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PET/CT imaging of the human bradykinin 1 receptor using radiolabeled peptides for cancer detection Amouroux, Guillaume Paul Victor 2016

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  PET/CT IMAGING OF THE HUMAN BRADYKININ 1 RECEPTOR USING RADIOLABELED PEPTIDES FOR CANCER DETECTION by  Guillaume Paul Victor Amouroux  B.Sc. of Cellular Biology and Physiology, Université Paul Sabatier Toulouse III, 2007 M.Sc. in Health Biology and Cancer Research, Université Paul Sabatier Toulouse III, 2009  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)  August 2016   © Guillaume Paul Victor Amouroux, 2016ii  Abstract  Many compounds mimicking endogenous molecules have been used as a starting point to develop targeted diagnostic and therapeutic radiotracers. In particular, radiolabeled peptidomimetics, in association with positron emission tomography combined with computed tomography (PET/CT), are powerful tools to detect cancer with high sensitivity. Peptide-based radiotracers have the advantage of combining favorable pharmacokinetics that allow the use of short-lived isotopes, with a flexible modular design that offers a high versatility for functionalization, making them optimal for developing targeted imaging probes. The bradykinin receptors, which are powerful mediators of inflammation, have been shown to be highly expressed in many common cancers, notably breast and prostate cancers. The purpose of this project was to evaluate the human Bradykinin Receptor 1 (hB1R) as a potential target for cancer imaging and radionuclide therapy. Analogs of [des-Arg10] Kallidin (KD) were synthesized and labeled with 68Ga or 18F. Following determination of their affinity for hB1R, selected tracers were evaluated in vitro and in vivo using hB1R expressing cells to select optimal radiotracers to imaging by positron emission tomography. The replacement of key amino acids at peptidase cleavage points by unnatural aminoacids improved the stability of the radiolabeled [des-Arg10]KD analogs in vitro and in vivo. Such peptides were used successfully for h1BR imaging by PET/CT in preclinical models. The use of hydrophilic and in particular cationic linker significantly improved tumour accumulation of various bradykinin analogues.  Tracers combining the most favorable features gave high tumour to normal tissue contrast, by combining specific and high tumour uptake with low background and rapid clearance. The accumulation of agonist and antagonist radiotracers in tumours was also compared. In summary, we developed several promising bradykinin receptor ligands, as radiolabeled probes for cancer imaging. iii  Preface  The work presented in the Chapters 3, 4, 5, 6 and 7 was conducted at the BC Cancer Agency Research Center. The chemistry, radiochemistry and corresponding data analysis was done by Drs. Jinhe Pan and Zhengxing Zhang. Silvia Jenni performed all the in vitro binding experiments. Joseph Lau performed the biological part of the in vitro plasma stability studies. Dr. Chengcheng Zhang performed the in vitro calcium release experiments and corresponding data analysis. Navjit Hundal-Jabal and Nadine Colpo operated the PET/CT scan for all the in vivo  imaging experiments.  A version of the material presented in Chapter 3 has been presented at the Society of Nuclear Medicine and Molecular Imaging Annual Meeting 2014; Saint-Louis MO, USA, and subsequently published as: Lin KS, Pan J, Amouroux G, Turashvili G, Mesak F, Hundal-Jabal N, Pourghiasian M, Lau J, Jenni S, Aparicio S and Bénard F (2015). “In Vivo Radioimaging of Bradykinin Receptor B1, a Widely Overexpressed Molecule in Human Cancer”, Cancer Research Jan 15; 75(2):387–93. Immunohistochemistry on tissue microarray was performed by Gulisa Turashvili in collaboration with CTAG at the British Columbia Cancer Agency. Dr Félix Mesak created the cell constructs and contributed to the in vitro and in vivo biological experiments. I created the protocol for plasma membrane purification used for hB1R membrane expression assessment in HEK293T::hB1R. I performed all the in vivo biodistribution and static imaging experiments and corresponding data analysis. Dr. Felix Mesak performed the in vivo dynamic imaging experiments. I contributed to the reviewing of the article written by Dr. François Bénard and Kuo-Shyan Lin. A version of the material presented in Chapter 4 has been presented at the European Association of Nuclear Medicine Annual Meeting 2014; Gothenburg, Sweden and subsequently iv  published as: Lin KS, Amouroux G, Pan J, Zhang Z, Jenni S, Lau J, Liu Z, Hundal-Jabal N, Colpo N and Bénard F (2015). “Comparative Studies of Three 68Ga-Labeled [Des-Arg10] Kallidin Derivatives for Imaging Bradykinin B1 Receptor Expression with PET”, Journal of Nuclear Medicine Jun 23; 56(4):622–627. Dr. Zhibo Liu performed the mass analysis. I performed all the in vitro internalization assays and corresponding data analysis. I performed the in vivo and ex vivo part of the metabolite studies. I performed all the in vivo biodistribution and imaging experiments and corresponding data analysis. I contributed in writing the manuscript with editorial help of Drs François Bénard and Kuo-Shyan Lin. A version of the material presented in Chapter 5 has been presented at the Society of Nuclear Medicine and Molecular Imaging 2014 Annual Meeting; Saint-Louis MO, USA, and subsequently published as: Amouroux G and Pan J, Jenni S, Zhang C, Zhang Z, Hundal-Jabal N, Colpo N, Liu Z, Bénard F and Lin KS (2015). “Imaging Bradykinin B1 Receptor with 68Ga-Labeled [des-Arg10]Kallidin Derivatives: Effect of the Linker on Biodistribution and Tumour Uptake”, Molecular Pharmaceutics Aug 4; 12, 2879-2888. Dr. Zhibo Liu performed the mass analysis. I performed all the in vitro internalization assays and corresponding data analysis. I performed all the in vivo and ex vivo part of the metabolite studies. I performed all the in vivo biodistribution and imaging experiments and corresponding data analysis. I contributed in writing the manuscript with editorial help of Drs François Bénard, Samuel Aparicio and Kuo-Shyan Lin. A version of the material presented in Chapter 6 has been presented at the Society of Nuclear Medicine and Molecular Imaging Annual Meeting 2014; Saint-Louis MO, USA and will be published as: Amouroux G, Zhang Z, Pan J, Jenni S, Zhang C, Hundal-Jabal N, Colpo N, Bénard F and Lin KS (2015). “Comparison of agonist/antagonist versions of a synthetic tracer for human bradykinin B1 receptor imaging using positron emission tomography” Journal to be decided. I performed all the in vitro internalization assays and corresponding data analysis. I v  performed all the in vivo and ex vivo part of the metabolite studies. I performed all the in vivo biodistribution and imaging experiments and corresponding data analysis. I wrote the manuscript with editorial help of Drs François Bénard and Kuo-Shyan Lin. A version of part of the material presented in Chapter 7 has been published as: Liu Z and Amouroux G, Zhang Z, Pan J, Hundal-Jabal N, Colpo N, Lau J, Perrin DM, Bénard F and Lin KS (2015). “F-trifluoroborate derivatives of [Des-Arg]Kallidin for imaging bradykinin B1 receptor expression with positron emission tomography” Journal of Nuclear Medicine Mar 5; 56:622-627.  Work is performed in collaboration of department of Chemistry at UBC. The chemistry and radiochemistry was conducted by Dr. Zhibo Liu at UBC and the BC Cancer Agency in collaboration with Drs Zhengxing Zhang and Jinhe Pan. A version of another part of the material presented in chapter 7 has been presented at the Society of Nuclear Medicine and Molecular Imaging Annual Meeting 2015; Baltimore MD, USA. As: Amouroux G, Pan J, Jenni S, Zhang Z, Zhang C, Hundal-Jabal N, Colpo N, Bénard F and Lin KS (2015). “Radiolabeled peptides targeting the human bradykinin B1 receptor: is a cationic linker always preferable?” Silvia Jenni performed all the in vitro binding experiments and corresponding data analysis. Joseph Lau performed the biological part of the in vitro plasma stability experiments. I performed all the in vivo biodistribution and imaging experiments and corresponding data analysis. I contributed in reviewing the manuscript written by Drs François Bénard and Kuo-Shyan Lin. All the animal studies performed in this thesis were under animal protocol A13-0032, “Radiolabeled peptides to improve cancer diagnosis by Positron emission tomography and Single-photon emission tomography” that was approved by the Institutional Animal Care Committee of the University of British Columbia and was performed in compliance with the Canadian Council on Animal Care Guidelines. vi  Table of contents  Abstract ........................................................................................................................................... ii Preface ............................................................................................................................................ iii Table of Contents .......................................................................................................................... vi List of Tables ................................................................................................................................. xv List of Figures ............................................................................................................................. xvii List of Abbreviations and Symbols ............................................................................................. xx Acknowledgements .................................................................................................................... xxvi Dedication ................................................................................................................................ xxviii Chapter 1: Introduction ................................................................................................................. 1 1.1 Molecular imaging in nuclear medicine ......................................................................... 1 1.2 Positron Emission Tomography (PET) .......................................................................... 2 1.3 Cancer specific PET radiotracers ................................................................................... 4 1.4 Kinin Kallikrein System (KKS) ..................................................................................... 5 1.4.1 Kininogens ...................................................................................................... 7 1.4.2 Kininogenases ................................................................................................. 7 1.4.3 Kinins .............................................................................................................. 8 1.4.4 Kininases ......................................................................................................... 8 1.5 KKS functions ................................................................................................................ 9 1.5.1 BKR ................................................................................................................. 9 1.5.2 KKS and cancer ............................................................................................. 11 1.5.3 hB1R expression in cancer ............................................................................ 11 1.6 BKR targeting .............................................................................................................. 16 vii  1.6.1 Modulation .................................................................................................... 16 1.6.2 Imaging .......................................................................................................... 17 1.7 hB1R targeting ............................................................................................................. 17 1.7.1 Peptide analog ............................................................................................... 17 1.7.2 Sequence ........................................................................................................ 18 1.7.3 Radiolabeling ................................................................................................ 18 1.7.4 Model ............................................................................................................ 20 1.8 Hypothesis and objectives ............................................................................................ 21 1.8.1 Specific aim 1 ................................................................................................ 21 1.8.2 Specific aim 2 ................................................................................................ 21 1.8.3 Specific aim 3 ................................................................................................ 22 Chapter 2: General Materials and Methods .............................................................................. 23 2.1 General ......................................................................................................................... 23 2.2 Peptide synthesis and chemistry ................................................................................... 24 2.2.1 Precursor synthesis ........................................................................................ 24 2.2.2 Synthesis of Nat-Ga labeled tracers .............................................................. 25 2.2.3 Synthesis of 68Ga labelled tracers .................................................................. 26 2.3 LogD7.4 determination .................................................................................................. 27 2.4 in vitro metabolic stability in mouse plasma ................................................................ 27 2.5 Cellular model for hB1R stable expression .................................................................. 28 2.6 Receptor binding assays ............................................................................................... 28 2.7 Fluorometric measurement of calcium release ............................................................. 29 2.8 Animal studies .............................................................................................................. 30 2.8.1 in vivo metabolic stability ............................................................................. 30 viii  2.8.2 Biodistribution and preclinical imaging ........................................................ 31 2.9 Statistical analysis ........................................................................................................ 33 Chapter 3: Importance the Metabolic Stability for in vivo Radioimaging of hB1R .............. 34 3.1 Background .................................................................................................................. 34 3.2 Materials and methods ................................................................................................. 35 3.2.1 Chemistry and radiochemistry ...................................................................... 35 3.2.1.1 Synthesis of DOTA-Ahx-[Leu9,desArg10] kallidin ........................ 35 3.2.1.2 Synthesis of DOTA-Ahx-[Hyp4,Cha6,Leu9,desArg10] kallidin ...... 35 3.2.1.3 Synthesis of DOTA-PEG2-[Hyp4,Cha6,Leu9,desArg10] kallidin ... 36 3.2.1.4 Synthesis of Ga-DOTA-Ahx-[Leu9, desArg10] kallidin ................. 36 3.2.1.5 Synthesis of Ga-DOTA-Ahx-[Hyp4,Cha6,Leu9,desArg10] kallidin  .................................................................................................................... 37 3.2.1.6 Synthesis of Ga-DOTA-PEG2-[Hyp4,Cha6,Leu9,desArg10] kallidin .   .................................................................................................................... 37 3.2.1.7 Synthesis of 68Ga-DOTA-Ahx-[Leu9,desArg10] kallidin ............... 37 3.2.1.8Synthesis of 68Ga-DOTA-Ahx-[Hyp4,Cha6,Leu9,desArg10] kallidin ........................................................................................................ 38 3.2.1.9 Synthesis of 68Ga-DOTA-PEG2-[Hyp4,Cha6,Leu9,desArg10] kallidin ........................................................................................................ 38 3.2.2 Stability in mouse plasma ............................................................................. 38 3.2.3 LogD7.4 measurements .................................................................................. 39 3.2.4 hB1R expression in established cell lines ..................................................... 39 3.2.5 Creation of hB1R expression model cells ..................................................... 39 3.2.6 Preparation of cell membranes ...................................................................... 39 ix  3.2.7 Receptor-binding assays ................................................................................ 40 3.2.8 Biodistributions and preclinical imaging ...................................................... 40 3.2.9 Peptidase inhibition with phosphoramidon or enalaprilat ............................. 40 3.2.10 Statistical analysis ....................................................................................... 41 3.3 Results .......................................................................................................................... 41 3.3.1 Radiochemistry and plasma stability ............................................................. 41 3.3.2 hB1R expression model and affinity ............................................................. 42 3.3.3 Cancer cell lines screening ............................................................................ 43 3.3.4 Biodistribution and imaging .......................................................................... 45 3.4 Discussion .................................................................................................................... 47 3.5 Conclusion .................................................................................................................... 50 Chapter 4: Effect of Linkers on PET/CT Imaging of hB1R With 68Ga labeled [des-Arg10] Kallidin Derivatives Tumour Uptake and Biodistribution ...................................................... 51 4.1 Background .................................................................................................................. 51 4.2 Materials and methods ................................................................................................. 52 4.2.1 Chemistry and radiochemistry ...................................................................... 52 4.2.1.1 Synthesis of DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10] kallidin  .................................................................................................................... 52 4.2.1.2 Synthesis of DOTA-Pip-[Hyp4,Cha6,Leu9,des-Arg10] kallidin ...... 52 4.2.1.3 Synthesis of Ga-DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]- -kallidin (P04115) ...................................................................................... 53 4.2.1.4 Synthesis of Ga-DOTA-Pip-[Hyp4,Cha6,Leu9,des-Arg10]-  -kallidin (P04168) ...................................................................................... 53 4.2.1.5 Synthesis of 68Ga-DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]- x  -kallidin (68GaP04115) ............................................................................... 53 4.2.1.6 Synthesis of 68Ga-DOTA-Pip-[Hyp4,Cha6,Leu9,des-Arg10]- -kallidin (68Ga-P04168) .............................................................................. 54 4.2.2 LogD7.4 measurements .................................................................................. 54 4.2.3 Stability in mouse plasma in vitro and in vivo .............................................. 54 4.2.4 in vitro competition assays ............................................................................ 54 4.2.5 Fluorometric measurement of calcium release .............................................. 55 4.2.6 Biodistribution and PET/CT imaging ........................................................... 55 4.2.7 Statistical analysis ......................................................................................... 55 4.3 Results .......................................................................................................................... 55 4.3.1 Chemistry and radiochemistry ...................................................................... 55 4.3.2 in vitro/vivo plasma stability ......................................................................... 56 4.3.3 Binding affinity and hB1R signaling modulation ......................................... 56 4.3.4 Biodistribution and PET/CT imaging ........................................................... 59 4.4 Discussion .................................................................................................................... 61 4.5 Conclusion .................................................................................................................... 63 Chapter 5: Impact of the Aminoacid Sequence: Comparison of Three 68Ga-labeled [des-Arg10] Kallidin Derivatives .......................................................................................................... 64 5.1 Background .................................................................................................................. 64 5.2 Materials and methods ................................................................................................. 65 5.2.1 Chemistry and radiochemistry ...................................................................... 65 5.2.1.1 Synthesis of DOTA-PEG2-Lys-[Hyp4,Igl6,D-Igl8,Oic9,desArg10]- -kallidin ...................................................................................................... 65 5.2.1.2 Synthesis of DOTA-PEG2-Lys-[Hyp4,Cpg6,D-Tic8,Cpg9, xi  desArg10] kallidin ....................................................................................... 65 5.2.1.3 Synthesis of Ga-DOTA-PEG2-Lys-[Hyp4,Igl6,D-Igl8,Oic9,des- -Arg10] kallidin ........................................................................................... 66 5.2.1.4 Synthesis of Ga-DOTA-PEG2-Lys-[Hyp4,Cpg6,D-Tic8,Cpg9,des- -Arg10] kallidin ........................................................................................... 66 5.2.1.5 Synthesis of 68Ga-DOTA-PEG2 Lys-[Hyp4,Igl6,D-Igl8,Oic9,des- -Arg10] kallidin ........................................................................................... 66 5.2.1.6 Synthesis of 68Ga-DOTA-PEG2Lys-[Hyp4,Cpg6,D-Tic8,Cpg9, des-Arg10] kallidin ...................................................................................... 67 5.2.2 LogD7.4 measurements .................................................................................. 67 5.2.3 Stability in mouse plasma in vitro ................................................................. 67 5.2.4 in vitro competition assays ............................................................................ 67 5.2.5 Biodistribution and PET/CT imaging ........................................................... 68 5.2.6 Statistical analysis ......................................................................................... 68 5.3 Results .......................................................................................................................... 68 5.3.1 Chemistry and plasma stability ...................................................................... 68 5.3.2 hB1R affinity in vitro .................................................................................... 69 5.3.3 Biodistribution and imaging .......................................................................... 71 5.4 Discussion .................................................................................................................... 74 5.5 Conclusion .................................................................................................................... 77 Chapter 6: Importance of Ligand Activity: Comparison of Synthetic Agonist and Antagonist Tracers for Human Bradykinin B1 Receptor PET Imaging ................................ 78 6.1 Background .................................................................................................................. 79 6.2 Materials and methods ................................................................................................. 79 xii  6.2.1 Chemistry and radiochemistry ....................................................................... 79 6.2.1.1 Synthesis of DOTA-Ahx-[Hyp4,Cha6,D-Phe9,des-Arg10] kallidin (Z01115) ..................................................................................................... 79 6.2.1.2 Synthesis of NatGa-DOTA-Ahx-[Hyp4,Cha6,D-Phe9,des-Arg10]- -kallidin (NatGa-Z01115) ............................................................................. 80 6.2.1.3 Synthesis of 68Ga-DOTA-Ahx-[Hyp4,Cha6,D-Phe9,des-Arg10]- -kallidin (68Ga-Z01115) .............................................................................. 80 6.2.2 in vitro internalization assay .......................................................................... 80 6.2.3 LogD7.4 measurements ................................................................................... 81 6.2.4 Stability in mouse plasma in vitro and in vivo ............................................... 81 6.2.5 in vitro competition assays ............................................................................ 81 6.2.6 Fluorometric measurement of calcium release .............................................. 81 6.2.7 Biodistribution and PET/CT imaging ............................................................ 82 6.2.8 Statistical analysis .......................................................................................... 82 6.3 Results .......................................................................................................................... 82 6.3.1 Chemistry, radiochemistry and metabolic stability ....................................... 82 6.3.2 hB1R binding affinity and agonist vs antagonist cell signaling .................... 83 6.3.3 in vitro internalization assay .......................................................................... 85 6.3.4 Biodistribution and imaging .......................................................................... 86 6.4 Discussion .................................................................................................................... 89 6.5 Conclusion .................................................................................................................... 92 Chapter 7: Impact of the Labeling Approach on hB1R PET Tracers: NODA, Al-fluoride and F-trifluoroborate Derivatives of [Des-Arg10] Kallidin for Imaging Bradykinin B1 Receptor Expression with PET ................................................................................................... 94 xiii  7.1 Background .................................................................................................................. 94 7.2 Materials and methods ................................................................................................. 95 7.2.1 Peptide synthesis ............................................................................................ 95 7.2.1.1 Synthesis of DOTA-Pip-B9958 .................................................... 95 7.2.1.2 Synthesis of NODA-Mpaa-Pip-B9958 ......................................... 96 7.2.1.3 Synthesis of azidoacetyl-Mta-Pip-B9958 ..................................... 96 7.2.1.4 Synthesis of AlOH-NODA-Mpaa-Pip-B9958 .............................. 97 7.2.2 Cold labelling................................................................................................. 97 7.2.2.1 Synthesis of Ga-DOTA-Pip-B9958 (Z02176) .............................. 97 7.2.2.2 Synthesis of Ga-NODA-Mpaa-Pip-B9958 (Z02137) ................... 98 7.2.2.3 Synthesis of AmBF3-Mta-Pip-B9958 (L08060) ........................... 98 7.2.2.4 Synthesis of AlF-NODA-Mpaa-Pip-B9958 (Z04139) ................. 99 7.2.3 Radiochemistry .............................................................................................. 99 7.2.3.1 Synthesis of 68Ga-DOTA-Pip-B9958 (68Ga-Z02176) .................. 99 7.2.3.2 Synthesis of 68Ga-NODA-Mpaa-Pip-B9958 (68Ga-Z02137) ..... 100 7.2.3.3 Synthesis of 18F-AmBF3-Mta-Pip-B9958 (18F-L08060) ............ 100 7.2.3.4 Synthesis of 18F-AlF-NODA-Mpaa-Pip-B9958 (18F-Z04139) ... 101 7.2.4. LogD7.4 measurements ................................................................................ 101 7.2.5 Stability in mouse plasma in vitro and in vivo ............................................. 102 7.2.6 in vitro competition assays .......................................................................... 102 7.2.7 Biodistribution and PET/CT imaging .......................................................... 102 7.2.8 Statistical analysis ........................................................................................ 102 7.3 Results ........................................................................................................................ 103 7.3.1 Chemistry and radiochemistry ..................................................................... 103 xiv  7.3.2 in vitro hB1R binding affinity and plasma stability .................................... 104 7.3.3 in vivo stability ............................................................................................. 104 7.3.4 in vivo PET/CT imaging and biodistribution ............................................... 105 7.4 Discussion .................................................................................................................. 108 7.5 Conclusion .................................................................................................................. 113 Chapter 8: Summary and Conclusion ...................................................................................... 114 Summary .......................................................................................................................... 121 Bibliography ............................................................................................................................... 123                 xv  List of Tables  Table 1.1: Kinins and radiolabeled synthetic analogs design. ...................................................... 20 Table 3.1: Radiolabeling, LogD7.4 and pharmacokinetics data of 68Ga-labeled [des-Arg10]KD derivativesa ..................................................................................................................................... 42 Table 3.2: Biodistribution of 68Ga-P03083 in mice 60 min p.i. with cold labeled standard (blocking), with peptidase inhibitors and without compared to 68Ga-SH01078 and 68Ga-P03034  ........................................................................................................................................................ 45 Table 4.1: Radiolabeling, LogD7.4 and in vitro plasma stability data of 68Ga-labeled [des-Arg10]KD derivativesa .................................................................................................................... 56 Table 4.2: Amino acid sequences and hB1R binding affinities of bradykinin and related peptides  ........................................................................................................................................................ 57 Table 4.3: Biodistribution and uptake ratios of 68Ga-labeled [des-Arg10]KD derivatives in tumour-bearing micea ..................................................................................................................... 59 Table 5.1: Radiolabeling, LogD7.4 and pharmacokinetics data of 68Ga-labeled [des-Arg10]KD derivativesa ..................................................................................................................................... 69 Table 5.2: Amino acid sequences and hB1R binding affinities of bradykinin and related peptides .   ........................................................................................................................................................ 70 Table 5.3: Biodistribution and uptake ratios of 68Ga-P03034, 68Ga-P04158 and 68Ga-Z02090 in tumour-bearing micea ..................................................................................................................... 71 Table 6.1: Radiolabeling, LogD7.4 and pharmacokinetics data of 68Ga-labeled [des-Arg10]KD derivativesa ..................................................................................................................................... 83 Table 6.2: Amino acid sequences and hB1R binding affinities of bradykinin and related peptides  ........................................................................................................................................................ 84 xvi  Table 6.3: Biodistribution and uptake ratios of 68Ga-labeled [des-Arg10]KD derivatives in tumour bearing micea .................................................................................................................................. 87 Table 7.1: Overall charge, radiolabeling and LogD7.4 data of radiolabeled hB1R-targeting tracers  ...................................................................................................................................................... 107 Table 7.2: Biodistribution data of radiolabeled hB1R-targeting tracers at 1-h p.i. in tumour-bearing mice. *Non-decay-corrected ........................................................................................... 107 Table 7.3: Biodistribution data (1-h p.i.) of 68Ga-Z02176 and 18F-Z04139 in tumour-bearing mice used for imaging or biodistribution study .................................................................................... 108 Table 8.1: Data of affinity, overall charge, and selected tissue uptake of previously reported hB1R-targeting peptides. apEC50. bpIC50. ..................................................................................... 118               xvii  List of Figures  Figure 1.1: Beta plus decay followed by positron/electron annihilation (Left), and coincident detection of the annihilation photon by the two sensors (gamma cameras) positioned on the same line of response (Right) .................................................................................................................... 3 Figure 1.2: The Kinin Kallikrein System (KKS) and kinin metabolism in human ......................... 6 Figure 1.3: Immunohistochemistry against hB1R in primary breast cancer core ......................... 15 Figure 3.1: Epifluorescence images of HEK283T::hB1R: RFP expression showing successful lentiviral transduction with hB1R sequence carrying the RFP reporter gene (Right).................... 43 Figure 3.2: Saturation assay on HEK293T plasma membrane with the hB1R antagonist [desArg10, Leu9]KD. A: HEK293T:hB1R. B: HEK293T WT ....................................................... 43 Figure 3.3: Comparison of different common cancer cell lines purified plasma membrane and hB1R physiological expression control. Competition between [desArg10, Leu9KD] and [3H-desArg10KD]. MCF7: Breast cancer; PC3 and LNCaP: Prostate cancer; HTR-8/SVneo: Non-pathogenic hB1R+ control trophoblastic cell line .......................................................................... 44 Figure 3.4: Effect of the metabolic stability on the 68Ga labelled kinin analogs biodistribution 60 min post injection. Red arrow: negative tumour; Green arrow: hB1R+ tumour. From left to right: Natural: 68Ga-P03083; 68Ga-P03083 in coinjection with enalaprilat (EP); 68Ga-P03083 in combination with phosphoramidon; Synthetic: 68Ga-SH01078 (n=6) ........................................... 46 Figure 4.1: Representative displacement curves of [3H]-[Leu9, des-Arg10]KD by SH01078, P03034, P04115 and P04168 ......................................................................................................... 57 Figure 4.2: Calcium release in HEK293T::hB1R cells induced by hB1R-targeting peptides: (A) [des-Arg10]KD, (B) [Leu9, des-Arg10]KD, (C) SH01078, (D) P03034, (E) P04115, and (F) P04168. Data are presented as mean ± SD (n = 3) ......................................................................... 58 xviii  Figure 4.3: Maximum intensity projection (MIP) PET/CT images of 68Ga-labeled [des-Arg10]KD derivatives at 1-h p.i. in mice bearing both hB1R+ (red arrows) and hB1R- (yellow arrows) tumours without (top row) or with (bottom row) co-injection of the non-radioactive standard .... 60 Figure 5.1: Representative displacement curves of [3H]-[Leu9, des-Arg10]KD by P03034, P041858 and Z02090 ..................................................................................................................... 70 Figure 5.2: PET/CT images of 68Ga-P03034, 68Ga-P04158, and 68Ga-Z02090 in mice bearing hB1R+ (yellow arrows) and hB1R- (red arrows) tumours without (top) or with (bottom) the corresponding cold standard ........................................................................................................... 73 Figure 5.3:  Comparison of the uptake of 68Ga-labeled hB1R-targeting tracers between hB1R+ and hB1R- tumours in mice in control (A) and blocked (B) conditions. *P < 0.05. ***P < 0.001 ...   ........................................................................................................................................................ 74 Figure 6.1: Representative displacement curve of [3H]-[Leu9, des-Arg10]KD by SH01078 and Z01115 (n=3) .................................................................................................................................. 84 Figure 6.2: Calcium release in HEK293T::hB1R cells after induction by hB1R-targeting peptides: (A) [des-Arg10]KD, (B) [Leu9, des-Arg10]KD, (C) Z01115, (D) SH01078. Data are presented as mean ± SD (n=3) ....................................................................................................... 85 Figure 6.3: Representative internalization curve of 68Ga-SH01078 and 68Ga-Z01115. Data are displayed as mean ± SD (n=3) and significance of differences between control and blocked groups: *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001 ............................................................. 86 Figure 6.4: Comparison of uptake of 68Ga-labeled [des-Arg10]KD derivatives in hB1R+ and hB1R- tumours in mice in the (A) control and (B) blocked groups. NS and **** indicate the p value is >0.05 and <000.1 respectively .......................................................................................... 88 Figure 6.5: Maximum Intensity Projection (MIP) of a 10 min PET/CT scan obtained 1h after injection of 3.7MBq of  68Ga-[des-Arg10]KD derivatives. Mice are bearing both hB1R+ (right xix  shoulder, red arrow) and hB1R- (left shoulder, yellow arrow) tumours were injected without (top row) or with (bottom row) co-injection of the non-radioactive standard (100µg) and anesthetized with isoflurane inhalation ............................................................................................................... 89 Figure 7.1: Representative radio-HPLC chromatograms of 68Ga-Z02176 and 18F-Z04139 from QC (upper chromatograms) and mouse plasma samples (lower chromatograms) taken at 5 min post-injection ................................................................................................................................ 105 Figure 7.2: Representative PET/CT Maximum Intensity Projections (MIP) of NSG mice bearing both hB1R- (left shoulder, green arrow) and hB1R+ (right shoulder, red arrow) tumors 60 min p.i. of 68Ga-Z02176 (A), 68Ga-Z02037 (B), 18F-Z04139 (C), or 18F-L08060(D) ......................... 106                xx  List of Abbreviations and Symbols   2D  Two dimensions 3D  Three dimensions 4D  Four dimensions %ID/g  Percentage of the injected dose per gram of tissue  β+   Positron  μ   Micro  ˚C   Degree Celsius  A  Amper ACE   Angiotensin conversion enzyme  Ahx  Aminohexanoic Acid Akt  Protein kinase B (Ak strain Thymoma) AmBF3 Ammoniomethyltrifluoroborate ANOVA Analysis of Variance APP  Amino Peptidase P APN  Amino Peptidase N B1R  Bradykinin 1 receptor B2R  Bradykinin 2 receptor BBB  Blood Brain Barrier BK  Bradykinin BKR  Bradykinin Receptor Bq   Becquerel  BSA   Bovine serum albumin  xxi  C   Carbon  CHO  Chinese hamster ovary Ci   Curie  COX  Cyclo Oxygenase CPM  Carboxy Peptidase M CPN  Carboxy Peptidase N CT   Computed tomography  CTA   Clinical Trial Application  CTAG  BCCA’s Centre for Translational and Applied Genomics  Cu   Copper  DCFBC  N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-fluorobenzyl-L-cysteine  DIEA   N,N-Diisopropylethylamine  DMEM  Dulbecco’s Modified Eagle Media  DMF   N,N-Dimethylformamide  DMSO  Dimethylesulfoxide  DNA  Deoxyribonucleic Acid DOTA  1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid e+  positron e-   electron EGFR  Epidermal growth factor receptor EP  Enalaprilat ERK  Extracellular signal Regulated Kinase ESI-MS ElectroSpray Ionization Mass Spectrometry F   Fluorine  xxii  FBS   Fetal bovine serum  FDA   US Food and Drug Administration  FDG   Fluorodeoxyglucose  Fmoc   9-Fluorenylmethyloxycarbonyl  Ga  Gallium Ge  Germanium GFP  Green fluorescent protein GPCR   G protein coupled receptor  hB1R  human Bradykinin 1 Receptor hB2R  human Bradykinin 2 Receptor HBSS  Hank’s balanced salt solution HBTU  2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium HEK293  Human embryonic kidney cell line  HEPES 4-(2-hyrdoxyethyl)-1-piperazineethanesulfonic acid HER2  Human Epidermal growth factor Receptor 2 hK  human Kallikrein HMWK High Molecular Weight Kininogen HPLC   High performance liquid chromatography  IC50   half-maximal inhibitory concentration  IHC   Immunohistochemistry  IL1β   Interleukin 1 beta In   Indium  kBq/cc  Kilo Becquerel per cubic centimeter  KD  Kallidin xxiii  Kd   Affinity constant  keV   Kilo electron Volt  Ki   Inhibition constant  KKS  Kinin Kallikrein System L  Liter LMWK Low Molecular Weight Kininogen LOR  Line Of Response LSO  Lutetium Ortho Silicate mAb   Monoclonal antibody MALDI-MS Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry MBq   Mega Becquerel  MDP  Methyl diphosphonate  MeCN  Acetonitrile  MMP  Matrix Metallo Protease MRI   Magnetic Resonance Imaging  mRNA  messenger RNA  NatF  Native fluorine NatGa  Native Gallium NaOH   Sodium hydroxide  NEP  Neutral Endo Peptidase NET   Neuroendocrine tumour  NFκB  Nuclear factor kappa-light-chain enhancer/activated B cells  NMP  N-methylpyrrolidinone NO  Nitric Oxyde xxiv  NODA  1,4,7-triazacyclononane-1,4-diacetate NOTA   1,4,7-triazacyclononane-1,4,7-triacetic acid  PA  Phosphoramidon PBS   Phosphate buffered saline  PEG  Polyethylene glycol PET   Positron emission tomography  PET/CT Positron Emission Tomography/Computed Tomography pH  potential of Hydrogen p.i.   Post-injection  PI3K  Phosphoinositide 3-kinase PK  Pre Kallikrein PRRT  Peptide Receptor Radio Therapy PSA   Prostate specific antigen  PSMA  Prostate-specific membrane  qPCR   Quantitative polymerase chain reaction  RFP  Red fluorescent protein RFU  Relative Fluorescent Unit RGD  Arginylglycylaspartic acid RNA   Ribonucleic acid  ROI   Region of interest  RSV  Rous sarcoma virus RT-PCR  Reverse transcriptase polymerase chain reaction  SCLC  Small cell lung cancer SD   Standard deviation  xxv  SerPin  Serin Protease Inhibitor SPECT  Single photon emission computed tomography T  Tesla Tc   Technetium  TEA  Tris EDTA Acetic acid TFA   Trifluoroacetic acid  TMA  Tissue Micro Array TNFα  Tumour Necrosis Factor alpha TK  Tissue Kallikrein TRAP  Triazacyclononane-phosphinate TRIUMF  Canada’s national laboratory for particle and nuclear physics TRT   Targeted Radio Therapy V  Volts            xxvi  Acknowledgements  The completion of this thesis was possible thanks to the tremendous support I received from many people. I thank with affection the three members of my committee, Pr. François Bénard, Dr. Urs Häfeli, and Dr. Marcel Bally, for their kindness and the fabulous scientific inspiration they represent to me. I thank Pr. Bénard for offering me a second chance to become a PhD, for the humanity he welcomed me with, and for the unwavering support he has provided since. Pr. Bénard shared with simplicity his knowledge and vision of science, making me feel smarter after every time we interacted. I express my deepest gratitude to Dr. Häfeli for believing in me, and for the extraordinary support and mentoring he has given me throughout my studies at UBC. I thank Dr. Bally, who offered me guidance and support in critical times since the beginning of my doctorate studies. I thank the “Bénard lab” team for being such a driving force in my mind and my companions of many days. I thank Jennifer, Sean, Hwan, Joseph, Carlos, Hsiou-Ting and Chengcheng for their help and participation to my work in many aspects during those years. Thanks to Julius, Wade and Milan for feeding my curiosity with their knowledge and time in the cyclotron facility. Many thanks to Milena, Iulia, Cynthia and Julie for their positive energy and the great discussions we had. Thanks to Helen and Jutta for bringing their caring energy in the lab. Thanks to Nav and Nadine who taught me so many things about the in vivo imaging procedure from the first to the last of all these long days. I thank Dr. Kuo-Shyan Lin for his unstoppable efficiency and great support in the publication process. I thank Dr. Zhengxing Zhang and Dr. Jinhe Pan for the hard work they accomplished and the team effort we shared together. Thanks to Silvia and Gemma for being such terrific lab mates, team players and friends to me. xxvii  I thank the “Häfeli lab”, José, René, Dana, Tullio, Mehrdad, Regina and especially Dr. Kathy Saatchi, for their scientific input, human support, and time spent together. I also thank members of the BCCRC for helping me daily in the lab: Anthony, Shima, Carolyn, Gayle, Tina, Teresa, Elizabeth and Susan. I thank Dr Federico Rosell for his scientific expertise and hours spent with me at the spectrophotometers. I thank Dr. Wayne Vogl for his support and generosity during my studies at UBC. I thank with all my heart my dear Jordan, for the laughs, the love and all the care she gave me during my studies. Thank you for the happiness I felt getting back to you every day. I thank my father for teaching me about the world, for giving me the means to achieve this work, for having done the best he could. I thank myself for never giving up.              xxviii  Dedication          To Sébastien, Renaud, and Nicolas, For giving me a light that never turns off, and many stars to aim at.            1  Chapter 1: Introduction  1.1 Molecular imaging in nuclear medicine   Molecular imaging is a non-invasive technique which uses signal-emitting molecules to visualize cellular functions and molecular processes in vivo. In nuclear medicine, molecular imaging is based on the detection of the radioactive signal (scintigraphy) emitted by a radiolabeled molecule known as radiotracer. The two main imaging modalities used to detect these emissions are Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) scanning. The combination of those two imaging modalities with X-ray Computed Tomography (CT) in PET/CT and SPECT/CT improves the localization of the radioactive signal within the body. CT imaging assesses anatomical structures based on their density When overlapped with the radioactive signals from PET imaging, hybrid PET/CT instruments can map physiological processes in living subjects by measuring specific biochemical processes. The distribution of radioactivity is driven by the targeting moiety bound to the isotope. This molecular motif is designed to bind physiological molecules expressed at the cellular level and reflect a specific metabolic pathway. The best known PET radiopharmaceutical is the 2-deoxy-2-[18F]fluoro-D-glucose or 18F-FDG, an analog of glucose which accumulates at higher rate in cells with enhanced glycolytic activity1. New targeting compounds require the identification of novel specific markers associated with specific tissues and/or conditions. Successive structure activity studies are necessary to engineer the probes in order to achieve specific detection of a target protein, and optimal pharmacokinetics for their intended medical application2. Radioactivity-based molecular imaging using PET/CT is a well-established method that uses low amounts of positron emitters to monitor the time-activity course of a radiotracer. 2  This has notably been used to study drug pharmacokinetics in cancer3. Applied in clinical practice, molecular imaging helps plan, monitor and tailor treatment plans for patients, and reduces the incidence of unnecessary surgeries, thus improving cancer management4. Besides, targeted radiotherapy (TRT) benefits directly from the development of radioactive imaging probes for therapeutic application as an alternative treatment for chemoresistant cancers5,6.  1.2 Positron Emission Tomography (PET)   Positron Emission Tomography was first used in the 1950’s in order to detect brain tumors by Sweet and Brownell7,8. PET isotopes follow β+ decay by emitting a positron of specific energy and range depending on the radionuclide. Consecutively, the positron interacts with a surrounding electron which in turn emits two annihilation photons of 511keV in opposite directions. The signal recorded by a PET scanner corresponds to the coincident detection of the two 511 keV photons (gamma rays) by two opposite detectors present on the same line of response (LOR)9. PET cameras use scintillating materials to convert gamma photons into detectable light. Upon interaction with a 511keV photon, inorganic crystals (e.g. Lutetium Orthosilicate LSO) emit fluorescence that can be detected and amplified by photomultipliers (PMT). Originally, two sensors diametrically opposed were used to detect single lines of activity. The number of sensors was increased to full rings of detectors to create 2D images, and finally disposed in an annular shape to enable 3D acquisitions (Figure 1.1). In the 2000’s, the combination of PET with CT improved lesion localization by adding an anatomical map to the radioactive signal. PET imaging is also able to perform dynamic acquisitions (4D). More recently, modern scanners have the PMTs with silicon photomultipliers to enable faster, direct digital processing of the emitted signal10. For research purposes on small animals, a micro 3  PET/CT is used to evaluate novel positron emitting radiopharmaceuticals, the reduction of the size of the sensors having the advantage of improving images resolution (1-2 mm) in a context readily translatable to clinical applications11. Although SPECT and PET are related imaging techniques, PET remains the modality of choice for cancer imaging using radiopharmaceuticals. Coincidence detection of emitted photons in PET does not require the use of a collimator, making it highly sensitive, which leads to less noisy images. The spatial resolution obtained with PET scanners is also superior to SPECT, which enables the detection of smaller lesions. Besides, PET is quantitatively precise, as a consequence of more accurate attenuation correction of the radioactive signal collected12. As a result, the spatial and temporal resolutions of PET are superior to SPECT, resulting in better quality image for a shorter scanning time.   Figure 1.1: Beta plus decay followed by positron/electron annihilation (Left), and coincident detection of the annihilation photon by the two sensors (gamma cameras) positioned on the same line of response (Right).     4  1.3 Cancer specific PET radiotracers  PET imaging is used to image various physiological processes for both research and clinical applications13,14. Many probes used for cancer diagnosis by PET are based on the expected elevated metabolism of cancer cells. The glucose analog 18F-FDG is used as a marker of glucose consumption, the amino-acid analog 11C-methionine is used as marker of protein synthesis, and the nucleoside analog 18F-fluorothymidine (FLT) is used as marker of DNA synthesis15. In clinical practice, 18F-FDG is, by far, the dominant radiotracer used for diagnostic applications. A significant limitation with this approach is highlighted by the fact that some healthy tissues (brain, heart) have a high glucose utilization in basal (healthy) conditions. It is also well-known that false-positive results occur due to activated inflammatory cells, and false negative results occur with slow growing cancers or cancers that use alternate fuels other than glucose16. As a consequence, new imaging radiopharmaceuticals that target tissue specific molecular abnormalities (phenotypic variations) are necessary to overcome the non-specificity of metabolism-based targeting approaches17.  Antibodies are biomolecules designed for molecular pattern recognition, and are broadly used for molecular imaging with both PET and SPECT. Antibodies are highly stable in vivo and slowly excreted, making them more suitable for a labelling with long-lived isotopes, which leads to higher radiation exposure. First generation antibody based radiotracers used with SPECT for radioimmunoscintigraphy had limited diagnostic accuracy (e.g. ProstaScint, Cytogen Corporation), and have been largely abandoned in clinical practice today. However, the potential of new monoclonal antibodies is still investigated for applications in diagnosis, prognosis, and monitoring progression of cancer18, as well as for targeted therapeutic procedures (e.g. radioimmunotherapy, radioguided surgery)19,20,21. New detection systems using pre-targeting 5  approaches allow the use of full size antibodies conjugated with a secondary radiotracer labelled with a short-lived isotope21. The diminution of the molecular weight using affibodies or other constructs is another approach to overcome the long circulation time, non-target organs accumulation, low tumour penetration and slow excretion of antibodies. Small molecules and cytokines analogs can also be readily labelled with isotopes for diagnosis or radiation therapy. Radiolabeled peptidic analogs behave in a similar manner as the endogenous molecule they imitate, which expectedly results in a higher selectivity for the target of choice and predictable behavior. Peptidic analogs are typically unable to cross the blood brain barrier (BBB) and are normally rapidly excreted through the urinary tract, limiting off-target accumulation in other organs. However, peptidic analogs are prone to metabolic degradation in vivo. Peptide receptor radiotherapy (PRRT) is readily achievable nowadays22. Peptidic analogs can additionally function as active ligands and be used as targeted therapeutic drugs23,24. The human Bradykinin 1 Receptor (hB1R) is a potential cancer biomarker25, and the relative simplicity and great versatility of kinins make the optimization of a radiolabeled peptidic analog probe a promising idea for PET imaging26,27.  1.4 Kinin Kallikrein System (KKS)  The kinin-kallikrein system (KKS) is a vascular proteolytic cascade responsible for the release of bioactive kinins in the blood circulation. Kinins play a cardioprotective role, essentially by triggering vasodilatation in most vascular beds through the release of vasoactive hormones (NO, prostacyclin)28. Furthermore, the KKS is known to participate in humoral mediated proinflammatory responses through the modulation of vascular permeability to recruit inflammatory cells and induce tissue remodeling (via proteases, mitogenic signaling, and 6  angiogenesis). While this effect is beneficial during acute trauma, it becomes proinflammatory during severe trauma, and contributes to chronic pain, inflammation, and tissue destruction29. Bioactive kinins are released from their inactive precursors (kininogens) by the action of kininogenases (kallikreins). The short half-life required for the autacoid action of kinins is ensured by the kininase control step. Kininases inactivate kinins by rapidly hydrolyzing the peptides in tissues and circulation, and generate at the same time active kinin intermediates. The actions of the KKS are primarily mediated by the binding of bradykinin (BK) and kallidin (KD) to the two cell surface G-protein-coupled receptors bradykinin B1 and B2 (hB1R and hB2R)30.   Figure 1.2: The Kinin Kallikrein System (KKS) and kinin metabolism in human.    7  1.4.1 Kininogens  Kininogens are globulins released in the blood circulation by the liver and derived from the same unique gene transcribed into two alternative splicing variants31. Both kininogens subtypes have an identical amino-acid sequence from the N-terminal end and differ only in the C-terminus. The high molecular weight kininogen (HMWK) is the precursor of BK, and the low molecular weight kininogen (LMWK) is the precursor of KD32,33.  1.4.2 Kininogenases  Kininogenases are mainly represented by two types of serine proteases (kallikreins) that convert circulating kininogens into active kinins. Plasma kallikrein releases principally BK from HMWK, while tissue kallikreins hydrolyze both kininogens equivalently to release BK and KD32,34. The human plasma kallikrein (PK, or hKB1) is encoded by a single KLKB1 gene and synthesized predominantly in the liver as a zymogen called prekallikrein (PPL or PrehKB1) non-covalently complexed to HMWK. Tissue kallikreins (TK, or hK) are encoded by 15 KLK genes localized on the same chromosome and expressed as precursor enzymes (ProhK) in various exocrine organs (pancreas, intestine, brain, kidneys and salivary, sudoriparous and submandibular glands)29,31,35. The production of bradykinin by tissue kallikreins is specifically inhibited by an endogenous Serpin (Serine Protease inhibitor): kallistatin30.     8  1.4.3 Kinins  The kinin peptides, bradykinin (BK), kallidin (Lys-bradykinin or KD) and all the BK-related peptides are the bioactive compounds released by the KKS, and are known to be the most algogenic (pain-inducing) compounds produced by the body. In humans as well as in most mammals, the term “kinin” includes the BK nonapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) analogs (KD = Lys-BK) and their C-ter des-Arg metabolites32. Kinins are vasoactive and proinflammatory cytokines, whose activity depends on the enzymes responsible for their release and their degradation, and as well as on the expression of the two bradykinin receptors B1 and B2. BK and KD are the endogenous ligands of hB2R, while the elimination of their C-terminal Arg turns them into hB1R agonists36.  1.4.4 Kininases  The autocrine or paracrine mode of action of the active kinins depends on their local release by kallikreins and their short half-life. The second step of the KKS is named kininase control, and corresponds to the hydrolysis of kinins by two classes of kininases. This step is crucial for the proper functioning of the KKS, as it inactivates kinins by rapid degradation, and generates intermediates peptides metabolites potentially able to signal through the kinin receptors. Kinins are rapidly degraded by four main metallopeptidases separated in two subtypes: Kininases I and II30,32. Kininases II are represented by the angiotensin converting enzyme (ACE), the aminopeptidases P and N (APP and APN), and the neutral endopeptidase (NEP). Despite its name, the main function of ACE is kinin catabolism, and ACE is considered the main kinin inactivating enzyme in the blood circulation. ACE, a dipeptidyl carboxypeptidase, inactivates BK 9  by removing the C-terminal Phe8-Arg9 dipeptide, and subsequently removing the Ser6-Pro7 dipeptide. ACE cleaves similarly the Phe5-Ser6 bond in des-Arg9 BK, and releases the tripeptide Ser6-Pro7-Phe8 and the same Arg1-Pro2-Pro3-Gly4-Phe5 pentapeptide as from BK31,37. APP and APN are two plasmatic exopeptidases which cleave the N-terminal aminoacids of kinins in circulation. APP cleaves BK and des-Arg9BK Arg1 which release BK[2-9] and BK[2-8] respectively. APN cleaves KD and des-Arg10KD Lys1, releasing BK (hB2R agonist) and des-Arg9BK (hB1R agonist) respectively38. NEP (Neprilysin, or Endopeptidase 24.11), is a membrane bound kininase that functions mostly in tissues and endothelial tissues. NEP cleaves the Pro7-Phe8 and the Gly4-Phe5 bonds of BK and des-Arg9BK, and produces the Phe8-Arg9 dipeptide, Phe5-Ser6-Pro7 tripeptide and the Arg1-Pro2-Pro3-Gly4 tetrapeptide. Kininases I encompass two carboxypeptidases N and M (CPN , CPM) that convert the hB2R agonists BK and KD in two hB1R agonists des-Arg9BK and des-Arg10KD by removal of their C-terminal Arg. CPN is synthesized in the liver and secreted in the blood, while CPM is a membrane bound enzyme expressed in the lungs and the kidneys31,37. The importance of these peptidases depends on the animal species, the analytical approach, the biological milieu, and the pathophysiological context. However, they are all present in a soluble form in biologic fluids37.  1.5 KKS functions  1.5.1 BKR  Kinins exert their pharmacological action through binding to the bradykinin B1 and B2 receptors (BKRB1 and BKRB2, aka hB1R and hB2R). Bradykinin receptors are G protein coupled receptors (GPCR) anchored in the plasma membrane of human cells by seven transmembrane 10  domains. While bradykinin (BK) and kallidin (KD) are hB2R agonists, their metabolites des-Arg9BK and desArg10KD, generated by the carboxypeptidases N and M, are hB1R agonists36,39. However, the hB2R main ligand is bradykinin (BK), while the hB1R main ligand is desArg10KD. Despite the close similarity of their respective agonists, hB1R and hB2R share only 36% of homology at the aminoacid level, making their expression pattern and pharmacology very distinct and complementary39. This difference is reflected by the expression pattern of bradykinin receptors. hB2R is constitutively and ubiquitously expressed by tissues under healthy conditions, and is responsible for the early phase of the inflammatory response mediated by the KKS, including vasodilatation and vascular permeability increase32. hB1R expression is minimal in normal physiological conditions, and is only locally induced following sustained inflammatory stimuli (infection, tissue trauma, toxins, anoxia) by cytokines (IL1β, TNFα)39,40,41,42 that trigger the binding of NFκB-like factor to the hB1R gene promoter42. Besides, hB2R and hB1R activation stimulates each other’s expression levels43. hB2R is responsible for the acute phase of the inflammatory response; hB1R is responsible for the chronic phase. During an inflammatory episode, kinin generation and receptor cross induction act synergistically to form and maintain an autocrine stimulation loop that shifts bradykinin receptors directory from hB2R to hB1R44. In contrast to hB2R, hB1R cannot be desensitized, and its expression is also induced by its agonist desArg10KD45. In addition to this, hB2R activation triggers its endocytosis, while hB1R endocytosis is delayed upon binding to its ligand46,47. As a consequence, hB1R is thought to take over hB2R signaling and lead the chronic phase of inflammation by triggering pain, modulating permeability of vascular endothelia, immune cell recruitment, angiogenesis, and mitogenesis in tissues expressing hB1R48,49. The pattern of expression of hB1R and its functions are not limited to the inflammatory context, and also plays a role in the early embryonic stages and wound healing50,51. Local cellular 11  proliferation, migration and invasion are markers of these healthy physiological phenomena as well as of cancer, and require hB1R activation. These characteristics indicate that hB1R is a candidate of choice to target cancer associated chronic inflammation, without the disadvantage of potentially binding the ubiquitous hB2R.  1.5.2 KKS and cancer  The different stages of cancer development are known to be associated with remodeling of the surrounding tissues, through tumour volume increase, angiogenesis and tissue invasion, suggesting that the inflammatory response elicited by the host in these instances could be a marker for cancer detection27. Many elements of the Kinin Kallikrein System (KKS) are detected routinely in cancer patient fluids and have a prognostic value. In particular, hB1R and the elements of the KKS responsible for the production of its agonist are consistently found among the KKS elements modulated in a broad selection of cancers as highlighted below. The inducible expression pattern of the hB1R strongly indicates that it could be used as a marker to detect tumours using radioactive probes for non-invasive visualization.  1.5.3 hB1R expression in cancer  Many cancers express one or more elements of the KKS. Upregulation of kallikrein 4-11 and 14 gene expression correlates with invasion and metastasis of ovarian cancer52. In accordance with these findings, the serum levels of these kallikreins are used as ovarian cancer biomarkers for diagnosis (hK6, hK10, hK11), prognosis (hK6, hK11), and monitoring (hK6 and hK11). Ovarian cancer cell cytosolic levels of hK10 are also used for prognosis53,54,55,56,57. Specifically 12  glycosylated kininogens are used as hepatocellular carcinoma serum marker58 in patients, while lower levels of BK and des-Arg9BK metabolites are observed in the serum from bladder cancer patients59. High levels of kinins are found in the ascitic fluid of gastric tumours60, which is associated with a 20% reduction of the plasma activity of active prekallikreins and kininases compared to healthy context61. hB1R and hB2R protein levels are increased in colorectal cancer cells in vitro62 and correlate with the immunohistochemical staining obtained with patients tissues. hB1R and hB2R are overexpressed, with a more prominent staining of hB1R in tubular adenomas, while hB2R staining is more intense in hyperplasic polyps63. This difference suggests that hB1R might play a role in abnormal cellular transformation, whereas hB2R might play a protective role63. Levels of mRNA coding for hB1R, hB2R and kininogens are higher in melanomas64, while IHC studies performed on clear-cell carcinoma65 and esophageal squamous cell carcinoma66 demonstrate higher expression of tissue kallikrein, hB1R and hB2R. Using immunofluorescence microscopy, hK1,2,5-9, hKB1; hB1R and hB2R were detected at high levels in malignant pleural mesothelioma67. Correspondingly, IHC performed on lung cancer sections of various grades shows the overexpression of two precursor enzymes responsible for the release of active kinins from kininogens (Tissue ProKallikrein proHK1, Plasma PreKallikrein preHKB1), in association with the dual overexpression of hB1R and hB2R (respectively: adenocarcinoma, 70 and 74%; squamous cell carcinoma, 82 and 85%; large cell carcinoma, 68 and 58%; small cell carcinoma, 76 and 80%; carcinoid tumour, 88% for both)68. Also, hB1R and hB2R expression in human chondrosarcoma tumours is increased compared to normal cartilage.  In vitro, expression of α2β1 integrin by these cells and migration are directly dependent on the activation of those two receptors69.  The levels of glandular kallikrein (hK2) and Prostate Serum Antigen (PSA or hK3) in tissues and serum are increased in breast cancer patients52,70. Higher serum levels of BK and [des-13  Arg9]-BK metabolites are also found in these patients59. hB1R and the main KD generating kallikrein hK1 are produced at a basal level by estrogen sensitive and insensitive breast cancer cell lines and tumours71,72. In vitro, estrogen-sensitive breast cancer cells stimulated by KD downregulate hK10 (growth suppressor related) gene expression, but upregulate hK6 and hK11 (invasiveness and proliferation stimulations respectively) gene expression. Furthermore, the stimulation of these cells with KD triggers the release of kininogens, hK1 and hK6 through an hB1R-dependent pathway73. In accordance with this, hB1R and EGFR transactivation stimulate the proliferation of estrogen sensitive and insensitive breast cancer cells and trigger the release of MMP 2 and 973,74. Activation of hB1R and/or hB2R has been reported to promote the phosphorylation of extracellular regulated kinases ERK1/2 in estrogen sensitive cells in vitro and stimulate their proliferation74. A tissue microarray (TMA) study performed by the same team showed that hB1R was expressed in a high proportion of ductal breast carcinomas in situ as well as invasive ductal breast carcinomas (fibroadenomas (3/4), ductal carcinoma in situ (4/4), invasive ductal carcinoma (11/13))74. Studies on invasive breast carcinoma from patients confirmed hB1R upregulation by IHC using transcript screening and demonstrated the highest transcription of the hB1R gene in patient samples in the case of estrogen receptor negative tumours75.  Another TMA performed by our group (Figure 1.2) confirms these observations: 76% of the breast cancer patients’ biopsies expressed hB1; 50 % showed strong expression, 17% moderate expression and 7% low but significant expression.  The elevation of hK3 concentration in serum and tissue is used for diagnosis, prognosis and monitoring of prostate cancer. hK11 levels in serum are used only for diagnosis and prognosis of the disease52,70,76. The glandular kallikrein hK2 is also overexpressed in prostate cancer tissue77,78 and is able to release active kinins79 from kininogens. Immunohistochemical staining of 16 prostate cancer biopsies showed that hB2R was ubiquitously expressed in normal 14  and cancerous prostate tissues, while hB1R was only detected in all prostatic intraepithelial neoplasia and malignant lesions but not in benign prostate tissues80. In vitro stimulation of the endogenous hB1R expressed by androgen-insensitive and PSA negative prostate cancer cells promotes their growth, migration and invasion80, through ERK phosphorylation. While both hB1R and hB2R are expressed in those cell lines, the growth stimulation in the non-metastatic DU145 cells is only stimulated by the hB1R agonist, while hB2R is inactive. On the contrary, the proliferation of the highly metastatic PC3 cells is stimulated by both hB1R and hB2R agonists and is antagonized upon inactivation of either of the two receptors. In these cells, the absence of the usual markers and the hB1R common expression make it a potential alternative target for treatment81.  hB1R has been detected in other cancer types, including astrocytic tumours82 and glioblastoma83. Human glioma cell brain xenografts and the associated vasculature express hB1R, which enhances tumour blood perfusion84. Besides, stimulation of hB1R by KD triggers glioma cells migration and COX2 release in vitro83. In glioma cell lines co-overexpressing hB1R and hB2R, stimulation by one of the two receptor agonists enhanced proliferation85 through phosphorylation of PI3K/Akt and ERK1/2. In a study of precursor lesions for gallbladder cancers, hB1R and HER2 were not detected in normal epithelium. However, they were coexpressed in intestinal adenomas (hB1R: 100%; HER2: 17%), and both were highly detected in faster proliferating intestinal metaplasias (hB1R: 100%; HER2: 91.7%) and carcinomas in situ (hB1R: 100%; HER2: 90%). hB1R and HER2 expression did not follow each other in pyloric adenoma (hB1R: 67%; HER2: 0%) and were barely detectable in invasive carcinoma (hB1R: 8.3%; HER2: 33%). Interestingly, the increased coexpression of HER2 and hB1R was not due to gene amplification, and strongly suggested a common relocalization process from the cytoplasm to the plasma membrane86.  15  Taken together, these findings suggest that hB1R is a biomarker of cancer development.  While many cancer promoting receptors are constitutively activated following genetic mutations, the modulation of KKS and hB1R upregulation correspond to the normal physiological response to tissue injury, which is hijacked by cancer cells to stimulate their own survival and proliferation. hB1R overexpression in cancer seems to correlate with tumour grade, and is associated with many well-known cancer promoting receptors. hB1R activation stimulates cancer progression in many aspects, but depending on cancer types and grades, this effect is independent, cooperative, or dependent on these co-activated or co-expressed receptors. While many elements of the KKS are used already used routinely for cancer screening and monitoring, hB1R inducibility confers the potential to be a biomarker of chronic inflammation in cancer. Consequently, hB1R has been proposed as a promising therapeutic target in cancer23.  Figure 1.3: Immunohistochemistry against hB1R in primary breast cancer core.    16  1.6 BKR targeting  1.6.1 Modulation  Bradykinin peptidomimetics have been successfully patented to target hB2R87, and with the well described relationship between their ligands, it is likely that a similar strategy to design a strict hB1R targeting peptide will be successful88. As expected, the binding of kinin analogs to their cognate receptors can be used to develop stable antagonist kinin analogs as therapeutic agents89. The well described hB2R peptide antagonist HOE14087 and a nonpeptide hB1R antagonist are both able to induce cellular death in two glioma cell lines, triggering necrosis and apoptosis85. However, HOE140 has been found to act as a mitogenic stimulator on certain tumours expressing hB2R90. The hB2R peptidic dimer antagonist CU201 acts as a biased agonist91 by inducing apoptosis and completely inhibiting the proliferation of small cell lung cancer92 and prostate cancer93 in vitro and in vivo. In addition to this, CU201 acts synergistically with antitumoral drugs to inhibit SCLC cell growth in vitro and in vivo. While CU201 behaves as a hB1R and hB2R dual antagonist, it becomes a hB2R agonist at high concentrations94. A hB1R/hB2R dual antagonist has been synthesized to treat chronic inflammatory disorders and was found to be very active in vitro and in vivo95. Finally, in prostate cancer, a natural hB1R antagonist inhibited both hB1R and hB1R-hB2R activation by KD and subsequent proliferation81,96 of prostate cancer, which suggests that hB1R-targeting molecules could be used for cancer detection.    17  1.6.2 Imaging  The first radiolabeled kinin analog developed for in vivo imaging of hB2R was 99mTc-HOE140. Originally designed to visualize chronic inflammation in mice97, HOE140 is thought to potentially act as an anti-inflammatory drug87. The same aim was tackled using 11C-labeled sulfonamide to detect hB1R expressed at chronic inflammatory areas in mice. Despite a promising specific binding to hB1R in vivo, this tracer yielded significant non-displaceable background radioactivity in both target and healthy tissues98. Fluorescent KD derivatives targeting hB1R have been described for confocal microscopy99,100,101, flow cytometry, receptor internalization studies and confirmed the potential of this approach to generate hB1R active targeting cargoes. These last results confirm that kinin peptidic analogs tolerate modification at their N-terminal end without loss of affinity, and support the idea of coordinating such probes with various labels for hB1R detection102.  1.7 hB1R targeting  1.7.1 Peptide analog  The selective binding to hB1R in vivo using peptidomimetics has been shown to be possible with unnatural amino acid scaffolds able to resist the kininase control degradation step89. A high level of kinins during cancer development, and specifically at the tumour site, has been shown to correlate with a higher consumption of the kinin degrading enzymes61,61. In order to image efficiently hB1R in vivo, the metabolic stability appears as a strict requirement to reach the tumour site with a functional probe, while its affinity might drive the final binding event to hB1R 18  in competition with elevated endogenous autocrine stimulation. While the combinations of isotope/chelator/amino-acid sequence are too numerous to be all investigated, we will select each building block in accordance with the literature and put them in perspective to infer the most favorable properties for hB1R binding and evaluate them in vivo.  1.7.2 Sequence  As previously mentioned, each of the hB1R and hB2R has a cognate set of kinins to activate it. hB1R has a higher affinity for kinins lacking the C-terminal arginine41 and carrying a N-terminal Lys, whereas binding to hB2R does not. The favorable effect of the N-terminal Lys on hB1R binding depends on the additional positive charge carried by the Lys, but also on the spatial orientation that requires the L-isomer to fulfill its function103. Importantly, the exchange of the last amino acid of des-Arg10-KD from a Phe to a Leu shifts its activity from agonist to antagonist104. The hB1R targeting peptidic sequences will be established following the [desArg10]KD aminoacid sequence. In order to improve the metabolic stability of the peptidic sequence in vitro and vivo, unnatural aminoacids will be used to limit the recognition of cleavage sites by aminoacid specific peptidases (Table 1.1).  1.7.3 Radiolabeling  In order to match the short biological half-life of the endogenous kinins and the efficiency of the renal clearance, short lived isotopes are recommended to label KD peptidic analogs. The quicker decay allows the injection of higher doses of radioactivity without exceeding the acceptable radiation exposure to a patient. As a consequence, increased amounts of radiation can 19  be detected in a reduced amount of time. 18F and 11C are currently the most common isotopes used for PET clinical imaging. The 20.3 min half-life of 11C limits its application for tumour imaging, while 18F labelled compounds are subject to defluorination and can elicit non-target uptake in the bone or the gastrointestinal tract105. Another major drawback of 11C and 18F is the necessity of a proton beam (i.e. cyclotron) to be generated within transportation distance of the isotope half-life106. The growing interest in PET/MRI imaging has to be taken into account to select the proper isotope. In comparison with CT, MRI improves the visualization of soft tissues, making it more indicated to localize tumours in vivo. In addition, despite short scanning times, the absence of X-ray exposure is another key element to minimize patient radiation exposure. Interestingly, the application of magnetic fields over 5T has been shown to improve the PET scan resolution. Strong magnetic fields reduce the range of the two high energy positron emitters 82Rb and 68Ga, but not 18F and 11C 10. The long positron range and 75s half-life of 82Rb are not compatible with tumour imaging. Thus, our isotope of choice will be the radiometal 68Ga. 68Ga has a half-life of 68.3 min, and offers the advantage to be readily available from generators that have a long half-life of approximately 9 months. In addition, 68Ga can be coordinated with various chelators, DOTA being the most used for this isotope. High temperature and low pH used during the labelling are not expected to affect the tracer’s integrity107. However the impact on the tracer activity will be studied as a shift from antagonist to agonist after conjugation of a radiometal to DOTA has been reported with some somatostatin analogs108. Finally, the importance of the N-ter lysine as well as the impact of C-ter modifications suggest that a spacer between the radiometal chelator and the targeting moiety should be added by the use of a covalent linker. Alternative 68Ga- chelators and radiofluorination will be performed in the latter steps to compare the most favorable 68Ga- labeled radiotracer with their 18F homologs. Similarity between isotopes suggests the use of NODA for an alternative 68Ga labelling, while the recently 20  reported one step 18F-labeling approach via 18F-19F isotope exchange reaction on an ammoniomethyltrifluoroborate (AmBF3) moiety will be tested with our probes109,110. Peptide Isotope Chelator Linker Spacer Sequence Chapter 1 2 3 4 5 6 7 8 9 10 Bradykinin (BK) - - - - Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 Kallidin (KD) - - - Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 [Leu9,desArg10]KD - - - Lys Arg Pro Pro Gly Phe Ser Pro Leu - 1 P03083 68Ga DOTA Ahx Lys Arg Pro Pro Gly Phe Ser Pro Leu - 3 Z01115 68Ga DOTA Ahx Lys Arg Pro Hyp Gly Cha Ser Pro Leu - 6 SH01078 68Ga DOTA Ahx Lys Arg Pro Hyp Gly Cha Ser Pro Leu - 3,4,6 P04115 68Ga DOTA Gly-Gly Lys Arg Pro Hyp Gly Cha Ser Pro Leu - 4 P03034 68Ga DOTA dPEG2 Lys Arg Pro Hyp Gly Cha Ser Pro Leu - 3,4,5 P04168 68Ga DOTA Pip Lys Arg Pro Hyp Gly Cha Ser Pro Leu - 4 B9858 - - - Lys Lys Arg Pro Hyp Gly Igl Ser D-Igl Oic 5 B9958 - - - Lys Lys Arg Pro Hyp Gly Cpg Ser D-Tic Cpg 5,7 P04158 68Ga DOTA dPEG2 Lys Lys Arg Pro Hyp Gly Igl Ser D-Igl Oic 5 Z02090 68Ga DOTA dPEG2 Lys Lys Arg Pro Hyp Gly Cpg Ser D-Tic Cpg 5 Z02137 68Ga NODA Mpaa-Pip Lys Lys Arg Pro Hyp Gly Cpg Ser D-Tic Cpg 7 Z02176 68Ga DOTA Pip Lys Lys Arg Pro Hyp Gly Cpg Ser D-Tic Cpg 7 Z04139 18F Al-NODA Mpaa-Pip Lys Lys Arg Pro Hyp Gly Cpg Ser D-Tic Cpg 7 L08060 18F AmBF3 Mta-Pip Lys Lys Arg Pro Hyp Gly Cpg Ser D-Tic Cpg 7 Table 1.1: Kinins and synthetic radiolabeled analog design.  1.7.4 Model  Our base assay to compare the affinities of our radiotracers for hB1R in vitro relies on commercial plasma membranes purified from CHO cells transformed to constitutively express hB1R. While this assay offers consistency to evaluate our radiopeptides affinities, it limits our evaluation to a strict in vitro artificial and non-functional system. Preliminary results aimed at evaluating prostate (PC3, LNCap, VCAP) and breast (MCF7, T47D) cancer cell lines for hB1R expression revealed that none of these cells plasma membranes were able to bind the [3H-desArg10KD]. Plasma membranes purified from immortalized human trophoblastic HTR-8/SVneo cells were used as a non-pathologic positive control51, and bound significant levels of [3H-desArg10KD] in vitro. However, several parameters suggested that creating a different 21  expression model for live cell assays in vitro and in vivo were still necessary. First, the maximum binding achieved with the HTR-8SVneo cell membranes was less than half that measured with CHO membranes. Besides, despite the fact that hB1R expression is induced by inflammatory events, the binding to HTR-8SVneo cell membranes demonstrated that hB1R expression can also correspond to an inherited phenotype (e.g. differentiation stage), and suggest that other acquired expression patterns might exist in those cells and trigger effects which are not part of cancer progression, supported by the fact that HTR-8SVneo cells are already known to express hB2R51. As a consequence, we will aim to test our radiotracers in a synthetic hB1R overexpression system in a human cell line, offering a live cell system conveyable from in vitro signaling assays to in vivo imaging in a context as human as possible.  1.8 Hypothesis and objectives   The work presented here is based on the hypothesis that the human bradykinin B1 receptor is a specific biomarker of early cancer development and can be detected non-invasively in vivo with a radiolabeled synthetic peptide and a 3D imaging technique like PET/CT. The final aim is to develop highly specific probes to target cancer associated chronic inflammation for diagnostic and therapeutic purposes. To this end, three specific aims are proposed.       22  1.8.1 Specific aim 1   Design, synthesize and radiolabel des-Arg10 KD analogs conjugated with different spacers and radiometal chelators, and select the best compounds according to their in vitro binding properties.  1.8.2 Specific aim 2   Test the tracers selected in aim 1 for their in vitro biological activity and preliminary study of their in vivo pharmacokinetics to identify the suitable best candidates to be used for in vivo studies.    1.8.3 Specific aim 3   Evaluate in vivo the radiotracers selected in aim 2 by biodistribution studies and PET imaging with mice bearing hB1R expressing and negative tumour xenografts.        23  Chapter 2: General Materials and Methods  2.1 General  All chemicals and solvents were obtained from commercial sources, and used without further purification. 68Ga was obtained from a 68Ge/68Ga Eckert & Ziegler (Berlin, Germany) IGG100 generator or an iThemba Labs generator (Somerset West, South Africa) eluted with a total of 4 mL of 0.1 M HCl. The elution that contained the activity was mixed with 2 mL of concentrated HCl and passed through a DGA resin column. The column was then washed by 3 mL of 5 M HCl, dried by passage of air, and 68Ga was eluted off with 0.5 mL water. Fluoride was produced by the 18O(p, n)18F reaction using an Advanced Cyclotron Systems Inc. (Richmond, Canada) TR19 cyclotron.18F-fluoride Trap & Release Columns were purchased from ORTG Inc. (Oakdale, TN), and the C18 light Sep-Pak cartridges (1cc, 50 mg) were obtained from Waters (Milford, MA). hB1R targeting peptides were synthesized using a solid phase approach on an AAPPTec (Louisville, KY) Endeavor 90 peptide synthesizer. The collected HPLC eluates containing the desired peptide were lyophilized using a Labconco (Kansas City, MO) FreeZone 4.5 Plus freeze-drier. Mass analyses were performed using a Bruker (Billerica, MA) Autoflex MALDI-TOF system and a Bruker Esquire-LC/MS system with an ESI ion source. Purification and quality control of cold and radiolabeled peptides were performed on an Agilent high-performance liquid chromatography (HPLC) System equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (set at 220 nM), and a Bioscan (Washington, DC) NaI scintillation detector. The radio-detector was connected to a Bioscan B-FC-1000 Flow-count System, and the output from the Bioscan Flow-count system was fed into an Agilent 35900E Interface which converted the analog signal to a digital signal. The operation of the Agilent 24  HPLC system was controlled using Agilent ChemStation software. Peptide purification and analysis were performed using a semi-preparative column (Phenomenex C18, 5 µm, 250 x 10 mm) and an analytical column (Eclipse XOB-C18, 5 µm, 150 x 4 mm). The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA, except when described otherwise. CHO-K1 cell membranes overexpressing recombinant hB1R were obtained from PerkinElmer (Waltham, MA). Radioactivity of radiolabeled peptides was measured using a Capintec (Ramsey, NJ) CRC®-25R/W dose calibrator, and the radioactivity of mouse tissues collected from in vivo studies were counted using a Packard (Meriden, CT) Cobra II 5000 Series auto-gamma counter. Male immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were obtained from an in-house breeding colony at the Animal Resource Centre of the BC Cancer Agency Research Centre (Vancouver, BC, Canada). PET/CT imaging experiments were conducted using a Siemens (Erlangen, Germany) Inveon microPET/CT scanner.   2.2 Peptide synthesis and chemistry  2.2.1 Precursor synthesis  This peptide was synthesized via the Nα-Fmoc solid phase peptide synthesis strategy starting from Fmoc-Leu-Wang resin. The resin was treated with 20% piperidine (1 x 5 min and 1 x 10 min) in DMF to remove the Nα-Fmoc protecting group. The following Fmoc-protected amino acids (3 equivalents), including Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-L-Phe-OH, Fmoc-D-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cha-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Oic-OH, Fmoc-D-Igl-OH, Fmoc-Igl-OH, Fmoc-Cpg-OH, Fmoc-D-Tic-OH, Fmoc-6-Ahx-OH, Fmoc-PEG2-OH, Fmoc-4-amino-(1-carboxymethyl)piperidine 25  (Fmoc-Pip-OH), the chelator DOTA tri‐t‐butyl ester, and N-propargyl-N,N-dimethylammoniomethyl-trifluoroborate (synthesized as previously reported109 were subsequently coupled according to sequence.  The coupling was conducted in NMP with standard in situ activating reagent HBTU (3 equivalents) in the presence of DIEA (6 equivalents). At the end of the elongation, the peptide was deprotected and simultaneously cleaved from the resin by treatment with a 95/2.5/2.5 TFA/H2O/TIS (triisopropylsilane) mixture for 4 h at 20°C. After filtration, the peptide was precipitated by the addition of cold diethyl ether to the TFA solution. The precipitated crude peptides were collected by centrifugation, and were purified by HPLC using a semi preparative column (Phenomenex C18, 5 µm, 250 x 10 mm) with a specific A/B at 4.5 mL/min flow rate. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time was measured and the mass calculated and compared to the value obtained by enhanced product ion (EPI) mass spectrometry (MS). The HPLC eluates containing the desired peptide were collected, pooled, and lyophilized.  2.2.2 Synthesis of Nat-Ga labeled tracers  To obtain gallium conjugates, a solution of the DOTA-conjugated peptide (2-4 µmol) and GaCl3 (5 equivalents) in 500 µL of sodium acetate buffer (0.1M, pH 4.0) was incubated at 80°C for 15 min. The reaction mixture purified with the semi-preparative column with a 80/20 A/B mix at 4.5 mL/min flowrate, and the eluate fraction containing the compound was collected, pooled and lyophilized.   26  2.2.3 Synthesis of 68Ga labelled tracers  The purified 68GaCl3 solution (0.5 mL in water) was added to a 4-mL glass vial preloaded with 0.7 mL of HEPES buffer (2 M, pH 5) and the chelator conjugated precursor. All 68Ga labeling procedures were performed in a conventional microwave oven, using a 1 min reaction time (700W). Microwave labeling was used because of previous experience achieving high yields in a short time using similar peptides. It is likely that similar results can be obtained with conventional heating. The high specific activity required for preclinical imaging was guaranteed by HPLC purification using the semi-preparative column eluted with tracer-specific CH3CN/PBS ratios and flow rates. The fraction of the eluate that contained the radioactive product was collected, diluted with water (50 mL), and passed through a C18 Sep‐Pak cartridge pre-washed with ethanol (10 mL) and water (10 mL).  The 68Ga‐labeled product was eluted off the cartridge with ethanol (0.4 mL), and diluted with saline (10/1) for plasma stability assays and imaging/biodistribution studies. The quality control was performed by HPLC on the analytical column eluted with different CH3CN/PBS ratios and flow rates than for purification, and the retention time was measured. The specific activity of the radiolabeled peptides was measured using the analytical HPLC system and was calculated by dividing the injected radioactivity (~37 MBq) of final products by the mass of the peptides in the injected solution. The mass of the radiolabeled peptides was estimated by comparing the UV absorbance obtained from injection with a previously prepared standard curve.     27  2.3 LogD7.4 determination  The distribution coefficient (LogD7.4) of radiolabeled tracers was determined by “shake flask” methodology. Aliquots (2 µL ~ 0.1 mCi) of the radiolabeled tracer were added to a vial containing 3 mL of octanol mixed with 3 mL of 0.1 M phosphate buffer (pH 7.4). The vial was agitated on a vortex for 1 min and then spun down at 5000 rpm (1228g) for 10 min. After centrifugation, the two phases were separated. Aliquots of the octanol (1 mL) and the buffer (1 mL) layers were dispatched in wells and radioactivity counted in a gamma counter. The mean value of the LogD at pH 7.4 was calculated using the following equation: LogD7.4 = log10[(counts in octan-1-ol phase)/(counts in buffer phase)].  2.4 In vitro metabolic stability in mouse plasma  Radiotracer stability was determined by incubating 400 µL of balb/c mouse plasma (Innovative Research, Novi, MI) with 100 µL   aliquots (~ 0.1 mCi) of the radiolabeled peptides for 5, 15, 30, and 60 min at 37°C. At each time point, the assay was stopped by quenching the metabolic activity using 500µL of CH3CN, and the samples were spun down for 20 min (1228g). The supernatant was filtered through a syringe filter (0.2 µm) before it was subjected to HPLC. The radioactive metabolites in suspensions were measured using a semi-preparative HPLC system (Agilent) with the same conditions of elution as described for the preparation of the radiotracer they originate from. The peak areas of the radiotrace were used to calculate the purity of the radiotracer and the stability was obtained by comparing the purity at a certain time to the time zero purity.   28  2.5 Cellular model for hB1R stable expression  hB1R in vitro cellular assays and in vivo imaging were performed using human embryonic kidney cells (HEK293T Wild Type, Clontech Laboratories). Green Fluorescent Protein (GFP) transduction was performed using the Lenti-XTM Expression System (Clontech Laboratories) with the cloning vector pGIPz(TurboGFP). For a positive control, GFP+ HEK293T::WT were transfected using a lentiviral vector carrying the hB1R open reading frame constitutively expressed under super Cytomegalovirus (suCMV) promoter control, and a blasticidin (Bsd)-Red Fluorescent Protein (RFP) fusion marker under a Rous sarcoma virus (RSV) promoter. Transfection was performed using pre-made inducible lentiviral particles at 1×107 Inclusion Forming Units per mL (IFU/mL) (GenTarget, cat. no. LVP291). HEK293T::WT cells were used as a negative control for hB1R expression. Selection and expression of reporter genes were confirmed by fluorescence microscopy using a Nikon Eclipse TE2000 E confocal microscope.  2.6 Receptor binding assays  The affinity of the peptides for hB1R was measured by competitive binding assays on plasma membranes purified from Chinese hamster ovary (CHO) cells overexpressing the recombinant human hB1R receptor. Control kallidin (KD), H-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu-OH or (Leu9,des-Arg10)-KD (MW 998.19) was obtained from Bachem Americas Inc. (Torrance, CA; cat.no. H-2582). Radiochemical KD used was (Leu9, des-Arg10)KD-[3,4-prolyl-3,4-3H(N)] (American Radiolabeled Chemicals Inc., St. Louis, MO;  cat. no. ART1609, lot no. 111103, specific activity: 83.8 Ci/mmol, stock dilution 1.0 mCi/mL; or Perkin Elmer Inc., Boston, MA; cat.no. NET1096250UC, lot no. 1586889, specific activity: 76.0 Ci/mmol, stock 29  dilution: 1.0 mCi/mL). Briefly, 96-well Multi-Screen plates with glass fiber filters and Polyvinylidene difluoride (PVDF) supports (Millipore) were presoaked with 0.5% of cold polyethyleneimine (Sigma-Aldrich). After 30 min, wells were washed once with 50 mM of Tris-HCl, pH 7.4. The wells were loaded with an assay buffer containing 50 mM of Tris-HCl, pH 7.4, and 5 mM of MgCl2. Varying concentration of nonradioactive control [des-Arg10]KD (H-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-OH,  MW 1032.21, Cat. No. H-3122, Bachem Americas Inc., Torrance, CA) or of the peptides of interest were added in the presence of 4.8 nM of [3H]-(des-Arg10, Leu9)-KD. hB1R membranes were added to each well to a final protein concentration of 50 µg of membrane protein/well. The MultiScreen plate was incubated at 37oC for 15 min with gentle agitation at 300 rpm. The assay was stopped by aspirating the reaction solution through a PVDF membrane filter, followed by washing with ice-cold 50 mM Tris-HCl pH 7.4. The filter membranes were dried prior to adding scintillation liquid, and the activity in the plates was measured using a 1450 MicroBeta Counter (PerkinElmer, Shelton, CT). Data analysis was performed with GraphPad Prism 5, using a one-site competitive binding model. The inhibition constant Ki was calculated from the IC50 using the Cheng-Prusoff equation.  2.7 Fluorometric measurement of calcium release  A FLIPR® Calcium 6 Assay Kit (Molecular Devices, Sunnyvale, CA) was used according to the manufacturer’s protocol to monitor the increase in intracellular Ca2+ concentration upon binding of the 68Ga-labeled peptides to hB1R. 24h hours before the experiment, 5.104 HEK293TWT and HEK293T::hB1R cells were seeded in each well of a microplate (96-wells, clear bottom, black wall plates (Product no: 3603, Corning Inc., Corning, NY) to reach 90% confluence at the time of the assay. ATP and PBS were used as positive and negative controls for 30  intracellular calcium release, respectively. [des-Arg10]KD and [Leu9, des-Arg10]KD (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu-OH, MW 904.04, Cat. No. H-1965) from Bachem Americas Inc. (Torrance, CA) were used as the agonist and antagonist control ligands, respectively. 100 μL of loading buffer (calcium-sensitive dye in HBSS, 20 mM HEPES, pH 7.4, 100 μL) were added to each well and incubated for 30 min at 37 °C under 5% CO2. After the incubation, the cells were placed in a FlexStation® 3 Benchtop Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA). The baseline of the fluorescent signal was established for 20 seconds, then the vehicle (PBS) or the vehicle containing the cold peptide were added to the well to reach final concentrations of 0.5, 5 and 50 nM. The fluorescent signal was then monitored for 100 s. The Relative Fluorescence Intensity (RFI) was expressed as Relative Fluorescence Unit (RFU= Max RFI – Min RFI) to compare the calcium release induced by the radiotracer with [des-Arg10]KD.  2.8 Animal studies  Animal maintenance and experiments were conducted in accordance with the Canadian Council on Animal Care guidelines and approved by the University of British Columbia Animal Care Committee.   2.8.1 in vivo metabolic stability  For in vivo plasma stability studies, mice (n=3) were sedated with 2% isoflurane, and injected with ~ 15 MBq of 68Ga-labeled peptides via the tail vein. The mouse was left roaming then sedated with 2% isoflurane and euthanized by CO2 asphyxiation 5 min post-injection (p.i.). The blood was promptly drawn via cardiac puncture, and mixed with acetonitrile to reach a final 31  concentration of acetonitrile at ~ 10 – 15%. The mixture was centrifuged for 15 min at 7500g, and the supernatant was collected and analyzed with an Agilent radio-HPLC system consisting of a model 1260 Infinity pump, a model 1260 Infinity UV absorbance detector (set at 220 nm), a Bioscan NaI scintillation detector, and a semi-preparative column (Phenomenex C18, 5 µm, 250 x10 mm) eluted with 18/82 MeCN/PBS (pH 7.1) at a flow rate of 4.5 mL/min.  2.8.2 Biodistribution and preclinical imaging  Biodistribution and PET/CT imaging studies were performed using mice (n=4 to 10 per group) bearing hB1R positive and negative tumours. Wild-type HEK293T and HEK293T::hB1R xenografts were inoculated by subcutaneous injection of 1x106 cells on the left and right dorsal flank of each mouse. After 10 to 14 days of growth, the mice displayed 2 palpable tumours of 4 to 7 mm diameter. Mice were sedated with 2% isoflurane inhalation prior to the intravenous injection of the radiotracer in the tail vein. Mice used only for biodistribution studies were injected with 0.5-0.8 MBq of radiotracer, while the mice used for PET/CT imaging prior to biodistribution were injected with 5-8 MBq of radiotracer. Blocking experiments were performed by coinjection of 100 μg of the corresponding cold standard labelled with the native isotopes of Gallium and Fluorine (e.g. NatGa, NatF).  Mice used for biodistribution were allowed to recover and roam freely in their cages for 60 min. After full recovery for 60 min of uptake, the mice were anesthetized by isoflurane inhalation, followed by CO2 inhalation. Blood was promptly withdrawn, and the organs of interest were harvested, rinsed with saline, blotted dry and inserted into pre-weighed tubes. The radioactivity of the collected mouse tissues was counted using a Cobra II gamma counter (Packard), normalized to the injected dose using a standard curve and expressed as the percentage 32  of the injected dose per gram of tissue (%ID/g). After counting, the tubes were weighed again to deduce the weight of each tissue sample. Mice used for static PET imaging were allowed to recover and roam freely in their cages for 45 min. At this time, the mice were sedated with 2% isoflurane inhalation and positioned in the scanner. A baseline CT scan was obtained for localization and attenuation correction prior to the PET acquisition, using 60 kV x-rays at 500 μA and 3 sequential bed positions with 33% overlap over a 220 degree continuous rotation. A single static emission scan was acquired with the PET at 55 min for 10 min. During the acquisition, the mouse body temperature was maintained by the heated carbon-fiber bed. PET data were acquired in list mode, reconstructed using the 3d-OSEM-MAP algorithm with CT-based attenuation correction, and co-registered for alignment.  In some mice, dynamic PET scanning was performed to determine the time-activity course of the radiopharmaceuticals in the organs of interest. At that point, the mice were sedated with 2% isoflurane inhalation, placed in the scanner, and prior to PET acquisition, an attenuation correction CT scan was obtained as described above. The dynamic acquisition of 60 min was started at the time of intravenous injection of the radiolabeled peptides in the same manner as described above. The list mode data were rebinned into time intervals (12 x 10, 6 x 30, 5 x 60, 6 x 300, and 2 x 600 seconds) to obtain tissue time-activity curves. We noticed an overall high background, a higher renal accumulation and lower urinary excretion in these mice kept under prolonged isoflurane sedation. As a consequence, these animals were not used for biodistribution experiments or for the final analysis of tumour uptake by imaging at 60 min. The mice were euthanized following the PET acquisition, and the organs harvested for biodistribution.   33  2.9 Statistical analysis  The statistical analyses were performed using Microsoft Excel software and Prism 6 software (GraphPad). Biodistribution data were analyzed by two-way ANOVA, with the organs of interest as a factor, and the two different radiotracers as a second factor. Tukey’s multiple comparison test was used to compare the uptake in the tumours and organs between the different groups (e.g. 68Ga-Z01115 vs 68Ga-SH01078). P values were calculated using a Student’s t-test (unpaired, one-tailed, two-tailed), and an adjusted p value of less than 0.05 was considered statistically significant. The unpaired one tailed test was used to compare hB1R+ tumour uptake and hB1R+ tumour-to-background (blood, muscle and hB1R- tumor) ratios between the control and blocked mice, whereas a paired one tailed test was used to compare the uptake in hB1R+ and hB1R- tumours in the same mice. Comparison of uptake in hB1R- tumours and other tissues between control and blocked mice was performed using unpaired, two-tailed test.            34  Chapter 3: Importance of Metabolic Stability for in vivo Radioimaging of hB1R  3.1 Background   This study aims at evaluating radiolabeled KD analogs to visualize hB1R expression in vivo using PET/CT. Considering that hB1R is an inducible receptor, the experimental conditions may not trigger or maintain the expression of hB1R at the cell membrane. Therefore, the HEK293T Wild Type (HEK293TWT) cell line was chosen as a negative control for hB1R expression, and transformed to constitutively hB1R (HEK293T::hB1R) as a positive control. In the same manner as the inducible expression of hB1R, its endogenous ligand KD is locally generated and actively degraded to preserve its local action. In order to resist active enzymatic degradation, cleavage-sensitive amino acids were replaced with unnatural analogs to generate two metabolically stable radiolabeled KD analogs. The effect of these substitutions was compared to the effect of two different protease inhibitors co-administered with a natural KD analog. Three derivatives based on the [Leu9, desArg10]KD sequence were radiolabeled with 68Ga through the DOTA chelator. A linker was inserted between the chelator and the targeting motif in order to increase the hydrophilicity and maintain the binding affinity of the original unconjugated sequence. Ahx and dPEG2 were used as spacers of similar lengths according to their known overall innocuity and biocompatibility and compared in vitro with the endogenous hB1R ligand to determine the impact of the conjugation to the chelator and the amino acid substitutions on the overall hB1R binding ability. Ahx is a hydrophobic amino acid, while PEG2 is a hydrophilic linker, which can enhance the solubility of peptides. The natural hB1R antagonist 68Ga-P03083 (68Ga-DOTA-Ahx-[Leu9, desArg10]KD) was evaluated in vivo in the absence and presence of two 35  protease inhibitors. Phosphoramidon is a potent inhibitor of the peptidase neprilysin (neutral endopeptidase or NEP)111,112, and Enalaprilat is a powerful inhibitor of the angiotensin-converting enzyme (ACE) and aminopeptidase P (APP)113. The effect of protease inhibition on 68Ga-P03083 biodistribution was evaluated and compared to the unnatural analogs 68Ga-SH01078 (68Ga-DOTA-Ahx-[Hyp4, Cha6, Leu9, desArg10]KD) and 68Ga-P03034 (68Ga-DOTA-PEG2-[Hyp4, Cha6, Leu9, desArg10]KD) to assess the potential benefit of increasing the metabolic stability of the peptidic motif on hB1R visualisation by PET in vivo.  3.2 Materials and methods  3.2.1 Chemistry and radiochemistry  3.2.1.1 Synthesis of DOTA-Ahx-[Leu9, desArg10]kallidin:   HPLC purification of the crude peptide was performed with a gradient ranging from 85/15 A/B to 70/30 A/B in 30 min at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of DOTA-Ahx-[Leu9, desArg10]KD was 16.3 min, and the yield of the peptide synthesis was 22%. MS (EPI) calculated for C69H112N18O19 (M + 2H)2+ was 749.42; found 749.66.  3.2.1.2 Synthesis of DOTA-Ahx-[Hyp4, Cha6, Leu9, desArg10]kallidin:  HPLC purification of the crude peptide was performed under isocratic conditions with 80/20 A/B (pH 7.1) at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 36  0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of DOTA-Ahx-[Hyp4, Cha6, Leu9, desArg10]KD was 17.0 min, and the yield of the peptide synthesis was 47 %. MS (EPI) calculated for C70H120N18O22 (M + 2H)2+ 760.48, found 760.69.  3.2.1.3 Synthesis of DOTA-PEG2-[Hyp4, Cha6, Leu9, desArg10]kallidin:  HPLC purification of the crude peptide was performed with a gradient condition of 80/20 A/B to 77/23 A/B in 30 min at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of DOTA-PEG2-[Hyp4,Cha6, Leu9, desArg10]KD was 16.5 min, and the yield of the peptide synthesis was 42 %. MS (EPI) calculated for C70H120N18O22 (M + 2H)2+ is 783.44, found 783.57.  3.2.1.4 Synthesis of Ga-DOTA-Ahx-[Leu9, desArg10]kallidin:  For Ga-DOTA-Ahx-[Leu9,desArg10]KD (hereafter referred as P03083), the reaction mixture was purified by HPLC using a semi-preparative column eluted with 81/19 A/B (pH 7.1) at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of P03083) was 20.8 min, and the yield of the labelling reaction was 97%. MS (EPI) calculated for GaC69H109N18O19 (M + 2H)2+ is 782.37, found 782.66.     37  3.2.1.5. Synthesis of Ga-DOTA-Ahx-[Hyp4, Cha6, Leu9, desArg10]kallidin:  For Ga-DOTA-Ahx-[Hyp4,Cha6,Leu9,desArg10]KD (hereafter referred as SH01078), the reaction mixture was purified by HPLC using a semi-preparative column eluted with 77/23 A/B (pH 7.1)at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of SH01078 was 8.3 min, and the yield of the labelling reaction was 92%. MS (EPI) calculated for GaC70H117N18O22 (M + 2H)2+ is 793.43, found 793.66.  3.2.1.6 Synthesis of Ga-DOTA-PEG2-[Hyp4, Cha6, Leu9, desArg10]kallidin:  For Ga-DOTA-PEG2-[Hyp4,Cha6,Leu9,desArg10]KD (hereafter referred as P03034), the reaction mixture was purified by HPLC using a semi-preparative column eluted with 80/20 A/B (pH 7.1) at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of P03034 was 20.0 min, and the yield of the labelling reaction was 80%. MS (EPI) calculated for GaC70H117N18O22 (M + 2H)2+ is 816.39, found 816.53.  3.2.1.7 Synthesis of 68Ga-DOTA-Ahx-[Leu9, desArg10]kallidin:  68Ga-P03083 was synthesized using 50 µg of the unlabeled precursor, and purified by HPLC using a semi-preparative column eluted with 17/83 C/D (pH 7.1) at a flow rate of 4.5 mL/min. The HPLC solvents were C: CH3CN and D: PBS. Quality control was performed by 38  HPLC using an analytical column eluted with 16/84 CH3CN/PBS at a flow rate of 2 mL/min. The retention time of 68Ga-P03083 was 6.8 min.  3.2.1.8 Synthesis of 68Ga-DOTA-Ahx-[Hyp4, Cha6, Leu9, desArg10]kallidin:  68Ga-SH01078 was synthesized using 100 µg of the unlabeled precursor, and purified by HPLC using a semi-preparative column eluted with 18/82 C/D (pH 7.1) at a flow rate of 4.5 mL/min. The HPLC solvents were C: CH3CN and D: PBS. Quality control was performed by HPLC using an analytical column eluted with 19/81 CH3CN/PBS at a flow rate of 2 mL/min. The retention time of 68Ga-SH01078 was 6.1 min.  3.2.1.9 Synthesis of 68Ga-DOTA-PEG2-[Hyp4, Cha6, Leu9, desArg10]kallidin:  68Ga-P03034 was synthesized using 50 µg of the unlabeled precursor, and purified by HPLC under the same conditions as 68Ga-SH01078. Quality control was performed by HPLC using the analytical column eluted with 18/82 C/D at a flow rate of 2 mL/min. The HPLC solvents were C: CH3CN and D: PBS. The retention time of 68Ga-P03034 was 7.7 min.   3.2.2 Stability in mouse plasma  Radiotracer stability was assessed following the procedure described in Chapter 2: Materials and Methods (2.4). The metabolite suspension was loaded onto the HPLC column and eluted with 17/83 CH3CN/PBS (pH 7.1), 23% MeCN (0.1% TFA), and 23% MeCN (0.1% TFA) at a flow rate of 4.5 mL/minute, for 68Ga-P03083, 68Ga-SH01078, and 68Ga-P03034, respectively. 39  The retention times of 68Ga-P03083, 68Ga-SH01078, and 68Ga-P03034 were 16.6, 12.5, and 12.1 minutes, respectively.  3.2.3 LogD7.4 measurements  LogD7.4 measurements were performed according to the procedure described in Chapter 2: Material and Methods (2.3).  3.2.4 hB1R expression in established cell lines  HTR-8/SVneo cells were provided with courtesy of Pr. Charles H. Graham, School of Medicine, Department of Molecular Sciences, Queen’s University, Kingston, ON, Canada. MCF7, LNCaP and PC3 cells lines were purchased from ATCC.   3.2.5 Creation of hB1R expressing cells  hB1R expressing cells were generated according to the procedure described in the Chapter 2: Materials and Methods (2.5).  3.2.6 Preparation of cell membranes  The successful transduction of hB1R was measured by a saturation assay on HEK293T::GFP::hB1R cell membranes. Briefly, the cells were disrupted using a Dounce homogenizer in the presence of protease inhibitor cocktail and DNase1. Cell membranes were 40  isolated by sequential centrifugation. Protein concentration was determined using a Bradford assay. 50 μg of membrane protein per well was used for the saturation assay. Progressively higher concentrations of radioactive [3H]-[des-Arg10, Leu9]KD were used (range 0.05 – 20 nM), with and without the presence of excess competitor (30 μM Lys-(Des-Arg9)-Bradykinin, Bachem). GraphPad Prism 5 was used to calculate the binding affinity (Kd) and receptor concentration (Bmax), normalized to fmol/mg of protein.  3.2.7 Receptor-binding assays  The affinity of the peptides for hB1R was measured according to the procedure described in Chapter 2: Materials and Methods (2.6).  3.2.8 Biodistributions and preclinical imaging  Animal studies were performed according to the procedures described in Chapter 2: Materials and Methods (2.8.2).  3.2.9 Peptidase inhibition with phosphoramidon or enalaprilat  To determine the effects of peptidase inhibition on radiotracer uptake in target tissues, groups of mice injected with 68Ga-P03083 were coinjected with either 0.3 mg of phosphoramidon or 0.25 mg of enalaprilat in normal saline. Imaging and/or biodistribution was performed 55 to 60 minutes later, as described in Chapter 2: Materials and Methods (2.8).  41  3.2.10 Statistical analysis  Statistical analysis was performed as described in section 2.9 (Chapter 2: Materials and Methods 2.9). Tukey’s multiple comparison test was used to compare the uptake in the tumours and organs between the following groups: P03083 injected without peptidase inhibition, P03083 injected with enalaprilat, and P03083 injected with phosphoramidon, 68Ga-SH01078 and 68Ga-P03034. An adjusted P value of less than 0.05 was considered significant.  3.3 Results  3.3.1 Radiochemistry and plasma stability  The radiochemistry and plasma stability data are reported in Table 1. Because of the use of HPLC for purification, all the radiopeptides were obtained wit high-specific activities, suitable for use in mice for receptor imaging. 68Ga-SH01078, 68Ga-P03034, and 68Ga-P03083 were highly hydrophilic (LogD7.4 ≤ -2.69), 68Ga-P03083 being the most hydrophilic of the three. The removal of the C-terminal arginine followed by the exchange of the C-terminal D-Phe for a Leu in the des-Arg10 KD sequence did not affect the in vitro affinity of KD for hB1R. Unexpectedly, the addition of the DOTA chelator and the Ahx spacer at the N-terminus to obtain 68Ga-P03083 improved the affinity for h1BR by more than 3 fold, from 8.9 nM to 2.6 ± 0.7 nM. The replacement of the 4th and 6th amino acid, from proline and phenylalanine to hydroxyproline and cyclohexylalanine respectively, reduced the Ki of 68Ga-SH01078 for hB1R by more than 10 fold, from 2.6 ± 0.7 nM to 28 ± 4.9 nM. Exchange of the Ahx spacer for PEG2 slightly the affinity of 68Ga-P03034 for hB1R to 16 ± 1.9 nM. in vitro stability tests showed a relatively good stability of 42  all the peptides, with improvement by the use of unnatural amino acids for 68Ga-P03034 and 68Ga-SH01078.  Table 3.1: Radiolabeling, LogD7.4 and stability data of 68Ga-labeled [des-Arg10]KD derivativesa. aData are presented as mean ± SD.  3.3.2 hB1R expression model and affinity  The successful integration of the lentiviral vector carrying the hB1R gene was confirmed by the RFP emission detected by confocal microscopy (Figure 1). HEK293T Wild Type did not show any red fluorescence. Saturation assays were performed with the purified plasma membrane of these two cell lines using [3H]-(des-Arg10,Leu9)-KD and confirmed the correct localization and binding of hB1R in the HEK293T:hB1R cells. The maximal binding capacity (Bmax) was 451 ± 88 fmol/mg of protein for the HEK293T:hB1R cell membranes and 56 ± 48 fmol/mg of protein for the Wild Type HEK293T cell membranes (Figure 2). The binding affinities (Kd) of [3H]-[Leu9,des-Arg10]KD to these two cell membranes were 1.9 ± 0.5 and 1.6 ± 0.2 nM, respectively. The competitive assays performed with our tracers on hB1R expressing CHO cells plasma membranes showed a dose dependent  binding inhibition, with a corresponding constant (Ki) of 2.6 ± 0.7 nM for P03083, 27.8 ± 4.9 nM for SH01078 and 16.0 ± 1.9 nM for P03034. Tracers Overall charge Radiochemical yield (%, decay-corrected) Radiochemical purity (%) Specific activity (GBq/µmol, n≥3) LogD7.4 (n=3) Plasma stability (% intact, n=3) in vitro 5 min 15 min 30 min 60 min 68Ga-P03083 +1 70 ± 16 (n=3) > 99 185 ± 33 -2.83 ± 0.13 96 94 92 87 68Ga-P03034 +1 69 ± 8 (n=8) > 99 222 ± 37 -2.76 ± 0.11 99 97 94 91 68Ga-SH01078 +1 56 ± 20 (n=7) > 99 189 ± 59 -2.69 ± 0.25 99 99 99 99 43   Figure 3.1: Epifluorescence images of HEK283T::hB1R: RFP expression showing successful lentiviral transduction with an hB1R sequence carrying the RFP reporter gene (Right).   Figure 3.2: Saturation assay on HEK293T plasma membrane with the hB1R antagonist [desArg10, Leu9]KD. A: HEK293T:hB1R. B: HEK293T WT.  3.3.3 Cancer cell line screening  Plasma membranes from prostate (PC3, LNCaP) and breast (MCF7) cancer cell lines were purified and tested for hB1R ligand binding affinity using a competition assay. We used the HEK293TWT as a negative control, and HEK293T:hB1R as a reference for overexpression. The three cancer models LnCAP, PC3 and MCF7 membranes did not bind [3H-desArg10KD] 44  significantly. Cell membranes from the non-pathologic cell line HTR-8/SVneo cells bound [3H-desArg10KD] significantly (Figure 3.3).  Figure 3.3: Comparison of different common cancer cell lines purified plasma membrane and hB1R physiological expression control. Competition between [desArg10, Leu9KD] and [3H-desArg10KD]. MCF7: Breast cancer; PC3 and LNCaP: Prostate cancer; HTR-8/SVneo: Non-pathogenic hB1R+ control trophoblastic cell line.          45  3.3.4 Biodistribution and imaging  Tissue (%ID/g) 68Ga-P03083 68Ga-P03083 + EPa 68Ga-P03083 + PAb 68Ga-SH01078 68Ga-P03034 Control (n=6) Control (n=7) Control (n=4) Control (n=10) Blocked (n=6) Control (n=7) Blocked (n=5) Blood 0.12 ± 0.08 0.12 ± 0.05 0.10 ± 0.09 0.29 ± 0.08 0.25 ± 0.06 0.40 ± 0.28 0.31 ± 0.19 Plasma 0.53 ± 0.24 0.54 ± 0.25 0.39 ± 0.22 0.44 ± 0.13 0.37 ± 0.09 0.59 ± 0.37 0.48 ± 0.3 Fat 0.07 ± 0.03 0.09 ± 0.03 0.09 ± 0.04 0.07 ± 0.03 0.07 ± 0.04 0.06 ± 0.02 0.08 ± 0.08 Testes 0.11 ± 0.05 0.15 ± 0.05 0.11 ± 0.05 0.09 ± 0.02 0.12 ± 0.06 0.13 ± 0.09 0.12 ± 0.11 Large intestine 0.14 ± 0.05 0.39 ± 0.37 0.12 ± 0.03 0.12 ± 0.04 0.12 ± 0.03 0.11 ± 0.11 0.14 ± 0.02 Small intestine 0.27 ± 0.06 0.3 ± 0.16 0.20 ± 0.07 0.22 ± 0.12 0.09 ± 0.02 0.10 ± 0.02 0.16 ± 0.1 Spleen 0.17 ± 0.05 0.24 ± 0.11 0.17 ± 0.08 0.13 ± 0.03 0.11 ± 0.03 0.18 ± 0.17 0.11 ± 0.03 Liver 0.19 ± 0.06 0.19 ± 0.04 0.21 ± 0.10 0.14 ± 0.02 0.14 ± 0.04 0.17 ± 0.09 0.16 ± 0.06 Pancreas 0.08 ± 0.04 0.1 ± 0.03 0.07 ± 0.03 0.07 ± 0.02 0.07 ± 0.04 0.06 ± 0.06 0.06 ± 0.01 Adrenal glands 0.13 ± 0.12 0.15 ± 0.08 0.11 ± 0.01 0.10 ± 0.04 0.07 ± 0.01 0.12 ± 0.13 0.15 ± 0.17 Kidney 4.95 ± 0.86 6.44 ± 1.74 5.40 ± 0.81 3.66 ± 1.26 3.42 ± 0.62 4.28 ± 1.89 3.19 ± 0.72 Lungs 0.50 ± 0.33 0.71 ± 0.44 0.34 ± 0.13 0.27 ± 0.06 0.22 ± 0.05 0.5 ± 0.33 0.20 ± 0.05 Heart 0.13 ± 0.03 0.18 ± 0.05 0.13 ± 0.05 0.12 ± 0.03 0.08 ± 0.01 0.13 ± 0.07 0.09 ± 0.02 hB1R- tumour 0.20 ± 0.08 0.26 ± 0.09 0.30 ± 0.04 0.34 ± 0.15 0.22 ± 0.09 0.31 ± 0.12 0.28 ± 0.1 hB1R+ tumour 0.79 ± 0.22 0.85 ± 0.16 2.77 ± 1.22 2.01 ± 0.46 0.42 ± 0.04 2.36 ± 0.64 0.43 ± 0.08 Skin 0.24 ± 0.20 0.37 ± 0.29 0.19 ± 0.10 0.24 ± 0.11 0.15 ± 0.04 0.31 ± 0.17 0.15 ± 0.08 Muscle 0.11 ± 0.04 0.11 ± 0.05 0.08 ± 0.03 0.08 ± 0.03 0.06 ± 0.02 0.12 ± 0.11 0.06 ± 0.02 Bone 0.15 ± 0.05 0.12 ± 0.05 0.08 ± 0.03 0.09 ± 0.01 0.08 ± 0.02 0.08 ± 0.09 0.09 ± 0.03 Brain 0.02 ± 0.01 0.02 ± 0.00 0.01 ± 0.00 0.02 ± 0.01 0.01  0.00 0.01 ± 0.01 0.02 ± 0.02 aEP = Enalaprilat, bPA = phosphoramidon Table 3.2: Biodistribution of 68Ga-P03083 in mice 60 min p.i. with cold labeled standard (blocking), with peptidase inhibitors and without, compared to 68Ga-SH01078 and 68Ga-P03034.  46   Figure 3.4: Effect of the metabolic stability on the 68Ga labelled kinin analogs biodistribution 60 min post injection. Green arrow: negative tumour; Red arrow: hB1R+ tumour. From left to right: Natural: 68Ga-P03083; 68Ga-P03083 in coinjection with enalaprilat (EP); 68Ga-P03083 in combination with phosphoramidon; Synthetic: 68Ga-SH01078 (n=10); 68Ga-SH01078 in coinjection with 100 µg of cold labelled standard (n=6).  The results of the biodistribution experiments are presented in the Table 3.2. All the 68Ga-labeled tracers were cleared exclusively through the renal pathway and yielded a strong accumulation of the activity in the kidneys and the bladder urine. No significant amount of activity was detected in other organs, and PET images confirmed the similar minimal background of these tracers (Figure 4). 60 min after injection, 68Ga-P03083 uptake in the hB1R+ tumour was 4 times higher than in the negative tumour. However, the contrast was not suitable for proper visualization on the PET acquisition. Coinjection with enalaprilat increased only the kidney uptake of the 68Ga-P03083. Coinjection with phosphoramidon also increased the kidney accumulation, and increased significantly (P<0.0001) the radiotracer uptake in the hB1R+ tumour, reaching 2.77 ± 1.22 %ID/g, equivalent to 9 times the negative tumour uptake, and 3.5 times higher than in hB1R+ tumour uptake without peptidase inhibitor. The unnatural analogs 47  68Ga-SH01078 and 68Ga-P03034 with no peptidase inhibitors gave similar results as the natural analog with phosphoramidon, and accumulated significantly more (>2%ID/g) in the hB1R+ tumour than in the negative tumour. The average uptake ratios of hB1R+ tumour to plasma, muscle and negative tumour were 7.5 ± 1.5, 35.7 ± 2.3, 9.15 ± 3.1 when 68Ga-P03083 was coinjected with phosphoramidon. For 68Ga-SH01078 and 68Ga-P03034, those ratios were 7.8 ± 2.2, 30.2 ± 7.4, 7.9 ± 2.3 and 5.6 ± 3.4, 23.3 ± 14.3, and 6.3 ± 4.1, respectively. No significant uptake in the hB1R- tumour was measured during unblocked and blocked studies, while the blocking experiments performed with 68Ga-SH01078 and 68Ga-P03034 showed inhibition of uptake in hB1R+ tumour. The time activity study showed early and stable accumulation of 68Ga-P03034 in the hB1R expressing tumour, followed by the progressive clearance of the activity from non-target organs. The kidney activity was not affected by the blocking, remaining superior to 3%ID/g for the 60 min of the experiment with all the tracers tested.  3.4 Discussion  While hB1R expression is reported in many tumour samples from patients, none of the cancer cell lines tested in vitro bound hB1R ligands, supporting the fact that hB1R overexpression in cancers depends on a response from the host surrounding tissues to the tumour development and not on the acquisition of a mutation in genes related to hB1R expression and signaling pathway. The immortalized human trophoblastic HTR8-SVneo cells were able to bind [3H-desArg10KD] and could be used as a physiological model of hB1R expression42. The inflammatory cytokine IL-1β and desArg10-KD have been shown to induce hB1R expression in competent cells in vitro5, 7, and could be used on cancer cell lines in a second phase of screening. 48  As a consequence, we created an artificial hB1R expression model by permanently transfecting hB1R- HEK293T cells with a lentiviral vector expressing constitutively hB1R. The lack of expression of hB1R by the HEK293TWT and the constitutive expression of hB1R in the HEK293T::hB1R confirmed that these cells could be used as models for hB1R expression and binding in vitro and in vivo. The labelling motif DOTA-Ahx significantly improved the affinity of the KD amino acid sequence for h1BR. However, the use of unnatural amino acids decreased the affinity of 68Ga-P03034 and 68Ga-SH01078 for hB1R in vitro. However, their metabolic stability, as assessed by an in vitro plasma stability assay, showed some improvement with the unnatural amino acid substitutions. Considering the short half-life of kinins, we observed relatively good in vitro stability after 60 min of incubation, even with the natural KD sequence. This suggests that the peptidases regulating the human kinins activity are not reflected in this assay, making it less suitable to assess in vivo stability. The PEG2 linker improved the affinity of 68Ga-P03034 for hB1R in comparison with 68Ga-SH01078, confirming that each modification can impact receptor binding and should be investigated.  The fast renal clearance of the three tracers corresponded to the expected excretion pathway of small hydrophilic peptides, filtered through the glomerulus, and efficiently reabsorbed in the proximal tubule by endocytosis through various receptors, including megalin and cubilin114.  However, 68Ga-P03083 with phosphoramidon, 68Ga-P03034 and 68Ga-SH01078 were able to achieve high-contrast images, with low background activity and specific hB1R mediated tumour uptake, demonstrating the feasibility of imaging hB1R in vivo by PET/CT with metabolically stable radiopeptides. While their absolute tumour uptake did not exceed 3%ID/g, the improvement of tumour accumulation of the three radiopeptides correlated with the expected 49  resistance to peptidase activity, thus increasing the level of intact probes in circulation, as previously observed with DOTA labelled gastrin analogs coinjected with phosphoramidon115. The similar background achieved with P03083 with or without protease inhibition demonstrate that the degradation of our probes doesn’t affect the background accumulation. 68Ga-P03083, in the absence of peptidase inhibitors, was not suitable for imaging hB1R expressing tumours. This confirmed the presence of additional kinin degrading enzymes in vivo compared to in vitro.  Despite low affinities for hB1R in vitro, 68Ga-SH01078 and 68Ga-P03034 efficiently visualized hB1R tumours in vivo (>2%ID/g) and demonstrate that the improvement of stability can counterbalance the loss of affinity. While 68Ga-SH01078, 68Ga-P03034 and 68Ga-P03083 yielded similar levels of radioactivity in the kidneys, coinjection of 68Ga-P03083 with the protease inhibitors increased the renal accumulation of radioactivity. It is likely that the renal radioactivity is caused by tubular reabosorption of radiolabeled peptide or peptide fragments rather than free 68Ga or 68Ga-DOTA. Tubular reabsorption and retention of radiolabeled peptides/peptide fragments by the megalin and tubulin complexes has been reported with other peptides116. These two multiligand transporters are dedicated to amino acids and peptides reabsorption and bind equally active peptides or metabolites independently of their sequence. Thus, the competition for reabsorption is not a specific mechanism, and can be inhibited by excess amounts of non-radioactive peptides or peptidic fragments. As an example, in the case of two identical peptides being 10% stable (A) or 90% stable (B) in vivo 5 min post-injection, the injection of 10 units of A generates 9 non-radioactive peptides while the injection of 10 units of B generates 1 non-radioactive peptide. In the case of coadministration of 100 times more cold standard (1000 units total), this difference translates in 900 cold peptides units from the A compound versus 100 from the B compound. In addition, this example would correspond to the consequence of a single cleavage of the peptidic sequence. Considering the numerous cleavage 50  sites and dedicated kininases, multiple cleavage of the same sequence is likely to be occurring, which amplifies the amount of non-radioactive peptides binding the kidney endocytic transporters. As a consequence, it is possible that the in vivo degradation of P03034 generates sufficient quantities of non-radioactive small peptide fragments to compete with the reabsorption process, leading to lower renal accumulation of radioactivity. For stable peptides, presumably a greater proportion of intact radiolabeled peptides find their way to the renal tubules, where they accumulate in the kidneys31,117.  The natural and synthetic peptidic analogs tolerated coordination with the chelate with acceptable deterioration of their affinity for hB1R in vitro. The substitutions with unnatural amino acids mimicked the effect of the protease inhibitor PA on natural amino acid sequence degradation, providing sufficient stability to visualize the hB1R+ tumour. The low background and the quick renal excretion remained similar throughout the different experimental conditions. We confirmed using an artificial hB1R expression model that stable KD analogs can be engineered and used as hB1R targeting agents in vivo.  3.5 Conclusion  We synthesized 68Ga-labeled peptidic analogs of KD in order to visualize hB1R in vivo by PET/CT. The replacement of cleavage sensitive amino acids by synthetic analogs provided sufficient metabolic stability to image hB1R in expressing tumours. The readily available 68Ga radioisotope was quickly coordinated with the DOTA-labeled precursors, making this approach promising for clinical applications in human subjects.   51  Chapter 4: Effect of Linkers on PET/CT Imaging of hB1R With 68Ga labeled [des-Arg10] Kallidin Derivatives Tumour Uptake and Biodistribution  4.1 Background  The successful imaging of the hB1R in vivo by Positron Emission Tomography (PET) using radiolabeled KD analogs requires metabolic stability in vivo. We showed previously that amino acid substitutions and addition of DOTA-radiometal to the [Leu9, des-Arg10]KD sequence reduced the overall binding affinity for hB1R compared to the natural ligand. However, these modifications improved the resistance of the peptides to peptidase degradation sufficiently to achieve hB1R visualization using PET. In a second set of experiments, we evaluated other 68Ga-DOTA-PEG2 labelled analogs of [Leu9- desArg10]KD using various unnatural amino acids, and demonstrated that the new derivatives had significantly improved in vivo tumour uptake. In this chapter, we studied the effects of the linker motif inserted between the targeting moiety and the radiolabeling moiety on hB1R targeting properties of [Leu9, desArg10]KD analogs in vitro and in vivo. The previously described 68Ga-SH01078 and 68Ga-P03034 (Ahx and dPEG2 linker, respectively) were compared to 68Ga-P04115 (Glycine repeat or Gly2) and 68Ga-P04168 (4-amino-(1-carboxymethyl) piperidine or Pip), synthesized using the same 68Ga-DOTA-linker-[desArg10, Hyp4, Cha6, Leu9]KD construct. Besides the fact that these linkers were similar in lengths, the hydrophobic Gly2 was chosen as a highly flexible and commonly used dual amino-acid linker. The Pip linker was chosen for its positive charge and the reported known benefits of a cationic linker on tumour accumulation of radiopharmaceuticals targeting another G-coupled receptor, the gastrin releasing peptide receptor (GRPR)118,119,120,121,122,123,124.  52  4.2 Materials and methods  4.2.1 Chemistry and radiochemistry  4.2.1.1 Synthesis of DOTA-Gly-Gly-[Hyp4, Cha6, Leu9, des-Arg10]kallidin.   HPLC purification of the crude peptide was performed with 80/20 A/B at a flow rate of 4.5 mL/minute. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of DOTA-Gly-Gly-[Hyp4, Cha6, Leu9, des-Arg10]KD was 16.7 min, and the yield of the peptide synthesis was 26%. MALDI-MS: calculated [M+H]+ for DOTA-Gly-Gly-[Hyp4, Cha6, Leu9, des-Arg10]KD C67H114N19O21 m:z is 1520.8, found 1520.9. ESI-MS: found [M+H]+ m/z 1521.0.  4.2.1.2 Synthesis of DOTA-Pip-[Hyp4, Cha6, Leu9, des-Arg10]kallidin.   The HPLC purification of the crude peptide was performed with 81/19 A/B at a flow rate of 4.5 mL/minute. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of DOTA-Pip-[Hyp4, Cha6, Leu9, desArg10]KD was 13.0 min, and the yield of the peptide synthesis was 26%. MALDI-MS: calculated [M+H]+ for DOTA-Pip-[Hyp4, Cha6, Leu9, desArg1 0] KD C70H120N19O20 m/z is 1546.9, found m/z 1546.9. ESI-MS: found [M+H]+ m/z 1546.4.    53  4.2.1.3 Synthesis of Ga-DOTA-Gly-Gly-[Hyp4, Cha6, Leu9, des-Arg10]kallidin (P04115).   For Ga-DOTA-Gly-Gly-[Hyp4, Cha6, Leu9, des-Arg10]KD (hereafter referred as P04115), the reaction mixture was purified by HPLC using a semi-preparative column eluted with 80/20 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of P04115 was 16.7 min, and the yield of the labelling reaction was 88%. MALDI-MS: calculated [M+H]+ for GaC67H111GaN19O21 m/z was 1586.7, found m/z 1586.8. ESI-MS: found [M+H]+ 1586.8.  4.2.1.4 Synthesis of Ga-DOTA-Pip-[Hyp4, Cha6, Leu9, des-Arg10]kallidin (P04168).   For Ga-DOTA-Pip-[Hyp4, Cha6,L eu9, des-Arg10]KD (hereafter referred as P04168), the reaction mixture was purified by HPLC using a semi-preparative column eluted with 80/20 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of P04168 was 15.3 min, and the yield of the labelling reaction was 89%. MALDI-MS: calculated [M+H]+ for GaC70H117GaN19O20 m/z was 1612.8, found m/z 1612.8. ESI-MS: found [M+H]+ 1613.1.  4.2.1.5 Synthesis of 68Ga-DOTA-Gly-Gly-[Hyp4, Cha6, Leu9, des-Arg10]kallidin (68Ga-P04115).    68Ga-P04115 was synthesized using 100 μg of the unlabeled precursor, and purified by HPLC using a semi preparative column eluted with 18/82 C/D (pH 7.1) at a flow rate of 4.5 54  mL/minute. The HPLC solvents were C: CH3CN and D: PBS. The retention time of 68Ga-P04115 was 15.7 min.  4.2.1.6 Synthesis of 68Ga-DOTA-Pip-[Hyp4, Cha6, Leu9, des-Arg10]kallidin (68Ga-P04168).   68Ga-P04168 was synthesized using 70 μg of the unlabeled precursor, and purified by HPLC using a semi preparative column eluted with 18/82 C/D (pH 7.1) at a flow rate of 4.5 mL/minute. The HPLC solvents were C: CH3CN and D: PBS. The retention time of 68Ga- P04168 was 16.7 min.  4.2.2 LogD7.4 measurements   LogD7.4 measurements were performed according to the procedure described in Chapter 2: Material and Methods (2.3).  4.2.3 Stability in mouse plasma in vitro and in vivo   Metabolic stability in mouse plasma was measured according to the procedure described in Chapter 2: Material and Methods (2.4; in vitro) and (2.8.1; in vivo).  4.2.4 in vitro competition assays   These assays were performed according to the procedure described in Chapter 2: Material and Methods (2.6). 55  4.2.5 Fluorometric measurement of calcium release  These assays were performed according to the procedure described in Chapter 2: Material and Methods (2.7).  4.2.6 Biodistribution and PET/CT imaging  Preclinical imaging was performed according to the procedure described in Chapter 2: Material and Methods (2.8.2).  4.2.7 Statistical analysis  Statistical analyses were performed according to the procedure described in Chapter 2: Material and Methods (2.9).  4.3 Results  4.3.1 Chemistry and radiochemistry   The radiochemistry and plasma stability data are reported in Table 4.1. The DOTA-labeled peptidic precursors of P04115 and P04168 were obtained with a 26% yield synthesis, while the yields of the labeling with the non-radioactive gallium were 88 and 89% for Ga-P04115 and Ga-P04168, respectively. After HPLC purification, the radiolabeled compounds were obtained with good yields (≥55%), purities (≥99%) and specific activities (≥111 GBq/µmol) that 56  were suitable for PET/CT imaging. 68Ga-P04115, 68Ga-P04168, 68Ga-P03034 and 68Ga-SH01078 were highly hydrophilic (-2.99 ≤ LogD7.4  ≤ -2.69). Table 4.1: Radiolabeling, LogD7.4 and in vitro plasma stability data of 68Ga-labeled [des-Arg10]KD derivativesa. aData are presented as mean ± SD. bThe data for 68Ga-SH01078 were reported previously   4.3.2 in vitro/vivo plasma stability  The stability of these tracers in mouse plasma was evaluated after 60 min in vitro and 5 min in vivo. The four 68Ga-labeled analogs were stable in vitro, ranging from 84% of 68Ga-P04115 to 99% 68Ga-SH01078 remaining intact after 60 min. in vivo experiments showed the low stability of the peptides after intravenous injection, ranging from 8, 9 and 11% of 68Ga-P03034, 68Ga-SH01078 and 68Ga-P04115 respectively, to 17% of 68Ga-P04168 remaining intact after 5 min (Table 1).  4.3.3 Binding affinity and hB1R signaling modulation  Competition assays confirmed that the four different NatGa-labeled peptides were able to efficiently the binding of [3H]-[des-Arg10]KD to the hB1R expressed on CHO membranes (Figure 4.2). By competitive binding assays, Ki values of 11.42 ± 2.51, 27.81 ± 4.93, 15.98 ± Tracers Overall charge Radiochemical yield (%, decay-corrected) Radiochemical purity (%) Specific activity (GBq/µmol, n≥3) LogD7.4 (n=3) Plasma stability (% intact, n=3) in vitro in vivo 5 min 15 min 30 min 60 min 5 min 68Ga-SH01078b + 1 56 ± 20 (n=4) > 99 189 ± 59 -2.69 ± 0.25 99 99 99 99 9 ± 2 68Ga-P03034b + 1 69 ± 8 (n=7) > 99 222 ± 37 -2.76 ± 0.11 99 97 94 91 8 ± 2 68Ga-P04115 + 1 55 ± 12 (n=9) > 99 130 ± 67 -2.99 ± 0.22 92 89 87 84 11 ± 3 68Ga-P04168 + 2 58 ± 14 (n=9) > 99 111 ± 59 -2.82 ± 0.08 98 97 94 88 17 ± 4 57  1.94 and 3.56 ± 0.22 nM were measured for P04115, SH01078, P03034 and P04168 respectively. This result suggests a potential effect of peptide charge in hB1R binding in vitro (Table 2). Table 4.2: Amino acid sequences and hB1R binding affinities of bradykinin and related peptides. Peptide Radiolabel complex Linker hB1R binding domain Ki (nM) (mean ± SD) Ref Bradykinin    Arg Pro Pro Gly Phe Ser Pro Phe Arg 5.7a 125 Kallidin (KD)    Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 7.4a 125 1 2 3 4 5 6 7 8 9 10   [des-Arg10]KD   Lys Arg Pro Pro Gly Phe Ser Pro Phe  9.7a 125 [Leu9, des-Arg10]KD   Lys Arg Pro Pro Gly Phe Ser Pro Leu  8.9b 125 P03083 Ga-DOTA Ahx Lys Arg Pro Pro Gly Phe Ser Pro Leu  2.6 ± 0.7 - SH01078 Ga-DOTA Ahx Lys Arg Pro Hyp Gly Cha Ser Pro Leu  27.8 ± 4.9 - P03034 Ga-DOTA dPEG2 Lys Arg Pro Hyp Gly Cha Ser Pro Leu  16.0 ± 1.9 - P04115 Ga-DOTA Gly-Gly Lys Arg Pro Hyp Gly Cha Ser Pro Leu  11.4 ± 2.5 - P04168 Ga-DOTA Pip Lys Arg Pro Hyp Gly Cha Ser Pro Leu  3.6 ± 0.2 - apEC50. bpIC50.    Figure 4.1: Representative displacement curves of [3H]-[Leu9, des-Arg10]KD by SH01078, P03034, P04115 and P04168.  The endogenous hB1R agonist [3H]-[des-Arg10]KD triggered in the HEK293T::hB1R cells a dose dependent release of calcium into the intracellular compartment at 5 nM (145 ± 20 58  RFU) and 50 nM (178 ± 25 RFU). The corresponding natural antagonist [3H]-[Leu9, des-Arg10]KD  triggered a moderate calcium release at 50 nM only (64 ± 34 RFU). Up to 5 nM, none of the four 68Ga-labeled peptides were able to elicit a significant calcium release upon binding to hB1R on HEK293T::hB1R in vitro. Similarly to the antagonist control, 68Ga- P03034 and 68Ga-P04168 triggered a slight release of calcium at 50 nM (44 ± 8 and 24 ± 16 RFU, respectively), while 68Ga-P04115 and 68Ga-SH01078 did not (Figure 3).  Figure 4.2: Calcium release in HEK293T::hB1R cells induced by hB1R-targeting peptides: (A) [des-Arg10]KD, (B) [Leu9, des-Arg10]KD, (C) SH01078, (D) P03034, (E) P04115, and (F) P04168. Data are presented as mean ± SD (n = 3).       59  4.3.4 Biodistribution and PET/CT imaging Table 4.3: Biodistribution and uptake ratios of 68Ga-labeled [des-Arg10]KD derivatives in tumour-bearing micea. Tissue (%ID/g) 68Ga-SH01078 68Ga-P03034 68Ga-P04115 68Ga-P04168 Control (n = 10) Blocked (n = 6) Control (n = 7) Blocked (n = 5) Control (n = 8) Blocked (n = 4) Control (n = 12) Blocked (n = 5) Blood 0.29 ± 0.08 0.25 ± 0.06 0.4 ± 0.28 0.31 ± 0.19 0.39 ± 0.17 0.35 ± 0.11 0.3 ± 0.13 0.22 ± 0.06 Fat 0.07 ± 0.03 0.07 ± 0.04 0.06 ± 0.02 0.08 ± 0.08 0.07 ± 0.03 0.08 ± 0.02 0.06 ± 0.03 0.04 ± 0.01 Testes 0.09 ± 0.02 0.12 ± 0.06 0.13 ± 0.09 0.12 ± 0.11 0.14 ± 0.06 0.21 ± 0.20 0.09 ± 0.04 0.08 ± 0.03 Large intestine 0.12 ± 0.04 0.12 ± 0.03 0.11 ± 0.11 0.14 ± 0.02 0.19 ± 0.15 0.15 ± 0.09 0.11 ± 0.04 0.07 ± 0.02 Small intestine 0.22 ± 0.12 0.09 ± 0.02 0.1 ± 0.02 0.16 ± 0.1 0.26 ± 0.21 0.17 ± 0.08 0.22 ± 0.13 0.14 ± 0.08 Spleen 0.13 ± 0.03 0.11 ± 0.03 0.18 ± 0.17 0.11 ± 0.03 0.24 ± 0.13 0.20 ± 0.06 0.14 ± 0.05 0.21 ± 0.1 Liver 0.14 ± 0.02 0.14 ± 0.04 0.17 ± 0.09 0.16 ± 0.06 0.14 ± 0.02 0.15 ± 0.05 0.11 ± 0.02 0.11 ± 0.02 Pancreas 0.07 ± 0.02 0.07 ± 0.04 0.06 ± 0.06 0.06 ± 0.01 0.09 ± 0.01 0.08 ± 0.02 0.07 ± 0.02 0.05 ± 0.01 Adrenal glands 0.10 ± 0.04 0.07 ± 0.01 0.12 ± 0.13 0.15 ± 0.17 0.23 ± 0.20 0.18 ± 0.10 0.08 ± 0.06 0.07 ± 0.02 Kidney 3.66 ± 1.26 3.42 ± 0.62 4.28 ± 1.89 3.19 ± 0.72 4.02 ± 2.40 3.06 ± 0.92 4.09 ± 1.0 3.53 ± 0.29 Lungs 0.27 ± 0.06 0.22 ± 0.05 0.5 ± 0.33 0.20 ± 0.05 0.30 ± 0.08 0.27 ± 0.09 0.26 ± 0.06 0.26 ± 0.04 Heart 0.12 ± 0.03 0.08 ± 0.01 0.13 ± 0.07 0.09 ± 0.02 0.15 ± 0.06 0.14 ± 0.04 0.12 ± 0.04 0.09 ± 0.03 hB1R- tumour 0.34 ± 0.15 0.22 ± 0.09 0.31 ± 0.12 0.28 ± 0.01 0.28 ± 0.10 0.46 ± 0.02** 0.31 ± 0.14 0.15 ± 0.03 hB1R+ tumour 2.01 ± 0.46 0.42 ± 0.04*** 2.36 ± 0.64 0.43 ± 0.08*** 1.96 ± 0.83 0.48 ± 0.07** 4.24 ± 1.18 0.63 ± 0.15*** Muscle 0.08 ± 0.03 0.06 ± 0.02 0.12 ± 0.11 0.06 ± 0.02 0.09 ± 0.01 0.14 ± 0.12 0.06 ± 0.01 0.05 ± 0.01 Bone 0.09 ± 0.01 0.08 ± 0.02 0.08 ± 0.09 0.09 ± 0.03 0.12 ± 0.05 0.20 ± 0.12 0.09 ± 0.04 0.08 ± 0.03 Brain 0.02 ± 0.01 0.01 ± 0.00 0.01 ± 0.01 0.02 ± 0.02 0.02 ± 0.01 0.02 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 B1R+T:B1R-T 6.7 ± 2.5 2.2 ± 0.7** 7.4 ± 3.2 1.7 ± 0.8** 7.6 ± 3.9 1.1 ± 0.2** 17.8 ± 9.5 4.4 ± 1.7** B1R+T:Blood 7.5 ± 2.3 1.7 ± 0.4*** 7.1 ± 1.6 1.7 ± 0.6** 6.4 ± 3.8 1.5 ± 0.4* 15.9 ± 6.8 3.0 ± 1.2** B1R+T:Muscle 27.7 ± 8.7 7.4 ± 2.1*** 28.0 ± 9.7 8.3 ± 4.0* 26.1 ± 8.9 5.4 ± 3.6** 78.1 ± 28.5 12 ± 1.6*** B1R+T:Liver 15.1 ± 4.7 3.2 ± 0.8*** 14.9 ± 5.6 2.9 ± 1.0** 14.9 ± 5.2 3.6 ± 1.4** 42.9 ± 14.2 5.9 ± 2.4*** B1R+T:Kidney 0.6 ± 0.2 0.13 ± 0.0*** 0.6 ± 0.2 0.1 ± 0.0** 0.7 ± 0.4 0.2 ± 0.1* 1.1 ± 0.5 0.2 ± 0.0** aStudies were performed at 1 h p.i. with (blocked) or without (control) co-injection of non-radioactive standard. Data are displayed as mean ± SD, and significance of differences between control and blocked groups: *p < 0.05; **p < 0.01; ***p < 0.001.  The biodistribution data of these four 68Ga-labeled [des-Arg10]KD derivatives in tumour-bearing mice are summarized in Table 4.3. All the tracers showed similar low background uptake values in non-target organs 1h after injection (<1%ID/g), with the exception of the kidneys and the hB1R expressing tumour. The average uptake in the hB1R+ tumour was 1. 96 ± 0.83, 2.01 ± 0.46, 2.36 ± 0.64 and 4.24 ± 1.18 % ID/g of organ for 68Ga-P04115, 68Ga-SH01078, 68Ga-P03034 and 68Ga-P04168 respectively, while their average negative tumour uptake remained inferior to 0.46 %ID/g. The average kidney uptake values achieved by 68Ga-P04115 and 68Ga-P04168 (4.02 ± 2.4 and 4.09 ± 1.0 %ID/g, respectively) were in a similar range as those achieved by 68Ga-P03034 and 68Ga-SH01078 (4.28 ± 1.89 and 3.66 ± 1.26, respectively). As previously observed 60  with 68Ga-SH01078 and 68Ga-P03034, the blocking experiments with 68Ga-P04168 and 68Ga-P04115 significantly decreased the uptake in hB1R+ tumours, leading to similar uptake values as those of non-target organs (0.42 ± 0.04, 0.43 ± 0.08, 0.63 ± 0.15 and 0.48 ± 0.07 %ID/g, respectively). Representative PET/CT images obtained 1 h p.i. are shown in Figure 4.3. The minimal background on PET images was consistent with the biodistribution data for the four 68Ga-labeled analogs tested. Consistently, the activity concentrated at the hB1R expressing tumour, the kidneys and the bladder. The blocked condition with the cold standards reduced the uptake in the hB1R+ tumour but not the signal to the kidneys and the bladder only.  Figure 4.3: Maximum intensity projection (MIP) PET/CT images of 68Ga-labeled [des-Arg10]KD derivatives at 1-h p.i. in mice bearing both hB1R+ (red arrows) and hB1R- (yellow arrows) tumours without (top row) or with (bottom row) co-injection of the non-radioactive standard. 61  4.4 Discussion  The relatively higher average specific activity of 68Ga-SH01078 and 68Ga- P03034 (189−222 GBq/nmol) in comparison with 68Ga-P04115 and 68Ga-P04168 (130−111 GBq/nmol) possibly reflected the diminution of performance of the 68Ge−68Ga generator used for this study. Most of the work with 68Ga-SH01078 and 68Ga-P03034 was performed shortly after receiving the 68Ge/68Ga generator, whereas 68Ga-P04115 and 68Ga-P04168 were prepared using 68Ga eluted from the same generator at a much later time point, resulting in a relatively lower specific activity. Due to the fact that these four tracers shared the same receptor binding domain, their logD7.4 values were consistent with the predicted LogD7.4 values of these linkers obtained from ACS SciFinder: Ahx, −2.47; Pip, −2.57; dPEG2, −3.66; Gly-Gly, −4.97. Although our previous observations demonstrate the limited predictive values of these assays, the four tracers achieved high stabilities in vitro. In vivo stability values achieved by these four tracers were also all different, but mainly contrasted from the in vitro ones by being drastically lower (≤17%), and this only 5 min after circulation in vivo. These results echo the ones from the previous chapter by confirming that the difference in stability observed in vitro reflect an actual difference in stability, but cannot be extrapolated directly to in vivo stability. The four tracers bound hB1R in vitro with an affinity in the nanomolar range, confirming the fact that these [des-Arg10]KD derivatives tolerate various N-terminal modifications88,101,126,127. 68Ga-P04168 gave the highest binding affinity for hB1R, which suggests that the addition of a positive charge is beneficial to hB1R binding in vitro in this system. More generally, this also confirms that in vitro, apparent differences in stability have no impact on the inhibition constant for hB1R. Calcium release assays were performed to assess the biological activity of the four radiopeptides, and potential changes in the agonist/antagonist profile following their 62  modifications108. The natural hB1R agonist [des-Arg10]KD and antagonist [Leu9, des-Arg10]KD did not trigger calcium release in HEK293WT cells and confirmed the absence of hB1R in these cells. In contrast, the endogenous agonist elicited a significant calcium release at 5 and 50 nM in HEK::hB1R, while the natural antagonist did not, confirming the expression of a functional hB1R in these cells. However, at a 50 nM concentration, the natural antagonist [Leu9, des-Arg10]KD triggered a moderate calcium release suggesting a partial agonist effect at high doses. 68Ga-P04115 and 68Ga-SH01078 behaved as pure antagonists toward hB1R at both concentrations tested. 68Ga-P04168 and 68Ga-P03034 behaved as partial agonists by triggering a slightly higher signal at 50nM, corresponding to 15-36% of the calcium mobilization triggered by [des-Arg10]KD at the same concentration, and suggest that higher affinity tracers are more prone to trigger hB1R signaling at high concentrations. The biodistribution data and the PET/CT images acquired with the four 68Ga-labeled tracers confirmed that there was significant uptake in the hB1R expressing tumour and low uptake in non-target organs with these derivatives. The four tracers were cleared exclusively through the renal pathway, and accumulated in the kidneys. The superior uptake of 68Ga-P04168 in the hB1R expressing tumour improved the contrast to non-target organs, confirming the beneficial effect of the Pip linker on the imaging properties of our design. This result also confirms that despite their overall low stability in vivo, a 10 fold increase of affinity for hB1R upon addition of a positive charge was effective in improving tumour accumulation of the radiotracer. With no significant uptake in the hB1R negative tumour, blocking studies confirmed the specificity for hB1R expression by lowering the positive tumour uptake to the same range as the non-visible tumour. 68Ga-P04168 and 68Ga-P03034 uptake in the hB1R+ tumour was still significantly higher than the hB1R negative one under these conditions, while 68Ga-P04168 63  blocking did not completely abolish the contrast between the hB1R expressing tumour and the non-target organs, likely due to incomplete receptor occupancy by the blocking dose.  4.5 Conclusion  In summary, our findings confirm the important role played by the linker in the pharmacokinetics of our radiolabeled analogs in vivo. The addition of a positive charge on the linker improved tumour accumulation of a bradykinin analogue. This suggests that the use of such a linker, combined with a metabolically stable peptide sequence with additional unnatural amino acids could generate even more effective radiotracers for hB1R PET/CT imaging.               64  Chapter 5: Impact of the Aminoacid Sequence: Comparison of Three 68Ga-labeled [Des-Arg10] Kallidin Derivatives  5.1 Background  We previously demonstrated that improving the in vivo metabolic stability is required to detect hB1R efficiently in vivo by PET/CT with peptidic analogs of KD. hB1R expressing tumour visualization could be effectively achieved using unnatural amino acids. Compounds using the PEG2 linker tended to show higher tumour accumulation than the Ahx linker. The introduction of the novel unnatural amino acid Igl (D-(2-indanyl)glycine) was reported to dramatically the in vivo stability of the KD analog B9430 (D-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-Arg), while it retained its full antagonist properties and nanomolar affinities towards both hB1 and hB2R. Removal of the C-terminal Arginine of B9430 generated a similarly potent hB1R and hB2R antagonists B9858 (Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic)89, and B9958 (Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-D-Tic-Cpg)102. This led us to believe that these two peptidic analogs could be readily labeled using DOTA-dPEG2, and achieve similar metabolic stability in vivo as their closely related analog B9430. B9858 and B9958 were labeled with 68Ga coordinated to the DOTA chelator, coupled at the N-terminal extremity of the peptide with the PEG2 linker, generating 68Ga-P04158 and 68Ga-Z02090, respectively. These two radiotracers were compared with the previously described 68Ga-P03034 (68Ga-DOTA-dPEG2-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu) for imaging hB1R overexpressing tumour xenografts in mice using PET/CT.    65  5.2 Materials and methods  5.2.1 Chemistry and radiochemistry  5.2.1.1 Synthesis of DOTA-PEG2-Lys-[Hyp4, Igl6, D-Igl8, Oic9, desArg10]kallidin:  The HPLC purification of the crude peptide was performed under isocratic conditions with 72/28 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of DOTA‐dPEG2‐B9858 was 16.8 min, and the yield of the peptide synthesis was 21 %. MS (ESI): calculated [M+H]+ for DOTA‐dPEG2‐B9858 C87H134N20O23 is 1827.0, found 1828.4.  5.2.1.2 Synthesis of DOTA-PEG2-Lys-[Hyp4, Cpg6, D-Tic8, Cpg9, desArg10]kallidin:  The HPLC purification of the crude peptide was performed under isocratic conditions with 77/23 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of DOTA-dPEG2‐ B9958 was 13.2 min, and the yield of the peptide synthesis was 13 %.  MS (ESI): calculated [M+H]+ for DOTA‐dPEG2‐B9958 C80H130N20O23 is 1739.0, found 1740.4.     66  5.2.1.3 Synthesis of Ga-DOTA-PEG2- Lys-[Hyp4, Igl6, D-Igl8, Oic9, desArg10]kallidin:  For Ga-DOTA-PEG2 Lys-[Hyp4,Igl6,D-Igl8,Oic9,desArg10]KD (hereafter referred as P04158), the reaction mixture was purified by HPLC using the semi-preparative column eluted with 72/28 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of P04158 was 19.3 min, and the yield of the labelling reaction was 99%. MS (ESI): calculated [M + 2H]2+ for P04158 C87H131GaN20O23 is 1893.9, found 947.6.   5.2.1.4 Synthesis of Ga-DOTA-PEG2- Lys-[Hyp4, Cpg6, D-Tic8, Cpg9, desArg10]kallidin:  For Ga-DOTA-PEG2-Lys-[Hyp4,Cpg6,D-Tic8,Cpg9,desArg10]KD (hereafter referred as Z02090), the reaction mixture was purified by HPLC using a semi-preparative column eluted with 77/23 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of Z02090 was 17.0 min, and the yield of the labelling reaction was 89%. MS (ESI): calculated [M + H]+ for Ga‐DOTA‐dPEG2‐B9958 C80H127GaN20O23 is 1805.9, found 1805.5.   5.2.1.5 Synthesis of 68Ga-DOTA-PEG2 Lys-[Hyp4, Igl6, D-Igl8, Oic9, desArg10]kallidin:  68Ga-P04158 was synthesized using 100 mg of the unlabeled precursor, and purified by HPLC using a semi preparative column eluted with 69/31 C/D (pH 7.1) at a flow rate of 4.5 mL/minute. The HPLC solvents were C: CH3CN and D: PBS. The retention time of 68Ga-P04158 was 14.1 min. 67  5.2.1.6 Synthesis of 68Ga-DOTA-PEG2 Lys-[Hyp4, Cpg6, D-Tic8, Cpg9, desArg10]kallidin:  68Ga-Z02090 was synthesized using 40 mg of the unlabeled precursor, and purified by HPLC using a semi preparative column eluted with 21/79 C/E (pH 7.3) at a flow rate of 4.5 mL/minute. The HPLC solvents were C: CH3CN and E: TEA-Phosphate Buffer. The retention time of 68Ga-Z02090 was 12.8 min.  5.2.2 LogD7.4 measurements  LogD7.4 measurements were performed according to the procedure described in Chapter 2: Material and Methods (2.3).  5.2.3 Stability in mouse plasma in vitro  Metabolic stability in mouse plasma was measured according to the procedure described in Chapter 2: Material and Methods (2.4; in vitro) and (2.8.1; in vivo).  5.2.4 in vitro competition assays  These assays were performed according to the procedure described in Chapter 2: Material and Methods (2.6).    68  5.2.5 Biodistribution and PET/CT imaging  Preclinical imaging was performed according to the procedure described in Chapter 2: Material and Methods (2.8.2).  5.2.6 Statistical analysis  Statistical analyses were performed according to the procedure described in Chapter 2: Material and Methods (2.9).  5.3 Results  5.3.1 Chemistry and plasma stability  The identity of the three peptides was confirmed by MALDI-MS and ESI-MS analyses. Using HPLC, all the radiolabeled compounds were obtained with good yields (≥67%), purities (≥ 99%) and high-specific activities (≥ 185 GBq/µmol), suitable for use in mice for receptor imaging. The chemistry, radiolabeling and stability data are summarized in Table 5.1. 68Ga-P04158, 68Ga-Z02090, and 68Ga-P03034 were very hydrophilic (LogD7.4 ≤ -2.5), 68Ga-P03034 being the most hydrophilic of the three. Incubation in mouse plasma for 60 min revealed 68Ga-P04158 as the most stable (94%) of the three tracers in vitro, and 68Ga-Z02090 as the less stable (81%).  69  Table 5.1: Radiolabeling, LogD7.4 and pharmacokinetics data of 68Ga-labeled [des-Arg10]KD derivativesa. aData are presented as mean ± SD. bThe data for 68Ga-SH01078 were reported previously  5.3.2 hB1R affinity in vitro   The affinities and aminoacid sequences are summarized in the Table 5.2. The three tracers compete in a dose dependent manner with the binding of [3H]-[Leu9, des-Arg10]KD to hB1R expressing CHO cell membranes. The addition of a N-terminal lysine, and the respective replacement of the 4th and 8th prolines with Hyp and Igl and the 6th and 9th Phe with Igl D-(2-indanyl)glycine and Oic (octahydroindole-2-carboxylic acid) decreased the overall affinity of B9858 for hB1R in vitro in comparison with KD, while the conjugation of the Ga-DOTA-PEG2 label to the N-terminal end of B9858 led to improved affinity for hB1R. The addition of an N-terminal lysine and the respective replacement of the 4th and 8th proline with hydroxyproline and D-tetrahydroisoquinoline-3-carboxylic acid and the 6th and 9th phenylalanine with a cyclopentylglycine improved drastically the overall affinity of the unlabeled peptidic analog B9958 for hB1R in vitro in comparison with KD and B9858, the most potent inhibitor of the selection. Conjugation of the Ga-DOTA-PEG2 label to the N-terminal end of B9958 decreased slightly its affinity for hB1R but was still the highest of all the 68Ga-labeled compounds. The inhibition constants for P04158 and Z02090 were 1.5 ± 1.9 and 1.1 ± 0.8 nM, respectively.   Tracers Overall charge Radiochemical yield (%, decay-corrected) Radiochemical purity (%) Specific activity (GBq/µmol, n≥3) LogD7.4 (n=3) Plasma stability (% intact, n=3) in vitro 5 min 15 min 30 min 60 min 68Ga-P03034b +1 69 ± 8 (n=8) > 99 222 ± 37 -2.76 ± 0.11 99 97 94 91 68Ga-P04158 +2 76 ± 7 (n=8) > 99 89 ± 22 -2.50 ± 0.16 99 97 96 94 68Ga-Z02090 +2 67 ± 10 (n=3) > 99 100 ± 33 -2.71 ± 0.12 93 91 88 81 70  Table 5.2: Amino acid sequences and hB1R binding affinities of bradykinin and related peptides. Peptide Radiolabel complex Linker hB1R binding domain Ki (nM) (mean ± SD) Ref Bradykinin     Arg Pro Pro Gly Phe Ser Pro Phe Arg 5.7a 125 Kallidin (KD)     Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 7.4a 125  1 2 3 4 5 6 7 8 9 10   [des-Arg10]KD    Lys Arg Pro Pro Gly Phe Ser Pro Phe  9.7a 125 [Leu9, des-Arg10]KD    Lys Arg Pro Pro Gly Phe Ser Pro Leu  8.9b 125 P03083 Ga-DOTA Ahx  Lys Arg Pro Pro Gly Phe Ser Pro Leu  2.6 ± 0.7 - SH01078 Ga-DOTA Ahx  Lys Arg Pro Hyp Gly Cha Ser Pro Leu  27.8 ± 4.9 - B9858   Lys Lys Arg Pro Hyp Gly Igl Ser D-Igl Oic  10.1b 89 B9958   Lys Lys Arg Pro Hyp Gly Cpg Ser D-Tic Cpg  0.089 102 P04158 Ga-DOTA dPEG2 Lys Lys Arg Pro Hyp Gly Igl Ser D-Igl Oic  1.5 ± 1.9  Z02090 Ga-DOTA dPEG2 Lys Lys Arg Pro Hyp Gly Cpg Ser D-Tic Cpg  1.1 ± 0.8  P03034 Ga-DOTA dPEG2  Lys Arg Pro Hyp Gly Cha Ser Pro Leu  16.0 ± 1.9 - P04115 Ga-DOTA Gly-Gly  Lys Arg Pro Hyp Gly Cha Ser Pro Leu  11.4 ± 2.5 - P04168 Ga-DOTA Pip  Lys Arg Pro Hyp Gly Cha Ser Pro Leu  3.6 ± 0.2 - apEC50. bpIC50.    Figure 5.1: Representative displacement curves of [3H]-[Leu9, des-Arg10]KD by P03034, P041858 and Z02090.    71  5.3.3 Biodistribution and imaging Table 5.3: Biodistribution and uptake ratios of 68Ga-P03034, 68Ga-P04158 and 68Ga-Z02090 in tumour-bearing micea. Tissue (%ID/g) 68Ga-P03034 68Ga-P04158 68Ga-Z02090 Control (n=7) Blocked (n=5) Control (n=8) Blocked (n=4) Control (n=7) Blocked (n=5) Blood 0.40 ± 0.28 0.31 ± 0.19 1.1 ± 0.41 0.36 ± 0.1 0.12 ± 0.02 0.30 ± 0.22 Plasma 0.59 ± 0.37 0.48 ± 0.3 1.83 ± 0.67 2.44 ± 0.97 0.75 ± 0.21 1.22 ± 0.6 Fat 0.06 ± 0.02 0.08 ± 0.08 0.17 ± 0.06 0.2 ± 0.12 0.06 ± 0.02 0.09 ± 0.0 Testes 0.13 ± 0.09 0.12 ± 0.11 0.32 ± 0.11 0.34 ± 0.12 0.15 ± 0.05 0.2 ± 0.04 Large intestine 0.11 ± 0.11 0.14 ± 0.02 0.4 ± 0.14 0.46 ± 0.17 0.19 ± 0.06 0.27 ± 0.16 Small intestine 0.10 ± 0.02 0.16 ± 0.1 0.43 ± 0.08 0.62 ± 0.18 0.24 ± 0.08 0.54 ± 0.24 Spleen 0.18 ± 0.17 0.11 ± 0.03 0.45 ± 0.13 0.51 ± 0.13 0.29 ± 0.12 0.45 ± 0.24 Liver 0.17 ± 0.09 0.16 ± 0.06 0.69 ± 0.11 0.79 ± 0.18 0.39 ± 0.22 0.34 ± 0.1 Pancreas 0.06 ± 0.06 0.06 ± 0.01 0.24 ± 0.06 0.31 ± 0.12 0.11 ± 0.02 0.16 ± 0.07 Adrenal glands 0.12 ± 0.13 0.15 ± 0.17 1.16 ± 0.86 0.68 ± 0.44 0.35 ± 0.17 0.27 ± 0.09 Kidney 4.28 ± 1.89 3.19 ± 0.72 68.35 ± 8.17 52.68 ± 2.85 48.35 ± 8.64 46.78 ± 7.17 Lungs 0.5 ± 0.33 0.20 ± 0.05 0.85 ± 0.19 1.28 ± 0.44 0.45 ± 0.09 0.52 ± 0.04 Heart 0.13 ± 0.07 0.09 ± 0.02 0.48 ± 0.15 0.59 ± 0.22 0.21 ± 0.06 0.27 ± 0.03 hB1R- tumour 0.31 ± 0.12 0.28 ± 0.1 0.61 ± 0.18 0.67 ± 0.21 0.49 ± 0.18 0.5 ± 0.16 hB1R+ tumour 2.36 ± 0.64 0.43 ± 0.08 17.2 ± 5.04 1.44 ± 0.12 15.23 ± 3.68 0.86 ± 0.15 Skin 0.31 ± 0.17 0.15 ± 0.08 0.63 ± 0.27 0.85 ± 0.26 0.37 ± 0.06 0.48 ± 0.25 Muscle 0.12 ± 0.11 0.06 ± 0.02 0.29 ± 0.07 0.45 ± 0.26 0.12 ± 0.03 0.13 ± 0.02 Bone 0.08 ± 0.09 0.09 ± 0.03 0.43 ± 0.07 0.5 ± 0.17 0.21 ± 0.07 0.23 ± 0.09 Brain 0.01 ± 0.01 0.02 ± 0.02 0.03 ± 0.01 0.03 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 hB1R+ T hB1R- T 7.4 ± 3.22 1.74 ± 0.8 30.12 ± 10.53 2.33 ± 0.76 36.52 ± 22.66 1.86 ± 0.77 hB1R+ T to blood 7.06 ± 1.57 1.7 ± 0.73 17.71 ± 7.66 4.17 ± 1.09 129.37 ± 32.38 4.81 ± 4.29 hB1R+ T to muscle 28.0 ± 9.67 8.28 ± 3.98 61.97 ± 18.83 4.38 ± 3.00 130.48 ± 33.62 6.72 ± 1.55 hB1R+ T to liver 14.9 ± 5.57 2.89 ± 1.03 24.85 ± 5.67 1.89 ± 0.5 46.46 ± 20.97 2.76 ± 0.73 hB1R+ T to kidney 0.57 ± 0.17 0.13 ± 0.04 0.25 ± 0.07 0.03 ± 0.00 0.32 ± 0.09 0.02 ± 0.0 aStudies were performed at 1 h p.i. with (blocked) or without (control) co-injection of non-radioactive standard. Data are displayed as mean ± SD, and significance of differences between control and blocked groups: *p < 0.05; **p < 0.01; ***p < 0.001.  The 1-h biodistribution data are summarized in Table 5.3. All the 68Ga-labeled tracers accumulated strongly in the kidneys and the bladder urine and in no other non-target organ. As confirmed by PET imaging (Figure 5.2), all compounds were cleared from the blood circulation exclusively through the renal pathway. Despite the low background activity observed with the three tracers, 68Ga-P03034 had lower background activity, close to 68Ga-Z02090, while 68Ga-P04158 showed higher background activity. The uptake ratio values to hB1R- tumour, blood, 72  muscle, liver and kidney confirmed the high contrast visualization of the hB1R expressing tumour, kidney, and bladder with the three 68Ga-labeled probes. 68Ga-Z02090 achieved higher contrast than 68Ga-P04158, both significantly higher than the contrast obtained with 68Ga-P03034. 68Ga-P04158 and 68Ga-Z02090 achieved superior tumour (17.2 ± 5.04 and 15.23 ± 3.68 %ID/g respectively), and renal uptake (68.35 ± 8.17 and 48.35 ± 8.64 %ID/g respectively) in comparison with 68Ga-P03034 (2.36 ± 0.64 and 4.28 ± 1.89 %ID/g respectively).  68Ga-P04158 and 68Ga-Z02090 accumulated in the hB1R+ tumour 7.6 ± 1.3 and 7.1 ± 2.5 times more than 68Ga-P03034. The hB1R+ tumour to negative tumour, muscle, liver and kidney uptake ratios obtained with 68Ga-P04158 and 68Ga-Z02090 were on average 2.1 ± 1.5 (4.1, 2.2, 1.7 and 0.5 respectively) and 3.5 ± 2.1 (5.2, 4.8, 3.5 and 0.6) times higher than the ratios obtained with 68Ga-P03034, respectively. 68Ga-Z02090 and 68Ga-P04158 average hB1R+ tumour to blood were 19.3 and 2.4 times higher than 68Ga-P03034. Plasma activity levels were higher with 68Ga-P04158 and 68Ga-Z02090, and were higher upon co-administration with a blocking dose of unlabeled peptide (Figure 5.3). None of the three 68Ga-labeled tracers accumulated significantly in the hB1R- tumour during unblocked or blocked studies. Blocking experiments inhibited efficiently the binding of the three tracers to the h1BR+ tumour to uptake levels similar to the ones seen in hB1R- tumours. 73   Figure 5.2: PET/CT images of 68Ga-P03034, 68Ga-P04158, and 68Ga-Z02090 in mice bearing hB1R+ (Red arrows) and hB1R- (Green arrows) tumours without (top) or with (bottom) the corresponding cold standard.  74   Figure 5.3:  Comparison of the uptake of 68Ga-labeled hB1R-targeting tracers between hB1R+ and hB1R- tumours in mice in control (A) and blocked (B) conditions. *P < 0.05. ***P < 0.001.  5.4 Discussion  Conjugation of the Ga-DOTA-PEG2 label to the N-terminus of B9858 and B9958 peptides was tolerated and preserved the affinity of the unlabeled peptides, achieving higher affinity than the unlabeled natural sequence of [Leu9, desArg10]KD. 68Ga labeling gave excellent radiolabeling yields (>67%), and the specific activity was high due to purification by high-performance liquid chromatography. In order to keep the conditions reproducible, we chose to use high specific activity radiotracers for all experiments. Recently, triazacyclononane-phosphinate (TRAP) chelators were shown to be superior to traditional chelators, DOTA and NOTA (1,4,7-triazacyclononane-triacetic acid), for 68Ga labeling128. Specific activity values of approximately 5,000 GBq/µmol were routinely obtained using 1 GBq of 68Ga for the preparation of 68Ga-TRAP(RGD)3, using only 0.1 nmol of peptide. The effect of specific activity on tumour 75  accumulation with hB1R tracers is unknown. Replacing the DOTA chelator of P04158 and Z02090 with a TRAP chelator could be an alternative manner to generate hB1R-targeting probes for radiolabeling with 68Ga in high specific activities without the HPLC purification step. The diminution of the hydrophilicity between 68Ga-P03034, 68Ga-Z02090 and more importantly 68Ga-P04158 is a consequence of the replacement of the 8th and 9th aminoacid by more lipophilic unnatural analogs68Ga-Z02090 achieved a similar logD7.4 as 68Ga-P03034, while the hydrophilicity of 68Ga-P04158 was significantly lower. The labelling being identical, this suggests that the affinity for hB1R in vitro was affected by the hydrophilicity of the probe. The additional N-terminal lysine and the corresponding additional positive charge in 68Ga-P04158 and 68Ga-Z02090 improved their affinity for hB1R in vitro. The hB1R+ tumour uptake was not fully predicted by the in vitro plasma stability data. In vitro plasma stability assays are not necessarily predictive of the in vivo stability of a peptide, and are primarily useful as a first test to determine the suitability of a peptide for in vivo testing. Stability differences observed in vitro between the three tracers might not be directly extrapolated to their in vivo stability. 68Ga-Z02090 and 68Ga-P04158 accumulated similarly in the hB1R expressing tumours, demonstrating that the difference of hydrophilicity had no impact on the binding to hB1R in vivo. Besides, 68Ga-Z02090 and 68Ga-P04158 uptake in the hB1R+ tumour was significantly higher than 68Ga-P03034, and gave higher absolute contrast to background as well, demonstrating the benefit of increasing the affinity of the peptide to image hB1R in vivo. Differences in in vivo stability could also contribute to higher tumour uptake with 68Ga-Z02090 and 68Ga-P04158. The negative tumour uptake and the blocking studies confirmed the hB1R specific binding of these radiotracers in vivo. Despite a lower absolute uptake in the hB1R-expressing tumour, 68Ga-Z02090 achieved significantly higher contrast than 68Ga-P04158 in vivo. The same comparison with 68Ga-P03034 76  showed that the contrast to solid organs doesn’t increase by the same factor as the hB1R+ tumour uptake of 68Ga-Z02090 and 68Ga-P04158. This indicates that non-specific uptake in solid tissues are not a function of the affinity for hB1R but its metabolic stability.  The contrast to blood of 68Ga-P04158 and 68Ga-Z02090 was 2.4 and 19.3 times higher than 68Ga-P03034. However, 68Ga-P04158 and 68Ga-Z02090 uptake in the hB1R+ tumour were 7.6 ± 1.3 and 7.1 ± 2.5 higher than 68Ga-P03034. This demonstrates that the relative contrast to blood improves with 68Ga-Z02090, and decreases with 68Ga-P04158, in comparison with 68Ga-P03034 and suggests the existence of a positive correlation between the relative proportion of tracer present in the blood circulation and the logD7.4 values. As expected, the additional positive charge associated with the extra lysine at the N-terminus of 68Ga-P04158 and 68Ga-Z02090 increased their accumulation in kidneys114 in comparison with 68Ga-P03034. 68Ga-P04158 accumulated significantly less in the kidney during blocking studies, whereas 68Ga-P03034 and 68Ga-Z02090 kidney uptake were not affected. We previously observed the increased kidney accumulation of the positively charged analog 68Ga-P04168 in Chapter 4 (4.09 ± 1.0 %ID/g). The additional positive charge of 68Ga-P04168 was carried by a Pip linker and not an extra Lys. This lower kidney uptake value in comparison with 68Ga-P04158 and 68Ga-Z02090 suggests that the accumulation in the kidney is mainly dependent on the amino acid sequence, more particularly the additional lysine, and not the additional positive charge. The displaceable kidney uptake of 68Ga-P04158 correlates with its lower hydrophilicity, and supports the impact of this parameter on non-specific binding in vivo. Considering the fact that kidney reabsorption is not ligand specific, the decreased kidney accumulation of 68Ga-P04158 during blocking conditions could only be achieved by generating more unlabeled fragments from the blocking dose which compete with the labeled tracer for kidney reabsorption.  77  This suggests that 68Ga-P04158 is degraded at a higher rate in solid tissues, potentially as a consequence of its lower hydrophilicity, which seems to be associated with higher non-specific uptake. On the other hand, 68Ga-P03034 and 68Ga-Z02090 are more hydrophilic and accumulate less in non-target solid organs. Thus, the amount of tracer in circulation is increased, which enhances its renal clearance from the blood compartment, reducing the blood activity of 68Ga-Z02090 and 68Ga-P03034 one hour after the injection in comparison with 68Ga-P04158. This observation links hydrophilicity profile and stability in vivo, confirming the need to perform in vivo plasma stabilities.  5.5 Conclusion  Overall these results confirm that hB1R binding in vivo was significantly improved by substitutions of unnatural amino acids and the addition of an N-terminal lysine and a positive charge. 68Ga-P04158 and 68Ga-Z02090 generated specific and high-contrast images of hB1R+ tumour xenografts in mice.         78  Chapter 6: Importance of Ligand Activity: Comparison of Synthetic Agonist and Antagonist Tracers for Human Bradykinin B1 Receptor PET Imaging  6.1 Background  We previously described the synthesis of various 68Ga-labelled hB1R unnatural antagonists based on the [Leu9, des-Arg10]KD. The peptidase sensitive amino-acids of the sequence were replaced by unnatural analogues, and a spacer was inserted between the 68Ga chelator DOTA and the N-terminus of the peptide. Our efforts aimed initially to engineer hB1R antagonists for non-invasive detection of cancer, as these ligands are less likely to create undesirable effects upon systemic administration to patients. However, several studies have shown differences between agonist and antagonist peptides for cancer imaging129,130. Until a few years ago, the prevailing theory was that agonists were superior for imaging, since they commonly triggered receptor internalization. However, the recent demonstration that antagonist peptides will bind to the inactive form of g-coupled receptors, and therefore to more binding sites, has challenged this concept131.  The hB1R system does not behave like a classical g-coupled receptor in the sense that it is not clear that the receptor is significantly internalized upon ligand binding. hB1R activation by its cognate agonist is not followed by receptor phosphorylation nor endocytosis47,132. Instead, hB1R stimulation is followed by a reversible lateral translocation in the plasma membrane99 to caveolae-related rafts, while the internalization remains minimal133. However, endocytosis is believed to be necessary for hB1R signaling134. hB1R is constitutively expressed and internalized without agonist stimulation via a clathrin-dependent pathway and targeted to the lysosomal compartment for degradation. In other terms, hB1R binding with its agonist transiently stabilizes the receptor at the plasma membrane by inhibiting its endocytosis46. However, considering existing differences 79  observed in other receptor systems, we felt it was important to compare radiotracer internalization and tumour accumulation between agonists and antagonists in our model system of hB1R expressing tumours.  hB1R agonist and antagonists are identical except for the exchange of the 9th aminoacid from Leu to Phe, responsible for shifting the probe activity from an hB1R antagonist to an agonist. In this chapter, we compared the agonist and antagonist homologues of the same radiotracer to target hB1R in vivo using a KD peptidic analog. The ninth aminoacid of the antagonist 68DOTA-Ahx-[Hyp4, Cha6, Leu9, des-Arg10]KD (68Ga-SH01078) was replaced by a D-Phenylalanine and generated the agonist 68Ga-Ahx-[Hyp4, Cha6, D-Phe9, desArg10] KD (68Ga-Z01115). Both tracers were compared in vitro as well as in vivo using PET/CT in tumour bearing mice. Their effect on second-messenger signal (via time-resolved calcium release assays) was also investigated.  6.2 Materials and methods  6.2.1 Chemistry and radiochemistry  6.2.1.1 Synthesis of DOTA-Ahx-[Hyp4, Cha6, D-Phe9, des-Arg10]kallidin (Z01115)  The HPLC purification of the crude peptide was performed with a gradient of 20-27 % MeCN (0.1% TFA) in H2O (0.1% TFA) in 30 min at a flow rate of 4.5 mL/minute. The retention time of DOTA-Z01115 was 20.1 min, and the yield of the peptide synthesis was 35%. MS (EPI): calculated [M+H]+ for DOTA-Z01115 C72H116N18O20 [M+H]+ is 1553.9, found 1553.1.  80  6.2.1.2 Synthesis of NatGa-DOTA-Ahx-[Hyp4, Cha6, D-Phe9, des-Arg10]kallidin (NatGa-Z01115)  For Ga-DOTA-Ahx-[Hyp4, Cha6, D-Phe9, des-Arg10] KD (hereafter referred as Z01115), the reaction mixture was purified by HPLC using a semi-preparative column eluted with a gradient of 20-27 % MeCN (0.1% TFA) in H2O (0.1% TFA) in 30 min at a flow rate of 4.5 mL/min. The retention time of Z01115 was 21.8 min, and the yield of the labelling reaction was 94%. MS (EPI): calculated for Ga-DOTA-Z01115 GaC72H114N18O20 [M + H]+ is 1620.8, found 1621.3.  6.2.1.3 Synthesis of 68Ga-DOTA-Ahx-[Hyp4, Cha6, D-Phe9, des-Arg10]kallidin (68Ga-Z01115)  68Ga-Z01115 was synthesized using 25 µg of the unlabeled precursor, and purified by HPLC using a semi preparative column eluted with 79/21 Phosphate Buffer/MeCN (pH 7.5) at a flow rate of 4.5 mL/minute. The retention time of 68Ga-Z01115 was 14.7 min.  6.2.2 in vitro internalization assay  The experiments were performed in triplicates at 30, 60 and 90 min incubation at 37 °C. HEK293T::hB1R cells were plated in 6 wells poly L-lysine coated plates at a density of 200,000 cells per well and incubated 48h before the assay time. DMEM containing 10% FBS was used as the culture medium. Before the assay, cells were washed with PBS and incubated with FBS free DMEM for 60 min. Media was then removed and replaced with 1 mL of FBS free media containing 2.5 pmol of 68Ga-labeled tracers per well. In separate wells with the same setup, the 81  cold standard) was added at final concentration of 1 μM for the determination of nonspecific binding. Each set up was done in triplicate and parallel with and without 1 μM of phosphoramidon to evaluate the impact on the cellular internalization in vitro. After incubation at 37 °C to the pre-determined time point, the cells were washed twice with ice-cold PBS followed by two acid washes (0.2M acetic acid, 0.5M NaCl, pH 2.7) for 10 min on ice to measure the membrane-bound fraction. To measure the internalized activity, the cells were lysed with 0.3M NaOH, the solution was counted on a gamma counter and data decay-corrected to measure the % of the injected dose of tracer bound and internalized per 100,000 cells.  6.2.3 LogD7.4 measurements   LogD7.4 measurements were performed according to the procedure described in Chapter 2: Material and Methods (2.3).  6.2.4 Stability in mouse plasma in vitro and in vivo   Metabolic stability in mouse plasma was measured according to the procedure described in Chapter 2: Material and Methods (2.4; in vitro) and (2.8.1; in vivo).  6.2.5 in vitro competition assays   These assays were performed according to the procedure described in Chapter 2: Material and Methods (2.6).  82  6.2.6 Fluorometric measurement of calcium release   These assays were performed according to the procedure described in Chapter 2: Material and Methods (2.7).  6.2.7 Biodistribution and PET/CT imaging  Preclinical imaging was performed according to the procedure described in Chapter 2: Material and Methods (2.8.2).  6.2.8 Statistical analysis  Statistical analyses were performed according to the procedure described in Chapter 2: Material and Methods (2.9).  6.3 Results  6.3.1 Chemistry, radiochemistry and metabolic stability  The radiochemical and plasma stability data of the two 68Ga-labeled radiopeptides are summarized in Table 6.1. DOTA-Ahx-[Hyp4, Cha6, D-Phe9, des-Arg10]KD was synthesized with a 35% yield. After complexation with the non-radioactive Ga, Z01115 was obtained with a 94% yield. The identity of this peptide was confirmed by MS analysis. The decay-corrected radiochemical yield of 68Ga-Z01115 labelling was 74% ± 5 (n=3), radiochemical purity >99%, 83  and specific activity 155.4 ± 88.8 GBq/µmol (n=3). At pH 7.4, octanol:water distribution coefficient was measured at  -4.04 ± 0.2 (n = 3), and 93.1 % stable after 60 min in mouse plasma in vitro. Metabolic stability in vivo was 50.7 ± 4.5 % 5 min after injection. Table 6.1: Radiolabeling, LogD7.4 and pharmacokinetic data of 68Ga-labeled [des-Arg10]KD derivativesa. aData are presented as mean ± SD.   6.3.2 hB1R binding affinity and agonist vs antagonist cell signaling  The binding affinity of the two radiopeptides for hB1R was tested by a competition binding assay. A representative displacement curve is shown Fig.6.2. Both SH01078 and Z01115 inhibited in a dose-dependent manner the binding of [3H]-[Leu9, des-Arg10]KD to hB1R-expressing Chinese hamster ovary (CHO) cell membranes. The calculated inhibition constant (Ki) values of SH01078 and Z01115 were 27.8 ± 4.9 and 25.4 ± 5.06 nM, respectively. The quantification of the intracellular calcium release in HEK293T::hB1R cells induced by hB1R-targeting peptides is shown in Fig. 6.3. The endogenous agonist [des-Arg10]KD triggered a calcium release in a dose dependent manner. The relative fluorescent intensity (RFI) was measured at: 85 ± 52, 145 ± 20 and 178 ± 25 RFU at 0.5, 5 and 50 nM, respectively. The synthetic hB1R antagonist [Leu9, des-Arg10]KD triggered a significant calcium release only at the highest concentration (50 nM), reaching an average RFI of 64 ± 34 RFU. The antagonist derivative SH01078 was not able to elicit a significant calcium release, even at the highest concentration (50 nM). The agonist derivative Z01115 activated the hB1R signaling in a similar Tracers Overall charge Radiochemical yield (%, decay-corrected) Radiochemical purity (%) Specific activity (GBq/µmol, n≥3) LogD7.4 (n=3) Plasma stability (% intact, n=3) in vitro in vivo 5 min 15 min 30 min 60 min 5 min 68Ga-SH01078 +1 56 ± 20 (n=4) > 99 189 ± 59 -2.69 ± 0.3 99 99 99 99 9 ± 2 68Ga-Z01115 +1 74 ± 5 (n=3) > 99 155.4 ± 89 -3.81 ± 0.19 99.5 98.3 96.9 93.1 50.7 ± 4.5 84  manner as the agonist control [des-Arg10]KD. Average RFI was measured at: 75 ± 25, 132 ± 29 and 181 ± 35 RFU at 0.5, 5 and 50 nM respectively. Table 6.2: Amino acid sequences and hB1R binding affinities of bradykinin and related peptides. Peptide Radiolabel complex Linker hB1R binding domain Ki (nM) (mean ± SD Ref Bradykinin (BK)    Arg Pro Pro Gly Phe Ser Pro Phe Arg 5.7a 125 Kallidin (KD)    Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg 7.4a 125 1 2 3 4 5 6 7 8 9 10   [des-Arg10]KD   Lys Arg Pro Pro Gly Phe Ser Pro Phe  9.7a 125 [Leu9,des-Arg10]KD   Lys Arg Pro Pro Gly Phe Ser Pro Leu  8.9b 125 Z01115 Ga-DOTA Ahx Lys Arg Pro Hyp Gly Cha Ser Pro D-Phe  25.4 ± 5.1 - SH01078 Ga-DOTA Ahx Lys Arg Pro Hyp Gly Cha Ser Pro Leu  27.8 ± 4.9 - apEC50.  bpIC50.   Figure 6.1: Representative displacement curve of [3H]-[Leu9, des-Arg10]KD by SH01078 and Z01115 (n=3).  85   Figure 6.2: Calcium release in HEK293T::hB1R cells after induction by hB1R-targeting peptides: (A) [des-Arg10]KD, (B) [Leu9, des-Arg10]KD, (C) Z01115, (D) SH01078. Data are presented as mean ± SD (n=3).  6.3.3 in vitro internalization assay  Measurements of internalized and bound fractions of 68Ga-SH01078 and 68Ga-Z01115 by HEK::hB1R cells are shown in Figure 6.4. For both tracers, no significant difference was observed between the phosphoramidon and the non-treated conditions in terms of uptake or repartition (in vitro vs in vivo). For both tracers, over 80% of the maximum total uptake is reached at 30 min, and the total uptake is mainly represented by the bound fraction to the plasma membrane. 68Ga-Z01115 bound in a significantly higher proportion to hB1R compared to 68Ga-SH0178 (0.55%ID/100,000 cells vs 0.09%ID/100,000 cells respectively). The internalized fraction at 30, 60 and 90 min represented was 25.9, 30 and 28.9% of the total bound 68Ga-86  SH01078 respectively, and increased from 8.6 to 18.3, and finally 28% of the total bound 68Ga-Z01115, respectively.   Figure 6.3: Representative internalization curve of 68Ga-SH01078 and 68Ga-Z01115. Data are displayed as mean ± SD (n=3) and significance of differences between control and blocked groups: *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001.  6.3.4 Biodistribution and imaging  The biodistribution of the 68Ga-SH01078 and 68Ga-Z01115 was measured an hour after injection (Table 6.3). Both radiotracers selectively bound hB1R expressing tumour and were exclusively excreted through the renal pathway, as shown by the strong radioactive signal detected in the kidneys and the bladder. The average uptake level of all the other organs was <0.5% ID/g, resulting in a low background and high contrast images for hB1R visualization. Quantification of the uptake by hB1R expressing tumour with 68Ga-SH01078 and 68Ga-Z01115 showed a significant 2.8 fold difference in favor of the agonist (p < 0.001), reaching 2.02 ± 0.48 % and 5.65 ± 0.59 % ID/g, respectively (Figure 6.4). Average uptake by wild-type HEK293T 87  tumours was low at 0.25 ± 0.13 % and 0.34 ± 0.16 % for 68Ga-SH01078 and 68Ga-Z01115, respectively. Tumour to blood, to muscle and to wild-type tumour ratios were 7.46 ± 2.27, 28.28 ± 7.52, 2.2 ± 0.69 and 92.22 ± 59.55, 82.92 ± 34.99, 24.26 ± 14.83, for 68Ga-SH01078 and 68Ga-Z01115, respectively. Blocking experiments were performed using the non-radioactive gallium conjugated compounds, and confirmed the high in vivo specificity of our tracers for hB1R by abolishing the binding of the corresponding radioactive compound (Figure 6.5).  Table 6.3: Biodistribution and uptake ratios of 68Ga-labeled [des-Arg10]KD derivatives in tumour bearing micea. Tissue (% ID/g) 68Ga-Z01115 68Ga-SH01078 Control (n=9) Blocked (n=4)  Control (n=10) Blocked (n=6) Blood 0.24 ± 0.09 0.22 ± 0.20  0.29 ± 0.08 0.25 ± 0.06  Plasma 0.37 ± 0.10 0.34 ± 0.30  0.44 ± 0.13 0.37 ± 0.09  Red Blood Cells 0.06 ± 0.02 0.03 ± 0.03  0.06 ± 0.01 0.05 ± 0.00  Fat 0.05 ± 0.01 0.04 ± 0.04  0.07 ± 0.03 0.07 ± 0.04  Testes 0.15 ± 0.10 0.07 ± 0.06  0.09 ± 0.02 0.12 ± 0.06  Large intestine 0.15 ± 0.06 0.15 ± 0.13  0.12 ± 0.04 0.12 ± 0.03  Small intestine 0.18 ± 0.06 0.28 ± 0.27  0.22 ± 0.12 0.09 ± 0.02  Spleen 0.20 ± 0.13 0.20 ± 0.15  0.13 ± 0.03 0.11 ± 0.03  Liver 0.18 ± 0.09 0.24 ± 0.27  0.14 ± 0.02 0.14 ± 0.04  Pancreas 0.07 ± 0.02 0.06 ± 0.04  0.07 ± 0.02 0.07 ± 0.04  Adrenal glands 0.17 ± 0.09 0.14 ± 0.13  0.10 ± 0.04 0.07 ± 0.01  Kidney 4.63 ± 1.27 4.34 ± 2.68  3.66 ± 1.26 3.42 ± 0.62  Lungs 0.21 ± 0.04 0.25 ± 0.19  0.27 ± 0.06 0.22 ± 0.05  Heart 0.12 ± 0.04 0.11 ± 0.09  0.12 ± 0.03 0.08 ± 0.01  Left tumour 0.25 ± 0.13 0.17 ± 0.14  0.34 ± 0.15 0.22 ± 0.09  Right tumour 5.65 ± 0.59 0.45 ± 0.26 **** 2.02 1± 0.46 0.42 ± 0.04 **** Skin 0.25 ± 0.06 0.20 ± 0.18  0.24 ± 0.11 0.15 ± 0.04  Muscle 0.06 ± 0.01 0.07 ± 0.05  0.08 ± 0.03 0.06 ± 0.02  Bone 0.10 ± 0.05 0.10 ± 0.08  0.09 ± 0.01 0.08 ± 0.02  Brain 0.02 ± 0.01 0.01 ± 0.01  0.02 ± 0.01 0.01 ± 0.00  hB1R+T:hB1R-T 24.26 ± 14.83 3.69 ± 2.48 *** 6.92 ± 2.54 2.20 ± 0.69 ** hB1R+T:Blood 24.44 ± 12.85 20.82 ± 13.42 *** 7.46 ± 2.27 1.74 ± 0.35 **** hB1R+T:Muscle 82.92 ± 34.99 8.48 ± 5.67 **** 29.28 ± 7.52 7.37 ± 2.05 **** hB1R+T:Liver 31.62 ± 19.23 2.68 ± 1.65 *** 15.12 ± 4.74 3.15 ± 0.77 **** hB1R+T:Kidney 1.20 ± 0.58 0.10 ± 0.03 **** 0.63 ± 0.17 0.13 ± 0.02 **** aStudies were performed 1h p.i. with (blocked) or without (control) co-injection of non-radioactive standard. Data are displayed as mean ± SD, and significance of differences between control and blocked groups: *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001.  88   Figure 6.4: Comparison of uptake of 68Ga-labeled [des-Arg10]KD derivatives in hB1R+ and hB1R- tumours in mice in the (A) control and (B) blocked groups. NS and **** indicate the p value is >0.05 and <000.1 respectively.  89   Figure 6.5: Maximum Intensity Projection (MIP) of a 10 min PET/CT scan obtained 1h after injection of 3.7MBq of  68Ga-[des-Arg10]KD derivatives. 6.4 Discussion  The inducible expression of hB1R makes it an attractive target for non-invasive imaging. The accumulation of data demonstrating its expression and implication in many cancers as well as following tissue damage, was the rationale to develop new hB1R targeting compounds. In previous chapters, we evaluated the importance of the amino acid sequence and spacer for hB1R specific binding in vivo using peptidic analogs of the endogenous ligand [des-Arg10]KD. hB1R imaging is only achievable with probes that resist peptidase inactivation in vivo. An additional N-90  terminal lysine improved the binding affinity and the stability in vivo, potentially by enhancing the interaction with the 4 extracellular domains of hB1R103 structurally, and possibly electrostatically by the additional positive charge. The single introduction of a positive charge by use of the Pip linker preserved (Z02090, P04158) or improved (P04168 vs SH01078) the binding of peptides to hB1R in vivo. Finally, the increase of the overall hydrophilicity appears as a beneficial factor for hB1R imaging, while the link with the stability and the non-specific binding remains unclear. 68Ga-Z01115 and 68Ga-SH01078 radiochemical and chemical properties are very similar, and only significantly differ by their logD7.4. The D-Phe9 amino acid replacing the Leu9 in 68Ga-SH01078 makes 68Ga-Z01115 the most hydrophilic tracer we tested. Despite the fact that the DOTA-Ahx labelling improved the hB1R in vitro affinity of desArg10Leu9KD (compared to P03083), the replacement of amino acids by unnatural analogs decreased the affinity of the peptide derivatives significantly. Z01115 and SH01078 achieved similar Ki, demonstrating that replacing the Leu9 by a D-Phe9 did not affect the affinity for hB1R in vitro.  As previously observed, the in vitro stability assays were not predictive of the real fate of the radiotracers in vivo. However, according to our previous observations, the significant difference of stability between 68Ga-Z01115 and 68Ga-SH01078 is still expected to reflect actual differences in the resistance of these probes to their degradation in vivo. Indeed, the in vivo stabilities of 68Ga-Z01115 and 68Ga-SH01078 were very different. 68Ga-Z01115 was far more stable than 68Ga-SH01078 and its three other analogs 68Ga-P04115, 68Ga-P04168 and 68Ga-P03034, and this suggests a significant role of the C-terminal amino acid on metabolic stability in vivo. The poor correlation between the in vitro and in vivo stability values observed in the previous chapter is confirmed here, and suggests that 68Ga-Z01115 is more stable than the antagonist derivatives tested previously.  91  It has been reported previously that the DOTA labelling of an antagonist peptidic can shift it to an agonist108. While SH01078 was unable to stimulate endoplasmic reticulum calcium release, Z01115 confirmed its agonist nature by stimulating intracellular calcium release similarly to desArg10KD. The cellular uptakes of 68Ga-SH01078 and 68Ga-Z01115 were significantly different. Both tracers reached their maximum uptake at the 30 min time point. Interestingly, about 80% of this uptake corresponds to membrane bound tracer. However, 68Ga-Z01115 maximum uptake was significantly higher than 68Ga-SH01078. The internalized fraction of 68Ga-Z01115 increased significantly until 90 minutes, and reached a higher value than the total uptake of 68Ga-SH01078 at this time point. The differences in cell uptake observed between the two radiotracers might be due to the greater hydrophilicity of 68Ga-Z01115, as in this system, residual radioactivity that might bind non-specifically to the plastic used in cell incubation wells might compete with cell binding. While phosphoramidon was able to protect 68Ga-P03083 degradation in vivo, the absence of effect on cellular uptake of both tracers confirms that the proteases responsible for our tracers’ degradation in vivo were not present in vitro. PET images and biodistribution data were consistent. 68Ga-Z01115 uptake by the hB1R+ tumour was 2.8 times superior to 68Ga SH01078, and gave a proportionally higher contrast, similar to other antagonist analogs using this peptide sequence. This demonstrates that strong variations in hydrophilicity did not correlate directly with the background activity, and differences in in vivo stability may be the predominant factor that affected tumour accumulation. 68Ga-Z01115 uptake in the hB1R+ tumour was higher than the high affinity and positively charged 68Ga-P04168, demonstrating that a lack of additional positive charges and a lower affinity for hB1R in vitro were not sufficient to predict lower hB1R+ tumour accumulation in vivo.  92  However, 68Ga-Z01115 and 68Ga-P04168 were the most stable compounds tested to date, and this confirms that improved stability in vivo is essential to high uptake in hB1R expressing tumours. The absence of uptake in the hB1R- tumour was confirmed with a significant decrease in radiotracer uptake in the positive tumour uptake following co-administration of an excess of unlabeled compound for both tracers. Kidney uptake was not significantly different between the two tracers. 68Ga-Z01115 did not accumulate in brain tissue despite its activity, and was the only tracer we tested that accumulated significantly more in the hB1R expressing tumour than in the kidneys, making it a promising compound for application in human patients. Our results do not establish a clear link between the agonist and antagonist properties of the radiotracers and their tumour accumulation in vivo. The significant difference in in vitro cell uptakes could be due to the higher hydrophilicity of the agonist that was tested. The higher in vivo tumour accumulation could be related to the improved metabolic stability caused by the substitution of D-Phe for Leu at the N-terminus of the peptide. An alternative model to compare the effects of the agonist/antagonist properties of the peptide would have been to substitute L-Phe for the terminal leucine, but even in this case, differences in susceptibility to peptidases are likely to overwhelm the effect of the agonist/antagonist properties of the peptides.   6.5 Conclusion  Substitution of the terminal leucine by D-Phe in a derivative of [Leu9,des-Arg10]KD led, as expected, to an agonist compound that triggered calcium release in a model system of hB1R expressing cells. Compared to a compound with a similar peptide sequence, the D-Phe9 derivative was surprisingly more hydrophilic, and had improved metabolic stability in vivo. This led to improved tumour accumulation in hB1R expressing HEK293T cells. Due to differences in 93  metabolic stability between the two compounds, it cannot be concluded that the improved tumour accumulation is related to the agonist/antagonist properties of the peptides.                      94  Chapter 7: Impact of the Labeling Approach on hB1R PET Tracers: NODA, Al-fluoride and F-trifluoroborate Derivatives of [Des-Arg10] Kallidin for Imaging Bradykinin B1 Receptor Expression   7.1 Background  In this study we optimized the design of KD peptidic derivatives by combining the most favorable linkers and amino acid sequences determined previously and compared the effect of different radiolabeled chelates on the hB1R imaging properties in vivo using PET. We previously tested a series of the hB1R antagonist [Leu9, des-Arg10]KD analogs, and demonstrated the positive impact of unnatural aminoacids on metabolic stability (68Ga-P03083 vs 68Ga-SH01078), required to achieve proper hB1R detection. Subsequently we sequentially investigated the effect of various linkers (Chapter 5) and aminoacid sequences (Chapter 4) to improve our radiotracers’ imaging capabilities. We identified the addition of a positive charge (68Ga-P04168 vs 68Ga-P03034) and the B9958 aminoacid sequence (68Ga-Z02090 vs 68Ga-P04158, 18F-L08060 vs 18F-P08064135 as superior features for in vivo imaging of hB1R. As a consequence, we selected the positively charged linker Pip in combination with the [Lys-[Hyp4, Cpg6, D-Tic8, Cpg9, des-Arg10]KD peptide for derivatization and radiolabeling102. The goal of this study was to synthesize 68Ga- and 18F-labeled peptides derived from the Pip-B8858 antagonist and compare the effect of different radiolabel chelates and isotopes to investigate potential improvement of tumour uptake and tumour-to-background contrasts. For 68Ga-labeling, we compared the use of NODA with the previously used DOTA, supposing that the structure similarity between these two chelators would allow similar labelling properties. For 18F-labeling, we compared two strategies: formation of aluminum 18F-fluoride (18F-AlF)136 and the one-step 18F-19F isotope exchange reaction on an 95  ammoniomethyl-trifluoroborate moiety105,109,137,138. Both strategies could be performed directly in aqueous solution, and have been successfully applied for the design of 18F-labeled peptide-based PET tracers105,139,140,141. Herein, we present the synthesis and evaluation of four new tracers: 18F-AmBF3-Mta-Pip-B9958 (18F-L08060, AmBF3-Mta: (4-(N-trifluoroborylmethyl-N,N-dimethylammonio)methyl-1,2,3-triazole-1-acetic acid, Pip: 4-amino-(1-carboxymethyl)piperidine), 68Ga-DOTA-Pip-B9958 (68Ga-Z02176), 68Ga-NODA-Mpaa-Pip-B9958 (Z02137; Mpaa: 4-methylphenylacetic acid), and 18F-AlF-NODA-Mpaa-Pip-B9958 (Z04139).  7.2 Material and methods  7.2.1 Peptide synthesis  7.2.1.1 Synthesis of DOTA-Pip-B9958  The assembly of Fmoc-protected Pip-B9958 sequence (Fmoc-Pip-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Cpg-Ser(tBu)-D-Tic-Cpg) was synthesized via the Nα-Fmoc solid-phase peptide synthesis strategy starting from 2-chlorotrityl chloride resin. The crude peptide was purified from the reaction mixture by HPLC using a semi-preparative column eluted with gradient 20/80 A/B to 30/70 A/B in 30 min at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of DOTA-Pip-B9958 was 20.4 min, and the yield of the synthesis was 18 %. ESI-MS: calculated [M+2H]2+ for DOTA-Pip-B9958 C80H129N21O21 is 861.5, found 861.7.  96  7.2.1.2 Synthesis of NODA-Mpaa-Pip-B9958  The assembly of Fmoc-protected Pip-B9958 sequence (Fmoc-Pip-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Pro-Hyp(tBu)-Gly-Cpg-Ser(tBu)-D-Tic-Cpg) was performed as described above. 4-(Bromomethyl)phenylacetic acid (3 equivalents) was activated with DIC (3 equivalents) in 5 mL NMP and then coupled to the N-terminus. Subsequently, 1,4,7-triazacyclononane-1,4-bis(t-butyl acetate) (3 equivalents) was coupled to the peptide sequence by secondary amine alkylation in 5 mL NMP. The crude peptide was purified by HPLC using a semi-preparative column eluted with 24/76 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of NODA-Mpaa-Pip-B9958 was 18.7 min, and the yield of the synthesis was 10 %. MALDI-MS: calculated [M+H]+ for NODA-Mpaa-Pip-B9958 C83H128N20O19 is 1710.0, found 1710.0.  7.2.1.3 Synthesis of azidoacetyl-Mta-Pip-B9958  The assembly of azidoacetyl-Mta-Pip-B9958 sequence was performed as described above. After elongation, azidoacetic acid (40 equivalents) was pre-activated with DIC (20 equivalents) in DCM for 10 min, filtered, and then coupled to the peptide sequence to provide the azide functional group at the N-terminus for click reaction. The peptide was deprotected and cleaved from the resin using a cocktail of trifluoroacetic acid/ water/ triisopropylsilane/ ethanedithiol/ thioanisol/ phenol (81.5:5:1:2.5:5:5). After filtration to remove resin, the crude product was precipitated by the addition of cold diethyl ether. The crude product was filtered, dried, and purified by HPLC using a gradient condition of 79/21 A/B to 77/23 A/B in 30 min at a flow rate of 4.5 mL/minute. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN 97  containing 0.1% TFA. The yield of azidoacetyl-Pip-B9958 synthesis was 20%. ESI-MS: calculated [M+H]+ for azidoacetyl-Pip-B9958 C66H105N20O15 is 1417.8, found 1418.2.  7.2.1.4 Synthesis of AlOH-NODA-Mpaa-Pip-B9958  NODA-Mpaa-Pip-B9958 (6.4 mg, 3.7 μmol) was dissolved in 0.3 mL NaOAc buffer solution (2 mM, pH 7.0) containing AlCl3 (1.5 mg, 11.2 μmol), and the mixture was stirred at 110 °C for 30 min. The HPLC purification of the crude peptide was performed with a gradient condition of 75/25 A/B to 72/28 A/B in 20 min at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of AlOH-NODA-Mpaa-Pip-B9958 was 10.0 min, and the yield of the synthesis was 61%. ESI-MS: calculated [M+2H]2+ for AlOH-NODA-Mpaa-Pip-B9958 C83H127AlN20O20 is 876.5, found 876.7.  7.2.2 Cold labelling  7.2.2.1 Synthesis of Ga-DOTA-Pip-B9958 (Z02176)  Z02176 was synthesized according to the procedures described in Chapter 5 for the synthesis of P04168. For Ga- DOTA-Pip-B9958 (hereafter referred as Z02176), the reaction mixture was purified by HPLC using a semi-preparative column eluted with a gradient 20/80 A/B to 30/70 A/B in 30 min at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of Z02176 was 20.6 min, and labelling reaction produced Z02176 quantitatively. ESI-MS: calculated [M+2H]2+ for Z02176 C80H126GaN21O21 is 894.4, found 894.1. 98  7.2.2.2 Synthesis of Ga-NODA-Mpaa-Pip-B9958 (Z02137)  Z02137 was synthesized according to the procedures described above for the synthesis of P05022. For Ga-NODA-Mpaa-Pip-B9958 (hereafter referred as Z02137), the reaction mixture was purified by HPLC using a semi-preparative column eluted with 24/76 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of Z02137 was 15 min, and the yield of the labelling reaction was 43%. ESI-MS: calculated [M+2H]2+ for Z02137 C83H126GaN20O19 is 888.9, found 888.6.  7.2.2.3 Synthesis of AmBF3-Mta-Pip-B9958 (L08060)  For the synthesis of AmBF3-Mta-Pip-B9958 (hereafter referred as L08060) An Eppendorf tube (1.5 mL) was loaded with a mixture of N-propargyl-N,N-dimethylammoniomethyl-trifluoroborate (3.5 mg, 22.6 μmol), CuSO4 (1.0 M, 5.0 μL), sodium ascorbate (1.0 M, 12.5 μL) and 5% NH4OH (MeCN/H2O = 1:1, 50 μL) and Azidoacetyl-Pip-Mta-B9958 (2.8 mg, 2.0 μmol). The reaction was allowed to proceed for 2 h at 45 °C, purified by HPLC using a semi-preparative column (Agilent Eclipse XDB-C18, 5 μm, 250 x 9.2 mm) eluted with successive gradients (From 0 to 2 min, 5 to 20% B; from 2 to 7 min, 20 to 35% B; from 7 to 15 min, 35 to 45% B; from 15 to 16 min, 45 to 100% B) at a flow rate of 3 mL/min to obtain 1.5 mg of AmBF3-B9958 (47%). The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. ESI-MS: [M+H]+ for AmBF3-B9958 C72H116BF3N21O15 is 1582.9, found 1583.1.    99  7.2.2.4 Synthesis of AlF-NODA-Mpaa-Pip-B9958 (Z04139)  For AlF-NODA-Mpaa-Pip-B9958 synthesis, AlOH-NODA-Mpaa-Pip-B9958 (2 mg, 1.1 μmol) and NaF (48 µg, 1.1 μmol) were dissolved in 0.2 mL NaOAc buffer solution (2 mM, pH 7.0) and 0.2 mL ethanol, and stirred at 105 °C for 15 min. AlF-NODA-Mpaa-Pip-B9958 was purified by HPLC using the semi-preparative column eluted with 77/23 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of AlF-NODA-Mpaa-Pip-B9958 was 19.5 min, and the yield was 65%. ESI-MS: calculated [M+2H]2+ for AlF-NODA-Mpaa-Pip-B9958 C83H126AlFN20O19 is 877.5, found 877.7.  7.2.3 Radiochemistry  7.2.3.1 Synthesis of 68Ga-DOTA-Pip-B9958 (68Ga-Z02176)  68Ga-Z02176 was prepared according to the procedures described in the Chapter 5 for the synthesis of 68Ga-P04168 by using 20 µg of DOTA-Pip-B9958 as the radiolabeling precursor. 68Ga-Z02176 was purified by HPLC using a semi-preparative column eluted with 20/80 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: CH3CN (0.1 M, pH 7.4) and B: PBS.The retention time of 68Ga-Z02176 was 19.6 min. The quality control was performed by HPLC using the same semi-preparative column eluted with the same conditions.    100  7.2.3.2 Synthesis of 68Ga-NODA-Mpaa-Pip-B9958 (68Ga-Z02137)  68Ga-Z02137 was prepared according to the procedures described in the Chapter 5 for the synthesis of 68Ga-P04168 by using 25 µg of NODA-Mpaa-Pip-B9958 as the radiolabeling precursor. The reaction mixture was purified by HPLC using a semi-preparative column eluted with 24/76 A/B at a flow rate of 4.5 mL/min. The retention time of 68Ga-Z02137 was 15.0 min. Quality control was performed using an analytical column eluted with 24/76 A/B at a flow rate of 2 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of 68Ga-Z02137 was 10.0 min.  7.2.3.3 Synthesis of 18F-AmBF3-Mta-Pip-B9958 (18F-L08060)  19F-AmBF3-Mta-Pip-B9958 (100 nmol) was dissolved in a mixture of aqueous pyridazine-HCl buffer (20 μL, 1M, pH2) and DMF (20 μL) in a 4.5-mL Falcon tube. No carrier-added 18F-fluoride was obtained by bombarding H218O with 18 MeV protons, followed by trapping on an anion exchange (9 mg, QMA, chloride form) column. The 18F-fluoride was eluted off the column with 100 μL saline into the Falcon tube containing 19F-AmBF3- B9958. The tube was placed in a heating block, and heated at 80 °C for 15 min. The reaction mixture was subsequently quenched with 5% aqueous NH4OH (2 mL), and loaded onto a C18 light Sep-Pak cartridge. Free 18F-fluoride was removed by washing the Sep-Pak cartridge with deionized water (2 mL x 2). 18F-AmBF3-B9958 was then eluted off the cartridge with 4:1 ethanol/saline (0.5 mL), and diluted with saline for in vitro plasma stability, biodistribution and PET/CT imaging studies. A small sample was removed for quality control analysis by HPLC using a semi-preparative column (Phenomenex Jupiter-C18, 10 μm, 250 x 4.6 mm) eluted with successive gradients (0 to 2 101  min, 5% B; 2 to 7 min, 5 to 20% B; 7 to 15 min, 20 to 100% B). The chemical identity of 18F-AmBF3-Mta-Pip-B9958 was confirmed by co-injecting the radiolabeled product with their respective non-radiolabeled standard.  7.2.3.4 Synthesis of 18F-AlF-NODA-Mpaa-Pip-B9958 (18F-Z04139)  H218O containing 18F-fluoride was passed through a short anion exchange Trap & Release column (pre-activated with 5 mL brine), and the column was washed with de-ionized water (1 mL × 2). The 18F-fluoride was eluted with saline (100 µL) into a vial pre-loaded with ethanol (110 µL) and AlOH-NODA-Mpaa-Pip-B9958 (1 mM, 10 µL) solution in NaOAc buffer (2 mM, pH 4.4). The reaction mixture was incubated at 105 °C for 15 min and then purified by HPLC using a semi-preparative column ((Phenomenex Jupiter-C18, 10 μm, 250 x 4.6 mm) eluted with 76/24 A/B at a flow rate of 4.5 mL/min. The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. The retention time of 18F-Z04139 was 15.5 min. Quality control was performed on an analytical column eluted with 24/76 A/B at a flow rate of 2 mL/min. The retention time of 18F-Z04139 was 10.0 min.   7.2.4. LogD7.4 measurements  LogD7.4 measurements were performed according to the procedure described in Chapter 2: Materials and Methods (2.3).    102  7.2.5 Stability in mouse plasma in vitro and in vivo  Metabolic stability in mouse plasma was measured according to the procedure described in Chapter 2: Material and Methods (2.4; in vitro) and (2.8.1; in vivo).  7.2.6 in vitro competition assays  These assays were performed according to the procedure described in Chapter 2: Material and Methods (2.6).  7.2.7 Biodistribution and PET/CT imaging  Preclinical imaging was performed according to the procedure described in Chapter 2: Material and Methods (2.8.2).  7.2.8 Statistical analysis  Statistical analyses were performed according to the procedure described in Chapter 2: Material and Methods (2.9).      103  7.3 Results  7.3.1 Chemistry and radiochemistry  The radiolabeling data and logD7.4 are summarized in Table 7.1. Azidoacetyl-Mta-Pip-B9958, DOTA-Pip-B9958 and NODA-Mpaa-Pip-B9958 were assembled on solid phase using N-Fmoc protected amino acids, and obtained in 20, 18 and 10% yields, respectively. AlOH-NODA-Mpaa-Pip-B9958 was prepared by incubating NODA-Mpaa-Pip-B9958 with AlCl3 in acetate buffer (2 mM, pH 7.0), and obtained in 61% yield. After conjugation with alkyne and HPLC purification, AmBF3-B9958 was obtained in 47% yield. The cold standards of Z02176 and Z02137 were obtained in 100 and 43%, respectively, by reacting their respective precursor with GaCl3 in acetate buffer (0.1 M, pH 4.0). Cold Z04139 (Fig. 7.1) was prepared by incubating AlOH-NODA-Mpaa-Pip-B9958 with NaF in 1:1 ethanol/acetate buffer (2 mM, pH 7.0), and was obtained in 65% yield. The identities of the peptides were confirmed by the single peak obtained with mass spectrometry analysis. 18F-Z04139, 68Ga-Z02176 and 68Ga-Z02137 were prepared with average decay-corrected radiochemical yields ranging from 24 to 47%, and > 99% radiochemical purity. 18F-labeling was performed via an 18F-19F isotope exchange reaction, and the final purified product was obtained within 30 min of synthesis time. Starting with 21.5-33.3 GBq of 18F-fluoride, 18F-L08060 was obtained in 27.9 ± 5.4 % (n = 3) non-decay-corrected radiochemical yield. The average specific activities of these four tracers ranged from 74 to 261 GBq/µmol. 18F-Z04139, 68Ga-Z02176 and 68Ga-Z02137 were highly hydrophilic with average LogD7.4 (D: distribution coefficient) in the range of -4.15 - 3.35 (L08060 non-measured).    104  7.3.2 in vitro hB1R binding affinity and plasma stability  L08060, Z02176, Z02137 and Z04139 inhibited effectively and in a dose-dependent manner the binding of [3H]-[Leu9,des-Arg10]KD to hB1R expressing CHO cells membranes in vitro, yielding the following Ki values of 0.5 ± 0.0, 2.5 ± 0.8, 2.6 ± 0.7, and 14.0 ± 7.4 nM, respectively. The representative displacement curves are shown in Fig. 7.2. The in vitro stability of 18F-AmBF3-B9958 was assessed after 60 min at 37 °C in mouse plasma. At the end of the assay HPLC analysis showed > 95% of 18F-AmBF3-B9958 was intact, and no significant metabolites were observed.  7.3.3 in vivo stability  The in vivo stability of 68Ga-Z02176 and 18F-Z04139 were evaluated in mice (n = 3) 5 min after tail-vein injection. Blood was sampled after euthanasia; the plasma was isolated, and injected into a HPLC column to check the percentage of intake tracers. Representative radio-HPLC chromatograms are shown in Fig. 7.1. Both 68Ga-Z02176 and 18F-Z04139 were fairly stable in vivo in mice with 93.7 ± 0.6 and 95.8 ± 1.0 %, respectively, remaining intact at 5 min p.i. 105   Figure 7.1: Representative radio-HPLC chromatograms of 68Ga-Z02176 and 18F-Z04139 from QC (upper chromatograms) and mouse plasma samples (lower chromatograms) taken at 5 min post-injection.   7.3.4 in vivo PET/CT imaging and biodistribution  The 1-h static PET/CT images of 18F-L08060, 68Ga-Z02176 68Ga-Z02137, and 18F-Z04139 are shown in Figure 7.2. All tracers showed low background except in the kidney and bladder, corresponding to the renal excretion pathway. In addition, hB1R+ tumours were also clearly visualized in the images. The uptake in the hB1R expressing tumour was higher with 68Ga-Z02176 and 18F-Z04139, followed by 68Ga-Z02137, and lowest with 18F-L08060. Co-injection of their respective cold standard (100 µg) effectively blocked the uptake of all the tracers into the hB1R positive tumour. The results are summarized in Table 7.2.  106   Figure 7.2: Maximum Intensity Projection (MIP) of a 10 min PET/CT scan obtained 1h after injection of  68Ga-Z02176, 68Ga-Z02037, 18F-Z04139, or 18F-L08060 (Left to right). Mice are bearing both hB1R+ (right shoulder, red arrow) and hB1R- (left shoulder, yellow arrow) tumours were injected without (top row) or with (bottom row) co-injection of the non-radioactive standard (100µg) and anesthetized with isoflurane inhalation.  The 1-h post-injection (p.i.) biodistribution data of the four tracer were consistent with the PET/CT images. While the uptake was minimal in all the other non-target organs, the four tracers accumulated significantly in the hB1R expressing tumours and were excreted exclusively through the renal pathway, yielding a high uptake in the kidneys. 68Ga-L08060 uptake in the kidney was the lowest (30.8 ± 6.4 %ID/g), while the accumulation of the three other tracers ranged from 85.2 to 106 %ID/g. The uptake in hB1R+ tumour was the highest with 68Ga-Z02176 (28.9 ± 6.21 %ID/g) and 18F-Z04139 (22.6 ± 3.41 %ID/g), followed by 68Ga-Z02137 (14.0 ± 4.86 %ID/g) and 68Ga-L08060 (3.7 ± 1.1 %ID/g). The uptake ratios to hB1R negative tumour, blood and muscle 107  increased accordingly. Co-injection with 100 μg of cold standard significantly reduced the uptake (< -88%) of the four tracers in the hB1R+ tumours, while the uptake in hB1R- tumours remained lower than in the blood during control and blocked conditions. With the exception of the blood and the hB1R positive tumour, the uptake of the four tracers in the blocked mice remained steady in all the non-target organs in comparison with the control mice. At the difference of the three other tracers during blocked conditions, 68Ga-Z02176 uptake in the blood was significantly increased, while the kidney accumulation was significantly decreased. Table 7.1: Overall charge, radiolabeling and LogD7.4 data of radiolabeled hB1R-targeting tracers. Tracers Overall charge Radiochemical yield (%, decay-corrected) Radiochemical purity (%) Specific activity (GBq/µmol, n ≥ 3) LogD7.4 Ki (nM) 68Ga-Z02176 +3 46 ± 2 (n = 4) > 99 210 ± 72 -4.15 ± 0.04 2.5 ± 0.8 68Ga-Z02137 +4 47 ± 11 (n = 3) > 99 261 ± 74 -3.35 ± 0.03 2.6 ± 0.7 18F-Z04139 +3 32 ± 11 (n = 3) > 99 100 ± 8 -3.90 ± 0.21 14 ± 7.4 18F-L08060 +3 24 ± 2 (n = 3)* > 99 74 n/a 0.5 ± 003  Table 7.2: Biodistribution data of radiolabeled hB1R-targeting tracers at 1-h p.i. in tumour-bearing mice. * non-decay-corrected. Tissue (%ID/g) 68Ga-Z02176 68Ga-Z02137 18F-L08060 18F-Z04139 Control (n = 9) Blocked (n = 5) Control (n = 9) Blocked (n = 4) Control (n = 8) Blocked (n = 4) Control (n = 9) Blocked (n = 5) Blood 0.55 ± 0.15 0.84 ± 0.11** 0.46 ± 0.16 0.51 ± 0.21 0.34 ± 0.14 0.40 ± 0.03 0.43 ± 0.15 0.65 ± 0.14 Fat 0.10 ± 0.04 0.17 ± 0.04** 0.10 ± 0.04 0.09 ± 0.03 0.07 ± 0.04 0.06 ± 0.01 0.09 ± 0.03 0.13 ± 0.03** Testes 0.19 ± 0.07 0.30 ± 0.11* 0.16 ± 0.05 0.20 ± 0.07 0.14 ± 0.05 0.11 ± 0.02 0.16 ± 0.04 0.24 ± 0.03 Large intestine 0.23 ± 0.09 0.30 ± 0.12 0.22 ± 0.10 0.20 ± 0.07 0.32 ± 0.24 0.22 ± 0.04 0.25 ± 0.10 0.22 ± 0.04 Small intestine 0.33 ± 0.17 0.36 ± 0.09 0.25 ± 0.08 0.29 ± 0.05 0.4 ± 0.21 0.19 ± 0.09 0.36 ± 0.13 0.54 ± 0.37 Spleen 0.41 ± 0.25 0.32 ± 0.01 0.42 ± 0.28 0.54 ± 0.26 0.45 ± 0.32 0.2 ± 0.09 0.29 ± 0.07 0.35 ± 0.06 Liver 0.43 ± 0.18 0.49 ± 0.23 0.48 ± 0.12 0.50 ± 0.13 0.26 ± 0.08 0.22 ± 0.05 0.47 ± 0.10 0.64 ± 0.22 Pancreas 0.14 ± 0.05 0.18 ± 0.04 0.14 ± 0.06 0.13 ± 0.04 0.09 ± 0.03 0.09 ± 0.01 0.12 ± 0.04 0.15 ± 0.03 Kidney 90.9 ± 22.8 51.2 ± 2.70** 85.2 ± 12.1 70.2 ± 13.5 30.82 ± 6.42 26.54 ± 4.61 101 ± 14.4 106 ± 18.4 Lungs 0.69 ± 0.25 0.83 ± 0.23 0.79 ± 0.30 0.65 ± 0.20 0.4 ± 0.21 0.33 ± 0.06 0.52 ± 0.12 0.68 ± 0.13 Heart 0.25 ± 0.08 0.31 ± 0.07 0.28 ± 0.16 0.30 ± 0.08 0.18 ± 0.10 0.16 ± 0.04 0.23 ± 0.07 0.30 ± 0.05 B1R- tumour 0.38 ± 0.17 0.35 ± 0.06 0.31 ± 0.06 0.38 ± 0.21 0.23 ± 0.09 0.19 ± 0.07 0.31 ± 0.02 0.44 ± 0.24 B1R+ tumour 28.9 ± 6.21 0.89 ± 0.13*** 14.0 ± 4.86 1.06 ± 0.47*** 3.66 ± 1.07 0.47 ± 0.13*** 22.6 ± 3.41 2.05 ± 0.81*** Muscle 0.19 ± 0.07 0.18 ± 0.04 0.14 ± 0.04 0.22 ± 0.05* 0.10 ± 0.04 0.09 ± 0.02 0.14 ± 0.04 0.21 ± 0.07* Bone 0.30 ± 0.11 0.34 ± 0.07 0.30 ± 0.09 0.36 ± 0.07 0.6 ± 0.61 0.47 ± 0.12 0.21 ± 0.05 0.24 ± 0.06 Brain 0.03 ± 0.01 0.02 ± 0.01 0.03 ± 0.02 0.02 ± 0.01 0.01 ± 0.01 0.01 ± 0.0 0.03 ± 0.01 0.02 ± 0.01 Ratios:         B1R+T:B1R-T 85.4 ± 30.1 2.63 ± 0.70*** 47.5 ± 19.6 1.89 ± 1.35** 18.52 ± 9.03 2.67 ± 0.71*** 74.5 ± 15.1 5.16 ± 2.40*** B1R+T:Blood 56.1 ± 17.3 1.08 ± 0.20*** 34.3 ± 15.2 1.31 ± 0.95** 12.38 ± 5.08 6.27 ± 1.44*** 58.0 ± 20.9 3.31 ± 1.50*** B1R+T:Muscle 167  ± 57.6 5.00 ± 1.23*** 103  ± 30.2 5.23 ± 2.76*** 40.51 ± 17.03 5.41 ± 2.41*** 173 ± 42.9 10.8 ± 5.10*** Significance of differences between control and blocked groups: *p < 0.05; **p < 0.01; ***p < 0.001. 108  Table 7.3: Biodistribution data (1-h p.i.) of 68Ga-Z02176 and 18F-Z04139 in tumour-bearing mice used for imaging or biodistribution study. Tissue (%ID/g)                              68Ga-Z02176                          18F-Z04139 Imaged mice (n = 3) Biodistribution mice (n = 6) Imaged mice (n = 3) Biodistribution mice (n = 6) Blood 0.54 ± 0.23 0.55 ± 0.12 0.52 ± 0.19 0.39 ± 0.13 Fat 0.09 ± 0.05 0.11 ± 0.03 0.09 ± 0.02 0.12 ± 0.08 Testes 0.17 ± 0.06 0.20 ± 0.08 0.17 ± 0.04 0.15 ± 0.04 Large intestine 0.24 ± 0.08 0.22 ± 0.10 0.33 ± 0.03 0.21 ± 0.11 Small intestine 0.23 ± 0.10 0.38 ± 0.18 0.27 ± 0.03 0.40 ± 0.13 Spleen 0.31 ± 0.15 0.46 ± 0.28 0.29 ± 0.08 0.29 ± 0.07 Liver 0.43 ± 0.20 0.43 ± 0.19 0.50 ± 0.07 0.46 ± 0.11 Pancreas 0.15 ± 0.09 0.14 ± 0.03 0.13 ± 0.04 0.12 ± 0.04 Adrenal glands 0.09 ± 0.04 0.12 ± 0.03 0.20 ± 0.04 0.17 ± 0.05 Kidney 65.3 ± 8.51 104  ± 14.6** 88.4 ± 7.36 108  ± 12.7 Lungs 0.78 ± 0.36 0.65 ± 0.21 0.54 ± 0.12 0.51 ± 0.13 Heart 0.26 ± 0.13 0.25 ± 0.06 0.25 ± 0.06 0.21 ± 0.07 hB1R- tumour 0.30 ± 0.14 0.42 ± 0.19 0.31 ± 0.03 0.31 ± 0.02 hB1R+ tumour 21.3 ± 1.66 32.8 ± 2.76*** 22.2 ± 3.49 22.7 ± 3.69 Muscle 0.18 ± 0.09 0.19 ± 0.06 0.16 ± 0.03 0.13 ± 0.04 Bone 0.26 ± 0.13 0.31 ± 0.10 0.23 ± 0.05 0.21 ± 0.05 Brain 0.02 ± 0.01 0.03 ± 0.01 0.03 ± 0.00 0.02 ± 0.01 Ratios:     B1R+T:B1R-T 76.9 ± 23.5 89.7 ± 34.1 72.4 ± 17.3 76.1 ± 15.8 B1R+T:Blood 43.0 ± 14.3 62.6 ± 15.6 50.6 ± 31.3 61.7 ± 16.0 B1R+T:Muscle 132  ± 49.3 184  ± 57.1 150  ± 54.8 185  ± 35.2 Significance of differences between groups of imaging and biodistribution mice: *p < 0.05; **p < 0.01; ***p < 0.001.  7.4 Discussion  The four tracers were labelled with similar radiochemical properties, with the exception of higher specific activities achieved with the 68Ga-labelled tracers in comparison with the 18F-labelled ones. This first of all demonstrates that 68Ga coordination through NODA is possible and equivalent to DOTA. The isolated radiochemical yield and specific activity obtained with 18F-L08060 were similar to the ones achieved by 18F-Z04139, and consistent with the previously reported AmBF3 conjugates including AmBF3-TATE and Rhodamine-bisRGD-AmBF3 (105 = 26, 102 = 28). 109  Features of the 18F-L08060 labeling strategy include: (1) one-step reaction in aqueous solution without the final azeotropic drying step, (2) relative ease of purification via solid-phase extraction without HPLC, (3) short radiosynthesis/purification time (within 30 min), (4) good radiochemical yields, (5) high purity (> 99%), and (6) high specific activity. In addition, commercial azidoacetic acid could be directly coupled when preparing the azidoacetyl-B9958, and the apparent stability of the AmBF3-Mta-Pip-B9958 construct through the relative harsh conditions of synthesis suggests that the entire synthesis could be simplified and performed on solid phase. The affinities of the four tracers were all in the nanomolar range (Ki = 0.5 -14 nM), confirming the versatility of these tracers for a broad range of modifications. 18F-Z04139 Ki was significantly higher than the three other analogs, while 18F-L08060 yielded the lowest Ki, demonstrating that the method of coordination to the isotope affects the tracer binding to its target, as already observed with somatostatin analogs108,142. 68Ga-Z02137 achieved a higher affinity for hB1R in vitro than 18F-Z04139, demonstrating that the negative impact of the NODA-Mpaa modification is counterbalanced by the additional positive charge, confirming the positive impact of this parameter on the hB1R affinity. This also demonstrated that even the most distant modifications do affect the targeting properties of our analogs. No significant metabolites were observed in plasma stability studies indicating that B9958 sequence and the AmBF3 moiety are stable in mouse plasma.  Biodistribution data and PET/CT images showed that the four tracers were quickly cleared from most tissues/organs through the renal excretion pathway. This demonstrates that the 68Ga-NODA complex is as stable as 68Ga-DOTA in vivo, and thus could be directly applied to existing 18F-AlF-NODA-conjugates to generate 68Ga-labeled tracers as well143,144,145. In addition to the lack of brain uptake indicating that these tracers are not able to cross the blood brain barrier 110  (BBB), the bone uptake was steadily low during control and blocked conditions (0.21 - 0.6 %ID/g), indicating the absence of defluorination of 18F-Z04139 and 18F-L08060. Co-injection of 100 μg of cold standard reduced by more than 88% the uptake of the four tracers in hB1R+ tumours, demonstrating the specific uptake in the hB1R expressing cells in vivo. However, significantly higher uptake of the four tracers in hB1R+ than hB1R- tumours was still noticeable in blocked mice biodistribution with 18F-Z04139 (2.05 ± 0.81 %ID/g) and  68Ga-Z02137 (1.06 ± 0.47 %ID/g). These results indicate that the Pip-B9958 targeting moiety is highly selective for hB1R in vivo, and suggests that the NODA-Mpaa-Pip motif is the most favorable labelling motif to preserve the high specificity of the peptide for hB1R in vivo. PET images confirmed this observation, and revealed that the hB1R expressing tumours were still clearly visualized in blocked images with 18F-Z04139, despite the fact that it did not achieve the highest affinity for hB1R in vitro nor the highest uptake in the h1BR+. This demonstrates that a parameter other than the strict affinity for hB1R drives the accumulation of 18F-Z04139 in the positive tumour. The co-injected mass (100 μg) of non-radioactive standard was > 500-fold of the mass of the radiolabeled tracer, and should theoretically block most of hB1R available for binding, as previously described with similar tracers. Besides, the four tracers compared here gave similar background in control and blocked conditions, demonstrating that the difference observed is not due to a difference in non-specific accumulation, and suggests that the coordination with AlF improves the stability of 18F-Z04139 in vivo. Kidney uptake values were elevated with the four tracers, and consistent with the high values previously obtained with 68Ga-Z02090, despite significant variations between the different tracers and the different conditions tested. We previously demonstrated that the increase of kidney uptake is not a function of increased positive charge (68Ga-P04168 vs 68Ga-SH01078/68Ga-P030304/68Ga-P04115/68Ga-P03083), but a function of the additional N-ter lysine 111  present in the 68Ga-P04158 and 68Ga-Z02090. This result was confirmed here by the fact that 68Ga-Z02137 had the highest positive charge while the uptake in the kidney was not the highest. However, this suggested that the relative kidney uptake of radioactivity was also a function of the metabolic stability of the radiotracers in vivo. Blocking studies impacted the accumulation of the four tracers in different manners, and supported this hypothesis. Coinjection of the corresponding cold standard decreased the kidney accumulation of 18F-L08060 by 13.9%, 68Ga-Z02137 by 17.6%, and 68Ga-Z02176 by 43.7% (p < 0.01), while 18F-Z04139 uptake was not decreased (+ 5%).  Considering the fact that the receptor mediated peptide reabsorption in the proximal tubule is amino-acid and not sequence specific, and the fact that our four analogs share the same exact amino-acid sequence, the competition that occurs in the kidney during blocking studies is only a matter of ratio between the amounts of cold and radioactive amino acids. This ratio can be modified if the stability varies significantly: a highly unstable tracer will generate more unlabeled peptidic fragments than a stable one, and thus will compete more efficiently with the radiolabeled fragment reabsorption. As a consequence, the addition of saturating amounts of cold standard can generate exponentially more competition if the compound is unstable. In accordance with the in vivo metabolic stabilities, 68Ga-Z02176 is slightly less stable and the corresponding kidney accumulation is the most affected by the cold standard. At the opposite, 18F-Z04139 kidney uptake is unchanged during blocking, and corresponds to the more stable compound of the two. The four radiolabeled Pip-B9958 derivatives achieved high contrast between the hB1R+ tumour and the non-target organs. The relative increases of hB1R+ tumour uptake were proportional to the increase of the contrast for each tracer, confirming that the background achieved with these four tracers was reproducibly similar. 18F-L08060 achieved the highest affinity for hB1R in vitro (30 times higher than 18F-Z04139), but also the lowest uptake in the 112  hB1R expressing tumour (6 times less than 18F-Z04139), demonstrating that a high affinity is not sufficient to achieve proper imaging in vivo. This was confirmed by the significant difference of affinities for hB1R observed between 68Ga-Z02176 and 18F-Z04139 in vitro, despite the fact that they achieved the two highest uptakes in the hB1R expressing tumour. Following our hypothesis, this suggests that 68Ga-Z02176 is more specific but less stable than 18F-Z04139 for hB1R binding in vivo. The previous observations demonstrate that the four tracers achieve sufficient stabilities and affinities for hB1R in vivo to noticeably the competition with a massive amount of cold standard. The imaged mice received a higher dose of tracer, and were imaged at 55 min p.i., and euthanized afterwards for tissue collection. The biodistribution mice were the mice that received a lower dose of the tracer, and euthanized at 1-h p.i. for tissue collection. For 68Ga-Z02176, the imaged and biodistribution mice received 5.86 - 8.40 and 0.58 - 1.48 MBq of the tracer, respectively. For 18F-Z04139, the imaged and biodistribution mice received 8.06 - 8.16 and 1.32 - 2.30 MBq of the tracer, respectively. The proportions of radioactivity accumulated in the kidney with those two tracers were similar. Despite a limited number (n = 3 for the imaged mice) the biodistribution data showed that uptake of 68Ga-Z02176 in the hB1R+ tumour was significantly lower  (p < 0.001) in imaged mice (21.3 ± 1.66 %ID/g) than that in biodistribution mice (32.8 ± 2.76 %ID/g). However, no uptake difference was observed for mice receiving 18F-Z04139. Since all mice were euthanized at between 60-65 min after injection, timing should not be the cause for the uptake difference. Indeed, while all the mice were euthanized between 60-65 min after injection, mice used for imaging were anesthetized and maintained under anesthesia 20 min before the ones used exclusively for biodistribution. This potentially could significantly affect the uptake of our analogs in the hB1R+ tumour in imaged mice, but this is probably unlikely given that anesthesia was started fairly late, after much of the peptide had time to distribute and clear 113  from the plasma. Another possible cause was the higher (4- to 5-fold) injected dose (mass) received by the imaged mice compared with the biodistribution mice. To confirm this hypothesis, a more comprehensive imaging/biodistribution study in mice with various amount of injected mass of 68Ga-Z02176 is needed.  However, the dose dependent contrast between the hB1R+ tumour and the non-target organs suggests that 68Ga-Z02176 might be susceptible to specific activity, and this parameter may need to be controlled to maximize the potential of this radiotracer to detect hB1R expressing tumours in vivo.   7.5 Conclusion  In this chapter, we compared four coordination methods using the most favorable features determined previously to design a radiolabeled [Leu9, desArg10]KD based antagonist for hB1R imaging using PET. The results confirmed the benefit of combining an additional positive charge to the B9958 amino-acid sequence containing an extra lysine at the N-terminus, and the broad variety of isotopes and applications that can be used with these probes. Despite the fact that DOTA and AlF-NODA-Mpaa coordinated tracers achieved high uptake (> 22.6 %ID/g) and similar contrasts, the corresponding 68Ga-Z02176 and 18F-Z04139 had excellent properties for the diagnosis of hB1R expressing tumours. This work also demonstrates that the coordination method significantly impacts the accumulation of hB1R targeted peptides in vivo.     114  Chapter 8: Summary and Conclusion  The objective of this thesis was to design, synthesize and evaluate novel radiopeptides able to bind selectively to the hB1R in order to target cancer associated chronic inflammation for noninvasive diagnostic and therapeutic purposes. The sequences and characteristic values of the different tracers studied is presented in Table 8.1. Various radioisotopes and chelates attached targeting moieties can be used for PET and SPECT imaging of specific molecules expressed in cancer cells. These tools can be used in combination with X-ray tomography for anatomical localization. PET/CT is an imaging modality that is now routinely used to detect and stage many cancers, as well as localize sites of disease recurrence. PET/CT clinical imaging uses mainly 18F for 18F-FDG synthesis. 18F emits a low energy positron (0.65 MeV) with a short particle range (mean range in water  0.6 mm), which maintains the spatial resolution close to the typical resolution of modern PET/CT scanners ( 4.5 mm)146. However, the conjugation of 18F with peptides using classical radiofluorination chemistry is time consuming, which leads to significant loss of available radiotracer due to the 110 min half-life147. As a consequence, we performed the labelling of our peptides using the DOTA chelator coordinated with 68Ga. 68Ga has a shorter half-life than 18F (68 min), and higher positron energy (1.9 MeV) with a longer positron range (mean range in water  2.2 mm) which degrades spatial resolution. However, 68Ga is available through a long-lived radionuclide generator from its parent 68Ge radioisotope. Despite high prices and the need of pre-elutions to limit zinc accumulation, such generators can conveniently be purchased every 9 months, thus avoiding the need for a cyclotron in the vicinity.  Labelling with 68Ga is significantly quicker than classical fluorination, and this radioisotope is a useful alternative to 18F labelling. A recently introduced radiolabeling technique takes advantage of the high affinity of 115  fluoride for aluminum, to chelate 18F-Al complexes using the NODA chelator148, which can also be used for radiolabeling with 68Ga 149. Finally, we prepared an 18F labelled KD analog via the novel single-step 18F-19F exchange reaction on an ammoniomethyl-trifluoroborate motif (AmBF3)150.  In Chapter 3, we evaluated the impact of metabolic stability on the suitability of [Leu9, des-Arg10]KD analogs to enable in vivo imaging of hB1R expressing tumours by PET imaging. Natural and unnatural peptidic analogs were labelled with 68Ga using DOTA, and compared in vitro and in vivo for their ability to bind HEK293T::hB1R cells. The in vitro affinity for hB1R was improved by the labelling (68Ga-P03083). The substitution of aminoacids at specific cleavage points in the peptide (68Ga-SH01078 and 68Ga-P03034) decreased binding affinity, which remained in the nanomolar range. All the analogs were quickly and exclusively cleared from the circulation through the renal pathway. Despite achieving similar high yields and specific activities, the natural sequence of 68Ga-P03083 was able to achieve contrast between the hB1R expressing tumour and the background tissues only in presence of phosphoramidon, a peptidase inhibitor. Replacement of amino-acids with unnatural analogs improved the metabolic stability of 68Ga-SH01078 and 68Ga-P03034 sufficiently to enable visualization of hB1R expressing tumours, demonstrating the feasibility of this approach. In Chapter 4, we synthesized and compared three DOTA-PEG2 analogs carrying different amino-acid sequences, and evaluated them in vitro and in vivo to determine the most favorable unnatural amino-acid sequence for hB1R imaging. 68Ga-P04158 and 68Ga-Z02090 were synthesized with similar radiochemical properties as 68Ga-P03034, and achieved a higher affinity in vitro for hB1R, as expected from the high affinity of the unconjugated peptides from which they were derived. While following the previously observed renal excretion pathway, the two new radiotracers accumulated drastically more than 68Ga-P03034 in the kidney, but also achieved 116  significantly higher uptake and contrast relative to background in hB1R expressing tumours, with favorable imaging properties compared to the peptides studies in Chapter 3. In Chapter 5, we investigated the effect of different spacing linkers using the same amino-acid sequence and the same chelator to improve the binding to hB1R in vitro and in vivo. The four tracers were synthesized with similar radiochemical properties, and behaved as hB1R antagonists in vitro. However, the cationic linker improved the in vitro affinity for hB1R, as well as the uptake in h1BR expressing tumours, while renal excretion was not affected. In addition, 68Ga-P04168 achieved a higher metabolic stability in vivo. This confirmed the overall beneficial impact of an additional positive charge on the pharmacokinetics of KD analogs, and in particular on hB1R binding in vivo, supporting the importance of systematically evaluating each building block of these radiotracers. In Chapter 6, we applied the previously described solid-phase synthesis and DOTA labelling on an agonist analog of [Leu9, desArg10]KD and compared it with its antagonist homolog in vitro and in vivo. The two tracers varied only by the last C-terminus amino-acid, and were synthesized with similar radiochemical properties. The two tracers achieved identical affinities for h1BR in vitro. In addition, 68Ga-Z01115 triggered hB1R signaling in the same manner as the endogenous agonist [des-Arg10]KD, confirming its activity after radiolabeling. The unnatural agonist analog 68Ga-Z01115 was significantly more stable than 68Ga-SH01078 in vivo, and achieved a significantly higher uptake in hB1R expressing tumours in vivo, and improved contrast compared to non-target organs and tissues. In Chapter 7, we combined the most favorable features for hB1R binding evaluated in the previous chapters, and compared the effect of three alternative labelling techniques on hB1R binding in vitro and in vivo. Two 18F and two 68Ga labelled Pip-B9958 analogs were synthesized and compared in vitro and in vivo. Radiolabeling of the four peptides was achieved in 117  approximately the same time, and each compound was available in sufficient yield and specific activity for PET/CT imaging. The four resulting tracers, despite significant variations, bound hB1R with nanomolar affinities. Biodistribution and PET imaging showed maximal uptake in the hB1R expressing tumour and maximal contrast with 68Ga-DOTA-Pip-B9958 (68Ga-Z02176) and 18F-Al-NODA-Mpaa-Pip-B9958 (18F-Z04139). 68Ga-Z02176 had the highest affinity and lowest stability in vivo while 18F-Z04139 had the lowest affinity and the highest stability in vivo. The best tracers we obtained achieved subnanomolar binding affinity values for hB1R in vitro. Improved affinity did not necessarily translate into higher tumour accumulation, suggesting that improving the metabolic stability is the main parameter to obtain an optimal contrast between target and non-target organs in vivo. While different time points might provide higher contrasts using the same tracers, coadministration of phosphoramidon has already been used successfully to enhance the imaging properties of existing imaging tracers and was easily applicable for our purpose151. The results from Chapter 4 and 7 suggest that the stability of our analogs could also be improved by a structural modification consisting in extending the length of the spacing motif by combining or repeating the most favorable linkers described (e.g. PEG2 or Pip repeat, PEG2-Pip, Pip-PEG2). Expression of hB1R at the plasma membrane has recently been shown to require the receptor to form homo-oligomers during its cytoplasmic maturation152, and suggests that dimers of our existent tracers could be superior imaging agents, and also have a therapeutic effect on lung92 and prostate cancer93.     118  Peptide Sequence Ki (nM) Overall charge LogD7.4 Plasma stability (% intact, 5 min p.i.) Tissue uptake (1-h p.i., %ID/g) Chapter in vitro in vivo hB1R+ tumor Kidney Bradykinin (BK) Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 5.7a +1 - - < 3% - - 1 Kallidin (KD) Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 7.4a +2 - - < 3% - - 1 [Leu9,desArg10]KD Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu 8.9b +1 - - - - - 1 68Ga-P03083 68Ga-DOTA-Ahx-[Leu9,des-Arg10]KD 2.6 ± 0.7 +1 -2.8 ± 0.1 96 - 0.8 ± 0.2 5.0 ± 0.9 3 68Ga-Z01115 68Ga-DOTA-Ahx-[Hyp4,Cha6,des-Arg10]KD 25.4 ± 5.1 +1 -3.8 ± 0.2 99.5 50.7 ± 4.5 5.7 ± 0.6 4.6 ± 1.3 6 68Ga-SH01078 68Ga-DOTA-Ahx-[Hyp4,Cha6,Leu9,des-Arg10]KD 28 ± 4.9 +1 -2.7 ± 0.3 99 9 ± 2 2.0 ± 0.5 3.7 ± 1.3 3,4,6 68Ga-P04115 68Ga-DOTA-GlyGly-[Hyp4,Cha6,Leu9,des-Arg10]KD 11 ± 2.5 +1 -3.0 ± 0.2 92 11 ± 3 2.0 ± 0.8 4.0 ± 2.4 4 68Ga-P03034 68Ga-DOTA-dPEG2-[Hyp4,Cha6,Leu9,des-Arg10]KD 16 ± 1.9 +1 -2.8 ± 0.1 99 8 ± 2 2.4 ± 0.6 4.3 ± 1.9 3,4,5 68Ga-P04168 68Ga-DOTA-Pip-[Hyp4,Cha6,Leu9,des-Arg10]KD 3.6 ± 0.2 +2 -2.8 ± 0.1 98 17 ± 4 4.3 ± 1.2 4.1 ± 1.0 4 B9858 Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic 10.1b +2 - - - - - 5 B9958 Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-D-Tic-Cpg 0.089 +2 - - - - - 5,7 68Ga-P04158 68Ga-DOTA-dPEG2-B9858 1.5 ± 1.9 +2 -2.5 ± 0.2 99 - 17.2 ± 5.0 68.4 ± 8.2 5 68Ga-Z02090 68Ga-DOTA-dPEG2-B9958 1.1 ± 0.8 +2 -2.7 ± 0.1 93 - 15.3 ± 3.7 48.4 ± 8.7 5 68Ga-Z02137 68Ga-NODA-Mpaa-Pip-B9958 2.6 ± 0.7 +4 -3.4 ± 0.0 - - 14 ± 4.9 85.2 ± 12.1 7 68Ga-Z02176 68Ga-DOTA-Pip-B9958 2.5 ± 0.8 +3 -4.2 ± 0.0 - 93.7 ± 0.6 28.9 ± 6.2 90.9 ± 22.8 7 18F-Z04139 18F-Al-NODA-Mpaa-Pip-B9958 14 ± 7.4 +3 -3.9 ± 0.2 - 95.8 ± 1.0 22.6 ± 3.4 101 ± 14.4 7 18F-L08060 18F-AmBF3-Mta-Pip-B9958 0.5 ± 0.3 +3  95 - 3.7 ± 1.1 30.8 ± 6.4 7  Table 8.1: Data of affinity, overall charge, and selected tissue uptake and B1R+ tumor-to-background contrast ratios of previously reported B1R-targeting peptides. apEC50. bpIC50. 119  Our successive studies demonstrate a lack of correlation between in vitro plasma stability assays and in vivo metabolic stability and indicate that our observations may differ drastically when applied to humans. The consistent lack of relevance of the successive results is well exemplified by the superior in vitro stability achieved by the antagonist 68Ga-SH01078 in comparison with the agonist 68Ga-Z01115, while the latter one achieved a five folds higher metabolic stability in vivo. This demonstrates that the set of kininases present in vivo is different and broader, and cannot be replicated using frozen plasma. Besides, it is not known if the homology between the human KD and the murine BK and KD is sufficient to conclude that murine circulating kinins compete with the binding of our tracers to the hB1R, or that the lack of side organ uptake observed in mice can be confidently repeated in human. In addition to this, it is possible that the four unnatural aminoacid sequences we used to generate KD analogs used for hB1R targeting are equipotent hB2R ligands89. As a consequence, BK analogs designed in the same manner could represent a new set of potent hB2R targeting agents. The expression model we used allowed the direct comparison of a hB1R positive and negative tumours originating from the same cell line. Such artificial models are commonly used in preclinical studies for some radiotracers such as somatostatin receptor ligands142, but due to the artificial expression of the hB1R, may not reflect the actual expression levels that may be encountered in the clinical setting. hB1R is an inducible receptor, its expression at the plasma membrane can be triggered in competent cells using its natural agonist [desArg10]KD or inflammatory cytokines like TNFα and  IL-1β39,46. Considering that hB1R overexpression in cancer is also induced by the host inflammatory response and not by a dysregulation of cancer cells phenotype, evaluation of human cancer cell lines hB1R expression level should be performed in the presence of an inflammatory stimulus to closely the actual tumoral context. 120  All the tracers evaluated followed the same rapid renal excretion and accumulated significantly in the kidney, while the uptake in all the non-target organs was consistently minimal, including in the hepatobiliary excretion tract (liver and intestines). This clearance pathway limits advantageously the prolonged exposure of the patient abdomen to radiation; it also allows the detection of primary and/or metastatic lesions within this area, making these tracers ideal for diagnostic PET/CT imaging. However, the elevated renal uptake places the kidney as the dose-limiting organ for application of these tracers in Peptide Receptor Radiotherapy (PRRT) using alpha or beta emitter and targeted chemotherapeutic therapies. While radiation mitigation using amifostine gives promising results, the reabsorption of peptidic analogs can be reduced by increasing the urinary excretion of small peptides with a co-injection of amino-acids or colloidal peptide mixtures. Different biocompatible amino-acid or peptide blends provide excess amounts of unlabeled amino-acids that compete with the receptor mediated reabsorption of the radioactive peptides and amino-acids that takes place in the proximal tubule114,153,154,155,156. While our first aim was to target cancer associated chronic inflammation, hB1R targeting using a peptidic antagonist is potentially applicable to all the pathologies known to be associated with hB1R overexpression, such as inflammation or infection. hB1R is a potential target of interest for cardiovascular disease treatment157,158, as it is expressed in human atheroma. Even if this receptor does not seem to have any effect on vasomotricity and fibrinolysis in this pathology159, the expression of hB1R could be used to detect early stages of this pathology160,161,162. Recently, it has been shown that Amyloid-β peptide deposition in cerebral vasculature (Amyloid angiopathy) atherosclerosis was regulated by hB1R163 and could be used to treat Alzheimer’s disease and other neurodegenerative pathologies. Potential applications include cancer imaging as well as imaging other pathophysiological conditions such as infection164,165,166 121  and diabetes167,168 that have been reported to induce upregulation of B1R expression in animal models. Translation to phase I clinical trials will require first the assessment of the toxicity of the selected radiopharmaceutical. Such compounds carry molecular motifs and radioactive isotopes, and thus require differentiating the toxicity induced by the radiations and the toxicity induced by chemical reactions. Radiation exposure of tissues can be generated from mice biodistribution studies with 68Ga-Z02176, and extrapolated to human to evaluate the absorbed dose during PET imaging with this radiopharmaceutical. Chemical toxicity evaluations have to be performed to determine the pharmacological dose limitations before first-in-man studies. The stability of the isotope coordination and of the peptidic moiety are required to determine the toxicity resulting from the radiopharmaceutical degradation. Cold standards can be readily used to evaluate the toxicity strictly related to the chemical interactions with the radiopharmaceutical as well as the cellular pathways involved169.  Imaging radiotracers are typically used as single dose and would not be expected to be tested for long-term chemical toxicity (teratogenicity, carcinogenicity, mutagenicity)170. Finally, interspecies differences have to be expected and anticipated to assess the most adapted toxicity assays to be performed171.  Summary  In summary, we successfully imaged hB1R expressing tumours by PET/CT using radiolabeled metabolically stable analogs of [desArg10]Kallidin, the prototypical hB1R ligand. The radiotracers we synthesized had suitable pharmacokinetics for application in human patients as a targeted diagnostic imaging agent. hB1R targeting peptides could be used to detect non-122  invasively the early and late stages of cancer development in tissues (e.g. prostate and breast), and monitor the efficacy of anticancer therapy. The evaluation of hB1R expression level can also be used to anticipate the treatment response of tumours or damaged tissues to hB1R-targeted drugs.                      123  Bibliography 1. Seam P, Juweid ME, Cheson BD: The role of FDG-PET scans in patients with lymphoma. Blood 2007; 110(10):3507-3516. 2. Saha GB. Fundamentals of nuclear pharmacy: Sixth edition. Springer 2010. 3. Van der Veldt AA, Smit EF, and Lammertsman AA. Positron Emission Tomography as a method for measuring drug delivery to tumors in vivo: the example of [11C]docetaxel. Frontiers in Oncology 2013; 3:208. 4. Juweid ME, and Cheson BD. Positron-emission tomography and assessment of cancer therapy. The New England Journal of Medicine 2006; 354(5):496-507. 5. Juweid ME, Stroobants S, Hoekstra OS, Mottaghy FM, Dietlein M, Guermazi A, Wiseman GA, Kostakoglu L, Scheidhauer K, Buck A, Naumann R, Spaepen K, Hicks RJ, Weber WA, Reske SN, Schwaiger M, Schwartz LH, Zijlstra JM, Siegel BA, and Cheson BD. Use of positron emission tomography for response assessment of lymphoma: consensus of the imaging subcommittee of international harmonization project in lymphoma. Journal of Clinical Oncology 2007; 25(5):571-578. 6. Lardinois D, Weder W, Hany TF, Kamel EM, Korom S, Seifert B, von Schulthess GK, and Steinert HC. Staging of non-small-cell lung cancer with integrated positron-emission tomography and computed tomography. The New England Journal of Medicine 2003; 348(25):2500-2507. 7. Sweet WH, and Brownell GL. Localization of brain tumors with positron emitters. Nucleonis 1953 11:40-45. 8. Sweet WH, and Brownell GL. Localization of intracranial lesions by scanning with positron-emitting arsenic. The Journal of American Medical Association 1955 157(14):1183-1188. 9. Saha GB. Basics of PET imaging. Springer 2005.  10. Lewellen TK. Recent Developments in PET detector technology. Physics in Medical Biology 200 53(17):287-317. 11. Kuntner C., and Stout D. Quantitative preclinical PET imaging: opportunities and challenges. Frontiers in Physics 2014; 2:12. 12. Rahmim A and Zaidi H. PET vs SPECT: strengths, limitations and challenges. Nuclear medicine communications 2008; 29:193-207. 13. Jacobs AH, Li H, Winkeler A, Hilker R, Knoess C, Ruger A, Galldiks N, Schaller B, Sobesky J, Kracht L, Monfared P, Klein M, Vollmar S, Bauer B, Wagner R, Graf R, Wienhard K, Herholz K, and Heiss WD. PET-based molecular imaging in neuroscience. European Journal of Nuclear Medicine and Molecular Imaging 2003; 30:1051-1065. 14. Meyer JH. Imaging the serotonin transporter during major depressive disorder and antidepressant treatment. Journal of Psychiatry and Neurosciences 2007; 32:86-102. 15. Paquette M, Tremblay S, Bénard F, and Lecomte R. Quantitative hormone therapy follow-up in an ER+/ERalphaKD mouse tumor model using FDG and [11C]-methionine PET imaging. European Journal of Nuclear Medicine and Molecular Imaging Research 2012; 2:61. 16. Chang J.M., Lee H.J., Goo J.M., Lee HY., Lee J.J., Chung JK., Im JG. False positive and false negative FDG-PET scans in various thoracic diseases. Korean Journal of Radiology 2006; 7:57-69. 124  17. Jonson SD and Welch MJ. PET imaging of breast cancer with fluorine-18 radiolabeled estrogens and progestins. The Quarterly Journal of Nuclear Medicine and Molecular Imaging 1998; 42(1):8-17. 18. Van Kruchten M, Hospers GAP, Glaudemans AWJM, Hollema H, Arts HJG, and Reyners AKL. Positron emission tomography imaging of oestrogen receptor-expression in endometrial stromal sarcoma supports oestrogen receptor-targeted therapy: Case report and review of the literature. European Journal of Cancer 2013; 49:3850-3855. 19. Smith TAD. Towards detecting the HER-2 receptor and metabolic changes induced by HER-2-targeted therapies using medical imaging. The British Journal of Radiology 2010; 83: 638-644. 20. Weineisen M, Schottelius M, Simecek J, Baum RP, Yildiz A, Beykan S, Kulkarni HR, Lassmann M, Klette I, Eiber M, Schwaiger M, and Wester HJ. 68Ga-and 177Lu-labeled PSMA I&T: optimization of a PSMA targeted theranostic concept and first proof of concept human studies. Journal of Nuclear Medicine 2015; 56(8):1169-1176. 21. Maurer T, Weirich G, Schottelius M, Weineisen M, Frisch B, Okur A, Kübler H, Thalgott M, Nassir Navab, Schwaiger M, West HJ, Gschwend JE, and Eiber M. Prostate-specific membrane antigen-radioguided surgery for metastatic lymph nodes in prostate cancer. European Urology 2015; 6193:6198. 22. Bison AM, Konijnenberg MW, Melis M, Pool SE, Bernsen MR, Teunissen JJM, Kwekkeboom DJ, and De Jong M. Peptide receptor radionuclide therapy using radiolabeled somatostatin analogs: focus on future developments. Clinical and Translational Imaging 2014; 2:55-66. 23. Da Costa PLN, Sirois P, Tannock IF, and Chammas R. The role of kinin receptors in cancer and therapeutic opportunities. Cancer Letters 2014; 345:27-38. 24. Valdivia-Silva J, Medina-Tamayo J, and Garcia-Zepeda EA. Chemokine-derived peptides: novel antimicrobial and antineoplastic agents. International Journal of Molecular Sciences 2015; 16:12958-12985. 25. Kashuba E, Bailey J, Allsup D, and Cawkwell L. The kinin-kallikrein system: physiological roles, pathophysiology and its relationship to cancer biomarkers. Biomarkers 2013; 18:279-93. 26. Stewart JM. Bradykinin antagonists: discovery and development. Peptides 2004; 25:527-532. 27. Figueroa CD, Ehrenfeld P, and Bhoola KD. Kinin receptors as targets for cancer therapy. Expert Opinion in Therapeutic Targets 2012; 16(3):299-312. 28. Su BJ. Different cross-talk sites between the renin-angiotensin and the kallikrein-kinin systems.  Journal of Renin-Angiotensin-Aldosterone System 2013; 1-10. 29. Stadnicki A. Intestinal tissue kallikrein-kinin system in inflammatory bowel disease. Inflammatory Bowel Diseases 2011; 17(2):645-655. 30. Phipps JA and Feener EP. The kallikrein-kinin system in diabetic retinopathy: lessons for the kidney. Kidney International 2008; 73:114-1119. 31. Griffon C, Miternique-Grosse A, Hudlett P, and Stephan D. Système kinine-kallicréine et maladies cardiovasculaires: renaissance d’une entité centenaire. Médecine Thérapeutique: Cardiologie 2005; 1(1):35-47. 32. Moreau ME, Garbacki N, Molinaro G, Brown NJ, Marceau F and Adam A. The kallikrein-kinin system: current and future pharmacological targets. Journal of Pharmacological Sciences 2005; 99:6-38. 125  33. Bhoola KD, Figueroa CD, and Worthy K. Bioregulation of kinins; kallikreins, kininogens, and kininases. Pharmacological Reviews 1992; 44(1):1-80. 34. Abraham WM, Scuri M, and Farmer SG. Peptide and non-peptide bradykinin receptor antagonists: role in allergic airway disease. European Journal of Pharmacology 2006; 533:215-221. 35. Yousef GM and Diamandis EP. The new human tissue kallikrein gene family: structure, function, and association to disease. Endocrine Reviews 2001; 22(2): 184-204. 36. Leeb-Lundberg LMF, Marceau F, Muller-Esterl W, Pettibone DJ, and Zuraw BL. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacological Reviews 2005; 57:27–77. 37. Kuoppala A, Linstedt KA, Saarinen J, Kovanen PT, and Kokkonen JO. Inactivation of bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma. American Journal of Physiology: Heart and Circulatory Physiology 2000; 278:1069-1074. 38. Barnes PJ, Chung KF, and Page CP. Inflammatory Mediators of Asthma: An Update. Pharmacological Reviews 1998; 50(4):517-596. 39. Menke JG, Borkowski JA, Bierilo KK, MacNeil T, Derrick AW, Schneckg KA, Ransoms RW, Strader CD, Linemeyer DL, and Hess JF. Expression Cloning of a Human B1 Bradykinin Receptor. The Journal of Biological Chemistry 1994; 269(34):21583-21586. 40. Ni A, Chao L and Chao J. Transcription factor nuclear factor κB regulates the inducible expression of the human B1 receptor gene in inflammation. The Journal of Biological Chemistry 1998; 273(5):2784-2791. 41. Bellucci F, Cucchi P, Santicioli P, Lazzeri M, Turini D, and Meini S. Characterization of kinin receptors in human cultured detrusor smooth muscle cells. British Journal of Pharmacology 2007; 150:192-199. 42. Phagoo SB, Reddi K, Anderson KD, Leed-Lundberg LMF and Warburton D. Bradykinin B1 receptor up-regulation by interleukin-1β and B1 agonist occurs through independent and synergistic intracellular signaling mechanisms in human lung fibroblasts. Journal of Pharmacology and Experimental Therapeutics 2001; 298(1):77-85. 43. Rodrigues ES, Silva RF, Martin RP, Oliveira SM, Nakaie CR, Sabatini RA, Merino VF, Pesquero JB, Bader M, and Shimuta SI. Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors. Peptides 2013; 42:1-7. 44. Phagoo SB, Poole S and Leed-Lundberg LMF. Autoregulation of bradykinin receptors: agonists in the presence of interleukin-1beta shift the repertoire of receptor subtypes from B2 to B1 in human lung fibroblasts. Molecular Pharmacology 1999; 56:325-333. 45. Guevara-Lora I, Florkowska M, and Kozik A. Bradykinin-related peptides up-regulate the expression of kinin B1 and B2 receptor genes in human promonocytic cell line U937. Acta Biochimica Polonica 2009; 56(3):515-522. 46. Enquist J, Skröder C, Whistler JL, and Leed-Lundberg LMF. Kinins Promote B2 Receptor Endocytosis and Delay Constitutive B1 Receptor Endocytosis. Molecular Pharmacology 2007; 71:494-507. 47. Austin CE, Faussner A, Robinson HE, Chakravarty S, Kyle DJ, Bathon JM, and Proud D. Stable Expression of the Human Kinin B1 Receptor in Chinese Hamster Ovary 126  Cells: Characterization of ligand binding and effector pathways. The Journal of Biological Chemistry 1997; 272(17):11420-11425. 48. Campos MM, Leal PC, Yunes RA, and Calixto JB. Non-peptide antagonists for kinin B1 receptors: new insights into their therapeutic potential for the management of inflammation and pain. Trends in Pharmacological Sciences 2006; 27:646-651. 49. Calixto JB, Medeiros R, Fernandes ES, Ferreira J, Cabrini DA, Campos MM. Kinin B1 receptors: key G-protein-coupled receptors and their role in inflammatory and painful processes. British Journal of Pharmacology 2004; 143:803-818. 50. Valdes G, Kaufmann P, Corthorn J, Erices R, Brosnihan KB, and Joyner-Grantham J. Vasodilatators factors in the systemic and local adaptations to pregnancy. Reproductive Biology and Endocrinology 2009; 7:79. 51. Erices R, Corthorn J, Lisboa F and Valdès G. Bradykinin promotes migration and invasion of human immortalized trophoblasts. Reproductive Biology and Endocrinology 2011; 9:97. 52. Diamandis EP and Yousef GM. Human tissue kallikreins: a family of new cancer biomarkers. Clinical Chemistry 2002; 48(8):1198-1205.  53. Diamandis EP, Yousef GM, Soosaipillai AR, Grass L, Porter A, Little S, and Sotiropoulou G. Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications. Clinical Biochemistry 2000; 33:369–75. 54. Diamandis EP, Yousef GM, Soosaipillai AR, Bunting P. Human kallikrein 6 (zyme/protease M/neurosin): a new serum biomarker of ovarian carcinoma. Clinical Biochemistry 2000; 33:579-583. 55. Luo LY, Katsaros D, Scorilas A, Fracchioli S, Piccinno R, Rigault de la Longrais IA, Howarth DJC, and Diamandis EP. Prognostic value of human kallikrein 10 expression in epithelial ovarian carcinoma. Clinical Cancer Research 2001; 7:2372–9. 56. Luo L, Bunting P, Scorilas A, Diamandis EP. Human kallikrein 10: a novel tumor marker for ovarian carcinoma? Clinica Chinica Acta 2001; 306:111–8. 57. Diamandis EP, Okui A, Mitsui S, Luo LY, Soosaipillai A, Grass L, Nakamura T, Howarth DJC, and Yamaguchi N. Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma. Cancer Research Res 2002; 62:295–300. 58. Wang M, Long RE, Comunale MA, Junaidi O, Marrero J, Di Bisceglie AM, Block TM, and Mehta AS. Novel fucosylated biomarkers for the early detection of hepatocellular carcinoma. Cancer Epidemiology, Biomarkers and Prevention 2009; 18(6):194-1921. 59. Villanueva J, Shaffer DR, Philip J, Chaparro CA, Erdjument-Bromage H, Olshen AB, Fleisher M, Lilja H, Brogi E, Boyd J, Sanchez-Carbayo M, Holland EC, Cordon-Cardo C, Scher HI, and Tempst P. Differential exoprotease activities confer tumor-specific serum peptidome patterns. Journal of Clinical Investigation 2006; 116:271–284. 60. Maeda H, Mastumura Y and Kato H. Purification and identification of [Hydroxyprolyl3] Bradykinin in ascitic fluid from a patient with gastric cancer. The Journal of Biological Chemistry 1988; 263(31):16051-16054. 61. Mastumura Y, Maruo K, Kimura M, Yamamoto T, Konno T, and Maeda H. Kinin-generating cascade in advanced cancer patients and in vitro study. Journal of Cancer Research 1991; 82(6):732-41. 62. Wang G, Ye Y, Zhang X, and Song J. Bradykinin stimulates IL-6 production and cell invasion in colorectal cancer cells. Oncology Reports 2014; 32:1709-1714. 127  63. Zelawski W, Machnik G, Nowaczyk G, Plewka D, Lorenc Z, Sosada K and Stadnicki A. Expression and localization of kinin receptors in colorectal polyps. International Immunopharmacology 2006; 6:997-1002. 64. Fujita M, Andoh T, Ohashi K, Akira A, Saiki I, and Kuraishi Y. Roles of kinin B1 and B2 receptors in skin cancer pain produced by orthotopic melanoma inoculation in mice. European Journal of Pain 2010; 14:588-594. 65. Moodley R, Snyman C, Odhav B and Bhoola KD. Visualization of transforming growth factor-β1, tissue kallikrein, and kinin and transforming growth factor-β receptors on human clear-cell renal carcinoma cells. The Journal of Biological Chemistry 2005; 386:375-382. 66. Dlamini ZD and Bhoola KD. Upregulation of tissue kallikrein, kinin B1 receptor, and kinin B2 receptor in mast and giant cells infiltrating esophageal squamous cell carcinoma. Journal of Clinical Pathology 2005; 58:915-922. 67. Chee J, Singh J, Naran A, Misso NL, Thompson PJ and Bhoola KD. Novel expression of kallikreins, kallikrein-related peptidases and kinin receptors in human pleural mesothelioma. The Journal of Biological Chemistry 2007; 388:1235-1242. 68. Bhoola KD, Thompson PJ, Misso NL, Naran A and Chee J. Expression of tissue and plasma kallikreins and kinin B1 and B2 receptors in lung cancer. The Journal of Biological Chemistry 2008; 389:1225-1233. 69. Yang WH, Chang JT, Hsu SF, Li TM, Cho DY, Huang CY, Fong YC and Tang CH. Bradykinin enhances cell migration in human chondrosarcoma cells through BK receptor signaling pathways. Journal of Cellular Biochemistry 2010; 109:82-92. 70. Rittenhouse HG, Finlay JA, Mikolajczyk SD, and Partin AW. Human Kallikrein 2 (hK2) and prostate-specific antigen (PSA): two closely related, but distinct, kallikreins in the prostate. Critical Reviews in Clinical Laboratory Sciences 1998; 35:275–368. 71. Hsieh ML, Charlesworth MC, Goodmanson M, Zhang S, Seay T, Klee GG, Tindall DJ and Young CYF. Expression of human prostate-specific glandular kallikrein protein (hK2) in the breast cancer cell line T47D. Cancer Research 1997; 57:2651-2656. 72. Ehrenfeld P, Manso L, Pavicic MF, Matus CE, Borquez C, Lizama A, Sarmiento J, Poblete MT, Bhoola KD, Naran A, and Figueroa CD. Bioregulation of kallikrein-related peptidases 6,10 and 11 by the kinin B1 receptor in breast cancer cells. Anticancer Research 2014; 34:6925-6938. 73. Ehrenfeld P, Conejeros I, Pavicic MF, Matus CE, Gonzales CB, Quest AFG, Bhoola KD, Poblete MT, Burgos RA and Figueroa CD. Activation of kinin B1 receptor increases the release of metalloproteases-2 and -9 from both estrogen-sensitive and –insensitive breast cancer cells. Cancer Letters 2011; 301:106-118. 74. Molina L, Matus CE, Astroza A, Pavicic F, Tapia E, Toledo C, Perez JA, Nualart F, Gonzales CB, Burgos RA, Figueroa CD, Ehrenfeld P, and Poblete MT. Stimulation of the bradykinin B1 receptor induces the proliferation of estrogen-sensitive breast cancer cells and activates the ERK1/2 signaling pathway. Breast Cancer Research and Treatment 2009; 118:499-510. 75. Esseghir S, Reis-Flho JS, Kennedy A, James M, O’Hare MJ, Jeffery R, Poulsom R and Isacke CM. Identification of transmembrane protein as potential markers and therapeutic targets in breast cancer by a screen for signal sequence encoding transcripts. The Journal of Pathology 2006; 210:420-430. 128  76. Egidi MG, Cochetti G, Serva MR, Guelfi G, Zampinin D, Mechelli L, and Mearini E. Circulating microRNAs and kallikreins before and after radical prostatectomy: are they really prostate cancer markers? BioMed Research International 2013; 1-11. 77. Charlesworth MC, Young CYF, Klee GG, Saedi MS, Mikolajczyk SD, Finlay JA and Tindall DJ. Detection of a prostate-specific protein, human glandular kallikrein (hK2), in sera of patients with elevated prostate-specific antigen levels. Urology 1997; 49:487-493. 78. Mikolajczyk SD, Millar LS, Marker KM, Rittenhouse HG, Wolfert RL, Marks LS, Charlesworth MC, and Tindall DJ. Identification of a novel complex between human kallikrein 2 and protease inhibitor -6 in prostate cancer tissue. Cancer Research 1999; 59:3927-3930. 79. Charlesworth MC, Young CYF, Miller VM and Tindall DJ. Kininogenase activity of prostate-derived human glandular kallikrein (hK2) purified from seminal fluid. Journal of Andrology 1999; 20(2):220-229. 80. Taub JS, Guo R, Leed-Lundberg LMF, Madden JF and Daaka Y. Bradykinin receptor subtype 1 expression and function in prostate cancer. Cancer Research 2003; 63:2037-2041. 81. Barki-Harrington L, Bookout AL, Wang G, Lamb ME, Leed-Lundberg LMF and Daaka Y. Requirement for direct cross-talk between B1 and B2 kinin receptors for the proliferation of androgen-insensitive prostate cancer PC3 cells. Biochemical Journal 2003; 371:581-587. 82. Raidoo DM, Sawant S, Mahabeer R, and Bhoola KD. Kinin receptors are expressed in human astrocytic tumor cells. Immunopharmacology. 1999; 43:255–263. 83. Lu DY, Leung YM, Huang SM and Wong KL. Bradykinin induced cell migration and COX-2 production mediated by the bradykinin B1 receptor in glioma cells. Journal of Cellular Biochemistry 2010; 1(10):141-150. 84. Côté J, Bovenzi V, Savard M, Dubuc C, Fortier A, Neugebauer W, Tremblay L, Müller-Esterl W, Tsanaclis AM, Lepage M, Fortin D and Gobeil Jr, F. Induction of selective blood-tumor barrier permeability and macromolecular transport by a biostable kinin B1 receptor agonist in a glioma rat model. PLoS One 2012; 7(5):1-17. 85. Nicoletti NF, Erig TC, Zanin RF, Brandao Pereira TC, Bogo MR, Campos MM, and Morrone FB. Mechanisms involved in kinin-induced glioma cells proliferation: the role of ERK1/2 and PI3K/Akt pathways. Journal of Neuro-Oncology 2014; 120:235-244. 86. Toledo C, Matus CE, Barraza X, Arroyo P, Ehrenfeld P, Figueroa CD, Bhoola, K.D., Del Pozo, M., and Poblete, M.T. Expression of HER2 and bradykinin B1 receptors in precursor lesions of gallbladder carcinoma. World Journal of Gastroenterology 2012; 21,18 (11):1208-1215. 87. Charignon D, Späth P, Martin L, and Drouet C. Icatibant, the bradykinin B2 receptor antagonist with target to the interconnected kinin systems. Expert Opinion on Pharmacotherapy 2012; 13 (15):2233-2247. 88. Levesque L, Harvey N, Rioux F, Drapeau G, and Marceau F. Development of a binding assay for the B1 receptors for kinins. Immunopharmacology 1995; 29:141-147. 89. Stewart JM, Gera L, Chan DC, Whalley ET, Hanson WL, and Zusack JS. Potent, long-acting bradykinin antagonists for a wide range of applications. Canadian Journal of Physiology and Pharmacology 1997; 75:719-724. 129  90. Drube S, and Liebmann C. In various tumor cell lines the peptide bradykinin B2 receptor antagonist, Hoe 140 (Icatibant), may act as mitogenic agonist. British Journal of Pharmacology 2000; 131:1553–1560. 91. Chan D, Gera L, Stewart J, Helfrich B, Verella-Garcia M, Johnson G, Baron A, Yang J, Puck T, and Bunn Jr P. Bradykinin antagonist dimer, CU201, inhibits the growth of human lung cancer cell lines by a “biased agonist” mechanism. Proceedings of the National Academy of Sciences 2002; 99(7):4608-4613. 92. Chan DC, Gera L, Stewart JM, Helfrich B, Zhao TL, Feng WY, Chan KK, Covey JM and Bunn, Jr PA. Bradykinin antagonist dimer, CU201, inhibits the growth of human lung cancer cell lines in vitro and in vivo and produces synergistic growth inhibition in combination with other antitumor agents. Clinical Cancer Research 2002; 8:1280-1287. 93. Stewart JM, Chan DC, Simkeviciene V, Bunn PA, Jr., Helfrich B, York EJ, Taraseviciene-Stewart L, Bironaite D, and Gera L. Bradykinin antagonists as new drugs for prostate cancer. International Immunopharmacology 2002; 2:1781-1786. 94. Morissette G, Houle S, Gera L, Stewart JM, and Marceau F. Antagonist, partial agonist and antiproliferative actions of B-9870 (CU201) as a function of the expression and density of the bradykinin B1 and B2 receptors. British Journal of Pharmacology 2007; 150:369-379.  95. Cheronis JC, Whalley ET, Allen LG, Loy SD, Elder MW, Duggan MJ, Gross KL and Blodgett JK. Design, synthesis, and in vitro activity of bis(succinimido)hexane peptide heterodimers with combined B1 and B2 antagonist activity. Journal of Medicinal Chemistry 1994; 37:348-355. 96. Naidu N, Botha JH, and Naidoo S. B1 but not B2R bradykinin receptor agonist promote DU145 prostate cancer cell proliferation and migration. African Health Sciences 2014; 14(3):657-662. 97. Stahl W, Breipohl G, Kuhlmann L, Steinstrasser A, Gerhards HJ, and Scholkens BA. 99m Technetium-labeled HOE 140: a potential bradykinin B2 receptor imaging agent. Journal of Medicinal Chemistry 1995; 38:2799-801.  98. Fuchs K, Fischer K, Schwenck J, Kesenheimer C, Abbas S, Stiller D, Hauel NH, Jung B, Kneilling M, and  Pichler BJ. in vivo PET imaging of bradykinin receptor 1 (B1R) expression in a mouse model of chronic inflammation. Proceedings of the 2011 World Molecular Imaging Congress, S865. 99. Bawolak MT, Gera L, Morissette G, Bouthillier J, Stewart JM, Gobeil LA, Lodge R, Adam A, and Marceau F. Fluorescent ligands of the bradykinin B1 receptors: pharmacologic characterization and application to the study of agonist-induced receptor translocation and cell surface receptor expression. Journal of Pharmacology and Experimental Therapeutics 2009; 329:159-68. 100. Gera L, Roy C, Bawolak MT, Charest-Morin X, Marceau F. N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes. Peptides 2012; 34:433-46. 101. Talbot S, Theberge-Turmel P, Liazoghli D, Senecal J, Gaudreau P, and Couture R. Cellular localization of kinin B-1 receptor in the spinal cord of streptozotocin-diabetic rats with a fluorescent [Nα-Bodipy]-des-Arg9-bradykinin. Journal of Neuroinflammation 2009; 6:11. 130  102. Gera L, Stewart JM, Fortin JP, Morissette G, and Marceau F. Structural modification of the highly potent peptide bradykinin B1 receptor antagonist B9958. International Immunopharmacology 2008; 8:289-292.  103. Fathy DB, Kyle DJ and Leeb-Lundberg LMF. High-affinity binding of peptide agonists to the human B1 bradykinin receptor depends on interaction between the peptide N-terminal L-lysine and the fourth extracellular domain of the receptor. Molecular Pharmacology 2000; 57:171-179. 104. Regoli D, and Barabé J. Pharmacology of bradykinin and related peptides. Pharmacological Reviews 1980; 32:1. 105. Liu Z, Pourghiasian M, Bénard F, Pan J, Lin Ks, and Perrin DM. Preclinical evaluation of high-affinity 18F-trifluoroborate octreotate derivative for somatostatin receptor imaging. JNM 2014; 55:1499-1505. 106. Lasne MC, Perrio C, Rouden J, Barre L, Roeda D, Dolle F, and Crouzel C. Chemistry of β+-emitting compounds based on fluorine-18. Topics in Current Chemistry 2002; 222:201–258. 107. Pourghiasian M, Liu Z, Pan J, Zhang Z, Colpo N, Lin KS, Perrin MD, and Bénard F. 18F-AmBF3-MJ9: a novel radiofluorinated bombesin derivative for prostate cancer imaging. Bioorganic and Medicinal Chemistry 2015; 23:1500-1506. 108. Reubi JC, Erchegy J, Cescato R, Waser B, and Rivier JE. Switch from antagonist to agonist after addition of a DOTA chelator to a somastostatin analog. European Journal of Nuclear Medicine 2010; 37(8):1551-1558. 109. Liu Z, Radtke M, Wong M, Lin KS, Yapp D, and Perrin DM. Dual mode fluorescent 18F-PET tracers: efficient modular synthesis of rhodamine-[cRGD]2-[18F]-organotrifluoroborate, rapid and high yielding 18F-labeling at high specific activity with correlated in vivo PET Imaging and ex vivo fluorescence. Bioconjugate Chemistry 2014; 25:1951–1962. 110. Liu Z, Li Y, Lozada J, Pan J, Lin KS, Schäffer P, And Perrin DM. Rapid, one-step, high yielding 18F-labeling of an aryltrifluoroborate bioconjugate by isotope exchange at very high specific activity. Journal of Labelled Compounds and Radiopharmaceuticals 2012; 55:491-496. 111. Modin A, Pernow J and Lundberg JM. Phosphoramidon inhibits the vasoconstrictor effects evoked by big endothelin-1 but not the elevation of plasma endothelin-1 in vivo. Life Sciences 1991; 49:1619-1625. 112. Kukkola PJ, Savage P, Sakane Y, Berry JC, Bilci NA, Ghai RD and Jeng AY. Differential structure-activity relationships of phosphoramidon analogues for inhibition of three metalloproteases: endothelin-converting enzyme, neutral endopeptidase, and angiotensin-converting enzyme. Journal of Cardiovascular Pharmacology 1995; 26:65-68. 113. Sakamoto K, Sugimoto K, and Fujimara A. Different inhibition of enalaprilat on bradykinin metabolizing enzymes. Life Sciences 2000; 67:2159-2165. 114. Vegt E, Melis M, Eek A, de Visser M, Brom M, Oyen WJ, Gotthardt M, de Jong M, and Boermann OC. Renal uptake of different radiolabeled peptides is mediated by megalin: SPECT and biodistribution studies in megalin-deficient mice. European Journal of Nuclear Medicine and Molecular Imaging 2011; 38:623-32. 115. Nock BA, Maina T, Krenning EP, and De Jong M. "To serve and protect": enzyme inhibitors as radiopeptide escorts promote tumor targeting. The Journal of Nuclear Medicine 2014; 55:121-7. 131  116. Rolleman E.J., Melis M., Valkema R., Boerman O.C., Krenning E.P., de Jong M. Kidney protection during peptide receptor radionuclide therapy with somatostatin analogues. European Journal of Nuclear Medicine and Molecular Imaging 2010; 37:1018-1031. 117. Ura N, Carretero OA, and Erdös EG. Role of renal endopeptidase 24.11 in kinin metabolism in vitro and in vivo. Kidney International 1987; 32:507-513. 118. Guo H, and Miao Y. Introduction of an 8-aminooctanoic acid linker enhances uptake of 99mTc-labeled lactam bridge-cyclized α-MSH peptide in melanoma. The Journal of Nuclear Medicine 2014; 55:2057-2063. 119. De Visser M, Benard HF, Erion JL, Schmidt MA, Srinivasan A, Waser B, Reubi JC, Krenning EP, and De Jong M. Novel 111In-labeled bombesin analogues for molecular imaging of prostate tumours. European Journal of Nuclear Medicine and Molecular Imaging 2007; 34:1228-1238. 120. Lane SR, Nanda P, Rold TL, Sieckman GL, Figueroa SD, Hoffman TJ, Jurisson SS, and Smith CJ. Optimization, biological evaluation and microPET imaging of copper-64-labeled bombesin agonist, [64Cu-NO2A-(X)-BBN(7−14)NH2], in a prostate tumor xenografted mouse model. Nuclear Medicine and Biology 2010; 37:751-761. 121. Richter S, Wuest M, Krieger SS, Rogers BE, Friebe M, Bergmann R, and Wuest F. Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer. Nuclear Medicine and Biology 2013; 40:1025-1034. 122. Leyton L, Iddon L, Perumal M, Indrevoll B, Glaser M, Robins E, George AJT, Cuthbertson A,  Luthra SK, and Aboagye EO. Targeting somatostatin receptors: Preclinical evaluation of novel 18F-fluoroethyltriazole-Tyr3-octreotate analogs for PET. The Journal of Nuclear Medicine 2011; 52:1441-1448. 123. Yang M, Gao H, Zhou Y, Ma Y, Quan Q, Lang L, Chen K,  Niu G, Yan Y, and Chen X. 18F-Labeled GRPR agonists and antagonists: A comparative study in prostate cancer imaging. Theranostics 2011; 1:220-229. 124. Kahkonen E, Jambor I, Kemppainen J, Lehtio K, Gronroos TJ, Kuisma A, Luoto P, Sipila HJ, Tolvanen T, Alanen K, Silen J, Kallajoki M, Roivainen A, Schafer N, Schibli R, Dragic M, Johayem A, Valencia R, Borkowski S, and Minn H. in vivo imaging of prostate cancer using [68Ga]-labeled bombesin analog BAY86-7548. Clinical Cancer Research 2013; 19:5434-5443. 125. Regoli D., Allogho S.N., Rizzi A., Gobeil F. Jr. Bradykinin receptors and their antagonists. European journal of pharmacology 1998; 348:1-10. 126. Barth M, Bondoux M , Luccarini JM, Peyrou V, Dodey P, Pruneau D, Massardier C, and Paquet JL. From bradykinin B2 receptor antagonists to orally active and selective bradykinin B1 receptor antagonists. The Journal of Medicinal Chemistry 2012; 55:2574-2584. 127. Regoli D. Toward a new anti-inflammatory and analgesic agent. Proceedings of the National Academy of Sciences 2000; 97(14):7676-7677. 128. Notni J, Pohle K, and Wester HJ. Comparative gallium-68 labeling of TRAP-NOTA-, and DOTA-peptides: practical consequences for the future of gallium-68-PET. European Journal of Nuclear Medicine and Molecular Imaging Research 2012; 2:28. 132  129. Yang M., Gao H., Zhou Y., Ma Y., Quan Q., Lang L, Chen K, Niu G, Yan Y, and Chen X. 18F-labeled GRPR agonists and antagonists: a comparative study in prostate cancer imaging. Theranostics 2011; 1:220-229. 130. Nanda P.K., Wienhoff B.E., Rold T.L., Sieckman G.L., Szczodroski A.F., Hoffman T.J., Rogers B.E., and Smith C.J. Positron-emission tomography (PET) imaging agents for diagnosis of human prostate cancer: agonist vs. antagonist ligands. In Vivo 2012 26:583-592. 131. Ginj M., Zhang H., Waser B., Cescato R., Wild D., Wang X., Erchergyi J., Rivier J., Mäcke H.R., and Reubi JC. Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors. Proceedings of the National Academy of Sciences 2006; 103:16436-16441. 132. Faussner A., Bathon J.M., and Proud D. Comparisons of the responses of B1 and B2 kinin receptors to agonist stimulation. Immunopharmacology 1999; 45:13-20. 133. Marceau F., Sabourin T., Houle S., Fortin JP., Petitclerc E., Molinaro G., and Adam A. Kinin receptors: functional aspects. International Immunopharmacology 2002; 2:1729-1739 134. Enquist J., Sanden C., Skröder C., Mathis S.A., Leed-Lundberg L.M.F. Kinin-stimulated B1 receptor signaling depends on receptor endocytosis whereas B2 receptor signaling does not. Neurochemistry Research 2013. 135. Liu Z, Amouroux G, Zhang Z, Pan J, Hundal-Jabal N, Colpo N, Lau J, Perring DM, Bénard F, and Lin KS. (18)F-trifluorobrate derivatives of [des-Arg(10)]kallidin for imaging bradykinin B1 receptor expression with positron emission tomography. Molecular Pharmaceutics 2015; 12(3):974-982. 136. D’Souza CA, McBride WJ, Sharkey RM, Todaro LJ, Goldenberg DM. High-yielding aqueous 18F-labeling of peptides via Al18F chelation. Bioconjugate Chemistry 2011; 22:1793-1803. 137. Liu Z, Li Y, Lozada J, Pan J, Lin KS, Schäffer P, And Perrin DM. Rapid, one-step, high yielding 18F-labeling of an aryltrifluoroborate bioconjugate by isotope exchange at very high specific activity. Journal of Labelled Compounds and Radiopharmaceuticals 2012; 55:491-496. 138. Liu Z, Pourghiasian M, Radtke MA, Lau J, Pan J, Yapp D, Lin KS, Bénard F, and Perrin DM. An organotrifluoroborate for broadly applicable one-step 18F-labelling. Angewandte Chemie International Edition. 2014; 53:11876-11880. 139. Laverman P, D’Souza CA, Eek A, McBride WJ, Sharkey RM, Oyen WJ, Goldenberg DM, and Boerman OC. Optimized labeling of NOTA-conjugated octreotide with F-18. Tumour Biology 2012; 33:427-424. 140. Chatalic KLS, Franssen GM, Van Weerden WM, McBride WJ, Laverman P, De Blois E, Hajjaj B, Brunel L, Goldenbger DM, Fehrentz JA, Martinez J, Boerman OC, and De Jong M. Preclinical comparison of Al18F- and 68Ga-labeled gastrin-releasing peptide receptor antagonists for PET imaging of prostate cancer. The Journal of Nuclear Medicine 2014; 55:2050-2056. 141. Li Y, Liu Z, Harwig CW, Pourghiasian M, Lau J, Lin KS, Schäffer P, Bénard F, and Perrin DM. 18F-Click labeling of a bombesin antagonist with an alkyne-18F-ArBF3 -: in vivo PET imaging of tumors expressing the GRP-receptor. American Journal of Nuclear Medicine and Molecular Imaging 2013; 3: 57-70. 133  142. Fani M, Braun F, Waser B, Beetschen K, Cescao R, Erchegyu J, Rivier JE, Weber WA, Maecke HR, and Reubi JC. Unexpected sensitivity of sst2 antagonists to N-terminal radiometal modifications. The Journal of Nuclear Medicine 2012; 53:1481-1489. 143. Shetty D, Jeong JM, Kim YJ, Lee JY, Hoigebazar L, Lee YS, Lee DS, and Chung JK. Development of a bifunctional chelating agent containing isothiocyanate residue for one step F-18 labeling of peptides and application for RGD labeling. Bioorganic and Medicinal Chemistry 2012; 20:5941-5947. 144. Lütje S, Franssen GM, Sharkey RM, Laverman P, Rossi EA, Goldenberg DM, Oyen WJG, Boerman OC, and McBride WJ. Anti-CEA antibody fragments labeled with [18F]AlF for PET imaging of CEA-expressing tumors. Bioconjugate Chemistry 2014; 25:335-341. 145. Hoigebazar L, Jeong JM, Lee J-Y, Shetty D, Yang BY, Lee YS, Lee DS, Chung JK, and Lee MC. Syntheses of 2-nitroimidazole derivatives conjugated with 1,4,7-triazacyclononane-N,N’-diacetic acid labeled with F-18 using an aluminum complex method for hypoxia imaging. Journal of Medicinal Chemistry 2012; 55:3155-3162. 146. Okarvi SM. Recent progress in fluorine-18 labelled peptide radiopharmaceuticals. European journal of nuclear medicine 2001, 28(7):929-938. 147. Wester HJ, Hamacher K, and Stocklin G. A comparative study of N.C.A. fluorine-18 labeling of proteins via acylation and photochemical conjugation. Nuclear medicine and biology 1996, 23(3):365-372. 148. Wang W., Liu Z., and Li Z. One-step 18F labeling of non-peptidic bivalent integrin αvβ3 antagonist for cancer imaging. Bioconjugate chemistry (2014)  26:24-28. 149. Wängler C., Wängler B., Lehner S., Eisner A., Todica A., Bartenstein P., Hacker M., and Schirrmacher R. A universally applicable 68Ga-labeling technique for proteins. The Journal of Nuclear Medicine (2011) 52(4):586-591. 150. Lu Z., Li Y., Lozada J., Wong M.Q., Greene J., Lin K.S., Yapp D., and Perrin D.M. Kit-like 18F-labeling of RGD-19F-arytrifluoroborate in high yield and at extraordinarily high specific activity with preliminary in vivo tumour imaging. Nuclear Medicine and Biology (2013)  40(6):841-849 151. De Jong M, Maina-Nock T, Kaloudi A, Krenning EP, and Nock BA. [111In-DOTA]MG11 revisited: successful visualization of CCK2R-expressing cancer following a new concept. European Journal of Nuclear Medicine and Molecular Imaging 2013; 40(2):127. 152. Sanden C and Leed-Lundberg LMF. Kinin B1 receptor homo-oligomerization is required for receptor trafficking to the cell surface. International Immunopharmacology 2013; 15:121-128. 153. DePalatis LR, Frazier KA, Cheng RC, and Kotite NJ. Lysine reduces renal accumulation of radioactivity associated with injection of the [177Ly]alpha-[2-(4-aminophenyl) ethyl]-1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic acid-CC49 Fab radioimmunoconjugate. Cancer Research 1995; 55:5288-5295. 154. Melis M, Bijster M, De Visser M, Konijnenberg MW, De Swart J, Rolleman EJ, Boerman OC, Krenning EP, and De Jong M. Dose-response effect of gelofusine on renal uptake and retention of radiolabeled octreotate in rats with CA20948 tumours. European Journal of Nuclear Medicine and Molecular Imaging 2009; 36:1968-1976. 155. Melis M, Valkema R, Krenning EP, and De Jong M. Reduction of renal uptake of radiolabeled octreotate by amifostine coadministration. The Journal of Nuclear Medicine 2012; 53:749-753. 134  156. Bernard BF, Krenning EP, Breeman WAP, Rolleman EJ, Bakker WH, Visser TJ, Mäcke H, and De Jong M. D-lysine reduction of indium-111 octreotide and yttrium-90 octreotide renal uptake. The Journal of Nuclear Medicine 1997; 38:1929-1933. 157. Duchene J and Ahluwalia A. The kinin B1 receptor and inflammation: new therapeutic target for cardiovascular disease.  Current Opinion in Pharmacology 2009; 9:125-131. 158. Tschope C, Heringer-Walther S, Koch M, Spillmann F, Wendorf M, Leitner E, Schultheiss HP, and Walther T. Upregulation of bradykinin B1-receptor expression after myocardial infarction. British Journal of Pharmacology 2000; 129:1537−1538. 159. Cruden NLM, Lang NN, MacGillivray TJ, Uren NG, Fox KAA, and Newby D. Vasomotor and fibrinolytic responses to kinin receptor agonists in the atherosclerotic human lower limb. Heart Vessels 2012; 27:179-185. 160. Merino VF, Todiras M, Mori MA, Sales VMT, Fonseca RG, Saul V, Tenner K, Bader M, and Pesquero JB. Predisposition to atherosclerosis and aortic aneurysms in mice deficient in kinin B1 receptor and apolipoprotein. European Journal of Nuclear Medicine and Molecular Imaging 2009; 87:953−963. 161. Duchene J, Cayla C, Vessillier S, Scotland R, Yamashiro K, Lecomte F, Syed I, Vo P, Marrelli A, Pitzalis C, Cipollone F, Schanstra J,  Bascands JL, Hobbs AJ, Perretti M, and Ahluwalia A. Laminar shear stress regulates endothelial kinin B1 receptor expression and function: Potential implication in atherogenesis. Arteriosclerosis, Thrombosis, and Vascular Biology 2009; 29:1757−1763. 162. Raidoo DM, Ramsaroop R, Naidoo S, Muller-Esterl W, and Bhoola KD. Kinin receptors in human vascular tissue: their role in atheromatous disease. Immunopharmacology 1997; 36:153–160. 163. Passos GF, Medeiros R, Cheng D, Vasilevko V, LaFerla FM, and Cribbs DH. The Bradykinin B1 Receptor Regulates Aβ Deposition and Neuroinflammation in Tg-SwDI Mice. American Journal of Pathology 2013; 182:1740-1749. 164. Pesquero JB, Araujo RC, Heppenstall PA, Stucky CL, Silva JA Jr, Walther T, Oliveira SM, Pesquero JL, Paiva AC, Calixto JB, Lewin GR, and Bader M. Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors. Proceedings of the National Academy of Sciences of the United States of America 2000; 97:8140–8145. 165. Nicolau M, Feltrin MR, and Regoli D. Induction of bradykinin B1 hypotensive receptors in rats by lipopolysaccharide. Canadian Journal of Physiology and pharmacology 1996; 74:337–340. 166. Schremmer-Danninger E, Offner A, Siebeck M, and Roscher AA. B1 bradykinin receptors and carboxypeptidase M are both upregulated in the aorta of pigs after LPS infusion. Biochemical and Biophysical Research Communications 1998; 243:246–252. 167. Ongali B, Campos MM, Petcu M, Rodi D, Cloutier F, Chabot JG, Thibault G, and Couture R. Expression of kinin B1 receptors in the spinal cord of streptozotocin-diabetic rat. NeuroReport 2004; 15:2063−2466. 168. Midaoui AE, Ongali B, Petcu M, Rodi D, de Champlain J, Neugebauer W, and Couture R. Increases of spinal kinin receptor binding sites in two rat models of insulin resistance. Peptides 2005; 26:1323−1330. 169. Mazrin D. Preclinical evaluation of radiopharmaceutical toxicological prerequisite. Nuclear Medicine and Biology 1998 25(8):733-736. 135  170. Koziorowski J, Behe M, Decrisoforo C, Ballinger J, Elsing P, Ferrari V, Kolen Peitl P, Todde S, and Mindt TL. Position paper on requirements for toxicological studies in the specific case of radiopharmaceuticals. European Journal of Nuclear Medicine and Molceular imaging – Radiopharmacy and Chemistry 2016; 1:1. 171. FitzGerald R. Preclinical safety testing of diagnostic and therapeutic radiopharmacuticals – regulatory requirements. ESSR 2014: 17th European Symposium on Radiopharmacy and Radiopharmaceuticals 2014. 

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