Medicine, Faculty of
DSpace
UBCV
Dude, Iulia Gabriela
Master of Science - MSc
University of British Columbia
Somatostatin receptors (sstrs) are G-protein coupled receptors that modulate hormone secretions. Their overexpression on neuroendocrine tumours (NETs) has enabled successful imaging of these cancers with radioactive peptides using positron emission tomography. Luminal A breast cancers also overexpress sstr, but with a lower and more heterogeneous density than NETs. Recently, several authors demonstrated higher tumour binding with sstr antagonists compared to agonists. Antagonists are hypothesized to bind to target receptors in multiple configurations, thus labeling more binding sites than high-affinity agonists. This property could result in better visualization of tumours with lower sstr density, such as breast cancers. We hypothesized that the somatostatin agonist peptides TOC and TATE and antagonist peptide JR11 could be radiolabeled with ⁶⁸Ga and ¹⁸F, and that antagonist radiotracers will show higher tumour uptake when evaluated in vivo using the human breast cancer xenograft ZR-75-1. We synthesized the literature compounds ⁶⁸Ga-DOTATOC, ⁶⁸Ga-DOTATATE and antagonist ⁶⁸Ga-NODAGA-JR11 and evaluated binding affinity and tumour uptake. The agonist ⁶⁸Ga-DOTATOC had the highest tumour uptake (18.4 ± 2.87 %ID/g), and the antagonist ⁶⁸Ga-NODAGA-JR11, the lowest (12.1 ± 0.78 %ID/g). We radiolabeled the same peptide analogues with ¹⁸F using the easy and robust AmBF₃ method. ¹⁸F is an excellent isotope for peptide radiolabeling, having low positron energy (634 keV), a half-life well-matched to peptide kinetics (110 min), and an established cyclotron production method. We synthesized the compounds ¹⁸F-AmBF₃-TOC, ¹⁸F-AmBF3-TATE and ¹⁸F-AmBF3-JR11 and found that all were kinetically stable (determined by in vivo plasma stability studies) and successfully visualized the tumour with high intensity and contrast at both 1 hour and 2 hours post-injection (p.i.). At 1 h p.i. uptake means were between 10.0 – 13.4 %ID/g for all three radiotracers, and differences were not statistically significant. Imaging with ¹⁸F- and ⁶⁸Ga-labeled JR11 antagonists did not confer any additional advantages compared to agonists in this tumour model. However, the high tumour uptake of both ⁶⁸Ga- and ¹⁸F-labeled antagonists is striking, despite their significantly poorer binding affinity (inhibition contrast (Ki) > 18 nM) compared to agonist (Ki < 1.5 nM). With modifications to improve affinity, antagonist somatostatin analogues may be very promising for breast cancer visualization.
PRECLINICAL EVALUATION OF SOMATOSTATIN ANALOGUES FOR BREAST CANCER IMAGING USING POSITRON EMISSION TOMOGRAPHY by Iulia Gabriela Dude B.Sc., The University of Waterloo, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Interdisciplinary Oncology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2017 © Iulia Gabriela Dude, 2017 ii Abstract Somatostatin receptors (sstrs) are G-protein coupled receptors that modulate hormone secretions. Their overexpression on neuroendocrine tumours (NETs) has enabled successful imaging of these cancers with radioactive peptides using positron emission tomography. Luminal A breast cancers also overexpress sstr, but with a lower and more heterogeneous density than NETs. Recently, several authors demonstrated higher tumour binding with sstr antagonists compared to agonists. Antagonists are hypothesized to bind to target receptors in multiple configurations, thus labeling more binding sites than high-affinity agonists. This property could result in better visualization of tumours with lower sstr density, such as breast cancers. We hypothesized that the somatostatin agonist peptides TOC and TATE and antagonist peptide JR11 could be radiolabeled with 68Ga and 18F, and that antagonist radiotracers will show higher tumour uptake when evaluated in vivo using the human breast cancer xenograft ZR-75-1. We synthesized the literature compounds 68Ga-DOTATOC, 68Ga-DOTATATE and antagonist 68Ga-NODAGA-JR11 and evaluated binding affinity and tumour uptake. The agonist 68Ga-DOTATOC had the highest tumour uptake (18.4 ± 2.87 %ID/g), and the antagonist 68Ga-NODAGA-JR11, the lowest (12.1 ± 0.78 %ID/g). We radiolabeled the same peptide analogues with 18F using the easy and robust AmBF3 method. 18F is an excellent isotope for peptide radiolabeling, having low positron energy (634 keV), a half-life well-matched to peptide kinetics (110 min), and an established cyclotron production method. We synthesized the compounds 18F-AmBF3-TOC, 18F-AmBF3-TATE and 18F-AmBF3-JR11 and found that all were kinetically stable (determined by in vivo plasma stability studies) and successfully visualized the tumour with high intensity and contrast at both 1 hour and 2 hours post-injection (p.i.). At 1 h p.i. uptake means iii were between 10.0 – 13.4 %ID/g for all three radiotracers, and differences were not statistically significant. Imaging with 18F- and 68Ga-labeled JR11 antagonists did not confer any additional advantages compared to agonists in this tumour model. However, the high tumour uptake of both 68Ga- and 18F-labeled antagonists is striking, despite their significantly poorer binding affinity (inhibition contrast (Ki) > 18 nM) compared to agonist (Ki < 1.5 nM). With modifications to improve affinity, antagonist somatostatin analogues may be very promising for breast cancer visualization. iv Preface A version of Chapter 3 has been accepted for publication [I Dude, Z Zhang, J Rousseau, N Hundal-Jabal, N Colpo, H Merkens, KS Lin, F Bénard. Evaluation of Agonist and Antagonist Radioligands for Somatostatin Receptor Imaging of Breast Cancer using Positron Emission Tomography. European Journal of Nuclear Medicine and Molecular Imaging (EJNMMI) Radiopharmacy and Chemistry]. Z Zhang and I were the lead members on this project. Z Zhang performed the chemical and radiochemical synthesis of cold standards and radiotracers. I conducted the biological evaluation experiments (binding assays, qPCR, biodistribution). H Merkens assisted with animal procedures and monitoring. N Colpo, N Hundal-Jabal and J Rousseau performed PET/CT image acquisition. KS Lin and F Bénard were the supervisory authors and were both heavily involved in experimental design and manuscript input. The manuscript was drafted and finalized by me with critical input from Z Zhang, KS Lin and F Bénard. A version of Chapter 4 is currently being prepared for submission [I Dude, Z Zhang, C Zhang, N Hundal-Jabal, N Colpo, H Merkens, D Perrin, KS Lin, F Bénard. Synthesis and Preclinical Evaluation of 18F-labeled Ammoniomethyl- Trifluoroborate Somatostatin Analogues for Breast Cancer Imaging]. In this work, Z Zhang was responsible for chemistry and radiochemistry. I was responsible for all aspects of the biological evaluation. C Zhang provided assistance with internalization assays and in vivo stability measurements (Figure 4.3 and Figure 4.4). H Merkens assisted with animal procedures and monitoring. N Hundal-Jabal and N Colpo performed PET/CT image acquisition. D Perrin, KS Lin, and F Bénard were the supervisory authors. KS Lin, and F Bénard were heavily involved in both experimental design and v manuscript input. The manuscript was drafted and finalized by me with critical input from Z Zhang, KS Lin and F Bénard. The research in this thesis was conducted under animal protocol A11-0238, “Radiolabeled peptides to improve cancer diagnosis by positron emission tomography and single-photon emission tomography” which was approved by the Institutional Animal Care Committee of the University of British Columbia 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 ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations ................................................................................................................. xiii Acknowledgements .................................................................................................................. xviii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 1.1 Nuclear Medicine ............................................................................................................ 1 1.1.1 SPECT and Planar Scintigraphy ................................................................................. 3 1.1.2 Positron Emission Tomography .................................................................................. 3 1.1.2.1 18F-FDG in Oncology ......................................................................................... 5 1.1.2.2 Impact of PET on Cancer Care ........................................................................... 5 1.2 Peptide Radiopharmaceuticals ........................................................................................ 6 1.2.1 Peptide Radiopharmaceuticals for Cancer Imaging .................................................... 7 1.2.2 Antagonist Peptide Radiopharmaceuticals ............................................................... 10 1.2.3 Peptide Receptor Radionuclide Therapy .................................................................. 10 1.2.4 Peptide Radiolabeling ............................................................................................... 11 1.2.4.1 68Ga Radiolabeling ............................................................................................ 12 1.2.4.2 18F Radiolabeling .............................................................................................. 15 vii 1.3 Somatostatin Receptors and Somatostatin Radiotracers ............................................... 17 1.3.1 Somatostatin Receptor Signal Transduction ............................................................. 18 1.3.2 Somatostatin Receptor Expression in Cancer ........................................................... 19 1.3.2.1 Neuroendocrine Tumours ................................................................................. 19 1.3.2.2 Breast Tumours ................................................................................................. 21 1.3.3 Somatostatin Radiotracers ........................................................................................ 22 1.3.3.1 Overview of Somatostatin Radiotracers ........................................................... 22 1.3.3.2 Somatostatin Agonists versus Antagonists ....................................................... 23 1.3.3.3 Somatostatin Peptide Receptor Radionuclide Therapy ..................................... 25 1.4 Breast Cancer ................................................................................................................ 27 1.4.1 Molecular Subtypes of Breast Cancer....................................................................... 28 1.4.2 Breast Cancer Imaging .............................................................................................. 29 1.4.2.1 Nuclear Imaging................................................................................................ 31 1.4.2.2 Somatostatin Receptor Imaging ........................................................................ 32 1.5 Thesis Objectives .......................................................................................................... 33 1.5.1 Rationale ................................................................................................................... 33 1.5.2 Hypothesis................................................................................................................. 33 1.5.3 Specific Aims ............................................................................................................ 34 Chapter 2: Materials and Methods ............................................................................................35 2.1 Reagents and Instrumentation ....................................................................................... 35 2.2 Peptide and Precursor Synthesis ................................................................................... 35 2.3 Competition Binding Assays ........................................................................................ 37 2.4 Radiolabeling ................................................................................................................ 39 viii 2.4.1 68Ga Radiolabeling .................................................................................................... 39 2.4.2 18F Radiolabeling ...................................................................................................... 40 2.5 Internalization Assay .................................................................................................... 40 2.6 Quantitative Polymerase Chain Reaction ..................................................................... 41 2.7 Cell Culture ................................................................................................................... 42 2.8 Animal Studies .............................................................................................................. 43 2.8.1 17β-Estradiol Pellet Implant and Tumour Inoculation ............................................. 43 2.8.2 In Vivo Plasma Stability ............................................................................................ 44 2.8.3 Biodistribution Studies.............................................................................................. 44 2.8.4 PET/CT Imaging ....................................................................................................... 45 2.9 Statistics ........................................................................................................................ 45 Chapter 3: Comparison of 68Ga Agonists and Antagonist Somatostatin Radiotracers for Breast Cancer Imaging ................................................................................................................46 3.1 Introduction ................................................................................................................... 46 3.2 Materials and Methods .................................................................................................. 48 3.3 Results ........................................................................................................................... 48 3.3.1 Transcriptional Sstr Expression ................................................................................ 48 3.3.2 Binding Affinity and Radiolabeling.......................................................................... 49 3.3.3 Tumour and Organ Uptake ....................................................................................... 50 3.4 Discussion ..................................................................................................................... 53 3.5 Conclusion .................................................................................................................... 56 Chapter 4: Synthesis and Preclinical Evaluation of 18F-labeled Ammoniomethyl Trifluoroborate Somatostatin Analogues for Breast Cancer Imaging ...................................58 ix 4.1 Introduction ................................................................................................................... 58 4.2 Materials and Methods .................................................................................................. 59 4.3 Results ........................................................................................................................... 60 4.3.1 Chemistry and Radiochemistry ................................................................................. 60 4.3.2 In Vitro Binding Affinity and Internalization Studies .............................................. 60 4.3.3 In Vivo Plasma Stability ............................................................................................ 62 4.3.4 Tumour and Organ Uptake ....................................................................................... 63 4.4 Discussion ..................................................................................................................... 67 4.5 Conclusion .................................................................................................................... 70 Chapter 5: Conclusion and Future Direction ............................................................................71 5.1 Significance and Summary of Results .......................................................................... 71 5.2 Thesis Limitations ......................................................................................................... 73 5.3 Future Direction ............................................................................................................ 75 5.3.1 Peptide Receptor Radionuclide Therapy in Breast Cancer ....................................... 75 5.3.2 Clinical Translation of 18F-AmBF3-TATE ............................................................... 76 5.4 Concluding Remarks ..................................................................................................... 77 Bibliography .................................................................................................................................78 Appendices ....................................................................................................................................92 Appendix A qPCR Parameters.................................................................................................. 92 Appendix B Relationship between Peptide Mass and Organ Uptake ...................................... 94 x List of Tables Table 1.1 Commonly used radionuclides for imaging and therapy ................................................ 2 Table 1.2 Receptors targeted with radio-peptides in human tumours ............................................ 9 Table 1.3 Commonly used 68Ga chelators for peptide radiolabeling ........................................... 14 Table 1.4 Al-F, Si-F and B-F 18F labeling methods ...................................................................... 17 Table 1.5 Somatostatin analogues and corresponding peptide sequences .................................... 25 Table 1.6 Clinicopathological surrogate markers for breast cancer ............................................. 29 Table 3.1 Biodistribution of 68Ga-NODAGA-JR11, 68Ga-DOTATOC, and 68Ga-DOTATATE in NOD scid gamma tumour-bearing mice ....................................................................................... 52 Table 4.1 Radiochemistry and in vivo plasma stability results for compounds 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE ..................................................................................... 60 Table 4.2 Biodistribution of 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE at 1 and 2 h p.i. in ZR-75-1 tumour bearing mice ...................................................................................... 66 xi List of Figures Figure 1.1 Principles of SPECT and PET imaging ......................................................................... 4 Figure 1.2 Simplified representation of somatostatin receptor intracellular signaling ................. 19 Figure 1.3 Comparison of agonist and antagonist somatostatin radiotracers ............................... 27 Figure 3.1 Chemical structures of 68Ga-DOTATOC, 68Ga-DOTATATE and 68Ga-NODAGA-JR11. ............................................................................................................................................. 47 Figure 3.2 Relative transcriptional expression of sstr subtypes normalized to housekeeping gene HPRT1. ......................................................................................................................................... 48 Figure 3.3 Representative competition binding assays of natGa peptides ..................................... 49 Figure 3.4 Representative PET maximum intensity projection images of 68Ga compounds in a ZR-75-1 tumour model ................................................................................................................. 50 Figure 3.5 ZR-75-1 tumour uptake at 1 h p.i. with 68Ga radiotracers ........................................... 51 Figure 3.6 Organ uptake and tumour-to-background ratios with 68Ga-radiotracers at 1 h p.i.. .... 51 Figure 4.1 Chemical structures of 18F-AmBF3-TOC, 18F-AmBF3-TATE and 18F-AmBF3-JR11 59 Figure 4.2 Representative competition binding assays of 18F radiotracers ................................... 61 Figure 4.3 Internalized fractions of 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE on ZR-75-1 cells ........................................................................................................................... 61 Figure 4.4 Representative radio-HPLC chromatographs of plasma samples collected from mice treated with 18F tracers at 5 min and 15 min p.i. ........................................................................... 62 Figure 4.5 Coronal PET/CT images of 6 different ZR-75-1 tumour bearing mice injected with 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE and imaged 1 and 2 h p.i............... 64 Figure 4.6 ZR-75-1 tumour uptake with 18F radiotracers at 1 h and 2 h p.i. ................................ 64 Figure 4.7 Background uptake and tumour ratios with 18F-radiotracers at 1 h p.i. and 2 h p.i..... 65 xii Figure 5.1 Methodological differences between competition binding assays done using filtration versus autoradiography methods................................................................................................... 74 xiii List of Abbreviations %ID/g: Percent injected dose per gram of tissue 111In-pentetreotide: 111In-DTPA-D-Phe1-octreotide 125I-SRIF14: 125I-Tyr11-somatostatin-14 18F-DOPA: 18F-dihydroxyphenylalanine 18F-FDG: 2-deoxy-2-[18F]fluoro- D-glucose 18F-FES: 16-α-[18F] fluoro-17-β-estradiol 18F-FLT: 18F-fluoro-L-thymidine 18F-FMISO: 18F-fluoromisonidazole 1-Na1: 1-naphthyl-alanine 2D: 2-dimensional 3D: 3-dimensional 68Ga-DOTANOC: 68Ga-DOTA-Na13-octreotide 68Ga-DOTATATE: 68Ga-DOTA-Tyr3-octreotate 68Ga-DOTATOC: 68Ga-DOTA-Phe1-Tyr3-octreotide 99mT-sestamibi: 99mTc-methoxyisobutylisonitrile AmBF3: Ammonimethyl-trifluroborate ANOVA: Analysis of variance Aph(Hor): Amino-Phe-hydroorotic acid bp: Base pair(s) Bq: Becquerel BSA: Bovine serum albumin CCK: Cholecytokinin cDNA: Coding DNA Cpa: 4-Cl-pheny-alanine CT: Computed tomography Ct: Threshold cycle CXCR4: Chemokine receptor 4 d: Day(s) D-Aph (Cbm): D-4-amino-carbamoyl-phenylalanine xiv DI: Deionized DMEM: Dulbecco’s modified Eagle’s medium DMF: Dimethylformamide DNA: Deoxyribonucleic acid DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DTPA: Diethylenetriamine pentaacetic acid E2: 17β-estradiol EC: Electron capture EDDA: Ethylenediamine-N,N’-diacetic acid ER: Estrogen receptor eV: Electron volt(s) FBS: Fetal bovine serum FDA: Food and Drug Administration g: Gram(s) GEP-NET: Gasteroenteropancreatic neuroendocrine tumour GLP: Good laboratory practice GLP-1R: Glucagon-like peptide-1 receptor GPCR: G-protein coupled receptor GRPR: Gastrin-releasing peptide receptor GTP: Guanosine tri-phosphate h: Hour(s) HEK: Human embryonic kidney HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HER2: Human epidermal growth factor 2 HPLC: High performance liquid chromatography HPRT1: Hypoxanthine phosphoribosyltransferase 1 HYNIC: N-hydroxysuccinimidyl hydrazinonicotinamide IC50: Half-maximal inhibitory concentration IEX: Isotope exchange reaction IGF-1: Insulin-like growth factor-1 xv IHC: Immunohistochemistry IT: Isomeric transition k: Kilo Ki: Inhibition constant L: Litre(s) LET: Linear energy transfer LOR: Line of response M: Mega m: Meter(s) m: Milli M: Molar MC1R: Melanocortin 1 receptor MeCN: Acetonitrile MIBG: Metaiodobenzylguanidine min: Minute(s) mol: Mole(s) MRI: Magnetic resonance imaging mRNA: Messenger RNA n: Nano NaCl: Sodium Chloride NaI: Sodium Iodide NET: Neuroendocrine tumours NMP: N-methyl-2-pyrrolidone NOD scid gamma: NOD.Cg-PrkdcsciIl2rgtm1Wjl/SzJ NODAGA: 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid NOPO:1,4,7-triazacyclononane-1,4-bis(methylene(hydroxymethyl)phosphinic acid) NOTA: 1,4,7-triazacyclononane-1,4,7-trisacetic acid NPY: Neuropeptide Y NTR1: Neurotensin-1 receptor Octreotide LAR: Octreotide long-acting release xvi p.i.: Post-injection p: pico PBS: Phosphate buffered saline PCR: Polymerase chain reaction PEM: Positron emission mammography PET: Positron emission tomography PR: Progesterone receptor PRRT: Peptide receptor radionuclide therapy PTP: Phosphotyrosine phosphatase PTPη: Density-enhanced phosphatase 1 QC: Quality control qPCR: Quantitative polymerase chain reaction RCY: Radiochemical yield RGD: Arg-Gly-Asp RNA: Ribonucleic acid RPMI: Roswell Park Memorial Institute medium s: Second(s) SHP-1 and -2: Src-homology phosphatase type 1 and type 2 SiFA: Silicon-fluoride acceptor SPECT: Single photon emission computed tomography SRIF-14 and SRIF-28: Somatotropin-release inhibiting factor-14 and -28 (or somatostatin-14 and -28) sstr1-5: Somatostatin receptor subtype 1-5 T1/2: Half-life TFA: Trifluoroacetic acid Thr(ol): Threoninol TRAP: (3,30,3”-(((1,4,7-triazonane-1,4,7-triyl)tris(methylene))tris(hydroxy-phosphoryl))tripropanoic acid Tricine: N-[tris(hydroxyl-methyl)-methyl]glycine VPAC: Vasoactive intestinal peptide receptor xvii α: Alpha particle (2 protons, 2 neutrons) β-: Beta minus particle (electron) β+: Beta plus particle (positron) γ: Gamma ray μ: Micro xviii Acknowledgements I would like to thank Dr. Franҫois Bénard for his guidance and advice. Your breadth of knowledge, creativity, and work proficiency is inspiring to a young graduate. Secondly, I would like to thank Dr. Kuo-Shyan Lin for his invaluable teaching efforts and insights into all research related matters. Your tenacious drive and extraordinary work efficiency is both admirable and inspiring. Thank you to the members of my supervisory committee, Dr. Urs Hafeli and Dr. Paul Schaffer for meaningful discussions. Thank you Dr. David Perrin, for a productive collaboration. My sincerest gratitude goes to the members of the Bénard and Lin lab, past and present. I would like to acknowledge Dr. Zhengxing Zhang (Johnson) for his indispensable contribution to this project. I am grateful to have worked with such a reliable and diligent researcher. I would also like to thank Gemma for her immense kindness, Julie for her cheerful nature, Joseph for valuable advice, and Chengcheng for perspective in both research and life. Thank you Guillaume, Ting, Hwan, Maral, Carlos, Milena, Marin, Martha, Jinhe, Etienne, and Frederic for both your guidance and your friendship. Thank you Silvia, Helen and Jutta for fostering my hands-on lab skills. Thank you Nav and Nadine for your help and expertise in animal imaging. Thank you Gayle, Tina and the ARC staff for animal assistance. Thank you Amy, Ester and Adrian from the Huntsman and Aparicio labs for your help with DNA/RNA work. Lastly, I want to acknowledge my parents for their unyielding efforts to provide the best for their children. I am in awe of the courage and perseverance they displayed through the early years of our immigration to Canada. I also want to acknowledge my partner and best friend, D’arcy, without whom, this work would not have been possible. Thank you for your support and for sharing every one of my hardships and successes with me throughout this journey. xix Dedication Pentru cei mai buni parinți Mihaela și Mihai Dude 1 Chapter 1: Introduction 1.1 Nuclear Medicine Nuclear medicine is a medical practice that uses radionuclides for both imaging and therapy of various diseases. Specific molecular mechanisms or physiological processes can be targeted by probing key proteins (i.e., receptors, enzymes, and transporters) with radioactive compounds, known as radiopharmaceuticals. A radiopharmaceutical is comprised of a biological component (i.e., small-molecule, peptide, or antibody) specific for a certain protein or disease state, and a radioactive isotope which can have either photon emissions for diagnostic purposes, particle emissions for therapeutic purposes, or a combination of both. For imaging, patients undergoing a nuclear medicine scan will be administered (usually intravenously) a radiopharmaceutical and scanned by either positron emission tomography (PET) or single photon emission tomography (SPECT) based on the decay properties of the radionuclide in use1. Based on its biological characteristics, the radiopharmaceutical homes to various organs or lesions, and its spatial localization is determined by detection of the radioactive emissions. Nuclear medicine scans provide functional information on physiological processes (i.e., metabolism, proliferation, hypoxia, angiogenesis, apoptosis), protein expression profiles or presence of disease biomarkers2. PET and SPECT instruments are commonly paired with anatomical imaging modalities such as computed tomography (CT)3 or magnetic resonance imaging (MRI)4, allowing for co-registration of functional and anatomical information. Table 1.1 outlines various isotopes used in nuclear medicine. 2 Table 1.1 Commonly used radionuclides for imaging and therapy. Table adapted with permission from Fani and Maecke5 with some modifications6-10. Isotopes for single photon emission computed tomography (SPECT) Radionuclide T1/2 Decay Mode (%) Eγ (keV) (%) Principal Production Mode 99mTc 6.02 h IT (100), γ 141 (91) 99Mo/99mTc generator 111In 2.83 d EC (100) Auger, γ 171 (90) 245 (94) Cyclotron, 111Cd(p,n)111In 67Ga 3.26 d EC (100) Auger, γ 93 (39) 185 (21) 300 (17) Cyclotron, 68Zn(p,2n)67Ga 123I 13.2 h EC (100), γ 15 (84) 27 (71) 31 (16) Cyclotron, 124Te(p,2n)123I Isotopes for positron emission tomography (PET) Radionuclide T1/2 Decay Mode (%) Eβ+ (keV) (%) Principal Production Mode 11C 20.4 min β+ (99.8) 385 (99.8) Cyclotron, 14N(p,α)11C 18F 110 min β+ (97) EC (3) 634 (97) Cyclotron, 18O(p,n)18F 68Ga 68 min β+ (89) EC (11) 1899 (88) 68Ge/68Ga generator 89Zr 3.27 d β+ (23) EC (77) 897 (23) Cyclotron, 89Y(p,n)89Zr 124I 4.15 d EC (77) β+ (23) 3160 (24) 2556 (25) 2137 (11) 1535 (12) 866 (11) Cyclotron, 124Te(p,n)124I Isotopes for radionuclide therapy Radionuclide T1/2 Decay Mode (%) Eβ- (keV) (%) Principal Production Mode 90Y 2.67 d β- (100) 2280 (99.9) 90Sr/90Y Generator 177Lu 6.71 d β-, γ 498 (79) 385 (9) 177 (12) Reactor, 176Lu(n,γ)177Lu 188Re 17 h β-, γ 2120 (71.1) 1965 (25.6) 1487 (1.6) 188W/188Re generator 211At 7.2 h α (42) EC (58) Eα = 5870 (42) Cyclotron, 209Bi(α,2n)211At β+ decay is always accompanied with 511 keV annihilation gamma emission. IT = isomeric transition. EC = electron capture. 3 1.1.1 SPECT and Planar Scintigraphy SPECT and planar scintigraphy modalities rely on the detection of gamma photons emitted from the radioactive nucleus1. Planar scintigraphy scans are 2D projections acquired by detecting the incoming radioactive signal on a large-scale detector, called an Anger or gamma camera, placed near the patient. SPECT uses the same principle, but acquires several 2D projections at different angles around the patient, which are reconstructed to create a tomographic representation1. Both modalities require collimation of the incoming gamma rays, done by mounting a collimator (thick sheet of lead containing thousands of small pin holes) parallel to the Anger camera, which allows only photons travelling perpendicular to the detector to be registered1. Collimation ensures a representative spatial distribution of the radiotracer, but significantly reduces detection sensitivity leading to low counts and high noise levels1. Among its many applications, SPECT is clinically used in the detection of bone metastases (99mTc-phosphate compounds), coronary artery disease (99mTc-sestamibi), brain abnormalities (99mTc-ethyl cysteinate dimer/99mTc-exametazime) and cancer11. Oncological applications include diagnosis of neuroendocrine/neurological tumours using 123I-metaiodobenzylguanidine (123I-MIBG), an analogue of norepinephrine. 111In-radiolabeled antibodies detecting tumour specific antigens such as 111In-capromab-pentetide in prostate cancer and 111In-ibritumomab tiuxetan in non-Hodgkin lymphoma are also used11. 1.1.2 Positron Emission Tomography PET scanning can detect signals from positron emitting isotopes. A positron, or β+ particle, is the anti-matter counterpart of the electron and is emitted from certain unstable nuclei. When emitted, it travels a short distance in the tissue before annihilating with a nearby electron. The annihilation event produces two 511 keV photons, which are ejected 180° apart and will be 4 simultaneously detected by a pair of sensors mounted around the patient1 (See Figure 1.1). When two photons are detected in coincidence (within a 5-15 ns window), the annihilation can be localized along the imaginary line connecting the affected sensors, called the “line of response” (LOR). Coincidence events from many LORs are reconstructed to create a 3D image1. This system obviates the need for collimation, allowing PET to have improved sensitivity compared to SPECT. However, PET image resolution is limited by the distance between the emission and annihilation sites, which depends on the kinetic energy of the positron1. Overall clinical PET scanners have a higher sensitivity and resolution compared to SPECT1. Examples of PET radiotracers include 11C-choline for recurrent prostate cancer, 18F-dihydroxyphenylalanine (18F-DOPA) for brain imaging, 68Ga-DOTA-Phe1-Tyr3-octreotide for neuroendocrine tumours, 18F-NaF for bone lesions, and 18F-fluoromisonidazole (18F-FMISO) for visualization of hypoxia12. However, the most frequently used PET radiotracer is 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG), an analogue of glucose with the C-2 hydroxyl replaced by 18F13. Clinical applications for 18F-FDG are primarily in oncology, with moderate use in cardiology and neurology14,15. Figure 1.1 Principles of SPECT and PET imaging. SPECT detects only photons emitted parallel to the Anger camera. PET detects 511 keV photons emitted in coincidence.1 5 1.1.2.1 18F-FDG in Oncology Tumour uptake of 18F-FDG is driven by the Warburg effect, a phenomenon resulting in overexpression of glucose transporters and propensity of aerobic glycolysis in malignant versus non-malignant tissues13,16. Like glucose, 18F-FDG is actively transported into the cytoplasm and engages in the first step of glycolysis: conversion to 18F-FDG-6-phosphate by hexokinase. The next step cannot be performed, as it requires the C-2 oxygen (replaced by 18F), and thus 18F-FDG-6-phosphate continues to accumulate in the cell providing an enhanced PET signal13. 18F-FDG is used extensively in clinical care, for initial staging and evaluation of recurrent disease in lung cancers, melanoma, colorectal carcinoma, lymphomas, head and neck cancers, esophageal cancer, breast cancer and gynecological cancers (cervical and ovarian)17. 18F-FDG plays a major role in predicting early response to therapy and informing clinical management. However, 18F-FDG suffers from poor detection of small lesions (< 7 mm) or negative results in tumours with low metabolic rates (differentiated neuroendocrine tumours, prostate cancer, and hepatocellular carcinoma)17. 18F-FDG is not a tumour-specific tracer and will also accumulate in other metabolically active regions, such as benign inflammatory processes17. The development of tumour-specific radiotracers is currently an active and prolific area of research. 1.1.2.2 Impact of PET on Cancer Care Currently, the clinical use of PET in cancer care is dominated by 18F-FGD imaging. The National Oncologic PET Registry collected data from 22,975 studies (1,178 centers) across the United States, and found that 18F-FDG PET imaging changed the clinical course of 36.5% of cancer cases18. In patients with planned biopsy before imaging, 18F-FDG PET obviated the need for any further invasive procedures in 70% of cases18. 6 As of 2015, Canada has 45 publicly funded PET scanners (and 7 privately funded), all mainly used for oncology applications19. The British Columbia Cancer Agency in Vancouver, British Columbia is equipped with two scanners and has the capacity to perform 7,775 scans/year. 18F-FDG PET imaging will continue to be an important cancer management tool, especially in Canada’s aging population. In most developed countries, PET imaging is a standard of care, with approximately 2,000 PET scanners installed in the United States (as of 2009)20 and 724 in Europe (as of 2014)21. Aside from 18F-FDG, several radiopharmaceuticals specific for unique cancer proteins have been developed and evaluated2. Similar to current drug development approaches, radiotracer development is inspired by the “magic bullet” approach – synthesizing tracers that target and visualize key proteins and biomarkers, providing information on the patient’s unique molecular phenotype or potential response to treatment22. As an example, radiotracers targeting the somatostatin receptors in neuroendocrine tumours can visualize the extent of disease, but can also predict response to therapy with somatostatin-receptor specific agents23. 1.2 Peptide Radiopharmaceuticals Regulatory peptides (2-50 amino acids in size) act with high affinity and specificity on their respective cell surface receptors to modulate physiological processes. The majority of peptide receptors are G-protein coupled receptors (GPCRs), and their aberrant overexpression has been well documented in several cancer types24,25. Peptides are excellent imaging probes. Their small size enables efficient tumour penetration, fast clearance from non-target organs, and rapid excretion25,26. They have minimal side effects, no immunogenicity, and can be tuned to bind with high affinity to target receptors25,26. Peptides can be easily modified to accommodate a radiolabeling prosthetic, or 7 chemically engineered to modulate pharmacokinetics25,26. Challenges include a short biological half-life due to degradation by proteolytic enzymes, high specific activity requirements to avoid receptor saturation, high radiation dose to excretory organs (commonly kidneys and bladder), and a sensitive structure-activity relationship easily perturbed by the addition of a radiolabeling moiety25,26. 1.2.1 Peptide Radiopharmaceuticals for Cancer Imaging Table 1.2 outlines specific examples of receptors targeted with radio-peptides; somatostatin receptors (sstrs) being the most common. The SPECT analogue 111In-pentetreotide (OctreoScan, Mallinckrodt) was approved by the U.S. Food and Drug Administration (FDA) for imaging somatostatin-positive neuroendocrine tumours (NETs) in 199427. This strategy will likely be replaced by the higher-resolution PET procedure using 68Ga-DOTATATE (NetSpot, Advanced Accelerator Applications USA, Inc), which recently gained FDA approval in 201628. Several other experimental somatostatin analogues are in preclinical phases26. Gastrin-releasing peptide receptors (GRPR) are overexpressed on prostate and breast tumours. The GRPR-specific compounds 68Ga-RM229, 68Ga-SBS30, 18F-BAY-86436731 and 64Cu-CB-TE2A-AR0632 have been explored in clinical studies with promising results, albeit in small sample sizes. A notable challenge of imaging prostate lesions and pelvic lymph nodes with these radio-peptides is the high background uptake due to bladder and urine excretion33. Neoangiogenesis, a hallmark of tumour growth and metastasis34, can be visualized by targeting the ανβ3 integrin receptor overexpressed on active endothelial cells. The tri-peptide core sequence Arg-Gly-Asp (RGD), specific for ανβ3, has been used in the development of angiogenesis-specific PET tracers: 18F-Galacto-RGD, 18F-Fluciclatide, 18F-RGD-K5, 18F-FPPRGD2, 18F-Alfatide, 68Ga-NOTA-RGD and 68Ga-NOTA-PRGD2 which have been evaluated 8 in clinical studies35. Heteromultivalent RGD peptide tracers, in which RGD is conjugated to either a somatostatin or bombesin analogue, have also been explored. These compounds increase tumour avidity by simultaneously targeting co-expressed receptors36. Other receptors visualized clinically with 111In-, 99mTc-, 123I-, and 68Ga- labeled analogues include cholecystokinin (CCK) receptor expressed on medullary thyroid carcinomas37,38, glucagon-like peptide-1 receptor (GLP-1R) overexpressed on insulinomas39-41, vasoactive intestinal peptide receptor overexpressed in many epithelial neoplasms42, and neurotensin receptor (NTR1) in ductal pancreatic adenocarcinoma43. Peptide and small-molecule radiotracers (labeled with 18F, 68Ga and 64Cu) specific to chemokine receptor 4 (CXCR) have also been developed44, and a select few were evaluated in multiple myeloma45 or glioma46 patients. Development of PET and SPECT analogues targeting neuropeptide Y receptor (NPY), and melanocortin 1 receptor (MC1R) are currently in preclinical stages47,48.9 Table 1.2 Receptors targeted with radio-peptides in human tumours. Table adapted with permission from Fani et al.5 Target Receptor Peptide Overexpression on Tumour Type Status Somatostatin receptors (sstr1-5) Somatostatin Neuroendocrine and gastroenteropancreatic tumours, non-Hodgkin’s lymphoma, paragangliomas, breast, small cell lung cancer Clinical Use Gastrin-releasing peptide receptor (GRPR) Bombesin/GRP Prostate, breast, pancreas, gastric, small cell lung cancer, colorectal cancer Clinical Trials Cholecystokinin B/gastrin receptor (CCK2/CCK-B) CCK/gastrin Medullary thyroid cancer, small cell lung cancer, gastrointestinal stromal cancer, stromal ovarian cancer, astrocytomas Clinical Trials Glucagon-like peptide-1 receptor (GLP-1) Exendin Insulinomas, gastrinomas, phaeochromocytomas, paragangliomas and medullary thyroid carcinomas Clinical Trials ανβ3-integrin RGD Neoangiogenic vessels in brain, lung, ovary, breast, skin Clinical Trials Vasoactive intestinal peptide receptors (VPAC1 VPAC2) Vasoactive Intestinal Peptide Neuroendocrine tumours, brain tumours, adenocarcinoams of the pancreas, prostate, breast, colon, stomach and liver Studies in Patients Melanocortin 1 receptor (MC1R) α-MSH Melanomas Preclinical Neurotensin receptor (NTR1) Neurotensin Small cell lung cancer, colon, exocrine pancreatic cancer, Ewing’s sarcoma, breast, prostate Studies in Patients Chemokine receptor 4 (CXCR4) CXCR4 Lymphatic system, lung, breast and prostate cancer Clinical Trials 10 1.2.2 Antagonist Peptide Radiopharmaceuticals Upon binding to target receptors, agonist peptides stimulate downstream effects, and in some cases, trigger internalization of the peptide-receptor complex. Antagonist peptides, which also bind with high affinity and specificity, do not initiate signal transduction or receptor internalization. Until recently, agonist radiopeptides were preferred to antagonists, as internalization of the peptide-receptor complex was believed essential for successful imaging. However, recent studies using antagonist radiotracers to target sstr and GRPR (which both require receptor internalization for signal transduction) demonstrate otherwise49,50. In a preclinical study, Ginj et al. showed that 111In-labeled somatostatin antagonists outperformed agonists in targeting sstr-expressing tumours, having higher tumour uptake and contrast despite similar binding affinities49. In vitro binding assays revealed that antagonists bound to more sites on the tumour tissue resulting in an overall higher tumour uptake49. This phenomenon was also observed in in vitro studies with 5-HT2A51 and corticotropin releasing factor receptors52. Similarly, the GRPR antagonist 99mTc-Demobesin 1 was a superior imaging agent compared to the agonist 99mTc-Demobesin 4, having higher tumour uptake and contrast in mice bearing tumours from the human prostate cell line PC350. It is believed that agonist binding is modulated by the degree of receptor/G-protein coupling, as opposed to antagonists which are independent of this effect and hence can bind receptors in multiple conformations52. These studies suggest that antagonist radio-peptides may hold more value than originally believed. 1.2.3 Peptide Receptor Radionuclide Therapy Peptide receptor radionuclide therapy (PRRT) is a treatment approach that uses cancer-targeting peptides labeled with therapeutic isotopes to deliver a lethal dose of radiation to the tumour specifically26. Only isotopes with high linear energy transfer (LET) emissions can be 11 used for PRRT, to effectively damage DNA and induce tumour cell death. LET is defined as the amount of energy deposited per unit length on the radiation path24,53. High LET emissions include beta (β-), alpha (α) and Auger electron emissions (see Table 1.1)24. Currently, the most frequently used therapeutic isotopes are 177Lu and 90Y, which are both β- emitters. 177Lu β- particles (Eβ- (max) = 177, 385 and 498 keV) have a short-range of ~ 2 mm in tissue, which is appropriate for small tumours or metastases26. 90Y has a long-range emission (Eβ- (max) = 2280 keV) of ~12 mm in tissue, which is desirable for larger tumours with heterogeneous receptor distribution or hypoxic regions26. The primary toxicological concerns of PRRT are of the bone marrow (mild and reversible), and the kidneys (the main dose-limiting organ)54. Partial re-absorption of the tracer in the tubular cells of the kidneys causes high renal exposure and radiotoxicity. Kidney protection strategies, such as amino acid infusions (L-lysine and L-arginine), are employed and very effective, however these agents cause severe nausea, vomiting and discomfort54. 1.2.4 Peptide Radiolabeling Peptides have relatively fast pharmacokinetics and require radiolabeling with short half-life isotopes to minimize patient exposure (when used for diagnostic purposes). In this respect, 68Ga (t1/2 = 68 min) and 18F (t1/2 = 110 min) are ideal isotopes, however, several other radiometals have been previously used47,55. Other considerations include type of emission (γ or β+), positron range, γ energy, isotope cost, isotope availability and radiolabeling methods5. The radiolabeling moiety must not interfere with receptor binding, and must be able to conjugate the radioisotope with high thermodynamic and kinetic stability5. The radio-peptides investigated in this thesis are labeled with the isotopes 18F and 68Ga, therefore only 18F- and 68Ga- radiochemistry will be further discussed. 12 1.2.4.1 68Ga Radiolabeling 68Ga is a positron-emitting radionuclide, generally produced using a 68Ge/68Ga generator. The high positron emission abundance (90%) and minimal gamma emissions of 68Ga enables easier data collection, lower activities needed and lower patient exposure55. 68Ga has a high positron energy (1,880 keV) translating to ~8.9 mm max range in water56, which compromises resolution and detection of small lesions. The 68Ge/68Ga generator has a shelf life of approximately 1 year (68Ge T1/2 = 271 d), and can be easily mobilized to hospitals or research settings55. 68Ga is easily eluted with HCl solution, while the 68Ge remains bound on the stationary-phase of the generator column55. 68Ga benefits from robust and thermodynamically stable coordination chemistry with macrocyclic ligands, most commonly 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA)47,55 (Table 1.3). DOTA is the most frequently used, and binds 68Ga with fairly stable hexadentate co-ordination (although the ideal denticity of DOTA is octadentate)47,55. Protected forms of DOTA compatible with standard peptide synthesis methods can be purchased commercially, and enables simple peptide conjugation47. Additionally, DOTA has the advantage of stably binding other trivalent and divelent lanthanide isotopes (111In3+, 90Y3+, 177Lu3+, and 64Cu2+), allowing the same chelated biomolecule to be used for longer time-point imaging or therapy55. NOTA has a smaller cavity size compared to DOTA, and can bind 68Ga with perfect hexadentate co-ordination, forming an extremely thermodynamically and kinetically stable complex47. Conjugation of NOTA to a peptide is possible by introducing a coupling moiety at the alpha position of one carboxylate arm. 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid 13 (NODAGA) is a glutamic acid functionalized version of NOTA that maintains hexadentate co-ordination and excellent stability47. Other 68Ga chelators currently gaining popularity are TRAP (3,30,3”-(((1,4,7-triazonane-1,4,7-triyl)tris(methylene))tris(hydroxy-phosphoryl))tripropanoic acid) and NOPO (1,4,7-triazacyclononane-1,4-bis(methylene(hydroxymethyl)phosphinic acid)- 7-(methylene(2-carboxyethyl)phosphinic acid))57,58. Both exhibit very high 68Ga-radiolabelling efficiency over a broad pH range (0.5-5) and without the need for heating57. 68Ga binding to TRAP and NOPO is very specific and relatively unaffected by metal ion contaminants potentially present in the 68Ge/68Ga generator eluate57. 14 Table 1.3 Commonly used 68Ga chelators for peptide radiolabeling. Chelator 68Ga Radio-Chemistry Advantages Disadvantages Ref DOTA 37-90 ºC, 10-30 min, pH 4.0 - 5.5, RCY > 90% -Binds multiple lanthanide radiometals -Bifunctional without loss of 68Ga affinity -Large cavity size not ideal for 68Ga binding -Sensitive to trace metal contaminants 57-59 NOTA 95 ºC, 10 min, pH 4.0 - 5.5, RCY > 95 -Optimal cavity size for 68Ga enables excellent stability -One carboxyl arm required for peptide conjugation, which lowers overall hydrophilicity of tracer -Sensitive to trace metal contaminants 57-59 NODAGA 95 ºC, 25 min, pH 4.0 - 5.5 RCY = 95 -Bifunctional without loss of 68Ga affinity -Excellent 68Ga stability -Sensitive to trace metal contaminants 57-59 TRAP TRAP 25 ºC, 5 min, pH 3, RCY = 99% -Bifunctional -Excellent 68Ga affinity -Room temperature radiolabeling -Robust against generator impurities -Functional over a wide pH range (0.5 – 5) -Increases hydrophilicity of radiotracer -Trimeric (TRAP) and monomeric (NOPO) applications -Difficult to synthesize -Not extensively used in clinical applications yet 57,58,60,61 NOPO RCY: Radiochemical yield 15 1.2.4.2 18F Radiolabeling 18F is an excellent PET isotope with 97% positron emission, an ideal half-life of 110 min, and a low positron range (634 keV, ~2.3 mm max range in water56) enabling good image resolution62. 18F is cyclotron-produced by bombardment of an enriched 18O-water target with high-energy protons, resulting in high levels of radioactivity62. The slightly longer half-life of 18F compared to 68Ga allows more time for radiolabeling, quality control and distribution to imaging centers lacking a cyclotron facility63. Direct nucleophilic substitution with 18F at a carbon center is often done at high temperatures and non-aqueous conditions, which are unsuitable for peptides. Typically, 18F-fluoride requires drying to promote reactivity, as water molecules form a tight hydration sphere around the 18F anion inhibiting its nucleophilic action62-64. Drying is accomplished by repetitive and time-consuming azeotropic distillation62-64. To avoid exposing the peptide to highly basic and non-aqueous solvents, a prosthetic group is first radiolabeled, followed by conjugation to the peptide under milder conditions. This requires multiple reaction steps with adequate purification for each62-64. There have been a number of advances in 18F radiochemistry that reduced or eliminated the need for aziotropic drying. These approaches rely on 18F binding to boron, silicon or aluminum elements conjugated to the peptide cold-standards62-65. Al-18F chelation scaffolding developed by McBride et al. is a convenient labeling method reminiscent of radiometal approaches66. The Al-18F complex is stably bound by macrocyclic chelator NOTA, with minimal in vivo defluorination. Another approach is Silicon-Fluoride Acceptor chemistry (SiFA), which involves functionalization of the peptide with a Si-F moiety, followed by isotope exchange reaction (IEX) for radiolabeling67. Using IEX obviates the need for extensive purification, as the 16 precursor is chemically identical to the radiotracer; however, IEX requires large amounts of starting 18F to achieve acceptable specific activity64. A disadvantage of SiFA chemistry is the poor hydrolytic stability of the Si-F bond, requiring stabilization by bulky and hydrophobic tert-butyl groups. Hydrophobic-reducing auxiliaries (like polyethylene glycol and carbohydrate linkers) are subsequently needed to modulate the pharmacokinetics and excretion mechanism65,67. Lastly, the Perrin group has developed a radiolabeling approach using an ammoniomethyl-trifluroborate (AmBF3) moiety that can be click-reacted to the N- or C-terminus of a peptide resulting in a chemically identical non-radioactive standard. Radiolabeling is done by IEX within 25-30 minutes, without the need for azeotropic drying or high performance liquid chromatography (HPLC) purification68-70. We have employed the latter method to radiolabel several biomolecules and show that this construct is stable, efficient, and does not severely interfere with specific receptor binding70. Work using this radiolabeling method is described in Chapter 4 of this thesis. 17 Table 1.4 Al-F, Si-F and B-F 18F labeling methods Radiosynthon Chemisty Advantages Disadvantages Ref Al18F-NODA-MPAA Ligand exchange and solid-phase extraction 100 ºC, 20 min, RCY = 5-20% -Can be prepared as a lyophilized kit -One step aqueous radiolabeling -Metabolically stable -Variable yields and specific activity -Heating is not ideal for sensitive biomolecules 63,65,71 Si-18FA IEX and solid-phase extraction 25 ºC, 15 min, RCY = 80-95% -Little precursor amount needed -High RCY - Simple purification -Si-F is prone to hydrolysis and requires shielding by hydrophobic tert-butyl groups -Requires large amounts of starting 18F 63,65,68 AmBF3 IEX and solid-phase extraction 80 ºC, 25 min, RCY = 20-25% -One step aqueous radiolabeling -Metabolically stable -Fast radiolabeling and purification -Requires large amounts of starting 18F and HPLC purification to achieve high specific activity -Moderate RCY 63,65,70 RCY: Radiochemical yield 1.3 Somatostatin Receptors and Somatostatin Radiotracers Somatostatin receptors (sstr) are GPCRs expressed on cells of the brain, gut, pituitary, endocrine and exocrine pancreas, adrenals, thyroid, kidneys, and immune system72. There are five subtypes (sstr1-5) encoded by non-allelic genes on different chromosomes72. Sstrs have two known endogenous ligands, namely somatotropin-release inhibiting factor-14 and -28 (SRIF-14 and SRIF-28). SRIF-14 and SRIF-28 are comprised of 14 or 28 amino acids respectively73. Both bind with nanomolar affinity to all five receptor subtypes, and act as inhibitors of both endocrine (growth hormone, insulin, glucagon, gastrin, cholecystokinin, 18 vasoactive intestinal peptide, and secretin) and exocrine secretions (gastric acid, intestinal fluid, and pancreatic enzymes)73. SRIF analogues, notably octreotide, have been used to regulate hormone hypersecretion associated with various diseases, such as acromegaly and functional neuroendocrine tumours73. 1.3.1 Somatostatin Receptor Signal Transduction As is characteristic of GPCRs, ligand binding to sstr induces a conformational change in the receptor that stimulates guanosine tri-phosphate (GTP) to bind to the α-subunit of the G-protein. The G-protein is a trimeric complex (α, β and γ), which dissociates into two parts upon GTP binding: the GTP-bound α subunit, and the β/γ dimer. Both parts can interact with various second messenger enzymes to initiate signaling cascades74. For somatostatin receptors, the intracellular pathways that are triggered are subtype-, cell- and organ-dependent, and predominantly regulate hormone secretions, cell proliferation and neurotransmission. The inhibition of adenylyl cyclase and reduction of intracellular Ca2+ through co-ordination of Ca2+ and K+ channels are responsible for the anti-secretory effects. The activation of phosphotyrosine phosphatases (PTPs), notably Src-homology phosphatase type 1 (SHP-1), type 2 (SHP-2), and density-enhanced phosphatase 1 (PTPη) account for the apoptotic and anti-mitogenic effects. In neuronal cells, SRIFs stimulate neuronal K+ channels and inhibit Ca2+ channels leading to reduced action potential. See Figure 1.2 for a simplified schematic of intracellular SRIF signaling pathways73,75. Dimerization of somatostatin receptors is both constitutive and ligand-dependent, and can occur between receptors of the same or different species75. Once activated, sstr is phosphorylated and binds β-arrestin, which desensitizes the receptor by blocking its interaction with G-proteins. 19 Desensitized receptors are internalized into vesicles where they are dephosphorylated and recycled back to the cell membrane75. Figure 1.2 Simplified representation of somatostatin receptor intracellular signaling. Somatostatin decreases hormone sections by simultaneous inhibition of adenylyl cyclase and decreased intracellular Ca2+. Growth inhibitory effects are mediated by phosphotyrosine phosphatases73,75. 1.3.2 Somatostatin Receptor Expression in Cancer Somatostatin receptors are overexpressed on several different tumour types, most notably neuroendocrine tumours (NETs). Sstrs are also present (although at lower density) on breast tumours, brain cancers (meningiomas, astrocytomas, and medulloblastomas), renal cell carcinomas, hepatomas, gastric carcinomas, non-Hodgkin’s lymphoma, and some immune malignancies (sarcoidosis and tuberculosis)76-78. 1.3.2.1 Neuroendocrine Tumours NETs are a heterogeneous group of malignancies that arise from gland-forming neuroendocrine cells (such as those of the pituitary, parathyroid, paraganglia and adrenal 20 medulla), or from diffusely distributed neuroendocrine cells (found in gastrointestinal tract, pancreas, lung, thymus, thyroid and skin)79. NETs are rare and often benign, but can become metastatic at later stages. Some NETs, known as “functioning NETs”, produce excessive hormone secretions causing symptoms such as sweating, flushing, diarrhea and bronchospasm80. From 1994 to 2009, the incidence of NETs increased from 2.48 to 5.86 per 100,000 people in Ontario, Canada81 with similar findings observed worldwide82. The 5- and 10- year survival rates of NETs are 78% and 63%, respectively23,83. Sstr overexpression was reported by Reubi et al. on several NETs, such as pituitary adenomas, gasteroenteropancreatic NETs (GEP-NETs), medullary thyroid carcinomas, pheochromocytomas, paragangliomas, neuroblastomas and small cell lung cancers with neuroendocrine features84,85. Sstr2 was the most commonly overexpressed subtype, although heterogeneous expression of all five subtypes was observed86. Treatment with somatostatin analogues can alleviate symptoms of functioning NET and elicit anti-proliferative effects. However SRIF-14 and -28 have limited therapeutic potential due to their short plasma half-life of 2-3 min73,75. Shortening the endogenous peptides and incorporating unnatural amino acids resulted in the development of more kinetically stable analogues73,75. The most notable is octreotide, which is 8-amino acid long and has a plasma half-life of 1.5-2 hours73,75. The depot formulation, octreotide long-acting release (LAR), is often administered as an intramuscular injection once a month, and is well tolerated and very efficient at managing hormone secretions in patients with GEP-NETs and pituitary adenoma75. Octreotide LAR also prolongs progression-free survival in well-differentiated GEP-NETs due to its inhibitory growth effects80. 21 1.3.2.2 Breast Tumours Sstr overexpression has been documented in 15-100% of human breast cancers87. The high variability among reports is due to different receptor detection assays (scintigraphy, autoradiography, cross-linking experiments), the innate intra-tumour heterogeneity of sstr2 expression on breast cancer tissue88, and tumour sampling from a diverse patient population. Sstr2 is the most commonly observed subtype at both the transcriptional and translation level89, however expression of sstr1, 3 and 5 has also been reported75,90-93. Orlando et al. demonstrated that sstr2 is differently expressed on malignant breast cancer94. A 6-fold upregulation in receptor mRNA was observed on neoplastic tissue compared to corresponding normal tissue94. In 1989, Reubi et al. analyzed samples from a small cohort of 36 primary breast cancers and found that all samples with expression of estrogen and progesterone receptors (ER and PR respectively) were also sstr-positive95. This finding was later confirmed in larger studies86,88,89. Overexpression of sstr2 mRNA was found to correlate with a positive ER and PR status, a luminal A molecular profile, negative lymph node involvement, a favorable genomic grade index, and an overall good prognosis86,88,89. There is also evidence of a functional interaction between sstr2 and 17β-estradiol stimulation. In the ER-positive breast cancer cell line, T47D, sstr2 mRNA and protein expression levels were regulated by 17β-estradiol in a dose and time dependent manner96,97. This was attributed to a 1.5 kilo base-pairs response element in the 5’ flanking region of the human sstr2 gene in T47D DNA98. Treatment of recurrent breast cancer with octreotide LAR has also been explored99. In addition to the anti-proliferative effect of octreotide, sstr activation suppresses the release of insulin-like growth factor-1 (IGF-1). It was speculated that combined treatment with anti-estrogen therapy and octreotide LAR would better control tumour growth by simultaneously 22 inhibiting the mitogenic effects of both 17β-estradiol and IGF-1. However, when evaluated in a phase III clinical trial with 203 advanced breast carcinoma patients, there was no significant difference between patients treated with tamoxifen and octreotide LAR, or tamoxifen alone99. 1.3.3 Somatostatin Radiotracers Somatostatin radiotracers can image NETs by binding to the overexpressed somatostatin receptors on the tumour cell surface. Sstr imaging enables detection of tumour masses, extent of metastases, disease staging and stratification of patients into likely responders to octreotide treatment or PRRT23. The next sections will review the progression of somatostatin receptor probes in nuclear medicine. 1.3.3.1 Overview of Somatostatin Radiotracers Somatostatin receptor imaging has been in practice for the last 20 years, with a steady improvement in radiotracer synthesis and image quality100. The first evaluated sstr probe was the iodinated compound [123I]-Tyr3-octreotide. Although initially promising, [123I]-Tyr3-octreotide had poor tumour visualization capacity due to high intestinal accumulation and hepatobiliary excretion, and was expensive, difficult and time consuming to radiolabel101. Replacement of 123I with diethylenetriamine pentaacetic acid (DTPA)-chelated 111In lead to the development of 111In-DTPA-D-Phe1-octreotide (111In-pentetreotide), which gained FDA approval in 1994 and was ubiquitously used for NET imaging thereafter27,100. However, the high-energy γ emission of 111In compromised resolution and caused high patient dose100. This lead to development of 99mTc-labeled somatostatin tracers by conjugation of 99mTc to somatostatin analogues via chelator N-hydroxysuccinimidyl hydrazinonicotinamide (HYNIC) and co-ligands N-[Tris(hydroxyl-methyl)-methyl]glycine (tricine) or ethylenediamine-N,N’-diacetic acid (EDDA)100. 99mTc is an excellent isotope for SPECT imaging, with ideal photon energy of 140 keV and convenient 23 generator production1. Both 99mTc-EDDA/HYNIC-TOC and 99mTc-EDDA/HYNIC-TATE could detect more lesions with higher uptake compared to 111In-pentetreotide100. The next generation of somatostatin analogues was radiolabeled with 68Ga and benefits from imaging with the higher resolution modality PET55. Currently, the three most clinically relevant somatostatin PET tracers are 68Ga-DOTA-Phe1-Tyr3-octreotide (68Ga-DOTATOC), 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTATATE) and 68Ga-DOTA-NaI3-octreotide (68Ga-DOTANOC), which mainly differ in their in vitro sstr subtype binding affinity. 68Ga-DOTATOC has high affinity to sstr2 and moderate affinity to sstr5, 68Ga-DOTATATE has exceptional affinity to sstr2 and 68Ga-DOTANOC has high affinity to sstr2, sstr3 and sstr5100. However, no significant differences were observed between the three radiotracers in patient studies102,103. 1.3.3.2 Somatostatin Agonists versus Antagonists Original reports of somatostatin antagonists come from Bass and colleagues in 1996 who demonstrated that switching the stereochemistry of amino acids at position 1 and 2 of the octreotide peptide converted the agonist binder into an antagonist binder104. Bass et al. introduced the high-affinity antagonist BASS (or sstr-ANT), which was later radiolabeled with 111In and evaluated preclinically49. 111In-DOTA-sstr2-ANT showed high sstr2 binding affinity (IC50 = 9.4 nM) but was unable to trigger receptor internalization. When compared with the agonist 111In-DTPA-TATE (IC50 = 1.3 nM), 111In-DOTA-sstr2-ANT had twice the in vivo tumour uptake at both 4 h and 24 h post-injection (p.i.)49. In vivo uptake was even more striking for the sstr3 antagonist 111In-DOTA-sstr3-ODN-8 in sstr3-expressing xenografts (60% ID/g, 1 h p.i.)49. The improved uptake was attributed to a higher number of receptor binding sites available for the antagonist compared to the agonist, as determined by binding assays49. Further in vitro autoradiography using human samples from different sstr-expressing cancer types, confirmed 24 these initial results105. The antagonist 177Lu-DOTA-BASS, bound more sites on the tumour cell surface compared to agonist 177Lu-DOTATATE, even for tumours with low or heterogeneous sstr density105. The first clinical study of antagonist radiotracers compared 111In-DOTA-BASS with 111In-DOTA-octreotide in 5 patients with NETs. The antagonist identified 25 out of 28 lesions, and the agonist identified 17 out of 28 lesions106. Several other somatostatin antagonists were developed and tested for binding affinity107. As expected, the choice of radiometal and chelating moiety affected the binding affinity and pharmacokinetics of antagonist peptides108,109. However, the combination of radiometal 68Ga, chelator NODAGA and peptide JR11 (68Ga-NODAGA-JR11) had a particularly good binding affinity (IC50 = 1.2 nM) and striking tumour uptake (30.7%ID/g at 1 h p.i.) in sstr2-positive xenografts108. A phase I/II clinical trial (NCT02162446) in metastatic GEP-NETs patients showed that absolute tumour uptake is comparable between antagonist 68Ga-NODAGA-JR11 (68Ga-OPS202) and agonist 68Ga-DOTATOC. However, contrast is improved for the antagonist due to lower uptake in the liver, pancreas and gastro-intestinal tract110 25 Table 1.5 Somatostatin analogues and corresponding peptide sequences. Table adapted with permissions from Fani and Maecke5,111. Endogenous Peptide SIRF-14 Ala-Gly-cyclo(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys) Agonists Octreotide D-Phe-cyclo(Cys-Phe-D-Trp-Lys-Thr-Cys)-Thr(ol) TOC D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr(ol) TATE D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr NOC D-Phe-cyclo(Cys-1-Na1-D-Trp-Lys-Thr- Cys)-Thr(ol) Antagonists BASS (sstr2-ANT) p‐NO2‐Phe‐cyclo(D‐Cys‐Tyr‐D‐Trp‐Lys‐Thr‐Cys)‐D‐Tyr‐NH2 LM3 p‐Cl‐Phe‐cyclo(D‐Cys‐Tyr‐D‐Aph(Cbm)‐Lys‐Thr‐Cys)‐D‐Tyr‐NH2 JR10 p‐NO2‐Phe‐cyclo(D‐Cys‐Tyr‐D‐Aph(Cbm)‐Lys‐Thr‐ Cys)‐D-Tyr‐NH2 JR11 Cpa‐cyclo(D‐Cys‐Aph(Hor)‐D‐Aph(Cbm)‐Lys‐Thr‐Cys)‐D‐Tyr‐NH2 Amino acids in bold indicated substitutions made on the original octreotide sequence. Thr(ol): Threoninol, 1-Na1: 1-naphthyl-alanine, D-Aph (Cbm): D-4-amino-carbamoyl-phenylalanine, Cpa: 4-Cl-pheny-alanine, Aph(Hor): amino-Phe-hydroorotic acid 1.3.3.3 Somatostatin Peptide Receptor Radionuclide Therapy Initial attempts at PRRT were done with high doses of 111In-pentetreotide, speculating that the Auger electron emissions of 111In would elicit an anti-tumour response. Although patients experienced symptomatic improvements, there was no tumour regression and it was concluded that the tissue penetration range of Auger electrons was insufficiently short (max 10 μm) to be used for PRRT26,112. 26 90Y can deliver higher energy β- radiation into tumour tissues (Eβ- (max) = 2280 keV, ~ 12 mm tissue range). Several clinical studies have evaluated the radio-therapeutic 90Y-DOTATOC using different protocols and patient populations. It was estimated that approximately 10-30% of patients with NETs experienced a positive anti-tumour response upon 90Y-DOTATOC treatments, with GEP-NETs having the most favorable response112. 177Lu has a lower β- energy (Eβ- (max) = 498 keV), and a shorter β- emission range (~ 2 mm in tissue) compared to 90Y, making it useful for smaller tumours. Treatments with 177Lu-DOTA-Tyr3-octreotate (177Lu-DOTATATE) were effective for patients with high 111In-pentetreotide uptake and low tumour burden. 177Lu-DOTATATE also improved symptomatic conditions and quality of life112. In a study of 131 GEP-NET patients, treatment with 177Lu-DOTATATE resulted in complete or partial remission in 28% of patients, minor response in 19%, stable disease in 35% and tumour progression in 18%113. Recently, the NETTER1-trial, a phase III multi-centric clinical trial of 230 metastatic midgut GEP-NET patients evaluated the tolerability of 177Lu-DOTATATE and compared its efficacy to octreotide LAR114. 177Lu-DOTATATE was well tolerated and showed a significant improvement in progression-free survival and overall survival compared to octreotide LAR114. Somatostatin antagonists have also recently been explored for PRRT. The antagonist 177Lu-DOTA-JR11 has excellent sstr2 binding affinity (IC50 = 0.73)108. When evaluated in a preclinical model, 177Lu-DOTA-JR11 delivered a 4.4-fold higher radiation dose to the tumour compared to 177Lu-DOTATATE115. Pilot clinical studies in 4 patients with advanced NETs showed that 177Lu-DOTA-JR11 delivered a 1.7-10.6 fold higher dose to the tumour compared to 177Lu-DOTATATE, with only minor and reversible adverse effects116. The tolerability and 27 efficacy of 177Lu-DOTA-JR11 (177Lu-OPS201) in unresectable GEP-NETs is currently being investigated in a phase I/II clinical trial (NCT02592707). Figure 1.3 Comparison of agonist and antagonist somatostatin radiotracers. Planar scans of the same NET patient injected with 850 MBq 177Lu-DOTA-JR11 (A) and 990 MBq 177Lu-DOTATATE (B) at 24 and 72 h post-injection, showing higher tumour uptake with the antagonist. This research was originally published in JNM. Wild D, Fani M, Fischer R, Del Pozzo L, Kaul F, Krebs S, Fischer R, Rivier J, Reubi J, Maecke R. Comparison of Somatostatin Receptor Agonist and Antagonist for Peptide Receptor Radionuclide Therapy: A Pilot Study. J Nucl Med. 2014;55:1248-1252. © by the Society of Nuclear Medicine and Molecular Imaging, Inc. Figure used with permission116. 1.4 Breast Cancer Breast cancer is the 3rd most common cancer among Canadians117 and 2nd worldwide118. In Canada, breast cancers accounts for 25.8% of cancers among women, with 25,700 new cases in 2016 (1:9 women)117. From 1986 to 2016 Canada experienced a 44% decline in breast cancer mortality. In 2016, there were 4,900 breast cancer-related deaths among women, and an estimated 3.3% (1:30) lifetime probability of dying from breast cancers117. 28 Breast cancer develops from the cells of the ducts and lobules119. If the cancer mass has not broken through the basement membrane, the cancer is considered localized or in situ, and can be treated by surgical resection with excellent prognosis119. Once the cells have broken through the basement membrane, they are considered invasive and may have a more aggressive phenotype. Based on the type of disease, its molecular markers, stage and level of differentiation breast cancers can be treated with hormone therapies, chemotherapy, surgery, radiation and immunotherapy120. 1.4.1 Molecular Subtypes of Breast Cancer Breast cancer is a complex and heterogeneous disease not adequately represented by histological grading and current clinical parameters121,122. Gene expression profiling (GEP) studies have provided considerable information on the molecular characteristics of breast cancers, and demonstrated that they can be grouped into four distinct subtypes predictive of risk factors, prognosis and treatment sensitivity121,122. The four subtypes are Luminal A, Luminal B, Human Epidermal Growth Factor 2 (HER2)-enriched and Basal-like121,122. GEP is not readily available for all patients, and therefore surrogate histological biomarkers (mainly estrogen receptor (ER), progesterone receptor (PR) and HER2) are often used to stratify patients 121,122. Luminal A breast cancers are the most common (40 – 70%122-125). They have low histological grade, low mitotic activity, low recurrence rate, and are responsive to hormone therapies. Pathological biomarkers are ER-positive, PR-positive, HER2-negative and low Ki-67 index (proliferating cell nuclear antigen, a marker for proliferation)122. They are often sstr-positive, although sstr status is not typically evaluated in clinical practice. Luminal B cancers are less common (15 – 20%122-125) and have a more aggressive phenotype. By immunohistochemistry (IHC) analysis, they are ER-positive, can be HER2 positive or negative 29 and have high Ki-67 index122. HER2-enriched breast cancers (5 – 15%122-125) have a high histological grade, high proliferative index, and high propensity for metastasis. They respond well to chemotherapy and treatment with monoclonal antibody, Trastuzumab. By IHC, they show HER2 overexpression, and are ER-negative and PR-negative122. Basal-like cancers (10 – 20%122-125) have a high histological grade, high mitotic index and rapidly metastasize to brain and lungs. They are ER-negative, PR-negative and HER2-negative122. Table 1.6 Clinicopathological surrogate markers for breast cancer.122 Intrinsic Subtype Clinicopathological Surrogate Luminal A ER and PR positive HER2 negative Low Ki-67 Luminal B ER positive HER2 positive or negative High Ki-67 HER2-enriched HER2-positive or overexpressed ER and PR negative Basal-like ER and PR negative HER2 negative 1.4.2 Breast Cancer Imaging The most commonly used imaging modalities in breast cancer detection and monitoring are mammography, ultrasound and magnetic resonance imaging (MRI). Nuclear medicine procedures are used less frequently, and only in cases of recurrent or metastatic disease. Mammography detects abnormal micro-calcification by irradiating the breast with low dose X-rays. The overall sensitivity of mammography depends on the density of the breast, as highly fibroglandular tissue can obscure potential cancerous lesions. Mammography is the most 30 common screening and detection tool for breast cancer126-128. In British Columbia, asymptomatic women with average risk of breast cancer, 50-69 years old, are encouraged to undergo mammography screening biennially129,130. When a lesion is suspected, a higher dose diagnostic mammography is prescribed127. Ultrasonography is an excellent and complementary adjunct detection tool to mammography. High frequency sound is transmitted into the breast tissue, and the image is generated based on the quality of the reflected sound waves128. Ultrasound is used in patients with dense breast parenchyma. It is commonly used to differentiate between solid masses and cysts and, in combination with mammography, can also characterize lesions as either malignant or benign127. Because ultrasound can acquire images in real time, it is often used for guiding invasive procedures such as biopsy127. MRI is used as a supplemental tool in cases of inconclusive mammography or ultrasonography127. MRI images the magnetic properties of hydrogen atoms found in water molecules of soft tissues and fat131. MRI is highly sensitive, however, lacks specificity for tumours smaller than 3 mm127. It is a valuable tool for patients with silicone breast implants, or when mammography and ultrasonography are inconclusive. The latter includes cases of breast-conserving surgery, known carcinoma, metastasis, extensive postoperative scaring and very dense breast parenchyma127. As a screening tool, it is only used in high-risk patients, such as those with a first-degree relative with proven BRCA1 or BRCA2 mutations131. Nuclear medicine procedures are used less for diagnosis, but play a bigger role in monitoring recurrent and metastatic disease132, and will be further discussed in the next section. 31 1.4.2.1 Nuclear Imaging Imaging procedures for breast cancer using SPECT radiotracers are 99mTc-methoxyisobutylisonitrile (99mT-sestamibi) and 99mTc-diphosphonates (for bone metastases). 99mT-sestamibi concentrates in breast cancers due to an increase in blood flow, increased density of mitochondria and cancer cell membrane hyperpolarization133. 99mT-sestamibi is used to evaluate patients with a palpable breast abnormality when mammography is negative or inconclusive131,133. Several PET tracers have been investigated for breast cancer imaging132. The most common is 18F-FDG, which images the upregulated aerobic glucose metabolism characteristic of advanced tumours. 18F-FDG-PET was recommended as a routine procedure in patients suspected of recurrent or metastatic disease132,134. 18F-fluoro-L-thymidine (18F-FLT) is a radiotracer used to image DNA synthesis. The degree of 18F-FLT uptake reflects the proliferation rate of tumours. There is currently no established role for 18F-FLT in routine cancer care, however, it may be valuable in predicting early response to chemotherapy. In a small clinical study (n = 14) a decrease in 18F-FLT uptake correlated with a positive chemotherapy response, while no correlation was found for 18F-FDG132,135. Lastly, the radiotracer 16-α-[18F] fluoro-17-β-estradiol (18F-FES) is a ligand for ER and can be used to image ER-positive cancers132. 18F-FES uptake correlates well with estrogen receptor density (as confirmed by IHC) and may play an important role in assessing ER status of lesions that are difficult to biopsy, or in evaluating response to anti-estrogen therapy132. Imaging of localized breast lesions using PET radiotracers may benefit from the improved resolution of positron emission mammography (PEM). PEM uses a pair of detectors above and below the breast that scan along the tissue and detect 511 keV photons emitted in 32 coincidence. Compared to whole-body PET, which has a resolution of 5-7 mm at full width half-maximum, PEM has a resolution of 2.4 mm136. In a recent study, Kalinyak et al. demonstrated that PEM detected 92% of the tumours studied compared to PET/CT which detected 87% and whole-body PET which detected 56%137. 1.4.2.2 Somatostatin Receptor Imaging The expression of somatostatin receptors in breast cancer samples and cell lines has been documented since the early 1990’s87, motivating the use of somatostatin radiotracers for breast cancer visualization138. Radiotracers 111In-pentetreotide, 99mTc-depreotide and 99mTc-octreotide have been used for this purpose in several studies138. Sensitivity ranged from 69-100%, and specificity from 22-100%. 93-100% of visualized lesions were sstr-positive as confirmed by ex vivo scintigraphy, in vitro autoradiography, IHC, or mRNA studies138. Previous studies faced two major limitations in successful detection of somatostatin-positive breast cancers, namely small lesion size and low sstr2 density138. The former can be addressed by improvements in imaging resolution and sensitivity. The PET radiotracers 68Ga-DOTATATE, 68Ga-DOTATOC and 68Ga-DOTANOC are more sensitive compared to SPECT tracers100, and somatostatin antagonists may be even better49,106. Additionally, the use of PEM instrumentation could further better resolution 137. The latter issue, namely of sstr density, can be improved by patient stratification. Luminal A breast cancers, characterized by expression of ER and PR receptors, show a higher expression of sstr, and thus would be eligible for somatostatin imaging. Somatostatin receptor imaging can be used to monitor response to anti-hormone therapies, and to determined extent of disease to sentinel lymph nodes. Imaging can guide biopsy, or in some cases clarify the nature of lymph node lesions obviating the need for further 33 surgical intervention. Sstr positive cancers that develop resistance to anti-estrogen therapies, as is the case with ~40% of patients139, may be eligible for PRRT with somatostatin analogues, which have been shown to be well tolerated in NET patients114. 1.5 Thesis Objectives 1.5.1 Rationale Somatostatin antagonists may be superior imaging agents compared to agonists, as demonstrated in recent reports with antagonist 68Ga-NODAGA-JR11108,110. However, preclinical studies evaluating 68Ga-NODAGA-JR11 and other somatostatin radiotracers in vivo typically use tumour models with high sstr2 density, such as human embryonic kidney cells (HEK) transfected with sstr249,108,109. Although this model may adequately represent tumours with high sstr density, such as NETs, it may not represent the lower sstr density observed in breast cancers. The ZR-75-1 cell line is an invasive ductal carcinoma derived from the metastatic site of a 63-year-old woman140. Based on IHC biomarkers, ZR-75-1 cells classify as having a luminal A molecular subtype141, however, GEP analysis more accurately characterizes this model as Luminal B142. Previous studies from our lab showed that ZR-75-1 xenografts express sstr and could be imaged with somatostatin ligand 68Ga-DOTATATE143. 1.5.2 Hypothesis We hypothesized that agonist peptides TOC, TATE and antagonist peptide JR11 can be radiolabeled with 68Ga and 18F in high yield, purity and specific activity, and that ZR-75-1 xenografts imaged with antagonist tracers have higher tumour uptake and contrast compared with agonist tracers. 34 1.5.3 Specific Aims Aim 1: Synthesize and radiolabel the literature compounds 68Ga-DOTATOC, 68Ga-DOTATATE and 68Ga-NODAGA-JR11. Inject compounds in ZR-75-1 tumour-bearing animals and compare tumour uptake, tumour-to-normal tissue ratios, uptake in excretory organs and general biodistribution. Aim 2: Use the AmBF3 method described by Perrin and colleagues69,70 to synthesize the compounds AmBF3-TOC, AmBF3-TATE and AmBF3-JR11 and radiolabel with 18F. Determine the binding affinity, plasma stability, internalization capacity and ZR-75-1 tumour uptake of 18F-fluorinated somatostatin ligands. 35 Chapter 2: Materials and Methods 2.1 Reagents and Instrumentation All reagents and solvents were purchased from commercial sources and used without further purification except when otherwise specified. C18 Sep-Pak cartridges (1cc, 50 mg) were obtained from Waters Corporation and pre-washed with ethanol followed by deionized (DI) water prior to use. 18F-fluoride Trap & Release Columns (ORTG Inc.) were pre-washed with saturated NaCl solution followed by DI water. Peptide synthesis was performed on an Endeavor 90 peptide synthesizer (AAPPTec) using Fmoc protection strategy. Mass analyses were performed using a Bruker Esquire-liquid chromotography/mass spectrometry system with an electrospray ionization ion source, a Bruker Autoflex MALDI-TOF spectrometer or a TripleTOF 5600 mass spectrometer (AB/Sciex). High performance liquid chromatography (HPLC) purification and quality control were performed with a semi-preparative column (Phenomenex C18, 5 µ, 250 × 10 mm) or an analytical column (Phenomenex C18, 5 µ, 250 × 4.6 mm) respectively, on an Agilent 1260 infinity platform. HPLC systems were equipped with Infinity Diode Array Detector (DAD, UV to VIS) and/or a Bioscan NaI scintillation detector. 68Ga was eluted from a 50 mCi 68Ge/68Ga generator (iThemba LABS) and purified according to reported methods144. 18F-Fluoride was produced by 18O(p,n)18F reaction using a TR19 cyclotron purchased from Advanced Cyclotron Systems Inc. The activity was measured using a Capintec CRC®-25R/W dose calibrator. 2.2 Peptide and Precursor Synthesis All peptides were synthesized on solid support using standard Fmoc chemistry. The peptides described in Chapter 3, namely DOTATATE, DOTATOC, NODAGA-JR11 and the 36 non-radioactive standards of their natGa complexes were prepared according to literature procedures108,145. The peptides evaluated in Chapter 4 were AmBF3-TATE, which was prepared as previously described by Liu and Pourghiasian et al.146, AmBF3-TOC and AmBF3-JR11, which were prepared as described herein. For TOC, H-Threoninol(But)-2-ClTrt-resin was swelled successively in dichloromethane and dried under N2. Amino acids Fmoc-Cys(ACM)-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(ACM)-OH and Fmoc-D-Phe-OH (3 equivalents, eq.) pre-activated with HBTU/HOBt (3 eq.) and N,N-Diisopropylethylamine (6 eq.) in N-methyl-2-pyrrolidone (NMP) were sequentially coupled. The Fmoc group was removed by 20% piperidine in dimethylformamide (DMF). For JR11 synthesis, Fmoc-D-Tyr-Rink amide MBHA resin was swelled and deprotected with 20% piperidine in 15 mL x 2 DMF, followed by sequential amino acid coupling of Fmoc-Cys(ACM)-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Aph(Cbm)-OH, Fmoc-Aph(Hor)-OH, Fmoc-D-Cys(ACM)-OH and Fmoc-Cpa-OH. Fmoc-D-Aph(Cbm)-OH and Fmoc-Aph(Hor)-OH were synthesized according to literature procedures147. All peptides were cyclized by incubation with thallium(III) trifluoroacetate (1 eq. to the cystine on resin) for 90 min in 15 mL DMF. Azidoacetic acid pre-activated with 1 eq. diisopropylcarbodiimide was coupled to the N-terminus of both TOC and JR11 in NMP. Azido containing peptides were deprotected and simultaneously cleaved from the resin by treating with 4 mL 90/2.5/2.5/5 trifluoroacetic acid (TFA)/H2O/triisopropylsilane/phenol for 4 h at room temperature. Peptides were filtered into 10 times the volume of cold diethyl ether and collected by centrifugation. The crude peptides were washed thoroughly with 12 mL x 3 cold ether, dried and purified by HPLC. N3-TOC was eluted with 71% H2O (0.1% TFA) and 29% acetonitrile (MeCN) (0.1% TFA) at a flow rate of 4.5 37 mL/min. The retention time was 21.4 min, and yield was 27%. The calculated m/z by MALDI-MS was 1118.4 and the measured m/z was 1118.0. Similarly, N3-JR11 was eluted with 30-35% MeCN (0.1% TFA) in H2O (0.1% TFA) at a flow rate of 4.5 mL/min. The retention time was 10.1 min, and yield was 19%. The ESI-MS calculated m/z was 1385.5, and measured m/z was 1385.8. N-propargyl-N,N-dimethylammoniomethyltrifluoroborate was synthesized as described previously146 and click-reacted (3 eq. to peptide) with N3-TOC (3.5 mg, 3.1 µmol) or N3-JR11 (8.1 mg, 5.9 µmol) in CuSO4 (1.0 M, 5.0 µL), sodium ascorbate (1.0 M, 12.5 µL) and 5% NH4OH (MeCN/H2O=1:1, 100 µL). The reaction mixture was incubated at 45°C for 2 h and directly purified by HPLC using the semi-preparative column. AmBF3-TOC was eluted with 74% H2O (0.1% TFA) and 26% MeCN (0.1% TFA) at a flow rate of 4.5 mL/min. The retention time was 17.3 min and yield was 54%. ESI-MS calculated m/z was 1283.5, and measured m/z was 1283.6. AmBF3-JR11 was eluted with 25-30% MeCN (0.1% TFA) in H2O (0.1% TFA) at a flow rate of 4.5 mL/min. The retention time was 13.1 min and the yield was 62%. The ESI-MS calculated m/z was 1550.6 and measured mass was 1550.7. 2.3 Competition Binding Assays The in vitro binding affinity of our tested compounds to sstr2 was determined using a membrane-based competition binding assay. The assay measures binding affinity by evaluating the ability of non-radioactive standards to compete with a radioactive reference ligand for sstr2 binding sites on purified cell membranes overexpressing human sstr2. The assay is performed by co-incubation of purified membranes with non-radioactive standards (at varying concentrations) and the radioactive ligand (at a fixed concentration). The half-maximal inhibitory concentration (IC50) is determined by measuring the amount of bound radioactive ligand in each condition148. 38 In our assay, purified Chinese hamster ovary-K1 membranes overexpressing human sstr2 (Perkin Elmer) were incubated with radio-ligand 125I-Tyr11-Somatostatin-14 (125I-SRIF14, Perkin Elmer) and competing non-radioactive ligands in a 96-well, 1.2 μm glass fibre filter plate (EMD Millipore). Prior to assay, the plate filters were pre-soaked in 200 μL 0.1% polyethylenimine (Sigma) for 1 h at ambient temperature. Following pre-incubation, membranes (25 μg/well), 125I-SRIF14 (0.05 nM) and various concentrations of competing non-radioactive standards (10 μM to 1 pM) were diluted in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, 1 mM CaCl2, 0.5% bovine serum albumin (BSA)) and incubated for 1 h at 27°C with moderate shaking (200 μL reaction volume). Once complete, the incubation mixture was aspirated through the filters, followed by 6 washes with 200 μL ice-cold wash buffer (50 mM Tris-HCl pH 7.4, 0.2% BSA). Each filter was removed and counted on a PerkinElmer WIZARD 2480 gamma counter. The data was fit to a one-site Fit-Ki curve in GraphPad Prism v6.07, which calculated the inhibition constant, Ki. Unlike IC50, the Ki is an absolute value that does not depend on the radioactive ligand concentration or its binding affinity to receptors. Ki is calculated from IC50 using the Cheng-Prusoff equation149: i IC501+ A EC50 where [A] is the radioactive ligand concentration, and EC50 is the concentration of radioactive-ligand at half receptor saturation149. The endogenous ligand SRIF-28 (Bachem) was used in our assay as a control. To ensure that the concentration of our non-radioactive standards, and hence our determination of Ki, was accurate, our samples underwent amino acid analysis at SPARC BioCentre (Toronto Hospital for Sick Children), where peptides were hydrolyzed and comprising amino acids were separated on 39 HPLC. The peptide content was calculated by comparing the concentration of selected amino acids to known standards. 2.4 Radiolabeling 2.4.1 68Ga Radiolabeling 68Ga was eluted from the 68Ge/68Ga generator and purified as previously described144. 68GaCl3 in 0.5 mL DI water was added into an 8 mL glass vial preloaded with peptide precursor and HEPES buffer solution (0.7 mL, pH 5). The vial was then sealed with a screw cap and heated in a Danby microwave oven (model number: DMW7700WDB; power set to “2”) for 1 min. We used isocratic HPLC to separate the 68Ga-bound peptides from 68Ga-unbound peptides to ensure consistent high specific activity. The reaction mixture was cooled and directly injected into HPLC. The 68Ga-labeled peptide was collected and diluted with 50 mL 0.05 M ammonium formate solution and then passed through C18 light Sep-Pak cartridge. The product was eluted with 0.4 mL 90% ethanol in saline and formulated in saline for animal studies. HPLC solvents were 81% triethylamine-phosphate buffer and 19% MeCN for 68Ga-DOTATATE, 79% phosphate buffered saline (PBS) buffer and 21% MeCN for 68Ga-DOTATOC, and 77% PBS buffer and 23% MeCN for 68Ga-NODAGA-JR11. For purification, the flow rate was 4.5 mL/min. The retention times were 20.4, 19.2 and 14.3 min for 68Ga-DOTATATE, 68Ga-DOTATOC and 68Ga-NODAGA-JR11, respectively. For quality control (QC), the flow rate was 2 mL/min and retention times were 7.4, 6.8 and 5.3 min for 68Ga-DOTATATE, 68Ga-DOTATOC and 68Ga-NODAGA-JR11, respectively. The specific activity of 68Ga-DOTATATE, 68Ga-DOTATOC and 68Ga-NODAGA-JR11 was measured using the same analytical HPLC system. It was calculated by dividing the injected radioactivity by the tracer quantity, which was 40 interpolated from a UV standard curve. The standard curve was constructed with cold-standard compounds natGa-DOTATATE, natGa-DOTATOC and natGa-NODAGA-JR11. 2.4.2 18F Radiolabeling Radiolabeling was performed as described by Liu and Pourghiasian et al.146. Briefly, cyclotron produced 18F-fluoride was trapped on an 18F-fluoride Trap & Release Column and eluted into a reaction vial preloaded with 100 nmol precursor in aqueous solution (15 µL DMF, 15 µL 1 M pyridazine-HCl buffer pH = 2.0, and 50 nmol KHF2). The mixture was incubated at 80°C for 5 minutes, and again for another 15 min under vacuum. The reaction was quenched with 2 mL 5% NH4OH in water and purified using both Sep-Pak and HPLC. All HPLC purifications were done on a semi-preparative column with flow rate 4.5 mL/min, and used isocratic conditions comprised of MeCN (0.1% TFA) and H2O (0.1% TFA). 18F-AmBF3-TOC was purified with 29% MeCN; 18F-AmBF3-TATE with 28% MeCN, and 18F-AmBF3-JR11 with 26% MeCN. The specific activity of 18F-fluorinated compounds was measured using the analytical HPLC system. It was calculated by dividing the radioactivity of the injected tracer by its mass, which was interpolated from a UV absorbance standard curve constructed with cold-standard compounds AmBF3-TATE, AmBF3-TOC and AmBF3-JR11. 2.5 Internalization Assay Internalization assays, performed for 18F-AmBF3-TATE, 18F-AmBF3-TOC and 18F-AmBF3-JR11, were used to determine the internalization capacity of radiotracers by determining the membrane-bound or cytosol-internalized fractions. Roughly 3 – 5 x 105 cells/well (ZR-75-1 model) were seeded onto a 24-well plate (Corning) and grown overnight. On the day of the experiment, growth media was replaced with 500 μL reaction buffer (RPMI with 4.8 mg/mL HEPES, 1 mg/mL penicillin/streptomycin, and 2 mg/mL BSA) and incubated for at least 1 h at 41 37°C. Approximately 500,000 cpm (~15 kBq) of radiolabeled peptide was added to each well and incubated for 15, 30, 45, and 60 min in triplicates at 37°C. Following incubation, the reaction media was removed and cells were washed twice with 500 μL/well ice-cold PBS. Two acid washes (0.2 M acetic acid, 0.5 M NaCl pH 2.6), each 200 μL/well, were performed for 10 min on ice, and collected to isolate the membrane-bound fraction. Finally, cells were washed once more with ice-cold PBS and trypsinized to collect the internalized fraction. Both fractions were counted on a WIZARD Wallac gamma counter and results were expressed as percent internalized of total bound (% internalized/total bound). 2.6 Quantitative Polymerase Chain Reaction Quantitative polymerase chain reaction (qPCR) measures the transcriptional expression of various genes by amplifying coding DNA (cDNA) with specific DNA primers. We determined the baseline transcriptional expression of sstr1, sstr2, sstr3, sstr4 and sstr5 in ZR-75-1 cells relative to reference gene hypoxanthine phosphoribosyltransferase 1 (HPRT1) using qPCR. Total RNA from ZR-75-1 cells was purified using the GenEluteTM Mammalian total RNA miniprep kit (Sigma), treated with amplification grade DNase I (Sigma), and measured using a NanoDropTM spectrophotometer. 2.0 μg of total ZR-75-1 RNA was reverse transcribed in a 20 μL reaction using SuperScript® VILOTM cDNA synthesis kit (Invitrogen). qPCR was set up in 384-well plates, in a total volume of 10 μL; each reaction containing 1 μL template cDNA, 500 μM forward and reverse primers, 250 μM probe, and 1X SsoAdvancedTM universal probes supermix (BioRad). Each reaction was performed in triplicates and repeated 3 times. Predefined primers (forward and reverse) and probes for all six genes were purchased from IDT. The QuantstudioTM 6K Flex Real-Time PCR system (Thermo Fisher Scientific) was used for amplification and detection. The concentration of each target was determined by interpolating 42 the Ct value (cycle at which fluorescence reached a certain threshold) from respective standard curves of known concentrations. To construct the standard curves, RNA from cell lines with known expression of sstr subtypes was purified and reverse-transcribed as described above. Target sstr genes were PCR amplified using Q5® high-fidelity DNA polymerase (New England BioLabs) as per the manufacturer’s instructions, using the same primers as the qPCR reactions (without the fluorogenic probe). PCR products were separated on a 2% agarose gel, and target bands were extracted and purified using the Monarch® DNA gel extraction kit (New England BioLabs). The amount of DNA was quantified using Qubit® dsDNA HS assay kit (Thermo Fisher) and the number of copies/μL was calculated using the following formula: copies μL DNA Concentration ( g μL )amplicon length (bp) 50 g mol .022 1023 copies mol Standard curves were constructed from 10-fold serial dilutions (105 copies/μL to 1 copy/μL) and assayed by qPCR in triplicates. All standard curves were repeated 2 times (3 times for HPRT1). Sstr1 transcripts were amplified from HeLa cells, sstr2 from ZR-75-1 cells, sstr3 from Jurkat cells, sstr4 from the ChantestTM human sstr4 receptor cell line (irradiated cells, Charles river), and sstr5 and HPRT1 from HEK-sstr5 cells. Standard curves, primer and probe information, and cycling conditions for both PCR and qPCR can be found in Appendix A. 2.7 Cell Culture All imaging and biodistribution studies were performed using the human breast carcinoma, ER-positive cell model ZR-75-1140 (ATCC). In addition, HeLa cells (gifted from Dr. Sam Aparicio), Jurkat cells (ATCC) and sstr5-transfected HEK-293 cells (HEK-sstr5, gifted from Dr. Stefan Schultz) were used for qPCR standard curve construction. ZR-75-1 cells were cultured in RPMI 1640 media + GlutaMAXTM (Life Technologies) and supplemented with 10% 43 fetal bovine serum (FBS) (Seradigm). Jurkat cells were grown in the same base media, and contained 10% heat-inactivated FBS and 1 mM sodium pyruvate. HeLa cells were grown in DMEM + GlutaMAXTM (Life Technologies) with 10% FBS. HEK-sstr5 cells were grown in DMEM + GlutaMAXTM with 10% FBS, and contained 0.5 mg/mL G418 to maintain sstr5 expression. All cell cultures were exposed to 100 I.U./mL penicillin/streptomycin (Life Technologies) and grown in a humidified atmosphere at 37°C. 2.8 Animal Studies All animal studies were done in compliance with the Canadian Council on Animal Care guidelines and were approved by the Animal Care Committee of University of British Columbia. 2.8.1 17β-Estradiol Pellet Implant and Tumour Inoculation Immunodeficient female NOD scid gamma mice (NOD.Cg-PrkdcsciIl2rgtm1Wjl/SzJ or NSG) were obtained from an in-house breeding colony at BC Cancer Research Centre and Jackson Laboratory. To sustain the growth of the ER-positive ZR-75-1 cell model, animals were administered a slow-release 17β-estradiol pellet (E2) (Innovative Research of America) subcutaneously in the dorsal space of the neck. An initial pellet dose of 1.7 mg/60 days was used, however, this dose was reduced to 0.72 mg/60 days to minimize E2-related urinary retention and bladder stone formation150,151. Animals evaluated with compounds 68Ga-DOTATATE, 68Ga-DOTATOC and 68Ga-NODAGA-JR11 (Chapter 3) received the higher pellet dose of 1.7 mg/60-day. However, studies done with compounds 18F-AmBF3-TOC, 18F-AmBF3-TATE and 18F-AmBF3-JR11 (Chapter 4) received an equal mixture of 0.72 and 1.7 mg/60-day release pellets. Among these groups, animals evaluated at 1 h p.i. with the three different 18F tracers received a similar combination of 0.72 and 1.7 mg/60-day release pellets. However, animals evaluated at 2 h p.i. and 1 h p.i. with blocking received the lower 0.72 mg/60-day dose. 3-5 days post pellet-44 implant, 1 x 107 ZR-75-1 cells were re-suspended in a mixture of PBS and Matrigel (Corning) and inoculated subcutaneously on the right shoulder. Tumours were grown for 5 - 6 weeks, until they reached a size of 7 - 11 mm in diameter. 2.8.2 In Vivo Plasma Stability In vivo plasma stability studies were done for 18F-fluorinated tracers, 18F-AmBF3-TOC, 18F-AmBF3-TATE and 18F-AmBF3-JR11, to determine metabolic stability at 5 min or 15 min p.i. Naïve mice were injected intravenously with either 10 MBq (for 5 min time point) or 20 MBq (for 15 min time point) tracer and allowed to roam freely in their cage. Following euthanization, blood was collected by cardiac puncture, mixed with an equal volume of MeCN to precipitate proteins, and centrifuged for 15 min. The supernatant was collected and quantified on HPLC using an analytical column with 2 mL/min flow rate, and the following isocratic conditions in H2O (0.1% TFA): 27% MeCN (0.1% TFA) for 18F-AmBF3-TOC and 18F-AmBF3-TATE, and 26% MeCN (0.1% TFA) for 18F-AmBF3-JR11. 2.8.3 Biodistribution Studies Mice were sedated with 2% isoflurane in oxygen, at a flow-rate of 2 L/min, and administered a radiotracer dose of 1 - 3 MBq intravenously. Biodistribution studies were either done at 1 h p.i. or 2 h p.i. For evaluation of 18F radiotracers (Chapter 4), we also performed blocking studies to determine radioligand specificity for the tumour. For these studies, 50 μg (AmBF3-TOC) or 100 μg (AmBF3-TATE or AmBF3-JR11) of the non-radioactive standards were co-injected with the respective radiotracer. After 1 or 2 h uptake time, animals were sedated with 5% isoflurane (2 L/min) and euthanized by CO2 asphyxiation. Blood was promptly collected by cardiac puncture and weighed. All internal organs were harvested, rinsed in PBS, blotted dry and weighed. Organs were measured in a Wallac WIZARD2 gamma counter (Perkin 45 Elmer). Measured activity was normalized to the injected dose and the respective weight of the organ and expressed as percent injected dose per gram of tissue (%ID/g). 2.8.4 PET/CT Imaging Tumour-bearing mice were injected intravenously with 8 - 9 MBq of radio-peptide. Static PET images were acquired 55 min post-injection for 10 min using an Inveon microPET/CT scanner (Siemens) as described previously144. A baseline CT scan was used for localization and attenuation correction. Mice were promptly euthanized after imaging, and biodistribution studies were undertaken as described above. 2.9 Statistics Statistical analyses were performed using GraphPad Prism v6.07 software. Ordinary 1-way analysis of variance (ANOVA) was used in all statistical comparisons. P values ≥ 0.05, < 0.05, < 0.01 and < 0.001 were denoted as ns (non-significant), *, ** and *** respectively. 46 Chapter 3: Comparison of 68Ga Agonists and Antagonist Somatostatin Radiotracers for Breast Cancer Imaging 3.1 Introduction Several studies have reported on the superiority of somatostatin antagonist radiotracers for imaging of sstr2-positive tumours49,105,106,108-110. Antagonists are hypothesized to effectively bind to both active and inactive configurations of their target receptors, while agonists bind only when the receptor is in an active state52. Thus antagonist probes label more sites on the cell surface, resulting in overall higher uptake despite no internalization ability52. When imaging tumours with low or heterogeneous sstr density, such as breast cancers, antagonists may be the preferred choice as to maximize the number of binding sites labeled. Using in vitro autoradiography, Cescato et al. observed that binding of the antagonist 177Lu-DOTA-BASS to human breast cancer sections was 11-fold higher than the agonist 177Lu-DOTA-TATE105. The antagonist PET/CT radiotracer 68Ga-NODAGA-JR11 (or 68Ga-OPS202) showed excellent tumour uptake and biodistribution in both preclinical108 and pilot clinical110 studies. The aim of this chapter, in accordance with Aim 1, is to compare the potent antagonist, 68Ga-NODAGA-JR11, with two commonly used agonists, 68Ga-DOTATATE and 68Ga-DOTATOC, for in vivo breast cancer imaging using an sstr2-postive breast cancer xenograft model (ZR-75-1). Herein we synthesized and evaluated the binding affinity of non-radioactive standards natGa-DOTATATE, natGa-DOTATOC and natGa-NODAGA-JR11 to sstr2. We radiolabeled the above compounds with 68Ga, and compared their in vivo tumour uptake and biodistribution in ZR-75-1 47 tumour-bearing mice. Additionally, we determined the baseline transcriptional profile of sstr1-5 subtypes in ZR-75-1 cells. Figure 3.1 Chemical structures of 68Ga-DOTATOC, 68Ga-DOTATATE and 68Ga-NODAGA-JR11. 48 3.2 Materials and Methods The materials and methods used in this chapter are listed in Chapter 2. Relevant sections are those describing peptide synthesis (Section 2.2), binding affinity (Section 2.3), radiolabeling (Section 2.4.1), q-PCR (Section 2.6), cell culture (Section 2.7), 17β-estradiol pellet implant and tumour inoculation (Section 2.8.1), biodistribution (Section 2.8.3), PET/CT imaging (Section 2.8.4) and statistics (Section 2.9). 3.3 Results 3.3.1 Transcriptional Sstr Expression We calculated the target gene/HPRT1 copy number ratio for all five somatostatin subtypes and found predominant expression of sstr2. The normalized expression of sstr2 to HPRT1 was 0.055 ± 0.0083 (n = 3), and < 0.00005 for all other subtypes (n = 3 each, p < 0.001) (Figure 3.2). For all subtypes except sstr2, we calculated < 10 copies/μL of target transcript in our cDNA preparation. In comparison, we identified 12,915.5 ± 2,218.2 copies/μL (n = 4) HPRT1 transcripts, and 680.8 ± 148.0 (n = 3) copies/μL sstr2 transcripts. Figure 3.2 Relative transcriptional expression of sstr subtypes normalized to housekeeping gene HPRT1. 49 3.3.2 Binding Affinity and Radiolabeling natGa-DOTATOC and natGa-DOTATATE had inhibition constants (Ki) in the low nanomolar range (0.9 ± 0.1 nM, n = 4, and 1.4 ± 0.3 nM, n = 3 respectively), while the Ki of natGa-NODAGA-JR11 was much higher (25.9 ± 0.2 nM, n = 3, p < 0.001). The SRIF-28 control had a Ki of 3.7 ± 1.7 nM (n = 5) in our assays. Representative inhibition curves are shown in Figure 3.3. Multiple batches of 68Ga-DOTATOC, 68Ga-DOTATATE and 68Ga-NODAGA-JR11 were prepared in good radiochemical yield (61 ± 5%, 62 ± 8% and 68 ± 13% respectively, n = 3), purity (> 98%) and high specific activity (251.6 ± 33.9, 197.3 ± 85.2 and 138.8 ± 2.6 MBq/nmol respectively, n = 3). The particular tracer preparations used for animals studies corresponded to radiochemical yields of 62%, 66% and 58% respectively, and specific activities of 281.2, 218.3 and 136.9 MBq/nmol for 68Ga-DOTATOC, 68Ga-DOTATATE and 68Ga-NODAGA-JR11 respectively. Figure 3.3 Representative competition binding assays of natGa peptides. Peptides natGa-DOTATOC, natGa-DOTATATE, natGa-NODAGA-JR11 and SRIF-28 control were run with purified sstr2-overexpressing membranes and competing radioactive ligand 125I-SRIF14. 50 3.3.3 Tumour and Organ Uptake A full overview of tracer biodistribution is presented in Table 3.1. Among the three tested tracers, we found that the antagonist 68Ga-NODAGA-JR11 has the lowest tumour uptake (12.2 ± 0.78 %ID/g) compared to agonists 68Ga-DOTATOC (18.4 ± 2.87 %ID/g, p < 0.001) and 68Ga-DOTATATE (15.2 ± 2.20 %ID/g, ns) (see Figures 3.4 and 3.5). The antagonist 68Ga-NODAGA-JR11 also had the lowest tumour-to-blood (15.6 ± 2.20) and tumour-to-muscle (45.2 ± 11.6) ratios compared to 68Ga-DOTATOC (41.1 ± 5.68, p < 0.001 and 172 ± 55.3, p < 0.01, respectively) and 68Ga-DOTATATE (44.6 ± 11.7, p < 0.001 and 152.0 ± 60.8, p < 0.01, respectively) (see Figure 3.6). The excretion profile of all three tracers was predominantly renal, with high uptake in the kidneys and bladder. The agonist 68Ga-DOTATATE had the lowest kidney uptake (8.44 ± 1.73 %ID/g), followed by 68Ga-DOTATOC (9.27 ± 1.73 %ID/g) and lastly by 68Ga-NODAGA-JR11 (14.1 ± 1.65 %ID/g, p < 0.001). Figure 3.4 Representative PET maximum intensity projection images of 68Ga compounds in a ZR-75-1 tumour model. A. 68Ga-NODAGA-JR11, B. 68Ga-DOTATOC and C. 68Ga-DOTA-TATE. Note that image C is of a mouse treated with 68Ga-DOTATATE, but not included in the biodistribution analysis as it received a lower pellet dose of 0.72 mg/60 d compared to the others. 51 Figure 3.5 ZR-75-1 tumour uptake at 1 h p.i. with 68Ga radiotracers. *** p < 0.001. Figure 3.6 Organ uptake and tumour-to-background ratios with 68Ga-radiotracers at 1 h p.i. 68Ga-NODAGA-JR11 had statistically higher uptake in the blood, muscle and kidneys compared to the other two radiotracers, resulting in lower tumour-to-blood, tumour-to-muscle and tumour-to-kidney ratios. ** p < 0.01, *** p < 0.001. 52 Table 3.1 Biodistribution of 68Ga-NODAGA-JR11, 68Ga-DOTATOC, and 68Ga-DOTATATE in NOD scid gamma tumour-bearing mice. Organ Uptake (%ID/g) 68Ga-NODAGA-JR11 (n= 5) 68Ga-DOTATOC (n= 6) 68Ga-DOTATATE (n = 6) Mean SD Mean SD Mean SD Blood 0.80 0.10 0.45 0.09 *** 0.35 0.06 *** Fat 0.23 0.11 0.18 0.09 0.28 0.14 Ovaries 0.68 0.22 0.51 0.07 0.89 0.35 Uterus 0.86 0.22 0.54 0.11 0.84 0.10 Intestine 0.90 0.20 2.39 0.30 7.35 0.44 Stomach 1.37 0.78 2.75 1.25 8.29 3.06 Pancreas 9.29 2.03 11.01 1.32 51.56 5.47 Spleen 0.39 0.05 0.46 0.11 0.84 0.38 Kidneys 14.12 1.65 9.27 1.73 *** 8.44 1.73 *** Adrenals 2.00 0.65 9.52 3.77 15.20 7.26 Liver 0.99 0.12 0.71 0.17 1.95 0.52 Lungs 4.72 0.71 22.93 6.12 28.66 2.94 Heart 0.46 0.06 0.30 0.04 0.67 0.13 Tumour 12.21 0.78 18.44 2.87 *** 15.22 2.20 Muscle 0.28 0.07 0.11 0.02 *** 0.11 0.04 *** Bone 0.26 0.04 0.20 0.03 0.25 0.02 Brain 0.05 0.02 0.03 0.01 0.03 0.01 Tumour to Normal Tissue Ratios 68Ga-NODAGA-JR11 68Ga-DOTATOC 68Ga-DOTATATE Mean SD Mean SD Mean SD Blood 15.6 2.20 41.1 5.68 *** 44.6 11.7 *** Muscle 45.2 11.6 172 55.3 ** 152 60.8 ** Kidney 0.88 0.12 2.01 0.24 *** 1.88 0.49 *** Organ uptake is expressed as mean ± standard deviation in units of percent injected dose per gram of tissue (%ID/g). Statistical comparisons were performed between mean organ uptake of 68Ga-NODAGA-JR11 and 68Ga-DOTATOC and between mean organ uptake of 68Ga-NODAGA-JR11 and 68Ga-DOTATATE. P values < 0.05, < 0.01 and < 0.001 were expressed as *, **, and *** respectively. 53 3.4 Discussion In this study, we used a human breast cancer cell model with endogenous sstr2 expression to compare tumour uptake of the antagonist 68Ga-NODAGA-JR11 with two routinely used agonists 68Ga-DOTATOC and 68Ga-DOTATATE. Most studies evaluating sstr tracers in vivo typically used a rat pancreatic cell model, i.e. AR42J152, or HEK cells transfected with somatostatin receptors49,108,109, which may not adequately represent a breast cancer phenotype. We looked at the transcriptional expression of all five sstr subtypes in ZR-75-1 cells and found predominant expression of sstr2, with negligible expression of the other four subtypes (Figure 3.2). Previous studies have reported a strong correlation between sstr mRNA and protein expression, suggesting that transcriptional studies are sufficient for profiling this receptor family89,91,153. Breast carcinoma samples typically overexpress sstr289,91,153, however, co-expression of multiple subtypes is common72,74. We evaluated the binding affinity of natGa-DOTATOC, natGa-DOTATATE and natGa-NODAGA-JR11 to human sstr2 in a well-established, filtration-based, competition binding assay. For the agonists natGa-DOTATOC, natGa-DOTATATE and SRIF-28 control, our inhibition constants (Ki) were 0.9 ± 0.1 nM, 1.4 ± 0.3 nM and 3.7 ± 1.7 nM respectively. Our Ki values were comparable to the IC50 values obtained by Reubi et al. (Ki values were not reported), which were 2.5 ± 0.50 nM, 0.20 ± 0.04 nM and 2.7 ± 0.30 nM, respectively154. As IC50 values are dependent on the concentration of substrates used in a specific assay, they are not reproducible between laboratories, and it is recommended that Ki values are calculated. Although natGa-DOTATATE was reported to have very high affinity to sstr2 (12 fold higher than natGa-DOTATOC)154, we did not observe this in 54 our experiments. This may explain, in part, why the diagnostic performance of both peptides is similar in clinical studies, with perhaps a slight advantage for 68Ga-DOTATOC103. The binding affinity of the antagonist natGa-NODAGA-JR11 was significantly lower in our studies (Ki = 25.9 ± 0.2 nM) compared to the IC50 reported by Fani et al. (IC50 = 1.2 ± 0.2 nM)108. Differences between our studies and literature reports could be partially attributed to different methodology and assay conditions. We used a filtration-based binding assay, whereas others used an autoradiography approach108,154. We attempted to minimize the impact of assay conditions by calculating the Ki instead of IC50, and ensured accurate concentration of our tested peptides by performing amino acid analysis. A direct comparison between classical protein binding assays and autoradiography methods would be valuable to improve our understanding of the structure-activity relationship of these ligands. When evaluated in vivo, all compounds targeted the tumour and sstr-positive organs to varying degrees. Tumour uptake was highest for 68Ga-DOTATOC (18.4 ± 2.9 %ID/g), followed by 68Ga-DOTATATE (15.2 ± 2.2 %ID/g, ns) and finally 68Ga-NODAGA-JR11 (12.2 ± 0.8 %ID/g, p < 0.001). In contrast, sstr2 positive organs such as pancreas, adrenals, intestines and stomach showed the highest uptake with 68Ga-DOTATATE. We wanted to determine if specific activity played a confounding role in our analysis. Unlike enzymes or protein transporters, peptide receptors saturate at lower amounts, which can have an impact on uptake in tumour or receptor-positive organs155-157. To reach the minimum activity requirements for PET/CT image acquisition (typically 5 – 10 MBq per mouse) with low specific activity peptides, a higher amount of non-55 radioactive compound will also be co-injected. The co-injected unlabeled or cold-standard peptides may compete for binding sites, saturating receptors and effectively decreasing organ uptake155-157. De Jong et al. demonstrated that 111In-DOTATOC uptake in the tumour and sstr2-positive organs was a function of the total injected peptide amount, and not specific activity directly157. A negative correlation between peptide mass and uptake has, in fact, been observed in several studies, with the maximum tumour uptake reached between 10 - 100 pmol/mouse156,157. In other sstr2-positive organs, maximum uptake is reached at even lower peptide amounts, presumably due to lower absolute receptor quantities (lower density or more regionally concentrated) in these organs156,157. Studies with 111In-DOTATOC in CA20948-bearing rats (sstr-positive rat pancreatic tumour model) evaluated the relationship between tumour uptake and injected peptide mass and found that a dose of 0.5 μg (equivalent to ~30 pmol/mouse) resulted in optimal tumour uptake at 24 h p.i.157. In our data set, we found that 68Ga-DOTATOC (highest tumour uptake) and 68Ga-NODAGA-JR11 (lowest tumour uptake) were injected with comparable peptide amounts (33.0 ± 33.5 pmol/mouse and 40.3 ± 21.5 pmol/mouse respectively) allowing for a fair comparison between the two. Interestingly we saw a slight (but statistically significant) negative correlation between uptake and peptide mass in sstr-positive tissues imaged with 68Ga-NODAGA-JR11 (tumour, pancreas, adrenals, intestine) but not those imaged with 68Ga-DOTATOC (Appendix B Figure B.1) indicating that at lower doses, the antagonist might outperform the agonist. 56 For 68Ga-DOTATATE, the range of injected peptide in this group was very narrow and much lower than the other two tracers (15.6 ± 4.4 pmol/mouse) (Appendix B Figure B.1). We observed that low capacity organs such as pancreas, adrenals, intestine and stomach, but not high capacity sites like the tumour, showed elevated uptake with 68Ga-DOTATATE compared to both 68Ga-DOTATOC and 68Ga-NODAGA-JR11, indicating this might be a peptide-mass effect. In our studies, tumour-to-blood and tumour-to-muscle ratios were lowest for 68Ga-NODAGA-JR11 (15.6 ± 2.20 and 45.2 ± 11.6 respectively) compared to 68Ga-DOTATOC (41.1 ± 5.68, p < 0.001 and 172 ± 55.3, p < 0.001 respectively) and 68Ga-DOTA-TATE (44.6 ± 11.7, p < 0.001 and 152 ± 60.8, p < 0.01 respectively). 68Ga-NODAGA-JR11 had a ~2 fold higher uptake in the blood and muscle compared to the other two agonists, accounting for the lower tumour contrast. All three tested peptides had predominant renal clearance. Exogenous E2 pellets are known to cause hydronephrosis and urine retention150,151, thus we expected higher than normal kidney uptake due to the indirect effects of E2. 3.5 Conclusion To evaluate the feasibility of using antagonist radiotracers for breast cancer diagnosis, we compared the tumour uptake and biodistribution of two well-known agonists, 68Ga-DOTATOC and 68Ga-DOTATATE, and one potent antagonist, 68Ga-NODAGA-JR11, in vivo using a human breast cancer xenograft model with endogenous sstr2 expression. We found that 68Ga-DOTATOC had the highest tumour uptake (18.4 ± 2.9 %ID/g), followed by 68Ga-DOTATATE (15.2 ± 2.2 %ID/g) and 68Ga-NODAGA-JR11 (12.21 ± 0.78 %ID/g). The high uptake of 68Ga-NODAGA-JR11 is still surprising, 57 considering its significantly poorer binding affinity (25.9 ± 0.2 nM compared to 0.9 ± 0.1 for 68Ga-DOTATOC and 1.4 ± 0.3 nM for 68Ga-DOTATATE). Peptide mass effects may have played a role in this study. Although we injected comparable amounts of 68Ga-DOTATOC and 68Ga-NODAGA-JR11, we noticed a slight (but statistically significant) negative correlation between uptake and injected peptide mass in sstr2-positive tissues (tumour, pancreas, intestine, stomach) for 68Ga-NODAGA-JR11 but not for 68Ga-DOTATOC. Indeed, tracer uptake is a complex function of several concurrent factors including binding affinity, internalization capacity, peptide mass, and receptor density. Although these studies selected 68Ga-DOTATOC as the best tracer, this may not be the case when injecting even smaller peptide doses. 58 Chapter 4: Synthesis and Preclinical Evaluation of 18F-labeled Ammoniomethyl Trifluoroborate Somatostatin Analogues for Breast Cancer Imaging 4.1 Introduction 68Ga is traditionally the isotope of choice for radiolabeling somatostatin analogues due to its convenient generator production (although limited in quantity) and reliable chelator radiochemistry55. However, 18F also offers several advantages and in some cases might be preferred to 68Ga. 18F is routinely produced in multi Curie amounts in medical cyclotrons, has excellent imaging properties such as high positron emission (97%), low positron energy (0.64 MeV), and an ideal half-life of 110 minutes62,63. Compared to 68Ga (t1/2 = 68 min), 18F affords more time for radiolabeling, quality control and distribution of radiopharmaceuticals to other imaging centers62,63. However, 18F-fluoride’s poor aqueous reactivity has previously limited its use for peptide tracers, as traditional radiolabeling methods required harsh reaction conditions and lengthy multi-step approaches62-64. Perrin and colleagues report a simple labeling approach that uses aqueous 18F in an isotope exchange reaction (IEX) to radiolabel an organotrifluoroborate moiety conjugated to the N-terminus of the peptide69,70,146. In accordance with Aim 2, we used this method to synthesize the non-radioactive standards AmBF3-TOC, AmBF3-TATE and AmBF3-JR11 and evaluated their binding affinity to sstr2. We radiolabeled these constructs with 18F (Figure 4.1) and performed imaging and biodistribution at both 1 h post-injection (p.i.) and 2 h p.i., along with accompanying in vitro internalization assays and in vivo plasma stability. Based on recent reports 59 describing the superiority of antagonists49,106,108, we hypothesized that 18F-AmBF3-JR11 will be the best tracer for tumour visualization. Furthermore, we believe these studies will highlight the potential of AmBF3-peptides for breast cancer visualization and demonstrate their utility for centres that lack a 68Ga/68Ge generator, are close enough to a cyclotron to receive daily 18F shipments, or prefer to work with 18F due to 18F-FDG familiarity. Figure 4.1 Chemical structures of 18F-AmBF3-TOC, 18F-AmBF3-TATE and 18F-AmBF3-JR11 4.2 Materials and Methods The materials and methods used in this chapter are listed in Chapter 2. Relevant sections are those describing peptide synthesis (Section 2.2), binding affinity (Section 2.3), 18F radiolabeling (Section 2.4.2), internalization assay (Section 2.5), cell culture (Section 2.7), 17β-60 estradiol pellet implant and tumour inoculation (Section 2.8.1), in vivo plasma stability (Section 2.8.2), biodistribution (Section 2.8.3), PET/CT imaging (Section 2.8.4) and statistics (Section 2.9). 4.3 Results 4.3.1 Chemistry and Radiochemistry Peptides were successfully synthesized on solid support and modified with an azide at the N-terminus. Radiolabeling of 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE was achieved in > 97% radiochemical purity, 55.5 ± 18.5 to 122.1 ± 62.9 MBq/nmol specific activity and 13 ± 5 to 28 ± 9% decay-corrected radiochemical yield (RCY). The chemical structures of the three radiotracers are shown in Figure 4.1, and individual radiochemical data is summarized in Table 4.1. Table 4.1 Radiochemistry and in vivo plasma stability results for compounds 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE. Tracer RCY (%) (n = 4) Radiochemical Purity (%) (n = 4) Specific Activity (MBq/nmol) (n = 4) Plasma Stability (% Intact) 5 min (n = 2) 15 min (n = 2) 18F-AmBF3-JR11 18 ± 7 99 ± 1 55.5 ± 18.5 > 95 > 95 18F-AmBF3-TOC 13 ± 5 97 ± 1 55.5 ± 3.7 > 95 > 95 18F-AmBF3-TATE 28 ± 9 97 ± 2 122.1 ± 62.9 > 95 > 95 4.3.2 In Vitro Binding Affinity and Internalization Studies All trifluoroborate-modified peptides retained their in vitro binding affinity to sstr2. Representative inhibition curves are illustrated in Figure 4.2. SRIF-28 was run as a control under the same assay conditions and had an inhibition constant (Ki) of 3.7 ± 1.7 nM (n = 5). AmBF3-61 TATE had the highest binding affinity, with a Ki of 0.6 ± 0.3 nM, followed by AmBF3-TOC (1.3 ± 0.2 nM, ns) and lastly AmBF3-JR11 (18.8 ± 1.0 nM, p < 0.001). Figure 4.2 Representative competition binding assays of 18F radiotracers. AmBF3-TATE, AmBF3-TOC, AmBF3-JR11 and SRIF-28 control were run with competing radioactive ligand 125I-SRIF14 and human-sstr2 purified membranes. In vitro internalization assays confirmed that radiotracers 18F-AmBF3-TOC and 18F-AmBF3-TATE maintained agonist activity, and localized to both surface and internalized cell fractions. 18F-AmBF3-JR11 was predominantly found on the cell surface, indicating antagonist activity (see Figure 4.3). Figure 4.3 Internalized fractions of 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE on ZR-75-1 cells. ** p < 0.01, *** < p < 0.001 62 4.3.3 In Vivo Plasma Stability All three radiotracers demonstrated excellent in vivo plasma stability, with > 95% of the compound stable at 15 min p.i. (see Table 4.1 for results, and Figure 4.4 for representative radio-HPLC chromatograms of mouse plasma). This analysis could not be performed at later time points, as peptide radiotracers clear rapidly from circulation and thus would not be detected at longer time points. Figure 4.4 Representative radio-HPLC chromatographs of plasma samples collected from mice treated with 18F tracers at 5 min and 15 min p.i. Columns (from left to right) represent tracers 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE. Rows (from top to bottom) represent tracer quality control (QC), 5 min time point and 15 min time point. 63 4.3.4 Tumour and Organ Uptake All three tracers successfully targeted sstr-positive organs such as tumour, pancreas, stomach, intestine, adrenal glands and lung158-160; see Figure 4.5, Figure 4.6 and Table 4.2 for biodistribution results. At 1 h p.i., tumour uptake was highest for 18F-AmBF3-JR11 (13.4 ± 2.15 %ID/g) compared to 18F-AmBF3-TOC (9.96 ± 3.69 %ID/g, ns) and 18F-AmBF3-TATE (10.2 ± 3.26 %ID/g, ns) (Figure 4.6). At 2 h p.i, tumour uptake was highest for 18F-AmBF3-TATE (15.3 ± 3.24 %ID/g), followed by 18F-AmBF3-JR11 (13.1 ± 3.47 %ID/g, ns) and 18F-AmBF3-TOC (11.9 ± 2.63 %ID/g, ns) (Figure 4.6). As expected, all three radiotracers progressively cleared from background organs, resulting in improved tumour contrast at 2 h p.i. compared to 1 h p.i. (Figure 4.7). At the later time point, both agonists 18F-AmBF3-TOC and 18F-AmBF3-TATE showed significantly better contrast compared to the antagonist 18F-AmBF3-JR11 (Figure 4.7 and Table 4.2). At 2 h p.i. tumour-to-blood and tumour-to-muscle ratios were 36.9 ± 4.5 and 100 ± 9.5 respectively for 18F-AmBF3-JR11, compared to 63.5 ± 11.3 (p < 0.05) and 230 ± 46.2 (p < 0.01) for 18F-AmBF3-TOC and 76.4 ± 20.5 (p < 0.001) and 187 ± 85.7 (p < 0.05) for 18F-AmBF3-TATE. A co-injection of excess non-radioactive standard resulted in a tumour blocking efficiency of 86 – 96% for all three radiotracers, indicating tracer specificity. In vivo de-fluorination was minimal, indicated by the low bone uptake (< 0.31 %ID/g). See Figure 4.5 for PET/CT images of mice injected with the three tested compounds. 64 Figure 4.5 Coronal PET/CT images of 6 different ZR-75-1 tumour bearing mice injected with 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE and imaged 1 and 2 h p.i. t = tumour, k = kidneys. Figure 4.6 ZR-75-1 tumour uptake with 18F radiotracers at 1 h and 2 h p.i. No statistical differences found between any groups. 65 Figure 4.7 Background uptake and tumour ratios with 18F-radiotracers at 1 h p.i. and 2 h p.i. Kidney uptake at both 1 h and 2 h p.i. is significantly lower for 18F-AmBF3-TATE compared to both other radiotracers. Uptake with 18F-AmBF3-JR11 at 2 h p.i. is slightly higher in blood and muscle compared to the other two tracers. * p < 0.05, *** p < 0.001. 66 Table 4.2 Biodistribution of 18F-AmBF3-JR11, 18F-AmBF3-TOC and 18F-AmBF3-TATE at 1 and 2 h p.i. in ZR-75-1 tumour bearing mice. Uptake is expressed as mean ± SD in units of %ID/g. 18F-AmBF3-JR11 18F-AmBF3-TOC 18F-AmBF3-TATE 1h p.i (n=9) 2h p.i. (n=6) 1h p.i. Blocked (n=4) 1h p.i. (n=12) 2h p.i. (n=6) 1h p.i. Blocked (n=3) 1h p.i. (n=11) 2h p.i. (n=6) 1h p.i. Blocked (n=3) Blood 0.93 ± 0.15 0.35 ± 0.07 0.84 ± 0.25 0.76 ± 0.16 0.20 ± 0.06 2.90 ± 1.78 0.67 ± 0.25 0.22 ± 0.09 0.54 ± 0.10 Fat 0.21 ± 0.04 0.11 ± 0.02 0.30 ± 0.12 0.21 ± 0.09 0.06 ± 0.02 0.39 ± 0.04 0.20 ± 0.09 0.13 ± 0.13 0.09 ± 0.01 Uterus 1.12 ± 0.13 0.53 ± 0.08 1.12 ± 0.14 0.77 ± 0.21 0.21 ± 0.05 1.66 ± 0.37 0.64 ± 0.19 0.28 ± 0.07 0.51 ± 0.07 Intestine 3.94 ± 0.37 4.77 ± 0.80 2.50 ± 1.21 3.25 ± 0.47 4.43 ± 0.76 3.51 ± 0.08 3.19 ± 0.80 6.00 ± 1.28 1.85 ± 0.21 Stomach 2.03 ± 0.97 1.86 ± 1.04 0.21 ± 0.07 1.08 ± 0.42 1.23 ± 0.28 0.39 ± 0.07 1.85 ± 1.25 3.63 ± 1.55 0.31 ± 0.32 Pancreas 14.2 ± 4.64 8.75 ± 1.78 0.34 ± 0.08 4.41 ± 1.65 3.31 ± 0.87 0.58 ± 0.02 10.8 ± 7.48 12.4 ± 2.72 0.16 ± 0.06 Spleen 0.59 ± 0.09 0.50 ± 0.13 0.47 ± 0.15 0.45 ± 0.10 0.22 ± 0.08 0.70 ± 0.10 0.30 ± 0.09 0.18 ± 0.02 0.14 ± 0.03 Kidneys 11.6 ± 1.28 11.3 ± 0.93 12.5 ± 3.37 14.4 ± 1.82 13.2 ± 1.36 33.8 ± 10.5 6.89 ± 3.16 2.40 ± 0.43 5.09 ± 0.91 Adrenals 2.62 ± 0.81 1.65 ± 0.86 0.57 ± 0.24 2.24 ± 0.97 2.05 ± 0.65 0.98 ± 0.09 2.47 ± 1.49 3.47 ± 1.16 0.25 ± 0.03 Liver 3.83 ± 0.50 3.40 ± 0.12 2.86 ± 1.15 0.87 ± 0.08 0.46 ± 0.09 1.59 ± 0.07 0.59 ± 0.17 0.32 ± 0.07 0.36 ± 0.01 Lungs 7.23 ± 2.04 3.75 ± 0.48 1.35 ± 1.17 4.44 ± 2.50 5.47 ± 1.53 2.98 ± 0.36 6.17 ± 4.76 6.31 ± 2.78 0.60 ± 0.12 Heart 0.43 ± 0.16 0.26 ± 0.06 0.38 ± 0.12 0.39 ± 0.10 0.13 ± 0.04 0.67 ± 0.13 0.30 ± 0.09 0.17 ± 0.04 0.19 ± 0.04 Tumour 13.4 ± 2.15 13.1 ± 3.47 1.52 ± 1.17 9.96 ± 3.69 11.9 ± 2.63 1.40 ± 0.08 10.2 ± 3.26 15.3 ± 3.24 0.36 ± 0.15 Muscle 0.23 ± 0.05 0.13 ± 0.02 0.18 ± 0.07 0.19 ± 0.05 0.05 ± 0.01 0.55 ± 0.14 0.17 ± 0.07 0.09 ± 0.04 0.11 ± 0.01 Bone 0.31 ± 0.10 0.26 ± 0.05 0.19 ± 0.17 0.31 ± 0.08 0.14 ± 0.01 0.56 ± 0.00 0.22 ± 0.13 0.15 ± 0.03 0.18 ± 0.02 Brain 0.03 ± 0.01 0.02 ± 0.00 0.26 ± 0.29 0.03 ± 0.01 0.02 ± 0.00 0.05 ± 0.01 0.04 ± 0.02 0.03 ± 0.00 0.02 ± 0.00 Tumour to Background Ratios Blood 14.8 ± 3.15 36.9 ± 4.54 1.89 ± 1.24 14.0 ± 6.22 63.5 ± 11.3 0.61 ± 0.31 16.1 ± 7.45 76.4 ± 20.5 0.65 ± 0.17 Muscle 60.2 ± 16.4 100 ± 9.53 10.9 ± 9.47 58.7 ± 27.6 230 ± 46.2 2.71 ± 0.85 61.6 ± 28.5 187 ± 85.7 3.31 ± 1.36 Kidney 1.17 ± 0.24 1.14 ± 0.25 0.13 ± 0.09 0.73 ± 0.23 0.91 ± 0.25 0.05 ± 0.02 1.57 ± 0.58 6.38 ± 0.82 0.07 ± 0.02 67 4.4 Discussion The AmBF3 method was previously used to synthesize several tracers, including 18F-AmBF3-TATE, which showed high tumour uptake at 1 h p.i. (10.11 ± 1.67%ID/g) in the AR42J rat pancreatic tumour model146. Herein, we report two additional sstr2-targeting radiotracers, 18F-AmBF3-TOC and 18F-AmBF3-JR11 and compared them to 18F-AmBF3-TATE in the human breast cancer xenograft, ZR-75-1. We hypothesized that the antagonist 18F-AmBF3-JR11 would show the highest tumour uptake and contrast among the three peptides. We assessed the in vitro binding affinity of the three non-radioactive standards to sstr2 and found that AmBF3-TATE and AmBF3-TOC maintained high binding affinity, with a Ki of 0.6 ± 0.3 nM and 1.3 ± 0.2 nM respectively; both having better affinities then the endogenous ligand SRIF-28 (Ki = 3.7 ± 1.7 nM). AmBF3-JR11 had a much lower binding affinity (Ki = 18.8 ± 1.0 nM, p < 0.001) than both AmBF3-TOC and AmBF3-TATE. This affinity is also lower than that reported by Fani et al. for DOTA-JR11 (IC50 = 0.72 ± 0.12 nM) but comparable to natGa-DOTA-JR11 (IC50 = 29 ± 2.7 nM)108. Despite poor binding affinity, Fani et al. showed that tumour uptake with 68Ga-DOTA-JR11 was still promising, and in fact, higher than with high-affinity agonist 68Ga-DOTATATE108. As hypothesized by others, it is possible that the number of receptor sites available for antagonist binding can compensate for lower binding affinity, resulting in high tumour uptake49,50,52,105. We used a cell-based internalization assay to validate the agonist/antagonist identity of our AmBF3-modified peptides. 18F-AmBF3-TOC and 18F-AmBF3-TATE maintained their agonist capacity, and internalized into the ZR-75-1 cells as early as 15 min post-treatment. Similarly, 18F-AmBF3-JR11 remained an antagonist, and was not able to internalize into the cytoplasm (Figure 4.3). 68 We evaluated our 18F-labed radiotracers in vivo at both 1 h and 2 h p.i. in ZR-75-1 tumour bearing mice. All three compounds targeted sstr-positive tissues such as tumour, pancreas, and adrenal glands with high and specific uptake. At 1 h p.i., tumour uptake was highest for 18F-AmBF3-JR11 (13.4 ± 2.15 %ID/g) compared to 18F-AmBF3-TATE (10.3 ± 3.26 %ID/g) and 18F-AmBF3-TOC (9.96 ± 3.69%ID/g), although differences were not statistically significant (see Figure 4.6). Despite low binding affinity, 18F-AmBF3-JR11 uptake was comparable to 18F-AmBF3-TOC and 18F-AmBF3-TATE, in line with the current model of antagonist binding. Tumour-to-normal-tissue ratios were comparable among all three tracers at 1 h p.i. (Figure 4.7). We evaluated the metabolic stability of all three radiotracers, and found that all had excellent in vivo plasma stability (> 95% intact after 15 min p.i.) suggesting that imaging at a longer time point is feasible and could result in better tumour contrast. At 2 h p.i., tumour uptake was 15.3 ± 3.24 %ID/g for 18F-AmBF3-TATE, followed by 13.1 ± 3.47 %ID/g for 18F-AmBF3-JR11 and 11.9 ± 2.63 %ID/g for 18F-AmBF3-TOC (not statistically significant). All tracers showed significantly higher tumour-to-background radios from 1 h to 2 h p.i. due to wash-out from non-specific binding sites (Figure 4.7). Similar to the 68Ga-derivatives discussed in Chapter 3, we were interested in analysing the effects of injected mass on the biodistribution of 18F-labelled tracers. For 1 h p.i. studies, 18F-AmBF3-TOC, 18F-AmBF3-TATE and 18F-AmBF3-JR11 were injected in comparable amounts (152.5 ± 116.6, 95.9 ± 77.5 and 111.5 ± 76.1 pmol/mouse respectively), enabling an accurate comparison between radiotracers. Similar to 68Ga-DOTATOC, we found that 18F-AmBF3-TOC tumour uptake was the most tolerant to peptide mass differences, while 18F-AmBF3-JR11 and 69 18F-AmBF3-TATE showed a saturation effect in tumour and other sstr-positive organs (Appendix B, Figure B.2). At 2 h p.i.,18F-AmBF3-JR11 no longer had the highest tumour uptake (although differences were not statistically significant). In line with this observation, the injected peptide content of 18F-AmBF3-JR11 was higher (94.3 ± 33.85 pmol/mouse) than that of 18F-AmBF3-TOC (53.6 ± 25.3 pmol/mouse, p < 0.05) and 18F-AmBF3-TATE (23.5 ± 10.3 pmol/mouse, p < 0.001), possibly accounting for the lower tumour uptake. Although all radiotracers were HPLC-purified to achieve the highest possible specific activity, variability in radiolabeling reactions resulted in differences in specific activities for 2 h p.i. experiments, and therefore differences in amounts of co-injected cold peptide. Therefore, a direct comparison of tumour uptake at this later time point is not possible. These studies still provide valuable information on the uptake in background organs, which did not appear to be mass dependant in our studies (data not shown) or other reports157,161. Overall, we found that background uptake at 2 h p.i. was generally higher with 18F-AmBF3-JR11 compared to the other two tracers (Figure 4.7). Blood uptake was 0.35 ± 0.15 %ID/g with 18F-AmBF3-JR11 compared to 0.20 ± 0.06 %ID/g for 18F-AmBF3-TOC (p < 0.01) and 0.22 ± 0.09 %ID/g for 18F-AmBF3-TATE (p < 0.05). Muscle uptake was lowest for 18F-AmBF3-TOC (0.05 ± 0.01 %ID/g, p < 0.05), and kidney uptake was lowest for 18F-AmBF3-TATE (2.40 ± 0.043 %ID/g, p < 0.001). Liver uptake was significantly higher with 18F-AmBF3-JR11 at both 1 h and 2 h p.i. (3.83 ± 0.50 %ID/g, p < 0.001 and 3.40 ± 0.12 %ID/g, p < 0.001 respectively), which, in a clinical setting, could compromise the visibility of sstr-positive metastases in the liver (common for both NETs79 and breast cancers162). 70 The excretion profile of all three tracers is predominantly renal, resulting in high kidney uptake (7-14 %ID/g at 1 h p.i.). Our animals were treated with exogenous E2 pellets to support the growth of the ER-positive tumour, which are known to cause hydronephrosis and urine retention150,151. Thus, we expect kidney and bladder uptake to be unusually high. 18F-AmBF3-TATE had the lowest kidney uptake at both 1 h p.i. (6.89 ± 3.16%ID/g, p < 0.001), and 2 h p.i. (2.40 ± 0.43 %ID/g, p < 0.001). A fast wash-out from excretory organs, as seen with 18F-AmBF3-TATE is favorable, especially for patients with renal sensitivities. 4.5 Conclusion We evaluated three 18F-radiolabeled somatostatin analgoues for breast cancer visualization and found that the two agonists 18F-AmBF3-TOC and 18F-AmBF3-TATE and one antagonist 18F-AmBF3-JR11 were very comparable in terms of tumour uptake and contrast. At 1 h p.i. 18F-AmBF3-JR11 had the highest tumour uptake (13.4 ± 2.15 %ID/g), followed by 18F-AmBF3-TATE (10.3 ± 3.26 %ID/g) and 18F-AmBF3-TOC (9.96 ± 3.69 %ID/g). All tracers were stable in vivo and tumour uptake was sustained at 2 h p.i. 18F-AmBF3-JR11 (similar to 68Ga-NODAGA-JR11) suffers from increased non-specific uptake in background organs, compromising tumour contrast. Overall, we did not find imaging with the antagonist 18F-AmBF3-JR11 to be superior then with the other two agonists. However, we did find that 18F-AmBF3-TATE had lower kidney uptake, and could provide an advantage to patients with renal sensitivities or those intolerant to amino acid treatment. 71 Chapter 5: Conclusion and Future Direction 5.1 Significance and Summary of Results Our studies were motivated by the long-standing knowledge that breast cancers overexpress sstr2, and could hence be imaged using somatostatin analogs87-93. Breast cancers with high sstr expression have a Luminal A molecular profile and are ER/PR-positive163,164. Imaging with sstr analogues can provide surrogate information on the cancer phenotype, monitor response to anti-hormone therapies, and determine the extent of metastatic disease to sentinel and auxiliary lymph nodes. We believe that somatostatin analogues radiolabeled with PET isotopes would be promising imaging agents for breast cancer visualization. Most somatostatin analogues are labeled with the radio-metal 68Ga, although 18F has many desirable properties, and in some cases might be preferred. While both 68Ga and 18F have short half-lives well-matched with peptide pharmacokinetics, 18F has a lower positron range enabling better image resolution62. Additionally, 18F is readily produced in all nuclear medicine facilities equipped for 18F-FDG production, and by virtue of its slightly longer half-life, can also be distributed from a centralized radio-pharmacy to other imaging centres nearby62. The main objectives of this thesis were to compare agonists and antagonist radiotracers labeled with 68Ga and 18F for breast cancer imaging. We hypothesized that agonists TOC and TATE and antagonist JR11 can be radiolabeled with 68Ga and 18F, and that antagonist radiotracers will have a higher tumour uptake in breast cancer xenografts (ZR-75-1 cell line) compared to agonists. Our first aim was to synthesize compounds 68Ga-DOTATOC, 68Ga-DOTATATE and 68Ga-NODAGA-JR11, and compare their tumour uptake and biodistribution in ZR-75-1 tumour 72 bearing mice. As described in Chapter 3, the three compounds were achieved in high radiochemical yield, purity and specific activity. When evaluated in vivo, 68Ga-DOTATOC had the highest tumour uptake, followed by 68Ga-DOTATATE and finally by 68Ga-NODAGA-JR11. Tumour-to-background-tissue ratios were significantly higher with agonist compared to the antagonist. Our second aim was to use the AmBF3 method described by Perrin and colleagues69 to synthesize and radiolabel compounds 18F-AmBF3-TOC, 18F-AmBF3-TATE and 18F-AmBF3-JR11 (Chapter 4). All three compounds showed excellent metabolic stability, with > 95% of the radiolabeled compounds intact after 15 min in mouse circulation. This enabled sustained tumour uptake at 2 h p.i. The antagonist 18F-AmBF3-JR11 had similar tumour uptake, and tumour-to-background ratios compared to agonists 18F-AmBF3-TOC and 18F-AmBF3-TATE at 1 h p.i. Due to differences in injected mass, we could not properly compare tumour uptake at 2 h p.i., however we did notice that uptake in background organs was higher with 18F-AmBF3-JR11 compare to the other two, potentially compromising tumour contrast. We accept our first hypothesis, namely that somatostatin analogs TOC, TATE and JR11 can be radiolabeled with 68Ga and 18F in sufficiently high yield, purity and specific activity to enable imaging. We could not accept our second hypothesis, stating that ZR-75-1 tumour uptake is higher in mice imaged with antagonist analogues compared to agonists. In Chapter 3 we observed that 68Ga-NODAGA-JR11 had the lowest tumour uptake and contrast compared to the other two agonists; in Chapter 4 we found all three 18F-peptides to be comparable. We do not deny that antagonists are superior to agonists for tumour imaging, but perhaps the difference is not as striking in this tumour model. The high tumour uptake of antagonists 68Ga-NODAGA-JR11 and 18F-AmBF3-JR11 is still surprising, considering their low binding affinity. Further 73 evaluation of binding affinity, normalization of injected peptide mass and validation of in vivo sstr2 expression levels will be important, as addressed in the limitations section below. 5.2 Thesis Limitations Based on previous literature reports, we did not expect to find such low sstr2 binding affinity for our JR11-based peptides. The Ki of natGa-NODAGA-JR11 and AmBF3-JR11 in our studies was 25.8 ± 0.2 nM and 18.8 ± 1.0 nM respectively, which is 7.0-fold and 5.1-fold higher than the Ki for SRIF-28 (3.7 ± 1.7 nM). In contrast, previous studies using an autoradiography approach reported an IC50 of 0.7 ± 0.1 nM for DOTA-JR11107 and 1.2 ± 0.2 nM for natGa-NODAGA-JR11108, which is 3.9-fold and 2.3-fold lower than the IC50 observed for SRIF-28 (2.7 ± 0.3 nM)154 using the same method. Although unlikely, it could be hypothesized that our JR11 based probes were structurally different than those reported in literature, leading to the lower binding affinity and tumour uptake. The JR11 peptide and 68Ga-NODAGA-JR11 radiotracer were prepared according to literature procedures, molecular weights were validated by mass spectroscopy, and the stereochemistry of in-house modified amino acids (Aph(Hor) and D-Aph(Cbm)) was confirmed using an optical rotation test. It is more likely that differences in binding affinity are due to the different assay methods. We used a membrane filtration assay (explained in Section 2.3), however previous studies use an autoradiography approach. A direct comparison between these two methods will be valuable in elucidating the inconsistencies observed in binding affinity (Figure 5.1). We have worked towards this goal in our lab, however, we experienced low and unspecific binding of our referenced radioactive ligand 125I-Tyr11-SRIF14 to sstr2-positive tumour sections. More work is needed towards optimizing assay conditions, potentially using the radioactive ligand 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28, employed in most autoradiography applications reported in literature107,108,154. 74 Figure 5.1 Methodological differences between competition binding assays done using filtration versus autoradiography methods. Autoradiography information (right panel) was based on methods described by Fani et al.108. When evaluating tumour and organ uptake of peptide radiopharmaceuticals, it is important to also consider the total mass of injected peptide155,156. When imaging relatively low-density target systems, such as peptide receptors, the degree of receptor-occupancy will influence tumour uptake, especially when receptors approach saturation155,156. As a general rule, tumour uptake will increase with decreasing peptide mass, up to a limit of 10-100 pmol per mouse156. We observed saturation effects with 68Ga-NODAGA-JR11, 18F-AmBF3-TOC, 18F-AmBF3-JR11 75 and 18F-AmBF3-TATE in tumour and sstr2-positive organs such as pancreas, adrenals, intestine and stomach (Appendix B, Figures B.1, B.2 and B.3). In future studies, normalizing the injected peptide content between animals will enable direct comparisons between tracers with comparable receptor-occupancy levels. Finally, we did not fully consider the functional relationship between 17β-estradiol (E2) and sstr2 expression. The ZR-75-1 breast cancer model used in our studies is ER-positive and requires exogenous E2 administration to grow in vivo. We used subcutaneously implanted slow-release E2 pellets to sustain tumour growth in vivo. Although pellets are intended to deliver a steady amount of E2, Ingburg et al. observed that mice treated with high-dose pellets, such as the ones used in our studies, showed very high variability in E2 plasma levels week-to-week151. This variability may have affected the sstr2 expression levels on ZR-75-1 cells in vivo. Previous studies have reported on the upregulation of sstr2 mRNA in response to E2 in ZR-75-1 cells96,97,165, however, no upregulation was seen at the protein level96,165. In future studies it is recommended to validate the sstr2 protein levels of ZR-75-1 xenografts by flow cytometry or western blotting at the time of imaging to ensure consisted sstr2 expression between different radiotracer groups. 5.3 Future Direction 5.3.1 Peptide Receptor Radionuclide Therapy in Breast Cancer Therapy with 177Lu-DOTATATE and 177Lu-DOTA-JR11 has been compared in both animal models115, and pilot clinical studies106. Both studies demonstrated that the antagonist 177Lu-DOTA-JR11 delivered a higher radiation dose to the tumour compared to the agonist 177Lu-DOTATATE. We have shown that ZR-75-1 tumour xenografts can be successfully targeted with diagnostic somatostatin radiotracers, however, we have yet to evaluate the 76 therapeutic potential of 177Lu-labeled somatostatin compounds in the same model. As 68Ga-DOTATOC showed the highest tumour uptake in our ZR-75-1 studies at 1 h p.i., a comparison between 177Lu-DOTATOC and 177Lu-DOTA-JR11 would be worthwhile. ZR-75-1 tumour xenografts can be grown as reported herein, although care must be taken to ensure consistent pellet doses and validation of sstr2 protein expression. Biodistribution studies can be performed with 177Lu-DOTATOC and 177Lu-DOTA-JR11 at different time points. As 177Lu has a long half-life (t1/2 = 6.7 d), SPECT imaging and biodistribution can be done as early as 4 h p.i. and up to 7 d p.i. Dalm and colleagues demonstrated that a peptide dose of 0.5μg/30MBq resulted in the highest absorbed tumour dose115. For treatment studies, mice can be administered a single dose of 0.5 μg 177Lu-radiotracer once tumours reach ~500 mm3 in volume. Monitoring of tumour size, mouse weight and general health status can be done daily until a humane end-point is reached. 5.3.2 Clinical Translation of 18F-AmBF3-TATE All three 18F-radiolabeled tracers described in Chapter 4 would be excellent candidates for clinical translation, as all had comparable tumour uptake, low background and excellent in vivo tracer stability. Because 18F-AmBF3-TATE showed the fastest kidney wash-out, and significantly lower liver uptake than 18F-AmBF3-JR11, 18F-AmBF3-TATE should be the first of such tracers to be pursued for clinical translation, as imaging would result in a lower dose-burden for patients. To obtain approval for a clinical trial application, the radiotracer and non-radioactive standard must be prepared under good laboratory practice (GLP) conditions and evaluated for acute toxicity and dosimetry in rodents166. Toxicological studies are performed by administrating a single dose of the non-radioactive equivalent (AmBF3-TATE) at 100 times the expected peptide mass required for imaging. Animals will be inspected for changes in body weight, blood 77 chemistry, urine, and histology of key organs (kidneys, liver, pancreas, bone marrow, intestine, adrenal glands, stomach)167. Single-dose studies are sufficient, as patients will be administered the radiotracer only at the time of imaging, potentially weeks or months apart166. Dosimetry data will be acquired by performing biodistribution studies with 18F-AmBF3-TATE in healthy rodents over multiple time points. The uptake in organs will be used to calculate the predicted human effective radiation doses (mSv) using the OLINDA/EXM software168. 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Table A.1 Standard curve parameters Target Efficiency (%) (n=2) Curve Fit (R2) (n=2) Sstr1 101.6 0.9969 Sstr2 107.5 0.9941 Sstr3 112.5 0.9937 Sstr4 104.7 0.9970 Sstr5 113.0 0.9910 HPRT1 (n=3) 125.09 0.9575 93 Table A.2 PCR cycling conditions Denaturation 98 °C 30 sec 40 Cycles: Denaturation 98 °C 10 sec Annealing sstr1: 60 °C sstr2: 57 °C sstr3: 58 °C sstr4: 57 °C sstr5: 60 °C HPRT1: 57 °C 10 sec Extension 72 °C 20 sec Final Extension 72 °C 2 min Hold 4 °C Table A.3 qPCR cycling conditions Hot Start 95 °C 15 sec 40 cycles Denaturation 95 °C 10 sec Annealing /Extension sstr1: 60 °C sstr2: 57 °C sstr3: 58 °C sstr4: 58 °C sstr5: 60 °C HPRT1: 58 °C 60 sec 94 Appendix B Relationship between Peptide Mass and Organ Uptake Figure B.1 Correlation between injected peptide mass and uptake in sstr-positive organs and blood with 68Ga-NODAGA-JR11, 68Ga-DOTATOC and 68Ga-DOTATATE. * p < 0.05, **p < 0.01. 95 Figure B.2 Correlation between injected peptide mass and uptake in sstr-positive organs and blood with 18F-AmBF3-TOC, 18F-AmBF3-JR11, and 18F-AmBF3-TATE at 1h p.i. * p < 0.05, **p < 0.01. 96 Figure B.3 Correlation between injected peptide mass and uptake in sstr-positive organs and blood with 18F-AmBF3-TOC, 18F-AmBF3-JR11, and 18F-AmBF3-TATE at 2 h p.i. * p < 0.05, **p < 0.01.
Thesis/Dissertation
eng
Interdisciplinary Oncology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Preclinical evaluation of somatostatin analogues for breast cancer imaging using positron emission tomography.
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