peptide receptors expressed in human cancers are in bold). ...................................................................................................... 17 Table 2.1 The Lipophilicity of bombesin derivatives............................................................... 34 Table 2.2 Biodistribution studies of [18F]-ALK-BBN in nude mice bearing PC3 tumor 1 h post-injection. Data presented as %ID/g of tissues in unblocked (n=11) and blocking with co-injection (n=5). ......................................................................................................................... 39 Table 2.3 Biodistribution studies of [18F]-ALK-BBN-PEG in nude mice bearing PC3 tumor 1 h post- injection. ....................................................................................................................... 40 Table 2.4 Biodistribution studies of [18F]-PEG-BBN-PEG in nude mice bearing PC3 tumor 1 h post-injection. . ...................................................................................................................... 41 Table 3.1 Biodistribution and tumor-to-background ratios of 18F-AmBF3-MJ9 in NSG mice bearing PC-3 tumors. . .............................................................................................................. 65 Table 4.1 Biodistribution of 18F-AMBF3-TATE in AR42J tumor-bearing mice. . .................. 85 Table 5.1 Biodistribution Results of 68Ga-DOTA-TATE in immunocompromised mice bearing MCF-7 tumors at 1 h post-injection. ......................................................................... 116 Table 5.2 Biodistribution Results of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 in immuno-compromised mice bearing Zr-75-1 tumors at 1 h post-injection............................ 117 xiv  Table 5.3  Biodistribution Results of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 in immunocompromised mice bearing HEK-SST2a tumors at 1 h post-injection. .................... 118 Table 5.4 Tumor–to–normal-tissue ratios in Zr-75-1 and HEK-SST2a tumor xenografts at 1h post-injection. ......................................................................................................................... 118 Table 6.1 Binding affinity of NPY analogs in human breast and neuroblastoma tumor cells. ................................................................................................................................................ 139 Table 6.2 Biodistribution of [18F]-ALK-BVD15 in SK-N-MC and MCF-7 tumor-bearing mice at 1 h post-injection.. .............................................................................................................. 141  xv  List of Figures  Figure 1.1 Positron annihilation and detection by PET  ............................................................. 3 Figure 1.2 An example of a micro PET/CT for small animal imaging. ..................................... 4 Figure 1.3 A 1.5 × 0.7-cm peripancreatic lymph node uptake of 111In-capromab pendetide (ProstaScint) visualized by single photon emission computed tomography (SPECT) combined with 16-slice multidetector computed tomography (CT) (Precedence, Philips Healthcare, Andover, MA) scanner.  ........................................................................................................... 10 Figure 1.4 11C-choline positron emission tomography/CT showing focal . ............................. 10 Figure 1.5 Principle of in vivo peptide receptor targeting of cancer. . ..................................... 19 Figure 1.6 The main steps involved in development of a radiopharmaceutical ....................... 21 Figure 2.1 Competition binding assay of 19F-ALK-BBN-PEG (A) and 19F-PEG-BBN-PEG (B) in GRPR positive PC-3 cells. . ................................................................................................. 35 Figure 2.2 Immunohistochemical staining of GRPR expression in PC-3 tumors. 40X magnification. ........................................................................................................................... 35 Figure 2.3 HPLC traces of metabolic stability of [18F]-ALK-BBN-PEG at 30, 60 and 120 min incubation at 37 ºC in mouse plasma. ....................................................................................... 37 Figure 2.4 Biodistribution of the three BBN derivatives in the intestine at 1 h post-injection….. .............................................................................................................................. 42 Figure 2.5 Biodistribution of [18F]-ALK-BBN-PEG and [18F]-PEG-BBN-PEG in the (A) tumor and (B) pancreas at 1 h post-injection. ........................................................................... 42 Figure 2.6 Tumor to blood ratios of [18F]-ALK-BBN-PEG and [18F]-PEG-BBN-PEG at 15, 30, and 60 min post-injection. .................................................................................................. 43 xvi  Figure 2.7 Dynamic PET/CT imaging of [18F]-ALK-BBN in PC-3 tumor-bearing mice at 1 h- post-injection.  .......................................................................................................................... 44 Figure 2.8 Dynamic PET/CT imaging of [18F]-ALK-BBN-PEG in PC-3 tumor-bearing mice at (A) 20 min-unblocked, (B) 1 h-unblocked , and (C) preblocked 1h post-injection. . .............. 44 Figure 2.9 Dynamic µPET/CT imaging of [18F]-PEG-BBN-PEG in PC-3 tumor-bearing mice at (A) 20 min-unblocked, (B) 1 h-unblocked , and (C) co-injection 1 h post-injection.  ......... 45 Figure 2.10 Time activity curves of PC-3 tumors over 1 h imaging of [18F]-ALK-BBN-PEG (A), and [18F]-PEG-BBN-PEG (B). .......................................................................................... 46 Figure 2.11 Time activity curves of blood over 1 h imaging of [18F]-ALK-BBN-PEG (A), and [18F]-PEG-BBN-PEG (B). ........................................................................................................ 46 Figure 3.1 A representative displacement curve of [125I-Tyr4]bombesin by AmBF3-MJ9. . ... 60 Figure 3.2 Radio-HPLC analysis of purified 18F-AmBF3-MJ9. Only one single peak was observed on both radio- (upper) and UV-VIS (lower) chromatograms. .................................. 61 Figure 3.3 Radio-HPLC analysis of metabolic stability of 18F-AmBF3-MJ9 in mouse plasma after 0 (A), 1 (B), and 2 h (C) incubation at 37 ˚C. .................................................................. 62 Figure 3.4 Percentages of membrane-bound and internalized radioactivity after incubating 18F-AmBF3-MJ9 with PC-3 cells for up to 90 min. ........................................................................ 63 Figure 3.5 Small-animal PET/CT images of 18F-AmBF3-MJ9 in PC-3 prostate tumor bearing mice at 1 h (A) and 2 h (B) p.i., and at 1 h p.i. with pre-injection of 100 µg of BAY86-7548 (C). . .......................................................................................................................................... 66 Figure 4.1 Representative example of a competitive binding assay for 19F-AmBF3-TATE-: x-axis: log[AmBF3-TATE], y-axis: counts bound. . .................................................................... 81 xvii  Figure 4.2 HPLC traces of Sep-Pak purified 18F-AmBF3-TATE; top – the UV trace measured at 277 nm; bottom – radioactivity trace. ................................................................................... 82 Figure 4.3 Plasma stability assay of 18F-AmBF3-TATE; radiotraces shown for 0, 60 and 120 min. ........................................................................................................................................... 83 Figure 4.4 Internalization of 18F-AmBF3-TATE in AR42J cells in 120 min incubation. ........ 84 Figure 4.5  PET/CT (up) and PET (low) images of 18F-AmBF3-TATE PET using AR42J tumor-bearing mice at 60 min post-injection: unblocked (left) and blocked (right). . ............. 87 Figure 4.6 Time activity curves of tumor in unblocked and blocked imaging studies with 18F-AmBF3-TATE. ......................................................................................................................... 88 Figure 4.7 (A)Time activity curves indicating blood, liver, kidney clearance and tumor uptake, and (B) uptake of hottest clusters. ............................................................................................ 89 Figure 5.1 Competitive binding curve of (A) NOTA-BASS and (B) Ga-NOTA-BASS in SST2a membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11. .................................. 108 Figure 5.2 Competitive binding curve of Ga-DOTA-TATE in SST2a membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11. ........................................................................... 109 Figure 5.3 Competitive binding curve of Ga-NODAGA-LM3 in SST2a membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11. ........................................................................... 109 Figure 5.4 Competitive binding curve of Ga-DOTA-TATE in Zr-75-1 breast tumor membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11............................................... 110 Figure 5.5 Competitive binding curve of Ga-DOTA-TATE in MCF-7 breast tumor membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11............................................... 110 Figure 5.6 internalization results of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 in HEK-SST2a cells at 15, 30, 60, 120 min incubation at 37 ⁰C. ........................................................ 111 xviii  Figure 5.7 Flow cytometry analysis of HEK-SST2a cells gated on the living cells. ............. 112 Figure 5.8 Flow cytometry analysis of Zr-75-1 cells gated on the living cells. ..................... 113 Figure 5.9 Flow cytometry analysis of MCF-7 cells gated on the living cells. ...................... 114 Figure 5.10 Flow cytometry analysis of HEK-SST2a, Zr-75-1, and MCF-7 cells................. 114 Figure 5.11  Small-animal PET/CT images of 68Ga-DOTA-TATE at 1 h after injection showing potential of agonist radiotracers for in vivo imaging of Zr-75-1 breast tumors with an excellent tumor to background contrast. Left: unblocked, Right: blocked, B: bladder, K: kidney, T: tumor, I: intestine. ................................................................................................. 120 Figure 5.12 Small-animal PET/CT images of 68Ga-NODAGA-LM3 at 1 h after injection showing potential of antagonist radiotracers for in vivo imaging of Zr-75-1 breast tumors with an excellent tumor to background contrast. Left: unblocked, Right: blocked, B: bladder, K: kidney, T: tumor, I: intestine. ................................................................................................. 121 Figure 5.13 Small-animal PET/CT images of 68Ga-DOTA-TATE (left) and 68Ga-NODAGA-LM3 (right) in HEK293-SST2a tumor bearing mice 1 hour post-injection showing very good tumor to background contrast. ................................................................................................ 122 Figure 5.14 An example of a time activity curve of tumors in dynamic 68Ga-DOTA-TATE imaging. .................................................................................................................................. 123 Figure 5.15 An example of a time activity curve of 68Ga-DOTA-TATE in kidney, blood, and liver in Zr-75-1 tumor bearing mice. ...................................................................................... 123 Figure 5.16 An example of time activity curve of Zr-75-1 tumor in dynamic 68Ga-NODAGA-LM3 imaging. ......................................................................................................................... 124 Figure 5.17 Time activity curves of blood, liver and kidney in Zr-75-1 tumor-bearing mice with 68Ga-NODAGA-LM3. .................................................................................................... 124 xix  Figure 6.1 Saturation binding assays of (A) SK-N-MC neuroblastoma tumor cells and (B) MCF-7 human breast cancer cell line. .................................................................................... 140 Figure 6.2 Biodistribution of [18F]-ALK-BVD15 in SK-N-MC tumor-bearing mice 1 h post-inj (n=5). ................................................................................................................................. 142 Figure 6.3 Biodistribution of [18F]-ALK-BVD15 in MCF-7 tumor-bearing mice 1 h post-inj (unblocked: n=7, blocked: n=3). ............................................................................................ 142 Figure 6.4 Unblocked dynamic PET/CT imaging of [18F]-ALK-BVD15 in MCF-7 tumor-bearing mice at 1 hour post-inj. (A) Fused PET/CT, (B) PET image, I: Intestine, L: liver , T: tumor. ...................................................................................................................................... 143 Figure 6.5 Unblocked dynamic PET/CT imaging of [18F]-ALK-BVD15 in SK-N-MC tumor-bearing mice at 1 hour post-inj. (A) Fused PET/CT, (B) PET image, B: Bladder, I: Intestine, L: liver , T: tumor. .................................................................................................................. 143 Figure 6.6 Immunohistochemical staining of neuroblastoma SK-N-MC tumor, X400. ........ 144 Figure 6.7 19F-ALK-BVD15 peptide and decomposition peaks at 15, 60 and 120 min incubation in mouse plasma. .................................................................................................. 145 Figure 6.8 Plasma stability of 19F-ALK-BVD15 demonstrating the half life of the peptide in mouse plasma. ........................................................................................................................ 146 Figure 6.9  19F-ALK-BVD15 is cleaved in two steps, from arginine, and isoleucine. ........... 146 xx   List of Schemes  Scheme 2.1 Radiosynthesis of [18F]-ALK-BBN by click chemistry approach. ....................... 28 Scheme 2.2  Radiosynthesis of [18F]-ALK-BBN-PEG by click chemistry approach. ............. 29 Scheme 2.3 Radiosynthesis of [18F]-PEG-BBN-PEG by click chemistry approach. ............... 29 Scheme 3.1 Synthesis of AmBF3-MJ9 and radiosynthesis of 18F-AmBF3-MJ9. ..................... 60 Scheme 4.1 N3-TATE is condensed with N-propargyl-N,N-dimethyl-ammoniomethyltrifluoroborate (1) to provide the precursor AmBF3-TATE (2).  Precursor 2 is labeled by isotope exchange to provide the isotopolog 18F-2 at high specific activity for tracer studies. ...................................................................................................................................... 76 Scheme 5.1 Structures of three somatostatin derivatives. ...................................................... 107 Scheme 6.1 Radiosynthesis of [18F]-AK-BVD15 by click chemistry. ................................... 133  xxi  List of Abbreviations and Symbols  %ID/g   Percentage of the injected dose per gram of tissue β+   Positron µ   Micro ˚C   Degree Celsius ACE    Angiotensin conversion enzyme  BASS   A somatostatin antagonist BBN   Bombesin Bq   Becquerel BSA   Bovine serum albumin C   Carbon Ci   Curie CT                               Computed tomography CTA    Clinical Trial Application CTAG   BCCA’s Centre for Translational and Applied Genomics Cu   Copper DCFBC   N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine  DIEA   N,N-Diisopropylethylamine DMEM  Culture media DMF   N,N-Dimethylformamide DMSO   Dimethylesulfoxide DOTA   1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid xxii  DRE    Digital rectal examination ER   Estrogen receptor F   Fluorine FBS   Fetal bovine serum FDA    US Food and Drug Administration FDG   Fluorodeoxyglucose Fmoc   9-Fluorenylmethyloxycarbonyl GPCR   G protein coupled receptor GRPR   Gastrin releasing peptide receptor h   Hour (s) HDP   99mTc-hydroxymethylene diphosphonate HEK293  Human embryonic kidney cell line HPLC   High performance liquid chromatography IC50   half-maximal inhibitory concentration  IHC   Immunohistochemistry In   Indium kBq/cc   Kilo Becquerel per cubic centimeter Kd   Affinity constant keV   Kilo electron volts Ki   Inhibition constant  LM3   A somatostatin antagonist mA   Milliampere mAb    Monoclonal antibody xxiii  MBq   Mega becquerel MDP    99mTc-Methyl diphosphonate MeCN   Acetonitrile MRI   Magnetic Resonance Imaging mRNA   Messenger RNA N   Nitrogen n   Nano NaOH                          Sodium hydroxide NET                             Neuroendocrine tumour NOTA   1,4,7-triazacyclononane-1,4,7-triacetic acid NODAGA  1,4,7-Triazonine-1(2-carboxyethyl),4,7-triacetic acid NPY   Neuropeptide Y PBS   Phosphate buffered saline PET   Positron emission tomography p.i.   Post-injection PSA   Prostate specific antigen PSMA    Prostate-specific membrane  qPCR   Quantitative polymerase chain reaction RNA   Ribonucleic acid ROI   Region of interest RT-PCR   Reverse transcriptase polymerase chain reaction  SD   Standard deviation SPECT  Single photon emission computed tomography xxiv  SST1-5  Somatostatin receptors 1 through 5 SST2a   Somatostatin receptor 2, isoform  Tc   Technetium TFA   Trifluoroacetic acid TRIUMF  Canada’s national laboratory for particle physics    xxv  Acknowledgements  This thesis would not have been possible without the guidance and the help of a number of individuals who contributed in different ways and extended their valuable assistance in the preparation and completion of this study. First and foremost, my highest gratitude must go to my supervisor, Professor Dr. François Bénard, for his immense knowledge and vision, guidance, inspiration, support and compassion. I could not have asked for a better supervisor. I wish to thank my committee, Drs. Anna Celler, Marcel Bally, and Urs Hafeli, for their guidance and feedbacks. Dr. Kuo-Shyan Lin for his help and advice and being always available for my never-ending chemistry questions. Also, many thanks to the collaborators, Drs. Brigitte Guerin, Tom Ruth, David Perrin, James Inkster, and Zhibo Liu. Special thanks to the “Bénard lab”; Nav and Nadine for their help with PET imaging and being the best companion in crazy long days of animal studies. Jinhe and Johnson for their help with radiolabeling. Silvia, Gemma, Joseph, Guillaume, Chengcheng, Iulia and Hwan for all their help, support and friendship that made these six years so memorable. Jennifer, Mike, Wade, Julius at the cyclotron operation site. I would also like to thank the former members of the Bénard lab, Felix and Simon for their initial help in this project.  My sincere appreciation goes to Teresa and Gayle who have always helped me with the mice, and the members of the department of Molecular Oncology. xxvi  My enduring gratitude goes to my formers supervisors in the Netherland: Raymond, Adrian, Conchita, Connie and Fiona who introduced me to research, inspired and encouraged me to continue my studies in this field. And last but not the least, I cannot thank enough my mom and dad, and my brother who have always believed in me, made many sacrifices and supported me throughout my life by all means, especially during the years I have been abroad to pursue a higher education.  I would like to thank Mahbod for encouraging me to follow my dreams; Ali, for his unconditional support, encouragement and patience specially during the last stressful months; Mani for all his follow-ups, help, feedbacks and motivation; Amal and Sharmin for giving me hope and advice during the writing process; Aghigh for always being there for me even from far away; and all my other friends in Canada and around the globe for their moral support in these challenging years.       xxvii  Dedication        To my parents, for their endless encouragement and support during my academic journey1  Chapter 1: Introduction  1.1 Molecular imaging in nuclear medicine Molecular imaging is a medical imaging technique that provides detailed functional information of biochemical events in the body. This technique therefore serves as a crucial tool for understanding chemical and biological processes within the body. In contrast to medical imaging modalities such as X-rays, computed tomography (CT), and ultrasound allow physicians to study physical structures, molecular imaging detects and measures metabolic changes in the body by gamma cameras. The current strategy used in molecular imaging is to first identify a target molecule in a specific tissue or its disease state in a living organism. A high-affinity probe to this target is then developed, and  used to detect the distribution and pharmacodynamics of the molecule [1] .  Various techniques used in molecular imaging include: Magnetic Resonance Imaging (MRI), optical imaging, ultrasound, Raman spectroscopy, and nuclear medicine techniques. Here, only the application of molecular imaging in nuclear medicine will be discussed. Molecular imaging in the field of nuclear medicine employs use of small amounts of radioactive materials called radiopharmaceuticals or radiotracers. The use of suitable radiotracers or specific probes facilitates the detection and characterization of malignancies under physiological and pathological conditions, making it applicable in the field of oncology for the diagnosis or treatment of cancer. Two major imaging modalities in nuclear medicine for cancer imaging are Positron Emission Tomography (PET) and Single Photon Emission Tomography (SPECT). The radiopharmaceuticals used for molecular imaging are usually 2  analogs to some natural chemical compounds within the body. A well-known example of such analogs is a glucose analog, 2-deoxy-2-[18F]fluoro-D-g1ucose or 18F-FDG, as a no-carrier added radiopharmaceutical being employed for diagnostic purposes in PET. As a glucose analog, 18F-FDG is taken up by normal cells with high glucose consumption such as those in the brain, heart, and kidney. It can also be taken up by abnormal cells, and thereby identify regions with abnormal glucose metabolism, which is frequently the case in cancer cells. 1.2 Positron emission tomography (PET) PET was introduced in the late 1950’s as a functional imaging technique. PET is based on detection in coincidence of the two 511-keV annihilation photons that originate from β+ decay of radiotracer that is injected into the patients. Positrons are annihilated in body tissue and produce two 511-keV annihilation photons that are emitted in opposite directions. These two photons are detected by two detectors in coincidence. Data collected over many angles around the patient are used to reconstruct the image of the activity distribution in the section of interest (Figure 1.1). The 511-keV photons are converted to light photons in the detector and a pulse is formed in photo-multiplier (PM) tubes, or silicon photomultipliers, or avalanche photodiodes,  and analyzed in a similar manner to conventional gamma cameras [1].     3   Figure 1.1 Positron annihilation and detection by PET (Adopted from Basics of PET imaging 2005, and  Vander Veldt et al. 2013 )[2, 3].  To provide a more accurate diagnosis, dual modality scanners such as PET/CT scanners have been developed, in which functional PET images are fused with anatomical CT images. Both scanners are mounted on a common gantry with the CT unit in the front and the PET unit in the back. For research animal imaging, micro PET scanners with a smaller bore and therefore a better spatial resolution of 1-2 mm are used. An example of a micro PET/CT for preclinical applications is shown in Figure 1.2.  4   Figure 1.2 An example of a micro PET/CT for small animal imaging.  Combined PET/CT instruments have improved diagnosis for several malignancies such as lung cancer, lymphoma and colorectal cancer [4-9].  In order to develop the most effective radiopharmaceutical, it is necessary to consider physical properties of a radionuclide and to identify the right target. Whereas long-lived isotopes are used for therapy in nuclear medicine, short-lived isotopes are beneficial for diagnostic purposes. The characteristics of the most common radionuclides used in PET studies are summarized in Table 1.1.   5  Table 1.1 The characteristics of the most common radionuclides used in PET. (Adopted from Basics of PET imaging by Gopal B. Saha) [2]           Nuclide Physical  Mode of γ-ray Abundance half-life  decay energy (%) (t1/2) (%) (keV)   11C 20.4 min β+ (100) 511 200 13N 10 min β+ (100) 511 200 15O 2 min β+ (100) 511 200 18F 110 min β+ (97)  511 194   EC (3)   68Ga 68 min β+ (89)  511 178   EC (11)   94mTc 52 min β+ (70)  511 140   EC (30) 871 94    1521 4.5    1868 5.7 124I 4.2 d β+ (23)  511 46   EC (77) 603 61    1691 10.4 82Rb 75 s β+ (95)  511 190   EC (5) 777 13.4  64Cu 12.7 h β+ (61) 653  18   β- (39) 578 37       Short-lived radionuclides include 11C, 13N, 68Ga, 18F, and 64Cu. PET isotopes 18F and 68Ga, have half-lives of 110 and 68 minutes, respectively. This provides enough time after radiosynthesis for administration to the patient and the distribution of the isotope in target tissues by blood circulation. In addition to the half-life of the isotopes, nuclear  6  properties such as decay mode and their decay energy make them suitable for preclinical and clinical imaging of cancers.  1.3 Management of low metabolic rate cancers in nuclear medicine As mentioned in section 1.1, 18F-FDG is a very sensitive PET radiopharmaceutical currently used for diagnosis and treatment follow-ups in the clinic. Despite the fact that this radiopharmaceutical can detect rapidly growing cancers with high metabolic rate of glucose utilization, it is not a cancer-specific tracer. 18F-FDG uptake has been described in a number of non-neoplastic inflammatory lesions like sarcoidosis, tuberculosis, fungal infection and abscesses [10, 11]. In addition, its specificity and sensitivity for tumors with low metabolic rate, such as lobular breast carcinoma and prostate cancer, is limited [12, 13]. Thus, it is very important to develop a PET radiopharmaceutical that specifically targets and detects such cancers for which the application of 18F-FDG is limited. 1.3.1 Prostate cancer Prostate cancer is the most common non-skin cancer among men worldwide [14]; in the United States it is the second leading cause of cancer mortality in men after lung cancer [15]. Given that the disease is age-related and that life expectancy is on the rise, its incidence is bound to increase and become a major health problem [16]. Therefore, early detection of prostate cancer is essential for successful management of this disease [17]. Screening has been promoted as a way of detecting prostate cancer in the early stages, helping to decrease overall and disease-specific mortality [18]. The standard screening method for early detection of prostate cancer that was introduced in the clinic in 1980’s is screening for a prostate specific 7  antigen (PSA). PSA screening can be done with or without digital rectal examination (DRE). However, due to its low specificity, PSA does not differentiate between a low and a high grade of cancer. This insensitivity is a cause for concern, as it increases the potential for over-diagnosis and overtreatment, which would negatively affect the quality of life of patients [19]. Functional imaging techniques such as PET, SPECT, and MRI are promising tools in the management of prostate cancer, especially during therapy [17]. However, the lack of a PET or SPECT radiopharmaceutical that can help manage prostate cancer from its early detection through to the staging and therapy phase poses a serious challenge towards this end [17].  1.3.1.1 Common procedures in nuclear medicine for detection of prostate cancer 1.3.1.1.1 PSMA targeting agents The prostate specific membrane antigen (PSMA) is overexpressed in malignant prostate cancer and is the most investigated imaging agent for prostate cancer [20-23]. PSMA targeting by monoclonal antibody (mAb) is well studied in small animal tumor models and also in clinical studies [17]. 111In-capromab also called 111In-Prostascint is the only FDA approved radiolabeled monoclonal antibody for PSMA targeting (Figure 1.3) [24, 25]. It targets the intracellular epitope of PSMA and has shown potential in localizing the metastasis in soft tissues [26]. Another mAb targeting PSMA that is studied in phased clinical trials is 111In-J591 that targets the extracellular epitope of PSMA [27-32]. 111In-J591 has been considered a superior anti-PSMA imaging agent compared to the other mAb targeting agents [27]. N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine (DCFBC) is a low-molecular-weight, urea-based inhibitor of PSMA which targets a more accessible, external binding domain of 8  PSMA, rather than an intracellular domain. The first in-human clinical study with this novel PSMA targeting agent showed potential in detection of metastatic prostate cancer [33]. 1.3.1.1.2 Non-prostate specific agents Metabolic PET imaging agents such as 18F-FDG, 11C/18F-Choline (Figure 1.4), and 11C/18F-Acetate are used for the detection of glucose, choline and acetate metabolism, respectively, to evaluate the occurrence of distant metastasis in prostate cancer. There is limited expression of glucose transport 1 in primary prostate tumor cells, therefore, accumulation of 18F-FDG in prostate cancer sites is limited [12]. The prostate tumor is usually characterized by increased choline metabolism in the cells [34].11C-Choline is a PET imaging agent that is used for recurrent prostate cancer and its metastasis [35]. The prostate tumor is usually characterized by increased choline metabolism in the cells [34]. In prostate epithelial cells, a metabolic transition from citrate producing normal cells to citrate oxidizing malignant cells occurs that also leads to an increased turnover of acetate [12]. This prostate specific citrate metabolism could contribute to high accumulation of 11C-Acetate in prostate cancer cells and its metastasis [36-39]. 18F labeled version of Choline and Acetate [40-42] is available when there is no onsite cyclotron in a clinical setting. However, because of the differences in pharmacokinetics of 11C and 18F labeled compounds the results from these two radiopharmaceuticals are not the same [17]. 1.3.1.1.3 Androgen receptor imaging agents Dihydrotestostrone is directly associated with expression of androgen receptors that play an important role in tumor growth even in castration-resistant prostate cancer [43, 44]. 18F-FDHT 9  is a PET imaging radiotracer that targets androgen receptors and has shown promise in monitoring the effects of anti-androgen therapy in prostate cancer [17]. 1.3.1.1.4 Protein synthesis imaging agents Radiolabeled amino acids such as 11C-Methionine could detect protein synthesis and amino acid transport in tumor proliferation [45] and are used as  PET imaging agents for prostate cancer detection. In recent years the investigational synthetic L-leucine analogue (anti1 amino-3-18F-fluorocyclobutane-1-carboxylic acid, in brief 18F-FACBC) has been proposed as a possible alternative radiopharmaceutical to detect prostate cancer relapse [46]. 1.3.1.1.5 Bone imaging agents Whole body SPECT or PET scans are performed in prostate cancer imaging for monitoring progression of metastatic bone disease.  99mTc-phosphonate such as MDP and HDP are SPECT radiotracers that are absorbed into the bone matrix. The exchange rate of calcium phosphate and phosphonate tracer in bone matrix shows osteoblast activity, which can in turn be indicative of bone metastasis [17].  A PET tracer that works in a similar manner and rapidly absorbs into the bone matrix is 18F-NaF. 18F-NaF imaging reveals fluoride ion uptake by the bone, which indicates abnormalities that are related to bone metabolic disorders, including bone metastasis in prostate cancer [17].  10    Figure 1.3 A 1.5 × 0.7-cm peripancreatic lymph node uptake of 111In-capromab pendetide (ProstaScint) visualized by single photon emission computed tomography (SPECT) combined with 16-slice multidetector computed tomography (CT) (Precedence, Philips Healthcare, Andover, MA) scanner. Transverse images of SPECT alone (left); CT alone (middle), and SPECT/CT fusion (right). Arrows indicate where the 111In-ProstaScint uptake is in relation to its anatomical location from SPECT, CT, and SPECT/CT images [47].     Figure 1.4 11C-choline positron emission tomography/CT showing focal (A) and multifocal (B) lesion distribution of prostate cancer within the gland (arrows) [48].   11  1.3.2 Breast cancer Breast cancer is the most common type of cancer in women after skin cancer, and is a major cause of mortality and morbidity among women worldwide. The risk of breast cancer increases with age and it is more common after the age of 40 [49]. According to the American cancer society, about 232,340 new cases of invasive breast cancer will be diagnosed among women in 2013. In addition, 64,640 cases of in situ breast cancer, and about 39,620 deaths from breast cancer are estimated (Table 1.2). Breast cancer is the second cause of cancer death after lung cancer [49].   Table 1.2 Age-specific female breast cancer incidence and mortality rates in the US in 2013 (compiled from American Cancer Society, Surveillance and Health Services Research, 2013)          Age In-situ cases Invasive cases Mortality     < 40 1900 10980 1020 < 50 65048 9104 780 50-64 26770 84210 11970 > 65 22220 99220 22870 All ages 64640 232340 39620         Source: Total estimated cases are based on 1995-2009 incidence rates from 49 states as reported by the North American Association for Central Cancer Registries. Total estimated deaths are based on data from US Mortality Data, 1995-2009, National Center for Health Statistics, Centers for Disease Control and Prevention.      Imaging plays a key role at all stages of breast cancer treatment, from screening of symptomatic and asymptomatic cases to the treatment of those with an established tumor. 12  After diagnosis, imaging has a major role in tumor staging and treatment assessment of breast cancer [13]. Mammography is the most widely used imaging modality for early detection of breast cancer in women after the age of 40. Screening mammography identifies suggestive areas, and focused mammographic images are used for characterizing them [50].  However, this technique is not sensitive and specific in younger women who have dense breast tissues [51]. Moreover, mammography has a high false-positive rate, requiring further imaging before proceeding to biopsy [52]. Ultrasound and Magnetic Resonance Imaging (MRI) are the secondary imaging modalities in detection of breast lesions. Breast ultrasound has a specificity of only 34% for breast cancers [53]. The application of MRI has been studied in younger women and has shown small lesions of 2-3 mm can be detected by MRI. However, it is not always able to characterize the nature of the lesion even with the use of contrast agents, which indicates poor specificity as its major limitation. Such limitations in the detection of breast lesions by common imaging modalities motivate the application of nuclear medicine to breast cancer diagnosis. Nuclear medicine techniques can provide functional information along with anatomical information for diagnosis of breast cancer, which can be advantageous over conventional imaging modalities [54, 55]. 1.3.2.1 Common procedures in nuclear medicine for detection of breast cancer 1.3.2.1.1 SPECT imaging Imaging techniques such as bone scintigraphy with 99mTc-phosphonate are used to identify site of cancer and bone metastasis [56]. In symptomatic patients scintimammography is applied where radiopharmaceuticals such as 99mTc-MIBI and 99mTc-tetrofosmin are used [13]. 99mTc-MDP scintimammography can be used to identify the extent of progression of a diagnosed 13  case of breast cancer, and also to identify bone metastasis via bone scans. In addition, researchers have reported that the myocardial imaging agent 99mTc-sestamibi is taken up in breast tumors [57, 58]. 1.3.2.1.2 PET imaging PET has low accuracy in detection of non-palpable breast tumors or lesions smaller than 8 mm [13]. 18F-FDG uptake is seen in breast tumors especially those that are more advanced [59]. However, the degree of such uptake can be limited in some well-differentiated breast tumors and lobular carcinomas [13]. False-positive results have been reported in infectious and inflammatory lesions due to the accumulation of FDG in those cells. Although 18F-FDG has limitation in diagnosis of breast cancer, it has great potential for tumor staging and detection of soft tissue lesions and nodal, skeletal, and visceral metastasis [60]. Clinical studies have shown 18F-FDG is a superior technique to conventional imaging procedures in localizing tumor lesions at the site of cancer [55].  Two other radiotracers used in breast cancer applications are 18F-fluoroestradiol and 18F-fluoro-L-thymidine. 18F-fluoroestradiol is an estrogen analog used to demonstrate the estrogen status of a breast cancer [50].18F-fluoro-L-thymidine uptake by cells indicates cell proliferation, and therefore serves as a primary indicator of tumor aggressiveness. Therefore, 18F-fluoro-L-thymidine may be an excellent marker of response to breast cancer therapy [61]. 1.4 Regulatory peptides and peptide receptors Peptides are comprised of a few to dozens of amino acids linked with peptide bonds. In comparison to antibodies, peptides have smaller size, less than 100 amino acids with a 14  molecular mass of < 10000 Daltons. In nuclear medicine small peptides with less than 30 amino acids and molecular mass of < 3500 Da have been the most favorable [62]. In comparison to antibodies, they show lower or no toxicity and immunogenicity, faster clearance from blood and target tissues, and more rapid tissue and tumor permeability [63]. Unlike proteins, peptides generally do not have a tertiary structure. Moreover, they can exist naturally or be synthesized as novel molecules and are easy to link to chelators and be radiolabeled [64]. Due to the mentioned advantages of peptides over other proteins or antibodies, there has been a significant growth in the development of radiolabeled peptides in recent years [65-67].   Regulatory peptides have earned increasing attention as a class of molecules developed for cancer specific targeting in cancer imaging. That is mainly due to them playing a modulatory role in various regions of body such as the brain, gastrointestinal tract, endocrine, vascular, or lymphoid systems at very low concentrations [68]. Regulatory peptides mediate their functions usually through G-protein–coupled receptors (GPCR) [66].  Peptides can play a role in important intracellular systems. For instance their role in MAPK pathways is known in cell proliferation and apoptosis. Also, they not only play a role in normal conditions but also in pathological processes such as inflammation and cancer [69, 70] , which garners them further attention in the field of cancer research. Peptide receptors are often expressed in many primary human cancers and in some cases they are overexpressed in tumors, in comparison to their expression in normal tissue adjacent to the neoplasm and/or in its normal tissue of origin [71]. This is the key reason why regulatory peptide receptors have recently become very important in cancer imaging [72]. 15  The expression of peptide receptors has been demonstrated in different tumors by means of different techniques. Radioligand binding studies, immunohistochemistry, in situ hybridization, Northern blotting, RNAse protection and (quantitative) reverse transcriptase polymerase chain reaction (RT-PCR) are the most common among these techniques [73].  Some of the most important regulatory peptides in molecular imaging, and their corresponding receptors that are overexpressed on tumors are listed in Table 1.3 [74]. Gastrin releasing peptide receptors (GRPR), Somatostatin receptors, and neuropeptide Y1 receptors (NPY1R), are the mainly receptors focused on in this thesis as they have shown to be significantly overexpressed in many cancers, namely prostate and breast cancer [66]. Bombesin derivatives that bind to the gastrin releasing peptide receptors (GRPRs) have been broadly investigated in nuclear medicine. GRP receptors are overexpressed in several types of cancer such as prostate cancer, breast cancer, lung cancer and gastrointestinal cancers [75-80]. Mattei et al., 2014 reported that high densities of GRPR are present in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) biopsy specimens and cell lines [81].  Carrol et al. showed that the GRPR regulates colon cancer cellular differentiation and impairs cellular metastasis [82]. It is also reported that transactivation of the epidermal growth factor receptor in head and neck squamous cell carcinoma is regulated by GRPR [83]. Human endocrine tumors often express the somatostatin receptors (SSTR 1–5) with different intensities among individual tumors. The expression of somatostatin receptors has been well studied for their distribution in pituitary adenomas, brain tumors, adrenal tumors and neuroendocrine tumors in gastrointestinal tract (NET) [84]. Somatostatin receptor subtype 2a (SST2a) is the most frequently expressed SSR subtype in human somatostatin receptor 16  positive tumors. Several studies confirmed a high incidence of SST2A-expressing tumors by using antibodies against synthetic peptide sequences of somatostatin receptor subtypes [85-88]. Schultz et al. demonstrated that the level and the pattern of somatostatin receptor expression very much varied between individual breast carcinomas, but that SST2a was present in 85% of the breast tumors [86]. Neuropeptide Y (NPY) receptors are primarily expressed in some of the endocrine tumors, epithelial cancers, and also in embryonal tumors [89]. Presence of NPY receptors are seen in steroid hormone producing tumors, such as adrenal cortical adenomas, carcinomas,  ovarian granulosa cell tumors, Sertoli-Leydig cell tumors, and in catecholamine producing tumors such as pheochromocytomas and paragangliomas [90, 91]. Among the epithelial cancers tested to date, an NPY receptor expression is found mainly in breast carcinomas, renal cell carcinomas, and ovarian adenocarcinomas [90, 92, 93]. The expression of NPY receptors is extremely high in incidence and shows the highest density of NPY expression in breast carcinomas compared to the other NPY positive tumors.  Renal cell carcinomas express NPY receptors in moderate incidence and density compared to breast carcinoma, and ovarian adenocarcinomas show a relatively low NPY receptor expression [90].  Embryonal tumors, especially neuroblastomas and nephroblastomas, are characterized by a high NPY receptor incidence and a moderate receptor density [91, 93].    17  Table 1.3 Peptide receptor expression patterns.  Common peptide receptors expressed in human cancers are in bold (Adopted from Fani et al. 2012). Peptide Receptor subtypes Tumor expression Somatostatin sst1, sst2, sst3, sst4,sst5 Neuroendocrine tumors (gas-troenteropancreatic tumors), lymphoma, paraganglioma, carcinoids, breast, brain, renal, small cell lung cancer, me-dullary thyroid cancer Bombesin/GRP  (Gastrin releasing peptide)  BB1 (NMB-R), BB2 (GRPR), BB3, BB4 Prostate, breast, pancreas, gastric, colorectal,  small cell lung cancer VIP (Vasoactin intestinal peptide) VPAC1, VPAC2 Adenocarcinomas of breast, prostate, stomach and liver; neuroendocrine tumors CCK (cholecystokinin) /Gastrin CCK1, CCK2 Medullary thyroid cancer, small cell lung cancer, gas-trointestinal stromal tumor, stromal ovarian cancer,  astrocytomas Neurotensin NTR1, NTR2, NTR3 Small cell lung cancer, colon, exocrine ductal pancreatic cancer, Ewing sarcoma, meningioma, astrocytoma, breast, prostate cancer Neuropeptide Y Y1, Y2,Y3, Y,4, Y5 Breast, renal, ovarian carcinoma, Neuroblastoma tumor RGD (Arg-Gly-Asp) αvβ3-integrin Glioma, breast, prostate cancer Extendin Glucagon-like peptide 1             (GLP-1) Insulinomas, gastrinomas, pheochromocytomas, para-gangliomas and medullary thyroid carcinomas LHRH (luteinizing hormone-releasing hormone) LHRH-R Prostate, breast cancer Substance P NK1, NK2, NK3 Glial tumors (glioblastoma, medullary thyroid cancer), pancreas, breast, small cell lung cancer (α-MSH) α-melanocyte-stimulating hormone  MC1-5R Melanoma α-M2 α-M2-R Breast cancer 18  1.5 Peptide-based radiopharmaceuticals for cancer imaging  Molecular imaging techniques are increasingly being used in different aspects of cancer management, including the localization and staging of disease and in therapy follow-up [74]. By far the most sensitive imaging methods are the ones using nuclear probes for SPECT and PET, for which a variety of imaging probes have been developed [74]. Peptide based probes (peptide based radiopharmaceuticals) were introduced into the clinic more than two decades ago [94, 95]. In peptide based cancer imaging, a stable peptide analog is linked to a chelator that can bind PET or SPECT radioisotopes such as 18F, 68Ga, 111In, 99mTc, 64Cu, etc. The radiolabeled peptide is intravenously injected into a patient with a potential tumor, distributing the radiotracer through the body. If the patient has a tumor expressing specific peptide receptors in high quantities, the radiotracer will selectively bind to these receptors and will be taken up by the cells via receptor-ligand internalization. Internalization will result in accumulation of radioactivity in the tumor in comparison to the rest of normal tissues in the body, which can be detected by a nuclear medicine technique such as PET [71]. The principle of peptide receptor targeting of cancer is shown in Figure 1.5. Usually, uptake in the tumor is rapid and specific. However, due to the urinary excretion of the radiotracer, uptake in kidney and bladder is also observed [72]. Small tumors of few millimeters in diameter are often successfully identified, suggesting the excellent sensitivity of this type of cancer imaging [72].  19    Figure 1.5 Principle of in vivo peptide receptor targeting of cancer. The radiolabeled peptide (P) is injected intravenously into the patient and distributed in the body. If the patient has a tumor with cancer cells expressing the corresponding peptide receptor (P-R), the radiopeptide will bind to it and internalize with the receptor into the cell (arrows) where the radioactivity will accumulate. Imaging by a nuclear medicine technique such as PET will detect the radioactivity accumulated in the tumor, whereas the remaining radioactivity in the body will be cleared through the kidneys (Adopted from Ruebi 2003).  1.5.1 Development of peptide-based radiopharmaceuticals Designing a peptide based probe for molecular imaging is challenging [96]. Since most receptors have high affinity for their ligands and are active at nanomolar concentrations, it is crucial to have a radiopharmaceutical with high specific activity. Even very low concentrations of imaging agents may saturate a receptor, leading to a deteriorated visualization of receptor expression and an increase in non-specific binding [74]. Therefore, micro molar to picomolar concentrations of imaging probes are often favorable in nuclear medicine [96]. It is also important to use a minimum amount of peptide to reduce any possible pharmacologic side-effects in humans [96]. Other important factors to be considered include the choice of radionuclide, and the labeling position or location of the radiolabel. This is 20  because when smaller biomolecules such as peptides are used, radiometals may affect binding to receptor and metabolism in vivo [96].  To prevent the loss of binding affinity and biological activity of the radiolabeled peptide, it is important for the chelating site to be far from the binding site [64, 97]. Unmodified peptides mostly have a short biological half-life due to rapid proteolysis in plasma. This is one of the major obstacles in successful in vivo applications. To minimize rapid degradation of radiopharmaceuticals before reaching the target tissues, most peptides need to be synthetically modified [64, 98]. Lots of research has been dedicated to developing suitable metabolically stable peptides for clinical application.  The use of more stable D-amino acids for L-amino acids, the use of pseudo-peptide bonds, the inclusion of amino alcohols and insertion of unnatural amino acids or amino acid residues with modified side chains, are a few examples of molecular modifications used for making a more stable peptide without compromising the receptor binding affinity and biological activity of the peptide [64]. For instance, natural somatostatin has a very short half-life of 2-3 minutes in plasma, whereas its modified synthetic peptide derivative, octreotide has a half-life of 1.5-2 hours making it suitable for clinical use [96]. Another concern related with radiolabeled peptides is their often high uptake and retention by the kidneys and potential nephrotoxicity for therapeutic peptides [99-102]. Some procedures have been successfully developed and applied to manage this issue. It has been shown that infusion of mix of amino acids, such as lysine and arginine can reduce the nephrotoxicity [103]. The use of the cytoprotective drug amifostine [104], and low dose of plasma expander succinylated gelatin and gelatin based gelofusine have been recently shown to inhibit the renal uptake of octreotide pharmaceuticals [105, 106].  21  In summary, several characteristics are important for developing a radiolabeled peptide to be used as a tumor imaging agent: it must bind with high affinity to a target receptor that is found mainly on cancer cells and not on normal tissues, be specific to its target and not bind to non-target tissues, be metabolically stable to reach the tumor lesions in an intact state, and be cleared rapidly from the blood and non-target tissues in order to minimize the background radioactivity and patient dose [74]. The main steps involved in developing a radiopharmaceutical for clinical application are described in Figure 1.6.   Figure 1.6 The main steps involved in development of a radiopharmaceutical (Adopted from Fani et al. 2012) 22  1.6 Hypothesis and objectives The hypothesis of this thesis is that tumors overexpressing GRP, SST2a, and NPY1 receptors can be targeted by novel peptide based radiopharmaceuticals for cancer imaging. These radiopharmaceuticals could therefore be used in PET imaging to improve breast and prostate cancer diagnosis. The overall objective is to develop a novel and highly specific diagnostic approach using radiolabeled peptides for PET imaging to localize primary lesions, metastases, and sites of recurrence of breast and prostate cancers. In order to test this hypothesis and meet the objective of this thesis the following specific aims are proposed. 1.6.1 Specific aim 1 To prepare different derivatives of GRP, SST2a, and NPY1 peptides bearing different chelators and perform in vitro studies such as receptor binding assays to identify the promising candidates that have high binding affinities to the receptors and suitable metabolic stability in plasma. 1.6.2 Specific aim 2 Radiolabel promising candidates identified under Aim 1 with 18F or 68Ga and perform biological evaluations with them to identify the suitable best candidates to be used for in vivo studies. 23  1.6.3 Specific aim 3 Perform in vivo studies, including biodistribution and PET imaging studies, by using breast and prostate cancer models with the most promising candidates identified under Aim 2.   24  Chapter 2: GRP receptors, click chemistry approach 2.1 Background Bombesin and the gastrin releasing peptide (GRP) are members of a family of brain-gut peptides that play roles in cancer [107]. Bombesin is a 14-amino acid peptide present in amphibian tissues originally isolated from a skin of the frog Bombina bombin in 1971 [79]. GRP is a mammalian counterpart that consists of 27 amino acids isolated from the porcine stomach in 1979 [108].   Bombesin and GRP mediate their actions by membrane bound G protein coupled receptors and are comprised of at least 4 receptor subtypes including, neuromodin B receptor (BB1), GRP receptor (BB2), bombesin like receptor 3 (BB3), BB4 subtypes [109-112]. Bombesin and GRP share similar carboxy-terminal; therefore they exhibit similar biological activities [71]. GRP acts mainly in the central and enteric nervous systems and regulates a wide range of physiological processes, including thermoregulation, satiety, gastric and pancreatic secretion, nervous system stimulation, smooth muscle contraction, immune function and release of peptide hormones [113, 114]. Among all of the functions of GRP, the one related to cancer is the most studied. It is confirmed that primary human tumors and cancer cell lines can synthesize GRP and bombesin [115] and they regulate the cell growth in some malignant cell lines [113, 114]. Presence of GRP receptors in cancer cells was confirmed by measuring mRNA levels in different human neoplasms such as prostate, breast, lung and gastrointestinal tract cancers [116-118]. Moreover, it is shown that GRP receptors stimulate the growth of small cell lung cancer as part of an autocrine feedback mechanism that involves the expression of these peptides and their receptors in tumor cells [107]. Stimulation of 25  proliferation was also reported in breast and pancreatic cancers [119, 120] and neuroblastoma cell line [121] or PC-3 androgen independent human prostate cancer cells [122]. Studies have demonstrated that the level of GRP receptor correlates positively with the presence of estrogen receptors [123]. In vivo studies in nude mice xenografts showed that the growth of human prostate carcinoma and glioblastoma is inhibited by GRP receptor antagonists [122, 124]. Ruebi et al. reported that GRP receptor expression is the most detected in prostate and breast tumors [75, 125]. 30 of 30 invasive prostatic carcinomas, 26 of 26 of prostatic intraepitelial neoplasias, and two third of invasive ductal carcinomas and ductal carcinoma in situ in neoplastic epithelial mammary cells were GRP receptor positive [75, 125]. Peptide receptors are important targets for imaging and diagnosis of cancer [71, 126-128]. Radiolabeled bombesin (BBN) analogs that bind to GRP receptors have generated great interest as cancer imaging probes due to overexpression of GRPR in several cancers such as prostate, breast, lung and gastrointestinal cancers [75, 76, 116, 125, 129, 130]. Several publications have introduced different analogs of the natural GRP receptor ligand bombesin, a 14 amino-acid neuropeptide, labeled with radioisotopes such as 99mTc, 111In and 68Ga, mainly for the diagnosis of GPR receptor positive tumors that have shown high tumor uptake in prostate tumor models [131-138]. Prostate cancer, in particular, has attracted most of the attention as GRPR was reported to be overexpressed in 100% of malignant lesions but not in the surrounding normal prostate tissues [125]. Even though, 18F with a suitable half-life of 109.8 minutes is considered as an optimal radionuclide for PET imaging, fewer publications have reported on 18F labeling of bombesin peptides [139-141].  26  The 18F- labeling of model azide- modified peptide and oligonucleotide sequences with prosthetic [18F]FPy5yne via an efficient, chemo-selective Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was previously described [142-144].  It was predicted that the first radioactive product [18F]F-ALK-[D-Tyr6, βAla11,Thi13,Nle14]bombesin(6-14) ([18F]-ALK-BBN) would  be highly lipophilic and have a tendency for predominant hepatobiliary excretion in vivo that leads to unfavorable pharmacokinetic behavior.  Based on prior results [116, 145], we hypothesized that increasing the hydrophilicity of the bombesin analog may lead to derivatives with improved sensitivity, specificity, and pharmacokinetics for the optimal targeting of GRP receptor positive tumors. Polyethylene glycol (PEG) spacers can be used to enhance the hydrophilicity of peptides that undergo undesirable hepatobiliary clearance in vivo [146]. PEGylation also protects peptides from plasma enzymes which reduces degradation and increases the half-life in blood. This specially applies when the PEG size is larger than 30 kDa [147-151]. Therefore, alternative water-soluble 18F analogs of [D-Tyr6, βAla11,Thi13,Nle14]BBN(6-14) (hereafter referred to as BBN) were synthesized in which short PEG moieties were introduced both through further modification of the N3 peptide precursor, as well as through conjugation to a PEGylated 18F prosthetic group [145].  In this study three radiofluorinated derivatives of BBN with and without the addition of PEG groups were compared and evaluated in vitro and in vivo to determine whether changing the hydrophilicity of 2-fluoropyridine conjugated peptides can increase GRP receptor-mediated binding affinity and overall pharmacokinetics in PC-3 tumor-bearing mice. 27  2.2 Materials and methods 2.2.1 Cell culture Human prostate cancer cell line (PC-3) was obtained from the American Type Culture Collection and cultured in HAM’S/F-12 medium under a humidified atmosphere with 5% CO2 at 37 ˚C. Culture media was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. All culture reagents purchased from Sigma Aldrich or Invitrogen. 2.2.2 Peptide synthesis and radiosynthesis of bombesin derivatives Gastrin-releasing peptide receptor ligands F-ALK-BBN, F-ALK-BBN-PEG, and F-PEG-BBN-PEG were prepared from their azide- modified precursors via copper-catalyzed azide-alkyne conjugation to the acetylene-bearing 2-fluoropyridines FPy5yne [144] and PEG-FPyKYNE as previously described [143], [145]. Radiolabeling of [18F]-ALK-BBN was performed by first synthesizing [18F]FPy5yne, followed by copper-catalyzed cycloaddition to the azide-substituted peptide [(N3-(CH2)-CO)]-[D-Tyr6, βAla11,Thi13,Nle14]BBN(6-14). Radiolabeling of  [18F]-ALK-BBN-PEG was conducted by conjugating [18F]FPy5yne to [(N3-(CH2)-CO)]-[PEG4-Lys][D-Tyr6, βAla11,Thi13,Nle14]BBN(6-14), while [18F]-PEG-BBN-PEG was obtained by cycloaddition of [18F]-PEG-FPyKYNE to [(N3-(CH2)-CO)]-[PEG4-Lys][D-Tyr6, βAla11,Thi13,Nle14]BBN(6-14) [143], [145].  Schemes 2.1, 2.2 and 2.3 demonstrate radiosynthesis of the three BBN derivatives by click chemistry.28    Scheme 2.1 Radiosynthesis of [18F]-ALK-BBN by click chemistry approach.  29   Scheme 2.2  Radiosynthesis of [18F]-ALK-BBN-PEG by click chemistry approach.   Scheme 2.3 Radiosynthesis of [18F]-PEG-BBN-PEG by click chemistry approach. 30  2.2.3 LogD7.4 measurement The partition coefficient at pH 7.4 (LogD7.4) was measured by adding aliquots of the 18F-labeled peptides in a vial containing 1:1 mixture of octanol and phosphate buffered solution (10 mL, 20mM) at pH 7.4. The solution was vortexed for 3 minute at room temperature, then centrifuged for 3 minutes at 11227 g. Samples (1 ml) were withdrawn from the buffer and octanol layers, and counted on a gamma counter to calculate the LogD7.4.  2.2.4 Metabolic stability study [18F]-ALK-BBN-PEG or [18F]-PEG-BBN-PEG were incubated with BALB/c mouse plasma (Innovative Research; Cat#: IMS-BCN-N-25mL) at a final concentration of 4 MBq/0.3 mL for 5, 15, 30, 60, and 120 minutes at 37°C. Plasma proteins were precipitated by adding 0.3 ml of 75% MeCN followed by vortexing.  Samples were then filtered through a 0.45 µm filter and loaded onto the analytical HPLC equipped with radioactivity detector to check the stability of the peptide.  The percentage of intact peptide for each incubation time was determined by Agilent ChemStation software. 2.2.5 Immunohistochemistry Tumors were harvested from nude mice bearing PC-3 tumors and fixed in 4% paraformaldehyde in PBS for 48 hours. Tissues were embedded in paraffin after dehydration and 4 µm sections were mounted onto poly-L-lysine slides. Immunostaining was performed by the Centre of Translational Applied Genomics (CTAG) at the BC Cancer Agency. Staining was done on the Ventana DiscoveryXT. Slides underwent a pretreatment process for epitope retrieval using Protease1 for 4 min. The primary antibody GRPR (Novus Biologicals NB100-31  74434) was used at a 1:200 dilution for 1 hour at room temperature. The secondary antibody, Ultramap anti-Rb HRP was applied for 16 minutes followed by the Ultramap DAB detection kit. The stained sections were examined and photographed with a Leica EC3 microscope. 2.2.6 In vitro receptor binding assays Competition assays were performed using PC-3 cells to measure the affinity of the bombesin derivative peptides to GRP receptors. Cells were cultured in 24-well plates at a density of 105 cells per well and grown to 75% confluent. Two hours prior to the experiment, cells were washed with phosphate-buffered saline (PBS) and incubated with RPMI 1640 containing 2 mg/mL BSA, 4.8 mg/mL HEPES, 1 U/mL penicillin G and 1 μg/mL streptomycin. Then 0.04 nM of [125I]Tyr4-Bombesin (81.4TBq/mmol, Perkin Elmer, Canada) and various concentration of cold BBN peptides (10-6 to 10-13 M) by serial dilutions from stock solution (10% EtOH in water) was added to the cells in triplicates and incubated under agitation at 37 ˚C for 45 minutes. The cells were then washed 3 times with ice-cold PBS and trypsinized and counted by a gamma counter (Cobra-II Auto Gamma, Canberra Packard Canada). Data were analyzed by GraphPad Prism 6.01 software, and the inhibition constant (Ki) of the peptides was calculated using the software. 2.2.7 Biodistribution studies in tumor-bearing mice The animal protocol used for the animal studies was approved by the Institutional Animal Care Committee of the University of British Columbia and was performed in compliance with the Canadian Council on Animal Care Guidelines.  8-10 weeks old male nude mice purchased from Simonsen laboratories were used for animal studies. For the induction of tumor 32  xenografts, male nude mice were inoculated subcutaneously with 5 x 106 PC-3 cancer cells on the dorsal flank. The PC-3 tumor cells were freshly expanded in sterilized PBS/matrigel mixture prior to inoculation. The tumors were allowed to grow 4-6 weeks to reach a suitable size (5-7mm in diameter) for the in vivo studies.  In unblocked biodistribution studies, 10-20 µCi (0.37-0.74 MBq) of radiotracer was injected via the caudal lateral tail vein under 2% isoflurane anaesthesia. To determine the specificity of the in vivo uptake in receptor positive tissues, 100 µg of non-radioactive bombesin (Sigma B4272) as a blocking agent was either co-injected with radiotracer, pre-injected (10 min) or post-injected (10 min) to an additional group of mice. Mice were humanely euthanized by carbon dioxide asphyxiation and dissected 1 hour post-injection. Tissues of interest were collected, rinsed with PBS, dried and counted in a gamma counter (Cobra-II Auto Gamma, Canberra Packard Canada). The tissue weight and associated count per minute were used to calculate the percentage injected dose per gram of tissue (%ID/g).  2.2.8 PET/CT imaging and kinetic analysis One hour dynamic PET/CT imaging was performed using the Siemens Inveon multimodality small animal PET/CT scanner. Tumor-bearing animals were anaesthetized using 1.5-2% isoflurane and the caudal lateral tail vein was catheterized. Mice were placed onto the imaging bed while anaesthetized. A 10 minute CT attenuation scan was first performed, and then followed by a 60 minute dynamic PET imaging session. 100 µCi (3.7 MBq) of radiotracer was injected via catheter 30 seconds after PET acquisition started. In blocking studies, 100 µg of unlabeled bombesin was used for competition with the radiotracers. Mice were euthanized after termination of scans.  33  The PET data was reconstructed on the Inveon Acquisition Workplace (IAW) by using the MicroQ software program.  The data was first histogrammed using the original data. The data from the histogram and the attenuation map from the CT scan were used in the reconstruction. Time activity curves from the region of interests (ROI) were generated from the 1 hour dynamic imaging data. 2.2.9 Statistical analysis Quantitative data were expressed as Mean ± SD, and means were compared using 1-way ANOVA. Correlation statistics were performed using linear regression analysis. P values of 0.05 or less were considered statistically significant.  2.3 Results 2.3.1 Chemistry and radiochemistry The decay-corrected radiochemical yield of [18F]-ALK-BBN was 34±17% (n = 8) from [18F]FPy5yne. A 9:1 mixture of (2-hydroxypropyl)-β-cyclodextrin solution (10 % HPβCD in PBS) and DMSO was employed for in vivo studies. The decay-corrected radiochemical yield of [18F]-ALK-BBN-PEG was 31±19% (n = 11), and the specific activity was 1.2 ± 0.7 Ci/µmol at the end-of-synthesis calculated from HPLC analysis by using the area under the UV peak versus a standard curve. Specific activity of [18F]-PEG-BBN-PEG at end-of-synthesis was 2.1±1 Ci/µmol. The decay-corrected radiochemical yield of [18F]-PEG-BBN-PEG was 25 ± 13% DC (n = 4). PEGylated radiotracers were formulated in 10% EtOH in isotonic saline, for in vivo studies. 34  2.3.2 LogD7.4 measurements The lipophilicity of [18F]-ALK-BBN was assessed relative to PEGylated derivatives ([18F ]-ALK-BBN-PEG and [18F]-PEG-BBN-PEG). As expected, log D 7.4 values decreased with an increase in ethylene oxide groups in the targeting vectors as shown in Table 2.1.   Table 2.1 The Lipophilicity of bombesin derivatives. BBN derivative log D[7.4] [18F]-ALK-BBN 1.40 ± 0.01 [18F]-ALK-BBN-PEG -0.92 ± 0.03 [18F]-PEG-BBN-PEG -1.12 ± 0.01   2.3.3 In vitro binding assays Competition binding assays were performed with all three non-radioactive BBN derivatives to compare their binding affinities for GRPR in PC-3 cells. Inhibition constant (Ki) for ALK-BBN was 15 ± 8 nM, whereas sub-nanomolar affinities with Ki of 0.2 ± 0.1 nM (n=3) was achieved for ALK-BBN-PEG and PEG-BBN-PEG. Binding assays of PEGylated peptides are shown in Figure 2.1.  35   Figure 2.1 Competition binding assay of 19F-ALK-BBN-PEG (A) and 19F-PEG-BBN-PEG (B) in GRPR positive PC-3 cells. Curves were fitted to competition binding data by nonlinear regression and normalized by Graph Pad Prism 6.01 software.  2.3.4 Immunohistochemistry To validate GRPR expression in PC-3 tumors, histological tissue analysis was performed. As shown in Figure 2.2 membranous expression of GRPR is confirmed in PC-3 tumor xenografts.   Figure 2.2 Immunohistochemical staining of GRPR expression in PC-3 tumors. 40X magnification. 36  2.3.5 Metabolic stability in mouse plasma The stability of the PEGylated 18F labeled bombesin analogs was investigated in BALB/c mouse plasma at various incubation times from 0 to 120 min at 37 ˚C.  [18F]-ALK-BBN-PEG was rapidly degraded upon incubation in plasma (Figure 2.3). [18F]-PEG-BBN-PEG remained 88.3% intact after 2 h incubation in plasma.                    37     Figure 2.3 HPLC traces of metabolic stability of [18F]-ALK-BBN-PEG at 30, 60 and 120 min incubation at 37 ºC in mouse plasma. 38  2.3.6 Biodistribution studies in tumor bearing mice In vivo evaluation of non-PEGylated and PEGylated 18F labeled bombesin derivatives was determined by biodistribution studies and dynamic µPET/CT imaging in PC-3 tumor-bearing mice.  Tables 2.2, 2.3, and 2.4 summarize the biodistribution studies of three bombesin derivatives in PC-3 tumor-bearing mice at 1 h post-injection. The non-PEGylated derivative, [18F]-ALK-BBN was mostly retained in intestine with %ID/g of 10.2 ± 4.8 at 1 h post-injection. Pancreas uptake was 0.8 ± 0.6 %ID/g and minimal tumor uptake was seen at 1 h post injection. Blocking studies were performed by coinjection of 100 µg of unlabeled bombesin led to >95% reduction in accumulation within the intestine. However, uptake of the tumors and pancreas were increased >50% and 30% respectively. Biodistribution studies of PEGylated derivatives showed that the uptake in the gut was reduced (Figure 2.4), and tumor and pancreas uptake was improved (Figure 2.5 The %ID/g of tumors at 15, 30 and 60 min post-injection were 2.0 ± 0.3, 1.4 ± 0.5 and 1.0 ± 0.1 in [18F]-ALK-BBN-PEG, and 2.0 ± 0.1, 1.9 ± 0.4 and 1.1 ± 0.5 in [18F]-PEG-BBN-PEG. Uptake of [18F]-PEG-BBN-PEG in pancreas was higher compared to [18F]-ALK-BBN-PEG (10.17 ± 2.68 Vs. 5.38 ± 1.24, P < 0.05). Renal excretion was slower in [18F]-ALK-BBN-PEG.  As shown in Figure 2.6, tumor-to-blood ratio of [18F]-ALK-BBN-PEG and [18F]-PEG-BBN-PEG was 4.8 ± 1.2 and 2.4 ± 0.6 respectively.  Blocking studies were performed by pre- and post-blocking of PEGylated derivatives besides common way of co-injection. %ID/g of pancreas diminished in range of 60-90% in all blocking studies except for post-blocking of [18F]-PEG-BBN-PEG that resulted in 14% 39  increase in uptake (Figure 2.5). In tumors the best blocking result was achieved by pre-blocking of [18F]-ALK-BBN-PEG.  Table 2.2 Biodistribution studies of [18F]-ALK-BBN in nude mice bearing PC3 tumor 1 h post-injection. Data presented as %ID/g of tissues in unblocked (n=11) and blocking with co-injection (n=5).   Tissue [18F]-ALK-BBN     Unblocked Co-injection           Blood 0 ± 0 0 ± 0  Plasma 0.1 ± 0 0.2 ± 0.2  Fat 0 ± 0 0.1 ± 0.1  Seminal glands 0 ± 0 0.2 ± 0.3  Testes 0.1 ± 0.1 0 ± 0  Large intestine 10.2 ± 4.9 0.3 ± 0.6  Small intestine 0.6 ± 0.7 0.1 ± 0.1  Spleen 0.2 ± 0.1 0.6 ± 0.3  Liver 0.7 ± 0.5 1.3 ± 0.8  Pancreas 0.8 ± 0.6 1.2 ± 1.4  Adrenal glands 0.6 ± 0.4 0.9 ± 1.1  Kidney 0.5 ± 0.5 0.7 ± 0.2  Lung 0.5 ± 0.5 1.4 ± 1.6  Heart 0.1 ± 0.1 0.1 ± 0  Tumor 0.1 ± 0.1 0.2 ± 0.1  Skin 0.1 ± 0.1 0.1 ± 0.1  Muscle 0.1 ± 0.1 0 ± 0  Bone 0.3 ± 0.2 0.2 ± 0.2  Brain 0 ± 0 0 ± 0             40  Table 2.3 Biodistribution studies of [18F]-ALK-BBN-PEG in nude mice bearing PC3 tumor 1 h post- injection. Data presented as %ID/g of tissues in unblocked (n=6) and blocked (n=3) studies. Tissue [18F]-ALK-BBN-PEG      Unblocked Pre-blocked Post-blocked Co-injection               Blood 0.2 ± 0.0 0.4 ± 0.1 0.4 ± 0.4 1.9 ± 2.2  Plasma 0.8 ± 0.4 0.9 ± 0.6 1.5 ± 1.4 1.2 ± 1.2  Fat 0.1 ± 0.0 0.3 ± 0.4 0.2 ± 0.1 1.2 ± 1.0  Seminal glands 0.2 ± 0.1 0.1 ± 0.1 0.5 ± 0.3 0.2 ± 0.2  Testes 0.3 ± 0.3 0.2 ± 0.1 0.5 ± 0.7 0.2 ± 0.2  Large intestine 5.7 ± 7.3 2.3 ± 4.7 2.0 ± 2.3 0.3 ± 0.6  Small intestine 2.4 ± 2.3 8.4 ± 18.6 0.4 ± 0.4 1.6 ± 2.3  Spleen 0.4 ± 0.1 0.4 ± 0.2 3.2 ± 4.9 1.4 ± 1.3  Liver 1.5 ± 0.7 1.9 ± 0.3 3.9 ± 2.6 0.6 ± 0.6  Pancreas 4.2 ± 3.5 1.3 ± 1.0 2.5 ± 1.6 1.0 ± 0.3  Adrenal glands 1.3 ± 0.8 1.3 ± 1.2 15.2 ± 0.1 1.3 ± 0.8  Kidney 5.1 ± 7.9 4.4 ± 5.0 1.9 ± 0.1 5.8 ± 5.5  Lung 0.4 ± 0.2 0.5 ± 0.3 0.4 ± 0.3 1.3 ± 1.8  Heart 0.3 ± 0.1 0.3 ± 0.1 0.6 ± 0.4 0.6 ± 0.7  Tumor 1.1 ± 0.2 0.7 ± 0.3 0.1 ± 0.5 2.0 ± 0.9  Skin 0.5 ± 0.3 0.5 ± 0.3 0.2 ± 0.4 2.3 ± 2.5  Muscle 0.2 ± 0.1 0.2 ± 0.1 0.8 ± 0.0 0.4 ± 0.1  Bone 0.3 ± 0.1 0.7 ± 0.7 0.2 ± 0.2 0.5 ± 0.6  Brain 0.1 ± 0.0 0.0 ± 0.0 0.3 ± 0.4 0.1 ± 0.1                      41  Table 2.4 Biodistribution studies of [18F]-PEG-BBN-PEG in nude mice bearing PC3 tumor 1 h post-injection. Data presented as %ID/g of tissues in unblocked (n=4) and blocked (n=3) studies.  Tissue [18F]-PEG-BBN-PEG      Unblocked Pre-blocked Post-blocked Co-injection               Blood 0.5 ± 0.3  1.2 ± 0.6  0.7 ± 0.7  0.5 ± 0.1   Plasma 0.6 ± 0.3  2.9 ± 0.8  1.8 ±1.9   1.3 ± 1.0   Fat 0.3 ± 0.2  0.8 ± 1.0  2.4 ± 3.7  0.2 ± 0.1   Seminal glands 0.4 ± 0.3  1.0 ± 0.7  0.3 ± 0.1  1.0 ± 1.1   Testes 0.4 ± 0.2  0.5 ± 0.2  0.3 ± 0.2  0.2 ± 0.1   Large intestine 2.1 ± 1.2  1.3 ± 1.0  2.0 ± 0.9  0.5 ± 0.4   Small intestine 2.0 ± 1.7  1.7 ± 0.9  4.0 ± 3.7  1.1 ± 0.2   Spleen 0.9 ± 0.6  0.8 ± 0.3  1.0 ± 0.3  0.4 ± 0.1   Liver 1.4 ± 0.3  3.3 ± 0.6  1.9 ± 1.8  2.3 ± 0.2   Pancreas 10.2 ± 5.4  1.3 ± 0.8  11.6 ± 0.4  1.0 ± 0.5   Adrenal glands 3.0 ± 2.6  3.7 ± 1.7  3.7 ± 1.9  0.7 ± 0.0   Kidney 2.7 ± 1.7  26.2 ± 29.7  16.8 ± 22.4  34.4 ± 45.2   Lung 1.0 ± 0.4  1.3 ± 0.9  1.0 ± 1.1  1.6 ± 0.9   Heart 0.4 ± 0.3  0.9 ± 0.3  0.4 ± 0.3   0.5 ± 0.4   Tumor 1.1 ± 0.5  2.2 ± 0.5  1.6 ± 1.0  1.0 ± 0.3   Skin 0.6 ± 0.4  1.8 ± 0.6  1.1 ± 0.8  0.6 ± 0.2   Muscle 0.8 ± 1.0  0.7 ± 0.5  0.4 ± 0.3  0.3 ± 0.2   Bone 0.5 ± 0.3  1.0 ± 0.5  2.0 ± 0.3  0.4 ± 0.3   Brain 0.2 ± 0.1  0.3 ± 0.2  0.2 ± 0.1  0.1 ± 0.1                       42   Figure 2.4 Biodistribution of the three BBN derivatives in the intestine at 1 h post-injection.    Figure 2.5 Biodistribution of [18F]-ALK-BBN-PEG and [18F]-PEG-BBN-PEG in the (A) tumor and (B) pancreas at 1 h post-injection.      43   Figure 2.6 Tumor to blood ratios of [18F]-ALK-BBN-PEG and [18F]-PEG-BBN-PEG at 15, 30, and 60 min post-injection.  2.3.7 PET/CT imaging and kinetic analysis Nude mice bearing PC-3 tumors on the shoulders underwent dynamic PET/CT imaging for an hour in all unblocked and blocked studies. PET/CT imaging of nude mice bearing PC-3 tumors demonstrated that [18F]-ALK-BBN was rapidly excreted through hepatobiliary tract (Figure 2.7). PET/CT studies of both PEGylated derivatives provided clear visualization of PC-3 tumors in mice (Figure 2.8 and 2.9).  Better tumor to background ratio was observed for [18F]-ALK-BBN as expected from 2 fold higher tumor-to-blood ratio obtained in biodistribution studies. The specificity of radiotracers was confirmed by blocking studies as shown in Figure 2.8 and 2.9 (C).   Kinetic analysis of [18F]-ALK-BBN-PEG showed that the highest tumor uptake (2.8 ± 0.5%ID/g) was achieved at 15-20 min post-injection (Figure 2.10).  44   Figure 2.7 Dynamic PET/CT imaging of [18F]-ALK-BBN in PC-3 tumor-bearing mice at 1 h- post-injection. Arrows represent tumors.     Figure 2.8 Dynamic PET/CT imaging of [18F]-ALK-BBN-PEG in PC-3 tumor-bearing mice at (A) 20 min-unblocked, (B) 1 h-unblocked , and (C) preblocked 1h post-injection. Arrows represent tumors.     45   Figure 2.9 Dynamic µPET/CT imaging of [18F]-PEG-BBN-PEG in PC-3 tumor-bearing mice at (A) 20 min-unblocked, (B) 1 h-unblocked , and (C) co-injection 1 h post-injection. Arrows represent tumors.             46    Figure 2.10 Time activity curves of PC-3 tumors over 1 h imaging of [18F]-ALK-BBN-PEG (A), and [18F]-PEG-BBN-PEG (B).      Figure 2.11 Time activity curves of blood over 1 h imaging of [18F]-ALK-BBN-PEG (A), and [18F]-PEG-BBN-PEG (B).   47  2.4 Discussion Prostate cancer is the most common malignancy occurring in men. Due to widespread use of the PSA test for screening, the number of cases diagnosed has increased significantly in recent years, without significant changes in the mortality rate. It has been recently shown in clinical studies that peptides targeting GRP receptors could be used to detect a large proportion of prostate cancers and nodal metastases. The ability to localize primary cancers in the prostate gland could be useful to guide treatment localization in research protocols exploring the benefits of focal therapy as an alternative to active surveillance in men with low risk prostate cancers. The ability to detect local recurrence or regional metastases when the PSA value rises on follow-up could be useful to guide radiation or surgical excision as potential treatment modalities. In the process of developing novel bombesin derivatives for prostate cancer imaging, we chose 18F because of its desirable characteristics for PET imaging such as suitable half-life of 109.8 min and low positron range.  To radiolabel the bombesin derivatives, we used prosthetic groups conjugated to the peptides using copper-catalyzed cycloaddition (‘click chemistry’). This radiolabeling method was not simple to implement, as the prosthetic group had to be radiolabeled and purified prior to cycloaddition to the peptide. We successfully conjugated two different alkynes to PEGylated and non-PEGylated bombesin derivatives.  [18F]-ALK-BBN exhibited unfavorable pharmacokinetics in vivo, including very fast blood clearance and hepatobiliary excretion. This was to some extent expected because of lipophilic nature of this bombesin derivate. The uptake in the tumor and pancreas was minimal. Coinjection of the unlabeled BBN reduced the accumulation of radioactivity in the intestine 48  but increased the uptake in pancreas and tumor, likely due to a slower plasma clearance by competition with elimination pathways or peptidase degradation. In our first attempt to improve in vitro characterization and pharmacokinetics of our radiotracers in vivo one PEGylated group was added to F-ALK-BBN. PEGylation has been reported to increases the overall hydrophilicity of the compound, and also reduces the proteolysis and increases the half-life of the peptides in blood [146]. Moreover, enlarging the molecular weight of peptides can lead to reduced kidney filtration and enhance the permeability and retention effect in the tumor [146]. In vitro binding assay of 19F-ALK-BBN-PEG showed an excellent Ki (0.2 nM). Good tumor visualization was achieved in dynamic PET/CT imaging with the highest uptake of the tumors at 15-20 min post-injection. This early peak uptake followed by progressive decrease suggests that inactivation by peptidases or release of the radioactive moiety is causing a progressive loss of imaging signal in tumors.  Tumors were clearly delineated at 1 h post-injection with less background as [18F]-ALK-BBN-PEG was cleared from liver more rapidly. Pancreas uptake was also improved compared to non-PEGylated derivative (4.2 vs. 0.8 %ID/g). Since blocking by coinjection of unlabeled bombesin resulted in saturation of GRPR with non-PEGylated derivative, we carried out pre-blocking and post-blocking studies (10 min before or after injection of radiotracer) along with coinjection, to demonstrate how different distribution time of unlabeled bombesin in blood can affect blocking. Interestingly, pre-blocking was the most successful. Kinetic analyses of coinjection and post-blocking showed that although tumors were blocked more effectively at the earlier time points compared to pre-blocked, co-injection increased uptake in 1 hour and 49  post-blocked reached almost a plateau after 15 min post-injection whereas in pre-blocking uptake decreased over the course of 1 h imaging (Figure 2.11). We hypothesized that addition of a second PEGylated group can further improve the overall pharmacokinetics by making the peptide more hydrophilic and stable which was confirmed by the LogD7.4 values and plasma stability studies. In vivo studies showed a 7-fold increase in uptake of pancreas. Tumor uptake at 1 h post-injection was very similar to that of [18F]-ALK-BBN-PEG. Tumor visualization was not improved with [18F]-PEG-BBN-PEG. This can be due to a 2-fold higher tumor-to-blood ratio. Specificity of the tracer was confirmed in blocking studies by co-injection of unlabeled bombesin. Post-blocking resulted in saturation of GRPR in both pancreas and tumor. In pre-blocking pancreas uptake was reduced <90% but an increase in %ID/g of tumor was observed. 2.5 Conclusion An improved GRP receptor-mediated tumor uptake was achieved by using both PEGylated 18F labeled bombesin derivatives and less excretion through the hepatobiliary tract was observed. Slower blood elimination was observed with [18F]-PEG-BBN-PEG. However [18F]-ALK-BBN-PEG showed better tumor visualization in PET/CT imaging, with higher tumor to blood ratios.  50  Chapter 3: GRP receptors, trifluoroborate isotope exchange reaction approach 3.1 Background A variety of bombesin analogs, including both agonists and antagonists, have been radiolabeled and evaluated for GRP receptor-targeted prostate cancer imaging and therapy [133, 152-159]. Recent studies suggested that radiolabeled bombesin antagonists may have higher tumor uptake in comparison to agonists, despite their low internalization rate [160]. Among bombesin antagonists, the potent RM26 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) has been widely derivatized for radiolabeling with 64Cu/68Ga and 111In for imaging GRP receptor-expressing prostate cancer with positron emission tomography (PET) and single photon emission computed tomography (SPECT), respectively [161-163]. Recent successful detection of GRP receptor-expressing prostate cancer in clinical studies using RM26 derivatives (68Ga-BAY86-7548 [164] and 64Cu-CB-TE2A-AR06 [165]) further demonstrated the potential usefulness of radiolabeled GRPR antagonists for prostate cancer imaging. Among commercially available imaging radioisotopes, 18F is the most widely used for clinical PET imaging because of its ideal nuclear properties including high positron branching fraction (97%), low positron energy (0.64 MeV), and a suitable physical half-life of 109.7 min [166]. Due to the popularity of 18F-labeled 2-fluorodeoxyglucose for cancer imaging, 18F is available daily in multi-Curie quantities from most medical cyclotron facilities. This facile and on-demand production underscores the importance of developing 18F-labeled BBN-based PET probes for a more efficient application in clinic for prostate cancer imaging. 51  Previously we reported a facile one-step 18F-labeling approach via 18F-19F isotope exchange reaction on an ammoniomethyl-trifluoroborate (AmBF3) moiety [167] that could be conjugated to a number of peptides and other bioconjugates. To explore the generality of this approach for the design of peptide-based 18F-labeled tracers, we report the synthesis and biological evaluation of a novel GRP receptor antagonist 18F-AmBF3-MJ9 (Scheme 3.1) for prostate cancer imaging. 3.2 Materials and methods All chemicals and solvents were obtained from commercial sources, and used without further purification. [125I-Tyr4]Bombesin (81.4 TBq/mmol) was purchased from Perkin Elmer (Waltham, MA). Peptide sequence of MJ9 (Piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) was synthesized using an AAPPTEC (Louisville, KY) Endeavor 90 peptide synthesizer. Mass analyses were performed using a Bruker (Billerica, MA) Esquire-LC/MS system with ESI ion source. Purification and quality control of 18F-AmBF3-MJ9 were performed on an Agilent (Santa Clara, CA) HPLC System equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector, and a Bioscan (Washington DC) NaI scintillation detector. The radio-detector was connected to a Bioscan B-FC-1000 Flow-count system, and the output from the Bioscan Flow-count system was fed into an Agilent 35900E Interface which converted the analog signal to digital signal. The operation of the Agilent HPLC system was controlled using the Agilent ChemStation software. The following gradients were used for HPLC purification and quality control. Method A: Agilent Eclipse XDB-C18 5 µm 9.2 x 250 mm semi-prep column; Solvent A: 0.1% TFA water; solvent B: MeCN; 0 to 15 min, 20 to 40% B; 15 to 20 min, 40 to 20% B; flow rate: 4.5 mL/min. Method 52  B: Agilent Eclipse XDB-C18 5 µm 9.2 x 250 mm semi-prep column; 0 to 2 min, 5 to 20% B; 2 to 5 min, 20 to 30% B; 5 to 20 min, 30 to 50% B; 20 to 22 min, 50 to 5% B; flow rate: 3 mL/min. Method C: Phenomenex Jupiter 10 µm C18 4.6 x 250 mm analytical column; 0 to 2 min, 5 to 5% B; 2 to 7 min, 5 to 20% B; 7 to 12 min, 20 to 100% B; 12 to 18 min, 100 to 100%; 18 to 20 min, 100 to 5% B; flow rate: 2 mL/min. 18F-fluoride was produced by the 18O(p, n)18F reaction using an Advanced Cyclotron Systems Inc. (Richmond, Canada) TR19 cyclotron. 18F-fluoride Trap & Release Columns were purchased from ORTG Inc. (Oakdale, TN) and C18 light Sep-Pak cartridges were obtained from Waters (Mississauga, Canada). Radioactivity of 18F-AmBF3-MJ9 were measured using a Capintec (Ramsey, NJ) CRC®-25R/W dose calibrator, and the radioactivity of mouse tissues collected from biodistribution studies were counted using a Packard (Waltham, MA) Cobra II 5000 Series auto-gamma counter. PET imaging experiments were conducted using a Siemens (Malvern, PA) Inveon microPET/CT scanner. 3.2.1 HPLC analysis The following gradients were used for HPLC purification and quality control: Method A: Agilent Eclipse XDB-C18 5 µm 9.2 x 250 mm semi-prep column. Solvent A: 0.1% TFA water; solvent B: MeCN; 0 to 15 min: 20% to 40% B, 15 to 20 min, 40% to 20% B. Flow rate: 4.5 mL/min, column temperature: 19 to 21 °C. Method B: Agilent Eclipse XDB-C18 5 µm 9.2 x 250 mm semi-prep column. Solvent A: 0.1% TFA water; solvent B: MeCN; 0 to 2 min: 5% to 20% B, 2 to 5 min, 20% to 30% B, 5 to 20 min, 30% to 50%, 20 to 22 min, 50% to 5% B. Flow rate: 3 mL/min, column temperature: 19 to 21 °C. Method C: Phenomenex Jupiter 10 µm C18 300Å 4.6 x 250 mm analytical column. Solvent A: 0.1% TFA water; solvent B: 53  MeCN; 0 to 2 min: 5% to 5% B, 2 to 7 min, 5% to 20% B, 7 to 12 min, 20% to 100%, 12 to 18 min, 100% to 100%, 18 to 20 min, 100% to 5%, B. Flow rate: 2 mL/min, column temperature: 19 to 21 °C. 3.2.2 Synthesis of AmBF3-MJ9 3.2.2.1 Synthesis of N-propargyl-N,N-dimethyl-ammoniomethyl boronylpinacolate N-propargyl-N,N-dimethyl-ammoniomethyl-boronylpinacolate (alkynyl-AMB(pin)) was synthesized by condensation of iodomethyl-boronylpinacolate and propargylamine by standard alkylative amine quaternization. Briefly, a flame-dried round bottom flask was charged with N,N-dimethylpropargylamine (98 µL, 1.0 mmol) and 2 mL anhydrous diethyl ether under argon atmosphere to which Iodomethyl-pinacolboronate (165 µL, 0.9 mmol) was added drop-wise at room temperature. On stirring, the solution became cloudy followed by the formation of a white precipitate, which was the desired product. The resulting precipitate was filtered and washed with cold diethyl ether. Then the residue was dried under high vacuum to give a fluffy white powder in 95% yield. 1H NMR (300 MHz, CD3CN) δ 4.40 (d, 2H), 3.31 (s, 2H), 3.22 (s, 6H), 3.21 (t, 1H), 1.27 (s, 12H); ESI: calculated: 224.1; found: 224.1. 3.2.2.2 Synthesis of azidoacetyl-MJ9 The sequence of MJ9, Piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, was first constructed on resin using the solid-phase approach as previously described [134]. Then, 2-azidoacetic acid (5 equivalents) was coupled to the N-terminus via in situ activation using HCTU/HOBT (5 equivalents) and DIEA (10 equivalents) in NMP. The peptide was cleaved and de-protected from the resin by treatment with a TFA/water/triisopropylsilane/1,2-54  ethanedithiol/thioanisole/phenol (81.5:5:1:2.5:5:5) cocktail for 4 h. After precipitation by the addition of cold ether, the crude product was filtered, dried, and purified by HPLC using Method A. Azidoacetyl-MJ9 was obtained in 48% yield. MS (ESI) calculated for M+: 1335.7; found: 1335.7.  3.2.2.3 Synthesis of AmBF3-MJ9 N-propargyl-N,N-dimethylammonio-methylboronylpinacolate 1 (5.0 mg, 22.3 µmol) was converted to the corresponding trifluoroborate (alkynyl-AmBF3 2) by treating with KHF2 (3 M, 30 µL in water), HCl (4 M, 30 µL), deionized water (20 µL) and DMF (60 µL) at 45oC for 2 h, and then quenched by NH4OH (28%, 10 µL). The crude reaction was directly used for click conjugation to azidoacetyl-MJ9 without further purification. A mixture of azidoacetyl-MJ9 (5.0 mg, 3.75 µmol), CuSO4 (1.0 M, 5.0 µL), sodium ascorbate (1.0 M, 12.5 µL) and 5% NH4OH (MeCN/H2O = 1:1, 50 µL) was added, and the new mixture was warmed up to 45 °C for 2 h. Purification was performed by HPLC using Method B to obtain 2.5 mg (45% yield) of AmBF3-MJ9. MS (ESI) calculated for M+: 1500.8; found: 1501.8 [M+H]+. For convenience and to ensure reproducibility, the purified AmBF3-MJ9 was dissolved in ethanol and aliquoted in quantities of ~110 μg (~75 nmol) for future radiolabeling experiments.  3.2.3 In vitro receptor binding assays Competition binding assays were performed using GRPR-expressing human prostate cancer PC-3 cells. The cells were cultured in Ham’s F-12 medium under a humidified atmosphere with 5% CO2 at 37 ˚C. The culture media was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured in 55  24-well plates at a density of 0.1 million cells per well and grown to 75% confluence. Two hours prior to the experiment, the cells were washed with phosphate-buffered saline (PBS) and incubated with RPMI 1640 containing 2 mg/mL BSA, 4.8 mg/mL HEPES, 1 U/mL penicillin G and 1 μg/mL streptomycin. A final concentration of 4.2 pM of [125I-Tyr4]Bombesin and 10-5 to 10-12 M of  AmBF3-MJ9 were added to wells (triplicate) containing the cells, and the mixture was incubated under agitation at 37 ˚C for 45 min. The cells were then washed 3 times with ice-cold PBS, trypsinized and the cell-bound radioactivity was counted using a gamma counter. The IC50 and Ki values of AmBF3-MJ9 were calculated by non-linear curve fitting using GraphPad Prism 6.01.  3.2.4 Radiolabeling Just prior to radiosynthesis, AmBF3-MJ9 was resuspended in aqueous pyridazine-HCl buffer (50 µL, pH = 2) in a polypropylene Falcon tube. No carrier-added 18F-fluoride (~37 GBq) was obtained by bombardment of H218O with 18-MeV proton, followed by trapping on an anion exchange resin (9 mg, QMA, chloride form) column. The 18F-fluoride was eluted off with 100 µL isotonic saline into the reaction tube containing AmBF3-MJ9 (~75 nmol). The tube was heated at 80 °C for 20 min, and quenched with 5% NH4OH (2 mL). The mixture was loaded onto a C18 light Sep-Pak cartridge, and free 18F-fluoride was removed by flushing twice with 4 mL DI water. The desired 18F-AmBF3-MJ9 was then eluted off the column with 0.5 mL 1:1 ethanol/saline, and diluted with saline for plasma stability, internalization, biodistribution and PET/CT imaging studies. A small sample was removed for quality control by HPLC using Method C. 56  3.2.5 Metabolic stability study in mouse plasma 18F-AmBF3-MJ9 (20 µL) was added to the mouse plasma (500 µL) and the mixture was incubated at 37 °C for 0, 1 and 2 h. At the end of incubation at each time point, the reaction was quenched by adding 70% MeCN (1mL) to the plasma solution. The quenched reactions were centrifuged for 20 min, and the supernatants were filtered through a 0.45 micron filter and analyzed by HPLC using Method C.  3.2.6 Internalization studies The experiments were performed in triplicates at 30, 60 and 90 min incubation at 37 °C according to previously published procedures [168].  Briefly, PC-3 cells were plated in 6-well plates at a density of 0.8-1 million cells per well and incubated overnight. DMEM containing 1% FBS was used as a culture medium. On the day of the experiment, the cells were washed with PBS and 1 ml of culture medium containing 2.5 pmol of 18F-AmBF3-MJ9 was added to each well. In separate wells with the same setup, BAY86-7548 (Ga-DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, also known as RM2)  to a final concentration of 1 µM) was added for the determination of nonspecific binding. After incubation at 37 °C to the pre-determined time point, the cells were washed twice with ice-cold PBS followed by two acid washes (Glycine-HCl, pH 2.8) for 10 min on ice. To measure the internalized activity, the cells were lysed with 1M NaOH, the solution was counted on a gamma counter. 57  3.2.7 Biodistribution studies in tumor-bearing mice The animal protocol used for the animal studies was approved by the Institutional Animal Care Committee of the University of British Columbia and all experiments were performed in compliance with the Canadian Council on Animal Care Guidelines. 8-10 weeks old male immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) were obtained from an in-house breeding colony at the Animal Resource Centre of the BC Cancer Research Centre. Mice were inoculated subcutaneously in the dorsal flank with 5 × 106 PC-3 cells prepared in sterilized PBS/matrigel mixture. The tumors were allowed to grow for approximately 4 weeks to reach 5 – 7 mm in diameter for in vivo experiments.  For biodistribution study, 0.37-0.74 MBq of 18F-AmBF3-MJ9 was injected via the caudal lateral tail vein while mice were under brief isoflurane sedation. The radioactivity in the syringe was measured before and after injection to determine the total amount of administrated 18F-AmBF3-MJ9. To confirm the specific uptake of 18F-AmBF3-MJ9 in GRPR-expressing tissues, 100 µg of BAY86-7548 was pre-injected 15 min prior to the radiotracer as a blocking agent to an additional group of mice. Mice were euthanized at pre-determined time points (30 min, 1h and 2h) by isoflurane anesthesia followed by CO2 breathing. Tissues of interest were collected, rinsed with PBS, blotted dry, weighted and counted in a gamma counter. The gamma counter was cross-calibrated with the dose calibrator using an 18F standard curve. The tissue weight, associated counts per minute and the calibration factor were used to calculate the percentage injected dose per gram of tissue (%ID/g). 58  3.2.8 PET/CT imaging  Static (1 h and 2 h) PET/CT imaging was performed using a Siemens Inveon microPET/CT scanner. The PC-3 tumor-bearing mice were briefly sedated for i.v. injection of 18F-AmBF3-MJ9 as described above, and then allowed to recover and roam freely in their cages. For blocking study 100 µg of BAY86-7548 was administered 15 min prior to the injection of the radiotracer. At the pre-determined time point, the mice were sedated with 2% isoflurane inhalation and placed in the scanner. A baseline CT scan was obtained for localization and attenuation correction using 60 kV x-rays at 500 μA, using 3 sequential bed position with 33% overlap and 220 degree continuous rotation. The mice were kept warm by a heating pad during acquisition. A single static emission scan was acquired for 15 min. The mice were euthanized after termination of scans, and the organs harvested for biodistribution. PET image data were reconstructed using the Inveon Acquisition Workplace using two iterations of the ordered subset expectation maximization algorithm followed by 18 iterations of the maximum a posteriori algorithm, using a beta factor of 0.1.  3.3 Results 3.3.1  Synthesis of AmBF3-MJ9 The synthesis of AmBF3-MJ9 is illustrated in Scheme 3.1. The AmBF3-conjugated alkyne 2 was prepared by treating N-propargyl-N,N-dimethyl-ammoniomethyl boronylpinacolate 1[167] with KHF2 and the crude mixture was used directly for subsequent coupling to the azidoacetyl-MJ9 without further purification. Azidoacetyl-MJ9 was synthesized using solid-phase peptide synthesis approach, and obtained after HPLC purification in 48% yield. The 59  click coupling of alkyne 2 and azidoacetyl-MJ9 generated the desired AmBF3-MJ9 in 45% yield. 3.3.2 In vitro binding assay Competition binding assays were performed to determine the binding affinity of AmBF3-MJ9 for GRPR using GRPR-expressing PC-3 prostate cancer cells and the radioligand [125I-Tyr4]bombesin. A representative displacement curve of [125I-Tyr4]bombesin by AmBF3-MJ9 is shown in Figure 3.1. The IC50 was calculated to be 0.6 ± 0.1 nM (n = 3) and the corresponding Ki was 0.5 ± 0.1 nM. 3.3.3 Radiochemistry Starting with ~37.0 GBq of 18F-fluoride, ~7.4 GBq of 18F-AmBF3-MJ9 was reproducibly obtained within 25 min (23 ± 5%, n=3). Purified 18F-AmBF3-MJ9 was re-injected into HPLC for quality control. As shown in Figure 3.2, only one peak was observed on both radio- and UV-VIS chromatograms. The radiochemical purity and specific activity of 18F-AmBF3-MJ9 were > 99% and 100 ± 32 GBq/µmol, respectively.  60   Scheme 3.1 Synthesis of AmBF3-MJ9 and radiosynthesis of 18F-AmBF3-MJ9.  Figure 3.1 A representative displacement curve of [125I-Tyr4]bombesin by AmBF3-MJ9. IC 50: 0.6 nM (n=3), Ki: 0.5 nM (n=3).  61   Figure 3.2 Radio-HPLC analysis of purified 18F-AmBF3-MJ9. Only one single peak was observed on both radio- (upper) and UV-VIS (lower) chromatograms.  3.3.4 Metabolic stability in mouse plasma 18F-AmBF3-MJ9 was incubated in mouse plasma for up to 2 h at 37 °C, and analyzed by HPLC to assess its en vivo stability. As shown in Figure 3.3, there was no detectable decomposition on radio chromatogram for all the selected time points (0, 1 and 2 h).  62   Figure 3.3 Radio-HPLC analysis of metabolic stability of 18F-AmBF3-MJ9 in mouse plasma after 0 (A), 1 (B), and 2 h (C) incubation at 37 ˚C. 3.3.5 Internalization studies An internalization assay was conducted using PC-3 cells to assess if the binding of 18F-AmBF3-MJ9 to GRPR triggers the internalization of receptor. As shown in Figure 3.4, no significant change of the membrane-bound fraction was observed over time, and the numbers were 5.48 ± 0.15, 5.84 ± 0.12, and 5.32 ± 0.20 % at 30, 60 and 90 min, respectively. The internalized fraction was 2.37 ± 0.29 % at 30 min and 1.54 ± 0.27 % at 90 min.  63   Figure 3.4 Percentages of membrane-bound and internalized radioactivity after incubating 18F-AmBF3-MJ9 with PC-3 cells for up to 90 min.  3.3.6 Biodistribution and imaging studies Biodistribution of 18F-AmBF3-MJ9 was assessed in NSG mice bearing PC-3 tumor xenografts. As summarized in Table 3.1, higher accumulation was observed in GRPR-expressing PC-3 tumor, pancreas, and intestine. The uptake values for PC-3 tumor, pancreas and intestine were 2.80 ± 0.77, 8.04 ± 3.64 and 4.27 ± 5.03 %ID/g at 0.5 h;  1.37 ± 0.25, 4.16 ± 1.34 and 6.42 ± 3.13% ID/g at 1 h; and 2.20 ± 0.13, 4.79 ± 2.34 and 8.67 ± 11.5% ID/g at 2 h, respectively. Pre-blocking with 100 µg of a potent GRPR antagonist BAY86-7548[164] led to ~ 90% reduction in intestinal and pancreatic uptake , and ~ 60% reduction of uptake in PC-3 tumor at 1 h p.i. Tumor-to-background (blood, muscle, liver and kidney) ratios increased significantly over time, and the tumor-to-blood and tumor-to-muscle ratios reached 15.6 ± 14.6 and 75.4 ± 5.53, respectively, at 2 h p.i. The PET/CT images of 18F-AmBF3-MJ9 in PC-3 tumor-bearing mice were consistent with post-mortem biodistribution data (Table 3.1). As shown in Figure 2.16, low background uptake and high tumor-to-background ratios of 18F-AmBF3-MJ9 were observed at 1 h (Figure 3.5-A) and 2 h (Figure 3.5-B) p.i. The radioactivity 64  was excreted mainly through the renal pathway. Pre-blocking with BAY86-7548 (Figure 3.5-C) significantly reduced tumor uptake and tumor-to-background contrasts at 1 h p.i.                     65  Table 3.1 Biodistribution and tumor-to-background ratios of 18F-AmBF3-MJ9 in NSG mice bearing PC-3 tumors. Uptake data are presented as %ID/g of tissues (n=3).  Tissues 0.5 h 1 h 1 h pre-blocked 2 h Blood 2.10 ± 0.94 0.56 ± 0.37 0.48 ± 0.27 0.32 ± 0.35 Fat 0.25 ± 0.11 0.07 ± 0.03 0.12 ± 0.07 0.05 ± 0.05 Seminal gland 0.34 ± 0.13 0.70 ± 1.05 0.08 ± 0.04 0.16 ± 0.24 Testis 0.42 ± 0.05 0.25 ± 0.15 0.25 ± 0.18 0.13 ± 0.15 Intestine 4.27 ± 5.03 6.42 ± 3.13 0.25 ± 0.14 8.67 ± 11.5 Spleen 1.12 ± 0.29 0.33 ± 0.13 0.32 ± 0.09 0.26 ± 0.09 Liver 2.64 ± 0.56 0.81 ± 0.16 1.05 ± 0.34 0.33 ± 0.07 Pancreas 8.04 ± 3.64 4.16 ± 1.34 0.42 ± 0.48 4.79 ± 2.34 Adrenal gland 1.16 ± 0.12 0.50 ± 0.32 0.22 ± 0.15 0.30 ± 0.11 Kidney 6.60 ± 0.97 2.43 ± 0.39 6.45 ± 4.35 1.46 ± 0.29 Lung 2.04 ± 1.01 0.97 ± 0.20 0.73 ± 0.30 0.41 ± 0.10 Heart 0.54 ± 0.09 0.13 ± 0.02 0.18 ± 0.08 0.06 ± 0.02 Tumor 2.80 ± 0.77 1.37 ± 0.25 0.54 ± 0.27 2.20 ± 0.13 Muscle 0.39 ± 0.02 0.07 ± 0.03 0.13 ± 0.06 0.03 ± 0.00 Bone 0.70 ± 0.23 0.28 ± 0.08 0.27 ± 0.20 0.18 ± 0.01 Brain 0.07 ± 0.01 0.02 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 Target-to-nontarget ratios     Tumor-to-blood 1.53 ± 0.71 3.33 ± 2.06 1.23 ± 0.31 15.6 ± 14.6 Tumor-to-muscle 7.23 ± 1.59 25.7 ± 19.4 4.04 ± 0.18 75.4 ± 5.53 Tumor-to-liver 1.08 ± 0.31 1.77 ± 0.60 0.50 ± 0.11 6.73 ± 1.12 Tumor-to-kidney 0.43 ± 0.09 0.58 ± 0.20 0.09 ± 0.02 1.54 ± 0.24   66    Figure 3.5 Small-animal PET/CT images of 18F-AmBF3-MJ9 in PC-3 prostate tumor bearing mice at 1 h (A) and 2 h (B) p.i., and at 1 h p.i. with pre-injection of 100 µg of BAY86-7548 (C).  The unit of color bars is in %ID/g ranging from 0-3%ID/g. B: bladder, I: intestine, K: kidney, T: tumor. 67  3.4 Discussion Development of radiolabeled bombesin derivatives for imaging GRPR-expressing cancers has been an active research area in nuclear medicine since the late 1990s [169, 170]. However, the focus has gradually shifted from labeling agonists with SPECT isotopes [169, 170] such as 99mTc and 111In to labeling antagonists with PET isotopes [161-163, 171, 172] including 68Ga, 64Cu and 18F. The advantages of PET include higher sensitivity and resolution compared to SPECT which leads to improved image quality and the ability to detect smaller lesions. In terms of pharmacological properties, it has recently been demonstrated by Cescato et al.[160] that radiolabeled GRP receptor antagonists were superior to agonists for in vivo imaging.  Although PET imaging provides many advantages over SPECT and while 18F is often the preferred PET isotope, labeling peptides with 18F is not trivial, and often involves several lengthy reaction/separation steps, resulting in low radiochemical yields and low specific activities. To meet the challenge of peptide labeling, McBride et al. [173] introduced a novel strategy for labeling peptides with 18F via the complexation of [Al18F]2+ in aqueous solution with a polyaminocarboxylate chelator on the peptide. Such facile strategy eliminates the tedious water drying step, and the final 18F-labeled product could be simply purified by solid-phase extraction using a C18 Sep-Pak cartridge. This approach was quickly adopted for the preparation of 18F-labeled peptides including GRPR-targeting RM26 derivatives Al18F-NOTA-P2-RM26 [171] and Al18F-NODAGA-RM1 [172].    In this study, we introduced a novel GRPR-targeting peptide 18F-AmBF3-MJ9 for PET imaging of prostate cancer. AmBF3-MJ9 was designed for labeling via the 18F-19F isotope exchange reaction on AmBF3 recently reported by Perrin et al. [167, 174] The AmBF3-68  conjugated alkyne 2 (refer to Scheme 3.1) was coupled to an RM26 derivative, azidoacetyl-MJ9, via Cu-catalyzed 2+3 dipolar cycloaddition to yield AmBF3-MJ9. As expected, AmBF3-MJ9 retained high binding affinity (Ki = 0.5 ± 0.1 nM) to GRPR. The modification at the N-terminus of BBN derivatives has been shown to be tolerable, and the resulting peptides generally retained high binding affinity to GRPR [125, 156, 163, 165, 168, 171, 172]. 18F labeling of AmBF3-MJ9 was performed via a one-step 18F-19F isotope exchange reaction in aqueous solution resulting in an overall radiochemical yield of 23 ± 5% (n=3). Similar to the NOTA-Al18F labeling, isotope exchange labeling eliminates tedious drying and HPLC purification steps, and the final 18F-labeled products could be obtained in <30 min of overall reaction/purification time. Recently, the conditions for labeling peptides via NOTA-[Al18F]2+ complexation have been optimized, and up to 97% radiochemical yield could be obtained in the presence of 80% (v/v) acetonitrile or ethanol [175, 176]. Nevertheless, for such radiolabeling the ratio of Al3+, starting 18F-fluoride and peptide precursor needs to be taken into account to ensure high radiochemical yield. Despite a lower radiochemical yield herein, isotope exchange offers a simpler radiolabeling approach that does not require coordinated ratios of multiple components.   18F-AmBF3-MJ9 was metabolically stable in mouse plasma after 2 h incubation at 37 ºC. This is consistent with high metabolic stability reported for other radiolabeled RM26 derivatives [172, 177]. Internalization study showed that the fraction (5.32 – 5.84%) of membrane bound 18F-AmBF3-MJ9 remained relatively constant between 30 – 90 min incubation, indicating its fast binding kinetics to GRPR. The internalized fraction accounts for ~30% of total cell-associated radioactivity at 30 min, and gradually decreased to ~22% at 90 min. The extent of 69  internalization (22- 30%) of 18F-AmBF3-MJ9 is within similar range (14 – 35%) of reported radiolabeled RM26 derivatives [162, 163, 171, 177]. Interestingly, for reported 68Ga- and 111In-labeled RM26 derivatives [163], the percentages of internalized fraction increased over time indicating the residualizing characteristics of 68Ga and 111In radiolabels after being internalized into cells. In the case of 18F-AmBF3-MJ9, the internalized fraction slightly decreased over time (2.37 ± 0.29 % at 30 min to 1.54 ± 0.27 % at 90 min).  In vivo biodistribution (Table 3.1) and imaging (Fig. 2.16) studies in PC-3 tumor-bearing mice showed excellent tumor visualization and higher tumor contrast ratios at later time points. As shown in Table 2.5, tumor-to-blood, tumor-to-muscle, and tumor-to-liver ratios increased from 1.53 to 15.6 (10-fold), 7.23 to 75.4 (10-fold), and 1.08 to 6.73 (6-fold), respectively from 0.5 to 2 h p.i. Pre-blocking with BAY86-7548 significantly reduced the uptake of 18F-AmBF3-MJ9 in GRPR-expressing PC-3 tumors, pancreas, and intestine, confirming the uptake in these tissues was GRPR-mediated. Only minimal bone uptake (0.18 ± 0.01 %ID/g) was observed indicating that there was no significant in vivo defluorination of 18F-AmBF3-MJ9.  The absolute PC-3 tumor uptake value was lower with 18F-AmBF3-MJ9 (2.20 ± 0.13 %ID/g at 2 h p.i.) than the reported 18F-labeled RM26 derivatives, Al18F-NOTA-P2-RM26 [171] (5.53 ± 0.75 %ID/g at 3 h p.i.) and Al18F-NODAGA-RM1 [172] (5.25 ± 0.84 %ID/g at 2 h p.i.). Although inferior to Al18F-NOTA-P2-RM26, the tumor-to-background contrasts of 18F-AmBF3-MJ9 were similar to those of Al18F-NODAGA-RM1. Most importantly, we demonstrated that reliable radiochemical yields (~ 20%) and high specific activity (~100 GBq/µmol) of 18F-AmBF3-MJ9 could be obtained starting with ~37 GBq of 18F-fluoride. These numbers are consistent with the radiochemical yield and specific activity of previously 70  reported somatostatin receptor targeting tracer, 18F-AmBF3-TATE [174]. The final radioactivity of 18F-AmBF3-MJ9 was obtained in ~7 GBq which is enough for multiple doses, and could be potentially delivered for use at multiple sites from a single production batch. Al18F-NOTA-P2-RM26 and Al18F-NODAGA-RM1 were prepared starting with lower quantities (1 – 4 GBq) of 18F-fluoride. Considering the widespread availability of 18F, and the straightforward labeling technique using the 18F-19F isotope exchange reaction, 18F-AmBF3-MJ9 is a promising probe for non-invasive imaging of GRPR in humans. The in vivo data supports further studies on the potential utility of this radiotracer for detecting prostate cancers and other GRP receptor positive cancers in human subjects.  3.5 Conclusion  18F-AmBF3-MJ9 was synthesized and its potential evaluated as a GRP receptor-targeting PET tracer. 18F-AmBF3-MJ9 demonstrated excellent GRPR binding affinity, high plasma stability, and favorable pharmacokinetics. In addition, specific tumor targeting and excellent tumor-to-background contrasts further suggest that 18F-AmBF3-MJ9 is a promising radiotracer for GRPR-targeting imaging.      71  Chapter 4: Somatostatin receptors, trifluoroborate isotope exchange reaction approach 4.1 Background Somatostatin consists of a family of a 14-amino acid (somatostatin-14) and a 28-amino acid (somatostatin-28) peptide. It is considered  a neurotransmitter, neurohormone, or a normal hormone acting via autocrine or paracrine mechanisms in several organ systems, such as the central nervous system, the hypothalamopituitary system, the gastrointestinal tract, the exocrine and endocrine pancreas, and the immune system [178]. The action of natural somatostatin or synthetic somatostatin (octreotide) is mediated by high affinity somatostatin receptors located on the plasma membrane of the target cells. Five human somatostatin receptor sub-types (sst1, sst2, sst3, sst4, sst5) have been cloned and characterized [179, 180].  Inhibition of adenylylcyclase cAMP- protein kinase A pathway and modulation of potassium channels by somatostatin are two of the most widely studied and documented systems [178]. The inhibitory action of somatostatin through MAPK pathways, as an important aspect of somatostatin signaling has been well studied [70]. MAPK activation plays an important role in cell proliferation [181], therefore inhibition of this pathway could be linked to the antiproliferative effect of somatostatin. The role of somatostatin in stimulating apoptotic mechanisms in sst2- or sst3- expressing cells [182, 183] is another notable antiproliferative mechanism. Other signaling pathways regulated by somatostatin include the somatostatin induced stimulation of phospholipase A2 [184], and the activation of phosphotyrosine 72  phosphatases [185]. Pharmacological studies of somatostatin showed that all five human subtypes bind somatostatin-14 and somatostatin-28 with a high affinity. However, there are differences in the binding affinities of analogs of somatostatin; for instance, octreotide is bound with high affinity to the sst2 and sst5 receptor subtypes and with a moderate affinity to sst3, but not to sst1 and sst4 [180]. There are also differences in internalization capabilities of the different receptor subtypes [178, 186]. Sst3 and sst2 internalize much better than sst1 upon ligand binding [186]. Sst5 not only internalizes after ligand binding but can also trigger a massive recruitment of sst5 receptors from intracellular stores to the membrane [187].  Over the past 30 years there has been considerable interest in developing high-affinity somatostatin derived ligands that bind SST2 receptors, notably for radionuclide therapy [188]. To diagnose and monitor patients with SSTR2-positive tumors, radiotracers based on the somatostatin family of peptides [189-194], notably octreotate (TATE) and octreotide, have been labeled with various radioisotopes for noninvasive imaging. 111In-diethylenetriaminepentaacetic acid-pentetreotide (Octreoscan; Mallinckrodt) is the current clinical standard for imaging neuroendocrine tumors [195-197]. 99mTc derivatives such as 99mTc-depreotide [198] and 99mTc-hydrazinonicotinyl-Tyr3-octreotide have also been used [199] but are not commercialized in North America.  For PET imaging, 68Ga, 64Cu, and 18F along with various radio-prosthetics have been conjugated to various octreotide derivatives [200-205]. Of these, certain 68Ga ligands such as 68Ga-DOTA-TOC, 68Ga-DOTA-TATE, and 68Ga-DOTA-NOC have shown promise for neuroendocrine tumor imaging [206-208] and are used in clinical trials as well as under the local practice of pharmacy, particularly in Europe. Nevertheless, 68Ga-PET imaging is not 73  widely available because of the limited daily availability of 68Ga (~50 mCi) and the lack of Food and Drug Administration–approved 68Ge/68Ga generators [209]. 18F-fluoride presents several attractive properties for imaging [210, 211] and is produced on a daily basis in large quantities in hundreds of cyclotrons in hospitals and radiopharmacies worldwide. Yet the challenges of labeling peptides with 18F-fluoride are significant: the low chemical reactivity of 18F-fluoride in water [212] and short half-life (109.8 min) challenge 18F labeling of peptides that are generally soluble only in water or aqueous cosolvents. Hence, fluoride must be dried and reacted in dry solvents at high temperature to radiolabel a radioprosthetic that is then conjugated to the peptide in at least one additional step. Although such multistep 18F-labeling reactions are commonplace [213], the relatively short half-life of 18F-fluoride often hampers the clinical application of multistep reactions, particularly in terms of ensuring specific activity > 37 GBq/mmol (1 Ci/mmol) [214]. Given these challenges, an SSTR2 ligand that is easily labeled with 18F-fluoride in high yield and at high specific activity would facilitate SSTR2 imaging by PET. Toward these ends, new 18F-octreotate derivatives, such as 18F-SiFA and Al-18F-NOTA, have been labeled in one step and imaged with relative success [176, 215, 216]. Similarly, aryltrifluoroborate prosthetics, when conjugated to various peptides, allow 1-step aqueous radiofluorination in high yield and very high specific activity [217-219]. Recently, we identified a new ammoniomethyl-BF3 (AmBF3) that undergoes facile 18F-19F isotope exchange. To test the efficacy of boron-based 18F labeling, we conjugated AmBF3 to octreotate by means of a simple chemical synthesis to obtain AmBF3-TATE, which as a precursor is 18F-labeled by isotope exchange (Compound 2, Scheme. 4.1).We report the radiosynthesis and preclinical evaluation of this new derivative. 74  4.2 Materials and methods Reagents and solvents were purchased from Advanced Chemtech, Sigma-Aldrich, Combi-Blocks, or Novabiochem. The AR42J cell line was purchased from ATCC. 18F-fluoride Trap & Release Columns were purchased from ORTG Inc. (Oakdale, TN) and C18 Sep-Pak cartridges (1cc, 50 mg) were obtained from Waters. An Endeavor 90 peptide synthesizer (Aaptec) was applied to synthesize the peptide. 4.2.1 HPLC Analysis The following gradients were used for HPLC purification and quality control: Method A: Agilent Eclipse XDB-C18 5 µm 9.2 x 250 mm semi-prep column. Solvent A: 0.1% TFA water; solvent B: MeCN; 0 to 15 min: 20% to 40% B, 15 to 20 min, 40% to 20% B. Flow rate: 4.5 mL/min, column temperature: 19 to 21 °C. Method B: Agilent Eclipse XDB-C18 5 µm 9.2 x 250 mm semi-prep column. Solvent A: 0.1% TFA water; solvent B: MeCN; 0 to 2 min: 5% to 20% B, 2 to 5 min, 20% to 30% B, 5 to 20 min, 30% to 50%, 20 to 22 min, 50% to 5% B. Flow rate: 3 mL/min, column temperature: 19 to 21 °C. Method C: Phenomenex Jupiter 10m C18 300Å 4.6 x 250 mm analytical column. Solvent A: 0.1% TFA water; solvent B: MeCN; 0 to 2 min: 5% to 5% B, 2 to 7 min, 5% to 20% B, 7 to 15 min, 20% to 100%, 15 to 20 min, 100% to 5% B. Flow rate: 2 mL/min, column temperature: 19 to 21 °C. 4.2.2 Peptide synthesis and 18F labeling The TATE peptide was synthesized as previously described [220]. Briefly, the NHS-ester of bromoacetic acid was coupled to the N-terminus followed by successive treatment with sodium azide to provide a TATE derivative suitable for click-conjugation. The peptide was 75  deprotected and cleaved from the resin by treatment with trifluoroacetic acid supplemented with trimethylsilane scavengers. Purification by HPLC with a semi-preparative column with Method A provided the final peptide in quantities of ~10 mg. The calculated mass was 1131.2 and measured at 1131.4 by mass spectrometry. The purity of the peptide was >99%. 4.2.3 Synthesis of N-propargyl-N, N-dimethyl-ammoniomethyl-boronylpinacolate N-propargyl-N,N-dimethyl-ammoniomethyl-boronylpinacolate (alkynyl-AMB(pin)) was synthesized by condensation of iodomethyl-boronylpinacolate and propargylamine by standard alkylative amine quaternization [221]. Briefly, a flame-dried round bottom flask was charged with N,N-dimethylpropargylamine (98 µL, 1.0 mmol) and 2 mL anhydrous diethyl ether under argon atmosphere to which Iodomethyl-pinacolboronate (165 µL, 0.9 mmol) was added drop-wise at room temperature. On stirring, the solution became cloudy followed by the formation of a white precipitate, which was the desired product. The resulting precipitate was filtered and washed with cold diethyl ether. Then the residue was dried under high vacuum to give a fluffy white powder in 95% yield. 1H NMR (300 MHz, CD3CN) δ 4.40 (d, 2H), 3.31 (s, 2H), 3.22 (s, 6H), 3.21 (t, 1H), 1.27 (s, 12H); ESI: calculated: 224.1; found: 224.1. 4.2.4 Synthesis of AmBF3-TATE The N-propargyl-N, N-dimethylammonio-methylboronylpinacolate (5.0 mg, 22.3 µmol) was converted to the corresponding trifluoroborate (alkynyl-AMBF3) through the addition of KHF2 (3 M, 30 µL in water), HCl (4M, 30 µL in water), deionized water (20 µL) and DMF (60 µL) at 45oC for 2 hours, and then quenched by NH4OH (conc., 10 µL). The crude reaction was directly used for click conjugation to TATE azide without further purification: a mixture 76  of TATE-azide (4.0 mg, 3.4 µmol), CuSO4 (1.0 M, 5.0 µL), sodium ascorbate (1.0 M, 12.5 µL) and 5% NH4OH (MeCN/H2O=1:1, 50 µL) was added, and the new mixture was warmed up to 45 °C for 2 hours. The overall synthetic route is shown in scheme 3.1. Purification was performed by Agilent 1100 HPLC system with Gradient B to isolate 2.3 mg of chemically pure AmBF3-TATE. The identity and purity was confirmed with LC-MS: Calculated: 1296.5; Obtained: 1296.4. For convenience and to ensure reproducibility, the purified 19F-AmBF3-TATE was diluted in ethanol and aliquoted in quantities of ~60 μg (~50 nmol) for radiolabeling.     Scheme 4.1 N3-TATE is condensed with N-propargyl-N,N-dimethyl-ammoniomethyltrifluoroborate (1) to provide the precursor AmBF3-TATE (2).  Precursor 2 is labeled by isotope exchange to provide the isotopolog 18F-2 at high specific activity for tracer studies.  77  4.2.5 In vitro binding assays  Membranes from Chinese Hamster Ovary cells (CHO-K1) transfected with sst2r and [125I]Tyr-somatostatin-14 were obtained commercially from PerkinElmer, Canada. A standard filtration binding assay was performed in 96-well filtration plates (MultiScreen, Millipore) to determine the binding affinity (Ki) of AmBF3-TATE. Briefly, 0.25 µL of the membranes per well were incubated with the 125I-labeled standard at a concentration of 0.05 nM, and increasing concentrations of non-radioactive AmBF3-TATE (competitor), in buffer consisting of 25 mM Hepes pH 7.4, 10 mM MgCl2, 1 mM CaCl2 and 0.5% BSA. After incubation at 37 ºC for 1 h, the wells were aspirated and washed 8 times with 50 mM of ice-cold wash buffer (TRIS-HCL pH 7.4) over GF/B filters. The filters were removed and counted by a gamma counter (Cobra II, Packard). The experiment was repeated in triplicate. Data were fitted to a one-site competition model (GraphPad Prism 6.1 software) to calculate the inhibition constant (Ki). 4.2.6 Metabolic stability in mouse plasma The purified 18F-AmBF3-TATE in its saline formulation was assayed for plasma stability. For a plasma stability assay 20 µL of 18F-AmBF3-TATE was added to mouse plasma (500 µL) and incubated at 37 °C for 0, 60 and 120 min. Following incubation at each time point, the reaction was quenched by adding 1 mL MeCN to precipitate insoluble proteins from the solution. The quenched reactions were centrifuged at 13,000 RPM at 4 °C to remove insoluble material. The supernatant was aspirated by pipette, filtered, and analyzed by HPLC using Method C. 78  4.2.7 Radiolabeling Just prior to radiosynthesis, AmBF3-TATE was resuspended in ~50 µL aqueous pyridazine-HCl buffer (pH = 2) in a vial (polypropylene Falcon Tube). No carrier-added 18F-fluoride 29.6-37 GBq (800-1000 mCi) was obtained by bombardment of H218O with 18 MeV protons, followed by trapping on an anion exchange resin (9 mg, QMA, chloride form, prewashed with deionized water). The 18F-fluoride was eluted with 70-100 µL isotonic saline into the reaction vial containing AmBF3-TATE. The vial was placed in a heating block set at 80 °C for 20 min whereupon the reaction was quenched by the injection of 2 mL 5% NH4OH in water. The reaction mixture was loaded onto a C18 light cartridge. The impurities (i.e. free 18F-fluoride) were removed by flushing with 2 mL saline. Radiochemically pure 18F-AmBF3-TATE was then released into a glass vial by elution with 0.5 mL 1:1 ethanol/saline to provide 7.4 GBq (200 mCi) tracer. This solution was formulated into isotonic saline (5 mL) for imaging. A small sample was removed for quality control analysis by HPLC.  4.2.8 Internalization studies The experiments were performed in triplicates at 30, 60 and 90 min incubation at 37 °C according to previously published procedures [168].  Briefly, AR42J cells were plated in 6-well plates at a density of 0.8-1 million cells per well and incubated overnight. DMEM containing 1% FBS was used as a culture medium. On the day of the experiment, the cells were washed with PBS and 1 ml of culture medium containing 2.5 pmol of 18F-AmBF3-TATE was added to each well. In separate wells with the same setup, AmBF3-TATE to a final concentration of 1 µM was added for the determination of nonspecific binding. After incubation at 37 °C to the pre-determined time point, the cells were washed twice with ice-79  cold PBS followed by two acid washes (Glycine-HCl, pH 2.8) for 10 min on ice. To measure the internalized activity, the cells were lysed with 1M NaOH, the solution was counted on a gamma counter. 4.2.9 Animal models and biodistribution studies All animal studies were performed in accordance with the Canadian Council on Animal Care Guidelines and were approved by the animal care committee of the University of British Columbia. 107 Rat pancreatic adenocarcinoma cells (AR42J) were freshly expanded in PBS/matrigel mixture and inoculated subcutaneously in female immunocompromised mice (nod scid IL2r-γ-null, bred in house). The tumors were grown for 2 weeks until they reached 5-7 mm in diameter. While under 2% isoflurane anesthesia, the mice were injected via the tail vein with 0.37-0.74 MBq (10-20 µCi) of 18F-AMBF3-TATE (n=5). To demonstrate the specificity of uptake in vivo in receptor positive tissues, 100 µg of Ga-DOTA-TATE was pre-injected 15 minutes prior to 18F-AmBF3-TATE injection as a blocking control cohort (n=4). Sixty minutes following injection, the mice were anesthetized with isoflurane and euthanized by carbon dioxide. The organs were harvested, rinsed with saline, blotted dry and collected in previously weighted tubes. The tubes containing the organs were counted in a gamma counter (Cobra-II, Packard). The tissue weight and associated count per minute were used to calculate the percentage injected dose per gram of tissue (%ID/g). 4.2.10 PET/CT imaging The imaging studies were acquired using a multimodality PET/CT system (Inveon, Siemens). For imaging, a baseline low-dose CT scan was obtained for localization and attenuation 80  correction. ~3.7 MBq of radiotracer was injected in the caudal lateral tail vein of tumor-bearing mice. A dynamic scan was acquired in list-mode for 60 minutes while the animal was kept warm by a heated pad on the scanner bed. Ga-DOTA-TATE (100 µg per mouse) was pre-injected as a blocking agent in some animals. Following scanning, the mice were euthanized, while under anesthesia, by CO2 inhalation. The images were reconstructed by an iterative reconstruction algorithm (3D OSEM/MAP) using the Inveon Acquisition Workplace Software (Siemens), applying normalization, dead time, random and attenuation corrections. The uptake of tumor and tissues of interest were determined by region of interest (ROI), and the %ID/g was calculated (assuming a tissue density of 1.0 g/cc). The mean %ID/g was calculated by drawing a ROI to match the tumor contours visible on CT. The peak %ID/g was calculated from the hottest 2x2 voxel cluster within the tumor. 4.3 Results 4.3.1 In vitro binding assay The inhibition constant (Ki) of AmBF3-TATE using human SSTR2 receptors expressed on CHO membranes was 0.1 ± 0.03 nM. Using identical assay conditions and the same lot of membranes, the binding affinity of Ga-DOTA-TATE was 0.7 ± 0.2 nM. A representative competitive binding assay curve is shown in Figure 4.1.   81   Figure 4.1 Representative example of a competitive binding assay for 19F-AmBF3-TATE-: x-axis: log[AmBF3-TATE], y-axis: counts bound. The assay was run with triplicate data points.    4.3.2 Radiosynthesis Starting with 29.6-37 GBq (800-1000 mCi) of 18F-fluoride, ~7.4 GBq (200 mCi) of 18F-AMBF3-TATE was reproducibly obtained within 25 min (25±3%, n=5). Purified 18F-AMBF3-TATE was re-injected into HPLC for quality analysis (Figure 4.2). As expected, only one peak was observed in both radioactive mode and UV mode. As ~7.4 GBq (~200 mCi) of 2 was obtained starting with 50 nmol of precursor, a simple calculation suggests that the specific activity is 148 GBq/µmol. To validate this calculation, a standard curve was employed and showed that the specific activity was >111 GBq/µmol (>3 Ci/µmol).    82   Figure 4.2 HPLC traces of Sep-Pak purified 18F-AmBF3-TATE; top – the UV trace measured at 277 nm; bottom – radioactivity trace.  4.3.3 Metabolic stability in mouse plasma For plasma stability, 18F-AmBF3-TATE was incubated in mouse plasma for 120 min with no detectable loss of fluoride or other decomposition (Figure 4.3).  83   Figure 4.3 Plasma stability assay of 18F-AmBF3-TATE; radiotraces shown for 0, 60 and 120 min (not decay corrected).  4.3.4 Internalization studies Internalization assay was studied in rat pancreatic cancer (AR42J) cells to identify agonist or antagonist properties of 18F-Am BF3-TATE. The internalized fraction displayed as percentage of applied radioactivity reached 3.8% at 30 min, and reached a plateau (4.6 %) within 120 84  min. Membrane bound was 3.8% at 30 min and 6.8% at 120 min incubation at 37 °C (Figure 4.4).   Figure 4.4 Internalization of 18F-AmBF3-TATE in AR42J cells in 120 min incubation.  4.3.5 Biodistribution studies The biodistribution data of 18F-AmBF3-TATE at 1 hour post-injection are summarized in Table 4.1. The uptake in AR42J xenograft tumors in the unblocked mice was 10.11 ± 1.67% ID/g. As expected in the blocking controls, the presence of excess competitor caused a substantial reduction in apparent tumor uptake: 0.32 ± 0.21% ID/g. Hence blocking efficiency was 97%. The uptake values of blood and muscle (1 hour post-injection) were low: 0.40 ± 0.31% ID/g and 0.11 ± 0.03% ID/g, respectively, which corresponded to very high tumor-to-blood and tumor-to-muscle ratios of 25.1 ± 1.0 and 89.0 ± 3.1 respectively. Negligible bone uptake was detected (0.46 ± 0.17% ID/g, 1 hour after injection), indicating no in vivo defluorination. 85   Table 4.1 Biodistribution of 18F-AMBF3-TATE in AR42J tumor-bearing mice. Data represent mean ± SD of the %ID/g (unblocked: n=6, blocked: n=4).  Tissues Unblocked Blocked Blood 0.40  ± 0.31 0.32 ± 0.15 Plasma 0.72  ± 0.71 0.92 ± 0.16 Uterus 0.26  ± 0.05 0.51 ± 0.11 Large intestine 2.28  ± 2.64 4.27 ± 6.20 Small intestine 3.23  ± 1.58 1.82 ± 1.70 Spleen 0.42  ± 0.19 0.31 ± 0.11 Liver 0.39  ± 0.05 0.41 ± 0.14 Pancreas 2.81  ± 1.49 0.20 ± 0.01 Adrenal gland 0.54 ± 0.18 0.28 ± 0.07 Kidney 4.90  ± 1.54 4.50 ± 3.54 Lungs 1.85  ± 0.83 0.79 ± 0.26 Heart 0.17  ± 0.05 0.88 ± 1.12 Tumor 10.11  ± 1.67 0.32 ± 0.21 Muscle 0.11 ± 0.03 0.11 ± 0.09 Bone 0.46 ± 0.17 0.54 ± 0.36 Brain 0.03 ± 0.01 0.22 ± 0.33  86  4.3.6 PET/CT imaging The tracer accumulated intensely in the AR42J tumor (Figure 4.5 - left) and uptake was clearly specific as evidenced by the lack of observed uptake in the tumor of the mice receiving the competitor (Figure 4.5 - right). Based on the imaging studies, the average of the tumor uptake based on the whole tumor ROI was 10.2 ± 2.1 %ID/g. The average of the peak tumor uptake based on the hottest voxel cluster was 23.6 ± 3.0 %ID/g. In contrast, the average uptake of the liver, blood pool and muscle were 0.83 ± 0.16, 0.47 ± 0.12 and 0.09 ± 0.03 %ID/g, respectively. Excretion was predominantly renal with significant clearance to the bladder and low kidney retention. Some excretion via the hepatobiliary tract was noted and liver clearance was fast, leading to high tumor-to-liver ratios. Bone uptake was undetectable and there was low background activity in blood and muscle, resulting in very high contrast images.    87    Figure 4.5  PET/CT (up) and PET (low) images of 18F-AmBF3-TATE PET using AR42J tumor-bearing mice at 60 min post-injection: unblocked (left) and blocked (right). The tracer specifically accumulated into the tumor (T) while the remainder rapidly cleared via the kidneys (K) to the bladder (B). Some intestine (I) and gallbladder (G) accumulation occurs due to rapid hepatobiliary excretion.  88  4.3.7 Kinetic analysis Time activity curves (TAC) of uptake in tumor and other tissues are presented in Figure 4.6. Time dependent tumor uptake increased to a peak voxel cluster value ~24% ID/g.  As expected, uptake in non-target tissues rapidly declined after reaching the peak value at early time point soon after intravenous administration (Figure 4.7).   Figure 4.6 Time activity curves of tumor in unblocked and blocked imaging studies with 18F-AmBF3-TATE.     89   Figure 4.7 (A)Time activity curves indicating blood, liver, kidney clearance and tumor uptake, and (B) uptake of hottest clusters.  4.4 Discussion For radiosynthesis, isotope exchange labeling on the organotrifluoroborate prosthetic, sub-90  milligram quantities of precursor were used, and labeling was completed in ~20 minutes with a high specific activity of 148 Ci/µmol. Since the precursor was isotopically identical to the radiolabeled product, HPLC purification of precursor was not necessary to remove unincorporated free fluoride. In addition to the operational simplicity of this method, notably all operations were performed in a completely shielded hot-cell, in which Curie-level labeling was performed to enable multiple human doses in a single run. As increased market demand for 18F-FDG has coincided with improvements in cyclotron output to provide multi-Curie-levels of 18F-fluoride [222], the methodology is readily applicable to existing production facilities.  Whereas good radiosynthetic features are a requirement for tracer use, the value of any tracer ultimately lies in the in vivo image data and corroborating biodistribution data. Among the published TATE-based radiotracers, Ga-DOTA-TATE has the best-reported affinity for SSTR2 to date. Using identical assay conditions, 18F-AmBF3-TATE had better binding affinity than Ga-DOTA-TATE to the SST2a receptor. This was both unanticipated and significant. The sensitivity of somatostatin analogs, either agonists or antagonists, to substitutions at the N-terminus as well as to the radiometal is well documented [223, 224]. Here the octreotate was modified by adding AmBF3 via copper-catalyzed click conjugation, and we expected a potential decrease in the binding affinity to the SSTR2. Instead, we observed a 7-fold better binding affinity compared to 68Ga-DOTA-TATE using assays performed in the exact same condition. This suggests that other zwitterionic moieties at the N-terminus may improve affinity. Based on PET/CT imaging, 18F-AmBF3-TATE exhibited high receptor mediated uptake in a preclinical murine model of SSTR2-positive cancer with very 91  low background activity in non-target tissues. The in vivo imaging data was consistent with the biodistribution data verified the high tumor uptake values. While liver uptake of radiometalated octreotide derivatives is typically low, this is not always the case for 18F-labelled peptides. Liver uptake and in particular non-specific uptake is often observed and may preclude clinical detection of liver metastasis. Interestingly, as shown in PET/CT and biodistribution, the liver uptake of 18F-AmBF3-TATE was very low, resulting higher tumor-to-liver ratio (26.21 ± 0.79, 1 hour after injection) compared to other reported 18F-labelled TATE analogs (0.25-5.0, 2 hours after injection) [225].  A plasma stability assay was accomplished by incubating 18F-AmBF3-TATE at 37 °C for 120 min, the decomposition was negligible and the intact radiotracer was still dominant in the plasma. Consistent with this finding, minimal bone uptake (0.46 ± 0.13% ID/g, 1 hour after injection) was observed in both PET/CT and biodistribution, resulting high tumor-to-bone ratio of up to 21.3 ± 3.6. This low non-specific bone uptake is particularly encouraging for the detection of bone metastasis. Moreover this result highlights the general stability of such alkyltrifluoroborate radioprosthetics, an observation that should augur well for the development of other peptide tracers based on the same zwitterionic ammoniomethyl-BF3. 4.5 Conclusion We report an ammoniomethyl-BF3 octreotate derivative that was radiolabeled with 18F in high yield and high specific activity using a facile exchange reaction using minute quantities of precursor peptide, without HPLC purification. This methodology provides for rapid multidose production of a high affinity 18F-labeled octreotate in a single production run that should be easily amenable to automation. In addition to radiosynthetic ease, the biological evaluation of 92  18F-AmBF3-TATE indicates that this tracer provides good stability, optimal pharmacokinetics, excellent binding affinity, and very high tumor to non-target tissue ratios for in vivo imaging of SSTR positive tumors.            93  Chapter 5:  Evaluation of the potential of positron-emitting somatostatin analogs to detect breast cancer 5.1 Background Breast cancer is one of the most occurring cancers in females in North America [49]. High expression of somatostatin receptor (SST) subtype 2a (SST2a) has been shown in a variety of malignancies including neuroendocrine tumors and breast cancer. Reubi et al.[226] showed SST2a receptors are highly expressed in 31 out of 32 cases of breast cancer by autoradiography. Other investigators have shown a very high prevalence of SST mRNA expression in breast cancers [227]. SST expression is known to be modulated by the estrogen receptor, and SST2a imaging has been proposed as a surrogate marker of estrogenic activity [228]. However, most of reported studies on the development of novel radiotracers for imaging SST2a expression used mice bearing tumors derived from either SST2a transfected cell lines or rat pancreatic cancer cell line (AR42J).  The diagnostic performance of somatostatin receptor scintigraphy with single photon emitters in women with primary or metastatic breast cancer has been suboptimal to date. This could be due to insufficient sensitivity of the imaging probes, insufficient spatial resolution of conventional imaging by single photon emission computed tomography, or both. The purpose of this study was to evaluate whether radioligands used for PET imaging of SST2a could be suitable for the detection of breast cancer, and whether antagonists are preferable to agonists. Two ER-positive human breast cancer cell lines were screened, and xenograft models were evaluated for SST2a detection by PET/CT imaging. We compared 94  68Ga-DOTA-TATE as an established agonist with 68Ga-NODAGA-LM3 and 68Ga-NOTA-BASS as antagonists. 5.2 Materials and methods Reagents and solvents were purchased from Advanced Chemtech, Sigma-Aldrich, Combi-Blocks, or Novabiochem. A 68Ga-generator was purchased from iThemba , and C18 Sep-Pak cartridges (1cc, 50 mg) were obtained from Waters. An Endeavor 90 peptide synthesizer (Aaptec) was used to synthesize the peptides. The human breast cancer cell line ZR-75-1 was obtained from American Type Culture Collection and MCF-7 as a gift from Dr. C. Kent Osborne from Baylor College of Medicine. HEK293-SST2a cells were provided by Dr. S. Shulz from Jena University Hospital, Germany. 60-day release 17β-estradiol was purchased from Innovative Research of America. Filter plates were obtained from Millipore and 125I-Tyr-somatostatin-14 (2200 Ci/mmol) was purchased from Perkin Elmer, Canada. All culture reagents purchased from Sigma Aldrich or Invitrogen. All chemicals and solvents were obtained from commercial sources, and used without further purification. Peptides were synthesized using solid phase approach on an Aapptec (Louisville, KY) Endeavor 90 peptide synthesizer. The collected HPLC eluates containing the desired peptide were lyophilized using a Labconco (Kansas City, MO) FreeZone 4.5 Plus freeze-drier. MS analyses of peptides were performed at the Genome Sciences Centre of BC Cancer Agency on a AB/Sciex 4000 QTrap mass spectrometer coupled to a Agilent (Santa Clara, CA) 1100 liquid chromatograph system. Purification and quality control of cold and 68Ga-labeled peptides were performed on an Agilent HPLC System equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (set at 220 nM), and a Bioscan (Washington, 95  DC) NaI scintillation detector. The radio-detector was connected to a Bioscan B-FC-1000 Flow-count System, and the output from the Bioscan Flow-count system was fed into an Agilent 35900E Interface which converted the analog signal to digital signal. The operation of the Agilent HPLC system was controlled using the Agilent ChemStation software. The HPLC columns used were a semi-preparative column (Phenomenex C18, 5 µ, 250 × 10 mm) and an analytical column (Eclipse XOB-C18, 5 µ, 150 × 4 mm). The HPLC solvents were A: H2O containing 0.1% TFA, and B: MeCN containing 0.1% TFA. An Eckert & Ziegler (Berlin, Germany) IGG100 68Ga generator was used to obtain 68Ga. Radioactivity of 68Ga-labeled peptides was measured using a Capintec (Ramsey, NJ) CRC®-25R/W dose calibrator, and the radioactivity of mouse tissues collected from biodistribution studies were counted using a Packard (Meriden, CT) Cobra II 5000 Series auto-gamma counter.  5.2.1 Peptide synthesis  The Ga-DOTA-TATE, Ga-NODAGA-LM3 and Ga-NOTA-BASS were prepared by Fmoc solid-phase synthesis as described previously [229], as follows: 5.2.1.1 NOTA-BASS  NOTA-BASS was synthesized via the Nα-Fmoc solid-phase peptide synthesis strategy starting from Fmoc-D-Tyr(tBu)-Rink Amide MBHA resin. The resin was treated with 20% piperidine (1×5 min and 1×10 min) in DMF to remove the Nα-Fmoc protecting group. The following Fmoc-protected amino acids (3 equivalents) including Fmoc-Cys(Acm)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-D-Cys(Acm)-OH, and Fmoc-p-NO2-Phe-OH were subsequently coupled to the sequence according to their 96  order. The coupling was carried out in NMP with standard in situ activating reagent HBTU (3 equivalents) in the presence of DIEA (6 equivalents). After the peptide sequence was synthesized, thallium(III) trifluoroacetate (2 equivalents) in DMF was used to remove the Cys’s side-chain protecting group, Acm, and form disulfide, concomitantly. Bromoacetic acid (40 equivalents) was pre-activated with DIC (20 equivalents) in DCM for 10 min, filtered, and coupled with the cyclic peptide, successively. At the end of the synthesis, di-tert-butyl 2,2'-(1,4,7-triazacyclononane-1,4-dilyl)diacetate (1.7 equivalents) with DIEA (5 equivalents) in NMP was coupled to the N-terminus of the peptide. At the end of elongation, the peptide was de-protected and simultaneously cleaved from the resin by the treatment of 90/2.5/2.5/5 TFA/H2O/TIS/phenol for 2 h at room temperature. After filtration, the peptide was precipitated by the addition of cold diethyl ether to the TFA solution. The precipitated crude peptides were collected by centrifugation, and purified by HPLC using the semi-preparative column eluted with a gradient condition of 80/20 A/B to 65/35 A/B in 30 min at a flow rate of 4.5 mL/min. The retention time of NOTA-BASS was 19.5 min. The HPLC eluates containing the desired peptide were collected, pooled, and lyophilized. The yield of the peptide was 20 %.  5.2.1.2 NatGa-NOTA-BASS A solution of NOTA-BASS (2.4 µmol) and GaCl3 (35.0 µmol) in 300 µL acetonitrile and 2 mL sodium acetate buffer (0.05 M, pH 5.0) was incubated at room temperature for 30 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with a gradient condition of 80/20 A/B to 65/35 A/B in 30 min at a flow rate of 4.5 mL/min. The 97  retention time of Ga-NOTA-BASS was 15.6 min. The yield of this reaction was 99%. MS (EPI) calculated for Ga-NOTA-BASS GaC66H82N15O18S2 (M + 2H)2+ 753.74, found 753.90.  5.2.1.3 DOTA-TATE DOTA-TATE was synthesized via the Nα-Fmoc solid-phase peptide synthesis strategy starting from Fmoc-Thr(tBu)-Wang resin. The resin was treated with 20% piperidine (1×5 min and 1×10 min) in DMF to remove the Nα-Fmoc protecting group. The following Fmoc-protected amino acids (3 equivalents) including Fmoc-Cys(Acm)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH,  and Fmoc-D-Phe-OH were subsequently coupled to the sequence according to their order. The coupling was carried out in NMP with standard in situ activating reagent HBTU (3 equivalents) in the presence of DIEA (6 equivalents). After the peptide sequence was synthesized, thallium(III) trifluoroacetate (2 equivalents) in DMF was used to remove the Cys’s side-chain protecting group, Acm, and form disulfide, concomitantly. DOTA tri-t-butyl ester (3 equivalents) was pre-activated with DCC (3 equivalents) and N-Hydroxysuccinimide (3.6 equivalents) in DMF for 30 min, filtered, and coupled with the cyclic peptide, successively. At the end of elongation, the peptide was de-protected and simultaneously cleaved from the resin by the treatment of 90/2.5/2.5/5 TFA/H2O/TIS/phenol for 4 h at room temperature. After filtration, the peptide was precipitated by the addition of cold diethyl ether to the TFA solution. The precipitated crude peptides were collected by centrifugation, and purified by HPLC using the semi-preparative column eluted with 80/20 A/B in 30 min at a flow rate of 4.5 mL/min. The retention time of DOTA-TATE was 20.6 min. The HPLC eluates containing the desired 98  peptide were collected, pooled, and lyophilized. The yield of the peptide was 22 %. MS (EPI) calculated for DOTA-TATE C65H90N14O19S2 (M + H)+ 1435.6, found 1436.1.   5.2.1.4 NatGa-DOTA-TATE A solution of DOTA-TATE (2.0 µmol) and GaCl3 (10.0 µmol) in 500 µL sodium acetate buffer (0.1 M, pH 4.0) was incubated at 80 °C for 15 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 80/20 A/B 30 min at a flow rate of 4.5 mL/min. The retention time of Ga-DOTA-TATE was 29.9 min. The yield of this reaction was 57%. MS (EPI) calculated for Ga-DOTA-TATE GaC65H87N14O19S2 (M + H)+ 1501.5, found 1501.4.  5.2.1.5 NODAGA-LM3 Ga-NODAGA-LM3 was synthesized via the Nα-Fmoc solid-phase peptide synthesis strategy starting from Fmoc-D-Tyr(tBu)-Rink Amide-MBHA resin. The resin was treated with 20% piperidine (1×5 min and 1×10 min) in DMF to remove the Nα-Fmoc protecting group. The following Fmoc-protected amino acids (3 equivalents) including Fmoc-Cys(Acm)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Aph(Cbm(tBu))-OH, Fmoc-Tyr(tBu)-OH, Fmoc-D-Cys(Acm)-OH, Fmoc-p-Cl-Phe-OH, and the chelator NODAGA tri-t-butyl ester (synthesized according to literature [230] were subsequently coupled to the sequence according to their order. The coupling was carried out in NMP with standard in situ activating reagent HBTU (3 equivalents) in the presence of DIEA (6 equivalents). At the end of elongation, the peptide was de-protected and simultaneously cleaved from the resin by the treatment of 95/2.5/2.5 TFA/H2O/TIS for 1 h at room temperature and 3 h at 45 oC. After 99  filtration, the peptide was precipitated by the addition of cold diethyl ether to the TFA solution. The precipitated crude peptides were collected by centrifugation, and purified by HPLC using the semi-preparative column eluted with a gradient condition of 20-30 % MeCN (0.1% TFA) in H2O (0.1% TFA) in 30 min at a flow rate of 4.5 mL/min. The retention time of Ga-NODAGA-LM3 was 20.5 min. The HPLC eluates containing the desired peptide were collected, pooled, and lyophilized. The yield of the peptide was 36 %. MS (EPI) calculated for Ga-NODAGA-LM3 C68H90ClN15O19S2 (M + H)+ 1520.6, found 1521.1.   5.2.1.6 NatGa-NODAGA-LM3 A solution of NODAGA-LM3 (2.6 µmol) and GaCl3 (13.1 µmol) in 500 µL sodium acetate buffer (0.1 M, pH 4.2) was incubated at 80 °C for 15 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 20-30 % MeCN (0.1% TFA) in H2O (0.1% TFA) in 30 min at a flow rate of 4.5 mL/min. The retention time of Ga-NODAGA-LM3 was 19.6 min. The yield of this reaction was 56%. MS (EPI) calculated for Ga-NODAGA-LM3 GaC68H87ClN15O19S2 (M + H)+ 1586.5, found 1586.8.  5.2.2 Cell culture and membrane isolation Human ER positive breast cancer cell lines ZR-75-1 and MCF-7 were cultured in RPMI 1640 and DMEM media respectively under a humidified atmosphere with 5% CO2 at 37˚C. Culture medium was supplemented with 10% fetal bovine serum, 2 mM L-glutamin, 100 U/mL penicillin, and 100 μg/mL streptomycin. SST2a transfected HEK293 cells were used as control and cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. 100  To isolate ZR-75-1 and MCF-7 plasma membranes, the cells were harvested with a policeman at 4 ⁰C in presence of DNAse1 and protease inhibitors. Briefly, cells were disrupted using a Dounce homogenizer and after sequential centrifugation plasma membranes were collected at 48200 g. The pellet was resuspended in the assay buffer and snap frozen. The protein concentration was quantified using Bradford reagent. 5.2.3 In vitro binding assays  Filtration competitive binding assays were performed in 96-well filtration plates (MultiScreen, Millipore) to measure the binding affinity of NOTA-BASS, NatGa-NOTA-BASS,  DOTA-TATE, NatGa-DOTA-TATE, 68 Ga-DOTA-TATE, NODAGA-LM3, NatGa-NODAGA-LM3, and 68Ga-NODAGA-LM3.  0.2 µg of membrane from Chinese Hamster Ovary cells (CHO-K1) transfected with sst2r (Perkin Elmer, Canada), or 4-10 µg of Zr-75-1 and MCF-7 membranes per well were used for the assay followed by perkin elmer protocol. Briefly, membrane, 0.05 nM of [125I]Tyr-somatostatin-14 (Perkin Elmer, Canada) and increasing concentration of 10-5 to 10-12 M of  the peptide in an assay buffer (25 mM Hepes pH 7.4, 10 mM MgCl2, 1 mM CaCl2 and 0.5% BSA) plated in 96-well GF/B filter plate in triplicates.  After incubation under agitation at 27 ˚C for 60 minutes, the wells were washed 6 times with ice-cold 50 mM TRIS-HCl pH 7.8.  Membrane filters were punched and counted by a gamma counter (Cobra-II Auto Gamma, Canberra Packard Canada). The experiment was repeated in triplicate. Data were fitted to a one-site competition model (GraphPad Prism 6.1 software) to calculate the inhibition constant (Ki).  101  5.2.4 Radiolabeling 68Ga was eluted from 68Ge/68Ga generator, and purified using a DGA resin (Eichrom). Briefly, the eluate from the generator was mixed with HCl to a final concentration of 4M HCl, and passed over the DGA resin, which trapped the 68Ga on the resin. 68Ga was eluted with 0.5 – 1.0 mL of water.   5.2.4.1 68Ga-DOTA-TATE The 68Ga generator was eluted with a total of 4 mL of 0.6 M HCl. The elution which contained the activity was mixed with 2 mL concentrated HCl. The mixture was passed through a DGA resin column and the column was washed by 3 mL 5 M HCl. After the column was dried by passage of air, 68Ga was eluted with 0.5 mL water. The purified 68Ga solution was added to a 4-mL glass vial preloaded with 0.5 mL of HEPES buffer (2 M, pH 5.0) and 25 µg DOTA-TATE. The radiolabeling reaction was carried out under microwave for 1 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 81/19 MeCN/TEA-Phosphate Buffer (pH 7.29) at a flow rate of 4.5 mL/min. The retention time of 68Ga-DOTA was 19.1 min.  5.2.4.2 68Ga-NODAGA-LM3 The 68Ga generator was eluted with a total of 4 mL of 0.6 M HCl. The elution which contained the activity was mixed with 2 mL concentrated HCl. The mixture was passed through a DGA resin column and the column was washed by 3 mL 5 M HCl. After the column was dried by passage of air, 68Ga was eluted with 0.5 mL water. The purified 68Ga solution was added to a 4-mL glass vial preloaded with 0.5 mL of HEPES buffer (2 M, pH 5.0) and 50 102  µg NODAGA-LM3. The radiolabeling reaction was carried out under microwave for 1 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 76/24 Phosphate Buffer/MeCN (pH 7.5) at a flow rate of 4.5 mL/min. The retention time of 68Ga-NODAGA-LM3 was 15.3 min.  5.2.5 Flow cytometry The expression of SST2a in HEK-SST2a, Zr-75-1 and MCF-7 cells was confirmed by flow-cytometry analysis. Briefly, 1 million cells detached with non-enzymatic buffer (MP Bio #91676949) and were re-suspended in 3% BSA in phosphate buffer containing 2.5 µg of anti-SST2a antibodies (MAB4224, R&D) and incubated for 30 min on ice. Subsequently cells were washed 3 x times by centrifugation at 400g for 5 min. FITC anti mouse antibody (ab6785) was added in a dilution of 1:5000 and incubated on ice for 20 mins in the dark. The cells were washed as stated above and acquired with a cell analyzer (BD LSRFortessa) and analyzed using the FlowJo data analysis software (FlowJo). 5.2.6 Internalization studies The internalization of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 was studied in HEK293-SST2a cells, as previously described [231-233]. Briefly, HEK293-SST2a cells were seeded in 6-well plates to a density of 0.8 to 1 million cells per well and incubated overnight. DMEM containing 1% FBS was used as culture medium. On the day of experiment, the cells were washed with PBS, the radiotracer was added to the medium (2.5 pmol/mL), and the cells were incubated at 37 ˚C. For non-specific binding, 1 µM of unlabeled DOTA-TATE in addition to the radiotracer was added to corresponding wells. The internalization was stopped at 0.25, 0.5, 103  1, and 2 h by removing the medium and washing the cells with ice-cold PBS. To distinguish between membrane bound (acid releasable) and internalized (acid resistant) fraction, the cells were treated twice for 10 min with ice-cold glycine solution, pH 2.8. Finally, the cells were detached with 1M NaOH and counted by a gamma counter (Cobra II, Packard). Internalization was expressed as percentage of the radioactivity added to the wells. 5.2.7 Animal models and biodistribution studies The animal studies were approved by the Institutional Animal Care Committee of the University of British Columbia and were conducted in compliance with the Canadian Council on Animal Care Guidelines.  6-8 weeks old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD-scid gamma or NSG) mice were obtained from the Animal Resource Centre (ARC) of the BC Cancer Agency Research Centre. 60-day release estrogen pellets (1.7 mg/pellet) were implanted subcutaneously in the flank of the mice 3-5 days before tumor inoculation. ZR-75-1 or MCF-7 tumor cells were freshly expanded in sterilized PBS/matrigel mixture prior to inoculation. The mice were inoculated subcutaneously with 5x106 breast tumor cells on the dorsal flank. The tumors were allowed to grow 4 weeks to reach a suitable size (5-7 mm in diameter) for in vivo studies.  In unblocked biodistribution studies, 10-20 µCi (0.37-0.74 MBq) of radiotracer (68Ga-DOTA-TATE or 68Ga-NODAGA-LM3 or 68Ga-NOTA-BASS) were injected via tale vein under 2% isoflurane anesthesia. Blocking studies by injection of 100 µg of DOTA-TATE were performed to determine the specificity of the radioligand to the receptors in an additional group of mice. One hour following injection, the mice were euthanized by isoflurane anesthesia followed by carbon dioxide inhalation. Tissues of interest were collected, rinsed 104  with PBS, blotted dry and counted in a gamma counter. The tissue weight and associated count per minute were used to calculate the percentage injected dose per gram of tissue (%ID/g).  Control studies were carried out by using HEK293-SST2a tumor-bearing mice and the mentioned radiotracers under the same conditions. No estrogen pellet was used for tumor induction as this cell line is not ER dependent.   5.2.8 PET/CT imaging Dynamic PET/CT imaging was performed in the Siemens Inveon multimodality small animal PET/CT scanner. Tumor-bearing animals were anesthetized using 1.5-2% isoflurane and the tail vein was catheterized. A 10 minute CT attenuation scan was followed by a 60 minute dynamic PET acquisition, performed in list mode. In unblocked studies, each mouse was injected with 100 µCi (3.7 MBq) of radiotracer via catheter 30 seconds after PET acquisition started. In blocking studies 100 µg of unlabeled DOTA-TATE was co-injected. The mice were euthanized after termination of scans.  The PET data were reconstructed using the Inveon Acquisition Workplace (IAW).  The list-mode data was first histogrammed. The data from the histogram and the attenuation correction from the CT scan were used in the reconstruction, using the maximum a posteriori (MAP) reconstruction algorithm. Time activity curves from the region of interests (ROI) were generated from the 1 h dynamic imaging data. The uptake of tumor and tissues of interest were determined by ROI, and the %ID/g was calculated (assuming a tissue density of 1.0 105  g/cc). The mean %ID/g was calculated by drawing a ROI to match the tumor contours visible on CT. The peak %ID/g was calculated from the hottest 2x2 voxel cluster within the tumor. 5.3 Results 5.3.1 Peptide synthesis The chemical structures of Ga-DOTA-TATE, Ga-NODAGA-LM3 and Ga-NOTA-BASS are shown in Scheme 5.1. Peptides were obtained with 95% purity or greater as determined by HPLC and mass Spectrometry. 5.3.2 Radiolabeling The decay-corrected radiochemical yield of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 was > 69% (n=3). Radiochemical purity of > 99% was achieved for the labeled peptides as determined by radio HPLC. The specific activity of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 was 2.23 ± 0.60 Ci/µmol and 2.60 ± 0.82 Ci/µmol respectively. 106  5.3.3 In vitro binding assays Filtration assay was performed using breast cancer membranes obtained from Zr-75-1 cells as well as SST2a membranes to determine the ki of the three peptides. With SST2a membrane, ki of NOTA-BASS and Ga-NOTA-BASS was 13.9 ± 3.6, and 158 ± 110 nM respectively. Ga-DOTA-TATE had a binding affinity of 0.7 ± 0.2 nM, while ki of Ga-NODAGA-LM3 was found 10.7 nM. Examples of a binding curve of NOTA-BASS, Ga-NOTA-BASS, Ga-DOTA-TATE, and Ga-NODAGA-LM3 with SST2a membrane are shown in Figure 5.1-5.3.  Ga-DOTA-TATE showed a high binding affinity of 0.6 nM to SST2a receptors by using Zr-75-1 membranes (Figure 5.4). The same experiment did not achieve a binding curve with MCF-7 membranes (Figure 5.5).   107    Scheme 5.1 Structures of three somatostatin derivatives.      108     Figure 5.1 Competitive binding curve of (A) NOTA-BASS and (B) Ga-NOTA-BASS in SST2a membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11.  109   Figure 5.2 Competitive binding curve of Ga-DOTA-TATE in SST2a membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11.   Figure 5.3 Competitive binding curve of Ga-NODAGA-LM3 in SST2a membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11. 110   Figure 5.4 Competitive binding curve of Ga-DOTA-TATE in Zr-75-1 breast tumor membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11.  Figure 5.5 Competitive binding curve of Ga-DOTA-TATE in MCF-7 breast tumor membranes in the presence of 0.05 nM of SST14-[125I]-Tyr11.   111  5.3.4 Internalization studies The internalization kinetics of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 were studied in HEK293-SST2a cells. Within 30 min, the internalized fraction of 68Ga-DOTA-TATE reached 5.96% and increased over time, and up to 12.77% of the total added activity was internalized after two hours (Figure 5.6).  The internalized fraction of 68Ga-NODAGA-LM3 was 0.56% at 15 min and increased to 2.20% after 2 h of incubation.  This result confirmed the agonist nature of 68Ga-DOTA-TATE and antagonist characteristics of 68Ga-NODAGA-LM3. (Figure 5.6) Nonspecific internalization was less than 1%, indicating that the internalization process is specific and receptor-mediated.   Figure 5.6 internalization results of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 in HEK-SST2a cells at 15, 30, 60, 120 min incubation at 37 ⁰C.  112  5.3.5 Flow cytometry As shown in Figures 5.7-5.10, Zr-75-1 cells express SSTR2 (23.7%) while MCF-7 cells show a very low expression of SST2R (3.65%).    Figure 5.7 Flow cytometry analysis of HEK-SST2a cells gated on the living cells where forward and side scatter data are shown on top and forward vs. scatter data are shown on the bottom. 113    Figure 5.8 Flow cytometry analysis of Zr-75-1 cells gated on the living cells where forward and side scatter data are shown on top and forward vs. scatter data are shown on the bottom. 114    Figure 5.9 Flow cytometry analysis of MCF-7 cells gated on the living cells where forward and side scatter data are shown on top and forward vs. scatter data are shown on the bottom.    Figure 5.10 Flow cytometry analysis of HEK-SST2a, Zr-75-1, and MCF-7 cells  115  5.3.6 Biodistribution studies Tables 5.1 and 5.2 summarize the biodistribution of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3, 1 h post-injection in Zr-75-1 tumor-bearing mice. Due to the low binding affinity of Ga-NOTA-BASS to Zr-75-1 membranes, in vivo studies were not performed with 68Ga-NOTA-BASS.  A high tumor uptake was observed with 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 with 19.64 ± 4.89 and 23.63 ± 8.68 %ID/g respectively. The uptake in the pancreas was 31.36 ± 19.62%ID/g for 68Ga-DOTA-TATE and 24.78 ± 16.68%ID/g for 68Ga-NODAGA-LM3. The accumulation in blood, muscle, bone, and brain were lower than 1% in both tracers. HEK293-SST2a tumor-bearing mice were used as a control study (Tables 5.3), with a similar biodistribution of both radiotracers, but lower tumor uptake (p < 0.05). The uptake in pancreas was lower with 68Ga-DOTA-TATE and higher with 68Ga-NODAGA-LM3 (P > 0.05). The tumor-to-blood, -muscle, -liver and -kidney ratios of Zr-75-1 tumor bearing mice were 36.95, 55.87, 16.99 and 1.75 %ID/g respectively with 68Ga-DOTA-TATE, and 17.54, 45.69, 9.71, and 1.22 %ID/g respectively for 68Ga-NODAGA-LM3 (Table 5.4). In blocking studies, the uptake in tumors and pancreas were reduced by >99%. MCF-7 tumor bearing mice exhibited much lower uptake with 68Ga-DOTA-TATE and %ID/g of 1.25 ± 0.27 in tumors. As a result we did not pursue the study with 68Ga-NODAGA-LM3.    116  Table 5.1 Biodistribution Results of 68Ga-DOTA-TATE in immunocompromised mice bearing MCF-7 tumors at 1 h post-injection.  Tissue 68Ga-DOTA-TATE     Unblocked Blocked           Blood 0.72 ± 0.3 0.32 ± 0.05  Plasma 1.73 ± 1.18 0.61 ± 0.07  Uterus 1.08 ± 0.47 0.27 ± 0.23  Large Intestine 1.81 ± 0.43 0.15 ± 0.04  Small Intestine 3.72 ± 4.07 0.14 ± 0.05  Spleen 1.23 ± 1.1 0.12 ± 0.05  Liver 0.99 ± 0.12 0.15 ± 0.1  Pancreas 24.64 ± 18.03 0.18 ± 0.12  Adrenal glands 5.71 ± 4.22 0.56 ± 0.62  Kidney 11.33 ± 3.02 3.4 ± 2.93  Lungs 10.71 ± 8.71 3.57 ± 5.43  Heart 0.47 ± 0.16 0.19 ± 0.11  Skin 1.36 ± 0.39 0.71 ± 0.32  Tumor 1.25 ± 0.27 0.17 ± 0.14  Muscle 0.2 ± 0.11 0.21 ± 0.23  Bone 0.32 ± 0.25 0.3 ± 0.41  Brain 0.05 ± 0.02 0.01 ± 0.01                 117  Table 5.2 Biodistribution Results of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 in immuno-compromised mice bearing Zr-75-1 tumors at 1 h post-injection.  Tissue 68Ga-DOTA-TATE   68Ga-NODAGA-LM3   Unblocked Blocked   Unblocked Blocked                 Blood 0.56 ± 0.27 0.99 ± 0.55  1.28 ± 0.78 0.36 ± 0.48  Plasma 0.92 ± 0.34 1.87 ± 1.28  2.22 ± 1.43 1.29 ±  Uterus 1.36 ± 0.83 1.24 ± 0.62  1.88 ± 1.07 1.48 ± 0.52  Large Intestine 2.82 ± 1.51 0.56 ± 0.15  2.72 ± 1.61 0.71 ± 0.18  Small Intestine 3.87 ± 3.42 0.46 ± 0.17  2.06 ± 1.17 0.69 ± 0.1  Spleen 1.11 ± 0.78 0.32 ± 0.01  1.55 ± 0.91 0.77 ± 0.47  Liver 1.07 ± 0.42 0.49 ± 0.08  2.73 ± 1.25 1.34 ± 0.51  Pancreas 31.36 ± 19.62 0.33 ± 0.07  24.78 ± 16.68 0.43 ± 0.15  Adrenal glands 8.81 ± 5.4 0.51 ± 0.03  5.07 ± 3.35 1.03 ± 0.62  Kidney 14.9 ± 11.13 11.09 ± 1.73  20.88 ± 10.77 19.11 ± 1.6  Lungs 21.86 ± 15.06 1.02 ± 0.18  12.19 ± 9.89 2.74 ± 0.88  Heart 0.64 ± 0.28 0.42 ± 0.19  0.99 ± 0.46 0.51 ± 0.17  Skin 1.63 ± 1.15 1.17 ± 0.43  2.73 ± 1.71 2.48 ± 1.21  Tumor 19.64 ± 4.89 0.61 ± 0.13  23.63 ± 8.68 1.19 ± 0.24  Muscle 0.37 ± 0.15 0.21 ± 0.04  0.53 ± 0.2 0.24 ± 0.07  Bone 1.31 ± 0.58 0.26 ± 0.09  0.93 ± 0.5 0.71 ± 0.58  Brain 0.31 ± 0.31 0.04 ± 0.02  0.09 ± 0.04 0.04 ± 0.01                  118  Table 5.3  Biodistribution Results of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 in immuno-compromised mice bearing HEK-SST2a tumors at 1 h post-injection.  Tissue 68Ga-DOTA-TATE   68Ga-NODAGA-LM3   Unblocked Blocked   Unblocked Blocked                 Blood 1.51 ± 2.09 0.34 ± 0.26  1.14 ± 0.4 2.07 ± 1.08  Plasma 2.67 ± 3.68 0.57 ± 0.39  2.06 ± 0.54 3.61 ± 1.9  Uterus 4.25 ± 7.33 0.5 ± 0.1  1.73 ± 0.72 3.8 ± 2.83  Large Intestine 1.58 ± 0.52 0.17 ± 0.14  1.93 ± 0.67 2.23 ± 1.31  Small Intestine 3.45 ± 1.65 0.28 ± 0.08  3.02 ± 1.97 2.3 ± 2.02  Spleen 2.2 ± 1.39 1.43 ± 1.8  2.97 ± 0.99 4.45 ± 5.08  Liver 2.98 ± 4.97 0.24 ± 0.14  2.06 ± 0.52 2.88 ± 1.95  Pancreas 18.14 ± 15.23 0.2 ± 0.04  27.19 ± 3.41 1.49 ± 1.35  Adrenal glands 15.78 ± 17.95 2.05 ± 3.1  4.01 ± 0.87 7.47 ± 10.34  Kidney 4.47 ± 4.23 4.65 ± 4.06  16.87 ± 3.84 31.42 ± 6.01  Lungs 8.61 ± 6.72 1.85 ± 2.14  12.69 ± 0.98 6.16 ± 3.82  Heart 0.35 ± 0.12 0.21 ± 0.05  0.83 ± 0.28 2.08 ± 2.17  Tumor 19.34 ± 3.79 0.43 ± 0.22  16.35 ± 4.04 3.87 ± 2.84  Muscle 0.18 ± 0.07 0.13 ± 0.07  0.47 ± 0.1 2.38 ± 1.74  Bone 0.47 ± 0.18 0.04 ± 0.05  0.75 ± 0.26 0.69 ± 0  Brain 0.06 ± 0.02 0.02 ± 0.01  0.07 ± 0.02 0.7 ± 0.57                  Table 5.4 Tumor–to–normal-tissue ratios in Zr-75-1 and HEK-SST2a tumor xenografts at 1h post-injection.             Radiotracer 68Ga-DOTA-TATE   68Ga-NODAGA-LM3  Mouse models ZR-75-1 SST2a   ZR-75-1 SST2a Tumor to Blood 36.95 ± 19.05 24.85 ± 18.39  17.54 ± 3.12 15.17 ± 4.14 Tumor to Liver 16.99 ± 6.70 11.90 ± 6.41   9.71 ± 2.36 8.36± 2.97 Tumor to Kidney 1.75 ± 0.91 26.40 ± 35.66   1.22 ± 0.31 0.99 ± 0.28 Tumor to Muscle 55.87 ± 29.04 98.65 ± 22.56   45.69 ± 11.17 35.58 ± 8.13       119  5.3.7 PET/CT imaging and kinetic analysis Excellent tumor visualization with low background at 1 h post-injection was observed with 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 in Zr-75-1 tumor bearing mice 1 hour after injection (Figure 5.11 and 5.12). Mice bearing HEK-SST2a tumors used as positive controls also exhibited excellent tumor visualization with both radiotracers as shown in Figure 5.13. Imaging studies with 68Ga-DOTA-TATE in MCF-7 tumor-bearing exhibited higher background and lower tumor uptake, supporting the in vitro results. Therefore we did not carry on with imaging of 68Ga-NODAGA-LM3. Time activity curves of tumors, blood, liver and kidney in Zr-75-1 and MCF-7 tumor-bearing mice with 68Ga-DOTA-TATE are shown in Figures 5.14, and 5.15. Time activity curves of tumors, blood, liver and kidney in Zr-75-1 tumor-bearing mice with 68Ga-NODAGA-LM3 are shown in Figures 5.16, and 5.17.  120    Figure 5.11  Small-animal PET/CT images of 68Ga-DOTA-TATE at 1 h after injection showing potential of agonist radiotracers for in vivo imaging of Zr-75-1 breast tumors with an excellent tumor to background contrast. Left: unblocked, Right: blocked, B: bladder, K: kidney, T: tumor, I: intestine.  121    Figure 5.12 Small-animal PET/CT images of 68Ga-NODAGA-LM3 at 1 h after injection showing potential of antagonist radiotracers for in vivo imaging of Zr-75-1 breast tumors with an excellent tumor to background contrast. Left: unblocked, Right: blocked, B: bladder, K: kidney, T: tumor, I: intestine. 122    Figure 5.13 Small-animal PET/CT images of 68Ga-DOTA-TATE (left) and 68Ga-NODAGA-LM3 (right) in HEK293-SST2a tumor bearing mice 1 hour post-injection showing very good tumor to background contrast. B: bladder, K: kidney, T: tumor.   123   Figure 5.14 An example of a time activity curve of tumors in dynamic 68Ga-DOTA-TATE imaging.  Figure 5.15 An example of a time activity curve of 68Ga-DOTA-TATE in kidney, blood, and liver in Zr-75-1 tumor bearing mice.  124   Figure 5.16 An example of time activity curve of Zr-75-1 tumor in dynamic 68Ga-NODAGA-LM3 imaging.  Figure 5.17 Time activity curves of blood, liver and kidney in Zr-75-1 tumor-bearing mice with 68Ga-NODAGA-LM3. 125  5.4 Discussion Most somatostatin targeting peptides currently being used in nuclear medicine are agonists because of their capability to internalize into the cells. Somatostatin receptor antagonists were introduced a few years ago [194, 234, 235]. Preclinical and preliminary clinical studies using radiolabeled somatostatin-based antagonists showed excellent results for neuroendocrine tumor imaging, with high tumor uptake and improved tumor-to-background ratios compared to agonists [194, 235].  Antagonist radioligands appear to bind to more receptor sites and might have a slower dissociation rate from the receptor compared to agonist radioligands [224, 236]. In 2011, Fani et al showed high potential of two radio-antagonists (64Cu-64 and 68Ga-NODAGA-LM3) in comparison with their corresponding agonists in nude mice bearing HEK293-SST xenografts [229]. In a later study, they prepared a new somatostain antagonist, 68Ga-1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid-JR11 (68Ga-DOTA-JR11), where 68Ga-NODAGAJR11 exhibited 58% higher tumor accumulation and 100% higher kidney accumulation than the agonist 68Ga-DOTA-TATE  by PET imaging [223]. Most preclinical studies conducted to evaluate radioligands targeting SST used mice bearing tumors derived from transfected cell lines or a rat pancreatic cancer cell line (AR42J).  In this study we compared and evaluated three 68Ga labeled SST agonist and antagonists, 68Ga-DOTA-TATE, 68Ga-NOTA-BASS and 68Ga-NODAGA-LM3 in immuno-compromised 126  xenografts of breast cancer. Mice bearing SST2a transfected HEK293 cells were used as positive controls for in vivo studies in parallel with Zr-75-1 and MCF-7 tumor-bearing mice. 68Ga-labeled DOTA-TATE, NODAGA-LM3, and NOTA-BASS were prepared with high labeling yield and specific activity. Agonist and antagonist properties of 68Ga-DOTA-TATE and 68Ga-NODAGA-LM3 were confirmed by internalization experiments using HEK293-SST2a cells. All three peptides showed good binding affinity with the Zr-75-1 membranes, with Ga-DOTA-TATE showing the lowest (most favorable) Ki. Considering the good binding affinity Ga-DOTA-TATE this result was expected. The Ki of Ga-NOTA-BASS however was not very favorable at >100 nM therefore in vivo studies were not carried out with this antagonist peptide. MCF-7 membranes did not exhibit SST2a displacement in in vitro binding assays, and FACS analysis of MCF-7, Zr-75-1 and HEK293-SST2a cells confirmed the very low frequency of expression of SST2a receptors these cells compared to the other two cell lines. The Zr-75-1 cell line, despite its lower SST2a expression in comparison with SST2a transfected HEK293 cells, demonstrated an excellent binding affinity to SST2R, close to the Ki achieved with SST2a membranes. In vitro binding assays and FACS analysis provided the suitability of Zr-75-1 as a SST2a expressing breast cancer model for PET imaging.  The 68Ga-DOTA-TATE uptake in MCF-7 tumors was low (1.25 ± 0.27 %ID/g). However in blocking studies ~>99% reduction (P < 0.0001) in tumor uptake was achieved suggesting presence of SSTR expression in MCF-7 tumor cells. 127  A comparison of the imaging properties of 68Ga-NODAGA-LM3 and 68Ga-DOTA-TATE evaluated in vivo demonstrated slightly higher tumor uptake with the antagonist (23.63 Vs. 19.64 % ID/g) and lower pancreas uptake in Zr-75-1 tumor bearing mice. Interestingly tumor-to-blood and -muscle ratio of 68Ga-DOTA-TATE was 2.1 and 1.2 fold higher than the antagonist 68Ga-NODAGA-LM3 and provided superior tumor-to-background in PET imaging. The data presented in this study displayed a higher tumor uptake, tumor-to-blood and tumor-to-muscle ratios with both radiotracers 1-hour post-injection in the Zr-75-1 breast cancer model compared to the control SST2a transfected HEK tumor model as a common animal model of SST2a imaging. Ga-DOTA-TATE exhibited 18-fold better binding affinity than Ga-NODAGA-LM3. The fact that in this study they both had almost the same imaging properties suggest that the better binding affinity overcome potential advantages of antagonists versus agonists. As evidenced by a high expression of SST2a receptors demonstrated by flow cytometry studies and favorable binding to somatostatin-14 using in vitro receptor binding assays, the Zr-75-1 cell line is a promising breast cancer model for SST imaging. Although other studies reported a better overall pharmacokinetics with antagonist radiotracers, we obtained better tumor to background in both control and breast cancer models using 68Ga-DOTA-TATE compared to a promising antagonist, 68Ga-NODAGA-LM3.  Whether similar results are observed with newer antagonists such as 68Ga-NODAGA-JR11 will need to be experimentally confirmed in future studies. 5.5 Conclusion A high tumor to background ratio was achieved in the Zr-75-1 tumor model with 128  somatostatin receptor agonists (68Ga-DOTA-TATE) and antagonists (68Ga-NODAGA-LM3). Despite similar absolute tumor uptake, the tumor-to-muscle ratio was higher with 68Ga-DOTA-TATE compared to 68Ga-NODAGA-LM3, which perhaps reflects the superior binding affinity of 68Ga-DOTA-TATE rather than differences in agonist/antagonist properties.  The Zr-75-1 human tumor cell line is a promising breast cancer model for SST2a imaging by positron emission tomography.  129  Chapter 6: Neuropeptide Y1 receptors  6.1 Background Neuropeptide Y (NPY) is one of the regulatory peptides and part of a NPY family consisting of NPY, peptide YY (PYY), and pancreatic polypeptide (PP) [237]. NPY is a 36 amino-acid neurohormonal amide peptide with large number of tyrosine residues, and the most abundantly expressed peptide of the pancreatic polypeptide family in mammalian systems [237]. It is a neurotransmitter and mainly located in the central and peripheral nervous system [237].  NPY function in central nervous system includes stimulation of feeding behavior and anxiety inhibition [238-240]. It also plays role in memory and blood pressure regulation [241]. NPY actions mediated by the peripheral nervous system include vasoconstriction and regulation of the gastrointestinal motility and secretion, insulin release, and renal function [238, 242-245]. NPY role in reproduction at the level of the hypothalamic–hypophyseal axis that is modulated by LHRH and LH secretion is extensively investigated in rodents, but is less clear in humans [246-248]. The functions of the NPY family are mediated via the various NPY receptor subtypes that belong to the G protein-coupled receptor (GPCR) super family. Five receptor subtypes are characterized by now. While Y1, Y2, Y4, and Y5 are physiologically expressed in humans, Y6 has been demonstrated only at the mRNA level. A Y3 receptor subtype has not yet been proven to exist [237]. The level of neuropeptide receptors is partly regulated by estradiol and progesterone in the 130  hypothalamus [249]. Y1 and Y2 antagonists are developed for potential treatment of feeding disturbances and anxiety [250-252].  Unlike other regulatory peptides, NPY has not often linked with human cancer [71] and little was known about the role of NPY receptor in tumor biology until the last decade. Studies have shown that NPY induced a dose-dependent inhibition of tumor cell growth of over 40% in a Y1-expressing human neuroblastoma cell line [92].  Expression of NPY1 receptor has also been reported in ovarian tumors [90], adrenal tumors [91], some renal carcinomas and nephroblastomas [93]. Studies on several human prostate cancer cell lines including PC-3, LNCap and DU145 suggesting that the growth of prostate cancer cells are regulated by activation of NPY1R through NPY [253]. Moreover, Reubi et al. demonstrated an incidence of significant overexpression of NPY1 receptor  in 58% to 85% of breast tumors including lobular carcinomas, and showed a dose dependent inhibitory effect of NPY on the growth of breast tumors [92, 254].  Amlal et al. also confirmed the presence of NPY1 receptors on the MCF-7 human breast cancer cell line, and that the NPY1 receptor expression is up-regulated by estrogen in ER positive cell lines [255]. They also showed that NPY inhibits estrogen-induced cell proliferation.   According to published data, normal breast tissue expresses only Y2 receptors whereas many cases of breast cancer overexpress NPY1 with only focal expression of Y2 in small number of cases. Therefore, we hypothesized that NPY1 receptor is a potential candidate for peptide receptor targeting with radiopharmaceuticals for specific tumor imaging by PET. To date, not many examples of radiolabeled NPY analogs have been reported [256, 257]. Modified NPY analogs have been labeled with different radiometal/chelating agent 131  combination. For instance, Beck-Sickinger group obtained stable compounds that bind to Y1 and Y2 by using 99mTc/2-picolylamine-N,N-diacetic acid [256] and 111In/DOTA [257].  The objective of this study was to develop a potent NPY analog that has high binding affinity to NPY1 receptors in breast cancer tumors, and radiolabel with 18F for breast cancer imaging. This is believed to offer a better accuracy of diagnosis compared to conventional breast cancer examinations such biopsy and mammography combined with ultrasound. Daniel et al.  developed a truncated NPY analog, BVD15 ([Pro30, Tyr32, Leu34]NPY(28-36)-NH2) that exhibited agonist activity atY2 and Y4, but also very high affinity for NPY1 [257]. Therefore BVD15 was used as the template peptide to design a novel 18F labeled tracer. Some modifications such as substitution of a single amino acid in BVD15 analog could result in improving the binding affinity to NPY1. In our group substitutions at Ile4 of BVD15 was carried out, where an azide peptide (Ile-Asn-Pro-Lys(–CO–CH2–N3)-Tyr-Arg-Leu-Arg-Tyr-NH2) was substituted with an alkyne-bearing prosthetic group 18/19FFPy5yne. This derivative of NPY1 analog so called as ALK-BVD15 was labeled with 18F via click chemistry and evaluated in vitro and in vivo in MCF-7 human breast cancer model. The human neuroblastoma cell line, SK-N-MC, was used as a control cell line in this study. These cells are known NPY1 receptor positive cells and pharmacological studies have shown that only Y1 and not Y2 is expressed in this cell line [258-260]. This makes SK-N-MC tumor model suitable for a parallel comparative study along with MCF-7 breast cancer model.  132  6.2 Materials and methods 6.2.1 Cell culture Human ER positive breast cancer cell line (MCF-7) was obtained from Dr. C.K. Osborne’s lab, Baylore College of Medicine, Houston, Texas. Human neuroblastoma cell line (SK-N-MC) was obtained from American Type Culture Collection and used as a control cell line in all experiments. MCF-7 cells were cultured in DMEM medium and SK-N-MC cells in EMEM medium under a humidified atmosphere with 5% CO2 at 37 ˚C. Culture media was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. All culture reagents purchased from Sigma Aldrich or Invitrogen. 6.2.2 Peptide synthesis and radiosynthesis of NPY1 derivative Synthesis and radiosynthesis details related to the preparation of 18F prosthetic groups and 18F peptide analogues, along with the non-radioactive syntheses of 19F standards and [18F]fluorination precursors is reported in PhD dissertation of James A Inkster [261]. Briefly,  18F- and 19F-ALK-BVD-15 were prepared via copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation of an azide-substituted peptide (Ile-Asn-Pro-Lys(-CO-CH2-N3)-Tyr-Arg-Leu-Arg-Tyr-NH2) with alkyne-bearing prosthetic 18/19FFPy5yne (scheme 6.1).     133    Scheme 6.1 Radiosynthesis of [18F]-AK-BVD15 by click chemistry.  6.2.3 Immunohistochemistry Neuroblastoma tumors were harvested from nude BALB/c mice bearing SK-N-MC tumors and fixed in 4% paraformaldehyde in PBS for 48 h. Tissues were embedded in paraffin after dehydration and 4 µm sections were mounted onto poly-L-lysine slides. Immunostaining was performed by the Centre of Translational Applied Genomics (CTAG) at the BC Cancer 134  Agency. Staining was done on the Ventana DiscoveryXT. Slides underwent a pretreatment process for epitope retrieval using Protease1 for 4 min. The primary antibody NPY was used at a 1:200 dilution for 1 h at room temperature. The secondary antibody was applied for 16 min followed by the Ultramap DAB detection kit. The stained sections were examined and photographed with a Leica EC3 microscope. 6.2.4 In vitro receptor binding assays Competition assays were performed using MCF-7 and SK-N-MC cell lines. The cells were cultured in 24-well plates at a density of 105 cells per well and grown to 75% confluence. 2 h prior to the experiment, cells were washed with PBS and incubated with RPMI 1640 containing 2 mg/mL BSA, 4.8 mg/mL HEPES, 1 U/mL penicillin G and 1 μg/mL streptomycin.  0.02 nM of [125I]-PYY (2200Ci/mmol, Perkin Elmer, Canada) and various concentration of cold NPY or 19F-ALK-BVD15 peptides (10-5 to 10-12 M) was further added to the cells in triplicates and incubated under agitation at 37 ˚C for 45 minutes. The cells were then washed 3 times with ice-cold phosphate-buffered saline (PBS) and trypsinized and counted by a gamma counter (Cobra-II Auto Gamma, Canberra Packard Canada). Data were analyzed by GraphPad Prism 6.01 software, and the inhibition constant (Ki) of the peptides was calculated using the Cheng and Prusoff’s equation from the IC50 and the radioligand concentration.  Saturation assays were performed to determine the receptor density of the two cell lines. Briefly the cells were incubated with increasing concentrations of radioligand ([125I]-PYY), 135  and 0.1 mM of the cold NPY at 37 ˚C for 45 minutes. Then the same procedure as competition binding assays was followed to determine the Bmax values by Graphpad Prism software. 6.2.5 Biodistribution studies in tumor-bearing mice The animal protocol used in the animal studies was approved by the Institutional Animal Care Committee of the University of British Columbia and was performed in compliance with the Canadian Council on Animal Care Guidelines.  6-8 weeks old female nude mice purchased from Simonsen laboratories were used for animal studies. For the induction of tumor xenografts, female nude mice were inoculated subcutaneously with 10 million MCF-7 or SK-N-MC tumor cells in the dorsal flank. The tumor cells were freshly expanded in sterilized PBS/matrigel mixture prior to inoculation. The tumors were allowed to grow 4 weeks to reach a suitable size (5-7mm in diameter) for in vivo studies. In ER+ breast cancer model (MCF-7), 60-day release estradiol pellet was implanted subcutaneously in the base of tail of the female mice 3-5 days before the tumor inoculation.  Followed by pellet implantation, the mice were injected with 1mg/kg of metacam for 3 days and polysporin was applied to the wound every 12 hours if necessary. In unblocked biodistribution studies, 10-20 µCi (0.37-0.74 MBq) of 18F-ALK-BVD15 was injected via the tale vein under 2% isoflurane anaesthesia. To determine the specificity of the in vivo uptake in receptor positive tissues, 100 µg of unlabeled NPY1 as a blocking agent was co-injected with 18F-ALK-BVD15 to an additional group of mice. The mice were humanely euthanized by carbon dioxide and dissected 1 hour post-injection. Tissues of interest were collected, rinsed with PBS, dried and counted in a gamma counter (Cobra-II Auto Gamma, 136  Canberra Packard Canada). The tissue weight and associated counts per minute were used to calculate the percentage injected dose per gram of tissue (%ID/g).  6.2.6 PET/CT imaging and kinetic analysis One hour dynamic PET/CT imaging was performed in the Siemens Inveon multimodality small animal PET/CT scanner. Tumor-bearing animals were anaesthetized using 1.5-2% isoflurane and the tail vein was catheterized. The mice were placed onto the imaging bed while anaesthetized. 10 minute CT attenuation scan followed by a 60 minute Dynamic PET was carried out. 100 µCi (3.7 MBq) of 18F-ALk-BVD15 was injected via catheter 30 seconds after PET acquisition started. In blocking studies 100 µg of unlabeled NPY1 was used. The mice were euthanized after termination of scans.  The PET data was reconstructed using the Inveon Acquisition Workplace (IAW) by using the MicroQ software program.  The data was first histogrammed using the original data. The data from the histogram and the attenuation map from the CT scan were used in the reconstruction. Time activity curves from the region of interests (ROI) were generated from 1 h dynamic imaging data. 6.2.7 Metabolic stability study in mouse plasma Plasma stability studies were performed with the cold peptide, 19F-ALK-BVD15, at the concentration of 0.3 mM in BALB/c mouse plasma. Samples in triplicates were incubated at 37˚C, at 5 time points. After incubation for 0, 5, 15, 60, 120 minutes, the reaction was stopped by adding 100 µl of 75% MeCN followed by vortexing. In addition, a standard sample (0.3 137  mM of peptide solution) and a blank sample (Plasma and 75%MeCN) was prepared for HPLC as control samples. The samples were centrifuged at 13,000 rpm, at 4˚C for 20 min. The supernatants were transferred to new tubes and dried using Helium gas (except for the standard).  Then the 50 -60 µL of H2O was added to the evaporated peptides, vortexed and spun shortly and loaded onto the analytical HPLC to check the stability of the peptide.  The percentage of the intact peptide for each incubation time was determined by Agilent ChemStation software. The peptide peaks and the decomposition peaks at each time points were collected to be analyzed by Mass spectrometry. 6.2.8 Liquid chromatography tandem ms analysis Liquid chromatography tandem MS (LC-MS/MS), were performed on a 4000 QTrap (Applied Biosystems/Sciex, Foster City, CA USA) with nano-electrospray ionization source. The instrument was coupled to a high-performance liquid chromatography system (Agilent 1000 Nano-HPLC; Agilent, SantaClara, CA USA). Samples were desalted online using a reverse-phase trap column (Agilent, Zorbax, 300SB-C18, 5 m, 5 x 0.3mm) for 2.5 minute in solvent A ( 0.1% formic acid) then directed onto a reverse phase analytical column (75 m x 15 cm, packed in-house with 3 um-diameter Reprosil-Pur C18; Dr. Maisch, Ammerbuch-Entringen, Germany) and coupled to an uncoated fused silica emitter tip (20 µm inner diameter, 10 m tip; New Objective, Woburn, MA USA). Chromatographic separation was achieved using a 300 nl/min flow rate and a linear gradient of 0 to 23% solvent B (90% acetonitrile, 0.1% formic acid) over 8 minute followed by a 4 minute gradient from 23 to 39% solvent B. 138  For these experiments the 4000 QTrap parameters were as following: ESI source at 2000 V, interface heater at 200 °C, baseline pressure at 4.8x10-5 torr and with nitrogen (99.999%, Praxair, Danbury, CT, USA) flow for nebulizer gas (0.5 ml/min) and curtain gas (2 L/min). Data were collected using a 400-1600 m/z Enhanced MS scan followed by an Enhanced Resolution scan to select the top three +2 and +3 ions for collisional-induced dissociation and a final Enhanced Product Ion MS scan.  6.2.9 Statistical analysis Quantitative data are expressed as mean ± SEM, and means are compared using 1-way ANOVA. Correlation statistics were performed using linear regression analysis. P values of 0.05 or less were considered statistically significant. 6.3 Results 6.3.1 Chemistry and radiochemistry  The average preparative bioconjugate yields of 18F-ALK-BVD15 using DMF or DMSO were 2.7% DC (n=1), and 15.5 ± 10.6 % DC (n=2) respectively. The specific activity was 69.22 ± 37.84 GBq/µmol.   The radiotracer was formulated in 10 % EtOH in isotonic saline, for the in vivo biodistribution and PET imaging studies. 6.3.2 In vitro binding assays  Table 4.1 summarizes the results of competitive binding assays of different NPY analogs using NPY1 receptor positive cell lines, MCF-7 and SK-N-MC. In vitro binding assays with the commercial NPY1 peptide showed a very good binding to both cell lines with Ki of 3-4 139  nM. A lower Ki of 31 ± 18 nM for MCF-7 cells and 14.8 ± 5.8 nM for SK-N-MC cells was obtained with 19F-ALK-BVD15. Saturation assays demonstrated that Bmax values for MCF-7 and SK-N-MC were 2716 and 18387 respectively (Figure 6.1).  Table 6.1 Binding affinity of NPY analogs in human breast and neuroblastoma tumor cells.  Cell line Radioligand Peptide IC50 (nM) Ki (nM) MCF-7 [125I]-PYY NPY1 3.6 ± 1.4 3 ± 1.2 MCF-7 [125I]-PYY BVD15 22 ± 1.6 18.4 ± 13.8 MCF-7 [125I]-PYY 19F-ALK-BVD15 44 ± 29 31 ± 18 SK-N-MC [125I]-PYY NPY1 4.7 ± 2.9 3.9 ± 2.4 SK-N-MC [125I]-PYY 19F-ALK-BVD15 80 ± 8.9 14.8 ± 5.8         140    Figure 6.1 Saturation binding assays of (A) SK-N-MC neuroblastoma tumor cells and (B) MCF-7 human breast cancer cell line.  6.3.3 Biodistribution studies Biodistribution studies were carried out in nude mice bearing MCF-7 and SK-N-MC tumors, where SK-N-MC tumor model was used a control. 10-20 µCi of 18F-ALK-BVD15 was injected into each mouse with or without blocking agent (100 µg of NPY1).  The percentage 141  injected dose per grams (%ID/g) of tissues was calculated 1 hour post-injection. Table 6.2 and Figure 6.2-3 summarize unblocked and blocked experiments using breast and neuroblastoma tumor models.  18F-ALK-BVD15 was mostly retained in the gastrointestinal tract in both tumor models and had a suitable biodistribution in the rest of tissues, with low accumulation of activity in blood, muscle, and kidney. However no significant uptake in tumors was observed with %ID/g of 0.12 ± 0.07 in MCF-7 tumor-bearing mice, and 0.5 ± 0.3 in SK-N-MC tumor-bearing mice. Blocking with unlabeled NPY1, resulted in reduced uptake in SK-N-MC tumors but not in MCF-7 tumors. Due to a very low accumulation of radioactivity in the tumors in unblocked studies, receptor mediated uptake cannot be interpreted.  Table 6.2 Biodistribution of [18F]-ALK-BVD15 in SK-N-MC and MCF-7 tumor-bearing mice at 1 h post-injection. Data are shown as %ID/g of tissues.             Tissue SK-N-MC tumor-bearing mice MCF-7 tumor-bearing mice   Unblocked Blocked Unblocked Blocked               Red blood cells 0.16 ± 0.15 0.69 ± 0.3 0.44 ± 0.7 0.27 ± 0.37  Plasma 0.5 ± 0.57 1.56 ± 0.6 1.11 ± 1.91 0.74 ± 1.06  Liver 1.84 ± 2.02 6.02 ± 1.86 3.44 ± 2.59 2.85 ± 1.84  Kidney 9.67 ± 4.69 39.92 ± 61.8 0.22 ± 0.17 0.1 ± 0.01  Tumor 0.49 ± 0.36 0.43 ± 0.18 0.12 ± 0.07 0.12 ± 0.03  Muscle 0.08 ± 0.08 0.18 ± 0.18 0.07 ± 0.03 0.06 ± 0               142   Figure 6.2 Biodistribution of [18F]-ALK-BVD15 in SK-N-MC tumor-bearing mice 1 h post-inj (n=5).   Figure 6.3 Biodistribution of [18F]-ALK-BVD15 in MCF-7 tumor-bearing mice 1 h post-inj (unblocked: n=7, blocked: n=3). 143  6.3.4 PET/CT imaging One hour dynamic PET/CT imaging of breast cancer and neuroblastoma models are shown in Figure 6.5. Imaging results were consistent with biodistribution study results. The background signal as well as the tumor signal was low in MCF-7 tumor bearing mice (Figure 6.4). SK-N-MC tumor-bearing mice exhibited a higher uptake intensity, mostly at the rim of the tumors (Figure 6.5).   Figure 6.4 Unblocked dynamic PET/CT imaging of [18F]-ALK-BVD15 in MCF-7 tumor-bearing mice at 1 hour post-inj. (A) Fused PET/CT, (B) PET image, I: Intestine, L: liver , T: tumor.   Figure 6.5 Unblocked dynamic PET/CT imaging of [18F]-ALK-BVD15 in SK-N-MC tumor-bearing mice at 1 hour post-inj. (A) Fused PET/CT, (B) PET image, B: Bladder, I: Intestine, L: liver , T: tumor. 144  6.3.5 Immunohistochemistry Despite having a good in vitro binding affinity of 19F-ALK-BV15 to SK-N-MC cells and literature evidence of expressing NPY1 receptors, we did not achieve a good in vivo tumor uptake. Therefore, neuroblastoma tumors were harvested from SK-N-MC tumor-bearing mice and immunohistochemical staining was performed ex vivo to further confirm the NPY1R expression in this tumor model. As shown in Figure 6.6 NPY1 receptor expression is evident in SK-N-MC tumors.    Figure 6.6 Immunohistochemical staining of neuroblastoma SK-N-MC tumor, X400.  6.3.6 Metabolic stability in mouse plasma Due to unavailability of 18F-ALK-BVD15, plasma stability studies were performed using the cold analog, 19F-ALK-BVD15, in mouse plasma. The peptide was incubated in plasma in various time points up to 2 hours.  HPLC analysis demonstrated that only 30% of 19F-ALK-145  BVD15 was stable after 1 hour (Figure 6.7).  The half-life of the peptide in plasma was calculated to be 21 minutes (Figure 6.8).     Figure 6.7 19F-ALK-BVD15 peptide and decomposition peaks at 15, 60 and 120 min incubation in mouse plasma.       146   Figure 6.8 Plasma stability of 19F-ALK-BVD15 demonstrating the half-life of the peptide in mouse plasma. 6.3.7 Mass spectrometry analysis To identify the decomposition products and location of the peptide cleavage, mass spectrometry analysis was carried out. The peptide peak and the decomposition peak at each time point was collected and analyzed by LC MS/MS. The most abundant peaks at each time point was chosen and fragmented. The results suggest that 19F-ALK-BVD15 was primarily being cleaved at the C-terminus of Arginine residue. At a later stage Isoleucine and Asparagine were also cleaved (Figure 6.9). The click site (19F-ALK) was found intact in all samples.  Figure 6.9  19F-ALK-BVD15 is cleaved in two steps, from arginine, and isoleucine. 147  6.4 Discussion Breast cancer is the most common cancer among women with an estimation of 40,000 deaths per year according to the American Cancer Society. Similar to other types of cancer, the best way to fight and manage breast cancer is to detect the disease at its early stage and when it has not spread. Currently, the diagnosis of breast cancer is by observation of clinical symptoms, physical examination using biopsy and mammography which sometimes carried out in combination with ultrasound. PET/CT imaging is commonly used as a diagnostic imaging technique to stage tumors and treatment follow-up. However the common radiopharmaceutical 18F-FDG, is not sensitive enough to detect cancers with low metabolic rate such as breast cancer.  Therefore, there is a need for a powerful tool for early and accurate detection of breast cancer. Peptide receptor imaging has shown promise in specific tumor targeting. Thus, in this study we aimed to target neuropeptide Y1 receptors known to be overexpressed in breast cancer by 18F labeling and use of PET/CT imaging in animal model of breast cancer. To this end human ER + breast cancer cell line, MCF-7 was used for in-vitro validation and in vivo tumor model. As a control model we used human neuroblastoma cell line SK-N-MC. We synthesized a NPY analog, an azide-substituted peptide (Ile-Asn-Pro-Lys(-CO-CH2-N3)-Tyr-Arg-Leu-Arg-Tyr-NH2) with alkyne-bearing prosthetic 18/19FFPy5yne. As described in chapter 2, click chemistry as a well-established method in our group was used to prepare 19F-ALK-BVD-15, and for coupling of the 18F labeled prosthetic group to N3-ALK-BVD15 (Scheme 6.1).  148  In vitro receptor binding assays demonstrated that19F-ALK-BVD15 has a good binding affinity to NPY1 receptors in both cancer breast and neuroblastoma tumor cell lines. Number of NPY1 receptor was also confirmed in both cell lines with less density of receptors in MCF-7 cells compared to the control neuroblastoma cell line.  [18F]-ALK-BVD15 had a suitable specific activity of 69.22 ± 37.84 GBq/µmol for PET imaging studies. Despite good in vitro binding, 18F-ALK-BVD15 exhibited unfavorable pharmacokinetics in vivo in the both control and breast cancer model. In biodistribution and PET/CT imaging studies, radioactivity was mainly retained in the GI tract and no receptor mediated tumor uptake was observed. Although the lipophilic nature of 18F-ALK-BVD15 could contribute to faster hepatobiliary excretion, it was also expected to help retention in the tumors. To investigate the reasons behind the obtained unfavorable pharmacokinetics, extensive plasma stability studies were initiated. A short plasma half-life of ~20 minute was found for 19F-ALK-BVD15 which is not optimal for imaging studies. The analysis was performed to further identify the peptide cleaving sites in plasma. Mass spectrometry analysis demonstrated that 19F-ALK-BVD15 undergoes proteolysis at two stages where arginine and isoleucine are cleaved. By prolonging plasma half-life, the pharmacokinetic profile of peptide based radiopharmaceutical may improve [262]. Several strategies have been reported to overcome the low stability of the peptides in blood or to prolong the plasma half-life of such compounds [262]. One strategy is to modify the peptide sequence. A drug used for treatment of gastrointestinal tumors, octreotide, is a well-known example of a targeted modification, where the metabolic 149  stability was improved from few minutes to 1.5 hours [263]. In case of octreotide, the overall amino acid sequence of somatostatin was shortened from 14 to 8 and L-amino acids were replaced with D-amino acids [263]. Another strategy is the covalent attachment of polyethylene glycol (PEG) to the peptide terminus. As discussed in chapter two, PEGylation of peptides has shown to improve the half-life in blood by protecting the peptide from enzymatic degradation and therefore reducing proteolysis. However we did not pursue this strategy as we still needed to undergo the lengthy click chemistry method for 18F labeling.  Head to tail cyclization of peptides and proteins by formation of an amide bond between C- and N- terminus has been reported to prevent exopeptidases [264]. It is worth to mention that this alteration may lead to loss of activity of the peptide, especially in case of BVD15 which both N and C terminus are required for binding to the receptors. In order to modify peptides correctly and enhance their metabolic stability in the most efficient way, it is important to know what enzyme caused peptide cleavage. Therefore it is necessary to test against common peptidases and proteases present in human blood, liver and kidney.  Phosphoramidon is a peptidase inhibitor that has recently shown promise for in vivo imaging when co-injected with radiopharmaceutical. Phosphoramidon inhibits endopeptidase 24.11 and appears to be a potent inhibitor of angiotensin conversion enzyme (ACE) and other peptidases [265]. Nock et al, demonstrated that by co-administration of phosphoramidon and [111In-DOTA]MG11 the level of circulating intact radiotracer was significantly improved which led 150  to  better tumor visualization in mice [266].  In 2014, our group achieved very good tumor visualization in mice bearing bradykinin 1 receptor positive tumors by co-injection of phosphoramidon to protect a peptide from peptidase activity [267].  Even though phosphoramidon is not an FDA approved drug and human toxicity is still unknown, it will be interesting to explore the effect of this drug on metabolic stability of our BVD15 analogs.   Our group successfully synthesized an NPY analog by substitution of Har6 (Homoarginine) with Arg6 (arginine) where first cleavage occurred to improve in vivo stability. Different chelators could be added to this derivative of BVD15.  DOTA chelation was chosen for 68Ga labeling as our second approach to 18F labeling. DOTA-[Har6]BVD15 in addition has the potential of being a SPECT imaging radiotracer when other radio-metals such as 111In and 44Sc are used. In preliminary studies, [Har6]BVD15 showed a very good binding affinity with Ki of ~3nM and about 5 fold higher than 19F-ALK-BVD15 using SK-N-MC cells. In vitro characteristics of this BVD15 analog yet need to be established by comprehensive binding assays and plasma stability studies before proceeding to in vivo imaging studies. 6.5 Conclusion  In vitro binding assays showed a good binding affinity of 19F-ALK-BVD15 to SK-N-MC cells and a higher number of receptors compared to MCF-7 cells. 18F-ALK-BVD15 had a suitable biodistribution but low receptor-mediated tumor uptake was seen in NPY1R positive tumors. Mouse plasma stability data suggest that low uptake in tumors could be due to a rapid degradation of peptide in blood. Therefore, various strategies such as peptide modification and 151  use of peptidase inhibitors need to be explored in order to improve the overall metabolic stability of BVD15 derivatives in vivo. 152  Chapter 7: Summary and conclusion The purpose of this thesis was to prepare and characterize novel diagnostic peptide radiopharmaceuticals that can bind with high affinity to GRP, SST2a, or NPY1 receptors that are commonly overexpressed in human prostate and breast cancers. Such radiolabeled peptides have the potential to be employed as diagnostic agents in the clinic for non-invasive tumor detection by PET/CT imaging following their intravenous injection into a patient. While conventional imaging modalities such as MRI and CT are limited in sensitivity to detect the presence of early stage tumors and metastases, particularly in normal sized lymph nodes, PET imaging also remains limited in sensitivity to detect slower growing prostate cancers and lobular breast carcinomas. The development and availability of PET radiopharmaceutical with a high sensitivity and specificity that could target peptide receptors that are overexpressed in the two most common occurring cancers could significantly improve the treatment outcome for patients suffering from breast and prostate cancers by improving detection, staging, and providing an effective method to detect recurrence. 18F is the desirable radioisotope for diagnostic PET/CT applications. Across Canada, 18F is widely available with an increasing number of sites involved in 18F-FDG production. Another advantage of using 18F for peptide radiolabeling includes an optimal spatial resolution because of its low positron energy of 0.64 MeV [268]. Furthermore, its half-life of 110 minutes is adequate to allow radiosynthesis and imaging procedures that can take up to a few hours, thus making kinetic studies possible.  153  The major drawback of labeling peptides with 18F is the laborious and time consuming preparation of the 18F labeling agents [269]. To manage this constraint, we initially used a click chemistry method for the coupling of an 18F- radiolabeled prosthetic group to the peptide. This work was in collaboration with Dr. Tom Ruth’s group at TRIUMF, where a new prosthetic group derivatized from 2-nitro (or 2-trimethylammonium) pyridine and bearing an alkyne moiety was proposed. The coupling reaction involves a CuI catalyzed formation of 1,2,3-triazole using Huisgen 1,3-dipolar cycloaddition of terminal alkynes with azides [270]. This reaction by having the advantage of being regioselective [269],  does not require the protection of other functional groups within the peptide sequence [271].  The click chemistry approach was used in chapter 2 for radiolabeling of GRP analogs for imaging of prostate tumors, and in chapter 4 to radiolabel an NPY analog for breast cancer imaging. The objective of chapter 2 was to determine whether changing the hydrophilicity of 2-fluoropyridine conjugated peptides can increase GRP receptor-mediated binding affinity and overall pharmacokinetics in PC-3 tumor-bearing mice. Therefore, three radiofluorinated derivatives of bombesin with and without the addition of PEG groups were compared and evaluated in vitro, and in vivo. Given the high lipophilicity of the first radioactive product, [18F]F-ALK-[D-Tyr6, βAla11,Thi13,Nle14]bombesin(6-14) ([18F]-ALK-BBN), it was predominantly excreted from the hepatobiliary tract, leading to unfavorable pharmacokinetic behavior in PC-3 tumor bearing mice. Thus, alternative water-soluble 18F analogs of [D-Tyr6, βAla11,Thi13,Nle14]BBN(6-14) were synthesized in which short PEG moieties were introduced both through further modification of the N3 peptide precursor, as well as through conjugation 154  to a PEGylated 18F prosthetic group. Excellent sub-nanomolar binding affinities were achieved with PEGgylated peptides, 19F-ALK-BBN-PEG and19F-PEG-BBN-PEG. Biodistribution and PET imaging studies showed improved GRP receptor-mediated tumor uptake with PEGylated 18F labeled bombesin derivatives and reduced excretion through the hepatobiliary tract. The blood elimination was slower with 18F-PEG-BBN-PEG. However, 18F-ALK-BBN-PEG showed better tumor visualization in PET/CT imaging, with higher tumor to blood ratios at 1 hour post-injection. In chapter 6, an azide peptide (Ile-Asn-Pro-Lys(–CO–CH2–N3)-Tyr-Arg-Leu-Arg-Tyr-NH2) was substituted with an alkyne-bearing prosthetic group 18/19FFPy5yne. This derivative of NPY1 analog, so called as ALK-BVD15, was labeled with 18F via click chemistry, and its potential for specific targeting of breast tumors was evaluated in vitro and in vivo by using NPY1R-positive tumor models. In vitro properties of 19F-ALK-BVD15 were tested on MCF-7 human breast cancer and SK-N-MC human neuroblastoma cell lines. Both cell lines showed good binding affinity and receptor density, with MCF-7 exhibiting lower number of NPY1 receptors compared to the control cell line SK-N-MC.  Despite good in vitro binding and a suitable specific activity of 18F-ALK-BVD15 for PET imaging studies, we did not observe receptor-mediated tumor uptake in breast and neuroblastoma tumor bearing mice. Data from metabolic stability studies in mouse plasma suggest that the low uptake in tumors was due to a rapid degradation of peptide in blood. To overcome the short plasma half-life of the BVD15 analog, various strategies such as modification in the peptide sequence or the use of peptidase inhibitors need to be further explored.  155  Radiolabeling of the peptides with 18F via click chemistry required two steps, in addition to an HPLC (high-performance liquid chromatography) purification step required at the end of each step, making the process very long. Given the short half-life of 18F, and a specific activity of higher than 1 Ci/mmol required for PET imaging, the total radiosynthesis procedure of almost 3 hour was not optimal for efficient in vivo studies and clinical PET imaging applications.  Due to the labeling challenges with 18F, we carried out a different 18F labeling method in collaboration with Dr. David Perrin’s group at UBC, where a simple one-step 18F-labeling approach via 18F-19F isotope exchange reaction on an ammoniomethyl-trifluoroborate (AmBF3) moiety was introduced. The fact that AmBF3 could be conjugated to a number of peptides and other bioconjugates, and that the final radiolabeled peptide could be obtained in less than 30 minutes, makes this technique quite attractive for PET imaging applications. In addition, radiolabeling with 18F using the exchange reaction requires only small quantities of precursor peptide, does not require HPLC purification, and produces high yields and high specific activities. These advantages prompted our exploration of the applicability of this approach to the design of peptide-based 18F-labeled tracers for prostate and breast cancer imaging in chapters 3 and 4. In chapter 3, a novel GRPR-targeting peptide 18F-AmBF3-MJ9 for PET imaging of prostate cancer was synthesized and biologically evaluated, demonstrating AmBF3-MJ9’s ability to maintain high binding affinity to GRPR. 18F labeling of AmBF3-MJ9 was performed via a one-step 18F-19F isotope exchange reaction in aqueous solution resulting in a good overall radiochemical yield and excellent specific activity. 18F-AmBF3-MJ9 demonstrated excellent GRPR binding affinity, high plasma stability, and favorable pharmacokinetics. Moreover, 156  specific tumor targeting and excellent tumor-to-background contrasts further suggest that 18F-AmBF3-MJ9 is a promising radiopharmaceutical for GRPR-targeting imaging by PET. In chapter 4, 18F-AMBF3-TATE was introduced as an SST2a receptor imaging agent. To investigate the characteristics of 18F-AmBF3-TATE with the common TATE radiotracers, Ar42J tumor was used as a model for SST2a receptor positive tumors. Surprisingly, AmBF3-TATE had better binding affinity than Ga-DOTA-TATE to the SST2a receptor. Radiosynthesis resulted in high yield and specific activity as expected.  The biological evaluation of 18F-AmBF3-TATE showed that this tracer offers good metabolic stability, optimal pharmacokinetics, and very high tumor-to-non-target tissue ratios for in vivo imaging. 18F-AmBF3-TATE exhibited high receptor mediated uptake in a preclinical mouse model of SST2a receptor-positive cancer with very low background activity in non-target tissues. The objective of chapter 5 was to evaluate whether radiotracers used for PET imaging of SST2a could be suitable for the detection of breast cancer, and whether antagonists are preferable to agonists. To this end, two ER-positive human breast cancer cell lines (Zr-75-1 and MCF-7) were screened, and their xenograft models were evaluated for SST2a detection by PET/CT imaging. Mice bearing SST2a transfected HEK293 cells were used as positive controls for in vivo studies. Ga-DOTA-TATE (an established agonist) was compared with Ga-NODAGA-LM3 and Ga-NOTA-BASS as antagonists. The binding affinity obtained with Ga-NOTA-BASS was not optimal. Therefore, we did not pursue 68Ga labeling and in vivo evaluations with this compound. 68Ga-labeled DOTA-TATE, and NODAGA-LM3 were prepared with high labeling yield and specific activity. A high tumor-to-background ratio was achieved in the Zr-75-1 tumor model 157  with somatostatin receptor agonists (68Ga-DOTA-TATE) and antagonists (68Ga-NODAGA-LM3). Despite similar absolute tumor uptake, the tumor to muscle ratio was higher with 68Ga-DOTA-TATE compared to 68Ga-NODAGA-LM3, which perhaps reflects the superior binding affinity of 68Ga-DOTA-TATE rather than differences in agonist/antagonist properties.  The MCF-7 cell line and tumor model did not show promising results. The Zr-75-1 human tumor cell line is a promising breast cancer model for SST2a imaging by positron emission tomography.  In summary, we achieved encouraging results with the trifluoroborate exchange reaction for detection of GRP and somatostatin receptor positive tumors, suggesting the potential for these radiopharmaceuticals to be used in PET imaging of prostate and breast cancer. Furthermore, these results encourage the application of 18F-AmBF3 -TATE imaging in the Zr-75-1 human breast tumor model as a future task to be compared to other SST antagonists.  158  Bibliography  1. Saha GB: Fundamentals of nuclear pharmacy, Sixth edn: Springer; 2010. 2. Saha GB: Basics of PET imaging: Springer; 2005. 3. van der Veldt AA, Smit EF, Lammertsma AA: Positron Emission Tomography as a Method for Measuring Drug Delivery to Tumors in vivo: The Example of [(11)C]docetaxel. Frontiers in oncology 2013, 3:208. 4. Juweid ME, Cheson BD: Positron-emission tomography and assessment of cancer therapy. The New England journal of medicine 2006, 354(5):496-507. 5. Juweid ME, Stroobants S, Hoekstra OS, Mottaghy FM, Dietlein M, Guermazi A, Wiseman GA, Kostakoglu L, Scheidhauer K, Buck A et al: Use of positron emission tomography for response assessment of lymphoma: consensus of the Imaging Subcommittee of International Harmonization Project in Lymphoma. Journal of clinical oncology 2007, 25(5):571-578. 6. Lardinois D, Weder W, Hany TF, Kamel EM, Korom S, Seifert B, von Schulthess GK, Steinert HC: Staging of non-small-cell lung cancer with integrated positron-emission tomography and computed tomography. The New England journal of medicine 2003, 348(25):2500-2507. 7. Seam P, Juweid ME, Cheson BD: The role of FDG-PET scans in patients with lymphoma. Blood 2007, 110(10):3507-3516. 8. von Schulthess GK, Steinert HC, Hany TF: Integrated PET/CT: current applications and future directions. Radiology 2006, 238(2):405-422. 9. Weber WA: Positron emission tomography as an imaging biomarker. Journal of clinical oncology 2006, 24(20):3282-3292. 10. Larson SM: Cancer or inflammation? A Holy Grail for nuclear medicine. Journal of nuclear medicine 1994, 35(10):1653-1655. 11. Konishi J, Yamazaki K, Tsukamoto E, Tamaki N, Onodera Y, Otake T, Morikawa T, Kinoshita I, Dosaka-Akita H, Nishimura M: Mediastinal lymph node staging by FDG-PET in patients with non-small cell lung cancer: analysis of false-positive 159  FDG-PET findings. Respiration; international review of thoracic diseases 2003, 70(5):500-506. 12. Hofer C, Laubenbacher C, Block T, Breul J, Hartung R, Schwaiger M: Fluorine-18-fluorodeoxyglucose positron emission tomography is useless for the detection of local recurrence after radical prostatectomy. European urology 1999, 36(1):31-35. 13. Buscombe JR, Holloway B, Roche N, Bombardieri E: Position of nuclear medicine modalities in the diagnostic work-up of breast cancer. The quarterly journal of nuclear medicine and molecular imaging 2004, 48(2):109-118. 14. Parkin DM, Bray FI, Devesa SS: Cancer burden in the year 2000. The global picture. Eur J Cancer 2001, 37 Suppl 8:S4-66. 15. Jemal A, Siegel R, Xu J, Ward E: Cancer statistics, 2010. CA: a cancer journal for clinicians 2010, 60(5):277-300. 16. Cho D, Di Blasio CJ, Rhee AC, Kattan MW: Prognostic factors for survival in patients with hormone-refractory prostate cancer (HRPC) after initial androgen deprivation therapy (ADT). Urologic oncology 2003, 21(4):282-291. 17. Mari Aparici C, Seo Y: Functional imaging for prostate cancer: therapeutic implications. Seminars in nuclear medicine 2012, 42(5):328-342. 18. Lin K, Lipsitz R, Miller T, Janakiraman S: Benefits and harms of prostate-specific antigen screening for prostate cancer: an evidence update for the U.S. Preventive Services Task Force. Annals of internal medicine 2008, 149(3):192-199. 19. Stamey TA, Caldwell M, McNeal JE, Nolley R, Hemenez M, Downs J: The prostate specific antigen era in the United States is over for prostate cancer: what happened in the last 20 years? The Journal of urology 2004, 172(4 Pt 1):1297-1301. 20. Minner S, Wittmer C, Graefen M, Salomon G, Steuber T, Haese A, Huland H, Bokemeyer C, Yekebas E, Dierlamm J et al: High level PSMA expression is associated with early PSA recurrence in surgically treated prostate cancer. The Prostate 2011, 71(3):281-288. 21. Slovin SF: Emerging role of immunotherapy in the management of prostate cancer. Oncology 2007, 21(3):326-333; discussion 334, 338, 346-328. 160  22. Ross JS, Sheehan CE, Fisher HA, Kaufman RP, Jr., Kaur P, Gray K, Webb I, Gray GS, Mosher R, Kallakury BV: Correlation of primary tumor prostate-specific membrane antigen expression with disease recurrence in prostate cancer. Clinical cancer research 2003, 9(17):6357-6362. 23. Burger MJ, Tebay MA, Keith PA, Samaratunga HM, Clements J, Lavin MF, Gardiner RA: Expression analysis of delta-catenin and prostate-specific membrane antigen: their potential as diagnostic markers for prostate cancer. International journal of cancer Journal international du cancer 2002, 100(2):228-237. 24. Bander NH: Technology insight: monoclonal antibody imaging of prostate cancer. Nature clinical practice Urology 2006, 3(4):216-225. 25. Sodee DB, Ellis RJ, Samuels MA, Spirnak JP, Poole WF, Riester C, Martanovic DM, Stonecipher R, Bellon EM: Prostate cancer and prostate bed SPECT imaging with ProstaScint: semiquantitative correlation with prostatic biopsy results. The Prostate 1998, 37(3):140-148. 26. Taneja SS: ProstaScint(R) Scan: Contemporary Use in Clinical Practice. Reviews in urology 2004, 6 Suppl 10:S19-28. 27. Pandit-Taskar N, O'Donoghue JA, Morris MJ, Wills EA, Schwartz LH, Gonen M, Scher HI, Larson SM, Divgi CR: Antibody mass escalation study in patients with castration-resistant prostate cancer using 111In-J591: lesion detectability and dosimetric projections for 90Y radioimmunotherapy. Journal of nuclear medicine 2008, 49(7):1066-1074. 28. Bander NH, Trabulsi EJ, Kostakoglu L, Yao D, Vallabhajosula S, Smith-Jones P, Joyce MA, Milowsky M, Nanus DM, Goldsmith SJ: Targeting metastatic prostate cancer with radiolabeled monoclonal antibody J591 to the extracellular domain of prostate specific membrane antigen. The Journal of urology 2003, 170(5):1717-1721. 29. Milowsky MI, Nanus DM, Kostakoglu L, Vallabhajosula S, Goldsmith SJ, Bander NH: Phase I trial of yttrium-90-labeled anti-prostate-specific membrane antigen monoclonal antibody J591 for androgen-independent prostate cancer. Journal of clinical oncology 2004, 22(13):2522-2531. 161  30. Morris MJ, Divgi CR, Pandit-Taskar N, Batraki M, Warren N, Nacca A, Smith-Jones P, Schwartz L, Kelly WK, Slovin S et al: Pilot trial of unlabeled and indium-111-labeled anti-prostate-specific membrane antigen antibody J591 for castrate metastatic prostate cancer. Clinical cancer research 2005, 11(20):7454-7461. 31. Patri AK, Myc A, Beals J, Thomas TP, Bander NH, Baker JR, Jr.: Synthesis and in vitro testing of J591 antibody-dendrimer conjugates for targeted prostate cancer therapy. Bioconjugate chemistry 2004, 15(6):1174-1181. 32. Vallabhajosula S, Goldsmith SJ, Hamacher KA, Kostakoglu L, Konishi S, Milowski MI, Nanus DM, Bander NH: Prediction of myelotoxicity based on bone marrow radiation-absorbed dose: radioimmunotherapy studies using 90Y- and 177Lu-labeled J591 antibodies specific for prostate-specific membrane antigen. Journal of nuclear medicine 2005, 46(5):850-858. 33. Cho SY, Gage KL, Mease RC, Senthamizhchelvan S, Holt DP, Jeffrey-Kwanisai A, Endres CJ, Dannals RF, Sgouros G, Lodge M et al: Biodistribution, tumor detection, and radiation dosimetry of 18F-DCFBC, a low-molecular-weight inhibitor of prostate-specific membrane antigen, in patients with metastatic prostate cancer. Journal of nuclear medicine 2012, 53(12):1883-1891. 34. Hara T, Kosaka N, Kishi H: PET imaging of prostate cancer using carbon-11-choline. Journal of nuclear medicine 1998, 39(6):990-995. 35. Kotzerke J, Volkmer BG, Neumaier B, Gschwend JE, Hautmann RE, Reske SN: Carbon-11 acetate positron emission tomography can detect local recurrence of prostate cancer. European journal of nuclear medicine and molecular imaging 2002, 29(10):1380-1384. 36. Oyama N, Akino H, Kanamaru H, Suzuki Y, Muramoto S, Yonekura Y, Sadato N, Yamamoto K, Okada K: 11C-acetate PET imaging of prostate cancer. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2002, 43(2):181-186. 37. Oyama N, Miller TR, Dehdashti F, Siegel BA, Fischer KC, Michalski JM, Kibel AS, Andriole GL, Picus J, Welch MJ: 11C-acetate PET imaging of prostate cancer: 162  detection of recurrent disease at PSA relapse. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2003, 44(4):549-555. 38. Soloviev D, Fini A, Chierichetti F, Al-Nahhas A, Rubello D: PET imaging with 11C-acetate in prostate cancer: a biochemical, radiochemical and clinical perspective. European journal of nuclear medicine and molecular imaging 2008, 35(5):942-949. 39. Vavere AL, Kridel SJ, Wheeler FB, Lewis JS: 1-11C-acetate as a PET radiopharmaceutical for imaging fatty acid synthase expression in prostate cancer. Journal of nuclear medicine 2008, 49(2):327-334. 40. Ponde DE, Dence CS, Oyama N, Kim J, Tai YC, Laforest R, Siegel BA, Welch MJ: 18F-fluoroacetate: a potential acetate analog for prostate tumor imaging--in vivo evaluation of 18F-fluoroacetate versus 11C-acetate. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2007, 48(3):420-428. 41. Matthies A, Ezziddin S, Ulrich EM, Palmedo H, Biersack HJ, Bender H, Guhlke S: Imaging of prostate cancer metastases with 18F-fluoroacetate using PET/CT. European journal of nuclear medicine and molecular imaging 2004, 31(5):797. 42. Bauman G, Belhocine T, Kovacs M, Ward A, Beheshti M, Rachinsky I: 18F-fluorocholine for prostate cancer imaging: a systematic review of the literature. Prostate cancer and prostatic diseases 2012, 15(1):45-55. 43. Beattie BJ, Smith-Jones PM, Jhanwar YS, Schoder H, Schmidtlein CR, Morris MJ, Zanzonico P, Squire O, Meirelles GS, Finn R et al: Pharmacokinetic assessment of the uptake of 16beta-18F-fluoro-5alpha-dihydrotestosterone (FDHT) in prostate tumors as measured by PET. Journal of nuclear medicine 2010, 51(2):183-192. 44. Larson SM, Morris M, Gunther I, Beattie B, Humm JL, Akhurst TA, Finn RD, Erdi Y, Pentlow K, Dyke J et al: Tumor localization of 16beta-18F-fluoro-5alpha-dihydrotestosterone versus 18F-FDG in patients with progressive, metastatic prostate cancer. Journal of nuclear medicine 2004, 45(3):366-373. 45. Lutje S, Boerman OC, van Rij CM, Sedelaar M, Helfrich W, Oyen WJ, Mulders PF: Prospects in radionuclide imaging of prostate cancer. The Prostate 2012, 72(11):1262-1272. 163  46. Schuster DM, Savir-Baruch B, Nieh PT, Master VA, Halkar RK, Rossi PJ, Lewis MM, Nye JA, Yu W, Bowman FD et al: Detection of recurrent prostate carcinoma with anti-1-amino-3-18F-fluorocyclobutane-1-carboxylic acid PET/CT and 111In-capromab pendetide SPECT/CT. Radiology 2011, 259(3):852-861. 47. Aparici CM, Carlson D, Nguyen N, Hawkins RA, Seo Y: Combined SPECT and Multidetector CT for Prostate Cancer Evaluations. American journal of nuclear medicine and molecular imaging 2012, 2(1):48-54. 48. Reske SN, Blumstein NM, Neumaier B, Gottfried HW, Finsterbusch F, Kocot D, Moller P, Glatting G, Perner S: Imaging prostate cancer with 11C-choline PET/CT. Journal of nuclear medicine 2006, 47(8):1249-1254. 49. Society AC: Breast cancer. In.; 2014. 50. Prekeges J: Breast imaging devices for nuclear medicine. Journal of nuclear medicine technology 2012, 40(2):71-78. 51. Kopans DB: Screening for breast cancer among women in their 40s. The Lancet Oncology 2010, 11(12):1108-1109. 52. Taillefer R: Clinical applications of 99mTc-sestamibi scintimammography. Seminars in nuclear medicine 2005, 35(2):100-115. 53. Breast Cancer Screening  54. Bombardieri E, Crippa F, Baio SM, Peeters BA, Greco M, Pauwels EK: Nuclear medicine advances in breast cancer imaging. Tumori 2001, 87(5):277-287. 55. Bombardieri E, Crippa F: PET imaging in breast cancer. The quarterly journal of nuclear medicine 2001, 45(3):245-256. 56. Bombardieri E, Aktolun C, Baum RP, Bishof-Delaloye A, Buscombe J, Chatal JF, Maffioli L, Moncayo R, Morteimans L, Reske SN: Bone scintigraphy: procedure guidelines for tumour imaging. European journal of nuclear medicine and molecular imaging 2003, 30(12):BP99-106. 57. Khalkhali I, Mena I, Jouanne E, Diggles L, Venegas R, Block J, Alle K, Klein S: Prone scintimammography in patients with suspicion of carcinoma of the breast. Journal of the American College of Surgeons 1994, 178(5):491-497. 164  58. Aktolun C, Bayhan H, Kir M: Clinical experience with Tc-99m MIBI imaging in patients with malignant tumors. Preliminary results and comparison with Tl-201. Clinical nuclear medicine 1992, 17(3):171-176. 59. Lee JH, Rosen EL, Mankoff DA: The role of radiotracer imaging in the diagnosis and management of patients with breast cancer: part 1--overview, detection, and staging. Journal of nuclear medicine 2009, 50(4):569-581. 60. McDonough MD, DePeri ER, Mincey BA: The role of positron emission tomographic imaging in breast cancer. Current oncology reports 2004, 6(1):62-68. 61. Lee JH, Rosen EL, Mankoff DA: The role of radiotracer imaging in the diagnosis and management of patients with breast cancer: part 2--response to therapy, other indications, and future directions. Journal of nuclear medicine 2009, 50(5):738-748. 62. Zhang H: Design, Synthesis, and Preclinical Evaluation of Radiolabeled Bombesin Analogues for the Diagnosis and Targeted Radiotherapy of Bombesinreceptor Expressing Tumors. Basel; 2007. 63. Okarvi SM: Recent developments in 99Tcm-labelled peptide-based radiopharmaceuticals: an overview. Nuclear medicine communications 1999, 20(12):1093-1112. 64. Okarvi SM: Peptide-based radiopharmaceuticals: future tools for diagnostic imaging of cancers and other diseases. Medicinal research reviews 2004, 24(3):357-397. 65. Goldenberg DM: Advancing role of radiolabeled antibodies in the therapy of cancer. Cancer immunology, immunotherapy : CII 2003, 52(5):281-296. 66. Ellis RJ, Kim E, Foor R: Role of ProstaScint for brachytherapy in localized prostate adenocarcinoma. Expert review of molecular diagnostics 2004, 4(4):435-441. 67. Kahn D, Austin JC, Maguire RT, Miller SJ, Gerstbrein J, Williams RD: A phase II study of [90Y] yttrium-capromab pendetide in the treatment of men with prostate cancer recurrence following radical prostatectomy. Cancer biotherapy & radiopharmaceuticals 1999, 14(2):99-111. 165  68. Reubi JC: Neuropeptide receptors in health and disease: the molecular basis for in vivo imaging. Journal of nuclear medicine 1995, 36(10):1825-1835. 69. Villalba M, Bockaert J, Journot L: Pituitary adenylate cyclase-activating polypeptide (PACAP-38) protects cerebellar granule neurons from apoptosis by activating the mitogen-activated protein kinase (MAP kinase) pathway. The Journal of neuroscience 1997, 17(1):83-90. 70. Cattaneo MG, Amoroso D, Gussoni G, Sanguini AM, Vicentini LM: A somatostatin analogue inhibits MAP kinase activation and cell proliferation in human neuroblastoma and in human small cell lung carcinoma cell lines. FEBS letters 1996, 397(2-3):164-168. 71. Reubi JC: Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocrine reviews 2003, 24(4):389-427. 72. Reubi JC, Maecke HR: Peptide-based probes for cancer imaging. Journal of nuclear medicine 2008, 49(11):1735-1738. 73. Hofland LJ, Lamberts SW: Somatostatin receptor subtype expression in human tumors. Annals of oncology 2001, 12 Suppl 2:S31-36. 74. Fani M, Maecke HR, Okarvi SM: Radiolabeled peptides: valuable tools for the detection and treatment of cancer. Theranostics 2012, 2(5):481-501. 75. Gugger M, Reubi JC: Gastrin-releasing peptide receptors in non-neoplastic and neoplastic human breast. The American journal of pathology 1999, 155(6):2067-2076. 76. Halmos G, Wittliff JL, Schally AV: Characterization of bombesin/gastrin-releasing peptide receptors in human breast cancer and their relationship to steroid receptor expression. Cancer research 1995, 55(2):280-287. 77. Moody TW, Bertness V, Carney DN: Bombesin-like peptides and receptors in human tumor cell lines. Peptides 1983, 4(5):683-686. 78. Reubi JC, Wenger S, Schmuckli-Maurer J, Schaer JC, Gugger M: Bombesin receptor subtypes in human cancers: detection with the universal radioligand (125)I-[D-TYR(6), beta-ALA(11), PHE(13), NLE(14)] bombesin(6-14). Clinical cancer research 2002, 8(4):1139-1146. 166  79. Anastasi A, Erspamer V, Bucci M: Isolation and structure of bombesin and alytesin, 2 analogous active peptides from the skin of the European amphibians Bombina and Alytes. Experientia 1971, 27(2):166-167. 80. Heppeler A, Froidevaux S, Eberle AN, Maecke HR: Receptor targeting for tumor localisation and therapy with radiopeptides. Current medicinal chemistry 2000, 7(9):971-994. 81. Mattei J, Achcar RD, Cano CH, Macedo BR, Meurer L, Batlle BS, Groshong SD, Kulczynski JM, Roesler R, Dal Lago L et al: Gastrin-releasing peptide receptor expression in lung cancer. Archives of pathology & laboratory medicine 2014, 138(1):98-104. 82. Carroll RE, Matkowskyj KA, Chakrabarti S, McDonald TJ, Benya RV: Aberrant expression of gastrin-releasing peptide and its receptor by well-differentiated colon cancers in humans. The American journal of physiology 1999, 276(3 Pt 1):G655-665. 83. Lui VW, Thomas SM, Zhang Q, Wentzel AL, Siegfried JM, Li JY, Grandis JR: Mitogenic effects of gastrin-releasing peptide in head and neck squamous cancer cells are mediated by activation of the epidermal growth factor receptor. Oncogene 2003, 22(40):6183-6193. 84. Pisarek H, Stepien T, Kubiak R, Borkowska E, Pawlikowski M: Expression of somatostatin receptor subtypes in human thyroid tumors: the immunohistochemical and molecular biology (RT-PCR) investigation. Thyroid research 2009, 2(1):1. 85. Hofland LJ, Liu Q, Van Koetsveld PM, Zuijderwijk J, Van Der Ham F, De Krijger RR, Schonbrunn A, Lamberts SW: Immunohistochemical detection of somatostatin receptor subtypes sst1 and sst2A in human somatostatin receptor positive tumors. The Journal of clinical endocrinology and metabolism 1999, 84(2):775-780. 86. Schulz S, Schulz S, Schmitt J, Wiborny D, Schmidt H, Olbricht S, Weise W, Roessner A, Gramsch C, Hollt V: Immunocytochemical detection of somatostatin receptors sst1, sst2A, sst2B, and sst3 in paraffin-embedded breast cancer tissue using subtype-specific antibodies. Clinical cancer research 1998, 4(9):2047-2052. 167  87. Janson ET, Stridsberg M, Gobl A, Westlin JE, Oberg K: Determination of somatostatin receptor subtype 2 in carcinoid tumors by immunohistochemical investigation with somatostatin receptor subtype 2 antibodies. Cancer research 1998, 58(11):2375-2378. 88. Kimura N, Pilichowska M, Date F, Kimura I, Schindler M: Immunohistochemical expression of somatostatin type 2A receptor in neuroendocrine tumors. Clinical cancer research 1999, 5(11):3483-3487. 89. Korner M, Reubi JC: NPY receptors in human cancer: a review of current knowledge. Peptides 2007, 28(2):419-425. 90. Korner M, Waser B, Reubi JC: Neuropeptide Y receptor expression in human primary ovarian neoplasms. Laboratory investigation; a journal of technical methods and pathology 2004, 84(1):71-80. 91. Korner M, Waser B, Reubi JC: High expression of neuropeptide y receptors in tumors of the human adrenal gland and extra-adrenal paraganglia. Clinical cancer research 2004, 10(24):8426-8433. 92. Reubi JC, Gugger M, Waser B, Schaer JC: Y(1)-mediated effect of neuropeptide Y in cancer: breast carcinomas as targets. Cancer research 2001, 61(11):4636-4641. 93. Korner M, Waser B, Reubi JC: Neuropeptide Y receptors in renal cell carcinomas and nephroblastomas. International journal of cancer Journal international du cancer 2005, 115(5):734-741. 94. Krenning EP, Bakker WH, Breeman WA, Koper JW, Kooij PP, Ausema L, Lameris JS, Reubi JC, Lamberts SW: Localisation of endocrine-related tumours with radioiodinated analogue of somatostatin. Lancet 1989, 1(8632):242-244. 95. Otte A, Mueller-Brand J, Dellas S, Nitzsche EU, Herrmann R, Maecke HR: Yttrium-90-labelled somatostatin-analogue for cancer treatment. Lancet 1998, 351(9100):417-418. 96. Mankoff DA, Link JM, Linden HM, Sundararajan L, Krohn KA: Tumor receptor imaging. Journal of nuclear medicine 2008, 49 Suppl 2:149S-163S. 97. Langer M, Beck-Sickinger AG: Peptides as carrier for tumor diagnosis and treatment. Current medicinal chemistry Anti-cancer agents 2001, 1(1):71-93. 168  98. Behr TM, Gotthardt M, Barth A, Behe M: Imaging tumors with peptide-based radioligands. The quarterly journal of nuclear medicine 2001, 45(2):189-200. 99. Virgolini I, Traub T, Novotny C, Leimer M, Fuger B, Li SR, Patri P, Pangerl T, Angelberger P, Raderer M et al: New trends in peptide receptor radioligands. The quarterly journal of nuclear medicine 2001, 45(2):153-159. 100. Valkema R, Pauwels SA, Kvols LK, Kwekkeboom DJ, Jamar F, de Jong M, Barone R, Walrand S, Kooij PP, Bakker WH et al: Long-term follow-up of renal function after peptide receptor radiation therapy with (90)Y-DOTA(0),Tyr(3)-octreotide and (177)Lu-DOTA(0), Tyr(3)-octreotate. Journal of nuclear medicine 2005, 46 Suppl 1:83S-91S. 101. Imhof A, Brunner P, Marincek N, Briel M, Schindler C, Rasch H, Macke HR, Rochlitz C, Muller-Brand J, Walter MA: Response, survival, and long-term toxicity after therapy with the radiolabeled somatostatin analogue [90Y-DOTA]-TOC in metastasized neuroendocrine cancers. Journal of clinical oncology 2011, 29(17):2416-2423. 102. Teunissen JJ, Kwekkeboom DJ, de Jong M, Esser JP, Valkema R, Krenning EP: Endocrine tumours of the gastrointestinal tract. Peptide receptor radionuclide therapy. Best practice & research Clinical gastroenterology 2005, 19(4):595-616. 103. Rolleman EJ, Valkema R, de Jong M, Kooij PP, Krenning EP: Safe and effective inhibition of renal uptake of radiolabelled octreotide by a combination of lysine and arginine. European journal of nuclear medicine and molecular imaging 2003, 30(1):9-15. 104. Rolleman EJ, Forrer F, Bernard B, Bijster M, Vermeij M, Valkema R, Krenning EP, de Jong M: Amifostine protects rat kidneys during peptide receptor radionuclide therapy with [177Lu-DOTA0,Tyr3]octreotate. European journal of nuclear medicine and molecular imaging 2007, 34(5):763-771. 105. Vegt E, Wetzels JF, Russel FG, Masereeuw R, Boerman OC, van Eerd JE, Corstens FH, Oyen WJ: Renal uptake of radiolabeled octreotide in human subjects is efficiently inhibited by succinylated gelatin. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2006, 47(3):432-436. 169  106. van Eerd JE, Vegt E, Wetzels JF, Russel FG, Masereeuw R, Corstens FH, Oyen WJ, Boerman OC: Gelatin-based plasma expander effectively reduces renal uptake of 111In-octreotide in mice and rats. Journal of nuclear medicine 2006, 47(3):528-533. 107. Cuttitta F, Carney DN, Mulshine J, Moody TW, Fedorko J, Fischler A, Minna JD: Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature 1985, 316(6031):823-826. 108. McDonald TJ, Jornvall H, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V: Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochemical and biophysical research communications 1979, 90(1):227-233. 109. Spindel ER, Giladi E, Brehm P, Goodman RH, Segerson TP: Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin-releasing peptide receptor. Molecular endocrinology 1990, 4(12):1956-1963. 110. Wada E, Way J, Shapira H, Kusano K, Lebacq-Verheyden AM, Coy D, Jensen R, Battery J: cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor. Neuron 1991, 6(3):421-430. 111. Fathi Z, Corjay MH, Shapira H, Wada E, Benya R, Jensen R, Viallet J, Sausville EA, Battey JF: BRS-3: a novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. The Journal of biological chemistry 1993, 268(8):5979-5984. 112. Nagalla SR, Barry BJ, Creswick KC, Eden P, Taylor JT, Spindel ER: Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4). Proceedings of the National Academy of Sciences of the United States of America 1995, 92(13):6205-6209. 113. Mantey S, Frucht H, Coy DH, Jensen RT: Characterization of bombesin receptors using a novel, potent, radiolabeled antagonist that distinguishes bombesin receptor subtypes. Molecular pharmacology 1993, 43(5):762-774. 114. Yegen BC: Bombesin-like peptides: candidates as diagnostic and therapeutic tools. Current pharmaceutical design 2003, 9(12):1013-1022. 170  115. Moody TW, Pert CB, Gazdar AF, Carney DN, Minna JD: High levels of intracellular bombesin characterize human small-cell lung carcinoma. Science 1981, 214(4526):1246-1248. 116. Sun B, Halmos G, Schally AV, Wang X, Martinez M: Presence of receptors for bombesin/gastrin-releasing peptide and mRNA for three receptor subtypes in human prostate cancers. The Prostate 2000, 42(4):295-303. 117. Carroll RE, Carroll R, Benya RV: Characterization of gastrin-releasing peptide receptors aberrantly expressed by non-antral gastric adenocarcinomas. Peptides 1999, 20(2):229-237. 118. Saurin JC, Rouault JP, Abello J, Berger F, Remy L, Chayvialle JA: High gastrin releasing peptide receptor mRNA level is related to tumour dedifferentiation and lymphatic vessel invasion in human colon cancer. Eur J Cancer 1999, 35(1):125-132. 119. Nelson J, Donnelly M, Walker B, Gray J, Shaw C, Murphy RF: Bombesin stimulates proliferation of human breast cancer cells in culture. British journal of cancer 1991, 63(6):933-936. 120. Wang QJ, Knezetic JA, Schally AV, Pour PM, Adrian TE: Bombesin may stimulate proliferation of human pancreatic cancer cells through an autocrine pathway. International journal of cancer Journal international du cancer 1996, 68(4):528-534. 121. Kim S, Hu W, Kelly DR, Hellmich MR, Evers BM, Chung DH: Gastrin-releasing peptide is a growth factor for human neuroblastomas. Annals of surgery 2002, 235(5):621-629; discussion 629-630. 122. Milovanovic SR, Radulovic S, Groot K, Schally AV: Inhibition of growth of PC-82 human prostate cancer line xenografts in nude mice by bombesin antagonist RC-3095 or combination of agonist [D-Trp6]-luteinizing hormone-releasing hormone and somatostatin analog RC-160. The Prostate 1992, 20(4):269-280. 123. Pagani A, Papotti M, Sanfilippo B, Bussolati G: Expression of the gastrin-releasing peptide gene in carcinomas of the breast. International journal of cancer Journal international du cancer 1991, 47(3):371-375. 171  124. Kiaris H, Schally AV, Sun B, Armatis P, Groot K: Inhibition of growth of human malignant glioblastoma in nude mice by antagonists of bombesin/gastrin-releasing peptide. Oncogene 1999, 18(50):7168-7173. 125. Markwalder R, Reubi JC: Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer research 1999, 59(5):1152-1159. 126. Lee S, Xie J, Chen X: Peptides and peptide hormones for molecular imaging and disease diagnosis. Chemical reviews 2010, 110(5):3087-3111. 127. Schottelius M, Wester HJ: Molecular imaging targeting peptide receptors. Methods 2009, 48(2):161-177. 128. Wild D, Frischknecht M, Zhang H, Morgenstern A, Bruchertseifer F, Boisclair J, Provencher-Bolliger A, Reubi JC, Maecke HR: Alpha- versus beta-particle radiopeptide therapy in a human prostate cancer model (213Bi-DOTA-PESIN and 213Bi-AMBA versus 177Lu-DOTA-PESIN). Cancer research 2011, 71(3):1009-1018. 129. Reubi JC, Korner M, Waser B, Mazzucchelli L, Guillou L: High expression of peptide receptors as a novel target in gastrointestinal stromal tumours. European journal of nuclear medicine and molecular imaging 2004, 31(6):803-810. 130. Toi-Scott M, Jones CL, Kane MA: Clinical correlates of bombesin-like peptide receptor subtype expression in human lung cancer cells. Lung Cancer 1996, 15(3):341-354. 131. Hoffman TJ, Gali H, Smith CJ, Sieckman GL, Hayes DL, Owen NK, Volkert WA: Novel series of 111In-labeled bombesin analogs as potential radiopharmaceuticals for specific targeting of gastrin-releasing peptide receptors expressed on human prostate cancer cells. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2003, 44(5):823-831. 132. Maecke HR, Hofmann M, Haberkorn U: (68)Ga-labeled peptides in tumor imaging. Journal of nuclear medicine 2005, 46 Suppl 1:172S-178S. 133. Mansi R, Wang X, Forrer F, Kneifel S, Tamma ML, Waser B, Cescato R, Reubi JC, Maecke HR: Evaluation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic 172  acid-conjugated bombesin-based radioantagonist for the labeling with single-photon emission computed tomography, positron emission tomography, and therapeutic radionuclides. Clinical cancer research 2009, 15(16):5240-5249. 134. Mansi R, Wang X, Forrer F, Waser B, Cescato R, Graham K, Borkowski S, Reubi JC, Maecke HR: Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours. European journal of nuclear medicine and molecular imaging 2011, 38(1):97-107. 135. Nock B, Nikolopoulou A, Chiotellis E, Loudos G, Maintas D, Reubi JC, Maina T: [99mTc]Demobesin 1, a novel potent bombesin analogue for GRP receptor-targeted tumour imaging. European journal of nuclear medicine and molecular imaging 2003, 30(2):247-258. 136. Rogers BE, Bigott HM, McCarthy DW, Della Manna D, Kim J, Sharp TL, Welch MJ: MicroPET imaging of a gastrin-releasing peptide receptor-positive tumor in a mouse model of human prostate cancer using a 64Cu-labeled bombesin analogue. Bioconjugate chemistry 2003, 14(4):756-763. 137. Schroeder RP, van Weerden WM, Bangma C, Krenning EP, de Jong M: Peptide receptor imaging of prostate cancer with radiolabelled bombesin analogues. Methods 2009, 48(2):200-204. 138. Schweinsberg C, Maes V, Brans L, Blauenstein P, Tourwe DA, Schubiger PA, Schibli R, Garcia Garayoa E: Novel glycated [99mTc(CO)3]-labeled bombesin analogues for improved targeting of gastrin-releasing peptide receptor-positive tumors. Bioconjugate chemistry 2008, 19(12):2432-2439. 139. Hohne A, Mu L, Honer M, Schubiger PA, Ametamey SM, Graham K, Stellfeld T, Borkowski S, Berndorff D, Klar U et al: Synthesis, 18F-labeling, and in vitro and in vivo studies of bombesin peptides modified with silicon-based building blocks. Bioconjugate chemistry 2008, 19(9):1871-1879. 140. Li ZB, Wu Z, Chen K, Ryu EK, Chen X: 18F-labeled BBN-RGD heterodimer for prostate cancer imaging. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2008, 49(3):453-461. 173  141. Zhang X, Cai W, Cao F, Schreibmann E, Wu Y, Wu JC, Xing L, Chen X: 18F-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer. Journal of nuclear medicine 2006, 47(3):492-501. 142. Inkster JA, Adam MJ, Storr T, Ruth TJ: Labeling of an antisense oligonucleotide with [(18)F]FPy5yne. Nucleosides, nucleotides & nucleic acids 2009, 28(11):1131-1143. 143. Inkster JAH, Guerin B, Ruth TJ, Adam MJ: Radiosynthesis and bioconjugation of [F-18]FPy5yne, a prosthetic group for the F-18 labeling of bioactive peptides. Journal of labelled compounds & radiopharmaceuticals 2008, 51(13-14):444-452. 144. Kuhnast B, Hinnen F, Tavitian B, Dolle F: [F-18]FPyKYNE, a fluoropyridine-based alkyne reagent designed for the fluorine-18 labelling of macromolecules using click chemistry. Journal of labelled compounds & radiopharmaceuticals 2008, 51(9-10):336-342. 145. Inkster J, Lin KS, Ait-Mohand S, Gosselin S, Benard F, Guerin B, Pourghiasian M, Ruth T, Schaffer P, Storr T: 2-Fluoropyridine prosthetic compounds for the 18F labeling of bombesin analogues. Bioorganic & medicinal chemistry letters 2013, 23(13):3920-3926. 146. Dapp S, Garcia Garayoa E, Maes V, Brans L, Tourwe DA, Muller C, Schibli R: PEGylation of (99m)Tc-labeled bombesin analogues improves their pharmacokinetic properties. Nuclear medicine and biology 2011, 38(7):997-1009. 147. Fee CJ: Size comparison between proteins PEGylated with branched and linear poly(ethylene glycol) molecules. Biotechnology and bioengineering 2007, 98(4):725-731. 148. Chen X, Park R, Hou Y, Khankaldyyan V, Gonzales-Gomez I, Tohme M, Bading JR, Laug WE, Conti PS: MicroPET imaging of brain tumor angiogenesis with 18F-labeled PEGylated RGD peptide. European journal of nuclear medicine and molecular imaging 2004, 31(8):1081-1089. 149. Harris JM, Chess RB: Effect of pegylation on pharmaceuticals. Nature reviews Drug discovery 2003, 2(3):214-221. 174  150. Lee LS, Conover C, Shi C, Whitlow M, Filpula D: Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds. Bioconjugate chemistry 1999, 10(6):973-981. 151. Caliceti P, Veronese FM: Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Advanced drug delivery reviews 2003, 55(10):1261-1277. 152. Graham MM, Menda Y: Radiopeptide imaging and therapy in the United States. Journal of nuclear medicine 2011, 52 Suppl 2:56S-63S. 153. Ambrosini V, Fani M, Fanti S, Forrer F, Maecke HR: Radiopeptide imaging and therapy in Europe. Journal of nuclear medicine 2011, 52 Suppl 2:42S-55S. 154. Ait-Mohand S, Fournier P, Dumulon-Perreault V, Kiefer GE, Jurek P, Ferreira CL, Benard F, Guerin B: Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates. Bioconjugate chemistry 2011, 22(8):1729-1735. 155. Lantry LE, Cappelletti E, Maddalena ME, Fox JS, Feng W, Chen J, Thomas R, Eaton SM, Bogdan NJ, Arunachalam T et al: 177Lu-AMBA: Synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer. Journal of nuclear medicine 2006, 47(7):1144-1152. 156. Schroeder RP, van Weerden WM, Krenning EP, Bangma CH, Berndsen S, Grievink-de Ligt CH, Groen HC, Reneman S, de Blois E, Breeman WA et al: Gastrin-releasing peptide receptor-based targeting using bombesin analogues is superior to metabolism-based targeting using choline for in vivo imaging of human prostate cancer xenografts. European journal of nuclear medicine and molecular imaging 2011, 38(7):1257-1266. 157. Ho CL, Liu IH, Wu YH, Chen LC, Chen CL, Lee WC, Chuang CH, Lee TW, Lin WJ, Shen LH et al: Molecular imaging, pharmacokinetics, and dosimetry of In-AMBA in human prostate tumor-bearing mice. Journal of biomedicine & biotechnology 2011, 2011:101497. 175  158. Lane SR, Nanda P, Rold TL, Sieckman GL, Figueroa SD, Hoffman TJ, Jurisson SS, Smith CJ: Optimization, biological evaluation and microPET imaging of copper-64-labeled bombesin agonists, [64Cu-NO2A-(X)-BBN(7-14)NH2], in a prostate tumor xenografted mouse model. Nuclear medicine and biology 2010, 37(7):751-761. 159. Maddalena ME, Fox J, Chen J, Feng W, Cagnolini A, Linder KE, Tweedle MF, Nunn AD, Lantry LE: 177Lu-AMBA biodistribution, radiotherapeutic efficacy, imaging, and autoradiography in prostate cancer models with low GRP-R expression. Journal of nuclear medicine 2009, 50(12):2017-2024. 160. Cescato R, Maina T, Nock B, Nikolopoulou A, Charalambidis D, Piccand V, Reubi JC: Bombesin receptor antagonists may be preferable to agonists for tumor targeting. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2008, 49(2):318-326. 161. Durkan K, Jiang Z, Rold TL, Sieckman GL, Hoffman TJ, Bandari RP, Szczodroski AF, Liu L, Miao Y, Reynolds TS et al: A heterodimeric [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2] alphavbeta3/GRPr-targeting antagonist radiotracer for PET imaging of prostate tumors. Nuclear medicine and biology 2014, 41(2):133-139. 162. Varasteh Z, Rosenstrom U, Velikyan I, Mitran B, Altai M, Honarvar H, Rosestedt M, Lindeberg G, Sorensen J, Larhed M et al: The effect of mini-PEG-based spacer length on binding and pharmacokinetic properties of a 68Ga-labeled NOTA-conjugated antagonistic analog of bombesin. Molecules 2014, 19(7):10455-10472. 163. Varasteh Z, Velikyan I, Lindeberg G, Sorensen J, Larhed M, Sandstrom M, Selvaraju RK, Malmberg J, Tolmachev V, Orlova A: Synthesis and characterization of a high-affinity NOTA-conjugated bombesin antagonist for GRPR-targeted tumor imaging. Bioconjugate chemistry 2013, 24(7):1144-1153. 164. Kahkonen E, Jambor I, Kemppainen J, Lehtio K, Gronroos TJ, Kuisma A, Luoto P, Sipila HJ, Tolvanen T, Alanen K et al: In vivo imaging of prostate cancer using [68Ga]-labeled bombesin analog BAY86-7548. Clinical cancer research 2013, 19(19):5434-5443. 176  165. Wieser G, Mansi R, Grosu AL, Schultze-Seemann W, Dumont-Walter RA, Meyer PT, Maecke HR, Reubi JC, Weber WA: Positron emission tomography (PET) imaging of prostate cancer with a gastrin releasing peptide receptor antagonist--from mice to men. Theranostics 2014, 4(4):412-419. 166. Ross TL, Ametamey SM, Khalil MM: Basic Sciences of Nuclear Medicine. Heidelberg: Springer; 2011. 167. Liu Z, Pourghiasian M, Radtke MA, Lau J, Pan J, Dias GM, Yapp D, Lin KS, Benard F, Perrin DM: An Organotrifluoroborate for Broadly Applicable One-Step F-Labeling. Angew Chem Int Ed Engl 2014. 168. Zhang H, Chen J, Waldherr C, Hinni K, Waser B, Reubi JC, Maecke HR: Synthesis and evaluation of bombesin derivatives on the basis of pan-bombesin peptides labeled with indium-111, lutetium-177, and yttrium-90 for targeting bombesin receptor-expressing tumors. Cancer research 2004, 64(18):6707-6715. 169. Breeman WA, Hofland LJ, de Jong M, Bernard BF, Srinivasan A, Kwekkeboom DJ, Visser TJ, Krenning EP: Evaluation of radiolabelled bombesin analogues for receptor-targeted scintigraphy and radiotherapy. International journal of cancer Journal international du cancer 1999, 81(4):658-665. 170. Baidoo KE, Lin KS, Zhan Y, Finley P, Scheffel U, Wagner HN, Jr.: Design, synthesis, and initial evaluation of high-affinity technetium bombesin analogues. Bioconjugate chemistry 1998, 9(2):218-225. 171. Varasteh Z, Aberg O, Velikyan I, Lindeberg G, Sorensen J, Larhed M, Antoni G, Sandstrom M, Tolmachev V, Orlova A: In vitro and in vivo evaluation of a (18)F-labeled high affinity NOTA conjugated bombesin antagonist as a PET ligand for GRPR-targeted tumor imaging. PloS one 2013, 8(12):e81932. 172. Liu Y, Hu X, Liu H, Bu L, Ma X, Cheng K, Li J, Tian M, Zhang H, Cheng Z: A comparative study of radiolabeled bombesin analogs for the PET imaging of prostate cancer. Journal of nuclear medicine 2013, 54(12):2132-2138. 173. McBride WJ, Sharkey RM, Karacay H, D'Souza CA, Rossi EA, Laverman P, Chang CH, Boerman OC, Goldenberg DM: A novel method of 18F radiolabeling for PET. Journal of nuclear medicine 2009, 50(6):991-998. 177  174. Liu Z, Pourghiasian M, Benard F, Pan J, Lin KS, Perrin DM: Preclinical Evaluation of a High-Affinity 18F-Trifluoroborate Octreotate Derivative for Somatostatin Receptor Imaging. Journal of nuclear medicine 2014, 55(9):1499-1505. 175. Laverman P, McBride WJ, Sharkey RM, Goldenberg DM, Boerman OC: Al(18) F labeling of peptides and proteins. Journal of labelled compounds & radiopharmaceuticals 2014, 57(4):219-223. 176. Laverman P, D'Souza CA, Eek A, McBride WJ, Sharkey RM, Oyen WJ, Goldenberg DM, Boerman OC: Optimized labeling of NOTA-conjugated octreotide with F-18. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2012, 33(2):427-434. 177. Jamous M, Tamma ML, Gourni E, Waser B, Reubi JC, Maecke HR, Mansi R: PEG spacers of different length influence the biological profile of bombesin-based radiolabeled antagonists. Nuclear medicine and biology 2014, 41(6):464-470. 178. Patel YC: Somatostatin and its receptor family. Frontiers in neuroendocrinology 1999, 20(3):157-198. 179. Reisine T, Bell GI: Molecular biology of somatostatin receptors. Endocrine reviews 1995, 16(4):427-442. 180. Hoyer D, Bell GI, Berelowitz M, Epelbaum J, Feniuk W, Humphrey PP, O'Carroll AM, Patel YC, Schonbrunn A, Taylor JE et al: Classification and nomenclature of somatostatin receptors. Trends in pharmacological sciences 1995, 16(3):86-88. 181. Seger R, Krebs EG: The MAPK signaling cascade. FASEB journal 1995, 9(9):726-735. 182. Guillermet J, Saint-Laurent N, Rochaix P, Cuvillier O, Levade T, Schally AV, Pradayrol L, Buscail L, Susini C, Bousquet C: Somatostatin receptor subtype 2 sensitizes human pancreatic cancer cells to death ligand-induced apoptosis. Proceedings of the National Academy of Sciences of the United States of America 2003, 100(1):155-160. 183. Sharma K, Srikant CB: Induction of wild-type p53, Bax, and acidic endonuclease during somatostatin-signaled apoptosis in MCF-7 human breast cancer cells. International journal of cancer Journal international du cancer 1998, 76(2):259-266. 178  184. Schweitzer P, Madamba S, Siggins GR: Arachidonic acid metabolites as mediators of somatostatin-induced increase of neuronal M-current. Nature 1990, 346(6283):464-467. 185. Colas B, Cambillau C, Buscail L, Zeggari M, Esteve JP, Lautre V, Thomas F, Vaysse N, Susini C: Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells. European journal of biochemistry / FEBS 1992, 207(3):1017-1024. 186. Nouel D, Gaudriault G, Houle M, Reisine T, Vincent JP, Mazella J, Beaudet A: Differential internalization of somatostatin in COS-7 cells transfected with SST1 and SST2 receptor subtypes: a confocal microscopic study using novel fluorescent somatostatin derivatives. Endocrinology 1997, 138(1):296-306. 187. Stroh T, Jackson AC, Sarret P, Dal Farra C, Vincent JP, Kreienkamp HJ, Mazella J, Beaudet A: Intracellular dynamics of sst5 receptors in transfected COS-7 cells: maintenance of cell surface receptors during ligand-induced endocytosis. Endocrinology 2000, 141(1):354-365. 188. Kwekkeboom DJ, de Herder WW, van Eijck CH, Kam BL, van Essen M, Teunissen JJ, Krenning EP: Peptide receptor radionuclide therapy in patients with gastroenteropancreatic neuroendocrine tumors. Seminars in nuclear medicine 2010, 40(2):78-88. 189. Antunes P, Ginj M, Walter MA, Chen J, Reubi JC, Maecke HR: Influence of different spacers on the biological profile of a DOTA-somatostatin analogue. Bioconjugate chemistry 2007, 18(1):84-92. 190. Breeman WA, de Jong M, Kwekkeboom DJ, Valkema R, Bakker WH, Kooij PP, Visser TJ, Krenning EP: Somatostatin receptor-mediated imaging and therapy: basic science, current knowledge, limitations and future perspectives. European journal of nuclear medicine 2001, 28(9):1421-1429. 191. Ginj M, Schmitt JS, Chen J, Waser B, Reubi JC, de Jong M, Schulz S, Maecke HR: Design, synthesis, and biological evaluation of somatostatin-based radiopeptides. Chemistry & biology 2006, 13(10):1081-1090. 179  192. Kwekkeboom DJ, Kam BL, van Essen M, Teunissen JJ, van Eijck CH, Valkema R, de Jong M, de Herder WW, Krenning EP: Somatostatin-receptor-based imaging and therapy of gastroenteropancreatic neuroendocrine tumors. Endocrine-related cancer 2010, 17(1):R53-73. 193. Lamberts SW, Bakker WH, Reubi JC, Krenning EP: Somatostatin-receptor imaging in the localization of endocrine tumors. The New England journal of medicine 1990, 323(18):1246-1249. 194. Wild D, Fani M, Behe M, Brink I, Rivier JE, Reubi JC, Maecke HR, Weber WA: First clinical evidence that imaging with somatostatin receptor antagonists is feasible. Journal of nuclear medicine 2011, 52(9):1412-1417. 195. Buchmann I, Henze M, Engelbrecht S, Eisenhut M, Runz A, Schafer M, Schilling T, Haufe S, Herrmann T, Haberkorn U: Comparison of 68Ga-DOTATOC PET and 111In-DTPAOC (Octreoscan) SPECT in patients with neuroendocrine tumours. European journal of nuclear medicine and molecular imaging 2007, 34(10):1617-1626. 196. Krausz Y, Keidar Z, Kogan I, Even-Sapir E, Bar-Shalom R, Engel A, Rubinstein R, Sachs J, Bocher M, Agranovicz S et al: SPECT/CT hybrid imaging with 111In-pentetreotide in assessment of neuroendocrine tumours. Clinical endocrinology 2003, 59(5):565-573. 197. Storch D, Behe M, Walter MA, Chen J, Powell P, Mikolajczak R, Macke HR: Evaluation of [99mTc/EDDA/HYNIC0]octreotide derivatives compared with [111In-DOTA0,Tyr3, Thr8]octreotide and [111In-DTPA0]octreotide: does tumor or pancreas uptake correlate with the rate of internalization? Journal of nuclear medicine 2005, 46(9):1561-1569. 198. Virgolini I, Leimer M, Handmaker H, Lastoria S, Bischof C, Muto P, Pangerl T, Gludovacz D, Peck-Radosavljevic M, Lister-James J et al: Somatostatin receptor subtype specificity and in vivo binding of a novel tumor tracer, 99mTc-P829. Cancer research 1998, 58(9):1850-1859. 199. Gabriel M, Decristoforo C, Donnemiller E, Ulmer H, Watfah Rychlinski C, Mather SJ, Moncayo R: An intrapatient comparison of 99mTc-EDDA/HYNIC-TOC with 180  111In-DTPA-octreotide for diagnosis of somatostatin receptor-expressing tumors. Journal of nuclear medicine 2003, 44(5):708-716. 200. Gabriel M, Decristoforo C, Kendler D, Dobrozemsky G, Heute D, Uprimny C, Kovacs P, Von Guggenberg E, Bale R, Virgolini IJ: 68Ga-DOTA-Tyr3-octreotide PET in neuroendocrine tumors: comparison with somatostatin receptor scintigraphy and CT. Journal of nuclear medicine 2007, 48(4):508-518. 201. Guo Y, Ferdani R, Anderson CJ: Preparation and biological evaluation of (64)Cu labeled Tyr(3)-octreotate using a phosphonic acid-based cross-bridged macrocyclic chelator. Bioconjugate chemistry 2012, 23(7):1470-1477. 202. Leyton J, Iddon L, Perumal M, Indrevoll B, Glaser M, Robins E, George AJ, Cuthbertson A, Luthra SK, Aboagye EO: Targeting somatostatin receptors: preclinical evaluation of novel 18F-fluoroethyltriazole-Tyr3-octreotate analogs for PET. Journal of nuclear medicine 2011, 52(9):1441-1448. 203. Poethko T, Schottelius M, Thumshirn G, Hersel U, Herz M, Henriksen G, Kessler H, Schwaiger M, Wester HJ: Two-step methodology for high-yield routine radiohalogenation of peptides: (18)F-labeled RGD and octreotide analogs. Journal of nuclear medicine 2004, 45(5):892-902. 204. Sprague JE, Peng Y, Sun X, Weisman GR, Wong EH, Achilefu S, Anderson CJ: Preparation and biological evaluation of copper-64-labeled tyr3-octreotate using a cross-bridged macrocyclic chelator. Clinical cancer research 2004, 10(24):8674-8682. 205. Wester HJ, Schottelius M, Scheidhauer K, Meisetschlager G, Herz M, Rau FC, Reubi JC, Schwaiger M: PET imaging of somatostatin receptors: design, synthesis and preclinical evaluation of a novel 18F-labelled, carbohydrated analogue of octreotide. European journal of nuclear medicine and molecular imaging 2003, 30(1):117-122. 206. Henze M, Schuhmacher J, Hipp P, Kowalski J, Becker DW, Doll J, Macke HR, Hofmann M, Debus J, Haberkorn U: PET imaging of somatostatin receptors using [68GA]DOTA-D-Phe1-Tyr3-octreotide: first results in patients with meningiomas. Journal of nuclear medicine 2001, 42(7):1053-1056. 181  207. Kayani I, Conry BG, Groves AM, Win T, Dickson J, Caplin M, Bomanji JB: A comparison of 68Ga-DOTATATE and 18F-FDG PET/CT in pulmonary neuroendocrine tumors. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2009, 50(12):1927-1932. 208. Poeppel TD, Binse I, Petersenn S, Lahner H, Schott M, Antoch G, Brandau W, Bockisch A, Boy C: 68Ga-DOTATOC versus 68Ga-DOTATATE PET/CT in functional imaging of neuroendocrine tumors. Journal of nuclear medicine 2011, 52(12):1864-1870. 209. Banerjee SR, Pomper MG: Clinical applications of Gallium-68. Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine 2013, 76:2-13. 210. Kemerink GJ, Visser MG, Franssen R, Beijer E, Zamburlini M, Halders SG, Brans B, Mottaghy FM, Teule GJ: Effect of the positron range of 18F, 68Ga and 124I on PET/CT in lung-equivalent materials. European journal of nuclear medicine and molecular imaging 2011, 38(5):940-948. 211. Laforest R, Liu X: Image quality with non-standard nuclides in PET. The quarterly journal of nuclear medicine and molecular imaging 2008, 52(2):151-158. 212. Chang-Guo Zhan DAD: Hydration of the Fluoride Anion:  Structures and Absolute Hydration Free Energy from First-Principles Electronic Structure Calculations. J Phys Chem 2004(108 (11), pp 2020–2029). 213. Chin FT, Shen B, Liu S, Berganos RA, Chang E, Mittra E, Chen X, Gambhir SS: First experience with clinical-grade ([18F]FPP(RGD(2)): an automated multi-step radiosynthesis for clinical PET studies. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging 2012, 14(1):88-95. 214. Cai H, Conti PS: RGD-based PET tracers for imaging receptor integrin alphav beta3 expression. Journal of labelled compounds & radiopharmaceuticals 2013, 56(5):264-279. 215. Laverman P, McBride WJ, Sharkey RM, Eek A, Joosten L, Oyen WJ, Goldenberg DM, Boerman OC: A novel facile method of labeling octreotide with (18)F-fluorine. Journal of nuclear medicine 2010, 51(3):454-461. 182  216. Wangler C, Waser B, Alke A, Iovkova L, Buchholz HG, Niedermoser S, Jurkschat K, Fottner C, Bartenstein P, Schirrmacher R et al: One-step (1)(8)F-labeling of carbohydrate-conjugated octreotate-derivatives containing a silicon-fluoride-acceptor (SiFA): in vitro and in vivo evaluation as tumor imaging agents for positron emission tomography (PET). Bioconjugate chemistry 2010, 21(12):2289-2296. 217. Liu Z, Li Y, Lozada J, Schaffer P, Adam MJ, Ruth TJ, Perrin DM: Stoichiometric leverage: rapid 18F-aryltrifluoroborate radiosynthesis at high specific activity for click conjugation. Angew Chem Int Ed Engl 2013, 52(8):2303-2307. 218. Liu Z, Li Y, Lozada J, Wong MQ, Greene J, Lin KS, Yapp D, Perrin DM: Kit-like 18F-labeling of RGD-19F-arytrifluroborate in high yield and at extraordinarily high specific activity with preliminary in vivo tumor imaging. Nuclear medicine and biology 2013, 40(6):841-849. 219. Liu Z LY, Lozada J, Lin K-S, Schaffer P, Perrin DM. Rapid: One-Step, High Yielding 18F-Labeling of an Aryltrifluoroborate Bioconjugate by Isotope Exchange at Very High Specific Activity. J Lab Comp Radiopharm 2012(14:491-497). 220. Lewis JS, Srinivasan A, Schmidt MA, Anderson CJ: In vitro and in vivo evaluation of Cu-64-TETA-Tyr(3)-octreotate. A new somatostatin analog with improved target tissue uptake. Nuclear Medicine and Biology 1999, 26(3):267-273. 221. Matteson DS, Majumdar D: IODOMETHANEBORONIC ESTERS AND AMINOMETHANEBORONIC ESTERS. Journal of Organometallic Chemistry 1979, 170(2):259-264. 222. Eberl S, Eriksson T, Svedberg O, Norling J, Henderson D, Lam P, Fulham M: High beam current operation of a PETtrace cyclotron for 18F- production. Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine 2012, 70(6):922-930. 223. Fani M, Braun F, Waser B, Beetschen K, Cescato R, Erchegyi J, Rivier JE, Weber WA, Maecke HR, Reubi JC: Unexpected sensitivity of sst2 antagonists to N-183  terminal radiometal modifications. Journal of nuclear medicine 2012, 53(9):1481-1489. 224. Reubi JC, Schar JC, Waser B, Wenger S, Heppeler A, Schmitt JS, Macke HR: Affinity profiles for human somatostatin receptor subtypes SST1-SST5 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use. European journal of nuclear medicine 2000, 27(3):273-282. 225. Leyton J, Iddon L, Perumal M, Indrevoll B, Glaser M, Robins E, George AJT, Cuthbertson A, Luthra SK, Aboagye EO: Targeting Somatostatin Receptors: Preclinical Evaluation of Novel F-18-Fluoroethyltriazole-Tyr(3)-Octreotate Analogs for PET. Journal of Nuclear Medicine 2011, 52(9):1441-1448. 226. Reubi JC, Waser B, Schaer JC, Laissue JA: Somatostatin receptor sst1-sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands. European journal of nuclear medicine 2001, 28(7):836-846. 227. Kumar U, Grigorakis SI, Watt HL, Sasi R, Snell L, Watson P, Chaudhari S: Somatostatin receptors in primary human breast cancer: quantitative analysis of mRNA for subtypes 1--5 and correlation with receptor protein expression and tumor pathology. Breast cancer research and treatment 2005, 92(2):175-186. 228. Van Den Bossche B, D'Haeninck E, De Vos F, Dierckx RA, Van Belle S, Bracke M, Van de Wiele C: Oestrogen-mediated regulation of somatostatin receptor expression in human breast cancer cell lines assessed with 99mTc-depreotide. European journal of nuclear medicine and molecular imaging 2004, 31(7):1022-1030. 229. Fani M, Del Pozzo L, Abiraj K, Mansi R, Tamma ML, Cescato R, Waser B, Weber WA, Reubi JC, Maecke HR: PET of somatostatin receptor-positive tumors using 64Cu- and 68Ga-somatostatin antagonists: the chelate makes the difference. Journal of nuclear medicine 2011, 52(7):1110-1118. 230. Eisenwiener KP, Prata MI, Buschmann I, Zhang HW, Santos AC, Wenger S, Reubi JC, Macke HR: NODAGATOC, a new chelator-coupled somatostatin analogue labeled with [67/68Ga] and [111In] for SPECT, PET, and targeted therapeutic 184  applications of somatostatin receptor (hsst2) expressing tumors. Bioconjugate chemistry 2002, 13(3):530-541. 231. Antunes P, Ginj M, Zhang H, Waser B, Baum RP, Reubi JC, Maecke H: Are radiogallium-labelled DOTA-conjugated somatostatin analogues superior to those labelled with other radiometals? European journal of nuclear medicine and molecular imaging 2007, 34(7):982-993. 232. Fani M, Mueller A, Tamma ML, Nicolas G, Rink HR, Cescato R, Reubi JC, Maecke HR: Radiolabeled bicyclic somatostatin-based analogs: a novel class of potential radiotracers for SPECT/PET of neuroendocrine tumors. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2010, 51(11):1771-1779. 233. Jiang G, Stalewski J, Galyean R, Dykert J, Schteingart C, Broqua P, Aebi A, Aubert ML, Semple G, Robson P et al: GnRH antagonists: a new generation of long acting analogues incorporating p-ureido-phenylalanines at positions 5 and 6. Journal of medicinal chemistry 2001, 44(3):453-467. 234. Bass RT, Buckwalter BL, Patel BP, Pausch MH, Price LA, Strnad J, Hadcock JR: Identification and characterization of novel somatostatin antagonists. Molecular pharmacology 1996, 50(4):709-715. 235. Ginj M, Zhang H, Waser B, Cescato R, Wild D, Wang X, Erchegyi J, Rivier J, Macke HR, Reubi JC: Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors. Proceedings of the National Academy of Sciences of the United States of America 2006, 103(44):16436-16441. 236. Vauquelin G, Van Liefde I: Slow antagonist dissociation and long-lasting in vivo receptor protection. Trends in pharmacological sciences 2006, 27(7):356-359. 237. Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, Westfall T: XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacological reviews 1998, 50(1):143-150. 185  238. Pedrazzini T, Seydoux J, Kunstner P, Aubert JF, Grouzmann E, Beermann F, Brunner HR: Cardiovascular response, feeding behavior and locomotor activity in mice lacking the NPY Y1 receptor. Nature medicine 1998, 4(6):722-726. 239. Colmers WF, Bleakman D: Effects of neuropeptide Y on the electrical properties of neurons. Trends in neurosciences 1994, 17(9):373-379. 240. Wettstein JG, Earley B, Junien JL: Central nervous system pharmacology of neuropeptide Y. Pharmacology & therapeutics 1995, 65(3):397-414. 241. Inui A: Neuropeptide Y: a key molecule in anorexia and cachexia in wasting disorders? Molecular medicine today 1999, 5(2):79-85. 242. Michel MC, Rascher W: Neuropeptide Y: a possible role in hypertension? Journal of hypertension 1995, 13(4):385-395. 243. Playford RJ, Cox HM: Peptide YY and neuropeptide Y: two peptides intimately involved in electrolyte homeostasis. Trends in pharmacological sciences 1996, 17(12):436-438. 244. Sheikh SP: Neuropeptide Y and peptide YY: major modulators of gastrointestinal blood flow and function. The American journal of physiology 1991, 261(5 Pt 1):G701-715. 245. Wang ZL, Bennet WM, Wang RM, Ghatei MA, Bloom SR: Evidence of a paracrine role of neuropeptide-Y in the regulation of insulin release from pancreatic islets of normal and dexamethasone-treated rats. Endocrinology 1994, 135(1):200-206. 246. Jain MR, Pu S, Kalra PS, Kalra SP: Evidence that stimulation of two modalities of pituitary luteinizing hormone release in ovarian steroid-primed ovariectomized rats may involve neuropeptide Y Y1 and Y4 receptors. Endocrinology 1999, 140(11):5171-5177. 247. McDonald JK: Role of neuropeptide Y in reproductive function. Annals of the New York Academy of Sciences 1990, 611:258-272. 248. Raposinho PD, Broqua P, Pierroz DD, Hayward A, Dumont Y, Quirion R, Junien JL, Aubert ML: Evidence that the inhibition of luteinizing hormone secretion exerted by central administration of neuropeptide Y (NPY) in the rat is predominantly mediated by the NPY-Y5 receptor subtype. Endocrinology 1999, 140(9):4046-4055. 186  249. Ruscica M, Dozio E, Motta M, Magni P: Relevance of the neuropeptide Y system in the biology of cancer progression. Current topics in medicinal chemistry 2007, 7(17):1682-1691. 250. Rudolf K, Eberlein W, Engel W, Wieland HA, Willim KD, Entzeroth M, Wienen W, Beck-Sickinger AG, Doods HN: The first highly potent and selective non-peptide neuropeptide Y Y1 receptor antagonist: BIBP3226. European journal of pharmacology 1994, 271(2-3):R11-13. 251. Balasubramaniam AA: Neuropeptide Y family of hormones: receptor subtypes and antagonists. Peptides 1997, 18(3):445-457. 252. Dumont Y, Cadieux A, Doods H, Pheng LH, Abounader R, Hamel E, Jacques D, Regoli D, Quirion R: BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist. British journal of pharmacology 2000, 129(6):1075-1088. 253. Ruscica M, Dozio E, Boghossian S, Bovo G, Martos Riano V, Motta M, Magni P: Activation of the Y1 receptor by neuropeptide Y regulates the growth of prostate cancer cells. Endocrinology 2006, 147(3):1466-1473. 254. Reubi C, Gugger M, Waser B: Co-expressed peptide receptors in breast cancer as a molecular basis for in vivo multireceptor tumour targeting. European journal of nuclear medicine and molecular imaging 2002, 29(7):855-862. 255. Amlal H, Faroqui S, Balasubramaniam A, Sheriff S: Estrogen up-regulates neuropeptide Y Y1 receptor expression in a human breast cancer cell line. Cancer research 2006, 66(7):3706-3714. 256. Langer M, La Bella R, Garcia-Garayoa E, Beck-Sickinger AG: 99mTc-labeled neuropeptide Y analogues as potential tumor imaging agents. Bioconjugate chemistry 2001, 12(6):1028-1034. 257. Zwanziger D, Khan IU, Neundorf I, Sieger S, Lehmann L, Friebe M, Dinkelborg L, Beck-Sickinger AG: Novel chemically modified analogues of neuropeptide Y for tumor targeting. Bioconjugate chemistry 2008, 19(7):1430-1438. 187  258. Sheikh SP, O'Hare MM, Tortora O, Schwartz TW: Binding of monoiodinated neuropeptide Y to hippocampal membranes and human neuroblastoma cell lines. The Journal of biological chemistry 1989, 264(12):6648-6654. 259. Fuhlendorff J, Gether U, Aakerlund L, Langeland-Johansen N, Thogersen H, Melberg SG, Olsen UB, Thastrup O, Schwartz TW: [Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist. Proceedings of the National Academy of Sciences of the United States of America 1990, 87(1):182-186. 260. Sheikh SP, Hakanson R, Schwartz TW: Y1 and Y2 receptors for neuropeptide Y. FEBS letters 1989, 245(1-2):209-214. 261. Inkster JA: New Approaches to the Labelling of Biological Targeting Vectors with 18F Simon Fraser University; 2013. 262. Werle M, Bernkop-Schnurch A: Strategies to improve plasma half life time of peptide and protein drugs. Amino acids 2006, 30(4):351-367. 263. Harris AG: Somatostatin and somatostatin analogues: pharmacokinetics and pharmacodynamic effects. Gut 1994, 35(3 Suppl):S1-4. 264. Marastoni M, Salvadori S, Scaranari V, Spisani S, Reali E, Traniello S, Tomatis A: Synthesis and activity of new linear and cyclic peptide T derivatives. Arzneimittel-Forschung 1994, 44(9):1073-1076. 265. Matsas R, Kenny AJ, Turner AJ: The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11. The Biochemical journal 1984, 223(2):433-440. 266. Nock BA, Maina T, Krenning EP, de Jong M: "To serve and protect": enzyme inhibitors as radiopeptide escorts promote tumor targeting. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2014, 55(1):121-127. 267. Lin KS, Pan J, Amouroux G, Turashvili G, Mesak F, Hundal-Jabal N, Pourghiasian M, Lau J, Jenni S, Aparicio S et al: In vivo radioimaging of bradykinin receptor B1, a widely overexpressed molecule in human cancer. Cancer research 2014. 268. Okarvi SM: Recent progress in fluorine-18 labelled peptide radiopharmaceuticals. European journal of nuclear medicine 2001, 28(7):929-938. 188  269. Wester HJ, Hamacher K, Stocklin G: A comparative study of N.C.A. fluorine-18 labeling of proteins via acylation and photochemical conjugation. Nuclear medicine and biology 1996, 23(3):365-372. 270. Rostovtsev VV, Green LG, Fokin VV, Sharpless KB: A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew Chem Int Ed Engl 2002, 41(14):2596-2599. 271. Angell Y, Burgess K: Ring closure to beta-turn mimics via copper-catalyzed azide/alkyne cycloadditions. The Journal of organic chemistry 2005, 70(23):9595-9598.        

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