{"@context":{"@language":"en","Affiliation":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","AggregatedSourceRepository":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","Campus":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","Creator":"http:\/\/purl.org\/dc\/terms\/creator","DateAvailable":"http:\/\/purl.org\/dc\/terms\/issued","DateIssued":"http:\/\/purl.org\/dc\/terms\/issued","Degree":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","DegreeGrantor":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","Description":"http:\/\/purl.org\/dc\/terms\/description","DigitalResourceOriginalRecord":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","FullText":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","Genre":"http:\/\/www.europeana.eu\/schemas\/edm\/hasType","GraduationDate":"http:\/\/vivoweb.org\/ontology\/core#dateIssued","IsShownAt":"http:\/\/www.europeana.eu\/schemas\/edm\/isShownAt","Language":"http:\/\/purl.org\/dc\/terms\/language","Program":"https:\/\/open.library.ubc.ca\/terms#degreeDiscipline","Provider":"http:\/\/www.europeana.eu\/schemas\/edm\/provider","Publisher":"http:\/\/purl.org\/dc\/terms\/publisher","Rights":"http:\/\/purl.org\/dc\/terms\/rights","RightsURI":"https:\/\/open.library.ubc.ca\/terms#rightsURI","ScholarlyLevel":"https:\/\/open.library.ubc.ca\/terms#scholarLevel","Supervisor":"http:\/\/purl.org\/dc\/terms\/contributor","Title":"http:\/\/purl.org\/dc\/terms\/title","Type":"http:\/\/purl.org\/dc\/terms\/type","URI":"https:\/\/open.library.ubc.ca\/terms#identifierURI","SortDate":"http:\/\/purl.org\/dc\/terms\/date"},"Affiliation":[{"@value":"Medicine, Faculty of","@language":"en"}],"AggregatedSourceRepository":[{"@value":"DSpace","@language":"en"}],"Campus":[{"@value":"UBCV","@language":"en"}],"Creator":[{"@value":"Wang, Lei","@language":"en"}],"DateAvailable":[{"@value":"2025-03-19T19:37:40Z","@language":"en"}],"DateIssued":[{"@value":"2025","@language":"en"}],"Degree":[{"@value":"Doctor of Philosophy - PhD","@language":"en"}],"DegreeGrantor":[{"@value":"University of British Columbia","@language":"en"}],"Description":[{"@value":"Gastrin-releasing peptide receptor (GRPR) is overexpressed in multiple cancers, making it a promising target for diagnosis and therapy. However, most clinically evaluated GRPR-targeted radiopharmaceuticals show high accumulation in the pancreas and limited metabolic stability, which may restrict their diagnostic and therapeutic applications. This dissertation focuses on developing radiolabeled GRPR-targeting ligands with reduced pancreas uptake and improved metabolic stability to enhance diagnostic and therapeutic efficacy for GRPR-expressing tumors. \r\nInspired by a series of GRPR antagonists published by Schally\u2019s group, we first developed four radiolabeled GRPR antagonists based on the [Leu\u00b9\u00b3\u03c8Thz\u00b9\u2074]Bombesin(7-14) sequence and three radiolabeled GRPR agonists by restoring the reduced peptide bond between residues 13-14 (Leu\u00b9\u00b3\u03c8Thz\u00b9\u2074) in the [Leu\u00b9\u00b3\u03c8Thz\u00b9\u2074]Bombesin(7-14) sequence with a normal amide bond. The antagonist [\u2076\u2078Ga]Ga-TacsBOMB2 and agonist [\u2076\u2078Ga]Ga-TacBOMB2 showed good tumor uptake and minimal pancreas uptake. Then we modified our lead agonist and antagonist candidates (TacBOMB2 and TacsBOMB2) with unnatural amino acid substitution to strengthen the bonds at the potential cleavage sites of neutral endopeptidase (NEP) to improve the stability. Our findings indicate that the Tle\u00b9\u2070 and NMe-His\u00b9\u00b2 substitutions markedly improve the stability of the GRPR agonist TacBOMB2 without affecting its binding affinity. Though, with no significant improvement in stability, NMe-Gly\u00b9\u00b9 substitution was shown to improve the tumor uptake and provide a better tumor-to-background contrast ratios for [\u2076\u2078Ga]Ga-TacsBOMB5 derived from TacsBOMB2. Then we labeled our lead candidates with \u00b9\u2077\u2077Lu for further evaluations. However, shorter tumor retention and lower absorbed radiation doses in PC-3 tumor xenograft were observed for all our \u00b9\u2077\u2077Lu-labeled GRPR-targeted ligands compared with the clinically validated [\u00b9\u2077\u2077Lu]Lu-RM2, indicating further optimizations are still needed to prolong the tumor retention for therapeutic applications. To avoid the oxidation of Thz, we replaced the Thz\u00b9\u2074 with Pro\u00b9\u2074 to potentially prolong the shelf-life of our top GRPR-targeted radiopharmaceuticals. GRPR antagonist, [\u2076\u2078Ga]Ga-ProBOMB5 (DOTA-Pip-[DPhe\u2076,NMe-Gly\u00b9\u00b9,Leu\u00b9\u00b3\u03c8Pro\u00b9\u2074]Bombesin(6-14)), showed great tumor uptake, minimal accumulation in pancreas, and excellent tumor-to-background contrast ratios among all the novel GRPR-targeted tracers in this dissertation. Our results are encouraging to support clinical translation of [\u2076\u2078Ga]Ga-ProBOMB5 as a diagnostic radiotracer for detecting GRPR-expressing lesions, particularly the lesions in or adjacent to the pancreas.","@language":"en"}],"DigitalResourceOriginalRecord":[{"@value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/90509?expand=metadata","@language":"en"}],"FullText":[{"@value":" DEVELOPMENT OF NOVEL STABLE GRPR-TARGETING RADIOPHARMACEUTICALS WITH LOW PANCREAS UPTAKE FOR CANCER DIAGNOSIS AND THERAPY by Lei Wang M.D., Tongji University, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Interdisciplinary Oncology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2025 \u00a9 Lei Wang, 2025 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Development of novel stable GRPR-targeting radiopharmaceuticals with low pancreas uptake for cancer diagnosis and therapy submitted by Lei Wang in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Interdisciplinary Oncology Examining Committee: Dr. Kuo-Shyan Lin, Professor, Department of Radiology, UBC Supervisor Dr. Fran\u00e7ois B\u00e9nard, Professor, Department of Radiology, UBC Supervisory Committee Member Dr. Yuzhuo Wang, Professor, Department of Urologic Sciences, UBC University Examiner Dr. Urs H\u00e4feli, Emeritus Professor, Faculty of Pharmaceutical Sciences, UBC University Examiner Dr. Eric W. Price, Associate Professor, Department of Chemistry, University of Saskatchewan External Examiner Additional Supervisory Committee Members: Dr. Arman Rahmim, Professor, Department of Radiology, UBC Supervisory Committee Member Dr. David M. Perrin, Professor, Department of Chemistry, UBC Supervisory Committee Member iii Abstract Gastrin-releasing peptide receptor (GRPR) is overexpressed in multiple cancers, making it a promising target for diagnosis and therapy. However, most clinically evaluated GRPR-targeted radiopharmaceuticals show high accumulation in the pancreas and limited metabolic stability, which may restrict their diagnostic and therapeutic applications. This dissertation focuses on developing radiolabeled GRPR-targeting ligands with reduced pancreas uptake and improved metabolic stability to enhance diagnostic and therapeutic efficacy for GRPR-expressing tumors. Inspired by a series of GRPR antagonists published by Schally\u2019s group, we first developed four radiolabeled GRPR antagonists based on the [Leu13\u03c8Thz14]Bombesin(7-14) sequence and three radiolabeled GRPR agonists by restoring the reduced peptide bond between residues 13-14 (Leu13\u03c8Thz14) in the [Leu13\u03c8Thz14]Bombesin(7-14) sequence with a normal amide bond. The antagonist [68Ga]Ga-TacsBOMB2 and agonist [68Ga]Ga-TacBOMB2 showed good tumor uptake and minimal pancreas uptake. Then we modified our lead agonist and antagonist candidates (TacBOMB2 and TacsBOMB2) with unnatural amino acid substitution to strengthen the bonds at the potential cleavage sites of neutral endopeptidase (NEP) to improve the stability. Our findings indicate that the Tle10 and NMe-His12 substitutions markedly improve the stability of the GRPR agonist TacBOMB2 without affecting its binding affinity. Though, with no significant improvement in stability, NMe-Gly11 substitution was shown to improve the tumor uptake and provide a better tumor-to-background contrast ratios for [68Ga]Ga-TacsBOMB5 derived from TacsBOMB2. Then we labeled our lead candidates with 177Lu for further evaluations. However, shorter tumor retention and lower absorbed radiation doses in PC-3 tumor xenograft were observed for all our 177Lu-labeled GRPR-targeted ligands compared with the clinically validated [177Lu]Lu-iv RM2, indicating further optimizations are still needed to prolong the tumor retention for therapeutic applications. To avoid the oxidation of Thz, we replaced the Thz14 with Pro14 to potentially prolong the shelf-life of our top GRPR-targeted radiopharmaceuticals. GRPR antagonist, [68Ga]Ga-ProBOMB5 (DOTA-Pip-[DPhe6,NMe-Gly11,Leu13\u03c8Pro14]Bombesin(6-14)), showed great tumor uptake, minimal accumulation in pancreas, and excellent tumor-to-background contrast ratios among all the novel GRPR-targeted tracers in this dissertation. Our results are encouraging to support clinical translation of [68Ga]Ga-ProBOMB5 as a diagnostic radiotracer for detecting GRPR-expressing lesions, particularly the lesions in or adjacent to the pancreas. v Lay Summary Gastrin-releasing peptide receptor (GRPR) is a protein found overexpressed on the surface of many cancer cells, making it an important target for cancer detection and therapy. This research focuses on improving tools to help doctors detect and treat certain cancers expressing GRPR, like prostate and breast cancer. The study is centered on developing new radiopharmaceuticals, which are radioactive drugs designed to target GRPR. However, GRPR is also present in healthy tissues like the pancreas, which can cause the drugs to accumulate there, leading to side effects. This research aims to design radiopharmaceuticals that specifically target cancer cells while avoiding the pancreas. Additionally, the new drugs are designed to be stable in the body, ensuring they stay intact long enough to effectively find tumors during scans and kill cancer cells. If successful, this could lead to better and safer ways to detect and treat cancer. vi Preface All the animal experiments in this thesis were performed in accordance with the animal protocol A20-0113, 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. The research outlined in this thesis was also conducted under the biosafety (B20-0011) and radiation safety (RS20-0001) protocols. Section 3.1 was adapted from my own published manuscript, [Wang, L.], Zhang, Z., Merkens, H., Zeisler, J., Zhang, C., Roxin, A., Tan, R., B\u00e9nard, F. and Lin, K.S. 68Ga-Labeled [Leu13\u03c8Thz14]Bombesin(7-14) derivatives: promising GRPR-targeting PET tracers with low pancreas uptake. Molecules, 2022, 27, 3777. I served as the principal investigator for this work and responsible for data collection, analysis, and the manuscript composition. I conducted the peptide synthesis, purification, and characterization with LC\/MS. Helen Merkens and I performed most of the cell culture, PC-3 tumor inoculation, mice monitoring, and biodistribution studies. Dr. Jutta Zeisler and I conducted the in vitro binding assay. Dr. Chengcheng Zhang, Dr. Zhengxing Zhang, and I performed the calcium release assays and in vivo stability tests. Dr. Zhengxing Zhang performed the radiolabeling experiments and trained me on the peptide synthesis, purification, and characterization. Ruiyan Tan also performed the biodistribution studies and cell culture. Dr. Aron Roxin performed and analyzed the nuclear magnetic resonance (NMR) spectroscopy analysis. Nadine Colpo performed the PET images acquisition. Dr. Kuo-Shyan Lin and Dr. Fran\u00e7ois B\u00e9nard were supervisory authors and were involved in the conceptualization for this work. Dr. Kuo-Shyan Lin and I drafted this manuscript, and Dr. Aron Roxin was involved in the review and editing. Section 3.2 was adapted from my own published manuscript, [Wang, L.], Bratanovic, I.J., Zhang, Z., Kuo, H.T., Merkens, H., Zeisler, J., Zhang, C., Tan, R., B\u00e9nard, F. and Lin, K.S. 68Ga-vii labeled [Thz14]Bombesin(7-14) analogs: Promising GRPR-targeting agonist PET tracers with low pancreas uptake. Molecules, 2023, 28, 1977. I was the lead researcher on this work. I was responsible for data collection, analysis, and the manuscript composition. Dr. Ivica Jerolim Bratanovic and I performed the peptide synthesis, purification, and characterization. Helen Merkens and I performed the cell culture, PC-3 tumor inoculation, and mice monitoring. Dr. Jutta Zeisler and I conducted the in vitro binding assay. Dr. Chengcheng Zhang, Dr. Zhengxing Zhang, and I performed the calcium release assays and in vivo stability tests. Dr. Zhengxing Zhang and Dr. Hsiou-Ting Kuo conducted the radiolabeling experiments. Ruiyan Tan and I performed the biodistribution studies. Nadine Colpo performed the PET images acquisition. Dr. Kuo-Shyan Lin and Dr. Fran\u00e7ois B\u00e9nard were supervisory authors and were involved in the conceptualization for this work. Dr. Kuo-Shyan Lin and I drafted this manuscript. A version of Section 4.1 was published in EJNMMI Radiopharmacy and Chemistry. [Wang, L.], Kuo, H.T., Zhang, Z., Zhang, C., Chen, C.C., Chapple, D., Wilson, R., Colpo, N., B\u00e9nard, F. and Lin, K.S. Unnatural amino acid substitutions to improve in vivo stability and tumor uptake of 68Ga-labeled GRPR-targeted TacBOMB2 derivatives for cancer imaging with positron emission tomography. EJNMMI Radiopharmacy and Chemistry, 2024, 9, 8. I was the lead researcher on this work and responsible for data collection, analysis, and the manuscript composition. I conducted the peptide synthesis, purification, and characterization. I also performed the cell culture, PC-3 tumor inoculation, mice monitoring, and in vitro binding assay. Dr. Chengcheng Zhang and I performed the calcium release assays. Dr. Zhengxing Zhang, Dr. Hsiou-Ting Kuo, Dr. Chao-Cheng (Jimmy) Chen, and Dr. Devon Chapple conducted the radiolabeling experiments and in vivo stability tests. Ryan Wilson and I performed the biodistribution studies. Nadine Colpo performed and trained me the PET images acquisition. Dr. Kuo-Shyan Lin and Dr. Fran\u00e7ois viii B\u00e9nard were supervisory authors and were involved in the conceptualization for this work. Dr. Kuo-Shyan Lin and I were drafted this manuscript. Section 4.2 was adapted from my own published manuscript, [Wang, L.], Chen, C.C., Zhang, Z., Kuo, H.T., Zhang, C., Colpo, N., Merkens, H., B\u00e9nard, F. and Lin, K.S. Synthesis and evaluation of novel 68Ga-Labeled [D-Phe6,Leu13\u03c8Thz14]Bombesin(6-14) analogs for cancer imaging with positron emission tomography. Pharmaceuticals, 2024, 17, 621. I served as the principal investigator for this work and responsible for data collection, analysis, and the manuscript composition. I conducted the peptide synthesis, purification, and characterization with LC\/MS. Helen Merkens performed the cell culture, PC-3 tumor inoculation, and mice monitoring. I conducted the in vitro binding assay. Dr. Chengcheng Zhang and I performed the calcium release assays. Dr. Chao-Cheng (Jimmy) Chen, Dr. Hsiou-Ting Kuo, and Dr. Zhengxing Zhang and I performed the radiolabeling experiments and in vivo stability tests for this work. Pauline Ng and Wing Sum Lau performed the biodistribution studies together with me. Nadine Colpo and I performed the PET images acquisition. Dr. Kuo-Shyan Lin and Dr. Fran\u00e7ois B\u00e9nard were supervisory authors and were involved in the conceptualization for this work. The manuscript was drafted and finalized by me with critical input from Dr. Kuo-Shyan Lin. Chapter 5 contains data that is currently unpublished. I served as the principal investigator for this work and responsible for data collection, analysis, and the manuscript composition. I conducted the synthesis, purification, and characterization with LC\/MS for the non-radioactive standards. I also performed the cell culture, PC-3 tumor inoculation, mice monitoring, and the in vitro evaluations. Dr. Devon Chapple, and Dr. Hsiou-Ting Kuo performed the radiolabeling experiments and logD7.4 tests for this work. Ryan Wilson, Pauline Ng and Wing Sum Lau performed the biodistribution studies together with me. Dr. Sara Kurkowska performed the ix dosimetry analysis. Nadine Colpo and I performed the SPECT\/CT images acquisition. Dr. Kuo-Shyan Lin, Dr. Fran\u00e7ois B\u00e9nard, and Dr. Carlos Uribe were supervisory authors and were involved in the conceptualization for this work. Section 6.1 was adapted from my own published manuscript, [Wang, L.], Kuo, H.T., Chapple, D.E., Chen, C.C., Kurkowska, S., Colpo, N., Uribe, C., B\u00e9nard, F. and Lin, K.S. Synthesis and evaluation of 68Ga- and 177Lu-labeled [Pro14]Bombesin(8-14) derivatives for detection and radioligand therapy of gastrin-releasing peptide receptor-expressing cancer. Molecular Pharmaceutics, 2024. https:\/\/doi.org\/10.1021\/acs.molpharmaceut.4c00952. I was the lead researcher on this work and responsible for data collection, analysis, and the manuscript composition. I conducted the synthesis, purification, and characterization with LC\/MS for the ligands and their non-radioactive standards. I also performed the cell culture, PC-3 tumor inoculation, mice monitoring, in vivo stability tests, and the in vitro evaluations. Dr. Hsiou-Ting Kuo, Dr. Devon Chapple, and Dr. Chao-Cheng Chen performed the radiolabeling experiments and logD7.4 tests for this work. Ryan Wilson, Pauline Ng and Wing Sum Lau performed the biodistribution studies together with me. Dr. Sara Kurkowska performed the dosimetry analysis. Nadine Colpo and I performed the SPECT\/CT images acquisition. Dr. Kuo-Shyan Lin, Dr. Fran\u00e7ois B\u00e9nard, and Dr. Carlos Uribe were supervisory authors and were involved in the conceptualization for this work. A version of Section 6.2 was published in Molecules. [Wang, L.], Kuo, H.T., Chen, C.C., Chapple, D., Colpo, N., Ng, P., Lau, W.S., Jozi, S., B\u00e9nard, F. and Lin, K.S. Synthesis and Evaluation of the first 68Ga-labeled C-terminal hydroxamate-derived gastrin-releasing peptide receptor-targeted tracers for cancer imaging with positron emission tomography. Molecules, 2024, 29, 3102. I served as the principal investigator for this work and responsible for data collection, x analysis, and the manuscript composition. Shireen Jozi and I conducted the synthesis, purification, and characterization with LC\/MS. I performed the cell culture, PC-3 tumor inoculation, mice monitoring, and the in vitro evaluations. Dr. Devon Chapple and Dr. Chao-Cheng Chen conducted the radiolabeling experiments and logD7.4 tests for this work. Pauline Ng and Wing Sum Lau conducted the biodistribution studies together with me. Nadine Colpo and I performed the PET\/CT images acquisition. Dr. Kuo-Shyan Lin and Dr. Fran\u00e7ois B\u00e9nard were supervisory authors and were involved in the conceptualization for this work. xi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ......................................................................................................................... xi List of Tables .............................................................................................................................. xix List of Figures .............................................................................................................................xxv List of Symbols ..................................................................................................................... xxxviii List of Abbreviations ............................................................................................................. xxxix Acknowledgements .................................................................................................................... xlii Dedication .................................................................................................................................. xliv Chapter 1: Introduction ................................................................................................................1 1.1 Nuclear Medicine ............................................................................................................ 1 1.1.1 Single Photon Emission Computed Tomography ................................................... 2 1.1.2 Positron Emission Tomography .............................................................................. 3 1.2 Radiopharmaceuticals ..................................................................................................... 5 1.2.1 Antibody-based Radiopharmaceuticals .................................................................. 6 1.2.1.1 Principle of Antibody-based Radiopharmaceuticals ........................................... 6 1.2.1.2 Clinical Applications .......................................................................................... 8 1.2.1.3 Advantages and Limitations ............................................................................... 9 1.2.2 Peptide-based Radiopharmaceuticals ...................................................................... 9 1.2.2.1 Principle of Peptide-based Radiopharmaceuticals ............................................ 10 xii 1.2.2.2 Clinical Applications ........................................................................................ 11 1.2.2.3 Advantages and Limitations ............................................................................. 12 1.2.3 Small Molecule-based Radiopharmaceuticals ...................................................... 13 1.2.3.1 Principle and Clinical Applications .................................................................. 13 1.2.3.2 Advantages and Limitations ............................................................................. 15 1.2.4 Nanoparticle-based and Nucleic Acids-based Radiopharmaceuticals .................. 15 1.3 Gastrin-releasing Peptide Receptor ............................................................................... 16 1.3.1 Molecular Basis of GRPR and Ligands ................................................................ 17 1.3.1.1 Structure of GRPR ............................................................................................ 17 1.3.1.2 Gastrin-releasing Peptide and Bombesin .......................................................... 20 1.3.1.3 Signaling Pathways ........................................................................................... 22 1.3.2 GRPR Expression in Cancers ............................................................................... 25 1.3.2.1 Prostate Cancer ................................................................................................. 26 1.3.2.2 Breast Cancer .................................................................................................... 28 1.3.3 Development of GRPR-targeted ligands .............................................................. 31 1.3.3.1 Historical Development .................................................................................... 31 1.3.3.2 Limitations of GRPR-targeted Radiopharmaceuticals ...................................... 34 1.3.3.2.1 High Pancreas Uptake Issue ........................................................................ 34 1.3.3.2.2 Low Metabolic Stability ............................................................................. 36 1.4 Hypothesis and Aims .................................................................................................... 37 Chapter 2: Materials and Methods ............................................................................................39 2.1 Reagents and Instrumentation ....................................................................................... 39 2.2 Synthesis of Fmoc-Leu\u03c8Thz-OH ................................................................................. 40 xiii 2.3 Peptide Synthesis .......................................................................................................... 42 2.3.1 Synthesis of DOTA-Conjugated Precursors ......................................................... 42 2.3.2 Synthesis of GRPR-targeted peptides ................................................................... 44 2.4 Synthesis of Nonradioactive Standards ........................................................................ 44 2.4.1 Synthesis of Nonradioactive Ga-complexed Standards ........................................ 44 2.4.2 Synthesis of Nonradioactive Lu-complexed Standards ........................................ 45 2.5 Cell culture .................................................................................................................... 45 2.6 Fluorometric Calcium Release Assay ........................................................................... 46 2.7 In Vitro Competition Binding Assay ............................................................................ 46 2.8 Radiolabeling ................................................................................................................ 47 2.8.1 68Ga Radiolabeling ................................................................................................ 47 2.8.2 177Lu Radiolabeling ............................................................................................... 47 2.9 The logD7.4 Measurements ............................................................................................ 48 2.10 Animal Studies .............................................................................................................. 48 2.10.1 PET imaging and Biodistribution Studies............................................................. 48 2.10.2 SPECT imaging and Biodistribution Studies ........................................................ 49 2.10.3 In vivo Stability Studies ........................................................................................ 50 2.11 Dosimetry ...................................................................................................................... 50 2.12 Statistical Analysis ........................................................................................................ 52 Chapter 3: Novel GRPR-targeting Ligands with High Binding Affinity to Increase the Tumor Uptake ..............................................................................................................................53 3.1 68Ga-Labeled [Leu13\u03c8Thz14]Bombesin(7-14) Derivatives: Promising GRPR-targeting PET Tracers with Low Pancreas Uptake .................................................................................. 53 xiv 3.1.1 Introduction ........................................................................................................... 53 3.1.2 Materials and Methods .......................................................................................... 56 3.1.3 Results ................................................................................................................... 57 3.1.3.1 Peptide Synthesis and Radiolabeling ................................................................ 57 3.1.3.2 Binding Affinity, Antagonist Characterization and Hydrophilicity ................. 59 3.1.3.3 PET Imaging and Ex Vivo Biodistribution ........................................................ 61 3.1.3.4 In vivo Stability ................................................................................................. 65 3.1.4 Discussion ............................................................................................................. 67 3.1.5 Conclusions ........................................................................................................... 74 3.2 68Ga-labeled [Thz14]Bombesin(7-14) analogs: Promising GRPR-targeting Agonist PET Tracers with Low Pancreas Uptake .......................................................................................... 76 3.2.1 Introduction ........................................................................................................... 76 3.2.2 Materials and Methods .......................................................................................... 80 3.2.3 Results ................................................................................................................... 80 3.2.3.1 Chemistry and Radiochemistry ......................................................................... 80 3.2.3.2 Agonist Characterization, Binding Affinity, and Hydrophilicity ..................... 81 3.2.3.3 PET Imaging and ex vivo Biodistribution ......................................................... 83 3.2.3.4 In vivo Stability ................................................................................................. 87 3.2.4 Discussion ............................................................................................................. 89 3.2.5 Conclusions ........................................................................................................... 94 Chapter 4: Modifying the Amino Acids Around the Cleavage Sites to Increase the Stability of GRPR-targeted Peptides .........................................................................................................95 xv 4.1 Unnatural Amino Acid Substitutions to improve in vivo stability and tumor uptake of 68Ga-labeled GRPR-targeted TacBOMB2 Derivatives for Cancer Imaging with Positron Emission Tomography .............................................................................................................. 95 4.1.1 Introduction ........................................................................................................... 95 4.1.2 Materials and Methods .......................................................................................... 98 4.1.3 Results ................................................................................................................... 99 4.1.3.1 Chemistry and Radiochemistry ......................................................................... 99 4.1.3.2 GRPR Binding Affinities of LW01085 and its Derivatives ........................... 104 4.1.3.3 GRPR Binding Affinities of Ga-TacBOMB2 Derivatives ............................. 105 4.1.3.4 Confirmation of Agonist Characteristics of Ga-TacBOMB2 Derivatives ...... 108 4.1.3.5 PET Imaging and Biodistribution ................................................................... 109 4.1.3.6 The logD7.4 Measurement and in vivo Stability .............................................. 114 4.1.4 Discussion ........................................................................................................... 118 4.1.5 Conclusions ......................................................................................................... 122 4.2 Synthesis and Evaluation of Novel 68Ga-labeled [D-Phe6,Leu13\u03c8Thz14]Nombesin(6-14) Analogs for Cancer Imaging with Positron Emission Tomography ....................................... 123 4.2.1 Introduction ......................................................................................................... 123 4.2.2 Materials and Methods ........................................................................................ 126 4.2.3 Results ................................................................................................................. 127 4.2.3.1 Syntheses of GRPR-targeted Ligands ............................................................. 127 4.2.3.2 Binding Affinity, Antagonist Characterization, and Hydrophilicity .............. 128 4.2.3.3 PET Imaging and ex vivo Biodistribution ....................................................... 130 4.2.3.4 In vivo Stability ............................................................................................... 135 xvi 4.2.4 Discussion ........................................................................................................... 137 4.2.5 Conclusions ......................................................................................................... 142 Chapter 5: Synthesis and Evaluation of 177Lu-labeled [Thz14]Bombesin(6-14) derivatives for radioligand therapy of gastrin-releasing peptide receptor-expressing cancer ...............143 5.1 Introduction ................................................................................................................. 143 5.2 Materials and Methods ................................................................................................ 146 5.3 Results ......................................................................................................................... 146 5.3.1 Peptide Synthesis and Radiolabeling .................................................................. 146 5.3.2 Binding Affinity, Agonist\/Antagonist Characterization and Hydrophilicity Measurement ....................................................................................................................... 147 5.3.3 SPECT imaging .................................................................................................. 150 5.3.4 Ex vivo Biodistribution ....................................................................................... 153 5.3.5 Dosimetry ............................................................................................................ 159 5.4 Discussions ................................................................................................................. 162 5.5 Conclusions ................................................................................................................. 166 Chapter 6: Pro derivatives and hydroxamate derivatives .....................................................168 6.1 Synthesis and Evaluation of 68Ga- and 177Lu-labeled [Pro14]Bombesin(8-14) Derivatives for Detection and Radioligand Therapy of Gastrin-releasing Peptide Receptor-expressing Cancer ................................................................................................................... 168 6.1.1 Introduction ......................................................................................................... 168 6.1.2 Materials and Methods ........................................................................................ 172 6.1.3 Results ................................................................................................................. 172 6.1.3.1 Peptide Synthesis and Radiolabeling .............................................................. 172 xvii 6.1.3.2 Hydrophilicity, Agonist\/Antagonist Characteristics, and Binding Affinity ... 174 6.1.3.3 PET Imaging and Ex vivo Biodistribution ...................................................... 176 6.1.3.4 SPECT\/CT Imaging and Ex vivo Biodistribution ........................................... 180 6.1.3.5 Radiation Dosimetry ....................................................................................... 185 6.1.3.6 In vivo Stability ............................................................................................... 188 6.1.4 Discussion ........................................................................................................... 189 6.1.5 Conclusions ......................................................................................................... 196 6.2 Synthesis and Evaluation of First 68Ga-labeled C-terminal Hydroxamate-derived GRPR-targeted Tracers for Cancer Imaging with Positron Emission Tomography ............... 198 6.2.1 Introduction ......................................................................................................... 198 6.2.2 Materials and Methods ........................................................................................ 201 6.2.3 Results ................................................................................................................. 201 6.2.3.1 Peptide Synthesis and Radiolabeling .............................................................. 201 6.2.3.2 Binding Affinity .............................................................................................. 203 6.2.3.3 Antagonist Characterization and Hydrophilicity Measurement ..................... 203 6.2.3.4 PET Imaging ................................................................................................... 204 6.2.3.5 Ex vivo Biodistribution ................................................................................... 205 6.2.4 Discussion ........................................................................................................... 209 6.2.5 Conclusions ......................................................................................................... 212 Chapter 7: Conclusions and Future Directions ......................................................................213 7.1 Concluding remarks .................................................................................................... 213 7.2 Future Directions ........................................................................................................ 216 7.2.1 Clinical Translation of [68Ga]Ga-ProBOMB5 .................................................... 216 xviii 7.2.2 Design, Synthesis and Evaluation of GRPR-targeting Ligands with High Binding Affinity to Increase the Tumor Uptake and Minimize the Pancreas Uptake ...................... 217 7.2.3 Radiolabeling the Most Promising Candidate with 177Lu or 225Ac, and Evaluating Their Potential for Radioligand Therapy ............................................................................ 218 Bibliography ...............................................................................................................................219 xix List of Tables Table 1.1 Radioisotopes commonly utilized in nuclear medicine. ................................................. 1 Table 1.2 Examples of peptide-based radiopharmaceuticals utilized in cancer imaging and therapy.. ......................................................................................................................................... 11 Table 1.3 Amino acid sequences of BBN, GRP, and NMC share the same seven carboxyl-terminal amino acids ..................................................................................................................... 20 Table 1.4 Examples of GRPR-targeted radiopharmaceuticals. .................................................... 33 Table 3.1 HPLC purification conditions and MS characterizations of TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5 and TacsBOMB6. .................................................... 57 Table 3.2 HPLC purification conditions and MS characterizations of Ga-complexed TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5 and TacsBOMB6. ............................. 58 Table 3.3 HPLC conditions for the purification and quality control of 68Ga-labeled TacsBOMB2, TacsBOMB3, TacsBOMB5, and TacsBOMB6. ........................................................................... 58 Table 3.4 Biodistribution and uptake ratios of 68Ga-labeled GRPR-targeting tracers in PC-3 tumor-bearing mice. ...................................................................................................................... 65 Table 3.5 HPLC purification conditions and MS characterizations of TacBOMB2, TacBOMB3, and TacBOMB4. ........................................................................................................................... 80 Table 3.6 HPLC purification conditions and MS characterizations of Ga-TacBOMB2, Ga-TacBOMB3, and Ga-TacBOMB4. ............................................................................................... 81 Table 3.7 HPLC conditions for the purification and quality control of 68Ga-labeled TacBOMB2 TacBOMB3, and AMBA. ............................................................................................................. 81 Table 3.8 Biodistribution and uptake ratios of 68Ga-labeled GRPR-targeting tracers in PC-3 tumor-bearing mice. ...................................................................................................................... 86 xx Table 4.1 Peptide sequences and purities of GRPR-targeted peptides. ........................................ 99 Table 4.2 HPLC purification conditions and MS characterizations of GRPR-targeted peptides...................................................................................................................................................... 100 Table 4.3 Peptide sequences and purities of DOTA-conjugated GRPR-targeted peptides. ....... 101 Table 4.4 HPLC purification conditions and MS characterizations of DOTA-conjugated GRPR-targeted peptides. ........................................................................................................................ 102 Table 4.5 Peptide sequences and purities of Ga-complexed DOTA-conjugated GRPR-targeted peptides. ...................................................................................................................................... 102 Table 4.6 HPLC purification conditions and MS characterizations of Ga-complexed DOTA-conjugated GRPR-targeted peptides. .......................................................................................... 103 Table 4.7 HPLC conditions for the purification and quality control of 68Ga-labeled LW01107, LW01108, LW01110, LW01142, LW02021, and LW02040. .................................................... 104 Table 4.8 Biodistribution and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice at 1 h post-injection. ................................................................................... 113 Table 4.9 Biodistribution and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice. .................................................................................................................... 114 Table 4.10 LogD7.4 values and in vivo stability of GRPR-targeted tracers. ............................... 115 Table 4.11 MS characterizations, yields and HPLC purification conditions of LW01158, LW01160, LW01186, and LW02002. ........................................................................................ 127 Table 4.12 MS characterizations, yields and HPLC purification conditions of Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002. ............................................................................ 128 Table 4.13 HPLC conditions for the purification and quality control of 68Ga-labeled LW01158, LW01186, and LW02002. .......................................................................................................... 128 xxi Table 4.14 Biodistribution and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice. .................................................................................................................... 132 Table 5.1 MS characterizations, yields and HPLC purification conditions of Lu-TacsBOMB5, Lu-LW01110, and Lu-LW01142. ............................................................................................... 146 Table 5.2 HPLC conditions for the purification and quality control of 177Lu-labeled TacsBOMB5, LW01110, LW01142, RM2, and AMBA. ........................................................... 147 Table 5.3 Biodistribution and tumor-to-organ ratios of [177Lu]Lu-TacsBOMB5 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. ............................................................... 155 Table 5.4 Biodistribution and tumor-to-organ ratios of [177Lu]Lu-LW01110 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. ............................................................... 156 Table 5.5 Biodistribution and tumor-to-organ ratios of [177Lu]Lu-LW01142 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection.. .............................................................. 157 Table 5.6 Biodistribution and tumor-to-organ ratios of [177Lu]Lu-RM2 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. ............................................................................ 158 Table 5.7 Biodistribution and tumor-to-organ ratios of [177Lu]Lu-AMBA in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. ............................................................................ 158 Table 5.8 Estimated radiation absorbed doses in mice for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA. ............................... 160 Table 5.9 Estimated radiation absorbed doses in adult human male for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA. ............... 161 Table 6.1 HPLC purification conditions and MS characterizations of ProBOMB5, LW02056, and LW02057. ............................................................................................................................. 172 xxii Table 6.2 HPLC purification conditions and MS characterizations of Ga-ProBOMB5, Ga-LW02056, Ga-LW02057, Lu-ProBOMB5 and Lu-RM2. .......................................................... 173 Table 6.3 HPLC conditions for the purification and quality control of 68Ga-labeled ProBOMB5, LW02056, and LW02057, and 177Lu-labeled ProBOMB5. ........................................................ 173 Table 6.4 Biodistribution and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice. .................................................................................................................... 180 Table 6.5 Biodistribution and tumor-to-organ uptake ratios of [177Lu]Lu-ProBOMB5 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h. ........................................................................... 184 Table 6.6 Biodistribution and tumor-to-organ uptake ratios of [177Lu]Lu-RM2 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h. ...................................................................................... 184 Table 6.7 Absorbed dose per unit of injected activity in mice for [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 ........................................................................................................................... 186 Table 6.8 Estimated absorbed doses in adult human males for [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 ........................................................................................................................... 187 Table 6.9 HPLC purification conditions and MS characterizations of LW02075 and LW02050...................................................................................................................................................... 202 Table 6.10 HPLC purification conditions and MS characterizations of Ga-LW02075 and Ga-LW02050 .................................................................................................................................... 202 Table 6.11 HPLC conditions for the purification and quality control of [68Ga]Ga-LW02075, [68Ga]Ga-LW02050, and [68Ga]Ga-SB3. .................................................................................... 202 Table 6.12 Biodistribution and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice. .................................................................................................................... 207 xxiii List of Figures Figure 1.1 Schematic representation of SPECT and PET detection. .............................................. 3 Figure 1.2 (A) Graphic representation of true (a), scatter (b), and random events (c). (B) Diagram illustrates noncollinearity effect in coincidence imaging. .............................................................. 4 Figure 1.3 Random and site-specific immuno-PET\/SPECT bioconjugation sites of mAbs. .......... 7 Figure 1.4 Principle of in vivo peptide receptor targeting of cancer. ............................................ 10 Figure 1.5 (A) Schematic representation of the murine GRPR showing the postulated transmembrane topology. (B) Transmembrane hydropathy plot of the human GRPR. ............... 18 Figure 1.6 Peptide agonists binding to GRPR. ............................................................................. 21 Figure 1.7 Signaling pathways regulated by BBN\/GRP. .............................................................. 23 Figure 1.8 Maximum-intensity-projection PET images from [68Ga]Ga-PSMA-11 and [68Ga]Ga-RM2 PET scan pairs. .................................................................................................................... 27 Figure 1.9 (A) Chemical structures of ProBOMB1 and ProBOMB2, (B) Comparison of the in vivo PET images of [68Ga]Ga-NeoBOMB1, [68Ga]Ga-ProBOMB1, and [68Ga]Ga-ProBoMB2 at 1h post-injectionin PC-3 tumor-bearing mice. .............................................................................. 35 Figure 3.1 Chemical structures of (A) TacsBOMB2, TacsBOMB3, and TacsBOMB4, (B) TacsBOMB5, (C) TacsBOMB6, and (D) RM2. ........................................................................... 55 Figure 3.2 Displacement curves of [125I-Tyr4]Bombesin by Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, Ga-TacsBOMB6, and Ga-RM2 generated using GRPR-expressing PC-3 cells. ................................................................................................................... 59 Figure 3.3 Intracellular calcium efflux in PC-3 cells induced by various tested ligands. ............ 60 xxiv Figure 3.4 Representative maximum intensity projection PET images of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 acquired at 1h post-injection in mice bearing PC-3 tumor xenografts.. ....................................... 62 Figure 3.5 Uptake of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, and [68Ga]Ga-TacsBOMB6 in PC-3 tumor xenografts and major organs\/tissues of mice at 1h post-injection.. ............................................................................................................................... 63 Figure 3.6 Comparison of 68Ga-TacsBOMB5 and 68Ga-RM2 uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1h post-injection. ................................................................. 64 Figure 3.7 Comparison of [68Ga]Ga-TacsBOMB5 with\/without co-injection of nonradioactive standard on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1h post-injection. ........................................................................................................................................ 64 Figure 3.8 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-TacsBOMB2 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. ........................................................................................................................................ 66 Figure 3.9 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-TacsBOMB5 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. ........................................................................................................................................ 66 Figure 3.10 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-RM2 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. 67 Figure 3.11 The proposed chemical structure of the observed by-product from cleavage of protected TacsBOMB2 off the Rink Amide MBHA resin. .......................................................... 69 Figure 3.12 MS analysis of the observed by-product. .................................................................. 70 xxv Figure 3.13 Chemical structures of (A) TacsBOMB2, TacsBOMB3, and TacsBOMB4; (B) TacsBOMB5; (C) TacBOMB2, TacBOMB3, and TacBOMB4; and (D) AMBA. ...................... 79 Figure 3.14 Intracellular calcium efflux in PC-3 cells induced by various tested ligands. .......... 82 Figure 3.15 Displacement curves of [125I-Tyr4]Bombesin by Ga-TacBOMB2, Ga-TacBOMB3, Ga-TacBOMB4, and Ga-AMBA generated using GRPR-expressing PC-3 cells. ....................... 83 Figure 3.16 Representative PET images of [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA acquired at 1 h post-injection in mice bearing PC-3 tumor xenografts. .......... 84 Figure 3.17 Uptake of [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA in PC-3 tumor xenografts and major organs\/tissues of mice at 1 h post-injection. .......................... 85 Figure 3.18 Comparison of tumor-to-organ contrast ratios of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-AMBA obtained from PC-3 tumor-bearing mice at 1 h post-injection. ....................... 85 Figure 3.19 Comparison of [68Ga]Ga-TacBOMB2 with\/without co-injection of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6\u201314) on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. ................................................................................. 87 Figure 3.20 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-TacBOMB2 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. ........................................................................................................................................ 88 Figure 3.21 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-TacBOMB3 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. ........................................................................................................................................ 88 Figure 3.22 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-AMBA in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. ........................................................................ 89 xxvi Figure 4.1 Chemical structures and GRPR binding affinities of (A) LW01085 and its derivatives with an unnatural amino acid substitution at (B) His12, (C) Val10, (D) Ala9, (E) Gln7, (F) Val10-Gly11, and (G) Trp8. ...................................................................................................................... 98 Figure 4.2 Chemical structures and GRPR binding affinities of (A) Ga-TacBOMB2 and its derivatives with an unnatural amino acid substitution at (B) His12, (C) Val10, (D) Trp8, (E) Val10 and His12, (F) Gln7, Val10 and His12, (G) Gln7, Val10, Gly11 and His12, and (H) Trp8, Val10 and His12. ........................................................................................................................................... 106 Figure 4.3 Displacement curves of [125I-Tyr4]Bombesin by (A) Ga-LW01107, (B) Ga-LW01108, (C) Ga-LW01110, (D) Ga-LW01142, (E) Ga-LW01143, and (F) Ga-LW01149 generated using GRPR-expressing PC-3 cells. ..................................................................................................... 107 Figure 4.4 Displacement curves of [125I-Tyr4]Bombesin by (A) Ga-LW02021, (B) Ga-LW02023, (C) Ga-LW02025, and (D) Ga-LW02040 generated using GRPR-expressing PC-3 cells. ........ 108 Figure 4.5 Intracellular calcium efflux in PC-3 cells induced by GRPR-targeted ligands. Cells were incubated with DPBS or 50 nM of Ga-complexed GRPR-targeted ligand, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), bombesin, or ATP. ............................................................ 109 Figure 4.6 Representative PET images of (A) [68Ga]Ga-LW01107, (B) [68Ga]Ga-LW01108, (C) [68Ga]Ga-LW01110, (D) [68Ga]Ga-LW02040, (E) [68Ga]Ga-LW02021 and (F) [68Ga]Ga-LW01142 in mice bearing PC-3 tumor xenografts. .................................................................... 110 Figure 4.7 Uptake of [68Ga]Ga-LW01142 at 1 and 3 h post-injection in PC-3 tumor-bearing mice. ............................................................................................................................................ 112 Figure 4.8 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01107 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. ...................................................................................................................................... 116 xxvii Figure 4.9 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01108 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. ...................................................................................................................................... 116 Figure 4.10 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01110 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. ...................................................................................................................................... 117 Figure 4.11 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01142 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. ...................................................................................................................................... 117 Figure 4.12 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW02040 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. ...................................................................................................................................... 118 Figure 4.13 Chemical structures of (A) Ga-TacsBOMB2, (B) Ga-LW01158, (C) Ga-LW01160, (D) Ga-LW01186, (E) Ga-LW02002, and (F) Ga-RM2. ............................................................ 125 Figure 4.14 Displacement curves of [125I-Tyr4]Bombesin by Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002 generated using GRPR-expressing PC-3 cells. ........................... 129 Figure 4.15 Intracellular calcium efflux in PC-3 cells induced by Ga-LW01158, Ga-LW01186, Ga-LW02002, bombesin, ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), ATP, and DPBS...................................................................................................................................................... 130 Figure 4.16 Representative PET images of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 acquired at 1 h post-injection in mice bearing PC-3 tumor xenografts. .... 131 Figure 4.17 Uptake of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 in PC-3 tumor xenografts and major organs\/tissues of NRG mice at 1 h post-injection. ..................... 133 xxviii Figure 4.18 Tumor-to-organ uptake ratios of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-02002 obtained from PC-3 tumor-bearing mice at 1 h post-injection. ....................... 134 Figure 4.19 Comparison of [68Ga]Ga-LW01158 with\/without co-injection of 100 \u00b5g of nonradioactive Ga-LW01158 on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. Error bars indicate standard deviation. ............................................. 134 Figure 4.20 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01158 in (A) mouse plasma and (B) urine samples collected at 15 min post-injection. ...................................................................................................................................... 135 Figure 4.21 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01186 in (A) mouse plasma and (B) urine samples collected at 15 min post-injection. ...................................................................................................................................... 136 Figure 4.22 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW02002 in (A) mouse plasma and (B) urine samples collected at 15 min post-injection. ...................................................................................................................................... 136 Figure 5.1 Chemical structures of (A) Lu-TacsBOMB5, (B) Lu-LW01110, (C) Lu-LW01142, (D) Lu-RM2, and (E) Lu-AMBA. .............................................................................................. 145 Figure 5.2 (A) Displacement curves of [125I-Tyr4]Bombesin by Lu-TacsBOMB5, Lu-LW01110, Lu-LW011142, Lu-RM2, and Lu-AMBA generated using GRPR-expressing PC-3 cells. (B) Comparison of the binding affinities of Lu-TacsBOMB5, Lu-LW01110, Lu-LW011142, Lu-RM2, and Lu-AMBA. ................................................................................................................. 148 Figure 5.3 Intracellular calcium efflux in PC-3 cells induced by Lu-TacsBOMB5, Lu-LW01110, Lu-LW011142, bombesin, ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), ATP, and DPBS...................................................................................................................................................... 149 xxix Figure 5.4 Longitudinal SPECT\/CT images of [177Lu]Lu-TacsBOMB5 acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. . 151 Figure 5.5 Longitudinal SPECT\/CT images of [177Lu]Lu-LW01110 acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. ............ 151 Figure 5.6 Longitudinal SPECT\/CT images of [177Lu]Lu-LW01142 acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. ............ 152 Figure 5.7 Longitudinal SPECT\/CT images of [177Lu]Lu-RM2 acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. ............ 152 Figure 5.8 Longitudinal SPECT\/CT images of [177Lu]Lu-AMBA acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. ............ 153 Figure 5.9 Comparison of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA on their uptake in (A) PC-3 tumor xenografts and (B) the pancreas in mice at 1, 4, 24, 72, and 120 h post-injection. ......................................................... 155 Figure 5.10 (A) Comparison of the radiation absorbed doses for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA in organs of interest in mice; (B) comparison of the estimated radiation absorbed doses for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA in organs of interest in adult human male. ..................................................................... 161 Figure 6.1 Chemical structures of previously reported TacsBOMB5, LW01110, and LW01142 and their Pro14 derivatives. .......................................................................................................... 171 Figure 6.2 Intracellular calcium release in PC-3 cells induced by Ga-ProBOMB5, Lu-ProBOMB5, Ga-LW02056, Ga-LW02057, bombesin, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), ATP, and DPBS. ................................................................................... 175 xxx Figure 6.3 Displacement curves of (A) Ga-ProBOMB5, Ga-LW02056, and Ga-LW02057, and (B) Lu-ProBOMB5 and Lu-RM2 generated using GRPR-expressing PC-3 cells and [125I-Tyr4]Bombesin as the radioligand. ............................................................................................. 176 Figure 6.4 The displacement curve of Ga-ProBOMB5 generated using murine Swiss 3T3 cells and [125I-Tyr4]Bombesin as the radioligand. ............................................................................... 176 Figure 6.5 Representative PET images of [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 acquired at 1 h post-injection in mice bearing PC-3 tumor xenografts. The blocked mouse was co-injected with 100 \u03bcg of nonradioactive Ga-ProBOMB5. ...................... 177 Figure 6.6 (A) Comparison of [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 on their uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. (B) Comparison of [68Ga]Ga-ProBOMB5 with\/without co-injection of the nonradioactive standard (100 \u00b5g) on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. ............................................................................... 179 Figure 6.7 Longitudinal SPECT\/CT images of (A) [177Lu]Lu-ProBOMB5 and (B) [177Lu]Lu-RM2 in PC-3 tumor-bearing NRG mice. .................................................................................... 181 Figure 6.8 Comparison of [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 on their uptake in (A) PC-3 tumor xenografts and (B) the pancreas in mice at 1, 4, 24, 72, and 120 h post-injection. (C) Comparison of [177Lu]Lu-ProBOMB5 with\/without co-injection of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection.. ................................................................................................................... 183 Figure 6.9 Comparison of [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 on the absorbed dose in PC-3 tumor xenografts and major organs\/tissues in mice. ......................................................... 186 xxxi Figure 6.10 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-ProBOMB5 in mouse plasma (top) and urine (bottom) samples collected at 15 min post-injection. .............................................................................................................................. 188 Figure 6.11 Representative radio-HPLC chromatograms from analysis of intact fraction of [177Lu]Lu-ProBOMB5 in mouse plasma (top) and urine (bottom) samples collected at 15 min post-injection. .............................................................................................................................. 189 Figure 6.12 Chemical structures of (A) SB3, (B) LW02075, and (C) LW02050. ...................... 200 Figure 6.13 Displacement curves of [125I-Tyr4]Bombesin by Ga-LW02075, Ga-LW02050, and Ga-SB3 generated using GRPR-expressing PC-3 cells. ............................................................. 203 Figure 6.14 Intracellular calcium mobilization in PC-3 cells induced by Ga-LW02075, Ga-LW02050, Bombesin, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), ATP, and DPBS. ..... 204 Figure 6.15 Representative PET images of [68Ga]Ga-SB3, [68Ga]Ga-LW02075, and [68Ga]Ga-LW02050 acquired at 1 h post-injection in mice bearing PC-3 tumor xenografts. .................... 205 Figure 6.16 Uptake of [68Ga]Ga-SB3, [68Ga]Ga-LW02075, and [68Ga]Ga-LW02050 in tumors and major organs\/tissues of PC-3 tumor-bearing mice at 1 h post-injection. ............................. 208 Figure 6.17 Comparison of [68Ga]Ga-LW02050 with\/without co-injection of its nonradioactive standard (100 \u03bcg) on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. ........................................................................................................................... 208 xxxii List of Symbols \u03b1 = alpha \u03b2+ = beta plus (positron of nuclear origin) \u03b2- = beta minus (electron of nuclear origin) \u03b3 = gamma \u00b5 = micro \u00b0C = degree Celsius \u2265 = greater than or equal to < = smaller than > = greater than \u00b1 = both plus and minus operations % = percentage xxxiii List of Abbreviations %ID\/g Percent injected dose per gram of tissue Ab Antibody ASTRO American Society for Radiation Oncology ATP Adenosine triphosphate AUA American Urological Association BBB Blood-brain barrier BBN Bombesin BCa Breast cancer BCF Bifunctional chelator BRCA1 BReast CAncer gene 1 BRCA2 BReast CAncer gene 2 CAF Cancer-associated fibroblasts CEA Carcinoembryonic antigen CT Computed tomography CXCR4 C-X-C chemokine receptor type 4 DAG Diacylglycerol DCM Dichloromethane DIEA N,N-diisopropylethylamine DMF Dimethylformamide DNMs DNA-based nanomachines DODT 2,2\u2032-(ethylenedioxy)diethanethiol DOTA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid DPBS Dulbecco's phosphate-buffered saline EC Extracellular EGFR Epidermal growth factor receptor EOS End of synthesis ER Estrogen receptor ERK Extracellular signal-regulated kinase ESI-MS Electrospray ionization mass spectrometry FA Formic acid FAP Fibroblast activation protein FDG Fludeoxyglucose FNAs Functional nucleic acids GABA Gamma-aminobutyric acid GBq Gigabecquerel GCP II Glutamate carboxypeptidase II GEP-NETs Gastroenteropancreatic neuroendocrine tumors GLP Good Laboratory Practice GPCR G-protein coupled receptor GRP Gastrin-releasing peptide GRPR Gastrin-releasing peptide receptor xxxiv HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate HEPES N-2-hydroxyethylpiperazine-N\u2019-2-ethanesulfonic acid HER2 Human epidermal growth factor receptor-2 HMPAO d, l-hexamehylpropyleneamine oxime HOAt 1-Hydroxy-7-azabenzotriazole HPLC High performance liquid chromatography IC Intracellular IC50 Half maximal inhibitory constant ICAM-1 Intercellular adhesion molecule 1 Ig Immunoglobulin IgGs Immunoglobulin type 1 Abs IND Investigational new drug IP3 Inositol 1,4,5-trisphosphate IT Isomeric transition JNK Jun N-terminal kinase LOR Line of response LPPS Liquid-phase peptide synthesis LTM Lower transmembrane regions mAbs Monoclonal antibodies MAPK Mitogen-activated protein kinase MBq Megabecquerel mCRPC Metastatic castration-resistant prostate cancer MeOH Methanol MPS Mononuclear phagocytic system MRI Magnetic resonance imaging MS Mass spectrometry N3S Dmgly-L-Ser-L-Cys(acm) NSCLC Non-small cell lung carcinoma NEP Neutral endopeptidase 24.11 NF-\u03baB Nuclear factor \u03baB NHL Non-Hodgkin lymphoma NMB Neuromedin B NMC Neuromedin C NMR Nuclear magnetic resonance spectroscopy NPY1R Neuropeptide Y receptor Y1 NRG mice NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl\/SzJ mice NSG mice NOD.Cg-Prkdc scid Il2rg tm1Wjl \/SzJ mice OAR Organ at risk PA Phosphoramidon pABzA-DIG p-aminomethylaniline-diglycolic acid PCa Prostate cancer PD-L1 Programmed death ligand-1 PEG Poly(ethylene glycol) xxxv PET Positron emission tomography Pip 4-amino-(1-carboxymethyl)piperidine PIP2 Phosphatidylinositol 4,5-bisphosphate PKC Protein kinase C PLC Phospholipase C PR Progesterone receptor PRRT Peptide receptor radionuclide therapy PSMA Prostate-specific membrane antigen RCY Radiochemical yield RES Reticuloendothelial system RFUs Relative fluorescent units RGS Regulators of G protein signaling ROCK Rho-associated protein kinase SAR Structure activity relationship SCLC Small cell lung cancer SLN Solid lipid nanoparticles SPECT Single photon emission computed tomography SPPS Solid-phase peptide synthesis SST Somatostatin SSTRs Somatostatin receptors TFA Trifluoroacetic acid Thz Thiazoline-4-carboxylic acid TIACs time-integrated activity coefficients TIS Triisopropylsilane TMs Transmembrane regions TPM Transcripts per million U.S. FDA United States Food and Drug Administration UTM Upper transmembrane regions VCAM-1 Vascular cell adhesion molecule-1 VIP-R1 Vasoactive intestinal polypeptide receptor 1 xxxvi Acknowledgements First and foremost, I would like to express my deepest gratitude to my supervisor, Prof. Dr. Kuo-Shyan Lin, for his invaluable guidance, encouragement, and support throughout this journey. Since our very first meeting, his warm smile and kind words have given me the confidence to embark on this life-changing chapter in a new country. I appreciated the great opportunity provided by him to pursue my graduate degree in such a great team. His expertise, patience, and passion for science have not only been pivotal to this thesis but have also profoundly influenced my growth as a researcher. I would also like to sincerely thank Prof. Dr. Fran\u00e7ois B\u00e9nard, who is also one member of my supervisory committee, for his insightful advice, continuous support, and valuable contributions to my research. The precious knowledge and experiences I gained from him during the weekly lab meetings, yearly supervisory meetings, and my comprehensive exam has been crucial to the completion of this work and my career development. I am also grateful to the other two members of my supervisory committee, Prof. Dr. David M. Perrin and Prof. Dr. Arman Rahmim, for their insightful feedback, constructive criticism, and unwavering support throughout the process, particularly on chemistry and physics. All the warm suggestions and advice provided by both Dr. Perrin and Dr. Rahmim has been invaluable to me. I would like to extend my heartfelt thanks to my colleagues and fellow students in Lin lab, Benard lab, and Lau lab. Their collaboration and friendship made the challenging moments more manageable and the successes more enjoyable. Special thanks to Dr. Zhengxing (Johnson) Zhang, Dr. Chengcheng Zhang, Dr. Hsiou-Ting Kuo, Dr. Chao-Cheng Chen, Dr. Devon Chapel, Dr. Joseph Lau, Helen Merkens, Dr. Jutta Zeisler, Nadine Colpo, Dr. Sara Kurkowska, Dr. Carlos Uribe, Ruiyan Tan, Ryan Wilson, Wing Sum Lau, Pauline Ng, Dr. Aron Roxin, Antonio Wong, xxxvii Shireen Jozi, Aaron Ma, and Dr. Jinhe Pan, for their invaluable help with experiments and for always being there when I needed advice. To Arsyangela Verena, Dr. Bryan Fraser, Feiyuan (Kelly) Lu, Dr. Ingrid Bloise, Itzel Astiazar\u00e1n Rasc\u00f3n, Dr. Julie Rousseau, Dr. Lily Li, Dr. Lupe Jaraquemada-Pelaez, Sabrina Skyba-Lewin, Dr. Sara Harsini, Dr. Shreya Bendre, Dr. Xinchi Hou, your unwavering support, encouragement, and sense of humor have been a source of strength throughout this journey. Thank you for always being there for me, both in and outside of academia, and for making this experience so much more enjoyable. Finally, I would like to thank Canadian Institutes of Health Research, BC Cancer Research Centre, Alpha-9 Oncology Inc, and China Scholarship Council for providing the financial support that made this research possible. To everyone who contributed in one way or another to this thesis, your support has meant more to me than words can express. Thank you! xxxviii Dedication To my parents, Fang Mi and Chaoyi Wang, my beloved sister, Pengpeng Wang, and my partner, Tianhaozhe Sun\u2014thank you for your endless love, unwavering support, patience, and the countless sacrifices you\u2019ve made. I am forever grateful.1 Chapter 1: Introduction 1.1 Nuclear Medicine Nuclear medicine which integrates principles of physics, chemistry, and biology, is a medical specialty that utilizes radiopharmaceuticals to diagnose, manage, and treat disease, and is widely used in cardiology, neurology, to oncology departments 1. With the development of advanced technologies and novel radiopharmaceuticals, nuclear medicine now plays an increasingly critical role in personalized medicine, offering innovative solutions that enhance patient outcomes. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are two primary imaging modalities used in nuclear medicine for revealing physiological processes, including metabolism, hypoxia, apoptosis, and proliferation and giving people better understanding of metabolic and functional disorders 2, 3. Nowadays, SPECT and PET are typically fused with other anatomical imaging methods, such as computed tomography (CT) or magnetic resonance imaging (MRI), to achieve acquisition with both functional and anatomical information for precise localization of lesions and better disease management 4-6. As illustrated in Table 1.1, various radioisotopes are utilized for different purposes in nuclear medicine. Additionally, more novel isotopes are discovered for better imaging or therapeutic application, such as the promising potent theranostic pairs 155Tb\/161Tb and 203Pb\/212Pb 7, 8. Table 1.1 Radioisotopes commonly utilized in nuclear medicine. This table was adapted from Saha, G.B. (2004) 1 and Conti, M., et al.9. Radioisotopes used for SPECT imaging Isotope Half-life Decay types (%) E\u03b3 max (MeV) (%) 67Ga 3.3 d EC (100) 0.393 (5) 99mTc 6.0 h IT (100) 0.140 (90) 111In 2.8 d EC (100) 0.245 (94) 123I 13.2 h EC (100) 0.159 (83) Radioisotopes used for PET imaging 2 Isotope Half-life Decay types (%) E\u03b2+ max (MeV) (%) 11C 20.4 min \u03b2+ (100) 0.960 (100) 13N 10 min \u03b2+ (100) 1.199 (100) 15O 2 min \u03b2+ (100) 1.732 (100) 18F 110 min \u03b2+ (97) EC (3) 0.634 (97) 68Ga 68 min \u03b2+ (89) EC (11) 1.899 (88) 89Zr 3.3 d \u03b2+ (23) EC (77) 0.902 (23) \u03b2--particle emitting isotopes used for radiotherapy Isotope Half-life Decay types (%) E\u03b2- max (MeV) (%) 67Cu 2.6 d \u03b2- (100) 0.562 (20) 131I 8.0 d \u03b2- (100) 0.606 (90) 177Lu 6.7 d \u03b2- (100) 0.498 (80) \u03b1-particle emitting isotopes used for radiotherapy Isotope Half-life Decay types (%) E\u03b1 max (MeV) (%) 225Ac 9.9 d \u03b1 (100) 5.830 (51) *IT: isomeric transition; EC: electron capture. 1.1.1 Single Photon Emission Computed Tomography Single photon emission computed tomography (SPECT) is based on the detection of gamma photons (\u03b3) emitted by radioactive source. SPECT system primarily consists of a gamma camera with one to three NaI(Tl) scintillation detectors heads mounted on a gantry and an on-line computer for data acquisition and processing 1. The SPECT imaging relies on the use of radiopharmaceuticals with gamma-emitting radionuclides that are introduced into the patient\u2019s body, typically by intravenous injection. The radiopharmaceuticals then distribute in the body and emits \u03b3 rays as the radionuclides decay. Collimators, which are lead or tungsten plates with holes and placed in front of the scintillation crystal to allow the \u03b3 rays to pass through while blocking scattered \u03b3 photons to ensure the spatial distribution of the radiotracer is representative, play a key role in SPECT image quality (Figure 1.1A). The \u03b3 photons loss caused by collimators, on the other hand, results in a reduction of the instrument\u2019s sensitivity and spatial resolution 10, 11. Depending on the type of focusing, collimators are classified as different types. The most commonly utilized 3 collimators are parallel hole collimator, pinhole collimator, diverging collimator, and converging collimator 1. The collimated \u03b3 rays are then detected by the NaI(Tl) detectors rotating around the patient to capture the \u03b3 rays from multiple angles and converted the signal into light photons with respective energy levels. The images are reconstructed to represent the tracer\u2019s distribution and provide functional information about the target tissues. Figure 1.1 Schematic representation of SPECT and PET detection. (A) Linear SPECT detector with collimators; dotted arrows = photons that are absorbed by the collimators, solid arrows = photons that reach the detector. (B) Circular ring of PET detectors detects photons arriving simultaneously in opposite directions. This figure was distributed under the Creative Commons Attribution 4.0 International License (CC-BY license) and adapted from Khan, A.A. and de Rosales, R.T. (2021) 10. 1.1.2 Positron Emission Tomography Positron emission tomography (PET) relies on the detection of the coincidence of two 511 keV photons that are emitted during the annihilation of a positron (\u03b2+) from a positron emitting radionuclide (Figure 1.1B). During the positron decay, a proton inside the PET radionucleus decays into a neutron and a positron which is ejected out of the nucleus and encounter an electron after a short distance travel. The electron and positron annihilate and emit two 511-keV \u03b3 photons 4 going nearly opposite directions from each other (Figure 1.1B) 10. Different from the mounting detectors of SPECT, the scintillation crystal detectors of the PET system are surrounding the patient and detecting all the coincident 511-keV \u03b3 photons emitted from the annihilation without the use of collimator. Thus, PET system can detect more decay events without any reduction of the \u03b3 photons due to collimators, which leads to a higher sensitivity compared with SPECT 1, 10, 11. Detectors then register these photons, and by determining the location and angle of the emission, the scanner can reconstruct the biodistribution of the radiopharmaceuticals to reveal functional information of patients. Figure 1.2 (A) Graphic representation of true (a), scatter (b), and random events (c). (B) Diagram illustrates noncollinearity effect in coincidence imaging. This figure was originally published in JNM. Gabriele Tarantola, Felicia Zito and Paolo Gerundini. PET Instrumentation and Reconstruction Algorithms in Whole-Body Applications. J Nucl Med. 2003, 44 (5) 756-769. \u00a9 SNMMI. 12 Despite the higher sensitivity compared to SPECT, PET also has its own limitations. As mentioned earlier that the PET system assigns to coincidence events a line of response (LOR) for detection of the coincident 511-keV \u03b3 photons emitted from the annihilation as shown by \u201ca\u201d in Figure 1.2A. However, some emitted events interact in the body, followed by being absorbed or A. B.bac5 deflected by a certain angle before detection, which result in scatter events (\u201cb\u201d in Figure 1.2A) and cause error or noise at the final images 12-14. Additionally, random coincidence events caused by the detection of unrelated annihilation photons within the coincidence time window will superimpose a low-frequency noise on true events (\u201cc\u201d in Figure 1.2A) 12-14. Moreover, as the physical state of the atoms interacting with positrons, non-collinearity of annihilation photons happens and leads to deviations from 180\u00b0 between the trajectories of the two emitted photons as shown in Figure 1.2B. The non-zero net momentum for an emitted positron and the electron with which it annihilates causes a small relative angle (at maximum, \u00b1 0.25\u00b0) of the emission of the two 511-keV photons, which leading to a small variation around the correct direction of the detected LOR 11, 13, 14. 1.2 Radiopharmaceuticals A radiopharmaceutical, which may be a radioactive element such as 133Xe or a radioactive labeled compound such as 99mTc-labeled compound, used for the diagnosis and therapeutic treatment of human diseases, including cardiological diseases, neurological diseases, and cancers 1. A radiopharmaceutical consists of a radionuclide and a pharmaceutical which is chosen based on its preferential biodistribution in organs\/tissues or its participation in the physiologic function of the organs\/tissues. This dissertation is focused on the radiopharmaceuticals used in cancer imaging and therapy. Various of biomolecules have been used in the development of radiopharmaceuticals to target different receptors expressed on the surface of cancer cells. Each class of biomolecules has distinct biological properties and matches with the physical properties of the radionuclide for optimal performance. Based on the type of the biomolecules, radiopharmaceuticals are mainly categorized as peptide-based, antibody-based, small molecule-6 based, and other subtypes including nanoparticle-based radiopharmaceuticals and nucleic acids-based radiopharmaceuticals 15-17. 1.2.1 Antibody-based Radiopharmaceuticals Antibody (Ab) was the first biological carrier utilized in radiopharmaceuticals 18. Because of the high affinity and specificity toward the target antigens overexpressed on tumor cells, antibodies are considered excellent agents for the development of radiopharmaceuticals, particularly monoclonal antibodies (mAbs) and their derivatives 19. 1.2.1.1 Principle of Antibody-based Radiopharmaceuticals Antibody, also known as an immunoglobulin (Ig), is a distorted Y-shaped protein of which the molecular weight is 140-160 kDa. The two Fab fragments (arms) contain the identical antigen binding sites at their tips and connect to the Fc fragment (stem) with flexible hinges (Figure 1.3). The Fab region recognises specific antigen, while the Fc region mediates the immune response by interacting with effector cells 20. Radiolabeled Abs are designed to target specific antigens expressed on the surface of cancer cells and deliver the radiation to the tumor site by conjugating the antibodies with specific radioisotopes. 7 Figure 1.3 Random and site-specific immuno-PET\/SPECT bioconjugation sites of mAbs. This figure was distributed under the Creative Commons Attribution 4.0 International License (CC-BY license) and adapted from Dewulf J. et al. (2020) 21. The Ab radiolabeling strategies are developed based on the nature of the radioisotope and the Ab. For the radiolabeling of radiometals such as 89Zr and 64Cu, a bifunctional chelating agent is needed, while for non-metallic radionuclides such as 123I, the radiolabeling is usually performed by direct electrophilic aromatic substitution on histidine or tyrosine residues (Figure 1.3). Immunoglobulin type 1 Abs (IgGs) are the most commonly utilized Abs for targeted cancer therapy and cancer immunotherapy among five classes of Abs. The slow tissue distribution and long circulation time (t1\/2 = 1-3 weeks) make full-length IgG mAbs desirable for the development of therapeutic tumor targeting, and contribute to the high exposure and accumulation of the Abs at the target site 22. Thus, long-lived radionuclides (e.g., 89Zr, 124I\/123I, and 131I) are preferred for full-length Abs, particularly for late timepoint imaging and therapeutic applications 23-25. Smaller antibody fragments derived from full-length IgG Abs, such as antigen-binding fragments (Fab, 8 (Fab\u2019)2, Fab\u2019, and scFv), have also been used for the development of cancer diagnostic applications at earlier time points (1-12 h) owing to their shorter half-life compared with full-size Abs 26, 27. Hence, short-lived radionuclides are more suited, such as 64Cu 28, 29. Besides, the coupling of radiolabel\/chelator to various cysteines and lysines in Ab may generate different conjugates and give rise to a reduction of binding affinity if the coupling occurs at regions required for antigen recognition. Some site-specific modifications. including click chemistry, enzymatic reactions, and biorthogonal transformations, are performed on the cysteines and lysines located outside of antigen-recognition regions to prevent loss of binding affinity 21. 1.2.1.2 Clinical Applications Currently, several antibody-based radiopharmaceuticals have been approved by the United States Food and Drug Administration (U.S. FDA) and dozens of novel antibody-based radiopharmaceuticals are in preclinical and clinical trials. There are two U.S. FDA approved diagnostic tracers, 111In-satumomab pendetide (111In-OncoScint CR103) and 99mTc-Arcitumomab (99mTc-IMMU-4). 111In-satumomab pendetide was the first U.S. FDA approved monoclonal antibody-based radiopharmaceutical for tumor imaging, including colon and ovarian cancer. The target of 111In-satumomab pendetide is TAG-72 which is a tumor-associated antigen and expressed by many adenocarcinomas. 111In-satumomab pendetide is reactive with most colorectal and ovarian cancers, as well as other cancers 30. 99mTc-Arcitumomab (99mTc-IMMU-4), approved by U.S. FDA in 1999, is made by conjugating a murine anti-carcinoembryonic antigen (CEA) monoclonal antibody Fab\u00b4 fragment with 99mTc. However, it is no longer marketed in the United States for colorectal cancer screening or management 31. There are also two U.S. FDA approved therapeutic antibody-based radiopharmaceuticals, 90Y-ibritumomab tiuxetan (Zevalin) and 131I-9 tositumomab (Bexxar). Both of them are radiolabeled anti-CD20 murine mAbs for the treatment of relapsed or refractory, low-grade or follicular B cell Non-Hodgkin lymphoma (NHL) 32, 33. 1.2.1.3 Advantages and Limitations Because of the great specificity, excellent affinity, relatively extended circulation time, and the ability to activate mAb-mediated cell killing, antibody-based radiopharmaceuticals are very promising agents for cancer diagnosis and radiotherapy, particularly for those tumors with low receptor expression 19, 34, 35. Moreover, in comparison with the synthesis of the cancer-specific mAbs\u2019 small molecule counterparts, the process of generating mABs is relatively straightforward 36. However, full-length mAbs have some disadvantages due to their large size, including slow tissue penetration and clearance, low diffusivity within solid tumors, and potential off-target effects 37. As a result of slow pharmacokinetics, the use of long-lived radionuclides is required which leads to a higher radiation burden for the patient (20-40 mSv per scan) 34. To solve this issue, smaller sized antibody fragments derived from full-length mAbs were developed. Although the pharmacokinetics of antibody fragments are improved, a reduction of the in vivo stability and more nonspecific accumulation in healthy tissues were observed 27. Thus, further investments are still needed for the development of novel antibody-based radiopharmaceuticals. 1.2.2 Peptide-based Radiopharmaceuticals Peptides are short chains of amino acids that can bind specifically to receptors overexpressed in certain pathological conditions, particularly in cancer cells. The overexpression of some peptide receptors in numerous cancers and relatively low expression in physiological organs\/tissue make these peptide receptors promising targets for cancer imaging and targeted radionuclide therapy, such as somatostatin receptors (SSTRs) and GRPRs 38. Thus, peptides have become one of the widely used biological carriers for developing radiopharmaceuticals for cancer 10 diagnosis and targeted radiotherapy due to ease of synthesis and radiolabeling and the desirable pharmacokinetic characteristics, such as high binding affinity to receptors and rapid clearance from non-target tissue 39, 40. 1.2.2.1 Principle of Peptide-based Radiopharmaceuticals As shown in Figure 1.4, after labeling with a radioisotope, the resulting radiolabeled peptide (P) will be administered to the patient, typically through intravenous injection, and then distributed throughout the body. During the distribution, the radiolabeled peptide will bind to the corresponding peptide receptor (P-R) overexpressed on the surface of the cancer cells and accumulate in the tumor and enable imaging or delivering therapeutic radiation. Depending on the agonist\/antagonist property of the targeting peptide, the resulting radiopharmaceuticals will be accumulated on the surface or internalized with the receptor into the cancer cells (arrows) 38. Figure 1.4 Principle of in vivo peptide receptor targeting of cancer. P: radiolabeled peptide; P-R: peptide receptor. This figure was adapted from Reubi, JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev, Volume 24, Issue 4, 1 August 2003, Pages 389\u2013427 38; permission conveyed through Copyright Clearance Center, Inc. 11 There are two main methods for synthesizing peptides: liquid-phase peptide synthesis (LPPS) and solid-phase peptide synthesis (SPPS) 41. As LPPS technique is relatively more complicated, time consuming, and skill intensive, SPPS method is currently widely used in the synthesis of most peptide-based radiopharmaceuticals, including Fmoc SPPS, and Boc SPPS 42, 43. The peptides are synthesized by coupling the amino acids sequentially to an insoluble resin, which serves as a solid support, with fewer purification steps and minimal optimization of the reaction conditions. Additionally, a bifunctional chelator (BFC) and a spacer group can be attached into the peptide sequence during synthesis. SPPS provides high yield and is suitable for small to medium scale synthesis, which is good for the drug development 41. 1.2.2.2 Clinical Applications Some peptide-based radiopharmaceuticals have been approved by U.S. FDA for cancer imaging and peptide receptor radionuclide therapy (PRRT). Both first U.S. FDA approved peptide-based diagnostic radiopharmaceutical and therapeutic radiopharmaceutical are somatostatin (SST) analogs. [111In]In-pentetreotide (Octreoscan) was approved by the U.S. FDA in 1994 as the first radiopharmaceutical for imaging of SSTR-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs) 44. Meanwhile, [177Lu]Lu-DOTATATE (Lutathera) is the first radiopharmaceutical for PRRT approved by U.S. FDA for the treatment of SSTR-positive GEP-NETs 45. More and more peptide-based radiopharmaceuticals were developed, evaluated with preclinical\/clinical trials, and translated into clinic 46-52. Table 1.2 Examples of peptide-based radiopharmaceuticals utilized in cancer imaging and therapy. This table was adapted from Zhang, T., et al.53. Target Radiopharmaceuticals Cancer Phase Ref. Diagnosis SSTR [111In]In-pentetreotide [68Ga]Ga-DOTA-TOC [68Ga]Ga-DOTA-TATE NETs GEP-NETs Approved 44, 54, 55 12 GRPR [68Ga]Ga-RM2 Prostate cancer Breast cancer Clinical 47 NCT02559115 Therapy SSTR [177Lu]Lu-DOTA-TATE GEP-NETs Approved 45 GRPR [177Lu]Lu-RM2 [177Lu]Lu-AMBA Prostate cancer Clinical 46, 48 CXCR4 [177Lu]Lu-Pentixather [90Y]Y-Pentixather Multiple myeloma Clinical 49 NK-1R [90Y]Y-DOTAGA-SP [213Bi]Bi-DOTA-SP [225Ac]Ac-DOTA-SP Glioma Clinical 50-52 1.2.2.3 Advantages and Limitations Peptide-based radiopharmaceuticals are advantageous due to their small molecules, high specificity, rapid blood clearance, excellent permeability, low immunogenicity, and low toxicity 38, 56. Due to the fast pharmacokinetics of peptides, radioisotopes with short half-life are preferred to match the biological half-life of peptides for the development of diagnostic radiopharmaceuticals, such as 68Ga and 18F. Furthermore, peptide-based radiopharmaceuticals are easy to synthesize and modify chemically, and easy to be radiolabelled 38. However, the natural amino acid sequence of peptides can be rapidly degraded by peptidases due to the cleavage of peptide bonds, leading to the quick renal excretion and limiting their therapeutic efficacy. Peptidases are expressed on many normal organs and tissues, and usually have very defined specificities. Exopeptidases degrade peptides from either the C- or N-terminus, while endopeptidases cleave the specific bonds between the natural L-amino acids. Thus, unnatural amino acids substitution, such as introducing D-amino acids, and modification of the terminal amino acids, such as end-capping, are widely used by researchers to improve the metabolic stability of peptide-based radiopharmaceuticals 57. Moreover, some well-defined novel peptide modifications have been developed for improving in vivo stability, such as heterodimers and cyclic peptides 58, 59. With considered efforts, some peptide-based radiopharmaceuticals were developed 13 showing greatly improved metabolic stability compared with their natural endogenous peptide and even translated into clinic, such as [177Lu]Lu-DOTA-TATE (Lutathera) as the first radiopharmaceutical for PRRT approved by U.S. FDA for SSTR-expressing GEP-NETs 45. 1.2.3 Small Molecule-based Radiopharmaceuticals Small molecules are typically less than 500-900 Da in size and can interact with molecular targets such as enzymes, transporters, or receptors 60, 61. Their small size enables rapid tissue penetration, making them ideal carriers in nuclear medicine for both diagnosis and therapy. Although a lot of novel radiopharmaceuticals have been developed during the past decades, including antibody-based, peptide-based, nanoparticle-based, and nucleic acids-based tracers, small molecules are still playing an important role in the radiopharmaceutical development and marketplaces. 1.2.3.1 Principle and Clinical Applications Multi-step organic synthesis method and solid-phase synthesis method are needed for small molecule-based radiopharmaceuticals. Like other radiopharmaceuticals, the radiolabeled small molecules distribute in patient\u2019s body and localize to their targets for cancer imaging or therapy. The small molecules can be designed to target metabolic processes, hypoxic regions, or specific oncogenic pathways in tumors. Fludeoxyglucose F18 ([18F]FDG), a glucose analog developed in the 1970s and approved by the U.S. FDA in the 1990s, is still the most widely used PET tracer to diagnose and monitor various conditions, such as neurological, oncological, cardiological, and inflammatory conditions 62-64. Cancer cells are generally having higher glucose metabolism compared to normal cells because of the increasing activity of glucose transporters, higher phosphorylation activity, and reducing phosphatase activity. Thus, [18F]FDG will accumulate in the cancer cells with high 14 glucose metabolism. However, the glucose metabolism of cancer is variable due to the type, stage, and location, which may give rise to increased or decreased [18F]FDG uptake. Additionally, inflammatory cells also have high glucose metabolism and may be shown as a positive observation under PET imaging 65, 66. Besides glucose, hypoxia is another potential target for detecting malignancies, as reduced levels of oxygen (hypoxia) are found in many malignancies but rarely seen in normal tissues or organs. Moreover, hypoxia plays a key role in radioresistance due to reduced oxygen-mediated fixation of DNA damage and hypoxia induced factor 1\u03b1 (HIF1\u03b1)-mediated cell survival 67, 68. [18F]Fluoromisonidazole ([18F]FMISO), an investigational new drug (IND) exempt from the FDA, is the most commonly studied PET imaging agent for hypoxia 69, 70. In hypoxic tumor cells, the nitro group on the imidazole ring undergoes electron reduction to form reactive radicals. These radicals then further reduce forming covalent bonds with the biomolecules such as glutathione 71. Small molecule inhibitors were also developed to serve as alternatives to mAb for binding to specific antigens overexpressing in cancer cells, such as prostate-specific membrane antigen (PSMA). During the past decades, an increasing number of novel small molecule-based radiopharmaceuticals have been developed by mimicking PSMA substrates, \u03b3-glutamyl folic acid derivatives and the neuropeptide N-acetylaspartylglutamate 72. Some PSMA-targeted small molecule-based radiopharmaceuticals have been approved by U.S. FDA, including [18F]DCFPyL and [68Ga]Ga-PSMA-1173-75. In addition, [177Lu]Lu-PSMA-617 was approved by the U.S. FDA in 2022 for the treatment of PSMA-positive metastatic castration-resistant prostate cancer (mCRPC) after androgen receptor pathway inhibition and taxane-based chemotherapy 76. The development of novel small molecule-based radiopharmaceuticals targeting fibroblast activation protein (FAP) expressed by cancer-associated fibroblasts (CAFs) for cancer imaging 15 and therapy is rapidly evolving 77. [68Ga]Ga-FAPI-04, a quinoline-based FAP-targeting tracer, was verified to show faster pharmacokinetics, better tumor uptake, and higher tumor-to-background contrast ratios compared to [18F]FDG in clinical trials 78, 79. 1.2.3.2 Advantages and Limitations In contrast to larger molecules, such as peptide-based radiopharmaceuticals and antibody-based radiopharmaceuticals, small molecule-based radiopharmaceuticals are more likely to pass through the blood-brain barrier (BBB) and can be used for neurological studies and brain tumor imaging 80. Moreover, the rapid tissue penetration of small molecule-based radiopharmaceuticals results in reducing toxicity compared to other large sized radiopharmaceuticals 81. However, non-specific binding and rapid clearance can generate background noise which makes it hard to achieve high target-to-background ratios. Moreover, the multi-step organic synthesis is needed for some small molecules, which is more complicated and time consuming compared with other synthetic procedures, such as the SPPS for peptide-based radiopharmaceuticals. 1.2.4 Nanoparticle-based and Nucleic Acids-based Radiopharmaceuticals Beyond the three most commonly utilized radiopharmaceutical categories, newer classes of radiopharmaceuticals are emerging nowadays, including nanoparticle-based and nucleic acid-based radiopharmaceuticals. These novel approaches aim to improve the precision, safety, and efficacy of radiopharmaceuticals by exploiting the unique properties of these biomolecules. Nanoparticles are particles smaller than 1 \u00b5m, typically made from either biodegradable or non-biodegradable polymers, or from lipids, such as solid lipid nanoparticles (SLN). Due to the large surface-to-volume ratios, great surface functionalization possibilities, and the large capacity, nanoparticles are extensively used for biomedical applications 82. However, those nanoparticles over 5-10 nm show high uptake in liver and kidney due to the high levels of accumulation in 16 mononuclear phagocytic system (MPS, before also known as the reticuloendothelial system, RES), which limits their clinical applications. To solve this issue, hydrophilic poly(ethylene glycol) (PEG) chains were introduced to the surface of the nanoparticles to reduce the MPS capture 83. Almeida group reported a 99mTc-labled SLN (200 nm) with a lipophilic chelator d,l-hexamehylpropyleneamine oxime (HMPAO) showing significant accumulation in the lymphatics after inhalation 84. To improve nanoparticle\u2019s specificity to tumor, some traditional targeting biomolecules are introduced to the surface of nanoparticles, such as peptides and antibodies 85. A novel antiangiogenesis therapy using an anti-Flk-1 antibody coated 90Y-labeled nanoparticles was reported by Knox group showing the potential of being a novel therapeutic agent for the treatment of a variety of tumor types 86. Functional nucleic acids (FNAs), such as aptamers, DNAzymes, and DNA-based nanomachines (DNMs), are functional oligonucleotides playing a key role in cancer diagnosis and therapy 17, 87. With high specificity, good binding affinity, and intermediated size (8-15 kDa) compared with other ligands, FNAs are also promising vectors for the design of radiopharmaceuticals 88. Schmidt group developed a 99mTc-labeled RNA aptamer (TTA1) to target the extracellular matrix protein tenascin-C which is overexpressed in several solid tumors and evaluated it in glioblastoma (U251) and breast cancer (MDA-MB-435) tumor-bearing mice 89. Rapid uptake by tumors and fast clearance from normal organs\/tissues were observed for 99mTc-labeled TTA1 in both U251 and MDA-MB-435 mouse models, suggesting its potential in cancer imaging and therapeutic applications 89. 1.3 Gastrin-releasing peptide receptor Gastrin-releasing peptide receptor (GRPR), also known as BB2, belongs to the mammalian bombesin (BBN) receptor family together with other two receptors: the neuromedin B (NMB) 17 receptor (BB1) and the orphan receptor bombesin receptor subtype 3 (BRS-3, BB3) 90. GRPR is a G-protein coupled receptor (GPCR) and widely distributed in human body. GRPR plays a critical role in various physiological processes, including the regulation of gastrointestinal motility, the release of hormones, and the stimulation of cell proliferation 90-92. Furthermore, overexpression of GRPR has been observed in various solid malignancies, most notably prostate cancer, breast cancer, and small-cell lung cancer, making it an attractive target for both diagnostic and therapeutic applications in oncology 93-96. 1.3.1 Molecular Basis of GRPR and Ligands 1.3.1.1 Structure of GRPR The GRPR, a glycosylated seven-transmembrane GPCR with 384 amino acids, is expressed on gastric, respiratory, endocrine, muscle and nervous systems 97. Structurally, GRPR consists of seven transmembrane helices, with an extracellular ligand-binding domain and an intracellular domain responsible for signal transduction (Figure 1.5). It has been reported that the human GRPR (Figure 1.5B) shows high homology (90% amino acid identities) with the murine GRPR (Figure 1.5A) 90, 98, 99. Thus, both human PC-3 prostate adenocarcinoma cells and murine Swiss 3T3 fibroblast cells have been widely used by researchers for the evaluation of GRPR-targeted ligands in the past decades. 18 Figure 1.5 (A) Schematic representation of the murine GRPR showing the postulated transmembrane topology, sites for NH2-linked glycosylation (Y-like symbols), possible palmitoylated cysteines in the cytoplasmic tail, and the key amino acids for high-affinity GRP interaction or signaling (dark circles) or interaction with GRPR-targeted ligands (shaded circles). This figure was originally published in Pharmacological reviews by Jensen, R.T., Battey, J.F., Spindel, E.R., and Benya, R.V. 90. The permission conveyed through Copyright Clearance Center, Inc. (B) Transmembrane hydropathy plot of the human GRPR showing the extracellular side above, and the cytoplasmic side below, with the three glycosylation sites (Y-like symbols), and the key amino acids positions in human GRPR for determining affinity for GRPR-targeted ligand (dark circles). EC: extracellular; IC: intracellular; UTM: upper transmembrane regions; LTM: lower transmembrane regions. This figure was originally published in Biochemical pharmacology by Uehara, H., Hocart, S.J., Gonz\u00e1lez, N., Mantey, S.A., Nakagawa, T., Katsuno, T., Coy, D.H. and Jensen, R.T 99. Copyright \u00a9 2024 Elsevier B.V. and permission conveyed through Copyright Clearance Center, Inc. As shown in Figure 1.5A, four potential glycosylation sites of murine GRPR were determined by Benya, R.V. and colleagues by converting Asn (N) to Gln (Q)100. An inhibition of GRPR cell surface expression was observed when Asn24 or Asn191 were changed by site-directed mutagenesis, and the deglycosylation at Asn191 was speculated responsible for the G protein coupling. Furthermore, the elimination of all three NH2-terminal sites (N5, 20, 24) markedly attenuated the chronic desensitization and down-regulation. Thus, the four potential glycosylation Y YYA. B.19 sites were considered playing an important role in the trafficking of GRPR to the cell surface, ligand binding, G protein coupling, chronic desensitization, and down-regulation 100. In contrast, three potential glycosylation sites of human GRPR were reported by the Jensen group as shown in Figure 1.5B 99. The higher molecular mass of the murine GRPR (82 \u00b1 2 kDa) compared to the human GRPR (60 \u00b1 1 kDa) may be due to the additional glycosylated asparagine (N) site (Figure 1.5). In the extracellular (EC) loops and upper transmembrane regions (UTM), some key amino acids for high-affinity GRP interaction or signaling were determined by using different GRPR-targeted ligands and shown in dark circles or shade circles in Figure 1.5 101-107. Tokita, K., et al. reported a GRPR antagonist, JMV594 ([D-Phe6,Sta13]Bombesin(6-14)), and its pseudopeptide analog, JMV641 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu\u03c8(CHOH-CH2)-(CH2)2-CH3), showing potent binding affinities toward GRPR (2.2 \u00b1 0.1nM and 0.46 \u00b1 0.03 nM, respectively) 108. Their work suggested that Thr297, Phe302, and Ser305 in the fourth EC domain of murine GRPR are critical for the GRPR selectivity. Furthermore, their receptor modeling results demonstrated that all Thr297, Phe302, and Ser305 faced inward within 5 \u212b of the putative binding pocket, suggesting their important roles in ligand binding 108. The Jensen group reported a BBN analog, Univ.Lig [D-Tyr6,\u03b2-Ala11,Phe13,Nle14]Bombesin(6-14) with high affinity to GRPR 99. Their research indicated that the positively charged amino acids in EC-domains, particularly in EC-domain 4, attribute to the high affinity for Univ.Lig. Meanwhile, Asp97, Ile283, Tyr284, Arg287 were confirmed playing an important role in both charge-charge interactions and interaction with a tyrosine residue, and further contributing to the high binding affinity (Figure 1.5B) 99. In addition, other studies suggested that Gln120, Pro198, Arg287 and Ala307 are essential for GRPR binding with GRPR agonists, while Thr296, Phe301 and Ser304 are essential for binding with GRPR antagonists 101, 108. 20 For the intracellular (IC) domain, both human GRPR and murine GRPR consist of a DRY (Asp-Arg-Tyr) sequence in the second intracellular loop which is considered playing a key role in binding of the heterotrimeric G protein in most GPCRs. Meanwhile, multiple serine (S) and threonine (T) residues were found in the intracellular tail and were reported to be able to bind the protein kinase C (PKC) and responsible for the down-regulation of the receptor 90, 99, 109. 1.3.1.2 Gastrin-releasing Peptide and Bombesin Bombesin (BBN) is a biologically active 14-amino acids peptide (Table 1.3) isolated by Erspamer, V. and colleagues from the skin of the European fire-bellied toad Bombina bombina in 1970s 110, 111. The 27-amino acids peptide gastrin-releasing peptide (GRP, Table 1.3), the corresponding mammalian counterpart, was isolated from porcine non-antral gastric and intestinal tissue in 1979 112. GRP was later shown to have a growth stimulatory effect on various tissues as a neuroendocrine peptide 113, 114. The decapeptide of GRP (Table 1.3), neuromedin C (NMC, also known as GRP-10 or GRP18\u201327), was later isolated from porcine spinal cord 115. All three natural ligands share the same seven carboxyl-terminal amino acids which is considered a conserved domain crucial for the biological activity (Table 1.3) 90. Table 1.3 Amino acid sequences of BBN, GRP, and NMC share the same seven carboxyl-terminal amino acids (in blue). Name Sequence BBN pGlu1-Gly2-Arg3-Leu4-Gly5-Thr6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Met14-NH2 GRP H-Ala1-Pro2-Val3-Ser4-Val5-Gly6-Gly7-Gly8-Thr9-Val10-Leu11-Thr12-Lys13-Met14-Tyr15-Pro16-Arg17-Gly18-Asn19-His20-Trp21-Ala22-Val23-Gly24-His25-Leu26-Met27-NH2 NMC H-Gly18-Asn19-His20-Trp21-Ala22-Val23-Gly24-His25-Leu26-Met27-NH2 21 Figure 1.6 Peptide agonists binding to GRPR. (A) Cross-section of the peptide-binding pocket of GRPR was shown as forest green surface, with GRP as pink ribbons and side chains of W21G, V23G, and M27G displayed as pink sticks. (B and C) Structural alignment of GRP (green) and [D-Phe6,\u03b2-Ala11,Phe13,Nle14]Bombesin(6-14) (gray), with enlarged views of detailed interactions at N19G and F1B. (D) The TM bottom pocket. (E) The hydrophobic cavities around the helical fragment of GRP. (F) The EC loops. This figure was distributed under the Creative Commons Attribution 4.0 International License (CC-BY license) and adapted from Peng, S. et al. (2023) 116. Structure activity relationship (SAR) studies of BBN indicated that the aromatic amino acids (Trp8 and His12) are essential in biologic activity for GRPR 90. Peng, S. et al. reported the active state cryo-electron microscopy structures of GRPR bound to the endogenous GRP and a BBN analog [D-Phe6,\u03b2-Ala11,Phe13,Nle14]Bombesin(6-14) 116. Their data suggested that the C-22 termini of GRP inserted deeply into the helical bundle at the orthosteric ligand-binding pocket involving all EC loops and transmembrane regions (TMs) except TM1 and TM5, and the N-termini pointing out of the cavity (Figure 1.6A and B). The GRP binds in three distinct regions: the bottom pocket, the hydrophobic cavities, and the extracellular side. The residues H25G, L26G, and M27G at the C-terminus of GRP penetrate the bottom pocket by forming extensive hydrogen bonds and hydrophobic interactions (Figure 1.6D-F) 116. Similarly, the BBN analog [D-Phe6,\u03b2-Ala11,Phe13,Nle14]Bombesin(6-14) was shown binding to the GRPR at the same ligand-binding cavity with GRP ((Figure 1.6A-C) 116. Upon binding of its agonist ligand (GRP, BBN, and their agonist derivatives), GRPR undergoes conformational changes that activate associated G-proteins, leading to the activation of the downstream signaling pathways which regulate various cellular functions, including stimulating the release of gastrin, smooth muscle contraction, and promoting the proliferation of various cell types 90. 1.3.1.3 Signaling Pathways After binding with GRP, BBN, or their agonist analogs, GRPR is activated and triggers the downstream GRP\/GRPR signalling which is involved in the pathophysiological processes of many diseases, such as inflammation-related diseases, cardiovascular diseases, neurological diseases, and cancers 117-119. Studies showed that GRP\/GRPR participates in the occurrence and development of inflammation-related diseases by inducing neutrophil migration and activating the PI3K, PKC, and mitogen-activated protein kinase (MAPK) signalling pathways 120, 121. GRP has also been reported to upregulate the intercellular adhesion molecule 1 (ICAM-1) and induces vascular cell adhesion molecule-1 (VCAM-1), leading to the development of cardiovascular diseases such as myocardial 23 infarction 122, 123. Furthermore, GRPR signalling dysfunction has been reported playing a role in central nervous system (CNS) diseases, including itching, memory disorders associated with neurodegenerative diseases, and brain tumors 124. GRPR was also shown involved in the increasing of the gamma-aminobutyric acid (GABA) releasing, resulting in a readjustment of the memory storage system and memory disorder 125. Moreover, GRP\/GRPR signalling was reported being activated in many cancers and involved in several signalling pathways, including phospholipase C (PLC)\/PKC pathway and activation of nuclear factor \u03baB (NF-\u03baB) promoting gene expression 118, 119, 126-128. Figure 1.7 Signaling pathways regulated by BBN\/GRP. Adapted with permission from O. Laukkanen, M. and Domenica Castellone, M. Gastrin-releasing peptide receptor targeting in cancer treatment: emerging 24 signaling networks and therapeutic applications. Current Drug Targets. 2016, 17(5), 508-514 118. Permission conveyed through Copyright Clearance Center, Inc. In this dissertation, we will focus on the signalling pathways related to the pathophysiological processes of cancers (Figure 1.7). Gaq and Ga12\/13 are the primary G subunits coupled to GRPR and initiate the downstream signaling pathways after GRPR agonist binds to the receptor. The activated Gaq subunit triggers phospholipase C-\u03b2 (PLC-\u03b2) followed by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is involved in the regulation of calcium release from intracellular stores. The increasing of the in intracellular calcium concentration then would give rise to the activation of calcium-dependent enzymes, modulation of ion channels, and regulation of gene transcription 129. Meanwhile, the other second messenger, DAG, further activates the following PKC which stimulates downstream Raf1 and leads to the stimulation of the MAPK pathway, particularly the extracellular signal-regulated kinase (ERK) cascade. Subsequently, the activated ERK translocates to the nucleus, phosphorylates transcription factors and regulates the gene expression associated with cell proliferation, differentiation, and survival 130, 131. In addition, the binding of Ga12\/13 subunits to regulators of G protein signaling (RGS) GTPase activates protein family including p115-RhoGEF (p115), PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG) and leads to the activation of the small G protein Rho family (Figure 1.7) 132-135. The activation of Rho signaling then leads to the reorganization of actin cytoskeleton that controls cellular migration and cancer metastasis. Furthermore, Rho signaling is involved in c-Jun N-terminal kinase (JNK), p38, and Rho-associated protein kinase (ROCK) 25 regulation, which transduces signals to the nucleus regulating transcription factors and regulates gene expression associated with cancer cell proliferation, survival and differentiation 134, 135. In contrast, several novel GRPR antagonists have been developed showing the capability to inhibit the signal pathway after binding to GRPR 136-138. The tumor growth inhibition might be resulted from the reduction of epidermal growth factor receptor (EGFR) levels and the attenuation of oncogene expression 90, 139, 140. However, the mechanisms of the signaling pathway inhibition are still not completely understood 93, 141. Overall, the overexpression of GRPR in cancer cells results in enhancing the downstream signaling that promotes cancer cell proliferation and survival, making it an important target for both imaging and therapy 90. 1.3.2 GRPR Expression in Cancers GRPR and its messenger RNA (mRNA) were found overexpressed in several cancers and involved in cancer cell proliferation and facilitating malignant neoplasm development, including but not limited to prostate cancer (PCa), breast cancer (BCa), lung cancer, colorectal cancer, glio-\/neuroblastomas, and head and neck squamous cell cancers 90, 92, 93, 142-150. Mattei et al. analyzed the expression of GRPR in 200 non-small cell lung carcinoma (NSCLC) and 38 small cell lung carcinoma (SCLC) cases, reporting GRPR overexpression in 62.5% of NSCLC cases and 52.6% of SCLC cases 126. Additionally, Egloff et al. demonstrated that GRPR mRNA expression in histologically normal bronchial epithelial cells is significantly associated with lung cancer 151. Here, we focus on two solid malignancies, prostate cancer (PCa) and bladder cancer (BCa), both of which exhibit GRPR mRNA overexpression based on The Cancer Genome Atlas (TCGA) dataset, with expression levels of 0.6 transcripts per million (TPM) and 1.5 TPM, respectively. 26 1.3.2.1 Prostate Cancer Prostate cancer is the most common malignancy worldwide in men with high mortality rates 152. Hence, accurate diagnosis and staging of PCa is of upmost importance. The expert opinion from the American Urological Association (AUA) and American Society for Radiation Oncology (ASTRO) Guideline (2022) on clinically localized prostate cancer suggested that: \u201cin patients with PCa at high risk for metastatic disease with negative conventional imaging, clinicians may obtain molecular imaging to evaluate for metastases\u201d 153. As mentioned in Section 1.1.2, several PSMA-targeted radiotracers have been approved by FDA for staging for patients at high risk of metastasis and shown higher accuracy, better sensitivity and specificity compared with conventional imaging tools 73-75. PSMA, also known as glutamate carboxypeptidase II (GCP II), is a type II membrane-bound binuclear zinc metallopeptidase which was found overexpressed on the cell surface of PCa, especially in advanced stages of the disease 154, 155. A reduction of PSMA expression for PCa cells was reported related to the cancer progression from the androgen-dependent to the androgen-independent stage, while an increasing PSMA expression was observed related to androgen deprivation treatments 156, 157. Variations in PSMA expression in PCa cells during disease progression pose challenges for the use of PSMA-targeted radiotracers in diagnosis and radiotherapy. This creates a need for radiopharmaceuticals that target alternative biomarkers overexpressed in PCa cells, especially for patients with low or absent PSMA expression. The overexpression of the GRPR was observed in PCa samples at both the mRNA level and the protein level93, 158. GRPR mRNA was found overexpressed in 100% of early stages PCa tissues and around 60% of late stages PCa tissues. Thus, GRPR-targeted radiopharmaceuticals have been explored extensively in preclinical and clinical studies for the detection of GRPR-positive prostate tumors. 27 The variant PSMA and GRPR expressions in PCa cares can be also observed in two metastatic cell lines for PCa. PC-3 cell line, initiated from a bone metastasis of a grade IV prostatic adenocarcinoma, is androgen-independent with high GRPR expression but no PSMA expression 96, 159. In contrast, LNCaP cell line, isolated from a supraclavicular lymph node of a patient with metastatic prostate carcinoma, is confirmed androgen-dependent, with high PSMA expression but lacking GRPR expression 160, 161. As the paired [68Ga]Ga-PSMA-11 and [68Ga]Ga-RM2 scans shown in Figure 1.8, some metastatic lesions were visualized only by either [68Ga]Ga-PSMA-11 (B) or [68Ga]Ga-RM2 (A), while some lesions could be detected by both tracers (C) 159. A study compared PSMA- and GRPR-targeted PET results from 50 patients with biochemically recurrent PCa published by Baratto, L. et al., showed that both PSMA- and GRPR-targeted PET tracers detected 70 lesions in 32 patients, while 43 lesions in 18 patients were identified on only one scan. [68Ga]Ga-RM2 detected seven additional lesions in four patients, while PSMA-targeted tracer detected 36 additional lesions in 13 patients 162. Figure 1.8 Maximum-intensity-projection PET images from [68Ga]Ga-PSMA-11 and [68Ga]Ga-RM2 PET scan pairs show no uptake on [68Ga]Ga-PSMA-11 PET and focal uptake (arrow) in retroperitoneal lymph nodes on [68Ga]Ga-RM2 PET (A), focal uptake (arrow) in retroperitoneal lymph nodes on [68Ga]Ga-PSMA-28 11 PET and no uptake on [68Ga]Ga-RM2 PET (B), and focal uptake (arrows) in seminal vesicle on both [68Ga]Ga-PSMA-11 PET and [68Ga]Ga-RM2 PET (C). This figure was originally published in JNM. Iagaru, A., Will GRPR compete with PSMA as a target in prostate cancer? J Nucl Med. 2017, 58(12), 1883-1884. \u00a9 SNMMI 159. Furthermore, the lack of salivary or lacrimal gland uptake gives GRPR-targeted radiopharmaceuticals an advantage compared with PSMA-targeted radiopharmaceuticals, particularly in therapeutic applications. Meanwhile, it has been reported that GRPR expression is significantly upregulated in early-stage and androgen-dependent prostate cancer, making it a promising target for early diagnosis and imaging 163, 164. 1.3.2.2 Breast Cancer Breast cancer (BCa) is reported to be the most common malignancy worldwide in women 152. Conventional radiological modalities such as mammography, ultrasonography, and MRI play a major role in the diagnosis and staging of BCa. However, disadvantages such as over-diagnosis and false-positive findings were also reported on the conventional modalities 165, 166. With advances in nuclear medicine, molecular imaging and targeted radiotherapy have become increasingly vital in the diagnosis, staging, and management of BCa. [18F]FDG PET-CT imaging has been utilized for staging of BCa patients with recurrent, suspected, or documented stage IV disease showing good sensitivity and specificity 167, 168. Multiple biomarkers were reported being involved in BCa, such as immunohistochemical markers including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), genomic markers including BReast CAncer gene 1 and 2 (BRCA1, BRCA2), and immunomarker programmed death ligand-1 (PD-L1)] 167. Some biomarkers have been used as the target for BCa diagnosis and therapy in preclinical or clinical studies, such as ER-targeted radiotracer [18F]FES 29 ([18F]fluoroestradiol) and HER-2-targeted radiotracer [99mTc]Tc-HYNIC-H6 169, 170. [18F]FES was approved by the U.S. FDA in 2020, under the trade name Cerianna\u2122, to detect ER-positive\u2009lesions as an adjunct to biopsy in patients with recurrent or metastatic breast cancer 171. Some GPCRs including somatostatin receptor (SSTR), C-X-C chemokine receptor type 4 (CXCR4), neuropeptide Y receptor Y1 (NPY1R), and vasoactive intestinal polypeptide receptor 1 (VIP-R1) were also found overexpressed in BCa and are considered as promising targets for the cancer diagnosis and treatment 172-176. SSTRs were shown variably expressed at the mRNA level in BCa samples with 91% showing SSTR1, 98% SSTR2, 96% SSTR3, 76% SSTR3, and 54% SSTR5. Good correlation between SSTR mRNA and protein expression with 68-89% for all five SSTR subtypes was also revealed 172. Dalm et al. measured the CXCR4 mRNA level by quantitative reverse transcriptase polymerase chain reaction in 915 primary breast cancer tissues and revealed that the overexpression of CXCR4 mRNA was found associated in ER-negative BCa tumors 174. The CXCR4 expression in breast cancer was determined using immunohistochemistry which showing 67% of invasive tumors showed high nuclear staining and 41% of tumors showed cytoplasmic staining 177. Besides the above mentioned biomarkers, GRPR is also shown considerably high receptor density (9,819\u2009\u00b1\u2009530\u2009dpm\/mg of tissue) in 74% of the analyzed breast cancer specimens, making it an attractive target for the development of diagnostic and targeted therapeutic radiopharmaceuticals 178. A strong positive correlation was observed in BCa tissues between high ER expression and GRPR overexpression by Halmons, G. and colleagues 179. A tissue microarray of 1,432 primary tumors from BCa patients reported by Morgat, C., et al. showed that GRPR was overexpressed in 83.2% of ER-positive tumors. Considering the molecular subtypes of breast cancer, GRPR was found overexpressed in 86.2% of luminal A-like tumors, 70.5-82.8% of luminal 30 B-like tumors, 21.3% of HER2-enriched tumors, and 7.8% of triple-negative tumors. Furthermore, high GRPR expression was also found in metastatic lymph nodes in 94.6% of cases for those GRPR-overexpressing BCa patients 180. A study reported by Gugger, M. and Reubi, J.C. elucidated the role of GRP as a stimulatory growth factor in human BCa and suggested that the overexpression and activation of GRPR contributes to BCa cell proliferation 145. In addition, GRPR mRNA overexpression was found significantly correlated with ER-positive BCa tumors 174. These studies highlighted the favorable characteristics of GRPR, making it an attractive target for imaging and therapy in ER-positive BCa. Thus, novel GRPR-targeted ligands have been developed for the diagnosis and therapy of BCa. Several BBN analogs reported by the Schally group, such as RC-3095((D-Tpi6,Leu13\u03c8Leu14)Bombesin(6-14)) and RC-3940-II((Hca6,Leu13\u03c8Thz14)Bombesin(6-14)), were confirmed to be GRPR antagonists and shown the ability to inhibit the proliferation of BCa cells 181-184. Some GRPR-targeted radiopharmaceuticals have also been evaluated in preclinical studies and even translated into clinical evaluations. Maina, T., et al. reported a clinical study showing 50% breast tumors were successfully visualized in patients with advanced BCa by using a GRPR antagonist tracer, [68Ga]Ga-SB3 185. A study reported by Stoykow, C., et al. also indicated that 13 out of 18 BCa patients were successfully visualized by another GRPR antagonist tracer, [68Ga]Ga-RM2 47. Some therapeutic clinical trials are underway by using GRPR-targeted radiopharmaceuticals to treat BCa patients, e.g. [177Lu]Lu-NeoB was used in combination with Ribociclib and Fulvestrant for ER+, HER2- and GRPR+ advanced BCa (NCT05870579). Overall, GRPR is a very promising target for BCa diagnosis and radiotherapy. 31 1.3.3 Development of GRPR-targeted ligands The overexpression of GRPR in malignancies makes it a valuable target for molecular imaging and therapy. The same heptapeptide sequence at the C-terminus (Trp-Ala-Val-Gly-His-Leu-Met-NH2), which is shared by the two natural GRPR-targeted ligands, GRP and BBN, has been widely used as the template for designing GRPR-targeted pharmaceuticals for decades worldwide 46, 47, 111, 162, 186-192. Some derivatives of GRP and BBN have been labeled with radioisotopes for cancer diagnosis with SPECT and PET and radiotherapeutic applications 46, 187, 189, 191, 193, 194. 1.3.3.1 Historical Development Initial attempts on the development of GRPR-targeted radioligands focused on the native amino acid sequences of BBN and GRP 195-199. Van de Wiele, C. and coworkers coupled bombesin(7-14) with an N3S-chelator via a Gly-5-aminovaleric acid spacer and developed a radiolabeled GRPR agonist ([99mTc]Tc-RP527). This tracer showed low lung, heart, and liver uptake, and allowed early imaging of the supradiaphragmatic region with SPECT 195, 196. Dijkgraaf, I. et al. developed 18F- and 68Ga-labeled NOTA-8-Aoc-Bombesin(7-14)NH2 which showed 2.15 \u00b1 0.55 and 1.24 \u00b1 0.26 %ID\/g uptake in PC-3 tumor xenografts, respectively 197. AMBA is a bombesin-based peptide agonist with a DOTA chelator and has very potent binding affinity to GRPR. AMBA has been labelled with different radioisotopes and evaluated in preclinical and clinical studies 198, 199. [177Lu]Lu-AMBA was reported to have therapeutic effect in several prostate cancer tumors models derived from PC-3 and DU145 cell lines with different GRPR expression levels 199. However, a phase I escalation study of seven patients with metastatic castration-resistant prostate cancer revealed severe adverse effects (including abdominal cramps and vomiting) under high doses of [177Lu]Lu-AMBA (1.4-4.0 \u03bcg\/kg), and high accumulation in the pancreas 200. 32 Then the development of GRPR-targeted radiopharmaceuticals shifted toward using antagonist sequences as targeting vectors to prevent short term adverse effects and avoid the mitogenic effect of GRPR agonists 96, 201. SAR studies demonstrated that the two amino acids at the C-terminus (Leu-Met) play an essential role in the receptor activation and internalization, but they are not necessarily needed for the receptor binding 90. Hence, most GRPR antagonists were designed by modifying the two C-terminal amino acids or introducing a pseudopeptide bond between the two amino acids, which could potentially improve the flexibility of the C-terminus by shortening of hydrogen bonding 90, 108, 202-204. The bombesin analogs developed by the Schally group with a thiazoline-4-carboxylic acid (Thz14) substitution and a reduced peptide bond (CH2-N) between residues 13-14 (Leu13\u03c8Thz14) were confirmed to be GRPR antagonists, and some of them showed very potent GRPR binding affinities and the ability to inhibit the growth of several preclinical tumor models 137, 181, 182, 205, 206. Maecke, H.R. and colleagues developed a potent DOTA-conjugated antagonist, RM2, by replacing the C-terminal Leu-Met with Sta-Leu 202, 207, 208. The 68Ga- and 177Lu-labeled RM2 have been validated in the clinic for imaging and therapy for GRPR-positive cancers 47. In addition, a series of very potent radiolabeled GRPR antagonists were developed and showed very promising results in preclinical and\/or clinical studies, including radiolabeled SB3 and NeoB derivatives 185, 191, 209. Furthermore, it has been revealed that radiolabeled GRPR antagonists could potentially have higher tumor uptake due to their better in vivo stability and more binding sites than those available for radiolabeled GRPR agonists 210, 211. Cescato, R. et al. reported that with comparable GRPR binding affinities, the GRPR antagonist ligand ([99mTc]Demobesin 1) showed a significantly higher uptake in PC-3 tumor xenografts than a GRPR agonist ligand ([99mTc]Demobesin 4) 203. On the other hand, the internalization of GRPR 33 agonists upon binding to the receptor may lead to a longer tumor retention 90, 96, 212, which may be preferable for the development of radiotherapeutic agents. Beside the most widely studied peptide-based GRPR-targeted ligands, there are also some nonpeptide-based ligands, including small molecules and antibodies 136, 213. Ashwood, V., et al. reported a small molecule-based GRPR antagonist, PD176252, which showed very potent binding affinity to both GRPR (Ki = 1.0 nM) and NMBR (Ki = 0.15 nM), and the capability to inhibit the proliferation of lung cancer cells 136, 214. A GRP-targeted murine monoclonal antibody, 2A11, was used to inhibit the growth of SCLC cells in vitro and in athymic nude mice. 2A11 was further evaluated in clinical studies and shown antitumor activity in a minority of treated patients due to the interruption of the GRP autocrine growth factor loop 213. However, to our best knowledge, nonpeptide-based ligands have not been utilized in the design of GRPR-targeted radiopharmaceuticals yet. Table 1.4 Examples of GRPR-targeted radiopharmaceuticals. Agonist\/ Antagonist Name Ref agonist RP527 (N3S-Gly-5Ava-Bombesin(7-14)) 195, 196 NOTA-8-Aoc-Bombesin(7-14)NH2 197 Demobesin 4 (N4-[Pro1,Tyr4,Nle14]Bombesin) 203 AMBA (DOTA-G-4-aminobenzoyl-Bombesin(7-14)) 198, 199 antagonist RM2 (DOTA-Pip-[DPhe6,Sta13,Leu14]Bombesin(6-14)) 202, 207, 208 SB3 (DOTA- p-aminomethylaniline-diglycolic acid-[DPhe6,Leu13-NHEt]Bombesin(6-13)) 185 Demobesin 1 (N4-diglycolate-[D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14)) 203 NeoB (DOTA-pABzA-DIG-[DPhe6,His12-NH-CH[CH2- CH(CH3)2]2]Bombesin(6-12)) 191, 209 ProBOMB1 (DOTA-pABzA-DIG-[DPhe6,Leu13\u03c8Pro14-NH2]Bombesin(6-14)) 215, 216 34 ProBOMB2 (DOTA-Pip-[DPhe6,Leu13\u03c8Pro14-NH2]Bombesin(6-14)) 217 1.3.3.2 Limitations of GRPR-targeted Radiopharmaceuticals Though some GRPR-targeted radiopharmaceuticals have been translated into the clinic and shown promise for cancer theranositics, their clinical translation still faces several challenges, such as high pancreatic accumulation and limited in vivo stability 46, 189-191, 193. 1.3.3.2.1 High Pancreas Uptake Issue One of the main challenges in developing GRPR-targeted radiopharmaceuticals is reducing off-target uptake, especially in the pancreas. Due to the high physiologically expressing levels of GRPR in pancreas, high accumulation of GRPR-targeting radiopharmaceuticals in the pancreas was observed in both preclinical animal models and in patients for most currently reported GRPR-targeted radioligands, including radiolabeled RM2, AMBA, NeoB, and SB3 derivatives 46, 189-191, 193, 218. The most widely studied radiolabeled GRPR antagonist, RM2, was reported showing high accumulations in pancreas after labeled with different isotopes including 68Ga, 111In, and 177Lu 202. A study reported by Bakker, I.L., et al. showed that the highest absorbed dose (0.198 mGy\/MBq) was detected in the pancreas after administration of [68Ga]Ga-SB3 (187.4 \u00b1 40.0 MBq) 218. Similar result was also observed in radiolabeled GRPR-targeted agonists that an extremely high uptake in the pancreas (SUV up to 54.9) was observed after the administration of the radiolabeled GRPR agonist, [68Ga]Ga-AMBA 189. Although no clear absorbed dose limit for radiation therapy for the pancreas has been reported yet 219, 220, it has been reported that the pancreatic tissue might be structurally and functionally altered at doses starting at 30 to 40 Gy and the pancreas should be considered as an organ at risk (OAR) 221, 222. Hence, the high pancreas uptake not only limits the detection of cancer lesions located in or adjacent to the pancreas, but also lowers the maximum tolerated dose for targeted radioligand therapy. Development of novel GRPR-targeted 35 radiopharmaceuticals with low pancreas uptake is desirable for better imaging and therapeutic applications. As mentioned in Section 1.3.3.1, the Schally group reported some promising (Leu13\u03c8Thz14)Bombesin analogs showing very potent binding affinity to GRPR and the ability to inhibit the tumor growth 181, 182. Inspired by the reduced-peptide-bond-containing antagonist sequence of RC-3950-II (D-Phe-[Leu13\u03c8Thz14]Bombesin(7-14)), our group developed two GRPR-targeting ligands, ProBOMB1 and ProBOMB2 by replacing the C-terminal Thz14 with Pro14 (Figure 1.9A). Figure 1.9 (A) Chemical structures of ProBOMB1 and ProBOMB2, (B) Comparison of the in vivo PET images of [68Ga]Ga-NeoBOMB1, [68Ga]Ga-ProBOMB1, and [68Ga]Ga-ProBoMB2 at 1h post-injectionin PC-3 tumor-bearing mice. t: tumor; l: liver; p: pancreas; b: bowel; bl: bladder. The PET images of [68Ga]Ga-NeoBOMB1 and [68Ga]Ga-ProBOMB1 were distributed under the Creative Commons Attribution 4.0 International License (CC-BY license) and adapted from Joseph L. et al. (2019) 216. The PET image of [68Ga]Ga-ProBoMB2 was originally published in JNM. Bratanovic, I.J., Zhang, C., Zhang, Z., Kuo, H.T., Colpo, N., Zeisler, J., Merkens, H., Uribe, C., Lin, K.S. and B\u00e9nard, F., A radiotracer for molecular imaging and therapy of gastrin-releasing peptide receptor\u2013positive prostate cancer. JNM, 2022, 63(3), 424-430. \u00a9 SNMMI 217. HNNHHNNHHNNHHNNHNNONH2OHNNOOOONHOOH2NOOHNONNN NOHOOOHOHOProBOMB2HNNHHNNHHNNHHNHN NONH2OHNNOOOONHOOH2NOOONHOONNN NOOHOHOOOHNHProBOMB168Ga-NeoBOMB11h p.i.68Ga-ProBOMB11h p.i.68Ga-ProBOMB21h p.i.100 05tblA. B.6 7 8 9 10 11 12 13 1436 ProBOMB1 has a p-aminomethylaniline-diglycolic acid (pABzA-DIG) linker between the DOTA chelator and D-Phe-[Leu13\u03c8Pro14]Bombesin(7-14) sequence, while ProBOMB2 has a 4-amino-(1-carboxymethyl)piperidine (Pip) spacer instead 216, 217. As shown in Figure 1.9B, both [68Ga]Ga-ProBOMB1 and [68Ga]Ga-ProBOMB2 showed good uptake in PC-3 tumors but much less uptake in normal organs\/tissues especially the pancreas than the clinically validated [68Ga]Ga-NeoBOMB1 (4.68 \u00b1 1.26%ID\/g and 1.20 \u00b1 0.42%ID\/g, respectively, vs 123 \u00b1 28.4 %ID\/g) at 1h post-injection 216, 217. Though the mechanism for the reduction in the pancreas uptake of [68Ga]Ga-ProBOMB1 and [68Ga]Ga-ProBOMB2 is still unclear, these results demonstrated that the D-Phe-[Leu13\u03c8Pro14]Bombesin(7-14) is a promising sequence for the design of novel GRPR-targeted ligands with low pancreas accumulation 216, 217. 1.3.3.2.2 Low Metabolic Stability Besides high pancreas uptake, the other main limitation of the currently reported GRPR-targeted radiopharmaceuticals is their metabolic instability. The limited in vivo stability of GRPR-targeted ligands is mainly due to the enzymatic degradation especially by neutral endopeptidase 24.11 (NEP). NEP is widely expressed in vivo, including in vasculature walls, major organs and tissues, and cleaves linear peptides at the N-terminal sides of hydrophobic amino acids 201, 223. Studies have revealed that the potential cleavage sites for the clinically validated radiolabeled RM2 and AMBA analogs include His12-Leu13, Ala9-Val10, Trp8-Ala9 and Gln7-Trp8 190, 224. The radiometabolites released after the enzyme cleavage then lose the ability to interact with GRPR, leading to low diagnostic sensitivity, short tumor retention, and limited therapeutic efficacy. Previously, researchers tried to improve the in vivo stability of GRPR-targeted radiopharmaceuticals indirectly by co-injection of phosphoramidon (PA), an enzyme inhibitor of NEP 223, 225, 226. A study reported by Bakker, I.L. and coworkers showed that co-injection of 300 \u03bcg 37 PA with [111In]In-SB3 resulted in twice higher intact peptide in vivo and higher tumor uptake values at 1, 4, and 24 h post-injection (19.7\u2009\u00b1\u20093.5 vs 10.2\u2009\u00b1\u20091.5, 17.6\u2009\u00b1\u20095.1 vs 8.3\u2009\u00b1\u20091.1, 6.5\u2009\u00b1\u20093.3 vs 3.1\u2009\u00b1\u20091.9 % ID\/g, P\u2009<\u20090.0001) 225. However, application of radiopharmaceuticals without co-administration of enzyme inhibitors is preferred as the majority of the enzyme inhibitors were found having potential toxicity 227. Hence, more efforts have been put on the structural modifications of BBN\/GRP derivatives, such as C-terminal modifications and unnatural amino acids substitutions 228-232. Heimbrook, D.C. et al. developed potent GRPR antagonists based on the N-acetyl GRPR(20-27) sequence by replacing the Leu26-Met27 region of GRP with an alkyl group or an alkyl ether 228. Some of these C-terminally modified peptides were found to have higher stability in vitro and the ability to block GRP-stimulated mitogenesis in Swiss 3T3 mouse fibroblasts 228. The Wester group developed a potent and in vivo stable GRPR-targeted antagonist, AMTG, derived from RM2 by replacing Trp8 with \u03b1Me-Trp8. [177Lu]Lu-AMTG showed a significantly increased intact fraction in urine samples at 30 min post-injection (68.2 \u00b1 3.1%) compared with that of [177Lu]Lu-RM2 (0.5 \u00b1 0.1%) 231. NMe-His12 substitution was successfully used by Horwell, et al. for improving the stability of BBN without reducing its GRPR binding affinity 232. 1.4 Hypothesis and Aims The overall goal of this dissertation is to develop novel GRPR-targeted radiopharmaceuticals with good in vivo stability and minimal pancreas uptake for imaging and radiotherapy of GRPR-expressing cancer. Our first hypothesis is that modifying the C-terminal AA13 and AA14 of [68Ga]Ga-ProBOMB2 or the peptide bond between AA13 and AA14 can retain or improve the binding of the GRPR-targeting radiopharmaceuticals towards GRPR and retain minimized uptake in pancreas. As introducing unnatural amino acids is a common strategy to 38 improve in vivo stability of peptides 233, 234, our second hypothesis is that substituting the natural amino acids at the cleavage sites with unnatural amino acids can improve the in vivo stability of the GRPR-targeting radiopharmaceuticals. For my thesis project, there are four specific objectives: Objective 1: Design, synthesis and evaluation of GRPR-targeting radioligands with high binding affinity to increase the tumor uptake and minimize the pancreas uptake. Objective 2: Unnatural amino acid substitutions for the amino acids at the cleavage sites to increase the in vivo stability. Objective 3: Radiolabeling promising candidates with 177Lu, and evaluating the potential of the resulting radioligands with biodistribution studies and dosimetry calculations. Objective 4: Design, synthesis and evaluation of Pro derivatives and hydroxamate derivatives of the potent candidates. 39 Chapter 2: Materials and Methods 2.1 Reagents and Instrumentation RM2, its Ga-complexed analog, and its Lu-complexed analog were synthesized following previously reported procedures 202, 207, 208. SB3 and its Ga-complexed analog were synthesized following published procedures 235. AMBA, its Ga-complexed analog and Lu-complexed analog were synthesized following published procedures 199, 236. Fmoc-Leu\u03c8Pro-OH was kindly provided by Alpha-9 Oncology Inc. All other chemicals and solvents were purchased from commercial sources and used without further purification. GRPR-targeted peptides were synthesized using solid phase approach on an AAPPTec (Louisville, KY, USA) Endeavor 90 peptide synthesizer. Purification and quality control of DOTA-conjugated peptides and their natGa\/68Ga-complexed analogs were performed on Agilent (Santa Clara, CA, USA) HPLC systems equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (220 nm), and a Bioscan (Washington, DC, USA) NaI scintillation detector. The operation of Agilent HPLC systems was controlled using the Agilent ChemStation software. The HPLC columns used were a semi-preparative column (Luna C18, 5 \u00b5m, 250 \u00d7 10 mm) and an analytical column (Luna C18, 5 \u00b5m, 250 \u00d7 4.6 mm) purchased from Phenomenex (Torrance, CA, USA). The collected HPLC eluates were lyophilized using a Labconco (Kansas City, MO, USA) FreeZone 4.5 Plus freeze-drier. Low resolution mass spectrometry was acquired using a Waters (Milford, MA, USA) Acquity QDa mass spectrometer with the equipped 2489 UV\/Vis detector and e2695 Separations module. C18 Sep-Pak cartridges (1 cm3, 50 mg) were purchased from Waters (Milford, MA, USA). 68Ga was eluted from an ITM Medical Isotopes GmbH (Munich, Germany) generator and purified according to the previously published procedures using a N,N,N\u2019,N\u2019-tetra-n-octyldiglycolamide (DGA resin, normal) column from 40 Eichrom Technologies LLC (Lisle, IL, USA) 237. 177LuCl3 was purchased from Isotopia Molecular Imaging Ltd (Petah Tikva, Israel) and ITM Medical Isotopes GmbH (Munich, Germany). Radioactivity of 68Ga-labeled peptides and 177Lu-labeled peptides was measured using a Capintec (Ramsey, NJ, USA) CRC\u00ae-25R\/W dose calibrator, and the radioactivity of mouse tissues collected from biodistribution studies were counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter or a Hidex (Turku, Finland) automatic gamma counter. Calcium release assays were performed on a FlexStation 3 microplate reader (Molecular Devices, CA, USA). Internal dosimetry calculations were performed using the organ level internal dose assessment (OLINDA, Hermes Medical Solutions, version 2.2.3) software. 2.2 Synthesis of Fmoc-Leu\u03c8Thz-OH Scheme 1: Synthesis of Fmoc-Leu\u03c8Thz-OH hydrochloride (3). Fmoc-Leu\u03c8Thz-OH hydrochloride was synthesized following the reaction steps depicted in Scheme 1. (4R)-t-Butyl 4-thiazolidinecarboxylate hydrochloride (1) was synthesized following literature procedures 238. Synthesis of Fmoc-Leu\u03c8Thz-OtBu (2) Solution 1: Fmoc-Leucinol (3.79 g, 11.1 mmol) was converted to the aldehyde with Dess-Martin periodinane (5.87 g, 13.8 mol) in dichloromethane (70 mL) under ice\/water bath for 4 h. The reaction mixture was then mixed with saturated NaHCO3 aqueous solution (130 mL) and 41 sodium thiosulfate (13.0 g) and stirred for 30 min before being extracted with dichloromethane (130 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, concentrated in vacuo to ~ 20 mL in volume. Solution 2: Compound 1 (1.72 g, 7.62 mmol) was dissolved in saturated NaHCO3 aqueous solution (35 mL) and the mixture was extracted with ethyl acetate (100 mL \u00d7 2). The organic phases were combined, dried over anhydrous magnesium sulfate, evaporated in vacuo to obtain colorless oil. The oil was mixed with acetic acid (400 \u03bcL, 7.0 mmol) in dichloromethane (30 mL). Solutions 1 and 2 were mixed and the mixture was stirred for 30 min at room temperature. Sodium triacetoxyborohydride (5.41 g, 25.5 mmol) was added into the mixture and stirred for 20 h. Saturated NaHCO3 aqueous solution (100 mL) was added and stirred for 10 min. The mixture was extracted with ethyl acetate (100 mL \u00d7 2). The organic phases were combined, dried over anhydrous MgSO4 and purified by flash column chromatography eluted with 1:3 diethyl ether\/hexanes to obtain 2 as a white solid (2.13 g, 63% yield). ESI-MS: m\/z calculated for [M+H]+ of 2 C29H38N2O4S 511.7; found 511.5. 1H NMR (600 MHz, CDCl3) \u03b4 7.76 (d, J = 7.5 Hz, 2H, Ar-H), 7.60 (t, J = 6.5 Hz, 2H, Ar-H), 7.39 (t, J = 7.4 Hz, 2H, Ar-H), 7.30 (t, J = 7.3 Hz, 2H, Ar-H), 4.49 \u2013 4.35 (m, 2H, OCH2), 4.22 (t, J = 6.6 Hz, 1H, OCH2CH), 4.17 \u2013 3.96 (m, 2H, SCH2N), 3.93 \u2013 3.76 (m, 2H, SCH2CH), 3.18 (d, J = 8.7 Hz, NHCH), 3.08 \u2013 2.97 (m, 1H, COOCH), 2.59 \u2013 2.43 (m, 2H, NHCH2CH), 1.71-1.60 (m, 1H, CH3CH), 1.45 (s, 9H), 1.40 \u2013 1.32 (m, 2H, CH3CHCH2), 0.93 (2, 6H, CH3). Synthesis of Fmoc-Leu\u03c8Thz-OH (3) Compound 2 was dissolved in a mixture of dichloromethane (25 mL) and trifluoroacetic acid (75 mL), and stirred for 3 h at room temperature. After concentrated in vacuo, the residue was dissolved in ethyl acetate (80 mL) and mixed with 4M HCl in 1,4-dioxane dioxane (3 mL). After 42 being stirred for 10 min, the volatile solvents were removed in vacuo. Diethyl ether (250 mL) was added to the residue and the mixture was stirred for 30 min. White solid was collected by filtration to obtain 1.38 g of 3 (70% yield) as a white solid. ESI-MS: m\/z calculated for [M+H]+ of 3 C25H30N2O4S 455.6; found 455.4. 1H NMR (600 MHz, MeOD) \u03b4 7.81 (d, J = 7.5 Hz, 2H, Ar-H), 7.68 (dd, J = 10.3, 7.6 Hz, 2H, Ar-H), 7.41 (t, J = 7.4 Hz, 2H, Ar-H), 7.36 \u2013 7.30 (m, 2H, Ar-H), 4.73 \u2013 4.62 (m, 1H, OCH2CH), 4.53 \u2013 4.50 (m, 2H, OCH2), 4.29 \u2013 4.21 (m, 2H, SCH2N), 4.07 \u2013 3.91 (m, 1H, NHCH), 3.54 \u2013 3.39 (m, 2H, SCH2CH), 3.29 \u2013 3.22 (m, 2H, NHCH2CH), 3.03 (dd, J = 13.0, 9.6 Hz, 1H, COOHCH), 1.64 \u2013 1.58 (m, 1H, CH3CH), 1.46 \u2013 1.27 (m, 2H, CH3CHCH2), 0.94 (dd, J = 12.7, 6.6 Hz, 6H, CH3). 2.3 Peptide synthesis 2.3.1 Synthesis of DOTA-Conjugated Precursors All the GRPR-targeted DOTA-conjugated precursors were synthesized on solid phase using Fmoc peptide chemistry. (1) For the GRPR-targeted ligands with a C-terminal reduced peptide bond (Leu13\u03c8AA14), Sieber resin (0.05 mmol, 0.104 g) was treated with 20% piperidine in N,N-dimethylformamide (DMF) to remove Fmoc protecting group. After removal of the Fmoc protecting group, Fmoc-Leu\u03c8Thz-OH (3)\/Fmoc-Leu\u03c8Pro-OH (3 eq.), Fmoc-protected amino acids (5 eq.), Fmoc-4-amino-(1-carboxymethyl)piperidine (5 eq.) were pre-activated with HATU (3-5 eq.), HOAt (3-5 eq.), and N,N-diisopropylethylamine (DIEA, 9-15 eq.) before being sequentially coupled to the resin. Then DOTA(tBu)3 (5 eq.) pre-activated with HATU (5 eq.) and DIEA (25 eq.) was coupled to the resin. TacsBOMB6 was synthesized following similar procedures with the addition of Fmoc-cysteic acid before the coupling of DOTA(tBu)3. (2) For the GRPR-targeted ligands with an amide bond between Leu13-AA14, Rink Amide MBHA resin (0.05 mmol, 0.125 g) was treated with 20% piperidine in N,N-dimethylformamide (DMF) to remove the 43 Fmoc-protecting group. Fmoc-protected amino acids (5 eq.) and Fmoc-4-amino-(1-carboxymethyl)piperidine (5 eq.) were pre-activated with HATU (5 eq.), HOAt (5 eq.), and N,N-diisopropylethylamine (DIEA, 9 eq.) and then sequentially coupled to the resin. Then, DOTA(tBu)3 (5 eq.), pre-activated with HATU (5 eq.) and DIEA (25 eq.), was coupled to the N-terminus of the peptides. (3) For LW02075 and LW02050 with a C-terminal hydroxamate, Fmoc-hydroxylamine-2-Cl-Trt resin (0.1 mmol) was treated with 20% piperidine in N,N-dimethylformamide (DMF) to remove the Fmoc protecting group. After removing the Fmoc protecting group, Fmoc-protected amino acids (5 eq.) were pre-activated with HATU (5 eq.), HOAt (5 eq.), and N,N-diisopropylethylamine (DIEA, 15 eq.), before being sequentially coupled to the resin. Pre-activated with HATU (5 eq.), HOAt (5 eq.), and DIEA (15 eq.), Fmoc-p-aminomethylaniline-diglycolic acid (Fmoc-pABzA-DIG, 5 eq.) linker and Fmoc-4-amino-(1-carboxymethyl)piperidine (Fmoc-Pip-OH linker, 5 eq.) linker were coupled to the resins for LW02075 and LW02050, respectively. At the end, DOTA(tBu)3 (5 eq.) pre-activated with HATU (5 eq.) and DIEA (25 eq.) was coupled to the N-terminus. The peptides were deprotected and simultaneously cleaved from the resin with a mixture of trifluoroacetic acid (TFA, 81.5%), triisopropylsilane (TIS 1.0%), water (5%), 2,2\u2032-(ethylenedioxy)diethanethiol (DODT, 2.5%), thioanisole (5%), and phenol (5%) for 4 h at room temperature. The cleaved peptides were filtrated and then precipitated by the addition of cold diethyl ether. The crude peptides were collected by centrifugation and purified with HPLC (semi-preparative column; flow rate: 4.5 mL\/min). The eluates containing the desired peptides were collected and lyophilized. The HPLC conditions, retention times, isolated yields, purities, and MS confirmations of these GRPR-targeted DOTA-conjugated precursors are provided in their respective chapters. 44 2.3.2 Synthesis of GRPR-targeted peptides All GRPR-targeted peptides in Chapter 4 Section 4.1 were synthesized on solid phase using Fmoc peptide chemistry. Rink Amide MBHA resin (0.05 mmol, 0.125g) was treated with 20% piperidine in N,N-dimethylformamide (DMF) to remove Fmoc protecting group. Fmoc-protected amino acids (3 eq.) and Fmoc-4-amino-(1-carboxymethyl)piperidine (3 eq.) were pre-activated with HATU (3 eq.), HOAt (3 eq.), and N,N-diisopropylethylamine (DIEA, 9 eq.) and then sequentially coupled to the resin. For DOTA coupling, DOTA(tBu)3 (5 eq.) pre-activated with HATU (5 eq.) and DIEA (25 eq.) was coupled to the N-terminus. The peptides were deprotected and simultaneously cleaved from the resin with a mixture of trifluoroacetic acid (TFA, 81.5%), triisopropylsilane (TIS 1.0%), water (5%), 2,2\u2032-(ethylenedioxy)diethanethiol (DODT, 2.5%), thioanisole (5%), and phenol (5%) for 2 h at room temperature. The cleaved peptides were filtered and then precipitated by the addition of cold diethyl ether. The crude peptides were collected by centrifugation and purified with HPLC (semi-preparative column; flow rate: 4.5 mL\/min). The eluates containing the desired peptides were collected and lyophilized. The HPLC conditions, retention times, isolated yields, purities, and MS confirmations of these GRPR-targeted peptides are provided in Chapter 4 Section 4.2. 2.4 Synthesis of Nonradioactive Standards 2.4.1 Synthesis of Nonradioactive Ga-complexed Standards The nonradioactive Ga-complexed standards were prepared using a solution of the DOTA-conjugated precursor mixed and incubated with GaCl3 (5 eq.) in NaOAc buffer (0.1 M, 500 \u00b5L, pH 4.2-4.5) at 80 \u00b0C for 15 min. The reaction mixture was then purified via HPLC (semi-preparative column, flow rate: 4.5 mL\/min). The HPLC eluates containing the desired peptide were collected and lyophilized. The HPLC conditions, retention times, isolated yields and MS 45 confirmations of all the nonradioactive Ga-complexed standards are provided in their respective chapters. 2.4.2 Synthesis of Nonradioactive Lu-complexed Standards The nonradioactive Lu-complexed standards were synthesized by using a solution of the precursor solution mixed and incubated with LuCl3 (10 eq.) in NaOAc buffer (0.1 M, 500 \u00b5L, pH 4.5) at 90 \u00b0C for 30 min. The reaction mixture was then purified via HPLC (semi-preparative column, flow rate: 4.5 mL\/min). The HPLC eluates containing the desired peptides were collected and lyophilized. The HPLC conditions, retention time, isolated yield and MS confirmation of all the nonradioactive Lu-complexed standards are provided in their respective chapters. 2.5 Cell culture The PC-3 prostate adenocarcinoma cell line and murine Swiss 3T3 fibroblast cell line were both obtained from ATCC (via Cedarlane, Burlington, Canada), and verified pathogen-free via IMPACT Rodent Pathogen Test (IDEXX BioAnalytics, Columbia, MO, USA). The cell culture for both cell lines was performed in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) containing 10% FBS, penicillin (100 U\/mL) and streptomycin (100 \u03bcg\/mL) at 37 \u00b0C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO2. The PC-3 cells were rinsed with Dulbecco's phosphate-buffered saline (DPBS) and harvested after 1-min trypsinization when grown to 80-90% confluence. No DPBS rinse was performed for Swiss 3T3 cells. The cell concentration was counted in triplicate using a hemocytometer and a manual laboratory counter, or a Moxi mini automated cell counter (ORFLO Technologies, Ketchum, ID, USA). 46 2.6 Fluorometric Calcium Release Assay The antagonist\/agonist characteristics of the nonradioactive standards listed in Chapter 3-5 were determined following previously published procedures 216, 217. A 96-well clear bottom black plate was seeded with 5 \u00d7 104 PC-3 cells in 100 \u03bcL growth media per well 24 h prior to the assay. The loading buffer (100 \u03bcL\/well) containing a calcium-sensitive dye (FLIPR Calcium 6 assay kit from Molecular Devices, San Jose, CA, USA) was added into the 96-well plate, followed by 1 h incubation at 37 \u00b0C. After that, the plate was transferred into a FlexStation 3 microplate reader (Molecular Devices, San Jose, CA, USA). Nonradioactive Ga-complexed standard (50 nM), nonradioactive Lu-complexed standard (50 nM), [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) (50 nM, antagonist control), bombesin (50 nM, agonist control), adenosine triphosphate (ATP, 50 nM, positive control), or DPBS (blank control) was added, and the fluorescent signals were acquired for 2 min (\u03bbEx = 485 nm; \u03bbEm = 525 nm; n = 3). The relative fluorescent units (RFU = max \u2013 min) were calculated to determine the agonistic\/antagonistic characteristics for the tested ligands. 2.7 In vitro Competition Binding Assay PC-3 cells were seeded at 2 \u00d7 105 cells\/well in 24-well poly-D-lysine plates 24-48 h prior to the experiment, and Swiss 3T3 cells were seeded at 1 \u00d7 105 cells\/well in 24-well poly-D-lysine plates 48 h prior to the experiment. The growth medium was replaced with 400 \u03bcL of reaction medium (RPMI 1640 containing 2 mg\/mL BSA, and 20 mM HEPES). After 1 h incubation at 37 \u00b0C, nonradioactive Ga\/Lu-complexed standard in 50 \u03bcL of decreasing concentrations (10 \u03bcM to 1 pM), and 50 \u03bcL of 0.01 nM [125I-Tyr4]Bombesin were added to wells followed by incubation with moderate agitation for another 1 h at 37 \u00b0C. After that, the cells in each well were washed with ice-cold DPBS twice gently, harvested by trypsinization, and measured for radioactivity on a 47 Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter. Data were analyzed using nonlinear regression (one binding site model for competition assay) with GraphPad (San Diego, CA, USA) Prism 10 software (Version 10.1.1). 2.8 Radiolabeling 2.8.1 68Ga Radiolabeling The gallium-68 radiolabeling experiments were performed following previously published procedures 237, 239, 240. Purified 68Ga in 0.5 mL water was added into a 4-mL glass vial preloaded with 0.7 mL of HEPES buffer (2 M, pH 5.0) and 10 \u03bcL precursor solution (1 mM). The radiolabeling reaction was conducted by 100 \u00b0C microwave heating for 1 min before being purified by HPLC using the semi-preparative column. The eluate fraction containing the radiolabeled product was collected, diluted with water (50 mL), and passed through a C18 Sep-Pak cartridge that was pre-washed with ethanol (10 mL) and water (10 mL). After washing the C18 Sep-Pak cartridge with water (10 mL), the 68Ga-labeled product was eluted off the cartridge with ethanol (0.4 mL), and diluted with DPBS for imaging and biodistribution studies. Quality control was performed using the analytical column. The details of the HPLC conditions and retention times are provided in each of the following chapters. 2.8.2 177Lu Radiolabeling The Lutetium-177 radiolabeling experiments were conducted following literature procedures 217. 177LuCl3 in solution was added into a 4-mL glass vial preloaded with 0.7 mL of sodium acetate buffer (0.1 M, pH 4.5) and 10 \u03bcL precursor solution (1 mM), and incubated at 95 \u2103 for 15 min. 177Lu-labeled compounds were isolated from uncomplexed 177Lu and precursors by using HPLC with the semi-preparative column. The collection and solid-phase extraction procedures of 177Lu-labeled compounds were the same as those for 68Ga-labeled products. Quality 48 control was performed using the analytical column. The details of the HPLC conditions and retention times are provided in each of the following chapters. 2.9 The logD7.4 Measurements The logD7.4 values of all the 68Ga labeled GRPR-targeted ligands and 177Lu labeled GRPR-targeted ligands were measured using the shake flask method as previously reported 237. Briefly, an aliquot (~2 MBq) of the radiolabeled peptide was added to a 15 mL falcon tube containing 3 mL of n-octanol and 3 mL of DPBS (pH 7.4), followed by 1-min vortex. The resulting mixture was then centrifuged at 5,000 rpm for 15 min. Samples of the n-octanol (1 mL) and buffer (1 mL) layers were collected and counted with a gamma counter. The logD7.4 values were calculated using the following equation: \ud835\udc59\ud835\udc5c\ud835\udc54\ud835\udc37!.# =\t \ud835\udc59\ud835\udc5c\ud835\udc54$%[counts&'()*&+\t-.)\/0counts123304\t-.)\/0 ] 2.10 Animal Studies 2.10.1 PET imaging and biodistribution studies PET imaging and ex vivo biodistribution studies were performed using male NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl\/SzJ (NRG) mice following previously published procedures 241. The experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. The mice were anaesthetized by inhalation of 2.5% isoflurane in oxygen, and implanted subcutaneously with 5 \u00d7 106 PC-3 cells (100 \u00b5L; 1:1 DPBS\/Matrigel) behind the left shoulder. When the tumor grew to 5-8 mm in diameter over 3-4 weeks, the mice were used for PET\/CT imaging and biodistribution studies. 49 PET\/CT imaging experiments were carried out using a Siemens (Knoxville, TN, USA) Inveon micro PET\/CT scanner. Each PC-3 tumor-bearing mouse was injected with ~3-6 MBq of 68Ga-labeled tracer through a lateral caudal tail vein under 2.5% isoflurane in oxygen anesthesia, followed by recovery and roaming freely in its cage during the uptake period. At 50 min post-injection, a 10-min CT scan was conducted first for localization and attenuation correction after segmentation for reconstructing the PET images, followed by a 10-min static PET imaging acquisition. For biodistribution studies, the mice were injected with the radiotracer (~2-4 MBq) as described above. For blocking, the mice were co-injected with 100 \u03bcg of nonradioactive Ga-complexed nonradioactive standards for GRPR antagonist tracers or with 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) for GRPR agonist tracers. At 1 h post-injection, the mice were euthanized by CO2 inhalation. Blood was withdrawn by cardiac puncture, and organs\/tissues of interest were collected, weighed and counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter. Uptake values were expressed as the percentage of the injected dose per gram of tissue (%ID\/g). 2.10.2 SPECT imaging and biodistribution studies SPECT\/CT Imaging experiments were performed using an MI Labs (Houten, The Netherlands) U-SPECT-II\/CT scanner with a custom-made ultra-high sensitivity big mouse collimator (2 mm pinhole size). The PC-3 tumor-bearing mice were sedated (2.5% isoflurane in O2) and injected with 177Lu labeled tracer through a lateral caudal tail vein. The mice were imaged at 1, 4, 24, 72, and 120 h post-injection. At each time point, a 5-min CT scan was obtained using 615\u2009\u03bcA and 60\u2009kV parameters for localization and attenuation, followed by 2 \u00d7 30-min static SPECT scans acquired in list mode with an energy window centered around 208 keV. The U-50 SPECT II software was used to reconstruct data, and the images were decay corrected to the time of injection with PMOD v 3.402 (PMOD Technologies GmbH, Fallanden, Switzerland). For ex vivo biodistribution studies, the mice were injected with ~2-6 MBq of the radioligand. At 1, 4, 24, 72, and 120 h post-injection, the mice were anesthetized with 2% isoflurane and euthanized by CO2 inhalation. Blocking study was conducted at 1 h post-injection via co-injection of the radioligand with 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14). Blood was drawn via cardiac puncture, and the organs\/tissues of interested were collected, weighed, and counted using an automatic gamma counter (PerkinElmer). Uptake values were expressed as the percentage of the injected dose per gram of tissue (%ID\/g). 2.10.3 In vivo stability studies For in vivo stability studies, the 68Ga-labeled tracer or 177Lu-labeled ligand was injected via a lateral caudal vein into healthy male NRG mice or male NOD.Cg-Prkdc scid Il2rg tm1Wjl \/SzJ (NSG) mice (n = 3). At 15 min post-injection, mice were sedated and euthanized, and the urine and blood samples were collected. The plasma was extracted from the whole blood sample (500 \u03bcL) by the addition of CH3CN (500 \u03bcL), followed by 1-min vortex, 20-min centrifugation and the separation of supernatant (plasma) from the blood cells and precipitated proteins. The plasma and urine samples were analyzed via radio-HPLC by using the conditions for quality control of the radiolabeled ligands respectively as listed in each chapter. 2.11 Dosimetry The uptake values (%ID\/g) obtained from the biodistribution data were decayed to the appropriate time point and fitted to mono- or bi-exponential equations using SciPy library 242 integrated into an in-house Python script (Python Software Foundation v.3.10.12). The best fit was selected based on maximizing the coefficient of determination (R2) and minimizing the residuals. 51 Time-activity curves calculated from the parameters obtained from the best fit for each organ were then integrated and normalised to injected activity to acquire time-integrated activity coefficients (TIACs) per unit gram, and subsequently multiplied by the mass of model tissue (25-g or 30-g mouse phantom). The TIACs were corrected for tumor sink effect following formula adopted in the report by Cicone et al. as shown below: 243 \ud835\udc47\ud835\udc3c\ud835\udc34\ud835\udc365,'&440'(07(\ud835\udc5c\ud835\udc5f\ud835\udc54\ud835\udc4e\ud835\udc5b)= \ud835\udc47\ud835\udc3c\ud835\udc34\ud835\udc365(\ud835\udc5c\ud835\udc5f\ud835\udc54\ud835\udc4e\ud835\udc5b) + 9\ud835\udc47\ud835\udc3c\ud835\udc34\ud835\udc365(\ud835\udc61\ud835\udc62\ud835\udc5a\ud835\udc5c\ud835\udc5f) \u00d7 \ud835\udc47\ud835\udc3c\ud835\udc34\ud835\udc365(\ud835\udc5c\ud835\udc5f\ud835\udc54\ud835\udc4e\ud835\udc5b)\ud835\udc47\ud835\udc3c\ud835\udc34\ud835\udc365(\ud835\udc4a\ud835\udc35)\u2212\ud835\udc47\ud835\udc3c\ud835\udc34\ud835\udc365(\ud835\udc61\ud835\udc62\ud835\udc5a\ud835\udc5c\ud835\udc5f)A The TIAC values were input in OLINDA (Hermes Medical Solutions, v2.2.3) 244 which has pre-calculated dose factors for mouse models. Depending on the average body weight of mice used in the biodistribution studies, we used either the 30-g or 25-g mouse model for the dosimetry calculation 245. The mouse biodistribution data were extrapolated to humans using the method proposed by Kirschner, et al. using the following equation: 246 \ud835\udc47\ud835\udc3c\ud835\udc34\ud835\udc368(\ud835\udc5c\ud835\udc5f\ud835\udc54\ud835\udc4e\ud835\udc5b) = \ud835\udc47\ud835\udc3c\ud835\udc34\ud835\udc369(\ud835\udc5c\ud835\udc5f\ud835\udc54\ud835\udc4e\ud835\udc5b) \u00d7 Bm(organ)8\/\ud835\udc4a\ud835\udc358m(organ):\/\ud835\udc4a\ud835\udc35:H where m(organ)H and m(organ)M are masses of human and animal organs, respectively, and WB represents total-body mass. The human TIACs calculated using the above equation were input into OLINDA and dosimetry results were assessed for ICRP 89 Adult Male Model 247. The %ID\/g value for the blood was assumed to be that for the heart contents of the phantom. Finally, the TIAC for the tumor was also calculated based on the biodistribution data, and the values were input into the sphere model available in OLINDA 248. 52 2.12 Statistical analysis Statistical analyses were performed via Student\u2019s t-test using the Microsoft (Redmond, WA, USA) Excel software or two-way ANOVA tests using the GraphPad Prism software. The unpaired two-tailed test was used to compare biodistribution data between two tracers. The unpaired one-tailed test was used to compare the biodistribution data of unblocked and blocked groups. Two-way ANOVA was used to compare the binding affinities for nonradioactive standards (n > 2). Statistically significant difference was considered when the adjusted p value was < 0.05 (* p < 0.05, ** p < 0.01 and *** p < 0.001). 53 Chapter 3: Novel GRPR-targeting ligands with high binding affinity to increase the tumor uptake 3.1 68Ga-labeled [Leu13\u03c8Thz14]Bombesin(7-14) derivatives: promising GRPR-targeting PET tracers with low pancreas uptake The following section is an adaption of the following published paper: Wang, L., Zhang, Z., Merkens, H., Zeisler, J., Zhang, C., Roxin, A., Tan, R., B\u00e9nard, F. and Lin, K.S. 68Ga-labeled [Leu13\u03c8Thz14]Bombesin(7-14) derivatives: promising GRPR-targeting PET tracers with low pancreas uptake. Molecules, 2022, 27, 3777. https:\/\/doi.org\/10.3390\/molecules27123777. The compounds disclosed in this report are covered by a patent application (PCT\/CA2019\/051620). Zhengxing Zhang, Jutta Zeisler, Fran\u00e7ois B\u00e9nard and Kuo-Shyan Lin are listed as inventors of this filed patent. 3.1.1 Introduction As a member of G protein-coupled receptors, the GRPR is expressed and regulates many physiological functions in the central nervous system, gastrointestinal tract, pancreas, adrenal cortex tissues, and others 90. Moreover, GRPR is also overexpressed in several malignancies including melanoma, prostate, breast, and lung cancers 93, 142-147. GRPR can be coupled with phospholipase C, followed by PKC activation, which regulates cell cycle, cell proliferation, and implicates in the malignant neoplasms\u2019 development 90. GRPR is also associated with the growth of human prostate carcinoma and pancreatic cancer by autocrine loop with GRP 149, 150. Overexpression of GRPR in malignant tissues has prompted the development of GRPR-targeting radiopharmaceuticals for better management of GRPR-expressing cancer 46, 47, 162, 189-191, 193, 249, 250 . To date, several radiolabeled GRPR-targeting ligands (i.e., AMBA, RM2, and NeoBOMB1), 54 based on the amphibian GRPR analog bombesin, have been introduced into the clinic for cancer diagnosis and radioligand therapy 46, 47, 162, 189-191. However, high accumulations of reported GRPR-targeting radiopharmaceuticals in normal organs, particularly in the pancreas, were found in patients and preclinical animal models 46, 189, 191, 193. The high uptake of GRPR-targeting radiopharmaceuticals in the pancreas not only affects lesion detection, but also limits the maximum tolerated dose for targeted radioligand therapy application. 55 Figure 3.1 Chemical structures of (A) TacsBOMB2, TacsBOMB3, and TacsBOMB4, (B) TacsBOMB5, (C) TacsBOMB6, and (D) RM2. The Schally group reported a series of GRPR antagonists based on the bombesin(7-14) sequence which replaced Met14 with Thz14 and introduced a reduced peptide bond (CH2-N) between residues 13-14 (Leu13\u03c8Thz14) 181, 182. Some of those antagonists showed very potent HNNHHNNHHNNHHNNNSO NH2OHNNOOOONHOOH2NOHNONNN NOHOOOHOHONH O ONHNHNOXTacsBOMB2: X = D-Phe TacsBOMB3: X = D-2-Nal TacsBOMB4: X = D-TpiHNNHNNHHNNHHNNHNNSO NH2OHNNOOOONHOOH2NOOHNONNN NOHOOOHOHOHNNHHNNHHNNHHNNHNNSO NH2OHNNOOOONHOOH2NOOHNNHONNN NOHOOOHOHOOSOHOO(A)(B)(C)(D)TacsBOMB5TacsBOMB6HNNHHNNHHNNHHNNHNOHNNOOOONHOOH2NOOHNONNN NHOOOHOHOOOHNHONH2ORM26 7 8 9 10 11 12 13 1456 GRPR binding affinities (low pM) and the ability to inhibit the growth of several preclinical tumor models 137, 205, 206. On the basis of these studies, our group attempted to develop GRPR-targeting tracers based on those reported reduced-peptide-bond-containing antagonist sequences. 68Ga-labeled ProBOMB1 with a pABzA-DIG linker between the DOTA chelator and D-Phe-[Leu13\u03c8Pro14]Bombesin(7-14) sequence showed comparable uptake in PC-3 tumor but much less uptake in normal organs\/tissues especially the pancreas than the clinically validated [68Ga]Ga-NeoBOMB1 (4.68 \u00b1 1.26 vs 123 \u00b1 28.4 %ID\/g at 1h post-injection) 209. Replacing the pABzA-DIG linker in [68Ga]Ga-ProBOMB1 with 4-amino-(1-carboxymethyl)piperidine (Pip) generated [68Ga]Ga-ProBOMB2 which retained good uptake in PC-3 tumors but with reduced uptake in normal organs\/tissues especially pancreas and intestines, leading to further improvement in imaging contrast 216, 217. ProBOMB1 and ProBOMB2 were derived from the RC-3950-II (D-Phe-[Leu13\u03c8Thz14]Bombesin(7-14)) sequence by replacing the C-terminal Thz with Pro. In this study we synthesized Ga-complexed D-Phe-[Leu13\u03c8Thz14]Bombesin(7-14)-derived TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5, and TacsBOMB6 (Figure 3.1) with an unmodified C-terminal Leu13\u03c8Thz14-NH2. Their antagonist characteristics were investigated using in vitro fluorescence based calcium release assay. Their potential for imaging GRPR expression was evaluated by in vitro competition binding, PET imaging and ex vivo biodistribution studies in a preclinical PC-3 prostate cancer model in mice, and were compared with the clinically validated GRPR antagonist, [68Ga]Ga-RM2. 3.1.2 Materials and Methods The materials and methods described in this section are provided in Chapter 2. Relevant sections are those describing reagent and instrumentation (Section 2.1), synthesis of Fmoc-57 Le\u03c8Thz-OH (Section 2.2), synthesis of DOTA-conjugated precursors (Section 2.3.1), synthesis of nonradioactive Ga-complexed standards (Section 2.4.1), cell culture (Section 2.5), fluorometric calcium release assay (Section 2.6), in vitro competition binding assay (Section 2.7), 68Ga radiolabeling (Section 2.8.1), logD7.4 measurements (Section 2.9), animal studies (Section 2.10), PET imaging and biodistribution studies (Section 2.10.1), in vivo stability studies (Section 2.10.3), and statistical analysis (Sections 2.12). 3.1.3 Results 3.1.3.1 Peptide Synthesis and Radiolabeling DOTA-conjugated TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5, and TacsBOMB6 were obtained in 14-49% yields, and their nonradioactive Ga-complexed standards were obtained in 57-75% yields. The HPLC conditions for their purification and MS characterizations are provided in Table 3.1 and Table 3.2, respectively. Gallium-68 labeling was conducted in HEPES buffer (2 M, pH 5.0). After HPLC purification, 68Ga-labeled TacsBOMB2, TacsBOMB3, TacsBOMB5, and TacsBOMB6 were obtained in in 42-59% decay-corrected radiochemical yields with > 66 GB\/\u00b5mol molar activity and > 92% radiochemical purity. The HPLC conditions and retention times are provided in the Table 3.3. Table 3.1 HPLC purification conditions and MS characterizations of TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5 and TacsBOMB6. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) TacsBOMB2 25% CH3CN and 0.1% TFA in H2O 18.7 34 [M+2H]2+ 792.4 [M+2H]2+ 792.8 TacsBOMB3 28% CH3CN and 0.1% TFA in H2O 15.8 49 [M+2H]2+ 817.4 [M+2H]2+ 817.8 TacsBOMB4 28% CH3CN and 0.1% TFA in H2O 16.6 38 [M+2H]2+ 817.9 [M+2H]2+ 818.2 58 TacsBOMB5 25% CH3CN and 0.1% TFA in H2O 18.3 32 [M+2H]2+ 799.4 [M+2H]2+ 799.7 TacsBOMB6 29% CH3CN and 0.1% TFA in H2O 14.6 14 [M+2H]2+ 892.9 [M+2H]2+ 893.4 Table 3.2 HPLC purification conditions and MS characterizations of Ga-complexed TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5 and TacsBOMB6. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Ga-TacsBOMB2 26% CH3CN and 0.1% TFA in H2O 12.5 72 [M+2H]2+ 825.9 [M+2H]2+ 826.0 Ga-TacsBOMB3 28% CH3CN and 0.1% TFA in H2O 18.3 67 [M+2H]2+ 850.9 [M+2H]2+ 850.7 Ga-TacsBOMB4 28% CH3CN and 0.1% TFA in H2O 18.8 68 [M+2H]2+ 851.4 [M+2H]2+ 851.2 Ga-TacsBOMB5 25% CH3CN and 0.1% TFA in H2O 18.3 75 [M+2H]2+ 832.9 [M+2H]2+ 832.8 Ga-TacsBOMB6 29% CH3CN and 0.1% TFA in H2O 14.6 57 [M+2H]2+ 926.4 [M+2H]2+ 926.4 Table 3.3 HPLC conditions for the purification and quality control of 68Ga-labeled TacsBOMB2, TacsBOMB3, TacsBOMB5, and TacsBOMB6. FA: formic acid. Compound name HPLC conditions Retention time (min) [68Ga]Ga-TacsBOMB2 Semi-Prep 20% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 12.6 QC 23% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 7.2 [68Ga]Ga-TacsBOMB3 Semi-Prep 21% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 35.8 QC 26% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 8.8 [68Ga]Ga-TacsBOMB5 Semi-Prep 20% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 10.7 QC 23% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 5.1 [68Ga]Ga-TacsBOMB6 Semi-Prep 29% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 14.6 59 QC 28% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 6.7 3.1.3.2 Binding Affinity, Antagonist Characterization and Hydrophilicity The binding affinities of Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, Ga-TacsBOMB6 and Ga-RM2 were measured by a cell-based binding assay using GRPR-expressing PC-3 prostate cancer cells. Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, Ga-TacsBOMB6, and Ga-RM2 inhibited the binding of [125I-Tyr4]Bombesin to PC-3 cells in a dose-dependent manner (Figure 3.2). The calculated Ki values for Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, Ga-TacsBOMB6, and Ga-RM2 were 7.08 \u00b1 0.65, 4.29 \u00b1 0.46, 458 \u00b1 38.6, 6.09 \u00b1 0.95, 5.12 \u00b1 0.57, and 1.51 \u00b1 0.24 nM, respectively (n = 3). Figure 3.2 Displacement curves of [125I-Tyr4]Bombesin by Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, Ga-TacsBOMB6, and Ga-RM2 generated using GRPR-expressing PC-3 cells. Error bars indicate standard deviation. 60 The antagonist characteristics of Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, and Ga-TacsBOMB6 were confirmed via intracellular calcium release assays using PC-3 cells (Figure 3.3). ATP (50 nM) and bombesin (50 nM) as positive controls induced Ca2+ efflux corresponding to 334 \u00b1 39.0 and 754 \u00b1 38.3 relative fluorescence units (RFU), respectively, compared with 14.9 \u00b1 4.93 RFU for the blank control, Dulbecco's phosphate buffered saline (DPBS). For 50 nM of Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, and Ga-TacsBOMB6, 12.9 \u00b1 3.33, 7.57 \u00b1 3.17, 9.30 \u00b1 3.74, 24.0 \u00b1 3.43, 23.0 \u00b1 0.06 RFU were observed, respectively, which were not higher than 25.3 \u00b1 1.92 RFU obtained from the antagonist control, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) (50 nM). Figure 3.3 Intracellular calcium efflux in PC-3 cells induced by various tested ligands. Error bars indicate standard deviation. The logD7.4 values of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 were \u20132.39 \u00b1 0.13, \u20131.75 \u00b1 0.04, \u20132.52 \u00b1 0.05, \u20132.55 \u00b1 0.16, and \u20132.76 \u00b1 0.03, respectively (n = 3). 61 3.1.3.3 PET Imaging and Ex vivo Biodistribution The PC-3 tumor xenografts were clearly visualized in PET images acquired at 1h post-injection using [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 (Figure 3.4). All of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 were excreted primarily through the renal pathway. [68Ga]Ga-TacsBOMB3 had a higher liver uptake than [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2. With a cysteic acid as part of the linker, [68Ga]Ga-TacsBOMB6 had a lower liver uptake than [68Ga]Ga-TacsBOMB3, but its liver uptake was still higher than [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB5, and [68Ga]Ga-RM2. A very high pancreas uptake was observed for [68Ga]Ga-RM2, but not [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, or [68Ga]Ga-TacsBOMB6. Co-injection with 100 \u03bcg of nonradioactive Ga-TacsBOMB5 increased the overall background level of [68Ga]Ga-TacsBOMB5 especially the uptake in kidneys, and made the PC-3 tumor xenograft almost indistinguishable from the surrounding tissues. 62 Figure 3.4 Representative maximum intensity projection PET images of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 acquired at 1h post-injection in mice bearing PC-3 tumor xenografts. t: tumor; l: liver; k: kidney; p: pancreas; bl: urinary bladder. Biodistribution studies were conducted at 1h post-injection with 68Ga-labeled TacsBOMB2, TacsBOMB3, TacsBOMB5, TacsBOMB6, and RM2 in PC-3 tumor-bearing mice. The results are provided in Figure 3.5-3.7 and Table 3.4, and are consistent with the observations from their PET images. Tumor uptake values for [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 were 10.2 \u00b1 2.27, 6.84 \u00b1 1.66, 15.7 \u00b1 2.17, 6.63 \u00b1 0.40, and 10.5 \u00b1 2.03 %ID\/g, respectively. Pancreas uptake values for [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 were 2.81 \u00b1 0.78, 7.26 \u00b1 1.00, 1.98 \u00b1 0.10, 6.50 \u00b1 0.36, and 41.9 \u00b1 10.1 %ID\/g, respectively. [68Ga]Ga-TacsBOMB3 and [68Ga]Ga-TacsBOMB6 had higher liver uptake values (21.5 \u00b1 5.04 and 12.5 \u00b1 0.88 %ID\/g, respectively) than the other tracers ([68Ga]Ga-TacsBOMB2: 2.61 \u00b1 0.70 %ID\/g; [68Ga]Ga-TacsBOMB5: 0.64 \u00b1 0.11 %ID\/g; [68Ga]Ga-RM2: 0.84 \u00b1 0.55 %ID\/g). Uptake values of brain, muscle, bone, heart, and spleen were < 1 % ID\/g for all evaluated tracers. Compared with [68Ga]Ga-RM2, [68Ga]Ga-TacsBOMB5 had a significantly higher tumor uptake but a lower uptake in most major organs especially in the pancreas, leading to higher tumor-to-organ (bone, muscle, blood, kidney and pancreas) uptake ratios (Figure 3.6 and Table 3.4). 63 Figure 3.5 Uptake of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, and [68Ga]Ga-TacsBOMB6 in PC-3 tumor xenografts and major organs\/tissues of mice at 1h post-injection. Error bars indicate standard deviation. 64 Figure 3.6 Comparison of 68Ga-TacsBOMB5 and 68Ga-RM2 uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1h post-injection. Error bars indicate standard deviation. *p < 0.05, **p < 0.01. Co-injection of nonradioactive Ga-TacsBOMB5 reduced the average uptake of [68Ga]Ga-TacsBOMB5 in the PC-3 tumor xenografts by 83% (15.7 down to 2.60 %ID\/g at 1h post-injection), confirming its specific uptake in tumors. In addition, a significant reduction on the average uptake of [68Ga]Ga-TacsBOMB5 was also found in the pancreas (1.98 down to 0.78 %ID\/g at 1h post-injection), which indicates its specific uptake in the pancreas. On the contrary, the average uptake values of [68Ga]Ga-TacsBOMB5 in other major organs were increased at 1h post-injection with the co-injection of nonradioactive Ga-TacsBOMB5 (Figure 3.7and Table 3.4). Figure 3.7 Comparison of [68Ga]Ga-TacsBOMB5 with\/without co-injection of nonradioactive standard on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1h post-injection. Error bars indicate standard deviation. *p < 0.05, **p < 0.01. 65 Table 3.4 Biodistribution (mean \u00b1 SD, n = 4) and uptake ratios of 68Ga-labeled GRPR-targeting tracers in PC-3 tumor-bearing mice. The mice in the blocked group were co-injected with 100 \u00b5g of nonradioactive Ga-TacsBOMB5. Tissue (%ID\/g) [68Ga]Ga-TacsBOMB2 [68Ga]Ga-TacsBOMB3 [68Ga]Ga-TacsBOMB5 [68Ga]Ga-TacsBOMB6 [68Ga]Ga-RM2 1 h 1 h 1 h 1 h blocked 1 h 1 h Blood 0.76 \u00b1 0.21 2.16 \u00b1 0.28 0.76 \u00b1 0.08 2.57 \u00b1 0.68 1.86 \u00b1 0.12 0.64 \u00b1 0.10 Fat 0.09 \u00b1 0.03 0.19 \u00b1 0.08 0.11 \u00b1 0.01 0.58 \u00b1 0.22 0.25 \u00b1 0.05 0.05 \u00b1 0.03 Testes 0.19 \u00b1 0.05 0.33 \u00b1 0.08 0.23 \u00b1 0.02 1.06 \u00b1 0.13 0.60 \u00b1 0.16 0.18 \u00b1 0.03 Small intestine 1.04 \u00b1 0.30 2.15 \u00b1 0.47 0.66 \u00b1 0.09 1.63 \u00b1 0.62 1.62 \u00b1 0.07 5.08 \u00b1 1.05 Large intestine 0.37 \u00b1 0.16 0.66 \u00b1 0.13 0.41 \u00b1 0.09 1.16 \u00b1 0.41 0.50 \u00b1 0.04 2.19 \u00b1 0.67 Spleen 0.47 \u00b1 0.17 0.68 \u00b1 0.15 0.30 \u00b1 0.03 0.84 \u00b1 0.26 0.84 \u00b1 0.17 0.44 \u00b1 0.26 Pancreas 2.81 \u00b1 0.78 7.26 \u00b1 1.00 1.98 \u00b1 0.10 0.78 \u00b1 0.31 6.50 \u00b1 0.36 41.9 \u00b1 10.1 Stomach 0.32 \u00b1 0.08 1.10 \u00b1 0.18 0.40 \u00b1 0.15 0.63 \u00b1 0.20 0.57 \u00b1 0.07 3.87 \u00b1 2.80 Liver 2.61 \u00b1 0.70 21.5 \u00b1 5.04 0.64 \u00b1 0.11 1.87 \u00b1 0.35 12.5 \u00b1 0.88 0.84 \u00b1 0.55 Adrenal glands 0.57 \u00b1 0.40 1.81 \u00b1 0.72 0.58 \u00b1 0.10 0.85 \u00b1 0.32 1.27 \u00b1 0.33 3.01 \u00b1 0.91 Kidneys 2.51 \u00b1 0.59 4.49 \u00b1 0.51 3.52 \u00b1 0.41 22.9 \u00b1 9.41 3.84 \u00b1 0.43 2.57 \u00b1 0.48 Heart 0.27 \u00b1 0.08 0.66 \u00b1 0.09 0.24 \u00b1 0.03 0.87 \u00b1 0.26 0.58 \u00b1 0.04 0.19 \u00b1 0.03 Lungs 0.75 \u00b1 0.52 3.05 \u00b1 1.29 0.55 \u00b1 0.07 2.13 \u00b1 0.61 1.95 \u00b1 0.96 0.62 \u00b1 0.26 PC-3 tumor 10.2 \u00b1 2.27 6.84 \u00b1 1.66 15.7 \u00b1 2.17 2.60 \u00b1 0.42 6.63 \u00b1 0.40 10.5 \u00b1 2.03 Bone 0.19 \u00b1 0.06 0.42 \u00b1 0.06 0.10 \u00b1 0.04 0.70 \u00b1 0.35 0.26 \u00b1 0.07 0.11 \u00b1 0.03 Muscle 0.15 \u00b1 0.05 0.28 \u00b1 0.05 0.20 \u00b1 0.08 0.91 \u00b1 0.37 0.35 \u00b1 0.14 0.14 \u00b1 0.06 Brain 0.05 \u00b1 0.03 0.06 \u00b1 0.03 0.03 \u00b1 0.01 0.08 \u00b1 0.02 0.05 \u00b1 0.00 0.03 \u00b1 0.01 Tumor\/bone 61.3 \u00b1 25.0 17.0 \u00b1 5.84 175 \u00b1 82.4 4.37 \u00b1 1.99 27.5 \u00b1 7.98 96.5 \u00b1 27.1 Tumor\/muscle 70.1 \u00b1 14.2 26.0 \u00b1 9.92 82.3 \u00b1 19.2 3.21 \u00b1 1.18 20.5 \u00b1 6.45 80.8 \u00b1 27.5 Tumor\/blood 14.0 \u00b1 3.48 3.28 \u00b1 1.19 20.6 \u00b1 2.96 1.05 \u00b1 0.25 3.58 \u00b1 0.24 16.5 \u00b1 3.06 Tumor\/kidney 4.10 \u00b1 0.46 1.55 \u00b1 0.50 4.48 \u00b1 0.69 0.13 \u00b1 0.05 1.73 \u00b1 0.13 4.13 \u00b1 0.73 Tumor\/pancreas 3.70 \u00b1 0.55 0.98 \u00b1 0.37 7.95 \u00b1 1.40 3.77 \u00b1 1.50 1.02 \u00b1 0.05 0.25 \u00b1 0.04 3.1.3.4 In vivo Stability Both [68Ga]Ga-TacsBOMB2 and [68Ga]Ga-TacsBOMB5 were shown to be sufficiently stable in vivo in NRG mice (n = 3), with 83.3 \u00b1 1.45% and 67.1 \u00b1 4.76%, respectively, remaining intact in plasma at 15 min post-injection (Figures 3.8-3.9). These values are not significantly different from the intact fraction of [68Ga]Ga-RM2 (71.9 \u00b1 10.4%, Figure 3.10). On the other hand, no intact [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB5, or [68Ga]Ga-RM2 was detected in the mouse urine samples collected at 15 min post-injection (Figures 3.8-3.10). 66 Figure 3.8 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-TacsBOMB2 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. Figure 3.9 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-TacsBOMB5 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. 0 5 1001020304050Time (min)Radioactivity (mV)0 5 10050100150Time (min)Radioactivity (mV)A.B.0 5 10020406080Time (min)Radioactivity (mV)0 5 10050100150Time (min)Radioactivity (mV)A.B.67 Figure 3.10 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-RM2 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. 3.1.4 Discussion To the best of our knowledge, this is the first report on the development of GRPR-targeting tracers based on the [Leu13\u03c8Thz14]Bombesin(7-14) sequence. The designs of TacsBOMB2, TacsBOMB3, and TacsBOMB4 (Figure 3.1) were based on the potent GRPR antagonists, RC-3950-II (D-Phe-[Leu13\u03c8Thz14]Bombesin(7-14)), RC-3965-II (D-2-Nal-[Leu13\u03c8Thz14]Bombesin(7-14)), and RC-3910-II ((D-Tpi-[Leu13\u03c8Thz14]Bombesin(7-14))), respectively, reported by the Schally group 181, 182. TacsBOMB5 is an NMe-Gly11 derivative of TacsBOMB2 as replacing Gly11 by NMe-Gly has been previously reported to improve the metabolic stability of GRPR-targeting tracers 229, 230. TacsBOMB6 with the addition of a cysteic acid between the Pip linker and the DOTA chelator was designed to improve the hydrophilicity of TacsBOMB3. At our first attempt, the amino acids, Pip linker and DOTA chelator for the synthesis of TacsBOMB2 were sequentially coupled to the Rink Amide MBHA resin. However, after 0 5 1001020304050Time (min)Radioactivity (mV)0 5 100100200300400Time (min)Radioactivity (mV)A.B.68 treating the resin with trifluoroacetic acid for cleavage followed by precipitation with diethyl ether, very little crude product was obtained (data not shown). After checking with MS analysis, the major peak of the isolated crude product showed a molecular weight ~300 dalton higher than the expected product TacsBOMB2 (see Figures 3.11-3.12). This is consistent with the observation by Yraola et al. 251, and it is likely due to the protonation of the reduced peptide bond (a tertiary amine) between Leu13 and Thz14 by trifluoroacetic acid, which strengthened the amide bond between Thz14 and the Rink Amide MBHA resin. Therefore, instead of cleavage at the C-N bond where Thz14 was coupled to the Rink Amide MBHA resin, a labile C-N bond on the resin component was cleaved instead (Figure 3.11). To fix this problem, a more acid labile Sieber resin was used and the desired TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5, and TacsBOMB6 were successfully isolated and characterized (Table 3.1). 69 Figure 3.11 The proposed chemical structure of the observed by-product from cleavage of protected TacsBOMB2 off the Rink Amide MBHA resin. The motif in brown comes from the cleavage of another C-N bond on the Rink Amide MBHA resin. 70 Figure 3.12 MS analysis of the observed by-product. The 942.41 and 629.01 m\/z peaks are the observed [M+2H]2+ and [M+3H]3+ peaks of the by-product with a calculated molecular weight of 1881.92 (see the Figure 3.11). 71 The average Ki values of Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB5, and Ga-TacsBOMB6 were comparable (4.29 to 7.08 nM). This suggests that replacing D-Phe in Ga-TacsBOMB2 with D-2-Nal to obtain Ga-TacsBOMB3, replacing Gly11 in Ga-TacsBOMB2 with NMe-Gly to obtain Ga-TacsBOMB5, and the addition of a cysteic acid between the Pip linker and DOTA chelator of Ga-TacsBOMB3 to obtain Ga-TacsBOMB6 do not have a major effect on their GRPR binding affinity. However, replacing D-Phe in Ga-TacsBOMB2 with D-Tpi to obtain Ga-TacsBOMB4 resulted in a significant loss of binding affinity (Ki =7.08 \u00b1 0.65 vs 458 \u00b1 38.6 nM). This is likely due to the rigidity of the secondary amino group of D-Tpi, which prohibits free rotation of the added Ga-DOTA complex and Pip linker and, therefore, affects its binding to GRPR. Calcium release assays revealed that compared to the antagonist control, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) (50 nM), there was no higher calcium efflux observed by 50 nM of Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, and Ga-TacsBOMB6, confirming their antagonistic characteristics. This suggests that the addition of the Ga-DOTA complex and Pip linker to the N-terminus of the reported GRPR antagonists RC-3950-II (Ga-TacsBOMB2), RC-3965-II (Ga-TacsBOMB3) and RC-3910-II (Ga-TacsBOMB4) does not change their antagonistic characteristics. Similarly, replacing Gly11 with NMe-Gly (Ga-TacsBOMB5) and the addition of a cysteic acid between the Pip linker and DOTA chelator (Ga-TacsBOMB6) do not change their antagonistic characteristics either. The logD7.4 measurements confirmed the hydrophilic properties of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB4, [68Ga]Ga-TacsBOMB5, and [68Ga]Ga-TacsBOMB6. Replacing D-Phe in [68Ga]Ga-TacsBOMB2 with a bulkier D-2-Nal in [68Ga]Ga-TacsBOMB3 increased the lipophilicity (logD7.4 = \u22122.39 \u00b1 0.13 vs \u22121.75 \u00b1 0.04). The addition of 72 a cysteic acid to [68Ga]Ga-TacsBOMB3 resulted in a more hydrophilic [68Ga]Ga-TacsBOMB6 (logD7.4 = \u22121.75 \u00b1 0.04 vs \u22122.55 \u00b1 0.16) as expected. The reduction on the average logD7.4 values from [68Ga]Ga-TacsBOMB2 (\u22122.39 \u00b1 0.13) to [68Ga]Ga-TacsBOMB5 (\u22122.52 \u00b1 0.05) was unexpected as Gly in [68Ga]Ga-TacsBOMB2 is less lipophilic than NMe-Gly in [68Ga]Ga-TacsBOMB5. This suggests that the change in logD7.4 value cannot be determined by the individual replaced amino acid but has to take into account the interaction of the replaced amino acid with the remaining components of the peptide. PET imaging and biodistribution studies (Figures 3.4-3.5 and Table 3.4) confirmed good GRPR targeting capabilities of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, and [68Ga]Ga-TacsBOMB6, as PC-3 tumors were clearly visualized in their PET images. Compared to [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3 had a lower tumor uptake (10.2 \u00b1 2.27 vs 6.84 \u00b1 1.66 %ID\/g), likely due to its more lipophilic nature which also resulted in a higher liver uptake (2.61 \u00b1 0.70 vs 21.5 \u00b1 5.04). The addition of a cysteic acid did lower the liver uptake of [68Ga]Ga-TacsBOMB6 (12.5 \u00b1 0.88 %ID\/g) but no improvement in tumor uptake (6.63 \u00b1 0.40 %ID\/g) was observed. Replacing Gly11 in [68Ga]Ga-TacsBOMB2 with NMe-Gly resulted in [68Ga]Ga-TacsBOMB5 which showed 54% increase in tumor uptake (10.2 \u00b1 2.27 vs 15.7 \u00b1 2.17 %ID\/g) and superior tumor-to-background contrast ratios. To demonstrate the potential for clinical translation of [68Ga]Ga-TacsBOMB5 to detect GRPR-expressing cancers, we conducted head-to-head comparison with the clinically validated [68Ga]Ga-RM2. As shown in Figures 3.4-3.6 and Table 3.4, compared to [68Ga]Ga-RM2, [68Ga]Ga-TacsBOMB5 had a higher tumor uptake (10.5 \u00b1 2.03 vs 15.7 \u00b1 2.17 %ID\/g), a much lower pancreas uptake (41.9 \u00b1 10.1 vs 1.98 \u00b1 0.10 %ID\/g) and higher tumor-to-normal organ uptake ratios, especially the tumor-to-pancreas ratio (0.25 \u00b1 0.04 vs 7.95 \u00b1 1.40). The relatively 73 lower average pancreas uptake (1.98-7.26 %ID\/g, Table 3.4) of [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, and [68Ga]Ga-TacsBOMB6 is consistent with the observation from [68Ga]Ga-ProBOMB1 and [68Ga]Ga-ProBOMB2 derived from D-Phe-[Leu13\u03c8Pro14]Bombesin(7-14) 216, 217. Our data suggest that D-Phe-[Leu13\u03c8Pro14]Bombesin(7-14) and D-Phe-[Leu13\u03c8Thz14]Bombesin(7-14) are promising peptide sequences for the design of GRPR-targeting radiopharmaceuticals with low pancreas uptake, especially for radioligand therapy application to minimize toxicity to the pancreas. In vivo stability studies were conducted to tease out if the higher tumor uptake of [68Ga]Ga-TacsBOMB5 than [68Ga]Ga-RM2 was the results of improved stability from the NMe-Gly replacement. As shown in Figures 3.8-3.10, [68Ga]Ga-TacsBOMB5 was not more stable in vivo than [68Ga]Ga-RM2 against peptidase degradation as their intact fractions in plasma at 15 min post-injection were 67.1 \u00b1 4.76 and 71.9 \u00b1 10.4%, respectively. In addition, GRPR binding affinity of [68Ga]Ga-TacsBOMB5 was not better than [68Ga]Ga-RM2 either as their Ki values were 5.12 \u00b1 0.57 and 1.51 \u00b1 0.24 nM, respectively. One possible explanation is the much lower uptake of [68Ga]Ga-TacsBOMB5 in the pancreas when compared to [68Ga]Ga-RM2 (1.98 \u00b1 0.10 vs 41.9 \u00b1 10.1 %ID\/g), enabling more chances for the circulating [68Ga]Ga-TacsBOMB5 to bind to GRPR in PC-3 tumors. The blocking study (Figure 3.7 and Table 3.4) showed that the average uptake of [68Ga]Ga-TacsBOMB5 in PC-3 tumors was reduced by 83% with the co-injection of nonradioactive standard, confirming its specific uptake in tumors. In addition, the average uptake of [68Ga]Ga-TacsBOMB5 in the pancreas also reduced by 60%, suggesting there is some specific uptake of [68Ga]Ga-TacsBOMB5 in the pancreas as well. This is in agreement with the observation that the pancreas is probably the highest GRPR-expressing normal organ 90, 93, 142. However, compared to the 74 clinically validated [68Ga]Ga-RM2 and [68Ga]Ga-NeoBOMB1, [68Ga]Ga-TacsBOMB5 had ~50% more uptake in PC-3 tumors (10.5 \u00b1 2.03 and 9.83 \u00b1 1.48 %ID\/g, respectively vs 15.7 \u00b1 2.17 %ID\/g), but only a small fraction of uptake in mouse pancreas (41.9 \u00b1 10.1 and 122 \u00b1 28.4 %ID\/g, respectively vs 1.98 \u00b1 0.10 %ID\/g) 216. This suggests that the extremely high uptake of [68Ga]Ga-RM2 and [68Ga]Ga-NeoBOMB1 might not be entirely mediated by GRPR, and possibly by some other off-targets as well. However, it cannot rule out the possibility that compared to [68Ga]Ga-RM2 and [68Ga]Ga-NeoBOMB1, [68Ga]Ga-TacsBOMB5 might be more selective for the human GRPR expressed in PC-3 tumors than the mouse GRPR expressed in mouse pancreas. Further clinical studies of [68Ga]Ga-TacsBOMB5 are needed to validate if the observations from the mouse model can be translated to humans. 3.1.5 Conclusions Modifications on the reported potent GRPR antagonists, D-Phe-[Leu13\u03c8Thz14]Bombesin(7-14) and D-2-Nal-[Leu13\u03c8Thz14]Bombesin(7-14), do not affect their GRPR-targeting capability, and the resulting 68Ga-labeled TacsBOMB2, TacsBOMB3, TacsBOMB5 and TacsBOMB6 can clearly visualize GRPR-expressing PC-3 tumors in PET images. Among them, [68Ga]Ga-TacsBOMB5 shows superior tumor uptake and tumor-to-background contrast ratios than the clinically validated [68Ga]Ga-RM2. Most importantly, the pancreas uptake of [68Ga]Ga-TacsBOMB5 is only a small fraction (~5%) of that of [68Ga]Ga-RM2, so [68Ga]Ga-TacsBOMB5 should be more sensitive for detecting GRPR-expressing pancreatic cancer and other cancer lesions adjacent to the pancreas. Due to the low pancreas uptake of 68Ga-labeled tracers derived from D-Phe-[Leu13\u03c8Thz14]Bombesin(7-14) and D-2-Nal-[Leu13\u03c8Thz14]Bombesin(7-14), these two peptide sequences are promising for the design of 75 GRPR-targeting radiopharmaceuticals, especially for radioligand therapy application to minimize toxicity to the pancreas. 76 3.2 68Ga-labeled [Thz14]Bombesin(7-14) analogs: promising GRPR-targeting agonist PET tracers with low pancreas uptake The following section is an adaption of the following published paper: Wang, L., Bratanovic, I.J., Zhang, Z., Kuo, H.T., Merkens, H., Zeisler, J., Zhang, C., Tan, R., B\u00e9nard, F. and Lin, K.S. 68Ga-labeled [Thz14]Bombesin(7-14) analogs: promising GRPR-targeting agonist PET tracers with low pancreas uptake. Molecules, 2023, 28, 1977. https:\/\/doi.org\/10.3390\/molecules28041977. The compounds disclosed in this report are covered by a recent US patent application (PCT\/CA2023\/050401). Lei Wang, Zhengxing Zhang, Chengcheng Zhang, Ivica Jerolim Bratanovic, Fran\u00e7ois B\u00e9nard, and Kuo-Shyan Lin are listed as inventors of this filed patent application. 3.2.1 Introduction The gastrin-releasing peptide receptor (GRPR) is a transmembrane G protein-coupled receptor (GPCR) which is expressed in central nervous system, gastrointestinal tract and pancreas 90, and regulates a variety of physiological functions such as synaptic plasticity, hormone secretion, smooth muscle contraction, and cell proliferation 90-92. Furthermore, GRPR is shown to be overexpressed in a variety of malignancies 93, 142-147 and involved in a large array of pathophysiological conditions, such as the associations with some neurochemical alterations in neurological disorders, the development of malignant neoplasms, and the proliferation of cancer cells in several cancer types 90, 92, 148-150. Overexpression of GRPR in malignant tissues makes it a promising target for the design of targeted radiopharmaceuticals for the diagnosis and radioligand therapy of GRPR-expressing cancer. Gastrin-releasing peptide (GRP) and bombesin (BBN) are two natural GRPR ligands. GRP and BBN share the same heptapeptide sequence at the C-terminus which has been used as the 77 targeting vector for the design of GRPR-targeting radiopharmaceuticals for cancer diagnosis and radioligand therapy for decades 46, 47, 162, 188-192. Some of the reported GRPR-targeting radioligands have been evaluated in the clinic 46, 47, 162, 189-191. However, all clinically evaluated GRPR-targeting radioligands show an extremely high uptake in the pancreas 46, 189, 191, 193. The high pancreas uptake limits the application of these GRPR-targeting radiopharmaceuticals for detecting cancer lesions adjacent or located in the pancreas, and lowers the maximum tolerated dose for targeted radioligand therapy to minimize toxicity. The Schally group reported a series of GRPR-targeting ligands based on the bombesin(7-14) sequence by substituting Met14 with Thz14 (thiazoline-4-carboxylic acid) and introducing a reduced peptide bond (CH2-N) between residues 13-14 (Leu13\u03c8Thz14) 181, 182. These ligands were confirmed to be GRPR antagonists, and some were proved to have very promising binding affinities towards GRPR (Ki at pM scale) and the ability to inhibit cancer cell proliferation 137, 205, 206. Inspired by their work, our group recently reported a series of 68Ga-labeled DOTA-conjugated GRPR-targeting radioligands derived from the reported [Leu13\u03c8Thz14]Bombesin(7-14), (Figure 3.13A-B) 252. Compared with the clinically validated [68Ga]Ga-RM2, [68Ga]Ga-TacsBOMB2 showed comparable PC-3 tumor uptake and tumor-to-background contrast ratios, while [68Ga]Ga-TacsBOMB5 showed superior PC-3 tumor uptake and tumor-to-background contrast ratios. Most importantly, at 1 h post-injection the pancreas uptake values of [68Ga]Ga-TacsBOMB2 (2.81 \u00b1 0.78 %ID\/g) and [68Ga]Ga-TacsBOMB5 (1.98 \u00b1 0.10 %ID\/g) were much lower than that of [68Ga]Ga-RM2 (41.9 \u00b1 10.1 %ID\/g) 252. The development of GRPR-targeting radiopharmaceuticals has been focused on the use of antagonist sequences as the targeting vector in the past decade partly due to their higher in vivo stability 210, potentially higher tumor uptake due to more binding sites than those available for 78 agonists 211, and\/or less short term adverse effects 96, 201. However, agonists can be internalized upon binding to GRPR and potentially lead to a longer tumor retention 90, 96, 212, which might be preferable especially for use for the development of radiotherapeutic agents. We hypothesized that (1) replacing the reduced peptide bond (Leu13\u03c8Thz14) in our previously reported Ga-TacsBOMB2, Ga-TacsBOMB3 and Ga-TacsBOMB4 (Figure 3.13A) with an amide bond would restore their GRPR agonist characterizations, and (2) the resulting 68Ga-labeled [Thz14]Bombesin(7-14) derivatives might retain the low pancreas uptake characteristics observed from their Leu13\u03c8Thz14 analogs. Thus, in this study we synthesized [Thz14]Bombesin(7-14)-derived TacBOMB2, TacBOMB3, and TacBOMB4 (Figure 3.13C), by replacing the reduced peptide bond (CH2-N) between residues 13-14 (Leu13\u03c8Thz14) with a normal amide bond. Their agonist properties were determined using in vitro fluorescence based calcium release assay. Their potential for imaging GRPR expression was evaluated by in vitro competition binding, positron emission tomography (PET) imaging and ex vivo biodistribution studies in a preclinical prostate cancer model, and compared with a clinically validated GRPR agonist tracer, [68Ga]Ga-AMBA (Figure 3.13D). 79 Figure 3.13 Chemical structures of (A) TacsBOMB2, TacsBOMB3, and TacsBOMB4; (B) TacsBOMB5; (C) TacBOMB2, TacBOMB3, and TacBOMB4; and (D) AMBA. The reduced peptide bond (inside the dashed HNNHHNNHHNNHHNNNSO NH2OHNNOOOONHOOH2NOHNONNN NOHOOOHOHONH O ONHNHNOXTacsBOMB2: X =HNNHNNHHNNHHNNHNNSO NH2OHNNOOOONHOOH2NOOHNONNN NOHOOOHOHOTacsBOMB5TacsBOMB3: X = TacsBOMB4: X =ABHNNHHNNHHNNHHNNNSO NH2OHNNOOOONHOOH2NOHNONNN NOHOOOHOHONH O ONHNHNOXTacBOMB2: X =HNNHHNNHHNNHHNNHOHNNOOOONHOOH2NOAMBATacBOMB3: X = TacBOMB4: X =CDOO O NH2SHNONHONNN NOHOOHOHOO6 7 8 9 10 11 12 13 1480 brown circle) for the compounds in (A) is replaced with an amide bond (inside the dashed brown circle) for the compounds in (C). 3.2.2 Materials and Methods The materials and methods described in this section are provided in Chapter 2. Relevant sections are those describing reagent and instrumentation (Section 2.1), synthesis of DOTA-conjugated precursors (Section 2.3.1), synthesis of nonradioactive Ga-complexed standards (Section 2.4.1), cell culture (Section 2.5), fluorometric calcium release assay (Section 2.6), in vitro competition binding assay (Section 2.7), 68Ga radiolabeling (Section 2.8.1), logD7.4 measurements (Section 2.9), animal studies (Section 2.10), PET imaging and biodistribution studies (Section 2.10.1), in vivo stability studies (Section 2.10.3), and statistical analysis (Sections 2.12). 3.2.3 Results 3.2.3.1 Chemistry and Radiochemistry DOTA-conjugated TacBOMB2, TacBOMB3, and TacBOMB4 were obtained in 30-55% yields, and their nonradioactive Ga-complexed standards were obtained in 58-82% yields. The HPLC conditions for their purification and MS characterizations are provided in Table 3.5 and 3.6. Gallium-68 labeling was conducted in HEPES buffer (2 M, pH 5.0). After HPLC purification, 68Ga-labeled TacBOMB2, TacBOMB3, and AMBA were obtained in 51-80% decay-corrected radiochemical yields with 234-322 GBq\/\u00b5mol molar activity and > 95% radiochemical purity. The HPLC conditions for their purification and quality control are provided in Table 3.7. Table 3.5 HPLC purification conditions and MS characterizations of TacBOMB2, TacBOMB3, and TacBOMB4. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) TacBOMB2 23% CH3CN and 0.1% TFA in H2O 11.3 30 [M+2H]2+ 799.4 [M+2H]2+ 799.6 81 TacBOMB3 25% CH3CN and 0.1% TFA in H2O 13.0 38 [M+2H]2+ 824.4 [M+2H]2+ 824.9 TacBOMB4 25% CH3CN and 0.1% TFA in H2O 15.9 55 [M+2H]2+ 824.9 [M+2H]2+ 825.1 Table 3.6 HPLC purification conditions and MS characterizations of Ga-TacBOMB2, Ga-TacBOMB3, and Ga-TacBOMB4. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Ga-TacBOMB2 23% CH3CN and 0.1% TFA in H2O 12.0 82 [M+2H]2+ 833.9 [M+2H]2+ 833.7 Ga-TacBOMB3 25% CH3CN and 0.1% TFA in H2O 13.9 63 [M+2H]2+ 858.9 [M+2H]2+ 858.5 Ga-TacBOMB4 25% CH3CN and 0.1% TFA in H2O 17.3 58 [M+2H]2+ 859.4 [M+2H]2+ 858.8 Table 3.7 HPLC conditions for the purification and quality control of 68Ga-labeled TacBOMB2 TacBOMB3, and AMBA. FA: formic acid. Compound name HPLC conditions Retention time (min) [68Ga]Ga-TacBOMB2 Semi-Prep 19% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 12.1 QC 21% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 5.8 [68Ga]Ga-TacBOMB3 Semi-Prep 19% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 17.0 QC 22% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 4.7 [68Ga]Ga-AMBA Semi-Prep 19% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 24.8 QC 18.5% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 6.5 3.2.3.2 Agonist Characterization, Binding Affinity, and Hydrophilicity Ga-TacBOMB2, Ga-TacBOMB3, and Ga-TacBOMB4 were confirmed to be GRPR agonists via intracellular calcium release assays using PC-3 cells (Figure 3.14). ATP (50 nM), as a positive control, and bombesin (50 nM), as an agonist control, induced Ca2+ efflux corresponding 82 to 334 \u00b1 39.0 and 754 \u00b1 38.3 relative fluorescence units (RFUs), respectively. For 50 nM of Ga-TacBOMB2, Ga-TacBOMB3, and Ga-TacBOMB4, 361 \u00b1 46.8, 378 \u00b1 87.8, and 121 \u00b1 52.3 RFUs were observed, respectively, which were significantly higher than the 14.9 \u00b1 4.93 and 25.3 \u00b1 1.92 RFUs recorded from the blank control (Dulbecco\u2019s phosphate-buffered saline, DPBS) and the antagonist control ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), 50 nM), respectively. Figure 3.14 Intracellular calcium efflux in PC-3 cells induced by various tested ligands. Error bars indicate standard deviation. Ga-TacBOMB2, Ga-TacBOMB3, Ga-TacBOMB4, and Ga-AMBA inhibited the binding of [125I-Tyr4 ]Bombesin to PC-3 cells in a dose-dependent manner (Figure 3.15). The calculated Ki values for Ga-TacBOMB2, Ga-TacBOMB3, Ga-TacBOMB4, and Ga-AMBA were 7.62 \u00b1 0.19, 6.02 \u00b1 0.59, 590 \u00b1 36.5, and 0.99 \u00b1 0.08 nM, respectively (n = 3). The hydrophilicity of [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA were measured using the shake flask method, and their logD7.4 values were calculated to be \u22123.21 \u00b1 0.03, \u22122.55 \u00b1 0.03, and \u22123.66 \u00b1 0.29, respectively (n = 3). 83 Figure 3.15 Displacement curves of [125I-Tyr4]Bombesin by Ga-TacBOMB2, Ga-TacBOMB3, Ga-TacBOMB4, and Ga-AMBA generated using GRPR-expressing PC-3 cells. Error bars indicate standard deviation. 3.2.3.3 PET Imaging and Ex vivo Biodistribution The PC-3 tumor xenografts were clearly visualized in PET images acquired at 1 h post-injection using [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA (Figure 3.16). Both [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 were primarily excreted via the renal pathway, with low uptake in most background organs\/tissues. [68Ga]Ga-TacBOMB2 had a better tumor-to-background contrast than [68Ga]Ga-TacBOMB3 and [68Ga]Ga-AMBA. [68Ga]Ga-AMBA showed a very high pancreas uptake, while the pancreases were invisible in the PET images of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3. Co-injection with 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) significantly decreased the uptake of [68Ga]Ga-TacBOMB2 in PC-3 tumor xenografts. 84 Figure 3.16 Representative PET images of [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA acquired at 1 h post-injection in mice bearing PC-3 tumor xenografts. t: tumor; k: kidney; p\/i: pancreas\/intestines; bl: urinary bladder. Biodistribution studies were performed at 1 h post-injection with 68Ga-labeled TacBOMB2, TacBOMB3, and AMBA in PC-3 tumor-bearing mice. Biodistribution results are consistent with the observations from their PET images and are provided in Figures 3.17-3.19 and Table 3.8. PC-3 tumor uptake values for [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA were 5.95 \u00b1 0.50, 5.09 \u00b1 0.54, and 6.69 \u00b1 1.03 %ID\/g, respectively. Pancreas uptake values for [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA were 1.30 \u00b1 0.14, 2.41 \u00b1 0.72, and 62.4 \u00b1 4.26%ID\/g, respectively. Intestine uptake values for [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA were 0.60 \u00b1 0.12, 1.06 \u00b1 0.26, and 8.62 \u00b1 4.26%ID\/g, respectively. Uptake values for the brain, muscle, fat, bone, liver, stomach, heart, and spleen were < 1%ID\/g for both [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3. 85 Figure 3.17 Uptake of [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA in PC-3 tumor xenografts and major organs\/tissues of mice at 1 h post-injection. Error bars indicate standard deviation (n = 4). Figure 3.18 Comparison of tumor-to-organ contrast ratios of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-AMBA obtained from PC-3 tumor-bearing mice at 1 h post-injection. Error bars indicate standard deviation (n = 4). * p < 0.05; ** p < 0.01. 86 Table 3.8 Biodistribution (mean \u00b1 SD, n = 4) and uptake ratios of 68Ga-labeled GRPR-targeting tracers in PC-3 tumor-bearing mice. The mice in the blocked group were co-injected with 100 \u00b5g of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14). * and ** indicate p < 0.05 and < 0.01, respectively, when comparing the 1 h and 1 h blocked data of [68Ga]Ga-TacBOMB2. Tissue (%ID\/g) [68Ga]Ga-TacBOMB2 [68Ga]Ga-TacBOMB3 [68Ga]Ga-AMBA 1 h 1 h blocked 1 h 1 h Blood 0.39 \u00b1 0.12 0.55 \u00b1 0.19 1.06 \u00b1 0.51 0.58 \u00b1 0.07 Fat 0.10 \u00b1 0.03 0.07 \u00b1 0.02 0.19 \u00b1 0.18 0.08 \u00b1 0.02 Testes 0.16 \u00b1 0.05 0.21 \u00b1 0.16 0.37 \u00b1 0.06 0.20 \u00b1 0.03 Intestines 0.60 \u00b1 0.12 0.28 \u00b1 0.07** 1.06 \u00b1 0.26 8.62 \u00b1 1.00 Spleen 0.27 \u00b1 0.10 0.18 \u00b1 0.05 0.50 \u00b1 0.32 2.49 \u00b1 2.16 Pancreas 1.30 \u00b1 0.14 0.15 \u00b1 0.03** 2.41 \u00b1 0.72 62.4 \u00b1 4.26 Stomach 0.54 \u00b1 0.27 0.13 \u00b1 0.06* 0.82 \u00b1 0.37 2.32 \u00b1 0.60 Liver 0.29 \u00b1 0.13 0.29 \u00b1 0.06 0.65 \u00b1 0.19 0.43 \u00b1 0.05 Kidneys 2.43 \u00b1 0.16 1.99 \u00b1 0.59 4.61 \u00b1 2.72 5.70 \u00b1 2.45 Heart 0.14 \u00b1 0.02 0.16 \u00b1 0.04 0.35 \u00b1 0.18 0.22 \u00b1 0.02 Lungs 0.39 \u00b1 0.10 0.39 \u00b1 0.10 1.63 \u00b1 0.75 0.62 \u00b1 0.06 PC-3 tumor 5.95 \u00b1 0.50 0.92 \u00b1 0.22** 5.09 \u00b1 0.54 6.69 \u00b1 1.03 Bone 0.26 \u00b1 0.11 0.10 \u00b1 0.03* 0.19 \u00b1 0.09 0.33 \u00b1 0.13 Muscle 0.13 \u00b1 0.08 0.13 \u00b1 0.04 0.24 \u00b1 0.12 0.17 \u00b1 0.01 Brain 0.03 \u00b1 0.01 0.02 \u00b1 0.00 0.04 \u00b1 0.02 0.04 \u00b1 0.01 Tumor\/bone 28.9 \u00b1 18.3 8.94 \u00b1 1.49* 29.9 \u00b1 8.50 22.6 \u00b1 9.13 Tumor\/muscle 57.9 \u00b1 32.8 7.14 \u00b1 1.40* 23.5 \u00b1 9.61 39.5 \u00b1 7.46 Tumor\/blood 16.4 \u00b1 5.64 1.71 \u00b1 0.18** 5.61 \u00b1 2.33 11.7 \u00b1 2.10 Tumor\/intestines 10.5 \u00b1 3.06 3.33 \u00b1 0.67** 4.93 \u00b1 0.84 0.79 \u00b1 0.22 Tumor\/kidney 2.46 \u00b1 0.33 0.47 \u00b1 0.04** 1.34 \u00b1 0.58 1.36 \u00b1 0.73 Tumor\/pancreas 4.64 \u00b1 0.77 6.22 \u00b1 0.67* 2.21 \u00b1 0.40 0.11 \u00b1 0.01 Although the tumor uptake values of [68Ga]Ga-AMBA and [68Ga]Ga-TacBOMB2 were comparable, [68Ga]Ga-TacBOMB2 showed better tumor-to-organ uptake ratios in some major organs\/tissues, such as bone, muscle, blood, kidney, pancreas, and intestine (Figure 3.18 and Table 3.8). The tumor-to-pancreas uptake ratio of [68Ga]Ga-AMBA (0.11 \u00b1 0.01) was much lower than that of [68Ga]Ga-TacBOMB2 (4.64 \u00b1 0.77, p < 0.01). Similarly, the tumor-to-intestine uptake ratio of [68Ga]Ga-AMBA was 0.79 \u00b1 0.22, which was also much lower than that of [68Ga]Ga-TacBOMB2 (10.5 \u00b1 3.06, p < 0.01). 87 Figure 3.19 Comparison of [68Ga]Ga-TacBOMB2 with\/without co-injection of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6\u201314) on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. Error bars indicate standard deviation (n = 4). * p < 0.05; ** p < 0.01. The co-injection of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) reduced the uptake of [68Ga]Ga-TacBOMB2 in PC-3 tumor xenografts by 85% (from 5.95 \u00b1 0.50 to 0.92 \u00b1 0.22%ID\/g, p < 0.01) at 1 h post-injection. Similarly, a significant reduction in the average uptake of [68Ga]Ga-TacBOMB2 was also observed in the intestines, pancreas, and stomach (Figure 3.19). 3.2.3.4 In vivo Stability [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA were shown to have limited in vivo stability in NRG mice (n = 3) (Figure 3.20-3.22). Only 12.7 \u00b1 2.93% of [68Ga]Ga-TacBOMB2 was found intact in plasma at 15 min post-injection, which was significantly lower than the intact fraction of [68Ga]Ga-AMBA (39.4 \u00b1 10.8%, p = 0.01). The difference between the intact fraction of [68Ga]Ga-TacBOMB3 (27.3 \u00b1 4.84%) and [68Ga]Ga-AMBA was not statistically significant (p = 0.15). Conversely, no intact [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, or 88 [68Ga]Ga-AMBA was detected in the mouse urine samples collected at 15 min post-injection (Figure 3.20-3.22). Figure 3.20 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-TacBOMB2 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. Figure 3.21 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-TacBOMB3 in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. 89 Figure 3.22 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-AMBA in mouse plasma (A) and urine (B) samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. 3.2.4 Discussion The Schally group published a series of GRPR antagonists with picomolar binding affinity, including RC-3950-II (D-Phe-[Leu13\u03c8Thz14]Bombesin(7-14)), RC-3965-II (D-2-Nal-[Leu13\u03c8Thz14]Bombesin(7-14)), and RC-3910-II ((D-Tpi-[Leu13\u03c8Thz14]Bombesin(7-14))) 181, 182. Based on these three peptides, our group developed three Ga-complexed, DOTA-conjugated, GRPR-targeting ligands, Ga-TacsBOMB2, Ga-TacsBOMB3, and Ga-TacsBOMB4 (Figure 3.13A), respectively 252. All Ga-TacsBOMB2, Ga-TacsBOMB3, and Ga-TacsBOMB4 ligands were also confirmed to be GRPR antagonists. In this study, we replaced their C-terminal reduced peptide bond (Leu13\u03c8Thz14) with a normal amide bond and investigated whether the resulting Ga-TacBOMB2, Ga-TacBOMB3, and Ga-TacBOMB4 (Figure 3.13C) restored agonist characteristics as well as their potential for PET imaging. 90 Intracellular calcium release assays revealed that all Ga-TacBOMB2, Ga-TacBOMB3, and Ga-TacBOMB4 ligands induced significantly more intracellular Ca2+ efflux compared to the antagonist control, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) and the blank control, DPBS (Figure 3.14). This observation confirmed the agonist property of Ga-TacBOMB2, Ga-TacBOMB3, and Ga-TacBOMB4, which are different from those previously reported GRPR antagonists, Ga-TacsBOMB2, Ga-TacsBOMB3, and Ga-TacsBOMB4 252. This confirms that replacing the C-terminal-reduced peptide bond (Leu13\u03c8Thz14) of Ga-TacsBOMB2, Ga-TacsBOMB3, and Ga-TacsBOMB4 with a normal amide bond restores their agonist characteristics. As the internalization of GRPR agonists after receptor binding may potentially result in a longer tumor retention period 90, 96, 212, radiolabeled [Thz14]Bombesin(7-4)-derived, GRPR-targeting ligands may be preferable to [Leu13\u03c8Thz14]Bombesin(7-14) derivatives, especially for radioligand therapy applications. We further determined the binding affinities of these three GRPR-targeting ligands by conducting an in vitro competition binding assay. The Ki values of Ga-TacBOMB2 (7.62 \u00b1 0.19 nM) and Ga-TacBOMB3 (6.02 \u00b1 0.59 nM) were comparable, while Ga-TacBOMB4 showed a much poorer binding affinity to GRPR (Ki = 590 \u00b1 36.5 nM). This finding is consistent with our previous report that replacing D-Phe in Ga-TacsBOMB2 with D-2-Nal doesn\u2019t affect the binding affinity towards GRPR, while replacing D-Phe with D-Tpi leads to a significantly lower binding to GRPR. One possible explanation is that the free rotation of the Pip linker and the Ga-DOTA complex is compromised by the rigidity of the secondary amino group of D-Tpi, which results in a significant loss of binding affinity to GRPR. As Ga-TacBOMB4 showed inferior binding affinity to GRPR, we labeled only TacBOMB2 and TacBOMB3 with 68Ga for further in vivo evaluation. The hydrophilicity of 91 [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 was estimated by measuring their logD7.4 values and was then compared with that of [68Ga]Ga-AMBA. [68Ga]Ga-AMBA was confirmed to be the most hydrophilic tracer (logD7.4 = \u22123.66 \u00b1 0.29), followed by [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3, with logD7.4 values of \u22123.21 \u00b1 0.03 and \u22122.55 \u00b1 0.03, respectively. Based on our previous study, we found that replacing D-Phe in [68Ga]Ga-TacsBOMB2 with a bulkier D-2-Nal reduced the hydrophilicity (the average logD7.4 value increased by 0.64 from \u22122.39 \u00b1 0.13 for [68Ga]Ga-TacsBOMB2 to \u22121.75 \u00b1 0.04 for [68Ga]Ga-TacsBOMB3) 252. The reduction in hydrophilicity was also observed in this study by replacing D-Phe in [68Ga]Ga-TacBOMB2 with a bulkier D-2-Nal. The average logD7.4 value increased by 0.66 from \u22123.21 \u00b1 0.03 for [68Ga]Ga-TacBOMB2 to \u22122.55 \u00b1 0.03 for [68Ga]Ga-TacBOMB3, which is consistent with the 0.64 increase in the previous report, by converting [68Ga]Ga-TacsBOMB2 to [68Ga]Ga-TacsBOMB3. Similarly, we also observed that the amide bond derivatives ([68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3) were more hydrophilic than their corresponding reduced-peptide-bond derivatives ([68Ga]Ga-TacsBOMB2 and [68Ga]Ga-TacsBOMB3). The logD7.4 value reduced by ~0.80 when replacing a reduced peptide bond with an amide bond (a 0.82 reduction, from \u22122.39 \u00b1 0.13 for [68Ga]Ga-TacsBOMB2 to \u22123.21 \u00b1 0.03 for [68Ga]Ga-TacBOMB2; a 0.80 reduction, from \u22121.75 \u00b1 0.04 for [68Ga]Ga-TacsBOMB3 to \u22122.55 \u00b1 0.03 for [68Ga]Ga-TacBOMB3). Both [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 clearly visualized the PC-3 tumor xenografts in their PET images, which confirms the good targeting capabilities of both tracers to GRPR-expressing tumors (Figure 3.16). The biodistribution results of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 were consistent with the observations from their PET images. Both [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 showed good uptake in PC-3 tumor xenografts, with uptake values of 5.95 \u00b1 0.50 and 5.09 \u00b1 0.54 %ID\/g, respectively, which are comparable to 92 that of [68Ga]Ga-AMBA (6.69 \u00b1 1.03%ID\/g). However, the pancreas uptake values of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 were much lower than that of [68Ga]Ga-AMBA (1.30\u20132.41 %ID\/g vs 62.4 %ID\/g) (Figure 3.17 and Table 3.8). This suggests that the [Thz14]Bombesin(7-14) pharmacophore is a promising targeting vector for the design of GRPR-targeting radiopharmaceuticals with low pancreas uptake. Our data suggest that replacing the Leu13\u03c8Thz14 reduced peptide bond in [68Ga]Ga-TacsBOMB2 and [68Ga]Ga-TacsBOMB3 not only results in agonist ligands ([68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3) with preserved good GRPR binding affinity, but also their low pancreas uptake characteristics. Compared with [68Ga]Ga-AMBA, [68Ga]Ga-TacBOMB2 also showed lower background uptake, resulting in better tumor-to-background contrast ratios (Figures 3.16 and 3.18, and Table 3.8). This suggests that [68Ga]Ga-TacBOMB2 is a better imaging tracer than [68Ga]Ga-AMBA to detect GRPR-expressing malignant lesions. Interestingly, our previous study showed that [68Ga]Ga-TacsBOMB3 had significantly higher liver uptake (21.5 \u00b1 5.04%ID\/g) 252, while the liver uptake value of [68Ga]Ga-TacBOMB3 was only 0.65 \u00b1 0.19%ID\/g. This is most likely due to the increased hydrophilicity of [68Ga]Ga-TacBOMB3 (logD7.4: \u22122.55 \u00b1 0.03 for [68Ga]Ga-TacBOMB3 and \u22121.75 \u00b1 0.04 for [68Ga]Ga-TacsBOMB3). A blocking study was performed for [68Ga]Ga-TacBOMB2 on PC-3 tumor-bearing mice to confirm targeting specificity by co-injecting 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14). An 85% reduction in the average uptake of [68Ga]Ga-TacBOMB2 in PC-3 tumor xenografts was observed with the co-injection of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), confirming its specific uptake in tumors (Figure 3.19 and Table 3.8). Moreover, the average uptake values of [68Ga]Ga-TacBOMB2 in the pancreas and stomach were also reduced by 88% and 76%, respectively, indicating that there was specific uptake of [68Ga]Ga-93 TacBOMB2 in the pancreas and stomach as well, which is consistent with the reported physiological expression of GRPR in both organs 90, 93, 142. In vivo stability studies revealed that [68Ga]Ga-AMBA was more stable than [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 in mouse plasma, as their intact fractions were 39.4 \u00b1 10.8, 12.7 \u00b1 2.93 and 27.3 \u00b1 4.84%, respectively, at 15 min post-injection (Figures 3.20-3.22). This indicates that the slightly higher tumor uptake of [68Ga]Ga-AMBA may also owing to its better in vivo stability other than its better binding affinity toward GRPR. When comparing [68Ga]Ga-TacBOMB2 with our previously reported [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB2 was much more stable in vivo, with 83.3 \u00b1 1.15% remaining intact at 15 min post-injection. This suggests that replacing the reduced peptide bond (Leu13\u03c8Thz14) with an amide bond results in potential cleavage site(s) for endogenous peptidases, leading to a reduction in stability. However, this also emphasizes that there is potential for improvement for [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 if their in vivo stability can be enhanced, likely by substituting some of the amino acids in the targeting sequences with their unnatural amino acids analogs. In addition to RC-3950-II and RC-3965-II for the design of [68Ga]Ga-TacsBOMB2\/[68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacsBOMB3\/[68Ga]Ga-TacBOMB3, respectively, there are other potent [AA13\u03c8AA14]Bombesin-derived antagonists reported by the Schally group and others 253, 254. Our data suggest that these [AA13\u03c8AA14]Bombesin-derived antagonists can be used directly for the design of GRPR-targeting antagonist radioligands, or alternatively, by replacing the (AA13\u03c8AA14) reduced peptide bond with an amide bond for the design of GRPR-targeting agonist radioligands. 94 3.2.5 Conclusions Replacing the (Leu13\u03c8Thz14) reduced peptide bond in the previously reported GRPR antagonist tracers, [68Ga]Ga-TacsBOMB2 and [68Ga]Ga-TacsBOMB3, with an amide bond retains their high GRPR binding affinity, but the resulting [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 become GRPR agonists. Similar to [68Ga]Ga-TacsBOMB2 and [68Ga]Ga-TacsBOMB3, the derived [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 agonist PET tracers also retain in vivo GRPR-targeting capabilities, as demonstrated by their good tumor uptake and tumor-to-background contrast ratios in imaging and biodistribution studies. Compared with the clinically validated agonist PET tracer [68Ga]Ga-AMBA, [68Ga]Ga-TacBOMB2 has comparable tumor uptake but higher tumor-to-background contrast ratios. Therefore, [68Ga]Ga-TacBOMB2 is promising for clinical development to detect GRPR-expressing tumors with PET. Due to the agonist characteristics, potentially longer tumor retention, and negligible pancreatic uptake of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3, [Thz14]Bombesin(7-14) is a promising vector for the design of GRPR-targeting radiopharmaceuticals, particularly for radioligand therapy applications to minimize toxicity to the pancreas. 95 Chapter 4: Modifying the amino acids around the cleavage sites to increase the stability of GRPR-targeted peptides 4.1 Unnatural amino acid substitutions to improve in vivo stability and tumor uptake of 68Ga-labeled GRPR-targeted TacBOMB2 derivatives for cancer imaging with positron emission tomography The following section is an adaption of the following published paper: Wang, L., Kuo, H.T., Zhang, Z., Zhang, C., Chen, C.C., Chapple, D., Wilson, R., Colpo, N., B\u00e9nard, F. and Lin, K.S. Unnatural amino acid substitutions to improve in vivo stability and tumor uptake of 68Ga-labeled GRPR-targeted TacBOMB2 derivatives for cancer imaging with positron emission tomography. EJNMMI Radiopharmacy and Chemistry, 2024, 9, 8. https:\/\/doi.org\/10.1186\/s41181-024-00241-7. The compounds disclosed in this report are covered by a recent patent application (PCT\/CA2023\/050401). Kuo-Shyan Lin, Fran\u00e7ois B\u00e9nard, Lei Wang, Zhengxing Zhang and Chengcheng Zhang are also entitled to potential royalties upon commercialization of patented compounds. 4.1.1 Introduction Gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor, expressed in pancreas, gastrointestinal tract, and central nervous system, and involved in physiological functions such as synaptic plasticity, hormone secretion, and smooth muscle contraction 90-92. Overexpression of GRPR has been reported to induce cancer cell proliferation and facilitate malignant neoplasm development 90, 92, 93, 142-150. The overexpression of GRPR in various tumors makes it a promising target for the design of targeted radiopharmaceuticals for diagnosis and radioligand therapy of GRPR-expressing cancers. 96 Two natural ligands, gastrin-releasing peptide (GRP) and bombesin (BBN) show high binding affinity towards GRPR and share the same heptapeptide sequence (Trp-Ala-Val-Gly-His-Leu-Met-NH2) at the C-terminus 111, 186, 187. The C-terminal heptapeptide of GRP and BBN has been used as a template for designing GRPR-targeted radiopharmaceuticals for decades 46, 47, 162, 188-192. The derivatives of GRP and BBN have been radiolabeled for imaging with single photon emission computed tomography (SPECT) and positron emission tomography (PET), and some of them have also been radiolabeled with beta and alpha emitters for radiotherapeutic applications 46, 187, 189, 191, 193, 194. However, the current clinically validated GRPR-targeted radioligands show an extremely high uptake in the pancreas 46, 189-191, 193, which not only limits the detection of cancer lesions located in or adjacent to the pancreas, but also lowers the maximum tolerated dose for targeted radioligand therapy. Our group recently reported 68Ga-labeled TacsBOMB2 based on a known pseudopeptide-bond-containing antagonist sequence [Leu13\u03c8Thz14]Bombesin(7-14), which showed significantly lower pancreas uptake than the clinically validated [68Ga]Ga-RM2 252. Replacing the reduced peptide bond (Leu13\u03c8Thz14) with an amide bond restores the GRPR agonist characterizations and the derived [68Ga]Ga-TacBOMB2 retained high GRPR binding affinity and low uptake in mouse pancreas 255. The development of GRPR-targeted radiopharmaceuticals has been focused on using antagonist sequences as targeting vectors because of their potentially higher tumor uptake due to higher in vivo stability 210 and more binding sites than those available for agonists 211, and\/or less short term adverse effects 96, 201. However, agonists can be internalized upon binding to GRPR and lead to a longer tumor retention 90, 96, 212, which might be preferable especially for the development of radiotherapeutic agents. The in vivo instability of GRPR-targeted ligands is caused by enzymatic degradation especially by neutral endopeptidase 24.11 (NEP) 201, 223. The reported cleavage sites 97 including His12-Leu13, Trp8-Ala9 and Gln7-Trp8 for AMBA derivatives and Trp8-Ala9, Ala9-Val10 and Gln7-Trp8 for RM2 derivatives 190, 224. We hypothesized that (1) replacing amino acids at the potential cleavage sites of our previously reported GRPR agonist [68Ga]Ga-TacBOMB2 ([68Ga]Ga-DOTA-Pip-D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Thz14-NH2) with unnatural amino acids can improve in vivo stability and retain the agonist characteristics; and (2) the resulting stabilized [68Ga]Ga-TacBOMB2 derivatives can also retain the minimal pancreas uptake characteristics. Thus, in this study we first synthesized the GRPR-targeted sequence of TacBOMB2 (LW01085, D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Thz14-NH2, Figure 4.1) and systematically substituted the amino acids (Gln7, Trp8, Ala9, Val10, Gly11 and His12) at its potential cleavage sites with an unnatural amino acid. The derivatives with high GRPR binding affinity were coupled with the DOTA chelator and 4-amino-(1-carboxymethyl)piperidine (Pip) linker. The binding affinities of their Ga-complexed standards were further confirmed by in vitro competition assays, and their agonist characteristics were confirmed by calcium release assays. Finally, the lead candidates were radiolabeled with 68Ga and evaluated by PET imaging and ex vivo biodistribution studies using the GRPR-expressing PC-3 prostate cancer model. 98 Figure 4.1 Chemical structures and GRPR binding affinities (Ki, mean \u00b1 SD, n = 3) of (A) LW01085 and its derivatives with an unnatural amino acid substitution at (B) His12, (C) Val10, (D) Ala9, (E) Gln7, (F) Val10-Gly11, and (G) Trp8. The potential cleavage sites of LW01085 are pointed by black arrows. 4.1.2 Materials and Methods The materials and methods described in this section are provided in Chapter 2. Relevant sections are those describing reagent and instrumentation (Section 2.1), synthesis of DOTA-conjugated precursors (Section 2.3.1), synthesis of GRPR-targeted peptides (Section 2.3.2), synthesis of nonradioactive Ga-complexed standards (Section 2.4.1), cell culture (Section 2.5), 99 fluorometric calcium release assay (Section 2.6), in vitro competition binding assay (Section 2.7), 68Ga radiolabeling (Section 2.8.1), logD7.4 measurements (Section 2.9), animal studies (Section 2.10), PET imaging and biodistribution studies (Section 2.10.1), in vivo stability studies (Section 2.10.3), and statistical analysis (Sections 2.12). 4.1.3 Results 4.1.3.1 Chemistry and Radiochemistry LW01085 and its derivatives were synthesized in 2.2 to 46% yields and > 95% purity (Tables 4.1 and 4.2). DOTA-conjugated GRPR-targeted peptides were obtained in 4.0 to 33% yields, and their nonradioactive Ga-complexed standards were obtained in 47 to 87% yields. The HPLC conditions for their purification, MS characterizations, and purity characterizations are provided in Tables 4.3 to 4.6. Table 4.1 Peptide sequences and purities of GRPR-targeted peptides. The substituted unnatural amino acids are in bold. Name Sequence Purity (%) LW01085 D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW01088 D-Phe-Gln-Trp-Ala-Val-Gly-NMe-His-Leu-Thz-NH2 98 LW01080 D-Phe-Gln-Trp-Ala-Tle-Gly-His-Leu-Thz-NH2 99 LW02016 D-Phe-Gln-Trp-Ala-cyclopropylglycine-Gly-His-Leu-Thz-NH2 98 LW02011 D-Phe-Gln-Trp-Ala-2,3-dehydro-Val-Gly-His-Leu-Thz-NH2 99 LW01083 D-Phe-Gln-Trp-Ala-2-Abu-Gly-His-Leu-Thz-NH2 96 LW02019 D-Phe-Gln-Trp-Ala-cyclobutaneacetic acid-Gly-His-Leu-Thz-NH2 98 LW01075 D-Phe-Gln-Trp-Aib-Val-Gly-His-Leu-Thz-NH2 99 LW01078 D-Phe-Gln-Trp-2-Abu-Val-Gly-His-Leu-Thz-NH2 99 LW02030 D-Phe-Hse-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 98 LW01128 D-Phe-Gln-Trp-Ala-3-amino-1-carboxymethylcarprolactame-His-Leu-Thz-NH2 95 LW01136 D-Phe-Gln-Trp(Me)-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW01137 D-Phe-Gln-6-Cl-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 98 LW01183 D-Phe-Gln-4-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW02009 D-Phe-Gln-2-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW01177 D-Phe-Gln-7-F-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 98 100 LW01191 D-Phe-Gln-D-Tpi-Ala-Val-Gly-His-Leu-Thz-NH2 96 LW01173 D-Phe-Gln-5-OH-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW02007 D-Phe-Gln-7-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW01171 D-Phe-Gln-6-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW01182 D-Phe-Gln-5-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 98 LW01175 D-Phe-Gln-6-F-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW01166 D-Phe-Gln-5-F-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW01180 D-Phe-Gln-4-F-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 96 LW02013 D-Phe-Gln-7-Aza-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW02015 D-Phe-Gln-Bta-Ala-Val-Gly-His-Leu-Thz-NH2 96 Table 4.2 HPLC purification conditions and MS characterizations of GRPR-targeted peptides. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) LW01085 23% CH3CN and 0.1% TFA in H2O 16.0 41 [M+H]+ 1071.5 [M+H]+ 1071.8 LW01088 23% CH3CN and 0.1% TFA in H2O 17.4 29 [M+H]+ 1085.5 [M+H]+ 1085.7 LW01080 26% CH3CN and 0.1% TFA in H2O 9.0 26 [M+H]+ 1085.5 [M+H]+ 1086.0 LW02016 25% CH3CN and 0.1% TFA in H2O 8.4 11 [M+H]+ 1069.5 [M+H]+ 1069.4 LW02011 25% CH3CN and 0.1% TFA in H2O 9.0 5.0 [M+H]+ 1069.5 [M+H]+ 1069.7 LW01083 22% CH3CN and 0.1% TFA in H2O 12.7 46 [M+H]+ 1057.5 [M+H]+ 1057.8 LW02019 26% CH3CN and 0.1% TFA in H2O 9.8 15 [M+H]+ 1083.5 [M+H]+ 1083.5 LW01075 25% CH3CN and 0.1% TFA in H2O 11.6 13 [M+H]+ 1085.5 [M+H]+ 1085.8 LW01078 25% CH3CN and 0.1% TFA in H2O 10.8 27 [M+H]+ 1085.5 [M+H]+ 1085.7 LW02030 25% CH3CN and 0.1% TFA in H2O 12.2 2.2 [M+H]+ 1044.5 [M+H]+ 1044.3 LW01128 24% CH3CN and 0.1% TFA in H2O 14.3 10 [M+H]+ 1083.5 [M+H]+ 1083.5 LW01136 27% CH3CN and 0.1% TFA in H2O 9.8 30 [M+H]+ 1085.5 [M+H]+ 1085.8 LW01137 28% CH3CN and 0.1% TFA in H2O 12.6 18 [M+H]+ 1105.5 [M+H]+ 1105.7 LW01183 26% CH3CN and 0.1% TFA in H2O 10.3 26 [M+H]+ 1085.5 [M+H]+ 1085.7 101 LW02009 26% CH3CN and 0.1% TFA in H2O 9.1 23 [M+H]+ 1085.5 [M+H]+ 1085.5 LW01177 25% CH3CN and 0.1% TFA in H2O 12.2 24 [M+H]+ 1089.5 [M+H]+ 1089.7 LW01191 27% CH3CN and 0.1% TFA in H2O 11.3 20 [M+H]+ 1083.5 [M+H]+ 1083.4 LW01173 22% CH3CN and 0.1% TFA in H2O 8.6 21 [M+H]+ 1087.5 [M+H]+ 1087.6 LW02007 27% CH3CN and 0.1% TFA in H2O 0.9 25 [M+H]+ 1085.5 [M+H]+ 1085.4 LW01171 26% CH3CN and 0.1% TFA in H2O 11.5 29 [M+H]+ 1085.5 [M+H]+ 1085.4 LW01182 26% CH3CN and 0.1% TFA in H2O 11.7 33 [M+H]+ 1085.5 [M+H]+ 1085.7 LW01175 25% CH3CN and 0.1% TFA in H2O 13.0 40 [M+H]+ 1089.5 [M+H]+ 1089.5 LW01166 25% CH3CN and 0.1% TFA in H2O 10.5 46 [M+H]+ 1089.5 [M+H]+ 1089.5 LW01180 25% CH3CN and 0.1% TFA in H2O 11.4 36 [M+H]+ 1089.5 [M+H]+ 1089.6 LW02013 18% CH3CN and 0.1% TFA in H2O 13.5 8.4 [M+H]+ 1072.5 [M+H]+ 1072.6 LW02015 24% CH3CN and 0.1% TFA in H2O 14.4 24 [M+H]+ 1088.5 [M+H]+ 1088.6 Table 4.3 Peptide sequences and purities of DOTA-conjugated GRPR-targeted peptides. The substituted unnatural amino acids are in bold. Name Sequence Purity (%) LW01107 DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-NMe-His-Leu-Thz-NH2 95 LW01108 DOTA-Pip-D-Phe-Gln-Trp-Ala-Tle-Gly-His-Leu-Thz-NH2 99 LW01149 DOTA-Pip- D-Phe-Gln-\u03b1-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 98 LW02021 DOTA-Pip-D-Phe-Gln-7-F-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 98 LW02023 DOTA-Pip-D-Phe-Gln-5-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 98 LW02025 DOTA-Pip-D-Phe-Gln-2-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 LW01110 DOTA-Pip-D-Phe-Gln-Trp-Ala-Tle-Gly-NMe-His-Leu-Thz-NH2 97 LW01142 DOTA-Pip-D-Phe-His-Trp-Ala-Tle-Gly-NMe-His-Leu-Thz-NH2 99 LW01143 DOTA-Pip-D-Phe-His-Trp-Ala-Tle-NMe-Gly-NMe-His-Leu-Thz-NH2 99 LW02040 DOTA-Pip-D-Phe-Gln-7-F-Trp-Ala-Tle-Gly-NMe-His-Leu-Thz-NH2 97 102 Table 4.4 HPLC purification conditions and MS characterizations of DOTA-conjugated GRPR-targeted peptides. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) LW01107 23% CH3CN and 0.1% TFA in H2O 14.2 19 [M+2H]2+ 806.4 [M+2H]2+ 807.3 LW01108 24% CH3CN and 0.1% TFA in H2O 10.9 26 [M+2H]2+ 806.4 [M+2H]2+ 807.0 LW01149 25% CH3CN and 0.1% TFA in H2O 8.2 4.0 [M+2H]2+ 806.4 [M+2H]2+ 806.6 LW02021 25% CH3CN and 0.1% TFA in H2O 8.9 33 [M+2H]2+ 808.4 [M+2H]2+ 808.6 LW02023 25% CH3CN and 0.1% TFA in H2O 12.2 19 [M+2H]2+ 806.4 [M+2H]2+ 806.6 LW02025 23% CH3CN and 0.1% TFA in H2O 13.7 16 [M+2H]2+ 806.4 [M+2H]2+ 806.5 LW01110 24% CH3CN and 0.1% TFA in H2O 14.9 11 [M+2H]2+ 813.4 [M+2H]2+ 813.6 LW01142 25% CH3CN and 0.1% TFA in H2O 12.4 17 [M+2H]2+ 817.9 [M+2H]2+ 818.2 LW01143 26% CH3CN and 0.1% TFA in H2O 12.0 16 [M+2H]2+ 824.9 [M+2H]2+ 825.3 LW02040 25% CH3CN and 0.1% TFA in H2O 14.7 21 [M+2H]2+ 822.4 [M+2H]2+ 822.6 Table 4.5 Peptide sequences and purities of Ga-complexed DOTA-conjugated GRPR-targeted peptides. The substituted unnatural amino acids are in bold. Name Sequence Purity (%) Ga-LW01107 Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-NMe-His-Leu-Thz-NH2 99 Ga-LW01108 Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Tle-Gly-His-Leu-Thz-NH2 99 Ga-LW01149 Ga-DOTA-Pip-D-Phe-Gln-\u03b1-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 97 Ga-LW02021 Ga-DOTA-Pip-D-Phe-Gln-7-F-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 Ga-LW02023 Ga-DOTA-Pip-D-Phe-Gln-5-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 98 Ga-LW02025 Ga-DOTA-Pip-D-Phe-Gln-2-Me-Trp-Ala-Val-Gly-His-Leu-Thz-NH2 99 Ga-LW01110 Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Tle-Gly-NMe-His-Leu-Thz-NH2 98 Ga-LW01142 Ga-DOTA-Pip-D-Phe-His-Trp-Ala-Tle-Gly-NMe-His-Leu-Thz-NH2 99 103 Ga-LW01143 Ga-DOTA-Pip-D-Phe-His-Trp-Ala-Tle-NMe-Gly-NMe-His-Leu-Thz-NH2 99 Ga-LW02040 Ga-DOTA-Pip-D-Phe-Gln-7-F-Trp-Ala-Tle-Gly-NMe-His-Leu-Thz-NH2 99 Table 4.6 HPLC purification conditions and MS characterizations of Ga-complexed DOTA-conjugated GRPR-targeted peptides. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Ga-LW01107 23% CH3CN and 0.1% TFA in H2O 14.2 73 [M+2H]2+ 839.9 [M+2H]2+ 840.1 Ga-LW01108 24% CH3CN and 0.1% TFA in H2O 13.2 59 [M+2H]2+ 839.9 [M+2H]2+ 840.2 Ga-LW01149 24% CH3CN and 0.1% TFA in H2O 13.2 72 [M+2H]2+ 839.2 [M+2H]2+ 840.2 Ga-LW02021 25% CH3CN and 0.1% TFA in H2O 14.4 82 [M+2H]2+ 841.9 [M+2H]2+ 842.5 Ga-LW02023 25% CH3CN and 0.1% TFA in H2O 13.1 84 [M+2H]2+ 839.9 [M+2H]2+ 840.1 Ga-LW02025 23% CH3CN and 0.1% TFA in H2O 16.8 81 [M+2H]2+ 839.9 [M+2H]2+ 840.2 Ga-LW01110 24% CH3CN and 0.1% TFA in H2O 15.8 54 [M+2H]2+ 846.9 [M+2H]2+ 847.0 Ga-LW01142 25% CH3CN and 0.1% TFA in H2O 11.1 53 [M+2H]2+ 851.4 [M+2H]2+ 851.9 Ga-LW01143 26% CH3CN and 0.1% TFA in H2O 12.8 47 [M+2H]2+ 858.4 [M+2H]2+ 858.8 Ga-LW02040 25% CH3CN and 0.1% TFA in H2O 16.8 87 [M+2H]2+ 855.9 [M+2H]2+ 855.5 Gallium-68 labeling was conducted in HEPES buffer (2 M, pH 5.0). After HPLC purification, 68Ga-labeled LW01107, LW01108, LW01110, LW01142, LW02021, and LW02040 were obtained in 30-81% decay-corrected radiochemical yields with \u2265 72.8 GBq\/\u00b5mol molar activity and > 95% radiochemical purity. The HPLC conditions for their purification and quality control are provided in Table 4.7. 104 Table 4.7 HPLC conditions for the purification and quality control of 68Ga-labeled LW01107, LW01108, LW01110, LW01142, LW02021, and LW02040. FA: formic acid; TFA: trifluoroacetic acid. Tracer HPLC conditions Retention time (min) [68Ga]Ga-LW01107 Semi-Prep 16.5% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 23.2 QC 20% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 9.0 [68Ga]Ga-LW01108 Semi-Prep 18% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 10.8 QC 21% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 5.8 [68Ga]Ga-LW01110 Semi-Prep 18% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 21.2 QC 22% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 8.8 [68Ga]Ga-LW01142 Semi-Prep 16% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 17.3 QC 19% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 11.2 [68Ga]Ga-LW02021 Semi-Prep 17% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 32.7 QC 21% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 12.1 [68Ga]Ga-LW02040 Semi-Prep 19% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 27.1 QC 22% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 6.8 4.1.3.2 GRPR binding affinities of LW01085 and its derivatives As shown in Figure 4.1, the GRPR binding affinity (Ki) of LW01085 is 8.77 \u00b1 1.33 nM. Tle10 substitution improves binding affinity (LW01080: 3.56 \u00b1 0.72 nM) while NMe-His12 (LW01088: 9.00 \u00b1 1.99 nM), 2-Me-Trp8 (LW02009: 11.5 \u00b1 1.71 nM), 7-F-Trp8 (LW01177: 9.38 \u00b1 1.68 nM) and 5-Me-Trp8 (LW01182: 10.5 \u00b1 1.86 nM) substitutions lead to analogs with comparable binding affinities. The other substitutions generate analogs with either slightly reduced (Ki = 13.5-53.1 nM for LW02019, LW01078, LW01136, LW01183, LW02007, LW01175, 105 LW01166, LW01180 and LW02015) or greatly reduced binding affinities (Ki > 150 nM for LW02016, LW02011, LW01083, LW01075, LW02030, LW01128, LW01137, LW01191, LW01173, LW01171 and LW02013). 4.1.3.3 GRPR binding affinities of Ga-TacBOMB2 derivatives As shown in Figures 4.2-4.4, compared with the previously reported Ga-TacBOMB2 (Ki = 7.62 \u00b1 0.19 nM) 255, the NMe-His12 and Tle10 substitutions, either alone, combined or combined with an additional His7 or 7-F-Trp8 substitution generate analogs with an enhanced binding affinity (Ga-LW01107: 2.98 \u00b1 0.69 nM; Ga-LW01108: 1.34 \u00b1 0.12 nM; Ga-LW01110: 1.39 \u00b1 0.03 nM; Ga-LW01142: 3.19 \u00b1 0.78 nM; Ga-LW02040: 2.87 \u00b1 0.09 nM). The substitutions of 7-F-Trp8, 5-Me-Trp8 and 2-Me-Trp8 lead to Ga-LW02021, Ga-LW02023 and Ga-LW02025, respectively, with a slightly reduced binding affinity (Ki = 13.6-14.9 nM). The derivatives containing an \u03b1Me-Trp8 (Ga-LW01149) or NMe-Gly11 substitution (Ga-LW01143) have poor binding affinities (Ki > 300 nM). 106 Figure 4.2 Chemical structures and GRPR binding affinities (Ki, mean \u00b1 SD, n = 3) of (A) Ga-TacBOMB2 and its derivatives with an unnatural amino acid substitution at (B) His12, (C) Val10, (D) Trp8, (E) Val10 and His12, (F) Gln7, Val10 and His12, (G) Gln7, Val10, Gly11 and His12, and (H) Trp8, Val10 and His12. 107 Figure 4.3 Displacement curves (n = 3) of [125I-Tyr4]Bombesin by (A) Ga-LW01107, (B) Ga-LW01108, (C) Ga-LW01110, (D) Ga-LW01142, (E) Ga-LW01143, and (F) Ga-LW01149 generated using GRPR-expressing PC-3 cells. 108 Figure 4.4 Displacement curves (n = 3) of [125I-Tyr4]Bombesin by (A) Ga-LW02021, (B) Ga-LW02023, (C) Ga-LW02025, and (D) Ga-LW02040 generated using GRPR-expressing PC-3 cells. 4.1.3.4 Confirmation of agonist characteristics of Ga-TacBOMB2 derivatives To confirm the agonist characteristics of Ga-TacBOMB2 derivatives, calcium release assays were conducted using PC-3 cells. As shown in Figure 4.5, Ga-LW01107, Ga-LW01108, Ga-LW01110, Ga-LW01142, Ga-LW02021, Ga-LW02023, Ga-LW02025, and Ga-LW02040 induced Ca2+ efflux corresponding to 1,004 \u00b1 32.0, 549 \u00b1 46.7, 521 \u00b1 43.7, 559 \u00b1 96.2, 178 \u00b1 53.7, 312 \u00b1 45.9, 197 \u00b1 50.3, and 242 \u00b1 44.1 relative fluorescence units (RFUs), respectively. The RFUs for the blank control (DPBS), antagonist control ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), and positive controls (ATP and bombesin) are 6.57 \u00b1 1.66, 38.2 \u00b1 7.20, 253 \u00b1 46.5 and 450 \u00b1 136, respectively. Therefore, all tested Ga-TacBOMB2 derivatives are confirmed to be GRPR agonists as they induced comparable or higher calcium release than the positive control ATP. 109 Figure 4.5 Intracellular calcium efflux in PC-3 cells induced by GRPR-targeted ligands. Cells were incubated with DPBS or 50 nM of Ga-complexed GRPR-targeted ligand, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), bombesin, or ATP. 4.1.3.5 PET imaging and biodistribution The capability of 68Ga-labeled TacBOMB2 derivatives to target GRPR in vivo was evaluated by PET imaging and biodistribution studies in mice bearing GRPR-expressing PC-3 tumor xenografts. As shown in Figure 4.6, all 68Ga-labeled tracers enabled visualization of PC-3 tumors with good tumor-to-background contrasts. These tracers were excreted mainly via the renal pathway and had only low to moderate uptake in the pancreas. Higher tumor uptake was observed by using [68Ga]Ga-LW01110, [68Ga]Ga-LW02040 and [68Ga]Ga-LW01142, followed by [68Ga]Ga-LW01107 and [68Ga]Ga-LW01108, and [68Ga]Ga-LW02021 had the lowest tumor uptake. [68Ga]Ga-LW01142 which showed high blood retention at 1 h post-injection was further evaluated at 3 h post-injection (Figure 4.6F). The tumor uptake of [68Ga]-LW01142 increased further at 3 h post-injection, leading to an enhanced tumor-to-background contrast. Co-injection 110 of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) reduced tumor uptake of both [68Ga]Ga-LW01110 and [68Ga]Ga-LW01142 at 1 h post- injection (Figures 4.6C and 4.6F). Figure 4.6 Representative PET images of (A) [68Ga]Ga-LW01107, (B) [68Ga]Ga-LW01108, (C) [68Ga]Ga-LW01110, (D) [68Ga]Ga-LW02040, (E) [68Ga]Ga-LW02021 and (F) [68Ga]Ga-LW01142 in mice bearing PC-3 tumor xenografts. Blocking study was performed by co-injection with 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14). t: tumor; k: kidney; p: pancreas; bl: urinary bladder. The biodistribution data of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice obtained at 1 h post-injection are provided in Table 4.8 and the previously reported data obtained from [68Ga]Ga-TacBOMB2, [68Ga]Ga-RM2 and [68Ga]Ga-AMBA are included for comparison 252, 255. In consistence with the observations from PET images, all 68Ga-labeled 111 LW01107, LW01108, LW01110, LW01142, LW02021, and LW02040 had significantly lower uptake in the pancreas (0.39 \u00b1 0.03, 9.32 \u00b1 1.97, 8.99 \u00b1 1.54, 4.40 \u00b1 0.27, 1.22 \u00b1 0.18 and 11.7 \u00b1 0.47 %ID\/g, respectively) than [68Ga]Ga-RM2 (41.9 \u00b1 10.1 %ID\/g) and [68Ga]Ga-AMBA (62.4 \u00b1 4.26 %ID\/g). [68Ga]Ga-LW01110 showed the highest tumor uptake (16.6 \u00b1 1.60 %ID\/g), followed by [68Ga]Ga-LW02040 (12.3 \u00b1 2.14 %ID\/g) and [68Ga]Ga-LW01142 (11.4 \u00b1 1.22 %ID\/g). [68Ga]Ga-LW01110 also had the best tumor-to-organ uptake ratios (134 \u00b1 16.7, 119 \u00b1 22.6, 24.7 \u00b1 4.17 and 5.10 \u00b1 0.39 for tumor-to-bone, tumor-to-muscle, tumor-to-blood and tumor-to-kidney, respectively). With the lowest uptake in the pancreas (0.39 \u00b1 0.03 %ID\/g), [68Ga]Ga-LW01107 showed the highest tumor-to-pancreas uptake ratio (17.9 \u00b1 1.10). As [68Ga]Ga-LW01142 had high blood pool uptake at 1 h post-injection (6.88 \u00b1 0.29 %ID\/g), its biodistribution was further evaluated at 3 h post-injection. The tumor uptake increased (11.4 \u00b1 1.22 to 15.3 \u00b1 2.45 %ID\/g) and the uptake in other organs\/tissues decreased at 3 h post-injection (Figure 4.6F and 4.7;Table 4.9), leading to enhanced tumor-to-background contrast ratios. The tumor-to-bone, tumor-to-muscle, tumor-to-blood, tumor-to-kidney, and tumor-to-pancreas ratios of [68Ga]Ga-LW01142 at 3 h post-injection were 91.6 \u00b1 12.2, 86.6 \u00b1 25.1, 6.40 \u00b1 1.70, 3.17 \u00b1 0.46 and 7.36 \u00b1 1.17, respectively. 112 Figure 4.7 Uptake (mean \u00b1 SD, n = 4) of [68Ga]Ga-LW01142 at 1 and 3 h post-injection in PC-3 tumor-bearing mice. Blocking studies of [68Ga]Ga-LW01110 and [68Ga]Ga-LW01142 were conducted at 1 h post-injection (Table 4.9). The results showed that co-injection of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) reduced their average tumor uptake values by 44% and 31%, respectively. The average pancreas uptake values of [68Ga]Ga-LW01110 and [68Ga]Ga-LW01142 were also reduced by 42% and 30%, respectively. 113 Table 4.8 Biodistribution and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice at 1 h post-injection. Data are presented as mean \u00b1 SD (n = 4). The data of [68Ga]Ga-TacBOMB2, [68Ga]Ga-RM2, and [68Ga]Ga-AMBA have been reported previously 252, 255, and are included here for comparison. Tissue (%ID\/g) [68Ga]Ga-TacBOMB2 [68Ga]Ga-LW01107 [68Ga]Ga-LW01108 [68Ga]Ga-LW01110 [68Ga]Ga-LW01142 [68Ga]Ga-LW02021 [68Ga]Ga-LW02040 [68Ga]Ga- RM2 [68Ga]Ga-AMBA Blood 0.39 \u00b1 0.12 0.30 \u00b1 0.04 0.59 \u00b1 0.18 0.69 \u00b1 0.14 6.88 \u00b1 0.29 0.53 \u00b1 0.19 0.80 \u00b1 0.08 0.64 \u00b1 0.10 0.58 \u00b1 0.07 Fat 0.10 \u00b1 0.03 0.05 \u00b1 0.01 0.06 \u00b1 0.02 0.09 \u00b1 0.02 0.32 \u00b1 0.08 0.05 \u00b1 0.02 0.05 \u00b1 0.01 0.05 \u00b1 0.03 0.08 \u00b1 0.02 Testes 0.16 \u00b1 0.05 0.10 \u00b1 0.02 0.16 \u00b1 0.03 0.16 \u00b1 0.07 1.28 \u00b1 0.19 0.36 \u00b1 0.46 0.26 \u00b1 0.07 0.18 \u00b1 0.03 0.20 \u00b1 0.03 Small intestine 0.60 \u00b1 0.12 0.31 \u00b1 0.01 2.29 \u00b1 0.54 2.11 \u00b1 0.48 1.70 \u00b1 0.10 0.63 \u00b1 0.12 2.42 \u00b1 0.13 5.08 \u00b1 1.05 8.62 \u00b1 1.00 Large intestine - 0.18 \u00b1 0.04 1.31 \u00b1 0.33 1.27 \u00b1 0.27 1.07 \u00b1 0.09 0.44 \u00b1 0.18 1.90 \u00b1 0.21 2.19 \u00b1 0.67 4.90 \u00b1 0.91 Spleen 0.27 \u00b1 0.10 0.13 \u00b1 0.02 0.40 \u00b1 0.30 0.29 \u00b1 0.05 0.88 \u00b1 0.09 0.84 \u00b1 1.39 0.28 \u00b1 0.04 0.44 \u00b1 0.26 2.49 \u00b1 2.16 Pancreas 1.30 \u00b1 0.14 0.39 \u00b1 0.03 9.32 \u00b1 1.97 8.99 \u00b1 1.54 4.40 \u00b1 0.27 1.22 \u00b1 0.18 11.7 \u00b1 0.47 41.9 \u00b1 10.1 62.4 \u00b1 4.26 Stomach 0.54 \u00b1 0.27 0.07 \u00b1 0.01 0.89 \u00b1 0.18 0.94 \u00b1 0.31 0.98 \u00b1 0.05 0.40 \u00b1 0.27 1.47 \u00b1 0.41 3.87 \u00b1 2.80 2.32 \u00b1 0.60 Liver 0.29 \u00b1 0.13 0.32 \u00b1 0.15 0.35 \u00b1 0.19 0.41 \u00b1 0.06 2.88 \u00b1 0.45 0.37 \u00b1 0.14 0.71 \u00b1 0.09 0.84 \u00b1 0.55 0.43 \u00b1 0.05 Adrenal glands 0.52 \u00b1 0.11 0.32 \u00b1 0.19 1.51 \u00b1 0.99 1.64 \u00b1 0.18 2.07 \u00b1 0.27 0.38 \u00b1 0.08 2.38 \u00b1 1.62 3.01 \u00b1 0.91 10.0 \u00b1 2.49 Kidneys 2.43 \u00b1 0.16 1.88 \u00b1 0.17 2.47 \u00b1 0.66 3.26 \u00b1 0.25 6.36 \u00b1 0.41 2.15 \u00b1 0.29 4.00 \u00b1 0.33 2.57 \u00b1 0.48 5.70 \u00b1 2.45 Heart 0.14 \u00b1 0.02 0.11 \u00b1 0.02 0.18 \u00b1 0.04 0.23 \u00b1 0.03 1.62 \u00b1 0.12 0.16 \u00b1 0.05 0.27 \u00b1 0.04 0.19 \u00b1 0.03 0.22 \u00b1 0.02 Lungs 0.39 \u00b1 0.10 0.28 \u00b1 0.04 0.49 \u00b1 0.22 0.59 \u00b1 0.12 4.59 \u00b1 0.81 0.49 \u00b1 0.15 2.36 \u00b1 1.46 0.62 \u00b1 0.26 0.62 \u00b1 0.06 PC-3 tumor 5.95 \u00b1 0.50 7.05 \u00b1 0.71 5.90 \u00b1 0.68 16.6 \u00b1 1.60 11.4 \u00b1 1.22 3.08 \u00b1 0.48 12.3 \u00b1 2.14 10.5 \u00b1 2.03 6.69 \u00b1 1.03 Bone 0.26 \u00b1 0.11 0.06 \u00b1 0.02 0.15 \u00b1 0.11 0.12 \u00b1 0.00 0.47 \u00b1 0.08 0.08 \u00b1 0.02 0.14 \u00b1 0.01 0.11 \u00b1 0.03 0.33 \u00b1 0.13 Muscle 0.13 \u00b1 0.08 0.07 \u00b1 0.02 0.17 \u00b1 0.11 0.14 \u00b1 0.03 0.52 \u00b1 0.07 0.18 \u00b1 0.04 0.18 \u00b1 0.03 0.14 \u00b1 0.06 0.17 \u00b1 0.01 Brain 0.03 \u00b1 0.01 0.01 \u00b1 0.00 0.02 \u00b1 0.00 0.03 \u00b1 0.00 0.15 \u00b1 0.02 0.02 \u00b1 0.01 0.03 \u00b1 0.00 0.03 \u00b1 0.01 0.04 \u00b1 0.01 Tumor\/bone 28.9 \u00b1 18.3 126 \u00b1 32.7 54.2 \u00b1 32.9 134 \u00b1 16.7 25.0 \u00b1 4.53 38.2 \u00b1 9.17 88.6 \u00b1 16.7 96.5 \u00b1 27.1 22.6 \u00b1 9.13 Tumor\/muscle 57.9 \u00b1 32.8 99.8 \u00b1 11.2 45.4 \u00b1 19.6 119 \u00b1 22.6 22.0 \u00b1 2.37 18.2 \u00b1 5.02 69.9 \u00b1 15.9 80.8 \u00b1 27.5 39.5 \u00b1 7.46 Tumor\/blood 16.4 \u00b1 5.64 23.9 \u00b1 1.80 11.0 \u00b1 4.24 24.7 \u00b1 4.17 1.66 \u00b1 0.18 6.51 \u00b1 2.60 15.4 \u00b1 3.51 16.5 \u00b1 3.06 11.7 \u00b1 2.10 Tumor\/kidney 2.46 \u00b1 0.33 3.75 \u00b1 0.19 2.57 \u00b1 0.96 5.10 \u00b1 0.39 1.81 \u00b1 0.28 1.44 \u00b1 0.23 3.08 \u00b1 0.62 4.13 \u00b1 0.73 1.36 \u00b1 0.73 Tumor\/pancreas 4.64 \u00b1 0.77 17.9 \u00b1 1.10 0.66 \u00b1 0.17 1.87 \u00b1 0.25 2.61 \u00b1 0.39 2.56 \u00b1 0.44 1.04 \u00b1 0.16 0.25 \u00b1 0.04 0.11 \u00b1 0.01 114 Table 4.9 Biodistribution (mean \u00b1 SD, n = 4) and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice. The mice in the blocked group were co-injected with 100 \u00b5g of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14). The significance of differences between groups (1 h vs 1 h blocked; 1 h vs 3 h): *p < 0.05; **p < 0.01; ***p < 0.001. 4.1.3.6 The logD7.4 measurement and in vivo stability The logD7.4 measurements were conducted for the 68Ga-labeled tracers, and as shown in Table 4.10, these 68Ga-labeled tracers are highly hydrophilic with average logD7.4 values in the range of \u22123.10 to \u22121.81. In vivo stability studies showed that, compared with the previously reported [68Ga]Ga-TacBOMB2 (12.7 \u00b1 2.93% intact at 15 min post-injection), Tle10 ([68Ga]Ga-LW01108) and NMe-His12 ([68Ga]Ga-LW01107) substitutions increased the intact tracer fraction Tissue (%ID\/g) [68Ga]Ga -LW01110 [68Ga]Ga -LW01142 1 h 1 h blocked 1 h 1 h blocked 3 h Blood 0.69 \u00b1 0.14 1.57 \u00b1 0.61* 6.88 \u00b1 0.29 5.48 \u00b1 0.44* 2.48 \u00b1 0.61*** Fat 0.09 \u00b1 0.02 0.19 \u00b1 0.06* 0.32 \u00b1 0.08 0.47 \u00b1 0.13 0.12 \u00b1 0.04** Testes 0.16 \u00b1 0.07 0.42 \u00b1 0.08** 1.28 \u00b1 0.19 0.90 \u00b1 0.33 0.66 \u00b1 0.20** Small intestine 2.11 \u00b1 0.48 1.25 \u00b1 0.39* 1.70 \u00b1 0.10 1.19 \u00b1 0.10*** 0.76 \u00b1 0.30*** Large intestine 1.27 \u00b1 0.27 0.79 \u00b1 0.21* 1.07 \u00b1 0.09 0.71 \u00b1 0.07*** 0.80 \u00b1 0.21* Spleen 0.29 \u00b1 0.05 0.52 \u00b1 0.07** 0.88 \u00b1 0.09 0.82 \u00b1 0.04 0.51 \u00b1 0.07*** Pancreas 8.99 \u00b1 1.54 5.23 \u00b1 2.42* 4.40 \u00b1 0.27 3.09 \u00b1 0.52** 2.14 \u00b1 0.67*** Stomach 0.94 \u00b1 0.31 0.45 \u00b1 0.16* 0.98 \u00b1 0.05 0.55 \u00b1 0.16** 0.32 \u00b1 0.08*** Liver 0.41 \u00b1 0.06 0.82 \u00b1 0.19** 2.88 \u00b1 0.45 3.31 \u00b1 0.27 1.50 \u00b1 0.31** Adrenal glands 1.64 \u00b1 0.18 1.02 \u00b1 0.20** 2.07 \u00b1 0.27 2.47 \u00b1 1.39 1.01 \u00b1 0.25*** Kidneys 3.26 \u00b1 0.25 7.09 \u00b1 1.16*** 6.36 \u00b1 0.41 7.27 \u00b1 0.66 4.88 \u00b1 1.00* Heart 0.23 \u00b1 0.03 0.46 \u00b1 0.12** 1.62 \u00b1 0.12 1.39 \u00b1 0.19 0.58 \u00b1 0.15*** Lungs 0.59 \u00b1 0.12 1.23 \u00b1 0.37** 4.59 \u00b1 0.81 3.80 \u00b1 0.78 2.70 \u00b1 0.28** PC-3 tumor 16.6 \u00b1 1.60 9.32 \u00b1 1.57*** 11.4 \u00b1 1.22 7.85 \u00b1 0.91** 15.3 \u00b1 2.45* Bone 0.12 \u00b1 0.00 0.40 \u00b1 0.35 0.47 \u00b1 0.08 0.39 \u00b1 0.05 0.17 \u00b1 0.03*** Muscle 0.14 \u00b1 0.03 0.73 \u00b1 0.73 0.52 \u00b1 0.07 0.64 \u00b1 0.16 0.18 \u00b1 0.05*** Brain 0.03 \u00b1 0.00 0.06 \u00b1 0.02* 0.15 \u00b1 0.02 0.10 \u00b1 0.01** 0.06 \u00b1 0.01*** Tumor\/bone 134 \u00b1 16.7 35.5 \u00b1 18.7*** 25.0 \u00b1 4.53 20.7 \u00b1 4.77 91.6 \u00b1 12.2*** Tumor\/muscle 119 \u00b1 22.6 21.4 \u00b1 12.2*** 22.0 \u00b1 2.37 13.4 \u00b1 5.41* 86.6 \u00b1 25.1** Tumor\/blood 24.7 \u00b1 4.17 6.62 \u00b1 2.48*** 1.66 \u00b1 0.18 1.43 \u00b1 0.16 6.40 \u00b1 1.70*** Tumor\/kidney 5.10 \u00b1 0.39 1.32 \u00b1 0.15*** 1.81 \u00b1 0.28 1.09 \u00b1 0.17** 3.17 \u00b1 0.46** Tumor\/pancreas 1.87 \u00b1 0.25 1.99 \u00b1 0.62 2.61 \u00b1 0.39 2.57 \u00b1 0.30 7.36 \u00b1 1.17*** 115 in mouse plasma to 35.3 \u00b1 0.93 and 66.2 \u00b1 12.4%, respectively (Figures 4.8-4.9 and Table 4.10). Further improvement was obtained by combining both Tle10 and NMe-His12 as \u2265 89% intact was observed for [68Ga]Ga-LW01110, [68Ga]Ga-LW01142 and [68Ga]Ga-LW02040 (Figures 4.10-4.12 and Table 4.10). No intact tracer was detected in mouse urine samples for all the tested GRPR-targeted ligands (Figure 4.8-4.12). Table 4.10 LogD7.4 values and in vivo stability of GRPR-targeted tracers. Data are presented as mean \u00b1 SD (n = 3). The data of [68Ga]Ga-TacBOMB2, [68Ga]Ga-RM2 and [68Ga]Ga-AMBA have been reported previously 252, 255 and are included here for comparison. Tracer logD7.4 (n = 3) Intact fraction (%) in plasma at 15 min post-injection [68Ga]Ga-TacBOMB2 \u22123.21 \u00b1 0.04 12.7 \u00b1 2.93 [68Ga]Ga-LW01107 \u22123.10 \u00b1 0.05 66.2 \u00b1 12.4 [68Ga]Ga-LW01108 \u22123.10 \u00b1 0.02 35.3 \u00b1 0.93 [68Ga]Ga-LW01110 \u22122.83 \u00b1 0.08 89.0 \u00b1 2.17 [68Ga]Ga-LW01142 \u22121.81 \u00b1 0.02 91.2 \u00b1 5.30 [68Ga]Ga-LW02021 \u22122.46 \u00b1 0.09 - [68Ga]Ga-LW02040 \u22122.48 \u00b1 0.12 92.9 \u00b1 2.30 [68Ga]Ga-RM2 \u22122.76 \u00b1 0.03 71.9 \u00b1 10.4 [68Ga]Ga-AMBA \u22123.66 \u00b1 0.03 39.4 \u00b1 10.8 116 Figure 4.8 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01107 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. The peak of intact tracer is pointed by a black arrow. Figure 4.9 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01108 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. The peak of intact tracer is pointed by a black arrow. 117 Figure 4.10 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01110 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. The peak of intact tracer is pointed by a black arrow. Figure 4.11 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01142 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. The peak of intact tracer is pointed by a black arrow. 118 Figure 4.12 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW02040 in mouse (A) plasma and (B) urine samples collected at 15 min post-injection. The peak of intact tracer is pointed by a black arrow. 4.1.4 Discussion In this study, we first compared LW01085 (D-Phe-[Thz14]Bombesin(7-14), the pharmacophore of our previously reported [68Ga]Ga-TacBOMB2) with 25 derivatives with an unnatural amino acid substitution at the potential cleavage sites (Figure 4.1). In vitro competition binding assays showed that NMe-His12 substitution (LW01088) is tolerable, which is consistent with a previous report by Horwell, et al. 232 that Ac-Bombesin(7-14) and Ac-[NMe-His12]Bombesin have similar GRPR binding affinities (Ki = 0.7 vs 0.4 nM). In addition, we discovered that 2-Me-Trp8 (LW02009), 7-F-Trp8 (LW01177), 5-Me-Trp8 (LW01182) and Tle10 (LW01080) substitutions also led to derivatives with enhanced or comparable GRPR binding affinities. Therefore, these unnatural amino acid substitutions were selected and subsequently compared\/combined with two reported unnatural amino acid substitutions (\u03b1Me-Trp8 and NMe-119 Gly11) for the design of Ga-DOTA-complexed Pip-linker-containing GRPR-targeted ligands (Figure 4.2). Despite being popularly used for the design of GRPR-targeted antagonist ligands 230, 256, NMe-Gly11 substitution was reported to cause > 30-fold reduction in GRPR binding affinity for an agonist sequence (Ki = 0.7 vs 25 nM for Ac-Bombesin(7-14) and Ac-[NMe-Gly11]Bombesin(7-14), respectively) 232. Consistent with the previous report, we also observed a dramatic reduction in GRPR binding affinity with the NMe-Gly11 substitution (Figures 4.2F and 4.2G, Ki = 3.19 vs 12,790 nM for Ga-LW01142 and Ga-LW01143, respectively). \u03b1Me-Trp8 substitution has been successfully used by the Wester group for the design of potent and stable radiolabeled GRPR-targeted antagonists derived from RM2 231. However, for agonist Ga-TacBOMB2, \u03b1Me-Trp8 substitution in Ga-LW01149 caused significant loss of binding affinity (Ki = 7.62 vs 342 nM, Figures 4.2A and 4.2D). Our data suggest that GRPR agonists and antagonists might bind to the receptors in different configurations as \u03b1Me-Trp8 and NMe-Gly11 substitutions which are commonly used for antagonist modifications hinder the binding of agonists to the receptors. His7 (the amino acid at the corresponding position in GRP), 2-Me-Trp8, 7-F-Trp8, 5-Me-Trp8, Tle10 and NMe-His12 substitutions, either alone or in combination, still led to GRPR-targeted ligands with potent binding affinities (Ki = 1.34-14.9 nM, Figure 4.2). This suggests that compared with the targeted peptide sequences presented in Figure 4.1, the addition of Ga-DOTA complex and the Pip linker does not affect their binding affinity. Similarly, based on the results of calcium release assays (Figure 4.5), His7, 2-Me-Trp8, 7-F-Trp8, 5-Me-Trp8, Tle10 and NMe-His12 substitutions, either alone or in combination, do not change their agonist characteristics. Subsequently, we radiolabeled potent candidates and evaluated their potential for prostate cancer imaging. As shown in Figure 4.6, all 68Ga-labeled tracers were successfully used to 120 visualize PC-3 tumor xenografts in their PET images, confirming good GRPR targeting capabilities of these tracers. A lower tumor uptake was observed for [68Ga]Ga-LW02021, which could be due to its relatively weaker GRPR binding affinity compared with those of others (Ki = 13.6 vs 1.34 to 3.19 nM, Figure 4.2). The clearance of these tracers was mainly via the renal pathway, consistent with the highly hydrophilic nature of these tracers (logD7.4 values \u2264 \u22121.81). A higher blood retention was observed for [68Ga]Ga-LW01142 at 1 h post-injection, which could be due to its relatively higher lipophilicity than other tracers (logD7.4 = \u22121.81 vs \u22122.46 to \u22123.10, Table 4.10). Ex vivo biodistribution studies were also conducted to better quantify uptake in tumors and normal organs\/tissues. As shown in Table 4.8, except [68Ga]Ga-LW02021 (3.08 \u00b1 0.48 %ID\/g at 1 h post-injection), all other evaluated tracers had comparable or improved uptake in PC-3 tumors when compared to that of the previously reported [68Ga]Ga-TacBOMB2 (5.95 \u00b1 0.05 %ID\/g). Notably, while Tle10 substitution led to [68Ga]Ga-LW01108 (5.90 \u00b1 0.68 %ID\/g) with a comparable tumor uptake, NMe-His12 led to [68Ga]Ga-LW01107 (7.05 \u00b1 0.71 %ID\/g) with an improved tumor uptake. Most importantly, the combination of both Tle10 and NMe-His12 with and without an addition substitution (His7 or 7-F-Trp8) led to [68Ga]Ga-LW01110 (16.6 \u00b1 1.66 %ID\/g), [68Ga]Ga-LW01142 (11.4 \u00b1 1.22 %ID\/g) and [68Ga]Ga-LW02040 (12.3 \u00b1 2.14 %ID\/g) with a further improved tumor uptake. Since 68Ga-labeled LW01107, LW01108, LW01110, LW01142 and LW02040 have comparable GRPR binding affinities (Ki = 1.34-3.19 nM), we suspected that the greatly improved tumor uptake for tracers with at least both Tle10 and NMe-His12 substitutions could be mainly due to their improved in vivo stability. In vivo stability studies were subsequently conducted to verify our hypothesis. As shown in Table 4.10, compared with the previously reported [68Ga]Ga-TacBOMB2 (12.7 \u00b1 2.93% intact 121 tracer at 15 min post-injection), Tle10 and NMe-His12 substitutions led to [68Ga]Ga-LW01108 (35.3 \u00b1 0.93% intact) and [68Ga]Ga-LW01107 (66.2 \u00b1 12.4% intact) with an improved in vivo stability. Combination of at least both Tle10 and NMe-His12 substitutions further led to [68Ga]Ga-LW01110, [68Ga]Ga-LW01142 and [68Ga]Ga-LW02040 with an average \u2265 89% intact tracer at 15 min post-injection. These data are consistent with the trend of their tumor uptake observed from the ex vivo biodistribution studies: [68Ga]Ga-TacBOMB2 \u2248 [68Ga]Ga-LW01108 < [68Ga]Ga-LW01107 < [68Ga]Ga-LW01110, [68Ga]Ga-LW01142 and [68Ga]Ga-LW02040. In addition, our in vivo stability data also suggest that His12-Leu13 is the major cleavage site of GRPR-targeted ligands, followed by Ala9-Val10, and then Gln7-Trp8\/Trp8-Ala9. This is also consistent with the fact that most of reported GRPR-targeted radioligands had modifications to avoid the cleavage at His12-Leu13 such as using Sta13 substitution for the RM2 derivatives 190, 202 and Leu13\u03c8Thz14 in our previously reported TacsBOMB derivatives 252. Contrary to the improved stability observed in plasma, no intact tracer was detected in urine samples even for ligands with both Tle10 and NMe-His12 substitutions (Figures 4.8-4.12). This is due to the facts that GRPR-targeted ligands are cleaved mainly by NEP and kidneys have the highest NEP expression level 257. Therefore, GRPR-targeted tracers which remain intact in plasma are completely metabolized by NEP in kidneys before being excreted into the urinary bladder. Compared with the clinically validated [68Ga]Ga-RM2 and [68Ga]Ga-AMBA 252, 255, our stabilized tracers ([68Ga]Ga-LW01110, [68Ga]Ga-LW01142 and [68Ga]Ga-LW02040) have not only higher tumor uptake, but also comparable or even higher tumor-to-background uptake ratios (Tables 4.8-4.9). Most importantly, they also have a much lower pancreas uptake than [68Ga]Ga-RM2 and [68Ga]Ga-AMBA (4.40-11.7 vs 41.9-62.4 %ID\/g at 1 h post-injection). Therefore, these tracers are expected to have a higher sensitivity for detecting cancer lesions in or adjacent to the 122 pancreas, and can achieve better treatment efficacy and cause less damage to the pancreas when radiolabeled with an \u03b1- or \u03b2-emitter for radiotherapeutic applications. 4.1.5 Conclusions We systematically replaced the amino acids (Gln7, Trp8, Ala9, Val10, Gly11 and His12) at potential cleavage sites of the previously reported sequence of [68Ga]Ga-TacBOMB2, and identified that Tle10 and NMe-His12 substitutions, either alone or in combination, led to derivatives with comparable\/enhanced GRPR binding affinities. In vivo stability and ex vivo biodistribution studies confirmed the improved stability resulted from unnatural amino acid substitutions, which further led to enhanced tumor uptake. With both Tle10 and NMe-His12 substitutions, the top candidate [68Ga]Ga-LW01110 has higher in vivo stability, tumor uptake and tumor-to-background uptake ratios than clinically validated [68Ga]Ga-RM2 and [68Ga]Ga-AMBA, and is promising for use for detecting GRPR-expressing tumors with PET. Due to the observed lower pancreas uptake and foreseeable longer tumor retention as being agonists, our optimized sequence, [Tle10,NMe-His12,Thz14]Bombesin(7-14), is a promising template for use for the design of GRPR-targeted radiotherapeutic agents. 123 4.2 Synthesis and evaluation of novel 68Ga-labeled [D-Phe6,Leu13\u03c8Thz14]Bombesin(6-14) analogs for cancer imaging with positron emission tomography The following section is an adaption of the following published paper: Wang, L., Chen, C.C., Zhang, Z., Kuo, H.T., Zhang, C., Colpo, N., Merkens, H., B\u00e9nard, F. and Lin, K.S. Synthesis and evaluation of novel 68Ga-labeled [D-Phe6,Leu13\u03c8Thz14]Bombesin(6-14) analogs for cancer imaging with positron emission tomography. Pharmaceuticals, 2024, 17, 621. https:\/\/doi.org\/10.3390\/ph17050621. The compounds disclosed in this report are covered by a recent patent application (PCT\/CA2023\/050401; filing date: March 23, 2023). Lei Wang, Zhengxing Zhang, Chengcheng Zhang, Fran\u00e7ois B\u00e9nard, and Kuo-Shyan Lin are listed as inventors in this filed patent application. 4.2.1 Introduction As a member of transmembrane G protein-coupled receptors, gastrin-releasing peptide receptor (GRPR) is expressed in pancreas, gastrointestinal tract, and central nervous system, and regulates a series of physiological functions such as hormone secretion, smooth muscle contraction, and synaptic plasticity 90-92. Moreover, GRPR is found overexpressed in a variety of malignancies including breast, prostate, lung, and colon cancers, and activation of GRPR leads to proliferation of cancer cells 93, 144, 145, 147. Thus, GRPR has been considered as a promising target for the design of targeted radiopharmaceuticals for diagnosis and radioligand therapy of GRPR-expressing cancers. Bombesin (BBN), isolated from the skin of the European frog Bombina bombina, is a natural exogenous ligand showing good binding affinity towards GRPR. The heptapeptide sequence at the C-terminus (Bombesin(8-14)) is the minimal sequence needed for binding to GRPR with a high affinity. Thus, this peptide sequence has been used for the design of GRPR-124 targeted radiopharmaceuticals for cancer diagnosis and radioligand therapy 46, 47, 188-192. Although several GRPR-targeted radiotracers have been evaluated in the clinic, the extraordinarily high pancreas uptake might limit the detection of pancreatic cancer, and the metastatic lesions of other cancers in and\/or adjacent to the pancreas. In addition, to avoid damage to the pancreas, the maximum tolerated dose might have to be lowered, and this could potentially lead to a suboptimal treatment efficacy for radiotherapeutic application 46, 189, 191, 193. Inspired by the potent GRPR antagonist, RC-3950-II ([D-Phe6, Leu13\u03c8Thz14]Bombesin(6-14)), reported by the Schally group 181, 182, our group synthesized and evaluated a 68Ga-labeled DOTA-conjugated RC-3950-II derivative, [68Ga]Ga-TacsBOMB2 (Figure 4.13A), for imaging GRPR-expressing cancer with positron emission tomography (PET) 252. The GRPR antagonist characteristics of Ga-TacsBOMB2 was confirmed via intracellular calcium release assay. A potent GRPR binding affinity (Ki) of Ga-TacsBOMB2 at low nM scale contributes to the good uptake of [68Ga]Ga-TacsBOMB2 in human prostate cancer PC-3 tumor xenografts (10.2 \u00b1 2.27 %ID\/g) at 1 h post-injection. Most importantly, the pancreas uptake value of [68Ga]Ga-TacsBOMB2 (2.81 \u00b1 0.78 %ID\/g) was much lower than that of the clinically validated GRPR tracer, [68Ga]Ga-RM2 (41.9 \u00b1 10.1 %ID\/g) in the same preclinical animal model 252. 125 Figure 4.13 Chemical structures of (A) Ga-TacsBOMB2, (B) Ga-LW01158, (C) Ga-LW01160, (D) Ga-LW01186, (E) Ga-LW02002, and (F) Ga-RM2. The unnatural amino acid substitutions on Ga-TacsBOMB2 derivatives are shown in brown. 126 Similar to most of the reported GRPR-targeted ligands, Ga-TacsBOMB2 could potentially be enzymatically degraded in vivo, especially by the neutral endopeptidase 24.11 (NEP, EC 3.4.24.11, neprilysin) 201, 223. The amide bonds between Gln7-Trp8, Trp8-Ala9, Ala9-Val10 and His12-Leu13 were identified as the cleavage sites of clinically validated GRPR-targeted radioligands derived from RM2 and AMBA 190, 224. In this study, we hypothesized that (1) the amide bonds between Gln7-Trp8, Trp8-Ala9, Ala9-Val10 and His12-Leu13 in Ga-TacsBOMB2 (Figure 4.13A) are also potential cleavage sites of peptidases; and (2) replacing the amino acids adjacent to the potential cleavage sites in Ga-TacsBOMB2 with a closely related unnatural amino acid could improve in vivo stability, and potentially retain the high GRPR binding affinity and the low pancreas uptake characteristics. Hence, in this study we synthesized Ga-labeled LW01158, LW01160, LW01186, and LW02002 (Figures 4.13B-4.13E), by replacing the natural amino acids adjacent to the cleavage sites with a closely related unnatural amino acid. We determined their antagonist\/agonist characteristics by in vitro fluorescence based calcium release assay. The potential of these ligands for detecting GRPR-expressing cancer was evaluated by in vitro competition binding assay, PET imaging and ex vivo biodistribution studies in PC-3 tumor-bearing mice. The biodistribution data of these novel tracers were compared with the previously reported data of [68Ga]Ga-RM2 (Figure 4.13F) obtained using the same preclinical tumor model 252. 4.2.2 Materials and Methods The materials and methods described in this section are provided in Chapter 2. Relevant sections are those describing reagent and instrumentation (Section 2.1), synthesis of Fmoc-Le\u03c8Thz-OH (Section 2.2), synthesis of DOTA-conjugated precursors (Section 2.3.1), synthesis of nonradioactive Ga-complexed standards (Section 2.4.1), cell culture (Section 2.5), fluorometric 127 calcium release assay (Section 2.6), in vitro competition binding assay (Section 2.7), 68Ga radiolabeling (Section 2.8.1), logD7.4 measurements (Section 2.9), animal studies (Section 2.10), PET imaging and biodistribution studies (Section 2.10.1), in vivo stability studies (Section 2.10.3), and statistical analysis (Sections 2.12). 4.2.3 Results 4.2.3.1 Syntheses of GRPR-targeted Ligands The yields for the synthesis of LW01158, LW01160, LW01186, and LW02002 were ranged from 8 to 32%, and the yields for the synthesis of their nonradioactive Ga-complexed standards were ranged from 76 to 81% (Table 4.11-4.12). The identities of all precursors and nonradioactive Ga-complexed standards were confirmed by MS analyses (Table 4.11-4.12). 68Ga-labeled LW01158, LW01186, and LW02002 were purified by HPLC and obtained in 16-61% decay-corrected radiochemical yields with 132-298 GBq\/\u00b5mol molar activity and > 92% radiochemical purity (Table 4.13). Table 4.11 MS characterizations, yields and HPLC purification conditions of LW01158, LW01160, LW01186, and LW02002. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) LW01158 26% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 14.1 32 [M+2H]2+ 799.4 [M+2H]2+ 799.6 LW01160 23% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 10.6 24 [M+2H]2+ 799.4 [M+2H]2+ 1599.2 LW01186 29% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 12.1 8 [M+2H]2+ 806.4 [M+2H]2+ 806.8 LW02002 27% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 14.5 17 [M+2H]2+ 806.4 [M+2H]2+ 806.7 128 Table 4.12 MS characterizations, yields and HPLC purification conditions of Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Ga-LW01158 26% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 13.2 76 [M+2H]2+ 832.9 [M+2H]2+ 833.4 Ga-LW01160 25% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 9.3 81 [M+2H]2+ 832.9 [M+2H]2+ 833.6 Ga-LW01186 29% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 13.5 81 [M+2H]2+ 839.9 [M+2H]2+ 839.5 Ga-LW02002 27% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 16.3 77 [M+2H]2+ 839.9 [M+2H]2+ 839.4 Table 4.13 HPLC conditions for the purification and quality control of 68Ga-labeled LW01158, LW01186, and LW02002. FA: formic acid. Compound name HPLC conditions Retention time (min) [68Ga]Ga-LW01158 Semi-Prep 20% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 12.5 QC 21.5% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 6.1 [68Ga]Ga-LW01186 Semi-Prep 22% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 11.6 QC 24% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 5.5 [68Ga]Ga-LW02002 Semi-Prep 20% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 14.3 QC 21.5% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 6.7 4.2.3.2 Binding Affinity, Antagonist Characterization, and Hydrophilicity As shown in Figure 4.14, the binding of [125I-Tyr4 ]Bombesin to PC-3 cells was inhibited by Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002 in a dose-dependent manner. 129 The calculated Ki values for Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002 were 5.11 \u00b1 0.47, 187 \u00b1 17.8, 6.94 \u00b1 0.95, and 11.0 \u00b1 0.39 nM, respectively (n = 3). Figure 4.14 Displacement curves of [125I-Tyr4]Bombesin by Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002 generated using GRPR-expressing PC-3 cells. Error bars indicate standard deviation. Since Ga-LW01160 had a poor GRPR binding affinity (Ki = 187 \u00b1 17.8 nM), next we determined the agonist\/antagonist characteristics only for the potent Ga-LW01158, Ga-LW01186, and Ga-LW02002. Ga-LW01158, Ga-LW01186, and Ga-LW02002 were confirmed to be GRPR antagonists by intracellular calcium release assays using PC-3 cells (Figure 4.15). ATP (50 nM, a positive control) and bombesin (50 nM, an agonist control) induced Ca2+ efflux corresponding to 222 \u00b1 21.7 and 499 \u00b1 73.4 relative fluorescence units (RFUs), respectively. For 50 nM of Ga-LW01158, Ga-LW01186, and Ga-LW02002, 12.6 \u00b1 2.22, 6.64 \u00b1 2.44, and 8.24 \u00b1 2.28 RFUs were observed, respectively, which were significantly lower than the values of ATP and bombesin. The blank control (Dulbecco\u2019s phosphate-buffered saline, DPBS) and the antagonist control ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), 50 nM) induced Ca2+ efflux with 7.37 \u00b1 2.23 and 38.2 \u00b1 7.20 RFUs, respectively. !\"# !\"\" !\"$ !% !& !' !( !)$#$G$($&$\"$$+I-.LIML1M23425IM.ST89.W;1L5<5L.=5M[5M-.I<.S\"#) ?!]A3G8=IB=1C5Ma4!+b$\"\")&a4!+b$\"\"($a4!+b$\"\"&(a4!+b$#$$#130 Figure 4.15 Intracellular calcium efflux in PC-3 cells induced by Ga-LW01158, Ga-LW01186, Ga-LW02002, bombesin, ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), ATP, and DPBS. Error bars indicate standard deviation (n = 3). The hydrophilicity of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 were determined by the shake flask method, and their logD7.4 values were calculated to be \u22121.98 \u00b1 0.10, \u22122.03 \u00b1 0.10, and \u22122.33 \u00b1 0.03, respectively (n = 3). 4.2.3.3 PET Imaging and ex vivo Biodistribution The PC-3 tumor xenografts were clearly visualized in PET images acquired at 1 h post-injection using [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 (Figure 4.16). All three tracers were primarily excreted via the renal pathway. [68Ga]Ga-LW01158 had the best tumor-to-background contrast among all three tracers. While [68Ga]Ga-LW01186 showed significant pancreas and liver uptake, the uptake in these two organs were much lower for [68Ga]Ga-LW01158 and [68Ga]Ga-LW02002. Co-injection with 100 \u03bcg of nonradioactive standard decreased the uptake of [68Ga]Ga-LW01158 in the PC-3 tumor xenograft to close to the background level. Ga-LW01158Ga-LW01186Ga-LW02002Bombesin[D-Phe6 , Leu-NHEt13 ,des-Met14 ]Bombesin(6-14) ATP DPBS020406080100300500700RFU (Max-Min)Calcium Release Assay131 Figure 4.16 Representative PET images of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 acquired at 1 h post-injection in mice bearing PC-3 tumor xenografts. t: tumor; k: kidney; p: pancreas; l: liver; bl: urinary bladder. Biodistribution studies of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 were also performed at 1 h post-injection in PC-3 tumor-bearing mice, and the results were consistent with the observations from their PET images (Figures 4.17-4.19 and Table 4.14). The previously reported biodistribution data of [68Ga]Ga-TacsBOMB2 and [68Ga]Ga-RM2 obtained from the same PC-3 tumor model are also included for comparison (Table 4.14) 252. Among all three TacsBOMB2-derived tracers, [68Ga]Ga-LW01158 had the highest tumor uptake (11.2 \u00b1 0.65 %ID\/g) which was comparable to those of [68Ga]Ga-TacsBOMB2 (10.2 \u00b1 2.27 %ID\/g, p = 0.46) and [68Ga]Ga-RM2 (10.5 \u00b1 2.03 %ID\/g, p = 0.56). The tumor uptake values of [68Ga]Ga-LW02002 and [68Ga]Ga-LW01186 were 8.32 \u00b1 1.20 and 5.87 \u00b1 0.64 %ID\/g, respectively. [68Ga]Ga-LW01186 showed the highest pancreas uptake at 1 h post-injection (14.1 \u00b1 1.90 %ID\/g), followed by [68Ga]Ga-LW01158 (12.0 \u00b1 1.41 %ID\/g) and [68Ga]Ga-LW02002 (2.36 \u00b1 0.36 %ID\/g). The pancreas uptake values for all three tracers were significantly lower than that of 132 [68Ga]Ga-RM2 (41.9 \u00b1 10.1 %ID\/g). In addition, [68Ga]Ga-LW01186 showed the highest liver uptake (22.1 \u00b1 3.19 %ID\/g), while the liver uptake values for both [68Ga]Ga-LW01158 and [68Ga]Ga-LW02002 were only 4.33 \u00b1 0.22 and 1.06 \u00b1 0.24 %ID\/g, respectively. Table 4.14 Biodistribution (mean \u00b1 SD, n = 4) and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice. The biodistribution data of [68Ga]Ga-TacsBOMB2 and [68Ga]Ga-RM2 have been reported previously 252, and are included here for comparison. The mice in the blocked group were co-injected with 100 \u00b5g of Ga-LW01158. *,**, and *** indicate p < 0.05, < 0.01 and < 0.001, respectively, when comparing the 1 h and 1 h blocked data of [68Ga]Ga-LW01158. Tissue (%ID\/g) [68Ga]Ga-TacsBOMB2 [68Ga]Ga-LW01158 [68Ga]Ga-LW01158 [68Ga]Ga-LW01186 [68Ga]Ga-LW02002 [68Ga]Ga- RM2 1 h 1 h 1 h blocked 1 h 1 h 1 h Blood 0.76 \u00b1 0.21 1.14 \u00b1 0.15 2.82 \u00b1 0.90* 1.83 \u00b1 0.31 0.94 \u00b1 0.08 0.64 \u00b1 0.10 Fat 0.09 \u00b1 0.03 0.07 \u00b1 0.01 0.27 \u00b1 0.10** 0.16 \u00b1 0.03 0.07 \u00b1 0.01 0.05 \u00b1 0.03 Testes 0.19 \u00b1 0.05 0.24 \u00b1 0.01 0.66 \u00b1 0.24* 0.31 \u00b1 0.05 0.20 \u00b1 0.10 0.18 \u00b1 0.03 Small intestine 1.04 \u00b1 0.30 2.46 \u00b1 0.30 1.17 \u00b1 0.39** 2.54 \u00b1 0.44 0.66 \u00b1 0.06 5.08 \u00b1 1.05 Large intestine 0.37 \u00b1 0.16 1.41 \u00b1 0.38 0.90 \u00b1 0.57 1.27 \u00b1 0.21 0.54 \u00b1 0.29 2.19 \u00b1 0.67 Spleen 0.47 \u00b1 0.17 0.60 \u00b1 0.42 0.67 \u00b1 0.21 1.09 \u00b1 0.33 0.46 \u00b1 0.13 0.44 \u00b1 0.26 Pancreas 2.81 \u00b1 0.78 12.0 \u00b1 1.41 0.66 \u00b1 0.28*** 14.1 \u00b1 1.90 2.36 \u00b1 0.36 41.9 \u00b1 10.1 Stomach 0.32 \u00b1 0.08 1.30 \u00b1 0.41 0.42 \u00b1 0.15** 0.83 \u00b1 0.13 0.32 \u00b1 0.11 3.87 \u00b1 2.80 Liver 2.61 \u00b1 0.70 4.33 \u00b1 0.22 4.25 \u00b1 0.64 22.1 \u00b1 3.19 1.06 \u00b1 0.24 0.84 \u00b1 0.55 Adrenal glands 0.57 \u00b1 0.40 1.37 \u00b1 0.35 1.06 \u00b1 1.06 2.75 \u00b1 0.74 0.58 \u00b1 0.07 3.01 \u00b1 0.91 Kidneys 2.51 \u00b1 0.59 2.98 \u00b1 0.34 21.1 \u00b1 11.0* 4.58 \u00b1 0.66 3.19 \u00b1 0.36 2.57 \u00b1 0.48 Heart 0.27 \u00b1 0.08 0.34 \u00b1 0.05 1.13 \u00b1 0.62* 0.56 \u00b1 0.09 0.33 \u00b1 0.06 0.19 \u00b1 0.03 Lungs 0.75 \u00b1 0.52 1.19 \u00b1 0.32 3.74 \u00b1 0.94** 3.82 \u00b1 1.00 5.42 \u00b1 3.13 0.62 \u00b1 0.26 PC-3 tumor 10.2 \u00b1 2.27 11.2 \u00b1 0.65 2.18 \u00b1 0.56*** 5.87 \u00b1 0.64 8.32 \u00b1 1.20 10.5 \u00b1 2.03 Bone 0.19 \u00b1 0.06 0.13 \u00b1 0.01 0.39 \u00b1 0.19* 0.23 \u00b1 0.02 0.15 \u00b1 0.03 0.11 \u00b1 0.03 Muscle 0.15 \u00b1 0.05 0.20 \u00b1 0.05 0.41 \u00b1 0.15* 0.24 \u00b1 0.04 0.15 \u00b1 0.02 0.14 \u00b1 0.06 Brain 0.05 \u00b1 0.03 0.03 \u00b1 0.00 0.08 \u00b1 0.02** 0.06 \u00b1 0.01 0.06 \u00b1 0.03 0.03 \u00b1 0.01 Tumor\/bone 61.3 \u00b1 25.0 86.6 \u00b1 12.0 6.18 \u00b1 1.67*** 25.6 \u00b1 4.94 58.8 \u00b1 15.2 96.5 \u00b1 27.1 Tumor\/muscle 70.1 \u00b1 14.2 58.0 \u00b1 12.5 5.53 \u00b1 0.84*** 24.9 \u00b1 4.63 56.4 \u00b1 9.92 80.8 \u00b1 27.5 Tumor\/blood 14.0 \u00b1 3.48 9.91 \u00b1 1.38 0.79 \u00b1 0.11*** 3.26 \u00b1 0.59 8.93 \u00b1 1.49 16.5 \u00b1 3.06 Tumor\/kidney 4.10 \u00b1 0.46 3.76 \u00b1 0.36 0.11 \u00b1 0.03*** 1.26 \u00b1 0.10 2.62 \u00b1 0.33 4.13 \u00b1 0.73 Tumor\/pancreas 3.70 \u00b1 0.55 0.94 \u00b1 0.15 3.43 \u00b1 0.58*** 0.42 \u00b1 0.04 3.60 \u00b1 0.86 0.25 \u00b1 0.04 133 Figure 4.17 Uptake of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 in PC-3 tumor xenografts and major organs\/tissues of NRG mice at 1 h post-injection (n = 4). Error bars indicate standard deviation. Among all three 68Ga-labeled TacsBOMB2 derivatives, [68Ga]Ga-LW01158 had the highest tumor uptake and higher tumor-to-background contrast ratios for most of the major organs\/tissues (Figure 4.18 and Table 4.14). [68Ga]Ga-LW01158 had better tumor-to-bone and tumor-to-kidney uptake ratios than [68Ga]Ga-LW02002 (86.6 \u00b1 12.0 vs 58.8 \u00b1 15.2, and 3.76 \u00b1 0.36 vs 2.62 \u00b1 0.33, respectively). However, with the lowest pancreas uptake, [68Ga]Ga-LW02002 had a higher tumor-to-pancreas uptake ratio than [68Ga]Ga-LW01158 (3.60 \u00b1 0.86 vs 0.94 \u00b1 0.15, p < 0.001). The tumor-to-bone, tumor-to-muscle, tumor-to-blood, tumor-to-kidney, and tumor-to-pancreas uptake ratios of [68Ga]Ga-LW01186 were the lowest among these 3 tracers with the values of 25.6 \u00b1 4.94, 24.9 \u00b1 4.63, 3.26 \u00b1 0.59, 1.26 \u00b1 0.10, and 0.42 \u00b1 0.04 (p < 0.01), respectively. BloodSmall intestineLarge intestineSpleenPancreasStomachLiverKidneyHeartLungsPC-3 tumorBoneMuscle0102030Uptake (%ID\/g) [68Ga]Ga-LW01158 [68Ga]Ga-LW01186 [68Ga]Ga-LW02002134 Figure 4.18 Tumor-to-organ uptake ratios of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-02002 obtained from PC-3 tumor-bearing mice at 1 h post-injection (n = 4). Error bars indicate standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. Co-injection with 100 \u03bcg of nonradioactive standard reduced the uptake of [68Ga]Ga-LW01158 in PC-3 tumor xenograft by 81% (11.2 \u00b1 0.65 to 2.18 \u00b1 0.56 %ID\/g, p < 0.001) at 1 h post-injection. Furthermore, a significant reduction on the uptake of [68Ga]Ga-LW01158 was also found in the pancreas (12.0 \u00b1 1.41 to 0.66 \u00b1 0.28 %ID\/g, p < 0.001), small intestine (2.46 \u00b1 0.30 to 1.17 \u00b1 0.39 %ID\/g, p < 0.01), and stomach (1.30 \u00b1 0.41 to 0.42 \u00b1 0.15 %ID\/g, p < 0.01) (Figure 4.19 and Table 4.14). Figure 4.19 Comparison of [68Ga]Ga-LW01158 with\/without co-injection of 100 \u00b5g of nonradioactive Ga-LW01158 on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. Error bars indicate standard deviation (n = 4). *p < 0.05; **p < 0.01; ***p < 0.001. Tumor\/bone Tumor\/muscle Tumor\/blood Tumor\/kidney Tumor\/pancreas0510152060100140180220Ratio[68Ga]Ga-LW01158[68Ga]Ga-LW01186[68Ga]Ga-LW02002\u2731\u2731\u2731\u2731 \u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731 \u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731 \u2731\u2731\u2731\u2731 \u2731\u2731\u2731\u2731\u2731 \u2731\u2731\u2731Blood FatTestesSmall intestineLarge intestineSpleenPancreasStomachLiverAdrenal glandKidneyHeartLungsPC-3 tumorBoneMuscleBrain01234510203040Uptake (%ID\/g)[68Ga]Ga-LW01158Co-injection with Ga-LW01158\u2731 \u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731\u2731135 4.2.3.4 In vivo Stability All [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 showed good in vivo stability in NRG mice (n = 3, Figures 4.20-4.22). There were 80.7 \u00b1 1.57% of [68Ga]Ga-LW01158, 76.5 \u00b1 2.91% of [68Ga]Ga-LW01186, and 76.6 \u00b1 7.00% of [68Ga]Ga-LW02002 remaining intact in plasma at 15 min post-injection. No intact tracer was detected in urine samples for either [68Ga]Ga-LW01158 or [68Ga]Ga-LW02002, while 43.6 \u00b1 3.46% of intact [68Ga]Ga-LW01186 was detected in urine samples at 15 min post-injection. Figure 4.20 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01158 in (A) mouse plasma and (B) urine samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. 136 Figure 4.21 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW01186 in (A) mouse plasma and (B) urine samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. Figure 4.22 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-LW02002 in (A) mouse plasma and (B) urine samples collected at 15 min post-injection. The peak pointed by an arrow is the intact tracer. 137 4.2.4 Discussion Our group previously reported the synthesis and evaluation of a GRPR-targeted tracer, [68Ga]Ga-TacsBOMB2 (Figure 4.13A), based on the potent GRPR antagonist, RC-3950-II ([D-Phe6,Leu13\u03c8Thz14]Bombesin(7-14)), reported by the Schally group 181, 182, 252. [68Ga]Ga-TacsBOMB2 showed good uptake (10.2 \u00b1 2.27 %ID\/g) in PC-3 tumor xenograft and minimum pancreas uptake (2.81 \u00b1 0.78 %ID\/g) at 1 h post-injection 252. In this study, we modified the GRPR-targeting sequence of [68Ga]Ga-TacsBOMB2 with unnatural amino acid substitutions and evaluated the potential of the resulting ligands for PET imaging. Recently our group systematically substituted the amino acids (Gln7, Trp8, Ala9, Val10, Gly11 and His12) at potential cleavage sites of a previously reported GRPR agonist tracer ([68Ga]Ga-TacBOMB2: [68Ga]Ga-DOTA-Pip-D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Thz14-NH2) with unnatural amino acids to improve in vivo stability 258. We identified that Tle10 and NMe-His12 substitutions significantly improved in vivo stability and retained good binding affinity, high PC-3 tumor uptake, and minimal pancreas uptake 258. Therefore, in this study, we replaced Val10 and His12 in [68Ga]Ga-TacsBOMB2 with Tle10 and NMe-His12, respectively, and evaluated the potential of the resulting Ga-LW01158 (Figure 4.13B) and Ga-LW01160 (Figure 4.13C), respectively, for GRPR targeting. We first determined the binding affinities of Ga-LW01158 and Ga-LW01160 by in vitro competition binding assay (Figure 4.14). The Ki value of Ga-LW01158 was 5.11 \u00b1 0.47 nM which was better than that of Ga-TacsBOMB2 (7.08 \u00b1 0.65 nM) 252. This observation is consistent with our previous finding that Tle10 substitution on the GRPR agonist Ga-TacBOMB2 improves the binding affinity 258. However, Ga-LW01160 showed a very poor binding toward GRPR (Ki = 187 \u00b1 17.8 nM), while the previously reported NMe-His12 substitution significantly improved the binding affinity of Ga-TacBOMB2 from 7.62 \u00b1 0.19 nM to 2.98 \u00b1 0.69 nM 255, 258. These data 138 demonstrate that Tle10 substitution is tolerable by both GRPR agonists and antagonists, while NMe-His12 substitution can only be applied to GRPR agonists without significantly reducing the binding affinity. One possible explanation for this observation is that GRPR agonists and antagonists might bind to the receptors in different configurations so that modifications on some specific amino acids are tolerable only by either antagonists or agonists. Next, we introduced an additional \u03b1Me-Trp8 substitution on LW01158 to obtain LW01186 (Figure 4.13D). \u03b1Me-Trp8 substitution has been successfully used by the Wester group for the design of the potent and in vivo stable GRPR-targeted antagonist AMTG derived from RM2 231. NMe-Gly11 substitution has also been reported for the design of GRPR-targeted ligands to improve in vivo stability 229, 230. Previously, we developed a GRPR antagonist, Ga-TacsBOMB5, by introducing the NMe-Gly11 substitution on Ga-TacsBOMB2 252. Though [68Ga]Ga-TacsBOMB5 was not metabolically more stable than [68Ga]Ga-TacsBOMB2, it had a better PC-3 tumor uptake and tumor-to-background imaging contrast than [68Ga]Ga-TacsBOMB2 at 1 h post-injection 252. Therefore, in this study we also combined NMe-Gly11 and Tle10 substitutions to generate Ga-LW02002 (Figure 4.13E). As expected, good GRPR binding affinities of both Ga-LW01186 and Ga-LW02002 were observed (Ki = 6.94 \u00b1 0.95 and 11.0 \u00b1 0.39 nM, respectively) (Figure 4.14). These data also support that the configurations of the GRPR binding with agonists and antagonists might be different. While \u03b1Me-Trp8 and NMe-Gly11 substitutions are tolerable by antagonists, we have shown previously that \u03b1Me-Trp8 and NMe-Gly11 substitutions significantly reduced the binding affinity of GRPR agonists. The GRPR antagonist characteristics of the three potent Ga-TacsBOMB2 derivatives were determined by in vitro intracellular calcium release assays (Figure 4.15). In comparison with the positive control (ATP) and agonist control (bombesin), Ga-LW01158, Ga-LW01186, and Ga-139 LW02002 induced significantly lower intracellular Ca2+ efflux. This indicates that Tle10 substitution on Ga-TacsBOMB2, either alone or in combination with \u03b1Me-Trp8 or NMe-Gly11 substitution, retains the antagonist characteristics. Imaging studies showed that the PC-3 tumor xenograft was clearly visualized in PET images by all three 68Ga-labeled tracers ([68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002), confirming their good in vivo GRPR targeting capabilities. All three 68Ga-labeled tracers were mainly excreted via the renal pathway owing to the hydrophilic nature of these tracers (logD7.4 values \u2264 \u22121.98). The ex vivo biodistribution data of [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 were consistent with the findings in their PET images (Figures 4.16-4.19 and Table 4.14). Among these [68Ga]Ga-TacsBOMB2 derivatives, [68Ga]Ga-LW01158 had the highest PC-3 tumor uptake (11.2 \u00b1 0.65 %ID\/g), compared with 5.87 \u00b1 0.64 %ID\/g for [68Ga]Ga-LW01186 and 8.32 \u00b1 1.20 %ID\/g for [68Ga]Ga-LW02002. This might be resulted from the fact that Ga-LW01158 has a better GRPR binding affinity than Ga-LW01186 and Ga-LW02002 (Ki = 5.11 \u00b1 0.47, 6.94 \u00b1 0.95, and 11.0 \u00b1 0.39 nM, respectively). This also indicates that by using the Ga-TacsBOMB2 pharmacophore, the combination of \u03b1Me-Trp8 or NMe-Gly11 substitution with the Tle10 substitution cannot further improve either the binding affinity to GRPR or increase the uptake in GRPR-expressing PC-3 tumor xenografts. [68Ga]Ga-LW02002 had relatively lower uptake values in liver, small intestine, and large intestine (1.06 \u00b1 0.24, 0.66 \u00b1 0.06, and 0.54 \u00b1 0.29 %ID\/g, respectively) than those of [68Ga]Ga-LW01158 and [68Ga]Ga-LW01186. This is consistent with its relatively higher hydrophilicity than other two tracers (logD7.4 = \u22122.33 \u00b1 0.03 vs \u22121.98 \u00b1 0.10 for [68Ga]Ga-LW01158 and \u22122.03 \u00b1 0.10 for [68Ga]Ga-LW01186). Based on the logD7.4 value of [68Ga]Ga-LW01186 (\u22122.03 \u00b1 0.10), its high liver uptake (22.1 \u00b1 3.19 %ID\/g) was unexpected. Although the cause of its high liver uptake 140 remains to be investigated, the high liver uptake could be one of the reasons leading to its lower uptake in PC-3 tumor xenografts (5.87 \u00b1 0.64 %ID\/g) when compared with [68Ga]Ga-LW01158 (11.2 \u00b1 0.65 %ID\/g) and [68Ga]Ga-LW02002 (8.32 \u00b1 1.20 %ID\/g). Compared with the previously reported biodistribution data of [68Ga]Ga-RM2 (Table 4.14) 252, all three [68Ga]Ga-TacsBOMB2 derivatives showed significantly lower uptake in the pancreas. This is consistent with our previous finding that [D-Phe6,Leu13\u03c8Thz14]Bombesin(6-14) is a promising pharmacophore for the design of GRPR-targeted radiopharmaceuticals with a minimal pancreas uptake. One possible explanation is that these three [68Ga]Ga-TacsBOMB2 derivatives are more selective for binding to the human GRPR expressed in PC-3 tumors in comparison with the mouse GRPR expressed in mouse pancreas. The low pancreas uptake of these three [68Ga]Ga-TacsBOMB2 derivatives also demonstrates that \u03b1Me-Trp8, NMe-Gly11, and Tle10 substitutions do not significantly increase the pancreas uptake of the resulting GRPR-targeted tracers. With a significantly lower uptake in the pancreas and a comparable tumor uptake compared with the clinically validated [68Ga]Ga-RM2, [68Ga]Ga-LW01158 is a promising radiopharmaceutical for detecting GRPR-expressing lesions with PET, especially for the lesions in or adjacent to the pancreas. Similarly, LW01158 might be promising for labeling with 177Lu for radioligand therapy to minimize toxicity to the pancreas. A blocking study (Figure 4.19 and Table 4.14) was conducted to tease out the specificity of our top candidate, [68Ga]Ga-LW01158. The uptake in GRPR-expressing PC-3 tumor xenografts was reduced by > 80% with the co-injection of 100 \u00b5g of nonradioactive standard, confirming the tumor uptake of [68Ga]Ga-LW01158 is specific. Moreover, significant reductions were also observed in pancreas (12.0 \u00b1 1.41 to 0.66 \u00b1 0.28 %ID\/g, p < 0.001), stomach (1.30 \u00b1 0.41 to 0.42 \u00b1 0.15 %ID\/g, p < 0.01), and small intestine (2.46 \u00b1 0.30 to 1.17 \u00b1 0.39 % ID\/g, p < 0.01). This is 141 in agreement with the physiological expression pattern of GRPR in normal tissue\/organs 90. In addition, a significantly increased uptake was observed in kidneys (2.98 \u00b1 0.34 to 21.1 \u00b1 11.0 %ID\/g, p < 0.01). This is most likely due to the competitive binding of the nonradioactive standard to the GRPR in PC-3 tumors, increasing the amount of free [68Ga]Ga-LW01158 to be metabolized and excreted via the renal pathway. Besides, the GRPR-targeted ligands are mainly metabolized by NEP which is highly expressed in kidneys 201, 223. Co-injection with a significant amount of nonradioactive standard could saturate the metabolism of [68Ga]Ga-LW01158 by NEP in kidneys, leading to a higher kidney absorption and retention of [68Ga]Ga-LW01158. In vivo stability studies revealed that all three [68Ga]Ga-TacsBOMB2 derivatives were relatively stable in vivo with 76.5 to 80.7% of tracer remaining intact in mouse plasma at 15 min post-injection. These values were comparable to that of the previously reported [68Ga]Ga-TacsBOMB2 (83.3 \u00b1 1.45%) 252 . This suggests that among the potential cleavage sites on the [68Ga]Ga-TacsBOMB2 pharmacophore by peptidases, the amide bond between His12-Leu13 is the major one. Since the amide bond between His12-Leu13 has already been stabilized by the introduction of a reduced peptide bond (Leu13\u03c8Thz14), no further improvements in in vivo stability were observed with the additional Tle10 substitution, either alone or in combination with \u03b1Me-Trp8 or NMe-Gly11 substitution. No intact tracer was detected in urine samples of [68Ga]Ga-LW01158, [68Ga]Ga-LW02002, and the previously reported [68Ga]Ga-TacsBOMB2 at 15 min post-injection. Interestingly, although [68Ga]Ga-LW01186 had a similar intact fraction in mouse plasma when compared with [68Ga]Ga-LW01158, [68Ga]Ga-LW02002, and [68Ga]Ga-TacsBOMB2, 43.6 \u00b1 3.46% of intact [68Ga]Ga-LW01186 was detected in urine samples at 15 min post-injection (Figure 4.21). This observation was consistent with a recent report by the Wester group that the \u03b1Me-Trp8 substitution 142 on [177Lu]Lu-RM2 significantly increased the intact fraction of the resulting [177Lu]Lu-AMTG in urine samples at 30 min post-injection (0.5 \u00b1 0.1% to 68.2 \u00b1 3.1%) 231. This suggests that \u03b1Me-Trp8 substitution greatly inhibits degradation of GRPR-targeted ligands by the peptidases expressed in kidneys. 4.2.5 Conclusions The Tle10 substitution, either alone or in combination with \u03b1Me-Trp8 or NMe-Gly11, on the GRPR binding sequence of Ga-TacsBOMB2 generates derivatives with retained good GRPR binding affinity, antagonist characteristics, and good in vivo stability. However, the substitution of His12 with NMe-His leads to a significant decrease in GRPR binding affinity. In comparison with the clinically validated [68Ga]Ga-RM2, [68Ga]Ga-LW01158 has a comparable tumor uptake but much less pancreas uptake. Therefore, [68Ga]Ga-LW01158 is promising for clinical development for detecting GRPR-expressing lesions with PET, particularly for lesions in or adjacent to the pancreas. With a superior tumor-to-pancreas uptake ratio, [68Ga]Ga-LW02002 might be more promising for detecting cancer lesions adjacent to and in the pancreas. 143 Chapter 5: Synthesis and evaluation of 177Lu-labeled [Thz14]Bombesin(6-14) derivatives for radioligand therapy of gastrin-releasing peptide receptor-expressing cancer The compounds disclosed in this chapter are covered by a recent US patent application (PCT\/CA2023\/050401). Lei Wang, Zhengxing Zhang, Chengcheng Zhang, Fran\u00e7ois B\u00e9nard, and Kuo-Shyan Lin are listed as inventors of this filed patent application. 5.1 Introduction Overexpressed in many solid tumors, gastrin-releasing peptide receptor (GRPR) is a promising target for diagnosis and radiotherapy of GRPR-expressing cancers. Previously, our group developed one GRPR antagonist ([68Ga]Ga-TacsBOMB5)252 and two GRPR agonists ([68Ga]Ga-LW01110 and [68Ga]Ga-LW01142) 258 derived from [Thz14]Bombesin(6-14) for detecting GRPR-expressing cancer with positron emission tomography (PET). All three tracers showed high accumulations in PC-3 tumor xenografts (11.4 to 15.7 %ID\/g) and minimal uptake in the pancreas (1.98 to 8.99 %ID\/g) at 1h post-injection 252. Lutetium-177, a \u03b2\u2212 particle emitter (E\u03b2(max) 497 keV (78.6%), E\u03b2(max) 384 keV (9.1%), and E\u03b2(max) 176 keV (12.2%); 6.65 days half-life) , is widely used in radiotherapy applications and well chelated with the DOTA chelator 259. In addition, [68Ga]Ga-\/[177Lu]Lu-labeled theranostic pair agents have been widely used in cancer diagnosis and radiotherapy, such as [68Ga]Ga-\/[177Lu]Lu-NeoBOMB1 and [68Ga]Ga-\/[177Lu]Lu-DOTA-TATE 45, 209. Thus, in this study, we replaced 68Ga with 177Lu and synthesized [177Lu]Lu-TacsBOMB5 (Figure 5.1A), [177Lu]Lu-LW01110 (Figure 5.1B), and [177Lu]Lu-LW01142 (Figure 5.1C), and compared them with two clinically validated GRPR-targeted radioligands, [177Lu]Lu-RM2 (Figure 5.1D) and [177Lu]Lu-AMBA (Figure 5.1E). 144 Our hypotheses are (1) replacing 68Ga with 177Lu retains the high uptake in PC-3 tumor xenografts and the minimal uptake in the pancreas; (2) the resulting [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 are promising for clinical translation for radiotherapeutic applications. 145 Figure 5.1 Chemical structures of (A) Lu-TacsBOMB5, (B) Lu-LW01110, (C) Lu-LW01142, (D) Lu-RM2, and (E) Lu-AMBA. The unnatural amino acid substitutions are shown in blue. HNNHNNHHNNHHNNHNNSO NH2OHNNOOOONHOOH2NOOHNONNN NOOOOOOLu-TacsBOMB5HNNHHNNHHNNHHNNHNOHNNOOOONHOOH2NOOHNONNN NOOOOOOOHNHONH2OLu-RM2HNNHNNHHNNHHNNHNNSO NH2OHNNOOOONHOOH2NOOHNONNN NOOOOOOOLu-LW01110HNNHNNHHNNHHNNHNNSO NH2OHNNOOOONHOOOHNONNN NOOOOOOOLu-LW01142HNNLuLuHNNHHNNHHNNHHNNHO NH2OHNNOOOONHOOH2NOSOOOOOOONNN NHNONHOLu-AMBALuLuLuA.B.C.D.E.6 7 8 9 10 11 12 13 14146 5.2 Materials and Methods The materials and methods described in this section are provided in Chapter 2. Relevant sections are those describing reagent and instrumentation (Section 2.1), synthesis of Fmoc-Leu\u03c8Thz-OH (Section 2.2), synthesis of DOTA-conjugated precursors (Section 2.3.1), synthesis of nonradioactive Lu-complexed standards (Section 2.4.2), cell culture (Section 2.5), fluorometric calcium release assay (Section 2.6), in vitro competition binding assay (Section 2.7), 177Lu radiolabeling (Section 2.8.2), logD7.4 measurements (Section 2.9), animal studies (Section 2.10), SPECT imaging and biodistribution studies (Section 2.10.2), in vivo stability studies (Section 2.10.3), dosimetry (Section 2.11), and statistical analysis (Sections 2.12). 5.3 Results 5.3.1 Peptide Synthesis and Radiolabeling The HPLC conditions and MS characterizations of TacsBOMB5, LW01110, and LW01142 were provided in Section 3.1.3.1 and Section 4.1.3.1. The Lu-complexed nonradioactive standards of TacsBOMB5, LW01110, and LW01142 were synthesized in 47 to 88% yields and > 97% purity (Table 5.1). Table 5.1 MS characterizations, yields and HPLC purification conditions of Lu-TacsBOMB5, Lu-LW01110, and Lu-LW01142. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Lu-TacsBOMB5 25% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 13.9 47 [M+2H]2+ 885.4 [M+2H]2+ 885.8 Lu-LW01110 24% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 18.0 88 [M+2H]2+ 899.4 [M+2H]2+ 899.6 Lu-LW01142 24% CH3CN and 0.1% TFA in H2O; flow rate 4.5 mL\/min 12.6 61 [M+2H]2+ 903.9 [M+2H]2+ 904.1 147 177Lu-labeled TacsBOMB5, LW01110, LW01142, RM2 and AMBA were obtained in 24 to 62% decay-corrected radiochemical yields with 165 to 348 GBq\/\u00b5mol molar activity and > 91% radiochemical purity. The HPLC conditions for their purification and quality control are provided in Table 5.2. Table 5.2 HPLC conditions for the purification and quality control of 177Lu-labeled TacsBOMB5, LW01110, LW01142, RM2, and AMBA. FA: formic acid. Compound name HPLC conditions Retention time (min) [177Lu]Lu-TacsBOMB5 Semi-Prep 22% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 9.5 QC 24% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 6.1 [177Lu]Lu-LW01110 Semi-Prep 19% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 16.1 QC 22% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 6.8 [177Lu]Lu-LW01142 Semi-Prep 16% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 10.9 QC 19% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 7.0 [177Lu]Lu-RM2 Semi-Prep 19% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 36.3 QC 26% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 6.8 [177Lu]Lu-AMBA Semi-Prep 21% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 7.0 QC 22% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 9.4 5.3.2 Binding Affinity, Agonist\/Antagonist Characterization and Hydrophilicity Measurement The binding of [125I-Tyr4 ]Bombesin to PC-3 cells was inhibited by Lu-TacsBOMB5, Lu-LW01110, Lu-LW01142, Lu-RM2, and Lu-AMBA in a dose-dependent manner (Figure 5.2A). The comparison of the binding affinity values (Ki) for Lu-TacsBOMB5, Lu-LW01110, Lu-148 LW01142, Lu-RM2, and Lu-AMBA were shown in Figure 5.2B. Lu-AMBA showed the best binding affinity (0.33 \u00b1 0.16 nM) followed by Lu-RM2 (1.19 \u00b1 0.16 nM). The binding affinity of Lu-LW01110 is comparable with that of Lu-LW01142 (3.07 \u00b1 0.15 vs 2.37 \u00b1 0.28 nM, p = 0.1462) and lower than the two clinically validated Lu-RM2 and Lu-AMBA. In contrast, Lu-TacsBOMB5 showed a significantly lower binding affinity (12.6 \u00b1 1.02 nM) compared with the other GRPR-targeted compounds, almost 10-fold lower than that of Lu-RM2 and 38-fold lower than that of Lu-AMBA. Figure 5.2 (A) Displacement curves of [125I-Tyr4]Bombesin by Lu-TacsBOMB5, Lu-LW01110, Lu-LW011142, Lu-RM2, and Lu-AMBA generated using GRPR-expressing PC-3 cells. (B) Comparison of the binding affinities of Lu-TacsBOMB5, Lu-LW01110, Lu-LW011142, Lu-RM2, and Lu-AMBA. ns, *,**, and *** indicate p > 0.05, < 0.05, < 0.01 and < 0.001, respectively. Error bars indicate standard deviation. 149 The agonist\/antagonist characterization for Lu-TacsBOMB5, Lu-LW01110, and Lu-LW011142 was performed by intracellular calcium release assay using PC-3 cells (Figure 5.3). Lu-LW01110 (50 nM), Lu-LW01142 (50 nM), bombesin (50 nM, an agonist control), and ATP (50 nM, a positive control) induced Ca2+ efflux corresponding to 238 \u00b1 13.0, 224 \u00b1 16.1, 235 \u00b1 15.2, and 150 \u00b1 40.7 RFUs, respectively, while Lu-TacsBOMB5 (50 nM), [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) (50 nM, an antagonist control), and DPBS (blank control) induced Ca2+ efflux with 7.56 \u00b1 1.04, 29.7 \u00b1 2.73, and 8.59 \u00b1 1.88 RFUs, respectively. Figure 5.3 Intracellular calcium efflux in PC-3 cells induced by Lu-TacsBOMB5, Lu-LW01110, Lu-LW011142, bombesin, ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), ATP, and DPBS. Error bars indicate standard deviation (n = 3). The hydrophilicity of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 were determined by the shake flask method following previously published procedures, and their logD7.4 values were \u22122.53 \u00b1 0.20, \u22122.76 \u00b1 0.10, and \u22122.28 \u00b1 0.14, respectively (n = 3). 150 5.3.3 SPECT imaging The longitudinal SPECT\/CT images of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA are shown in Figure 5.4-5.8. [177Lu]Lu-TacsBOMB5 enabled visualization of the PC-3 tumor xenografts in SPECT images at 1 h, 4 h, and 24 h post-injection with a scale bar denoting 0-10 %ID\/g. For [177Lu]Lu-LW01110 and [177Lu]Lu-LW01142, PC-3 tumor xenografts were visualized up to 120 h post-injection with at a 0-15 %ID\/g color bar scale. The two standards, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA enabled visualization of the PC-3 tumor xenografts in the SPECT images up to 72 h post-injection with a 0-15 %ID\/g color bar scale. Bladders were also clearly visualized at 1 h and 4 h post-injection for all five radioligands. Accumulation in the pancreas was also observed at 1 h and 4 h post-injection for [177Lu]Lu-RM2, and up to 72 h post-injection for [177Lu]Lu-AMBA. Co-injection with 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) significantly decreased accumulation of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 in the PC-3 tumor xenografts at 1 h post-injection, making the PC-3 tumors nearly invisible in the SPECT images. 151 Figure 5.4 Longitudinal SPECT\/CT images of [177Lu]Lu-TacsBOMB5 acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. The blocked mice were co-injected with 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14). t: tumor; bl: urinary bladder. Figure 5.5 Longitudinal SPECT\/CT images of [177Lu]Lu-LW01110 acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. The blocked mice were co-injected with 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14). t: tumor; bl: urinary bladder. 152 Figure 5.6 Longitudinal SPECT\/CT images of [177Lu]Lu-LW01142 acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. The blocked mice were co-injected with 100 \u03bcg of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14). t: tumor; bl: urinary bladder. Figure 5.7 Longitudinal SPECT\/CT images of [177Lu]Lu-RM2 acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. t: tumor; p: pancreas; bl: urinary bladder. 153 Figure 5.8 Longitudinal SPECT\/CT images of [177Lu]Lu-AMBA acquired from PC-3 tumor-bearing NRG mice. Acquisition time points are 1, 4, 24, 72, and 120 h post-injection. t: tumor; p: pancreas; bl: urinary bladder. 5.3.4 Ex vivo Biodistribution The ex vivo biodistribution results of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA are provided in Figure 5.9 and Tables 5.3-5.7. The tumor uptake values of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 were 8.71 \u00b1 0.53, 11.0 \u00b1 1.03, and 13.4 \u00b1 1.48 %ID\/g at 1 h post-injection, and decreased over time. In contrast, the tumor uptake values of [177Lu]Lu-RM2 and [177Lu]Lu-AMBA slightly increased from 7.73 \u00b1 0.96 %ID\/g (1 h post-injection) to 8.49 \u00b1 1.26 %ID\/g (4 h post-injection) and from 5.42 \u00b1 1.17 %ID\/g (1 h post-injection) to 6.66 \u00b1 1.04 %ID\/g (4 h post-injection), respectively, and then decreased to 2.10 \u00b1 0.15 and 1.09 \u00b1 0.42 %ID\/g at 120 h post-injection, respectively. A faster clearance from PC-3 tumor xenografts was observed for [177Lu]Lu-TacsBOMB5 compared with other GRPR-targeted tracers (Figure 5.9A and Table 5.3). The tumor uptake of [177Lu]Lu-TacsBOMB5 dropped to 1.77 \u00b1 0.27%ID\/g at 24 h post-injection and kept decreasing to less than 1 %D\/g at 120 h post-injection. In contrast, the tumor uptake values of 154 [177Lu]Lu-LW01110 and [177Lu]Lu-LW01142 were still higher than 2 %ID\/g at 120 h post-injection (Figure 5.9A, Table 5.4 and Table 5.5). [177Lu]Lu-AMBA had the highest pancreas uptake at all five time points from 83.8 \u00b1 6.06 %ID\/g at 1 h post-injection to 16.3 \u00b1 1.00 %ID\/g at 120 h post-injection (Figure 5.9B and Table 5.5). [177Lu]Lu-RM2 also had a very high uptake in the pancreas at 1 h post-injection (34.8 \u00b1 10.6 %ID\/g), but the pancreas uptake dropped rapidly to less than 1 %ID\/g at 24 h post-injection (Figure 5.9A and Table 5.4). All [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 showed overall lower pancreas uptake compared to [177Lu]Lu-RM2 and [177Lu]Lu-AMBA. For [177Lu]Lu-TacsBOMB5, the pancreas uptake was only 1.08 \u00b1 0.22, 0.21 \u00b1 0.01, 0.08 \u00b1 0.02, 0.04 \u00b1 0.01, and 0.02 \u00b1 0.00 %ID\/g, respectively, at 1 h, 4 h, 24 h, 72 h, and 120 h post-injection. An overall lower accumulation of [177Lu]Lu-TacsBOMB5 was observed in most normal organs across all five time points compared with the other four radioligands. The tumor-to-muscle and tumor-to-blood ratios of all five radioligands were increasing over time from 1 h to 72 h post-injection. The tumor-to-muscle and tumor-to-blood ratios of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 dropped from 72 h to 120 h post-injection due to the clearance of radioactivity from PC-3 tumor xenografts. Blocking studies of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 were conducted at 1 h post-injection (Table 5.3 to 5.5). The results showed that co-injection of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) reduced the average tumor uptake values of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 by 71%, 73%, and 62%, respectively. In addition, the pancreas uptake values of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 were also reduced by 45%, 76% and 58%, respectively, for the blocked mice. Significant reductions were also observed on intestine and 155 stomach uptake for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 (Tables 5.3-5.5). Figure 5.9 Comparison of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA on their uptake in (A) PC-3 tumor xenografts and (B) the pancreas in mice at 1, 4, 24, 72, and 120 h post-injection. Table 5.3 Biodistribution (mean \u00b1 SD, n = 5) and tumor-to-organ ratios of [177Lu]Lu-TacsBOMB5 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. The mice in the blocked group were co-injected with 100 \u00b5g of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) per mouse. *,**, and *** indicate p < 0.05, < 0.01 and < 0.001, respectively, when comparing the 1 h and 1 h blocked data of [177Lu]Lu-TacsBOMB5. Tissue (%ID\/g, n = 5) [177Lu]Lu-TacsBOMB5 1 h 4 h 24 h 72 h 120 h 1 h blocked Blood 1.77 \u00b1 0.14 0.85 \u00b1 0.03 0.21 \u00b1 0.02 0.07 \u00b1 0.01 0.03 \u00b1 0.01 0.45 \u00b1 0.11*** Fat 0.15 \u00b1 0.04 0.06 \u00b1 0.01 0.03 \u00b1 0.01 0.02 \u00b1 0.01 0.01 \u00b1 0.01 0.06 \u00b1 0.01*** Testes 0.45 \u00b1 0.11 0.23 \u00b1 0.04 0.12 \u00b1 0.02 0.08 \u00b1 0.04 0.08 \u00b1 0.02 0.18 \u00b1 0.02*** Small intestine 0.60 \u00b1 0.16 0.19 \u00b1 0.05 0.09 \u00b1 0.03 0.03 \u00b1 0.00 0.02 \u00b1 0.00 0.26 \u00b1 0.04** 156 Large intestine 0.31 \u00b1 0.09 0.42 \u00b1 0.09 0.42 \u00b1 0.25 0.10 \u00b1 0.08 0.03 \u00b1 0.02 0.13 \u00b1 0.04** Spleen 0.31 \u00b1 0.03 0.23 \u00b1 0.04 0.16 \u00b1 0.03 0.18 \u00b1 0.05 0.14 \u00b1 0.06 0.15 \u00b1 0.02*** Pancreas 1.08 \u00b1 0.22 0.21 \u00b1 0.01 0.08 \u00b1 0.02 0.04 \u00b1 0.01 0.02 \u00b1 0.00 0.59 \u00b1 0.11** Stomach 0.24 \u00b1 0.06 0.11 \u00b1 0.05 0.27 \u00b1 0.16 0.03 \u00b1 0.00 0.02 \u00b1 0.02 0.06 \u00b1 0.01*** Liver 0.67 \u00b1 0.07 0.54 \u00b1 0.04 0.29 \u00b1 0.02 0.20 \u00b1 0.02 0.16 \u00b1 0.07 0.29 \u00b1 0.05*** Adrenal glands 0.61 \u00b1 0.28 0.29 \u00b1 0.07 0.17 \u00b1 0.02 0.18 \u00b1 0.12 0.12 \u00b1 0.08 0.17 \u00b1 0.04* Kidneys 3.47 \u00b1 0.29 3.10 \u00b1 0.35 1.38 \u00b1 0.18 0.60 \u00b1 0.07 0.39 \u00b1 0.11 2.78 \u00b1 0.51* Heart 0.45 \u00b1 0.06 0.21 \u00b1 0.03 0.09 \u00b1 0.01 0.05 \u00b1 0.01 0.04 \u00b1 0.01 0.15 \u00b1 0.02*** Lungs 2.20 \u00b1 0.93 1.45 \u00b1 0.34 0.25 \u00b1 0.03 0.13 \u00b1 0.01 0.07 \u00b1 0.03 0.37 \u00b1 0.04** PC-3 tumor 8.71 \u00b1 0.53 4.80 \u00b1 0.32 1.77 \u00b1 0.27 1.09 \u00b1 0.19 0.66 \u00b1 0.23 2.53 \u00b1 0.34*** Bone 0.12 \u00b1 0.02 0.09 \u00b1 0.03 0.07 \u00b1 0.02 0.07 \u00b1 0.01 0.07 \u00b1 0.01 0.06 \u00b1 0.03** Muscle 0.22 \u00b1 0.01 0.10 \u00b1 0.02 0.03 \u00b1 0.00 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.13 \u00b1 0.03*** Brain 0.04 \u00b1 0.00 0.02 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.02 \u00b1 0.00*** Tumor to Normal Tissue Ratios Tumor\/muscle 39.6 \u00b1 2.10 49.9 \u00b1 8.90 51.5 \u00b1 6.89 53.6 \u00b1 7.29 48.2 \u00b1 20.3 20.6 \u00b1 3.63 Tumor\/blood 4.95 \u00b1 0.52 5.64 \u00b1 0.30 8.34 \u00b1 1.81 15.5 \u00b1 3.40 22.2 \u00b1 6.11 5.88 \u00b1 1.56*** Tumor\/kidney 2.52 \u00b1 0.27 1.56 \u00b1 0.18 1.29 \u00b1 0.19 1.80 \u00b1 0.23 1.70 \u00b1 0.32 0.94 \u00b1 0.21** Tumor\/pancreas 8.32 \u00b1 1.70 22.8 \u00b1 2.08 22.2 \u00b1 4.34 31.0 \u00b1 3.31 33.4 \u00b1 7.57 4.42 \u00b1 0.82*** Table 5.4 Biodistribution (mean \u00b1 SD, n = 5) and tumor-to-organ ratios of [177Lu]Lu-LW01110 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. The mice in the blocked group were co-injected with 100 \u00b5g of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) per mouse. *,**, and *** indicate p < 0.05, < 0.01 and < 0.001, respectively, when comparing the 1 h and 1 h blocked data of [177Lu]Lu-LW01110. Tissue (%ID\/g, n = 5) [177Lu]Lu-LW01110 1 h 4 h 24 h 72 h 120 h 1 h blocked Blood 0.69 \u00b1 0.06 0.04 \u00b1 0.01 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.58 \u00b1 0.16 Fat 0.07 \u00b1 0.02 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.07 \u00b1 0.02 Testes 0.23 \u00b1 0.04 0.06 \u00b1 0.05 0.02 \u00b1 0.01 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.30 \u00b1 0.16 Small intestine 2.01 \u00b1 0.13 0.56 \u00b1 0.20 0.30 \u00b1 0.10 0.09 \u00b1 0.03 0.03 \u00b1 0.00 0.49 \u00b1 0.14*** Large intestine 1.00 \u00b1 0.15 1.15 \u00b1 0.38 0.44 \u00b1 0.11 0.23 \u00b1 0.04 0.09 \u00b1 0.02 0.29 \u00b1 0.08*** Spleen 0.28 \u00b1 0.05 0.15 \u00b1 0.03 0.12 \u00b1 0.02 0.10 \u00b1 0.02 0.05 \u00b1 0.01 0.21 \u00b1 0.05 Pancreas 11.1 \u00b1 1.37 4.91 \u00b1 0.63 3.10 \u00b1 0.48 1.03 \u00b1 0.30 0.34 \u00b1 0.04 2.71 \u00b1 0.76*** Stomach 0.99 \u00b1 0.21 0.43 \u00b1 0.16 0.17 \u00b1 0.07 0.08 \u00b1 0.02 0.04 \u00b1 0.02 0.17 \u00b1 0.10*** Liver 0.40 \u00b1 0.05 0.23 \u00b1 0.07 0.13 \u00b1 0.04 0.07 \u00b1 0.01 0.04 \u00b1 0.01 0.26 \u00b1 0.05** Adrenal glands 1.22 \u00b1 0.38 1.02 \u00b1 0.33 0.98 \u00b1 0.18 0.41 \u00b1 0.06 0.25 \u00b1 0.05 0.54 \u00b1 0.24** Kidneys 3.77 \u00b1 0.58 2.67 \u00b1 0.49 1.46 \u00b1 0.42 0.52 \u00b1 0.10 0.20 \u00b1 0.02 3.56 \u00b1 0.77 Heart 0.22 \u00b1 0.01 0.06 \u00b1 0.06 0.03 \u00b1 0.00 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.20 \u00b1 0.06 Lungs 0.69 \u00b1 0.12 0.23 \u00b1 0.11 0.07 \u00b1 0.03 0.06 \u00b1 0.03 0.03 \u00b1 0.03 0.52 \u00b1 0.08* PC-3 tumor 11.0 \u00b1 1.03 10.1 \u00b1 1.40 6.90 \u00b1 1.34 4.72 \u00b1 1.42 2.23 \u00b1 0.33 2.99 \u00b1 0.42*** Bone 0.14 \u00b1 0.05 0.05 \u00b1 0.03 0.02 \u00b1 0.01 0.02 \u00b1 0.01 0.01 \u00b1 0.00 0.08 \u00b1 0.02 Muscle 0.15 \u00b1 0.02 0.03 \u00b1 0.01 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.14 \u00b1 0.03 157 Brain 0.03 \u00b1 0.00 0.03 \u00b1 0.02 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.02 \u00b1 0.00** Tumor to Normal Tissue Ratios Tumor\/muscle 76.2 \u00b1 10.1 392 \u00b1 101 651 \u00b1 95.0 647 \u00b1 311 776 \u00b1 314 21.5 \u00b1 3.27*** Tumor\/blood 15.9 \u00b1 1.55 234 \u00b1 53.9 1218 \u00b1 229 1331 \u00b1 177 1309 \u00b1 412 5.32 \u00b1 0.87*** Tumor\/kidney 2.96 \u00b1 0.43 3.27 \u00b1 0.89 4.86 \u00b1 0.64 8.92 \u00b1 1.62 11.3 \u00b1 1.27 0.85 \u00b1 0.10*** Tumor\/pancreas 0.99 \u00b1 0.11 1.87 \u00b1 0.37 2.22 \u00b1 0.25 4.58 \u00b1 0.68 6.60 \u00b1 1.14 1.17 \u00b1 0.31 Table 5.5 Biodistribution (mean \u00b1 SD, n = 5) and tumor-to-organ ratios of [177Lu]Lu-LW01142 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. The mice in the blocked group were co-injected with 100 \u00b5g of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) per mouse. *,**, and *** indicate p < 0.05, < 0.01 and < 0.001, respectively, when comparing the 1 h and 1 h blocked data of [177Lu]Lu-LW01142. Tissue (%ID\/g, n = 5) [177Lu]Lu-LW01142 1 h 4 h 24 h 72 h 120 h 1 h blocked Blood 1.67 \u00b1 0.21 0.08 \u00b1 0.01 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 1.34 \u00b1 0.35 Fat 0.11 \u00b1 0.04 0.02 \u00b1 0.01 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.14 \u00b1 0.04 Testes 0.32 \u00b1 0.18 0.05 \u00b1 0.01 0.03 \u00b1 0.01 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.48 \u00b1 0.16 Small intestine 1.32 \u00b1 0.42 0.29 \u00b1 0.08 0.14 \u00b1 0.03 0.05 \u00b1 0.01 0.02 \u00b1 0.02 0.54 \u00b1 0.10** Large intestine 0.76 \u00b1 0.16 0.81 \u00b1 0.21 0.30 \u00b1 0.12 0.13 \u00b1 0.05 0.07 \u00b1 0.04 0.31 \u00b1 0.08*** Spleen 0.47 \u00b1 0.07 0.17 \u00b1 0.04 0.14 \u00b1 0.05 0.09 \u00b1 0.02 0.09 \u00b1 0.02 0.40 \u00b1 0.04 Pancreas 4.45 \u00b1 0.83 1.57 \u00b1 0.28 1.06 \u00b1 0.26 0.68 \u00b1 0.10 0.48 \u00b1 0.08 1.88 \u00b1 0.42*** Stomach 0.60 \u00b1 0.24 0.11 \u00b1 0.04 0.16 \u00b1 0.07 0.10 \u00b1 0.05 0.02 \u00b1 0.01 0.17 \u00b1 0.06** Liver 0.71 \u00b1 0.08 0.33 \u00b1 0.05 0.28 \u00b1 0.07 0.12 \u00b1 0.03 0.08 \u00b1 0.02 0.57 \u00b1 0.15 Adrenal glands 1.43 \u00b1 0.50 0.59 \u00b1 0.06 0.41 \u00b1 0.08 0.20 \u00b1 0.05 0.17 \u00b1 0.09 0.75 \u00b1 0.33 Kidneys 6.86 \u00b1 0.81 5.51 \u00b1 0.54 2.64 \u00b1 0.94 0.60 \u00b1 0.17 0.30 \u00b1 0.07 7.44 \u00b1 1.71 Heart 0.45 \u00b1 0.06 0.06 \u00b1 0.01 0.02 \u00b1 0.01 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.43 \u00b1 0.14 Lungs 1.53 \u00b1 0.19 0.57 \u00b1 0.21 0.16 \u00b1 0.08 0.03 \u00b1 0.01 0.03 \u00b1 0.01 1.06 \u00b1 0.27* PC-3 tumor 13.4 \u00b1 1.48 11.8 \u00b1 2.24 7.07 \u00b1 1.23 4.18 \u00b1 0.6 2.25 \u00b1 0.71 5.03 \u00b1 0.43*** Bone 0.15 \u00b1 0.03 0.03 \u00b1 0.01 0.03 \u00b1 0.01 0.03 \u00b1 0.01 0.02 \u00b1 0.01 0.13 \u00b1 0.03 Muscle 0.22 \u00b1 0.04 0.03 \u00b1 0.01 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.23 \u00b1 0.11 Brain 0.03 \u00b1 0.01 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.03 \u00b1 0.01 Tumor to Normal Tissue Ratios Tumor\/muscle 60.1 \u00b1 5.97 454 \u00b1 53.3 672 \u00b1 161 736 \u00b1 189 588 \u00b1 92.8 25.2 \u00b1 10.1*** Tumor\/blood 8.08 \u00b1 0.92 162 \u00b1 52.0 841 \u00b1 162 1620 \u00b1 500 1800 \u00b1 724 3.91 \u00b1 0.87*** Tumor\/kidney 1.95 \u00b1 0.04 2.14 \u00b1 0.26 2.88 \u00b1 0.84 7.34 \u00b1 1.83 7.46 \u00b1 1.11 0.70 \u00b1 0.15*** Tumor\/pancreas 3.06 \u00b1 0.45 7.53 \u00b1 0.35 6.78 \u00b1 0.88 6.18 \u00b1 0.60 4.08 \u00b1 1.00 2.79 \u00b1 0.69 158 Table 5.6 Biodistribution (mean \u00b1 SD, n = 5) and tumor-to-organ ratios of [177Lu]Lu-RM2 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. Tissue (%ID\/g, n = 5) [177Lu]Lu-RM2 1 h 4 h 24 h 72 h 120 h Blood 0.55 \u00b1 0.09 0.07 \u00b1 0.02 0.01 \u00b1 0.00 0.12 \u00b1 0.11 0.00 \u00b1 0.00 0.14 \u00b1 0.05 0.00 \u00b1 0.00 Fat 0.07 \u00b1 0.06 0.01 \u00b1 0.01 0.01 \u00b1 0.01 0.02 \u00b1 0.02 0.00 \u00b1 0.00 Testes 0.14 \u00b1 0.02 0.04 \u00b1 0.01 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.01 \u00b1 0.00 Small intestine 5.91 \u00b1 1.32 1.13 \u00b1 0.44 0.09 \u00b1 0.02 0.08 \u00b1 0.08 0.01 \u00b1 0.00 Large intestine 3.14 \u00b1 0.83 1.25 \u00b1 0.45 0.24 \u00b1 0.06 1.50 \u00b1 1.60 0.03 \u00b1 0.02 Spleen 0.39 \u00b1 0.27 0.11 \u00b1 0.03 0.08 \u00b1 0.02 0.13 \u00b1 0.11 0.03 \u00b1 0.01 Pancreas 34.8 \u00b1 10.6 4.17 \u00b1 0.69 0.58 \u00b1 0.12 0.38 \u00b1 0.31 0.06 \u00b1 0.01 Stomach 3.08 \u00b1 1.83 0.96 \u00b1 0.43 0.14 \u00b1 0.06 0.19 \u00b1 0.29 0.01 \u00b1 0.00 Liver 0.62 \u00b1 0.54 0.19 \u00b1 0.03 0.10 \u00b1 0.01 0.06 \u00b1 0.01 0.03 \u00b1 0.01 Adrenal glands 2.32 \u00b1 0.85 1.04 \u00b1 0.41 0.86 \u00b1 0.37 0.75 \u00b1 0.31 0.29 \u00b1 0.10 Kidneys 3.44 \u00b1 0.64 1.83 \u00b1 0.34 0.96 \u00b1 0.16 0.41 \u00b1 0.07 0.14 \u00b1 0.03 Heart 0.18 \u00b1 0.06 0.03 \u00b1 0.01 0.02 \u00b1 0.01 0.01 \u00b1 0.01 0.01 \u00b1 0.00 Lungs 1.42 \u00b1 0.69 0.46 \u00b1 0.29 0.07 \u00b1 0.02 0.05 \u00b1 0.01 0.01 \u00b1 0.00 PC-3 tumor 7.73 \u00b1 0.96 8.49 \u00b1 1.26 6.53 \u00b1 0.84 4.40 \u00b1 1.05 2.10 \u00b1 0.15 Bone 0.14 \u00b1 0.04 0.02 \u00b1 0.01 0.03 \u00b1 0.03 0.03 \u00b1 0.01 0.02 \u00b1 0.01 Muscle 0.13 \u00b1 0.04 0.03 \u00b1 0.01 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.00 \u00b1 0.00 Brain 0.03 \u00b1 0.01 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 Tumor to Normal Tissue Ratios Tumor\/muscle 61.7 \u00b1 12.8 343 \u00b1 38.9 865 \u00b1 278 789 \u00b1 235 963 \u00b1 312 Tumor\/blood 14.3 \u00b1 2.15 131 \u00b1 36.0 1051 \u00b1 432 2049 \u00b1 1068 2532 \u00b1 1372 Tumor\/kidney 2.28 \u00b1 0.30 4.68 \u00b1 0.63 6.87 \u00b1 1.16 9.90 \u00b1 0.97 16.0 \u00b1 3.19 Tumor\/pancreas 0.23 \u00b1 0.06 1.79 \u00b1 0.50 11.4 \u00b1 1.38 15.2 \u00b1 5.89 38.1 \u00b1 5.62 Table 5.7 Biodistribution (mean \u00b1 SD, n = 5) and tumor-to-organ ratios of [177Lu]Lu-AMBA in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h post-injection. Tissue (%ID\/g, n = 5) [177Lu]Lu-AMBA 1 h 4 h 24 h 72 h 120 h Blood 0.65 \u00b1 0.17 0.05 \u00b1 0.01 0.02 \u00b1 0.00 0.12 \u00b1 0.11 0.01 \u00b1 0.00 0.14 \u00b1 0.05 0.00 \u00b1 0.00 Fat 0.08 \u00b1 0.04 0.02 \u00b1 0.01 0.01 \u00b1 0.00 0.01 \u00b1 0.01 0.00 \u00b1 0.00 Testes 0.26 \u00b1 0.05 0.06 \u00b1 0.01 0.03 \u00b1 0.00 0.02 \u00b1 0.00 0.01 \u00b1 0.00 Small intestine 8.42 \u00b1 0.56 8.40 \u00b1 0.98 4.59 \u00b1 0.44 2.39 \u00b1 0.71 1.19 \u00b1 0.23 Large intestine 5.41 \u00b1 0.52 7.81 \u00b1 1.09 4.69 \u00b1 0.43 1.57 \u00b1 0.35 0.86 \u00b1 0.39 Spleen 0.80 \u00b1 0.35 0.63 \u00b1 0.24 0.40 \u00b1 0.15 0.17 \u00b1 0.05 0.11 \u00b1 0.02 Pancreas 83.8 \u00b1 6.06 77.7 \u00b1 10.5 45.4 \u00b1 3.30 27.6 \u00b1 5.37 16.3 \u00b1 1.00 Stomach 3.23 \u00b1 2.32 2.39 \u00b1 0.65 2.36 \u00b1 0.77 0.88 \u00b1 0.25 0.44 \u00b1 0.10 Liver 0.46 \u00b1 0.10 0.75 \u00b1 0.65 0.22 \u00b1 0.06 0.09 \u00b1 0.03 0.05 \u00b1 0.01 159 Adrenal glands 20.4 \u00b1 3.91 31.2 \u00b1 6.49 12.5 \u00b1 1.35 2.78 \u00b1 0.87 0.70 \u00b1 0.06 Kidneys 7.70 \u00b1 0.76 7.01 \u00b1 1.27 2.91 \u00b1 0.08 1.76 \u00b1 0.28 0.87 \u00b1 0.13 Heart 0.27 \u00b1 0.08 0.08 \u00b1 0.01 0.04 \u00b1 0.00 0.02 \u00b1 0.00 0.01 \u00b1 0.00 Lungs 0.72 \u00b1 0.14 0.28 \u00b1 0.03 0.14 \u00b1 0.03 0.06 \u00b1 0.02 0.03 \u00b1 0.01 PC-3 tumor 5.42 \u00b1 1.17 6.66 \u00b1 1.04 3.97 \u00b1 1.39 2.03 \u00b1 0.30 1.09 \u00b1 0.42 Bone 0.47 \u00b1 0.04 0.37 \u00b1 0.08 0.21 \u00b1 0.01 0.12 \u00b1 0.02 0.07 \u00b1 0.03 Muscle 0.27 \u00b1 0.02 0.12 \u00b1 0.03 0.05 \u00b1 0.01 0.02 \u00b1 0.00 0.01 \u00b1 0.00 Brain 0.05 \u00b1 0.01 0.05 \u00b1 0.01 0.04 \u00b1 0.01 0.02 \u00b1 0.01 0.02 \u00b1 0.00 Tumor to Normal Tissue Ratios Tumor\/muscle 20.4 \u00b1 4.03 58.6 \u00b1 15.4 72.5 \u00b1 22.5 88.9 \u00b1 15.6 103 \u00b1 46.0 Tumor\/blood 8.86 \u00b1 3.05 135 \u00b1 22.4 180 \u00b1 64.3 264 \u00b1 53.9 391 \u00b1 157 Tumor\/kidney 0.70 \u00b1 0.14 0.96 \u00b1 0.10 1.26 \u00b1 0.47 1.16 \u00b1 0.12 1.24 \u00b1 0.42 Tumor\/pancreas 0.06 \u00b1 0.01 0.09 \u00b1 0.01 0.09 \u00b1 0.03 0.08 \u00b1 0.02 0.07 \u00b1 0.02 5.3.5 Dosimetry The calculated radiation absorbed doses in mice from [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA are presented in Table 5.8 and Figure 5.10A. [177Lu]Lu-RM2 had the highest absorbed dose in a 1-g PC-3 tumor xenograft (Unit Density Sphere Model) with 429 mGy\/MBq, followed by [177Lu]Lu-LW01110 and [177Lu]Lu-LW01142 with the same absorbed dose (312 mGy\/MBq). The absorbed dose in a 1-g PC-3 tumor xenograft from [177Lu]Lu-ProBOMB5 was 87.1 mGy\/MBq, slightly higher than that of [177Lu]Lu-AMBA (76.3 mGy\/MBq). For [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, and [177Lu]Lu-RM2, urinary bladder received the highest absorbed dose among all the target organs with 562, 1260, 1180, and 2190 mGy\/MBq, respectively. For [177Lu]Lu-AMBA, the pancreas received the highest absorbed dose (3380 mGy\/MBq) followed by urinary bladder (1310 mGy\/MBq). The absorbed doses in kidneys for all five tracers were higher than those in other normal organs, except for the pancreas, with the absorbed doses ranging from 116 to 294 mGy\/MBq. [177Lu]Lu-LW01110 also had a high absorbed dose in the pancreas at 348 mGy\/MBq, followed by [177Lu]Lu-RM2 with 316 mGy\/MBq, which were nearly two-fold of that for [177Lu]Lu-LW01142 (180 mGy\/MBq) and approximately 30-fold of that for [177Lu]Lu-160 TacsBOMB5 (11.6 mGy\/MBq). [177Lu]Lu-AMBA also had the highest absorbed doses in some major organs including large intestine, small intestine, and stomach wall, while [177Lu]Lu-TacsBOMB5 had the lowest absorbed doses in these major organs. By extrapolating the biodistribution data to the adult human male model, the estimated radiation absorbed doses for all five radioligands were calculated and presented in Table 5.9 and Figure 5.10B. The estimated absorbed doses for [177Lu]Lu-AMBA in pancreas, small intestine, stomach wall, colon, and rectum were approximately 4 to 16 times higher than those of [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, and [177Lu]Lu-RM2, respectively. The lowest estimated absorbed doses were obtained by using [177Lu]Lu-TacsBOMB5, and those values were 10 to 300 folds lower than those of [177Lu]Lu-AMBA. The effective whole-body doses of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA to an adult human male were 6.49E-03, 1.35E-02, 1.16E-02, 1.43E-02, and 3.2E-02 mSv\/MBq, respectively. Table 5.8 Estimated radiation absorbed doses (per unit of injected radioactivity (mGy\/MBq)) in mice for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA. Target Organ [177Lu]Lu-TacsBOMB5 [177Lu]Lu-LW01110 [177Lu]Lu-LW01142 [177Lu]Lu-RM2 [177Lu]Lu-AMBA PC-3 tumor xenograft 87.1 312 312 429 76.3 Brain 2.52 1.79 3.7 1.41 4.62 Large intestine 23.6 92.9 49.5 95.3 302 Small intestine 8.58 40.4 25 60.5 334 Stomach wall 5.49 35.8 42.7 56.4 218 Heart 12.2 2.71 4.65 3.18 5.19 Kidneys 116 177 294 171 249 Liver 34.4 20.2 38.8 35.4 25.4 Lungs 29 19.1 22.7 20.8 11.9 Pancreas 11.6 348 180 316 3380 Skeleton 28.6 8.3 14.5 3.23 31.1 Spleen 33.8 27.4 35.4 8.88 74.8 Testes 18.2 15.4 4.46 3.45 4.93 Thyroid 2.22 0.87 1.65 1.12 1.87 Bladder 562 1260 1180 2190 1310 Total body 11.5 20.9 20 27.5 89.6 161 Figure 5.10 (A) Comparison of the radiation absorbed doses for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA in organs of interest in mice; (B) comparison of the estimated radiation absorbed doses for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA in organs of interest in adult human male. Table 5.9 Estimated radiation absorbed doses (mGy\/MBq) in adult human male for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA. Target Organ [177Lu]Lu-TacsBOMB5 [177Lu]Lu-LW01110 [177Lu]Lu-LW01142 [177Lu]Lu-RM2 [177Lu]Lu-AMBA Adrenals 1.17E-02 6.13E-02 2.82E-02 1.22E-02 2.55E-01 Brain 1.02E-04 4.10E-04 9.04E-04 1.26E-04 1.08E-03 Esophagus 4.89E-04 5.08E-04 7.08E-04 5.30E-04 1.40E-03 Eyes 1.87E-04 1.97E-04 3.53E-04 2.20E-04 3.63E-04 162 Gallbladder wall 7.53E-04 8.12E-04 1.16E-03 9.13E-04 1.97E-03 Left colon 9.17E-03 3.82E-02 2.02E-02 3.25E-02 1.02E-01 Small intestine 2.80E-03 1.56E-02 9.57E-03 1.95E-02 1.07E-01 Stomach wall 6.36E-04 4.30E-03 5.38E-03 5.78E-03 1.99E-02 Right colon 4.83E-03 1.94E-02 1.05E-02 1.66E-02 5.12E-02 Rectum 4.58E-03 1.83E-02 9.97E-03 1.60E-02 4.71E-02 Heart 8.30E-03 1.63E-03 3.12E-03 1.70E-03 2.59E-03 Kidneys 4.97E-02 7.57E-02 1.27E-01 6.08E-02 8.11E-02 Liver 1.42E-02 8.32E-03 1.63E-02 1.23E-02 6.37E-03 Lungs 1.29E-02 9.27E-03 1.03E-02 8.32E-03 4.09E-03 Pancreas 3.83E-03 1.53E-01 7.79E-02 1.16E-01 1.25E+00 Prostate 5.94E-04 1.13E-03 1.22E-03 1.51E-03 1.55E-03 Salivary glands 1.99E-04 2.08E-04 3.67E-04 2.31E-04 3.80E-04 Red Marrow 9.58E-04 4.78E-04 7.84E-04 5.57E-04 7.56E-04 Skeleton 1.12E-03 6.57E-04 1.02E-03 6.38E-04 1.43E-03 Spleen 1.40E-02 1.01E-02 1.42E-02 1.68E-03 1.31E-02 Testes 6.88E-03 6.09E-03 1.10E-03 7.11E-04 1.16E-03 Thymus 4.11E-04 3.42E-04 5.31E-04 3.67E-04 6.61E-04 Thyroid 2.91E-04 2.76E-04 4.44E-04 2.98E-04 4.72E-04 Urinary bladder 5.57E-02 1.25E-01 1.17E-01 1.82E-01 1.10E-01 Total body 1.54E-03 2.39E-03 2.67E-03 2.69E-03 5.87E-03 Effective dose (mSv\/MBq) 6.49E-03 1.35E-02 1.16E-02 1.43E-02 3.20E-02 5.4 Discussions GRPR is a very promising target for cancer diagnosis and therapy 93-96. Previously, our group reported three very potent GRPR-targeted tracers derived from the [Thz14]Bombesin(6-14) sequence, including [68Ga]Ga-TacsBOMB5, [68Ga]Ga-LW01110, and [68Ga]Ga-LW01142. These three tracers showed high uptake in PC-3 tumor xenografts and minimal pancreas uptake at 1 h post-injection 252. In this study, we labeled TacsBOMB5, LW01110, and LW01142 with lutetium-177 to evaluate their potential for radiotherapeutic applications. All Lu-LW01110, Lu-LW01142, Lu-RM2, and Lu-AMBA showed excellent binding affinities toward GRPR with Ki values from 0.33 to 3.07 nM (Figure 5.2), while the binding affinity of Lu-TacsBOMB5 (Ki = 12.6 nM) was inferior to those of the other four ligands. A significant decrease was found in the GRPR binding affinity for Lu-TacsBOMB5 and Lu-LW01110 in comparison with their Ga-complexed analogs: Ki = 6.09 \u00b1 0.95 nM for Ga-163 TacsBOMB5 vs 12.6 \u00b1 1.02 nM for Lu-TacsBOMB5, p = 0.0013; Ki = 1.39 \u00b1 0.03 nM for Ga-LW01110 vs 3.07 \u00b1 0.15 nM for Lu-LW01110, p < 0.001 252, 258. In contrast, the binding affinity of Lu-AMBA is about 3 times better compared with that of Ga-AMBA (Ki = 0.33 \u00b1 0.16 vs 0.99 \u00b1 0.08 nM, p = 0.0030) 255. For LW01142 and RM2, no significant difference was observed between their Lu- and Ga-complexed analogs 252, 258. The antagonist characteristics of Lu-TacsBOMB5 and the agonist characteristics of Lu-LW01110 and Lu-LW01142 were confirmed by intracellular calcium release assay (Figure 5.3). The results are consistent with the antagonist\/agonist characteristics of Ga-complexed TacsBOMB5, LW01110, and LW01142 reported previously by our group 252, 258. These data indicate that replacing Ga with Lu retains the good binding affinity of these ligands and the antagonist\/agonist characteristics. The hydrophilicity nature of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 was determined by logD7.4 measurements with the logD7.4 values ranging from \u22122.28 to \u22122.76. The highly hydrophilic nature of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 likely contributed to their rapidly renal excretion as shown in the SPECT images presented in Figure 5.4-5.6. Similar to the PET images of [68Ga]Ga-TacsBOMB5, [68Ga]Ga-LW01110, and [68Ga]Ga-LW01142 252, all [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 had minimal uptake in the pancreas and enabled clear visualization of the PC-3 tumor xenografts in SPECT images with good tumor-to-background contrast at 1 h post-injection. On the contrary, the pancreas was clearly visualized in the SPECT images by [177Lu]Lu-RM2 at 1 h post-injection and by [177Lu]Lu-AMBA from 1 h up to 72 h post-injection. A faster clearance of [177Lu]Lu-TacsBOMB5 was observed in PC-3 tumor xenografts in comparison with other four radioligands. This might be due to the lowest binding affinity of Lu-TacsBOMB5 among all five radioligands 164 (Figure 5.2A). Complete biodistribution studies for all five time points corroborated and quantified the finding on the SPECT images (Figure 5.9 and Table 5.3-5.7). Consistent with the SPECT images, the tumor uptake of [177Lu]Lu-TacsBOMB5 was lower than those of [177Lu]Lu-LW01110 and [177Lu]Lu-LW01142 at 1 h post-injection, and decreased to less than 2 %ID\/g at 24 h post-injection. This might be due to the lower binding affinity of Lu-TacsBOMB5 (Ki = 12.6 \u00b1 1.02 nM) compared with those of Lu-LW01110 and Lu-LW01142 (Ki = 3.07 \u00b1 0.15 and 2.37 \u00b1 0.28 nM, respectively). Surprisingly, with very potent Ki values to GRPR, both [177Lu]Lu-RM2 and [177Lu]Lu-AMBA showed lower accumulations in PC-3 tumor xenografts at 1 h post-injection in comparison with the other three tracers. Meanwhile, very high pancreas uptake was observed for both [177Lu]Lu-RM2 and [177Lu]Lu-AMBA. One possible explanation is that both [177Lu]Lu-RM2 and [177Lu]Lu-AMBA might have better binding affinity to mouse GRPR expressed by the mouse pancreas compared to the human GRPR expressed by PC-3 tumor xenografts, particularly for [177Lu]Lu-AMBA. The overall lower pancreas uptake for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 indicated that replacing 68Ga with 177Lu retains the minimal pancreas uptake characteristics of our [Thz14]Bombesin(6-14) derivatives. Due to the rapid clearance from normal organ\/tissues, the tumor-to-background uptake ratios of all five radioligands increased over time. The nearly invisible PC-3 tumors in the SPECT images acquired from the blocking studies of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 and the significant reduction of the tumor uptake demonstrate the GRPR-targeting specificity of all three radioligands (Figure 5.4A-C). The biodistribution data obtained from the blocking studies of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 also confirmed their specificity for GRPR (Table 5.3-5.5). The significant uptake reductions of [177Lu]Lu-TacsBOMB5, [177Lu]Lu-165 LW01110, and [177Lu]Lu-LW01142 in pancreas, intestine, and stomach were also in agreement with the physiological expression of GRPR in normal organs\/tissues, particularly for the pancreas which is the normal organ with the highest GRPR expression 90. Corroborated with observations from the SPECT\/CT images, the high calculated radiation absorbed doses received by the urinary bladders for [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, [177Lu]Lu-RM2, and [177Lu]Lu-AMBA indicate that the renal pathway is the main excretion pathway for all five radioligands. With a longer tumor retention, [177Lu]Lu-RM2 had the highest absorbed dose received by the PC-3 tumor xenograft than the other four radioligands. However, with the best binding affinity to GRPR (0.33 \u00b1 0.16 nM), [177Lu]Lu-AMBA had the lowest absorbed dose to the PC-3 tumor xenograft but the highest absorbed dose to the pancreas. One possible explanation is that the binding of [177Lu]Lu-AMBA to the GPRP expressed in the mouse pancreas competes with its binding to the GRPR expressed in the PC-3 tumor xenograft. Compared to [177Lu]Lu-LW01110, [177Lu]Lu-LW01142, and [177Lu]Lu-RM2, less radiation absorbed dose was delivered to PC-3 tumor xenografts for [177Lu]Lu-TacsBOMB5. This is resulted from faster tumor clearance of [177Lu]Lu-TacsBOMB5 likely due to its lowest binding affinity to GRPR. Compared with the clinically validated [177Lu]Lu-RM2, [177Lu]Lu-LW01110 had lower absorbed dose in PC-tumor xenograft but higher absorbed doses in mouse pancreas, likely resulting from the slower clearance of [177Lu]Lu-LW01110 from the pancreas in comparison with [177Lu]Lu-RM2. For [177Lu]Lu-LW01142, the calculated absorbed dose in PC-3 tumor xenograft is 73% of that of [177Lu]Lu-RM2, while the absorbed dose in the pancreas is 57% of that of [177Lu]Lu-RM2. Furthermore, [177Lu]Lu-LW01142 also showed lower absorbed doses in intestine and stomach wall in mice and lower estimated absorbed doses in colon, intestine, stomach wall, and rectum in an average adult male compared to those of [177Lu]Lu-RM2. On the 166 other hand, higher absorbed doses for [177Lu]Lu-LW01142 were observed in some other normal organs in mice such as skeleton and spleen. Our data suggested that both [177Lu]Lu-TacsBOMB5 and [177Lu]Lu-LW01142 can reduce the radiation exposure to some normal organ\/tissues, particularly to the pancreas. However, the shorter tumor retention and lower overall absorbed dose delivered to tumors might limit their therapeutic applications and further optimizations are needed for both [177Lu]Lu-TacsBOMB5 and [177Lu]Lu-LW01142. 5.5 Conclusions In this study, we synthesized one GRPR antagonist ([177Lu]Lu-TacsBOMB5) and two GRPR agonist ([177Lu]Lu-LW01110 and [177Lu]Lu-LW01142) radioligands and performed SPECT\/CT imaging and biodistribution studies at 1 h, 4 h, 24 h, 72 h, and 120 h post-injection, and further conducted the dosimetry calculations for all three radioligands and compared them with the clinically validated GRPR antagonist ([177Lu]Lu-RM2) and agonist ([177Lu]Lu-AMBA) radioligands. All three ligands retained the same antagonist\/agonist characteristics as their 68Ga-labeled analogs, demonstrating that the substitution of the radiometal in these radioligands does not change their antagonist\/agonist characteristics. Consistent with the low pancreas uptake of their 68Ga-labeled analogs, all [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142 showed significant lower pancreas uptakes at 1 h post-injection than [177Lu]Lu-RM2 and [177Lu]Lu-AMBA. The longer retention of [177Lu]Lu-LW01110 in the pancreas gave rise to a higher radiation absorbed dose delivered to the pancreas compared to [177Lu]Lu-RM2. In contrast, both [177Lu]Lu-TacsBOMB5 and [177Lu]Lu-LW01142 showed lower absorbed doses in the pancreas and some other key organs suggesting that both radioligands can reduce the radiation exposure to some normal organs\/tissues, particularly to the pancreas. However, compared to the clinically validated [177Lu]Lu-RM2, faster clearance of [177Lu]Lu-TacsBOMB5 and [177Lu]Lu-167 LW01142 from the PC-3 tumor xenografts gave rise to lower radiation absorbed doses delivered to tumors. Thus, further optimizations are needed to prolong the tumor retention of both [177Lu]Lu-TacsBOMB5 and [177Lu]Lu-LW01142 for therapeutic applications. 168 Chapter 6: Pro derivatives and hydroxamate derivatives 6.1 Synthesis and evaluation of 68Ga- and 177Lu-labeled [Pro14]Bombesin(8-14) derivatives for detection and radioligand therapy of gastrin-releasing peptide receptor-expressing cancer The following section is an adaption of the following published paper: Wang, L., Kuo, H.T., Chapple, D.E., Chen, C.C., Kurkowska, S., Colpo, N., Uribe, C., B\u00e9nard, F. and Lin, K.S., 2024. Synthesis and Evaluation of 68Ga-and 177Lu-Labeled [Pro14]Bombesin(8-14) Derivatives for Detection and Radioligand Therapy of Gastrin-Releasing Peptide Receptor-Expressing Cancer. Molecular Pharmaceutics, 2024. https:\/\/doi.org\/10.1021\/acs.molpharmaceut.4c00952. 6.1.1 Introduction The gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor and is expressed in the pancreas, stomach, gastrointestinal tract, and nervous system. GRPR is involved in the regulation of a series of physiological functions, such as the secretion of gastrointestinal hormones and the contraction of smooth muscles 90. Furthermore, GRPR is also overexpressed in many solid cancers, notably prostate, breast, colon, and lung cancers, and is involved in the development of malignant neoplasm 93-96. Due to its overexpression in various cancers, GRPR is an attractive cancer imaging marker and therapeutic target. Bombesin (BBN) and gastrin-releasing peptide (GRP) are two naturally occurring peptides which bind to GRPR with high binding affinity 90. Over the past two decades, many potent GRPR-targeted radioligands derived from BBN and GRP have been reported. Some of them including radiolabeled RM2, NeoB, SB3, and AMBA have already been evaluated in the clinic for cancer detection or radioligand therapy 46, 162, 185, 189-191. However, the high physiologic accumulation of reported GRPR-targeted radioligands in the pancreas not only impairs the detection of lesions in 169 or adjacent to the pancreas, but could also limit the maximum tolerated dose for targeted radioligand therapy 90. Inspired by a potent BBN antagonist, [D-Phe6,Leu13\u03c8Thz14]Bombesin(6-14), reported by Schally et al. 181, 182, our group developed a 68Ga-labeled GRPR antagonist, [68Ga]Ga-TacsBOMB5 (68Ga-DOTA-Pip-[D-Phe6,NMe-Gly11,Leu13\u03c8Thz14]Bombesin(6-14), Figure 6.1), with an NMe-Gly11 substitution in the reported [D-Phe6,Leu13\u03c8Thz14]Bombesin(6-14) sequence. [68Ga]Ga-TacsBOMB5 showed a high uptake in GRPR-expressing PC-3 prostate cancer tumor xenografts and most importantly, a minimal uptake in the pancreas 252. In addition, our group also developed two 68Ga-labeled GRPR agonists, [68Ga]Ga-LW01110 (68Ga-DOTA-Pip-[D-Phe6,Tle10,NMe-His12,Thz14]Bombesin(6-14), Figure 6.1) and [68Ga]Ga-LW01142 (68Ga-DOTA-Pip-[D-Phe6,His7,Tle10,NMe-His12,Thz14]Bombesin(6-14), Figure 6.1) 258. Unlike the pseudopeptide bond (Leu13\u03c8Thz14) in [68Ga]Ga-TacsBOMB5, [68Ga]Ga-LW01110 and [68Ga]Ga-LW01142 have an amide bond between Leu13 and Thz14. Furthermore, [68Ga]Ga-LW01110 and [68Ga]Ga-LW01142 also have Tle10 and NMe-His12 substitutions to improve their in vivo stability258. Both [68Ga]Ga-LW01110 and [68Ga]Ga-LW01142 showed high uptake in PC-3 tumor xenografts and minimal uptake in the pancreas258. However, the 4-thiazolidinecarboxylic acid (Thz14) residue in these three 68Ga-labeled GRPR-targeted tracers is prone to oxidation in the final product formulation. This could lead to a shorter shelf-life of the resulting radioligands and could be problematic especially when radiolabeled with a longer half-life radionuclide such as 177Lu (t1\/2 = 6.7 days) and 225Ac (t1\/2 = 9.9 days) for therapeutic applications. To avoid the problem caused by the oxidation of Thz in the product formulation, in this study, we synthesized ProBOMB5, LW02056, and LW02057 (Figure 6.1) by replacing the Thz14 residue in the previously reported TacsBOMB5, LW01110, and LW01142, respectively with Pro14. 170 We hypothesized that (1) replacing Thz14 in the sequences of previously reported TacsBOMB5, LW01110 and LW01142 with Pro14 would retain their antagonist\/agonist characteristics and good binding affinity towards GRPR; and (2) the resulting radioligands would have minimal uptake in the pancreas. Hence, we first synthesized ProBOMB5, LW02056, and LW02057, and determined the GRPR binding affinity and antagonist\/agonist characteristics of their nonradioactive Ga-complexed standards. The potential of [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 for detecting GRPR-expressing cancer was evaluated by PET imaging and ex vivo biodistribution studies in PC-3 tumor-bearing mice. Finally, the lead candidate, ProBOMB5, was labeled with 177Lu and evaluated by SPECT imaging, ex vivo biodistribution studies, and dosimetry analysis, and compared with the clinically validated [177Lu]Lu-RM2. 171 Figure 6.1 Chemical structures of previously reported TacsBOMB5, LW01110, and LW01142 and their Pro14 derivatives. 6 7 8 9 10 11 12 13 14172 6.1.2 Materials and Methods The materials and methods described in this section are provided in Chapter 2. Relevant sections are those describing reagent and instrumentation (Section 2.1), synthesis of DOTA-conjugated precursors (Section 2.3.1), synthesis of nonradioactive Ga-complexed standards (Section 2.4.1), synthesis of nonradioactive Lu-complexed standards (Section 2.4.2), cell culture (Section 2.5), fluorometric calcium release assay (Section 2.6), in vitro competition binding assay (Section 2.7), 68Ga radiolabeling (Section 2.8.1), 177Lu radiolabeling (Section 2.8.2), logD7.4 measurements (Section 2.9), animal studies (Section 2.10), PET imaging and biodistribution studies (Section 2.10.1), SPECT imaging and biodistribution studies (Section 2.10.2), in vivo stability studies (Section 2.10.3), dosimetry (Section 2.11), and statistical analysis (Sections 2.12). 6.1.3 Results 6.1.3.1 Peptide Synthesis and Radiolabeling ProBOMB5, LW02056, and LW02057 were obtained in 12-34% yield (Table 6.1) and their nonradioactive Ga-complexed standards were obtained in 49-77% yield (Table 6.2). The Lu-complexed standards of ProBOMB5 and RM2 were synthesized in 38% and 47% yields, respectively (Table 6.2). Table 6.1 HPLC purification conditions and MS characterizations of ProBOMB5, LW02056, and LW02057. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Purity ProBOMB5 25% CH3CN and 0.1% TFA in H2O 18.7 34 [M+2H]2+ 790.4 [M+2H]2+ 790.6 >99% LW02056 22% CH3CN and 0.1% TFA in H2O 19.4 30 [M+2H]2+ 804.4 [M+2H]2+ 804.8 >99% LW02057 21% CH3CN and 0.1% TFA in H2O 21.1 12 [M+2H]2+ 808.9 [M+2H]2+ 809.3 >99% 173 Table 6.2 HPLC purification conditions and MS characterizations of Ga-ProBOMB5, Ga-LW02056, Ga-LW02057, Lu-ProBOMB5 and Lu-RM2. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Purity Ga-ProBOMB5 28% CH3CN and 0.1% TFA in H2O 15.8 49 [M+2H]2+ 823.9 [M+2H]2+ 823.9 >99% Ga-LW02056 22% CH3CN and 0.1% TFA in H2O 24.3 57 [M+2H]2+ 837.9 [M+2H]2+ 837.7 >99% Ga-LW02057 21% CH3CN and 0.1% TFA in H2O 24.8 77 [M+2H]2+ 842.4 [M+2H]2+ 842.7 >98% Lu-ProBOMB5 28% CH3CN and 0.1% TFA in H2O 16.6 38 [M+2H]2+ 876.4 [M+2H]2+ 876.6 >99% Lu-RM2 25% CH3CN and 0.1% TFA in H2O 13.1 47 [M+2H]2+ 906.4 [M+2H]2+ 906.6 >99% The 68Ga-labeled ProBOMB5, LW02056, and LW02057 were obtained in 36-54% decay-corrected radiochemical yield with > 105 GBq\/\u00b5mol molar activity and > 92% radiochemical purity. The 177Lu-labeled Lu-ProBOMB5 and RM2 were obtained in 24-57% decay-corrected radiochemical yield with > 143 GBq\/\u00b5mol molar activity and > 95% radiochemical purity. The HPLC conditions and retention times are provided in Table 6.3. Table 6.3 HPLC conditions for the purification and quality control of 68Ga-labeled ProBOMB5, LW02056, and LW02057, and 177Lu-labeled ProBOMB5. FA: formic acid. Compound name HPLC conditions Retention time (min) [68Ga]Ga-ProBOMB5 Semi-Prep 20% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 12.6 QC 23% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 7.2 [68Ga]Ga-LW02056 Semi-Prep 24% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 40.4 QC 29% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 7.9 174 [68Ga]Ga-LW02057 Semi-Prep 22% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 18.1 QC 28% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 7.4 [177Lu]Lu-ProBOMB5 Semi-Prep 21% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 35.8 QC 26% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 8.8 [177Lu]Lu-RM2 Semi-Prep 19% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 36.3 QC 26% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 6.8 6.1.3.2 Hydrophilicity, Agonist\/Antagonist Characteristics, and Binding Affinity The logD7.4 values of [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, [68Ga]Ga-LW02057, and [177Lu]Lu-ProBOMB5 were \u20132.74 \u00b1 0.41, \u20132.93 \u00b1 0.08, \u20132.39 \u00b1 0.12, and \u20132.60 \u00b1 0.08, respectively (n = 3). The antagonist\/agonist characteristics of Ga-ProBOMB5, Lu-ProBOMB5, Ga-LW02056, and Ga-LW02057 were determined via intracellular calcium release assays using PC-3 cells (Figure 6.2). Ga-ProBOMB5, Lu-ProBOMB5, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) (antagonist control), and DPBS (blank control) induced minimal Ca2+ release, corresponding to 34.1 \u00b1 10.1, 20.4 \u00b1 5.09, 65.0 \u00b1 8.23, and 22.9 \u00b1 9.61 RFU, respectively. On the other hand, Ga-LW02056, Ga-LW02057, bombesin (agonist control) and ATP (positive control) caused significant Ca2+ release, corresponding to 492 \u00b1 90.6, 470 \u00b1 31.4, 465 \u00b1 65.1, and 278 \u00b1 30.1 RFU, respectively. 175 Figure 6.2 Intracellular calcium release in PC-3 cells induced by Ga-ProBOMB5, Lu-ProBOMB5, Ga-LW02056, Ga-LW02057, bombesin, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), ATP, and DPBS. The binding affinities of Ga-ProBOMB5, Ga-LW02056, Ga-LW02057, Lu-ProBOMB5, and Lu-RM2 to GRPR were measured by a cell-based binding assay using GRPR-expressing human PC-3 prostate cancer cells. The binding affinity of Ga-RM2 (1.51 \u00b1 0.24 nM, n = 3) to human GRPR was measured using the same assay and reported in our previous work 252. In addition, the binding affinity of Ga-ProBOMB5 to murine GRPR was measured using GRPR-expressing Swiss 3T3 cells. All of the tested GRPR-targeted ligands inhibited the binding of [125I-Tyr4]Bombesin to PC-3 cells\/Swiss 3T3 cells in a dose-dependent manner (Figure 6.3 and 6.4). The calculated Ki values relative to the human GRPR for Ga-ProBOMB5, Ga-LW02056, Ga-LW02057, Lu-ProBOMB5, and Lu-RM2 were 12.2 \u00b1 1.89, 14.7 \u00b1 4.81, 13.8 \u00b1 2.24, 13.6 \u00b1 0.25, and 1.19 \u00b1 0.16 nM, respectively (n = 3), and the calculated Ki value to murine GRPR for Ga-ProBOMB5 was 14.4 \u00b1 1.52 nM (n = 3). !\"#AB&DEFDGH,#AB&DEFDG!\"#H-.L.GM!\"#H-.L.GND&OP4RSTU9#AW4! ;H4,#<=[?\"# ;]4R#F4?\"$ AD&OP4RSTBM#CabFdA9ADe.L..a..M..IJhiBF\"M#FSTb176 Figure 6.3 Displacement curves of (A) Ga-ProBOMB5, Ga-LW02056, and Ga-LW02057, and (B) Lu-ProBOMB5 and Lu-RM2 generated using GRPR-expressing PC-3 cells and [125I-Tyr4]Bombesin as the radioligand. Figure 6.4 The displacement curve of Ga-ProBOMB5 generated using murine Swiss 3T3 cells and [125I-Tyr4]Bombesin as the radioligand. 6.1.3.3 PET Imaging and Ex vivo Biodistribution All [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 were mainly excreted via the renal pathway and enabled clear visualization of PC-3 tumor xenografts in PET images acquired at 1 h post-injection (Figure 6.12). All of these tracers showed low uptake in most of the normal organs\/tissues as only tumor xenografts, kidneys and urinary bladder could be clearly visualized in PET images. [68Ga]Ga-ProBOMB5 showed a slightly better tumor-to-background contrast compared to [68Ga]Ga-LW02056 and [68Ga]Ga-LW02057. Co-injection with 100 \u03bcg of !\"# !\"\" !\"A !B !& !' !( !) !GA#AGA(A&A\"AA\"#A+I-.LIML1MOP4ORIM.ST89.W;1LR 0.05). Figure 6.8 Comparison of [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 on their uptake in (A) PC-3 tumor xenografts and (B) the pancreas in mice at 1, 4, 24, 72, and 120 h post-injection. (C) Comparison of [177Lu]Lu-ProBOMB5 with\/without co-injection of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. *p < 0.05, **p < 0.01. 184 Table 6.5 Biodistribution (mean \u00b1 SD, n = 5) and tumor-to-organ uptake ratios of [177Lu]Lu-ProBOMB5 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h. The mice in the blocked group were co-injected with 100 \u00b5g of [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14) per mouse. * and ** indicate p < 0.05 and < 0.01, respectively, when comparing the 1 h and 1 h blocked data of [177Lu]Lu-ProBOMB5. Tissue (%ID\/g, n = 5) [177Lu]Lu-ProBOMB5 1 h 4 h 24 h 72 h 120 h 1 h blocked Blood 0.57 \u00b1 0.18 0.03 \u00b1 0.01 0.00 \u00b1 0.00 0.12 \u00b1 0.11 0.00 \u00b1 0.01 0.14 \u00b1 0.05 0.00 \u00b1 0.00 0.52 \u00b1 0.26 Fat 0.07 \u00b1 0.04 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.07 \u00b1 0.04 Testes 0.23 \u00b1 0.05 0.03 \u00b1 0.01 0.02 \u00b1 0.01 0.02 \u00b1 0.01 0.01 \u00b1 0.00 0.18 \u00b1 0.05 Small intestine 0.54 \u00b1 0.19 0.06 \u00b1 0.02 0.04 \u00b1 0.02 0.04 \u00b1 0.04 0.01 \u00b1 0.00 0.29 \u00b1 0.10* Large intestine 0.45 \u00b1 0.13 0.31 \u00b1 0.09 0.19 \u00b1 0.09 0.31 \u00b1 0.32 0.37 \u00b1 0.61 0.22 \u00b1 0.12* Spleen 0.23 \u00b1 0.04 0.08 \u00b1 0.02 0.07 \u00b1 0.03 0.06 \u00b1 0.02 0.04 \u00b1 0.02 0.18 \u00b1 0.08 Pancreas 1.38 \u00b1 0.64 0.11 \u00b1 0.03 0.06 \u00b1 0.01 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.54 \u00b1 0.23* Stomach 0.41 \u00b1 0.29 1.17 \u00b1 1.57 0.21 \u00b1 0.30 0.20 \u00b1 0.24 0.03 \u00b1 0.02 0.08 \u00b1 0.04* Liver 0.46 \u00b1 0.14 0.22 \u00b1 0.11 0.14 \u00b1 0.05 0.09 \u00b1 0.03 0.06 \u00b1 0.04 0.26 \u00b1 0.11* Adrenal glands 0.76 \u00b1 0.28 0.23 \u00b1 0.10 0.34 \u00b1 0.14 0.14 \u00b1 0.10 0.06 \u00b1 0.05 0.23 \u00b1 0.10 Kidneys 3.29 \u00b1 0.93 1.96 \u00b1 0.34 0.80 \u00b1 0.07 0.25 \u00b1 0.04 0.14 \u00b1 0.06 4.07 \u00b1 1.59 Heart 0.22 \u00b1 0.06 0.03 \u00b1 0.01 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.16 \u00b1 0.07 Lungs 0.61 \u00b1 0.17 0.09 \u00b1 0.04 0.05 \u00b1 0.04 0.06 \u00b1 0.09 0.02 \u00b1 0.02 0.38 \u00b1 0.14* PC-3 tumor 8.09 \u00b1 1.70 3.29 \u00b1 0.56 1.48 \u00b1 0.29 0.72 \u00b1 0.12 0.37 \u00b1 0.15 2.42 \u00b1 0.90** Bone 0.13 \u00b1 0.06 0.03 \u00b1 0.02 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.09 \u00b1 0.05 Muscle 0.31 \u00b1 0.17 0.06 \u00b1 0.11 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.21 \u00b1 0.13 Brain 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.02 \u00b1 0.01 Tumor to Normal Tissue Ratios Tumor\/muscle 31.8 \u00b1 16.5 163 \u00b1 91.7 192 \u00b1 41.2 261 \u00b1 76.5 270 \u00b1 186 15.9 \u00b1 9.32 Tumor\/blood 14.8 \u00b1 3.56 106 \u00b1 27.4 356 \u00b1 89.0 421 \u00b1 297 402 \u00b1 192 4.86 \u00b1 0.77** Tumor\/kidney 2.57 \u00b1 0.67 1.69 \u00b1 0.18 1.73 \u00b1 0.23 2.86 \u00b1 0.16 2.75 \u00b1 0.83 0.60 \u00b1 0.06* Tumor\/pancreas 6.51 \u00b1 2.19 30.6 \u00b1 5.97 22.3 \u00b1 1.74 41.1 \u00b1 10.5 37.6 \u00b1 10.6 4.64 \u00b1 0.68 Table 6.6 Biodistribution (mean \u00b1 SD, n = 5) and tumor-to-organ uptake ratios of [177Lu]Lu-RM2 in PC-3 tumor-bearing mice at 1, 4, 24, 72, and 120 h. Tissue (%ID\/g, n = 5) [177Lu]Lu-RM2 1 h 4 h 24 h 72 h 120 h Blood 0.55 \u00b1 0.09 0.07 \u00b1 0.02 0.01 \u00b1 0.00 0.12 \u00b1 0.11 0.00 \u00b1 0.00 0.14 \u00b1 0.05 0.00 \u00b1 0.00 Fat 0.07 \u00b1 0.06 0.01 \u00b1 0.01 0.01 \u00b1 0.01 0.02 \u00b1 0.02 0.00 \u00b1 0.00 Testes 0.14 \u00b1 0.02 0.04 \u00b1 0.01 0.02 \u00b1 0.00 0.01 \u00b1 0.00 0.01 \u00b1 0.00 Small intestine 5.91 \u00b1 1.32 1.13 \u00b1 0.44 0.09 \u00b1 0.02 0.08 \u00b1 0.08 0.01 \u00b1 0.00 Large intestine 3.14 \u00b1 0.83 1.25 \u00b1 0.45 0.24 \u00b1 0.06 1.50 \u00b1 1.60 0.03 \u00b1 0.02 Spleen 0.39 \u00b1 0.27 0.11 \u00b1 0.03 0.08 \u00b1 0.02 0.13 \u00b1 0.11 0.03 \u00b1 0.01 185 Pancreas 34.8 \u00b1 10.6 4.17 \u00b1 0.69 0.58 \u00b1 0.12 0.38 \u00b1 0.31 0.06 \u00b1 0.01 Stomach 3.08 \u00b1 1.83 0.96 \u00b1 0.43 0.14 \u00b1 0.06 0.19 \u00b1 0.29 0.01 \u00b1 0.00 Liver 0.62 \u00b1 0.54 0.19 \u00b1 0.03 0.10 \u00b1 0.01 0.06 \u00b1 0.01 0.03 \u00b1 0.01 Adrenal glands 2.32 \u00b1 0.85 1.04 \u00b1 0.41 0.86 \u00b1 0.37 0.75 \u00b1 0.31 0.29 \u00b1 0.10 Kidneys 3.44 \u00b1 0.64 1.83 \u00b1 0.34 0.96 \u00b1 0.16 0.41 \u00b1 0.07 0.14 \u00b1 0.03 Heart 0.18 \u00b1 0.06 0.03 \u00b1 0.01 0.02 \u00b1 0.01 0.01 \u00b1 0.01 0.01 \u00b1 0.00 Lungs 1.42 \u00b1 0.69 0.46 \u00b1 0.29 0.07 \u00b1 0.02 0.05 \u00b1 0.01 0.01 \u00b1 0.00 PC-3 tumor 7.73 \u00b1 0.96 8.49 \u00b1 1.26 6.53 \u00b1 0.84 4.40 \u00b1 1.05 2.10 \u00b1 0.15 Bone 0.14 \u00b1 0.04 0.02 \u00b1 0.01 0.03 \u00b1 0.03 0.03 \u00b1 0.01 0.02 \u00b1 0.01 Muscle 0.13 \u00b1 0.04 0.03 \u00b1 0.01 0.01 \u00b1 0.00 0.01 \u00b1 0.00 0.00 \u00b1 0.00 Brain 0.03 \u00b1 0.01 0.01 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 0.00 \u00b1 0.00 Tumor to Normal Tissue Ratios Tumor\/muscle 61.7 \u00b1 12.8 343 \u00b1 38.9 865 \u00b1 278 789 \u00b1 235 963 \u00b1 312 Tumor\/blood 14.3 \u00b1 2.15 131 \u00b1 36.0 1051 \u00b1 432 2049 \u00b1 1068 2532 \u00b1 1372 Tumor\/kidney 2.28 \u00b1 0.30 4.68 \u00b1 0.63 6.87 \u00b1 1.16 9.90 \u00b1 0.97 16.0 \u00b1 3.19 Tumor\/pancreas 0.23 \u00b1 0.06 1.79 \u00b1 0.50 11.4 \u00b1 1.38 15.2 \u00b1 5.89 38.1 \u00b1 5.62 6.1.3.5 Radiation Dosimetry The radiation absorbed doses for mice from [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 are provided in Figure 6.9 and Table 6.7. The absorbed dose in a 1-g PC-3 tumor xenograft (Unit Density Sphere Model) from [177Lu]Lu-ProBOMB5 was 57.3 mGy\/MBq, while that from [177Lu]Lu-RM2 was 429 mGy\/MBq which is 6 times higher than that from [177Lu]Lu-ProBOMB5. Among all of the target organs, urinary bladder received the highest absorbed dose for both [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 with 920 and 2190 mGy\/MBq, respectively. The absorbed dose of [177Lu]Lu-ProBOMB5 in the pancreas was 5.81 mGy\/MBq, which is less than 2% of that of [177Lu]Lu-RM2 (316 mGy\/MBq). A lower absorbed dose was observed for [177Lu]Lu-ProBOMB5 in the majority of the normal organs compared with [177Lu]Lu-RM2, including the critical organs such as kidneys (60.5 vs 171 mGy\/MBq). 186 Figure 6.9 Comparison of [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 on the absorbed dose in PC-3 tumor xenografts and major organs\/tissues in mice. Table 6.7 Absorbed dose per unit of injected activity (mGy\/MBq) in mice for [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 Target Organ [177Lu]Lu-ProBOMB5 [177Lu]Lu-RM2 PC-3 tumor xenograft 57.3 429 Brain 0.77 1.41 Large intestine 99.5 95.3 Small intestine 2.9 60.5 Stomach wall 29.9 56.4 Heart 3.09 3.18 Kidneys 60.5 171 Liver 14.8 35.4 Lungs 6.2 20.8 Pancreas 5.81 316 Skeleton 5.02 3.23 Spleen 16.6 8.88 Testes 2.02 3.45 Thyroid 0.685 1.12 Bladder 920 2190 Total body 9.96 27.5 # Dosimetry data of [177Lu]Lu-RM2 have been listed previously in Table 5.8. The estimated absorbed doses for both [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 to an average adult male are provided in Table 6.8. The highest estimated absorbed dose was observed in the urinary bladder for both [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2, with 9.14E-02 and 1.82E-01 mGy\/MBq, respectively. The estimated absorbed dose of [177Lu]Lu-RM2 in the pancreas !\"#ABCD()GHG,-KL,GM1B-KC12C-K1P(,44B-KC12C-K1PC)(,5SBT,4481,GC9-:K1;2L-<1GLDKM2!,K5G1,2P=141C)KP>411K?12C12?S;G)-:H4,::1G?):,4BH):;@ABBB@B@@B@@@B@@@@Ca2)Ga1:Bb)21Bc(d;eIHgh!\"##$B&$B'()*+,-+.!\"##$B&$B'L-M187 (1.16E-01 mGy\/MBq) was around 60 times higher than that of [177Lu]Lu-ProBOMB5 (1.98E-03 mGy\/MBq). For [177Lu]Lu-ProBOMB5, the estimated absorbed doses in most of the key organs were lower than those of [177Lu]Lu-RM2, including the small intestine, stomach wall, kidneys, liver, lungs, and red marrow. The effective whole-body dose per injected radioactivity of [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 to an adult human male were 8.70E-03 and 1.43E-02 mSv\/MBq, respectively. Table 6.8 Estimated absorbed doses (mGy\/MBq) in adult human males for [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2 Target Organ [177Lu]Lu-ProBOMB5 [177Lu]Lu-RM2 Adrenals 1.13E-02 1.22E-02 Brain 4.15E-05 1.26E-04 Esophagus 3.15E-04 5.30E-04 Eyes 1.95E-04 2.20E-04 Gallbladder wall 5.66E-04 9.13E-04 Left colon 4.12E-02 3.25E-02 Small intestine 1.13E-03 1.95E-02 Stomach wall 3.91E-03 5.78E-03 Right colon 2.10E-02 1.66E-02 Rectum 1.98E-02 1.60E-02 Heart 1.45E-03 1.70E-03 Kidneys 2.60E-02 6.08E-02 Liver 6.22E-03 1.23E-02 Lungs 2.58E-03 8.32E-03 Pancreas 1.98E-03 1.16E-01 Prostate 8.74E-04 1.51E-03 Salivary glands 2.02E-04 2.31E-04 Red Marrow 3.36E-04 5.57E-04 Skeleton 4.59E-04 6.38E-04 Spleen 7.07E-03 1.68E-03 Testes 4.64E-04 7.11E-04 Thymus 2.56E-04 3.67E-04 Thyroid 2.26E-04 2.98E-04 Urinary bladder 9.14E-02 1.82E-01 Total body 1.35E-03 2.69E-03 Effective dose (mSv\/MBq) 8.70E-03 1.43E-02 188 6.1.3.6 In vivo Stability Both [68Ga]Ga-ProBOMB5 and [177Lu]Lu-ProBOMB5 were shown to be sufficiently stable in vivo in NRG mice (n = 3) at 15 min post-injection with 71.8 \u00b1 7.19% and 67.1 \u00b1 6.04%, respectively, remaining intact in mouse plasma (Figure 6.10 and Figure 6.11). The intact fraction of [68Ga]Ga-ProBOMB5 in mouse plasma at 15 min post-injection was not significantly different from that of the previously reported [68Ga]Ga-RM2 (71.9 \u00b1 10.4%, p = 0.99) 252. In addition, 71.8 \u00b1 1.92% of intact [68Ga]Ga-ProBOMB5 and 72.9 \u00b1 1.10% of intact [177Lu]Lu-ProBOMB5 were detected in the mouse urine samples collected at 15 min post-injection (Figure 6.10 and Figure 6.11). Figure 6.10 Representative radio-HPLC chromatograms from analysis of intact fraction of [68Ga]Ga-ProBOMB5 in mouse plasma (top) and urine (bottom) samples collected at 15 min post-injection. The peaks pointed by an arrow are the intact tracer. ! \" #!!\"#!#\"$!%&'()*'&+,-.\/&0.12&3&24)*'R,! \" #!!\"!#!!#\"!%&'()*'&+,-.\/&0.12&3&24)*'R,189 Figure 6.11 Representative radio-HPLC chromatograms from analysis of intact fraction of [177Lu]Lu-ProBOMB5 in mouse plasma (top) and urine (bottom) samples collected at 15 min post-injection. The peaks pointed by an arrow are the intact radioligand. 6.1.4 Discussion Previously, our group reported three potent GRPR-targeted tracers derived from the [Thz14]Bombesin(6-14) sequence, including [68Ga]Ga-TacsBOMB5, [68Ga]Ga-LW01110, and [68Ga]Ga-LW01142 (Figure 6.1). All of them showed high uptake in PC-3 tumor xenografts and minimal pancreas uptake at 1 h post-injection 252. However, the Thz residue is prone to oxidation to sulfoxide in the final product formulation, potentially leading to a shorter shelf-life for radioligands derived from the [Thz14]Bombesin(6-14) sequence. To avoid this problem, in this study we synthesized ProBOMB5, LW02056, and LW02057 by replacing the Thz14 residue in TacsBOMB5, LW01110, and LW01142, respectively, with Pro14. The hydrophilic nature of ! \" #!$\"!%!!%\"!&!!'()*+,)(-.\/01(2034(R(46+,)T.! \" #!!#!!$!!%!!&!!\"!!'()*+,)(-.\/01(2034(R(46+,)T.190 [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, [68Ga]Ga-LW02057, and [177Lu]Lu-ProBOMB5 was confirmed via logD7.4 measurements as all of their logD7.4 values were < \u22122.3. With a lower level of induced intracellular Ca2+ release in comparison with that of the antagonist control ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14)), Ga-ProBOMB5 and Lu-ProBOMB5 were confirmed to be GRPR antagonists (Figure 6.2). Similarly, both Ga-LW02056 and Ga-LW02057 had a comparable level of induced intracellular Ca2+ release compared to that of the agonist control (bombesin), confirming their GRPR agonist characteristics (Figure 6.2). Our data demonstrate that replacing the Thz14 residue in the previously reported Ga-TacsBOMB5, Ga-LW01110, and Ga-LW01142 with Pro14 retains their antagonist\/agonist characteristics. Additionally, this is also in agreement with our previous findings that the GRPR-targeted ligands with a C-terminal reduced peptide bond (Leu13\u03c8AA14) are GRPR antagonists, while replacing the reduced peptide bond (Leu13\u03c8AA14) with an amide bond restores their agonist characteristics 217, 252, 255, 258. Similarly, a series of bombesin derivatives with Leu13\u03c8Nle14, Leu13\u03c8Leu14, Leu13\u03c8Phe14, and D-Pro13\u03c8Phe14 at their C-termini were reported to be GRPR antagonists 204, 253, 254. One possible explanation is that replacing the carbonyl moiety (-C(O)-) of the amide bond at the 13-14 position with a methylene (-CH2-) eliminates the intramolecular hydrogen bonding between the carbonyl moiety and the -NH moiety of the amide bond at the 9-10 position in a b-turn 204, 260. The binding affinities of Ga-ProBOMB5, Ga-LW02056, and Ga-LW02057 toward human GRPR were determined via an in vitro competition binding assay using PC-3 cells. Compared to Ga-TacsBOMB5 (Ki = 6.09 \u00b1 0.95 nM) 252, a significant decrease in binding affinity was observed for Ga-ProBOMB5 (Ki = 12.2 \u00b1 1.89 nM). Similarly, a significant decrease in GRPR binding affinity was also observed for the two Pro14-derived agonists, Ga-LW02056 and Ga-LW02057. The Ki value of Ga-LW02056 (14.7 \u00b1 4.81 nM) was ~10-fold that of Ga-LW01110 (1.39 \u00b1 0.03 191 nM), and the Ki value of Ga-LW02057 (13.8 \u00b1 2.24 nM) was ~4-fold that of Ga-LW01142 (3.19 \u00b1 0.78 nM) 258. This finding demonstrates that replacing the Thz14 residue with Pro14 affects the binding of our GRPR-targeted ligands to GRPR. We further performed a competition binding assay for Ga-ProBOMB5 by using a murine fibroblast cell line, Swiss 3T3 cells, to determine its binding affinity toward murine GRPR. The comparable Ki values (14.4 \u00b1 1.52 vs 12.2 \u00b1 1.89 nM, p = 0.18) indicate that Ga-ProBOMB5 binds to human and mouse GRPR with similar affinities. This suggests that the low uptake of [68Ga]Ga-ProBOMB5 in the mouse pancreas is not due to the weak binding of [68Ga]Ga-ProBOMB5 to the mouse GRPR. Whether the high mouse pancreas uptake of RM2-derived radioligands is due to the binding to off-targets in the pancreas remains to be investigated. No statistically significant difference was observed between the Ki values of Lu-ProBOMB5 and Ga-ProBOMB5 (13.6 \u00b1 0.25 nM vs 12.2 \u00b1 1.89 nM, p = 0.27), indicating that replacing the metal chelated by the N-terminal DOTA from Ga to Lu has no impact on their overall binding to GRPR. PET imaging and ex vivo biodistribution studies in PC-3 tumor-bearing mice were conducted to evaluate the potential of [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 for cancer imaging. Consistent with their hydrophilic nature, renal excretion was confirmed to be the major extraction pathway for all three tracers based on the PET imaging and biodistribution data (Figure 6.5, Figure 6.6 and Table 6.4). The binding affinity of the antagonist ligand (Ga-ProBOMB5) was comparable to that of the two agonist ligands Ga-LW02056 and Ga-LW02057 (Ki = 12.2 \u00b1 1.89 nM vs 14.7 \u00b1 4.81 and 13.8 \u00b1 2.24 nM, respectively). However, [68Ga]Ga-ProBOMB5 had a tumor uptake significantly higher than [68Ga]Ga-LW02056 and [68Ga]Ga-LW02057 (Figure 6.5 and Table 6.4). A similar observation was also reported by Cescato et al. that despite not having a better GRPR binding affinity, the GRPR antagonist ligand 192 ([99mTc]Demobesin 1) had a significantly higher uptake in PC-3 tumor xenografts compared to that of a GRPR agonist ligand ([99mTc]Demobesin 4) 203. This could be due to the fact that GRPR belongs to the G protein-coupled receptors which are characterized by the presence of high- and low-affinity states to agonists depending on the coupling of GDP or GTP on the G-protein 261. Agonists preferably bind to G protein-coupled receptors at the high affinity state, whereas antagonists bind to G protein-coupled receptors at both high and low affinity states with equal potency. Consequently, more high-affinity G protein-coupled receptor binding sites are available for antagonists. Such a phenomenon has also been observed for other G protein-coupled receptors such as the corticotrophin-releasing factor receptor and somatostatin sst3 receptor 262. Compared with those of [68Ga]Ga-LW02056 and [68Ga]Ga-LW02057, the higher tumor uptake and superior tumor-to-organ uptake ratios of [68Ga]Ga-ProBOMB5 suggest that [68Ga]Ga-ProBOMB5 is a promising PET tracer for detecting GRPR-expression malignancies. We further compared the biodistribution data of [68Ga]Ga-ProBOMB5 with those of our previously reported [68Ga]Ga-TacsBOMB5. As shown in Table 6.4, although the tumor uptake of [68Ga]Ga-ProBOMB5 was slightly lower than that of [68Ga]Ga-TacsBOMB5 (12.4 \u00b1 1.35 %ID\/g vs 15.7 \u00b1 2.17 %ID\/g, p < 0.05), the tumor-to-muscle and tumor-to-blood uptake ratios of [68Ga]Ga-ProBOMB5 were 1.5 and 1.4-fold of those of [68Ga]Ga-TacsBOMB5, respectively. In addition, the tumor-to-bone, tumor-to-muscle, the tumor-to-blood, and the tumor-to-kidney uptake ratios of [68Ga]Ga-ProBOMB5 were 1.2 to 1.8-fold higher than previously reported data of [68Ga]Ga-RM2 generated by our group using the same tumor model (Table 6.4) 252. Most importantly, the tumor-to-pancreas uptake ratio of [68Ga]Ga-ProBOMB5 was 38.4-fold that of [68Ga]Ga-RM2 (9.60 \u00b1 2.49 vs 0.25 \u00b1 0.04). Since [68Ga]Ga-RM2 is already a well validated clinical tracer for detecting 193 GRPR-expressing cancer, these data further support that [68Ga]Ga-ProBOMB5 is a very promising PET tracer for clinical translation. A relatively faster clearance of [177Lu]Lu-ProBOMB5 from the PC-3 tumor xenografts was observed in both SPECT\/CT images and biodistribution data (Figure 6.7 and Figure 6.8A, and Table 6.5 and Table 6.6). Despite similar tumor uptake at 1 h post-injection ([177Lu]Lu-ProBOMB5: 8.09 \u00b1 1.70 %ID\/g; [177Lu]Lu-RM2: 7.73 \u00b1 0.96 %ID\/g), tumor uptake of [177Lu]Lu-ProBOMB5 dropped quickly to 3.29 \u00b1 0.56 %ID\/g at 4 h post-injection and 1.48 \u00b1 0.29 %ID\/g at 24 h post-injection. On the other hand, the average tumor uptake of [177Lu]Lu-RM2 increased slightly to 8.49 \u00b1 1.26 %ID\/g at 4 h post-injection and then decreased relatively slower to 2.10 \u00b1 0.15 %ID\/g at 120 h post-injection (Figure 6.8A and Table 6.6). The relatively faster clearance of [177Lu]Lu-ProBOMB5 from PC-3 tumor xenografts is most likely due to the inferior binding affinity of Lu-ProBOMB5 (13.6 \u00b1 0.25 nM) compared to that of Lu-RM2 (1.19 \u00b1 0.16 nM) (Figure 6.3B). Similar to the biodistribution data of [68Ga]Ga-ProBOMB5 and [68Ga]Ga-RM2, [177Lu]Lu-ProBOMB5 also showed a much less accumulation in the pancreas at all time points when compared with those of [177Lu]Lu-RM2 (Figure 6.8B, Table 6.5 and Table 6.6). This suggests that replacing the Thz14 residue in our previously reported [D-Phe6,Leu13\u03c8Thz14]Bombesin(6-14) analogs with Pro14 retains the minimal pancreas uptake characteristics. Surprisingly, more than 80% of [177Lu]Lu-RM2 were washed out from the pancreas within 3 h (from 34.8 \u00b1 10.6 %ID\/g at 1 h post-injection to 4.17 \u00b1 0.69 %ID\/g at 4 h post-injection). Meanwhile, an increase in the tumor uptake of [177Lu]Lu-RM2 was observed at 4 h post-injection. It is possible that [177Lu]Lu-RM2 cleared from the pancreas could be taken up by the tumor xenografts. Therefore, besides a higher GRPR binding affinity for [177Lu]Lu-RM2, the extremely high pancreas uptake of 194 [177Lu]Lu-RM2 and its subsequent release from the pancreas and reuptake into tumors might contribute to its longer tumor retention as well. Blocking studies were conducted for both [68Ga]Ga-ProBOMB5 and [177Lu]Lu-ProBOMB5 at 1 h post-injection (Figure 6.6B and Figure 6.8C). The significantly reduced tumor uptake of both [68Ga]Ga-ProBOMB5 and [177Lu]Lu-ProBOMB5 confirms their specific binding toward GRPR. Additionally, due to the expression of GRPR in the pancreas, a significant reduction in the pancreas uptake of both [68Ga]Ga-ProBOMB5 and [177Lu]Lu-ProBOMB5 was also observed. Corroborated with the SPECT\/CT images, the highest absorbed dose was received by the urinary bladder in the mouse model for both [177Lu]Lu-ProBOMB5 and [177Lu]Lu-RM2, which can be explained by the renal excretion of both radioligands (Figure 6.7 and Figure 6.9). The absorbed dose of [177Lu]Lu-ProBOMB5 in a 1-g PC-3 tumor (Unit Density Sphere Model) was only about one seventh of that of [177Lu]Lu-RM2 (57.3 vs 429 mGy\/MBq). This is consistent with the fact that [177Lu]Lu-RM2 has a longer tumor retention than [177Lu]Lu-ProBOMB5 (Table 6.7), which is most likely due to the inferior binding affinity of Lu-ProBOMB5 compared with that of Lu-RM2 (Ki = 13.6 \u00b1 0.25 nM vs 1.19 \u00b1 0.16 nM). In contrast, with a significantly lower accumulation of [177Lu]Lu-ProBOMB5 in the pancreas, the absorbed dose for [177Lu]Lu-ProBOMB5 in the pancreas is around 50 times lower than that of [177Lu]Lu-RM2. Moreover, the overall lower absorbed doses of [177Lu]Lu-ProBOMB5 to normal organs\/tissues are consistent with the less off-target accumulation of [177Lu]Lu-ProBOMB5 in normal organs\/tissues. The estimated absorbed whole-body doses for an average adult male are listed in Table 6.8. For [177Lu]Lu-RM2, the highest estimated absorbed dose was observed in the pancreas among all the target organs except urinary bladder, and was around two folds of the absorbed dose in kidneys (1.16E-01 vs 6.08E-02 mGy\/MBq) and near ten folds of the absorbed dose in liver (1.16E-01 vs 195 1.23E-02 mGy\/MBq). These data are consistent with the dosimetry data reported by Hoffman group that the radiation absorbed dose of [177Lu]Lu-RM2 in the pancreas is the highest among all target organs (2.4E-01 mGy\/MBq) 263. For comparison, the calculated absorbed doses of [177Lu]Lu-RM2 in kidneys and liver reported by the same group were 6.3E-02 and 9.8E-03 mGy\/MBq, respectively.263 This finding is also in agreement with the fact that pancreas is the main dose-limiting organ of GRPR-targeted radiopharmaceuticals 46, 191. In contrast, our [177Lu]Lu-ProBOMB5 delivered less than 2% of the estimated absorbed dose of [177Lu]Lu-RM2 in the pancreas (1.98E-03 mGy\/MBq), which is also 100 times lower than the published absorbed dose for [177Lu]Lu-NeoBOMB1 (2.82E-01 mGy\/MBq) 215. Moreover, compared to [177Lu]Lu-RM2, [177Lu]Lu-ProBOMB5 has lower estimated absorbed doses in critical organs, such as kidneys (6.08E-02 vs 2.6E-02 mGy\/MBq) and red marrow (5.57E-04 vs 3.36E-04 mGy\/MBq). This indicates that [177Lu]Lu-ProBOMB5 has the potential to reduce radiation exposure to normal organs\/tissues, particularly to the main dose-limiting pancreas. However, the shorter tumor retention and lower overall absorbed dose delivered to the tumor for [177Lu]Lu-ProBOMB5 limit its therapeutic application. Further optimizations to prolong tumor retention and improve the tumor absorbed dose are needed for [177Lu]Lu-ProBOMB5. The in vivo stabilities of [68Ga]Ga-ProBOMB5 and [177Lu]Lu-ProBOMB5 were determined in NRG mice at 15 min post-injection (Figure 6.10 and Figure 6.11). Both [68Ga]Ga-ProBOMB5 and [177Lu]Lu-ProBOMB5 were relatively stable in vivo. Compared with the previously reported [68Ga]Ga-TacsBOMB5 252, no significant difference was found for the intact fractions of [68Ga]Ga-TacsBOMB5 and [68Ga]Ga-ProBOMB5 in mouse plasma at 15 min post-injection (67.1 \u00b1 4.76 vs 71.8 \u00b1 7.19%, respectively, p = 0.40). This indicates that replacing the Thz14 residue in [68Ga]Ga-TacsBOMB5 with Pro14 has no impact on the overall stability in mouse 196 plasma. Surprisingly, 71.8% of both [68Ga]Ga-ProBOMB5 and [177Lu]Lu-ProBOMB5 remained intact in mouse urine samples collected at 15 min post-injection. This is contradictory to our previous report that showed complete degradation of [68Ga]Ga-TacsBOMB5 and [68Ga]Ga-ProBOMB2 in mouse urine samples collected at 15 min post-injection 217, 252. This observation suggests that the combination of Leu13\u03c8Pro14 and NMe-Gly11 substitutions may change the conformation of the GRPR-targeted peptides and greatly inhibit their degradation by the peptidases expressed in kidneys. 6.1.5 Conclusions In this study, we synthesized one GRPR antagonist (Ga-ProBOMB5) and two GRPR agonists (Ga-LW02056 and Ga-LW02057) by substituting the Thz14 residue in our previously reported potent GRPR-targeted ligands with Pro14. All three ligands retained the same antagonist\/agonist characteristics, demonstrating that the substitution of Thz14 with Pro14 does not change their antagonist\/agonist characteristics. Consistent with the low pancreas uptake of their previously reported Thz14 analogs, all [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 also showed a much lower pancreas uptake than that of [68Ga]Ga-RM2. [68Ga]Ga-ProBOMB5 had higher tumor uptake and tumor-to-organ contrast ratios than [68Ga]Ga-LW02056 and [68Ga]Ga-LW02057, and even the clinically validated [68Ga]Ga-RM2. This demonstrates that [68Ga]Ga-ProBOMB5 is a promising tracer for clinical translation to detect GRPR-expressing cancer. We also successfully labeled ProBOMB5 with 177Lu and compared it with [177Lu]Lu-RM2. Despite comparable tumor uptake at the earlier time point, faster clearance was observed for [177Lu]Lu-ProBOMB5 in PC-3 tumor xenografts, leading to a lower radiation absorbed dose delivered to tumors. Further optimizations are needed for [177Lu]Lu-ProBOMB5, especially on the 197 improvement of binding affinity, to prolong tumor retention for therapeutic applications. A recent report by Obeid et al. demonstrated that the insertion of a positive linker Arg-Arg to the RM2 sequence between DOTA and Pip greatly improves GRPR binding affinity, and the resulting [111In]In-AU-RM26-M4 had an extremely potent binding affinity (KD at low pM) 264. Therefore, we are currently investigating the use of the Arg-Arg linker to improve the GRPR binding affinities of radiolabeled ProBOMB5 derivatives. 198 6.2 Synthesis and evaluation of first 68Ga-labeled C-terminal hydroxamate-derived GRPR-targeted tracers for cancer imaging with positron emission tomography The following section is an adaption of the following published paper: Wang, L., Kuo, H.T., Chen, C.C., Chapple, D., Colpo, N., Ng, P., Lau, W.S., Jozi, S., B\u00e9nard, F. and Lin, K.S. Synthesis and evaluation of the first 68Ga-Labeled C-terminal hydroxamate-derived gastrin-releasing peptide receptor-targeted tracers for cancer imaging with positron emission tomography. Molecules, 2024, 29, 3102. https:\/\/doi.org\/10.3390\/molecules29133102. The compounds disclosed in this report are covered by a recent US patent application (PCT\/CA2024\/051215). Kuo-Shyan Lin, Fran\u00e7ois B\u00e9nard, Lei Wang, and Chao-Cheng Chen are listed as inventors in this filed patent application. 6.2.1 Introduction Gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor. It is expressed in some normal tissues and organs such as the pancreas, gastrointestinal tract, and nervous system, and regulates a series of physiological functions including stimulation of the contraction of smooth muscle cells and secretion of gastrointestinal hormones 90. GRPR is also found overexpressed in several solid malignancies, such as prostate, breast, colon, and lung cancers, and is involved in the malignant neoplasm\u2019s development by coupling with phospholipase C and activating protein kinase C 93, 142-145, 147. Owing to its overexpression in malignant tissues, GRPR is a promising target for cancer imaging and therapy. In the past two decades, many radiolabeled GRPR-targeted ligands have been developed, and some of them have been translated into the clinic for cancer diagnosis and radioligand therapy 46, 162, 189-191. [68Ga]Ga-SB3 (SB3: DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt; Figure 6.12A; pABzA-DIG: p-aminomethylaniline-diglycolic acid), developed by Maina 199 et al., is one of the most popular radiolabeled GRPR antagonists, and has been validated in the clinic for detecting prostate and breast cancer lesions 185. Although [68Ga]Ga-SB3 enabled clear visualization of prostate and breast cancer lesions in PET images, a very high pancreas uptake was observed in patients 185. In addition to [68Ga]Ga-SB3, a high pancreas uptake is observed by other clinically validated GRPR-targeted radioligands including derivatives of AMBA, RM2 and NeoB 189-191. The high uptake in pancreas not only affects lesion detection in or adjacent to the pancreas, but also significantly limits the maximum tolerated dose for targeted radioligand therapy application 90, 185. Most of the reported GRPR-targeted agonist and antagonist sequences contain a C-terminal amide. However, it seems that replacing the C-terminal amide with a C-terminal hydroxamate is tolerable, as a C-terminal hydroxamate-derived GRPR-targeted antagonist (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), reported by Devin et al., was shown to bind GRPR with a high affinity (Ki = 5.8 nM) 265. To the best of our knowledge, no GRPR-targeted radioligands containing a C-terminal hydroxamate have ever been reported. Therefore, the goal of this study was to evaluate the potential of C-terminal hydroxamate-derived GRPR-targeted radioligands for cancer imaging, and document the extent of their uptake in the pancreas. 200 Figure 6.12 Chemical structures of (A) SB3, (B) LW02075, and (C) LW02050. The differences in the chemical structures between SB3 and the C-terminal hydroxamate-derived LW02075 and LW02050 are shown in blue. We used the chemical structure of [68Ga]Ga-SB3 (Figure 6.12A) as a template since it has been used to successfully detect prostate and breast cancer lesions in the clinic 185. Additionally, the only difference between the GRPR-targeted sequence of [68Ga]Ga-SB3 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt) and the sequence reported by Devin et al. (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH) is at the C-terminus. Therefore, we synthesized LW02075 (Figure 6.12B) by replacing the C-terminal N-ethylamide in SB3 with a C-terminal hydroxamate. Previously, our group reported two GRPR-targeted tracers, [68Ga]Ga-ProBOMB1 and [68Ga]Ga-ProBOMB2, based on the same GRPR-targeted sequence, [D-Phe6,Leu13\u03c8Pro14]Bombesin(6-14) 216, 217. We HNNHHNNHHNNHHNNHOOHNNOOOONHOOH2NOONHONNN NOHOOOHOHOOSB3NHONHHNNHHNNHHNNHHNNHNNHOHNNOOOONHOOH2NOOHNONNN NOHOOOHOHOOOHHNNHHNNHHNNHHNNHOOHNNOOOONHOOH2NOONHONNN NOHOOOHOHOONHOHONHLW02075LW02050A.B.C.6 7 8 9 10 11 12 13201 demonstrated that substitution of the pABzA-DIG linker in [68Ga]Ga-ProBOMB1 with a 4-amino-(1-carboxymethyl)piperidine (Pip) linker retained high tumor uptake but significantly reduced uptake in normal organs, particularly in the pancreas and intestines 216, 217. Therefore, for comparison, we also synthesized LW02050 (Figure 6.12C) by replacing the pABzA-DIG linker in LW02075 with a Pip linker to potentially facilitate excretion of the tracer via the renal pathway and reduce its uptake in normal organs\/tissues. Both LW02075 and LW02050 were labeled with 68Ga and evaluated by biodistribution and PET imaging studies, and compared with the clinically validated [68Ga]Ga-SB3. 6.2.2 Materials and Methods The materials and methods described in this section are provided in Chapter 2. Relevant sections are those describing reagent and instrumentation (Section 2.1), synthesis of DOTA-conjugated precursors (Section 2.3.1), synthesis of nonradioactive Ga-complexed standards (Section 2.4.1), cell culture (Section 2.5), fluorometric calcium release assay (Section 2.6), in vitro competition binding assay (Section 2.7), 68Ga radiolabeling (Section 2.8.1), logD7.4 measurements (Section 2.9), animal studies (Section 2.10), PET imaging and biodistribution studies (Section 2.10.1), in vivo stability studies (Section 2.10.3), and statistical analysis (Sections 2.12). 6.2.3 Results 6.2.3.1 Peptide Synthesis and Radiolabeling LW02075 and LW02050 were obtained in 3.4% and 9.5% yields, respectively (Table 6.9) and their corresponding nonradioactive Ga-complexed standards were obtained in 25% and 82% yields, respectively (Table 6.10). 68Ga radiolabeling was conducted by microwave heating and, after HPLC purification (Table 6.11), the 68Ga-labeled tracers were obtained in 36-59% decay-202 corrected radiochemical yield with > 114 GBq\/\u00b5mol molar activity and > 97% radiochemical purity. Table 6.9 HPLC purification conditions and MS characterizations of LW02075 and LW02050. Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Purity LW02075 22% CH3CN and 0.1% TFA in H2O 28.6 3.4 [M+2H]2+ 789.9 [M+2H]2+ 790.1 >99% LW02050 20% CH3CN and 0.1% TFA in H2O 15.8 9.5 [M+2H]2+ 749.9 [M+2H]2+ 750.0 >99% Table 6.10 HPLC purification conditions and MS characterizations of Ga-LW02075 and Ga-LW02050 Compound name HPLC conditions Retention time (min) Yield (%) Calculated mass (m\/z) Found (m\/z) Purity Ga-LW02075 25% CH3CN and 0.1% TFA in H2O 17.3 25 [M+2H]2+ 822.9 [M+2H]2+ 823.5 >99% Ga-LW02050 20% CH3CN and 0.1% TFA in H2O 22.0 82 [M+2H]2+ 782.9 [M+2H]2+ 783.2 >99% Table 6.11 HPLC conditions for the purification and quality control of [68Ga]Ga-LW02075, [68Ga]Ga-LW02050, and [68Ga]Ga-SB3. FA: formic acid. Compound name HPLC conditions Retention time (min) [68Ga]Ga-LW02075 Semi-Prep 20% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 17.0 QC 21% CH3CN and 0.1% FA in H2O; flow rate 2.0 mL\/min 7.7 [68Ga]Ga-LW02050 Semi-Prep 15% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 15.8 QC 18% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 5.5 [68Ga]Ga-SB3 Semi-Prep 29% CH3CN and 0.1% FA in H2O; flow rate 4.5 mL\/min 22.5 QC 32% CH3CN and 0.1% FA in H2O; flow rate 2 mL\/min 11.0 203 6.2.3.2 Binding Affinity The Ki values of Ga-LW02075, Ga-LW02050, and Ga-SB3 were measured using GRPR-expressing PC-3 prostate cancer cells via a cell-based competition binding assay. Ga-SB3, Ga-LW02075, and Ga-LW02050 inhibited the binding of [125I-Tyr4]Bombesin towards PC-3 cells in a dose-dependent manner (Figure 6.13). The calculated Ki values for Ga-LW02075, Ga-LW02050, and Ga-SB3 were 1.39 \u00b1 0.54, 8.53 \u00b1 1.52, and 1.20 \u00b1 0.31 nM, respectively (n = 3). Figure 6.13 Displacement curves of [125I-Tyr4]Bombesin by Ga-LW02075, Ga-LW02050, and Ga-SB3 generated using GRPR-expressing PC-3 cells. 6.2.3.3 Antagonist Characterization and Hydrophilicity Measurement The antagonist characteristics of both Ga-LW02075 and Ga-LW02050 were confirmed by intracellular calcium release assay (Figure 6.14). ATP (50 nM, positive control) and bombesin (50 nM, agonist control) induced Ca2+ mobilization with 271 \u00b1 24.4 and 371 \u00b1 65.0 relative fluorescence units (RFUs), respectively. For 50 nM of Ga-LW02075 and Ga-LW02050, only 31.4 \u00b1 16.5 and 25.8 \u00b1 7.95 RFUs, respectively, were observed. These values were significantly lower than the values obtained by ATP and bombesin, and were close to those obtained using the blank -12 -11 -10 -9 -8 -7 -6 -5020406080100Log concentration [M]% Specific binding of [125 I-Tyr4]BombesinGa-SB3Ga-LW02075Ga-LW02050204 control (Dulbecco\u2019s phosphate-buffered saline, DPBS, 25.7 \u00b1 9.24 RFUs) and the antagonist control ([D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), 50 nM, 47.2 \u00b1 6.61 RFUs). The logD7.4 values of [68Ga]Ga-LW02075, [68Ga]Ga-LW02050, and [68Ga]Ga-SB3 were obtained using the shake flask method, and were \u22122.09 \u00b1 0.05, \u22122.30 \u00b1 0.14, and \u22122.47 \u00b1 0.09, respectively (n = 3). Figure 6.14 Intracellular calcium mobilization in PC-3 cells induced by Ga-LW02075, Ga-LW02050, Bombesin, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), ATP, and DPBS. 6.2.3.4 PET Imaging The PC-3 tumor xenografts were clearly visualized in PET images acquired at 1 h post-injection using [68Ga]Ga-SB3 and [68Ga]Ga-LW02050 (Figure 6.15). All [68Ga]Ga-SB3, [68Ga]Ga-LW02075, and [68Ga]Ga-LW02050 were excreted primarily through the renal pathway, as a very high uptake in urinary bladder was observed. A high pancreas uptake was also observed by both [68Ga]Ga-SB3 and [68Ga]Ga-LW02075, while the pancreas was nearly invisible in the PET image of [68Ga]Ga-LW02050. Co-injection with 100 \u03bcg of nonradioactive Ga-LW02050 significantly reduced the uptake of [68Ga]Ga-LW02050 in the PC-3 tumor xenograft to close to the background level. Ga-LW02075Ga-LW02050Bombesin[D-Phe6 ,Leu-NHEt13 ,des-Met14 ]Bombesin(6-14) ATP DPBS0100200300400500RFU (Max-Min)205 Figure 6.15 Representative PET images of [68Ga]Ga-SB3, [68Ga]Ga-LW02075, and [68Ga]Ga-LW02050 acquired at 1 h post-injection in mice bearing PC-3 tumor xenografts. t: tumor; p: pancreas; bl: urinary bladder. 6.2.3.5 Ex vivo Biodistribution Biodistribution studies were conducted for 68Ga-labeled SB3, LW02075 and LW02050 in PC-3 tumor-bearing mice (n = 4). The results of the biodistribution studies are provided in Figure 6.16, Figure 6.17 and Table 6.12, and are consistent with the observations from the PET images. Tumor uptake of [68Ga]Ga-LW02050 was comparable to that of [68Ga]Ga-SB3 (5.38 \u00b1 1.00 vs. 6.98 \u00b1 1.36 %ID\/g, p = 0.107), while [68Ga]Ga-LW02075 showed a significantly lower uptake in PC-3 tumor xenografts (3.97 \u00b1 1.71 %ID\/g). Consistent with the PET images, [68Ga]Ga-SB3 showed the highest pancreas uptake (37.3 \u00b1 6.90 %ID\/g), which was around two-fold greater than the pancreas uptake of [68Ga]Ga-LW02075 (17.8 \u00b1 5.24 %ID\/g). In contrast, the pancreas uptake of [68Ga]Ga-LW02050 was only 0.53 \u00b1 0.11 %ID\/g. Compared to [68Ga]Ga-LW02050, which had a low uptake in the liver (0.44 \u00b1 0.05 %ID\/g), small intestine (0.43 \u00b1 0.04 %ID\/g), and large intestine (0.34 \u00b1 0.15 %ID\/g), both [68Ga]Ga-SB3 and [68Ga]Ga-LW02075 showed a considerably 206 higher uptake in the liver (2.10 \u00b1 0.45 and 4.14 \u00b1 1.50 %ID\/g, respectively), small intestine (6.03 \u00b1 1.07 and 6.72 \u00b1 1.94 %ID\/g, respectively), and large intestine (2.20 \u00b1 0.89 and 1.71 \u00b1 0.47 %ID\/g, respectively). The tumor-to-bone, tumor-to-muscle, tumor-to-blood, tumor-to-kidney, and tumor-to-pancreas uptake ratios of [68Ga]Ga-LW02050 were 43.6 \u00b1 22.6, 44.4 \u00b1 14.1, 9.68 \u00b1 2.84, 1.92 \u00b1 0.41, and 10.7 \u00b1 4.17, respectively, significantly higher than those of [68Ga]Ga-LW02075 (30.6 \u00b1 18.9, 24.8 \u00b1 9.99, 5.51 \u00b1 1.49, 1.28 \u00b1 0.33, and 0.22 \u00b1 0.05, respectively). Consistent with the PET images, the co-injection of nonradioactive Ga-LW02050 (100 \u03bcg) reduced the average uptake of [68Ga]Ga-LW02050 in PC-3 tumor xenografts by 92%, from 5.38 %ID\/g down to 0.42 %ID\/g at 1 h post-injection (Figure 6.17 and Table 6.12). Furthermore, the average uptake of [68Ga]Ga-LW02050 in the pancreas was also reduced by 81% (0.53 %ID\/g down to 0.10 %ID\/g) by the co-injection of nonradioactive Ga-LW02050. A blocking study was also performed for [68Ga]Ga-LW02075. Similarly, co-injecting 100 \u03bcg of nonradioactive Ga-LW02075 reduced the average uptake of [68Ga]Ga-LW02075 in the PC-3 tumor xenografts by 54% (3.97 %ID\/g down to 1.83 %ID\/g). In addition, a significant reduction in the average pancreas uptake of [68Ga]Ga-LW02075 was observed. The average pancreas uptake of [68Ga]Ga-LW02075 declined by 91% from 17.8 %ID\/g to 1.55 %ID\/g (Table 6.12). 207 Table 6.12 Biodistribution (mean \u00b1 SD, n = 4) and uptake ratios of 68Ga-labeled GRPR-targeted tracers in PC-3 tumor-bearing mice. The mice in the blocked group were co-injected with 100 \u00b5g of their nonradioactive Ga-complexed standard (Ga-LW02075 or Ga-LW02050). Statistical analyses were conducted to compare uptake values and uptake ratios between baseline and blocked groups. * p < 0.05, ** p < 0.01, *** p < 0.001. Tissue (%ID\/g) [68Ga]Ga-SB3 [68Ga]Ga-LW02075 [68Ga]Ga-LW02050 1 h 1 h 1 h blocked 1 h 1 h blocked Blood 0.50 \u00b1 0.09 0.76 \u00b1 0.41 1.15 \u00b1 0.26 0.57 \u00b1 0.11 0.34 \u00b1 0.06* Fat 0.05 \u00b1 0.01 0.08 \u00b1 0.05 0.12 \u00b1 0.02 0.05 \u00b1 0.01 0.04 \u00b1 0.01 Testes 0.13 \u00b1 0.01 0.17 \u00b1 0.10 0.30 \u00b1 0.06 0.17 \u00b1 0.05 0.13 \u00b1 0.05 Small intestine 6.03 \u00b1 1.07 6.72 \u00b1 1.94 5.62 \u00b1 1.86 0.43 \u00b1 0.04 0.26 \u00b1 0.09* Large intestine 2.20 \u00b1 0.89 1.71 \u00b1 0.47 0.40 \u00b1 0.17* 0.34 \u00b1 0.15 0.19 \u00b1 0.17 Spleen 0.33 \u00b1 0.17 0.26 \u00b1 0.13 0.35 \u00b1 0.12 0.25 \u00b1 0.04 0.12 \u00b1 0.02** Pancreas 37.3 \u00b1 6.90 17.8 \u00b1 5.24 1.55 \u00b1 0.83*** 0.53 \u00b1 0.11 0.10 \u00b1 0.03*** Stomach 1.42 \u00b1 0.57 1.23 \u00b1 0.22 0.73 \u00b1 0.29* 0.34 \u00b1 0.14 0.04 \u00b1 0.02** Liver 2.10 \u00b1 0.45 4.14 \u00b1 1.50 5.92 \u00b1 1.69 0.44 \u00b1 0.05 0.29 \u00b1 0.20 Adrenal glands 2.07 \u00b1 0.66 4.42 \u00b1 2.41 1.02 \u00b1 0.59* 0.43 \u00b1 0.15 0.05 \u00b1 0.05** Kidneys 2.26 \u00b1 0.26 3.11 \u00b1 1.18 4.98 \u00b1 1.59 2.88 \u00b1 0.68 1.95 \u00b1 0.44 Heart 0.16 \u00b1 0.02 0.24 \u00b1 0.13 0.34 \u00b1 0.04 0.19 \u00b1 0.03 0.10 \u00b1 0.02** Lungs 1.18 \u00b1 0.23 0.75 \u00b1 0.41 0.94 \u00b1 0.17 0.55 \u00b1 0.06 0.33 \u00b1 0.09** PC-3 tumor 6.98 \u00b1 1.36 3.97 \u00b1 1.71 1.83 \u00b1 0.22* 5.38 \u00b1 1.00 0.42 \u00b1 0.08*** Bone 0.12 \u00b1 0.01 0.20 \u00b1 0.20 0.21 \u00b1 0.07 0.14 \u00b1 0.06 0.03 \u00b1 0.03* Muscle 0.18 \u00b1 0.10 0.21 \u00b1 0.18 0.27 \u00b1 0.14 0.13 \u00b1 0.02 0.06 \u00b1 0.02** Brain 0.04 \u00b1 0.01 0.03 \u00b1 0.01 0.03 \u00b1 0.01 0.03 \u00b1 0.01 0.01 \u00b1 0.00** Tumor\/bone 61.0 \u00b1 15.5 30.6 \u00b1 18.9 9.30 \u00b1 2.39 43.6 \u00b1 22.6 27.1 \u00b1 18.1 Tumor\/muscle 48.2 \u00b1 24.9 24.8 \u00b1 9.99 7.85 \u00b1 3.13* 44.4 \u00b1 14.1 8.28 \u00b1 2.70** Tumor\/blood 14.4 \u00b1 3.88 5.51 \u00b1 1.49 1.65 \u00b1 0.40** 9.68 \u00b1 2.84 1.23 \u00b1 0.14** Tumor\/kidney 3.09 \u00b1 0.51 1.28 \u00b1 0.33 0.39 \u00b1 0.09** 1.92 \u00b1 0.41 0.22 \u00b1 0.05*** Tumor\/pancreas 0.19 \u00b1 0.03 0.22 \u00b1 0.05 1.46 \u00b1 0.76* 10.7 \u00b1 4.17 4.30 \u00b1 0.83* 208 Figure 6.16 Uptake of [68Ga]Ga-SB3, [68Ga]Ga-LW02075, and [68Ga]Ga-LW02050 in tumors and major organs\/tissues of PC-3 tumor-bearing mice at 1 h post-injection. Figure 6.17 Comparison of [68Ga]Ga-LW02050 with\/without co-injection of its nonradioactive standard (100 \u03bcg) on the uptake in PC-3 tumor xenografts and major organs\/tissues in mice at 1 h post-injection. * p < 0.05, ** p < 0.01, *** p < 0.001. BloodSmall intestineLarge intestineSpleenPancreasStomachLiverKidneyPC-3 tumorBoneMuscle051020304050Uptake (%ID\/g)[68Ga]Ga-SB3[68Ga]Ga-LW02075[68Ga]Ga-LW02050209 6.2.4 Discussion Inspired by the potent GRPR antagonist sequence, D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, reported by Devin et al., we synthesized LW02075 (Figure 6.12B) by replacing the C-terminal ethylamide in SB3 (Figure 6.12A) with a C-terminal hydroxamate. The design of LW02050 (Figure 6.12C), replacing the pABzA-DIG linker in LW02075 with a more hydrophilic Pip linker, was based on data from our previously reported GRPR-targeted tracers, [68Ga]Ga-ProBOMB1 and [68Ga]Ga-ProBOMB2 216, 217. Compared to [68Ga]Ga-ProBOMB1 containing a pABzA-DIG linker, [68Ga]Ga-ProBOMB2 containing a Pip linker showed a comparable high tumor uptake but a significantly lower uptake in normal organs\/tissues, particularly in the pancreas. Accordingly, in this study, we also used a Pip linker instead of the pABzA-DIG linker to synthesize LW02050 for comparison. The average Ki(GRPR) of Ga-LW02075 was comparable with that of Ga-SB3 (1.39 \u00b1 0.54 vs. 1.20 \u00b1 0.31 nM, Figure 6.13). This indicates that replacing the C-terminal ethylamide in Ga-SB3 with a hydroxamate does not affect their GRPR binding affinity. In contrast, the average Ki(GRPR) of Ga-LW02050 (8.53 \u00b1 1.52 nM) containing the Pip linker was significantly inferior to those of Ga-SB3 and Ga-LW02075. This finding is unexpected as the Pip linker is also very popular for the design of GRPR-targeted radioligands, including the clinically validated [68Ga\/177Lu]Ga\/Lu-RM2 46, 47, 190. In addition, in our previous studies, we showed that the binding affinity of Ga-ProBOMB1 containing a pABzA-DIG linker was comparable to that of Ga-ProBOMB2 containing a Pip linker (Ki(GRPR) = 3.97 \u00b1 0.76 vs. 4.58 \u00b1 0.67 nM) 216, 217. The antagonist characteristics of Ga-LW02075 and Ga-LW02050 were confirmed by calcium release assay because, compared with the antagonist control, [D-Phe6,Leu-NHEt13,des-Met14]Bombesin(6-14), there was a slightly lower calcium mobilization induced by Ga-LW02075 210 and Ga-LW02050 (Figure 6.14). The confirmed antagonist characteristics indicates that the addition of Ga-DOTA-pABzA-DIG (in LW02075) or Ga-DOTA-Pip (in LW02050) to the N-terminus of the reported GRPR antagonist sequence, D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, does not change its agonist\/antagonist characteristics. The hydrophilic nature of both [68Ga]Ga-LW02075 and [68Ga]Ga-LW02050 was verified by their low logD7.4 values of \u22122.09 \u00b1 0.05 and \u22122.30 \u00b1 0.14, respectively. Compared to [68Ga]Ga-SB3 (logD7.4 = \u22122.47 \u00b1 0.09), [68Ga]Ga-LW02075 is slightly more lipophilic (p = 0.00043). This indicates that replacing the C-terminal ethylamide of [68Ga]Ga-SB3 with a hydroxamate led to a less hydrophilic [68Ga]Ga-LW02075. In contrast, replacing both the C-terminal ethylamide and pABzA-DIG linker in [68Ga]Ga-SB3 with a hydroxamate and a Pip linker, respectively, led to [68Ga]Ga-LW02050 with a comparable hydrophilicity (logD7.4 = \u22122.47 \u00b1 0.09 vs. \u22122.30 \u00b1 0.14, p = 0.112). This indicates that replacing the pABzA-DIG linker with a cationic Pip linker can increase the radioligands\u2019 hydrophilicity. The observations from PET images were consistent with the obtained biodistribution data (Figure 6.15, Figure 6.16, and Table 6.12). A good in vivo GRPR-targeting capability of [68Ga]Ga-SB3 and [68Ga]Ga-LW02050 was confirmed as PC-3 tumor xenografts were clearly visualized in the PET images, while [68Ga]Ga-LW02075 showed a lower uptake in PC-3 tumor xenografts. Although the GRPR binding affinity of Ga-LW02075 was significantly better than that of Ga-LW02050 (Ki = 1.39 \u00b1 0.54 vs. 8.53 \u00b1 1.52 nM, p = 0.0016), [68Ga]Ga-LW02075 showed a lower tumor uptake (3.97 \u00b1 1.71 vs. 5.38 \u00b1 1.00 %ID\/g). One possible explanation for these observations is the better hydrophilicity of [68Ga]Ga-LW02050 resulting from the incorporation of the more hydrophilic cationic Pip linker. All [68Ga]Ga-SB3, [68Ga]Ga-LW02075, and [68Ga]Ga-LW02050 were excreted primarily through the renal pathway with extremely high radioactivity accumulated 211 in the urinary bladders as observed from the PET images. A moderate hepatobiliary excretion of [68Ga]Ga-SB3 and [68Ga]Ga-LW02075 was also observed, as both tracers showed higher uptake values in the liver, small intestine, and large intestine when compared with those of [68Ga]Ga-LW02050. This is likely due to the incorporation of the cationic Pip linker in [68Ga]Ga-LW02050, leading to a lower extent of hepatobiliary excretion. The pancreas was also clearly visualized in the PET images of [68Ga]Ga-SB3 and [68Ga]Ga-LW02075, but not in the PET image of [68Ga]Ga-LW02050. This was also confirmed by the obtained biodistribution data: [68Ga]Ga-SB3 showed the highest pancreas uptake (37.3 \u00b1 6.90 %ID\/g) followed by [68Ga]Ga-LW02075 (17.8 \u00b1 5.24 %ID\/g), while the uptake of [68Ga]Ga-LW02050 in the pancreas was only 0.53 \u00b1 0.11 %ID\/g. The minimal uptake of [68Ga]Ga-LW02050 in the liver, intestine, and pancreas might also contribute to the higher uptake in PC-3 tumor xenografts compared to that of [68Ga]Ga-LW02075. The good tumor uptake of [68Ga]Ga-LW02050 and its minimal uptake in normal organs\/tissues result in excellent tumor-to-background imaging contrast (Figure 6.15). Our data support [68Ga]Ga-LW02050 as a promising PET tracer for detecting GRPR-expressing lesions, even for those adjacent to or in the pancreas. In addition, our findings suggest that LW02050 is a promising pharmacophore for the design of GRPR-targeted radiopharmaceuticals, especially when labeled with \u03b1- or \u03b2-emitters for therapeutic application to minimize toxicity to the pancreas. Blocking studies were performed for both [68Ga]Ga-LW02075 and [68Ga]Ga-LW02050 by the co-injection with 100 \u03bcg of their respective nonradioactive standard (Figure 6.15, Figure 6.17, and Table 6.12). Consistent with the observation from the PET image, the tumor uptake of [68Ga]Ga-LW02050 reduced by 92%, confirming its specific uptake in PC-3 tumor xenografts. The significant reduction in [68Ga]Ga-LW02050 in the pancreas (by 81%) is in agreement with the fact that the pancreas is the highest GRPR-expressing normal organ 90, 93, 142. For [68Ga]Ga-212 LW02075, there was 54% reduction in PC-3 tumor uptake and 91% reduction in pancreas uptake by the co-injection of 100 \u03bcg of nonradioactive Ga-LW02075. This suggests that [68Ga]Ga-LW02075 might have a higher binding affinity or be more selective toward the mouse GRPR expressed by the mouse pancreas than the human GRPR expressed by the PC-3 tumor xenografts. 6.2.5 Conclusions In this study, we report the first C-terminal hydroxamate-derived GRPR-targeted radioligands based on the reported potent GRPR antagonist, D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH. We demonstrated that the addition of Ga-DOTA-pABzA-DIG (in Ga-LW02075) or Ga-DOTA-Pip (in Ga-LW02050) to the N-terminus of D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH retains its high GRPR binding affinity. Compared with the closely related and clinically validated [68Ga]Ga-SB3, [68Ga]Ga-LW02050 showed comparable tumor uptake but a much less hepatobiliary excretion and especially a dramatically lower uptake in the pancreas. Our data suggest that [68Ga]Ga-LW02050 is a promising PET tracer for detecting GRPR-expressing lesions, even for lesions adjacent to or in the pancreas. Due to the minimal pancreas uptake of [68Ga]Ga-LW02050, LW02050 is also promising for the radioligand therapy application when labeled with a \u03b1- or \u03b2-emitters to minimize toxicity to the pancreas. 213 Chapter 7: Conclusions and Future Directions 7.1 Concluding remarks The overexpression of GRPR in various malignancies makes it a very potent target for cancer diagnosis and therapy 93, 142-147. However, most currently reported GRPR-targeted radiopharmaceuticals show extremely high accumulations in the pancreas and low in vivo metabolic stabilities, which significantly limit their clinical applications 46, 47, 162, 189-191. The studies in this thesis were focused on developing novel GRPR-targeted radiopharmaceuticals with minimal pancreas uptake and good metabolic stability to improve detection sensitivity and treatment efficacy for GRPR-expressing tumors. Inspired by a series of GRPR antagonists published by Schally\u2019s group having the replacement of Met14 with Thz14 and the introduction of a reduced peptide bond (CH2-N) between residues 13-14 (Leu13\u03c8Thz14) in the bombesin(7-14) sequence 181, 182, we developed a series of GRPR antagonists by adding a Pip linker and DOTA chelator on the reduced-peptide-bond-containing sequences 252. By restoring the reduced peptide bond between residues 13-14 (Leu13\u03c8Thz14) with an amide bond, we further developed several GRPR agonists 255. Among these novel GRPR-targeted radiopharmaceuticals, the antagonist [68Ga]Ga-TacsBOMB2 and the agonist [68Ga]Ga-TacBOMB2 showed good binding affinities to GRPR (low nM scale) and good tumor uptake. Most importantly, a minimal pancreas uptake was observed for both [68Ga]Ga-TacsBOMB2 and [68Ga]Ga-TacBOMB2. We further replaced the Gly11 in [68Ga]Ga-TacsBOMB2 with NMe-Gly11, and the resulting [68Ga]Ga-TacsBOMB5 showed a better tumor uptake in PC-3 tumor xenograft (15.7 \u00b1 2.17 %ID\/g) and an even lower pancreas uptake (1.98 \u00b1 0.10 %ID\/g) at 1 h post-injection. Our findings indicated that improving the binding affinity of the ligands to GRPR increases the tumor uptake. [Leu13\u03c8Thz14]Bombesin(7-14) and [Thz14]Bombesin(7-14) are two 214 promising vectors for the design of GRPR-targeting radiopharmaceuticals with minimal pancreas uptake. To prove our second hypothesis that substitution of the natural amino acids at the cleavage sites of our GRPR-targeted ligands with unnatural amino acids can improve their metabolic stability, we subsequently replaced the natural amino acids at the potential cleavage sites with unnatural amino acids. We identified that Tle10 and NMe-His12 substitutions in our GRPR agonist ligand, TacBOMB2, either alone or in combination, led to derivatives with comparable\/enhanced GRPR binding affinities and improved in vivo metabolic stabilities 258. LW01110 and LW01142 with both Tle10 and NMe-His12 substitutions were confirmed to be promising candidates to be labeled with long lived isotopes for further evaluations as radiotherapeutic agents. We then introduced these unnatural amino acids, chosen based on our previous studies on stabilizing the GRPR agonist candidates, into the GRPR antagonist TacsBOMB2 266. However, a significant decrease in GRPR binding affinity was observed for the derivative with NMe-His12 substitution. Meanwhile, comparable metabolic stabilities were observed for all three [68Ga]Ga-TacsBOMB2 derivatives when compared with [68Ga]Ga-TacsBOMB2. This finding suggests that the amide bond between His12-Leu13 is the major cleavage site on the [68Ga]Ga-TacsBOMB2 pharmacophore. For the third step, we labeled the leading candidates selected from previous studies with 177Lu and evaluated their potential with SPECT\/CT, biodistribution studies, and dosimetry analysis. We chose TacsBOMB5 as our antagonist candidate, and LW01110 and LW01142 as our agonist candidates. Despite comparable or higher tumor uptake at the earlier time points, rapid tumor clearance was observed for all [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110, and [177Lu]Lu-LW01142, resulting in lower radiation absorbed doses in PC-3 tumor xenograft compared with 215 that of [177Lu]Lu-RM2. Thus, further optimizations are still needed for 177Lu-labeled [Thz14]Bombesin derivatives to prolong the tumor retention for therapeutic applications. To avoid the oxidation of Thz and to prolong the shelf-life, we replaced the Thz14 in TacsBOMB5, LW01110, and LW01142 with Pro14 to obtain ProBOMB5, LW02056, and LW02057, respectively. Consistent with the low pancreas uptake observed from their previously reported Thz14 analogs and [68Ga]Ga-ProBOMB2 217, all [68Ga]Ga-ProBOMB5, [68Ga]Ga-LW02056, and [68Ga]Ga-LW02057 showed very low pancreas uptake. Among all three tracers and the clinical validated GRPR antagonist [68Ga]Ga-RM2, [68Ga]Ga-ProBOMB5 had the highest tumor uptake and tumor-to-organ contrast ratios. Moreover, though the tumor uptake of [68Ga]Ga-ProBOMB5 in PC-3 tumor xenografts is slightly less than that of its Thz14 analog [68Ga]Ga-TacsBOMB5, [68Ga]Ga-ProBOMB5 showed better tumor-to-organ\/tissue ratios. However, after being labeled with 177Lu, the resulting [177Lu]Lu-ProBOMB5 showed a faster clearance from PC-3 tumor xenografts compared to [177Lu]Lu-TacsBOMB5 and [177Lu]Lu-RM2, which gave rise to a lower radiation absorbed dose delivered to tumors. Similar to [177Lu]Lu-TacsBOMB5, [177Lu]Lu-LW01110 and [177Lu]Lu-LW01142, further optimizations are also needed to prolong the tumor retention of [177Lu]Lu-ProBOMB5 for therapeutic applications. Other than introducing Thz14 and Pro14 with or without a reduced peptide bond between residues 13-14, we also attempted to replace the C-terminal amide with a hydroxamate. Motivated by a potent C-terminal hydroxamate-derived GRPR antagonist (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, Ki = 5.8 nM) reported by Devin et al. 265, we used [68Ga]Ga-SB3 as a template and synthesized its C-terminal hydroxamate-derived [68Ga]Ga-LW02075 and [68Ga]Ga-LW02050 with a C-terminal hydroxamate substitution and also a cationic Pip linker instead. Our data 216 suggested that by C-terminal hydroxamate substitution retains the good binding affinity and antagonist characteristic, and decreases the pancreas uptake significantly. To sum up, among all our novel GRPR-targeted tracers, [68Ga]Ga-ProBOMB5 showed good tumor uptake, minimal accumulation in the pancreas, and excellent tumor-to-background contrast ratios. Our results are encouraging to support clinical translation of [68Ga]Ga-ProBOMB5 as a diagnostic radiotracer for detecting GRPR-expressing lesions, particularly the lesions in or adjacent to the pancreas. 7.2 Future Directions 7.2.1 Clinical Translation of [68Ga]Ga-ProBOMB5 As our lead diagnostic candidate with remarkable tumor-to-background contrast ratios, [68Ga]Ga-ProBOMB5 warrants clinical translations for PET imaging practices. First, the ProBOMB5 precursor and its Ga-complexed nonradioactive standard will be synthesized in accordance with the Good Laboratory Practice (GLP) regulations 267, 268. Based on the FDA guideline \u201cMicrodose radiopharmaceutical diagnostic drugs: nonclinical study recommendations guidance for industry\u201d, pharmacology studies (i.e., receptor\/target\/off-target profiling, imaging\/radiation dosimetry studies) and extended single-dose toxicity in one species should be conducted before the phase I clinical trial 269. Dosimetry study is one of the important pharmacology studies. The [68Ga]Ga-ProBOMB5 internal dosimetry estimates will be obtained by conducting an organ distribution analysis in non-tumor bearing immunocompetent mice (both sexes) over additional time points. The biodistribution results will be used to extrapolate the human effective radiation doses (mSv) and estimated absorbed radiation doses using OLINDA software following the published procedures 243-247. Single-dose toxicity study in rodent species (both sexes) will be conducted by administering an excessive dose of the Ga-ProBOMB5 (100 times the 217 expected mass required for imaging). Body weight, clinical signs, hematology, and histology of key organs will be determined during a 14-day observation. An interim necropsy will be performed on day 2. Furthermore, the shelf-life of [68Ga]Ga-ProBOMB5 will also be evaluated by measuring the radiochemical purity of [68Ga]Ga-ProBOMB5 over time after the end of synthesis (EOS). 7.2.2 Design, synthesis and evaluation of GRPR-targeting ligands with high binding affinity to increase the tumor uptake and minimize the pancreas uptake Obeid et al. recently reported that the insertion of a positive linker Arg-Arg to a radiolabeled RM2 analog between the DOTAGA chelator and the Pip linker greatly improves its binding affinity to GRPR (KD = 6.0 pM) and results in a higher uptake in PC-3 tumor xenografts 264. The study published by Marsouvanidis et al. also demonstrated that a positive charge residues at the N-terminal of bombesin(6-14) analogs improves the binding affinity by evaluating the impact of the presence of basic Arg17 and Lys13 in the native human GRP and Arg3 in amphibian BBN sequences 270. Furthermore, our previous findings showed that the GRPR-targeted ligand with a cationic Pip linker (ProBOMB2) showed better binding affinity and higher tumor uptake than the GRPR-targeted ligand with a neutral pABzA-DIG linker (ProBOMB1) 215-217. Thus, we will introduce positively charged amino acid(s) at the N-terminal of our lead candidates to improve their binding affinity toward GRPR. Our hypothesis is that the introduction of one or more positively charged amino acids at the N-terminal of our top candidates can give rise to a better binding affinity to GRPR, leading to higher tumor uptake and longer tumor retention. Therefore, our group will investigate the use of an additional Arg, Arg-Arg, or Lys linker to improve GRPR binding affinities of our reported radiolabeled [Thz14]Bombesin and [Pro14]Bombesin derivatives. In vitro and in vivo evaluations will be conducted to determine the potential of these radiolabeled derivatives as effective agents for diagnosis and radioligand therapy. 218 7.2.3 Radiolabeling the most promising candidate with 177Lu or 225Ac, and evaluating their potential for radioligand therapy The most promising 68Ga-labeled GRPR-targeted tracer will be converted to radiotherapeutic agents by replacing the 68Ga label with 177Lu or 225Ac. Biodistribution studies of these 177Lu- or 225Ac-labeled ligands will be conducted at multiple time-points in PC-3 tumor-bearing mice. The radiation absorbed doses of the 177Lu- or 225Ac-labeled ligands received by the PC-3 tumors and normal organs\/tissues in mice will be calculated using the OLINDA software, and their estimated absorbed doses in adult human males will be extrapolated using the method published by Kirschner, et al 246. The treatment efficacies of the lead candidates will also be evaluated using PC-3 tumor-bearing mice. 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Journal of Medicinal Chemistry 2013, 56 (21), 8579-8587. 238 ","@language":"en"}],"Genre":[{"@value":"Thesis\/Dissertation","@language":"en"}],"GraduationDate":[{"@value":"2025-05","@language":"en"}],"IsShownAt":[{"@value":"10.14288\/1.0448224","@language":"en"}],"Language":[{"@value":"eng","@language":"en"}],"Program":[{"@value":"Interdisciplinary Oncology","@language":"en"}],"Provider":[{"@value":"Vancouver : University of British Columbia Library","@language":"en"}],"Publisher":[{"@value":"University of British Columbia","@language":"en"}],"Rights":[{"@value":"Attribution-NonCommercial-NoDerivatives 4.0 International","@language":"*"}],"RightsURI":[{"@value":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/","@language":"*"}],"ScholarlyLevel":[{"@value":"Graduate","@language":"en"}],"Supervisor":[{"@value":"Lin, Kuo-Shyan","@language":"en"}],"Title":[{"@value":"Development of novel stable GRPR-targeting radiopharmaceuticals with low pancreas uptake for cancer diagnosis and therapy","@language":"en"}],"Type":[{"@value":"Text","@language":"en"}],"URI":[{"@value":"http:\/\/hdl.handle.net\/2429\/90509","@language":"en"}],"SortDate":[{"@value":"2025-12-31 AD","@language":"en"}],"@id":"doi:10.14288\/1.0448224"}